A Single Amino Acid Change in the Enhancer of Zeste Ortholog CURLY LEAF Results in Vernalization-Independent, Rapid Flowering in Arabidopsis 1[C][W][OA]

Many strains of Arabidopsis ( Arabidopsis thaliana ) require exposure to prolonged cold for rapid ﬂowering, a process known as vernalization. Vernalization in Arabidopsis results in the suppression of FLOWERING LOCUS C ( FLC ), a repressor of ﬂowering. In a screen for mutants that no longer require vernalization for rapid ﬂowering, we identiﬁed a dominant allele of the Enhancer of Zeste E(z) ortholog CURLY LEAF ( CLF ), clf-59 . CLF is a Polycomb Group gene, and the clf-59 mutant protein contains a proline-to-serine transition in a cysteine-rich region that precedes the SET domain. Mutant plants are early ﬂowering and have reduced FLC expression, but, unlike clf loss-of-function mutants, clf-59 mutants do not display additional pleiotropic phenotypes. clf-59 mutants have elevated levels of trimethylation on lysine 27 of histone H3 (H3K27me3) at FLC . Thus, clf-59 appears to be a gain-of-function allele, and this allele represses FLC without some of the components required for vernalization-mediated repression. In the course of this work, we also identiﬁed a marked difference in H3K27me3 levels at FLC between plants that contain and those that lack the FRIGIDA ( FRI ) gene. Furthermore, FRI appears to affect CLF occupancy at FLC ; thus, our work provides insight into the molecular role that FRI plays in delaying the onset of ﬂowering.

The switch from vegetative to reproductive growth is an important developmental transition in the life history of flowering plants. The proper timing of this switch is critical for reproductive success, especially in temperate climates where flower and seed production often needs to align with favorable weather conditions and/or the presence of pollinators. To ensure that flowering occurs at an optimal time of the year, plants have evolved mechanisms to sense and respond to seasonal environmental cues. One such cue is the prolonged cold of winter. The promotion of flowering by cold occurs through a process known as vernalization (for review, see Sung and Amasino, 2005;Dennis and Peacock, 2007).
Among accessions of Arabidopsis (Arabidopsis thaliana), there is variation in the requirement for vernalization. In winter-annual accessions, flowering is delayed unless plants undergo vernalization; summerannual accessions do not require prolonged cold for rapid flowering. Natural variation at two loci in Arabidopsis, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC), has a major influence on the requirement for vernalization (Napp-Zinn, 1979;Koornneef et al., 1994;Lee et al., 1994a). Many summer-annual accessions, including Columbia (Col), Wassilewskija (Ws), and Landsberg erecta, flower rapidly without a prolonged cold treatment because they carry mutations in FRI (Johanson et al., 2000). FRI acts to delay flowering through the up-regulation of FLC, a MADS box transcription factor that represses flowering. Vernalization leads to FLC repression in the presence of FRI and is thus able to supersede the ability of FRI to activate FLC (Michaels and Amasino, 1999;Sheldon et al., 1999). Thus, under natural conditions, the role of the FRI/FLC system is likely to repress flowering in autumn. The prolonged cold of winter then results in vernalization, which is manifest in the repression of FLC and rapid flowering in the spring.
Arabidopsis also contains a group of genes known collectively as the autonomous pathway that, in contrast to FRI, act as repressors of FLC. LUMINIDEPEN-DENS (LD), FCA, FLOWERING LOCUS D (FLD), and FVE are among this group of genes (Lee et al., 1994b;MacKnight et al., 1997;He et al., 2003;Ausin et al., 2004). FCA functions in RNA-mediated gene silencing (Baurle et al., 2007). FLD and FVE function in complexes that modify chromatin (He et al., 2003;Ausin et al., 2004). Mutations in autonomous pathway genes result in elevated FLC expression and delayed flowering (Michaels and Amasino, 2001). Similar to winterannual accessions with an active FRI allele, delayed flowering in autonomous pathway mutants can be suppressed through vernalization.
Chromatin modification appears to play a large role in setting the expression level of FLC. In genetic backgrounds that favor elevated FLC expression, active chromatin modifications accumulate at FLC, and several proteins required for the deposition of these modifications have been identified (for review, see Schmitz and Amasino, 2007). During the course of cold exposure, FLC expression is suppressed (Michaels and Amasino, 1999;Sheldon et al., 1999), and several signatures of silenced chromatin accumulate at FLC (Bastow et al., 2004;Sung and Amasino, 2004;Mylne et al., 2006;Schubert et al., 2006;Sung et al., 2006b;Finnegan and Dennis, 2007;Schmitz et al., 2008). One such signature is trimethylation on Lys-27 of histone H3 (H3K27me3). Changes in the amount and distribution of H3K27me3 at FLC both during and after cold treatment have been described (Schubert et al., 2006;Sung et al., 2006a;Finnegan and Dennis, 2007). H3K27me3 is carried out by Polycomb-Group (PcG) complexes that contain orthologs of Drosophila melanogaster Polycomb Repressive Complex 2 (PRC2) components and other plant-specific proteins, such as VERNALIZATION INSENSITIVE3 (VIN3; Wood et al., 2006;De Lucia et al., 2008). PcG proteins have also been shown to repress FLC in accessions that do not require vernalization for rapid flowering (Jiang et al., 2008).
The Arabidopsis protein CURLY LEAF (CLF) is an ortholog of the Drosophila PRC2 component Enhancer of Zeste [E(z)], a methyltransferase with specificity for H3K27 (Goodrich et al., 1997;Czermin et al., 2002;Muller et al., 2002). CLF was initially characterized as a suppressor of floral homeotic genes, including AGAMOUS (AG; Goodrich et al., 1997). In addition, recent work has indicated a role for CLF in the suppression of FLC and the floral promoter FT in a nonvernalized, rapid-flowering accession (Jiang et al., 2008). CLF has also been shown to play a role in the repression of FLC by vernalization (Wood et al., 2006).
Here, we describe a gain-of-function allele of CLF that was isolated in a screen for suppressors of delayed flowering in an autonomous pathway mutant background. This allele leads to elevated H3K27me3 at FLC chromatin, FLC mRNA repression, and rapid flowering in backgrounds that would otherwise require vernalization. In addition, we also demonstrate that the presence of FRI has a substantial effect on the degree of H3K27me3 accumulation and CLF occupancy at FLC.

A Mutation in CLF Suppresses FLC-Mediated Delayed Flowering via Reduced FLC Expression
Mutants with lesions in the autonomous pathway gene LD are delayed in flowering due to elevated expression of FLC (Michaels and Amasino, 2001). An ethyl methanesulfonate mutagenesis carried out in an ld-3 mutant background (Ws accession) resulted in the isolation of several mutants with rapid flowering phenotypes. One mutation suppressed delayed flowering in a semidominant manner ( Fig. 1A; the mutation is designated clf-59) and mapped to a region that included CLF (Goodrich et al., 1997). Given the dominant nature of this mutation, it was not possible to confirm gene identity by transgenic complementation with the wild-type gene product. Instead, the CLF genomic region was cloned from the mutant and introduced into an ld-3 background. Plants carrying the mutant transgene flowered more rapidly than nontransformed plants (an example of a transgenic versus parental line is shown in Fig. 1B). An introduced copy of wild-type CLF did not alter flowering time (data not shown). Thus, this semidominant mutation is an allele of CLF, which we designate clf-59.
Mutations in autonomous pathway genes lead to delayed flowering via elevated expression of FLC (Michaels and Amasino, 2001). Similarly, plants carrying a functional copy of FRI also display FLC-dependent late flowering (Koornneef et al., 1994;Lee et al., 1994a;Michaels and Amasino, 1999;Sheldon et al., 1999). To evaluate whether the effect on flowering was specific to ld, clf-59 was combined genetically with fca, another autonomous pathway mutant (MacKnight et al., 1997), and a Col line that contains a functional copy of FRI (FRI-Col; Lee et al., 1994a); in both cases, clf-59 suppressed late flowering in a semidominant manner (Fig.  1C). Rapid flowering was also observed when clf-59 was introduced transgenically into the autonomous pathway mutants fld (He et al., 2003) and fve (Ausin et al., 2004;Fig. 1D). Because autonomous pathway mutants and FRI-containing plants are delayed in flowering due to elevated FLC levels (Michaels and Amasino, 2001), rapid flowering in a clf-59 background could result from FLC repression. Indeed, ld-3, fca-1, and FRI-Col all displayed reduced levels of FLC mRNA when combined with clf-59 (Fig. 1E). Thus, rapid flowering in clf-59 results, at least in part, from decreased expression of FLC.

clf-59 Contains a Single Amino Acid Change in a Cys-Rich Motif
The CLF protein contains several conserved motifs (Fig. 2). The C5 motif functions in protein-protein interactions (Chanvivattana et al., 2004;Ketel et al., 2005), and the SET domain contains the histone methyltransferase catalytic site (Rea et al., 2000). In CLF and other E(z)-like proteins, a conserved Cys-rich region precedes the SET domain. clf-59 contains a single amino acid substitution within this region that converts a conserved Pro to a Ser (P704S; Fig. 2).
In addition to CLF, Arabidopsis contains two additional E(z)-like genes, SWINGER (SWN) and MEDEA (MEA; Grossniklaus et al., 1998;Chanvivattana et al., 2004). MEA is essential for endosperm development (Grossniklaus et al., 1998). SWN is partially redundant in function with both CLF and MEA (Chanvivattana et al., 2004;Wang et al., 2006). For example, CLF and SWN act redundantly in the silencing of FLC as a result of vernalization. Both clf and swn single mutants can become vernalized, but down-regulation of both genes simultaneously results in vernalization insensitivity (Wood et al., 2006). Given the pronounced effect of clf-59, it was of interest to determine whether the P-to-S substitution in SWN and MEA would also cause rapid flowering or possibly other developmental aberrations. Accordingly, this substitution was introduced into genomic clones of both SWN and MEA. The mutant genes were subsequently transformed into Col and FRI-Col backgrounds. All transgenic plants flowered synchronously with the nontransformed parents (data not shown). Thus, the P-to-S transition in MEA and SWN does not cause enhanced FLC repression. In addition, no visual phenotypic differences were observed in plants containing modified SWN or MEA at any stage of development. Possible reasons for lack of a phenotype in plants with modified SWN or MEA include, but are not limited to, wild-type CLF masking an effect of these modified transgenes or simply the lack of an effect of this substitution on SWN or MEA activity. As noted below, the only effect of the P-to-S transition in clf-59 that we observe is on flowering and FLC expression.

clf-59 Mutants Do Not Display Phenotypes Seen in Either clf or ag Loss-of-Function Mutants
Loss-of-function mutations in clf cause a number of phenotypes, including upward curling of leaves and abnormal flower development (Goodrich et al., 1997). These phenotypes are largely due to ectopic expression of the floral homeotic gene AG (Mizukami and Ma, 1992;Goodrich et al., 1997). On the other hand, plants that do not express AG fail to produce stamens and carpels (Bowman et al., 1989;Yanofsky et al., 1990). Given that CLF is a regulator of AG, it was of interest to look for altered AG expression in a clf-59 background. The presence of clf-59 does not result in hypersuppression AG similar to the effects of this allele on FLC, at either the mRNA or phenotypic level. Specifically, in a clf-59 background, expression of AG in either seedling (Fig. 3A) or inflorescence tissue (Fig.  3B) is indistinguishable from the wild type nor are there any ag reduction-of-function phenotypes in clf-59 mutants (Fig. 3, C and D). In addition, no phenotypes characteristic of clf loss of function (and the associated ectopic expression of AG) are observed in clf-59 mutants. Thus, the clf-59 protein does not appear to affect all targets of wild-type CLF.
The effect of a clf loss-of-function allele, clf-28, on flowering time and FLC expression was also examined. clf-28 hastened flowering in FRI-Col but to a much lesser degree than clf-59 ( Fig. 4A) and, unlike clf-59, did not have a significant effect on FLC expression (Fig. 4B). This distinction indicates that clf-59 is a gainof-function allele with respect to FLC expression as opposed to a dominant negative.
In the absence of FRI, clf-28 also hastened flowering despite slightly elevated FLC levels (Fig. 4, A and B).
Such FLC derepression in a loss-of-function clf mutant has been reported previously (Jiang et al., 2008). Early flowering in clf loss-of-function mutants likely results from derepression of FT (Jiang et al., 2008), a strong promoter of flowering that when overexpressed bypasses the repressive effects of FLC (Michaels et al., 2005).
Enrichment of H3K27me3 at FLC Chromatin Is Altered in clf-59 and Is Dependent on Genetic Background CLF catalyzes the methylation of H3K27. Previous studies have extensively examined the distribution of H3K27me3 across FLC and surrounding chromatin (Bastow et al., 2004;Sung and Amasino, 2004;Schubert et al., 2006;Finnegan and Dennis, 2007). The accumulation H3K27me3 at FLC that occurs as a result of vernalization initiates near the transcriptional start site and then spreads throughout the gene (Finnegan and Dennis, 2007); thus, we have focused our study on regions of FLC near the transcriptional start site. Levels of H3K27me3 were analyzed in nonvernalized FRI clf-59, Col, and FRI-Col using chromatin immunoprecipitation (ChIP). The level of H3K27me3 in FRI clf-59 was greater than that in FRI-Col, indicating that, in a FRI background, clf-59 is capable of elevating H3K27me3 levels in the absence of vernalization (Fig. 5, A and B).
When assaying H3K27me3 in clf-59 plants, a marked difference in H3K27me3 levels was observed between Col and FRI-Col (Fig. 5, A and B). To our knowledge, this is the first report of the effect of FRI on H3K27 methylation. Reduced H3K27me3 levels in the presence of FRI indicate that, in a genetic sense, FRI is a negative regulator of H3K27me3 at FLC. Moreover, autonomous pathway mutants also display reduced levels of H3K27me3 at FLC, which are comparable to levels seen in FRI-Col (Fig. 5, C and D). Thus, in backgrounds that lack FRI, the autonomous pathway is required for the elevated H3K27me3 levels found at FLC.
Given the elevated levels of H3K27me3 in Col relative to FRI-Col in nonvernalized plants, it was of interest to determine whether vernalization simply elevates H3K27me3 levels to those found in Col or if additional H3K27me3 accumulates in Col upon exposure to prolonged cold. H3K27me3 levels are in fact elevated in Col following cold treatment; however, the fold change is not as great as that seen in a FRI-Col background (Fig. 5E). In both vernalized and nonvernalized plants, FLC had higher levels of H3K27me3 in Col than in FRI-Col. However, it should be noted that the 30-d cold treatment given in these experiments is not saturating with respect to the acceleration of   Table S1. Real-time data shown are relative to an AG control.
flowering (Lee and Amasino, 1995). Thus, longer cold treatments may equalize the amount of H3K27me3 at FLC in vernalized Col and vernalized FRI-Col.

clf-59 Causes Rapid Flowering in the Absence of Genes Required for Vernalization
In addition to CLF, several other genes have been shown to play a role in the vernalization-mediated deposition and maintenance of H3K27me3 at FLC. These include the PcG gene VERNALIZATION2 (VRN2; Gendall et al., 2001;Bastow et al., 2004;Sung and Amasino, 2004) as well as VRN1 and the PHD domain proteins VIN3 and VIN3-LIKE1 (VIL1; also known as VRN5; Levy et al., 2002;Bastow et al., 2004;Sung and Amasino, 2004;Sung et al., 2006a;Wood et al., 2006;Greb et al., 2007;De Lucia et al., 2008). clf-59 was introduced into lines carrying mutations in these genes in order to evaluate whether any of these genes are required for the rapid flowering, gain-of-function phenotype in FRI clf-59. However, rapid flowering was observed in all tested lines: FRI vrn1-1, FRI vin3-1, FRI vil1, and fca vrn2-1 (Fig. 6, A and B), indicating that these genes, which are required for vernalization, are not required for the clf-59 phenotype.
Although the loss of vil1 did not affect the ability of clf-59 to repress FLC and cause early flowering (Fig.  6C), FRI vil1 clf-59 plants did display several pleiotropic phenotypes, including pale green leaves and downward leaf curling (Fig. 6, D and E), the opposite of that seen in clf loss-of-function mutants. Thus, VIL1 appears to be required for wild-type leaf morphology in the presence of clf-59.

Genetic Background Affects Wild-Type CLF Localization to FLC
As discussed above, FRI-Col and autonomous pathway mutants have relatively low levels of H3K27me3 at FLC chromatin when compared to Col (Fig. 5). In addition, it has been shown that clf loss-of-function mutants have reduced H3K27me3 levels at FLC chromatin in a Col background (Jiang et al., 2008). Because the presence of FRI or a clf mutation has a similar effect on H3K27me3 levels at FLC, the effect of FRI on CLF localization was investigated. A constitutively expressed, GFP-tagged version of CLF (GFP:CLF; Schubert et al., 2006) was introduced by crossing into a line containing FRI (FRI-Ws: all lines in the FRI/CLF localization experiments are in a Ws background). ChIP analysis using an anti-GFP antibody revealed that levels of CLF were enriched at FLC chromatin in Ws when compared to FRI-Ws (Fig. 7A). Such a reduction in CLF occupancy likely contributes to the lower levels of H3K27me3 at FLC seen in FRI-containing backgrounds (Fig. 5B).
GFP:CLF was also introduced into fca-9 and fld-3 backgrounds. In both cases, the autonomous pathway mutant displayed lower levels of CLF occupancy at FLC chromatin relative to the wild type (Fig. 7B). Thus, as with FRI, the fca and fld genetic backgrounds create a condition that leads to reduced occupancy of CLF protein at FLC chromatin.
We also examined the effect of prolonged cold on the enrichment of CLF at FLC. After a 35-d cold exposure, a difference in CLF occupancy at FLC could no longer be detected between plants that contain and those that lack FRI (Fig. 7C). This 35-d cold exposure was sufficient to substantially hasten flowering time in FRI-Ws (Fig. 7D). Thus, with vernalization, the presence of FRI no longer affects the amount of CLF that occupies FLC chromatin, and increased CLF occupancy at FLC after prolonged cold correlates with the vernalizationmediated increase in H3K27me3 at FLC.

DISCUSSION
We have identified a gain-of-function allele of CLF, clf-59, that reduces the level of FLC expression and thus eliminates the requirement for vernalization in winter-annual types of Arabidopsis. CLF is a PcG protein that functions in a complex, PRC2, which has conserved features in plants and animals. Plant genomes typically contain multiple copies of conserved PRC2 components [for example, Arabidopsis has three E(z) homologs: CLF, SWN, and MEA], and these components interact with members of the plantspecific, VIN3-like family of proteins (Wood et al., 2006;De Lucia et al., 2008). Thus, there is potential for much variability in the specific PcG complexes that assemble in plants. The clf-59 protein may either enhance the activity of a particular PcG complex or allow for the formation of a novel PcG complex that possesses high affinity for FLC chromatin.
clf-59 harbors a Pro-to-Ser amino acid transition in a Cys-rich region. Although the specific biochemical role of this region is not known, it contains two contiguous domains with a unique spacing of Cys residues called CXC domains as first described in ENX-1, the human homolog of E(z) (Hobert et al., 1996). Subsequent work has shown this domain to be important for the function of E(z) and E(z)-like proteins (Carrington and Jones, 1996;Kuzmichev et al., 2002;Ketel et al., 2005). Several otherwise unrelated proteins from plants and animals have been described that contain CXC domains separated by sequences of varying length, and these domains have been shown to bind zinc in vitro (Andersen et al., 2007). TSO1 is a CXC-containing protein in Arabidopsis that functions in the regulation of cell division, and both the tso1-1 and tso1-2 loss-of-function alleles contain amino acid substitutions within the CXC domain (Hauser et al., 2000;Song et al., 2000). Real-time data shown is relative to an AG control. Graphs in A and C represent the average of three experiments. Data in B represent a single experiment. Error bars represent SE. Samples were standardized to a Ws line that does not contain GFP. D, Flowering time of lines used in A and C both with (V) and without (NV) vernalization. The arrow indicates that .60 leaves were produced prior to flowering. Leaf counts represent the average of at least 10 plants. Error bars represent SE.
Both clf-59 and clf loss-of-function alleles hasten the onset of flowering; however, the two types of alleles do so via different mechanisms. clf-59 causes hyperrepression of FLC expression with no noticeable effect on AG. In contrast, clf loss-of function mutants cause derepression of several genes, including AG, FLC, and FT (Goodrich et al., 1997;Jiang et al., 2008), and it is likely that FT derepression leads to rapid flowering in clf loss-of-function mutants. This occurs despite elevated FLC expression, as ectopic FT expression can bypass the repressive effects of FLC (Michaels et al., 2005).
It is interesting to compare the degree of FLC derepression that we observe in a clf loss-of-function mutant to that reported previously (Jiang et al., 2008). In a Col background, Jiang et al. (2008) report a higher level of FLC derepression than we observe. In a delayed-flowering background, the difference is even greater: Jiang et al. (2008) report extensive derepression in an fca clf double mutant, whereas we do not observe derepression in FRI clf. Therefore, with respect to FLC derepression, clf loss-of-function mutants may have different effects in FRI compared to autonomous pathway mutant backgrounds.
The wild-type function of the autonomous pathway is to repress FLC expression (Michaels and Amasino, 2001). We find reduced levels of both H3K27me3 and CLF at FLC chromatin in several autonomous pathway mutants. Thus, one aspect of autonomous pathwaymediated FLC repression involves a direct or indirect effect on the occupancy of CLF at FLC chromatin. However, given the large degree of FLC derepression previously reported in fca clf mutants (Jiang et al., 2008), it is possible that a sufficient amount of CLF still occupies FLC chromatin in autonomous pathway mutant backgrounds as this would allow for some degree of derepression when CLF is genetically removed.
In this work, we present data that are consistent with FRI playing an antagonistic role with respect to PcG occupancy at FLC. We observe that H3K27me3 levels, a mark deposited by PcG protein complexes, were less abundant at FLC chromatin in a FRI-Col background relative to Col. We provide a possible explanation for the difference in H3K27me3 levels in the presence or absence of FRI by demonstrating that CLF, a PcG protein that catalyzes H3K27me3 deposition, is less abundant at FLC in FRI-Col relative to Col plants. Because CLF occupancy at FLC is reduced in a FRI background, one might expect a clf loss-of-function mutant to have less of an effect on FLC expression in a FRI-Col background; indeed, this is consistent with the lack of FLC derepression we observe in FRI clf plants. A molecular signature present in vernalized Arabidopsis plants is the accumulation of H3K27me3 at FLC (Schubert et al., 2006;Sung et al., 2006a;Finnegan and Dennis, 2007). Consistent with our results, the antagonism between FRI and CLF is relieved upon vernalization; CLF occupancy and H3K27me3 increases at FLC following exposure to prolonged cold. Genes required for vernalization, such as VIN3, may play a role in overcoming the antagonistic effects of FRI by helping to recruit CLF to FLC chromatin.
Chromatin modification has been shown in many systems to reinforce and/or maintain states of gene expression. Much of the data presented here and in other articles on FLC chromatin and flowering show correlations between the transcriptional level of FLC and particular chromatin modifications. We show, for example, that levels of H3K27me3, a repressive chromatin mark, and occupancy of CLF, a PcG protein, are decreased at FLC in two genetic situations in which FLC expression is elevated: the presence of FRI and the lack of autonomous pathway genes. The differential accumulation of marks such as H3K27me3 at FLC may very well be an indirect consequence of the effect that FRI and autonomous pathway genes have on FLC transcription. Indeed, just as we show that FRI leads to a reduction in CLF occupancy at FLC, a recent report has shown that FRI also correlates with the accumulation of a COMPASS complex component at FLC that modifies chromatin to an active state (Jiang et al., 2009). The specific biochemical role of FRI and the autonomous pathway components in FLC regulation remain to be determined.
The unique gain-of-function clf-59 allele results in an increase in the abundance of H3K27me3 at FLC in the presence of FRI. Thus, the clf-59 protein might be immune to the ability of FRI or loss of autonomous pathway components to reduce CLF occupancy at FLC chromatin in the absence of vernalization; thus, clf-59 may be able to accumulate to higher levels than wildtype CLF at FLC in these genetic backgrounds. Alternatively, clf-59 may produce a hyperactive protein that can elevate H3K27me3 levels without increased occupancy at FLC. It is intriguing that clf-59 can suppress FLC expression in nonvernalized plants to a level comparable to that found in the wild type after vernalization in the absence of proteins known to mediate the vernalization-induced silencing of FLC, including VRN2, VIN3, and VIL1. Future studies addressing the unique biochemical properties of the clf-59 protein may further our knowledge of FLC regulation and PcG targeting in general by providing a better understanding of the molecular events that either recruit or exclude PcG genes to FLC chromatin.

Plant Growth Conditions and Mutant Stocks
Plants were grown under cool-white fluorescent lights at 22°C in long-day photoperiods (16 h light: 8 h dark). Cold treatments were carried out at 4°C under cool-white fluorescent lights. The clf-59 mapping population was created by crossing ld-1 to ld-3 clf-59. The ld-1 and ld-3 mutants are from the Col and Ws-2 backgrounds, respectively (Lee et al., 1994b;Michaels and Amasino, 2001). F2 plants segregated 1:2:1 for the fully suppressed, partially suppressed, and the parental delayed flowering phenotypes, respectively. The latest flowering F2 plants were used to map the locus, as these plants were known to be homozygous Col at the locus of interest. A PCR-based derived cleaved amplified polymorphic sequence marker (Michaels and Amasino, 1998) was designed to detect the clf-59 mutation in both genetic studies and in transgenic plants. The marker was designed such that the wild-type CLF sequence contained an NcoI restriction site at the lesion that was absent in the clf-59. Primer sequences are listed in Supplemental Table S1. Other lines used in this work have been previously described. The fca-1 allele is from the Landsberg erecta but contains a Col allele of FLC (Sanda and Amasino, 1996). Alleles of other autonomous pathway mutants used in this work are in the Col background: fpa-7 (Michaels and Amasino, 2001), fca-9 (Bezerra et al., 2004), ld-1 (Redei, 1962), fld-3 (He et al., 2003), and fve-4 (Michaels and Amasino, 2001). clf-28 is from the Salk T-DNA collection (SALK_139371), and clf-52 is a T-DNA allele from the Ws-2 background (Noh and Amasino, 2003). The Ws-2 line with an introgressed FRI and the CLF:GFP line have been described previously (Noh and Amasino, 2003;Schubert et al., 2006). CLF:GFP is under the control of the cauliflower mosaic virus 35S promoter as described. CLF: GFP segregates as a single locus, and all lines containing this construct carried homozygous null mutations in the endogenous CLF gene. The CLF genomic region was amplified out of both ld-3 and ld-3 clf-59 using PCR. DNA fragments were then cloned into the binary vector pPZP221B using BamHI sites engineered into the primers. These primers and primers used to clone and make site-directed mutants in MEA and SWN are listed in Supplemental  Table S1.

Analysis of RNA Abundance
Tissue was harvested from 7-d-old seedlings. RNA was isolated using TRI reagent (Sigma-Aldrich). For northern analysis, 12 mg of total RNA was run on a 1% agarose denaturing formaldehyde gel and transferred to a nylon filter (Hybond). A DNA probe was used that was complementary to the 3# untranslated region of FLC. Real-time PCR was performed with the 7000 Real-Time PCR System (Applied Biosystems) using the DyNAmo Flash SYBR Green qPCR Kit (Finnzymes). The PCR parameters were as follows: one cycle of 15 min at 95°C, 40 cycles of 15 s at 95°C, 20 s at 58°C, and 45 s at 72°C. The constitutively expressed gene encoding the A3 subunit of protein phosphatase 2A was used as a control (Czechowski et al., 2005). PCR primer sequences are listed in Supplemental Table S1. All results presented are an average of at least three biological replicates.

ChIP
Tissue was harvested from 7-d-old seedlings. Chromatin samples were prepared as described (Gendrel et al., 2005). Antibodies used in this work were as follows: anti-GFP (Invitrogen catalog no. A-11122) and anti-H3K27me3 (Upstate catalog no. 07-449). Real-time PCR was done as described above. Changes at FLC chromatin are shown relative to an AG control. Because AG has abundant H3K27me3, it is possible that this mark at AG may behave similarly to that at FLC. However, observed patterns of H3K27me3 and CLF localization at FLC were also evident relative to an Actin control. Actin does not contain high levels of H3K27me3 (data not shown). PCR primer sequences are listed in Supplemental Table S1. All ChIP results presented are an average of at least two biological replicates unless noted otherwise.