Plant Stress Biology || Cold, Salinity, and Drought Stress

7

Cold, Salinity, and Drought Stress

Narendra Tuteja

Abstract

Genetically modified crops are emerging as a key weapon to fight the

negative impact of abiotic stresses on agricultural production. Among

abiotic stresses, cold mainly causes mechanical constraint to the membrane,

whereas salinity and drought exert their negative impact essentially by dis-

rupting the ionic and osmotic equilibrium of the cell. Cytosolic free Ca2þ

concentration ([Ca2þ ]cyt) has been found to increased in response to the

abiotic stress. The stress signal is first perceived at the membrane level by

the receptors and then transduced in the cell to switch on various stress-

responsive genes for mediating tolerance. The products of stress-inducible

genes function both in the initial stress response and in establishing plant

stress tolerance. Some genes have been reported to be upregulated in

response to more than one stress, indicating the presence of cross-talk

between the different stress signaling pathways. The generation of reactive

oxygen species represents a universal mechanism in cellular responses to

environmental stresses. Plants also accumulate a variety of osmoprotectants

that improve their ability to combat abiotic stresses. Understanding the

mechanism of abiotic stress tolerance is important for crop improvement. In

this chapter various aspects of cold, salinity, and drought stresses along with

the role of calcium are discussed.

7.1

Introduction

The world population is increasing at an alarming rate and is expected to reach

more than 9 billion by the end of 2050 (http://www.unfpa.org/swp/2007/presskit).

However, food productivity is decreasing due to the negative effect of various stress

factors. Minimizing these losses is a major area of concern for all nations. Among

these, abiotic stress is the principal cause of decreasing the average yield of major

Plant Stress Biology. Edited by H. HirtCopyright r 2009 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 978-3-527-32290-9

| 137

crops by more than 50%, which causes losses worth hundreds of millions of

dollars each year. In 2000, the United Nations Secretary-General, Kofi Annan,

called for a ‘‘Blue Revolution’’ and said that there was an urgent need for more

crops out of dry land. In 2007, the United Nations Food and Agriculture Orga-

nization warned of food shortages in new climates (‘‘food security is in danger’’).

Recently, in 2008, Neena Fedoroff, Science and Technology adviser to the United

States Secretary of State, emphasized the acute need for a ‘‘Second Green Revo-

lution.’’ Climate change and the decreased availability of fertile land will create a

problem for future crop production. In fact, these stresses threaten the sustain-

ability of agricultural industry. The challenge now is to produce additional food

under stress conditions and in less soil. Therefore, it is now necessary to obtain

stress-tolerant varieties to cope with this upcoming problem of food security.

It is important, first of all, to understand the notion of stress. Stress in physical

terms is defined as a mechanical force per unit area applied to an object. In bio-

logical systems stress can be defined as an adverse force, effect, or influence that

tends to inhibit normal systems from functioning [1, 2]. Various stress signals,

both abiotic as well as biotic, serve as elicitors for the plant cell. Abiotic stresses

include heat, cold, drought, salinity, wounding, heavy metals toxicity, excess light,

excess water (flooding), high speed wind, nutrient loss, anaerobic conditions, and

radiation. Biotic stresses include pathogens (bacteria, fungus, virus), herbivores,

weeds, insects, nematodes, and mycoplasma. Plants respond to stress as individual

cells and synergistically as a whole organism. In general, the stress signal is first

perceived by receptors of the plant cells. Following this the signal information is

transduced, resulting in the activation of various stress-responsive genes. The

products of these stress genes ultimately lead to a stress tolerance response or

plant adaptation, and help the plant to survive and surpass unfavorable conditions

[1, 2]. The response could also result in growth inhibition or cell death, which will

depend upon how many and which kinds of genes are up- or downregulated in

response to the stress. The various stress-responsive genes can be broadly cate-

gorized as early- and late-induced genes. Early genes are induced within minutes

of stress signal perception, which include various transcription factors. Late genes

include the major stress-responsive genes such as RD (RESPONSIVE TODEHYDRATION)/KIN (COLD INDUCED)/COR (COLD RESPONSIVE), whichencode and modulate the proteins needed for synthesis, for example, late

embryogenesis abundant (LEA)-like proteins, antioxidants, membrane-stabilizing

proteins, and osmolytes [2]. Overall, the stress response is a coordinated action of

many genes encoding signaling proteins/factors, including protein modifiers

(methylation, ubiquitination, glycosylation, etc.), adaptors, and scaffolds [2, 3].

In this chapter I have emphasized the general response to abiotic stress followed

by cold, salt, and drought stresses, and the reason for these stresses being injur-

ious for plants. Various genes involved in cold acclimation and their role towards

membrane stabilization are discussed. The role of calcium in relation to stress is

covered. Furthermore, the role of the salt overly sensitive (SOS) pathway in salt

tolerance and the role of glycine betaine (GB, N,N,N-trimethylglycine-betaine) as a

major osmolyte in response to salt stress are also described.

138 | 7 Cold, Salinity, and Drought Stress

7.2

Abiotic Stress Response and Stress-Induced Genes

A generic pathway in response to salinity, drought, and cold stresses is depicted in

Figure 7.1.

To sense these environmental signals, higher plants have evolved a complex

signaling network, which may also cross-talk. Stress signal transduction pathways

start with signal perception by receptors (phytochromes, histidine kinases,

receptor-like kinases, G-protein-coupled receptors (GPCR), hormone receptors,

etc.). Heterotrimeric G-proteins mediate the coupling of signal transduction from

activated GPCRs to generate secondary signaling molecules (inositol phosphatase,

reactive oxygen species (ROS), abscisic acid (ABA), etc.). These secondary mole-

cules can modulate the intracellular Ca2þ levels by receptor-mediated Ca2þ

Figure 7.1 Generic pathway under salinity, drought, and cold

stresses. Salinity and drought stresses mainly disrupt the

ionic and osmotic equilibrium of the cell. These stresses can

also cause injury to the cellular physiology, which leads to

metabolic dysfunctions followed by growth inhibition. Cold

stress mainly exerts its negative effect by disruption of

membrane integrity and solute leakage. Finally, in response to

all these stresses several stress-responsive genes are

upregulated whose products can directly or indirectly help the

plant in stress tolerance.

7.2 Abiotic Stress Response and Stress-Induced Genes | 139

release or can bypass Ca2þ in early signaling steps and initiate protein phos-

phorylation cascades (protein phosphatase, mitogen-activated protein kinase

(MAPK), calcium-dependent protein kinase (CDPK), SOS3/protein kinase S, etc.),

which activate specific stress-responsive genes for cellular protection through

transcription control (MYC/MTB, C-repeat binding (CBF)/dehydration-responsive

element binding (DREB) factors) [2, 3, 5, 6]. Salinity and drought exert their

influence on a cell mainly by disrupting the ionic and osmotic equilibrium [2].

Thus, excess of Naþ ions and osmotic changes in the form of turgor pressure are

the initial triggers, leading to a cascade of events, which can be grouped under

ionic and osmotic signaling pathways, the outcome of which is ionic and osmotic

homeostasis, leading to stress tolerance. These stresses are marked by symptoms

of stress injury, including chlorosis and necrosis, and may also exert negative

influences on cell division resulting in growth retardation of plants [2]. Reduction

in shoot growth, especially leaves, is beneficial for plants as it reduces the surface

area exposed for transpiration, hence minimizing water loss. Plants may also

sacrifice or shed older leaves, which is another adaptation in response to drought.

Stress injury may occur through denaturation of cellular proteins/enzymes or

through the production of ROS, Naþ toxicity, and disruption of membrane

integrity. In response to injury stress plants trigger a detoxification process, which

may include change in the expression of LEA/dehydrin-type gene synthesis of

molecular chaperones, proteinases, enzymes for scavenging ROS, and other

detoxification proteins. This process functions in the control and repair of stress-

induced damage, and results in stress tolerance. Cold stress mainly results in

disruption of membrane integrity, leading to severe cellular dehydration and

osmotic imbalances. Cold acclimation results in the triggering of various genes,

which result in a restructuring of the cellular membranes by changes in the lipid

composition and the generation of osmolytes, which prevent cellular dehydration

and enhances stress tolerance (Figure 7.1).

Plants suffer from dehydration or osmotic stress under drought, salinity, and

also under low-temperature conditions that cause reduced availability of water for

cellular function and maintenance of cellular turgor pressure. Prolonged periods

of dehydration lead to high production of ROS in the chloroplasts, causing irre-

versible cellular damage and photoinhibition. Overall, in response to all these

stresses several stress-responsive genes are upregulated whose products can

directly or indirectly help the plant through stress tolerance. Understanding the

molecular mechanism for abiotic stress tolerance is still a major challenge in

biology. Many chemicals are also critical for plant growth and development, and

play an important role in integrating various stress signals and controlling

downstream stress responses by modulating gene expression machinery and

regulating various transporters/pumps and biochemical reactions. Some of the

chemicals include calcium (Ca2þ ), cyclic nucleotides, polyphosphoinositides,

nitric oxide, sugars, ABA, jasmonates, salicylic acid, and polyamines [7].

Microarrays employing cDNAs or oligonucleotides are a powerful tool for ana-

lyzing the gene expression profiles of plants exposed to abiotic stresses. A 7000

full-length cDNA microarray was utilized to identify 299 drought-inducible genes,


54 cold-inducible genes, 213 high salinity-inducible genes, and 245 ABA-inducible

genes in Arabidopsis [8, 9]. More than half of these drought-inducible genes were

also induced by high salinity and/or ABA treatments, implicating significant

cross-talk between the drought, high salinity, and ABA response pathways.

Recently, Shinozaki and Yamaguchi-Shinozaki [10] summarized the gene net-

works involved in drought stress response and tolerance. By using transgenic

technology, Bhatnagar-Mathur et al. [11] have also described the recent progress in

the improvement of abiotic stress tolerance in plants, which includes a discussion

on the evaluation of abiotic stress responses and protocols for testing transgenic

plants for their tolerance under close-to-field conditions. Emerging evidence

indicates CDPKs sense the Ca2þ concentration changes in plant cells, and play

important roles in signaling pathways for disease resistance and various stress

responses. Among the 20 wheat CDPK genes studied, 10 were found to respond to

drought, salinity, and ABA treatment [12].

7.3

Cold Stress

Each plant has its own set of temperature requirements, which are optimum for its

proper growth and development. Deviation from optimum temperature may lead

to plant growth inhibition and yield loss. The cold stress experienced by plants can

be classified into two types: those occurring at (i) temperatures below freezing and

(ii) low temperatures above freezing (nonfreezing temperatures).This section

covers various aspects of cold stress.

7.3.1

Effect of Low-Temperature Stress on Plant Physiology

Many plants such as maize, soybean, cotton, tomato, and banana are sensitive to

nonfreezing temperatures (10–15 1C) and exhibit signs of injury [13–15]. Various

phenotypic symptoms in response to chilling stress include reduced leaf expan-

sion, wilting, and chlorosis, which may lead to necrosis. Low temperature can also

severely hamper the reproductive development of plants, as reported in rice [16].

Freezing temperatures can induce severe membrane damage, which is largely due

to the acute dehydration associated with freezing [14, 17]. The temperature at

which a membrane changes from a semifluid state to a semicrystalline state is

known as the transition temperature. Chilling-sensitive plants usually have a

higher transition temperature as compared to the chilling-resistant plants, which

have a lower transition temperature. An understanding of how freezing induces

plant injuries is essential for the development of frost-tolerant crops. The real

cause of freeze-induced injury to plants is ice formation rather than the low

temperatures. Ice formation in plants begins in the apoplastic space having rela-

tively low solute concentrations. This causes a mechanical strain on the cell wall

and plasma membrane leading to cell rupture [18]. Freezing temperatures exert

7.3 Cold Stress | 141

their effects largely by membrane damage due to severe cellular dehydration, but

certain additional factors including ROS also contribute to damage induced by

freezing. Overall, chilling ultimately results in loss of membrane integrity, which

leads to solute leakage. The integrity of intracellular organelles is also disrupted,

leading to the loss of compartmentalization and impairment of photosynthesis,

protein assembly, and general metabolic processes. The primary environmental

factor responsible for triggering increased tolerance against freezing is the phe-

nomenon known as ‘‘cold acclimation.’’ It is the process where certain plants

increase their freezing tolerance upon prior exposure to low nonfreezing tem-

peratures [2].

7.3.2

Cold Acclimation

Cold temperatures induce a number of alterations in cellular components,

including the extent of unsaturated fatty acids, the composition of glycerolipids,

changes in protein and carbohydrate composition, and the activation of ion channels

[2, 19]. For cold acclimation, membranes have to be stabilized against freeze injury,

which can be achieved through changes in the lipid composition and induction of

other nonenzymatic proteins that alter the freezing point of water. Accumulation

of sucrose and other simple sugars also contributes to the stabilization of mem-

branes as these molecules can protect membranes against freeze-damage. Low

temperatures activate a number of cold-inducible genes, such as those encoding

dehydrins, lipid transfer proteins, translation elongation factors, and the LEA pro-

teins [2, 14, 19]. Overall, cold acclimation results in protection and stabilization of

the integrity of cellularmembranes, enhancement of the antioxidativemechanisms,

increased intercellular sugar levels as well as accumulation of other cryoprotectants

including polyamines that protect intracellular proteins by inducing the genes

encoding molecular chaperones. All these modifications help plants to withstand

and surpass severe dehydration associated with freezing stress [2, 19].

7.3.3

Function of Cold-Regulated Genes in Freezing Tolerance

The Arabidopsis FAD8 gene [20] encodes a fatty acid desaturase that contributes

to freezing tolerance by altering the lipid composition. Cold-responsive genes

encoding molecular chaperones include heat shock protein genes spinach hsp70[21] and Brassica napus hsp90 [22], and contribute to freezing tolerance by sta-

bilizing proteins against freeze-induced denaturation. Many cold-responsive

genes encoding various signal transduction and regulatory proteins have been

identified and this list includes those for a MAPK [23], MAPK kinase kinase [24]

and genes for calmodulin-related proteins [25]. The largest class of cold-induced

genes encodes polypeptides that are homologs of LEA proteins – polypeptides

that are synthesized during late embryogenesis, just prior to seed desiccation

and also in seedlings in response to dehydration [26]. Other examples of


cold-responsive genes include COR15a, alfalfa Cas15, and wheat WCS120 [2].

The expression of COR genes has been shown to be critical for both chilling

tolerance and cold acclimation in plants [27]. Arabidopsis COR genes include

COR78/RD29, COR47, COR15a, COR6.6, and the genes encoding LEA-like

proteins [27]. These genes are induced by cold, dehydration, or ABA. The ana-

lysis of the promoter elements of COR genes revealed that they contain

dehydration-responsive elementDREs or C-repeats (CRTs) and some of them

contain ABA-responsive element-responsive elements (abscisic acid-responsive

element-responsive elementABREs) as well [28, 29]. Induction of the COR genes

was accomplished by overexpression of the transcription factor CBF [29]. CBF binds

to the CRT/DRE elements that are present in the promoters of the COR and other

cold-regulated genes. The overexpression of these regulatory elements resulted in

increased freezing and drought tolerance [30]. Lee et al., in 2001 [31], genetically

analyzed the HOS1 (HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE1)locus of Arabidopsis. HOS1 encodes a ring finger protein and is constitutively

expressed but is drastically downregulated within 10 min of cold stress. Genetic

analysis led to the identification of ICE1 (INDUCEROF CBF EXPRESSION1) as anactivator of CBF3 [32]. ICE1 encodes a transcription factor that specifically recog-

nizedMYC sequence on theCBF3 promoter. Transgenic lines overexpressing ICE1did not express CBF3 at warm temperatures but showed a higher level of expres-

sion for CBF3 as well as RD29 and COR15a at low temperatures. This study sug-

gests that cold-induced modification of ICE1 is necessary for it to act as an activator

of CBF3. Two CBF1-like cDNAs, CaCBFIA and CaCBFIB, have been cloned

and characterized from hot pepper [2]. These were induced in response to

low-temperature stress (4 1C), but not in response to wounding or ABA. The gene

expression as well as protein accumulation of Oryza sativa OsCDPK13 were

upregulated in response to cold. Cold-tolerant rice varieties exhibited higher

expression of OsCDPK13 than cold-sensitive varieties [33].

Proline has been shown to be an effective cryoprotectant and this is also one

of the major factors imparting freezing tolerance. The esk1 (eskimo1) gene is

known to play an important role in freezing tolerance. The concentration of free

proline [34] in esk1 mutants was found to be 30-fold higher than in the wild-type

plants. Sui et al. [35] have reported that overexpression of glycerol-3-phosphate

acyltransferase improves chilling tolerance in tomato. Recently, soybean

GmbZIP44, GmbZIP62, and GmbZIP78 genes have been shown to function as

negative regulators of ABA signaling, and their overexpression confers salt and

freezing tolerance in transgenic Arabidopsis [36]. Recently, O. sativa cold shock

domain protein OsCSP transcripts are reported to be transiently upregulated in

response to low-temperature stress and rapidly return to a basal levels of gene

expression [37]. OsCSP1 and OsCSP2 (O. sativa CSD protein) encode putative

proteins consisting of an N-terminal CSD and glycine-rich regions that are

interspersed by four and two CX2CX4HX4C (CCHC) retroviral-like zinc fingers,

respectively. In vivo functional analysis confirmed that OsCSPs could comple-

ment a cold-sensitive bacterial strain, which lacks four endogenous cold shock

proteins.

7.3 Cold Stress | 143

7.3.4

Calcium Signaling in Cold Stress Response

The generic pathway for the plant response to cold stress is shown in Figure 7.2.

The extracellular cold stress signal is first perceived by the membrane receptors/

sensors and then activates a complex intracellular signaling cascade including the

generation of secondary signals. Increases in cytosolic free Ca2þ ([Ca2þ ]cyt) arecommon to many stress-activated signaling pathways, including the response to

cold environments. In Arabidopsis [25] and alfalfa [38], cytoplasmic Ca2þ levels

increase rapidly in response to low temperature, largely due to an influx of Ca2þ

from extracellular stores. Through the use of pharmacological and chemical

reagents, it has been demonstrated that Ca2þ is required for the full expression of

some of the cold induced genes including the CRT/DRE controlled COR6 and

KIN1 genes of Arabidopsis [38]. Ca2þ release can occur primarily from extracellular

sources (apoplastic space) as addition of the calcium chelators EGTA (ethyle-

neglycol bis(b-aminoethyl ether)-N,N,Nu,Nu -tetraacetic acid) or BAPTA (O,Ou-bis(2-aminophenyl)ethyleneglycol-N,N,Nu,Nu -tetraacetic acid) was shown in many

cases to block Ca2þ effects (Figure 7.2). Ca2þ release may also result from acti-

vation of phospholipase C (PLC), leading to hydrolysis of phosphatidylinositol

bisphosphate (PIP2) to inositol triphosphate (IP3) and diacylglycerol (DAG), which

trigger the subsequent release of Ca2þ from intracellular Ca2þ stores [2, 3].

Furthermore, Ca2þ -binding proteins (Ca2þ sensors) can provide an additional

level of regulation in Ca2þ signaling. These Ca2þsensor proteins recognize and

decode the information provided in the Ca2þ signatures, and relay the informa-

tion downstream to initiate a phosphorylation cascades. These cascades regulate

the expression of genes, like SNOW and ICE1, which in turn regulate cold binding

factors to induce the DRE/CRT and ABRE regulatory elements to upregulate the

level of cold-responsive genes like COR, KIN, LT1, and RD [2]. The product of

these cold stress-responsive genes can provide cold stress tolerance directly or

indirectly (Figure 7.2). Overall, the cold stress response could be a coordinated

action of many genes, which may also cross-talk with each other.

7.4

Salinity Stress

In general, the term salinity means the presence of salts in the soil. These soils can

be categorized into two types: (i) sodic (or alkali) and (ii) saline (a third type can

also be referred to as saline/sodic soils). Sodic (or alkaline) soils contain high con-

centrations of free carbonate and bicarbonate and excess of sodium. The pH of this

soil is greater than 8.5 and sometimes up to 10.7. Saline soils are dominated by

sodium cations, and usually soluble chloride and sulfate anions, and the pH values

of these soils are much lower than in sodic soils. Generally, soil salinity arises due to

many factors such as (i) use of poor-quality irrigation water, (ii) unsustainable irri-

gation practices (heavy irrigation), (iii) high evaporation, and (iv) previous exposure

of the land to seawater. Seawater contains approximately 3% of NaCl and in terms of


Figure 7.2 Generic pathway for plant responses to cold stress.

The extracellular cold stress signal is first perceived by

membrane receptors/sensors, which activate PLC to hydrolyze

PIP2 to generate IP3 and DAG. These compounds increase the

level of Ca2þ ions in the cytosol, which is sensed by calcium

sensors to activate phosphorylation cascades. This pathway

then induces cis-regulatory elements, like SNOW and ICE1,

which in turn regulate cold binding factors, which in turn

induce DRE/CRT and ABRE regulatory elements to upregulate

cold-stress responsive genes like COR, KIN, LT1, and RD. The

product of these cold stress-responsive genes can provide

cold stress tolerance directly or indirectly.

7.4 Salinity Stress | 145

molarity of different ions, Naþ is about 460 mM, Mg2þ is 50 mM and Cl� around

540mM, along with smaller quantities of other ions [2, 3]. Many crop species are very

sensitive to soil salinity and are glycophytes, whereas salt-tolerant plants are known

as halophytes. In general, glycophytes cannot grow at 100 mM NaCl, whereas

halophytes can grow at salinities over 250 mM NaCl. The salinity-sensitive plants

restrict the uptake of salt and strive to maintain an osmotic equilibrium by the

synthesis of compatible solutes, such as proline, GB, and sugars. The salinity-tol-

erant plants have the capacity to sequester and accumulate salt in the cell vacuoles,

thus preventing the build up of salt in the cytosol and maintaining a high cytosolic

Kþ /Naþ ratio in their cells. Generally, salinity tolerance is inversely related to the

extent of Naþ accumulation in the shoot [3]. The basic physiology of high-salinity

stress and drought stress plants overlaps with each other. High salinity also leads to

increased cytosolic Ca2þ , which initiates the salinity stress signal transduction

pathways for stress tolerance as described above in Sections 7.2 and 7.3.4.

In Arabidopsis, the transcript profiles of various genes under salinity and other

stresses have been made openly available through several databases, such as TAIR,

NASC, and Genevestigator [39]. The various genes that have been reported to be

upregulated in response to salinity stress are listed in Ma et al. [39]. Earlier,we have shown a novel role of a DNA helicase and G-proteins in salinity

stress tolerance [40, 41]. Recently, it has been shown that overexpression of the

trehalose-6-phosphate phosphatase gene OsTPP1 confers salt, osmotic, and ABA

tolerance in rice, and results in the activation of stress-responsive genes [42]. The

conservation in the mechanisms of salt responses and stress tolerance has been

observed between bryophytes and higher plants [43]. Some of the web sites for

further study on salinity stress include:

i. Affymetrix microarray data (http://www.arabidopsis.org/

portals/expression/microarray/ATGenExpress.jsp).

ii. The abiotic transcript profile data (Affymetrix microarray

data) (http//www.weigelworld.org/resources/microarray/

ATGenExpress/).

iii. Glass microarray data (http://ag.arizona.edu/microarray/),

the resulting GPR files can be analyzed by TIGR-TM4

(http://www.tm4.org/).

7.4.1

Negative Impact of Salinity Stress

The adverse effects observed in response to high salinity stress are:

1. Salinity stress interferes with plant growth and development as it can also lead

to physiological drought conditions and ion toxicity, and therefore causes both

hyperionic and hyperosmotic stresses that lead to plant demise [44, 45].

2. High salt deposition in the soil leads to a low water potential in the soil. This

makes it increasingly difficult for the plant to acquire water as well as

nutrients.


3. High salt also decreases the soil porosity and thereby reduces soil aeration.

4. Salinity causes ion-specific stresses resulting in altered Kþ /Naþ ratios.

External Naþ can negatively impact intracellular Kþ influx.

5. Kþ ions are one of the essential elements required for growth. Alterations in

Kþ ions (due to the impact of high salinity stress) can disturb the osmotic

balance, function of stomata, and function of some enzymes.

6. Salinity leads to the accumulation of Naþ and Cl� in the cytosol, which can be

ultimately detrimental for the cell. The Naþ can dissipate the membrane

potential and therefore facilitate the uptake of Cl– down the gradient.

7. Higher concentrations of sodium ions (above 100 mM) are toxic to cell

metabolism, and can inhibit the activity of many essential enzymes, cell

division and expansion, membrane disorganization, and osmotic imbalance

[44, 46].

8. Higher concentrations of sodium ions can also lead to a reduction in

photosynthesis, and increase in the production of ROS and polyamines [47].

9. High salinity can also injure cells in transpiring leaves, which leads to growth

inhibition. This is the salt-specific or ion-excess effect of salinity, which causes

the toxic effects of salt inside the plant. Salt can concentrate in old leaves that

subsequently die – a process that can be crucial for the survival of a plant [48].

10. High salinity affects the cortical microtubule organization and helical growth

in Arabidopsis [49].

7.4.2

Calcium Signaling and SOS Pathways in Relation to Salinity Stress

High salinity results in increased cytosolic Ca2þ that is transported from the

apoplast as well as the intracellular compartments [50]. This transient increase in

cytosolic Ca2þ initiates the stress signal transduction leading to salt adaptation. As

described in Section 7.3.4, this Ca2þ release occurs primarily from extracellular

sources (apoplastic space), but also from the activation of PLC, leading to hydro-

lysis of PIP2 to IP3 and subsequent release of Ca2þ from intracellular Ca2þ stores

[2, 3]. The Ca2þ -binding proteins sense and relay the information downstream to

initiate phosphorylation cascades, leading to gene expression [5].

Wu et al. [51] commenced a mutant screen for Arabidopsis plants, which were

over-sensitive to salt stress. As a result of this screen, three genes SOS1, SOS2, andSOS3 were identified. SOS3 (also known as AtCBL4) encodes a calcineurin B-like

protein (CBL, Ca2þ sensor), which is a Ca2þ -binding protein, and senses the

change in cytosolic Ca2þ concentration and transduces the signal downstream.

The SOS pathway results in the exclusion of excess Naþ ions out of the cell via the

plasma membrane Naþ /Hþ antiporter and helps in reinstating cellular ion

homeostasis. The discovery of SOS genes paved the way for the elucidation of a

novel pathway linking Ca2þ signaling in response to salt stress [52, 53]. SOS genes

(SOS1, SOS2, and SOS3) were genetically confirmed to function in a common

pathway of salt tolerance [54].


In the SOS pathway, the salinity stress signal is perceived by an unknown

hypothetical plasma membrane sensor. The resulting cytoplasmic Ca2þ pertur-

bation is sensed by SOS3 followed by transduction of the signal to the downstream

components. The myristoylation motif of SOS3 results in the recruitment of the

SOS3–SOS2 complex to the plasma membrane, where SOS2 phosphorylates and

activates SOS1 [55]. SOS1 is a Naþ /Hþ antiporter and sos1 mutants are hyper-

sensitive to salt and show an impaired osmotic/ionic balance. The SOS pathway also

seems to have other branches, which help to remove the excess of Naþ ions out of

the cell and thereby maintain the cellular ion homeostasis. In Arabidopsis, Naþ

entry into root cells during salt stress appears to be mediated by AtHKTI, a low-

affinity Naþ transporter, which blocks entry of Naþ [2, 45]. SOS2 also interacts and

activates the vacuolar NHX (Naþ /Hþ exchanger), resulting in sequestration of

excess Naþ ions and pushing it into vacuoles, and thereby further contributes to

Naþ ion homeostasis. Some other Ca2þ -binding proteins like calnexin and cal-

modulin also sense the increased level of Ca2þ and can interact and activate NHX.

Overexpression ofAtNHX1 substantially enhanced salt tolerance ofArabidopsis [56].TheHþ /Ca2þ antiporter CAX1 has been identified as an additional target for SOS2

activity reinstating cytosolic Ca2þ homeostasis. This reflects that the components of

the SOS pathway may cross-talk and interact with other branching components to

maintain cellular ion homeostasis, which helps in salinity tolerance.

So far, the main avenue in breeding crops for salt tolerance has been to reduce

Naþ uptake and transport from roots to shoots. It has been demonstrated that

retention of cytosolic Kþ could also be considered as another key factor in con-

ferring salt tolerance in plants. Recently, Zepeda-Jazo et al. [57] have shown that

the expression of NORC was significantly lower in salt-tolerant genotypes. NORC

is capable of mediating Kþ efflux coupled to Naþ influx, suggesting that the

restriction of its activity could be beneficial for plants under salt stress.

7.4.3

ABA and Transcription Factors in Salinity Stress Tolerance

ABA is a phytohormone that regulates plant growth and development, and also plays

an important role in the plant’s response to abiotic stresses including salinity stress

(reviewed in [2, 3, 45, 58]). The role of ABA in salinity stress was confirmed by a study

of Zhu’s group where it was shown that ABA-deficient mutants performed poorly

under salinity stress [59].ABA levels are known to be inducedunder stress conditions,

which is mainly due to the induction of the enzymes responsible for ABA biosynth-

esis. The induction of osmotic stress-responsive genes imposed by salinity is trans-

mitted through either ABA-dependent or ABA-independent pathways, although

some others are only partially ABA-dependent [60]. However, the components

involved in these pathways often cross-talk through Ca2þ with other stress signaling

pathways. The transcript accumulation of RD29A gene is reported to be regulated in

both anABA-dependent andABA-independentmanner [61]. Proline accumulation in

plants can be mediated by both ABA-dependent and ABA-independent signaling

pathways [45]. The salinity stress-induced upregulation of transcripts of PDH45 (PEA


DNA HELICASE45) follows an ABA-dependent pathway [40] while CBL and CBL-

interacting protein kinase frompea followed the ABA-independent pathway [62]. The

role of Ca2þ in ABA-dependent induction ofP5CS (PYRROLINE-5-CARBOXYLATESYNTHASE) during salinity stress has been reported [63]. Overall, the ABA-depen-

dent pathways are involved essentially in osmotic stress gene expression.

The transcriptional regulatory network of cis-acting elements and transcription

factors involved in ABA and salinity stress-responsive gene expression has been

described [3]. The ABA-dependent salinity stress signaling activates basic leucine

zipper transcription factors called ABRE-binding proteins, which binds to ABRE

elements to induce the stress responsive gene RD29A. Transcription factors like

DREB2A and DREB2B activate the DRE cis elements of osmotic stress genes, and

thereby are involved inmaintaining the osmotic equilibrium of the cell. Some genes

such asRD22 lack the typicalCRT/DREelements in their promoter, suggesting their

regulation by some other mechanism. The MYC/MYB transcription factors,

RD22BP1 andAtMYB2, could bindMYCRSandMYBRSelements, respectively, and

help in the activation of RD22 [2, 3]. Overall, these transcription factors may also

cross-talk with each other for their maximal response to stress tolerance.

7.4.4

Water Stress due to Salinity

One of the consequences of salinity stress is the loss of intracellular water. To

prevent water loss from the cell and protect the cellular proteins, plants accu-

mulate many metabolites that are also known as ‘‘compatible solutes.’’ These

solutes do not inhibit the normal metabolic reactions [64]. Frequently observed

metabolites with an osmolyte function are sugars, mainly fructose and sucrose,

alcohols, and complex sugars like trehalose and fructans. In addition, charged

metabolites such as GB, proline, and ectoine also accumulate. The accumulation

of these osmolytes facilitates the osmotic adjustment [65]. Water moves from

sites of high water potential to low water potential, and accumulation of

osmolytes decreases the water potential inside the cell and therefore prevents

intracellular water loss.

7.4.5

Proline and GB in Salinity Stress

Proline and GB are two major osmoprotectant osmolytes that are synthesized by

many plants (but not all) in response to stress including salinity stress [66]. In

higher plants the amino acid proline is synthesized by glutamic acid by the actions

of two enzymes P5CS and P5CR (PYRROLINE-5-CARBOXYLATE REDUCTASE).

Overexpression of the P5CS gene in transgenic tobacco resulted in increased

production of proline and salinity/drought tolerance [67]. The exogenous appli-

cation of proline also provided osmoprotection and facilitated growth of salinity-

stressed plants. Proline can also protect cell membranes from salinity-induced

oxidative stress by upregulating activities of various antioxidants [68]. It is reported


that salt stress enhances proline utilization in the apical region of barley roots [69].

The function of proline is thought to be an osmotic regulator under water stress

and its transport into cells is mediated by a proline transporter. However, recently,

Ueda et al. [70] have reported that altered expression of barley HvProT (Hordeumvulgare proline transporter) causes different growth responses in Arabidopsis, as itleads to the reduction in biomass production and decreased proline accumulation

in leaves. Impaired growth of HvProT transformed plants was restored by exo-

genously adding proline, which suggested that growth reduction was caused by a

deficiency of endogenous proline.

In plants where GB is not produced, transgenic plants overexpressing

GB-synthesizing genes showed production of sufficient GB to tolerate stresses

including salinity stress. GB is synthesized from choline by the action of choline

monooxygenase and betaine aldehyde dehydrogenase enzymes. The over-

expression of the genes encoding betaine aldehyde decarboxylase from the halo-

phyte Suaeda liaotungensis improved salinity tolerance in tobacco plants. The codA(choline dehydrogenase) gene from Arthrobacter globiformis helped salinity tolerance

in rice (see [66] and references therein). Overexpression of N-methyl transferase in

cyanobacteria and Arabidopsis resulted in accumulation of GB and improved

salinity tolerance [71]. It is also reported that foliar application of GB to low- or

nonaccumulating plants helped in improving the growth of plants under salinity

stress conditions as reported in Zea mays [72]. In plants, betaine is synthesized

upon abiotic stress via choline oxidation, in which choline monooxygenase is a key

enzyme. Although it had been thought that betaine synthesis is well regulated to

protect abiotic stress, recently it has been shown that exogenous supply of pre-

cursors such as choline, serine, and glycine in the betaine-accumulating plant

Amaranthus further enhances the accumulation of betaine under salt stress, but

not under normal conditions [73]. Recently, Waditee et al. [74] have shown

that expression of Aphanothece 3-phosphoglycerate dehydrogenase in Arabidopsisplants enhances levels of betaine by providing serine as precursor for both choline

oxidation and glycine methylation pathways.

7.4.6

ROS in Salinity Stress

ROS typically result from the excitation of O2 to form singlet oxygen (1O2) or

transfer of one, two, or three electrons to O2 to form superoxide radical (O21�),

hydrogen peroxide (H2O2), or a hydroxyl radical (OH�) respectively. The enhanced

production of ROS during stresses can pose a threat to plants because they are

unable to detoxify effectively by the ROS scavenging machinery. Unquenched ROS

spontaneously react with organic molecules and cause membrane lipid perox-

idation, protein oxidation, enzyme inhibition, DNA and RNA damage, and so on

[3, 66]. Oxidative stress arising under environmental stresses including salinity

may exceed the scavenging capacity of the natural defense system of plants. The

major ROS-scavenging mechanisms of plants include superoxide dismutase,

ascorbate peroxidase, catalase, and glutathione reductase, which help in


deactivation of active oxygen species in multiple redox reactions and thereby

contribute to the protective system against oxidative stress. ROS scavengers can

increase plant resistance to salinity stress. Overexpression of aldehyde dehy-

drogenase in Arabidopsis has been reported to confer salinity tolerance. Aldehyde

dehydrogenase catalyzes the oxidation of toxic aldehydes, which accumulate as a

result of side-reactions of ROS with lipids and proteins. The enhancement of

stress tolerance in transgenic tobacco plants has been shown by overexpressing

Chlamydomonas glutathione peroxidase in the chloroplast or cytosol (see [66] and

references therein).

7.5

Drought Stress

Water-deficit stress is known as drought stress, which reduces agricultural pro-

duction mainly by disrupting the osmotic equilibrium and membrane structure of

the cell. Climate models have indicated that drought stress will become more

frequent because of the long-term effects of global warming, which indicate the

urgent need to develop adaptive agricultural strategies for a changing environ-

ment. Actually, the water stress within the lipid bilayer results in displacement of

membrane proteins, which contributes to loss of membrane integrity, selectivity,

disruption of cellular compartmentalization, and loss of membrane-based enzyme

activity. The high concentration of cellular electrolytes due to the dehydration of

the protoplasm may also cause disruption of the cellular metabolism. To avoid

drought stress, plants close their stomata, repress cell growth and photosynthesis,

activate respiration, reduce leaf expansion, and start shedding older leaves to

reduce the transpiration area [10]. Relative root growth may be enhanced, which

facilitates the capacity of the root system to extract more water from deeper soil

layers. The components of drought and salt stress cross-talk as both these stresses

ultimately result in dehydration of the cell and an osmotic imbalance. Overall,

drought stress signaling encompasses three important parameters [75]:

1. Reinstating the osmotic as well as the ionic equilibrium of the cell to maintain

cellular homeostasis under the conditions of stress.

2. Control as well as repair of stress damage by detoxification.

3. Signaling to coordinate cell division to meet the requirements of the plant

under stress.

As a consequence of drought stress many changes occur in the cell, which

include changes in the expression level of LEA/dehydrin-type genes, and synthesis

of molecular chaperones that help in protecting the partner protein from degra-

dation and proteinases that function to remove denatured/damaged proteins.

Drought stress also leads to the activation of enzymes involved in the production

and removal of ROS [53, 76]. Overexpression of some genes has been now reported

to help plants in drought stress tolerance [10]. Some examples are mentioned

below.

7.5 Drought Stress | 151

Overexpression of barley group 3 LEA geneHVA1 in leaves and roots of rice and

wheat led to improved tolerance against osmotic stress as well as improved recovery

after drought and salinity stress [77]. Dehydrins are also known to accumulate in

response to both dehydration as well as low temperature stresses [78]. Over-

expression of the vacuolar Naþ /Hþ antiporter and Hþ -pyrophosphatase pump

has resulted in enhanced tolerance to both salinity [79, 80] and drought stress [81,

82]. These results suggest that the enhanced vacuolar Hþ -pumping in the trans-

genic plants provide an additional driving force for vacuolar sodium accumulation

via the vacuolar Naþ /Hþ antiporter. Brini et al. [83] reported that overexpression

of wheat Naþ /Hþ antiporter TNHX1 and Hþ -pyrophosphatase TVP1 improve

salt- and drought-stress tolerance inArabidopsis thaliana plants. Recently, Jung et al.[84] showed that overexpression of AtMYB44 enhanced stomatal closure and con-

fers dehydration stress tolerance in transgenic Arabidopsis. Recently, Jia et al. [85]have shown that a Ca2þ -binding protein calreticulin from wheat is involved in the

plant response to drought stress; TaCRT-overexpressing tobacco (Nicotiana ben-thamiana) plants grew better and exhibited less wilting under drought stress.

Plants produce compounds in roots that are transported to shoots via the xylem

sap. Some of these compounds are vital for signaling and adaptation to drought

stress. Recently, Alvarez et al. [86] observed metabolomic and proteomic changes

in the xylem sap of maize under drought stress. The application of these new

techniques provides insight into the range of compounds in sap, and how

alterations in their composition may lead to changes in development and signaling

during adaptation to drought.

7.5.1

Effect of Drought on Stomata and Photosynthesis

The first response of plants to drought stress is the closure of stomata to prevent

transpirational water loss [87]. The closure of stomata may result from direct

evaporation of water from the guard cells with no metabolic involvement and is

referred to as hydropassive closure. Stomatal closure may also be metabolically

dependent and involve processes that result in reversal of the ion fluxes that cause

stomatal opening. This process of stomatal closure, which requires ions and

metabolites, is known as hydroactive closure. Plant growth and response to a stress

condition is largely under the control of hormones. ABA promotes the efflux of

Kþ ions from the guard cells, which results in the loss of turgor pressure leading

to stomata closure. The closure of stomata does not always depend upon the

perception of water-deficit signals arising from leaves. In fact, stomata closure also

responds directly to soil desiccation even before there is any significant reduction

in leaf mesophyll turgor pressure. The fact that ABA can act as a long distance

communication signal between water-deficient roots and leaves, inducing the

closure of stomata, has been known for decades [88].

Stomatal closure in response to drought stress primarily results in a decline in

the rate of photosynthesis. Severe drought was reported to decrease ribulose-1,5-

bisphosphate carboxylase/oxygenase (RuBisCO) activity, which leads to limited


photosynthesis [89]. The photosystem II has been reported to decline under

drought conditions [90] and the decline in the rate of photosynthesis in drought

stress is primarily due to CO2deficiency [91]. Decreasing intracellular CO2 levels

also result in the over-reduction of components within the electron transport

chain and electrons get transferred to oxygen at photosystem I. This process

generates ROS including superoxide, H2O2 and hydroxyl radicals. These ROS

need to be scavenged by the plant as they may lead to photo-oxidation. Plant-

detoxifying systems, which include ascorbate and glutathione pools, control

the intracellular concentration of ROS. Under longer drought situations, plant

cells can undergo shrinkage, leading to mechanical constraints on cellular

membranes, which impairs the functioning of ions and transporters as well as

membrane-associated enzymes.

7.5.2

Sugars and other Osmolytes in Response to Drought Stress

To cope with drought stress plants need to perform osmotic adjustments whereby

they decrease their cellular osmotic potential by the synthesis/accumulation of

solutes including proline, glutamate, GB, carnitine, mannitol, sorbitol, fructans,

polyols, trehalose, sucrose, oligosaccharides, and inorganic ions like Kþ . Thesecompounds help plant cells to maintain their hydrated state, and therefore func-

tion to provide resistance against drought and cellular dehydration [92]. The

hydroxyl groups of sugar alcohols substitutes the OH group of water to maintain

the hydrophilic interactions with membrane lipids and proteins, and therefore

help to maintain the structural integrity of membranes. These stress-accumulated

solutes do not intervene with normal cellular metabolic processes. The accumu-

lation of simple sugars such as glucose and fructose increases invertase activity in

leaves of drought-challenged plants [93]. ABA has been implicated in enhancing

the activity and expression of vacuolar invertase [93]. ABA biosynthesis is also

directly controlled by glucose, as transcripts of several genes responsible for ABA

synthesis increase by glucose in Arabidopsis seedlings [94]. Cross-talk may exist

between the sugars and plant hormones such as ABA and ethylene. Glucose and

ABA signaling act in coordination for regulating plant growth and development. A

high concentration of ABA and sugars can inhibit growth under severe drought

stress, while low concentrations can promote growth.

Osmolytes function at low concentrations to protect macromolecules by stabi-

lizing tertiary structures or by scavenging ROS [95]. However, high accumulation

of osmolytes in transgenic plants can impair the growth in the absence of any

stress probably due to plant adaptation strategies to conserve water in acute stress

[2]. Therefore, a controlled synthesis of osmolytes is the main concern in

designing transgenic strategies for crop improvement. Oligosaccharides such as

raffinose and galactinol are among the sugars synthesized in response to drought.

These compounds seem to function as osmoprotectants rather than providing

osmotic adjustment. Mannitol is one of the most widely distributed sugar alco-

hols in nature, and functions to scavenge ROS and hydroxyl radicals, and also

7.5 Drought Stress | 153

stabilizes the macromolecular structure of enzymes [96]. Trehalose is a non-

reducing disaccharide of glucose, and has been shown to exert its positive

influence during drought by stabilizing membranes and macromolecules. Tre-

halose overexpression helps in the maintenance of an elevated capacity for pho-

tosynthesis primarily due to increased protection of photosystem II against photo-

oxidation [97]. Overexpression of P5CS from Vigna aconitifolia in tobacco, leads to

increased levels of proline and consequently improved growth under drought

stress [98].

7.5.3

Phospholipid Signaling in Drought Stress

Lipids are important membrane components and are also major targets of

environmental stresses including drought stress. The changes in the lipid com-

position may help to maintain membrane integrity and preserve cell compart-

mentalization under water stress conditions. Gigon et al. [99] have shown that in

response to drought, total leaf lipid contents decrease progressively. However, for

leaves with a relative water content as low as 47.5%, total fatty acids still repre-

sented 61% of control contents. The lipid content of extremely dehydrated leaves

rapidly increased after rehydration. In general, phospholipids from plant cell

membranes constitute a dynamic system that generates a multitude of signaling

molecules like IP3, DAG, and phosphatidic acid [53]. In response to stress, PLC is

activated, which catalyzes the hydrolysis of PIP2 into IP3 and DAG. IP3 releases

Ca2þ from internal stores, as described in Figure 7.2. Several studies have shown

that in various plant systems IP3 levels rapidly increase in response to hyper-

osmotic stress [2, 100]. IP3 levels also increase upon treatment with exogenous

ABA in Vicia faba guard cell protoplasts [101] and in Arabidopsis seedlings [102].

Arabidopsis AtPLC is also induced by salt and drought stress [103]. In guard cells,

IP3 induced a Ca2þ increase in the cytoplasm, and leads to stomatal closure and

thus retention of water in the cells [104]. PLD was shown to be rapidly activated in

response to drought stress in two plant species – Craterostigma plantagineum and

Arabidopsis [105, 106]. When drought stress-induced PLD activity was compared

between drought-resistant and -sensitive cultivars of cowpea, it was found that

PLD activities were higher in the drought sensitive cultivars [107].

7.6

Conclusions and Future Prospects

Plant adaptation to different stresses is dependent upon the activation of cascades

of molecular networks involved in stress perception, signal transduction, and

expression of specific stress-responsive genes. The maintenance of intracellular

ionic homeostasis is fundamental to the physiology of a living cell. It is vital for the

cell to keep the concentration of toxic ions below a threshold level and accumulate

essential ions. As stress imposes a major environmental threat to agriculture,


understanding the basic physiology and genetics of cells under stress is crucial for

developing any transgenic strategies. Plants have also evolved mechanisms to

respond at the morphological, anatomical, cellular, and molecular levels for

avoidance of and/or tolerance to various abiotic stresses. In response to stress,

plants respond by gene expression leading to cellular homeostasis and detox-

ification of toxins, ultimately aiming to recovery of growth. These adaptive

mechanisms can be investigated by molecular, biochemical, and physiological

studies.

Transgenic research has opened up a new opportunity in crop improvement

allowing the transfer of desirable gene(s) across species and genera for developing

transgenic plants with novel traits, such as built-in protection, improved nutri-

tional qualities, and so on. Physiological, biochemical, and molecular studies have

revealed that a number of genes are induced by abiotic stresses, and various

transcription factors are involved in the regulation of stress-inducible genes.

Functional genomic studies may provide tools for dissecting abiotic stress

responses in plants through which networks of stress perception, signal trans-

duction, and defense responses can be examined from transcriptomic through

proteomic to metabolomic profiles of stressed tissues. The major attempt to

enhance plant tolerance is the manipulation of genes that are either directly

involved in protection of cells against water loss or the genes that are involved in

regulating signal transduction pathways in response to water stress.

A deeper understanding of the transcription factors regulating these genes, the

products of the major stress responsive genes, and the cross-talk between different

signaling components should remain an area of intense research activity in the

future. The knowledge generated through these studies should be utilized in

producing transgenic plants that are able to tolerate stress conditions without

showing any growth and yield penalty. In the improvement of crops it is very

important to perturb the natural machinery as little as possible and activate the

stress genes at a correct time. Therefore, it is desirable that appropriate stress-

inducible promoters drive the stress genes as well as transcription factors, which

will minimize their expression in nonstressed conditions and thereby reduce yield

penalty. Attempts should be made to design suitable vectors for stacking relevant

genes of one pathway or complementary pathways to develop durable tolerance.

These genes should preferably be driven by a stress-inducible promoter to have

maximum beneficial effects. Additionally, due importance be given to the phy-

siological parameters such as the relative content of different ions present in

the soil as well as the water status of the crop in designing transgenic plants for the

future. A better understanding of the specific roles of various metabolites in stress-

tolerant plants will give rise to strategies for metabolic engineering of plant tol-

erance to abiotic stress.

Much effort is still required to uncover in detail each product of a gene induced

by cold, salinity, and drought stress, and their interacting partners, to understand

the complexity of the stress signal transduction pathways. The role of endogenous

small interfering RNAs in regulating these stresses is also an important area and

will further help to better understand the mechanisms of stress tolerance. The

7.6 Conclusions and Future Prospects | 155

determination of the upstream receptors or sensors that monitor the stress stimuli

as well as the downstream effectors that regulate the responses is essential and will

also expedite our understanding of the stress signaling mechanisms in plants.

Interconnected signal transduction pathways leading to multiple responses to

abiotic stresses have been difficult to study due to their complexity and the large

number of genes involved in the various defense responses. Understanding how

cells coordinate the activity of multiple signaling pathways to prevent unwanted

cross-talk remains a challenge. Transcriptome analyses based on microarrays have

also provided powerful tools for the discovery of stress-responsive genes. The

stress tolerance could also be enhanced by pyramiding various genes. This can be

done by either combining multiple genes of a single protective pathway or by

combining key regulatory genes of different protective pathways. Overall, a com-

bination of a good genetic background with multiple transgenes/allele mining and

promising performance in field conditions will reveal the success of the devel-

opment of abiotic stress-tolerant plants.

Acknowledgments

I thank Dr. Renu Tuteja for critical reading and scientific corrections, Mrs.

Suzanne Karvacic for English corrections, and Mr. Hung Dang Quang for his help

in the preparation of Figure 7.2. I also thank the Department of Biotechnology,

and the Department of Science and Technology, Government of India grants for

partial support. I apologize if some references could not be cited due to space

constraint.

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