Abstract:

Amylopectin is a major constituent of starch in plant tissues, physical and chemical analysis of granular starch have led to a widely accepted model for amylopectin structure called “cluster model”, in which amorphous and crystalline regions alternate with a defined periodicity. Within amylopectin the crystalline component is composed of parallel arrays of linear chains packed tightly in double helices. Branch linkages, which account for approximate 5% of the glucosyl linkages in amylopectin, are located at the root of each cluster in the amorphous region. This periodic clustering of branches allows for the alignment of the intervening linear chains and their dense packing into crystalline regions, thus providing an efficient mechanism for nutrient storage. Biochemical analysis of su-1 mutants of maize (Zea mays L.) and rice (Oryza sativa L.) endosperm indicate that debranching enzymes (DBE) are proposed to play an essential role in the final determination of amylopectin structure. The precise way in which debranching enzymes involved in amylopectin synthesis is difficult to assess because of the lack of information about the occurrence and nature of these enzymes in starch-synthesizing organs and relatively little is known about these enzymes in higher plants. To provide more information about the occurrence and possible role of debranching enzymes in starch synthesis, six wheat (Triticum aestivum L.) cultivars corresponding to Lumai 21 and Yanyou 361 (low amylopectin), Lumai 1 and Jining 13 (medium amylopectin), and Jinan16 and Nuomai 2 (high amylopectin) were used in the present study. Activity changes, activity difference and types of DBE were studied during grain filling period by using spectrophotometer, native-PAGE and HPLC. The DBEs were also purified by salting out with ammonium sulphate, DEAE-cellulose column chromatograthy, and gel filtration using sephadex G-100. The results showed that two types of DBE were identified during starch synthesis and they all reached the highest activity at the 15th day after anthesis, the activity changes of isoamylase appeared as a single-peak curve, but those of pullulanase appeared as an undulatory curve, with another high activity around the 25th day after flowering. The molecular weights of isoamylase and pullulanase were determined to be 83 and 100 kD by SDS-PAGE respectively. HPLC analysis showed that activities of starch debranching enzymes to amylopectin and pullulan were similar among the six wheat cultivars. Glucose and dextrin were identified as the major hydrolysis products of amylopectin by isoamylase and maltotriose was identified as a major hydrolysis product of pullulan by pullulanase.

Key words: Wheat, Starch Debranching-enzyme, Isoamylase, Pullulanase