Extended Data Fig. 6: Glucose-TOR specifically promotes the cytoplasm-to-nucleus translocation of FIE to enhance the PRC2 activity in the nucleus.
From: Glucose-driven TOR–FIE–PRC2 signalling controls plant development
a, A proposed model for the cytoplasm-to-nucleus translocation of FIE regulated by TOR. FIE mainly localizes in the cytoplasm at low TOR activity and its phosphorylation by glucose-activated TOR stimulates its nuclear entry to enhance PRC2 activity. Blocking TOR activity or mutation of the phosphorylation sites inhibited the nuclear translocation of FIE. b, Quantitative confocal imaging of GFP-FIE and GFP-FIE(SSTS/AAAA) in leaf primordia and roots. The cytoplasm/nucleus (C/N) signal intensity ratio of GFP-FIE or GFP-FIE(SSTS/AAAA) at the single-cell level was measured by quantitative confocal imaging using the Leica LAS-X software. WT seedlings (5 d) expressing GFP-FIE without (Ctrl) or with 10 μM of Torin2 or AZD treatment (24 h) or tor-es (10 μM estradiol for 3 d) were examined. Root elongation zone and root meristem zone are illustrated. c, Confocal images of GFP-tagged PRC2 components in the meristem zone of roots. Plants were imaged with or without (Mock) Torin2 treatment. d, Confocal images of GFP-tagged FIE and mutants in protoplasts. BF, bright field. NLS–mC denotes nuclear HY5–mCherry as a control for protoplasts co-transfection and nuclear localization. Scale bars, 10 μm. Images are representative of 10 protoplasts from three biological repeats. e, Immunoblot analysis of GFP-tagged FIE variants expressed in protoplast and transgenic plants. Tubulin was used for the loading control. Data are representatives of three biological replicates. f, g, The dynamics of GFP-FIE during glucose starvation. f, Confocal images of GFP-FIE from 2–6 d in the root elongation zone in glucose-free medium. g, Quantitative confocal imaging of GFP-FIE C/N ratio. h, i, j, Glucose stimulates dynamic nuclear translocation of GFP-FIE after starvation in the root elongation zones. h, Confocal images of GFP-FIE without or with 25 mM glucose stimulation for 6 h in starved seedlings (5 d). i, Quantitative confocal imaging of GFP-FIE C/N ratio. j, Time-lapse live imaging of glucose stimulated nuclear translocation of GFP-FIE in the root elongation zone. Representative images were taken from Supplementary Video at 1–4 h time points after 25 mM glucose stimulation. The experiment was repeated three times with similar results. b, f, i, In the boxplots, data were analysed from more than 15 cells from three experiments, and are expressed as mean ± s.d. Centre line, median; box limits, 25th and 75th percentiles; the whiskers indicate data's minimum and maximum; the points represent each individual value. Statistical significance was determined by unpaired two-sided Student’s t test. c, g, h, j, Images are representative of six seedlings from three biological repeats. Scale bar, 25 μm.
