Antisense suppression of l‐galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated l‐galactose synthesis

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Abstract
Summary: 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.