Antisense suppression of l-galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated l-galactose synthesis.
l-Galactose dehydrogenase (l-GalDH), a novel enzyme that oxidizes l-Gal to l-galactono-1,4-lactone (l-GalL), has been purified from pea seedlings and cloned from Arabidopsis thaliana. l-GalL is a proposed substrate for ascorbate biosynthesis in plants, therefore the function of l-GalDH in ascorbate biosynthesis was investigated by overexpression in tobacco and antisense suppression in A. thaliana. In tobacco the highest expressing lines had a 3.5-fold increase in extractable activity, but this did not increase leaf ascorbate concentration. Arabidopsis thaliana, transformed with an antisense l-GalDH construct, produced lines with 30% of wild-type activity. These had lower leaf ascorbate concentration when grown under high light conditions. l-Gal pool size increased in antisense transformants with low l-GalDH activity, and l-Gal concentration was negatively correlated with ascorbate. The results provide direct evidence for a role of l-GalDH in ascorbate biosynthesis. Ascorbate pool size in A. thaliana is increased by acclimation to high light, but l-GalDH expression was not affected. l-Gal accumulation was higher in antisense plants acclimated to high light, indicating that the capacity to synthesize l-Gal from GDP-mannose is increased. Because the only known function of l-GalL is ascorbate synthesis, these antisense plants provide an opportunity to investigate ascorbate function with minimal effects on carbohydrate metabolism. Measurements of other antioxidants revealed an increase in ascorbate- and pyrogallol-dependent peroxidase activity in low-ascorbate lines. As ascorbate is the major hydrogen peroxide-scavenging antioxidant in plants, this could indicate a compensatory mechanism for controlling hydrogen peroxide concentration.
Gatzek
Stephan
S
School of Biological Sciences, University of Exeter, Washington Singer Laboratories, Perry Road, Exeter EX4 4QG, United Kingdom. S.Gatzek@exeter.ac.uk
Wheeler
Glen L
GL
Smirnoff
Nicholas
N
D000975:Antioxidants / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D001205:Ascorbic Acid / Q000096:biosynthesis* / Q000235:genetics; D016373:DNA, Antisense / Q000235:genetics / Q000378:metabolism*; D005690:Galactose / Q000096:biosynthesis*; D005691:Galactose Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D008027:Light; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000201:enzymology; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
D000975:Antioxidants; D016373:DNA, Antisense; D005691:Galactose Dehydrogenases; C047959:galactose dehydrogenase; D001205:Ascorbic Acid; D005690:Galactose