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.

Ascorbate (vitamin C) is a potent antioxidant protecting plants against oxidative damage imposed by environmental stresses such as ozone and drought. Dehydroascorbate reductase (DHAR; EC 1.8.5.1) is one of the two important enzymes functioning in the regeneration of ascorbate (AsA). To examine the protective role of DHAR against oxidative stress, we developed transgenic tobacco plants overexpressing cytosolic DHAR gene from Arabidopsis thaliana. Incorporation of the transgene in the genome of tobacco plants was confirmed by polymerase chain reaction and Southern blot analysis, and its expression was confirmed by Northern and Western blot analyses. These transgenic plants exhibited 2.3–3.1 folds higher DHAR activity and 1.9–2.1 folds higher level of reduced AsA compared with non‐transformed control plants. The transgenic plants showed maintained redox status of AsA and exhibited an enhanced tolerance to ozone, drought, salt, and polyethylene glycol stresses in terms of higher net photosynthesis. In this study, we report for the first time that the elevation of AsA level by targeting DHAR overexpression in cytosol properly provides a significantly enhanced oxidative stress tolerance imposed by drought and salt.

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 H(2)O(2).

No abstract