Dynamic Change in Starch Biosynthetic Enzymes Complexes during Grain-Filling Stages in BEIIb Active and Deficient...

Starch is the predominant reserve in rice (Oryza sativa L.) endosperm, which is synthesized by the coordinated efforts of a series of starch biosynthetic-related enzymes in the form of a multiple enzyme complex. Whether the enzyme complex changes during seed development is not fully understood. Here, we investigated the dynamic change in multi-protein complexes in an indica rice variety IR36 (wild type, WT) and its BEIIb-deficient mutant (be2b) at different developmental stages. Gel permeation chromatography (GPC) and Western blotting analysis of soluble protein fractions revealed most of the enzymes except for SSIVb were eluted in smaller molecular weight fractions at the early developing stage and were transferred to higher molecular weight fractions at the later stage in both WT and be2b. Accordingly, protein interactions were enhanced during seed development as demonstrated by co-immunoprecipitation analysis, suggesting that the enzymes were recruited to form larger protein complexes during starch biosynthesis. The converse elution pattern from GPC of SSIVb may be attributed to its vital role in the initiation step of starch synthesis. The number of protein complexes was markedly decreased in be2b at all development stages. Although SSIVb could partially compensate for the role of BEIIb in protein complex formation, it was hard to form a larger protein complex containing over five proteins in be2b. In addition, other proteins such as PPDKA and PPDKB were possibly present in the multi-enzyme complexes by proteomic analyses of high molecular weight fractions separated from GPC. Two putative protein kinases were found to be potentially associated with starch biosynthetic enzymes. Collectively, our findings unraveled a dynamic change in the protein complex during seed development, and potential roles of BEIIb in starch biosynthesis via various protein complex formations, which enables a deeper understanding of the complex mechanism of starch biosynthesis in rice.