The missing step of the l-galactose pathway of ascorbate biosynthesis in plants, an l-galactose guanyltransferase, increases leaf ascorbate content
- *The Horticultural and Food Research Institute of New Zealand, PB 92160, Auckland 1142, New Zealand; and
- ‡The Horticultural and Food Research Institute of New Zealand, PB 3123, Hamilton 3240, New Zealand
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Edited by Charles J. Arntzen, Arizona State University, Tempe, AZ, and approved March 29, 2007 (received for review February 23, 2007)
Abstract
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.
Footnotes
- †To whom correspondence should be addressed. E-mail: wlaing{at}hortresearch.co.nz
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Author contributions: W.A.L. designed research; W.A.L., M.A.W., and J.C. performed research; J.C. and S.M.B. contributed new reagents/analytic tools; W.A.L. analyzed data; and W.A.L. wrote the paper.
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The authors declare no conflict of interest.
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This article is a PNAS Direct Submission.
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Data deposition: The sequence reported in this paper has been deposited in the GenBank database [accession no. EF379384 (EST 319998)].
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See Commentary on page 9109.
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This article contains supporting information online at www.pnas.org/cgi/content/full/0701625104/DC1.
- Abbreviations:
- GalT,
- d-galactose-1-phosphate-uridyltransferase;
- LCMS,
- liquid chromatography MS;
- SRM,
- selective reaction monitoring;
- SIM,
- selected ion monitoring;
- FW,
- Fresh Weight.
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Freely available online through the PNAS open access option.
- © 2007 by The National Academy of Sciences of the USA
