Indian Himalayan natural Arabidopsis thaliana accessions with abolished miR158 levels exhibit robust miR173-initiated trans-acting cascade silencing.

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.

Endophytic bacilli from Cyamopsis tetragonoloba (L.) Taub. induces plant growth and drought tolerance.

Cyamopsis tetragonoloba (L.) Taub. (guar) is a commercially important crop known for its galactomannan content in seeds. Drought stress is a significant global concern that compromises the productivity of major legumes including guar. The endophytic microbes associated with plants play a significant role in enhancing plant growth and modulating the impact of abiotic stress(s). The present study involved the isolation of 73 endophytic bacteria from the guar seeds of drought-tolerant (RGC-1002 and RGC-1066) and sensitive (Sarada and Varsha) varieties. Based on multiple PGP attributes and drought tolerance, at 50% PEG6000 w/v, 11 efficient isolates were selected and identified through 16S rRNA gene sequencing. Isolates belonging to ten different species of bacilli including Cytobacillus oceanisediminis, Mesobacillus fermenti, Peribacillus simplex from sensitive and Bacillus zanthoxyli, B. safensis, B. velezensis, B. altitudinis, B. licheniformis, B. tequilensis, and B. paralicheniformis isolated from tolerant varieties. A greenhouse experiment with a drought-sensitive guar variety demonstrated that inoculation of selected isolates showed comparatively better plant growth, higher relative water content (RWC), decreased carbon isotope discrimination ratio (Δ13C), increased chlorophyll, carotenoids, anthocyanin, and proline content, decreased malondialdehyde (MDA) and modulated defense enzymes as compared to their uninoculated controls. Tolerant variety isolates B. tequilensis NBRI14G and B. safensis NBRI10R showed the most promising results in improving plant growth and also drought stress tolerance in guar plants. This study represents for the first time that seed endophytic bacterial strains from guar can be utilized to develop the formulation for improving the productivity of guar under drought-stress conditions.

Endofungal Rhizobium species enhance arsenic tolerance in colonized host plant under arsenic stress.

Arsenic (As) is a toxic metalloid that is present in natural surroundings in many forms with severe consequences to sustainable agriculture and human health. Plant growth-promoting Rhizobia have been found involved in the induction of plant tolerance under various biotic and abiotic stresses. An endofungal Rhizobium species associated with arbuscular mycorrhizal fungi (AMF) Serendipita indica deploy beneficial role in the promotion of plant growth and tolerance against various biotic and abiotic stresses. In the current study, we have determined the role of endofungal Rhizobium species in protection of host plant growth under As stress. We observed that endofungal Rhizobium species strain Si001 tolerate AsV up to 25 mM and its inoculation enhances tomato seed germination and seedling growth. A hyper-colonization of Rhizobium species Si001 in tomato roots was observed under As stress and results in modulation of GSH and proline content with reduced ROS. Rhizobium species Si001 colonization in host plant recovered pigment contents (chlorophyll-a and chlorophyll-b up to 189.5% and 192%, respectively), photosynthesis (157%), and water use efficiency (166%) compared to As-treated plants. Interestingly, bacterial colonization results in 40% increased As accumulation in the root, while a reduction in As translocation from root to shoot up to 89% was observed as compared to As treated plants. In conclusion, endofungal Rhizobium species Si001 association with the host plant may improve plant health and tolerance against As stress with reduced As accumulation in the crop produce.

Exogenous application of methyl jasmonate alleviates arsenic toxicity by modulating its uptake and translocation in rice (Oryza sativa L.).

Methyl jasmonate (Me-JA) is a plant growth regulator known for modulating plant responses to various abiotic and biotic stresses. The unavoidable arsenic (As) contamination in rice (Oryza sativa) results in reduced crop yield and greater carcinogenic risk to humans. The present work examines the significance of Me-JA induced molecular signaling and tolerance towards arsenic toxicity in rice. The arsenite (AsIII; 25 µM) stress hampered the overall growth and development of the rice seedling. However, the co-application (25 µM AsIII+0.25 µM Me-JA) resulted in increased biomass, chlorophyll content, enhanced antioxidant enzyme activities as compared to AsIII treated plants. The co-application also demonstrated a marked decrease in malondialdehyde content, electrolyte leakage and accumulation of total AsIII content (root + shoot) as compared to AsIII treated plants. The co-application also modulated the expression of genes involved in downstream JA signaling pathway (OsCOI, OsJAZ3, OsMYC2), AsIII uptake (OsLsi1, OsLsi2, OsNIP1;1, OsNIP3;1), translocation (OsLsi6, and OsINT5) and detoxification (OsNRAMP1, OsPCS2, and OsABCC2) which revealed the probable adaptive response of the rice plant to cope up arsenic stress. Our findings reveal that Me-JA alleviates AsIII toxicity by modulating signaling components involved in As uptake, translocation, and detoxification and JA signaling in rice. This study augments our knowledge for the future use of Me-JA in improving tolerance against AsIII stress.

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.

Physiological performance of two contrasting rice varieties under water stress.

Two rice varieties PR-115 and Super-7 were imposed to water stress and different physiological traits were monitored to evaluate the performance of these varieties under drought. Under water stress condition although the relative water content, osmotic potential, chlorophyll content, photosynthesis rate, carbon discrimination and biomass decreased in both the varieties however, the reduction was more pronounced in Super-7 variety. Oryzanol a trans-ester of ferulic acid functions as antioxidant and it increased along with total phenolic and anthocyanin content in both the varieties under drought stress. However, gallic acid, 4 hydroxy benzoic acid, syringic acid and chlorogenic acid showed differential pattern in both of the varieties under water limiting conditions. Under drought, grain yield was penalized by 17 and 54% in PR-115 and Super-7 varieties, respectively in comparison to watered plants. Super-7 variety showed pronounced electrolyte leakage and MDA enhancement under water stress condition. High non photochemical quenching and reduction in Y(NO) and Y(II) indicated balanced energy management in tolerant PR-115 variety. The studies showed that PR-115 is a drought tolerant variety while Super-7 is drought sensitive in nature.

Alterations in plant anatomy and higher lignin synthesis provides drought tolerance in cluster bean [Cyamopsis tetragonoloba (L.) Taub.].

Four contrasting varieties of guar, RGC-1002 and RGC-1038, drought tolerant, while, Sarada and RGC-936, drought sensitive, were monitored in watered and drought. The water status, phenolics, plant anatomy and transcript level of genes related to anatomical traits were assessed. The study aimed to decipher the anatomical adaptations of guar plants in response to water stress. The physiological determinants, relative water content (RWC), water potential (ψ), and leaf membrane damage, declined under drought in all four varieties although, the decrement was lesser in the tolerant varieties. Furthermore, the tolerant cultivars subjected to water stress recorded higher accumulation of total phenolic content, anthocyanin and lignin, which efficiently scavenge the reactive oxygen species. The results suggest that the cultivars RGC-1002 and RGC-1038 are better able to resist drought-induced oxidative stress than Sarada and RGC-936. Moreover, leaf, petiole, stem and root anatomical traits viz. size of epidermal cell, parenchyma, width of cortex layer, and diameter of xylem vessels were narrowed in all the varieties although, the decrement was lesser in the tolerant varieties under drought. The expression analysis of genes revealed that drought-tolerant varieties showed enhanced mechanical support for water conduction by up-regulation of genes, Phenylalanine ammonia-lyase1 (PAL1), cinnamate-4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), cinnamoyl-CoA reductase (CCR), caffeoyl-CoA O-methyltransferase (CCOMT), and cinnamyl-alcohol dehydrogenase (CAD6) in water stress conditions. The alterations in physio-anatomical, biochemical and gene expression traits in tolerant guar varieties enabled them to maintain steady nutrient transport while reducing the risk of embolisms and increasing water-flow resistance for better survival in water stressed conditions.

Phytoremediation potential of Solanum viarum Dunal and functional aspects of their capitate glandular trichomes in lead, cadmium, and zinc detoxification.

In the present scenario, remediation of heavy metals (HMs) contaminated soil has become an important work to be done for the well-being of human and their environment. Phytoremediation can be regarded as an excellent method in environmental technologies. The present contemporary research explores the Solanum viarum Dunal function as a potential accumulator of hazardous HMs viz. lead (Pb), cadmium (Cd), zinc (Zn), and their combination (CHM). On toxic concentrations of Pb, Cd, Zn, and their synergistic exposure, seeds had better germination percentage and their 90d old aerial tissues accumulated Pb, Cd, and Zn concentrations ranging from 44.53, 84.06, and 147.29 mg kg-1 DW, respectively. Pattern of accumulation in roots was as Zn 70.08 > Pb 48.55 > Cd 42.21 mg kg-1DW. Under HMs treatment, positive modulation in physiological performances, antioxidant activities suggested an enhanced tolerance along with higher membrane stability due to increased levels of lignin, proline, and sugar. Phenotypic variations were recorded in prickles and roots of 120 d old HM stressed plants, which are directly correlated with better acclimation. Interestingly, trichomes of the plant also showed HM accumulation. Later, SEM-EDX microanalysis suggested involvement of S. viarum capitate glandular trichomes as excretory organs for Cd and Zn. Thus, the present study provides an understanding of the mechanism that makes S. viarum to function as potent accumulator and provides information to generate plants to be used for phytoremediation.

Endophytic fungus Serendipita indica reduces arsenic mobilization from root to fruit in colonized tomato plant.

The accumulation of arsenic in crop plants has become a worldwide concern that affects millions of people. The major source of arsenic in crop plants is irrigation water and soil. In this study, Serendipita indica, an endophytic fungus, was used to investigate the protection against arsenic and its accumulation in the tomato plant. We found that inoculation of S. indica recovers seed germination, plant growth and improves overall plant health under arsenic stress. A hyper-colonization of fungus in the plant root was observed under arsenic stress, which results in reduced oxidative stress via modulation of antioxidative enzymes, glutathione, and proline levels. Furthermore, fungal colonization restricts arsenic mobilization from root to shoot and fruit by accumulating it exclusively in the root. We observed that fungal colonization enhances the arsenic bioaccumulation factor 1.48 times in root and reduces the arsenic translocation factor by 2.96 times from root to shoot and 13.6 times from root to fruit compared to non colonized plants. Further, investigation suggests that S. indica can tolerate arsenic by immobilizing it on the cell wall and accumulating it in the vacuole. This study shows that S. indica may be helpful for the reduction of arsenic accumulation in crops grown in arsenic-contaminated agriculture fields.

A tau class glutathione-S-transferase (OsGSTU5) confers tolerance against arsenic toxicity in rice by accumulating more arsenic in root.

Arsenic (As) considered as one of the hazardous metalloid that hampers various physiological activities in rice. To study the mechanism of As tolerance in rice, one differentially expressed tau class glutathione-S-transferase (OsGSTU5) has been selected and transgenic rice plants with knockdown (KD) and overexpressing (OE) OsGSTU5 were generated. Our results suggested that KD lines became less tolerant to As stress than WT plants, while OE lines showed enhanced tolerance to As. Under As toxicity, OE and KD lines showed enhanced and reduced antioxidant activities such as, SOD, PRX and catalase, respectively indicating its role in ROS homeostasis. In addition, higher malondialdehyde content, poor photosynthetic parameters and higher reactive oxygen species (ROS) in KD plant, suggests that knockdown of OsGSTU5 renders KD plants more susceptible to oxidative damage. Also, the relative expression profile of various transporters such as OsABCC1 (As sequestration), Lsi2 and Lsi6 (As translocaters) and GSH dependent activity of GSTU5 suggests that GSTU5 might help in chelation of As with GSH and sequester it into the root vacuole using OsABCC1 transporter and thus limits the upward translocation of As towards shoot. This study suggests the importance of GSTU5 as a good target to improve the As tolerance in rice.

Physiological attributes associated with leaf spectral alterations in guar [Cyamopsis tetragonoloba (L.) Taub.] under drought.

Two contrasting guar varieties [Cyamopsis tetragonoloba (L.) Taub.], RGC-1002-a better drought tolerant than RGC-936-a comparatively sensitive variety, were subjected to 8 days of water stress. The physiological characteristics of gas exchange, pigments and the spectral properties of the leaves were monitored and assessed. The guar variety, RGC-1002 exhibited higher relative water content (RWC) and biomass accumulation in water stress condition as compared to RGC-936. The RGC-1002 variety also showed a higher rate of photosynthesis, stomatal conductance, and lesser reduction in chlorophyll content as compared to RGC-936 variety. On the 8th day of drought, the leaf anthocyanin levels were also higher in RGC-1002 than RGC-936. Increased levels of anthocyanin result in decrease in absorption of light, and an increase in reflectance, and transmittance. The scattering coefficient of leaf spectra was significantly increased in RGC-936 than in RGC-1002 leaves; this is due to an increase in the size of intercellular air spaces and shrinkage of cells as a result of water loss. The leaf reflectance was also observed to be significantly increased in RGC-936 than in RGC-1002 leaves. The physiological and leaf optical observations substantiate that RGC-1002 was better adapted to water stress than RGC-936 variety. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02674-2.

Bacterial endophytes modulates the withanolide biosynthetic pathway and physiological performance in Withania somnifera under biotic stress.

Despite the vast exploration of endophytic microbes for growth enhancement in various crops, knowledge about their impact on the production of therapeutically important secondary metabolites is scarce. In the current investigation, chitinolytic bacterial endophytes were isolated from selected medicinal plants and assessed for their mycolytic as well as plant growth promoting potentials. Among them the two most efficient bacterial endophytes namely Bacillus amyloliquefaciens (MPE20) and Pseudomonas fluorescens (MPE115) individually as well as in combination were able to modulate withanolide biosynthetic pathway and tolerance against Alternaria alternata in Withania somnifera. Interestingly, the expression level of withanolide biosynthetic pathway genes (3-hydroxy-3-methylglutaryl co-enzyme A reductase, 1-deoxy-D-xylulose-5-phosphate reductase, farnesyl di-phosphate synthase, squalene synthase, cytochrome p450, sterol desaturase, sterol Δ-7 reductase and sterol glycosyl transferases) were upregulated in plants treated with the microbial consortium under A. alternata stress. In addition, application of microbes not only augmented withaferin A, withanolide A and withanolide B content (1.52-1.96, 3.32-5.96 and 12.49-21.47 fold, respectively) during A. alternata pathogenicity but also strengthened host resistance via improvement in the photochemical efficiency, normalizing the oxidized and non-oxidized fraction, accelerating photochemical and non-photochemical quantum yield, and electron transport rate. Moreover, reduction in the passively dissipated energy of PSI and PSII in microbial combination treated plants corroborate well with the above findings. Altogether, the above finding highlights novel insights into the underlying mechanisms in application of endophytes and emphasizes their capability to accelerate biosynthesis of withanolides in W. somnifera under biotic stress caused by A. alternata.

Modulations in primary and secondary metabolic pathways and adjustment in physiological behaviour of Withania somnifera under drought stress.

In general medicinal plants grown under water limiting conditions show much higher concentrations of secondary metabolites in comparison to control plants. In the present study, Withania somnifera plants were subjected to water stress and data related to drought tolerance phenomenon was collected and a putative mechanistic concept considering growth responses, physiological behaviour, and metabolite content and gene expression aspects is presented. Drought induced metabolic and physiological responses as well as drastic decrease in CO2 uptake due to stomatal limitations. As a result, the consumption of reduction equivalents (NADPH2+) for CO2 assimilation via the calvin cycle declines significantly resulting in the generation of a large oxidative stress and an oversupply of antioxidant enzymes. Drought also results in the shifting of metabolic processes towards biosynthetic activities that consume reduction equivalents. Thus, biosynthesis of reduced compounds (isoprenoids, phenols and alkaloids) is enhanced. The dynamics of various metabolites have been discussed in the light of gene expression analysis of control and drought treated leaves. Gene encoding enzymes of pathways leading to glucose, fructose and fructan production, conversion of triose phosphates to hexoses and hexose phosphorylation were up-regulated in the drought stressed leaves. The down-regulated Calvin cycle genes were co-ordinately regulated with the down-regulation of chloroplast triosephosphate/phosphate translocator, cytoplasmic fructose-1,6-bisphosphate aldolase and fructose bisphosphatase. Expression of gene encoding Squalene Synthase (SQS) was highly upregulated under drought stress which is responsible for the diversion of carbon flux towards withanolides biosynthesis from isoprenoid pathway.

Expression of Rice CYP450-Like Gene (Os08g01480) in Arabidopsis Modulates Regulatory Network Leading to Heavy Metal and Other Abiotic Stress Tolerance.

Heavy metal (HM) toxicity has become a grave problem in the world since it leads to hazardous effects on living organisms. Transcriptomic/proteomic studies in plants have identified a large number of metal-responsive gene families. Of these, cytochrome-P450 (CYPs) family members are composed of enzymes carrying out detoxification of exogenous molecules. Here, we report a CYP-like protein encoded by Os08g01480 locus in rice that helps the plant to combat HM and other abiotic stresses. To functionally characterize CYP-like gene, cDNA and promoter were isolated from rice to develop Arabidopsis transgenic lines. Heterologous expression of Os08g01480 in Arabidopsis provided significant tolerance towards abiotic stresses. In silico analysis reveals that Os08g01480 might help plants to combat environmental stress via modulating auxin metabolism. Transgenic lines expressing reporter gene under control of Os08g01480 promoter demonstrated differential promoter activity in different tissues during environmental stresses. These studies indicated that differential expression of Os08g01480 might be modulating response of plants towards environmental stresses as well as in different developmental stages.