Opinion

TRENDS in Plant Science

Vol.10 No.5 May 2005

The future of C4 research maize,

Flaveria or Cleome?
Naomi J. Brown, Kate Parsley and Julian M. Hibberd
Department of Plant Sciences, Downing Street, University of Cambridge, Cambridge, UK CB2 3EA

C4 photosynthesis has evolved multiple times among

the angiosperms: the spatial rearrangement of the
photosynthetic apparatus, combined with alterations
to the leaf structure, allows CO2 to be concentrated
around Rubisco. Higher CO2 concentrations at Rubisco
decrease the rate of oxygenation and therefore reduce
the amount of energy lost through photorespiration. C4
plants are particularly prevalent in tropical and subtropical regions because they can sustain higher rates of
net photosynthesis; they also represent some of our
most productive crops. To date, most progress in
identifying genes crucial for C4 photosynthesis has
been made using maize and Flaveria. We propose that
Cleome, the most closely related genus containing C4
species to the C3 model Arabidopsis, be used together
with Arabidopsis resources to accelerate our progress in
elucidating the genetic basis of C4 photosynthesis.

Why C4 photosynthesis evolved and its global

importance
Photosynthesis is the basis of life on Earth and yet its
efficiency in most plants is compromised because Rubisco,
the primary carboxylating enzyme, is promiscuous [1].
When Rubisco evolved w3 billion years ago, the atmosphere of Earth contained high concentrations of CO2 but
low concentrations of O2, therefore there was little
selection pressure for Rubisco to discriminate between
them [2]. However, over geological time, O2 concentrations
increased and CO2 concentrations dropped. The inability
of Rubisco to distinguish between CO2 and O2 has
therefore led to an increase in the ratio of oxygenations
to carboxylations at its active site [2].
When oxygenation takes place at the active site of
Rubisco, carbon is lost from the regenerative phase of the
Calvin cycle because phosphoglycolate is produced [1].
Although some carbon present in phosphoglycolate is
retrieved via photorespiration, it is at the expense of ATP
and NADPH [3]. C4 photosynthesis (so called because the
initial product of photosynthesis is a four carbon compound) essentially eliminates the oxygenase activity of
Rubisco via anatomical, biochemical and ultrastructural
modifications to leaves [4]. Although there is large
variation in the mechanisms by which C4 plants spatially
concentrate CO2 around Rubisco [5], key features of any
C4 system include: initial fixation of bicarbonate by an
Corresponding author: Hibberd, J.M. (julian.hibberd@plantsci.cam.ac.uk).
Available online 2 April 2005

enzyme that does not possess oxygenase activity, the

subsequent release of CO2 in a separate compartment
where CO2 is refixed by Rubisco, and then regeneration of
the primary CO2 acceptor (Box 1). Typically, in C4 plants,
these stages are compartmentalized into different celltypes, although recent evidence indicates that compartmentation can occur within individual cells. For example,
a single-celled version of C4 photosynthesis has been
reported in both Borszczowia aralocaspica and Bienertia
cycloptera, members of the Chenopodiaceae (within the
Carophyllales, see Figure 1) [67], and also in a singlecelled marine diatom [8].
As temperature increases, the ratio of carboxylation to
oxygenation reactions decreases, therefore the energy
demands associated with photorespiration are greater in
warmer areas of the world. C4 plants are therefore most
abundant in certain regions of the tropics and subtropics,
where photorespiration can reduce photosynthesis of C3
plants by O30% [9]. In absolute numbers, C4 plants
represent fewer than 4% of all angiosperm species but
they dominate some of the most productive ecosystems on
Earth. Because of this, C4 plants exert a large affect on
climate and therefore global warming and human welfare
[2]. It has been estimated that C4 plants are responsible
for w2530% of global terrestrial productivity [10]. In
addition, the human food supply in many tropical areas is
largely based on C4 species such as maize and sorghum
[11]. As a consequence, to increase the productivity of rice
(a C3 crop that feeds the majority of people in many areas
of the world that have the highest rate of population
increase) there has been considerable interest in engineering rice to use the C4 pathway [1214].
Although the alterations to leaf anatomy and biochemistry needed for C4 photosynthesis appear complex, it is
estimated that C4 photosynthesis has evolved independently at least 45 times within 18 different families of land
plants (Figure 1) [15], implying that many, if not all, of the
genes necessary for C4 photosynthesis are already present
in C3 species.
Given the interest in understanding the genetic basis of
C4 photosynthesis, with perspectives that range from how
it evolved to the engineering of more efficient crops, it
seems timely to review progress made to date, to assess
why progress is limited in specific areas and to propose
approaches that should be productive in the future. To do
this, we will provide several examples illustrating what
we know about the genetic determinants of C 4

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Box 1. Summary of an idealized C4 pathway

C4 photosynthesis typically demands the following modifications (key
modifications to the biochemistry of photosynthesis are represented
by purple numerals 15 in Figure I).
In a compartment lacking Rubisco, CO2 is converted to HCOK
3 by
carbonic anhydrase (1) and then fixed via phoshoenolpyruvate
carboxylase (PEPC) (2). In most C4 species, this compartment is the
mesophyll, which possesses relatively low numbers of chloroplasts
per cell. PEPC does not possess oxygenase activity. Because of the
equilibrium constant for CO2 and HCOK
3 , PEPC operates while
saturated with substrate.
PEPC generates oxaloacetate (OAA), which is converted to malate via
malate dehydrogenase (3). Abundant plasmodesmata allow flux of C4
organic acids from mesophyll cells to bundle sheath. The exact form in
which carbon moves (e.g. malate or aspartate) varies between different
species of C4 plant.
CO2 is then released from organic acids in the bundle sheath by
one of three decarboxylase enzymes (4) to provide high concentrations of CO2 to Rubisco. This effectively reduces the oxygenation reaction of Rubisco. The decarboxylases can be located in
the cytoplasm (PEPCK), mitochondria (NAD-ME) or chloroplast
(NADP-ME). Different types of C4 plants have co-opted alternative
decarboxylases for this purpose. The bundle sheath cells of C4
species are often enlarged, with thick cells walls and abundant
chloroplasts compared with C3 species.
The remaining three-carbon backbone derived from the decarboxylated organic acid diffuses back to the mesophyll cells. In NADP-ME
and NAD-ME subtypes, pyruvate is rephosphorylated to produce
phoshoenolpyruvate (PEP) to enable the cycle to continue. Phosphorylation of pyruvate is catalysed by pyruvate orthophosphate
dikinase (PPDK) (5). PPDK regenerates the primary CO2 acceptor so that
the C4 cycle can continue.
In Bienertia cycloptera and Borzcscowia aralocaspica, the
C4-concentrating mechanism takes place in individual cells [6,7]. In
both cases this is because of polarity in the biochemical steps. For

photosynthesis and the models that have been used to

gain this knowledge.
Current understanding of C4 photosynthesis
Biochemistry of C4 photosynthesis
Several genera, including Atriplex, Amaranthus, Eleocharis, Flaveria, Sorghum and Zea, have been used to
study C4 photosynthesis [1622]. Much of the biochemistry needed for C4 photosynthesis was determined
using sorghum, sugarcane and maize. For example,
work with tropical grasses indicated that phosphoenolpyruvate carboxylase (PEPC) catalyses the first committed step of C4 photosynthesis [23]. More recently, its
reaction mechanism and structure have been determined [24] and comparative analysis of PEPC in C3 and
C4 species has provided insights into the evolution of the
protein and the regulation of the gene in C4 species. In
Flaveria, PEPC proteins from C3 and C4 species are 96%
identical at the amino acid level but their kinetics are
considerably different. For example, the Km for PEP in
C4 species is ten times higher than that in C3 species. In
addition, the C4 enzyme is more sensitive to activation
by glucose-6-phosphate, and has a lower sensitivity to
inhibition by malate [25]. Domain swapping has shown
that a 141 amino acid section towards the middle of the
protein and a single alanine-to-serine substitution at
position 774 are responsible for two-thirds of the C4
kinetics [2627]. These amino acids comprise !15% of
the entire protein.
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CO2

Mesophyll cell
HCO3

OAA

2
PEP

Malate

Pyruvate

4
CO2
Rubisco

Bundle sheath cell

TRENDS in Plant Science

Figure I.

example, in the case of B. aralocaspica, PEPC is found at the cell

periphery, whereas NAD-ME and Rubisco are located in a central
cytoplasmic compartment separated from the peripheral cytoplasm by
a large vacuole.

Substantial progress has also been made in determining the mechanisms responsible for PEPC accumulating
specifically in mesophyll cells of C4 species. In Flaveria,
regions of the PEPC promoter responsible for high levels
of expression of the uidA reporter in mesophyll cells have
been determined. Two regions, one distal and one
proximal to the translational start were identified. In
the distal region, a 41 bp segment that contains the
tetranucleotide sequence CACT is present in C4 but not in
C3 species [19]. In maize, PEPC accumulation appears to
be controlled at the level of transcription via differential
methylation of the promoter in mesophyll and bundle
sheath cells [28]. In the bundle sheath, exposure to light
induces demethylation of the promoter, which coincides
with transcript accumulation. Whereas in the mesophyll,
demethylation does not occur in the light and transcripts
do not accumulate [28].
PEPC allows four carbon organic acids to be produced
that then diffuse from the mesophyll to the bundle sheath
cells where decarboxylase enzymes release CO2 around
Rubisco. There are three decarboxylases, of which the best
characterized in C4 plants with respect to gene regulation
is NADP-dependent malic enzyme (NADP-ME). In C4
species of Flaveria, elements within the promoter control
expression in the bundle sheath, whereas elements near
the 5 0 end of the coding region and sequences within the 3 0
UTR regulate the amount of expression [2930].
One crucial feature of C4 photosynthesis is that the
accumulation of Rubisco is restricted to the bundle sheath;

Opinion

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Vol.10 No.5 May 2005

217

Ceratophyllales
Laurales
Magnoliales
Piperales

Angiosperms

Eudicots

Monocots (3)
Ranunculales
Proteales
Carophyllales (8)
Santales
Saxifragales
Gereniales

Eurosids I (2)
Eurosids II (1)

Capparoideae
Cleomoideae

Capparaceae

Brassicaceae
Cornales
Ericales

Euasterids I (3)
Euasterids II (1)
TRENDS in Plant Science

Figure 1. The evolution of C4 photosynthesis. Molecular phylogeny of the angiosperms: the major clades where C4 photosynthesis has evolved are highlighted in purple. The
phylogeny combines those from the angiosperm phylogeny group [49] with that of the Capparaceae [50]. Numbers in parentheses indicate the number of families in each
clade that contain C4 species. Eurosids II contains the Brassicaceae and the sister group Cleomoideae (originally classified as being within the Capparaceae), which contains
C4 species such as Cleome gynandra.

evidence indicates that both transcriptional and posttranscriptional mechanisms are important for this. In
maize, 238 bp, including the last 14 nucleotides that are
translated and the 3 0 UTR, combined with 463 bp in the
promoter act to repress RbcS expression in mesophyll cells
after transfer to the light [31]. However, there is also
evidence from run-on assays that transcription occurs in
mesophyll and bundle sheath protoplasts [32]. In addition,
post-transcriptional regulation of RbcS is important in the
C4 Amaranthus hirta. 5 0 and 3 0 UTRs from AhRbcS1A
enhance translation of the uidA transcript in the C4
species Flaveria bidentis and the C3 plant tobacco; in
F. bidentis, this leads to preferential accumulation of GUS
in the bundle sheath [17].
After CO2 is released around Rubisco, PEP, the primary
acceptor for bicarbonate in the mesophyll, is regenerated
to maintain the C4 cycle. In NADP-ME and NAD-ME
types, pyruvate orthophosphate dikinase (PPDK) is
needed to regenerate PEP from pyruvate. Studies with
rice and maize have provided most of our knowledge
regarding the regulation of PPDK. For example, in maize,
a single gene codes for the C4-specific isoform of PPDK, but
this gene possesses two promoters and the two different
transcripts generated result in two distinct proteins [33].
The first promoter, upstream of exon 1, generates the
longer C4 form of the protein, which possesses a transit
peptide that targets it to chloroplasts, whereas the second
promoter is found within the first intron and produces a
shorter cytosolic protein [33]. The same PPDK gene
structure is found in C3 species [34]. Elements within
the PPDK promoter direct expression in the mesophyll: gel
retardation assays, as well as nuclease protection, have
shown that a region of 27 nucleotides that is w300 bp
upstream from the translation start binds a trans-acting
factor that is likely to be responsible for mesophyll
expression [35].
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Comparative analysis of species such as maize and rice

or of Flaveria bidentis (C4) and Flaveria pringlei (C3) have
been useful in elucidating genetic alterations associated
with C4 photosynthesis. Analyses of promoter elements
from maize and Flaveria show that they direct expression
patterns in the same way that they do in C4 photosynthesis when placed in closely related C3 species [19,36,37].
For example, the promoter of PEPC from Flaveria
trinervia (C4) directs mesophyll-specific expression in
tobacco (C3). In addition, when intact genes from maize
are placed in rice, they are not only expressed in the same
cell-types as they would be in maize but are also expressed
at high levels [38,39]. Indeed, some of the biochemical
characteristics of C4 photosynthesis are already present in
C3 species [40], indicating that some cell-types of C3
species are able to accumulate large amounts of the
enzymes needed for C4 photosynthesis, which suggests
that the correct regulatory mechanisms for high level
expression of these genes are already present.
Development of C4 leaf anatomy and ultrastructure
Leaf development in C4 species is significantly different
from that in C3 species (Figure 2). Key alterations to C4
leaf structure include: thinner leaves, proliferation of
veins, wreaths of mesophyll cells around each bundle
sheath, abundant plasmodesmata linking mesophyll and
bundle sheath, inhibition of chloroplast development in
the mesophyll, enlarged bundle sheath cells with thick cell
walls and specific development of chloroplasts within the
bundle sheath. Significant progress has been made in
elucidating the genetic basis of the specific development of
chloroplasts within the bundle sheath of C4 leaf
development.
Analysis of sector development and comparisons of
normal maize leaves (C4) and husk leaves (C3) have shown
that positional information (rather than cell lineage) is

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(a)

Vol.10 No.5 May 2005

(b)

(c)
(d)

(e)

(f)

(g)

(h)

Figure 2. Growth habit and leaf characteristics of C4 (ad,f,h) and C3 (e,g) species of Cleome. (a) Seedling of Cleome gynandra (C4) illustrating its growth habit, (b) Flowering
spikelet of C. gynandra showing that multiple flower heads and seedpods are formed in a similar manner to Arabidopsis. (c,d) Mature and developing seedpod from
C. gynandra showing that many seeds are produced per pod. (e,f) Confocal images of transverse sections of mature leaves of the C3 species Cleome spinosa (e) and the C4
species C. gynandra (f), which shows the C4 anatomy. Note the large bundle sheath cells and low interveinal distance in C. gynandra. Veins are marked by red asterisks. (g,h)
Venation patterns in leaves of C. spinosa (g) and C. gyndandra (h). Note the proliferation of veins in the C4 species. Scale bars: (a,b)Z2 cm; (c,d)Z1 cm; (e,f)Z40 mm; (g,h)Z
200 mm.

crucial for the development of mesophyll and bundle

sheath cells [22,41]. It is thought that the vascular system
provides the information that controls the fate of bundle
sheath and mesophyll cells, although to date we do not
understand what these signals might be.
The bundle sheath defective mutants (bsd) have
provided key insights into processes regulating the
development of chloroplasts in the bundle sheath. Two
have been studied in detail. The bsd2 mutant possesses
swollen bundle sheath chloroplasts with little internal
membrane structure, whereas mesophyll chloroplasts
appear normal [42]. In bsd2 mutants, Rubisco protein
does not accumulate but large amounts of rbcL transcript
are associated with polysomes in both mesophyll and
bundle sheath cells. This is consistent with the wild-type
Bsd2 gene product being involved in the post-translational
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regulation of rbcL. In the mutant, failure of the mature

polypeptide to be released from ribosomes would inhibit
assembly of the Rubisco holoprotein and lead to accumulation of active O2 species in the chloroplast, which in turn
would destroy thylakoid membranes [43]. The bsd1
mutant, now known as golden2, has pale leaves: the
bundle sheath etioplasts contain fewer lamellae and
bundle sheath chloroplasts are smaller than the wild
type [44]. golden2 contains a mutation in a gene that codes
for a transcriptional activator that is a member of the
GARP family of transcription factors [45]. All angiosperms
investigated to date contain more than one Golden gene,
and so related members are known as Golden-like (GLK).
In Arabidopsis and rice, Golden-like genes have overlapping expression patterns and there is redundancy in
protein function [46,47]. Golden2 protein is thought to be

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TRENDS in Plant Science

involved in assembly or stabilization of Photosystem II. In

Arabidopsis, mutants lacking GLK2 possess normal
amounts of psbA transcript but no D1 protein, and the
transcript abundance of LHCB genes is lower. It therefore
appears that in maize, G2 has specifically been co-opted to
control the assembly of Photosystem II in bundle sheath
chloroplasts.
Overall, these mutants have provided key insights into
processes governing chloroplast development in bundle
sheath cells. However, much of the genetic basis for the
development of C4 leaves remains elusive.
Areas where progress is still needed
Because C4 photosynthesis has evolved many times, and
various lineages of C4 plants possess different anatomical
and biochemical arrangements that all result in the
concentration of CO2 around Rubisco [5], to understand
each of these mechanisms fully, detailed analysis would
need to be conducted in representative species from each
major lineage. However, there are phenotypes shared by
many of the C4 variants that remain poorly understood. For
example, little is known about the genetic basis of
chloroplast division and expansion within the bundle
sheath, the development of bundle sheath cells themselves,
the limited division and development of mesophyll chloroplasts, the proliferation of plasmodesmata between mesophyll and bundle sheath cells, the increased venation, and
the reduction in the thickness of C4 leaves (Figure 2).
The main reason for these significant gaps in our
understanding of C4 photosynthesis is probably the lack of
a genetically tractable model species with a short life

Vol.10 No.5 May 2005

219

cycle. Each of the models used to date has advantages and

disadvantages (Table 1). Maize is associated with the most
genetic resources and it has been immensely useful but its
large genome, large size and relatively long life span have
restricted progress. Because the life cycles of rice and
maize are relatively long, the time needed for comparative
experiments is therefore lengthened. Flaveria suffers from
the same drawbacks in terms of life cycle. In addition,
there are no internationally available genetic resources
associated with Flaveria and it is only distantly related
(Figure 1) to the two species of angiosperm that have had
their genomes sequenced, Arabidopsis thaliana and rice
(Oryza sativa). The phylogenetic distance between Flaveria and Arabidopsis complicates analysis. For example,
when the PEPC promoter from F. trinervia is placed in
Arabidopsis, mesophyll specificity is lost, even though in
tobacco it is maintained (P. Westhoff, personal communication). Flaveria and tobacco are in the Asterid clade
whereas Arabidopsis belongs to the Rosid clade, therefore
it seems likely that the phylogenetic distance does not
allow the regulatory elements associated with C4 photosynthesis to operate in Arabidopsis. An alternative
approach would be to use genetic resources and information from Arabidopsis to inform and therefore accelerate our understanding of C4 photosynthesis.
Proposal to use Arabidopsis and Cleome to determine
the genetic basis of C4 photosynthesis
Although Arabidopsis does not use C4 photosynthesis, we
argue that it can still be used to provide key insights into
how C4 photosynthesis has evolved. To understand how C4

Table 1. Advantages and disadvantages associated with the main genera (including Cleome) that have been used to study C4
photosynthesis and are therefore potential candidates for determining its genetic basis
Generaa
Amaranthus
Atriplex
Borszczowia

Cleome

Eleocharis

Flaveria

Sorghum
Zea

Advantages
Biochemical and molecular work conducted; contains C3 and
C4 species
Biochemical and genetic work conducted; contains C3 and C4
species; hybridization between species has been reported
Represents the single-celled C4 photosynthesis pathway;
involves fascinating mechanisms to target proteins to
specific populations of chloroplasts in single cells
Closely related to Arabidopsis; contains C3 and C4 species;
life cycle relatively short; potential for use of Arabidopsis
microarrays; insights from Arabidopsis should allow a
candidate gene approach and transfer of knowledge to the
maizerice system
Eleocharis vivipora is able to switch between C3 and C4

Biochemical, anatomical and developmental work

conducted; one species transformable; contains C3, C4 and
C3C4 intermediates; has provided key insights into the
regulation of PEPC
Used for much of early biochemical work
Biochemical, anatomical and developmental work conducted; large amount of genome sequence and mutant
resources; of major agronomic importance; phylogenetically
close to rice; intact maize genes are expressed in a C4
manner in rice

Disadvantages
Member of the Caryophyllales; few genetic resources and
currently not transformable
Member of the Caryophyllales; no genetic resources and
currently not transformable; little recent C4 work
Member of the Caryophyllales; no genetic resources and
currently not transformable; possesses few of the
developmental traits of C4 photosynthesis found in the
classical pathway
Currently no genetic resources, not transformable and no
mutants

Member of the Cyperaceae (i.e. not particularly close

phylogenetically to either Arabidopsis or the Oryzoideae);
no genetic resources and currently not transformable
Member of the Caryophyllales; no recognized genetic
resources; life cycle relatively long

Large plant with relatively large genome; genetic resources

that exist not being used for photosynthesis research
Large plant with large genome; life cycle relatively long;
although work with rice should inform maize research,
progress is slower than that with Arabidopsis

Given allocation of sufficient resources, all these genera could be used as models and therefore would shed additional light on the genetic basis of C4 photosynthesis.
However, unlimited resources are unlikely and so it is pertinent to ask which of these groups might provide the greatest gains in the shortest time. We consider using genera
that are in the Caryophyllales a disadvantage because of their phylogenetic distance from Arabidopsis. If the aim is to transfer the C4 pathway into rice, then all the dicot
genera have the drawback that understanding mechanisms in dicots might not translate directly into understanding the same processes in monocots.

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photosynthesis has evolved, we need to determine how

genes that have been co-opted into C4 photosynthesis are
regulated in C3 species, and to elucidate the exact role of
each protein in C3 plants. Because of the resources
associated with Arabidopsis, this should be a relatively
rapid process. A recent example is where the role of the
decarboxylase PEPCK has been investigated in Arabidopsis; PEPCK appears to have been recruited into C4
photosynthesis from a role in mobilizing lipid reserves
from cotyledons during post-germinative growth [48]. We
therefore argue that analysis of Arabidopsis could provide
key information relating to how the regulation of genes
and the function of proteins have altered as they have
been co-opted into the C4 pathway.
This logic led us to consider the feasibility of using the
most closely related C4 species to Arabidopsis for
comparative work. Within the angiosperms, Arabidopsis
is a member of the Eurosids II clade [49], which also
includes a genus of plants containing C4 species (Figure 1).
This genus is Cleome, traditionally part of the Capparaceae, but recent analysis led to the proposal that the
Capparaceae should be separated into two clades, the
subfamilies Capparoideae and Cleomoideae. This analysis
also indicates that the Cleomoideae and the Brassicaceae
are monophyletic, therefore the Cleomoideae and the
Brassicaceae are sister clades [50]. There are three known
C4 species of Cleome [51] and of these Cleome gynandra
has been the most studied [5254]. C. gynandra is
relatively small and its life cycle is relatively short, it is
self-fertile and produces plentiful amounts of seed
(Figure 2). Therefore, there are several potential advantages associated with using Cleome to study the genetic
basis of C4 photosynthesis compared with the other genera
that have been used as models to date (Table 1). In some
areas of the world, C. gynandra is used as a crop, producing
nutritious leaves with medicinal properties [55].
Because of the degree of relatedness of Arabidopsis and
Cleome, it should be possible for us to use many of the tools
and advantages associated with Arabidopsis. For
example, Arabidopsis genes known to be involved in
aspects of leaf development needed for C4 photosynthesis
could be used to clone homologues from Cleome and to
investigate whether alterations in their expression profiles or changes to interacting partners have occurred in
C4 species. Given the precedence from work with maize
and rice, and with Flaveria and tobacco, and because
Arabidopsis and Cleome are relatively closely related, it
seems likely that regulatory elements from Arabidopsis
will recognize genes from C. gynandra when they are
placed in Arabidopsis. If, for example, a promoter to a key
gene for the C4 pathway determines bundle sheath
expression in Arabidopsis as well as in Cleome, promoter
deletion analysis and isolation of trans-acting factors
would be relatively simple if Arabidopsis was used as a
fast throughput system to analyse patterns of expression.
Because gene sequences of Arabidopsis and Cleome are
relatively similar, Arabidopsis microarrays could be used
to monitor changes in global gene expression at key stages
of C4 leaf development. Analysis of candidate genes for
involvement in crucial components of the C4 pathway
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Vol.10 No.5 May 2005

could first be screened in Arabidopsis via knockout

mutants or similar resources.
For the potential of Cleome to be maximized, key
resources that are shared between laboratories working
on the genetic basis and evolution of C4 photosynthesis
should be generated. Resources should include:
A molecular phylogeny of the genus.
A repository of seed for as many species of Cleome as
is practical.
Accessible microarray databases so that laboratories
can share and make use of the large amount of data
concerning transcript abundance generated from
arrays.
cDNA libraries so that candidate genes for key C4
processes can be rapidly cloned using homologues
from Arabidopsis as probes.
A mutant resource so that more genes controlling
developmental aspects of C4 photosynthesis can be
isolated and basic genetic analysis undertaken.
Genetic markers and a mapping population to allow
mutant genes to be isolated.
A transformation system so that mechanisms regulating gene expression in a C4 species of Cleome can
be rapidly determined.
If these resources can be generated and work with
Arabidopsis and Cleome is used to inform our understanding of C4 photosynthesis in monocot systems (much
as Arabidopsis has been used for work with cereals),
Cleome offers the potential to revolutionize our understanding of how crucial processes within the efficient C4
photosynthesis pathway work and how the C4 photosynthesis pathway evolved.
Acknowledgements
We are grateful to Susan Stanley for technical support, Rowan Sage, John
Gray and Howard Griffiths for advice, and Elisabeth Truernit for confocal
expertise. We thank the BBSRC, the Royal Society and The Gatsby
Charitable Foundation for funding.

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11 Brown, H.A. (1999) Agronomic implications of C4 photosynthesis. In

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