The Starch-Debranching Enzymes Isoamylase and Pullulanase Are Both Involved in Amylopectin Biosynthesis in Rice Endosperm

Abstract

The activities of the two types of starch debranching enzymes, isoamylase and pullulanase, were greatly reduced in endosperms of allelic sugary-1 mutants of rice (Oryza sativa), with the decrease more pronounced for isoamylase than for pullulanase. However, the decrease in isoamylase activity was not related to the magnitude of the sugary phenotype (the proportion of the phytoglycogen region of the endosperm), as observed with pullulanase. In the moderately mutated line EM-5, the pullulanase activity was markedly lower in the phytoglycogen region than in the starch region, and isoamylase activity was extremely low or completely lost in the whole endosperm tissue. These results suggest that both debranching enzymes are involved in amylopectin biosynthesis in rice endosperm. We presume that isoamylase plays a predominant role in amylopectin synthesis, but pullulanase is also essential or can compensate for the role of isoamylase in the construction of the amylopectin multiple-cluster structure. It is highly possible that isoamylase was modified in some sugary-1 mutants such as EM-273 and EM-5, since it was present in significant and trace amounts, respectively, in these mutants but was apparently inactive. The results show that the Sugary-1 gene encodes the isoamylase gene of the rice genome.The super-cluster structure of amylopectin (Gallant et al., 1997) might have evolved as a fitting strategy for plant survival and must be accomplished by well-refined regulation of a network of numerous enzyme actions. The fine structure of amylopectin is distinct from that of glycogen in animals and bacteria in that glycogen is randomly branched, the branches are more numerous, and the chains are shorter compared with amylopectin. Biochemical analyses of sugary-type mutants of maize, rice (Oryza sativa), Arabidopsis, and Chlamydomonas reinhardtii have provided new insights needed to understand the mechanism of synthesis and construction of the amylopectin fine structure

The Effect of the Amount of Nitrogen Fertilizer on Starch Metabolism in Leaf Sheath of Japonica and Indica Rice Varieties during the Heading Period

“…In endosperm during grain fi lling of cereal plants, DBE probably play a crucial role in the starch synthesis (James et al, 1995, Nakamura et al, 1996Kubo et al, 1999;Burton et al, 2002;Dinges et al, 2003). As shown in Fig.…”

“…There are two types of DBE, pullulanase and isoamylase, that hydrolyze the α-1,6-glucosidic bond in amylopectin. In addition, it is accepted that DBE also plays an important role in starch synthesis (James et al, 1995;Nakamura et al, 1996;Kubo et al, 1999;Burton et al, 2002;Dinges et al, 2003). We examined the activity of pullulanase-type DBE in rice leaf sheath during the heading period (Fig.…”

“…However, analyses of mutants with the reduced starch content in endosperm in rice (Nakamura et al, 1996;Kubo et al, 1999), maize (James et al, 1995;Dinges et al, 2003), and barley (Burton et al, 2002) indicate that DBE plays a crucial role in the starch synthesis. Though a function of DBE in the rice leaf sheath is not clear, DBE may also be involved in the starch synthesis in leaf sheath.…”

Allelic variation at a single gene increases food value in a drought-tolerant staple cereal

“…Knockouts of maize and rice pullulanases result in the loss of pullulanase activity (PA) without readily detectible phenotypic changes in plant growth or grain phenotypes 16,18 . Pullulanase function overlaps partially with the de-branching isoamylases and it has been reasoned that pullulanase knockouts are still viable because of activity of the isoamylases 16,19 .…”

Distribution of Starch-degrading Enzymes in Rice Grains of Different Cultivars and Elution Behavior during Cooking

“…Both enzymes differ in their substrate specificity. Isoamylases can debranch glycogen, but scarcely attack pullulan, while pullulanase shows reverse specificity.Several studies have revealed that starch-debranching enzymes not only hydrolyze amylopectin in storage starch during germination, but also play essential roles in amylopectin biosynthesis (Ball et al, 1996;Kubo et al, 1999). In our previous studies, we revealed the distribution of starch-degrading enzymes in rice grains and their elution behavior during cooking with a specific antibody against each enzyme, including α-glucosidase, α-amylases, β-amylase, pullulanase and isoamylase, in Koshihikari cultivars (Tsuyukubo et al, 2010;Tsuyukubo et al, 2012).…”