Overexpression of Tamarix albiflonum TaMnSOD increases drought tolerance in transgenic cotton

Drought is a major environmental stress that limits cotton (Gossypium hirsutum L.) production worldwide. TaMnSOD plays a crucial role as a peroxidation scavenger. In this study, TaMnSOD cDNA of Tamarix albiflonum was overexpressed in the cotton cultivar fy11 by Agrobacterium tumefaciens-mediated transformation. The transformed plants were assessed by gDNA PCR, RT-PCR and DNA gel blot analysis. The physiological and biochemical characters of two independent transgenic lines and control plants were tested and compared, and the morphological traits (biomass, root and lateral root length, leaf number) were also detected after recovery from water-withholding stress. When water was withheld from pot-grown 6-week-old seedlings for 18 days (watering to 8 % of field capacity), transgenic cotton plants accumulated more proline and soluble sugar than wild-type plants (WT). The activity of antioxidant enzymes such as superoxide dismutase and peroxidase was enhanced in transgenic plants under drought stress. Cell membrane integrity was also considerably improved under water stress, as indicated by reduced malondialdehyde content relative to control plants. Furthermore, net photosynthesis, stomatal conductance and transpiration rate were increased in transgenic plants compared with wild type. Transgenic cotton showed increases in biomass as well as root and leaf systems compared with WT after 2 weeks recovery from stress. These results suggest that TaMnSOD transgenic cotton plants acquired improved drought tolerance through enhanced development of the root and leaf system and the regulation of superoxide scavenging.     

GbMPK3, a mitogen-activated protein kinase from cotton,enhances drought and oxidative stress tolerance in tobacco

Mitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules found in all eukaryotes, and play significant roles in developmental and environmental signal transduction. In this study, a MAPK gene (GbMPK3), which showed homologous to AtMPK3 and NtWIPK, was isolated from sea-island cotton (Gossypium barbadense) and induced during multiple abiotic stress treatments including salt, cold, heat, dehydration and oxidative stress. Transgenic tobacco (Nicotiana benthamiana) with constitutively higher expression of GbMPK3 was conferred with enhanced drought tolerance, reduced water loss during drought treatment and improved plant height and survival rates after re-watering. Additionally, the gene expression levels and enzymatic activity of antioxidant enzymes were more strongly induced with depressed hydrogen peroxide accumulation in GbMPK3-overexpressing tobacco compared with wild-type under drought condition. Furthermore, observation of seed germination and leaf morphology showed that tolerance of transgenic plants to methyl viologen was improved due to increased antioxidant enzyme expression, suggesting that GbMPK3 may positively regulate drought tolerance through enhanced reactive oxygen species scavenging ability.     

Expression of the Arabidopsis vacuolar H+-pyrophosphatase gene AVP1 in peanut to improve drought and salt tolerance

The Arabidopsis gene AVP1 encodes an H⁺-pyrophosphatase that functions as a proton pump at the vacuolar membranes, generating a proton gradient across vacuolar membranes, which serves as the driving force for many secondary transporters on vacuolar membranes such as Na⁺/H⁺-antiporters. Overexpression of AVP1 could improve drought tolerance and salt tolerance in transgenic plants, suggesting a possible way in improving drought and salt tolerance in crops. The AVP1 was therefore introduced into peanut by Agrobacterium-mediated transformation. Analysis of AVP1-expressing peanut indicated that AVP1-overexpression in peanut could improve both drought and salt tolerance in greenhouse and growth chamber conditions, as AVP1-overexpressing peanuts produced more biomass and maintained higher photosynthetic rates under both drought and salt conditions. In the field, AVP1-overexpressing peanuts also outperformed wild-type plants by having higher photosynthetic rates and producing higher yields under low irrigation conditions.     

Creating drought- and salt-tolerant cotton by overexpressing a vacuolar pyrophosphatase gene

Increased expression of an Arabidopsis vacuolar pyrophosphatase gene, AVP1, leads to increased drought and salt tolerance in transgenic plants, which has been demonstrated in laboratory and field conditions. The molecular mechanism of AVP1-mediated drought resistance is likely due to increased proton pump activity of the vacuolar pyrophosphatase, which generates a higher proton electrochemical gradient across the vacuolar membrane, leading to lower water potential in the plant vacuole and higher secondary transporter activities that prevent ion accumulation to toxic levels in the cytoplasm. Additionally, overexpression of AVP1 appears to stimulate auxin polar transport, which in turn stimulates root development. The larger root system allows AVP1-overexpressing plants to absorb water more efficiently under drought and saline conditions, resulting in stress tolerance and increased yields. Multi-year field-trial data indicate that overexpression of AVP1 in cotton leads to at least 20% more fiber yield than wild-type control plants in dry-land conditions, which highlights the potential use of AVP1 in improving drought tolerance in crops in arid and semiarid areas of the world.Key words: drought tolerance, proton pump, salt tolerance, transgenic cotton, vacuolar membraneDrought and salinity are major environmental factors that limit agricultural productivity in most parts of the world.¹ Climate change will likely make many places worse in terms of water availability and soil salinization,² which will have negative impacts on food production in world agriculture. Yet, the demand for more food will continue to rise because of the growing world population that may reach 9 billon people by 2050.³ Therefore, the primary challenge we face during this century is the production of more food under the constraints of limited water and fertilizer on marginal soils.Many genes that respond to abiotic stresses have been identified in the model plant Arabidopsis,⁴ and some of them were shown to play important roles in protecting plants under abiotic stress conditions.⁵ The Arabidopsis vacuolar pyrophosphatase gene AVP1 appears to be one of the most promising genes that may be used to improve drought- and salt-tolerance in crops.⁶ Roberto Gaxiola''s group first demonstrated that overexpression of AVP1 could lead to significantly improved drought- and salt-tolerance in transgenic Arabidopsis plants.⁷ Later when this gene was introduced into tomato⁸ and rice,⁹ similar tolerance phenotypes were observed. Overexpression of AVP1 in cotton, not only improved drought- and salt-tolerance in greenhouse conditions, but also increased fiber yield in dryland field conditions.⁶ AVP1-expressing cotton plants produced larger root systems and bigger shoot biomass than controls when grown under hydroponic conditions in the presence of up to 200 mM NaCl.⁶ In the greenhouse, AVP1-expressing cotton plants also produced more root and shoot biomass than controls when grown under saline conditions or reduced irrigation.⁶ The increased yield by AVP1-expressing cotton plants is due to more bolls produced, which in turn is due to larger shoot system that AVP1-expressing cotton plants develop under saline or drought conditions.⁶The larger root systems of AVP1-expressing cotton plants under saline and water-deficit conditions allow transgenic plants access to more of the soil profile and available soil water resulting in increased biomass production and yield. Li et al. showed that the larger root systems of AVP1-overexpressing Arabidopsis is caused by increased auxin polar transport in the root, which stimulates root development in AVP1-overexpressing Arabidopsis plants.¹⁰ Furthermore, a recent comparative study of transgenic Arabidopsis lines that produce enlarged leaves showed that auxin levels were increased by 50% in AVP1-overexpressing plants.¹¹ To test if altered auxin level is responsible for the observed larger root systems in AVP1-expressing cotton plants, we germinated wild-type and AVP1-expressing cotton plants in the absence or presence of the auxin polar transport inhibitor Naphthylphthalamic acid (NPA). Both wild-type and AVP1-expressing cotton plants developed robust lateral root systems in the absence of NPA (Fig. 1A). The presence of 50 µM NPA resulted in nearly complete inhibition of lateral root development in wild-type plants, while lateral root development in AVP1-expressing plants was reduced, it was significantly greater than wild-type (Fig. 1B). These data indicate that AVP1-overexpression could overcome the inhibitory effects of NPA on root development in AVP1-expressing cotton plants, suggesting that either increased auxin transport or higher auxin concentration in the root systems of AVP1-expressing cotton plants is responsible for the observed larger root systems, and eventually for the increased boll numbers and fiber yields under dryland field conditions.Open in a separate windowFigure 1Root development of wild-type and AVP1-expressing cotton plants in the absence and presence of auxin transport inhibitor NPA. (A) Phenotype of cotton roots after 10 days of growth in the absence of NPA. WT, Wild-type; 1, 5, 9, three independent AVP1-overexpressing cotton lines. (B) Phenotype of cotton roots after 10 days of growth in the presence of 50 µm NPA.Many genes that may play important roles under water-deficit conditions have been tested in laboratory conditions,^4,5 but very few have been tested vigorously in field conditions. A bacterial cold shock protein gene was shown to improve drought tolerance in maize based on multi-year and multi-place field trial experiments,¹² and it appears that this gene will likely gain approval for commercial release and become the first genetically engineered product that demonstrates improved drought tolerance in a major crop in the U.S. Another example of increased drought tolerance supported by multiple field trial experiments is through downregulation of farnesylation in transgenic canola plants.¹³ Downregulation of farnesyltransferase by antisense or RNAi techniques in transgenic canola leads to increased sensitivity to abscisic acid, consequently resulting in smaller guard cell aperture under drought conditions. These transgenic canola plants lose less water through transpiration and are more drought resistant. Data from more than 5 years of field studies in Canada consistently proved that this approach can indeed increase drought tolerance in transgenic canola. Our study with AVP1-expressing cotton over the last several years in field conditions is another example that genetic engineering approach can be an efficient tool in generating drought-tolerant crops. AVP1-expressing cotton plants can establish a larger shoot mass in dryland conditions (Fig. 2), which results in increased boll numbers and fiber production. Our approach is likely applicable to other major crops as well.Open in a separate windowFigure 2Wild-type and AVP1-expressing cotton plants grown in the dryland field condition. Plants were planted in the middle of may 2009 and the picture was taken in the middle of July 2009 at the USDA experimental Farm in Lubbock, Texas.     

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.     

An Ipomoea batatas Iron-Sulfur Cluster Scaffold Protein Gene,IbNFU1, Is Involved in Salt Tolerance

Iron-sulfur cluster biosynthesis involving the nitrogen fixation (Nif) proteins has been proposed as a general mechanism acting in various organisms. NifU-like protein may play an important role in protecting plants against abiotic and biotic stresses. An iron-sulfur cluster scaffold protein gene, IbNFU1, was isolated from a salt-tolerant sweetpotato (Ipomoea batatas (L.) Lam.) line LM79 in our previous study, but its role in sweetpotato stress tolerance was not investigated. In the present study, the IbNFU1 gene was introduced into a salt-sensitive sweetpotato cv. Lizixiang to characterize its function in salt tolerance. The IbNFU1-overexpressing sweetpotato plants exhibited significantly higher salt tolerance compared with the wild-type. Proline and reduced ascorbate content were significantly increased, whereas malonaldehyde (MDA) content was significantly decreased in the transgenic plants. The activities of superoxide dismutase (SOD) and photosynthesis were significantly enhanced in the transgenic plants. H₂O₂ was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbNFU1 up-regulated pyrroline-5-carboxylate synthase (P5CS) and pyrroline-5-carboxylate reductase (P5CR) genes under salt stress. The systemic up-regulation of reactive oxygen species (ROS) scavenging genes was found in the transgenic plants under salt stress. These findings suggest that IbNFU1gene is involved in sweetpotato salt tolerance and enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and activating ROS scavenging system.     

Expression of an Arabidopsis vacuolar H+‐pyrophosphatase gene (AVP1) in cotton improves drought‐ and salt tolerance and increases fibre yield in the field conditions

The Arabidopsis gene AVP1 encodes a vacuolar pyrophosphatase that functions as a proton pump on the vacuolar membrane. Overexpression of AVP1 in Arabidopsis, tomato and rice enhances plant performance under salt and drought stress conditions, because up‐regulation of the type I H⁺‐PPase from Arabidopsis may result in a higher proton electrochemical gradient, which facilitates enhanced sequestering of ions and sugars into the vacuole, reducing water potential and resulting in increased drought‐ and salt tolerance when compared to wild‐type plants. Furthermore, overexpression of AVP1 stimulates auxin transport in the root system and leads to larger root systems, which helps transgenic plants absorb water more efficiently under drought conditions. Using the same approach, AVP1‐expressing cotton plants were created and tested for their performance under high‐salt and reduced irrigation conditions. The AVP1‐expressing cotton plants showed more vigorous growth than wild‐type plants in the presence of 200 mm NaCl under hydroponic growth conditions. The soil‐grown AVP1‐expressing cotton plants also displayed significantly improved tolerance to both drought and salt stresses in greenhouse conditions. Furthermore, the fibre yield of AVP1‐expressing cotton plants is at least 20% higher than that of wild‐type plants under dry‐land conditions in the field. This research indicates that AVP1 has the potential to be used for improving crop’s drought‐ and salt tolerance in areas where water and salinity are limiting factors for agricultural productivity.     

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.     

Cloning of Gossypium hirsutum Sucrose Non-Fermenting 1-Related Protein Kinase 2 Gene (GhSnRK2) and Its Overexpression in Transgenic Arabidopsis Escalates Drought and Low Temperature Tolerance

The molecular mechanisms of stress tolerance and the use of modern genetics approaches for the improvement of drought stress tolerance have been major focuses of plant molecular biologists. In the present study, we cloned the Gossypium hirsutum sucrose non-fermenting 1-related protein kinase 2 (GhSnRK2) gene and investigated its functions in transgenic Arabidopsis. We further elucidated the function of this gene in transgenic cotton using virus-induced gene silencing (VIGS) techniques. We hypothesized that GhSnRK2 participates in the stress signaling pathway and elucidated its role in enhancing stress tolerance in plants via various stress-related pathways and stress-responsive genes. We determined that the subcellular localization of the GhSnRK2-green fluorescent protein (GFP) was localized in the nuclei and cytoplasm. In contrast to wild-type plants, transgenic plants overexpressing GhSnRK2 exhibited increased tolerance to drought, cold, abscisic acid and salt stresses, suggesting that GhSnRK2 acts as a positive regulator in response to cold and drought stresses. Plants overexpressing GhSnRK2 displayed evidence of reduced water loss, turgor regulation, elevated relative water content, biomass, and proline accumulation. qRT-PCR analysis of GhSnRK2 expression suggested that this gene may function in diverse tissues. Under normal and stress conditions, the expression levels of stress-inducible genes, such as AtRD29A, AtRD29B, AtP5CS1, AtABI3, AtCBF1, and AtABI5, were increased in the GhSnRK2-overexpressing plants compared to the wild-type plants. GhSnRK2 gene silencing alleviated drought tolerance in cotton plants, indicating that VIGS technique can certainly be used as an effective means to examine gene function by knocking down the expression of distinctly expressed genes. The results of this study suggested that the GhSnRK2 gene, when incorporated into Arabidopsis, functions in positive responses to drought stress and in low temperature tolerance.     

Overexpression of wheat Na+/H+ antiporter TNHX1 and H+-pyrophosphatase TVP1 improve salt- and drought-stress tolerance in Arabidopsis thaliana plants

Transgenic Arabidopsis plants overexpressing the wheat vacuolarNa⁺/H⁺ antiporter TNHX1 and H⁺-PPase TVP1 are much more resistantto high concentrations of NaCl and to water deprivation thanthe wild-type strains. These transgenic plants grow well inthe presence of 200 mM NaCl and also under a water-deprivationregime, while wild-type plants exhibit chlorosis and growthinhibition. Leaf area decreased much more in wild-type thanin transgenic plants subjected to salt or drought stress. Theleaf water potential was less negative for wild-type than fortransgenic plants. This could be due to an enhanced osmoticadjustment in the transgenic plants. Moreover, these transgenicplants accumulate more Na⁺ and K⁺ in their leaf tissue thanthe wild-type plants. The toxic effect of Na⁺ accumulation inthe cytosol is reduced by its sequestration into the vacuole.The rate of water loss under drought or salt stress was higherin wild-type than transgenic plants. Increased vacuolar soluteaccumulation and water retention could confer the phenotypeof salt and drought tolerance of the transgenic plants. Overexpressionof the isolated genes from wheat in Arabidopsis thaliana plantsis worthwhile to elucidate the contribution of these proteinsto the tolerance mechanism to salt and drought. Adopting a similarstrategy could be one way of developing transgenic staple cropswith improved tolerance to these important abiotic stresses. Key words: H⁺-pyrophosphatase, Na⁺/H⁺ antiporter, salt and drought tolerance, sodium sequestration, transgenic Arabidopsis plants     

A Novel α/β-Hydrolase Gene IbMas Enhances Salt Tolerance in Transgenic Sweetpotato

Salt stress is one of the major environmental stresses in agriculture worldwide and affects crop productivity and quality. The development of crops with elevated levels of salt tolerance is therefore highly desirable. In the present study, a novel maspardin gene, named IbMas, was isolated from salt-tolerant sweetpotato (Ipomoea batatas (L.) Lam.) line ND98. IbMas contains maspardin domain and belongs to α/β-hydrolase superfamily. Expression of IbMas was up-regulated in sweetpotato under salt stress and ABA treatment. The IbMas-overexpressing sweetpotato (cv. Shangshu 19) plants exhibited significantly higher salt tolerance compared with the wild-type. Proline content was significantly increased, whereas malonaldehyde content was significantly decreased in the transgenic plants. The activities of superoxide dismutase (SOD) and photosynthesis were significantly enhanced in the transgenic plants. H₂O₂ was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbMas up-regulated the salt stress responsive genes, including pyrroline-5-carboxylate synthase, pyrroline-5-carboxylate reductase, SOD, psbA and phosphoribulokinase genes, under salt stress. These findings suggest that overexpression of IbMas enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and increasing reactive oxygen species scavenging capacity.     

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.     

SlAGO4A,a core factor of RNA-directed DNA methylation (RdDM) pathway,plays an important role under salt and drought stress in tomato

In Arabidopsis, it has been clarified that AGO4 protein is implicated in a phenomenon termed RNA-directed DNA methylation (RdDM). Previously, four orthologs of AtAGO4 were cloned in tomato, designated as SlAGO4A–SlAGO4D. Here, we studied the role of the SlAGO4A gene in regulating salt and drought tolerance in tomato. SlAGO4A-down-regulating (AS) transgenic tomato plants showed enhanced tolerance to salt and drought stress compared to wild-type (WT) and SlAGO4A-overexpressing (OE) transgenic plants, as assessed by physiological parameters such as seed germination rate, primary root length, chlorophyll/proline/MDA/soluble sugar/RWC content, and survival rate. Moreover, several genes involved in ROS scavenging and plant defense, including CAT, SOD, GST, POD, APX, LOX, and PR1, were up- or down-regulated consistently under salt and drought stress. Notably, expression levels of some DNA methyltransferase genes and RNAi pathway genes were significantly lower in AS plants than in WT. Taken together, our results suggest that SlAGO4A gene plays a negative role under salt and drought stress in tomato probably through the modulation of DNA methylation as well as the classical RNAi pathway. Hence, it may serve as a useful biotechnological tool for the genetic improvement of stress tolerance in crops.     

Coexpression of ScNHX1 and ScVP in Transgenic Hybrids Improves Salt and Saline-Alkali Tolerance in Alfalfa (Medicago sativa L.)

Salt and saline-alkali are major environmental factors limiting the growth and productivity of alfalfa, the most economically important forage legume worldwide. In this study, alfalfa plants transgenic for both ScNHX1 (encoding vacuolar membrane Na⁺/H⁺ antiporter from Suaeda corniculata) and ScVP (encoding vacuolar H⁺-PPase from S. corniculata) were produced by cross-pollination. Transgenic alfalfa plants coexpressing ScVP/ScNHX1 showed enhanced salt and saline-alkali tolerance to 300 or 200 mM NaCl with 100 mM NaHCO₃ treatments, compared with wild-type plants. In addition, ScVP/ScNHX1-coexpressing alfalfa plants accumulated more Na⁺ in leaves and roots than wild-type plants and showed increased tolerance to higher salt and saline-alkali stress. Using the fluorescent carboxy-SNARF probe, the intracellular pH was visualized in the transgenic and wild-type plants under salt and saline-alkali stress. The results showed that the overnight treatment caused a massive change in pH in ScVP/ScNHX1-coexpressing alfalfa plants and they showed that there was significantly higher vacuolar alkalization under salt stress compared with wild-type plants. However, saline-alkali treatment enhanced vacuolar acidification more in the wild-type plants than in transgenic plants. Taken together, our results indicate that coexpression of multiple, effective genes in transgenic plants can enhance resistance to salt and saline-alkali stress.     

Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation

Pyrroline-5-carboxylate reductase (P5CR) lies at the converging point of the glutamate and ornithine pathways and is the last and critical enzyme in proline biosynthesis. In the present study, a P5CR gene, named IbP5CR, was isolated from salt-tolerant sweetpotato line ND98. Expression of IbP5CR was up-regulated in sweetpotato under salt stress. The IbP5CR-overexpressing sweetpotato (cv. Kokei No. 14) plants exhibited significantly higher salt tolerance compared with the wild-type. Proline content and superoxide dismutase and photosynthetic activities were significantly increased, whereas malonaldehyde content was significantly decreased in the transgenic plants. H₂O₂ was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbP5CR up-regulated pyrroline-5-carboxylate synthase gene and down-regulated proline dehydrogenase and P5C dehydrogenase genes under salt stress. The systemic up-regulation of reactive oxygen species (ROS) scavenging genes was found in the transgenic plants under salt stress. These findings suggest that overexpression of IbP5CR increases proline accumulation, which enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and activating ROS scavenging system. This study indicates that IbP5CR gene has the potential to be used for improving salt tolerance of plants.     

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.     

BcPMI2, isolated from non-heading Chinese cabbage encoding phosphomannose isomerase,improves stress tolerance in transgenic tobacco

Phosphomannose isomerase (PMI) is an enzyme that catalyses the first step of the l-galactose pathway for ascorbic acid (AsA) biosynthesis in plants. To clarify the physiological roles of PMI in AsA biosynthesis, the cDNA sequence of PMI was cloned from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) and overexpressed in tobacco transformed with Agrobacterium tumefaciens. The AsA and soluble sugar contents were lower in 35S::BcPMI2 tobacco than in wild-type tobacco. However, the AsA level in BcPMI2-overexpressing plants under stress was significantly increased. The T₁ seed germination rate of transgenic plants was higher than that of wild-type plants under NaCl or H₂O₂ treatment. Meanwhile, transgenic plants showed higher tolerance than wild-type plants. This finding implied that BcPMI2 overexpression improved AsA biosynthetic capability and accumulation, and evidently enhanced tolerance to oxidative and salt stress, although the AsA level was lower in transgenic tobacco than in wild-type tobacco under normal condition.