cDNA cloning and gene expression of ascorbate oxidase in tobacco

A cDNA clone for ascorbate oxidase (AAO) has been isolated from a cDNA library of tobacco (Nicotiana tabacum) cells. The identity of the amino acid sequence deduced from tobacco AAO cDNA to that from pumpkin AAO cDNA was 68%, which was much lower than the identity (80%) between pumpkin and cucumber AAO. AAO activity in tobacco cells was much lower than that in pumpkin cells, whereas the immunoreactive protein in tobacco cells was more abundant than that in pumpkin cells. We suppose that AAO protein in tobacco cells may be less active than that in pumpkin cells. Genomic Southern blotting suggested that AAO in tobacco was encoded by a single-copy gene. Northern blotting revealed that mRNA of AAO was highly expressed in young and growing tissues of tobacco plant.     

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.     

Overexpression of Ks-type dehydrins gene OeSRC1 from Olea europaea increases salt and drought tolerance in tobacco plants

Molecular Biology Reports - Agricultural production is greatly affected by environmental stresses, such as cold, drought and high-salinity. It is possible to produce tolerant genotypes by...     

Changes in ascorbate oxidase gene expression and ascorbate levels in cell division and cell elongation in tobacco cells

The biological function of ascorbate oxidase (AAO) was not yet clarified, although it was suggested that AAO may be involved in cell growth. We investigated AAO expression and ascorbate metabolism during non-synchronous, synchronous, and elongation cultures of tobacco BY-2 cells. In non-synchronous culture, AAO mRNA was abundant in logarithmic growth phase. Ascorbate content greatly increased during the growth, whereas dehydroascorbate content was slightly increased. In synchronous division culture, AAO mRNA was detected in all phases, but the levels were quite low in G1 phase. Ascorbate content was high in all phases, whereas dehydroascorbate content was low, especially in G1 phase. In elongation culture, the levels of AAO mRNA increased during elongation of the cells. AAO activity in the culture medium, as well as ascorbate and dehydroascorbate contents in the cells, also increased during the elongation. We propose that AAO expression and production of dehydroascorbate are under the control of the cell cycle and that AAO may function apoplastically as an ascorbate oxidizer in the process of cell elongation.     

Overexpression of the AtSTK gene increases salt, PEG and ABA tolerance in Arabidopsis

AtSTK (At5g02800), which is a serine-threonine protein kinase gene of Arabidopsis thaliana, was cloned, and its function was studied. The study found that the overexpression of AtSTK could significantly improve the ability of A. thaliana to tolerate salt, PEG, and ABA stresses. RT-PCR analysis revealed that the expression of the AtSTK gene could be obviously induced by salt, PEG, and ABA. The examination of the physiological characteristics showed that the overexpression of AtSTK in Arabidopsis significantly reduced the plasma membrane permeability, significantly increased the proline content, and decreased the MDA content. These changes may reflect the physiological mechanisms through which AtSTK overexpression improves stress resistance in Arabidopsis. In addition, the overexpression of the AtSTK gene significantly antagonised the inhibitory effect of high concentrations of exogenous ABA on Arabidopsis seed germination. The subcellular localisation results showed that AtSTK is located in both the cytosol and the nucleus. The examination of its tissue-specific expression showed that AtSTK is expressed in various Arabidopsis tissues and is particularly strongly expressed in the vessels. The signalling pathway analysis indicated that AtSTK might transfer the salt stress signal in Arabidopsis through the MAPK pathway.     

Overexpression of an Arabidopsis peroxisomal ascorbate peroxidase gene in tobacco increases protection against oxidative stress.

The Arabidopsis gene APX3 that encodes a putative peroxisomal membrane-bound ascorbate peroxidase was expressed in transgenic tobacco plants. APX3-expressing lines had substantial levels of APX3 mRNA and protein. The H2O2 can be converted to more reactive toxic molecules, e.g. .OH, if it is not quickly removed from plant cells. The expression of APX3 in tobacco could protect leaves from oxidative stress damage caused by aminotriazole which inhibits catalase activity that is found mainly in glyoxysomes and peroxisomes and leads to accumulation of H2O2 in those organelles. However, these plants did not show increased protection from oxidative damage caused by paraquat which leads to the production of reactive oxygen species in chloroplasts. Therefore, protection provided by the expression of APX3 seems to be specific against oxidative stress originated from peroxisomes, not from chloroplasts, which is consistent with the hypothesis that APX3 is a peroxisomal membrane-bound antioxidant enzyme.     

An Arabidopsis mitochondrial uncoupling protein confers tolerance to drought and salt stress in transgenic tobacco plants

Background

Plants are challenged by a large number of environmental stresses that reduce productivity and even cause death. Both chloroplasts and mitochondria produce reactive oxygen species under normal conditions; however, stress causes an imbalance in these species that leads to deviations from normal cellular conditions and a variety of toxic effects. Mitochondria have uncoupling proteins (UCPs) that uncouple electron transport from ATP synthesis. There is evidence that UCPs play a role in alleviating stress caused by reactive oxygen species overproduction. However, direct evidence that UCPs protect plants from abiotic stress is lacking.

Methodology/Principal Findings

Tolerances to salt and water deficit were analyzed in transgenic tobacco plants that overexpress a UCP (AtUCP1) from Arabidopsis thaliana. Seeds of AtUCP1 transgenic lines germinated faster, and adult plants showed better responses to drought and salt stress than wild-type (WT) plants. These phenotypes correlated with increased water retention and higher gas exchange parameters in transgenic plants that overexpress AtUCP1. WT plants exhibited increased respiration under stress, while transgenic plants were only slightly affected. Furthermore, the transgenic plants showed reduced accumulation of hydrogen peroxide in stressed leaves compared with WT plants.

Conclusions/Significance

Higher levels of AtUCP1 improved tolerance to multiple abiotic stresses, and this protection was correlated with lower oxidative stress. Our data support previous assumptions that UCPs reduce the imbalance of reactive oxygen species. Our data also suggest that UCPs may play a role in stomatal closure, which agrees with other evidence of a direct relationship between these proteins and photosynthesis. Manipulation of the UCP protein expression in mitochondria is a new avenue for crop improvement and may lead to crops with greater tolerance for challenging environmental conditions.     

Overexpression of SOD2 increases salt tolerance of Arabidopsis

The yeast (Schizosaccharomyces pombe) SOD2 (Sodium2) gene was introduced into Arabidopsis under the control of the cauliflower mosaic virus 35S promoter. Transformants were selected for their ability to grow on medium containing kanamycin. Southern- and northern-blot analyses confirmed that SOD2 was transferred into the Arabidopsis genome. There were no obvious morphological or developmental differences between the transgenic and wild-type (wt) plants. Several transgenic homozygous lines and wt plants (control) were evaluated for salt tolerance and gene expression. Overexpression of SOD2 in Arabidopsis improved seed germination and seedling salt tolerance. Analysis of Na+ and K+ contents of the symplast and apoplast in the parenchyma cells of the root cortex and mesophyll cells in the spongy tissue of the leaf showed that transgenic lines accumulated less Na+ and more K+ in the symplast than the wt plants did. The photosynthetic rate and the fresh weight of the transgenic lines were distinctly higher than that of wt plants after NaCl treatment. Results from different tests indicated that the expression of the SOD2 gene promoted a higher level of salt tolerance in vivo in transgenic Arabidopsis plants.     

Overexpression of the acerola (Malpighia glabra) monodehydroascorbate reductase gene in transgenic tobacco plants results in increased ascorbate levels and enhanced tolerance to salt stress

Monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) is a key enzyme of the ascorbate (AsA)-glutathione cycle that maintains reduced pools of AsA and serves as an important antioxidative enzyme. Previously, we have cloned MDHAR cDNA from acerola (Malpighia glabra), a plant that accumulates abundant amount of AsA. In this study, MDHAR cDNA from acerola was introduced into tobacco plants using an Agrobacterium-mediated gene delivery system. Transgenic tobacco plants accumulated greater amounts of AsA and showed higher MDHAR activity than the control plants. Lipid peroxidation and chlorophyll degradation, which were stimulated in control plants, were restrained in transgenic plants subjected to salt stress. These results indicate that overexpression of acerola MDHAR imparts greater tolerance to salt stress.     

Transformation of Arabidopsis with the codA gene for choline oxidase enhances freezing tolerance of plants

Arabidopsis thaliana was transformed with the codA gene from Arthrobacter globiformis, which encodes choline oxidase, the enzyme that synthesizes glycinebetaine from choline. The transformation enabled the plants to accumulate glycinebetaine in chloroplasts, and significantly enhanced the freezing tolerance of plants. Furthermore, the photosynthetic machinery of transformed plants was more tolerant to freezing stress than that of wild-type plants. Exogenous application of glycinebetaine also increased the freezing tolerance of wild-type plants, suggesting that the presence of glycinebetaine in transformed plants had enhanced their ability to tolerate freezing stress. Northern blotting analysis revealed that the enhancement of freezing tolerance was not related to the expression of four cold-regulated genes. These results suggest that engineering of the biosynthesis of glycinebetaine by transformation with the codA gene might be an effective method for enhancing the freezing tolerance of plants.     

Overexpression of the rice Osmyb4 gene increases chilling and freezing tolerance of Arabidopsis thaliana plants

The expression of the gene Osmyb4, detected at low level in rice (Oryza sativa) coleoptiles grown for 3 days at 29 degrees C, is strongly induced by treatments at 4 degrees C. At sublethal temperatures of 10 and 15 degrees C, its expression in rice seedlings is already evident, but this effect cannot be vicariated by other stresses or ABA treatment. We demonstrate by transient expression that Myb4 transactivates the PAL2, ScD9 SAD and COR15a cold-inducible promoters. The Osmyb4 function in vivo is demonstrated overexpressing its cDNA in Arabidopsis thaliana plants (ecotype Wassilewskija) under the control of the constitutive CaMV 35S promoter. Myb4 overexpressing plants show a significant increased cold and freezing tolerance, measured as membrane or Photosystem II (PSII) stability and as whole plant tolerance. Finally, in Osmyb4 transgenic plants, the expression of genes participating in different cold-induced pathways is affected, suggesting that Myb4 represents a master switch in cold tolerance.     

Over-expression of ascorbate peroxidase in tobacco chloroplasts enhances the tolerance to salt stress and water deficit

The role of APX (ascorbate peroxidase) in protection against oxidative stress was examined using transgenic tobacco plants. The full length cDNA, coding Arabidopsis thaliana L. APX fused downstream to the chloroplast transit sequence from A. thaliana glutathione reductase, was cloned into appropriate binary vector and mobilized into Agrobacterium tumefaciens C58C2. Leaf discs were infected with the Agrobacterium and cultured on medium supplied with kanamycin. The incorporation of the gene in tobacco genome was confirmed by Southern dot blot hybridization. Transgenic lines were generated, and the line Chl-APX5 shown to have 3.8-fold the level of APX activity in the wild-type plants. The isolated chloroplasts from this line showed higher APX activity. During early investigation, this line showed enhanced tolerance to the active oxygen-generating paraquat and sodium sulphite. The first generation of this line, also, showed enhanced tolerance to salt, PEG and water stresses, as determined by net photosynthesis. The present data indicate that overproducing the cytosolic APX in tobacco chloroplasts reduces the toxicity of H₂O₂.     

Overexpression of Arabidopsis thaliana LTL1, a salt-induced gene encoding a GDSL-motif lipase, increases salt tolerance in yeast and transgenic plants

Genes involved in the mechanisms of plant responses to salt stress may be used as biotechnological tools for the genetic improvement of salt tolerance in crop plants. This would help alleviate the increasing problem of salinization of lands cultivated under irrigation in arid and semi-arid regions. We have isolated a novel halotolerance gene from Arabidopsis thaliana, A. thaliana Li-tolerant lipase 1 (AtLTL1), on the basis of the phenotype of tolerance to LiCl conferred by its expression in yeast. AtLTL1 encodes a putative lipase of the GDSL-motif family, which includes bacterial and a very large number of plant proteins. In Arabidopsis, AtLTL1 expression is rapidly induced by LiCl or NaCl, but not by other abiotic stresses. Overexpression of AtLTL1 increases salt tolerance in transgenic Arabidopsis plants, compared to non-transformed controls, allowing germination of seeds in the presence of toxic concentrations of LiCl and NaCl, and stimulating vegetative growth, flowering and seed set in the presence of NaCl. These results clearly point to a role of AtLTL1 in the mechanisms of salt tolerance. In addition, we show that AtLTL1 expression is also activated, although only transiently, by salicylic acid (SA), suggesting that the lipase could also be involved in defence reactions against pathogens.     

Overexpression of the wheat salt tolerance-related gene TaSC enhances salt tolerance in Arabidopsis

A novel gene named TaSC was cloned from salt-tolerant wheat. Northern blot showed that the expression of TaSC in salt-tolerant wheat was up-regulated after salt stress. Real-time quantitative PCR analyses showed that TaSC expression was induced by salt and ABA in wheat. Localization analysis showed that TaSC proteins were localized to the plasma membrane in transgenic Arabidopsis thaliana. The overexpression of TaSC in Col-0 and atsc (SALK_072220) Arabidopsis strains resulted in increased salt tolerance of the transgenic plants. TaSC overexpression in Col-0 and atsc signi?cantly up-regulated the expression of AtFRY1, AtSAD1, and AtCDPK2. AtCDPK2 overexpression in atsc rescued the salt-sensitive phenotype of atsc. The TaSC gene may improve plant salt tolerance by acting via the CDPK pathway.