Expression of B. subtilis Phytase gene driven by fruit specific E8 promoter for enhanced minerals, metabolites and phytonutrient in cucumber fruit.

The fruit nutrigenomics is an interesting and important research area towards nutrition enhancement. The phytic acid is one of the major antinutrient compound, present in seeded fruits and crops. It hinders the absorption of iron (Fe), zinc (Zn), magnesium (Mg), potassium (K) and calcium (Ca), causing mineral deficiencies. In the present study, the BsPhy gene was overexpressed in the cucumber fruits using the tomato fruit specific E8 and constitutive CaMV 35S promoter. The E8 promoter imparted heterologous expression of GUS gene in cucumber fruits, furthermore, the fruit specific expression of E8 promoter with BsPhy gene was confirmed in transgenics (E8::BsPhy) using anti rabbit-phytase antibody. The physio-biochemical analysis of transgenics revealed, maximum phytase activity in E8::BsPhy cucumber fruits at 10 days after anthesis (DAA) compared to 35S::BsPhy and wild-type (WT) fruits. Consequently, E8::BsPhy fruits also showed increased amount of inorganic phosphorus (Pi), total phosphorus (P), minerals (Zn, Fe, Mg, K, Ca), total carotenoid and other macronutrients at 10 DAA compared to 35S::BsPhy fruits. The metabolite profiling of fruits (10 DAA) showed increased sugars, amino acids, sugar acids and polyols, in both E8::BsPhy and 35S::BsPhy transgenics suggesting higher phytate metabolism, compared to WT fruits. Interestingly, both the transgenic fruits showed higher fruit biomass and yield along with improved nutritional quality, which can be attributed to increased P and Zn contents in transgenic fruits, compared to WT fruits. Our findings reveal that the BsPhy gene enhances minerals and macronutrients in transgenic cucumber fruits making it nutritious and healthy.

The AlRabring7 E3-Ub-ligase mediates AlRab7 ubiquitination and improves ionic and oxidative stress tolerance in Saccharomyces cerevisiae.

The maintenance of ROS homeostasis, membrane biogenesis and recycling of molecules are common stress responses involving specific and complex regulatory network. Ubiquitination is an important and common mechanism which facilitates environmental adaptation in eukaryotes. In the present study we have cloned the AlRabring7, an E3-Ub-ligase, previously identified as AlRab7 interacting partner. The role of AlRabring7 for ubiquitinating AlRab7 and facilitating stress tolerance is analysed. The AlRabring7, with an open-reading frame of 702 bp encodes a protein of 233 amino acids, with RING-HC domain of 40 amino acids. In silico analysis shows that AlRabring7 is a C3HC4-type RING E3 Ub ligase. The protein - protein docking show interaction dynamics between AlRab7-AlRabring7-Ubiquitin proteins. The AlRab7 and AlRabring7 transcript showed up-regulation in response to different salts i.e: NaCl, KCl, CaCl2, NaCl + KCl, NaCl + CaCl2, imposing ionic as well as hyperosmotic stress, and also with oxidative stress by H2O2 treatment. Interestingly, the AlRabring7 showed early transcript expression with maximum expression in shoots on combinatorial stresses. The AlRab7 showed delayed and maximum expression with NaCl + CaCl2 stress treatment. The AlRab7 complements yeast ypt7Δ mutants and restored the fragmented vacuole. The in vitro ubiquitination assay revealed that AlRabring7 function as E3 ubiquitin ligase and mediates AlRab7 ubiquitination. Overexpression of AlRab7 and AlRabring7 independently and when co-transformed enhanced the growth of yeast cells during stress conditions. Further, the bimolecular fluorescence complementation assay shows the in planta interaction of the two proteins. Our results suggest that AlRab7 and AlRabring7 confers enhanced stress tolerance in yeast.

SbMYB15 transcription factor mitigates cadmium and nickel stress in transgenic tobacco by limiting uptake and modulating antioxidative defence system.

Plants require different inorganic minerals in an appropriate amount for growth; however, imbalance can limit growth and productivity. Heavy metal accumulation causes toxicity and generates signalling crosstalk with reactive oxygen species (ROS), phytohormones, genes and transcription factors (TFs). The MYB (myeloblastoma) TFs participate in plant processes such as metabolism, development, cell fate, hormone pathways and responses to stresses. This is the first report towards characterisation of R2R3-type MYB TF, SbMYB15, from succulent halophyte Salicornia brachiata Roxb. for heavy metal tolerance. The SbMYB15 showed >5-fold increased transcript expression in the presence of CdCl2 and NiCl2•6H2O. The constitutive overexpression of SbMYB15 conferred cadmium and nickel tolerance in transgenic tobacco, with improved growth and chlorophyll content. Further, the transgenics showed reduced generation of reactive oxygen species (H2O2 and O2•-) as compared with the wild-type (WT) with both Cd2+ and Ni2+ stress. Transgenics also showed low uptake of heavy metal ions, increased scavenging activity of the antioxidative enzymes (CAT and SOD) and higher transcript expression of antioxidative genes (CAT1 and MnSOD). Thus, the present study signifies that SbMYB15 can be deployed for developing heavy metal tolerance in crop plants via genetic engineering.

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.