Starch Biosynthetic Protein Complex Formation in Rice ss2a be2b (+) Double Mutant Differs from Their Parental Single Mutants

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 (+).