The sugary-type isoamylase in wheat: tissue distribution and subcellular localisation

To gain an increased understanding of the role of isoamylase (EC 3.2.1.68) in amylopectin synthesis, we studied the tissue-specific distribution and subcellular localisation of this enzyme in wheat (Triticum aestivum L.). A cDNA for wheat isoamylase was isolated from an endosperm-specific library and the missing 5' end was amplified by anchored polymerase chain reaction. Isoamylase transcripts were detected in reproductive and vegetative tissues, with the highest levels occurring in developing kernels. Wheat kernels were then dissected into embryo, endosperm, pericarp and chlorophyll layer, and subjected to protein blot analysis. Isoamylase was most abundant in the endosperm. Within the endosperm, the vast majority of isoamylase was soluble. A much smaller amount of the enzyme was associated with starch granules. Isoamylase was not trapped within starch granules and was absent from dry seeds. Isoamylase was also present in green tissue, which suggests a role in the synthesis of both reserve and leaf starches.