Two genes in Arabidopsis thaliana encoding GDP-L-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability.
Plants synthesize ascorbate from guanosine diphosphate (GDP)-mannose via L-galactose/L-gulose, although uronic acids have also been proposed as precursors. Genes encoding all the enzymes of the GDP-mannose pathway have previously been identified, with the exception of the step that converts GDP-L-galactose to L-galactose 1-P. We show that a GDP-L-galactose phosphorylase, encoded by the Arabidopsis thaliana VTC2 gene, catalyses this step in the ascorbate biosynthetic pathway. Furthermore, a homologue of VTC2, At5g55120, encodes a second GDP-L-galactose phosphorylase with similar properties to VTC2. Two At5g55120 T-DNA insertion mutants (vtc5-1 and vtc5-2) have 80% of the wild-type ascorbate level. Double mutants were produced by crossing the loss-of-function vtc2-1 mutant with each of the two vtc5 alleles. These show growth arrest immediately upon germination and the cotyledons subsequently bleach. Normal growth was restored by supplementation with ascorbate or L-galactose, indicating that both enzymes are necessary for ascorbate generation. vtc2-1 leaves contain more mannose 6-P than wild-type. We conclude that the GDP-mannose pathway is the only significant source of ascorbate in A. thaliana seedlings, and that ascorbate is essential for seedling growth. A. thaliana leaves accumulate more ascorbate after acclimatization to high light intensity. VTC2 expression and GDP-L-galactose phosphorylase activity rapidly increase on transfer to high light, but the activity of other enzymes in the GDP-mannose pathway is little affected. VTC2 and At5g55120 (VTC5) expression also peak in at the beginning of the light cycle and are controlled by the circadian clock. The GDP-L-galactose phosphorylase step may therefore play an important role in controlling ascorbate biosynthesis.
Dowdle
John
J
School of Biosciences, University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter EX4 4QD, UK.
Ishikawa
Takahiro
T
Gatzek
Stephan
S
Rolinski
Susanne
S
Smirnoff
Nicholas
N
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001205:Ascorbic Acid / Q000096:biosynthesis*; D018507:Gene Expression Regulation, Developmental / Q000528:radiation effects; D018506:Gene Expression Regulation, Plant / Q000528:radiation effects; D018525:Germination / Q000235:genetics / Q000502:physiology; D008027:Light; D008969:Molecular Sequence Data; D009154:Mutation; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D036226:Seedlings / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D029681:Arabidopsis Proteins; C522629:GDP-L-galactose phosphorylase, Arabidopsis; D010744:Phosphoric Monoester Hydrolases; D001205:Ascorbic Acid