Ectopic over-expression of peroxisomal ascorbate peroxidase (SbpAPX) gene confers salt stress tolerance in transgenic peanut (Arachis hypogaea)

Peroxisomal ascorbate peroxidase gene (SbpAPX) of an extreme halophyte Salicornia brachiata imparts abiotic stress endurance and plays a key role in the protection against oxidative stress. The cloned SbpAPX gene was transformed to local variety of peanut and about 100 transgenic plants were developed using optimized in vitro regeneration and Agrobacterium mediated genetic transformation method. The T₀ transgenic plants were confirmed for the gene integration; grown under controlled condition in containment green house facility; seeds were harvested and T₁ plants were raised. Transgenic plants (T₁) were further confirmed by PCR using gene specific primers and histochemical GUS assay. About 40 transgenic plants (T₁) were selected randomly and subjected for salt stress tolerance study. Transgenic plants remained green however non-transgenic plants showed bleaching and yellowish leaves under salt stress conditions. Under stress condition, transgenic plants continued normal growth and completed their life cycle. Transgenic peanut plants exhibited adequate tolerance under salt stress condition and thus could be explored for the cultivation in salt affected areas for the sustainable agriculture.     

Overexpression of a GST gene (ThGSTZ1) from Tamarix hispida improves drought and salinity tolerance by enhancing the ability to scavenge reactive oxygen species

Although plant glutathione transferase (GST) genes are reported to be involved in responses to abiotic stress, few GST genes have been functionally characterized in woody halophytes. In the present study, a GST gene from Tamarix hispida, designated ThGSTZ1, was cloned and functionally characterized. Expression of ThGSTZ1 was downregulated by drought and salinity stress, and abscisic acid. Transgenic Arabidopsis thaliana plants with constitutive expression of ThGSTZ1 showed increased survival rates under drought and salinity stress. These transgenic Arabidopsis plants exhibited increased levels of GST, glutathione peroxidase, superoxide dismutase and peroxidase activity, along with decreased malondialdehyde content, electrolyte leakage rates and reactive oxygen species (ROS) levels under salt and drought stress conditions. Transgenic T. hispida that transiently overexpressed ThGSTZ1 showed increased GST and GPX activities under NaCl and mannitol treatments, as well as improved ROS scavenging ability. These results suggest that ThGSTZ1 can improve drought and salinity tolerance in plants by enhancing their ROS scavenging ability. Therefore, ThGSTZ1 represents a candidate gene with potential applications for molecular breeding to increase stress tolerance in plants.     

RhEXPA4, a rose expansin gene, modulates leaf growth and confers drought and salt tolerance to Arabidopsis

Drought and high salinity are major environmental conditions limiting plant growth and development. Expansin is a cell-wall-loosening protein known to disrupt hydrogen bonds between xyloglucan and cellulose microfibrils. The expression of expansin increases in plants under various abiotic stresses, and plays an important role in adaptation to these stresses. We aimed to investigate the role of the RhEXPA4, a rose expansin gene, in response to abiotic stresses through its overexpression analysis in Arabidopsis. In transgenic Arabidopsis harboring the Pro _RhEXPA4 ::GUS construct, RhEXPA4 promoter activity was induced by abscisic acid (ABA), drought and salt, particularly in zones of active growth. Transgenic lines with higher RhEXPA4 level developed compact phenotypes with shorter stems, curly leaves and compact inflorescences, while the lines with relatively lower RhEXPA4 expression showed normal phenotypes, similar to the wild type (WT). The germination percentage of transgenic Arabidopsis seeds was higher than that of WT seeds under salt stress and ABA treatments. Transgenic plants showed enhanced tolerance to drought and salt stresses: they displayed higher survival rates after drought, and exhibited more lateral roots and higher content of leaf chlorophyll a under salt stress. Moreover, high-level RhEXPA4 overexpressors have multiple modifications in leaf blade epidermal structure, such as smaller, compact cells, fewer stomata and midvein vascular patterning in leaves, which provides them with more tolerance to abiotic stresses compared to mild overexpressors and the WT. Collectively, our results suggest that RhEXPA4, a cell-wall-loosening protein, confers tolerance to abiotic stresses through modifying cell expansion and plant development in Arabidopsis.     

An obesity-like gene MdTLP7 from apple (Malus × domestica) enhances abiotic stress tolerance

Tubby-like proteins (TLPs) are found in a broad range of multicellular organisms. In mammals, genetic mutation of tubby or other TLPs can result in certain disease phenotypes related to animal specific characters: obesity, retinal degeneration, hearing loss, et al. Plants also harbor a large number of TLP genes, but the information in plants is far more limited. We identified a highly up-regulated obesity-like gene, MdTLP7, in our previous study of apple differential gene expression profile under chilling, indicating its possible role in plant abiotic stress tolerance. cDNA of MdTLP7 was amplified and expressed in Escherichia coli. In the solid and solution medium, the rate of growth and the quantity of the cell carrying MdTLP7 gene were significantly more than that of empty vector under salt and temperature stresses. To identify the functional region, serial deletion from both N-terminus and C-terminus of MdTLP7 was performed. In 415 amino acid polypeptide chain of MdTLP7, a middle conservative fragment (120–310 amino acid residues) played vital roles in stress tolerance. This fragment was involved in β barrel of Tubby domain according to the model of Tubby domain. All above results suggested MdTLP7 confers stress-tolerance to E. coli cell against abiotic stresses.     

Overexpression of a trehalose-6-phosphate synthase/phosphatase fusion gene enhances tolerance and photosynthesis during drought and salt stress without growth aberrations in tomato

Trehalose is a non-reducing disaccharide of glucose that confers tolerance against abiotic stresses in many diverse organisms, including higher plants. It was previously reported that overexpression of the yeast trehalose-6-phosphate synthase gene in tomato results in improved tolerance against abiotic stresses. However, these transgenic tomato plants had stunted growth and pleiotropic changes in appearance. In this study, transgenic tomato plants were generated by the introduction of a gene encoding a bifunctional fusion of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase genes from Escherichia coli under the control of the CaMV35S promoter. Transgenic plants accumulated higher levels of trehalose in their leaves and exhibited enhanced drought and salt tolerance and photosynthetic rates under salt stress conditions than wild-type plants. All of the transgenic plants had normal growth patterns and appearances. Therefore, the system described in this study can be used for practical application of the gene in crop improvement.     

MdVHP1 encodes an apple vacuolar H(+)-PPase and enhances stress tolerance in transgenic apple callus and tomato

Vacuolar H⁺-translocating inorganic pyrophosphatase (VHP, EC 3.6.1.1) is an electrogenic proton pump, which is related to growth as well as abiotic stress tolerance in plants. In this study, a VHP gene MdVHP1 was isolated from apple. The alignment of nucleotide and amino acid sequences showed that it encoded a type I VHP protein. It expressed in vegetative and reproductive organs, and its expression was induced by salt, PEG-mediated osmotic stress, cold and heat in apple in vitro shoot cultures. MdVHP1 expression showed a similar pattern in different apple tissues, but different change dynamics in response to abiotic stresses, compared with MdVHP2 (another MdVHP gene in apple). MdVHP1 overexpression enhanced tolerance to salt, PEG-mimic drought, cold and heat in transgenic apple calluses, which was related to an increased accumulation of proline and decreased MDA content compared with control calluses. In addition, MdVHP1 overexpression confers improved tolerance to salt and drought in transgenic tomato, along with an increased ion accumulation, high RWC and low solute potential compared with wild type. These results indicate that MdVHP1 is an important regulator for plant tolerance to abiotic stresses by modulating internal stores of ions and solutes.     

Ectopic expression of pigeonpea cold and drought regulatory protein (CcCDR) in yeast and tobacco affords multiple abiotic stress tolerance

Pigeonpea (Cajanus cajan L.) cold and drought regulatory protein encoding gene (CcCDR) has been introduced into yeast and tobacco for its functional validation. In yeast, expression of CcCDR imparted marked tolerance against abiotic stresses exerted by PEG and NaCl. Transgenic tobacco lines, expressing CcCDR under the control of CaMV35S and rd29A promoters, when subjected to mannitol, NaCl and cold (4 °C) stress, developed into healthy plants with profuse root system, increased biomass, root length and chlorophyll content in contrast to the weak-stunted wild-type plants. Transgenic plants also showed increased levels of proline, reducing sugars and endogenous abscisic acid (ABA) content. Exogenous ABA treatment resulted in increased hypersensitivity and decreased stomatal aperture size of transgenic plants compared to wild type. Localization studies confirmed that CcCDR could enter the nucleus as revealed by intense fluorescence, indicating its plausible interaction with various nuclear proteins. The overall results amply demonstrate the intrinsic effect of CcCDR in bestowing multiple abiotic stress tolerance at cellular and whole plant levels. Accordingly, the multipotent CcCDR seems promising as a prime candidate gene to fortify crop plants with abiotic stress tolerance.     

Developing Transgenic Jatropha Using the SbNHX1 Gene from an Extreme Halophyte for Cultivation in Saline Wasteland

Jatropha is an important second-generation biofuel plant. Salinity is a major factor adversely impacting the growth and yield of several plants including Jatropha. SbNHX1 is a vacuolar Na⁺/H⁺ antiporter gene that compartmentalises excess Na⁺ ions into the vacuole and maintains ion homeostasis. We have previously cloned and characterised the SbNHX1 gene from an extreme halophyte, Salicornia brachiata. Transgenic plants of Jatropha curcas with the SbNHX1 gene were developed using microprojectile bombardment mediated transformation. Integration of the transgene was confirmed by PCR and Rt-PCR and the copy number was determined by real time qPCR. The present study of engineering salt tolerance in Jatropha is the first report to date. Salt tolerance of the transgenic lines JL2, JL8 and JL19 was confirmed by leaf senescence assay, chlorophyll estimation, plant growth, ion content, electrolyte leakage and malondialdehyde (MDA) content analysis. Transgenic lines showed better salt tolerance than WT up to 200 mM NaCl. Imparting salt tolerance to Jatropha using the SbNHX1 gene may open up the possibility of cultivating it in marginal salty land, releasing arable land presently under Jatropha cultivation for agriculture purposes. Apart from this, transgenic Jatropha can be cultivated with brackish water, opening up the possibility of sustainable cultivation of this biofuel plant in salty coastal areas.     

Isolation and functional characterization of Salt overly sensitive 1 (SOS1) gene promoter from Salicornia brachiata

Soil salinity is a major abiotic stress and salt overly sensitive (SOS) pathway plays an important role in imparting tolerance to salinity by reinstating cellular ionic equilibrium. Salt overly sensitive 1 (SOS1) gene of SOS pathway has been implicated in increasing salt tolerance in plants. In this study, a 734 bp fragment of SOS1 promoter (SbUSOS1) was isolated from a halophyte Salicornia brachiata Roxb. In silico analysis of SbUSOS1 predicted several cis-acting regulatory elements such as DOF motif, GT elements, ABRE-like sequence, and root specific motifs. Functional validation of SbUSOS1 into tobacco stems and leaves using the GUS reporter gene showed that this promoter is induced by salt stress (250 mM NaCl) but not by ABA (500 μM) and cold (4 °C) stresses. This study indicated that SbUSOS1 was functional with predicted cis-acting elements that could be responsible for its salt-inducible nature. It can be used for the development of salt stress tolerant transgenic plants.     

A Medicago truncatula EF-Hand Family Gene,MtCaMP1, Is Involved in Drought and Salt Stress Tolerance

Background

Calcium-binding proteins that contain EF-hand motifs have been reported to play important roles in transduction of signals associated with biotic and abiotic stresses. To functionally characterize gens of EF-hand family in response to abiotic stress, an MtCaMP1 gene belonging to EF-hand family from legume model plant Medicago truncatula was isolated and its function in response to drought and salt stress was investigated by expressing MtCaMP1 in Arabidopsis.

Methodology/Principal Findings

Transgenic Arabidopsis seedlings expressing MtCaMP1exhibited higher survival rate than wild-type seedlings under drought and salt stress, suggesting that expression of MtCaMP1 confers tolerance of Arabidopsis to drought and salt stress. The transgenic plants accumulated greater amounts of Pro due to up-regulation of P5CS1 and down-regulation of ProDH than wild-type plants under drought stress. There was a less accumulation of Na⁺ in the transgenic plants than in WT plants due to reduced up-regulation of AtHKT1 and enhanced regulation of AtNHX1 in the transgenic plants compared to WT plants under salt stress. There was a reduced accumulation of H₂O₂ and malondialdehyde in the transgenic plants than in WT plants under both drought and salt stress.

Conclusions/Significance

The expression of MtCaMP1 in Arabidopsis enhanced tolerance of the transgenic plants to drought and salt stress by effective osmo-regulation due to greater accumulation of Pro and by minimizing toxic Na⁺ accumulation, respectively. The enhanced accumulation of Pro and reduced accumulation of Na⁺ under drought and salt stress would protect plants from water default and Na⁺ toxicity, and alleviate the associated oxidative stress. These findings demonstrate that MtCaMP1 encodes a stress-responsive EF-hand protein that plays a regulatory role in response of plants to drought and salt stress.     

Expression of Arabidopsis NDPK2 increases antioxidant enzyme activities and enhances tolerance to multiple environmental stresses in transgenic sweetpotato plants

Transgenic sweetpotato (Ipomoea batatas L. cv. Yulmi) plants expressing the Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) gene under the control of an oxidative stress–inducible peroxidase (SWPA2) promoter (referred to as SN plants) were developed and evaluated for enhanced tolerance of SN plants under various abiotic stress conditions. The level of AtNDPK2 expression and NDPK activity in SN plants following methyl viologen (MV) treatment was positively correlated with the plant’s tolerance to MV. Interestingly, we observed that antioxidant enzyme activities such as peroxidase, ascorbate peroxidase, and catalase increased in MV-treated SN plants. In addition, SN plants showed enhanced tolerance to cold, high salinity, and drought stresses by an increase in the activity of H₂O₂ scavenging enzymes. These results indicate that overexpression of AtNDPK2 in sweetpotato might efficiently modulate oxidative stress from various environmental stresses.