The timing of the floral transition has significant consequences for reproductive success in plants. Plants gauge both environmental and endogenous signals before switching to reproductive development. Many temperate species only flower after they have experienced a prolonged period of cold, a process known as vernalization, which aligns flowering with the favourable conditions of spring. Considerable progress has been made in understanding the molecular basis of vernalization in Arabidopsis. A central player in this process is FLC, which blocks flowering by inhibiting genes required to switch the meristem from vegetative to floral development. Recent data shows that many regulators of FLC alter chromatin structure or are involved in RNA processing.

Introduction

The switch to flowering is a major developmental transition in the plant life cycle (Simpson and Dean,2002). Plants initially undergo a period of vegetative development, characterised by the iterative production of leaves from the shoot meristem (Poethig,1990). Later in development, the meristem undergoes a change in fate and enters reproductive development, producing flowers and differentiating the germ line. Plant species exhibit great variability in flowering-time, and the timing of this floral switch is controlled by multiple environmental and endogenous cues (Battey,2000; Izawa et al.,2003; Simpson and Dean,2002). This enables plants to align their life history with favourable environmental conditions.

Genetic analysis of Arabidopsis thaliana has identified numerous pathways that control the timing of the floral transition(Fig. 1, Table 1 and Table 2). Downstream of many of the floral pathways are a set of floral pathway integrator genes(Kardailsky et al., 1999; Kobayashi et al., 1999; Samach et al., 2000; Lee et al., 2000; Moon et al., 2003a; Hepworth et al., 2002; Nilsson et al., 1998; Blazquez et al., 2000) (see Fig. 1 and Table 1). It is the activation of these floral pathway integrator genes that triggers the floral transition. In turn, the integrators activate a set of genes known as floral meristem identity (FMI) genes, which encode proteins that promote floral development,not only by positively regulating genes required for flower development, but also by repressing AGAMOUS-LIKE 24 (AGL24), a promoter of inflorescence fate (Yu et al.,2004).

Fig. 1.

Pathways controlling flowering-time in Arabidopsis. The flowering-time pathways control the expression of the floral pathway integrators SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1(SOC1), FT and LEAFY (LFY). These genes encode proteins that activate the floral meristem identity (FMI) genes APETALA1 (AP1), APETALA2 (AP2), FRUITFULL (FUL), CAULIFLOWER (CAL) and LFY, which convert the vegetative meristem to a floral fate. Recent expression data has indicated that FUL may also act as a floral integrator (Schmid et al.,2004). The photoperiod, gibberellin, light-quality and ambient-temperature pathways activate floral pathway integrators. The CONSTANS(CO) transcription factor functions in the photoperiod pathway; long-day photoperiods promote flowering by circadian clock (CLOCK) dependent and independent mechanisms, which control the activity of CO. Activation of flowering is antagonised by the floral repressors encoded by (shown in green) FLOWERING LOCUS C (FLC), FLOWERING LOCUS M(FLM), TERMINAL FLOWER1 (TFL1), TERMINAL FLOWER2 (TFL2), SHORT VEGETATIVE PHASE (SVP), TARGET OF EAT1 (TOE1), TARGET OF EAT2(TOE2), SCHNARCHZAPFEN (SNZ), SCHLAFMUTZE(SMZ) and EMBRYONIC FLOWER1/2 (EMF1, EMF2). TFL1 may also be downstream of CO, as it is induced after CO activation (Simon et al.,1996). FLC expression is controlled by a number of different pathways. The genes shown in purple, FRIGIDA(FRI), FRIGIDA-LIKE1 (FRL1), FRIGIDA-LIKE2(FRL2), PHOTOPERIOD INSENSITIVE EARLY FLOWERING1(PIE1), AERIAL ROSETTE1 (ART1), EARLY UNDER SHORT DAYS4 (ESD4), VERNALIZATION INDEPENDENCE3(VIP3) and VERNALIZATION INDEPENDENCE4 (VIP4),encode proteins that promote FLC expression and delay flowering. FLC expression is downregulated in response to prolonged cold by proteins encoded by the genes (shown in blue) VERNALIZATION INSENSITIVE3 (VIN3), VERNALIZATION1 (VRN1) and VERNALIZATION2 (VRN2), and also by proteins encoded by the genes of the autonomous pathway (red): FCA, FY, LUMINIDEPENDENS(LD), FLOWERING LOCUS D (FLD), FVE, FLOWERING LOCUS K (FLK) and FPA. The distinction between potential transcriptional and post-transcriptional functions of genes of the autonomous pathway is not made here, but is shown more clearly in Fig. 3.

Fig. 1.

Pathways controlling flowering-time in Arabidopsis. The flowering-time pathways control the expression of the floral pathway integrators SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1(SOC1), FT and LEAFY (LFY). These genes encode proteins that activate the floral meristem identity (FMI) genes APETALA1 (AP1), APETALA2 (AP2), FRUITFULL (FUL), CAULIFLOWER (CAL) and LFY, which convert the vegetative meristem to a floral fate. Recent expression data has indicated that FUL may also act as a floral integrator (Schmid et al.,2004). The photoperiod, gibberellin, light-quality and ambient-temperature pathways activate floral pathway integrators. The CONSTANS(CO) transcription factor functions in the photoperiod pathway; long-day photoperiods promote flowering by circadian clock (CLOCK) dependent and independent mechanisms, which control the activity of CO. Activation of flowering is antagonised by the floral repressors encoded by (shown in green) FLOWERING LOCUS C (FLC), FLOWERING LOCUS M(FLM), TERMINAL FLOWER1 (TFL1), TERMINAL FLOWER2 (TFL2), SHORT VEGETATIVE PHASE (SVP), TARGET OF EAT1 (TOE1), TARGET OF EAT2(TOE2), SCHNARCHZAPFEN (SNZ), SCHLAFMUTZE(SMZ) and EMBRYONIC FLOWER1/2 (EMF1, EMF2). TFL1 may also be downstream of CO, as it is induced after CO activation (Simon et al.,1996). FLC expression is controlled by a number of different pathways. The genes shown in purple, FRIGIDA(FRI), FRIGIDA-LIKE1 (FRL1), FRIGIDA-LIKE2(FRL2), PHOTOPERIOD INSENSITIVE EARLY FLOWERING1(PIE1), AERIAL ROSETTE1 (ART1), EARLY UNDER SHORT DAYS4 (ESD4), VERNALIZATION INDEPENDENCE3(VIP3) and VERNALIZATION INDEPENDENCE4 (VIP4),encode proteins that promote FLC expression and delay flowering. FLC expression is downregulated in response to prolonged cold by proteins encoded by the genes (shown in blue) VERNALIZATION INSENSITIVE3 (VIN3), VERNALIZATION1 (VRN1) and VERNALIZATION2 (VRN2), and also by proteins encoded by the genes of the autonomous pathway (red): FCA, FY, LUMINIDEPENDENS(LD), FLOWERING LOCUS D (FLD), FVE, FLOWERING LOCUS K (FLK) and FPA. The distinction between potential transcriptional and post-transcriptional functions of genes of the autonomous pathway is not made here, but is shown more clearly in Fig. 3.

Table 1.

Floral promotive genes

Gene nameProtein functionReference
Floral pathway integrators   
   SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1/AGAMOUS-LIKE20(SOC1/AGL20) MADS-box transcription factor Samach et al., 2000; Lee et al., 2000  
   FT Putative kinase inhibitor Kardailsky et al., 1999; Kobayashi et al., 1999  
   LEAFY (LFY) Plant specific transcription factor Weigel et al., 1992; Nilsson et al., 1998  
Photoperiodic pathway   
   CONSTANS (CO) B-box transcription factor Putterill et al., 1995  
Light-quality pathway   
   PHYTOCHROME AND FLOWERING TIME1 (PFT1) Nuclear protein Cerdan and Chory, 2003  
Autonomous pathway   
   FCA RNA-binding protein Macknight et al., 1997  
   FY Polyadenylation factor Simpson et al., 2003  
   FPA RNA-binding protein Schomburg et al., 2001  
   FLOWERING LOCUS K (FLK) RNA-binding protein Lim et al., 2004  
   FVE MSI4 Ausin et al., 2004  
   FLOWERING LOCUS D (FLD) HDAC-associated protein He et al., 2003  
   LUMINIDEPENDENS (LD) Homeodomain protein Lee et al., 1994a  
Vernalization pathway   
   VERNALIZATION INSENSITIVE3 (VIN3) Protein with fibronectin repeats and PHD domain Sung and Amasino, 2004  
   VERNALIZATION1 (VRN1) B3 domain DNA-binding protein Levy et al., 2002  
   VERNALIZATION2 (VRN2) Su(z) 12-like polycomb protein Gendall et al., 2001  
Gene nameProtein functionReference
Floral pathway integrators   
   SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1/AGAMOUS-LIKE20(SOC1/AGL20) MADS-box transcription factor Samach et al., 2000; Lee et al., 2000  
   FT Putative kinase inhibitor Kardailsky et al., 1999; Kobayashi et al., 1999  
   LEAFY (LFY) Plant specific transcription factor Weigel et al., 1992; Nilsson et al., 1998  
Photoperiodic pathway   
   CONSTANS (CO) B-box transcription factor Putterill et al., 1995  
Light-quality pathway   
   PHYTOCHROME AND FLOWERING TIME1 (PFT1) Nuclear protein Cerdan and Chory, 2003  
Autonomous pathway   
   FCA RNA-binding protein Macknight et al., 1997  
   FY Polyadenylation factor Simpson et al., 2003  
   FPA RNA-binding protein Schomburg et al., 2001  
   FLOWERING LOCUS K (FLK) RNA-binding protein Lim et al., 2004  
   FVE MSI4 Ausin et al., 2004  
   FLOWERING LOCUS D (FLD) HDAC-associated protein He et al., 2003  
   LUMINIDEPENDENS (LD) Homeodomain protein Lee et al., 1994a  
Vernalization pathway   
   VERNALIZATION INSENSITIVE3 (VIN3) Protein with fibronectin repeats and PHD domain Sung and Amasino, 2004  
   VERNALIZATION1 (VRN1) B3 domain DNA-binding protein Levy et al., 2002  
   VERNALIZATION2 (VRN2) Su(z) 12-like polycomb protein Gendall et al., 2001  
Table 2.

Floral repressive genes

GeneProtein functionReference
Activators of FLC   
   FRIGIDA (FRI) Novel protein Johanson et al., 2000  
   FRIGIDA-LIKE1 (FRL1) Novel protein related to FRIGIDA Michaels et al., 2004  
   FRIGIDA-LIKE2 (FRL2) Novel protein related to FRIGIDA Michaels et al., 2004  
   VERNALIZATION INDEPENDENCE3 (VIP3) Novel WD-repeat protein Zhang et al., 2003  
   VERNALIZATION INDEPENDENCE4 (VIP4) Protein with homology to the yeast transcriptional activator, Leo1p Zhang and van Nocker, 2002  
   EARLY IN SHORT DAYS4 (ESD4) Nuclear protease regulating SUMOylation Murtas et al., 2003  
   AERIAL ROSETTE1 (ART1) Not yet identified Poduska et al., 2003  
   PHOTOPERIOD INSENSITIVE1 (PIE1) SWI/SNF-helicase-like protein Noh and Amasino, 2003  
Floral repressors   
   FLOWERING LOCUS C (FLC) MADS-box transcription factor Michaels and Amasino, 1999; Sheldon et al., 1999  
   FLOWERING LOCUS M (FLM) MADS-box transcription factor Scortecci et al., 2001  
   SHORT VEGETATIVE PHASE (SVP) MADS-box transcription factor Hartmann et al., 2000  
   TARGET OF EAT1/2 (TOE1/2) AP2-like transcription factor Aukerman and Sakai, 2003  
   SCHNARCHZAPFEN/SCHLAFMUTZE (SNZ/SMZ) AP2-like transcription factor Schmid et al., 2004  
   TFL1 Putative kinase inhibitor Bradley et al., 1997  
   TFL2/LHP1 Heterochromatin protein1 (HP1)-like protein Gaudin et al., 2001; Kotake et al., 2003  
   EMBRYONIC FLOWER1 Novel protein Aubert et al., 2001  
   EMBRYONIC FLOWER2 Su(z) 12-like polycomb protein Yoshida et al., 2001  
GeneProtein functionReference
Activators of FLC   
   FRIGIDA (FRI) Novel protein Johanson et al., 2000  
   FRIGIDA-LIKE1 (FRL1) Novel protein related to FRIGIDA Michaels et al., 2004  
   FRIGIDA-LIKE2 (FRL2) Novel protein related to FRIGIDA Michaels et al., 2004  
   VERNALIZATION INDEPENDENCE3 (VIP3) Novel WD-repeat protein Zhang et al., 2003  
   VERNALIZATION INDEPENDENCE4 (VIP4) Protein with homology to the yeast transcriptional activator, Leo1p Zhang and van Nocker, 2002  
   EARLY IN SHORT DAYS4 (ESD4) Nuclear protease regulating SUMOylation Murtas et al., 2003  
   AERIAL ROSETTE1 (ART1) Not yet identified Poduska et al., 2003  
   PHOTOPERIOD INSENSITIVE1 (PIE1) SWI/SNF-helicase-like protein Noh and Amasino, 2003  
Floral repressors   
   FLOWERING LOCUS C (FLC) MADS-box transcription factor Michaels and Amasino, 1999; Sheldon et al., 1999  
   FLOWERING LOCUS M (FLM) MADS-box transcription factor Scortecci et al., 2001  
   SHORT VEGETATIVE PHASE (SVP) MADS-box transcription factor Hartmann et al., 2000  
   TARGET OF EAT1/2 (TOE1/2) AP2-like transcription factor Aukerman and Sakai, 2003  
   SCHNARCHZAPFEN/SCHLAFMUTZE (SNZ/SMZ) AP2-like transcription factor Schmid et al., 2004  
   TFL1 Putative kinase inhibitor Bradley et al., 1997  
   TFL2/LHP1 Heterochromatin protein1 (HP1)-like protein Gaudin et al., 2001; Kotake et al., 2003  
   EMBRYONIC FLOWER1 Novel protein Aubert et al., 2001  
   EMBRYONIC FLOWER2 Su(z) 12-like polycomb protein Yoshida et al., 2001  

The multiple pathways that regulate the floral pathway integrators in Arabidopsis are classified as promotion, enabling and resetting pathways (Boss et al., 2004). Those that promote the floral transition are currently defined as the photoperiod, gibberellin, ambient-temperature and light-quality pathways(Fig. 1 and Table 1). Many angiosperms flower in response to the changing length of the day and night as the year progresses – this is called photoperiodism. Long day photoperiods promote flowering in Arabidopsis by activating the B-box transcription factor CONSTANS (CO), which is required for the upregulation of the floral integrator genes (Putterill et al., 1995; Suarez-Lopez et al., 2001; Samach et al.,2000; Blazquez and Weigel,2000). CO mRNA exhibits rhythmic, diurnal expression controlled by the circadian clock(Suarez-Lopez et al., 2001). This rhythm is reinforced through different photoreceptors acting on CO protein stability (Valverde et al.,2004). Phytochromes and cryptochromes are two groups of photoreceptors: PHYTOCHROME B (PHYB) promotes degradation of CO protein,whereas PHYTOCHROME A (PHYA), CRYPTOCHROME1 (CRY1) and CRYPTOCHROME2 (CRY2)stabilise it. These antagonistic activities result in the accumulation of CO only in the evening (Valverde et al.,2004; Yanovsky and Kay,2002). The hormone gibberellin promotes Arabidopsisflowering by upregulating the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1; also known as AGL20)(Wilson et al., 1992; Moon et al., 2003a) and LEAFY (LFY) (Blazquez et al., 1998; Blazquez and Weigel, 2000). The light-quality and ambient-temperature pathways appear to control FT expression, and activation of this integrator by the light-quality pathway requires the nuclear protein PHYTOCHROME AND FLOWERING TIME1 (PFT1) (Blázquez et al., 2003; Cerdan and Chory,2003; Halliday et al.,2003). After flowering, a series of genes in the resetting pathway appear to be required to reset the expression states of floral genes during formation of the gametes or during embryo development. Perturbation of their function results in ectopic expression of floral genes and premature flowering(Kinoshita et al., 2001; Moon et al., 2003b).

In contrast to the promotion pathways, enabling pathways determine the activity of repressors of the floral pathway integrators(Fig. 1, Table 2). The expression of the floral repressor FLC is regulated by several independent pathways. FLC is upregulated by a number of genes, including FRIGIDA(FRI), and is downregulated by prolonged periods of cold, a process known as vernalization. In temperate environments, the long period of cold temperature experienced over the winter can promote flowering, aligning reproductive development with spring and summer conditions. Once acquired, the vernalized state is `remembered' by the plant during subsequent growth,suggestive of an epigenetic basis. Several proteins, classified as the autonomous promotion pathway, act in parallel to vernalization and also repress FLC (Table 1). The autonomous pathway was named because of its lack of involvement in either the photoperiodic- or gibberellin-promotive floral pathways. This review focuses on recent work addressing the control of FLC expression in response to the prolonged periods of cold experienced during vernalization. Chromatin regulation and RNA processing have emerged as key mechanisms that modulate expression of the floral repressor FLC. We will speculate on how the different pathways controlling FLC expression may be integrated at the molecular level.

The floral repressor, FLC

FLC is a MADS-box transcriptional repressor, expressed predominantly in shoot and root apices and vasculature, that quantitatively represses flowering by repressing the floral pathway integrators(Michaels and Amasino, 1999; Sheldon et al., 1999; Michaels and Amasino, 2001). The mechanism by which it does this is not well understood, although a MADS-box binding site within the promoter of SOC1 is required(Hepworth et al., 2002).

Natural Arabidopsis accessions vary in their requirement for a vernalization treatment before flowering. Accessions are isogenic Arabidopsis backgrounds collected from a single location and maintained in a seed bank. Rapid cycling accessions, such as the laboratory strains Columbia and Landsberg erecta, flower early without a vernalization treatment. By contrast, many wild accessions flower much later,unless they receive a vernalization treatment; these are termed winter annual backgrounds (Fig. 2). Allelic variation at FLC contributes to natural variation in vernalization requirement, with weak alleles leading to a rapid-cycling habit(Fig. 2)(Michaels et al., 2003; Gazzani et al., 2003). Interestingly, the phenotypes of plants with naturally occurring weak FLC alleles appear to be caused by changes in the regulation of expression rather than alterations of protein function. The rapid-cycling Landsberg erecta and Da accessions contain FLC alleles with independent transposon insertions within the large FLC intron 1(Michaels et al., 2003; Gazzani et al., 2003). Sequences important for FLC regulation and expression have been mapped to this intron, which may account for the effects of these insertions(Sheldon et al., 2002; He et al., 2003).

Fig. 2.

Schematic representations of Arabidopsis plants summarizing the genetic control of vernalization requirement and response. The flowering phenotype of Arabidopsis is represented as either a rapid cycler(e.g. top right), which produces a flowering inflorescence, or as a winter annual accession (e.g. top left), which continues to produce rosette leaves. Rapid-cycling accessions do not require a vernalization treatment to flower early and are commonly used as laboratory backgrounds. By contrast, the majority of Arabidopsis accessions are winter annuals, which flower late unless they have been exposed to a prior vernalization treatment. Typically, 6 weeks of growth at 4°C produces a saturated vernalization response in Arabidopsis. Growth habit is indicated either with (+VRN)or without (–VRN) a vernalization treatment. When both FRI and FLC are active, the plant is vernalization responsive, as is found in many winter annual accessions. Mutations in either fri or flc can lead to rapid cycling. A vernalization-responsive FRI FLC accession is rendered insensitive to vernalization by a vrnmutation. Finally, a rapid-cycling fri FLC genotype becomes a winter annual background in the presence of an autonomous pathway mutation such as fca.

Fig. 2.

Schematic representations of Arabidopsis plants summarizing the genetic control of vernalization requirement and response. The flowering phenotype of Arabidopsis is represented as either a rapid cycler(e.g. top right), which produces a flowering inflorescence, or as a winter annual accession (e.g. top left), which continues to produce rosette leaves. Rapid-cycling accessions do not require a vernalization treatment to flower early and are commonly used as laboratory backgrounds. By contrast, the majority of Arabidopsis accessions are winter annuals, which flower late unless they have been exposed to a prior vernalization treatment. Typically, 6 weeks of growth at 4°C produces a saturated vernalization response in Arabidopsis. Growth habit is indicated either with (+VRN)or without (–VRN) a vernalization treatment. When both FRI and FLC are active, the plant is vernalization responsive, as is found in many winter annual accessions. Mutations in either fri or flc can lead to rapid cycling. A vernalization-responsive FRI FLC accession is rendered insensitive to vernalization by a vrnmutation. Finally, a rapid-cycling fri FLC genotype becomes a winter annual background in the presence of an autonomous pathway mutation such as fca.

There are five close homologues of FLC in the Arabidopsisgenome, and these are called MADS AFFECTING FLOWERING1(MAF1) to MAF5(Ratcliffe et al., 2003; Ratcliffe et al., 2001), with MAF1 also referred to as FLM(Scortecci et al., 2001) or AGL27 (Alvarez-Buylla et al.,2000). FLM is a floral repressor; however, it does not appear to be involved with the vernalization pathway(Scortecci et al., 2001). MAF2 is also a floral repressor and maf2 mutants show a pronounced vernalization response when subjected to short periods of cold that would not affect wild-type plants (Ratcliffe et al.,2003). MAF3 and MAF4 may act as floral repressors; however, the expression of MAF5 is increased by vernalization, so MAF5 may play an opposite role to FLCduring vernalization (Ratcliffe et al.,2003).

Activation of FLC

A key activator of FLC expression is FRI(Fig. 1, Table 1). Pioneering genetic analysis performed by Klaus Napp-Zinn (University of Cologne) in the 1950s identified allelic variation at FRI as the major determinant of flowering-time variation between rapid-cycling and winter annual accessions. Active FRI alleles confer late flowering and a vernalization requirement for early-flowering(Napp-Zinn, 1955; Napp-Zinn, 1957). It is striking, given that so many genes regulate FLC, that the winter annual habit can be mapped as a single gene trait to FRI. FRI represses flowering by upregulating FLC RNA levels(Michaels and Amasino, 1999; Sheldon et al., 2000) and,consistent with this, loss of FLC function eliminates the ability of FRI to delay flowering (Michaels and Amasino, 2001) (Fig. 2). Map-based cloning of FRI revealed that it encodes a novel protein with coiled-coil domains, but gave no indication as to the mechanism by which it upregulates FLC(Johanson et al., 2000). Analysis of natural Arabidopsis accessions identified at least nine independent loss-of-function mutations in FRI(Gazzani et al., 2003; Johanson et al., 2000; Le Corre et al., 2002). Hence,evolution of the rapid-cycling growth habit in some strains of Arabidopsis may have evolved multiple times through the loss of FRI. Genetic analysis of natural variation in flowering time has also identified AERIAL ROSETTE1 (ART1) from the extremely late-flowering accession Sy-0; ART1 acts synergistically with FRI to upregulate FLC (Poduska et al.,2003).

Recently, an increasing number of FLC activators have been identified by the analysis of early-flowering mutants. Two FRIGIDA-LIKE (FRL)genes, FRL1 and FRL2, are required for the upregulation of FLC expression by FRI (Michaels et al., 2004). Although FRI, FRL1 and FRL2 are related at the amino acid sequence level, they appear not to be functionally redundant(Michaels et al., 2004). The VERNALIZATION INDEPENDENCE (VIP) genes are also required for high FLCexpression (Zhang et al.,2003; Zhang and van Nocker,2002). The VIP4 protein exhibits homology with the yeast Leo1p protein, a component of the Paf complex, which is required for chromatin modification and transcriptional activation(Zhang et al., 2002; Porter et al., 2002). The VIP3 protein encodes WD repeats that typically mediate protein-protein interactions(Zhang et al., 2003). Hence,the VIP proteins may represent a complex that is required for FLCtranscription and chromatin regulation. PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1 (PIE1) also provides a link to chromatin regulation,as it encodes a protein that activates FLC and has homology to ATP-dependent chromatin-remodelling proteins of the ISWI and SWI2/SNF2 family(Noh and Amasino, 2003). A more tenuous link to chromatin regulation may be EARLY IN SHORT DAYS 4 (ESD4), which encodes a nuclear protease that upregulates FLC and is required for the regulation of SUMOylation (SMALL UBIQUITIN-RELATED MODIFIER) in Arabidopsis(Murtas et al., 2003; Reeves et al., 2002). SUMOylation is a recently discovered modification of histones and may be a part of the `histone code' (see Box 1) (Shiio and Eisenman,2003). Many other proteins, however, are also SUMOylated, so ESD4 may function to regulate the levels, activity or compartmentalization of an FLC regulator. Interestingly, mutations in VIP3, PIE1 and ESD4 suppress the high FLC expression caused by either dominant FRI alleles or mutations in the autonomous pathway(Zhang et al., 2003). By contrast, FRL1 and FRL2 are specifically required for the activation of FLC via FRI(Michaels et al., 2004).

Repression of FLC by vernalization

FLC mRNA levels are downregulated by vernalization. In nature,winter provides the necessary cold and results in the alignment of flowering with the favourable conditions of spring. At the molecular level, FLCregulation by the cold shows similarities with many of the physiological properties of vernalization (Chouard,1960; Lang, 1965). The vernalization response is strongly quantitative, with increasing durations of cold leading to progressively accelerated flowering once the plants return to ambient temperatures (Chouard,1960; Lang, 1965). The downregulation of FLC RNA is also a quantitative process, with longer periods of cold exposure leading to progressively lower FLCmRNA expression (Michaels and Amasino,1999; Sheldon et al.,2000). For annual plants (those that germinate and flower within one year), the vernalization response is saturated after several weeks of cold and, once established, the vernalized state is stable though subsequent growth at ambient temperatures, although it is reset after meiosis(Chouard, 1960; Lang, 1965). Similarly,repression of FLC levels is achieved after several weeks of cold and is then maintained at low levels throughout subsequent development, whilst being reset in the next generation(Sheldon et al., 2000). FLC repression must therefore be `remembered' through mitotic proliferation until flowering occurs. Furthermore, grafting experiments reveal that the site of cold perception during vernalization is the shoot apex and this is a region of FLC expression(Wellensiek, 1962; Wellensiek, 1964; Sung et al.,2004).

The maintenance of FLC repression following vernalization indicates that this gene is epigenetically silenced. Epigenetic silencing of genes is mediated by numerous covalent modifications of both the DNA and histones (Box 1)(Fischle et al., 2003; Bird, 2002). Early work on the control of vernalization focused on the role of DNA cytosine methylation(Finnegan et al., 1998). However, recent data has demonstrated a more important role for histone modifications at FLC chromatin during vernalization(Sung and Amasino, 2004; Bastow et al., 2004). Specific residues of histone H3 tails are modified by acetylation and methylation, and changes in these modifications serve as part of a `histone-code' specifying active or repressed gene activity states(Fischle et al., 2003). Vernalization increases histone H3 deacetylation in the 5′-region of FLC very early after exposure to the cold, a modification typically associated with gene repression (Sung and Amasino, 2004). Vernalization also induces increased methylation of histone H3 lysine residues 9 and 27, modifications associated with repressed gene states (Sung and Amasino,2004; Bastow et al.,2004). In animal systems, deacetylation is typically a prelude to acquisition of histone methylation(Fischle et al., 2003). Furthermore, histone methylation marks can act as signals to recruit further mediators of gene silencing (Orlando,2003). Interestingly, the histone marks observed at the FLC locus appear to be localised to specific regions of the gene(Bastow et al., 2004), at the 5′ end of the gene and within intron 1, co-localising with sequences already known to be involved in the regulation of FLC by vernalization (Sheldon et al.,2002; He et al.,2003).

Box 1. The histone code

graphic

The N-terminal tails of histones H3 and H4 undergo extensive post-translational modifications, including acetylation, phosphorylation,methylation, ubiquitination, SUMOylation and ADP-ribosylation (Wolffe, 1998;Lachner et al., 2003). These modifications have diverse consequences for gene expression and chromosomal organisation. Importantly, histone tail modifications can be inherited through cell division and thus they facilitate epigenetic control. Once acquired, several of these marks are bound by further proteins, which leads to the reinforcement of expression states or chromatin structure. Methylation and acetylation are two intensively studied modifications with well-defined roles in the control of gene expression states.

The N-terminal tail of histone H3 has four lysine residues – K4, K9,K27 and K36 – that are capable of being methylated by histone methyltransferases (HMTases) (Wolffe, 1998; Lachner et al., 2003). There is also diversity in whether particular lysines acquire mono-, di- or tri-methylation, and the relative importance of these marks may differ between organisms (Jackson et al.,2004). Typically, methylation at K9 and K27 is associated with gene repression, whereas methylation at K4 is associated with gene activation(Wolffe, 1998; Lachner et al., 2003). Methylation of K9 and K27 leads to the binding of the chromodomain proteins HP1 and POLYCOMB, respectively (indicated as `heterochromatic proteins' in the figure), which then results in mitotically stable, heterochromatic gene silencing(Bannister et al., 2001; Cao et al., 2002). Acetylation of histone H3 and H4 is generally present in active chromatin. In the case of H3, K9 acetylation and methylation are mutually exclusive marks, and deacetylation must occur first as a prelude to methylation and silencing(Wolffe, 1998; Lachner et al., 2003). The acetylation state of histone tails is determined by the relative activities of histone acetyltransferase (HAT)and histone decatylases (HDAC) acting at a particular locus. Recently, histone SUMOylation has also been demonstrated to mediate gene repression. SUMO is a small peptide related to ubiquitin, which when attached to H4 leads to gene silencing (Shiio and Eisenman,2003).

Genetic screens for mutants compromised in vernalization have identified trans-factors that mediate repression of FLC in response to the cold(Chandler et al., 1996; Sung and Amasino, 2004). The earliest acting gene is VERNALIZATION INSENSITIVE 3 (VIN3),which encodes a protein with a plant homeodomain (PHD) and fibronectrin type III repeats (Sung and Amasino,2004). PHD domains have been found in proteins associated with chromatin-remodelling complexes and can bind phosphoinositides, whereas fibronectin repeats are often involved in protein-protein interactions(Sung and Amasino, 2004). In vin3 mutants, the vernalization-mediated decrease in histone acetylation and increase in H3 K9 and K27 methylation does not occur, and thus FLC is not repressed by vernalization(Sung and Amasino, 2004). Intriguingly, VIN3 expression increases with cold, and only significantly accumulates after a period of cold sufficient to trigger vernalization (Sung and Amasino,2004). The VIN3 expression domain also overlaps with that of FLC (Sung and Amasino,2004). Hence, upregulation of VIN3 expression is an early step during the vernalization-signalling pathway. Understanding how prolonged cold induces expression of VIN3 is a key question for future research.

A second class of gene involved in the vernalization response is represented by the genes VERNALIZATION1 (VRN1) and VERNALIZATION2 (VRN2)(Chandler et al., 1996; Gendall et al., 2001; Levy et al., 2002). The vrn1 and vrn2 mutants are distinct from vin3 in that initial repression of FLC expression by the cold still occurs(Gendall et al., 2001; Levy et al., 2002). However,when vrn1 and vrn2 mutants return to ambient temperatures, FLC repression is not maintained and FLC RNA levels progressively increase (Chandler et al.,1996; Gendall et al.,2001; Levy et al.,2002). Unlike VIN3, expression of VRN1 and VRN2 is not upregulated by cold, and hence VIN3 may provide a cold-induced activity that recruits them to FLC(Sung and Amasino, 2004; Gendall et al., 2001; Levy et al., 2002). Furthermore, VRN1 and VRN2 are not required for the VIN3-mediated FLCdeacetylation early in vernalization (Sung and Amasino, 2004). The VRN2 protein shows homology to the Drosophila Polycomb protein Suppressor of Zeste 12 (Su(z)12)(Gendall et al., 2001). The Polycomb-Group (PcG) proteins function to maintain epigenetic gene activity states throughout Drosophila embryogenesis and cell proliferation(Orlando, 2003). Su(z)12 acts in a PcG complex, PRC2, with histone methyltransferase activity directed against histone H3 lysines 27 and 9(Kuzmichev et al., 2002; Muller et al., 2002). Hence,VRN2 is likely to mediate stable repression of FLC activity by a PcG-like mechanism. Indeed, in vrn2 mutants, increased histone H3 methylation of lysines 27 and 9 does not occur at FLC during vernalization (Sung and Amasino,2004; Bastow et al.,2004). This indicates that elements of the `histone-code' involved in developmental gene regulation are highly conserved between plants and animals. By contrast, the VRN1 protein is plant-specific and carries two B3 domains, which mediate non-sequence specific DNA binding in vitro(Levy et al., 2002). Unlike VRN2, VRN1 is required only for increases in histone H3 lysine 9 methylation,and not for methylation of lysine 27 (Sung and Amasino, 2004; Bastow et al., 2004). This suggests that VRN1 may function either downstream or independently of VRN2 during FLC repression. Overexpression of VRN1 revealed a vernalization-independent function for VRN1, mediated predominantly through the floral pathway integrator FT, and demonstrated that VRN1 requires vernalization-specific factors to target FLC (Levy et al.,2002).

Repression of FLC by autonomous pathway genes

The autonomous pathway acts in parallel to vernalization to repress FLC expression (Koornneef et al.,1991; Simpson and Dean,2002). In the absence of FRI, this pathway is the major regulator of FLC levels and therefore confers a vernalization requirement (Koornneef et al.,1991). Mutants in the autonomous pathway are late-flowering because of elevated levels of FLC mRNA, and this late-flowering is vernalization responsive (Koornneef et al., 1991; Sheldon et al.,2000; Michaels and Amasino,2001) (Fig. 1 and Table 1). Although all members of this pathway act to limit FLC expression, genetic analysis has revealed that they have distinct functions. Two epistasis groups – FCA, FY and FPA, FVE – have been found using double mutants, although the significance of this is not yet fully understood(Koornneef et al., 1998). The ld and fld mutations are strongly suppressed by the FLC allele in Ler, the background in which the other mutations were isolated, so epistasis analysis of these genes has not yet been performed (Lee et al., 1994b; Sanda and Amasino, 1996).

HDACs in the flowering response

FLD encodes a protein with homology to a human protein that functions in the histone deacetylase 1,2 (HDAC1/2) co-repressor complex(He et al., 2003). Histone deacetylation mediated by this complex is commonly associated with gene repression (He et al., 2003). The FLD protein carries an N-terminal SWIRM domain, such as that found in chromatin remodelling enzymes, in addition to a polyamine oxidase domain(He et al., 2003). In fld mutants, the 5′-end of FLC displays hyperacetylation of histone H4 (He et al.,2003), indicating that FLD is required to deacetylate FLCchromatin and thereby repress its expression. Intriguingly, removal of a 295-base pair region of FLC intron 1 prevents this regulation and results in high FLC expression, independent of FLD activity(He et al., 2003). Thus, this FLC intronic region may contain cis sequences required for recruitment of a HDAC complex. Currently, the identity of the HDAC that functions with FLD is unknown.

The Arabidopsis genome encodes four HDAC1/2 homologs but late-flowering mutations in these genes have yet to be identified(Pandey et al., 2002). However, an antisense construct designed to target multiple HDACs does result in delayed flowering, which may be due to a failure to repress FLC(Tian and Chen, 2001). Analysis of histone H4 acetylation status in the other autonomous mutants revealed a similar hyperacetylation phenotype only in fve(He et al., 2003). FVE encodes the nuclear WD-repeat protein, MSI4(Ausin et al., 2004). There are five MSI-related proteins in Arabidopsis, which display homology to the mammalian Retinoblastoma Associated Protein46 (RbAp46) and RbAp48 proteins(Ausin et al., 2004). MSI-like proteins are typically found in complexes involved in chromatin assembly and histone modification, and FVE was demonstrated to co-immunoprecipitate with plant Rb (Retinoblastoma protein) (Ausin et al., 2004). In other systems, Rb functions in histone deacetylase complexes, which again is consistent with the histone hyperacetylation of FLC observed in fve and fld mutants(Ausin et al., 2004; He et al., 2003). In addition to a histone H4 hyperacetylation phenotype, analysis in fve mutants also revealed hyperacetylation of histone H3, indicating that both histones are deacetylated by this pathway (Ausin et al., 2004). Hence, FVE and FLD are likely to act together in a HDAC complex to repress FLC expression(Ausin et al., 2004; He et al., 2003). It will be important to determine if this HDAC complex is specifically targeted to FLC or whether it performs broader functions that are covered by redundancy. How this deacetylase activity integrates with the epigenetic modifications directed by vernalization is also an interesting question.

RNA processing

Mutations in the autonomous pathway gene FCA display no effect on FLC acetylation status (He et al., 2003). Indeed, FCA appears to be genetically distinct from FVE (Koornneef et al., 1998). FCA encodes a plant-specific, nuclear RNA-binding protein (Macknight et al.,1997). In addition to two RNA recognition motif (RRM) domains, FCA possesses a C-terminal WW protein interaction domain(Macknight et al., 1997; Sudol and Hunter, 2000). This domain mediates interaction with another component of the autonomous pathway,FY (Simpson et al., 2003). In contrast to FCA, FY is highly conserved throughout eukaryotes and displays homology to the yeast polyadenylation factor, Pfs2p(Ohnacker et al., 2000; Simpson et al., 2003). Pfs2p carries seven WD repeats and acts as a scaffold protein within the large CPF(cleavage and polyadenylation factor) complex(Ohnacker et al., 2000). The CPF complex is required for 3′-cleavage and polyadenylation of pre-mRNA transcripts, and strong mutations in polyadenylation factors, including PFS2, are lethal because of a failure to correctly express RNA polymerase II transcripts (Ohnacker et al., 2000). In addition to these WD repeats, FY possesses a novel C-terminal domain with which FCA interacts. FY may perform a generic function in RNA processing, while also functioning in regulated polyadenylation through interaction with FCA. FPA encodes a second plant-specific RRM domain protein within the autonomous pathway(Schomburg et al., 2001). Although FPA is required for the regulation of FLC, the level at which it functions is unknown. Finally, FLK is the most recently identified member of the autonomous pathway and encodes a nuclear KH-type RNA-binding protein (Lim et al.,2004). Hence, multiple RNA-binding proteins are required for repression of FLC expression by the autonomous pathway. Determining whether this reflects a cascade of post-transcriptional regulators or a complex of RNA-binding factors will require further analysis of proteins of the autonomous pathway.

Currently there is no evidence that FCA/FY, FPA or FLK directly regulates FLC mRNA processing. However, FCA expression itself is complex and exhibits an autoregulatory mechanism involving polyadenylation site choice (Macknight et al.,1997; Macknight et al.,2002; Quesada et al.,2003). There are four FCA transcripts, and intron 3 is a major site of alternative processing. Premature cleavage and polyadenylation within this intron generates the truncated, non-functional FCA-βtranscript (Macknight et al.,1997; Macknight et al.,2002). FCA negatively autoregulates its own expression by promoting intron 3 polyadenylation(Quesada et al., 2003). This regulation also requires the functional interaction between FCA and FY,demonstrating that these proteins mediate alternative 3′-end processing(Macknight et al., 1997; Macknight et al., 2002; Quesada et al., 2003). Hence,FCA may function as a novel trans-regulator of polyadenylation site choice via interaction with the core 3′-processing factor FY. An intriguing aspect of FCA autoregulation is its tissue specificity. Premature polyadenylation is inhibited in meristematic regions relative to non-meristematic regions (Macknight et al., 2002; Quesada et al.,2003). The mechanism by which this occurs is currently unknown but might also have a consequence for the regulation of FLC. FPA and FLK appear not to be required for FCA intron 3 regulation(Lim et al., 2004; Quesada et al., 2003). Hence,the proteins of the autonomous pathway appear to have partially redundant activities that repress FLC by distinct mechanisms. It is not known whether the chromatin regulation and RNA processing activities of the autonomous pathway are integrated during the control of FLCexpression, although chromatin modification and 3′-processing interact functionally in yeast (Alen et al.,2002).

Integration of the pathways regulating FLC expression

Plants need to monitor their environmental conditions during growth and development, and acquire sufficient resources to complete reproductive development. The FLC activators are considered to function early in development to ensure high levels of FLC and floral repression at germination,thus avoiding precocious flowering before resources have accumulated. The repressors of FLC expression may be downregulated early in development for the same reason. This appears to be the case for FCA,as production of the active FCA transcript via a change in polyadenylation site usage increases significantly in meristems 4-5 days after germination (Macknight et al.,2002). Indeed, bypassing this control on FCA overrides FRI repression of flowering(Quesada et al., 2003). However, the precise temporal expression of many FLC activators, and when their functions are required in flowering control, remains to be determined.

The interaction between FLC activators and repressors effectively determines whether a plant adopts a winter annual or rapid-cycling habit. It is possible that this interaction is determined by the antagonistic effects of the different pathways on FLC chromatin. PIE1 and VIP proteins are FLC upregulators that may act to promote active chromatin, whereas FVE and FLD act to deacetylate histones, thus promoting a silent chromatin state. The roles of the multiple RNA-binding proteins (FCA, FPA, FLK), and the polyadenylation factor FY, in repressing FLC raises some interesting possibilities. They may function to repress FLC directly or by regulating components of the activation pathway. Alternatively, the recent demonstrations of non-coding RNA acting in chromatin regulation means that they may play a role in generating RNA intermediates that feed back to regulate FLC chromatin (Volpe et al., 2002; Zilberman et al.,2003).

The onset of winter perturbs the steady-state FLC expression by the induction of VIN3 after several weeks of cold, potentially initiating a chain of epigenetic modifications at the FLC locus. An early step in this sequence appears to be histone deacetylation(Fig. 3), and the stable maintenance of FLC repression involves the activities of VRN1, VRN2 and histone methylation. In animals, histone methylation recruits further proteins required to maintain gene repression(Orlando, 2003), although the identity of such factors in plants and during vernalization remains unknown. FLC expression then remains low during subsequent development and flowering, but at some stage during meiosis, gametogenesis or early embryogenesis, FLC expression is reset. The epigenetic modifications at FLC established during vernalization, or by the activity of the autonomous pathway, are erased, allowing high FLC expression in the young seedlings and determining a requirement for vernalization in each generation. This molecular sequence accounts for flowering in annual plants. Many plants, however, are perennials, that is they live for many years with only a proportion of the apical meristems undergoing the transition to flowering each year. Whether similar mechanisms are involved in controlling flowering in perennials remains to be established.

Fig. 3.

Model for the regulation of the floral repressor FLC throughout the Arabidopsis life cycle. During seedling growth, a group of genes encode proteins that function as activators of FLC expression (shown in purple); these genes include FRI, FRL1, FRL2, ESD4, ART1, PIE1,VIP3 and VIP4. These proteins may maintain FLCchromatin in an active state (indicated by an open structure and the presence of active histone tail modifications shown in green). The autonomous pathway functions antagonistically to the activators to repress FLCexpression. The RNA-binding proteins FCA, FPA and FLK, and the polyadenylation factor FY, may function post-transcriptionally to achieve this and are shown in red. The FVE/FLD proteins act with a putative histone deacetylase (HDAC;all shown in orange) to promote an inactive FLC chromatin state,represented by a closed structure with inactive histone tail modifications(red). FLC is also repressed by exposure to long periods of cold(vernalization). The proteins acting in the vernalization pathway are shown in pink. Prolonged cold induces VIN3 expression, which promotes an inactive FLC chromatin state. Subsequently, the VRN1 and VRN2 proteins are recruited to FLC, and are required for the methylation of FLC histones and the maintenance of silencing. These marks may promote the association of silencing factors with FLC chromatin that reinforce its repression. During meiosis, gametogenesis or early embryogenesis, FLC repression is overcome, thus resetting its expression in the next generation.

Fig. 3.

Model for the regulation of the floral repressor FLC throughout the Arabidopsis life cycle. During seedling growth, a group of genes encode proteins that function as activators of FLC expression (shown in purple); these genes include FRI, FRL1, FRL2, ESD4, ART1, PIE1,VIP3 and VIP4. These proteins may maintain FLCchromatin in an active state (indicated by an open structure and the presence of active histone tail modifications shown in green). The autonomous pathway functions antagonistically to the activators to repress FLCexpression. The RNA-binding proteins FCA, FPA and FLK, and the polyadenylation factor FY, may function post-transcriptionally to achieve this and are shown in red. The FVE/FLD proteins act with a putative histone deacetylase (HDAC;all shown in orange) to promote an inactive FLC chromatin state,represented by a closed structure with inactive histone tail modifications(red). FLC is also repressed by exposure to long periods of cold(vernalization). The proteins acting in the vernalization pathway are shown in pink. Prolonged cold induces VIN3 expression, which promotes an inactive FLC chromatin state. Subsequently, the VRN1 and VRN2 proteins are recruited to FLC, and are required for the methylation of FLC histones and the maintenance of silencing. These marks may promote the association of silencing factors with FLC chromatin that reinforce its repression. During meiosis, gametogenesis or early embryogenesis, FLC repression is overcome, thus resetting its expression in the next generation.

Conclusions

Multiple mechanisms have evolved to ensure the fine control of FLClevels and thus the timing of the transition to flowering. Considerable progress has been made towards elucidating the molecular mechanisms involved,but several important questions remain. Is VIN3 expression really the cold-induced trigger that initiates the chromatin changes at FLC? How do these changes overcome the function of activators such as FRI, and how do genes of the autonomous pathway fit into the molecular picture? Understanding the mechanisms involved in the resetting of FLC expression may provide insights into fundamental aspects of epigenetic reprogramming in plants and animals. The power of forward genetics, together with the exploitation of natural variation, will undoubtedly be key to unravelling many of these questions, and will provide answers as to how the different Arabidopsis reproductive strategies have been selected.

Recent progress in wheat has also identified key regulators determining the vernalization requirement in cereals (Yan et al., 2003; Trevaskis et al., 2003; Yan et al.,2004). The genes identified are so far distinct from those identified in Arabidopsis. Wheat VRN1 functions as a floral promoter and is a MADS-box protein with homology to APETALA1(Yan et al., 2003). Wheat VRN2 contains a CCT domain (a 43-amino acid region with homology to Arabidopsis proteins CO, CO-LIKE and TOC1), and it functions to repress directly or indirectly the expression of wheat VRN1(Yan et al., 2004). Vernalization progressively reduces levels of wheat VRN2 RNA,preventing repression of VRN1 and promoting flowering. The involvement of distinct proteins in cereals and Arabidopsis implies that different pathways have evolved to regulate the vernalization requirement. However, it will be interesting to determine whether chromatin regulation of these targets also mediates the epigenetic memory of winter in wheat. Together, work in cereals and Arabidopsis should allow the manipulation of vernalization, a key agricultural trait.

Acknowledgements

Thanks to all members of the Dean group, past and present, for helpful discussions, and especially Gordon Simpson for his comments on the manuscript.

References

Alen, C., Kent, N. A., Jones, H. S., O'Sullivan, J., Aranda, A. and Proudfoot, N. J. (
2002
). A role for chromatin remodeling in transcriptional termination by RNA polymerase II.
Mol. Cell
10
,
1441
-1452.
Alvarez-Buylla, E. R., Liljegren, S. J., Pelaz, S., Gold, S. J.,Burgeff, C., Ditta, G. S., Verara-Silva, F. and Yanofsky, M. F.(
2000
). MADS-box gene evolution beyond flowers: expression in pollen, endosperm, guard cells, roots and trichomes.
Plant J.
24
,
1
-11.
Aubert, D., Chen, L., Moon, Y. H., Martin, D., Castle, L. A.,Yang, C. H. and Sung, Z. R. (
2001
). EMF1, a novel protein involved in control of shoot architecture and flowering in Arabidopsis.
Plant Cell
13
,
1865
-1875.
Aukerman, M. J. and Sakai, H. (
2003
). Regulation of flowering time and floral organ identity by a MicroRNA and its APETALA2-like target genes.
Plant Cell
15
,
2730
-2741.
Ausin, I., Alonso-Blanco, C., Jarillo, J. A., Ruiz-Garcia, L. and Martinez-Zapater, J. M. (
2004
). Regulation of flowering time by FVE, a retinoblastoma-associated protein.
Nat. Genet.
36
,
162
-166.
Bannister, A. J., Zegerman, P., Partridge, J. F., Miska, E. A.,Thomas, J. O., Allshire, R. C. and Kouzarides, T. (
2001
). Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain.
Nature
410
,
120
-124.
Bastow, R., Mylne, J. S., Lister, C., Lippman, Z., Martienssen,R. A. and Dean, C. (
2004
). Vernalization requires epigenetic silencing of FLC by histone methylation.
Nature
427
,
164
-167.
Battey, N. H. (
2000
). Aspects of seasonality.
J. Exp. Bot.
51
,
1769
-1780.
Bird, A. (
2002
). DNA methylation patterns and epigenetic memory.
Genes Dev.
16
,
6
-21.
Blázquez, M. A. and Weigel, D. (
2000
). Integration of floral inductive signals in Arabidopsis.
Nature
404
,
889
-892.
Blázquez, M. A., Green, R., Nilsson, O., Sussman, M. R. and Weigel, D. (
1998
). Gibberellins promote flowering of Arabidopsis by activating the LEAFY promoter.
Plant Cell
10
,
791
-800.
Blázquez, M., Ahn, J. H. and Weigel, D.(
2003
). A thermosensory pathway controlling flowering time in Arabidopsis thaliana.
Nat. Genet.
33
,
168
-171.
Boss, P., Bastow, R., Mylne, J. M. and Dean, C.(
2004
). Multiple pathways in the decision to flower: enabling,promoting and resetting.
Plant Cell
(in press).
Bradley, D., Ratcliffe, O., Vincent, C., Carpenter, R. and Coen,E. (
1997
). Inflorescence commitment and architecture in Arabidopsis.
Science
275
,
80
-83.
Cao, R., Wang, L., Wang, H., Xia, L., Erdjument-Bromage, H.,Tempst, P., Jones, R. S. and Zhang, Y. (
2002
). Role of histone H3 lysine 27 methylation in Polycomb-group silencing.
Science
298
,
1039
-1043.
Cerdan, P. D. and Chory, J. (
2003
). Regulation of flowering time by light quality.
Nature
423
,
881
-885.
Chandler, J., Wilson, A. and Dean, C. (
1996
). Arabidopsis mutants showing an altered response to vernalization.
Plant J.
10
,
637
-644.
Chouard, P. (
1960
). Vernalization and its relations to dormancy.
Ann. Rev. Plant Physiol.
11
,
191
-237.
Finnegan, E. J., Genger, R. K., Kovac, K., Peacock, W. J. and Dennis, E. S. (
1998
). DNA methylation and the promotion of flowering by vernalization.
Proc. Natl. Acad. Sci. USA
95
,
5824
-5829.
Fischle, W., Wang, Y. and Allis, C. D. (
2003
). Binary switches and modification cassettes in histone biology and beyond.
Nature
425
,
475
-479.
Gaudin, V., Limbault, M., Pouteau, S., Juul, T., Zhao, G.,Lefebvre, D. and Grandjean, O. (
2001
). Mutations in LIKE HETEROCHROMATIN PROTEIN 1 affect flowering time and plant architecture in Arabidopsis.
Development
128
,
4847
-4858.
Gazzani, S., Gendall, A. R., Lister, C. and Dean, C.(
2003
). Analysis of the molecular basis of flowering time variation in Arabidopsis accessions.
Plant Physiol.
132
,
1107
-1114.
Gendall, A. R., Levy, Y. Y., Wilson, A. and Dean, C.(
2001
). The VERNALIZATION 2 gene mediates the epigenetic regulation of vernalization in Arabidopsis.
Cell
107
,
525
-535.
Halliday, K. J., Salter, M. G., Thingnaes, E. and Whitelam, G. C. (
2003
). Phytochrome control of flowering is temperature sensitive and correlates with expression of the floral integrator FT.
Plant J.
33
,
875
-885.
Hartmann, U., Hohmann, S., Nettesheim, K., Wisman, E., Saedler,H. and Huijser, P. (
2000
). Molecular cloning of SVP:a negative regulator of the floral transition in Arabidopsis.
Plant J.
21
,
351
-360.
He, Y., Michaels, S. D. and Amasino, R. M.(
2003
). Regulation of flowering time by histone acetylation in Arabidopsis.
Science
302
,
1751
-1754.
Hepworth, S. R., Valverde, F., Ravenscroft, D., Mouradov, A. and Coupland, G. (
2002
). Antagonistic regulation of flowering-time gene SOC1 by CONSTANS and FLC via separate promoter motifs.
EMBO J.
21
,
4327
-4337.
Izawa, T., Takahashi, Y. and Yano, M. (
2003
). Comparative biology comes into bloom: genomic and genetic comparison of flowering pathways in rice and Arabidopsis.
Curr. Opin. Plant Biol.
6
,
113
-120.
Jackson, J. P., Johnson, L., Jasencakova, Z., Zhang, X.,PerezBurgos, L., Singh, P. B., Cheng, X., Schubert, I., Jenuwein, T. and Jacobsen, S. E. (
2004
). Dimethylation of histone H3 lysine 9 is a critical mark for DNA methylation and gene silencing in Arabidopsis thaliana.
Chromosoma
112
,
308
-315.
Johanson, U., West, J., Lister, C., Michaels, S., Amasino, R. and Dean, C. (
2000
). Molecular analysis of FRIGIDA,a major determinant of natural variation in Arabidopsis flowering time.
Science
290
,
344
-347.
Kardailsky, I., Shukla, V. K., Ahn, J. H., Dagenais, N.,Christensen, S. K., Nguyen, J. T., Chory, J., Harrison, M. J. and Weigel,D. (
1999
). Activation tagging of the floral inducer FT.
Science
286
,
1962
-1965.
Kinoshita, T., Harada, J. J., Goldberg, R. B. and Fischer, R. L. (
2001
). Polycomb repression of flowering during early plant development.
Proc. Natl. Acad. Sci USA
98
,
14156
-14161.
Kobayashi, Y., Kaya, H., Goto, K., Iwabuchi, M. and Araki,T. (
1999
). A pair of related genes with antagonistic roles in mediating flowering signals.
Science
286
,
1960
-1962.
Koornneef, M., Hanhart, C. J. and Van der Veen, J. H.(
1991
). A genetic and physiological, analysis of late flowering mutants in Arabidopsis thaliana.
Mol. Gen. Genet.
229
,
57
-66.
Koornneef, M., Alonso-Blanco, C., Blankestijn-de Vries, H.,Hanhart, C. J. and Peeters, A. J. (
1998
). Genetic interactions among late-flowering mutants of Arabidopsis.
Genetics
148
,
885
-892.
Kotake, T., Takada, S., Nakahigashi, K., Ohto, M. and Goto,K. (
2003
). Arabidopsis TERMINAL FLOWER2 gene encodes a HETEROCHROMATIN PROTEIN1 homolog and represses both FLOWERING LOCUS T to regulate flowering time and several floral homeotic genes.
Plant Cell Physiol.
44
,
555
-564.
Kuzmichev, A., Nishioka, K., Erdjument-Bromage, H., Tempst, P. and Reinberg, D. (
2002
). Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein.
Genes Dev.
16
,
2893
-2905.
Lang, A. (
1965
). Physiology of flower initiation. In
Encyclopedia of Plant Physiology
, vol.
15
(ed. W. Ruhland), pp.
1380
-1536. Berlin: Springer-Verlag.
Le Corre, V., Roux, F. and Reboud, X. (
2002
). DNA polymorphism at the FRIGIDA gene in Arabidopsis thaliana: extensive nonsynonymous variation is consistent with local selection for flowering time.
Mol. Biol. Evol.
19
,
1261
-1271.
Lee, H., Suh, S. S., Park, E., Cho, E., Ahn, J. H., Kim, S. G.,Lee, J. S., Kwon, Y. M. and Lee, I. (
2000
). The AGAMOUS-LIKE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis.
Genes Dev.
14
,
2366
-2376.
Lee, I., Aukerman, M. J., Gore, S. L., Lohman, K. N., Michaels,S. D., Weaver, L. M., John, M. C., Feldmann, K. A. and Amasino, R. M.(
1994a
). Isolation of LUMINIDEPENDENS: a gene involved in the control of flowering time in Arabidopsis.
Plant Cell
6
,
75
-83.
Lee, I., Michaels, S. D., Masshardt, A. S. and Amasino, R. M. (
1994b
). The late-flowering phenotype of FRIGIDAand mutations in LUMINIDEPENDENS is suppressed in the Landsberg erecta strain of Arabidopsis.
Plant J.
6
,
903
-909.
Levy, Y. Y., Mesnage, S., Mylne, J. S., Gendall, A. R. and Dean,C. (
2002
). Multiple roles of Arabidopsis VRN1 in vernalization and flowering time control.
Science
297
,
243
-246.
Lim, M.-H., Kim, J., Kim, Y.-S., Chung, K.-S., Seo, Y.-H., Lee,I., Kim, I., Kim, J., Hong, C. B., Kim, H.-J. et al. (
2004
). A new Arabidopsis gene, FLK, encodes an RNA binding protein with K homology motifs and regulates flowering via FLOWERING LOCUS C.
Plant Cell
16
,
731
-740.
Macknight, R., Bancroft, I., Page, T., Lister, C., Schmidt, R.,Love, K., Westphal, L., Murphy, G., Sherson, S., Cobbett, C. et al.(
1997
). FCA, a gene controlling flowering time in Arabidopsis, encodes a protein containing RNA-binding domains.
Cell
89
,
737
-745.
Macknight, R., Duroux, M., Laurie, R., Dijkwel, P., Simpson, G. and Dean, C. (
2002
). Functional significance of the alternative transcript processing of the Arabidopsis floral promoter FCA.
Plant Cell
14
,
877
-888.
Michaels, S. D. and Amasino, R. M. (
1999
). FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering.
Plant Cell
11
,
949
-956.
Michaels, S. D. and Amasino, R. M. (
2001
). Loss of FLOWERING LOCUS C activity eliminates the late-flowering phenotype of FRIGIDA and autonomous pathway mutations but not responsiveness to vernalization.
Plant Cell
13
,
935
-941.
Michaels, S. D., He, Y., Scortecci, K. C. and Amasino, R. M.(
2003
). Attenuation of FLOWERING LOCUS C activity as a mechanism for the evolution of summer-annual flowering behavior in Arabidopsis.
Proc. Natl. Acad. Sci. USA
100
,
10102
-10107.
Michaels, S. D., Bezerra, I. C. and Amasino, R. M.(
2004
). FRIGIDA-related genes are required for the winter-annual habit in Arabidopsis.
Proc. Natl. Acad. Sci. USA
101
,
3281
-3285.
Moon, J., Suh, S. S., Lee, H., Choi, K. R., Hong, C. B., Paek,N. C., Kim, S. G. and Lee, I. (
2003a
). The SOC1MADS-box gene integrates vernalization and gibberellin signals for flowering in Arabidopsis.
Plant J.
35
,
613
-623.
Moon, Y. H., Chen, L., Pan, R. L., Chang, H. S., Zhu, T.,Maffeo, D. M. and Sung, Z. R. (
2003b
). EMF genes maintain vegetative development by repressing the flower program in Arabidopsis.
Plant Cell
15
,
681
-693.
Muller, J., Hart, C. M., Francis, N. J., Vargas, M. L.,Sengupta, A., Wild, B., Miller, E. L., O'Connor, M. B., Kingston, R. E. and Simon, J. A. (
2002
). Histone methyltransferase activity of a Drosophila Polycomb group repressor complex.
Cell
111
,
197
-208.
Murtas, G., Reeves, P. H., Fu, Y. F., Bancroft, I., Dean, C. and Coupland, G. (
2003
). A nuclear protease required for flowering-time regulation in Arabidopsis reduces the abundance of SMALL UBIQUITIN-RELATED MODIFIER conjugates.
Plant Cell
15
,
2308
-2319.
Napp-Zinn, K. (
1955
). Genetische Grundlagen des Kältebedürfnisses bei Arabidopsis thaliana (L.) Heynh.
Naturwissenschaften
42
,
650
.
Napp-Zinn, K. (
1957
). Untersuchungen über das Vernalisationsverhalten einer winterannuellen Rasse von Arabidopsis thaliana.
Planta
50
,
170
-177.
Nilsson, O., Lee, I., Blázquez, M. A. and Weigel, D.(
1998
). Flowering-time genes modulate the response to LEAFY activity.
Genetics
150
,
403
-410.
Noh, Y.-S. and Amasino, R. M. (
2003
). PIE1, an ISWI family gene, is required for FLC activation and floral repression in Arabidopsis.
Plant Cell
15
,
1671
-1682.
Ohnacker, M., Barabino, S. M., Preker, P. J. and Keller, W.(
2000
). The WD-repeat protein Pfs2p bridges two essential factors within the yeast pre-mRNA 3′-end-processing complex.
EMBO J.
19
,
37
-47.
Orlando, V. (
2003
). Polycomb, epigenomes, and control of cell identity.
Cell
112
,
599
-606.
Pandey, R., Muller, A., Napoli, C. A., Selinger, D. A., Pikaard,C. S., Richards, E. J., Bender, J., Mount, D. W. and Jorgensen, R. A.(
2002
). Analysis of histone acetyltransferase and histone deacetylase families of Arabidopsis thaliana suggests functional diversification of chromatin modification among multicellular eukaryotes.
Nucl. Acids Res.
30
,
5036
-5055.
Poduska, B., Humphrey, T., Redweik, A. and Grbic, V.(
2003
). The synergistic activation of FLOWERING LOCUS Cby FRIGIDA and a new flowering gene AERIAL ROSETTE 1underlies a novel morphology in Arabidopsis.
Genetics
163
,
1457
-1465.
Poethig, R. S. (
1990
). Phase change and the regulation of shoot morphogenesis in plants.
Science
250
,
923
-930.
Porter, S. E., Washburn, T. M., Chang, M. and Jaehning, J. A. (
2002
). The yeast Pafl-RNA polymerase II complex is required for full expression of a subset of cell cycle-regulated genes.
Eukaryot. Cell
1
,
830
-842.
Putterill, J., Robson, F., Lee, K., Simon, R. and Coupland,G. (
1995
). The CONSTANS gene of Arabidopsispromotes flowering and encodes a protein showing similarities to zinc finger transcription factors.
Cell
80
,
847
-857.
Quesada, V., Macknight, R., Dean, C. and Simpson, G. G.(
2003
). Autoregulation of FCA pre-mRNA processing controls Arabidopsis flowering time.
EMBO J.
22
,
3142
-3152.
Ratcliffe, O. J., Nadzan, G. C., Reuber, T. L. and Riechmann, J. L. (
2001
). Regulation of flowering in Arabidopsis by an FLC homologue.
Plant Physiol.
126
,
122
-132.
Ratcliffe, O. J., Kumimoto, R. W., Wong, B. J. and Riechmann, J. L. (
2003
). Analysis of the Arabidopsis MADS AFFECTING FLOWERING gene family: MAF2 prevents vernalization by short periods of cold.
Plant Cell
15
,
1159
-1169.
Reeves, P. H., Murtas, G., Dash, S. and Coupland, G.(
2002
). early in short days 4, a mutation in Arabidopsis that causes early flowering and reduces the mRNA abundance of the floral repressor FLC.
Development
129
,
5349
-5361.
Samach, A., Onouchi, H., Gold, S. E., Ditta, G. S.,Schwarz-Sommer, Z., Yanofsky, M. F. and Coupland, G. (
2000
). Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis.
Science
288
,
1613
-1616.
Sanda, S. L. and Amasino, R. M. (
1996
). Ecotype-specific expression of a flowering mutant phenotype in Arabidopsis thaliana.
Plant Physiol.
111
,
641
-644.
Schmid, M., Uhlenhaut, N. H., Godard, F., Demar, M., Bressan,R., Weigel, D. and Lohmann, J. U. (
2004
). Dissection of floral induction pathways using global expression analysis.
Development
130
,
6001
-6012.
Schomburg, F. M., Patton, D. A., Meinke, D. W. and Amasino, R. M. (
2001
). FPA, a gene involved in floral induction in Arabidopsis, encodes a protein containing RNA-recognition motifs.
Plant Cell
13
,
1427
-1436.
Scortecci, K. C., Michaels, S. D. and Amasino, R. M.(
2001
). Identification of a MADS-box gene, FLOWERING LOCUS M, that represses flowering.
Plant J.
26
,
229
-236.
Sheldon, C. C., Burn, J. E., Perez, P. P., Metzger, J., Edwards,J. A., Peacock, W. J. and Dennis, E. S. (
1999
). The FLF MADS box gene: a repressor of flowering in Arabidopsisregulated by vernalization and methylation.
Plant Cell
11
,
445
-458.
Sheldon, C. C., Rouse, D. T., Finnegan, E. J., Peacock, W. J. and Dennis, E. S. (
2000
). The molecular basis of vernalization: the central role of FLOWERING LOCUS C (FLC).
Proc. Natl. Acad. Sci. USA
97
,
3753
-3758.
Sheldon, C. C., Conn, A. B., Dennis, E. S. and Peacock, W. J. (
2002
). Different regulatory regions are required for the vernalization-induced repression of FLOWERING LOCUS C and for the epigenetic maintenance of repression.
Plant Cell
14
,
2527
-2537.
Shiio, Y. and Eisenman, R. N. (
2003
). Histone sumoylation is associated with transcriptional repression.
Proc. Natl. Acad. Sci USA
100
,
13225
-13230.
Simon, R., Igeno, M. I. and Coupland, G.(
1996
). Activation of floral meristem identity genes in Arabidopsis.
Nature
384
,
59
-62.
Simpson, G. G. and Dean, C. (
2002
). Arabidopsis, the Rosetta stone of flowering time?
Science
296
,
285
-289.
Simpson, G. G., Dijkwel, P. P., Quesada, V., Henderson, I. and Dean, C. (
2003
). FY is an RNA 3′-end processing factor that interacts with FCA to control the Arabidopsis floral transition.
Cell
13
,
777
-787.
Suarez-Lopez, P., Wheatley, K., Robson, F., Onouchi, H.,Valverde, F. and Coupland, G. (
2001
). CONSTANS mediates between the circadian clock and the control of flowering in Arabidopsis.
Nature
410
,
1116
-1120.
Sudol, M. and Hunter, T. (
2000
). NeW wrinkles for an old domain.
Cell
103
,
1001
-1004.
Sung, S. and Amasino, R. M. (
2004
). Vernalization in Arabidopsis thaliana is mediated by the PHD finger protein VIN3.
Nature
427
,
159
-164.
Tian, L. and Chen, Z. J. (
2001
). Blocking histone deacetylation in Arabidopsis induces pleiotropic effects on plant gene regulation and development.
Proc. Natl. Acad. Sci. USA
98
,
200
-205.
Trevaskis, B., Bagnall, D. J., Ellis, M. H., Peacock, W. J. and Dennis, E. S. (
2003
). MADS box genes control vernalization-induced flowering in cereals.
Proc. Natl. Acad. Sci. USA
100
,
13099
-13104.
Valverde, F., Mouradov, A., Soppe, W. J., Ravenscroft, D.,Samach, A. and Coupland, G. (
2004
). Photoreceptor regulation of CONSTANS protein in photoperiodic flowering.
Science
303
,
1003
-1006.
Volpe, T. A., Kidner, C., Hall, I. M., Teng, G., Grewal, S. I. and Martienssen, R. A. (
2002
). Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi.
Science
297
,
1833
-1837.
Weigel, D., Alvarez, J., Smyth, D. R., Yanofsky, M. F. and Meyerowitz, E. M. (
1992
). LEAFY controls floral meristem identity in Arabidopsis.
Cell
69
,
843
-859.
Wellensiek, S. J. (
1962
). Dividing cells as the locus for vernalization.
Nature
195
,
307
-308.
Wellensiek, S. J. (
1964
). Dividing cells as the prerequisite for vernalization.
Plant Physiol.
39
,
832
-835.
Wilson, R. N., Heckman, J. W. and Somerville, C. R.(
1992
). Gibberellin is required for flowering in Arabidopsis under short days.
Plant Physiol.
100
,
403
.
Yan, L., Loukoianov, A., Tranquilli, G., Helguera, M., Fahima,T. and Dubcovsky, J. (
2003
). Positional cloning of the what vernalization gene VRN1.
Proc. Natl. Acad. Sci. USA
100
,
6263
-6268.
Yan, L., Loukoianov, A., Blechi, A., Tranquilli, G.,Ramakrishna, W., SanMiguel, P., Bennetzen, J. L., Echenique, V. and Dubcovsky,J. (
2004
). The wheat VRN2 gene is a flowering repressor down-regulated by vernalization.
Science
303
,
1640
-1644.
Yanovsky, M. J. and Kay, S. A. (
2002
). Molecular basis of seasonal time measurement in Arabidopsis.
Nature
419
,
308
-312.
Yoshida, N., Yanai, Y., Chen, L., Kato, Y., Hiratsuka, J., Miwa,T., Sung, Z. R. and Takahashi, S. (
2001
). EMBRYONIC FLOWER2,a novel polycomb group protein homolog, mediates shoot development and flowering in Arabidopsis.
Plant Cell
13
,
2471
-2481.
Yu, H., Ito, T., Wellmer, F. and Meyerowitz, E. M.(
2004
). Repression of AGAMOUS-LIKE 24 is a crucial step in promoting flower development.
Nat. Genet.
36
,
157
-162.
Zhang, H. and van Nocker, S. (
2002
). The VERNALIZATION INDEPENDENCE 4 gene encodes a novel regulator of FLOWERING LOCUS C.
Plant J.
31
,
663
-673.
Zhang, H., Ransom, C., Ludwig, P. and van Nocker, S.(
2003
). Genetic analysis of early flowering mutants in Arabidopsis defines a class of pleiotropic developmental regulator required for expression of the flowering-time switch FLOWERING LOCUS C.
Genetics
164
,
347
-358.
Zilberman, D., Cao, X. and Jacobsen, S. E.(
2003
). ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation.
Science
299
,
716
-719.