Abstract
The evolutionarily conserved target of rapamycin (TOR) kinase acts as a master regulator that coordinates cell proliferation and growth by integrating nutrient, energy, hormone and stress signals in all eukaryotes1,2. Research has focused mainly on TOR-regulated translation, but how TOR orchestrates the global transcriptional network remains unclear. Here we identify ethylene-insensitive protein 2 (EIN2), a central integrator3,4,5 that shuttles between the cytoplasm and the nucleus, as a direct substrate of TOR in Arabidopsis thaliana. Glucose-activated TOR kinase directly phosphorylates EIN2 to prevent its nuclear localization. Notably, the rapid global transcriptional reprogramming that is directed by glucose–TOR signalling is largely compromised in the ein2-5 mutant, and EIN2 negatively regulates the expression of a wide range of target genes of glucose-activated TOR that are involved in DNA replication, cell wall and lipid synthesis and various secondary metabolic pathways. Chemical, cellular and genetic analyses reveal that cell elongation and proliferation processes that are controlled by the glucose–TOR–EIN2 axis are decoupled from canonical ethylene–CTR1–EIN2 signalling, and mediated by different phosphorylation sites. Our findings reveal a molecular mechanism by which a central signalling hub is shared but differentially modulated by diverse signalling pathways using distinct phosphorylation codes that can be specified by upstream protein kinases.
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Data availability
The RNA-seq raw data for the glucose–EIN2-regulated transcriptome have been deposited in the NCBI Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) under the accession number: GSE114505. The RNA-seq raw data for the ethylene-regulated transcriptome can be downloaded from the NCBI SRA (https://www.ncbi.nlm.nih.gov/sra/) under the accession number: SRP017925. Source gel data for all immunoblots and radiograms (Figs. 1c–g, 2b, c, Extended Data Figs. 1f, 2c, 3b, k, 4b, 6e) are provided in Supplementary Fig. 1. Source data are provided with this paper.
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Acknowledgements
We thank J. Sheen, T. D. Xu and X. Y. Zhu for critical reading of the manuscript, all members of the Xiong laboratory for discussion, C. B. Xiang for ERF1-OX seeds, W. F. Wang for the BZR1 antibody, R. X. Li for the CFP-HDEL plasmid and ABRC for the T-DNA insertional and point mutation mutants. This research was supported by funding from the National Natural Science Foundation of China (grant 31870269 to Y.X. and 31800199 to Yanlin Liu; the Chinese Academy of Sciences; and the Basic Forestry and Proteomics Research Centre, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University.
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Y.X., L.F., Yanlin Liu, S.-D.Y. and Y.-H.C. designed the experiments, and L.F. and Yanlin Liu performed most of the experiments and prepared the figures. G.Q., Yaxing Li, P.W., C.P., Z.Z. and C.C.L.W. carried out proteomics analyses. H.Z., Z.X. and R.L. conducted RNA-seq data analyses. X.Z., Y.W., S.Y. and L.F. generated the EIN2 transgenic lines. S.-D.Y. and Y.-H.C. provided ethylene-related mutants. Y.X., L.F. and Yanlin Liu wrote the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Non-canonical role of EIN2, EIN3 and EIL1 in glucose–TOR signalling.
a, Quantification data of Fig. 1a. C2H4, ethylene. b, c, Glucose-promoted hypocotyl elongation depends on TOR kinase. Wild-type and tor-es lines were germinated for 4 days in the dark without or with glucose (Glc, 0.5% w/v). Representative images (b) and hypocotyl length (c). Scale bar, 1 mm. d, e, Glucose–TOR-regulated hypocotyl elongation is independent of ethylene sensing and biosynthesis. The tor-es seedlings were germinated in the dark without or with oestradiol (Est), 1-MCP (100 nl l−1) or AVG (1 μM) for 4 days. Representative images (d) and hypocotyl length (e). Scale bar, 1 mm. f, Oestradiol induces TOR silencing, as revealed by western blot. Experiments were repeated three times with similar results. g, h, Representative images (g) and hypocotyl length (h) of wild-type (Ler) and gin2 seedlings germinated in the dark with or without various chemical treatments for 4 days. Scale bar, 1 mm. i, Quantification data of Fig. 1b. j, Quantification data of Fig. 1c. n = 3 biological repeats. k, l, Representative images (k) and hypocotyl length (l) of wild-type (Col-0) and mutant seedlings germinated in the dark with or without various chemical treatments for 4 days. Scale bars, 1 mm. Data were analysed from 45 (a, c, i), 35 (e), 30 (h) or 52 (l) seedlings for each genotype and treatment from 3 experiments, and expressed as mean ± s.d. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test (a, e, h, i, j, l) or by two-sided Student’s t-test (c); n.s. not significant; P values are indicated. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 2 Different dosage response curves between wild-type, etr1-1, ein2-5 and ein3 eil1 in response to rapamycin, Torin2 and AZD8055.
a, b, Representative images (a) and hypocotyl length (b) of wild type (Col-0) and mutant seedlings germinated in the dark with different concentration of rapamycin, Torin2 or AZD8055 for 4 days. Scale bars, 1 mm. Data were analysed from 30 (rapamycin and Torin2 treatment) or 90 (AZD8055 treatment) seedlings for each genotype and treatment from 3 experiments, and are expressed as mean ± s.d. Statistical significance was determined by two-sided Student’s t-test; P values are indicated. c, Rapamycin, Torin2 or AZD8055 inhibits TOR kinase activity in a concentration-dependent manner. p35S::S6K1-HA transgenic seedlings were germinated with different concentrations of rapamycin, Torin2 or AZD8055 in the dark for 4 days. TOR kinase activities were analysed based on phosphorylation of the conserved TOR substrate (pThr449 of S6K1) by western blot. Experiments were repeated three times with similar results. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 3 Glucose–TOR–EIN2 regulates EIN3 stability and the expression of ERFs to control hypocotyl elongation.
a, TOR interacts with EIN2-C, as revealed by yeast two-hybrid analyses. b, Inactivation of TOR triggers EIN3 accumulation depending on EIN2. p35S::EIN3-GFP/ein3 eil1 and p35S::EIN3-GFP/ein2-5 seedlings were treated with rapamycin (10 μM) or ACC (10 μM) for the indicated times. Tubulin (TUB) was used as the loading control. c–e, Ethylene (C2H4) and Torin2 activate the expression of both overlapped and distinct ERF genes. The 4-d-etiolated wild-type seedlings (c, d) or mutants (e) were treated with ethylene (c) or Torin2 (d, e) for 2 h, then the hypocotyls were collected for RT–qPCR analyses. Data are mean ± s.d.; n = 3 biological repeats. f, g, Representative images (f) and hypocotyl length (g) of wild-type (Col-0) and ERF1-OX seedlings germinated in the dark with different concentrations of Torin2 for 4 days. Scale bar, 1 mm. h, erf-c mutant sequences. The erf-c mutants were generated by CRISPR–Cas9 technology. Green arrows indicate the position of the deletion or insertion of a nucleotide; red letters indicate amino acids resulting from the frameshift translation; * indicates stop codon. i, j, Representative images (i) and hypocotyl length (j) of wild-type (Col-0) and erf-c seedlings germinated in the dark with or without Torin2 (1 μM) for 4 days. Scale bar, 1 mm. k, l, TOR regulation of BZR1 stability is independent from the EIN2–EIN3/EIL1 axis. The 3-d-etiolated seedlings were treated with Torin2 for 2 days. Tubulin was used as the loading control. Representative images (k) and relative BZR1 abundance (l). Data are mean ± s.d., n = 3 biological repeats. m, The non-canonical role of EIN2 in glucose–TOR-signalling-mediated hypocotyl elongation. In g, j, data were analysed from 45 seedlings for each genotype and treatment from 3 experiments, and are expressed as mean ± s.d. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test (g) or by two-sided Student’s t-test (c, d, e, j, l); n.s. not significant; P values are indicated. In a, b, experiments were repeated three times with similar results. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 4 Glucose–TOR and ethylene–CTR1 regulate the nuclear localization of EIN2 through distinct phosphorylation sites.
a, Thr657 is conserved among EIN2 orthologues of A. thaliana, O. sativa and Z. mays. b, TOR kinase directly phosphorylates OsEIN2-C and ZmEIN2-C, as shown by an in vitro kinase assay. Torin2, 1 μM. Experiments were repeated three times and similar results were obtained. c–j, Different nucleus accumulation patterns and quantification of relative GFP fluorescent signal intensity between the nucleus and the cytosol of OsEIN2–GFP and ZmEIN2–GFP (c, d), EIN2(WT)–GFP (e, f), EIN2(T657A)–GFP and EIN2(T657D)–GFP (g, h), and EIN2(S645D)–GFP and EIN2(S924D)–GFP (i, j) in Arabidopsis leaf mesophyll protoplasts. Mesophyll protoplasts transiently transfected with various EIN2 constructs were treated with or without rapamycin (10 μM) or ACC (10 μM) for 3 h. Scale bars, 5 μm. NLS-mC denotes nuclear HY5–mCherry. In d, f–h data were analysed from the indicated numbers of cells from three experiments, and are expressed as mean ± s.d. Centre line, median; box limits, 25th and 75th percentiles; whiskers extend to 1.5 × IQR from the 25th and 75th percentiles. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test; n.s, not significant; P values are indicated. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 5 Glucose–TOR- and ethylene signalling regulate hypocotyl elongation through distinct phosphorylation sites in EIN2.
a, b, Different patterns of nuclear accumulation of EIN2(WT)–GFP, EIN2(T657D)–GFP and EIN2(S645D)–GFP in hypocotyl cells. Seedlings were germinated in the dark for 4 days, then treated with Torin2 (1 μM) or ethylene (C2H4, 100 ppm) (a), or rapamycin (10 μM) (b) for 4 h. Arrows indicate nuclei. Scale bars, 10 μm. c, Quantification of the relative GFP fluorescent signal intensity between the nucleus and the cytosol of EIN2(WT)–GFP, EIN2(T657D)–GFP and EIN2(S645D)–GFP in b. See quantification data of a in Fig. 2e. d, Quantification data of Fig. 2f. e, f, EIN2(S645D)–GFP but not EIN2(T657D)–GFP restores ein2-5 sensitivity to rapamycin-inhibited hypocotyl elongation. Seedlings were germinated in the dark with or without rapamycin (10 μM) for 4 days. Representative images (e) and hypocotyl length (f). Scale bar, 1 mm. In c, data were analysed from the indicated numbers of hypocotyl cells from three experiments, and are expressed as mean ± s.d. Centre line, median; box limits, 25th and 75th percentiles; whiskers extend to 1.5 × IQR from the 25th and 75th percentiles. In d, f, data were analysed from 45 (d) and 40 (f) seedlings for each genotype and treatment from three experiments, and are expressed as mean ± s.d. Statistical significance was determined by two-sided Student’s t-test (c) or by one-way ANOVA with post hoc Tukey test (d, f); n.s, not significant; P values are indicated.
Extended Data Fig. 6 Full-length EIN2 is translocated to the nucleus in response to inhibition of TOR.
a, b, EIN2–GFP and GFP–EIN2 complement the response of ein2-5 seedlings to treatment with Torin2 and ethylene (C2H4). Seedlings were germinated on solid half-strength MS medium with or without Torin2 (1 μM) or ethylene (100 ppm) in the dark for 4 days. Representative images (a) and hypocotyl length (b). Scale bar, 1 mm. Data were analysed from 22 seedlings for each genotype and treatment from 3 experiments, and are expressed as mean ± s.d. c, Different nucleus accumulation patterns of EIN2(WT)–GFP and GFP–EIN2(WT); see quantification data in Fig. 2g. Scale bars, 500 μm (left); 10 μm (right). d, EIN2 accumulates in the nucleus after inhibition of TOR. Arabidopsis mesophyll protoplasts transiently co-transfected with mCherry–EIN2–GFP and CFP–HDEL (an ER marker) were treated with or without rapamycin (10 μM) or Torin2 (1 μM) for 3 h. Scale bars, 5 μm. Experiments were repeated three times with similar results. e, f, Full-length EIN2 is translocated to the nucleus in response to inhibition of TOR. Four-day-etiolated pEIN2::EIN2WT-3XHA seedlings were treated with or without Torin2 (1 μM) for the indicated times, then total and nuclear fractions of hypocotyl protein were prepared and subjected to western blot (e). Tubulin (total protein loading control), calnexin homologue 1 or 2 (CNX1/2, ER marker) and histone H3 (nuclear marker) were used as controls to monitor the loading and purity of the total and nuclear fraction. f, Quantification of e. Data are mean ± s.d., n = 3 biological repeats. For b, f, statistical significance was determined by one-way ANOVA with Tukey’s post hoc test; n.s, not significant; P values are indicated. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 7 Role of EIN2 in glucose–TOR-mediated activation of the root meristem.
a, Inhibition of TOR triggers nuclear localization of EIN2–GFP in root meristem cells. Seedlings (4 DAG) were treated with Torin2 (1 μM) for 4 h. Arrows indicate nuclei. Scale bars, 25 μm (left); 10 μm (right). See Fig. 3a for quantification data. b–f, EIN2 is involved in glucose-promoted primary root growth (b, d) and cell proliferation activity indicated by EdU staining (c, e) and S-phase gene expression (f; RT–qPCR analysis; mean ± s.d., n = 3 biological repeats). Scale bars, 1 mm (b); 20 μm (c). g, h, Quantification data of Fig. 3f, g. i, EIN2 Thr657 is required for glucose-promoted S-phase gene expression. RT–qPCR analysis; mean ± s.d.; n = 3 biological repeats. For d, e, g, h, data were analysed from 45 (d), 30 (e), 45 (g) and 30 (h) of roots for each genotype from 3 experiments, and are expressed as mean ± s.d. In all of the bar charts, statistical significance was determined by two-sided Student’s t-test; n.s, not significant; P values are indicated.
Extended Data Fig. 8 EIN2(T657D) and EIN2(S645D) distinguish ethylene- and glucose–TOR-regulated cell proliferation.
a, b, EIN2(T657D) and EIN2(S645D) distinguish ethylene-regulated cell proliferation, indicated by EdU staining of the root apical meristem. Scale bar, 50 μm. Seedlings were germinated in glucose-free liquid half-strength MS medium for 4 days, then transferred to solid half-strength MS medium with glucose (15 mM) and ethylene (C2H4, 100 ppm) for 4 h. Representative EdU staining images (a) and quantification of EdU intensity (b). c–f, EIN2(T657D) and EIN2(S645D) distinguish glucose-TOR-regulated cell proliferation. Scale bars, 50 μm. Seedlings were germinated in glucose-free liquid half-strength MS medium for 4 days, then treated with glucose (15 mM) and Torin2 (1 μM) for 2 h. Representative EdU staining images and quantification of the EdU intensity of ethylene-insensitive mutants (c, d) and pEIN2::EIN2WT-GFP/ein2-5, pEIN2::EIN2T657-GFP/ein2-5 and pEIN2::EIN2S645D-GFP/ein2-5 transgenic plants (e, f). For b, d, f, data were analysed from 30 roots for each genotype and treatment from 3 experiments, and are expressed as mean ± s.d. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test (b) or by two-sided Student’s t-test (d, f); n.s, not significant; P values are indicated.
Extended Data Fig. 9 E2Fa acts downstream of EIN2 to promote primary root growth.
a, RT–qPCR analyses of E2Fa expression in glucose-depleted roots of wild-type (Col-0), ein2-5, ein3 eil1 and etr1-1 seedlings. Data are mean ± s.d.; n = 3 biological repeats. b–g, Overexpression of E2Fa in oestradiol-inducible E2Fa-GFP transgenic seedlings (b) resulted in longer roots (c, e), increased cell proliferation activities indicated by EdU staining (d, f) and S-phase gene expression revealed by RT–qPCR analysis (g; mean ± s.d.; n = 3 biological repeats). Scale bars, 20 μm (b); 1 mm (c); 25 μm (d). Data were analysed from 45 (#1 −Est), 43 (#1 +Est), 38 (#2 −Est) and 37 (#2 +Est) roots (e) or from 38 (#1 −Est), 38 (#1 +Est), 34 (#2 −Est) and 33 (#2 +Est) roots (f) from 3 experiments, and are expressed as mean ± s.d. In all of the bar charts, statistical significance was determined by two-sided Student’s t-test; P values are indicated.
Extended Data Fig. 10 The glucose–TOR–EIN2 axis mediates nuclear signalling to modulate plant growth.
a, RT–qPCR analyses of transcriptome variation in response to TOR inhibition and ethylene treatments. Wild-type seedlings and various mutants and transgenic lines were germinated in 0.5% glucose (w/v) for 4 days, then treated with or without Torin2 (1 μM) or ethylene (C2H4, 100 ppm) for 2 h. The numbers in the heat map indicate the relative fold changes. b, Representative images and hypocotyl length of wild type (Col-0) and various mutants germinated in the dark with or without AZD8055 (1 μM) or Torin2 (1 μM) for the indicated times. Scale bars, 1 mm. Data were analysed from 30 seedlings for each genotype and treatment from 3 experiments, and are expressed as mean ± s.d. Statistical significance was determined by two-sided Student’s t-test; n.s, not significant; P values are indicated. c, Proposed model for the glucose–TOR–EIN2 axis in plant growth and development. Glucose-activated TOR directly phosphorylates EIN2 to inhibit its nuclear shuttling. In glucose-depleted conditions, dephosphorylated EIN2 moves into the nucleus, and controls root meristem activation and hypocotyl cell elongation through different downstream effectors.
Supplementary information
Supplementary Figure
This file contains Supplementary Figure 1, the source gel data.
Supplementary Table 1
Glucose-controlled genes. The data includes both glucose up-and down-regulated (FDR<0.01) genes, which are visualized in Fig. 3b-e.
Supplementary Table 2
Glucose-EIN2 controlled genes. The data includes glucose insensitive and less sensitive genes in ein2-5, which are visualized in Fig. 3b-e.
Supplementary Table 3
Differential regulated genes between ein2-5 and WT under glucose-depletion condition, which are visualized in Fig. 3b-e.
Supplementary Table 4
Transcriptome comparison between glucose/ethylene-regulated genes. The data includes ethylene specific, glucose/TOR specific, and ethylene/glucose common-regulated genes, which are visualized in Fig. 3h.
Supplementary Table 5
Primers used for plasmid constructs, RT-qPCR and mutagenesis.
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Fu, L., Liu, Y., Qin, G. et al. The TOR–EIN2 axis mediates nuclear signalling to modulate plant growth. Nature 591, 288–292 (2021). https://doi.org/10.1038/s41586-021-03310-y
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DOI: https://doi.org/10.1038/s41586-021-03310-y
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