LHP1, the Arabidopsis homologue of HETEROCHROMATIN PROTEIN1, is required for epigenetic silencing of FLC

Abstract

Vernalization is the acceleration of flowering by prolonged cold that aligns the onset of reproductive development with spring conditions. A key step of vernalization in Arabidopsis is the epigenetic silencing of FLOWERING LOCUS C (FLC), which encodes a repressor of flowering. The vernalization-induced epigenetic silencing of FLC is associated with histone deacetylation and H3K27me2 and H3K9me2 methylation mediated by VRN/VIN proteins. We have analyzed whether different histone methyltransferases and the chromodomain protein LIKE HETEROCHROMATIN PROTEIN (LHP)1 might play a role in vernalization. No single loss-of-function mutation in the histone methyltransferases studied disrupted the vernalization response; however, lhp1 mutants revealed a role for LHP1 in maintaining epigenetic silencing of FLC. Like LHP1, VRN1 functions in both flowering-time control and vernalization. We explored the localization of VRN1 and found it to be associated generally with Arabidopsis chromosomes but not the heterochromatic chromocenters. This association did not depend on vernalization or VRN2 function and was maintained during mitosis but was lost in meiotic chromosomes, suggesting that VRN1 may contribute to chromatin silencing that is not meiotically stable.

Fig. 1.

FLC expression in different histone methyltransferase mutants. (A) Northern blot analyzing FLC expression in fca-1, vrn1-2 fca-1, and kyp-2 fca-1. Seedlings were grown for 19 days (NV) or vernalized 4 weeks as imbibed seeds and harvested after 16 days growth (+V). The blot was subsequently stripped and probed with 18S rDNA as a loading control. (B) Northern blot analyzing FLC expression in Columbia, suvh, suvr, vrn1-3, and vin3-5 single mutants in Columbia either nonvernalized (0) or vernalized as seeds for 2 or 5 weeks on soil and harvested after 15 days of growth. The blot was subsequently stripped and probed with TUBULIN (TUB) as a loading control. (C) RT-PCR performed on total RNA extracted from plants that were nonvernalized or vernalized for 2 weeks as seeds and harvested after 12 days of growth. Plants from two different seed lots of suvh2 were included.

Fig. 2.

Effect of lhp1 mutations on FLC repression by vernalization. Merged FLC/luciferase (pseudocolor) and photographic (black/white) images taken of Col FRI FLC/luciferase grown for 3 weeks either without vernalization (NV) or after 6 weeks vernalization (+V) (A) and lhp1-3 FRI FLC/luciferase nonvernalized (NV) or vernalized (+V) (B). (C and D) Northern blot time-course analysis of FLC expression in lhp1-3 single mutants and Columbia (C) or lhp1-3 FRI FLC/luciferase and Col FRI FLC/luciferase (D). Plants were harvested from plates at three stages of nonvernalized growth (NV6–NV19 days), immediately after 6 weeks cold (V0) and three stages of growth after vernalization (V6–V19). Blots were subsequently stripped and probed with TUBULIN (TUB) as a loading control.

Fig. 3.

VRN1/GFP associates with mitotic chromosomes and not heterochromatin. (A) A genomic VRN1/GFP construct was produced by mutating the VRN1 stop codon into a BamHI site for in-frame fusion with GFP coding sequence. (B) GFP expression in root tissue of VRN1/GFP vrn1-2 fca-1. (C) Time series showing association of VRN1/GFP with chromosomes during mitosis, images taken at 30-second intervals. (D) Immunodetection in wild-type Ler using VRN1 (green) and H3K4me2 (red) antibodies. (E) Immunodetection in wild-type Ler using VRN1 (green) and H3K9me2 (red) antibodies. (F) Quantitative line profiles of DAPI (blue), VRN1 (green), and H3K4me2 (red). (G) Quantitative line profiles of DAPI (blue), VRN1 (green), and H3K9me2 (red). (Scale bars, 5 μm.)

Fig. 4.

Immunodetection of anther preparations. (A) DAPI staining (Left, white) and VRN1 immunodetection (Right, green). The larger meiotic cells, which are weakly stained with VRN1 antibody, are marked by arrows. (B) The H3K4me2 antibody (red) is able to penetrate and label the somatic binucleate tapetum nuclei (above, binucleate and stronger DAPI) and mid-prophase meiotic nuclei (below, mononucleate and weaker DAPI stain). (C) The VRN1 antibody (green) labels somatic (above) but not midprophase meiotic nuclei (below). (D) The H3K9me2 antibody (red) labels the chromocenters of somatic (above and Right) and mid-prophase meiotic nuclei (below, Left). (E) The VRN1 antibody (green) labels the tapetum nuclei (below) but not the nuclei of microspores (above). (F) Selected AtGenExpress data (34) showing drop in VRN1 expression in mature pollen. (Scale bars, 5 μm.)