Variation in the epigenetic silencing of FLC contributes to natural variation in Arabidopsis vernalization response

Abstract

Vernalization, the cold-induced acceleration of flowering, involves the epigenetic silencing of the floral repressor gene FLOWERING LOCUS C (FLC). We investigated the molecular basis for variation in vernalization in Arabidopsis natural accessions adapted to different climates. A major variable was the degree to which different periods of cold caused stable FLC silencing. In accessions requiring long vernalization, FLC expression was reactivated following nonsaturating vernalization, but this reactivation was progressively attenuated with increasing cold exposure. This response was correlated with the rate of accumulation of FLC histone H3 Lys 27 trimethylation (H3K27me3). Thus, variation in epigenetic silencing of FLC appears to have contributed to Arabidopsis adaptation.

Figure 1.

Variation in vernalization response and FLC expression of the selected accessions. (A) Flowering time was assayed as final LN. Data is mean of at least three experiments with standard error. Nonvernalized plants (NV) of four Scandinavian accessions did not flower when the experiments were terminated in 5 mo since germination, whereas Edi-0 and H51 completed flowering but were still very late. (B) FLC expression after different lengths of cold. (W) Weeks of cold period. (C) VIN3 expression in the accessions assayed after different periods of cold. Semiquantitative PCR analysis was used to analyze VIN3 RNA levels (Supplemental Material). Twenty-day-old nonvernalized (NV) whole seedlings were compared with 7-d-old seedlings exposed to cold for 2, 4, 6, and 8 wk and up to 14 wk for Lov-1, Ull-2-5, and H51 and then harvested in the cold.

Figure 2.

Variation in the maintenance of repression of FLC. FLC expression was assayed by Northern hybridization of RNA extracted from whole seedlings. (A) Nonvernalized (NV) plants grown for different days were compared with seedlings grown for 7 d, vernalized for 4 wk (4W), and then transferred to 23°C for 0, 10, 20, or 30 d. (B) Repression of FLC following different periods of cold. Thirty-day-old nonvernalized plants (at a developmental stage similar to the 30-d-post-vernalization plants) were compared with seedlings grown at 23°C in long-day photoperiods for 0, 10, 20, and 30 d following each cold treatment (4W, 6W, 8W, 10W, 12W). The 30-d value is missing for H51 as the plants had already flowered.

Figure 3.

QTL analysis to map genes contributing to the variation in vernalization response. Each chromosome is shown separately with the markers used indicated as triangles. LOD (Logarithm of the odds) score is calculated by the MapMaker Version 3.0b with LOD 3 considered significant. The map positions of candidate genes are indicated.

Figure 4.

ChIP analysis (A) Genomic structure of Arabidopsis FLC and the region examined—FLC-A (as indicated). (B) PCR analysis of DNA immunoprecipitated with anti-trimethyl-histone H3 Lys 27 (H2K27me3). (C) Quantification of the H3K27me3 enrichment on FLC locus relative to the heterochromatic locus Ta3 (Johnson et al. 2002). The graph comprises the quantification of two ChIP experiments (error bars are standard deviation of six replicates). Chromatin was prepared from nonvernalized seedlings (20 d old) and seedlings that had been vernalized 5 wk following 7 d pregrowth at 23°C and then grown for 15 d after vernalization at 23°C.

Figure 5.

Model proposing that quantitative accumulation of epigenetic modifications in FLC contributes to natural variation of vernalization response. Three phases of vernalization are envisaged: an insensitive phase, a phase where silencing occurs (and histone modifications accumulate), and a phase where FLC is fully silenced. The dashed and solid lines, estimated from the ChIP analysis shown in Figure 4C, represent the accumulation of epigenetic marks in Edi-0 and Lov-1, respectively. Due to a high initial level and higher accumulation rate, full silencing would be reached after 4 wk of cold in Edi-0 and 10–12 wk of cold in Lov-1.