Vernalization: Winter and the Timing of Flowering in Plants

Transcription

1 Annu. Rev. Cell Dev. Biol : First published online as a Review in Advance on August 13, 2009 The Annual Review of Cell and Developmental Biology is online at cellbio.annualreviews.org This article s doi: /annurev.cellbio Copyright c 2009 by Annual Reviews. All rights reserved /09/ $20.00 Vernalization: Winter and the Timing of Flowering in Plants Dong-Hwan Kim, 1 Mark R. Doyle, 2 Sibum Sung, 1 and Richard M. Amasino 2 1 Section of Molecular Cell and Developmental Biology and the Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas Department of Biochemistry, University of Wisconsin, Madison, Wisconsin ; kdhinchrist@mail.utexas.edu, markdoyle@biochem.wisc.edu, sbsung@mail.utexas.edu, amasino@biochem.wisc.edu Key Words Arabidopsis, cereals, histone modifications, epigenetics, Polycomb, Trithorax Abstract Plants have evolved many systems to sense their environment and to modify their growth and development accordingly. One example is vernalization, the process by which flowering is promoted as plants sense exposure to the cold temperatures of winter. A requirement for vernalization is an adaptive trait that helps prevent flowering before winter and permits flowering in the favorable conditions of spring. In Arabidopsis and cereals, vernalization results in the suppression of genes that repress flowering. We describe recent progress in understanding the molecular basis of this suppression. In Arabidopsis, vernalization involves the recruitment of chromatin-modifying complexes to a clade of flowering repressors that are silenced epigenetically via histone modifications. We also discuss the similarities and differences in vernalization between Arabidopsis and cereals. 277

2 Contents INTRODUCTION Floral Integrators and Environmental Sensing Vernalization in Arabidopsis The Autonomous Pathway Vernalization as an Epigenetic Switch Genetic Analysis of the Vernalization Response Vernalization-Mediated Changes in FLC Chromatin Polycomb Repression Complexes and Vernalization-Mediated FLC Repression Activation of FLC Is Associated with Specific Chromatin Modifications Generational Resetting of FLC Other Targets of Vernalization in Arabidopsis Vernalization in Cereals FUTURE DIRECTIONS INTRODUCTION Like all organisms, species of flowering plants have evolved mechanisms to maximize their reproductive success. One component of optimizing this success is the proper timing of the transition from vegetative to reproductive growth. Proper timing is important for many reasons; the following are a few examples. A wide range of plants require cross-pollination for successful reproduction, and thus flowering must occur synchronously within individuals of the same species. For many plants, flowering must also occur at a time when pollinators are present. In many parts of the world, flowering must coincide with weather conditions that will permit flowers, which are relatively delicate structures, to develop properly, followed by conditions that will permit seeds to mature properly. To ensure that flowering occurs within a particular season, plants rely primarily on environmental cues. One such cue is photoperiod, the relative change in the length of day and night that occurs throughout the course of a year. Plants that respond to lengthening days and flower in the spring or early summer are known as long-day (LD) plants. Short-day (SD) plants flower in the late summer or autumn in response to shortening days and lengthening nights (Thomas & Vince-Prue 1997). In temperate climates, many winter-annual, biennial, and perennial plants also use cold as an environmental cue to flower at the proper time of year. Certain plant species need to experience a period of winter cold to overcome a block to flowering. This occurs through a process known as vernalization. A requirement for vernalization is an adaptation to temperate climates that prevents flowering prior to winter and permits flowering in the favorable conditions of spring. Many vernalization-requiring plants are LD plants. A LD requirement coupled to a vernalization requirement further ensures that precocious flowering does not occur during the decreasing day lengths of fall and favors flowering as day length increases during spring. The physiology of vernalization in a wide range of species has been intensively studied for many decades, and there are several comprehensive reviews of vernalization physiology (e.g., Bernier et al. 1981, Chouard 1960, Lang 1965). The effective temperature and the length of cold exposure required to achieve the vernalized state vary among different plant species and different varieties within a species. This variation is expected for a response that provides an adaptation to a range of different environmental niches. A vernalization response occurs only at temperatures around or above freezing; temperatures below freezing are not effective, which is not surprising because vernalization is an active process that requires changes in gene expression during cold exposure, as discussed below. The focus of this article is to discuss recent progress in understanding the molecular basis of vernalization. The link between classic physiological studies and more recent molecular genetic analyses have been reviewed elsewhere (Amasino 2004, Sung & Amasino 2005). 278 Kim et al.

3 Floral Integrators and Environmental Sensing Flowers result from the expression of regulatory genes known as floral meristem-identity genes, which specify that certain cells in the growing tips of the plant (the shoot apical meristems) differentiate into a floral meristem and ultimately form a flower (Coen & Meyerowitz 1991). Many of these genes are conserved throughout the plant kingdom including MADS-box transcription factors like APETALA1 (AP1), FRUITFUL (FUL), and LEAFY (LFY), which is a protein unique in the plant lineage (reviewed in Soltis et al. 2007). Upstream of the floral meristem-identity genes are a group of genes known as floral integrators such as FT and FD (Figure 1); i.e., the floral integrators are involved in the regulation of meristem-identity genes. The floral integrators are so named because their expression is in turn regulated by flowering pathways that sense environmental cues, such as photoperiod and cold, and/or developmental cues such as the levels of the hormone gibberellin; i.e., the regulation of these genes serves to integrate a range of environmental and developmental cues (Figure 1). There is not a precise demarcation between a floral integrator and a meristem-identity gene, and a gene can have properties of both. For example, LFY is a key meristem-identity gene, but because it is activated directly by gibberellin (Figure 1) (Blazquez et al. 1998), it can also be considered a floral integrator. In this review, we discuss how the circuitry of photoperiod and vernalization pathways are connected to floral integrators in the well-studied model Arabidopsis thaliana. Studies of the photoperiodic control of flowering indicated the existence of a common floral stimulus in plants, sometimes referred to as florigen (Zeevaart 1976). Physiological studies revealed that the floral stimulus was produced in leaves exposed to inductive photoperiods and traveled to the meristem and caused flowering. Recent studies in Arabidopsis and rice have made a strong case that florigen, or at least a component of the floral stimulus, is the floral Floral integrator genes Inductive photoperiod pathway CO Floral meristem identity genes Figure 1 FD SEP3 FT FUL Autonomous pathway FLC SOC1 AP1 Flowering FLC clade LFY Vernalization pathway Gibberellins Outline of flowering pathways in Arabidopsis. Components and genetic interactions that promote flowering are shown in blue; those that repress flowering are shown in red. Flowering is repressed in Arabidopsis by FLOWERING LOCUS C (FLC) and FLC relatives designated the FLC clade. FLC represses the expression of the floral integrator genes FD, FT, and SUPPRESSOR OF CONSTANS 1 (SOC1). These floral integrator genes are promoters of flowering. One of the floral integrator genes, FT, is induced by the photoperiod pathway through CONSTANS (CO); FT protein is a mobile flowering signal that partners with FD protein in the meristem to activate SOC1 and the floral meristem-identity genes SEPALATA (SEP), FRUITFUL (FUL), and APETALA 1 (AP1) [for review see Turck et al. (2008)]. Thus, FLC and the photoperiod pathway act antagonistically. The floral meristem-identity genes specify the formation of floral meristems that will develop into flowers. The basal level of FLC expression is set in part by the level of repression that results from the action of autonomous pathway genes. In response to exposure to the prolonged cold of winter, the vernalization pathway provides further FLC repression by chromatin remodeling. The gibberellin class of plant hormones promotes flowering by activating SOC1 and the floral meristem-identity gene LEAFY (LFY). integrator FT. The FT gene is expressed in leaves, and the protein travels to the meristem where it interacts with another integrator, FD, to initiate the floral transition (reviewed in Turck et al. 2008, Zeevaart 2008). FT-like genes are ubiquitous in plants and have been found to regulate flowering in a variety of species including wheat and poplar (reviewed in Turck et al. 2008). [Note: Several genes discussed in this review such as FT, FD, FVE, FCA, FY, and FPA were given two- or three-letter designations but not longer names (Koornneef et al. 1991).] Vernalization 279

4 There is also a significant level of conservation in the molecular mechanisms plants use to sense photoperiod. Arabidopsis is a facultative LD plant; i.e., it flowers most rapidly in LD, but it also flowers eventually in SD. In Arabidopsis, the perception of day length is a function of coincidence between light and expression of the circadian-regulated gene CONSTANS (CO) (reviewed in Turck et al. 2008). In LD-grown Arabidopsis plants, CO transcription extends into the daylight phase. Light stabilizes CO protein, which in turn leads to increased expression of FT (Figure 1) (Corbesier et al. 2007, Hepworth et al. 2002, Wenkel et al. 2006). The photoperiodic induction system, in which CO levels are affected by day length and translated into the regulation of FT (or FT homologs such as Hd3a in rice or VRN3 in cereals), appears to be well conserved among flowering plants (reviewed in Turck et al. 2008). Much of what we know about the molecular mechanism of the vernalization response comes from studies of Arabidopsis and temperate cereals. These studies of vernalization indicate that this process is not conserved at a biochemical level like the photoperiodic flowering pathway. However, as discussed in detail below, the floral integrator FT/VRN3 is one of the targets of the vernalization pathway in both Arabidopsis and temperate cereals, and vernalization alleviates the repression of FT/VRN3 expression. Thus, the control of FT/VRN3 expression may be conserved as an integration point of the photoperiod and vernalization pathways. In Arabidopsis, there are additional floral integrators including the MADS-box genes SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), AGAMOUS-LIKE 19 (AGL19), and AGL24 (Lee et al. 2000, Liu et al. 2008, Michaels et al. 2003, Samach et al. 2000, Schonrock et al. 2006). FT and FD partner to activate SOC1 in the meristem, and SOC1 expression is also controlled by vernalization, as discussed below. Thus, SOC1 regulation integrates inputs from multiple flowering pathways (Figure 1). SOC1 also functions with FUL to maintain the meristem in a floral state (i.e., as an inflorescence meristem) (Melzer et al. 2008), and therefore, SOC1 can also be considered a meristem-identity gene. SOC1 and AGL24 positively regulate each other (Liu et al. 2008, Michaels et al. 2005), interact at the protein level, and are both required for proper activation of LFY (Lee et al. 2008). Indeed, there is an intricate network of positive feedback loops among floral integrators and meristem-identity genes to ensure that once flowering is initiated in Arabidopsis, it is maintained in the absence of environmental and developmental cues. It is beyond the scope of this review to discuss the details of these loops. Hereafter we focus on vernalization and note the connections to the integrators. Vernalization in Arabidopsis The identification of genes involved in the vernalization response in Arabidopsis began with the study of natural variation in flowering found among different isolates collected from a range of locations worldwide. Most commonly used lab strains of Arabidopsis do not require vernalization to flower rapidly; however, many isolates flower very late unless first vernalized (Burn et al. 1993, Clarke & Dean 1994, Lee et al. 1993, Napp-Zinn 1987). The rapid-flowering types are sometimes referred to as summer annuals, which indicates that their life cycle is likely to be completed in one growing season. Plants with a strong vernalization requirement are often called winter annuals, because they may not complete their life cycle until the second growing season after an intervening winter. However, the actual life cycle in nature is clearly influenced by environment as well as genotype; in one environment a genotype might take two seasons to flower, whereas in a different environment the same genotype might flower in a single season (e.g., Wilczek et al. 2009). In Arabidopsis, studies of natural variation demonstrated that the vernalization requirement is conferred by two dominant genes, FRIGIDA (FRI ) and FLOWERING LOCUS C (FLC ) (Koornneef et al. 1994, Lee et al. 1994). FRI encodes a nuclear protein found only in plants ( Johanson et al. 2000) that may interact 280 Kim et al.

5 with the mrna cap-binding protein to regulate the level of FLC mrna (Geraldo et al. 2009). FLC encodes a MADS-box DNA binding protein that functions as a repressor of flowering (Michaels & Amasino 1999, Sheldon et al. 1999). Genetic studies have shown that in the context of vernalization FRI acts solely to upregulate FLC (Michaels & Amasino 2001). The level of FLC expression is the primary determinant of the vernalization requirement in Arabidopsis. FLC represses flowering, in part, by repressing the expression of floral integrators such as FT, FD, and SOC1 (Helliwell et al. 2006, Hepworth et al. 2002, Lee et al. 2000, Samach et al. 2000, Searle et al. 2006). FLC binds to promoter regions of SOC1 and FD and to the first intron of FT (Searle et al. 2006). This binding likely attenuates the ability of the photoperiod pathway to activate these integrators. Indeed, the expression of integrators from constitutive promoters bypasses the repressive effects of FLC and the vernalization requirement (Lee et al. 2000, Michaels et al. 2005). There are many examples of MADS-box proteins acting in multimeric complexes with other MADS-box proteins (de Folter et al. 2005, Honma & Goto 2001, Pelaz et al. 2000). Recent studies show that FLC interacts directly with another MADS-box protein SHORT VEGETATIVE PHASE (SVP) (Fujiwara et al. 2008, Li et al. 2008). Genetic studies indicate that this interaction is biologically relevant: loss of SVP provides partial suppression of the FLC-mediated delay in flowering (Li et al. 2008). However, the loss of SVP cannot fully suppress the FLC-mediated delay of flowering, which indicates that FLC may redundantly interact with other MADSbox proteins. Although SVP and other interaction partners may be critical for FLC-mediated repression of flowering, SVP levels or activity do not appear to be involved in the regulation of flowering like FLC. For example, SVP mrna levels are not affected by vernalization, whereas vernalization results in the stable repression of FLC (Bastow et al. 2004, Michaels & Amasino 1999, Sheldon et al. 1999, Sung & Amasino 2004). Moreover, overexpression of FLC alone is sufficient to cause extremely late flowering (Michaels & Amasino 2001), which indicates that the levels of binding partners are not limiting in vivo. The circadian clock has an influence on SVP levels; SVP protein accumulates to higher levels in a double mutant of two clock components, LATE ELONGATED HYPOCOTYL (LHY ) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), than in wild type (Fujiwara et al. 2008). Thus, there may be cross talk in the circuitry between the photoperiod pathway and FLCdependent repression in Arabidopsis; however, no effect of different photoperiods on SVP levels has been reported. The Autonomous Pathway Most commonly used types of Arabidopsis are null mutants for FRI (e.g., Columbia, Landsberg, Wassilewskija) and thus do not require vernalization for rapid flowering. Historically, a group of late-flowering mutants derived from such rapid-flowering accessions have been referred to as the autonomous pathway (AP) of floral promotion (Henderson & Dean 2004). AP mutants are characterized by delayed flowering in both LD and SD, which is in contrast to photoperiod-pathway mutants that show delayed flowering in inductive LD photoperiods only. The flowering behavior of AP mutants is similar to FRI-containing winter-annual accessions (i.e., both have strong FLC expression, are late flowering, and require vernalization for rapid flowering) (Michaels & Amasino 2001). To date, eight AP genes have been identified: LUMINIDEPENDENS (LD), FCA, FY, FPA, FLOWERING LOCUS D (FLD), FVE, FLK, and REF6 (Noh et al. 2004, Simpson 2004). FCA, FPA, FY, and FLK encode proteins that are predicted to be involved in RNA metabolism (Lim et al. 2004, Macknight et al. 1997, Schomburg et al. 2001, Simpson et al. 2003); however, there is no evidence to suggest that these components directly interact with FLC mrna. FVE, FLD, and REF6 have domains common to chromatin-modifying components. FVE (also known as MSI4) is a member of the Vernalization 281

6 MSI1-like protein family (MSI1 MSI5) in Arabidopsis (Ausin et al. 2004, Kim et al. 2004); MSI proteins are found in several chromatinmodifying complexes in eukaryotes (Hennig et al. 2005). FLD and REF6 are predicted to encode two different types of histone demethylases (He et al. 2003, Jiang et al. 2007, Noh et al. 2004). The repression of flowering by AP genes acts primarily through FLC because flc null mutants completely suppress the delayedflowering phenotypes of AP mutants (Michaels & Amasino 2001). Recent studies demonstrate that many AP genes are also involved in the regulation of other genes that are not involved in flowering (Baurle et al. 2007, Veley & Michaels 2008). Indeed, most of the AP genes do not appear to be specific to flowering, nor do they comprise a pathway in the typical sense. Instead, these genes are involved in a range of repressive mechanisms that act on FLC and other genes. For example, the RNA-binding proteins FPA and FCA are broadly required for RNAmediated silencing, which raises the possibility that their role in FLC repression involves RNA-mediated silencing (Baurle et al. 2007). In addition, all tested AP mutants showed derepression of certain transposons (Baurle et al. 2007, Veley & Michaels 2008), and double mutant analysis among the AP mutants revealed a wide range of nonflowering phenotypes (Veley & Michaels 2008). It is interesting to note that genetic analysis showed FCA requires FVE for the repression of certain transposons (Baurle & Dean 2008), whereas FLD is necessary for FCA to repress FLC (Liu et al. 2007). Thus, there appears to be a range of mechanisms and combinatorial interactions by which AP genes affect gene expression. Recent work provides additional support for the involvement of RNA-mediated silencing in FLC repression. Double mutants of dicer-like 1 and dicer-like 3 (dcl1/3) have phenotypes similar to that of AP mutants: elevated levels of FLC expression and delayed flowering that is suppressed by both flc mutations and vernalization (Schmitz et al. 2007). Because late flowering in dcl1/3 and AP mutants is overcome by vernalization, FLC repression by these genes occurs via a vernalization-independent mechanism. Perhaps RNA-mediated silencing plays a role in setting the prevernalization basal level of FLC expression. Notably, there is an intermediate-size, noncoding RNA that arises from beyond 3 UTR of FLC (Swiezewski et al. 2007). Mutations in the region of this RNA have a small effect on flowering relative to dcl1/3 or other AP mutants, and the relationship of this RNA to other flowering pathways is not known. Vernalization as an Epigenetic Switch Chouard (1960) provided a useful definition of vernalization as the acquisition or acceleration of the ability to flower by a chilling treatment. As defined, vernalization does not necessarily induce flowering. Rather, prolonged chilling provides competence to flower. As noted above, in many vernalization-requiring plant species, other endogenous and/or environmental conditions, such as inductive photoperiods, are also required for flowering. A classic experiment illustrates that this competence to flower can be stable through mitotic cell divisions. Lang and Melchers used a biennial variety of Hyoscyamus niger (henbane) that requires both vernalization and inductive photoperiods (LD) to initiate flowering (reviewed by Lang 1965). When plants are first exposed to cold (vernalized) and then kept in a noninductive photoperiod (SD) in a normal, warm growth temperature, the plants grow vegetatively but do not flower. However, when the previously vernalized plants are moved to inductive photoperiods, even after many months in noninductive conditions, flowering occurs. Thus, the vernalization-mediated change in competence is stable through a large number of mitotic divisions in the apical meristem. Mitotic stability in the absence of the inducing signal (cold in the case of vernalization) is arguably an epigenetic switch (e.g., Amasino 2004, Dennis & Peacock 2007, Wu & Morris 2001). The molecular basis of competence in Arabidopsis is the mitotically stable repression of FLC and some FLC relatives discussed below. 282 Kim et al.

7 It is worth noting that not all plant species exhibit behavior that fits the stable acquisition of competence model described above; for example, there are species that need to flower during cold exposure presumably because competence is not stably maintained through mitotic cell divisions after cold exposure ceases (Bernier et al. 1981). Indeed, there are mutants in Arabidopsis in which FLC repression occurs during cold exposure but it is not stably maintained upon a return to warm; not surprisingly, these mutants affect chromatin-modifying proteins (Gendall et al. 2001, Levy et al. 2002, Mylne et al. 2006, Sung et al. 2006a). Genetic Analysis of the Vernalization Response As discussed above, winter-annual accessions of Arabidopsis exhibit a delayed-flowering phenotype if they are not vernalized, and most of the natural variation in the vernalization requirement is due to allelic variation at FRI and FLC. The molecular mechanism by which FLC is epigenetically repressed by vernalization in Arabidopsis has been addressed by molecular genetic analyses. Using winterannual accessions as a parental line, one can screen for mutants that can no longer be vernalized; such vernalization-insensitive mutants have lesions in genes that participate in the vernalization pathway. To date, these screens have revealed five genes: VERNALIZA- TION 1 (VRN1), VRN2, VERNALIZATION INSENSITIVE 3 (VIN3), VRN5/ VIN3-LIKE 1(VIL1), and atprmt5 (Bastow et al. 2004, Gendall et al. 2001, Greb et al. 2007, Schmitz et al. 2008, Sung & Amasino 2004, Sung et al. 2006b). As expected, given the nature of the genetic screen, all mutants fail to stably repress FLC by vernalization. All of these genes are constitutively expressed except for VIN3. VIN3 is expressed specifically during a vernalizing cold treatment, and expression is completely abolished when plants are returned to a warm temperature (Sung & Amasino 2004). The cold induction and transient nature of VIN3 expression indicates that VIN3 may be a part of the trigger to set in motion the molecular events that stably repress FLC during vernalization. Vernalization-Mediated Changes in FLC Chromatin The stable nature of the vernalized state is consistent with a role for chromatin modification of target genes. In Arabidopsis, FLC repression is the molecular basis of vernalizationinduced competence. Thus, FLC is a likely target for chromatin modification. The first molecular evidence for chromatin modification in Arabidopsis vernalization came from the identification of VRN2 and VIN3. VRN2 encodes a homolog of Suppressor of zeste (Su(z)12), a component of Polycomb repression complex 2 (PRC2) that was first identified in animals (Gendall et al. 2001). VIN3 encodes a PHD (plant homeodomain) protein (Sung & Amasino 2004); the PHD domain is commonly found in a wide range of complexes that are involved in chromatin-level regulation (Mellor 2006). Vernalization results in an increase in two repressive histone modifications at FLC chromatin (Figure 2): histone H3 Lys 9 (H3K9) and histone H3 Lys 27 (H3K27) methylation (Bastow et al. 2004, Sung & Amasino 2004). In vin3 and vrn2 mutants, H3K9 and H3K27 methylation are not enriched at FLC following a sufficient cold treatment, and FLC expression is not repressed (Bastow et al. 2004, Sung & Amasino 2004). The loss of VRN1, which encodes a DNA-binding protein containing two plant-specific B3 domains, shows distinctive chromatin modifications from those of vin3 and vrn2 mutants. Whereas methylation of both H3K9 and H3K27 are affected in vin3 and vrn2 mutants, only H3K9 methylation fails to occur in vrn1 during and after vernalization (Bastow et al. 2004, Sung & Amasino 2004), which suggests the two methylation events can occur independently and that VRN1 is involved specifically in H3K9 methylation (Figure 2). VIL1/VRN5 was identified independently by a yeast two-hybrid screen for VIN3- interacting proteins (Sung et al. 2006b) and by a Vernalization 283

8 a K4 H3 PRC2 core VRN2 H2B K36 M ATX K4 K4 K4 K4 1/2 M PAF1 K27 K36 complex K36 K36 EFS PRC2 core VRN2 Pol II Z Z Z K36 SWR1 complex FIE1 ARP6 ARP4 FLC active HUB1 HUB2 Ubiquitinase Annu. Rev. Cell Dev. Biol : Downloaded from Next generation b c H3 H2B VIN3 Winter cold (vernalization) FLC repression initiates VIL1/VRN5 PRC2 core H3K27Me and H3K9Me density increases VIL1/VRN5 VIN3 PRC2 M K27 core K9 K9 K9 H3 VRN2 K27 K27 VRN1 VRN1 VRN1 VRN1 H2B K27 LHP1 VIN3 Induced K27 VIN3 No longer expressed VRN2 M Winter cold (vernalization) Return to warm K9 K27 M H3K4triMe H3K26triMe H2Bub1 H3K9triMe H3K27triMe Methyl group d Mitotically stable repression VIL1/VRN5 PRC2 core VIL1/VRN5 PRC2 core K9 K9 K9 H3 VRN2 VRN2 K27 K27 K27 VRN1 VRN1 VRN1 VRN1 K27 LHP1 K27 K9 HMTase LHP1 K9 HMTase LHP1 K9 HMTase LHP1 284 Kim et al.

9 genetic screen for mutants with a reduced vernalization response (Greb et al. 2007). VIN3 and VIL1/VRN5 belong to a small family of proteins that contain a PHD domain, a fibronectin III (FNIII) domain, and a conserved C-terminal region. Whereas the PHD and FNIII domains are found throughout eukaryotes, the C-terminal domain is unique in plants and mediates protein-protein interactions between VIN3 and VIL1/VRN5 (Greb et al. 2007, Sung et al. 2006b). Similar to VIN3, VIL1/VRN5 is also required for vernalization-mediated histone modifications at FLC chromatin including methylation of both H3K9 and H3K27 (Greb et al. 2007, Sung et al. 2006b). Recent reports claim that yet another member of the family, VIL2/VEL1, is part of a VIL1-containing complex that is physically located at FLC after vernalization-mediated repression (De Lucia et al. 2008). However, this repression is not impaired when VIL2/VEL1 levels are decreased either by mutation (Kim, Schmitz, Amasino, and Sung, unpublished work) or by RNA interference (Greb et al. 2007), which indicates possible functional redundancy of VIL2/VEL1 with other members of the VIN3/VEL protein family. Recent studies demonstrated that specific PHD domains associate with specific histone modifications, which include methylated histone H3K4 (Mellor 2006), nonmethylated histone H3 (Lan et al. 2007), methylated histone H3K9 (Karagianni et al. 2008), and methylated histone H3K36 (Shi et al. 2007). The diverse binding activities of PHD domains make them excellent candidates for readers of histone modifications. As expected, amino acid sequence variation within PHD domains is responsible for variations in their specificity (Li et al. 2007). In the VIN3/VEL family there are variations in amino acid sequences within the PHD domains, and thus, if VIN3/VEL family members indeed bind modified histone tails, there may be variation within the family as to which modifications are bound. One possibility is that VIN3 binds to modifications associated with active FLC chromatin and in doing so initiates the transition to a repressed state. After a certain degree of conversion to the repressed state is achieved and modifications associated with active FLC chromatin are replaced by repressive marks, VIN3 is no longer necessary, as in spring, when other positive feedback loops of histone modification maintain repression (Figure 2). However, the specificity of various members of the VIN3/VEL protein family for modified histones and the significance of any variability in modifying FLC chromatin remain to be investigated. The mechanism of how FLC is targeted for repression remains a major unanswered question. The induction of VIN3 is a necessary first Figure 2 Vernalization-mediated changes in FLC chromatin. (a) Prior to cold exposure, FLC is actively expressed. The complexes that maintain this active chromatin conformation include the PAF complex, which methylates histone 3 tails at lysine 4 and 36 (H3K4triMe and H3K36triMe), a SWR1-like complex, which deposits a histone 2A variant in the nucleosomes of FLC chromatin, and H2B ubiquitinases like HUB1 and HUB2 that ubiquitinate histone 2B tails (H2Bub1). Although FLC is in an active state, there are repressive complexes present such as Polycomb Repression Complex 2 and some degree of lysine 27 methylation of histone 3 (H3K27triMe a repressive modification) (b) During cold exposure, FLC repression is initiated. VIN3 is induced, VIN3 and VIL1/VRN5 associate with the Polycomb complex, the density of repressive chromatin modifications such as lysine 27 methylation of histone 3 increases, and repressors such as LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) assemble on FLC chromatin. (c) As vernalization proceeds, the density of repressive modifications, particularly H3K27triMe and lysine 9 methylation of histone 3 [H3K9triMe; mediated by an unknown H3K9 methyltransferase (HMTase)] increases. (d ) Eventually, a mitotically stable state of repression that no longer requires VIN3 is achieved. This mitotically stable state is likely to involve positive feedback loops in which the repressive chromatin modifications serve to recruit the chromatin-modifying complexes including VRN1 to maintain a repressive state. As the FLC locus passes to the next generation, the active chromatin state represented in (a) is re-established. Vernalization 285

10 step, but how this translates into a change in the activities of chromatin-modifying complexes at the FLC locus is not at all understood. Even if the PHD domain of VIN3 binds to specific histone modifications characteristic of FLC in the pre-vernalized state (see below), such modifications are not likely to be unique to FLC chromatin. Clearly, there is much to learn about targeting specificity. In addition to vernalization-mediated methylation at H3K9 and H3K27 at FLC chromatin, histone arginine methylation also plays a role in maintaining stable repression of FLC (Schmitz et al. 2008). Mutations in a Type II protein arginine methyltransferase gene atprmt5, reduced the vernalization response. atprmt5 is required for the symmetric methylation of Arg 3 of histone H4 (smeh4r3), and the level of this methylation is increased at FLC chromatin by vernalization. Furthermore, smeh4r3 appears to be required for the vernalization-mediated enrichment of both H3K9 and H3K27 methylation (Schmitz et al. 2008). Polycomb Repression Complexes and Vernalization-Mediated FLC Repression As discussed above, the identification of an Arabidopsis homolog of Su(z)12, VRN2, in a vernalization-insensitive mutant screen suggested the involvement of a PRC2-like complex in FLC repression (Gendall et al. 2001). In animals, there are two distinct PRC complexes, PRC2 and PRC1 (reviewed in Schuettengruber et al. 2007). Polycomb group (PcG) proteins were first identified in Drosophila as essential components for maintaining HOMEOTIC (HOX) genes in a repressed state. The PRC2 complex is involved in the initial di- or trimethylation of H3K27 at target chromatin. Then, in many systems, methylated H3K27 is bound by the PRC1 complex, which acts to maintain chromatin in a repressed state and to maintain H3K27 methylation through mitotic cell divisions (Cao et al. 2002, Muller et al. 2002). Core components of PRC2 are well conserved from plants to animals (Hsieh et al. 2003), but, as discussed below, PRC1 components are not. In Drosophila, core components of PRC2 consist of Enhancer of zeste [E(z)], Extra Sex Combs (ESC), and Suppressor of zeste Su(z)12 (Cao et al. 2002). Arabidopsis and other plants contain multiple copies of many PRC2 components (Hsieh et al. 2003, Wood et al. 2006). For example, in Arabidopsis, there are three homologs of the histone methyltransferase E(z). These include CURLY LEAF (CLF), SWINGER (SWN), and MEDEA (MEA) (Hsieh et al. 2003). CLF was first identified as a repressor of the floral homeotic gene AGAMOUS (AG) (Goodrich et al. 1997), whereas MEA was identified in a screen for mutants that have altered endosperm development (Grossniklaus et al. 1998). SWN is partially redundant with CLF (Chanvivattana et al. 2004). None of the Arabidopsis E(z) homologs were identified in screens for vernalizationinsensitive mutants, which is likely due to the functional redundancy among these proteins with regard to FLC repression. In fact, VRN2 is the only PRC2 component identified as being involved in vernalization from mutant screens. Biochemical approaches revealed that Arabidopsis PRC2 complexes include VIN3 during cold exposure (De Lucia et al. 2008, Wood et al. 2006). PRC2 complexes in Arabidopsis can also include VIL1/VRN5; purification of proteins associated with a VIL1/VRN5-TAP fusion revealed components of PRC2, which include VRN2, SWN, MSI1, and FERTILIZATION INDEPENDENT ENDOSPERM (FIE; an Arabidopsis ESC homolog) (De Lucia et al. 2008). The theme of PHD domain-containing proteins associating with PRC2 may be widespread in eukaryotes; PHD domain proteins also associate with PRC2 in Drosophila and humans (Cao et al. 2008a, Nekrasov et al. 2007, Sarma et al. 2008). Analyses of the occupancy of PRC2 components at FLC chromatin showed that VRN2 is already present at FLC chromatin prior to vernalization (De Lucia et al. 2008). In cold, VIN3 is induced and VIN3 protein is present at 286 Kim et al.

11 a region encoding the first intron of FLC. Association of VIL1/VRN5 is VIN3-dependent, and thus VIN3 may serve as a guiding factor for the recruitment of VIL1/VRN5 to FLC (De Lucia et al. 2008). Whereas the enrichment of VIL1/VRN5 is restricted to the first intron of FLC in cold temperatures, VIL1/VRN5 spreads throughout the FLC chromatin when plants return to warm temperatures. This spreading may require warm temperatures, or warm temperatures may simply increase the rate of these chromatin changes. Presumably, the spreading of VIL1/VRN5 across FLC chromatin maintains the repressed state of FLC (Figure 2) (De Lucia et al. 2008). Although the presence of PRC2 components in plants is well established, Arabidopsis lacks components with an overall similarity to PRC1 components (Hsieh et al. 2003, Schubert et al. 2005). Instead, LIKE- HETEROCHROMATIN PROTEIN 1 (LHP1) likely binds to and stably maintains the PRC2-mediated modified histones at FLC following vernalization (Mylne et al. 2006, Sung et al. 2006a). In lhp1 mutants, the active histone mark, H3K4triMe, is only transiently reduced when plants are kept in cold (Sung et al. 2006a). Interestingly, lhp1 mutants were not found in screens for vernalization-insensitive mutants because the loss of LHP1 also results in the derepression of flowering promoters such as FT, and thus lhp1 mutants are early flowering without vernalization (Kotake et al. 2003, Sung et al. 2006a). LHP1 is likely to play a broad role in gene repression in plants and may be part of a protein complex that serves a similar role to that of PRC1 in animals. Activation of FLC Is Associated with Specific Chromatin Modifications As discussed above, FLC repression is associated with dynamic changes in histone composition, which are initiated during prolonged cold exposure. Conversely, the high levels of FLC that create a vernalization requirement are associated with active histone marks. Mutant screens for the loss of the vernalization requirement (i.e., plants that flower early without cold) in winter-annual Arabidopsis have revealed many of the components required for FLC activation. H3K4triMe is associated with active chromatin in most eukaryotes (Schneider et al. 2004). A specific Saccharomyces cerevisiae HM- Tase, Set1, is responsible for mono- to trimethylation of H3K4, and mutations in set1 cause diverse phenotypic defects such as slow growth and rdna derepression (Briggs et al. 2001, Fingerman et al. 2005). Yeast Set1 is an essential component of a complex called COMPASS (complex proteins associated with Set1) (Krogan et al. 2003). Another complex, the RNA polymerase II-Associated Factor 1 (PAF1)-containing complex, is necessary for H3K4triMe enrichment at target chromatin. These two complexes, COMPASS and PAF1, physically associate to coordinate the transcription of target genes (Krogan et al. 2003). Screens for rapidly flowering mutants identified two Arabidopsis homologs of the yeast PAF1 complex components PAF1 and CTR9: EARLY FLOWERING 7 (ELF7) and ELF8, respectively (He & Amasino 2005). ELF7 and ELF8 are required for high FLC expression and for H3K4triMe enrichment at FLC chromatin (He et al. 2004). Other Arabidopsis genes that encode relatives of PAF1 and COMPASS complex components are VERNALIZATION INDEPENDENCE 3 (VIP3; Arabidopsis homolog of human hski8), VIP4 (Arabidopsis homolog of yeast Leo1), VIP5 (Arabidopsis homolog of yeast Rtf1), ARABIDOPSIS TRITHORAX-LIKE 1 (ATX1), and ATX2 (see below for roles of ATX1 and ATX2) (Alvarez- Venegas et al. 2003, He et al. 2004, Oh et al. 2004, Pien et al. 2008, Saleh et al. 2008, Zhang et al. 2003, Zhang & van Nocker 2002). Mutations in these genes typically cause reduced H3K4triMe deposition at FLC chromatin and early-flowering phenotypes similar to elf7 and elf8 (Alvarez-Venegas et al. 2003, He et al. 2004, Pien et al. 2008, Saleh et al. 2008, Zhang & van Nocker 2002). Methylation of H3K4 at FLC chromatin appears to be mediated by ATX1 and ATX2, which are homologous to Drosophila Trithorax (Trx). Trx is a H3K4 Vernalization 287

12 methyltransferase that belongs to the Trithorax group of proteins (TrxG), which are activators of Drosophila homeotic genes (a role opposite to that of PcG proteins) (Pien et al. 2008, Saleh et al. 2008). ATX1 mediates trimethylation on H3K4, whereas ATX2 dimethylates H3K4 in vitro (Saleh et al. 2008). The involvement of TrxG- and PcG-like genes in the regulation of FLC indicates that FLC is controlled by an evolutionarily conserved mechanism that involves a dynamic balance between PcG and TrxG. A recent study reveals that this dynamic balance occurs throughout the genome in plants (Oh et al. 2008). In Drosophila, TrxG activator proteins as well as components of PRC2 repressive complexes are constitutively bound to HOX target chromatin; i.e., both repressive and activating modifiers can reside at the same locus (Papp & Muller 2006). As discussed above, PRC2 is detectable at FLC chromatin prior to vernalization (De Lucia et al. 2008). Furthermore, H3K27triMe is present at FLC chromatin prior to vernalization when FLC is actively transcribed, but the level of H3K27triMe is much lower than when FLC is repressed (Pien et al. 2008). This indicates that a threshold level of H3K27triMe enrichment is necessary to establish repressed FLC chromatin. Another Trx-like SET domain protein is also required for proper FLC activation. EARLY FLOWERING IN SHORT DAYS (EFS) encodes a SET domain protein, which mediates di- and trimethylation of histone H3 Lys 36 (H3K36) on target chromatin (Xu et al. 2008). Mutants with EFS lesions exhibit reduced FLC expression and early flowering similar to mutations in PAF1-COMPASS components (Kim et al. 2005, Xu et al. 2008, Zhao et al. 2005). In winter-annual strains of Arabidopsis, EFS may also be required for the enrichment of H3K4triMe at FLC chromatin (Kim et al. 2005). Thus, the three Arabidopsis HMTases, ATX1, ATX2, and EFS, appear to act coordinately to mediate the transcriptional activation of FLC. Monoubiquitination at lysine 123 of Histone H2B (H2Bub1), like H3K4, is a histone modification associated with active transcription. In yeast, a complex that contains RAD6 (with E2-ubiquitin-conjugating activity) and BRE1 (with E3-ubiquitin ligase activity) acts to monoubiquitinate histone H2B at specific target chromatin (Tenney & Shilatifard 2005). H2Bub1 is an important prerequisite for the proper enrichment of H3K4triMe and for transcriptional activity of target genes (Wood et al. 2003). In Arabidopsis, H2B monoubiquitination of FLC chromatin is also required for proper activation of FLC. Investigators identified two Arabidopsis BRE1 homologs, HI- STONE MONOUBIQUITINATION 1 (HUB1) and HISTONE MONOUBIQUITINATION 2 (HUB2) (Cao et al. 2008b, Gu et al. 2009), and found that mutations in either hub1 or hub2 result in early flowering and the loss of H3K4triMe enrichment at the promoter region of FLC. Arabidopsis has three RAD6 homologs: UBIQUITIN-CONJUGATING ENZYME 1, 2, and 3 (AtUBC1, AtUBC2, and AtUBC3) (Cao et al. 2008b, Gu et al. 2009). AtUBC1 and AtUBC2 are involved in flowering and act redundantly for the enrichment of H2Bub1 at FLC chromatin, whereas AtUBC3 does not play a role in FLC activation (Xu et al. 2009). Although the enrichment of H2Bub1 is required for the proper activation of FLC, over-enrichment of H2Bub1 results in the failure of FLC activation; a mutation in a H2B deubiquitinase, UBIQUITIN-SPECIFIC PROTEASE26 (UBP26 ), results in a rapidflowering phenotype due to the loss of FLC expression (Schmitz et al. 2009). Interestingly, ubp26 mutants have a reduced level of H3K36 trimethylation at FLC chromatin without altered enrichment of H3K4triMe (Schmitz et al. 2009). Thus, altered levels of H2Bub1 at FLC chromatin result in a phenotype similar to atx1, atx2, and efs mutants, which indicates the presence of an intricate regulatory loop among histone modifiers in FLC activation. In addition to modifications of histone proteins, the exchange of certain histone variants also plays a role in achieving proper levels of FLC expression. The replacement of with its variant Z is mediated by the SWR1 288 Kim et al.

13 complex in yeast (Kobor et al. 2004, Mizuguchi et al. 2004). Z deposition by the SWR1 complex is required for transcriptional regulation, maintenance of heterochromatic barriers, and genome stability in yeast (Kamakaka & Biggins 2005). Arabidopsis homologs of SWR1 components are also involved in the activation of FLC (Reyes 2006). PHOTOPERIOD- INDEPENDENT EARLY FLOWERING 1 (PIE1) is the Arabidopsis gene most homologous to SWR1; PIE1 is required for FLC activation and mediates Z deposition onto target chromatin including FLC (Deal et al. 2007, Noh & Amasino 2003). Other homologs of SWR1 complex components in Arabidopsis include ACTIN-RELATED PROTEIN 4 (AtARP4) and SUPPRESSOR OF FRIGIDA 3 (SUF3) (also known as AtARP6 and ESD1) (Choi et al. 2005, Deal et al. 2005, Kandasamy et al. 2005, Martin-Trillo et al. 2006). Mutations in any of these homologs cause an earlyflowering phenotype and a reduction in FLC expression. Thus, FLC activation, and the resulting creation of a vernalization requirement in Arabidopsis, requires deposition of Z via PIE1, AtARP4, and AtARP6/ESD1/SUF3 (Choi et al. 2005; Deal et al. 2005, 2007; Noh & Amasino 2003). Generational Resetting of FLC Although FLC repression in Arabidopsis is stably maintained throughout mitotic cell divisions following vernalization, FLC is reactivated at some point as the locus is passed to the next generation. This reactivation re-establishes the requirement for vernalization (Figure 2). The resetting of FLC expression in each generation distinguishes this type of epigenetic repression from heritable (i.e., not reset) epigenetic silencing that involves DNA methylation and small interfering RNAs (sirnas) (e.g., Henderson & Jacobsen 2007). Changes in DNA methylation do not appear to be involved in the resetting of FLC expression (Finnegan et al. 2005). Studies of expression of a vernalized FLC locus can determine the time by which FLC resetting must have occurred (Sheldon et al. 2008, Choi et al. 2009). However, it is important to note that a locus can be reset prior to actual expression; for example, key chromatin changes can occur before the transcription factors necessary for expression are present. Expression studies reveal that FLC is transiently expressed during male gametogenesis (Sheldon et al. 2008), but there is no expression during female gametogenesis (Sheldon et al. 2008, Choi et al. 2009). Whether this transient expression during male gametogenesis represents resetting or a transient state in which there is some expression of repressed gene remains to be determined. In the next generation, FLC is clearly re-expressed in the mid- to late stages of embryo development (i.e., in the early stages of the next generation) (Sheldon et al. 2008, Choi et al. 2009). Interestingly, the earliest stages of FLC expression during early embryogenesis are dependent on the presence of PIE1 but not on the presence of FRI; however, later maintenance of FLC expression requires both FRI and PIE1 (Choi et al. 2009). Perhaps reduced levels of some of the key players in FLC repression in pollen, including VRN1 and LHP1, may contribute to the reactivation of FLC during gametogenesis (Mylne et al. 2006). Indeed, a majority of FLC regulators, which includes FLC activators as well as repressors, are poorly expressed in pollen (Choi et al. 2009). Perhaps any FLC reactivation mechanism that occurs in male gametogenesis might be expected to apply to the female gamete as well. The mechanism of FLC resetting will be an interesting area to explore. Other Targets of Vernalization in Arabidopsis In Arabidopsis there are five paralogs of FLC (often called the FLC clade) FLOWER- ING LOCUS M (FLM)/MADS AFFECT- ING FLOWERING 1 (MAF1), MAF2, MAF3, MAF4, and MAF5. FLM/MAF1, MAF2, and MAF4 act as floral repressors (Ratcliffe et al. 2001, 2003; Scortecci et al. 2001). It is likely that these proteins repress the expression of floral integrators in a manner that is biochemically redundant with that of FLC (Figure 1). Vernalization 289

14 Figure 3 In the presence of FRI, FLC provides the majority of the repressive activity, but in rapidflowering lines that lack FRI, other clade members such as FLM/MAF1 can provide a greater amount of repression than FLC under certain environmental conditions (Ratcliffe et al. 2001, Scortecci et al. 2001, Werner et al. 2005). Likewise, when FRI is present, the repression of FLC accounts for the majority of the vernalization response. However, vernalization clearly promotes flowering in flc null mutants in SD. This vernalization response has been referred to as an FLC-independent vernalization pathway (Michaels & Amasino 2001). The repression of FLC paralogs is likely to account for some, and possibly all, of the vernalization Inductive photoperiod (LD) PPD1 Floral integrator genes FDL VRN3 Non-inductive photoperiod (SD) b c Floral meristem identity genes VRN2 VRN1 Flowering a Vernalization VRN1 Outline of flowering pathways in cereals. As in Figure 1, components and genetic interactions that promote flowering are shown in blue; those that repress flowering are shown in red. The photoperiod pathway in cereals activates the floral integrator VRN3 (an Arabidopsis FT homolog), which in turn activates the floral meristem-identity gene VRN1 (an Arabidopsis AP1/FUL homolog). However, in non-vernalized plants, VRN2 represses VRN3 and attenuates photoperiod-pathway activation of VRN3. Vernalization alleviates this VRN3 repression by suppressing VRN2 expression. VRN2 is suppressed by VRN1, and VRN1 is induced by cold exposure. The dual role of VRN1 in promoting the floral transition as a meristem-identity gene and in repressing VRN2 is depicted by placing VRN1 at two different locations in the diagram. Note that the induction of VRN1 by cold initiates a positive feedback loop: (a) VRN1 represses VRN2, (b) repression of VRN2 allows the photoperiod pathway to activate VRN3, and (c) VRN3 activates the expression of VRN1. In addition, non-inductive SD represses VRN2 providing an additional pathway to downregulate VRN2 during the SD of winter. response that remains in flc null mutants. Given the presumably functionally overlapping FLC paralogs, it is perhaps simpler to think of vernalization as repressing FLC and several other FLC-like genes rather than being comprised of FLC-dependent and independent pathways. Indeed, at least one other member of the FLC clade, FLM/MAF1, undergoes the same VIN3- dependent, vernalization-mediated chromatin changes as FLC (Sung et al. 2006b). Whether there is an effect of vernalization in Arabidopsis that is independent of repression of all members of the FLC clade remains to be determined. For example, it will be interesting to determine whether the induction of genes such as AGL19 (Alexandre & Hennig 2008, Schonrock et al. 2006) or AGL24 (Michaels et al. 2003) by vernalization is independent of not only FLC but of other FLC clade members as well. Vernalization in Cereals The cereals of the grass subfamily Pooideae, particularly wheat and barley, are the only other group of plants in which vernalization has been characterized molecularly. There are winter varieties of wheat and barley that possess a clear vernalization requirement and spring varieties that flower without vernalization. As in Arabidopsis, the genetic differences between winter and spring varieties have been explored, and genes responsible for the winter/spring difference have been cloned (reviewed in Colasanti & Coneva 2009, Distelfeld et al. 2009, Trevaskis et al. 2007). As discussed below, although some aspects of the circuitry of the vernalization pathway are similar in Arabidopsis and cereals, the differences indicate that the respective vernalization pathways evolved independently. Genetic studies comparing winter and spring cultivars of wheat and barley have identified three loci that play a role in the vernalization response: VRN1, VRN2, and VRN3 (Figure 3). VRN1 and VRN2 are not homologous to the Arabidopsis genes with the same name. VRN1 encodes a MADS-box transcription factor that resembles the Arabidopsis floral meristem-identity genes AP1 and FUL 290 Kim et al.

15 (Danyluk et al. 2003, Preston & Kellogg 2006, Trevaskis et al. 2003, Yan et al. 2003) and is required for flowering (Shitsukawa et al. 2007). VRN2 encodes a CCT-domain protein that has no clear homolog in Arabidopsis (Yan et al. 2004). VRN2 acts as a repressor of flowering by blocking the expression of VRN3, which is the wheat/barley homolog of FT (Yan et al. 2006). Constitutive expression of VRN3 from a promoter not subject to VRN2-mediated repression bypasses the vernalization requirement (Yan et al. 2006), just as the constitutive expression of FT bypasses FLC repression of flowering and the vernalization requirement in Arabidopsis (Michaels et al. 2005). In cereals, the LD floral promotion pathway functions similarly to that in Arabidopsis: Upon exposure to LD, a CO-like gene, PPD1, activates VRN3 (Turner et al. 2005). VRN3, in turn (and in conjunction with proteins homologous to the Arabidopsis floral integrator FD), activates the floral meristem-identity gene, VRN1 (Li & Dubcovsky 2008). VRN2, as noted above, acts to repress the expression of VRN3 under LD conditions. This repression prevents the activation of VRN3 by PPD1 and establishes a requirement for vernalization in LD (Hemming et al. 2008). VRN2 expression, like that of FLC, is repressed by cold (Yan et al. 2004). As discussed above, the induction of VIN3 by a long exposure to cold is key to FLC repression in Arabidopsis. In wheat, prolonged cold elevates the expression level of VIN3-like genes; however, the change in expression is not as defined as that seen for Arabidopsis VIN3, and none of the wheat homologs to date have been linked to the control of flowering time (Fu et al. 2007). In cereals, VRN1 is also induced by a vernalizing cold exposure, and VRN1 acts to repress VRN2 expression (Loukoianov et al. 2005, Trevaskis et al. 2006). Thus, VRN1 serves a similar role to VIN3 in cereal vernalization, but that does not rule out the existence of other components that play a VIN3-like role in cereals. Thus, there are three general similarities in the vernalization circuitry between cereals and Arabidopsis:(a) a block to flowering results from a repressor (FLC in Arabidopsis or VRN2 in wheat) that represses a homologous floral integrator in both groups of plants (FT/VRN3), (b) the repressors of flowering (FLC or VRN2) are downregulated in the cold, which permits FT/VRN3 to be expressed, and (c) downregulation of FLC and VRN2 is mediated by upregulation in the cold of VIN3 and VRN1 in Arabidopsis and cereals, respectively (Figures 1 and 3). There are, however, some distinct differences in both the vernalization circuitry and components between Arabidopsis and cereals. As discussed above, the repressors (FLC and VRN2) are not related proteins. With respect to circuitry, the VRN1 gene in cereals plays a dual role in the regulation of flowering. VRN1 is both a promoter of flowering downstream of VRN3 and a cold-activated repressor upstream of VRN2. This dual role of VRN1 in cereals creates a situation not found in Arabidopsis: a positive flowering feedback loop that involves vernalization components (Figure 3). Such a positive feedback loop ensures that once flowering initiates, it continues in the absence of environmental cues. The regulation of flowering in Arabidopsis also incorporates positive feedback loops as discussed above, but these involve components that are downstream of vernalization such as the reciprocal reinforcement of LFY and AP1 expression (Figure 1) (Sablowski 2007). Another circuitry difference is the convergence of photoperiod and vernalization on the floral repressor VRN2 in cereals (Figure 3). In addition to being repressed by cold, VRN2 expression is also governed by day length. In SD, VRN2 expression is greatly reduced, and this reduction occurs rapidly upon transfer of LD-grown plants to SD (Dubcovsky et al. 2006, Trevaskis et al. 2006). Cold and SD appear to repress VRN2 by distinct mechanisms. VRN2 repression via cold is mediated by VRN1, but VRN1 is not expressed in SD (Dubcovsky et al. 2006, Trevaskis et al. 2006). Wheat and barley are LD plants, so even though SD removes VRN2, LD is still required to activate VRN3. SD and cold are indicators of winter, and cereals can apparently use both as cues to remove a Vernalization 291

16 block to flowering, whereas in Arabidopsis only cold is a winter cue. Grasses such as wheat and barley may have evolved in a region where winters can be too mild to induce floral competence. Thus, there may be adaptive value in linking a photoperiod response with vernalization because, regardless of severity, winter always coincides with short days. That vernalization alleviates repression of the homologous genes FT in Arabidopsis and VRN3 in cereals is perhaps not surprising. As discussed above, FT-like genes are ubiquitous in plants and have been found to initiate the transition to flowering in all angiosperms examined (Turck et al. 2008). Because the role of FT/VRN3 homologs as a key flowering initiator is conserved among flowering plants, it makes a logical target for the repression of flowering; i.e., selection for this target may be an example of convergent evolution in cereals and Arabidopsis. It is important to note that vernalization has been more thoroughly studied in Arabidopsis than in cereals. As more of the molecular components of vernalization in grasses are identified, it is possible that additional similarities between the vernalization processes in these two groups may be revealed. FUTURE DIRECTIONS In cereals and Arabidopsis, the only two systems in which vernalization has been studied at a molecular level, the first known molecular event of vernalization is the induction of genes by cold. Upstream of these genes there must be a biochemical process that serves as a cold sensor. The mechanisms of cold sensing and subsequent gene activation are not known. There are a variety of possibilities for how cold sensing might operate during vernalization [see Sung & Amasino (2005) for a partial list], but in the absence of data these possibilities remain speculative. Indeed, little is known about the molecular basis of any cold-induced process in plants. A challenge for the future will be to understand how plants sense cold. Another challenge will be to explore the range of vernalization mechanisms that exist in flowering plants. There are some striking differences between the circuitry and components of vernalization in Arabidopsis and cereals. Perhaps the lack of conservation in vernalization pathways compared with the photoperiod pathway is not surprising. Flowering plants began to diversify less than 200 mya (Solds et al. 2008) when the climate was warmer and the locations of continents were quite different than at present. The major groups of angiosperms arose before continental drift, and a changing climate created environments in which a vernalization response would have had adaptive value. In contrast, evolving a mechanism to sense photoperiod would have had adaptive value much earlier as seasonal changes in day length occur nearly everywhere. Moreover, the molecular bases of other long-term cold responses such as bud dormancy remain to be determined. In many perennials, buds become dormant in the fall season and, once dormant, buds of many species must be exposed to a long period of cold before they become competent to exit dormancy and resume growth (e.g., Chouard 1960). Exploring the cold response pathways in a range of species will hopefully provide insight into the molecular mechanisms that plants have evolved to coordinate their life cycles with the changing seasons. DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review. ACKNOWLEDGMENTS R.M.A. is grateful to the National Institutes of Health, the National Science Foundation, the U.S. Department of Agriculture National Research Initiative Competitive Grants Program, the 292 Kim et al.

17 College of Agricultural and Life Sciences, and the Graduate School of the University of Wisconsin for their generous support of our flowering research. S.S. is grateful to the College of Natural Sciences and the Institute for Cellular and Molecular Biology of the University of Texas for their generous start-up support. We apologize to those in the flowering field whose work was not cited due to length limits. Annu. Rev. Cell Dev. Biol : Downloaded from LITERATURE CITED Alexandre CM, Hennig L FLC or not FLC: the other side of vernalization. J. Exp. Bot. 59: Alvarez-Venegas R, Pien S, Sadder M, Witmer X, Grossniklaus U, Avramova Z ATX-1, an Arabidopsis homolog of Trithorax, activates flower homeotic genes. Curr. Biol. 13: Amasino R Vernalization, competence, and the epigenetic memory of winter. Plant Cell 16: Ausin I, Alonso-Blanco C, Jarillo JA, Ruiz-Garcia L, Martinez-Zapater JM Regulation of flowering time by FVE, a retinoblastoma-associated protein. Nat. Genet. 36: Bastow R, Mylne JS, Lister C, Lippman Z, Martienssen RA, Dean C Vernalization requires epigenetic silencing of FLC by histone methylation. Nature 427: Baurle I, Dean C Differential interactions of the autonomous pathway RRM proteins and chromatin regulators in the silencing of Arabidopsis targets. PLoS One 3:e2733 Baurle I, Smith L, Baulcombe DC, Dean C Widespread role for the flowering-time regulators FCA and FPA in RNA-mediated chromatin silencing. Science 318: Bernier G, Kinet JM, Sachs RM, eds The Control of Floral Evocation and Morphogenesis. Boca Raton: CRC Blazquez MA, Green R, Nilsson O, Sussman MR, Weigel D Gibberellins promote flowering of Arabidopsis by activating the LEAFY promoter. Plant Cell 10: Briggs SD, Bryk M, Strahl BD, Cheung WL, Davie JK, et al Histone H3 lysine 4 methylation is mediated by Set1 and required for cell growth and rdna silencing in Saccharomyces cerevisiae. Genes Dev. 15: Burn JE, Bagnall DJ, Metzger JD, Dennis ES, Peacock WJ DNA methylation, vernalization, and the initiation of flowering. Proc. Natl. Acad. Sci. USA 90: Cao R, Wang H, He J, Erdjument-Bromage H, Tempst P, Zhang Y. 2008a. Role of hphf1 in H3K27 methylation and Hox gene silencing. Mol. Cell. Biol. 28: Cao R, Wang L, Wang H, Xia L, Erdjument-Bromage H, et al Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298: Cao Y, Dai Y, Cui S, Ma L. 2008b. Histone H2B monoubiquitination in the chromatin of FLOWERING LOCUS C regulates flowering time in Arabidopsis. Plant Cell 20: Chanvivattana Y, Bishopp A, Schubert D, Stock C, Moon YH, et al Interaction of Polycomb-group proteins controlling flowering in Arabidopsis. Development 131: Choi J, Hyun Y, Kang MJ, In Yun H, Yun JY, et al Resetting and regulation of FLOWERING LOCUS C expression during Arabidopsis reproductive development. Plant J. 57: Choi K, Kim S, Kim SY, Kim M, Hyun Y, et al SUPPRESSOR OF FRIGIDA3 encodes a nuclear ACTIN-RELATED PROTEIN6 required for floral repression in Arabidopsis. Plant Cell 17: Chouard P Vernalization and its relations to dormancy. Annu. Rev. Plant Physiol. 11: Clarke JH, Dean C Mapping FRI, a locus controlling flowering time and vernalization response in Arabidopsis thaliana. Mol. Gen. Genet. 242:81 89 Coen ES, Meyerowitz EM The war of the whorls: genetic interactions controlling flower development. Nature 353:31 37 Colasanti J, Coneva V Mechanisms of floral induction in grasses: something borrowed, something new. Plant Physiol. 149:56 62 Corbesier L, Vincent C, Jang S, Fornara F, Fan Q, et al FT protein movement contributes to longdistance signaling in floral induction of Arabidopsis. Science 316: Danyluk J, Kane NA, Breton G, Limin AE, Fowler DB, Sarhan F TaVRT-1, a putative transcription factor associated with vegetative to reproductive transition in cereals. Plant Physiol. 132: Vernalization 293

18 de Folter S, Immink RG, Kieffer M, Parenicova L, Henz SR, et al Comprehensive interaction map of the Arabidopsis MADS box transcription factors. Plant Cell 17: De Lucia F, Crevillen P, Jones AM, Greb T, Dean C A PHD-polycomb repressive complex 2 triggers the epigenetic silencing of FLC during vernalization. Proc. Natl. Acad. Sci. USA 105: Deal RB, Kandasamy MK, McKinney EC, Meagher RB The nuclear actin-related protein ARP6 is a pleiotropic developmental regulator required for the maintenance of FLOWERING LOCUS C expression and repression of flowering in Arabidopsis. Plant Cell 17: Deal RB, Topp CN, McKinney EC, Meagher RB Repression of flowering in Arabidopsis requires activation of FLOWERING LOCUS C expression by the histone variant H2 A.Z. Plant Cell 19:74 83 Dennis ES, Peacock WJ Epigenetic regulation of flowering. Curr. Opin. Plant Biol. 10: Distelfeld A, Li C, Dubcovsky J Regulation of flowering in temperate cereals. Curr. Opin. Plant Biol. 12: Dubcovsky J, Loukoianov A, Fu D, Valarik M, Sanchez A, Yan L Effect of photoperiod on the regulation of wheat vernalization genes VRN1 and VRN2. Plant Mol. Biol. 60: Fingerman IM, Wu CL, Wilson BD, Briggs SD Global loss of Set1-mediated H3 Lys4 trimethylation is associated with silencing defects in Saccharomyces cerevisiae. J. Biol. Chem. 280: Finnegan JE, Kovac KA, Jaligot E, Sheldon CC, James Peacock W, Dennis ES The downregulation of FLOWERING LOCUS C (FLC) expression in plants with low levels of DNA methylation and by vernalization occurs by distinct mechanisms. Plant J. 44: Fu D, Dunbar M, Dubcovsky J Wheat VIN3-like PHD finger genes are up-regulated by vernalization. Mol. Genet. Genomics 277: Fujiwara S, Oda A, Yoshida R, Niinuma K, Miyata K, et al Circadian clock proteins LHY and CCA1 regulate SVP protein accumulation to control flowering in Arabidopsis. Plant Cell 20: Gendall AR, Levy YY, Wilson A, Dean C The VERNALIZATION 2 gene mediates the epigenetic regulation of vernalization in Arabidopsis. Cell 107: Geraldo N, Baurle I, Kidou SI, Hu X, Dean C FRIGIDA delays flowering in Arabidopsis via a cotranscriptional mechanism involving direct interaction with the nuclear cap binding complex. Plant Physiol. doi: /pp Goodrich J, Puangsomlee P, Martin M, Long D, Meyerowitz EM, Coupland G A Polycomb-group gene regulates homeotic gene expression in Arabidopsis. Nature 386:44 51 Greb T, Mylne JS, Crevillen P, Geraldo N, An H, et al The PHD finger protein VRN5 functions in the epigenetic silencing of Arabidopsis FLC. Curr. Biol. 17:73 78 Grossniklaus U, Vielle-Calzada JP, Hoeppner MA, Gagliano WB Maternal control of embryogenesis by MEDEA, a Polycomb group gene in Arabidopsis. Science 280: Gu X, Jiang D, Wang Y, Bachmair A, He Y Repression of the floral transition via histone H2B monoubiquitination. Plant J. 57: He Y, Amasino RM Role of chromatin modification in flowering-time control. Trends Plant Sci. 10:30 35 He Y, Doyle MR, Amasino RM PAF1-complex-mediated histone methylation of FLOWERING LOCUS C chromatin is required for the vernalization-responsive, winter-annual habit in Arabidopsis. Genes Dev. 18: He Y, Michaels SD, Amasino RM Regulation of flowering time by histone acetylation in Arabidopsis. Science 302: Helliwell CA, Wood CC, Robertson M, James Peacock W, Dennis ES The Arabidopsis FLC protein interacts directly in vivo with SOC1 and FT chromatin and is part of a high-molecular-weight protein complex. Plant J. 46: Hemming MN, Peacock WJ, Dennis ES, Trevaskis B Low-temperature and daylength cues are integrated to regulate FLOWERING LOCUS T in barley. Plant Physiol. 147: Henderson IR, Dean C Control of Arabidopsis flowering: the chill before the bloom. Development 131: Henderson IR, Jacobsen SE Epigenetic inheritance in plants. Nature 447: Hennig L, Bouveret R, Gruissem W MSI1-like proteins: an escort service for chromatin assembly and remodeling complexes. Trends Cell Biol. 15: Kim et al.

19 Hepworth SR, Valverde F, Ravenscroft D, Mouradov A, Coupland G Antagonistic regulation of flowering-time gene SOC1 by CONSTANS and FLC via separate promoter motifs. EMBO J. 21: Honma T, Goto K Complexes of MADS-box proteins are sufficient to convert leaves into floral organs. Nature 409: Hsieh TF, Hakim O, Ohad N, Fischer RL From flour to flower: how Polycomb group proteins influence multiple aspects of plant development. Trends Plant Sci. 8: Jiang D, Yang W, He Y, Amasino RM Arabidopsis relatives of the human lysine-specific Demethylase1 repress the expression of FWA and FLOWERING LOCUS C and thus promote the floral transition. Plant Cell 19: Johanson U, West J, Lister C, Michaels S, Amasino R, Dean C Molecular analysis of FRIGIDA, a major determinant of natural variation in Arabidopsis flowering time. Science 290: Kamakaka RT, Biggins S Histone variants: deviants? Genes Dev. 19: Kandasamy MK, McKinney EC, Deal RB, Meagher RB Arabidopsis ARP7 is an essential actin-related protein required for normal embryogenesis, plant architecture, and floral organ abscission. Plant Physiol. 138: Karagianni P, Amazit L, Qin J, Wong J ICBP90, a novel methyl K9 H3 binding protein linking protein ubiquitination with heterochromatin formation. Mol. Cell. Biol. 28: Kim HJ, Hyun Y, Park JY, Park MJ, Park MK, et al A genetic link between cold responses and flowering time through FVE in Arabidopsis thaliana. Nat. Genet. 36: Kim SY, He Y, Jacob Y, Noh YS, Michaels S, Amasino R Establishment of the vernalization-responsive, winter-annual habit in Arabidopsis requires a putative histone H3 methyl transferase. Plant Cell 17: Kobor MS, Venkatasubrahmanyam S, Meneghini MD, Gin JW, Jennings JL, et al A protein complex containing the conserved Swi2/Snf2-related ATPase Swr1p deposits histone variant H2 A.Z into euchromatin. PLoS Biol. 2:E131 Koornneef M, Blankestijn-de Vries H, Hanhart C, Soppe W, Peeters T The phenotype of some lateflowering mutants is enhanced by a locus on chromosome 5 that is not effective in the Landsberg erecta wild-type. Plant J. 6: Koornneef M, Hanhart CJ, Van Der Veen JH A genetic and physiological analysis of late flowering mutants in Arabidopsis thaliana. Mol. Gen. Genet. 229:57 66 Kotake T, Takada S, Nakahigashi K, Ohto M, Goto K Arabidopsis TERMINAL FLOWER 2 gene encodes a heterochromatin protein 1 homolog and represses both FLOWERING LOCUS T to regulate flowering time and several floral homeotic genes. Plant Cell Physiol. 44: Krogan NJ, Dover J, Wood A, Schneider J, Heidt J, et al The Paf1 complex is required for histone H3 methylation by COMPASS and Dot1p: linking transcriptional elongation to histone methylation. Mol. Cell 11: Lan F, Collins RE, De Cegli R, Alpatov R, Horton JR, et al Recognition of unmethylated histone H3 lysine 4 links BHC80 to LSD1-mediated gene repression. Nature 448: Lang A Physiology of flower initiation. In Encyclopedia of Plant Physiology, ed. W Ruhland, pp Berlin: Springer-Verlag Lee H, Suh SS, Park E, Cho E, Ahn JH, et al The AGAMOUS-LIKE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis. Genes Dev. 14: Lee I, Bleecker A, Amasino R Analysis of naturally occurring late flowering in Arabidopsis thaliana. Mol. Gen. Genet. 237: Lee I, Michaels SD, Masshardt AS, Amasino RM The late-flowering phenotype of FRIGIDA and mutations in LUMINIDEPENDENS is suppressed in the Landsberg erecta strain of Arabidopsis. Plant J. 6:903 9 Lee J, Oh M, Park H, Lee I SOC1 translocated to the nucleus by interaction with AGL24 directly regulates leafy. Plant J. 55: Levy YY, Mesnage S, Mylne JS, Gendall AR, Dean C Multiple roles of Arabidopsis VRN1 in vernalization and flowering time control. Science 297: Li C, Dubcovsky J Wheat FT protein regulates VRN1 transcription through interactions with FDL2. Plant J. 55: Vernalization 295

20 Li D, Liu C, Shen L, Wu Y, Chen H, et al A repressor complex governs the integration of flowering signals in Arabidopsis. Dev. Cell 15: Li H, Fischle W, Wang W, Duncan EM, Liang L, et al Structural basis for lower lysine methylation state-specific readout by MBT repeats of L3MBTL1 and an engineered PHD finger. Mol. Cell 28: Lim MH, Kim J, Kim YS, Chung KS, Seo YH, et al A new Arabidopsis gene, FLK, encodes an RNA binding protein with K homology motifs and regulates flowering time via FLOWERING LOCUS C. Plant Cell 16: Liu C, Chen H, Er HL, Soo HM, Kumar PP, et al Direct interaction of AGL24 and SOC1 integrates flowering signals in Arabidopsis. Development 135: Liu F, Quesada V, Crevillen P, Baurle I, Swiezewski S, Dean C The Arabidopsis RNA-binding protein FCA requires a lysine-specific demethylase 1 homolog to downregulate FLC. Mol. Cell 28: Loukoianov A, Yan L, Blechl A, Sanchez A, Dubcovsky J Regulation of VRN-1 vernalization genes in normal and transgenic polyploid wheat. Plant Physiol. 138: Macknight R, Bancroft I, Page T, Lister C, Schmidt R, et al FCA, a gene controlling flowering time in Arabidopsis, encodes a protein containing RNA-binding domains. Cell 89: Martin-Trillo M, Lazaro A, Poethig RS, Gomez-Mena C, Pineiro MA, et al EARLY IN SHORT DAYS 1 (ESD1) encodes ACTIN-RELATED PROTEIN 6 (AtARP6), a putative component of chromatin remodelling complexes that positively regulates FLC accumulation in Arabidopsis. Development 133: Mellor J It takes a PHD to read the histone code. Cell 126:22 24 Melzer S, Lens F, Gennen J, Vanneste S, Rohde A, Beeckman T Flowering-time genes modulate meristem determinacy and growth form in Arabidopsis thaliana. Nat. Genet. 40: Michaels SD, Amasino RM FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering. Plant Cell 11: Michaels SD, Amasino RM 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: Michaels SD, Ditta G, Gustafson-Brown C, Pelaz S, Yanofsky M, Amasino RM AGL24 acts as a promoter of flowering in Arabidopsis and is positively regulated by vernalization. Plant J. 33: Michaels SD, Himelblau E, Kim SY, Schomburg FM, Amasino RM Integration of flowering signals in winter-annual Arabidopsis. Plant Physiol. 137: Mizuguchi G, Shen X, Landry J, Wu WH, Sen S, Wu C ATP-driven exchange of histone Z variant catalyzed by SWR1 chromatin remodeling complex. Science 303: Muller J, Hart CM, Francis NJ, Vargas ML, Sengupta A, et al Histone methyltransferase activity of a Drosophila Polycomb group repressor complex. Cell 111: Mylne JS, Barrett L, Tessadori F, Mesnage S, Johnson L, et al LHP1, the Arabidopsis homologue of HETEROCHROMATIN PROTEIN1, is required for epigenetic silencing of FLC. Proc. Natl. Acad. Sci. USA 103: Napp-Zinn K Vernalization: environmental and genetic regulation. In Manipulation of Flowering, ed. JG Atherton, pp London: Butterworths Nekrasov M, Klymenko T, Fraterman S, Papp B, Oktaba K, et al Pcl-PRC2 is needed to generate high levels of H3-K27 trimethylation at Polycomb target genes. EMBO J. 26: Noh B, Lee SH, Kim HJ, Yi G, Shin EA, et al Divergent roles of a pair of homologous jumonji/zincfinger-class transcription factor proteins in the regulation of Arabidopsis flowering time. Plant Cell 16: Noh YS, Amasino RM PIE1, an ISWI family gene, is required for FLC activation and floral repression in Arabidopsis. Plant Cell 15: Oh S, Park S, van Nocker S Genic and global functions for Paf1 C in chromatin modification and gene expression in Arabidopsis. PLoS Genet. 4:e Oh S, Zhang H, Ludwig P, van Nocker S A mechanism related to the yeast transcriptional regulator Paf1c is required for expression of the Arabidopsis FLC/MAF MADS box gene family. Plant Cell 16: Kim et al.

21 Papp B, Muller J Histone trimethylation and the maintenance of transcriptional ON and OFF states by trxg and PcG proteins. Genes Dev. 20: Pelaz S, Ditta GS, Baumann E, Wisman E, Yanofsky MF B and C floral organ identity functions require SEPALLATA MADS-box genes. Nature 405:200 3 Pien S, Fleury D, Mylne JS, Crevillen P, Inze D, et al Arabidopsis TRITHORAX1 dynamically regulates FLOWERING LOCUS C activation via histone 3 lysine 4 trimethylation. Plant Cell 20: Preston JC, Kellogg EA Reconstructing the evolutionary history of paralogous APETALA1/ FRUITFULL-like genes in grasses (Poaceae). Genetics 174: Ratcliffe OJ, Kumimoto RW, Wong BJ, Riechmann JL Analysis of the Arabidopsis MADS AFFECTING FLOWERING gene family: MAF2 prevents vernalization by short periods of cold. Plant Cell 15: Ratcliffe OJ, Nadzan GC, Reuber TL, Riechmann JL Regulation of flowering in Arabidopsis by an FLC homologue. Plant Physiol 126: Reyes JC Chromatin modifiers that control plant development. Curr. Opin. Plant Biol. 9:21 27 Sablowski R Flowering and determinacy in Arabidopsis. J. Exp. Bot. 58: Saleh A, Alvarez-Venegas R, Yilmaz M, Le O, Hou G, et al The highly similar Arabidopsis homologs of trithorax ATX1 and ATX2 encode proteins with divergent biochemical functions. Plant Cell 20: Samach A, Onouchi H, Gold SE, Ditta GS, Schwarz-Sommer Z, et al Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis. Science 288: Sarma K, Margueron R, Ivanov A, Pirrotta V, Reinberg D Ezh2 requires PHF1 to efficiently catalyze H3 lysine 27 trimethylation in vivo. Mol. Cell. Biol. 28: Schmitz RJ, Hong L, Fitzpatrick KE, Amasino RM DICER-LIKE 1 and DICER-LIKE 3 redundantly act to promote flowering via repression of FLOWERING LOCUS C in Arabidopsis thaliana. Genetics 176: Schmitz RJ, Sung S, Amasino RM Histone arginine methylation is required for vernalization-induced epigenetic silencing of FLC in winter-annual Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 105: Schmitz RJ, Tamada Y, Doyle MR, Zhang X, Amasino RM Histone H2B deubiquitination is required for transcriptional activation of FLOWERING LOCUS C and for proper control of flowering in Arabidopsis. Plant Physiol. 149: Schneider R, Bannister AJ, Myers FA, Thorne AW, Crane-Robinson C, Kouzarides T Histone H3 lysine 4 methylation patterns in higher eukaryotic genes. Nat. Cell Biol. 6:73 77 Schomburg FM, Patton DA, Meinke DW, Amasino RM FPA, a gene involved in floral induction in Arabidopsis, encodes a protein containing RNA-recognition motifs. Plant Cell 13: Schonrock N, Bouveret R, Leroy O, Borghi L, Kohler C, et al Polycomb-group proteins repress the floral activator AGL19 in the FLC-independent vernalization pathway. Genes Dev. 20: Schubert D, Clarenz O, Goodrich J Epigenetic control of plant development by Polycomb-group proteins. Curr. Opin. Plant Biol. 8: Schuettengruber B, Chourrout D, Vervoort M, Leblanc B, Cavalli G Genome regulation by Polycomb and Trithorax proteins. Cell 128: Scortecci KC, Michaels SD, Amasino RM Identification of a MADS-box gene, FLOWERING LOCUS M, that represses flowering. Plant J. 26: Searle I, He Y, Turck F, Vincent C, Fornara F, et al The transcription factor FLC confers a flowering response to vernalization by repressing meristem competence and systemic signaling in Arabidopsis. Genes Dev. 20: Sheldon CC, Burn JE, Perez PP, Metzger J, Edwards JA, et al The FLF MADS box gene: a repressor of flowering in Arabidopsis regulated by vernalization and methylation. Plant Cell 11: Sheldon CC, Hills MJ, Lister C, Dean C, Dennis ES, Peacock WJ Resetting of FLOWERING LOCUS C expression after epigenetic repression by vernalization. Proc. Natl. Acad. Sci. USA 105: Shi X, Kachirskaia I, Walter KL, Kuo JH, Lake A, et al Proteome-wide analysis in Saccharomyces cerevisiae identifies several PHD fingers as novel direct and selective binding modules of histone H3 methylated at either lysine 4 or lysine 36. J. Biol. Chem. 282: Shitsukawa N, Ikari C, Shimada S, Kitagawa S, SakaMoto K, et al The einkorn wheat (Triticum monococcum) mutant, maintained vegetative phase, is caused by a deletion in the VRN1 gene. Genes Genet. Syst. 82: Vernalization 297

22 Simpson GG The autonomous pathway: epigenetic and post-transcriptional gene regulation in the control of Arabidopsis flowering time. Curr. Opin. Plant Biol. 7: Simpson GG, Dijkwel PP, Quesada V, Henderson I, Dean C FY is an RNA 3 end-processing factor that interacts with FCA to control the Arabidopsis floral transition. Cell 113: Solds DE, Bell CD, Kim S, Soltis PS Origin and early evolution of angiosperms. In Year in Evolutionary Biology 2008, pp Oxford: Blackwell Soltis DE, Ma H, Frohlich MW, Soltis PS, Albert VA, et al The floral genome: an evolutionary history of gene duplication and shifting patterns of gene expression. Trends Plant Sci. 12: Sung S, Amasino RM Vernalization in Arabidopsis thaliana is mediated by the PHD finger protein VIN3. Nature 427: Sung S, Amasino RM Remembering winter: toward a molecular understanding of vernalization. Annu. Rev. Plant Biol. 56: Sung S, He Y, Eshoo TW, Tamada Y, Johnson L, et al. 2006a. Epigenetic maintenance of the vernalized state in Arabidopsis thaliana requires LIKE HETEROCHROMATIN PROTEIN 1. Nat. Genet. 38: Sung S, Schmitz RJ, Amasino RM. 2006b. A PHD finger protein involved in both the vernalization and photoperiod pathways in Arabidopsis. Genes Dev. 20: Swiezewski S, Crevillen P, Liu F, Ecker JR, Jerzmanowski A, Dean C Small RNA-mediated chromatin silencing directed to the 3 region of the Arabidopsis gene encoding the developmental regulator, FLC. Proc. Natl. Acad. Sci. USA 104: Tenney K, Shilatifard A A COMPASS in the voyage of defining the role of trithorax/mll-containing complexes: linking leukemogenesis to covalent modifications of chromatin. J. Cell Biochem. 95: Thomas B, Vince-Prue D, eds Photoperiodism in Plants. San Diego: Academic Trevaskis B, Bagnall DJ, Ellis MH, Peacock WJ, Dennis ES MADS box genes control vernalizationinduced flowering in cereals. Proc. Natl. Acad. Sci. USA 100: Trevaskis B, Hemming MN, Dennis ES, Peacock WJ The molecular basis of vernalization-induced flowering in cereals. Trends Plant Sci. 12: Trevaskis B, Hemming MN, Peacock WJ, Dennis ES HvVRN2 responds to daylength, whereas HvVRN1 is regulated by vernalization and developmental status. Plant Physiol. 140: Turck F, Fornara F, Coupland G Regulation and identity of florigen: FLOWERING LOCUS T moves center stage. Annu. Rev. Plant Biol. 59: Turner A, Beales J, Faure S, Dunford RP, Laurie DA The pseudo-response regulator Ppd-H1 provides adaptation to photoperiod in barley. Science 310: Veley KM, Michaels SD Functional redundancy and new roles for genes of the autonomous floralpromotion pathway. Plant Physiol. 147: Wenkel S, Turck F, Singer K, Gissot L, Le Gourrierec J, et al CONSTANS and the CCAAT box binding complex share a functionally important domain and interact to regulate flowering of Arabidopsis. Plant Cell 18: Werner JD, Borevitz JO, Warthmann N, Trainer GT, Ecker JR, et al Quantitative trait locus mapping and DNA array hybridization identify an FLM deletion as a cause for natural flowering-time variation. Proc. Natl. Acad. Sci. USA 102: Wilczek AM, Roe JL, Knapp MC, Cooper MD, Lopez-Gallego C, et al Effects of genetic perturbation on seasonal life history plasticity. Science 323: Wood A, Schneider J, Dover J, Johnston M, Shilatifard A The Paf1 complex is essential for histone monoubiquitination by the Rad6-Bre1 complex, which signals for histone methylation by COMPASS and Dot1p. J. Biol. Chem. 278: Wood CC, Robertson M, Tanner G, Peacock WJ, Dennis ES, Helliwell CA The Arabidopsis thaliana vernalization response requires a Polycomb-like protein complex that also includes VERNALIZATION INSENSITIVE 3. Proc. Natl. Acad. Sci. USA 103: Wu C, Morris JR Genes, genetics, and epigenetics: a correspondence. Science 293: Xu L, Menard R, Berr A, Fuchs J, Cognat V, et al The E2 ubiquitin-conjugating enzymes, AtUBC1 and AtUBC2, play redundant roles and are involved in activation of FLC expression and repression of flowering in Arabidopsis thaliana. Plant J. 57: Kim et al.

23 Xu L, Zhao Z, Dong A, Soubigou-Taconnat L, Renou JP, et al Di- and tri- but not monomethylation on histone H3 lysine 36 marks active transcription of genes involved in flowering time regulation and other processes in Arabidopsis thaliana. Mol. Cell. Biol. 28: Yan L, Fu D, Li C, Blechl A, Tranquilli G, et al The wheat and barley vernalization gene VRN3 is an orthologue of FT. Proc. Natl. Acad. Sci. USA 103: Yan L, Loukoianov A, Blechl A, Tranquilli G, Ramakrishna W, et al The wheat VRN2 gene is a flowering repressor down-regulated by vernalization. Science 303: Yan L, Loukoianov A, Tranquilli G, Helguera M, Fahima T, Dubcovsky J Positional cloning of the wheat vernalization gene VRN1. Proc. Natl. Acad. Sci. USA 100: Zeevaart JA Leaf-produced floral signals. Curr. Opin. Plant Biol. 11: Zeevaart JAD Physiology of flower formation. Annu. Rev. Plant Physiol. 27: Zhang H, Ransom C, Ludwig P, van Nocker S 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: Zhang H, van Nocker S The vernalization independence 4 gene encodes a novel regulator of flowering locus C. Plant J. 31: Zhao Z, Yu Y, Meyer D, Wu C, Shen WH Prevention of early flowering by expression of FLOWERING LOCUS C requires methylation of histone H3 K36. Nat. Cell Biol. 7: Vernalization 299

24 Contents Chromosome Odds and Ends Joseph G. Gall 1 Small RNAs and Their Roles in Plant Development Xuemei Chen 21 From Progenitors to Differentiated Cells in the Vertebrate Retina Michalis Agathocleous and William A. Harris 45 Mechanisms of Lipid Transport Involved in Organelle Biogenesis in Plant Cells Christoph Benning 71 Innovations in Teaching Undergraduate Biology and Why We Need Them William B. Wood 93 Membrane Traffic within the Golgi Apparatus Benjamin S. Glick and Akihiko Nakano 113 Molecular Circuitry of Endocytosis at Nerve Terminals Jeremy Dittman and Timothy A. Ryan 133 Many Paths to Synaptic Specificity Joshua R. Sanes and Masahito Yamagata 161 Mechanisms of Growth and Homeostasis in the Drosophila Wing Ricardo M. Neto-Silva, Brent S. Wells, and Laura A. Johnston 197 Vertebrate Endoderm Development and Organ Formation Aaron M. Zorn and James M. Wells 221 Signaling in Adult Neurogenesis Hoonkyo Suh, Wei Deng, and Fred H. Gage 253 Vernalization: Winter and the Timing of Flowering in Plants Dong-Hwan Kim, Mark R. Doyle, Sibum Sung, and Richard M. Amasino 277 Quantitative Time-Lapse Fluorescence Microscopy in Single Cells Dale Muzzey and Alexander van Oudenaarden 301 Mechanisms Shaping the Membranes of Cellular Organelles Yoko Shibata, Junjie Hu, Michael M. Kozlov, and Tom A. Rapoport 329 The Biogenesis and Function of PIWI Proteins and pirnas: Progress and Prospect Travis Thomson and Haifan Lin 355 Annual Review of Cell and Developmental Biology Volume 25, 2009 vii