"SummaryPlants in nature are frequently exposed to a plethora of stress conditions such aslow temperature, salt, drought, wounding, heat, heavy metal toxicity, and inadequateminerals, result in crop failure. Drought is one of the major abiotic stresses that arereducing average yields of crop plants by more than 50%. Groundnut is a rainfed cropand its productivity is highly vulnerable to rainfall deviations and displays hugefluctuation between years constrained by moisture stress in most of the cultivated areas.The crops grown under rain fed conditions (moisture stress), besides water mining,intrinsic cellular level tolerance (CLT) mechanisms to sustain the cell metabolism isimportant. Even with higher water mining abilities under drought plants experiencedecreased water status hence improving CLT mechanisms is important.Severalupstream regulatory genes (TFs) have identified that regulate many transcriptionalprocesses and involved in many mechanisms which play an important roles in plantadaptation to stress. There are considerable evidences to show that manipulation of TFscan bring about tolerance at cellular level under stressful condition. Therefore, targetedmanipulation of specific TFs involved in stress tolerance is considered as a relevantapproach for crop improvement towards stress tolerance. Among the several stressresponsive TFs, WRKY TFs compose one of the largest families of plant-specificregulatory proteins which are known to involve in various developmental events and111 Summarytheir major function appears to help the plant to cope with various stresses.WRKY TFsare studied in plants response to biotic stresses but limited studies have been attemptedin response to abiotic stress. In view of this, attempts have been made to improvecellular tolerance in plants by over expressing WRKY TFs.The major objective of the study is to identify the drought stress responsiveWRKY genes from a dry land crop (horsegram) and to study its functional relevance inground nut by transgenic approach in response to drought stress. In order to achieve this objective, initially expression of eight horsegramWRKY TF genes (WRKY3, WRKY12, WRKY 33, WRKY 57, WRKY 53, WRKY 65,and WRKY75) were studied in response to drought stress by RTPCR analysis. Amongeight WRKY TFs studied, WRKY3 showed significantly high level of expression inhorsegram under drought stress conditions and could be considered as a putativecandidate gene to improve drought stress tolerance in relatively stress adaptive cropslike groundnut.Cloning and analysis of WRKY3 TF Full length clone of WRKY3 TF gene was amplified from the horsegram,MuWRKY3 sequence was submitted to NCBI database (#KM520390.1). The in silicoanalysis of MuWRKY3 revealed that it belongs to WRKY TF family and showed thesimilarity with GmWRKY. Multiple alignment of MuWRKY3 gene with other WRKY3genes indicate that the homology of MuWRKY3 and those WRKY3 proteins from otherplant species are significantly difference except at the WRKY conserved region.Phylogenetic tree analysis has revealed that MuWRKY3 and GmWRKY3 belong to thesame category. The conserved domain search of MuWRKY3 amino acids sequence hasshown the presence of two WRKY domains and belongs to group I of the WRKY TFsuperfamily. 112 SummaryGateway cloning and subcellular localizationThe gateway cloning strategy provided an option to develop the binary constructfor subcellular localization studies in model system tobacco. The sub-cellularlocalization study of MuWRKY3 along with yellow floroscent protein (35S:MuWRKY3::YFP) in N. benthamianarevealed that MuWRKY3 was nuclear-localizedprotein serving as a TF.MuWRKY3 plant expression vector constructionThe MuWRKY3 gene was successfully cloned under 35SCaMV promoter and apoly A terminator. The whole cassette was sub cloned to pCAMBIA2301. The binaryvector expressing 35SCaMV: MuWRKY3 transferred to the Agrobacterium EHA105 forplant transformation.Development of transgenic groundnut plants Agrobacterium-mediated in planta transformation The groundnut grown under rain fed conditions is affected by drought stress andhence improving the cellular level tolerance mechanism to cope up with drought stressand it is crucial to realize the potential yields. In view of this developing droughttolerant groundnut transgenics has phenomenal significance. Therefore drought tolerantgroundnut transgenics was developed to adopted rainfed conditions.Few groundnutgenotypes are less amenable to gene transformation using in vitro techniques and it isvery essential to have an efficient standardized protocol for gene transformation. As weselected groundnut K6 cultivar for transgenic development, since non-availability ofefficient transformation protocol, we standardized a genotype and tissue cultureindependent in planta transformation method to develop groundnut transgenics. Sincethe primary putative transformants (T ) being chimeric in nature. The analysis of0 transgenic plants was carried out in the T generation. After acclimatization, healthy1 113 Summaryplantlets are transferred to earthen pots containing soil mix, (soil and manure in 3:1ratio) and allowed to grow in a greenhouse. The putative transformants in T generation are identified by screening for1 kanamycin tolerance. Molecular analysis by PCR for MuWRKY3, GUS and NptIIconfirmed the integration of gene. Putative PCR positives lines from T1 generationwere further advanced to successive generations based on the stability of the integratedgene and stress response.Molecular characterization of transgenic groundnut plantsTransgene integration and expression in transgenic groundnut plants werechecked at the molecular level. The stable integration of gene in transgenic plants wasanalyzed by amplification of genomic DNA with gene specific, GUS and NtpII specificprimers. Southern blot analysis of transgenic groundnut plants The stable integration and copy numbers of the groundnut transgenics wereassessed using southern blot analysis in T generation. Three out of five transgenic3 groundnut lines showed the stable integration with single copy insertion in one of thelines. Characterization of T groundnut transgenic plants for drought tolerance 3 Osmolyte accumulation in transgenic plantsOsmolytes accumulation can increase the survival rate of the plant duringdrought stress. Accumulation of proline and total soluble sugars were measured inplants after severe drought stress treatment. High proline and soluble sugar content intransgenic lines suggest that MuWRKY3 can participate in osmolyte accumulationthereby increasing the tolerance to drought stress.114 SummaryEffect of drought on ROS production in transgenic plants?- Reactive oxygen species like Superoxide anion (O ) and Hydrogen peroxide2 (H O ) are produced as a result of stress which leads to oxidative damage. The2 2 ?- production of O and H O ions under drought stress was high in wild type compared to2 2 2transgenic plants.In situ histochemical localization has shown the production of ROS in?- wild-type plants as detected by the more intense development of blue (O ) and brown2 color spots (H O ) compared to that of transgenic lines. 2 2 Enzymatic antioxidants and lipid peroxidation in transgenic plantsThe SOD activity was determined by measuring its ability to inhibit thephotochemical reduction of NBT. And the activity of APX was recorded as the amountof ascorbate oxidized. The production of antioxidative enzymes like SOD and APXwere relatively high in transgenic lines when compared to wild type under droughtstress conditions. The levels of lipid peroxidation was determined as 2-thiobarbituric acid reactivesubstances (TBARS) in groundnut during drought stress. The concentration of MDAwas lesser in transgenic lines when compared to wild type, indicating the less lipidperoxidation. qRT-PCR analysis of wild type and transgenic groundnut plantsThe qRT-PCR analysis was performed with gene specific primers at differenttime points 24h, 48h and 72hto check the expression level of WRKY3 TF gene underdrought stress conditions in wild-type and transgenic plants. The increase in thetranscript levels of MuWRKY3 gene was found to be more in the transgenic plant whencompared to wild type under drought stress. Among three time points studied theexpression level MuWRKY3 was found to be high after 72hr of stress imposition. 115 SummaryUpregulation of stress-induced gene like SOD, APX, CAT, MIPS, LEA andHSP, indicates that increase in the level MuWRKY3 under drought stress, in turn,increasing the expression of other stress responsive genes. Conclusions? A stress-responsive novel MuWRKY3 TF gene was isolated from horsegram, adrought-tolerant dryland crop.? The nucleotide sequence of full lengthMuWRKY3 TF gene(Genbank #KM520390) contained a complete open reading frame of 1476 bp and encodes aprotein with 491 amino acids having MW 53.7 kDa and pI of 6.12.? Sub-cellular localization assay has revealed the presence of MuWRKY3 protein inthe nucleus.? A binary vector pCAMBIA2301expressing 35S:MuWRKY3::PolyA was constructedfor plant transformation. Transgenic groundnut plants expressing35S:MuWRKY3::PolyA were generated by following a standardizedAgrobacterium-mediated in planta transformation. ? T1 generation plants are screened for the kanamycin resistance and putativetransformants were advanced for next generation.?PCR analysis was taken as the first line of proof for the detection of putativetransformants and Gus histochemical study had revealed the expression of thetransgene in the transgenic plants and the southern blotting analysis using biotinlabeled GUS gene confirmed the stable integration of the transgene.?Physiological and qRT-PCR analysis has revealed that expression of MuWRKY3gene in transgenic plants has improved the drought tolerance capacity.116 Summary? The groundnut transgenic plants expressing MuWRKY3 showed enhanced droughttolerance by up-regulation of stress-responsive genes like SOD, APX, CAT, MIPS,LEA, and HSP, better antioxidant efficacy and higher cellular tolerance.? In this study, the overexpression of MuWRKY3 transcription factor imparted higheradaptability to drought stress in the transgenic groundnut plants as revealed byseveral physiological and molecular assays. In conclusion, our results clearly demonstrated that MuWRKY3 is a stress- inducible transcription factor, and the MuWRKY3 was up-regulated in horsegram bydrought, dehydration, salt and heat stresses. MuWRKY3 could enhance drought stresstolerance in transgenic groundnut plants. These functions are achieved by maintainingbetter relative water content, higher proline and soluble sugar contents, and bysignificantly reducing plant ROS and MDA contents in transgenic groundnut plants.MuWRKY3confers drought stress tolerance by regulating the expression of stressrelated genes (such as SOD, APX, CAT, MIPS, HSP and LEA) thus protecting plantsfrom stress caused damage. However, further studies are needed to demonstratewhether these upregulated stress responsive genes are direct targets of MuWRKY3 andexploring the specific and direct genes regulated by MuWRKY3, together with itsinteracting partners will reveal the exact mechanisms of plant responses to abioticstresses. Therefore, MuWRKY3 could be a possible candidate gene for use in geneticengineering approaches to confer stress tolerance in crop plants for sustained growthand productivity under drought conditions. 117 "