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

UDP-galactose 4'-epimerase (UDPG epimerase) catalyses the reversible conver sion 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 legu mes. Using functional complementation of a UDPG epimerase deficient Escheri chia coli mutant (PL-2) by a cDNA expression library from immature guar (Cy amopsis tetragonoloba) seeds, galactose metabolising colonies with UDPG epi merase activities comparable to wild type level were obtained. Two cDNA clo nes (GEPI42 and GEPI48) encoding two different UDPG epimerases were isolate d. Re-transformation of PL-2 by plasmid DNA, isolated from either of the tw o clones, resulted in numerous galactose-metabolising colonies, all with hi gh UDPG epimerase activities. GEPI42 and GEPI48 encoded proteins with 354 a nd 350 amino acid residues, respectively, corresponding to deduced molecula r weights of 39 286 and 38 373 Dalton, respectively. The amino acid sequenc e identity was 66.9%. Southern analysis of genomic guar DNA confirmed the o rigin and distinctness of the two UDPG epimerase genes. Analysis by immunoh istochemistry showed the presence of significant levels of UDPG epimerase a ntigen in the endosperm of immature seeds with rapid galactomannan biosynth esis. In the endosperm of seeds close to maturity where galactomannan depos ition 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, suggesti ng that one or both may be involved in this process. (C) 1999 Elsevier Scie nce Ireland Ltd. All rights reserved.