genetic technologies for the identification of plant genes (genetic technologies for the...

Genetic technologies for the identification of plant genescontrolling environmental stress responses

Csaba PapdiA, Mary Prathiba JosephA, Imma Pérez SalamóA, Sabina VidalB

and László SzabadosA,C

AInstitute of Plant Biology, Biological Research Centre, 6726-Szeged, Temesvári krt. 62, Hungary.BFacultad de Ciencias, Universidad de la República, Iguá 4225, CP 11400, Montevideo, Uruguay.CCorresponding author. Email: [email protected]

Abstract. Abiotic conditions such as light, temperature, water availability and soil parameters determine plant growth anddevelopment. The adaptation of plants to extreme environments or to sudden changes in their growth conditions is controlledby awell balanced, genetically determined signalling system,which is still far from being understood. The identification andcharacterisation of plant geneswhich control responses to environmental stresses is an essential step to elucidate the complexregulatory network, which determines stress tolerance. Here, we review the genetic approaches, which have been used withsuccess to identify plant geneswhich control responses to different abiotic stress factors.Wedescribe strategies and conceptsfor forward and reverse genetic screens, conventional and insertion mutagenesis, TILLING, gene tagging, promotertrapping, activation mutagenesis and cDNA library transfer. The utility of the various genetic approaches in plant stressresearch we review is illustrated by several published examples.

Additional keywords: abiotic stress, activation, Arabidopsis, cold, drought, forward genetics, gene, genetic screen, genetagging, heat stress mutagenesis, osmotic stress, oxidative stress, reverse genetics, salinity, variability.

Introduction

Abiotic stresses, especially drought, extreme temperatures andsoil salinity, have increasing agronomic, economic andecologicalimpacts in Europe and worldwide. Recent studies indicate acorrelation between the increased frequency of extremeenvironmental events and global warming, underlining anurgent need for protective measures including the developmentand introduction of new crop cultivarswith enhanced tolerance toenvironmental stresses (Boyer 1982; Etterson and Shaw 2001;Mittler 2006). Most of our knowledge of the control of abioticstress responses has derived from molecular genetic analysisof the model plant Arabidopsis thaliana (L.). Arabidopsis isnot particularly stress tolerant and has limited capacity towithstand harmful effects of extreme environmentalconditions. Nevertheless, the contribution of this model plantto the advance of plant stress biology cannot be underestimated.Information on abiotic stress signal transduction has beengenerated almost exclusively using the Arabidopsis model.Arabidopsis became the model plant for plant biology becauseit has numerous advantageous features, which allow the efficientcombination of genetic and molecular analysis (Redei 1975;Meyerowitz 1989). The publication of the whole Arabidopsisgenome sequence in 2000 (The Arabidopsis Genome Initiative2000a), and the development of genomic tools for Arabidopsisresearch have allowed the adaptation of systems biologyapproaches to this species, accelerating research on differentaspects of plant biology, including abiotic stress responses

(Weigel et al. 2000; Bohnert et al. 2006; Koiwa et al. 2006).Information using Arabidopsis can easily be validated in otherspecies, and technical advances can be adapted to research anddevelopment programs focusing on crop plants.

Plant responses to environmental stresses are controlled byinterconnected signalling networks, which regulate theexpression of large sets of target genes. Receptor moleculesinvolved in primary perception of abiotic stress stimuli are atpresent largely unknown in higher plants, with the exceptionof AtHK1, a putative two-component hybrid histidine kinaseosmosensor, which is implicated in osmosensing under saltstress (Urao et al. 1999). G-protein complexes have beensuggested to function as receptors in sensing stress signals(Misra et al. 2007; Pandey et al. 2009), but the transmission ofprimary stress signals, sensed by receptors to downstreameffector molecules is poorly understood. One of the earliestevents in the process is the release of intercellular calcium thattriggers further stress specific adaptation responses (Knight 1999;Sanders et al. 2002). Numerous further downstream signallingcomponents have been identified including hydrogen peroxide,phospholipid secondary messengers, and cytosolic Ca2+

oscillation, which controls the activity of CDPK, SnRK andMAPK-type protein kinase phosphorylation cascades (Knight1999; Zhu 2002, 2003; Wang 2004; Bartels and Sunkar2005). Abscisic acid (ABA) is a major ‘stress’ hormone thatplays a central role in the control of abiotic stress responses. ABAcontrols gas exchange and photosynthesis by triggering stomatal

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closure, induces the synthesis of seed storage proteins and lipids,promotes seed desiccation tolerance and dormancy, inhibitstransition from embryonic stage to germination, and regulatesnumerous other aspects of plant development. ABA stimulatesthe expression ofmany drought and osmotic stress-induced genesthrough a complex signalling mechanism. ABA perception andsignalling appear to involve a branching pathway, in which Ca2+

release, reactive oxygen species (ROS), and protein kinases andphosphatases are the most important components (Leung andGiraudat 1998; Finkelstein et al. 2002; Wang and Zhang 2008).The ABA dependent and independent signalling cascadescontrol the activity of a large set of transcription factors andregulate the expression of downstream target genes through atleast six different pathways (Zhu 2002; De Smet et al. 2006;Yamaguchi-Shinozaki and Shinozaki 2006; Nakashima et al.2009).

In this paper, we review the genetic and genomic approacheswhich have been used in plants to dissect the regulation ofabiotic stress responses. Although most examples are obtainedfrom Arabidopsis, references to other plants are made whererelevant advances have been made (Table S1 available as anAccessory Publication to this paper).

Mutant isolation and forward genetic approaches

In order to perform genetic analysis, genetic variation has tobe identified by specific screening procedures (Fig. 1). Inforward genetic strategies, genetic variation is generated firstthrough chemical, physical or biological mutagenesis, andthe mutagenised plant population is screened for the phenotypeof interest. Alternatively, natural variation can provide valuableresourcesofgeneticpolymorphism. InArabidopsis, ethylmethane-sulfonate (EMS) is the most frequently used chemical mutagen,which predominantly generates point mutations (Rédei andKoncz 1992; Kim et al. 2006). Physical mutagenesis can beperformed by gamma irradiation and by fast neutrons, whichusually generates small deletions (Rédei and Koncz 1992; Liand Zhang 2002; Shikazono Suzuki et al. 2005). The number ofmutagenised plants to be screened depends on the mutationfrequency, which depends on the mutagenesis treatment. Afterlow mutagen doses, large number of mutagenised plants have tobe screened. High mutagen doses can be more efficient, butundesired mutations need to be removed later by repeatedbackcrossing of the mutant line with parental lines (Rédei andKoncz 1992). To isolate mutations in genes which control stressresponses, mutagenesis is usually followed by genetic screensdesigned to identify mutants with enhanced or reduced toleranceto controlled environmental stress conditions. In order to screenlarge numbers of plants, and increase the chances of identifying thehighest number of mutants, the screening procedure should beas simple as possible (Page and Grossniklaus 2002; Alonso andEcker 2006). For these reasons, in vitro-based screens have oftenbeen developed.

Forward genetic screens in Arabidopsis represent a powerfuland unbiased approach to identify mutants with altered stressresponses. Identification of the corresponding gene requiresmap-based cloning, which is often a tedious and time consumingprocedure. For mapping, mutant plants are crossed with anotherecotype and a small population of F2 plants are genotyped to

determine genetic linkage to molecular markers representingeach chromosomal region. Fine-scale mapping is performed ona larger population of F2 plants narrowing linkage to a 30–50 kbpregion. Localisation of the mutation is done by sequencing this

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Fig. 1. Schematic representation of several genetic strategies used toidentify plant genes. (a) Simplified structure of a plant gene consisting of50 regulatory sequences, transcribed region with exons and introns, and30 regulatory sequences. (b) Point mutations modify but do not destroygene structure, resulting in modified gene product. (c) Deletions remove agene or part of it, which results in loss of the gene function. (d) Insertionsinterrupt and destroy gene structures, leading to loss-of-function mutations.(e) Gene traps can inactivate genes and simultaneously create in situ genefusions with the reporter gene of the insertion element. ( f ) Activatormutagenesis uses strong constitutive enhancers or promoters in theinsertion element, and usually generates gain-of-function mutations byactivating the transcription of flanking genes. (g) Random cDNA transferuses a cDNAlibrary cloned in a plant expressionvector,where transcriptionofthe inserted cDNA is controlled by the promoter of the expression cassette.The result is dominant gain or loss-of-function phenotype, depending on theintegrity of the inserted cDNA.

Genetic identification of plant genes in response to stress Functional Plant Biology 697

region of the mutant and wild type chromosomal DNA (Jander2006). The efficiency of map-based cloning depends on theavailability of a detailed genetic map, and saturating thegenome with PCR-based markers facilitates mapping andcloning of mutant loci. Thousands of markers, Insertion/Deletion (InDel) polymorphism and Single NucleotidePolymorphism (SNPs) are available for fine mapping inArabidopsis, making map-based cloning feasible within a year(Cho et al. 1999; Peters et al. 2003; Jander 2006). Recently, DNAmicroarray technologies have been adapted for mappingmutations in forward genetic studies. Oligonucleotide arrayswere originally designed for transcript profiling, but have beenadapted for high density genotyping to detect polymorphisms,mutation mapping and mapping and cloning of quantitative traitloci (QTL) (Spiegelman et al. 2000; Borevitz et al. 2003; Hazenet al. 2005). Although the detection of single base substitutionsis limited, deletions, as caused by fast neutrons can easily bedetected. Natural variation or induced mutations with InDelpolymorphism can be recognised with higher precision withspecific genotyping arrays (Salathia et al. 2007). To improvethe efficiency of deletion mapping, a novel tiling array, AtMap1was constructed and used for mapping several deletion mutantsand the detection of natural polymorphism which involvesdeletions (Nagano et al. 2008).

Genome-wide mapping of mutations can be performed inplant species which have a well defined genetic map. Highdensity linkage maps have been established for most of theimportant crops, such as barley (Hordeum vulgare L.; Horiet al. 2003; Wenzl et al. 2006), wheat (Triticum aestivum L.;Dilbirligi et al. 2006; Peleg et al. 2008), rice (Oryza sativa L.;Harushima et al. 1998), Sorghum sp. (Draye et al. 2001; Menzet al.2002), potato (Solanum tuberosumL.;Tanksley et al.1992),tomato (Lycopersicom esculentum L.; Tanksley et al. 1992;Saliba-Colombani et al. 2000), Brassica sp. (Pradhan et al.2003; Sun et al. 2007), soybean (Glycine max L.; Xia et al.2007), papaya (Carica papaya L.; Ma et al. 2004), Lotus sp.(Wang et al. 2008) and Rose sp. (Yan et al. 2005). Althoughgene identification in non-model species is usually a difficulttask due to technical reasons, i.e. large genomes, low markerdensities, lack of genomic sequence information, etc., microarraytechnology offers a powerful strategy to identify deletions andfacilitate cloning of mutant alleles in complex genomes such asCitrus (Ríos et al. 2008).

Recent advances in massively parallel sequencingtechnology offer new and robust strategies to identify geneticpolymorphism and for mutation mapping (Shendure and Ji 2008;Ansorge 2009). Combination of bulked segregant analysisand deep sequencing of F2 generation mutant plants willfacilitate mapping of Arabidopsis mutations in a short time atrelatively low cost (Ossowski et al. 2008; Lister et al. 2009).Withfurther improvement of sequencing technology the mapping ofmutations and polymorphism in plant specieswith large genomeswill also be possible.

Examples for genetic screens and gene identification

In order to identify salt hypersensitive Arabidopsismutants, Zhu(2002) screened mutagenised M2 generation seedlings forimpaired root curling and elongation on inverted vertical plates

containing culture medium with sublethal concentrations ofNaCl (Ishitani et al. 1997). Differences in root growth onthese conditions could be scored easily. Identification andmolecular analysis of the Salt Overly Sensitive (sos) mutantshave confirmed the utility of this screening procedure and leadto the discovery of a regulatory pathway which is essentialto maintain ion homeostasis (Zhu 2002; Chinnusamy et al.2006). The so-called SOS pathway detects and transducesCa2+ signals generated by salt stress. SOS3, a calcineurinB-type calcium binding protein is able to detect changes inintracellular Ca2+ concentrations caused by high salinity(Ishitani et al. 2000). SOS3 interacts with the SnRK3-typeSOS2 protein kinase (Halfter et al. 2000; Liu et al. 2000;Gong et al. 2002a), which activates several membrane iontransporters such as the plasmamembrane Na+/H+ antiporterSOS1 (Wu et al. 1996; Shi et al. 2000), the H+/Ca2+ antiporterCAX1 (Cheng et al. 2004), or the vacuolar H+-ATPase(Batelli et al. 2007). Conservation of the SOS pathway in riceand wheat suggests that these factors are important for salttolerance through maintaining Na+/K+ homeostasis in dicotsand monocots alike (Martínez-Atienza et al. 2007; Mullanet al. 2007). In a similar forward genetic approach, inhibitionof lateral root development by osmotic stress was employed toisolate 30Arabidopsismutants with altered sensitivity to droughtand abscisic acid (Xiong et al. 2006). Analysis of the DroughtInhibition of lateral root Growth (dig) mutants revealedthat responses of root growth to osmotic stress are linked todrought tolerance in the whole plant.

A salt hypersensitive mutant was mapped with a microarray-based strategy and the mutation was identified as a 523 bpgenomic deletion in the AtHKT1 gene (Gong et al. 2004b).Segregation analysis and mutant complementation confirmedthat the deletion and inactivation of AtHKT1 was responsiblefor the salt sensitivity of the mutant.

Genetic screens for deregulated expression of stress-responsive reporter gene constructs provide an alternativeoption to isolate mutations in regulatory genes that eitheractivate or repress the activity of the reporter gene (Xionget al. 1999b). As multiple regulatory networks control theexpression of stress-induced genes (Yamaguchi-Shinozaki andShinozaki 2006; Nakashima et al. 2009) diverse reporter geneconstructs can be designed to dissect each of these regulatorycascades. Combined with non-destructive bioluminescenceimaging, alterations of the cold, drought and ABA-responsiveRD29A-LUC reporter gene activity led to the identification ofcos, los and hos mutants, corresponding to constitutive, low orhigh expression of osmotically responsive genes, respectively(Ishitani et al. 1997; Chinnusamy et al. 2002). Such luciferasescreens were employed to identify important regulatorycomponents of salt, cold and ABA signalling pathways(Ishitani et al. 1998; Xiong et al. 1999a; Xiong et al. 2001;Lee et al. 2002; Zhu et al. 2004). The RD29A-LUC reportersystem was used for example to identify several alleles of thetranscriptional activator FIERY2, which regulates the expressionof genes with DRE/CRT-type cis elements in their promoters(Xiong et al. 2002). The Arabidopsis RCI2A gene is induced byseveral abiotic stresses through an ABA-dependent pathway.Alterations in RCI2A-LUC activity were used to identifymutants with impaired tolerance to freezing, dehydration, salt

698 Functional Plant Biology C. Papdi et al.

stress and ABA sensitivity (Medina et al. 2005). The 2-cysteineperoxiredoxin A (2CPA) gene encodes an important chloroplastantioxidant enzyme. 2CPA transcription is controlled by redoxbalance, and accumulation of ROS. A transgenic Arabidopsisline, expressing the 2CPA-LUC reporter gene was used toidentify the Arabidopsis redox imbalanced (rimb) mutants,where transcriptional regulation of the 2CPA gene, andnumerous other redox-controlled genes were affected (Heiberet al. 2007).

Abscisic acid has a central role in controlling droughtresponses, stomatal closure, root growth, expression ofnumerous stress-induced genes and the accumulation ofprotective compounds such as proline. The isolation andcharacterisation of Arabidopsis mutants defective in ABAsynthesis, perception or signal transduction is thereforeessential for understanding the genetic basis of droughttolerance. The first ABA response mutants were identified byABA insensitive or hypersensitive germination, seedling or rootgrowth. The dominant abi1 and abi2 mutants show ABA-resistant germination and pleiotropic defects in ABA responsesin whole plants (Koornneef et al. 1984). Map-based cloning leadto the molecular characterisation of ABI1 and ABI2 genes, whichencode similar PP2C type serine-threonine phosphatases and areimportant components of whole-plant responses to water deficitand osmotic stress (Leung et al. 1994, 1997; Meyer et al. 1994).Other ABA signalling mutants have been isolated in similargermination screens, including the ABA insensitive abi3, abi4,and abi5 mutants, which lead to the identification of thecorresponding genes encoding different transcription factors,essential for the activation of different classes of ABA-regulated genes (Giraudat et al. 1992; Finkelstein et al. 1998;Finkelstein and Lynch 2000). ABA hypersensitive mutants havebeen identified in several genetic screens, many of them based onthe inhibition of germination by ABA. Examples include theenhanced response toABA (era1) mutant (Cutler et al. 1996), thegrowth control byABA (gca1-gca8) mutants (Himmelbach et al.1998) and the ABA hypersensitivity (abh1) mutant (Hugouvieuxet al. 2001), which displays pleiotropic ABA responses such asenhanced guard cell response and drought tolerance. ERA1encodes the b subunit of farnesyl transferase, a lipid transferenzyme, while ABH1 and other ABA response loci encodeRNA binding proteins, suggesting that they are involved inRNA processing.

Infrared thermal imaging offers an alternative approach toisolate ABA insensitive mutants, in which genetic defectsspecifically affect the ABA sensitivity of stomatal guard cells.Changes in leaf temperature indicate alterations in ABA-dependent stomatal opening and respiration, which can bevisualised by infrared thermography. Differences in leaftemperature led to the isolation of the open stomata mutantsost1 andost2,whichdisplayed lower leaf temperatures andhighertranspirational water loss than wild type plants (Merlot et al.2002). Map-based cloning revealed that the OST1 locus encodesa SnRK2-type protein kinase, which mediates the closureof stomatal aperture triggered by ABA (Mustilli et al. 2002).Advances in the non-invasive infrared thermal imagingtechnology offer an efficient screening tool to identify novelmutants defective in regulation of stomatal opening and droughttolerance (Wang et al. 2004).

Natural variation and quantitative trait loci

Naturally occurring variation is one of themost important geneticresources for plant breeding and is available in practically all plantspecies. Responses to environmental conditions depend onnumerous genes and are typically controlled by QTLs, whichrepresent heritable variability of traits and characters. QTLscorrespond to natural alleles, which otherwise have smalleffects on the tolerance to abiotic constrains. Mapping of suchQTLs may lead to identification and cloning of importantregulatory genes or allelic variants and provide geneticmarkers for molecular breeding or cloned genes for geneticengineering to improve stress tolerance. Thus, improvement ofcrop performance in suboptimal or extreme environmentalconditions will benefit from the identification andcharacterisation of such adaptive QTLs (Collins et al. 2008).Hundreds of reports have described QTLs which can affectabiotic stress tolerance (Flowers et al. 2000; Koornneef et al.2004; Munns et al. 2006; Kliebenstein 2009). However, geneticdissection of QTLs, mapping the genes and alleles thatcontribute to stress adaptation as well as the identification ofpolymorphism at the nucleotide level has been hindered for along time by the lack of adequate tools. With the use ofrecombinant inbred lines (RILs) the dissection and analysis ofQTLs became possible in Arabidopsis as well as in severalimportant crops (Koornneef et al. 2004; Collins et al. 2008;O’Neill et al. 2008). Nevertheless, QTL mapping remains atedious and time-consuming task. Next-generation sequencingof natural accessions can reveal sequence variability in genomescale and will facilitate the large scale identification of SNP-sand InDels in different ecotypes and varieties (Ossowski et al.2008) (http://1001genomes.org/, verified 7 July 2009). Thecomprehensive analysis of natural polymorphism and variationin stress tolerance will facilitate the understanding of geneticbases of adaptation to extreme environmental conditions.

QTLs regulating responses to different abiotic stress factorshave been identified in several plant species. For example, inter-and intra-specific variability in salt tolerance has allowed theidentification of several QTLs that contribute to this trait inplants. Salt tolerance is determined by several physiologicalcomponents such as sodium transport and exclusion, toleranceto osmotic and oxidative stress, and tolerance to ion toxicity attissue and cell level (Munns and Tester 2008). Ion homeostasisis a key element of salt tolerance, and is determined by iontransport, exclusion or accumulation (Serrano and Rodriguez-Navarro 2001). Several major and minor QTLs have beenmapped in different plants, and used to identify importantcomponents controlling Na accumulation. High-throughputionomic profiling, coupled with microarray-based geneticanalysis lead to the identification of novel natural variants of theNa+ transporter AtHKT1, which controls salt tolerance inArabidopsis (Rus et al. 2006). Natural variation of sodiumaccumulation in shoots and salt tolerance was shown to dependon the expression patterns of AtHKT1 alleles in differentArabidopsis accessions. Polymorphism of the AtHKT1 genein salt tolerant and sensitive wild populations of Arabidopsistherefore confirmed the importance of this transporter in salttolerance (Rus et al. 2006). Nax1/TmHKT1;4 and Nax2/TmHKT1;5 salt tolerance loci of wheat have been identified by


QTL mapping and were shown to control sodium transport(Lindsay et al. 2004; James et al. 2006). Nax1/TmHKT1;4 is aNa+ transporter (HKT7) gene (Huang et al. 2006). Fine mappingwith microsatellite markers suggested that the high-affinity K+

transporter TmHKT1,5 (HKT8) was the candidate gene forNax2 and the homoeologous Kna1/TaHKT1;5 in bread wheat(Byrt et al. 2007). SKC1 is a major QTL for salt tolerance in rice,which affects sodium accumulation and transport. Map-basedcloning of the SKC1 locus lead to the identification ofOsHKT1,5,which is an orthologue of the wheat TmHKT1,5 gene andfunctions as a Na+-selective transporter (Ren et al. 2005).Physiological data confirmed that SKC1 regulates K+/Na+

homeostasis and is important in salt tolerance (Ren et al. 2005).Mapping and analysis of HKT1 alleles in several plant species,confirmed that control of Na+ accumulation and K+ uptakethrough specific transporters is a general mechanism for salttolerance (Platten et al.2006; Rodriguez-Navarro andRubio 2006;Gupta et al. 2008; Huang et al. 2008; Munns and Tester 2008).

Transpiration efficiency is an important component ofwater use and drought tolerance. QTLs have been identifiedfor transpiration efficiency in several plant species, withoutthe contributing genes having been identified. ERECTA wasthe first Arabidopsis gene, which was mapped as a mainQTL, and regulates transpiration efficiency by controlling leafphotosynthesis efficiency and stomatal conductance (Masle et al.2005). ERECTA encodes a LRR-type receptor-like kinase (RLK)and was previously known to control inflorescence development(Torii et al. 1996) and resistance against bacterial wilt (Godiardet al. 2003).

Cold tolerance correlates with geographical distribution ofa species and is an important component of adaptation toclimates with low temperatures. Freezing tolerance iscontrolled by seven QTLs in Arabidopsis. QTL mappingrevealed that the C-repeat Binding Factor (CBF) locus is themost important component in cold acclimation (Alonso-Blancoet al. 2005). CBF genes encode transcriptional activators whichbind to C-repeat or DRE promoter elements and activate a largeset of stress-induced genes. Low freezing tolerance of the Cviecotype was associated with a low expression level of CBF2caused by a promoter deletion (Alonso-Blanco et al. 2005).Nevertheless correlation of freezing tolerance and CBF geneexpression levels in contrasting Arabidopsis ecotypes couldnot be confirmed in more recent studies, indicating that coldtolerance is determined by complex network of genes (Le et al.2008; McKhann et al. 2008). In barley, QTL mapping identifiedthe CBF genes as important determinants of cold and droughttolerance (Tondelli et al. 2006). Frost tolerance in diploid wheat(Triticum monococcum L.) is controlled by multiple QTLs.Several CBF genes were mapped into QTLs which determinesfrost survival, suggesting that wheat CBF genes are importantfor cold acclimation and resistance to freezing temperatures(Miller et al. 2006; Knox et al. 2008). Differences inexpression levels of several CBF genes were shown toinfluence frost tolerance in wheat (Vágújfalvi et al. 2005).

Insertion mutagenesis

Traditional mutagenesis has generated numerous mutants whichare valuable resources for genetic analysis. Identification of the

corresponding gene is however often difficult due to thetedious work of map-based cloning. Linking of molecular andphysiological studies with genetic analysis is easier whenmutations are generated with insertions of DNA elements.Mobile genetic elements can be endogenous or introducedtransposons or the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens, which is covalently integrated into the genome ofthe host cell (Bancroft et al. 1992; Zupan and Zambryski1995; Azpiroz-Leehan and Feldmann 1997; Meissner et al.2000). Integration of a DNA fragment with the size ofseveral thousand base pairs can disrupt the sequence of agene and effectively eliminate or reduce its activity (Fig. 1).Chromosomal loci are not onlymutated by T-DNAor transposoninsertions but also labelled by these molecular tags, which canbe detected by standard molecular methods (Koncz et al. 1992;Azpiroz-Leehan and Feldmann 1997; Martienssen 1998).

The most frequently used insertion elements in Arabidopsisgenetics are the Ac/DS or En/I type transposable elements(Bancroft et al. 1992; Martienssen 1998; Wisman et al. 1998),or theAgrobacteriumT-DNA (Feldmann et al. 1989;Koncz et al.1989; Koncz et al. 1992; Azpiroz-Leehan and Feldmann 1997).A. tumefaciens is a Gram negative soil bacterium, which is able totransfer and covalently insert a fragment of its large Ti plasmid(T-DNA) into the genomic DNA of the infected host cell (Schellet al. 1979; Caplan et al. 1983; Zupan and Zambryski 1995;Zupan et al. 2000). Integration of the T-DNA into the genome ofthe host cell takes place via illegitimate recombination, whichdoes not require sequence homology of the target genomic region(Tinland and Hohn 1995; Hansen and Chilton 1999; Zupan et al.2000). Consequently, distribution of the T-DNA integration sitesin theArabidopsisgenome is nearly random, although integrationfrequency is lower in centromeric regions, andhigher in gene-richchromosomal regions (Szabados et al. 2002; Alonso et al. 2003).Moreover, T-DNA integration in promoter and 50 untranslatedregions of actively transcribed plant genes is more frequentthan in intergenic regions (Szabados et al. 2002; Li et al.2006). Preference for integration in promoter regions can beexplained by the interaction of the TATA binding protein withthe Agrobacterium VirD2 protein, which is phosphorylated byPolII-CTD interacting CDKD-type kinases (Bako et al. 2003).To make use of the Agrobacterium T-DNA transfer mechanism,numerous plant transformation vectors have been generated inthe last two decades, which have been used in several insertionmutagenesis programs (Bevan 1984; McBride and Summerfelt1990; Düring 1994; Koncz et al. 1994; Fuse et al. 1995; Hellenset al. 2000; Szabados and Koncz 2003; Alvarado et al. 2004;Calderon-Villalobos et al. 2006; Komari et al. 2006; Thole et al.2007).

Establishment of efficient transformation proceduresor identification of transposable elements allows theimplementation of large-scale insertion mutagenesis in plantspecies. Arabidopsis can easily be transformed by dipping orby vacuum infiltration of floral buds with the Agrobacteriumsuspension, carrying the appropriate vector, and subsequentlyselecting the progenies for antibiotic or herbicide resistance,determined by the T-DNA marker gene (Bechtold et al. 1993;Bechtold andPelletier 1998;Bent 2000). Inplanta transformationof Arabidopsis can be scaled up easily, and is suitablefor generating large number of transgenic lines for T-DNA


tagging programs (Parinov and Sundaresan 2000; Sussman et al.2000; Weigel et al. 2000; Bouché and Bouchez 2001; Ríos et al.2002; Szabados et al. 2002;Alonso et al. 2003; Rosso et al. 2003;Koiwa et al. 2006). Among the monocotyledonoues plants,the most efficient Agrobacterium-mediated transformation hasbeen developed for rice (Hiei et al. 1994), leading to large-scaleT-DNA insertion mutagenesis programs (Jeong et al. 2002; Anet al. 2005; Hsing et al. 2007; Wan et al. 2009). In the legumemodel plant Medicago truncatula (L.), the Trt1 retrotransposonhas been used for insertion mutagenesis (Tadege et al. 2005;Benlloch et al. 2006; Tadege et al. 2008).

T-DNA insertions usually lead to loss-of-function, recessivemutations, which show Mendelian segregation among T2

generation plants. In order to isolate mutants with alteredstress responses, forward genetic screens can be employed andT2 generation transgenic plants can be screened eitherindividually or in pooled stocks to identify mutants displayingenhanced or reduced stress tolerance (Alonso and Ecker 2006;Koiwa et al. 2006; Schneider and Leister 2006). Progenies ofselected plants are subsequently tested for co-segregation of theobserved phenotype and the T-DNA insert. Multiple T-DNAinsertions may occur during the transformation procedure, thusthe determination of linkage between a T-DNA insert and amutation require special care. Once linkage of the mutationand the T-DNA insertion is confirmed, T-DNA insertion sitescan be recovered by PCR amplification of the T-DNA junctions.PCR fragments can subsequently be sequenced and the insertionsites can be exactly mapped by sequence homology searches(Earp et al. 1990; Liu et al. 1995; Mathur et al. 1998; Szabadosand Koncz 2003).

In order to identify tagged Arabidopsis, mutants with alteredsalt tolerance, T-DNA insertion lines were generated in differentArabidopsis genetic backgrounds, and large forward geneticscreens of T-DNA mutagenised lines were carried out (Koiwaet al. 2006). Employing a root bending assay, number of saltand osmotic stress sensitive mutants were identified, includingnew alleles of sos1. New mutations have been identified inloci encoding proteins with different cellular functions such assignalling (protein phosphatase, transcription factors), vesiculartrafficking (syntaxin), protein glycosylation and solute transport(Koiwa et al. 2006). Using the RD29A-LUC reporter gene andluciferase imaging screens, several mutants with enhancedluciferase activity were identified, including alleles of cpl. TheArabidopsis CPL genes encodes isoforms of Polymerase IICTD phosphatase, suggesting that CTD phosphorylationcontrols stress signal transduction (Koiwa et al. 2002). Inanother study, mutants with salt tolerance phenotypes wereisolated by screening for enhanced capacity for germinationand growth on high salt medium. In the salt tolerant sto1mutant the insertion inactivated the NCED3-like geneencoding an isoform of 9-cis-epoxicarotenoid dioxygenase, akey enzyme inABAbiosynthesis (Ruggiero et al.2004). The sto1mutant was more tolerant to salt in vitro, but was susceptible fordehydration due to its inability to accumulate ABA. Screeningfor stress hypersensitivity in our T-DNA mutant collection(Szabados et al. 2002) led to the identification of a semi-dwarftagged mutant with enhanced sensitivity to ABA, salt, osmoticand oxidative stress. In the ppr40 mutant, the T-DNA waslocalised in the coding region of the PPR40 gene, which

encodes for a mitochondrial pentatricopeptide repeat (PPR)domain protein, controlling electron flow through Complex IIIin the mitochondrial electron transport chain (Zsigmond et al.2008).

Gene and promoter trapping

Promoter and enhancer trap technologies use promoterlessreporter genes facing the boundaries of T-DNA or transposoninsertions. Promoter trap insertions in transcribed chromosomalloci may generate in situ fusions between plant genes andreporter genes, which can be identified by monitoring theactivity of the reporters (Teeri et al. 1986; Koncz et al. 1989;Kertbundit et al. 1991). Enhancer traps use reporter genes fusedto minimal promoter elements to facilitate the activation ofreporter genes by flanking plant enhancer sequences (Klimyuket al. 1995; Campisi et al. 1999; Ito et al. 2004). Plant gene fusiontechnologies were first demonstrated in tobacco and Arabidopsisusing a promoterless kanamycin phosphotransferase (npt II;Teeri et al. 1986; Koncz et al. 1989) and the b-glucuronidase(GUS; uidA) reporter genes (Jefferson et al. 1987; Kertbunditet al. 1991; Fobert et al. 1994). Using T-DNA or transposoninsertions, gene and promoter trapping programs have been usedto identify tagged genes, promoters and enhancers with tissuespecific activity in crop species such as rice (Chin et al. 1999;Acosta-Garcia et al. 2004; Ito et al. 2004), barley (Lazarow andLutticke 2009). The GUS reporter is suitable for sensitivehistochemical detection and analysis of cell and tissue specificexpression patterns, but is unsuitable for nondestructivescreening purposes, which is essential for the identification ofenvironmentally controlled gene activities. The light-emittingbacterial (LUX) or firefly (LUC) luciferase reporter enzymes canbe detected in vivo by bioluminescence imaging with sensitiveCCD cameras and are better suited as real-time reporters forin planta gene expression studies (Riggs and Chrispeels 1987;Jiang et al. 1992; Millar et al. 1992). The luciferase reportersystem has been used in random promoter and gene trappingexperiments in tomato (Meissner et al. 2000), Arabidopsis(Yamamoto et al. 2003; Alvarado et al. 2004; Calderon-Villalobos et al. 2006) and banana (Musa sp.; Remy et al.2005). Recently a dual reporter system has been developed, inwhich a fused GUS and LUC reporter gene is employed for thedetection of the activity of the trapped gene (Koo et al. 2007). Inthis system, the luciferase allows the sensitive and non-invasivedetection of the gene activity, while GUS activity can bevisualised in histochemical assays to analyse tissue and cellspecific expression patterns. A Gal4-mediated LUC/GFP/GUSreporter system has been used for enhancer trapping inArabidopsis, to identify specific enhancers and to study genesilencing (Engineer et al. 2005).

Using gene or promoter trap technologies, characterisation ofthe mutant phenotype and expression analysis of the tagged genecan be performed simultaneously with the same line. Transposontagging with a DS-GUS gene trap construct has been used toidentify 15Arabidopsisgenes, induced by anoxia (Baxter-Burrellet al. 2003). Several tagged genes were shown to control theactivity of the anoxia-responsive alcohol dehydrogenase (ADH)gene. Two mutants were hypersensitive to low-oxygenenvironment, suggesting that the corresponding genes were


important for such stress tolerance. Screening for cold and salt-induced GUS expression in transgenic rice lines transformed bythe pGA2144 promoter trap vector, a stress-responsiverice orthologue of the Arabidopsis brassinosteroid insensitive(BIN2) gene was identified, which encodes a GSK3/SHAGGY-like kinase (OsGSK1). Knockout mutants of the OsGSK1gene showed cold, salt, drought and heat tolerance, whileoverexpression of the full-length cDNA lead to stunted growth(Koh et al. 2007). OsGSK1 is therefore a negative regulator ofbrassinosteroid signalling, which controls stress responses aswell.

The promoterless firefly luciferase has been used as reporterin promoter trap experiments to tag stress and ABA regulatedArabidopsis genes in our laboratory (Fig. 2). Analysis ofluciferase expression patterns showed, that stress-regulatedgenes were tagged in 24 lines. Although several such taggedlines had detectable background luminescence without anytreatment, luciferase activity increased considerably in severalpromoter trap lines when seedlings were treated by salt andosmotic stress or by abscisic acid (Fig. 2). Characterisation ofthe transcriptional fusion between the ABC transporter geneAtWBC11 and the luciferase reporter revealed induction ofAtWBC11 with high salt, osmotic and ABA treatments, anddownregulation of ABA-induced expression with gibberellin(Alvarado et al. 2004). Subsequent characterisation of theAtWBC11 gene revealed its function in cutin and waxsecretion and confirmed the importance of this transporter indefence against osmotic and salt stress (Panikashvili et al. 2007).Recently a gene trap approach was used to identify Arabidopsisgenes expressed in stomatal guard cells, and five lines wererecovered with stomata specific activity of the promoterlessGUS reporter gene. Statistical analysis of promoter regions instomata specific gene trap insertions revealed the presence ofthe (A/T)AAAG motif, a characteristic regulatory element instomatal specific genes. Characterisation of the homozygoustagged mutant of the Pleiotropic Drug Resistance 3 locusrevealed that the AtPDR3 gene controls ABA-induced stomatalclosure, and subsequently drought tolerance (Galbiati et al.2008). Using the promoterless GUS reporter, a gene trapsystem has recently been established in rice. The system wassuitable to identify stress and hormone-induced rice genes(Chen et al. 2008).

Activation tagging

While T-DNA insertions usually result in loss-of-functionphenotypes due to inactivation of the tagged gene, gain-of

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Fig. 2. Identification of salt-induced luciferase gene fusions in promotertrap experiments. (a) Screening for salt-induced luciferase activity intransgenic Arabidopsis seedlings transformed by pTluc (Alvarado et al.2004). T1 generation seedlings were selected on hygromycin-containingmedium, and tested for bioluminescence. Low luciferase activity wasdetected in the shoot apex of one seedling (lum/ctr), which was enhancedby transferring the seedlings to high salt medium for 12 h (lum/NaCl,germination medium supplemented by 200mM NaCl). (b) Luciferaseactivity in T2 generation seedlings of a selected promoter trap line,induced with salt or ABA. Two-week-old seedlings were transferred tofresh germination medium (control), or to media supplemented by 200mM

NaCl (NaCl) and50mMABA(ABA). Images are shownat 0 h, 6 h or 15 h aftertransfer. Note: that luminescence increases after salt or ABA treatments.(c) Kinetics of the salt andABA-dependent luciferase activity in the promotertrap line shown in Fig. 2b. Luminescence increased until 6 h after transferringseedlings tomediumsupplemented byNaCl andABA, andgradually declinedlater.


function mutations can be generated by activation of plant geneswith strong transcriptional enhancers or promoters in the insertionelement (Koncz et al. 1994;Weigel et al. 2000; Jeong et al. 2002;Szabados and Koncz 2003; Koiwa et al. 2006; Wan et al. 2009).Activation tagging induces ectopic expression of genes adjacentto the insertion sites, and can be used for generation of dominantmutations that can reveal the function of unique genes as well asindividual members in a gene family (Weigel et al. 2000;Nakazawa et al. 2003). Ectopic expression of stress-inducedregulatory genes such as transcription factors can lead tooverexpression of downstream stress-responsive genes andmay increase tolerance to extreme environmental conditions.Activation tagging with strong promoters is not only capableto activate transcription of flanking genomic sequences but alsocan generate antisense transcripts and therefore can be used forgene suppression, generating loss-of-function type mutations(Ülker et al. 2008). Functional identification of the cytokininreceptor kinase (Kakimoto 1996), and various microRNA genes(Palatnik et al. 2003) illustrates the potential of activationtagging. While most activation tagging has employed T-DNA-derived vectors, transposon-based vector systems have also beencreated for monocots, including cereals (Ayliffe et al. 2007; Quet al. 2008). A large tagged rice population was generated with aT-DNA activation tagging vector, containing double CaMV35Senhancer at the right border (Wan et al. 2009). A combinedpromoter trap/activation tagging T-DNA vector was also used inrice, which contained multiple enhancers of the CaMV35Spromoter, able to activate the genes adjacent to the insertionsite (Jeong et al. 2002). The maize (Zea mays L.) Ac/Dstransposable element system was also employed for activationtagging in rice (Qu et al. 2008) and barley (Ayliffe et al. 2007).

Activation tagging was used to isolate several Arabidopsisgain-of-functionmutantswith enhanced tolerance to high salinityanddrought, leading to the identificationofnumerousnovelgenescontrolling such traits. The Shine (SHN) gene was identified inactivation tagging screens and was shown to control droughttolerance through regulating cuticular wax composition(Aharoni et al. 2004). The SHN gene encodes an AP2/EREBPtype transcription factor, which has an important role inconstructing plant protective layers in cuticle, wounding,abscission and dehiscence. By screening for drought and salttolerance of Arabidopsis activation tagged lines, the sdt1mutantwas isolated and was found to survive lethal levels of salt anddrought stresses (Li et al. 2005). Isolation of dominant mutationsconferring enhanced salt tolerance in direct genetic screensconfirmed the utility of this approach for salt stress research(Koiwa et al. 2006). The edt1 mutant was identified using asimilar approach, andwas shown to exhibit drought tolerance as aresult of the enhanced expression of a homeodomain-STARTtranscription factor (Yu et al. 2008). Similarly, mutants withincreased tolerance to chilling were also identified in tobaccousing activation tagging (Ahad et al. 2003). While isolation ofrecessive mutations in tetraploid species, such as tobacco(Nicotania tabacum L.), is very difficult, activation tagginggenerates dominant mutations, which can be identified easilyin all type of plants regardless of the ploidy level. TheCDT-1geneof Craterostigma plantagineum Hochst. was identified byactivation tagging, screening for desiccation tolerance in calluscultures. Constitutive overexpression of CDT-1 could enhance

desiccation tolerance andwas able to upregulate the expression ofseveral dehydration and ABA-induced transcripts in the absenceof ABA (Furini et al. 1997). CDT-1 was shown to be similar toretroelements and directs the synthesis of 21 bp short interferingRNA (siRNA). Recovery after dehydration in C. plantagineumwas suggested to depend on the capacity to synthesise siRNA(Hilbricht et al. 2008).

Combination of activation tagging with screening forinduction (or repression) of promoter driven luciferasereporters provides a more specific technique to identify and tagregulatory genes. Screening for enhanced luminescence of apathogen responsive PR1-luciferase reporter gene was usedfor tagging of the Arabidopsis ADR1 pathogen resistance gene(Grant et al. 2003). The activated ADR1 gene was later shown toconfer drought tolerance through a salicylic acid-dependentpathway, suggesting that biotic and abiotic signalling canshare common components and have functional overlaps(Chini et al. 2004).

Activation tagging offers a unique strategy for dissection ofstress signalling pathways through mutant suppression screens.The salt hypersensitive sos3–1 mutation inactivates the SOS3gene encoding a CBL-type calcium sensor, which controls theactivity of the SnRK3-type SOS2 protein kinase (Halfter et al.2000; Ishitani et al. 2000; Gong et al. 2004a). Activation taggingwas used to identify suppressor or enhancer mutations of thesos3–1 mutant. Among the 16 lines that suppressed the saltsensitive phenotype of sos3–1, a new mutant allele of theAtHKT1 gene was isolated which is an important Na+

transporter mediating salt influx into plant cells (Koiwa et al.2006).

Complementary DNA library transformation

Transfer and expression of plant cDNA libraries in yeast is afunctional equivalent of multicopy suppressor screens developedto identify yeast genes able to suppress salt sensitivity (Benderand Pringle 1991; Masson and Ramotar 1998).

AnArabidopsis cDNAlibrary in ayeast expressionvectorwasused to identify lithium tolerant yeast transformants and lead tothe identification of SAL1, which participates to maintain salttolerance (Quintero et al. 1996). Suppressor screen of the saltsensitive G19 yeast mutant with a rice full-length cDNAexpression library lead to the identification of Shaker familyK+ channel KAT1 (OsKAT1) gene, which participates inmaintaining cation homeostasis in rice during salt stress(Obata et al. 2007). Several Arabidopsis genes that couldenhance antioxidant defences and improve tolerance tooxidative stress were identified using Arabidopsis cDNAlibrary transfer to yeast and subsequent selection for diamide(Kushnir et al. 1995).

Transfer and expression of cDNA libraries in plants providea novel strategy to screen for gain-of-function phenotypes.An Arabidopsis cDNA library driven by the constitutiveCaMV35S promoter has been used to generate transgenicArabidopsis lines, showing altered developmental traits(LeClere and Bartel 2001). The Full-length cDNA Over-eXpressing gene hunting system (FOX) uses a normalised full-length cDNA collection under the control of the constitutiveCaMV35S promoter, and offers a technique to identify novel


gain-of-function phenotypes in Arabidopsis (Ichikawa et al.2006). A similar FOX-type cDNA overexpression system isavailable for rice (Nakamura et al. 2007). These cDNAlibraries were used in different genetic screens to identifyseveral important regulatory factors in hormonal and stresssignalling. Large-scale transformation of Arabidopsis rootswith a cDNA library lead to the identification of the ESR1gene, encoding a putative AP2/EREBP transcription factor,overexpression of which stimulates cytokinin-independentplant regeneration (Banno et al. 2001). By screening acollection of cDNA overexpressing plants for ABAindependent germination, Kuhn et al. (2006) identified theAtPP2AC gene, encoding an unknown protein phosphatase,which plays an important regulatory role in ABA-controlledclosure of stomata and thereby controls drought tolerance(Kuhn et al. 2006). A modified version of the FOX system hasbeen used to screen for salt tolerant phenotypes, leading to theidentification of several lines with enhanced tolerance to highsalinity. One of the identified lines overexpressed the bZIP-typetranscription factor AtbZIP60 which was subsequently shown tocontrol the expression of at least 29 Arabidopsis genes andimprove salt tolerance through modulating stress signaltransduction (Fujita et al. 2007).

The Controlled cDNA Overexpression System (COS) wascreated in our laboratory to overcome the problems causedby the constitutive gene activation. High level and constitutiveexpression of stress regulatory genes can disturb cell proliferationand development resulting in dwarf and sterile plants(Kasuga et al. 1999; Gilmour et al. 2000). The COS librarywas created in a Gateway version of the oestradiol-induciblepER8 plasmid, in which transcription of the inserted cDNAis strictly controlled by the addition of the inducer (Zuo et al.2000). The COS system was used to identify a set of cDNAsconferring dominant stress-tolerance phenotypes in threedifferent genetic screens: tolerance to salt stress, insensitivitytoABAand activation of the stress-inducedADH1-LUC reportergene (Papdi et al.2008). Screening forABA insensitivity resultedin the identification of 26 transgenic lines, which showedoestradiol-dependent germination and growth on culturemedium supplemented by inhibitory concentration of ABA(Fig. 3). Complementary DNA inserts were recovered andidentified in selected lines. We showed that overexpressionof HSP17.6 cDNA conferred ABA insensitivity whereasactivation of the 2-alkenal reductase 2AER resulted inimproved salt tolerance. The ADH1-LUC reporter gene wasused in combination with the COS activation to identify noveltranscriptional regulators of the ADH1 gene. Bioluminescencewas detected in several Arabidopsis lines when seedlings weretreated with oestradiol in the absence of any stress or hormoneeffect (Fig. 4). In line ADH121, oestradiol-dependent activationof the ERF/AP2-type transcription factor RAP2.12 sustainedhigh-level ADH1-LUC bioluminescence, and increased ADHenzyme activity in the roots (Papdi et al.2008). The cDNA librarytransformation approach usually creates dominant, gain-of-function phenotypes, but may also produce dominant loss-of-function phenotypes, which result from co-suppression ofendogenous genes by overexpression of truncated or antisensecDNAs (LeClere and Bartel 2001). An important advantageof the cDNA library transformation is that gene identification

in a selected line is extremely fast. A cDNA insert can beamplified by PCR from genomic DNA templates usingprimers that anneal to the vector T-DNA sequences flankingthe cDNA cloning site. The isolated cDNA is subsequentlysequenced and the identity of the insert is determined byhomology searches of sequence databases. In the COS systemeach cDNA was flanked by attB1 and attB2 recombination sites,therefore PCR fragments could easily be recloned in aGATEWAY entry vector and subsequently transferred intoany destination vector for recurrent transformation and othermolecular and biochemical analysis (Fig. 6; Papdi et al. 2008).

Complementary DNA library transformation is not restrictedto interspecific experimentation, but can also be used to transforma cDNA to a plant species different from the RNA source. In suchscenario cDNAs from any plant species, including naturalvariants of drought, salt or cold tolerant plant species can betransferred and tested in Arabidopsis or other model species.Ectopic expression of rice full length cDNA library (Rice FOX)in Arabidopsis offers a genetic resource to identify and analyserice genes in a heterologous system (Kondou et al. 2009).Overexpression of the rice cDNA library in Arabidopsis andsubsequent screen for thermotolerance lead to the identificationof the NAC type transcription factor ONAC063, which couldimprove tolerance to high temperature, salinity and osmoticstress (Yokotani et al. 2009). Overexpression of ONAC063 inArabidopsis upregulated several stress-responsive genes,suggesting that NAC-type transcription factors are importantfor stress defences and have conserved function in differentplant species. Complementary DNA expression libraries wererecently created from salt cres (Thellungiella halophylaC.A. Meyer.), a salt tolerant relative of Arabidopsis, and weresubsequently used to generate transgenic Arabidopsis lines.Screening for salt tolerance of transgenic Arabidopsisseedlings expressing Thellungiella cDNAs, resulted in theidentification of several lines showing enhanced salt tolerance(I. Pérez-Salamó and L. Szabados, unpubl. data; Fig. 5).Identification of several novel small protein coding genes,which could confer salt tolerance to Arabidopsis, confirmedthe potential of this approach (Du et al. 2008). Using atargeted interspecific gene transfer strategy, it is possible toidentify natural sequence variations in regulatory genes thatcan confer tolerance to different biotic or abiotic stresses.

Epigenetic regulation of stress responses

Gene silencing is an important regulatory mechanism thatcontrols gene activity at the transcriptional and posttranscriptional level (Baulcombe 2004). Post transcriptionalgene silencing is regulated by micro RNAs (miRNA) andsmall interfering RNA (siRNA), which are generated byenzymes of the Dicer family and processed with the RNA-induced silencing complex (RISC) (Tang 2005). siRNA of the24 nt size class control transcriptional gene silencing throughDNA and histone methylation (Matzke and Birchler 2005).Transcriptional gene silencing (TGS) has recently beenidentified as an important regulator of stress responses. Therepressor of silencing 1 and 3 (ros1 and ros3) mutants wereisolated by screening for suppressed activity of the RD29A-LUCreporter. In the ros1 mutant DNA hypermethylation in the


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promoter region of RD29A-LUC transgene and the endogenousRD29A gene lead to TGS. Map-based cloning of the ROS1 generevealed that it encoded a bifunctional glycosylase/lyase protein

which can repress transcriptional gene silencing bydemethylating the target promoter DNA (Gong et al. 2002b).Suppressor mutant screens of ros1 lead to the identification of

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Fig. 4. Screening for pADH1-LUC activation in Arabidopsis seedlings transformed by the COS cDNAlibrary. (a) Identification of an Arabidopsis seedling with bioluminescence imaging, which shows luciferaseactivity upon oestradiol spraying (indicated by an arrow). (b) Segregation of oestradiol-dependentluminescence in a T2 generation seedlings in a COS line. Twenty four out of 38 plants showluminescence, suggesting that activated bioluminescence in this line is dominant.

Fig. 5. Screening for salt tolerance in transgenicArabidopsis seedlings overexpressingThellungiella genes.Wild type Arabidopsis plants were transformed with a random cDNA library of Thellungiella halophyla,constructed in the inducible plant expression vector pER8-GW (Papdi et al. 2008). T1 generation seedlingswere screened for enhanced salt tolerance in the presence of the inducer (4mM estradiol). Seedlings withsuperior growth and survival (labelled with arrows) were rescued, and subsequently transferred to soil forfurther growth.


rpa2 and ago6–1mutants, on base of enhanced bioluminescenceof RD29A-LUC reporter gene. The ros1 ago6–1 double mutantshowed reduced DNA methylation and transcriptional genesilencing of RD29A (Zheng et al. 2007), while in theros1rpa2 double mutant silencing of RD29A-LUC was notaffected (Kapoor et al. 2005). The ros3 mutation causesconsiderable reduction of RD29A-LUC activity by promotingTGS through hypermethylation of the RD29A promoter regions.The ROS3 protein has RNA recognition motif (RRM) and canbind small RNA which may direct demethylation of targetpromoter sequences (Zheng et al. 2008). Genetic analysisshowed, that ROS1 and ROS3 act in the same pathway tocontrol DNA methylation and transcriptional gene silencing.Histone acetylation in chromatin is important to defineeukaryotic gene activity. Genetic screen to identify regulatorsofRD29A-LUC reporter gene lead to the identification ofHOS15,aWD40-type regulator of histone acetylation.HOS15was shown

to interact with and reduce histone H4 acetylation while analysisof the hos15mutant showed that thewild type gene is essential forcold tolerance (Zhu et al. 2008).

Recent studies show that gene silencing, mediated bymiRNAor siRNA can play an important role in controlling abiotic stressresponses. Stress-regulated miRNA and siRNA were identifiedin different plant species including Arabidopsis (Sunkar and Zhu2004; Zhou et al. 2008), rice (Zhao et al. 2007, 2009), maize(Zhang et al. 2008),Phaseolus (Arenas-Huertero et al. 2009) andPopulus spp. (Lu et al. 2005, 2008). miRNA-mediated genesilencing can control cold tolerance (Lu Sun et al. 2008; Zhouet al. 2008), phosphate starvation (Fujii et al. 2005; Bari et al.2006), heat stress (Lu et al. 2008), UV-B responses (Zhou et al.2007), drought and salt tolerance (Arenas-Huertero et al. 2009;Zhao et al. 2009), adaptation to anaerobic conditions (Zhanget al. 2008), and to mechanical stresses (Lu et al. 2008). TheArabidopsis CSD1 andCSD2 genes are encoding the detoxifying

Fig.6. Flowchart of theCOScDNAlibrary transformationandscreening strategies to identify stressregulatory functions in Arabidopsis.


superoxide dismutase (SOD) enzymes and were shown to beregulated by miR398, which repress the activity of CSD genes inthe absence of stress (Sunkar et al. 2006). Post transcriptionalgene silencingwas shown to control the activity of a NF-YA typetranscription factor which regulates the expression of numerousstress-related genes (Zhao et al. 2009). The involvement ofseveral class of microRNA in control of ABA-mediated stressresponses was recently confirmed in Arabidopsis and rice(Han et al. 2004; Jung and Kang 2007; Reyes and Chua 2007;Zhao et al. 2007), showing that miRNAs and epigeneticregulation is implicated in modulating ABA signalling.Endogenous siRNA was shown to control the expression ofP5C dehydrogenase gene (P5CDH), which regulates prolinedegradation in mitochondria, suggesting that small RNA canregulate metabolic responses to environmental stresses (Borsaniet al. 2005). Genetic screens for mutants deficient in phosphatenutrition or activation of the Pi starvation induced reporter gene(AtIPS1-GUS) lead to the identification of miR399, whichcontrols the activity of Pi-responsive genes and phosphatemetabolism (Fujii et al. 2005; Bari et al. 2006; Chiou et al.2006). Small RNA can therefore function as master regulators ofstress responses by silencing a well defined group of stress-responsive and defence genes. Stress-induced small RNAs canrepress the activity of target genes, while small RNAs which aredownregulated during stress may enhance the activity of a groupof stress-induced genes (Shukla et al. 2008).

Reverse genetics

In forward genetics, a mutant is isolated first on base of aphenotype, and the corresponding gene is assigned when themutated locus is identified either by map-based cloning or bymapping the insertion site. In reverse genetic approachesmutantsare identified or created for selected genes or gene families inorder to understand their function. Reverse genetics is a typicalanalytical tool for the post-genomic era, when information onwhole genome sequence is available and precise gene predictionsallow the systematic analysis of all genes in a genome.

Allele replacement by homologous recombination isconsidered as ideal tool for targeted modification of endogenousgenes (Mortensen 2006). Gene targeting with homologousrecombination is a method of choice to engineer mouse genes(Evans et al. 2001), but has limited success in plants. Lowfrequency of recombination into target sequences, too manyrandom integration events, non-homologous end joining, lack ofgene specific selection system prevent this technique fromwidespread use in higher plants (Puchta 2002; Hanin andPaszkowski 2003; Reiss 2003). Gene targeting via homologousrecombination have been used for engineering Arabidopsis plantsfor herbicide resistance (Hanin et al. 2001). Engineering of theacetolactate synthase (ALS) gene by introducing two amino acidchanges by gene targeting resulted in herbicide tolerant rice(Endo et al. 2007). Modification of the stress-induced ADH2gene by gene targeting have been reported in rice when strongpositive selection was applied (Terada et al. 2007; Johzuka-Hisatomi et al. 2008). However, despite sporadic success,genome modification by homologous recombination is not aroutine technology for higher plants, and further technologicalimprovements are needed for widespread use for reverse

genetics (Iida and Terada 2005; Johzuka-Hisatomi et al. 2008).Physcomitrella patens is a moss which exhibits high frequency ofhomologous recombination and ismore suitable for gene targetingthan higher plants (Schaefer and Zryd 1997; Kamisugi et al. 2005,2006). The haploid dominant stage of the plant life cycle facilitatesthephenotypiccharacterisationof theresultingmutants.Thusallelereplacement and directed mutagenesis is more accessible for thisspecies.Genome sequence ofPhyscomitrella is available (Rensinget al. 2008), making this plant an ideal model for reverse geneticapproaches. Targeted gene disruption in Physcomitrella involvesthe generation of transgenic lines that have replaced a specificgenomic locus by a targeting construct consisting of a selectioncassette flanked by two stretches of the genomic sequence. Theefficiency of allele replacement has been shown to be dependenton the length and the symmetry of the genomic sequencespresent in the construct (Kamisugi et al. 2005, 2006; Quatranoet al. 2007). When these parameters are taken into account, it ispossible to achieve levels of gene targeting comparable withthose shown in Saccharomyces cereviceae (Schaefer 2001).

Physcomitrella is also known for high degree of toleranceto different abiotic stresses such as high salinity, dehydrationand osmotic stress (Kroemer et al. 2004; Frank et al. 2005).This plant survives dehydration to water content as low as 2%(Oldenhof et al. 2006), and is able to rapidly recover fromprolonged periods of high salinity (Saavedra et al. 2006).Mosses often exhibit desiccation tolerance of vegetativetissues, in contrast with higher plants, in which this trait isusually restricted to reproductive structures such as pollen andseeds (Oliver 1996). Desiccation tolerant plants have protectivemechanisms that limit and repair stress-induced tissue damageand allow them to survive and recover from anhydrobiosis(Alpert and Oliver 2002). The functional analysis ofPhyscomitrella genes involved in stress responses may lead tothe identification of candidate genes for engineering stresstolerance in crops, as well as helping to address fundamentalquestions in plant biology, such as the function and conservationof plant molecular mechanisms involved in abiotic stresstolerance. In this context, the study of PP2C-dependentsignalling in Arabidopsis and Physcomitrella suggestedevolutionarily conserved ABA signalling pathways betweenmosses and higher plants (Komatsu et al. 2009). Knockoutmutants of the dehydrin gene were engineered by genetargeting and used to reveal the importance of the dehydrins inosmotic stress tolerance (Saavedra et al. 2006). As dehydrins areencoded by multigene families in higher plants, similargenetic strategies could not be implemented for the functionalanalysis of these genes in Arabidopsis or other plants.

The genome sequence of Arabidopsis has been available since2000 (The Arabidopsis Genome Initiative 2000b). Although genetargeting cannot be routinely used inArabidopsis, alternative toolsare available for reverse genetic analysis. Large-scale T-DNA andtransposon insertion mutagenesis programs lead to the applicationof reverse genetic strategies for different model species such asArabidopsis, rice andM. truncatula. One possibility is the creationof pooled genomic DNA templates from T-DNA and transposoninsertion mutant collections which serve as PCR templates toscreen for insertions in known genes (Martienssen 1998;Sussman et al. 2000; Mahalingam and Fedoroff 2001; Ríos et al.2002). Mutations are identified by PCR screening of the DNA


templates with combination of gene and T-DNA border specificprimers. A collection of 90 000 Arabidopsis lines (consisting of~116000 T-DNA inserts) was shown to be suitable for detectinga mutation in any gene with 77% probability (Ríos et al. 2002).Saturation mutagenesis and large-scale mapping of T-DNAinsertion sites on the genome allows the easy identificationof mutations for any genes. Insertions are localised throughinternet-based databases and mutant lines are obtained frompublic strain collections. Such resources are most abundantfor Arabidopsis, making it possible to identify mutations fornearly all of Arabidopsis genes (Samson et al. 2002; Szabadoset al. 2002; Alonso et al. 2003; Rosso et al. 2003). Among themonocotyledonous plants large collections of indexed T-DNA ortransposon insertion mutants have been established for rice(An et al. 2005; van Enckevort et al. 2005; Chern et al. 2007;Hsing et al. 2007; Piffanelli et al. 2007). The rice reverse geneticsdatabase OryGenesDB (http://orygenesdb.cirad.fr/index.html,verified 7 July 2009) has been established to identify flankingsequence tags (FSTs) for each rice genes and to support ricefunctional genomics programs (Droc et al. 2006, 2009). TheOryza Tag Line mutant database (http://urgi.versailles.inra.fr/OryzaTagLine/, verified 7 July 2009) contains phenotypic dataof30 000T-DNAenhancer trap lines,complementedbyGUS/GFPexpression data (Larmande et al. 2008). Such genetic resourcesallow the application of systematic reverse genetic screens toidentify mutants for specific traits, including stress responses(Alonso and Ecker 2006; Ülker et al. 2008).

Identification of putative stress responsive genes is possible byanalysis of global datasets on stress-dependent gene expression(Kant Gordon et al. 2008). Large scale transcript profilingexperiments showed, that expression of thousands ofArabidopsis genes is modified during different environmentalstresses, suggesting that these genes are involved in stressresponses (Chen et al. 2002; Seki et al. 2002; Oono et al.2003; Hannah et al. 2005; Vanderauwera et al. 2005; Gadjevet al. 2006; Robinson and Parkin 2008). Functional analysis ofstress-regulated Arabidopsis and rice genes is therefore possibleby reverse genetic approaches where identification ofmutants formost genes is feasible.

Asystematic useof a reverse genetic strategyhas recentlybeendescribed identifying and analysing a large set of Arabidopsisgenes, which regulate responses to multiple abiotic stresses.Multiple Stress Regulatory genes (MSTR) have been firstidentified by bioinformatics analysis of microarray data.Insertion mutants were subsequently identified for the MSTRgenes and their stress sensitivity and function in regulatingstress responses was analysed. Among the 16 identified MSTRgenes, the highest score was assigned to the Circadian ClockAssociated 1 (CCA1) transcription factor, which has a structuralhomologue, Late ElongatedHypocotyl (LHY), previously knownto be part of circadian oscillator (Schaffer et al. 1998; Wang andTobin 1998). Functional analysis of cca1 and lhymutants showedthat the double mutant has greater sensitivity to differentenvironmental stresses than single mutants or wild type plants,

Fig.7. Simplifieddepictionofgenetic strategiesused to identifyandcharacterise stressgenes inplants. Important steps are shown ineachprocedure, highlightingthe most critical, time-consuming or bottleneck step. Numerous modifications of the core technologies exist and adaptations to particular plants, conditions orpurposes have been published.


suggesting that the CCA1 and LHY genes control not onlycircadian clockbut also abiotic stress responses (Kant et al.2008).

Reverse genetic strategies can also be based on chemical orphysical mutagenesis as well as on natural variability. As analternative to sequence-indexed insertion collections, Targeting-induced local lesions in genomes (TILLING) has been adoptedin various plant species to identify point mutations and singlenucleotide polymorphism (SNP) in defined genes (Greene et al.2003; Till et al. 2003; Henikoff et al. 2004; Barkley and Wang2008). For TILLING, gene fragments from mutated plants areamplified with primers labelled by fluorescent dyes, and theproducts are denatured and annealed to form heteroduplexes.These molecules are in turn recognised and cleaved by the CelIendonuclease and the resulting DNA fragments are analysed ondenaturing polyacrylamide genes (Till et al. 2006b). A high-throughput TILLING program has been established for theArabidopsis community which can identify series of pointmutations in any Arabidopsis gene (Till et al. 2003, 2006a).As this technology does not use transgenic plants, it can be veryattractive to screen for point mutations in crop plants. TILLINGhas been used to identify mutants in maize (Henikoff et al. 2004;Till et al. 2004), melon (Cucumis melo L.; Nieto et al. 2007),mungbean (Vigna radiate L.; Barkley et al. 2008) and rice(Till Cooper et al. 2007; Suzuki et al. 2008) among otherspecies. Moreover, the TILLING approach has been used fordetection of natural variation in numerous plant species(ecotilling) (Comai et al. 2004; Henikoff et al. 2004; Gilchristet al. 2006). For example, ecotilling was employed for theidentification of allelic variants of melon elF4E factor, whichcontrols susceptibility to biotic stresses such as virus (Nieto et al.2007). TILLING has also been useful to identify mutant allelesin stress-related Arabidopsis genes. Using this approach,hot5 alleles were isolated and used to study heat acclimationin Arabidopsis (Lee et al. 2008). Similarly, swi3b mutantsshowed ABA insensitive germination and reduced expressionof ABA induced genes, suggesting that SWI3B in the wildtype plant functions as positive regulator of ABA signalling(Saez et al. 2008).

Reverse genetic strategies are particularly useful for theanalysis of multigenic families, where forward genetic screensdo not result in recognisable phenotypes due to functionalredundancy. Mutants can be identified for each member of agene family in sequence indexed insertion line collections or byTILLING. Double and multiple mutant lines can subsequentlybe generated by crossing the single mutants, and tested forphenotypic alterations. Numerous examples show, that doublemutants but not single mutants of a gene family can produce aphenotype. The SNF1-Related Protein Kinase 2 gene familyconsist of 10 member genes in Arabidopsis (Hrabak et al.2003), which are implicated in stress and ABA signaltransduction (Boudsocq et al. 2004). Single knockout mutantsof SnRK2.2 and SnRK2.3 genes had no recognisable phenotype,while the snrk2.2/snrk2.3 double mutant showed strong ABAinsensitivity. Altered seed dormancy, proline accumulation,water loss in detached leaves and reduced expression ofnumerous stress and ABA-induced genes in the double mutantsuggested that the SnRK2.2 and SnRK2.3 genes are majorregulators of ABA signalling and control drought tolerance(Fujii et al. 2007). Due to their functional redundancy, forward

screens for ABA insensitivity could not have recovered singlesnrk2.2 or snrk2.3 mutants.

Conclusion

Dissections of regulatory mechanisms controlling responses toenvironmental stresses or functional analysis of genes which areimplicated in defence mechanisms require genetic analysis.Variability can be generated by chemical, physical, insertionmutagenesis and gene transfer or derived from naturalpopulations. Identification of genetic variability which affectstress responses requires phenotypic screens which are able todistinguish between plants with differences in stress toleranceor reverse genetic technologies, which can identify mutants ingiven genes. All these strategies and technologies have certainadvantages and inconveniences, which have to be taken intoconsideration (Fig. 7).

Chemical and physical mutagenesis is easy and can generatelarge numbers of single nucleotide changes or deletions withminimal effort. Forward genetic screens require larger effortwhich ultimately leads to the isolation of either loss or gain offunction mutants, depending on the type of mutation. Majoreffort is required for the mapping and cloning of the genethat is affected by the mutation. Except for the Arabidopsismodel, map-based cloning is a challenging program formost plant species. Advanced microarray technologies andnew generation sequencing technologies can improve theefficiency of positional cloning in many species.

Natural variability offers a large resource of polymorphism,which is often explored to identify traits with adaptationvalue. Natural variability is often based on minor geneticchanges, generating small quantitative alterations in responsesto environmental conditions. However, QTL mapping andcloning is a time-consuming effort, and identification of thepolymorphism at molecular level is tedious.

Insertion mutagenesis facilitates easy identification oftagged mutants through PCR amplification and mapping ofinsertion sites. Insertion mutagenesis is a real alternative inplants which have suitable transposable elements or efficienttransformation systems. The generation of tens of thousands ofT-DNA insertions requires large-scale genetic transformation,which is feasible for Arabidopsis but is a bottleneck for mostplant species, where in planta transformation methods arenot available. Endogenous transposons or the maize Ac/Dssystem offer an alternative for insertion mutagenesis. Insertionmutagenesis most often generates loss-of function mutants, withthe exception of activation tagging, which can generate gain-offunction phenotypes through the ectopic activation of the genesthat are flanking the insertion element.

Variability can be generated by random cDNAtransformation, which creates mainly gain-of functionphenotypes based on ectopic (or controlled) expression. Wheninterspecific cDNA transfer is used, genetic polymorphism ofspecific plant species can be explored to identify genes withadaptive values. Creation of the cDNA library is critical step,while establishment of the transformed plant population is a time-consuming procedure, making this approach generally lessattractive. On the other hand, gene identification and genecloning from selected plants is easy and straight forward, as


the inserts are flanked by known vector sequences, facilitatingPCR amplification of the cDNA.

Variability for known genes can be identified by reversegenetic approaches. Gene targeting through homologousrecombination is routinely available for Physcomitrella but notfor higher plants. Nevertheless, several reverse genetictechnologies are available for several species using traditionalor insertionmutagenesis. Searching the sequence-tagged T-DNAdatabases is the most common procedure to find Arabidopsismutants. Although services of a similar scale are not availablefor other plants, smaller collections have been established forsome species. TILLING, identification of InDels and insertionsby PCR offer an opportunity to identify mutants in numerousplants, including crops. Most of these strategies rely on theestablishment of large, characterised mutant collections whichrequires manpower and extensive funding. Once the mutantplatforms (T-DNA insertion database, TILLING collection)exist, mutant identification is easy and fast.

Research inplant stress biologyhas used all these technologiesextensively and numerous examples are available from bothmodel and crop species. Genetic analysis has become anessential part of the functional characterisation of genescontrolling stress responses and made it possible to dissectcomplex regulatory pathways. The genetic strategies reviewedhave been used and will be used to identify and characterisemaster genes which can improve tolerance of crop plants toharmful environmental conditions such as salinity, drought orextreme temperatures (see Table S1).

Acknowledgements

We are indebted to Paul Lazzeri, Csaba Koncz and László Kozma Bognár forcareful reading and corrections. This work was supported by OTKA grantno. T-46552 and K-68226, EU grant no. FP6–020232–2, ICGEB-TWASProgram CRP.PB/URU06–01. Csaba Papdi and Mary Prathiba Joseph weresupported by fellowships of the Biological Research Centre.

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Manuscript received 26 February 2009, accepted 11 June 2009


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