Drought stress has adverse effects on growth, water relations, photosynthesis and yield of groundnut. WRKY transcription factors (TFs) are the plant-specific TFs which regulate several down-stream stress-responsive genes and play an essential role in plant biotic and abiotic stress responses. We found that WRKY3 gene is highly up-regulated under drought stress conditions and therefore isolated a new WRKY3TF gene from a drought-adapted horsegram (Macrotyloma uniflorum Lam. Verdc.). Conserved domain studies revealed that protein encoded by this gene contains highly conserved regions of two WRKY domains and two C2H2 zinc-finger motifs. The fusion protein localization studies of transient MuWRKY3-YFP revealed its nuclear localization. Overexpression of MuWRKY3 TF gene in groundnut (Arachis hypogaea L.) showed increased tolerance to drought stress compared to wild-type (WT) plants. MuWRKY3 groundnut transgenics displayed lesser and delayed wilting symptoms than WT plants after 10-days of drought stress imposition. The transgenic groundnut plants expressing MuWRKY3 showed less accumulation of malondialdehyde, hydrogen peroxide (H2O2), and superoxide anion (O2∙-), accompanied by more free proline, total soluble sugar content, and activities of antioxidant enzymes than WT plants under drought stress. Moreover, a series of stress-related LEA, HSP, MIPS, APX, SOD, and CAT genes found up-regulated in the transgenic groundnut plants. The study demonstrates that nuclear-localized MuWRKY3 TF regulates the expression of stress-responsive genes and the activity of ROS scavenging enzymes which results in improved drought tolerance in groundnut. We conclude that MuWRKY3 may serve as a new putative candidate gene for the improvement of stress resistance in plants.

The NAC family being the largest plant-specific transcription factors functions in diverse and vital physiological processes during development. NAC proteins are known to be crucial in imparting tolerance to plants against abiotic stresses, such as drought and salinity, but the functions of most of them are still elusive. In this study, we report for the first time expression of the MuNAC4, a member of NAC transcription factor from horsegram (Macrotyloma uniflorum) conferring drought tolerance. The groundnut (Arachis hypogaea) transgenics were generated using recombinant MuNAC4 binary vector transformation approach. Molecular analysis of these transgenic lines confirmed the stable gene integration and expression of the MuNAC4 gene. Twelve lines of T5 generation exhibited significantly enhanced tolerance to drought stress with proliferated lateral root growth as compared to wild types. Transgenics exposed to long-term desiccation stress assays showed increased lateral roots and greenish growth. The physiological parameters analysis also suggests that overexpression of MuNAC4 plays a significant role in improving the water stress tolerance of transgenic groundnut, reducing the damage to membrane structures and enhancing osmotic adjustment and antioxidative enzyme regulation under stress. This study validates MuNAC4 as an important candidate gene for future phytoengineering approaches for drought tolerance in crop plants.

Drought is one of the major environmental constraints affecting the crop productivity worldwide. One of the agricultural challenges today is to develop plants with minimized water utilization and reduced water loss in adverse environmental conditions. Epicuticular waxes play a major role in minimizing water loss. Epicuticular wax covers aerial plant parts and also prevents non-stomatal water loss by forming the outermost barrier from the surfaces. Epicuticular wax content (EWC) variation was found to be affiliated with drought tolerance of groundnut cultivars. In the current study, a fatty acid elongase gene, KCS1, which catalyzes a rate limiting step in the epicuticular wax biosynthesis was isolated from drought tolerant cultivar K-9 and overexpressed in drought sensitive groundnut cultivar (K-6) under the control of CaMV35S constitutive promoter. Transgenic groundnut plants overexpressing AhKCS1 exhibited normal growth and displaying greenish dark shiny appearance. Environmental scanning electron microscopy (ESEM) revealed the excess of epicuticular wax crystal depositions on the transgenic plant leaves compared to non-transgenic wild type plants. The findings were further supported by gas chromotography–mass spectroscopic analysis (GC-MS) that revealed enhanced levels of fatty acids, secondary alcohols, primary alcohols, aldehydes, alkanes, and ketones in transgenics compared to wild types. The AhKCS1 overexpressing transgenic groundnut plants exhibited increase in the cuticular wax content, reduction of water loss, lower membrane damage, decreased MDA content, and high proline content compared to that of non-transgenic groundnut plants. Our findings suggest that the AhKCS1 gene plays a major role in combating drought stress by preventing non-stomatal water loss in drought sensitive groundnut cultivar (K-6).

Polyamines can regulate the expression of antioxidant enzymes and impart plants tolerance to abiotic stresses. A comparative analysis of polyamines, their biosynthetic enzymes at kinetic and at transcriptional level, and their role in regulating the induction of antioxidant defense enzymes under salt stress condition in two foxtail millet (Setaria italica L.) cultivars, namely Prasad, a salt-tolerant, and Lepakshi, a salt-sensitive cultivar was conducted. Salt stress resulted in elevation of free polyamines due to increase in the activity of spermidine synthase and S-adenosyl methionine decarboxylase enzymes in cultivar Prasad compared to cultivar Lepakshi under different levels of NaCl stress. These enzyme activities were further confirmed at the transcript level via qRT-PCR analysis. The cultivar Prasad showed a greater decrease in diamine oxidase and polyamine oxidase activity, which results in the accumulation of polyamine pools over cultivar Lepakshi. Generation of free radicals, such as O 2 (·-) and H2O2, was also analyzed quantitatively. A significant increase in O 2 (·-) and H2O2 in the cultivar Lepakshi compared with cultivar Prasad was recorded in overall pool sizes. Further, histochemical staining showed lesser accumulation of O 2 (·-) and of H2O2 in the leaves of cultivar Prasad than cultivar Lepakshi. Our results also suggest the ability of polyamine oxidation in regulating the induction of antioxidative defense enzymes, which involve in the elimination of toxic levels of O 2 (·-) and H2O2, such as Mn-superoxide dismutase, catalase and ascorbate peroxidase. The contribution of polyamines in modulating antioxidative defense mechanism in NaCl stress tolerance is discussed.

Drought and salt stress are two major environmental constraints that limit the productivity of agriculture crops worldwide. WRKY transcription factors are the plantspecific transcription factors that regulate several developmental events and stress responses in plants. The WRKY domain is defined by a 60-amino acid conserved sequence named WRKYGQK at N-terminal and a Zinc Finger-like motif at the Cterminal. WRKY genes are known to respond several stresses which may act as negative or positive regulators. The function of most of the WRKY transcription factors from non-model plants remains poorly understood. This investigation shows the expression levels of eight WRKY transcription factor genes from horsegram plant under drought and salt stress conditions. The increase in mRNA transcript levels of WRKY transcription factor genes was found to be high in drought stressed plants compared to salt-stressed plants. The levels of MDA which indicates the lipid peroxidation were less in drought stress. More ROS is produced in salt stress conditions compared to drought. The results show that the expression of WRKY transcription factors in drought stress conditions is reducing the adverse effect of stress on plants. These results also suggest that, during abiotic stress conditions such as drought and salt stress, WRKY transcription factors are regulated at the transcription level.