Overexpression of <Emphasis Type="Italic">CsNMAPK</Emphasis> in tobacco enhanced seed germination under salt and osmotic stresses

In this research, biological function of CsNMAPK, encoding a mitogen-activated protein kinase of cucumber, was investigated under salt and osmotic stresses. Northern blot analysis showed that the expression of CsNMAPK was induced by salt and osmotic stresses in the cucumber root. In order to determine whether CsNMAPK was involved in plant tolerance to salt and osmotic stresses, transgenic tobacco plants constitutively overexpressing CsNMAPK were generated. Northern and Western blot analysis showed that strong signals were detected in the RNA and protein samples extracted from transgenic lines, whereas no signal was detected in the wild type tobacco, indicating that CsNMAPK was successfully transferred into tobacco genome and overexpressed. The results of seed germination showed that germination rates of transgenic lines were significantly higher than wild type under high salt and osmotic stresses. In addition, seed growth of transgenic lines was much better than wild type under salt and osmotic stresses. These results indicated that overexpression of CsNMAPK positively regulated plant tolerance to salt and osmotic stresses.     

Ovexpression of a Vacuolar H<Superscript>+</Superscript>-ATPase c Subunit Gene Mediates Physiological Changes Leading to Enhanced Salt Tolerance in Transgenic Tobacco

H⁺-ATPase subunit c (VHA-c) is involved in the adaptation to environmental stresses, including salt, drought, and heavy metals. However, it remains unclear whether VHA-c can induce a physiological response related to stress tolerance. To investigate this possibility, we generated transgenic tobacco lines overexpressing a V-ATPase subunit c (LbVHA-c1) gene from Limonium bicolor (Bunge) Kuntze. Compared with wild-type (WT) tobacco, superoxide dismutase (SOD) and peroxidase (POD) activities in the transgenic plants were significantly enhanced under salt stress conditions. The level of malondialdehyde (MDA) in the transgenic plants was significantly lower than that in WT plants grown under salt stress conditions. Moreover, the transgenic plants displayed obviously better growth than the WT plants under salt stress. These results suggest that LbVHA-c1 may confer stress tolerance through enhancing POD and SOD activities, and by protecting membranes from damage by decreasing lipid peroxidation under salt stress.     

Overexpression of tomato <Emphasis Type="Italic">tAPX</Emphasis> gene in tobacco improves tolerance to high or low temperature stress

In order to investigate the function of chloroplast ascorbate peroxidase under temperature stress, the thylakoid-bound ascorbate peroxidase gene from tomato leaf (TtAPX) was introduced into tobacco. Transformants were selected for their ability to grow on medium containing kanamycin. RNA gel blot analysis confirmed that TtAPX in tomato was induced by chilling or heat stress. Over-expression of TtAPX in tobacco improved seed germination under temperature stress. Two transgenic tobacco lines showed higher ascorbate peroxidase activity, accumulated less hydrogen peroxide and malondialdehyde than wild type plants under stress condition. The photochemical efficiency of photosystem 2 in the transgenic lines was distinctly higher than that of wild type plants under chilling and heat stresses. Results indicated that the over-expression of TtAPX enhanced tolerance to temperature stress in transgenic tobacco plants.     

Cloning and Functional Characterization of a Novel Glutathione <Emphasis Type="Italic">S</Emphasis>-Transferase Gene from <Emphasis Type="Italic">Limonium bicolor</Emphasis>

Plant glutathione S-transferases (GSTs) are involved in protecting plants against both diverse biotic and abiotic stresses. In the present study, a novel GST gene (LbGST1) was cloned from Limonium bicolor (Bunge) Kuntze (Plumbaginaceae). To characterize its function in salt tolerance, tobacco lines transformed with LbGST1 were generated. Compared with wild-type (WT) tobacco, transgenic plants overexpressing LbGST1 exhibited both GST and glutathione peroxidase activities. Moreover, superoxide dismutase, peroxidase (POD), and catalase activities in transgenic plants were significantly higher than those in WT plants, particularly when grown under conditions of salt stress. Similarly, levels of proline in transgenic plants were also higher than those in WT plants grown under NaCl stress conditions. Whereas, Malondialdehyde contents in transgenic plants were lower than those in WT plants under NaCl conditions. Furthermore, Na⁺ content in transgenic plants was lower than that in WT plants under these stress conditions. Subcellular localization analysis revealed that the LbGST1 protein was localized in the nucleus. These results suggested that overexpression of LbGST1 gene can affect many physiological processes associated with plant salt tolerance. Therefore, we hypothesize that LbGST1 gene can mediate many physiological pathways that enhance stress resistance in plants.     

Co-transfer of LEA and bZip Genes from Tamarix Confers Additive Salt and Osmotic Stress Tolerance in Transgenic Tobacco

In this study, the additive effects of a late embryogenesis abundant (LEA) gene and a basic leucine zipper (bZIP) gene on salt and osmotic stress in Tamarix plants were analyzed. The constructs containing one or both of the LEA and bZIP genes were transformed into tobacco. Northern blot analysis showed the genes were overexpressed under the control of the CaMV 35S promoter in both dual and single gene-transgenic tobacco lines. The effects of salt and osmotic stress in transgenic tobacco plant were investigated. Following exposure to NaCl, mannitol, and PEG6000 stress, dual gene-transgenic lines showed higher seed generation and growth rates than single gene-transgenic lines and the wild-type. In response to NaCl stress, the dual gene-transgenic lines showed lower malondialdehyde and higher leaf chlorophyll content than single gene-transgenic lines and the wild-type. These results suggested that the co-expression of LEA and bZIP resulted in an additive enhancement of stress tolerance in dual gene-transgenic tobacco.     

Expression of Indica rice <Emphasis Type="Italic">OsBADH1</Emphasis> gene under salinity stress in transgenic tobacco

Glycine betaine has been reported as an osmoprotectant compound conferring tolerance to salinity and osmotic stresses in plants. We previously found that the expression of betaine aldehyde dehydrogenase 1 gene (OsBADH1), encoding a key enzyme for glycine betaine biosynthesis pathway, showed close correlation with salt tolerance of rice. In this study, the expression of the OsBADH1 gene in transgenic tobacco was investigated in response to salt stress using a transgenic approach. Transgenic tobacco plants expressing the OsBADH1 gene were generated under the control of a promoter from the maize ubiquitin gene. Three homozygous lines of T₂ progenies with single transgene insert were chosen for gene expression analysis. RT-PCR and western blot analysis results indicated that the OsBADH1 gene was effectively expressed in transgenic tobacco leading to the accumulation of glycine betaine. Transgenic lines demonstrated normal seed germination and morphology, and normal growth rates of seedlings under salt stress conditions. These results suggest that the OsBADH1 gene could be an excellent candidate for producing plants with osmotic stress tolerance.     

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.     

Improved salt tolerance in tobacco plants by co-transformation of a betaine synthesis gene <Emphasis Type="Italic">BADH</Emphasis> and a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene <Emphasis Type="Italic">SeNHX1</Emphasis>

Three types of transgenic tobacco plants were acquired by separate transformation or co-transformation of a vacuolar Na⁺/H⁺ antiporter gene, SeNHX1, and a betaine synthesis gene, BADH. When exposed to 200 mM NaCl, the dual gene-transformed plants displayed greater accumulation of betaine and Na⁺ than their wild-type counterparts. Photosynthetic rate and photosystem II activity in the transgenic plants were less affected by salt stress than wild-type plants. Transgenic plants exhibited a greater increase in osmotic pressure than wild-type plants when exposed to NaCl. More importantly, the dual gene transformed plants accumulated higher biomass than either of the single transgenic plants under salt stress. Taken together, these findings indicate that simultaneous transformation of BADH and SeNHX1 genes into tobacco plants can enable plants to accumulate betaine and Na⁺, thus conferring them more tolerance to salinity than either of the single gene transformed plants or wild-type tobacco plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Salinity and drought tolerance of mannitol-accumulating transgenic tobacco

Tobacco plants (Nicotiana tabacum L.) were transformed with a mannitol-1-phosphate dehydrogenase gene resulting in mannitol accumulation. Experiments were conducted to determine whether mannitol provides salt and/or drought stress protection through osmotic adjustment. Non-stressed transgenic plants were 20–25% smaller than non-stressed, non-transformed (wild-type) plants in both salinity and drought experiments. However, salt stress reduced dry weight in wild-type plants by 44%, but did not reduce the dry weight of transgenic plants. Transgenic plants adjusted osmotically by 0.57 MPa, whereas wild-type plants did not adjust osmotically in response to salt stress. Calculations of solute contribution to osmotic adjustment showed that mannitol contributed only 0-003-0-004 MPa to the 0.2 MPa difference in full turgor osmotic potential (π_o) between salt-stressed transgenic and wild-type plants. Assuming a cytoplasmic location for mannitol and that the cytoplasm constituted 5% of the total water volume, mannitol accounted for only 30–40% of the change in π_o of the cytoplasm. Inositol, a naturally occurring polyol in tobacco, accumulated in response to salt stress in both transgenic and wild-type plants, and was 3-fold more abundant than mannitol in transgenic plants. Drought stress reduced the leaf relative water content, leaf expansion, and dry weight of transgenic and wild-type plants. However, π_o was not significantly reduced by drought stress in transgenic or wild-type plants, despite an increase in non-structural carbohydrates and mannitol in droughted plants. We conclude that (1) mannitol was a relatively minor osmolyte in transgenic tobacco, but may have indirectly enhanced osmotic adjustment and salt tolerance; (2) inositol cannot substitute for mannitol in this role; (3) slower growth of the transgenic plants, and not the presence of mannitol per se, may have been the cause of greater salt tolerance, and (4) mannitol accumulation was enhanced by drought stress but did not affect π_o or drought tolerance.     

The garlic NF-YC gene, <Emphasis Type="Italic">AsNF-YC8</Emphasis>, positively regulates non-ionic hyperosmotic stress tolerance in tobacco

To investigate the relationship between nuclear factor Y (NF-Y) and stress tolerance in garlic, we cloned a NF-Y family gene AsNF-YC8 from garlic, which was largely upregulated at dehydrate stage. Expression pattern analyses in garlic revealed that AsNF-YC8 is induced through abscisic acid (ABA) and abiotic stresses, such as NaCl and PEG. Compared with wild-type plants, the overexpressing-AsNF-YC8 transgenic tobacco plants showed higher seed germination rates, longer root length and better plant growth under salt and drought stresses. Under drought stress, the transgenic plants maintained higher relative water content (RWC), net photosynthesis, lower levels of malondialdehyde (MDA), and less ion leakage (IL) than wild-type control plants. These results indicate the high tolerance of the transgenic plants to drought stress compared to the WT. The transgenic tobacco lines accumulated less reactive oxygen species (ROS) and exhibited higher antioxidative enzyme activities compared with wild-type (WT) plants under drought stress, which suggested that the overexpression of AsNF-YC8 improves the antioxidant defense system by regulating the activities of these antioxidant enzymes, which in turn protect transgenic lines against drought stress. These results suggest that AsNF-YC8 plays an important role in tolerance to drought and salt stresses.     

Overexpression of suadea salsa <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase gene promotes salt tolerance in transgenic tobacco

The suadea salsa full-length S-adenosylmethionine synthetase (SsSAMS2) was introduced into tobacco (Nicotiana tabacum L.) by Agrobacterium tumefaciens-mediated transformation. The gene transformation and expression in tobacco were confirmed by PCR, RT-PCR and Northern blotting analysis. Several transgenic lines (ST lines) overexpressing SsSAMS2 gene under the control of cauliflower mosaic virus 35S promoter showed more seeds number and weight, and accumulated higher free total polyamines (PAs) than wild-type plants (WT lines) and transformants with blank vector (BT lines). Salt stress-induced damage was attenuated in these transgenic plants, in the symptom of maintaining higher photosynthetic rate and biomass. These results that the transgenic plants overexpressing suadea salsa SAMS2 are more tolerant to salt stress than wild-type plants suggest that PAs may play an important role in contributing salt tolerance to plants.     

Ectopic overexpression of vacuolar and apoplastic <Emphasis Type="Italic">Catharanthus roseus</Emphasis> peroxidases confers differential tolerance to salt and dehydration stress in transgenic tobacco

CrPrx and CrPrx1 are class III peroxidases previously cloned and characterized from Catharanthus roseus. CrPrx is known to be apoplastic in nature, while CrPrx1 is targeted to vacuoles. In order to study their role in planta, these two peroxidases were expressed in Nicotiana tabacum. The transformed plants exhibited increased peroxidase activity. Increased oxidative stress tolerance was also observed in transgenics when treated with H₂O₂ under strong light conditions. However, differential tolerance to salt and dehydration stress was observed during germination of T1 transgenic seeds. Under these stresses, the seed germination of CrPrx-transformed plants and wild-type plants was clearly suppressed, whereas CrPrx1 transgenic lines showed improved germination. CrPrx-transformed lines exhibited better cold tolerance than CrPrx1-transformed lines. These results indicate that vacuolar peroxidase plays an important role in salt and dehydration stress over cell wall-targeted peroxidase, while cell wall-targeted peroxidase renders cold stress tolerance.     

Transgenic tomato plants overexpressing chloroplastic monodehydroascorbate reductase are resistant to salt- and PEG-induced osmotic stress

RNA gel hybridization showed that the expression of monodehydroascorbate reductase (MDHAR) in the wild type (WT) tomato was decreased firstly and then increased under salt- and polyethylene glycol (PEG)-induced osmotic stress, and the maximum level was observed after treatment for 12 h. WT, sense transgenic and antisense transgenic tomato plants were used to analyze the antioxidative ability to cope with osmotic stresses. After salt stress, the fresh mass (FM) and height of sense transgenic lines were greater than those of antisense lines and WT plants. Under salt and PEG treatments, sense transgenic plants showed a lower level of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), a higher net photosynthetic rate (P _N), and the maximal photochemical efficiency of PSII (F_v/F_m) compared with WT and antisense transgenic plants. Moreover, sense lines maintained higher ascorbate peroxidase (APX) activity than WT and antisense plants under salt- and PEG-induced osmotic stress. These results indicate that chloroplastic MDHAR plays an important role in alleviating photoinhibition of PSII by elevating ascorbate (AsA) level under salt- and PEG-induced osmotic 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.     

Overexpression of monodehydroascorbate reductase in transgenic tobacco confers enhanced tolerance to ozone,salt and polyethylene glycol stresses

Ascorbate (AsA) is a major antioxidant and free-radical scavenger in plants. Monodehydroascorbate reductase (MDAR; EC 1.6.5.4) is crucial for AsA regeneration and essential for maintaining a reduced pool of AsA. To examine whether an overexpressed level of MDAR could minimize the deleterious effects of environmental stresses, we developed transgenic tobacco plants overexpressing Arabidopsis thaliana MDAR gene (AtMDAR1) in the cytosol. Incorporation of the transgene in the genome of tobacco plants was confirmed by PCR and Southern-blot analysis and its expression was confirmed by Northern- and Western-blot analyses. These transgenic plants exhibited up to 2.1-fold higher MDAR activity and 2.2-fold higher level of reduced AsA compared to non-transformed control plants. The transgenic plants showed enhanced stress tolerance in term of significantly higher net photosynthesis rates under ozone, salt and polyethylene glycol (PEG) stresses and greater PSII effective quantum yield under ozone and salt stresses. Furthermore, these transgenic plants exhibited significantly lower hydrogen peroxide level when tested under salt stress. These results demonstrate that an overexpressed level of MDAR properly confers enhanced tolerance against ozone, salt and PEG stress.     

Effect of water stress on yield and nutrition quality of tomato plant overexpressing StAPX

We investigated the effect of water stress on yield and quality of tomato plants overexpressing Solanum lycopersicum thylakoid-bound ascorbate peroxidase gene (StAPX). APX activity, hydrogen peroxide content, net photosynthetic rate of tomato leaves, and yield and nutrition quality of tomato fruits were measured under soil moisture 70, 60, and 50 % of full field capacity. Results show that the capability of APX for scavenging hydrogen peroxide induced by water stress was higher in the transgenic than the wild type (WT) plants. The yield of fruits of the transgenic tomato plants was higher than that of WT plants under water stress and the fruit nutrition quality was not different. These results indicate that overexpression of StAPX might improve water stress tolerance in the transgenic tomato plants.     

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.     

Marker-free transgenic durum wheat cv. Karim expressing the AlSAP gene exhibits a high level of tolerance to salinity and dehydration stresses

We have recently isolated the AlSAP (stress-associated protein) gene from the halophyte grass Aeluropus littoralis and demonstrated that AlSAP expression improves tolerance to continuous salt and drought stresses in transgenic tobacco. To extend these findings to an important crop, we generated marker-free transgenic durum wheat plants of the commercial cv. Karim expressing the AlSAP gene. The integration and expression of AlSAP in T3 homozygous plants were ascertained by Southern, Northern and Western blotting respectively. AlSAP wheat lines exhibited improved germination rates and biomass production under severe salinity and osmotic stress conditions. Following a long-term salt or drought stress greenhouse trial, AlSAP lines produced normally filled grains whereas wild-type (WT) plants either died at the vegetative stage under salt stress or showed markedly reduced grain filling under drought stress. Measurements of the RWC (relative water content) and endogenous Na⁺ and K⁺ levels in leaves of AlSAP plants, showed a lower water loss rate and a higher Na⁺ accumulation in senescent-basal leaves, respectively, compared to those of WT plants. Taken together, these results extend to cereals the high potential of the AlSAP gene for engineering effective drought and salt tolerance.     

Transgenic cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) seedlings expressing a tobacco glutathione <Emphasis Type="Italic">S</Emphasis>-transferase fail to provide improved stress tolerance

Transgenic cotton (Gossypium hirsutum L.) lines expressing the tobacco glutathione S-transferase (GST) Nt107 were evaluated for tolerance to chilling, salinity, and herbicides, antioxidant enzyme activity, antioxidant compound levels, and lipid peroxidation. Although transgenic seedlings exhibited ten-fold and five-fold higher GST activity under normal and salt-stress conditions, respectively, germinating seedlings did not show improved tolerance to salinity, chilling conditions, or herbicides. Glutathione peroxidase (GPX) activity in transgenic seedlings was 30% to 60% higher under normal conditions, but was not different than GPX activity in wild-type seedlings under salt-stress conditions. Glutathione reductase, superoxide dismutase, ascorbate peroxidase, and monodehydroascorbate reductase activities were not increased in transgenic seedlings under salt-stress conditions, while dehydroascorbate reductase activity was decreased in transgenic seedlings under salt-stress conditions. Transgenic seedlings had 50% more oxidized glutathione when exposed to salt stress. Ascorbate levels were not increased in transgenic seedlings under salt-stress conditions. Malondialdehyde content in transgenic seedlings was nearly double that of wild-type seedlings under normal conditions and did not increase under salt-stress conditions. These results show that expression of Nt107 in cotton does not provide adequate protection against oxidative stress and suggests that the endogenous antioxidant system in cotton may be disrupted by the expression of the tobacco GST.