[Advances in Botanical Research] Plant Responses to Drought and Salinity Stress - Developments in a Post-Genomic Era Volume 57 || Drought Stress

Drought Stress: Molecular Genetics and Genomics Approaches

MELDA KANTAR, STUART J. LUCAS AND HIKMET BUDAK1

Biological Sciences and Bioengineering Program, Faculty of Engineering

and Natural Sciences, Sabanci University, Istanbul, Turkey

I. I

1C

AdvanCopyr

ntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

orresponding author: E-mail: [email protected]

ces in Botanical Research, Vol. 57 0065-2296/1ight 2011, Elsevier Ltd. All rights reserved. DOI: 10.1016/B978-0-12-387692-8

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II. T he Molecular Biology of Drought . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 47
A
. G rowth Responses ..... .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. 4 47 B . S ignalling Pathways .... .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. 4 48 C . C ompatible Solutes..... .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. 4 54 D . P rotective Proteins ..... .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. 4 54 E . A ntioxidants.... .. .. .. ... .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. 4 58 F . O ther related Molecules ... .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. 4 59
III. T
ranscriptional Regulation of Drought. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 60 IV. P ost-Transcriptional Regulation of Drought . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 62
A
. M icroRNAs .... .. .. .. ... .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. 4 62 B . O ther Post-Transcriptional Mechanisms .... .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. 4 63 C . P ost-Translational Modifications .... ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. 4 63
V. M
olecular Methods of Drought Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 64 A . M odel Organisms.... ... .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. 4 64 B . C onventional Breeding .... .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. 4 65 C . I dentification of QTLs and Marker-Assisted Breeding ..... .. .. .. .. .. .. .. 4 65 D . ‘O mics’ Studies... .. .. ... .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. 4 66 E . T ransgenic Approaches and Functional Studies.... .. .. ... .. .. .. .. .. .. .. .. 4 70 F . B ioinformatics and Databases.... .. .. ... .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. .. .. 4 71
VI. C
onclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 72 R eferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 73
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446 M. KANTAR ET AL.

ABSTRACT

Agriculture faces a constant challenge to increase crop production annually inresponse to human population growth. As land and water resources become limiting,high-yielding crops even in environmentally stressful conditions will be essential.Drought is the single largest abiotic stress factor leading to reduced crop yields, andas such, has been a target of research for some decades. Recently, however, the rapidadvance of molecular biological, transgenic and functional genomics technologies hasfacilitated significant progress in identifying some aspects of the drought response inplants. This chapter summarizes the current state of knowledge of the molecularevents that take place when a plant is under drought stress, starting with the mechan-isms by which the plant perceives drought and the intracellular signalling pathwaysthat are engaged in initiating the drought response. Next, the functional importanceof various biomolecules that are synthesized or activated to protect the plant fromcellular damage during drought are considered. The differing capacity of varieties ofthe same species to respond to drought stress is associated with differing geneexpression patterns, so the mechanisms by which drought-responsive gene expressionis regulated are discussed at the transcriptional and post-transcriptional levels.A large number of genes and gene products have been implicated in the droughtresponse, but identifying which are most useful for breeding drought-resistant cropvarieties remains a significant technical challenge. The second half of the chapter,therefore, surveys the molecular methods that are currently in use for droughtresearch, and ways in which they can be applied to accelerate breeding for droughtresistance. Particular focus is given to post-genomic techniques—transcriptomics,proteomics and metabolomics—assessing the relative strengths and weaknesses ofeach approach and how to make use of the large datasets they produce.

I. INTRODUCTION

Increasing demands on land, water and petroleum mean that simply plough-

ing in more resources is not feasible—we need to produce more from the

existing resources. This is not a new situation; 50 years ago, population

growth threatened to overtake food production. At that point, it was discov-

ered that semi-dwarf mutants of wheat produced much more grain than their

taller relatives. Selective breeding for this and other important traits over the

past half century has led to steady annual increases in grain production, the

so-called Green Revolution.

Unfortunately, this growth may no longer be sufficient to meet future

demand (Tester and Langridge, 2010). Climate change, with the prospect

of increasing environmental stresses, makes stabilizing yields as much of a

challenge as increasing them. This is an important aspect especially for crops

which are of great economic importance (Habash et al., 2009). Drought,

arguably the biggest single abiotic stress factor impacting agricultural pro-

duction (Ergen and Budak, 2009), is increasing globally, affects more than

10% of arable land and reduces average crop yield by more than 50%

MOLECULAR GENETICS AND GENOMICS APPROACHES 447

(Bray et al., 2000). Ironically, the semi-dwarfism trait that boosted grain

yields in the past makes wheat more vulnerable to drought in many cases.

Therefore, the next Green Revolution must develop plant varieties that

produce high yields even under environmental stresses such as drought.

In this chapter, we present an overview of recent gains in understanding of

how plants acclimatize to drought, before outlining new molecular

approaches that promise to facilitate rapid development of drought-tolerant

varieties.

II. THE MOLECULAR BIOLOGY OF DROUGHT

Plants have frequently evolved in habitats where drought occurs, and so have

developed multiple strategies to cope with drought stress. Drought tolerance

is defined as the ability of a plant to live, grow and reproduce satisfactorily

with limited water supply. Tolerance strategies can be divided into resistance

mechanisms, which enable plants to survive dehydration, and avoidance

mechanisms, which are growth habits that prevent the exposure of plant to

osmotic stress, such as deeper rooting or a shorter growth season.

The capacity of a plant to tolerate drought depends largely on the drought-

adaptation mechanisms present within its genome, and how efficiently it can

activate them. Unfortunately, the domestication of modern crops has greatly

reduced the genetic diversity of elite cultivars, and may even have promoted

accumulation of deleterious mutations in their stress response mechanisms

(Tang et al., 2010). Exposure to drought stress in plants leads to cellular

dehydration, causing decreased cytosolic and vacuolar volumes and osmotic

stress. Drought responses of plants includes attenuated growth, altered gene

expression, changes in hormone levels, accumulation of osmoprotective

solutes and proteins, increased levels of antioxidants and suppression of

core metabolism. Drought tolerance is a quantitative trait, with a complex

phenotype including all of these responses and involving a number of genes.

A. GROWTH RESPONSES

Plant response in relation to growth varies according to the tissue, mode/

severity/time scale of the stress and species of concern. Mild osmotic stress

can cause growth arrest in leaves and stems, but no inhibition in root growth.

The growth arrest can be a mechanism for either energy conservation with

reduced metabolism for better subsequent recovery or a support for osmotic

adjustment (Osorio et al., 1998; Sharp et al., 1988; Westgate and Boyer,

1985). Several lines of evidence have supported the role of cyclin-dependent


kinases (CDKs) and cyclin-dependent kinase inhibitors (ICKs) in the regula-

tion of cell division under drought conditions (Schuppler et al., 1998). There

is also evidence linking ICKs with abscisic acid (ABA)-dependent mecha-

nism of drought (Kang et al., 2002; Wang et al., 1998). In a recent study, it

has been shown that Arabidopsis thaliana MYB, discussed further in this

chapter, limits cell expansion since its constitutive expression results in a

dwarf phenotype and small cells (Cominelli et al., 2008). However, drought-

triggered growth sustenance of roots can be an adaptive mechanism for water

uptake. Expansin genes, involved in cell wall loosening, a parameter involved

in cell expansion, were shown to alter their expression patterns in response to

water deficit (Jones and McQueen-Mason, 2004; Wu et al., 2001).

B. SIGNALLING PATHWAYS

1. Signal perception

Plants perceive drought prior to initiating a signalling cascade for appropriate

response. A number of plant osmosensor candidates were proposed.

A receptor-like protein, NTC7, was suggested in a study inwhich its transcripts

were induced in response to osmotic stress and its overexpression induced

osmotic stress tolerance (Tamura et al., 2003). Additionally, Arabidopsis cyto-

kinin receptor, Cre1 (cytokinin response 1), which has a similar structural

organization to yeast osmosensor SLN1 was proposed as an osmosensor

(Reiser et al., 2003). Further, Arabidopsis homologue of SLN1, a plasma

membrane nonethylene receptor histidine kinase, ATHK1, was shown to com-

plement yeast SLN1mutant (Urao et al., 1999).Recently another study showed

that AHK1/ATHK1 positively regulates ABA-related drought response while

other nonethylene receptor kinases called cytokine receptors (CK) including

AHK2, AHK3 and CRE1 are involved in drought-related negative regulation

(Tran et al., 2007b). Further analysis of ATHK1 revealed that it is involved in

drought response not only during early vegetative stages of growth but also

during seed formation. This research showed that it is co-regulated with several

Arabidopsis response regulators and its overexpression induced water deficit

tolerance (Wohlbach et al., 2008). Recently, Oryza sativa receptor-like kinase

(RLK), OsSIK1, was cloned, characterized in relation to kinase activity, and

transgenic work has shown that it is involved in drought tolerance modulating

stomata and activating antioxidative system (Ouyang et al., 2010).

2. Signal transduction

Signalling pathways consist of signalling molecules and a network of protein

interactions which are mediated by reversible phosphorylation in response to

environmental factors including drought. Several components of the signal

TR-4 hTR-4 h

TTD-4 h

TTD-8 h

1,4,5-trifosfatsignaltransductionmechanism

Secondary signal mechanismsbased on the changes on Caconcentration

ABA synthesis

Increase in TF depending on ABA(bZIP, HD-ZIP)

Increase in TF (EREBP)

Ethylenesynthesis

1,4,5-trifosfatsignaltransductionmechanism

Fig. 1. A proposed model for the ABA and ethylene syntheses of wild emmerwheat, TRt (a tolerant genotype) and TTD (a sensitive genotype) under shockdrought stress (4 and 8 h stress).


transduction have been identified although their interactions and positions

along the pathway remain unknown. Differences in signalling between

related genotypes can effect their drought response (Fig. 1).

3. MAPKinases

Mitogen-activated protein kinase (MAPK) cascade includes three protein

kinases (MAPK, MAPKK and MAPKKK) which are activated by serial

phosphorylation, resulting in specific localization of the module in cell com-

partments, phosphorylating and regulating transcription factors and other

proteins. In Arabidopsis, using sequence information, a number of MAPKi-

nases were identified (Ichimura et al., 2002). Some MAPKinases were tran-

scriptionally up-regulated and others were shown to be post-translationally

activated by drought stress (Ichimura et al., 2000; Jonak et al., 1996;

Mizoguchi et al., 1996). Additionally, ADR1, a CC–NBS–LRR gene which

is a homologue of serine/threonine protein kinases was shown to confer

dehydration tolerance consistent with dehydration responsive gene expres-

sion (Chini et al., 2004). Recently, a rice drought-hypersensitive mutant

(dsm1) of a putative MAP kinase kinase kinase (MAPKKK) was identified.

DSM1 protein was shown to belong to Raf-like MAPKKK, localize in the

nucleus, induced in response to water deficit/ABA and confer seedling

drought resistance. It was also proposed as an early signalling component,

a regulator of scavenging of reactive oxygen species (ROS; Ning et al., 2010).


Taking into consideration the above evidence that one MAPKinase can

respond to different stress conditions and there are different numbers of

proposed and identified MAPKinases from each of the three categories up

to date, there should be a convergence in the signalling of MAPK cascade,

possibly different stress factors activating MAPKinases to different levels

(Bartels and Sunkar, 2005).

4. SNF1-like kinases

SNF-1-like kinases, classified into three families, SnRK1, SnRK2 and

SnRK3 are another family of protein kinases which are activated by the

phosphorylation of their serine or threonines (Halford and Hardie, 1998).

In various plant species, several SNF-1 like kinases were predicted and

shown to be expressed in response to dehydration or ABA, including Arabi-

dopsis OPEN STOMATA1 (OST1) protein kinase (Bartels and Sunkar,

2005). Arabidopsis OST1 protein kinase was shown to be involved as a

positive regulator of ABA-induced stomatal closure and regulated negatively

as a substrate of protein phosphatases 2C (PP2C) HAB1. There are lines of

evidence on ABA-bound receptor inhibiting protein phosphatases resulting

in activation of OST1 (Mustilli et al., 2002; Vlad et al., 2009). A recent study

brings evidence for the involvement of distinct phosphorylation mechanisms

in the activation of the two subgroups of SnRK2s. This can be related to their

ABA responsiveness because members of SnRK2 are responsive to osmotic

stress, but only some to ABA (Vlad et al., 2010). In line with these evidences,

analysis of the phosphoproteome in response to ABA treatment leads to the

identification of increases in the phosphorylation states of SNF1-related

kinases after ABA treatment (Kline et al., 2010).

5. Phosphatases

Phosphatases are classified based on their substrates into two major groups

phosphoprotein (serine/threonine) phosphatases (PPases) including PP1,

PP2A, PP2B and PP2C; tyrosine phosphatases (PTPases), receptor-like,

intracellular or dual specific. Phosphatases aid in counteracting the action

of kinases as noted above (Bartels and Sunkar, 2005).

As noted above, there is intense research on the negative role of serine

threonine PP2Cs includingABI1, ABI2 andHAB1 inABA signalling. Studies

with ABI1 and ABI2 mutants have shown in guard cells that ABA activation

of Caþ-permeable channels requires intermediate steps of first ABI1 action,

then ROS, finally ABI2 action (Murata et al., 2001). Two recent independent

studies have revealed substrates of HAB1 as OST1 (as noted above) and

PYL5 from the Bet v1-like superfamily, which was shown to be a cytosolic

and nuclear ABA receptor that activates ABA signalling through direct


inhibition of HAB1 and ABI enhancing drought tolerance (Santiago et al.,

2009; Vlad et al., 2009). In another study, PTPases were shown to be involved

in stomatal closure, downstream of Ca2þ signalling, most probably aiding in

dephosphorylation of an unidentified protein, resulting in subsequent ion flux

from guard cells and stomatal aperture (MacRobbie, 2002).

Moreover, there are several independent studies on various pyrophospha-

tases. One of them is plastidial soluble pyrophosphatases (psPPs), which is

involved in plastidial pyrophosphate (PPi) degradation for continuation of

several metabolic pathways. This work involves transient down-regulation of

psPPs in the leaves of tabacco (Nicotiana benthamiana) resulting in drought

sensitivity, probably resulting from inability of plants in ABA synthesis

(George et al., 2010). In another study, overexpression of vacuole mem-

brane-bound proton-translocating inorganic pyrophosphatase (H(þ)-

PPase), AVP1, was shown to induce drought tolerance in Arabidopsis and

tomato (Lycopersicon esculentum). In tomato, increased pyrophosphate-

driven cation transport into root vacuoles was also observed (Park et al.,

2005). Increased (H(þ)-PPase) activity is thought to acidify the vacuoles

triggering secondary active transport of ions into vacuoles resulting in a

decrease in vacuolar osmotic potential powering water uptake.

6. Phospholipid signalling

One recognized class of osmotic stress-signalling secondary messengers are

phospholipid-derived signalling molecules of the phosphoinositide pathway

which are cleaved from membrane phospholipids by phospholipases. Several

phospholipid-derived secondary messengers, especially inositol 1,4,5-

triphosphate (IP3), diacylglycerol (DAG) and phosphatidic acid (PA), were

shown to be drought related. Phospholipases in the context of drought are

phospholipase C (PLC) and phospholipase D (PLD). PLC cleaves phospho-

lipid phosphatidylinositol 4,5-bisphosphate (PIP2), which is synthesized by a

phosphatidylinositol-kinase, PIP5K, into IP3 and the membrane protein

DAG. PIP5K, PIP, PLC and IP3 levels were shown to be induced in response

to water deficit or ABA in several plant species (El-Maarouf et al., 2001;

Hirayama et al., 1995; Kopka et al., 1998; Mikami et al., 1998; Pical et al.,

1999; Takahashi et al., 2001). Two independent studies together support that

phospholipid signalling involving this pathway is activated through both

ABA-dependent and -independent mechanisms. The current hypothesis is

that drought activation of PLC leads to higher IP3 levels, a subsequent

release of Ca2þ from intracellular stores to cytoplasm, and activation of

Kþ ion channels resulting in stomatal closure (Staxen et al., 1999;

Takahashi et al., 2001). In addition, its initial synthesis, an additional regu-

latory mechanism for inositol phosphate levels, involves the action of


5-phosphatases (5Ptases) or inositol polyphosphate 1-phosphatases. Arabi-

dopsis SAL1 belongs to the latter group mentioned and recently drought-

tolerant Sal1 mutants along with omics studies have supported it as a negative

regulator of both ABA-independent and also -dependent drought-response

pathways (Wilson et al., 2009). In another recent study, an inositol phosphate-

lacking transgenic plant was generated by the expression of inositol polypho-

sphate 5-phosphatase (InsP 5-ptase; Perera et al., 2008). These plants exhibited

higher drought tolerance and ABA-induced stomatal closure. Moreover,

SAL1 was suggested as a regulator of an ABA-independent pathway since

expression of dehydration-responsive element binding protein (DREB), which

will be discussed below, is induced in transgenic plants (Wilson et al., 2009).

Another important secondarymessenger is PLDwhich cleaves phospholipases

producing PA which contains a Ca2þ-binding domain and can activate PLC

(Katagiri et al., 2001). PLDs were shown to be drought or ABA induced in

several plant species (El-Maarouf et al., 2001; Frank et al., 2000; Jacob et al.,

1999; Katagiri et al., 2001; Sang et al., 2001). Interaction of PLD with ABA

effectors supports that PLD is involved in ABA-dependent pathway. There is

supportive evidence of the role of PLD in stomatal closure such as its interac-

tion with ABA effectors including ABI1 (Gampala et al., 2001; Jacob et al.,

1999; Sang et al., 2001). One study has also shown the simultaneous accumu-

lation of PLD and PA in water deficit (Katagiri et al., 2001). Non-specific

phospholipase C (NPC4) hydrolyses phospholipids in a calcium-dependent

manner, producing DAG. In another recent study, transgenic studies of this

messenger reveal that it is converted to PA and involved in ABA sensitivity,

drought tolerance and stomatal closure (Peters et al., 2010).

7. Secondary messengers and calcium

Calcium functions (Ca2þ) as a secondary messenger since different extracel-

lular stimuli eliciting specific and Ca2þ signatures results in a variety of

intracellular response.

Several Ca2þ sensors were implicated to be involved in drought signalling.

Spatial and temporal dynamics of Ca2þ transients in response to drought were

studied revealing increase in cytosolic Ca2þ due to release of Ca2þ from the

vacuole and cell-type specificity of Ca2þ transients (Kiegle et al., 2000; Knight

et al., 1997). One Ca2þ sensor is calcium-dependent protein kinases (CDPKs)

which was reported to be drought induced and the importance of specific

CDPK isoforms in mediating the effects of stress was demonstrated (Ozturk

et al., 2002; Seki et al., 2002). Arabidopsis genome encodes 34 CDPKs and

recently one of them, CPK10, was shown to confer drought tolerance via its

interactionwithaheat shockprotein (HSP)aiding inABA/Ca(2þ) inhibitionof

the inward K(þ) currents resulting in guard cell stomatal closure (Zou et al.,


2010). Another Ca2þ sensor is calmodulin which is Ca2þ-binding protein

activated by increased calcium concentrations and then modulates Ca2þ con-

centrations further by activating specific kinases. A family of calmodulin-

binding transcription activators were first discovered in drought-stressed

Brassica napus (Bouche et al., 2002). Further research revealed osmotic stress

orABA-activated calmodulins inArabidopsis and rice (O. sativa) and suggested

it as a negative regulator of osmotic stress (Frandsen et al., 1996; Perruc et al.,

2004). Another Ca2þ-binding protein is calcineurin B-like protein (CBLs), and

CBL1 was shown to be the only drought induced CBL among 10 identified in

ArabidopsisCBLs (Kudla et al., 1999). Further transgenic studies supported the

role of CBL1 in conferring drought tolerance (Albrecht et al., 2003).

There is also lines of research on annexins which are a family of Ca2þ-dependent membrane-binding proteins. There is supportive evidence that

certain annexins may be targets of abiotic stress-induced cytoplasmic Ca2þ.Their up-regulation in response to ABA or osmotic stress is well documen-

ted. In a recent transgenic study, annexin AnnAt1 was shown to reduce

accumulation of H2O2, and confer increased drought tolerance. AnnAt1

was also shown to be highly susceptible to oxidation-driven S-glutathionyla-

tion, which decreases its Ca2þ affinity and occurs after ABA treatment. This

mechanism probably aids in post-translational regulation of annexins in their

modulation of ROS (Konopka-Postupolska et al., 2009).

Another area of research comprises Arabidopsis AKT1 and Grapevine

(Vitis vinifera) VvK1.1 which are homologous K(þ) channels and were

shown to be regulated by CBL-interacting protein kinase (CIPK) and

Ca(2þ)-sensing CBL partners. Kþ channel expression was shown to be sensi-

tive to drought and ABA. Loss of function of Arabidopsis CIPK23 and over-

expression of rice CIPK12 increased drought tolerance (Cuellar et al., 2010).

8. Salicylic acid and nitric oxide

The role of salicylic acid (SA) in drought signalling is based on the observa-

tion of reduced necrosis in the seedlings of SA-deficient transgenic lines in

comparison to wild types in the presence of mannitol. The general view is that

SA amplifies the effects of water deficit by enhancing the generation of ROS

in photosynthesis (Borsani et al., 2001). Supportive data was established with

studies on Arabidopsis lesion-mimic mutants which misregulate programmed

cell death and used for studying hypersensitive response. A line with consti-

tutive expression of pathogenesis-related (PR) genes 22 (cpr22) was shown to

have elevated levels of SA which was further induced with water deficit.

In the mutant, elevated SA levels were shown to change ABA levels and

ABA-related gene expression. Mutant exhibited reduced responsiveness to

ABA and suppressed responses to drought (Mosher et al., 2010). In a recent


study, functional characterization of phenylalanine ammonia-lyase (PAL)

was undertaken. PAL catalyses the first step of the phenylpropanoid path-

way producing flavonoids, precursors of several secondary metabolites. The

study revealed Arabidopsis pal1 pal2 mutants as drought tolerant and pal1,

pal2, pal3, pal4 mutants as SA deficient, probably resulting from alterations

in their signalling pathways (Huang et al., 2010).

Nitric oxide (NO) is another signalling molecule which was suggested to

have a role in drought tolerance although the nature of its role is unclear.

Exogenous application of NO was shown to confer dehydration tolerance;

conversely, mutants of NO biosynthetic enzymes exhibited reduced NO

content and resistance to dehydration (Lozano-Juste and Leon, 2010; Mata

and Lamattina, 2001). In the initial study, NO application triggered higher

accumulation of late embryogenesis-abundant (LEA) transcripts, which will

be discussed later in detail, and increased stomatal closure. In the latter

study, ABA hypersensitivity of the mutants deficient in NO synthesis sup-

ported the role of NO in ABA-dependent stomatal closure.

C. COMPATIBLE SOLUTES

Compatible solutes are nontoxic molecules that accumulate in the cytoplasm

in response to drought stress and do not interfere with metabolism. Major

compatible solutes are sugars (sucrose, hexose, raffinose-type oligosaccar-

ides, trehalose); sugar alcohols including cyclic polyols (pinitol, D-ononitol);

glycine betaine; and amino acids, most importantly proline.

Current hypothesis on their mode of action ranges from conferring osmot-

ic adjustment, scavenging ROS, stabilizing proteins and cell structures and

adaptive value of metabolic pathways. The accumulation of several compati-

ble solutes was observed to be drought induced and engineering the synthesis

of compatible solutes has been relatively successful (Bartels and Sunkar,

2005). A recent striking evidence is several truncated/recombinant transcripts

of betaine aldehyde dehydrogenase (BADH), the enzyme involved in glycine

betaine synthesis, were observed in monocots. Surrounding the deletion/

insertion sites of these transcripts, sequence similarities, named short, direct

repeats (SDR), were detected (Niu et al., 2007). These can possibly be

recognition sites for post-transcriptional silencing.

D. PROTECTIVE PROTEINS

1. Late embryogenesis-abundant proteins

LEA proteins are a diverse group of proteins expressed normally during

embryogenesis, or in vegetative tissues, in response to ABA or drought stress.

Accumulation of LEA proteins correlates with ABA levels and desiccation


tolerance (Dure et al., 1981; Ergen et al., 2009; Galau et al., 1986). Evidence

from expression profiles and overexpression studies supports a role for LEA

proteins as protective molecules in water deficiency. LEA proteins were

grouped based on conserved structural features (Dure, 1993; Dure et al.,

1989). Group 1 LEA proteins harbour high hydrophilicity and are thought to

be soluble aiding in water binding or replacement. The group 2 (dehydrins)

and group 4 LEA proteins may contribute to the maintenance of protein and

membrane structures. Early response to dehydration proteins of the dehydrin

family was recently shown to have disordered 3D structure enabling them to

maintain in low water concentrations, probably enabling them to act as

chaperones in high ionic strength (Kovacs et al., 2008; Mouillon et al.,

2008). Additionally, a research on spatial and temporal accumulation pat-

terns of group 4 LEA proteins was conducted. This was further followed by a

bioinformatics analysis revealing origination of subgroups of group 4 LEA

with gene duplication events. Generation of transgenic plants confirmed that

the role of group 4 LEA proteins is indispensable in dehydration tolerance

and recovery (Olvera-Carrillo et al., 2010). The group 3 and group 5 LEA

proteins which harbour the most hydrophobicity are thought to sequester

ions, which accumulate due to water deficit. A supporting evidence came

from a recent study in which Arabidopsis LEA5 was shown to confer ABA-

dependent protection against oxidative stress by decreasing photosynthesis

(Mowla et al., 2006). Moreover, lately Medicago truncatula seed desiccation

tolerance (DT) was linked to 11 mostly seed-specific LEA proteins from

different groups (Boudet et al., 2006).

Homeostasis of indole-3-acetic acid (IAA), the main form of active auxin

in plants, is maintained through the conjugation of free IAA to sugars, amino

acids or methyl groups. GH3 family are responsible for converting active

IAA to its inactive form via the conjugation of IAA with amino acids.

Recently, OsGH3.13, which encodes IAA-amido synthetase, exclusively in-

duced by drought stress, was cloned from a gain-of-function mutant tld1-D.

It was shown that activation of TLD1/OsGH3.13 in tld1-D mutant rice

results in down-regulation of IAA leading to the accumulation of LEA

proteins (Zhang et al., 2009).

2. Aquaporins

Changes in water flow is crucial to drought and the rate of water flux into or

out of cells can be determined either by diffusion resulting from water

potential gradient or by aquaporin proteins facilitating osmosis by forming

water-specific pores which increase water permeability of the membrane

shown by aquaporin antisense experiments (Kaldenhoff et al., 1998; Siefritz

et al., 2002). Aquaporins are members of a large superfamily of membrane


spanning proteins called the major intrinsic proteins (MIPs) including tono-

plast intrinsic proteins (TIPs) and plasma membrane intrinsic proteins (PIPs;

Weig et al., 1997). There are several reports that aquaporin genes are induced

by dehydration enhancing water uptake (Fray et al., 1994; Guerrero et al.,

1990; Sarda et al., 1999; Yamada et al., 1997; Yamaguchi-Shinozaki et al.,

1992) and some showing aquaporins are reduced by dehydration which can

allow water conservation (Johansson et al., 1998; Smart et al., 2001; Yamada

et al., 1997). Likewise, most classes of Arabidopsis MIPs are up-regulated in

response to drought, but only some are down-regulated. A recent expression

profiling study of Arabidopsis PIPs revealed that this regulation is consistent

through accessions excluding the special case for three PIP genes. The

relation of PIP genes to drought was further supported by linking variation

of drought-related PIP expression to leaf water content and demonstrating

the presence of drought stress response elements in the promoters of two

PIPs (Alexandersson et al., 2010). Additional supportive evidence on the

interspecies conservation of the aquaporin gating mechanism came from a

recent work on structural dynamic simulations of spinach aquaporin

SoPIP2;1 (Tornroth-Horsefield et al., 2006).

Recently, 10 PIPs from rice were cloned, classified into OsPIP1 andOsPIP1

based on amino acid sequence similarity and shown to be drought responsive

having distinct roles in response to stress with transgenic studies (Guo et al.,

2006). In V. vinifera roots, there are also two classes of PIPs with different

functions: VvPIP2;2 functions as a water channel andVvPIP1;1 interacts with

it to induce water permeability. In a recent work, it was shown that water-

induced diurnal changes in VvPIP1;1 expression follow the same trend with

the amplitude of changes in hydraulic conductance-related variables. This

supports that VvPIP1;1 may dynamically account for water transport capaci-

ty across roots to meet transpirational demand occurring on a diurnal basis in

response to water stress (Vandeleur et al., 2009). Another study supported the

role of PIPs in root hydraulic conductivity which was dropped in the roots of

transgenic plants with low level of PIPs. In this study, antisense RNA was

used to knock down ABA biosynthesis, resulting in transcript and protein

level down-regulation of four PIPs in root tissue, suggesting ABA promotes

root water uptake through aquaporins (Parent et al., 2009).

3. Ion channels

Aside from water uptake and efflux, transporters that regulate ion flow are

also implicated in the drought response, especially in the regulation of

stomatal opening and closure. One player in this sense is proton (Hþ)-ATPases in guard cells, which are known to drive hyperpolarization of the

plasma membrane to initiate stomatal opening. It was previously observed


that Hþ-ATPase activity is diminished by ABA. Recently, mutations in the

OST2 locus which encode Arabidopsis H(þ)-ATPase AHA1 causing consti-

tutive activity were reported (Merlot et al., 2007). Another protein implicated

in this response is Arabidopsis glycine-rich protein 7 (GRP7), which is abun-

dantly expressed in guard cells and involved in regulation of stomatal clo-

sure. When overexpressed, it conferred freezing tolerance on plants but

retarded their germination and growth under drought or high salinity con-

ditions (Kim et al., 2008).

Anion efflux from guard cells precedes stomatal closure and involves slow,

weak voltage-dependent, deactivating (S-type) and rapid (R-type) anion

channels. Recently, SLAC1 gene has been shown to encode a slow, voltage-

independent anion channel component. In a later study, AtALMT12, a

member of the aluminium-activated malate transporter, was found to repre-

sent a guard cell voltage-dependent R-type anion channel. Plants lacking the

transporter were shown to be impaired in stomatal closure in response to

ABA (Meyer et al., 2010).

Ca(2þ)-independent OST1 was identified as an interaction partner of

SLAC1 leading to its activation via phosphorylation. In one study, OST1

was shown to interact with ABI1 which deactivates the SLAC1/OST1 com-

plex via dephosphorylation (Geiger et al., 2009). Similarly, PP2CA was

shown to inhibit the activity of SLAC1 by either direct interaction with

SLAC1 itself or interaction with OSTI leading to inhibition of the kinase

independently of phosphatase activity (Lee et al., 2009b). In another study,

mutants lacking calcium-dependent protein kinases (CDPKs) were observed

to be impaired in ABA stimulation of ion channels. So a Ca(2þ)-sensitive

CDPK was found to interact with SLAC1 and activate it in an ABI1-

dependent manner. Overall, the CDPK and OST1 branch of ABA signal

transduction in guard cells seem to converge on the level of SLAC1 under the

control of the ABI1/ABA-receptor complex (Geiger et al., 2010).

Recently, a K(þ) channel from the Shaker family, named VvK1.1, was

identified from grape (V. vinifera). It was found to be the counterpart of the

Arabidopsis AKT1 channel with similar functional properties such as Kþ

uptake from soil, regulation by CIPK and Ca(2þ)-sensing CBL partners.

Unlike AKT1, its expression was found to be sensitive to drought and ABA

supporting its role in K(þ) loading upon drought stress (Cuellar et al., 2010).

4. Heat Shock Proteins

HSPare important for efficient cellular functions since they are chaperones that

aid in folding and assembly of correctly structured proteins during synthesis,

their maintenance by preventing aggregation by binding and stabilizing

denatured proteins and in the removal and disposal of non-functional and


degraded proteins (Lee et al., 1995). Low water content impairs protein

structure and HSPs are usually only present in vegetative tissues under stress

conditions. HSPs were shown to be dehydration induced in several plants

(Alamillo et al., 1995; Campalans et al., 2001; Coca et al., 1996; Wehmeyer

and Vierling, 2000). Their role in conferring tolerance was shown with

transgenic studies and (Sun et al., 2001) chaperone binding protein from

HSP70 protein family is involved in targeting andwas shown to be water deficit

induced and shown to confer tolerance to drought (Alvim et al., 2001; Cascardo

et al., 2000).

Since HSPs are dehydration-induced genes, there has been research on the

transcriptional mechanisms of drought regulating its expression. One report

revealed DREB 2A, discussed in chapter 7, activates HS-related gene expres-

sion (Sakuma et al., 2006). This was supported by a study in sunflower

(Helianthus annuus) in which small stress protein (sHSP) is activated by the

binding of DREB2 to sHSP promoter and interacting with its known regula-

tor heat stress factor A9 (HaHSFA9; Diaz-Martin et al., 2005). Very recent-

ly, HSP was also identified as an interacting partner of CDPKs and involved

in ABA and Ca(2þ)-mediated stomatal closure leading to drought tolerance

(Zou et al., 2010).

E. ANTIOXIDANTS

Drought stress leads to increased accumulation of ROS, generated mostly in

chloroplast and to some extend in mitochondria, causing oxidative stress.

MajorROSmolecules are singlet oxygen, superoxide anion radicals, hydroxyl

radicals and hydrogen peroxide (H2O2). To detoxify ROS, plants can intrin-

sically develop different types of antioxidants reducing oxidative damage and

conferring drought tolerance. ROS scavengers are either nonenzymatic

(ascorbate (vitamin C), glutathione, tocopherol (vitamin E), flavonoids, alka-

loids, carotenoids) or enzymatic containing superoxide dismutase, peroxi-

dases and catalase. Free radical-mediated lipid peroxidation results in

complex, highly reactive and toxic aldehydes, which are scavenged by either

aldehyde dehydrogenases or aldose/aldehyde reductases. There are lines of

evidence revealing the involvement of these enzymes in drought response

(Kirch et al., 2001; Mundree et al., 2000; Oberschall et al., 2000; Ozturk

et al., 2002; Seki et al., 2002; Sunkar et al., 2003). The osmotic stress involve-

ment of peroxiredoxins which detoxify toxic peroxides was also shown (Seki

et al., 2001; Mowla et al., 2002). Peroxiredoxin is also a potential target of

DREB1A (Kasuga et al., 1999). Thioredoxins function as hydrogen donors,

and their role in water deficit was studied (Broin et al., 2000; Pruvot et al.,

1996; Rey et al., 1998). Peptide-methionine sulphoxide reductases (MsrA) can


counteract the damage caused by the modification of methionine-containing

proteins to methionine sulphoxide [Met(O)] making them vulnerable to pro-

tease degradation and causing loss of function. Their involvement in drought

stress was confirmed with expression and transgenic studies (Moskovitz et al.,

1999; Rodrigo et al., 2002).

Recently, it was demonstrated that changes in the auxin substrate of a

H2O2-responsive enzyme involved in anthocyanin production conferred

drought tolerance (Tognetti et al., 2010). In another study, mutants with

reduced anthocyanin levels were more dehydration tolerant (Huang et al.,

2010). These data support a role of anthocyanins, which are antioxidants, in

drought tolerance related to its interplay with ROS and phytohormones.

Further independent studies have raised new insights into the regulation of

ROS homeostasis in conferring drought tolerance. The implicated molecules

include glutathione peroxidase and cellulase-synthase-like protein (Zhu

et al., 2010). Squalene epoxidase implicated in sterol biosynthesis was also

shown to have a role the localization of NADPH oxidases required for

regulation of ROS (Pose et al., 2009). Isoprene, a product of photosynthesis,

which was also proposed to have antioxidant activity, was suggested to have

a role in drought response (Fortunati et al., 2008).

F. OTHER RELATED MOLECULES

A number of other small molecules have putative roles in the drought

response, although most of these await further confirmation. Recently, an

Arabidopsis mutant was identified with lower content of dolichols, a type of

polyisoprenoid, and increased drought tolerance (Zhang et al., 2008). Subtle

differences were observed between tocopherol (vitamin E) mutant and con-

trols in tolerance to drought stress, but the main focus of the study was on

low-temperature stress (Maeda et al., 2006). Lipocalins are small ligand-

binding proteins and recently an A. thaliana lipocalin AtCHL was function-

ally characterized. Its transcript and protein were found to be induced upon

drought and ABA.With transgenic studies, it was shown that AtCHL aids in

coping with stress conditions, especially damage upon photo-oxidative stress

induced by drought (Levesque-Tremblay et al., 2009).

Recently, an Arabidopsis mutant defective in fatty acid elongase condens-

ing enzyme was isolated. This defective protein was shown to be involved in

suberin biosynthesis which is a polyester found in cell walls. It was transcrip-

tionally activated upon polyethylene glycol-induced drought (Franke et al.,

2009). Also, two transgenic Arabidopsis plants were engineered with altered

enzyme levels resulting in high/low glucosamine (GlcN) levels. Decreased


GlcN resulted in enhanced sensitivity to drought stress, consistent with

previous findings of GlcN-induced ROS generation (Chu et al., 2010).

In transgenic Arabidopsis with higher levels of methyl jasmonate (MeJA)

and wild-type plants with drought-induced MeJA levels, ABA was also

increased. This suggests that MeJA induces ABA biosynthesis under drought

conditions. With further expression analysis, genes that were regulated by

both were identified, including genes involved in ABA or MeJA biosynthesis

in rice (Kim et al., 2009a).

III. TRANSCRIPTIONAL REGULATION OF DROUGHT

Many plants can gradually acclimatize to drought conditions, indicating that

the genetic basis of tolerance is also present in non-tolerant plants to some

extent, and changes in expression of drought-related genes leads to tolerance

acquisition (Zhu, 2001). The expression of many drought-induced gene pro-

ducts is regulated at the transcriptional level. Two transcriptional regulation

circuits induced by drought have been studied in detail, and are labelled the

ABA-dependent and -independent pathways reviewed in detail previously in

this book (in Chapters 6 and 7). However, transcriptional regulation in re-

sponse to drought is complex; the two pathways can overlap since cis elements

for both are known to reside in some genes. It is also highly possible that there

are other cis elements yet to be discovered (Seki et al., 2002). Further, recent

studies demonstrated that transcriptional regulation of abiotic stress converges

with biotic stress regulation, as well as that several additional transcription

factors are involved in drought transcriptional regulation.

In particular, overexpression of a NAC family transcription factor,

SNAC1, was shown to significantly improve drought resistance in rice

(Hu et al., 2006). Another rice NAC, OsNAC6, similarly conferred resistance

to drought as well as to rice blast infection (Nakashima et al., 2007) while

OsNAC10 gave enlarged roots and improved yield during drought under

field conditions (Jeong et al., 2010). Examination of NAC genes in Arabi-

dopsis showed that ATAF1 could be induced both by ABA and by drought in

ABA-deficient mutants, and its overexpression conferred greatly enhanced

drought tolerance (WU et al., 2009). One gene that contains the NAC

recognition sequence, ERD1, also contains a zinc-finger homeodomain

(ZFHD) recognition sequence, and yeast-1-hybrid screening identified tran-

scription factor ZFHD1, which was drought inducible, interacted with NAC

proteins and gave a drought-tolerant phenotype on overexpression (Tran

et al., 2007a).


In maize, the B3 domain-containing transcription factor Viviparous1

(Vp1) was found to be inducible by drought stress and ABA, an effect

mediated by an ABRE and coupling element-binding motifs in its promoter

(Cao et al., 2007). ArabidopsisNFYA5 was also ABA-induced and conferred

drought resistance, but in this case did not contain an ABRE; instead, the

induction was achieved by down-regulation of miR169 (Li et al., 2008b).

Another member of the NFY family, AtNFYB1, was found to confer

improved performance under drought conditions, as did an orthologous

maize transcription factor (Nelson et al., 2007). Additionally, during a screen

for gain-of-function mutants leading to increased drought tolerance, an HD-

START transcription factor was identified (Yu et al., 2008).

All of these factors are likely to act downstream of AREB or DREB-

mediated induction, but there are also proteins that function to modulate

either or both pathways. Transcriptional co-activators are proteins which

enhance the binding of transcription factors to the basal transcription

machinery, and overexpression of one such, MBF1c, enhanced osmotic and

heat stress tolerance (Suzuki et al., 2005). The plant-specific histone deace-

tylase HD2, whose function is not well understood, was found to be re-

pressed by ABA and when overexpressed confer an ABA-insensitive

phenotype (Sridha and Wu, 2006). Among nonethylene receptor histidine

kinases, AHK1/ATHK1 has been shown to up-regulate AREB1, ANAC and

DREB2A, while AHK2, AHK3 and CRE1 seem to be negative regulators of

the same pathways (Tran et al., 2007a,b). Loss-of-function mutants for the

disease-response regulator OCP3 were also found to gain improved drought

resistance in an ABA-dependent manner (Ramirez et al., 2009). WRKY

transcription factor ABO3 was isolated from a screen for mutations giving

altered ABA sensitivity, and shown to modulate the transcription of AREB1/

ABF2 (Ren et al., 2010). Transgenic plants with disrupted IP3 signalling

showed basal up-regulation of DREB2A and a subset of its targets, suggest-

ing that IP3 is a negative regulator of this pathway; surprisingly, the trans-

genic guard cells also had altered responsiveness to ABA, suggesting that

both pathways contribute to the regulation of stomatal closure (Perera et al.,

2008). Again in guard cells, overexpression of a novel nuclear protein NPX1

was shown to decrease stomatal response to ABA, and it was proposed that

this was due to transcriptional repression of an ABA-inducible NAC tran-

scription factor, TIP1 (Kim et al., 2009b).

Another important signal for stomatal closure is the H2O2 level. Recently,

a zinc-finger transcription factor DST (drought and salt tolerance) was

cloned and was shown to negatively regulate stomatal closure by modulating

expression of genes related to H2O2 homeostasis (Huang et al., 2009). Con-

versely, ABA was shown to increase H2O2 production by signalling through


AtMKK1 and AtMPK6 to enhance catalase expression (Xing et al., 2008).

Overexpressing AtMKK1 conferred an increase in drought tolerance.

In summary, the plant response to drought stress involves changes in the

transcriptional levels of literally hundreds of genes, most of which are regu-

lated by multiple transcription factors. Therefore it is perhaps unsurprising

that for many different transcription factors, constitutive overexpression

or loss of function result in a changed drought phenotype. Productive

future research directions should include temporal studies to determine the

sequence in which different transcriptional units are activated during

drought, as well as dissecting the downstream pathways to determine which

transcriptional programmes improve drought resistance without negatively

impacting yield.

IV. POST-TRANSCRIPTIONAL REGULATION OFDROUGHT

A. MICRORNAS

Plant genes involved in responses to stresses such as drought may also be

regulated at the post-transcriptional level, and microRNAs (miRNAs) are

small regulatory non-coding RNA molecules that have a major role in post-

transcriptional regulation, usually by repressing target transcripts. The

involvement of miRNAs in drought is supported by several lines of evidence:

(1) Several elements related to miRNA metabolism have been shown to be

involved in ABA signalling (Nishimura et al., 2005; Xiong et al., 2001). (2) In

plants, miRNAs are involved in several cellular processes including response

to environmental stress (Chen et al., 2005; Dugas and Bartel, 2004; Jones-

Rhoades et al., 2006; Zhang et al., 2006). (3) Several studies show changes in

the expression level of multiple miRNAs in response to abiotic or biotic stress

(Sunkar and Zhu, 2004). Further, analysis of the Sorghum bicolor genome

has indicated that recent duplication of miRNA genes may contribute to this

species’ drought tolerance (Paterson et al., 2009).

Recently, miRNA expression profiling in response to drought was

performed for O. sativa, Populus trichocarpa, Arabidopsis and Triticum

dicoccoides (Kantar et al., 2010a,b; Liu et al., 2008; Lu et al., 2008; Zhao

et al., 2007). Arenas-Huertero and his team constructed a small RNA library

of drought and ABA-treated Phaseolus vulgaris (Arenas-Huertero et al.,

2009). Independent studies showed drought stress responsiveness of miR-

NAs in Triticum aestivum, Hordeum vulgare, Brachypodium distachyon and

Euphorbiaceae (Jia et al., 2009; Kantar et al., 2010a,b; Wei et al., 2009; Yao


et al., 2010; Zeng et al., 2010). Analysis of Arabidopsis transcriptome under

drought stress with the whole-genome tiling techniques has also revealed

several unannotated non-coding stress-induced RNA molecules including

miRNAs, splicing and processing variants (Matsui et al., 2008; Zeller et al.,

2009). The overall results indicate that miRNA expression profiles are con-

served to some extent in different species, but further studies are required

because the details of their response often differ (Sunkar, 2010).

B. OTHER POST-TRANSCRIPTIONAL MECHANISMS

There are other possible mechanisms of post-transcriptional regulation,

although none have yet been demonstrated to have a role in drought

responses. In several cereal species, numerous transcripts with truncations

and/or recombination around SDRs in the 50 exonic region of the two BADH

genes were detected. These unusually processed transcripts were not found in

dicotyledonous plants, so this may represent a cereal-specific regulation

mechanism for biosynthesis of the important osmoprotectant glycine betaine

(Niu et al., 2007).

C. POST-TRANSLATIONAL MODIFICATIONS

Ubiquitination is a eukaryotic post-translational protein modification that is

mediated by the cascade of E1, E2 and E3 ubiquitin (Ub) ligases, most often

targeting ubiquitinated proteins for degradation by the 26S proteasome. It is

involved in regulating numerous cellular functions and has been implicated

in various aspects of the drought response. For example, an E3 Ub ligase

from hot pepper, Rma1H1 and its Arabidopsis orthologue were both shown

to confer strongly increased drought tolerance when overexpressed in Arabi-

dopsis. This phenotype was mediated by targeting the aquaporin isoform

PIP2:1 for proteasomal degradation, thus reducing water loss (Lee et al.,

2009a). In contrast, E3 Ub ligases PUB22 and PUB23 had a negative effect

on drought responses, apparently by interacting with the proteasome regula-

tor RPN12a (Cho et al., 2008). Two further E3 Ub ligases, SDIR1 and

AtAIRP1, have been shown to be ABA and drought inducible and act as

positive regulators of the ABA signalling pathway, acting upstream of ABFs

(Ryu et al., 2010; Zhang et al., 2007).

SUMOylation enzymatically resembles ubiquitination, with the difference

that the peptide added to the modified protein is SUMO rather than ubiqui-

tin. In the Arabidopsis genome, SIZ1 is the only E3 SUMO ligase. During

drought stress, plants accumulated higher levels of SUMOylated proteins,

while null mutant siz1-3 plants were highly drought sensitive. Subsequent


analysis of the entire transcriptome under drought stress in wild-type and

siz1-3 mutants indicated identified 262 drought-inducible genes that required

SIZ1 for their induction, including components of the ABA response, and

some that were induced independently of ABA or DREB2A (Catala

et al., 2007).

Finally, C-terminal modification of proteins by isoprenylcysteine methyl-

transferase (ICMT) seems to inhibit ABA signalling, although which pro-

teins are isoprenylated remains unclear. Overexpression of isoprenylcysteine

methylesterase (ICME), which removes isoprenylation, increased ABA sen-

sitivity, with ABA inducing ICME expression in a positive feedback mecha-

nism (Huizinga et al., 2008).

V. MOLECULAR METHODS OF DROUGHTRESEARCH

Drought-sensitive and -resistance model plants, conventional breeding,

marker-assisted breeding, omics studies, transgenics and functional methods

are all major research areas with the aim of developing drought-tolerant

crops to increase crop productivity. In two recent independent reviews,

conventional and molecular tools of drought research are discussed (Fleury

et al., 2010; Reynolds and Tuberosa, 2008).

A. MODEL ORGANISMS

Studies on water deficit have advanced by focusing on drought-tolerant

plants, the most remarkable of which are ‘resurrection plants’ such as Cra-

terostigma plantagineum (Bartels and Salamini, 2001). These can tolerate

desiccation in environments with long arid periods, appearing dried out

and dead but recovering rapidly during seasonal rainfall. Interestingly,

a relative of C. plantagineum that is not found in arid environments,Lindernia

brevidens, exhibits the same desiccation tolerance, showing that this adapta-

tion does not severely impact growth in non-arid habitats (Phillips et al.,

2008). The fern Mohria caffrorum has been proposed as a valuable model

organism for verifying protection mechanisms because it cycles seasonally

between desiccation-tolerant and -sensitive phenotypes (Farrant et al., 2009).

Equally valuable is the wild germplasm of wild relatives of crop species

which have adapted to a broad range of environments and contain rich

genetic diversity (Nevo and Chen, 2010). For example, the progenitors of

cultivated wheat and barley, T. dicoccoides and Hordeum spontaneum, have

drought-response traits which have been identified and transferred to


cultivated species leading to improved drought tolerance. To collect and

preserve the genetic diversity of wild relatives, seed germplasm banks have

been established as a source of genes for improving agricultural crops

(Tanksley and McCouch, 1997). Other crops including soybean are also

major lines of research (Manavalan et al., 2009).

B. CONVENTIONAL BREEDING

Utilization of this wild germplasm requires first screening for a wild donor

line with the desired trait (such as drought resistance) and then crossing this

line with an elite cultivar with good agronomic characteristics. Traditionally,

screening was carried out by observation of phenotypes (conventional breed-

ing). By crossing two lines with different advantages and shortcomings,

selecting for physiological traits and interspecies/intergeneric-wide crossing/

backcrossing techniques, conventional breeding has played an important role

in the past century for improving drought stress tolerance (reviewed by

Ashraf, 2010). Drought-tolerant lines of crops such as peanut, common

bean, safflower, chickpea, wheat, tall fescue, soybean, wheatgrass, barley

and maize have been developed using conventional breeding techniques.

One challenge to applying this approach is identifying phenotypes that

correlate well with drought tolerance. Recently through an Arabidopsis

genetic screen, Xiong et al. (2006) proposed that drought/ABA inhibition

of lateral root growth is a good indicator of drought tolerance. Meanwhile in

Brassica rapa, early flowering was shown to be rapidly evolved adaptation to

escape drought (Franks et al., 2007).

C. IDENTIFICATION OF QTLS AND MARKER-ASSISTED BREEDING

Conventional breeding has major limitations, including the need for multiple

backcrosses to eliminate undesirable traits, restriction to loci that give a

clearly observable phenotype and inadequacy if the gene pool lacks sufficient

variation in the trait of interest. Therefore, the focus is currently on marker-

assisted breeding, which allows ‘pyramiding’ of desirable traits for more

rapid crop improvement with less input of resources.

Marker-assisted breeding is of great value for drought-related studies

due to the complex and additive polygenic nature of the trait (Mohammadi

et al., 2005; Thi Lang and Chi Buu, 2008; Zhao, 2002). This approach is made

possible by the development of DNA markers that can be used to construct

detailed genetic linkage maps. A plethora of different types of DNA markers

that have been described for stress tolerance such as RFLPs, RAPDs, CAPS,

PCRindels, AFLPs, microsatellites (SSRs) and SNPs (reviewed by Ashraf


et al., 2008). A fundamental application of linkage maps is to localize the

sequences encoding drought-related, quantitatively inherited traits (quantita-

tive trait loci, QTLs). QTL mapping and identification of drought-associated

QTLs has been performed for several crops including maize, wheat, barley,

cotton, sorghum and rice (Bernier et al., 2008; Quarrie et al., 1994; Sanchez

et al., 2002; Saranga et al., 2001; Sari-Gorla et al., 1999; Teulat et al., 1997).

However, the complexity of the intergenic and gene–environment interac-

tions in drought makes robust identification of QTLs difficult, as shown by

the contrasting results of similar QTL mapping studies in rice (Babu et al.,

2003; Bernier et al., 2008; Kamoshita et al., 2008; Kumar et al., 2007).

Of several crops, rice has been the major focus of marker-assisted breeding

for drought tolerance, which led to the release of the first highly drought-

tolerant rice variety, Birsa Vikas Dhan 111 (PY 84; Bernier et al., 2009;

Steele, 2009). Selection of QTLs to breed drought-tolerant crop cultivars

has also been performed in pearl millet, cotton, maize, barley and sorghum

(Baum et al., 2003; Bidinger et al., 2007; Harris et al., 2007; Levi et al., 2009a,

b; Ribaut and Ragot, 2006; Serraj et al., 2005). QTL analysis is now being

used alongside genomic approaches. For example, Street et al. (2006) com-

bined phenotyping and QTL analysis of a poplar mapping population with

transcript microarrays to identify drought-inducible genes that co-localized

with QTLs known to be associated with drought resistance.

D. ‘OMICS’ STUDIES

The complex nature of the drought response means that, to understand it, the

plant must be viewed as a complete system, rather than just looking at

individual components. This is made possible by the use of ‘omics’ techni-

ques, that examine all or representative subset of a plant’s genes, transcripts,

proteins or metabolites (Urano et al., 2010). The availability of genome

sequence data for crop species is undoubtedly important for drought

research, but as the drought response is largely mediated by changes in

protein expression, the current focus is on other ‘omic’ methods such as

transcriptomics, proteomics and metabolomics to elucidate plant responses

to drought. In general, the most technically straightforward and by far the

most widely used approaches are those which survey RNA transcripts.

1. Transcriptomics

Transcriptomics techniques include microarrays (e.g. AtGenExpress; Kilian

et al., 2007) and next-generation sequencing-based profiling methods

(e.g. RNA-Seq), which measure the abundance of thousands of transcripts

in parallel. To date, over 100 publications have used microarrays to describe


the transcriptomes of a total of 28 species exposed to osmotic stress (reviewed

by Deyholos, 2010). With their high potential, microarrays have several

limitations including working with a predefined probe set and a high rate

of false positives necessitating confirmation of results with RT-PCR or other

techniques. Some of the limitations such as sensitivity, resolution and narrow

probe sets have been overcome with high-throughput sequencing. To date, a

few such analyses of plants have been reported, in addition to previous

reports of serial analysis of gene expression (SAGE) studies of several abiotic

stresses (Barakat et al., 2009; Byun et al., 2009; Eveland et al., 2008; Molina

et al., 2008; Moon et al., 2007). High-throughput sequencing is also the most

powerful method available for detecting novel small, non-coding RNAs such

as miRNAs (reviewed by Unver et al., 2009) and natural antisense transcript

RNAs (Zhou et al., 2009).

A major weakness of transcriptomics techniques is that steady-state tran-

script levels often do not accurately represent gene or protein expression

because of post-transcriptional regulation (Branco-Price et al., 2008;

Kawaguchi et al., 2004). One approach to offset this is by the selection of

polysome (i.e. multiple ribosomes) associated transcripts during RNA

extraction, by immunopurification of epitope-tagged ribosomes (Arava

et al., 2003; Ederth et al., 2009; Mustroph et al., 2009; Zanetti et al., 2005).

In transcriptomics studies, the exact stress treatment applied can have a large

effect, as shown by comparative analyses of different drought treatments

(Bray, 2004; Talame et al., 2007). Moreover, the importance of consistency in

tissue sampling was demonstrated by a microarray analysis of the effects of

water stress in four serial, transverse segments cut from the maize root apex

(Spollen et al., 2008). Methods of selecting individual cells or tissues for

transcript analysis, such as fluorescence-activated cell sorting (FACS) and

microdissection, are also being developed (Deyholos, 2010). Comparative

transcriptome analysis of related species, genotypes or ideally near isogenic

lines differing in their drought tolerance is a powerful strategy and has been

reported in Arabidopsis, rice, wheat, sugarcane and Andean potato (Aprile

et al., 2009; Ergen et al., 2009; Mane et al., 2008; Mohammadi et al., 2007,

2008; Rabello et al., 2008; Rodrigues et al., 2009; Wong et al., 2006). For

example, von Korff et al. (2009) undertook profiling of allele-specific expres-

sion variations in different barley hybrids, revealing that expression levels of

the same stress-related genes frequently varied between hybrids because of

variation between different alleles in their cis-regulatory sequences. In anoth-

er transcriptomics study, Ergen et al. generated a subtractive cDNA library

from drought applied and control wheat tissues sequencing 13,000 expressed

sequence taqs (ESTs; Ergen and Budak, 2009).


As we have noted above, drought stress responses primarily involve tran-

scriptional regulation of gene expression. This can depend on the interaction

of transcription factors with cis-regulatory sequences, or post-transcriptional

regulatory mechanisms such as miRNA function. One use of microarrays is

to identify cis-regulatory elements adjacent to transcripts co-regulated by

drought (Ma and Bohnert, 2008). Transcriptomic approaches are also useful

in describing the phenotype associated with a mutation in a transcription

factor (Fowler and Thomashow, 2002; Hu et al., 2006; Jiang and Deyholos,

2009; Jiang et al., 2009; Li et al., 2008a,b; Maruyama et al., 2004; Sakuma

et al., 2006; Yokotani et al., 2009), alongside validation techniques such as

chemically inducible transcription factors and chromatin immunoprecipita-

tion (Waters et al., 2009). Further, transcript profiling can confirm whether

other endogenous or exogenous signalling molecules affect the drought

response by detecting changes in expression of known drought-induced

genes (de Torres-Zabala et al., 2007; Kim et al., 2009a; Perera et al., 2008;

Suzuki et al., 2005; Tran et al., 2007a,b; Wilson et al., 2009).

The relative ease with which a microarray, once set up, can be probed with

multiple samples allows time-course analysis of the drought response, which

provides greater resolution by identifying time-specific expression patterns as

well as core genes up-regulated throughout the test period (Harb et al., 2010;

Wilkins et al., 2009, 2010).

In theory, it should be straightforward to characterize novel, protein-

coding, stress-related genes with transcriptomic approaches. An overview

of drought-induced transcriptomics results indicates a decrease in the abun-

dance of transcripts related to primary energy metabolism, photosynthesis

and protein synthesis, and an increase in stress-signalling, transport

protein, hydrophilic, osmoprotective and antioxidative-related transcripts

(Bray, 2002; Catala et al., 2007; Gong et al., 2005; Jia et al., 2006; Jiang

and Deyholos, 2006; Sahi et al., 2006). Transcriptome analysis of a

triple knockout mutant for AREB/ABFs identified drought-regulated genes

that had not been detected in single or double knockouts (Yoshida et al.,

2010).

However, despite its theoretical utility, no pathway or confirmed major

component of the stress response was first identified through transcriptomics

(Munns and Tester, 2008; Takeda andMatsuoka, 2008; Yamaguchi-Shinozaki

and Shinozaki, 2006). This could be due to the limitations of transcriptomics

highlighted above, especially the lack of correlation between transcript levels

and function. So transcriptomics is an indispensable tool especially when

studying gene networks, but it should be used as only an initial screening

tool of validated with subsequent functional assays. Transcriptome data

should be integrated with other types of linkage or expression data to identify


candidate genes. For example, drought stress-induced transcript expres-

sion profiles have been used to identify candidate genes associated with

QTLs (Diab et al., 2008; Golkari et al., 2009; Gorantla et al., 2005; Street

et al., 2006).

2. Proteomics and metabolomics

Protein and metabolite profiling, typically carried out using separation techni-

ques such as 2D gel electrophoresis or chromatography followed by analysis by

mass spectrometry (MS), allow direct assessment of changing levels of proteins

or metabolites. Protein abundance is not perfectly correlated with functional

activity because of post-translational modifications, localization and associa-

tion with othermolecules, but is generally more representative of function than

transcript levels. For example, a protein phosphatase profiling strategy was

important in establishing the role of SNF1-related kinases in the ABA signal-

ling pathway, by showing that one such kinase, OST1, is a target for the PP2C

HAB1 (Vlad et al., 2009). Further, use of 14N/15N labelling and MS to profile

the phosphorylation state ofArabidopsis proteins followingABA treatment not

only identified the proteins known to be involved in the ABA signalling

pathway but also 20 proteins not previously known to be regulated by

ABA (Kline et al., 2010). Meanwhile, metabolite profiling and the measure-

ment of 30 different enzyme activities found that Arabidopsis drought adapta-

tion did not involve major changes to carbon metabolism, with Kþ and

organic acids being the main contributors to osmotic adjustment (Hummel

et al., 2010).

In M. truncatula seed radicles, proteomic analysis highlighted 15 polypep-

tides linked to DT (Boudet et al., 2006). A further proteomic analysis of

Medicago root nodules was able to identify subsets of both plant and

bacterial proteins involved in the drought response (Larrainzar et al.,

2007). Assessment of mild drought stress effect on key metabolites showed

rapid accumulation of respiratory substrated succinate and sucrose as well as

antioxidant enzymes (Naya et al., 2007). This was taken to indicate a loss of

bacterial respiration caused by oxidative damage, leading to the loss in

nitrogen fixation in root nodules under even mild drought stress.

Recently, proteomic analysis has also been carried out in bread wheat

(Peng et al., 2009) identifying 93 root and 65 leaf proteins that show differ-

ential expression under stress, and demonstrating the substantial overlap

between responses to drought and salt stress.

Despite their limitations, microarrays tend to identify an order of magni-

tudemore gene products than reported in proteomic studies (Baginsky, 2009).

Therefore, integration of transcriptomic data with proteomic and metabolo-

mic studies can be an effective way of confirming the inferences derived from


both methods. For example, metabolomic profiling of an Arabidopsis

NCED3 knockout mutant indicated that during drought, accumulation of

osmoprotective amino acids was dependent on ABA synthesis, whereas that

of raffinose was not. Additional transcriptomics studies revealed that biosyn-

thesis of branched-chain amino acids during drought was controlled by ABA

at the transcriptional level (Urano et al., 2009). A combination of transcrip-

tomic and metabolomic approaches were used to investigate the ability of

A. thaliana leaves to grow and develop at a similar rate to controls in spite of

mild osmotic stress (Skirycz et al., 2010). Under these conditions, ethylene

and gibberellin but not ABA-based regulatory circuits were implicated, and

changes in mitochondrial function were shown to be important. In particular,

overexpression of a mitochondrial alternative oxidase led to increased

drought resistance. Accordingly, metabolite and transcript profiling in the

absence of alternative oxidase 1a (Giraud et al., 2008) showed that these

plants suffered acute sensitivity to drought stress and widespread perturba-

tions in the expression of stress response genes. However, datasets produced

by ‘omic’ methods are not always easy to interpret. In a comprehensive study

of Populus euphratica drought acclimation combining transcript, protein and

metabolite profiling under arid field conditions, no correlation was found

between levels of drought-responsive proteins and their corresponding tran-

scripts (Bogeat-Triboulot et al., 2007).

E. TRANSGENIC APPROACHES AND FUNCTIONAL STUDIES

Transgenic methodology is an alternative to ongoing breeding programmes,

and has the advantage of transferring only the desired genes from one species

to another. Potentially, this allows only the drought tolerance genes from

stress adapted wild relatives to be incorporated into cultivated lines, without

adjacent loci that might reduce yield. Additionally, transgenic plants provide

the clearest way to study the function of a candidate drought-resistance gene,

allowing modification of a single gene on an otherwise identical genetic

background. Functional relevance of the candidate gene can be demon-

strated by confirming that loss-of-function and overexpressing mutants

have opposite phenotypes. These functional studies are most frequently

carried out in A. thaliana, due to the availability of a large collection of

sequence-tagged insertional knockout mutants (e.g. Salk Lines), its rapid

generation time and ease of genetic transformation (Alonso et al., 2003).

However, there are certainly functional differences in the drought response

between Arabidopsis and some crop species, particularly monocots; for this

reason, efforts are underway to develop other transgenic systems, such as the

model grass B. distachyon (Vogel et al., 2006). Of course, even validation in a


model system does not guarantee that a transgene will perform identically in

a crop plant. For example, transgenic tomato plants overexpressing NCED3

to increase ABA biosynthesis surprisingly did not appear to be more resistant

to soil drying than controls, but rather showed better water-use efficiency

under well-watered conditions (Thompson et al., 2007). In other cases, the

performance of a gene in Arabidopsis does correlate well with its action in a

crop plant, such as transgenic expression of the AP2-type transcription factor

HARDY in rice (Karaba et al., 2007). Many of the studies described in the

above text used transgenic techniques to elucidate the function of all kinds of

drought-related genes, and are detailed under the relevant sections.

Many other functional approaches have also proved valuable in dissecting

drought tolerance:

. X-ray crystallography was important in demonstrating the function of

the ABA receptor PYL1 (Miyazono et al., 2009; Nishimura et al., 2009)

and the conformational changes undergone by spinach aquaporin

(Tornroth-Horsefield et al., 2006).. Protein–protein interactions have been investigated using yeast two-hy-

brid, in vitro pull-down, in vivo co-immunoprecipitation experiments

(Cho et al., 2008) and isothermal titration calorimetry (Santiago et al.,

2009).. Use of reporter gene constructs to detect changes in expression (Li et al.,

2008b; Zhang et al., 2007).. Transactivation assays using yeast assay and yeast one hybrid systems

(Guan et al., 2009; Liao et al., 2008).. Colocalization with GFP-tagged proteins (Wormit et al., 2006).. Physiological assays such as stomatal bioassay (Perera et al., 2008) and

patch clamp (Meyer et al., 2010).. Protein-promoter element binding by Northern blot (Ren et al., 2010)

and electrophoretic mobility shift assays (Cao et al., 2007).. Quantitative PCR analysis (Kim et al., 2009a).. Fourier transform infrared spectroscopy to detect changes in LEA pro-

teins (Boudet et al., 2006).

F. BIOINFORMATICS AND DATABASES

Some of the online resources and/or tools that have proved useful or appli-

cable in drought-related research are The Generation Challenge Programme

(GCP, a comparative plant stress-responsive gene catalogue), AthaMap,

MultiGO and DRASTIC-INSIGHTS. Their applications include elucidat-

ing orthologous/paralogous relationships; identifying co-regulated genes,


deducing functional gene sets from clustered expression data; and construct-

ing signal transduction pathways (Button et al., 2006; Galuschka et al., 2007;

Kankainen et al., 2006; Wanchana et al., 2008).

Online databases contain valuable material for drought analysis. For

O. sativa, Lee and colleagues have assembled available transcriptomic data

into a database called RiceArrayNet, and using it demonstrated a correlation

between expression patterns of different abiotic stress-inducible genes (Lee

et al., 2009c). Tools are now being developed to aid in mining these data-

bases; for instance, Geisler et al. (2006) developed an algorithm to verify

putative cis-regulatory elements by correlating their occurrence in genomic

DNA sequences with results of transcriptome profiling studies, and con-

firmed that it worked for the ABRE and DRE/C-repeat in Arabidopsis.

Similarly with bioinformatic tools, Arabidopsis genes that are differentially

regulated by drought were shown to contain unusually high levels of G-

quadruplexes, which are tandem stretches of guanines that can associate in

hydrogen-bonded arrays stabilized by K(þ) ions (Mullen et al., 2010).

The complexity and multivariate nature of drought stress also makes com-

puter simulation of drought responses an attractive approach, and there is now

enough data to construct useful models (reviewed by Tardieu and Tuberosa,

2010). These frequently focus on one aspect of the drought response. For

example, a dynamic model of guard cell signal transduction network for

ABA-induced stomatal closure, including 40 previously identified network

components, has been assessed. This can be used as a tool for the identification

of candidate manipulations for conferring drought tolerance (Li et al., 2006).

More recently, photosynthesis in the C3 plants, which can thrive in optimal

environments, was modelled using method called Minimization of Metabolic

AdjustmentDynamicFluxBalanceAnalysis (M_DFBA).Then its performance

was assessed under drought conditions showing highly cooperative regulation

resulting in robust photosynthesis even under stress (Luo et al., 2009).

VI. CONCLUSION

Drought research has been underway for many years, but the past decade has

seen an explosion of the molecular tools and techniques available to tackle

this and other intractable biological questions. The best prospect for facing

future demands on agriculture will be to combine the best of traditional

breeding techniques with the latest innovations and complementary research

in model organisms.


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[Advances in Botanical Research] Plant Responses to Drought and Salinity Stress - Developments in a Post-Genomic Era Volume 57 || Drought Stress