Overexpression of ß-Ketoacyl Co-A Synthase1 Gene Improves Tolerance of Drought Susceptible Groundnut (Arachis hypogaea L.) Cultivar K-6 by Increased Leaf Epicuticular Wax Accumulation

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)

A Novel WRKY Transcription Factor, MuWRKY3 (Macrotyloma uniflorum Lam. Verdc.) Enhances Drought Stress Tolerance in Transgenic Groundnut (Arachis hypogaea L.) Plants

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

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<p>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.</p