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Antagonistic control of seed dormancy in rice by two bHLH transcription factors

Abstract

Preharvest sprouting (PHS) due to lack of seed dormancy seriously threatens crop production worldwide. As a complex quantitative trait, breeding of crop cultivars with suitable seed dormancy is hindered by limited useful regulatory genes. Here by repeatable phenotypic characterization of fixed recombinant individuals, we report a quantitative genetic locus, Seed Dormancy 6 (SD6), from aus-type rice, encoding a basic helix-loop-helix (bHLH) transcription factor, which underlies the natural variation of seed dormancy. SD6 and another bHLH factor inducer of C-repeat binding factors expression 2 (ICE2) function antagonistically in controlling seed dormancy by directly regulating the ABA catabolism gene ABA8OX3, and indirectly regulating the ABA biosynthesis gene NCED2 via OsbHLH048, in a temperature-dependent manner. The weak-dormancy allele of SD6 is common in cultivated rice but undergoes negative selection in wild rice. Notably, by genome editing SD6 and its wheat homologs, we demonstrated that SD6 is a useful breeding target for alleviating PHS in cereals under field conditions.

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Fig. 1: Identification of SD6.
Fig. 2: ICE2 interacting with SD6 positively regulates rice seed dormancy.
Fig. 3: ABA8OX3 is a direct target of SD6 and ICE2.
Fig. 4: OsbHLH048 attenuates seed dormancy downstream of SD6 and ICE2.
Fig. 5: The temperature response of SD6 and ICE2.
Fig. 6: Natural variations of SD6.
Fig. 7: Proposed working model and application of SD6 in rice and wheat.

Data availability

Sequence data from this study can be found in the MSU database (http://rice.plantbiology.msu.edu/) under the following accession numbers: SD6 (LOC_Os06g06900), ICE2 (LOC_Os01g70310), NCED2 (LOC_Os12g24800), ABA8OX3 (LOC_Os09g28390), OsACTIN1 (LOC_Os03g50885), OsbHLH048 (LOC_Os02g52190) and OsUBIQUITIN2 (LOC_Os02g06640). Sequence for constructing the phylogenetic tree of ICE2 (Extended Data Fig. 8) can be found in The Arabidopsis Information Resource database (https://www.arabidopsis.org/) or the MSU database (http://rice.plantbiology.msu.edu/) under the following accession numbers: AtICE1 (AT3G26744), AtICE2 (AT1G12860), AtbHLH57 (AT4G01460), ZOU (AT1G49770), SD6 (LOC_Os06g06900), OsICE1 (LOC_Os11g32100), OsICE2 (LOC_Os01g70310), Rc (LOC_Os07g11020), LOC_Os06g44320, LOC_Os03g08930, LOC_Os10g23050, LOC_Os09g29360, LOC_Os02g52190, LOC_Os04g35010, LOC_Os08g38210, LOC_Os01g18870, LOC_Os04g51070, LOC_Os07g36460, LOC_Os02g02820, LOC_Os04g41570, LOC_Os03g56950, LOC_Os04g52770, LOC_Os04g35000, LOC_Os01g13460, LOC_Os03g26210 and LOC_Os07g43530. For Extended Data Fig. 10, HORVU7Hr1G026560 can be found in phytozome v12.1 (https://phytozome-next.jgi.doe.gov/) by selecting the genome of Hordeum vulgare r1, and TaSD6-A1 (TraesCS7A02G126900), TaSD6-B1 (TraesCS7B02G026300), TaSD6-D1 (TraesCS7D02G124700), BRADI_1g48400v3, Zm00001d018416, SORBI_3004G336700 and AET7Gv20319600 can be found in EnsemblPlants database (https://plants.ensembl.org/index.html).

Further information and requests for resources and reagents should be directed to and will be fulfilled by C.C. (ccchu@genetics.ac.cn) and C.G. (cxgao@genetics.ac.cn). Source data are provided with this paper.

Code availability

All software used in the study are publicly available on the Internet as described in Methods and Reporting Summary.

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Acknowledgements

We thank Jiayang Li (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences (CAS)) for providing the SCYNER and SCYCER vectors, Yaoguang Liu (College of Life Sciences, South China Agricultural University) for providing the CRISPR–Cas9 vector system. We also thank Zhiming Feng (Yangzhou University) for providing a field photograph of the rice PHS. This work was supported by the grants from G2P project of the Ministry of Science and Technology of China (2020YFE0202300), the Strategic Priority Research Program of CAS (XDA24020000), the Key Research Program of Frontier Sciences, CAS (QYZDJ-SSW-SMC014) and the National Natural Science Foundation of China (32172059).

Author information

Authors and Affiliations

Authors

Contributions

F.X., J.T. and C.C. conceived the project, designed the experiments and analyzed the data. F.X. and X.C. performed most of the experiments with the help of S.G., S.W. and B.L. who performed the wheat experiments. H.W. and S.O. performed the population genetic analyses. Y.Q. helped create rice mutants. C.C. and C.G. supervised the project. F.X., J.T., S.W., H.W., C.G. and C.C. wrote the manuscript with contributions from all authors.

Corresponding authors

Correspondence to Caixia Gao or Chengcai Chu.

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Competing interests

The authors declare no competing interests.

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Peer review information

Nature Genetics thanks Makoto Matsuoka, Yongzhong Xing, and George W Bassel for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Characters of CSSSL Q27.

a, Germination performance of seeds in freshly harvested CSSSL Q27 panicles. Scale bar 1 cm. Data are presented as mean values ± s.d. Each analysis was repeated with four panicle replicates and photographs were taken after six days imbibition. b, Germination performance of breaking dormancy seeds (one week 45°C treatment) of CSSSL Q27. Scale bar 1 cm. Data are presented as mean values ± s.d. Germination test in b was repeated with four panicle replicates and photographs were taken after four days imbibition. c,d, Electrophoretogram of detected fragment of molecular markers S1 to S10 in Q27 (c) and NIL-SD6 (d). The upper- and lower-boundaries are 100 bp and 250 bp, respectively.

Source data

Extended Data Fig. 2 SNP analysis of SD6.

a, SNPs between SD6Nip and SD6Kasa. b, Align of SD6Nip and SD6Kasa. Red, nuclear localization signal (NLS); light blue, basic domain; purple, helix-loop-helix (HLH) domain; yellow, Beta strand.

Extended Data Fig. 3 Identification of sd6 and ice2 mutants.

a, Schematic representation of SD6 CRISPR-Cas9 mutants. b, The expression of SD6 in sd6 mutants and SD6 overexpression plants. c, The germination percentage of breaking dormancy seeds (one week 45°C treatment) of sd6 mutants and SD6 overexpression plants. d, The expression level of SD6 in Nipponbare and NIL-SD6 seeds detected by qRT-PCR. e, Schematic representation of ICE2 CRISPR-Cas9 mutants. f,g, The expression of ICE2 in ice2 mutants (f) and ICE2 overexpression plants (g). Analysis in b,d,f,g, was repeated with three biological replicates. Germination test in c was repeated with four biological replicates. Data are presented as mean values ± s.d.

Extended Data Fig. 4 The ABA content in SD6- and ICE2-overexpression lines.

a, The ABA content in freshly harvested 21-25 DAP seeds of SD6-overexpression lines (SO2-7 and SO3-6). b, The ABA content in freshly harvested 21-25 DAP seeds of ICE2-overexpression lines (IO1-2 and IO1-8). Each analysis was repeated with three biological replicates. Data are presented as mean values ± s.d. All P values are based on two-tailed Student’s t-tests.

Extended Data Fig. 5 ABI3 and ABI5 were not the directly targets of SD6 and ICE2.

a, Yeast one-hybrid interactions between SD6 and ICE2 proteins with two elements, ‘E-box variant’ and ‘Triple G-box’. The interaction between p53 proteins and p53 binding elements was used as a positive control, and mutated versions of the E-box and G-box were used as negative controls. b, Schematic representation of the indicated genes: Vertical light blue bar represents the start codon ATG; Vertical red bar represents the stop codon TGA; Vertical orange bars represent G-box motifs; vertical gray bars represent E-box motifs; horizontal thin black lines represent amplicons assayed for SD6 and ICE2 binding. c,d, Relative ChIP-qPCR enrichment of the indicated promoter regions of ABI3 and ABI5 for SD6 (c) and ICE2 (d). Flag-GFP was served as a negative control. Fragment of ACTIN1 gene body region was used as interval negative control. e,f, The expression level of ABI3 and ABI5 in freshly harvested 21-25 DAP seeds of ZH11, sd6 mutants, and ice2 mutants. Each analysis was repeated with three biological replicates. Data are presented as mean values ± s.d.

Extended Data Fig. 6 SD6 and ICE2 mutually impaired their regulation of ABA8OX3.

a,b, pA8-P1-LUC (a, only containing G-box motif) and pA8-P2-LUC (b, only containing E-box motif) expression in rice protoplasts co-transformed with SD6Nip and ICE2. Each analysis was repeated with four biological replicates. Data are presented as mean values ± s.d. All P values are based on two-tailed Student’s t-tests.

Extended Data Fig. 7 Identification of ABA8OX3 and OsbHLH048 CRISPR-Cas9 mutants.

a, Schematic representation of ABA8OX3 CRISPR-Cas9 mutants. bd, The expression of ABA8OX3 (b), SD6 (c), and ICE2 (d) in aba8ox3 mutants. e, Schematic representation of OsbHLH048 CRISPR-Cas9 mutants. f, The expression of OsbHLH048 in its mutants. Each analysis was repeated with three biological replicates. Data are presented as mean values ± s.d.

Extended Data Fig. 8 Phylogenetic analysis of ICE2 and expression profiles of ICE1 and ICE2.

a, Phylogenetic tree of SD6, ICE2 and its homologous proteins in Oryza sativa (ICE1) Arabidopsis (AtICE1 and AtICE2), Rc, AtbHLH57 and its homologous proteins in Oryza sativa, ZOU, and other bHLHs in rice. b, The spatio-temporal expression pattern of ICE1 and ICE2 in various tissues/organs throughout entire growth in the field. Data obtained from RiceXPro database (http://ricexpro.dna.affrc.go.jp/).

Extended Data Fig. 9 Germination perfomance of sd6 mutants in Huaidao5 background.

a,b, Germination perfomance (a) and germination percentage (b) of seeds in freshly harvested 31-35 DAP panicles of sd6 mutants in Huaidao5 (Huai5) background. Each analysis was repeated with four biological replicates and photographs were taken after 5-day imbibition. Scale bar 1 cm. Box plots denote median (horizontal line) with minima to maxima whiskers. All P values are based on two-tailed Student’s t-tests.

Extended Data Fig. 10 Characters of TaSD6.

a, Phylogenetic analysis of SD6 in different cereal crops by the Neighbor-Joining method. Branch lengths represented extents of homology between branches. b, Characterization of SD6 homologs in wheat. Exons of TaSD6 are shown as blue squares, and the target site of CRISPR/Cas9 is marked with red lines in the conserved region encoding the helix-loop-helix DNA-binding domain. c, PCR/RE analysis of seven mutants at the target site of TaSD6. PCR was performed using A, B, and D sub-genome-specific primers and the products were digested with restriction enzyme Alw44I. d, Detailed sequence of the triple-recessive bi-allelic mutant (T0-1) at the target site. The red lowercase letters and short lines represent 1-bp insertions and deletions, respectively. e, Seed germination rates of wild type and the triple-recessive homozygous mutant (tasd6) at each day after imbibition in 2021. Germination test in e was performed with eight biological replicates. Data are presented as mean values ± s.d. f, Seed germination status of wild type and the triple-recessive homozygous mutant (tasd6) after 4-day imbibition. g, Seed germination rates of dormancy-released wild type and tasd6 via after-ripening at each day after imbibition in 2021. Germination test in g was performed with four biological replicates. h, Spike spouting rate of wild type and tasd6 after 6-day imbibition in 2021. Each spike was harvested and incubated under high humidity. i, Grain number per spike of wild type and tasd6. Analysis in h and i was repeated with at least twenty-five biological replicates. Scale bar 1 cm. Box plots denote median (horizontal line) with minima to maxima whiskers. All P values are based on two-tailed Student’s t-tests.

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Supplementary Tables 1–5.

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Source Data Fig. 2

Unprocessed western blots.

Source Data Extended Data Fig. 1

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Source Data Extended Data Fig. 10

Unprocessed gels.

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Xu, F., Tang, J., Wang, S. et al. Antagonistic control of seed dormancy in rice by two bHLH transcription factors. Nat Genet 54, 1972–1982 (2022). https://doi.org/10.1038/s41588-022-01240-7

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