Poster: Genomic & proteomic resources

Abs # 897: Functionally defining genes for pyridoxine-5-phosphate oxidase and pyridoxal reductase in Arabidopsis thaliana

Presenter:	Manning, Mark A, mannima@auburn.edu
Authors	Manning, Mark A (A)   Ellis, Melissa  (A)   Oliver, Melanie  (A) (B)  Cherry, Joe H. (A)   Locy, Robert D (A)
Affiliations:	(A): Auburn University (B): Unviersity of Alabama at Birmingham

Plants are an important source of vitamin B6 (pyridoxal-5-phosphate, PLP) for human nutrition, and PLP has emerged as an interesting signaling molecule during environ-mental stresses in plants. Thus, genetic manipulation of food-plant vitamin content and plant stress tolerance requires that we understand the genes involved in the biosynthesis of PLP in plants. However, the biochemical pathway of PLP synthesis in eukaryotic organisms, and plants in particular, is largely undetermined. The last step in this pathway must involve the conversion of pyridoxine-5-phosphate (PNP) or pyridoxine (PN) into PLP or pyridoxal (PL) respectively. In bacteria, fungi, and animals these two reactions are catalyzed by PNP oxidase (PPOX) and PL reductase (PR). Two putative genes from Arabidopsis thaliana thought to code for a pyridoxal-5-phosphate oxidase (PPOX) and a pyridoxal reductase (PR) have been identified using bioinformatic analysis of Arabidopsis genomic sequences. These sequences have been obtained by PCR amplification using an Arabidopsis cDNA library as template. The cloned sequences have been placed in a yeast shuttle vector, and transformed into a Saccharomyces double deletion mutant strain containing mutations in the YBR035c (PPOX) and YPR 127w (PR) genes. This double mutant is unable to make PLP using either native PPOX or PR. The phenotype of the double mutant separately expressing either Arabidopsis PPOX or PR will be examined to functionally demonstrate that the cloned Arabidopsis sequences are in fact PPOX and PR genes.