Abstract
N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic mRNAs, yet how plants recognize this chemical modification to swiftly adjust developmental plasticity under environmental stresses remains unclear. Here we show that m6A mRNA modification and its reader protein EVOLUTIONARILY CONSERVED C-TERMINAL REGION 8 (ECT8) act together as a key checkpoint for negative feedback regulation of abscisic acid (ABA) signalling by sequestering the m6A-modified ABA receptor gene PYRABACTIN RESISTANCE 1-LIKE 7 (PYL7) via phase-separated ECT8 condensates in stress granules in response to ABA. This partially depletes PYL7 mRNA from its translation in the cytoplasm, thus reducing PYL7 protein levels and compromising ABA perception. The loss of ECT8 results in defective sequestration of m6A-modified PYL7 in stress granules and permits more PYL7 transcripts for translation. This causes overactivation of ABA-responsive genes and the consequent ABA-hypersensitive phenotypes, including drought tolerance. Overall, our findings reveal that m6A-mediated sequestration of PYL7 by ECT8 in stress granules negatively regulates ABA perception, thereby enabling prompt feedback regulation of ABA signalling to prevent plant cell overreaction to environmental stresses.
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Data availability
The nanopore DRS data described in this Article have been deposited in the NCBI Sequence Read Archive database with the accession number PRJNA974737. The Arabidopsis reference transcriptome (TAIR10) was used for gene annotation and obtained from the Arabidopsis Information Resource (http://www.arabidopsis.org/). All remaining data are in the main paper or the supplementary materials. Source data are provided with this paper.
References
Zaccara, S., Ries, R. J. & Jaffrey, S. R. Reading, writing and erasing mRNA methylation. Nat. Rev. Mol. Cell Biol. 20, 608–624 (2019).
Sharma, B., Prall, W., Bhatia, G. & Gregory, B. D. The diversity and functions of plant RNA modifications: what we know and where we go from here. Annu. Rev. Plant Biol. 74, 53–85 (2023).
Shen, L., Ma, J., Li, P., Wu, Y. & Yu, H. Recent advances in the plant epitranscriptome. Genome Biol. 24, 43 (2023).
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).
Ruzicka, K. et al. Identification of factors required for m6A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI. N. Phytol. 215, 157–172 (2017).
Shen, L. et al. N6-methyladenosine RNA modification regulates shoot stem cell fate in Arabidopsis. Dev. Cell 38, 186–200 (2016).
Shen, L. Functional interdependence of m6A methyltransferase complex subunits in Arabidopsis. Plant Cell 35, 1901–1916 (2023).
Wong, C. E. et al. Shaping the landscape of N6-methyladenosine RNA methylation in Arabidopsis. Plant Physiol. 191, 2045–2063 (2023).
Xu, T. et al. FIONA1-mediated m6A modification regulates the floral transition in Arabidopsis. Adv. Sci. 9, e2103628 (2022).
Wang, C. et al. FIONA1 is an RNA N6-methyladenosine methyltransferase affecting Arabidopsis photomorphogenesis and flowering. Genome Biol. 23, 40 (2022).
Duan, H. C. et al. ALKBH10B is an RNA N6-methyladenosine demethylase affecting Arabidopsis floral transition. Plant Cell 29, 2995–3011 (2017).
Martinez-Perez, M. et al. Arabidopsis m6A demethylase activity modulates viral infection of a plant virus and the m6A abundance in its genomic RNAs. Proc. Natl Acad. Sci. USA 114, 10755–10760 (2017).
Wei, L. H. et al. The m6A reader ECT2 controls trichome morphology by affecting mRNA stability in Arabidopsis. Plant Cell 30, 968–985 (2018).
Scutenaire, J. et al. The YTH domain protein ECT2 is an m6A reader required for normal trichome branching in Arabidopsis. Plant Cell 30, 986–1005 (2018).
Arribas-Hernandez, L. et al. An m6A–YTH module controls developmental timing and morphogenesis in Arabidopsis. Plant Cell 30, 952–967 (2018).
Song, P. et al. Arabidopsis N6-methyladenosine reader CPSF30-L recognizes FUE signals to control polyadenylation site choice in liquid-like nuclear bodies. Mol. Plant 14, 571–587 (2021).
Hou, Y. et al. CPSF30-L-mediated recognition of mRNA m6A modification controls alternative polyadenylation of nitrate signaling-related gene transcripts in Arabidopsis. Mol. Plant 14, 688–699 (2021).
Arribas-Hernandez, L. et al. Principles of mRNA targeting via the Arabidopsis m6A-binding protein ECT2. Elife 10, e72375 (2021).
Arribas-Hernandez, L. et al. The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation. ELife 10, e72377 (2021).
Shao, Y., Wong, C. E., Shen, L. & Yu, H. N6-methyladenosine modification underlies messenger RNA metabolism and plant development. Curr. Opin. Plant Biol. 63, 102047 (2021).
Yu, Q. et al. RNA demethylation increases the yield and biomass of rice and potato plants in field trials. Nat. Biotechnol. 39, 1581–1588 (2021).
Shen, L. & Yu, H. Epitranscriptome engineering in crop improvement. Mol. Plant 14, 1418–1420 (2021).
Tang, J., Yang, J., Duan, H. & Jia, G. ALKBH10B, an mRNA m6A demethylase, modulates ABA response during seed germination in Arabidopsis. Front. Plant Sci. 12, 712713 (2021).
Tang, J. et al. The RNA N6-methyladenosine demethylase ALKBH9B modulates ABA responses in Arabidopsis. J. Integr. Plant Biol. 64, 2361–2373 (2022).
Zhu, J. K. Salt and drought stress signal transduction in plants. Annu. Rev. Plant Biol. 53, 247–273 (2002).
Raghavendra, A. S., Gonugunta, V. K., Christmann, A. & Grill, E. ABA perception and signalling. Trends Plant Sci. 15, 395–401 (2010).
Park, S. Y. et al. Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science 324, 1068–1071 (2009).
Chen, K. et al. Abscisic acid dynamics, signaling, and functions in plants. J. Integr. Plant Biol. 62, 25–54 (2020).
Chantarachot, T. & Bailey-Serres, J. Polysomes, stress granules, and processing bodies: a dynamic triumvirate controlling cytoplasmic mRNA fate and function. Plant Physiol. 176, 254–269 (2018).
Maruri-Lopez, I., Figueroa, N. E., Hernandez-Sanchez, I. E. & Chodasiewicz, M. Plant stress granules: trends and beyond. Front. Plant Sci. 12, 722643 (2021).
Buchan, J. R. & Parker, R. Eukaryotic stress granules: the ins and outs of translation. Mol. Cell 36, 932–941 (2009).
Zhang, H. et al.A comprehensive online database for exploring approximately 20,000 public Arabidopsis RNA-seq libraries. Mol. Plant 13, 1231–1233 (2020).
Waadt, R. et al. Plant hormone regulation of abiotic stress responses. Nat. Rev. Mol. Cell Biol. 23, 680–694 (2022).
Chong, P. A. & Forman-Kay, J. D. Liquid–liquid phase separation in cellular signaling systems. Curr. Opin. Struct. Biol. 41, 180–186 (2016).
Meszaros, B., Erdos, G. & Dosztanyi, Z. IUPred2A: context-dependent prediction of protein disorder as a function of redox state and protein binding. Nucleic Acids Res. 46, W329–W337 (2018).
Kosmacz, M. et al. Protein and metabolite composition of Arabidopsis stress granules. N. Phytol. 222, 1420–1433 (2019).
Ries, R. J. et al. m6A enhances the phase separation potential of mRNA. Nature 571, 424–428 (2019).
Fu, Y. & Zhuang, X. m6A-binding YTHDF proteins promote stress granule formation. Nat. Chem. Biol. 16, 955–963 (2020).
Pratanwanich, P. N. et al. Identification of differential RNA modifications from nanopore direct RNA sequencing with xPore. Nat. Biotechnol. 39, 1394–1402 (2021).
Zhao, Y. et al. ABA receptor PYL9 promotes drought resistance and leaf senescence. Proc. Natl Acad. Sci. USA 113, 1949–1954 (2016).
Protter, D. S. W. & Parker, R. Principles and properties of stress granules. Trends Cell Biol. 26, 668–679 (2016).
Gonzalez-Guzman, M. et al. Arabidopsis PYR/PYL/RCAR receptors play a major role in quantitative regulation of stomatal aperture and transcriptional response to abscisic acid. Plant Cell 24, 2483–2496 (2012).
Koornneef, M., Reuling, G. & Karssen, C. The isolation and characterization of abscisic acid‐insensitive mutants of Arabidopsis thaliana. Physiol. Plant. 61, 377–383 (1984).
Pan, W. et al. The UBC27–AIRP3 ubiquitination complex modulates ABA signaling by promoting the degradation of ABI1 in Arabidopsis. Proc. Natl Acad. Sci. USA 117, 27694–27702 (2020).
Antoni, R. et al. PYRABACTIN RESISTANCE1-LIKE8 plays an important role for the regulation of abscisic acid signaling in root. Plant Physiol. 161, 931–941 (2013).
Zhang, Y. et al. Phase separation of HRLP regulates flowering time in Arabidopsis. Sci. Adv. 8, eabn5488 (2022).
Ma, X. et al. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol. Plant 8, 1274–1284 (2015).
Chen, Y. et al. SHAGGY-like kinase 12 regulates flowering through mediating CONSTANS stability in Arabidopsis. Sci. Adv. 6, eaaw0413 (2020).
Wu, F. H. et al. Tape-Arabidopsis Sandwich—a simpler Arabidopsis protoplast isolation method. Plant Methods 5, 16 (2009).
Cheng, P. et al. RNA N6-methyladenosine modification promotes auxin biosynthesis required for male meiosis in rice. Dev. Cell 57, 246–259 (2022).
Li, H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34, 3094–3100 (2018).
Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Loman, N. J., Quick, J. & Simpson, J. T. A complete bacterial genome assembled de novo using only nanopore sequencing data. Nat. Methods 12, 733–735 (2015).
Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).
Liao, Y., Smyth, G. K. & Shi, W. The R package Rsubread is easier, faster, cheaper and better for alignment and quantification of RNA sequencing reads. Nucleic Acids Res. 47, e47 (2019).
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
Wu, T. et al. clusterProfiler 4.0: a universal enrichment tool for interpreting omics data. Innovation 2, 100141 (2021).
Supek, F., Bosnjak, M., Skunca, N. & Smuc, T. REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS ONE 6, e21800 (2011).
Sorenson, R. & Bailey-Serres, J. Selective mRNA sequestration by OLIGOURIDYLATE-BINDING PROTEIN 1 contributes to translational control during hypoxia in Arabidopsis. Proc. Natl Acad. Sci. USA 111, 2373–2378 (2014).
Acknowledgements
We thank E. Gutierrez (University of Seville) and P. Bozhkov (Swedish University of Agricultural Sciences) for providing the Rbp47b–GFP plasmid. We thank the Protein and Proteomics Centre in the Department of Biological Sciences, National University of Singapore for the LC–MS/MS service, and the Genome Institute of Singapore, A*STAR for the nanopore DRS service. This work was supported by the National Research Foundation Competitive Research Programme (grant no. NRF-CRP22-2019-0001) and intramural research support from Temasek Life Sciences Laboratory and National University of Singapore.
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X.W., L.S. and H.Y. conceived the study and designed the experiments. X.W., T.S., Y.Z., C.E.W., J.M., Y.S. and C.H. performed the experiments. S.Z. analysed the nanopore DRS data. X.W., L.S. and H.Y. wrote the paper, and all authors commented on the paper.
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Nature Plants thanks Hunseung Kang, Jorge Lozano-Juste 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 Characterization of the m6A reader gene ECT8.
a, Heatmap showing expression levels of m6A reader genes under abiotic stresses from public RNA-seq datasets. b, Analysis of ECT8 expression in pyr1 pyl1,2,4 (left) and abi1-1 (right) mutants. In b, e and j, TUB2 was used as an internal control, and the levels in mock-treated wild-type (WT) (b, j) and WT (e) plants are set as 1.0. Values are means ± s.d. (n = 3 biologically independent replicates). In b, f and i, asterisks indicate statistically significant differences between WT plants and mutants (two-tailed Student’s t-test, P < 0.01). ns, not significant. c, Diagram shows gene structure of ECT8. Black boxes and lines represent exons and introns, respectively. T-DNA insertions are indicated by triangles. d, Full-length ECT8 transcripts are undetectable in ect8 mutants revealed by RT-PCR. TUB2 was amplified as a control. Data are representative of three independent experiments. e, ECT8 expression in various genetic backgrounds. In e, h and j, different letters at the top of each column indicate significant differences at P < 0.05 determined by one-way ANOVA with Tukey’s HSD test. f, Seed germination rates of ect8 mutants under ABA treatment. Values are means ± s.d. (n = 10 biologically independent replicates). g, Analysis of ABA-mediated inhibition of root growth in ect8 mutants. Scale bar, 1 cm. h, Quantification of the primary root length in g. Values are means ± s.d. (n = 15 biologically independent replicates). i, Relative water loss in WT and ect8. Values are means ± s.d. (n = 10 biologically independent replicates). j, Expression levels of EM6 in various genetic backgrounds. k, Analysis of ECT8 expression in young seedlings (Sg), roots (Rt), rosette leaves (RL), cauline leaves (CL), floral buds (FB), open flowers (OF), stems (St) and siliques (Sil). Levels of gene expression are normalized to TUB2 expression, and shown relative to the maximal expression level set as 100%. Values are means ± s.d. (n = 3 biologically independent replicates). l, ECT8 expression patterns in developing pECT8:GUS seedlings revealed by GUS staining. Scale bar, 2.5 mm. The exact P values (b, e, f, h, i and j) are provided in Supplementary Table 10.
Extended Data Fig. 2 Formation of ECT8-GFP condensates under various abiotic stresses.
a, ABA upregulates ECT8 protein levels. Total protein extracted from 7-day-old ect8-1 gECT8-GFP seedlings mock-treated (Mock) or treated with 50 µM ABA for different time periods was examined by Western blot analysis using anti-GFP antibody. Levels of ECT8-GFP protein normalized to RbcL stained with Ponceau S are indicated below the blots and shown relative to the level at 0 h in each blot set as 1.00. Data shown in a, b and d are representative of three independent experiments. b, Subcellular localization of ECT8-GFP in root tip cells of 7-day-old ect8-1 gECT8-GFP seedlings mock-treated or treated with 50 µM ABA, 300 mM mannitol, 300 mM NaCl for 6 h, and by heat stress (37 °C) for 1 h. BF, bright field. Merge, merge of GFP and BF images. The boxed regions in leftmost panels are enlarged and shown as zoom-in images. Scale bars, 10 µm. c, Quantification of the number of ECT8-GFP condensates per cell. Box plots display medians (lines), interquartile ranges (boxes) and whiskers (extending 1.5 × the interquartile ranges) of the number of condensates per cell. Left panel shows the results in root tip cells (b) and protoplasts (d) mock-treated (n = 15) or treated with ABA (n = 15). Right panel shows the results in root tip cells (b) untreated (Control; n = 15) or treated with other stresses (n = 10 each). ND, not detected. d, Subcellular localization of ECT8-GFP in 35 S:ECT8-GFP protoplasts mock-treated and treated with 50 µM ABA. White arrows indicate ECT8-GFP condensates. BF, bright field. DAPI, fluorescence of 4′, 6′-diamino-2-phenylindol. Merge, merge of GFP, DAPI, and BF images. Scale bars, 10 µm. e, FRAP assay of a subregion of an ECT8-GFP condensate in an ect8-1 gECT8-GFP root cell. The white circle indicates the bleached area. Scale bar, 0.1 µm. f, FRAP recovery plot of the ECT8-GFP condensate shown in e. The value at the beginning of photobleaching was set as 0. Values are means ± s.d. (n = 8 individual condensates from three independent experiments).
Extended Data Fig. 3 IDR2 is required for phase separation of ECT8.
a, Prediction of intrinsically disordered regions (IDRs) of ECT8. Top, schematic diagrams of truncated ECT8 proteins that are fused with GFP at the N-terminus. Numbers at the top indicate the positions of different domains of ECT8. Bottom, prediction of IDRs by IUPred2A. b, Differential interference contrast images show the droplets formed by GFP-ECT8, but not by GFP itself, with the addition of PEG-8000. Scale bars, 20 µm. c, In vitro phase separation of GFP-ECT8 shown as the formation of droplets at the indicated concentrations of GFP-ECT8 in the presence of PEG-8000. BF, bright field. Merge, merge of GFP and BF images. Scale bars, 20 µm. The experiments in b, c were repeated three times independently with similar results. d, FRAP of GFP-ECT8 droplets in vitro. White arrowheads indicate the bleached droplet. Scale bar, 10 µm. e, FRAP recovery plot of GFP-ECT8 droplets in d. In e, g and k, the value at the beginning of photobleaching was set as 0. Values are means ± s.d. (n = 10 in e, n = 8 in g, n = 10 in k from three independent experiments). f, FRAP of a subregion of a GFP-ECT8 droplet in vitro. The white circle indicates the bleached area. Scale bar, 1 µm. g, FRAP recovery plot of the subregion of the GFP-ECT8 droplet shown in f. h, Expression of recombinant GFP-ECT8 and its truncated forms shown in a. Arrowheads indicate the expected proteins. i, Examination of in vitro phase separation of GFP-ECT8 and its truncated forms shown as the formation of droplets in the presence of PEG-8000. Scale bars, 100 µm. Data are representative of three independent experiments. j, FRAP of a subregion of a GFP-ECT8IDR2 droplet in vitro. The white circle indicates the bleached area. Scale bar, 1 µm. k, FRAP recovery plot of the subregion of the GFP-ECT8IDR2 droplet shown in j.
Extended Data Fig. 4 Phase separation of ECT8 conferred by IDR2 is critical for ECT8 function in drought response.
a, Analysis of ECT8 expression in wild-type (WT) plants, ect8-1, ect8-1 gECT8-GFP, and two ect8-1 gECT8ΔIDR2-GFP lines (#6 and #16). TUB2 was used as an internal control, and the level in WT plants is set as 1.0. Values are means ± s.d. (n = 3 biologically independent replicates). In a, d, different letters at the top of each column indicate significant differences at P < 0.05 determined by one-way ANOVA with Tukey’s HSD test. The exact P values (a, d) are provided in Supplementary Table 10. b, Western blot analysis of ECT8-GFP and ECT8ΔIDR2-GFP in the indicated transgenic plants. Data are representative of three independent experiments. c, Drought response of plants in various genetic backgrounds. Scale bar, 1 cm. d, Analysis of seedling survival rates of the different genotypes as shown in c. Values are means ± s.d. (n = 10 biologically independent replicates). e, Comparison of the formation of condensates in root tip cells between 7-day-old ect8-1 gECT8-GFP and ect8-1 gECT8ΔIDR2-GFP #16 treated with mannitol for 6 h. Scale bars, 10 µm. f, Quantification of the number of condensates per cell in ABA-treated samples as shown in e (right) and Fig. 2g (left). In f, h, box plots display medians (lines), interquartile ranges (boxes), and whiskers (extending 1.5 × the interquartile ranges) of the number of condensates per cell in different genotypes (n = 20 individual cells from three independent experiments). ND, not detected. g, Subcellular localization of ECT8-GFP and ECT8ΔIDR2-GFP in 35 S:ECT8-GFP and 35 S:ECT8ΔIDR2-GFP protoplasts, respectively, mock-treated (Mock) and treated with 50 µM ABA. White arrows indicate ECT8-GFP condensates. Scale bars, 10 µm. h, Quantification of the number of condensates per cell in ABA-treated samples as shown in g.
Extended Data Fig. 5 ECT8 condensates are associated with SG components.
a, Identification of the interactome of ECT8 in response to ABA treatment. Protein extracts from 7-day-old ect8-2 gECT8-FLAG seedlings mock-treated or treated with 50 µM ABA for 6 h were co-immunoprecipitated using anti-FLAG beads, and the associated proteins were subjected to LC-MS/MS analysis. The identified proteins in response to ABA are color-coded for functional categories. Red triangles indicate the components also found as the SG components in Arabidopsis identified previously36. b, Venn diagram showing the number of overlapping proteins between the ECT8 interactome in response to ABA and the SG components in Arabidopsis identified previously36. c, Colocalization of ECT8-GFP and Rbp47b-RFP in Arabidopsis protoplasts mock-treated (Mock) or treated with 50 µM ABA for 6 h. BF, bright field. Merge, merge of RFP and GFP images. Scale bars, 10 µm. Data are representative of three independent experiments. d, CoIP shows the interaction between ECT8 and Rbp47b in ect8-2 gECT8-FLAG 35 S:Rbp47b-RFP seedlings. The input and coimmunoprecipitated protein were detected by anti-RFP and anti-FLAG antibodies. The experiments were repeated three times independently with similar results. e, Analysis of formation of SGs marked by Rbp47b-RFP in wild-type (WT) and ect8-1 protoplasts upon mock and ABA treatment for 6 h. Scale bars, 10 µm. f, Quantification of the number of SGs per cell as shown in e. Box plots display medians (lines), interquartile ranges (boxes), and whiskers (extending 1.5 × the interquartile ranges) of the number of SGs per cell in the two genotypes (n = 25 individual cells from three independent experiments). There is no statistically significant difference (ns) between the two genotypes (two-tailed Student’s t-test). The exact P values are provided in Supplementary Table 10.
Extended Data Fig. 6 Phase separation of ECT8 and its effect on drought response are dependent on its m6A binding capacity.
a, Dot blot analysis of m6A levels in mRNA purified from in vitro pull-down assays. Data shown in a, c and d are representative of three independent experiments. b, ECT8 expression in various genetic backgrounds. TUB2 served as an internal control, and the level in WT plants is set as 1.0. Values are means ± s.d. (n = 3 biologically independent replicates). In b, f, different letters at the top of each column indicate significant differences at P < 0.05 determined by one-way ANOVA with Tukey’s HSD test. c, Western blot analysis of ECT8-GFP and mECT8-GFP in indicated plants. d, Dot blot analysis of m6A levels in mRNA purified from in vivo RIP assays. e, Drought response of various plants. Scale bar, 1 cm. f, Analysis of survival rates of different genotypes as shown in e. Values are means ± s.d. (n = 10 biologically independent replicates). g, Comparison of the formation of condensates in root tip cells treated with mannitol for 6 h. Scale bars, 10 µm. h, Subcellular localization of ECT8-GFP and mECT8-GFP in 35 S:ECT8-GFP and 35 S:mECT8-GFP protoplasts, respectively, mock-treated and treated with 50 µM ABA for 6 h. White arrows indicate condensates. Scale bars, 10 µm. i, Quantification of the number of condensates per cell in ABA-treated samples as shown in h. j, Subcellular localization of ECT8-GFP in WT and fip37-4 LEC1:FIP37 protoplasts transformed with 35 S:ECT8-GFP mock-treated and treated with 50 µM ABA for 6 h. White arrows indicate condensates. Scale bars, 10 µm. k, Quantification of the number of condensates per cell in ABA-treated samples shown in j. In i, k, box plots display medians (lines), interquartile ranges (boxes), and whiskers (extending 1.5 × the interquartile ranges) of the number of condensates per cell (n = 20 in i, n = 25 in k from three independent experiments), asterisks indicate statistically significant differences (two-tailed Student’s t-test, P < 0.01). The exact P values (b, f, i and k) are provided in Supplementary Table 10.
Extended Data Fig. 7 ECT8 affects the m6A RNA methylome in SGs.
a, Dot blot analysis of m6A levels in cellular mRNA and SG-enriched mRNA purified from 7-day-old wild-type seedlings treated with 50 µM ABA for 6 h. Data shown in a-c are representative of three independent experiments. b, c, Dot blot analysis of m6A levels in SG-enriched mRNA (b) and cellular mRNA (c) purified from 7-day-old wild-type and ect8-1 seedlings treated with 50 µM ABA for 6 h. d, Pie chart showing the percentages of hypomethylated m6A sites in SG-enriched mRNA of ect8-1 along transcripts divided into 5′ UTR, Stop (100 bp upstream or downstream of the stop codon), other CDS, and 3′ UTR regions. UTR, untranslated region; CDS, coding region. e, Frequency of the top ten 5-nt k-mers for hypomethylated m6A sites in SG-enriched mRNA of ect8-1 revealed by PEKA analysis. The consensus m6A motifs in plants, RRACH (R = G/A; H = A/U/C) are highlighted in red. f, Frequency of numbers of m6A sites per transcript for hypomethylated genes in SG-enriched mRNA of ect8-1. g, Top 10 GO terms for the hypomethylated genes in SG-enriched mRNA of ect8-1. Circle size and color indicate the number of genes and P-value of GO terms, respectively. h, Distribution of changes in expression of differentially expressed genes (DEGs) with and without hypomethylated m6A sites in SG-enriched mRNA of ect8-1. DEGs were determined by log2 (fold change) > 0.5 or < −0.5 and P < 0.05 in SG-enriched mRNA of ect8-1 versus wild-type seedlings.
Extended Data Fig. 8 Examination of ECT8 expression in transgenic lines used for RIP-qPCR analysis.
a, Western blot analysis using anti-FLAG antibody shows the expression of ECT8-FLAG in protein extracts (Input) from 7-day-old ect8-2 gECT8-FLAG seedlings mock-treated (Mock) and treated with 50 µM ABA for 6 h and the corresponding immunoprecipitated fractions (anti-FLAG IP). b, c, Western blot analysis using anti-RFP and anti-GFP antibodies shows the expression of Rbp47b-RFP (b) and ECT8-GFP (c) in total protein extracts (Total), stress granule (SG)-enriched fractions, and the supernatant fractions generated in the process of SG isolation from 7-day-old 35 S:Rbp47b-RFP and ect8-1 gECT8-GFP seedlings mock-treated (-) or treated with 50 µM ABA for 6 h (+). The different fractions extracted from 7-day-old wild-type (WT) seedlings were used as controls. In a-c, Rubisco large subunit (RbcL) stained with Ponceau S serves as loading controls, the experiments were repeated three times independently with similar results.
Extended Data Fig. 9 ECT8 binds to PYL7 and affects its localization in SG.
a, m6A-IP-qPCR analysis of m6A modification on PYL7 transcripts in total RNA extracted from 7-day-old wild-type (WT) and ect8-1 seedlings mock-treated and treated with 50 µM ABA for 6 h. Levels of m6A enrichment are shown relative to the level in mock-treated WT seedlings (at P2) set as 1.0. Values are means ± s.d. (n = 3 biologically independent replicates). In a, b and g, different letters at the top of each column indicate significant differences at P < 0.05 determined by one-way ANOVA with Tukey’s HSD test. b, RIP-qPCR assay shows direct binding of ECT8-FLAG to PYL7 transcripts. PYL7 enrichment levels are shown relative to the level of ACT2 (negative control) in mock-treated WT seedlings set as 1.0. Values are means ± s.d. (n = 3 biologically independent replicates). c, Colocalization of ECT8-GFP and Rbp47b-RFP in SGs purified from 7-day-old ect8-1 gECT8-GFP 35 S:Rbp47b-RFP seedlings under ABA treatment. ECT8-GFP, GFP fluorescence of ECT8-GFP; Rbp47b-RFP, RFP fluorescence of Rbp47b-RFP; Merge, merge of GFP and RFP; Alexa Fluor 647, fluorescence of Alexa Fluor 647 channel (serve as an autofluorescence control); BF, bright field. Scale bar, 20 µm. Data are representative of three independent experiments. d, qPCR analysis of PYL7 expression in SG-enriched RNA extracted from various genetic backgrounds under ABA treatment. Levels of gene expression are normalized to TUB2 expression and shown relative to the level in WT plants set as 1.0. Values are means ± s.d. (n = 3 biologically independent replicates); ns, not significant (two-tailed Student’s t-test, P < 0.01). e, Alignment of WT and pyl7-2 sequences containing the CRISPR/Cas9 target sites. The 20 bp CRISPR/Cas9 target sequences (targets 1 and 2) adjacent to the underlined protospacer adjacent motifs (PAMs) are indicated in red. f, Drought response of plants in various genetic backgrounds. Scale bar, 1 cm. g, Analysis of seedling survival rates of the different genotypes as shown in f. Values are means ± s.d. (n = 10 biologically independent replicates). The exact P values (a, b, d and g) are provided in Supplementary Table 10.
Extended Data Fig. 10 ABA sensitivity analysis of plants in different genetic backgrounds.
a, ABA sensitivity analysis of plants defective in ECT8 and several ABA receptor genes grown on MS medium mock-treated or treated with 0.5 µM ABA. Scale bar, 0.5 cm. b, Analysis of cotyledon greening percentages of the different genotypes as shown in a. c, ABA sensitivity analysis of plants defective in ECT8 and ABI grown on MS medium mock-treated or treated with 0.5 µM ABA. Scale bars, 0.5 cm. d, Analysis of cotyledon greening percentages of the different genotypes as shown in c. In b, d, values are means ± s.d. (n = 10 biologically independent replicates). Approximately 30 seeds were analyzed in each replicate. Different letters at the top of each column indicate significant differences at P < 0.05 determined by one-way ANOVA with Tukey’s HSD test. The exact P values are provided in Supplementary Table 10.
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Wu, X., Su, T., Zhang, S. et al. N6-methyladenosine-mediated feedback regulation of abscisic acid perception via phase-separated ECT8 condensates in Arabidopsis. Nat. Plants 10, 469–482 (2024). https://doi.org/10.1038/s41477-024-01638-7
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DOI: https://doi.org/10.1038/s41477-024-01638-7
