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Targeting a gene regulatory element enhances rice grain yield by decoupling panicle number and size

Nature Biotechnology volume 40, pages 1403–1411 (2022)Cite this article

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Abstract

Crop genetic improvement requires balancing complex tradeoffs caused by gene pleiotropy and linkage drags, as exemplified by IPA1 (Ideal Plant Architecture 1), a typical pleiotropic gene in rice that increases grains per panicle but reduces tillers. In this study, we identified a 54-base pair cis-regulatory region in IPA1 via a tiling-deletion-based CRISPR–Cas9 screen that, when deleted, resolves the tradeoff between grains per panicle and tiller number, leading to substantially enhanced grain yield per plant. Mechanistic studies revealed that the deleted fragment is a target site for the transcription factor An-1 to repress IPA1 expression in panicles and roots. Targeting gene regulatory regions should help dissect tradeoff effects and provide a rich source of targets for breeding complementary beneficial traits.

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Fig. 1: Tiling deletions in IPA1 CRRs overcame tradeoffs.
Fig. 2: IPA1-Pro10 showed simultaneously increased tiller number and panicle size.
Fig. 3: ipa1-pro10 was a semi-dominant allele.
Fig. 4: An-1 directly bound to IPA1 promoter and negatively regulated its expression.
Fig. 5: Deletion of An-1 binding site-containing fragment was responsible for increased panicle size and root thickness.
Fig. 6: Diverse transcriptional regulation of IPA1 in determining different traits.

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All data supporting the findings of this study are available in the article, supplementary information and source data files. Source data are provided with this paper.

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Acknowledgements

We thank C. Sun (China Agricultural University) for providing the NIL-An-1 and NIL-an-1 seeds. This work was supported by the National Natural Science Foundation of China (31788103 to J.L. and H.Y. and 32122064 to H.Y.), the Chinese Academy of Sciences (XDA24030504 to J.L.) and the Hainan Excellent Talent Team (to J.L.).

Author information

Authors and Affiliations

  1. State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China

    Xiaoguang Song, Xiangbing Meng, Hongyan Guo, Qiao Cheng, Yanhui Jing, Mingjiang Chen, Guifu Liu, Bing Wang, Yonghong Wang, Jiayang Li & Hong Yu

  2. University of Chinese Academy of Sciences, Beijing, China

    Qiao Cheng, Yonghong Wang, Jiayang Li & Hong Yu

  3. College of Life Sciences, Shandong Agricultural University, Tai’an, China

    Yonghong Wang

  4. Hainan Yazhou Bay Seed Laboratory, Sanya, China

    Jiayang Li

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  1. Xiaoguang Song
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Contributions

X.S. performed most of the experiments. X.S., X.M. and H.G. designed the CRISPR target and constructed the plasmid library. H.G. and X.M. transformed rice. X.S., X.M., H.G. and Q.C. characterized the genotypes and phenotypes of the edited lines. X.M., Y.J., M.C. and G.L. contributed to the rice materials. X.S., H.G., B.W., Y.W. and H.Y. analyzed the data. X.S., J.L. and H.Y. conceived and designed experiments and wrote the manuscript.

Corresponding author

Correspondence to Hong Yu.

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Nature Biotechnology thanks Xiangdong Fu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Design of gRNAs in the tiling-deletion screen.

Red bars, CRISPR-Cas9 target sites in each vector. Blue bars, regions between CRISPR-Cas9 target sites on same vector. TSS, transcription start site. TTS, transcription termination site. The positions in promoter and 5’ UTR were relative to the transcription start site. The positions in 3’ UTR and downstream region were relative to the transcription termination site.

Extended Data Fig. 2 Morphological phenotypes of edited lines.

Upper panel, plants at the mature stage; lower panel, their corresponding panicles. Bars = 10 cm.

Extended Data Fig. 3 Correlations between the panicle weight and tiller number in two years.

a,b, Scatter plot of panicle weight (a) and tiller number (b) of 21 edited lines in 2019 and 2020. R2 values were calculated using linear regression.

Source data

Extended Data Fig. 4 Stem diameter of ZH11, IPA1-Pro10, ipa1-5D, and ipa1-12 plants.

a, Cross-sections of internodes of ZH11, IPA1-Pro10, ipa1-5D, and ipa1-12. Bars = 1 mm. b-d, Statistical analysis of internode diameters in a. Values indicate means ± s.d. (n = 10 plants). Exact P values are shown; Tukey’s HSD test (b-d).

Source data

Extended Data Fig. 5 IPA1 expression levels in different tissues of ZH11 and IPA1-Pro10.

a, IPA1 expression levels in panicles at different stages of ZH11 and IPA1-Pro10. b,c, IPA1 expression levels in tiller bud (b) and root (c) of ZH11 and IPA1-Pro10. d, IPA1 expression profile in different tissues. Shoot base, young leaf, and root were sampled from 2-week-old seedings. Old leaf and stem were sampled from plants after heading stage. IPA1 expression levels in 0.5-cm panicle (a), tiller bud (b), root (c), and shoot base (d) of ZH11 were set to one. Values indicate means ± s.d. (n = 3 biological replicates in a-c and n = 3 technical replicates in d). Exact P values are shown; two-sided Student’s t-test.

Source data

Extended Data Fig. 6 Thousand-grain weight and days to heading of ZH11 and IPA1-Pro10 in paddy field.

a, Statistical analysis of thousand-grain weight. b, Statistical analysis of days to heading. Values indicate means ± s.d. (n = 3 biological replicates). Exact P values are shown; two-sided Student’s t-test.

Source data

Extended Data Fig. 7 Predicted An-1 binding sites in the promoters of SPL14 orthologs.

The An-1 binding sites were predicted in the promoters of IPA1 homologous genes, including Aegilops tauschii SPL17 (LOC109731696), Brachypodium distachyon SPL14 (LOC100836973), Sorghum bicolor SPL14 (LOC8064025), Zea mays SBP8 (GRMZM2G160917) and SPL15 (GRMZM2G460544), Setaria italica SPL14 (LOC101773844), Arabidopsis thaliana SPL9 (AT2G42200) and SPL15 (AT3G57920), and Nicotiana tabacum SPL9 (LOC107809759).

Extended Data Fig. 8 An-1 expression levels in different tissues of ZH11 and IPA1-Pro10.

a, An-1 expression levels in panicles at different stages of ZH11 and IPA1-Pro10. b,c, An-1 expression levels in tiller bud (b) and root (c) of ZH11 and IPA1-Pro10. d, An-1 expression profile in different tissues. Shoot base, young leaf, and root were sampled from 2-week-old seedings. Old leaf and stem were sampled from plants after heading stage. An-1 expression levels in 0.5-cm panicle (a), tiller bud (b), root (c), and shoot base (d) of ZH11 were set to one. Values indicate means ± s.d. (n = 3 biological replicates in a-c and n = 3 technical replicates in d). Exact P values are shown; two-sided Student’s t-test.

Source data

Extended Data Fig. 9 IPA1 expression levels in different tissues of NIL-An-1 and NIL-an-1.

a, IPA1 expression levels in panicles at different stages of NIL-An-1 and NIL-an-1. b, An-1 expression in the tiller buds of NIL-An-1 and NIL-an-1. c, IPA1 expression profile in different tissues of NIL-An-1 and NIL-an-1. Shoot base, young leaf, and root were sampled from 2-week-old seedings. Old leaf and stem were sampled from plants after heading stage. Gene expression levels in 0.5-cm panicle (a), tiller bud (b), and shoot base (c) of NIL-An-1 were set to one. Values indicate means ± s.d. (n = 3 biological replicates in a and b, n = 3 technical replicates in c). Exact P values are shown; two-sided Student’s t-test.

Source data

Extended Data Fig. 10 Panicle phenotypes of IPA1-Pro11, IPA1-Pro12 and IPA1-Pro13.

a-h, Panicle morphology (a), panicle weight (b), grain number per primary branch (c), secondary branch per primary branch (d), primary branch number (e), secondary branch number (f), thousand-grain weight (g), and grain setting rate (h) of plants with wild-type (–/–), heterozygous (+/–), and homozygous (+/+) ipa1-pro11 alleles. i-p, Panicle morphology (i), panicle weight (j), grain number per primary branch (k), secondary branch per primary branch (l), primary branch number (m), secondary branch number (n), thousand-grain weight (o), and grain setting rate (p) of plants with wild-type, heterozygous, and homozygous ipa1-pro12 alleles. q-x, Panicle morphology (q), panicle weight (r), grain number per primary branch (s), secondary branch per primary branch (t), primary branch number (u), secondary branch number (v), thousand-grain weight (w), and grain setting rate (x) of plants with wild-type, heterozygous and homozygous ipa1-pro13 alleles. Bars = 10 cm. Values indicate means ± s.d. (n = 15 plants). Exact P values are shown; two-sided Student’s t-test.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–6 and Supplementary Tables 1 and 6

Reporting Summary

Supplementary Tables 2–5

Supplementary Table 2: 39 vectors used in the tiling-deletion screen. Supplementary Table 3: Genotyping result of 792 T0 plants. Supplementary Table 4: Agronomic traits of 21 edited lines. Supplementary Table 5: Predicted transcription factor binding sites in the promoter of IPA1.

Supplementary Data 1

Statistical Source Data for Supplementary Figs. 5b,c and 6b,c

Supplementary Data 2

Unprocessed gel for Supplementary Fig. 5a

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Song, X., Meng, X., Guo, H. et al. Targeting a gene regulatory element enhances rice grain yield by decoupling panicle number and size. Nat Biotechnol 40, 1403–1411 (2022). https://doi.org/10.1038/s41587-022-01281-7

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