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.
Laing
William A
WA
Horticultural and Food Research Institute of New Zealand, PB 92160, Auckland 1142, New Zealand. wlaing@hortresearch.co.nz
Wright
Michele A
MA
Cooney
Janine
J
Bulley
Sean M
SM
D029042:Actinidia / Q000201:enzymology / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D001205:Ascorbic Acid / Q000096:biosynthesis / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018515:Plant Leaves / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
D013403:Sugar Phosphates; D009713:Nucleotidyltransferases; C020842:guanylyltransferase; D001205:Ascorbic Acid; D005690:Galactose