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Chalk5 encodes a vacuolar H+-translocating pyrophosphatase influencing grain chalkiness in rice

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An Erratum to this article was published on 28 May 2014

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Abstract

Grain chalkiness is a highly undesirable quality trait in the marketing and consumption of rice grain1,2,3,4,5,6. However, the molecular basis of this trait is poorly understood. Here we show that a major quantitative trait locus (QTL), Chalk5, influences grain chalkiness, which also affects head rice yield and many other quality traits. Chalk5 encodes a vacuolar H+-translocating pyrophosphatase (V-PPase) with inorganic pyrophosphate (PPi) hydrolysis and H+-translocation activity. Elevated expression of Chalk5 increases the chalkiness of the endosperm, putatively by disturbing the pH homeostasis of the endomembrane trafficking system in developing seeds, which affects the biogenesis of protein bodies and is coupled with a great increase in small vesicle-like structures, thus forming air spaces among endosperm storage substances and resulting in chalky grain. Our results indicate that two consensus nucleotide polymorphisms in the Chalk5 promoter in rice varieties might partly account for the differences in Chalk5 mRNA levels that contribute to natural variation in grain chalkiness.

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Figure 1: Map-based cloning of Chalk5.
Figure 2: Effects of Chalk5 on grain chalkiness and head rice yield.
Figure 3: Expression profile and natural variation of Chalk5.
Figure 4: Subcellular localization and biochemical analysis of the Chalk5 protein.
Figure 5: Effects of Chalk5 on the subcellular structures of rice seed endosperm.

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NCBI Reference Sequence

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NCBI Reference Sequence

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  • 07 May 2014

    In the version of this article initially published, there were errors in the layout of Figure 2 that resulted in truncations of the right portion of Figure 2b and the bottom portion of Figure 2c. These errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank Q. Zhang, L. Jiang, J. Chen, W. Yao, H. Zhou, L. Wang, J. Cao, X. Lian, Y. Jiang, L. Sun, P. Li, C. Xiao, L. Qiu, P. Chen, Q. Zhang and G. Gao for editing, suggestions and assistance. This work was supported by grants from the National 863 Project (2012AA10A303), the National Program on the Development of Basic Research (2011CB100200), the National Program on Research & Development of Transgenic Plants (2013ZX08009003-004), the earmarked fund for the China Agriculture Research System (CARS-01-03) and the National Natural Science Foundation of China (31171523 and 31300992) and by the Bill and Melinda Gates Foundation.

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Authors and Affiliations

Authors

Contributions

Y.L. performed most of the experiments, including fine mapping, gene cloning, genetic transformation, expression analysis, electron microscopy analysis, enzyme assays and other functional analyses. C.F., Y.X. and L.L. conducted the QTL primary mapping analysis and developed the NILs. P.Y., B.Y. and B.P. performed part of the quality analysis. G.W., W.X., X.L., C.X. and J.X. participated in sequencing and rice germplasm analysis. Y.H. and Y.L. designed and supervised the study, analyzed the data and wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Yuqing He.

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Integrated supplementary information

Supplementary Figure 1 Distribution of chalkiness rate of the three genotypic classes of 288 random individuals from BC3F2.

The homozygous genotypes for the H94 allele, the ZS97 allele and their heterozygous genotype were determined using C88 and RM574.

Supplementary Figure 2 Grain chalkiness rate and the Chalk5 transgene in three T2 families of ZpZc transgenic plants from Zhonghua 11 harboring a construct with the coding region from Zhenshan 97 (high grain chalkiness) driven by the native promoter of Zhenshan 97.

Chalkiness rate is the rate of white belly grains in total grains (>200 grains per plant). Black and white bars are positive and negative transgenic plants detected by GUS primers, respectively. The P values produced by two-tailed t test are 4.4 × 10−13, 8.8 × 10−13 and 1.1 × 10−8 for the ZpZc-26, ZpZc-3 and ZpZc-6 families, respectively.

Supplementary Figure 3 Grain chalkiness rate and the Chalk5 transgene in three T2 families of OX transgenic plants from Zhonghua 11 harboring a construct with the coding region from Zhenshan 97 (high grain chalkiness) driven by the 35S promoter.

Chalkiness rate is the rate of white belly grains in total grains (>200 grains per plant). Black and white bars are positive and negative transgenic plants detected by GUS primers, respectively. The P values produced by two-tailed t test are 8.6 × 10−12, 1.1 × 10−13 and 2.4 × 10−10 for the OX-37, OX-27 and OX-9 families, respectively.

Supplementary Figure 4 Mature plants of the Chalk5 NILs, and ZpZc and OX transgene-positive and transgene-negative plants in T2.

Supplementary Figure 5 Number (per 660 μm2) and mean area of starch granules in the fourth and fifth cell layers from aleurones of the endosperm bellies of NIL(H94) and NIL(ZS97) at 14 and 20 d.a.f.

All P values are produced by two-tailed t test.

Supplementary Figure 6 A proposed model for the putative role of Chalk5 in the regulation of grain chalkiness formation.

PVC/MVB, prevacuolar compartment/multivesicular body; PSV, protein storage vacuole; VLS, vesicle-like or vacuole-like structures; SG, starch granule; PB, protein body. The up and down arrows with thin lines represent increases and decreases in corresponding items, respectively.

Supplementary Figure 7 Relationship between grain width and chalkiness rate caused by the tight linkage of Chalk5, GS5 and GW5/qSW5.

(a) Tight linkage pattern and genotypes of Chalk5, GS5 and GW5 in NIL(ZS97) and NIL(H94) and in Class A and Class B from the Sub1 population (indica cultivars) of the minicore collection (Fig. 3). (b,c) Grain width, 1,000-grain weight and grain yield per plant in NIL(ZS97), NIL(H94), Class A and Class B. All data for NILs are based on a field experiment with a randomized complete block design with four replications. All data are given as means ± s.e.m. n is the plant number for NIL(ZS97) and NIL(H94) or the accession number for Class A and Class B. P values are produced by Duncan test. (d) Correlation of grain width with grain white belly rate in the Sub1 population of the minicore collection. ***P < 0.0001.

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Li, Y., Fan, C., Xing, Y. et al. Chalk5 encodes a vacuolar H+-translocating pyrophosphatase influencing grain chalkiness in rice. Nat Genet 46, 398–404 (2014). https://doi.org/10.1038/ng.2923

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