Cold stress

In this section, we have emphasized on various aspects of cold stress, which includes aVect of
cold on plants physiology, cold acclimation and its role in providing freeze-tolerance, function of
cold-regulated genes in cold acclimation, negative regulation of cold stress and the role of
calcium in relation to cold stress. All these topics would help in our better understanding of cold
induced cellular changes and its aVect on gene expression.

Affect of cold on plants physiology

Each plant has its unique set of temperature requirements, which are optimum for its proper
growth and development. A set of temperature conditions, which are optimum for one plant may
be stressful for another plant. Many plants, especially those, which are native to warm habitat,
exhibit symptoms of injury when exposed to low non-freezing temperatures [3]. These plants
including maize (Zea mays), soybean (Glycine max), cotton (Gossypium hirsutum), tomato (Lycopersicon
esculentum) and banana (Musa sp.) are in particular sensitive to temperatures below 10-15 C and
exhibit signs of injury see [3-5]. The symptoms of stress induced injury in these plants appear
from 48 to 72 h, however, this duration varies from plant to plant and also depend upon the
sensitivity of a plant to cold stress. Various phenotypic symptoms in response to chilling stress
include reduced leaf expansion, wilting, chlorosis (yellowing of leaves) and may lead to necrosis
(death of tissue). Chilling also severely hampers the reproductive development of plants for
example exposure of rice plants to chilling temperature at the time of anthesis (floral opening)
leads to sterility in flowers [6].
The major malicious eVect of freezing is that it induces severe membrane damage [7,8]. This
damage is largely due to the acute dehydration associated with freezing. Membrane lipids are
primarily composed of two kinds of fatty acids unsaturated as well as saturated fatty acids.
Unsaturated fatty acids have one or more double bonds between two carbon atoms (CH=CH
) whereas saturated fatty acids are fully saturated with hydrogen atoms ( CH2CH2). It is a
well-known fact that lipids containing saturated fatty acids solidify at temperatures higher than
those containing unsaturated fatty acids. Therefore, the relative proportion of unsaturated fatty
acids in the membrane strongly influences the fluidity of the membrane [8]. The temperature at
which a membrane changes from semi fluid state to a semi crystalline state is known as the
transition temperature. Chilling sensitive plants usually have a higher proportion of saturated
fatty acids and, therefore, a higher transition temperature. Chilling resistant species on the other
hand are marked by higher proportion of unsaturated fatty acids and correspondingly a lower
transition temperature.
The success of many crops rests on their ability to withstand the freezing temperature of late
spring or early autumn frost. Therefore tolerance to freezing temperatures is in particular
important for the sustainability of agricultural crops. As understanding the basics of a disease is
essential for its cure, in the same way understanding of how freezing induces its injurious effects
on plants is essential for the development of frost tolerant crops. The real cause of freeze-induced
injury to plants is the ice formation rather than low temperatures. It is noteworthy to mention
here that dehydrated tissues such as seeds and fungal spores can survive at very low temperatures
without any symptoms of injury. Even cryopreservation is a common method for storage of
seeds and other biological materials, which is based on the fact that water essentially solidifies
without the formation of ice crystals.
Ice formation in plants, begins in the apoplastic space as it has relatively lower solute
concentration. As the vapor pressure of ice is much lower than water at any given temperature,
ice formation in the apoplast establishes a vapor pressure gradient between the apoplast and
surrounding cells. The unfrozen cytoplasmic water migrates down the gradient from the cell
cytosol to the apoplast, which contributes to the enlargement of existing ice crystals and causes a
mechanical strain on the cell wall and plasma membrane leading to cell rupture [9,10]. Freeze
induced cellular dehydration results in multiple forms of membrane damage including
expansion-induced-cell lyses and fracture lesions [8,11] and lamellar-to-hexagonal-II phase
transition. Although freeze exerts its effect largely by membrane damage due to severe cellular
dehydration, certain additional factors may also contribute to damage induced by freeze.
ROS produced in response to freeze stress contributes to membrane damage. Chilling
sensitive plants characteristically exhibits structural injuries and may suffer from metabolic
dysfunction when chilled [12]. Overall, chilling ultimately results in loss in membrane integrity,
which leads to solute leakage. The integrity of intracellular organelles is also disrupted leading to
the loss of compartmentalization, reduction and impairing of photosynthesis, protein assembly
and general metabolic processes. The primary environmental factors responsible for triggering
increased tolerance against freezing, is the phenomenon known as cold acclimation. It is the
process where certain plants increase their freezing tolerance upon prior exposure to low non-
freezing temperatures.
Cold acclimation and its role in providing freeze-tolerance

The primary function of cold acclimation is to stabilize the membranes against freeze injury.
Acclimation results in increase in proportion of unsaturated fatty acids and thereby a drop in
transition temperature [13,14]. It functions to prevent the expansion-induced lyses and formation
of hexagonal II phase lipids in rye and other plants [8,11].
Cold acclimation results in physical and biochemical restructuring of cell membranes through
changes in the lipid composition and induction of other non-enzymatic proteins that alter the
freezing point of water. Addition of solutes decreases the freezing point of water to a more neg-
ative value, thus preventing ice formation.
Low temperatures induce a number of alterations in cellular components, including the extent
of unsaturated fatty acids [15], the composition of glycerolipids [16], changes in protein and
carbohydrate composition and the activation of ion channels [17]. Accumulation of sucrose and
other simple sugars that occurs with cold acclimation also contributes to the stabilization of
membrane as these molecules can protect membranes against freeze-damage. Freezing tolerance
is a multigenic trait. Low temperatures activate a number of cold-inducible genes [18], such as
those that encode dehydrins, lipid transfer proteins, translation elongation factors and the late-
embryogenesis-abundant proteins [19]. Moreover, intercellular ice formation can cause a
mechanical strain on cell wall and membrane leading to cell rupture [9,10]. There is also
substantiation that protein denaturation occurs in plants at low temperature which could also
result in cellular damage [20].
Overall, cold acclimation results in protection and stabilization of the integrity of cellular
membranes, enhancement of the antioxidative mechanisms, increased intercellular sugar levels
as well as accumulation of other cryoprotec- tants including polyamines that protect the
intracellular proteins by inducing the genes encoding molecular chaperones [21]. All these
modifications help the plant to withstand and surpass the severe dehydration associated with
freezing stress.
Function of cold-regulated genes in cold acclimation

Considerable efforts have been directed towards determining the nature of cold-inducible
genes and establishing their role in freezing tolerance. The Arabidopsis FAD8 gene [22] encodes a
fatty acid desaturase that contributes to freezing tolerance by altering the lipid composition.
Cold-responsive genes encoding molecular chaperones including a spinach hsp70 gene [23],
and a Brassica napus hsp90 gene [24], contribute to freezing tolerance by stabilizing proteins against
freeze-induced denaturation. Many cold-responsive genes encoding various signal transduction
and regulatory proteins have been identified and this list includes the mitogen-activated protein
(MAP) kinase [25], MAP kinase, kinase, kinase (MAPKKK) [26] and the calmodulin-related
proteins [27]. These proteins might contribute to freezing tolerance as well as tolerance to other
stresses by controlling or regulating the expression and activity of the major stress genes as well
their proteins.
The largest class of cold induced genes encodes polypeptides that are homologs of LEA
proteins and the polypeptides that are synthesized during the late embryogenesis phase, just prior
to seed desiccation and also in the seedlings in response to dehydration stress [28-30]. These LEA
like proteins are mainly hydrophilic, many have relatively simple amino-acid composition, and
are composed largely of a few amino acids with repeated amino acid sequence motifs. Many of
these proteins are predicted to contain regions capable of forming amphipathic a helices. The
examples of cold responsive genes include: COR15a, [31], alfalfa Cas15 [32], and wheat WCS120
[33]. The expression of COR genes has been shown to be critical for both chilling tolerance and
cold acclimation in plants [34]. Arabidop- sis COR genes include: COR78/RD29, COR47, COR15a,
COR6.6 and encode LEA like proteins [34]. These genes are induced by cold, dehydration or
ABA. COR15A polypeptide is targeted to the chloroplast. Formation of hexagonal II phase lipids
is a major cause of membrane damage in non-acclimated plants. COR 15a expression decreases
the propensity of the membranes to form hexagonal II phase lipids in response to freezing [8,11].
The analysis of the promoter elements of COR genes revealed that they contain DRE
(dehydration responsive elements) or CRT (C-repeats) and some of them contain ABRE (ABA-
responsive element) as well [35,36]. Induction of the COR genes was accomplished by over-
expression of transcription factor CBF (CRT/DRE binding factor) [36]. CBF binds to the
CRT/DRE elements present in the promoter of the COR genes and other cold-regulated genes.
The over-expression of these regulatory elements not only resulted in increased freezing
tolerance but also an increase to drought tolerance [37]. This finding provides strong support that
a fundamental role of cold-inducible genes is to protect the plant cells against cellular
dehydration. Lee et al. [38] genetically analyzed HOS1 (high expression of osmotically responsive
genes) locus of Arabidopsis. The hos1 mutation resulted in sustained and super induction of CBF2,
CBF3 and their target regulatory genes during cold stress. Therefore, HOS1 was identified as a
negative regulator of COR genes by modulating the expression level of CBFs. [39]. HOS1 gene
encodes a ring finger protein and is constitutively expressed but gets drastically down-regulated
within 10 min of cold stress. Genetic analysis led to the identification of ICE1 (inducer of CBF
expression 1) as an activator of CBF3 [39]. ICE1 encoded a transcription factor that specifically
recognized MYC sequence on the CBF3 promoter. Transgenic lines overexpressing ICE1 did not
express CBF3 at warm temperature but showed a higher level of expression for CBF3 as well as
RD29 and COR15a at low temperatures. This study suggests that cold induced modification of ICE1
is necessary for it to act as an activator of CBF3 in planta.
Recently two CBF1-like cDNAs CaCBFIA and CaCB- FIB have been cloned and characterized
[40] from hot pepper. These were induced in response to low temperature stress (4C) and not in
response to wounding or ABA. Two-hybrid screening led to the isolation of a homeodo- main
leucine zipper (4D-Zip) protein that interacts with CaCBFIB. The expression of 4D-Zip was
elevated by low temperature and drought [40]. Calcium-dependent protein kinases (CDPKs) play
an important role in the signal transduction and recently the function of OsCDPK13 (Oryza sativa
CDPK 13) has been characterized [41]. The gene expression as well as protein accumulation of
OsCDPK13 were up-regulated in response to cold and gibberellin (GA) but suppressed under
salt and drought stress and also in response to ABA. The overexpressing transgenic lines of
OsCDPK13 had higher recovery rates following cold stress in comparison with the vector
control rice. Cold-tolerant rice varieties exhibited higher expression of OsCDPK13 than the cold
sensitive ones. Antisense OsCDPK13 transgenic lines were shorter in comparison with the vector
control lines. Moreover, dwarf mutants of rice also had lower level of OsCDPK13 than in wild
type [41]. However, there has been no mention of the sensitivity of OsCDPK13 antisense lines in
response to cold stress [41]. We however expect that these antisense lines should be
hypersensitive to cold stress as the gene has been shown to play an important role in mediating
tolerance in response to cold stress which is evident due to higher recovery rates following cold
stress than the vector control lines.
Negative regulation of cold stress

Mutagenesis study resulted in the identification of a gene, eskimo l (esk1), which has a major
effect on freezing tolerance. These plants were more freeze tolerant than the wild type plants
without cold acclimation. The concentration of free proline [42] in the esk1 mutant was found to
be 30-fold higher than in the wild-type plants. Proline has been shown to be an effective
cryoprotectant and this is also one of the major factors imparting freezing tolerance. In addition
to the total sugars, which were elevated, the expression of RAB18 cold-responsive LEA II gene was
also found to be elevated three fold. This suggests that ESK1 may act as a negative regulator.
Significantly, the esk 1 mutation did not appear to affect the expression of COR genes. This
suggests that multiple signaling pathways are involved in response to cold stress and they may
cross talk with each other as well as with genes involved in other stresses.
Role of calcium in relation to cold stress

Calcium is an important messenger in a low temperature signal transduction pathway. The

change in cytosolic calcium levels is a necessary first step in a temperature sensing mechanism,
which enables the plant to withstand future cold stress in a better way. In both Arabidopsis [17,27]
and alfalfa [43] cytoplasmic calcium levels increase rapidly in response to low temperature,
largely due to an influx of calcium from extracellular stores. Through the use of pharmacological
and chemical reagents, it has been demonstrated that calcium is required for the full expression
of some of the cold induced genes including the CRT/DRE controlled COR6 and KIN1 genes of
Arabidopsis [17,32,43]. For example, Ca2+ chelators such as BAPTA and Ca2+ channel blockers
such as La3+ inhibited the cold-induced influx of calcium and resulted in the decreased
expression of the cold inducible Cas15 gene and blocked the ability of alfalfa to acclimate in cold.
In addition Cas15 expression can be induced at a much higher temperature, i.e., 25 C by treating
the cells with A23187, a Ca2+ ionophore that causes a rapid influx of calcium [43].

Salinity stress

Salinity is a major environmental stress and is a substantial constraint to crop production.

Increased salinization of arable land is expected to have devastating global effects, resulting in
30% land loss within next 25 years and up to 50% by the middle of 21st century [44]. High
salinity causes both hyperionic and hyperosmotic stress and can lead to plant demise. Sea water
contains approximately 3% of NaCl and in terms of molarity of different ions, Na+ is about 460
mM, Mg2+ is 50mM and CE around 540 mM along with smaller quantities of other ions. Salinity
in a given land area depends upon various factors like amount of evaporation (leading to increase
in salt concentration), or the amount of precipitation (leading to decrease in salt concentration).
Weathering of rocks also affects salt concentration. Inland deserts are marked by high salinity as
the rate of evaporation far exceeds the rate of precipitation. Agricultural lands that have been
heavily irrigated are highly saline. As drier areas in particular need intense irrigation, there is
extensive water loss through a combination of both evaporation as well as transpiration. This
process is known as evapotranspiration and as a result, the salt delivered along with the irrigation
water gets concentrated, year-by-year in the soil. This leads to huge losses in terms of arable land
and productivity as most of the economically important crop species are very sensitive to soil
salinity. These salt sensitive plants, also known as glycophytes include rice (Oryza sativa), maize
(Zea mays), soybean (Glycine max) and beans (Phaseolus vulgaris). High salt concentration (Na+) in
particular which deposit in the soil can alter the basic texture of the soil resulting in decreased
soil porosity and consequently reduced soil aeration and water conductance. The basic
physiology of high salt stress and drought stress overlaps with each other. High salt depositions
in the soil generate a low water potential zone in the soil making it increasingly difficult for the
plant to acquire both water as well as nutrients. Therefore, salt stress essentially results in a water
deficit condition in the plant and takes the form of a physiological drought. The major ions
involved in salt stress signaling, include Na+, K+, H+ and Ca2+. It is the interplay of these ions,
which brings homeostasis in the cell.
In this section, we have emphasized on various aspects of salinity stress, which includes the
reasons why salinity stress is injurious to plant cells, generic function of K+, role of Ca2+ and
SOS pathway in relation to imparting salt stress tolerance, loss of water due to salinity stress and
the role of glycine betaine as a major osmolyte. Moreover, the role of DNA unwinding enzymes,
i.e., helicases, imparting salinity stress tolerance have also been discussed.
Maladies caused by salt stress on plant cells arise from the following
 (1) Disruption of ionic equilibrium: Influx of Na+ dissipates the membrane potential and
facilitates the uptake of CE down the chemical gradient.
(2) Na+ is toxic to cell metabolism and has deleterious effect on the functioning of some of the
enzymes [45].
(3) High concentrations of Na+ causes osmotic imbalance, membrane disorganization,
reduction in growth, inhibition of cell division and expansion.
(4) High Na+ levels also lead to reduction in photosynthesis and production of reactive oxygen
species [4648].
Where sodium (Na+) is deleterious for plant growth, K+ is one of the essential elements and is
required by the plant in large quantities.

Generic functions of K+

(1) K+ is required for maintaining the osmotic balance.

(2) K+ has a role in opening and closing of stomata.
(3) K+ is an essential co-factor for many enzymes like the pyruvate kinase, whereas Na+ is not.

Movement of salt into roots and to shoots is a product of the transpirational flux required to
maintain the water status of the plant [48,49]. As common proteins transport Na+ and K+, Na+
competes with K+ for intracellular influx [45,50,51]. Many K+ transport systems have some
affinity for Na+, i.e., Na+/K+ symporters. Thus external Na+ negatively impacts intracellular K+
influx. Most cells maintain relatively high K+ and low concentrations of Na+ in the cytosol. This
is achieved through a coordinated regulation of transporters for H+, K+, Ca2+ and Na+.
The plasma membrane H+-ATPases serves as the primary pump that generates a proton
motive force driving the transport of other solutes including Na+ and K+. Increased ATPase-
mediated H+ translocation across the plasma membrane is a component of the plant cell response
to salt imposition [52,53]. K+ and Na+ influx can be differentiated physiologically into two
categories, one with high affinity for K+ over Na+ and the other for which there is lower K+/Na+
selectivity. The Na+/K+ transporter and K+ transporters with dual high and low affinity may
contribute substantially to Na+ influx.
Role of Ca2+ in relation to salt stress

For decades it has been shown that another ion, Ca2+ has role in providing salt tolerance to
plant. Externally supplied Ca2+ reduces the toxic effects of NaCl, presumably by facilitating
higher K+/Na+ selectivity [54-56]. High salinity results in increased cytosolic Ca2+ that is
transported from the apoplast as well as the intracellular compartments [57]. This transient
increase in cytosolic
Ca2+ initiates the stress signal transduction leading to salt adaptation.
The search to identify genes involved in providing salt tolerance commenced in 1998, by Liu
and Zhu [56] where several mutants were screened and SOS (salt overly sensitive) genes were
identified through positional cloning. Briefly, SOS pathway results in the exclusion of excess Na+
ions out of the cell via the plasma membrane Na+/H+ antiporter and helps in reinstating cellular
ion homeostasis. The discovery of SOS genes paved the way for elucidation of a novel pathway
linking the Ca2+ signaling in response to a salt stress [58,59].
SOS3 gene encodes a Ca2+ binding protein with 4 EF hand Ca2+ binding motifs and a
myristoylation sequence (MGXXXST/K) at the N-terminus of the protein. In response to Ca2+
perturbation SOS3 changes its conformation and transduces the signal downstream by interacting
with an effector kinase. Mutation in SOS3 (sos 3-1), which results in the reduction of its Ca2+
binding ability also impairs the cellular ionic equilibrium and renders the plant hypersensitive to
salt stress [60]. This defect can be partially rescued by addition of high levels of Ca2+ in the
growth medium [56]. Ca2+ sensors differ in their affinity with which they bind Ca2+ and this
difference is an important parameter in distinguishing and decoding various Ca2+ sensors. In
comparison with Ca2+ sensors like calmodulin and caltrac- tin, SOS3 binds Ca2+ with a
relatively low affinity.
SOS2 gene was isolated through the genetic screening of mutants oversensitive to salt stress
in Arabidopsis. The mRNA level of SOS2 was shown to be up-regulated in response to salt stress
in the roots [61]. SOS2/CIPK24 encodes a novel serine/threonine protein kinase with an N
terminal catalytic and C terminal regulatory domain. Whereas the N terminal domain shares
sequence homology with sucrose non-fermenting kinases (SNF), the C terminal domain is
unique to this class of kinases and harbors a 21 amino acid FISL/NAF motif [62]. FISL motif acts
as an autoinhibitory domain and interacts with the catalytic domain thereby keeping the enzyme
in an OFF state under normal conditions. SOS3 interacts with SOS2 via FISL motif and relieves
the protein from autoinhibition thereby making the kinase active. SOS3 activates SOS2 protein
kinase activity in a calcium- dependent manner [63]. SOS2 could be constitutively activated by
the deletion of FISL motif [64] and this deletion resulted in SOS2 acting independent of SOS3.
Ara- bidopsis plants with double mutant genotype (sos3/sos2) showed no additive effects towards
salt sensitivity, this indicates that SOS3 and SOS2 function in the same pathway [63]. Constitutively
over-expressed SOS2 under the control of CaMV35S promoter could rescue the salt
hypersensitive phenotype of both sos3 and sos2 mutants, thereby further supporting the
functioning of SOS3 and SOS2 in the same Ca2+ mediated pathway during salt stress [59,65].
SOS1 gene was identified as the target of SOS3-SOS2 pathway by genetic analysis of sos1
mutants of Arabidopsis.
Osmotic as well as ionic balance was impaired in sos1 mutants and they exhibited
hypersensitivity towards salt stress. SOS genes (SOS1, SOS2 and SOS3) were genetically
confirmed to function in a common pathway of salt tolerance [58]. SOS1 gene was cloned and
predicted to encode a 127-kDa protein with a N terminal region composed of 12 trans-membrane
domains and a C terminal region with a long hydrophilic cytoplasmic tail [66]. The trans-
membrane region of SOS1 shared substantial sequence homology to the plasma membrane
Na+/H+ antiporter isolated from bacteria and fungi [66].
The SOS pathway is depicted in Fig. 2. The perception of salt stress by an unknown
hypothetical plasma membrane sensor elicits cytoplasmic Ca2+ perturbations. This perturbation
in the cytosolic Ca2+ levels is sensed by SOS3, which transduces the signal to the down stream
components. The myristoylation motif of SOS3 results in the recruitment of SOS3-SOS2
complex to the plasma membrane, where SOS2 phosphorylates and activates SOS1 (a plasma
membrane Na+/H+ antiporter) [67]. The excess Na+ ions are expelled out of the cell and cellular
ion homeostasis is restored. SOS pathway regulates Na+ ion homeostasis by interacting with
other regulatory proteins and seems to have additional branches. AtHKT1 is a low affinity Na+
transporter and seems to mediate Na+ entry into the root cells of Arabidopsis during a salt stress
[68]. Remarkably, mutation in Athkt1 also suppresses the sos3 mutation [69] suggesting that SOS3-
SOS2 complex functions to down regulate HKT1 gene expression or inactivate the HKT1
protein during salt stress, thereby preventing the Na+ entry and its build up in the cell [59]. SOS3
and SOS2 seem to negatively regulate the activity of AtHKT1 under salt stress.
In addition to controlling SOS1 activity resulting in efflux of excess Na+ ions, SOS3-SOS2
complex also seems to function in sequestration of excess Na+ ions in the intracellular
compartments. SOS2 is shown to interact with vacuolar Na+/H+ antiporter and influence the
Na+/H+ exchange activity significantly [70]. Recently, further cross talk in the SOS pathway was
explored and it was shown that SOS2 interacted with the N terminus of CAX1 (H+/ Ca2+)
antiporter and regulated its activity [65]. This activation of CAX1 via SOS2 was however
independent of SOS3 and resulted in maintenance of Ca2+ homeostasis. SOS pathway may also
influence the functioning of other membrane proteins in sequestration of excess Na+ ions in
other sub-cellular compartments.
Overall, osmotic homeostasis after salt stress is mediated by Na+ efflux across the plasma
membrane and/or by its compartmentalization into the vacuoles. The energy for these reactions is
provided by H+-ATPases that serve as primary pumps. Plant cDNAs encoding NHE (Na+/H+
exchanger)-like proteins similar to mammalian sodium/ proton exchangers were isolated and can
functionally complement a yeast mutant deficient for the endomembrane Na+/H+ transporter,
NHX1 [71,72]. The AtNHX1 gene encodes a tonoplast Na+/H+ antiporter and functions in