climate change and plant abiotic stress tolerance || plant environmental stress responses for...

Part Two

Abiotic Stress Tolerance and Climate Change

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Climate Change and Plant Abiotic Stress Tolerance, First Edition. Edited by Narendra Tuteja and Sarvajeet S. Gill.� 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

4

Plant Environmental Stress Responses for Survival

and Biomass Enhancement

Yuriko Osakabe, Keishi Osakabe, and Kazuo Shinozaki

Abstract

Environmental stresses caused by climate change, such as drought, high salinity,low and high temperature, and light conditions, are predicted to become moresevere and widespread. The environmental stresses decrease plant photosyntheticcapacity and cause excess light stresses, and lead to defects in plant growth andbiomass productivity. Plants with long-term growth periods, such as perennialcrops and woody plants, are particularly damaged by long-term stresses. Manyplant species have evolved complex mechanisms for growth adjustment andadaptation to various environmental conditions. Elucidation of the molecularmechanisms involved in water stress tolerance and optimization of water-useefficiency that define the crop quality as the ratio of biomass are major breedingtargets for crop improvement under drought stress conditions. In this chapter, wehighlight the molecular mechanisms that control plant stress responses for growthadaptation and development. In addition, we summarize the major strategies ofphoto-protective mechanisms in chloroplasts used to prevent excess light damage.Finally, we discuss progress in genetic engineering aimed at breeding improvedenvironmental stress tolerance in plants, including crops and woody species, forenhanced biomass production.

4.1

Introduction

Plant biomass is primarily a product of photosynthesis, a process requiring carbondioxide (CO2), water, minerals, and solar radiation. Plant biomass productivity isseverely affected by adverse environmental stresses resulting from climate change.Globally, drought stress is a major agricultural problem, and “drought tolerance” istherefore a key objective for breeding crops with increased survivability and growthunder stress conditions. “Water-use efficiency” (WUE) and “water productivity”define the crop quality as the ratio of biomass, and are crucial breeding targets forcrop improvement under drought stress conditions. Many plant species have

Climate Change and Plant Abiotic Stress Tolerance, First Edition. Edited by Narendra Tuteja and Sarvajeet S. Gill.� 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

81

evolved complex mechanisms for growth adjustment and adaptation to variousenvironmental conditions. During stress conditions, plants must allocate energy toreprogram their stress-signaling networks for survival [1–5]. Enhanced tolerance towater stress is an important trait for the breeding of many crops and woody plants.Water stress affects various cellular and molecular events, including stomatalclosure, metabolic processes, and expression of several genes involved not only instress tolerance, but also the stress response. Thus, the signaling system of plantsfor adaptation and survival under environmental stress conditions has beenextensively studied. Elucidation of the molecular mechanisms involved in waterstress tolerance is essential for improving the tolerance and adaptation of crops andwoody plants to drought stress conditions.Plant biomass is mainly derived from carbon captured by photosynthesis. Light is

an essential energy source for photosynthesis. However, excess light has harmfuleffects on plants, such as photooxidation of chlorophyll, which leads to increasedproduction of highly reactive intermediates that cause damage to biologicalmacromolecules and decrease plant productivity. Abiotic stresses, such as drought,high salinity, temperature stress, and nutrient deprivation, decrease photosyntheticcapacity and cause excess light stresses. Furthermore, when plants suffer fromdrought stress, a decrease in leaf water potential and stomatal opening leads toreduced CO2 availability, and therefore lower photosynthetic rates. Additionally,molecular studies have demonstrated that drought and high-salinity stresses causedownregulation of photosynthesis gene expression [4–7], and therefore a decreasein plant growth. Limitation of these effects may constitute a strategy for enhancingplant adaptation to stress.Response to water stress is regulated by an orchestrated, but complex, signaling

network, involving cross-talk with other signaling pathways [2]. In this chapter, wewill summarize the molecular mechanisms involved in plant stress response andgrowth adaptation to water stress. Drought and salinity stress signaling andabscisic acid (ABA) signaling are integrated into complex regulatory networks. Thebasis of core ABA signaling involving the ABA receptor complex consisting of ABAreceptor family PYR/PYL/RCAR, protein phosphatase PP2Cs, and Snf1-relatedprotein kinases 2 (SnRK2s) is described [8].In this chapter, we will focus on the essential signaling networks of water stress

responses associated with ABA signaling. In addition, we will summarize themajor strategies of photoprotective mechanisms in chloroplasts, to prevent excesslight damage during water stress. Finally, we will discuss progress in geneticengineering, aimed at breeding improved environmental stress tolerance in plants,including crops and woody species, for enhanced biomass production.

4.2

Stomatal Responses in the Control of Plant Productivity

Adjustment of growth and development, through changes in stomatal and rootactivity, is one of the most important plant adaptation systems to drought. Plants

82 4 Plant Environmental Stress Responses for Survival and Biomass Enhancement

control stomatal responses to assimilate CO2 through photosynthesis and also toprevent excess desiccation. During drought stress, ion and water transport systemsin the plasma membrane and tonoplast are used to control turgor pressure changesin guard cells and trigger stomatal closure. The plant hormone ABA has beenshown to play a critical role in drought stress. ABA is rapidly produced underdrought and salinity stress conditions, and induces the signaling cascades ofstomatal closure. By controlling the internal water status, the ABA signaling systemaffects plant tolerance and adaptation to stress. CO2 also acts as a signalingmolecule in stomatal responses. Increased CO2 concentrations in the leaves inducestomatal closure, whereas low CO2 concentrations and high humidity triggerstomatal opening. Therefore, WUE is also affected by increased atmospheric CO2

concentrations [9]. Stomatal closure is linked to heat stress, because decreasedstomatal conductance causes an increase in leaf temperature. Thus, drought,elevated CO2 concentrations, and high temperature become interrelated and affectphotosynthetic activity. Elucidation of the stomatal control mechanisms is thereforecrucial to the enhancement of plant biomass production and crop yields underconditions of global climate change.

4.2.1

ABA Biosynthesis and Transport

Drought stress induces ABA accumulation in plants. The transcription of 9-cis-epoxy carotenoid dioxygenase 3 (NCED3) from Arabidopsis, which catalyzes the firststep of ABA biosynthesis from carotenoids, is induced by drought stress. Moreover,nced3 mutant plants showed reduced water loss and decreased drought tolerance,suggesting that NCED3 functions as a key gene for ABA biosynthesis duringdrought stress [10]. The NCED3 gene and its protein are expressed and localized invascular parenchymal cells [11–13]. It has been suggested that ABA is the root-to-shoot signal that induces stomatal closure in response to drought stress in the soil.Christmann et al. demonstrated an increase in ABA levels in the leaves when waterstress applied to the roots resulted in reduced water status in the shoots. Incontrast, when the water stress applied to the roots did not affect the water status inthe shoots, ABA levels in the leaves were not increased [14,15]. Under water stressconditions, the site of ABA biosynthesis is considered to be mainly in leaf vasculartissues and the synthesized ABA is transported to guard cells to trigger stomatalclosure. ABA is a weak acid and may therefore be transported from conditions oflow pH to conditions of high pH without ABA transporters via a passive diffusionmechanism in response to pH changes. Alternatively, ABA may be transported byspecific transporters during stress.Recently, two members of the membrane-localized ABC transporter family –

ABCG25 and ABCG40 – were independently isolated from Arabidopsis showingdecreased ABA sensitivity during germination and stomatal closure [16,17].ABCG25 was revealed to have ABA export activity, whereas ABCG40 was shown tofunction during ABA import into plant cells. ABCG25 was expressed mainly invascular tissues, and was induced by ABA and drought stress [16]. ABCG40 was

4.2 Stomatal Responses in the Control of Plant Productivity 83

expressed in guard cells [17]. These findings led to the development of a model inwhich the ABA synthesized in response to drought stress is transported to theoutside of vascular cells by ABCG25 and imported to the guard cells via ABCG40activity. Subsequently, ABA signaling in the cytosol induces stomatal closure. Lossof function of ABCG25 and ABCG40 was shown to be inconsistent with thephenotypes of ABA-deficient mutants [18], suggesting that the ABA transportsystem contains redundant functions and additional transporters, or passivetransport with a pH gradient function [18]. ABA receptors on the plasmamembrane (GTG1 and GTG2) have been reported [19]; the functions of thesereceptors may also explain the redundant phenotypes of ABCG40 mutants [18].Recently, Seo et al. identified another ABA importer, ABA-importing transporter

(AIT)-1, which has been characterized as the low-affinity nitrate transporter,NRT1.2 [20]. To isolate ABA transporters, a modified yeast two-hybrid screeningsystem was used, in which Arabidopsis cDNAs were screened as the componentscapable of inducing interactions between the ABA receptor PYR family and PP2Cunder low ABA concentrations [20]. The ait1/nrt1.2 mutants exhibited openstomata phenotypes and decreased sensitivity to ABA during seed germination orpostgermination growth, whereas AIT1/NRT1.2 overexpression resulted in ABAhypersensitivity. Interestingly, AIT1/NRT1.2 is expressed at the site of ABAbiosynthesis (i.e., vascular tissues), suggesting that ABA import systems in thevasculature are important for the regulation of water stress responses. Further-more, cross-talk between ABA/drought stress and nitrate signaling may occur.

4.2.2

Signal Mediation of Stomatal Aperture

During drought stress, guard cells perceive increased ABA levels; their turgor andvolume are subsequently reduced, and the stomata close [9,21]. ABA is known totrigger the production of reactive oxygen species (ROS), which induce an increasein cytosolic [Ca2þ]cyt and thereby activate two distinct types of anion channels: slow-activating sustained (S-type) and rapid transient (R-type). These anion channels areactivated differentially; S-type anion channels generate slow anion efflux, while R-type anion channels are activated transiently. Anion efflux causes membranedepolarization, and leads to a decrease in inward Kþ channels (KAT1/KAT2) andHþ-ATPases, which control cell turgor during stomatal opening. Membranedepolarization also activates outward Kþ channels, such as GORK (gated outwardlyrectifying Kþ channel), thereby resulting in Kþ efflux from guard cells. The anionsand Kþ effluxes from guard cells lead to a loss of guard cell turgor, followed bystomatal closure [22–25]. Hþ-ATPase has a proton efflux activity, which is inducedby blue light and low CO2 concentrations. This proton efflux activity results inhyperpolarization of the guard cell plasma membrane, thereby causing stomatalopening. Membrane hyperpolarization activates inward Kþ channels and induceswater uptake into guard cells (Figure 4.1).Recent studies have identified the various ion transport systems involved in

stomatal responses. SLAC1 (slow anion channel-associated 1) has been isolated by


means of screening for ozone-sensitive or CO2-insensitive mutants [24,25]. Theslac1mutant exhibited reduced stomatal closure to ABA, CO2, Ca

2þ, and ozonetreatments, and its guard cells showed impaired Ca2þ and ABA activation of S-typeanion channels. The SLAC1 gene encodes a 10-transmembrane domain protein,with a similar structure to bacterial dicarboxylate/malate transporters [24,25]. Theactivity of SLAC1 as an anion channel was estimated using Xenopus oocyte systems[26,27], suggesting that SLAC1 functions as a major S-type anion channel in guardcells [25]. Current findings indicate the direct activation of S-type anion channels byABA. SLAC1 is directly activated by SRK2E/OST1/SnRK2.6, which is involved in theABA-signaling complex of ABA receptor, PYR family, and PP2Cs [26,27], or by thecalcium-dependent protein kinases, CPK21 and CPK23 [28]. SRK2E also inhibitsKAT1 activity through phosphorylation [29]. These results suggest that thecomplicated, but direct, control mechanisms of ion channels by ABA signaling mayplay an important role in enhancing the signaling system during stomatal responses.Arabidopsis mutant AHA1/OST2 (Arabidopsis Hþ-ATPase 1/open stomata 2) has

been identified. The ost2-1 and ost2-2 dominant mutants exhibited constitutive Hþ-ATPase activity, ABA insensitivity, and stomatal closure defects [30]. The ATP-binding cassette(ABC) protein AtMRP5 (multidrug resistance protein 5) has beenshown to function in ABA-induced stomatal closure [31,32]. A loss-of-functionstudy suggested that AtMRP5 functions as a regulator of several guard cell signaltransduction mechanisms, rather than directly as an ion channel [33]. The

Figure 4.1 Schematic illustration of plant water stress responses. Stomatal response, ROS

scavenging, metabolic changes, and photosynthesis activity are affected under water stress and

adjust plant growth rates.

4.2 Stomatal Responses in the Control of Plant Productivity 85

disruption of the AtMRP5 homologous gene, AtMRP4, impairs stomatal opening[34]. The vacuolar Kþ channels also contribute to stomatal closure, through Kþ

release from the vacuoles [35]. TPK1 (two pore Kþ channel 1) mediates guard cellVK channel currents; moreover, ABA-induced stomatal closure is decreased in thetpk1 mutant [36]. The redundant phenotypes in the loss-of-function analyses of theion transport systems mentioned above suggest the existence of additionalpathways in the stomatal responses of plants.The signaling systems involved in CO2-mediated stomatal closure have not yet

been fully elucidated. Increased CO2 concentrations activate anion channels,thereby causing membrane depolarization and triggering the activity of outwardKþ channels. An HT1 (high leaf temperature) mutant, which impaired CO2-inducedstomatal responses, has been identified. The HT1 gene encodes a protein kinase,indicating the requirement for phosphorylation activity in CO2-mediated stomatalsignaling [37]. The ht1 mutants respond to ABA and blue light, suggesting thatHT1 functions in the upstream pathway during these stomatal responses.ABCGB14, a member of membrane-localized ABC transporters, has been shownto be involved in malate transport during CO2-induced stomatal closure. However,no CO2 sensors have yet been isolated, and the signaling mechanisms involvedinCO2-mediated stomatal responses remain unclear [38].During stomatal closure, second messengers also play signaling roles. ROS,

calcium ions, phospholipids, and nitric oxide (NO) are induced by ABA, and act assignaling molecules during stomatal closure [39]. ABA-mediated stomatal closureinvolves Ca2þ-dependent and also Ca2þ-independent signaling pathways. Severalcalcium-dependent protein kinases (CDPKs) have been identified as being involvedin ABA signaling. Double mutations of the CPK3 and CPK6 genes led to decreasedABA-induced activation of Ca2þ channels, ABA-/Ca2þ-induced activation of S-typeanion channels, and stomatal closure [40]. The double mutants for CPK3 and CPK6showed impaired ABA sensitivity in stomatal closure, resulting in decreasedtolerance to drought stress. ABA acts as a trigger for hydrogen peroxide (H2O2)production, through plasma membrane-localized NADPH oxidases [40]. TwoArabidopsis genes for NADPH oxidase (AtrbohD and AtrbohF) have been identifiedas functional NADPH oxidases during stomatal closure. Mutant analysis of thesegenes revealed that they abolished ABA-induced ROS production and [Ca2þ]cytincreases, and thereby stomatal closure [41]. Recently, it was reported that SRK2E isable to phosphorylate AtrbohF, but not AtrbohD in vitro [42], suggesting the directactivation of ROS production by core ABA signaling.

4.2.3

Guard Cell Development

Many aspects of stomatal morphology and physiology have evolved to optimizegaseous exchange, photosynthesis, and WUE under drought stress conditions.Stomatal differentiation in the epidermis of plants is initiated by a series ofasymmetric cell divisions, and involves cell–cell communication to establishnumbers and arrangement [43]. The erecta (ER) family, which encodes leucine-rich


repeat, receptor-like kinases (LRR-RLKs) and consists of ER, erecta-like 1 and 2(ERL1 and ERL2), mediates various plant developmental processes, includingstomatal development [44]. The multiple mutant plants of the ER family exhibitedabnormal stomatal phenotypes, with a high density of mis-patterned stomata [45].The ER family interacts with the LRR receptor-like protein, TMM (too manymouths) and controls stomatal patterning in a synergistic manner [46]. Recentfindings suggest that the different types of receptor–ligand pairs between ER/TMMand epidermal patterning factors (EPFs) function in the specification of differentprocesses involved in stomatal development [47–50].The downstream pathway of ER signaling has been characterized as a mitogen-

activated protein kinase (MAPK) cascade, which includes MAPKKK YODA, MAPKkinases MKK4, MKK5, MKK7, and MKK9, and MAPKs MPK3 and MPK6 [51].They target basic helix–loop–helix (bHLH) transcription factors, SPEECHLESS(SPCH), MUTE, and FAMA, to control stomatal development [43]. ICE1/SCRM1and SCRM2, which encode another bHLH subfamily and interact physically withSPCH, MUTE, and FAMA, are also involved in stomatal development [43].Stomatal development may also be regulated by environmental factors, such as

light and CO2 [52]. The amount of light energy available during stomataldevelopment affects stomatal density and stomatal index. The photoreceptor, PhyB,and a bHLH transcription factor, PIF4, play major roles in stomatal development[53]. Stomatal numbers are further affected by the atmospheric CO2 concentration.Research on stomatal characteristics and CO2 concentrations over long geologicaltime periods indicates that stomatal density is negatively correlated with CO2 levels,whereas stomatal size is positively correlated [54,55]. In addition, Lake andWoodward reported that aba mutant plants, which are defective in ABA biosynth-esis, display higher stomatal densities [56]. The regulation of stomatal conductanceand transpiration rates is linked to stomatal development, and stomatal develop-ment is controlled by systemic signaling in response to environmental conditions[52]. However, the molecular relationship between the ER-mediated signalingcascades and environmental factors remains unclear. Elucidation of the modulationof signaling pathways by environmental factors will facilitate the breeding of plantswith enhanced adaptation to adverse growth conditions.

4.3

Signaling and Transcriptional Control in Water Stress Tolerance

4.3.1

Signaling Mediation by Membrane-Localized Proteins

Receptor and sensor proteins localized to the membranes play an essential role invarious signaling processes of multicellular organisms. These membrane-boundreceptor proteins convey information to their cytoplasmic target proteins viacatalytic processes such as protein kinase activity. AHK1, a histidine kinase in thetwo-component signaling system, mediates osmotic stress signaling in prokaryotes

4.3 Signaling and Transcriptional Control in Water Stress Tolerance 87

and has been shown to function as an osmosensor; overexpression of histidinekinase enhanced tolerance to drought stress in Arabidopsis [57,58]. Furthermore,ahk1 mutants exhibited decreased sensitivity to ABA, and also downregulation ofABA and stress-responsive gene expression, indicating that the osmosensor AHK1acts as a positive regulator of osmotic stress signaling [58,59]. The AHK1downstream cascades are potentially controlled by AHPs and ARRs in the multipleHis–Asp phospho-relay; however, the factors that receive the signals from AHK1,and also the precise signaling cascades, remain to be determined. In contrast, thephytohormone cytokinin receptor histidine kinases, AHK2, AHK3, and AHK4,have been shown to negatively regulate ABA and water stress signaling [58,60,61].Multiple mutants of ahk2, ahk3, and ahk4 showed increased sensitivity to ABA, andalso enhanced tolerance to cold, salt, and drought stresses [58,60]. These findingssuggest the existence of cross-talk among ABA, cytokinin, and stress-signalingpathways [58].Receptor-like kinases (RLKs), which form a large gene family in plants, contain

Ser/Thr kinase as a cytosolic domain, while having structural elements similar toanimal receptor tyrosine kinases. In Arabidopsis, the RLK family includes morethan 600 members, with the LRR-RLKs constituting the largest group [62]. Severalreceptor-like kinases that are localized to the plasma membrane are also known tobe involved in the early steps of osmotic stress signaling in Arabidopsis [63–68], rice(Oryza sativa) [69], Medicago truncatula [70], and Glycine soja [71]. These RLKspossess a variety of extracellular domains (e.g., LRR, an extensin-like domain, or acysteine-rich domain), indicating that various environmental stimuli may activatethe RLK-mediated signaling pathways. There is increasing evidence that RLKs canplay either a positive or a negative regulatory role in abiotic stress responses.RPK1 (receptor-like protein kinase 1) is an LRR-RLK, the expression of which is

induced by ABA, dehydration, high salt, and low temperature. Loss of function ofRPK1 revealed ABA insensitivity and reduced expression levels of various waterstress-responsive genes, indicating positive regulation of ABA/stress signaling byRPK1. Microarray analysis of the Arabidopsis RPK1 loss-of-function mutantidentified a number of downregulated stress-related genes, including ROS-relatedgenes [63]. ROS production is activated during biotic and abiotic stresses, includingpathogen attack, excess light, osmotic stress, heavy metal stress, and herbicides.ROS act as important second messengers for stress-responsive signal transductionpathways [41,72–76]. Various water stress-responsive genes, including ROS-relatedgenes, were consistently upregulated in Arabidopsis RPK1-overexpressing plants[65]. RPK1 transgenic plants exhibited increased tolerance to drought and oxidativestress, suggesting that RPK1 controls ROS homeostasis, and thereby the mechan-isms regulating water and oxidative stress response in Arabidopsis. In anindependent study, Lee et al. [67] reported that RPK1 also functions in ABA-dependent leaf senescence. RPK1 has mainly been identified in the genomes ofBrassica species, suggesting a specific regulatory function in this genus. In contrast,the orthologous protein, RPK2/TOAD2, with high similarity in the kinase domain,was identified in diverse plant species (Figure 4.2) [77]. RPK2 controls cell fate inanthers [78], embryo development [79], and stem cell homeostasis in the shoot


Figure 4.2 Signaling pathway of stomatal

development and closure. (a) A meristemoid

mother cell (MMC) that is differentiated from

a protodermal cell in the developing leaf

divides asymmetrically and enters the

stomatal lineage to create a meristemoid. The

meristemoid differentiates into a guard

mother cell (GMC), which divides once

symmetrically and produces two guard cells.

(b) Model of EPF peptide ligands and

receptors. EPF1 and EPF2 bind to TMM and

ER family receptors, and inhibit stomatal

development. STOMAGEN promotes

stomatal development and may compete with

the binding EPF1/2 and the receptors. (c)

Model of the signaling pathway in stomatal

closure. Light activates proton (Hþ)-ATPases(e.g., OST2) in guard cells and this initiates

inward-rectifying Kþ channels (e.g., KAT1)

that have an essential role in stomatal

opening. During water-deficit stress, ABA

binds to the receptor PYR/PYL family and

forms the receptor complex with PP2Cs,

which can bind to SnRK2s to inhibit their

kinase activity and act as negative regulators

in the signaling pathway. The activated

SRK2E/OST1 then inhibits KAT1, and

phosphorylates and activates NADPH

oxidase to produce H2O2 that is the second

messenger to promote Ca2þ release.

Moreover, SRK2E/OST1 and CPK

phosphorylate and activate the S-type anion

channels, such as SLAC1, which triggers

membrane depolarization and induces Kþ

outward rectifying channel (e.g., GORK1)

activation.


apical meristem through the mediation of CLV3 [80,81]. These findings indicatethat RPK2 is one of the important RLKs governing plant development in variousspecies.Recent research has demonstrated that other RLK members are involved in water

stress signaling. The proline-rich extensin-like receptor kinase 4 (PERK4), fromArabidopsis, functions as a positive regulator in ABA responses [64]. The perk4mutant exhibited decreased sensitivity to ABA during seed germination, seedlinggrowth, and root tip growth, and also ABA-induced [Ca2þ]cyt increases in the roots.This finding suggested that PERK4 is an ABA- and Ca2þ-activated protein kinase,involved at an early stage of ABA signaling, to control the inhibition of root cellelongation [64]. A cysteine-rich RLK (CRK), CRK36 (an abiotic stress-inducible CRKfrom Arabidopsis [82]) was identified by coexpression analyses and yeast two-hybridscreening as a potential interacting factor with ARCK1, which encodes a receptor-like cytoplasmic kinase (RLCK) [68]. CRK36 acts as a negative regulator of osmoticstress and ABA signaling. The knockdown of CRK36 resulted in increasedsensitivity to ABA and osmotic stress during postgerminative growth, and also theupregulation of ABA-responsive genes (such as late embryogenesis abundant (LEA)genes), oleosin, ABA-responsive transcription factors, ABA-insensitive 4 (ABI4), andABI5. This finding indicates that CRK36 physically interacts with and phosphor-ylates ARCK1. Thus, by forming a complex, CRK36 and ARCK1 may control ABAand osmotic stress responses through a negative feedback mechanism [68].

4.3.2

Stress-Responsive Transcription

Environmental stress responses are regulated by multiple signaling pathways thatmediate the expression of various stress-responsive genes. These genes function instress responses and also in stress tolerance [1,83]. Recent transcriptome andproteome analyses have focused on responses to osmotic stress in various plantspecies. The results have indicated the involvement of general processes in alteringthe gene expression of plants, including crops and woody plants, such asArabidopsis, rice, maize, wheat, barley, sorghum, soybean, tomato, chickpea, cotton,poplar, loblolly pine, grapevine, and cassava [84–101]. Most of these works haveused macro- or microarrays and recently they identified the mass of transcriptomesusing next-generation sequencing. The isolated transcriptomes showed similarfunctional genes and also species-specific responsive genes. Key genes involved instress tolerance and stress signaling, such as osmoprotectant biosynthesis genes,LEA and chaperone genes, ROS homeostasis-related genes, ABA biosynthesis andsignaling genes, and ion homeostasis and signaling genes, together with theirtranscription factors, have been identified in those transcriptome studies.To investigate drought stresses encountered by field crops, a controlled moderate

drought treatment system has been developed in Arabidopsis [91]. This system hasenabled the detection of ABA accumulation, the induction of genes related to ABAsignaling, ion channels, and ROS scavengers, and the decrease of stomatalconductance as early responses to drought stress. The stress-responsive genes


upregulated during the early stage of this system also involve cell wall expansions,which may function in cell wall adjustment. In addition, the ABA–jasmonateantagonistic pathway was detected in drought stress response; at the late stage ofmoderate drought, plants altered their jasmonate–ABA balance and showedreduced growth [91]. Another approach to developing an experimental model forcombined stresses in the natural environment, particularly heat and droughtstresses, has been to use transcriptome analysis [102]. This technique also providesclues to understanding stress response in leaves, because stomatal closure duringdrought stress results in elevated leaf temperatures.Woody plants are severely affected by environmental stresses during their

lengthy growth periods. Damage to forest trees resulting from environmentalstresses caused by climate change is an important issue globally. To understand theadaptive responses of forest trees, transcriptome and proteome studies have beenconducted on several species [4,5]. A transcriptome study of responses to thevarious levels of drought stress in loblolly pine revealed the differential expressionof heat shock protein (HSP), LEA, and phenylpropanoid biosynthetic genes [103].HSPs primarily function as molecular chaperones during stress, and playimportant roles not only in thermotolerance, but also in adaptation to variousenvironmental stresses [4,5,104,105]. In Populus euphratica, a salt-tolerant poplar,the ionic/osmotic homeostasis-related and HSP genes were shown to beupregulated during stress [106]. Proteomic analysis of two Populus cathayanaaccessions (native species to wet and dry regions of China) identified drought-responsive proteins involved in the regulation of transcription and translation,photosynthesis, ROS scavenging, and HSPs, and also enzymes involved in redoxhomeostasis and secondary metabolism [107]. In this way, transcriptome studiesenable the identification of molecular gene expression responses that are commonamong plant species. Thus, the control of osmotic stress responses in woody plantsis associated with ROS signaling, including production and scavenging.

4.3.3

Key Transcription Factors

Key stress-responsive transcription factors, including the MYB, MYC, AP2/EREB,bZIP, NAC, and WRKY families, have been identified and shown to controldownstream stress-responsive gene expression. These transcription factors func-tion in drought tolerance of several plant species, including crops and woody plants[4,5,108–111]. The drought-responsive cis-element (DRE/CRT/LTRE, 50-TACCGA-CAT-30) has been identified in the promoter regions of drought-, salinity-, and coldstress-inducible genes. DREB1/CBF (DRE-binding protein 1/CRT-binding factor)and DREB2 [112,113], the transcription factors that specifically recognize the DRE/CRT sequence, have been identified and shown to form the DREB/CBF family,which is a subfamily of the plant-specific AP2 (apetala 2)/ERF transcription factorfamily [114]. Drought and salinity stresses are partially mediated by ABA, whichinduces expression of various genes through the ABA-responsive cis-element,called ABRE. DRE/CRT and ABRE have been identified in many stress-responsive


gene promoters, suggesting that ABRE also plays a role in stress-responsivetranscription [1]. Here, we will focus on the transcription factor families identifiednewly as being involved in drought stress responses.Recently, novel types of transcription factors with critical functions in drought

stress responses have been identified. A C2H2-type transcription factor, DST(drought and salt tolerance), controls the expression of genes involved in H2O2

homeostasis, and mediates H2O2-induced stomatal closure and abiotic stresstolerance in rice [115]. A transcription factor that is structurally related to humanNF-X1 (nuclear transcription factor X-box binding 1) from Arabidopsis was shownto contribute to salt and defense responses [116,117]. A drought stress-induciblenuclear transcription factor Y, NFYA5, controls stomatal aperture and droughttolerance [118]. The GRAS/SCL-type transcription factor gene, PeSCL7, has beenisolated from P. euphratica, a salt-tolerant species. PeSCL7 was induced during theearly stages of severe salt stress; overexpression of this gene in Arabidopsis inducedenhanced tolerance to drought and salt stresses [119]. Several transcription factorsalso regulate stomatal closure during drought stress. SNAC1 (stress-responsiveNAC1) is expressed in rice guard cells; overexpression of this gene inducedenhanced ABA sensitivity, stomatal closure, and drought and salt tolerance [120].AtMYB60 and AtMYB61 are expressed mainly in guard cells, and are characterizedas important transcription factors for the regulation of stomatal aperture anddrought tolerance. AtMYB60 is a negative regulator of stomatal closure, whereasAtMYB61 is a positive regulator; loss of function of AtMYB61 induced stomatalopening [121,122]. OCP3, which encodes a homeodomain transcription factorfamily, also plays a role in ABA-induced stomatal closure and drought resistance[123]. Further elucidation of the molecular targets of these transcription factors anddownstream key factors by means of transcriptome and proteome studies willprovide an in-depth understanding of the regulation networks of plant stressresponses. This knowledge will facilitate the genetic engineering of useful cropsand woody plants.

4.4

Protection Mechanisms of Photosynthesis During Water Stress

Photosynthesis, which provides energy and essential metabolites to controlplant growth and productivity, is primarily affected by water stress. Waterstress affects photosynthesis directly (through decreased CO2 availabilitybecause of stomatal closure [6,124,125] or by means of changes in photosyn-thetic metabolism [126]) and also indirectly (through ROS production [127]).Under water stress conditions, when photosynthesis is downregulated, excesslight has a negative effect on photosynthesis [5]. Plants have evolved a range ofdirect and indirect mechanisms for sensing and protecting against excess light[127]. Here, we will focus on cross-talk responses between water stress andexcess light stress, and the transcriptional regulation of photosynthetic genesduring these stresses.


Excess light is also associated with the production of ROS such as H2O2,superoxide (O2

��), and singlet oxygen (1O2), which are generated by distinctphotochemical and biochemical processes. The generation of 1O2 is promoted bytriplet-excited-state chlorophyll under conditions of excess light. An Arabidopsisfluorescent (flu) mutant accumulated protochlorophyllide released 1O2 upon a dark-to-light shift [128]. Microarray analysis of the flu mutant and plants treated withmethyl viologen showed distinct 1O2 and H2O2/O2 transcriptional responses, inwhich a large number of nuclear genes were induced by 1O2 [129]. H2O2

upregulates the expression of many genes that overlap with those in plantssubjected to various environmental stresses, such as methyl viologen, heat, cold,and drought [130,131]. The expression of cytosolic ascorbate peroxidases (APXs),which play a role in cytosolic H2O2 scavenging, responds to early excess light stressand the redox state of plastoquinone [132]. In Arabidopsis, the APX2 gene was alsoinduced by drought stress and ABA [133]. Loss-of-function mutants of APXexhibited a protective role for chloroplast proteins under excess light conditions[127]. These findings suggest that APXs function in ROS scavenging duringresponse to excess light and also to water stress. Further studies are required tomore fully elucidate the interaction of cytosolic ROS scavenging by APXs and theprotective mechanism of photosynthesis.ROS molecules can additionally act as signals for transcriptional changes. Several

transcription factors have been shown to be involved in the response to excess lightand ROS. Zinc finger transcription factors, ZAT10 and ZAT12, are induced byexcess light acclimation and ROS treatment in Arabidopsis, and regulate theresponse to oxidative stresses [102,134]. ZAT12 is also induced by variousenvironmental stresses, such as salinity, heat, cold, and wounding, suggesting thatit too is involved in multiple and cross-talk pathways in stress responses [127].Excess light regulates nuclear gene expression by chlorophyll intermediates. Mg-protoporphyrin IX (Mg-Proto) has been proposed as a retrograde plastid-to-nucleussignal. The genomes uncoupled (gun) mutants, gun4 and gun5, impair the generationof Mg-Proto, which has been shown to act as a signal to repress LHCB geneexpression in Arabidopsis [135–137]. LHCB expression was also repressed underthe control of GUN1 and ABI4, which encodes a transcription factor and isinvolved in ABA signaling [138]. These factors are thought to be involved inmultiple retrograde signaling pathways. Moulin et al. [139] re-examined theproposed role of Mg-Proto and other chlorophyll intermediates as signalingmolecules, and reported that none of the chemicals could be detected in ROS-induced plant materials under conditions in which nuclear gene expression wasrepressed. They hypothesized that the extremely short-lived Mg-Proto (which isaccumulated light-dependently) may generate 1O2 under excess light conditionsand that a much more complex ROS signal may be generated during chloroplastdestruction. There is evidence for the regulation of nuclear gene expression by 1O2

[129] and H2O2 [140], and a role for these ROS, either individually or incombination, requires further investigation. These studies implicate a complex butcoordinated molecular mechanism of photosynthesis protection under waterstresses to adapt and survive to the stresses.

4.4 Protection Mechanisms of Photosynthesis During Water Stress 93

Transcriptome and proteome studies have indicated that photosynthesis-relatedgenes are downregulated during stress responses in Arabidopsis, rice, and poplar[83,98,106]. Recent research has focused on the photosynthetic acclimationprocesses during mild water stress to identify adaptive responses in the naturalenvironment. Transcriptome profiles of loblolly pine under drought stress werecorrelated with physiological data, showing photosynthetic acclimation to milddrought stress and inhibition of photosynthesis under severe drought stress[92,103]. Under long-term or mild stress conditions that mimic the naturalenvironment, plants acclimate to the stress by means of various molecular andphysiological processes, which control plant growth and development [91,125,141].Further investigation of the mechanisms involved in these studies may facilitate anunderstanding of coordinated growth control regulation during stress and alsoacclimation to long-term stress.

4.5

Metabolic Adjustment During Water Stress

Several chemical hydrophilic compounds that are synthesized and accumulatedduring water stress are known as osmolytes. These osmolytes, which function tomaintain cell turgor, and also stabilize proteins and cell structures during stress,include raffinose family oligosaccharides, sucrose, trehalose, sorbitol, proline, andglycine betaine [142–144]. The expression levels of genes encoding enzymesinvolved in the biosynthesis of osmolytes have been found to be upregulated duringstress; moreover, overexpression of the stress-inducible osmolyte synthetic genes intransgenic plants enhanced stress tolerance [142–144]. Here, we will summarizerecent advances in metabolomics studies to detect stress-responsive metabolites.We will focus on several plant cell biomacromolecules, particularly cell wallcompounds with roles in water stress responses.

4.5.1

Metabolomic Study of Primary Metabolites

Metabolomic approaches have been used to explore the composition of compoundsunder water stress conditions [84,145–153]. Metabolomics in temperature stresshas been conducted using gas chromatography-mass spectroscopy (GC-MS) andGC-time-of-flight (TOF)-MS [147,154], and revealed an overlapping of the majorityof metabolites in response to low and high temperature conditions; proline,glucose, fructose, galactinol, and raffinose functioned during tolerance totemperature stresses. Transgenic plants that overexpressed DREB1A/CBF3 weretolerant of drought and cold stress [155–157], and accumulated more galactinol andraffinose than did wild-type plants [158,159]. Metabolomic investigation ofDREB1A/CBF3 revealed similarity between low-temperature-responsive metabo-lomes (e.g., monosaccharides, oligosaccharides, and sugar alcohols), suggestingthat their levels are controlled by DREB1A/CBF3 [146,159]. Comparison of


metabolites between cold-treated DREB1A/CBF3-overexpressing plants and con-stitutively active form of DREB2A (DREB2A-CA)-overexpressing transgenic plantsshowed that DREB2A overexpression did not increase the accumulation of low-temperature-responsive metabolites [159]. This finding suggests that the accumula-tion of low-temperature metabolites is tightly controlled by transcription factorsand that stress-specific metabolic pathways function during various stresses.The model halophyte was used for metabolome analysis to identify salt stress

responses and genome diversity [150]. In comparison with Arabidopsis, Thellungiellamaintained higher levels of osmolytes in the presence, and also the absence, of saltstress. Transcriptome analysis revealed that several stress-related genes wereupregulated in Thellungiella, even in the absence of salt stress [150]. Comprehen-sive metabolomic analysis of the salt stress response was performed with anArabidopsis cell culture [152]. GC-MS, liquid chromatography (LC)-MS, and time-course profiling of the changes in metabolites under salt stress conditions revealedthat the short-term responses to salt stress included induction of the methylationcycle for the supply of methyl groups, the phenylpropanoid pathway for ligninproduction, and glycine betaine production [152]. Long-term responses involvedcoinduction glycolysis and sucrose metabolism, and also coreduction of themethylation cycle. Using the woody plant, grapevine, Cramer et al. studied theearly and late changes in transcript and metabolite profiles. GC-MS profiling andanion-exchange chromatography showed that different metabolites were accumu-lated in response to salt and drought stress, and that glucose, malate, and prolinewere accumulated in higher quantities during drought stress than during saltstress. In addition, metabolomic differences were shown to be correlated withdifferences in transcriptomes [84]. Integrated metabolomic and transcriptomeanalysis of drought stress responses in the NCED3 mutant was performed usingGC-TOF-MS, CE-MS, and DNA microarrays [160]. The results indicated thatmetabolite profiling during drought stress regulates the accumulation of variousamino acids (e.g., branched-chain amino acids, saccharopine, proline, andagmatine) and sugars (e.g., glucose and fructose). The expression levels ofdrought/ABA-inducible genes for the key amino acid biosynthetic enzymes werecorrelated with the metabolite profiles. These findings demonstrate the existence ofa metabolic network during drought stress and the key role of ABA in regulatingmetabolic changes during stress responses [160].

4.5.2

Cell Wall Compounds

Vascular tissue structure and function have evolved alongside stomatal function inplants to optimize gaseous exchange, photosynthesis, and WUE. Recent studiessuggest that disruption of cellulose synthase genes, which are involved insecondary cell wall formation, affect osmotic stress responses in Arabidopsis. Themajority of assimilated carbon is accumulated and stored in the secondary cell wallof woody plants, mainly as cellulose and lignin. A cellulose synthase mutant fromArabidopsis, cesA8 (irx1), exhibited collapsed xylem cells and reduced water loss in

4.5 Metabolic Adjustment During Water Stress 95

response to a reduction in water flow through the xylem and therefore increasedtolerance to drought [161]. Similarly, the cesA7 (irx3-5) mutant exhibited reducedstomatal pore width and collapsed xylem cells [162]. The induction of collapsedwalls in the tracheid cells of pine needles was observed under drought stressconditions [163]. Together, these findings suggest that the genes involved incellulose biosynthesis may affect water use in plants. Lignin provides hydrophobi-city and rigidity to the thickening cell wall, for water transport and mechanicalsupport. Lignin production has been the target of genetic engineering, to facilitatethe production of woodpulp and biofuel [164]. Lignification is also induced byvarious types of biotic and abiotic stress (e.g., wounding, pathogen attack, anddrought), and inhibits further growth of invading pathogens [165–169]. Water losscontrols the cell turgor pressure and affects the cell wall flexibility by either relaxingor tightening the wall structure. During water stress, cell wall flexibility wasaffected by the levels of lignin and phenolic compounds [170]. Dehydration causesincreased enzyme activity during lignin biosynthesis in white clover [171]. Theexpression levels of the genes encoding cinnamoyl-CoA reductase, which isinvolved in lignin biosynthesis, increased after drought and, furthermore, localizedchanges in lignification were involved in acclimation to drought stress [172]. Thesefindings suggest that cell wall modifications are involved in the reprogramming ofdevelopmental processes in response to stress.

4.6

Future Perspective

Unlike animals, sessile plants constantly face environmental stresses throughouttheir life cycles. Plants have evolved adaptive mechanisms to these stresses in orderto survive and grow. The characteristics of different plant species in response tostress conditions, such as stress tolerance and WUE, are important traits forobtaining high biomass productivity. The mechanisms involved, identification ofkey factors, and development of new genetic and biochemical technologies toengineer superior plants have been extensively documented. Genome editing usingcustom-designed restriction endonucleases, such as zinc finger nucleases (ZFN) orTAL-effector nucleases (TALEN) [173–176], is rapidly becoming a crucial technol-ogy for the development of superior plants. Double-stranded DNA breaks mediatedby ZFNs or TALENs can markedly enhance the production of mutations (smallinsertions or deletions) at specific genomic locations and also gene targeting. Thesetechnologies will enable the modification and regulation of key genes involved ingrowth and development under environmental stress conditions, and therebycontribute to a more detailed understanding of the gene functions involved instress responses. Identification of target genes in model plants such as Arabidopsiswill enable breeding and precise manipulation of specific crops or woody plants.Further studies using new molecular approaches, including elucidation of thegenetic variation of significant traits, will facilitate the engineering of plants withincreased tolerance and WUE under conditions of climate change.


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