Overexpression of <Emphasis Type="Italic">IrlVHA</Emphasis>-<Emphasis Type="Italic">c</Emphasis>, A Vacuolar-Type H<Superscript>+</Superscript>-ATPase c Subunit Gene from <Emphasis Type="Italic">Iris lactea</Emphasis>, Enhances Salt Tolerance in Tobacco

Vacuolar-type H⁺-ATPase (V-ATPase), a multi-subunit endomembrane proton pump, plays an important role in plant growth and response to environmental stresses. In the present study, transgenic tobacco that overexpressed the V-ATPase c subunit gene from Iris lactea (IrlVHA-c) was used to determine the function of IrlVHA-c. Quantitative PCR analysis showed that IrlVHA-c expression was induced by salt stress in I. lactea roots and leaves. Subcellular localization of green fluorescent protein (GFP) as marker combined with FM4-64 staining showed that the IrlVHA-c-GFP was localized to the endosomal compartment in tobacco cells. Compared with the wild-type, the IrlVHA-c transgenic tobacco plants exhibited greater seed germination rates, root length, fresh weight, and higher relative water content (RWC) of leaves under salt stress. Furthermore, the IrlVHA-c transgenic tobacco leaves have lower stomatal densities and larger stomatal apertures than wild-type. Under salt stress, superoxide dismutase (SOD) activity in the transgenic tobacco was significantly enhanced. Moreover, the level of malondialdehyde (MDA) in the transgenic tobacco was significantly lower than that in wild-type plants under salt stress. Taken together, these results suggested that the IrlVHA-c plays an important role in salt tolerance in transgenic tobacco by influencing stomatal movement and physiological changes.     

An aquaporin gene from halophyte <Emphasis Type="Italic">Sesuvium portulacastrum</Emphasis>, <Emphasis Type="Italic">SpAQP1</Emphasis>, increases salt tolerance in transgenic tobacco

Key message

SpAQP1 was strongly induced by salt in an ABA-independent way, promoted seed germination and root growth in transgenic tobaccos and increased salt tolerance by increasing the activities of antioxidative enzymes.

Abstract

Aquaporin (AQP) plays crucial roles in the responses of plant to abiotic stresses such as drought, salt and cold. Compared to glycophytes, halophytes often have excellent salt and drought tolerances. To uncover the molecular mechanism of halophyte Sesuvium portulacastrum tolerance to salt, in this study, an AQP gene, SpAQP1, from S. portulacastrum was isolated and characterized. The amino acid sequence of SpAQP1 shared high homology with that of plant plasma membrane intrinsic proteins (PIPs) and contained the distinct molecular features of PIPs. In the phylogenic tree, SpAQP1 was evidently classified as the PIP2 subfamily. SpAQP1 is expressed in roots, stems and leaves, and was significantly induced by NaCl treatment and inhibited by abscisic acid (ABA) treatment. When heterologously expressed in yeast and tobacco, SpAQP1 enhanced the salt tolerance of yeast strains and tobacco plants and promoted seed germination and root growth under salt stress in transgenic plants. The activity of antioxidative enzymes including superoxide dismutase, peroxidase and catalase was increased in transgenic plants overexpressing SpAQP1. Taken together, our studies suggested that SpAQP1 functioned in the responses of S. portulacastrum to salt stress and could increase salt tolerance by enhancing the antioxidative activity of plants.

     

A new <Emphasis Type="Italic">Em</Emphasis>-like protein from <Emphasis Type="Italic">Lactuca sativa</Emphasis>, <Emphasis Type="Italic">LsEm1</Emphasis>, enhances drought and salt stress tolerance in <Emphasis Type="Italic">Escherichia coli</Emphasis> and rice

Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these genes may be involved in the enhanced drought and salt tolerance of transgenic rice. Thus, this work paves the way for improvement in tolerance of crops by genetic engineering breeding.     

Improving tobacco freezing tolerance by co-transfer of stress-inducible <Emphasis Type="Italic">CbCBF</Emphasis> and <Emphasis Type="Italic">CbICE53</Emphasis> genes

Cold stress is one of the major limitations to crop productivity worldwide. We investigated the effects of multiple gene expression from cold tolerant Capsella bursa-pastoris in transgenic tobacco (Nicotiana tabaccum) plants. We combined CblCE53 and CbCBF into a reconstruct vector by isocaudomers. Plant overexpression of CbICE53 under the stress inducible CbCOR15b promoter and CbCBF under a constitutive promoter showed increased tolerance to both chilling and freezing temperatures in comparison to wild-type plants, according to the electrolyte leakage and relative water content. The expressions of endogenous cold-responsive genes in transgenic tobacco (NtDREB1, NtDREB3, NtERD10a and NtERD10b) were obviously upregulated under normal and low temperature conditions. These results suggest that the CbICE53 + CbCBF transgenic plants showed a much greater cold tolerance as well as no dwarfism and delayed flowering. Thus they can be considered as a potential candidate for transgenic engineering for cold tolerant tobacco.     

Salt stress induced soybean <Emphasis Type="Italic">GmIFS1</Emphasis> expression and isoflavone accumulation and salt tolerance in transgenic soybean cotyledon hairy roots and tobacco

Effects of isoflavones on plant salt tolerance were investigated in soybean (Glycine max L. Merr. cultivar N23674) and tobacco (Nicotiana tabacum L.). Leaf area, fresh weight, net photosynthetic rate (Pn), and transpiration rate (Tr) of soybean N23674 plants treated with 80 mM NaCl were significantly reduced, while a gene (GmIFS1) encoding for 2-hydroxyisoflavone synthase was highly induced, and isoflavone contents significantly increased in leaves and seeds. To test the impact of isoflavones to salt tolerance, transgenic soybean cotyledon hairy roots expressing GmIFS1 (hrGmIFS1) were produced. Salt stress slightly increased isoflavone content in hairy roots of the transgenic control harboring the empty vector but substantially reduced the maximum root length, root fresh weight, and relative water content (RWC). The isoflavone content in hrGmIFS1 roots, however, was significantly higher, and the above-mentioned root growth parameters decreased much less. The GmIFS1 gene was also transformed into tobacco plants; plant height and leaf fresh weight of transgenic GmIFS1 tobacco plants were much greater than control plants after being treated with 85 mM NaCl. Leaf antioxidant capacity of transgenic tobacco was significantly higher than the control plants. Our results suggest that salt stress-induced GmIFS1 expression increased isoflavone accumulation in soybean and improved salt tolerance in transgenic soybean hairy roots and tobacco plants.     

Increase in Salt Tolerance of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> by <Emphasis Type="Italic">TaDi19</Emphasis>

The gene expression profile chip of salt-resistant wheat mutant RH8706-49 under salt stress was investigated. The overall length of the cDNA sequence of the probe was obtained using electronic cloning and RT-PCR. An unknown gene induced by salt was obtained, cloned, and named TaDi19 (Triticum aestivum drought-induced protein). No related report or research on the protein is available. qPCR analysis showed that gene expression was induced by many stresses, such as salt. Arabidopsis thaliana was genetically transferred using the overexpressing gene, which increased its salt tolerance. After salt stress, the transgenic plant demonstrated better physiological indicators (higher Ca²⁺ and lower Na⁺) than those of the wild-type plant. Results of non-invasive micro-test technology indicate that TaDi19-overexpressing A. thaliana significantly effluxed Na⁺ after salt treatment, whereas the wild-type plant influxed Na⁺. Chelating extracellular Ca²⁺ resulted in insignificant differences in salt tolerance between overexpressing and wild-type A. thaliana. Subcellular localization showed that the gene encoding protein was mainly located in the cell membrane and nucleus. TaDi19 was overexpressed in wild-type A. thaliana, and the transgenic lines were more salt-tolerant than the control A. thaliana. Thus, the wheat gene TaDi19 could increase the salt tolerance of A. thaliana.     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

A <Emphasis Type="Italic">Vitis vinifera</Emphasis> xanthine dehydrogenase gene, <Emphasis Type="Italic">VvXDH</Emphasis>, enhances salinity tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Xanthine dehydrogenase (EC1.1.1.204; XDH) plays an important role in purine catabolism that catalyzes the oxidative hydroxylation of hypoxanthine to xanthine and of xanthine to uric acid. Long attributed to its role in recycling and remobilization of nitrogen, recently, XDH is implicated in plant stress responses and acclimation, such research efforts, however, have thus far been restricted to Arabidopsis XDH-knockdown/knockout studies. This study, using an ectopic overexpression approach, is expected to provide novel findings. In this study, a XDH gene from Vitis vinifera, named VvXDH, was synthesized and overexpressed in Arabidopsis, the transgenic Arabidopsis showed enhanced salt tolerance. The VvXDH gene was investigated and the results demonstrated the explicit role of VvXDH in conferring salt stress by increasing allantoin accumulation and activating ABA signaling pathway, enhancing ROS scavenging in transgenic Arabidopsis. In addition, the water loss and chlorophyll content loss were reduced in transgenic plants; the transgenic plants showed higher proline level and lower MDA content than that of wild-type Arabidopsis, respectively. In conclusion, the VvXDH gene has the potential to be applied in increasing allantoin accumulation and enhancing the tolerance to abiotic stresses in Arabidopsis and other plants.     

Overexpression of <Emphasis Type="Italic">MuHSP70</Emphasis> gene from <Emphasis Type="Italic">Macrotyloma uniflorum</Emphasis> confers multiple abiotic stress tolerance in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A 70-KD heat shock protein (HSP70) is one of the most conserved chaperones. It is involved in de novo protein folding and prevents the aggregation of unfolded proteins under lethal environmental factors. The purpose of this study is to characterise a MuHSP70 from horsegram (Macrotyloma uniflorum) and elucidating its role in stress tolerance of plants. A MuHSP70 was cloned and characterised from a natural drought stress tolerant HPK4 variety of horsegram (M. uniflorum). For functional characterization, MuHSP70 was overexpressed in transgenic Arabidopsis. Overexpression of MuHSP70 was found to provide tolerance to the transgenic Arabidopsis against various stresses such as heat, cold, drought, salinity and oxidative stress. MuHSP70 transgenics were observed to maintain the shoot biomass, root length, relative water content, and chlorophyll content during exposure to multi-stresses relative to non-transgenic control. Transgenic lines have further shown the reduced levels of MDA, H₂O₂, and proteolytic activity. Together, these findings suggest that overexpression of MuHSP70 plays an important role in improving abiotic stress tolerance and could be a crucial candidate gene for exploration in crop improvement program.     

A chalcone synthase gene <Emphasis Type="Italic">AeCHS</Emphasis> from <Emphasis Type="Italic">Abelmoschus esculentus</Emphasis> regulates flavonoid accumulation and abiotic stress tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Chalcone synthase (CHS) is one of the key enzymes in flavonoid biosynthesis pathway in plants. However, the roles of AeCHS gene from Abelmoschus esculentus in flavonoid accumulation and tolerance to abiotic stresses have not been studied. In this study, the AeCHS gene was cloned from Abelmoschus esculentus. The open reading frame contained 1170 nucleotides encoding 389 amino acids. The coding region of AeCHS was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis to obtain transgenic plants. Overexpression of AeCHS increased the production of downstream flavonoids and the expression of related genes in the flavonoid biosynthesis pathway. It also improved resistance to salt and mannitol stresses during seed germination and root development. Further component and enzymatic analyses showed the decreased content of H₂O₂ and malondialdehyde and the increased activities of superoxide dismutase (SOD) and peroxidase (POD) in transgenic seedlings. Meanwhile, the expression level of AtSOD and AtPOD genes was up-regulated against salt and osmotic stresses. Together, our finding indicated that changing the expression level of AeCHS in plants alters the accumulation of flavonoids and regulates plantlet tolerance to abiotic stress by maintaining ROS homeostasis. The AeCHS gene has the potential to be used to increase the content of valuable flavonoids and improve the tolerance to abiotic stresses in plants.     

A novel <Emphasis Type="Italic">TaSST</Emphasis> gene from wheat contributes to enhanced resistance to salt stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

In this research, through the analyzing of the Triticum aestivum salt-tolerant mutant gene expression profile, under salt stress. A brand new gene with unknown functions induced by salt was cloned. The cloned gene was named Triticum aestivum salt stress protein (TaSST). GenBank accession number of TaSST is ACH97119. Quantitative polymerase chain reaction (qPCR) results exhibited that the expression TaSST was induced by salt, abscisic acid (ABA), and polyethylene glycol (PEG). TaSST could improve salt tolerance of Arabidopsis-overexpressed TaSST. After salt stress, physiological indexes of transgenic Arabidopsis were better compared with WT (wild-type) plants. TaSST was mainly located in the cytomembrane. qPCR analyzed the expression levels of nine tolerance-related genes of Arabidopsis in TaSST-overexpressing Arabidopsis. Results showed that the expression levels of SOS3, SOS2, KIN2, and COR15a significantly increased, whereas the expression of the five other genes showed no obvious change. OsI_01272, the homologous gene of TaSST in rice, was interfered using RNA interference (RNAi) technique. RNAi plants became more sensitive to salt than control plants. Thus, we speculate that TaSST can improve plant salt tolerance.     

Morphological changes and increase of resistance to oxidative stress by overexpression of the <Emphasis Type="Italic">LebZIP2</Emphasis> gene in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

The tomato bZIP2-encoding gene was inserted into the Nicotiana benthamiana genome using Agrobacterium-mediated transformation to characterize resistance to oxidative stress and two herbicides, glyphosate and paraquat. We produced transgenic tobacco plants using the LebZIP2 gene, which were then utilized to examine salt stress and herbicide resistance through oxidative mechanisms. Transgenic LebZIP2-overexpressing plants were examined using specific primers for selection marker genes (PCR using genomic DNA) and target genes (RT-PCR). Based on microscopic examination, we observed an increase in leaf thickness and cell number in transgenic plants. The electrolyte leakage of leaves suggested that LebZIP2-overexpressing lines were weak tolerant to NaCl stress and resistant to methyl viologen. During our analysis, transgenic lines were exposed to different herbicides. Transgenic plants showed an increased tolerance based on visual injury, as well as an increased biomass. Based on these results, the LebZIP2 gene may be involved in oxidative stress tolerance and cell development in plants.