Isolation and expression of two cDNA clones encoding UDP-galactose epimerase expressed in developing seeds of the endospermous legume guar

Abstract

UDP-galactose 4′-epimerase (UDPG epimerase) catalyses the reversible conversion of UDP- d-glucose to UDP- d-galactose. This compound is a precursor for the biosynthesis of various galactosides and cell wall polymers, including galactomannan which is the main storage polysaccharide in endospermous legumes. Using functional complementation of a UDPG epimerase deficient Escherichia coli mutant (PL-2) by a cDNA expression library from immature guar ( Cyamopsis tetragonoloba) seeds, galactose metabolising colonies with UDPG epimerase activities comparable to wild type level were obtained. Two cDNA clones (GEPI42 and GEPI48) encoding two different UDPG epimerases were isolated. Re-transformation of PL-2 by plasmid DNA, isolated from either of the two clones, resulted in numerous galactose-metabolising colonies, all with high UDPG epimerase activities. GEPI42 and GEPI48 encoded proteins with 354 and 350 amino acid residues, respectively, corresponding to deduced molecular weights of 39 286 and 38 373 Dalton, respectively. The amino acid sequence identity was 66.9%. Southern analysis of genomic guar DNA confirmed the origin and distinctness of the two UDPG epimerase genes. Analysis by immunohistochemistry showed the presence of significant levels of UDPG epimerase antigen in the endosperm of immature seeds with rapid galactomannan biosynthesis. In the endosperm of seeds close to maturity where galactomannan deposition has ceased, no antigen was detected. These data indicate that one or both of the two cloned UDPG epimerase genes are expressed in guar endosperm at a developmental stage where galactomannan biosynthesis occurs, suggesting that one or both may be involved in this process.