Comparative transcriptomic analysis and antioxidant defense mechanisms in clusterbean (Cyamopsis tetragonoloba (L.) Taub.) genotypes with contrasting drought tolerance.

To understand drought tolerance mechanism(s) in clusterbean (Cyamopsis tetragonoloba), we conducted physiological, biochemical, and de novo comparative transcriptome analysis of drought-tolerant (RGC-1002) and drought-sensitive (RGC-1066) genotypes subjected to 30 days of drought stress. Relative water content (RWC) was maintained in tolerant genotype but was reduced in sensitive genotype. Leaf pigment concentrations were higher in tolerant genotype. Net photosynthesis was significantly decreased in sensitive genotype but insignificant reduction was found in tolerant genotype. Enzymatic antioxidant (GR, APX, DHAR) activities were enhanced in tolerant genotype, while there were insignificant changes in these enzymes in sensitive genotype. The ratios of antioxidant molecules (ASC/DHA and GSH/GSSG) were higher in tolerant genotype as compared to sensitive genotype. In sensitive genotype, 6625 differentially expressed genes (DEGs) were upregulated and 5365 genes were downregulated. In tolerant genotype, 5206 genes were upregulated and 2793 genes were downregulated. In tolerant genotype, transketolase family protein, phosphoenolpyruvate carboxylase 3, temperature-induced lipocalin, and cytochrome oxidase were highly upregulated. Moreover, according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the drought tolerance may be attributed to upregulated starch and sucrose metabolism-related genes in tolerant genotype. Finally, quantitative real-time PCR confirmed the reproducibility of the RNA-seq data.

Chickpea glutaredoxin (CaGrx) gene mitigates drought and salinity stress by modulating the physiological performance and antioxidant defense mechanisms.

Glutaredoxins (Grxs) are short, cysteine-rich glutathione (GSH)-mediated oxidoreductases. In this study, a chickpea (Cicer arietinum L.) glutaredoxin [LOC101493651 (CaGrx)] gene has been selected based on screening experiments with two contrasting varieties of chickpea, PUSA-362 (drought-tolerant) and ICC-1882 (drought-sensitive) under drought and salinity. The tolerant variety showed higher CaGrx gene expression, as compared to less in the sensitive variety, under both the stresses. The CaGrx gene was then over-expressed in Arabidopsis thaliana and were exposed to drought and salinity. The over-expression of CaGrx elevated the activity of glutaredoxin, which induced antioxidant enzymes (glutathione reductase; GR, glutathione peroxidase; GPX, catalase; CAT, ascorbate peroxidase; APX, glutathione-S-transferase; GST, superoxide dismutase; SOD, monodehydroascorbate reductase; MDHAR, and dehydroascorbate reductase; DHAR), antioxidants (GSH and ascorbate) and stress-responsive amino acids (cysteine and proline). Enhancement in the antioxidant defense system possibly administered tolerance in transgenics against both stresses. CaGrx reduced stress markers (H2O2, TBARS, and electrolyte leakage) and enhanced root growth, seed germination, and survival against both stresses. The physiological parameters (net photosynthesis; P N, water use efficiency; WUE, stomatal conductance; g s, transpiration; E, electron transport rate; ETR, and photochemical quenching; qP), chlorophylls and carotenoids, were improved in the transgenics during both stresses, that maintained the photosynthetic apparatus and protected the plants from damage. The enhanced activity of the cysteine biosynthesis enzyme, o-acetylserine (thiol) lyase (OAS-TL), increased the cysteine level in the transgenics, which elevated glutathione biosynthesis to maintain the ascorbate-glutathione cycle under both stresses. This investigation verified that the CaGrx gene provides tolerance against salinity and drought, maintaining physiological and morphological performances, and could be exploited for genetic engineering approaches to overcome both the stresses in various crops. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-00999-z.

Over-expression of chickpea metallothionein 1 gene confers tolerance against major toxic heavy metal stress in Arabidopsis.

Heavy metals are ubiquitously present in nature, including soil, water, and thus in plants, thereby causing a potential health risk. This study has investigated the role and efficiency of the chickpea metallothionein 1 (MT1) gene against the major toxic heavy metals, i.e., As [As(III) and As(V)], Cr(VI), and Cd toxicity. MT1 over-expressing transgenic lines had reduced As(V) and Cr(VI) accumulation, whereas Cd accumulation was enhanced in the L3 line. The physiological responses (WUE, A, Gs, E, ETR, and qP) were noted to be enhanced in transgenic plants, whereas qN was decreased. Similarly, the antioxidant molecules and enzymatic activities (GSH/GSSG, Asc/DHA, APX, GPX, and GRX) were higher in the transgenic plants. The activity of antioxidant enzymes, i.e., SOD, APX, GPX, and POD, were highest in the Cd-treated lines, whereas higher CAT activity was observed in As(V)-L1 and GRX in Cr-L3 line. The stress markers TBARS, H2O2, and electrolyte leakage were lower in transgenic lines in comparison to WT, while RWC was enhanced in the transgenic lines, and the transcript of MT1 gene was accumulated in the transgenic lines. Similarly, the level of stress-responsive amino acid cysteine was higher in transgenic plants as compared to WT plants. Among all the heavy metals, MT1 over-expressing lines showed a highly increased accumulation of Cd, whereas a non-significant effect was observed with As(III) treatment. Overall, the results demonstrate that Arabidopsis thaliana transformed with the MT1 gene mitigates heavy metal stress by regulating the defense mechanisms in plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01103-1.

Overexpression of rice glutaredoxin genes LOC_Os02g40500 and LOC_Os01g27140 regulate plant responses to drought stress.

Glutaredoxins (Grxs) are small (10-15 kDa) glutathione (GSH) - dependent redox proteins. The role of Grxs are well documented in tolerance to heavy metal stress in prokaryotic and mammalian systems and a few plant genera, but is poorly understood in plants against drought. In the present study, two rice glutaredoxin (Osgrx) genes (LOC_Os02g40500 and LOC_Os01g27140) responsible for tolerance against heavy metal stress have been studied for investigating their role against drought. Each glutaredoxin gene was over-expressed in Arabidopsis thaliana to reveal their role in drought stress. The relative expression of both Osgrx genes was higher in the transgenic lines. Transgenic lines of both Osgrxs showed longer roots, higher seed germination, and survival efficiency during drought stress. The physiological parameters (PN, gs, E, WUE, qP, NPQ and ETR), antioxidant enzymes (GRX, GR, GPX, GST, APX, POD, SOD, CAT, DHAR, and MDHAR), antioxidant molecules (ascorbate and GSH) and stress-responsive amino acids (cysteine and proline) levels were additionally increased in transgenic lines of both Osgrxs to provide drought tolerance. The outcomes from this study strongly determined that each Osgrx gene participated in the moderation of drought and might be utilized in biological engineering strategies to overcome drought conditions in different crops.

Over-expression of chickpea glutaredoxin (CaGrx) provides tolerance to heavy metals by reducing metal accumulation and improved physiological and antioxidant defence system.

Glutaredoxins (Grxs) are small multifunctional redox proteins. Grxs have glutathione-dependent oxidoreductase activity in the presence of glutathione reductase and NADPH. The role of Grxs is well studied in heavy metal tolerance in prokaryotic and mammalian systems but not in plant genera. In the present study, a chickpea glutaredoxin (CaGrx) gene (LOC101493651) has been investigated against metal stress based on its primary screening in chickpea which revealed higher up-regulation of CaGrx gene under various heavy metals (AsIII-25 µM, AsV-250 µM, Cr(VI)-300 µM, and Cd-500 µM) stress. This CaGrx gene was overexpressed in Arabidopsis thaliana and investigated various biochemical and physiological performances under each metal stress. Transgenic plants showed significant up-regulation of the CaGrx gene during qRT-PCR analysis as well as longer roots, higher seed germination, and survival efficiency during each metal stress. The levels of stress markers, TBARS, H2O2, and electrolyte leakage were found to be less in transgenic lines as compared to WT revealed less toxicity in transgenics. The total accumulation of AsIII, AsV, and Cr(VI) were significantly reduced in all transgenic lines except Cd, which was slightly reduced. The physiological parameters such as net photosynthetic rate (PN), stomatal conductance (gs), transpiration (E), water use efficiency (WUE), photochemical quenching (qP), and electron transport rate (ETR), were maintained in transgenic lines during metal stress. Various antioxidant enzymes such as glutaredoxin (GRX), glutathione reductase (GR), glutathione peroxidase (GPX), glutathione-S-transferase (GST), ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase (CAT), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), antioxidant molecules (ascorbate, GSH) and stress-responsive amino acids (proline and cysteine) levels were significantly increased in transgenic lines which provide metal tolerance. The outcome of this study strongly indicates that the CaGrx gene participates in the moderation of metal stress in Arabidopsis, which can be utilized in biotechnological interventions to overcome heavy metal stress conditions in different crops.

Over-expression of CarMT gene modulates the physiological performance and antioxidant defense system to provide tolerance against drought stress in Arabidopsis thaliana L.

Drought is one of the major abiotic stresses which negatively affect plant growth and crop yield. Metallothionein (MTs) is a low molecular weight protein, mainly involved in metal homeostasis, while, its role in drought stress is still to be largely explored. The present study was aimed to investigate the role of MT gene against drought stress. The chickpea MT based on its up-regulation under drought stress was overexpressed in Arabidopsis thaliana to explore its role in mitigation of drought stress. The total transcript of MT gene was up to 30 fold higher in transgenic lines. Arabidopsis plants transformed with MT gene showed longer roots, better efficiency of survival and germination, larger siliques and higher biomass compared to WT. The physiological variables (A, WUE, G, E, qP and ETR) of WT plants were reduced during drought stress which recovered in transgenic Arabidopsis lines. The enzymatic and non-enzymatic antioxidant (APX, GPX, POD, GR, GRX, GST, CAT, MDHAR, ASc and GSH) levels were also enhanced in transgenic lines to provide tolerance. Simultaneously, drought responsive amino acids, i.e. proline and cysteine contents were higher in transgenic lines. Overall, the results suggest that MT gene is actively involved in the mitigation of drought stress and could be the choice for genetic engineering strategy to overcome drought stress.