The missing step of the l -galactose pathway of ascorbate biosynthesis in plants, an l -galactose guanyltransferase, increases leaf ascorbate content

The gene for one postulated enzyme that converts GDP-L-galactose to L-galactose-1-phosphate is unknown in the L-galactose pathway of ascorbic acid biosynthesis and a possible candidate identified through map-based cloning is the uncharacterized gene At4g26850. We identified a putative function for At4g26850 using PSI-Blast and motif searching to show it was a member of the histidine triad superfamily, which includes D-galactose uridyltransferase. We cloned and expressed this Arabidopsis gene and the homologous gene from Actinidia chinensis in Escherichia coli and assayed the expressed protein for activities related to converting GDP-L-galactose to L-galactose-1-P. The expressed protein is best described as a GDP-L-galactose-hexose-1-phosphate guanyltransferase (EC 2.7.7.), catalyzing the transfer of GMP from GDP-l-galactose to a hexose-1-P, most likely D-mannose-1-phosphate in vivo. Transient expression of this A. chinensis gene in tobacco leaves resulted in a >3-fold increase in leaf ascorbate as well as a 50-fold increase in GDP-L-galactose-D-mannose-1-phosphate guanyltransferase activity.