Salt stress alleviation in transgenic Vigna mungo L. Hepper (blackgram) by overexpression of the glyoxalase I gene using a novel Cestrum yellow leaf curling virus (CmYLCV) promoter

A reproducible and efficient transformation system utilizing the nodal regions of embryonal axis of blackgram (Vigna mungo L. Hepper) has been established via Agrobacterium tumefaciens. This is a report of genetic transformation of Vigna mungo for value addition of an agronomic trait, wherein the gene of interest, the glyoxalase I driven by a novel constitutive Cestrum yellow leaf curling viral promoter has been transferred for alleviating salt stress. The overexpression of this gene under the constitutive CaMV 35S promoter had earlier been shown to impart salt, heavy metal and drought stress tolerance in the model plant, tobacco. Molecular analyses of four independent transgenic lines performed by PCR, Southern and western blot revealed the stable integration of the transgene in the progeny. The transformation frequency was ca. 2.25% and the time required for the generation of transgenic plants was 10–11 weeks. Exposure of T1 transgenic plants as well as untransformed control plants to salt stress (100 mM NaCl) revealed that the transgenic plants survived under salt stress and set seed whereas the untransformed control plants failed to survive. The higher level of Glyoxalase I activity in transgenic lines was directly correlated with their ability to withstand salt stress. To the best of our knowledge this is the only report of engineering abiotic stress tolerance in blackgram. Prasanna Bhomkar, Chandrama P. Upadhyay are contributed equally. An erratum to this article can be found at     

Overexpression of Rice CBS Domain Containing Protein Improves Salinity, Oxidative, and Heavy Metal Tolerance in Transgenic Tobacco

We have recently identified and classified a cystathionine ??-synthase domain containing protein family in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa L.). Based on the microarray and MPSS data, we have suggested their involvement in stress tolerance. In this study, we have characterized a rice protein of unknown function, OsCBSX4. This gene was found to be upregulated under high salinity, heavy metal, and oxidative stresses at seedling stage. Transgenic tobacco plants overexpressing OsCBSX4 exhibited improved tolerance toward salinity, heavy metal, and oxidative stress. This enhanced stress tolerance in transgenic plants could directly be correlated with higher accumulation of OsCBSX4 protein. Transgenic plants could grow and set seeds under continuous presence of 150?mM NaCl. The total seed yield in WT plants was reduced by 80%, while in transgenic plants, it was reduced only by 15?C17%. The transgenic plants accumulated less Na⁺, especially in seeds and maintained higher net photosynthesis rate and Fv/Fm than WT plants under NaCl stress. Transgenic seedlings also accumulated significantly less H₂O₂ as compared to WT under salinity, heavy metal, and oxidative stress. OsCBSX4 overexpressing transgenic plants exhibit higher abiotic stress tolerance than WT plants suggesting its role in abiotic stress tolerance in plants.     

MusaDHN-1, a novel multiple stress-inducible SK3-type dehydrin gene, contributes affirmatively to drought- and salt-stress tolerance in banana

Dehydrins are highly hydrophilic proteins involved in playing key adaptive roles in response to abiotic stress conditions having dehydration as a common component. In the present study, a novel banana SK(3)-type dehydrin, MusaDHN-1, was identified and later characterized using transgenic banana plants to investigate its functions in abiotic stress tolerance. Expression profiling in native banana plants demonstrated that MusaDHN-1 was induced in leaves by drought, salinity, cold, oxidative and heavy metal stress as well as by treatment with signalling molecules like abscisic acid, ethylene and methyl jasmonate. Promoter analysis carried out by making a MusaDHN-1 promoter: β-glucuronidase fusion construct reconfirmed the abiotic stress inducibility of MusaDHN-1. Transgenic banana plants constitutively overexpressing MusaDHN-1 were phenotypically normal and displayed improved tolerance to drought and salt-stress treatments in both in vitro and ex vitro assays. Enhanced accumulation of proline and reduced malondialdehyde levels in drought and salt-stressed MusaDHN-1 overexpressing plants further established their superior performance in stressed conditions. This study is the first to report generation of transgenic banana plants engineered for improved drought and salt-stress tolerance.     

Physiological evaluation of drought stress tolerance and recovery in Verbascum sinuatum plants treated with methyl jasmonate,salicylic acid and titanium dioxide nanoparticles

Abstract

The genus Verbascum L. (Scrophulariaceae) includes medicinal plants, which have several bioactive compounds especially saponins. The possible recovery ability of Verbascum sinuatum from drought stress conditions was assessed by using salicylic acid (SA), methyl jasmonate (MJA) and titanium dioxide nanoparticles (TiO₂NPs) as plant growth regulators (PGRs) in liquid culture media. Thirty days-old plants were exposed to different concentrations of polyethylene glycol (PEG-6000) for creating artificial drought conditions (0, ?0.3, and ?0.6?MPa osmotic potential) and also treated with 200?µM methyl jasmonate (MJA), 100?µM salicylic acid (SA) and 20?ppm TiO₂ nanoparticles (TiO₂NPs). Results showed that the growth parameters and the content of photosynthetic pigments decreased at higher drought level (?0.6?MPa). However, SA and TiO₂NPs alleviated the adverse effects of drought stress by increasing water stress tolerance through promotion of enzymatic and nonenzymatic antioxidant defense systems. MJA negatively affected the growth parameters and increased the content of malondialdehyde (MDA), hydrogen peroxide (H₂O₂) and total saponin and also the activity of peroxidase (POD) and polyphenol oxidase (PPO). Based on the results obtained from this study, the recovery treatments mainly affected the defense-related metabolism in Verbasum sinuatum plants.     

Cotton metallothionein GhMT3a, a reactive oxygen species scavenger, increased tolerance against abiotic stress in transgenic tobacco and yeast

A cDNA clone encoding a 64-amino acid type 3 metallothioneinprotein, designated GhMT3a, was isolated from cotton (Gossypiumhirsutum) by cDNA library screening. Northern blot analysisindicated that mRNA accumulation of GhMT3a was up-regulatednot only by high salinity, drought, and low temperature stresses,but also by heavy metal ions, abscisic acid (ABA), ethylene,and reactive oxygen species (ROS) in cotton seedlings. Transgenictobacco (Nicotiana tabacum) plants overexpressing GhMT3a showedincreased tolerance against abiotic stresses compared with wild-typeplants. Interestingly, the induced expression of GhMT3a by salt,drought, and low-temperature stresses could be inhibited inthe presence of antioxidants. H₂O₂ levels in transgenic tobaccoplants were only half of that in wild-type (WT) plants undersuch stress conditions. According to in vitro assay, recombinantGhMT3a protein showed an ability to bind metal ions and scavengeROS. Transgenic yeast overexpressing GhMT3a also showed highertolerance against ROS stresses. Taken together, these resultsindicated that GhMT3a could function as an effective ROS scavengerand its expression could be regulated by abiotic stresses throughROS signalling. Key words: Abiotic stress, antioxidant, GhMT3a, ROS, transgenic tobacco, yeast     

Ectopic expression of wheat expansin gene TaEXPA2 improved the salt tolerance of transgenic tobacco by regulating Na+/K+ and antioxidant competence

High salinity is one of the most serious environmental stresses that limit crop growth. Expansins are cell wall proteins that regulate plant development and abiotic stress tolerance by mediating cell wall expansion. We studied the function of a wheat expansin gene, TaEXPA2, in salt stress tolerance by overexpressing it in tobacco. Overexpression of TaEXPA2 enhanced the salt stress tolerance of transgenic tobacco plants as indicated by the presence of higher germination rates, longer root length, more lateral roots, higher survival rates and more green leaves under salt stress than in the wild type (WT). Further, when leaf disks of WT plants were incubated in cell wall protein extracts from the transgenic tobacco plants, their chlorophyll content was higher under salt stress, and this improvement from TaEXPA2 overexpression in transgenic tobacco was inhibited by TaEXPA2 protein antibody. The water status of transgenic tobacco plants was improved, perhaps by the accumulation of osmolytes such as proline and soluble sugar. TaEXPA2‐overexpressing tobacco lines exhibited lower Na⁺ but higher K⁺ accumulation than WT plants. Antioxidant competence increased in the transgenic plants because of the increased activity of antioxidant enzymes. TaEXPA2 protein abundance in wheat was induced by NaCl, and ABA signaling was involved. Gene expression regulation was involved in the enhanced salt stress tolerance of the TaEXPA2 transgenic plants. Our results suggest that TaEXPA2 overexpression confers salt stress tolerance on the transgenic plants, and this is associated with improved water status, Na⁺/K⁺ homeostasis, and antioxidant competence. ABA signaling participates in TaEXPA2‐regulated salt stress tolerance.