Maydica 50 (2005): 549-558

DROUGHT TOLERANCE IN MAIZE A. Moreno, V. Lumbreras, M. Pagès* IBMB-CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain

Received February 16, 2005

ABSTRACT - Water-deficit stress caused by drought, soil salinity and low-temperatures, affects negatively plant growth and development and it is one of the major causes for crop yield reductions in farmed regions around the world. Indeed, limited availability of water resources for agronomical uses in many regions generates important problems in a context where it is essential to raise crop productivity to insure food needs of an increasing world population (). Development of crop varieties with less dependence on water and more tolerant to drought should contribute to ameliorate this problem. Genetic engineering seems to be a more rapid and efficient approach to improve stress tolerance than the traditional breeding and marker-assisted selection strategies. Due to the complexity of the molecular mechanisms of plant stress responses, research into this area together with the study of genetic modification of stress tolerant traits will provide the necessary tools for a successful application of genetic engineering (WANG et al., 2003). KEY WORDS: Drought; Maize; ABA; Genetic engineering.

INTRODUCTION Maize (Zea mays) is one of the most important cereal crops in the world. In developing countries, maize is used for human consumption, while in the developed world is mainly employed for animal feed and industrial use (http://www.fao.org /docrep/T0395E /T0395E02.htm). Historically, maize improvements in grain yield have been attributed in part to the selection of parental inbred lines and their hybrid progenies, with an enhanced performance under water-deficit stress conditions (HOISINGTON et al., 1999; BRUCE et al., 2002; FARINHA et al., 2004). This review summarizes the recent advances

* For correspondence (fax: +34 93 204 59 04; e.mail: [email protected]).

in the knowledge of the molecular mechanisms underlying plant water-stress responses and the potential application of genetic engineering to improve crop production. Finally, several strategies to obtain transgenic maize lines with enhanced drought tolerance are discussed. Plant responses to water-deficit stress Drought, soil salinity and low-temperatures are manifested in the plant primarily as osmotic stress. Water-deficit results in a loss of cell turgor and in the alteration of cellular constituents that may lead to the destabilization of the different membrane systems, protein aggregation and disruption of normal metabolism (WALTERS et al., 2002). Limited water availability is often accompanied by oxidative stress due to the photoreduction of oxygen and concomitant production of reactive oxygen species (ROS), such as superoxides (O2-) and peroxides (H2O2) that interact with proteins, lipids and nucleic acids causing permanent damage to enzymes, membranes and chromosomes (SMIRNOFF, 1998; WALTERS et al., 2002). Water-deficit stress leads to a series of physiological and molecular responses that will enable plants to overcome this unfavourable situation. Almost all plants can tolerate extremes of osmotic stress at some stages of their life cycle. For example, embryos of cereal seeds can sustain reductions in water content of about 80%, whereas such severe desiccation results in cell death in other plant tissues. This fact makes the cereal embryo an important model system for the understanding of water-deficit stress tolerance. During vegetative growth, plants have evolved two major mechanisms to endure dry periods: water-deficit avoidance and water-deficit tolerance. The first strategy allows the plant to reduce water loss from leaves by regulating stomatal function, or to increase water absorption by adapting root architecture. The second one, permits plants to sustain osmotic stress through the

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re-establishment of cellular homeostasis and the structural and functional protection of proteins and membranes (JENSEN et al., 1996). The phytohormone abscisic acid (ABA) plays a major role in regulating several developmental and physiological processes in the plant such as seed development and germination, and mediating the response of vegetative tissues to osmotic stress. ABA levels increase in late embryo development shortly before the onset of desiccation and in vegetative tissues under water-deficit stress conditions (ZEEVART and CREELMAN, 1988). ABA mediates both stomatal closure and water-deficit tolerance responses by regulating changes in the activity of signalling molecules, ion channels, and changes in gene expression (FINKELSTEIN et al., 2002; HIMMELBACH et al., 2003) (Fig 1). Many genes that respond to water-deficit stress are expressed during normal seed maturation, can be induced in immature embryos and unstressed vegetative tissues by the exogenous application of ABA. The products of these genes are thought to function, directly or indirectly, in protecting cells from dehydration (BRAY, 1993; BOHNERT et al., 1995; INGRAM and BARTELS, 1996), and can be classified into two groups: functional proteins and regulatory proteins. The first group includes proteins that partici-

FIGURE 1 - Drought signal transduction pathways.

pate in stress tolerance: aquoporins and ion channels, enzymes required for the biosynthesis of various compatible solutes, osmoprotectans such as late embryogenesis abundant (Lea) proteins and chaperones, and detoxification enzymes. The second group contains proteins involved in dehydration/ABA signal transduction cascades and gene expression regulators: metabolic enzymes, protein kinases and phosphatases, and transcription factors (SHINOZAKI and YAMAGUCHI-SHINOZAKI, 1997) (Fig 1). Recently, genetic screenings have allowed the identification of a set of genes induced in maize seedlings during waterdeficit stress. Among them, enzymes of amino acid and carbohydrate metabolism, kinases and transcription factors, have been proposed to be involved in drought signalling pathway (ZHENG et al., 2004). Drought and ABA perception and signalling cascades A few data is known about the plant mechanisms for sensing dehydration or ABA. A first perception event triggers a cellular signal transduction pathway that will translate a physical or hormonal signal into a molecular response. There are several aspects of cellular water loss that can be sensed by the plant, such as the decrease on turgor, changes in cell volume or membrane area and alterations in

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cell wall-plasma membrane connections (BRAY, 1993). One protein of Arabidopsis has been proposed to act as an osmosensor, the trans-membrane hybrid-type histidine kinase, AtHK1. This molecule is thought to relay changes in osmotic potential outside the cell to one or more transduction pathways inside the cell, leading to the expression of droughtinducible genes (URAO et al., 1999). Despite many efforts during years, the identification of ABA receptor candidates has resulted unsuccessful. However, physiological and molecular studies suggest the existence of ABA-sensing mechanisms presumable involving stereospecific ABA receptors, both soluble and localized in the plasma membrane (FINKELSTEIN et al., 2002; HIMMELBACH et al., 2003) (Fig 1). Once the physical or hormonal signal has been perceived, it is transduced into a signalling cascade that will allow the plant to give an appropriate response. Second messengers and several classes of proteins such as G proteins, phospholipases, protein kinases and phosphatases have been described to participate in dehydration/ABA signalling (SHINOZAKI and YAMAGUCHI-SHINOZAKI, 1997) (Fig 1). In leaves of maize seedlings drought-induced ABA accumulation causes an increased generation of ROS that enhances the activities of antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase. ABA-induced ROS production is mainly carried out by the plasma membrane-bound NADPH oxidase, which transfers electrons from cytoplasmic NADPH to O2 to form O2- followed by dismutation of O2- to H2O2 (JIANG and ZHANG, 2002). Ca2+ has also been shown to be involved in ABA-induced antioxidant defence in leaves of maize seedlings, acting in the upstream as well as downstream of ROS production (JIANG and ZHANG, 2003). Guard cells of Arabidopsis have been extensively used as a simple biological system for analyzing ABA responses. ABA induces cytosolic Ca2+elevations through extra cellular Ca2+ influx and release from intracellular stores. Ca2+ is triggered by second messengers such as ROS, cyclic ADP ribose (cADPR), inositol triphosphate (IP3) and myo-inositol hexakiphosphate (IP6). Cytosolic Ca2+ increase activate anion channels (S- and R-types) that mediate anion release from guard cells and subsequent plasma membrane depolarization, activating outward-rectifying K+ (K+out) channels and results in K+ efflux from guard cells. Both anion and K+ effluxes contribute to the loss of guard cells turgor, leading to stomata closure (SCHROEDER et al., 2001). Another phospholipidderived molecule, the phosphatidic acid (PA), is

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emerging as an important second messenger. Dehydration and ABA induce the expression and activity of phospholipases D (PLD) and the accumulation of PA (FAN et al., 1997; KATAGIRI et al., 2001). In guard cells, PA mimics the ABA action by inhibiting partially the K+ uptake channels (K+in) and inducing a 50% of stomata closure (JACOB et al., 1999). In addition, the ABA-dependent production of phospholipid sphingosine-1-phosphate (S1P) stimulates stomata closure in response to drought and requires G protein a subunit 1 for action (GPA1) (NG et al., 2001; COURSOL et al., 2003). Loss of function of gpa1 gene disrupts ABA-inhibition of guard cell K+in channels and plants present increased leaf transpiration (WANG et al., 2001). The Rho-like small G protein ROP10 negatively regulates ABA-mediated stomata closure and requires farnesylation and plasma membrane localization to exert its action (ZHENG et al., 2002). Reversible protein phosphorylation is one of the major mechanisms for mediating cellular responses. Many kinases have been implicated in dehydration and/or ABA signalling affecting stomata function and/or gene expression. The sucrose non-fermenting 1-related protein kinases 2 (SnRK2) family members, ABA-induced protein kinase 1 (PKABA1) of wheat and ABA-activated protein kinase (AAPK) of Vicia are positive regulators of ABA-mediated gene expression and stomata closure, respectively (GOMEZ-CADENAS et al., 2001; JOHNSON et al., 2002; LI et al., 2002). AAPK and open stomata 1 (OST1)/SnRK2-type protein kinase E (SRK2E) of Arabidopsis are likely orthologs, since both AAPK and OST1/SRK2E mutants do no close stomata pores in response to ABA and drought (MUSTILLI et al., 2002; YOSHIDA et al., 2002). Over expression of another member of the SnRK2 family, SRK2C in Arabidopsis confers drought tolerance by up-regulating several stress-responsive genes (UMEZAWA et al., 2004). Studies on SnRK2 protein kinases activity regulation have been carried out in different plant models. All members identified are activated by hyperosmotic stress, but only some of them are also activated by ABA (MIKOLAJCZYK et al., 2000; BOUDSOCQ et al., 2004; KOBAYASHI et al., 2004). Another group of protein kinases that has been described to be activated by osmotic stress are the mitogen-activated protein kinases (MAPK) (ICHIMURA et al., 2000; MIKOLAJCZYK et al., 2000). Transgenic tobacco plants expressing a constitutively active form of Nicotiana protein kinase (NPK1), a MAP kinase kinase kinase (MAPKKK), display enhanced tolerance to multiple environmental stresses by inducing oxidative stress-

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responsive genes (KOVTUN et al., 2000). Ca2+-regulated protein kinases also play an important role in signalling osmotic stress. Ca2+-dependent protein kinases (CDPK) are induced by water-deficit stress (URAO et al., 1994) and over expression of OsCDPK7 results in increased osmotic stress tolerance in rice (SAIJO et al., 2000). Finally, members of the calcineurin B-like protein (CBL)-interacting protein kinases (CIPK) (also known as SnRK3), do not bind Ca2+ by themselves, but instead each interacts with a specific member of the CBL/SCaBP (SOS2-like Ca2+binding protein) family of Ca2+ sensors, that have been described to be involved in high salinity and ABA signalling (QIU et al., 2002; GUO et al., 2002; KIM et al., 2003). On the other hand, protein phosphatases belonging to the PP2C family, ABA-insensitive 1 and 2 (ABI1 and ABI2), act as negative regulators of ABA responses (MERLOT et al., 2001). Arabidopsis mutants, abi1 and abi2, show a pleiotropic phenotype alterations including abnormal stomata regulation and excessive water loss under drought conditions (KOORNNEEF et al., 1984; LEUNG et al., 1997), due to a reduction of ABA-induced cytosolic Ca2+ elevations in guard cells (GETHYN et al., 1999). Regulation of gene expression by drought and ABA Transcription control is a key process during drought stress responses. As was mentioned above, plants increase endogenous ABA levels in response to drought and many drought-inducible genes are induced by exogenous application of ABA, indicating the role of the phytohormone in mediating drought responses at the transcriptional level. For example, the maize Lea genes responsive to aba 17 (rab17) and rab28 which are up-regulated during the late embryogenesis and by water-deficit stress in vegetative tissues, are also induced by ABA treatment (VILARDELL et al., 1990; PLA et al., 1991). In Arabidopsis, more than half of the drought-inducible genes are also induced by high-salinity and/or ABA, suggesting the existence of a significant cross-talk among these responses (SEKI et al., 2002a,b). On the other hand, ABA deficient or insensitive mutants have been used to elucidate how ABA regulates specific gene expression. Expression analysis in maize viviparous mutants deficient (vp2) or insensitive to ABA (vp1), revealed that rab17 and rab28 mRNA accumulation is reduced but not absent (PLA et al., 1989, 1991), suggesting that different signal transduction pathways control ABA-mediated gene expression. Similarly, in Arabidopsis ABA-

deficient (aba) or ABA-insensitive (abi) Arabidopsis mutants some of the stress-inducible genes did not require an accumulation of endogenous ABA under water-deficit conditions, supporting the idea of the existence of ABA-dependent and ABA-independent mechanisms during adaptation to drought stress (BRAY, 1993; INGRAM and BARTELS, 1996; SHINOZAKI and YAMAGUCHI-SHINOZAKI, 1997). Different studies have provided evidence of physiological roles of Rab proteins in stress tolerance. Over expression of maize rab17 in Arabidopsis resulted in higher survival rates during salt stress and faster recovering from mannitol treatment (FIGUERAS et al., 2004). Several groups of transcription factors are involved in dehydration- and/or ABA-responsive gene expression. DRE/CRT-binding proteins (DREB/CBF) contain a conserved DNA-binding motif, the APETALA2 (AP2) domain, also found in the ERF family of transcription factors which binds the dehydrationresponsive element (DRE) (OKAMURO et al., 1997). Members of the DREB/CBF family have been cloned in maize, named DRE-binding protein 1 (DBF1) and DBF2. These two proteins bind to the DRE2 motif on the rab17 promoter. DBF1 was transcriptionally induced by dehydration, NaCl and ABA, while DBF2 showed a constitutive expression profile (KIZIS and PAGES, 2002). Preliminary data indicate that over expression of DBF1 in Arabidopsis results in plants that are significantly more tolerant to drought and salt stresses than control plants (A. SALEH and M. PAGES, unpublished results). In Arabidopsis, DREB/CBF members are classified into two groups, DREB1 and DREB2, based on low sequence similarity outside the AP2 domain. In addition, both groups are differentially regulated: DREB1 genes are quickly and transiently induced by cold stress, whereas DREB2 genes are induced by dehydration and high-salinity. Transgenic Arabidopsis plants that over express the DREB1A gene showed enhanced drought, high-salinity and cold tolerance. However, these transgenic plants displayed severe growth retardation under normal conditions (LIU et al., 1998). The same phenotype was observed when the maize DREB1A homolog was over express in Arabidopsis (QIN et al., 2004). It has been shown that the use of the stress-inducible promoter rd29A minimizes the negative effects on the growth of the transgenic plants and enhances drought, high-salinity and cold tolerance to a greater extent than the 35S cauliflower mosaic virus (CaMV) (KASUGA et al., 1999). The basic-domain leucine zipper (bZIP) family of

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transcription factors bind to the ABA-responsive element (ABRE) and activate ABA-dependent gene expression in response to drought and high-salinity stresses (UNO et al., 2000). In maize, EmBP2 and ZmBZ-1, two new basic-domain leucine zipper (bZIP) family members are involved in the expression of the rab28 by binding to the ABRE motifs on its promoter region, and this effect was modulated by ABA (NIEVA et al., 2005). Transgenic Arabidopsis plants over expressing the ABRE-binding factor 3 (ABF3) or ABF4 genes showed reduced transpiration rate and enhanced drought tolerance (KANG et al., 2002). Maize VP1 and Arabidopsis ABI3 proteins are homologous transcriptional activators essential for ABA action during seed development (MCCARTY et al., 1991; GIRAUDAT et al., 1992). VP1 and ABA have a synergistic effect on transcription through the ABRE elements of the rab28 promoter. However, there was found a strong protein binding even in the absence of VP1, thus it was proposed that VP1 binds indirectly to the ABRE through protein-protein contacts with ABRE binding factors previously bound to this motif, and stimulates transcription by means of the acidic activator domain (BUSK and PAGES, 1997). MYB and MYC proteins also activate ABA-dependent gene expression in response to dehydration by binding to specific cis-acting elements, but in contrast to the bZIP/ABRE system, protein biosynthesis is required (ABE et al., 1997). AtMYB2 and AtMYC2 have been shown to bind the cis-acting elements of the rd22 promoter and activate its expression. Over expression of both AtMYB2 and AtMYC2 genes improved osmotic-stress tolerance of transgenic plants (ABE et al., 2003). Maize genetic engineering of drought tolerance traits Drought stress tolerance is a complex trait that involves a wide range of proteins. Genetic engineering of plants for drought tolerance is a longstanding goal of agricultural biotechnology. Multiple examples of the genetically modified drought tolerant plants are non-agronomical plants like tobacco and Arabidopsis. This fact is related to their greater facility in transforming and reduced life cycles compared with crop plants. Moreover, among the crop plants genetically modified for drought tolerance, only a few have been tested in field. An ideal genetically modified drought-tolerant maize crop should posses a high grain yield potential and stability under both water-deficit and wellwatered conditions (BRUCE et al., 2002; DUNWELL,

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2002). Unfortunately, drought-tolerance traits are not always associated with a better grain yield. For instance, root architecture plays an important role in water acquisition, and therefore is a significant component of drought tolerance. However, no correlation seems to exist between root development and effects of drought on yield. Comparison of recombinant inbred lines differing in seedling root architecture indicated that lines with a poorer early root development yielded better than lines with an extensive adventitious and lateral root development (unpublished results referenced in BRUCE et al., 2002). These results are in accordance with the observation that improved grain yield under drought stress was accompanied by reduced root biomass (BOLAÑOS et al., 1993). For maize, the critical developmental period of determining grain yield centres on flowering and early seed filling (BOYER and WESTGATE, 2004). It has been shown the importance of a steady photoassimilates flow during both ovule/pollen and seed development. Water-deficit stress inhibits photosynthesis, likely by inducing stomata closure and limiting CO2 availability. Moreover, dehydration affects negatively the activity of carbon metabolic enzymes such as acid invertase, which plays a central role in providing the necessary sugars for growth to the developing ear (ZINSELMEIER et al., 1999). Therefore, an important factor determining grain yield of crops is the maintenance of the photosynthetic activity and thus carbon assimilation rate. Maize is a well adapted plant to warm climates, since it has evolved the C4 photosynthetic carbon assimilation metabolism in addition to the C3 pathway to achieve high photosynthetic capacity and high water- and nitrogenuse efficiencies. In the C4 pathway, the initial fixation of CO2 occurs in the cytosol of mesophyll cells by phosphoenolpyruvate carboxylase (PEPC) to form a C4 acid, the oxaloacetate (OAA). OAA is reduced by NADP-malate dehydrogenase (NADPMDH) to malate, which is pumped up into the chloroplasts of bundle sheath cells and decarboxylated by NADP-malic enzyme (NADP-ME). Releasing of CO2 at the vicinity of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) allows plants to avoid the loss of fixed carbon by photorespiration (MIYAO, 2003). This natural ability of maize to optimize CO2 fixation under water-deficit conditions can be used to improve drought tolerance by gene transference. The C4-PEPC enzyme has been shown to exert significant control on the photosynthetic carbon flux (BAILEY, 2000). Maize

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plants over expressing the C4-PEPC gene of Sorghum bicolor showed an initial gain in CO2 assimilation rate over control plants, that progressively diminished during mild drought treatment until reach wild-type values. In contrast, intrinsic water use efficiency (WUE=CO2 assimilation rate/stomatal conductance) became 30% higher than control plants towards the end of the experiment, suggesting the involvement of C4-PEPC in enhancing photosynthetic CO2 assimilation rate under moderate drought conditions (JEANNEAU et al., 2002). Unfortunately, no data about the grain yield of these mutant and transgenic maize lines have been reported. Maintenance of the photosynthetic activity and thus carbon assimilation rate appears to be also a good approach to enhance drought tolerance. Photosynthetic activity increases during leaf expansion and declines with leaf age until it reaches a low level prior to the onset of leaf senescence. Adverse environmental conditions such as drought induce loss of leaf function and premature onset of senescence in older leaves (THOMAS and HOWARTH, 2000). Several hormones such as ethylene control the onset of the senescence program in leaves. Drought promotes increasing 1-aminocyclopropane-1-carboxylic acid (ACC) synthesis and its conversion to ethylene (APELBAUM and YANG, 1981). Inhibition of ethylene synthesis reduces the drought-induced loss of chlorophyll and prevents drought-induced senescence (BELTRANO et al., 1999). Maize knockout mutant deficient in ACC synthase (ACS), the first enzyme in the ethylene biosynthetic pathway, exhibited a reduction of up to 90% of foliar ethylene and delayed leaf senescence. Mutant leaves exhibited retained chlorophyll and protein content, and maintained normal rates of transpiration, CO2 assimilation and stomatal conductance longer than wildtype leaves under both normal and drought conditions (YOUNG et al., 2004). Ability to protect enzymes and cellular membranes and to recover from a water-deficit situation by osmotic adjustment enhances plant capacity to survive and produce grain. Plants have evolved the capacity to synthesize and accumulate compatible solutes or osmolytes as an important mechanism to adapt to osmotic stress. Osmolytes can be classified into three major groups: amino acids (e.g. proline), quaternary amines (e.g. glycine betaine, dimethysulfoniopropionate), and polyols/sugars (e.g. mannitol, trehalose). Compatible solutes accumulation function primarily to generate the driving gradient for water uptake and thus to maintain cell turgor. Being

non-toxic, compatible solutes can be accumulated to osmotically significant high levels without disrupting metabolism. Recently, several studies indicated that compatible solutes can also act as osmoprotectants by directly stabilizing proteins and membranes. However, some species are able to accumulate such compounds more efficiently than others. For that reason, genetic engineering of osmolyte-producing enzyme systems from plants and microbes can be used as a target for improving water-deficit tolerance (HOLMBERG and BÜLOW, 1998; NUCCIO et al., 1999; CUSHMAN and BOHNERT, 2000; WANG et al., 2003). In maize, levels of glycine betaine varie among different inbred lines (BRUNK et al., 1989). Positive correlation has been found between the level of endogenous glycine betaine and the degree of tolerance to salt in maize (SANEOKA et al., 1995). Transgenic maize lines over expressing the betA gene from Escherichia coli encoding choline dehydrogenase, a key enzyme in the biosynthesis of glycine betaine from choline, accumulated high levels of glycine betaine and were more tolerant to drought. According with the idea that glycine betaine has a protective rather than an osmotic effect, transgenic plants showed less cell membrane damage and greater activity of photosystem II than the nontransgenic plants after an osmotic treatment. The enhanced glycine betaine synthesis may also protect the enzymes associated with sugar and amino acid metabolism, leading to higher increases in total soluble sugar and free amino acids in transgenic lines than in control plants in response to dehydration. Most importantly, the grain yield of transgenic plants is significantly higher than that of control plants after drought stress, which may be due to a less inhibition of the development of reproductive organs (QUAN et al., 2004). Since drought tolerance implies a complex regulation of physiological and molecular mechanisms, it is thought that the transference of several genes encoding functional proteins, or the transference of one gene encoding a regulatory protein offers advantage in the achievement of a high level of waterdeficit or other types of stress tolerance (HOLMBERG and BÜLOW, 1998; NUCCIO et al., 1999; WANG et al., 2003). SHOU et al. (2004a) expressed in maize a cDNA fragment encoding the kinase domain of the tobacco NPK1 under the control of a constitutive promoter. NPK1 is a MAPKKK previously described to be involved in activating an oxidative signal cascade and leading to drought, salinity and freezing toler-

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FIGURE 2 - Strategy to identify potential genes to improve crop drought tolerance.

ance in transgenic tobacco plants (KOVTUN et al., 2000). Under drought conditions, transgenic plants showed higher photosynthesis rates than did the wild-type plants, suggesting that NPK1 induced a mechanism that protected photosynthetic machinery from dehydration damage, maybe by switching on the oxidative pathway that leads activation of genes such as glutathione S-transferase (GST) and heat shock proteins (HSPs). The detrimental effects on cell division, embryogenesis and seed development observed previously in transgenic tobacco NPK1 plants (KOVTUN et al., 1998) were not observed in transgenic maize lines, probably due to that the transgenic plants selected in this experiment had relatively low expression of the transgene (SHOU et al., 2004b). In addition, drought-stressed transgenic plants produced grains with similar weight to those under watered conditions, while grain weight of drought-stressed wild-type plants was reduced when compared with their non-stressed counterparts. Future perspectives Large-scale sequencing of expressed sequence tags (EST) and genomic DNA is the first step to identify those genes involved in drought responses

that can be used as targets for engineered drought tolerance. Extensive EST collections already exist for maize and are available in the public domain at GenBank (http://www.ncbi.nlm.nih.gov/dbEST/) and MaizeGDB (http://www.maizegdb.org). Maize genomic DNA sequencing projects are under way (MESSING et al., 2004) and an integrated genetic/physical map is being produced (http://www.maizemap.org; http://www.genome.arizona.edu/fpc/maize/). Microarray technology using cDNAs or oligonucleotides, among other transcriptprofiling techniques, offers a high-throughput tool to describe gene expression profiles under drought conditions and identify components involved in water-deficit responses. Those genomic approaches can be extended with proteomic tools, since recent developments in mass spectrometry and the creation of algorithms for automatic spot quantification, along with a better resolution and reproducibility of twodimensional SDS-PAGE gels, have simplified protein studies (Fig 2). For example, genomic and proteomic analyses of drought-sensitive and -tolerant maize varieties are powerful approaches to identify genes/proteins involved in water-deficit stress responses and tolerance.

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The generation and screening of large T-DNA or transposon insertional mutant collections is necessary to complement data obtained by both genomic and proteomic approaches. In maize, knockout lines derived by insertional mutagenesis will be made and available in databases (http://www.maizegdb.org/; http://mtm.cshl.org). In parallel, employment of gene silencing technology (e.g. RNA interference mediated gene silencing) will make possible the recovering of knockout phenotypes for genes with redundant function. Once potential target genes for engineering stress tolerance have been identified, the first step is to analyse their effects in non-agronomical plants. For example, tobacco and Arabidopsis transgenic plants allow a rapid analysis for drought tolerance, and also can be used to check that ectopic expression of the particular gene has not metabolic imbalance consequences that could result in undesirable traits. This will require gene manipulation guided by thorough analysis of metabolic fluxes and pool sizes. In vivo nuclear magnetic resonance (NMR) spectroscopy is being applied successfully in plants to monitor enzymatic rate and pool size data (AUBERT et al., 1998).

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