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 InformationThe online version contains supplementary material available at 10.1007/s12298-021-00999-z.     

Overexpressing <Emphasis Type="Italic">SgNCED1</Emphasis> in Tobacco Increases ABA Level,Antioxidant Enzyme Activities,and Stress Tolerance

Abscisic acid (ABA) regulates plant adaptive responses to various environmental stresses. 9-cis-epoxycarotenoid dioxygenase (NCED) is the key enzyme of ABA biosynthesis in higher plants. A NCED gene, SgNCED1, was overexpressed in transgenic tobacco plants which resulted in 51–77% more accumulation of ABA in leaves. Transgenic tobacco plants decreased stomatal conductance, transpiration rate, and photosynthetic rate but induced activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate-peroxidase (APX). Hydrogen peroxide (H₂O₂) and nitric oxide (NO) in leaves were also induced in the transgenic plants. Compared to the wild-type control, the transgenic plants improved growth under 0.1 M mannitol-induced drought stress and 0.1 M NaCl-induced salinity stress. It is suggested that the ABA-induced H₂O₂ and NO generation upregulates the stomatal closure and antioxidant enzymes, and therefore increases drought and salinity tolerance in the transgenic plants.     

An osmotin from the resurrection plant Tripogon loliiformis (TlOsm) confers tolerance to multiple abiotic stresses in transgenic rice

Osmotin is a key protein associated with abiotic and biotic stress response in plants. In this study, an osmotin from the resurrection plant Tripogon loliiformis (TlOsm) was characterized and functionally analyzed under abiotic stress conditions in T. loliiformis as well as in transgenic Nicotiana tabacum (tobacco) and Oryza sativa (rice) plants. Real‐time PCR analysis on mixed elicitor cDNA libraries from T. loliiformis showed that TlOsm was upregulated a 1000‐fold during the early stages of osmotic stresses (cold, drought, and salinity) in both shoots and roots but downregulated in shoots during heat stress. There was no change in TlOsm gene expression in roots of heat‐stressed plants and during plant development. The plasma membrane localization of TlOsm was showed in fluorescent‐tagged TlOsm tobacco plants using confocal laser scanning microscopic analysis. Transgenic rice plants expressing TlOsm were assessed for enhanced tolerance to salinity, drought and cold stresses. Constitutively expressed TlOsm in transgenic rice plants showed increased tolerance to cold, drought and salinity stress when compared with the wild‐type and vector control counterparts. This was evidenced by maintained growth, retained higher water content and membrane integrity, and improved survival rate of TlOsm‐expressing plants. The results thus indicate the involvement of TlOsm in plant response to multiple abiotic stresses, possibly through the signaling pathway, and highlight its potential applications for engineering crops with improved tolerance to cold, drought and salinity stress.     

Overexpression of osmotin gene confers tolerance to salt and drought stresses in transgenic tomato (Solanum lycopersicum L.)

Abiotic stresses, especially salinity and drought, are major limiting factors for plant growth and crop productivity. In an attempt to develop salt and drought tolerant tomato, a DNA cassette containing tobacco osmotin gene driven by a cauliflower mosaic virus 35S promoter was transferred to tomato (Solanum lycopersicum) via Agrobacterium-mediated transformation. Putative T0 transgenic plants were screened by PCR analysis. The selected transformants were evaluated for salt and drought stress tolerance by physiological analysis at T1 and T2 generations. Integration of the osmotin gene in transgenic T1 plants was verified by Southern blot hybridization. Transgenic expression of the osmotin gene was verified by RT-PCR and northern blotting in T1 plants. T1 progenies from both transformed and untransformed plants were tested for salt and drought tolerance by subjecting them to different levels of NaCl stress and by withholding water supply, respectively. Results from different physiological tests demonstrated enhanced tolerance to salt and drought stresses in transgenic plants harboring the osmotin gene as compared to the wild-type plants. The transgenic lines showed significantly higher relative water content, chlorophyll content, proline content, and leaf expansion than the wild-type plants under stress conditions. The present investigation clearly shows that overexpression of osmotin gene enhances salt and drought stress tolerance in transgenic tomato plants.     

The pepper late embryogenesis abundant protein CaLEA1 acts in regulating abscisic acid signaling,drought and salt stress response

As sessile organisms, plants are constantly challenged by environmental stresses, including drought and high salinity. Among the various abiotic stresses, osmotic stress is one of the most important factors for growth and significantly reduces crop productivity in agriculture. Here, we report a function of the CaLEA1 protein in the defense responses of plants to osmotic stress. Our analyses showed that the CaLEA1 gene was strongly induced in pepper leaves exposed to drought and increased salinity. Furthermore, we determined that the CaLEA1 protein has a late embryogenesis abundant (LEA)_3 homolog domain highly conserved among other known group 5 LEA proteins and is localized in the processing body. We generated CaLEA1‐silenced peppers and CaLEA1‐overexpressing (OX) transgenic Arabidopsis plants to evaluate their responses to dehydration and high salinity. Virus‐induced gene silencing of CaLEA1 in pepper plants conferred enhanced sensitivity to drought and salt stresses, which was accompanied by high levels of lipid peroxidation in dehydrated and NaCl‐treated leaves. CaLEA1‐OX plants exhibited enhanced sensitivity to abscisic acid (ABA) during seed germination and in the seedling stage; furthermore, these plants were more tolerant to drought and salt stress than the wild‐type plants because of enhanced stomatal closure and increased expression of stress‐responsive genes. Collectively, our data suggest that CaLEA1 positively regulates drought and salinity tolerance through ABA‐mediated cell signaling.     

Differential protection of photosynthetic capacity in trehalose-and lea protein-producing transgenic plants under abiotic stresses

We previously demonstrated that both trehalose and LEA protein protect plants from damage by drought, salt, and heat. Here, we compared their effectiveness in preserving photosynthetic capacity under those abiotic stresses. Upon dehydration, the Pmax (maximal photosynthetic rate) of O₂ evolution decreased similarly in both nontransformants andotsA plants. Contrastingly, Pmax was maintained at a considerably higher level inCaLEA6 plants. However, no significant differences in Chl fluorescence parameters were observed between transformants and nontransformants. Under salinity stress,CaLEA6 plants were also better thanotsA plants in terms of their values for Pmax, photochemical efficiency of PSII(Fv/Fm), and photochemical quenching (qP). After heat bothotsA andCaLEA6 plants maintained a higher Pmax as well as more favorable Chl fluorescence parameters, although the latter transformant performed slightly better overall. Therefore, despite the comparable effectiveness of trehalose and LEA protein in enhancing tolerance against those abiotic stresses, they confer differential protection in maintaining photosynthetic capacity. Compared with trehalose, the CaLEA6 protein appears to be a more universal and effective agent under those stresses.     

Expression of cold and drought regulatory protein (CcCDR) of pigeonpea imparts enhanced tolerance to major abiotic stresses in transgenic rice plants

Main conclusion

Transgenic rice expressing pigeonpea Cc CDR conferred high-level tolerance to different abiotic stresses. The multiple stress tolerance observed in CcCDR -transgenic lines is attributed to the modulation of ABA-dependent and-independent signalling-pathway genes.

Stable transgenic plants expressing Cajanus cajan cold and drought regulatory protein encoding gene (CcCDR), under the control of CaMV35S and rd29A promoters, have been generated in indica rice. Different transgenic lines of CcCDR, when subjected to drought, salt, and cold stresses, exhibited higher seed germination, seedling survival rates, shoot length, root length, and enhanced plant biomass when compared with the untransformed control plants. Furthermore, transgenic plants disclosed higher leaf chlorophyll content, proline, reducing sugars, SOD, and catalase activities, besides lower levels of MDA. Localization studies revealed that the CcCDR-GFP fusion protein was mainly present in the nucleus of transformed cells of rice. The CcCDR transgenics were found hypersensitive to abscisic acid (ABA) and showed reduced seed germination rates as compared to that of control plants. When the transgenic plants were exposed to drought and salt stresses at vegetative and reproductive stages, they revealed larger panicles and higher number of filled grains compared to the untransformed control plants. Under similar stress conditions, the expression levels of P5CS, bZIP, DREB, OsLEA3, and CIPK genes, involved in ABA-dependent and-independent signal transduction pathways, were found higher in the transgenic plants than the control plants. The overall results amply demonstrate that the transgenic rice expressing CcCDR bestows high-level tolerance to drought, salt, and cold stress conditions. Accordingly, the CcCDR might be deployed as a promising candidate gene for improving the multiple stress tolerance of diverse crop plants.

     

Overexpression of Rat Neurons Nitric Oxide Synthase in Rice Enhances Drought and Salt Tolerance

Nitric oxide (NO) has been shown to play an important role in the plant response to biotic and abiotic stresses in Arabidopsis mutants with lower or higher levels of endogenous NO. The exogenous application of NO donors or scavengers has also suggested an important role for NO in plant defense against environmental stress. In this study, rice plants under drought and high salinity conditions showed increased nitric oxide synthase (NOS) activity and NO levels. Overexpression of rat neuronal NO synthase (nNOS) in rice increased both NOS activity and NO accumulation, resulting in improved tolerance of the transgenic plants to both drought and salt stresses. nNOS-overexpressing plants exhibited stronger water-holding capability, higher proline accumulation, less lipid peroxidation and reduced electrolyte leakage under drought and salt conditions than wild rice. Moreover, nNOS-overexpressing plants accumulated less H₂O₂, due to the observed up-regulation of OsCATA, OsCATB and OsPOX1. In agreement, the activities of CAT and POX were higher in transgenic rice than wild type. Additionally, the expression of six tested stress-responsive genes including OsDREB2A, OsDREB2B, OsSNAC1, OsSNAC2, OsLEA3 and OsRD29A, in nNOS-overexpressing plants was higher than that in the wild type under drought and high salinity conditions. Taken together, our results suggest that nNOS overexpression suppresses the stress-enhanced electrolyte leakage, lipid peroxidation and H₂O₂ accumulation, and promotes proline accumulation and the expression of stress-responsive genes under stress conditions, thereby promoting increased tolerance to drought and salt stresses.     

Erratum to: Constitutive expression of CaXTH3, a hot pepper xyloglucan endotransglucosylase/hydrolase, enhanced tolerance to salt and drought stresses without phenotypic defects in tomato plants (Solanum lycopersicum cv. Dotaerang)

The hot pepper xyloglucan endo-trans-gluco-sylase/hydrolase (CaXTH3) gene that was inducible by a broad spectrum of abiotic stresses in hot pepper has been reported to enhance tolerance to drought and high salinity in transgenic Arabidopsis. To assess whether CaXTH3 is a practically useful target gene for improving the stress tolerance of crop plants, we ectopically over-expressed the full-length CaXTH3 cDNA in tomato (Solanum lycopersicum cv. Dotaerang) and found that the 35S:CaXTH3 transgenic tomato plants exhibited a markedly increased tolerance to salt and drought stresses. Transgenic tomato plants exposed to a salt stress of 100 mM NaCl retained the chlorophyll in their leaves and showed normal root elongation. They also remained green and unwithered following exposure to 2 weeks of dehydration. A high proportion of stomatal closures in 35S:CaXTH3 was likely to be conferred by increased cell-wall remodeling activity of CaXTH3 in guard cell, which may reduce transpirational water loss in response to dehydration stress. Despite this increased stress tolerance, the transgenic tomato plants showed no detectable phenotype defects, such as abnormal morphology and growth retardation, under normal growth conditions. These results raise the possibility that CaXTH3 gene is appropriate for application in genetic engineering strategies aimed at improving abiotic stress tolerance in agriculturally and economically valuable crop plants.     

Functional roles of the pepper antimicrobial protein gene, <Emphasis Type="Italic">CaAMP1</Emphasis>, in abscisic acid signaling,and salt and drought tolerance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Biotic signaling molecules including abscisic acid (ABA) are involved in signal transduction pathways that mediate the defense response of plants to environmental stresses. The antimicrobial protein gene CaAMP1, previously isolated from pepper (Capsicum annuum), was strongly induced in pepper leaves exposed to ABA, NaCl, drought, or low temperature. Because transformation is very difficult in pepper, we overexpressed CaAMP1 in Arabidopsis. CaAMP1-overexpressing (OX) transgenic plants exhibited reduced sensitivity to ABA during the seed germination and seedling stages. Overexpression of CaAMP1 conferred enhanced tolerance to high salinity and drought, accompanied by altered expression of the AtRD29A gene, which is correlated with ABA levels and environmental stresses. The transgenic plants were also highly tolerant to osmotic stress caused by high concentrations of mannitol. Together, these results suggest that overexpression of the CaAMP1 transgene modulates salt and drought tolerance in Arabidopsis through ABA-mediated cell signaling. The nucleotide sequence data reported here have been deposited in the GenBank database under the accession number AY548741.     

Overexpression of a <Emphasis Type="Italic">Panax ginseng</Emphasis> tonoplast aquaporin alters salt tolerance,drought tolerance and cold acclimation ability in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

Water movement across cellular membranes is regulated largely by a family of water channel proteins called aquaporins (AQPs). Since several abiotic stresses such as, drought, salinity and freezing, manifest themselves via altering water status of plant cells and are linked by the fact that they all result in cellular dehydration, we overexpressed an AQP (tonoplast intrinsic protein) from Panax ginseng, PgTIP1, in transgenic Arabidopsis thaliana plants to test its role in plant’s response to drought, salinity and cold acclimation (induced freezing tolerance). Under favorable conditions, PgTIP1 overexpression significantly increased plant growth as determined by the biomass production, and leaf and root morphology. PgTIP1 overexpression had beneficial effect on salt-stress tolerance as indicated by superior growth status and seed germination of transgenic plants under salt stress; shoots of salt-stressed transgenic plants also accumulated greater amounts of Na⁺ compared to wild-type plants. Whereas PgTIP1 overexpression diminished the water-deficit tolerance of plants grown in shallow (10 cm deep) pots, the transgenic plants were significantly more tolerant to water stress when grown in 45 cm deep pots. The rationale for this contrasting response, apparently, comes from the differences in the root morphology and leaf water channel activity (speed of dehydration/rehydration) between the transgenic and wild-type plants. Plants overexpressed with PgTIP1 exhibited lower (relative to wild-type control) cold acclimation ability; however, this response was independent of cold-regulated gene expression. Our results demonstrate a significant function of PgTIP1 in growth and development of plant cells, and suggest that the water movement across tonoplast (via AQP) represents a rate-limiting factor for plant vigor under favorable growth conditions and also significantly affect responses of plant to drought, salt and cold stresses.     

Transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> Plants Expressing Grape Glutathione S-Transferase Gene (<Emphasis Type="Italic">VvGSTF13</Emphasis>) Show Enhanced Tolerance to Abiotic Stress

Although glutathione S-transferase (GST, EC 2.5.1.18) is thought to play important roles in abiotic stress, limited information is available regarding the function of its gene in grapes. In this study, a GST gene from grape, VvGSTF13, was cloned and functionally characterized. Transgenic Arabidopsis plants containing this gene were normal in terms of growth and maturity compared with control plants but had enhanced resistance to salt, drought, and methyl viologen stress. The increased tolerance of the transgenic plants correlated with changes in activities of antioxidative enzymes. Our results indicate that the gene from grape plays a positive role in improving tolerance to salinity, drought, and methyl viologen stresses in Arabidopsis.