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A photoregulatory mechanism of the circadian clock in Arabidopsis

Nature Plants volume 7, pages 1397–1408 (2021)Cite this article

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A Publisher Correction to this article was published on 22 October 2021

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Abstract

Cryptochromes (CRYs) are photoreceptors that mediate light regulation of the circadian clock in plants and animals. Here we show that CRYs mediate blue-light regulation of N6-methyladenosine (m6A) modification of more than 10% of messenger RNAs in the Arabidopsis transcriptome, especially those regulated by the circadian clock. CRY2 interacts with three subunits of the METTL3/14-type N6-methyladenosine RNA methyltransferase (m6A writer): MTA, MTB and FIP37. Photo-excited CRY2 undergoes liquid–liquid phase separation (LLPS) to co-condense m6A writer proteins in vivo, without obviously altering the affinity between CRY2 and the writer proteins. mta and cry1cry2 mutants share common defects of a lengthened circadian period, reduced m6A RNA methylation and accelerated degradation of mRNA encoding the core component of the molecular oscillator circadian clock associated 1 (CCA1). These results argue for a photoregulatory mechanism by which light-induced phase separation of CRYs modulates m6A writer activity, mRNA methylation and abundance, and the circadian rhythms in plants.

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Fig. 1: CRYs mediate blue-light regulation of epitranscriptomes.
Fig. 2: Multivalent interaction of the CRY2/writer complex.
Fig. 3: Blue light elicits LLPS of CRY2.
Fig. 4: CRYs and MTA regulate CCA1 mRNA metabolism and the circadian clock.

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Data availability

All data supporting the findings of this study are available in the main text or the supplementary table. Biological materials used in this study are available from C.L. on reasonable request. The m6A-seq and RNA-seq data reported in this study have been deposited in the NCBI Gene Expression Omnibus under accession number GSE152466 which is fully available. The RNA-seq data for WT and cry1cry2 sample in the dark condition have been reported previously (GSE80350)26. Source data are provided with this paper.

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References

  1. Sanchez, S. E., Rugnone, M. L. & Kay, S. A. Light perception: a matter of time. Mol. Plant 13, 363–385 (2020).

    Article  CAS  PubMed  Google Scholar 

  2. Patke, A., Young, M. W. & Axelrod, S. Molecular mechanisms and physiological importance of circadian rhythms. Nat. Rev. Mol. Cell Biol. 21, 67–84 (2020).

    Article  CAS  PubMed  Google Scholar 

  3. Webb, A. A. R., Seki, M., Satake, A. & Caldana, C. Continuous dynamic adjustment of the plant circadian oscillator. Nat. Commun. 10, 550 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Wang, Q. & Lin, C. Mechanisms of cryptochrome-mediated photoresponses in plants. Annu. Rev. Plant Biol. 71, 103–129 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Cashmore, A. R. Cryptochromes: enabling plants and animals to determine circadian time. Cell 114, 537–543 (2003).

    CAS  PubMed  Google Scholar 

  6. Sancar, A. Cryptochrome: the second photoactive pigment in the eye and its role in circadian photoreception. Annu. Rev. Biochem. 69, 31–67 (2000).

    Article  CAS  PubMed  Google Scholar 

  7. Bailey-Serres, J., Zhai, J. & Seki, M. The dynamic kaleidoscope of RNA biology in plants. Plant Physiol. 182, 1–9 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Parker, M. T. et al. Nanopore direct RNA sequencing maps the complexity of Arabidopsis mRNA processing and m(6)A modification. eLife; https://doi.org/10.7554/eLife.49658 (2020).

  9. Wang, C. Y., Yeh, J. K., Shie, S. S., Hsieh, I. C. & Wen, M. S. Circadian rhythm of RNA N6-methyladenosine and the role of cryptochrome. Biochem. Biophys. Res. Commun. 465, 88–94 (2015).

    Article  CAS  PubMed  Google Scholar 

  10. Fustin, J. M. et al. RNA-methylation-dependent RNA processing controls the speed of the circadian clock. Cell 155, 793–806 (2013).

    Article  CAS  PubMed  Google Scholar 

  11. Anderson, S. J. et al. N6-Methyladenosine inhibits local ribonucleolytic cleavage to stabilize mRNAs in Arabidopsis. Cell Rep. 25, 1146–1157 e1143 (2018).

    Article  CAS  PubMed  Google Scholar 

  12. Wang, X. et al. N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505, 117–120 (2014).

    Article  PubMed  CAS  Google Scholar 

  13. Zaccara, S., Ries, R. J. & Jaffrey, S. R. Reading, writing and erasing mRNA methylation. Nat. Rev. Mol. Cell Biol. 20, 608–624 (2019).

    Article  CAS  PubMed  Google Scholar 

  14. Shen, L., Liang, Z., Wong, C. E. & Yu, H. Messenger RNA modifications in plants. Trends Plant Sci. 24, 328–341 (2019).

    Article  CAS  PubMed  Google Scholar 

  15. Zhao, B. S., Roundtree, I. A. & He, C. Post-transcriptional gene regulation by mRNA modifications. Nat. Rev. Mol. Cell Biol. 18, 31–42 (2017).

    Article  CAS  PubMed  Google Scholar 

  16. Dominissini, D., Moshitch-Moshkovitz, S., Salmon-Divon, M., Amariglio, N. & Rechavi, G. Transcriptome-wide mapping of N(6)-methyladenosine by m(6)A-seq based on immunocapturing and massively parallel sequencing. Nat. Protoc. 8, 176–189 (2013).

    Article  CAS  PubMed  Google Scholar 

  17. Wang, Z. Y. & Tobin, E. M. Constitutive expression of the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene disrupts circadian rhythms and suppresses its own expression. Cell 93, 1207–1217 (1998).

    Article  CAS  PubMed  Google Scholar 

  18. Liu, Q. et al. Molecular basis for blue light-dependent phosphorylation of Arabidopsis cryptochrome 2. Nat. Commun. 8, 15234 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Arribas-Hernandez, L. & Brodersen, P. Occurrence and functions of m(6)A and other covalent modifications in plant mRNA. Plant Physiol. 182, 79–96 (2020).

    Article  CAS  PubMed  Google Scholar 

  20. Ruzicka, K. et al. Identification of factors required for m6A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI. New Phytol. 215, 157–172 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Zhong, S. et al. MTA is an Arabidopsis messenger RNA adenosine methylase and interacts with a homolog of a sex-specific splicing factor. Plant Cell 20, 1278–1288 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Chen, Y. et al. Regulation of Arabidopsis photoreceptor CRY2 by two distinct E3 ubiquitin ligases. Nat. Commun. 12, 2155 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Yu, X. et al. Arabidopsis cryptochrome 2 completes its posttranslational life cycle in the nucleus. Plant Cell 19, 3146–3156 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Liu, Q. et al. Photooligomerization determines photosensitivity and photoreactivity of plant cryptochromes. Mol. Plant 13, 398–413 (2020).

    Article  CAS  PubMed  Google Scholar 

  25. Liu, H. et al. Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in Arabidopsis. Science 322, 1535–1539 (2008).

    Article  CAS  PubMed  Google Scholar 

  26. Wang, Q. et al. Photoactivation and inactivation of Arabidopsis cryptochrome 2. Science 354, 343–347 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Yu, X. et al. Formation of nuclear bodies of Arabidopsis CRY2 in response to blue light is associated with its blue light-dependent degradation. Plant Cell 21, 118–130 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Mas, P., Devlin, P. F., Panda, S. & Kay, S. A. Functional interaction of phytochrome B and cryptochrome 2. Nature 408, 207–211 (2000).

    Article  CAS  PubMed  Google Scholar 

  29. Sabari, B. R. et al. Coactivator condensation at super-enhancers links phase separation and gene control. Science https://doi.org/10.1126/science.aar3958 (2018).

  30. Bracha, D. et al. Mapping local and global liquid phase behavior in living cells using photo-oligomerizable seeds. Cell 175, 1467–1480 e1413 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Shin, Y. et al. Spatiotemporal control of intracellular phase transitions using light-activated optodroplets. Cell 168, 159–171 e114 (2017).

    Article  CAS  PubMed  Google Scholar 

  32. Dignon, G. L., Best, R. B. & Mittal, J. Biomolecular phase separation: from molecular driving forces to macroscopic properties. Annu. Rev. Phys. Chem. 71, 53–75 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Alberti, S., Gladfelter, A. & Mittag, T. Considerations and challenges in studying liquid–liquid phase separation and biomolecular condensates. Cell 176, 419–434 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Wang, Q. et al. The blue light-dependent phosphorylation of the CCE domain determines the photosensitivity of Arabidopsis CRY2. Mol. Plant 8, 631–643 (2015).

    Article  CAS  PubMed  Google Scholar 

  35. Shalitin, D. et al. Regulation of Arabidopsis cryptochrome 2 by blue-light-dependent phosphorylation. Nature 417, 763–767 (2002).

    Article  CAS  PubMed  Google Scholar 

  36. Devlin, P. F. & Kay, S. A. Cryptochromes are required for phytochrome signaling to the circadian clock but not for rhythmicity. Plant Cell 12, 2499–2510 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Somers, D. E., Devlin, P. F. & Kay, S. A. Phytochromes and cryptochromes in the entrainment of the Arabidopsis circadian clock. Science 282, 1488–1490 (1998).

    Article  CAS  PubMed  Google Scholar 

  38. Bodi, Z. et al. Adenosine methylation in Arabidopsis mRNA is associated with the 3ʹ end and reduced levels cause developmental defects. Front. Plant Sci. 3, 48 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Guo, H., Yang, H., Mockler, T. C. & Lin, C. Regulation of flowering time by Arabidopsis photoreceptors. Science 279, 1360–1363 (1998).

    Article  CAS  PubMed  Google Scholar 

  40. Legris, M., Ince, Y. C. & Fankhauser, C. Molecular mechanisms underlying phytochrome-controlled morphogenesis in plants. Nat. Commun. 10, 5219 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Leivar, P. & Quail, P. H. PIFs: pivotal components in a cellular signaling hub. Trends Plant Sci. 16, 19–28 (2011).

    Article  CAS  PubMed  Google Scholar 

  42. Bhat, S. S. et al. mRNA adenosine methylase (MTA) deposits m(6)A on pri-miRNAs to modulate miRNA biogenesis in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 117, 21785–21795 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Aschoff, J. Exogenous and endogenous components in circadian rhythms. Cold Spring Harb. Symp. Quant. Biol. 25, 11–28 (1960).

    Article  CAS  PubMed  Google Scholar 

  44. Jang, G. J., Yang, J. Y., Hsieh, H. L. & Wu, S. H. Processing bodies control the selective translation for optimal development of Arabidopsis young seedlings. Proc. Natl Acad. Sci. USA 116, 6451–6456 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Godoy Herz, M. A. et al. Light regulates plant alternative splicing through the control of transcriptional elongation. Mol. Cell 73, 1066–1074 e1063 (2019).

    Article  CAS  PubMed  Google Scholar 

  46. Wu, S. H. Gene expression regulation in photomorphogenesis from the perspective of the central dogma. Annu. Rev. Plant Biol. 65, 311–333 (2014).

    Article  CAS  PubMed  Google Scholar 

  47. Paik, I., Yang, S. & Choi, G. Phytochrome regulates translation of mRNA in the cytosol. Proc. Natl Acad. Sci. USA 109, 1335–1340 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Juntawong, P. & Bailey-Serres, J. Dynamic light regulation of translation status in Arabidopsis thaliana. Front. Plant Sci. 3, 66 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Wang, X. et al. SKIP is a component of the spliceosome linking alternative splicing and the circadian clock in Arabidopsis. Plant Cell 24, 3278–3295 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Mockler, T. C., Guo, H., Yang, H., Duong, H. & Lin, C. Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in the regulation of floral induction. Development 126, 2073–2082 (1999).

    Article  CAS  PubMed  Google Scholar 

  51. Harmoko, R. et al. RNA-dependent RNA polymerase 6 is required for efficient hpRNA-induced gene silencing in plants. Mol. Cells 35, 202–209 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Clough, S. J. Floral dip: agrobacterium-mediated germ line transformation. Methods Mol. Biol. 286, 91–102 (2005).

    CAS  PubMed  Google Scholar 

  53. Wu, F. H. et al. Tape-Arabidopsis Sandwich – a simpler Arabidopsis protoplast isolation method. Plant Methods 5, 16 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

  55. Fang, X. et al. Arabidopsis FLL2 promotes liquid–liquid phase separation of polyadenylation complexes. Nature 569, 265–269 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Rapsomaniki, M. A. et al. easyFRAP: an interactive, easy-to-use tool for qualitative and quantitative analysis of FRAP data. Bioinformatics 28, 1800–1801 (2012).

    Article  CAS  PubMed  Google Scholar 

  57. Tseng, Q. et al. A new micropatterning method of soft substrates reveals that different tumorigenic signals can promote or reduce cell contraction levels. Lab Chip 11, 2231–2240 (2011).

    Article  CAS  PubMed  Google Scholar 

  58. Lund, F. W. et al. SpatTrack: an imaging toolbox for analysis of vesicle motility and distribution in living cells. Traffic 15, 1406–1429 (2014).

    Article  CAS  PubMed  Google Scholar 

  59. Ries, R. J. et al. m(6)A enhances the phase separation potential of mRNA. Nature 571, 424–428 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Zielinski, T., Moore, A. M., Troup, E., Halliday, K. J. & Millar, A. J. Strengths and limitations of period estimation methods for circadian data. PLoS ONE 9, e96462 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  61. Moore, A., Zielinski, T. & Millar, A. J. in Plant Circadian Networks: Methods and Protocols (ed. Staiger, D.) 13–44 (Springer, 2014).

  62. Hutchison, A. L. et al. Improved statistical methods enable greater sensitivity in rhythm detection for genome-wide data. PLoS Comput. Biol. 11, e1004094 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  63. Chantarachot, T. et al. DHH1/DDX6-like RNA helicases maintain ephemeral half-lives of stress-response mRNAs. Nat. Plants 6, 675–685 (2020).

    Article  CAS  PubMed  Google Scholar 

  64. Sorenson, R. S., Deshotel, M. J., Johnson, K., Adler, F. R. & Sieburth, L. E. Arabidopsis mRNA decay landscape arises from specialized RNA decay substrates, decapping-mediated feedback, and redundancy. Proc. Natl Acad. Sci. USA 115, E1485–E1494 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Pfaffl, M. W. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res. 29, e45 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Trcek, T., Larson, D. R., Moldon, A., Query, C. C. & Singer, R. H. Single-molecule mRNA decay measurements reveal promoter-regulated mRNA stability in yeast. Cell 147, 1484–1497 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  68. Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5, 621–628 (2008).

    Article  CAS  PubMed  Google Scholar 

  69. Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Luo, G. Z. et al. Unique features of the m6A methylome in Arabidopsis thaliana. Nat. Commun. 5, 5630 (2014).

    Article  CAS  PubMed  Google Scholar 

  71. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  72. Yang, Y., Li, Y., Sancar, A. & Oztas, O. The circadian clock shapes the Arabidopsis transcriptome by regulating alternative splicing and alternative polyadenylation. J. Biol. Chem. 295, 7608–7619 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Romanowski, A., Schlaen, R. G., Perez-Santangelo, S., Mancini, E. & Yanovsky, M. J. Global transcriptome analysis reveals circadian control of splicing events in Arabidopsis thaliana. Plant J. 103, 889–902 (2020).

    Article  CAS  PubMed  Google Scholar 

  74. Li, S. et al. CGDB: a database of circadian genes in eukaryotes. Nucleic Acids Res. 45, D397–D403 (2017).

    CAS  PubMed  Google Scholar 

  75. Covington, M. F., Maloof, J. N., Straume, M., Kay, S. A. & Harmer, S. L. Global transcriptome analysis reveals circadian regulation of key pathways in plant growth and development. Genome Biol. 9, R130 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Acknowledgements

We thank J. Bailey-Serres for research discussions and manuscript editing, R. G. Fray for providing the ABI3::MTA/mta line, R. McClung and X. Xu for providing pCCA1::LUC and pTOC1::LUC reporter constructs and reporter lines, the UCLA-FAFU Joint Research Center on Plant Proteomics and the UCLA-MCDB/BSCRC Microscopy Core for institutional support. Works in the authors’ laboratories are supported in part by the National Natural Science Foundation of China (31970265 to Q.W.), Natural Science Foundation of Fujian Province (2019J06014 to Q.W.), the National Institutes of Health (R01GM056265 to C.L.) and UCLA Sol Leshin Programme (to C.L.).

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  1. These authors contributed equally: Xu Wang, Bochen Jiang, Lianfeng Gu.

Authors and Affiliations

  1. Basic Forestry and Proteomics Research Center, Fujian Agriculture and Forestry University, Fuzhou, China

    Xu Wang, Lianfeng Gu, Yadi Chen & Qin Wang

  2. Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA, USA

    Xu Wang, Bochen Jiang, Manuel Mora, Mulangma Zhu, Eliace Noory & Chentao Lin

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Contributions

C.L. and X.W. designed the study and wrote the paper. L.G. performed bioinformatics analyses. X.W., B.C., Y.C., M.M., M.Z., E.N. and Q.W. performed the experiments and analysed the data. Q.W. contributed materials/analysis tools.

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Correspondence to Qin Wang or Chentao Lin.

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Extended data

Extended Data Fig. 1 CRY-mediated photoresponsive epitranscriptomic changes in plants.

a, The heatmaps showing relative m6A abundance for all genes (epitranscriptome, top) or clock-controlled genes (CCG, bottom) in wild type (WT) and cry1cry2 mutants grown in the dark (D) or blue light conditions (B). b, Genomic visualization of m6A density maps of core clock genes detected in m6A-seq. CRY1 and CRY2 detected only in WT but not cry1cry2 samples are shown as the negative control. c, Hierarchical clustering analysis of different m6A-seq samples indicates no unusual sample variances. d, m6A abundance of individual sites of selected genes was analyzed by m6A-IP-qPCR. The relative m6A level in each gene was calculated by normalizing the m6A-IP to input signals. Data are shown as Mean ± SD from 3 independent experiments. D, dark; B, blue light (30 μmol m-2 s-1).

Source data

Extended Data Fig. 2 Epitranscriptomes of selected GO group of genes.

a–j, The scatter plots showing photoresponsive changes of m6A abundance and RNA abundance (left), and metagene profiles showing photoresponsive changes of m6A density (right) of mRNAs categorized as the TAIR10 GO groups.

Extended Data Fig. 3 CRYs interacts with subunits of m6A mRNA methyltransferase in human cells.

a-c, co-IP assays showing interactions between CRY2 and MTA (a), MTB (b) and FIP37 (c) in HEK293T cells which were illuminated with blue light (BL; 100 μmol m-2 s-1) for indicated time (0-60 min). d, co-IP assay showing the interaction of CRY2D387A mutant and MTA in HEK293T cells with or without blue light irradiation. e, co-IP assay showing interactions between MTA and CRY1 in HEK293T cells. BL, blue light (100 μmol m-2 s-1). Three independent experiments are performed for a, two independent experiments for b-e, showing similar results.

Source data

Extended Data Fig. 4 Photoresponsive condensation of the CRY2-MTA complex in Arabidopsis.

a, Time-lapse images showing the partial colocalization of MTA and CRY2 in CRY2 photobodies over the time of blue laser illumination in Arabidopsis protoplasts. Scale bar= 2 μm. Two independent experiments are performed showing similar results. b, Quantification of partition coefficient of CRY2-YFP and MTA-DsRed in the assay shown in (a). The data is presented as mean ± SD (n = 15 independent measurements from 5 nuclei). c, Fluorescence profiles of CRY2-YFP and MTA-DsRed over the white line shown in (a). The arrow heads indicate the locations of photobodies. d, Quantification of partition coefficient of CRY2D387A-YFP and MTA-DsRed in the assay shown in (a). The data is presented as mean ± SD (n = 15 independent measurements from 5 nuclei). e, Fluorescence profiles of CRY2D387A-YFP and MTA-DsRed over the white line shown in (a). f, FRAP of CRY2/MTA BiFC photobodies in protoplasts. White circle indicates the region for photobleaching. Quantification of FRAP is shown in Fig. 2m. Scale bar= 2 μm. g, Negative controls of BiFC assays. Plasmids expressing mRFP are used to monitor protoplasts transformation efficiency. The arrow in the cell indicates the location of nucleus. Scale bar = 5μm. Two independent experiments are performed showing similar results. h, m6A-IP-qPCR assay showing the relative abundance of m6A on the 3’UTR of CCA1 mRNA in etiolated wild-type seedlings treated with mock (H2O) or 1,6-hexanediol (10%) in response to blue light. The data is shown as Mean ± SD (n = 6 technical replicates from 2 biological repeats). i, m6A-IP-qPCR assay showing the relative abundance of m6A on the 3’UTR of CCA1 mRNA in wild-type (WT) and indicated genotypes grown in the Dark or Blue light (30 µmol m-2 s-1). The data is shown as Mean ± SD (n = 6 technical replicates from 2 biological repeats).

Source data

Extended Data Fig. 5 LLPS of CRY2 in plant cells.

a, FRAP of CRY2 photobodies in CRY2-GFP transgenic plants, the quantification of FRAP is shown in Fig. 3c. Scale bar = 2μm. b, Time-lapse images showing light-elicited formation of CRY2-YFP photobodies in protoplasts. Light-insensitive CRY2D387A-YFP was used as the negative control. Quantification of partition coefficient for both CRY2-YFP and CRY2D387A-YFP fluorescence is shown in Fig. 3e. Scale bar = 2μm. c, FRAP of CRY2-YFP photobodies in Arabidopsis protoplasts. Protoplasts transfected with CRY2-YFP were illuminated with 488 nm laser for 30 s before FRAP. Time-lapse images before and after FRAP are shown on the left, white arrow heads point at the photobodies for photobleaching. Quantification of FRAP assay is shown on the right. Double exponential fit (the dark line) of normalized and averaged recovery curves is shown (n = 5 independent experiments; Mean ± SD). Scale bar = 2μm. d, Representative motion trajectories of CRY2-GFP photobodies in the live or fixed plant nucleus. The time-lapse images were taken every 3 s for 10 min. The dashed lines in the images are the outlines of the nucleus. Scale bar= 5 μm. e, The ensemble-averaged MSD (Mean Square Displacement) curves of tracked CRY2-GFP photobodies (as shown in d) in live or fixed samples. Shaded areas represent standard error (SE) of the mean (n = 4 and 3 independent cells for the live and fixed sample, respectively). Fitting of the MSD for live sample to the anomalous diffusion model yields the Diffusion constant (D), anomalous exponent (α; α < 1 indicates the subdiffusive motion of the particles) and the best fitted curve (black line) which are shown in the graph.

Source data

Extended Data Fig. 6 Wavelength specificity of LLPS of CRY2-DsRed in Arabidopsis protoplasts.

a, Time-lapse images showing CRY2-DsRed distribution pattern in the nucleus under different wavelengths in protoplasts. Protoplasts transiently expressing CRY2-DsRed were kept in the dark and illuminated with different lasers during imaging. Scale bar=2 µm. b, Quantification of PC of CRY2-DsRed under different wavelengths (n = 16 measurements for each time point from 3 nuclei; in each box, the center line denotes median value; boxes extend from the 25th to the 75th percentile of each group’s distribution of values; vertical extending lines denote the range from minima to maxima; the open circles denote the values ranging from minima to maxima).

Source data

Extended Data Fig. 7 CRY2 photobodies are reversible in response to light-dark cycles in Arabidopsis protoplasts.

Time-lapse images of CRY2-DsRed expressed in protoplasts. Blue triangle indicates that 488 nm laser was on during imaging. Quantification of this experiment is shown in Fig. 3d. Scale bar=2 µm.

Extended Data Fig. 8 PPK1-dependent phosphorylation is essential to maintain CRY2 photobodies in liquid phase.

a, Representative images showing the FRAP of CRY2 photobody in HEK293T cells with or without co-expression of PPK1 or PPK1D267N. The quantification of FRAP assays is shown in Fig. 3j. The dashed circle outlines the nucleus and the small solid circle inside the nucleus indicates the region for photobleaching. Scale bar = 2μm. b, The mobile fraction of CRY2 photobodies at 57 s after photobleaching (mean ± SD; n = 5 independent experiments). p value is calculated in two-tailed Students’ t-test.

Source data

Extended Data Fig. 9 C-terminus of CRY2 is necessary to maintain the photobodies in liquid phase in both human cells and Arabidopsis protoplasts.

a, Representative images showing the FRAP of CRY2, CRY2N509 (N509) and CRY2N489 (N489) photobodies in the presence of PPK1 in HEK293T cells. The dashed circle outlines the nucleus and the small solid circle inside the nucleus indicates the region for photobleaching. The quantification of FRAP assays is shown in Fig. 3m. Scale bar = 2μm. b, The mobile fraction of CRY2, N509 and N489 photobodies at 57 s after photobleaching in HEK293T cells (mean ± SD; n = 5 independent experiments). p value is calculated in two-tailed Students’ t-test. c, Images showing the FRAP of CRY2, N509 and N489 photobodies in Arabidopsis protoplasts. The circle inside the nucleus indicates the region for photobleaching. The quantification of FRAP assays is shown in Fig. 3n. Scale bar = 2μm. d, The mobile fraction of CRY2, N509 and N489 photobodies at 57 s after photobleaching in protoplasts (mean ± SD; n = 6 independent experiments). p value is calculated in two-tailed Students’ t-test.

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Extended Data Fig. 10 Genotyping of ABI3::MTA/mta plants.

a, The schematic diagram of the T-DNA insertion mta mutant (SALK_074069). The exons (boxes), introns (lines) of the MTA gene and the T-DNA insert (opened triangle) are shown. The positions of primers used for genotyping the mutant locus are marked on MTA gene structure. The positions of RP (right primer) or LP (left primer) of the MTA gene, and primer LBb1.3 in the left border of T-DNA insert are indicated. b, Representative image of WT, mta (short for ABI3::MTA/mta) and cry1cry2 plants grown in long day conditions for 50 days. c, Genomic PCR of the MTA locus in WT and ABI3::MTA/mta plants using genomic DNA as the templates. The sizes of the PCR products were 965 bp (amplified with LP + RP) or 444-744 bp (amplified with LBb1.3+RP). Four plants (#1-#4) were randomly selected from a ABI3::MTA/mta population for genotyping. M, DNA marker.

Supplementary information

Reporting Summary

Supplementary Video 1

CRY2-YFP photobodies formation under blue light in the nucleus of Arabidopsis protoplast.

Supplementary Video 2

FRAP of CRY2-GFP photobodies in the nucleus of transgenic Arabidopsis plants.

Supplementary Video 3

Dynamics of CRY2-DsRed photobodies in response to light/dark cycle in the nucleus of Arabidopsis protoplast.

Supplementary Video 4

Movement of CRY2-GFP photobodies in the nucleus of transgenic Arabidopsis plants.

Supplementary Table 1

Epitranscriptome and transcriptome data.

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Wang, X., Jiang, B., Gu, L. et al. A photoregulatory mechanism of the circadian clock in Arabidopsis. Nat. Plants 7, 1397–1408 (2021). https://doi.org/10.1038/s41477-021-01002-z

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