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33 Maize: Physiological and Molecular Approaches for Improving Drought Tolerance Ishwar Singh, Thirunavukkarasu Nepolean, Rajyalakshmi Ambika Rajendran, and Mariko Shono Maize is a C 4 crop with a high rate of photosynthetic activity, leading to high grain and biomass yield. It is predominantly a cross-pollinating species, a feature that has contributed to its broad genetic and morphological variability and geographical adaptability. Economically, the most important types of maize are grown for grain and fodder or silage production. However, in the tropics, grain is primarily used for human consumption. FAO predicts that an additional 60 Mt of maize grain will be needed from the annual global harvest by 2030. The demand for maize as an animal feed will continue to grow faster than the demand for its use as a human food, particularly in Asia, where a doubling of production is expected from the present level of 165 Mt to almost 400 Mt in 2030. 33.1 Introduction One of the focal points of global food security is the ability to produce the crop all year round in variable climatic conditions, including unpredictable rainfall, low soil moisture, excess soil moisture, excess heat, and so on. Among all, drought poses a major hindrance to this objective in many tropical countries [1]. The ability of plants to tolerate moisture stress condition is crucial for sustaining the agricultural production worldwide. Recent studies of molecular and genomic experiments have increased the understanding of the regulatory and functional networks controlling the drought stress response and have led to practical approaches for developing drought tolerance in plants [2]. From an application point of view, it is important to select genotypes that are able to optimize water harvest and use water efciently, while maximizing yield in relation to the dynamics of the drought episodes prevailing in each target environment. The objective of this review is to consolidate the current emerging trends of physiology, molecular breeding, and functional genomics that would be inuential in integrating breeding and genetic engineering approaches for development of drought-tolerant genotypes in maize. Improving Crop Resistance to Abiotic Stress, First Edition. Edited by Narendra Tuteja, Sarvajeet Singh Gill, Antonio F. Tiburcio, and Renu Tuteja Ó 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA. j 751

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33Maize: Physiological and Molecular Approachesfor Improving Drought ToleranceIshwar Singh, Thirunavukkarasu Nepolean, Rajyalakshmi Ambika Rajendran,and Mariko Shono

Maize is aC4 cropwith a high rate of photosynthetic activity, leading to high grain andbiomass yield. It is predominantly a cross-pollinating species, a feature that hascontributed to its broad genetic and morphological variability and geographicaladaptability. Economically, themost important types ofmaize are grown for grain andfodder or silage production. However, in the tropics, grain is primarily used forhuman consumption. FAO predicts that an additional 60Mt of maize grain will beneeded from the annual global harvest by 2030. The demand for maize as an animalfeed will continue to grow faster than the demand for its use as a human food,particularly inAsia, where a doubling of production is expected from the present levelof 165Mt to almost 400Mt in 2030.

33.1Introduction

One of the focal points of global food security is the ability to produce the crop all yearround in variable climatic conditions, including unpredictable rainfall, low soilmoisture, excess soil moisture, excess heat, and so on. Among all, drought posesamajor hindrance to this objective inmany tropical countries [1]. The ability of plantsto tolerate moisture stress condition is crucial for sustaining the agriculturalproduction worldwide. Recent studies of molecular and genomic experiments haveincreased the understanding of the regulatory and functional networks controllingthe drought stress response and have led to practical approaches for developingdrought tolerance in plants [2].

From an application point of view, it is important to select genotypes that are able tooptimizewater harvest andusewater efficiently, whilemaximizing yield in relation tothe dynamics of the drought episodes prevailing in each target environment. Theobjective of this review is to consolidate the current emerging trends of physiology,molecular breeding, and functional genomics that would be influential in integratingbreeding and genetic engineering approaches for development of drought-tolerantgenotypes in maize.

Improving Crop Resistance to Abiotic Stress, First Edition.Edited by Narendra Tuteja, Sarvajeet Singh Gill, Antonio F. Tiburcio, and Renu Tuteja� 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.

j751

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33.2Basic Concept of Drought Tolerance and its Significance

Drought is a meteorological term and can be defined as the absence of adequatemoisture necessary for normal plant growth and to complete the life cycle.Drought inagriculture is due to shortage of water in the root zone, resulting in yield reduction,which is the principal concern of this chapter. The lack of adequate moisture leadingtowater stress is a commonoccurrence in rain-fed areas, brought about by infrequentrains as well as inadequate and poor irrigation [3]. Growth and other processes areprogressively retarded as soil water content decreases below field capacity. However,most of the plant physiological processes are influenced directly by plant water stressand only indirectly by soil and atmospheric water stress. As much as 17–60% loss inmaize yield could be recorded per year in tropical countries due to moisture stress.Hence, the conservation of the soil moisture is essential in the dry season of the cropgrowth [4].

The term drought tolerance relates to ultimate yield rather than to the capacity ofthe plant to survive in water-limited conditions [5]. Tolerance consists of droughtavoidance and dehydration tolerance that are ultimately measured by the reproduc-tive success of the species [6]. Those plants able to reproduce are represented in thenext generation. For grain crops, the measure is similar but determined as yield perunit area of land. Drought avoidance strategies in plants include deep rooting traits,conservative use of available water to ensure that the grain filling is completed, croplife cycle tomatch rainfall, and short-duration genotypes to escape from the drought.Dehydration tolerance involves plants� ability to partially dehydrate but remain viableand resume growth when water is available.

33.3Impact of Drought on Phonological Phases of Maize

Moisture stress particularly affects the ability of the maize plant to produce grain atthree critical stages of growth: early in the growing season (seedling emergence), atflowering, and during mid-to-late grain filling. Moisture stress during flowering andpollination leads to pollen and silk sterility, inadequate partitioning of source, andimproper mobilization from source to sink, which in turn cause maximum yieldpenalty (Figure 33.1). In fact, silking or the onset of the reproductive stage is themostsensitive stage and will result in 100% yield loss when moisture stress is accom-panied with heat stress [7]. In India, moisture stress, particularly at reproductivestage, has been identified as the most important limiting factor of maize productionand productivity.

In tropics, moisture stress at the beginning of a season can damage plant stands;however, crop can revivewhen it gets water. On the other hand, grain yield reductionsfrom mid-to-late grain filling are not nearly as severe as those produced by a similarstress during flowering.

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Reduction of plant water status to the wilting point during the preflowering,flowering, and postflowering stages results in yield reduction by 25, 50, and 21%,respectively [9]. Silk growth, kernel size, andnumber appear to depend directly on theflowof photosynthates produced during the 3weeks of extreme sensitivity period thatbracket flowering [10]. Drought also lessens the capacity of developing kernels to useavailable assimilates because of the impaired functioning of a key enzyme acidinvertase [11, 12]. Once kernels enter the linear phase of biomass accumulation about2–3 weeks after pollination, they develop the capacity to access reserve assimilatesstored in the stem and husk. If kernels successfully reach this stage, they willnormally grow to at least 30% of the weight of kernels on unstressed plants, even inthe presence of severe moisture stress [13]. Plants also respond and adapt to waterdeficit at both cellular and molecular levels, for instance, by accumulation ofosmolytes and proteins specifically involved in stress tolerance. The physiologicalmechanisms involved in cellular and whole-plant responses to water stress, there-fore, generate considerable interest.

33.3.1Physiological, Morphological, and Metabolic Changes Induced by Drought

During drought, certain morphological, physiological, and metabolic changes occurin response to drought that allow the plant to avoid water loss by continuous wateruptake at reduced water potential or to tolerate a reduced tissue water content [14].Numerous physiological and biochemical changes occur in response to droughtstress in various plant species. Changes in protein expression, accumulation, andsynthesis have been observed in many plant species as a result of plant exposure todrought stress during growth. The physiological responses of plants to a deficit ofwater include leafwilting, a reduction in leaf area, leaf abscission, and the stimulationof root growth by directing nutrients to the underground parts of the plants.

Figure 33.1 Impact of drought stress at different stages of maize development [8]. Reprinted from[8] with kind permission from ASA, CSSA, and SSSA Book Publishing.

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Plant cells are required to maintain water balance. Tomaintain this water balance,plants absorb water when water potential is negative. Cells can decrease their waterpotential through the accumulation of solutes, such as sugars, amino acids, organicacids, and ions, especially potassium (Kþ ). As cellular enzymes are severely inhibitedby the presence of ions, these must be removed from the cytosol (the ground fluidsubstance of the cell) and stored in special storage cell organelles, the vacuoles. Plantsresort to many adaptive strategies in response to abiotic environmental stresses suchas dehydration and excessive osmotic pressure. These adaptive mechanisms includechanges in physiological, morphological, and biochemical processes.

33.3.2Physiological Changes

Physiological changes at the cellular level associated with drought stress includeaccumulation of osmolytes, turgor loss, reduction in photosynthetic activity, andchanges in membrane fluidity. Abscisic acid (ABA) production is also induced andleads to a further loss of stomatal turgor. The resulting stomatal closure causes aconcomitant decrease in CO2 availability in the leaves, and hence in assimilatesavailability to the plant [15].

Duringmoisture stress, the xylem vessels give up contents such as ABA to the leafapoplast, thereby increasing thehormone concentration in this compartment. ABA iscarried with the transpiration stream inside the leaf around and through themesophyll cells so that it reaches the stomatal guard cells of the epidermis thatcontain ABA receptors with external (and possibly internal) loci in their plasmamembranes. Once bound, the hormone induces an internal signal transductioncascade, usually involving increase in both externally and internally sourced cyto-plasmic calcium, which eventually reduces the osmotic potential of guard cells vialoss of Kþ and Cl� with stomatal closure as a consequence [16].

Although the photosynthetic machinery has a range of photoprotective mechan-isms to dissipate excess light energy, the continued exposure of leaves to excessiveexcitation energy can lead to photoreduction of oxygen and the generation of highlytoxic reactive oxygen species (ROS), such as superoxides and peroxides. These freeradicals are harmful compounds causing chemical damage to DNA and proteins andcan therefore have lethal effects on cellular metabolism [17]. Plants have evolvedseveral strategies to dealwithROS, including theproduction of chemical antioxidantssuch as ascorbic acid, glutathione, and a-tocopherol that directly remove potentiallydamaging electrons from the ROS, and also peroxidases and superoxide dismutasesthat scavenge the electrons enzymatically [18].

Another adaptive mechanism for protection against drought is the maintenanceof turgor during periods of drought by adjusting the osmotic pressure of cells. Thereare two main routes whereby this can be achieved. First, the cell can sequesterions into cellular compartments. Second, specialized osmolytes such as proline,glycine-betaine, mannitol, trehalose, ononitol, and ectoine can be synthesized toreadjust cellular osmotic potential. These osmolytes are also active in scavengingROS, especially if they are targeted to the chloroplast [19]. Other specialized organic

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molecules can be used to protect cellular membranes against physical damage andproteins against unfolding. Dehydration induces the partitioning of amphiphilicmolecules such as glycosylated flavonols and hydroquinones into membranes; thesecompounds increase membrane fluidity and depress phase transition tempera-tures [15]. During extreme desiccation, tolerant plants synthesize large amounts ofnonreducing disaccharides, such as trehalose, which can substitute for water bysatisfying hydrogen bonding requirements of polar amino acid residues at proteinsurfaces and maintain the folded active states of the proteins.

Maturation proteins, which are induced in response to ABA or dehydration, alsoprotect plants under stress by stabilizing cellmembranes.Heat shock proteins (Hsps)and molecular chaperones, as well as late-embryogenesis abundant (LEA) proteinfamilies, are involved in plant abiotic stress tolerance [20]. High temperature, salinity,and drought stress can cause denaturation and dysfunction of many proteins. Hspsand LEA proteins help to protect against stress by controlling the proper folding andconformation of both structural (i.e., cell membrane) and functional (i.e., enzymes)proteins. Proteins synthesized in response to drought stress are called dehydrins(dehydration induced) and belong to the group II LEA proteins. The dehydrin familyof proteins accumulates in a wide range of plant species under dehydration stress,which range in size from 9 to 200 kDa. Dehydrin proteins have been characterized ashydrophilic, heat stable, macromolecular stabilizer, free of cysteine and tryptophan,responsive to ABA, prevent denaturation of cellular proteins, and rich in lysine. Theyaccumulate along with other LEAproteins in response to a particular stress and havebeen proposed to play an important role in membrane protein stability and osmoticadjustment. A proposed role of dehydrin-like proteins in drought stress has been theprotection of cells from dehydration stress. Dehydrin-like proteins may also have arole similar to compatible solutes in osmotic adjustment. Another possible role ofstress proteins is to bind with the ions accumulated (ion sequestering) under droughtstress and to control solute concentration in the cytoplasm.

33.3.3Morphological Changes

Plants exposed to sublethal abiotic stress conditions exhibit a broad range ofmorphogenic responses that include inhibition of cell elongation, localized stimu-lation of cell division, and alterations in cell differentiation status [21]. As such, abioticstress stimuli negatively affect plant growth and development through the arrest ofthe cell cycle machinery. Abiotic stress perception activates signaling cascades thatstimulate cell cycle checkpoints, resulting in an impaired G1-to-S transition, slowingdown of DNA replication, and delayed entry into mitosis [22]. Water stress inducesmeristem shortening in leaves of maize and prolongs the cell cycle duration as aresult of reduced CDK activity [23]. In plant tissues, water potential and content aremaintained close to the unstressed level by increasing uptake or limiting loss, so thatloss and uptake rates of water remain balanced. Such a balance is achieved in theshort term mainly by developmental and morphological traits, such as stomatalclosure that is paralleled by a decreased photosynthetic rate [24]. Indeed, stomatal

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closure in response to drought stress restricts CO2 entry into leaves, therebydecreasing CO2 assimilation and water loss from the leaves and affecting mesophyllmetabolism [25].

In the longer term, the root/shoot ratio, the tissuewater storage capacity, the cuticlethickness, and the water permeability are perceived to be the potentially importanttarget traits, of which change in root growth is the most crucial for crop plants tomaximize water uptake [26]. Highly water-stressed maize plants respond by rollingthe leaf early in the day. The stress-inducedmorphogenic response is postulated to bepart of a general acclimatization strategy, whereby plant growth is redirected todiminish stress exposure [7].

33.3.4Metabolic Changes

The plant defense response to drought stress is associated with the synthesis ofosmoprotectants, osmolytes, or compatible solutes. The accumulation of severalorganic solutes according to the metabolic responses has drawn much attention.

Solutes that accumulate in the cytosol and do not interfere with enzymaticreactions comprise sugar alcohols (mannitol and sorbitol), the amino acid proline,free amino acids, and sugars in roots and shoots, and glycine-betaine. The synthesisof these compounds by the plant enhances tolerance to drought [27]. Osmoprotec-tants are small neutral molecules that are nontoxic to the cell at molar concentrationand that stabilize proteins and cell membranes against the denaturing effect of stressconditions on cellular functions [28].

The compatible solutes may be classified into two categories: (1) nitrogen-contain-ing compounds such as proline and other amino acids, quaternary ammoniumcompounds, and polyamines; (2) hydroxy compounds, such as sucrose, polyhydricalcohols, and oligosaccharides [29]. The plant�s response to drought is accompaniedby the activation of genes involved in the perception of drought stress and in thetransmission of the stress signal. One group of genes encodes proteins to protect thecells from the effects of desiccation. These genes include those that govern theaccumulation of compatible solutes, passive transport across membranes, energy-requiring water transport systems, and protection and stabilization of cell structuresfrom desiccation and damage by ROS [30]. The second group of genes activatedby drought consists of regulatory proteins that further regulate the transduction ofthe stress signal and modulate gene expression. At least four independent stress-responsive genetic regulatory pathways are known to exist in plants, forming a highlycomplex and redundant gene network [2, 30]. Two of the pathways are dependent onthe hormone ABA, while the other two are ABA independent. These pathways arealso implicated in the perception and response to additional stress factors, includingcold, high temperature, and salinity.

Mannitol is a major photosynthetic product in many algae and higher plants,enhancing tolerance to water deficit-induced stress primarily through osmoticadjustment [31]. Its mechanisms are likely to involve the scavenging of hydroxyl

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radicals (OH�) and the stabilization of macromolecules [32]. Water deficit alters thesynthesis and partitioning of metabolically important carbohydrates in plants. Someof these effects on carbohydrate metabolism might be required for the photosyn-thetic assimilation of carbon and its conversion to metabolically usable forms. Otherstress-induced changes in carbon metabolism might reflect adaptations for stresstolerance [33]. For example, raffinose family oligosaccharides (RFOs), such asraffinose, stachyose, and galactinol, play important roles in the desiccation toleranceof plants. Raffinose accumulates in vegetative tissues under drought stress [34].RFO biosynthesis requires the presence of galactinol, which is formed by galactinolsynthase (GolS) from UDP-Gal and myoinositol. Galactinol is the galactosyl donorfor the biosynthesis of raffinose from Suc by raffinose synthase (RafS). Becausegalactinol has not been assigned any function in plants other than acting as agalactosyl donor for RFOs synthesis, GolS potentially catalyzes a metabolic key stepfor RFO synthesis. Overexpression of one of them causes an increase in endog-enous galactinol and raffinose, as well as an improvement in drought tolerance [35].Fructans are polyfructose molecules that are soluble carbohydrates and are locatedin the vacuoles of many plants. Fructan metabolism plays a significant role indrought stress tolerance in plants [36]. As these compounds are soluble, they mightplay a role in the osmotic adjustment of natural fructan accumulators by varying thedegree of polymerization of the fructan pool. Trehalose (a-D-glucopyranosyl-1, 1-a-D-glucopyranoside) is an innocuous, scentless, nonreducing disaccharide andmelliferous nonreducing disaccharide containing two glucose residues bound in ana,a-1,1-glycosidic linkage. It is the nonreducing nature of trehalose that determinesits high stability to acid, alkali, and heat. Trehalose can become glass state structureby combining two water molecules. Its hydroscopic property is more than threetimes of sucrose, maltose, glucose, and fructose. In cells, the high tolerance oftrehalose to dehydration provides protection to proteins and biomembranes fromdrying, freezing, and heating. Accumulation of proline is a widespread plantresponse to environmental stresses, including low water potential. Proline has aclear role as an osmoticum. In particular, because of its zwitterionic, high hydro-philic characteristics, proline acts as a �compatible solute,� that is, one that canaccumulate to high concentrations in the cell cytoplasm without interfering withcellular structure or metabolism. There is presently no clear agreement on thefunction of drought-induced accumulation, although a role in osmoregulationseems likely. Other functions of proline accumulation have also been proposed,including statabilization of macromolecules, a sink of carbon and nitrogen for useafter relief of water deficit, radical detoxification, and regulation of cellular redoxstatus by proline metabolism [37]. Glycine-betaine, a quaternary ammoniumcompound, is a very effective compatible solute. In higher plants, glycine-betaineis synthesized from choline (Cho) via betaine aldehyde (BA). Glycine-betainebalances the osmotic pressure between outside and inside of cells to cope up withosmotic stress and hence maintains turgor. Moreover, glycine-betaine also protectsphysiological processes such as photosynthesis and protein synthesis underdrought conditions [38].

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33.4Role of ABA in Drought Tolerance

ABA, a plant stress hormone, induces the closure of leaf stomata (microscopic poresinvolved in gas exchange), thereby reducing water loss through transpiration anddecreasing the rate of photosynthesis. These responses improve the water useefficiency of the plant on the short term. ABA plays an important role in seedmaturation and dormancy, as well as in the adaptation of vegetative tissues to abioticenvironmental stresses such as drought and high salinity [39].

33.4.1ABA-Dependent Signaling

Drought stress induces de novo synthesis of the phytohormone ABA that plays animportant role in the adaptation of vegetative tissues to abiotic stresses, such asdrought and high salinity, by promoting stomatal closure in guard cells [40]. ManyABA-inducible genes contain a conserved, ABA-responsive, cis-acting element,designated ABRE (PyACGTGGC), in their promoter regions. Reversible proteinphosphorylation is an early and centrally regulated event in ABA signal transduction,at least in the guard cells. Upon drought stress, the ABA-responsive 42 kDa kinasesare activated, thereby phosphorylating the conserved regions of ABA-responsiveelement binding protein (AREB)/ABFs. Several SNF1-related protein kinases 2(SnRK2s) such as ABA-activated protein kinase (AAPK) [41] and OST1/SRK2Ein Arabidopsis [42] were reported as AAPKs. All these kinases phosphorylate in vitro,a motif in the so-called �Constant� subdomains found among basic-leucine zipper(b-ZIP) transcription factors (TFs), includingAREB1, AREB2, andABI5 [43]. Someb-ZIP TFs may also be the targets of calcium-dependent protein kinases (CPKs).

Abiotic stress activates the production of intracellular ROS. When the increase inROS is relatively small, the housekeeping antioxidant capacity is recruited to reset theoriginal balance between ROS production and scavenging, thus reestablishing theredox homeostasis [44]. Otherwise, ROS is sensed bymembrane-localized kinases thateventually activate the MAPK (Figure 33.2). MAPK regulates gene expression byaltering the TF activity through phosphorylation of serine and threonine residues,whereas ROS is regulated by oxidation of cysteine residues [45]. Changes in geneexpression play an important role in plant drought stress response, and many stress-induced genes are known or presumed to play roles in drought resistance. Formany ofthese genes, the hormone ABA is a key signaling intermediate controlling theirexpression in anABA-dependent or ABA-independentmanner (Figure 33.3), as shownlargely by the analysis ofABA-deficient andABA-insensitivemutants inArabidopsis [46].

33.4.2ABA-Independent Signaling

The TFs DREB1 and DREB2 (Figure 33.4) are important in the ABA-independentdrought-tolerant pathways that induce the expression of stress response genes.

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Ox

ROS SENSOR

ROS extracellular

MAPKKK

MAPKKS/T S/T

MAPKKS/T S/T

activeinactive

MAPKT Y

MAPKT Y

activeinactive

Transcription Factor

S/TP CTranscription

Factoractiveinactive

S/TP C

Gene Expression

PPase

OxC

ROS intracellular

Figure 33.2 Cellular ROS signaling in plants [45].

Figure 33.3 ABA-dependent and ABA-independent signaling in plants [46].

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Overexpression of the native form of DREB1, and of a constitutively active form ofDREB2, increases the tolerance of transgenic Arabidopsis plants to drought, highsalinity, and cold. Although these genes were initially identified inArabidopsis plants,their presence and role in stress tolerance have been reported in many otherimportant crops includingmaize, indicating that thiswould be a conserved, universalstress defense mechanism in plants.

Abundance of cationic peroxidases induces lignin biosynthesis in the xylemvessels and induces cell wall stiffening that might strengthen the xylem vessels andprevent any further cell expansion either to better withstand the tension occurringduringwater stress or to restrict water loss from internal tissues [47]. Furthermore, inwater-stressed plants, the levels of many amino acids in the sap increase transientlyand a number of amino acids also accumulate only under severe water stress.Metabolic changes associated with drought stress include modifications in soluteconcentration and protein–protein and protein–lipid interactions [48]. Production ofphytoalexins, activation of the general phenylpropanoid pathway, and induction oflignin biosynthesis have evolved as adaptation mechanisms to water deficit. Salicylicacid, methyl salicylate, jasmonic acid, methyl jasmonate, and other small moleculesproduced as a result of stress can also serve as signalingmolecules activating systemicdefense and acclimatization responses [49], whereas others protect plants fromoxidative damage associated with a variety of stresses, such as ascorbic acid,glutathione, tocopherols, anthocyanins, and carotenoids, by scavenging the gener-ated active oxygen intermediates. Knowledge of the physiological and biochemicalresponses to severe water deficit conditions has been exploited for drought stresstolerance in plants.

Figure 33.4 Effect of cold, salt loading, and dehydration on expression of stress-related genesleading to striking improvements in plant tolerance [40].

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33.5Developing Drought-Tolerant Maize

Unlike relatively static abiotic stresses such as soil salinity or acidity, drought stress inmost maize growing areas is strongly dependent upon stochastic weather processes.Transient stresses give rise to complex genotype� year, genotype� season andgenotype� season� year, genotype� season� year�management interactionssince stress tolerance varies among genotypes and throughout the season of thecropping period. With the advent of combine yield monitors, within-field spatialvariation in yield has become much more obvious to the farmer and often relates tovariation in soil texture and plant available water.

The application of genetics to improve drought tolerance and provide yield stabilityis an important part of the solution to stabilizing global maize production. This doesnot imply that agronomic interventions that aim tomaximize water availability at keygrowth stages are not critically important since genetic solutions are unlikely to closemore than30%of the gapbetweenpotential and realized yieldunderwater stress [50].However, improved genetics can be conveniently packaged in a seed and thereforemore easily and completely adopted than improved agronomic practices that dependmore heavily on input availability, infrastructure, access tomarkets, and skills in cropand soil management. Fortunately, under stressful conditions, the performanceadvantage of modern elite germplasm over its less improved and older counterpartsbecomes larger, and much of the observed genetic gain in yield during the past 30years has been attributed to greater stress tolerance rather than to an increase in yieldpotential per se [51–53]. Physiology, coupled with genomics, offers promise ofimproving the rate of gain for key traits, and especially those such as droughttolerance that are difficult to phenotype, the baseline for comparisonmust be the rateof improvement obtained through established selection systems. Thus, it is instruc-tive to consider rates of gain in drought tolerance resulting from conventionalselection in a large hybrid development program that relies on extensive multi-environment testing to identify superior progenies.

To do this, however, the association between genotype and phenotype must bebetter understood and quantified so that our ability to predict phenotypic perfor-mance from genetic information for many traits observed in an array of environ-ments is greatly improved. Genomics, or the study of the function and structure ofspecific genetic sequences accompanied by high-throughput laboratory-based anal-ysis of DNA [54], is considered a key to comprehending gene–phenotype associationsat the level of candidate genes and sequences. This will be critically important forquantitative traits such as drought tolerance, where performance is regulated bymany loci and subject to multiple genotype� environment (G�E), gene� gene(G�G) interactions (epistasis), and gene� gene� environment (G�G�E)interactions.

Identification and measurement of secondary traits associated with grain yieldprovides a guide to specificmechanisms that contribute to grain yield under drought.Thus,water depletion patterns, leaf rolling, and canopy temperatures are indicative ofroot exploration and water extraction capacity, and chlorophyll concentration is a

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measure of functional stay green [55]. Some secondary traits are associated withspecific developmental stages such asflowering, while others, such as photosyntheticrate, are indicative of plant growth throughout the life cycle of the crop. Ideally,secondary traits should be correlated with grain yield under stress, highly heritable,easy to measure, and stable over time. A short anthesis-silking interval (ASI) isindicative of general tolerance to reduced photosynthesis per plant at flowering inmany cases [56]. To understand the genotypic differences in water acquisition, adistinction must be made between the fine lateral roots, usually with diameterssmaller than 0.8mm [57], and their larger parental axile roots. In the fibrous rootsystem of maize, axile roots emerge from the stem, guaranteeing a wide vertical andhorizontal distribution of the root system, away from the plant basis, while lateralroots are ofmajor importance for the efficient short-distance exploitation ofwater andnutrients [58]. A series of correlated phenotypes that have associations with grainyield under drought conditions are precocity, plant stature, chlorophyll content, rootmorphology and conductivity, glucose, sucrose, dehydrin, ABA, and ABA glucoseester measured on leaves, ear tips, and silks harvested at different developmentalstages.

Drought tolerance that impacts crop yield can be assessed reliably only in the field.Managed stress environments, where the severity and timing of drought stress arecontrolled in a manner relevant to target environment conditions, are essential forapproaches aimed at achieving genetic progress for drought tolerance. Accuratewater management in the absence of rain allows stress intensity to be adjusted so theexpression of genetic variability for key secondary traits ismaximized and the patternof stress, targeted at specific growth stages, can be repeated. The detection ofgenotype� stress level interactions for drought tolerance provides essential evidencefor the presence (and absence) of unique, adaptive mechanisms among genotypes.Generation of such interactions requires the application of relatively severe stresslevels that, in some cases, are more severe than those experienced in the targetpopulation of environments. A well-watered control is generally needed to monitorfor losses in yield potential associated with selection for stress tolerance.

Comparison of performance in these contrasting environments provides thecritical data required to predict yield stability of genotypes. Care must be exercised,however, when designing water stress regimes to ensure that the genetic correlationbetween the managed stress environments and the target population environmentremains positive and reasonably large. Under managed stress environments, stan-dard plot management techniques often require adjustment to enhance uniformitywithin trials. Particularly critical is the establishment of uniform stands to ensureevenness of water availability per plant. As plants remove soil water, differences inroot volume per plant and in transpiring leaf area can exaggerate plant-to-plantvariability. Blocking by flowering date is important in maize because of its suscep-tibility to stress imposed at flowering. If entries vary widely in time to silk, the most�tolerant� may simply be those that flower earlier than the mean and thus escapestress that intensifies with time. Finally, time trends in data can occur when variablessuch as canopy temperature are measured using handheld infrared thermo-meters [59]. As soil water is depleted, spatial variability generated by differences in

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soil texture becomes increasingly obvious and can obscure genotypic differences.The use of uniform land is obviously the best solution, and knowledge of thesepatterns generated over time can be used to select the most uniform plot sites. Theuse of incomplete block designs such as row/column or alpha (0, 1) designs [60] oraugmented designs provides amethod to adjust data for the effects ofwithin-replicatespatial variation when dealing with large entry numbers [55]. Spatial trends in dataarise from soil heterogeneity aswell as unintentionalmanagement factors that can beidentified within a linear mixed model analysis framework. In addition, data can beadjusted for these effects by incorporating appropriate model terms and identifyingappropriate variance structures for spatial trends [61]. Linear mixed models can beused for estimating variance components and determining best linear unbiasedpredictors (BLUPs) for genotypes from unbalanced data sets.

33.6Modern Tools to Improve Drought Tolerance in Maize

Integrating molecular approaches of the latest advances in biotechnology, genomicresearch, andmolecularmarker applications with conventional plant breeding, plantphysiology, and biochemistry could increase significantly the potential for geneticgain under water-limited conditions (Figure 33.5).

Figure 33.5 Development of drought-tolerant maize genotypes by integration of classical andmodern tools.

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Changing maize genetic improvement from an empirical to a knowledge-basedprocess involves investing heavily in the use of high-throughput recombinant DNAtechnology, genomics, and bioinformatics tools. The utility of these tools has, in turn,been increased by advances in DNA sequencing capacity and in database develop-ment and management. The availability of organized dense genetic maps based onmolecular markers and the awareness of the particulate nature of the inheritance ofquantitative traits have fostered an interest in the genetic dissection of droughttolerance. Ideally, this involves associating genetic variation at the sequence levelwithobserved phenotypic variation and ensuring that those specific sequences impartingtolerance are present in subsequent generations.

Geneticmapping with densemarkermaps can be used to identify the number andgenetic positions of quantitative trait loci (QTL) associated with a specific trait underdrought stress. In addition, this process can be used to estimate effects of thesegregating QTL and their contributions to trait variation (individually and incombinedQTLmodels) and to obtain estimates of their stability across environments(QTL� environment interactions) and across genetic backgrounds (QTL� geneticbackground interactions).

The responses of the genotypes to drought stress are governed by the activity ofseveral genes involved in diverse pathways, including �constitutive� QTL and�adaptive� QTL. DNA-based markers located in such genomic (bin) locations couldpotentially serve as informative �anchor� markers for molecular marker-assistedselection (MAS) as well as functional genomics. In addition to the consensus QTL,analysis of individual genes, transcriptome profiling, and in silico mapping leads toidentification of specific candidate genes with significant influence over droughtstress tolerance in maize, many of which colocalize with the consensus QTL fordrought tolerance. Lebreton et al. [62]were thefirst to attempt to applyQTL analysis toobtain genetic insights into the drought tolerance response in maize. Since then, anumber of reports of QTL associated with specific traits under drought stress havebeen indicated using diversemapping populations [63–66]. The reports have targetedgrain yield and its components, ASI, root traits, andmeasures of plant water use andstatus, such as stomatal conductance, and leaf and xylem ABA content [67].

Progress of trait is attributed to the number of major QTL identified per trait, themagnitude of observed phenotypic variance that they generally express individually,their interaction with the environment, and difficulty of epistasis evaluation [68]. Forcomplex traits such as drought tolerance, many QTL identified in elite linesdeveloped by breeding programs are likely to be context dependent due to the effectsof several gene and environmental interactions. Therefore, although we can men-delize quantitative traits, the value of the QTL alleles will need to be determined forthe specific situation to which they are to be applied. Capacity for precisionphenotyping under repeatable but representative levels of stress in the field islagging far behind the capacity to generate genomic information and will limitprogress in generating gene–phenotype associations for traits.

MAS experiments based only on the QTL involved in the expression of yieldcomponents would be inefficient because only a few of the QTL are stable acrossenvironments. A MAS experiment should consider the QTL involved in the expres-

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sion of secondary traits of interest correlated with the yield under drought. If it ispossible, the selected QTL should be stable across environments and account for alarge percentage of the phenotypic variance. Therefore, an efficient MAS strategyshould take into account the most suitable QTL from different traits as an index [69].Many studies clearly indicate the colocation of the QTL influenced including keytraits such as grain yield, ASI, root traits, and ABA accumulation across differentgenetic backgrounds [5]. In maize, MAS has been used to introgress QTL alleles forreducing ASI.

Compared to modern cultivars, which are usually selected for high-input envir-onments where water supply is often not a major limiting factor, wild species showmorphophysiological features for survival and adaptation to drought conditions.When considering exploiting alleles fromwild species for drought adaptive features,a careful evaluation in terms of yield, once they are backcrossed in elite accessions, isimportant [68].

Information aboutQTL in tropicalmaize can be applied to increase heritability andfavorable gene action to design optimum transgenic strategies for crop improve-ment. Marker-assisted selection also accelerates the use of transgenes in commercialcultivars, typically achieved through marker-assisted backcrossing [70]. An updatedcompilation ofmappedQTL andmajor genes associated with abiotic stress toleranceincluding drought inmaize and other plants is available at www.plantstress.com [71].Other useful Web resources are www.generationcp.org, http://rarge.gsc.riken.jp/,and http://rootgenomics.missouri.edu/ [5]. Identification of universal drought QTLand putative candidate genes could be valuable for further analysis andutilization [72].

33.7Functional Genomics of Drought Tolerance

Functional genomics usesmostlymultiplex techniques tomeasure the abundance ofmany or all gene products such as mRNAs or proteins within a biological sample.Functional genomics includes function-related aspects of the genome itself such asmutation and polymorphism (e.g., single-nucleotide polymorphic (SNP)) analysis, aswell as measurement of molecular activities. The latter comprise a number of�omics� such as transcriptomics (gene expression), proteomics (protein expression),phosphoproteomics (a subset of proteomics), and metabolomics (analysis ofmetabolites).

Functional genomics provides important information for evaluating stress per-ception, signal transduction, and defensive responses, the role of potential candidategenes, and the pathways inwhich they are involved. Intensive studies have already ledto the discovery of promoter regulatory elements, such as DRE (dehydration-responsive elements) or ABRE (ABA-responsive elements), involved in both dehy-dration and low-temperature-induced gene expression [73], aswell as identification ofseveral key transcriptional factors interacting with such promoters [74]. With theadvent of genomics-related technologies, necessary tools to identify the key gene

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networks that respond to drought stress and those relating to the regulation ofadoptive events occurring during stress are now becoming available [75].

Cloning a drought-related QTL substantially contributes toward a better under-standing of the genetic and functional basis of the response of a plant to drought.Furthermore, the sequence responsible for the QTL becomes available for geneticengineering andmining for themost desirable alleles within germplasm collections.Until now, two approaches have been mainly used for the molecular dissection of aQTL: positional cloning and association mapping [76].

Positional cloning begins with the production of a large population in a near-isogenic lines (NILs) where only the target QTL segregates. A large number ofprogenies (>1000) capturing all possible recombination events and molecularmarkers in the target region enable us to identify the genetic and physical intervalcosegregating with the QTL. The availability of the genome sequence facilitates theconnection between genetic and physical information. When the genome sequenceis not available, genomic libraries (e.g., BAC clones) are used. Amaize domesticationlocus, teosinte glume architecture (tga1), which encodes a transcriptional regulator,was the first maize gene positionally cloned, using a population of over 3000individuals [77]. Among the quantitative traits affecting drought tolerance, particularattention has been devoted to the concentration of ABA, in view of its pivotal role inregulating other molecular and morphophysiological processes involved in theadaptive responses. Differences among NIHs (near-isogenic hybrids) for leaf ABAand othermorphophysiological traitswere not affected bywater regimes.On average,the QTL allele for high leaf ABA markedly reduced stomatal conductance and rootlodging [78]. Candidate genes or sequences that cosegregate with the QTL are thenfunctionally tested with reverse genetics tools (e.g., knockout mutants, RNAi, andtargeting induced local lesions in genomes (TILLING)) and ectopic expression.

Association mapping based on linkage disequilibrium (LD) seeks to establish astatistical association between allelic (or haplotype) variation at a locus and thephenotypic value of a trait across a large enough sample of unrelated accessions [79].The LD approach offers two distinct advantages: the survey of multiple alleles in asingle analysis and avoidance of the time-consuming preparation of mappingpopulations. In the presence of high LD (�100 kb or more), association mappingcan provide only coarse mapping information; however, when LD is low (�10 kb orless), the resolution power is sufficiently high to assign a QTL to an intervalcontaining one or a few genes. Analysis of candidate genes has already providedinteresting results [79, 80]. Association mapping should greatly benefit from theintroduction of high-throughput platforms that are able to profile a multiplex of SNPmarkers. Techniques such as EcoTILLING [81] are also available to streamline theidentification and scoring of new alleles at target genes or sequences.

Major challenges for gene discovery include the following:

. The large size of the maize genome

. Variation in genome size and gene order

. The high incidence of multicopy genes

. Transposons and other repetitive sequencesmakingup a large portion of the genome

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The wealth of active transposable elements residing in themaize genome plays animportant role in functional genomics. In addition to serving as molecular tags formutated genes, these transposons tend to knock out genes into which they areinserted. Mutant libraries constructed using transposon tagging, T-DNA insertion,and chemical and physical mutagenesis provide materials that can be screened forbase changes in any genes by reverse genetics methods such as TILLING [82].

Strategies have been developed so that subtle changes such as point mutationgenerated by EMS can be detected easily. In the basic TILLING method, pollen ismutagenized by treatment with EMS. These mutagenized lines serve as a generalreverse genetic resource. Weil andMonde [83] provided a detailed protocol for maizeTILLING including TILLING mutagenesis, tissue collection, DNA preparation, 2Dpooling, and detailed TILLING workflows. As the maize genome is completelysequenced, advances in reverse genetics technologies including TILLING, EcoTIL-LING, and massively parallel DNA resequencing provide excellent methods foridentifying mutations in a wide variety of traits and biological processes [84].EcoTILLING is a way to survey how much allelic diversity there is for a given genetarget and where that diversity is located (intron versus exon).

Gene expression experiments have identified several hundred genes that areinduced or repressed during drought. Both cDNA- and oligonucleotide-based geneexpression profiling platforms are used to examine the effects of drought stress onyield potential of maize [85]. The expression QTL (eQTL) mapping involves expres-sion profiling as measured by mRNA transcript abundance for a large number ofgenes that are each treated as a quantitative phenotype likely to be conveyed bymultiple genes and influenced by environmental factors. These expressional profilesthen constitute a marker-based fingerprint of each individual in a segregatingpopulation and can be subjected to conventional QTL analysis [86], albeit interpretedin the spatially and temporally specific context in which the data are collected. Theprocedure is called mapping of eQTL, that is, genetic locus where allelic variationaffects the level of gene expression. Overlapping expression profiles and coordinateexpression indicated that genes relevant to stress resistance and such data sets forman excellent resource for identifying candidate genes [87] through positional cloningor association mapping. Transcription profiling has increasingly become an impor-tant genomics tool for gene functional analysis. Expression patterns of some genes inseveral stress response-associated pathways, including abscisic acid, jasmonic acid,and phenylalanine ammonia lyase, are positively responsive to drought stress.However, the cost of profiling the large number of samples required to identifyeQTL is still too high for routine application of this approach. The expression ismostoften quantified in terms of the amount ofmRNA in amicroarray-based analysis, butthe same principle has been applied to genetic control of the protein level. Micro-arrays have become an important technology for the global analysis of geneexpression. Implemented in the context of a well-designed experiment, cDNA andoligonucleotide arrays can provide high-throughput simultaneous analysis of tran-script abundance for hundreds, if not thousands, of genes. Microarrays are beingused to assess gene expression in plants exposed to the experimentalmanipulation ofair temperature and soil water content in the root zone. Analysis often includes

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characterizing transcript profiles for multiple posttreatment sampling periods andcategorizing genes with common patterns of response using hierarchical clusteringtechniques. In addition, microarrays are also providing insights into developmentalchanges in gene expression associated with root elongation in maize [88].

The identification of candidate genes for the QTL and the elucidation of theirfunctional role can be facilitated by combining QTL maps with the so-calledfunctional maps (i.e., maps enriched with genes potentially involved in controllingthe target trait or with fully annotated genomic sequences). An important part of newgene discovery through ESTand genome sequencing is the annotation of those genesto assign putative functions [89]. In the absence of empirical data for a particular genein the appropriate target organism, gene annotation software can predict a functionusing data from rice, Arabidopsis, and other organisms based on similarities forintron/exon sequence and structure plus likely protein domains (www.maizese-quence.org). One can BLASTmaize sequences against the cereal and other speciesdatabases to generate predicted functional information. In forward and reversegenetics approaches, maize is characterized by excellent mutagenesis resources inthe form of well-studied transposon systems, and new techniques for inducedmutations are also being applied [90].

Maize is one of the model systems for genetic research. The genome size of themaize is 2300Mb, which is fivefold larger than rice but eightfold smaller thanwheat [91]. The sequencing of maize was recently completed by a U.S.-basedconsortium of researchers [92]. The complete genome of B73, an important com-mercial crop variety, was decoded. The 2.3 billion base sequence – the largest geneticblueprint worked out for any plant species – includes more than 32 000 protein-coding genes spread across 10 chromosomes of maize. The transposable elementsare the most abundant parts of the sequence, spanning almost 85% of the genomeand dispersed nonuniformly across the genome. The Maize Genome SequencingConsortium has generated a reference genome sequence that was integrated withboth physical and geneticmaps. Using a previously published integrated genetic andphysical map, combined with genomic sequence, new sequence-based geneticmarkers, and an optical map, the researchers have picked a minimum tiling path(MTP) of 16 910 bacterial artificial chromosome (BAC) and fosmid clones that wereused to sequence the maize genome. The new integrated physical and genetic mapcovered 2120Mb (93%) of the 2300Mb genome, of which 405 contigs were anchoredto the genetic map, totaling 2103.4Mb (99.2% of the 2120Mb physical map). Usingall available physical, sequence, genetic, and optical data, a golden path (AGP) ofchromosome-based pseudomolecules, referred to as the B73 reference genomesequence version, was generated [93].

The completion of the maize genome sequence provides the most essentialresource to move easily from gene to mutant phenotype and back. There are severalmethods such as gene cloning, gene expression profiling, TILLING/EcoTILLINGand transposon tagging, and SNP haplotypes to target loci for experimentallydetermining gene function [94]. Traditionally, gene discovery inmaize has employedtransposon tagging, ESTsearches, and comparative genomics, and due to increase ingenomic resources, positional cloning is increasingly being used for both qualitative

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and quantitative traits. Now with the physical maps for maize, the large numbers ofavailable markers, and conservation of synteny across the cereal genomes, it isfeasible to consider a less time-consuming chromosome walk rather than cloning bytransposon tagging [95].

The length and breadth of the utility of genome sequencing in crop research hasincreased with the availability of new-generation sequencing (NGS) technologies.This new-generation techniques will increase the throughput tremendously whilereducing the cost multiple times [96, 97]. The data generated through NGS techni-ques have opened new era of maize genome analysis and provide sequenceinformation of the germplasm that are genetically diverse so as to uncover thegenetic potential of the unexplored genotypes. Getting enormous amount of datacheaply in no time has extended its applications beyond just reading the order ofbases. Microarrays are being replaced by sequence-basedmethod in gene expressionstudies. The ability to sequence the whole genome of many organisms will allowlarge-scale comparative and evolutionary studies [98]. Sequencing technology isdeveloping very rapidly and already the third-generation sequencing platforms arebeing made available such as �real-time sequencing� (www.pacificbiosciences.com).

Genomics and bioinformatics allow us to investigate sequence colinearity in themain crops [99] and compare their gene order and content with those of modelspecies whose genomes have been sequenced, such as rice and Arabidopsis. Thecolinearity between Arabidopsis and maize [100] has been eroded to such an extentthat theArabidopsis sequence does not appear to be ofmuchhelp for the identificationof related genes in maize. Conversely, comparative mapping between rice andmaize [101] as well as other cereals [102] provides valuable opportunities to exploithigh-resolution collinear maps to facilitate the positional cloning of maize QTL andidentify candidate genes and to establish whether sequences with high homology areso because they represent orthologous loci.

Transcriptomics, proteomics, and gene expression studies have identified theactivation and regulation of several stress-related transcripts and proteins that aregenerally classified into two major groups. One group is involved in signalingcascades and in transcriptional control, whereas members of the other groupfunction in membrane protection, as osmoprotectants, as antioxidants, and as ROSscavengers [103]. Manipulation of genes that protect and maintain cellular functionsor that maintain the structure of cellular components has been the major target ofattempts to produce plants that have enhanced stress tolerance [104].

Progress in the mass-scale profiling of the transcriptome, proteome, and meta-bolome has allowed a more holistic approach in investigations of drought tolerancebased on the measurement of the concerted expression of thousands of genes andtheir products. High-throughput mRNAprofiling has been applied to investigate thechanges in gene expression in response to dehydration [105]. An example of howtranscriptome analysis can advance our understanding of the physiology underpin-ning drought-related traits has been recently provided by the expression profiling ofprimary root apices in maize [106]. Collectively, the transcriptome profiling experi-ments conducted on drought-stressed plants have highlighted the central role of TFswhile unveiling the complex hierarchy of the regulatory network that differentially

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modulates the expression of dehydration signature genes in a tissue-specificmanner.In this respect, laser capturedmicrodissection is amajor technical breakthrough: thetechnique allows the profiling of specific cell types [107] – a feature particularlyimportant when investigating genes encoding for TFs expressed weakly and in a celltype-specific fashion.

The importance of metabolic changes during plant responses to abiotic stresssuggests that detailed metabolite profiling may provide valuable insights into stressresponse mechanisms. Deciphering gene function can also be facilitated by infor-mation gathered through profiling the proteome and metabolome. Profiling theproteome of a mapping population offers the opportunity to identify PQL (proteinquantity loci) influencing protein quantity. In water-stressed maize, the Asr1 gene, aputative TF, has been shown to colocalize with a PQL for the ASR1 protein and aQTLfor ASI and leaf senescence [108]. With regard to metabolomics, the presenttechnology enables the profiling of �2000 metabolites in a single sample [109]. Thesusceptibility of early developing grain to water stress is a major problem in maize,where a shortage of assimilate supply has been indicated as the likely cause forinsufficient grain filling and sterility [110]. In this respect, invertase activity in thedeveloping kernel has been shown to be an important limiting factor for grain yield inmaize exposed to drought [111]. Among the QTL for invertase activity described inmaize, one is mapped near Ivr2, an invertase-encoding gene [112]. Furthermore,collocation between the activities of two enzymes (sucrose-P synthase and ADP-glucose pyrophosphorylase) involved in carbohydrate metabolism and correspond-ing structural genes has been reported in young maize plants subjected to waterdeficit [113]. The increase in Hsp expression under conditions of abiotic stress wasstudied extensively using functional genomics and proteomics in different plantspecies [114]. Genomics-based approaches can contribute novel information toidentify candidate genes and elucidate their functions and regulation underwater-limited conditions.

33.8Genetic Engineering Approaches for Improving Drought Tolerance

Although not a crop plant, Arabidopsis has played a vital role in the elucidation of thebasic processes underlying stress tolerance, and the knowledge obtained has beentransferred to a certain degree to important food plants. Many of the genes known tobe involved in stress tolerance have been isolated initially fromArabidopsis [115]. Twogeneral strategies for the metabolic engineering of abiotic stress tolerance have beenproposed: increased production of specific desired compounds or reduction in thelevels of unwanted (toxic) compounds [116]. However, modulation of a singleenzymatic step is usually regulated by the tendency of cell systems to restorehomeostasis, thus limiting the potential of this approach. Targeting multiple stepsin the same pathway could help to control metabolic fluxes in a more predictablemanner [117]. Effort heavily depends on the development and utilization of drought-tolerant germplasm resources, which is far from plentiful and bottlenecks maize

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improvement. Therefore, germplasm enhancement and development is founda-tionally important for maize improvement [118, 119]. Transgenic operation is auseful technology to overcome reproductive isolation among species and utilizebeneficial exotic genes.

Acquired plant tolerance to abiotic stress can be achieved both by geneticengineering and by conventional plant breeding combined with the use ofmolecularmarkers utilizing QTL, Hsp, LEA, and ROS (Figure 33.6). The tolerance of thetransgenic offspring is not strong enough to meet the requirement of maizeproduction, as the mechanism of these exotic genes is not adaptive to physiologicalmetabolism of maize. It is the key step to explore new exotic gene with strongtolerance and adaptive mechanism to maize [121–123]. Seed of improved cultivarshas shown itself to be an effective means of delivering conventional and transgenictraits that contribute to improved yield and its stability.

Many loci for genes that control tolerance to abiotic stress in plants have beenidentified by genetic analysis [124, 125]. However, many genes that control agro-nomically important traits remain to be identified and modified to generate newvarieties with desirable traits. There is evidence that transgenic plants in which theexpression of a single gene has been modified have enhanced tolerance to abioticstress [126]. Ideally, modification of a single gene should confer tolerance to morethan one form of abiotic stress [127].

Figure 33.6 Approaches to improve stress tolerance [120].

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SOD, dehydration-responsive element binding (DREB), and some other stress-tolerant genes have been used to transform maize for drought tolerance improve-ment. A new sequence of trehalose synthase gene TPS1 was cloned from Saccha-romyces cerevisiae using the method of homologous amplification. Sequence analysisshowed that its similarity to formerly reported sequence of gene TPS1was as high as99.3%. The putative protein of this sequence had the same conserved contigswith theprotein sequences of trehalose synthases in many eukaryotic and prokaryoticorganisms. This sequence was used as exotic gene to construct a stress-inducibleexpression vector and transform embryonic calli of maize mediated by Agrobacter-ium. After screening and regeneration, one fertile plant was detected to be positiveusing specific PCR amplification and sequencing of the amplified product [128].Quan et al. [129] transformed maize with betA gene from Escherichia coli encodingcholine dehydrogenase, a key enzyme in the biosynthesis of glycine-betaine. Thetransgenic maize plants accumulated higher levels of glycine betaine and were moretolerant to drought stress than wild-type plants (nontransgenic) at germination andthe young seedling stage [130].

Multiple transgenic approaches include the use of polyol compounds such asmannitol [131] and sorbitol [132]; dimethylsulfonium compounds such as dimethyl-sulfoniopropionate and glycine-betaine [127]; sugars such as sucrose, trehalose [133],galactinol [34], ononitol [134], and fructan [135]; or amino acids such as proline [136]and ectoine that serve as osmolytes and osmoprotectants [7]. Therefore, it has beenhypothesized that engineering the introduction of osmoprotectant synthesis path-ways is a potential strategy for improving the stress tolerance of crop plants [48]. Thegenetic engineering of metabolic pathways for the production of osmolytes, such asmannitol, fructans, trehalose, proline, and glycine betaine, among others, mightincrease resistance to drought, but themechanism by which these osmolytes provideprotection is not completely understood [137]. Usually, the osmolytes are localized inthe cytoplasm of plants. The active accumulation of osmolytes decreases the osmoticpotential of cell andmaintains cell turgor. Other responses, such as the production ofscavenging ROS and the induction of chaperone-like activities that protect proteinstructure and metabolic detoxification, are also being reported during droughtstress [138].

Metabolic engineering has allowed the introduction of biosynthetic pathways ofglycine-betaine from microorganisms into maize. Indeed, maize accumulatedhigher levels of glycine-betaine when transformed with the beta gene from E. colithat encodes choline dehydrogenase, a key enzyme in the biosynthesis of glycine-betaine [129]. An assortment of genes with diverse functions are induced orrepressed by these stresses [139]. Most of their gene products may function in stressresponse and tolerance at cellular level. Significantly, the introduction ofmany stress-inducible genes via gene transfer resulted in improved plant stress tolerance [2, 140].

Genetic engineering of plants for tolerance to extreme abiotic stresses could beachieved by the regulated expression of stress-induced TFs, which, in turn, wouldregulate the expression of a large number of relevant downstream genes [141]. TFshave been used to elicit multiple biochemical and developmental pathways thatregulate drought tolerance, thereby improving performance during drought under

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laboratory and greenhouse conditions [2]. The best stress-responsive TFs are the C-repeat binding factor (CBF)/dehydration-responsive element binding proteins thatbelong to the AP2/ethylene-responsive element binding protein family [142]. Thesefactors enhance or modulate the expression of genes with a CBF/DRE box in theirpromoters and define a major stress tolerance pathway, in addition to the ABAbiosynthesis/response pathway. On the other hand, the promoter of the earlyresponse to dehydration 1 (ERD1) gene contains cis-acting element(s) involved inABA-independent stress-responsive gene expression [143]. Positive reports on theuse of TFs to improve drought resistance in model and crop plants are based onlaboratory and greenhouse conditions rather than field conditions; hence, the use ofTFs in enhancing drought tolerance in crop plants should be consideredwith caution.Therefore, there is a need for understanding the basic molecular mechanismsinfluencing drought tolerance and grain yield under field stress conditions [7]. Tilldate, various genes and TFs crucial for stress tolerance have been studied [144].

Recently, drought tolerance in transgenic maize plants under field conditions hasbeen enhanced through overexpression of NF-YB [144], which is part of a ubiquitousTF composed of three distinct subunits, NF-YA (HAP2), NFYB (HAP3), and NF-YC(HAP5) [145]. The NF-Ycomplex is also known as theHAP or the CAATcomplex thatacts in concert with other regulatory factors to modulate gene expression in a highlycontrolled manner. The overexpression of a maize CAAT box TF (ZmNFYB2)imparted significant tolerance to drought, resulting in increased yield. Most impor-tant, in field trials, the transgenic lines gave higher grain yields than control linesunder drought conditions [7]. Engineering upstream signaling components ofdrought stress pathways might be another promising way to obtain drought stresstolerance. Indeed, constitutive expression of NPK1, a tobacco MAPKKK, in maizeenhanced drought tolerance, as demonstrated by higher photosynthesis rates andhigher kernel weight in the transgenic plants than those of the nontransgeniccontrols under greenhouse dehydration conditions [146]. The stress adaptationresponses contribute to a yield advantage in maize that is grown within droughtenvironments. The application of this technology is therefore expected to have themost significant impact on severely water-limited maize production systems [144].

33.9Conclusions

The occurrence of drought varies unpredictably according to years, seasons, places,and within fields, so maize genotypes able to withstand stress throughout their lifecycles at no cost to yield potential are the need of the hour. The use of genetics andgenomics within an integrated framework that relies heavily upon critical input fromdisciplines such as plant breeding, crop physiology, crop modeling, and precise fieldphenotyping is sought. This integration of quantitative knowledge arising fromdiverse, but complementary, disciplines will allow researchers to more fully under-stand genes associated with drought tolerance in maize and more accurately predictthe consequences of modulating expression levels of those genes.

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Knowledge-based approach can improve maize production for the drought-proneweather and to developmore focusedfield screening techniques that increase rates ofgain for yield and its stability under conditions of variable and unpredictable waterstress. It is thus essential to test newly developed genotypes tomultiple stresses and tocarry out extensivefield studies under a large range of conditions that assess toleranceas absolute yield increases. As a number of measures are in place to ensure the safeand responsible design of field tests, especially the transgenic approach, excessiveprecaution should not become a barrier to using all the tools available to us for amoresustainable agriculture.

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