Gradually decreasing starch branching enzyme expression is responsible for the formation of heterogeneous starch granules

Rice endosperm is mainly occupied by homogeneous polygonal starch from inside to outside. However, morphologically different (heterogeneous) starches have been identified in rice mutants. How these heterogeneous starches form remains unknown. A high-amylose rice line (TRS) generated through the antisense inhibition of starch branching synthase (SBE) I and IIb contains four heterogeneous starches: polygonal, aggregate, elongated, and hollow starch; these starches are regionally distributed in the endosperm from inside to outside. Here, we investigated the relationship between SBE dosage and both the morphological architecture and regional distribution of heterogeneous starches in TRS endosperm. The dynamic accumulation and molecular structure analysis of starch indicated that the gradually increasing amylose content in heterogeneous starches was mainly due to amylose increase and amylopectin decrease. The analysis of granule-bound starch synthesis-related enzymes showed that gradually decreasing SBEI, SBEIIa and SBEIIb were responsible for the regular alteration of molecular structure in heterogeneous granules, including amylose increases, amylopectin reductions, and amylopectin structural changes. Immunostaining analysis revealed that the gradually decreasing SBEs acting on the formation of the four heterogeneous granules was mainly due to the spatial distribution of the three SBEs in the endosperm. In addition, different SBE dosages in heterogeneous granules also caused the alterations of some other starch synthesis-related enzymes. The above results suggested that the decreased amylopectin in starch might remove steric hindrance and provide extra space for amylose accumulation when granule-bound starch synthase I amount was not elevated. Furthermore, extra amylose possibly led to morphological changes in heterogeneous granules.