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Relative importance of branching enzyme isoforms in determining starch fine structure and physicochemical properties of indica rice

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Down-regulation of starch branching enzymes alters fine structure and starch properties, especially the B-type crystalline pattern and extremely high amylose content identified in the BEIIb-deficiency mutant in the indica rice.

Abstract

The relative importance of the starch branching enzymes in determining the molecular fine structure and starch functional properties were uncovered in this study. An indica rice, Guangluai 4 with high amylose content (AC) and high gelatinization temperature (GT) was used to generate the clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein-9 (Cas9) knockout lines. Five mutant lines were identified including be1-1, be1-2, be2a-1, be2a-2 and be2b-1, and analysis of western blot showed the CRISPR/Cas9 system was successful in inducing mutations in the targeted genes. AC of be2b-1 (34.1%) was greater than that of wild type (WT) (27.4%) and other mutants. Mutations of either BEI or BEIIa did not alter the starch crystallite pattern (A-type). The BEIIb deficiency caused an opaque endosperm phenotype, changed the crystallite pattern from A- to B-type, and dramatically increased the degree of ordered structure, the relative proportion of amylose chains and intermediate to long amylopectin chains, average chain length of amylopectin molecules as well as GT. The BEIIa deficiency had no effect on the proportion of amylose chains, the length of amylopectin intermediate-long chains, conclusion temperature and enthalpy of gelatinization. Down-regulation of BEI increased the proportion of shortest amylopectin chains (fa) but decreased the proportion of long amylopectin chains (fb2 and fb3), leading to a lower GT. It is concluded that the relative importance in determining starch fine structures and functionality was in the order of BEIIb > BEI > BEIIa. Our results provide new information for utilizations of BE-deficient mutants in rice quality breeding.

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Acknowledgements

This work was financially supported by the National Key Research and Development Program of China (2016YFD0400104), the Natural Science Foundation of China (31871531), and Zhejiang Province (LZ21C130003) (for J.B.), and the Postdoctoral International Exchange Program (for P.T.). We sincerely thank Dr. Xin Zhou, Miss Ruihan Qiu and Xiaoyu Chen for their help in genotyping.

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  1. Piengtawan Tappiban and Yaqi Hu have contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Nuclear Agricultural Sciences of Ministry of Agriculture and Zhejiang Province, Institute of Nuclear Agricultural Sciences, College of Agriculture and Biotechnology, Zhejiang University, Zijingang Campus, Hangzhou, 310058, China

    Piengtawan Tappiban, Yaqi Hu, Jiajia Zhao, Yining Ying, Zhongwei Zhang, Feifei Xu & Jinsong Bao

  2. Department of Applied Bioscience, College of Agriculture and Biotechnology, Zhejiang University, Zijingang Campus, Hangzhou, 310058, China

    Jiaming Deng

  3. Hainan Institute of Zhejiang University, Yazhou District, Sanya, 572025, China

    Jinsong Bao

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  1. Piengtawan Tappiban
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Contributions

PT, YH, JZ and JZ performed mRNA and protein analyses; YH, JZ and YY performed DNA genotyping; YH and ZZ performed the analyses of starch; FX designed the gRNA and obtained the mutants; PT, ZZ and JB wrote the article; and all authors read and approved the final manuscript.

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Correspondence to Feifei Xu or Jinsong Bao.

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Tappiban, P., Hu, Y., Deng, J. et al. Relative importance of branching enzyme isoforms in determining starch fine structure and physicochemical properties of indica rice. Plant Mol Biol 108, 399–412 (2022). https://doi.org/10.1007/s11103-021-01207-y

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