The Arabidopsis thaliana vernalization response requires a polycomb-like protein complex that also includes VERNALIZATION INSENSITIVE 3

Abstract

In Arabidopsis thaliana, the promotion of flowering by cold temperatures, vernalization, is regulated via a floral-repressive MADS box transcription factor, FLOWERING LOCUS C (FLC). Vernalization leads to the epigenetic repression of FLC expression, a process that requires the polycomb group (PcG) protein VERNALIZATION 2 (VRN2) and the plant homeodomain protein VERNALIZATION INSENSITIVE 3 (VIN3). We demonstrate that the repression of FLC by vernalization requires homologues of other Polycomb Repressive Complex 2 proteins and VRN2. We show in planta that VRN2 and VIN3 are part of a large protein complex that can include the PcG proteins FERTILIZATION INDEPENDENT ENDOSPERM, CURLY LEAF, and SWINGER. These findings suggest a single protein complex is responsible for histone deacetylation at FLC and histone methylation at FLC in vernalized plants. The abundance of the complex increases during vernalization and declines after plants are returned to higher temperatures, consistent with the complex having a role in establishing FLC repression.

Fig. 1.

Epitope tagging constructs, protein expression, and complementation of mutant phenotypes. (A) Outline of the T-DNA region of pFLAG-GW (pCH252; ref. 24), pHA-GW, and pTAP-GW. RB, T-DNA right border; 35S, CaMV 35S promoter; npt II, neomycin phosphotransferase; hpt, hygromycin phophotransferase; Tnos, nopaline synthase terminator; cm^R, chloramphenicol-resistance gene; ccdB, conditional cell death B gene; 3X F, 3X FLAG epitope; LB, T-DNA left border; attR1 and attR2, Gateway recombination sites; 6xHA, 6x HA epitope; bar, phosphinothricin-resistance gene; TAP, tandem affinity purification epitope. (B) Additional peptide sequence added to proteins expressed from pHA-GW. (C) Western blot detection of VRN2-FLAG, SWN-FLAG, CLF-FLAG, FIE-HA, SWN-HA, CLF-HA, VIN3-HA, and VRN2-TAP in transformed Arabidopsis plants. Estimates of fusion protein size are indicated based on migration relative to protein standard markers. (D) Leaf number at bolting of fca-1, vrn2-1 fca-1, vrn2-1 fca-1 + VRN2-FLAG (T2 plants), vrn2-1 fca-1 + VRN2-TAP (T3 plants), Col^FRISf2, vin3-4, and vin3-4 + VIN3-FLAG (T2 plants). Seed from transgenic lines and control lines were vernalized on plates for 4 weeks then transferred to LD. Values are the average rosette leaf number at flowering, error bar is SD. Experiments with VRN2-FLAG (open columns), VRN2-TAP (gray columns), and VIN3-FLAG (black columns) were conducted separately (NF; not flowered). (E) The FIE-HA construct was used to transform heterozygous fie-11 (25), and the phenotypes of seeds in the siliques of the resulting T1 plants scored as normal or shriveled. Seed from heterozygous FIE/fie-11 plants are 50% shriveled (26) due to the maternal embryo lethality of fie. FIE/fie-11 plants carrying the FIE-HA transgene are 74% wild type, showing that the fie-11 mutation is complemented (see Supporting Text, which is published as supporting information on the PNAS web site).

Fig. 2.

Expression of VRN2-FLAG, SWN-HA, CLF-HA, FIE-HA, and VIN3-HA proteins. (A) Western blot analysis of total protein extracts from plants expressing VRN2-FLAG, SWN-HA, CLF-HA, FIE-HA, or VIN3-HA grown for 14 LD (NV), 12 LD + 4-week vernalization (V), and 12 LD + 4-week vernalization + 2 LD, (V + 2). Blots were reprobed with anti-tubulin antibody to confirm uniform loading. (B) Quantitative RT-PCR analysis of expression of endogenous VRN2, SWN, CLF, FIE, and VIN3 mRNAs in wild-type C24 plants (error bars are SD) and RT-PCR analysis of VRN2-FLAG mRNA, (Vx2, a PCR with twice the amount of cDNA added to demonstrate a 2-fold difference in mRNA content could be detected). (C) Time course of VRN2-FLAG, FIE-HA, and VIN3-HA protein expression. Plants were grown for 12 LD then transferred to 4°C, samples were taken after 0, 1, 8, 16, and 32 days at 4°C and 32 days at 4°C followed by 2 LD. (D) Organ specificity of VRN2-FLAG and VIN3-HA protein expression. Proteins extracted from shoot tips, leaves (cotyledons and first leaf pair), and roots of plants grown for 14 LD (NV) or 12 LD followed by 32 days at 4°C (V).

Fig. 3.

Size exclusion chromatography analysis of VRN2, FIE, SWN, CLF, and VIN3 epitope-tagged proteins in native extracts before and after vernalization. Protein extracts from plants before vernalization (NV) or after 4-week cold treatment (V) and separated through Superdex S200. Size markers (kilodaltons) are indicated along the top (V_o, void volume).

Fig. 4.

Coimmunoprecipitation of proteins associated with VRN2. (A) FLAG immunoprecipitates from vrn2-1 fca-1 + VRN2-FLAG lines retransformed with FIE-HA (1), CLF-HA (2), SWN-HA (3), and VIN3-HA (4) probed with anti-HA antibody. Plants grown for 12 LD (NV), 12 LD + 4-week vernalization (V), and 12 LD + 4-week vernalization + 2 LD (V + 2). HA tag control is a line expressing the relevant HA-tagged protein but not the VRN2-FLAG protein. Western blots (WB) are probed with anti-HA antibody. (B) Input protein extracts and FLAG immunoprecipitates treated with micrococcal nuclease (+) or untreated (−) probed with anti-HA antibody (this experiment was carried out by using independent transgenic lines to those used in A), blots are probed with anti-HA-HRP antibody-enzyme conjugate. (C) HA immunoprecipitates treated with micrococcal nuclease (+) or untreated (−) probed with anti-FLAG-HRP antibody-enzyme conjugate (by using the same plant lines as in B). (D) Calmodulin-agarose purified proteins from vrn2-1 fca-1 + VRN2-TAP plants probed with anti-FIE antibodies (also detects protein A epitope in VRN2-TAP). A single box separated by black lines are from a single image of a Western blot. ∗, IgG detected by anti-mouse secondary antibody is visible in blots probed with anti-FLAG.

Fig. 5.

PcG proteins are required for vernalization-mediated repression of FLC. (A) Average leaf number at flowering of three T2 siFIE lines grown in LD conditions (NV) or 12 LD followed by 4 weeks of vernalization and returned to LD conditions (V) and FLC mRNA extracted after 12 LD (NV) or 12 LD plus 4 weeks vernalization (V) for the same lines. (B) Average leaf number at flowering and FLC, SWN, and CLF mRNA content of C24 wild-type and T2 plants carrying the siCLF-SWN transgene. Plants were either grown in LD conditions (NV) or vernalized for 4 weeks as a seed before growth under LD conditions (V). mRNAs were quantified by quantitative RT-PCR normalized to FDH. ∗, 3 of 19 line 1 V plants not flowered, 7 of 7 line 2 NV plants not flowered, and 10 of 12 line 2 V plants not flowered (NF) at the termination of the experiment; nd, not determined. (C) FLC and FIE mRNAs (measured by quantitative RT-PCR, normalized to FDH) in C24 wild-type, weak, and strong T1 siFIE plants grown for 19 LD (NV) or vernalized 4 weeks as a seed and grown for 19 LD (V + 19). Plants were killed for RNA isolation, and flowering time was not determined.