Biochemical consequences of ascorbate deficiency were studied in the leaf tissue of Arabidopsis thaliana ascorbate-deficient vtc mutants with a view of understanding the relationship between ascorbate, stress response and metabolism. Ascorbate is an important antioxidant and is also a cofactor for 2-oxoglutarate-dependent dioxygenases, which are involved in the biosynthesis of a number of metabolites. The response of wild type (Col-0) and vtc1, vtc2-1, vtc2-2 and vtc3-1 mutants to high light intensity, wounding and salinity was investigated using a metabolomics and proteomics approach. Metabolite profiling and comparative proteomics were performed by liquid chromatography-quadrupole time of flight mass spectrometry (LC-QToF MS) and targeted analysis of plant hormones and flavonoids by liquid chromatography triple-quadrupole mass spectrometry (LC-QQQ-MS). These combined analyses revealed the effect of ascorbate deficiency and stress on metabolites and cell wall proteins. LC-QToF-MS based untargeted metabolite profiling methodologies were developed for analysis of metabolites on a large scale. Using this method about 3000-5000 metabolites (mass-retention time pairs) could be reproducibly detected in A. thaliana leaf extract and aligned between samples. Approximately 1000 metabolites were differentially expressed between WT and vtc mutants in different experiments. Of these, twenty eight compounds were confirmed to be differentially expressed by LC-QQQ-MS between WT and vtc mutants, and eight of these compounds were positively identified and validated with standards. The plant hormones abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA) have all been implicated in plant stress responses and differences in their accumulation in some of the vtc mutants have been reported. A systematic study of the response to stress of these hormones in several vtc mutants was carried out using LC- QQQ- MS. While some of the mutants showed increased SA and SA-glycoside accumulation, stress-induced ABA and JA accumulation was generally unaffected. Methods for identifying the metabolites in a targeted manner by LC- QQQ-MS was developed and were shown that all vtc mutants were impaired in the accumulation of anthocyanin in response to HL treatment. In strong contrast to anthocyanin, flavonol glycosides were not affected by ascorbate deficiency. Therefore, ascorbate deficiency has a specific effect on the anthocyanin biosynthesis. Ascorbate occurs in the plant cell wall and isolation of apoplastic fluid showed that all vtc mutants have decreased apoplastic ascorbate compared to WT. Ionically-bound proteins were from the cell wall of A. thaliana leaves. Peroxidase specific activity in this fraction tended to be higher in vtc mutants than WT. High light intensity also increased peroxidase activity in WT and vtc mutants. To determine which peroxidase isoenzyme caused increased peroxidase activity, ionically-bound cell wall N-glycosylated proteins were isolated by Concanavalin A chromatography and analysed by LC-QToF-MS. Comparison of WT and vtc2-2 grown in low light and high light identified 937 peptides significantly different between WT and vtc2-2 and some are also affected by light intensity. Specifically, peroxidases 33 and 34 had increased abundance in vtc2-2. The results show that ascorbate deficiency causes a detectable change in the metabolome of A. thaliana leaves, with specific effects on anthocyanin accumulation being detected. Ascorbate deficiency also influences the expression of cell wall proteins. Peroxidase activity is increased, and this response could be related to the increased pathogen resistance reported in vtc mutants.

Vitamin C (ascorbic acid) is one of the most essential organic compounds required by the human body for normal metabolic function. Unfortunately, this valuable nutrient is not produced in the human body but most plants and animal can produce this molecule. Although ascorbic acid was not isolated until the early part of the twentieth century, it was known that eating limes and other citrus fruits could ward off the affects of scurvy as early as the 1500's. Ascorbate serves many critical functions in plants as well as the human body. In both, it works as a cofactor in the production of hydroxyproline-rich compounds and helps protect molecules such as proteins, lipids and fatty acids from oxidation. Although the biochemical pathway in animals has been known since the 1950's (Jackel et al., 1950), the exact process by which ascorbic acid is made in plants has eluded scientists. It was shown in 1963 that the inversion of the hexose carbon chain, which occurs in the animal pathway, is not a possible mode of synthesis in plants (Loewus, 1963). As an alternative, a non-inversion pathway was proposed, which achieves ascorbic acid using D-mannose and L-galactose as intermediates, referred to as the Smirnoff-Wheeler pathway (Wheeler et al., 1998). It was shown that transforming lettuce (cv. Grand Rapids and Black Seeded Simpson) and tobacco (cv. Xanthi) with the terminal enzyme in the animal biosynthetic pathway (GLO; L-gulono-gamma-lactone oxidase) increases the ascorbic acid content between 4 and 7 fold. It was also shown through feeding studies that wild type tobacco plants had elevated ascorbate levels when fed the animal precursor (Jain and Nessler, 2000). These data suggest that at least part of the animal pathway could be present in plants, along with the Smirnoff-Wheeler (1998) pathway. To further investigate this discovery, wild type and ascorbic acid-deficient Arabidopsis thaliana were transformed with the glo. Homozygous lines of these transformants were generated and the ascorbic acid levels were compared to the untransformed wild type and mutant plants. Although the wild type plants containing glo did not show a significant increase in ascorbic acid production, all five of the vtc mutant lines had an increased ascorbic acid content relative to wild type level. These data suggest that an alternative pathway is present in plants that does not require many of the steps in the published Smirnoff-Wheeler (1998) pathway to produce ascorbic acid. / Master of Science