Molecular cloning and expression of GDP-D-mannose-3",5"-epimerase during fruit ripening in acerola

Man is unable to synthesize an essential compound L-ascorbic acid (Vitamin C; AsA), and so depends on external sources such as fruits and vegetables for its supply. GDP-D-mannose 3 '',5 ''-epimerase (GMEase) is one of the vital enzymes in the D-Mannose/L-galactose pathway proposed for L-ascorbic acid biosynthesis in plants. We have cloned and sequenced a cDNA encoding GMEase from acerola (Malpighia glabra L.), designated MgGMEase. The full-length cDNA was 1675 bp and contained an open reading frame of 1128 bp encoding a peptide of 376 amino acids with a predicted molecular mass of 42.5 kD and an isoelectric point of 5.80. The predicted gene sequence is highly conserved among plant species with greater than 89% identity at the protein level with the GMEase of Arabidopsis thaliana and Oryza sativa, and 44% and 42% with the sugar epimerase BlmG genes of Salinibacter ruber and Streptomyces verticillus, respectively. Q-PCR analysis revealed the abundance of MgGMEase transcripts in all organs examined, especially the fruit, which also had the highest amount of ascorbic acid. The expression of MgGMEase transcript shown by northern blot analysis and its relative mRNA levels revealed a high correlation with the changing ascorbic acid contents of acerola fruits as ripening progressed.