Introduction

Plant science has never been more important. The growing and increasingly prosperous human
population needs abundant safe and nutritious food, shelter, clothes, fibre, and renewable energy,
and needs to address the problems generated by climate change, while preserving habitats. These
global challenges can only be met in the context of a strong fundamental understanding of plant
biology and ecology, and translation of this knowledge into field‐based solutions.

Plant science is beginning to address these grand challenges, but it is not clear that the full range
of challenges facing plant science is known or has been assessed. What questions should the next
generation of plant biologists be addressing? To start to answer this question we set out to compile
a list of 100 important questions facing plant science research.

We had three main goals.

 1 We aimed to stimulate discussion amongst the plant science and related communities, and
identify areas of research that would have a substantial impact.
 2 We hoped to encourage plant scientists to think beyond the limits of their own sphere of research
and consider the most important research that could possibly be carried out.
 3 We sought to illustrate the importance and potential of plant science to the broader public.

This paper addresses aims 1 and 2, but questions were selected with all three aims in mind. This
is intended to be a starting point. Research priorities and challenges change continuously and
unpredictably as new concerns and needs arise, and new knowledge is revealed, and it will be
important to review and reassess this list in the future. Here we present, with brief explanations
of their significance, our list of the important questions facing plant science research today.

Methods
Questions were invited online over a 3‐month period
at http://www.100plantsciencequestions.org.uk/index.php. The website was publicized by
email using distribution lists of plant scientists in the UK and abroad, on websites aimed at plant
scientists and farmers, and in a press release, which led to coverage by some news websites and
newspapers. The questions submitted to the website are listed in full
at http://www.100plantsciencequestions.org.uk/viewquestions.php, along with the names of
the people who submitted them, apart from a few cases where submitters chose to be anonymous.
The online consultation process allowed input from contributors with a range of nationalities and
experience. The full list of 350 questions was provided in advance to a panel of 15 individuals
(Steve Barnes, Ruth Bastow, Mark Chase, Matthew Clarke, Claire Grierson, Alastair Fitter, Don
Grierson, Keith Edwards, Graham Jellis, Jonathan Jones, Sandy Knapp, Giles Oldroyd, Guy
Poppy, Paul Temple and Roger Williams) representing the academic, commercial and public
service communities that produce or benefit from plant science research, and able to take part in
a 2‐d workshop at Bristol (UK) in 2009. During the process the list was reduced to 96 questions
by mutual agreement, which we hope will stimulate more local variants particularly adapted to
research and societal priorities in both the developing and developed world. Before the panel
meeting the full list of 350 submitted questions was roughly organized into groups according to
topic. Each panel member independently selected their top 20 questions and these lists were
combined. During this process other possible questions under each topic were suggested and
considered for inclusion. Each question selected by a panel member was discussed by the whole
panel, along with other questions that addressed similar issues. The most important question on
each topic was agreed upon by the whole panel and a final wording chosen. In some cases the
panel decided that a new question was required, and the panel worked together to produce the
wordings for these new questions.
 Results and Discussion
As plant science is a broad and diverse field, we provide brief explanations of the background,
context and prospects for addressing each question with the aim of making the questions
accessible to the broadest possible audience.

There is no ideal way to divide the questions into topic areas. Many questions inevitably and
desirably span more than one category, and some particularly substantial topics merit multiple
questions. For the purposes of this paper, the panel decided to categorize the questions into five
broad areas that reflect the breadth and depth of plant research discussed during the 2‐d workshop:
Society, Environment and adaptation, Species interactions, Understanding and utilizing plant
cells, and Diversity.

A. Society (18)
Here we consider the overall significance of plants and plant science to human society in general.
We open with 10 questions that the panel felt encapsulated the most burning societal issues that
should be addressed by plant science, followed by other societal questions selected by the panel.
More specific biological questions in plant science follow in later sections.

The 10 questions most important to society

 A1.

How do we feed our children’s children?

By 2050 the world population will have reached c. 9 billion people. This will represent a tripling
of the world population within the average lifetime of a single human being. The population is
not only expanding, but also becoming more discerning, with greater demands for energy‐
intensive foods such as meat and dairy. Meeting these increasing food demands over the years to
come requires a doubling of food production from existing levels. How are we going to achieve
this? Through the cultivation of land currently covered in rainforests, through enhanced
production from existing arable land or by changing people’s habits to change food consumption
patterns and reduce food waste? The reality is probably a combination of all three. However, if
we are to reduce the impact of food production on the remaining wilderness areas of the planet
then we need significant investment in agricultural science and innovation to ensure maximum
productivity on existing arable land.

 A2.

Which crops must be grown and which sacrificed, to feed the billions?

The majority of agricultural land is used to cultivate the staple food crops wheat (Triticum
aestivum), maize (Zea mays) and rice (Oryza sativa), the oil‐rich crops soy (Glycine max), canola
(Brassica napus), sunflower (Helianthus spp.) and oil palm (Elaeis guineensis) and commodity
crops such as cotton (Gossypium spp.), tea (Camellia sinensis) and coffee (Coffea spp.). As the
world population expands and meat consumption increases, there is a growing demand for staples
and oil‐rich crops for both human needs and animal feed. Without significant improvements in
yields of these basic crop plants, we will experience a squeeze on agricultural land. It is therefore
essential that we address the yield gap; the difference between future yield requirements and
yields available with current technologies, management and gene pools. Otherwise we may be
forced to choose between production of staple food crops to feed the world population and the
production of luxury crops, such as tea, coffee, cocoa (Theobroma cacao), cotton, fruits and
vegetables.
 A3.

When and how can we simultaneously deliver increased yields and reduce the
environmental impact of agriculture?

The first green revolution of the late 1950s and early 1960s generated unprecedented growth in
food production. However, these achievements have come at some cost to the environment, and
they will not keep pace with future growth in the world population. We need creative and
energetic plant breeding programmes for the major crops world‐wide, with a strong public sector
component. We need to explore all options for better agronomic practice, including better soil
management and smarter intercropping, especially in the tropics. Finally, we need to be able to
deploy existing methods of genetic modification that reduce losses to pests, disease and weeds,
improve the efficiency of fertilizer use and increase drought tolerance. We also need to devise
methods to improve photosynthetic efficiency, and move the capacity for nitrogen fixation from
legumes to other crops. These are all desirable and, with public support, feasible goals.

 A4.

What are the best ways to control invasive species including plants, pests and pathogens?

Invasive species are an increasingly significant threat to our environment, economy, health and
well‐being. Most are nonindigenous (evolved elsewhere and accidentally introduced) and have
been removed from the constraints regulating growth in their native habitat. The best method of
control is to prevent establishment in the first place or to quickly identify establishment and adopt
an eradication programme. However, if an invasive species becomes established many of the
options for removal can cause environmental damage, for example chemical control or
mechanical excavation. Biological control (introduction of a natural predator/pathogen) can work
well as long as the control organism targets only the invasive species. Otherwise there is a risk
that the control organism might also become an invasive species. Alternatives, such as
manipulating existing natural enemies and/or the environment to enhance biological control, are
also being developed. Sustainable solutions are required if we are to deal with the continually
growing problem of invasive species.

 A5.

Considering two plants obtained for the same trait, one by genetic modification and one by
traditional plant breeding techniques, are there differences between those two plants that
justify special regulation?

The products of traditional plant breeding are subject to no special regulations, even though the
wild sources of germplasm often used by breeders may contain new components that have not
been assessed before. A plant derived by genetic modification, however, is highly regulated, even
though the target genotype and the modification itself may both be highly characterized and
accepted as innocuous for their intended use. This is a major exception to the norm for safety
regulation in food and other areas, which is normally based on the properties of the object being
regulated. It is important for food safety and for the public’s perception of science and technology
in general to establish whether there are any objective differences between these groups of
products that justify the different approaches to their regulation.

 A6.

How can plants contribute to solving the energy crisis and ameliorating global warming?

Plants use solar energy to power the conversion of CO2 into plant materials such as starch and
cell walls. Plant material can be burnt or fermented to release heat energy or make fuels such as
 ethanol or diesel. There is interest in using algae (unicellular aquatic plants) to capture
CO2 emissions from power stations at source. Biomass cellulose crops such as Miscanthus ×
giganteus (Poaceae) are already being burnt with coal at power stations. There is understandable
distaste for using food crops such as wheat and maize for fuel, but currently 30% of the US maize
crop is used for ethanol production, and sustainable solutions are being found. Sugarcane
(Saccharum officinarum) significantly reduces Brazil’s imports of fossil fuels. Agave (Agavea
fourcroydes) in hot arid regions can provide very high yields (> 30 T ha−1) of dry matter
with low water inputs compared with other crops. To ameliorate global warming, CO 2 must be
taken out of the air and not put back. There is considerable interest in ‘biochar’ in which plant
material is heated without air to convert the carbon into charcoal. In this form, carbon cannot
readily re‐enter the air, and, if added to the soil, can increase fertility. Carbon markets do not
currently provide sufficient incentive for farmers to grow crops simply to take CO2 out of the air.

 A7.

How do plants contribute to the ecosystem services upon which humanity depends?

Ecosystem services are those benefits we human beings derive from nature. They can be loosely
divided into supporting (e.g. primary production and soil formation), provisioning (e.g. food,
fibre and fuel), regulating (e.g. climate regulation and disease regulation) and cultural (e.g.
aesthetic and recreational) services. Plants are largely responsible for primary production and
therefore are critical for maintaining human well‐being, but they also contribute in many other
ways. The Earth receives virtually no external inputs apart from sunlight, and the regenerative
processes of biological and geochemical recycling of matter are essential for life to be sustained.
Plants drive much of the recycling of carbon, nitrogen, water, oxygen, and much more. They are
the source of virtually all the oxygen in the atmosphere, and they are also responsible for at least
half of carbon cycling (hundreds of billions of metric tons per year). The efficiency with which
plants take up major nutrients, such as nitrogen and phosphorus, has major impacts on agricultural
production, but the application of excess fertilizers causes eutrophication, which devastates
acquatic ecosystems. Plants are already recognized as important for sustainable development (e.g.
plants for clean water) but there are many other ways that plants might contribute. A combined
approach of understanding both the services provided by ecosystems and how plants contribute
to the functioning of such ecosystems will require interdisciplinary collaboration between plant
scientists, biogeochemists, and ecologists.

 A8.

What new scientific approaches will be central to plant biology in the 21st Century?

Biologists now have a good general understanding of the principles of cell and developmental
biology and genetics, and how plants function, change, and adapt to their environment.
Addressing the questions in this list, including those related to generating crops that can deal with
future challenges, will require detailed knowledge of many more processes and species. New
high‐throughput technologies for analysing genomes, phenotypes, protein complements, and the
biochemical composition of cells can provide us with more detailed information in a week than
has ever been available before about a particular process, organism or individual. This is
delivering a deluge of information that is both exhilarating and daunting. The challenge is to
develop robust ways of analysing and interpreting this mountain of data to answer questions and
deliver new insights. The skill sets required to make full use of the new information extend far
beyond those previously expected from biologists. There is general agreement that we need a new
era of collaboration between all types of plant scientists, geographers, geologists, statisticians,
mathematicians, engineers, computer scientists, and other biologists to evaluate complex data,
find new relationships, develop and test hypotheses, and make discoveries. Challenges include
 understanding complex traits and interactions with the environment, generating ‘designer crops’,
and using modelling to predict how different genotypes will cope with alterations in the climate.

 A9.

(a) How do we ensure that society appreciates the full importance of plants?

Plants are fundamental to all life on Earth. They provide us with food, fuel, fibre, industrial
feedstocks, and medicines. They render our atmosphere breathable. They buffer us against
extremes of weather and provide food and shelter for much of the life on our planet. However,
we take plants and the benefits they confer for granted. Given their importance, should we not
pay plants greater attention and give higher priority to improving our understanding of them?
Awareness could be increased through the media, school education, and public understanding of
science activities, but a major step‐change in activity will be required to make a substantial
difference.

 A9.

(b) How can we attract the best young minds to plant science so that they can address Grand
Challengesfacing humanity such as climate change, food security, and fossil fuel
replacement?

Everyone knows that we need doctors, and the idea that our best and brightest should go into
medicine is embedded in our culture. However, even more important than medical care is the
ability to survive from day to day; this requires food, shelter, clothes, and energy, all of which
depend on plants. Beyond these essentials, plants are the source of many other important products.
As is clear from the other questions on this list, plant scientists are tackling many of the most
important challenges facing humanity in the 21st Century, including climate change, food
security, and fossil fuel replacement. Making the best possible progress will require exceptional
people. We need to radically change our culture so that ‘plant scientist’ (or, if we can rehabilitate
the term, ‘botanist’) can join ‘doctor’, ‘vet’ and ‘lawyer’ in the list of top professions to which
our most capable young people aspire.

 A10.

How do we ensure that sound science informs policy decisions?

It is important that policy decisions that can affect us all, for example environmental protection
legislation, are based on robust and objective evidence underpinned by sound science. Without
this, the risk of unintended consequences is severe. Ongoing dialogue between policy makers and
scientists is therefore critical. How do we initiate and sustain this dialogue? How do we ensure
that policy makers and scientists are able to communicate effectively? What new mechanisms are
needed to enable scientists to respond to the needs of policy makers and vice versa?

The Supporting Information, Notes S1, provides explanato ry text for the remaining questions.

 A11.

How can we translate our knowledge of plant science into food security?
Plant science clearly has a role to play in the food security agenda on the supply side of the equation by enhancing
crop nutrient content and productivity. Most progress is likely to be made if the gap between plant and crop
physiology is more strongly bridged, and the tools and concepts available at the field level are utilized. But to be
most effective this approach needs to be better integrated with the social, environmental, economic and political
factors that also influence the food system
 "Plant science can help to bolster supply, by raising the availability and nutritional content of crops, and
increasing the efficiency of 'post-farm-gate' practices," they write and continue to underline the clear need to
increase food production per unit land area.
To this end, cropping systems must be designed to use water, nutrients and sunlight more efficiently. Efficiency
gains made thus far are the result of crop breeding and better management. However, the actual production of
food occurs in the field and not the laboratory and this must be recognized by plant scientists.
The researchers illustrate how plant science can contribute with a line of examples. First of all further
improvements in water-use efficiency can be made by better matching crop water demand to supply, and by
reducing water loss. And improving resistance to the major pests and diseases, such as cereal rusts, late-blight
of potatoes, corn-borers in maize, and bacterial and fungal diseases of rice, is key to raising production per unit
land.
Next the global prevalence of nutrient deficiency necessitates an increase in the nutrient content of crops,
especially the essential protein, amino acid and micronutrient content. Molecular approaches can help in this
respect, as illustrated by work to enhance vitamin A content in rice and protein quality in maize.
And finally could crops for example also be modified to increase the efficiency of post-farm-gate food chain
activities, such as food processing and storage. For instance, the gluten content of wheat could be modified to
reduce staling in bread, and thereby food waste.
And fruits, vegetables and grains could be modified to retain more beneficial compounds, such as antioxidants,
when undergoing processing operations such as drying, dehydration and canning; staple crops such as cassava,
maize and sweet potato have already been bred to increase retention of provitamin A carotenoid after
processing, cooking and storage. And such modifications will be especially important given the increased risk
of reduced vitamin and micronutrient concentration in crops with climate change.

 A12.

Which plants have the greatest potential for use as biofuels with the least effects on
biodiversity, carbon footprints and food security?
Among terrestrial plants "Jatropha" has good potential for biodiesel production. It has high yield, less
maintenance requirement, and is free from food vs. feed debate.
However, in recent times, microalgae as a source of triglyceride feedstock have gained huge
attention worldwide because of its higher productivity with high oil content.
Biodiesel, a promising substitute as an alternative fuel has gained significant attention due to the predicted
shortness of conventional fuels and environmental concern. The utilization of liquid fuels such as biodiesel
produced from Jatropha oil by transesterification process represents one of the most promising options for the
use of conventional fossil fuels. The Jatropha oil is converted into jatropha oil methyl ester known as biodiesel
prepared in the presence of homogeneous acid catalyst. The physical properties such as density, flash point,
Kinematic viscosity, Cloud point and Pour point were found out for Jatropha oil and Jatropha methyl ester. The
same characteristics study was also carried out for the diesel fuel for obtaining the base line data for analysis.
The values obtained from the Jatropha methyl ester is closely matched with the values of conventional diesel
and can be used in the existing diesel engine without any modification.

This study, based on an interdisciplinary scientific assessment2 of the world’s agro-ecological productivity and
socio-economic conditions at national, regional and global levels, highlights that current polices supporting
firstgeneration biofuels production and consumption need to be reconsidered in the light of direct and indirect
impacts on food security, agriculture and the environment. The results of the study indicate that first-generation
biofuels development as has been promoted by national policies is conflicting with goals of achieving food
security, results in only modest increases of agricultural value added in developing countries, achieves net
greenhouse gas savings only after 2030, creates additional risks of deforestation and threats to biodiversity. The
target of achieving a ten percent biofuels share in transport fuel at the global level can be met but this causes
about a fifteen percent increase in the number of people at risk of hunger (i.e., and increase 140-150 million
people at risk of hunger as compared to 2008 numbers). In particular the poor urban population, subsistence
farmers and the landless in developing countries will bear the brunt. Moreover anticipated greenhouse gas
savings from biofuels use can only be expected after 30 to 50 years and that is about the time when climate
change impacts will result in increased agricultural vulnerability, particularly in a number of developing
countries. To avoid negative impacts of biofuels on food security, any use of firstgeneration biofuels would
need to be preceded by concerted research efforts to increase agricultural productivity. The foremost priority is
to ensure that future food demand is met and only then any surplus production would be available for biofuels.
Among the first-generation feedstocks, sustainable sugar cane production under rain-fed conditions in former
pastures and grassland areas offers environmentally and economically an attractive biofuel option as
demonstrated in the case of Brazil. 38 OFID PAMPHLET SERIES 38 OFID PAMPHLET SERIES 38 39
Second-generation biofuels produced on land other than cultivated land required for food and feed productions
may offer opportunities for the development of environmentally cleaner and economically competitive biofuels.
 However this will depend on the timely delivery of efficient and effective second-generation conversion
technologies as well as advances in feedstock production and land use regulation. Food security, energy security
and climate change mitigation are all critical to social, economic and environmental sustainability, not only at
the national level but also globally. A successful resolution of these challenging issues requires the goodwill
and commitment of all nations to work together. Biofuel development polices have a direct impact on these
triple challenges and yet it is national polices with national interests that have been the driving force of setting
biofuel targets. The global and spatial agro-ecological and socio-economic methodology and assessments
presented in this study provide the analytical means and science-based knowledge to evaluate policy options
towards making the right choices that recognize the pitfalls and mobilize the opportunities to make progress
towards achieving national and global sustainable development. For more than thirty years there have been
countless debates on the concerns of feeding cereals to livestock in a world where over one-sixth of the
population has lived with chronic hunger and debilitating poverty. There is a risk that we might end up for the
next thirty years debating the fallacy of feeding cereals to cars. This time the situation though is different as the
entire world’s population will be affected if we fail to deal with the challenges of coping with climate change,
providing clean energy and ensuring food security, all of which are interrelated and need to be tackled together.

 A13.

Can crop production move away from being dependent on oil‐based technologies?
“Eating Oil” was the title of a book which was published in 1978 following the first oil crisis in 1973 (1). The aim
of the book was to investigate the extent to which food supply in industrialized countries relied on fossil fuels.
In the summer of 2000 the degree of dependence on oil in the UK food system was demonstrated once again
when protestors blockaded oil refineries and fuel distribution depots. The fuel crises disrupted the distribution
of food and industry leaders warned that their stores would be out of food within days. The lessons of 1973
have not been heeded.
Today the food system is even more reliant on cheap crude oil. Virtually all of the processes in the modern food
system are now dependent upon this finite resource, which is nearing its depletion phase.
Moreover, at a time when we should be making massive cuts in the emissions of greenhouse gases into the
atmosphere in order to reduce the threat posed by climate change, the food system is lengthening its supply
chains and increasing emissions to the point where it is a significant contributor to global warming.
The organic sector could be leading the development of a sustainable food system. Direct environmental and
ecological impacts of agriculture ‘on the farm’ are certainly reduced in organic systems. However, global trade
and distribution of organic products fritter away those benefits and undermine its leadership role.
Not only is the contemporary food system inherently unsustainable, increasingly, it is damaging the
environment.
The systems that produce the world’s food supply are heavily dependent on fossil fuels. Vast amounts of oil and
gas are used as raw materials and energy in the manufacture of fertilisers and pesticides, and as cheap and
readily available energy at all stages of food production: from planting, irrigation, feeding and harvesting,
through to processing, distribution and packaging. In addition, fossil fuels are essential in the construction and
the repair of equipment and infrastructure needed to facilitate this industry, including farm machinery,
processing facilities, storage, ships, trucks and roads. The industrial food supply system is one of the biggest
consumers of fossil fuels and one of the greatest producers of greenhouse gases.
Ironically, the food industry is at serious risk from global warming caused by these greenhouse gases, through
the disruption of the predictable climactic cycles on which agriculture depends. But global warming can have
the more pronounced and immediate effect of exacerbating existing environmental threats to agriculture, many
of which are caused by industrial agriculture itself. Environmental degradation, water shortages, salination, soil
erosion, pests, disease and desertification all pose serious threats to our food supply, and are made worse by
climate change. But many of the conventional ways used to overcome these environmental problems further
increase the consumption of finite oil and gas reserves. Thus the cycle of oil dependence and environmental
degradation continues.
Industrial agriculture and the systems of food supply are also responsible for the erosion of communities
throughout the world. This social degradation is compounded by trade rules and policies, by the profit driven
mindset of the industry, and by the lack of knowledge of the faults of the current systems and the possibilities
of alternatives. But the globalisation and corporate control that seriously threaten society and the stability of
our environment are only possible because cheap energy is used to replace labour and allows the distance
between producer and consumer to be extended.
However, this is set to change. Oil output is expected to peak in the next few years and steadily decline
thereafter. We have a very poor understanding of how the extreme fluctuations in the availability and cost of
both oil and natural gas will affect the global food supply systems, and how they will be able to adapt to the
decreasing availability of energy. In the near future, environmental threats will combine with energy scarcity to
cause significant food shortages and sharp increases in prices – at the very least. We are about to enter an era
where we will have to once again feed the world with limited use of fossil fuels. But do we have enough time,
 knowledge, money, energy and political power to make this massive transformation to our food systems when
they are already threatened by significant environmental stresses and increasing corporate control?
The modern, commercial agricultural miracle that feeds all of us, and much of the rest of the world, is completely
dependent on the flow, processing and distribution of oil, and technology is critical to maintaining that flow.
* Oil refined for gasoline and diesel is critical to run the tractors, combines and other farm vehicles and
equipment that plant, spray the herbicides and pesticides, and harvest/transport food and seed
* Food processors rely on the just-in-time (gasoline-based) delivery of fresh or refrigerated food
* Food processors rely on the production and delivery of food additives, including vitamins and minerals,
emulsifiers, preservatives, colouring agents, etc. Many are oil-based. Delivery is oil-based
* Food processors rely on the production and delivery of boxes, metal cans, printed paper labels, plastic trays,
cellophane for microwave/convenience foods, glass jars, plastic and metal lids with sealing compounds. Many
of these are essentially oil-based
* Delivery of finished food products to distribution centres in refrigerated trucks. Oil-based, daily, just-in-time
shipment of food to grocery stores, restaurants, hospitals, schools, etc., all oil-based; customer drives to grocery
store to shop for supplies, often several times a week
https://www.resilience.org/stories/2005-04-01/why-our-food-so-dependent-oil/

 A14.

How can we use plant science to prevent malnutrition?

“What I find most amazing about plants is that they’re a great source for thousands and thousands of chemicals,” says Dr.
Asaph Aharoni of the Weizmann Institute of Science’s Department of Plant Sciences. “A single plant can produce 10,000 to
20,000 metabolites, or active compounds, which we can use for nutrition and in products like drugs and cosmetics.”

Dr. Aharoni studies how plants regulate the production of metabolites during development and under conditions of stress, such
as insect attack or exposure to ultraviolet (UV) radiation. His goal is to identify novel genes that are associated with, for
example, better nutritional quality and enhanced resistance to insect pests. In the future, his findings could help scientists
develop better crops. “We’re interested in how we can help address a major challenge: improving the nutritional quality of
plants and solving problems of malnutrition around the world,” he says.

For a number of years, Dr. Aharoni has been investigating the process of tomato fruit maturation and ripening. During this
process, the tomato fruit accumulates large amounts of carotenoids such as lycopene and beta-carotene. These compounds are
antioxidants and are important in human nutrition. The body converts beta-carotene, for instance, into vitamin A, a nutrient that
plays a key role in vision, bone growth, and regulating the immune system.

To understand how tomato ripening is triggered, Dr. Aharoni and his team alter the expression of certain genes that are involved
in the process. “One of the ‘mutant’ tomatoes we created is highly interesting,” he says. “When we over-express a particular
gene that is a regulator of the ripening process, it induces ripening and an accumulation of carotenoids in the sepals [a leaf-like
part of the plant where carotenoids do not normally build up].”

Dr. Aharoni explains that research on the regulation of the pathways that generate lycopene and beta-carotene could lead to the
engineering of crops with higher levels of these compounds. He notes that, according to a report by the World Health
Organization, improved vitamin A nutrition could prevent approximately 1-2 million deaths annually among children under five
years of age. Vitamin A deficiency greatly increases the risk of a child’s dying from an infectious disease such as measles.

His team is also working to learn how plants regulate the production of thiamin (vitamin B1). This vitamin helps the body
convert carbohydrates into energy and is essential for the functioning of the nervous system, muscles, and heart. A few years
ago, Dr. Aharoni and his colleagues identified a mechanism that regulates the levels of thiamin in plants. “This mechanism
works like a thermostat. If we manipulate it, we can increase the amount of thiamin a plant produces. We were able to do this in
a model plant called Arabidopsis thaliana, and now we hope to do it in rice.” Most of the naturally occurring thiamin in rice is
in the outer parts of the grain, which are removed during the polishing process. As a result, in some developing countries—for
example, in Asia, where polished white rice is the staple food—thiamin deficiency is a common health problem.

In recent work, Dr. Aharoni’s team discovered the gene that produces a variety of pink-skinned tomato. Pink tomatoes, which
are particularly popular in the Middle East, have a slightly sweeter taste, a thinner skin, and contain less lycopene than red
tomatoes. The scientists found that the relevant gene is a sort of “master switch” that regulates an entire network of other genes,
overseeing the production and quantities of many metabolites in the tomato fruit. Now researchers can use this gene, known as
 SIMYB12, as a marker to predict the future color of a fruit in the very early stages of development, before the plant has even
flowered. This could accelerate the creation of new tomato varieties, a process that normally takes a decade or more.

Dr. Aharoni received his MSc in agricultural science from the Hebrew University of Jerusalem and his PhD in plant sciences
from Wageningen University in The Netherlands, joining the Weizmann Institute in 2004. Dr. Aharoni says he realized early in
his career that he was drawn to basic research. “As a result, in my lab today, we’re tackling questions using basic research and
applying what we’re learning to improve plant nutrition and our quality of life,” he says.

The Weizmann Institute is an ideal place to do such work, as it has a long history of research aimed at enhancing the nutritional
quality of food crops, increasing their resistance to disease, and reducing the use of harmful fertilizers and herbicides. In coming
years, this work will be one of the Institute’s highest priorities as its scientists respond to pressing global issues such as climate
change, hunger, obesity, and malnutrition.

https://www.weizmann-usa.org/news-media/feature-stories/fighting-hunger-and-malnutrition-with-powerful-
plants

 A15.

How can we use knowledge of plants and their properties to improve human health?
The concept of growing crops for health rather than for food or fiber is slowly
changing plant biotechnology and medicine. Rediscovery of the connection between plants
and health is responsible for launching a new generation of botanical therapeutics that
include plant-derived pharmaceuticals, multicomponent botanical drugs, dietary
supplements, functional foods and plant-produced recombinant proteins. Many of these
products will soon complement conventional pharmaceuticals in the treatment,
prevention and diagnosis of diseases, while at the same time adding value to agriculture.
Such complementation can be accelerated by developing better tools for the efficient
exploration of diverse and mutually interacting arrays of phytochemicals and for the
manipulation of the plant's ability to synthesize natural products and complex proteins.
This review discusses the history, future, scientific background and regulatory issues
related to botanical therapeutics.

 A16.

How do plants and plant communities (morphology, colour, fragrance, sound, taste
etc.) affect human well‐being?

Health and well-being benefits of plants

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Concentration and Memory. Being around plants helps people concentrate better in the home and
workplace. Studies show that tasks performed while under the calming influence of nature are performed
better and with greater accuracy, yielding a higher quality result. Moreover, being outside in a natural
environment can improve memory performance and attention span by twenty percent.
Keeping ornamental plants in the home and in the workplace increases memory retention and
concentration. The calming influence of natural environments is conducive to positive work
environments by increasing a person’s ability to concentrate on the task at hand. Work performed
under the natural influence of ornamental plants is normally of higher quality and completed with a
much higher accuracy rate than work done in environments devoid of nature. Going outside or being
under the influence of plants can increase memory retention up to twenty percent, a recent University
of Michigan study showed (Sewach). The effect of nature in the home and in the workplace serves to
stimulate both the senses and the mind, improving mental cognition and performance. (Bisco Werner
1996; Brethour 2007; Frank 2003; Pohmer 2008; Serwach 2008; Shibata, 2001, 2004; Yannick 2009)

Educational Programs / Special Events. Parks and botanical gardens often play host to educational
programs and special events, which contribute to the cultural awareness and education of the community
(children especially). This raises environmental consciousness and appreciation.
Installing a park or botanical garden in a community has many direct benefits to residents, but an
auxiliary benefit of having such a naturalized landmark in the community is the special events and
cultural opportunities it brings to people who might not otherwise be exposed. Botanical gardens and
zoos often create educational programs for children in order to teach them how the value of being
environmentally-conscious and conserving the environment. They can also impact adults in the
community as well, creating a cultural awareness of the importance of natural environments. Parks
and gardens foster an appreciation for nature that often instills in residents a sense of responsibility
for the caring of and protection of the environment. (Appleseed, Inc. 2009, Dubey 2007, Nadel 2005,
Phipps Botanical Gardens and Conservatory 2010)

Flowers Generate Happiness. Having flowers around the home and office greatly improves people’s
moods and reduces the likelihood of stress-related depression. Flowers and ornamental plants increase
levels of positive energy and help people feel secure and relaxed.
Keeping flowers around the home and in the workplace greatly reduces a person’s stress levels.
Natural aesthetic beauty is soothing to people, and keeping ornamental flowers around the home
environment is an excellent way to lower levels of stress and anxiety. People who keep flowers in
their home feel happier, less stressed, and more relaxed. As a result of the positive energy they
derive from the environment, the chances of suffering from stress-related depression are decreased
as well. Overall, adding flowers to your home or work environment reduces your perceived stress
levels and makes you feel more relaxed, secure, and happy. Flowers can help you achieve a more
optimistic outlook on your life, bringing you both pleasing visual stimulation and helping you to
increase your perceived happiness.

(Brethour 2007, Collins 2008, Dunnet 2000, Etcoff 2007, Frank 2003, Haviland-Jones 2005, Hartig
2010, McFarland 2010, Rappe 2005, Waliczek 2000)

Health and Recreation. Access to parks and recreational activities is positively correlated with rates of
physical activity, which improves mood and contributes to overall healthiness. Health care costs are
subsequently reduced.
Parks and urban green spaces impact people’s health by providing them with an inexpensive (often
free) and convenient recreational service. There is a positive correlation between the presence of a
park in a neighborhood and the level of physical activity of the residents; people are much more likely
to exercise when there is a no-cost, aesthetically pleasing area or facility for them to use. As a result,
residents of neighborhoods with beautiful parks are much healthier; their increase in exercise makes
them less susceptible to physical ailments and more resilient against minor illnesses. As a result,
these residents do not spend as much each year on health care and medical treatment, because they
require fewer of these services Healthy people are happier people; residents who exercise often have
excellent overall health and therefore have a more positive mental outlook. The presence of parks in
neighborhoods encourages residents to exercise, thus improving their physical state and enabling
them to more fully enjoy their lives.

(Appleseed, Inc. 2009, Mitchell, 2008, Bisco Werner 1996, Brethour 2007, Fjeld 2000, Frank 2003,
Sallis 1995, Shoemaker 2009, The Trust for Public Land 2008, Wolf 2004b)

Accelerates Healing Process. The presence of plants in hospital recovery rooms and/or views of
aesthetically-pleasing gardens help patients to heal faster, due to the soothing affects of ornamental
horticulture.
Shrubs, trees, and flowers have a practical application in hospitals: the presence of plants in patient
recovery rooms greatly reduces the time necessary to heal. The soothing effects of ornamental
flowers and plants are so great that simply having daily views of flowers and other ornamental plants
in landscaped areas outside patient recovery room significantly speed up recovery time. Another
technique to decrease recovery time is horticulture therapy, where patients care for and nurture
plants themselves. Patients who physically interact with plants experience a significantly reduced
recovery time after medical procedures. (Brethour 2007, Frank 2003, Friend 2008, Lohr 2000, Park,
2009, Pennsylvania Landscape and Nursery Assn. 2009, Ulrich 1984)

Improves Relationships/Compassion. Research shows that people who spend extended lengths of time
around plants tend to have better relationships with others. This is due to measurable increases in feelings
of compassion; another effect of exposure to ornamental plants.
Ornamental plants affect the levels of compassion that people feel for others. Studies have shown
that people who spend more time around plants are much more likely to try and help others, and
often have more advanced social relationships. People who care for nature are more likely to care for
others, reaching out to their peers and forming shared bonds resulting from their common interests.
Extended exposure to nature and wildlife increases people’s compassion for each other as it
increases people’s compassion for the environment in which they live. In short, being around plants
can help to improve relationships between people and increase their concern and empathy toward
others. (Brethour 2007, Etcoff 2007, Frank 2003, Hagen 2009, Haviland-Jones 2005, Pohmer 2008,
Rappe 2005)
Improved Human Performance/Energy. Spending time in natural environments makes people better at
doing their jobs. It also increases energy levels and feelings of vitality.
Spending time in nature gives people an increased feeling of vitality, increasing their energy levels
and making them feel more animated. Their performance levels are, in turn, increased by this
improved state of mind. Natural environments induce a positive outlook on life, making people feel
more alive and active. When people experience increased vigor, they put more of themselves and
their energy into their work. Plants can help people to improve their performance at work and at home
by increasing their perceived vitality and giving them more feelings of added energy. (Bernstein 2009,
Brethour 2007, Bringslimark 2007, Dravigne 2008, Etcoff 2007, Kaplan 1995, Kuo 2001a,
Pennsylvania Landscape and Nursery Assn. 2009, Rappe 2005, Shoemaker 1992, Univ. of
Rochester 2010)

Learning. Research shows that children who spend time around plants learn better. In addition, being
around natural environments improves the ability of children with Attention Deficit Disorder to focus,
concentrate, and engage more with their surrounding environment.
Keeping plants in a child’s learning environment enhances learning capabilities by helping them to
focus and concentrate. This improves their ability to learn new things and makes it easier for them to
absorb and retain information. Ornamental plants are conducive to generating a positive learning
environment, reducing children’s tendency towards distraction and helping them to be better able
concentrate on school work. Specifically for children with problems paying attention, adding plants to
the classroom can have a dramatic positive effect on the way they learn. For example, children with
Attention Deficit Disorder, learning in a natural environment can help them to engage more in the
classroom, improving their focus and concentration on the task at hand. The soothing effects of
natural aesthetic beauty help to minimize the distractions that would otherwise occupy their minds. By
altering the environment in which children learn, plants can help them to learn better. (Faber Taylor
2001a, Frank 2003, Kellert 2002, Kuo 2004, Lieberman 1998)

Medicinal Properties. Cultivating plants is beneficial to humankind because of the many medicinal
properties of trees and foliage plants.
One of the more obvious benefits of plants and trees is that many of them have valuable medicinal
properties. Cultivating plants helps humanity because it provides opportunities for additional scientific
studies of the possible positive medicinal values of plants. Natural herbal remedies are simple and
holistic methods for treating common illnesses and maladies. Some can be made in the home and
are a natural way to treat minor complaints. By cultivating plants we can continue to cultivate our
knowledge of the natural world and arm ourselves with more defenses against disease and infection.
(Brethour 2007)

Mental Health. Studies have proven that people who spend more time outside in nature have better mental
health and a more positive outlook on life.
People who spend more time outside in nature have a significantly more positive outlook on life than
people who spend a great deal of time indoors. Communing with the natural world increases people’s
feelings of vitality and energy, and consequently has a large positive effect on their overall mental
health. Being outside around trees and ornamental horticulture is proven to improve people’s mental
health, and give them a more positive outlook on their lives. People who spend time outside every
day are less likely to be depressed or stressed, and thus have fewer burdens on their mental health.
(Barnicle 2003, Faber Taylor 2001b, Grinde 2009, McFarland 2010, Morikami Museum and Japanese
Gardens 2009, Shoemaker 2009, Wolf 2004b)

Perceived Quality of Life. People associate beautifully landscaped areas with a higher quality of life. This
is important in attracting businesses and sustaining growth in the community.
Beautiful natural landscapes not only improve the aesthetics of the community, they also affect
resident’s perceived quality of life. People associate living in areas with a great deal of natural beauty
with a higher quality of life. A high quality of life, in turn, benefits the entire community, because
residents spend more money and positively affect the economy and social pulse of the town and can
also attract new businesses. Thriving communities are ones in which natural beauty is appreciated as
a part of an overall high quality of life, which is why installing landscaping is crucial to both the
success and happiness of the individual and the public. (Brethour 2007, Bisco Werner 1996,
McFarland 2010, Nadel 2005, Phipps Botanical Gardens and Conservatory 2010, Wolf 2004b, Younis
2008)

Reduce Community Crime / Community Cohesion. Neighborhoods with beautiful parks tend to have
less crime. This is due in part to the effect that parks have on a community; parks give people a reason to
come together and become a tight-knit community. People who care about their neighborhood parks are
much more likely to get politically involved when businesses threaten to downsize them. Increasing
political activism in the community is another positive outcome of cultivating a love for neighborhood
parks.
Neighborhoods with beautiful parks and landscaping have reduced crime rates. This is due to the
increase in community cohesion that occurs as a neighborhood rallies around a beautiful local
landmark. When residents feel greater pride in the beauty of where they live, they are much less likely
to detract from it (either by graffiti or endangering people within it). Communities that choose to clean
up their parks and beautify crime-ridden neighborhoods have less crime and fewer criminals to deal
with. Parks can positively affect the community be reducing criminal acts and bringing residents
together. Cohesion in the community is critical to the success of the community as a whole, and this
can be achieved through unifying people around a park or botanical garden. Parks decrease
incentives for people to commit crimes in the community, and at the same time help to bring
neighbors together. They can also increase local political activism. As businesses and urban
expansion threatens to downsize parks in the community, more and more people are banding
together in a political effort to save their parks. Parks inspire people to come together and fight for
what they know is holding them together as a community. (Appleseed, Inc. 2009; Austin 2002; Bisco
Werner 1996; Brethour 2007; Brown 2010; Brunson 1998; Frank 2003; Gorham 2009; Harnik 2009;
Inerfield 2002; Kuo 2001b, 2001c, 2003; Pennsylvania Landscape and Nursery Assn. 2009; The
Trust for Public Land 2008; Wolf 2004b)

Reduce Stress. Studies show that people who spend time cultivating plants have less stress in their lives.
Plants soothe human beings and provide a positive way for people to channel their stress into nurturing.
Participation in gardening and landscaping activities is an effective way to reduce levels of stress.
Studies have shown that people who nurture plants and garden have less mental distress than
others. Gardening provides people with a positive way to channel their stress and frustration into
something beautiful that provides them with comfort and joy. Part of the effects of gardening come
from the satisfaction people get from nurturing and helping a living thing grow. Plants and gardening
soothe people because they help them turn their stressful feelings into something positive which
gives them pleasure. By helping them transform their stress into a more positive emotion, gardening
also gives people an excellent coping mechanism for their daily frustrations. Nurturing plants reduces
stress levels and gives people a way to cope with their negative feelings. (Mitchell, 2008, Brethour
2007, Bringslimark 2007, Frank 2003, Kohlleppel 2002, McFarland 2010, Pohmer 2008, Ulrich 1991,
Waliczek 2000)

Therapeutic Effects of Gardening. Gardening can act as therapy for people who have undergone trauma.
The act of nurturing something is a way for people to work through the issues surrounding traumatic events
and improve their mental health.
Gardening can have therapeutic effects on people who have undergone trauma, either mental or
physical. The act of nurturing a plant can provide victims with a way to work through their issues and
heal their wounds, whether they are on the surface of the skin or deeper. Cultivating plants also
improves their mental states and helps to put them in a better place for recovering. Gardening is a
therapeutic tool that can be used to help put people in a better psychological state during recovery
and help them to work past the mental barriers that could impede their healing. (Aldous 2000,
Barnicle 2003, Brethour 2007, Collins 2008, Morikami Museum and Japanese Gardens 2009,
Pohmer 2008, Rappe 2005, Stoneham 1995)

Traffic Safety / Driver Satisfaction. Beautifying road ways can have the dual effect of increasing driver
satisfaction with the roadside landscape and creating a natural median. Drivers are much less likely to
accidentally drive over a median if there is a landscaped area between oncoming lanes of traffic.
Beautifying traffic medians not only improves the aesthetics of the roadways, it also affects driver
attitudes. Studies show that drivers are more at ease on roadways with natural landscaping, and are
much more inclined to think positively about the community that they are driving through if the
roadways are beautiful. Furthermore, adding trees to roadways creates a sort of natural obstruction
which could reduce the likelihood of cars crossing medians into oncoming traffic lanes. This improves
driver safety and makes the community a safer place for everyone to live in. Landscaped areas
between oncoming lanes of traffic could decrease the number of accidents occurring due to drivers
crossing the median and make the road a safer place. (Wolf, 2001b, 2001c, 2006)

Upgrade Effect. As parts of the community begin to improve their urban green spaces, other areas will be
forced to stay competitive and beautify their areas as well. The upgrade effect benefits the entire
community, as neighborhoods and businesses encourage each other to landscape and beautify the
community.
As more businesses and neighborhoods take on the task of beautifying their surroundings, other
competing areas will be forced to follow suit. In other words, as a community works to better itself,
other parts of the area will be forced to upgrade as well to keep drawing people in; this phenomenon
is known as the upgrade effect. The upgrade effect positively affects everyone, because it keeps
communities from ignoring the benefits of landscaping and developing green spaces, it forces
competition and keeps the area looking beautiful. Neighborhoods will be encouraging each other to
keep beautifying the landscaping, setting off a cycle of self-improvement that has positive ripple
effects outwards to all sectors of the community. (Bisco Werner 1996, Brethour 2007)
 https://ellisonchair.tamu.edu/health-and-well-being-benefits-of-plants/

 A17.

How can we use plants and plant science to improve the urban environment?
Urban planning is a vital process in determining the functionality of future cities. It is predicted that at least two
thirds of the world’s citizens will reside in towns and cities by the middle of this century, up from one third in
the middle of the previous century. Not only is it essential to provide space for work and dwelling, but also for
their well-being. Well-being is inextricably linked with the surrounding environment, and natural landscapes
have a potent positive effect. For this reason, the inclusion and management of urban green infrastructure has
become a topic of increasing scientific interest. Elements of this infrastructure, including green roofs and
façades are of growing importance to operators in each stage of the planning, design and construction process in
urban areas. Currently, there is a strong recognition that “green is good”. Despite the positive recognition of
urban greenery, and the concerted efforts to include more of it in cities, greater scientific attention is needed to
better understand its role in the urban environment. For example, many solutions are cleverly engineered
without giving sufficient consideration to the biology of the vegetation that is used. This review contends that
whilst “green is good” is a positive mantra to promote the inclusion of urban greenery, there is a significant
opportunity to increase the contribution of plant science to the process of urban planning through both green
infrastructure, and biomimicry.

Introduction

This review has been approached by considering key environmental parameters which pose opportunities and
challenges in the built environment; namely, light, heat, water and carbon dioxide. In each section, the
opportunities to use plants in situ, or to learn from them through biomimicry, are discussed in relation to an
over-arching question. Current research regarding the ability of urban environments to respond to the challenges
posed by fluctuations in these environmental parameters is then discussed and opportunities for interdisciplinary
learning between plant science and building related disciplines are presented. Green space has long been
associated with well-being. Plants in cities provide us colour and character in our streets, and a range of
ecosystem services such as shading, cooling, control of storm water run-off, and CO2 fixation. Cities are
efficient in their provision of infrastructure and public services as well as a concentration of opportunities for
employment, business and inter-personal relationships. As a result, urban centres are growing around the globe,
and it has been predicted that more than 70% of the human population will live in one by the year 2050 [1]. As
urbanisation increases globally, we need our urban plants to do more than decorate the city. Plants being sessile
are highly capable of successful environmental adaptation, including tolerance to extremes of heat, light, water
and CO2. Plant science has traditionally sought to understand the biology of plants and to exploit this through
agriculture and horticulture. Increasingly, however, the expertise of plant scientists is also likely to be important
for the growing of plants in new environments and for design inspiration. Plants can therefore provide both
direct and indirect solutions, the latter through biomimicry. Plants can help to provide thermal comfort, energy
savings, storm water mitigation or carbon sequestration in urban environments. A better knowledge of how
green spaces interact with the built environment, and how people utilise them is vital to maximising the health
and wellbeing of those living in the city. The diversity, and longevity of functional ecosystems within a city
which require little maintenance and provide a greater range of ecosystem services will be vital to the success of
urban greening schemes. In Europe and the US, the integration of plants into urban environments is being led by
urban planners and policy makers where there is a strong recognition of the importance of green space [2–4].
Thus far, the consideration of how to incorporate plants into many urban settings on a large scale could be
characterised as “green is good”, with less consideration given to precisely what kind of green is best.
Ecological research has had a significant impact on the use of plants in the urban environment for example in
the promotion of biodiversity [5–8] and in terms of biological suitability [9]. Meanwhile, there is a need for a
greater contribution from plant scientists in the evaluation of which plant should be used for each given function
and how plants respond biologically, to the challenges that urban environments pose such as increased heat,
highly transient drought and flooding events and elevated CO2.

This review has argued that there are opportunities for a greater integration of plant science in building
disciplines to stimulate further innovation in urban design and planning. In each section, current research and
opportunities were discussed from the perspective of an over-arching question concerning the management of
key environmental parameters in urban environments. In order to summarise the discussions and research
herein, we are, in this conclusion, identifying important areas for each environmental parameter (light, heat,
water, and CO2) where plant research has enhanced the adaptation of the built environment to environmental
 parameters, or where there is a need for further research to develop the impact of plant science on urban design
and planning.
https://orca.cf.ac.uk/96209/1/buildings-06-00048.pdf

 A18.

How do we encourage and enable the interdisciplinarity that is necessary to achieve the
UN’s Millennium Development Goals which address poverty and the environment?

B. Environment and adaptation (21)

Plants have evolved to cope with changes in their environment but their adaptability has not
necessarily been preserved as crops have been developed from wild species. Assessing and
utilizing the capacity of plants to adapt should help to increase the use of more marginal land for
cultivation, and enhance agricultural production despite changes in climate.

 B1.

How can we test if a trait is adaptive?

How do we know whether a trait of an organism is an adaptation or

not?
Paul Lucas, Ph.D in Biochemistry
Answered Mar 24, 2018

Stephen Jay Gould and Richard Dawkins argued this point for decades. The argument only stopped with Gould’s
death. Gerhard Adam is correct on the difference between “adaptation” and “adaptive”.

First, an adaptation is inherited. To take Gerhard’s example, the ability to manipulate abstract ideas is inherited
(for all people), while the ability to do mathematics is usually not.

Or, another example. Having good balance while bipedal is inherited, but the ability to perform on a balance beam
in gymnastics is not.

Gould argued that not all traits were adaptations or even adaptive. His favorite example was human male nipples.
In human females, nipples are definitely an adaptation. but in males it is a side effect of developmental biology:
part of the developmental program is activated in males.

Another example is the spur on one of the ankle bones of pandas. In the forelimbs of pandas, there is an extension
of a wrist bone to make a serviceable “thumb”. That is an adaptation to help the panda eat bamboo. However,
there is no functionality to the spur on the ankle bone. Instead, the same genes are involved in limb development
for fore and hind limbs. Those activated genes produce the spur on the ankle bone.

So, the second criteria is looking for a way that the trait aids the individual in the struggle for existence, or did so in
the past. If there is such a way, then the trait is considered adaptive. If there is no known functionality to aid in the
struggle for existence, then the trait is possibly not adaptive. Some traits — such as the vermiform appendix —
were possibly adaptations to some past selection pressure, but are not now in the current environment.

https://www.quora.com/How-do-we-know-whether-a-trait-of-an-organism-is-an-adaptation-or-not

 B2.

What is the role of EPIGENETIC PROCESSES in modulating response to the environment

during the life span of an individual?
Plants depend on epigenetic processes for proper function. Epigenetics has been defined as "the study of changes in gene
function that are mitotically and/or meiotically heritable and that do not entail a change in DNA sequence" (Wu et al.
 2001). Epigenetic examines proteins' interactions with DNA and its associated components, including histones and various
modifications such as methylation, which alter the rate or target of transcription. Epigenetic mechanisms are required for
proper regulation while epi-alleles and epi-mutants, much like their genetic complements, describe changes in phenotype
associated with distinct epigenetic circumstance. There has been scientific enthusiasm for the study of epigenetics in
plants because of their long-standing importance in agriculture.
Plants still need to protect themselves from harmful pathogens, just like animals, and using pesticides becomes harder
and harder as the number of crops increases. Professor of Plant Environmental from the Institute for Sustainable Food
and the Grantham Centre Signalling, Jurriaan, researched on improving plants’ immune systems so that they can deal
with pathogens and other threats by using traits inherited through epigenetic modifications, rather than relying on
pesticides. Epigenetics is quite hard to define, but Jurriaan refers to epigenetic traits as those that are stable, heritable,
and cannot be explained by variation in DNA sequence.

Unlike genetic modifications, which occur due to a change in the genetic sequence code of DNA, epigenetic modifications
don't change the sequence code at all. Instead, they involve biochemical modifications to the DNA or the proteins
associated with the DNA (collective referred to as the chromatin).

Some chromatin are packed very tightly, others less so, giving rise to differences in the transcription or transcriptional
responsiveness of genes. This means that genes in very tightly packed chromatin are often not expressed, whereas genes
in very lightly packed chromatin can be expressed to much higher levels. These changes cause plants to become
phenotypically different (have different physical characteristics), while remaining genotypically identical (have the same
genetic code).

 B3.

Are there untapped potential benefits to developing perennial forms of currently annual
crops?

PDF-perennial

 B4.

Can we generate a step‐change in C 3 crop yield through incorporation of a C 4 or

intermediate C 3 /C 4 or crassulacean acid metabolism (CAM) mechanism?

C3, C4, and CAM plants

How the C4 and CAM pathways help minimize photorespiration.
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Key points:
 Photorespiration is a wasteful pathway that occurs when the Calvin cycle enzyme
rubisco acts on oxygen rather than carbon dioxide.
 The majority of plants are \text C_3C3start text, C, end text, start subscript, 3, end
subscript plants, which have no special features to combat photorespiration.
 \text C_4C4start text, C, end text, start subscript, 4, end subscript plants minimize
photorespiration by separating initial \text {CO}_2CO2start text, C, O, end text, start
subscript, 2, end subscript fixation and the Calvin cycle in space, performing these steps
in different cell types.
 Crassulacean acid metabolism (CAM) plants minimize photorespiration and save water
by separating these steps in time, between night and day.
Introduction
High crop yields are pretty important—for keeping people fed, and also for keeping
economies running. If you heard there was a single factor that reduced the yield of wheat
by 20\%20%20, percent and the yield of soybeans by 36\%36%36, percent in the United
States, for instance, you might be curious to know what it was^11start superscript, 1, end
superscript.
As it turns out, the factor behind those (real-life) numbers is photorespiration. This
wasteful metabolic pathway begins when rubisco, the carbon-fixing enzyme of the Calvin
cycle, grabs \text O_2O2start text, O, end text, start subscript, 2, end subscript rather
than \text {CO}_2CO2start text, C, O, end text, start subscript, 2, end subscript. It uses up
fixed carbon, wastes energy, and tends to happens when plants close their stomata (leaf
pores) to reduce water loss. High temperatures make it even worse.
Some plants, unlike wheat and soybean, can escape the worst effects of photorespiration.
The \text {C}_4C4start text, C, end text, start subscript, 4, end subscript and CAM
pathways are two adaptations—beneficial features arising by natural selection—that
allow certain species to minimize photorespiration. These pathways work by ensuring
that Rubisco always encounters high concentrations of \text{CO}_2CO2start text, C, O,
end text, start subscript, 2, end subscript, making it unlikely to bind to \text O_2O2start
text, O, end text, start subscript, 2, end subscript.
In the rest of this article, we'll take a closer look at the \text C_4C4start text, C, end text,
start subscript, 4, end subscript and CAM pathways and see how they reduce
photorespiration.
\text C_3C3start text, C, end text, start subscript, 3, end
subscript plants
A "normal" plant—one that doesn't have photosynthetic adaptations to reduce
photorespiration—is called a \text {C}_3C3start text, C, end text, start subscript, 3, end
subscript plant. The first step of the Calvin cycle is the fixation of carbon dioxide by
rubisco, and plants that use only this "standard" mechanism of carbon fixation are
called \text C_3C3start text, C, end text, start subscript, 3, end subscript plants, for the
three-carbon compound (3-PGA) the reaction produces^22squared. About 85\%85%85,
percent of the plant species on the planet are \text C_3C3start text, C, end text, start
subscript, 3, end subscript plants, including rice, wheat, soybeans and all trees.
 Image of the C3 pathway. Carbon dioxide enters a mesophyll cell and is fixed
immediately by rubisco, leading to the formation of 3-PGA molecules, which contain
three carbons.
\text C_4C4start text, C, end text, start subscript, 4, end
subscript plants
In \text C_4C4start text, C, end text, start subscript, 4, end subscript plants, the light-
dependent reactions and the Calvin cycle are physically separated, with the light-
dependent reactions occurring in the mesophyll cells (spongy tissue in the middle of the
leaf) and the Calvin cycle occurring in special cells around the leaf veins. These cells are
called bundle-sheath cells.
To see how this division helps, let's look at an example of \text C_4C4start text, C, end
text, start subscript, 4, end subscript photosynthesis in action. First, atmospheric \text
{CO}_2CO2start text, C, O, end text, start subscript, 2, end subscript is fixed in the
mesophyll cells to form a simple, 444-carbon organic acid (oxaloacetate). This step is
carried out by a non-rubisco enzyme, PEP carboxylase, that has no tendency to bind \text
O_2O2start text, O, end text, start subscript, 2, end subscript. Oxaloacetate is then
converted to a similar molecule, malate, that can be transported in to the bundle-sheath
cells. Inside the bundle sheath, malate breaks down, releasing a molecule of \text
{CO}_2CO2start text, C, O, end text, start subscript, 2, end subscript. The \text
{CO}_2CO2start text, C, O, end text, start subscript, 2, end subscript is then fixed by
rubisco and made into sugars via the Calvin cycle, exactly as in \text C_3C3start text, C,
end text, start subscript, 3, end subscript photosynthesis.
 In the C4 pathway, initial carbon fixation takes place in mesophyll cells and the Calvin
cycle takes place in bundle-sheath cells. PEP carboxylase attaches an incoming carbon
dioxide molecul to the three-carbon molecule PEP, producing oxaloacetate (a four-carbon
molecule). The oxaloacetate is converted to malate, which travels out of the mesophyll
cell and into a neighboring bundle-sheath. Inside the bundle sheath cell, malate is broken
down to release CO_22start subscript, 2, end subscript, which then enters the Calvin
cycle. Pyruvate is also produced in this step and moves back into the mesophyll cell,
where it is converted into PEP (a reaction that converts ATP and Pi into AMP and PPi).
This process isn't without its energetic price: ATP must be expended to return the three-
carbon “ferry” molecule from the bundle sheath cell and get it ready to pick up another
molecule of atmospheric \text {CO}_2CO2start text, C, O, end text, start subscript, 2, end
subscript. However, because the mesophyll cells constantly pump \text{CO}_2CO2start
text, C, O, end text, start subscript, 2, end subscript into neighboring bundle-sheath cells
in the form of malate, there’s always a high concentration of \text{CO}_2CO2start text,
C, O, end text, start subscript, 2, end subscript relative to \text O_2O2start text, O, end
text, start subscript, 2, end subscript right around rubisco. This strategy minimizes
photorespiration.
The \text C_4C4start text, C, end text, start subscript, 4, end subscript pathway is used in
about 3\%3%3, percent of all vascular plants; some examples are crabgrass, sugarcane
and corn. \text C_4C4start text, C, end text, start subscript, 4, end subscript plants are
common in habitats that are hot, but are less abundant in areas that are cooler. In hot
conditions, the benefits of reduced photorespiration likely exceed the ATP cost of
moving \text {CO}_2CO2start text, C, O, end text, start subscript, 2, end subscript from
the mesophyll cell to the bundle-sheath cell.

CAM plants
Some plants that are adapted to dry environments, such as cacti and pineapples, use
the crassulacean acid metabolism (CAM) pathway to minimize photorespiration. This
name comes from the family of plants, the Crassulaceae, in which scientists first
discovered the pathway.
 Image of a succulent.
Image credit: "Crassulaceae," by Guyon Morée (CC BY 2.0).

Instead of separating the light-dependent reactions and the use of \text{CO}_2CO2start

text, C, O, end text, start subscript, 2, end subscript in the Calvin cycle in space, CAM
plants separate these processes in time. At night, CAM plants open their stomata,
allowing \text {CO}_2CO2start text, C, O, end text, start subscript, 2, end subscript to
diffuse into the leaves. This \text{CO}_2CO2start text, C, O, end text, start subscript, 2,
end subscript is fixed into oxaloacetate by PEP carboxylase (the same step used by \text
C_4C4start text, C, end text, start subscript, 4, end subscript plants), then converted to
malate or another type of organic acid^33cubed.
The organic acid is stored inside vacuoles until the next day. In the daylight, the CAM
plants do not open their stomata, but they can still photosynthesize. That's because the
organic acids are transported out of the vacuole and broken down to
release \text{CO}_2CO2start text, C, O, end text, start subscript, 2, end subscript, which
enters the Calvin cycle. This controlled release maintains a high concentration
of \text{CO}_2CO2start text, C, O, end text, start subscript, 2, end subscript around
rubisco^44start superscript, 4, end superscript.
 CAM plants temporally separate carbon fixation and the Calvin cycle. Carbon dioxide
diffuses into leaves during the night (when stomata are open) and is fixed into
oxaloacetate by PEP carboxylase, which attaches the carbon dioxide to the three-carbon
molecule PEP. The oxaloacetate is converted to another organic acid, such as malate. The
organic acid is stored until the next day and is then broken down, releasing carbon
dioxide that can be fixed by rubisco and enter the Calvin cycle to make sugars.
The CAM pathway requires ATP at multiple steps (not shown above), so like \text
{C}_4C4start text, C, end text, start subscript, 4, end subscript photosynthesis, it is not an
energetic "freebie." ^33cubed However, plant species that use CAM photosynthesis not
only avoid photorespiration, but are also very water-efficient. Their stomata only open at
night, when humidity tends to be higher and temperatures are cooler, both factors that
reduce water loss from leaves. CAM plants are typically dominant in very hot, dry areas,
like deserts.
Comparisons of \text C_3C3start text, C, end text, start
subscript, 3, end subscript, \text C_4C4start text, C, end
text, start subscript, 4, end subscript, and CAM plants
\text C_3C3start text, C, end text, start subscript, 3, end subscript, \text C_4C4start text,
C, end text, start subscript, 4, end subscript and CAM plants all use the Calvin cycle to
make sugars from \text {CO}_2CO2start text, C, O, end text, start subscript, 2, end
subscript. These pathways for fixing \text {CO}_2CO2start text, C, O, end text, start
subscript, 2, end subscript have different advantages and disadvantages and make plants
suited for different habitats. The \text C_3C3start text, C, end text, start subscript, 3, end
subscript mechanism works well in cool environments, while \text C_4C4start text, C,
end text, start subscript, 4, end subscript and CAM plants are adapted to hot, dry areas.
Both the \text {C}_4C4start text, C, end text, start subscript, 4, end subscript and CAM
pathways have evolved independently over two dozen times, which suggests they may
give plant species in hot climates a significant evolutionary advantage^55start superscript,
5, end superscript.
Separation of initial \text
{CO}_2CO2start text, C, O,
end text, start subscript, 2, end
subscript fixation and Calvin Stomata Best adapted
Type cycle open to
\text C_3C3start
text, C, end text,
start subscript, 3, Cool, wet
end subscript No separation Day environments
\text C_4C4start Between mesophyll and bundle- Hot, sunny
text, C, end text, sheath cells (in space) Day environments
 Separation of initial \text
{CO}_2CO2start text, C, O,
end text, start subscript, 2, end
subscript fixation and Calvin Stomata Best adapted
Type cycle open to
start subscript, 4,
end subscript

Very hot, dry

CAM Between night and day (in time) Night environments

https://www.khanacademy.org/science/biology/photosynthesis-in-plants/photorespiration--c3-c4-cam-
plants/a/c3-c4-and-cam-plants-agriculture

 B5.

How do plants regulate the proportions of storage reserves laid down in various plant parts?

Storage Reserve Accumulation in Arabidopsis: Metabolic and Developmental

Control of Seed Filling
Sébastien Baud,1 Bertrand Dubreucq, Martine Miquel, Christine Rochat, and Loïc Lepiniec
Author information Copyright and License information Disclaimer
This article has been cited by other articles in PMC.

Abstract
Go to:

INTRODUCTION
In seed plants, or spermaphyta, seed development is a key process linking two sporophytic generations that
enables the life cycle to temporarily pause, allowing survival and dispersion (Bewley, 1997; Vicente-Carbajosa
and Carbonero, 2005). A seed must package together all of the genetic material and nutrients required to allow
successful propagation of the species. Seed formation is an intricate process that requires the coordinated
growth of three tissues of distinct origins. The embryo and the endosperm are zygotic tissues, the development
of which is initiated by the double fertilisation of the embryo sac. The embryo and the endosperm are protected
by maternally-derived integuments, which constitute the seed coat. Seed development can be divided into
embryo morphogenesis and maturation, the latter being characterised by storage compound accumulation,
acquisition of tolerance to desiccation, growth arrest and the entry into a dormancy period broken upon
germination (Harada, 1997). Beyond the diversity of size, shape and means of dispersal, one common element
in plant seeds is the storage of reserve compounds that will be mobilised to fuel post-germinative seedling
growth until seedling photosynthesis can be efficiently established. These components usually consist of starch,
triacylglycerols (TAGs) and specialised storage proteins (SSPs), the relative proportions of which vary greatly
depending on the species considered. Storage compounds contribute up to 90% of the seed dry weight and they
also constitute the economic value of seeds in most field crops. Given the importance of seeds in the human and
animal diet, centuries of agricultural research have been directed at improving the qualitative and quantitative
traits associated with seed components. More recently, some molecular genetic approaches have been
developed to modify both the quality and quantity of seed products (Mazur et al., 1999). Today, there is a
tremendous interest in understanding the genetic controls of seed development and metabolism.
In oleaginous species such as Brassica napus (rapeseed), one of the world's major oilseed crops, storage
products consist of proteins and TAGs synthesised mainly in the embryo. As a Brassicaceae, Arabidopsis
thaliana constitutes an excellent model system to investigate storage compound accumulation in oilseeds. The
development of extensive tools for its genetics and molecular dissection together with the emergence of
analytical procedures adapted to its very small seeds have led to major advances in isolating and characterising
the factors involved in the metabolic and developmental control of seed filling.
Go to:

THE SEED MATURATION PROCESS IN A. THALIANA

Seed Development
Seed development comprises two major phases: embryo morphogenesis (EM) and maturation (Figure 1A). In
the model plant A. thaliana, several studies have been carried out to provide a complete description of this
developmental process (Baud et al., 2002; Fait et al., 2006; Goldberg et al., 1994; Jenik et al., 2007). When
plants are grown under a natural night and day regime, seed development takes roughly three weeks, the
pollination event being referred to as day 0. Due to the autogamy of A. thaliana, this event cannot be directly
observed. Yet, day 0 can be easily identified on the shoot since pollination is concomitant with the appearance
of the petals that form a white dip at the extremity of the flower bud. Embryo morphogenesis is initiated by the
double fertilisation of the embryo sac that gives rise to the zygote (2n) and endosperm (3n). The zygote firstly
divides asymmetrically, the resulting apical and basal cells giving rise to the embryo and its suspensor,
respectively. Through a series of cell divisions, the embryo progressively acquires the basic plan of the plant. At
the end of EM, 6 days after pollination (DAP), torpedo-shaped embryos exhibit a polarity along an apical-basal
axis. At the apical end of this axis, the shoot meristem is flanked by the cotyledons and is connected to the basal
root meristem by the hypocotyl and root. Superimposed on this axis are radial and bilateral symmetries.
Together with different cell fate patterning events, these symmetries lay the foundations for all post-embryonic
development (Jenik et al., 2007; Jürgens, 2001). During EM, the triploid endosperm develops in two steps: a
coenocytic stage followed by a cellularised and differentiated stage (Berger et al., 2006; Olsen, 2001). Seeds
exhibit a white or pale yellow colour and their water content is high (> 80%).
 Open in a separate window
Figure 1.
Seeds of Arabidopsis thaliana
(A) Schematic overview of seed development in A. thaliana adapted from Baud et al. (2002). The relative volume of
endosperm and embryo within the seed are presented throughout the seed developmental process. Embryos are
represented at the globular, torpedo, early-bent cotyledon, and upturned-U stages. A time course of carbohydrate and
major storage compound (e.g. triacylglycerols and seed storage proteins) content is shown in parallel. E.M., embryo
morphogenesis. (B) Composition of mature A. thaliana seeds (Wassilewskija accession). (C) Fatty acid composition of
mature A. thaliana seeds (Wassilewskija accession). PUFA, polyunsatu-rated fatty acids; VLCFA, very long chain fatty
acids.
The maturation phase is characterised by the accumulation of storage compounds and the acquisition of
dormancy and desiccation tolerance (Goldberg et al., 1994). This process can be conveniently divided into three
stages (Figure 1A). From 7 to 10 DAP early maturation corresponds to the embryo growth phase resulting in the
formation of a fully developed embryo that fills the seed (Raz et al., 2001). During this phase, the ratio between
the embryo volume and the endosperm volume is gradually reversed: the embryo goes through a period of
cellular division and expansion whereas the endosperm is degraded and reduced to one cell layer surrounding
the embryo. This rapid increase in embryo volume is accompanied by the accumulation of photosynthetic
pigments, so that early maturing seeds turn green. Early maturing embryos exhibit elevated starch
concentrations, while the synthesis and accumulation of storage lipids and proteins are initiated (Baud and
Graham, 2006; Hills, 2007). During the second phase of the maturation process, ranging from 11 to 16 DAP,
starch level drops regularly whereas high rates of fatty acid and protein synthesis can be measured in the
embryo. These sustained biosynthetic activities result in a steady increase in seed dry weight throughout this
phase (Baud et al., 2002). Finally, late maturation occurs between 17 and 20 DAP. Storage compound synthesis
ends while the embryo becomes metabolically quiescent and tolerant to desiccation. In late-maturing seeds, the
water content declines sharply from 32% to less than 10%. In spite of this drastic loss of water, syntheses
continue, with raffinose and stachyose being specifically stored during this late maturation stage (Baud et al.,
2002; Fait et al., 2006). The accumulation of such oligosaccharides, together with the increase detected in both
sucrose and trehalose levels, may participate to the protection of membranes and proteins, thus contributing to
the acquisition of tolerance desiccation (Bailly et al., 2001; Hoekstra et al., 2001).

Composition of Mature Dry Seeds

Seeds from the accessions Landsberg erecta (Ler) or Wassilewskija (Ws) usually weigh 20 μg when produced
in a greenhouse, whereas seeds from the Cape Verde Islands (Cvi) accession weigh 35 μg (Alonso-Blanco et al.,
1999; Baud et al., 2002). If Cvi seeds are almost twice as heavy as Ler seeds, the accession Cvi yields on
average about 40% fewer seeds than Ler. Seed size is determined both by maternal and non-maternal genetic
factors, and the final cell number and cell size of the seed coat and the zygotic tissues can exhibit significant
variations (Alonso-Blanco et al., 1999). Some authors have proposed that a cross talk between maternal and
zygotic controls may constitute the primary regulator of the coordinated control of seed size in A.
thaliana (Garcia et al., 2005). TRANSPARENT TESTA GLABRA2 (TTG2; At2g37260), KIP RELATED
PROTEIN2 (KRP2; At3g50630), and the HAIKU (IKU) genetic pathway that involves
the IKU1 locus, IKU2 (At3g19700), and MINISEED3 (At1g55600), participate into this complex regulatory
network (Garcia et al., 2003; Luo et al., 2005). Likewise, APETALA2 (At4g36920) has been shown to function
outside the boundaries of flower meristem and flower organ development to participate in the regulation of seed
mass (Jofuku et al., 2005; Ohto et al., 2005).
In dry seeds, cells of the embryo and endosperm are packed full of protein storage vacuoles (PSVs) and oil
bodies. The SSPs and TAGs thus accumulated account for roughly 30–40% of the seed dry weight (DW) each
(Figure 1B; Baud et al., 2002). PSVs are often localised in the centre of the embryonic cell, close to the nucleus,
whereas oil bodies, which occupy about 60% of the cell volume in the cotyledons of mature embryos, are rather
found at the periphery of the cell (Mansfield and Briarty, 1992). Two types of SSPs are stored in PSVs referred
to as 2S albumins and 12S globulins (for a review, see Fujiwara et al., 2002). The TAGs stored in oil bodies
consist of esters of glycerol and fatty acids (Figure 1C; Miquel and Browse, 1995). When analysing the seed
composition of 360 A. thaliana accessions, the modal oil content is found to be 38% of DW, with most
accessions studied lying within the range 33–43% (O'Neill et al., 2003). Despite coming from diverse
geographical locations, seeds of all the ecotypes analysed so far contain identical fatty acids, but exhibit
reproducible variations in the relative proportions of those fatty acid species (Millar and Kunst, 1999). For
instance, very long chain fatty acid (VLCFA) content ranges from 13 to 21% of total fatty acids while
polyunsatu-rated fatty acid (PUFA) content ranges from 53 to 66% of total fatty acids (O'Neill et al., 2003). The
separation of mature Columbia-0 seeds into embryo and endosperm has revealed that fatty acids in the
endosperm tissues compose approximately one-tenth of those present in whole seeds (Penfield et al., 2004).
Interestingly, if all the fatty acids detected in the embryo are also present in the endosperm, the latter contains
proportionally high levels of 16:1Δ9 (or 16:1n7, palmitoleic acid), 18:1Δ11 (or 18:1n7, vaccenic acid), and
20:1Δ13 (or 20:1n7, paullinic acid) long chain fatty acids (LCFAs), accounting for more than 50% of the total n7
monoun-saturated fatty acid present in the whole seed. Around 20% of endosperm fatty acids are n7
monounsaturated compared with only 2% in the embryo (Penfield et al., 2004). Contrarily to SSPs or TAGs,
oligosaccharides like sucrose, raffinose, and stachyose account for only 2% of the mature seed DW. Yet,
variations in the content of those components can be observed among various accessions, with Cvi seeds storing
very low quantities of oligosac-charides of the raffinose series for instance (Bentsink et al., 2000).
Go to:

SEED STRUCTURE AND NUTRIENT SUPPLY

Post-phloem Transport
Seeds are mostly heterotrophic organs, dependent on nutrients supplied by the parent plant for their growth and
development (Zhang et al., 2007). Nutrients are imported through the phloem, and nutrient loading of seeds is a
spatially and temporally dynamic process (Patrick and Offler, 2001). The seed anatomy in A. thaliana is
different from that described for well-characterised legumes and cereals (Weber et al., 2005). In A. thaliana, the
unique vascular bundle terminates at the end of the funiculus. There is no vascular tissue within the seed and
most of the nucellar cells have degenerated in the fully developed seed. The seed coat consists of five cell
layers: the innermost endothelial layer, followed by two cell layers each of inner and outer integuments
(Lepiniec et al., 2006). Two symplastic domains, corresponding to the outer and the inner integuments, have
been identified (Stadler et al., 2005). The outer integument allows movement of unloaded nutrients and
represents a symplastic extension of the funicular phloem. The transfer between the outer integument and the
inner integument, between the inner integument and the endosperm, and between the endosperm and the
embryo might be apoplastic (Kim and Zambryski, 2005). Three apoplastic borders may consequently separate
the phloem from the maturing embryo; carrier-mediated transports may be required to transport nutrients across
these borders. While the outer integument may be compared, to a certain extent, to the vascular compartment in
the seed coat of grain legumes, the inner integument is speculated to function as the ground tissue of legume
grains, which releases the nutrients from the testa (Stadler et al., 2005). During seed development, dominant
sinks for nutrient loading thus shift from integuments early in development, to filial tissues during later stages
of development. Released nutrients are mainly accumulated in endosperm during EM. Then, during early
maturation, the endosperm functions as a nutrient source for the growing embryo (Hill et al., 2003). During EM,
the embryo and its suspensor form a single symplast (Kim et al., 2005a; Stadler et al., 2005) and the suspensor
is assumed to have a nutritive function for the young embryo (Yeung and Meinke, 1993). This function is most
likely limited to the early steps of embryo development (globular stage) since the connectivity between
suspensor and embryo stops with the specification of the hypophysis (triangular stage). Then, the suspensor
begins to degenerate (heart stage). As the maturing embryo develops, the functional aperture of plasmodesmata
between embryonic cells is down regulated so that four subdomains of symplastic transport are formed within
the embryo (torpedo stage): the shoot apex subdomain, the cotyledon subdomain, the hypocotyl subdomain, and
the root subdomain (Kim et al., 2005a).

Seed Transporters
Sucrose represents the major form in which photosynthetically assimilated carbon is transported in plants.
Because of the absence of symplastic linkage between maternal and filial tissues in A. thaliana, sucrose must
cross several apoplastic borders to reach filial tissues. At each of the apoplastic boundaries both export steps
into the apoplastic space and import steps into the next symplastic isolated cell layer are required. The
mechanisms involved in export processes are not well understood. Among the various transport mechanisms
that can contribute to an efficient uptake on the import side of the apoplastic border, energy dependent, proton-
symporting carrier proteins have been shown to play a role of the utmost importance (Patrick et al.,
1995; Schmidt et al., 2007; Sauer, 2007). The complete sequencing of the A. thaliana genome revealed nine
putative sucrose transporters. At least three of them are expressed in developing seeds. AtSUC3 (At2g02860)
exhibits a widespread expression pattern that includes the embryo suspensor and embryonic root tip; its
implication in uptake of sucrose by the maturing embryo remains to be demonstrated (Meyer et al.,
2004). AtSUC5 (At1g71890), which is induced in the endosperm during EM and early maturation, plays an
important but transient role in the nutrition of filial tissues during early seed development (Baud et al., 2005).
The case of AtSUC2 (At1g22710) is more ambiguous: although transcriptomic data have pointed out its up
regulation in maturing seeds (Ruuska et al., 2002), neither its detailed expression pattern nor its function in
maturing seeds has been elucidated so far.
Given the complexity of amino acid transport and the high number of putative transporters in A. thaliana, our
understanding of amino acid supply to maturing seeds is still very partial. Within the amino acid transporter
superfamily 1 (ATF1), two subfamilies, the amino acid permeases (AAPs) and lysine histidine transporter
(LFTs), encode functional transporters with a broad substrate range. In the second superfamily, the amino acid-
polyamine-choline (APC) superfamily, transporters of the cationic amino acid transporter-family (CATs) were
characterised as more specific for cationic and neutral amino acids (Schmidt et al., 2007). To date, two
members of the AAP subfamily are thought to be involved in amino acid supply to the developing
seed. AtAAP1 (At1g58360) is expressed during EM and early maturation in the filial tissues (Hirner et al.,
1998). Likewise, AtAAP8 (At1g10010) is induced in developing seeds (Okumoto et al., 2002) and plays a
critical role for the uptake of amino acids like aspartate and glutamate during embryo morphogenesis and
growth (Schmidt et al., 2007). Beyond AAPs, the peptide transporter AtPTR2 (At2g02040) may participate in
the supply of reduced organic nitrogen compounds to young developing seeds (Song et al., 1997).
Data concerning the transport and storage of minerals during the seed maturation process are even scarcer.
Mineral deposits are located inside the PSVs of mature seeds, in inclusions called globoids (Gillespie et al.,
2005). They are composed of phytin, a salt of phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate) and its
cations (mostly Mg, K, and Ca) (Lott et al., 1995; Otegui et al., 2002). In A. thaliana, the seed iron is also stored
in globoids of the PSVs, predominantly in the provascular strands of the embryo (Kim et al., 2006; Lanquar et
al., 2005). AtOPT3 (At4g16370), a peptide transporter, and the vacuolar iron uptake transporter VIT1
(At2g01770) have been shown to play a critical role in iron nutrition of developing seeds (Kim et al.,
2006; Stacey et al., 2002; Stacey et al., 2007). The exact mechanism involved remains to be characterised.
Finally, the ATNRT2.7 (At5g14570) nitrate transporter was recently shown to control nitrate accumulation in
the PSVs of late maturing seeds (Chopin et al., 2007). In dry seeds, N from nitrate represents less than one
thousandth of seed nitrogen and may not be regarded as a storage form of nitrogen. However, the role of nitrate
as a signalling molecule during germination may be important.
Go to:

METABOLIC FLUXES IN THE MATURING EMBRYO

A Set of Complementary Tools Adapted to Investigate Seed Metabolism

The emergence of A. thaliana as a major plant model system together with the development of extensive tools
for its genetic and molecular dissection has led to major advances in the understanding of many developmental
and physiological processes, including seed development and maturation (Girke et al., 2000). The complete
sequencing of the A. thaliana genome (Arabidopsis Genome Initiative, 2000) has provided considerable insights
into the nature and number of proteins involved in the metabolic pathways required for storage compound
synthesis. These data have been further improved by the careful annotation of genes related to seed metabolism
by researchers who have knowledge of these biological processes (Beisson et al., 2003). The use of large-scale
chemical mutagenesis experiments and the emergence of T-DNA mutant collections have then allowed to
isolate and characterise new actors (enzymes, transcription factors) of the seed maturation process. The
exploitation of natural variation for seed composition among a collection of seeds originating from a wide range
of geographical locations throughout the world has proved highly valuable for elucidating the regulation of
adaptive traits (O'Neill et al., 2003). Due to their very small size, seeds of A. thaliana have posed a technical
challenge that has led to the emergence of experimental procedures adapted to this material. Sets of ESTs from
developing seeds (White et al., 2000) and microarrays displaying seed-expressed genes (Girke et al.,
2000; Buckhout and Thimm, 2003) have been produced. Sensitive and high throughput metabolite assays for
the precursors of starch and fatty acid synthesis have been developed (Gibon et al., 2002). Probing in
vivo metabolism by stable isotope labelling of storage lipids and proteins in developing embryos of B. napus has
then allowed obtaining maps of the metabolic fluxes through central metabolism (Schwender and Ohlrogge,
2002; Schwender et al., 2004b). Finally, considering that the three tissues composing the seed are very different
and should, if possible, be analysed separately (Hill et al., 2003), techniques like in situ hybridisation,
immunolocalisation, and in situ histochemistry (Baud and Graham, 2006) have provided key elements to
understand seed metabolism and physiology. However, high throughput procedures adapted to seed material are
not yet available for these techniques.

Primary Metabolism of Maturing Embryos

In B. napus, the main organic compounds furnished to the maturing embryos consist of sucrose, glucose, Gln,
Glu, and Ala (Schwender and Ohlrogge, 2002; Schwender et al., 2006). In sink organs, incoming sucrose can be
cleaved via two distinct pathways involving either invertase (EC 2.4.1.13) or sucrose synthase (SUS; EC
3.2.1.26). In several species, SUS activity is a marker for storage accumulation activity (Craig et al., 1999).
Among the six members of the AtSUS gene family, two isoforms are strongly induced during early- (SUS2,
At5g49190) or mid-maturation (SUS3; At4g02280) (Baud et al., 2004b). However, recent results demonstrate
that this pair is not essential for seed filling (Bieniawska et al., 2007) and that the invertase pathway participates
to the cleavage of incoming sucrose, at least in maturing embryos of Brassica napus (Schwender et al., 2003).
Cleavage of sucrose generates a hexose-phosphate pool that fuels three distinct biochemical pathways (Figure
2): (i) the transient starch biosynthesis pathway in the plastids (see below), (ii) the oxidative pentose phosphate
pathway (OPPP), and (iii) the cytosolic and plastidial glycolytic pathways. These hexose-phosphates are
thought to be massively metabolised through the glycolytic pathway before utilisation for fatty acid synthesis.
In oil seeds, maturing embryos do have a complete glycolytic pathway in the cytosol and in the plastids (Dennis
and Miernyk, 1982; Kang and Rawsthorne, 1994). The extent to which both pathways are utilised in the
conversion of carbohydrates into precursors of fatty acid biosynthesis is highly debated. Transcriptomic data
suggest that a major route involves the cytosolic glycolytic pathway up to phosphoenolpyruvate (PEP) (Ruuska
et al., 2002; White et al., 2000), this compound being imported into the plastid before subsequent conversion to
pyruvate (Kubis et al., 2004). The essentially irreversible transphosphorylation from PEP and ADP to pyruvate
and ATP is catalysed by pyruvate kinase (PK, EC 2.7.1.40) (Valentini et al., 2000). Among the 14 putative
isoforms of PK present in the genome of A. thaliana, three genes encode subunits α(PKp-α, At3g22960), β1
(PKp-β1, At5g52920), and β2 (PKp-β2, At1g32440) of plastidic PK (PKp). The plastid enzyme prevalent in
developing seeds likely has a subunit composition of 4α4β1, and the analysis of pkpα-pkpβ1 mutant seeds that
appear to be severely depleted in oil (up to 60%) clearly establishes the importance of the plastid route in the
conversion of PEP into precursors of fatty acid synthesis (Andre et al., 2007; Baud et al., 2007b). The
subsequent oxidative decarboxylation of pyruvate to produce acetyl-CoA, CO2, and NADH is catalysed by the
pyruvate dehydrogenase complex (PDC). The PDC complex is a large multienzyme structure composed of three
primary component enzymes, pyruvate dehydrogenase (PDH or E1 component; EC 1.2.4.1), dihydrolipoamide
acetyl-transferase (E2; EC 2.3.1.12), and dehydrolipoamide dehydrogenase (E3; EC 1.8.1.4) (Johnston et al.,
1997). PDH is a heterodimer composed of αand βsubunits encoded by
genes E1α(At1g01090), E1β1 (At1g30120), and E1β2 (At2g34590), while E2 is a homotrimer and E3 a
homodimer (Randall and Miernyk, 1990). Both PTLPD1 (At3g16950) and PTLPD2 (At4g16155) genes for the
E3 component are induced in maturing seeds (Drea et al., 2001; Lutziger and Oliver, 2000). Disruption
of plE2 (At3g25860), the gene for the E2 component causes an early embryo lethal phenotype (Lin et al., 2003).
During the formation of acetyl-CoA by the PDC complex, one-third of the carbon of precursors for fatty acids is
released as CO2, suggesting that without refixation, a substantial fraction of the carbon entering seeds of B.
napus would be lost (Schwender and Ohlrogge, 2002). In green maturing embryos, ribulose 1,5-bisphosphate
carboxylase/oxygenase (RuBisCO) is able to act without the Calvin cycle to refix this CO2, thus increasing the
efficiency of carbon use in maturing embryos (Schwender et al., 2004a). This metabolic route involves the
conversion of hexose phosphates to ribulose-1,5-bisphosphate (Ru-1,5-P) by the non-oxidative reactions of the
OPPP together with phosphoribulokinase (EC 2.7.1.19), and the subsequent conversion of Ru-1,5-P and CO2 to
3-phosphoglycerate (3-PGA) by RuBisCO. Although increasing activities of phosphoenolpyruvate carboxylase
(PEPc, EC 4.1.1.31) can be monitored in maturing embryos of Brassica campestris and Brassica napus (Junker
et al., 2007; King et al., 1998; Sebei et al., 2006), refixation of CO2 into oxaloacetate, and then into seed
proteins is limited in green developing embryos (Schwender et al., 2004a). In maturing embryos of Brassica
napus, mitochondrial carbon metabolism is largely “unconventional” (Schwender et al., 2006). Flux around the
TCA cycle is absent and mitochondrial substrate oxidation contributes little to ATP production. Most
mitochondrial metabolic flux is devoted to cytosolic fatty acid elongation, since essentially all the citrate formed
in the mitochondria is exported and used for the production of cytosolic acetyl-CoA by ATP citrate lyase. Most
of the oxaloacetate produced is committed to the synthesis of Asp and amino acids derived from it. Maturing
embryos import nitrogen as amino acids (mainly Gln and Ala) used directly for SSPs synthesis but also for the
synthesis of other amino acids via transamination/deamination reactions (Schwender et al., 2006). As a
consequence, alpha-ketoglutarate (KG) derives from Gln and not from anaplerotic flux from PEP via
oxaloacetate (OAA).

Open in a separate window

Figure 2.
Simplified scheme of central metabolism in maturing embryos of Arabidopsis thaliana.
This scheme is adapted from White et al. (2000), Schwender et al. (2004), and Schwender et al. (2006). The arrow
thicknesses are proportional to net fluxes of carbon, based on biochemical data and transcriptional profiling of maturing
seeds. Black stars indicate metabolites that are directly imported from the apoplastic space. Letters in a diamond indicate
metabolic links with pathways presented on figures 3 and 4. ADP-Glc, adenosine diphosphoglucose; AcCoA, acetyl-
coenzyme A; 1,3-BPG, 1,3-bisphosphoglycerate; DHAP, dihydroxyacetone-3-phosphate; E-4-P, erythrose-4-phosphate;
Fru, fructose; Fru-1,6-P, fructose-1,6-bisphosphate; Fru-6-P, fructose-6-phosphate; GAP, glyceraldehyde-3-phosphate;
Glc, glucose; Glc-1-P, glucose-1-phosphate; Glc-6-P, glucose-6-phosphate; KG, alpha-ketoglutarate; OAA, oxaloacetate;
6-PG, 6-phosphogluconate; 6-PGL, 6-phosphogluconolactone; PEP, phospho-enolpyruvate; 2-PGA, 2-phosphoglycerate;
3-PGA, 3-phosphoglycerate; R-5-P, ribose-5-phosphate; Ru-1,5-P, ribulose-1,5-bisphosphate; Ru-5-P, ribulose-5-
phosphate; S-7-P, sedoheptulose-7-phosphate; UDP-Glc, uridine diphosphoglucose; Xu-5-P, xylulose-5-phosphate.

Light, Phosynthesis, Oxygen, and Seed Maturation

Fatty acid synthesis, which occurs in the plastids, not only depends on the supply of precursors but also requires
large amounts of ATP and reductant. The OPPP is regarded as one of the major sources of nicotinamide adenine
dinucleotide phosphate (NADPH) in non-photosynthetic tissues as well as in photosynthetic tissues
experiencing darkness (Neuhaus and Emes, 2000). The two dehydrogenases in the OPPP, namely glucose-6-
phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH, EC
1.1.1.44) use NADP+ as a cofactor and generate two moles of NADPH during the conversion of glucose-6-
phosphate to ribulose-5-phosphate (Wakao and Benning, 2005). However, the study of carbon fluxes in
developing B. napus embryos cultured in vitro has established that the reducing power produced by the OPPP
accounts for at most 44% of the NADPH and 22% of total reductant needed for fatty acid synthesis (Schwender
et al., 2003). Considering the limited role for OPPP in NADPH production and the absence of respiratory TCA
flux in embryonic tissues exhibiting high ATP demands, other sources are required to supply these needs of
reductant and ATP in oilseeds. An alternative for NADPH and ATP production within plastids of green seeds is
photosynthesis, provided that enough light is available. Recent studies have confirmed the importance of light
passing through the silique wall on green oilseed metabolism (Goffman et al., 2005; Ruuska et al.,
2004; Schwender et al., 2004a). In B. napus, only 20–30% of incident light penetrate the silique wall (Eastmond
et al., 1996; King et al., 1998) and the pigment composition of developing seeds reflects shade adaptation
(Ruuska et al., 2004). Yet, seed photosynthesis contributions quantitatively match the major demand for
NADPH and ATP for fatty acid synthesis and other plastid metabolism, and increasing light can improve
significantly the efficiency of oil synthesis in developing embryos (Goffman et al., 2005). Seeds from higher
light tend to be bigger and to exhibit an increased C:N ratio, light preferentially enhancing oil over protein
storage (Li et al., 2006). If green oilseeds maximise oil synthesis during light periods, those syntheses appear to
be drastically reduced during the night, thus avoiding large losses of carbon (Goffman et al., 2005; King et al.,
1998).
Several reports document an inhibition of embryo growth and seed storage compound accumulation when
plants are grown at low external oxygen (Kuang et al., 1998; Porterfield et al., 1999). Even when plants are
cultured at ambient O2 levels, recent studies on B. napus suggest that internal oxygen concentrations can limit
metabolism in seeds placed under low or mild light (Vigeolas et al., 2003). Thus, green oilseed embryos can
experience hypoxia, as described in developing legumes (Rolletschek et al., 2002). Low O2 is accompanied by a
decrease in the energy state within seeds and decreased metabolic fluxes to storage TAG biosynthesis. On the
contrary, under high light, photosystem activity may release sufficient O2 to prevent hypoxia, so that oilseed
metabolism is not restricted by low O2 concentrations (Goffman et al., 2005). In green oilseeds, photosystems
thus play a dual role, supplying NADPH and ATP for energetically expensive fatty acid syntheses and
preventing hypoxia (Ruuska et al., 2004).

Starch Transient Accumulation in Early Maturing Embryos

Transient starch deposition occurs in early maturing embryos of A. thaliana (Baud and Graham, 2006) and B.
napus (da Silva et al., 1997). In embryos of A. thaliana, starch content peaks at 10 DAP and then drops
regularly during late maturation, resulting in the almost complete disappearance of starch in mature dry seeds
(Baud et al., 2002; Focks and Benning, 1998). The route of starch synthesis has been mostly studied in
developing rapeseed embryos (Rawsthorne, 2002). The starch accumulated in developing embryos originates
from carbon imported into the embryo (da Silva et al., 1997). Within the plastids, glucose-6-phosphate (Glc-6-
P) is metabolised to glucose-1-phosphate (Glc-1-P) by phosphoglucomutase (EC 2.7.5.1; Caspar et al.,
1985; Periappuram et al., 2000) followed by the conversion of Glc-1-P to adenosine diphosphoglucose (ADP-
Glc) by ADP-Glc pyrophosphorylase (AGPase, EC 2.7.7.27). AGPase is regarded as the key regulatory element
of the starch biosynthetic pathway in embryos of B. napus seeds (Vigeolas et al., 2004). The subsequent
incorporation of the glucose moiety into the starch granule is catalysed by starch synthases (da Silva et al.,
1997; Kang and Rawsthorne, 1994). Starch degradation may be specifically catalysed by plastidial isoforms of
α-(EC 3.2.1.1) and β-amylases (EC 3.2.1.2) and phosphorylases (EC 2.4.1.1; da Silva et al., 1997).
Interestingly, the ESTs of starch metabolism in developing seeds of A. thaliana represent an example of the
apparent coordinate expression of genes encoding enzymes of the same metabolic pathway (White et al., 2000).
Together with enzymatic activities associated with starch metabolism, these expression data demonstrate that
the changes in starch content throughout embryo development reflect the net balance between synthetic and
degradative capacity rather than a synthetic phase followed by a degradative phase (da Silva et al., 1997). The
role of this transient accumulation of starch in oilseeds is still a matter of debate. It has been proposed that
starch constitutes an important carbon source required to sustain lipid synthesis during the phase of rapid oil
deposition (Norton and Harris, 1975). Starch degradation may also provide precursors for the synthesis of
sucrose and oligosaccharide (raffinose, stachyose) accumulated in seeds during late maturation to achieve
desiccation tolerance (Leprince et al., 1990). Alternatively, the development of an early capacity for starch
synthesis has been proposed to contribute to the establishment of the embryo as a sink organ prior to the onset
of oil deposition (da Silva et al., 1997). This last hypothesis has been corroborated by the characterisation of
transgenic B. napus lines exhibiting an embryo-specific reduction in AG-Pase activity (Vigeolas et al., 2004).
Starch synthesis was inhibited in the developing embryos of these plants. This phenotype was accompanied
both by a decrease in the rate of sucrose degradation and an inhibition of glycolysis and storage lipid synthesis
in early- but not in late-maturing seeds. These results demonstrate that starch synthesis is required to establish
the growing embryo as a sink during early-maturation. However, the final size, weight, and lipid content of
these transgenic seeds were unmodified, indicating that seed filling is largely compensated for during mid-and
late-maturation and making it unlikely that starch serves as an important carbon source for lipid or sucrose
synthesis during the maturation process, at least under optimal growth conditions (Vigeolas et al., 2004).
Go to:

SYNTHESIS OF TRIACYLGLYCEROLS

Production of Glycerol Backbones

Glycerol-3-phosphate (Gly-3-P) species constitute the backbones of TAG molecules. Measurements of Gly-3-P
levels in developing A. thaliana (Gibon et al., 2002) or B. napus (Vigeolas and Geigen-berger, 2004) seeds
indicate that the rate of Gly-3-P provision is limiting during the rapid TAG accumulation phase. Together with
the reported Km of the first enzyme catalysing the acylation of Gly-3-P in rapeseed, these measurements suggest
that the availability of this substrate could restrict the overall rate of TAG accumulation (Perry et al., 1999).
This hypothesis has been further demonstrated in B. napus by injecting various concentrations of glycerol in
maturing seeds. These injections have led to an increase in Gly-3-P within 28 h and have specifically stimulated
the sucrose flux into the TAG biosynthetic pathway (Vigeolas and Geigenberger, 2004). In plants, two enzymes
can synthesise Gly-3-P. Glycerol kinase (GlyK; E.C. 2.7.1.30) is a cytosolic enzyme that converts glycerol into
Gly-3-P (Huang, 1975; Sadava and Moore, 1987). Ubiquitously expressed in plant tissues, this enzyme is
thought to be involved in the assimilation of glycerol (Eastmond, 2004) rather than in the supply of the Gly-3-P
backbones used for TAG biosynthesis. The second route leading to Gly-3-P synthesis consists in the conversion
of dihydroxyacetone phosphate (DHAP) and NADH to Gly-3-P and NAD+. This reaction is catalysed by Gly-3-
P dehydrogenase (Gly3PDH; E.C. 1.2.1.12). In seeds like in leaves, both cytosolic and plastidial Gly3PDH
isoforms have been identified (Kirsch et al., 1992; Sharma et al., 2001). The analyses of A. thaliana knock out
mutants defective in the plastidic isoform of Gly3PDH encoded by gene At2g40690 (SFD1/GLY1) have ruled
out the hypothesis of a significant involvement of this isoform in TAG biosynthesis (Miquel et al., 1998; Nandi
et al., 2004). The lack of phenotype of sfd1/gly1 seeds may be explained by the presence of a redundant isoform
of Gly3PDH (At5g40610) in the plastids (Wei et al., 2001). Alternatively, the supply of Gly-3-P for oil
synthesis may be carried out by cytosolic isoforms of Gly3PDH. A transgenic approach recently carried out
in B. napus has strengthened this last hypothesis. The expression of a yeast gene coding for cytosolic Gly3PDH
(gpd1) in transgenic oilseed rape using the seed-specific napin promoter has led to a three- to fourfold increase
in the level of Gly-3-P in developing seeds, resulting in a 40% increase in the final seed oil content (Vigeolas et
al., 2007).

Synthesis of Fatty Acids

Fatty acids are produced in the plastids from acetyl-CoA and mal-onyl-CoA (Figure 3). A multisubunit
heteromeric acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) catalyses the ATP-dependent formation of malonyl-
CoA from acetyl-CoA and bicarbonate (Konishi et al., 1996). This reaction is the first committed step of de
novo fatty acid biosynthesis and is regarded as a major control point of the pathway (Thelen and Ohlrogge,
2002a). ACCase contains three functional domains and catalyses a two step-reaction. Carboxylation of a biotin
cofactor covalently bound to the central-carboxyl carrier domain (BCCP) is catalysed by the biotin carboxylase
domain (BC). Transfer of the carboxyl group from carboxy-biotin to acetyl-CoA occurs at the
carboxyltransferase domain (CT). The BC subunit is encoded by gene At5g35360 (CAC2, Sun et al., 1997). The
CT domain is composed of associated nonidentical CTαand CTβsubunits encoded by At2g38040 and
AtCg00500, respectively. Two isoforms of the BCCP protein are encoded by genes At5g15530 (BCCP2) and
At5g16390 (BCCP1). The malonyl-CoA produced by plastidial ACCase constitutes the carbon donor for each
cycle of the fatty acid biosynthetic process. However, the malonyl group has to be transferred from CoA to a
protein cofactor, or acyl carrier protein (ACP) before entering the fatty acid synthesis pathway. This transfer is
catalysed by a mal-onyl-CoA: acyl carrier protein malonyltransferase (EC 2.3.1.39).
Figure 3.
Fatty acid synthesis in the plastids of maturing embryos of Arabidopsis thaliana.
This scheme is adapted from Miquel and Browse (1995) and Somerville et al. (2000). Fatty acids are grown by sequential
acylation of two-carbon units. The first condensation reaction is catalysed by ketoacyl-ACP synthase III (KASIII). During
the following cycles, the condensation reaction is catalysed by KASI. Finally, the conversion of 16:0 to 18:0 is catalysed
by KASII. ACP, acyl-carrier protein; CoA, coenzyme A. Letters in a diamond indicate metabolic links with pathways
presented on figures 2 and 4
The de novo biosynthesis of fatty acids is performed by fatty acid synthase (FAS), an easily dissociable
multisubunit complex consisting of monofunctional enzymes (Brown et al., 2006), including 3-ketoacyl-ACP
synthase (EC 2.3.1.41; KAS), 3-ketoacyl-ACP reductase (EC 1.1.1.100), 3-hydroxyacyl-ACP dehydratase (EC
4.2.1.*), and enoyl-ACP reductase (EC 1.3.1.9; MOD1, At2g05990) (Mou et al., 2000). Acetyl-CoA is used as
the starting unit and malonyl-ACP as the elongator. The malonyl-thioester enters into a series of condensation
reactions with acetyl-CoA, then acyl-ACP acceptors. The initial condensation reaction is catalysed by KAS III
(2:0 to 4:0-ACP; At1g62640), the subsequent condensations by KAS isoforms I (4:0-ACP to 16:0-ACP;
At5g46290) and II (16:0-ACP to 18:0-ACP; FAB1, At1g74960, Pidkowich et al., 2007). Each condensation
step is followed by a reduction of the carbonyl group, a dehydration step, and a reduction of the trans-2 double
bond to obtain a saturated acyl chain that is two carbons longer than at the start of the cycle. Some 16:0-ACP is
released from the FAS machinery, whereas molecules that are elongated to 18:0-ACP are efficiently desaturated
by a stromal stearoyl-ACP desaturase (EC 1.14.19.2) (Browse and Somerville, 1991). Thus, 16:0-ACP and
18:1-ACP constitute the main products of plastidial fatty acid synthesis (Miquel and Browse, 1995). Following
their synthesis, these long-chain acyl groups are hydrolysed by an acyl-ACP thioesterase (FAT) that releases
free fatty acids. The FATA class (EC 3.1.2.14) has highest in vitro activity for 18:1-ACP whereas FATB (EC
3.1.2.*; At1g08510; Bonaventure et al., 2003) prefer saturated acyl groups but also have activity for unsaturated
acyl-ACPs (Salas and Ohlrogge, 2002). On the outer membrane of the chloroplast envelope, these free fatty
acids are finally activated to coenzyme A (CoA) esters by an acyl-CoA synthetase and exported to the
endoplasmic reticulum (ER).

Fatty Acid Modifications

Synthesis of very long chain fatty acids.
The seeds of A. thaliana contain approximately 30% of very long chain fatty acids (VLCFAs; 20–24 carbons),
eicosenoic acid (C20:1) being the predominant VLCFA species (23 mol%). VLCFA are released in the ER as
CoAs after sequential addition of two-carbon moieties from malonyl-CoA at the carboxyl end of pre-existing
18-carbon fatty acids (Figure 4). Fatty acid elongase, which catalyses VLCFA biosynthesis, is a membrane-
bound multienzyme complex. Analogous to de novo fatty acid synthesis, each cycle of fatty acid elongation
involves four enzymatic reactions: (i) condensation of malonyl-CoA with a long chain acyl-CoA is catalysed by
a 3-ke-toacyl-CoA synthase (KCS, condensing enzyme); (ii) reduction to β-hydroxyacyl-CoA is made by a 3-
ketoacyl-CoA reductase; (iii) dehydration to an enoyl-CoA is catalysed by a 3-hydroxyacyl-CoA dehydrase;
(iv) and an enoyl-CoA reductase catalyses the reduction of the enoyl-CoA, resulting in an elongated acyl-CoA
(von Wettstein-Knowles, 1982). The initial step in fatty acid elongation, the iterative condensation of acyl-CoA
units with malonyl-CoA by KCS, is rate limiting. The KCS also plays a key role in determining the chain length
of fatty acids found in seed oils (Blacklock and Jaworski, 2006; Katavic et al., 2001; Millar and Kunst, 1997).
Among the large family of condensing enzymes found in the genome of A. thaliana, a seed-specific KCS
(FAE1, At4g34520) has been identified (James et al., 1995; Roscoe et al., 2001; Rossak et al., 2001; Wu et al.,
2007) that is responsible for the biosynthesis of C20 and C22 fatty acids for seed storage lipids. Several studies
of the structure/function basis of KCS catalysis and substrate specificity have been reported. Site-directed muta-
genesis experiments have enlightened the importance of Cys-223 and four His residues for the enzyme activity
(Ghanevati and Jaworski, 2001), while domains found in the amino terminal one-third of the protein excluding
the transmembrane domain have been shown to impart chain-length substrate specificity (Blacklock and
Jaworski, 2002). In contrast with the KCS enzymes, it has been proposed that the other three enzyme activities
of the elongase complex exhibit broad substrate specificities and are shared by all the plant VLCFA
biosynthetic pathways (Millar and Kunst, 1997). This hypothesis has been partially confirmed by the recent
isolation of an A. thaliana gene encoding an enoyl-CoA reductase (ECR; At3g55360) based on its similarity to
the yeast TSC13 gene (Kohlwein et al., 2001). The ECR gene is ubiquitously expressed and the corresponding
mutants exhibit pleiotropic phenotypes including a significant reduction in VLCFA content of seed TAGs
(Zheng et al., 2005). However, considering the amount of VLCFAs still accumulated in these mutant lines, it is
very likely that other ECR participate in VLCFA elongation in A. thaliana. So far, they have not been
identified. Likewise, the genes encoding the other reductase and the dehydratase of the elongase complex
remain to be isolated.
 Open in a separate window
Figure 4.
Simplified scheme of the reactions involved in triacylglycerol biosynthesis in seeds of Arabidopsis thaliana.
Fatty acids are synthesised in the plastids, and 16:0 and 18:1 are massively exported toward the cytosolic compartment as
CoA esters. Glycerol backbones are derived from cytosolic dihydroxyacetone phosphate (DHAP) through the action of
glycerol-3-phosphate dehydrogenase. The enzymes of the Kennedy pathway catalyse triacylglycerol assembly in the
endoplasmic reticulum. The contribution of the acyl-CoA independent phospholipid:diacylglycerol acyltransferase
(PDAT) is presented. ER, endoplasmic reticulum; FAD2, oleoyl desaturase; FAD3, linoleoyl desaturase. Letters in a
diamond indicate metabolic links with pathways presented on figures 2 and 3
Because cellular membranes are impermeable to acetyl-CoA and malonyl-CoA, the synthesis of these
compounds is thought to occur in each cellular compartment where they are required (Liedvogel, 1986). The
malonyl-CoA species used for VLCFA biosynthesis are consequently produced in the cytosolic compartment,
where they are derived from citrate through the sequential action of ATP-citrate lyase (ACL; EC 4.1.3.8) and
acetyl-CoA carboxylase (ACCase; EC 6.4.1.2). ACL catalyses the ATP-dependent reaction of citrate and
coenzyme A to form acetyl-CoA and oxaloacetic acid (Fatland et al., 2002). The enzyme is a heterooctamer
consisting of ACLA and ACLB subunits, each subunit being encoded by a small gene family (Fatland et al.,
2005). ACCase then catalyses the ATP-dependent formation of malonyl-CoA from acetyl-CoA and bicarbonate
(Konishi and Sasaki, 1994). Like the plastidial ACCase involved in the de novo fatty acid synthesis, the
cytosolic ACCase contains three catalytic domains necessary to catalyse the two-step carboxylation of acetyl-
CoA (Ke et al., 2000). However, the cytosolic isoform is a multifunctional homodimeric form composed of
subunits >200 kDa (Alban et al., 2000) encoded by the ACC1 gene (At1g36160; Baud et al., 2003; Yanai et al.,
1995). This gene is ubiquitously expressed, corresponding acc1 mutant embryos are totally deprived of
VLCFAs and the mutation is embryo lethal (Baud et al., 2003; Baud et al., 2004).

Fatty acid desaturation.

In A. thaliana, desaturated fatty acid species like linoleic (18:2Δ9cis, 12cis) and linolenic acids (18:3Δ9cis, 12cis, 15cis)
account for 45–50% of the seed fatty acid content. The synthesis of these two fatty acids proceeds by sequential
desaturation of oleic acid (18:1Δ9cis) to linoleic by Δ12 oleate desaturase (FAD2), and then linoleic acid to
linolenic acid by ω-3-fatty acid desaturase (FAD3) (Figure 4). The cloning of FAD2 (At3g12120) was allowed
by a T-DNA tagging method (Okuley et al., 1994). FAD3 (At2g29980) was isolated both by map-based
chromosome walking (Arondel et al., 1992) and T-DNA tagging (Yadav et al., 1993). Both genes are
significantly up-regulated in maturing seeds. The corresponding desaturases are inserted co-translationally into
the ER (MacCartney et al., 2004), where they act on fatty acids esterified to phosphatidylcholine (PC), and
utilise cytochrome b5 as an intermediate electron donor. Considering that cytochrome b5 is reduced in turn by
cytochrome b5 reductase, three proteins are required for the overall desaturation reaction (Somerville et al.,
2000).

Triacylglycerol Assembly
TAG assembly takes place within the ER, with Gly-3-P and acyl-CoAs as the primary substrates (Stymne and
Stobart, 1987). Several pathways leading to TAG biosynthesis have been described to date (Figure 4). One
known as the Kennedy pathway is relatively straightforward (Kennedy, 1961). Fatty acids are sequentially
transferred from CoA to positions sn-1 and sn-2 of Gly-3-P. Gly-3-P is first acylated by a glycerol-3-phosphate
acyltransferase (G3PAT; EC 2.3.1.15) to form lysophosphatidic acid (LPA), which in turn is acylated by
lysophosphatidic acid acyltransferases (LPAAT; EC 2.3.1.51) to produce the central metabolite phosphatidic
acid (PA). Dephosphorylation of PA by phosphatidic acid phosphohydrolase (PAP; EC 3.1.3.4) releases
diacylglycerol (DAG). In the final step of TAG assembly, a third fatty acid is transferred to the vacant
position sn-3 of DAG by diacylglycerol acyl-transferases (DAGAT; EC 2.3.1.20; Cao et al., 1986; Cao et al.,
1987). Two ER-localised and structurally unrelated enzymes exhibiting DAGAT activity have been described
in A. thaliana that are encoded by genes At2g19450 (TAG1) and At3g51520, respectively (Hobbs et al.,
1999; Routaboul et al., 1999; Zou et al., 1999). Fatty acids produced in the plastid are not always immediately
available for TAG biosynthesis, however. Instead, the acyl chains can enter into phosphatidylcholine (PC) (and
into phosphatidyl ethanolamine to a lesser extent), where they become desaturated or otherwise modified
(Browse and Somerville, 1991). In this case, PC is synthesised from DAG by CDP-choline: diacylglycerol
cholinephosphotransferase (CPT; EC 2.7.8.2) and acyl residues are then further desaturated by fatty acid
desaturases (FAD). The CPT reaction being rapidly reversible (Vogel and Browse, 1996), the DAG moiety of
PC can be liberated by hydrolysis at any moment, thus becoming available for TAG final assembly by DAGAT
(Stymne and Stobart, 1987). However, it is to be noted that fatty acids from PC may also become available for
TAG synthesis by acyl exchange between a fatty acid attached to CoA (from the acyl-CoA pool) and a fatty
acid on PC, this reversible reaction being catalysed by acyl CoA: lysophosphatidylcholine acyltransferase
(LPCAT, EC 2.3.1.23). Recently, a second mechanism of TAG synthesis has been described. In this acyl-CoA
independent pathway, a phospholipid such as PC or phosphatidyl ethanolamine can donate its sn-2 acyl group to
DAG resulting in formation of TAG (Ståhl et al., 2004). This transacylase activity is catalysed by a
phospholipid: diacylglycerol acyltransferase (PDAT; EC 2.3.1.158).
The TAG biosynthetic pathway has many reactions in common with the synthesis of membrane lipids, DAG
being also a precursor for membrane PC. This observation raises the question of the enzymes involved in TAG
biosynthesis in seeds. Special isoforms may be involved in this metabolic process. Alternatively, some
housekeeping isoforms may be up-regulated (Beisson et al., 2003). Eight GPAT genes and five LPAAT genes
have been found in the genome of A. thaliana. Most of the corresponding enzymes are probably targeted to the
ER but to date, none of them has been shown to play a clear role in seed TAG biosynthesis (Beisson et al.,
2007; Kim et al., 2005b). A detailed characterisation of these two gene families will be required to get further
insights into the understanding of fatty acid fluxes into TAGs during seed maturation. Beyond the identification
of the isoforms involved in TAG formation, the characterisation of their ER localisation will contribute to
elucidate cellular aspects of storage oil formation. One of the longstanding questions in plant lipid metabolism
has been whether there was a specific region of the ER specifically dedicated to TAG synthesis. Recent
evidence suggests that this is likely to be the case (Shockey et al., 2006).

Storage of TAGs in Oil Bodies

TAGs are accumulated in subcellular structures called oil bodies. Seed oil bodies are simple organelles
comprising a matrix of TAGs surrounded by a phospholipid monolayer where the aliphatic chains are oriented
to the TAG lumen and the phosphate groups toward the cytosol (Yatsu and Jacks, 1972). Oil bodies are derived
from the ER and the exact mechanism of their biogenesis remains a matter of debate (Murphy and Vance,
1999; Robenek et al., 2004) (Figure 5). It has been proposed that TAGs accumulate within the lipid bilayer of
the ER membrane from where they are budded of, enclosed by a protein-bearing phospholipids monolayer
originating from the cytosolic leaflet of the ER membrane. Recent observations suggest an entirely different
mechanism of lipid droplet biogenesis involving the elaboration of droplets within ER cups, rather than between
the leaflets of the ER membrane, with adipophilin clusters in the cytoplasmic leaflet of the ER transferring
lipids from the ER to the droplet surface (Robenek et al., 2006). A specific subset of proteins is embedded in the
lipid monolayer surrounding oil bodies that represents 1–4% of the weight of seed oil bodies (Huang,
1992; Tauchi-Sato et al., 2002; Tzen and Huang, 1992). Recent proteomic efforts have provided a detailed
characterisation of the set of proteins imbedded in the half membrane unit of A. thaliana oil bodies (Jolivet et
al., 2004). The oil body proteome consists of five distinct oleosins (S1/OLEO5, At3g01570; S2/OLEO4,
At3g27670; S3/OLEO1, At4g25140; S4/OLEO2, At5g40420; S5/OLEO3, At5g51210), one caleosin (CLO1,
At4g26740; Poxleitner et al., 2006), one steroleosin (HSD1, At5g50600/At5g50700; d'Andréa et al., 2007; Li et
al., 2007a), a putative aquaporin (TIP3.2, At1g17810), and a glycosylphosphatidylinositol (GPI)-anchored
protein (At1g54860). The most abundant oil-body associated proteins are oleosins and much of the oil-body
surface may be covered by these proteins (Tzen and Huang, 1992). These alkaline, amphipatic proteins contain
three structural domains: an N-terminal amphipatic domain, a central hydrophobic domain, and a C-terminal
amphipatic domain. The central domain is organised around a unique 12-amino acid motif called a proline knot
and is inserted into the oil body TAG matrix (Abell et al., 1997). The N- and C-terminal domains are proposed
to reside on the oil-body surface (Frandsen et al., 2001). The accumulation of oleosins during maturation
determines the size of oil bodies (Siloto et al., 2006). During late maturation, when the seed water content
sharply decreases, oil bodies experience cytoplasmic compression. They are forced into contact with each other
in the periphery of embryonic cells. Yet they resist coalescence and remain as individual units. The oleosins
embedded in the phospholipids monolayer form a steric barrier and maintain oil bodies as small individual units
by means of steric hindrance and electronegative repulsion (Leprince et al., 1998; Murphy and Vance, 1999).
Oil bodies thus provide a high surface-to-volume ratio that optimises access by lipases during reserve re-
mobilisation.

Figure 5.
Sorting and trafficking of reserve compounds in maturing seeds of Arabidopsis thaliana.
This scheme is adapted from Otegui et al. (2006). CCV, clathrin coated vesicles; ER, endoplasmic reticulum; MVB,
multivesicular bodies; TGN, trans Golgi network; PSV, protein storage vacuole.
Go to:

SEED STORAGE PROTEINS

Diversity of Seed Storage Proteins

Seeds of A. thaliana contain two predominant classes of SSPs named after those of B. napus (for a review,
see Fujiwara et al., 2002). Legumin-type globulins are referred to as 12S globulin or cruciferin (Li et al.,
2007b; Sjodahl et al., 1991), and napin-type albumins are referred to as 2S albumin or arabin (Krebbers et al.,
1988; van der Klei et al., 1993). The 12S globulins accumulate as complexes consisting of six αand six
βsubunits linked via disulfide bonds. The mature αand βpolypeptides are proteolytically processed from a single
precursor. In the A. thaliana genome of Columbia ecotype, four genes encoding 12S precursors have been
identified (Gruis et al., 2002; Gruis et al., 2004; Pang et al., 1988),
including CRA1 (At5g44120), CRB (At1g03880), CRC (At4g28520), and CRU2 (At1g03890). The 2S albumins
accumulate as heterodimers consisting of two subunits (also called large and small chains) generated by
cleavage of a precursor and linked by disulfide bridges (Guerche et al., 1990; Krebbers et al., 1988). Five genes
encoding 2S precursors have been reported, referred to
as At2S1 (At4g27140), At2S2 (At4g27150), At2S3 (At4g27160), At2S4 (At4g27170), and At2S5 (At5g54740).
Additional minor SSPs encoded by a vicilin-like gene (At2g18540) and two globulin-like genes (At1g07750
and At4g36700) may be stored in the seeds of A. thaliana (Fujiwara et al., 2002). Other proteins such as late
abundant embryogenesis (LEAs) proteins may account for a limited amount of nitrogen stored in the seed
(Fujiwara et al., 2002; Wise et al., 2004).

Synthesis of Seed Storage Proteins

SSPs are actively synthesised on the rough ER as precursor forms and then are transported into PSVs by a
vesicle-mediated pathway. Upon arrival at the PSV, the precursors are converted into their respective mature
forms by limited proteolysis at specific sites. Prolegumin-type globulins are believed to be inserted co-
translationally into the ER lumen, where the formation of disulfide bonds and assembly into trimers occur
(Muntz, 1998). After transport to PSVs, proteolytic processing at a conserved Asn-Gly peptide bond by an
asparaginyl endopeptidase converts the proform into the disulfide-linked mature αand βpolypeptides. This
cleavage is concomitant with a further assembly of the trimer protein complexes into hexamers that sediment as
a 12S complex (Barton et al., 1982). The proteolytic processing of pronapin-type albumins appears to be more
complex, requiring removal of three propeptide regions to generate the two disulfide-linked mature
polypeptides (Krebbers et al., 1988). Several of the proteolytic steps also involve a conserved Asn residue
(Krebbers et al., 1988). Yet, additional aspartic endopeptidases may be involved in the processing of the
propeptides (D'Hondt et al., 1993). If the post-translational polypeptide cleavage events leading to the synthesis
of mature SSPs are well described, the function of such a complex processing is less clear. The protein
conformational changes thus triggered might enable dense packaging and long-term stable storage of reserves
within PSV, a compartments that presumably contains proteolytic enzymes for the mobilisation of SSPs during
germination (Gruis et al., 2004). Work to isolate the Asn-specific endopeptidases responsible for the proteolytic
processing of prolegumin-type globulins and pronapin-type albumins has led to the identification of an Asn-
specific subclass (C13; EC 3.4.22.34) of the Cys endopeptidase family, referred to as the vacuolar processing
enzymes (VPEs) (Hara-Nishimura et al., 1991; Hara-Nishimura et al., 1995). In A. thaliana, the VPE gene
family is composed of four genes (Gruis et al., 2002; Kinoshita et al., 1995a; Kinoshita et al., 1995b; Kinoshita
et al., 1999), namely αVPE (At2g25940), βVPE (At1g62710), γVPE (At4g32940), and δVPE (At3g20210).
These genes exhibit distinct but partially overlapping expression patterns, and at least three of these VPEs are
involved in SSPs processing (Gruis et al., 2004). The degree of involvement of these three genes
(βVPE > γVPE > αVPE) appears to be correlated with the abundance of their respective transcripts during seed
maturation. Interestingly, the complete removal of VPE function in the αvpe βvpe γvpe δvpe quadruple mutant
results in a total shift of SSPs accumulation from wild-type processed polypeptides to a finite number of
alternatively processed forms cleaved at sites other than the conserved Asn residues targeted by VPEs. This
demonstrates that VPE-mediated processing of SSPs is not compulsory for the successful depositions of these
SSPs. The emergence of alternatively processed peptides then reveals Asn-independent proteolytic activities
(e.g. aspartic protease; Hirawai et al., 1997) probably less active than VPE activities in maturing seeds of the
wild type (Gruis et al., 2004).

Protein Storage Vacuoles

Most SSPs are synthesised in the ER as precursors that are transported into PSVs, where they are converted into
mature forms (Figure 5). PSVs are electron dense vacuolar compartments delimited by a lipid bilayer, the
tonoplast. The internal environment of PSVs, the matrix, contains not only soluble storage proteins, but also
intra-organellar inclusions called globoids, that correspond to spherical inclusions of phytate (Gillespie et al.,
2005). In A. thaliana, a Golgi-dependent pathway is responsible for the transport of the soluble SSPs and their
processing enzymes to the PSVs (Otegui et al., 2006). Yet, storage proteins and processing enzymes are
segregated into different cisternal domains during Golgi trafficking. SSPs become sequestered into dense
aggregates formed in the specialised marginal buds of the cis-Golgi cisternae that progress through the stack as
the cisternae mature (Hillmer et al., 2001). Upon reaching the trans-Golgi network, the buds containing protein
aggregates give rise to electrondense vesicles (DVs). Processing enzymes (β-VPE, aspartic protease) may be
packaged in clathrin-coated vesicles that ultimately fuse to the DVs to produce multivesicular bodies (MVBs),
which act as prevacuolar compartments. The proteolytic processing of 2S albumins begins in the MVBs,
leading to conformational changes that decrease their solubility and make them more resistant to further
proteolysis inside the PSVs (Gruis et al., 2004). If SSP aggregation plays a key role in protein sorting, vacuolar
sorting receptors are also required for selective recognition and sorting of these proteins (Hinz et al., 2007).
Two different types of receptors are currently in discussion in the plant literature. RMR1 (for receptor
homology region transmembrane domain ring H2 motif protein; At5g66160) may function as the main receptor
for PSV-destinated storage proteins (Park et al., 2005). The interaction between RMR1 and 2S albumin- and/or
12S globulin-type SSPs remains to be demonstrated. RMR proteins are not recycled from the MVBs and must
accompany their attached cargo to the PSVs (Hinz et al., 2007). Some storage proteins are thought to escape
this sorting machinery and to reach the trans Golgi, where a second type of receptor called VSR-1/ATELP1
(At3g52850) might catch them. The VSR-1/ATELP1 protein may thus be required for a complete and efficient
targeting of SSPs (Shimada et al., 2003). Unlike RMR1, the VSR-1 receptor, which binds SSPs in a Ca2+-
dependent manner, has been postulated to be recycled back from MVBs to the trans-Golgi network by a
retromer-mediated mechanism (Olivusson et al., 2006; Fuji et al., 2007) that could involve the MAIGO1
(MAG1/VPS29, At3g47810) protein (Shimada et al., 2006).
Go to:

DEVELOPMENTAL CONTROL OF SEED FILLING

Hormones and Sugars

ABA immunomodulation in transgenic tobacco seeds has clearly established the key role played by this
hormone in triggering the maturation process (Phillips et al., 1997). Since then, it has been shown that the ABA-
to-GA ratio regulates seed maturation (Finklestein et al., 2002). At the beginning of the maturation phase, the
seed ABA content steadily increases, and the resulting elevated ABA-to-GA ratio inhibits embryo growth and
germination, induces seed dormancy, and promotes maturation (Nambara and Marion-Poll, 2005). If early
maturing embryos are removed from the seed, thus alleviating the ABA effect, they can proceed through the
germination phase and develop into seedlings (Vicente-Carbajosa and Carbonero, 2005). The regulatory
mechanisms involving ABA and GA during seed development and maturation yet remain to be fully elucidated
(Finkelstein et al., 2002).
The carbohydrate status has been proposed as a control element of seed development in legume seeds: a high
glucose-to-sucrose ratio is correlated with cell divisions (embryo morphogenesis) whereas a high sucrose-to-
glucose ratio triggers storage product synthesis (maturation) (Weber et al., 1997). Similar variations of the
carbohydrate status have been measured on whole seeds at the onset of the maturation phase in A.
thaliana (Baud et al., 2002; Ohto et al., 2005). However, the role of the hexose-to-sucrose ratio in the control of
the switch to storage product accumulation in oilseeds remains highly controversial (Tomlinson et al., 2004).
The elucidation of the putative regulatory role of sucrose and hexoses in the developing A. thaliana seed will
require a fine characterisation of the biochemistry and enzymology of sugar metabolism linked to a better
understanding of the ultrastructure of the seed (Hill et al., 2003). Trehalose-6-phosphate (T6P) is also thought to
play a role of the utmost importance in developing A. thaliana embryos. Mutants disrupted in the TREHALOSE-
6-PHOSPHATE SYNTHASE1 (TPS1) gene (At1g78580) do not develop past late torpedo or early cotyledon
stage, accumulate limited quantities of storage compounds and consequently produce wrinkled seeds (Eastmond
et al., 2002; Gomez et al., 2005; Gomez et al., 2006). To date, the molecular mechanism involved in this
regulatory process remains to be elucidated. If the role of sugar metabolism in the regulation of storage function
is not understood, it is clear that sugar-sensing pathways interact with hormonal signalling (Avonce et al.,
2004; Leon and Sheen, 2003).

Master Regulators
The isolation and characterisation of mutants skipping the maturation phase and displaying a viviparious
behaviour has enabled the identification of master genes that determine a state of competence under which
maturation gene expression programmes occur (Vicente-Carbajosa and Carbonero, 2005; Wobus and Weber,
1999). In A. thaliana, four loci have been identified, namely FUSCA3 (FUS3, At3g26790), ABSCISIC ACID
INSENSITIVE3 (ABI3, At3g24650) and LEAFY COTYLEDON1 (LEC1, At1g21970) and 2 (LEC2, At1g28300),
that control a wide range of seed-specific characters and play a role of the utmost importance in seed
maturation. FUS3, ABI3 and LEC2 encode related transcription factors of the B3-domain family (Giraudat et
al., 1992; Gazzarrini et al., 2004; Luerssen et al., 1998; Meinke et al., 1994; Stone et al., 2001),
whereas LEC1 encodes a protein homologous to the HAP3 subunit of the CAAT box-binding protein (Lee et
al., 2003; Lotan et al., 1998). The fus3, abi3, lec1 and lec2 mutants share common phenotypes such as reduced
accumulation of storage compounds, and exhibit specific phenotypes such as the lack of chlorophyll
degradation, anthocyanin accumulation, intolerance to dessication, or defects in cotyledon identity (see Table 1
in To et al., 2006). These master regulators exhibit partially overlapping expression patterns that include
crosstalk and complex feedback regulations (Gutierrez et al., 2007; Santos Mendoza et al., 2005; To et al.,
2006). The four regulators act in concert with hormone signalling (auxin, ABA, GA), epigenetic mechanisms
involving the chromatin-remodelling factor (CHD3) PICKLE (PKL, At2g25170), and possibly other regulators
like LEC1-Like (At5g47670) and TAN (At4g29860) (Santos Mendoza et al., 2008, and references therein).
These B3-type transcription factors appear to be involved in a local, intricate, and highly redundant gene
regulation network governing most seed maturation aspects, including storage compound synthesis. Due to
partial functional redundancy, genetic analyses have failed to determine the specific function of each
regulator in planta. More recently, the use of inducible systems, coupled to quantitative RT-PCR experiments
and/or transcriptomic analyses have finally unravelled some of the target genes of these master regulators
(Kagaya et al., 2005) (Figure 6). LEC2 exerts a direct control over At2S1, At2S2, At2S3, At2S4, CRA1 and 2S-
like (At5g54740) gene expression (Braybrook et al., 2006; Kroj et al., 2003). Likewise, FUS3 and ABI3
contribute directly to the induction of storage protein gene expression (Ezcurra et al., 1999; Reidt et al., 2000).
LEC2 has then been shown to trigger the expression of genes encoding oleosins
like S3/OLEO1 and HSD1 (Braybrook et al., 2006; Santos Mendoza et al., 2005). Using in vitro approaches
(with FUS3, LEC2 and ABI3) and yeast one-hybrid assays (with LEC2 and FUS3), it was established that these
B3-type regulatory proteins can recognise and bind RY motifs (CATGCA) present in the promoters of their
target genes (Braybrook et al., 2006; Kroj et al., 2003; Monke et al., 2004; Reidt et al., 2000). However, RY
elements alone are not always sufficient to confer the proper expression pattern of target genes during the
maturation phase, and additional regulatory cis-acting elements like G boxes (CACGTG) may be required
(Ezcurra et al., 1999; Kurup et al., 2000; Nakashima et al., 2006; Vicente-Carbajosa and Carbonero, 2005).
These G-box elements may be bound by bZIP transcription factors like AtbZIP10 (At4g02640) or AtbZIP25
(At3g54620) that participate into the synergistic activation of target gene expression (Lara et al., 2003).

Open in a separate window

Figure 6.
Model for the control of storage compound synthesis and accumulation in maturing seeds of Arabidopsis thaliana.
Precursors for fatty acid synthesis are derived from sucrose through the glycolysis, the OPPP and the RuBisCO shunt.
Fatty acids synthesised in the plastids are then exported toward the cytosol in the form of acyl-CoAs, acylated on a
glycerol backbone to form triacylglycerides, which are ultimately stored in oil bodies. Amino acids required for the
synthesis of seed storage proteins are either directly imported from the maternal tissues or synthesised/modified in the
embryo. Solid arrows represent positive transcriptional regulations. Target genes encoding actors of the metabolic
network are placed in the inner ring of the scheme. ACCase, heteromeric acetyl-CoA carboxylase; FA, fatty acids; OPPP,
oxidative pentose phosphate pathway; PDHp, plastidial pyruvate dehydrogenase complex; PKp, plastidial pyruvate
kinase; SSP, seed storage proteins; TAG, triacylglycerides.

Transcriptional Regulation of the Metabolic Network

Recent studies have clearly established that master regulators like LEC2 or FUS3 can trigger directly the
transcription of SSP or oleosin genes. On the contrary, there were no clues to explain how the master regulators
could directly impact on primary metabolism and fatty acid biosynthesis. These observations have led to
hypothesise that part of the seed maturation process is indirectly regulated via secondary transcription factors
able to trigger their own transcription program (Gutierrez et al., 2007). WRINKLED1 (WRI1; At3g54320) was
thus shown to be a target of LEC2 (Baud et al., 2007a) that encodes a transcription factor of the APETALA2-
ethylene responsive element-binding protein (AP2-EREBP) family (Cernac and Benning, 2004).
In wri1 maturing seeds, primary metabolism is compromised (Baud and Graham, 2006; Focks and Benning,
1998), rendering maturing embryos unable to efficiently convert sucrose into TAGs. Recent studies have led to
the isolation of putative targets of WRI1 (Baud et al., 2007a; Ruuska et al., 2002). These include several genes
encoding enzymes of late glycolysis (PKp-α, PKp-β1, PDH-E1α) and the plastidial fatty acid biosynthetic
machinery (MOD1). Taken together, these data illustrate how WRI1 specifies the regulatory action of a master
regulator toward the metabolic network involved in the production of storage fatty acids (Figure 6).
Interestingly, LEC1 may participate in the regulation of WRI1 expression (Casson and Lindsey, 2006) and
sucrose may play a role in triggering induction of WRI1 (Masaki et al., 2005).
Go to:

METABOLIC ENGINEERING
Metabolic engineering can be defined as the redirection or modulation of flux through a metabolic pathway,
resulting in an increase in the concentration of an existing compound or the accumulation of a novel product
(Kinney, 2006). In A. thaliana, several attempts have led to successes in flux redirection, especially in the
production of modified plant oils. Most of these attempts rely on the manipulation of lipid metabolism. To date,
the protein-to-oil ratio and the factors controlling this ratio in oilseeds have been poorly investigated. Some
modifications of this ratio have been reported as side effects of strategies specifically designed to enhance TAG
production. The recent discovery that the over-expression, in A. thaliana, of two Dof-type transcription factor
genes from soybean, GmDof4 and GmDof11, induces the transcription of several genes of the fatty acid
biosynthetic network whilst down-regulating CRA1, may pave the way for further investigations in this
direction (Wang et al., 2007).

Modification of Protein Content

The introduction of an “extra” member of the 2S albumin SSP family in transgenic A. thaliana has little or no
effect on the expression of the genuine members of the family (Guerche et al., 1990). Data concerning the
impact of this strategy on the overall amount of SSP stored are missing. An enhancement of the nitrogen status
accompanied by an elevation of the seed protein content has been obtained in transgenic lines overexpressing
constitutively ASN1 (At3g47340), the major expressed gene for Asn synthase (EC 6.3.5.4) (Lam et al., 2003).
This is at least partially due to an increased transport of Asn from source to sink via the phloem and illustrates
how seed protein content depends on amino acid supply (Hernandez-Sebastia et al., 2005; Rolletschek et al.,
2005).
The structural features of SSPs require a specific amino acid composition for each storage protein. To be
efficiently incorporated into SSPs, the content in free amino acids in plant seeds should fit the amino acid
composition of SSPs. This requirement may involve a supercomposite regulatory network of amino acid
metabolism (Galili and Höfgen, 2002). Several attempts have concentrated on the manipulation of amino acid
balance in seeds to increase the content of “essential” amino acids like lysine or me-thionine that contribute
significantly to the nutritional quality of plant products. Efforts to improve lysine production in seeds have
firstly utilised bacterial dihydrodipicolinate synthase (DHPS) enzymes that are much less sensitive to lysine
feedback inhibition than their plant counterparts (Falco et al., 1995; Mazur et al., 1999). Transgenic rapes over
expressing DHPS have significantly elevated free lysine in mature seeds, but extreme free lysine
overproduction cause problems of seed germination. Another approach to enhance the level of a given amino
acid has consisted in transforming plants with genes encoding SSPs that are rich in the desired amino acid. If
the expression of lysine-rich protein has been mostly performed in maize, several attempts have been made to
accumulate methionine-rich 2S storage proteins like the Brazil nut 2S albumin in rapeseed (Altenbach et al.,
1992; De Clercq et al., 1990). However, most of the genetic engineering strategies that have been recently
developed to increase and/or modify the protein content of seeds have been conducted on legume (Rolletschek
et al., 2005) and cereal species (Bhalla, 2006; Shewry, 2007).

Modification of Oil Content

A first set of attempts has concentrated on the manipulation of total oil levels in seeds of A. thaliana and B.
napus. Over-expression of individual genes involved in de novo fatty acid synthesis in the plastid has not
allowed stimulating fatty acid production (Dehesh et al., 2001; Thelen and Ohlrogge, 2002b). The over-
expression of the WRI1 transcription factor, regarded as central regulator of seed fatty acid synthesis, slightly
increased the oil content of transgenic lines (Cernac and Benning, 2004). Whereas the manipulation of fatty acid
synthesis seemed to have limited impact on the amount of oil stored, increased supply of glycerol backbones led
to a significant improvement of the final seed oil content. The seed specific over-expression of a yeast gene
coding for cytosolic Gly3PDH thus resulted in a 40% increase in oil content of transgenic rape seeds (Vigeolas
 et al., 2007). Likewise, studies investigating the over-expression of GPAT (Jain et al., 2000), LPAAT (Taylor et
al., 2002; Zou et al., 1997) and DAGAT (Jako et al., 2001) demonstrated that the acylating steps of the Kennedy
pathway exert significant control over the amount of oil stored in seeds.
A second set of attempts has focused on modifying the balance of fatty acids naturally occurring in seeds of A.
thaliana or B. napus. Vegetable oils derived from the seeds of crop plants serve as easily extracted resource for
both food and a variety of industrial applications. For many food applications, vegetable oils are made semisolid
by hydrogenation. Unfortunately, this process increases saturated fatty acid content and also results in the
production of trans-unsaturated fatty acids that have been associated with coronary heart disease (Broun et al.,
1999). Vegetable oils with a reduced amount of trans-unsaturated fatty acids are consequently desirable to
improve human health (Thelen and Ohlrogge, 2002). This can be achieved by down regulating endogenous
stearoyl-ACP desaturase gene in B. Napus (Knutzon et al., 1992). For plant oils to be economically viable for
the manufacture of specialty chemicals, these oils need to be highly enriched in fatty acid of interest (for a
review, see Jaworski and Cahoon, 2003). When fatty acids of interest are naturally found at low levels in crop
species, strategies can be developed to enhance their relative proportion by modulating the expression of
appropriate endogenous genes (Dörmann et al., 2000) or by over-expressing related genes originating from
other plant species (Dehesh et al., 1996; Hawkins and Kridl, 1998; Jones et al., 1995). Attempts have also been
made to store intermediate species of the fatty acid biosynthetic pathway that are not accumulated in wild-type
seeds. The expression of a California bay thioesterase in the seeds of A. thaliana or B. napus thus results in the
accumulation of laurate (12:0) up to 24 and 58% of total fatty acids, respectively, by short-circuiting the acyl
chain elongation process (Voelker et al., 1992; Voelker et al., 1996). The additional introduction of a laurate-
specific coconut LPAAT in transgenic B. napus lines further increases laurate accumulation, up to 67%
(Knutzon et al., 1999).
Finally, many unusual fatty acids found in non-agronomic species exhibit properties that are suitable for
industrial applications, including appropriate chain length, appropriate positioning and configuration of double
bonds, presence of hydroxyl or epoxy groups. Biotechnological efforts have therefore focused on isolating
genes that encode the enzymes required for the synthesis of these unusual fatty acids for transfer to A.
thaliana and then/or to crop species like rape (Voelker and Kinney, 2001). In addition to the search for naturally
occurring enzymes, there is potential for the rational design of enzymes to produce novel fatty acids with
desired functionalities (Jaworski and Cahoon, 2003). The introduction, in A. thaliana, of acyl-ACP desaturases
like the naturally occurring Thunbergia Δ6 16:0-ACP desaturase (Schultz and Ohlrogge, 2001) and an
engineered castor Δ9 18:0-ACP desat-urase with improved specificity toward 16:0-ACP (Cahoon and Shanklin,
2000) illustrate these complementary approaches. Recent efforts to produce unusual fatty acids have mainly
focused on divergent forms of the Δ12-oleic acid desaturase (FAD2) that catalyse a wide range of fatty acid
modifications (Cahoon and Kinney, 2005), including hydroxylation (Lu et al., 2006; Smith et al., 2003),
epoxygenation, and double bond conjugation (Cahoon et al., 2006). A greater challenge has been the production
of very-long-chain polyunsaturated fatty acids (VLCPUFA) like eicosapentaenoic acid (EPA, 20:5) and
docosahexaenoic acid (DHA, 22:6) in oilseed crops (Cahoon et al., 2007). Engineering of EPA and DHA
biosynthetic pathways in brassicaceae species has required the introduction of four and six genes, respectively,
that have been isolated from several marine algal, thraustochytrid, mammalian, and fungal sources (Abbadi et
al., 2004; Robert et al., 2005; Wu et al., 2005). Despite some successes like laurate or EPA productions (for a
review see Davies, 2007), the amounts of unusual fatty acids that accumulate in transgenic lines are often
considerably less than those found in tissues from the natural sources of these fatty acids. This illustrates the
week efficiency of unsual fatty acid fluxes into TAGs in transgenic plants (Cahoon et al., 2007). The high levels
of β-oxidation and glyoxylate enzyme activities found in developing transgenic seeds further restrict the
production of high levels of unusual fatty acids (Larson et al., 2002; Moire et al., 2004). Improving our
knowledge of fatty acid fluxes is now necessary to introduce, in addition to the enzymes necessary for the
synthesis of unusual fatty acids, the enzymes that will assure a proper and efficient channelling of these fatty
acids into TAGs (Graham et al., 2007).
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3243342/

 B6.

What is the theoretical limit of productivity of crops and what are the major factors
preventing this being realized?

Factors limiting and preventing emerging farmers to progress to

commercial agricultural farming in the King William's Town area of the
Eastern Cape Province, South Africa
Khapayi M.I; Celliers P. R.II

I
PhD candidate, Department of Agriculture and Game Management, Nelson Mandela Metropolitan
University, Port Elizabeth, South Africa. E-mail: musa.khapayi@nmmu.ac.za
II
Senior lecturer, Department of Agriculture and Game Management, Nelson Mandela Metropolitan
University, Port Elizabeth, South Africa Email:Phillip.Celliers2@nmmu.ac.za

Correspondence

ABSTRACT

The aim of the study was to investigate the main limiting factors that prevent emerging farmers from
progressing from subsistence to commercial agricultural farming in the Eastern Cape Province. The study
was conducted in the King William's Town area by means of a structured questionnaire survey. A sample
of 50 households was drawn from the research area which was chosen owing to its uniqueness with regard
to agricultural potential. A descriptive analysis technique was employed to investigate the main limiting
factors faced by farming households in migrating towards commercial agricultural markets. The findings
demonstrated that the specific limiting factors emerging farmers face are poor physical infrastructure such
as poor roads, lack of transportation to the markets from the farms, lack of marketing skills and
information, poor market infrastructure, and high transaction costs, insufficient land availability to expand
production, lack of agricultural implements to better production, poor production and farm management
skills, as well as low education levels which results in an inability to interpret market information to be
used in production planning and marketing. The results from the study highlighted that the government
has a crucial role to play in increasing market participation of emerging farmers through encouraging
group marketing, upgrading of roads to enable smooth accessibility of farmers to output markets and the
establishment of local point sales in farming rural areas. Finally the study recommended that government
provides planned workshops to all farmers in order to equip them with marketing knowledge.

Keywords: Emerging farmer; Constraints; Eastern Cape Province

1. INTRODUCTION

A number of studies have been conducted on the topic of commercialisation of emerging farmers with the
aim of broadening the knowledge on the challenges that limit such migration from subsistence to
commercial farming. Despite the valuable knowledge generated by these studies, there is still a
remarkable scarcity of scientific information describing a more detailed picture of major challenges that
affect emerging farmers. The South African government has in the past 18 years implemented several
policies and programmes as well as increased the budget spent on the agricultural sector supporting
emerging farmers (Department of Agriculture Forestry and Fisheries, 2010; Frequin, Anseeuw & Da'Haese,
2012; National Treasury, 1996, 2004, 2005, 2008, 2010 and Aliber & Hall, 2012). However to-date there
is inadequate evidence that these attempts had been successful (Frequin, Anseeuw & Da'haese, 2012;
National Treasury, 1996, 2004, 2005, 2008, 2010; Aliber & Hall, 2012).

Indeed programmes and other attempts by government and development agencies have exacerbated
rather than alleviated the difficulties emerging farmers face (Frequin et al, 2012). To-date emerging
farmers still living below the poverty line are faced with difficulties in migrating into the commercial
agricultural sector (Frequin, et.al 2012; Aliber & Hall, 2012). The failure of several attempts by
government to integrate emerging farmers into the commercial agricultural economy has increased the
need for a well-grounded scientific knowledge and a thorough understanding of these challenges that
emerging farmers' face.

A better understanding of specific factors that limit the development of emerging farmers is crucial in
order to effectively prepare policies, development strategies, programmes and models aimed at
supporting and enhancing the transition of emerging farmers into commercial agricultural farming. South
Africa can no longer afford to run the risk of development programmes and policy intervention aimed at
emerging farmers that do not work seemingly not because they are not working but because the
challenges emerging farmers face were not correctly identified. Given the prominence of the topic the
main objective of this article is to provide a scientific understanding of the challenges, issues and barriers
emerging farmers face at transitioning into commercial farming as well as provide the relationship limiting
factors have with the commercialisation of emerging farmers. Commercialisation of emerging farmers is a
crucial topic for the current times in South Africa. The present study is primarily focused on the factors
that limit the transition of emerging farmers in the King William's Town area from successfully
participating in commercially viable markets. In the study it is assumed that commercialisation of
subsistence agriculture implies increased participation in remunerative agricultural commercial output
markets.

1.1 Issues and challenges for emerging farmers in South Africa

The South African agricultural economy has little room for emerging farmers. There is no strong support
system available to support previously disadvantaged farmers (Chikazunga & Paradza, 2012:3-4), causing
such farmers to be unable to take advantage of the various opportunities that the South African
government has been instituting (Moloi, 2010:46 & Anyike, 2011). According to a study by Chikazunga &
Paradza (2012:4), South African agricultural economy grew rapidly under the previous South African
government owing to strong state subsidies and support programmes aimed at supporting commercial
farmers. Similar support programmes and state subsidies were seen as encouraging the agricultural
economy of the United States of America and Europe (Chikazunga et.al, 2012:4). Currently South African
agriculture depends heavily on world markets for marketing agricultural products (Chikazunga et al.
2012:3-4). The removal of marketing boards' state subsidies along with the de-regulation of the
agricultural sector subsequent to the democratic transition in 1994 caused serious problems for
commercial farmers in particular to previously disadvantaged farmers. By 1997 interest rate subsidies and
export subsidies had ended completely and by late 1998 all marketing control boards were privatised with
only the sugar industry continuing to have price support from the government (Chikazunga et al. 2012:3-
4). Many emerging farmers face difficulties in accessing formal agricultural markets. As a result formal
markets do not interest emerging farmers. Lack of market participation is a common feature of emerging
farmers world-wide and is identified by Bie'nabe & Vermuelen (2011:494) as a constraint to emergent
farmer development. In South African underdeveloped rural areas emerging farmers find it difficult to
participate in commercial markets because of a range of constraints (Makura & Mokoena 2001:455;
Wynne & Lyne, 2003:566). Attempts by farmers to market their commodity are mostly affected by poor
infrastructure, inadequate property rights (Wynne & Lyne, 2003:566; Wynne & Lyne, 2004:9), low
education levels amongst the farmers, lack of credit access, absence of innovative production implements
needed in-order to increase yield of commodity produced and poor entrepreneurial skills needed to make
the efforts of the farmers a success (Bie'nabe & Vermuelen, 2011:494). Research conducted by the
National Emergent Red Meat Producer's Organisation (2004) identified a number of skills shortages among
emerging farmers such as a major constraint of growth. NERPO (2004) suggested that the new South
African government must improve its efforts in attracting young people into the agricultural industry. Poor
financial and social capital and limited access to legal resources make it difficult for emerging farmers to
change negative market factors individually. As a result emerging farmers continue to be trapped in a
cycle of operating within the given market from which their agricultural activities do not receive rewards
(Makhura et al. 2001:456).

2. METHODOLOGY

It has been noted in the literature that some researchers use the term methods and methodology
interchangeable (Hussey & Hussey, 1997:35). Many researchers consider methodology as an overall study
approach that is undertaken and methods as various means by which data is collected and analysed.
According to Mason (2002:30) the concept of methodology is separated from method. A method is part of
the methodological strategy (Mason, 2003:30). Saunders, Fernandes & Kosnes (2009:3) explain
methodology as the theory of how a research study should be undertaken and that it entails the study
design and the methods that are used for data collection and analysis.

The approach taken in this study is to include all aspects of the research process under the heading of
methodology. Therefore the research design, the approach taken in this study, the type of data collection
methods selected and the means of data analysis are all considered to be part of the study's methodology.

To ensure that the survey will get to the heart of the research problem and enable the researcher to
answer the research questions a pilot study was conducted in Port Elizabeth, Rocklands and Uitenhange.
The pilot study was also conducted to pre-test the questionnaires used for the present study data
collection. The main survey to obtain the primary data for this study was conducted in King William's Town
an area situated in the central region of the Eastern Cape Province. Agriculture in this area is the most
used enterprise for household survival followed by operating shebbeens and taverns and the State grant
(Moloi, 2010:47). Most inhabitants in the identified area relied on farming for household survival. Data
was collected using structured questionnaires. A survey of households provided much needed information
on the demographics of the group of farmers such as their socio-economic characteristics. The survey
involved 50 households selected in the research area.

In selecting a suitable representative sample the researcher followed a two-stage sampling process, in
which the first stage involved selecting the survey area. This was followed by selecting the total number of
emerging farmers engaged in agriculture in the area. It was found that the total number of agricultural
households in the area is 43624 (Statistics South Africa & Department of Agriculture, 2000; Statistics
South Africa, 2010; Statistics South Africa, 2011). This consisted of mainly commercial farmers (Statistics
South Africa & Department of Agriculture, 2000; Statistics South Africa, 2010; Statistics South Africa,
2011). According to Statistics South Africa (2011), the country lacks information on smallholder and
subsistence agriculture. The current list of farmers being used to conduct surveys is mainly confined to
commercial agriculture. Until now agricultural censuses and surveys have largely concentrated on
commercial agriculture and have ignored small-scale and subsistence agriculture (Statistics South Africa,
2010; 2011). Thus a total number of subsistence farmers in the survey area could not be realized from
Statistics South Africa or the Department of Agriculture.

The researcher established that emerging farmers in the area had formed associations as farmers. A list of
emerging farmers' associations and the members of the associations was obtained from a non-
government organization (NGO) aimed at assisting emerging farmers in the area. The researcher
approached the chairman of the NGO and explained the objectives and the purpose of the study. No
objections were made by the chairman. The researcher attempted to interview all the farmers on the list
totalling to 124, but many farmers declined to participate in the study for a variety reason; some farmers
were farming for household consumption where only surplus was marketed; some farmers were involved
in other businesses, thus farming part-time; and some farmers were inactive owing to various constraints.
These farmers could not participate in the study. The target group of the study was on emerging farmers
that depend on the farm sources for household survival, farming full-time and producing mainly for the
market. The researcher applied criteria of availability, willingness to cooperate and a sampling size of 50
was realised amongst those farmers available and willing to participate.

In general researchers prefer probabilistic or random sampling methods over non-probabilistic ones. They
consider the former to be more accurate and rigorous. However, in applied social research there are
circumstances where it is not feasible, practical or theoretically sensible to apply a random sampling
method (Trochim, 2006a: 361). A non-probabilistic method was used in the present study. According to
Trochim (2006b:10) purposive sampling is one of the methods of non-probability sampling. It is
approached with a specific plan in mind and targets a specific sample. The sampling procedure is further
subcategorised as snowballing. In snowball sampling the researcher identifies the participants that meet
the criteria for inclusion, and the participants are then asked to recommend others who they may know,
who also meet the criteria for inclusion in the study. Although this method would hardly lead to
representative samples, there are times when it may be the best method available (Trochim, 2006:10). In
the case of the present study, farmers had formed associations as mentioned earlier, which made it easy
for participants to recommend others as they knew each other well.

A questionnaire was used to collect data. According to Truckman (2000) and Van Niekerk (2002:35),
questionnaire survey methods make it possible to measure what a person knows and the type of
information he/she has, the values and beliefs of the person and the attitudes towards what the
questionnaire is about. When conducting a questionnaire survey it is better to use an administered
questionnaire for better results (Van Niekerk, 2002:36). The questionnaire survey can be used in three
different ways namely: personal interviews, telephonic interviews and mail interviews (Randela, 2005:9).
The present study made use of personal interviews because they enable the interviewer to observe
behaviour that the questionnaire is not designed to detect. The questionnaire was relevant to the
objectives of the study and the respondents involved in the study.

The questionnaire consisted of section A and section B. Section A contained questions on demographic
characteristics and socioeconomic factors. While section B contained questions covering farming skills of
the respondents and production and marketing challenges the respondents face. The questions in the
questionnaire were designed in English but during the interviews they were translated in isiXhosa by the
researcher, the language of the survey area and understood by the participants. The researcher
understands that most of emerging farmers have low education levels and people express their views
better when they express them in their own language. The respondents were asked to select the
challenges that affect them the most from all the common challenges that were listed in the
questionnaire. All data collected were based on the main factors affecting the migration of emerging
farmers into commercial markets in the survey area.

2.1 Data analysis

The data collected from the questionnaire in this study was coded by the researcher by assigning a
numerical value in order to facilitate easier workability on the SPSSx program version 21 and Statistical
version 11. The Microsoft Office Excel 2010 software package was used to capture the coded data. This
made it easier for the researcher to check for mistakes before analysing the data in SPSSx and Statistical.
The Institute for Statistical Consultation at the Nelson Mandela Metropolitan University carried out the
processing of the data, using the statistical Package for Social Science and Statistical, software used for
statistical analyses. The percentages calculated were based on the total number of farmers who responded
to that particular question. The farmers that did not respond to a particular question were excluded from
the calculation of percentage values for that question. When a farmer selected more than one answer or
gave more than one method to a question, percentages were calculated for each group of similar answers.

3. RESULTS AND DISCUSSIONS

The information that follows result from a descriptive analysis of the data collected. The results are
presented using descriptive statistics frequencies, counts, charts, percentages and standard deviations. All
respondents were interviewed on their farms by appointment. Interviewing the respondents in their farms
permitted the researcher to observe their farming areas. The respondents were asked to motivate their
answers to verify whether they understood what they were being asked. In this regard all the results in
the present study are assumed to be correct and valid.
The study used data collected from a sample of 50 emerging farmers using a questionnaire survey. All 50
farmers were engaged in livestock rearing; about 30 of whom were also engaged in crop production.

Figure 1 below shows the distribution of the farmers by their level of education. The education levels of
the farmers were low. Figure 1 shows that 12 % of farmers had less than grade 8 school level; 32 % had
grade 8; 18 % had grade 9; 16 % had grade 10; 18 % had grade 11; and 4 % had completed grade 12.
This means that 62 % of the farmers had less than a grade 10 school level. None of the heads of
households had tertiary education.

3.1 Farming skills

Twenty-six items were used to measure the level of adequacy of the farmers on farming and management
skills necessary to operate crop and livestock production farms. The farmers were measured according to
their line of production. Farmers solely engaged in livestock production were asked to respond to livestock
production questions and management questions in the questionnaire only. The farmers that were also
engaged in crop production were asked to respond to the livestock and crop production questions since
they were also engaged in livestock production as well as the management questions. The questions were
designed using four point Likert scale. Likert scale is a psychometric scale commonly used in researches
which employs questionnaire as a survey instrument. After the questionnaires were completed each item
was analysed and presented separately. In this study experience is equal to knowledge. It is assumed that
if a farmer has knowledge of a certain aspect it will be easier to find experience along the way. Similarly if
a farmer has experience about certain aspects then the farmer will have knowledge on that experience.

3.2 Crop production skills

Twelve items were used to measure the level of adequacy of the respondents on crop production skills
necessary to operate a crop production enterprise. The results are shown on Table 1. For most of the
items the farmers perceived their level of adequacy to be inadequate or they did not know. The farmers
said that all the skills listed were important for them to know as farmers and knowing them would
contribute towards their development to commercial farming and accessing lucrative markets. The farmers
further claimed that given an opportunity to learn the skills they would learn them. Limited knowledge as
well as lack of skills in crop production among farmers constrains crop production, particularly in small
scale irrigation systems. The following table shows the distribution of the sampled farmers experience on
skills need for successful crop production.

As depicted in Table 1, majority of the sampled farmers namely 57% had no experience in mulching and
33% had inadequate experience. The farmers did not practise mulching on their farms owing to
insufficient knowledge about mulching and its application. Only 7 % had adequate experience and 3% had
an outstanding experience.

For seed bed and care skills 47 % of the farmers had inadequate experience and 50 % had adequate
experience. The farmers with inadequate experience in seed bed and care skills were buying their
seedlings from commercial farmers and nurseries. Only 3% of the farmers had an outstanding experience
in producing their own seedlings.

A total of 57% of the farmers were not irrigating their farms, 43% had inadequate knowledge about
irrigation equipment and were also not irrigating in their farms owing to lack of irrigation equipment. The
farmers claimed that the irrigation equipment is expensive to buy and could not afford to buy it. The
majority of the farmers were using hosepipes and watering cans to water their crops.

Pests can cause mechanical damage to the produce affecting yield quality and resulting to yield loss.
Weeds compete with crops for nutrients and can habour pests with a negative effect on the planted crop.
The sampled farmers had no integrated pest or weed management measures in place to control pests and
weeds. When asked about the use of chemical pesticides, the farmers claimed that they were afraid of
using pesticides owing to lack of understanding on the use of chemical pesticides.

A correct application of fertilizer on crops normally boosts production and the possibility of the farmer to
be engaged in high value markets. The majority of the farmers namely 17% had no experience of using
fertilisers, 37% had inadequate experience and 47% had adequate experience. The farmers revealed that
they cannot afford to buy chemical fertilisers owing to high prices.

Harvesting is the final stage of the production cycle where the invested capital yields income to pay debts
and to buy production inputs. If harvesting is not well planned and managed the capital invested will not
yield the profit needed to buy production inputs. The sampled farmers claimed that they do not plan prior
the harvesting period. They did not even know that they had to plan for harvesting.

Poor packing of produce can cause mechanical damage. Mechanical damage causes rotting of produce,
resulting in poor yield quality. The majority of consumers look at the appearance of produce before buying
it (Raleting, 2011:68). The majority of the farmers were packing their produce in available plastic bags
during the harvesting time.

3.3 Livestock rearing skills

Seven items were used to measure the farmers' level of experience in livestock skills necessary to operate
a livestock enterprise. The results are shown in Table 2. For most of the items farmers perceived their
level of experience to be either no experience or inadequate experience. Calf rearing is an important
activity on a livestock farm because young calves are the future of the herd. Well reared healthy calves
produce high yielding healthy adults. The majority of the sampled respondents namely 44 % had no
experience in calf rearing and 52 % had inadequate experience in calf rearing while 2 % had adequate
experience in calf rearing. The majority of the farmers 48 % claimed not to plan for the weaning of their
calves, 46 % had adequate experience in calf weaning and 6 % had outstanding experience in calf
weaning and claimed to plan for weaning of their calves as well as to keep records of everything such as
the mother of the calf and the father.

The majority of sampled households were not able to identify sick animals, diagnose them or solve the
health problem prior to it becoming a severe condition. The respondents relied mostly on other
neighbouring farmers and animal technicians that rarely visit the farms. However they were able to
recognise animals with foot-rot and mastitis. The hygiene of livestock and the farm helps to prevent
disease build up and outbreaks among livestock. Calf pneumonia can be minimised by cleaning the houses
where calves are kept. Hygienic feeding equipment helps to prevent the transmission of disease. Trimming
and cleaning hooves of livestock help to prevent foot rot disease among livestock. The majority of the
farmers (98 %) claimed not to be informed about animal and farm hygiene and as a result it was not a
concern of theirs. The majority of farmers had no systematic breeding programme or approach they were
following for breeding their livestock. The farmers had no experience or inadequate experience of breeding
skill. With artificial insemination skill farmers can breed their own desired breeds. This is a cheaper
method than buying breeds from certified breeders. A total of 88 % of the farmers claimed not to have
experience on artificial insemination skill; 8 % perceived their level of experience on artificial insemination
skill to be inadequate and 4 % of the farmers perceive their level of experience in artificial insemination
skills to be adequate. The following table shows the distribution of the farmer's experience on skills
necessary for livestock production.

3.4 Management skills

Seven items were used to measure the level of adequacy of the sampled farmers in different farming
management skills necessary to operate a livestock or crop production farm. The frequencies for these
items are shown in Table 3. For most of the items the farmers perceived their experience to be inadequate
or adequate. However, there were some farmers with no knowledge in certain management skills although
some farmers had outstanding experience in certain management skills. The farmers agreed that skills
such as business management, marketing skills, banking skills, labour management and record keeping
are critical for successful farming and knowing them could contribute towards the success of their farms.

Educating small scale farmers on management skills need to compliment the policies which are geared
towards small scale farmers' development. Agribusiness requires some knowledge of how the commodity
has been produced, the amount and brand of fertiliser and the chemicals applied. All these are achievable
through good management skills.

3.5 Marketing challenges

Seven items were used to identify the marketing challenges faced by the farmers. The frequencies for the
items are shown in Table 4. The findings showed that the sampled farmers are faced with marketing
challenges such as insufficient market facilities, scale pens and loading ramps for livestock farmers,
insufficient market information, low prices, cheap food imports coming from other countries and high
transaction costs. Even farmers who managed to produce products of good quality were not realising good
profits from their produce owing to insufficient markets being available. The harvests of the farmers were
lost after harvesting because of spoilage. Many of the farmers tried to sell their produce to big
supermarkets but were told that their produce does not meet the requirements specified by the
supermarkets and lack certificate for Good Agricultural Practises. The following table shows the
distribution of marketing challenges that mostly affect the sampled farmers.

3.6 Transportation costs

Amongst the marketing challenges the farmers face were high transportation costs. Marketing transport is
important as it links the farmers to the markets or consumers on time. The availability of one's own
market transport influences the delivery time of produce to the markets, unlike the case of farmers who
depend on hired transport or public transport to transport their produce. Transport availability determines
the quality of the delivered produce. Unreliable transport can lead to the late delivery of produce. In the
case of emerging farmers who lack storage facilities late delivery of produce can result in loss of produce
quality and rendering the producer unreliable to the buyer. Table 5 shows the transportation cost of
emerging farmers per year. The majority of the farmers were spending between R 3000 to above R 5000
on transportation per annum.

Farmers that are close to the road and have access to transport are better integrated to the markets as
compared to farmers that are not. The majority of the farmers' localities were situated far from public
roads and were serviced by gravel roads which were not well maintained and impenetrable during rainy
conditions.

3.7 Marketing channels

Emerging farmers use different types of marketing channels to market their produce. Each marketing
channel has associated costs such as transportation costs, profits and prices of produce. Before choosing a
marketing channel a farmer has to consider these costs. The farmers' choice of marketing channel can
pose problems and result in lower earnings. In general, the income of the farmer can be determined by
the choice of marketing channel used. The farmers claimed to be using more than one marketing channel:
in that case they were allowed to choose more than one answer from the questionnaire. The results were
treated separately and are presented separately in Figure 2. Therefore the percentages in Figure 2 are
expected not to add up to 100 % as one answer was chosen more than once from the questionnaire. The
majority of the sampled farmers appeared to be using informal markets to market their commodity, such
as neighbours, rural consumers, farm gates, local traders and urban spot markets. The following figure
shows the distribution of marketing channels used by the farmers.
3.8 Poor access to market information

As shown in figure 3 the majority of the sampled farmers (55 %) did not have access to market
information. Such farmers are unlikely to participate in marketing because they are not well informed of
what is happening in the markets. The farmers were not well informed of market prices, products in
supply or the products in demand. Only 45 % of the farmers had access to market information. The
majority of the farmers with access to market information relied on family members, self-research and
other farmers for market information. The farmers claimed that the information was not timely, was
sometimes biased and was unreliable making the usefulness of the information doubtful.

3.9 Support services

The provision of support services remain one of the major important interventions in the agricultural
sector for rural development, commercialisation, food security, poverty alleviation and income generation
of emerging farmers. The commercialisation of emerging farmers cannot be achieved without appropriate
farmer support services. With adequate access to farmer support services, emerging agriculture can
contribute to an increased agricultural growth, rural development and have a positive impact on the farm
income. When the respondents were asked about support services various answers were given. The
majority of the sampled farmers namely 64 % claimed to be receiving support services for their farming
enterprises (see Table 6) while 36 % claimed not to be receiving any support service but had to rely on
their own resources. When the farmers' were asked about extension services stated that the extension
officer visited them once in a while. None of them could recall the visit routine.
When asked about their source of support services the farmers appeared to have more than one source of
support and more than one type of support they were receiving. The majority of the farmers were
receiving support from the government in the form of water, bales during droughts and medicine during
disease outbreaks. Other farmers claimed that the department of agriculture assisted them with poultry
structures and some farmers with piggery structures. The community NGO's were also assisting with
market support. The NGO's were buying some of the farmers' commodity for feeding schemes and care
homes.

The availability of land by the farmers seems to be also a concern in market participation. It is important
that farmers have enough land to produce if they are to participate in commercial agricultural markets.
The study found that 72 % of the farmers are producing on land less than 10 hectares. Only 28 % of the
farmers are producing on land more than 10 hectares. This demonstrates that insufficient land availability
in South Africa is still a challenge that many emerging farmers face. This has negative implications for
sustainability and farm income, especially for livestock emerging farmers who depend on the availability of
land for grazing and expansion of livestock production.

4. CONCLUSIONS

The emergent agricultural sector in South Africa has the potential to contribute to the growth of rural
areas, and the reduction of unemployment, poverty and inequalities. The potential of emerging farmers to
participate in this sector is untapped. Emerging farmers do not participate in markets that yield high
returns. In order for emerging farmers to contribute to rural development and transit into the commercial
farming sector the above mentioned aspects need to be addressed effectively. The main objective of the
present study was to identify the challenges that prevent the commercialisation of emerging farmers into
commercial agricultural markets. The study identified seven specific challenges to emerging farmers in the
area. These were low education levels, lack of farming skills on crop and livestock production, poor
management skills, high transportation cost, lack of market information, poor support services from the
government, and participating in low remunerative marketing channels.

The majority of the produce produced by the farmers is sold to informal markets with low market value.
Some of the farmers used more than one marketing channel. The distance to output markets is an
important factor. Long distances to the market can be discouraging to farmers who want to
commercialise. All the remunerative markets were located far away from the localities of the farmers. This
implied that farmers had to travel long distances to formal markets on gravel road with their commodity
loaded on poor transportation. Poor infrastructures and a lack of transportation infrastructure affect the
quality of produce thus causing farmers' produce to be uneconomical or to lose quality.
 The finding also showed that most respondents suffer from a lack of market information owing to a lack of
communication, tools and support services from the government and extension officers. The majority of
the farmers relied on word-of-mouth, family and self-research for information regarding market prices
which in most cases was biased not accurate or up-to-date. Marketing information is very important for
the market participation of emerging farmers. The availability of market information with regard to prices
can boost the confidence of a farmer in marketing his or her produce. It can also help a farmer to choose
marketing channels which ensure a better profit. The availability of market information helps farmers to
make informed decisions about the marketing channels in which to participate. Farmers with no access to
market information often make poor decisions. Market information helps the farmer by enhancing his or
her bargaining power. The availability of market information about market variables such as prices and
products in high supply and high demand in the market is important to the market performance of
emerging farmers.

A business in general requires someone who is open-minded and has a quick understanding mind, skills
such as record keeping and banking skills, labour management and the ability to choose a profitable
enterprise and production method for that enterprise. Agricultural production methods in particular are
dynamic and require someone who is current with developments and changes. All these requirements are
achievable through education. When people are uneducated they become victims of being cheated and
once people are cheated they refuse to adopt further innovation or change even if it is beneficial to them.
Chemicals such as pesticides and herbicides and integrated pest management methods need someone
who will understand them and their instructions because they can be dangerous to humans and produce
resulting in loss of produce. However this does not mean that uneducated farmers or farmers with low
education levels cannot be successful commercial farmers. The majority of the farmers in the present
study were uneducated or had low education levels. This contributes to their lack of participation in formal
markets.

Infrastructure such as roads, communication lines and farming facilities needed to be upgraded as it was
restricting emerging farmers from commercialising to commercial farming. Fencing was required in the
localities of the farmers. Irrigation equipment for crop farmers is important and needed for vegetable
farming as water is a fundamental necessity. The unavailability of cultivation infrastructure such as
cultivation tractors and ploughing implements was found to limit the farmers' productivity. The majority of
the respondents claimed to be using the little money they have to hire these implements when needed.
The implements of those who had them were old and poor in quality.

The farmers needed training in various production skills of which could boost their productivity and farm
income. Skills such as training in the development of marketing strategies could help them access and
secure marketing channels. Overcoming the challenges emerging farmers face can induce the farmers to
move towards commercial agricultural systems. In order for emerging farmers to withstand both local and
international competition, the South African government needs to consider support policies and regulation
that are necessary to stimulate growth among emerging farmers. The State has a crucial role to play in
increasing market participation of emerging farmers through encouraging group marketing, the upgrading
of roads to enable smooth accessibility of farmers to output markets and the establishment of local point
sales in farming rural areas. The government should enact laws and implement policies that are
favourable to emerging farmers. Finally, the present study recommends that government provides
planned workshops to all farmers in order to equip them with marketing knowledge.

5. RECOMMENDATIONS

Many limiting factors that affect the migration of emerging farmers into commercial agricultural farming
throughout the Eastern Cape Province have been discussed in this paper. The main factors are low
education levels among the farmers in order to understand the dynamics of agriculture, poor
management, lack of farming skills, poor access to formal remunerative markets, high transportation
costs to formal markets, poor market information and insufficient support services from the government.
It was found that the government needs to take a leading role in investing in these support services.
Access to productive land, production inputs, infrastructure, extension services, and value adding facilities
in the location of the farmers, market information and transport logistics has been found to be the key
factors influencing emerging farmers' participation in remunerative agricultural markets.

http://www.scielo.org.za/scielo.php?script=sci_arttext&pid=S0301-603X2016000100003

 B7.

What determines seed longevity and dormancy?

 Natural Variation for Seed Longevity and Seed
Dormancy Are Negatively Correlated in
Arabidopsis
Thu-Phuong Nguyen, Paul Keizer, Fred van Eeuwijk, Sjef Smeekens, Leónie Bentsink
Published December 2012. DOI: https://doi.org/10.1104/pp.112.206649

 Article

 Figures & Data

 Info & Metrics

 PDF

 © 2012 American Society of Plant Biologists. All Rights Reserved.

Abstract

Dormancy is a state of metabolic arrest that facilitates the survival of organisms during environmental conditions incompatible
with their regular course of life. Many organisms have deep dormant stages to promote an extended life span (increased
longevity). In contrast, plants have seed dormancy and seed longevity described as two traits. Seed dormancy is defined as a
temporary failure of a viable seed to germinate in conditions that favor germination, whereas seed longevity is defined as seed
viability after dry storage (storability). In plants, the association of seed longevity with seed dormancy has not been studied in
detail. This is surprising given the ecological, agronomical, and economic importance of seed longevity. We studied seed
longevity to reveal its genetic regulators and its association with seed dormancy in Arabidopsis (Arabidopsis thaliana).
Integrated quantitative trait locus analyses for seed longevity, in six recombinant inbred line populations, revealed five
loci: Germination Ability After Storage1 (GAAS1) to GAAS5. GAAS loci colocated with seed dormancy loci, Delay Of
Germination (DOG), earlier identified in the same six recombinant inbred line populations. Both GAAS loci and their colocation
with DOG loci were validated by near isogenic lines. A negative correlation was observed, deep seed dormancy correlating with
low seed longevity and vice versa. Detailed analysis on the collocating GAAS5 and DOG1 quantitative trait loci revealed that
the DOG1-Cape Verde Islands allele both reduces seed longevity and increases seed dormancy. To our knowledge, this study is
the first to report a negative correlation between seed longevity and seed dormancy.
Dormancy describes a state of apparent metabolic arrest during which the normal progression of life activities and development
is dramatically reduced or brought to a halt. Dormancy facilitates the survival of organisms during environmental conditions
that cannot support the regular course of life. Many organisms have dormant stages, which in different species have different
names, such as dauer stage in Drosophila spp., diapause in water flea and fish embryos, akinetes in cyanobacteria, spores in
yeast (Saccharomyces cerevisiae), and dormancy in plant seeds and flower buds. Organisms can enter the dormant state due to
environmental cues such as a lack of water by undergoing desiccation, low temperature, or through developmentally
programmed arrest, as occurs, for example, in yeast spores and plant seeds. In most organisms, dormancy has been related to an
extension of their life span (increasing longevity; Lubzens et al., 2010).
In contrast to most other organisms described above, in plant seeds dormancy and longevity have been described as two
separate traits. Seed dormancy is defined as a temporal failure of a seed to germinate in conditions that favor germination
(Bewley, 1997). Seed dormancy can be overcome by environmental cues (i.e. seed dry storage [after ripening] and cold
stratification). Seed dormancy has been studied extensively (Finch-Savage and Leubner-Metzger, 2006; Holdsworth et al.,
2008), and recently, a quantitative trait locus (QTL) analysis in combination with transcriptome analyses in Arabidopsis
(Arabidopsis thaliana) has revealed that natural variation for seed dormancy is controlled by independent genetic and molecular
pathways (Bentsink et al., 2010).
Seed longevity is defined as seed viability after seed dry storage (storability) and, therefore, describes the total seed life span
(Rajjou and Debeaujon, 2008). This storability period includes both the dormant and nondormant states. During seed storage,
seeds deteriorate, lose vigor, and, as a result, become more sensitive to stresses during germination, and ultimately die. The rate
of this aging depends on the seed moisture content, temperature, and initial seed quality (Walters, 1998; Walters et al., 2005).
Seed longevity is a quantitative trait for which variation is present among naturally occurring accessions. QTLs for seed
longevity have been identified after natural aging in Arabidopsis (Bentsink et al., 2000; Clerkx et al., 2004b), lettuce (Lactuca
sativa; Schwember and Bradford, 2010), and rice (Oryza sativa; Sasaki et al., 2005) and after artificial aging imposed by a
controlled deterioration test (CDT) in Arabidopsis (Bentsink et al., 2000; Clerkx et al., 2004b), rice (Miura et al., 2002), and
wheat (Triticum aestivum; Landjeva et al., 2009). The ability of the CDT to predict seed longevity was shown by the
colocalization of major QTLs after artificial and natural aging in two different studies using the Arabidopsis recombinant
inbred line (RIL) populations Landsberg erecta (Ler)/Cape Verde Islands (Cvi; Bentsink et al., 2000) and Ler/Shakdara
(Sha; Clerkx et al., 2004b). Besides these genetic analyses, also proteome studies in Arabidopsis have shown that similar
molecular events occur during natural and artificial (CDT) aging (Rajjou et al., 2008). In contrast with this, Schwember and
Bradford (2010) did not find overlap between seed longevity QTLs under conventional and controlled deterioration storage
conditions in lettuce.
The genetic basis of seed longevity is unclear. However, there are several groups of mutants that have altered seed longevity.
The majority of mutants with known effects on seed longevity are the seed developmental mutants. Mutations in the key
regulators of seed maturation lead to rapid loss of viability upon storage, as has been shown for leafy cotyledon1 (lec1)
and abscisic acid intensitive3 (abi3) mutants (Ooms et al., 1993; Clerkx et al., 2004a; Sugliani et al., 2009). Another group of
mutants with a seed longevity phenotype consists of the testa mutants. The seed coat or testa acts as a structural barrier to
protect the embryo and seed reserves from biotic and abiotic stresses. The testa-defective mutants, including transparent
testa (tt) and aberrant testa shape (ats; Debeaujon et al., 2000), display considerably reduced seed longevity. Moreover,
mutations in protection and repair systems that prevent seed vigor loss lead to decreased seed longevity. Arabidopsis mutants
affected in vitamin E (lipophilic antioxidant) biosynthesis, vte1 and vte2, exhibited significantly reduced seed longevity (Sattler
et al., 2004). Waterworth et al. (2010) showed that DNA LIGASEVI and DNA LIGASEIV, which are essential to maintain
genome integrity in plants, are major determinants of Arabidopsis seed quality and longevity. The atlig6 mutant and atlig6
atlig4 double mutant are more sensitive to controlled seed aging than wild type.
Proteins and enzymes are also described as factors that may determine seed longevity. Heat stress transcription factor-
overaccumulating seeds of transgenic Arabidopsis display enhanced accumulation of Heat Stress Protein and improved
tolerance to aging (Prieto-Dapena et al., 2006). Protein repair appears to play a key role in the long-term survival of seeds in
the dry state. PIMT (for protein L-isoaspartyl methyltransferase), which limits and repairs age-damaged aspartyl and asparaginyl
residues in proteins, has been associated with greater seed longevity because it is highly accumulated in sacred lotus seed
(Nelumbo nucifera), one of the world’s longest living seeds (1,300 years; Shen-Miller, 2002). Overexpression of PIMT1 in
Arabidopsis enhanced both seed longevity and germination vigor, whereas reduced PIMT1 expression led to increased
sensitivity to aging treatments and loss of seed vigor under stressful germination conditions (Ogé et al., 2008).
However, PIMT exhibited a decreased activity in naturally aged barley (Hordeum vulgare) seeds (Mudgett et al., 1997). In
addition, enzymes playing roles in the detoxification of reactive oxygen species, such as glutathione peroxidase and glutathione
reductase (Bailly et al., 1996), and toxic cyanide compounds, such as β-mercaptopyruvate sulfurtransferase (Rajjou et al.,
2008), are important to prolong seed longevity.
Given the important precondition of many organisms to become dormant before exposure to and survival of long-term
(desiccation) stress, as well as the ecological, agronomical, and economic importance of seed longevity, it is surprising that the
association of seed longevity and seed dormancy has not been studied in much detail. The current idea is that seed dormancy
and seed longevity are positively correlated. This hypothesis is based mainly on the performance of the earlier
mentioned lec1, abi3 (Ooms et al., 1993; Clerkx et al., 2004a; Sugliani et al., 2009), tt, and ats mutants (Debeaujon et al.,
2000) but also on the loss-of-function mutant in the DOG1 gene (Bentsink et al., 2006) and the green seed mutant (enhancer
of abi3-1; Clerkx et al., 2003). All these mutants have a reduced dormancy level that correlates with reduced seed longevity.
Here, we study natural variation for seed longevity in order to reveal its genetic regulators and their possible association with
seed dormancy. We have performed integrated QTL analyses for seed longevity, measured as germination ability after storage
at ambient conditions, in six RIL populations. These populations were derived from crosses between the Arabidopsis standard
laboratory accession Ler and the accessions Cvi, Antwerpen (An-1), St. Maria do Feira (Fei-0), Kashmir (Kas-2), Kondara
(Kond), and Sha, which were previously used for seed dormancy analyses (Bentsink et al., 2010). The major seed
longevity QTLs colocated with the earlier identified seed dormancy QTLs. QTLs and colocation have been validated by near-
isogenic lines (NILs). The results are discussed in the context of the current knowledge of seed dormancy and seed longevity.

RESULTS

Seed Longevity of the Parental Accessions and Their RIL Populations

Seed longevity was measured as germination ability after seed dry storage at ambient conditions, which is referred to as “natural
aging” in this work. We have used seeds of seven accessions (Ler, An-1, Cvi, Fei-0, Kas-2, Kond, and Sha) and
six RIL populations that were constructed from crosses between Ler and the other accessions for this study. The RILs were
grown between 2002 and 2005 (Supplemental Table S1). After harvest, seed dormancy behavior was measured until
germination reached 100% (Bentsink et al., 2010). In early 2010 (after 4–7 years of dry storage), the same RILs were assessed
for seed longevity (germination ability after aging) using the Germinator tool developed by Joosen et al. (2010). As a result of
aging, the maximal germination percentage (Gmax) decreased. Overall, the six populations exhibited variation
for Gmax (ranging from 0% to 100%; Fig. 1A). Transgression beyond values of both parents was observed in all populations,
indicating that both parental lines carried allele-increasing and -decreasing seed longevity (Fig. 1A). The Ler/Kas-2 population
harvested in 2002 was the most aged population; approximately one-third of the RILs germinated to less than 50%, whereas the
majority of the RILs in Ler/Fei-0 (harvested in 2004) and Ler/Kond (harvested in 2003) populations had a Gmax higher than
75%.

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Figure 1.
Frequency distributions of seed longevity presented by four germination parameters in six RIL populations. The
different RIL populations are indicated at the top. The x axis contains the trait values for Gmax (%; A), AUC (B), t10totS (h;
C), and t50Gmax (h; D). Arrowheads depict the values of parental lines (black arrowheads for Ler and gray arrowheads for
other accessions).
The Ler parent, which was grown together with each population, had a Gmax ranging from 54% to 100% (Supplemental Table
S1). The Ler parent grown with the Ler/Cvi population harvested in 2005 had a much lower germination ability (Gmax of
80%) than the longer stored Ler parents grown in earlier years (2003 and 2004). This might be the consequence of different
growing environments, since it is known that environmental conditions during seed maturation strongly affect seed quality
(Contreras et al., 2008).
Seed germination after storage is not only analyzed by Gmax but also by other germination parameters, such as germination
rates (time to reach 10% germination of the total number of seeds [t10totS] and time to reach 50% germination of the total
number of germinated seeds [t50Gmax]) and area under the curve (AUC; a parameter that describes the germination curve
based on the germination rate and the Gmax), which were also measured by the Germinator tool. There was a lot of variation
for these three additional parameters in the six populations: t10totS ranged from 30 to 120 h, t50Gmax from 30 to 150 h,
and AUC from 0 to 85 (Fig. 1, B–D). For these germination parameters, we do not have the initial values, since the Germinator
tool that allows scoring of large populations was not developed at the time the seeds were harvested. Moreover, as a result of
aging, a reduction of germination behavior becomes first apparent in a lower germination rate (t10totS and t50Gmax), followed
by a decrease of Gmax, which are both reflected by a reduction in AUC. For this reason, these parameters might still contain
valuable information for the seed longevity analyses. Therefore, we will focus our study mainly on QTLs found for Gmax but
will compare these also with QTLs identified for AUC and use these together to identify colocation with seed dormancy.
Integrated QTL Analyses for Seed Longevity in Six RIL Populations
In order to identify loci controlling seed longevity, mixed-model QTL analyses were performed as described by Bentsink et al.
(2010). The six RIL populations were explored simultaneously, and the different allele effects were examined for
each QTL and every population. In total, five QTLs for seed longevity measured as Gmax were identified. We named
these QTLs Germination Ability After Storage1 (GAAS1) to GAAS5 (Fig. 2). These loci showed strong additive effects
accounting for an average of 23.3% of the total explained variance (Table I); no epistatic interaction among loci was detected.
The mapping results revealed three major loci, GAAS1, GAAS2, and GAAS5, which were also detected for the other parameters
(Table I; Supplemental Table S3). The genome-wide significance values of the major QTLs, calculated on a −10Log(P) scale
using a final QTL model after backward selection from the Composite Interval Mapping model, were 15.6, 20.5, and 7.2,
respectively, while those of the minor QTLs were below 7.2.

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Figure 2.
Integrated QTL analyses for seed longevity expressed as Gmax in six RIL populations. A, Genome-wide profiling of simple
interval mapping with a genome-wide threshold of 2.74 on the −10Log(P) scale. B, Composite interval mapping with a fixed
cofactor revealed five QTL loci (GAAS1–GAAS5). Cofactor positions are depicted by black vertical bars. The confidence
interval of each locus is presented by the blue columns: the darker the color, the more significant the QTL. C, QTL effects for
every single population. Orange indicates that the Ler allele increases seed longevity (Gmax), and cyan indicates that
the Ler allele decreases seed longevity. The intensity of the color corresponds to the size of the QTL effect: the higher the
intensity, the stronger the effect. Black vertical bars indicate the significance of the QTL effect in that population. D,
Confirmation of the major seed longevity loci (GAAS1, GAAS2, GAAS3, and GAAS5). The genotypes of NILGAAS1-Cvi,
NILGAAS2-An-1, NILGAAS3-Kas-2, and NILGAAS5-Sha as well as Ler are schematically presented. Orange indicates
the Ler alleles, and cyan indicates the alleles of the other accessions; missing marker data are indicated in light gray. Seed
longevity (Gmax) values of the four NILs and Ler after natural aging for 5 years are indicated at the right. The Gmax values of
the NILs are significantly different from that of Ler (P < 0.05).
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Table I.QTLs for Gmax in six populations, as obtained by integrated analyses comprising composite interval mapping
and backward selection
The genome-wide threshold in composite interval mapping was 2.74 on the −10Log(P) scale, with P representing
the P value. QTL name, chromosome, position, significance expressed on a −10Log(P) scale, and a ±1.5 dropoff interval on the
−10Log(P) scale (support interval) are presented. P values are taken from the final multi-QTL model after backward selection.
Dropoff intervals are assessed on the composite interval mapping profile. QTL allele substitution effects (Gmax) are given in
the right part of the table. A negative value indicates that Ler is decreasing the Gmax, whereas a positive value indicates
that Ler increases the Gmax. Significant effects are indicated in boldface; these are the effects of the QTLs indicated in Figure
1. In the bottom part of the table, for each population's mean Gmax, the explained variance and the average of explained
variance by main-effect QTLs are presented in percentages. The last column shows the range of explained effects of every
locus in percentages.
The major locus GAAS2 explained 1.9% to 15.5% of the phenotypic variation in the individual populations and had a significant
effect in almost all populations (Table I; Fig. 2). The Ler allele of this locus decreased seed longevity (lower Gmax) in all
cases. GAAS1, the second strongest QTL, accounting for 0.6% to 12.5% of the effect in the individual populations, only showed
a significant effect in the Ler/Cvi population; also for this QTL, the Ler allele decreased seed longevity. GAAS5, explaining
0.4% to 5.4% of phenotypic variation in the single populations, had a significant effect in two of the six populations (Ler/An-
1 and Ler/Cvi); for this QTL, the Ler allele increased seed longevity.
Seven additional minor QTLs identified for the other parameters (AUC, t10totS, and t50Gmax) are
named GAAS6 to GAAS12 (Supplemental Table S2).
Confirmation of the GAAS Loci
To characterize the GAAS loci, NILs carrying single genomic fragments of different accessions into the Ler genetic background
were used (Table II). The germination behavior of these lines was analyzed after 5 years of seed dry storage. We could confirm
four of the five GAAS (Gmax) loci. For the two strongest QTLs, GAAS1 and GAAS2, we had only one NIL each available
(NILGAAS1-Cvi and NILGAAS2-An-1, respectively). The seeds of these lines performed significantly better after storage when
compared with their genetic background Ler (Table II; Fig. 2D). GAAS3 was validated by NILGAAS3-Kas-2, which showed a
significant reduction in Gmax when compared with Ler. For the GAAS5 locus, we had five NILs available (NILGAAS5-Cvi,
NILGAAS5-Fei-0, NILGAAS5-Kas-2, NILGAAS5-Kond, and NILGAAS5-Sha), of which only NILGAAS5-Fei-0 and
NILGAAS5-Sha showed a significant reduction for Gmax in comparison with Ler. Nongerminating seeds were stained with
2,3,5-triphenyl tetrazolium chloride to determine whether they were dead (Moore, 1985). We concluded that the seeds were
deteriorated, since the majority of the seeds did not stain (Supplemental Fig. S1).
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Table II.Confirmation of GAAS loci by NILs
The germination behavior of a set of NILs grown in 2007 (QTL locus, NIL name, and original name [Bentsink et al., 2010])
after 5 years of natural aging is presented by Gmax, AUC, t10totS, t50Gmax, and percentage of normal seedlings. Average
values and SE (as indicated for each NIL) are indicated. Germination behaviors that are significantly different from that
of Ler are indicated by asterisks (*P < 0.05, **P < 0.01).

Comparison between Seed Longevity and Seed Dormancy

In order to explore the relationship between seed longevity and seed dormancy, we investigated the overlap between QTLs that
have been mapped for these two traits. Integrated QTL analyses for both seed longevity and seed dormancy (Delay of
Germination [DOG]) have been performed in the same populations (Bentsink et al., 2010), which allows a neat comparison
between those two traits. Four of the five GAAS loci identified for Gmax overlapped with DOG loci
(GAAS2/DOG22, GAAS3/DOG6, and GAAS5/DOG1) or mapped in very close proximity (GAAS1/DOG2; Fig. 3A). All
five GAAS Gmax QTLs were also identified using AUC as a longevity parameter, which can be explained by the high
correlation between these two parameters (r2 = 0.75–0.90; Supplemental Table S3). AUC showed the highest variation (Fig.
1B), which provided more statistical power in the QTL analyses and led to the identification of four
additional QTLs (Supplemental Table S3). Seven of the nine GAAS AUC QTLs colocated with seed dormancy QTLs (Fig.
3A). The three additional genomic regions that showed colocation are GAAS7/DOG20, GAAS10/DOG5,
and GAAS11/DOG4 (Fig. 3A). Unexpectedly, a negative relationship between seed dormancy and seed longevity was observed.
Deep dormancy correlated with low storability and shallow dormancy with high storability, shown by the direction of the
arrows in Figure 3A. Since we had NILs available for most of the QTLs, we were able to analyze this correlation in more
detail (Fig. 3, B and C). The NILs were grown in two independent experiments in the greenhouse (in 2006 and 2007) and
analyzed for their seed dormancy (directly after harvest) and seed longevity behavior (after 5 years of storage; Table
II; Supplemental Table S4). The negative correlation between seed longevity (Gmax) and seed dormancy (days of seed dry
storage required to reach 50% germination [DSDS50]) that was identified by the QTL analyses was proven to be very
significant, given the very high correlation coefficients (r2) of 0.66 (P = 0.03) and 0.89 (P < 0.01), respectively, for the two
experiments (Fig. 3, B and C).

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Figure 3.
Colocation and correlation between seed longevity (GAAS) and seed dormancy (DOG) QTLs. A, Integrated composite interval
mapping profiles of seed longevity (AUC; top) and seed dormancy (DSDS50; bottom) performed in the six RIL populations.
The confidence interval of each locus is presented by the gray columns: the darker the color, the more significant the QTL.
Cofactor positions are depicted by arrows: arrows pointing up indicate that the Ler allele is increasing the trait value, and when
the arrows point down, Ler decreases the trait value. B and C, Correlation of seed longevity (germination percentage) and seed
dormancy (DSDS50) in NILs. B, Experiment harvested in 2006 including NILDOG1/GAAS5-Sha, NILDOG22/GAAS2-An-1,
NILDOG2/GAAS1-Cvi, NILDOG6/GAAS3-Fei-0, NILDOG6/GAAS3-Kas-2, NILDOG20/GAAS7-Fei-0, and Ler. C,
Experiment harvested in 2007 including NILDOG20/GAAS7-Fei-0, NILDOG1/GAAS5-Sha, NILDOG1/GAAS5-Kond,
NILDOG1/GAAS5-Kas-2, NILDOG22/GAAS2-An-1, NILDOG2/GAAS1-Cvi, NILDOG6/GAAS3-Kas-2, and Ler.
Next we investigated the correlation between seed longevity and seed dormancy in the RILs; however, no correlation was
found (r2 between 0.01 and 0.12 for the six populations). This lack of correlation might be explained by the fact that total
genetic variation was not fully explained by the identified QTLs (Table I) and by the low variation for seed longevity
in RIL populations (Fig. 1). Furthermore, we have ended up with a random combination of seed longevity and dormancy loci in
the RILs due to the broken linkage (which is the nature of this type of population). This results in a random combination of
phenotypes, since some of these loci have a stronger longevity effect and others a stronger dormancy effect. To remove the
above-mentioned noise, we have performed correlation analyses on RILs that have been selected for either seed longevity
increasing or decreasing alleles at the position of the strongest four QTLs (GAAS1, GAAS2, GAAS3, and GAAS5) in two of the
populations that show the largest variation for seed longevity (Ler/Kas-2) or the maximum possibility of QTL colocation
within a population (Ler/Cvi; Supplemental Table S5 and S6). These analyses showed again the negative relationship between
seed longevity and dormancy (Supplemental Fig. S4), with r2 of 0.58 for Ler/Kas-2 (P < 0.01) and 0.78 for Ler/Cvi (P < 0.01).
Seed Longevity and Dormancy Are Regulated by One Gene at the Position of GAAS5/DOG1
The colocation between seed longevity and seed dormancy QTLs can be caused by a single gene or by separate linked genes.
The genetic nature of the colocation can only be studied when the genes underlying the QTLs are identified. So far, the only
identified QTL is the dormancy locus DOG1 (collocating with GAAS5). We used transgenic lines that carry
the DOG1 Cvi allele in the Ler genetic background to investigate whether these lines, in addition to their increased dormancy
level, also showed a seed longevity phenotype. The Gmax of seeds (Ler, NILGAAS5/DOG1-Cvi, and two independent
transformants) that had been naturally aged for 7 years was still above 70% and did not reveal clear differences between any of
the genotypes (Supplemental Fig. S3). In order to accelerate the aging, we stored these seeds at 75% relative humidity for 59 d
and analyzed the Gmax at several time points during this storage. After 59 d of storage in 75% relative humidity, all seeds of all
genotypes were completely deteriorated. However, the two transformants were significantly less storable than the Ler control,
which shows that DOG1 does not only control seed dormancy but also seed longevity (Fig. 4; Supplemental Fig. S3). This
result supports the negative correlation between seed longevity and seed dormancy.

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Figure 4.
Seed dormancy and longevity phenotypes of Ler, NILDOG1/GAAS5-Cvi, and two independent transformants. Seed dormancy
measured as DSDS50 and longevity after 7 years of storage and 45 d in 75% relative humidity (germination percentage) is
shown for Ler, NILDOG1/GAAS5-Cvi, and two independent transformants (SR3-1 and SR3-2), which contain
the DOG1 Cvi allele in the Ler genetic background.

DISCUSSION

In order to study natural variation for seed longevity, we used seed batches that had been stored for 4 to 7 years at ambient
conditions. Seed longevity had been analyzed in Arabidopsis populations previously, using artificial (CDT; Bentsink et al.,
2000; Clerkx et al., 2004b; Joosen et al., 2012) and natural (Bentsink et al., 2000) aging. Bentsink et al. (2000) showed that
naturally aged seeds of the Ler/Cvi RIL population that had been stored for 4 years led to the detection of one QTL on
chromosome 1 (GAAS1 region). This locus was also the major QTL in our experiment here for the Ler/Cvi population.
However, with this long-term-aged seed, we were able to identify two additional QTLs (GAAS2 and GAAS5) in
this Ler/Cvi genetic background. GAAS1 and GAAS2 or colocating QTLs were also detected after artificial aging in
the Ler/Cvi, Ler/Sha, and Bayreuth/Sha (not for GAAS2) RIL populations (Bentsink et al., 2000; Clerkx et al.,
2004b; Joosen et al., 2012). GAAS5 appears to be specific for natural aging and does not show a QTL after controlled
deterioration in the Ler/Cvi and Ler/Sha populations, which indicates that the CDT does not completely mimic natural aging.
Overall, we were able to identify many more loci than in earlier work (Bentsink et al., 2000; Clerkx et al., 2004b; Joosen et
al., 2012), probably due to the much longer time that the seeds had been stored and because of the integrated approach on the
multiple populations that was taken.
The parents of every population were grown together with the RIL populations. Ler differed the most from Fei-0 and Kas-
2 accessions for seed longevity (Supplemental Table S2). In both cases, Ler seeds were better storable, which might be
explained by the fact that the Ler allele contributes to better storability for almost all GAAS loci except for GAAS2 (Fig. 2). The
seed longevity of Ler was not very different from those of the accessions An-1, Cvi, Kond, and Sha, but transgressions beyond
these parents were identified for those populations (Fig. 1), which resulted in the identification of seed longevity QTLs for
which alleles of both parents contributed to better storability (Fig. 2).
GAAS2 and GAAS5 had significant allelic effects in more than one population (Table I; Supplemental Table S2), which may
indicate the importance of these seed longevity loci under natural selection. For GAAS1 and GAAS2, the Ler allele decreased
seed longevity, while the GAAS5 Ler allele increased seed longevity. Epistatic interactions between the seed longevity loci were
not identified, which indicated that natural variation for seed longevity in these populations is determined by additive loci.
However, part of the differences might result from genotype-environment interactions due to differences in the growing
environments of each population.

Candidate Genes

Several GAAS genomic regions identified here contain genes previously associated with seed longevity. Most obvious is the
colocation of GAAS2 with the vitamin E locus. Sattler et al. (2004) have shown that vitamin E (tocopherol) is essential for seed
longevity in an artificial aging assay, as it prevents lipid oxidation during seed germination. Genetic analysis of seed vitamin E
levels in the Cvi/Ler and Columbia/Ler populations exhibited a common QTL on the top of chromosome 3 (GAAS2 region),
namely QVE7 and QVE8, respectively (Sattler et al., 2004).
The DNA ligase AtLIG4 coincides within the confidence interval of GAAS6 in the middle of chromosome 1. Repair of DNA
damage in seeds to maintain genome integrity is one of the mechanisms to prevent seed deterioration (Waterworth et al.,
2010). DNA damage is associated with single and double strand breaks, which can be rejoined by DNA ligase. Two DNA ligase
genes, AtLIG4 and AtLIG6, were shown to be involved in DNA repair upon seed imbibition in Arabidopsis. Mutations in these
genes lead to decreased seed viability and seed germination vigor after a CDT.
PIMT genes might be the underlying loci for GAAS3 and GAAS5. PIMT1 and PIMT2, which are located on the lower arms of
chromosome 3 and 5, respectively, play a role in the repair of age-related protein damage in Arabidopsis (Ogé et al., 2008). The
accumulation of the PIMT1 enzyme enhances resistance to seed vigor loss induced by CDT.
Colocation between Seed Longevity QTLs and Seed Dormancy QTLs
Several QTL studies have been performed for seed longevity (Bentsink et al., 2000; Miura et al., 2002; Clerkx et al.,
2004b; Sasaki et al., 2005; Landjeva et al., 2009; Schwember and Bradford, 2010) and seed dormancy (Bentsink et al.,
2007); however, none of these discuss the relationship (colocation and/or correlation) of both traits. Therefore, we report, to our
knowledge for the first time, a negative correlation between seed longevity and seed dormancy QTLs. Lower storability levels
correlated with higher seed dormancy levels, and conversely, better storability correlated with lower seed dormancy. This
finding is unexpected, since current seed literature only describes correlations of low longevity with low dormancy and high
longevity with high dormancy. However, these correlations were mainly based on mutants such as lec1, abi3, tt, ats, dog1, and
the green seed mutant. We assume that these contrasting observations are based on the nature of the mutations. The induced
mutants all have defective seed maturation and consequently did not become dormant and desiccation tolerant, as these features
are acquired during seed maturation. Very likely, these mutants represent artifacts that do not survive in nature. Moreover, none
of the earlier mentioned seed longevity mutants (i.e. atlig4, atlig6, pimt1, and pimt2) have been investigated for their seed
dormancy behavior. Such an analysis would reveal whether the same processes could affect seed longevity and seed dormancy
and thereby also provide insight in the underlying mechanisms. We expect that the natural variants that we used in our study
display the ecologically relevant germination behavior. The GAAS5/DOG1-Fei-0 allele (Supplemental Fig. S2) is special in that
it has lower longevity and lower seed dormancy when compared with Ler. The Fei-0 DOG1 allele has an opposite allelic effect
as compared with the other alleles for this QTL (Cvi, Kas-2, Kond, Sha), indicating that this allele is even weaker than
the Ler allele, which is not a null allele (Bentsink et al., 2006, 2010). The Fei-0 allele of DOG1, therefore, might result in a
nonfunctional DOG1 gene, since it has a similar phenotype (lack of dormancy and low storability) to the dog1 mutant
(Bentsink et al., 2006).
The colocation between seed longevity and seed dormancy QTLs can be caused by a single gene or by separate linked genes,
and this remains to be investigated. Detailed analyses on GAAS5/DOG1 showed that DOG1 is controlling both seed longevity
and seed dormancy. DOG1 has been cloned, and the Ler transformant containing the DOG1 allele of Cvi shows
complementation of the seed dormancy phenotype of NILDOG1 (Bentsink et al., 2006) and also reduction in seed longevity
(Fig. 4). This result shows that GAAS5 is actually the same locus as DOG1 and that the DOG1-Cvi allele leads to both higher
seed dormancy and lower seed longevity. The molecular mechanism by which DOG1 controls seed dormancy is still unclear;
however, recently it has been proposed that DOG1 protein abundance in freshly harvested seeds acts as a timer for seed
dormancy release (Nakabayashi et al., 2012). How the DOG1 protein affects seed storability remains to be investigated.
The novel observation of a negative correlation between dormancy and longevity strongly suggests that seeds are able to extend
their life span either by dormancy (and dormancy cycling) or by an active longevity mechanism. Selection for the different
mechanisms could be based on the natural environments in which the seeds are dispersed, dry environments resulting in active
longevity mechanisms and humid environments resulting in dormancy cycling during which aging damage may be prevented or
repaired. The presence of loci that either improve longevity or increase seed dormancy within one accession will allow adaptive
plasticity, resulting in the expression of the optimal phenotype over a range of environments (i.e. dry to humid; Simons, 2011).

CONCLUSION

We performed integrated QTL analyses on natural variation that exists for seed longevity after natural aging. We used
six RIL populations that were stored between 4 and 7 years at ambient conditions. The major QTLs could be confirmed
by NILs that also had been stored for 5 years. Seed longevity and dormancy data revealed a negative correlation, which
contrasts with the common notion in seed biology research. High storability correlated with shallow seed dormancy, and low
storability correlated with high levels of seed dormancy.

MATERIALS AND METHODS

Plant and Seed Materials

RILs
The six RIL populations derived from crosses between the Arabidopsis (Arabidopsis thaliana) standard laboratory
accession Ler and the accessions An-1, Cvi, Fei-0, Kas-2, Kond, and Sha were used. These populations have been analyzed
previously for seed dormancy as described by Bentsink et al. (2010). The RILs were grown and harvested between 2003 and
2005 as described by Bentsink et al. (2010). Seeds of every RIL were stored in 6- × 13-cm cellophane flat bags at room
temperature without humidity control until seed longevity was analyzed (Table I).
NILs
The NILs we used in this work were originally developed by the introgression of the identified dormancy QTL regions into
the Ler genetic background (Table II; Supplemental Table S3; Alonso-Blanco et al., 2003; Bentsink et al., 2010). In 2006,
seven NILs, NILGAAS5-Sha, NILGAAS2-An-1, NILGAAS1-Cvi, NILGAAS3-Cvi, NILGAAS3-Fei-0, NILGAAS3-Kas-2, and
NILGAAS5-Fei-0, were grown and harvested together with Ler. In 2007, nine NILs, NILGAAS1-Cvi, NILGAAS2-An-1,
NILGAAS3-Kas-2, NILGAAS5-Cvi, NILGAAS5-Fei-0, NILGAAS5-Kas, NILGAAS5-Kond, NILGAAS5-Sha, and Ler were
grown and harvested. Growing and harvesting methods were as described by Bentsink et al. (2010), and the storage method
was as described for the RILs.

The DOG1 Transformants

The two transformants, SR3-1 and SR3-2, containing the DOG1 Cvi allele into the Ler genetic background were obtained as
described by Bentsink et al. (2006). These lines and their control lines (Ler and NILDOG1-Cvi) were grown as in the study
of Bentsink et al. (2006) and stored as described for the RILs.

Seed Longevity Measurement

Natural Aging

Seed longevity was evaluated as germination ability after several years of storage in natural conditions. We have used the same
seed batches described by Bentsink et al. (2010). The germination percentage after dormancy release was 100% for all lines, as
was described by these authors. Germination assays after aging were performed according to Joosen at al. (2010) over a period
of 7 d. Briefly, six samples, 50 to 200 seeds each, were sown on two layers of blue germination papers equilibrated with 43 mL
of demineralized water in plastic trays (15 × 21 cm). Trays were piled and wrapped in a closed and transparent plastic bag.
Germination was incubated in a 22°C incubator under continuous light (30 W m−2).
Seed longevity was confirmed by viability test with 2,3,5-triphenyl tetrazolium chloride according to the International Seed
Testing Association (Moore, 1985). After 7 d of germination assay, nongerminated seeds were taken out for staining. Seeds
were punched gently by sharp forceps to make staining solution easily penetrate the embryo, placed on filter paper (Sartorius
filter discs 3hw) soaked with 1% 2,3,5-triphenyl tetrazolium chloride, sealed, and incubated at 28°C for 2 d. Seeds that are
viable stain red, and seeds that are dead do not stain.

Artificial Aging

In order to accelerate aging, we stored Ler, NILDOG1-Cvi, and the two independent transformants (SR3-1 and SR3-2) above a
saturated NaCl solution in a closed desiccator (relative humidity of 75%) for 59 d. At 0, 13, 31, 38, 45, 52, and 59 d, we
performed germination assays as described above. Two-way ANOVA (P < 0.05) was performed in order to identify the
differences in Gmax between the lines.

Seed Dormancy Measurement

Germination assays during after-ripening were performed on the same seed lots as described for the seed longevity
measurements. The data and methods used are presented by Bentsink et al. (2010). Germination data were fitted to logistic
curves by nonlinear regression analysis to determine DSDS50 as described by Bentsink et al. (2010).

Germination Parameters

Images from germination assays were taken twice per day over a 7-d period. Automatic scoring and curve fitting were analyzed
by the Germinator package (Joosen et al., 2010). Four parameters, Gmax, AUC, t10totS, and t50Gmax, representing the
germination ability were extracted. Gmax is the final germination percentage at the end of the germination assay.
The AUC parameter was measured at 120 h after sowing. AUC describes the germination curve by combining germination rate
and Gmax. The Germinator curve-fitting script, a part of the Germinator package, enables one to calculate averages and to
perform statistical Student’s t test. At the end of the germination assay, the percentage of normal seedlings was scored.
Integrated QTL Analyses
Integrated QTL analyses of the six RIL populations were carried out for every germination parameter defined above. The
analysis method was performed as described by Bentsink et al. (2010).

Supplemental Data
 The following materials are available in the online version of this article.

 Supplemental Figure S1. Viability test for seeds after natural aging with 2,3,5-triphenyl tetrazolium chloride.
 Supplemental Figure S2. Seed germination behavior of Ler, dog1 mutant, and NILDOG1/GAAS5-Fei-0 during seed dry
storage.
 Supplemental Figure S3. Seed life span of Ler, NILDOG1/GAAS5-Cvi, and two independent transformants.
 Supplemental Figure S4. Correlation analyses between seed longevity and seed dormancy of the RILs.
 Supplemental Table S1. Details of the six RIL populations.
 Supplemental Table S2. QTLs for AUC, t10totS, and t50Gmax in six RIL populations.
 Supplemental Table S3. Correlation coefficient of germination parameters: Gmax, AUC, t10totS, and t50Gmax in six RIL
populations.
 Supplemental Table S4. Seed longevity and dormancy phenotypes of NILs of two independent experiments.
 Supplemental Table S5. Genotypes of the RILs selected for seed longevity increasing and decreasing alleles for the Ler/Kas-2
population.
 Supplemental Table S6. Genotypes of the RILs selected for seed longevity increasing and decreasing alleles for the Ler/Cvi
populations.

Acknowledgments

We thank Corrie Hanhart and Maarten Koornneef (Laboratory of Genetics, Wageningen University) for storing the seeds of
the RIL populations over all these years. We also thank Henk Hilhorst (Laboratory of Plant Physiology, Wageningen
University), Bas Dekkers (Department of Molecular Plant Physiology, Utrecht University), and two anonymous reviewers for
useful comments and critical reading of the manuscript.

Footnotes

 The author responsible for distribution of materials integral to the findings presented in this article in accordance with the
policy described in the Instructions for Authors (www.plantphysiol.org) is: Leónie Bentsink (l.bentsink@uu.nl).
 www.plantphysiol.org/cgi/doi/10.1104/pp.112.206649
 ↵1 This work was supported by the Dutch Technology Foundation, which is the applied science division of the Netherlands
Organization for Scientific Research and the Technology Program of the Ministry of Economic Affairs (to T.-P.N. and L.B.),
by the Generation Challenge Program (Molecular Breeding Platform activity no. 2.2.1), and by the Centre of BioSystems
Genomics (project nos. BB9 and BB12 to P.K. and F.v.E.).
 ↵[OA] Open Access articles can be viewed online without a subscription.
 ↵[W] The online version of this article contains Web-only data.

Glossary

QTL
quantitative trait locus
CDT
controlled deterioration test
RIL
recombinant inbred line

http://www.plantphysiol.org/content/160/4/2083

 B8.

How can we control flowering time?

Control of Flowering Time

Interacting Pathways as a Basis for Diversity
 Aidyn Mouradov, Frédéric Cremer, George Coupland
Published May 2002. DOI: https://doi.org/10.1105/tpc.001362

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 American Society of Plant Biologists

INTRODUCTION

Flowering is controlled by environmental conditions and developmental regulation. The complexity of this regulation is created
by an intricate network of signaling pathways. Arabidopsis is an excellent model system in which to approach this complexity,
because it responds to many of the environmental conditions that control flowering in other species, and genetic tools are well
developed. Studies in Arabidopsis have led to the identification of components within individual signaling pathways that affect
flowering, and to their positioning within molecular hierarchies. Furthermore, distinct signaling pathways are known to
converge on the activation of the same flowering-time genes. This convergence of pathways on a common set of genes may
enable the integration of different responses, so that the plant can produce a coordinated flowering response under conditions in
which multiple environmental parameters are changing simultaneously. Also, genetic analysis of Arabidopsis varieties showing
natural variation in flowering time has demonstrated how the activity of these pathways can be altered in nature and how
balancing the effects of different environmental stimuli on flowering time is important in plants adapting to growth in different
geographical locations.

At present, the full complexity of the flowering network can only be approached in Arabidopsis where the necessary tools are
available, and extensive efforts are being made to describe related pathways. For example, photoreception, circadian clock
regulation, growth regulator synthesis and response, chromatin structure, and response to low temperatures play important roles
in flowering-time control and are studied extensively in Arabidopsis. Nevertheless, there is a need to understand how the full
diversity in flowering responses is generated. For example, Arabidopsis responds to photoperiod, but all ecotypes are long-day
plants that flower earlier under long than short days, whereas many other species show the reverse response. Also,
all Arabidopsis thaliana ecotypes are annual plants, and understanding the perennial habit will require a different model species.
To understand the diversity in flowering responses, there is a need to look to other species, and here we compare detailed
information from Arabidopsis with the emerging picture of the genetic control of flowering in short-day plants.
The control of flowering has been reviewed frequently over the last few years (Koornneef et al., 1998b; Simpson et al.,
1999; Reeves and Coupland, 2000; Samach and Coupland, 2000; Araki, 2001). However, progress has been rapid, and in
the following review we emphasize results that have emerged recently. In particular, we summarize the current understanding of
the signaling pathways involved in flowering control in Arabidopsis, and stress how alterations in the balance of activity
between pathways can give rise to dramatically different flowering behaviors, for example between ecotypes. We also address
recent progress in describing how these pathways function in species that show different responses to environmental conditions
than does Arabidopsis.

PATHWAYS CONTROLLING FLOWERING TIME IN ARABIDOPSIS

The Photoperiod Response Pathways

One of the most important factors controlling flowering time in temperate regions is the duration of the daily light period, or
photoperiod. Arabidopsis is a facultative long-day plant, which flowers earlier under long days but eventually flowers under
short days. Under laboratory conditions, Arabidopsis will flower in response to a single long day (Corbesier et al., 1996).
Molecular-genetic approaches have identified genes required for the daylength response, and some of these encode regulatory
proteins specifically involved in the regulation of flowering, while others encode components of light signal transduction
pathways or are involved in circadian clock function. A representation of the relationships among these processes is shown
in Figure 1 and is described in the following sections.
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Figure 1.
Signaling Pathways Involved in the Regulation of Flowering by Photoperiod in Arabidopsis.
A diagram showing the putative relationships among genes involved in the photoperiod pathway. On the basis of the
phenotypes of known mutants, genes shown in red generally repress flowering, whereas those in green promote it. Small upright
arrows indicate the role of the genes as determined by overexpression in transgenic plants. Arrows between genes represent a
promotive effect, whereas perpendicular lines represent a repressive effect, and simple lines represent protein–protein
interactions. Arrows from the clock indicate that the expression of the gene is circadian clock controlled. Arrows to the clock
indicate that the gene lengthens period length, while perpendicular lines indicate that it shortens period length. Numbers
between brackets refer to the following publications, which support the indications provided in the diagram. (1) (Somers et al.,
1998b) (2) (Swarup et al., 1999) (3) (Wang and Tobin, 1998) (4) (Schaffer et al., 1998) (5) (Mizoguchi et al.,
2002) (6) (Goto et al., 1991) (7) (Samach et al., 2000) (8) (Onouchi et al., 2000) (9) (Alabadi et al., 2001) (10) (Strayer et
al., 2000) (11) (Martinez-Garcia et al., 2000) (12) (Nelson et al., 2000) (13) (Jarillo et al., 2001) (14) (Millar et al.,
1995a) (15) (Somers et al., 2000) (16) (Sugano et al., 1999) (17) (Zagotta et al., 1992) (18) (Sugano et al., 1998) (19) (Liu et
al., 2001b) (20) (Hicks et al., 2001) (21) (Covington et al., 2001) (22) (Fowler et al., 1999) (23) (Park et al.,
1999) (24) (Fowler et al., 1999; Park et al., 1999) (25) (Johnson et al., 1994) (26) (Bagnall et al., 1995) (27) (Guo et al.,
1998) (28) (Putterill et al., 1995) (29) (Simon et al., 1996) (30) (Mozley and Thomas, 1995) (31) (Ahmad et al.,
1998) (32) (Koornneef et al., 1980) (33) (Suarez-Lopez et al., 2001) (34) (Somers et al., 1998a).

A Pathway That Promotes Flowering of Arabidopsis in Response to Long Days

Among the flowering-time mutants of Arabidopsis, there is a group that is late flowering in long days but similar or identical to
the wild type under short days. These mutants are weakly, or not at all, sensitive to vernalization, and were proposed to define a
genetic pathway that promotes flowering specifically in response to long
days. CONSTANS (CO), CRYPTOCHROME2/FHA (CRY2), GIGANTEA (GI), FT and FWA are part of this long day–promoting
pathway (Koornneef et al., 1991). CO is the latest acting of the known genes that is specific to this pathway; all of the other
genes also act in other pathways or have more general effects (Figure 1). FT and FWA act downstream of CO and in other
pathways (Kardailsky et al., 1999; Kobayashi et al., 1999; Onouchi et al., 2000), whereas GI and CRY2 act upstream
of CO (Guo et al., 1998; Suarez-Lopez et al., 2001).
The CO gene was proposed to encode a protein with two zinc fingers loosely related to those of GATA transcription factors
(Putterill et al., 1995) and contains a carboxy-terminal domain called CCT because it is present in CO, CO-like (COL), and
TIMING OF CAB 1 (TOC1) proteins (Strayer et al., 2000; Robson et al., 2001). Comparison of 16 Arabidopsis COL genes
that code for proteins sharing the zinc fingers and CCT domains (Robson et al., 2001) demonstrated that the zinc fingers are
most similar to the B-box, a type of zinc finger identified in animal proteins and believed to mediate protein–protein
interactions (Borden, 1998). The CCT domain is sufficient and necessary for nuclear localisation of GFP:CO or GFP:TOC1
fusion proteins (Strayer et al., 2000; Robson et al., 2001), but may have additional roles in protein–protein interactions
(Kurup et al., 2000).
When constitutively expressed from a cauliflower mosaic virus 35S promoter, CO induces early flowering and loss of
photoperiod sensitivity (Onouchi et al., 2000). Mutations in the FT, SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1,
also called AGAMOUS-LIKE 20 [AGL20]) and FWA genes partially suppress 35S::CO (Onouchi et al., 2000). In
addition, FT and SOC1 expression levels are increased in 35S::CO plants or 35S::CO:GR plants in which CO activity is induced
by dexamethasone (Samach et al., 2000), indicating that these genes act downstream of CO. In agreement with
this, 35S::FT can suppress the effect of co mutations (Kardailsky et al., 1999; Kobayashi et al., 1999). However, no mutations
were recovered that totally suppress the 35S::CO early-flowering phenotype, suggesting that parallel pathways are activated
by CO (Onouchi et al., 2000).
The CO transcript level shows a diurnal rhythm in long days, with a broad biphasic peak between 12 and 24 h after dawn and
maximum levels 16 and 24 h after dawn. This peak is narrower in short days, ending 4 h earlier (Suarez-Lopez et al., 2001).
Plants entrained in long days show a circadian rhythm in CO transcript level when transferred to continuous light (LL),
indicating that this rhythm is controlled by the circadian clock (Suarez-Lopez et al., 2001).
FT is an early target of CO (Samach et al., 2000), and its transcript level follows a circadian rhythm that peaks 20 h after dawn
in long days (Suarez-Lopez et al., 2001). This peak is absent in the co mutant. In addition, the circadian clock–related
mutants, LATE ELONGATED HYPOCOTYL (LHY), GI, and EARLY FLOWERING3 (ELF3), show an altered rhythm
in CO transcript level that correlates with their flowering phenotype. Overall, CO appears to mediate between the circadian
clock and the flowering-time gene FT (Suarez-Lopez et al., 2001). This model suggests that the circadian clock acts within the
long-day pathway to regulate the expression of downstream genes such as CO and FT.
Two general models have been proposed for how flowering may be regulated by daylength (Thomas and Vince-Prue,
1997; Samach and Coupland, 2000; Samach and Gover, 2001). The external coincidence model proposes that light acts as an
external signal that interacts with a light-sensitive rhythm at certain times of day. In long-day plants, such as Arabidopsis, if the
plant is exposed to light at the crucial phase of the rhythm, flowering is promoted. Another model, the internal coincidence
model, suggests that in photoperiods that are inductive, two rhythms are brought into the same phase and interact to promote
flowering, whereas in noninductive photoperiods, these rhythms are out of phase. Observing the activation
of FT by CO, Suarez-Lopez et al. (2001) proposed that the expression pattern of CO may represent a light-sensitive rhythm
that is exposed to light only under long-day conditions that promote flowering, and that post-transcriptional activation of CO by
light may lead to the activation of FT transcription and ultimately to flowering. This proposal represents a version of the
external coincidence model.
Candidates for photoreceptors that might be involved in the post-transcriptional activation of CO protein under long days are
CRY2 and PHYTOCHROME A (PHYA). On the basis of the observation that phyA mutants are slightly late flowering in long
days (Johnson et al., 1994) and transgenic plants overexpressing PHYA flower earlier than the wild type in short days (Bagnall
et al., 1995), PHYA is a photoreceptor that promotes flowering. Similarly, the cry2 mutant is late flowering in long days and
transgenic plants overexpressing CRY2 are early flowering in short days, indicating that CRY2 is involved in sensing
photoperiod (Guo et al., 1999). Furthermore, a novel allele of CRY2 was recently isolated by positional cloning of a
quantitative trait locus for early flowering identified between two Arabidopsis ecotypes (El-Assal et al., 2001). This new allele
causes early flowering under short days because of a single amino acid substitution that impairs the light-induced
downregulation of CRY2 under short days. Mutation in CO suppresses the early flowering caused by the CRY2 allele,
suggesting that CRY2 does regulate flowering through CO. PHYA and CRY2 are therefore good candidates for photoreceptors
that perceive the photoperiod in the external coincidence model.

The Circadian Clock and the Central Oscillator

Circadian rhythms have a period length (the duration of one cycle) of ∼24 h. These rhythms do not require daily transitions
from light to dark, but continue under constant conditions. Circadian rhythms have been observed at different levels of
organization, from leaf movement to stomatal aperture, CO2 assimilation, or gene transcription. The mechanism that generates
these rhythms is often described in three interrelated sections. These are input pathways that synchronise the clock mechanism
to daily cycles of light and dark, a central oscillator that generates the 24-h time-keeping mechanism, and output pathways that
regulate particular processes (Roenneberg and Merrow, 2000; McClung, 2001). The control of flowering
via CO and FT represents one such output pathway (Suarez-Lopez et al., 2001), whereas many others have been described in
detail using global gene expression assays (Harmer et al., 2000; Schaffer et al., 2001).
One output pathway, represented by the CHLOROPHYLL A/B BINDING PROTEIN2 (CAB2) gene, has enabled detailed
analysis of clock regulation. The timing of CAB (toc) mutants were isolated from transgenic plants carrying
the LUCIFERASE (LUC) reporter gene under the control of the CAB2 promoter (Millar et al., 1995a). The toc1 mutant showed
a 2- to 3-h shorter period length for CAB transcription and other gene expression rhythms, leaf movement (Millar et al.,
1995a), and stomatal conductance (Kreps and Simon, 1997; Somers et al., 1998b). The toc1 mutant flowered early in short
days (Kreps and Simon, 1997; Somers et al., 1998b), and this phenotype could be rescued by growing the plants in daily
cycles of 21 h rather than 24 h, a reduction of 3 h corresponding to the shorter period length observed in toc1 (Strayer et al.,
2000). This supports the proposal that shortened circadian period length is the cause of the early-flowering phenotype
of toc1 mutants.
The TOC1 protein shows two characteristic domains. The first of these, at the N terminus, is similar to the receiver domain of
response regulators from two-component signal transduction systems. However, invariant residues of this motif, including the
Asp residue that is normally phosphorylated in response regulators, are substituted in TOC1, suggesting that TOC1 does not
function as a classic response regulator. Indeed, TOC1, also known as APRR1, and other pseudo–response regulators, are not
phosphorylated in an in vitro assay (Makino et al., 2000). The second motif, at the C terminus, is the CCT domain, shared with
the CO family of transcriptional regulators. TOC1 transcript level cycles in light/dark cycles with a peak in the evening and
shows a circadian rhythm in LL (continuous light; Strayer et al., 2000). In the toc1 mutant, the period of the circadian rhythm
of TOC1 expression in LL is shorter, indicating that TOC1 controls its own rhythm of expression through a feedback loop. The
rhythm in TOC1 transcript level, the period-shortening effect of the toc1 mutation with the identical influence of the mutation
on multiple outputs, and the participation of TOC1 in a feedback loop make it a candidate component of the oscillator.
Other likely components of the oscillator are the CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED
HYPOCOTYL (LHY) genes, which are also involved in the photoperiodic induction of flowering. These genes encode highly
similar single Myb domain DNA binding proteins and are each regulated by the circadian clock, with a peak in their expression
soon after dawn (Schaffer et al., 1998; Wang and Tobin, 1998). The overexpression of LHY or CCA1 disrupts all circadian
rhythms tested, such as those in leaf movement and gene expression rhythms in different phases, including those
in LHY or CCA1 expression (Schaffer et al., 1998; Wang and Tobin, 1998). Recent reports, nevertheless, suggest
that LHY overexpression may not disrupt rhythms in expression of the circadian clock–regulated gene EARLY FLOWERING
3 (ELF3), which would be an unexpected exception to the effect of LHY (Hicks et al., 2001). Loss-of-function alleles
of LHY or CCA1 cause circadian period length to be shortened by ∼3 h, and inactivation of both genes prevents the maintenance
of circadian rhythms under constant conditions (Green and Tobin, 1999; Mizoguchi et al., 2002). The observations
that CCA1 and LHY present a circadian rhythm of expression, that they regulate their own transcripts through a feedback loop,
and that they both suppress all observed rhythms when constitutively expressed suggest that they could be components of the
oscillator. There is also a close relationship between the effect of LHY and CCA1 on clock regulation, and flowering time.
Overexpression of either gene causes late flowering under long-day conditions, whereas loss-of-function alleles
of CCA1 and LHY, which, like the toc1 mutant, show a shortening of the period length in LL, also resulted in early flowering in
short days (Mizoguchi et al., 2002).
The proposal that TOC1 and LHY/CCA1 may be part of a central oscillator was recently strengthened by the demonstration that
they participate in an autoregulatory loop (Alabadi et al., 2001). LHY and CCA1 bind to the TOC1 promoter in vitro and
reduce TOC1 expression when overexpressed, whereas in toc1 mutants, the period length of LHY and CCA1 expression is
shorter and their level of expression is reduced. Therefore, TOC1, LHY and CCA1 may act in a negative feedback loop, in
which LHY expression and CCA1 expression rise in the morning and repress the expression of TOC1. According to this model,
LHY and CCA1 feed back to repress their own expression, and as their protein levels fall, the expression of TOC1 rises. TOC1,
in turn, promotes the expression of LHY and CCA1, so initiating another cycle (Alabadi et al., 2001). This feedback loop may
regulate flowering time by determining the time of day that flowering-time genes in the long-day pathway, such as CO and GI,
are expressed (Figure 1).

Entraining the Clock

Circadian rhythms must be synchronized with the daily rhythm of light and dark and of temperature. The phase of circadian
rhythms can therefore be reset by environmental conditions such as light and temperature that fluctuate during day/night cycles.
Mutations that affect entrainment of circadian rhythms by light signals have been described, and some of them directly affect
photoreceptors, whereas others affect light signal transduction. Many of these mutations also have severe effects on flowering
time, but for many of them, it is not clear whether their effect on flowering time is due to an impairment of circadian clock
entrainment or to another aspect of light signal transduction. The intensity and quality of light will also affect flowering
independently of the effects of light on circadian clock regulation, and the role of the blue light–receptor cryptochromes is
reviewed in this volume (Lin, 2002).
For example, a general effect of phytochromes on flowering can be deduced from the hy1 and hy2 mutants, which are impaired
in the synthesis of the chromophore of all phytochromes. Both mutations cause severe early flowering in short days and in long
days (Goto et al., 1991), indicating that phytochromes generally are repressors of flowering. However, only a part of this effect
is likely to be due to the long circadian period also caused by these mutations (Millar et al., 1995b).
The photoreceptors PHYA, PHYB, CRY1, and CRY2 all influence clock entrainment under specific light conditions. However,
the quadruple mutant phyA phyB cry1 cry2 still shows robust circadian rhythms that can be entrained, indicating that either
another phytochrome or an unknown photoreceptor is also part of the clock input machinery (Yanovsky et al., 2000). The
influence of PHYA and CRY2 on clock entrainment occurs only at low fluence rates (Somers et al., 1998a), suggesting that the
late-flowering phenotype associated with mutations in these two photoreceptors probably is not caused by a direct effect on
circadian rhythms (see also section describing the long-day pathway).
Mutations in a PHYB-interacting protein, ZEITLUPE (ZTL), also have dramatic effects on clock function and flowering time.
A semidominant mutation in ZTL caused late flowering in long days and a 3-h increase in the period length of gene expression
rhythms (Millar et al., 1995a; Somers et al., 2000), whereas the period of leaf movements was lengthened by 5 to 6 h. The
lengthening of the period in CAB transcription, as followed using a CAB::LUC reporter, was shown to be strongly dependent on
light intensity, but no effect of light quality was observed (Somers et al., 2000), whereas leaf movements were arrhythmic
under red light (Jarillo et al., 2001). Constitutive overexpression of ZTL also gave a late-flowering phenotype in long days
(Nelson et al., 2000). The ZTL protein contains a PAS domain, an F-box, and six repeated kelch motifs that are predicted to
form a β-propeller. The β-propellers are believed to be implicated in protein–protein interactions (Adams et al., 2000). The F-
box interacts with SKP1 within the Skp1p-Cdc53p-F-box protein (SCF) complex that acts as a ubiquitin ligase implicated in the
ubiquitination of target proteins (Patton et al., 1998). The PAS domain mediates protein–protein interactions and has been
found in a group of blue light photoreceptors (Briggs and Huala, 1999). The combination of domains within ZTL suggested
that it might recruit proteins for degradation by the proteosome in a way that is influenced by light. Protein–protein interactions
have been described between ZTL and PHYB and between ZTL and CRY1 (Jarillo et al., 2001). Because the transcript level
of ZTL is not regulated by the clock and its effect is dependent on light intensity (Somers et al., 2000), ZTL may affect
circadian rhythms by the impairment of a light input pathway.
ZTL is a member of a family of three genes that also includes FKF1 and LKP2 (Somers et al., 2000). The fkf1 mutant shares the
late-flowering phenotype of ztl. However, its transcript level shows a circadian rhythm, and an fkf1 null allele has no effect on
the period length of CAB and CCA1 expression; only a change in the form of the peak was observed (Nelson et al., 2000). The
late-flowering phenotype of fkf1 can be rescued by vernalization and gibberellin (GA) application, suggesting that fkf1 may be
associated with the autonomous flowering pathway (Nelson et al., 2000). The association of F-box proteins with circadian
clock function is consistent with the importance of protein instability in circadian clock regulation.
Target protein recognition by F-box proteins is strictly dependent on their phosphorylation (del Pozo and Estelle, 2000). CCA1
and LHY can be bound by CKB3, a regulatory subunit of the protein kinase CK2 (Sugano et al., 1998, 1999). Transgenic
plants overexpressing CKB3 show a period length of CCA1 and LHY expression under LL that is shorter by ∼4 h, and are early
flowering under long or short days. The period shortening was also detected in the rhythms in expression of several other genes,
which peak at different times of the day, indicating the importance of CK2 in the regulation of the circadian clock. CK2 activity
is increased in the transgenic plants, and this activity has been shown to phosphorylate CCA1 and LHY in vitro. Thus, in plants
overexpressing CKB3, CCA1 may be phosphorylated more rapidly, perhaps causing a shorter period length due to an increased
rate of degradation of the protein. This may involve F-box proteins and the ubiquitin pathway.
The elf3 mutant was isolated as an early-flowering mutant insensitive to photoperiod (Zagotta et al., 1992), and this phenotype
is likely to be due, at least in part, to increased expression of CO (Suarez-Lopez et al., 2001). elf3 shows similarities
to phyB mutants, including elongated hypocotyls and petioles, early flowering, and defects in the red light response (Reed et
al., 2000). ELF3 encodes a nuclear protein proposed to act as a transcriptional activator (Hicks et al., 2001; Liu et al., 2001b).
The ELF3 protein interacts with PHYB and requires PHYB for the regulation of hypocotyl elongation but not for the control of
flowering time, indicating that although ELF3 is part of the PHYB signaling pathway, they control flowering time independently
(Liu et al., 2001b). ELF3 transcript level is circadian clock regulated, and peaks at the beginning of the subjective night (Hicks
et al., 2001). The elf3 mutant is arrhythmic for leaf movements and gene expression rhythms when grown in LL, but not in
continuous darkness (DD). It also shows increased sensitivity to red light, as measured by light-induced expression of CAB2 in
etiolated seedlings, which is independent of circadian clock control, or induction of CAB2 during the night, a process regulated
by the circadian clock. ELF3-overexpressing plants show a reduced sensitivity to acute light induction and an increased period
length of circadian rhythms. Taken together, these data suggest that ELF3 is a repressor of light signaling that represses light
input to the clock during the night. It also inhibits phytochrome-induced responses such as the acute induction
of CAB (McWatters et al., 2000; Covington et al., 2001). ELF3 illustrates the complexity of the function of genes that
simultaneously control circadian rhythms and light regulation while they are themselves influenced by the clock.
The gi mutant is late flowering in long days, and gi seedlings show a loss of sensitivity to red light but not to far-red light,
typical of a disruption of the PHYB signal transduction pathway (Huq et al., 2000). However, how this role
for GI in PHYB signal transduction relates to its late-flowering phenotype is unclear because in contrast
to gi mutants, phyB mutants are early flowering. Mutant alleles of GI also cause alterations in period length of gene expression
rhythms. The GI protein contains six putative transmembrane domains (Fowler et al., 1999; Park et al., 1999), suggesting a
membrane protein, but GUS:GI and GFP:GI fusion proteins were targeted to the nucleus (Park et al., 1999). The GI transcript
level is circadian clock regulated. In the elf3 mutant, the GI transcript level is increased in long days and short days, and the
rhythm is disrupted in LL, suggesting that GI acts after ELF3 (Fowler et al., 1999). The late flowering of gi mutants is at least
in part due to reduced amplitude of CO expression, but how this relates to the roles of GI in light signal transduction or in
regulating circadian clock period length is unknown.

The Vernalization Response Pathway

Exposure to low temperatures for several weeks will often accelerate flowering. Susceptibility to this treatment can differ
markedly between varieties of a species. For example, many naturally occurring Arabidopsis varieties will flower very late if
they are not exposed to a vernalization treatment but flower early if exposed to low temperatures for 4 to 8 weeks (Michaels
and Amasino, 2000). Such a requirement for vernalization is associated with a winter annual growth habit. In nature, these
varieties do not flower until they have been exposed to winter conditions. They typically germinate in the summer, grow
vegetatively through the winter until the following spring, and then flower, often in response to appropriate daylengths during
the spring or summer. This is in contrast to summer annuals, which germinate and flower in the same summer without a
requirement for vernalization. The genetic control of vernalization was addressed by crossing winter annual varieties that
require vernalization with summer annual varieties that do not. These varieties differed at two loci, FLOWERING LOCUS
C (FLC) and FRIGIDA (FRI), and dominant alleles at these loci in the winter annual are required to confer a vernalization
requirement (Burn et al., 1993a; Lee and Amasino, 1993; Clarke and Dean, 1994).
FLC encodes a MADS box transcription factor (Michaels and Amasino, 1999b; Sheldon et al., 1999). The mRNA and protein
of FLC are expressed at much higher levels in vernalization-requiring winter annual varieties of Arabidopsis than in early-
flowering summer annual varieties. In addition, expression of FLC in summer annual varieties from the 35S promoter causes a
dramatic late-flowering phenotype. Therefore, FLC encodes a repressor of flowering, and high-level expression
of FLC correlates with the vernalization requirement of winter annual varieties (Michaels and Amasino, 1999b; Sheldon et
al., 1999, 2000). The association between FLC and vernalization was strengthened by demonstration that the abundance
of FLC mRNA falls when the plants are exposed to cold, and that this reduction occurs progressively in a way that is consistent
with the progressive effect on flowering time. For example, treatment of plants at 4°C for 14 days causes a partial reduction
in FLC expression, and incomplete vernalization as measured by flowering time (Sheldon et al., 2000). Flowering-time genes
whose expression is repressed by FLC have been isolated (see below, Integration of Flowering Pathways).
Although FLC plays a central role in vernalization, and much of this response involves reductions in FLC mRNA, this probably
does not explain the whole response to vernalization. Mutants carrying null alleles of FLC still show a response to vernalization
(Michaels and Amasino, 2001). There is a clade of MADS box transcription factors closely related to FLC, and partial
redundancy between these might explain the ability of flc mutants to respond to vernalization (Ratcliffe et al., 2001; Scortecci
et al., 2001). Inactivation of at least one of these transcription factors, FLM, causes early flowering, and its overexpression
delays flowering (Scortecci et al., 2001). However, none of these genes are regulated by vernalization in a way similar to FLC.
There is also suggestive physiological and genetic evidence that vernalization does not simply act to reduce the expression of an
inhibitor of flowering such as FLC, but that it more actively promotes flowering. For example, vernalization of flc mutants in a
Columbia background causes early flowering in short days, suggesting that promotion of flowering by vernalization can
overcome the requirement for long-day induction of flowering (Michaels and Amasino, 2001). Similarly, vernalization causes
a co ga1 fca triple mutant to flower surprisingly early, suggesting that, even in this background, in which the three major
pathways are impaired, vernalization will promote early flowering (Reeves and Coupland, 2001). However, the molecular
nature of a pathway that promotes flowering via vernalization but independently of FLC is not known (Figure 2) .

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Figure 2.
The Effects of Vernalization and the Autonomous Pathway on Flowering Time, Emphasizing the Central Role of FLC.
The autonomous pathway genes and vernalization promote flowering by repressing FLC expression. Once FLC expression is
reduced by vernalization, it is stably repressed by the VRN2 gene product. Vernalization promotes flowering independently
of FLC as well as by FLC repression. HOS1 seems to act as a repressor of the vernalization pathway. FLC expression is
promoted by FRI. FLC represses flowering and, at least in part, this occurs through repressing the flowering time
genes SOC1 and FT. The data used to derive this model are described in detail in the text.
The other locus at which dominant alleles confer a vernalization requirement is FRI. The product of this gene somehow
increases FLC mRNA abundance (Michaels and Amasino, 1999b; Sheldon et al., 1999). That this effect on FLC expression is
required for FRI to delay flowering is supported by the observation that loss-of-function flc mutations suppress the effect
of FRI on flowering time. The biochemical function of FRI protein is unknown, but it contains coiled-coil domains that may be
involved in protein–protein interactions (Johanson et al., 2000). Most early-flowering varieties of Arabidopsis carry one of two
deletions that disrupt the open reading frame of FRI, suggesting that these early-flowering varieties are derived from late-
flowering ones by inactivation of the FRI gene (Johanson et al., 2000).
The molecular-genetic analysis of FRI/FLC suggests that vernalization acts by reducing FLC expression in response to
extended exposure to cold. The effect of vernalization was previously shown to be stable through mitosis (Michaels and
Amasino, 2000), and once FLC expression is reduced, it remains low even in plant organs that are formed subsequent to the
vernalization treatment (Michaels and Amasino, 1999b; Sheldon et al., 1999). Similarly, the effect of vernalization is reset
during meiosis, and high-level expression of FLC is also restored in the progeny of vernalized plants. FLC expression therefore
shows features in common with the vernalized state. These features of vernalization show similarities to epigenetic control of
gene expression, and led to the proposal that regulation of gene expression by methylation may be the basis of vernalization
(Burn et al., 1993b). Consistent with this proposal, demethylating agents caused early flowering of late-flowering mutants of
Arabidopsis, transient reductions in methylation occurred in vernalized plants, and reductions in methylation in transgenic
plants carrying an antisense methyltransferase construct caused early flowering of late-flowering plants that respond to
vernalization (Finnegan et al., 1998). FLC expression was also reduced in a transgenic line carrying the antisense methylase
transgene (Sheldon et al., 1999).
A genetic approach was also taken to identifying genes required for the vernalized state. In this case, vernalization-requiring
plants (fca mutants; see below, The Autonomous Pathway) were mutagenised to identify mutations that prevented early
flowering after vernalization (Chandler et al., 1996). The resulting vernalization (vrn) mutants appeared to prevent
downregulation of FLC expression by exposure to cold, because when fca vrn double mutant plants were vernalized and then
grown at normal temperatures for 2 to 3 weeks, FLC mRNA was expressed strongly in the double mutant but reduced to low
levels in the fca mutant (Sheldon et al., 1999). However, recently the role of VRN2 was described in more detail (Gendall et
al., 2001). These experiments demonstrated that FLC mRNA levels in an fca vrn2 double mutant were as low as in
an fca mutant immediately after vernalization, but that as plants were grown at normal growth temperatures, the abundance
of FLC mRNA rose in the double mutant but not in fca. This suggests that the role of VRN2 is not the reduction
of FLC expression in response to low temperatures, but the maintenance of the vernalized state (Gendall et al., 2001). Cloning
of VRN2 showed that its product is related in sequence to a Drosophila Polycomb group protein, Su(z)12, that regulates gene
expression by modifying chromatin structure (Birve et al., 2001; Gendall et al., 2001). A similar role for VRN2 was indicated
by the demonstration of altered chromatin structure around FLC in vrn2 mutants. The epigenetic regulation of vernalization
may, then, be mediated by alterations in chromatin structure; how this relates to the effect of methylation is not clear.
The analysis of VRN2 gene function demonstrated that this protein is required for the maintenance of the vernalized state but not
for the initial response to cold. A gene that appears to regulate the response to cold is HIGH EXPRESSION OF OSMOTICALLY
RESPONSIVE GENES 1 (HOS1) (Lee et al., 2001). Mutations in this gene were identified by isolating mutants that showed
enhanced expression of the cold-inducible gene RD29 in response to cold stress. The hos1 mutants were also early flowering,
and showed reduced expression of FLC (Ishitani et al., 1998; Lee et al., 2001). These observations are consistent with the idea
that HOS1 encodes a negative regulator of cold signaling, and that in the absence of HOS1, enhanced activity of this pathway
leads to increased expression of cold-induced genes such as RD29 and reduced expression of FLC, which is normally repressed
by cold (Lee et al., 2001). The hos1 mutant provides a genetic link between vernalization and cold stress. The HOS1 gene
encodes a RING finger protein that may serve as a ubiquitin ligase; these have been implicated as negative regulators of a
number of other plant signal transduction pathways, but a role in cold signal transduction has not been demonstrated (Bachmair
et al., 2001). Further genes involved in vernalization and required for cold perception and/or response to the cold will surely
emerge from further genetic screens, and the indication, based on the analysis of HOS1, that there may be similarities to the
mechanism of cold stress is a tantalizing one.

The Autonomous Pathway

The autonomous pathway was identified via a group of mutants that are late flowering under all photoperiods, and are highly
responsive to vernalization (Martinez-Zapater and Somerville, 1990; Koornneef et al., 1991). These mutants
include fca, fy, fpa, luminidependens (ld), and fve. The similarity between these mutants was further emphasized by the
demonstration that they all contain much higher levels of FLC mRNA than do wild-type plants or late-flowering mutants
affected in the long-day or GA pathways (Michaels and Amasino, 1999b; Sheldon et al., 1999). Thus, autonomous pathway
mutations appear to delay flowering by causing increased expression of FLC, and therefore in wild-type plants this pathway can
be considered to negatively regulate FLC expression (Figure 2). Such a role for autonomous pathway mutants was also
demonstrated genetically, because loss-of-function flc alleles suppress the late-flowering phenotype of autonomous pathway
mutants (Michaels and Amasino, 2001).
Nevertheless, the interaction of autonomous pathway mutants with naturally occurring alleles of FLC differs.
The fca, fy and fpa mutations cause late flowering in a Landsberg erecta background, and the FLC allele present in this ecotype
is therefore sensitive to loss-of-function alleles in these autonomous pathway genes. However, the ld mutation does not cause
late flowering in Landsberg erecta, but does in other ecotypes such as Columbia, suggesting that the Columbia but not the
Landsberg allele of FLC responds to loss-of-function of LD (Koornneef et al., 1994; Lee et al., 1994a). These allelic
differences in the regulation of FLC remain to be explained.
Although all of the autonomous pathway mutations act by increasing FLC expression, genetic evidence suggests that they may
not all act in a simple linear pathway (Figure 2). For example, fca fpa double mutants are much later flowering than would be
expected as a result of a simple additive effect of these mutations, and combining fpa and fy mutations appears to be lethal,
indicating a much wider role for these genes in plant development than simply regulating flowering time (Koornneef et al.,
1998a).
Several of the genes within the autonomous pathway have been cloned. The predicted FCA protein contains two copies of an
RNA binding domain, the RNP (also referred to as RNA recognition motif or consensus sequence RNA-binding domain), and a
WW protein–protein interaction domain (Macknight et al., 1997). FCA was shown to bind RNA in vitro. Strikingly, FPA also
encodes an RNA binding protein containing RNP motifs, suggesting that post-transcriptional regulation may play a general role
in the pathway (Schomburg et al., 2001). The LD protein contains a homeobox and putative nuclear localization sequences,
and may encode a transcription factor (Lee et al., 1994b). All three genes are expressed in a similar pattern, with maximal
expression at the apex of the plant and inflorescences, and low-level expression in mature leaves and roots. Overexpression
of FPA causes severe early flowering under short days, whereas increased expression of FCA had relatively minor effects on
flowering time, but in this experiment, post-transcriptional regulation of FCA RNA splicing prevented large increases in the
abundance of the fully processed FCA mRNA (Macknight et al., 1997; Schomburg et al., 2001).
The manner in which these proteins regulate FLC expression is not known. However, plants containing meristem sectors
of fca homozygous mutant cells within the L2 and L3 layers of otherwise wild-type plants flowered at the same time as did the
wild type. This suggests that FCA wild-type cells, either in adjacent L1, L2, or L3 cells within the meristem or in other tissues
of the plant, were able to correct the phenotype of the fca mutant cells (Furner et al., 1996). Thus non–cell autonomous factors
may act downstream of FCA.

The GA Pathway

The growth regulator GA promotes flowering of Arabidopsis (Wilson et al., 1992; Putterill, et al., 1995; Blazquez et al.,
1998). This was initially demonstrated by applications of exogenous GA (Langridge, 1957), and has been more recently
studied using mutations that disrupt either GA biosynthesis or signaling (Wilson et al., 1992). These mutations also have effects
on many other aspects of plant growth and development, including stem elongation, germination, and floral development. In
this section, we summarize the impact of GA on flowering (Figure 3) , and GA signaling is thoroughly reviewed by Olszewski
et al. (2002).

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Figure 3.
GA-Signaling Pathway That Regulates Flowering in Arabidopsis.
Activation of a hypothetical transmembrane receptor by GA inhibits repressors of GA signaling. These repressors are encoded
by the RGA, GAI, and RGL genes. The SPY gene also represses GA signaling and genetically acts upstream of RGA and GAI. It
may act to promote the activity of GAI/RGA/RGL by GlcNAc modification, in which case GA signaling may inhibit
GAI/RGA/RGAL by repressing SPY function. PHOR1 has not been described in Arabidopsis, but has been shown to be
involved in GA signaling in potato. Its possible involvement in ubiquitination and protein degradation leads to the tentative
proposal that it is involved in the demonstrated degradation of the repressing protein RGA in response to GA. The floral
meristem identity gene LFY is upregulated at the transcriptional level by GA. The flowering-time gene SOC1 is also
upregulated by GA, whereas FPF1 and GA-MYB were proposed to mediate between GAs and the regulation of flowering time.
These three genes may therefore act downstream of GAI/RGA/RGL but upstream of LFY. The data underlying this scheme are
described in detail in the text.
Several mutations affecting GA biosynthesis have been identified in Arabidopsis, and the mutant genes cloned. Roles for
the GA1, GA4 and GA5 genes in regulating flowering time have been described. GA1 encodes copalyl diphosphate synthase, an
enzyme that catalyzes the first committed step in GA biosynthesis (Sun and Kamiya, 1994). The corresponding mutant, ga1-3,
is unable to flower in short days, and is later flowering than is the wild type under long days (Wilson et al., 1992). These plants
are also severely dwarfed, do not germinate in the absence of exogenous GA, and exhibit reduced apical dominance.
In contrast to ga1, the ga4 and ga5 mutations have less-severe effects, giving rise to semidwarf plants that produce fertile
flowers with normal siliques (Koornneef and van der Veen, 1980). The ga4 and ga5 mutants are defective in GA 3β-
hydroxylase and GA 20-oxidase activity, respectively (Talon et al., 1990). GA 20-oxidase is regulated by environmental or
physiological changes, suggesting that it may be involved in a key regulatory step in GA biosynthesis (Xu et al., 1995).
Expression of this gene increases when plants are transferred from short days to long days, and therefore high-level expression
correlates with conditions that induce early flowering (Xu et al., 1997). Furthermore, transgenic plants containing elevated
levels of GA 20-oxidase also contained more GA4 and flowered earlier than did wild-type control plants under both long-day
and short-day conditions (Huang et al., 1998; Coles et al., 1999). This suggests that GA levels are limiting on flowering time,
and is consistent with previous observations that application of exogenous GA causes early flowering of wild-type plants.
A role for GA in the control of flowering is also suggested by studying genes involved in GA signaling. Three related genes
with key roles in GA signaling, GIBBERELLIC ACID INSENSITIVE (GAI), REPRESSOR OF GA1-3 (RGA) and RGA-LIKE
1(RGL1) have overlapping functions. GAI and RGA proteins share 71% sequence identity with each other (Peng et al.,
1997; Silverstone et al., 1998), and 61% identity to RGL1 (Sanchez-Fernandez et al., 1998; Wen and Chang, 2002). These
proteins belong to the plant-specific GRAS (for GAI, RGA, SCARECROW) family of regulatory proteins (Pysh et al.,
1999; Bolle et al., 2000). All GRAS family members contain highly conserved central (VHIID) and carboxy-terminal (RVER)
regions (Silverstone et al., 1998). RGA, GAI and RGL1 also contain a conserved sequence near their N termini that was called
DELLA after the amino acids making up the region, although in RGL1 an alanine-to-valine substitution changes the sequence
to DELLV (Wen and Chang, 2002). This sequence is not present in most GRAS family members (Peng et al.,
1997; Silverstone et al., 1998). GAI, RGA and RGL1 are believed to inhibit GA responses in the absence of active GA, and
GA relieves this inhibition.
A prediction of this model is that GA responses would occur in the absence of GA if GAI, RGA, and RGL1 genes were
inactivated. This prediction was recently confirmed by constructing ga1 mutant plants, which contain very low amounts of
active GA, in which RGA and GAI were also inactivated. The introduction of rga gai loss-of-function alleles completely
rescued the stem growth defects of ga1-3 and suppressed the nonflowering phenotype of ga1 mutants so that the triple mutant
flowered slightly earlier than did the wild type under short days (Dill and Sun, 2001; King et al., 2001). This, together with the
observation that rga gai double loss-of-function mutants are slightly earlier flowering than wild-type plants, indicates that the
late-flowering phenotype shown by GA biosynthetic mutants is due to active repression of flowering by GAI and RGA. This is
an important observation, because the rga gai mutations do not rescue all aspects of the ga1 phenotype. For example, the rga
gai ga1 triple mutant retains the germination and impaired floral development phenotype of ga1. These aspects of
the ga1 phenotype may be negatively regulated by RGL1 and other related genes, such as RGL2 and RGL3 (Wen and Chang,
2002). Indeed, flower development of transgenic plants in which RGL1 expression was reduced by co-suppression was resistant
to the effects of inhibitors of GA biosynthesis (Wen and Chang, 2002). Unlike GAI and RGA, expression of RGL1 is almost
restricted to the inflorescence. In flowers, RGL1 expression was localized to ovules and developing anthers.
However, RGL1 may also be involved in sepal and petal development. In transgenic plants overexpressing a modified RGL1
protein lacking the DELLA domain, sepals, petals, and stamens were underdeveloped and the flowers were male sterile. RGL1
may therefore play a role in repressing GA responses in the inflorescence, where apparently GAI and RGA are less important.
The DELLA sequence appears to play an important role in the mechanism by which GA inhibits the function of GAI, RGA, and
RGL1. This was suggested by the analysis of a dominant gai allele carrying a deletion within the DELLA domain, because this
mutant form inhibited shoot growth and delayed flowering even in the presence of GA (Peng et al., 1997). Deletion of the
DELLA domain may therefore cause GAI to become insensitive to GA, so that it continues to repress shoot growth and
flowering even in the presence of GA (Peng et al., 1997). Further evidence for this model comes from recent analysis of RGA.
The RGA protein accumulates in the nucleus in the absence of GA, but in the presence of GA is rapidly degraded (Silverstone
et al., 2001). However, this effect is prevented by removal of the DELLA domain (Dill et al., 2001). Therefore, regulation
of RGA by GA may be caused by RGA degradation through a mechanism that requires the DELLA domain of RGA. Transgenic
plants expressing a modified RGL1 protein lacking the DELLA domain also exhibited repression of GA responses and were
phenotypically similar to GA-deficient plants (Wen and Chang, 2002). However, unlike GFP:RGA, GFP:RGL1 was not
degraded upon GA treatment, suggesting this might not be a universal mechanism for the regulation of these DELLA proteins.
The SPY locus encodes another negative regulator of GA responses that influences flowering time. Mutations in SPY cause
partial suppression of the effects of reduced GA levels, whether these are due to mutations in GA biosynthetic genes or the
presence of GA biosynthesis inhibitors (Jacobsen and Olszewski, 1993). The spy mutant is early flowering, probably due to
the effects of increased activity of the GA-signaling pathway. Cloning of SPY (Jacobsen et al., 1996) and its homolog in
barley, HvSPY (Robertson et al., 1998), revealed that SPY is highly similar to Ser/Thr O-linked N-acetylglucosamine
transferases in rat and humans (Kreppel et al., 1997). The manner in which this enzyme acts to regulate GA responses is not
known, but genetically it acts upstream of GAI, and may be required for GAI/RGA activity.
Other proteins implicated in GA signal transduction and flowering time regulation are PHOR1 (PHOTOPERIOD
RESPONSIVE 1), FPF1, and SHI. PHOR1 was initially identified in potato, and has not been incorporated into the signal
transduction pathway defined in Arabidopsis (Amador et al., 2001). However, PHOR1 is transported into the nucleus in
response to GA. This protein belongs to a family of armadillo-related helical repeat proteins, which serve as scaffolding
proteins on which other proteins and/or nucleotides can assemble, and contains a protein domain related to that present in
components of the ubiquitination system. The GA-responsive nuclear import of PHOR1 and its relationship to the
ubiquitination system suggest that it could act upstream of RGA and could be involved in its degradation in the nucleus in
response to GA, although there is no direct demonstration of this (Figure 3). FPF1 is upregulated in the shoot meristem at the
transition to flowering, and when overexpressed causes early flowering (Kania et al., 1997; Melzer et al., 1999). On the basis
of genetic tests performed by combining transgenes that overexpress FPF1 with mutations that delay flowering, FPF1 was
proposed to promote flowering via the GA pathway. FPF1 encodes a small protein, whose biochemical function is unknown.
Finally, the dominant shi mutation causes a phenotype similar to a GA-deficient mutant, including late flowering, and is caused
by overexpression of a zinc finger protein (Fridborg et al., 1999, 2001).
Several studies have described genetic interactions between the GA pathway and other flowering-time pathways. For example,
genetic analysis suggests that the GA pathway probably acts in parallel to the long-day pathway because there is redundancy
between mutations affecting the two pathways. The effect of mutations that impair the GA pathway is strongest under short
days, and combining ga1 with mutations that impair the long-day pathway, such as co, produced double mutants that often did
not flower under long days (Putterill et al., 1995; Reeves and Coupland, 2001). This suggests that in short days, where the
long-day pathway is not active, GA is the major flowering pathway and loss of function of this pathway can prevent flowering.
Under long days, the effect of inactivation of the GA pathway is less severe because of the activity of the long-day pathway.
There is also recent evidence for a connection between FPA, a gene encoding an RNA binding protein that acts in the
autonomous pathway, and GA (Meier et al., 2001). Two late-flowering mutants, fpa1-3 and fpa1-4, markedly increased activity
of a GA5::LUC transgene. These plants showed elevated levels of GA19 and GA4, which is consistent with overexpression of
the GA5 gene (Coles et al., 1999). fpa1-3 and fpa1-4 plants also show reduced sensitivity to GA levels for floral promotion and
germination (Meier et al., 2001). Finally, the early flowering caused by vernalization was proposed to be due to the GA
pathway (Sheldon et al., 1999). This model indicated that FLC acts to repress GA activity at the apex and thereby to repress
flowering, and vernalization overcomes this repression by reducing FLC levels. However, ga1-3 FRI FLC plants, which never
flower under long days without vernalization, flower as early as do ga1-3 single mutants after cold induction, suggesting the
ability to respond to vernalization under long days does not require GA (Michaels and Amasino, 1999a). Although this
suggested that the GA pathway is not the basis of vernalization, the recent demonstration that expression of the flowering-time
gene SOC1 is repressed by FLC and promoted by GA or the long-day pathway indicates that the GA pathway does regulate the
same flowering-time genes as the other flowering-time pathways (Borner et al., 2000; Lee et al., 2000; Samach et al., 2000)
(see also below, section on pathway integration).
One way in which GAs promote flowering is by increasing the transcriptional activity of the floral meristem identity gene
LEAFY (LFY). Expression of LFY::GUS is reduced in mutants defective in GA biosynthesis, such as ga1-3, and increased in
mutants with constitutive GA signaling, such as spy and 35S::FPF1 (Blazquez et al., 1998; Melzer et al.,
1999). Overexpression of LFY also restores flowering of ga1-3 in short days (Blazquez et al., 1998). The effect of GA
on LFY transcription appears to act through a promoter motif that is similar to the consensus binding site for MYB transcription
factors (Blazquez and Weigel, 2000). A particular MYB protein, AtMYB33, which resembles a MYB transcription factor of
barley that mediates activation of amylase gene expression by GA, and is upregulated at the apex during floral initiation was
proposed to interact with this LFY promoter motif in vivo (Gocal et al., 2001). Although LFY transcription is activated by
GA, 35S::LFY ga1-3 plants still produced more leaves than did 35S::LFY plants, suggesting that GA plays an additional role in
the regulation of flowering time (Blazquez et al., 1998).

Chromatin Structure and Floral Repression in Arabidopsis

Arabidopsis mutants have been described that flower without forming any adult leaves, but progress directly from embryonic
development to flowering (Sung et al., 1992; Yang et al., 1995; Kinoshita et al., 2001). The original representative of this
class of mutants was embryonic flower 1 (emf 1), which formed a reduced inflorescence and abnormal flowers that lacked petals
without first forming any rosette (Sung et al., 1992). A second mutant with a similar phenotype, emf2, was identified
subsequently (Yang et al., 1995). The embryonic flower mutations are recessive, and were interpreted as identifying genes that
are required either to promote vegetative growth or to repress flowering during embryo and seedling development. The floral
meristem identity gene APETALA1 (AP1) and the AGAMOUS (AG) gene, which specifies carpel and stamen identity in the
flower, are ectopically expressed in germinating seedlings of emf mutants (Chen et al., 1997). However, the mutant phenotype
of emf2 was not reduced in severity when combined in double mutants with the ap1, ap2, or lfy mutations, suggesting that
ectopic expression of each of these genes is not essential for the emf phenotype.
A mechanism of action of the EMF genes was suggested by their cloning. The predicted EMF2 protein contains a zinc finger,
an N-terminal basic domain and a C-terminal acidic domain (Yoshida et al., 2001). This protein shows similarity to two
Arabidopsis proteins, VRN2 and FERTILISATION INDEPENDENT SEED 2 (FIS2), which were previously identified by
mutations, and to a Drosophila protein, Su(z)12. The Su(z)12 gene encodes a Polycomb group (PcG) protein involved in the
repression of homeobox gene expression during development of Drosophila (Birve et al., 2001). The VRN2 gene is required for
maintenance of the vernalized state and continued repression of FLC expression after vernalization (Gendall et al., 2001), and
mutations in FIS2 allow partial development of the seed without fertilization (Chaudhury et al., 1997).
The similarity of EMF2, VRN2 and FIS2 to Su(z)12 suggests that these three plant proteins may act in a way similar to PcG
proteins of animals. These proteins act in large protein complexes to repress transcription by altering chromatin structure. The
EMF2 protein may then act as part of a protein complex that during embryo and seedling development represses the expression
of genes that promote reproductive development.

The importance of PcG genes in repressing reproductive development was also emphasized by the demonstration that another
Arabidopsis PcG gene, FERTILISATION INDEPENDENT ENDOSPERM (FIE), which is related to the PcG protein EXTRA
SEX COMBS OF Drosophila, represses reproductive development in the seedling (Kinoshita et al., 2001). Loss-of-
function fie alleles were originally described because they allow partial endosperm formation prior to fertilization. However, the
effect of the mutation in the seedling or adult was not described, because maternal FIE alleles are essential for embryo
development (Ohad et al., 1999). By expressing FIE from a defective FIE promoter that allows expression during seed
development but not in the germinated seedling, it could be demonstrated that FIE is required for the repression of flowering in
the seedling (Kinoshita et al., 2001). Plants homozygous for fie and expressing FIE from such a defective promoter initiate
reproductive development as seedlings and resemble emf mutants.
The effects of fie mutations on embryo and seed development are similar to those of
the mea and fis2 mutations. MEDEA (MEA) encodes a SET-domain PcG group protein while FIS2 encodes a Su(z)12 homolog
(see above), and MEA/FIS2/FIE probably form a protein complex that regulates seed development (Luo et al., 1999; Spillane
et al., 2000; Yadegari et al., 2000). However, mea and fis2 mutant seedlings do not initiate reproductive development in the
seedling. Therefore, FIE may interact in the seedling with other proteins related to MEA and FIS2 to generate a protein complex
that represses reproductive development. One candidate for such a protein is CURLY LEAF (CLF), a SET-domain protein
implicated in the repression of homeotic gene expression during vegetative growth, and clf mutants show an early-flowering
phenotype (Goodrich et al., 1997). The analysis of mutants that initiate flowering prematurely in the seedling therefore
illustrates a requirement for active transcriptional repression to prevent reproductive development occurring so early that no
vegetative development can take place. The activation of flowering by environmental conditions such as photoperiod and
vernalization must be able to overcome these repression mechanisms and ensure that flower development is initiated at
appropriate times.
There is also evidence that chromatin modeling can affect flowering by changing methylation patterns. The decreased DNA
methylation (ddm1) mutation affects a gene encoding a SWI2/SNF2–like protein that is most closely related to genes of the
SNF2 family implicated in chromatin remodeling (Jeddeloh et al., 1999). Repeated self fertilization of ddm1 mutants creates
epigenetic alleles, some of which affect flowering time (Kakutani et al., 1996). One of the loci affected by these alleles
is FWA (Kakutani, 1997). Epigenetic alleles of FWA result in late flowering due to reduced methylation in the vicinity of the
gene, which causes increased and ectopic expression of FWA (Soppe et al., 2000). FWA encodes a homeodomain containing
transcription factor (Soppe et al., 2000), whose ectopic expression delays flowering. Analysis of FWA, together with the other
loci causing early and late flowering in the ddm background, suggests that methylation levels regulate the expression of a
number of genes involved in flowering-time regulation.

Integration of Arabidopsis Flowering Pathways

Separate genetic pathways regulate flowering in response to different environmental signals, but these pathways eventually
converge to regulate the expression of the same downstream genes. For example, all of the flowering-time pathways ultimately
lead to the transcriptional activation of the same set of floral identity genes that act within the floral primordia (Pineiro and
Coupland, 1998). LFY is the earliest of the known floral identity genes to be expressed, and directly activates at least one of the
later genes, AP1 (Wagner et al., 1999). Plants carrying fusions of the LFY promoter to the GUS marker gene were used to
demonstrate that LFY expression responds both to the long-day flowering pathway and to GA. Furthermore, deletion of a
putative MYB transcription factor binding site within the LFY promoter prevented activation by GA, but not by the long-day
pathway (Blazquez and Weigel, 2000). Plants homozygous for the lfy mutation and carrying a LFY transgene in which this
GA-responsive element was deleted appeared wild type under long days but exhibited the lfy mutant phenotype under short
days, where LFY expression requires the GA pathway. This conditional activation of the mutant LFY transgene demonstrated
that activation of LFY by each of these two flowering pathways is separable. Thus the GA and long-day pathways converge on
the LFY promoter, rather than both pathways activating an earlier acting gene that in turn increases the expression
of LFY (Blazquez and Weigel, 2000).
Convergence of the long-day and autonomous pathways has also been studied. These pathways are clearly separate until
the FLC and CO genes (Figure 4) . For example, expression of the floral repressor FLC, a component of the autonomous
pathway, delays flowering but does not reduce expression of CO, a component of the long-day pathway (Suarez-Lopez et al.,
2001). Similarly, mutations in the CO gene do not affect expression of FLC (Sheldon et al., 1999). Nevertheless, flowering-
time genes have been identified that act downstream of both CO and FLC. The SOC1 (or AGL20) gene that encodes a MADS
box protein is both activated by CO and repressed by FLC, suggesting that the pathways represented by these genes converge
on SOC1 (Borner et al., 2000; Lee et al., 2000; Samach et al., 2000; Michaels and Amasino, 2001). SOC1 is also regulated
by GA, and therefore is a common target of all three flowering pathways (Borner et al., 2000). Mutations that
inactivate SOC1 delay flowering, while SOC1 overexpression causes early flowering and suppresses the effect of mutations in
the long-day and autonomous pathways. Expression of the Sinapis alba ortholog of SOC1 is increased by daylength, as well as
by applications of GA and cytokinin (Bonhomme et al., 2000). This is consistent with the results from Arabidopsis, and in
addition indicates that SOC1 responds to cytokinins. Expression of FT, a second flowering-time gene regulated by more than
one pathway, is reduced by mutations that impair the function of the long-day and autonomous pathways and activated by
overexpression of CO, indicating that FT acts downstream of both of these pathways (Kardailsky et al., 1999; Kobayashi et
al., 1999; Samach et al., 2000).

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Figure 4.
Overview of the Relationships among Arabidopsis Flowering Pathways.
The pathways described in detail in Figures 1 to 3 are combined to emphasize their relationships. In particular, the effects of the
integration of the photoperiod, GA, and vernalization pathways on the regulation of expression of FT and SOC1 is illustrated.
The data underlying this model are described in detail in the text.
The repression of SOC1 and FT expression by FLC, even in the presence of wild-type CO represents an important aspect of the
adaptation of flowering time to changing seasons. This adaptation involves responses to several environmental stimuli,
including temperature and daylength, which need to be balanced to produce a coherent response. For example, the winter annual
growth habit requires repressing the induction of flowering by daylength until the plant has been exposed to winter
temperatures, after which the plant must respond to lengthening daylength the following spring. The antagonism
between FLC and CO in the activation of downstream genes such as SOC1 and FT can explain such an adaptation because the
activation of these genes by CO may be prevented by FLC during the first summer, but reduction in FLC expression during
winter would allow CO to activate expression of the downstream genes during the long photoperiods of the following summer.
The analysis of SOC1 expression demonstrates convincingly that this gene represents a point of convergence of several
flowering-time pathways. Although SOC1 expression increases rapidly within the floral meristem in response to inductive
conditions, such as exposure to inductive photoperiods, the manner in which its function is related to increases in expression of
the floral meristem identity genes is not understood.
The relationship between FT and floral meristem identity gene expression has been studied at the genetic and molecular levels.
The ft mutation did not reduce the expression of LFY::GUS expression, suggesting that FT does not act upstream
of LFY (Nilsson et al., 1998). Furthermore, the late flowering caused by ft mutations was epistatic to the early flowering
of 35S::LFY. Finally, ft lfy double mutants showed strongly synergistic effects on floral development, whereas ft ap1 double
mutants did not (Ruiz-Garcia et al., 1997). All of these results were taken as evidence that FT does not act upstream of LFY to
regulate its expression, but rather acts in a parallel pathway. In particular, FT might activate the expression of other floral
meristem identity genes such as AP1 (Ruiz-Garcia et al., 1997). Thus the primary activation of LFY and AP1 may occur by
parallel pathways, and subsequent direct activation of AP1 by LFY may be required to rapidly amplify floral meristem identity
gene expression in young primordia. Amplification of floral meristem identity gene expression was proposed on the basis of the
phenotypes of plants carrying mutations in more than one of these genes (Bowman et al., 1993).
FT is a member of a small gene family in Arabidopsis that also contains TERMINAL FLOWER 1 (TFL1). The function of this
family of proteins in plants is not known. However, as was shown for the Antirrhinum ortholog
of TFL1, CENTRORADIALIS (CEN), they have homology to mammalian phosphatidylethanolamine binding proteins (PEBP),
which were originally shown to bind phospholipids (Bradley et al., 1997). These proteins are also known as Raf1 kinase
inhibitor proteins because of their ability to prevent Raf1 phosphorylation. The crystal structure of CEN confirmed its
relationship with PEBP, and in particular demonstrated that it also contained the ligand binding site that is believed to interact
with phosphate groups (Banfield and Brady, 2000). The sequence of FT is therefore consistent with it controlling flowering by
regulating a phosphorylation cascade.

Conservation of Flowering Pathways in Plants Showing Responses Different from Those of Arabidopsis

Plants that flower in response to photoperiod are classified as short day, long day or day-neutral. Arabidopsis is a facultative
long-day plant, and genetic and biochemical analyses have identified a pathway responsible for early flowering in response to
inductive long photoperiods, and a GA-mediated pathway that promotes flowering under short days. However, GAs have no
florigenic effect in most short-day plants in noninductive photoperiods, and whereas daylength perception appears to occur
mainly under darkness in short-day plants, it seems to occur predominately during the day in long-day plants (Thomas and
Vince-Prue, 1997). These observations suggest that there may be major differences in the mechanisms of flower induction in
the long-day plant Arabidopsis compared with most short-day plants, and that genetic models based on Arabidopsis may not be
sufficient to explain photoperiodic responses in short-day plants. However, recent molecular-genetic experiments in short-day
plants have demonstrated striking parallels between these and Arabidopsis.
Molecular and genetic approaches were used to study the transition from the vegetative to the reproductive phase in the classic
short-day plants, rice, Pharbitis nil, and tobacco. In rice, flowering time (or heading date) is critical for the adaptation to
different cultivation areas and cropping seasons and may be affected by environmental conditions such us photoperiod,
temperature, and light intensity. There are several major genes affecting heading date that relate to vegetative growth or
photoperiod sensitivity. Five quantitative trait loci, HEADING DATE 1 (Hd1) to Hd5, that control heading date were identified
by comparing genetic differences between a japanica rice variety (Nipponbare) and an indica variety (Kasalath) (Yano et al.,
1997). Genetic interactions among three loci involved in photoperiod sensitivity, Hd1, Hd2, and Hd3 identified epistatic
interactions between some combinations, suggesting that they act in the same genetic pathway (Lin et al., 2000).
Hd1 was cloned on the basis of its map position. Sequence analysis revealed that Hd1 encodes a B-box zinc finger protein
containing a C-terminal CCT domain and exhibits a high degree of similarity to the Arabidopsis CO gene (Yano et al., 2000).
Experiments with various rice lines showed that the presence of a functional Nipponbare Hd1 allele was associated with a
stronger photoperiod response, causing early heading under short days and later heading under long days. Lines homozygous
for the recessive Kasalath Hd1 allele in a Nipponbare background flowered extremely early under long-day field conditions.
Thus, the CO/Hd1 gene is important in photoperiod response in long-day and short-day plants. However, unlike CO in
Arabidopsis, Hd1 appears to have two roles in rice, promoting heading under short days and delaying it under long days.
Detailed comparison of the sequences of wild-type and mutant forms of CO or Hd1 also suggests that there may be differences
in the biochemical mechanism of CO and Hd1 function. For example, co-3 is a strong mutant allele that converts a His residue
predicted to be involved in zinc binding within the second B-box to a Tyr residue (Robson et al., 2001). Strikingly, the active
Ginbouzu allele of Hd1 has exactly the same change of His to Tyr, suggesting that in rice this does not impair protein function.
Similarly, the co-1 allele of Arabidopsis has a deletion of three residues within the second B-box, while the active Nipponbare
allele has a deletion of 36 bp that removes two of these residues. Taken together, these data suggest that the second B-box is
essential for CO but not for Hd1 function. Detailed comparisons of COL proteins among species may help in determining their
biochemical function. Although a COL gene displaying a high level of homology was found in gymnosperms (GenBank
accession number AF001136), suggesting conservation of these sequences, there is also evidence that the family evolved
rapidly (Lagercrantz and Axelsson, 2000).
Among the other major genes that affect heading date and are related to vegetative growth or photoperiod sensitivity in rice
are Hd6 and SE5, which were identified as the α subunit of protein kinase CK2 (Takahashi et al., 2001) and a putative heme
oxygenase (Izawa et al., 2000), respectively. Hd6 is a quantitative trait locus involved in rice photoperiod sensitivity, which
was also detected in backcross progeny derived from a cross between the Nipponbare and Kasalath varieties (Yamamoto et al.,
2000). The Nipponbare Hd6 allele is nonfunctional. Introduction of the functional Kasalath allele delayed heading date of
Nipponbare. CK2 belongs to the family of messenger-independent serine/threonine kinases that are present in all eukaryotic
cells examined to date. This also suggests a relationship with photoperiod response in Arabidopsis. The expression of CK2
antisense RNA in Arabidopsis affected the expression of some light-regulated genes (Lee et al., 1999). CK2 interacts with and
phosphorylates the Arabidopsis CCA1 protein in vitro (Sugano et al., 1998; Yamamoto et al., 2000). Overexpression of CK2
shortened the period of rhythmic expression of the CCA1 and LHY genes, and caused early flowering in both long- and short-
day conditions (Sugano et al., 1999). Therefore, protein kinase CK2 may have a role in the photoperiod pathway of
Arabidopsis and rice.
Finally, the rice photoperiodic sensitivity 5 (se5) mutation prevents the delay in flowering caused by exposure to noninductive
long-day conditions (Izawa et al., 2000). SE5 encodes a putative heme oxygenase, which shows 70% identity to HY1 from
Arabidopsis within the heme oxygenase domain (Davis et al., 1999; Muramoto et al., 1999). Both hy1 and se5 mutants are
deficient in phytochrome responses such as coleoptile responses to light pulses and seedling growth under continuous red and
far-red light. The hy1 mutant of Arabidopsis also flowers very early under noninductive conditions, emphasizing the
involvement of the phytochromes in the repression of flowering under noninductive conditions in both long-day and short-day
plants.
The Japanese Morning Glory, P. nil cv Violet, has also been used extensively as a model system for the physiology of flowering
in short-day plants (Takeba and Takimoto, 1966; Vince-Prue and Gressel, 1985). A P. nil homolog of
the CO gene, PnCO was isolated using differential display to identify genes with increased expression under short-day
conditions (Liu et al., 2001a). The PnCO mRNA is inefficiently spliced, but the fully processed cDNA under the control of the
cauliflower mosaic virus 35S promoter in Arabidopsis co-1 complements the late-flowering phenotype of co-1 mutants,
confirming that PnCO has the same biochemical function as CO. PnCO, like CO, is under circadian clock control (Liu et al.,
2001a). In continuous inductive darkness, the level of PnCO mRNA reaches a peak between 16 and 20 h, but it is not clear how
this circadian clock control of PnCO regulates flowering in response to short days. Much lower accumulation of PnCO mRNA
was detected after the noninductive 8, 10, and 12 h of darkness. Interestingly, the daily cycle in Arabidopsis CO expression
under short days also shows a broad peak between 16 and 20 h with a strong peak at 20 h (Suarez-Lopez et al., 2001). In
Arabidopsis, circadian clock control is thought to regulate exposure of CO protein to light, and post-transcriptional regulation of
CO by light is proposed to promote flowering in long days. A night break (NB), 5 min of red light given 8 h into 14 h of
darkness, inhibits flowering of P. nil. However, the level of PnCO RNA is only slightly reduced under these conditions,
suggesting that the NB does not inhibit flowering by reducing transcription of PnCO. However, CO protein is very unstable in
light (Suarez-Lopez et al., 2001), and P. nil PnCO protein could also be unstable and a target for degradation in response to
NB.
The mechanism by which CO and its orthologs in short-day plants mediate the photoperiodic response is not clear. In
Arabidopsis, CO promotes flowering under long-day conditions, activating at least two flowering-time genes, SOC1 and FT,
which have major roles in promoting flowering time (Samach et al., 2000). Light-activated post-transcriptional regulation
of CO could be a part of this signal transduction pathway. It remains possible that differences in photoperiod-regulated activity
of CO in short-day plants lie in the interaction of CO with different sets of proteins and subsequent upregulation of the target
flowering-time genes. In addition, in short-day plants, CO may act in protein complexes that actively repress flowering under
long days. The fact that the Hd1 protein is involved in two opposite processes—activation of heading under short-day
conditions and inhibition of heading under long-day conditions—supports this suggestion.
Isolation and characterization of homologs of the key genes involved in photoperiod-mediated transition to flowering in short-
day plants as well as expression of the homologous genes in transgenic long-day and short-day plants will enable comparison of
the molecular mechanisms that regulate the floral transition in short-day and long-day plants. In a study of this type,
the SOC1 homolog from mustard, MADSA, was overexpressed in long-day (Nicotiana sylvestris), day-neutral (N. tabacum), and
short-day tobacco (N. tabacum cv Maryland Mammoth) species (Borner et al., 2000). Analysis of the transgenic plants showed
that only in the short-day cultivar could overexpression of MADSA overcome the photoperiodic barrier of floral induction.
 MADSA overexpression did not cause long-day and day-neutral cultivars to flower under noninductive photoperiod, suggesting
a difference in the behavior of long- and short-day plants at the molecular level, even in downstream processes.

PERSPECTIVES

Genetic analysis in Arabidopsis has enabled the isolation of genes that control flowering time, and the identification of
interacting pathways that promote flowering in response to different environmental conditions. However, our present
understanding of these pathways represents only a skeletal framework, and future work in this area will concentrate on
understanding the biochemical function of pathway components and the manner in which the signaling pathways convey
information that ultimately regulates flowering time. Furthermore, the incorporation into these models of classic observations,
such as the graft transmissibility of flowering signals formed in the leaf, is a major challenge.

The complexity of the data regarding the network of flowering pathways will also increase further. Some environmental effects
on flowering have not been placed within the pathway structure. For example, it is not clear whether the effects of nutrient
availability or light quality on flowering act through the pathways described here or through further pathways, although high
sucrose levels delay flowering and reduce FT expression (Ohto et al., 2001). There is also physiological evidence that
vernalization may act partly through an additional pathway that has so far not been described genetically (Figure 2). In
addition, although there is strong genetic and molecular evidence for the separation of the photoperiod and autonomous
pathways, as shown in Figure 4, there are also indications of interactions between the pathways at unexpected levels. For
example, FLC, a major repressor within the autonomous pathway, influences circadian period length, a feature of the
photoperiod pathway (Swarup et al., 1999).
Explaining the diversity in flowering-time responses also represents a great challenge. The analysis of natural variation in
flowering time between ecotypes of Arabidopsis indicates that modifiying the balance of the activity of the described flowering
pathways can create different reproductive strategies, such as winter annual or summer annual types (Michaels and Amasino,
2000). For example, in comparing winter annual and summer annual varieties of Arabidopsis, changing the relative activities of
the vernalization and photoperiod pathways generates a vernalization requirement. It seems likely that altering the balance of
the activity of distinct pathways on the activation of genes such as FT and SOC1 that integrate the different pathways may be a
more general mechanism of generating diversity in flowering behavior. However, there are also fundamental differences among
species in the roles of pathways. The comparison of photoperiod responses in long-day and short-day plants demonstrates that
for this example of diversity in flowering behavior, the pathway described in Arabidopsis is likely to be relevant for species that
behave very differently. However, the function of key pathway components is likely to be altered in short-day plants to change
their target genes or the activity of the protein complexes in which they act. Similar changes may explain why GA promotes
flowering in Arabidopsis but represses flowering in trees and some short-day plants. There are flowering strategies, however,
for which Arabidopsis may be a less-effective model. For example, although Arabidopsis genes can be used to drive very early
flowering of trees and to overcome developmental delays of flowering (Weigel and Nilsson, 1995; Pena et al., 2001), studies
of an annual plant such as Arabidopsis will probably not reveal the molecular mechanisms underlying many of the unique
features of perennial plants (Battey, 2000).

Footnotes

 Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.001362.

 ↵1 These authors contributed equally to this work.
 Received December 21, 2001.
 Accepted March 4, 2002.
 Published May 1, 2002.

http://www.plantcell.org/content/14/suppl_1/S111

 B9.

How do signalling and cross‐talk between the different plant hormones operate?
Abstract

Plant growth and development is influenced by mutual interactions among plant hormones. The five classical
plant hormones are auxins, cytokinins, gibberellins, abscisic acid and ethylene. They are small diffusible
molecules that easily penetrate between cells. In addition, newer classes of plant hormones have been identified
such as brassinosteroids, jasmonic acid, salicylic acid and various small proteins or peptides. These hormones
also play important roles in the regulation of plant growth and development. This review begins with a brief
summary of the current findings on plant hormones. Based on this knowledge, a conceptual model about
interactions among plant hormones is built so as to link and develop an understanding of the diverse functions
of different plant hormones as a whole in plants.

Introduction
Every stage of the plant's life cycle is regulated by plant hormones. In general, plant biological activity is
manipulated by more than one hormone, thus the biological phenomenon often reflects the combined interplay
of several different hormones. Meanwhile, unlike animals which can escape from harsh environments, plants
can only survive through adjusting various biological activities when encountering biotic and abiotic stresses.
During these situations, plant hormones also cooperate to modify biological responses for the formation and
maintenance of plant stress tolerance.1 In this review we aim to develop a model that can be used as a
foundation to investigate the interactions of plant hormones that allow plants to survive the onslaught of various
environmental factors.

Hormones are compounds that work at low concentrations where they are able to signal and control the
response, growth and development of living organisms via circulating through part or all of the organisms. The
action of hormones involves processes of signal transduction. Signal transduction is a relay involving the
conversion of intracellular or extracellular signals into cellular responses. Such signaling processes can be
separated into three types according to transduction distances: (1) long distant (plant) or endocrine (animal)
signaling where signal cells and target cells are at a long distance apart; (2) paracrine signaling where signal
cells and target cells are adjacent cells; (3) autocrine signaling where signal cells and target cells are the same
cells.2

The signaling process embodies synthesis of signal molecules (ligands), transport of signal molecules, binding
of signal molecules with receptors, development of cellular responses and removal/degradation of signal
molecules. A conformational change of receptor via ligand-binding plays a critical role in initiating the signal
transduction process. Most receptors are present on the cell surface although some are localized in the cytosol.
Furthermore, cell surface receptors can be classified as: receptors with intrinsic enzyme activities (e.g., receptor
kinases), protein coupled receptors (e.g., G protein-coupled receptors) and ligandgated ion channel
receptors.3 The cell surface receptors recognize and perceive stimuli (ligands) in the extracellular environment,
thereafter they interact with the ligands and transduce the extra-cellular signal into the cells.

Usually when an extracellular signal molecule binds to its receptor, the initial cellular reaction involves
activating the production of a second messenger or an intracellular signaling mediator, which consequently
triggers a series of cellular responses. Substances acting as second messengers can vary in their chemical
composition. They include lipids, sugar, ions, nucleotides or gases, such as Ca2+, cAMP, cGMP, cyclic ADP-
ribose (cADPR), small GTPases (small or monomeric G proteins), 1,2-diacylglycerol (DAG), inositol-1,4,5-
trisphosphate (IP3), NO, inositol phospholipids (phosphoinositides) and phosphatidic acid. Intracellular
signaling mediators refer to several classes of intracellular signal proteins. They are mainly different kinds of
enzymes including kinases, phosphatases, phospholipases, phosphodiesterases and so on.2,3 Notably, some of
these intracellular signaling molecules are shared between different signaling pathways. Hence these molecules
are most likely to serve as crosstalk points in the signaling network communication within the cell.

Auxin, cytokinin, gibberellin (GA), abscisic acid (ABA) and ethylene are well accepted as five classes of
classic plant hormones. Nowadays, evidence has accumulated to extend this concept to include brassinosteroids,
jasmonic acid and salicylic acid. Furthermore some biologically active peptides are also found to be key
signaling players in many aspects of plant life, like systemin, polaris, phytosulfokines (PSKs), ENOD40,
CLAVATA3 (CLV3), S-locus cysteine-rich proteins (SCPs) and plant natriuretic peptides (PNPs).4–
6 Therefore it is likely that further newcomers may be added to this list in the future. One such example is
strigolactones, which are essential for root colonization by arbuscular mycorrhizae but are also important
hormonal compounds involved in inhibiting shoot branching.7 To understand signaling cross-talk among plant
hormones, it is particularly important to elucidate the signaling mechanism of each plant hormone first. The
main functions and signaling pathways of the major plant hormones are briefly reviewed as a prelude to
developing a model to unravel the possible interactions among different hormones in terms of the dynamic
maintenance of a normal plant.

Auxin

Auxin is an indispensable hormone for plant growth and development. It is synthesized from actively growing
tissues such as shoot meristems, leaf primordia, young expanding leaves, developing seeds, fruits and pollens. A
number of biological processes are regulated by auxin: cell division, cell expansion, ion fluxes, root initiation,
phototropism, geotropism, apical dominance, ethylene production, fruit development, parthenocarpy, abscission
and sex expression. One major cellular effect of auxin is to cause hyperpolarization of the plasma membrane.
This is a necessary requirement for various auxin-triggered biological actions.1,8,9 Meanwhile, auxin also
seems able to stimulate GA biosynthesis and suppress ethylene and ABA biosynthesis.10,11

Auxin has been one of the plant hormones attracting researchers to hunt for its perception mechanism(s).
Several reports indicate that the levels of lysophospholipid and Ca2+ were increased after auxin addition.9 Thus
these two components may be intracellular signals for auxin. Receptors for auxin-binding are separated into two
types: soluble and those on the outer plasma membrane. Some soluble auxin-binding proteins appear to be
directly linked with the plasma membrane ATPase as auxin binding can rapidly cause proton pumping,12 while
others directly regulate gene expression and include TIR1, the auxin receptor. Auxin affects gene expression via
interaction with the auxin responsive element (AuxRE) which contains the characteristic nucleotide sequence
TGTCTC and is located in the promoter region of auxin-inducible genes. Transcription factors bind to AuxRE
and are members of the auxin responsive factor (ARF) family which can be repressed by the Aux/IAA family of
transcription factors. Aux/IAA is a target for ubiquitin degradation by TIR1 which is the auxin receptor and a
member of the Skp1/Cullin/F-box (SCF) ubiquitin ligase (E3) family. The targeted Aux/IAA is degraded in the
26S proteasome after being attached to polyubiquitin chains and this process is accelerated by auxin
application.8,9,13,14

The ubiquitin/proteasome system degrades targeted proteins. It plays a crucial role in maintaining proteomic
plasticity in plants where it occupies nearly 6% of the proteome.15 In this system, the 76 amino acids protein
ubiquitin acts as a covalent molecular tag, and is attached to a substrate by the catalytic activities of three
enzymes named: E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating protein) and E3 (ubiquitin ligase),
and this last group includes four different types of ligase enzyme (RING, HECT, U-Box and SFC). Ubiquitin
chains are thus formed tagging the protein for degradation. The polyubiquitylated substrate is delivered to a
proteasome via ubiquitinbinding proteins, and then is deubiquitylated and digested by various proteases and/or
proteolytic complexes. The free ubiquitin molecules are then released and become available for another
attachment. TIR1 and other auxin-signaling F-box proteins are the receptors for auxin which raises the question:
are other members of the F-box protein family (>700 in Arabidopsis alone) functioning as plant hormone
receptors? Many activities of plant hormones are reported to be closely linked with the ubiquitin/proteasome
system.14–16

Cytokinin

Cytokinins have many functional activities throughout the life of plants. Cytokinins occur at highest
concentrations in meristematic regions, roots, young leaves, developing fruits and seeds. Their main functions
include regulation of cell division, induction of organ differentiation, control of stomatal movement, delay of
chlorophyll breakdown and attenuation of leaf senescence.1,9 Two different kinds of cytokinin hormonal
activities are present in plants. One is the local (paracrine/autocrine) activity that mainly regulates cell division;
whereas the other is the long distance signaling (endocrine) that acts as a root to shoot signal to control the
functional response of above ground organs.17,18

It is clear now that cytokinin is perceived by membrane located histidine kinase receptors. Signal transduction
operates through a bicomponent system where cytokinins bind to the histidine kinases and then histidine
phosphotransfer proteins transmit the signal to nuclear response regulators, leading to gene activation or
repression.17,19 Moreover, cytokinins also influence gene expression in chloroplasts, revealing that its
receptors may also be located in this subcellular site.9 Some research shows that auxin pretreatment may
upregulate the expression of cytokinin receptors.20 Signaling manipulation of cytokinin shares some similarity
with the auxin control mechanism(s). Cytokinin responsive genes are positively stimulated by transcription
factors. These transcription factors are counteracted by a family of specific proteins which are also subjected to
degradation by the ubiquitin recognizing proteasome system. Cytokinin promotes the degradation of these
specific proteins.21,22 The removal of specific short-lived proteins may be essential feature of many hormone
responsive processes. The unstable proteins possibly serve as transcriptional repressors.

Gibberellin

Gibberellins (GAs) stimulate plant elongation, promote flowering and release seed/tuber dormancy. It is
believed that gibberellins are produced and function in the same cell acting as an autocrine signal.1 GAs are
synthesized from geranylgeranyl diphosphate through several intermediate steps catalyzed by enzymes in
plastids and the cytosol and bioactive GAs are deactivated by epoxidation or methylation processes.23 The
regions of GA action are mainly located in the meristematic zone and transition zone.9,24,25

GA signaling also demonstrates the importance of protein degradation in plant hormone regulation. GA binds to
a soluble, nuclear-localized receptor GA-INSENSITIVE DWARF1 (GID1) that in turn binds to members of a
family of transcription repressors called DELLA after their signature Asp-Glu-Leu-Leu-Ala (DELLA) motif.
This in turn activates the F-box proteins SLEEPY1 (SLY1)/GID2 culminating in ubiquitin mediated
degradation of DELLA and transcription of GA responsive genes. In low GA levels, DELLA proteins sequester
and inactivate PHYTOCHROME INTERACTING FACTORS (PIFs), which are basic helix-loop-helix
transcription factors.14,15,26 The discovery that DELLA proteins sequester PIFs is particularly exciting as it
provides a biochemical mechanism underpinning the involvement of GA in light regulated seedling
development. Following GA perception, early responses of plants include the increase of intracellular
Ca2+/calmodulin, decrease of intracellular pH and elevation of the second messenger cGMP.27

GA seems to cross-talk with several other hormones. In seeds, the GA-induced Ca2+/calmodulin signaling
regulates the synthesis and secretion of hydrolase; while ABA blocks the expression of hydrolase.28 This may
explain the antagonistic interaction between GA and ABA. GA shows overlap or downstream effects with auxin
in numerous developmental reactions.24,29 One of the ultimate effects of GA is inducing cell elongation. The
pre-requirement of cell elongation is cell wall plasticity and cytoplasmic protein synthesis.30 To achieve these
results, the cooperation of other hormones such as auxin may be an underlying requirement. A recent study
shows that auxin and DELLA proteins independently regulate the GA synthesis pathway and promote the
accumulation of bioactive GAs.31 In addition, combined positive or negative interplays between GA and
ethylene also exist, depending on internal and external conditions, tissue types and developmental
stages.24 Transcript meta-analysis suggests that applying exogenous ethylene to plants represses expression of
genes involved in GA metabolism while some ethylene synthesis genes are upregulated by GA
treatment.24 Overall, GA functions in a complicated context with many interacting factors.

Abscisic acid

Abscisic acid (ABA) is a “stress hormone,” named for its roles in response to stress environments. One notable
effect of ABA is causing stomatal closure and preventing water loss by transpiration.32 Besides the traits of
stress response, ABA is also associated with normal physiological manipulations like storage of compounds,
dehydration at later stages of embryogenesis, seed maturation, dormancy formation and abscission.9 ABA is a
carotenoid derivative produced in chloroplasts and other plastids and its synthesis is increased under stress
conditions such as drought, salinity and cold.1,33 ABA is also circulated throughout the plant as an inactive
glucose ester that can be rapidly released into the active by β-glucosidases present in the apoplast and
endoplasmic reticulum33 allowing a combination of long distance and paracrine/autocrine effects to occur.

Multiple candidates for ABA receptors have been reported but the core signaling pathway involves
PYRABACTIN RESISTANCE (PYR)/REGULATORY COMPONENT OF ABA RECEPTOR (RCAR) family
of ABA receptors. The PYR/RCAR receptors are found in the cytoplasm as an inactive dimer that dissociates
upon ABA binding to inhibit PROTEIN PHOSPHATASE 2C (PP2C) activity and thereby allow SNF1-
RELATED PROTEIN KINASE 2 (SnRK2) to activate various downstream effectors including ion channels
and transcription factors. However other candidates including membrane bound proteins are still potential ABA
receptors (reviewed in refs. 34–39); therefore ABA is capable of inducing different signaling effects.
Phosphatidic acid and IP3 have been reported to be involved in the ABA signaling pathway where they probably
act as second messengers. For instance in guard cells, IP3 activates Ca2+ channels within the endoplasmic
reticulum and vacuoles, leading to the release of Ca2+ into the cytosol from internal stores.9 Along with the
increase of Ca2+ level, Ca2+ inhibits the plasma membrane H+-ATPase to prevent K+ uptake and further drive K+,
Cl−-efflux.40 As a result, guard cells are closed. Obviously, multiple signaling pathways are involved in ABA
transduction, demonstrating that ABA functions in a network rather than in a chain. Signal molecules involved
in ABA signaling include: increases in pH, phosphatidic acid, IP3, Ca2+, NO, cADPR, mitogen activated protein
kinase (MAPK) cascades and so on.9

Interestingly, ABA biosynthesis is largely regulated by its endogenous levels via a positive feedback
system.33,41 This kind of synthesis pattern can further stimulate ABA accumulation and represents a critical
step of stress adaptation, since ABA plays an important role in stress responses by mediating considerable gene
expression. ABA-responsive gene promoters often contain cis-acting elements. The cis-acting elements
commonly have a G-box ACGT core motif. Most proteins binding to the DNA sequences with this ACGT core
motif share a basic region that adjoins to a leucine zipper domain.34,42 However, these leucine zipper proteins
are particular targets for ubiquitin mediated degradation involving RING E3 ligases.43 ABA can promote or
protect proteins from ubiquitin mediated degradation.14,34

Ethylene

Ethylene is a gaseous hormone that activates fruit maturation, stimulates germination, accelerates senescence,
inhibits elongation, increases horizontal growth, initiates adventitious roots and programs cell death. Ethylene is
produced from 1-aminocyclopropane-1-carboxylic acid (ACC) and the production of this compound is rate-
limited by the activity of ACC synthases, which are short-lived enzymes whose stability is increased by
ethylene itself in a feed forward process.15 However, the production of ethylene is tissue-specific and depends
upon the stage of plant development.1,44 Ethylene levels may also increase or decrease in response to abiotic
and biotic stresses.9

Ethylene receptors like cytokinin receptors are members of the bicomponent regulatory system family. Ethylene
binds to the receptors via a copper co-factor and ethylene binding induces a mitogen-activated protein kinase
(MAPK) cascade.9,44 Ethylene receptors are localized in endoplasmic reticulum membranes. Since ethylene is
a gas, such a binding site is in accord with the diffusion of ethylene in either aqueous or lipid environment. The
ubiquitin 26S proteosome system plays a key part in ethylene responses. For instance the ETYLENE
INSENSITIVE3 (EIN3) is constitutively synthesized and degraded by SCF E3 ligases co-assembled with F-box
proteins in the absence of ethylene. The presence of ethylene inhibits EIN3 degradation and allows it to act as a
transcription factor which means that a very rapid and robust response will occur when ethylene levels
rise.15 In addition, microarray studies demonstrated that a high level of coordination in gene expression was
present with ethylene, jasmonic acid and salicylic acid treatments. This implicates close linkages among these
hormones.45

Brassinosteroid

Brassinosteroid is a plant steroid hormone. Brassinosteroids have been detected in pollens, leaves, shoots,
flowers, stems and seeds, but not roots.1 They affect cell expansion and division, tissue differentiation,
reproductive development and stress resistance.46 Brassinosteroids act in a paracrine/autocrine manner with
limited signal transport between tissues and organs.47

Unlike animal steroids that are perceived by nuclear receptors, brassinosteroids are perceived by the leucine rich
repeat receptor like kinases (LRR RLK), which are members of the family of membrane localized receptor
kinases containing extracellular leucine rich repeat regions. Brassinosteroids bind to a subdomain of these
repeats, thereby initiating intracellular signal transduction via activating a kinase cascade beginning with
receptor autophosphorylation and culminating in altered gene expression.48 The sites of phosphorylation of the
brassinosteroid receptor BRASSINOSTEROID INSENSITIVE1 (BRI1) and its downstream interacting partners
are relatively well characterized.49,50 The homeostatic levels of brassinosteroids are manipulated by
brassinosteroid dependent transcriptional controls whose genes are involved in brassinosteroid metabolism.
Such controls lower brassinosteroid production by reducing the transcription of biosynthetic genes or increasing
the transcription of catabolic genes, when brassinosteroid levels are too high.51

Jasmonic Acid

Jasmonic acid and its derivatives such as methyl jasmonate are cyclic fatty acid-derived regulators synthesized
from linolenic acid.45 Jasmonic acid levels are the highest in areas of active division such as stem apex, young
leaves, immature fruits and root tips.1 Jasmonic acid and its bioactive derivatives and precursors have various
activities in plants. Firstly, jasmonic acid can inhibit germination in non-dormant seeds but stimulate
germination of dormant seeds. Secondly, jasmonic acid also inhibits root-growth and tuber formation. Thirdly,
insufficiency of jasmonic acid affects anther or ovule development and results in sterile flower organs. Fourthly,
jasmonic acid may be associated with senescence.52

Many frontline plant defense genes are induced by jasmonic acid, which mediate numerous transcriptional
responses to wounding, herbivory and pathogenesis.53 The signaling of jasmonic acid is via the ubiquitin
system as the jasmonic acid receptor is CORONATINE INSENSITIVE1 (COI1) which is an F-box protein
related to the auxin receptor TIR1. The other component of this system is the JA-ZIM domain (JAZ) repressor
proteins which inhibit MYB2 directed transcription. JA binds COI1 and this assembles with JAZ to form an
SCF complex that tags JAZ with ubiquitin for degradation via the 26S proeasome and so releases the
transcription factors.14,15,54 Jasmonic acid is also a potent lipid regulator, playing a central role in regulating
the biosynthesis of many secondary metabolites, so that plants can tolerate a wide variety of biotic and abiotic
stresses.55

Salicylic Acid

Salicylic acid is a phenol hormone critical for signaling innate immunity in plants. It functions in flowering
promotion, thermogenesis and disease resistance. Salicylic acid has been identified in leaves and reproductive
structures.1

Like the immune system of vertebrates and insects, plants usually undergo apoptotic-like cell death in the sites
surrounding pathogen entry, causing a hypersensitive response in the nearby region. During this period, plants
continuously develop a broad range and long lasting defense to further similar infections, known as systemic
acquired resistance.56 Salicylic acid is an endogenous signal for the activation of both local and systemic
resistance and induces the production of “pathogenesis-related proteins” such as catalase, peroxidase and
hydrolase.56,57 Microarray data demonstrates that salicylic acid treatments share a large number of coinduced
and co-repressed genes with jasmonic acid treatments.58 Therefore, salicylic acid and jasmonic acid display
cross regulation in gene expression, both being responsible for inducing the production of resistance related
proteins.

The salicylic acid receptor may be a member of the large gene family, receptor like protein kinases (RLKs).
Salicylic acid is able to induce the expression of numerous RLKs. The salicylic acid responsive RLKs often
contain C-X8-C-X2-C motifs in the putative extracellular domain. These RLK genes also contain the TTGAC
sequence in the promoter regions.59 Members of the RLK family bind to other ligands, such as brassinosteroids
and several of the peptides discussed below.

Peptide Signaling Molecules

Peptide signaling molecules (or hormones) are a relatively new class of growth regulators in plants used across
all the kingdoms that form a relatively ancient adaptation of paracrine/autocrine communication between cells.
Several of the peptide signaling molecules have distinct roles in development where they regulate cell
differentiation and organogenesis or modulating general growth in response to the environment. Other peptide
signaling molecules seem to act as endogenous danger signals.6,60 Some relatively well known signaling
peptides include: systemin, polaris, phytosulfokine (PSK), ENOD40, CLAVATA3 and the CLE family, S-locus
cysteine rich proteins (SCP), PEP1, Rapid Alkalization Factor (RALF) and plant natriuretic peptide (PNP).
Systemin is important in the induction of defense responses to wounding in Solanaceae species and is intimately
associated with jasmonic acid signaling.61 Polaris and PSKs are required for proper root growth and vascular
development. The expression of polaris is suppressed by ethylene and induced by auxin. PSK promotes cell
proliferation and longevity and PSK-α appears to act in a cooperative manner with CLE41-44 to regulate vessel
development. ENOD40 is associated with legume nodule formation. CLAVATA3 and other members of the
CLE family regulate development of shoot and root apical meristems. SCP regulates pollen determination.
PEP1 and RALF (and systemin) act as danger signals to stimulate the innate immune response in plants in
response to pathogen attack. PNP plays a role in water and solute balance. Hence peptide hormones are
involved in various biological processes, regulating growth, development, reproduction and defense responses
of plants.4–6,60,62,63

Conserved domains of peptide hormones are found between animals and plants, indicating evolutionary
parallels occurred in both kingdoms. However, receptors for peptide signaling molecules that have so far been
identified are significantly different. In animals peptide hormones are generally perceived by tyrosine kinases
and/or G protein-coupled receptors; whereas in plants peptide receptors are mainly serine/threonine kinases of
the LRR RLK family which contain an extracellular domain with leucine-rich repeats, a transmembrane domain
and a cytoplasmic kinase domain. These include the receptors of CLAVATA3, PSK and PEP1.6,64 Another
interesting feature is that several of these receptors also contain a predicted guanylate cyclase domain embedded
within their kinase domain,65 and in some instances this has been shown to have some catalytic activity.66 This
indicates that multiple signaling systems may be controlled by the ligand binding the receptor.

Peptide signaling molecules are often generated from the cleavage of their precursor polypeptides. Proteolysis is
the common cleavage manner by various peptidases located within the cell and also extracellular. A polypeptide
can be processed into multiple peptides, each with independent functions. The pep-tides can also undergo a
series of post-translational modification such as hydroxyprolination, tyrosine sulphation or glycosylation before
they can function properly. Such complicated maturation processes are usually completed in the endoplasmic
reticulum or Golgi apparatus.6,67,68

Model of Hormone Interaction

Plants need to maintain homeostasis of water, ion and other nutrients for survival. Various themes can be
elucidated from the above discussion where plants have developed common signaling processes. One such
process involves the ubiquitin 26 proteasome as a means to remove abnormal and useless proteins or, in the
case of hormone signaling, proteins that are no longer required.14,15 Another common theme is that many of
the plant hormone receptors identified to date are intracellular, which suggests that plants also have developed
transport systems to not only to allow the hormone to move over long distances (where it does) but also to enter
the destination cells. Auxin transport proteins are relatively well characterized and recently ATP binding
cassette (ABC) transporters for ABA have been identified.35,39 Also another feature to emerge is that
antagonist and competitive interactions are also able to reduce the dominance of one hormone. An example is
the interplay between ABA and other hormones such as GA and auxin32 which is discussed in more detail
below. Thus there is the possibility of several hormone signals acting on one response pathway simultaneously.
The final output of a physiological activity can be viewed as a combined result of the effects of several different
hormones that are raised in response to various biological stimuli. This results in signaling interaction networks
which include positive and/or negative feedback loops rather than simple linear signaling pathways. The above
overview was used to develop a hypothetical conceptual model (Fig. 1) about the signaling network of plant
hormones. This overview has been developed employing the traditional Chinese thoughts regarding mutual
restraint and mutual promotion within the natural world. The model is intended to build up a framework or
guideline for research thinking and to assist in the development of testable hypotheses.

Since hormonal influences mainly affect regulation of gene expression and/or the interaction among signaling
pathways, it is therefore essential to adopt two approaches to unravel the action of plant hormones. Firstly, a
particular developmental process of plants is generally controlled by some key players in a temporal and spatial
manner. Thus a single hormone influencing the quantitative and qualitative changes of gene expression
becomes a crucial concern in a hormone study. Microarray studies are now identifying coordinated expression
of genes by one or more hormones in time and spatially separated studies. This data can be used to develop
critical points that need to be investigated to confirm hormone interaction and we will use an example to
demonstrate how the model predicts such interactions. Secondly, in a systemic view, any biological
phenomenon of plants is due to the mixed responses of different signaling factors operating via a complex and
flexible network. Discovering the points of cross talk among various signaling pathways is a central feature to
dissecting the integration of hormones. There appears to be mutual restraint and mutual promotion within plant
hormones signaling networks and where this is described, it has influenced the development of the model.
Hence during the dissection of a plant hormone signaling pathway, conflicting and complementary effects from
other plant hormones should be monitored simultaneously (this is indicated by the dashed intersecting arrows
in Fig. 1) to obtain a view of hormonal interaction in the plant as a whole.

How effective is this model in practice? We use a recent publication69,70 looking at whole plant responses in a
spatial and temporal manner to test the model. The model predicts that the ethylene group will provide a
fundamental state for the plant to respond to ABA and that ABA in turn does this for the auxin group. The
model also predicts that the actions of the ethylene and ABA groups can be competed with or counteracted by
GA for instance although the auxin group will compete/antagonize with the ethylene group and the cytokinin
group with ABA. Skirycz et al.69 used microarray and metalabolomic analysis to probe leaf growth and
development under mild growth limiting osmotic stress conditions. They found growing leaves with dividing
and expanding cells were regulated by ethylene and GAs but not ABA, whereas mature leaves were regulated
by ABA under these conditions.69,70 That is contrary to the current dogma, ABA did not affect the leaves
undergoing division although it contributed to the response in the expanding cells and was the main player in
mature leaves. These exciting results demonstrate that different hormonal responses are employed at different
developmental stages to adapt young leaves to stress. Furthermore, the model independently developed in this
review (Fig. 1) also predicts that these hormones could be candidates involved in the plant's adaptations to this
environmental situation. Naturally, the model will need to be examined and modified further in practice.

Conclusion

The last decade has revealed a lot about plant hormone signaling pathways. Receptors for several hormones
have been identified at the genetic level and confirmed biochemically and in some cases structural information
has been gained. The common theme of protein degradation generally involving the ubiquitin 26S proteasome
system has been clearly established. Microarray experiments are revealing coordinated interactions among plant
hormones. The functional activity and signaling mechanism of plant hormones is a very complicated system
where the entire biological response is the combined result of different hormone actions. We have used the
available data to develop a conceptual model to describe how hormones interact. This model can be used to
develop testable hypotheses and further insights into the mechanisms of maintenance of plant homeostasis by
continuing to explore coordinated regulation by different hormones.

Acknowledgments

This work was supported by the Australian Research Council's Discovery project funding scheme
(DP0878194). Y.H.W. was supported by an Australian Postgraduate Award.

Abbreviations

ABA abscisic acid

ACC
1-aminocyclopropane-1-carboxylic acid

cADPR
cyclic ADP-ribose

ARF
auxin responsive factor

AuxRE
auxin responsive element

CLV3
CLAVATA3

DAG
1,2-diacylglycerol
 GA
gibberellin

IP3
inositol 1,4,5-trisphosphate

LRR RLK
leucine rich repeat receptor like kinase

MAPK
mitogen activated protein kinase

PNP
plant natriuretic peptide

PSK
phytosulfokine

RALF
rapid alkalization factor

RLK
receptor like kinase

SCF
Skp1/Cullin/F-box

SCP
S-locus cysteine-rich protein

Figures and Tables

Figure 1 A conceptual model of potential interactions amongst plant hormones. The hormones within
the same circle have complementary or similar actions. The solid arrows point to a new circle
containing hormones whose action has been promoted or substantially prepared by the hormones in the
previous circle. The dashed lines link the hormones that have competitive or antagonistic interactions
in plants.
 https://www.tandfonline.com/doi/full/10.4161/psb.6.4.14558

Hormonal cross-talk in plant

development and stress responses
Sergi Munné-Bosch* and Maren Müller

 Departament de Biologia Vegetal, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain

In contrast to animals, plants can continuously cease and resume growth. This flexibility
in their architecture and growth patterns is partly achieved by the action of plant
hormones. Plant hormones are structurally diverse compounds that act usually at
nanomolar concentrations and include five groups of the so-called “classic” hormones,
namely auxins, cytokinins, gibberellins, abscisic acid, and ethylene. Jasmonates,
salicylates, strigolactones, brassinosteroids, polyamines, and some peptides were
recognized as new families of plant hormones. Hormones build a signaling network and
mutually regulate several signaling and metabolic systems, which are essential both for
plant development and plant responses to biotic and abiotic stresses. Although earlier
work greatly advanced our knowledge of how hormones affect plant growth and
development and stress responses focusing on a single compound, it is now evident that
physiological processes are regulated in a complex way by the cross-talk of several
hormones. In this Research Topic, we aim at collecting a comprehensive set of original
research and review papers focused on hormonal crosstalk in plants.
The goal of this Research Topic is to bring together recent work of experts studying
hormonal crosstalk in plant development and stress response. Understanding how
hormones and genes interact to coordinate plant growth is a major challenge in
developmental biology. The activities of auxin, ethylene, and cytokinin depend on the
cellular context and exhibit either synergistic or antagonistic interactions. Liu et al.
(2013) use experimentation and network construction to elucidate the role of the
interaction of the POLARIS peptide (PLS) and the auxin efflux carrier PIN proteins in the
crosstalk of three hormones (auxin, ethylene, and cytokinin) in Arabidopsis root
development. Naidoo et al. (2013) elegantly describe the transcriptional response of PR
genes (EgrPR2, EgrPR3, EgrPR4, EgrPR5, and EgrLOX) identified in Eucalyptus
grandis in response to SA and methyl jasmonate (MeJA) treatment. Blanco-Ulate et al.
(2013) analyzed a transcriptome study of tomato fruit infected with Botrytis cinerea in
order to profile the expression of genes for the biosynthesis, modification and signal
transduction of ethylene, salicylic acid, jasmonic acid, and abscisic acid, hormones that
may be not only involved in ripening, but also in fruit interactions with pathogens. The
changes in relative expression of key genes during infection and assays of susceptibility of
fruit with impaired synthesis or perception of these hormones were used to formulate
hypotheses regarding the involvement of these regulators in the outcome of the tomato
fruit–B. cinerea interaction.

A series of reviews also add to the current knowledge of hormonal cross-talk in the
regulation of plant development and stress responses. Arc et al. (2013) review our current
knowledge of ABA crosstalk with ethylene and NO, both volatile compounds that have
been shown to counteract ABA action in seeds and to improve dormancy release and
germination. McAtee et al. (2013) review current evidence on the topic and elegantly
describe the hormonal cross-talk in the developing seed and its surrounding fruit tissue
during fruit development. Denancé et al. (2013) address novel insights on the regulatory
roles of the ABA, SA, and auxin in plant resistance to pathogens and describe the complex
interactions among their signal transduction pathways. The strategies developed by
pathogens to evade hormone-mediated defensive responses are also reviewed. Based on
these data it is also discussed how hormone signaling could be manipulated to improve the
resistance of crops to pathogens. From another perspective, Daszkowska-Golec and
Szarejko (2013) review recent findings on phytohormone crosstalk, including changes in
signaling pathways and gene expression that impact on modulating stress response
through the closing or opening of stomata. da Costa et al. (2013) review current evidence
indicating a clear hormonal cross-talk in the regulation of adventitious rooting. Cheng et
al. (2013) review current evidence on the recently discovered phytohormone class,
strigolactones and their cross-talk with other plant hormones—such as auxin, cytokinin,
abscisic acid (ABA), ethylene (ET), and gibberellins (GA)—during different physiological
processes. Finally, O'Brian and Benkkova discuss the complex hormonal cross-talk in
plant responses to environmental stress, with a focus on cytokinins and other hormones,
such as abscisic acid, jasmonates, salicylates, ethylene, and auxin. Of particular interest is
the discussion of the impact of this research in the biotechnological industry.
 In conclusion, taken together these original and review articles reflect the explosion of
interest and considerable progress that has recently been made in the dynamic field of
plant biology, with a particular focus on better understanding hormonal cross-talk in plant
development and stress responses. It will be intriguing to see how future work on
hormonal cross-talk in plants will continue. We hope that the articles that have been
compiled will provide new insights into this topic and shed new light concerning the
complex but exciting phenomenon of hormonal cross-talk in plant development and stress
responses.

https://www.frontiersin.org/articles/10.3389/fpls.2013.00529/full

 B10.

Can we develop salt/heavy metal/drought‐tolerant crops without creating invasive plants?

PDF Tolerant crop

https://nmwrri.nmsu.edu/wp-content/uploads/2015/FacultyGrants2014-2015/PicchioniFinalReport.pdf

 B11.

Can plants be better utilized for large‐scale remediation and reclamation efforts on
degraded and/or toxic land?

Phytoremediation of heavy metal polluted soils and water: Progresses and

*
perspectives
Mohammad Iqbal Lone,1,2 Zhen-li He,†‡,1,3 Peter J. Stoffella,1 and Xiao-e Yang3
Author information Article notes Copyright and License information Disclaimer
This article has been cited by other articles in PMC.

ABSTRACT
Go to:

INTRODUCTION
Land and water are precious natural resources on which rely the sustainability of agriculture and the civilization
of mankind. Unfortunately, they have been subjected to maximum exploitation and severely degraded or
polluted due to anthropogenic activities. The pollution includes point sources such as emission, effluents and
solid discharge from industries, vehicle exhaustion and metals from smelting and mining, and nonpoint sources
such as soluble salts (natural and artificial), use of insecticides/pesticides, disposal of industrial and municipal
wastes in agriculture, and excessive use of fertilizers (McGrath et al., 2001; Nriagu and Pacyna, 1988;
Schalscha and Ahumada, 1998). Each source of contamination has its own damaging effects to plants, animals
and ultimately to human health, but those that add heavy metals to soils and waters are of serious concern due to
their persistence in the environment and carcinogenicity to human beings. They cannot be destroyed
biologically but are only transformed from one oxidation state or organic complex to another (Garbisu and
Alkorta, 2001; Gisbert et al., 2003). Therefore, heavy metal pollution poses a great potential threat to the
environment and human health.
In order to maintain good quality of soils and waters and keep them free from contamination, continuous efforts
have been made to develop technologies that are easy to use, sustainable and economically feasible.
Physicochemical approaches have been widely used for remedying polluted soil and water, especially at a small
scale. However, they experience more difficulties for a large scale of remediation because of high costs and side
effects. The use of plant species for cleaning polluted soils and waters named as phytoremediation has gained
increasing attention since last decade, as an emerging cheaper technology. Many studies have been conducted in
this field in the last two decades. Numerous plant species have been identified and tested for their traits in the
uptake and accumulation of different heavy metals. Mechanisms of metal uptake at whole plant and cellular
levels have been investigated. Progresses have been made in the mechanistic and practical application aspects of
phytoremediation. They were reviewed and reported in this paper.
Go to:

SOURCES OF HEAVY METALS AND SOIL WATER POLLUTION

Land and water pollution by heavy metals is a worldwide issue. All countries have been affected, though the
area and severity of pollution vary enormously. In Western Europe, 1 400 000 sites were affected by heavy
metals (McGrath et al., 2001), of which, over 300 000 were contaminated, and the estimated total number in
Europe could be much larger, as pollution problems increasingly occurred in Central and Eastern European
countries (Gade, 2000). In USA, there are 600 000 brown fields which are contaminated with heavy metals and
need reclamation (McKeehan, 2000). According to government statistics, coal mine has contaminated more
than 19 000 km of US streams and rivers from heavy metals, acid mine drainage and polluted sediments. More
than 100 000 ha of cropland, 55 000 ha of pasture and 50 000 ha of forest have been lost (Ragnarsdottir and
Hawkins, 2005). The problem of land pollution is also a great challenge in China, where one-sixth of total
arable land has been polluted by heavy metals, and more than 40% has been degraded to varying degree due to
erosion and desertification (Liu, 2006). Soil and water pollution is also severe in India, Pakistan and
Bangladesh, where small industrial units are pouring their untreated effluents in the surface drains, which
spread over near agricultural fields. In these countries raw sewage is often used for producing vegetables near
big cities.
Heavy metals that have been identified in the polluted environment include As, Cu, Cd, Pb, Cr, Ni, Hg and Zn.
The sources of various heavy metals are listed in Table Table1.1. The presence of any metal may vary from site
to site, depending upon the source of individual pollutant. Excessive uptake of metals by plants may produce
toxicity in human nutrition, and cause acute and chronic diseases. For instance, Cd and Zn can lead to acute
gastrointestinal and respiratory damages and acute heart, brain and kidney damages. High concentrations of
heavy metals in soil can negatively affect crop growth, as these metals interfere with metabolic functions in
plants, including physiological and biochemical processes, inhibition of photosynthesis, and respiration and
degeneration of main cell organelles, even leading to death of plants (Garbisu and Alkorta, 2001;
Schmidt, 2003; Schwartz et al., 2003). Soil contamination with heavy metals may also cause changes in the
composition of soil microbial community, adversely affecting soil characteristics (Giller et al., 1998; Kozdrój
and van Elsas, 2001; Kurek and Bollag, 2004).

Table 1
Different sources of heavy metals

Heavy Sources
metals

As Semiconductors, petroleum refining, wood preservatives, animal feed additives, coal power plants, herbicides,
volcanoes, mining and smelting (Nriagu, 1994; Walsh et al., 1979)

Cu Electroplating industry, smelting and refining, mining, biosolids (Liu et al., 2005)

Cd Geogenic sources (Baize, 1997), anthropogenic activities (Nriagu and Pacyna, 1988), metal smelting and refining,
fossil fuel burning, application of phosphate fertilizers, sewage sludge (Alloway, 1995; Kabata-Pendias, 2001)

Cr Electroplating industry, sludge, solid waste, tanneries (Knox et al., 1999)

 Heavy Sources
metals

Pb Mining and smelting of metalliferous ores, burning of leaded gasoline, municipal sewage, industrial wastes enriched in
Pb, paints (Gisbert et al., 2003; Seaward and Richardson, 1990)

Hg Volcano eruptions, forest fire, emissions from industries producing caustic soda, coal, peat and wood burning
(Lindqvist, 1991)

Se Coal mining, oil refining, combustion of fossil fuels, glass manufacturing industry, chemical synthesis (e.g., varnish,
pigment formulation)

Ni Volcanic eruptions, land fill, forest fire, bubble bursting and gas exchange in ocean, weathering of soils and geological
materials (Knox et al., 1999)

Zn Electroplating industry, smelting and refining, mining, biosolids (Liu et al., 2005)

Open in a separate window

Go to:

TECHNOLOGIES FOR THE RECLAMATION OF POLLUTED SOILS

The cleaning of contaminated soils from heavy metals is the most difficult task, particularly on a large scale.
The soil is composed of organic and inorganic solid constituents, water and mixture of different gases present in
various proportions. The mineral components vary according to parent materials on which the soil had been
developed under a particular set of climatic conditions. Therefore, soils vary enormously in physical, chemical
and biological properties. Soil water movement is controlled by physical properties, such as soil structure and
texture. The soil moisture has great bearing in controlling solute movement, salt solubility, chemical reactions
and microbiological activities and ultimately the bioavailability of the metal ions. A successful
phytoremediation program, therefore, must take into consideration variations in soil properties of the specific
site.
Different approaches have been used or developed to mitigate/reclaim the heavy metal polluted soils and waters
including the landfill/damping sites. These may be broadly classified into physicochemical and biological
approaches.
The physicochemical approach includes excavation and burial of the soil at a hazardous waste site,
fixation/inactivation (chemical processing of the soil to immobilize the metals), leaching by using acid solutions
or proprietary leachants to desorb and leach the metals from soil followed by the return of clean soil residue to
the site (Salt et al., 1995), precipitation or flocculation followed by sedimentation, ion exchange, reverse
osmosis and microfiltration (Raskin et al., 1996). The physicochemical approaches are generally costly and
have side effects (Raskin et al., 1997; McGrath et al., 2001).
Biological approaches of remediation include: (1) use of microorganisms to detoxify the metals by valence
transformation, extracellular chemical precipitation, or volatilization [some microorganism can enzymatically
reduce a variety of metals in metabolic processes that are not related to metal assimilation], and (2) use of
special type of plants to decontaminate soil or water by inactivating metals in the rhizosphere or translocating
them in the aerial parts. This approach is called phytoremediation, which is considered as a new and highly
promising technology for the reclamation of polluted sites and cheaper than physicochemical approaches
(Garbisu and Alkorta, 2001; McGrath et al., 2001; Raskin et al., 1997).
Phytoremediation, also referred as botanical bioremediation (Chaney et al., 1997), involves the use of green
plants to decontaminate soils, water and air. It is an emerging technology that can be applied to both organic
and inorganic pollutants present in the soil, water or air (Salt et al., 1998). However, the ability to accumulate
heavy metals varies significantly between species and among cultivars within species, as different mechanisms
of ion uptake are operative in each species, based on their genetic, morphological, physiological and anatomical
characteristics. There are different categories of phytoremediation, including phytoextraction, phytofiltration,
phytostabilization, phytovolatization and phytodegradation, depending on the mechanisms of remediation.
Phytoextraction involves the use of plants to remove contaminants from soil. The metal ion accumulated in the
aerial parts that can be removed to dispose or burnt to recover metals. Phytofiltration involves the plant roots or
seedling for removal of metals from aqueous wastes. In phytostabilization, the plant roots absorb the pollutants
from the soil and keep them in the rhizosphere, rendering them harmless by preventing them from leaching.
Phytovolatization involves the use of plants to volatilize pollutants from their foliage such as Se and Hg.
Phytodegradation means the use of plants and associated microorganisms to degrade organic pollutants
(Garbisu and Alkorta, 2001). Some plants may have one function whereas others can involve two or more
functions of phytoremediation.
Go to:

PLANT SPECIES FOR PHYTOREMEDIATION

To identify plant populations with the ability to accumulate heavy metals, 300 accessions of 30 plant species
were tested by Ebbs et al.(1997) in hydroponics for 4 weeks, having moderate levels of Cd, Cu and Zn. The
results indicate that many Brasssica spp. such as B. juncea L., B. juncea L. Czern, B. napus L. and B. rapa L.
exhibited moderately enhanced Zn and Cd accumulation. They were also found to be most effective in
removing Zn from the contaminated soils. To date, more than 400 plant species have been identified as metal
hyperaccumulators, representing less than 0.2% of all angiosperms (Brooks, 1998; Baker et al., 2000). The plant
species that have been identified for remediation of soil include either high biomass plants such as willow
(Landberg and Greger, 1996) or those that have low biomass but high hyperaccumulating characteristics such
as Thlaspi and Arabidopsis species. On worldwide basis, the number of species identified to have ability to
accumulate one or more metals >1000 mg/kg dry weight is listed in Table Table22 (Reeves, 2003).

Table 2
The number of plant species that are reported to have hyperaccumulation traits (metal concentration >1000
mg/kg dry weight) (Reeves, 2003)

Metals Number of species

As 04

Cd 01

Co 34

Cu 34

Pb 14

Ni >320

Se 20

The hyperaccumulators that have been most extensively studied by scientific community
include Thlaspi sp., Arabidopsis sp., Sedum alfredii sp. (both genera belong to the family of Brassicaceae and
Alyssum). Thlaspi sp. are known to hyperaccumulate more than one metal, i.e., T. caerulescens for Cd, Ni, Pb
and Zn, T. goesingense for Ni and Zn, T. ochroleucum for Ni and Zn, and T. rotundifolium for Ni, Pb and Zn
(Prasad and Freitas, 2003). Among the genus Thlaspi, the hyperaccumulator plant Thlaspi
caerulescens received much attention and has been extensively studied as potential candidates for Cd and Zn
contaminated soils. Robinson et al.(1998) found T. caerulescens as hyperaccumulator for Cd and Zn could
remove as high as 60 kg Zn/ha and 8.4 kg Cd/ha. It can accumulate as high as 2600×10−6 Zn without showing
any injury (Brown et al., 1995) and extract up to 22% of soil exchangeable Cd from the contaminated site. It
also showed remarkable Cd tolerance (Sneller et al., 2000; Escarre et al., 2000; Lombi et
al., 2000). T. caerulescens has higher uptake of Cd due to specific rooting strategy and a high uptake rate
resulting from the existence in this population of Cd-specific transport channels or carriers in the root
membrane (Schwartz et al., 2003).
Go to:

METAL HYPERACCUMULATION IN VARIOUS PLANT SPECIES

The hyperaccumulation of metals in various plant species has been extensively investigated and to date
substantial progress has been made. It becomes clear that different mechanisms of metal accumulation,
exclusion and compartmentation exist in various plant species. In T. caerulescens, Zn is sequestered
preferentially in vacuoles of epidermal cells in a soluble form (Frey et al., 2000). In A. halleri leaves, Zn was
found to be accumulated in the mesophyll cells (Kupper et al., 2000; Zhao et al., 2000; Sarret et al., 2002).
Cosio et al.(2004) investigated the mechanisms of Zn and Cd accumulation in three different plant species
through ion compartmentation by measuring the short term 109Cd and 65Zn uptake in mesophyll protoplast
of T. caerulescens “Ganges” and A. halleri. Their study suggests the existence of regulation mechanism on the
plasma membrane of leaf mesophyll protoplast.
Puschenreiter et al.(2003) investigated chemical changes in the rhizosphere of
hyperaccumulators T. goesingense and T. caerulescens and the metal excluder T. arvense with a rhizosphere
bag experiment on the contaminated and non-contaminated soils. Hyperaccumulation and depletion of labile Zn
in the rhizosphere were observed for T. goesingense grown on the contaminated soil. In the non-contaminated
soil, Zn was accumulated but labile Zn in the rhizosphere was not changed. Nickel present in background
concentration in both soils was accumulated by T. goesingenseonly when grown on non-contaminated soil. In
contrast, labile Ni in the rhizosphere increased in both soils, suggesting a general tendency of Ni mobilization
by T. goesingense. Uneo et al.(2004a) studied the interaction between Zn and Cd in T. caerulescens in solution
culture and in pot soil. Results from long term (4 weeks) and short term (1 week) solution culture experiments
indicate that Cd accumulation in the shoot was not affected by the supply of a 4~10-fold excess of Zn, whereas
the Cd concentration of the roots decreased with increasing Zn concentrations in the solution. The results
suggest that the Ganges ecotype of T. caerulescens displayed different uptake systems for Cd and Zn and that
Cd competed with Zn uptake while Zn did not compete with Cd uptake. Uneo et al.(2004b) investigated the
uptake of Cd and Zn by T. caerulescens (the Ganges ecotype) from enriched soil with different insoluble and
soluble sources of Cd and Zn. The data show that there was no significant differences in the shoot Cd
concentration between the treatments with soluble or insoluble Cd compounds, even though Cd concentration in
the soil solution was in the order of CdSO4>>CdCO3>CdS. Thlaspi caerulescens grown on the ZnS-enriched
soil accumulated up to 6 900 mg Zn/kg in the shoots, although Zn accumulation was 1.5 times higher with the
addition of more soluble compounds Zn3(PO4)2 or ZnSO4. These results indicate that the Ganges ecotype
of T. caerulescens is able to utilize insoluble Cd and Zn compounds in soils.
Whiting et al.(2000) found that the plants from T. carerulescens population that accumulated Cd also showed
increased root biomass and root length after allocation into Cd-enriched soil, whereas plants from the
population that did not accumulate Cd showed no such increase.
T. caerulescens was grown with H. vulgare and L. heterophyllum in the field to examine the effect of
rhizosphere interaction on metal uptake. The data show that the Cd concentration in H. vulgare was increased
by a factor of 2.4 when it was grown along the sides of T. caerulescens without a barrier. In contrast, the uptake
of Zn by H. vulgare was significantly decreased, probably through metal depletion within the zone of the Zn-
hyperaccumulator rhizosphere. These results suggest that T. caerulescens may alter conditions in the shared
rhizospheres and thereby affect the availability of selected metals to neighboring plants (Gove et al., 2002). On
the other hand, when S. alfredii was intercropped with a grain crop, Z. mays, heavy metals (Zn and Cu)
accumulated in the grains were significantly reduced, as compared to monoculture cropping, and the
intercropping improved the growth of both plant species (Liu et al., 2005).
Studies on the role of rhizosphere process in metal hyperaccumulation of Ni in T. geosingense Halacsy by
Wenzel et al.(2002) indicate that root exudates of organic ligands may contribute to Ni hyperaccumulation
in T. geosingense Halacsy. This was attributed to the ligand-induced dissolution of Ni bearing minerals in the
rhizosphere of T. geosingense and appeared to be less effective in the rhizosphere of excluder Silene
vulgaris and Rumex acetosella growing on the same site.
Sedum alfredii Hance was identified in China as hyperaccumulator for Cd and Zn and has been intensively
investigated by various researchers in their studies conducted in hydroponics and/or the uncontaminated and
contaminated soils (Li H. et al., 2005; Li T.Q. et al., 2005a; Liu et al., 2005; Xiong et al., 2004; Yang et
al., 2004; 2006). The data show that the concentrations of Cd and Zn in leaves and stems increased with
increasing Cd and Zn supply levels. The distributions of the metals in different plant parts decreased in the
order: stem>leaf>root for Zn and leaf>stem>root for Cd. These results indicate that S. alfredii has an
extraordinary ability to tolerate Cd/Zn toxicities, and to absorb and hyperaccumulate Cd and Zn under a range
of Cd/Zn combining levels. The uptake and accumulation of Cd by the mined and the non-mined ecotypes
of S. alfredii indicated that the plants of the mined ecotype (ME) have higher tolerance to Cd than those of the
non-mined ecotypes (NME) in terms of dry matter yield (Xiong et al., 2004).
Zinc compartmentation studies involving hyperaccumulating and non-hyperaccumulating S. alfredii plants
using radioactive tracer flux technique indicate that S. alfredii H. can accumulate Zn in shoots over 2% of dry
weight. Leaf and stem Zn concentrations of the hyperaccumulating ecotype (HE) were 24- and 28-fold higher,
respectively, than those of the non-hyperaccumulating ecotype (NHE), whereas 1.4-fold more Zn was
accumulated in the roots of the NHE. Approximately 2.7-fold more Zn was stored in the root vacuoles of the
NHE, and thus became unavailable for loading into the xylem and subsequent translocation to shoots. These
results also indicate that the altered Zn transport across tonoplast in the root and the stimulated Zn uptake in the
leaf cells are the major mechanisms involved in the strong Zn hyperaccumulation observed in S. alfredii H.
(Yang et al., 2006).
The root morphology and Zn2+ uptake kinetics of HE and NHE of S. alfredii H. were investigated using
hydroponic methods and the radiotracer flux technique. The results indicate that the root length, root surface
area and root volume of NHE decreased significantly with increasing Zn2+ concentration in growth media,
whereas the root growth of HE was not adversely affected, and even promoted, by 500 µmol/L Zn2+. The
concentrations of Zn2+ in both ecotypes of S. alfredii H. were positively correlated with root length, root surface
area and root volumes, but no such correlation was found with root diameter. The uptake kinetics for 65Zn2+ in
the roots of both ecotypes of S. alfredii were characterized by a rapid linear phase during the first 6 h and a
slower linear phase during the subsequent period of investigation. The concentration-dependent uptake kinetics
of the two ecotypes of S. alfredii could be characterized by the Michaelis-Menten equation, with
the V max (maximum uptake speed) for 65Zn2+ influx being 3-fold greater in the HE than that in the NHE,
indicating that enhanced absorption into the root was one of the mechanisms involved in Zn hyperaccumulation.
A significantly larger V max value suggested that there was a higher density of Zn transporters per unit membrane
area in HE roots (Li H. et al., 2005).
Li T.Q. et al.(2005b) investigated the root morphological and physiological response of the HE of S. alfridii H.
from the mined area and the NHE of S. alfridii from the agricultural area to the supplied Zn and Pb in
hydroponics. The results show that Zn concentrations in the leaves and the stems of HE were 34 and 41 times
higher, whereas Pb concentrations were 1.9 and 2.4 times higher, respectively than those of the NHE when
grown at 1224 µmol/L Zn and/or 200 µmol/L Pb. The study also shows that the tolerance and
hyperaccumulation of the HE of S. alfridii H. to Zn and Pb appear to be closely related to its high adaptation of
root growth, morphology and physiology to Pb and Zn toxicity. Through its root excretion of some special
substances, the plant can activate Pb and Zn in the mined soil, thus increasing their mobilization and
bioavailability.
The Alyssum species has been extensively studied for the hyperaccumulation of Ni. Kupper et al.(2001) studied
the Ni uptake and cellular compartmentation in three Ni
hyperaccumualtors: A. bertolonii (Desv), A. lesbiacum (Candargy), and T. goesingense (Halacsy). These three
species showed similar hyperaccumulation of Ni, but T. goesingense was less tolerant to Ni than the other two
species. Addition of 500 mg Ni/kg to a nutrient-rich growth medium significantly increased shoot biomass of all
species. X-ray microanalysis of frozen-hydrated tissues of leaves and stems of all species showed that Ni in all
species was distributed preferentially in the epidermal cells, most likely in the vacuoles of the leaves and stem.
Kidd and Monterroso (2005) investigated the efficiency of Alyssum
serpyllifolium ssp. lusitanicum (Brassicaceae) for use in phytoextraction of polymetal-contaminated soils. The
plant was grown on two mine spoil soils, one contaminated with Cr (283 mg/kg) and the other moderately
contaminated with Cr (263 mg/kg), Cu (264 mg/kg), Pb (1433 mg/kg) and Zn (377 mg/kg). The results suggest
that A. serpyllifolium could be suitable for phytoextraction uses in polymetal-contaminated soils, provided that
Cu concentrations were not phytotoxic.
Among different fern species, three accessions of P. vitta, two cultivars
of P. cretica, P. longifolia and P. umbrosa were grown with 0~500 mg As/kg added to the substrate. The results
show that in addition to P. vitta, P. cretica, P. longifolia and P. umbrosa also hyperaccumulate As to a similar
extent. This study identified three new species of As hyperaccumulators in the Pteris genus (Zhao et al., 2002).
In another study, the speciation and distribution of As of Brake fern was investigated by Zhang et al.(2002),
which was grown for 20 weeks in As contaminated soil. The results show that As recoveries of 85% to 100%
were obtained from most parts of the plant (rhizomes, fiddle heads, young fronds and old fronds), and for roots,
the corresponding value was approximately 60%. The result also demonstrates the ability of Blake fern as As
hyperaccumulator, which can transfer As rapidly from soil to above ground biomass with minimal As
concentration in the roots. As is found to be predominantly as inorganic species. Caille et al.(2005) conducted a
pot experiment with 0~500 mg/kg As added as arsenate and another short term (8 h) uptake experiment with
5×10−6 arsenate under phosphorus sufficient conditions, and grew hyperaccumulator Pteris vitta and the
nonhyperaccumulator Pteris tremula. The results show that in both experiments P. vitta accumulated much
more As than P. tremula without any visual toxicity symptoms.
Go to:

PHYTOREMEDIATION OF POLLUTED WATER

Rhizofiltration is the removal of pollutants from the contaminated waters by accumulation into plant biomass.
Several aquatic species have been identified and tested for the phytoremediation of heavy metals from the
polluted water. These include sharp dock (Polygonum amphibium L.), duck weed (Lemna minor L.), water
hyacinth (Eichhornia crassipes), water lettuce (P. stratiotes), water dropwort [Oenathe javanica (BL) DC],
calamus (Lepironia articulate), pennywort (Hydrocotyle umbellate L.) (Prasad and Freitas, 2003). The roots of
Indian mustard are found to be effective in the removal of Cd, Cr, Cu, Ni, Pb and Zn, and sunflower can remove
Pb, U, Cs-137 and Sr-90 from hydroponic solutions (Zaranyika and Ndapwadza, 1995; Wang et al., 2002;
Prasad and Freitas, 2003).
The potential of duck weed was investigated by Zayed et al.(1998) for the removal of Cd, Cr, Cu, Ni, Pb and Se
from nutrient-added solution and the results indicate that duck weed is a good accumulator for Cd, Se and Cu, a
moderate accumulator for Cr, but a poor accumulator of Ni and Pb. Dos Santos and Lenzi (2000) tested aquatic
macrophyte (Eiochhornia crassipes) in the elimination of Pb from industrial effluents in a green house study
and found it useful for Pb removal. Water hyacinth possesses a well-developed fibrous root system and large
biomass and has been successfully used in wastewater treatment systems to improve water quality by reducing
the levels of organic and inorganic nutrients. This plant can also reduce the concentrations of heavy metals in
acid mine water while exhibiting few signs of toxicity. Water hyacinth accumulates trace elements such as Ag,
Pb, Cd, etc. and is efficient for phytoremediation of wastewater polluted with Cd, Cr, Cu and Se (Zhu et
al., 1999).
Wang et al.(2002) conducted a pot experiment to test five wetland plant species, i.e., sharp dock, duckweed,
water hyacinth, water dropwort and calamus for their possible use in remedying the polluted waters. The results
show that sharp dock was a good accumulator of N and P. Water hyacinth and duckweed strongly accumulated
Cd with a concentration of 462 and 14200 mg/kg, respectively. Water dropwort achieved the highest
concentration of Hg, whereas the calamus accumulated Pb (512 m/kg) substantially in its roots. Ingole and
Bhole (2003) conducted hydroponic studies to investigate the uptake of As, Cr, Hg, Ni, Pb and Zn by water
hyacinth from the aqueous solution at the concentrations ranging from 5 to 50 mg/L, and observed that in
aqueous solutions containing 5 mg/L of As, Cr and Hg, the maximum uptake was 26, 108 and 327 mg/kg dry
weight of water hyacinth, respectively.
Among the ferns, Pteris vitta commonly known as Brake fern has been identified as As hyperacccumulator for
As contaminated soils and waters. It can accumulate up to 7500 mg As/kg on a contaminated site (Ma et
al., 2001) without showing toxicity symptoms. One fern cultivar is available commercially for As
phytoremediation and has been successfully used in field trials (Salido et al., 2003).
Li H. et al.(2005) conducted a laboratory study in hydroponics to test different levels of Cd on the growth and
Cd uptake by three hydrophytes: Gladiolous, Isoetes taiwaneneses Dwvol and Echinodorus amazonicus. The
data show that the biomass of all the plants decreased with an increase in Cd concentration from 5 to 20 mg/L.
However, Cd toxic effect was greater on Isoetes taiwaneneses Dwvol and Echinodorus amazonicus than that
on Gladiolous. In addition, the accumulation of Cd was higher in Gladiolous than the other two plants. Zhang et
al. (2005) investigated the efficiency of Cu removal from the contaminated water by Elsholtzia
argyi and Elsholtzi splendens in hydroponics. The results show that Elsholtzia argyi showed better Cu
phytofiltration than Elsholtzi splendens, which was associated with better ability to higher Cu concentrations
and translocation to shoots.
Go to:

ENHANCEMENT OF PHYTOREMEDIATION BY CHEMICAL AND BIOLOGICAL

APPROACHES
In order to cope with heavy metal contaminated soils, various phytoremediation approaches (phytostabilization,
phytoimmobilization and phytoextraction) can be applied. However, the choice will depend on many factors,
such as plant tolerance to pollutants, soil physicochemical properties, agronomic characteristics of the plant
species, climatic conditions (rainfall, temperature), and additional technologies available for the recovery of
metals from the harvested plant biomass. It appears that both chemical and biological approaches are passing
through their infancy and need more efforts for their effective use in the future.
The solubility of heavy metals in the polluted soils can be increased by using organic and inorganic agents, thus
enhancing the phytoextraction capabilities of many plant species. Ebbs et al.(1997) amended the contaminated
soil with Grower-Power, a commercial soil amendment that improves soil structure and fertility, and the
removal of Zn by plant shoots was doubled to more than 30 000 mg Zn/pot (4.5 kg). Other applied enhancement
materials include ethylene diamine tetraacetic acid (EDTA), citric acid, elemental sulfur or ammonium sulfate.
Increases greater than 100 folds in Pb concentration in the biomass of crops were reported when EDTA was
 applied to the contaminated soils (Cunningham and Berti, 2000). Uranium, cadmium and zinc concentrations in
plant biomass were increased by the application of citric acid, elemental sulfur or ammonium sulfate,
respectively (Schmidt, 2003). In addition to the chelating material, the plant roots excrete metal-mobilizing
substances called phytosiderophores. Other exudates include mugenic and deoxymugeneic acids from barley
and corn, and avenic acid from oats (Welch and Norvell, 1993). Plant roots can increase metal bioavailability by
exuding protons that acidify the soil and mobilize the metals. The lowering of soil pH decreases the adsorption
of heavy metals and increases their concentrations in the soil solution. Soil microbes associated with plant roots
are also helpful in the phytoextraction of the heavy metals in soils through the degradation of organic pollutants.
These include several strains of bacillus and psedumonos, which increase the Cd accumulation in Brassica
juncea seedlings (Salt et al., 1995).
Scott Angle et al.(2003) determined the effect of high soil moisture content on the growth and
hyperaccumulation of Ni in three different species, including Alyssum murale and Berkheya coddii and Zn
hyperaccumulator T. caerulescens cultivar AB300 and AB336. The results show that hyperaccumulators grew
well under high soil moisture content and the biomass of all the tested species was generally greater at higher
soil moistures and inhibited at lower soil moistures. These results suggest that for successful phytoremediation
of metal polluted soils, a strategy should be developed to combine a rapid screening of plant species possessing
the ability to accumulate heavy metals with agronomic practices that enhance shoot biomass production and/or
increase metal bioavailability in the rhizosphere.
Go to:

CONCLUSIONS AND PERSPECTIVES

The contamination of heavy metals to the environment, i.e., soil, water, plant and air is of great concern due to
its potential impact on human and animal health. Cheaper and effective technologies are needed to protect the
precious natural resources and biological lives. Substantial efforts have been made in identifying plant species
and their mechanisms of uptake and hyperaccumulation of heavy metals in the last decade. There are genetic
variations among plant species and even among the cultivar of the same species. The mechanisms of metal
uptake, accumulation, exclusion, translocation, osmoregulation and copartmentation vary with each plant
species and determine its specific role in phytoremediation. Variations exist for hyperaccumulation of different
metals among various plant species and within populations. These variations do not correlate with either the
metal concentration in the soil or the degree of metal tolerance in the plant (Pollard et al., 2002). In order to
develop new crop species/plants having capabilities of metal extraction from the polluted environment,
traditional breeding techniques, hybrid generation through protoplast fusions, and production of mutagens
through radiation and chemicals are all in progress. With the development of biotechnology, the capabilities of
hyperaccumulators may be greatly enhanced through specific metal gene identification and its transfer in certain
promising species. This can play a significant role in the extraction of heavy metals from the polluted soils. The
use of cleaning technologies is site-specific due to spatial and climatic variations and is not economically
feasible everywhere. Therefore, cheaper technologies are being sought for practical use. Nevertheless, the
recent advances in plant biotechnology have created a new hope for the development of hyperaccumulating
species. However, much research work is needed in this respect such as metal uptake studies at cellular level
including efflux and influx of different metal ions by different cell organelles and membranes. Rhizosphere
studies under the control and field conditions are also needed to examine the antagonistic and synergistic effects
of different metal ions in soil solution and the polluted waters. In depth soil microbial studies are required to
identify the micro-organisms highly associated with metal solubility or precipitations. To date the available
methods for the recovery of heavy metals from plant biomass of hyperaccumulators are still limited. Traditional
disposal approaches such as burning and ashing are not applicable to volatile metals; therefore, investigations
are needed to develop new methods for effective recovery of metals from the hyperaccumulatior plant biomass.
Go to:

ACKNOWLEDGMENTS
Dr. M.I. Lone is obliged to the Vice Chancellor, University of Arid Agriculture, Rawalpindi, Pakistan, for
sending him to USA for training in phytoremediation and Dr. Z.L. He and Dr. P.J. Stoffella for hosting this
training program at the University of Florida, USA.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2266886/

 B12.

How can we translate our knowledge of plants and ecosystems into ‘clever farming’
practices?
 PDF clever farming
http://www.fao.org/climate-smart-agriculture-sourcebook/production-resources/module-b1-crops/b1-
overview/en/?type=111

 B13.

Can alternatives to monoculture be found without compromising yields?

PDF alternative to monoculture

https://www.panna.org/sites/default/files/imported/files/PANalternativesToGEcrops.pdf

 B14.

Can plants be bred to overcome dry land salinity or even reverse it?ware for
optimizing fertilization and yields!
Home / SMART Library / How to Prevent and Manage Soil Salinity

HOW TO PREVENT AND MANAGE SOIL

SALINITY
There are various practices that you can apply, in order to prevent soil salinity or manage salinity
problems once they already occur.

1. Select a crop that fits the conditions in your field

 Soil type - water infiltration capacity, how much air does the soil contain ,how much water will be needed to wash the soil in
order to avoid salinity build up.
Does your soil have special drainage problems?

For example, it is better avoid planting a salt sensitive crop in a soil which is not well drained.
 The microclimate conditions in the field - parameters such as wind direction and solar radiation may affect water consumption
of the crop.
 The agricultural history of the field - did salts accumulate in the soil during a previous crop?
 Irrigation water quality - Check the quality of the available source water.
What kind of salts does it contain and what is the total level of salts in it?
 Type of irrigation system and its distribution - what type of irrigation system are you going to use?
Is it flood irrigation, sprinklers, pivot or drip irrigation?

Each type of irrigation system has its own water distribution pattern, depending also on the soil properties.
Make sure the emitters are set in the appropriate spacing, to allow uniform irrigation depending on your soil type.
2. Know the leaching requirement for your crop
Irrigation water amounts must coincide the growing stage of your crop.
Apply the minimum needed to flush salts from soil. This means that you always have to give a little more water than the crop
consumption, to allow leaching of salts below the root zone.
Heavier soils require larger water applications than lighter soils, in order to avoid salinity buildup.

The leaching requirement is expressed as:

LR = Water leached / Water applied

A general equation to calculate the leaching requirement is

LR (%) =ECiw/(5ECth-ECiw)
Where ECiw is the EC of the irrigation water, and ECth is the threshold salinity measured in the saturated soil extract, above
which yield begins do decline (both in ds/m).

The total amount of water to be applied is AW = ET/(1-LR)

Where AW is the amount of water to be applied and ET is the water consumption based on evapotranspiration.

3. Keep the right Intervals between irrigations

Irrigation regimen and intervals must be appropriate to the soil conditions and to growth stage of the crop.
Frequent and shallow (superficial) applications result in salt accumulation in the root zone, while larger applications, in longer
intervals, will flush the salts below the root zone.

4. Use appropriate fertilizers types

The fertilizers type and their quantities should coincide with to the requirements of the crop and with nutrients which are
already in the soil. There are fertilizers which contain salts which are not taken up by plants in large amounts, such as chlorides.
These salts tend to accumulate in the soil.

5. Have your soil tested periodically

Soil analysis gives you a better indication of the salt content in the soil, without which you'll be only guessing.
Guessing often comes close enough, but in many cases growers realize there's a salinity problem only after yields are decreased
or crop quality is reduced.

A practical approach in order to prevent salinity buildup early enough is sampling the soil 5 times over a growing period of 8
months (a test every 6 weeks or so). It is recommended to do at least one water analysis as well.
The tests will indicate any change in soil content, allowing you to adjust the fertilization and irrigation regimen as needed.

This is the cheapest, most practical way to follow up on salinity status, keeping your crop quality and yield at optimal level.

6. And if after all that, you still face a salinity problem...

When you identify a salinity problem during the growing season, it is recommended to flush the field, even if it means risking
some crop damage, rather than allowing further deterioration of the crop due to salinity.

Flushing applications should be carefully planned according to the crop conditions and growth stage.
In light soils, which drain easily, the impact of flushing on the crop is usually insignificant.

In heavy soils, water infiltration and drainage problems may be encountered, resulting in excess of water and lack of air to the
roots. Flushing heavy soils is a prolonged process and its final result is difficult to anticipate in advance.

Therefore, extra care should be taken when growing on heavy soils, as to not reach salinity buildup at all, or at least identify the
problem early enough, when salts levels are still relatively easy to flush.

If all else fails and flushing is the chosen course of action, in heavier soils, not more than the maximal water amount that can be
absorbed by the soil should be applied, and the longest intervals possible should be maintained. In the meantime, fertilization
should be based only on Nitrogen and only the minimum amount should be applied.
 The water used for flushing should be the highest quality possible, because the purpose of the flushing process is to decrease the
soil salinity to the levels of the irrigation water.

https://www.smart-fertilizer.com/articles/manage-soil-salinity

 B15.

Can we develop crops that are more resilient to climate fluctuation without yield loss?

PDF clever farming

http://www.fao.org/climate-smart-agriculture-sourcebook/production-resources/module-b1-crops/b1-
overview/en/?type=111

 B16.

Can we understand (explain and predict) the succession of plant species in any habitat, and
crop varieties in any location, under climate change?
Beemnet Mengesha Kassahun
Kyungpook National University
Interesting
Dear Prashant Balasaheb Pawar, one of the starting point for initiating a research idea or activity is challenge/problem of production
and productivity. As you know, climate change is one of the pertinent challenge/problem of the globe that have an impact on production
and productivity of crops. So, for addressing the problem, we do have no option except for developing varieties that can adapt and or
outperform with the changing climates. For this, scientists are applying their efforts for developing improved varieties using conventional
to modern breeding tools and positive results are also observed.

The magnitude and direction of climate change on yields depends on crop types and locations under some
circumstances. Our simulations studies show that dry areas will benefit from climate change and climate change is
likely to have positive impacts on C4 crops (e.g. sugarcane) due to CO2 fertilization. However, for most cereal crops
like wheat, climate change will likely have negative effects on yields mainly due to the shortage of growth stages as
a result of global warming. On the other hand, flowering time earlier (moving forward to a cooler period) caused by
increased temperature would make it possible to avoid drought stress in the reproductive stage. Crop modelling
simulation studies suggest it is more urgent to develop heat-tolerance cultivars than to develop drought-resistant
cultivars to mitigate future climate change.
Bin Wang
New South Wales Department of Primary Industries

https://www.researchgate.net/post/Can_we_develop_crops_that_are_more_resilient_to_climate_fluctuation_wit
hout_yield_loss

 B17.

To what extent are the stress responses of cultivated plants appropriate for current and
future environments?

Plant responses to environmental stresses—from

gene to biotechnology
Mohammad Abass Ahanger, Nudrat Aisha Akram, Muhammad Ashraf, Mohammed Nasser
Alyemeni, Leonard Wijaya, Parvaiz Ahmad
Author Notes
AoB PLANTS, Volume 9, Issue 4, July 2017, plx025, https://doi.org/10.1093/aobpla/plx025
 Published:

27 June 2017
Article history

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Abstract
Increasing global population, urbanization and industrialization are increasing the rate of conversion of arable land
into wasteland. Supplying food to an ever-increasing population is one of the biggest challenges that agriculturalists
and plant scientists are currently confronting. Environmental stresses make this situation even graver. Despite the
induction of several tolerance mechanisms, sensitive plants often fail to survive under environmental extremes. New
technological approaches are imperative. Conventional breeding methods have a limited potential to improve plant
genomes against environmental stress. Recently, genetic engineering has contributed enormously to the development
of genetically modified varieties of different crops such as cotton, maize, rice, canola and soybean. The identification
of stress-responsive genes and their subsequent introgression or overexpression within sensitive crop species are now
being widely carried out by plant scientists. Engineering of important tolerance pathways, like antioxidant enzymes,
osmolyte accumulation, membrane-localized transporters for efficient compartmentation of deleterious ions and
accumulation of essential elements and resistance against pests or pathogens is also an area that has been intensively
researched. In this review, the role of biotechnology and its successes, prospects and challenges in developing stress-
tolerant crop cultivars are discussed.

Introduction

In plant biology, the transgenic approach has emerged as an important tool to adapt crops to rapidly changing
environmental conditions. The use of transgenic crops has increased considerably over the past decade. The
primary step before proceeding with transgenics is the identification of genes serving as key regulators of
different metabolic pathways, including osmolyte synthesis, ion homeostasis through selective ion uptake,
antioxidant defence system and other frontline defence pathways (Ahmad et al. 2012).

Genome editing has revolutionized plant biotechnology by providing plant scientists with the option of selection
or incorporation of genes of interest into desired species or cultivars (Tzfira et al. 2012). A particular stress
alters the expression of specific genes in a species-dependent fashion. It causes differences in the efficiency of
signal perception and subsequent transcriptional alterations leading to elicitation of a specific response and
adaptation and finally enhanced stress tolerance. The microarray hybridization technique (employing cDNAs or
oligonucleotides) is the main technique used to isolate a set of desired genes. Recently, rice microarrays using
oligonucleotides (22 000 oligoarray) have been produced based on full-length cDNA information through the
Rice Genome Program of Japan. A number of companies such as Axon Instruments, Inc., Amersham
Biosciences, MWG Biotech AG, Genetic Analysis Technology Consortium, Clontech Laboratories, Azign
Bioscience A/S, Mergen Ltd., Invitrogen, Promega, QIAGEN, Stratagene and QIAGEN Operon are providing
these arrays. The services provided by Agilent Company Ltd. are now being used to evaluate and understand
responses to abiotic stresses in rice through transcriptomic studies (Ban and Moriguchi 2010). Limitations in
terms of the availability of sophisticated cost-intensive apparatus and materials are common in many countries’
laboratories (Ban and Moriguchi 2010).

Suppression Subtraction Hybridization (SSH) is an extremely powerful and widely used technique for
separating cDNA or genomic DNA (Ban and Moriguchi 2010; Ding et al. 2014; Ma et al. 2017). Transgenes
are introduced into plants by biological or physical methods. Successful and efficient transformation demands
specific criteria to be met including regeneration capacity and competence of target tissues, efficient DNA
delivery method and precautions for avoiding somaclonal variations and sterility. Several techniques fulfil these
requirements, e.g. protoplast transformation, biolistics or microprojectile bombardment and Agrobacterium-
mediated transformation (Rodrigues et al. 2012; Fei et al. 2015).

In this review, we summarize stress-responsive genes and their subsequent introgression or overexpression
within other crop species. In addition, engineering of important pathways involved in the oxidative defence
system, osmoprotection, ion transportation and resistance against pathogens is explored. The role of
biotechnology and its successes, prospects and challenges in developing stress-tolerant crop cultivars are
discussed.

Responses of Transgenic Plants to Different Stresses

The past decade has extensively increased our understanding of ways to improve stress tolerance through the
transgenic approach (Bhatnagar-Mathur et al. 2008; Gilliham et al. 2016; Wang et al. 2016). The majority of
transgenic plants has been tested against different abiotic factors only in growth chambers, greenhouses or
under controlled conditions (Ashraf and Foolad 2007). Few studies are available in the literature in which
abiotic stress tolerant transgenic plants were tested under true field conditions (Table 1).
Table 1.
Transgenic plants showing resistance to various environmental stresses through expression of
genes.
Growth
Plant/crop Possible conditions
species Gene role Tolerance to Reference

Mitochondrial penta Increase in

Arabidopsis tricopeptide repeat domain proline, Growth Zsigmond
thaliana protein (PPR40) mitochondria Salinity stress chamber et al. 2012
 Growth
Plant/crop Possible conditions
species Gene role Tolerance to Reference

l respiration.
Decrease in
SOD, APX,
lipid
peroxidation

Decreased
ROS,
decreased
Na+ and
increased
potassium in
shoot,
increased
Arabidopsis AOX Glasshouse Smith et
thaliana Ataox1 activity Salinity stress al. 2009

Higher
Arabidopsis P5CS genes from common proline Greenhous Chen et
thaliana bean content Salinity stress e al. 2013

Efficient
scavenging
of ROS by
Arabidopsis β-lycopene cyclase gene enhanced
and SeLCY from Salicornia carotenoid Greenhous Chen et
tobacco europaea contents Salinity stress e al. 2011

Increase in
germination,
chlorophyll
and osmotic
constituents
like sugars
with a
concomitant
Dehydration-responsive decrease in Greenhous Jamoussi e
Tobacco RD22 gene of Vitis vinifera Na uptake Salinity stress e t al. 2014

ABA
biosynthesis
and
Arabidopsis Zeaxanthin epoxidase xanthophyll Salinity, Controlled Park et
thaliana (ZEP) cycle drought conditions al. 2008

Salinity, Hydroponi
Six-fold osmotic and cs under
Brassica γ-Tocopherol methyl increase in γ- heavy metal controlled Yusuf et
juncea transferase (γ-TMT) tocopherol stress conditions al. 2010

Increased
proline,
RWC, Salinity and Controlled Goel et
Tomato Osmotin germination drought conditions al. 2010
 Growth
Plant/crop Possible conditions
species Gene role Tolerance to Reference

GmWRKY13, Drought,
Arabidopsis GmWRKY21 and Lateral root salinity and Controlled Zhou et
thaliana GmWRKY54 development cold conditions al. 2008

Maintained
sufficient
CaXTH3, a hot pepper chlorophyll
xyloglucan even at
endotransglucosylase/hydro 100 mM Drought and Controlled Choi et
Tomato lase NaCl salinity conditions al. 2011

Glycine
betaine
accumulation
, maintained
cell
membrane
integrity,
photosyntheti
c activity.

Reduced
ROS
production
and quick
ROS
scavenging
by increased Salinity,
Sweet activity of oxidative and
potato Chloroplastic BADH gene free radical- low
(Ipomoea from Spinacia scavenging temperature Growth Fan et
batatas) oleracea (SoBADH) enzymes stress chamber al. 2012

Increase
RWC, leaf
and root
growth, Greenhous Amara et
Maize Rab28 LEA lower MDA Water stress e al. 2013

Higher
WUE,
photosyntheti
c rates, SOD
and APX
activity.
Cu/Zn sod (cytsod) Reduced
Nicotiana from Spinacia oleracea and lipid
tabacum cv. cytosolic apx1 (cytapx) peroxidation, Greenhous Faize et
Xanthi) from Pisum sativum H2O2 Water stress e al. 2011

PtrABF, a bZIP Decreased Growth Huang et

Tobacco transcription factor ROS Drought chamber al. 2010
 Growth
Plant/crop Possible conditions
species Gene role Tolerance to Reference

Increased
activities of
SOD, CAT
and CAT

Overexpressi
on of stress
responsive
genes

High RWC,
less
membrane
Arabidopsis leakage and Drought and Growth Wang et
thaliana TaPLDα chlorosis osmotic stress chamber al. 2014

Increased
SOD and
APX
activities and
Medicago sativa (alfalfa) proline Mannitol,
helicase 1(MH1), homolog content; NaCl, methyl
Arabidopsis of the pea DNA helicases osmotic viologen and Growth Luo et
thaliana 45 (PDH45) adjustment ABA chamber al. 2009

Overexpressi
on of
MtSAP1 Temperature
enhanced stress, drastic
stress osmotic and Controlled Charrier et
Tobacco MtSAP1 tolerance salinity stress conditions al. 2013

S-adenosylmethionine Osmotic stress,

decarboxylase oxidative
stress,
Gene (SAMDC) Increased temperature
from Dianthus polyamine stress, acid Room Wi et
Tobacco caryophyllus biosynthesis stress conditions al. 2006

Greenhous
Heavy metal e
Arabidopsis phytochelatin detoxificatio Cadmium hydroponic Pomponi e
Tobacco synthase gene (AtPCS1) n stress s t al. 2006

High
chlorophyll
and
tryptophan

Synthase β
enzyme
activity, low
Arabidopsis Tryptophansynthase beta1 lipid Cadmium Controlled Sanjaya et
and tomato (AtTSB1) peroxidation stress conditions al. 2008
 Growth
Plant/crop Possible conditions
species Gene role Tolerance to Reference

Reduced
lipid
peroxidation
and
electrolyte
leakage,
Maintained
Fescue 2-Cys peroxiredoxins (2- chlorophyll Methyleviolog Growth Kim et
plants Cys Prx) fluorescence en, heat stress chamber al. 2010

Cytosolic copper/zinc Methylviologe

superoxide dismutase Higher n, oxidative
(CuZnSOD) from Avicennia activity of stress, salinity Growth Prashanth
Indica rice marina SOD stress chamber et al. 2008

Antioxidant Methyl
gene viologen
regulation (MV),
and high oxidative
Solanum antioxidant stress, high
tuberosum Nucleoside diphosphate enzyme temperature Growth Tang et
L. kinase 2 (AtNDPK2) activity and salt stress chamber al. 2008

Higher
activities
SOD, CAT, Methyl
APX and viologen (MV)
GR. and high
Increased temperature
Wheat OXO (oxalate photosyntheti oxidative Greenhous Wan et
Tobacco oxidase) gene c efficiency. stress e al. 2009

Increased
proline and
Arabidopsis decreased Glasshouse Zhao et
thaliana OsSPX1 MDA Cold stress al. 2009

Increased
expression of
stress
Arabidopsis associated High Controlled Yokotani e
thaliana OsHsfA2e TF from rice genes temperature conditions t al. 2008

Modulates
salicylic acid
mediated
system
acquired
resistance;
Cross-talk
NPR1 (non-expressor of with
pathogenesis related genes jasmonate Oxidative Culture Srinivasan
Tobacco 1, AtNPR1 pathway stress room et al. 2009
 Growth
Plant/crop Possible conditions
species Gene role Tolerance to Reference

High
activities of
SOD and
CAT, and
increased
Arabidopsis tocopherol Oxidative Growth Xi et
thaliana MSD1, CAT1, HPT1 content stress chamber al. 2010

High glycine
betaine
synthesis,
maintained
photosynthes
is through
protection to
PSII; higher
codA gene activity of
Arabidopsis from Arthrobacter APX and Controlled Alia et
thaliana globiformis CAT Light stress conditions al. 1999

Drought stress

Adaptation to water stress conditions is one of the major challenges for plant scientists and biotechnologists in
the current scenario of rapid climate change. Scientists are increasing their efforts to elucidate various climate
triggered metabolic processes at cellular and gene levels (Chaves et al. 2003). Research trials tailoring the plant
genome for water stress tolerance and enhanced yield carried out all over the globe have increased with the
premier goal of more crop per drop (Medici et al. 2014). There is a growing trend to improve the water use
efficiency (WUE) of crops to enable the more efficient use of available water (Al-Karaki 2000).

The difference between transgenic and conventional approaches for achieving improved water stress tolerance
is considerable. One viable transgenic approach is the engineering of genes of important metabolic and
defensive pathways, e.g. osmoprotectant synthesizing pathways and antioxidant defence systems (Ashraf
2009; Wang et al. 2016). Several stress inducible genes have been identified through microarrays, but as yet
their function within the molecular mechanisms for crop stress response and tolerance still needs to be
deciphered. For example, the production of the phytohormone abscisic acid (ABA) causes stomatal closure and
induces the expression of stress responsive genes (Tuteja 2007). However, how they function is not known.
In Arabidopsis thaliana wild type and abi11 mutant seedlings, Hoth et al. (2002) identified ∼1354 genes that
were up- or down-regulated following ABA treatment, with most of them coding for signal transduction.

Pinheiro and Chaves (2011) state that during lowered stomatal conductance, in combination with sustained
irradiance, relatively more CO2 is available within intercellular sites, because during the Calvin cycle the
consumption of light is slowed down while production rates of reducing power are increased under such
conditions. These changes slow down the photosynthetic rate through photoinhibition, which may serve as a
defensive mechanism for plants following the C3 pathway through thermo-regulated energy dissipation and
light harvesting complexes (Ruban et al. 2012). Plants with upregulated photosynthetic pathways exhibit a high
rate of photosynthesis (Gu et al. 2013).
There are three major pathways of CO2 assimilation and fixation: C3, C4 and crassulacean acid metabolism
(CAM) pathways. C4 plants can minimize photorespiration by separating initial CO2 fixation and the Calvin
cycle in different cell types, and CAM plants can fix carbon at night, and are more tolerant to drought stress due
to their more efficient carbon fixation and specialized anatomical features (Ashraf and Harris 2013).
Recently, Ashraf and Harris (2013) comprehensively described the progress made during the last two decades in
producing transgenic lines of different C3 crops with enhanced photosynthetic performance either due to
introgression of genes encoding C4 enzymes into C3 plants or overexpression of C3 enzymes or transcription
factors (TF). Usually, C4 and CAM plants are best adapted to arid environments, because they have higher
photosynthetic efficiency as well as WUE as compared with C3 plants (Fischer and Turner 1978).
Likewise, Kim et al. (2014) reported that overexpression of Capsicum annuum drought stress responsive 6
(CaDSR6) in Arabidopsis plants led to higher tolerance to drought as compared with wild type plants. Saad et
al. (2013) also showed that the stress-responsive NAC1 (SNAC1) gene controlled signalling of sucrose
phosphate synthase type 2C protein phosphatases, 1-phosphatidylinositol-3-phosphate-5-kinase as well as
regulatory components of ABA receptor in wheat plants under drought stress. Overall, a variety of genes
contributing to drought tolerance in plants have been explored and characterized in Arabidopsis. However, few
of these genes have been tested in other crops, and only under controlled or laboratory conditions instead of
natural field conditions.

Salinity stress

Salt is a premier environmental stress that affects plant growth and development adversely through induction of
ion toxicity, reduced water uptake, hormonal disturbance and oxidative stress (Ashraf and McNeilly
2004; Athar et al. 2008; Tuna et al. 2007; Siddiqi et al. 2007; Ashraf and Foolad 2013). As with other abiotic
stresses, several tolerance responses are triggered in the plants to avoid high salinity-induced deleterious effects.
One response used to avoid saline stress is compartmentation and the exclusion of deleterious ions (Na+ and Cl-)
from sensitive tissues like the mesophyll (where sodium toxicity is induced by competing for K+ binding sites)
and their diversion into the apoplast or vacuole (Sperling et al. 2014).

Maintenance of high potassium and retention of deleterious ions or solutes within a root or apoplastic regions
are the major tolerance strategies, and hence a high K/Na ratio is maintained through the efficient function of
transporters (Shabala and Cuin 2008). Pandolfi et al. (2012) suggested that short-term exposure and acclimation
of glycophytes to a lower salt concentration can help withstand prolonged exposure to a higher concentration.
The plant acclimates through a set of physiological mechanisms including controlled xylem ion loading and
efficient Na+ compartmentation (Pandolfi et al. 2012).

For example, plants overexpressing the ion transporter genes show high salinity tolerance as in halophytes
(Flowers and Colmer 2008). Overexpression of Na+/H+ antiporter (AlNHX) from the
halophyte, Aeluropuslittoralis, in tobacco enhanced the salinity tolerance of tobacco by maintaining a suitable
level of Na+ and K+/Na+ ratio (Zhang et al. 2008). Overexpression of vacuolar ATPase subunit c1 (SaVHAc1)
gene from Spartinaalterniflora enhanced rates of photosynthesis and cell wall expansion, improved the
K+/Na+ ratio and led to a higher relative water content (RWC) in rice (Baisakh et al. 2012). Overexpression of
the wheat transporter geneTaNHX2 enhanced the salt tolerance of C.annuum by improving the K+/Na+ ratio
(Bulle et al. 2016). These studies indicate that genes contributing towards tolerance to high salinity in
halophytic grasses could be better engineered to achieve enhanced tolerance of sensitive cash crops.

Cold stress
Plant survival under low temperature depends on the physiological and molecular responses triggered by the
plant on exposure to low temperature (Sergeant et al. 2014; John et al. 2016). These can be confounded by
photoperiod response as cold is often associated with extreme latitudes. Water availability, growth and
development, energy metabolism and photoperiod are amongst the important factors that determine the
deacclimation and reacclimation of plants to cold stress (Thomashow 1999). Compatible solutes, membrane
proteins, antioxidants and expression of cold responsive genes have a significant role in cold tolerance
(Kalberer et al. 2006). Cold stress alters the expression of putative cold responsive genes coding for an array of
important proteins, for example enzymes involved in respiration and the metabolism of carbohydrates,
phenylpropanoids, lipids, antioxidants and those coding for chaperones and antifreeze proteins. Several other
genes involved in regulating intriguing tolerance mechanisms are involved in freezing-induced dehydration
(John et al. 2016). Altered gene expression and subsequent production of specific proteins during cold tolerance
play an important role in the distribution and survival of plants as well as yield (Sanghera et al. 2011).

Interspecific and intergeneric hybridization-dependent conventional breeding has not been fully successful in
developing cold tolerant crop cultivars. However, biotechnological and molecular approaches, including
genome sequencing and alteration of the genome for transgenic development, provide an opportunity to
understand and access the complex cold tolerance mechanisms operating at the transcriptional as well as the
translational levels (John et al. 2016). Altered gene expression has increased the level of several metabolites
that have a protective role under cold stress. Amongst the low-temperature-induced genes that have been
isolated to date, the expression of most of them has been reported to be regulated by cold binding
factor/dehydration responsive element binding transcription factors (CBF/DREB TFs; Sanghera et al. 2011).

Cold stress induces the expression of several proteins, e.g. proteins of methionine pathways and membrane
stabilizing proteins. The methionine metabolism pathway has an important role in the biosynthesis of essential
metabolites including polyols and polyamines, which play a role in cold acclimation. Although their actual role
in cold tolerance is not fully known, their accumulation in plants has been reported in response to cold stress
(John et al. 2016). Overexpression of methionine sulphoxide reductase A (MsrA), an important enzyme in the
regulation of methionine metabolism, increases resistance to oxidative damage at low temperatures. For
example, Arabidopsis plants with a mutation in methionine sulphoxide reductase B3 (MsrB3) were more
sensitive to low temperature than their respective wild-type and MsrB3 transgenic plants. MsrB3 plays a
ubiquitous role in eliminating reactive oxygen species (ROS) and methionine sulphoxide (MetO) accumulating
in the endoplasmic reticulum during cold stress (Kwon et al. 2007).

Cold stress is believed to damage photosynthetic machinery, including photosystems and photosynthetic
pigments, by altering the expression of photosynthetic genes (Oquist and Huner 2003). Han et
al. (2010) isolated the violaxanthin de-epoxidase gene (LeVDE), a gene regulated by temperature rhythms,
from Lycopersicon esculentum. Overexpression of this gene increased non-photochemical quenching, Fv/Fmand
quantum yield, oxidizable P700, and the activity of the xanthophyll cycle and alleviated PSI and PSII
photoinhibition under temperature stress. Recently, similar observations have been reported in transgenic
tobacco by introgression of LeLUT1 (carotenoid epsilon-ring hydroxylase gene from tomato), which reduced
ROS production and hence maintained membrane integrity (Miller et al. 2010). Transgenic plants that
overexpress these stress responsive genes benefit from their key roles in alleviating photoinhibition and photo-
oxidation, which in turn decrease the sensitivity of the plant’s photosynthetic apparatus to cold (Zhou et
al. 2013). The gene AtICE1, which is responsible for stimulating the expression of CBF/DREB
in Arabidopsis under cold stress, was introgressed in rice thereby enhancing tolerance to cold stress (Dian-jun et
al. 2008). Transgenic A. thaliana overexpressing CcCDR, a potent cold and drought regulatory protein gene,
conferred enhanced tolerance to cold, salinity and low temperature by improving various physio-biochemical
attributes, such as increased antioxidant activity and accumulation of osmolytes (Tamirisa et al. 2014).

By using suppression subtractive hybridization (SSH), Guo et al. (2013) identified the genes up- or down-
regulated in ABA-pre-treated pepper seedlings incubated at 6 °C for two days. It has been observed that
50.68 % of unigenes showed similarities to genes with known functions while 49.32 % showed fewer
similarities or unknown functions. The expression level of ten genes was at least 2-fold higher in the ABA-pre-
treated seedlings than in non-treated (control) plants under chilling stress, which suggested that ABA negatively
or positively regulates the genes in pepper plants under cold stress.

Cold induces accumulation of oligosaccharides and galactosyl synthase activity. Galactinol synthase mediates
the synthesis of galactinol, which serves as a donor of galactosyl during the synthesis of oligosaccharides of the
raffinose family (Zhou et al. 2013). Photinia serrulata overexpressing the galactinol synthase gene (AmGSl)
from a cold tolerant tree, Ammopiptanthus mongolicus, exhibited enhanced cold tolerance (Song et al. 2013).
Galactinol and raffinose are active scavengers of hydroxyl radicals. The role of galactinol synthase in drought
and salinity is well documented (Nishizawa et al. 2008), yet very few reports are available pertaining to its
possible role in cold tolerance. One of the few studies is by Zhou et al. (2013), who introgressed and
overexpressed MfGolS1 in tobacco, which resulted in increased cold tolerance through improved formation of
galactinol, stachyose and raffinose. Elucidation of mechanisms of tolerance to cold stress in cold tolerant
grasses at biochemical and molecular levels can be very helpful in improving our understanding of putative cold
responsive genes and their subsequent introgression for enhancing the tolerance of economic crops to cold
stress.

High temperature

High temperature reduces a number of growth and physiological processes including seed germination,
subsequent development, reproductive processes and photosynthesis, which have adverse effects on the overall
yield of a crop (Gillooly et al. 2001). For example, impaired reproductive growth by high temperature results in
inhibition of pollen grain swelling leading to anther indehiscence and perturbed pollen dispersal, which
ultimately adversely affects seed production (Das et al. 2014). Understanding the high temperature tolerance
mechanisms at physiological, biochemical and molecular levels in the light of global warming is essential for
further successful efforts in developing high temperature tolerant crop cultivars. Genetic and molecular
mechanisms for circumventing high-temperature-induced deleterious changes play an essential role in plant
survival under such conditions. Sensing of high temperature stress and developing tolerance is highly complex,
involving networks operating in different cellular compartments. Different putative sensors, e.g. histone sensors
located in the nucleus, protein sensors in the endoplasmic reticulum and cytoplasm and a plasma membrane
channel initiating inward calcium flux, mediate activation of heat stress responsive genes involved in
thermotolerance (Mittler et al. 2012).

Genome modification for thermotolerance in crop plants is of immense concern because of its direct influence
on the mechanisms involved in the reprogramming of the proteome, transcriptome, metabolome and lipidome.
Molecular chaperones, e.g. heat shock proteins (HSPs), have a key role in mitigating the deleterious effects
induced by heat stress (Xu et al. 2013). Reduction in the levels of HSPs causes developmental abnormalities
(Kotak et al. 2007). Five major highly conserved HSP families have been recognized that differ in their
respective molecular masses. Under normal metabolism, the HSPs are involved in several processes including
protein folding, assembly, translocation as well as degradation, signalling and cell cycle control (Young et
al. 2001). However, under stress conditions, the HSPs interact with other co-chaperones to bring about
refolding of proteins in order to re-establish protein conformation and cellular homoeostasis, thereby protecting
plant cellular functioning.

The essence behind the successful acclimation of plants to high temperature depends on the massive
accumulation of transcripts coding for HSPs and ROS detoxifying enzymes like ascorbate peroxidase (APX).
For example, Zea mays and Arabidopsis mutants for HSP100 showed retarded growth and adaptation to high
temperature (Hong and Vierling 2000; Nieto-Sotelo et al. 2002). Similarly, silencing of chloroplast
HSP100/ClpB protein gene expression in tomato reduced heat stress tolerance (Yang et al. 2006). Reports
pertaining to the sensitivity of crop plants to high temperature as a result of mutation/silencing of HSPs (Bita
and Gerats 2013) help our understanding of how essential these HSPs are for plants in triggering expression of
heat responsive genes. Thermotolerance in plants can be better achieved by manipulating the detoxification
pathways of ROS, e.g. Shi et al. (2001) cloned the peroxisomal APX-encoding gene, HvAPX1, and its
introgression within Arabidopsis enhanced heat stress tolerance by increasing APX activity, thereby exhibiting
low lipid peroxidation. The phospholipid hydroperoxide glutathione peroxidase encoding gene
from L.esculentum, LePHGPx, protects yeast cells from lethal effects. However, its introgression and
overexpression protected tomato from lethal temperature and salinity levels by reducing apoptosis levels
(Chen et al. 2004).

Until recently, the genes that have been identified or introgressed in different genetically modified (GM) plants
mainly relate to the regulation of the oxidative defence system. However, multiple other plant metabolic
systems and activities are affected by changes in temperature, and have the potential to be tackled in transgenic
crops. Moreover, a rise in ambient temperature as already visible over the last 10 years is a continuing challenge
for crop productivity, creating the need to develop stress tolerant plants with heat tolerance.

Fungicide and herbicide stress resistance

A variety of pesticides, herbicides and fungicides are frequently used to control crop loss due to pathogen attack
(Yoon et al. 2013). Excessive use of these chemicals has a considerable negative impact on crop growth and
yield (Chen 2006). Use of pesticides, fungicides and herbicides has become an integral part of modern
agriculture (Aktar et al. 2009; Mattah et al. 2015). Residues of sprayed pesticides and fungicides residing on the
fruits or seeds have a direct impact on human health. Scientists are continuously endeavouring to develop
alternative chemicals to replace the commonly used chemicals so that threats to plants, animals as well as the
environment can be minimized (Aktar et al. 2009; Mahmood et al. 2014; Mattah et al. 2015). Crops vary in
their degree of sensitivity towards a particular pesticide, herbicide or fungicide, and extreme conditions in terms
of heavy use of these chemicals can lead to crop death because of their direct interference with the metabolic
processes of the plant (Mahmood et al. 2014; http://www.irac-online.org).

Synthetic pesticides, herbicides and fungicides are effective, but excessive use can generate environmental
pollution, development of resistance and non-degradable residues. For example, chemical fungicides used for
the treatment of plant diseases have diverse mechanisms of action involving the mitochondrial respiratory
chain, inhibition of sterol biosynthesis as well as microtubule assembly, resulting in some limitations related to
their toxicity and resistance in plants. A number of stress response pathways such as the cell wall integrity and
high-osmolarity glycerol pathway are triggered by stimuli such as changes in osmolarity, cell wall instability
and production of ROS (Hayes et al. 2014). There is, however, an emerging fear globally about the mis/over use
of synthetic chemicals particularly on food crops because of their potential effects on the environment and
human health (Al-Samarrai et al. 2012; Yoon et al. 2013). So, the introduction of bio
pesticides/fungicides/herbicides is necessary (Yoon et al. 2013).
Elucidating and understanding the molecular mechanisms of chemicals used to control pests, and the
development of pest-resistant crops and other alternative ecologically sound methods, are important so that
chemical-dependent agriculture can be replaced with safer productive alternatives to agrochemicals. The
development of crops that are resistant to pests and fungi and improving plant tolerance to a particular chemical
pesticide or fungicide are being discussed in this regard (Mahmood et al. 2014). In addition to causing crop
damage, many insects and pests have developed resistance to these chemicals, e.g. in pests, resistance mediated
through enhanced activities of complex multigene enzymes like glutathione-S-transferase, esterases and
cytochrome P450s is well reported in the literature (Bass and Field 2011). Engineering of crop plants by
introducing genes involved in these important defence mechanisms from animals, bacteria and pests could be a
useful part of xenobiotic strategies (Abhilash et al. 2009).

Qianet al. (2014) demonstrated that introgression of α-momorcharin (α-MC), a ribosome-inactivating protein
(RIP) isolated from Momordicacharantia seeds, enhanced the tolerance of rice to Magnaporthe grisea induced
blast. In another study, Zhu et al. (2013) showed that pre-treatment of tobacco plants with α-MC (0.5 mg mL-1)
increased resistance to Bipolaris maydis, Fusarium graminearum, Aspergillus oryzae, Aspergillus
niger and Sclerotinia sclerotiorum, thereby favouring the antifungal and antiviral activity of α-MC.

Use of fungal cell wall degrading enzymes to enhance fungal resistance has been widely practiced, e.g.
introgression of rice chitinase cDNA into cucumber enhanced its resistance to Botrytis cinerea by suppressing
the growth of fungi (Kishimoto et al. 2002). In Brassica juncea, Rhizoctonia solani infection induces the
expression of BjCHI1, a chitinase enzyme. Transgenic Solanum tuberosum L. overexpressing
either BjCHI1 or BjCHI1 and HbGLU (Hevea brasiliensis β-1,3-glucanase) exhibited significant inhibition of
fungal growth (Chye et al. 2005). Chye et al. (2005) suggested that co-expression of proteins can effectively
degrade fungal cell wall producing elicitors by initiating epidermal cell collapse and thus restricting further
hyphal penetration. They also noted that there are small-sized proteins, e.g. plant defensins, that play an active
role in plant defence against a variety of diseases. Of the various plant defensins, NaD1 from Nicotiana alata is
a well-characterized antifungal protein and its overexpression increased the resistance of cotton to Fusarium
oxysporum and Verticillium dahlia, resulting in an enhanced survival rate and yield (Gaspar et al. 2014).

Amongst the most commonly used herbicides are glyphosate and bromoxynil (3,5-dibromo-4-
hydroxybenzonitrile). Glyphosate restricts growth by reducing aromatic amino acid biosynthesis while
bromoxynil prevents photosynthesis by affecting PSII activity (Stalker et al. 1988). The development of
glyphosate resistant crops would help plants resist the glyphosate and thus reduce yield losses. Research efforts
have been successful in identifying and characterizing glyphosate resistance genes and to date various 5-
enolpyruvylshikimate-3-phosphate (EPSP) resistant genes have been identified. Shah et al. (1986) developed
glyphosate resistant petunia using the cauliflower mosaic virus 35S promoter, resulting in 20-fold amplification
of the ESPS synthase gene. Similarly, Tian et al. (2013) developed a transgenic rice cultivar through the
incorporation of MdEPSPS, a gene conferring glyphosate resistance, in Malus domestica, which they identified
after five rounds of DNA shuffling and screening; amongst the eight mutations in the amino acid sequence of
this gene only two were identified as site directed and important for glyphosate resistance. Stalker et
al. (1988) isolated and cloned the bxn gene from the soil bacterium Klebsiella ozaenae. This gene codes for
nitrilase and mediates conversion of bromoxynil to its primary metabolite form (3,5-dibromo-4-hydroxybenzoic
acid), and when introduced into tobacco, enhanced bromoxynil resistance. Recently, Iwakami et
al. (2014) isolated two cytochrome P450 genes of CYP81A, i.e. CYP81A12 and CYP81A21, from a noxious
weed Echinochloa phyllopogon that is resistant to the herbicides bensulphuron-methyl and penoxsulam, and
developed transgenic Arabidopsis expressing either of these genes that showed enhanced herbicide resistance
through the O-demethylation of herbicides. These results indicate that the characterization and understanding of
molecular mechanisms and the development of resistant crops can help withstand the devastating effects of
pathogens and pests and provide an important alternative to chemical dependent agriculture.

Nutrient stress

Changes in environmental conditions have a direct influence on nutrient uptake and assimilation in plants
(Lopez-Arredondo et al. 2013). Amongst the various nutrient deficiencies, commonly reported deficiencies
include those of iron, zinc and calcium, while other mineral deficiency disorders are believed to be rare (Taiz
and Zeiger 2010). Most chemical fertilizers, which are enriched with desired nutrients, may improve biomass,
but their role in improving the nutritional value for consumption is minimized either through leaching, surface
run-off, volatilization or microbial consumption. Increasing nutrient use efficiency (NUE) amongst crops
through efficient means is crucial to prevent mineral losses. Extensive contributions from conventional breeding
with regard to improving NUE in crops have been made during the past few decades (Ashraf et al. 2011), but
such achievements through advanced molecular techniques have not been numerous. Efficient working of
transporters and enzymes involved in nutrient assimilation is essential for achieving enhanced nutrient uptake,
and this has a direct influence on the crop yield status. For example, overexpression of glutamine synthetase
gene (GS1) in wheat plants led to increased nitrogen accumulation in shoot and grains (Habash et al. 2001),
whereas overexpression of GS1-3 led to enhanced (30 %) kernel number in maize (Martin et al. 2006). At the
molecular level, there are very few reports in the literature pertaining to the mechanisms and associated genes
involved in nutrient transport and assimilation. However, it is widely accepted that TFs and associated kinases
are involved in these processes (Canales et al. 2014).

Efficient working of the ammonium transporter, OsAMT1, helps transgenic rice plants to achieve and maintain
sufficient levels of ammonium, the major source of nitrogen for rice. This suggests the role of this transporter in
enhancing NUE, growth and yield under optimal as well as suboptimal nitrogen conditions (Ranathunge et
al. 2014). NRT1.1 functions as a nitrate sensor and can enhance high to low affinity nitrate transporters in the
protein kinase CIPK23 dependent phosphorylation and dephosphorylation of intracellular threonine, thereby
changing NRT1.1’s ability to mediate efficient nitrate transport (Parker and Newstead 2014). Moreover, nodule
inception (NIN)-like protein (NLP) TFs are the master regulators of nitrate response, and upon binding with the
nitrate responsive cis-element activate nitrate-responsive transcription, which is further modulated by nitrate
signalling at the post-translational level. Suppression of NLP function results in impeded expression of several
nitrate-inducible genes (Konishi and Yanagisawa 2013). Castaings et al. (2009) reported that nlp7 mutants show
impaired nitrate signal transduction, and that its expression pattern and function in sensing nitrogen are closely
associated with each other. Kuo and Chiou (2011) suggested that micro-RNAs have a putative rolein regulating
the nutrient starvation genes at post-transcriptional levels.

Nutrient rich cultivars can be selectively developed from the existing germplasm or through genetic
manipulation. Tailoring of the genetic makeup of crops for improved nutrient levels is gaining interest as a
means to reduce malnutrition. Microarray and sequence based transcription profiling technology to study gene
expression changes in response to nutrient stress can yield meaningful results (Lee et al. 1999). Transient
changes in gene expression in nutrient starved plants are well documented. Bi et al. (2007) reported the
differential expression of genes under mild nitrogen stress that were acting as putative regulators of nitrogen
stress responses in Arabidopsis. An Arabidopsis mutant defective in developing proper nitrogen stress responses
showed altered transcriptional responses to nitrogen limitation because of the absence of a key regulatory
gene, NLA (Peng et al. 2007). In rice and maize, a systems approach is being adopted, mainly aiming at
profiling genes at transcriptional levels in response to individual or combined nutrient stress.
Genetic engineering approaches for enhancing NUE range from increasing the solubility of mineral nutrients
and remobilization within the plant to transport and accumulation within storage organs. Recently, Zhou et
al. (2014) developed transgenic soybean in which constitutive overexpression of GmEXPB2 (β-expansin)
increased leaf expansion and improved phosphate efficiency. Overexpression of expansin genes,
e.g. HvEXPB1 in barley (Kwasniewski and Szarejko 2006) and OsEXPA17 in rice (Yu et al. 2011), improved
phosphate uptake through induction of better root hair growth even under phosphate-deficient conditions.
Remobilization of nutrients within a plant is critical for their survival, and manipulation of transporter genes for
efficient remobilization of nutrients is an important strategy. For example, overexpression of
the GmPT1 transporter gene enhanced phosphate remobilization, yield and related attributes like phosphorus
use efficiency and quantum yield in soybean (Song et al. 2014). To enhance NUE through genetic
manipulations, a thorough understanding about the factors governing efficient mineral uptake and
remobilization within the plant is necessary. The use of methods such as transcription profiling, analysis of
mutants defective in their response to mineral deficiency and investigation of plants showing normal growth
under nutrient stress are pre-requisites.

The biofortification of important seed crops for optimal accumulation of micronutrients has been the subject of
intensive research for the last few decades (Akram et al. 2009; Akram and Ashraf 2011). For example, during a
study on sunflowers, Akram et al. (2009) found that a foliage spray of potassium sulphate significantly
improved shoot and leaf K+ contents, while no change was observed in leaf and root Mg2+, Ca2+ or N content
under non-stress and saline conditions. Similarly, soil and foliar application of Zn and Fe at the rate of 4.0 and
2.0 mg kg−1, respectively, had a significant effect on plant available nutrients and nutrient concentration in wheat
grain and straw (Naz et al. 2015).

The transgenic approach has been preferred over the conventional one for the biofortification of important
crops, because it is a convenient and time and labour saving approach for overcoming nutrient deficiency
problems (Zhu et al. 2007). Rice plants overexpressing the iron storage protein ferritin were reported to have
increased seed iron content (Lucca et al. 2001). Adoption of GM crops is a time-consuming process as a
number of policies and laws need to be adopted before the commercial release of such cultivars or varieties can
occur. A cultivar or variety that is transgenic for one of the nutrients could involve up- or down-regulation of a
cascade of genes involved therein, which could cause human health problems. A number of government
agencies are working mostly in developed countries to review these issues. The transgenic approach is
considered as an extremely useful tool in basic plant science research, but understanding of the gene networks
and molecular physiology of plant responses to deficiency or excess of nutrients is a pre-requisite.

Heavy metals

Heavy metal pollution and contamination is a major determinant of the global distribution of plant species as
well as agricultural productivity, particularly in countries where the economy relies heavily on industry
(Khan et al. 2014, 2015). Many plants show symptoms of heavy metal toxicity when a metal concentration
surpasses a specific threshold level. In general, necrosis and stunted shoot growth are the first visible symptoms
of heavy metal exposure (Liu et al. 2014).

Heavy metals interfere with the growth and physiology of plants in several ways. Heavy metals like lead (Pb)
and cadmium (Cd) reduce the number of mitochondrial cristae leading to impaired oxidative phosphorylation.
Upon binding to nucleic acids, heavy metals promote the aggregation and condensation of chromatin as well as
impaired replication and transcription (Youssef and Azooz 2013). The affinity of Pb and Cd to sulphohydryl
groups of enzymes leads to their inactivation. In addition, heavy metal stress leads to the enhanced production
and accumulation of ROS and thus oxidative stress, which usually upsets the normal metabolism (Groppa et
al. 2012). Phytoremediation is being extensively promoted as a means to remediate environmental contaminants
such as heavy metals (Glick 2003). Moreover, rhizoremediation, which involves plants as well as their
rhizospheric microbes, either naturally occurring or introduced, also helps to degrade or lower the levels of
contaminants and promote normal plant growth (Gerhardt et al. 2009; Qadir et al. 2014).

Most heavy metal salts are hydrophilic and easily soluble in wastewater, so they are difficult to separate by
physical separation methods. At low levels of heavy metals, physico-chemical methods can be ineffective or
costly. Alternative methods include biosorption or bioaccumulation for the removal of heavy metals. The use of
microorganisms and plants for remediation purposes is thus an effective strategy to overcome or minimize
heavy metal pollution (Dixit et al. 2015). Despite the potential of these strategies to contribute to reclamation of
contaminated soils, detailed information on the underlying mechanisms is not available in the literature and
efforts to transform these strategies from successful laboratory or greenhouse trials to field natural sites are
highly challenging. Two factors that make these strategies not very effective are (i) the multiple stress factors
available in the field are not employed under laboratory and greenhouse studies and (ii) there is a lack of
efficient and adequate methodologies and techniques that can be employed to ascertain whether or not the
concentrations of different contaminants are decreasing (Gerhardt et al. 2009).

Phytoremediation is an ecofriendly, cost-effective and non-invasive strategy that is now being used extensively
to clean up heavy metals from the environment or render them harmless. Various mechanisms, e.g. chelation,
trafficking, compartmentation, etc. are employed for detoxification of toxic heavy metals and metalloids
(Guo et al. 2008; Khan et al. 2014). Moreover, production of higher levels of high affinity ligands like
phytochelatins (PCs) and metallothioneins (MTs), and cysteine rich thiol-reactive peptides, mediates
detoxification by binding with toxic metals and metalloids (Gasic and Korban 2007; Guo et al. 2008; Pal and
Rai 2010). Formation of PSc-metal or MT-metal complexes and their subsequent sequestration into the vacuole
are essential for heavy metal tolerance. The enzyme phytochelatin synthase (PCS) mediates the synthesis of PCs
using GSH or γ-glutamyl cysteine as a substrate (Cobbett and Goldsbrough 2002; Wunschmann et al. 2007).
Effective research has been performed regarding genes encoding PCs, with several genes being cloned to date,
e.g. OsPCS1, TaPCS1, AtPCS1 and CePCS1 from rice, wheat and Arabidopsis (Ha et al. 1999; Vatamaniuk et
al. 1999; Gasic and Korban 2007), and BjPCS1 and AsPCS1 from the metal tolerant plants B.juncea and Allium
sativum, respectively (Heiss et al. 2003; Zhang et al. 2005).

Guo et al. (2008) demonstrated that simultaneous overexpression of AsPCS1 and GSH1 (from A. sativum and S.
cerevisiae) enhanced the tolerance of A. thaliana to heavy metals and metalloids. They further reported that
single-gene transgenic lines showed higher tolerance and accumulated more Cd and As than wild type plants,
while dual gene transgenic lines exhibited even more tolerance and accumulated (2-fold) more Cd and As
compared with single gene transformants. The elevated production of GSH and PCs resulted in accumulation
and tolerance to Cd and As (Li et al. 2004). Plants used in phytoextraction should have an inherent capacity to
accumulate and tolerate high contents of metalloids in aboveground biomass (Pajevic et al. 2016). In addition,
they should have fast growing adaptability and biomass, and ideally be repulsive towards herbivores so that
transmission of toxic metals to various components of the food chain can be avoided. Besides these basic
characteristics, hyper-accumulator plants have should have a profusely branched root system, wide geographical
distribution and be easy to cultivate as well as harvest.

Genetic manipulations for developing specific morphological characteristics supported by unique anatomical
efficiencies for the accumulation of metalloids are being intensively studied (Kotrba et al. 2009). Tolerant plant
species show increased uptake and metal binding capacity at intracellular sites, and efficient sequestration into
the vacuole for deposition and detoxification that is controlled in a highly regulated manner by a set of gene
products (Peng et al. 2014). For instance, yeast protein YCF1 mediates the sequestration of Pb and Cd into the
vacuole. Transgenic A. thaliana plants overexpressing YCF1 were found to be tolerant to Pb and Cd (Song et
al. 2003). In addition, enhanced translocation of metals to aboveground plant parts via the apoplast or symplast,
and their subsequent extrusion to metabolically less active tissues like trichomes, was also found to contribute
to enhanced metal tolerance as well as remediation (Clemens et al. 2002).

The role of PCs and MTs in heavy metal detoxification has been well documented. Gonzalez-Mendoza et
al. (2007) reported that increased expression of AvPCS and AvMt2 in Avicennia germinans under Cd and Cu
stress indicates that the PCs and MTs are involved in a coordinated detoxification response mechanism
employed for removal of non-essential metals. Scientists are continuously striding towards enhancing the
detoxifying potential of crop plants through manipulating the genes coding for PCs and MTs, e.g. Nicotiana
tabacum (Sylwia et al. 2010) and B.juncea (Gasic and Korban 2007) overexpressing AtPCS1 (PCS) showed
improved tolerance to Cd by maintaining higher levels of PCs in the cytosol and vacuole. Arabidopsis
thaliana overexpressing PCs1 showed higher resistance to Cd and arsenic (As) and accumulated more biomass
(Verbruggen et al. 2009). Moreover, concentrations of Cd and As decreased while that of thiol peptide
increased in shoot biomass (Li et al. 2004). Besides increasing tolerance, transgenic plants accumulated less
metal content in the aboveground biomass (Gasic and Korban 2007).

Overexpression of AtPCS1 in N.tabacum harbouring the Agrobacterium rhizogenes rolB oncogene enhanced its
tolerance to Cd; tolerance was further enhanced when the culture was supplemented with GSH (Pomponi et
al. 2006). Transgenic plants showing increased expression of O-acetylserine (thiol) lyase (OASTL), a key
enzyme that catalyzes cysteine formation (from sulphide and O-acetylserine) and a key limiting step in the
production of GSH, showed high tolerance to heavy metals (Ning et al. 2010). Nicotiana tabacum plants
expressing the wheat (Triticum aestivum) OASTL gene, cys1, and exposed to SO2 maintained high levels of Cys
and GSH as well as higher rates of accumulation of Cu/Zn SOD transcripts.

The advancement in transgenic approaches (individual or combination) could be a successful means to promote
phytoextraction of toxic metalloids (Se and As) and metals, particularly Cu, Pb and Cd in the aboveground plant
organs and to promote tissue up-take involving metal transporters, high production of enzymes and the
production of metal-detoxifying chelators including PCs and MTs. Advances in the mechanistic basis of a
transgenic approach would help provide a better understanding of the genetic basis of resistance or tolerance
and hyperaccumulation of metals and metalloids, means of translocation and other environmental factors
influencing phytoremediation, because these all hinder its implementation.

Conclusions and Future Prospects

Several intrinsic protective mechanisms are triggered in plants when they are exposed to various environmental
stresses (Sadiq et al. 2017). Deciphering the regulatory mechanisms involved in initiating these tolerance
pathways has remained under intensive research for decades. Testing of the validity of these assumptions has
provided new insights towards the better understanding and elucidation of stress-induced changes in plants.
Plant physiologists and biochemists have remained the key players in elucidating the basics of these
mechanisms, which are now being extensively explored at the genetic and molecular levels using various
molecular, genomic and biotechnological approaches.

The identification and selection of key stress responsive genes and their subsequent introgression for developing
resistant crop cultivars through conventional breeding protocols are time-consuming. Plant biotechnology,
 despite being costly in comparison with conventional breeding, is very efficient. Several stress responsive genes
have been identified and successfully introduced into other crops to create transgenic crops with enhanced stress
tolerance. However, it is important to point out here that during the development of a transgenic crop variety,
care is taken to introduce genes that result in enhanced tolerance to multiple stresses, specifically at the whole
plant level. This requires the development of sets of markers designed to enhance stress tolerance.

The advantages of biotechnology in the development of transgenic plants for efficient crop varieties are
undoubtedly enormous, but their commercialization after proper field testing is still an unavoidable reality. In
addition, risk assessment of transgenic plants/crops is one of the preliminary steps required before the release or
use of transgenic plants. The set standard all over the world explains the risk and official registration of plants
and plant products has to be under taken. In addition, the risks to the environment from the transgenic crop
plants must be examined with many field tests prior to commercialization, with institutional assessments,
decisions on plants or varieties and adequate management practices in place to tackle inherent risks. For
decision making, risk assessment must be followed in a scientific, sound and transparent manner. There are
many operational governmental regulations in many countries for the safety assessment of GM crops.
Furthermore, there are some international agreements that regulate the cultivation and commercialization of
transgenic plants and their derivatives. All over the world, the major objective of these regulations and risk
assessment strategies is focused on protecting the environment and human/animal health. The adoption of
transgenic plants entirely depends on the assessments of the risks or benefits, regulatory approval, cost and time
period, commercialization as well as the economic status, requirements and values of different countries.

https://academic.oup.com/aobpla/article/9/4/plx025/3894178

 B18.

Are endogenous plant adaption mechanisms enough to keep up with the pace of man‐made
environmental change?

Adapt, move or die: How biodiversity reacted to past climate change

Date:
August 30, 2018
Source:
Faculty of Science - University of Copenhagen
Summary:
A new paper reviews current knowledge on climate change and biodiversity. In the past, plants and animals reacted
to environmental changes by adapting, migrating or going extinct. These findings point to radical changes in
biodiversity due to climate change in the future.

Share:
FULL STORY

A new paper reviews current knowledge on climate change and biodiversity. In the past, plants
and animals reacted to environmental changes by adapting, migrating or going extinct. These
findings point to radical changes in biodiversity due to climate change in the future. The paper is
published in the scientific journal Trends in Ecology and Evolution by an international group of
scientists led by the Center for Macroecology, Evolution and Climate, University of Copenhagen.
ADVERTISEMENT
 Nature is reacting to climate change. We see altered behaviour and movement among plants and animals; flowers change
flowering period and owls get darker body colour, due to warmer winters. So, how does the future for biodiversity look like?
Will plants and animals be able to adjust quickly enough to survive the changing temperatures, precipitation and seasons?
Lead-author of a new study Professor David Bravo-Nogues from Center for Macroecology, Evolution and Climate, University
of Copenhagen, explains,
"We compiled an enormous amount of studies of events, which we know influenced biodiversity during the past million
years. It turns out species have been able to survive new conditions in their habitat by changing either their behaviour or body
shape. However, the current magnitude and unseen speed of change in nature may push species beyond their ability to
adapt."
Too fast changes leave species small chances
Until now, scientists thought species' main reaction to climatic changes was to move. However, the new study shows that
local adaptation to new conditions seems to have played a key role in the way species survived. Species adapt when the
whole population change, e.g. when all owls get darker body colour. This happens slowly over a long period of time.
Coauthor Stephen Jackson, director of the US Geological Survey's Southwest Climate Adaptation Science Center, elaborates,
"From fossils and other biological "archives" we have access to a nearly limitless number of case studies throughout Earth's
history. This provide us with valuable knowledge of how climate changes of various rates, magnitudes, and types can affect
biodiversity."
Past extinctions help to protect future biodiversity
The new study might give us the answer to decode how biodiversity changes under climate change. This knowledge can
inform policy-makers in order to implement effective conservation schemes in the future. Some species, when failed to adapt
or move fast enough, like the orange-spotted filefish, have already gone extinct due to climate change. Co-author Francisco
Rodriguez-Sanchez from the Spanish Research Council (CSIC), says,
"We know animals and plants have prevented extinction by adapt or migrate in the past. However, the models we use today
to predict future climate change, foresee magnitudes and rates of change, which have been exceptionally rare in the last
million years. Thus, we need to expand our knowledge and improve our prediction models. Also, we must recognise the
limitations of the models, because they are used to inform politicians and decision-makers about effects of climate change
on biodiversity."

https://www.sciencedaily.com/releases/2018/08/180830143138.htm

 B19.

How can we improve our cultivated plants to make better use of finite resources?

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Improving crop productivity and resource use

efficiency to ensure food security and environmental
quality in China
Mingsheng Fan, Jianbo Shen, Lixing Yuan, Rongfeng Jiang, Xinping Chen, William J.
Davies, Fusuo Zhang
Journal of Experimental Botany, Volume 63, Issue 1, January 2012, Pages 13–
24, https://doi.org/10.1093/jxb/err248
 Published:

30 September 2011
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Abstract
In recent years, agricultural growth in China has accelerated remarkably, but most of this growth has been driven by
increased yield per unit area rather than by expansion of the cultivated area. Looking towards 2030, to meet the
demand for grain and to feed a growing population on the available arable land, it is suggested that annual crop
production should be increased to around 580 Mt and that yield should increase by at least 2% annually. Crop
production will become more difficult with climate change, resource scarcity (e.g. land, water, energy, and nutrients)
and environmental degradation (e.g. declining soil quality, increased greenhouse gas emissions, and surface water
eutrophication). To pursue the fastest and most practical route to improved yield, the near-term strategy is
application and extension of existing agricultural technologies. This would lead to substantial improvement in crop
and soil management practices, which are currently suboptimal. Two pivotal components are required if we are to
follow new trajectories. First, the disciplines of soil management and agronomy need to be given increased emphasis
in research and teaching, as part of a grand food security challenge. Second, continued genetic improvement in crop
varieties will be vital. However, our view is that the biggest gains from improved technology will come most
immediately from combinations of improved crops and improved agronomical practices. The objectives of this paper
are to summarize the historical trend of crop production in China and to examine the main constraints to the further
increase of crop productivity. The paper provides a perspective on the challenge faced by science and technology in
agriculture which must be met both in terms of increased crop productivity but also in increased resource use
efficiency and the protection of environmental quality.
Food security, environmental quality, genetic improvement, integrated soil-crop systems
management, resource use efficiency
Issue Section:
FOOD SECURITY
Trends in crop production

Increased crop production and yield

Over the last 50 years there has been remarkable growth in agricultural production in China. This
has created the so-called ‘Miracle in China’ with 7% of the world's arable land feeding 22% of the
world's population.

Chinese cereal production has increased steadily from 83.4 Mt in 1961 to 474.2 Mt in 2009 (Fig.
1A), accounting for 9.5% of total global cereal production in 1961 and 21.8% in 2009. The net
increase over this period is 390.8 Mt with an annual growth rate of 3.7%, which is substantially
higher than the world mean growth rate in cereal production of 2% during the same period. In
2009, China was responsible for approximately 29.1% of global rice production, 20% of maize, and
16.9% of wheat production (National Bureau of Statistics of China, 1950–2010; FAO, 2010). The
success of crop production in China has impacted on both global food supply and on natural
resource use and availability and both of these changes have received global recognition.

Fig. 1.

Open in new tabDownload slide

Production and cultivation areas of cereal crops (rice+wheat+maize) (A), grain yields of rice, wheat, and maize (B) and trends in all
fertilizer and N fertilizer consumption in China from 1961 to 2009. The consumption is the apparent whole consumption in China
based on the calculation of balance (production+imports–exports). Source: China Agriculture Yearbook. FAO STAT electronic
databases (http://apps.fao.org)

Historically, cereal production has been dominant in the south of the country and practised less in
the north of China. However, over the last few decades the balance has shifted to some extent.
From 1980 to 2008, total cereal cultivation area decreased by 3.77 Mha in the Yangtze River Basin,
by 3.27 Mha in south China, and by 0.81 Mha in South-West China, where rice-based cropping
systems are dominant. In contrast, the cereal cultivation area increased by 5.42 M ha on the North
China Plain and in North-East China. Total cereal production in the north increased from 129 Mt in
1980 to 283.5 Mt in 2008, which accounted for 41.4% of the national total cereal production in
1980 and 57.5% in 2008 (National Bureau of Statistics of China, 1950–2010). As a result, the
North China Plain and the North-East of China have become important cereal production and food-
commodity supply regions. In these regions, however, water availability for agriculture is
becoming a major issue for the nation.

The increase in total crop production in China has arisen mainly as a result of increases in yield per
unit area rather than from increases in the cultivated area. For example, from 1961 to 2009 there
was a 3.2-fold increase in the productivity of rice (from 2041 kg ha−1 to 6585 kg ha−1), an 8.5-fold
increase in the productivity of wheat (from 557 kg ha−1 to 4739 kg ha−1), and a 4.6-fold increase in
the productivity of maize (from 1139 kg ha−1 to 5258 kg ha−1) (Fig. 1B). Over the same period the
total cultivated area of cereals increased by only 30% (from 65.5 Mha in 1961 to 85.1 Mha in 2009)
(National Bureau of Statistics of China, 1950–2010; Fig. 1A).

Intensification of crop production contributes to increased yield

Intensification of crop production over the last 50 years has come to be known as the ‘green
revolution’ and has been achieved by the use of modern high-yielding varieties while greater
benefits have been realized from chemical fertilizers, irrigation, and weed and pest control.

The consumption of fertilizer in China has increased linearly since 1961. The total consumption of
chemical fertilizers exceeded 64 Mt in 2009, and this is nearly 35% of the total global fertilizer
consumption. Use of nitrogen (N) fertilizer has increased from 0.5 Mt in 1961 to 46.6 Mt in 2009
(Consumption=production+import–export, Revised from National Bureau of Statistics of China,
1950–2010, Fig. 1C). The area of irrigated farmland has expanded by 32% since the 1970s and the
effective area of irrigated farmland has now reached 58.5 Mha. This is 48% of the total arable land
area in China, but it produces 75% of the national grain production and 90% of the products from
cash crops (National Bureau of Statistics of China, 1950–2010). Chemical use increased from 0.76
Mt in 1991 to 1.76 Mt in 2005 (National Bureau of Statistics of China, 1950–2010). As the second-
largest producer and consumer of pesticides, use in China accounts for 14% of the world total and
the country has now become a net exporter (Liu and Diamond, 2005). Without the use of synthetic
fertilizers, irrigatio, and chemicals, China's food production could not have increased at the rates
recorded.

Figure 2 shows that intensification of maize production has occurred with time. Improvements in
maize varieties and cropping techniques have contributed to increased grain yield per unit area
since 1960s in China (Li and Wang, 2009). In the 1960s, double-cross hybrids were dominant and
use was combined with planting technologies focused on improving the condition of farmland.
Planting density was also increased at this time. Since the 1970s, single-cross hybrids have been
extensively used. The main trends in the development of breeding strategies to increase maize yield
potential included selection for disease-resistance traits, the improvement of above-ground plant
architecture, stay-green and late-maturing characteristics. Planting technologies were characterized
by the use of chemical fertilizers, irrigation, weed and pest control, higher planting density, and soil
quality improvement. As management intensity increased, planting technologies shifted from the
use of novel individual techniques to more technical integration. For example, chemical fertilizer
use was largely just N fertilization in the 1970s with both N and P additions common in the 1980s
and use of combined NPK fertilizers used much more often in the 1990s and 2000s. Chemical
fertilizer rate has increased greatly since the 1990s.

Fig. 2.

Open in new tabDownload slide

The main varieties and techniques of maize production from the 1960s to the 2000s in China. In the 1960s, double-cross hybrids were
dominant with main planting technologies characterized by improving farmland condition and increasing planting density. Since the
1970s, single-cross hybrids have been extensively used. The main trends in the development of breeding strategies to increase maize
yield potential including disease-resistant gene selection, plant canopy architecture improvement, stay green, and late-maturing.
Planting technologies were characterized by the use of chemical fertilizers, irrigation, weed and pest control, higher plant density, and
soil quality improvement. However, increased management intensity was observed: planting technologies had shifted from individual
techniques to technical integration. For example, chemical fertilizer types were used from sole N in the 1970s to N and P in the 1980s,
and even to the combination of NPK in the 1990s and 2000s. Chemical fertilizer rate has greatly increased since the 1990s. (Adapted
from Li and Wang, 2009, and reproduced by kind permission of the Chinese Academy of Agricultural Sciences.)

Declining rates of yield increase

Despite the achievement of increased crop production and grain yield per unit area, annual growth
rates of cereal yields are gradually declining. For example, the average growth rate of cereal yields
decreased from 4% in the 1970s to 1.9% in the 1990s. Over the last 10 years, rice and maize yields
have shown declining or stagnant trends in most provinces in China. Inappropriate crop
management practices, especially poor nutrient, soil, and water management, are likely to be
responsible (Dawe et al., 2000; Peng et al., 2002; Ladha et al., 2003; Zhang et al., 2007; Peng,
2011). Nevertheless, wheat yields have increased in most regions. This may be due to increasing
rainfall in the autumn and winter in northern China providing better conditions for wheat growth
and increased incentives for farmers to plant grass, fruit trees, and other alternative crops in some
regions with low wheat yields.

The main constraints in further improving crop production

If it is assumed that dietary trends in China continue and that the Chinese population will stabilize
at around 1.6 billion after another 20 years, the demand for grain can only be met and the
population fed on the remaining arable land if annual crop production can be increased to around
580 Mt. To achieve this, grain yield in China must increase by 2% annually over the next 20 years
(Fan et al., 2010). However, further increases in crop production will be more problematic than has
been the case for the last 50 years. The availability of water and good soil are major limiting factors
for China. Agricultural inputs must be reduced, especially N and phosphorus (P) fertilizer, overuse
of which have led to environmental problems such as increased greenhouse gas emissions and
severe water pollution in parts of China. Furthermore, climate change will also aggravate crop
stresses such as heat, drought, salinity, and submergence in water.

Limited arable land and poor soil quality

A long history of arable farming and steady increases in human population have led to the depletion
of arable land reserves in China (Li and Sun, 1990). For example, the population has more than
doubled since the 1950s to its current level of 1.3 billion but the total arable land area has expanded
by only 29% to the current 134.5 Mha (National Bureau of Statistics of China, 1950–2010). The
per capita arable land area is 0.1 ha at present, which is 45% of the world average (Wang et al.,
2009a). China has used almost every piece of available land for agriculture. The potential to
increase grain area will therefore be limited in the future, and more food will need to be produced
from the same amount of (or even less) land. Now, it is clear that it will become more important to
adopt technological and policy measures to improve the sustainability of agriculture as well as to
increase grain yield per unit area of arable land.

Most arable land in China has poor soil quality, so that it is difficult to achieve high crop yields.
For instance, in North-East China, grain yields on low productivity soils were less than 1500 kg
ha−1, but the corresponding average value was 7595 kg ha−1 on high productivity land (Fan et al.,
2010). The areas of high, medium, and low productivity land account for 28.7, 30.1, and 41.2% of
the total arable land in China, respectively (Wang, 2005). Soil organic matter (SOM), a key
indicator of soil quality, is still low in Chinese cropping systems although recent studies show SOM
in croplands has increased since the 1980s (Xie et al., 2007; Huang and Sun, 2006; Lu et al.,
2009; Piao et al., 2009). The average content of SOM in topsoil from cropland is 10 g kg −1 in China
compared with 25-40 g kg−1 in European countries and the United States (Fan et al., 2010). Soil
degradation, a reduction in soil quality as a result of human activities, is a very serious problem in
China. Of the total degraded land area in the world estimated to be 1964 Mha (Oldeman et al.,
1991), degraded land in China comprises 145 Mha or 7.4% of the world total (Lal, 2002). The
average thickness of topsoil in China over a 50-year period progressively decreased from 22.9 cm
in the 1930s to 17.6 cm in the 1980s (Lindert, 2000). Some soils are likely to be even thinner now
due to the intensity of erosional and depositional processes.

To enhance crop production in China with efficient resource utilization, improvement in soil
quality is critical. By definition, low quality soil has a lower resource buffer than exists in good soil
and this decreases the margin of error for nearly all crop management practices. Improving the
recycling of organic manures such as animal and human excreta, crop straw and stalks, and green
manure can be an important step towards saving natural resources and, simultaneously, stabilizing
and optimizing soil quality in crop production systems. Novel soil management practices should be
developed and promoted in China. For example, biochar addition to soils is an ancient practice
which has recently begun to attract wider notice. Incorporation of biochar represents a means of
sequestrating carbon and there is increasing evidence that although there may be some negative
effects of incorporation, it can also reduce nutrient leaching and impact positively on the slow
release of nutrients to enhance crop yields (Marris, 2006; Lehmann, 2007). Zero-till or reduced till
practices, which have rarely been practised in China until now, have reportedly allowed sustained
yields with largely positive effects on ecosystem services in some parts of the world. Although
there is some doubt about positive effects on greenhouse gas emissions (Rochette, 2008), there are
reports of fewer weeds, more beneficial insects and improved water use efficiency resulting from
this practice (Hobbs et al., 2008). Reduced till may be especially beneficial on low productivity
land.

Water shortage

Of all China's environmental woes, the biggest threat to livelihoods and food security may be
looming water shortages (Li, 2010, Peng, 2011). China's total fresh water volume is 2.81×1012 m3,
with 2.7×1012 m3 of surface water and 0.83×1012 m3 of groundwater (The Ministry of Water
Resources of the People's Republic of China, 2009). Although this water resource is large in
absolute value, ranking sixth in the world, the per capita water resource is only 25% of the world
average (Wang et al., 2008). China is listed as one of the 13 countries which are shortest of water.
Moreover, the distribution of water resources is spatially and seasonally uneven. The north of the
country, similar in land area and population to the south, holds only 18% of the total water despite
having 65% of the total arable land. By contrast, the south receives water from summer rainfalls,
which is often ‘wasted’ through flooding (Piao et al., 2010).

Agricultural water use is a major part of all water used annually. However, increased water
shortage associated with overuse of surface water, declining groundwater levels and water pollution
is threatening the sustainability of agricultural production. The share of irrigation in total water use
in China has declined from 80% in 1980s to 65% in 2009 (The Ministry of Water Resources of the
People's Republic of China, 2009). Annual water shortage in agriculture amounts to 30×1010 m3 in
China. By 2030, China's total water deficit could reach 130×1010 m3 (Li, 2006).

The outlook for water shortage is especially dire on the North China Plain (NCP), one of the main
grain production areas in China. This plain comprises 33.8% of the national arable land, but only
has 3.85% of the national water resources. Over the past 40 years, NCP's water table has fallen
steadily as some 120×1010 m3 more water has been pumped from the land than the amount replaced
by rainfall (Li, 2010). The current plan for a northern diversion of the Yangtze would not, however,
benefit agriculture.

Agricultural water use efficiency (WUE) which is defined as grain produced per unit of water
consumed is still very low in China due to poor irrigation management practices (Wang et al.,
2002; Deng et al., 2006) and lack of investment in infrastructure (Xu and Zhao, 2001; Lohmar et
al., 2003). The average WUE of three main grain crops in China is 1.12 kg m−3 with 0.85 kg m−3 for
rice, 1.01 kg m−3 for wheat, and 1.51 kg m−3 for maize, respectively (Li and Peng, 2009). Zwart and
Bastiaanssen (2004), who reviewed 84 literature sources for experiments around the world which
are not older than 25 years, found that the average WUE of rice, wheat, and maize was 1.09, 1.09,
and 1.80 kg m-3, respectively. Thus, the overall WUE of grain production in China has fallen behind
the world average. This implies that there are tremendous opportunities for China to reduce water
consumption with no reduction or even an increase in grain yield (Wang et al., 2002; Hu et al.,
2006). However, increasing crop productivity in China still requires innovative approaches to water
saving in agriculture.
Low nutrient use efficiency and environmental pollution

Increase in fertilizer nutrient input has made a significant contribution to the improvement of crop
yields in China. Fertilizer consumption has increased almost linearly (Fig. 1C). China is currently
the world's largest consumer of fertilizer.

Unfortunately, since about 1990, the increase in grain production has been associated with a major
decline in fertilizer nutrient use efficiency, especially N, and with widespread environmental
damage. According to yearly data for grain yield and synthetic N consumption (National Bureau of
Statistics of China, 1950–2010), the partial factor productivity of applied N (PFP, the ratio of yield
to the amount of applied N) has been halved over the last 30 years. The recovery efficiency of N (%
fertilizer N recovered in above-ground crop biomass, REN) for cereal crops was 35% on average in
the 1990s. However, this value has gradually reduced since then and the current REN is 28.3% for
rice, 28.2% for wheat, and 26.1% for maize (Zhang et al., 2008,a), all of which are lower than the
world values (40–60%). Similarly, Ma (2006) reported that the contribution of synthetic N to
increased grain yield in China was 30.8% between 1978 and 1984 but declined to 10.4% between
1999 and 2003.

The low nutrient use efficiency may be attributed to fertilizer overuse and high nutrient loss
resulting from inappropriate timing and methods of fertilizer application, especially in high-
yielding fields. For example, the average amount of N applied for the winter wheat–summer maize
double-cropping system in the North China Plain increased from 143 kg ha −1 in 1967 to about 384
kg ha−1 in 1988 and 670 kg ha−1 in 2000 (Zhen et al., 2006). The average fertilizer N application rate
for rice of 150 kg ha−1 is higher than in most countries and as much as 67% above the global
average, but application rates of 150–250 kg N ha−1 are common in China and can reach 300 kg N
ha−1 in some places (Roelcke et al., 2004; Peng et al., 2010). Following an on-farm country-wide
survey, Li et al. (2010) found that N fertilizer rates for cereal crops still show an increasing trend:
the rates were 204 kg N ha−1 for wheat, 199 kg N ha−1 for maize, and 217 kg N ha−1 for rice in 2000.
In 2007, rates had increased to 229 kg N ha−1 for wheat, 237 kg N ha−1 for maize, and 231 N kg
ha−1 for rice. Fertilizer application is not often based on real-time nutrient requirements of the crop
and/or site-specific knowledge of soil nutrient status. For example, in rice production systems most
farmers apply N in two split dressings (basal and top-dressings) within the first 10 d of the rice
growing season (Fan et al., 2007). In the intensive wheat–maize system in China, applying large
amounts of N fertilizer before planting or at the early growth stage constitutes standard
management practice to ensure adequate N for the whole growing season, and this N supply rate is
usually about 50% of the total amount given (Cui et al., 2010). This large amount of basal
fertilizer-N is prone to loss over an extended period because the plants require time to develop their
root systems and a significant demand for N.

Irrational fertilizer utilization has led to substantial environmental pollution. For example, losses of
N and P through leaching and runoff have led to drinking water pollution which affects 30% of the
population and results in the eutrophication of 61% of lakes in the country. Annual synthetic
fertilizer N-induced N2O emission from Chinese croplands has increased from 120 Gg N2O-N
yr−1 in the 1980s to 210 Gg N2O-N yr−1 in the 1990s (Zou et al., 2010). Another case study shows
that soil pH in the major Chinese crop-production areas has declined significantly from the 1980s
to the 2000s because of excessive N fertilizer inputs (Guo et al., 2010).
In conclusion, rationalization of nutrient application to deliver greater nutrient use efficiency and
reduced environmental risks is urgently required in China. There is now overwhelming evidence
that the quantities of fertilizer applied could be reduced with no detrimental effect on yield. Crop
yields might even be increased by a reduced use of fertilizer (Wilkinson et al., 2007; Fan et al.,
2008). The great challenge ahead is to determine how crop productivity can be further increased to
feed a growing population while minimizing nutrient loss and any subsequent environmental
damage for China. In reality, achieving such a target represents one of the greatest scientific
challenges facing humankind (Tilman et al., 2002).

The impacts of climate change on agriculture

Climate change and its impacts on crop production are major forces with which China will have to
cope in the twenty-first century (Editorial Board of Science Press, 2007; Godfray et al., 2010).
Rising temperature, altered rainfall patterns, and more frequent extreme events will increasingly
affect crop production, often in those places that are already most vulnerable (Morton, 2007). In
China, the clear warming has occurred in recent decades. The average temperature has increased by
1.2 °C since 1961. Precipitation patterns show significant regional trends. The drier regions of
northeastern China (including North China and North-East China) are receiving less and less
precipitation in summer and autumn. By contrast, the wetter region of southern China is
experiencing more rainfall during both summer and winter (Piao et al., 2010). China is at risk from
heavy rainfalls, heat waves, and drought (Zhai et al., 2005; Su et al., 2008; Wei and Chen, 2009).

Countrywide, a 4.5% reduction in wheat yields is thought to be due to rising temperatures over the
period 1979–2000 (You et al., 2009). Maize yields may also have been sensitive to recent warming,
with data from eight Chinese provinces showing a negative response to rising temperature during
the period 1979–2002. By contrast, rice yields in the north east appear to have increased by 4.5–
14.6% per °C in response to night-time warming between 1951 and 2002 (Tao et al., 2008).
However, improvements in crop management have been so influential that they prevent a clear
conclusion on the net impact of historical climate change on agriculture in China (Piao et al.,
2010). For instance, the autonomous adoption of new crop varieties seems to have compensated for
the negative impact of climate change on both wheat and maize production in the North China
Plain (Liu et al., 2010).

IPCC global climate models suggest that the climate warming trend will continue and China's
average temperature is estimated to increase further by 1–5 °C by 2100 (Meehl et al., 2007). Cereal
yields may benefit globally from the synergy of climate change and the fertilizing effect of elevated
CO2 (Chavas et al., 2009; Xiong et al., 2009), but the impacts of climate change on crop production
is still largely uncertain (Piao et al., 2010). This is because the magnitude of the CO2 fertilization
effect on crop yield is still uncertain and a matter of debate (Baker, 2004; Bannayan et al.,
2005; Sakai et al., 2006; Li et al., 2007; Ma et al., 2007; Ziska, 2008) and not all the effects of
climate, for example, O3 pollution, are included in the current projections (Piao et al.,
2010; Wilkinson and Davies, 2010). Another important source of uncertainty in current projections
lies in the potential of crop production to adapt to climate change. This implies that the future
adverse effect of climate change might be ameliorated by developing and using improved
agronomic practices and improved crop germplasm (Lobell et al., 2008).
The way forward

Intensification leading to increased yields per unit area provided most of the recent doubling of
agricultural production. The potential for a further doubling in yields now attracts increasing
attention and research. The need to revitalize yield growth with few resources and in a sustainable
manner is not under question. Several conceptual frameworks have been proposed for such an
advance, such as ‘Ecological Intensification’ (Cassman, 1999), ‘Evergreen Revolution’
(Swaminathan, 2000), and ‘Sustainable Intensification’ (Baulcombe et al., 2009). However, the key
question is how are we to achieve this objective in the face of several constraints, including land
and water scarcity, environmental degradation, and climate change.

Two issues are emphasized which are crucial if crop productivity is to be increased with efficient
resource use while limiting environmental degradation (Fig. 3). The initial challenge is how to
apply good governance to change suboptimal crop and soil management practices using existing
agricultural sciences and technologies. At the same time, advances in crop productivity will be
needed. Two pivotal components are required to follow new trajectories: (i) the development of
integrated soil–crop systems management (ISSM), which will address key constraints in existing
crop varieties, and (ii) the production of new crop varieties that offer higher yields but use less
water, fertilizer or other inputs and are more resistant to drought, heat, submersion, and pests and
diseases.

Fig. 3.

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Conceptual model for optimal crop production to achieve synchronously increasing crop productivity, improving resource use
efficiency, and environmental protection in China. (A) The current status in crop productivity on farm fields. (B) Scenario of crop
productivity upon application of the existing technologies. (C) Scenario of crop productivity upon improved soil and crop
management such as integrated soil—crop systems management, in existing crop varieties. (D) Scenario of crop productivity upon
improved soil and crop management and improved crop varieties.

Application and extension of existing technologies countrywide

Despite the fact that cereal yields per unit area have shown a remarkable increase since 1961,
inappropriate crop management practices are still very common in China today. Available evidence
suggests that the yield gap between average farm yields and the regional variety test experiments
for major cereal crops are derived from factors such as: (i) low profitability of crop production; (ii)
limited access to new agricultural technologies, and (iii) poor soil and crop management by farmers
(Lobell et al., 2009; Fan et al., 2010). China has devoted great effort to developing easy-application
and low-cost technology in agriculture, and has recently made remarkable progress. For example,
since 2003, a strategy has been followed which promotes the integrated use of nutrients from
various resources and N management and emphasizes the synchronization of supply and crop
demand (Fan et al., 2008). Integrated nutrient management techniques can, on average, increased
grain yield by 9.2–14.6%, and raise N fertilizer partial productivity by 10.5–18.5%, compared with
conventional agricultural practice with cereal crops (Table 1). In a recent study, a triangular
transplanting pattern and split N fertilizer application in the South-West of China has led to 22%
increase in rice yields with improved REN of 119% (Fan et al., 2009). It has been well recognized
that the WUE can be improved with maintained or even increased crop yield by use of water-saving
techniques (Davies et al., 2010). Examples of these are alternate wetting and drying irrigation for
rice (Yang and Zhang, 2010), mulching (plastic film or crop straw) for both rice and upland crops
(Fan et al., 2005a, b;,Zhang et al., 2008b;,Wang et al., 2009b), deficit irrigation for upland crops
(Fereres and Soriano, 2007), and alternate furrow irrigation for maize (Du et al., 2010). Net
reductions in some greenhouse gas emission can potentially be achieved by changing agronomic
practices. For example, improving N management can greatly reduce greenhouse gas (GHG) (N2O
and CO2) emissions from Chinese croplands (Huang and Tang, 2010).

Table 1.
On-farm evaluation of performance of integrated nutrient management in yield and partial
productivity of N fertilizer (PFP-N) compared with farmers’ conventional practices in major cereal
cropping systems in 110 agricultural counties in China
Yield increase(kg Yield PFP-N
Year Crop Sites(no.) ha−1) increase(%) increase(%)

2008 Rice 171 590 9.2 11.0

Maize 139 629 10.7 10.5

Wheat 67 521 12.6 18.5

2009 Rice 267 785 10.2 14.1

Maize 257 1092 14.6 16.6

Wheat 115 518 9.9 12.8

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Our view is that the most effective near-term strategy for improving crop productivity for China is
application and extension where possible of existing technologies in current agricultural systems.
The situation is a little similar to the case of the African smallholder farmers, for example, in
Malawi, where maize yields were doubled, even tripled within 2–3 years on a national scale. This
was achieved through improved seed and fertilizer use, and good governance from an input subsidy
programme supported by both international agencies and local government (Denning et al., 2009).

However, the key issue is how existing agricultural technologies can be quickly accessed and
adopted by farmers? Currently, the efficiency of the agricultural technology extension system in
China is low. There have been serious difficulties such as lack of investment, and poor training of
technicians (Research Centre of Rural Economy of Ministry of Agriculture, 2005). Due to small-
scale farming, economic benefits derived from improved management practices generally do not
translate into economic incentives which induce farmers to adopt new technologies voluntarily.
Therefore, incentive measurements such as farming subsidies may be useful to encourage the
farmer to adopt new technologies and change inappropriate management practices. A multiple
approach of extension, involving official extension systems, enterprise and non-government
organizations (NGOs) such as farmers’ special associations should be pursued simultaneously to
promote the dissemination of technologies. A lack of appropriate extension services have been
identified as a problem in many farming systems around the world (Baulcombe et al., 2009)

Advances in crop production

Development of integrated soil–crop systems management:

Existing knowledge and technology can, to a certain extent, improve management practices of
farmers, but will be unlikely to increase production to the level that is needed to allow a response to
international challenges with a doubling of food production by 2030. Greater advances in crop
production, which must follow new trajectories, are needed during the next 20 years to ensure a
substantial increase in cereal yield and ensure food security. The science of crop and soil
management and agricultural practice needs to be given particular emphasis as part of a food
security grand challenge (Baulcombe et al., 2009). Despite the enormous importance of the subject
and the growing number of specific studies, a multi-disciplinary synthesis of novel understanding
and even the established understanding of plant science, agronomy, soil science, and agroecology is
scarce in China. The development of more ecologically-influenced agricultural systems that
integrate features of traditional agricultural knowledge and add new ecological knowledge into the
intensification process will be needed (Matson and Vitousek, 2006).

An ISSM approach is advocated here, addressing the key constraints to yield in existing crop
varieties. Such constraints may be low soil fertility, water shortage, low nutrient use efficiencies,
and impacts of climate change etc (Fig. 4). In this approach, advances are needed to help us
understand coupling mechanisms between plants and climate, plants and soil, plant/microbial
biology and ecology, and rhizosphere interaction and management (Zhang et al., 2010). In this
area, the key proposals are: (i) take all possible measures to improve soil quality, (ii) integrate the
utilization of various nutrient resources and match nutrient supply to crop requirements, and (iii)
integrate soil and nutrient management with high yielding cultivation systems (Zhang et al., 2011).

Fig. 4.

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Conceptual model of an integrated soil–crop systems management approach. Note: Temp., temperature; Prec. precipitation. (Figure
taken from Chen et al., 2011, and reproduced by kind permission of the National Academy of Sciences.)

Recently, an ‘integrated soil–crop systems of management for maize’ has been demonstrated. This
involves the use of the Hybrid-Maize simulation model to maximize the use of solar radiation and
the exploitation of temperature changes. To design crop and nutrient management for given
ecological conditions, it also uses a root-zone in-season N management strategy to synchronize N
supply from soil and fertilizer and the N demand of the crop (Chen et al., 2011). Current studies on
the NCP show that this ISSM system could generate 14.6 t h−1 maize grain yields with 265 kg N
ha−1 fertilizer application. This yield level is 2.4 times higher than that achieved by farmers’
practices, but the amount of N fertilizer applied is similar to farmers’ practice. Thus, N efficiency is
increased 2.4 times above farmers’ practices (Fig. 5).

Fig. 5.

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Performance of an integrated soil–crop systems management (ISSM) in the North China Plain. The ISSM uses the hybrid-maize
simulation model to maximize the use of solar radiation and temperature, a root-zone in-season N management strategy by
synchronizing the N supply from soil and fertilizer and the N demand of crop. Right: ISSM with grain yield of 14.6 t ha −1, and partial
factor productivity of applied N (PFP-N) of 56 kg kg−1, Left: farmer's practices (FP) with grain yield of 6 t ha−1, N fertilizer PFP of 20
kg kg−1.

The above research for maize has illustrated the potential for substantial improvements in yield
with higher input efficiency by ISSM approaches. Much more analysis is required for maize, and
also for other major crops to establish how yield can be increased as the result of genotype,
environment, and management interaction. This type of analysis permits an understanding of the
factors that lie behind regional and crop differences in limitations in yield improvement. These
insights can then be used to apply more targeted research and develop ISSM as needed.

Continued genetic improvement in crop varieties:

Improving yield potential of crop varieties through plant breeding will be a critical component for
future food security (Foulkes et al., 2010). Yield potential is defined as the yield of a crop cultivar
when grown in environments to which it is adapted, with nutrients and water non-limiting and pests
and diseases effectively controlled (Evans, 1996). When average farm yields reach about 80% of
the yield potential ceiling, it becomes more difficult for farmers to sustain yield increases through
fine-tuning in soil, crop, water, nutrient, and pest management (Cassman, 1999). Rice yield,
especially in those productive regions, appear to be at or near 80% of yield potential in China
(Cassman et al., 2003). Recently, it was found that an ISSM approach to management of rice led to
a significant increase in N use efficiency but only a small increase in grain yield. This may suggest
that further increases in rice yield will mostly depend on the improvement of yield potential.
However, there is less certainty over how close we are to delivering yield potential in Chinese
maize and wheat production. For example, in the last ten years, wheat yields increased by 2.7% per
year in China. Chen et al. (2011) reached nearly 12.8 t ha−1 maize managed by ISSM across several
ecological regions of China. This is twice the response to current farmers’ practices (see above).
Therefore, closing the current yield gaps for maize and wheat may be a priority for agricultural
researchers to ensure food security in China. For a long-term view, plant breeders still need to focus
on the traits with the greatest potential to increase wheat and maize yields.

In the context of global environmental changes and other constraints to increase yield for China
further, the efficient use of nutrient, especially N, and water have emerged as two key targets. New
crop varieties will need to be more efficient in their use of reduced levels of nutrients (Godfray et
al., 2010; Tester and Langridge, 2010). Crop varieties with increased tolerance to drought are also
required in many parts of the world but particularly in China (Morison et al. 2008).

In the last half-century, traditional plant breeding has occurred almost entirely under management
regimes that include fumigated soils with extravagant additions of nutrients and sufficient water
(Boyer, 1982). This has potentially selected against traits that allow plants to maintain high net
primary productivity (NPP) and yields under non-saturating nutrient conditions (Jackson and Koch,
1997). For example, breeding for increased rice yield potential has been focused on increasing
panicle size and improving lodging resistance with thick stems in China. Therefore, rice breeders in
China often select progenies with “tolerance to high N” in the breeding nursery with high N
application. As a result, the most recently released cultivars and hybrid combinations will not lodge
even at a very high N rate (Peng et al., 2002). Research to improve the yield potential of cereal
grains in low nutrient environments has been sporadic, with mixed results until a recent concerted
effort showed that it is possible to improve the yields of wheat and maize in low input
environments (Bänziger and Cooper, 2001; Drinkwater and Snape, 2007). Therefore, there is an
urgent need for Chinese breeders to invest more in the capacity to strengthen this strategy.

Traditional breeding methods need to be combined with advanced breeding technologies such as
marker-assisted selection (MAS) and genetic modification (GM). This allows for more efficient
 selection of favourite germplasms across multiple traits and accelerates the breeding cycles. In
China, the largest plant biotechnology capacity outside North America is now being built (Huang et
al., 2002). Since 2008, the Chinese government has already rolled out a $3.5 billion research and
development (R&D) initiative on GM plants (Stone, 2008). Challenges ahead are: (i) to identify the
candidate genes and traits valuable for breeding; (ii) incorporate these into elite cultivars and to
evaluate their performance under real agricultural field conditions (Zhang, 2007); and (iii) adopt
new approaches for generating GM crops to reduce the constraints on regulatory approvals and
increase consumer acceptance.

Conclusions

Global food production now faces greater challenges than ever before. There is no simple solution
to delivering increased crop productivity while improving resource use efficiency and protecting
environmental quality. In this review, the focus has been on science and technology, but a broad
range of options including social and economic factors such as technology extension and access to
technologies by farmers also needs to be pursued. The path from the application of existing
technologies to the delivery of improved soil–crop systems management and improved crops must
be explored step by step.

Above all, future work will require a mult-disciplinary approach that involves not just soil
scientists, agronomists, and farmers, but also ecologists, policy-makers, and social scientists. Our
strong view is that governments of the world must allocate more funds to both fundamental plant
science and applied crop research and, despite substantial current spending, China is no exception
to this. However, global co-operation is needed to avoid duplication of effort and low efficiency
and to ensure faster progress.

https://academic.oup.com/jxb/article/63/1/13/553113

 B20.

How do we grow plants in marginal environments without encouraging invasiveness?

PDF invasiveness

 B21.

How can we use the growing of crops to limit desert spreading?

6.2 What actions can be taken to prevent desertification?

Terracing prevents further gully erosion and stores surface runoff for olive production (Tunisia)
Source: MA
 The creation of a "culture of prevention" can go a long way toward protecting drylands when desertification is just
beginning and even when it is ongoing. It requires a change in governments’ and peoples’ attitudes. It has been shown
that dryland populations, building on long-term experience and active innovation, can stay ahead of desertification by
improving agricultural and grazing practices in a sustainable way.
Preventive actions include:
o Integrating land and water management to protect soils from erosion, salinization, and other forms of degradation.
o Protecting the vegetative cover, which can be a major instrument for soil conservation against wind and water erosion.
o Integrating the use of land for grazing and farming where conditions are favorable, allowing for a more efficient cycling
of nutrients within the agricultural systems.
o Applying a combination of traditional practices with locally acceptable and locally adapted land use technologies.
o Giving local communities the capacity to prevent desertification and to manage dryland resources effectively.
o Turning to alternative livelihoods that do not depend on traditional land uses, such as dryland aquaculture, greenhouse
agriculture and tourism-related activities, is less demanding on local land and natural resources, and yet
provides sustainable income.
o Creating economic opportunities in dryland urban centers and in areas outside of drylands.

Prevention

Truck with wood

Source: MA
The creation of a “culture of prevention” can go a long way toward protecting drylands from the onset
of desertification or its continuation.The culture of prevention requires a change in governments’ and peoples’
attitudes through improved incentives. Young people can play a key role in this process. Evidence from a growing body of
case studies demonstrates that dryland populations, building on long-term experience and active innovation, can stay
ahead of desertification by improving agricultural practices and enhancing pastoral mobility in a sustainable way. For
example, in many areas of the Sahel region, land users are achieving higher productivity by capitalizing on improved
organization of labor, more extensive soil and water conservation, increased use of mineral fertilizer and manure, and new
market opportunities (C22.3.1).
Integrated land and water management are key methods of desertification prevention. All measures that protect
soils from erosion, salinization, and other forms of soil degradation effectively prevent
desertification. Sustainable land use can address human activities such as overgrazing, overexploitation of plants,
trampling of soils, and unsustainable irrigation practices that exacerbate dryland vulnerability. Management strategies
include measures to spread the pressures of human activities, such as transhumance (rotational use) of rangelands and
well sites, stocking rates matched to the carrying capacity of ecosystems, and diverse species composition. Improved
water management practices can enhance water-related services. These may include use of traditional water-harvesting
techniques, water storage, and diverse soil and water conservation measures. Maintaining management practices for
water capture during intensive rainfall episodes also helps prevent surface runoff that carries away the thin, fertile,
moisture-holding topsoil. Improving groundwater recharge through soil-water conservation, upstream revegetation, and
floodwater spreading can provide reserves of water for use during drought periods
(C22.2.3, C22.4.3, C22.4.4, R6.2.2, R6.3.7).
Protection of vegetative cover can be a major instrument for prevention of desertification. Maintaining vegetative
cover to protect soil from wind and water erosion is a key preventive measure against desertification. Properly maintained
vegetative cover also prevents loss of ecosystem services during drought episodes. Reduced rainfall may be induced if
vegetation cover is lost due to overcultivation, overgrazing, overharvesting of medicinal plants, woodcutting, or mining
activities. This is usually coupled with the effect of reduced surface evapotranspiration and shade or increased albedo
(C22.2.3, C22.2.2, C13 Box 13.1).
In the dry subhumid and semiarid zones, conditions equally favor pastoral and cropping land use.Rather than
competitively excluding each other, a tighter cultural and economic integration between the two livelihoods can
prevent desertification. Mixed farming practices in these zones, whereby a single farm household combines livestock
rearing and cropping, allows a more efficient recycling of nutrients within the agricultural system. Such interactions can
lower livestock pressure on rangelands through fodder cultivation and the provision of stubble to supplement livestock
feed during forage scarcity (and immediately after, to allow plant regeneration) due to within- and between-years climatic
variability. At the same time, farmland benefits from manure provided by livestock kept on fields at night during the dry
season. Many West African farming systems are based on this kind of integration of pastures and farmland
(C22.2.6, R6.3.7).
Use of locally suitable technology is a key way for inhabitants of drylands at risk of desertification to work with
ecosystem processes rather than against them. Applying a combination of traditional technology with selective transfer
of locally acceptable technology is a major way to prevent desertification. Conversely, there are numerous examples of
practices—such as unsustainable irrigation techniques and technologies and rangeland management, as well as growing
crops unsuited to the agroclimatic zone—that tend to accelerate, if not initiate, desertification processes. Thus technology
transfer requires in-depth evaluation of impacts and active participation of
recipient communities (R.SDM, R17.2.4, R14.ES).
Local communities can prevent desertification and provide effective dryland resource management but are often
limited by their capacity to act. Drawing on cultural history and local knowledge and experience, and reinforced by
science, dryland communities are in the best position to devise practices to prevent desertification. However, there are
many limitations imposed on the interventions available to communities, such as lack of institutional capacity, access to
markets, and financial capital for implementation. Enabling policies that involve local participation
and community institutions, improve access to transport and market infrastructures, inform local land managers, and allow
land users to innovate are essential to the success of these practices. For example, a key traditional adaptation was
transhumance for pastoral communities, which in many dryland locations is no longer possible. Loss of such livelihood
options or related local knowledge limits the community’s capacity to respond to ecological changes and heightens the
risk of desertification (C22.ES, C22.6.4, R6.2.2, R17.3, R2.4.3).
Desertification can be avoided by turning to alternative livelihoods that do not depend on traditional land uses,
are less demanding on local land and natural resource use, yet provide sustainable income. Such livelihoods
include dryland aquaculture for production of fish, crustaceans and industrial compounds produced by microalgae,
greenhouse agriculture, and tourism-related activities. They generate relatively high income per land and water unit in
some places. Dryland aquaculture under plastic cover, for example, minimizes evaporative losses, and provides the
opportunity to use saline or brackish water productively. Alternative livelihoods often even provide their practitioners a
competitive edge over those outside the drylands, since they harness dryland features such as solar radiation, winter
relative warmth, brackish geothermal water, and sparsely populated pristine areas that are often more abundant than in
non-drylands. Implementation of such practices in drylands requires institution building, access to markets, technology
transfer, capital investment, and reorientation of farmers and pastoralists (C22.4.4).
Desertification can also be avoided by creating economic opportunities in drylands urban centers and areas
outside drylands. Changes in overall economic and institutional settings that create new opportunities for people to earn
a living could help relieve current pressures underlying the desertification processes. Urban growth, when undertaken with
adequate planning and provision of services, infrastructure, and facilities, can be a major factor in relieving pressures that
cause desertification in drylands. This view is relevant when considering the projected growth of the urban fraction in
drylands, which will increase to around 52% by 2010 and to 60% by 2030 (C22.5.2, C27.2.3).

https://www.greenfacts.org/en/desertification/l-3/6-prevention-desertification.htm#2p0

Rotation Plant Diseases With Crop

Managing
Managing Plant Diseases With Crop
Rotation
https://www.sare.org/Learning-Center/Books/Crop-Rotation-on-Organic-Farms/Text-Version/Physical-and-
Biological-Processes-In-Crop-Production/Managing-Plant-Diseases-With-Crop-Rotation
by Margaret Tuttle McGrath
Rotating land out of susceptible crops can be an effective and relatively inexpensive means for managing some
diseases. To successfully use crop rotation for disease management, however, requires understanding the life cycle
of the disease-causing organism (pathogen). Generally, the technique of using crop rotation for disease
management is to grow non-host plants until the pathogen in the soil dies or its population is reduced to a level
that will result in negligible crop damage. To manage a disease successfully with rotation, one needs to know (1)
how long the pathogen can survive in the soil, (2) which additional plant species (including weeds and cover crops)
it can infect or survive on, (3) other ways it can survive between susceptible crops, (4) how it can be spread or
reintroduced into a field, and (5) methods for managing other pathogen sources. For example, a pathogen that can
survive in the soil but can also disperse by wind may not be successfully managed by rotation if an infected planting
occurs nearby or the spores can disperse long distances.
SIDEBAR 3.1

Importance of Scientific Names in Disease Management

When designing a rotational sequence for managing a particular disease, focus should be on the pathogen’s scientific name,

because common names can be misleading. For example, powdery mildew, downy mildew, bacterial blight, and fusarium wilt

are usually caused by different pathogens in different crops. On the other hand, white mold, which occurs in several crops, is

caused by the same fungus that causes lettuce drop.

Knowing whether the pathogen exists as specialized strains that limit the host range is critical for designing disease-controlling

rotations. For example, all grasses get anthracnose; however, host specialization was recently found to occur in the fungal

pathogen causing this disease. Consequently, grass weeds do not play as important a role as alternate hosts for anthracnose in

corn as was originally thought. Strains of some fungi are called formae speciales (f. sp.); others are called pathovar (pv.). These

abbreviations occur in some of the pathogen scientific names listed in Appendix 3.

Appendix 3 lists sources of pathogen inoculum and recommended rotation periods for diseases of vegetable and
field crops in the greater northeastern United States (including the mid-Atlantic region). The number of years
needed to suppress a disease cannot be stated precisely for many diseases because of the impact of other factors
and lack of extensive research, but general guidelines have been developed from research and farm observations,
as well as knowledge of pathogen biology. While these periods are based on research and observations from
conventional production systems, they are generally applicable to organic systems because pathogen biology
doesn’t change. However, if the activity of beneficial soil microorganisms that suppress a pathogen is much higher
in an organic field than in a conventional field, the required rotation period might be shorter. On the other hand, if
more organic matter, such as an incorporated cover crop, is present in an organic system, those pathogens that can
survive on decomposing organic matter may be more difficult to manage. Knowing which weeds can host a disease
is important, as these weeds will need to be controlled during the rotation (see Appendices 3 and 5). Avoiding
reintroduction of the pathogen when the crop is planted again is also critical. For example, infested seed,
transplants, or soil on farm machinery can reintroduce a pathogen to a clean field.
The sections below describe the biological basis for managing plant diseases with crop rotation. First, several critical
aspects of pathogen biology are discussed. These dictate whether rotation is a potentially viable option for
managing a particular pathogen and the disease it causes. Second, how the characteristics of certain rotation crops
affect pathogens is considered: Good rotations do more than simply provide an unsuitable host. Third, the impact of
cover crops and incorporated green manures on diseases is considered. Fourth, various environmental and
management factors that affect the success of crop rotation for disease suppression are discussed. Finally, some
selected diseases that can be managed successfully with rotation are discussed, followed by some examples of
diseases that cannot be controlled by rotation. These examples help explain the factors that affect rotation success.
Although the focus here is on diseases of vegetable and field crops in the northeastern US, the principles are
broadly applicable, and many of the specific diseases discussed occur in other regions as well. Because the same
disease name is often applied to diseases caused by several different pathogens, scientific names are used
frequently in the following sections to avoid ambiguity (see sidebar 3.1).

Pathogen Characteristics that Determine the Success of Rotation and Length of

the Rotation Period

Rotation can effectively suppress a crop disease when the target pathogen is capable of surviving in the soil or on
crop debris for no more than a few years. Some fungal and bacterial pathogens can survive in soil only in crop
debris, and these are the most suitable pathogens to target for management with crop rotation because they
cannot survive once the debris has decomposed. Pathogens that survive on soil organic matter but for only a few
years can also be managed with crop rotation. These short-term residents of the soil are called soil invaders or soil
transients. Pathogens in this group vary in the length of time they can survive, and thus in the length of rotation
needed.
"Wind, irrigation water, or insects can spread the pathogen from infected crops and re-infest
the field after rotation."
Survival time partly reflects the type of plant host tissue infected. For example, the barley scald pathogen primarily
infects leaves and leaf sheaths, which decompose fairly rapidly. In contrast, the net blotch pathogen also infects
barley stems, including the nodes, which are more resistant to decay. Consequently, a longer rotation is needed to
manage net blotch than to manage scald. Infected seed, and also wind-dispersed spores for the net blotch
pathogen, are additional sources of these pathogens that need to be managed to ensure successful control through
rotation.
Similarly, managing the bacterial canker disease that affects tomatoes requires a longer rotation than is needed to
manage bacterial speck and bacterial spot. The canker-causing bacteria get inside tomato stems, whereas speck
and spot are restricted to rapidly decomposing leaves and fruit. All three pathogens can be seed-borne. Rotation is
only one aspect of a good control program. Managing other sources of bacterial pathogens is critical for success.
This is covered in detail in the section on specific diseases below.
A few fungal and bacterial pathogens are true soil inhabitants, able to grow on organic matter in the soil. Such
organisms are referred to as saprophytes. These are hard to manage with rotation. Examples of soil inhabitants are
the fungi Pythium, Rhizoctonia, and Fusarium and the bacteria Erwinia, Rhizomonas, and Streptomyces.
Several species of Pythium and Rhizoctonia are commonly found in most soils as part of the normal soil flora. These
fungi attack seeds and the roots and stems of tender seedlings, causing seed decay and damping-
off. Pythium species also cause fruit rot in cucurbits, and Rhizoctonia solani causes wirestem, bottom rot, and head
rot in crucifers. Although crop rotations will not completely control these fungi, reducing the pathogen population by
rotating with small grains can reduce losses in subsequent crops. Since these saprophytes can also use fresh plant
residues, incorporating large amounts of organic matter will stimulate their growth. Thus, crops planted too soon
after incorporating a cover crop could be severely affected by these fungi. Other types of organic matter, such as
leaves or incompletely decomposed compost, could have a similar effect. Some fungi that cause fusarium wilts can
survive in or on roots of plants that do not develop symptoms (“symptomless carriers”), and they can also grow as
saprophytes on plant debris and other partly decomposed organic matter.
Some fungal pathogens produce specialized structures that, like seeds, enable them to persist in a state of
dormancy. These structures help the pathogen survive periods when host plants are absent, as well as cold winter
temperatures and other adverse conditions. The maximum survival time varies among types of structures and
species of pathogen. Fungal structures capable of dormancy include oospores, sclerotia, chlamydospores, and
cleistothecia. Similarly, some pathogenic nematodes produce cysts. Recognizing that these terms refer to resting
structures is helpful when reading about plant diseases because they indicate that a pathogen can potentially
persist in soil.
Some pathogens are heterothallic, which means they produce the dormant structure only when individuals of
opposite mating types (the fungal equivalent of male and female) interact. This is important because presence or
absence of the different mating types can determine whether the pathogen persists in the soil. For example,
although the cucurbit downy mildew fungus, Pseudoperonospora cubensis, is potentially capable of producing
oospores, only one mating type occurs in the US; thus, it cannot produce oospores, and that prevents it from
surviving winter in the northeast US. In contrast, the onion downy mildew fungus, Peronospora destructor, does
produce oospores in the northeast US, and they can survive four to five years in soil. The situation can change,
however: Until recently only one mating type of the late blight fungus, Phytophthora infestans, existed in the US.
Now two mating types are present, and the pathogen can persist in soil as oospores. Phytophthora erythroseptica,
which causes pink rot in potato, is homothallic. Thus, it can produce oospores when just one mating type is present.
Sclerotia and chlamydospores are structures that can be produced without interaction between fungi of opposite
mating types. Sclerotia produced by Colletotrichum coccodes, which causes anthracnose and black dot in tomato,
survive at least eight years. Those formed by Sclerotinia sclerotiorum, the white mold fungus, can survive up to ten
years. Rhizoctonia spp. also produce sclerotia. Verticillium dahliae, which causes verticillium wilt, produces tiny
sclerotia (microsclerotia) that can survive up to 13 years. Fungi causing fusarium wilts can persist for many years as
chlamydospores.
The target pathogen should have a narrow host range for rotation to be successful. Peronospora farinose f.
sp. spinacia causes downy mildew only in spinach. Another spinach pathogen, Albugo occidentalis, causes white
rust in spinach and in some species of lambsquarters and goosefoot. However, the strains of fungi that attack the
different species are specialists, and this host specificity can prevent cross infection. Thus, white rust may occur
only on spinach or only on weeds in a field. In contrast, the fungus Sclerotinia sclerotiorum, which causes white
mold, can infect more than 360 plant species. Corn and cereals are among the few non-host crops that can be used
in rotation to decrease abundance of this pathogen. Weeds must be managed carefully, however, for this rotation
to be successful. In addition, rotation out of susceptible crops for at least five years is needed because this fungus
produces long-lived sclerotia. Effectiveness of a rotation can be compromised when nearby plantings have white
mold. Wind, irrigation water, or insects can spread the pathogen from infected crops and re-infest the field after
rotation.
When considering rotation to manage a fungal pathogen that produces wind-dispersed spores, it is critical to know
how far the spores can travel. Powdery mildew and downy mildew fungi produce spores that can disperse great
distances. These cucurbit pathogens can move up the entire eastern coast of the US each year, helped by the
sequential planting of these crops from south Florida to Maine as conditions become favorable. Clearly, pathogens
like these would be difficult to control with rotation. In contrast, downy mildew of onion can be controlled with
rotation, partly because the crop is not grown as extensively. Several other fungal pathogens that attack leaves,
including Alternaria, Cercospora, Septoria, and Stemphylium, are more effectively controlled by rotation because
they produce large spores. They disperse only short distances by wind, although they can leave the field on
equipment. Other fungal pathogens, such as Colletotrichum, produce spores on leaves and fruit that disperse by
splashing water. Bacteria are also dispersed by splashing water and in windblown water droplets.

“Corn, small grains, and other grasses are usually good crops to rotate with vegetable crops.”

New findings or changes in the pathogen can affect rotation guidelines. For example, a short rotation was initially
thought to be adequate for Phytophthora capsici, which causes blight in cucurbits, pepper, tomato, and eggplant.
Recently this pathogen was found on new hosts (lima bean, snap bean, purslane, and a few other weeds), which
may explain why short rotations have not been effective. Additionally, it may be able to move between fields more
easily than anticipated, possibly accounting for its occurrence in fields where susceptible crops had not been grown.
Recently, most cases of early blight in tomatoes have been shown to be caused by a different species
of Alternaria than the species causing early blight in potato. Consequently, early blight may not be more severe in
tomatoes that follow early blight–affected potatoes than in tomatoes that follow other crops.
As a group, plant-pathogenic nematodes are more difficult to manage with rotation than fungi and bacteria because
almost all exist for part of their lives in the soil. Only a few nematodes attack just leaves, rarely entering soil, and
none of these infect vegetable or grain crops.
None of the few soilborne viruses that affect vegetable or grain crops occur in the northeastern US. Most viruses
cannot persist in soil between crops because they survive only in living host tissue or vectors, and few viruses are
vectored by nematodes or soilborne fungi. However, some viruses can persist between crops in weeds—for
example, cucumber mosaic in chickweed and potato virus Y in dandelion (Appendix 5).

Beneficial Plants to Include in Rotations

The typical focus in designing a rotation for disease management is to alternate among crops that are susceptible to
different pathogens. Alternating crops in different families is a good starting point, but some pathogens attack crops
in two or more families. For example, Phytophthora capsici causes blight in cucurbits, peppers, and lima beans.
Corn, small grains, and other grasses are usually good crops to rotate with vegetable crops. Fusarium fruit rot,
however, has been more common in pumpkin following corn.
Some plants suppress pathogens in addition to being unsuitable hosts. These include some cover and green manure
crops, as well as cash crops. Including disease-suppressive species in a rotation sometimes reduces the time
needed before a particular cash crop can again be produced successfully. Examples include some legumes and
crucifers. These plants suppress pathogens by stimulating beneficial organisms in the soil and by producing toxic
chemicals. The specific mechanisms involved appear to vary with the crop and the pathogen. Depending on the
mechanism, the beneficial effect can disappear shortly after incorporation or last for years. Suppression can vary
with how well the pathogen is established in a field. Also, to achieve success, beneficial crops may need to be
grown more than once before a susceptible cash crop is replanted. It is important to remember that incorporating
large quantities of biomass in the form of green manure stimulates general microbial activity, which can include
pathogens like Pythium, as described earlier.
Including legumes such as clover, pea, bean, vetch, and lupine in crop rotations has been recognized as beneficial
for disease management since ancient times. Legumes stimulate the growth and activity of soil microbes, in
addition to increasing soil nitrogen and organic matter. Hairy vetch residue incorporated into soil reduces fusarium
wilt in watermelon and enhances crop growth. On the other hand, hairy vetch is a good host for root-knot
nematodes.
“The degree of control was better than that achieved by fumigating with chloropicrin, a
product used by conventional farmers."
Members of the mustard plant family (crucifers) release substances while decomposing that are toxic to some fungi,
nematodes, and even weeds; and they also stimulate beneficial microorganisms. One group of chemical breakdown
by-products from these plants is the volatile isothiocyanates. These originate from glucosinolates, which are
themselves harmless. Glucosinolate content varies among plants of the mustard family. White mustard, brown
mustard, and rapeseed have especially high concentrations. IdaGold is a yellow mustard variety bred for high
glucosinolate content. Glucoraphanin is a glucosinolate found at much higher levels in broccoli than in other
cruciferous plants. Using these plants to manage pests is called biofumigation. Research has been done in California
with mustard seeded in fall and incorporated in spring. In the northeast US, mustard would be seeded in early
spring, then incorporated several weeks later when it is in full flower and organic matter is at a maximum. Soil
temperature should be 59° to 77°F. Amending the soil with crucifer seed meal can similarly suppress disease.
Isothiocyanates are thought to be less damaging to beneficial soil organisms than conventional chemical fumigants.
These compounds can also be toxic to crops; thus, planting should be delayed about a month after incorporation.
Quantity of isothiocyanates produced can vary with soil type, as well as variety of crucifer. The degree of disease
control has been related to the quantity of isothiocyanates in some systems, but in other systems disease
suppression evidently is due to another mechanism. Beneficial microorganisms stimulated include myxobacteria
and Streptomyces.
Note that although a mustard or legume crop can suppress some pathogens, such crops may also promote
pathogens that attack plants in those families.

Examples of Specific Diseases Influenced By Crop Rotation

Carrot root dieback

The soilborne fungi Pythium spp. and Rhizoctonia solani can infect and kill the tip of carrot tap roots, causing them
to become forked or stubby. In severe cases, they kill the plants. A study in California showed that when carrots
were grown after alfalfa, populations of Pythium and Rhizoctonia were larger, and fewer marketable carrots were
produced. The study also revealed more misshapen carrots and a higher population of Pythium when barley
preceded carrots, but the barley residue may not have decomposed sufficiently before the carrots were planted.
Carrots and pathogen populations were normal when onions or a fallow period preceded the carrot crop. Another
reason not to grow alfalfa before carrots is that alfalfa is a host for the fungus causing cavity spot of
carrot, Pythium violae.
“Rotation that includes a fallow period can be the key for controlling some pathogens that have a wide
host range.”

Clubroot

The roots of mustard family crops that are attacked by the slime mold fungus Plasmodiophora brassicae become
greatly swollen. This pathogen can survive in soil for seven years in the absence of mustard family crops or weeds.
Clubroot has declined more quickly when tomato, cucumber, snap bean, and buckwheat were grown. Clubroot was
effectively controlled by growing summer savory, peppermint, garden thyme, or other aromatic perennial herb
crops for two or three consecutive years.

Verticillium wilt

Rotating among crop families is generally recommended, because crops within a family are typically susceptible to
the same diseases; however, growing broccoli immediately before cauliflower resulted in a reduction in verticillium
wilt, even though these plants are closely related. Broccoli produces more of a specific glucosinolate and stimulates
myxobacteria that reduce survival of Verticillium microsclerotia. The degree of control was better than that achieved
by fumigating with chloropicrin, a product used by conventional farmers. Fresh broccoli residue is more effective
than dry residue. The greatest reductions in pathogen microsclerotia occurred when soil temperatures were above
68°F. Verticillium wilt diseases of other crops in California were also suppressed by broccoli.

Verticillium wilt and scab of potato

Both these diseases were reduced when corn or alfalfa was grown the previous year rather than potato. Verticillium
wilt severity also was lower when a buckwheat green manure preceded potato than when canola or a fallow
preceded potato.

Lettuce drop and whitemold

Broccoli is also a good crop to grow in rotation with lettuce and crops susceptible to white mold. The number of
sclerotia of the lettuce drop fungus, Sclerotinia sclerotiorum, decreased after residue from a spring broccoli crop
was incorporated during the summer. This resulted in reduced incidence of lettuce drop in a fall lettuce crop. Similar
results were obtained in California when two consecutive crops of broccoli in one year were followed by two
consecutive crops of lettuce the next year. Density of sclerotia of Sclerotinia minor was lower following broccoli than
where broccoli was not grown, and broccoli was associated with lower incidence of white mold in subsequent crops.
In contrast, cover crops of Lana woollypod vetch, phacelia, and Austrian winter pea in California hosted S. minor,
and incidence of drop was higher where lettuce was grown after these cover crops were incorporated than in plots
that had been left fallow. This pathogen also caused disease on purple vetch but not on oilseed radish, barley, or
fava bean. Oilseed radish, however, is a host of the clubroot pathogen and root-knot nematodes. Phacelia and
purple vetch are also hosts of root-knot nematodes.
Other Factors Affecting Success of Rotation in Managing Disease
Rotation is more likely to be effective if the entire field is rotated out of susceptible crops rather than just the
section previously planted to the crop. When farm equipment is used throughout the field, infested soil or crop
debris can be moved from the contaminated section to other parts of the field. If a field is divided into management
units, rotation can be effective within a unit if cultivators and other farm equipment are cleaned before working in
another unit and water does not flow between units during heavy rainstorms.

“Rotations of at least five or seven years often prevent the pathogen population from building up to a
level that can cause economic damage.”
Time required for rotation to be effective can vary with disease severity and environmental conditions. When a
disease has been severe, a longer rotation may be needed to reduce the pathogen’s inoculum level sufficiently to
avoid economic loss. A two-year period between wheat crops is generally needed to reduce Septoria leaf spot;
however, just one year without wheat resulted in a similar reduction in disease severity when environmental
conditions were less favorable for this disease. Sclerotia can be sensitive to drying. Some pathogens, such
as Sclerotium rolfsii, which causes southern blight, are adapted to warm conditions and do not survive low soil
temperatures. That is why southern blight does not occur in most of the northeastern US.
Using other cultural practices with rotation can be the key to successfully controlling some pathogens. For example,
after rotating land out of tomatoes to reduce pathogen populations, staking and mulching the subsequent tomato
crop minimizes the potential for pathogen propagules remaining in the soil to disperse up onto the tomato plants.
For a similar reason, deeply burying infested crop debris and pathogen survival structures by moldboard plowing
reduces disease incidence. For this to work, the residue must be buried deeply enough that it is not pulled back up
during seedbed preparation and cultivation. Burying diseased material is especially useful against pathogens that
produce sclerotia and those that infect only aboveground plant tissue. However, deep, full inversion plowing
decreases soil health by burying beneficial organisms that live in the top few inches of the soil profile. The value of
incorporating debris is illustrated by corn diseases, in particular gray leaf spot, that are more common and more
severe under no-till production. Breaking up infested crop debris immediately after harvest—for example, with flail
chopping or repeated disking—can hasten decomposition of the debris, thereby reducing survival time for those
pathogens that cannot survive in soil without debris.
While several pathogens are more likely to cause disease in subsequent crops when infested crop debris is left on
the soil surface, there are exceptions. An in-depth study on survival of Colletotrichum coccodes, which causes
anthracnose and black dot in tomato, revealed that its sclerotia survive longer when buried shallowly in the soil than
when on the soil surface, probably because temperature and moisture vary more on the surface. The sclerotia of
this species also appear to survive longer when not associated with plant tissue, likely because the skin tissue of
tomato fruits becomes colonized by beneficial fungi that can parasitize the sclerotia. Roots are an important,
overlooked source of this pathogen. Tomato roots in several fields were found to be infected and to have sclerotia
when there were no symptoms on aboveground parts of the plants. Additionally, this fungus is pathogenic on roots
of other plants in the nightshade and cucurbit families, including several weeds (see Appendix 3). It can also survive
on the roots of numerous other non-host plants (symptomless carriers), including chrysanthemum, white mustard,
cress, cabbage, and lettuce. This could account for C. coccodes occurring on tomato roots in fields with no previous
history of tomato or other crops in the nightshade family.
Rotation that includes a fallow period can be the key for controlling some pathogens that have a wide host range.
Bacteria causing soft rot are generally not considered amenable to management by rotation because they are
common soil inhabitants with a wide host range. However, one of the common bacteria causing soft rot, Erwinia,
does not survive well in a field that is fallow and repeatedly tilled.
Some cultural practices can also negate the benefit of rotation. Incidence of scurf ( Monilochaetes infuscans) in
sweet potato can be increased when animal manure is applied. Diseases caused by the fungus Rhizoctonia
solani can be enhanced when undecomposed crop residue is present at planting. Potato and onion cull piles can be
sources of pathogen inoculum and thus need to be destroyed before planting the next crop. Tillage can spread
disease through-out a field from an initial few infested areas.

Some Diseases That Can Be Managed with Crop Rotation

Understanding the mechanisms that allow or prevent management of specific diseases by rotation can improve
success and avoid wasted effort. This section discusses some diseases that can be successfully managed by crop
rotation; the next section discusses some diseases that cannot. Other diseases for which rotation is or is not
effective are listed in Appendix 3.

Bacterial spot of pepper and tomato

The bacterium causing spot (Xanthomonas campestris pv. vesicatoria) can be effectively controlled with rotation
because this pathogen cannot survive in the soil once diseased plant debris decomposes. A minimum of two years
without a host crop is recommended.

Bacterial speck of tomato

This disease is more difficult to control with rotation than bacterial spot because the pathogen (Pseudomonas
syringae pv. tomato) can survive on roots and leaves of taxonomically diverse weeds. Therefore, success requires
good control of weeds and volunteer tomatoes during the rotation period. A study on survival of this bacterium
showed that it lived up to 30 weeks on crop debris but less than 30 days just in soil.
Bacteria causing spot and speck, as well as Clavibacter michiganensis subs. michiganensis, which causes bacterial
canker in tomato, can occur on seeds. Thus, subsequent crops should be planted with seed lots that have been
tested and shown to have no detectable pathogen to avoid reintroducing the pathogen into the field. The seed
should also be hot water treated, because bacteria could be present at a low, undetectable level. A description of
how to treat seed with hot water is
at http://vegetablemdonline.ppath.cornell.edu/NewsArticles/PepperLeafSpot.htm. These tomato diseases also affect
peppers, but resistant pepper varieties are available.
Volunteer tomato and pepper plants could grow from infested seed left in the field from a previous crop. Thus,
volunteers need to be destroyed during rotation to ensure successful suppression of bacterial diseases. Destroying
volunteer crop plants is also important for other seedborne bacterial diseases, in particular, bacterial fruit blotch of
watermelon. Bacterial pathogens of tomato can also survive on tomato stakes and planting supplies, so these
materials need to be replaced or disinfected before reuse.

Root-knot nematodes

Northern root-knot nematode (Meloidogyne hapla) is the most common species of root-knot nematode in the
northeastern US and the only one found during a recent study of vegetable soils in New York. The predominant
species in the mid-Atlantic region is southern root-knot nematode (M. incognita), although northern root-knot
nematode also occurs there. These nematodes have a large range of hosts that includes most vegetables. Growing
sorghum, small grains, or grasses or leaving a clean summer fallow between crops can reduce the nematode
population to a tolerable level. The effect of these crops on root-knot nematodes is short lived, so they should be
routinely incorporated into the cropping sequence. Weeds need to be controlled during the rotation to grasses,
because some, notably nutsedges, are good hosts for these nematodes. Varieties of alfalfa, common bean,
soybean, cowpea, peppers, and tomato are available with resistance to certain root-knot nematode species.
Some commonly used cover crops, notably hairy vetch, are good hosts of root-knot nematodes.

Some Diseases That Cannot Be Easily Managed with Crop Rotation

Fusarium wilts of crucifers, cucurbits, pea, spinach and tomato

These diseases are difficult to manage with rotation because the pathogens can persist for many years in soil in the
absence of their crop host. They persist as dormant chlamydospores and on roots of some non-host plants
(symptomless carriers). Rotations of at least five or seven years often prevent the pathogen population from
building up to a level that can cause economic damage. However, if the disease has been severe in a field, even
seven years may not be enough. Selecting resistant varieties is a more effective and practical means of controlling
fusarium wilt. Multiple races have been identified for many of its host-specific forms. Therefore, knowing what races
have occurred in an area is important when selecting resistant varieties. Fusarium wilt fungi also can be seed-borne
and are easily moved on infected transplants. They can also be easily moved between fields in soil on equipment.
These are the major ways they are brought onto a farm. Drought, mechanical damage, low soil pH, soil compaction,
 and other stress factors can predispose plants to infection by fusarium wilt fungi. Fortunately, the fusarium wilts in
various crops are caused by different strains of the fungus Fusarium oxysporum (see sidebar 3.1). Thus, for
example, healthy muskmelons can be grown in a field where fusarium wilt previously affected watermelon.

Rhizoctonia diseases

When environmental conditions are favorable (warm and wet), Rhizoctonia solani, a common fungus in most soils
worldwide, can become a serious pathogen on susceptible crops. This fungus is subdivided into several strains,
which further complicates management. It has a wide host range. Potatoes, beans, lettuce, and cabbage are among
the most important hosts. Other hosts include broccoli, kale, radish, turnip, carrot, cress, cucumber, eggplant,
pepper, tomato, and sweet potato. Symptoms produced on a host can vary with the time of infection. For example,
in crucifers it causes damping-off of seedlings, wirestem in young plants, bottom rot in midseason, and head rot as
heads mature. Although difficult to manage, rotating away from the most susceptible crops for at least three years
can be helpful; cereals are an especially good choice for rotation crops. Incorporating residue of host plants can
help.

C. Species interactions (15)

Cultivated plants interact directly with other species, including pathogens, pests, symbionts and
weeds. Some interactions are beneficial, whereas others can cause devastating agricultural losses.
It remains a challenge to control deleterious species without causing significant environmental
damage, and there is untapped potential in developing improved interactions with beneficial
species, such as mycorrhizal fungi.

 C1.

What are the best ways to control invasive species including plants, pests and pathogens?
https://www.researchgate.net/post/What_are_the_best_means_to_control_invasive_species_including_plants_p
ests_and_pathogens

YOGESH CHANDRA TRIPATHI

 Forest Research Institute Dehradun

What are the best means to control invasive

species including plants, pests and pathogens?
Invasive species, most of which are exotic, have become significant threat to our environment, economy, health and well-being. The
best way of control is to prevent establishment in the first place by adopting prompt eradication action. However, if an invasive species
is established, options like chemical control or mechanical excavation for removal can cause environmental damage. Target specific
biological control can work well; otherwise there is a risk that the control organism might also become an invasive species. Alternative,
such as manipulating existing natural enemies and/or the environment to enhance biological control, are also on hand. Despite all these
tackling the problem of invasive species remained a great challenge. Hence what should be the sustainable solution and practicable
strategy to deal with the continually growing problem of invasive species?

Jack M Gallup
Boehringer Ingelheim/Newport Labs, Worthington, MN
"Stop all unnecessary commerce on planet earth" some would say. Invariably it comes back to the sad fact that humans are stressing
the planet out - but that equation always works out in a bad way. We may have to live in our own filth after a while. Then, and only then,
we might learn to not be so stubborn in our seeming current inability to change. The sooner we see ourselves as the primary
problematic invasive species on the planet, the sooner funds and self-preserving ideas might someday be used correctly. We take risks
in the way we tamper with the balance of things... and sometimes, the consequences of mother nature are just that. Why do we always
tend to kick the dog that bites us for a good reason?? We are the victims of our own activities, and there may not be a convenient
solution to this problem. it will take discipline and a hell of a lot of trust, love and confidence in one another to do the right thing. Some
things never recover from the damage done to them.

I advise to check very carefully whether the targeted plant is an "invasive species" and not an "envrionmental
present & treasure". Prosopis juliflora for example is hated in East and West-Africa and disregarded, while soberly
 looking it is a highly valuable, multi-usable tree/shrub for forage, fuel, food, timber, eco-treasure--- if properly
managed....and coping wonderfully with climate-change. If search-machines do not reveal any positiv feature, the
well-established measures have to be applied. While applying traditional measure, pursue the ecological features
and interdependancies to trace probably a much cheaper, easier approach and method of control. Widened
creativity is an option.
YOGESH CHANDRA TRIPATHI
Forest Research Institute Dehradun
@ Jack M. Gallup and Didier Andrivon - You are very correct "Invasive species are also the part of over living ecosystem". Migration of
a species from his native place to a non-native place due to human activities that has already crossed some dangerous lines has now
become a great problem due to a wide diversity of economic and environmental loss. We all should take some responsibility for
reducing the problems caused by invasive species by adopting both proactive and reactive management. Some very simple steps
include careful selection of garden plants, use species that are not likely to be invasive. When travelling everyone should notify customs
officials at ports of entry if he/she bringing in food, fruit, plants, wood and animal products, fishing, hiking, and camping equipment.
These things can be quickly examined by experts and determined if they pose a risk or not. Biological control approach can be effective
as it re-associates natural enemies with the invader in the new area in which the pest is causing problems. Natural enemies like
predators, parasites, and pathogens have been used most commonly to successfully control invasive weeds, and insect pests. This
form of pest control can be very safe if the correct natural enemies are used, and once established, effective natural enemies can
provide permanent control of the invasive species.

 C2.

Can we provide a solution to intractable plant pest problems in order to meet increasingly
stringent pesticide restrictions?

Tips for Reducing Pesticide Impacts on

Wildlife
When used properly, pesticides can play a valuable role in controlling weeds, insects, and other pests.
On the other hand, they can harm wildlife if the user does not follow label directions. Wildlife
includes non-target birds, mammals, fish, aquatic invertebrates, insect pollinators, and plants.
This Web page provides tips for pesticide users in residential and agricultural settings, as well as tips
for certified pesticide applicators for ways to protect wildlife from potentially harmful effects of
pesticides. You will also find links to some additional sources of information on wildlife and habitat
protection.
On this page:

 Tips for household pesticide users

 Tips for farmers
 Tips for certified pesticide applicators
 Some resources for reducing pesticide effects on wildlife

Tips for Household Pesticide Users

 Follow all requirements on pesticide product labels.
 Store and dispose of pesticides properly. See instructions on pesticide product label for product- or
chemical-specific instructions. Read more about:
o Safe Disposal.
o Storing Pesticides Safely.
 Inspect pesticide containers regularly for leaks and corrosion.
 Mix pesticides, clean equipment and rinse containers in an area where pesticides and rinse water
cannot enter sewers or storm drains.
 Keep pesticides out of waters and areas near waters.
 Minimize potential harm to birds, beneficial insects, and fish by using pesticides only when
necessary.
 Treat only the specific areas needing treatment.
 Most insecticides are toxic to bees. When using them outdoors, apply at night when bees are not
actively foraging.
 Read our new labeling to improve protection for bees and find more information about the advisory
box.
 Use bait stations for rodent baits that are formulated with food (e.g., peanut butter or grain bait) or
place the bait where non-target wildlife cannot get to it.
 Use landscaping techniques that help increase native habitat and reduce the need for pesticides.

Top of Page
Tips for Farmers
 Follow all requirements on pesticide product labels.
 Keep pesticides out of storm drains and gutters.
 Consider the characteristics of the application site (soil texture, slope, organic matter) before
applying the pesticide.
 Be aware of the geology and the relative depth of the groundwater in your area.
 Know which pesticides leach into groundwater. Pesticides with groundwater contamination potential
will include the following statement on the label:

This chemical has properties and characteristics associated with chemicals detected in ground water.
This chemical may leach into ground water if used in areas where soils are permeable, particularly where
the water table is shallow.

 Implement an Integrated Pest Management plan, which uses cultural, mechanical, and biological
pest controls where possible. (Read more about IPM)
 If a spill occurs, contain and clean it up immediately.
 Where possible, leave a border of untreated vegetation between treated areas and areas where
wildlife may be present.
 Take care when planting treated seeds to prevent dust that could affect bees.
 Follow label precautions designed to protect pollinators and be aware of any hives in the area that
could be affected by spraying.
 Read our new labeling to improve protection for bees.

Top of Page
Tips for Certified Pesticide Applicators
 Follow all requirements on pesticide product labels.
 Maintain all application equipment in good working order and calibrate it regularly.
 Check equipment for leaks and malfunctions before use to minimize the potential for accidental
spills.
 Rinse pesticide application equipment and pesticide containers on a solid surface where it won’t
drain to waterways.
 If not specified on the label, apply when wind speed is between 3 and 10 mph.
 For ground boom applications, apply using a nozzle height of no more than 2 feet above the ground
or crop canopy, unless a greater height is required for efficacy or safety.
 Use a low pressure, large droplet sprayer, and spray close to the crop canopy or the ground.
 Do not spray if heavy rain is expected within 48 hours as the pesticide may wash away from the area
of application and into water bodies.
 Where possible, leave a vegetative buffer strip between the field and areas where wildlife may be
present, including downhill aquatic habitats. Be sure to follow any label requirements related to
buffers, as well.
 Make sure you get and maintain proper training and certification.

Top of Page
Some Resources for Reducing Pesticide Effects on
Wildlife
Note: The listed resources external to EPA do not necessarily represent EPA’s opinion. For EPA's
most current pesticide-related information, please consult EPA resources.
Pesticide Use Around Homes

 Pesticides and Water Quality EXIT-- Information from the University of California Agriculture and
Natural Resources about how consumer pesticides can affect creeks, rivers, and oceans.
 Biopesticides Fact Sheet -- EPA’s biopesticides fact sheet explains the purpose of biopesticides and
the benefits of their use.
 Protecting Wildlife from Pesticides EXIT-- Information from the National Pesticide Information
Center (NPIC) on pesticide impacts on wildlife and tips to minimize exposures.
 GreenScaping -- EPA brochure with lawn care tips, including reducing pesticide use.
 Healthy Lawn, Healthy Environment -- EPA brochure provides consumers with solutions for an
environmentally friendly yard.
 Backyard Conservation -- USDA’s Natural Resources Conservation Service (NRCS) publication that
highlights 10 conservation activities adapted from farms and ranches that can be used in your
backyard.

Information for Farmers and Certified Applicators

 USDA’s NRCS Fish and Wildlife information -- includes a link to "Fish and Wildlife Technology
Findings," which provides access to a variety of guidelines and projects related to conservation, as
well as a link to the Migratory Bird Habitat Initiative, and other related information.
 USDA’s NRCS Conservation Buffers to Reduce Pesticide Losses (PDF) (25 pp, 1.23 mb, about PDF) --
explains how conservation buffers can be used to trap and degrade pesticides.
 Pesticides and Wildlife: An Introduction to Testing, Registration, and Risk Management (PDF) (40 pp,
2MB, About PDF)EXIT-- a comprehensive guide from Purdue University’s Cooperative Extension
Service about pesticides and wildlife. It covers the impacts on wildlife, the pesticide review process as
it relates to wildlife, laws, and agencies that oversee the protection of wildlife and tips for reducing
pesticide risk to wildlife.
 Integrated Pest Management (IPM) in Agriculture –- EPA website that describes IPM as an effective
and environmentally sensitive approach to pest management in agriculture.
 Wildlife and Pesticides EXIT-- Virginia Department of Game and Inland Fisheries’ overview of the
potential pesticide effects on wildlife, plus tips to reduce wildlife impacts and when and where to
spray.

 https://www.epa.gov/safepestcontrol/tips-reducing-pesticide-impacts-wildlife

 C3.

Is it desirable to eliminate all pests and diseases in cultivated plants?

New Directions for Biosciences Research in Agriculture: High-Reward

Opportunities.
5Plant Diseases and Insect Pests
The damage to plants caused by competition from weeds and by other pests including viruses, bacteria, fungi,
and insects greatly impairs their productivity and in some instances can totally destroy a crop. Today,
dependable crop yields are obtained by using disease-resistant varieties, biological control practices, and by
applying pesticides to control plant diseases, insects, weeds, and other pests. In 1983, $1.3 billion was spent on
pesticides—excluding herbicides—to protect and limit the damage to crops from plant diseases, nematodes, and
insects. The potential crop losses in the absence of pesticide use greatly exceeds that value.
For about 100 years, breeding for disease resistance has been an important component of agricultural
productivity worldwide. But the successes achieved by plant breeding are largely empirical and can be
ephemeral. That is, because of a lack of basic information about the function of genes for resistance, studies are
often random rather than specifically targeted explorations. In addition, any results can be short-lived because
of the changing nature of pathogens and other pests as new genetic information is introduced into complex
agroecological systems.
An excellent example of the effect of genetic change is the sterile pollen trait bred into most major corn
varieties to aid in the production of hybrid seed. Plants containing Texas (T) cytoplasm transfer this male sterile
trait via the cytoplasm; it is associated with a particular type of mitochondrion. Unknown to breeders, these
mitochondria also carried vulnerability to a toxin produced by the pathogenic
fungus Helminthosporium maydis. The result was the corn leaf blight epidemic in North America in the
summer of 1970.
The methods used in the discovery of pesticide chemicals also have largely been empirical. With little or no
prior information on mode of action, chemicals are tested to select those that kill the target insect, fungus, or
weed but do not harm the crop plant or the environment.
Empirical approaches have produced enormous successes in controlling some pests, particularly weeds, fungal
diseases, and insects, but the struggle is continuous, since genetic changes in these pests can often restore their
virulence over a resistant plant variety or render the pest resistant to a pesticide. What is missing from this
apparently endless cycle of susceptibility and resistance is a clear understanding of both the organisms and the
plants they attack. As knowledge of pests—their genetics, biochemistry, and physiology, their hosts and the
interactions between them—increases, better-directed and more effective pest control measures will be devised.
This chapter identifies several research approaches to a better understanding of the fundamental biological
mechanisms that might be exploited to control plant pathogens and insects. Molecular biology offers new
techniques for isolating and studying the action of genes. The existence of susceptible and resistant host plants
and virulent and avirulent pathogens can be exploited to identify and isolate the genes that control the
interactions between host and pathogen. Studies of the fine structure of these genes can lead to clues about the
biochemical interactions that occur between the two organisms and to the regulation of these genes in the
pathogen and in the tissues of the plant. It should be possible in the future to improve the methods and
opportunities for the transfer of desirable traits for resistance into crop plants and, conversely, to create
pathogens that will be virulent against selected weeds or arthropod pests. An increased understanding of insect
neurobiology and the chemistry and action of modulating substances, such as the endocrine hormones that
regulate metamorphosis, diapause, and reproduction, will open new avenues for controlling insect pests by
disrupting their physiology and behavior at critical stages in the life cycle.
Go to:

Molecular Bases of Plant-Pathogen Interactions

The existence of susceptible and resistant cultivars implies specificity in plant diseases. One explanation for this
high specificity is a ''recognition'' mechanism between pathogen and host. Understanding the molecular bases
that determine this specificity in recognition or in the pathogen's ability to alter the host's metabolism should
yield new, definitive, and more efficient ways to prevent attacks on crop plants or to mitigate disease symptoms.
Based on our current, limited understanding of the types of interactions that occur between host plants and
pathogens, the mechanisms involved are varied and complex. Theoretically, a minimum of two criteria are
involved. The first is recognition. There may be preformed molecules in both host and parasite that can interact.
Second, there must be metabolic changes in the host or pathogen or both that are triggered by the initial
interaction step. Genetic mutations in either host or pathogen can change the specificity of molecular
interactions or their ability to trigger metabolic change.
The following presents discussions on research directed toward possible mechanisms involved in recognition
between host and pathogen and the metabolic changes that cause disease symptoms.

Molecular Determinants of Resistance and Susceptibility

It is widely held that some forms of resistance to fungal and bacterial pathogens are the result of a host plant's
ability to synthesize chemicals that inhibit the growth and development of the pathogen. During infection by a
pathogen, metabolic pathways in the plant are activated, leading to the detectable biosynthesis of the inhibitors.
A major class of inhibitors, called phytoalexins, are primarily low-molecular-weight, secondary plant
metabolites that possess wide-ranging activity against fungi and, to a lesser extent, bacteria. In the last two
decades, more than 100 phytoalexins have been identified. The induction of the biosynthesis of phytoalexins,
however, does not follow the specificity that most pathogens have for a specific cultivar. For example,
phytoalexin synthesis can be induced by abiotic agents, such as wounding or other stress conditions, in both
resistant and susceptible plants. Phytoalexins can be toxic to both virulent and avirulent pathogens. It appears
that phytoalexin synthesis might be a general, nonspecific type of active resistance.
An alternate approach, the study of susceptibility, has revealed mechanisms that show a high degree of
specificity. Many pathogens possess specific agents for virulence, such as toxins or enzymes, that determine the
course of events in susceptible plants. In the last five years, six host-specific or host-selective toxins have been
chemically characterized. These toxins affect only susceptible cultivars and are produced only by specific
pathogens that can attack these same susceptible cultivars. One well-studied example is the toxin produced by
the fungus Helminthosporium maydis, mentioned earlier. The H. maydis toxin disrupts energy generation in
susceptible mitochondria that characterize the T cytoplasm of corn. Normal mitochondria are resistant and are
unaffected by the toxin because they apparently lack a receptor site for it.
Genetic specificity also exists for resistance and susceptibility to plant viruses, but there is no information on
how such genes act. With respect to plant viruses the term resistance is used rather loosely. Quite often only the
appearance of disease symptoms is considered. Thus, a plant that supports virus replication but shows no
symptoms is considered to be resistant because it superficially appears to be so. More correctly, that plant
should be called tolerant.
Recent observations suggest that one type of resistance may involve the ability of viruses to spread from cell to
cell in their hosts. The continuum of responses ranges from rapid and complete invasion of the whole plant by
the virus to slow invasion to circumstances where the virus is unable to spread from an infected cell, even
though it might replicate well there. Accumulating evidence indicates that viruses induce the synthesis of
proteins that are necessary for the movement of viruses from cell to cell. The host, however, depending on its
genotype, can in some way interfere with this protein. Although the process is poorly understood, it may be, in
part, the basis of resistance of plants to viruses.
In a sense, viruses might be thought of as packages of genes; they are composed primarily of RNA or DNA, and
they can replicate only in a favorable host cell environment. Studies of the interactions between vital RNA or
DNA and genes in the host cell can lead not only to an understanding of how viruses function but also to the
development of viruses as gene-carrying vectors for genetic engineering.
An improved understanding of the basic concepts controlling resistance and susceptibility will result from
research based on interrelated approaches to the analysis of the genetics of these traits, the gene products, the
structure of the genes, and the methods that will permit their transfer between organisms.

Genetics
Continued breeding studies and genetic analysis of resistance traits in host plants and virulence traits in
pathogens provide the experimental systems needed to isolate and determine the properties of recognition
molecules involved in susceptibility or active resistance, such as phytoalexin biosynthesis.
Single-gene changes that confer resistance against a pathogen exist and are used in crop breeding to develop
improved cultivars. In other cases multiple genes appear to be involved in resistance, and complicate crop
breeding. The growing collection of data on the genetics of host plants and particularly of pathogens needs to be
strengthened. Such data are essential for identification of the genes that control the specificity of receptor
molecules, which determine resistance or susceptibility to bacteria, fungi, or viruses. Genetic analysis of some
important fungal pathogens, however, will be difficult because sexual reproduction does not occur, and the
modes of genetic reassortment and inheritance are unknown.
Many genetic approaches are now being initiated. For example, single-pathogen genes responsible for disease
reactions in two bacterial leaf-spot diseases, soybean blight and bacterial spot of tomato, are being isolated and
cloned. These techniques have potential for wide application.

Gene Products
The end product of most genes is a protein. There is little direct evidence for the role of any specific proteins in
controlling interactions between a host and a pathogen. Many potential candidates, however, can be
hypothesized. By analogy with animal systems, surface molecules, such as membrane glycoproteins, may
interact with low-molecular-weight messenger molecules, such as small carbohydrates released from cell walls.
Cell wall extracts from both hosts and pathogens have been shown to elicit some resistance responses. Both the
hydrolytic enzymes that release carbohydrate fragments from cell walls and the enzymes involved in the
biosynthesis of toxins or phytoalexins are gene products that may be selected for study.
Additional basic information is needed about the cellular interactions between host and pathogen during the
onset of resistance reactions. For example, the precise mechanisms employed by phytoalexins to exert their
effects on pathogens are unknown and need to be actively studied. Metabolic pathways for the biosynthesis of
phytoalexins must be clarified, and other compounds associated with resistance need to be identified. The
regulation and coordinated synthesis of the enzymes involved in these pathways must be detailed.
In addition, the phenomenon of acquired resistance in plants needs further study. Resistance can be localized or
can occur throughout the plant. Systemic resistance, however, may be of more practical value. This
phenomenon can appear after a host plant is inoculated with an avirulent strain of the bacterial, fungal, or vital
pathogen. This exposure somehow induces resistance properties so that when the plant is subsequently
challenged by one or more pathogenic strains, it will resist infection or exhibit only mild disease symptoms.
Acquired resistance is most actively being studied using Pseudomonas solanacearum, some strains of which
cause wilt and stem rot in tobacco, ginger, potato, tomato, and banana. Other avirulent strains only induce
resistance. The experimental approach is to find mutants of the avirulent strains that fail to induce the acquired
resistance. A comparison of the gene libraries of the active with the inactive mutants could lead to the
identification of the genes and gene products responsible for triggering the acquired resistance.

Gene Structure
Once the genes and gene products are identified, it is feasible to alter their activity by changing the structure of
the gene itself. The tools of molecular genetics can be used to study both the structure and activity of pathogen
genes for virulence and avirulence and host genes for resistance and susceptibility. Some progress has been
made recently with bacterial pathogens, particularly in characterizing some virulence factors such as pectolytic
enzymes. Much of the basic information on the molecular biology of fungal pathogens, however, is yet to be
acquired.
The functions of proteins coded for by vital genomes must also be established to aid in the understanding of
their possible roles in replication and pathogenesis.
It may now be possible to isolate genes for specific types of resistance, such as that characterized by so-called
hypersensitive lesions. For example, certain plant species and cultivars respond to infection by a pathogen by
rapidly undergoing cell necrosis at the site of infection. The hypersensitive lesion can effectively stop the spread
of a virus or confine the bacterial or fungal pathogen. In the latter two cases, the pathogen then dies.
This response is controlled in most cases by a single, dominant gene in the host plant. One approach to study of
the mechanism controlling development of the hypersensitive lesion would be to first isolate messenger RNA
from infected plants—those induced to give a hypersensitive response and those with a suppressed
hypersensitive response. The mRNA from the suppressed plants could be used to prepare complementary DNA.
This complementary DNA should recognize and hybridize with all the mRNAs from induced plants, except for
those involved in the hypersensitive response. In principle, the remaining free mRNAs could then be used to
probe a gene library of the hypersensitive plant for the gene that they can hybridize with. This gene should be
the one responsible for inducing the hypersensitive lesion.

Gene Transfer
The ultimate goal of research discussed in this section on genetics, gene products, and gene structure is the
routine transfer and expression of genes for resistance in agriculturally useful plants. As noted in the earlier
chapter on genetic engineering, some bacterial and vital pathogens may be developed as suitable carriers for the
transfer of genes into host plants. Current and prospective vectors take advantage of naturally occurring,
intimate associations between microorganisms and plants, both pathogenic and beneficial. An appreciable effort
is needed to identify and obtain suitable vectors in addition to the one successful vector, the Agrobacterium Ti
plasmid that can be used in some dicotyledonous plants.
The techniques necessary to manipulate vectors are available and will likely be refined and improved within the
next few years. It is, unfortunately, the lack of knowledge of the basic biology of plants and of the function,
transfer, and expression of genes that restricts progress in this area.
Go to:

Molecular Basis of Cellular Damage in Susceptible Hosts

Although it may appear that research on cellular damage and disease symptoms is a subset of the research
discussed previously on resistance and susceptibility, its intent is distinct, but of equal major importance.
Research emphasis in this area will yield insights into the biochemical mechanisms that result in cellular
damage, or disease, following successful pathogenic invasion. As yet there is no clear explanation of how major
symptoms, such as the yellowing and loss of chlorophyll in chlorosis or the tumors, galls, and morphological
changes caused by cellular growth distortion, are induced once a virulent pathogen becomes established in a
tissue. It may be possible to ameliorate symptoms or prevent crop damage directly by treatment, if the
biochemical details are known. The little-understood phenomenon of natural tolerance to disease is evidence
that such treatment should be possible. Indeed, the study of natural tolerance may be a valuable guide for
developing disease protection traits for crop improvement.
Easily observable disease symptoms, such as chlorosis, necrosis, and cellular growth distortions, can have a
number of diverse causes. Therefore, it is not possible to make progress on such generalized disease symptoms
without some indication of the kinds of pathogens involved. Some research approaches hold promise for
establishing general scientific principles of host-pathogen interactions.

Mode of Action of Toxins

Research in the last decade on purification and structural characterization has led to an acceptance of the
concept that toxins are the potent chemical agents of virulence in many important diseases caused by bacteria
and fungi. Only a small number of toxins have been chemically identified. Even fewer have a postulated target
or receptor site in the host cell, as was described earlier for Helminthosporium maydis. But even in these few
cases, it is not known how interference with the target site leads to cell damage. Much additional research is
needed on toxins—on their genetics, such as chromosomal versus plasmid inheritance; on their chemical
structure; on the pathways of biosynthesis in pathogens; and on their biochemical effects and role in
pathogenesis.

Nucleic Acid Interactions

It is clear that the mere replication of a virus or viroid within a plant does not determine whether that plant will
be diseased. There are many examples of strains that produce a great deal of virus, but with very little damage
to the host. On the other hand, some of the most serious plant diseases are caused by viruses that replicate very
sparingly.
Viruses, with their small genomes, have too little genetic information to code for the variety of proteins
necessary to account for the almost infinite number of symptom types. Thus, it seems likely that interactions
between the nucleic acid of the pathogen and that of the host initiate the disease process. Viroids, which are
RNA molecules that do not code for a protein product, can cause symptoms similar to those caused by viruses.
This lends support to the supposition that viruses as well as viroids interact directly with the genome of the host
plant.
Complete nucleic acid sequences are now available for several viroids; for satellite RNAs, which modify the
symptoms of their carrier viruses; and for a few plant viruses. Complete complementary DNA clones have been
made for some of these RNA agents and have been shown to be infectious. Because DNA is technically easier
to modify than RNA, such DNA clones provide the opportunity to make site-specific modifications in the
sequence of the nucleic acid by inserting or deleting short stretches of DNA. The effect of such changes on the
agent's ability to infect and on the symptoms produced can then be determined.
Using current methods the nucleotide sequences responsible for the disease syndrome should be identified.
Furthermore, these complementary DNA clones could also be used in hybridization studies to locate regions in
the host genome where the host and the virus, satellite RNA, or viroid sequences interact. As knowledge of the
fine structure of the host's genes increases, future studies should enable researchers to determine the specific
genes and processes that are perturbed by the presence of the pathogen.
If the DNA clones themselves are not infectious, the cloned viral or viroid DNA can be transcribed back to
RNA using any of several in vitro systems. Thus, site-specific modifications made in the DNA clone can be
transcribed into the RNA to test the effect of such changes on infectivity and disease symptoms. In this manner,
critical regions of the genome could be identified, which would aid in the understanding of their functions and
possibly the functions of viral-coded proteins.

Bacterial Interactions
Bacteria that cause diseases in plants cause symptoms, at least in part, by the production of various metabolites.
Relatively few of these substances have been identified. The metabolites include, but are not limited to, toxins,
polysaccharides, pectic enzymes, and plant hormones. All the bacterial toxins identified to date appear to be
general toxins affecting a wide spectrum of plants. Many of these plants are not considered to be host species
for the bacterial pathogen producing the toxin.
Other bacterial metabolites appear to have specific effects on host plant species. Bacterial polysaccharides,
which are associated with wilting of plants, can be released in amounts great enough to clog up transport
between plant cells, and may act by disrupting plant cell membrane functions. Soft rots, for example, are the
result of bacterial enzymes that degrade the cementing pectin layer between plant cells. The production of plant
hormones by bacteria disrupts the endogenous hormone balance in the host plant and can be part of the
mechanism leading to crown gall tumors and other abnormal growths.
The molecular and genetic bases of the synthesis of these pathogen metabolites and the basis of the symptoms
they cause in the host plant are largely unknown. There is increasing knowledge, however, about the genetics of
some of the bacterial virulence factors that contribute to the severity of a disease. For example, in crown gall,
which is caused by Agrobacterium, both bacterial chromosomal and plasmid genes are known to be required for
pathogenicity. The molecular genetics of crown gall is the most thoroughly studied of any plant disease.
In the genetic analyses of virulence in bacteria, two different approaches are currently being used. One is the
introduction of transposons into virulent strains of bacteria to create avirulent mutants. The transposon is used
as a probe to locate and isolate the turned-off virulence gene. DNA clones of virulence genes can be used for an
analysis of gene products. The second approach is molecular and genetic analyses of known or suspected
determinants of pathogenicity, such as cell surface components, hormones, toxins, and extracellular enzymes.
Both approaches hold promise for the elucidation of the biochemical steps in pathogenesis. There is an essential
need to have basic knowledge about the structure, function, and regulation of virulence factors in the pathogen
to provide a basis for directed plant breeding and to design effective inducers of plant resistance.

Research Status
It is important to recognize that considerable expertise and training in molecular biology are necessary for many
of the research approaches discussed in this section of the report. Progress is facilitated by individuals working
together in groups. Interactions with researchers in other laboratories are important sources of intellectual
stimulation as well as sources of technical expertise.
The tools of genetics and molecular biology offer some new methods for understanding the highly specific
interactions between host and pathogen. Studies of the molecular aspects of plant pathology must receive high
priority and emphasis within the ARS research programs on plant diseases.
Currently the ARS research centers are undertaking relatively little basic work in molecular plant pathology.
The ARS does have a few strong research programs in virology and in viroids, but very little work at the
molecular level is being conducted with bacteria or fungi. A single laboratory, at Beltsville, is studying plant
mycoplasmas.
To strengthen programs in the molecular basis of plant diseases, research investigations should emphasize:
 The molecular bases of the factors that determine whether a host-pathogen pair will result in a resistant
or a susceptible interaction.
  The basic concepts of the interaction between the host and the invading pathogen that result in disease.
This should lead to novel methods of preventing damage from disease, including natural plant tolerance.
 The transfer of resistance traits to normally susceptible plants through the development and subsequent
exploitation of vector systems that allow for gene transfer between plant species.
It is significant to note that very few laboratories in the world have undertaken studies to understand the
molecular basis of plant diseases from gene identification to disease symptoms.
Go to:

Modification of Microorganisms for Biological Control and Organic Pesticide

Disposal
The reliance on chemical-based pesticides, the increasing occurrence of pesticide resistance, particularly in
insect pests, and the potential of such agricultural chemicals for polluting the environment are of increasing
concern. The search for ways to address these concerns has led to greater emphasis on biological control.
Biological control methods involve the use of one organism to mitigate the undesirable effects of another. Two
complementary approaches are (1) the identification, biological characterization, and genetic engineering of
crop-enhancing microorganisms, especially those that can be applied to seeds or roots to promote improved
growth or yield; and (2) the development of genetically engineered microorganisms to remove organic pesticide
residues, such as herbicides.

Microbial Agents for Biological Control

Little commercialization of microorganisms for biological control has been done. Yet there is great potential for
research and development in this area. Knowledge of the basic biology of viruses, bacteria, fungi, nematodes,
insects, and weeds is essential for identifying and developing naturally occurring antagonists as biological
control agents. This includes knowledge of the growth and metabolism of the organisms obtained from both
laboratory and field studies. Fundamental knowledge of the biological basis of the control mechanism and the
ecology of both organisms involved is needed to successfully manipulate them to full advantage.
Interactions among several disciplines will be necessary. Soil physicists, meteorologists, computer modeling
experts, and analytical chemists, as well as biologists with expertise in areas such as ecology, microbiology, and
genetics will be essential contributors. The potential opportunities to use pathogens and pests against terrestrial
and aquatic weeds, and against pathogens of agronomic crops, forest trees, and ornamental plants are enormous.
Biological control is not generally expected to substitute totally for chemical control, but will supplement or be
integrated with it.

Control of Pathogens
The proven feasibility of using biological control in this area has spurred research. The
bacterium Agrobacterium radiobacter is commercially used to prevent infection of susceptible plants by a
related bacterium, A. tumefaciens, which causes tumorous galls to form on many plants. The
fungus, Peniophora gigantea, is used to control another fungus, Hetero-basidion [=Fomes] annosum, which
causes root rot of pine trees. In both these cases the control mechanism is not completely understood, but is
believed to result from competition between the control microorganism and the disease-causing organism for
specific binding sites on the host plant. Also, it may be that the control organism can elicit a resistance reaction
in the host. In the case of A. radiobacter, an antibiotic, agrocin 84, produced by the A. radiobacter has been
identified as the possible mechanism in that example of biological control.
Biological control is also illustrated by the epiphytic bacterium Pseudomonas syringae. Both pathogenic and
nonpathogenic strains of this bacterium are known to synthesize ice-nucleating proteins. When these bacteria on
the plant surface are killed by antibiotic treatments or displaced with mutants of P. syringae that have lost their
ability to synthesize ice-nucleating proteins, the plant can tolerate chilling to -7°C without frost damage. Mutant
strains have been produced both by mutagenesis using chemicals or ultraviolet irradiation and by removing the
ice-nucleation protein gene using genetic engineering techniques.

Control of Insect Pests

Alternative strategies for the control of insect pests need to be developed to augment the chemical and
biological approaches currently in use. Some success has been achieved with Bacillus thuringiensis, used
commercially for the biological control of some insects. This bacterium, when ingested, is lethal to the
caterpillar stage of many insects. The bacterium harbors a toxic crystalline structure that dissolves in the
alkaline hind-gut of susceptible caterpillars, resulting in disruption of digestion and death.
The use and genetic manipulation of insect pathogenic bacteria and viruses constitute a promising but
comparatively underdeveloped approach to insect control. The potential exists for genetically improving these
organisms to increase their pathogenicity, either by enhancing existing pathogenic traits or by introducing
desirable pathogenic characteristics.
Basic knowledge about potentially useful pathogens must be acquired. This includes identification of the
pathogen and characterization of the insect host. The specificity between pathogen and host and the techniques
for production and storage of candidate pathogens must also be studied. With this information the physiology,
biochemistry, and genetics of the host-pathogen interaction can then be investigated. More specific areas of
study include the molecular basis of processes such as recognition, virulence, and toxicity and the mechanisms
regulating gene function during these interactions.
Progress in this line of research is apparent from the work of many laboratories worldwide. Candidate
microorganisms identified by this research include baculoviruses and Bacillus thuringiensis. With recent
developments in insect cell culture, some of the fundamental processes detailed here, in principle, can be
directly probed in vitro with any of these microorganisms.

Control of Nematodes
Control of plant parasitic nematodes has been largely accomplished through the use of chemical nematocides,
many of which have now been shown to be harmful to the environment and have been withdrawn from use.
Biological control measures using resistant plant varieties and trap crops have been effective in some cases. A
trap crop can stimulate the hatching of nematode eggs but does not support nematode growth, thus reducing
nematode populations to harmless levels.
More information is needed on the basic biology of nematodes to provide directed approaches to their control,
using less toxic, target-specific substances. This might include the use of the hatching stimulants that are
apparently produced by plants and trigger nemarode eggs to hatch. The growing nematodes then perish in the
absence of a suitable host plant. Studies of nemarode pheromones and hormones could lead to methods for
controlling reproduction or development.

Plant Health Microorganisms

In recent years some information has been gathered on soil microorganisms, specifically, certain bacteria, that
can improve plant vigor and contribute to increased yields. The mechanisms by which such bacteria exert these
effects are essentially unknown, nor are their relationships to pathogens or other microorganisms in the
environment well understood. Indeed, candidate organisms suited for particular crops remain to be identified
and characterized. Such bacteria contribute a desirable and perhaps essential microflora for optimal plant
growth. While a range of microflora is known to be essential for human health, virtually nothing on a
comparable basis is known for plants.
Several mechanisms have been suggested that describe the effects of soil microorganisms on plant health.
Beneficial microbes may produce antibiotics that inhibit the growth of pathogens, or they may be involved in
the acquired resistance phenomenon. Recent evidence suggests that some plant growth-promoting bacteria
produce siderophores, iron-chelating molecules, that restrict the availability of this essential element to
pathogens and other members of the microflora.

Biological Degradation of Organic Pesticides

Timely and appropriate disposal of pesticide residues in water and soils is an important and attainable goal in
routine agricultural production practices. The biological degradation of pesticides is theoretically feasible. For
example, pseudomonads have been identified as being able to degrade the herbicide 2,4-dichlorophenoxyacetic
acid (2,4-D) to innocuous compounds. Lack of knowledge of the chemistry, the fate of breakdown products, and
the ecology of the organisms involved, however, is still a constraint to their use.
Both waste disposal of agricultural by-products and biomass reduction on an industrial scale are under intensive
investigation. The processes are not commercially feasible as yet, however, because of low yields and organism
management problems. These problems can be overcome using genetically engineered organisms, especially
bacteria, that are currently more amenable to manipulation than other microorganisms.

Research Status
The ARS laboratories are among those contributing to progress in biological control of plant pathogens and
insect pests. With increased emphasis, the ARS could be at the forefront of this research. The potential return
for the ARS extends beyond the control of plant pathogens and insect pests; it would involve the development
of general methodologies for gene transfer, cloning, and gene expression using microbial and insect systems.
Basic research on the microflora of the rhizosphere is also an area that ARS can strengthen.
There is enormous potential for the identification, development, and application of microorganisms that can
degrade pesticide residues and other toxic wastes. The ARS should expand its efforts—some of which are
exemplary—in these areas. It is high-risk, long-range research and requires the multidisciplinary base that is
already in place in some locations.
Specifically, the ARS should focus research toward:
  Exploring and identifying microbial agents that can control plant diseases and insect pests. Further, the
agency should seek conventional genetic or recombinant-DNA technologies to make these agents more
effective;
 Generating more knowledge of the basic biology of plant pathogenic nematodes to develop novel,
nonpesticide means of control by perturbing reproduction and development; and
 Developing unique microorganisms that will promote plant health and others that can be used to
detoxify or destroy organic pesticide pollutants.
Go to:

Molecular Basis of Pesticide Action

Pesticides are major tools in the production of food and fiber and in the maintenance of high standards of
veterinary, human, and plant health. Better pesticides are needed, relative to cost effectiveness, potency,
selectivity, persistence, environmental impact, and safety for domestic animals, humans, and plants. Most of the
early pesticides were discovered in industrial programs involving the synthesis and screening of thousands of
synthetic chemicals for safe and effective molecules. The emphasis in current discovery efforts favors research
on the natural chemicals produced by plants and microorganisms, such as occurred for the pyrethroids. Equally
important are investigations into the molecular basis of pesticide action.
Advances in bioregulation research provide new vistas in seeking enzyme or receptor targets for pesticide
action. Increasing fundamental knowledge of the function and regulation of communication systems within
living organisms focuses attention on new targets and greatly facilitates the molecular design of optimal
compounds for pest control. Greater diversity is needed in the targets for future pesticides, such as insecticides,
herbicides, nematocides, and fungicides to avoid or minimize the impact of pesticide resistance and toxicity
against non-target species. Susceptible and tolerant species often differ only in the sensitivity of their pesticide
receptor site or their facility for detoxifying the pesticide.
A clear definition of the mechanisms involved will provide the background for the next generation of improved
pesticides. New pesticides, in turn, provide unique probes to explore cellular entities such as enzymes,
receptors, and membranes.
The molecular basis for metabolic activation and detoxification must be defined. Using this background
knowledge genetic engineering can provide opportunities for modifying receptor sites and detoxification
mechanisms for improved animal and crop safety.

Research Status
Research on the molecular basis of pesticide action is carried out in many laboratories within industry,
universities, and the ARS. Industrial labs tend to focus on the modes of action of their proprietary compounds.
Universities more often use pesticides as probes for physiological and pharmacological investigations. The ARS
has placed considerable emphasis on the mechanism of pesticide action. The laboratory defining a new target
often reaps the benefit of finding alternative agents working at the same site or in the same way.
Research on pesticide mode of action requires the creative teamwork of biochemists, chemists, and geneticists
with adequate instrumentation and the appropriate environment to stimulate communication. This multi-
disciplinary approach and the requisite personnel are now in place in several ARS laboratories. The ARS should
increase its emphasis on the molecular basis of pesticide action, using the available expertise in microbial, plant,
and insect physiology, biochemistry, and natural products chemistry. Success in this program will serve as the
basis for improving animal health and for reducing crop losses during production and storage.
More specifically, the ARS should emphasize:
 Definition of the molecular basis for metabolic activation and detoxification of pesticides;
 Study of new targets for selective pesticide action;
 Identification of new natural chemicals important in regulating pest populations;
 Investigation of the basic molecular biology of vectors for gene transfer and elucidation of gene
regulation in insects; and
 Continued research on both insect genetics and on natural products chemistry.
Go to:

Insect Neurobiology and the Regulation of Development and Reproduction

The functional responsiveness of an insect is dependent on rapid chemical communications among its own cells
and between the individual and other insects. Intercellular communication is mediated primarily by the nervous
system, through substances such as neurotransmitters, neurohormones, and neuromodulators as well as by the
endocrine system, through hormones. The endocrine system is closely coupled to the functioning of the nervous
system. Communication between individuals is achieved through volatile chemicals called pheromones. Their
production and action is mediated by the nervous system.

Insect Neurobiology
The function of the nervous system makes it a logical focus for investigations of alternative means of insect
control that could potentially have considerable selectivity. Before investigations can be initiated, however,
basic information about the function of the insect nervous system must be obtained, specifically, information
about nervous processes involving chemical communication. This approach is the only potentially successful
avenue to the solution of applied research problems. For this reason a research emphasis in fundamental insect
neurobiology should be developed by the ARS.
Insect neurobiology is now experiencing a period of exponential growth. Despite the fact that the insect nervous
system has been used for many years as a model for studying certain neurophysiological processes, basic
research using modern techniques has only recently begun on the neurochemistry, neuroendocrinology,
neurogenetics, and neuropharmacology of the insect nervous system.
For example, the number of identified insect peptides with neurohormonal activity is fewer than 20. Only 4 of
these insect neurohormones have been purified and sequenced. These include the
neurotransmitter/neuromodulator proctolin, the two adipokinetic hormones, and cardiac accelerator peptides.
Proctolin is important in the stimulation of muscle contraction and is co-released with other neurotransmitters.
The adipokinetic hormones mobilize lipid for its metabolism by muscle in insect flight, and the cardiac
accelerator peptides control the heartbeat of the insect. It now appears that the structures of the
prothoracicotropic hormones, the primary effectors of insect metamorphosis and the first hormone of neural
origin described for any animal (1917), are finally being resolved. In addition, a new brain peptide that regulates
the production of pheromones has been described and promises to introduce a renaissance in pheromone
research.
Study of these and of yet-undiscovered hormones will aid in an understanding of the physiology of the insect,
its growth and development. Such studies will also define the mechanisms by which the central nervous system
integrates and regulates these processes. This understanding may allow scientists eventually to selectively
manipulate the neuroendocrine system, and thus control insects by altering their ability to fly, curtailing
metamorphosis, or disrupting sexual recognition. The study of neurohormones may not provide an immediate
answer to insect control. The resulting knowledge, however, will provide scientists with the sound foundation
necessary to propose and pursue new directed and applied research on the neural regulation of insect growth
and development.
The top scientific priority for neurobiological research on insects is the elucidation of the mechanisms by which
chemical communication directs and coordinates the growth, development, homeostasis, and reproduction of
insects. The basic information still lacking includes the identification of neural regulators and an elucidation of
their chemistry, synthesis, secretion, and metabolism.
Other opportunities for manipulation of insect pests include the neurohormones bursicon, diuretic hormone, and
egg development neurotropic hormone. Bursicon causes the insect skeleton to harden. Inhibition of the secretion
of this hormone would cause death. Manipulation of the diuretic hormone, which regulates water and salt
balance, might also result in death, through ionic imbalance and dehydration. Secretion of egg development
neurotropic hormone from the brain of the female mosquito is stimulated following a blood meal. The hormone
indirectly causes the ovary to mature the eggs. Manipulation of this reproductive hormone would prevent the
development of generations of mosquitoes.
These hormones are examples of the potential in this field. To realize this potential the hormones must be
studied extensively at the chemical, molecular, and physiological level.
At this point a major research program encompassing the physiology, biochemistry, and molecular biology of
these regulators can be initiated. Research should include the study of mechanisms of communication within the
nervous system, between organs and organ systems, and between individuals of the same species. Studies of
interorganismal communication should emphasize the neuroendocrine and neural bases of this process and
relate this communication to behavioral patterns in nature.
Knowledge gained from such a fundamental research program in insect neurobiology could be used in
conjunction with genetic engineering methodologies to investigate the basic molecular biology of vectors for
gene transfer and to elucidate gene regulation in insects. These new technologies could also aid in mapping the
insect genome, particularly the genes for regulatory peptides.
Peptides offer researchers an extremely important direct line of study; they probably are all products of single
genes. An understanding of these gene products or polyprotein precursors and their posttranslational processing
to a bioactive peptide is essential for the potential control of insects. (Posttranslational processing, which
follows the translation of RNA, is proving to be a fundamental mechanism that determines the protein nature of
the neurosecretion from a given cell.) A disruption of the synthesis or processing of neurohormones would be
lethal.
The long-term goal of this research is modification of the normal function of the insect nervous system to affect
viability. A research program on insect pathogens as vectors for gene transfer would clearly be important in
achieving this objective.

Endocrine Regulation of Metamorphosis, Diapause, and Reproduction

The postembryonic development of the insect involves a series of dramatic physiological and biochemical
transformations that culminates in its emergence from a pupa as an adult form with its own unique function. It is
generally accepted that these transformations and their associated metabolic processes all are directly or
indirectly under endocrine control, including production of hormones by neural tissue. The full extent of the
role of the endocrine system is not completely known, mainly because of a lack of knowledge of the hormones
involved, the molecular basis of the developmental and reproductive processes these hormones control, and
their mechanisms of action. The progress made in this field in recent years has largely been at a descriptive
level. Thus, basic research is needed to identify and chemically characterize insect hormones and to define at
the molecular level both their physiological function and their mechanism of action.
Although some insect hormones, such as the sesquiterpenoid juvenile hormones and the ecdysteroids, have been
intensively investigated, the extent of their involvement in regulating insect development and reproduction is
only now being realized. They are known to exist as structural and functional families of molecules, each acting
at a specific time during the life cycle of the insect. The multiple functions of these hormones provide multiple
avenues for pursuing control of the insect. Substantial stantially more research is needed, both in the above-
cited areas as well as on the mechanisms of their interaction at the level of the target gland and interendocrine
feedback control. Research studies must be designed to show how these hormones regulate one another's
synthesis and secretion to drive development and growth.
A virtually unknown family of insect regulators that control metamorphosis, diapause, and reproduction is the
peptides. Only a few have thus far been identified, and as has proved to be the case with vertebrates, there are
numerous peptide hormones involved in the control of embryogenesis, postembryonic development,
reproduction, and homeostasis. These peptides need to be characterized, their physiological functions defined,
and mechanisms of action elucidated.
The regulation of the synthesis, secretion, and metabolism of these insect hormones, whether peptide, steroid, or
other chemical structure, is another relatively unexplored research area of considerable significance and
potential application to the control of insects. The secretion of these hormones has consistently been shown to
be precisely regulated, frequently in response to discrete environmental cues such as photo-period, temperature,
and stress. The mechanisms by which these cues are transduced by the nervous system to elicit an endocrine
response are important areas for basic research in insect neurobiology.
Knowledge of the regulation of insect development and reproduction is applicable to the manipulation of these
systems for improved pest control. Some natural and synthetic chemicals, including insecticidal compounds,
alter growth and development by inhibiting the biosynthesis or action of juvenile hormones or ecdysteroids and
by governing the initiation and termination of diapause. Certain antibiotics and the highly insecticidal
benzoylphenyl ureas interrupt chitin synthesis necessary for the formation of the insect cuticle or skeleton.
Studies on insect genetics indicate the possibility of breeding sterile hybrids for use in pest control. Bacteria and
other microorganisms producing insecticidal materials and the plant itself may also be modified by selection
and genetic engineering to increase the impact of natural toxicants or feeding deterrents in host-insect pest
interactions. Further development of insect cell cultures and vectors for gene transfer in insects may permit the
introduction of deleterious effects into pest populations.
The benefit from research in insect neurobiology is not the potential control of insect pests alone. Although the
insect is a relatively simple system structurally, it is functionally complex, much like that of vertebrates. An
understanding of the insect endocrine system will lead to a further understanding of similar processes in all
eukaryotic organisms.

Research Status
The ARS is recognized worldwide for developing the sterile insect release method of control and for
investigations on insect genetics and ecdysteroids. The agency also is internationally recognized for natural
products research, particularly pheromone chemistry, and the application to insect development and
reproduction. This type of interdisciplinary research requires a coordinated team of entomologists,
physiologists, biochemists, and chemists.
There are a number of ARS laboratories currently conducting excellent research on the physiological and
chemical aspects of endocrine control of insect development and reproduction. By bolstering these existing
programs with the appropriate additions of scientists skilled in protein chemistry, basic biochemistry, and the
study of nuclear and membrane proteins as receptors, the ARS should be able to make substantial contributions
to this research area.
Although the ARS is becoming increasingly more involved in fundamental insect neurobiological research, this
program is not developing in a focused manner. While most of the research skills necessary for a major program
in insect neurobiology—chemistry, neurophysiology, behavior, biophysics, and physiology—are already in
 place within the ARS, additional expertise in neurochemistry, peptide chemistry, and biochemistry (mechanistic
aspects or chemical regulation), and immunology must be added. Generally, adequate instrumentation for this
research exists within the ARS. Analytical facilities are needed, however, for peptide and neurotransmitter
structural identification.
Of the few laboratories worldwide engaged in insect neurobiological research, a number are emerging as centers
of excellence. The comparative paucity of such centers, however, means that relatively few neurobiological
systems are currently being explored. Thus, the scientific opportunities in this field are enormous.
Unfortunately, the lack of basic information has created a situation wherein the most important areas of research
are high risk and will require considerable effort and resources. Such high-risk research is well suited for
government-supported organizations like the ARS.
To date, a multidisciplinary program in insect neurobiology does not exist. The ARS has an opportunity to
establish the first program of this kind. The success of such a program greatly depends upon the centralization
of research at a single site, preferably near a university or another research institute that has a strong program in
neurobiology.
ARS research should specifically focus on the following:
 Chemistry of the brain factors that control pheromone production and release, and their mechanisms of
action;
 Neural regulation of the synthesis, processing, and secretion of cerebral pheromonotropic peptides;
 The endocrine basis of insect reproduction, in particular, identification of the cerebral neuropeptides
involved and their target glands, and identification of the mechanisms regulating these glands;
 Mechanisms that regulate the synthesis of ecdysteroids and juvenile hormones, and the biosynthetic
pathways of these two hormome families; and
 Interhormonal endocrine feedback; regulation of insect growth, development, and reproduction; and the
roles and molecular mechanisms of the principal developmental hormones in regulating one another's
synthesis.
 https://www.ncbi.nlm.nih.gov/books/NBK216440/

 C4.

What is the most sustainable way to control weeds?

PDF sustainable_weed_management

 C5.

How can we simultaneously eradicate hunger and conserve biodiversity?

PDF eradicating hunger

 C6.

How can we move nitrogen‐fixing symbioses into nonlegumes?

PDF nitrogen fixation

 C7.

Why is symbiotic nitrogen fixation restricted to relatively few plant species?

PDF nitrogen fixation

 C8.

How can the association of plants and mycorrhizal fungi be improved or extended towards
better plant and ecosystem health?
 Benefits of Mycorrhizae
Mycorrhizae are soil fungi that benefit the soil in many ways. A healthy soil is important for a
water-wise landscape. Organic matter, drainage, and plant nutrients contribute to the fertility
and health of the soil and plants found therein.

Mycorrhizae literally means “fungus root” and describes a mutualistic association between
fungus and plant roots that exists in almost all plants. The plant supports the fungus by providing
carbohydrates needed for fungal growth, while the fungus helps the plant by increasing its root
surface area.

Potential Benefits of Mycorrhizae:

 Enhanced water and nutrient uptake

 Reduction of irrigation requirements
 Reduction need for fertilizer
 Increased drought resistance
 Increased pathogen resistance
 Increased plant health and stress tolerance
 Higher transplanting success
There are many types of mycorrihizae. Remember that most plants naturally contain
mycorrhizae and may not benefit from the addition of mycorrhizal fungi. However, in cases
where plants are being transplanted from or into sterile soil, adding mycorrhizal fungi may be of
benefit.

https://landscape-water-conservation.extension.org/benefits-of-mycorrhizae/

 C9.

How do plants communicate with each other?

Plant Talk
Plants communicate and interact with each other, both aboveground and
below, in surprisingly subtle and sophisticated ways.
Jan 1, 2014
DAN COSSINS
 ©
POP_JOP/ISTOCKPHOTO.COM

I t’s every plant’s worst nightmare. In the fall of 2009, in a Victorian greenhouse at the Cruickshank Botanic
Garden at the University of Aberdeen in Scotland, Zdenka Babikova sprinkled vegetation-devouring aphids on
eight broad bean plants and sealed each plant’s leaves and stems inside a clear plastic bag. This was no act of
malice, though; it was all in the name of science. Babikova, a PhD student at the University of Aberdeen, knew
that aphid-infested bean plants release odorous chemicals known as volatile organic compounds (VOCs) into
the air to warn their neighbors, which respond by emitting different VOCs that repel aphids and attract aphid-
hunting wasps. What she didn’t know was whether the plants were also sounding the alarm beneath the soil
surface.

Five weeks earlier, Babikova filled eight 30 cm–diameter pots with soil containing Glomus intraradices, a
mycorrhizal fungus that connects the roots of plants with its hyphae, the branching filaments that make up the
fungal mycelium. Like a subterranean swap meet, these hyphal networks facilitate the trade of nutrients
between fungi and plants. In each pot, Babikova planted five broad bean plants: a “donor” plant surrounded by
four “receiver” plants. One of the receivers was allowed to form root and mycorrhizal contact with the donor;
another formed mycorrhizal contact only, and two more had neither root nor mycorrhizal contact. Once the
mycorrhizal networks were well established, Babikova infested the donor plants with aphids and sealed each
plant in a separate plastic bag that allowed for the passage of carbon dioxide, water, and water vapor but
blocked larger molecules, such as the VOCs used for airborne communication.

Four days later, Babikova placed individual aphids or parasitoid wasps in spherical choice chambers to see how
they reacted to the VOC bouquets collected from receiver plants. Sure enough, only plants that had mycorrhizal
connections to the infested plant were repellent to aphids and attractive to wasps, an indication that the plants
were in fact using their fungal symbionts to send warnings.1

In last 15 years the idea that plants are communicating has become much more accepted. It’s exciting to
unravel all these different realms of plant communication.—Richard Karban, University of California, Davis

“When Zdenka first showed us those results it was quite a eureka moment,” says David Johnson, a soil ecologist
at Aberdeen who co-led the research. “There was a really striking difference between the insects’ responses to
plants with and without hyphal connections. We had more samples to test, but even at that point, it was pretty
clear that this is an effective signaling system.”

The remarkable conclusions from this study, published last May, are the latest shoots in a growing thicket of
data revealing the unexpectedly complex ways that plants exchange information with one another. Researchers
are unearthing evidence that, far from being unresponsive and uncommunicative organisms, plants engage in
regular conversation. In addition to warning neighbors of herbivore attacks, they alert each other to threatening
pathogens and impending droughts, and even recognize kin, continually adapting to the information they
receive from plants growing around them. Moreover, plants can “talk” in several different ways: via airborne
chemicals, soluble compounds exchanged by roots and networks of threadlike fungi, and perhaps even
ultrasonic sounds. Plants, it seems, have a social life that scientists are just beginning to understand.
“In the last 15 years the idea that plants are communicating has become much more accepted,” says Richard
Karban, an evolutionary ecologist at the University of California, Davis. “The evidence for that is now
substantial, and it’s exciting to unravel all these different realms of plant communication.”

Whispers on the wind

PLANT CHATTER: Volatile organic compounds (VOCs), first

theorized by plant scientists Jack Schultz and Ian Baldwin in the early 1980s, are now a well-known form of
plant communication. Maple tree saplings (pictured here) ramp up their own defenses in the presence of
herbivore-damaged neighbors.© EERIK/ISTOCKPHOTO.COMIn 1983, plant scientists Jack Schultz and Ian
Baldwin reported that intact maple tree saplings ramped up their defense systems when exposed to herbivore-
damaged maples. The injured trees, they suggested, were alerting neighbors to the presence of a predator by
releasing chemical signals into the air. But the plant research community didn’t buy it. The results were difficult
to replicate, critics pointed out, and many questioned how a trait that benefits neighboring plants but not the
emitter could be evolutionarily stable. By the late 1980s, “most ecologists felt these ideas had been debunked
and that it was time to move on,” says Karban.

A decade later, however, a trickle of more carefully designed experiments began to yield convincing evidence
to the contrary. In 2000, Karban showed that wild tobacco plants grown in close proximity to sagebrush plants
whose leaves had been clipped became resistant to herbivores, ostensibly in response to VOCs released by the
sagebrush.2 Other researchers soon reported similar VOC-induced defense responses—both intra- and
interspecies—in several other plants, including lima bean, broad bean, barley, and corn. And in 2006, Karban
showed that VOCs released by damaged sagebrush induce herbivore resistance in plants growing at distances of
up to 60 cm, well within the range of sagebrush neighbors in nature.3

By now the phenomenon of VOC-based plant communication is well described. In several cases, it has also
been demonstrated that volatile cues increase fitness in receiver plants. In one study, lima bean plants exposed
to herbivore-induced VOCs lost less leaf mass to herbivores and produced more new leaves than controls, for
example.4 But no experiment has yet demonstrated that volatile signaling between neighboring plants can
benefit the emitting plant, prompting some researchers to suggest that “eavesdropping” is a more accurate
description of what has been observed than “intentional” communication.

Either way, researchers who doubt that plants would have evolved to be altruistic have ruminated on the old
question of the evolutionary origins of the phenomenon. Perhaps the ability to synthesize and emit VOCs could
simply be an unavoidable consequence of non-communicative functions, such as repelling herbivorous insects
and attracting insects that parasitize those herbivores. Another possibility is that external communication
channels are merely an extension of within-plant signaling. In sagebrush, lima bean, and poplar, VOCs released
from damaged parts of a plant induce resistance in intact sections of the same plant, suggesting that each
individual plant uses the signals to coordinate its own physiological responses. “The interplant signaling we see
may be a result of plants co-opting that process,” explains Karban.

Alternatively, VOC-based signaling between plants may have been favored because it enhances the “extended
fitness” of the emitter by benefitting its related conspecifics: a strategy known as kin selection. This idea gained
support from a study published last February, in which Karban and colleagues found that airborne
communication was more effective among sagebrush plants that were closely genetically matched than among
those that were more distantly related.5 “If communication is more effective among kin, it is less likely that
plants are giving away information to potential competitors and more likely that it will benefit close relatives,”
says Karban. (See sidebar: “Foliage Family Values.”)

While the evolutionary explanation for volatile communication among plants remains open to debate,
researchers have been working to identify and chemically characterize VOCs and to figure out how they encode
messages. Gas chromatography–mass spectroscopy has unveiled various compounds that function as plant-to-
plant signals, and such work has made it increasingly clear that specific blends of those compounds dictate the
content of the messages. In 2011, for example, Japanese researchers found that in herbivore-damaged daisies,
eliminating any one compound from a mixture of five VOCs, which induces insecticide production in
neighbors, significantly reduced the expression of genes associated with insecticide biosynthesis in those
plants.6

“Individual compounds are the words,” says Jarmo Holopainen, an ecologist at the University of Eastern
Finland, “and these words are combined to make specific sentences.” Unfortunately, he adds, researchers know
little about what these volatile signals mean to a plant and how they are perceived. “We’ve made very little
progress in deciphering this chemical code.”

Root rumors

THE SECRET SOCIAL LIVES OF PLANTS: Contrary to the

long-held idea that plants are uncommunicative, recent research has made it clear that many species conduct
lively and informative conversations with one another. Scientists have revealed that plants communicate
through the air, by releasing odorous chemicals called volatile organic compounds (VOCs), and through the
soil, by secreting soluble chemicals into the rhizosphere and transporting them along thread-like networks
formed by soil fungi. And this is more than mere gossip: these signals warn neighbors of the many dangers
facing plants.
See full infographic: JPG | PDF© LOGAN PARSONSPlant gossip is not only spread on the breeze; the
rhizosphere crackles with chatter, too. Over the past few years, a team led by Ariel Novoplansky of Ben-Gurion
University of the Negev in Israel has produced compelling evidence that plants eavesdrop on hints of their
neighbors’ distress through their root systems.
Novoplansky’s team planted six garden pea plants so that each pot contained the roots of two different plants.
The researchers then subjected the first plant in each row to drought-like conditions and evaluated the response
of its neighbors by measuring the width of the microscopic pores, called stomata, on leaf surfaces, which close
in response to drought stress.

Fifteen minutes after drought was initiated, the stressed plant closed its stomata—as did its nearest unstressed
neighbor, suggesting some sort of drought warning sign had been passed between the two. After an hour, all
five neighbors, each more distant from the stressed plant—the only one that actually experienced drought-like
conditions—had also shuttered their stomata, indicating that they, too, received the message to prepare for
drought.7 Importantly, in a control setup where root contact between neighboring plants was blocked, pores
stayed open, indicating that the message was somehow being passed between roots.

“The finding that plants communicate stress cues via roots was itself novel,” says Novoplansky. “But for me
another aspect was more interesting and important: that unstressed neighbors not only responded as if exposed
to drought themselves, but also released more of the same cue, which was in turn perceived by further, more
remote, unstressed plants.”

Novoplansky’s team has since observed the same phenomenon in garden peas under more realistic, field-like
conditions and in three wild plant species, and he suspects that root-based stress cuing could be common in
nature.8 The mechanisms underlying this ability are still under investigation, but the cue is almost certainly
chemical, says Novoplanksy: when researchers extract soluble chemicals released into the soil by drought-
stressed roots and apply them to unstressed plants, they see the same response. His team is currently doing
metabolomic studies of root exudates to pinpoint the compound or combination of compounds that carry the
message. “The hottest candidate for the vector is abscisic acid (ABA), a hormone involved in plant responses to
drought and osmotic stress,” he says. “But I wouldn’t be surprised if it was something else.”

Meanwhile, other researchers are starting to explore another underground plant communication system, one in
which messages are sent through the labyrinth of hairlike fungal filaments that festoon the roots of the vast
majority of plants on the planet—including important crops such as wheat, rice, maize, and barley. These
mycorrhizal fungi are involved in an important mutualistic relationship: in exchange for sugars, they provide
plants with much-needed phosphorus and nitrogen. In many cases, the fungi connect the roots of neighboring
plants to form common mycelial networks (CMNs), which play a major role in recycling soil nutrients and
water. And as Aberdeen’s Johnson revealed last summer, CMNs are also a means of communication. The
stringy white hyphae act like fiber-optic cables, carrying information between plants of the same or even of
different species. (See “Fighting Microbes with Microbes,” The Scientist, January 2013.)
This idea first gained credence in 2010, when a team at South China Agricultural University in Guangzhou
showed that interplant connections via CMNs led to increased disease resistance in healthy tomato plants
connected to tomato plants that had previously been infected with the fungal pathogen that causes leaf
blight.9 The work of Johnson and Babikova, published last year, provided the first evidence that signals warning
of herbivore damage can also be transmitted via CMNs.

“Most research into mycorrhizal networks has focused on their role as biological marketplaces,” says Johnson,
“but we’ve now shown that they could have an additional role as a really effective way to transmit messages.”

Subsequent work by Johnson’s group found that CMN-mediated signals elicit behavioral changes within 24
hours of an aphid infestation.10 “That’s important because the signaling has to be quick to be ecologically
relevant,” says Johnson.

CMNs may also carry signals over far greater distances than airborne volatiles can. One 2009 study documented
a fungal network that wove its way through an entire forest, with each tree connected to dozens of others over
distances as great as 20 meters.11 Still, Johnson notes, communication within extensive mycelial networks such
as these has yet to be demonstrated in the field.

For Johnson and his colleagues, the next step is to use transcriptomics to see which plant genes are expressed in
response to aphid attacks. He says he may also compare proteomic analyses of CMN-connected roots with
unconnected roots to identify any compounds that differ between the two systems. But it won’t be easy, he says.
“Pinning down the molecules involved is going to be a slog.”

Sounding off

Monica Gagliano’s first attempt to publish evidence suggesting that plants might communicate with each other
using sounds was met with rank disbelief. Her manuscript was rejected by six different journals in 2010 and
2011. “It was dismissed as something that is not possible,” says Gagliano, an evolutionary ecologist at the
University of Western Australia in Perth.

Undeterred, Gagliano kept plugging away. In April 2012, after she had repeated her experiments and addressed
comments from seven different reviewers, PLOS ONE accepted the paper. The study showed that chili plant
seedlings grown next to fennel germinated more quickly than seedlings grown with conspecifics. Gagliano and
her colleagues suspect the chili plants are compensating for the presence of the fennel, which is known to
release chemicals that inhibit the growth of other plants. Remarkably, however, all known interplant
communication pathways—airborne volatiles, root contact, and common fungal networks—were blocked. The
results begged for an alternative explanation.

In an article reviewing the evidence suggestive of plants’ use of sound as a communication medium, Gagliano
and colleagues cited a study showing that the roots of young corn plants grown in water make clicking sounds,
and that when sounds in the same frequency range were played back to the roots, they responded by bending
toward the source.13

“We’ve shown that plants can recognize when they’re growing next to a ‘bad neighbor’ and change their
growth behavior accordingly, even when we remove all the channels of communication we know about,” says
Gagliano. “We also have some evidence that there is an emission [of sound] and a response of some sort. We
are not ruling out other possibilities, of course, but we think this other channel of communication might be
acoustic.”

Theoretically, she says, sound has several advantages over chemical signaling: it propagates faster and over
greater distances, and it can be generated with fewer energy costs.14 But behavioral ecologist Carel ten Cate of
the University of Leiden in the Netherlands points out that taking advantage of such putative benefits would
require sensory mechanisms yet to be described in plants.15 Plants do generate sounds at frequencies outside the
range of human hearing, but it’s not clear how they are produced or whether plants can detect sounds at all.
“There are many outstanding questions,” Gagliano admits.

Despite widespread skepticism in the plant research field, Gagliano has received some encouragement. UC
Davis’s Karban, for example, is cautiously enthusiastic. “She’s presenting results that don’t fit conventional
wisdom . . . but she is repeatedly getting results that are very difficult to explain based on the mechanisms we’re
currently aware of,” says Karban, noting the parallels between Gagliano’s struggles and his own situation in the
1990s, when the field was reluctant to entertain the idea that plants can communicate with airborne volatiles.
“Whether or not the explanation she favors is the right one,” he adds, “I think that she’s gotten results that, as a
field, we need to come to grips with.”

Applications for agriculture?

 PLANT SENSE: Young chili plants seem to know their
neighbors: chilis grown next to fennel germinate more quickly than those grown with other chili plants. The
question is, how do they do it? Curiously, the effect is still present even when all known interplant
communication pathways—airborne volatiles, root contact, and common fungal networks—are blocked, leading
Monica Gagliano of the University of Western Australia to suggest that the plants may somehow be “hearing”
acoustic clues.© URSULA ALTER/ISTOCKPHOTO.COMWhether they’re studying volatiles drifting on the
breeze or phytochemicals zipping through subterranean fungi, researchers are now bent on elucidating the
relevant receptors and deciphering the molecular lingua franca of plant communication. They could then begin
to clarify the ecological significance of the phenomenon and, potentially, help farmers grow hardier crops.

Understanding how plants perceive airborne volatile signals, for instance, could inform the genetic engineering
of crops that are hypersensitive to cues from sacrificial “beacon” plants that are deliberately damaged to emit
signals that trigger neighboring plants to activate their antipredator and/or antipathogen defenses. And if
researchers could pinpoint the compounds that act as vectors for stress cues passed between roots, they could
potentially “train” crop seedlings to better cope with drought and other stresses.

“I think it can [be a successful strategy], but we have to put in the work to make it happen,” says Novoplansky.
“You’re dealing with farmers’ livelihoods, so you have to be certain it will work in realistic agricultural
settings,” agrees Johnson, who collaborates with scientists at the UK’s James Hutton Institute and Rothamsted
Research, both of which are dedicated to increasing agricultural productivity. “Applying our limited knowledge
[of plant-communication mechanisms] to agriculture is a big jump,” he says, “but it is definitely on the
horizon.”

Regardless, one message has emerged loud and clear from those studying this new realm of botanical
interaction: despite not possessing eyes, ears, or a nervous system, plants are anything but uncommunicative.
Rather, the latest findings speak of a plant kingdom brimming with chatter. “When I did my PhD [in the late
1980s], all this stuff was considered very weird,” recalls Novoplansky. “Today, there is no doubt. We now
recognize that plants are capable of some very sophisticated exchanges of information with other plants. This
idea is not strange anymore.”

FOLIAGE FAMILY VALUES

In 2007, Susan Dudley of McMaster University in Ontario, Canada, and graduate student Amanda File
showed that a beach weed called sea rocket, which is common on the shores of the Great Lakes, senses
whether it’s growing among siblings or unrelated plants of the same species. When sea rocket detects
strangers, it allocates more resources toward sprouting nutrient-grabbing roots, but when it recognizes kin, it
graciously restrains itself.1

Since then, researchers have demonstrated kin recognition in several other plant species. The common model
organism Arabidopsis thaliana, for example, reins in root development when growing among brethren. Pale
jewelweed (Impatiens pallida), on the other hand, reduces leaf growth and elongates and branches its stems
in the presence of kin—likely to reduce shading of siblings in the woody areas where it grows, in which light
acquisition is a priority.2

Together with Harsh Bais of the University of Delaware, Dudley has also demonstrated that Arabidoposis’s
ability to recognize kin depends on the secretion of soluble chemicals from roots.3 The problem is, the
researchers have no idea which compounds are important. “Roots put out a lot of chemicals,” says Dudley,
“and we don’t know which matter; which are serving as name tags.”

Nevertheless, researchers have been thinking about the evolutionary dynamics underlying such adaptations.
“If plants can recognize their siblings and agree not to waste resources competing against them, then the
group as a whole benefits,” Dudley explains. But, she adds, precious few studies have measured whether
 family groups actually do benefit from kin-dependent behavioral changes. “If we want to test our
predictions, we have to see whether differential responses have fitness consequences.”

In 2012, Dudley and File documented some of the first evidence along those lines. They showed that
ragweed grown among kin develop larger common mycelial networks—through which plants and fungi
trade nutrients—than those grown among non-kin, and that ragweed plants with increased root colonization
were better protected against pathogens. Greater fungal abundance was also associated with higher leaf-
nitrogen levels, indicative of healthy plants.4 “We have more evidence [that plants benefit from kin
recognition] in the pipeline,” says Dudley. “Now we need to see what happens out in the field.”
https://www.the-scientist.com/features/plant-talk-38209

 C10.

How can we use our knowledge of the molecular biology of disease resistance to develop
novel approaches to disease control?
Molecular genetics and evolution of disease resistance in cereals
Summary
Cereal crops produce a large part of the globally consumed food and feed. Because of the constant presence of
devastating pathogens, the molecular characterization of disease resistance is a major research area and highly
relevant for breeding. There has been recent and accelerating progress in the understanding of three distinct
resistance mechanisms in cereals: resistance conferred by plasma membrane‐localized receptor proteins; race‐
specific resistance conferred by intracellular immune receptors; and quantitative disease resistance. Intracellular
immune receptors provide a particularly rich source for evolutionary studies, and have, for example, resulted in
the recent discovery of a novel detection mechanism based on integrated decoy domains. Evolutionary studies
have also revealed the origins of active resistance genes in both wild progenitors of today's cereals as well as in
cultivated forms. In addition, independent evolution of orthologous genes in related cereals has resulted in
resistance to different pathogen species. Quantitative resistance genes have been best characterized in wheat.
The quantitative resistance genes identified so far in wheat encode transporter proteins or unusual kinase
proteins. The recent discoveries in these three different resistance mechanisms have contributed to the basic
molecular understanding of cereal immunity against pathogens and have suggested novel applications for
resistance breeding.

I. Introduction
Plants live in close association with a vast number of diverse microbial organisms, including viruses, bacteria
and fungi. For the plant, these interactions can have different outcomes, ranging from beneficial, as in the case
of rhizobacteria that fix nitrogen, to harmful, as in the case of pathogens. Hence, plants have evolved amazing
strategies that allow them to perceive harmful microbes without compromising the ability to associate with
symbionts. Damage caused by plant pathogens is of particular importance in agriculture, where diseases are a
major reason for crop losses. In addition, secondary metabolites produced by certain fungal pathogens, for
example Fusarium and Aspergillus, can have toxic effects on livestock and humans. Cereal crops, including
wheat, rice, maize, barley, millet, sorghum, oat and rye, play a pivotal role in global food and feed production.
Indeed, most calories consumed today are directly or indirectly derived from cereals: directly through the
consumption of cereal grains and indirectly through animal feed. All cereals are close relatives that belong to
the grass family (Poaceae) and share their last common ancestor at 45–60 million yr ago (The International
Brachypodium Initiative, 2010). It is estimated that, on average, 10–15% of the global crop production is lost to
plant diseases (Chakraborty & Newton, 2011; Fisher et al., 2012). For cereals alone, a 10% loss amounts to a
total of c. 300 million tons each year (the statistics division of the Food and Agricultural Organization of the
United Nations (FAOstat)). If all cereal diseases could be eliminated, this would result in additional food for
1.7 billion people, assuming a consumption of c. 170 kg of cereals per capita per year (Food and Agricultural
Organization of the United Nations (FAO)). A better understanding of the molecular genetics and evolution of
natural disease resistance in cereals is therefore an important prerequisite to continuously improve cereal
cultivars for effective field resistance.
II. Immunity in plants
Plants lack an adaptive immune system comparable with that found in mammals. Instead, plants rely solely on
innate immune mechanisms that are mostly based on the perception of pathogen‐derived ligands by plant
receptor proteins. Intensive research efforts during the past two decades have led to the identification of a
plethora of immune receptors in plants, which can be broadly classified into two distinct types according to their
subcellular localization: (1) plasma membrane‐localized receptors with an extracellular ligand‐binding domain;
and (2) intracellular immune receptors (Jones & Dangl, 2006; Dodds & Rathjen, 2010; Thomma et al., 2011;
Cook et al., 2015). Most surface‐localized immune receptor proteins perceive the presence of pathogen
structures in the apoplast. These can comprise highly conserved microbial signatures that are characteristic for
entire pathogen classes. Examples of such ‘pathogen‐associated molecular patterns’ (PAMPs) are the bacterial
flagella or the fungal cell wall component chitin (Jones & Dangl, 2006; Dodds & Rathjen, 2010). Other plasma
membrane‐localized immune receptors perceive the presence of ligands that are specific to a single or a few
related pathogen species (Thomma et al., 2011; Cook et al., 2015), and others again are able to bind host plant‐
derived molecules that are released during pathogen penetration. The recognition of an appropriate ligand by the
corresponding receptor protein activates a defense response whose magnitude can range from a weak basal
resistance to death of the attacked cell. We discuss examples of surface‐localized immune receptors in cereals in
Section III..

To successfully colonize a host plant, pathogens rely on a set of virulence effectors that is tailored to
specifically disarm components of the host plant's immune response. Virulence effectors are often small
secreted proteins that can modify or degrade host proteins involved in basal immunity. The magnitude and
diversity of a pathogen's effector set defines its host range, that is the number of host species it can infect
(Schulze‐Lefert & Panstruga, 2011). Although some pathogens have a very narrow host range, others are able to
infect many different species. Many virulence effectors are injected into the plant cell and therefore circumvent
perception by surface‐localized immune receptors.

In response to cytoplasmic virulence effectors, plants have evolved a second line of defense. This second tier is
formed by intracellular immune receptors mostly belonging to the conserved protein family of nucleotide‐
binding, leucine‐rich repeat receptor (NLR) proteins (Dodds & Rathjen, 2010). Direct or indirect effector
perception by NLRs generally triggers a strong hypersensitive response (HR) that often leads to the death of the
infected cell. It was mainly research performed in the dicotyledonous model plant Arabidopsis thaliana (mouse‐
ear‐cress) that led to the initial discovery and characterization of surface‐localized immune receptors and NLR
proteins. Numerous studies have documented that these proteins also play an important role in cereals and we
discuss recent insights into NLR evolution in cereals in Sections IV. and V..

In addition, observations made in crops, particularly in cereals, have revealed resistance mechanisms that are
different from those described above. Such resistance mechanisms are often collectively referred to as
‘quantitative’. Some quantitative disease resistance (QR) genes provide durable, albeit partial, field resistance
against most or all races of a pathogen (St Clair, 2010; Ellis et al., 2014; Niks et al., 2015). The term ‘durable’
refers to disease resistance that is effective over a long period of time in an environment favorable to the disease
(Johnson, 1984), and the fact that QR genes often confer resistance to all races of a pathogen species, and
sometimes even against multiple pathogens, is referred to as ‘broad spectrum’. The durability, broad‐spectrum
specificity and lack of HR clearly differentiate such QR genes from NLR‐triggered immunity. A number of
public cereal breeding programs nowadays favor this resistance type, but there are still many breeding efforts
based on NLR‐based disease resistance. Repeated phenotypic observations by experienced pathologists in
different locations and over multiple years are necessary to reliably quantify the sometimes subtle phenotypic
effects of single QR genes. This can explain why this particular resistance type has so far mainly been described
in crops and not in model plants. In contrast with its importance in breeding, research on the genetic, molecular
and evolutionary basis of QR is still in its infancy.
III. Receptors in the plant plasma membrane – the importance of
monitoring the extracellular space
The extracellular environment plays a central role in host–pathogen interactions because it usually is the first
point of contact between the two organisms. It is therefore not surprising that the extracellular space is closely
monitored for the presence of pathogen‐derived ‘non‐self’ signatures by cell surface‐localized receptor‐like
kinases (RLKs) and receptor‐like proteins (RLPs) (Jones & Dangl, 2006; Dodds & Rathjen, 2010;
Thomma et al., 2011; Cook et al., 2015). Similar receptor proteins, named Toll‐like receptors (TLRs), are also
involved in innate immunity in humans (Nurnberger et al., 2004). In contrast with humans, however, RLKs and
RLPs massively increased in number during plant evolution, highlighting the importance of the innate immune
system in plants. Although humans only have 10 TLR genes, more than 100 genes encoding RLKs and RLPs
are usually found in plant genomes (Schwessinger & Ronald, 2012). One of the first plant RLKs identified was
XA21 of rice (Song et al., 1995) which confers resistance against most strains of the agronomically important
bacterial blight disease (Xanthomonas oryzae pv. oryzae). Xa21 consists of an extracellular leucine‐rich repeat
(LRR) domain and a cytoplasmic kinase domain. LRR domains have been shown to be involved in the
perception of proteinaceous elicitors. A recent key paper indeed identified a sulfated Xanthomonas peptide
named ‘required for activation of XA21 X’ (RaxX) as an elicitor of XA21. Interestingly, the closest homolog of
RaxX was not found in other bacteria (Pruitt et al., 2015). Instead, RaxX shows homology to a sulfated
signaling peptide of Arabidopsis, which led to the conclusion that RaxX might mimic plant proteins involved in
basal immunity.

Although extracellular LRRs mainly perceive peptide elicitors, other non‐LRR domains found in RLKs and
RLPs recognize different elicitor classes (Schwessinger & Ronald, 2012). For example, the extracellular
domains of the rice RLP CEBiP (chitin elicitor‐binding protein) and the RLK CERK1 contain LysM motifs.
These two proteins cooperatively perceive the fungal cell wall component chitin (Kaku et al., 2006;
Shimizu et al., 2010). In addition, several wall‐associated receptor‐like kinases (WAKs) have been shown
recently to confer resistance against fungal disease in rice and maize. Fine mapping of the head smut
(Sporisorium reilianum) resistance locus qHSR1 in maize identified ZmWAK as the causal resistance protein
(Zuo et al., 2015). Head smut is a soil‐borne fungus that infects maize roots and subsequently spreads to above‐
ground parts where symptoms develop. Interestingly, ZmWAK did not repress root penetration of the fungus
and maize lines with and without qHSR1 showed similar levels of fungal growth in roots. Instead, ZmWAK was
mainly expressed in the mesocotyl, where it repressed spread of the fungus to the above‐ground parts. It is
possible that strong expression of ZmWAK in roots would compromise the ability of maize plants to interact
with beneficial arbuscular mycorrhizal fungi and that is why maize evolved this amazing resistance mechanism
that allows for a certain degree of head smut infection. Similar to qHSR1, Hurni et al. (2015) identified the
WAK‐encoding gene Htn1 in maize that confers resistance against the fungal disease northern corn leaf blight
(Exserohilum turcicum). In addition to maize, several WAKs in rice have recently been reported to positively or
negatively influence basal defense against the fungal rice blast disease (Magnaporthe oryzae) (Li et al., 2009;
Delteil et al., 2016). Interestingly, although only 27 WAK‐like genes are found in the model plant Arabidopsis,
the rice genome contains > 120 WAK‐like genes.

Kinase domains can be classified into RD and non‐RD based on the presence of a conserved arginine residue
(R) at the catalytic site. The fusion of WAK domains to non‐RD kinase domains is specific to monocots (de
Oliveira et al., 2014). Although RD kinases are often associated with developmental processes, non‐RD kinases
are typically found in plant immune receptors (Dardick et al., 2012). Hence, the WAK gene family in monocots
has expanded, diversified and might play a very important role in fungal disease resistance, specifically in
cereals. Delteil et al. (2016) hypothesized that the rice blast resistance‐conferring WAK proteins OsWAK14,
OsWAK91, OsWAK92 and OsWAK112d trigger a basal defense response on perception of the fungal cell wall
component chitin.

Some RLKs and RLPs perceive highly conserved ligands and can confer resistance against a broad range of a
particular group of pathogen. The transfer of such immune receptors between species could therefore enhance
the durability of disease resistance. Indeed, the functional transfer of RLKs has been demonstrated recently in
several cases. The rice XA21 protein, for example, is functional in banana, where it confers resistance against
the bacterial banana Xanthomonas wilt disease (Tripathi et al., 2014). Likewise, the Arabidopsis LRR‐RLK
EFR, which perceives a bacterial elongation factor, is functional in transgenic wheat against the halo blight‐
conferring bacterial pathogen Pseudomonas syringae pv. oryzae (Schoonbeek et al., 2015). These examples
provide the first experimental evidence that the transfer of surface‐localized immune receptors across species is
a feasible strategy to improve disease resistance in cereals. In addition, targeted modifications of endogenous
RLKs and RLPs through genome editing might also be used to increase their resistance spectra or durability.
The molecular differences between susceptible and resistant alleles might be used as a starting point to identify
critical domains and amino acids for recognition.

IV. Inside the cell – the role of intracellular immune receptors in

cereals
The involvement of intracellular receptors, mostly NLRs, in effector recognition is probably the best studied
area of plant immunity. A complete summary of NLR‐based immunity in cereals would go beyond the scope of
this review. Rather, we focus on the evolutionary mechanisms that have shaped NLR specificity in cereals.
Specifically, we discuss NLR evolution in the Triticeae species wheat, barley and rye. This tribe of the grass
family is particularly interesting for evolutionary studies because it contains several closely related crop species
and different ploidy levels.

Most secreted virulence effectors of pathogens show high rates of diversifying selection. This has been shown,
for example, for the two closely related powdery mildew formae speciales of wheat and barley, where candidate
effector genes exhibit a higher rate of diversifying selection than most other genes (Wicker et al., 2013). The
rapid evolution of effectors allows the pathogen to escape recognition by the cognate NLR. This evolutionary
process can have dramatic consequences for cereal production and usually results in rapid breakdown of NLR‐
based disease resistance in the field. Ironically, the most popular NLR genes are most prone to resistance
breakdown because they are used in many cereal cultivars grown over large areas, factors that, in turn, favor
rapid adaptation and spread of virulent pathogen races. A fateful example was the emergence of new and highly
virulent stem rust races in wheat, once the popular stem rust resistance gene Sr31 was overcome. The Sr31‐
breaking race was first observed in Uganda in 1999 and was therefore termed Ug99 (Pretorius et al., 2000).
Ug99 spread over East Africa into the Middle East within only a few years. In this Ug99 lineage, rapid
diversification and adaptation to additional genes was later observed (Singh et al., 2015). In response to the
rapid evolution of pathogen effectors, many NLR genes are also under strong diversifying selection, which
results in the typical arms race between host and pathogen.

Generally, one would expect that the pathogen would have an evolutionary advantage over the host plant
because microbial organisms have relatively short generation times and produce enormous numbers of spores
(Fetch & McCallum, 2014). Assuming an equal mutation rate in both host and pathogen, we would expect that
the pathogen would evolve much more rapidly than the host plant. So, how does the host manage to not lose
ground in the constant tug of war with the pathogen, and why have all the resistance genes not been overcome
long since? An interesting and exciting solution to this evolutionary conundrum, coined ‘integrated domains’,
has recently gained wide attention (Fig. 1; Cesari et al., 2014; Wu et al., 2015; reviewed by Ellis, 2016;
Krattinger & Keller, 2016). Several independent studies have reported that protein domains mimicking effector
targets can be integrated into NLR immune receptors where they act as effector traps. For example, two rice
NLR proteins, RGA5 and Pik‐1, have been shown to contain an additional heavy metal‐associated domain
(HMA) that is not found in other NLRs. The HMA domains in these two immune receptors directly recognized
several rice blast effectors which triggered a defense response (Cesari et al., 2013; Maqbool et al., 2015). It is
assumed that HMA domain‐containing proteins are components of the basal immune response in rice and that
they are consequently targeted by pathogen effectors. Indeed, loss‐of‐function mutations in the HMA domain‐
containing protein Pi21 of rice resulted in durable and broad‐spectrum rice blast resistance, and it is tempting to
hypothesize that wild‐type Pi21 plays a role in basal defense (Fukuoka et al., 2009). By fusing an HMA domain
to an NLR, rice plants might have evolved a neat trick to trap HMA‐targeting effectors.
Figure 1
Open in figure viewerPowerPoint
Different models of indirect nucleotide‐binding, leucine‐rich repeat receptor (NLR) effector perception. (a)
Microbial effectors (red) target and modify components of the host's basal defense response (blue). In the
absence of an effector‐recognizing NLR, this results in suppressed basal immunity. (b) In the guard model, the
status of the basal defense component (guardee) is monitored by an NLR protein (gray). Changes to the guardee
introduced by virulence effectors are perceived by the NLR protein which triggers a hypersensitive response
(HR). (c) The decoy (purple) has often arisen through duplication of a basal defense gene. The decoy, however,
has lost its original function and its sole purpose is to trap effectors. In the classical decoy model, the decoy and
the corresponding NLR protein are encoded by two separate genes. (d) Recent reports have shown that decoy
domains can be directly integrated into the corresponding NLR, resulting in NLR‐IDs.
Two recent whole‐genome comparisons in different plant species have revealed that ‘integrated domain’ NLRs
(NLR‐IDs) are frequently found in many plant species. Between 3.5% and 10% of all NLRs contain additional
integrated domains (Kroj et al., 2016; Sarris et al., 2016). In addition to the HMA domain, Sarris et al. (2016)
identified 264 additional integrated domains in a total of 750 NLR‐IDs. Seventeen integrated domains were
found in at least two of the five cereal species included in their analysis (Table 1). Some of these integrated
domains have a known function in basal immunity, such as WRKY or protein kinase domains. Other domains
found in these NLR‐IDs have so far not been linked to plant immunity. The current assumption is that proteins
similar to the integrated domain play a role in basal defense and, consequently, that a knowledge of integrated
domains with unknown function could lead to the identification of novel effector targets. The systematic
analysis of NLR‐IDs could therefore lead to the possibility of the identification of further effector targets and to
a better understanding of host–pathogen interactions. Kroj et al. (2016) experimentally tested this hypothesis by
using the BED zinc finger domain which is frequently found in NLR‐IDs in different plant species. No direct
involvement of BED domain‐containing proteins in disease resistance has been demonstrated so far. The protein
encoded by the rice ZBED gene contains three BED domains which show homology to the respective integrated
domains in rice NLR‐IDs. Interestingly, rice lines overexpressing ZBED were more resistant to rice blast
disease, whereas ZBED mutants were more susceptible. Hence, Kroj et al. (2016) successfully used information
on integrated domains to establish a role of ZBED proteins in disease resistance.

Table 1. Integrated domains found in at least two of the five studied cereal species by Sarris et al. (2016):
barley (Hordeum vulgare), rice (Oryza sativa), sorghum (Sorghum bicolor), wheat (Triticum aestivum) and
maize (Zea mays)

Domain description Cereal species

Protein kinase domain Barley (6), rice (1), sorghum (3), wheat (13), maize (1)

Protein tyrosine kinase Barley (1),rice (1), sorghum (1), wheat (1)
 Domain description Cereal species

Jacalin‐like lectin domain Barley (2), rice (3), sorghum (1), wheat (2)

Thioredoxin Barley (1), rice (1), sorghum (2), wheat (3)

WRKY DNA‐binding domain Barley (1), sorghum (4), wheat (2)

BED zinc finger Barley (1), rice (2), wheat (1)

WD domain, G‐beta repeat Rice (1), sorghum (3)

B3 DNA‐binding domain Barley (1), rice (1)

VQ motif Rice (1), sorghum (1), wheat (1)

FNIP repeat Barley (1), sorghum (2)

Kelch motif Barley (2), wheat (4)

Protein phosphatase 2C Barley (1), wheat (2)

Cleavage site for pathogenic effector Barley (1), rice (1)

Glutaredoxin Barley (3), sorghum (1)

Phloem protein 2 Sorghum (1), maize (1)

DDE superfamily endonuclease Barley (2), wheat (1)

Exo70 exocyst complex subunit Barley (1), wheat (1)

 The numbers in parentheses indicate the number of nucleotide‐binding, leucine‐rich repeat receptor proteins
with the respective integrated domain (NLR‐IDs).

V. NLR evolution in Triticeae

In this section, we focus on the evolution of NLR specificities in the grass tribe Triticeae. The availability of
closely related species with different ploidy levels, as well as wild and domesticated forms, represents a
powerful system to address the evolution of NLR specificities.

1. The MLA‐like family of NLR proteins

An interesting example that illustrates the diversification of NLR genes is the mildew A (Mla)‐like family found
in Triticeae. Originally, Mla was described as an allelic series of resistance genes in barley and > 30 functional
alleles with different specificities against barley powdery mildew strains (Blumeria graminis f.sp. hordei) were
identified molecularly (Wei et al., 1999; Seeholzer et al., 2010). Subsequently, a functional ortholog of the
barley Mla gene was found in the diploid wheat Triticum monococcum. TmMLA1 showed 78% amino acid
identity with the barley HvMLA1 protein and conferred race‐specific resistance against wheat powdery mildew
(B. graminis f.sp. tritici) in a transient assay (Jordan et al., 2011). TmMLA1, however, showed no effect against
barley powdery mildew in barley. Even more remarkably, two additional Mla orthologs, Sr33 and Sr50, in
wheat provide resistance against fungal stem rust disease (Puccinia graminis f.sp. tritici)
(Periyannan et al., 2013; Mago et al., 2015). Sr50 was introgressed from rye into cultivated wheat
and Sr33 stems from the diploid wild wheat progenitor Aegilops tauschii. Mildew and rust fungi are only
distantly related and they belong to different phyla. Although wheat, barley and rye shared their last common
ancestor c. 9 million yr ago (Middleton et al., 2014), powdery mildew and rusts split c. 400 million yr ago
(Taylor & Berbee, 2006). The protein sequences of Sr33, TmMLA1 and Sr50 blast to the same MLA family
member in the barley genome, indicating that the different pathogen specificities of Sr33, TmMLA1 and Sr50
arose after wheat and barley shared their last common ancestor. These findings demonstrate that different
members of the same NLR family can rapidly diversify and adapt to perceive effectors of distantly related
fungal pathogens (Fig. 2). Whether the perception of virulence effectors by the MLA family is based on a direct
or indirect recognition is still unknown, and it is possible that the rapid adaptation to different pathogens is the
result of indirect recognition events (Fig. 1). Furthermore, even effectors with little sequence homology can
have very similar protein structures and hence might be recognized by the same or related NLR immune
receptors (Ellis, 2016).

Figure 2
Open in figure viewerPowerPoint
Closely related members of the mildew A (MLA)‐like immune receptor family perceive effector proteins of
distantly related fungal pathogens. Left: phylogenetic tree of MLA‐like family members from diploid wheat
(Tm), hexaploid wheat (Ta) and barley (Hv). Mildew‐perceiving members are indicated in gray and nucleotide‐
binding, leucine‐rich repeat receptor (NLR) proteins recognizing stem rust are marked in brown. The leucine‐
rich repeat (LRR) domain was used to construct the tree and the wheat Pm3A protein was used as outgroup to
root the tree. Bootstrap numbers at the forks indicate how many times the sequences to the right of the fork
occurred in the same group out of 100 trees. For barley, only three of the > 30 known functional MLA proteins
were included in the tree. Right: schematic representation of the divergent evolution of different MLA‐like
family members providing resistance to different fungal pathogens. Divergence times in million yr ago (Ma) are
based on the reports of Taylor & Berbee (2006), Wicker et al. (2013) and Middleton et al. (2014).

2. The wheat leaf rust resistance genes Lr1 and Lr10 originated in wheat
progenitors
The long agricultural history of wheat has produced an enormous genetic diversity within this species. Much of
this diversity is conserved in genebanks which contain more than half a million accessions (Mitrofanova, 2012).
This material represents a unique possibility to study the evolution and diversity of specific genes. In particular,
there are wild and domesticated species and genotypes, landraces and elite cultivars, all representing relatively
recent evolutionary events. In addition, very similar genomes are present in species of different ploidy levels.
Tapping the diversity stored in genebanks allows comparative approaches and the study of evolutionary events
at the molecular level. Bread wheat (Triticum aestivum) is a hexaploid species which resulted from two
hybridization events (Marcussen et al., 2014). First, two wild species, Triticum urartu and a now extinct relative
of Aegilops speltoides, hybridized and formed wild tetraploid wheat (T. turgidum ssp. dicoccoides). After
domestication of this wild form to cultivated tetraploid wheat (T. turgidum ssp. dicoccum), a second
hybridization occurred with the wild grass Ae. tauschii resulting in the hexaploid bread wheat (Fig. 3).

Figure 3
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Evolutionary history of different wheat disease resistance genes. The times of the different hybridization events
are based on Middleton et al. (2014). Ma, million years ago.
Many resistance genes have been introgressed from wild species into cultivated wheat (Baum et al., 1992). For
example, the Lr21 leaf rust resistance gene was introgressed from Ae. tauschii using a synthetic wheat.
Extensive sequence analyses revealed that the functional Lr21 allele is a chimera of two non‐
functional lr21 haplotypes (H1 and H2) (Huang et al., 2003, 2009). In bread wheat, only inactive lr21 alleles
are present, but an active resistance gene could be experimentally reconstituted by intramolecular recombination
of the inactive H1 and H2 haplotypes. This provides an indication that novel, active resistance genes might have
originally evolved in wild species by recombination of existing, non‐functional haplotypes. Such events are in
agreement with the proposed model for the recycling of resistance gene sequences resulting in the evolution of
molecular diversity (Holub, 2001).

In contrast with Lr21 and other wheat NLR genes, the origin of a large number of resistance genes first
described in hexaploid wheat is not known. Their origin should be determined by comparative analyses and
allele mining of diverse germplasm. The wheat leaf rust resistance gene Lr1 was first described by
Ausemus et al. (1946) in the hexaploid wheat cultivar Malakoff (Dyck & Samborski, 1968). Lr1 is located on
chromosome 5D of wheat and Ling et al. (2004) screened > 200 accessions of Ae. tauschii to search for an Lr1‐
type of gene in the D‐genome donor of bread wheat. Using well‐characterized leaf rust isolates, five
different Ae. tauschii accessions were identified which carried a resistance gene that was functionally identical
to Lr1 in bread wheat and mapped to the same genetic region as Lr1 on chromosome 5D, indicating an
orthologous resistance gene. Physical mapping and comparison with the later isolated Lr1 gene
(Cloutier et al., 2007) revealed that a specific polymorphic segment of 605 bp, encoding LRRs 9–15,
distinguished both the hexaploid wheat Lr1 as well as the Ae. tauschii ortholog from the susceptible allele
of Lr1 (Qiu et al., 2007). There are only very few polymorphisms between the Lr1 gene in hexaploid wheat and
the orthologous Lr1 genes in the five Ae. tauschii accessions with the Lr1 resistance phenotype, and it is likely
that the Lr1 gene in hexaploid wheat evolved in the diploid Ae. tauschii and was later introduced to bread wheat
by gene flow based on hybridization.
Similar studies have been performed on the origin of the Lr10 leaf rust resistance gene in wheat. It is located on
chromosome arm 1AS and was cloned from bread cv Thatcher Lr10 (Feuillet et al., 2003). A diversity study on
20 diploid (T. urartu; A‐genome donor) and tetraploid (wild and domesticated) wheat lines revealed that all
carried a sequence homologous to Lr10 (Loutre et al., 2009). It has been shown previously that the hexaploid
wheat genepool has two haplotypes at the Lr10 locus. One originated from a complex deletion event in the
original functional haplotype, followed by a large inversion, and therefore is evolutionary younger. This
deletion/inversion haplotype in hexaploid wheat was found in an identical form in the A genomes of diploid and
tetraploid wheat, and is therefore also an ancient haplotype (Isidore et al., 2005). Thus, identical
presence/absence haplotypes were present at the Lr10 locus at all three ploidy levels. Interestingly,
the Lr10 coding sequence in the tetraploid wheat cultivar Altar was identical to the bread wheat Lr10 gene, with
only two sequence polymorphisms in the intronic region. Using virus‐induced gene silencing, it was found that,
in addition to cultivar Altar, there were functional Lr10 genes in the two tetraploid cultivars Russello and Bufala
that are of Italian origin (Loutre et al., 2009). These studies demonstrated ancient diversity of the Lr10 locus
and the presence of an active Lr10 gene in tetraploid wheat. The evolutionary stability of a presence/absence
polymorphism of the Lr10 gene suggests a balanced polymorphism and maintenance of both haplotypes in the
genepool, as also suggested for other resistance genes, such as Rpm1 in Arabidopsis (Tian et al., 2003). In
conclusion, the Lr10 gene was most probably already present in a tetraploid wheat progenitor (Fig. 3).

The studies on Lr1 and Lr10 both revealed orthologous functional genes in diploid and tetraploid bread wheat
progenitor species. Thus, these two genes are most probably ancient and have been introduced into the bread
wheat genepool, either in the original hybridization resulting in hexaploid wheat or by gene flow (Fig. 3).
Clearly, this is one evolutionary route of bread wheat resistance gene evolution, but, as the next case study
shows, is not the only one.

3. The evolutionary history of functional Pm3 alleles in relation to wheat

evolution and domestication
The Pm3 powdery mildew resistance in hexaploid wheat germplasm was described originally as a multi‐allelic
series of 10 resistance alleles, Pm3a–j (McIntosh et al., 2008), each having a characteristic race specificity. The
gene is located on chromosome arm 1AS in a similar region of the chromosome arm as the Lr10 gene described
above. The Pm3 alleles were identified in cultivars originating from all over the world, with no clear center of
origin. After map‐based cloning of the first allele Pm3b (Yahiaoui et al., 2004), the remaining alleles were
isolated by PCR amplification and were found to be true alleles of the same gene in a cluster of Pm3‐like genes
(Srichumpa et al., 2005; Yahiaoui et al., 2006). Three of the alleles (Pm3h, Pm3i and Pm3j) turned out to be
identical to Pm3d, Pm3c and Pm3b, respectively.

The seven alleles Pm3a–g all encode classical NLR immune receptors and the encoded proteins are highly
similar to each other. Polymorphisms were either restricted to a few single amino acid polymorphisms, mostly
in the LRR region, or caused by short polymorphic blocks encoded by gene sequences probably derived from
gene conversion events (Yahiaoui et al., 2006). Large‐scale allele mining in diverse wheat germplasm
consisting of > 2000 accessions, identified by both a focused identification of germplasm strategy as well as
geographical considerations, revealed a total of nine additional functional Pm3 alleles (Pm3l–t)
(Bhullar et al., 2009, 2010b). In addition, these alleles are highly similar in amino acid sequence, indicating a
very recent common ancestor in hexaploid wheat. Indeed, a rough estimate of the allelic divergence time
suggested that they could have diverged as recently as a few thousand years ago, that is, after formation of
hexaploid wheat. Based on the relatively low sequence diversity of these 16 alleles, a Pm3 consensus sequence
could be derived (Yahiaoui et al., 2006; Bhullar et al., 2010b). Interestingly, a gene that is identical to this
consensus sequence exists in the bread wheat genepool, for example, in the Chinese landrace ‘Chinese Spring’,
which is powdery mildew susceptible. This Pm3 allele was called Pm3CS and is non‐functional, that is, it does
not confer resistance to any of the tested powdery mildew isolates.

Allele mining in 375 accessions representing wild and domesticated tetraploid wheat species, including 126
accessions from a highly diverse collection assembled by Ozkan et al. (2003, 2005), revealed considerable
diversity of Pm3 alleles in this genepool (Yahiaoui et al., 2006, 2009). With the exception of one haplotype,
haplotypes in tetraploid wheat were all different from haplotypes in hexaploid wheat, indicating an independent
evolution of Pm3 in hexaploid wheat after domestication. Only one new, functionally active resistance
allele, Pm3k, was identified in tetraploid genotypes. The Pm3k sequence is not present in the bread wheat
genepool analyzed so far (Bhullar et al., 2009; Yahiaoui et al., 2009). Thus, none of the 16
functional Pm3 alleles in bread wheat corresponds to the only active Pm3 allele in tetraploid wheat, confirming
independent evolution in the two species. As described above, there is a natural, susceptible allele in the bread
wheat genotype Chinese Spring, Pm3CS, which also corresponds to the consensus sequence of all
functional Pm3 alleles. Interestingly, the Pm3CS allele was also found in a total of six tetraploid wheat lines and
is actually the only common Pm3 allele between the tetraploid and hexaploid wheat genepools that diverged
only 10 000 yr ago (Yahiaoui et al., 2006). Moreover, the four wild tetraploid wheat genotypes which
contain Pm3CS originate from south‐eastern Turkey, from sites which are geographically close or identical to
the proposed sites for tetraploid wheat domestication (Ozkan et al., 2005; Yahiaoui et al., 2006). The best
explanation for these observations is based on a model in which the Pm3CS allele is the ancestral allele of all
hexaploid Pm3 alleles. Possibly, at this time, this was a functional allele providing resistance to powdery
mildew and was only later overcome once large plant populations containing this allele were grown in early
agriculture after bread wheat domestication. A wild tetraploid wheat line carrying this allele contributed
the Pm3 genomic region to some of the first domesticated emmer genotypes. Furthermore, one of these early
domesticated emmer wheats with Pm3CS was involved in the hybridization(s) with Ae. tauschii, resulting in a
hexaploid wheat line carrying Pm3CS. Based on this template allele, mutation and gene conversion events then
resulted in a larger set of new haplotypes, some of which were active against powdery mildew and therefore
selected by farmers and breeders.

In summary, the functional allelic diversity of Pm3 in bread wheat originated only after bread wheat
domestication, and therefore the evolution of active Pm3 genes is distinct from that of the ancient resistance
genes Lr1 and Lr10 (Fig. 3).

4. Modeling of Pm3 NLR protein structure reveals distinct evolutionary histories

in tetraploid and hexaploid wheat
Three dimensional modeling of the Pm3 protein was used to determine the possible location of polymorphic
amino acid residues compared with the susceptible consensus sequence (Sela et al., 2014). This analysis of
possible structural consequences of diversity suggested distinct evolutionary histories of Pm3 proteins in
tetraploid and hexaploid wheat. The structural model proposed a very long LRR 24 repeat which separated the
two regions of highest variability in the two wheat species. Most interspecies polymorphic amino acids occur in
LRRs 19–24 in the core motif LxxLxLxxN/C and also in the loop regions of LRRs 20, 22 and 22a which
connect the core motifs (Fig. 4). By contrast, the within‐species polymorphism hotspot in Pm3 variants of
hexaploid wheat is mostly confined to the core motif in the LRRs 25–28, specifically at solvent exposed
residues. This pattern could reflect differential selection pressure on Pm3 in the two wheat species. With the
exception of Pm3k, the Pm3 alleles in wild tetraploid wheat do not give resistance to current mildew races.
However, it is possible that they were functional before domestication and the location of the polymorphisms
indicates selection pressure during that time. After domestication, all the tetraploid Pm3 genes were overcome
because of the rapidly increasing acreage of wheat and the large pathogen populations. The new Pm3 resistance
alleles would then have evolved as a consequence of mutations in different LRR regions. This model implies
that both LRRs 19–24, as well as 25–29, are involved in specific binding of the avirulence gene product or a
putative guardee/decoy.
Figure 4
Open in figure viewerPowerPoint
Comparison of interspecies and within‐species polymorphisms in the powdery mildew resistance nucleotide‐
binding, leucine‐rich repeat receptor (NLR) protein Pm3 of tetraploid and hexaploid wheat (adapted from
Sela et al., 2014). The Pm3‐leucine‐rich repeat (LRR) domain backbone is shown in gray in full‐length (LRRs
1–28) (a) and LRRs 19–28 (b). Interspecies polymorphic residues between tetraploid and hexaploid wheat are
colored in green and within‐species polymorphic residues are colored in blue. The interspecies comparison was
made using tetraploid wild emmer wheat (Triticum dicoccoides) and hexaploid bread wheat (Triticum
aestivum), and the within‐species comparison was made using hexaploid bread wheat accessions.
In the last 2 yr, there has been significant progress in the characterization of the genetic basis of mildew
interactions on the pathogen side. Although all the work described above on the identification and
characterization of Pm3 alleles in tetraploid and hexaploid wheat was performed with mildew from bread
wheat, there is some evidence for mildew specialization on the two different wheat species (Ben‐
David et al., 2016; Menardo et al. 2016). The first Avr gene recognized by a Pm3 allele has been cloned
recently (AvrPm3a/f, recognized by Pm3a and Pm3f) (Bourras et al., 2015) and has been found to encode a
typical secreted protein. The molecular isolation of this Avr gene provides a start to test the interactions
suggested by structural modeling. The protein modeling data indicate that molecular diversity studies can
generate hypotheses for molecular functional studies, demonstrating the potential of such studies which goes
beyond a simple catalog of diversity.

5. Conservation of orthologous gene function after long divergence times in

evolution
It is still an open question how frequently orthologous genes in different cereal species evolve in parallel over a
long time to confer resistance against similar pathogens. The Mla locus in barley and wheat represents such a
case in which the same orthologous genes in different species confer resistance (Jordan et al., 2011;
Periyannan et al., 2013; Mago et al., 2015). Another well‐studied example relates to the Pm3 locus. It was
found that the rye gene Pm8 is an ortholog of wheat Pm3 (Hurni et al., 2013). Pm8 is active in wheat, but the
global use of this gene, which forms part of the yield‐increasing 1BL/1RS translocation, has resulted in the
occurrence of many virulent races. It is surprising that orthologous genes have a conserved function after many
million years of divergence, as resistance genes and resistance loci in general are very dynamic and can evolve
rapidly.
The functional conservation of orthologous resistance genes also has implications on our understanding of
interactions with the individual mildew forms that are specialized on the different hosts. The powdery mildew
form specialized on rye cannot infect wheat. Nevertheless, the conservation of activity of Pm8 towards wheat
powdery mildew suggests that the molecular activity recognized by Pm8 is functionally conserved in the two
specialized mildews. Therefore, a comparative molecular study of the Pm3/Pm8 interaction with mildew might
reveal evolutionary conserved elements that are essential for the pathogen and that could ultimately be used to
develop more durable resistance. Such pathogen‐informed strategies could ideally complement a whole set of
approaches focusing on resistance management on the host side, which have been described in two recent
excellent reviews (Burdon et al., 2014; Zhan et al., 2015).

6. Evolutionary differentiation of NLR genes can result in unexpected side

effects: hybrid necrosis and resistance suppression
Plant immune receptor‐type proteins have been implicated in hybrid necrosis, which occurs when two
incompatible genotypes are crossed, resulting in evolutionary steps towards plant speciation (Bomblies &
Weigel, 2007; Bomblies et al., 2007; Ispolatov & Doebeli, 2009; Chae et al., 2014). Hybrid necrosis was also
observed in crosses of some wheat genotypes. The two wheat genes Ne1 and Ne2 occur in different allelic
forms in the genepool and, when combined in the same genotype, result in hybrid necrosis. Therefore,
genepools with either the Ne1 or Ne2 gene are on an evolutionary trajectory to evolve into different species.
Based on genetic crosses and mutational studies, Zhang et al. (2016) have concluded recently that Lr13 and
the Ne2m allele may be the same gene and, based on the work in Arabidopsis, it is tempting to speculate
that Lr13 encodes an NLR‐type protein. Given the rapid progress in wheat genome sequencing, the cloning
of Lr13 seems to be within reach in the next few years.

In addition to negative interactions, the model of Ispolatov & Doebeli (2009) also predicts a class of hybrids
with a weakened resistance response. Interestingly, it has been observed previously that the introgression of
resistance genes from lower to higher ploidy levels frequently results in loss of resistance (Hsam &
Zeller, 2002; McIntosh et al., 2011). It has been speculated that this could be a result of suppression interactions
by non‐functional gene homoeologs of the introgressed active resistance genes. This would result in a dominant
negative genetic interaction. These hypotheses have been experimentally tested for the rye Pm8 resistance gene,
which was found to be suppressed in certain wheat genotypes, but not in others. It was found that suppression
depends on Pm3 alleles (Hurni et al., 2014). Suppression did not occur at the transcriptional or translational
level, but seemed to be caused by protein interactions resulting in protein dimers or multimers that were
incompetent for the induction of the immune response. Interestingly, this suppression was also observed among
some, but not all, of the Pm3 alleles when they were present in hybrid plants, but also in transgenic plants
homozygous for two different Pm3 alleles (Stirnweis et al., 2014). Thus, the products of cereal NLR genes,
being functional genes or non‐functional homologs, can contribute to a variety of molecular interactions which
result in plant phenotypes of evolutionary and agricultural importance. The molecular and structural basis of
such interactions remains unclear and represents one of the very attractive research topics in the field of NLR
genes.

VI. Quantitative disease resistance – why plasma membrane‐

localized receptors and NLRs are not sufficient to explain disease
resistance in cereals
Quantitative disease resistance (QR) is defined as host plant resistance that is partial and conferred by the joint
effect of several genes (St Clair, 2010; Niks et al., 2015). Although there is no causal link between the
completeness of resistance gene action and durability, it has been found that partial QR genes are often more
durable than NLR‐based immunity (Lagudah, 2011; Ellis et al., 2014). It is not surprising that very little is
known about partial QR in model plants and that the bulk of our knowledge on QR stems from cereals. The
partial effects of QR genes can be difficult to score and can depend on environmental conditions, as well as the
developmental stage of the plant. Breeders and pathologists, however, have exploited QR during decades of
cereal breeding. Access to breeding records and disease monitoring programs provides us with exact and rich
information on cereal cultivars that were grown over long periods and on large areas without resistance
breakdown. It is important to note that, despite the joint action of several partial resistance genes in one cultivar,
single QR genes can be mendelized and their effects scored as single genes in near‐isogenic backgrounds. One
example is the recessive rice blast resistance gene pi21 that has been discussed previously. pi21 was originally
identified in a quantitative trait locus (QTL) study as one of several QR genes in the Japanese upland rice
cultivar Owarihatamochi (Fukuoka & Okuno, 2001). Through repeated backcrosses, pi21 was transferred into a
near‐isogenic background which allowed the assessment of the phenotypic effect of pi21 as a single gene. Near‐
isogenic lines with the loss‐of‐function pi21 allele showed smaller rice blast lesions than lines with the
functional, susceptible Pi21 allele (Fukuoka et al., 2009). QTL analyses are statistical approaches and it is not
possible to draw any conclusions from QTL peaks with regard to the nature of the protein responsible for the
resistance phenotype. For example, it is feasible that membrane‐localized RLKs and RLPs can be identified in
QTL studies, and there are even examples of race‐specific NLR genes that were identified in QTL studies
(Paillard et al., 2012; Zhang et al., 2015). However, recent research has led to the identification of QR genes
that code for unusual resistance proteins. In the next two sections, we discuss three examples of such unusual
resistance proteins.

1. Spontaneous mutation events in transporter genes led to the emergence of

durable, multi‐pathogen resistance after wheat domestication
Hexaploid bread wheat contains a small, but important, group of genes that confer resistance against multiple
biotrophic fungal pathogens (Ellis et al., 2014). Two of these genes, Lr34 and Lr67, have been cloned recently
and encode for membrane‐localized transporter proteins: Lr34 for a full‐size ATP‐binding cassette (ABC)
transporter (Krattinger et al., 2009) and Lr67 for a hexose transporter (Moore et al., 2015). Both genes confer
race non‐specific and partial resistance against the three wheat rusts and powdery mildew. Lr34 can be
considered as one of the most durable sources of partial disease resistance known in cereals. A very interesting
observation was that the resistant versions of both Lr34 and Lr67 only differed by two critical amino acid
changes from their susceptible protein versions. The susceptible version of Lr67 showed a high affinity to
glucose in yeast uptake experiments, whereas glucose uptake was abolished in the resistant hexose transporter
version. The substrate of the Lr34 ABC transporter is still unknown.

Very interestingly, the resistant alleles of both genes were only found in cultivated hexaploid bread wheat, but
not in wild wheat progenitors (Krattinger et al., 2013; Moore et al., 2015). This indicates that the resistant
versions of both transporters evolved only after wheat domestication, most probably as a result of spontaneous
sequence changes (Fig. 3). Selection by ancient farmers might therefore have played an important role in
preserving these rare mutation events for modern breeding. It is possible that these rare alleles conferred a
selective disadvantage in natural populations and that they would therefore rapidly disappear again. Only under
human cultivation might such gene variants be beneficial. Based on the observations on Lr34 and Lr67, we
hypothesize that similar specific sequence changes in other transporter proteins might have the same effect. This
suggests the exciting possibility to artificially ‘create’ durable multi‐pathogen alleles through targeted
mutagenesis. It is therefore essential to obtain a better understanding of how exactly these mutations affect the
substrate specificities in the two transporters. Interestingly, Lr34‐like or Lr67‐like disease resistances have not
been described in diploid cereals, such as barley, rice or maize. Transformation experiments, however, have
shown that Lr67 and Lr34 can be functionally transferred into barley (Lr67/Lr34) and rice (Lr34), where they
confer partial resistance against diseases specific to barley (barley leaf rust, barley powdery mildew) and rice
(rice blast), respectively (Risk et al., 2013; Moore et al., 2015; Krattinger et al., 2016). These results indicate
that all components required for this type of disease resistance are present in diploid cereals as well.

2. Yr36 – a wheat broad‐spectrum resistance gene that was not incorporated into
modern cultivars
Another interesting example of a partial, broad‐spectrum resistance gene is Yr36, which was originally
identified in wild tetraploid emmer wheat (Triticum turgidum ssp. dicoccoides)
(Uauy et al., 2005). Yr36 confers race non‐specific resistance against fungal stripe rust disease and encodes a
WHEAT KINASE START1 (WKS1) protein with an N‐terminal non‐RD kinase domain fused to a START
lipid‐binding domain at the C‐terminus (Fu et al., 2009). Interestingly, there are two major splice variants of this
gene, resulting in a full‐length WKS1.1 protein and WKS1.2 with a complete kinase but a truncated START
domain. Only the WKS1.1, but not WKS1.2, variant conferred resistance to stripe rust (Gou et al., 2015). After
stripe rust infection, the WKS1.1 splice variant increased in abundance compared with the alternative variant.
WKS1.1 interacted with the thylakoid‐associated ascorbate peroxidase (tAPX) in the choloroplast. Ascorbate
peroxidases are involved in the detoxification of peroxides. Gou et al. (2015) concluded that the interaction of
WKS1.1 and tAPX might reduce the cell's ability to detoxify reactive oxygen species, which could trigger an
HR. As WKS1.1‐mediated signaling is much slower than a typical NLR‐triggered HR, Yr36‐containing wheat
plants only show a partial resistance phenotype. Interestingly, Yr36 was not introgressed into the cultivated
durum and bread wheat genepool, but was only found in wild emmer. Huang et al. (2016) studied the
distribution of Yr36 in 435 wild emmer accessions and only found the gene in accessions from the southern
regions of the Fertile Crescent, whereas accessions from the northern parts were absent for Yr36. Wheat
domestication is thought to have occurred in the northern parts of the Fertile Crescent, which would explain the
absence of Yr36 in domesticated wheats. Yr36 therefore represents an example of an important resistance gene
that escaped incorporation into the modern genepool through domestication and breeding. This highlights the
importance of mining wild progenitors of cultivated cereals and landraces for new sources of durable and broad‐
spectrum disease resistance (Tanksley & McCouch, 1997).

VII. Conclusions: evolutionary studies of cereal resistance genes

contribute to both basic research and breeding
Studies on the origin of resistance genes can identify geographic regions and/or genepools that are promising
for the identification of new functional resistance genes. For example, the diversity studies on Pm3 revealed that
both Turkey as well as the Himalayan mountain range are regions with high diversity of this gene
(Bhullar et al., 2009, 2010a,b). Furthermore, for genes that evolved after domestication, the bread wheat
genepool is most promising for the identification of additional alleles, with wild or domesticated relatives being
less promising. Nevertheless, it is also possible to find functional diversity there, as shown by the identification
of the rye Pm8 as an orthologue of Pm3, or the functional orthologs of Mla in wheat and rye. Wild relatives are
attractive to explore for ancient genes, because a higher level of allelic diversity can be expected in wild grass
genepools. Thus, it is highly important to isolate as many resistance genes as possible in the near future to be
able to perform allele mining in diverse genepools. Given the recent rapid progress in wheat genome
sequencing and the development of new technology based on rapid mutant identification
(Steuernagel et al., 2016), it can be expected that many more resistance genes will be cloned in the next few
years, resulting in large opportunities for gene studies and combination breeding and gene stacking. It has been
shown that a transgenic use of overexpressed Pm3 alleles in the field can increase resistance
(Brunner et al., 2011, 2012), but the real test for agricultural use would be the growth of such transgenic lines
on a large scale to determine a possible pathogen adaptation.

The observation of very recent events resulting in novel, active resistance genes is also encouraging for
emerging approaches to develop resistance genes with new specificities by gene modification. In cases such
as Pm3, Lr67 or Lr34, the short time period of domestication and the low natural mutation frequencies might
not have been sufficient to evolve all possible useful variants of these genes based on the available ‘template’
sequences that made it through the bottleneck of domestication. Thus, it can reasonably be assumed that, based
on a better molecular understanding, novel genes can be designed. Indeed, the use of sequence information
obtained from natural diversity studies has allowed Stirnweis et al. (2014) to make improved versions of
known Pm3 genes in the laboratory by modifying only two amino acids. Finally, there are some immediate
applications of several of the recent findings in practical breeding. For example, mutation screens for the
removal of suppressor genes which have possibly accumulated during evolution could awaken ‘sleeping’
resistance genes which might be highly useful. Systematic work in this direction is very promising and has not
yet been undertaken at a larger scale.
 Acknowledgements
We would like to thank Dr Thomas Wicker and Dr Teresa Koller for support with the preparation of the figures.
This work was supported by an Ambizione grant from the Swiss National Science Foundation to S.G.K. and a
grant from the same funding source to B.K. (#310030_163260).

https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.14097

 C11.

What are the mechanisms for systemic acquired resistance to pathogens?

Signal regulators of systemic acquired

resistance

Salicylic acid (SA) is an important phytohormone that plays a vital role in a number of
physiological responses, including plant defense. The last two decades have witnessed a
number of breakthroughs related to biosynthesis, transport, perception and signaling
mediated by SA. These findings demonstrate that SA plays a crictical role in both local and
systemic defense responses. Systemic acquired resistance (SAR) is one such SA-dependent
response. SAR is a long distance signaling mechanism that provides broad spectrum and
long-lasting resistance to secondary infections throughout the plant. This unique feature
makes SAR a highly desirable trait in crop production. This review summarizes the recent
advances in the role of SA in SAR and discusses its relationship to other SAR inducers.

Introduction
Plants being sessile are constantly exposed to a number of pathogenic microbes, which
based on their infectious lifestyles can be broadly divided into biotrophs and necrotrophs
(Glazebrook, 2005; Mengiste, 2012; Lai and Mengiste, 2013). Biotrophic pathogens rely on
nutrients from living host cells, whereas necrotrophic pathogens feed on dead cells. Plants
employ distinct immune responses to counter these pathogens and this aspect has been
covered in detail in several recent reviews (Spoel and Dong, 2012; Dangl et al., 2013). This
first layer of host defense involves the recognition of pathogen (or microbe) associated-
molecular patterns (PAMPs/MAMPs), such as bacterial flagellin, lipopolysaccharides, and
peptidoglycans. PAMPs are recognized by specialized transmembrane proteins in the
plant, termed pattern recognition receptors (PPRs). PRR-mediated recognition of PAMPs
triggers downstream signaling leading to the activation of basal resistance termed PAMP-
triggered immunity (PTI; Schwessinger and Ronald, 2012). PTI can be suppressed by
pathogen encoded effector proteins commonly known as avirulence (avr) factors (Göhre
and Robatzek, 2008; Cunnac et al., 2009; Bozkurt et al., 2011; Caillaud et al.,
2012; Marrtin and Kamoun, 2012; Cheong et al., 2013; Cui et al., 2013; Dangl et al.,
2013; Giraldo and Valent, 2013). The avr factors are in turn recognized by the host
encoded resistance (R) proteins, which confer more durable and robust resistance
termed R gene- or effector-triggered immunity (ETI; Bogdanove and Martin,
2000; Mackey et al., 2002, 2003; Gu et al., 2005; Jones and Dangl, 2006; Narusaka et al.,
2009; Cesari et al., 2013). ETI is generally associated with programmed cell death (PCD)
at the site of infection and this phenomenon is called hypersensitive response
(HR; Holliday et al., 1981; Dangl et al., 1996; Morel and Dangl, 1997).

Induction of local responses is associated with the transport of defense signals throughout
the plant resulting in broad-spectrum disease resistance against secondary infections. This
phenomenon, known as systemic acquired resistance (SAR), is conserved among diverse
plants and confers long-lasting resistance to unrelated pathogens (Chaturvedi et al.,
2008; Dempsey and Klessig, 2012; Fu and Dong, 2013; Kachroo and Robin, 2013; Lucas et
al., 2013; Shah and Zeier, 2013; Wendehenne et al., 2014). Several studies have shown that
the establishment of SAR involves the generation and transport of signals via phloem to
the uninfected distal tissues (Guedes et al., 1980; Tuzun and Kuc, 1985). Among the
signals contributing to SAR are salicylic acid (SA) and several components of the SA
pathway including the methylated derivative of SA (methyl SA,MeSA, Park et al., 2007).
Additionally, the diterpenoid dehydroabietinal (DA, Chaturvedi et al., 2012), the nine
carbon (C9) dicarboxylic acid azelaic acid (AzA, Jung et al., 2009), an amino acid
derivative pipecolic acid (Pip; Návarová et al., 2012), auxin (Truman et al., 2010), the
phosphorylated sugar glycerol-3-phosphate (G3P, Chanda et al., 2011; Mandal et al.,
2012; Yu et al., 2013), the free radicals nitric oxide (NO) and reactive oxygen species
(ROS; Wang et al., 2014a; El-Shetehy et al., 2015), galactolipids (Gao et al., 2014), factors
contributing to cuticle formation (Xia et al., 2009, 2010, 2012) and the lipid transfer
proteins (LTPs) DIR1 (Defective in Induced Resistance, Maldonado et al., 2002) and AZI1
(AzA insensitive, Jung et al., 2009), have all been proposed to serve as SAR signals. Here,
we review the role of SA in SAR and discuss its relationship to these various SAR signals.

SA Biosysnthesis and SAR

Salicylic acid biosynthesis occurs via the shikimic acid pathway, which forms two distinct
sub-branches both of which synthesize SA. These branched pathways, designated as
isochorismate synthase (ICS)- and the phenylalanine ammonia-lyase (PAL)-derived
pathways, utilize chorismate as the common precursor (Shah, 2003; Chen et al.,
2009; Kachroo and Kachroo, 2009; Yu et al., 2010; An and Mou, 2011; Dempsey et al.,
2011; Vlot et al., 2009; Singh et al., 2013; Figure 1). The first step of the PAL pathway
involves conversion of phenylalanine (Phe) to trans-cinnamic acid and this reaction is
catalyzed by PAL, a key enzyme of this pathway that is induced by pathogen infection.
The Arabidopsis genome encodes four PAL isoforms and PAL quadruple mutants or wild-
type plants treated with the PAL inhibitor, 2-aminoindan-2-phosphonic acid contain
reduced SA, show increased susceptibility to pathogens and are unable to induce SAR
(Yalpani et al., 1993; Mauch-Mani and Slusarenko, 1996; Pallas et al., 1996; Huang et al.,
2010). Although relative contributions of PAL versus ICS branches toward SA biosynthesis
vary between different plant species, at least in Arabidopsis majority of the pathogen-
induced SA appears to be derived from the ICS branch. The ICS branch involves
conversion of chorismate to isochorismate by ICS followed by coversion of isochorismate
to SA by isochorismate pyruvate lyase (IPL). The Arabidopsis genome encodes two
isoforms of ICS, of which ICS1 (SID2) accounts for ∼95% of basal- or pathogen-induced
SA (Strawn et al., 2007; Garcion et al., 2008). A mutation in ICS1 also impairs SAR
(Wildermuth et al., 2001; Jung et al., 2009; Chanda et al., 2011; Wang et al., 2014a),
suggesting that SA contributed by both PAL- and ICS-pathways is critical for the induction
and/or establishment of SAR. This together with the compromised SAR phenotype of
transgenic plants expressing bacterial salicylate hydroxylase (NahG; Vernooij et al., 1994),
an enzyme that catalyzes the conversion of SA to catechol, reemphasize the importance of
SA in SAR. It is unclear what factors govern the specific recruitment of the PAL or ICS
pathways for SA biosynthesis.
FIGURE 1

FIGURE 1. Simplified scheme for salicylic acid (SA) biosynthesis in plants.

Critical enzymes are shown in red. ICS, isochorismate synthase; IPL, isochorismate
pyruvate lyase; PAL, phenylalanine ammonia-lyase; BA2H, benzoic acid 2-hydroxylase.

Salicylic acid synthesized in the chloroplasts is exported out to the cytosol via EDS5, a
member of the MATE (Multidrug and Toxin Extrusion) transporter family, located in the
chloroplast envelope (Nawrath et al., 2002; Serrano et al., 2013). Notably, a mutation
in EDS5 results in complete shut down of SA biosynthesis rather than SA accumulation
within the chloroplasts. Thus, mutations in ICS1 and EDS5 similarly affect SA levels and
the corresponding mutants thereby exhibit overlapping defense defects. This is likely due
to negative feed-back regulation of ICS1 by SA (Fragnière et al., 2011; Serrano et al., 2013).
The triphosphate tunnel metalloenzyme 2 is a negative regulator of the SA feed-back loop
and functions in defense signal amplification (Ung et al., 2014). Pathogen induced
expression of ICS1 requires the binding of calmodulin binding protein CBP60g and its
homolog, non-calmodulin binding SARD1 (SAR Deficient 1) to the ICS1 promoter. CBP60g
and SARD1 specifically bind the GAAATTTTGG sequence in the ICS1 promoter (Truman
and Glazebrook, 2012). The induction of ICS1 and thereby SA biosynthesis is inhibited
in cbp60g sard1 double mutant, resulting in compromised SAR (Zhang et al., 2010).

Although a number of studies have demonstrated the critical requirement of SA in SAR, a

specific requirement for SA accumulation beyond basal levels during SAR has not been
established. For instance, plants lacking a functional R protein RPS2 accumulate normal
levels of SA in their distal tissues in response to infection by Pseudomonas syringae pv.
tomato expressing avrRpt2, yet these plants are compromised for SAR (Cameron et al.,
1999). Additionally, exogenous application of either G3P or AzA, which induce SAR in
wild-type plants, do not induce SA accumulation. However, neither G3P nor AzA can
confer SAR in ics1 (sid2) mutant plants, which contain significantly reduced basal- and
pathogen-induced SA. Thus, although SA is clearly critical for SAR, accumulation of SA
alone is insufficient to establish SAR. Furthermore, although SA has been shown to
accumulate to varying levels in the distal tissues of SAR induced plants (Table 1), there is
no evidence suggesting that this accumulation is essential for SAR.
TABLE 1
TABLE 1. Free and bound salicylic acid (SA) levels reported in distal tissues of
mock-and pathogen-inoculated plants.

In comparison to local tissues, the distal tissues of SAR-induced plants have been shown
to accumulate a broad range of SA ranging from as low as 10 ng/ g FW to ∼2.6 μg/g FW
(Table 1; Rasmussen et al., 1991; Yalpani et al., 1991; Meuwly and Métraux, 1993; Molders
et al., 1994; Vernooij et al., 1994; Lawton et al., 1995; Shulaev et al., 1995; Cameron et al.,
1999; Kiefer and Slusarenko, 2003; Mishina and Zeier, 2006; Attaran et al., 2009; Liu et
al., 2010, 2011; Xia et al., 2010; Chanda et al., 2011; Gao et al., 2014). The inability to
accumulate SA in distal tissues has also been suggested to contribute to impaired SAR
in ald1 (agd2-Like Defense response protein 1) and fmo1 (Flavin Monooxygenase 1)
mutants, both of which accumulate normal SA in the local tissue (Song et al.,
2004a,b; Mishina and Zeier, 2006). ALD1 encodes an aminotransferase that catalyzes the
biosynthesis of the SAR inducer Pip, (Song et al., 2004b; Návarová et al., 2012) and FMO1
has been suggested to function downstream of Pip (Návarová et al., 2012). Thus, other
factors besides SA might contribute to the SAR defect of ald1 and fmo1 mutants. One
possibility is that SAR can be induced via SA-independent factors so long as a minimum
basal level of SA can be maintained. Alternatively, SA accumulation in distal tissues might
contribute to the priming process resulting in the activation of stronger defense responses
upon secondary infections (Návarová et al., 2012; Gruner et al., 2013).

SA-Derivatives and SAR

A majority of the synthesized SA is converted and stored as biologically inactive
derivatives via glucosylation, methylation and amino acid conjugation since accumulation
of the acidic SA has adverse physiological consequences (Heil and Baldwin, 2002; Heidel
et al., 2004). These include SA 2-O-β-D-glucose (SAG), SA glucose ester (SGE), methyl SA
(MeSA), and SA-amino acid conjugates (Pierpoint, 1994; Vlot et al., 2009; Dempsey et al.,
2011). Most recently, SA was shown to be derivatized to 2,3-dihydroxybenzoic acid (2,3-
DHBA) and this reaction is catalyzed by SA 3-hydroxylase (S3H; Zhang et al., 2013). As
predicted s3h knockout plants contain very high levels of SA, while plants
expressing S3H gain-of-function mutations accumulate high amounts of 2,3-DHBA
(Zhang et al., 2013). SA derivatives serve as storage forms that can be converted back to
free SA (Hennig et al., 1993; Kawano et al., 2004; Kachroo and Kachroo, 2012). With the
exception of MeSA however, the exact role of SA derivatives in SAR remains unclear.

Methyl SA is a volatile and phloem mobile SA derivative, which accumulates in infected

and distal tissues in response to pathogen infection. Like MeSA, SA also accumulates in
the phloem of tobacco leaves infected with tobacco mosaic virus or Colletotrichum
lagenarium and in cucumber leaves infected with tobacco necrosis virus (Malamy et al.,
1990; Métraux et al., 1990; Park et al., 2007). For SAR, MeSA must be converted to SA in
the distal tissues between the 48–72 h period post primary infection. This time-frame
correlates with that of pathogen-inducible MeSA accumulation in infected and systemic
tissues. The biosynthesis of MeSA is catalyzed by SA methyltransferases (SAMT/BSMT),
and the conversion of MeSA back to SA is mediated by methyl esterase (MES; Chen et al.,
2003; Effmert et al., 2005; Koo et al., 2007). The tobacco MES was first identified based
on its ability to bind SA, and therefore designated as SA-binding protein 2 (SABP2; Kumar
and Klessig, 2003). Grafting studies in tobacco plants silenced for SABP2 have shown that
SABP2 activity in scions, but not root-stocks is required for normal SAR (Park et al.,
2007). Furthermore, the synthetic SA analog, 2,2,2,2′-tetra-fluoroacetophenone, which
inhibits the esterase activity of SABP2, also inhibits SAR (Park et al., 2009). As in tobacco,
homologs of SABP2 (AtMES9) and SAMT AtBSMT1 are required for SAR
in Arabidopsis (Liu et al., 2011). Thus, the ability to derivatize SA to MeSA and reconvert
MeSA back to SA are critical for SAR. Intriguingly, the requirement for AtBSMT1 in SAR
can be bypassed by prolonged exposure to light after pathogen inoculation (Attaran et al.,
2009; Liu et al., 2011). However, the role of light signaling in SAR and how it might
compensate for MeSA is unclear. It is also not known whether MeSA merely functions to
deliver SA to the distal tissues or has other function(s) in SAR. Notably, a certain
percentage of SA is always transported from the inoculated to distal tissues (Meuwly et al.,
1995; Kiefer and Slusarenko, 2003). The biological significance of this transport is unclear,
particularly in view of the fact that SA is not considered to be the mobile SAR signal since
wild-type tobacco scions grafted onto NahG root-stocks exhibit normal SAR (Vernooij et
al., 1994; Meuwly et al., 1995; Kiefer and Slusarenko, 2003).

Regulation of SA Accumulation and SAR

Besides proteins that directly contribute to SA biosynthesis (ICS and PAL) or transport
(EDS5), a number of other proteins have been identified that participate in pathogen
induced SA accumulation and thereby SAR. These include EDS1 (Enhanced Disease
Susceptibility 1), PAD4 (Phytoalexin Deficient 4), and NDR1 (Non-race-specific Disease
Resistance 1; Century et al., 1995, 1997; Falk et al., 1999; Jirage et al., 1999; McDowell et
al., 2000; Feys et al., 2001; Coppinger et al., 2004; Ishihara et al., 2008; Bhattacharjee et
al., 2011; Cacas et al., 2011; Heidrich et al., 2011; Knepper et al., 2011; Lu et al., 2013).
Unlike ICS1 and EDS5, mutations in EDS1, PAD4, or NDR1 cause partial reduction in SA
levels. EDS1 and PAD4 are lipase-like proteins, which together with another lipase-like
protein SAG101 (Senescence Associate Gene 101) form binary and ternary complexes (Feys
et al., 2005; Zhu et al., 2011). EDS1 interacts with PAD4 in both cytosol and nucleus, and
with SAG101 only in the nucleus. EDS1, PAD4, and SAG101 function cooperatively as well
as independently in pathogen defense (Feys et al., 2005; Venugopal et al., 2009; Rietz et
al., 2011; Zhu et al., 2011). For instance, all three proteins are required for R-mediated
resistance against Turnip crinkle virus (TCV) but only PAD4 is required for the SA-
mediated induction of the R gene which confers HR against TCV (HRT; Chandra-Shekara
et al., 2004, 2006, 2007). Interestingly, EDS1, but not PAD4 or SAG101, interacts with
HRT and potentiates HRT-mediated HR to TCV (Zhu et al., 2011). Similarly, only PAD4 is
required for resistance to the green peach aphid, whereas EDS1 and SAG101 are not
(Pegadaraju et al., 2005, 2007; Louis et al., 2010, 2012). Both nuclear and extranuclear
localization of EDS1 is important for its defense function (García et al., 2010). However,
the role of binary or ternary complex formation between EDS1, PAD4, and SAG101
proteins remains unknown. EDS1 was recently shown to participate in both SAR signal
generation in the local tissues as well as perception in the distal leaves (Breitenbach et al.,
2014).
The Arabidopsis genome encodes two isoforms of EDS1 that function redundantly and can
compensate for each other (Zhu et al., 2011). However, some Arabidopsis ecotypes, such
as Wassilewskija, Landsberg, and Dujon, contain only one functional EDS1 isoform, and
this is sufficient for normal resistance in these ecotypes. Like Arabidopsis, soybean also
contains two EDS1 isoforms. Interestingly, Arabidopsis eds1 mutant expressing the
soybean EDS1 orthologs is only partially restored in SA levels, but completely restored in
bacterial resistance (Wang et al., 2014b). This further questions the requirement for
increased SA accumulation during defense activation and raises the possibility that a
certain threshold of SA may be sufficient to induce appropriate defense responses. The
soybean EDS1 orthologs are unable to potentiate TCV coat protein-derived activation of
HRT even though they do interact with HRT (Wang et al., 2014b). This suggests that EDS1
orthologs in different plants may have evolved to perform overlapping as well as distinct
functions.

SA Signaling Components
Salicylic acid-mediated signaling leading to SAR is dependent on the ankyrin repeat
containing protein NPR1 [Non-expressor of Pathogenesis-Related (PR) genes] (Dong,
2004). Under basal or uninduced conditions, NPR1 exists as a cytosolic inactive oligomer
formed by intermolecular disulfide bonding (Mou et al., 2003). Reducing conditions
resulting from accumulation of SA cause dissociation of the NPR1 oligomer into active
monomers and the monomeric form of NPR1 is translocated into the nucleus (Kinkema et
al., 2000; Mou et al., 2003; Tada et al., 2008). Nuclear localization of NPR1 facilitates its
interaction with members of the TGACG motif binding (TGA) transcription factors that
belong to the basic leucine zipper (bZIP) protein family (Zhang et al., 1999; Després et al.,
2000; Niggeweg et al., 2000; Zhou et al., 2000; Chern et al., 2001; Fan and Dong,
2002; Kim and Delaney, 2002). This in turn enhances binding of the TGA factors to
promoter elements of NPR1-dependent target genes (Wang et al., 2006, 2011). Like NPR1,
TGA factors are also required for SAR; the tga2 tga5 tga6 triple mutant is non-responsive
to SA and is defective in SAR (Zhang et al., 2003). Recent studies have shown that NPR1
and TGA1 also undergo S-nitrosylation, which is necessary for the proper functioning of
NPR1 in immunity and increases the DNA binding activity of TGA1 (Tada et al.,
2008; Lindermayr et al., 2010). On the other hand, thiol S-nitrosylation has also been
shown to promote NPR1 oligomerization and thereby its inactivation (Tada et al., 2008).
The nuclear NPR1 is phosphorylated and degraded in a proteasome-dependent manner
(Spoel et al., 2009), and the turnover of NPR1 is essential for SAR establishment.
The Arabidopsis genome contains five paralogs of NPR1 (Liu et al., 2005). Like NPR1,
NPR3, and NPR4 also interact with TGA proteins (Zhang et al., 2006). The npr3
npr4 mutant plants accumulate elevated levels of NPR1 and are consequently defective in
SAR. NPR3 and NPR4 bind SA and function as adaptors of the Cullin 3 ubiquitin E3 ligase
to mediate NPR1 degradation in an SA-dependent manner (Fu et al., 2012). However, the
two differ in that NPR3 has higher affinity for SA than NPR4, and SA promotes the NPR1–
NPR3 interaction but inhibits the NPR1–NPR4 interaction. These contrasting effects
might offer a possible explanation for the nuances underlying NPR1-dependent immunity
under different levels of SA. For instance, high concentration of SA in infected tissues
might favor binding of NPR3 with SA, which would mediate degradation of the cell-death
suppressor NPR1, and initiate PCD and local immunity. On the other hand, lower SA levels
in the distal uninfected tissue would minimize NPR3-SA binding, thereby inhibiting PCD.
Interestingly, in yet another study, NPR1 was also shown to bind SA via the transition
metal copper (Wu et al., 2012; Manohar et al., 2015). The binding of SA to NPR was
suggested to induce a conformational change in NPR1 (Wu et al., 2012), which in turn is
important for NPR1-dependant PR1 expression.

NPR1 is also required for transgenerational SAR, which in turn involves epigenetic
changes (Jaskiewicz et al., 2011; Luna et al., 2012). NPR1 othologs have been characterized
from a number of plants including rice, tobacco, soybean, and cacao (Chern et al.,
2001, 2005, 2014; Ekengren et al., 2003; Zwicker et al., 2007; Sandhu et al., 2009; Shi et
al., 2010; Chen et al., 2013). Transgenic expression of Arabidopsis NPR1 confers enhanced
resistance in heterologous plants (Lin et al., 2004; Shi et al., 2010; Chen et al., 2012).
Conversely, transgenic expression of soybean orthologs can complement the Arabidopsis
npr1 mutation (Sandhu et al., 2009). Overexpression of NPR1 also enhances pathogen
resistance in monocots (Chern et al., 2005; Yuan et al., 2007). However, studies in rice
and barley suggest that NPR1 function may not be fully conserved in monocots and dicots
and that SA signaling and SAR in monocots might involve NPR1-independent pathways
(Shimono et al., 2007; Dey et al., 2014). Transcription analysis in distal tissues revealed
that bacteria-triggered SAR in barley was likely associated with jasmonic acid, ethylene
and ABA, rather than SA. In contrast, SAR in maize is associated with SA accumulation in
local and distal leaves (Balmer et al., 2013). Additionally, petiole exudates from pathogen
infected Arabidopsis plants induced SAR in wheat (Chaturvedi et al., 2008). This suggests
that SAR signaling in barley may not be similar to that in other monocots like maize and
wheat.

The stability of NPR1 is dependent on Mediator (MED) 16 [allelic to Sensitive to Freezing

(SFR) 6] (McKown et al., 1996; Warren et al., 1996), a subunit of the MED complex which
functions as a bridge between transcription factors and the general RNA polymerase II
transcriptional machinery (Zhang et al., 2012). A mutation in MED16 compromises SAR
and SA-induced defense responses but does not affect SA levels or nuclear localization of
NPR1. Thus, MED16 likely functions downstream of SA in the SAR pathway. Interestingly,
MED16 is also required for jasmonic acid/ethylene-responsive gene expression and
resistance to necrotrophic pathogens (Zhang et al., 2012). Thus, MED16 might function by
relaying signals from transcription factors that are specific to the SA and JA/ethylene
pathways. A mutation in another MED subunit, MED 15 (isolated in a screen for non-
recognition-of-the SA analog, BTH, nrb4), also attenuates SAR and SA responsiveness
(Canet et al., 2012). However, MED15 is not required for NPR1 stability or localization and
likely functions downstream of NPR1.

SA Versus Other SAR Inducers

Systemic acquired resistance is a complex phenomenon that involves the interplay of a
diverse group of chemicals and associated proteins, besides SA. Most of these molecules
can now be placed in one of two main branches that comprise the SAR pathway. One
branch involves SA and its signaling component NPR1, and the other branch involves the
free radicals NO and ROS, which function directly upstream of AzA, which in turn is
upstream of G3P (Wang et al., 2014a; Wendehenne et al., 2014; El-Shetehy et al., 2015).
Unlike G3P and AzA, exogenous application of Pip or DA induces SA accumulation in the
absence of pathogen infection (Chaturvedi et al., 2012; Návarová et al., 2012). Therefore,
Pip and DA likely function in the SA branch of SAR. The presence of two SAR branches is
supported by the fact that SA cannot restore SAR in mutants defective in NO, ROS, or G3P
biosynthesis, while NO/ROS cannot confer SAR on mutants defective in SA synthesis or
signaling. Furthermore, pharmacological inhibitors of NO synthesis or NO scavengers
attenuate SA-induced SAR in tobacco (Song and Goodman, 2001). Interestingly, unlike
SA, both NO and ROS function in a concentration dependent manner because they can
confer SAR only when present at an optimal concentration (Wang et al., 2014a). Free
radicals are well known to operate similarly in animal systems where too little or too much
can produce opposing physiological effects (Delledonne et al., 1998; Besson-Bard et al.,
2008; Wink et al., 2011). Free radicals are thought to participate in SAR by mediating the
oxidation of carbon (C) 18 unsaturated fatty acids (FAs) containing a double bond on C 9.
This results in the formation of 9-oxo nonanoic acid (ONA), which is converted to the di-
carboxylic acid AzA by the addition of a carboxylic group. AzA is unable to confer SAR on
mutants unable to synthesize G3P, indicating it functions upstream of G3P. Exogenous
AzA increases the expression of the G3P synthesizing GLY1 and GLI1 genes, which encode
G3P dehydrogenase and glycerol kinase, respectively. G3P operates in a feedback loop
with the LTPs DIR1 and AZI1 such that lack of DIR1 or AZI1 impairs pathogen-induced
G3P accumulation while lack of G3P results in reduced DIR1 and AZI1 transcripts (Yu et
al., 2013). DIR1 and AZI1 form homo- and hetromers suggesting that a complex
comprising these proteins might function in SAR. Perhaps such a complex or the
individual LTPs serve in transporting SAR essential signal(s) to the distal tissues. G3P
appears to be the logical choice for such a transported signal since it is a precursor for lipid
biogenesis. However, no direct interaction could be detected between G3P and DIR1
raising the possibility that G3P may be derivatized and this derivative may then be
transported from infected to distal tissues. Radiolabel feeding experiments showed that
G3P is indeed converted to an as yet unidentified derivative which can translocate from
infected to distal tissues in a DIR1-dependent manner (Chanda et al., 2011).

Recent studies have shown that the C 18 FAs which serve as precusors for AzA are derived
from the major plastidal lipids, monogalactosyldiacylglycerol (MGDG) and
digalactosyldiacylglycerol (DGDG), which comprise ∼80% of the total lipids in plants
(Zoeller et al., 2012; Gao et al., 2014). Thus, besides SA, NO, ROS and G3P, chloroplasts
also serve as an important site for AzA biosynthesis. Notably, both galactose sugars in
DGDG appear to be important for SAR since dgd1 plants producing α-glucose-β-galactose
diacylglycerol via transgenic expression of a bacterial glucosyltransferase, are not restored
in SAR even though they are partially restored in chloroplast function. Thus it appears that
the position of the hydroxyl group on C 4 of galactose may be important for SAR since
glucose and galactose are sterioisomeric sugars which differ only in the position of their
axial hydroxyl group at C 4.
Cross Talk between SA and NO Pathways in SAR
Monogalactosyldiacylglycerol and DGDG galactolipids also serve additional functions in
SAR. For instance, DGDG is required for SA and NO biosynthesis (Gao et al., 2014) and for
AzA responsiveness. Interestingly, in spite of their impaired SA and NO synthesis, petiole
exudates from pathogen-infected dgd1 plants were able to confer SAR in wild-type plants.
This suggests that dgd1 plants can make signals that are capable of inducing SA- and NO-
synthesis in plants with normal DGDG levels. These results show that SAR involves
DGDG-dependent retrograde signaling between the chloroplast and nucleus and
emphasizes the fact that the two branches of SAR are intricately linked (Gao et al., 2014).

In fact it is well known that there is cross talk between SA- and NO-mediated signaling.
For example, NO mediated S-nitrosylation of NPR1 can result in the oligomerization and
nuclear localization of NPR1 (Tada et al., 2008; Lindermayr et al., 2010). Moreover, SA
has been suggested to regulate chloroplast structure since exogenous SA can cause
swelling of grana thylakoids, coagulation of the stroma and increased chloroplast volume
(Uzunova and Popova, 2000; Rivas-San Vicente and Plasencia, 2011). Regulation of SA
and AzA levels by EDS1 is another case in point (Wittek et al., 2014). Together, these
results suggest that the parallel operation of the interlinked SA- and NO-pathways might
allow multiple points of regulation in fine tuning the optimal onset of SAR. This may be
particularly relevant for signals like NO and ROS, which are functional within specific
concentration ranges (Wang et al., 2014a).

Conclusion and Perspectives

Recent work on SAR has identified a number of chemical and protein signals and placed
them in a common pathway that comprises at least two parallel branches (Figure 2).
However, these studies also indicate the involvement of additional unknown signal(s) that
function upstream of the branchpoint separating SA-NPR1- and NO-ROS-AzA-G3P-
derived pathways. In addition, several chemical signals, including G3P and AzA, undergo
derivatization into unknown compounds and at least one of the G3P-derivative is SAR
bioactive (unpublished data). Identification of these signals should provide useful insights
into signaling events leading to the induction and establishment of SAR. Another area of
SAR research that has not received much attention is the transport and perception of
signals in the distal tissues. Although cuticle was implicated in the perception of SAR
signals (Xia et al., 2009), later studies on cuticle mutants have suggested that perception
might relate to the severity of cuticular damage or perhaps other unknown factors (Xia et
al., 2012). These aspects of SAR should provide exciting avenues for studying how SAR
overlaps with basic physiological processes and the distinct events that decide the onset of
SAR versus normal growth and development.
https://www.frontiersin.org/articles/10.3389/fpls.2015.00228/full

C12. When a plant resists a pathogen, what stops the pathogen growing?

How plants recognize pathogens and defend themselves.

 https://www.ncbi.nlm.nih.gov/pubmed/17876517
Abstract
Plants have an innate immunity system to defend themselves against pathogens. With the primary immune system,
plants recognize microbe-associated molecular patterns (MAMPs) of potential pathogens through pattern
recognition receptors (PRRs) that mediate a basal defense response. Plant pathogens suppress this basal defense
response by means of effectors that enable them to cause disease. With the secondary immune system, plants
have gained the ability to recognize effector-induced perturbations of host targets through resistance proteins (RPs)
that mediate a strong local defense response that stops pathogen growth. Both primary and secondary immune
responses in plants depend on germ line-encoded PRRs and RPs. During induction of local immune responses,
systemic immune responses also become activated, which predispose plants to become more resistant to
subsequent pathogen attacks. This review gives an update on recent findings that have enhanced our
understanding of plant innate immunity and the arms race between plants and their pathogens.

Plant Defense Mechanisms

Plant Defenses Against Herbivores

Plants defend against herbivores with mechanical wounding, barriers, secondary metabolites, and
attraction of parasitoids.

LEARNING OBJECTIVES

Identify plant defense responses to herbivores

KEY TAKEAWAYS

Key Points

 Many plants have impenetrable barriers, such as bark and waxy cuticles, or adaptations, such
as thorns and spines, to protect them from herbivores.
 If herbivores breach a plant’s barriers, the plant can respond with secondary metabolites, which
are often toxic compounds, such as glycol cyanide, that may harm the herbivore.
 When attacked by a predator, damaged plant tissue releases jasmonate hormones that promote
the release of volatile compounds, attracting parasitoids, which use, and eventually kill, the
predators as host insects.

Defense Responses Against Herbivores

Herbivores, both large and small, use plants as food and actively chew them. Plants have developed
a variety of strategies to discourage or kill attackers.

Mechanical Defenses

The first line of defense in plants is an intact and impenetrable barrier composed of bark and a waxy
cuticle. Both protect plants against herbivores. Other adaptations against herbivores include hard
shells, thorns (modified branches), and spines (modified leaves). They discourage animals by
causing physical damage or by inducing rashes and allergic reactions. Some Acacia tree species
have developed mutualistic relationships with ant colonies: they offer the ants shelter in their hollow
thorns in exchange for the ants’ defense of the tree’s leaves.
Acacia collinsii: The large thorn-like stipules of Acacia collinsii are hollow and offer shelter for ants, which in return protect the
plant against herbivores.
Modified leaves on a cactus: The spines on cactus plants are modified leaves that act as a mechanical defense against
predators.

Chemical Defenses

A plant’s exterior protection can be compromised by mechanical damage, which may provide an entry
point for pathogens. If the first line of defense is breached, the plant must resort to a different set of
defense mechanisms, such as toxins and enzymes. Secondary metabolites are compounds that are
not directly derived from photosynthesis and are not necessary for respiration or plant growth and
development.

Many metabolites are toxic and can even be lethal to animals that ingest them. Some metabolites are
alkaloids, which discourage predators with noxious odors (such as the volatile oils of mint and sage)
or repellent tastes (like the bitterness of quinine). Other alkaloids affect herbivores by causing either
excessive stimulation (caffeine is one example) or the lethargy associated with opioids. Some
compounds become toxic after ingestion; for instance, glycol cyanide in the cassava root releases
cyanide only upon ingestion by the herbivore. Foxgloves produce several deadly chemicals, namely
cardiac and steroidal glycosides. Ingestion can cause nausea, vomiting, hallucinations, convulsions,
or death.

Foxgloves: Foxgloves produce several deadly chemicals, namely cardiac and steroidal glycosides. Ingestion can cause nausea,
vomiting, hallucinations, convulsions, or death.

Timing

Mechanical wounding and predator attacks activate defense and protective mechanisms in the
damaged tissue and elicit long-distancing signaling or activation of defense and protective
mechanisms at sites farther from the injury location. Some defense reactions occur within minutes,
while others may take several hours. In addition, long-distance signaling elicits a systemic response
aimed at deterring predators. As tissue is damaged, jasmonates may promote the synthesis of
compounds that are toxic to predators. Jasmonates also elicit the synthesis of volatile compounds
that attract parasitoids: insects that spend their developing stages in or on another insect, eventually
killing their host. The plant may activate abscission of injured tissue if it is damaged beyond repair.

Plant Defenses Against Pathogens

Plants defend against pathogens with barriers, secondary metabolites, and antimicrobial compounds.

LEARNING OBJECTIVES

Identify plant defense responses to pathogens

KEY TAKEAWAYS

Key Points
  Many plants have impenetrable barriers, such as bark and waxy cuticles, or adaptations, such
as thorns and spines, to protect them from pathogens.
 If pathogens breach a plant’s barriers, the plant can respond with secondary metabolites, which
are often toxic compounds, such as glycol cyanide, that may harm the pathogen.
 Plants produce antimicrobial chemicals, antimicrobial proteins, and antimicrobial enzymes that
are able to fight the pathogens.

Defense Responses Against Pathogens

Pathogens are agents of disease. These infectious microorganisms, such as fungi, bacteria, and
nematodes, live off of the plant and damage its tissues. Plants have developed a variety of strategies
to discourage or kill attackers.

The first line of defense in plants is an intact and impenetrable barrier composed of bark and a waxy
cuticle. Both protect plants against pathogens.

A plant’s exterior protection can be compromised by mechanical damage, which may provide an entry
point for pathogens. If the first line of defense is breached, the plant must resort to a different set of
defense mechanisms, such as toxins and enzymes. Secondary metabolites are compounds that are
not directly derived from photosynthesis and are not necessary for respiration or plant growth and
development. Many metabolites are toxic and can even be lethal to animals that ingest them.

Additionally, plants have a variety of inducible defenses in the presence of pathogens. In addition to
secondary metabolites, plants produce antimicrobial chemicals, antimicrobial proteins, and
antimicrobial enzymes that are able to fight the pathogens. Plants can close stomata to prevent the
pathogen from entering the plant. A hypersensitive response, in which the plant experiences rapid cell
death to fight off the infection, can be initiated by the plant; or it may use endophyte assistance: the
roots release chemicals that attract other beneficial bacteria to fight the infection.

Mechanical wounding and predator attacks activate defense and protective mechanisms in the
damaged tissue and elicit long-distancing signaling or activation of defense and protective
mechanisms at sites farther from the injury location. Some defense reactions occur within minutes,
while others may take several hours.

https://courses.lumenlearning.com/boundless-biology/chapter/plant-defense-mechanisms/

 C13.

How do pathogens overcome plant disease resistance, and is it inevitable?

Plant disease resistance

From Wikipedia, the free encyclopedia
Jump to navigationJump to search
 Cankers caused by Chestnut blight, a disease that affects the chestnut tree

This diagram shows the process from fungi or bacterial attachment to the plant cell all the way to the specific type of response.
PTI stands for Pattern-Triggered Immunity and ETI stands for Effector-triggered immunity.
Plant disease resistance protects plants from pathogens in two ways: by pre-formed structures and chemicals, and
by infection-induced responses of the immune system. Relative to a susceptible plant, disease resistance is the
reduction of pathogen growth on or in the plant (and hence a reduction of disease), while the term disease
tolerance describes plants that exhibit little disease damage despite substantial pathogen levels. Disease outcome
is determined by the three-way interaction of the pathogen, the plant and the environmental conditions (an
interaction known as the disease triangle).
Defense-activating compounds can move cell-to-cell and systematically through the plant's vascular system.
However, plants do not have circulating immune cells, so most cell types exhibit a broad suite
of antimicrobial defenses. Although obvious qualitative differences in disease resistance can be observed when
multiple specimens are compared (allowing classification as “resistant” or “susceptible” after infection by the same
pathogen strain at similar inoculum levels in similar environments), a gradation of quantitative differences in disease
resistance is more typically observed between plant strains or genotypes. Plants consistently resist certain
pathogens but succumb to others; resistance is usually specific to certain pathogen species or pathogen strains.

Contents

 1Background
 2Common disease resistance mechanisms
o 2.1Pre-formed structures and compounds
o 2.2Inducible post-infection plant defenses
 3Immune system
o 3.1Pattern-triggered immunity
o 3.2Effector triggered immunity
o 3.3R genes and R proteins
o 3.4Effector biology
o 3.5Small RNAs and RNA interference
o 3.6Species-level resistance
 4Signaling mechanisms
o 4.1Perception of pathogen presence
o 4.2Transcription factors and the hormone response
o 4.3Regulation by degradation
 5Plant breeding for disease resistance
o 5.1GM or transgenic engineered disease resistance
 6Host range
 7Epidemics and population biology
 8See also
 9References
o 9.1Notes
o 9.2Further reading
 10External links
Background[edit]
Plant disease resistance is crucial to the reliable production of food, and it provides significant reductions in
agricultural use of land, water, fuel and other inputs. Plants in both natural and cultivated populations carry inherent
disease resistance, but this has not always protected them.
The late blight Irish potato famine of the 1840s was caused by the oomycete Phytophthora infestans. The world’s
first mass-cultivated banana cultivar Gros Michel was lost in the 1920s to Panama disease caused by the
fungus Fusarium oxysporum. The current wheat stem rust, leaf rust and yellow stripe rust epidemics spreading from
East Africa into the Indian subcontinent are caused by rust fungi Puccinia graminis and P. striiformis. Other
epidemics include Chestnut blight, as well as recurrent severe plant diseases such as Rice blast, Soybean cyst
nematode, Citrus canker.[1][2]
Plant pathogens can spread rapidly over great distances, vectored by water, wind, insects, and humans. Across
large regions and many crop species, it is estimated that diseases typically reduce plant yields by 10% every year in
more developed nations or agricultural systems, but yield loss to diseases often exceeds 20% in less developed
settings.[1]
However, disease control is reasonably successful for most crops. Disease control is achieved by use of plants that
have been bred for good resistance to many diseases, and by plant cultivation approaches such as crop rotation,
pathogen-free seed, appropriate planting date and plant density, control of field moisture, and pesticide use.

Common disease resistance mechanisms[edit]

Pre-formed structures and compounds[edit]

secondary plant wall

 Plant cuticle/surface
 Plant cell walls
 Antimicrobial chemicals (for example: glucosides, saponins)
 Antimicrobial proteins
 Enzyme inhibitors
 Detoxifying enzymes that break down pathogen-derived toxins
 Receptors that perceive pathogen presence and activate inducible plant defences[3]
Inducible post-infection plant defenses[edit]

 Cell wall reinforcement (cellulose, lignin, suberin, cell wall proteins)[4]

 Antimicrobial chemicals, including reactive oxygen species such as hydrogen peroxide or peroxynitrite, or more
complex phytoalexins such as genistein or camalexin
 Antimicrobial proteins such as defensins, thionins, or PR-1
 Antimicrobial enzymes such as chitinases, beta-glucanases, or peroxidases[4]
 Hypersensitive response - a rapid host cell death response associated with defence mediated by "Resistance
genes."(Bryant, Tracy 2008).

Immune system[edit]
The plant immune system carries two interconnected tiers of receptors, one most frequently sensing molecules
outside the cell and the other most frequently sensing molecules inside the cell. Both systems sense the intruder
and respond by activating antimicrobial defenses in the infected cell and neighboring cells. In some cases, defense-
activating signals spread to the rest of the plant or even to neighboring plants. The two systems detect different
types of pathogen molecules and classes of plant receptor proteins.[5][6]
The first tier is primarily governed by pattern recognition receptors that are activated by recognition of evolutionarily
conserved pathogen or microbial–associated molecular patterns (PAMPs or MAMPs). Activation of PRRs leads to
intracellular signaling, transcriptional reprogramming, and biosynthesis of a complex output response that limits
colonization. The system is known as PAMP-Triggered Immunity or as Pattern-Triggered Immunity (PTI).[6][7]
The second tier, primarily governed by R gene products, is often termed effector-triggered immunity (ETI). ETI is
typically activated by the presence of specific pathogen "effectors" and then triggers strong antimicrobial responses
(see R gene section below).
In addition to PTI and ETI, plant defenses can be activated by the sensing of damage-associated compounds
(DAMP), such as portions of the plant cell wall released during pathogenic infection.
Responses activated by PTI and ETI receptors include ion channel gating, oxidative burst, cellular redox changes,
or protein kinase cascades that directly activate cellular changes (such as cell wall reinforcement or antimicrobial
production), or activate changes in gene expression that then elevate other defensive responses
Plant immune systems show some mechanistic similarities with the immune systems of insects and mammals, but
also exhibit many plant-specific characteristics.[8] The two above-described tiers are central to plant immunity but do
not fully describe plant immune systems. In addition, many specific examples of apparent PTI or ETI violate
common PTI/ETI definitions, suggesting a need for broadened definitions and/or paradigms.[9]
Pattern-triggered immunity[edit]
PAMPs, conserved molecules that inhabit multiple pathogen genera, are referred to as MAMPs by many
researchers. The defenses induced by MAMP perception are sufficient to repel most pathogens. However, pathogen
effector proteins (see below) are adapted to suppress basal defenses such as PTI. Many receptors for MAMPs (and
DAMPs) have been discovered. MAMPs and DAMPs are often detected by transmembrane receptor-kinases that
carry LRR or LysM extracellular domains.[5]
Effector triggered immunity[edit]
Effector Triggered Immunity (ETI) is activated by the presence of pathogen effectors. The ETI response is reliant
on R genes, and is activated by specific pathogen strains. Plant ETI often causes an apoptotic hypersensitive
response.
R genes and R proteins[edit]
Plants have evolved R genes (resistance genes) whose products mediate resistance to specific virus, bacteria,
oomycete, fungus, nematode or insect strains. R gene products are proteins that allow recognition of specific
pathogen effectors, either through direct binding or by recognition of the effector's alteration of a host protein.[6] Many
R genes encode NB-LRR proteins (proteins with nucleotide-binding and leucine-rich repeat domains, also known as
NLR proteins or STAND proteins, among other names). Most plant immune systems carry a repertoire of 100-600
different R gene homologs. Individual R genes have been demonstrated to mediate resistance to specific virus,
bacteria, oomycete, fungus, nematode or insect strains. R gene products control a broad set of disease resistance
responses whose induction is often sufficient to stop further pathogen growth/spread.
Studied R genes usually confer specificity for particular strains of a pathogen species (those that express the
recognized effector). As first noted by Harold Flor in his mid-20th century formulation of the gene-for-gene
relationship, a plant R gene has specificity for a pathogen avirulence gene (Avr gene). Avirulence genes are now
known to encode effectors. The pathogen Avr gene must have matched specificity with the R gene for that R gene
to confer resistance, suggesting a receptor/ligand interaction for Avr and R genes.[8] Alternatively, an effector can
modify its host cellular target (or a molecular decoy of that target), and the R gene product (NLR protein) activates
defenses when it detects the modified form of the host target or decoy.[6]
Effector biology[edit]
Effectors are central to the pathogenic or symbiotic potential of microbes and microscopic plant-colonizing animals
such as nematodes.[10][11][12] Effectors typically are proteins that are delivered outside the microbe and into the host
cell. These colonist-derived effectors manipulate the host's cell physiology and development. As such, effectors
offer examples of co-evolution (example: a fungal protein that functions outside of the fungus but inside of plant cells
has evolved to take on plant-specific functions). Pathogen host range is determined, among other things, by the
presence of appropriate effectors that allow colonization of a particular host.[5] Pathogen-derived effectors are a
powerful tool to identify plant functions that play key roles in disease and in disease resistance. Apparently most
effectors function to manipulate host physiology to allow disease to occur. Well-studied bacterial plant pathogens
typically express a few dozen effectors, often delivered into the host by a Type III secretion apparatus.[10] Fungal,
oomycete and nematode plant pathogens apparently express a few hundred effectors.[11][12]
So-called "core" effectors are defined operationally by their wide distribution across the population of a particular
pathogen and their substantial contribution to pathogen virulence. Genomics can be used to identify core effectors,
which can then be used to discover new R gene alleles, which can be used in plant breeding for disease resistance.
Small RNAs and RNA interference[edit]
Plant sRNA pathways are understood to be important components of pathogen-associated molecular pattern
(PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI).[13][14] Bacteria‐ induced miRNAs in
Arabidopsis have been shown to influence hormonal signalling including auxin, abscisic acid (ABA), jasmonic acid
(JA) and salicylic acid (SA).[15][16] Advances in genome‐ wide studies revealed a massive adaptation of host miRNA
expression patterns after infection by fungal pathogens Fusarium virguliforme, [17] Erysiphe graminis,[18] Verticillium
dahliae,[19] and Cronartium quercuum,[20] and the oomycete Phytophthora sojae.[21] Changes to sRNA expression in
response to fungal pathogens indicate that gene silencing may be involved in this defense pathway. However, there
is also evidence that the antifungal defense response to Colletotrichum spp. infection in maize is not entirely
regulated by specific miRNA induction, but may instead act to fine-tune the balance between genetic and metabolic
components upon infection.[22]
Transport of sRNAs during infection is likely facilitated by extracellular vesicles (EVs) and multivesicular bodies
(MVBs).[23] The composition of RNA in plant EVs has not been fully evaluated, but it is likely that they are, in part,
responsible for trafficking RNA. Plants can transport viral RNAs, mRNAs, microRNAs (miRNAs) and small
interfering RNAs (siRNAs) systemically through the phloem.[24] This process is thought to occur through the
plasmodesmata and involves RNA-binding proteins that assist RNA localization in mesophyll cells. Although they
have been identified in the phloem with mRNA, there is no determinate evidence that they mediate long-distant
transport of RNAs.[25] EVs may therefore contribute to an alternate pathway of RNA loading into the phloem, or could
possibly transport RNA through the apoplast.[26] There is also evidence that plant EVs can allow for interspecies
transfer of sRNAs by RNA interference such as Host-Induced Gene Silencing (HIGS).[27][28] The transport of RNA
between plants and fungi seems to be bidirectional as sRNAs from the fungal pathogen Botrytis cinerea have been
shown to target host defense genes in Arabidopsis and tomato.[29]
Species-level resistance[edit]
In a small number of cases, plant genes are effective against an entire pathogen species, even though that species
that is pathogenic on other genotypes of that host species. Examples include barley MLO against powdery
mildew, wheat Lr34 against leaf rust and wheat Yr36 against wheat stripe rust. An array of mechanisms for this type
of resistance may exist depending on the particular gene and plant-pathogen combination. Other reasons for
effective plant immunity can include a lack of coadaptation (the pathogen and/or plant lack multiple mechanisms
needed for colonization and growth within that host species), or a particularly effective suite of pre-formed
defenses.[citation needed]

Signaling mechanisms[edit]
Perception of pathogen presence[edit]
Plant defense signaling is activated by the pathogen-detecting receptors that are described in an above
section.[5] The activated receptors frequently elicit reactive oxygen and nitric
oxide production, calcium, potassium and proton ion fluxes, altered levels of salicylic acid and other hormones and
activation of MAP kinases and other specific protein kinases.[8] These events in turn typically lead to the modification
of proteins that control gene transcription, and the activation of defense-associated gene expression.[7]
Transcription factors and the hormone response[edit]
Numerous genes and/or proteins as well as other molecules have been identified that mediate plant defense signal
transduction.[30][31] Cytoskeleton and vesicle trafficking dynamics help to orient plant defense responses toward the
point of pathogen attack.
Mechanisms of transcription factors and hormones[edit]
Plant immune system activity is regulated in part by signaling hormones such as:[32][33]

 Salicylic acid
 Jasmonic acid
 Ethylene
There can be substantial cross-talk among these pathways.[32]
Regulation by degradation[edit]
As with many signal transduction pathways, plant gene expression during immune responses can be regulated by
degradation. This often occurs when hormone binding to hormone receptors stimulates ubiquitin-associated
degradation of repressor proteins that block expression of certain genes. The net result is hormone-activated gene
expression. Examples:[34]

 Auxin: binds to receptors that then recruit and degrade repressors of transcriptional activators that stimulate
auxin-specific gene expression.
 Jasmonic acid: similar to auxin, except with jasmonate receptors impacting jasmonate-response signaling
mediators such as JAZ proteins.
 Gibberellic acid: Gibberellin causes receptor conformational changes and binding and degradation of Della
proteins.
 Ethylene: Inhibitory phosphorylation of the EIN2 ethylene response activator is blocked by ethylene binding.
When this phosphorylation is reduced, EIN2 protein is cleaved and a portion of the protein moves to the nucleus
to activate ethylene-response gene expression.
Ubiquitin and E3 signaling[edit]
Ubiquitination plays a central role in cell signaling that regulates processes including protein degradation and
immunological response.[35] Although one of the main functions of ubiquitin is to target proteins for destruction, it is
also useful in signaling pathways, hormone release, apoptosis and translocation of materials throughout the cell.
Ubiquitination is a component of several immune responses. Without ubiquitin's proper functioning, the invasion of
pathogens and other harmful molecules would increase dramatically due to weakened immune defenses.[35]
E3 signaling[edit]
The E3 Ubiquitin ligase enzyme is a main component that provides specificity in protein degradation pathways,
including immune signaling pathways.[34] The E3 enzyme components can be grouped by which domains they
contain and include several types.[36] These include the Ring and U-box single subunit, HECT, and CRLs.[37][38] Plant
signaling pathways including immune responses are controlled by several feedback pathways, which often include
negative feedback; and they can be regulated by De-ubiquitination enzymes, degradation of transcription factors
and the degradation of negative regulators of transcription.[34][39]

This image depicts the pathways taken during responses in plant immunity. It highlights the role and effect ubiquitin has in
regulating the pathway.

Plant breeding for disease resistance[edit]

Plant breeders emphasize selection and development of disease-resistant plant lines. Plant diseases can also be
partially controlled by use of pesticides and by cultivation practices such as crop rotation, tillage, planting density,
disease-free seeds and cleaning of equipment, but plant varieties with inherent (genetically determined) disease
resistance are generally preferred.[2] Breeding for disease resistance began when plants were first domesticated.
Breeding efforts continue because pathogen populations are under selection pressure for increased virulence, new
pathogens appear, evolving cultivation practices and changing climate can reduce resistance and/or strengthen
pathogens, and plant breeding for other traits can disrupt prior resistance.[40] A plant line with acceptable resistance
against one pathogen may lack resistance against others.
Breeding for resistance typically includes:

 Identification of plants that may be less desirable in other ways, but which carry a useful disease resistance trait,
including wild plant lines that often express enhanced resistance.
 Crossing of a desirable but disease-susceptible variety to a plant that is a source of resistance.
 Growth of breeding candidates in a disease-conducive setting, possibly including pathogen inoculation.
Attention must be paid to the specific pathogen isolates, to address variability within a single pathogen species.
 Selection of disease-resistant individuals that retain other desirable traits such as yield, quality and including
other disease resistance traits.[40]
Resistance is termed durable if it continues to be effective over multiple years of widespread use as pathogen
populations evolve. "Vertical resistance" is specific to certain races or strains of a pathogen species, is often
controlled by single R genes and can be less durable. Horizontal or broad-spectrum resistance against an entire
pathogen species is often only incompletely effective, but more durable, and is often controlled by many genes that
segregate in breeding populations.[2]
Crops such as potato, apple, banana and sugarcane are often propagated by vegetative reproduction to preserve
highly desirable plant varieties, because for these species, outcrossing seriously disrupts the preferred traits. See
also asexual propagation. Vegetatively propagated crops may be among the best targets for resistance
improvement by the biotechnology method of plant transformation to manage genes that affect disease resistance.[1]
Scientific breeding for disease resistance originated with Sir Rowland Biffen, who identified a single recessive gene
for resistance to wheat yellow rust. Nearly every crop was then bred to include disease resistance (R) genes, many
by introgression from compatible wild relatives.[1]
GM or transgenic engineered disease resistance[edit]
The term GM ("genetically modified") is often used as a synonym of transgenic to refer to plants modified using
recombinant DNA technologies. Plants with transgenic/GM disease resistance against insect pests have been
extremely successful as commercial products, especially in maize and cotton, and are planted annually on over 20
million hectares in over 20 countries worldwide[41] (see also genetically modified crops). Transgenic plant disease
resistance against microbial pathogens was first demonstrated in 1986. Expression of viral coat protein gene
sequences conferred virus resistance via small RNAs. This proved to be a widely applicable mechanism for
inhibiting viral replication.[42] Combining coat protein genes from three different viruses, scientists
developed squash hybrids with field-validated, multiviral resistance. Similar levels of resistance to this variety of
viruses had not been achieved by conventional breeding.
A similar strategy was deployed to combat papaya ringspot virus, which by 1994 threatened to
destroy Hawaii’s papaya industry. Field trials demonstrated excellent efficacy and high fruit quality. By 1998 the first
transgenic virus-resistant papaya was approved for sale. Disease resistance has been durable for over 15 years.
Transgenic papaya accounts for ~85% of Hawaiian production. The fruit is approved for sale in the U.S., Canada
and Japan.
Potato lines expressing viral replicase sequences that confer resistance to potato leafroll virus were sold under the
trade names NewLeaf Y and NewLeaf Plus, and were widely accepted in commercial production in 1999-2001, until
McDonald's Corp. decided not to purchase GM potatoes and Monsanto decided to close their NatureMark potato
business.[43] NewLeaf Y and NewLeaf Plus potatoes carried two GM traits, as they also expressed Bt-mediated
resistance to Colorado potato beetle.
No other crop with engineered disease resistance against microbial pathogens had reached the market by 2013,
although more than a dozen were in some state of development and testing.

Examples of transgenic disease resistance projects[1]

Publication
Crop Disease resistance Mechanism Development status
year

2012 Tomato Bacterial spot R gene from pepper 8 years of field trials

Bacterial blight and

2012 Rice Engineered E gene Laboratory
bacterial streak

2 years of field trials at time of

2012 Wheat Powdery mildew Overexpressed R gene from wheat
publication
 4 years of field trials at time of
2011 Apple Apple scab fungus Thionin gene from barley
publication

1 year of field trial at time of

2011 Potato Potato virus Y Pathogen-derived resistance
publication

12 years of field trials at time of

2010 Apple Fire blight Antibacterial protein from moth
publication

Multibacterial
2010 Tomato PRR from Arabidopsis Laboratory scale
resistance

2010 Banana Xanthomonas wilt Novel gene from pepper Now in field trial

2009 Potato Late blight R genes from wild relatives 3 years of field trials

2 years of field trials at time of

2009 Potato Late blight R gene from wild relative
publication

2 years of field trials at time of

2008 Potato Late blight R gene from wild relative
publication

Regulatory approvals, no
2008 Plum Plum pox virus Pathogen-derived resistance
commercial sales

2005 Rice Bacterial streak R gene from maize Laboratory

Resting lymphocyte kinase (RLK)

2002 Barley Stem rust Laboratory
gene from resistant barley cultivar

Approved and commercially sold

1997 Papaya Ring spot virus Pathogen-derived resistance since 1998, sold into Japan since
2012

Approved and commercially sold

1995 Squash Three mosaic viruses Pathogen-derived resistance
since 1994

Mammalian interferon-induced 3 years of field trials at time of

1993 Potato Potato virus X
enzyme publication

PRR transfer[edit]
Research aimed at engineered resistance follows multiple strategies. One is to transfer useful PRRs into species
that lack them. Identification of functional PRRs and their transfer to a recipient species that lacks an orthologous
receptor could provide a general pathway to additional broadened PRR repertoires. For example,
the Arabidopsis PRR EF-Tu receptor (EFR) recognizes the bacterial translation elongation factor EF-Tu. Research
performed at Sainsbury Laboratory demonstrated that deployment of EFR into either Nicotiana
benthamianaor Solanum lycopersicum (tomato), which cannot recognize EF-Tu, conferred resistance to a wide
range of bacterial pathogens. EFR expression in tomato was especially effective against the widespread and
devastating soil bacterium Ralstonia solanacearum.[44] Conversely, the tomato PRR Verticillium 1 (Ve1) gene can be
transferred from tomato to Arabidopsis, where it confers resistance to race 1 Verticillium isolates.[1]
Stacking[edit]
The second strategy attempts to deploy multiple NLR genes simultaneously, a breeding strategy known as stacking.
Cultivars generated by either DNA-assisted molecular breeding or gene transfer will likely display more durable
resistance, because pathogens would have to mutate multiple effector genes. DNA sequencing allows researchers
to functionally “mine” NLR genes from multiple species/strains.[1]
The avrBs2 effector gene from Xanthomona perforans is the causal agent of bacterial spot disease of pepper and
tomato. The first “effector-rationalized” search for a potentially durable R gene followed the finding that avrBs2 is
found in most disease-causing Xanthomonas species and is required for pathogen fitness. The Bs2 NLR gene from
the wild pepper, Capsicum chacoense, was moved into tomato, where it inhibited pathogen growth. Field trials
demonstrated robust resistance without bactericidal chemicals. However, rare strains
of Xanthomonas overcame Bs2-mediated resistance in pepper by acquisition of avrBs2 mutations that avoid
recognition but retain virulence. Stacking R genes that each recognize a different core effector could delay or
prevent adaptation.[1]
More than 50 loci in wheat strains confer disease resistance against wheat stem, leaf and yellow stripe rust
pathogens. The Stem rust 35 (Sr35) NLR gene, cloned from a diploid relative of cultivated wheat, Triticum
monococcum, provides resistance to wheat rust isolate Ug99. Similarly, Sr33, from the wheat relative Aegilops
tauschii, encodes a wheat ortholog to barley Mla powdery mildew–resistance genes. Both genes are unusual in
wheat and its relatives. Combined with the Sr2 gene that acts additively with at least Sr33, they could provide
durable disease resistance to Ug99 and its derivatives.[1]
Executor genes[edit]
Another class of plant disease resistance genes opens a “trap door” that quickly kills invaded cells, stopping
pathogen proliferation. Xanthomonas and Ralstonia transcription activator–like (TAL) effectors are DNA-binding
proteins that activate host gene expression to enhance pathogen virulence. Both the rice and pepper lineages
independently evolved TAL-effector binding sites that instead act as an executioner that induces hypersensitive host
cell death when up-regulated. Xa27 from rice and Bs3 and Bs4c from pepper, are such “executor” (or "executioner")
genes that encode non-homologous plant proteins of unknown function. Executor genes are expressed only in the
presence of a specific TAL effector.[1]
Engineered executor genes were demonstrated by successfully redesigning the pepper Bs3 promoter to contain two
additional binding sites for TAL effectors from disparate pathogen strains. Subsequently, an engineered executor
gene was deployed in rice by adding five TAL effector binding sites to the Xa27 promoter. The
synthetic Xa27 construct conferred resistance against Xanthomonas bacterial blight and bacterial leaf streak
species.[1]
Host susceptibility alleles[edit]
Most plant pathogens reprogram host gene expression patterns to directly benefit the pathogen. Reprogrammed
genes required for pathogen survival and proliferation can be thought of as “disease-susceptibility genes.”
Recessive resistance genes are disease-susceptibility candidates. For example, a mutation disabled
an Arabidopsis gene encoding pectate lyase (involved in cell wall degradation), conferring resistance to the powdery
mildew pathogen Golovinomyces cichoracearum. Similarly, the Barley MLO gene and spontaneously mutated pea
and tomato MLO orthologs also confer powdery mildew resistance.[1]
Lr34 is a gene that provides partial resistance to leaf and yellow rusts and powdery mildew in wheat. Lr34 encodes
an adenosine triphosphate (ATP)–binding cassette (ABC) transporter. The dominant allele that provides disease
resistance was recently found in cultivated wheat (not in wild strains) and, like MLO provides broad-spectrum
resistance in barley.[1]
Natural alleles of host translation elongation initiation factors eif4e and eif4g are also recessive viral-resistance
genes. Some have been deployed to control potyviruses in barley, rice, tomato, pepper, pea, lettuce and melon. The
discovery prompted a successful mutant screen for chemically induced eif4e alleles in tomato.[1]
Natural promoter variation can lead to the evolution of recessive disease-resistance alleles. For example, the
recessive resistance gene xa13 in rice is an allele of Os-8N3. Os-8N3 is transcriptionally activated byXanthomonas
oryzae pv. oryzae strains that express the TAL effector PthXo1. The xa13 gene has a mutated effector-binding
element in its promoter that eliminates PthXo1 binding and renders these lines resistant to strains that rely
on PthXo1. This finding also demonstrated that Os-8N3 is required for susceptibility.[1]
Xa13/Os-8N3 is required for pollen development, showing that such mutant alleles can be problematic should the
disease-susceptibility phenotype alter function in other processes. However, mutations in
the Os11N3 (OsSWEET14) TAL effector–binding element were made by fusing TAL effectors to nucleases
(TALENs). Genome-edited rice plants with altered Os11N3 binding sites remained resistant to Xanthomonas oryzae
pv. oryzae, but still provided normal development function.[1]
Gene silencing[edit]
RNA silencing-based resistance is a powerful tool for engineering resistant crops. The advantage of RNAi as a
novel gene therapy against fungal, viral and bacterial infection in plants lies in the fact that it regulates gene
expression via messenger RNA degradation, translation repression and chromatin remodelling through small non-
coding RNAs. Mechanistically, the silencing processes are guided by processing products of the double-stranded
RNA (dsRNA) trigger, which are known as small interfering RNAs and microRNAs.[45]

Host range[edit]
See also: Plant pathology
Among the thousands of species of plant pathogenic microorganisms, only a small minority have the capacity to
infect a broad range of plant species. Most pathogens instead exhibit a high degree of host-specificity. Non-host
plant species are often said to express non-host resistance. The term host resistance is used when a pathogen
species can be pathogenic on the host species but certain strains of that plant species resist certain strains of the
pathogen species. The causes of host resistance and non-host resistance can overlap. Pathogen host range is
determined, among other things, by the presence of appropriate effectors that allow colonization of a particular
host.[5] Pathogen host range can change quite suddenly if, for example, the pathogen's capacity to synthesize a
host-specific toxin or effector is gained by gene shuffling/mutation, or by horizontal gene transfer.[46] [47]
 Epidemics and population biology[edit]
Native populations are often characterized by substantial genotype diversity and dispersed populations (growth in a
mixture with many other plant species). They also have undergone of plant-pathogen coevolution. Hence as long as
novel pathogens are not introduced/do not evolve, such populations generally exhibit only a low incidence of severe
disease epidemics.[48]
Monocrop agricultural systems provide an ideal environment for pathogen evolution, because they offer a high
density of target specimens with similar/identical genotypes.[48] The rise in mobility stemming from modern
transportation systems provides pathogens with access to more potential targets.[48] Climate change can alter the
viable geographic range of pathogen species and cause some diseases to become a problem in areas where the
disease was previously less important.[48]
These factors make modern agriculture more prone to disease epidemics. Common solutions include constant
breeding for disease resistance, use of pesticides, use of border inspections and plant import restrictions,
maintenance of significant genetic diversity within the crop gene pool (see crop diversity), and constant surveillance
to accelerate initiation of appropriate responses. Some pathogen species have much greater capacity to overcome
plant disease resistance than others, often because of their ability to evolve rapidly and to disperse broadly.[48]
https://en.wikipedia.org/wiki/Plant_disease_resistance

 C14.

What are the molecular mechanisms for uptake and transport of nutrients?

PDF molecular uptake

 C15.

Can we use nonhost resistance to deliver more durable resistance in plants?

Current Understandings of Plant Nonhost

Resistance
Hyun-Ah Lee
,
Hye-Young Lee
,
Eunyoung Seo
,
Joohyun Lee
,
Saet-Byul Kim
,
Soohyun Oh
,
Eunbi Choi
,
Eunhye Choi
,
So Eui Lee
, and
Doil Choi
Affiliations
Published Online:12 Jan 2017https://doi.org/10.1094/MPMI-10-16-0213-CR
ABOUT

 SECTIONS




Abstract
Nonhost resistance, a resistance of plant species against all nonadapted pathogens, is considered the most
durable and efficient immune system of plants but yet remains elusive. The underlying mechanism of nonhost
resistance has been investigated at multiple levels of plant defense for several decades. In this review, we have
comprehensively surveyed the latest literature on nonhost resistance in terms of preinvasion, metabolic defense,
pattern-triggered immunity, effector-triggered immunity, defense signaling, and possible application in crop
protection. Overall, we summarize the current understanding of nonhost resistance mechanisms. Pre- and
postinvasion is not much deviated from the knowledge on host resistance, except for a few specific cases.
Further insights on the roles of the pattern recognition receptor gene family, multiple interactions between
effectors from nonadapted pathogen and plant factors, and plant secondary metabolites in host range
determination could expand our knowledge on nonhost resistance and provide efficient tools for future crop
protection using combinational biotechnology approaches.

Copyright © 2017 The Author(s). This is an open access article distributed under the CC BY-NC-
ND 4.0 International license.

Unlike mammals, plants are sessile and have no adaptive immune system. Although numerous pathogens attack
plants in nature, most plants are resistant to most pathogens. This phenomenon, termed nonhost resistance
(NHR), defines the resistance of an entire plant species against a specific parasite or pathogen (Heath 2000). In
other words, NHR is the plant resistance at species-specific level and the most durable resistance of plants. Due
to its durability, NHR has attracted increasing attention as a valuable strategy for improving crop resistance.
However, interspecific sterility has often hindered the elucidation of the genetic basis of NHR, slowing down its
deployment in the field. The mechanism of NHR is currently considered to rely on a complex combination of
constitutive and induced defense components (Fan and Doerner 2012; Lee et al. 2014; Niks and Marcel
2009).

Plants possess an elaborate suite of defense components to protect themselves against pathogen infection.
Encounters between plants and pathogens occur at the cuticle and cell-wall layer, which act as a physical barrier
(Tucker and Talbot 2001). Plants also establish a chemical barrier by accumulating a diverse array of
secondary metabolites constitutively or rapidly produced upon pathogen infection with toxic or inhibitory
effects (VanEtten et al. 1994). In addition, plants can recognize invading pathogens using membrane or
cytoplasmic receptors that induce two layers of defense response (Dodds and Rathjen 2010). First, surface-
localized receptors can perceive nonself or damaged-self signal, in the form of conserved pathogen structures
called pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs)
released during alteration of host cell integrity (Raaymakers and Van den Ackerveken 2016). These patterns
are recognized via pattern-recognition receptors (PRRs), mainly located at the plasma membrane, that induce a
first rise in plant defense level generally termed PAMP-triggered immunity (PTI) (Zipfel 2009). To suppress
PTI and dampen host defense, successful pathogens secrete a number of effector proteins into host cells (Dodds
and Rathjen 2010). In turn, plants induce a second rise in defense level, designated effector-triggered
immunity (ETI), via recognition of effectors by intracellular immune receptors called resistance (R) proteins,
which often belong to the nucleotide-binding domain and leucine-rich repeat-containing (NLR) family
(Maekawa et al. 2011). ETI is typically accompanied by a hypersensitive response (HR), a rapid programmed
cell death at the infection site to restrict the spread of pathogens (Jones and Dangl 2006).

From numerous studies using various approaches from cytology to omics techniques over the last decades, it
appears that the multiple defense components are involved in NHR in a complex manner. Most of the
cytological observations of pathogen penetration on nonhost plants were performed in the 1970s. These initial
observations show that often nonadapted pathogens stop growing during the very early stage of infection and
that several nonhost species exhibit HR cell death at the infection site (Crute and Johnson 1976; Heath
1974; Mendgen 1978). Later, it was suggested that NHR consists of complex defense factors including a
physical or chemical barrier and an active response (Heath 1981). Callose and phytoalexin accumulation
around the infection sites were observed and quantified in nonhost plants (Fink et al. 1990; Jahnen and
Hahlbrock 1988; Perumalla and Heath 1989). Transposon mutagenesis further allowed the identification of
pathogenicity factors that elicit HR on nonhost plants (Lindgren et al. 1986; Whalen et al. 1988).
Concomitantly, the durability of NHR has been recognized as an important strategy to improve crop resistance
(Niks 1987). Genes involved in NHR were identified, in the 1990s, from both plant (polygalacturonase) and
pathogen (INF1) (Allen 1991; Fillingham et al. 1992; Kamoun et al. 1998). Participation of both PTI and ETI
to NHR emerged from identification of Arabidopsis PRR EFR and of two NLR genes, including
maize Rxo1 and Arabidopsis WRR4 (Borhan et al. 2010; Lacombe et al. 2010; Zhao et al. 2004b). Later, with
respect to defense signaling, the concept that NHR shares signaling components with host resistance has been
supported by transcriptional and metabolite profiling approaches (Gill et al. 2015; Ishiga et al. 2015; Kanzaki
et al. 2003; Peart et al. 2002; Zellerhoff et al. 2010). Hence, there has been considerable progress toward
unraveling NHR. Here, we comprehensively review the multiple defense components involved in NHR and
summarize our current knowledge and prospects on this valuable form of plant resistance.

The role of preinvasion resistance.

The plant epidermis is generally protected by a waxy cuticle and the plant cell wall. Together, they form a
physical barrier that pathogens have to breach in order to infect plants. The preinvasive penetration barrier is the
first line of plant defense against pathogen attacks and is considered an important factor in NHR.

Nonadapted pathogens normally fail to penetrate nonhost plant cells when blocked by preformed physical
barriers present on the plant surface (Kamoun 2001; Pinosa et al. 2013). Components of leaf waxes can act as
an inductive cue for pathogens to develop infection structures and lack of appropriate signals in plants
contributes to NHR. Colletotrichum gloeosporioides that successfully induces appressorium on host avocado
cannot induce differentiation of appressorium on nonhost plants (Podila et al. 1993). This is the result of
difference in fatty alcohol composition of waxes, as nonhost plants contain a higher content of alcohols with
longer chains compared with host plants. Similarly, C26 aldehyde presence in wax of host barley cuticle plays an
important role in appressorium differentiation of Blumeria graminis f. sp. hordei (Tsuba et al. 2002). Nonhost
cabbage cuticle has higher C28 and C30 aldehyde contents, which are less efficient to trigger B.
graminis appressorium differentiation. Another component of preinvasive defense is the alfalfa IRG1 (Inhibitor
of rust germ tube differentiation 1) gene that encodes a C2H2 zinc finger transcription factor now called PALM1
(Palmate-like pentafoliata1) and regulates the expression of genes involved in wax biosynthesis. Reduction of
surface hydrophobicity due to altered proportion of primary alcohol in irg1 mutant cuticle inhibits the
differentiation of prepenetration structures of nonadapted rust pathogens (Uppalapati et al. 2012). Barley
epicuticular wax biosynthetic gene CYP96B22 also functions in penetration resistance of
nonadapted Magnaporthe oryzae (Delventhal et al. 2014). Of note, in addition to leaf waxes, membrane lipid
phosphatic acid is also associated with Arabidopsis NHR to pea fungal pathogen Erysiphe pisi (Pinosa et al.
2013).

Beside preformed structural defenses, induced preinvasive defense responses have an important role in NHR.
During the early phase of invasion, polarized rearrangement of microfilaments and microtubules is triggered by
attempted pathogen penetration and mediates defense-related responses, such as massive cytoplasmic
aggregation and cell-wall apposition at the infection site (Kobayashi et al. 1997a; Takemoto et al. 2003).
Evidence suggests that nonadapted pathogen invasion is hindered by these cytoskeleton and wall
rearrangements. For instance, treatment with actin polymerization inhibitor cytochalasin allows Erysiphe pisi to
penetrate epidermal cells of nonhost plants and to form haustoria (Kobayashi et al. 1997b). The level of actin
depolymerization is, indeed, correlated with the penetration efficiency of several nonadapted pathogens,
including Colletotrichum lagenarium and Alternaria alternata (Kobayashi et al. 1997a). In Arabidopsis,
nonadapted pathogens, including wheat powdery mildew B. graminis f. sp. tritici and Colletotrichum species,
also penetrate epidermal cells and develop haustoria under the cytochalasin treatment (Shimada et al. 2006).
Additionally, adhesion between plasma membrane and cell wall is important for penetration resistance.
Exogenous application of peptides carrying an Arg-Gly-Asp (RGD) motif of mammalian plasma membrane–
bound receptors causes a loss of connection between plasma membrane and cell wall in cowpea, which results
in downregulation of the cell wall–associated defense response and increased penetration of nonadapted fungal
pathogen (Mellersh and Heath 2001).

Taken together, cell wall–associated defense response plays an important role in NHR as well as in basal
resistance. In contrast to adapted pathogens that suppress wall defenses and successfully penetrate the host cells,
nonadapted pathogens might not have evolved a suppressor of preinvasion resistance.

The role of metabolic defense.

Plants produce secondary metabolites as chemical barriers to defend themselves against invading pathogens
(Hölscher et al. 2014; Lei et al. 2014). This diverse array of antimicrobial compounds can largely be divided
into two groups: i) phytoanticipins, a group of constitutive secondary metabolites and ii) phytoalexins that are
de novo synthesized and rapidly accumulated upon pathogen infection (Dixon 2001; Hammerschmidt
1999; VanEtten et al. 1994). Extensive studies about defensive secondary metabolites support the determinant
role in resistance for antimicrobial compounds in compatible host-pathogen interactions (Ahuja et al. 2012).
Antimicrobial phytochemicals are toxic for a broad range of fungi and detoxification mechanisms are required
to establish pathogenicity and virulence. For example, in tomato, the phytoanticipin α-tomatine binds to sterols
in fungal membrane, resulting in disruption of membrane integrity of pathogens and enhanced resistance
(Bangham and Horne 1962; Roddick 1976; Schulz and Sander 1957). However, several adapted pathogens
overcome α-tomatine by producing tomatinase, a glycosyl hydrolase. Tomatinase-deficient mutants
of Cladosporium fulvum and Fusarium oxysporum f. sp. lycopercisi are less virulent than the wild-type strain
(Ökmen et al. 2013; Pareja-Jaime et al. 2008; Roldán-Arjona et al. 1999; Sandrock and VanEtten 1998).

The causal agent of take-all disease of wheat, Gaeumannomyces graminis var. tritici, cannot cause disease on
nonhost oats due to its susceptibility to the phytoanticipin avenacin. However, Gaeumannomyces
graminis var. avenae, which produces avenacinase, overcomes the avenacin-mediated growth inhibition and
causes disease on oats (Bowyer et al. 1995; Osbourn et al. 1994; Papadopoulou et al. 1999). Similarly,
preformed sulforaphane plays a determinant role in NHR of Arabidopsis to bacteria Pseudomonas
syringae (Fan et al. 2011). Multiple sax genes of adapted P. syringae pv. maculicola function in detoxification
and efflux of sulforaphane. Nonadapted P. syringae strains transformed with saxCAB genes can
overcome Arabidopsis NHR. In Amaranthus gangeticus, accumulation of nicotinamide in roots prevents
colonization by nonadapted fungal pathogen Aphanomycese cochliodes (Islam et al. 2004).

Unlike constitutively produced phytoanticipins, phytoalexin synthesis is triggered by recognition of elicitors,

PAMPs or DAMPs, or effectors and is often mediated by activation of the mitogen-activated protein kinase
(MAPK) pathway (Graham et al. 1990; Kishi-Kaboshi et al. 2010; Raaymakers and Van den Ackerveken
2016; Umemoto et al. 1997). Arabidopsis oligogalacturonides released by polygalacturonase activity
of Botrytis cinerea are a class of DAMPs that induce production of phytoalexin camalexin via activation of
MAPK cascades and phosphorylation of WRKY33 (Ferrari et al. 2007; Mao et al. 2011; Ren et al. 2008).
Phytoalexins are also linked to other defense signaling mechanisms. For instance, indole glucosinolate is
required for callose deposition induced by flagellin elicitor peptide flg22, which plays a role as an effective
physical barrier at the sites of pathogen attack (Clay et al. 2009; Luna et al. 2011). Phytohormones including
ethylene (ET), jasmonate (JA), auxin, and cytokinin also control the synthesis of phytoalexins (Grosskinsky et
al. 2011; Huffaker et al. 2011; Matsukawa et al. 2013; Robert-Seilaniantz et al. 2011). In particular, JA acts
as a positive regulator and induces secondary metabolite accumulation through transcriptional regulation in a
wide range of plant species (De Geyter et al. 2012).

Similarly to host resistance, pathogen recognition in nonhost plants elicits phytoalexin accumulation. Alfalfa
infected with Asian soybean rust mounts a localized cell death in penetrated epidermal cells and induces the
synthesis of medicarpin and its intermediate metabolites that suppress rust appressorium formation (Ishiga et
al. 2015). The oligopeptide elicitor Pep-13, highly conserved among the oomycete genus Phytophthora, triggers
transcriptional reprogramming and accumulation of phytoalexin furanocoumarins in nonhost parsley (Brunner
et al. 2002; Nürnberger et al. 1994). In addition to Pep-13, necrosis-inducing Phytophthora protein 1 (NPP1)
elicits HR-like cell death and causes responses similar to Pep-13 in parsley, indicating that phytoalexin
induction is associated with recognition of multiple elicitors in nonhost (Hahlbrock et al. 2003). Recognition
of necrotrophic effector ToxA by nonhost wheat results in cell death and serotonin accumulation, which inhibits
sporulation of Stagonospora nodorum (Du Fall and Solomon 2013). In Arabidopsis, tryptophan (Trp)-derived
indole glucosinolates and camalexin are involved in NHR against biotroph powdery mildew and
hemibiotroph C. gloeosporioides (Hiruma et al. 2013; Lipka et al. 2005; Sanchez-Vallet et al.
2010). PENETRATION2 (PEN2) encodes a myrosinase involved in hydrolysis of indole glucosinolates (Lipka
et al. 2005). Arabidopsis pen2 mutants deficient in accumulation of an indole and a cysteine metabolite allow
frequent initiation of invasive growth of broad-spectrum nonadapted pathogens, including Phytophthora
infestans, B. graminis f. sp. hordei, and Magnaporthe oryzae (Bednarek et al. 2009; Lipka et al. 2005; Maeda
et al. 2009). In spite of enhanced entry rates of nonadapted pathogens in pen2 mutants, postinvasive resistance
associated with cell death blocks further infection, suggesting the involvement of the other defense components
in NHR. In the interaction between Arabidopsis and C. gloeosporioides, glutathione with Trp-derived
metabolites is required for pre- and postinvasive resistance (Hiruma et al. 2013). Mutants defective in both
glutathione synthesis and Trp metabolism allow enhanced invasive hyphae expansion of nonadapted
pathogen C. gloeosporioides. Furthermore, Arabidopsis RRS1/RPS4-mediated resistance to the adapted
pathogen C. higginisianum is compromised in these mutants, further supporting the idea that defense
compounds are shared between host and nonhost resistance.

Plants have evolved lineage-specific antimicrobial compounds as a consequence of the coevolutionary arms
race and adapted pathogens can cause disease by detoxification of these compounds (Kliebenstein
2012; Kroymann 2011). Probably, nonadapted pathogens had no chance to encounter defense metabolites
produced by nonhost plants and to prepare adequate weapons to overcome the obstacles, as illustrated by the
higher sensitivity of Phytophthora infestans to capsidiol induced in nonhost pepper compared with the adapted
pathogen Phytophthora capsici (Giannakopoulou et al. 2014; Jones et al. 1975). Thus, adaptation of
pathogens to specific antimicrobial compounds is one of the determinants of NHR and comprehensive analysis
of the role of chemical barriers in relation to other defense components could improve our understanding of the
NHR mechanisms.

The role of PTI.

Plant cells have the ability to discriminate self from nonself and activate defense signaling in response to
attempted pathogen invasion (Nürnberger and Brunner 2002). Sensing of potential invaders is mediated by
membrane-localized PRRs. In adapted host-pathogen interactions, several PAMPs, such as flagellin, chitin,
lipopolysaccharide, or extracellular adenosine triphosphate, have been identified and their mode of recognition
has been characterized (Couto and Zipfel 2016). As PAMPs are highly conserved and functionally essential for
microbe fitness, PAMP recognition and PTI are an efficient component of NHR.

The best-studied PAMP playing a role in NHR is the bacterial flagellin, the flagella building block (Hayashi et
al. 2001). P. syringae pv. tabaci mutants deficient for flagellin production cause disease symptoms on
nonhost Arabidopsis and tomato (Li et al. 2005; Taguchi et al. 2003). Conversely, the P.
syringae pv. tabaci ΔfliD mutant, which secretes large amounts of flagellin monomer, induces HR cell death on
the nonhost tomato (Shimizu et al. 2003). However, flagellin of an adapted P. syringae strain does not induce
HR on tobacco, in spite of identical amino acid sequence with flagellin of nonadapted P. syringae pv. glycinea,
which suggests the role of posttranslational modification of flagellin in recognition by nonhost plants (Taguchi
et al. 2003; Takeuchi et al. 2003). Flagellin glycosylation that is indispensable for flagella stabilization is
ubiquitous in most of phytopathogenic bacteria and plays an important role in bacterial virulence (Ichinose et
al. 2013; Taguchi et al. 2008, 2009). Glycosyltransferase-deficient mutants of P. syringae pv. glycinea can
multiply in nonhost Arabidopsis and tobacco while losing pathogenicity on host plants (Ishiga et al.
2005; Takeuchi et al. 2003). The findings that nonglycosylated flagellin induces a higher level of defense
responses on host plants than glycosylated flagellin suggest that pathogens might have evolved to evade
recognition by host plants through posttranslational modification (Taguchi et al. 2009). The modified PAMPs
to increase virulence in host plants might enable pathogens to be recognized by nonhost plants.

On the other hand, the flagellin receptor Flagellin-sensing 2 (FLS2) also mediates NHR (Zipfel et al. 2004).
Genetic screening of Arabidopsis natural variation for resistance to nonadapted bacteria reveals that FLS2
confers resistance to the bean pathogen P. syringae pv. phaseolicola (Forsyth et al. 2010).
Moreover, Arabidopsis fls2 mutants and FLS2-silenced Nicotiana benthamiana plants show enhanced
susceptibility to nonadapted P. syringae strains (Hann and Rathjen 2007). Nonhost plants also induce
downstream signaling upon flagellin perception. The flg22 strongly induces transcription
of NONHOST1 (NHO1) encoding a glycerol kinase that is required for Arabidopsis NHR against nonadapted P.
syringae pv phaseolicola, whereas the adapted P. syringae strain only transiently induces NHO1 expression (Li
et al. 2005; Lu et al. 2001). The induction of NHO1 is presumably essential because Arabidopsis plants
overexpressing NHO1 exhibit enhanced resistance to adapted P. syringae pv. tomato DC3000 (Kang et al.
2003). These results suggest that nonhost plants could trigger an immune response by PAMP recognition
similarly as during host resistance.

Based on the evolutionary model of NHR, the relative contribution of PTI to NHR increases in nonhost species
with a distant evolutionary relationship from the host due to lack of effector targets to suppress defense
response (Schulze-Lefert and Panstruga 2011). Interfamily or interspecies transfer of PRRs from nonhost
plants expands the recognition specificity, as shown by Arabidopsis EFR (EF-Tu receptor) transfer to tomato
and wild potato ELR (elicitor response) transfer to Solanum tuberosum (Du et al. 2015; Lacombe et al. 2010).
Thus, PTI in NHR as well as in host resistance provides broad-spectrum resistance to phytopathogens.

The role of ETI.

ETI is one of the major components of host resistance in plants (Cui et al. 2015). It is activated by direct or
indirect interaction between one or more pathogen effectors and one or more plant R proteins, often resulting in
HR. Previous studies proposed that NHR may be partially mediated by effectors and R genes even though
specific roles and mechanisms of ETI in NHR remain elusive (Stam et al. 2014).

Numerous cases of the effector recognition in nonhost plants have been reported (Table 1). For example, P.
syringae pv. tomato avrA homolog, P. syringae pv. phaseolicola avrPphD, and Xanthomonas.
oryzae pv. oryzicola AvrRxo1 induce HR on their respective nonhost plants (Arnold et al. 2001; Kobayashi et
al. 1989; Liu et al. 2014; Zhao et al. 2004b). Similarly, Bremia lactucae effector BLG01 carrying a GKLR
variant of the RXLR translocation motif induces HR in a backcross inbred line of nonhost Lactuca
saligna (Jeuken and Lindhout. 2002; Stassen et al. 2013). In addition, a recent study reported that several
RXLR effectors of Phytophthora infestans trigger HR in nonhost pepper accessions, suggesting multiple
interaction between effectors and putative target genes in NHR (Lee et al. 2014). Meanwhile, some effectors
have been suggested as major determinants of host range. P. syringae pv. tomato DC3000 HopQ1 deletion
mutant can cause bacterial speck symptoms in nonhost N. benthamiana (Wei et al. 2007). Similarly, mutation
of HopAS1 from P. syringae pv. tomato T1 confers enhanced virulence in nonhost Arabidopsis and reduced
virulence of host tomato (Sohn et al. 2012). Therefore, effectors might be involved in limitation of host range
and NHR.

Table 1. Effectors and nucleotide-binding domain and leucine-rich repeat (NLR) proteins in nonhost
interactions
View as HTML

The HR triggered by effectors in certain nonhost plants probably results from the activation of one or
more R genes and subsequent defense signaling, as observed in compatible host-pathogen interactions. Genetic
analysis reveals that necrotic phenotypes induced by AvrRPS4 and HopK1 from P. syringae cosegregate with
clusters of RGC4 (Resistance Gene Candidate4) homologs encoding NLRs in nonhost lettuce (Wroblewski et
al. 2009). In addition, maize NLR Rxo1 recognizes avrRxo1 from X. oryzae pv. oryzicola, which causes
bacterial leaf streak disease in rice (Zhao et al. 2004a, 2004b, 2005). In Arabidopsis, WRR4 encoding a
toll/interleukin1-receptor-NLR protein confers resistance against Albugo candida, which is a nonadapted
pathogen indicating that R genes are indeed involved in NHR, although the corresponding effector remains to
be elucidated (Borhan et al. 2008, 2010). By contrast, single mutants carrying nonfunctional copies of those
NLRs still show resistance to nonadapted pathogens, which opens the possibility of multiple R protein–effector
interactions in NHR (Borhan et al. 2008; Zhao et al. 2005). Finally, there are more unidentified R genes that
cosegregate with HR triggered by effectors in nonhost plants (Vega-Arreguín et al. 2014; Whalen et al. 1988).
This further highlights the importance of endogenous R genes and R gene-mediated responses in NHR.

Plants and pathogens are coevolving in a never-ending arms race. A recent concept of NHR in evolutionary
aspects suggests that relative contribution of ETI mediated by NLR proteins increases in the case of close
evolutionary relationship between host and nonhost plant (Schulze-Lefert and Panstruga 2011). Identification
of one or more plant factors recognizing one or more effectors from nonadapted pathogens and elucidation of
ETI mechanisms in NHR may shed light on how NHR is established.

Defense signaling in NHR.

After perception of pathogens, plants activate complex signaling including oxidative burst, MAPK cascade,
hormone biosynthesis, and transcriptional reprogramming (Boller and Felix 2009; Tsuda and Katagiri 2010).
On the basis of nonhost defense responses from several pathosystems, accumulation of reactive oxygen species
(ROS), induction of defense-related genes, salicylic acid (SA) and JA production, and callose deposition are
commonly associated with NHR, regardless of HR cell-death symptoms, suggesting overlaps with the host
defense response (An and Mou 2012; Cheng et al. 2012; Narusaka et al. 2005; Trujillo et al. 2004; Yun et
al. 2003; Zhang et al. 2011).

There are some evidences that PRR- or R gene–mediated signaling machinery used by host resistance also
function in NHR. The PRR FLS2 forms heteromeric complexes with coreceptors BAK1 (BRI1-associated
receptor kinase1) or related SERKs (somatic embryogenesis receptor kinase) (Heese et al. 2007; Chinchilla et
al. 2007; Roux et al. 2011). BAK1 transcript accumulation in Arabidopsis increases more than twofold in the
presence of nonadapted P. syringae pv. phaseolicola but decreases during host interaction (Kemmerling et al.
2007). Arabidopsis bak1 mutants are susceptible to nonadapted P. syringae pv. tabaci 6605 and Alternaria
brassicicola (Kemmerling et al. 2007; Roux et al. 2011). Similarly, silencing of BAK1 in N.
benthamiana enhances the growth of nonadapted P. syringae pv. tomato DC3000 (Heese et al. 2007).
In R gene–mediated signaling, a conserved chaperone complex comprising HSP90, SGT1, and RAR1 is
required for activation and stabilization of R proteins (Kadota et al. 2010). Silencing of HSP90 or SGT1 in N.
benthamiana compromises NHR to nonadapted pathogens such as P. cichorii or X.
axonopodis pv. vesicatoria (Kanzaki et al. 2003; Peart et al. 2002). EDS1 (Enhanced disease susceptibility 1)
is an essential regulator of R gene–mediated signaling. Mutation in Arabidopsis EDS1 enhances susceptibility
to nonadapted Albugo candida and Erwinia amylovora (Moreau et al. 2012; Parker et al. 1996). In addition,
HR triggered by conidia of nonadapted wheat powdery mildew (B. graminis f. sp. tritici) is significantly
reduced in eds1 mutants (Yun et al. 2003). In the nonhost Arabidopsis-Blumeria pathosystem, the mutation of
EDS1 interactors, such as PAD4 and SAG101 in the pen2 mutant, caused breakdown of NHR (Lipka et al.
2005). Further, one of the earliest downstream signaling events upon pathogen perception is activation of
MAPKs that act as a central hub in subsequent defense responses, such as ROS generation, defense-gene
activation, and hormone biosynthesis (Meng and Zhang 2013). As MAPK cascades are highly conserved
among eukaryotes, signal transduction in NHR is also mediated through MAPKs. Silencing
of NbMKK1, NbSIPK, or NbWIPK compromises NHR of N. benthamiana to P. cichorii (Sharma et al.
2003; Takahashi et al. 2007). Conversely, adapted P. syringae strains carry effectors such as AvrPto, AvrPtoB,
or HopAl1 to suppress MAPK cascade in host plants. Moreover, ectopic expression of AvrPto promotes the
growth of two nonadapted bacterial pathogens in Arabidopsis, suggesting that effectors that have been evolved
to suppress defense response could play a pivotal role for pathogens to overcome NHR (He et al. 2006; Zhang
et al. 2007).

Other early responses of plants upon pathogen perception include calcium influx and ROS production, which
both act as secondary messengers (Boller and Felix 2009). Increased cytosolic calcium level leads to activation
of calcium-dependent protein kinases and NADPH oxidase RbohD responsible for the rapid production of ROS
in Arabidopsis, which suggests the mechanistic link between calcium and ROS signaling (Ogasawara et al.
2008; Ranf et al. 2011; Segonzac and Zipfel 2011). ROS also mediates cellular signaling associated with
defense-related gene expression, HR, and phytoalexin production (O’Brien et al. 2012). However, only a few
studies have investigated the role of ubiquitous messenger Ca2+ and ROS in NHR. In N. benthamiana,
calreticulin that functions in Ca2+ binding and storage is essential for NHR to X. oryzae pv. oryzae (Li et al.
2012). Likewise, the mutation of calcium sensor CaM7 in Arabidopsis compromises NHR to Asian soybean
rust (Campe et al. 2016). On another hand, a hydrogen peroxide burst is observed at the site of cell-wall
apposition in barley upon tentative infection by nonadapted wheat powdery mildew infection (Hückelhoven et
al. 2001). In N. benthamiana, silencing of glycolate oxidase (Gox), which produces hydrogen peroxide in
peroxisome, compromises NHR to P. syringae pvs. tomato and glycinea as well as Pto-AvrPto interaction
(Rojas et al. 2012b). Arabidopsis gox mutants also display enhanced susceptibility to a nonadapted strain of P.
syringae, concomitantly to reduced accumulation of hydrogen peroxide and compromised HR. In addition,
treatment with flavodoxin, which prevents ROS formation in plastids, reduces HR and ROS accumulation in
tobacco, upon inoculation with nonadapted X. campestris pv. vesicatoria (Zurbriggen et al. 2009). These
results highlight that nonhost plants employ common factors of host resistance in an early stage of signal
transduction after receptor-dependent pathogen perception.

Pathogen perception activates signaling for hormone biosynthesis. The plant hormones ET, JA, and SA play an
important role as secondary messengers of plant defense response (Meng and Zhang 2013). Accumulating
evidence shows that defense hormones also contribute to NHR. Tobacco expressing the Arabidopsis etr1-1 gene
shows ET insensitivity and loses NHR against Pythium sylvaticum, demonstrating a role for ET in NHR
(Knoester et al. 1998). Nonadapted rust fungi, including Uromyces vignae and U. appendiculatus, produce
longer infection hyphae in an Arabidopsis sid2 mutant deficient for SA biosynthesis and in NahG-expressing
plants in which SA is degraded to catechol (Mellersh and Heath 2003). In Arabidopsis, Plant defensin
1.2 (PDF1.2), a marker gene of the JA/ET signaling pathway, is induced upon inoculation with nonadapted
barley powdery mildew, whereas host interaction between Arabidopsis and Erysiphe cichoracacearum only
triggers a moderate induction of PDF1.2 expression (Zimmerli et al. 2004). In spite of the effect of defense
hormones in plant resistance, most of the single mutants related to hormone signaling are not significantly
affected in NHR (Ham et al. 2007; Mishina and Zeier 2007). This accounts for the complex network of
defense hormone signaling and multiple layers of NHR.

Comparative analysis of transcription profiles following inoculation of adapted and nonadapted pathogens
reveals that plants activate fundamentally similar programs but that strong transcriptional activation or
repression occurs earlier in nonhost interaction (Zellerhoff et al. 2010; Zimmerli et al. 2004). According to
quantitative analysis of transcript abundance, most of the differentially expressed genes in nonhost interactions
are related to biotic or abiotic stress, the secondary metabolic pathway, or transcription factors, including
APETALA2/ET response element binding factor (ERF) (Daurelio et al. 2013; Moreau et al. 2012; Zellerhoff
et al. 2010). Transcription factors modulate different sets of defense-related genes and participate in NHR as
well as in host resistance. NbCD1, encoding an ERF, is induced by nonadapted P. cichorii and silencing
of NbCD1 compromises NHR of N. benthamiana to P. cichorii (Nasir et al. 2005). Similarly, NAC
transcription factor ATAF1 contributes to penetration resistance of Arabidopsis against the nonadapted B.
graminis f. sp. hordei via suppression of abscisic acid biosynthesis and signaling (Jensen et al. 2008; Wang et
al. 2009). In addition, calmodulin-binding transcription activators negatively regulate NHR to bacterial
pathogens, by downregulation of target genes including EDS1, CBP60G, and NDR1 and by subsequent ROS
accumulation suppression (Rahman et al. 2016). Changes of defense-related gene expression and transcription
factor activities could amplify defense signals in a positive feedback loop to enhance NHR.

It is interesting to note that various accessions of a nonhost species show different responses to the same
nonadapted pathogen or its effectors (Lee et al. 2014; Mellersh and Heath 2001; Wroblewski et al. 2009).
Given the overlaps of defense signaling components in host and nonhost resistance, identification of nonhost
receptors that mediate pathogen perception and induce defense response could be a vital element to utilize NHR
mechanisms for crop improvement.

Miscellaneous factors in NHR.

Last but not least, there are reports on involvement of unexpected plant components in NHR. For example,
sphingolipids are membrane components that are involved in signal transduction for cellular homeostasis and
stress responses such as apoptosis. Serine palmitoyltransferases, which catalyze the first step of sphingolipid
biosynthesis, comprise two different subunits, LCB1 and LCB2. N. benthamiana NHR to P. cichorii is
compromised in NbLCB1- and NbLCB2-silenced plants, suggesting involvement of sphingolipids in NHR
(Takahashi et al. 2009). A similar example is reported for membrane-attached protein, such as the small
GTPase (Ras) that regulate diverse processes including vesicular trafficking (Rojas et al. 2012a). Silencing
of ADP ribosylation factor 1 (ARF1), a member of the conserved Ras superfamily, enhances P. cichorii growth
on nonhost N. benthamiana (Coemans et al. 2008). In addition, silencing of RPL12 and RPL19, encoding
ribosomal proteins in N. benthamiana, leads to delay of HR and enhances bacterial growth of nonadapted
pathogens (P. syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria) (Nagaraj et
al. 2015). Moreover, overexpression of barley BI-1 (Bax inhibitor-1), which suppresses programmed cell death,
allows nonadapted B. graminis f. sp. hordei to penetrate the barley epidermal cells, indicating a connection
between cell-death regulation and NHR (Eichmann et al. 2004). Furthermore, alteration of chromatin
structures via DNA damage and nuclear protein modifications affects transcription initiation of pathogenesis-
related (PR) genes (Hadwiger 2015). DNase released from nonadapted Fusarium solani f. sp. phaseoli elicits
DNA fragmentation of pea in contact with Fusarium solani f. sp. phaseoli and induces a defense response
including PR gene expression and phytoalexin accumulation. Ubiquitination of histones and HMG A
transcription factor that binds to the PR gene promoter is also associated with nonhost response of pea
against Fusarium solani f. sp. phaseoli (Isaac et al. 2009). Therefore, the possibility remains that unexplored
plant components are involved in NHR against a wide diversity of pathogens.

Application of NHR for crop improvement.

NHR is considered a powerful source for durable resistance. Over the past two decades, there have been several
attempts to deploy NHR components to improve disease resistance. Introduction of master switches including
PRRs and R genes into host plants confers resistance through conserved downstream signaling. Interfamily
transfer of Arabidopsis EFR, which recognizes the bacterial PAMP elonglation factor Tu, confers elf18
responsiveness and enhances resistance to a wide-range of bacterial pathogens in transgenic tomato and N.
benthamiana (Lacombe et al. 2010). Similarly, N. benthamiana FLS2 was transferred into citrus lacking flg22
perception, resulting in enhanced resistance to citrus canker caused by X. citri (Hao et al. 2016). Receptor-like
protein ELR from the wild potato Solanum microdontum recognizes several elicitins of the Phytophthora genus
and transgenic S. tuberosum carrying ELR is resistant to Phytophthora infestans infection (Du et al. 2015).
With regard to R gene–mediated resistance, maize Rxo1 and Arabidopsis WRR4 have been reported to function
in other species. Transfer of Rxo1 into susceptible host rice confers resistance to X.
oryzae pv. oryzicola carrying the matching effector AvrRxo1 (Zhao et al. 2005). Similarly, WRR4 fully
functions in Brassica napus and Brassica juncea against the white rust Albugo candida (Borhan et al. 2010).

In addition, interspecific transfer of metabolic machinery that actively hinder pathogen growth enables host
plants to produce foreign antimicrobial compounds from common intermediate molecules. Stilbene synthase, a
key enzyme for resveratrol biosynthesis, was identified from grapevine and was then transferred into tobacco
(Hain et al. 1993). Transgenic tobacco producing resveratrol shows enhanced resistance to Botrytis cinerea. In
the same way, three genes for dhurrin synthesis of Sorghum bicolor were transferred into Arabidopsis, which
does not produce dhurrin (Tattersall et al. 2001). Dhurrin accumulation in transgenic Arabidopsis confers
resistance to the flea beetle Phylotreta nemorum, which has not encountered dhurrin.

Taken together, these studies have demonstrated the possibility of utilizing nonhost resistance sources across
species. Further understanding of NHR genetic basis could broaden the source of disease resistance as well as
elucidate the detailed mechanisms of plant resistance.

Conclusion and prospects.

During the long history of the arms race between hosts and pathogens, plants have evolved unique defense
systems (host resistance) against their pathogens, including physical and chemical barriers, PTI activated by
surface-localized PRRs, and ETI activated by intracellular NLR immune receptors. However, in nature, most
plant species still remain nonhosts for most of pathogens. Then, how could plants establish resistance against
previously unencountered pathogens? This critical question remains to be explained. Possibly, plants built up
overfull defense potential from the intensive arms race with devastating pathogens over a long period of time.
These furnished defense systems of plants could resist a broad spectrum of nonadapted pathogens as the results
of so-called “cold war effects.” On the pathogen side, they also may have evolved specialized machinery to
evade recognition or overcome host defense responses. However, the pathogens may not prepare weapons for
battle with unencountered plants.

In this review, we focused on understanding the differences in recognition of nonself and defense mechanisms
between hosts and nonhosts at the pre- and postpenetration levels, using all available literatures published
during recent decades. Overall, we found the current understanding of NHR mechanisms is not much deviated
from our knowledge on host resistance, except for few specific cases (Fig. 1). Several defense factors of host
resistance are also involved in NHR, but the relative contribution of the defense factors to determine NHR
varies depending on plant or pathosystem.
 Fig. 1.Mode of action in plant nonhost resistance. Relative contribution of defense components to nonhost
resistance (NHR) may vary depending on the pathogen species. Various pathogens cease to grow in nonhost
plants due to maldifferentiation of infection structures during preinvasion. In addition, nonhost pattern-
recognition receptors (PRRs) trigger defense response via pathogen-associated molecular pattern (PAMP)
recognition, which terminates pathogen infection. Conversely, nonadapted pathogens could have not evolved
effectors to modulate defense response or nonhost plants could not have effector targets utilized as host factors.
Effector-triggered immunity is also a major component of NHR and several evidences support multiple
resistance (R) protein–effector interactions. Although NHR largely shares defense signaling components with
host resistance, chemical barriers such as phytoalexin have evolved in a species-specific manner and play a
pivotal role as determinants of host range.

Download as PowerPoint

On that viewpoint, we suggest several topics for further understanding of the NHR mechanism and its
implication in crop improvement.

 i) Further characterization on the role of PRRs in plant NHR. Most plant genomes contain hundreds of
receptor-like protein kinase genes that could be potential PRR candidates, but only a few of them are
functionally characterized. High throughput screening using gain- or loss-of-function studies in PRR
gene families could provide a number of resistance sources for biotechnological application beyond the
species barrier.
 ii) Molecular characterization of multiple interactions among effectors of nonadapted pathogens and
nonhost plants. A recent study (Lee et al. 2014) strongly supports the idea that ETI caused by multiple
interactions between effectors and host factors underpins the NHR of plant species closely related to
host plants. Isolation and functional characterization of effector target proteins, including NLRs, could
serve as a valuable source of resistance for protecting plants against pathogens in combination with
PRRs, as suggested by Du et al. (2015).
 iii) Roles of plant secondary metabolites in host range determination. There is lineage-specific evolution
of antimicrobial metabolite synthesis in plants, such as isoflavonoids in Leguminosae, indole derivatives
in Cruciferae, and sesquiterpenes in Solanaceae plants. Current evidence reveals that preexisting or
inducible antimicrobial metabolites have critical roles in host range determination in multiple cases of
plant-pathogen interactions, as reviewed above. Revisits and further understanding on the roles of these
lineage-specific secondary metabolites in the plant kingdom could provide new insights in NHR from
previous knowledge.

NHR is considered the most durable and powerful control measure of plant disease. Further understanding of
the critical components exposed in this review will not only extend our knowledge about NHR but, also,
provide basic resources for protecting plants against pathogens by combinational biotechnology approaches.

https://apsjournals.apsnet.org/doi/10.1094/MPMI-10-16-0213-CR

D. Understanding and utilizing plant cells (25)

Plant structure and function depend on the composition and behaviour of plant cells. A lot of
progress has been made towards identifying cellular components (including DNA, RNA,
proteins, cell wall components and membranes) and understanding how they contribute to
specific processes (such as development, metabolism, and pathogen resistance). The early
questions in this section address frontiers in our understanding of plant cells, and potentially
timely applications are tackled later.

 D1.

How do plant cells maintain totipotency and how can we use this knowledge to improve
tissue culture and regeneration?

 D2.

How are growth and division of individual cells coordinated to form genetically
programmed structures with specific shapes, sizes and compositions?

 D3.
 How do different genomes in the plant talk to one another to maintain the appropriate
complement of organelles?

 D4.

How and why did multicellularity evolve in plants?

 D5.

How can we improve our understanding of programmed developmental gene regulation

from a genome sequence?

 D6.

How do plants integrate multiple environmental signals and respond?

 D7.

How do plants store information on past environmental and developmental events?

 D8.

To what extent do epigenetic changes affect heritable characteristics of plants?

 D9.

Why are there millions of short RNAs in plants and what do they do?

 D10.

What is the array of plant protein structures?

 D11.

How do plant cells detect their location in the organism and develop accordingly?

 D12.

How do plant cells restrict signalling and response to specific regions of the cell?

 D13.

Is there a cell wall integrity surveillance system in plants?

 D14.

How are plant cell walls assembled, and how are their strength and composition
determined?

 D15.

Can we usefully implant new synthetic biological modules in plants?

 D16.

To what extent can plant biology become predictive?

 D17.
 What is the molecular/biochemical basis of heterosis?

 D18.

How do we achieve high‐frequency targeted homologous recombination in plants?

 D19.

What factors control the frequency and distribution of genetic crossovers during meiosis?

 D20.

How can we use our knowledge about photosynthesis and its optimization to better harness
the energy of the sun?

 D21.

Can we improve algae to better capture CO2and produce higher yields of oil or hydrogen
for fuel?

 D22.

How can we use our knowledge of carbon fixation at the biochemical, physiological and
ecological levels to address the rising concentrationsof atmospheric CO2?

 D23.

What is the function of the phenomenal breadth of secondary metabolites?

 D24.

How can we use plants as the chemical factories of the future?

 D25.

How do we translateour knowledge of plant cell walls to produce food, fuel and fibre more
efficiently and sustainably?

E. Diversity (17)
It is currently estimated that there are at least a quarter of a million species of flowering plant in
the world, the vast majority of which have not been tested for useful properties. Questions in this
section address the need to identify plants with potential for human benefit that have yet to be
recognized, and to do so in a sustainable and responsible manner. The resulting knowledge and
natural resources could then be used to tackle new challenges as they arise.

 E1.

How much do we know about plant diversity?

 E2.

How can we better exploit a more complete understanding of plant diversity?

 E3.

Can we increase crop productivity without harming biodiversity?

 E4.
 Can we define objective criteria to determine when and where intensive or extensive
farming practices are appropriate?

 E5.

How do plants contribute to ecosystem services?

 E6.

How can we ensure the long‐term availability of genetic diversity within socio‐economically
valuable gene pools?

 E7.

How do specific genetic differences result in the diverse phenotypes of different plant
species? That is, why is an oak tree an oak tree and a wheat plant a wheat plant?

 E8.

Which genomes should we sequence and how can we best extract meaning from the
sequences?

 E9.

What is the significance of variation in genome size?

 E10.

What is the molecular and cellular basis of plants’ longevity and can plant life spans be
manipulated?

 E11.

Why is the range of life spans in the plant kingdom so much greater than in animals?

 E12.

What is a plant species?

 E13.

Why are some clades of plants more species‐rich than others?

 E14.

What is the answer to Darwin’s ‘abominable mystery’ of the rapid rise and diversification
of angiosperms?

 E15.

How has polyploidy contributed to the evolutionary success of flowering plants?

 E16.

What are the closest fossil relatives of the flowering plants?

 E17.
How do we best conserve phylogenetic diversity in order to maintain evolutionary
potential?

Conclusions
Plant science is central to addressing many of the most important questions facing humanity.
Secure food production and quality remain key issues for the world in the 21st Century, and the
importance of plants extends well beyond agriculture and horticulture as we face declining fossil
fuel reserves, climate change, and a need for more sustainable methods to produce fuel, fibre,
wood, and industrial feedstocks. There is also untapped potential in optimizing the nutritional
properties of foods, and in identifying novel plant products such as medicines. Tackling these
frontiers will require new scientific methods and collaborations as existing approaches are
delivering incomplete answers.

MANY OF THE MOST IMPORTANT QUESTIONS THAT WE HAVE IDENTIFIED CAN

ONLY BE ADDRESSED BY THE INTEGRATED EFFORTS OF SCIENTISTS WITH
DIVERSE EXPERTISE. For example, many require close cooperation between scientists
working to improve crops and those working on environmental stability and ecosystem services.
Funding mechanisms, scientific publishing and conferences could be more effective in supporting
and encouraging the efficient transfer of knowledge between different areas of plant science and
this should be addressed. In the longer term, changes to higher education may be required to
ensure that future researchers have the most suitable background knowledge and skill sets to
address the research challenges that they are likely to face.

As plant science becomes increasingly important, we need to attract the brightest and best to
careers in plant research. School education does not include the most interesting or relevant
aspects of plant science, and discourages young people from studying the subject at university.
This is indefensible in a world with such a strong requirement for outstanding plant scientists,
and steps should be taken to put it right.

Research moves quickly, and our questions and suggestions about the ways that they might be
tackled will require revision, but in the meantime we hope that they will stimulate discussion, and
encourage plant scientists to think beyond the limits of their own specific fields about the most
important research that can conceivably be carried out, the most promising approaches that can
be developed and applied, and the most significant discoveries that can possibly be made.

Acknowledgements
The 100 questions workshop and website were supported by funds from the New Phytologist
Trust, the Journal of Experimental Botany, The Gatsby Foundation, and the BBSRC‐funded plant
science network GARNet. The authors thank everyone who submitted a question to the website,
Professor Alastair Fitter, the Editor and the referees for helpful comments, and Dr Robert Payne
and Professor William Sutherland for inspiration.

Citing Literature
Supporting Information

Volume192, Issue1
October 2011
 Pages 6-12

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Citations:35

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Keywords

What is the overall health of the plant?

It is a good reminder to put into perspective overall plant health. Presumably you have found
something abnormal or you would not be continuing with the diagnosis, but step back for a moment
to consider overall health. This helps later in terms of what you will recommend and how important
various problems on the plant might be, but it also helps provide focus relative to how long
problems might have been present. Consider, relative to a healthy specimen of the same plant,
such questions as whether leaf size and color are normal, if the canopy is full or if the growth rate
is normal.
 Figure 8. The thinning canopy of this ash Figure 9. Annual growth rate. (Graphic drawn by Joe
tree indicates an overall decline in the Boggs.)
health of the tree.
For example, the terminal buds on many deciduous trees produce a distinct scar that delineates
seasonal growth. If you measure the space between terminal bud scars on the twig, you can tell
how much it has grown in recent years. It is a little tricky to know what a normal rate of growth is
and whether lower than normal rates necessarily mean the plant is unhealthy. However, declining
rates of growth over the past several years can be telling, and they can often even be traced to a
particular event, such as installation of new sewer lines or a new driveway. Conversely, pointing
out normal annual growth can also help allay fears that something major is wrong with the plant—
for example, on maple when all that is found is some tarry spots on the leaves.

Why are plants important in the ecosystem and to the survival of other organisms?

Plant Biology Questions

Bring on the tough stuff
1. If you pushed a small thumbtack into a tree’s trunk, what would happen to that
thumbtack in twenty years? Would it get higher, lower, become embedded in the tree or
fall out?

2. Why doesn’t cutting grass with a lawnmower kill the grass? Doesn’t it damage the
shoot apical meristem?

3. If you pick up a bottle of plant food or fertilizer, it usually has three numbers in a ratio
on it. What might these numbers refer to?

4. In some ecosystems, many of the leaves look the same, even though they are on
different plants. For example, in the desert most of the plants have prickly leaves, and
on mountaintops the plants have needles or small whitish leaves. Why do all the leaves
look the same in the same habitat?

5. Why are flowers so important for angiosperm reproduction?

6. Abscisic acid (ABA) inhibits seed germination. Why would it be advantageous for
prolonged cold temperatures to break down ABA and cause seeds to germinate?

Plant Biology Answers

1. Thumbtacks are small, so we will assume it only pushed into the outer layers of bark.
Over twenty years, the tree will shed its outermost bark layers, and the tree will continue
growing outward. Most likely, the thumbtack will fall off with the bark.

2. Grasses are monocots, and therefore have intercalary meristems. Even though the
lawnmower takes off the top of the grass stems and leaf blades, the intercalary
meristems further down the stem can produce new cells quickly. This allows the grass
to re-grow stems and leaves, and gives you summer job security if you want it.

3. The ratio on fertilizer bottles is the relative amount of the primary macronutrients:
nitrogen (N), phosphorus (P), and potassium (K). You will be looking at the N-P-K ratio.
Other nutrients included in the fertilizer are often listed somewhere else on the bottle,
but N, P and K are the most important. If the ratio is even, such as 10-10-10, there are
equal amounts of each primary macronutrient.

4. Many leaves are modified to provide an advantage to the plant in a particular

environment. In the desert, needle-shaped leaves don’t lose as much water as flat, broad
leaves because they have less surface area. On mountaintops, leaves are often lighter
in color to reflect the intense sunlight; they are also smaller to minimize water loss from
the winds present on mountains. Rainforest plants usually have broad leaves to
maximize the sunlight they receive, since they are experiencing intense competition for
light with all the other plants around. If organisms have features that make them look the
same but they aren’t closely related, it is called convergent evolution because the
organisms converge on one adaptive form through evolution.

5. Flowers attract pollinators, which is a huge development for angiosperms. Plants that
are wind-pollinated have to make a lot of pollen so that eventually some pollen grains
will end up on different individuals of the same species. Animal-pollinated plants can
make less pollen because they have a personal courier delivering pollen to other plants
of the same species. The bright colors of flower petals advertise to bees, butterflies,
hummingbirds and other pollinators that the plant is offering a nice sugary snack, and
animals often end up doing the pollination inadvertently because they have to brush
against the anthers on their way to the nectary.

6. ABA prevents seed germination, which is good when conditions aren’t right for plants
to grow. For example, the beginning of winter in the northern United States is not a good
time for plants since freezing temperatures can harm plants. However, over time cold
destroys ABA. This happens slowly enough that after three or four months, when ABA
levels are low enough that the seed starts to germinate, spring is just around the corner
and it’s safe to be a growing plant again.

References

1. The Future of Food: Plant science approaches to the global challenge of food security by James Turner
https://www.sheffield.ac.uk/aps/news/food-security-global-challenge-plant-science-1.814357

2. The role of plant science in food security

by University of Copenhagen https://phys.org/news/2015-11-role-science-food.html
3. http://pure.iiasa.ac.at/id/eprint/8984/1/XO-09-062.pdf
4.