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SUMMARY: Small RNAs (sRNAs) such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) are short 20–24‐nucleotide non‐coding RNAs. They are key regulators of gene expression in plants and other organisms. Several 22‐nucleotide miRNAs trigger biogenesis cascades of trans‐acting secondary siRNAs, which are involved in various developmental and stress responses. Here we show that Himalayan Arabidopsis thaliana accessions having natural mutations in the miR158 locus exhibit robust cascade silencing of the pentatricopeptide repeat (PPR)‐like locus. Furthermore, we show that these cascade sRNAs trigger tertiary silencing of a gene involved in transpiration and stomatal opening. The natural deletions or insertions in MIR158 led to improper processing of miR158 precursors, thereby blocking synthesis of mature miR158. Reduced miR158 levels led to increased levels of its target, a pseudo‐PPR gene that is targeted by tasiRNAs generated by the miR173 cascade in other accessions. Using sRNA datasets derived from Indian Himalayan accessions, as well as overexpression and knockout lines of miR158, we show that absence of miR158 led to buildup of pseudo‐PPR‐derived tertiary sRNAs. These tertiary sRNAs mediated robust silencing of a gene involved in stomatal closure in Himalayan accessions lacking miR158 expression. We functionally validated the tertiary phasiRNA that targets NHX2, which encodes a Na+‐K+/H+ antiporter protein, thereby regulating transpiration and stomatal conductance. Overall, we report the role of the miRNA–TAS–siRNA–pseudogene–tertiary phasiRNA–NHX2 pathway in plant adaptation. Significance Statement: A natural variant of the miR158 locus of west Himalayan Arabidopsis thaliana shows miRNA‐driven cleavage of a pseudogene, and abolished activity of the miRNA leads to tertiary phasiRNA biogenesis. One such phasiRNA targets a gene that regulates growth and development in A. thaliana. The present study describes negative regulation of phasiRNA biogenesis by a miRNA. [ABSTRACT FROM AUTHOR]

Plant defensins are mainly known for their antifungal activity. However, limited information is available regarding their function in abiotic stresses. In this study, a defensin gene, Ca-AFP , from Cicer arietinum , commonly known as chickpea, was cloned and transformed in Arabidopsis thaliana for its functional characterization under simulated water-deficit conditions. Under simulated water-deficit conditions (mannitol and polyethylene glycol-6000 induced), the transgenic A. thaliana plants had higher accumulation of the Ca-AFP transcript compared to that under non-stress condition and showed higher germination rate, root length, and biomass than the wild-type (WT) plants. To get further insights into the role of Ca-AFP in conferring tolerance to water-deficit stress, we determined various physiological parameters and found significant reduction in the transpiration rate and stomatal conductance whereas the net photosynthesis and water use efficiency was increased in the transgenic plants compared to that in the WT plants under water deficit conditions. The transgenic plants showed enhanced superoxide dismutase, ascorbate peroxidase, and catalase activities, had higher proline, chlorophyll, and relative water content, and exhibited reduced ion leakage and malondialdehyde content under water-deficit conditions. Overall, our results indicate that overexpression of Ca-AFP could be an efficient approach for conferring tolerance to water-deficit stress in plants. [ABSTRACT FROM AUTHOR]

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 (P N , g s , E , WUE, q P, 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. • LOC_Os02g40500 and LOC_Os01g27140 reduced the ROS generated under drought stress. • Osgrxs enhanced the level of the antioxidant molecules, antioxidant enzymes, and stress responsible amino acids. • Osgrxs enhanced the various physiological parameters against drought stress and protected the plant from damage. • Osgrxs enhanced the RWC and reduced the stress markers. [ABSTRACT FROM AUTHOR]

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, H 2 O 2, 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 (P N), stomatal conductance (g s), transpiration (E), water use efficiency (WUE), photochemical quenching (q P), 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. • CaGrx reduced metal accumulation in Arabidopsis under heavy metal stress. • CaGrx reduced the level of stress markers (TBARS, H 2 O 2, and electrolyte leakage). • CaGrx enhanced the level of antioxidant molecules, antioxidant enzymes, and stress-responsive amino acids. • CaGrx enhances the various physiological parameters against metal stress. • CaGrx protects the plant from oxidative damage. [ABSTRACT FROM AUTHOR]

Unravelling the plants responses towards rising atmospheric [CO 2 ] have largely been explored in genetical and morpho-physiological contexts. However, epigenetic factors including DNA methylation which play critical roles in adaptation are largely unexplored. Here we investigated the methylome, transcriptome and morpho-physiological responses of the two Arabidopsis thaliana populations evolved at high (3400 m amsl) and low elevation (700 m amsl) zones to elevated [CO 2 ] (e[CO 2 ]). We show that depending on the origin of the population, there were local level variations in DNA methylation when exposed to e[CO 2 ], but global methyl cytosine (mC) content remained unchanged. Further, there was loss of methylation and more protein coding genes were differentially methylated than transposable elements and non-coding RNA genes, in both the populations but more so in the low elevation one. The differentially methylated genes of the two populations belonged to distinct functional categories. More genes related to methylation machinery were down-regulated in the high elevation population than the low elevation one. Although there was no correlation between methylation and gene expression at the global scale but a few genes exhibited methylation dependent expression level. Finally the hyper- and hypo-methylated status of a few genes due to e[CO 2 ] treatment were validated. Overall, our data suggested the two populations responded differently towards e[CO 2 ] with respect to methylome remodeling, phenotypic and molecular plasticity. However, methylome remodeling and molecular plasticity were more prominent in the low elevation population. Understanding the evolution of epigenetic response towards e[CO 2 ] may help in future crop improvement strategies. • Elevated CO 2 affects DNA methylation remodeling in Arabidopsis thaliana depending on origin of the population. • Methylome remodeling and molecular plasticity of the low elevation population were more than the higher elevation one. • More protein coding genes were differentially methylated in response to elevated CO 2. • Elevated CO 2 induced global gene expression and methylation is not co-linear. [ABSTRACT FROM AUTHOR]