Overexpression of JcWRKY2 confers increased resistance towards Macrophomina phaseolina in transgenic tobacco.

WRKY proteins are plant-specific transcription factors (TFs), and form one of the largest families and are involved in plant development and responses to stress. The salicylic acid (SA) responsive WRKY family auto or cross-regulate the defence stress signalling pathways. In this study, we functionally validated the role of JcWRKY2 gene from biofuel crop Jatropha curcas towards improving resistance to tobacco transgenic against charcoal rot causing necrotrophic fungus, Macrophomina phaseolina. The microscopic studies revealed that JcWRKY2 participated in preventing the spread of infection in transgenic. The generation of H2O2 during M. phaseolina and combinatorial stress in transgenic induces the expression and activity of antioxidant enzymes. The transcript expression of SA biosynthetic (NtICS1) gene, pathogenesis-related (NtPR-10) gene and antioxidative enzymes (NtCAT1 and NtSOD) gene revealed that JcWRKY2 transgenic play a role in SA-mediated, antioxidative enzymes regulation during biotic challenges. The study highlights the potential of JcWRKY2 as an important regulator for plant biotic stress responses through the SA-dependent pathway.

Functional Validation of JcWRKY2, a Group III Transcription Factor Toward Mitigating Salinity Stress in Transgenic Tobacco.

The plants being sessile cannot escape from the adverse environmental stresses, hence get negatively affected in terms of their growth and yield. Transcriptional control simultaneously regulate different cellular processes, minimizing the deleterious effects of these stresses. The salicylic acid (SA)-inducible WRKY family of transcription factors auto or crossregulate the stress signaling in response to abiotic and biotic stresses, facilitating enhanced stress tolerance. In this study, we characterized the group III WRKY gene, JcWRKY2 from ecological and economical valued shrub Jatropha curcas. The JcWRKY2 tobacco transgenics showed improved physiological growth parameters, elevated chlorophyll content, improved antioxidative activities, and increased endogenous SA with both salt and SA stress. Interestingly, the pretreatment with SA and hydrogen peroxide facilitated improved germination of transgenic seeds with salinity stress. The transgenics showed differential regulation of antioxidative enzymes, calcium/calmodulin, dehydrins, and phospholipase genes with salt and SA stress. The increased SA content in transgenics on stress treatments, enhanced the antioxidant capacity leading to reduced susceptibility to stresses. Thus, JcWRKY2 transgenics participate in SA-mediated, improved antioxidative status during salinity stress with reduced reactive oxygen species damage.

Ectopic Expression of JcWRKY Transcription Factor Confers Salinity Tolerance via Salicylic Acid Signaling.

Plants, being sessile, have developed intricate signaling network to specifically respond to the diverse environmental stress. The plant-specific WRKY TFs form one of the largest TF family and are involved in diverse plant processes, involving growth, development and stress signaling through auto and cross regulation with different genes and TFs. Here, we report the functional characterization of a salicylic acid -inducible JcWRKY TF. The JcWRKY overexpression confers salinity tolerance in transgenic tobacco, as was evident by increased chlorophyll content and seed germination potential. The transgenic plants showed increased soluble sugar, membrane stability, reduced electrolyte leakage and generation of reactive oxygen species (H2O2 and [Formula: see text]) as compared to the wild type. Furthermore, the low SA treatment along with salinity improved the tolerance potential of the transgenics by maintaining ROS homeostasis and high K+/Na+ ratio. The transcript expression of SA biosynthetic gene ICS1 and antioxidative enzymes (CAT and SOD) showed upregulation during stress. Thus, the present study reflects that JcWRKY is working in co-ordination with SA signaling to orchestrate the different biochemical and molecular pathways to maneuvre salt stress tolerance of the transgenic plants.

Improved Shoot Regeneration, Salinity Tolerance and Reduced Fungal Susceptibility in Transgenic Tobacco Constitutively Expressing PR-10a Gene.

Plants in ecosystems are simultaneously exposed to abiotic and biotic stresses, which restrict plant growth and development. The complex responses to these stresses are largely regulated by plant hormones, which in turn, orchestrate the different biochemical and molecular pathways to maneuver stress tolerance. The PR-10 protein family is reported to be involved in defense regulation, stress response and plant growth and development. The JcPR-10a overexpression resulted in increased number of shoot buds in tobacco (Nicotiana tabacum), which could be due to high cytokinin to auxin ratio in the transgenics. The docking analysis shows the binding of three BAP molecules at the active sites of JcPR-10a protein. JcPR-10a transgenics showed enhanced salt tolerance, as was evident by increased germination rate, shoot and root length, relative water content, proline, soluble sugar and amino acid content under salinity. Interestingly, the transgenics also showed enhanced endogenous cytokinin level as compared to WT, which, further increased with salinity. Exposure of gradual salinity resulted in increased stomatal conductance, water use efficiency, photosynthesis rate and reduced transpiration rate. Furthermore, the transgenics also showed enhanced resistance against Macrophomina fungus. Thus, JcPR-10a might be working in co-ordination with cytokinin signaling in mitigating the stress induced damage by regulating different stress signaling pathways, leading to enhanced stress tolerance.

Molecular cloning and characterization of a group II WRKY transcription factor from Jatropha curcas, an important biofuel crop.

The WRKY family of transcription factors (TFs) play an intricate role in regulating the stress signaling pathways by autoregulation or may be by cross regulation through interaction with other proteins. Although WRKY TFs are considered to be plant specific, however, their presence has been reported from unicellular algae, slime mould, and gymnosperms. We have isolated the JcWRKY cDNA from an important biofuel crop Jatropha curcas growing in the wastelands of India. The JcWRKY gene has an ORF of 693 bp and encodes a 230 amino acids protein with estimated molecular mass of 25.25 kDa. JcWRKY shows close homology to FaWRKY1 and St-WRKY1. The JcWRKY contains seven potential phosphorylation sites, which might be involved in regulating its function. The transcript analysis revealed that the JcWRKY transcript gets upregulated in response to salinity, dehydration, salicylic acid (SA), methyl jasmonate (MeJa), and collar rot fungus Macrophomina. However, maximum expression is observed under SA, highlighting its role in enhancing systemic acquired resistance for disease tolerance. The JcWRKY recombinant protein showed binding to W-box of pathogenesis related-1 (PR-1) and iso1 (encoding isoamylase1) promoters. Overexpression of JcWRKY in Escherichia coli enhanced the growth of cells in NaCl, KCl, mannitol, sorbitol, SA, and MeJa treatments, indicating that it protects and promotes growth under ionic, osmotic, and chemical stresses. The enhancement in growth can be due to the regulation of stress responsive genes. Therefore, it can be used as an important gene for enhancing abiotic and biotic resistance in plants and to facilitate faster growth of E. coli cells under stress conditions for efficient expression.