The yeast HAL1 gene improves salt tolerance of transgenic tomato

Overexpression of the HAL1 gene in yeast has a positive effect on salt tolerance by maintaining a high internal K(+) concentration and decreasing intracellular Na(+) during salt stress. In the present work, the yeast gene HAL1 was introduced into tomato (Lycopersicon esculentum Mill.) by Agrobacterium tumefaciens-mediated transformation. A sample of primary transformants was self-pollinated, and progeny from both transformed and non-transformed plants (controls) were evaluated for salt tolerance in vitro and in vivo. Results from different tests indicated a higher level of salt tolerance in the progeny of two different transgenic plants bearing four copies or one copy of the HAL1 gene. In addition, measurement of the intracellular K(+) to Na(+) ratios showed that transgenic lines were able to retain more K(+) than the control under salt stress. Although plants and yeast cannot be compared in an absolute sense, these results indicate that the mechanism controlling the positive effect of the HAL1 gene on salt tolerance may be similar in transgenic plants and yeast.     

The cotton GhNHX1 gene encoding a novel putative tonoplast Na(+)/H(+) antiporter plays an important role in salt stress

A cDNA clone was isolated from cotton (Gossypium hirsutum) cDNA library and characterized with regard to its sequence, regulation in response to salt stress and functions in yeast mutants and transgenic tobacco plants. The clone, designated as GhNHX1, contains 2485 nucleotides with an open reading frame of 1629 nucleotides, and the deduced amino acid sequence showed high identities with other plant vacuolar-type Na(+)/H(+) antiporters. Northern blot analysis indicated that the mRNA accumulation of GhNHX1 was strongly induced by salt stress and abscisic acid in cotton seedlings. The expression of GhNHX1 in yeast Na(+)/H(+) antiporter mutant showed function complementation. The transgenic tobacco plants overexpressing GhNHX1 also had higher salt tolerance than the wild-type plants. The salt-induced mRNA level of GhNHX1 was 3 and 7 times higher in the salt-tolerant cotton cultivar ZM3 than those in the salt-sensitive cotton cultivars ZMS17 and ZMS12, respectively. Together, these results suggest that the products of the novel gene, GhNHX1, function as a tonoplast Na(+)/H(+) antiporter and play an important role in salt tolerance of cotton.     

<Emphasis Type="Italic">Xerophyta viscosa</Emphasis> Aldose Reductase,XvAld1, Enhances Drought Tolerance in Transgenic Sweetpotato

Sweetpotato is a significant crop which is widely cultivated particularly in the developing countries with high and stable yield. However, drought stress is a major limiting factor that antagonistically influences the crop’s productivity. Dehydration stress caused by drought causes aggregation of reactive oxygen species (ROS) in plants, and aldose reductases are first-line safeguards against ROS caused by oxidative stress. In the present study, we generated transgenic sweetpotato plants expressing aldose reductase, XvAld1 isolated from Xerophyta viscosa under the control of a stress-inducible promoter via Agrobacterium-mediated transformation. Our results demonstrated that the transgenic sweetpotato lines displayed significant enhanced tolerance to simulated drought stress and enhanced recuperation after rehydration contrasted with wild-type plants. In addition, the transgenic plants exhibited improved photosynthetic efficiency, higher water content and more proline accumulation under dehydration stress conditions compared with wild-type plants. These results demonstrate that exploiting the XvAld1 gene is not only a compelling and attainable way to improve sweetpotato tolerance to drought stresses without causing any phenotypic imperfections but also a promising gene candidate for more extensive crop improvement.     

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.     

Enhanced salinity tolerance and improved yield properties in Bangladeshi rice Binnatoa through <Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated transformation of <Emphasis Type="Italic">PgNHX1</Emphasis> from <Emphasis Type="Italic">Pennisetum glaucum</Emphasis>

Rice yield is severely affected by high-salt concentration in the vicinity of the plant. In an effort to engineer rice for improved salt tolerance Agrobacterium-mediated transformation of rice cv. Binnatoa was accomplished with the Pennisetum glaucum vacuolar Na⁺/H⁺ antiporter gene (PgNHX1) under the constitutive CaMV35S promoter. For the molecular analysis of putative transgenic plants, PCR and RT-PCR were performed. Transgenic rice plants expressing PgNHX1 showed better physiological status and completed their life cycle by setting flowers and seeds in salt stress, while wild-type plants exhibited rapid chlorosis and growth inhibition. Moreover, transgenic rice plants produced higher grain yields than wild-type plants under salt stress. Assessment of the salinity tolerance of the transgenic plants at seedling and reproductive stages demonstrated the potential of PgNHX1 for imparting enhanced salt tolerance capabilities and improved yield.     

Improved salt tolerance of transgenic tobacco expressing apoplastic yeast-derived invertase

We investigated the salt tolerance of transgenic tobacco, in which yeast invertase is expressed in the apoplastic (Apo-Inv) spaces. Whereas photosynthetic activities in wild-type tobacco in light were inhibited under salt stress, transgenic Apo-Inv tobacco maintained constant photosynthetic activities. The physical appearance of plants under salt stress also indicates that yeast invertase expression in the apoplastic space is beneficial for inducing salt tolerance. Apo-Inv tobacco had a much higher osmotic pressure increase in the cell sap than did wild-type tobacco under this type of stress. The physiological importance of sucrose metabolism under salt stress is discussed.     

Salt tolerance-related protein STO binds to a Myb transcription factor homologue and confers salt tolerance in Arabidopsis

Regulating the intracellular Na+/K+ ratio is an essential process for salinity tolerance. The yeast mutant, can, which is deficient in calcineurin, can not grow on medium containing Na+ because it is unable to regulate the intracellular Na+/K+ ratio. Expression of the STO gene of Arabidopsis thaliana in the can mutant complements the salt-sensitive phenotype. A protein of Arabidopsis, an H-protein promoter binding factor (HPPBF-1), that binds to STO protein was isolated. HPPBF-1 cDNA has a sequence encoding a Myb DNA binding-motif and its gene expression is induced by salt stress. Furthermore, HPPBF-1 protein is localized in the nucleus. Although, the expression level of STO is not induced under salt-stress conditions, overexpression of STO in a transgenic Arabidopsis plant gave it a higher salt tolerance than was observed in the wild type. When STO transgenic plants and wild-type plants were subjected to salt stress, root growth was increased by 33-70% in the transgenic plants under salt stress. These results suggest that STO is involved in salt-stress responses in Arabidopsis.     

Increase of glycinebetaine synthesis improves drought tolerance in cotton

The tolerance to drought stress of the homozygous transgenic cotton (Gossypium hirsutum L.) plants with enhanced glycinebetaine (GB) accumulation was investigated at three development stages. Among the five transgenic lines investigated, lines 1, 3, 4, and 5 accumulated significantly higher levels of GB than the wild-type (WT) plants either before or after drought stress, and the transgenic plants were more tolerant to drought stress than the wild-type counterparts from young seedlings to flowering plants. Under drought stress conditions, transgenic lines 1, 3, 4, and 5 had higher relative water content, increased photosynthesis, better osmotic adjustment (OA), a lower percentage of ion leakage, and less lipid membrane peroxidation than WT plants. The GB levels in transgenic plants were positively correlated with drought tolerance under water stress. The results suggested that GB may not only protect the integrity of the cell membrane from drought stress damage, but also be involved in OA in transgenic cotton plants. Most importantly, the seedcotton yield of transgenic line 4 was significantly greater than that of WT plants after drought stress, which is of great value in cotton production.     

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.     

The overaccumulation of glycinebetaine alleviated damages to PSII of wheat flag leaves under drought and high temperature stress combination

To analyze the physiological mechanisms underlying the increased tolerance to drought and high temperature stress combination by overproduction of glycinebetaine (GB) in wheat, a transgenic wheat line T6 and its wild-type (WT) Shi4185 were used. The transgenic line was generated by introducing a gene encoding betaine aldehyde dehydrogenase (BADH) into a wheat line Shi4185. The gene was cloned from Garden Orache (Atriplex hortensis L.). Wheat plants were exposed to drought (withholding irrigation), high temperature stress (40 °C), and their combination at the flowering stage. Analyses of oxygen-evolving activity and photosystem II (PSII) photochemistry, modulated chlorophyll fluorescence, rapid fluorescence induction kinetics, and the polyphasic fluorescence transients (OJIP) were used to evaluate PSII photochemistry in wheat plants. The results suggest that the PSII in transgenic plants showed higher resistance than that in wild-type plants under the stresses studied here, this increased tolerance was associated with an improvement in stability of the oxygen-evolving complex and the reaction center of PSII; streptomycin treatment can impair the protective effect of overaccumulated GB on PSII. The overaccumulated GB may protect the PSII complex from damage through accelerating D1 protein turnover to alleviate photodamage. The results also suggest that the PSII under combined high temperature and drought stress shows higher tolerance than under high temperature stress alone in both transgenic and wild-type plants.     

Over-expression of OsUGE-1 altered raffinose level and tolerance to abiotic stress but not morphology in Arabidopsis

OsUGE-1 is known to be induced by various abiotic stresses, but its exact function in plants is unclear. In the present study, OsUGE-1 was over-expressed in Arabidopsis, transgenic plants conferred tolerance to salt, drought and freezing stress without altering plant morphology. In addition, transgenic plants showed a higher level of the soluble sugar raffinose than did wild-type plants. Our results suggest that elevated level of raffinose with over-expressed OsUGE-1 resulted in enhanced tolerance to abiotic stress. Thus, the gene may be applied to improve tolerance to abiotic stress in crops.     

Heterology expression of the Arabidopsis C-repeat/dehydration response element binding factor 1 gene confers elevated tolerance to chilling and oxidative stresses in transgenic tomato

In an attempt to improve stress tolerance of tomato (Lycopersicon esculentum) plants, an expression vector containing an Arabidopsis C-repeat/dehydration responsive element binding factor 1 (CBF1) cDNA driven by a cauliflower mosaic virus 35S promoter was transferred into tomato plants. Transgenic expression of CBF1 was proved by northern- and western-blot analyses. The degree of chilling tolerance of transgenic T(1) and T(2) plants was found to be significantly greater than that of wild-type tomato plants as measured by survival rate, chlorophyll fluorescence value, and radical elongation. The transgenic tomato plants exhibited patterns of growth retardation; however, they resumed normal growth after GA(3) (gibberellic acid) treatment. More importantly, GA(3)-treated transgenic plants still exhibited a greater degree of chilling tolerance compared with wild-type plants. Subtractive hybridization was performed to isolate the responsive genes of heterologous Arabidopsis CBF1 in transgenic tomato plants. CATALASE1 (CAT1) was obtained and showed activation in transgenic tomato plants. The CAT1 gene and catalase activity were also highly induced in the transgenic tomato plants. The level of H(2)O(2) in the transgenic plants was lower than that in the wild-type plants under either normal or cold conditions. The transgenic plants also exhibited considerable tolerance against oxidative damage induced by methyl viologen. Results from the current study suggest that heterologous CBF1 expression in transgenic tomato plants may induce several oxidative-stress responsive genes to protect from chilling stress.     

Improvement of drought tolerance and grain yield in common bean by overexpressing trehalose-6-phosphate synthase in rhizobia

Improving stress tolerance and yield in crops are major goals for agriculture. Here, we show a new strategy to increase drought tolerance and yield in legumes by overexpressing trehalose-6-phosphate synthase in the symbiotic bacterium Rhizobium etli. Phaseolus vulgaris (common beans) plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene had more nodules with increased nitrogenase activity and higher biomass compared with plants inoculated with wild-type R. etli. In contrast, plants inoculated with an R. etli mutant in trehalose-6-phosphate synthase gene had fewer nodules and less nitrogenase activity and biomass. Three-week-old plants subjected to drought stress fully recovered whereas plants inoculated with a wild-type or mutant strain wilted and died. The yield of bean plants inoculated with R. etli overexpressing trehalose-6-phosphate synthase gene and grown with constant irrigation increased more than 50%. Macroarray analysis of 7,200 expressed sequence tags from nodules of plants inoculated with the strain overexpressing trehalose-6-phosphate synthase gene revealed upregulation of genes involved in stress tolerance and carbon and nitrogen metabolism, suggesting a signaling mechanism for trehalose. Thus, trehalose metabolism in rhizobia is key for signaling plant growth, yield, and adaptation to abiotic stress, and its manipulation has a major agronomical impact on leguminous plants.     

Trehalose accumulation in Azospirillum brasilense improves drought tolerance and biomass in maize plants

Bacteria of the genus Azospirillum increase the grain yield of several grass crops. In this work the effect of inoculating maize plants with genetically engineered Azospirillum brasilense for trehalose biosynthesis was determined. Transformed bacteria with a plasmid harboring a trehalose biosynthesis gene-fusion from Saccharomyces cerevisiae were able to grow up to 0.5 M NaCl and to accumulate trehalose, whereas wild-type A. brasilense did not tolerate osmotic stress or accumulate significant levels of the disaccharide. Moreover, 85% of maize plants inoculated with transformed A. brasilense survived drought stress, in contrast with only 55% of plants inoculated with the wild-type strain. A 73% increase in biomass of maize plants inoculated with transformed A. brasilense compared with inoculation with the wild-type strain was found. In addition, there was a significant increase of leaf and root length in maize plants inoculated with transformed A. brasilense . Therefore, inoculation of maize plants with A. brasilense containing higher levels of trehalose confers drought tolerance and a significant increase in leaf and root biomass. This work opens the possibility that A. brasilense modified with a chimeric trehalose biosynthetic gene from yeast could increase the biomass, grain yield and stress tolerance in other relevant crops.     

转AtNHX1基因杨树Tr品系的耐盐性研究

以不同盐分强度处理欧美107杨(Populus × euramericana ‘Neva’) (Wt)和转拟南芥液泡膜Na⁺/H⁺逆向转运蛋白基因AtNHX1欧美107杨新品系(Tr)幼苗, 揭示Tr和Wt两品系幼苗耐盐性的差异, 探索拟南芥液泡膜Na⁺/H⁺逆向转运蛋白基因AtNHX1对提高杨树耐盐能力的效应。结果表明: 低盐处理下, Wt植株生长明显受到抑制, 其干重显著低于对照, 盐分强度加大后, 抑制作用更大, 其干重只有对照的50%; 而Tr植株在低盐处理下干重与对照差异不显著, 高盐处理时其干重为对照的74%。同时, 不同盐度处理下, Tr的干重均显著高于Wt, 且随着盐度升高, 两品系间植株干重差异增大。盐处理后, Tr植株叶片叶绿素和类胡萝卜素的含量均显著高于Wt, 并能维持较高的净光合速率(P_n)和PSII最大光化学效率(F_v/F_m); 在盐处理下虽然Tr叶片和根系均较Wt积累了更多的Na⁺, 但同时也维持了更高的K⁺和K⁺/Na⁺比率, 而且叶片对K⁺选择性的运输明显高于Wt; 同时, Tr叶片MDA含量和电解质渗漏率显著低于Wt。可见, 在盐处理下转AtNHX1植株较未转基因植株维持了更高的生长量、光合色素、光合能力和叶片质膜稳定性, 说明AtNHX1的转入能够显著提高欧美107杨的耐盐性。     

Cloning of an H+-PPase gene from Thellungiella halophila and its heterologous expression to improve tobacco salt tolerance

An H(+)-pyrophosphatase (PPase) gene named TsVP involved in basic biochemical and physiological mechanisms was cloned from Thellungiella halophila. The deduced translation product has similar characteristics to H(+)-PPases from other species, such as Arabidopsis and rice, in terms of bioinformation. The heterologous expression of TsVP in the yeast mutant ena1 suppressed Na(+) hypersensitivity and demonstrated the function of TsVP as an H(+)-PPase. Transgenic tobacco overexpressing TsVP had 60% greater dry weight than wild-type tobacco at 300 mM NaCl and higher viability of mesophyll protoplasts under salt shock stress conditions. TsVP and AVP1, another H(+)-PPase from Arabidopsis, were heterologously expressed separately in both the yeast mutant ena1 and tobacco. The salt tolerance of TsVP or AVP1 yeast transformants and transgenic tobacco were improved to almost the same level. The TsVP transgenic tobacco lines TL3 and TL5 with the highest H(+)-PPase hydrolytic activity were studied further. These transgenic tobacco plants accumulated 25% more solutes than wild-type plants without NaCl stress and 20-32% more Na(+) under salt stress conditions. Although transgenic tobacco lines TL3 and TL5 accumulated more Na(+) in leaf tissues, the malondialdehyde content and cell membrane damage were less than those of the wild type under salt stress conditions. Presumably, compartmentalization of Na(+) in vacuoles reduces its toxic effects on plant cells. This result supports the hypothesis that overexpression of H(+)-PPase causes the accumulation of Na(+) in vacuoles instead of in the cytoplasm and avoids the toxicity of excessive Na(+) in plant cells.     

Development of flooding-tolerant Arabidopsis thaliana by autoregulated cytokinin production

Flooding is one of the most serious environmental stresses that affect plant growth and productivity. Flooding causes premature senescence which results in leaf chlorosis, necrosis, defoliation, cessation of growth and reduced yield. This study was conducted to determine the effects of autoregulated cytokinin production on the flooding tolerance of Arabidopsis thaliana plants. A chimeric gene containing the senescence-specific SAG12 promoter and the ipt gene coding for isopentenyl transferase, a rate-limiting enzyme in the cytokinin biosynthesis pathway, was constructed. The chimeric gene was introduced into Arabidopsis plants by Agrobacterium-mediated vacuum infiltration. Four transgenic lines were chosen for flooding tolerance determinations. DNA hybridization analysis and PCR confirmed that all four of the transgenic lines carried the ipt gene. The segregation of kanamycin resistance in the T₂ generation indicated 1 to 3 integration events. GUS expression and RT-PCR of the ipt gene confirmed the senescence-specificity of the SAG12 promoter. Morphologically, the transgenic lines appeared healthy and normal. Transgenic plants began to flower at the same time as wild-type plants, but the period from flowering to senescence was lengthened by 7 to 12 days. Tolerance of the transgenic plants to waterlogging and complete submergence was assayed in three independent experiments. All four transgenic lines were consistently more tolerant to flooding than wild-type plants. The results indicated that endogenously produced cytokinin can regulate senescence caused by flooding stress, thereby, increasing plant tolerance to flooding. This study provides a novel mechanism to improve flooding tolerance in plants.