RESUMO
Amylopectin, which consists of highly branched glucose polymers, is a major component of starch. Biochemical processes that regulate the elongation of glucose polymers and the generation and removal of glucose branches are essential for determining the properties of starch. Starch synthases (SSs) and branching enzyme (BE) mainly form complexes consisting of SSI, SSIIa, and BEIIb during endosperm development. Loss of BEIIb in rice is complemented by BEIIa, but the compensatory effects differ depending on the presence or absence of inactive BEIIb. To better understand these compensatory mechanisms, ss2a be2b (+) double mutant, which possessed truncated inactive SSIIa and inactive BEIIb, were analyzed. Soluble proteins separated by gel filtration chromatography showed that SSIIa and BEIIb proteins in the wild-type exhibited a broad range of elution patterns and only small amounts were detected in high molecular mass fractions. In contrast, most of truncated inactive SSIIa and inactive BEIIb from ss2a be2b (+) were found in high molecular mass fractions, and the SSI-SSIIa-BEIIb trimeric protein complex found in the wild-type was likely absent in ss2a be2b (+). Those SSIIa and BEIIb proteins in high molecular mass fractions in ss2a be2b (+) were also identified by mass spectrometry. Parental ss2a single mutant had negligible amounts of SSIIa suggesting that the truncated inactive SSIIa was recruited to high-molecular mass complexes in the presence of inactive BEIIb in ss2a be2b (+) double mutant. In addition, SSIVb might be involved in the formation of alternative protein complexes with < 300 kDa in ss2a be2b (+).
RESUMO
Starch biosynthetic enzymes form multi-protein complexes consisting of starch synthase (SS) I, SSIIa, and starch branching enzyme (BE) IIb, which synthesize amylopectin clusters. This study analyzed the starch properties in two double mutant rice lines lacking SSIIa and BEIIb, one of which expressed an inactive BEIIb protein. The ss2a be2b lines showed similar or greater seed weight than the be2b lines, and plant growth was not affected. The ss2a line showed increased short amylopectin chains resulting in a lower gelatinization temperature. Starch granule morphology and A-type crystallinity were similar between the ss2a line and the wild type, except for a mild chalky seed phenotype in the ss2a line. However, the starch phenotype of the ss2a be2b lines, which was similar to that of be2b but not ss2a, was characterized by increased long amylopectin chains, abnormal starch granules, and B-type crystallinity. The similarity in phenotype between the ss2a be2b and be2b lines may be attributed to the inability of the be2b mutants to generate short amylopectin branches, which serve as primers for SSIIa. Therefore, the presence or absence of SSIIa hardly affected the amylopectin structure under the be2b background. The amylose content was significantly higher in the ss2a be2b lines than in the be2b lines. Starch crystallinity was greater in ss2a be2b lines than in be2b lines, despite the fact that starch crystallinity is generally negatively correlated with amylose content. This suggests that the formation of a double helix between long amylopectin chains and amylose affects starch crystallinity in the ss2a be2b mutants.
