Genetic manipulation of Vitamin B-6 biosynthesis in tobacco and fungi uncovers limitations to up-regulation of the pathway

Transgenic expression of vitamin biosynthetic genes has been investigated for over-production of these dietary supplements in microorganisms and plants. In plants, successful efforts have been reported with Vitamins A, C, E and B-9, however information is lacking for other vitamins. Vitamin B-6 is an essential cofactor for numerous enzymatic reactions, and has also been shown to be a potent antioxidant involved in protecting phytopathogenic Cercospora fungi from their own toxin, cercosporin. In this report, we transformed and expressed two Vitamin B-6 biosynthetic genes (PDX1 and PDX2) isolated from Cercospora nicotianae in cercosporin-sensitive organisms, tobacco and the fungal species Aspergillus flavus and Neurospora crassa. Our goal was to determine if Vitamin B-6 levels could be increased by constitutive expression of these genes, and if over-production confers resistance to oxidative stresses induced by cercosporin and salinity stress. Elevated Vitamin B-6 levels were observed in one tobacco line. For other lines evaluated in this work, expression of PDX1 and PDX2 in transgenic organisms did not result in a significant increase in Vitamin B-6 content over controls. Analysis of gene expression in tobacco indicated that the lack of elevated B-6 content was not due to lack of enzymatic activity, but to down-regulation of the endogenous tobacco genes compounded with limited transgene expression. The single line with elevated B-6 levels had higher expression of both the PDX1 and PDX2 transgenes compared to the other lines, and the observed increase on Vitamin B-6 was correlated with higher enzyme activity. Consistent with our inability to elevate cellular B-6 levels, only small changes were observed in the response to either cercosporin or high salt, and most transgenic individuals were as susceptible as controls. Compared to tobacco lines transformed to express either PDX1 or PDX2 alone, half of the transgenic tobacco lines expressing both genes were impaired in seed germination and initial growth. However no correlation was observed between the observed phenotype and Vitamin B-6 levels in seeds. This is the first report on genetic engineering to manipulate the Vitamin B-6 pathway in plants. Our results suggest that genetic manipulation of the Vitamin B-6 biosynthetic pathway is possible but is limited by regulation of endogenous genes.