Involvement of the Histone Acetyltransferase ... - Plant Physiology

Involvement of the Histone Acetyltransferase AtHAC1 in the Regulation of Flowering Time via Repression of FLOWERING LOCUS C in Arabidopsis1[W][OA] WeiWei Deng2, ChunYan Liu2, YanXi Pei, Xian Deng, LiFang Niu, and XiaoFeng Cao* State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China 100101 (W.D., C.L., Y.P., X.D., L.N., X.C.); Graduate School, Chinese Academy of Sciences, Beijing, China 100039 (W.D., L.N.); and College of Life Science and Technology, Shanxi University, Taiyuan, China 030006 (Y.P.)

Histone acetylation is an important posttranslational modification correlated with gene activation. In Arabidopsis (Arabidopsis thaliana), the histone acetyltransferase AtHAC1 is homologous to animal p300/CREB (cAMP-responsive element-binding protein)-binding proteins, which are the main histone acetyltransferases participating in many physiological processes, including proliferation, differentiation, and apoptosis. The functions of p300/CREB-binding proteins in animals are well characterized, whereas little is known about the roles of AtHAC1 in developmental control in Arabidopsis. Lesions in AtHAC1 caused pleiotropic developmental defects, including delayed flowering, a shortened primary root, and partially reduced fertility. Analysis of the molecular basis of late flowering in hac1 mutants showed that the hac1 plants respond normally to day length, gibberellic acid treatment, and vernalization. Furthermore, the expression level of the flowering repressor FLOWERING LOCUS C (FLC) is increased in hac1 mutants, indicating that the late-flowering phenotype of hac1 mutants is mediated by FLC. Since histone acetylation is usually associated with the activation of gene expression, histone modifications of FLC chromatin are not affected by mutations in HAC1 and expression levels of all known autonomous pathway genes are unchanged in hac1 plants, we propose that HAC1 affects flowering time by epigenetic modification of factors upstream of FLC.

Posttranslational modifications of histone N termini play fundamental roles in maintaining chromatin structure and function. One such modification, histone acetylation, has been implicated in many biological processes. In humans, histone acetylation is catalyzed by distinct histone acetyltransferase (HAT) families including the p300/CREB (cAMP-responsive elementbinding protein)-binding protein (CBP) family, the TAFII-250 family, the MYST (MOZ, Ybf2/Sas3, Sas2, and Tip60) family, and the GNAT (GCN5-related N-terminal acetyltransferase) family. The p300/CBP family plays a major role in transcriptional regulation by promoting acetylation of both histones and nonhistone proteins, such as the transcription factors CREB, 1

This work was supported by National Basic Research Program of China (grant no. 2005CB522400 to X.C.), by National Natural Science Foundation of China (grant no. 30571032 to C.L., and nos. 30325015, 30430410, and 30621001 to X.C.), and by the Chinese Academy of Sciences (grant no. CXTD–S2005–2). 2 These authors contributed equally to the paper. * Corresponding author; e-mail [email protected]; fax 86–10– 64873428. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: XiaoFeng Cao ([email protected]). [W] The online version of this article contains Web-only data. [OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.095521 1660

p53, HIV-1 Tat protein, and Stat3 (Sterner and Berger, 2000; Liu et al., 2004). Therefore, p300/CBP are not only HATs, but also factor acetyltransferases, and both types of activity are physiologically important for p300/ CBP functions (Sterner and Berger, 2000). In addition, because of their transcriptional coactivator functions, p300/CBP are proposed to contribute to transcriptional activation by directly interacting with DNA-binding transcription factors and/or being part of coactivation complexes (Dai et al., 1996; Goldman et al., 1997; Seo et al., 2001). In general, the histone acetylation status of a gene correlates with its transcriptional activity (Cheung et al., 2000; Strahl and Allis, 2000). The p300/CBP family exists in most multicellular eukaryotes, including mammals, Drosophila melanogaster, and Arabidopsis (Arabidopsis thaliana; Sterner and Berger, 2000; Pandey et al., 2002). The essential roles for p300/CBP in early development have been demonstrated in human patients, mice, and Drosophila. For example, mutations within the human CBP cause the Rubinstein-Taybi syndrome, which is characterized by severe facial abnormalities, broad thumbs, broad big toes, mental retardation, and abnormal retinal development (Roelfsema et al., 2005). In mice, both p300 and CBP null mice exhibit embryonic lethality between 8.5 and 11.5 d of gestation (Yao et al., 1998; Roth et al., 2001). In addition to the essential roles in normal development, mutation of one allele of either CBP or p300 affects embryogenesis and pattern formation, indicating a requirement for proper gene dosage

Plant Physiology, April 2007, Vol. 143, pp. 1660–1668, www.plantphysiol.org Ó 2007 American Society of Plant Biologists

Histone Acetyltransferase AtHAC1 Regulates Flowering Time

et al., 2002; Yuan and Giordano, 2002), the functions of AtHACs in gene regulation and developmental control in Arabidopsis remain unknown. In this study, we isolated Arabidopsis mutants with T-DNA insertions in the HAC1 gene and investigated their effects on plant development. We showed here that HAC1 played an important role in vegetative and reproductive development, and lesions in HAC1 caused a late-flowering phenotype in Arabidopsis. hac1 mutants responded normally to day length, gibberellin (GA), and vernalization treatments, and displayed an increased level of transcripts of FLOWERING LOCUS C (FLC) and two additional MADS-box genes, MADS AFFECTING FLOWERING 4 (MAF4) and MAF5. Therefore, HAC1 is critical for the normal regulation of flowering time in Arabidopsis.

RESULTS Isolation and Molecular Characterization of hac1 Mutants

Figure 1. HAC1 gene structure, T-DNA insertion alleles, and mRNA expression. A, Structure of the HAC1 gene. The HAC1 gene consists of 17 exons. Exons were presented as black boxes, introns as black lines, and 3# untranslated regions as gray boxes. The locations of hac1-3, hac1-4, and hac1-5 alleles were shown as triangles either above (from SALK collection) or below (from GABI) the gene. B, Domains of HAC1. Domains of TAZ, ZZ, and HAT were marked as predicted by Pandey (Pandey et al., 2002). The T-DNA insertion sites on the genomic sequences of HAC1 were marked on the corresponding positions of the translated protein with their allele number. aa, Amino acid. C and D, HAC1 gene expression. HAC1 mRNA accumulation in seedlings was sequentially analyzed by northern blot using the full-length CDS (C) and the C terminus of HAC1 (D) as shown in Fig. 1B. The membrane was stripped to remove the probe before blotting with another probe. The same membrane was also probed with an FLC probe and an Actin probe (see Fig. 4A).

(Yao et al., 1998). In Drosophila, there is only one p300/CBP gene and the reduction of this activity alters gene expression such that multiple developmental processes are affected, leading to pleiotropic phenotypes (Lilja et al., 2003). In Arabidopsis, there are five p300/CBP HAT homologs, named AtHAC1 (or PCAT2), AtHAC2 (or PCAT1), AtHAC4 (or PCAT3), AtHAC5 (or PCAT4), and AtHAC12 (Bordoli et al., 2001; Pandey et al., 2002; Yuan and Giordano, 2002). All the AtHACs contain ZZ-type and TAZ-type zinc finger domains and a Cysrich HAT domain at the C termini. Both ZZ-type and TAZ-type zinc finger domains have been implicated in protein-protein interactions with transcription factors (Ponting et al., 1996) and the HAT domain of AtHAC1 confers HAT activity in vitro (Bordoli et al., 2001). Although the structural conservation and diversification of AtHACs have been characterized (Pandey Plant Physiol. Vol. 143, 2007

To investigate the roles of the CBP homologs in Arabidopsis (AtHAC1, AtHAC2, AtHAC4, AtHAC5, and AtHAC12), T-DNA insertion mutants from the SALK or GABI-Kat collection were screened for homozygotes. No obvious developmental defects were observed from single mutants of hac2, hac4, hac5, or hac12 under normal growth conditions. However, three different T-DNA insertions in HAC1 (named hac1-3 for SALK_080380, hac1-4 for SALK_082118, and hac1-5 for GABI-314B12) showed a late-flowering phenotype. Therefore, we focused our investigations on the functions of HAC1 gene. The HAC1 gene contains 17 exons and encodes a 1,691 amino acid polypeptide with distinct domains including two ZZ-type and two TAZ-type zinc finger domains (Fig. 1, A and B). These domains have been shown in other organisms to mediate protein-protein interactions with transcription factors (Ponting et al., 1996). Like CBP in animals, HAC1 also has a catalytic domain (HAT domain) important for acetyltransferase activity. Both the HAT domain and zinc finger domains are located in the C terminus (Pandey et al., 2002). The location of the T-DNA insertions is upstream of these conserved domains, in either the fifth exon (hac1-3) or the sixth exon (hac1-4 and hac1-5). The location of each insertion was confirmed by PCR and DNA sequencing of the junction regions. Since both hac1-4 and hac1-5 contain the T-DNA at a similar position and show identical phenotypes, we used hac1-3 and hac1-4 for the rest of the analysis. RNA-blot analysis was performed to confirm the efficient disruption of HAC1 mRNA in hac1 plants. When full-length coding sequence (CDS) of HAC1 was used as a probe, we did not detect the full-length transcript of HAC1 in hac1 mutants. However, a short transcript was observed in both hac1-3 and hac1-4 plants, indicating that neither hac1-3 nor hac1-4 was a 1661

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null allele (Fig. 1C). Because the main functional domains of HAC1 were located in the C terminus, we performed RNA-blot analysis using a probe specific to the C-terminal portion of the HAC1 gene (Fig. 1B) and no mRNA containing these conserved domains could be detected (Fig. 1D). Thus, our mutant alleles are unlikely to produce any functional protein. A backcross between the hac1 homozygote and a wild-type line resulted in wild-type phenotype in F1 plants. The self-pollinated F1 progeny displayed an approximately 3:1 ratio of wild-type and hac1 phenotype, indicating that hac1 is a recessive, single-gene mutation. In addition, different alleles of hac1 (hac1-3, hac1-4, and hac1-5) do not complement each other. For example, the hac1-3/hac1-4 trans-heterozygous F1 plants from a cross between homozygous hac1-3 and hac1-4 showed the same phenotype as hac1-3 and hac1-4 plants (Fig. 2, E and F), and no segregation was observed in the F2 progeny (data not shown). These data indicate that the hac1 mutant is recessive and the observed phenotypes of hac1 mutants are indeed caused by disrupting HAC1 activity. The hac1 Mutants Display Pleiotropic Developmental Defects in Arabidopsis

In addition to the late-flowering phenotype, we examined other developmental defects in hac1 plants. The primary roots of hac1 mutants were shorter than

those of the wild-type plants when measured after 6 d of growth (Fig. 2A). The hac1 mutants also had reduced fertility in the first few siliques (Fig. 2, B and C). In the partial sterile flowers, the pollen produced by hac1 mutants didn’t reach the stigmas due to the short stamens resulting in the reduction of pollination and fertility (data not shown). The first 1 to 4 siliques of hac1 were about 30% shorter than those of wild-type plants. Siliques 5 to 8 were longer than the first 1 to 4, but still shorter than those of wild type (Fig. 2C). Siliques formed after these were similar to those of wildtype plants. The most notable phenotype of hac1 plants is late flowering (Fig. 2, D–G). In Arabidopsis, flowering time is regulated by four major pathways, namely the photoperiod, GA, vernalization, and autonomous pathways (Simpson et al., 1999; Reeves and Coupland, 2000; Mouradov et al., 2002; Komeda, 2004; Amasino, 2005). To investigate the role of HAC1 in the regulation of the flowering time, we first tested whether the photoperiod pathway was affected in hac1 mutants. By comparing the flowering time of the wild-type accession Columbia (Col-0) with that of hac1 plants, we found that hac1 plants flowered later than wild type under both long day (LD) and short day (SD) when the days to bolting (from germination to floral bud formation) and the total leaf numbers at flowering were measured (Fig. 2, D, E, and G). We observed only eight hac1-3 and six hac1-4 plants flowering by 200 d under

Figure 2. Phenotypes of hac1 mutants. A, The primary roots of wild-type and hac1 plants grown on Murashige and Skoog medium for 6 d. B, The siliques from the primary stem of wild-type and hac1 plants. The triangles indicate the shortened siliques in hac1 plants. C, Statistical analysis of the first to fourth and fifth to eighth siliques length as indicated. Bars represent the SD of the silique length. For each line, 20 plants were scored. D, Flowering time of wild-type and hac1 mutants under LD and SD. The flowering time was measured by days to bolting (black boxes) and by total leaf numbers (gray boxes) at flowering. Bars represent the SD. For each line, at least 50 plants were scored except for eight hac1-3 and six hac1-4 under SD, respectively. E and F, Wild-type (Col) and hac1 plants grown under LD. Trans-HZ indicates F1 hac1-3/ hac1-4 trans-heterozygous plants from a cross between hac1-3 and hac1-4. G, Wild-type Col and hac1 mutants grown under SD.

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SD and the rest of the hac1 plants had not yet flowered. The experiment was terminated at this point. Since hac1 plants responded normally to day length, the lateflowering phenotype of hac1 plants is not mediated by the photoperiod pathway. The Late-Flowering Phenotype of hac1 Plants Is Rescued by GA and Vernalization Treatments

The growth regulator GA promotes flowering of Arabidopsis. Exogenous application of GA accelerates flowering in wild-type Arabidopsis, particularly under SD (Wilson et al., 1992; Blazquez et al., 1998). To evaluate the effect of GA on the flowering time of hac1 plants, we sprayed the plants with a 100 mM GA solution once a week under SD until they flowered. Like the wild-type controls, hac1 mutants showed acceleration of flowering by GA treatment (Fig. 3A). Vernalization is a process by which flowering is promoted by prolonged exposure to the cold of a typical winter. Mutants in the vernalization pathway no longer respond to prolonged cold to promote flowering (Gendall et al., 2001; Amasino, 2004; Bastow et al., 2004; Sung and Amasino, 2004b; Amasino, 2005; Kim et al., 2005; Sung and Amasino, 2005). To determine whether vernalization could reverse the late-flowering phenotype of hac1, the hac1 mutants and wild-type plants were grown at 4°C for 6 or 10 weeks before transferring to 23°C under LD. Similar to the wild-type plants, the hac1 mutants flowered much earlier after vernalization treatment (Fig. 3B). After vernalization for 10 weeks, hac1 mutants and wild-type plants

flowered almost at the same time (Fig. 3B). Therefore, the hac1 lesions do not affect the vernalization pathway. HAC1 Controls Flowering Time through the Autonomous Pathway

The autonomous and vernalization pathways converge on FLC, a MADS-box transcription factor, which is a central regulator of floral transition in Arabidopsis (Michaels and Amasino, 1999; Sheldon et al., 1999). To examine whether the expression of FLC was increased in hac1 mutants, RNA gel-blot analysis was performed using the FLC full-length CDS as a probe. In both hac1-3 and hac1-4 plants, FLC mRNA expression levels are increased (Fig. 4A). In Arabidopsis, there are five MADS-box genes highly related to FLC, named MAF1 to MAF5, respectively (Alvarez-Buylla et al., 2000; Ratcliffe et al., 2001, 2003; Scortecci et al., 2001, 2003). To test whether MAFs were also affected by lesions in HAC1, we employed reverse transcription (RT)-PCR to examine mRNA levels. Like FLC, the expression levels of MAF4 and MAF5 were increased in hac1 plants, while MAF1-3 expression levels were unchanged when grown under LD (Fig. 4B, left section). We quantified this effect using real-time PCR and found a 2- to 4-fold increase in mRNA levels of MAF4 and MAF5 in hac1 mutants (Fig. 4B, right section). Since hac1 mutants are late flowering under both LD and SD, GA and vernalization treatments reverse the late-flowering phenotype, and hac1 mutants have an increased level of FLC mRNA, HAC1 behaves as a member of the autonomous pathway.

Figure 3. Flowering time of wild-type and hac1 plants grown under different conditions. A, Effects of GA on flowering time. Bars represent the SD. For each wild-type (white box), hac1-3 (gray box), and hac1-4 (black box) line, 50 plants were scored except for the GA untreated control. In our assay, only seven hac1-3 and six hac1-4 plants sprayed with ethanol flowered at 200 d under SD and the experiment was terminated at this point. B, Vernalization effects. Wild-type and hac1 plants were grown under LD without vernalization (untreated) or after vernalization for 6 (V42d) or 10 weeks (V70d). Flowering time was measured by days to bolting (left section, black box) and by leaf numbers (right section) at flowering. Gray boxes represent cauline leaf number and white boxes represent rosette leaf number. Bars indicate the SD. At least 50 plants for each genotype were scored.


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Figure 4. FLC and autonomous pathway gene expression in wild-type and hac1 plants. A, The levels of FLC mRNA in wild-type and hac1 plants were determined by northern blot using FLC fulllength CDS probe (top section). Actin gene was used as a constitutive expression control (bottom section). B and C, The expression levels of MAF1-5 (B) and autonomous pathways regulators of flowering time (C) in wild-type and hac1 plants were analyzed by RT-PCR and quantitated by real-time PCR. UBQ gene was used as a control for constitutive expression. a and b represent different amplification cycles in RT-PCR. Bars represent the SD. 22DCT corresponds to the ratio of each gene expression versus UBQ.

The components of the autonomous pathway, including FCA (Macknight et al., 1997; Page et al., 1999; Quesada et al., 2003), FPA (Schomburg et al., 2001), FLK (Lim et al., 2004; Mockler et al., 2004), FVE (Ausin et al., 2004; Kim et al., 2004), FLD (Sanda and Amasino, 1996; Yang and Chou, 1999; He et al., 2003), LD (Lee et al., 1994; Aukerman et al., 1999), FY (Simpson et al., 2003; Henderson et al., 2005), and RELATIVE OF EARLY FLOWERING 6 (REF6; Noh et al., 2004), promote flowering by repressing FLC. If HAC1 was essential for the expression of one of these repressors of FLC, hac1 mutants would be predicted to reduce the expression of the repressor leading to an increase in FLC transcription. We tested this hypothesis using RTPCR and real-time PCR to compare the mRNA levels of FVE, FLD, FCA, FPA, LD, FY, FLK, and REF6 between wild-type and hac1 plants. The mRNA levels of all known autonomous genes were not significantly affected in hac1 plants (Fig. 4C).

The Late-Flowering Phenotype of hac1 Is Mainly FLC Dependent

To determine whether the late-flowering phenotype of hac1 plants was solely due to the increase of FLC expression, hac1 mutants were crossed with an flc null allele, flc-3 (Michaels and Amasino, 1999). If the effect of hac1 on flowering time was fully due to the upregulation of FLC expression, the late-flowering phenotype of hac1 mutants would be entirely suppressed by flc null mutant. Under LD, the flowering time of both hac1-3/flc-3 and hac1-4/flc-3 double mutants was earlier than that of hac1 plants (Fig. 5), indicating that 1664

the late-flowering phenotype of hac1 was mainly FLC dependent. Epigenetic modifications have been shown to play an essential role in regulating FLC expression. Two components of the autonomous pathway, FLD and FVE, negatively regulate FLC by participating in the deacetylation of chromatin (He et al., 2003; Ausin et al., 2004; Kim et al., 2004). The vernalization pathway also regulates FLC expression by chromatin acetylation and methylation (Bastow et al., 2004; Finnegan et al., 2004; He et al., 2004; Sung and Amasino, 2004a, 2004b; Amasino, 2005; He and Amasino, 2005). Furthermore, REF6, which encodes a nuclear protein with jumonji and zinc finger domains, represses FLC transcription through histone deacetylation (Noh et al., 2004). Thus, histone acetylation is an essential modification of FLC chromatin. Although it is clear that histone acetylation/deacetylation regulates FLC expression, the identity of the HAT responsible for this process is unknown. To investigate whether HAC1 acetylates histones at FLC, we performed chromatin immunoprecipitation assays using 12-d-old seedlings of hac1 mutant and wild-type plants. In addition to histone acetylation, di- and trimethylation of H3K4, which are hallmarks for active chromatin, were also analyzed (Bernstein et al., 2002; Alvarez-Venegas and Avramova, 2005). However, our chromatin immunoprecipitation experiments did not show significant changes of histone H3 acetylation or H3K4 methylation in regions A, B, or C at the FLC locus, suggesting that increased FLC expression in hac1 mutants was not associated with changes in these epigenetic modifications at the assayed regions (Supplemental Fig. S1). Plant Physiol. Vol. 143, 2007


Figure 5. FLC-dependent late flowering caused by lesions in HAC1. Flowering time of hac1-3/flc-3 (top section) and hac1-4/flc-3 (bottom section) double mutants. Wild-type (Col), flc-3, hac1/flc double-mutant and hac1 single-mutant plants were grown under LD. LN means leaf number. The flowering time was measured by days to bolting (black box), cauline leaf number (gray box), and rosette leaf number (white box) at bolting. Bars represent the SD. For each line, at least 50 plants were scored.

DISCUSSION

Histone acetylation is known to regulate flowering time in Arabidopsis (Tian and Chen, 2001; He et al., 2003; Ausin et al., 2004; Tian et al., 2005). However, the HATs involved have not been identified. Our studies provide a link between a HAT, HAC1, and floweringtime control in Arabidopsis. The late-flowering phenotype of hac1 mutants was FLC dependent. First, hac1 plants flowered later than Col under LD and SD, which is a characteristic of FLC repressing flowering (Fig. 2, D–G). Second, hac1 mutants had a proper vernalization response that promotes flowering by repressing the expression of FLC (Fig. 3B). Third, the late-flowering phenotype of hac1 mutants was suppressed by an flc null mutant (Fig. 5). Finally, the mRNA levels of FLC were increased in hac1 mutants (Fig. 4A). In addition to the increase of FLC expression, a portion of the late-flowering phenotype of hac1 muPlant Physiol. Vol. 143, 2007

tants could be due to other floral repressors because the flowering time of hac1/flc-3 double-mutant plants was still slightly later than that of flc-3 under LD (Fig. 5). Consistent with this hypothesis, the expression of two additional MADS-box genes in the FLC clade, MAF4 and MAF5, were also up-regulated in hac1 mutants (Fig. 4B). However, the up-regulation of MAF4 and MAF5 was not unique in hac1 mutants because some autonomous pathway mutants also resulted in increased expression of MAF4 and MAF5 (Supplemental Fig. S2). In addition to the late-flowering phenotypes, mutations in HAC1 caused pleiotropic developmental defects, including shortened primary roots and partially reduced fertility. These pleiotropic effects observed in hac1 may be caused by loss of acetylation at multiple target genes, similar to mutations in other genes responsible for chromatin modifications. The Arabidopsis genome encodes five HACs, whereas the number of p300/CBP proteins in animals is only one to two (Pandey et al., 2002). In Arabidopsis, HAC1, HAC4, HAC5, and HAC12 are highly related and could be functionally redundant, which may explain why the mutations in humans and mice p300/CBP are much more severe than that of the hac1 mutation in Arabidopsis. This hypothesis is supported by a recent study in which a much stronger flowering phenotype was observed with two different hac double mutants, hac1/ hac5 and hac1/hac12 (Han et al., 2007). In humans, the transcriptional regulation by p300/ CBP appears to be exerted through multiple mechanisms. p300/CBP have been shown to acetylate both nucleosomal histones and certain sequence-specific transcription factors, leading to transcriptional activation in most cases (Sterner and Berger, 2000; Liu et al., 2004). Furthermore, as transcriptional coactivators, p300/CBP are proposed to contribute to transcriptional activity by directly interacting with transcription factors and/or being part of the coactivator complex (Dai et al., 1996; Goldman et al., 1997; Seo et al., 2001). A variety of transcription factors have been demonstrated to interact with the bromodomain, HAT, KIX, and zinc finger domains of p300/CBP (Vo and Goodman, 2001). It is possible that HAC1 is also a factor acetyltransferase and that this contributes to the regulation of flowering time. In general, histone acetylation correlates with gene activation and when HAC1 is disrupted, one would expect that the expression of its direct target genes would be reduced. In hac1 mutants, the expression levels of FLC, MAF4, and MAF5 are increased (Fig. 4, A and B). Therefore, FLC, MAF4, and MAF5 may not be the direct targets of HAC1. Instead, HAC1 might regulate the expression of a trans-acting repressor of FLC, MAF4, and MAF5. Since the vernalization and autonomous pathway genes repress the expression of FLC, and vernalization rescues the late-flowering phenotype of hac1 mutants, it is unlikely that the targets of HAC1 are in the vernalization pathway. Therefore, we examined the mRNA expression of all known 1665

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autonomous pathway genes by RT-PCR and real-time PCR, but did not find any significant differences in hac1 plants (Fig. 4C). One possible explanation is that HAC1 acetylates the histones of an unknown component that represses FLC and/or MAF4/5. Alternatively, HAC1 might acetylate a known or an unknown component of the autonomous pathway to affect its activity at posttranslational level. A third possibility is that the direct target of HAC1 (or HAC1 itself) might be involved in the recruitment of the known or unknown autonomous pathway components to FLC chromatin. Further studies aimed at identifying the direct targets of HAC1 in the FLC-dependent pathway would help to distinguish between these possibilities.

For RNA gel-blot analysis, RNA was transferred onto N1 nylon membrane and probed with [a-32P]dCTP-labeled FLC full-length CDS fragment (NM_121052, from 110–700 bp), or HAC1 full-length CDS (NM_106550, from 1–5,076 bp) or C-terminal fragments from 1,873 to 5,076 bp, respectively. Blots were also probed with an Actin probe as a loading control.

Chromatin Immunoprecipitation The chromatin immunoprecipitation experiments were performed as described by Johnson et al. (2002) using 12-d-old seedlings. Anti-acetyl-histone H3 (AcH3; Upstate Biotechnology, 06-599), anti-dimethyl-histone H3 (H3K4m2; Upstate Biotechnology, 07-030), or anti-trimethyl-histone H3 (H3K4m3; Upstate Biotechnology, 07-473) were used to immunoprecipitate the chromatin. The amount of immunoprecipitated FLC chromatin was determined by semiquantitative PCR on three different regions of FLC locus as previously reported (Bastow et al., 2004). Primer sequences were listed in Supplemental Table S3.

Supplemental Data MATERIALS AND METHODS Plant Materials and Growth Conditions All the Arabidopsis (Arabidopsis thaliana) lines used in this study were in ecotype Col-0. Plants were grown at 23°C 6 1°C. The photoperiods for LD were 16 h of light followed by 8 h of darkness and 8 h of light followed by 16 h of darkness for SD. For flowering-time measurements, plants were sowed side by side and transferred into Versatile Environmental Test Chamber (MLR350H, SANYO) at 23°C 6 1°C. Flowering time was measured by counting the total number of rosette and cauline leaves at flowering and the days from germination to floral bud formation. For GA treatment, plants were grown on soil under SD, and a GA solution of 100 mM was sprayed once a week until flowering. Ethanol was sprayed as untreated control. For vernalization treatment, the hac1 mutants and wild-type plants were grown side by side at 4°C for 6 or 10 weeks under SD and then transferred to 23°C under LD. Nonvernalized control seeds were grown for 3 d under vernalization conditions.

Identification of T-DNA Insertion Mutants DNA was isolated from hac1-3, hac1-4, and hac1-5 mutants. T-DNA border primers cx0101 LBb1 or cx0102 LBa1 were used to amplify DNA from mutants obtained from the SALK collection and T-DNA border primer cx1475 was used for GABI T-DNA insertion mutant hac1-5. The gene-specific primers were cx0517 and cx0516 for hac1-3 and cx0515 and cx0514 for hac1-4. hac1-5 was amplified with cx0514 and cx0517. Primer sequences were listed in Supplemental Table S1.

The following materials are available in the online version of this article. Supplemental Figure S1. Chromatin immunoprecipitation of FLC in wildtype and hac1 plants. Supplemental Figure S2. Expression levels of MAF4, MAF5, and FLC in autonomous pathway mutants. Supplemental Table S1. Primers for genotyping of hac1 mutants. Supplemental Table S2. Primers for RT-PCR and real-time PCR analysis of flowering-time regulatory genes. Supplemental Table S3. Primers for chromatin immunoprecipitation analysis at FLC locus.

ACKNOWLEDGMENTS We thank Dr. R. Jorgensen for analysis of the HAC1 protein structure, Dr. L. Johnson for critical reading and comments on the manuscript, and Dr. R. Amasino for providing flc-3 seeds. We also thank the Arabidopsis Biological Resources Center at The Ohio State University and Bernd Weisshaar for providing SALK and GABI T-DNA insertion lines in the Col background. Received January 5, 2007; accepted January 31, 2007; published February 9, 2007.

LITERATURE CITED Root Growth Conditions Seeds of Col-0 and hac1 mutants were surface sterilized with 10% bleach for 15 min, washed with sterile water, and plated on 9 3 9-cm2 dishes containing 30 mL of agar-solidified culture medium. This medium contains 13 Murashige and Skoog basal medium with vitamins (PhytoTechnology Laboratories) supplemented with 3% Suc and 0.8% to 1% plant tissue culture agar. The plates were kept at 4°C for 3 d and then placed vertically to allow the downward growth of roots in a growth chamber under LD at 23°C. After 6 d, measurements of primary root length were made for each sample.

Analysis of Transcript Levels Transcript levels were measured either by real-time PCR and RT-PCR or by RNA gel blots. Total RNA extraction and RT-PCR were performed as previously described by Liu et al. (2005). Real-time PCR analysis was performed using the Chromo4 Real-Time PCR instrument (MJ) and SYBR Green I (Invitrogen, S-7567). Primer sequences were listed in Supplemental Table S2. The cycle number at which the amplification plot crosses a fixed threshold above baseline is defined as the threshold cycle (Ct). Specific gene expression was normalized to the internal control gene Ubiquitin (UBQ; Mockler et al., 2004) given by the formula 22DCt. DCt is the Ct of the target gene subtracted from the Ct of the UBQ gene.

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