Gain-of-function mutagenesis approaches in rice for

functional genomics and improvement of crop

productivityMazahar Moin, Achala Bakshi, Anusree Saha, Mouboni Dutta, and P. B. Kirti**Corresponding author: P. B. Kirti, Department of plant Sciences, University of Hyderabad, Hyderabad, 500046, India. Tel.: þ040-23134545;E-mail: pbkirti@yahoo.com

Abstract

The epitome of any genome research is to identify all the existing genes in a genome and investigate their roles. Varioustechniques have been applied to unveil the functions either by silencing or over-expressing the genes by targeted expres-sion or random mutagenesis. Rice is the most appropriate model crop for generating a mutant resource for functionalgenomic studies because of the availability of high-quality genome sequence and relatively smaller genome size. Rice hassyntenic relationships with members of other cereals. Hence, characterization of functionally unknown genes in rice willpossibly provide key genetic insights and can lead to comparative genomics involving other cereals. The current review at-tempts to discuss the available gain-of-function mutagenesis techniques for functional genomics, emphasizing the contem-porary approach, activation tagging and alterations to this method for the enhancement of yield and productivity of rice.

Key words: activation tagging; functional genomics; gain-of-function mutagenesis; rice; tissue-specific tagging; water-useefficiency (WUE)

Introduction

About 25 species of rice are found globally, of which Asian riceor Oryza sativa is widely cultivated and consumed. Rice, con-sidered as the poor’s staple cereal, is consumed by >3.2 billionpeople across the globe, feeding about 40% of the world popula-tion. Sustained or increased productivity of rice demands morearable land, irrigation facilities and manpower. Therefore, a bet-ter understanding of its genome function can facilitate the de-velopment of tailor-made varieties of agricultural importance.The past decade has been the decennium mirabilis in the rice gen-ome research with (1) the avalanche of complete genome se-quence, (2) development of tools and techniques for functional

genomic studies and (3) identification and characterization ofrelevant, candidate genes for agronomical traits in transgenicrice plants. Mutant populations are the indispensable tools formining the functions of plant genes. Mutants generated by theuse of chemical agents, high-energy radiations and T-DNA/transposable elements disrupt the function of genes in the tar-get genome. These gene-disruption technologies have the limi-tations that they induce recessive loss-of-function mutationsand are unable to produce a distinct mutant phenotype of gen-etically redundant genes.

As an alternative, several gain-of-function mutagenesisstrategies have been developed that use multiple enhancers or

Mazahar Moin is a Research Fellow in the Department of plant Sciences, University of Hyderabad. His research program is on activation tagging in rice foridentification of the candidate genes responsible for water-use-efficiency and the characterization of novel genes.Achala Bakshi is a Research Fellow in the Department of Plant Sciences, University of Hyderabad. She is working on activation tagging in rice and stress-responsive roles of novel candidate genes in rice.Anusree Saha is a Research Fellow in the Department of Plant Sciences, University of Hyderabad. She is working on seed-specific tagging of genes for yieldimprovement in rice.Mouboni Dutta is a Research Fellow in the Department of Plant Sciences, University of Hyderabad. She is working on the validation of genes identifiedthrough activation tagging in abiotic stress tolerance in rice.P B Kirti is a professor in the Department of Plant Sciences, University of Hyderabad. His research interests are on crop genetic manipulation for yield en-hancement and stresstolerance.

VC The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com

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strong promoters to activate the genes when inserted at theirneighboring sites. Gain-of-function mutant phenotypes overex-pressing a member of a gene family having many isoforms canbe visualized independently without the effect of other mem-bers of the same gene family. Thus, the roles of even redundantgenes can be elucidated as dominant mutations. The most suc-cessful gain-of-function mutagenesis approach in plants is acti-vation tagging through which for the first time the function of agene required for auxin-independent growth of tobacco plantswas identified [1]. In this system, a T-DNA/transposable elementcarrying multiple enhancers activates the expression of thenearby genes on insertion into the genome. Thus, the mRNA/transcript levels of the activated genes would be more than theirnative levels [2]. These enhancers, usually tetrameric repeats ofCaMV35S promoter, activate the expression of the gene(s) in ei-ther orientation and up to as far as 10 kb on either side of the siteof their insertion in the genome [3, 4]. The use of strong pro-moters such as CaMV35S would result in direct activation of thegene that comes under its control [4–6]. The individual CaMV35Spromoters cause ectopic overexpression of the tagged genes,whereas tetrameric enhancers do not lead to constitutive ex-pression, rather they increase the endogenous expression levelsof the target genes [5]. Thus, the phenotype resulting from animprovement of the endogenous expression would more likelyreflect the proper functioning of the activated gene [5, 6].

Activation tagging technology using Ac/Dssystem

A typical Ac/Ds-based activation tagging vector contains a Dselement carrying tandem repeats of CaMV35S promoter and anActivator transposase within the T-DNA but outside the Ds elem-ent in cis configuration [7]. In the presence of an Ac transposase,the Ds element tends to transpose and integrate randomlyacross the genome, thus activating the expression of the genesat the sites of insertions (Figure 1). Several factors determinethe success of activation tagging, such as the activity of Ac trans-posase, the location of the transposon in the genome, type of en-hancer elements used and the susceptibility of the target locus.The transposition of the Ds element depends on the promoterunder which Ac transposase is expressed. When Ac is expressedunder a constitutive CaMV35S promoter, the Ds element excisedearly in developmental stages in tobacco, Arabidopsis and rice[8–10]. Such transposition events occurring at early develop-mental stages are carried through germ line and result in thesame insertion patterns in the progenies of the next generation[11]. The Ds element transposed late in barley during the devel-opmental stages when the Ac element was expressed under thenative promoter and gave rise to independent transpositions[12]. The number of transposition events or transposition fre-quency of the Ds element was less when Ac expressed under aconstitutive as well as a native promoter. However, inArabidopsis, when the Ac element was expressed under octo-pine synthase promoter, the number of transposition eventsincreased drastically [13]. These varied transposition patterns ofthe Ds element indicate that it is not only promoter-dependentbut also species specific. The Ds also prefers to integrate at theregions with high gene densities, 72% of total insertions onchromosome-1 of rice located in the genic regions [11].

According to the recent release of The ArabidopsisInformation Resource (TAIR), the diploid genome size ofArabidopsis is 134 634 692 bp having 27 416 protein-codinggenes with an average size of 2940 bp and gene density of

4.35 kb/gene. In about 60% of its genome constituting the tran-scriptionally active area, insertion of the Ds in the genic regionis possible and expected. The rice genome has a 165 996 229 bptranscriptionally active area (including 546 008 43 bp TE-relatedgenic region and 111 395 386 bp non-TE-related genic region)from 373 245 519 bp of a non-overlapping sequence comprising44% of the total coding area. Insertion of the Ds in the coding re-gions underpins that it preferentially transposes to the genicsites [11]. Preferential insertion of these transposable elementsat the genic regions makes this technique more feasible and canbe extended to other crop systems as well for functional gen-omic studies. Table 1 lists the genes characterized in variousplant species using activation-tagging technology.

Activation-tagging technology using Cre-Loxsystem

An alternative to the Ac-Ds activation tagging is the use of Cre-Lox system [47]. In this approach, the pEnLox vector carriesCaMV35S enhancers flanked by lox sequences, which are a 34 bpsequence (an 8 bp asymmetric sequence flanked by a 13 bp pal-indrome), and the pCRE vector carries Cre recombinase. The Lox-Enhancer vector induces gain-of-function mutations at the siteof insertion, while pCRE vector is used to produce the Cre re-combinase protein required for site-specific recombination.These two vectors are either transformed independently fol-lowed by the crossing of the transgenic plants or the transgenicplants carrying any one vector (pEnLox) is re-transformed withthe other vector (pCRE).

The Cre recombinase causes excision of any DNA fragmentflanked by lox sites through site-specific recombination. Thisproperty has been well exploited to eliminate marker genesfrom plants for generating marker-free transgenic plants select-ively [48]. The binary vectors for marker-free transgenic riceplants carry a gene for Cre recombinase and a marker gene intandem flanked by lox sequences. Also, a promoterless reportergene is cloned in cis. When Cre is activated by constitutive, indu-cible or tissue-specific promoters [49, 50], it causes the excisionof the lox sequences with appropriate action and thus elimin-ates the marker gene. As a result of this, the reporter gene isbrought under the control of the native promoter and becomesactivated.

Activation tagging for mining novel genes

Activation-tagged populations have an important role in func-tional genomic studies. Though the sequencing of genomes pro-vides sufficient raw data for sequence annotation, the data donot assume significance unless the functional characterizationof the sequence is undertaken. Activation tagging identifiesgenes and their importance in a single step. The success of acti-vation tagging depends on the level of saturation of the genomewith the enhancer elements, thereby obtaining tags for as manycoding sequences present in the genome. Once such a popula-tion developed, it can be screened for all the possible pheno-types as desired and mined for identifying novel genes forstress amelioration and agronomically important traits.

Significance of enhancer-based activationtagging

Gain-of-function mutagenesis approaches uncover the functionof genes that remain unidentified by loss-of-function screens

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such as T-DNA or transposon tagging. It particularly applies tothe genes exhibiting redundancy. The advantage of enhancer-based activation-tagging over other methods of insertional mu-tagenesis is that they cause endogenous activation of genes oneither side of insertion throughout the plant genome. If an en-hancer inserts within a gene, this intragenic insertion tends toinactivate the target gene and at the same time activates theneighboring gene, which may lead to a complex phenotype.Hence, intergenic insertions are preferred over intragenic inser-tions in activation tagging. About 41.84% of T-DNA, 30.74% of Dsand 52% of dSpm mutations in mutant rice libraries are inter-genic insertions [50]. Hence, if enhancers are introduced intothe genome through T-DNA, Ds or particularly dSpm tags, a largenumber of genes can be activated through intergenic activation.Also, the number of insertions required to tag each gene withenhancers is less.

Gain-of-function mutagenesis through FOX-hunting system

Though the activation tagging approach has been a successfultechnology, it entails the development of a plethora of trans-genic plants to identify a gene function for the desired trait.Each gene activated by the enhancers in the tagged populationsneeds to be validated by cloning the identified gene under aconstitutive promoter and transforming back into the same sys-tem. This process cross-checks the phenotype that has resultedfrom the overexpression of the gene in the transgenic plantwith the phenotype identified in the original line with gene acti-vation by multiple enhancers. Also, enhancers can sometimesactivate more than one gene at a time simultaneously, thusleading to a complex phenotype where multiple genes becomeactivated in a single transgenic plant. In such cases, each acti-vated gene needs to be cloned and analyzed separately. To

overcome these limitations, an alternative and relatively recenttechnique that involves the cloning of full-length (Fl) cDNAs,called FOX-hunting system (Full-length cDNA Over-eXpressing)to generate dominant mutations was introduced [51]. This tech-nique was first initiated in Arabidopsis where full-length cDNAswere ectopically expressed under CaMV35S promoter, and waslater extended to rice and other model systems [51–53]. In brief,FOX-hunting method involves a large number of fl-cDNA mol-ecules (about 10 000–15 000) mixed in equimolar ratios to gener-ate a normalized fl-cDNA mixture. This mixture is digested andsubsequently ligated using compatible sites in a binary vector todevelop a fl-cDNA library, which is introduced into the plant,followed by confirmation through Polymerase Chain Reaction(PCR) amplification [51–53]. According to the latest re-port,>30 000 Arabidopsis transgenic lines expressing rice fl-cDNAs under CaMV35S promoter and Tobacco Mosaic Virus(TMV) omega sequence were generated and had been systemat-ically screened for various morphological traits and also toler-ance against a range of biotic and abiotic stresses [54].

GAL4-based tissue-specific transgeneactivation and activation tagging

GAL4 activation-tagging system, popularly called fly geneticist’s‘Swiss army knife’ [55] was extensively exploited in Drosophilawith >5000 GAL4 enhancer trap lines developed and character-ized. The yeast Gal4 DNA sequence codes for an 881-amino acidGAL4-activator protein, which binds to a 17 bp UpstreamActivation Sequence (UAS) present in the promoters ofgalactose-inducible genes [56]. The GAL4 activation tagging sys-tem involves the use of Gal4 gene sequence cloned under a min-imal promoter (MP). When introduced into the genome, the Gal4gene is brought under the control of native promoters so thatthe amount of GAL4 synthesized reflects the transcriptional

Figure 1. Transposon-based activation-tagging process. Activation tagging using 4� enhancers are widely used in forward genetics, in which enhancers activate the ex-

pression of genes up to as long as 10 kb on either side of the insertion. The Ac/Ds activation tagged plants needs to be stabilized through segregation or stable enhancer

carrying lines can also be generated directly by transforming vectors with 4� enhancers in T-DNA/Ds backbone, which can be used to cross with Ac carrying plants. (A

colour version of this figure is available online at: http://bfg.oxfordjournals.org.)

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activity of native genes visualized through GAL4-linked reportergenes like Green Fluorescent Protein (GFP) and b-glucuronidase(GUS). Depending on the site of Gal4 insertion and type of thenative gene promoter, the expression pattern of GAL4 variesspatially throughout the organism ranging from individual cellsto tissues [56]. This system also uses 5–6 tandem repeats of17 bp UAS, which controls the expression of the reporter gene.Thus, by selecting a transgenic line synthesizing high levels ofGAL4 in tissue-specific manner detected through GFP fluores-cence or GUS-staining and crossing or re-transforming it with aUAS line, tissue-specific activation tagging can be achieved. Thesignificance of this technique is that the transcriptional activityof native promoters in a plant is determined, which furtherhelps in selecting a suitable promoter by GAL4 expression fortissue-specific activation of the genes.

When the DNA binding domain of GAL4 activator fused witha Viral Protein16 (VP16) protein of Herpes Simplex Virus [57], theGAL4: VP16 (GV) hybrid transcription factor increases the ex-pression of target genes highly efficiently.

The GV-UAS-mediated tissue-specific transgene activationinvolved the use of two binary vectors. One consists of trans-activator gene GV under MP in cis with UAS-controlled reportergene GUS so that the expression of GV corresponds to the ex-pression of GUS; the other vector carries eGFP and the gene ofinterest (GOI) along with 5� UAS. After independent

transformation of these two vectors, the hybrid transgenic linesare produced by crossing the desired GV: MP carrying a driverand the pattern line exhibiting tissue-specific reporter gene ex-pression with GOI: UAS target lines, thus targeting the expres-sion of GOI to a specific tissue. By introducing UAS:uidA/gfp intoselected GV line, the transgene expression can be targeted tospecific cell/tissue type [57, 58]. Rice represents the largest GAL4resource among all the plants with the development of>145 000 GAL4 enhancer trap lines in three cultivars of japonica,viz., Zhonghua11, Zhonghua15 and Nipponbare [59, 60].By using GV: UAS activation tagging system, several dominantmutants with abnormal root phenotypes and their relatedgenes have been identified and characterized in Arabidopsis[61].

Transgene activation and activation taggingwith inducible and tissue-specific promoters

The expression of the transgene is regulated spatially and tem-porally by the use of tissue-specific (Table 2) and inducible/con-ditional promoters (Table 3), respectively. The spatiotemporal-specific expression of the genes overcomes the pleiotropic ef-fects that might arise from the use of strong constitutivepromoters.

Table 1. Genes identified through activation tagging in various plant species

Gene identified Origin Reference Function

Glutamate receptor-like Rice [14] Drought toleranceRac1 (Rac Immunity1) Rice [15] Resistance against rice blast fungusLesion mimic resembling ATPase Rice [16] Resistance against blast and blightRPL (ribosomal protein large) subunit gene 6 & 23A Rice [17] Water-use efficiencyNicotianamine synthase2 Rice [18] Zinc accumulation in seedsYellow stripe1-like16 Rice [19] Enhanced iron efficiencyOsAT1 Rice [20] Lesion mimic spotted leaf mutantedt1 Arabidopsis [21] Drought tolerance with improved root systemYucca Arabidopsis [22] Flavin Monooxygenase synthesisGa 2-ox Rice [23] Synthesis of gibberellin 2-oxidaseces101 Arabidopsis [24] Receptor-like kinaseasl1 (asymmetric leaves2 like1) Arabidopsis [25] Controls proximal-distal patterning in petalsasml2 Arabidopsis [26] Controls expression of sugar-inducible genesCtg Arabidopsis [27] Cold temperature germinating mutantsDVL1 Arabidopsis [28] Fruit developmentcdr1-d Arabidopsis [29] Resistance to virulent Pseudomonasetc1 (enhancer of triptychon), cpc1 (caprice) Arabidopsis [30] Trichome and root hair cell patterningasl5/lbd12 Arabidopsis [31] Epinastic leavesant1 Tomato [32] Transcriptional regulator of anthocyaninsga 2-oxidase Poplar [33] Synthesis of gibberellins 2-oxidase.orca3 Catharanthus [34] Regulates terpenoid indole alkaloid pathwayjaw-D Arabidopsis [5] Plant developmentvas (vascular tissue size) Arabidopsis [35] Increase in phloem cambial and pericycle cellspga6 (or wuschel) Arabidopsis [36] Role in embryogenesisSho Petunia [37] Cytokinin-specific effectsbak1 (bri1 associated receptor kinase1-1 dominant) Arabidopsis [38] Brassinosteroid signalingSturdy Arabidopsis [39] Controls inflorescence and branchingbri1 Arabidopsis [40] Receptor for brassinosteriodslep (leafy petiole) Arabidopsis [41] Member of AP2/EREBP transcription factoresc1D Arabidopsis [5] Late flowering mutantlhy (late elongated hypocotyl) Arabidopsis [42] Photoperiodic control of floweringpap1-D Arabidopsis [43] Production of anthocyanin pigmentshi (short Internodes) Arabidopsis [44] Suppression of gibberellin responseCdt1 Craterostigma [45] Drought toleranceft (flowering locus) Arabidopsis [46] Induce floweringAxi Arabidopsis [1] Auxin-independent plant growth

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Genes characterized in abiotic stress tolerancein rice

To sustain under the diverse environmental and climaticchanges, plants being sessile organisms have naturally evolvedwith armoury in the form of genetic loci within their germplasm.However, identification of these genetic elements that contributeto plants’ survivability in challenging conditions is of paramountimportance to further improve their productivity potential. Themost important abiotic factors that restrain the plant yield andproductivity are drought and salinity, both imposed throughwater scarcity. Hence, investigations into the mechanism of planttolerance to these two stresses assume significance worldwide.Water scarcity is inevitable on the planet earth in the comingyears; moreover, plant genomes should be tailored to resist water

stress and use the available water most efficiently. Rice and waterare the world’s two assets that are closely associated.

Rice requires extensive irrigation; 75% of rice productivitycomes from irrigated land, while<25% yield is achieved throughrain and deep water systems [92] with an estimate of rice receiv-ing 35% to 43% of total world’s irrigation water. Rice is sensitiveto water shortage, and if rice genetic system is engineered touse less water without compromising on its productivity, thesaved water could be efficiently used to irrigate all other cropsto enhance their productivity potential significantly.

Plants resistance to drought stress can improve by droughtavoidance or drought tolerance among which drought avoidancetends to conserve water by water-use efficiency (WUE). In naturalconditions, when plants experience water shortage, they tend toclose their stomatal apertures to avoid transpirational water loss,

Table 2. Tissue-specific promoters that enable the spatial activation of genes

Specificity Promoter type Function Reference

Seed Endosperm periphery GlutelinA-1 Seed storage [62]Aleurone and sub-aleurone layers GluB-3 Seed storageWhole endosperm GluB-5 Seed storageWhole endosperm GluC Seed storageCotyledons Sus3 sucrose synthase [63]Endosperm Rice pullulanase Starch debranching enzyme [64]

Root Primary and secondary roots Ethylmalonic encephalopathy protein 1 SOD activity [65]Root hairs Rice cellulose synthase-like D1 Root hair morphogenesis [66]Root hairs Expansin7 Root hair Elongation [67]Root Apical Meristem WUSCHEL-type homeobox Auxin signaling [68]Root tonoplast OsHMA3 Differential Cd accumulation [69]Root central cylinder OsMADS23/25/27/57 Active in external stimuli [70]

Leaf Mesophyll cells Leaf panicle2 Leucine-rich repeat reporter kinase [71]Mature leaves Chlorophyll a/b binding protein2 (CAB2) of PSII Photosynthesis [72]Mesophyll cells of leaf blade OsRbcS3 (Rubisco) Co2 assimilation [73]Vascular bundles of leaf sheath OsRbcS1 Co2 assimilation [73]Leaf blades and leaf sheath Maize Phospho Enol Pyruvate Carboxylase in rice Co2 assimilation [63]Whole leaf Lea3 and Uge1 Regulation of growth [74]Green tissue OsCHLH Chlorophyll biosynthesis [75]

Flower Tapetum and pollen Rice cysteine protease 1 [76]Anther Cytochrome P450 hydroxylase GA signaling pathway [77]Microspore mother cell Lipid transfer protein Pollen maturation [78]Pollen Rice immature pollen 1 Regulator of late pollen development [79]Anther wall MADS62/63/68 [80]Whole flower Rab21 and Wsi18 Regulation of growth [109]Anther Rice tapetum-specific gene Male fertility [81]

Table 3. Inducible/conditional promoters that activate genes under given treatments

Promoter Inducible agent Reference

OsNac6, LIP9, Oshox24 Drought, high salinity and ABA treatment [82]ZmGAPP Drought, salinity [83]LEA HVA1 ABA, salt, dehydration, cold [84]CYP76M7 Magnaporthe oryzae inducible [85]OsACO1 Heat inducible [86]MYBS3 Cold inducible [87]OsMPS Salt inducible [88]OsNac2 Drought, salt wound [89]OsASR5 Dehydration [90]OsLea14a Drought, salinity and ABA [82]ZFP179 ABA, PEG [91]

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which is followed by the development of long roots to absorb deepsoil moisture. The other features of WUE genotypes are early flower-ing, reduced biomass and seed yield [93]. High yielding WUE pheno-types can be produced through gain-of-function mutagenesisstrategies by overexpressing the genes having the potential to im-prove yields under water-deficit conditions. The overexpression ofWUE genes in rice generated plants with significantly higher bio-mass and yield under limited water conditions [94].

More than 1000 genes including transcription factors and kin-ases have been identified as having roles in response to dehydra-tion, salinity, heat and cold stresses in rice [95–97]. However,only 600 (60%) of them have been characterized in detail by gen-erating independent transgenic plants for each gene for the cor-responding stress tolerance. Heat shock proteins (OsHsp17.0,OsHsp23.7), group 3 late embryogenesis abundant (LEA) (OsLEA3-1, OsLEA3-2) proteins, transcription factors like basis Helix-Loop-Helix (bHLH) (OsHLHU8), basic Leucine Zipper (bZIP) (OsbZIP23/16/46/71), AP2/ERF APETALA2/Ethylene Responsive Factor(OsDREB1A/2A/1B, OsERF1/4a/10a, AP37), MYB (OsMYB2), NAC(SNAC1, OsNAC5/6/9/10), Zinc finger (OsZFP182/245/252), WRKY(OsWRKY30), kinases (calcium-dependent kinases, MAP kinases),osmoprotectants (OsOAT), water [98] ion channels [99] and detox-ifying enzymes are analyzed in abiotic stress tolerance.Overexpression of these endogenous genes in transgenic riceunder constitutive promoters resulted in increased yield underdehydration and salinity conditions. Members of bHLH [100],AP2/ERF [101], NAC [102], MYB [103], bZIP [104] have been wellcharacterized with their direct roles in defense to biotic and abi-otic stresses in rice. NAC, the largest plant transcription factorfamily, is characterized by a highly conserved DNA-binding do-main at the N-terminal end. Overexpression of members of NACtranscription family significantly enhanced drought and salt tol-erance in transgenic rice [40, 105]. The Arabidopsis (HARDY) HRDgene coding for an AP2/ERF-like transcription factor identified bya gain-of-function approach exhibited drought and salt toler-ance. Overexpression of HARDY gene identified in the activa-tion-tagged mutant population of Arabidopsis improved droughtresistance and WUE in the transgenic rice plants [94]. Also, over-expression of dehydration responsive element binding (DREB)transcription factors in rice conferred tolerance to drought, salin-ity and various other stresses [106].

Plants respond to the adverse environmental conditions byinitiating a series of signaling cascades that involve protein kin-ases including calcineurin B-like protein-interacting proteinkinases (CIPKs). Transgenic rice overexpressing CIPK12 andCIPK15 showed improved tolerance to drought and salt stress,respectively. About 125 drought-responsive genes were identi-fied using the expressed sequence tags generated from a cDNAlibrary of indica rice [107]. In addition to transcription factorsand kinases, which function in stress response as a network,candidate genes have also been characterized. Some of these in-clude Auxin efflux carrier (OsPIN3t), myoinositol oxygenase(OsMIOX), Ski-interacting protein (OsSKIPa), His phosphotransferprotein (OsAHP), Lipid transfer protein (OsDIL), Ornithine d-amino-transferase (OsOAT), Trehalose-6-phosphate synthase(OsTPS1) and Deeper Rooting 1 (OsDRO1) for drought stress toler-ance in rice [108–115]. Although these studies resulted in theidentification of genes for tolerance to various stresses, a searchfor identifying novel genes controlling different pathways thatare not hitherto reported continues. For example, transcriptionfactors and kinases work in a network activating multipledownstream proteins in a signaling cascade. A detailed investi-gation of each protein involved can further lead to the discoveryof genes for many unknown functions.

C4 rice and gain-of-function strategies toincorporate functional C4 shuttle

Rice productivity should be augmented by >35% in the next 25years [116], and higher yields are achieved by improving photo-synthesis and carbon assimilation processes. Because thephotosynthetic efficiency of C4 plants has evolutionarilyincreased by>40% than C3 plants [92], completely switching theso-called C3 photosynthetic system of rice into C4 is one solu-tion. The high photosynthetic efficiency of C4 plants is main-tained even under high temperature, high light intensity, lowCO2 (<50 ppm), reduced water and nitrogen availability, allbeing limiting factors for rice productivity. Hence, a C4 rice,which is a powerhouse of photosynthesis, would be morewater-use efficient in which carbon dioxide gradient maintainsthrough partially closed stomata (more CO2 absorbed per unit ofwater lost), radiation-use efficient carried through CO2-concen-trating mechanisms with one group of cells capturing CO2

(mesophyll) surrounded by another set of suberin-coated bun-dle sheath (BS) cells to boost its level. Also, a C4 plant is more ni-trogen-use efficient, as little amount of Rubisco is required perthe same amount of carbon fixed [117, 118]. These factors resultin dramatic increase in the yield by up to 50% per hectare.

The fully functional C4 photosynthetic system has graduallyevolved >66 times in 19 different families of angiosperms, indi-cating that a C3 plant can be conditioned into C4 [119]. This wasinitiated with the overexpression of four enzyme-coding genesin mesophyll cells of transgenic rice [120]. The overproductionof either of the enzymes independently did not increase therate of photosynthesis. However, overproduction of all the fourenzymes simultaneously had some positive effects on photo-synthesis, illustrating that incorporation of C4 pathway is nei-ther single-celled nor controlled by a single master gene.

The alternative approach is gain-of-function mutagenesisthrough activation tagging, which entails the development ofindependent enhancer-tagged events, followed by examiningeach line for C4-like leaf anatomy. Transgenic plants withincreased chloroplast content, particularly in BS cells, can be se-lected as a starter or progenitor line for C4 pathway. Again, theprobability of an enhancer upregulating each/every C4 gene pre-sent within a C3 plant (whose endogenous leaf expression isweak) independently in mesophyll and BS cells is less.Therefore, vectors with cell-specific promoters cloned in twocomponent transposon backbone is another proposal for driv-ing expression of as many genes contained in a specific cell. Forexample, PEP-carboxylase and pyruvte phosphate dikinase(PPDK) promoters from maize and PEP-carboxykinase promoterfrom Zoysia induced strong mesophyll cell (MC) and BS cell-specific expression in rice, respectively [121].

Future perspectives for rice improvementthrough functional genomic approaches

Among cereals and other crops, rice is perhaps the only crop thathas made vast advancements in the area of genomics and func-tional genomics within a decade’s time, because unlike mostother cereal crops, rice is a diploid organism with the smallestgenome size. Post-genome sequencing of rice, more than 30 data-bases were made publicly available that enable the genetic ma-nipulation. Development of high-yielding rice varieties thatutilize fewer resources such as water and yet maintain sus-tained/improved productivity, an important agronomical traitcalled WUE, could save a significant amount of irrigated water,which can be used to improve the productivity potential of other

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crops. It is either achieved by continuous crossing between high-yielding and low-water-susceptible varieties and looking for aprogenitor line with desired characters or through gain-of-function mutagenesis approaches such as activation or tissue-specific tagging. Breeding is a laborious process, as it involvesyears of continuous, conventional crossing and plant mainten-ance. In activation tagging, enhancer elements randomly activatethe genes from the site of insertions in the genome. After gener-ating a sufficiently large number of activation-tagged plants, theyare initially grown under limited water conditions. Those mu-tants exhibiting high-yield-potential-related phenotypic charac-ters such as increased tillering, panicles and seed yield underlimited water availability is isolated for further physiologicalscreening related to WUE such as photosynthetic measurementsand D13C, which is a proxy for WUE. The selected mutants arethen analyzed for the genes responsible for the trait. This ap-proach not only helps in developing high-yielding varieties butalso identifies the novel gene functions. This resource can furtherbe exploited in isolating mutants for any desired trait of agro-nomic importance such as tolerance to salt and biotic stresses,and so forth. Because it requires the development of a massivescale of stable and independent transgenic plants, it is a tediousprocess to extend this technology to all the rice varieties.

The Agrobacterium-mediated transformation process of ricedepends on the genotype and the type of explant used, with ind-ica rice varieties being less amenable to tissue culture manipula-tion than japonica varieties [122]. Hence, efficient, simple andhigh-throughput transformation protocols are required to gen-erate such a large-scale transgenic population in widely culti-vated indica varieties. Tissue-specific tagging is another optionto produce gain-of-function mutants that activate the genes ina selected tissue depending on the type of promoter used.Tissue-specific promoters can be cloned under dual Ac/Ds cas-settes, producing a limited number of primary transgenic plantsto tag as many number of genes in a tissue followed by stabiliz-ing the lines by segregation. If efficient genetic transformationstrategies are available, using a T-DNA insertion-based ap-proach to raise the tissue-specific stable activation lines directlyis feasible. GlutelinC is an ideal seed-specific promoter, as it isactive throughout the endosperm unlike other Glu promoters[62] for tagging and enhancing the expression of seed-specificgenes for traits with improved seed yield and WUE. By usingleaf-specific C4 promoters, C4 shuttle can be incorporated in C3rice by activating leaf-specific genes and simultaneously down-regulating the C3 genes. As discussed earlier, a C4 rice would bewater-use efficient, radiation-use efficient and nitrogen-use ef-ficient [118], thus increasing yields under limited resources.However, generating a fully functional C4 rice is a challengingtask, as a single master gene does not regulate this process; ra-ther it is controlled by multiple genes, each having a specificfunction. However, a C4 progenitor rice with features similar toa typical C4 plant can be produced either by targeting the ex-pression of selected genes encoding C4 enzymes and trans-porters under tissue-specific promoters or by random gain-of-function mutagenesis such as tissue-specific activation taggingand looking for C4-like leaf anatomy. Using C4-specific pro-moters specific for phosphoenolpyruvate carboxylase (PECP)and small subunit Rubisco genes, cell-specific and light-inducedexpression of reporter genes was observed in MC of C3 rice[123]. It provides a hint that these two promoters could be ex-ploited in the direction of leaf-specific activation of genes in ricefor increasing the photosynthetic performance and thus yield ofthe plant. Using activation tagging technology, we have recentlyidentified ribosomal protein large (RPL) subunit genes, RPL6 &

RPL23A as the promising candidates for the manipulation ofabiotic stress tolerance in rice [17]. Further, a detailed character-ization of rice RPL gene family revealed their significant role inboth biotic and abiotic stress responses [124].

Key Points

• The review is an appraisal on the existing gain-of-function mutagenesis approaches in rice with an em-phasis on activation tagging.

• An overview of the potential candidate genes identi-fied in the recent past for stress-amelioration in add-ition to the genes identified specifically through activa-tion tagging is provided.

• The review also presents the tissue-specific and indu-cible promoters identified for spatio-temporal and in-ducible expression of genes.

• More importantly, it highlights on the prospects of en-hancer-based activation tagging and tissue-specifictagging for manipulation of the rice genetic system forimproving yield under the conditions of limited re-source availability and efficient carbon fixation.

Acknowledgements

Authors are grateful to V Sundaresan (UC, Davis) for giftingthe activation tagging vector, pSQ5.

Funding

Our investigation on ‘Identification of candidate genes re-sponsible for high water-use efficiency through activationtagging in indica rice’ is funded by the Department ofBiotechnology, GOI, through grant number BT/PR13105/AGR/02/684/2009 to P.B.K. MM and AB acknowledge theDepartment of Biotechnology for providing the ResearchFellowships.

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