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Proc Natl Acad Sci U S A. May 19, 2009; 106(20): 8386–8391.
Published online May 4, 2009. doi:  10.1073/pnas.0903566106
PMCID: PMC2677093
Plant Biology

Vernalization-induced flowering in cereals is associated with changes in histone methylation at the VERNALIZATION1 gene

Abstract

Prolonged exposure to low temperatures (vernalization) accelerates the transition to reproductive growth in many plant species, including the model plant Arabidopsis thaliana and the economically important cereal crops, wheat and barley. Vernalization-induced flowering is an epigenetic phenomenon. In Arabidopsis, stable down-regulation of FLOWERING LOCUS C (FLC) by vernalization is associated with changes in histone modifications at FLC chromatin. In cereals, the vernalization response is mediated by stable induction of the floral promoter VERNALIZATION1 (VRN1), which initiates reproductive development at the shoot apex. We show that in barley (Hordeum vulgare), repression of HvVRN1 before vernalization is associated with high levels of histone 3 lysine 27 trimethylation (H3K27me3) at HvVRN1 chromatin. Vernalization caused increased levels of histone 3 lysine 4 trimethylation (H3K4me3) and a loss of H3K27me3 at HvVRN1, suggesting that vernalization promotes an active chromatin state at VRN1. Levels of these histone modifications at 2 other flowering-time genes, VERNALIZATION2 and FLOWERING LOCUS T, were not altered by vernalization. Our study suggests that maintenance of an active chromatin state at VRN1 is likely to be the basis for epigenetic memory of vernalization in cereals. Thus, regulation of chromatin state is a feature of epigenetic memory of vernalization in Arabidopsis and the cereals; however, whereas vernalization-induced flowering in Arabidopsis is mediated by epigenetic regulation of the floral repressor FLC, this phenomenon in cereals is mediated by epigenetic regulation of the floral activator, VRN1.

Keywords: epigenetic, MADS, intron, barley, chromatin

Plants respond to seasonal cues, such as temperature and day-length, to ensure that flowering coincides with favorable conditions. Prolonged exposure to low winter temperatures (vernalization) accelerates the progression from vegetative to reproductive growth in many plant species, including the temperate cereals (such as wheat and barley) and dicot species (such as Arabidopsis) (13). In both these lineages, plants retain a “memory” of the prolonged cold of winter, which stimulates flowering when days lengthen during spring (13). The memory of cold is then reset in the next sexual generation to ensure progeny are competent to respond to vernalization (13).

In Arabidopsis, the vernalization response is mediated by epigenetic regulation of the floral repressor, FLOWERING LOCUS C (FLC), which encodes a MADS-box transcription factor that represses genes involved in floral initiation, including SUPPRESSOR OF CONSTANS 1 and FLOWERING LOCUS T (FT) (1, 46). FLC is expressed before vernalization and delays flowering, but its expression is repressed by vernalization (1, 4). FLC remains repressed when plants are subsequently exposed to warm temperatures, allowing activation of FT, which promotes flowering (1, 4). The stable down-regulation of FLC by vernalization is associated with an increase in the levels of repressive histone modifications at FLC chromatin, such as histone H3 lysine 27 di- and trimethylation (H3K27me2, H3K27me3), histone H3 lysine 9 dimethylation, and histone H4 arginine 3 symmetrical dimethylation, as well as the loss of histone modifications associated with active transcription, such as histone H3 acetylation and histone H3 lysine 4 di- and trimethylation (H3K4me2, H3K4me3) (713). Repression of FLC by vernalization involves the vernalization-dependent association of Polycomb-Group (PcG) complexes to FLC chromatin, which are required for addition and maintenance of H3K27me3 at FLC (14, 15). Taken together, these studies indicate that vernalization induces an alteration of FLC chromatin state from actively transcribed to stably repressed (715). The cellular memory of transcriptional repression of FLC is maintained during successive cell divisions by mitotic inheritance of repressive histone modifications at the gene (11), but active FLC transcription is restored in progeny, ensuring that the next generation is competent to respond to vernalization (1, 4, 16).

In temperate cereals, the vernalization response is mediated by the stable induction of a floral promoter, VERNALIZATION1 (VRN1) (3, 1719). VRN1 encodes a FRUITFULL-like MADS-box transcription factor required for the initiation of reproductive development at the shoot apex (2022). In vernalization-requiring cereal plants, VRN1 is expressed at low levels and is induced by vernalization, with the level of expression being dependent on the length of cold exposure (1719, 2325). VRN1 expression remains high when plants are exposed to warm temperatures following vernalization, and promotes the transition to reproductive development (1719, 2325). VRN1 down-regulates the floral repressor VERNALIZATION2 (VRN2), and allows long-day induction of the floral activator FT to accelerate subsequent stages of floral development (3, 2426).

The vernalization response of VRN1 shows characteristics of epigenetic regulation, in that VRN1 is induced by vernalization, expression is maintained following vernalization, and the prevernalization level of VRN1 expression is reset in the next generation (1719, 2325). In this article we analyze the effect of vernalization on the levels of histone modifications at the barley (Hordeum vulgare) VRN1 gene (HvVRN1). Our study indicates that vernalization-induced flowering in cereals is mediated by epigenetic regulation of VRN1 chromatin state. Our results suggest that regulation of the histone methylation status of VRN1 chromatin is important for repression of VRN1 before vernalization, for activation of VRN1 by vernalization, and for maintaining a memory of vernalization following cold exposure.

Results

Activation of HvVRN1 by Vernalization Is Associated with a Gain of H3K4me3 and a Loss of H3K27me3 at HvVRN1 Chromatin.

The effect of vernalization on the levels of 2 histone modifications at HvVRN1 chromatin was measured in the vernalization-responsive barley variety Sonja. Levels of H3K4me3, a modification associated with epigenetic inheritance of active gene transcription (2730), and H3K27me3, a modification associated with long-term gene repression (28, 30, 31), were analyzed. The HvVRN1 gene has a large (10.8 kb) first intron containing regions associated with regulation of HvVRN1 expression (23, 3234). We analyzed 6 regions across the 5′ end of the HvVRN1 gene: the promoter (2 kb upstream of the translational start, region 1), exon 1 (region 2), and 4 sites along the length of the first intron (regions 3–6) (Fig. 1A). To assess both the immediate and long-term effects of vernalization on histone modifications at HvVRN1, we examined 2 developmental stages following seed-vernalization treatment: seedlings harvested immediately at the end of vernalization (see Fig. 1), and plants that had been transferred to normal glasshouse conditions at the end of vernalization treatment and allowed to develop to the third-leaf stage (approximately 2 weeks from the end of vernalization treatment) (Fig. 2).

Fig. 1.
The effect of vernalization on histone modifications at HvVRN1, HvVRN2, and HvFT1 in barley seedlings. (A) Diagram of the 5′ end of HvVRN1 showing the regions (1–6, short dashed lines) analyzed by ChIP, followed by quantitative real-time ...
Fig. 2.
The effect of seed vernalization on histone modifications at HvVRN1, HvVRN2, and HvFT1 in leaves postvernalization. (A–C) HvVRN1 (A), HvVRN2 (B), and HvFT1 (C) expression in nonvernalized (NV) and postvernalized (PV) leaves from the barley varieties ...

In Sonja seedlings, vernalization caused an increase in the levels of H3K4me3 at regions 2 and 3 of HvVRN1 (corresponding to exon 1 and the 5′ end of intron 1), but not regions 1, 4, 5, and 6 (see Fig. 1C); these results parallel the induction of HvVRN1 expression by vernalization (see Fig. 1B), and are consistent with previous findings that H3K4me3 occurs around the start of transcription in actively transcribing genes (28, 30). Levels of H3K27me3 were reduced by vernalization in regions 1 to 5, but not in region 6 (see Fig. 1E), indicating a decrease in H3K27me3 across the promoter, first exon, and first intron of HvVRN1 (average 63% reduction). The increase in H3K4me3 and loss of H3K27me3 at HvVRN1 suggest that vernalization promotes an active state of HvVRN1 chromatin.

Repression of HvVRN1 Expression Before Vernalization Is Associated with High Levels of H3K27me3.

In nonvernalized Sonja seedlings, high levels of H3K27me3 were detected at the HvVRN1 gene (see Fig. 1E), suggesting that the presence of H3K27me3 at HvVRN1 chromatin is associated with repression of the gene before vernalization. In some varieties of wheat and barley, deletions within the first intron of VRN1 are associated with activation of VRN1 expression without vernalization treatment (23, 3234). The barley variety Morex contains a large (5.2 kb) deletion in the first intron of HvVRN1 (i.e., regions 4 and 5 are absent) (see Fig. 1A) (32), and Morex exhibits significantly higher HvVRN1 expression in nonvernalized seedlings compared to Sonja (P < 0.05) (see Fig. 1B). In nonvernalized Morex seedlings, levels of H3K4me3 at regions 2 and 3 of HvVRN1 were significantly higher than in Sonja (P < 0.05, compare NV in Fig. 1 C and D), while levels of H3K27me3 at regions 1, 2, and 3 of HvVRN1 were significantly lower in Morex than Sonja (P < 0.05, compare NV in Fig. 1 E and F). These data show that the activation of HvVRN1 in nonvernalized Morex seedlings is associated with low levels of H3K27me3 at HvVRN1. Taken together, these results indicate that repression of HvVRN1 before vernalization is associated with high levels of H3K27me3 at the gene, and suggest that regions of the first intron could be important for determining levels of H3K27me3 at HvVRN1 before vernalization.

Similar to Sonja, vernalization caused an increase in HvVRN1 expression in Morex seedlings (see Fig. 1B), along with an increase in H3K4me3 and a reduction of H3K27me3 at regions 2 and 3 of HvVRN1 (see Fig. 1 D and F). These results show that in a variety with high basal levels of HvVRN1, vernalization further activates HvVRN1 chromatin state in seedlings.

Vernalization Does not Affect Levels of H3K4me3 and H3K27me3 at HvVRN2 and HvFT1 in Seedlings.

In addition to HvVRN1, the effect of vernalization on histone modifications at HvVRN2 and HvFT1, which regulate the long-day flowering response, was examined (3, 2426, 35). HvVRN2 and HvFT1 do not contain a large first intron, and our analysis was restricted to 1 region within exon 1 for these 2 genes. Additionally, Morex lacks the HvVRN2 locus (36, 37), so HvVRN2 was only analyzed in Sonja.

HvVRN2 and HvFT1 were expressed at low levels in seedlings (HvVRN2, undetectable; HvFT1, mean expression relative to ACTIN = less than 0.0002). In both nonvernalized and vernalized seedlings, HvVRN2 and HvFT1 contained low levels of H3K4me3 but were enriched for H3K27me3 (see Fig. 1 C–F). In contrast to HvVRN1, vernalization did not affect the levels of either histone modification at HvVRN2 or HvFT1 (see Fig. 1 C–F). These results agree with previous data showing that the seed-vernalization response in cereals involves cold-activation of VRN1 independently of day-length response pathways (3, 24).

Histone Modifications at HvVRN1 Are Maintained Postvernalization.

A key feature of the vernalization response in cereals is the maintenance of high VRN1 expression levels when plants are exposed to warm growth temperatures following vernalization (see Fig. 2A) (1719, 2325). In vernalization-requiring varieties, such as Sonja, this is associated with a rapid transition of the shoot apex from vegetative to reproductive development [supporting information (SI) Fig. S1]. Without vernalization, VRN1 expression remains low (see Fig. 2A) and the shoot apex remains vegetative (see Fig. S1). We examined the long-term effect of seed vernalization on the levels of H3K4me3 and H3K27me3 at HvVRN1 chromatin by measuring these histone modifications in plants grown from nonvernalized or vernalized seedlings. In Sonja, this revealed a similar pattern of histone modifications at HvVRN1 to that observed in vernalized seedlings; in postvernalized Sonja leaves, H3K4me3 levels were higher in regions 2 and 3 of HvVRN1 (see Fig. 2D), while H3K27me3 levels were lower across the gene (see Fig. 2F) compared to nonvernalized leaves. These data indicate that vernalization-induced changes in histone methylation at HvVRN1 are maintained in leaves following seed vernalization.

Developmental Induction of HvVRN1 Is Associated with High Levels of H3K4me3 and Low Levels of H3K27me3.

In contrast to Sonja, Morex plants rapidly initiate flowering and HvVRN1 is expressed in leaves without vernalization treatment (see Fig. 2A and Fig. S1) (23, 24). Morex plants derived from nonvernalized and vernalized seed had similar levels of HvVRN1 expression (see Fig. 2A), which suggests that the high level of HvVRN1 expression in these plants is because of developmental induction of HvVRN1 independent of vernalization treatment. Consistent with the high level of HvVRN1 expression in Morex, levels of H3K4me3 were high at regions 2 and 3 of HvVRN1, and levels of H3K27me3 were low across the gene in both nonvernalized and postvernalized leaves, and vernalization did not affect the levels of these modifications (see Fig. 2 E and G). These data show that the developmental induction of HvVRN1 in a variety that flowers without vernalization is associated with high levels of H3K4me3 and low levels of H3K27me3 at the gene.

HvVRN2 and HvFT1 Remain Enriched for H3K27me3 Postvernalization.

Expression of HvVRN2 and HvFT1 is influenced by exposure to long days following vernalization treatment (3, 2426, 35). Seed vernalization treatment caused a reduction in HvVRN2 expression in Sonja leaves (see Fig. 2B), while HvFT1 expression was induced but remained low (see Fig. 2C). HvFT1 expression was significantly higher in Morex compared to Sonja, regardless of vernalization treatment (P < 0.05) (see Fig. 2C), probably because of either the absence of HvVRN2, a repressor of HvFT1, in Morex (36, 37), or the particular HvFT1 allele present in Morex (35). Vernalization did not affect the levels of H3K4me3 or H3K27me3 at HvVRN2 or HvFT1 in leaves, and both genes were enriched for H3K27me3 (see Fig. 2 D–G). These data indicate that, unlike VRN1, regulation of expression of VRN2 and FT in response to vernalization is not associated with changes in the levels of H3K4me3 and H3K27me3.

Discussion

In this article, we have analyzed the effect of vernalization on histone modifications at the HvVRN1 gene. Our study demonstrates that induction of HvVRN1 transcription by vernalization involves changes in histone methylation at HvVRN1. Before vernalization, the occurrence of H3K27me3 at HvVRN1 is associated with repression of the gene. Vernalization causes increased levels of H3K4me3 and decreased levels of H3K27me3 at HvVRN1 chromatin, and these changes are retained postvernalization, suggesting that vernalization promotes an active state of HvVRN1 chromatin that is maintained following vernalization treatment. Our study suggests that the memory of vernalization in cereals involves epigenetic inheritance of histone modifications associated with active transcription at VRN1.

In plants and other organisms, maintenance of H3K4me3 and H3K27me3 levels is achieved through the action of Trithorax-group and PcG protein complexes, respectively (3843). Putative core Trithorax-group and PcG complex components and potential interacting proteins have been identified in cereals (44, 45), and it is likely that these complexes are involved in the addition and maintenance of H3K4me3 and H3K27me3 at VRN1. Repression of the floral activation genes AGL19 and FT in Arabidopsis involves PcG-mediated maintenance of H3K27me3 at these genes (40, 46). The occurrence of H3K27me3 at VRN1 and repression of the gene before vernalization may be mediated by PcG complexes, which may require PcG-binding elements within the gene, although such elements have not yet been defined in plants (42). In Arabidopsis, regions of the first intron of FLC are required for the maintenance of FLC repression following vernalization (47), and the vernalization-dependent association of a PcG complex to FLC occurs specifically within the first intron (15). Deletions within the first intron of VRN1 that are associated with high basal VRN1 expression may lack PcG-binding elements, resulting in lower H3K27me3 levels. The vernalization-induced loss of H3K27me3 at VRN1 may result from histone demethylase activity, but H3K27me3 demethylases have not yet been identified in plants.

VRN2 and FT are involved in determining flowering-time as part of the day-length response pathway in cereals (3, 2426, 35). Both VRN2 and FT are repressed in germinating seedlings and are marked by H3K27me3, suggesting that they are targets of PcG-mediated repression in this tissue. In Arabidopsis, PcG-mediated H3K27me3 at FT chromatin limits expression of FT, and mutations of PcG-complex components cause FT induction and precocious flowering (46). Here, we have shown that H3K27me3 enrichment at FT chromatin occurs in cereals, suggesting that PcG complexes play a conserved role in the repression of FT in both Arabidopsis and the cereals. In contrast to VRN1, vernalization did not affect the levels of H3K4me3 or H3K27me3 at VRN2 and FT, suggesting that vernalization directs changes in the chromatin state at VRN1 but not VRN2 or FT.

In conclusion, our study has shown that, like Arabidopsis, vernalization in barley results in an alteration of chromatin state at a key floral regulatory gene. However, in contrast to the PcG-mediated repression of FLC in Arabidopsis, vernalization promotes an active chromatin state of VRN1 in barley. A key question to be addressed in the future is how the plant's perception of cold directs the changes in the chromatin state of these genes.

Materials and Methods

Plant Material.

For seed vernalization treatments, barley seeds were sown in soil, pots were covered with aluminium foil, and then incubated at 2 °C ± 1 °C for 49 days as previously described (25); at this time-point, seedlings have germinated and show no signs of etiolation. Previous experiments showed that vernalization for 35 to 63 days accelerated flowering (25), and so a vernalization treatment of 49 days was chosen for the present study. Vernalized seedlings (above-ground coleoptiles plus internal leaves, ≈3 cm in length after 49 days vernalization) were harvested directly in the cold. Nonvernalized seedlings were derived from seeds sown in aluminium foil-covered pots of soil and germinated in the dark in a glasshouse at 19 °C ± 2 °C; seedlings were harvested 5 days after sowing, at which time the coleoptiles were the same length as vernalized seedlings. At this time-point, the shoot apex is vegetative (see Fig. S1). For the analysis of postvernalized leaves, pots of vernalized seed were transferred to sunlit glasshouses, the foil was removed, and plants were maintained under long days (16-h light/8-h dark, with supplementary lighting used when natural light levels dropped below 200 μE). Leaf tissue (the third leaf from each plant) was harvested in the middle of the light period at the third-leaf stage (Zadoks scale Z = 13) (48), which occurred approximately 2 weeks after vernalization. Nonvernalized leaf tissue was harvested from plants maintained in the same glasshouse from nonvernalized seed, also at the third-leaf stage.

RNA Isolation, cDNA Synthesis, and Quantitative PCR.

Total RNA was isolated using the Qiagen RNeasy Plant Miniprep kit (Qiagen), then treated with DNase (Promega) and used for cDNA synthesis with an oligo dT primer and SuperScript III (Invitrogen) according to the manufacturer's instructions. Quantitative real-time PCR was performed to measure levels of gene expression using a Rotor-Gene 3000 Real-Time Cycler (Corbett Research) as previously described (26). DNA concentrations were determined using the comparative quantification method as previously described (9). ACTIN was used for normalization of gene expression as described previously (24, 26). Primer pairs for HvVRN1, HvVRN2, HvFT1, and ACTIN have previously been described (24, 26). Expression results for each gene were measured in the same PCR run in Sonja and Morex.

Chromatin Immunoprecipitation.

For the ChIP analysis of leaf tissue, ChIP was performed essentially as previously described (49), with some modifications. Leaf tissue was harvested and immediately cross-linked under vacuum for 10 min in buffer containing 0.4 M sucrose, 10 mM Tris (pH 8), 1 mM EDTA, 1 mM PMSF, and 1% formaldehyde; then glycine was added to a final concentration of 0.1 M and the vacuum was reapplied for 5 min. Leaves were briefly rinsed in water, frozen in liquid nitrogen, and stored at –80 °C. For the isolation of chromatin, leaves (2 g) were ground in liquid nitrogen and added to 5-ml lysis buffer [50 mM Hepes (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF] containing 100 μl of Complete protease inhibitors (Roche, 1 tablet dissolved in 1-ml lysis buffer). Samples were subjected to sonication to shear the DNA into fragments of ≈500 bp, and then centrifuged for 1 min at 16,000 × g (4 °C) to pellet debris. The supernatant was removed to a fresh tube, a further 100 μl of Complete protease inhibitors was added, and precleared with Protein A agarose beads (Upstate) for 1 h at 4 °C on a rotary shaker. Following centrifugation for 2 min at 800 × g (4 °C), aliquots of the supernatant were taken for immunoprecipitation with the appropriate antibody. The extract and antibody was incubated overnight at 4 °C on a rotary shaker, then Protein A agarose beads were added and the incubation continued for an additional 2 h. The beads were then washed with 2× low salt buffer, 2× medium salt buffer, and 1× LNDET buffer, on a rotary shaker at 4 °C for 10 min per wash. A further 2× TE washes were performed for 5 min per wash, with a final 1× TE wash at room temperature. Immunoprecipitates were eluted from the beads with 2 × 150 μl incubations with 1% SDS, 0.1 M NaHCO3 on a rotary shaker at room temperature for 15 min. Reverse cross-linking was performed as previously described (49), followed by a DNA cleanup with the Qiagen Qiaquick PCR cleanup kit (Qiagen). DNA was then used for quantitative real-time PCR as described below. For the ChIP analysis of seedlings, nuclei were extracted from 2-g seedlings as previously described (50), and the resultant chromatin was used for immunoprecipitation, as described for leaf tissue. Antibodies recognizing H3K4me3 and H3K27me3 were obtained from Upstate Biotechnology. The antibody against histone H3 was purchased from Abcam.

The amount of genomic DNA precipitated in ChIP assays was quantified by quantitative real-time PCR as described above. Primer pairs are shown in Table S1; each was verified to amplify a single product consisting of the expected target sequence. For each primer pair, the amount of DNA precipitated using anti-H3K4me3 or anti-H3K27me3 antibodies was normalized to the amount precipitated by an anti-H3 antibody from the same sample to correct for differences in ChIP input DNA. The genes for ACTIN (enriched for H3K4me3) (Fig. S2) or BARLEY MADS 9 (51) (BM9, enriched for H3K27me3) (Fig. S3) were used for normalization to compare vernalized and nonvernalized samples (expression of these genes does not change with vernalization). No-antibody control reactions were performed in parallel with each antibody reaction to verify that the immunoprecipitated DNA was enriched for the control genes (ACTIN for H3K4me3, BM9 for H3K27me3, all for H3). Take-off values for ACTIN in the H3K4me3 immunoprecipitations and BM9 in the H3K27me3 immunoprecipitations were comparable (within 1 cycle) between Sonja and Morex samples, enabling a direct comparison of the normalized H3K4me3 and H3K27me3 levels between Sonja and Morex. The data presented is the relative amount of precipitated DNA normalized to either ACTIN (for H3K4me3) or BM9 (for H3K27me3), and each graph represents the mean of at least 3 biological replicate experiments ± SEM.

Statistical Analysis.

Data were analyzed using the Student's t test and deemed significant if P < 0.05.

Supplementary Material

Supporting Information:

Acknowledgments.

The authors thank Drs Megan Hemming, Weiwei Deng, Candice Sheldon, Ming Luo, and Mr. Aaron Greenup for helpful discussions and critical reading of the manuscript. S.N.O. was supported by a CSIRO Office of the Chief Exectutive Postdoctoral Fellowship.

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0903566106/DCSupplemental.

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