Expression Profiles and Protein Complexes of Starch Biosynthetic Enzymes from White-Core and Waxy Mutants Induced from High Amylose Indica Rice

doi:10.1016/j.rsci.2020.01.006

Abstract

Abstract:

Physicochemical properties of endosperm starches in milled rice determine its cooking and eating quality. Amylose is synthesized by granule-bound starch synthase I (GBSSI), whilst amylopectin is synthesized by the synergistic activities of starch synthases (SSs), branching enzymes (BEs) and debranching enzymes (DBEs). However, the complexes formed by starch biosynthetic enzymes are not well characterized. Gene expression profiles and protein complexes were determined in white-core (GM645) and waxy (GM077) mutants derived from a high amylose indica rice Guangluai 4 (GLA4). In GM645, genes including AGPS1, GBSSI, SSIIa, BEI, BEIIa, BEIIb, PUL, ISA1 and SP were significantly downregulated during seed development. In GM077, the expression levels of AGPL2, AGPS1, AGPS2b, SSIIIa, BEI, PUL and ISA1 were significantly upregulated. Co-immunoprecipitation assays revealed interactions of SSs-BEs, SSs-PUL and BEs-PUL in developing seeds. However, weak SSI-SSIIa interaction was detected in GM077, whilst SSI-PUL interaction was absent. Weak interaction signals for SSI-SSIIa, SSIIa-BEI, SSIIa-BEIIb, BEI-BEIIb and SSI-BEI were also observed in GM645. These results suggest that the protein-protein interactions for starch biosynthesis are modified in mutants, which provides insight into the mechanisms of starch biosynthesis, particularly in indica rice.

Key words: amylose, endosperm mutant, indica rice, protein-protein interaction, starch biosynthetic enzyme, waxy rice

Yaling Chen, Yuehan Pang, Jinsong Bao. Expression Profiles and Protein Complexes of Starch Biosynthetic Enzymes from White-Core and Waxy Mutants Induced from High Amylose Indica Rice[J]. Rice Science, 2020, 27(2): 152-161.

Figures/Tables 5

Fig. 1. Expression profiles of rice starch synthesis genes during seed development in GLA4 and two endosperm mutants. GLA4, Guangluai 4. ADP-glucose pyrophosphorylase (ADPase) genes: AGPL1, AGPL2, AGPS1 and AGPS2b; Granule-bound starch synthase (GBSS) gene: GBSSI; Soluble starch synthase (SS) genes: SSI, SSIIa and SSIIIa; Branching enzyme (BE) genes: BEI, BEIIa and BEIIb; Debranching enzyme (DBE) genes: ISAI and PUL; Starch phosphorylase (SP) gene: SP.Total RNAs were extracted from developing seeds at 5, 10 and 15 d after flowering (DAF). Data are Mean ± SD from three replicates. The asterisks indicate statistical significance between GLA4 and the mutants, as determined by the Student’s t-test (*, P < 0.05; **, P < 0.01).

Fig. 2. Western blotting of starch synthetic enzymes extracted from developing endosperms of GLA4, GM077 and GM645 at 5, 10, 15 d after flowering (DAF) was performed using antisera against GBSSI, SSI, SSIIa, BEI, BEIIb and PUL from soluble proteins.A, Starch granule-bound protein for GBSSI blot. B, Soluble proteins for SSI, SSIIa, BEI, BEIIb and PUL enzymes blots. Values in the first line under the gel are mean relative levels to the GLA4 at 5 DAF, while those in the second lines are the standard deviation. GLA4, Guangluai 4.

Table 1 Comparison of protein-protein interactions among starch synthetic related enzymes in wild type and mutant endosperms determined by co-immunoprecipitation assay.

Material	Reciprocal		One sided ^a		No signal
Material	Strong signal	Weak signal	Strong signal	Weak signal	No signal
GlA4	SSI-SSIIa, SSI-BEIIb, SSI-PUL, SSIIa-BEI, SSIIa-BEIIb, BEI-BEIIb, BEI-PUL, BEIIb-PUL		SSI-BEI, PUL-SSIIa
GM077	SSI-BEI, SSI-BEIIb, SSIIa-BEI, SSIIa-BEIIb, SSIIa-PUL, BEI-BEIIb, BEI-PUL, BEIIb-PUL	SSI-SSIIa*			SSI-PUL*
GM645	SSI-BEIIb, SSI-PUL, BEI-PUL, BEIIb-PUL	SSI-SSIIa, SSIIa-BEI, SSIIa-BEIIb, BEI-BEIIb	SSIIa-PUL	SSI-BEI*

Fig. 3. Protein-protein interactions between starch synthetic related enzymes in wild type and mutant endosperms by co-immunoprecipitation (Co-IP).Black arrows represent the western blotting signals. M, Protein marker; a, Guangluai 4 (GLA4); b, GM077; c, GM645.

Fig. 4. Hypothesis models for starch synthesis in high amylose indica rice Guangluai 4 (GLA4) and two endosperm mutants. The predicted protein-protein complexes in developing seed of wild type indica rice GLA4 were demonstrated referring to Nakamura (2002); Nakamura et al (2005) and Fujita (2014). Only the difference in the protein-protein interactions in GM077 and GM645 from GLA4 was shown. Alterations of chain length distribution in GM077 and GM645 referred to Kong et al (2014). The small rounds of different color represent glucosyl residues from different starch synthases. DP, Degree of polymerization.

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