Biochemical and Genetic Analysis of the Effects of Amylose-Extender Mutation in Rice Endosperm

Abstract

Biochemical analysis of amylose-extender (ae) mutant of rice (Oryza sativa) revealed that the mutation in the gene for starch-branching enzyme IIb (BEIIb) specifically altered the structure of amylopectin in the endosperm by reducing short chains with degree of polymerization of 17 or less, with the greatest decrease in chains with degree of polymerization of 8 to 12. The extent of such change was correlated with the gelatinization properties of the starch granules, as determined in terms of solubility in urea solution. The ae mutation caused a dramatic reduction in the activity of BEIIb. The activity of soluble starch synthase I (SSI) in the ae mutant was significantly lower than in the wild type, suggesting that the mutation had a pleiotropic effect on the SSI activity. In contrast, the activities of BEI, BEIIa, ADP-Glc pyrophosphorylase, isoamylase, isoamylase, pullulanase, and Suc synthase were not affected by the mutation. Therefore, it is stressed that the function of BEIIb cannot be complemented by BEIIa and BEI. These results strongly suggest that BEIIb plays a specific role in the transfer of short chains, which might then be extended by SS to form the A and B 1 chains of amylopectin cluster in rice endosperm.Starch is composed of two types of molecule, namely amylose and amylopectin. Amylose is an essentially linear molecule composed of ␣(134)-linked glucosidic chains, whereas amylopectin is a highly branched glucan with ␣(136) glucosidic bonds that connect linear chains. The ␣-1,4 chains of amylopectin consist of A chains that carry no additional chains, B chains that carry A chains or other B chains, and a C chain that includes the reducing terminus (Peat et al., 1952). Hizukuri (1986) proposed a cluster model for amylopectin. In this model, A and B 1 chains form a single cluster, whereas B 2 and B 3 chains extend to two and three clusters, respectively. Hanashiro et al. (1996) proposed that, in amylopectin, chains of degree of polymerization (DP) Ϲ12, 13 Ϲ DP Ϲ 24, 25 Ϲ DP Ϲ 36, and DP м 37 correspond to A chains, B 1 chains, B 2 chains, and B 3 and longer chains, respectively.

Gradually Decreasing Starch Branching Enzyme Expression Is Responsible for the Formation of Heterogeneous Starch Granules

“…S2A). A reduction in SSI activity was demonstrated previously in sbe2b-related mutants, including the sbe2b single mutant (Nishi et al, 2001), and ss1 L /be2b and ss3a/be2b double mutants Asai et al, 2014). For other starch biosynthesis-related enzymes, including SSIIa, SSIIIa, isoamylase1 (ISA1), ISA2, pullulanase, and Pho1, their mRNA levels, soluble protein amounts, and activity were not significantly different between these two materials ( Fig.…”

Starch accumulation, activities of key enzyme and gene expression in starch synthesis of wheat endosperm with different starch contents

“…Several enzymes influence the fine structure of amylopectin, mainly by altering its cluster structure, although the modes of changes in amylopectin cluster structure strongly depend on the individual enzyme affected. SBE-IIb plays a specific role in the formation of Achains of the amylopectin cluster (Nishi et al 2001), and the levels of BEIIb activity affect the extent of structural changes in the cluster (Tanaka et al 2004). In monocots, SBE-II is made up of two closely related but discrete gene products (SBE-IIa and SBE-IIb) (Sun et al 1998;Rahman et al 2001;McCue et al 2002).…”

Recent progress toward understanding the role of starch biosynthetic enzymes in the cereal endosperm

“…Interestingly, amylopectin from rice SBEIdeficient endosperm showed a significant reduction of chains with DP 12-21 and DP ≥ 37, together with an increase of chains with DP ≤ 10 and DP 24-34, indicating that rice SBEI synthesizes B1 chains and cluster-connecting B chains of amylopectin [79]. Although the SBEIIa activity was detected at a very early stage in the developing rice endosperm [80], the SBEIIa mutation in maize and rice only caused a slight reduction in the short-chain content of leaf starch and did not show any significant changes in the endosperm amylopectin CLDs [81,82]. SBEIIa might thus play a role in leaf starch biosynthesis and be involved in the initiation of starch biosynthesis.…”

“…SBEIIa might thus play a role in leaf starch biosynthesis and be involved in the initiation of starch biosynthesis. SBEIIb mutant was originally found to have a high amylose content [83], which was later shown in maize and rice to be because the higher amount of amylose is actually from the A (internal) amylopectin chains [80,84,85]. Moreover, a rice SBEIIb mutant had a 50% reduction of SSI activity, suggesting that SBEIIb and SSI may interact in vivo [80].…”