Please wait a minute...
Journal of Integrative Agriculture
Advanced Online Publication | Current Issue | Archive | Adv Search
SBEIIb is responsible for chalk2 phnotype by regulating formation of resistant starch in indica rice

Xinwei Li1*, Zihang Wang1*, Tianxiao Chen1, Shen Lin2, Guiai Jiao1, Shaoqing Tang1, Long Chen1, 3, Xiangjin Wei1#, Peisong Hu1#

1 State Key Laboratory of Rice Biology and breeding, China National Center for Rice Improvement, China National Rice Research Institute, Hangzhou 311400, China

2 Huaiyin Institute of Agricultural Science in Xuhuai region of Jiangsu, Huaian 223001, China

3 Environment-friendly Crop Germplasm Innovation and Genetic Improvement Key Laboratory of Sichuan Province, Chengdu 610066, China 

 Highlights 

1. A novel OsSBEIIb allele was identified in an indica rice mutant.

2. The OsSBEIIb mutation increases resistant starch (RS) content in raw rice flour, as well as in retrograded and heated rice.

3. The chalk2 mutant serves as a valuable indica rice germplasm for breeding high-RS rice varieties.

Download:  PDF in ScienceDirect  
Export:  BibTeX | EndNote (RIS)      
摘要  

高抗性淀粉稻米因其独特的健康功效而备受关注,其在改善2型糖尿病患者血糖控制、促进心血管健康及调节肠道菌群等方面具有重要应用前景。然而,高抗性淀粉水稻品种的选育仍存在诸多技术瓶颈。本研究从籼稻ZJ100的突变体库中鉴定出一个高垩白突变体chalk2,其直链淀粉与蛋白质含量显著升高,支链淀粉比例发生改变,同时总淀粉与脂质含量显著下降。抗性淀粉含量分析显示,chalk2中两类抗性淀粉(RS2RS3)含量较野生型大幅增加。chalk2突变体还表现出粒长、粒宽和粒厚减小,结实率下降,最终导致产量显著降低。通过连锁分析、Mut-Map分析和转基因互补实验,我们确定淀粉分支酶家族成员SBEIIb是导致chalk2表型的关键基因。SBEIIb是一个胚乳特异表达基因,编码蛋白定位于质体。进一步研究发现,chalk2突变体中SBEIIb的表达水平、酶活性和蛋白含量均显著降低,同时伴随多个淀粉合成代谢基因表达变化。这些结果表明,SBEIIb在调控籼稻淀粉组分及抗性淀粉形成过程中发挥核心作用。本研究揭示了SBEIIb调控籼稻中抗性淀粉形成的分子机制,为高抗性淀粉水稻的分子设计育种提供了重要理论依据和基因资源。



Abstract  

High-resistant starch rice is a valuable food for human health, especially for individuals with type 2 diabetes, as it supports effective blood sugar control and provides cardiovascular and intestinal benefits.  However, developing rice varieties with high resistant starch content remains a major challenge.  In this study, we identified a mutant, chalk2, with increased chalkiness from the mutant library of indica rice ZJ100.  The chalk2 mutants exhibited significantly higher amylose and protein contents, while total starch and lipid contents were reduced. Analysis of resistant starch in chalk2 revealed substantial increases in two resistant starch (RS) types RS2 and RS3.  Electron microscopy revealed abnormal starch granule development in the endosperm. The chalk2 mutant also showed reduced grain length, width, and thickness, as well as a decreased seed setting rate, which ultimately led to a significant reduction in grain yield.  Through physical localization, Mut-Map analysis, and transgene complementation, we found that SBEIIb was responsible for the chalk2 phynotypes, a member of the starch branching enzyme (SBE) family, specifically expressed in the endosperm.  Furthermore, the expression levels, enzyme activity, and protein abundance of SBEIIb were significantly reduced in chalk2 mutants.  These findings suggest that SBEIIb plays a crucial role in regulating the composition of starch and resistant starch formation in indica rice.

Keywords:  Oryza sativa       resistant starch        amylose        rice quality        rice yield  
Online: 18 April 2025  
Fund: 

This work was supported by the grants from the the National Natural Science Foundation of China (32372099 and 32188102), the Zhejiang Provincial Natural Science Foundation of China (LQ24C130007), the Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-CSCB-202402).  

About author:  Xinwei Li, Tel: +8617673653328, E-mail: lixinwei162013@163.com; #Correspondence Peisong Hu, Tel: +86-571-63370221, Fax: +86-571-63370221, E-mail: peisonghu@126.com; Xiangjin Wei, Tel: +86-571-63370080, Fax: +86-571-63370080, E-mail: weixiangjin@caas.cn *These authors contributed equally to this study.

Cite this article: 

Xinwei Li, Zihang Wang, Tianxiao Chen, Shen Lin, Guiai Jiao, Shaoqing Tang, Long Chen, Xiangjin Wei, Peisong Hu. 2025. SBEIIb is responsible for chalk2 phnotype by regulating formation of resistant starch in indica rice. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2025.04.026

Abe A, Kosugi S, Yoshida K, Natsume S, Takagi H, Kanzaki H, Matsumura H, Yoshida K, Mitsuoka C, Tamiru M. 2012. Genome sequencing reveals agronomically important loci in rice using MutMap. Nature Biotechnology, 30, 174-178.

Amagliani L, O’Regan J, Kelly A L, O’Mahony J A. 2016. Chemistry, structure, functionality and applications of rice starch. Journal of Cereal Science, 70, 291-300.

Annison G, Illman R J, Topping D L. 2003. Acetylated, propionylated or butyrylated starches raise large bowel short-chain fatty acids preferentially when fed to rats. The Journal of Nutrition, 133, 3523-3528.

Bao J, Zhou X, Xu F, He Q, Park Y J. 2017. Genome-wide association study of the resistant starch content in rice grains. Starch-Stärke, 69, 1600343.

Bird A R, Flory C, Davies D A, Usher S, Topping D L. 2004. A novel barley cultivar (Himalaya 292) with a specific gene mutation in starch synthase IIa raises large bowel starch and short-chain fatty acids in rats. The Journal of Nutrition, 134, 831-835.

Biswas S, Ibarra O, Shaphek M, Molina-Risco M, Faion-Molina M, Bellinatti-Della Gracia M, Thomson M J, Septiningsih E M. 2023. Increasing the level of resistant starch in ‘Presidio’ rice through multiplex CRISPR-Cas9 gene editing of starch branching enzyme genes. Plant Genome, 16, e20225.

Deng B W, Zhang Y N, Zhang F, Wang W S, Xu J L, Yu Z, Bao J S. 2024. Genome-wide association study of cooked rice textural attributes and starch physicochemical properties in indica rice. Rice Science, 31, 300-316.

Butardo Jr V M, Daygon V D, Colgrave M L, Campbell P M, Resurreccion A, Cuevas R P, Jobling S A, Tetlow I, Rahman S, Morell M, Fitzgerald M. 2012. Biomolecular analyses of starch and starch granule proteins in the high-amylose rice mutant Goami 2. Journal of Agricultural and Food Chemistry, 60, 11576-11585.

Butardo V M, Fitzgerald M A, Bird A R, Gidley M J, Flanagan B M, Larroque O, Resurreccion A P, Laidlaw H K, Jobling S A, Morell M K. 2011. Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA-and hairpin RNA-mediated RNA silencing. Journal of Experimental Botany, 62, 4927-4941.

Chisbert M, Castell A L, Vinoy S, Nazare J A. 2024. The impact of slowly digestible and resistant starch on glucose homeostasis and insulin resistance. Current Opinion in Clinical Nutrition and Metabolic Care, 27, 338-343.

Crofts N, Nakamura Y, Fujita N. 2017. Critical and speculative review of the roles of multi-protein complexes in starch biosynthesis in cereals. Plant Science, 262, 1-8.

Cummings J H, Englyst H N. 1991. Measurement of starch fermentation in the human large intestine. Canadian Journal of Physiology and Pharmacology, 69, 121-129.

DeMartino P, Cockburn D W. 2020. Resistant starch: Impact on the gut microbiome and health. Current Opinion in Biotechnology, 61, 66-71.

Fujita N, Miura S, Crofts N. 2022. Effects of various allelic combinations of starch biosynthetic genes on the properties of endosperm starch in rice. Rice (N Y), 15, 24.

Griebel S, Westerman R P, Adeyanju A, Addo-Quaye C, Craig B A, Weil C F, Cunningham S M, Patel B, Campanella O H, Tuinstra M R. 2019. Mutations in sorghum SBEIIb and SSIIa affect alkali spreading value, starch composition, thermal properties and flour viscosity. Theoretical and Applied Genetics, 132, 3357-3374.

Guo D, Ling X, Zhou X, Li X, Wang J, Qiu S, Yang Y, Zhang B. 2020. Evaluation of the quality of a high-resistant starch and low-glutelin rice (Oryza sativa L.) generated through CRISPR/Cas9-mediated targeted mutagenesis. Journal of Agricultural and Food Chemistry, 68, 9733-9742.

Huang L, Xiao Y, Zhao W, Rao Y, Shen H, Gu Z, Fan X, Li Q, Zhang C, Liu Q. 2024. Creating high-resistant starch rice by simultaneous editing of ss3a and ss3b. Plant Biotechnology Journal, 22, 787.

Ikegaya T, Ashida K. 2021. Genetic region responsible for the differences of starch properties in two glutinous rice cultivars in Hokkaido, Japan. Breeding Science, 71, 375-383.

Inukai T. 2017. Differential regulation of starch-synthetic gene expression in endosperm between indica and japonica rice cultivars. Rice, 10, 1-10.

Itoh K, Ozaki H, Okada K, Hori H, Takeda Y, Mitsui T. 2003. Introduction of Wx transgene into rice wx mutants leads to both high-and low-amylose rice. Plant and Cell Physiology, 44, 473-480.

Jang E H, Lee S J, Hong J Y, Chung H J, Lee Y T, Kang B S, Lim S T. 2016. Correlation between physicochemical properties of japonica and indica rice starches. LWT-Food Science and Technology, 66, 530-537.

Kang H G, Park S, Matsuoka M, An G. 2005. White-core endosperm floury endosperm-4 in rice is generated by knockout mutations in the C4-type pyruvate orthophosphate dikinase gene (OsPPDKB). The Plant Journal, 42, 901-911.

Kang H J, Hwang I K, Kim K S, Choi H C. 2006. Comparison of the physicochemical properties and ultrastructure of japonica and indica rice grains. Journal of Agricultural and Food Chemistry, 54, 4833-4838.

Kim M K, Park J, Kim D M. 2024. Resistant starch and type 2 diabetes mellitus: Clinical perspective. Journal of Diabetes Investigation, 15, 395-401.

Li J, Jiao G, Sun Y, Chen J, Zhong Y, Yan L, Jiang D, Ma Y, Xia L. 2021. Modification of starch composition, structure and properties through editing of TaSBEIIa in both winter and spring wheat varieties by CRISPR/Cas9. Plant Biotechnol Journal, 19, 937-951.

Long X K, Guan C M, Wang L, Jia L T, Fu X J, Lin Q L, Huang Z Y, Liu C. 2023. Rice storage proteins: Focus on composition, distribution, genetic improvement and effects on rice quality. Rice Science, 30, 207-221.

Luo J, Butardo Jr V M, Yang Q, Konik-Rose C, Colgrave M L, Millar A, Jobling S A, Li Z. 2020. The impact of the indica rice SSIIa allele on the apparent high amylose starch from rice grain with downregulated japonica SBEIIb. Theoretical and Applied Genetics, 133, 2961-2974.

Luo J, Jobling S A, Millar A, Morell M K, Li Z. 2015. Allelic effects on starch structure and properties of six starch biosynthetic genes in a rice recombinant inbred line population. Rice (N Y), 8, 15.

Miao M, Hamaker B R. 2021. Food matrix effects for modulating starch bioavailability. Annual Review of Food Science & Technology, 12, 169-191.

Miura S, Narita M, Crofts N, Itoh Y, Hosaka Y, Oitome N F, Abe M, Takahashi R, Fujita N. 2022. Improving agricultural traits while maintaining high resistant starch content in rice. Rice, 15, 28.

Nagamatsu S, Wada T, Matsushima R, Fujita N, Miura S, Crofts N, Hosaka Y, Yamaguchi O, Kumamaru T. 2022. Mutation in BEIIb mitigates the negative effect of the mutation in ISA1 on grain filling and amyloplast formation in rice. Plant Molecular Biology, 108, 497-512.

Nakamura Y. 2023. A model for the reproduction of amylopectin cluster by coordinated actions of starch branching enzyme isoforms. Plant Molecular Biology, 112, 199-212.

Nakamura Y, Ono M, Hatta T, Kainuma K, Yashiro K, Matsuba G, Matsubara A, Miyazato A, Mizutani G. 2020. Effects of BEIIb-deficiency on the cluster structure of amylopectin and the internal structure of starch granules in endosperm and culm of japonica-type rice. Frontiers in Plant Science, 11, 571346.

Nakamura Y, Steup M, Colleoni C, Iglesias A A, Bao J, Fujita N, Tetlow I. 2022. Molecular regulation of starch metabolism. Plant Molecular Biology, 108, 289-290

Nakamura Y, Utsumi Y, Sawada T, Aihara S, Utsumi C, Yoshida M, Kitamura S. 2010. Characterization of the reactions of starch branching enzymes from rice endosperm. Plant and Cell Physiology, 51, 776-794.

Nishi A, Nakamura Y, Tanaka N, Satoh H. 2001. Biochemical and genetic analysis of the effects of amylose-extender mutation in rice endosperm. Plant Physiology, 127, 459-472.

Ohdan T, Francisco Jr P B, Sawada T, Hirose T, Terao T, Satoh H, Nakamura Y. 2005. Expression profiling of genes involved in starch synthesis in sink and source organs of rice. Journal of Experimental Botany, 56, 3229-3244.

Pugh J E, Cai M, Altieri N, Frost G. 2023. A comparison of the effects of resistant starch types on glycemic response in individuals with type 2 diabetes or prediabetes: A systematic review and meta-analysis. Frontiers in Nutrition, 10, 1118229.

Raigond P, Ezekiel R, Raigond B. 2015. Resistant starch in food: A review. Journal of the Science of Food and Agriculture, 95, 1968-1978.

Sajilata M G, Singhal R S, Kulkarni P R. 2006. Resistant starch-A review. Comprehensive Reviews in Food Science and Food Safety, 5, 1-17.

Sawada T, Itoh M, Nakamura Y. 2018. Contributions of three starch branching enzyme isozymes to the fine structure of amylopectin in rice endosperm. Frontiers in Plant Science, 9, 1536.

Sun Y, Jiao G, Liu Z, Zhang X, Li J, Guo X, Du W, Du J, Francis F, Zhao Y. 2017. Generation of high-amylose rice through CRISPR/Cas9-mediated targeted mutagenesis of starch branching enzymes. Frontiers in Plant Science, 8, 298.

Takahashi T, Fujita N. 2017. Thermal and rheological characteristics of mutant rice starches with widespread variation of amylose content and amylopectin structure. Food Hydrocolloids, 62, 83-93.

Tang Z, Zhang D. 2023. Research progress on the molecular mechanism of starch accumulation in rice endosperm. Chinese Bulletin of Botany, 58, 612-621.

Tappiban P, Hu Y, Deng J, Zhao J, Ying Y, Zhang Z, Xu F, Bao J. 2022. Relative importance of branching enzyme isoforms in determining starch fine structure and physicochemical properties of indica rice. Plant Molecular Biology, 108, 399-412

Toyosawa Y, Kawagoe Y, Matsushima R, Crofts N, Ogawa M, Fukuda M, Kumamaru T, Okazaki Y, Kusano M, Saito K. 2016. Deficiency of starch synthase IIIa and IVb alters starch granule morphology from polyhedral to spherical in rice endosperm. Plant Physiology, 170, 1255-1270.

Turker B, Savlak N Y. 2015. Resistant starch: Types, sources, beneficial physiological effects and functional properties. Akademik Gida, 13, 354-359.

Utsumi Y, Utsumi C, Tanaka M, Takahashi S, Okamoto Y, Ono M, Nakamura Y, Seki M. 2022. Suppressed expression of starch branching enzyme 1 and 2 increases resistant starch and amylose content and modifies amylopectin structure in cassava. Plant Molecular Biology, 108, 413-427.

Wada T, Yamaguchi O, Miyazaki M, Miyahara K, Ishibashi M, Aihara T, Shibuta T, Inoue T, Tsubone M, Toyosawa Y. 2018. Development and characterization of a new rice cultivar,‘Chikushi-kona 85’, derived from a starch-branching enzyme IIb-deficient mutant line. Breeding Science, 68, 278-283.

Wang A, Jing Y, Cheng Q, Zhou H, Wang L, Gong W, Kou L, Liu G, Meng X, Chen M, Ma H, Shu X, Yu H, Wu D, Li J. 2023. Loss of function of SSIIIa and SSIIIb coordinately confers high RS content in cooked rice. Proceedings of the National Academy of Sciences of the United States of America, 120, e2220622120.  

Wang A Q, Cheng Q, Li W J, Kan M X, Zhang Y X, Meng X B, Guo H Y, Jing Y H, Chen M J, Liu G F, Wu D X, Li J Y, Yu H. 2024. Creation of high-resistant starch rice through systematic editing of amylopectin biosynthetic genes in rs4. Plant Biotechnology Journal, 23, 480-488. 

Wang F X, Mo J H, Hu X Z, Ma Z. 2024. The degradation mechanism of resistant starch by human gut microflora. Journal of Chinese Institute of Food Science and Technology, 24, 364-378.

Wang L, Liu L L, Zhao J L, Li C L, Wu H L, Zhao H X, Wu Q. 2023. Granule-bound starch synthase in plants: Towards an understanding of their evolution, regulatory mechanisms, applications, and perspectives. Plant Science, 336, 111843. 

Wei X, Jiao G, Lin H, Sheng Z, Shao G, Xie L, Tang S, Xu Q, Hu P. 2017. GRAIN INCOMPLETE FILLING 2 regulates grain filling and starch synthesis during rice caryopsis development. Journal of Integrative Plant Biology, 59, 134-153.

Wen J J, Li M Z, Nie S P. 2023. Dietary supplementation with resistant starch contributes to intestinal health. Current Opinion in Clinical Nutrition and Metabolic Care, 26, 334-340.

Yamanouchi H, Nakamura Y. 1992. Organ specificity of isoforms of starch branching enzyme (Q-enzyme) in rice. Plant and Cell Physiology, 33, 985-991.

Zhang Q. 2024. Research progress on the regulatory effect of resistant starch on type 2 diabetes mellitus and its mechanism. Journal of Food Safety and Quality, 15, 250-259.

Zhang Z, Hu Y, Zhao J, Zhang Y, Ying Y, Xu F, Bao J. 2022. The role of different Wx and BEIIb allele combinations on fine structures and functional properties of indica rice starches. Carbohydrate Polymers, 278, 118972.

No related articles found!
No Suggested Reading articles found!