Extended Data Fig. 9: VIN3 expression, H3K27me3 enrichment at FLC, and GFP-FIE nuclear translocation during vernalization.
From: Glucose-driven TOR–FIE–PRC2 signalling controls plant development
a, VIN3 Induction by vernalization is not regulated by TOR. Seedlings were treated at 4 °C to induce vernalization for the indicated days without (Ctrl) or with TOR inhibitors (1 μM Torin2 and INK128), or in the inducible tor-es mutant. Data are relative to the UBC21 (At5g25760) expression as a control gene and normalized to non-vernalized (NV) levels of ctrl plants. Data show mean ± s.d. from 3 biological replicates, two-way ANOVA with Tukey’s multiple comparisons test. b, TOR deficiency reduces H3K27me3 levels at FLC. Relative H3K27me3/H3 level normalized to that of ctrl plants under non-vernalized (NV) condition as 1. Black boxes, exons. Red boxes, selected regions for H3K27me3 ChIP-qPCR analyses. bp, base pairs. FLC-3 and FLC-4,5 are located in the nucleation and spreading regions of H3K27me3 at FLC, respectively. c, d, Prolonged cold treatment prompts the nuclear localization of GFP-FIE in the elongation zones of roots. c, Confocal images of GFP-FIE during vernalization. Images are representative of six seedlings from three biological repeats. Scale bar, 25 μm. d, Quantitative confocal imaging of GFP-FIE in the root elongation zone. The cytoplasm/nucleus (C/N) signal intensity ratio of GFP-FIE at the single-cell level was measured by quantitative confocal imaging using the Leica LAS-X software. In the boxplot, 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. e, Vernalization induced H3K27me3 levels at FLC are compromised in AAAA/fie-FRI. Relative H3K27me3/H3 level was normalized to that of fie/Col-FRI plants under non-vernalized (NV) condition as 1. Data in b, e show mean ± s.d. from 3 biological replicates, two-way ANOVA with Tukey’s multiple comparisons test; NV, non-vernalized. V, vernalization days at 4 °C. T, postcold days at 22 °C.
