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Improving yield-related traits by editing the promoter of the heading date gene Ehd1 in rice

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We developed an efficient promoter editing method to create different weak Ehd1 alleles in elite japonica rice variety ZJ8 with slightly delayed heading and improved yield for use in breeding.

Abstract

Heading date is an important agronomic trait of rice (Oryza sativa) that determines the planting areas and cultivation seasons of different varieties, thus affecting final yield. Early heading date 1 (Ehd1) is a major rice integrator gene in the regulatory network of heading date whose expression level is negatively correlated with heading date and grain yield. Some elite japonica varieties such as Zhongjia 8 (ZJ8) show very early heading with poor agronomic traits when planted in South China. This problem can be addressed by downregulating the expression of Ehd1. In this study, we analyzed the cis-regulatory elements in the Ehd1 promoter region. We then used CRISPR/Cas9-mediated editing to modify the Ehd1 promoter at multiple target sites in ZJ8. We rapidly identified homozygous allelic mutations in the T2 generation via long-read sequencing. We obtained several Ehd1 promoter mutants with different degrees of lower Ehd1 expression, delayed heading date, and improved yield-related traits. We developed an efficient promoter editing method to create different weak Ehd1 alleles for breeding selection. Using this method, a series of heading date materials from elite varieties can be created to expand the planting area of rice and improve grain yields.

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Data supporting the findings of this work are available within the paper and its Supplementary Information files.

References

  • Andrés F, Galbraith DW, Talón M, Domingo C (2009) Analysis of PHOTOPERIOD SENSITIVITY5 sheds light on the role of phytochromes in photoperiodic flowering in rice. Plant Physiol 151:681–690

    Article  PubMed  PubMed Central  Google Scholar 

  • Belhaj K, Chaparro-Garcia A, Kamoun S, Patron NJ, Nekrasov V (2015) Editing plant genomes with CRISPR/Cas9. Curr Opin Biotechnol 2:76–84

    Article  Google Scholar 

  • Boyle AP, Davis S, Shulha HP, Meltzer P, Margulies EH, Weng Z, Furey TS, Crawford GE (2008) High-resolution mapping and characterization of open chromatin across the genome. Cell 132:311–322

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Brambilla V, Martignago D, Goretti D, Cerise M, Somssich M, de Rosa M, Galbiati F, Shrestha R, Lazzaro F, Simon R, Fornara F (2017) Antagonistic transcription factor complexes modulate the floral transition in rice. Plant Cell 29:2801–2816

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Cai M, Chen S, Wu M, Zheng T, Zhou L, Li C, Zhang H, Wang J, Xu X, Chai J, Ren Y, Guo X, Zhang X, Lei C, Cheng Z, Wang J, Jiang L, Zhai H, Wang H, Zhu S, Wan J (2019) Early heading 7 interacts with DTH8, and regulates flowering time in rice. Plant Cell Rep 38:521–532

    Article  PubMed  CAS  Google Scholar 

  • Cerise M, Giaume F, Galli M, Khahani B, Lucas J, Podico F, Tavakol E, Parcy F, Gallavotti A, Brambilla V, Fornara F (2021) OsFD4 promotes the rice floral transition via florigen activation complex formation in the shoot apical meristem. New Phytol 229:429–443

    Article  PubMed  CAS  Google Scholar 

  • Chai J, Zhu S, Li C, Wang C, Cai M, Zheng X, Zhou L, Zhang H, Sheng P, Wu M, Jin X, Cheng Z, Zhang X, Lei C, Ren Y, Lin Q, Zhou S, Guo X, Wang J, Zhao Z, Wan J (2021) OsRE1 interacts with OsRIP1 to regulate rice heading date by finely modulating Ehd1 expression. Plant Biotechnol J 19:300–310

    Article  PubMed  CAS  Google Scholar 

  • Chen R, Deng Y, Ding Y, Guo J, Qiu J, Wang B, Wang C, Xie Y, Zhang Z, Chen J, Chen L, Chu C, He G, He Z, Huang X, Xing Y, Yang S, Xie D, Liu Y, Li J (2022) Rice functional genomics: decades’ efforts and roads ahead. Sci China Life Scin 65:33–92

    Article  Google Scholar 

  • Cho LH, Yoon J, Pasriga R, An G (2016) Homodimerization of Ehd1 is required to induce flowering in rice. Plant Physiol 170:2159–2171

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34:184–191

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Doi K, Izawa T, Fuse T, Yamanouchi U, Kubo T, Shimatani Z, Yano M, Yoshimura A (2004) Ehd1, a B-type response regulator in rice, confers short-day promotion of flowering and controls FT-like gene expression independently of Hd1. Genes Dev 18:926–936

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Hallikas O, Palin K, Sinjushina N, Rautiainen R, Partanen J, Ukkonen E, Taipale J (2006) Genome-wide prediction of mammalian enhancers based on analysis of transcription-factor binding affinity. Cell 124:47–59

    Article  PubMed  CAS  Google Scholar 

  • Hinnebusch AG (2005) Translational regulation of GCN4 and the general amino acid control of yeast. Annu Rev Microbiol 59:407–450

    Article  PubMed  CAS  Google Scholar 

  • Hu Y, Li S, Xing Y (2019) Lessons from natural variations: artificially induced heading date variations for improvement of regional adaptation in rice. Theor Appl Genet 132:383–394

    Article  PubMed  CAS  Google Scholar 

  • Jiang P, Wang S, Zheng H, Li H, Zhang F, Su Y, Xu Z, Lin H, Qian Q, Ding Y (2018) SIP1 participates in regulation of flowering time in rice by recruiting OsTrx1 to Ehd1. New Phytol 219:422–435

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Karst SM, Ziels RM, Kirkegaard RH, Sørensen EA, McDonald D, Zhu Q, Knight R, Albertsen M (2021) High-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing. Nat Methods 18:165–169

    Article  PubMed  CAS  Google Scholar 

  • Kim SL, Lee S, Kim HJ, Nam HG, An G (2007) OsMADS51 is a short-day flowering promoter that functions upstream of Ehd1, OsMADS14, and Hd3a. Plant Physiol 145:1484–1494

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Lee YS, Jeong DH, Lee DY, Yi J, Ryu CH, Kim SL, Jeong HJ, Choi SC, Jin P, Yang J, Cho LH, Choi H, An G (2010) OsCOL4 is a constitutive flowering repressor upstream of Ehd1 and downstream of OsphyB. Plant J 63:18–30

    PubMed  CAS  Google Scholar 

  • Li X, Xie Y, Zhu Q, Liu YG (2017) Targeted genome editing in genes and cis-regulatory regions improves qualitative and quantitative traits in crops. Mol Plant 10:1368–1370

    Article  PubMed  CAS  Google Scholar 

  • Li C, Li W, Zhou Z, Chen H, Xie C, Lin Y (2020) A new rice breeding method: CRISPR/Cas9 system editing of the Xa13 promoter to cultivate transgene-free bacterial blight-resistant rice. Plant Biotechnol J 18:313–315

    Article  PubMed  Google Scholar 

  • Liu L, Gallagher J, Arevalo ED, Chen R, Skopelitis T, Wu Q, Bartlett M, Jackson D (2021a) Enhancing grain-yield-related traits by CRISPR–Cas9 promoter editing of maize CLE genes. Nat Plants 7:287–294

    Article  PubMed  CAS  Google Scholar 

  • Liu X, Liu H, Zhang Y, He M, Li R, Meng W, Wang Z, Li X, Bu Q (2021b) Fine-tuning flowering time via genome editing of upstream open reading frames of Heading Date 2 in rice. Rice (New York, N.Y.) 14:69

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408

    Article  PubMed  CAS  Google Scholar 

  • Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen YL, Guo J, Chen L, Zhao X, Dong Z, Liu YG (2015) A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8:1274–1284

    Article  PubMed  CAS  Google Scholar 

  • Ma X, Zhu Q, Chen Y, Liu YG (2016) Crispr/cas9 platforms for genome editing in plants: developments and applications. Mol Plant 9:961–974

    Article  PubMed  CAS  Google Scholar 

  • Nemoto Y, Nonoue Y, Yano M, Izawa T (2016) Hd1, a CONSTANS ortholog in rice, functions as an Ehd1 repressor through interaction with monocot-specific CCT-domain protein Ghd7. Plant J 86:221–233

    Article  PubMed  CAS  Google Scholar 

  • Pennacchio LA, Rubin EM (2003) Comparative genomic tools and databases: providing insights into the human genome. J Clin Invest 111:1099–1106

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Rodríguez-Leal D, Lemmon ZH, Man J, Bartlett ME, Lippman ZB (2017) Engineering quantitative trait variation for crop improvement by genome editing. Cell 171:470–480

    Article  PubMed  Google Scholar 

  • Rogers SO, Bendich AJ (1985) Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Mol Biol 5:69–76

    Article  PubMed  CAS  Google Scholar 

  • Römer P, Recht S, Strauß T, Elsaesser J, Schornack S, Boch J, Wang S, Lahaye T (2010) Promoter elements of rice susceptibility genes are bound and activated by specific TAL effectors from the bacterial blight pathogen. Xanthomonas Oryzae Pv Oryzae New Phytol 187:1048–1057

    PubMed  Google Scholar 

  • Roudier F, Ahmed I, Bérard C, Sarazin A, Mary-Huard T, Cortijo S, Bouyer D, Caillieux E, Duvernois-Berthet E, Al-Shikhley L, Giraut L, Després B, Drevensek S, Barneche F, Dèrozier S, Brunaud V, Aubourg S, Schnittger A, Bowler C, Martin-Magniette ML, Robin S, Caboche M, Colot V (2011) Integrative epigenomic mapping defines four mainchromatin states in Arabidopsis. EMBO J 30:1928–1938

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Song YH, Shim JS, Kinmonth-Schultz HA, Imaizumi T (2015) Photoperiodic flowering:time measurement mechanisms in leaves. Annu Rev Plant Biol 66:441–464

    Article  PubMed  CAS  Google Scholar 

  • Song X, Meng X, Guo H, Cheng Q, Jing Y, Chen M, Liu G, Wang B, Wang Y, Li J, Yu H (2022) Targeting a gene regulatory element enhances rice grain yield by decoupling panicle number and size. Nat Biotechnol 40:1403–1411

    Article  PubMed  CAS  Google Scholar 

  • Takahashi Y, Teshima KM, Yokoi S, Innan H, Shimamoto K (2009) Variations in Hd1 proteins, Hd3a promoters, and Ehd1 expression levels contribute to diversity of flowering time in cultivated rice. Proc Natl Acad Sci U S A 106:4555–4560

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Thurman RE, Rynes E, Humbert R, Vierstra J, Maurano MT, Haugen E, Sheffield NC, Stergachis AB, Wang H, Vernot B, Garg K, John S, Sandstrom R, Bates D, Boatman L, Canfield TK, Diegel M, Dunn D, Ebersol AK, Frum T, Giste E, Johnson AK, Johnson EM, Kutyavin T, Lajoie B, Lee BK, Lee K, London D, Lotakis D, Neph S, Neri F, Nguyen ED, Qu H, Reynolds AP, Roach V, Safi A, Sanchez ME, Sanyal A, Shafer A, Simon JM, Song L, Vong S, Weaver M, Yan Y, Zhang Z, Zhang Z, Lenhard B, Tewari M, Dorschner MO, Hansen RS, Navas PA, Stamatoyannopoulos G, Iyer VR, Lieb JD, Sunyaev SR, Akey JM, Sabo PJ, Kaul R, Furey TS, Dekker J, Crawford GE, Stamatoyannopoulos JA (2012) The accessible chromatin landscape of the human genome. Nature 489:75–82

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Vicentini G, Biancucci M, Mineri L, Chirivì D, Giaume F, Miao Y, Kyozuka J, Brambilla V, Betti C, Fornara F (2023) Environmental control of rice flowering time. Plant Commun 4(5):100610

    Article  PubMed  PubMed Central  Google Scholar 

  • Voytas DF, Gao C (2014) Precision genome engineering and agriculture: opportunities and regulatory challenges. PLoS Biol 12:e1001877

    Article  PubMed  PubMed Central  Google Scholar 

  • Wei X, Xu J, Guo H, Jiang L, Chen S, Yu C, Zhou Z, Hu P, Zhai H, Wan J (2010) DTH8 suppresses flowering in rice, influencing plant height and yield potential simultaneously. Plant Physiol 153:1747–1758

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Wenger AM, Peluso P, Rowell WJ, Chang PC, Hall RJ, Concepcion GT, Ebler J, Fungtammasan A, Kolesnikov A, Olson ND, Töpfer A, Alonge M, Mahmoud M, Qian Y, Chin CS, Phillippy AM, Schatz MC, Myers G, DePristo MA, Ruan J, Marschall T, Sedlazeck FJ, Zook JM, Li H, Koren S, Carroll A, Rank DR, Hunkapiller MW (2019) Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome. Nat Biotechnol 37:1155–1162

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Wethmar K, Barbosa-Silva A, Andrade-Navarro MA, Leutz A (2014) uORFdb—a comprehensive literature database on eukaryotic uORF biology. Nucleic Acids Res 42:D60–D67

    Article  PubMed  CAS  Google Scholar 

  • Wittkopp PJ, Kalay G (2011) Cis-regulatory elements: molecular mechanisms and evolutionary processes underlying divergence. Nat Rev Genet 13:59–69

    Article  PubMed  Google Scholar 

  • Wu C, You C, Li C, Long T, Chen G, Byrne ME, Zhang Q (2008) RID1, encoding a Cys2/His2-type zinc finger transcription factor, acts as a master switch from vegetative to floral development in rice. Proc Natl Acad Sci 105:12915–12920

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Xie X, Ma X, Zhu Q, Zeng D, Li G, Liu YG (2017) CRISPR-GE: a convenient software toolkit for crispr-based genome editing. Mol Plant 10:1246–1249

    Article  PubMed  CAS  Google Scholar 

  • Xue W, Xing Y, Weng X, Zhao Y, Tang W, Wang L, Zhou H, Yu S, Xu C, Li X, Zhang Q (2008) Natural variation in Ghd7 is an important regulator of heading date and yield potential in rice. Nat Genet 40:761–767

    Article  PubMed  CAS  Google Scholar 

  • Yano M, Katayose Y, Ashikari M, Yamanouchi U, Monna L, Fuse T, Baba T, Yamamoto K, Umehara Y, Nagamura Y, Sasaki T (2000) Hd1, a major photoperiod sensitivity quantitative trait locus in rice, is closely related to the arabidopsis flowering time gene CONSTANS. Plant Cell 12:2473–2484

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Yao W, Li G, Yu Y, Ouyang Y (2018) funRiceGenes dataset for comprehensive understanding and application of rice functional genes. GigaScience 7:1–9

    Article  PubMed  Google Scholar 

  • Yoshitake Y, Yokoo T, Saito H, Tsukiyama T, Quan X, Zikihara K, Katsura H, Tokutomi S, Aboshi T, Mori N, Inoue H, Nishida H, Kohchi T, Teraishi M, Okumoto Y, Tanisaka T (2015) The effects of phytochrome-mediated light signals on the developmental acquisition of photoperiod sensitivity in rice. Sci Rep 5:7709

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Young SK, Wek RC (2016) Upstream open reading frames differentially regulate gene-specific translation in the integrated stress response. J Biol Chem 291:16927–16935

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Yuan T, Li X, Xiao J, Wang S (2011) Characterization of xanthomonas oryzae-responsive cis-acting element in the promoter of rice race-specific susceptibility gene xa13. Mol Plant 4:300–309

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Zhang A, Liu Y, Wang F, Li T, Chen Z, Kong D, Bi J, Zhang F, Luo X, Wang J, Tang J, Yu X, Liu G, Luo L (2019) Enhanced rice salinity tolerance via CRISPR/Cas9-targeted mutagenesis of the OsRR22 gene. Mol Breed 39:47

    Article  PubMed  CAS  Google Scholar 

  • Zhao J, Chen H, Ren D, Tang H, Qiu R, Feng J, Long Y, Niu B, Chen D, Zhong T, Liu YG, Guo J (2015) Genetic interactions between diverged alleles of Early heading date 1 (Ehd1) and Heading date 3a (Hd3a)/RICE FLOWERING LOCUS T1 (RFT1) control differential heading and contribute to regional adaptation in rice (Oryza sativa). New Phytol 208:936–948

    Article  PubMed  CAS  Google Scholar 

  • Zhao Z, Xie X, Liu W, Huang J, Tan J, Yu H, Zong W, Tang J, Zhao Y, Xue Y, Chu Z, Chen L, Liu YG (2022) STI PCR: An efficient method for amplification and de novo synthesis of long DNA sequences. Mol Plant 15:620–629

    Article  PubMed  CAS  Google Scholar 

  • Zhou J, Liu G, Zhao Y, Zhang R, Tang X, Li L, Jia X, Guo Y, Wu Y, Han Y, Bao Y, He Y, Han Q, Yang H, Zheng X, Qi Y, Zhang T, Zhang Y (2023) An efficient CRISPR-Cas12a promoter editing system for crop improvement. Nature Plants 9:588–604

    Article  PubMed  CAS  Google Scholar 

  • Zong W, Ren D, Huang M, Sun K, Feng J, Zhao J, Xiao D, Xie W, Liu S, Zhang H, Qiu R, Tang W, Yang R, Chen H, Xie X, Chen L, Liu YG, Guo J (2021) Strong photoperiod sensitivity is controlled by cooperation and competition among Hd1, Ghd7 and DTH8 in rice heading. New Phytol 229:1635–1649

    Article  PubMed  CAS  Google Scholar 

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Funding

This research was supported by grants from the National Nature Science Foundation of China (31871596, 31921004), the Major Program of Guangdong Basic and Applied Research (2019B030302006), and the Laboratory of Lingnan Modern Agriculture Project (NT2021002).

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  1. Shengting Li and Yanqiu Luo have contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory for Conservation and Utilization of Subtropical Agro-BioresourcesGuangdong Laboratory for Lingnan Modern AgricultureCollege of Life Sciences, South China Agricultural University, Guangzhou, 510642, China

    Shengting Li, Yanqiu Luo, Guangliang Wei, Wubei Zong, Wanyong Zeng, Dongdong Xiao, Han Zhang, Yingang Song, Yu Hao, Kangli Sun, Chen Lei, Xiaotong Guo, Bingqun Xu, Weitao Li, Zeqiang Wu, Yaoguang Liu, Xianrong Xie & Jingxin Guo

  2. Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Maoming, 525000, China

    Yanqiu Luo

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  1. Shengting Li
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  2. Yanqiu Luo
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Contributions

JG, XX conceived the work, analyzed the data, and revised the manuscript. SL, YL, and GW performed the experiments and prepared the manuscript. WZ, DX, HZ, YS, and YH helped with the experiments. WZ helped analyze some of the data. KS, CL, XG, BX, WL and ZW helped investigate the phenotypes. JG, XX, and YL revised the manuscript.

Corresponding authors

Correspondence to Xianrong Xie or Jingxin Guo.

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Li, S., Luo, Y., Wei, G. et al. Improving yield-related traits by editing the promoter of the heading date gene Ehd1 in rice. Theor Appl Genet 136, 239 (2023). https://doi.org/10.1007/s00122-023-04489-6

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