Fig 1.
Flowering time analysis of flc mutants.
(A) Flowering-related traits were measured at 22°C under LD, without vernalization. Means from 3 to 12 replicates per line are shown, with the number of measured lines indicated at the bottom of each graph. The black dashed horizontal line indicates Col-0. (B) Correlation between flowering times (S1 Table) and FLC expression levels (S2 Table) in unvernalized plants. Black dashed vertical line indicates an FLC relative expression level of 5 a.u., which divided the mutants into knockout (KO) and knockdown (KD) lines. To better illustrate the phenotypes of the KO mutants only, their values are shown as violin plots to the right in each subpanel. flc KO, DTF mean [±sd] 21.8 [±5.6], range 13.9 to 38.0, RLN 14.6 [±6.5], range 5.9 to 32.6. (C) Correlation of the mutant-versus-wild type phenotypic ratios (log2[DTFWildtype/DTFMutant]) versus the wild-type phenotypic values. Left, DTF, Pearson’s r, all lines r = 0.77, p < 2.2e−16, df = 108, KO only r = 0.81, p = 5.697e−10, df = 36. Middle, RLN, all lines r = 0.06, p = 0.58, df = 96; KO only r = 0.35, p = 0.037, df = 32. Right, CLN, all lines r = −0.017, p = 0.87, df = 96; KO only r = 0.28, p = 0.11, df = 32. Simple linear model values: multiple r2adj[p], log2(DTF ratio KO) − log(FLCWildtype): 0.30 [0.0002], log2(RLN ratio KO) − log(FLCWildtype): 0.18 [0.0068], log2(CLN ratio KO) − log(FLCWildtype): 0.02 [>0.05]. The dashed line indicates 0. (D) Correlation between vernalization sensitivity (log2) after eight weeks of vernalization and FLC expression levels before vernalization. Black dashed horizontal line indicates Col-0. Mutants: log2(vernalization sensitivity DTF): mean [±sd] 0.23 [±0.21], range −0.20 to 0.82; log2(vernalization sensitivity RLN): mean [±sd] 0.40 [±0.40], range −0.42 to 1.42; wild types: log2(vernalization sensitivity DTF): mean [±sd] 1.41 [±0.49], range 0.17 to 2.41; log2(vernalization sensitivity RLN): mean [±sd] 1.57 [±0.35], range 0.59 to 2.26; KO mutants only: log2(vernalization sensitivity DTF); mean [±sd] = 0.12 [±0.19]), range: −0.20 to 0.5; log2(vernalization sensitivity RLN): mean [±sd] = 0.20 [±0.38]), range: and −0.42 to 0.97. The distribution of vernalization sensitivities, as shown by the histograms, was analyzed separately for KO and KD populations. (E) Pearson’s r of flowering traits before and after vernalization. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; n.s., not significant. The data underlying this figure can be found in S1 Data and https://doi.org/10.5281/zenodo.15403194.
Fig 2.
Quantitative genetic analysis of FLC-independent flowering.
(A) Distribution of flowering time (DTF) of F2 individuals, including the mean value of the respective flc mutant parent lines. The numbers of F2 individuals per population are shown on top. The arrow indicates a single outlier of population C3-090, which was excluded in the correlation shown in C. (B) Correlation between the phenotypic range of the parents and of the F2 populations (range = F2(max) − F2(min)). Simple linear model, DTF, excluding a single outlier of population C3-090 (see panel A): r2adj = 0.65, p = 0.0006; including the outlier: r2adj = 0.37, p = 0.016; RLN, r2adj = 0.52, p = 0.0033; CLN, r2adj = 0.15, p = 0.109. (C) Flowering trait correlations in F2 populations. Range of Pearson’s r: RLN versus DTF 0.41 to 0.85; RLN versus CLN 0.31 to 0.80; DTF versus CLN 0.13 to 0.68; all p < 0.0001. (D) Summary of QTL analysis. On top, schematic representation of chromosomes, with black dots representing centromeres. The physical position of genes with a known role in flowering is shown below (S6 Table), with color indicating the published classification [33,34]. LOD scores were summed over a non-overlapping moving window of size 100 kb and are shown at the center of the window. The detected QTL clusters 1–4 are indicated on top, the widths of the gray boxes indicate the size of each cluster. Cluster 1 is a 2 Mb region (±1 Mb from QTL LOD peak at 24.675 Mb). Cluster 2 is a 7 Mb region (0–7 Mb) on chromosome 5. Clusters 3 and 4 are smaller and contain fewer QTL. The QTL intervals (95% Bayes interval) are shown as horizontal lines, and the physical positions of a priori flowering candidate genes are indicated as dashed vertical green lines. (E) Proportional explained additive variation (PEV) of QTL co-localizing with FT. (F) Correlation of FT expression levels with flowering traits. Mutants on the lower and wild types on the upper triangle. Pearson’s r, mutants, −0.57 to −0.45, p < 0.001, 109 d.f.; wild types, DTF: r = −0.34, p < 0.01, RLN r = −0.51, p < 0.001, 59 d.f. The significance of the correlations is indicated by *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; n.s., not significant. The data underlying this figure can be found in S1 Data and https://doi.org/10.5281/zenodo.15403194.
Fig 3.
Discovery of very early flowering genotypes in Italy.
(A) Comparison of FT phylogeny with flowering time at 16°C (FT16) [39] and FLC expression (FLC normCounts) [42]. The earliest flowering parents of the five F2 populations with a QTL at FT are indicated with an arrow. (B) Early-flowering plants in the wild (at the Angit site). (C) Southern Italian sampling sites. Schematic map on top shows the Angit site. Colors roughly indicate apparent flowering times. (D) Flowering time of progeny from wild plants and control genotypes in the greenhouse (22°C long days). “IDXXXX” is the wild-type strain ID from the 1001 Genomes Project (https://1001genomes.org). The subsequent digits are from a consecutive numbering system for lines selected in the T2 and T3 generations. Similar letters indicate no significant difference in total leaf number (RLN + CLN) (ANOVA with post hoc Tukey HSD, p.adj. < 0.05). The data underlying this figure can be found in S1 Data and https://doi.org/10.5281/zenodo.15403194.
Fig 4.
Growth trajectories of flc mutants.
(A) Projected rosette area (PRA) of 61 wild types and 112 mutants per day (x-axis). PRA of the 8376 wild type could not be determined. Differences between mutants and wild types on each day were not significant (n.s.) (Mann–Whitney U rank test, Bonferroni correction, p.adj. > 0.05). (B) Clustering by PRA (log2) of 110 mutants versus wild types. The number of days with significantly different projected rosette area measurements (Mann–Whitney-U-test, Benjamini–Hochberg correction, p.adj. > 0.05) is indicated. A schematic representation shows the cluster size differences. (C) Cluster mean values of the ratio on each day (dots) and over all days (horizontal lines, log2). The values between 25th and 75th percentiles are shown as ribbons. (D) FLC levels (log10) and flowering time of mutants in cluster 1–3. The data underlying this figure can be found in S1 Data and https://doi.org/10.5281/zenodo.15403194.
Fig 5.
Evidence for pleiotropic roles of FLC.
(A) Heatmap of the log2FC values of differentially expressed genes (DEGs) present in at least one contrast. Flowering time data (DTF) from the experiment shown in Fig 1A and 1B. (B) GO enrichment analysis. The top 5 hits (−log(FDR)) are shown with an FDR cutoff of 0.01. (C) Relative expression levels [a.u.] of AZI1 and NRT1.11 in all the wild types and the mutants. AZI1, mean wild types: 0.92 a.u., mean mutants: 0.47 a.u., NRT1.11, mean wild types: 0.47 a.u., mean mutants: 1.37 a.u. (D) Nitrogen isotope composition (δ15N [‰]) in a subset of 29 early flowering mutants and the corresponding wild types (means of three biological replicates). Mean [±se] mutants: 3.17‰ [±0.26]; wild types: 2.43‰ [± 0.46]; Mann–Whitney U rank test, p > 0.05. (E) Difference in δ15N (δ15Ndiff = δ15NWildtype − δ15NMutant) between wild type and the respective mutant. Turquoise indicates significant differences, two-sided Student's t test, Benjamini–Hochberg correction, p.adj. < 0.05. The data underlying this figure can be found in S1 Data and https://doi.org/10.5281/zenodo.15403194.