Glucose-TOR signalling reprograms the transcriptome and activates meristems

Abstract

Meristems encompass stem/progenitor cells that sustain postembryonic growth of all plant organs. How meristems are activated and sustained by nutrient signalling remains enigmatic in photosynthetic plants. Combining chemical manipulations and chemical genetics at the photoautotrophic transition checkpoint, we reveal that shoot photosynthesis-derived glucose drives target-of-rapamycin (TOR) signalling relays through glycolysis and mitochondrial bioenergetics to control root meristem activation, which is decoupled from direct glucose sensing, growth-hormone signalling and stem-cell maintenance. Surprisingly, glucose-TOR signalling dictates transcriptional reprogramming of remarkable gene sets involved in central and secondary metabolism, cell cycle, transcription, signalling, transport and protein folding. Systems, cellular and genetic analyses uncover TOR phosphorylation of E2Fa transcription factor for an unconventional activation of S-phase genes, and glucose-signalling defects in e2fa root meristems. Our findings establish pivotal roles of glucose-TOR signalling in unprecedented transcriptional networks wiring central metabolism and biosynthesis for energy and biomass production, and integrating localized stem/progenitor-cell proliferation through inter-organ nutrient coordination to control developmental transition and growth.

Figure 1. Photosynthesis controls the metabolic activation of root meristems

a, Photosynthesis promotes root growth. DAG, day-after-germination, Glc, glucose; L, light; C, CO₂; D, DCMU. b-e, Photosynthesis drives root meristem activation. Results of primary root length, meristem cell (MC) number, meristem size and S-phase entry. Means ± s.d., (n≥25). f, Glycolysis and mitochondrial bioenergetics stimulate root growth and meristem proliferation. 2-DG, 2-Deoxyglucose, AMA, Antimycin A, DNP, 2,4-dinitrophenol and CCCP, carbonylcyanide-m-chlorophenylhydrazone. g, Growth-hormone treatments. IAA, indole-3-acetic acid; BL, brassinosteroid; tZ, trans-zeatin; GA, gibberellins. Scale bar, 1 mm or 25 µm. Arrowheads: quiescent centre. Red arrow: transition zone.

Figure 2. Glucose-TOR signalling in root meristems

a, Root growth activation by glucose is TOR dependent. WT (Col or Ler), estradiol-inducible tor mutants (tor-es1, tor-es2) and gin2 seedlings at 3DAG were incubated without or with glucose (Glc) or rapamycin (Rap) for 24 h. Scale bar, 2 mm. b, Glucose activates endogenous TOR. TOR activation is detected using an anti-phospho-T449 antibody for S6K1 after 1 h glucose treatment without or with glycolysis inhibitor (2DG), mitochondrial blockers (AMA, DNP, CCCP) or rapamycin (Rap). c-d, TOR controls root meristem activation and S-phase entry. Scale bar, 25 µm.

Figure 3. Auxin and cytokinin signalling and root stem-cell maintenance are decoupled from TOR activation

a-b, Auxin signalling. c-d, Cytokinin signalling. Rapamycin (Rap), mitochondrial blocker (AMA). Primary auxin and cytokinin marker genes were activated by 1 h of indole-3-acetic acid (IAA) or trans-zeatin (tZ) treatment, and analysed by qRT-PCR. Means ± s.d., n=3. DR5::GFP or TCS::GFP was activated by 6 h of IAA or tZ treatment. Scale bar, 50 µm. e, f, Root stem-cell maintenance is TOR independent. PLT1::GFP, root stem-cell marker; WOX5::GFP: root quiescent centre marker. Scale bar, 20 µm.

Figure 4. Glucose-TOR signalling orchestrates transcriptome reprogramming

a-b, Glucose-TOR activated genes. c-d, Glucose-TOR repressed genes. 3DAG WT or tor seedlings were treated without or with glucose for 2 h. Hierarchical clustering analysis of glucose-TOR genes and five independent datasets (Glc2h, Glc4h, Sucrose, LowCO₂-light, lowCO₂-dark). Deep-pink/blue bar indicates novel glucose-TOR genes. The enriched functional categories highlighted in bold (Supplementary Tables 1, 3, 4). e, Hierarchical clustering analysis of glucose-TOR genes (Glc) and cell cycle genes (G1, S, G2, M). f, Glucose-TOR activated genes overlap with E2Fa target genes. g, Glucose-TOR activates S-phase genes. QRT-PCR analyses. Means ± s.d., n=3. *P<0.05.

Figure 5. TOR kinase phosphorylates and activates E2Fa

a, Ectopic E2Fa activation of S-phase genes requires glucose-TOR signalling in leaf cells. WT or tor protoplasts expressing E2Fa-HA or S6K1-FLAG were treated without or with rapamycin (Rap) or antimycin A (AMA). QRT-PCR analyses. P-T449 indicates endogenous TOR kinase activity. Protein blot analysis (inset). b-c, TOR kinase directly phosphorylates E2Fa and 4E-BP1. Torin1 specifically inhibits TOR kinase. Staurosporine (Stau) inhibits S6K1 kinase. d, TOR directly interacts with E2Fa by immunoprecipitation (IP) and Western (W) blot analysis.

Figure 6. TOR kinase controls the activity of E2Fa in transcriptional activation

a, TOR kinase phosphorylates the N-terminal domain of E2Fa. b, TOR kinase phosphorylation is critical for E2Fa activation of S-phase genes. c, E2Fa-DNA binding is not affected by TOR kinase phosphorylation. ChIP-qPCR analyses with P (promoter) or G (gene body) primers. Stars, putative E2Fa-binding motifs. Error bars (n=2). d-e, Glucose responses is diminished in e2fa root meristems. Scale bar, 1 mm or 20 µm. QRT-PCR analyses. f, Model of leaf-root coordination in glucose-TOR signalling. GSH, GLUTATHIONE, RGF, ROOT GROWTH FACTOR, UPB1, UPBEAT1. Means ± s.d., n=3. *P<0.05.