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Modulation of histone acetylation enables fully mechanized hybrid rice breeding

Nature Plants volume 10, pages 954–970 (2024)Cite this article

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Abstract

Hybrid rice has achieved high grain yield and greatly contributes to food security, but the manual-labour-intensive hybrid seed production process limits fully mechanized hybrid rice breeding. For next-generation hybrid seed production, the use of small-grain male sterile lines to mechanically separate small hybrid seeds from mixed harvest is promising. However, it is difficult to find ideal grain-size genes for breeding ideal small-grain male sterile lines without penalties in the number of hybrid seeds and hybrid rice yield. Here we report that the use of small-grain alleles of the ideal grain-size gene GSE3 in male sterile lines enables fully mechanized hybrid seed production and dramatically increases hybrid seed number in three-line and two-line hybrid rice systems. The GSE3 gene encodes a histone acetyltransferase that binds histones and influences histone acetylation levels. GSE3 is recruited by the transcription factor GS2 to the promoters of their co-regulated grain-size genes and influences the histone acetylation status of their co-regulated genes. Field trials demonstrate that genome editing of GSE3 can be used to immediately improve current elite male sterile lines of hybrid rice for fully mechanized hybrid rice breeding, providing a new perspective for mechanized hybrid breeding in other crops.

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Fig. 1: An ideal male sterile line (XQA) with small grains and a restorer line (DHZ) with large grains allow for fully mechanized hybrid rice breeding.
Fig. 2: GSE3 encodes an N-acetyltransferase-like protein that influences histone acetylation levels.
Fig. 3: GSE3 interacts physically and genetically with GS2 to control grain size and weight.
Fig. 4: GS2 recruits GSE3 to influence the histone status of target genes.
Fig. 5: Rapid generation of male sterile lines for mechanized production of hybrid seeds using genome editing technology in three-line and two-line systems.
Fig. 6: A strategy to rapidly improve current parental lines of hybrid rice for fully mechanized hybrid seed production.

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All data and materials are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank J. Xiao for help in the CUT&Tag assay and Y. Pan for help in the CUT&Tag data analysis. This work is supported by grants from the National Key Research and Development Program of China (nos. 2022YFF1002903-YHL, 2021YFF1000202-YHL and 2022YFD1200101-HLW), the STI2030-Major Project (nos. 2023ZD04068-JQH and 2023ZD0406902-HLW), the Hainan Seed Industry Laboratory (no. B21HJ0220-YHL), the Key Research and Development Program of Hainan (no. ZDYF2021XDNY165-YHL) and the strategic priority research programme of the Chinese Academy of Sciences (nos. XDA24010101 and XDB27010102).

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Author notes

  1. These authors contributed equally: Ke Huang, Yuexing Wang, Yingjie Li.

Authors and Affiliations

  1. Key Laboratory of Seed Innovation, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Ke Huang, Yingjie Li, Baolan Zhang, Limin Zhang, Penggen Duan, Ran Xu, Lijie Liu, Guozheng Zhang, Hao Zhang, Jianqin Hao & Yunhai Li

  2. Hainan Seed Industry Laboratory, Sanya, China

    Ke Huang & Yingjie Li

  3. State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou, China

    Yuexing Wang & Xudong Zhu

  4. College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, China

    Dekai Wang

  5. College of Advanced Agriculture, University of Chinese Academy of Sciences, Beijing, China

    Lijie Liu, Hao Zhang & Yunhai Li

  6. School of Breeding and Multiplication, Hainan University, Sanya, China

    Chenjie Wang, Nian Guo & Yuehua Luo

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  1. Ke Huang
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Contributions

Yunhai Li, K.H., Y.W. and X.Z. designed the project. Yunhai Li supervised and conceived the project. K.H., Y.W., Yingjie Li, L.L., P.D., R.X., G.Z., D.W., Y. Luo, C.W., N.G. and J.H. performed most of the experiments. B.Z. performed the rice genetic transformation. H.Z. and L.Z. analysed the RNA-seq data and CUT&Tag data. K.H. and Yunhai Li analysed the data. K.H. described the results. Yunhai Li and K.H. wrote the article.

Corresponding authors

Correspondence to Xudong Zhu or Yunhai Li.

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Extended data

Extended Data Fig. 1 The phenotype of parental lines of hybrid rice variety TYHZ (TFA X HZ) and XQHZ (XQA X DHZ).

a-b, Mature grains (a) and panicles (b) of the maintainer line TFB, the improved maintainer line XQB with small seeds, the restorer line HZ, and the improved restorer line DHZ with large seeds. c-f, The grain length (c), grain width (d), grain thickness (e), and 1,000 grain weight (f) of parental lines TFB, XQB, HZ, and DHZ. n = 60 (c-d), n = 40 (e), and n = 3 (f). g-h, The plant height (g) and panicle length (h) of parental lines TFB, XQB, HZ, and DHZ. n = 12 (g), n = 13 (TFB, XQB, HZ) and 10 (DHZ) in (h). Values in (c-h) are given as mean ± SD. **P < 0.01 compared with TFB or HZ by Student’s t-test. ns, not significant (Student’s unpaired t-test). Bars: 3 mm (a) and 5 cm (b).

Source data

Extended Data Fig. 2 Genetic complementation of ZH11-GSE3small grain.

a-b, The mature plants (a) and panicles (b) of ZH11, ZH11-GSE3small grain and genomic complementary lines ZH11-GSE3small grain,gGSE3. c-e, Tiller number (c), panicle length (d), and grain number per panicle (e) of ZH11, ZH11-GSE3small grain and genomic complementary lines ZH11-GSE3small grain,gGSE3. n = 11 (c) and n = 12 (d-e). Values in (c-e) are given as mean ± SD. **P < 0.01 compared with ZH11-GSE3small grain by Student’s t-test. Bars: 10 cm (a), 5 cm (b).

Source data

Extended Data Fig. 3 Loss-of-function mutations of GSE3 lead to small grains.

a, The target site in GSE3. Red letters represent the target sequence and blue letters represent the PAM sequence. The knockout line gse3-cri1 had a single base T insertion that caused a frame-shift, and gse3-cri2 had 30 bp base pair deletion. b, The knockout line gse3-cri1 was validated to have a single base T insertion in the second exon of GSE3 by sanger sequencing. c, The knockout line gse3-cri2 was validated to have the 30 bp deletion in the second exon of GSE3 by sanger sequencing. d, Mature rice grains of ZH11, gse3-cri1 and gse3-cri2. e-h, The grain length (e), grain width (f), grain thickness (g) and 1,000 grain weight (h) of ZH11, gse3-cri1 and gse3-cri2. n = 50 (e-f), n = 40 (g), and n = 3 (h). i-j, The tiller number (i) and grain number per plant (j) of ZH11, gse3-cri1 and gse3-cri2. n = 12 (i-j). Values in (e-j) are given as mean ± SD. **P < 0.01 compared with parental line (ZH11) by Student’s t-test. Bars: 3 mm (d).

Source data

Extended Data Fig. 4 The phenotypes of ideal m238.

a-c, Mature grains (a), plants (b), and panicles (c) of ZH11 and m238 mutant. d-g, The grain length (d), grain width (e), grain thickness (f), and 1,000 grain weight (g) of ZH11 and m238 mutant. n = 60 (d-e), n = 40 (f), and n = 3 (g). h-i, The tiller number (h) and grain number per panicle (i) of ZH11 and m238 mutant. n = 8 (h), and n = 12 (i). j, A single base substitution on the first exon of the GSE3 gene results in an amino acid change (Ser/Pro) in the m238. The red arrow indicates the location of the single base replacement. Values in (d-i) are given as mean ± SD. **P < 0.01 compared with ZH11 by Student’s t-test. Bars: 3 mm (a), 10 cm (b) and 5 cm (c).

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Extended Data Fig. 5 GSE3 interacts with GS2 in yeast cells and rice protoplasts.

a, GSE3 was divided into three fragments including GSE3-N (1-84), GSE3-GNAT (85-177) and GSE3-C (178-414), GSE3-GNAT (85-177) and GSE3-C (178-414) interacted with GS2 in yeast cells. The yeast cells were cultured on –LW (a medium that lacked tryptophan and leucine) and –LWHA (a medium that lacked tryptophan, leucine, histidine, and adenine). AD (pGADT7) and BD (pGBKT7) are the prey and bait vectors, respectively. b, GS2 was divided into five segments, including GS2-N-QLQ (1-112), GS2-QLQ (50-112), GS2-WRC (113-181), GS2-C (169-394) and GS2-QLQ-WRC-C(50-394). Only the segments that contain the QLQ domain can interact with GSE3-C (178-414). c, GSE3 interacts with GS2 in rice protoplasts. The plasmids 35 S:GFP-GSE3 and 35 S:MYC-GS2 were introduced to the rice protoplasts by PEG-mediated protoplast transformation method and incubated at room temperature for 15 h. The total proteins were extracted and immunoprecipitated with GFP-Trap-Agarose (gta-200, Chromotek). The immunoprecipitated proteins were detected with anti-GFP and anti-MYC antibodies, respectively. MYC-GS2 was detected in the immunoprecipitated GFP-GSE3 complex. IB: immunoblot. IP: immunoprecipitated.

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Extended Data Fig. 6 Simultaneous knockout of GRF3 and GRF4 forms small grains.

a, Sanger sequencing confirmed that the knockout lines grf3-cri1 grf4-cri2 and grf3-cri1 grf4-cri3 have a single T base insertion in the third exon of GRF3. The sequencing analysis also revealed a single T base deletion in the first exon of the GRF4 gene in the grf3-cri1 grf4-cri2 line, as well as a single C base deletion in the first exon of the GRF4 gene in the grf3-cri1 grf4-cri3 line. The gene structure of GRF3 and GS2/GRF4 and the target sites (red arrow) are shown. b, Mature rice grains of ZH11, knockout lines grf3-cri1 grf4-cri2 and grf3-cri1 grf4-cri3. c-e, Grain length (c), grain width (d), and 1,000 grain weight (e) of ZH11 and knockout lines grf3-cri1 grf4-cri2 and grf3-cri1 grf4-cri3. n = 50 (c-d) and n = 3 (e). f-g, The tiller number and grain number per panicle of ZH11, knockout lines grf3-cri1 grf4-cri2 and grf3-cri1 grf4-cri3. n = 10 (f-g). Values in (c-g) are given as mean ± SD. **P < 0.01 compared with parental line (ZH11) by Student’s t-test. Bars: 3 mm (b).

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Extended Data Fig. 7 The histone H4Ac (pan-acetyl) level in the promoter region of GSE3 and GS2 co-regulated genes.

ChIP-qPCR analysis showed the relative enrichment of histone H4Ac (pan-acetyl) at the promoter region of various genes, such as XIAO, GW6, and OsBZR1 in both ZH11 and ZH11-GSE3small grain young panicles. Data are shown as assay-site relatively fold enrichment of the signal from immunoprecipitation over the background. ChIP–qPCR was performed with three replicates. Values are given as mean ± SD. **P < 0.01, *P < 0.05 compared with ZH11.

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Extended Data Fig. 8 The phenotypes of TFB and TFBgse3-cri3.

a-c, The mature plants (a), panicles (b), and grains (c) of TFB and TFBgse3-cri3. d-g, The grain length (d), grain width (e), grain thickness (f), and 1,000 grain weight (g) of TFBgse3-cri3 compared with those of TFB. n = 60 (d-e), n = 40 (f), and n = 3 (g). h-i, The number of tillers per plant (h) and grain number per panicle (i) of TFB and TFBgse3-cri3. n = 13 (h), and n = 10 (i). j-m, The plant height (j), leaf length (k), leaf width (l), and panicle length (m) of TFB and TFBgse3-cri3. n = 13 (j-l), and n = 10 (m). n, The grain yield per plant of TFB and TFBgse3-cri3. n = 22. Values in (d-n) are given as mean ± SD. **P < 0.01 compared with TFB by Student’s t-test. ns, not significant (Student’s unpaired t-test). Bars: 10 cm (a), 5 cm (b), and 3 mm (c).

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Extended Data Fig. 9 The phenotypes of Y58S and Y58Sgse3-cri4.

a-b, The mature plants (a) and panicles (b) of Y58S and Y58Sgse3-cri4. c-d, The tiller number (c) and the grain number per panicle (d) of Y58S and Y58Sgse3-cri4. n = 12 (c), n = 9 (Y58S) and 10 (Y58Sgse3-cri4) in (d). e-h, The plant height (e), leaf length (f), leaf width (g), and panicle length (h) of Y58S and Y58Sgse3-cri4. n = 12 (e-g) and n = 10 (h). i, The grain yield per plant of Y58S and Y58Sgse3-cri4. n = 20. Values in (c-i) are given as mean ± SD. **P < 0.01, *P < 0.05 compared with Y58S by Student’s t-test. ns, not significant (Student’s unpaired t-test). Bars: 10 cm (a), 5 cm (b).

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Extended Data Fig. 10 The comparison of panicles and rice grain size between F1 hybrids plants YLY900 and YLY900 (gse3-cri4).

a, Mature panicles of the F1 hybrids plants YLY900 and YLY900 (gse3-cri4). b, Mature rice grains of the F1 hybrids plants YLY900 and YLY900 (gse3-cri4). c-f, Panicle length (c), number of primary branches (d), number of secondary branches (e) and grain number per panicle (f) of the F1 hybrids plants YLY900 and YLY900 (gse3-cri4). n = 12. g-j, Grain length (g), grain width (h), grain thickness (i) and 1,000 grain weight (j) of the F1 hybrids plants YLY900 and YLY900 (gse3-cri4). n = 60 (g-h), n = 40 (i), and n = 3 (j). Values in (c-j) are given as mean ± SD. **P < 0.01 compared with parental line by Student’s t-test. ns, not significant (Student’s unpaired t-test). Bars: 5 cm (a), 3 mm (b).

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Supplementary information

Supplementary Information

Supplementary Figs. 1–32 and Tables 1–4.

Reporting Summary

Supplementary Data 1

Co-upregulated and co-downregulated genes in ZH11-GSE3small grain and grf3-cri1 grf4-cri2.

Supplementary Data 2

Differentially expressed genes in ZH11-GSE3small grain versus ZH11.

Supplementary Data 3

Overlapping genes of the CUT&Tag data from GS2 and RNA-seq data from GSE3.

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Huang, K., Wang, Y., Li, Y. et al. Modulation of histone acetylation enables fully mechanized hybrid rice breeding. Nat. Plants 10, 954–970 (2024). https://doi.org/10.1038/s41477-024-01720-0

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