Engineering drought tolerance in plants: discovering and tailoring genes to unlock the future Taishi Umezawa1,2, Miki Fujita2,3, Yasunari Fujita4, Kazuko Yamaguchi-Shinozaki3,4,5 and Kazuo Shinozaki1,2,3 The ability of plants to tolerate drought conditions is crucial for agricultural production worldwide. Recent progress has been made in our understanding of gene expression, transcriptional regulation and signal transduction in plant responses to drought. Molecular and genomic analyses have facilitated gene discovery and enabled genetic engineering using several functional or regulatory genes to activate specific or broad pathways related to drought tolerance in plants. Several lines of evidence have indicated that molecular tailoring of genes has the potential to overcome a number of limitations in creating drought-tolerant transgenic plants. Recent studies have increased our understanding of the regulatory networks controlling the drought stress response and have led to practical approaches for engineering drought tolerance in plants. Addresses 1 Gene Discovery Research Group, RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Yokohama, Kanagawa 203-0045, Japan 2 Laboratory of Plant Molecular Biology, RIKEN Tsukuba Institute, 3-1-1 Kouyadai, Tsukuba, Ibaraki 305-0074, Japan 3 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan 4 Biological Resources Division, Japan International Research Center for Agricultural Sciences, 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan 5 Laboratory of Plant Molecular Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Corresponding author: Shinozaki, Kazuo (email@example.com)
for this purpose, one of the major research goals is to understand the molecular mechanisms underlying drought tolerance in plants. Drought triggers a wide variety of plant responses, including alterations in gene expression, the accumulation of metabolites such as the phytohormone abscisic acid (ABA) or osmotically active compounds, and the synthesis of specific proteins (e.g. largely hydrophilic proteins, proteins that function to scavenge oxygen radicals, chaperone proteins, etc. ). The basic strategy of genetic engineering for drought tolerance is to introduce functional genes that are directly involved in these events. With the advancement of DNA microarray technology, which allows high-throughput analysis of differential messenger RNA expression, several hundred stress-induced genes have been identified as candidate genes for genetic engineering. Among the genes identified, several were classified as regulatory genes, such as protein kinases and transcription factors, in addition to functional genes. Although the conventional approaches in both plant breeding and physiology are of great importance [3,4], the genetic engineering of key regulatory genes that govern a subset of stress-related genes appears to be one of the most promising strategies for enabling scientists to minimize the deleterious effects associated with drought (Figure 1). This review focuses on recent advances in the basic research of drought stress tolerance at the molecular level. In particular, we emphasize genetic engineering approaches in model plants using recently discovered or tailored genes related to drought tolerance.
Current Opinion in Biotechnology 2006, 17:113–122 This review comes from a themed issue on Plant biotechnology Edited by Nam-Hai Chua and Scott V Tingey Available online 21st February 2006 0958-1669/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2006.02.002
Introduction Growing in their natural environment, plants often encounter unfavorable environmental conditions that interrupt normal plant growth and productivity. Among such environmental stresses, drought is one of the greatest worldwide environmental constraints for agriculture . Therefore, engineering drought tolerance in plants has huge economical importance. To develop novel strategies www.sciencedirect.com
Functional proteins: classical and innovative approaches Recently, several successful attempts to engineer drought tolerance have transferred functional genes that encode enzymes associated with the synthesis of osmotically active compounds, transporters, chaperones and reactive oxygen species (ROS) scavengers, as shown in Table 1 (reviewed in [4,5]). In drought-tolerant transgenic plants, many genes involved in the synthesis of compatible solutes — organic compounds such as amino acids (e.g. proline), quaternary and other amines (e.g. glycinebetaine and polyamines) and a variety of sugars and sugar alcohols (e.g. mannitol, trehalose and galactinol) that accumulate during osmotic adjustment — have been used to date [4,6]. The most recent advances in this area include the finding of a novel synthetic pathway for glycinebetaine, one of the major osmoprotectants, in halophilic microorganisms [7,8,9]. Higher levels of glycine-betaine accumulation were detected in transgenic Arabidopsis Current Opinion in Biotechnology 2006, 17:113–122
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Strategies for the genetic engineering of drought tolerance. Molecular and genomic analyses have facilitated the discovery of genes involved in stress tolerance, which can then be used for genetic engineering. In this approach, a range of genes can be employed, including genes encoding functional proteins such as transporters and chaperones. For metabolic engineering, enzymatic fusions are commonly used to target multiple steps. In some cases, signal peptides are added to make proteins work in their correct organellar location. Recent studies have also shed light on the role of regulatory proteins, such as transcription factors and signaling factors, which have provided novel routes for engineering drought tolerance. The activity of such regulatory proteins can be controlled through point mutations, partial deletions, post-translational modifications (e.g. phosphorylation by kinases) or by the addition of activation or repression domains. Genes can also be regulated at the mRNA level (e.g. upregulated by sense overexpression transgenes and downregulated by antisense or RNAi). The intensity and time-space pattern of transgene expression can be driven by appropriate promoters (Pro).
plants expressing the betaine synthesis genes of the novel pathway, which acts via glycine, than in plants expressing a choline-oxidizing enzyme from the conventional cholinemediated betaine synthesis pathway . Interestingly, although the accumulation of glycine-betaine in these transgenic plants is still lower than in plants of species that normally accumulate glycine-betaine under conditions of stress, these transgenic plants showed significant augmentation of dehydration tolerance. The increased tolerance could be associated with the protective function of betaine molecules, which might be involved in the stabilization of proteins and cell structures and/or in scavenging of free-radicals, as well as the osmotic effects mediated by betaine molecules . One of the most important metabolites produced in response to drought stress is the plant hormone ABA . Recently, in addition to a key ABA biosynthesis gene (NCED3 ), a cytochrome P450 CYP707A family has been identified as ABA 80 -hydroxylases, which play a central role in regulating ABA levels during seed imbibiCurrent Opinion in Biotechnology 2006, 17:113â€“122
tion and dehydration stress conditions [12,13]. It is possible that these findings could enable us to control ABA levels under conditions of drought stress and may ultimately contribute to an advancement in the engineering of drought tolerance. Indeed, an insertional mutant of CYP707A3, which was expressed most abundantly among four CYP707A members under stress conditions, exhibited elevated drought tolerance with a concomitant reduction of transpiration rate . Not only metabolites, but some stress-responsive proteins such as the late-embryogenesis-abundant (LEA) class of proteins, have also been thought to function in the detoxification and alleviation of cell damage during dehydration . Overexpression of some LEA genes has been reported to result in enhanced tolerance to dehydration, although the precise mechanism is still unknown [4,10]. Recent biochemical analysis demonstrated that LEA proteins can prevent protein aggregation induced by desiccation as well as freezing . Taken together with recent computational studies [16,17], LEA proteins are www.sciencedirect.com
Engineering drought tolerance Umezawa et al. 115
Table 1 Engineering drought tolerance using functional proteins. Transgenic
Osmolyte metabolism Fructan SacB Trehalose TPS1 myo-Inositol IMT1 Proline P5CS
Tobacco Tobacco Tobacco Rice
B. subtilis b S. cerevisiae b Iceplant Mothbean
5–10% PEG (soil) Desiccation Water withholding Water withholding
Plant growth FW, survivability Photosynthesis Shoot growth
1995 1996 1997 1998
E. coli b
CaMV35SP CaMV35SP CaMV35SP AIPC-ABAinducible CaMV35SP
Fructan Glycine betaine
B. subtilis b A. pascens b
AtGolS2 TPSP (OtsA+OtsB) TPSP (OtsA+OtsB) mtlD ADC SPDS P5CS TPS1 GSMT+DMT
Sugar beet Arabidopsis/ canola/ tobacco Arabidopsis Rice
Arabidopsis E. coli b E. coli b
Wheat Rice Arabidopsis Petunia Tomato Arabidopsis,
E. coli b D. stramonium b C. ficifolia Arabidopsis, rice S. cerevisiae b A. halophytica b
Maize Ubi-1P Maize Ubi-1P CaMV35SP CaMV35SP CaMV35SP CaMV35SP
Survivability Photosynthesis, shoot growth Photosynthesis, shoot growth Limiting water supply Shoot growth, biomass 20% PEG (soil) Shoot growth Water withholding Shoot growth Water withholding Survivability Water withholding Shoot growth Water withholding Photosynthesis, shoot growth
CaMV35SP Water withholding ABA-inducible/ Water withholding rbcSP Maize Ubi-1P Water withholding
2003 2004 2004 2005 2005 2005
Protective proteins LEA HVA1 LEA HVA1 Chaperone BiP
Rice Wheat Tobacco
Barley Barley Soybean
Rice Act-1P Maize Ubi-1P CaMV35SP
1996 2000 2001
Heat shock protein AyHsp17.6A LEA HVA1
CaMV35SP Rice Act-1P
Chinese cabbage Canola
Water withholding Plant growth Limiting water supply Plant growth, biomass Water withholding Shoot growth, RWC, photosynthesis Water withholding Survivability, FW Water withholding Shoot growth, RWC, water potential Water withholding Shoot growth, survivability
Trehalose Mannitol Polyamines Polyamines Proline Trehalose Glycine betaine
Gene name a
Limiting water supply Leaf dry weight (productivity) Limiting water supply Biomass production Limiting water supply Shoot growth
    
f,g f,g e
2001 e,f 2004  2005 
ROS-scavenging proteins Detoxification MnSOD
N. plumbaginifolia CaMV35SP
Water withholding, field trial
Lipid peroxide NAD+ breakdown
CaMV35SP CaMV35SP (RNAi)
Water withholding Water withholding
Photosynthesis, electrolyte leakage, yield Photosynthesis FW, shoot growth
Survivability of callus
Ion transport ABA biosynthesis Stomata
AVP1 Arabidopsis AtNCED3 Arabidopsis Chl-NADP-ME Tobacco
Arabidopsis Arabidopsis Maize
Water withholding Water withholding Hydroponic culture, soil Water withholding
Plant growth, RWC Shoot growth Stomatal conductance, plant growth Survivability, transpiration rate
2001 e,f 2001 e 2002 
Agrobacterium pg5 CaMV35SP CaMV35SP Agrobacterium MAS Knockout
2000 e,f,g 2005 
a ADC, arginine decarboxylase; ALR, aldose/aldehyde reductase; AVP1, H+-pyrophosphatase; Chl-NADP-ME, chlorophyll-targeting NADP-malic enzyme; COX, choline oxidase; DMT, dimethylglycine methyltransferase; GolS, galactinol synthase; GSMT, glycine sarcosine; methyltransferase; IMT, myo-inositol O-methyltransferase; MnSOD, manganese superoxide dismutase; mtlD, mannitol-1-phosphate dehydrogenase; NCED, 9-cis epoxycarotenoid dioxygenase; OtsA, TPS, trehalose-6-phosphate synthase; OtsB, TPP, trehalose-6-phosphate phosphatase; PARP, poly(ADP-ribose) polymerase; P5CS, D1-pyrroline-5-carboxylate synthase; SPDS, spermidine synthase; TPS, trehalose-6-phosphate synthase; TPSP, TPS+TPP. bNon-plant species. cPromoter list and comment for mode of regulation (only in the case of downregulation of target genes). d PEG, polyethylene glycol; FW, fresh weight; RWC, relative water content; Chl, chlorophyll. References are cited in review articles: e; f, g ; h.
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proposed to function as chaperone-like protective molecules and act against cellular damage [4,10]. Thus, unraveling the mechanisms underlying the action of metabolites and proteins involved in drought tolerance could facilitate the creation of stress-tolerant plants. In addition, the recent progress of metabolome analysis should also contribute to the discovery of novel stress resistance systems that can be utilized for stress engineering .
Transcription factors for regulon biotechnology Transcriptome analyses using microarray technology, together with conventional approaches, have revealed that dozens of transcription factors (TFs) are involved in the plant response to drought stress [4,10]. Most of these TFs fall into several large TF families, such as AP2/ ERF, bZIP, NAC, MYB, MYC, Cys2His2 zinc-finger and WRKY. The expression of TFs regulates the expression of downstream target genes that are involved in the drought stress response and tolerance. Recent progress in TF study (Table 2), which has led to the engineering of drought tolerance using both transcriptional activators and repressors, is discussed below. Transcriptional activators that upregulate stress-responsive genes have been utilized to produce drought-tolerant transgenic plants. In addition to the enhancement of drought tolerance, overexpression of the DREB1/CBF3 (dehydration-responsive element binding protein/CRTbinding factor) TF in Arabidopsis controlled many stressinducible target genes [19â€“21] and increased tolerance to freezing and high salt exposure . In a more recent example, transgenic plants expressing a drought-responsive AP2-type TF, SHN1-3 or WXP1, induced several wax-related genes and resulted in enhanced cuticular wax accumulation and increased drought tolerance [23,24]. Thus, it has been shown that the overexpression of some drought-responsive transcription factors leads to the expression of downstream genes and the enhancement of drought tolerance (see  for a review). By contrast, overexpression of AREB1 (ABA-responsive element binding protein; ABF2) and DREB2A does not result in the significant induction of downstream genes nor cause any phenotypes related to drought tolerance [26,27]. There are two possible explanations for this observation. Firstly, downstream target genes governed by the TFs might not be directly involved in drought tolerance or, secondly, some modification could be required after transcription to induce the expression of downstream target genes. In the latter case, an altered form of AREB1 has been recently reported that overcomes this problem . Although overexpression of the intact AREB1 gene on its own is not sufficient to induce the expression of downstream genes, transgenic Current Opinion in Biotechnology 2006, 17:113â€“122
plants that express a constitutively active form of AREB1, created by deleting the regulatory sequence linking the activation and DNA-binding domains of the protein, did confer drought tolerance . In combination with lossof-function approaches, the downstream genes of AREB1, which encode LEA proteins and regulatory genes, were identified by microarray analysis . This is consistent with recent findings that a DNA-binding domain alone is sufficient to confer target gene specificity of a native TF . Overexpression of DREB2A carrying a small internal deletion of 30 amino acids induces the expression of downstream genes under untreated conditions and enhances drought tolerance (Y Sakuma et al., personal communication). Interestingly, the deleted region contains a transcriptional inhibitory region with a PEST sequence, which is generally known to play a role in the degradation of the protein, although the actual inhibitory mechanism of DREB2A is still unknown (Y Sakuma et al., personal communication). In addition to the deletion of inhibitory regions, the use of point mutations is an important strategy to produce active forms of transcriptional activators for engineering drought tolerance. A point mutation mimicking the phosphorylation of a rice bZIP transcription factor, TRAB1, from serine to aspartic acid at a phosphorylation site, significantly increased the level of transcriptional activation in the absence of the inducer ABA in a rice protoplast transient assay . Recently, transgenic plants expressing a phosphorylated active form of AREB1 with multisite mutations also resulted in the induction of many ABA-responsive genes without exogenous ABA application . These data suggest that TFs rendered constitutively active by point mutations could have the potential to contribute to the enhancement of drought tolerance by gene transfer. As recent genomics technologies have shown that various types of TFs are involved in the response to drought stress in plants, the creation of active forms of transcriptional activators is an attractive approach for the production of drought-tolerant plants. Along similar lines, an excellent review recently provided valuable insight into the creation of plants overexpressing TFs and considered how they can be used to analyse TF function . Transcriptional repressors that downregulate gene expression under conditions of stress have also been used to engineer drought tolerance. AtMYB60, an R2R3-MYB transcriptional repressor in Arabidopsis, functions in the regulation of stomatal movements. This gene is specifically expressed in guard cells and its expression is negatively regulated during drought stress . A null, TDNA insertion mutation in AtMYB60 results in the constitutive reduction of stomatal opening and minimizes wilting under water stress conditions. Interestingly, the atmyb60-1 mutation results in guard-cell-specific defects, www.sciencedirect.com
Engineering drought tolerance Umezawa et al. 117
Table 2 Engineering drought tolerance using transcription factors. Classification a
AP2/ERF family DREB1/CBF DREB1A/CBF3 DREB1/CBF DREB1A/CBF3
Water withholding Water withholding
DREB1/CBF DREB1/CBF DREB1/CBF DREB1/CBF AP2/ERF DREB1/CBF
DREB1B/CBF1 CBF4 ZmDREB1A DREB1C/CBF2 SHN1/WIN1 DREB1A/CBF3
Tomato Arabidopsis Arabidopsis Arabidopsis Arabidopsis Wheat
Arabidopsis Arabidopsis Maize Arabidopsis Arabidopsis Arabidopsis
Water withholding Water withholding Desiccation Desiccation Water withholding Water withholding
Plant growth Survivability Electrolyte leakage FW Survivability Plant growth
2002 2002 2004 2004 2004 2004
   
CaMV35SP Arabidopsis RD29AP CaMV35SP CaMV35SP CaMV35SP Knock out CaMV35SP Arabidopsis RD29AP Arabidopsis RD29AP Maize Ubi-1P
WXP1 DREB2A (active form with internal deletion)
M. truncatula Arabidopsis
CaMV35SP CaMV35SP, Arabidopsis RD29AP
Water withholding Water withholding
Photosynthesis, survivability Survivability Survivability
Basic leucine-zipper (bZIP) protein bZIP ABF3, AREB2/ABF4
CaMV35SP Maize Ubi-1P
Water withholding Water withholding
AREB1/ABF2 (active form with internal deletion) AREB1/ABF2 (phosphorylated active form)
Water withholding, dehydration
AtMYC2, AtMYB2 CpMYB10 AtMYB60
Arabidopsis Arabidopsis Arabidopsis
Arabidopsis C. plantagineum Arabidopsis
Zinc-finger protein Cys2His2-type ZPT2-3 Cys2His2-type CAZFP1
MYB/MYC MYB, MYC MYB R2R3-MYB
Gene name b
Plant growth, survivability Survivability Photosynthesis, survivability Survivability
CaMV35SP CaMV35SP Knock out
Mannitol treatment Water withholding Water withholding
Electrolyte leakage Survivability Plant growth, RWC
2002 2004 2005
Dehydration Water withholding
Survivability Survivability, Chl content Survivability, electrolyte leakage
AP2/ERF, APETALA2/ethylene-response factor; DREB1/CBF, dehydration-responsive element binding protein/C-repeat binding factor; NAC, NAM/ATAF/CUC. bCAZFP1, Capsicum annuum zinc-finger protein 1; SHN1/WIN1, shine1/wax inducer 1; STZ, salt-tolerance zinc finger protein; WXP1, wax production 1. cFW, fresh weight; RWC, relative water content; Chl, chlorophyll. d Promoter list and comment for mode of regulation (only in the case of downregulation of target genes). References are cited in review articles: e; f, g; h Y Sakuma, et al. unpublished.
with no apparent deleterious affects upon other developmental and physiological processes . This is very important when considering the yield of agricultural crops, because most transgenic plants that constitutively express TFs show growth retardation and alterations in basic metabolism . Although such undesirable traits can be improved to some extent through the use of stressinducible promoters that regulate the expression of TFs [22,33], the engineering of a stomatal response as a means www.sciencedirect.com
to reduce water loss is an attractive approach to confer drought tolerance in crops . Several plant zinc-finger transcriptional repressors carry repression domains and are involved in the downregulation of gene expression. A repression domain, SRDX, which consists of only 12 amino acids derived from the EAR motif of SUPERMAN, a TFIIIA-type zinc finger repressor, was recently shown to be useful for determining Current Opinion in Biotechnology 2006, 17:113â€“122
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the role of stress-responsive TFs, which often have functionally redundant homologs [27,35,36]. Overexpression of a chimeric repressor comprising a TF fused to the SRDX domain dominantly suppressed the expression of target genes, even in the presence of redundant TFs [27,35,36,37]. The repression domain SRDX is useful for identifying the role of TFs through the creation of a dominant loss-of-function mutant. Constitutive expression of a Cys2/His2-type zinc-finger transcriptional repressor, STZ, which was upregulated by dehydration, highsalt, cold stress and ABA treatment, increased tolerance to drought stress . Transgenic Arabidopsis plants expressing an STZ ortholog (CAZFP1), which normally functions as a transcriptional repressor in yeast, also showed tolerance to drought stress and exhibited resistance against bacterial infection . Overall, in addition to the conventional overexpression of TFs in regulon biotechnology, the modification of TFs might one day allow us to create TF-modified plants that confer novel traits.
Engineering drought tolerance using signaling factors Upstream of TFs, various signal transduction systems function in abiotic stress responses, involving protein phosphorylation and/or dephosphorylation, phospholipid metabolism, calcium sensing, protein degradation and so on (for reviews, see [4,10,40]). Although these complex signaling processes are not yet fully understood, several genes encoding signaling factors that function in the drought response have been identified [25,41,42]. Some of these factors are currently available for engineering drought tolerance (Table 3).
One of the merits for the manipulation of signaling factors is that they can control a broad range of downstream events, which results in superior tolerance for multiple aspects. For example, a tobacco mitogen-activated protein kinase kinase kinase (MAPKKK), NPK1, which was truncated for constitutive activation, activated an oxidative signal cascade and led to cold, heat, salinity and drought tolerance in transgenic plants [43,44]. Conversely, as shown in the case of farnesyltransferases, suppression of signaling factors can also effectively enhance tolerance to abiotic stress. In the regulation of stomatal closure by ABA, a b subunit of farnesyltransferase ERA1 functions as a negative regulator of ABA signaling [45,46]. Antisense downregulation of the a or b subunits of farnesyltransferase enhances response to ABA and drought tolerance of canola plants . An additional advantage of engineering signaling factors is that they can control the signal output involved in stress resistance. In many cases, signaling factors are activated or inactivated in response to various stress conditions. For example, an SNF1-related protein kinase 2 (SnRK2) was shown to be activated by ABA or osmotic stress, suggesting that SnRK2 might be important for signal transduction in the stress response [48–51]. There are ten members of the SnRK2 family in Arabidopsis (SRK2A– J/SnRK2.1–10) and ten members identified in the rice genome (SAPK1–10); almost all SnRK2s showed specific activation by ABA or hyperosmolarity [52,53]. The most crucial finding in SnRK2 function was established in guard cells: that is, fava bean ABA-activated protein kinase (AAPK) was essential for ABA-dependent stomatal
Table 3 Engineering drought tolerance using signaling factors. Classification a
Gene name b
Protein kinases CDPK OsCDPK7
Water withholding Limiting water supply
Shoot growth, Fv/Fm, wilting, gene expression Survivability Leaf number, kernel yield Survivability, gene expression
Others Calcium sensor
Agrobacterium MAS CaMV35SP
CaMV35SP/ RD29AP (antisense)
Water withholding, field test
Limiting water supply
Survivability, gene expression Senescence, Chl content, photosynthesis Survivability, gene expression Survivability, water loss, seed yield, oil content
CC-NBS-LRR, coiled-coil motif – nucleotide-binding site – leucine-rich repeat domains; CDPK, calcium-dependent protein kinase; GSK3/Shaggy, GSK3/Shaggy-like protein kinase; MAPKKK, mitogen-activated protein kinase kinase kinase. bADR1, activated disease resistance 1; ERA1, enhanced response to ABA 1; bNPK1, Nicotiana protein kinase 1. cPromoter list and comment for mode of regulation (only in the case of downregulation of target genes). dFv/Fm, variable fluorescence/maximum fluorescence. References are cited in review articles: e; f.
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closure . In Arabidopsis, the physiological function of SRK2E/OST1 (SnRK2.6), an ortholog of AAPK, has been confirmed in planta: srk2e or ost1 mutants showed hypersensitivity to drought because of their lack of transpirational control [54–56]. These data suggested that SnRK2 serves as a central hub to mediate ABA signaling in guard cells. In addition, one of the Arabidopsis SnRK2s, SRK2C (SnRK2.8), is an osmotic stress-activated protein kinase and its overexpression significantly improves drought tolerance in Arabidopsis . Under normal conditions, the activity of overexpressed SRK2C in complex with green fluorescent protein (SRK2C–GFP) was difficult to detect, despite considerable amounts of mRNA. By contrast, SRK2C–GFP was dramatically activated after drought stress, resulting in the enhancement of transcriptional events and drought tolerance in Arabidopsis. These data support the supposition that stress-dependent activation or deactivation of signal components might function as a molecular switch for the biotechnological manipulation of the stress response in plants. CBL/SCaBP (calcineurin B-like protein/SOS3-like calcium-binding protein) and its interacting partner CIPK/ PKS, which is identical to SnRK3, form a complex that is known to be an important regulator of signal transduction in the presense of ABA and under conditions of drought, salt or cold stress [58,59,60,61,62,63]. CBL1 is a member of the CBL/SCaBP family of proteins which are induced by cold, NaCl, ABA and hyperosmotic stress. Plants overexpressing CBL1 exhibited elevated drought and salt tolerance and upregulated expression of stressresponsive genes . Although SnRK3 seems to be important for engineering drought tolerance, it is not a simple case. The manipulation of SnRK3 for drought stress is complicated because its kinase activity is regulated by a Ca2+-dependent interaction of CBL/SCaBP proteins which sense internal or external stimuli. As demonstrated in the case of SOS2 or PKS18 kinases, mutations in the activation loop of the catalytic domain or deletion of the FISL motif, which is essential for CBL/ SCaBP interactions, enable the production of a constitutively active form of SnRK3 . It is thought that this technology will prove useful for the application of SnRK3 to genetic engineering. Although recent progress in this field has identified several signaling factors related to the drought stressresponse in plants, these findings also highlight the complexity of the signal transduction network with cross-talk, feedback and physical interactions delivering appropriate signals to suitable targets at the correct time. As with the yeast ‘scaffold’ hypothesis, signaling proteins often have multiple signal outputs, an occurrence that explains the different functions of the MAPKKK, Ste11p [65,66]. For example, AtMPK6 functions in at least two different MAPK cascades, MKK2-MPK6 and MKK4/ MKK5-MPK6, and each combination can transmit or www.sciencedirect.com
transduce different signals from cold or salt stress, and bacterial pathogens, respectively . Further functional or biochemical analyses will be required to gain a precise understanding of the functions of various signaling factors in plants, which can then be used to promote the engineering of drought tolerance using signaling factors.
Conclusions We have identified several factors related to the plant response to drought stress and many of these factors have already been shown to be effective for engineering drought tolerance in model plants (Tables 1–3). Such demonstrated success under experimental conditions has encouraged the use of this strategy to engineer drought tolerance in crop species (Figure 1). Since the application of functional genomics approaches, the identification of candidate genes related to drought response has increased, thus fueling this approach for genetic engineering. In future, the combination of transcriptomic, proteomic or metabolomic analyses will also be useful for gene discovery in the engineering of drought tolerance. As well as gene discovery, technical advances in applied research are also important for engineering drought-tolerant plants. Transformation methodologies should be developed, adapted and applied to many more plant species, because currently we are only able to produce transgenic plants with a limited selection of plant species. An effective expression system, including suitable promoters, will also be required for each plant species, because constitutive promoters such as the CaMV35S promoter are not always functional or can have negative effects on plant growth or development [22,68]. Recent studies have shed light not only on structural proteins, such as metabolic enzymes and LEA proteins, but also on regulatory proteins such as TFs and signaling factors that control a broad range of stress responses. Significant cross-talk and inter-connections are involved in stress signaling, such as positive and negative regulators or trans- and cis-acting factors [25,42,68]. Nevertheless, systematic approaches with molecular and genomic analyses — mainly using Arabidopsis or other model plants — will facilitate the resolution of such complex networks and lead to the discovery of additional stress factors or mechanisms. Furthermore, it is anticipated that several areas, such as post-transcriptional regulation involving protein modification, protein degradation and RNA metabolism, will emerge and assume importance in the future. Such basic studies will allow us to create novel approaches for the molecular tailoring of genes (e.g. modulation of the activity of target proteins with deletions, mutational or conformational changes as shown in the case of AREB1 or DREB2A). It is also hoped that these approaches will allow us to finetune drought tolerance according to the time and circumstances for the onset of this environmental constraint. Current Opinion in Biotechnology 2006, 17:113–122
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Acknowledgements This work was supported in part by a grant for Genome Research from RIKEN, the Program for Promotion of Basic Research Activities for Innovative Biosciences from the Bio-Oriented Technology Research Advancement Institution of Japan (BRAIN), the Special Coordination Fund of the Japan Science and Technology Agency (JST), and a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) to KS and KY-S. This work was also supported in part by Grant-in-Aids for Scientific Research for Young Scientists (B) 14760075 and 16770043 from MEXT to TU.
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Engineering drought tolerance in plants_ discovering and tailoring genes to unlock the future