Natural variation of OsGluA2 is involved in grain protein content regulation in rice

First author: Yihao Yang; Affiliations: Yangzhou University (扬州大学): Yangzhou, China

Corresponding author: Changjie Yan

Grain protein content (GPC) affects rice nutrition quality. Here, we identify two stable quantitative trait loci (QTLs), qGPC-1 and qGPC-10, controlling GPC in a mapping population derived from indica and japonica cultivars crossing. Map-based cloning reveals that OsGluA2, encoding a glutelin type-A2 precursor, is the candidate gene underlying qGPC-10. It functions as a positive regulator of GPC and has a pleiotropic effect on rice grain quality. One SNP located in OsGluA2 promoter region is associated with its transcript expression level and GPC diversity. Polymorphisms of this nucleotide can divide all haplotypes into low (OsGluA2LET) and high (OsGluA2HET) expression types. Population genetic and evolutionary analyses reveal that OsGluA2LET, mainly present in japonica accessions, originates from wild rice. However, OsGluA2HET, the dominant type in indica, is acquired through mutation of OsGluA2LET. Our results shed light on the understanding of natural variations of GPC between indica and japonica subspecies.

籽粒蛋白含量影响水稻的营养价值。本文，作者基于籼稻（indica）和粳稻（japonica）杂交的作图群体鉴定了两个数量性状位点qGPC-1和qGPC-10控制籽粒的蛋白含量。图位克隆揭示一个编码谷蛋白A2型前体的OsGluA2基因是qGPC-10位点的候选基因。OsGluA2基因正向调控籽粒的蛋白含量，并且对于水稻籽粒质量具有多效性影响。OsGluA2基因启动子区的一个SNP与其转录水平和籽粒蛋白含量多样性相关。该SNP的多态型能够将所有的单倍型分成低（OsGluA2LET）、高（OsGluA2HET）表达类型。群体遗传和进化分析显示来源于野生水稻的OsGluA2LET主要存在于粳稻品种中。然而，籼稻中占主要类型的OsGluA2HET是通过OsGluA2LET的突变获得的。本文的结果揭示了籼稻和粳稻两个亚种之间籽粒蛋白含量自然变异的分子基础。

Background

水稻（Oryza sativa L.）是世界上最重要的人类粮食作物，为世界人口提供超过21%的热量需求，为东南亚提供高达76%的热量摄入 [1]。随着生活水平的提升，人们越来越重视水稻籽粒的品质。水稻的籽粒品质主要由四个方面组成：籽粒外观、碾磨品质、营养品质、烹饪和食用品质 [2, 3]。其中，营养品质以及烹饪和食用品质是两个最重要的指标，受到消费者的广泛关注。胚乳的三种主要成份：淀粉（~70-80%）、蛋白（~7-10%）和脂类（~1%）在很大程度上决定了水稻籽粒的营养品质以及烹饪和食用品质 [4]。因此，研究淀粉、蛋白和脂类合成的分子机制成为水稻籽粒品质改良的先决条件。在最近十年的研究中，对于淀粉合成通路及调控淀粉动态的遗传网络已经有了很深入的理解 [5-8]。然而，另外一个影响水稻籽粒营养品质以及烹饪和食用品质的重要因素，即籽粒蛋白含量GPC的遗传基础还所知甚少。

水稻籽粒蛋白主要有两种：一种是功能蛋白（~10%），另外一种是种子贮藏蛋白（SSP，~90%）。根据溶度相关的物理特性将SSP分为四类：清蛋白、球蛋白、醇溶蛋白和谷蛋白 [9, 10]。其中，有以谷蛋白含量最为丰富，约占所有SSP的60-80% [11]。水稻谷蛋白相对于其它蛋白具有更高的赖氨酸含量和人体消化率，其营养价值更高 [12]。所以，任何水稻谷蛋白含量的较大变化对于籽粒的营养品质肯定会有影响。通过蛋白序列的相似性，谷蛋白又能进一步的分为四类：GluA、GluB、GluC和GluD，首先在细胞质中合成为57 kDa前体，然后再分裂为37–39 kDa酸性亚单位和22–23 kDa碱性亚单位 [13]。

亚洲栽培种水稻通常可以分为两个亚种：籼稻（indica）和粳稻（japonica）。研究表明，籼稻和粳稻栽培种的GPC变异都很大，分别是4.9%-19.3%和5.9%-16.5%，并且籼稻中的GPC均值通常要比粳稻高 [14, 15]。籼稻和粳稻之间GPC的差异很有可能是由于他们之间的遗传结构差异所导致的 [16, 17]。然而，籼稻和粳稻之间GPC差异的分子基础还不清楚。

对于水稻GPC的遗传机制已有很多的研究，通过各种水稻突变体鉴定了很多能够影响水稻GPC的调控基因 [11, 18-25]，同时还有数百个潜在关联的QTLs被报道 [26-34]。然而，GPC属于典型的数量性状，遗传结构比较复杂，并且容易受到环境的影响，尤其是在生长后期容易受到氮肥的影响。因此，通过不同的遗传群体鉴定的QTLs往往不一致，很少能重复鉴定到相同的QTL。目前为止，仅仅只克隆到了一个位于一号染色体长臂上的QTL，叫qPC1。qPC1编码一个氨基酸转运蛋白OsAAP6，并且是水稻GPC的正向调控子 [35]。除此以外，由于缺少靶基因，在水稻GPC育种改良方面的进展甚微。

本文中，作者通过QTL作图、基因克隆和功能验证阐释了水稻GPC变异的遗传机制，并且揭示了谷蛋白含量是导致籼稻和粳稻两者之间GPC差异的主要原因。作者鉴定了两个与水稻GPC相关的QTLs（qGPC-1和qGPC-10），这两个QTLs在不同环境条件下都很稳定。作者通过图位克隆的方法分离出了qGPC-10的功能基因OsGluA2，该基因编码一个谷蛋白A2型前体。作者通过遗传互补试验、CRISPR敲除试验和自然变异分析显示OsGluA2是水稻GPC的正向调控子。另外，本文的进化和群体遗传分析揭示OsGluA2很大程度上导致了籼稻和粳稻之间的遗传差异。

Reference

1. Sasaki, T. & Burr, B. International rice genome sequencing project: the effort to completely sequence the rice genome. Curr. Opin. Plant Biol. 3, 138–141 (2000).

2. Umemoto, T. Genes affecting eating and processing qualities. Rice Genomics Genet. Breed. 21, 417–434 (2018).

3. Hori, K. Genetic dissection and breeding for grain appearance quality in rice. Rice Genomics Genet. Breed. 22, 435–451 (2018).

4. Martin, M. & Fitzgerald, M. A. Proteins in rice grains influence cooking properties. J. Cereal Sci. 36, 285–294 (2002).

5. Nakamura, Y. Towards a better understanding of the metabolic system for amylopectin biosynthesis in plants: rice endosperm as a model tissue. Plant Cell Physiol. 43, 718–725 (2002).

6. Yan, C. J. et al. Genetic analysis of starch paste viscosity parameters in glutinous rice (Oryza sativa L.). Theor. Appl. Genet. 122, 63–76 (2011).

7. Tian, Z. et al. Allelic diversities in rice starch biosynthesis lead to a diverse array of rice eating and cooking qualities. Proc. Natl. Acad. Sci. USA 106, 21760–21765 (2009).

8. Fitzgerald, M. A. et al. Not just a grain of rice: the quest for quality. Trends Plant Sci. 14, 133–139 (2009).

9. Kawakatsu, T. et al. Characterization of a new rice glutelin gene GluD-1 expressed in the starchy endosperm. J. Exp. Bot. 59, 4233–4245 (2008).

10. Saito, Y. et al. Formation mechanism of the internal structure of type I protein bodies in rice endosperm: relationship between the localization of prolamin species and the expression of individual genes. Plant J. 70, 1043–1055 (2012).

11. Kusaba, M. et al. Low glutelin content1: a dominant mutant that suppress the glutelin multigene family via RNA silencing in rice. Plant Cell 15, 1455–1467 (2003).

12. Hamaker, B. R. & Griffin, V. K. Effect of disulfide bond-containing protein on rice starch gelatinization and pasting. Cereal Chem. 70, 377–380 (1993).

13. Kawakatsu, T. & Takaiwa, F. Cereal seed storage protein synthesis: fundamental processes for recombinant protein production in cereal grains. Plant Biotechnol. J. 8, 939–953 (2010).

14. Lin, R. et al. in Rice Germplasm Resources in China (ed. Ying, C.) 83–93 (Agricultural Science and Technology, Beijing, 1993).

15. Zhou, L. H. et al. Variation and distribution of seed storage protein content and composition among different rice varieties. Acta Agron. Sin. 35, 884–891 (2009).

16. Shenoy, V. V. et al. Inheritance of protein per grain in rice. Indian J. Genet. 51, 214–220 (1991).

17. Shi, C. et al. Genetic analysis for protein content in indica rice. Euphytica 107, 135–140 (1999).

18. Terao, T. & Hirose, T. Control of grain protein contents through SEMIDWARF1 mutant alleles: sd1 increases the grain protein content in Deegeo- woo-gen but not in Reimei. Mol. Genet. Genomics 290, 939–954 (2015).

19. Ren, Y. et al. GLUTELIN PRECURSOR ACCUMULATION3 encodes a regulator of post-Golgi vesicular traffic essential for vacuolar protein sorting in rice endosperm. Plant Cell 26, 410–425 (2014).

20. Tian, L. et al. Small GTPase Sar1 is crucial for proglutelin and alpha-globulin export from the endoplasmic reticulum in rice endosperm. J. Exp. Bot. 64, 2831–2845 (2013).

21. Wang, G. et al. Opaque7 encodes an acyl-activating enzyme-like protein that affects storage protein synthesis in maize endosperm. Genetics 189, 1281–1295 (2011).

22. Wang, Y. et al. OsRab5a regulates endomembrane organization and storage protein trafficking in rice endosperm cells. Plant J. 64, 812–824 (2010).

23. She, K. C. et al. A novel factor FLOURY ENDOSPERM2 is involved in regulation of rice grain size and starch quality. Plant Cell22, 3280–3294 (2010).

24. Wang, Y. et al. The vacuolar processing enzyme OsVPE1 is required for efficient glutelin processing in rice. Plant J. 58, 606–617 (2009).

25. Takemoto, Y. et al. The rice mutant esp2 greatly accumulates the glutelin precursor and deletes the protein disulfide isomerase. Plant Physiol. 128, 1212–1222 (2002).

26. Yano, K. et al. Genome-wide association study using whole-genome sequencing rapidly identifies new genes influencing agronomic traits in rice. Nat. Genet. 48, 927–934 (2016).

27. Xu, F. et al. Genome-wide association study of eating and cooking qualities in different subpopulations of rice (Oryza sativa L.). BMC Genomics 17, 663 (2016).

28. Yang, Y. H. et al. Identification of quantitative trait loci responsible for rice grain protein content using chromosome segment substitution lines and fine mapping of qPC-1 in rice (Oryza sativa L.). Mol. Breed. 35, 1–9 (2015).

29. Cheng, L. et al. Identification of stably expressed quantitative trait loci for grain yield and protein content using recombinant inbred line and reciprocal introgression line populations in rice. Crop Sci. 53, 1436–1446 (2013).

30. Zheng, L. et al. Genetic relationship between grain chalkiness, protein content, and paste viscosity properties in a backcross inbred population of rice. J. Cereal Sci. 56, 153–160 (2012).

31. Zheng, L. et al. Dynamic QTL Analysis of rice protein content and protein index using recombinant inbred lines. J. Plant Biol. 54, 321–328 (2011).

32. Liu, X. et al. Dissecting the genetic basis for the effect of rice chalkiness, amylose content, protein content, and rapid viscosity analyzer profile characteristics on the eating quality of cooked rice using the chromosome segment substitution line population across eight environments. Genome 54, 64–80 (2011).

33. Ye, G. et al. QTL mapping of protein content in rice using single chromosome segment substitution lines. Theor. Appl. Genet. 121, 741–750 (2010).

34. Zhao, K. et al. Genome-wide association mapping reveals a rich genetic architecture of complex traits in Oryza sativa. Nat. Commun. 2, 467 (2011).

35. Peng, B. et al. OsAAP6 functions as an important regulator of grain protein content and nutritional quality in rice. Nat. Commun. 5, 4847 (2014).

通讯：严长杰（http://nxy.yzu.edu.cn/art/2018/2/4/art_12481_383081.html）

研究方向：植物转基因技术及分子标记辅助育种、植物重要性状基因克隆与功能分析。

doi: https://doi.org/10.1038/s41467-019-09919-y

Journal: Nature Communications

Published date: April 26, 2019