Understanding the impact of starch synthase IIa on starch structure and function in cereal endosperm

Abstract

Starch synthase IIa (SSIIa) is one of the major starch biosynthetic enzymes in starch biosynthesis process in cereal endosperms. It catalyzes the synthesis of intermediate chains in amylopectin of the grain starch. During starch deposition it interacts with other starch synthases (SSs) and starch branching enzymes (SBEs) to form functional protein complexes. The defective SSIIa mutants of cereals in common showed significantly altered amylopectin chain length distribution (CLD), and pleiotropic effects inside the starch granules with reduced amount of starch granule bound proteins (GBPs). However, these effects observed in the mutants varied at different degrees in different cereals. In this study, SSIIa was chosen to investigate its impact on starch structure, functional properties and regulatory role in starch biosynthesis in the endosperms of barley, wheat and rice. The rice ssIIa mutants with japonica type SSIIa were obtained from a population of recombinant inbred lines (RILs) derived from an IR64 and Nipponbare hybrid. In addition, five other starch biosynthetic genes, Wx, SSI, SBEI, SBEIIa and SBEIIb, which could have potential allelic effects on starch structure and functional properties, were also investigated to understand the allelic recombination effect. Defective SSIIa hybrid populations of barley and wheat were used for screening ssIIa mutants for the quantitatively comparative analysis. The comparison was done at three levels: gene transcription, posttranslational regulation and starch structure. The allelic effect study showed that SSIIa and Wx had the most significant influences on starch structure and functional properties. The impact of Wxi and SSIIaj on starch functional properties was similar, having lower peak viscosity (PV) and breakdown (BD), higher final viscosity (FV) and setback (SB), lower amylopectin gelatinization temperatures (GTs) and higher amylose-lipid complex dissociation enthalpy compared with Wxj and SSIIai, respectively. Only SSIIaj haplotype produced a novel amylopectin structure with the most pronounced alteration in short chain and intermediate chain fractions. At similar levels of starch AC, the ratio of short chain/ short plus intermediate chain fraction percentage in debranched starch was negatively correlated to PV, BD and GTs. The remarkable changes in starch structure were also related with the abundance of GBPs in starch granules. SSI, SBEI, SBEIIa and SBEIIb had only limited allelic effects on starch structure and functional properties compared with SSIIa and Wx. The comparison analysis in the three cereals indicated that the total amount of SSI, SBEIIa and SBEIIb synthesized in the endosperm of the three ssIIa mutants was not affected by the SSIIa defection. The pleiotropic effects on GBPs in starch granules were due to the changes in protein partitions between amyloplast stroma and starch granules regulated by SSIIa. The order of changes (HvssIIa > TassIIa > OsssIIa) in the ssIIa mutants compared with corresponding wildtypes were consistent in the starch AC, amylopectin CLD, and starch biosynthetic enzyme partitions. These changes were related with the variation of SSI, SSIIa, SBEIIa and SBEIIb abundance inside starch granules and/or the dosage of SSIIa gene. The various SSIIa abundances inside starch granules were due to the differences in SSIIa mutations in different cereal mutants.