The goals of this work are to characterize the pathways for vitamin B6 biosynthesis in plants and to demonstrate a role for this vitamin in plant responses to environmental stress. Vitamin B6 is required in all organisms for its essential role as an enzyme co-factor. Previous work identified the major de novo biosynthetic pathway in fungi. These studies also provided evidence that pyridoxine and its vitamers quench singlet oxygen and superoxide and have antioxidant activity, results that support the hypothesis that B6 vitamers play a heretofore unidentified role in cellular stress responses and antioxidant defense. Preliminary work has demonstrated that genes in the de novo pyridoxine biosynthetic pathway in Arabidopsis are upregulated in response to stress caused by high light intensities, chilling, and drought. Tolerance or susceptibility to these stresses has been strongly linked in plants, and this coordinated response is also consistent with localization of pyridoxine biosynthesis in chloroplasts. This research will investigate the role of pyridoxine in tolerance to high light, chilling, and drought by overexpression of the de novo biosynthetic genes in Arabidopsis and characterizing the response to these stresses. Green fluorescent protein fusion constructs with epifluorescence and confocal microscopy will be used to determine the cellular localization of the pathway enzymes in cells. In addition, this research will characterize genes involved in the vitamin B6 salvage pathway in Arabidopsis, the pathway that interconverts between pyridoxine and pyridoxal and their phosphorylated derivatives. Analysis of relative vitamer levels of T-DNA insertion knockout mutants and/or overexpression lines will be used to confirm the salvage pathway genes. Genes will be assayed for regulation in response to high light, chilling, drought, and other oxidative stress-inducing conditions, and the response of knockout mutants or overexpression lines will confirm the possible role of the salvage pathway in oxidative stress responses. The results of this study will identify the genes involved in the vitamin B6 de novo and salvage pathways in a model plant, Arabidopsis, and demonstrate the cellular localization of the two pathways. This study will also provide evidence for a new function for this important vitamin in protecting plants and perhaps other organisms against environmental and oxidative stresses. This research has the potential to lead to the development of crop plants with improved ability to tolerate environmental stresses including chilling, drought, and high light. This project will also benefit teaching and the training of young scientists.
Vitamin B6 is an essential vitamin, required by all living organisms. Little is known, however, about how organisms synthesize this vitamin. This research will characterize the pathway for production of vitamin B6 in plants. Further, recent evidence suggests that vitamin B6 may play a role (similar to vitamins C and E) in protecting cells against toxicants and environmental stresses. This research will investigate the importance of this vitamin in tolerance of plants to environmental stresses, and has the potential to lead to the development of crop plants with improved resistance to cold, drought, and salinity.