Study of starch debranching enzymes in developing wheat kernels
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Starch debranching enzymes, which specifically hydrolyse a-1,6-glucosidic bonds in glucans containing both a-1,4 and a-1,6 linkages, are classified into two types: isoamylase (EC. 18.104.22.168) and limit dextrinase (EC. 22.214.171.124). The starch debranching enzymes are primarily involved in starch degradation during seed germination. Recently, however, starch debranching enzymes, particularly isoamylase, have been implicated in starch biosynthesis in many plant species. In this study, a 2,590-bp isoamylase cDNA, which encoded an 88-kDa isoamylase pre-protein containing the N-terminal transit peptide of 5 kDa, was isolated from a developing wheat kernel cDNA library. The recombinant protein produced in E. coli by expression of the cDNA exhibited isoamylase-type debranching enzyme activity. Accumulation of the 2.6-kb isoamylase transcripts in various tissues of wheat indicated the involvement of isoamylase in both starch synthesis in storage and non-storage organs, and starch degradation during seed germination. In the early stages during wheat kernel development, the presence of a 1.7-kb transcript was observed. The 1.7-kb transcript encoded a 51-kDa truncated isoamylase, whose starch debranching enzyme activity could not be detected in vitro. Southern blot analysis of genomic DNA of hexaploid wheat (Triticum aestivum L. cultivar CDC Teal) showed that isoamylase gene existed as a single copy in the wheat genome. Therefore, the production of various isoamylase transcripts and the involvement of isoamylase during two developmental stages of wheat plant may result from transcriptional modification of the existing gene and/or expression from a distantly related gene. Western blot analysis using rabbit antibodies raised against wheat isoamylase detected a single polypeptide of 83-kDa from developing wheat kernels. Accumulation of isoamylase polypeptide increased as the kernels developed from five to 15 days post-anthesis (dpa), and then started to decrease in 20-dpa kernels until it could not be detected in mature kernels. Similar to the presence of isoamylase, highest activity of limit dextrinase was observed in 15-dpa wheat kernels. According to the amounts of polysaccharides present at different stages during wheat kernel development, the concentration of starch increased as kernels matured, while the amylose/amylopectin ratio remained constant until the kernels reached a fully matured stage. The relationships between the concentrations of polysaccharides and the presence of isoamylase polypeptide, and the activity of limit dextrinase in developing wheat grains have suggested that debranching activities of starch debranching enzymes are essential to maintain a constant ratio of amylose and amylopectin during wheat kernel development.