Gradually Decreasing Starch Branching Enzyme Expression Is Responsible for the Formation of Heterogeneous Starch Granules

Abstract

Rice (Oryza sativa) endosperm is mainly occupied by homogeneous polygonal starch from inside to outside. However, morphologically different (heterogeneous) starches have been identified in some rice mutants. How these heterogeneous starches form remains unknown. A high-amylose rice line (TRS) generated through the antisense inhibition of starch branching synthase I (SBEI) and SBEIIb 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 the morphological architecture of heterogeneous starches in TRS endosperm from the view of the molecular structure of starch. The results indicated that their molecular structures underwent regular changes, including gradually increasing true amylose content but decreasing amylopectin content and gradually increasing the ratio of amylopectin long chain but decreasing the ratio of amylopectin short chain. Granule-bound starch synthase I (GBSSI) amounts in the four heterogeneous starches were not significantly different from each other, but SBEI, SBEIIa, and SBEIIb showed a gradually decreasing trend. Further immunostaining analysis revealed that the gradually decreasing SBEs acting on the formation of the four heterogeneous granules were mainly due to the spatial distribution of the three SBEs in the endosperm. It was suggested that the decreased amylopectin in starch might remove steric hindrance and provide extra space for abundant amylose accumulation when the GBSSI amount was not elevated. Furthermore, extra amylose coupled with altered amylopectin structure possibly led to morphological changes in heterogeneous granules.

Figure 1.

Dynamic deposition of four heterogeneous starch granules in TRS developing kernels in 2016. Whole sections of 2 μm thickness are sectioned transversely at the midregion of developing kernels and stained with safranine and I₂/KI. The endosperm after 7 DAF is divided into four regions, I, II, III, and IV, from the inner to the outer part. The four squared areas in the whole sections from the inner to the outer part are successively magnified, showing that the polygonal, aggregate, elongated, and hollow granules are gradually deposited regionally in the endosperm from the inner to the outer part. Bars = 1 mm for the whole sections and 10 μm for the magnified photographs.

Figure 2.

Changes of amylose, amylopectin, and starch contents in developing kernels in 2016. The contents are standardized by one seed. Values are means ± sd from three biological replicates. Asterisks highlight significant differences in amylose, amylopectin, and starch contents between TQ and TRS by Student’s t test (*, P < 0.05; **, P < 0.01; and ***, P < 0.001).

Figure 3.

Pleiotrophic effects of SBEI and SBEIIb down-regulation on other starch biosynthesis-related enzymes in developing endosperm at 10 DAF in 2016. A, Reverse transcription-PCR analysis of starch biosynthesis-related enzyme genes. The actin gene is used as a reference for comparative quantitation. Values are means ± sd from three biological replicates each with technical triplicates. Asterisks highlight significant differences in starch synthesis-related enzymes between TQ and TRS by Student’s t test (***, P < 0.001). B, Immunoblotting of starch biosynthesis-related enzymes. Anti-HSP82 antibody is used as a loading control. C, Native PAGE/activity staining of SBEs, SSs, Pho, and DBEs. D, GBSSI activities of starch granules isolated from TQ and TRS endosperm. Values are means ± sd from three biological replicates.

Figure 4.

Dynamic expression and deposition of amylopectin synthesis-related enzymes in soluble fraction (A) and granule-associated fraction (B) of developing kernels in 2016. The amount of soluble protein is standardized by HSP82, and granule-bound fraction is standardized by one seed.

Figure 5.

Starch components and amylopectin molecular structure of TQ and TRS developing kernels at 25 DAF in 2016. A to F, GPC profiles of isoamylase-debranched starch (solid lines) and purified amylopectin (dotted lines). Peak 1 and peak 2 consist of short and long branch chains of amylopectin, respectively. Peak 3 is a mixture of amylose and amylopectin extra-long chains. MW, Molecular weight. G, Chain-length distribution of amylopectin by FACE. H, Differences in amylopectin chain lengths between TQ and TRS starches.

Figure 6.

Distribution (A) and relative protein amount (B) of granule-bound proteins in starch granules from TQ and TRS developing kernels at 25 DAF in 2016. A, The protein amount is standardized by milligrams of starch. TRS-p, TRS-a, TRS-e, and TRS-h represent the polygonal, aggregate, elongated, and hollow starch granules from TRS, respectively. B, The relative protein amount is the proportion of protein in TRS to that in TQ. Values are means ± sd from three biological replicates. Values for the same protein indicated by different lowercase letters are significantly different (P < 0.05) as determined by one-way ANOVA and Tukey’s test.

Figure 7.

Immunostaining analysis of three SBEs in developing kernels at 10 DAF in 2016. SBEI, SBEIIa, and SBEIIb are broadly associated with all the granules in TQ (A, C, and E), while their fluorescence signals display a gradually decreased trend in endosperm from the inner to the outer part in TRS (B, D, and F). Bars = 1 mm for A to F and 10 μm for A1 to F4.