Molecular analysis of monodehydroascorbate reductase and dehydroascorbate reductase in plants
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Oxidative stress is an inevitable phenomenon in green plants because they generate toxic reactive oxygen species. Through normal metabolism plants produce copious amount of molecular oxygen in cellular organelles that carry out highly energetic electron transfer reactions, resulting in the reduction of oxygen to form reactive oxygen intermediates (ROI's). Therefore, scavenging systems to remove reactive oxygen species are necessary. Ascorbate is the primary soluble antioxidant compound that can scavenge ROI's in plant cells. Monodehydroascorbate reductase (MR, EC 18.104.22.168) and dehydroascorbate reductase (DR, EC 22.214.171.124) are the enzymes responsible for the regeneration of ascorbate. As little is known about the role of MR and DR in oxidative stress in plants, this study was focused on determining the protective roles of these enzymes against oxidative stress using molecular genetic approaches. In order to fulfill this goal, transgenic tobacco plants that overexpress cucumber MR were developed. Analyses of the stress tolerance of the MR overexpressing plants were also investigated. Some plants showed increased tolerance against photoinhibition and methyl viologen exposure. However, they showed no increased stress tolerance differences during germination under low temperature and salt stress. Identification of a tobacco MR using PCR was also performed. The nucleotide sequence and amino acid comparison of tobacco MR with other plant MRs showed significant similarity. It was shown that FAD"" and NAD* binding sites were highly conserved between species. In addition, the ascorbate regeneration systems in honey locust leaves were investigated in this research. The Sunburst variety of honey locust showed higher sensitivity to light. Leaves of these plants were green in the shade but yellow in the light. This photosensitivity could be caused by the loss of chlorophyll due to oxidative stress. Surprisingly, Sunburst honey locust leaves had less DR activity and more MR activity than wild type leaves. In this system, DR seems to be required ascorbate regeneration. Although Sunburst leaves had elevated MR leaves, this was not support to overcome the photo bleaching phenotype. From this research, it was possible to better understand the function of MR and DR in oxidative stress. Understanding the role of these enzymes could lead to development of stress resistant transgenic crops capable of increased production.