Vitamin B6 Metabolism in Arabidopsis thaliana

Abstract

Vitamin B6 is an important coenzyme in over one hundred different cellular reactions and processes, including those of amino acid metabolism, heme and chlorophyll biosynthesis, ethylene biosynthesis, fatty acid metabolism, transcriptional regulation and response to oxidative stress. There are six different forms of vitamin B6 which are termed vitamers and include pyridoxal (PL), pyridoxine (PN) and pyridoxamine (PM) and their phosphorylated vitamers, PLP, PNP and PMP respectively. Vitamin B6 is synthesized de novo by two different enzymatic pathways, the DXP-dependent pathway of E. coli and a few other bacteria, and the DXP- independent pathway found in almost all other organisms, except for animals, who are generally unable to make their own vitamin B6. In addition to the de novo pathways, another pathway is found in all organisms, and functions to convert the six different vitamer forms between each other. This pathway is called the “salvage pathway.†Research reported in this dissertation focuses on genes involved in vitamin B6 biosynthesis in the model plant Arabidopsis. In the first study, the four Arabidopsis de novo pathway genes (PDX1.1, PDX1.2, PDX1.3 and PDX2) were characterized for expression and response to environmental stress conditions. Under control conditions, PDX1.1 and PDX1.3 had the highest expression, with PDX1.2 the lowest, and PDX2 intermediate. PDX1.1, PDX1.3, and PDX2 were upregulated by high light, chilling, and drought. PDX1.2 was upregulated in response to ozone. PDX2 fused to the green fluorescent protein was used to localize the PLP synthase enzyme complex in the nucleus and cellular membranes. In the second study, two different Arabidopsis mutants of vitamin B6 metabolism, pdx1.3 and sos4, were characterized. pdx1.3 is deficient in one of the homologs of the PLP synthase complex, while sos4 lacks a functional pyridoxine kinase, which phosphorylates the non-phosphorylated vitamers within the salvage pathway. These two mutants were found to have significantly different levels of B6 vitamers. Compared to wild type plants, the pdx1.3 mutant contains 40% vitamin B6 of wild type plants, while the sos4 mutant has 150% vitamin B6. However, both mutants were shown to share a large number of phenotypes, including chlorosis, decreased plant growth, altered chloroplast ultrastructure, altered expression of carbohydrate metabolism and photosynthetic genes, and severely decreased root growth when grown on exogenous sucrose. Many of the phenotypes could be explained by a deficiency of vitamin B6 within the chloroplast, and it was hypothesized that chloroplasts of both mutants were deficient in vitamin B6. Assays of vitamin B6 content in chloroplasts of the pdx1.3 and sos4 mutants showed that as compared to wild type, both mutants had significantly reduced levels of phosphorylated vitamers in chloroplasts with no difference in the levels of non-phosphorylated vitamers. Therefore, even though sos4 mutants have a surplus of vitamin B6 in their leaf tissue, they are deficient in vitamin B6 in their chloroplasts, resulting in phenotypes similar to vitamin B6- deficient pdx1.3 mutants. This work identifies an essential role for pyridoxal kinase in maintenance of vitamin B6 within the chloroplast.

Another hypothesis that was considered to explain the common phenotypes in the pdx1.3 and sos4 mutants was that the PDX1.3 and SOS4 proteins interacted with each other or in a common pathway, and that a mutation in either one of the proteins would produce the same phenotype. This work did not identify an interaction between the PDX1.3 and SOS4 proteins, but did identify a possible novel protein interaction of the PDX1.3 protein with a relatively uncharacterized zinc-binding oxidoreductase (At3g28670). Further work needs to be performed to confirm and characterize this interaction and its possible role in the pdx1.3 mutant phenotype.