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.

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.

Response of tropical trees to elevated Ozone: a Free Air Ozone Enrichment study.

Tropospheric ozone (O3) has become one of the main urban air pollutants. In the present study, we assessed impact of ambient and future ground-level O3 on nine commonly growing urban tree species under Free Air Ozone Enrichment (FAOE) condition. During the study period, mean ambient and elevated ozone (EO3) concentrations were 48.59 and 69.62 ppb, respectively. Under EO3 treatment, stomatal density (SD) significantly decreased and guard cell length (GCL) increased in Azadirachta indica, Bougainvillea spectabilis, Plumeria rubra, Saraca asoca and Tabernaemontana divaricata, while SD increased and GCL decreased in Ficus benghalensis and Terminalia arjuna. Proline levels increased in all the nine plant species under EO3 condition. EO3 significantly reduced photosynthetic rate, stomatal conductance (gs), and transpiration rates (E). Only A. indica and N. indicum showed higher gs and E under EO3 treatment. Water use efficiency (WUE) significantly increased in F. benghalensis and decreased in A. indica and T. divaricata. Air Pollution Tolerance Index (APTI) significantly increased in Ficus religiosa and S. asoca whereas it decreased in B. spectabilis and A. indica. Of all the plant species B. spectabilis and A. indica were the most sensitive to EO3 (high gs and less ascorbic acid content) while S. asoca and F. religiosa were the most tolerant (lowgs and more ascorbic acid content). The sensitivity of urban tree species to EO3 is a cause of concern and should be considered for future urban forestry programmes. Our study should guide more such studies to identify tolerant trees for urban air pollution abatement.

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.

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.

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.

Impact of chronic elevated ozone exposure on photosynthetic traits and anti-oxidative defense responses of Leucaena leucocephala (Lam.) de wit tree under field conditions.

In this study, the impact of long term exposure of elevated ozone (+20 ppb above ambient) on photosynthetic traits and anti-oxidative defense system of Leucaena leucocephala, a tree of great economic importance, was studied in a Free Air Ozone Concentration Enrichment (O3-FACE) facility at different time intervals (6, 12, 18, and 24 months). Results showed that net photosynthesis, photosynthetic pigments and lipid peroxidation were significantly reduced after 6, 12 and 24 months of exposure to elevated ozone (eO3) whereas stomatal conductance and transpiration rate were significantly decreased after 12 months of exposure to eO3. Antioxidant enzymatic activities (catalase, ascorbate peroxidase and glutathione reductase) were significantly increased after 12 months of exposure to eO3. Ascorbate was increased significantly after 6 and 12 months of exposure to eO3 while reduced glutathione content declined significantly after 6 and 24 months of exposure to eO3. The study showed that there were several negative long lasting physiological and biochemical responses in Leucaena. The results provide evidence that Leucaena exhibited greater sensitivity to O3 during initial exposure (up to 12 months) but showed moderate tolerance by the end of the 2nd year.

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.

Integral terminal sliding mode control unified with UDE for output constrained tracking of mismatched uncertain non-linear systems.

This paper presents a novel integral terminal sliding mode control (ITSMC) algorithm compounded with Barrier Lyapunov's Function (BLF) for high precision output tracking objective of uncertain N-dimensional strict feedback class of non-linear systems subjected to non-infringement of system output constraint. Besides, the proposed algorithm also employs uncertainty and disturbance estimator (UDE) technique for abating the existence of unknown mismatched and matched uncertainties. Most of the preliminary SMC schemes encompass the UDE technique to cope with unknown matched uncertainties only. However, the presented method embodies the dynamic surface control (DSC) architecture to get rid of the matching architectural constraint in UDE approach as well as 'complexity explosion' concern in backstepping approach. Additionally, the introduced method also applies the notion of symmetric BLF to enforce strict bounds on output tracking deviation from its reference value. To emphasize on the strengths of devised methodology, two examples are simulated by comparing the derived outcomes with already existing techniques in literature.

Overexpression of Chickpea Defensin Gene Confers Tolerance to Water-Deficit Stress in Arabidopsis thaliana.

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.

Fractional order uncertainty estimator based hierarchical sliding mode design for a class of fractional order non-holonomic chained system.

This paper proposes a novel fractional order sliding mode control approach to address the issues of stabilization as well as tracking of an N-dimensional extended chained form of fractional order non-holonomic system. Firstly, the hierarchical fractional order terminal sliding manifolds are selected to procure the desired objectives in finite time. Then, a sliding mode control law is formulated which provides robustness against various system uncertainties or external disturbances. In addition, a novel fractional order uncertainty estimator is deduced mathematically to estimate and mitigate the effects of uncertainties, which also excludes the requirement of their upper bounds. Due to the omission of discontinuous control action, the proposed algorithm ensures a chatter-free control input. Moreover, the finite time stability of the closed loop system has been proved analytically through well known Mittag-Leffler and Fractional Lyapunov theorems. Finally, the proposed methodology is validated with MATLAB simulations on two examples including an application of fractional order non-holonomic wheeled mobile robot and its performances are also compared with the existing control approach.