Cloning and expression of GDP-D-mannose pyrophosphorylase gene and ascorbic acid content of acerola (Malpighia glabra L.) fruit at ripening stages

Abstract

Acerola (Malpighia glabra L.) is one of the richest natural sources of L-ascorbic acid (AsA; vitamin C). GDP-D-mannose pyrophosphorylase (GMP; EC 2.7.7.13) was found to play a major role in the proposed AsA biosynthetic pathway in plants, considering that Arabidopsis vtc1-1 mutant with point mutation in this gene has a highly reduced AsA content. GMP cDNA was isolated from acerola fruits, designated MgGMP, using rapid amplification of cDNA ends (RACE), and its expression was monitored during fruit ripening. The full-length cDNA was found to have an ORF of 1083bp encoding a polypeptide of 361 amino acids. In silico analysis of the predicted amino acid sequence showed a pI of 6.45 and molecular mass of 39.7kD. MgGMP showed over 80% amino acid sequence identity with other plant GMP homologues. The phylogenetic tree shows the close relation of MgGMP to the GMP of other plants as against those from parasite, yeasts and mammals. Southern analysis indicated that M. glabra contains not less than two copies of GMP genes. Northern blot analysis showed the transcript abundance of MgGMP in all the organs of acerola examined, with the fruit having the highest expression. The relative transcript abundance of MgGMP mRNA levels in the fruits changes as the ripening process progresses, with the unripe green fruits having the highest relative mRNA level, and the lowest was found in the fruits at advanced ripening stage. A strong correlation was also observed between the relative MgGMP mRNA levels and the AsA contents of acerola during fruit ripening.