Physical clustering of FLC alleles during Polycomb-mediated epigenetic silencing in vernalization

Abstract

Vernalization, the promotion of flowering by cold, involves Polycomb-mediated epigenetic silencing of FLOWERING LOCUS C (FLC). Cold progressively promotes cell-autonomous switching to a silenced state. Here, we used live-cell imaging of FLC-lacO to monitor changes in nuclear organization during vernalization. FLC-lacO alleles physically cluster during the cold and generally remain so after plants are returned to warm. Clustering is dependent on the Polycomb trans-factors necessary for establishment of the FLC silenced state but not on LIKE HETEROCHROMATIN PROTEIN 1, which functions to maintain silencing. These data support the view that physical clustering may be a common feature of Polycomb-mediated epigenetic switching mechanisms.

Keywords: FLC; Polycomb; epigenetic; nuclear organization; vernalization.

Figure 1.

Monitoring FLC in root nuclei using an FLC-lacO transgene. (A) The lacO array was inserted downstream from the polyadenylation site of FLC. (B) Arabidopsis root showing expression of LacI-YFP in nuclei predominantly in the differentiation zone (bar, 100 μm). (C–F) Representative fluorescence images of nuclei from region 2 showing multiple FLC foci. (G,H) Representative fluorescence images of nuclei from meristematic cells (from region 1) (bars, 5 μm).

Figure 2.

Cold-induced clustering of FLC-lacO. (A–D) Representative fluorescence images of Arabidopsis root cells (from region 2) (see Fig. 1B) in plants grown in cold for 2 wk (and imaged immediately afterward) showing clustering of FLC-lacO loci. (E,F) Clustering of FLC-lacO copies in meristematic cells in plants cold-treated for 2 wk. LacI-YFP expression was induced using a 1% ethanol vapor treatment for 1.5 h. (G) Quantification of the FLC-lacO foci (30 cells counted in three different roots) from cells in region 2. (H,I) Representative fluorescence images of nuclei from cells in region 2 expressing a lacO-only transgene. Plants were either nonvernalized (NV) or vernalized for 2 wk (+V). (J) Quantification of the lacO foci (30 cells counted in three different roots) from cells in region 2. (K) FLC-lacO transgene with the lacO array inserted at the BstEII restriction site in FLC intron 1 of the genomic FLC sequence. (L,M) Representative fluorescence images of FLC-lacO-Bst transgene in root cells from region 2 nonvernalized (L) and vernalized for 2 wk (M). (N) Quantification of the FLC-lacO-Bst foci from cells in region 2 (30 cells counted in three different roots) nonvernalized and vernalized for 2 wk (bars, 5 μm).

Figure 3.

The clustering of FLC-lacO is impaired in vrn2 and vrn5 but not in lhp1. (A) Region 2 root cells either nonvernalized (NV) or vernalized for 2 wk (+V). (B) vrn5 nonvernalized (NV) or vernalized for 2 wk (+V). (C) Quantification of FLC-lacO foci in 30 vrn2 cells in region 2. (D) Quantification of FLC-lacO foci in 30 vrn5 cells in region 2. (E) Images of FLC-lacO transgene in lhp1 background nonvernalized (NV) or vernalized for 2 wk (+V). (F) Quantification of FLC-lacO foci in 50 lhp1 cells in region 2. (G,H) Representative fluorescence images of nuclei from meristematic cells harboring the FLC-lacO from plants nonvernalized (NV) or vernalized for 2 wk (+V) in wild type (G) and vrn5 (H) (bars, 5 μm).

Figure 4.

The clustering of FLC-lacO increases quantitatively with increasing cold exposure. (A) Time course of FLC-lacO clustering after different weeks of cold (XT0). The number of foci (one, two, or more than two) was counted in 10 randomly selected nuclei in five different plants. (B) The same analysis but after cold the plants were grown for 7 d (T7) at 20°C before imaging. The number of foci (one, two, or more than two) was counted in 10 randomly selected nuclei in five different plants. (C) Quantification of the proportion of cells in region 2 showing one FLC-lacO cluster after 2 wk of cold and at post-cold time points T7 (7 d post-transfer from cold) and T14. (D) Quantification of cells in the meristematic region showing one FLC-lacO cluster. FLC-lacO clustering in the meristem was quantified in nonvernalized (NV) plants and plants vernalized for different lengths (2–5 wk) as well as at the respective T7 time point. For this analysis, at least three different plants were used for quantification at each time point. (E) Clustering dynamics parallel the cold-induced increases in H3K27me3 at the nucleation site. With increasing cold exposure, H3K27me3 levels increase at the nucleation site (end of exon 1–start intron 1), the timing of which is coincident with the reduction in FLC-lacO foci number. FLC is fully silenced after 6 wk of cold, and this is associated with high H3K27me3 and maintenance of the clustering during subsequent growth at warm temperatures. Gray data points represent the increase of H3K27me3 at the nucleation site normalized to H3 and SHOOT MERISTEMLESS (data from Angel et al. 2011). Black data points represent the frequency of one FLC–lacO cluster.