Genetic and molecular characterization of maize starch debranching enzymes

Starch is the primary carbohydrate reserve of plants, a significant source of food in the human diet, and an important industrial raw material. The arrangement of linear chains and branch linkages within amylopectin is responsible for the highly conserved architecture of the starch granule. This structure allows for efficient packaging of glucose and results in the unique functional properties of starch. It is known that starch synthases and branching enzymes catalyze alpha(1→4) chain elongation and introduction of alpha(1→6) branch linkages, respectively, but studies in several species suggests that starch debranching enzymes (DBE) are also involved in the process of starch synthesis. The overall goal of this research is to determine the role played in maize starch metabolism by each individual DBE. DBEs are broadly classified as either isoamylase- or pullulanase-type based upon their substrate specificity and sequence similarity to defined bacterial proteins. To determine the function(s) of the isoamylase-type DBE, an allelic series of mutations in the gene Su1 was characterized. The molecular defects of the su1-alleles were associated with accumulation of phytoglycogen at the expense of granular starch. The phenotypic severity was also correlated to an alteration in the fine structure of the residual amylopectin. Thus, the SU1 isoamylase-type DBE functions directly in starch synthesis by removing branches from an amylopectin precursor. cDNAs coding for two other isoamylase-type DBE genes, ZmIso2 and ZmIso3, were cloned and characterized with respect to mRNA transcript accumulation. This analysis indicated metabolic specialization for some DBE isoforms either during starch catabolism or anabolism. To investigate the function(s) of the pullulanase-type DBE, reverse genetics was used to identify a transposable element insertion in the maize Zpu1 gene. Characterization of this mutation demonstrated that pullulanase-type DBE is important for degradation of both leaf and endosperm starch. ZPU1 also acts during starch synthesis significantly reducing phytoglycogen accumulation in some genetic backgrounds. This suggests that the biosynthetic function of ZPU1 partially overlaps with the biosynthetic function of SU1. Together, these data indicate that isoamylase- and pullulanase-type DBEs are each involved in aspects of both starch synthesis and degradation