ANRV389-CB25-12 ARI 12 September 2009 8:41

Vernalization: and the Timing of Flowering in Plants

Dong-Hwan Kim,1 Mark R. Doyle,2 Sibum Sung,1 and Richard M. Amasino2

1Section of Molecular Cell and Developmental Biology and the Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas 78712 2Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706-1544; email: [email protected], [email protected], [email protected], [email protected]

Annu. Rev. Cell Dev. Biol. 2009. 25:277–99 Key Words First published online as a Review in Advance on Arabidopsis, , histone modifications, epigenetics, Polycomb, August 13, 2009 Trithorax The Annual Review of Cell and Developmental Biology is online at cellbio.annualreviews.org Abstract by University of Wisconsin - Madison on 08/09/12. For personal use only. This article’s doi: Plants have evolved many systems to sense their environment and to 10.1146/annurev.cellbio.042308.113411 modify their growth and development accordingly. One example is Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org Copyright c 2009 by Annual Reviews. vernalization, the process by which flowering is promoted as plants All rights reserved sense exposure to the cold temperatures of winter. A requirement for 1081-0706/09/1110-0277$20.00 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 in- volves the recruitment of chromatin-modifying complexes to a clade of flowering repressors that are silenced epigenetically via histone modifi- cations. We also discuss the similarities and differences in vernalization between Arabidopsis and cereals.

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the relative change in the length of day and Contents night that occurs throughout the course of a year. Plants that respond to lengthening days INTRODUCTION ...... 278 and flower in the spring or early summer are Floral Integrators and known as long-day (LD) plants. Short-day (SD) Environmental Sensing...... 279 plants flower in the late summer or autumn in Vernalization in Arabidopsis ...... 280 response to shortening days and lengthening The Autonomous Pathway ...... 281 nights (Thomas & Vince-Prue 1997). In tem- Vernalization as an Epigenetic perate climates, many winter-annual, biennial, Switch...... 282 and perennial plants also use cold as an en- Genetic Analysis of the vironmental cue to flower at the proper time Vernalization Response...... 283 of year. Certain plant species need to experi- Vernalization-Mediated Changes ence a period of winter cold to overcome a in FLC Chromatin ...... 283 block to flowering. This occurs through a pro- Polycomb Repression Complexes cess known as vernalization. A requirement for and Vernalization-Mediated vernalization is an adaptation to temperate cli- FLC Repression...... 286 mates that prevents flowering prior to winter Activation of FLC Is Associated and permits flowering in the favorable condi- with Specific Chromatin tions of spring. Many vernalization-requiring Modifications ...... 287 plants are LD plants. A LD requirement cou- Generational Resetting of FLC ..... 289 pled to a vernalization requirement further en- Other Targets of Vernalization sures that precocious flowering does not occur in Arabidopsis...... 289 during the decreasing day lengths of fall and Vernalization in Cereals ...... 290 favors flowering as day length increases during FUTURE DIRECTIONS ...... 292 spring. The physiology of vernalization in a wide range of species has been intensively studied INTRODUCTION for many decades, and there are several com- Like all organisms, species of flowering plants prehensive reviews of vernalization physiology have evolved mechanisms to maximize their re- (e.g., Bernier et al. 1981, Chouard 1960, Lang productive success. One component of optimiz- 1965). The effective temperature and the length ing this success is the proper timing of the tran- of cold exposure required to achieve the vernal- sition from vegetative to reproductive growth. ized state vary among different plant species and Proper timing is important for many reasons; different varieties within a species. This vari- by University of Wisconsin - Madison on 08/09/12. For personal use only. the following are a few examples. A wide range ation is expected for a response that provides of plants require cross-pollination for success- an adaptation to a range of different environ- Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org ful reproduction, and thus flowering must occur mental niches. A vernalization response occurs synchronously within individuals of the same only at temperatures around or above freezing; species. For many plants, flowering must also temperatures below freezing are not effective, occur at a time when pollinators are present. which is not surprising because vernalization is In many parts of the world, flowering must co- an active process that requires changes in gene incide with weather conditions that will permit expression during cold exposure, as discussed flowers, which are relatively delicate structures, below. The focus of this article is to discuss re- to develop properly, followed by conditions that cent progress in understanding the molecular will permit seeds to mature properly. basis of vernalization. The link between classic Toensure that flowering occurs within a par- physiological studies and more recent molec- ticular season, plants rely primarily on envi- ular genetic analyses have been reviewed else- ronmental cues. One such cue is photoperiod, where (Amasino 2004, Sung & Amasino 2005).

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Floral Integrators and Inductive photoperiod Autonomous Vernalization Environmental Sensing pathway pathway pathway result from the expression of regulatory genes known as floral -identity genes, which specify that certain cells in the growing CO FLC tips of the plant (the shoot apical ) dif- FLC clade ferentiate into a floral meristem and ultimately Gibberellins form a flower (Coen & Meyerowitz 1991). Many of these genes are conserved through- out the plant kingdom including MADS-box Floral integrator genes FD FT SOC1 transcription factors like APETALA1 (AP1), FRUITFUL (FUL), and LEAFY (LFY), which is a protein unique in the plant lineage (re- viewed in Soltis et al. 2007). Upstream of the floral meristem-identity genes are a group of Floral meristem identity genes SEP3FUL AP1 LFY genes known as floral integrators such as FT and FD (Figure 1); i.e., the floral integrators are Flowering involved in the regulation of meristem-identity Figure 1 genes. The floral integrators are so named be- cause their expression is in turn regulated by Outline of flowering pathways in Arabidopsis. Components and genetic interactions that promote flowering are shown in blue; those that repress flowering pathways that sense environmental flowering are shown in red. Flowering is repressed in Arabidopsis by cues, such as photoperiod and cold, and/or de- FLOWERING LOCUS C (FLC) and FLC relatives designated the FLC velopmental cues such as the levels of the hor- clade. FLC represses the expression of the floral integrator genes FD, FT, and mone gibberellin; i.e., the regulation of these SUPPRESSOR OF CONSTANS 1 (SOC1). These floral integrator genes are genes serves to integrate a range of environ- promoters of flowering. One of the floral integrator genes, FT, is induced by the photoperiod pathway through CONSTANS (CO); FT protein is a mobile mental and developmental cues (Figure 1). flowering signal that partners with FD protein in the meristem to activate There is not a precise demarcation between SOC1 and the floral meristem-identity genes SEPALATA (SEP), FRUITFUL a floral integrator and a meristem-identity gene, (FUL), and APETALA 1 (AP1) [for review see Turck et al. (2008)]. Thus, FLC and a gene can have properties of both. For ex- and the photoperiod pathway act antagonistically. The floral meristem-identity genes specify the formation of floral meristems that will develop into flowers. ample, LFY is a key meristem-identity gene, but The basal level of FLC expression is set in part by the level of repression that because it is activated directly by gibberellin results from the action of autonomous pathway genes. In response to exposure (Figure 1) (Blazquez et al. 1998), it can also to the prolonged cold of winter, the vernalization pathway provides further be considered a floral integrator. In this review, FLC repression by chromatin remodeling. The gibberellin class of plant hormones promotes flowering by activating SOC1 and the floral by University of Wisconsin - Madison on 08/09/12. For personal use only. we discuss how the circuitry of photoperiod and vernalization pathways are connected to floral meristem-identity gene LEAFY (LFY).

Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org integrators in the well-studied model Arabidop- integrator FT. The FT gene is expressed in sis thaliana. leaves, and the protein travels to the meris- Studies of the photoperiodic control of flow- tem where it interacts with another integrator, ering indicated the existence of a common flo- FD, to initiate the floral transition (reviewed in ral stimulus in plants, sometimes referred to as Turcket al. 2008, Zeevaart 2008). FT-like genes florigen (Zeevaart 1976). Physiological studies are ubiquitous in plants and have been found to revealed that the floral stimulus was produced regulate flowering in a variety of species includ- in leaves exposed to inductive photoperiods and ing and poplar (reviewed in Turck et al. traveled to the meristem and caused flower- 2008). [Note: Several genes discussed in this re- ing. Recent studies in Arabidopsis and rice have view such as FT, FD, FVE, FCA, FY, and FPA made a strong case that florigen, or at least a were given two- or three-letter designations but component of the floral stimulus, is the floral not longer names (Koornneef et al. 1991).]

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There is also a significant level of conser- and therefore, SOC1 can also be considered vation in the molecular mechanisms plants use a meristem-identity gene. SOC1 and AGL24 to sense photoperiod. Arabidopsis is a facultative positively regulate each other (Liu et al. 2008, LD plant; i.e., it flowers most rapidly in LD, but Michaels et al. 2005), interact at the protein it also flowers eventually in SD. In Arabidop- level, and are both required for proper activa- sis, the perception of day length is a function tion of LFY (Lee et al. 2008). Indeed, there is of coincidence between light and expression of an intricate network of positive feedback loops the circadian-regulated gene CONSTANS (CO) among floral integrators and meristem-identity (reviewed in Turck et al. 2008). In LD-grown genes to ensure that once flowering is initiated Arabidopsis plants, CO transcription extends into in Arabidopsis, it is maintained in the absence the daylight phase. Light stabilizes CO protein, of environmental and developmental cues. It is which in turn leads to increased expression of beyond the scope of this review to discuss the FT (Figure 1) (Corbesier et al. 2007, Hepworth details of these loops. Hereafter we focus on et al. 2002, Wenkel et al. 2006). The photope- vernalization and note the connections to the riodic induction system, in which CO levels are integrators. affected by day length and translated into the regulation of FT (or FT homologs such as Hd3a Vernalization in Arabidopsis in rice or VRN3 in cereals), appears to be well conserved among flowering plants (reviewed in The identification of genes involved in the ver- Turck et al. 2008). nalization response in Arabidopsis began with Much of what we know about the molecular the study of natural variation in flowering found mechanism of the vernalization response comes among different isolates collected from a range from studies of Arabidopsis and temperate cere- of locations worldwide. Most commonly used als. These studies of vernalization indicate that lab strains of Arabidopsis do not require vernal- this process is not conserved at a biochemical ization to flower rapidly; however, many isolates level like the photoperiodic flowering pathway. flower very late unless first vernalized (Burn However, as discussed in detail below, the flo- et al. 1993, Clarke & Dean 1994, Lee et al. ral integrator FT/VRN3 is one of the targets 1993, Napp-Zinn 1987). The rapid-flowering of the vernalization pathway in both Arabidopsis types are sometimes referred to as summer an- and temperate cereals, and vernalization alle- nuals, which indicates that their life cycle is viates the repression of FT/VRN3 expression. likely to be completed in one growing season. Thus, the control of FT/VRN3 expression may Plants with a strong vernalization requirement be conserved as an integration point of the pho- are often called winter annuals, because they toperiod and vernalization pathways. may not complete their life cycle until the sec- by University of Wisconsin - Madison on 08/09/12. For personal use only. In Arabidopsis, there are additional floral ond growing season after an intervening winter. integrators including the MADS-box genes However, the actual life cycle in nature is clearly Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org SUPPRESSOR OF OVEREXPRESSION OF influenced by environment as well as genotype; CONSTANS1 (SOC1), AGAMOUS-LIKE 19 in one environment a genotype might take two (AGL19), and AGL24 (Lee et al. 2000, Liu et al. seasons to flower, whereas in a different envi- 2008, Michaels et al. 2003, Samach et al. 2000, ronment the same genotype might flower in a Schonrock et al. 2006). FT and FD partner to single season (e.g., Wilczek et al. 2009). activate SOC1 in the meristem, and SOC1 ex- In Arabidopsis, studies of natural variation pression is also controlled by vernalization, as demonstrated that the vernalization require- discussed below. Thus, SOC1 regulation inte- ment is conferred by two dominant genes, grates inputs from multiple flowering pathways FRIGIDA (FRI ) and FLOWERING LOCUS C (Figure 1). SOC1 also functions with FUL to (FLC ) (Koornneef et al. 1994, Lee et al. 1994). maintain the meristem in a floral state (i.e., as FRI encodes a nuclear protein found only in an inflorescence meristem) (Melzer et al. 2008), plants ( Johanson et al. 2000) that may interact

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with the mRNA cap-binding protein to reg- is sufficient to cause extremely late flowering ulate the level of FLC mRNA (Geraldo et al. (Michaels & Amasino 2001), which indicates 2009). FLC encodes a MADS-box DNA bind- that the levels of binding partners are not lim- ing protein that functions as a repressor of flow- iting in vivo. ering (Michaels & Amasino 1999, Sheldon et al. The circadian clock has an influence on SVP 1999). Genetic studies have shown that in the levels; SVP protein accumulates to higher levels context of vernalization FRI acts solely to up- in a double mutant of two clock components, regulate FLC (Michaels & Amasino 2001). LATE ELONGATED HYPOCOTYL (LHY ) The level of FLC expression is the primary and CIRCADIAN CLOCK ASSOCIATED 1 determinant of the vernalization requirement (CCA1), than in wild type (Fujiwara et al. 2008). in Arabidopsis. FLC represses flowering, in part, Thus, there may be cross talk in the circuitry by repressing the expression of floral integra- between the photoperiod pathway and FLC- tors such as FT, FD, and SOC1 (Helliwell et al. dependent repression in Arabidopsis; however, 2006, Hepworth et al. 2002, Lee et al. 2000, no effect of different photoperiods on SVP lev- Samach et al. 2000, Searle et al. 2006). FLC els has been reported. 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 pho- The Autonomous Pathway toperiod pathway to activate these integrators. Most commonly used types of Arabidopsis Indeed, the expression of integrators from con- are null mutants for FRI (e.g., Columbia, stitutive promoters bypasses the repressive ef- Landsberg, Wassilewskija) and thus do not re- fects of FLC and the vernalization requirement quire vernalization for rapid flowering. Histori- (Lee et al. 2000, Michaels et al. 2005). cally, a group of late-flowering mutants derived There are many examples of MADS-box from such rapid-flowering accessions have been proteins acting in multimeric complexes with referred to as the autonomous pathway (AP) of other MADS-box proteins (de Folter et al. floral promotion (Henderson & Dean 2004). 2005, Honma & Goto 2001, Pelaz et al. AP mutants are characterized by delayed flow- 2000). Recent studies show that FLC inter- ering in both LD and SD, which is in contrast acts directly with another MADS-box pro- to photoperiod-pathway mutants that show de- tein SHORT VEGETATIVE PHASE (SVP) layed flowering in inductive LD photoperiods (Fujiwara et al. 2008, Li et al. 2008). Genetic only. The flowering behavior of AP mutants is studies indicate that this interaction is biologi- similar to FRI-containing winter-annual acces- cally relevant: loss of SVP provides partial sup- sions (i.e., both have strong FLC expression, pression of the FLC-mediated delay in flow- are late flowering, and require vernalization for by University of Wisconsin - Madison on 08/09/12. For personal use only. ering (Li et al. 2008). However, the loss of rapid flowering) (Michaels & Amasino 2001). SVP cannot fully suppress the FLC-mediated To date, eight AP genes have been identified: Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org delay of flowering, which indicates that FLC LUMINIDEPENDENS (LD), FCA, FY, FPA, may redundantly interact with other MADS- FLOWERING LOCUS D (FLD), FVE, FLK, box proteins. Although SVP and other interac- and REF6 (Noh et al. 2004, Simpson 2004). tion partners may be critical for FLC-mediated FCA, FPA, FY, and FLK encode proteins that are repression of flowering, SVP levels or activity predicted to be involved in RNA metabolism do not appear to be involved in the regulation of (Lim et al. 2004, Macknight et al. 1997, Schom- flowering like FLC. For example, SVP mRNA burg et al. 2001, Simpson et al. 2003); how- levels are not affected by vernalization, whereas ever, there is no evidence to suggest that vernalization results in the stable repression of these components directly interact with FLC FLC (Bastow et al. 2004, Michaels & Amasino mRNA. FVE, FLD, and REF6 have domains 1999, Sheldon et al. 1999, Sung & Amasino common to chromatin-modifying components. 2004). Moreover, overexpression of FLC alone FVE (also known as MSI4) is a member of the

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MSI1-like protein family (MSI1–MSI5) in Ara- vernalization, FLC repression by these genes bidopsis (Ausin et al. 2004, Kim et al. 2004); occurs via a vernalization-independent mech- MSI proteins are found in several chromatin- anism. Perhaps RNA-mediated silencing plays modifying complexes in eukaryotes (Hennig a role in setting the prevernalization basal level et al. 2005). FLD and REF6 are predicted to of FLC expression. encode two different types of histone demethy- Notably, there is an intermediate-size, non- lases (He et al. 2003, Jiang et al. 2007, Noh et al. coding RNA that arises from beyond 3UTR of 2004). FLC (Swiezewski et al. 2007). Mutations in the The repression of flowering by AP genes region of this RNA have a small effect on flow- acts primarily through FLC because flc null ering relative to dcl1/3 or other AP mutants, and mutants completely suppress the delayed- the relationship of this RNA to other flowering flowering phenotypes of AP mutants (Michaels pathways is not known. & Amasino 2001). Recent studies demonstrate that many AP genes are also involved in the reg- ulation of other genes that are not involved in Vernalization as an Epigenetic Switch flowering (Baurle et al. 2007, Veley & Michaels Chouard (1960) provided a useful definition of 2008). Indeed, most of the AP genes do not ap- vernalization as “the acquisition or acceleration pear to be specific to flowering, nor do they of the ability to flower by a chilling treatment.” comprise a pathway in the typical sense. In- As defined, vernalization does not necessarily stead, these genes are involved in a range of re- induce flowering. Rather, prolonged chilling pressive mechanisms that act on FLC and other provides competence to flower. As noted above, genes. For example, the RNA-binding proteins in many vernalization-requiring plant species, FPA and FCA are broadly required for RNA- other endogenous and/or environmental con- mediated silencing, which raises the possibil- ditions, such as inductive photoperiods, are also ity that their role in FLC repression involves required for flowering. A classic experiment il- RNA-mediated silencing (Baurle et al. 2007). lustrates that this competence to flower can be In addition, all tested AP mutants showed dere- stable through mitotic cell divisions. Lang and pression of certain transposons (Baurle et al. Melchers used a biennial variety of Hyoscyamus 2007, Veley & Michaels 2008), and double mu- niger (henbane) that requires both vernaliza- tant analysis among the AP mutants revealed tion and inductive photoperiods (LD) to initi- a wide range of nonflowering phenotypes ate flowering (reviewed by Lang 1965). When (Veley & Michaels 2008). It is interesting to plants are first exposed to cold (vernalized) note that genetic analysis showed FCA requires and then kept in a noninductive photoperiod FVE for the repression of certain transposons (SD) in a normal, warm growth temperature, by University of Wisconsin - Madison on 08/09/12. For personal use only. (Baurle & Dean 2008), whereas FLD is neces- the plants grow vegetatively but do not flower. sary for FCA to repress FLC (Liu et al. 2007). However, when the previously vernalized plants Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org Thus, there appears to be a range of mecha- are moved to inductive photoperiods, even after nisms and combinatorial interactions by which many months in noninductive conditions, flow- AP genes affect gene expression. ering occurs. Thus, the vernalization-mediated Recent work provides additional support for change in competence is stable through a large the involvement of RNA-mediated silencing in number of mitotic divisions in the apical meris- FLC repression. Double mutants of dicer-like 1 tem. Mitotic stability in the absence of the in- and dicer-like 3 (dcl1/3) have phenotypes sim- ducing signal (cold in the case of vernalization) ilar to that of AP mutants: elevated levels of is arguably an epigenetic switch (e.g., Amasino FLC expression and delayed flowering that is 2004, Dennis & Peacock 2007, Wu & Morris suppressed by both flc mutations and vernaliza- 2001). The molecular basis of competence in tion (Schmitz et al. 2007). Because late flower- Arabidopsis is the mitotically stable repression of ing in dcl1/3 and AP mutants is overcome by FLC and some FLC relatives discussed below.

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It is worth noting that not all plant species trigger to set in motion the molecular events exhibit behavior that fits the stable acquisition that stably repress FLC during vernalization. of competence model described above; for ex- ample, there are species that need to flower dur- ing cold exposure presumably because compe- Vernalization-Mediated Changes tence is not stably maintained through mitotic in FLC Chromatin cell divisions after cold exposure ceases (Bernier The stable nature of the vernalized state is et al. 1981). Indeed, there are mutants in consistent with a role for chromatin modifi- Arabidopsis in which FLC repression occurs dur- cation of target genes. In Arabidopsis, FLC re- ing cold exposure but it is not stably maintained pression is the molecular basis of vernalization- upon a return to warm; not surprisingly, these induced competence. Thus, FLC is a likely mutants affect chromatin-modifying proteins target for chromatin modification. The first (Gendall et al. 2001, Levy et al. 2002, Mylne molecular evidence for chromatin modification et al. 2006, Sung et al. 2006a). in Arabidopsis vernalization came from the iden- tification of VRN2 and VIN3. VRN2 encodes a homolog of Suppressor of zeste (Su(z)12), Genetic Analysis of the a component of Polycomb repression com- Vernalization Response plex 2 (PRC2) that was first identified in an- As discussed above, winter-annual accessions imals (Gendall et al. 2001). VIN3 encodes a of Arabidopsis exhibit a delayed-flowering phe- PHD (plant homeodomain) protein (Sung & notype if they are not vernalized, and most Amasino 2004); the PHD domain is commonly of the natural variation in the vernalization found in a wide range of complexes that are requirement is due to allelic variation at involved in chromatin-level regulation (Mellor FRI and FLC. The molecular mechanism by 2006). which FLC is epigenetically repressed by ver- Vernalization results in an increase in two nalization in Arabidopsis has been addressed repressive histone modifications at FLC chro- by molecular genetic analyses. Using winter- matin (Figure 2): histone H3 Lys 9 (H3K9) annual accessions as a parental line, one can and histone H3 Lys 27 (H3K27) methylation screen for mutants that can no longer be (Bastow et al. 2004, Sung & Amasino 2004). vernalized; such vernalization-insensitive mu- In vin3 and vrn2 mutants, H3K9 and H3K27 tants have lesions in genes that participate methylation are not enriched at FLC follow- in the vernalization pathway. To date, these ing a sufficient cold treatment, and FLC ex- screens have revealed five genes: VERNALIZA- pression is not repressed (Bastow et al. 2004, TION 1 (VRN1), VRN2, VERNALIZATION Sung & Amasino 2004). The loss of VRN1, by University of Wisconsin - Madison on 08/09/12. For personal use only. INSENSITIVE 3 (VIN3), VRN5/ VIN3-LIKE which encodes a DNA-binding protein con- 1(VIL1), and atPRMT5 (Bastow et al. 2004, taining two plant-specific B3 domains, shows Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org Gendall et al. 2001, Greb et al. 2007, Schmitz distinctive chromatin modifications from those et al. 2008, Sung & Amasino 2004, Sung et al. of vin3 and vrn2 mutants. Whereas methylation 2006b). As expected, given the nature of the ge- of both H3K9 and H3K27 are affected in vin3 netic screen, all mutants fail to stably repress and vrn2 mutants, only H3K9 methylation fails FLC by vernalization. All of these genes are to occur in vrn1 during and after vernalization constitutively expressed except for VIN3. VIN3 (Bastow et al. 2004, Sung & Amasino 2004), is expressed specifically during a vernalizing which suggests the two methylation events can cold treatment, and expression is completely occur independently and that VRN1 is involved abolished when plants are returned to a warm specifically in H3K9 methylation (Figure 2). temperature (Sung & Amasino 2004). The cold VIL1/VRN5 was identified independently induction and transient nature of VIN3 expres- by a yeast two- screen for VIN3- sion indicates that VIN3 may be a part of the interacting proteins (Sung et al. 2006b) and by a

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a M ATX K4 K4 K4 K4 K4 1/2 M PAF1 K27 H3 SWR1 K36 K36complex K36 K36 K36 complex EFSEFS FIE1 PRC2 PRC2 ARP6 core ARP4 core Pol II H2AZ H2AZ H2AZ VRN2 VRN2

H2B

HUB1 HUB2 FLC active Ubiquitinase

VIN3 Winter cold (vernalization) Induced

FLC repression initiates b VIL1/VRN5

VIN3 PRC2 H3K4triMe LHP1 K27 K27 core M H3K26triMe VRN2 H2Bub1 H3 K27 K27 H3K9triMe

H3K27triMe M Methyl group H2A H2A H2A H2A H2A H2A

H2B

Winter cold (vernalization)

Next generation H3K27Me and H3K9Me c density increases VIL1/VRN5 K9

HMTase VIN3 PRC2 M LHP1 LHP1 K27 core K9 K9 K9 VRN2 K9 H3 K27 K27 K27 VRN1 VRN1VRN1 VRN1

H2A H2A H2A H2A H2A H2A

by University of Wisconsin - Madison on 08/09/12. For personal use only. H2B

Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org VIN3 Return to warm No longer expressed

Mitotically stable repression d K9 K9 VIL1/VRN5 VIL1/VRN5 HMTase HMTase PRC2 PRC2 LHP1 LHP1 core core K9 K9 VRN2 VRN2 K9 H3 K27 K27 K27 VRN1 VRN1VRN1 VRN1

H2A H2A H2A H2A H2A H2A

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genetic screen for mutants with a reduced ver- histone H3 (Lan et al. 2007), methylated nalization response (Greb et al. 2007). VIN3 histone H3K9 (Karagianni et al. 2008), and and VIL1/VRN5 belong to a small family of pro- methylated histone H3K36 (Shi et al. 2007). teins that contain a PHD domain, a fibronectin The diverse binding activities of PHD domains III (FNIII) domain, and a conserved C-terminal make them excellent candidates for readers of region. Whereas the PHD and FNIII do- histone modifications. As expected, amino acid mains are found throughout eukaryotes, the sequence variation within PHD domains is re- C-terminal domain is unique in plants and sponsible for variations in their specificity (Li mediates protein-protein interactions between et al. 2007). In the VIN3/VEL family there are VIN3 and VIL1/VRN5 (Greb et al. 2007, Sung variations in amino acid sequences within the et al. 2006b). Similar to VIN3, VIL1/VRN5 is PHD domains, and thus, if VIN3/VEL fam- also required for vernalization-mediated his- ily members indeed bind modified histone tails, tone modifications at FLC chromatin including there may be variation within the family as to methylation of both H3K9 and H3K27 (Greb which modifications are bound. One possibil- et al. 2007, Sung et al. 2006b). Recent reports ity is that VIN3 binds to modifications associ- claim that yet another member of the fam- ated with active FLC chromatin and in doing ily, VIL2/VEL1, is part of a VIL1-containing so initiates the transition to a repressed state. complex that is physically located at FLC after After a certain degree of conversion to the re- vernalization-mediated repression (De Lucia pressed state is achieved and modifications asso- et al. 2008). However, this repression is not im- ciated with active FLC chromatin are replaced paired when VIL2/VEL1 levels are decreased by repressive marks, VIN3 is no longer neces- either by mutation (Kim, Schmitz, Amasino, sary, as in spring, when other positive feedback and Sung, unpublished work) or by RNA in- loops of histone modification maintain repres- terference (Greb et al. 2007), which indicates sion (Figure 2). However, the specificity of var- possible functional redundancy of VIL2/VEL1 ious members of the VIN3/VEL protein family with other members of the VIN3/VEL protein for modified histones and the significance of any family. variability in modifying FLC chromatin remain Recent studies demonstrated that specific to be investigated. PHD domains associate with specific histone The mechanism of how FLC is targeted for modifications, which include methylated hi- repression remains a major unanswered ques- stone H3K4 (Mellor 2006), nonmethylated tion. The induction of VIN3 is a necessary first

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Figure 2 by University of Wisconsin - Madison on 08/09/12. For personal use only. 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 Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org 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.

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step, but how this translates into a change in the conserved from plants to animals (Hsieh et al. activities of chromatin-modifying complexes at 2003), but, as discussed below, PRC1 compo- the FLC locus is not at all understood. Even nents are not. if the PHD domain of VIN3 binds to specific In Drosophila, core components of PRC2 histone modifications characteristic of FLC in consist of Enhancer of zeste [E(z)], Extra the pre-vernalized state (see below), such mod- Sex Combs (ESC), and Suppressor of zeste ifications are not likely to be unique to FLC Su(z)12 (Cao et al. 2002). Arabidopsis and other chromatin. Clearly, there is much to learn about plants contain multiple copies of many PRC2 targeting specificity. components (Hsieh et al. 2003, Wood et al. In addition to vernalization-mediated 2006). For example, in Arabidopsis, there are methylation at H3K9 and H3K27 at FLC three homologs of the histone methyltrans- chromatin, histone arginine methylation also ferase E(z). These include CURLY LEAF plays a role in maintaining stable repression (CLF), SWINGER (SWN), and MEDEA of FLC (Schmitz et al. 2008). Mutations in (MEA) (Hsieh et al. 2003). CLF was first iden- a Type II protein arginine methyltransferase tified as a repressor of the floral homeotic gene atPRMT5, reduced the vernalization gene AGAMOUS (AG) (Goodrich et al. 1997), response. atPRMT5 is required for the sym- whereas MEA was identified in a screen for mu- metric methylation of Arg 3 of histone H4 tants that have altered endosperm development (sMeH4R3), and the level of this methylation (Grossniklaus et al. 1998). SWN is partially is increased at FLC chromatin by vernalization. redundant with CLF (Chanvivattana et al. Furthermore, sMeH4R3 appears to be required 2004). None of the Arabidopsis E(z) homologs for the vernalization-mediated enrichment of were identified in screens for vernalization- both H3K9 and H3K27 methylation (Schmitz insensitive mutants, which is likely due to the et al. 2008). functional redundancy among these proteins with regard to FLC repression. In fact, VRN2 is the only PRC2 component identified as being Polycomb Repression Complexes involved in vernalization from mutant screens. and Vernalization-Mediated Biochemical approaches revealed that FLC Repression Arabidopsis PRC2 complexes include VIN3 As discussed above, the identification of an during cold exposure (De Lucia et al. 2008, Arabidopsis homolog of Su(z)12, VRN2, in a Wood et al. 2006). PRC2 complexes in vernalization-insensitive mutant screen sug- Arabidopsis can also include VIL1/VRN5; gested the involvement of a PRC2-like com- purification of proteins associated with a plex in FLC repression (Gendall et al. 2001). In VIL1/VRN5-TAP fusion revealed components by University of Wisconsin - Madison on 08/09/12. For personal use only. animals, there are two distinct PRC complexes, of PRC2, which include VRN2, SWN, MSI1, PRC2 and PRC1 (reviewed in Schuettengruber and FERTILIZATION INDEPENDENT Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org et al. 2007). Polycomb group (PcG) proteins ENDOSPERM (FIE; an Arabidopsis ESC were first identified in Drosophila as essen- homolog) (De Lucia et al. 2008). The theme of tial components for maintaining HOMEOTIC PHD domain-containing proteins associating (HOX) genes in a repressed state. The PRC2 with PRC2 may be widespread in eukaryotes; complex is involved in the initial di- or PHD–domain proteins also associate with trimethylation of H3K27 at target chromatin. PRC2 in Drosophila and humans (Cao et al. Then, in many systems, methylated H3K27 is 2008a, Nekrasov et al. 2007, Sarma et al. 2008). bound by the PRC1 complex, which acts to Analyses of the occupancy of PRC2 com- maintain chromatin in a repressed state and ponents at FLC chromatin showed that VRN2 to maintain H3K27 methylation through mi- is already present at FLC chromatin prior to totic cell divisions (Cao et al. 2002, Muller vernalization (De Lucia et al. 2008). In cold, et al. 2002). Core components of PRC2 are well VIN3 is induced and VIN3 protein is present at

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a region encoding the first intron of FLC. As- (i.e., plants that flower early without cold) in sociation of VIL1/VRN5 is VIN3-dependent, winter-annual Arabidopsis have revealed many and thus VIN3 may serve as a guiding factor of the components required for FLC activation. for the recruitment of VIL1/VRN5 to FLC H3K4triMe is associated with active chro- (De Lucia et al. 2008). Whereas the enrich- matin in most eukaryotes (Schneider et al. ment of VIL1/VRN5 is restricted to the first in- 2004). A specific Saccharomyces cerevisiae HM- tron of FLC in cold temperatures, VIL1/VRN5 Tase, Set1, is responsible for mono- to spreads throughout the FLC chromatin when trimethylation of H3K4, and mutations in set1 plants return to warm temperatures. This cause diverse phenotypic defects such as slow spreading may require warm temperatures, or growth and rDNA derepression (Briggs et al. warm temperatures may simply increase the 2001, Fingerman et al. 2005). Yeast Set1 is rate of these chromatin changes. Presumably, an essential component of a complex called the spreading of VIL1/VRN5 across FLC chro- COMPASS (complex proteins associated with matin maintains the repressed state of FLC Set1) (Krogan et al. 2003). Another complex, (Figure 2) (De Lucia et al. 2008). the RNA polymerase II-Associated Factor 1 Although the presence of PRC2 com- (PAF1)-containing complex, is necessary for ponents in plants is well established, Ara- H3K4triMe enrichment at target chromatin. bidopsis lacks components with an overall These two complexes, COMPASS and PAF1, similarity to PRC1 components (Hsieh et al. physically associate to coordinate the transcrip- 2003, Schubert et al. 2005). Instead, LIKE- tion of target genes (Krogan et al. 2003). HETEROCHROMATIN PROTEIN 1 Screens for rapidly flowering mutants iden- (LHP1) likely binds to and stably maintains the tified two Arabidopsis homologs of the yeast PRC2-mediated modified histones at FLC fol- PAF1 complex components PAF1 and CTR9: lowing vernalization (Mylne et al. 2006, Sung EARLY FLOWERING 7 (ELF7) and ELF8, et al. 2006a). In lhp1 mutants, the active histone respectively (He & Amasino 2005). ELF7 and mark, H3K4triMe, is only transiently reduced ELF8 are required for high FLC expression and when plants are kept in cold (Sung et al. 2006a). for H3K4triMe enrichment at FLC chromatin Interestingly, lhp1 mutants were not found in (He et al. 2004). Other Arabidopsis genes that screens for vernalization-insensitive mutants encode relatives of PAF1 and COMPASS because the loss of LHP1 also results in the complex components are VERNALIZATION derepression of flowering promoters such as INDEPENDENCE 3 (VIP3; Arabidopsis ho- FT, and thus lhp1 mutants are early flowering molog of human hSki8), VIP4 (Arabidopsis without vernalization (Kotake et al. 2003, Sung homolog of yeast Leo1), VIP5 (Arabidopsis et al. 2006a). LHP1 is likely to play a broad homolog of yeast Rtf1), ARABIDOPSIS by University of Wisconsin - Madison on 08/09/12. For personal use only. role in gene repression in plants and may be TRITHORAX-LIKE 1 (ATX1), and ATX2 (see part of a protein complex that serves a similar below for roles of ATX1 and ATX2) (Alvarez- Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org role to that of PRC1 in animals. 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). Mu- Activation of FLC Is Associated with tations in these genes typically cause reduced Specific Chromatin Modifications H3K4triMe deposition at FLC chromatin and As discussed above, FLC repression is associated early-flowering phenotypes similar to elf7 and with dynamic changes in histone composition, elf8 (Alvarez-Venegas et al. 2003, He et al. which are initiated during prolonged cold ex- 2004, Pien et al. 2008, Saleh et al. 2008, Zhang posure. Conversely, the high levels of FLC that & van Nocker 2002). Methylation of H3K4 create a vernalization requirement are associ- at FLC chromatin appears to be mediated ated with active histone marks. Mutant screens by ATX1 and ATX2, which are homologous for the loss of the vernalization requirement to Drosophila Trithorax (Trx). Trx is a H3K4

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methyltransferase that belongs to the Trithorax modification associated with active transcrip- group of proteins (TrxG), which are activators tion. In yeast, a complex that contains RAD6 of Drosophila homeotic genes (a role opposite (with E2-ubiquitin-conjugating activity) and to that of PcG proteins) (Pien et al. 2008, Saleh BRE1 (with E3-ubiquitin ligase activity) acts et al. 2008). ATX1 mediates trimethylation on to monoubiquitinate histone H2B at specific H3K4, whereas ATX2 dimethylates H3K4 in target chromatin (Tenney & Shilatifard 2005). vitro (Saleh et al. 2008). The involvement of H2Bub1 is an important prerequisite for the TrxG- and PcG-like genes in the regulation proper enrichment of H3K4triMe and for of FLC indicates that FLC is controlled by transcriptional activity of target genes (Wood an evolutionarily conserved mechanism that et al. 2003). In Arabidopsis, H2B monoubiq- involves a dynamic balance between PcG and uitination of FLC chromatin is also required TrxG. A recent study reveals that this dynamic for proper activation of FLC. Investigators balance occurs throughout the genome in identified two Arabidopsis BRE1 homologs, HI- plants (Oh et al. 2008). STONE MONOUBIQUITINATION 1 (HUB1) In Drosophila, TrxG activator proteins as and HISTONE MONOUBIQUITINATION 2 well as components of PRC2 repressive com- (HUB2) (Cao et al. 2008b, Gu et al. 2009), plexes are constitutively bound to HOX target and found that mutations in either hub1 or chromatin; i.e., both repressive and activating hub2 result in early flowering and the loss modifiers can reside at the same locus (Papp of H3K4triMe enrichment at the promoter & Muller 2006). As discussed above, PRC2 is region of FLC. Arabidopsis has three RAD6 detectable at FLC chromatin prior to vernal- homologs: UBIQUITIN-CONJUGATING ization (De Lucia et al. 2008). Furthermore, ENZYME 1, 2, and 3 (AtUBC1, AtUBC2, H3K27triMe is present at FLC chromatin prior and AtUBC3) (Cao et al. 2008b, Gu et al. to vernalization when FLC is actively tran- 2009). AtUBC1 and AtUBC2 are involved in scribed, but the level of H3K27triMe is much flowering and act redundantly for the enrich- lower than when FLC is repressed (Pien et al. ment of H2Bub1 at FLC chromatin, whereas 2008). This indicates that a threshold level of AtUBC3 does not play a role in FLC activation H3K27triMe enrichment is necessary to estab- (Xu et al. 2009). Although the enrichment of lish repressed FLC chromatin. H2Bub1 is required for the proper activation Another Trx-like SET domain protein is of FLC, over-enrichment of H2Bub1 results in also required for proper FLC activation. EARLY the failure of FLC activation; a mutation in a FLOWERING IN SHORT DAYS (EFS) en- H2B deubiquitinase, UBIQUITIN-SPECIFIC codes a SET domain protein, which medi- PROTEASE26 (UBP26 ), results in a rapid- ates di- and trimethylation of histone H3 Lys flowering phenotype due to the loss of FLC by University of Wisconsin - Madison on 08/09/12. For personal use only. 36 (H3K36) on target chromatin (Xu et al. expression (Schmitz et al. 2009). Interestingly, 2008). Mutants with EFS lesions exhibit re- ubp26 mutants have a reduced level of H3K36 Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org duced FLC expression and early flowering sim- trimethylation at FLC chromatin without ilar to mutations in PAF1-COMPASS compo- altered enrichment of H3K4triMe (Schmitz nents (Kim et al. 2005, Xu et al. 2008, Zhao et al. et al. 2009). Thus, altered levels of H2Bub1 at 2005). In winter-annual strains of Arabidopsis, FLC chromatin result in a phenotype similar to EFS may also be required for the enrichment atx1, atx2, and efs mutants, which indicates the of H3K4triMe at FLC chromatin (Kim et al. presence of an intricate regulatory loop among 2005). Thus, the three Arabidopsis HMTases, histone modifiers in FLC activation. ATX1, ATX2, and EFS, appear to act coordi- In addition to modifications of histone pro- nately to mediate the transcriptional activation teins, the exchange of certain histone variants of FLC. also plays a role in achieving proper levels of Monoubiquitination at lysine 123 of His- FLC expression. The replacement of H2A with tone H2B (H2Bub1), like H3K4, is a histone its variant H2AZ is mediated by the SWR1

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complex in yeast (Kobor et al. 2004, Mizuguchi 2008, Choi et al. 2009). However, it is impor- et al. 2004). H2AZ deposition by the SWR1 tant to note that a locus can be reset prior to complex is required for transcriptional regu- actual expression; for example, key chromatin lation, maintenance of heterochromatic barri- changes can occur before the transcription fac- ers, and genome stability in yeast (Kamakaka & tors necessary for expression are present. Ex- Biggins 2005). Arabidopsis homologs of SWR1 pression studies reveal that FLC is transiently components are also involved in the activa- expressed during male gametogenesis (Sheldon tion of FLC (Reyes 2006). PHOTOPERIOD- et al. 2008), but there is no expression during fe- INDEPENDENT EARLY FLOWERING 1 male gametogenesis (Sheldon et al. 2008, Choi (PIE1)istheArabidopsis gene most homolo- et al. 2009). Whether this transient expression gous to SWR1; PIE1 is required for FLC ac- during male gametogenesis represents resetting tivation and mediates H2AZ deposition onto or a transient state in which there is some ex- target chromatin including FLC (Deal et al. pression of repressed gene remains to be deter- 2007, Noh & Amasino 2003). Other homologs mined. In the next generation, FLC is clearly of SWR1 complex components in Arabidop- re-expressed in the mid- to late stages of em- sis include ACTIN-RELATED PROTEIN 4 bryo development (i.e., in the early stages of (AtARP4) and SUPPRESSOR OF FRIGIDA 3 the next generation) (Sheldon et al. 2008, Choi (SUF3) (also known as AtARP6 and ESD1) et al. 2009). Interestingly, the earliest stages of (Choi et al. 2005, Deal et al. 2005, Kandasamy FLC expression during early embryogenesis are et al. 2005, Martin-Trillo et al. 2006). Muta- dependent on the presence of PIE1 but not on tions in any of these homologs cause an early- the presence of FRI; however, later maintenance flowering phenotype and a reduction in FLC of FLC expression requires both FRI and PIE1 expression. Thus, FLC activation, and the re- (Choi et al. 2009). Perhaps reduced levels of sulting creation of a vernalization requirement some of the key players in FLC repression in in Arabidopsis, requires deposition of H2AZ pollen, including VRN1 and LHP1, may con- via PIE1, AtARP4, and AtARP6/ESD1/SUF3 tribute to the reactivation of FLC during game- (Choi et al. 2005; Deal et al. 2005, 2007; Noh togenesis (Mylne et al. 2006). Indeed, a majority & Amasino 2003). of FLC regulators, which includes FLC activa- tors as well as repressors, are poorly expressed in pollen (Choi et al. 2009). Perhaps any FLC Generational Resetting of FLC reactivation mechanism that occurs in male ga- Although FLC repression in Arabidopsis is stably metogenesis might be expected to apply to the maintained throughout mitotic cell divisions female gamete as well. The mechanism of FLC following vernalization, FLC is reactivated at resetting will be an interesting area to explore. by University of Wisconsin - Madison on 08/09/12. For personal use only. some point as the locus is passed to the next generation. This reactivation re-establishes the Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org requirement for vernalization (Figure 2). The Other Targets of Vernalization in Arabidopsis resetting of FLC expression in each generation distinguishes this type of epigenetic repression In Arabidopsis there are five paralogs of FLC from heritable (i.e., not reset) epigenetic silenc- (often called the FLC clade) - ing that involves DNA methylation and small ING LOCUS M (FLM)/MADS AFFECT- interfering RNAs (siRNAs) (e.g., Henderson & ING FLOWERING 1 (MAF1), MAF2, MAF3, Jacobsen 2007). Changes in DNA methylation MAF4, and MAF5. FLM/MAF1, MAF2, and do not appear to be involved in the resetting of MAF4 act as floral repressors (Ratcliffe et al. FLC expression (Finnegan et al. 2005). 2001, 2003; Scortecci et al. 2001). It is likely Studies of expression of a vernalized FLC that these proteins repress the expression of flo- locus can determine the time by which FLC ral integrators in a manner that is biochemi- resetting must have occurred (Sheldon et al. cally redundant with that of FLC (Figure 1).

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In the presence of FRI, FLC provides the ma- response that remains in flc null mutants. Given jority of the repressive activity, but in rapid- the presumably functionally overlapping FLC flowering lines that lack FRI, other clade mem- paralogs, it is perhaps simpler to think of ver- bers such as FLM/MAF1 can provide a greater nalization as repressing FLC and several other amount of repression than FLC under certain FLC-like genes rather than being comprised of environmental conditions (Ratcliffe et al. 2001, FLC-dependent and independent pathways. In- Scortecci et al. 2001, Werner et al. 2005). Like- deed, at least one other member of the FLC wise, when FRI is present, the repression of clade, FLM/MAF1, undergoes the same VIN3- FLC accounts for the majority of the vernaliza- dependent, vernalization-mediated chromatin tion response. However, vernalization clearly changes as FLC (Sung et al. 2006b). Whether promotes flowering in flc null mutants in SD. there is an effect of vernalization in Arabidopsis This vernalization response has been referred that is independent of repression of all mem- to as an FLC-independent vernalization path- bers of the FLC clade remains to be determined. way (Michaels & Amasino 2001). The repres- For example, it will be interesting to determine sion of FLC paralogs is likely to account for whether the induction of genes such as AGL19 some, and possibly all, of the vernalization (Alexandre & Hennig 2008, Schonrock et al. 2006) or AGL24 (Michaels et al. 2003) by ver- Inductive Non-inductive nalization is independent of not only FLC but photoperiod (LD) photoperiod (SD) Vernalization of other FLC clade members as well.

PPD1 VRN2 a VRN1 Vernalization in Cereals The cereals of the grass subfamily Pooideae, b particularly wheat and barley, are the only other group of plants in which vernalization has been characterized molecularly. There are winter va- Floral integrator genes FDL VRN3 rieties of wheat and barley that possess a clear vernalization requirement and spring varieties c that flower without vernalization. As in Ara- bidopsis, the genetic differences between win- Floral meristem identity genes VRN1 ter and spring varieties have been explored, and genes responsible for the winter/spring differ-

Flowering ence have been cloned (reviewed in Colasanti & Coneva 2009, Distelfeld et al. 2009, Trevaskis

by University of Wisconsin - Madison on 08/09/12. For personal use only. Figure 3 et al. 2007). As discussed below, although some Outline of flowering pathways in cereals. As in Figure 1, components and genetic interactions that promote flowering are shown in blue; those that aspects of the circuitry of the vernalization Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org repress flowering are shown in red. The photoperiod pathway in cereals pathway are similar in Arabidopsis and cereals, activates the floral integrator VRN3 (an Arabidopsis FT homolog), which in the differences indicate that the respective ver- turn activates the floral meristem-identity gene VRN1 (an Arabidopsis nalization pathways evolved independently. AP1/FUL homolog). However, in non-vernalized plants, VRN2 represses Genetic studies comparing winter and VRN3 and attenuates photoperiod-pathway activation of VRN3. Vernalization alleviates this VRN3 repression by suppressing VRN2 expression. VRN2 is spring cultivars of wheat and barley have iden- suppressed by VRN1, and VRN1 is induced by cold exposure. The dual role of tified three loci that play a role in the ver- VRN1 in promoting the floral transition as a meristem-identity gene and in nalization response: VRN1, VRN2, and VRN3 repressing VRN2 is depicted by placing VRN1 at two different locations in the (Figure 3). VRN1 and VRN2 are not ho- diagram. Note that the induction of VRN1 by cold initiates a positive feedback mologous to the Arabidopsis genes with the loop: (a) VRN1 represses VRN2, (b) repression of VRN2 allows the same name. encodes a MADS-box tran- photoperiod pathway to activate VRN3, and (c) VRN3 activates the expression VRN1 of VRN1. In addition, non-inductive SD represses VRN2 providing an scription factor that resembles the Arabidopsis additional pathway to downregulate VRN2 during the SD of winter. floral meristem-identity genes AP1 and FUL

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(Danyluk et al. 2003, Preston & Kellogg 2006, wheat) that represses a homologous floral in- Trevaskis et al. 2003, Yan et al. 2003) and is re- tegrator in both groups of plants (FT/VRN3), quired for flowering (Shitsukawa et al. 2007). (b) the repressors of flowering (FLC or VRN2) VRN2 encodes a CCT-domain protein that are downregulated in the cold, which permits has no clear homolog in Arabidopsis (Yan et al. FT/VRN3 to be expressed, and (c) downreg- 2004). VRN2 acts as a repressor of flowering by ulation of FLC and VRN2 is mediated by up- blocking the expression of VRN3, which is the regulation in the cold of VIN3 and VRN1 in wheat/barley homolog of FT (Yan et al. 2006). Arabidopsis and cereals, respectively (Figures 1 Constitutive expression of VRN3 from a pro- and 3). moter not subject to VRN2-mediated repres- There are, however, some distinct differ- sion bypasses the vernalization requirement ences in both the vernalization circuitry and (Yan et al. 2006), just as the constitutive expres- components between Arabidopsis and cereals. sion of FT bypasses FLC repression of flowering As discussed above, the repressors (FLC and and the vernalization requirement in Arabidopsis VRN2) are not related proteins. With respect (Michaels et al. 2005). to circuitry, the VRN1 gene in cereals plays a In cereals, the LD floral promotion pathway dual role in the regulation of flowering. VRN1 functions similarly to that in Arabidopsis: Upon is both a promoter of flowering downstream of exposure to LD, a CO-like gene, PPD1, acti- VRN3 and a cold-activated repressor upstream vates VRN3 (Turneret al. 2005). VRN3, in turn of VRN2. This dual role of VRN1 in cereals (and in conjunction with proteins homologous creates a situation not found in Arabidopsis:a to the Arabidopsis floral integrator FD), activates positive flowering feedback loop that involves the floral meristem-identity gene, VRN1 (Li & vernalization components (Figure 3). Such a Dubcovsky 2008). VRN2, as noted above, acts positive feedback loop ensures that once flow- to repress the expression of VRN3 under LD ering initiates, it continues in the absence of en- conditions. This repression prevents the acti- vironmental cues. The regulation of flowering vation of VRN3 by PPD1 and establishes a re- in Arabidopsis also incorporates positive feed- quirement for vernalization in LD (Hemming back loops as discussed above, but these involve et al. 2008). VRN2 expression, like that of FLC, components that are downstream of vernaliza- is repressed by cold (Yan et al. 2004). tion such as the reciprocal reinforcement of As discussed above, the induction of VIN3 by LFY and AP1 expression (Figure 1) (Sablowski a long exposure to cold is key to FLC repression 2007). in Arabidopsis. In wheat, prolonged cold elevates Another circuitry difference is the conver- the expression level of VIN3-like genes; how- gence of photoperiod and vernalization on the ever, the change in expression is not as defined floral repressor VRN2 in cereals (Figure 3). by University of Wisconsin - Madison on 08/09/12. For personal use only. as that seen for Arabidopsis VIN3, and none of In addition to being repressed by cold, VRN2 the wheat homologs to date have been linked to expression is also governed by day length. In Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org the control of flowering time (Fu et al. 2007). In SD, VRN2 expression is greatly reduced, and cereals, VRN1 is also induced by a vernalizing this reduction occurs rapidly upon transfer of cold exposure, and VRN1 acts to repress VRN2 LD-grown plants to SD (Dubcovsky et al. 2006, expression (Loukoianov et al. 2005, Trevaskis Trevaskis et al. 2006). Cold and SD appear to et al. 2006). Thus, VRN1 serves a similar role repress VRN2 by distinct mechanisms. VRN2 to VIN3 in vernalization, but that does repression via cold is mediated by VRN1, but not rule out the existence of other components VRN1 is not expressed in SD (Dubcovsky et al. that play a VIN3-like role in cereals. 2006, Trevaskis et al. 2006). Wheat and bar- Thus, there are three general similarities in ley are LD plants, so even though SD removes the vernalization circuitry between cereals and VRN2, LD is still required to activate VRN3. Arabidopsis:(a) a block to flowering results from SD and cold are indicators of winter, and cere- a repressor (FLC in Arabidopsis or VRN2 in als can apparently use both as cues to remove a

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block to flowering, whereas in Arabidopsis only There are a variety of possibilities for how cold cold is a winter cue. Grasses such as wheat and sensing might operate during vernalization [see barley may have evolved in a region where win- Sung & Amasino (2005) for a partial list], but ters can be too mild to induce floral compe- in the absence of data these possibilities remain tence. Thus, there may be adaptive value in speculative. Indeed, little is known about the linking a photoperiod response with vernaliza- molecular basis of any cold-induced process in tion because, regardless of severity, winter al- plants. A challenge for the future will be to un- ways coincides with short days. derstand how plants sense cold. That vernalization alleviates repression of Another challenge will be to explore the the homologous genes FT in Arabidopsis and range of vernalization mechanisms that exist VRN3 in cereals is perhaps not surprising. As in flowering plants. There are some striking discussed above, FT-like genes are ubiquitous differences between the circuitry and compo- in plants and have been found to initiate the nents of vernalization in Arabidopsis and cereals. transition to flowering in all angiosperms ex- Perhaps the lack of conservation in vernaliza- amined (Turck et al. 2008). Because the role tion pathways compared with the photoperiod of FT/VRN3 homologs as a key flowering ini- pathway is not surprising. Flowering plants be- tiator is conserved among flowering plants, it gan to diversify less than 200 mya (Solds et al. makes a logical target for the repression of flow- 2008) when the climate was warmer and the lo- ering; i.e., selection for this target may be an cations of continents were quite different than example of convergent evolution in cereals and at present. The major groups of angiosperms Arabidopsis. arose before continental drift, and a changing It is important to note that vernalization climate created environments in which a vernal- has been more thoroughly studied in Arabidop- ization response would have had adaptive value. sis than in cereals. As more of the molecular In contrast, evolving a mechanism to sense pho- components of vernalization in grasses are iden- toperiod would have had adaptive value much tified, it is possible that additional similarities earlier as seasonal changes in day length oc- between the vernalization processes in these cur nearly everywhere. Moreover, the molecu- two groups may be revealed. lar bases of other long-term cold responses such as bud remain to be determined. In many perennials, buds become dormant in the FUTURE DIRECTIONS fall season and, once dormant, buds of many In cereals and Arabidopsis, the only two sys- species must be exposed to a long period of tems in which vernalization has been studied cold before they become competent to exit at a molecular level, the first known molecu- dormancy and resume growth (e.g., Chouard by University of Wisconsin - Madison on 08/09/12. For personal use only. lar event of vernalization is the induction of 1960). Exploring the cold response pathways in genes by cold. Upstream of these genes there a range of species will hopefully provide insight Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org must be a biochemical process that serves as a into the molecular mechanisms that plants have cold sensor. The mechanisms of cold sensing evolved to coordinate their life cycles with the and subsequent gene activation are not known. 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. ANRV389-CB25-12 ARI 12 September 2009 8:41

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.

LITERATURE CITED Alexandre CM, Hennig L. 2008. FLC or not FLC: the other side of vernalization. J. Exp. Bot. 59:1127–35 Alvarez-Venegas R, Pien S, Sadder M, Witmer X, Grossniklaus U, Avramova Z. 2003. ATX-1, an Arabidopsis homolog of Trithorax, activates flower homeotic genes. Curr. Biol. 13:627–37 Amasino R. 2004. Vernalization, competence, and the epigenetic memory of winter. Plant Cell 16:2553–59 Ausin I, Alonso-Blanco C, Jarillo JA, Ruiz-Garcia L, Martinez-Zapater JM. 2004. Regulation of flowering time by FVE, a retinoblastoma-associated protein. Nat. Genet. 36:162–66 Bastow R, Mylne JS, Lister C, Lippman Z, Martienssen RA, Dean C. 2004. Vernalization requires epigenetic silencing of FLC by histone methylation. Nature 427:164–67 Baurle I, Dean C. 2008. 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. 2007. Widespread role for the flowering-time regulators FCA and FPA in RNA-mediated chromatin silencing. Science 318:109–12 Bernier G, Kinet JM, Sachs RM, eds. 1981. The Control of Floral Evocation and Morphogenesis. Boca Raton: CRC Blazquez MA, Green R, Nilsson O, Sussman MR, Weigel D. 1998. Gibberellins promote flowering of Arabidopsis by activating the LEAFY promoter. Plant Cell 10:791–800 Briggs SD, Bryk M, Strahl BD, Cheung WL, Davie JK, et al. 2001. Histone H3 lysine 4 methylation is mediated by Set1 and required for cell growth and rDNA silencing in Saccharomyces cerevisiae. Genes Dev. 15:3286–95 Burn JE, Bagnall DJ, Metzger JD, Dennis ES, Peacock WJ. 1993. DNA methylation, vernalization, and the initiation of flowering. Proc. Natl. Acad. Sci. USA 90:287–91 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. 28:1862–72 Cao R, Wang L, Wang H, Xia L, Erdjument-Bromage H, et al. 2002. Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298:1039–43 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 20:2586–602 Chanvivattana Y, Bishopp A, Schubert D, Stock C, Moon YH, et al. 2004. Interaction of Polycomb-group

by University of Wisconsin - Madison on 08/09/12. For personal use only. proteins controlling flowering in Arabidopsis. Development 131:5263–76 Choi J, Hyun Y, Kang MJ, In Yun H, Yun JY, et al. 2009. Resetting and regulation of FLOWERING LOCUS C expression during Arabidopsis reproductive development. Plant J. 57:918–31 Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org Choi K, Kim S, Kim SY, Kim M, Hyun Y, et al. 2005. SUPPRESSOR OF FRIGIDA3 encodes a nuclear ACTIN-RELATED PROTEIN6 required for floral repression in Arabidopsis. Plant Cell 17:2647–60 Chouard P. 1960. Vernalization and its relations to dormancy. Annu. Rev. Plant Physiol. 11:191–238 Clarke JH, Dean C. 1994. Mapping FRI, a locus controlling flowering time and vernalization response in . Mol. Gen. Genet. 242:81–89 Coen ES, Meyerowitz EM. 1991. The war of the whorls: genetic interactions controlling flower development. Nature 353:31–37 Colasanti J, Coneva V. 2009. 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. 2007. FT protein movement contributes to long- distance signaling in floral induction of Arabidopsis. Science 316:1030–33 Danyluk J, Kane NA, Breton G, Limin AE, Fowler DB, Sarhan F. 2003. TaVRT-1, a putative transcription factor associated with vegetative to reproductive transition in cereals. Plant Physiol. 132:1849–60

www.annualreviews.org • Vernalization 293 ANRV389-CB25-12 ARI 12 September 2009 8:41

de Folter S, Immink RG, Kieffer M, Parenicova L, Henz SR, et al. 2005. Comprehensive interaction map of the Arabidopsis MADS box transcription factors. Plant Cell 17:1424–33 De Lucia F, Crevillen P, Jones AM, Greb T, Dean C. 2008. A PHD-polycomb repressive complex 2 triggers the epigenetic silencing of FLC during vernalization. Proc. Natl. Acad. Sci. USA 105:16831–36 Deal RB, Kandasamy MK, McKinney EC, Meagher RB. 2005. 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 17:2633–46 Deal RB, Topp CN, McKinney EC, Meagher RB. 2007. 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. 2007. Epigenetic regulation of flowering. Curr. Opin. Plant Biol. 10:520–27 Distelfeld A, Li C, Dubcovsky J. 2009. Regulation of flowering in temperate cereals. Curr. Opin. Plant Biol. 12:178–84 Dubcovsky J, Loukoianov A, Fu D, Valarik M, Sanchez A, Yan L. 2006. Effect of photoperiod on the regulation of wheat vernalization genes VRN1 and VRN2. Plant Mol. Biol. 60:469–80 Fingerman IM, Wu CL, Wilson BD, Briggs SD. 2005. Global loss of Set1-mediated H3 Lys4 trimethylation is associated with silencing defects in Saccharomyces cerevisiae. J. Biol. Chem. 280:28761–65 Finnegan JE, Kovac KA, Jaligot E, Sheldon CC, James Peacock W, Dennis ES. 2005. 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. 44:420–32 Fu D, Dunbar M, Dubcovsky J. 2007. Wheat VIN3-like PHD finger genes are up-regulated by vernalization. Mol. Genet. Genomics 277:301–13 Fujiwara S, Oda A, Yoshida R, Niinuma K, Miyata K, et al. 2008. Circadian clock proteins LHY and CCA1 regulate SVP protein accumulation to control flowering in Arabidopsis. Plant Cell 20:2960–71 Gendall AR, Levy YY, Wilson A, Dean C. 2001. The VERNALIZATION 2 gene mediates the epigenetic regulation of vernalization in Arabidopsis. Cell 107:525–35 Geraldo N, Baurle I, Kidou SI, Hu X, Dean C. 2009. FRIGIDA delays flowering in Arabidopsis via a co- transcriptional mechanism involving direct interaction with the nuclear cap binding complex. Plant Physiol. doi: 10.1104/pp.109.137448 Goodrich J, Puangsomlee P, Martin M, Long D, Meyerowitz EM, Coupland G. 1997. 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. 2007. 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. 1998. Maternal control of embryogenesis by MEDEA, a Polycomb group gene in Arabidopsis. Science 280:446–50 Gu X, Jiang D, Wang Y, Bachmair A, He Y. 2009. Repression of the floral transition via histone H2B monoubiquitination. Plant J. 57:422–33 He Y, Amasino RM. 2005. Role of chromatin modification in flowering-time control. Trends Plant Sci. 10:30–35 by University of Wisconsin - Madison on 08/09/12. For personal use only. He Y, Doyle MR, Amasino RM. 2004. PAF1-complex-mediated histone methylation of FLOWERING LOCUS C chromatin is required for the vernalization-responsive, winter-annual habit in Arabidopsis. Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org Genes Dev. 18:2774–84 He Y, Michaels SD, Amasino RM. 2003. Regulation of flowering time by histone acetylation in Arabidopsis. Science 302:1751–54 Helliwell CA, Wood CC, Robertson M, James Peacock W, Dennis ES. 2006. 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. 46:183–92 Hemming MN, Peacock WJ, Dennis ES, Trevaskis B. 2008. Low-temperature and daylength cues are inte- grated to regulate FLOWERING LOCUS T in barley. Plant Physiol. 147:355–66 Henderson IR, Dean C. 2004. Control of Arabidopsis flowering: the chill before the bloom. Development 131:3829–38 Henderson IR, Jacobsen SE. 2007. Epigenetic inheritance in plants. Nature 447:418–24 Hennig L, Bouveret R, Gruissem W. 2005. MSI1-like proteins: an escort service for chromatin assembly and remodeling complexes. Trends Cell Biol. 15:295–302

294 Kim et al. ANRV389-CB25-12 ARI 12 September 2009 8:41

Hepworth SR, Valverde F, Ravenscroft D, Mouradov A, Coupland G. 2002. Antagonistic regulation of flowering-time gene SOC1 by CONSTANS and FLC via separate promoter motifs. EMBO J. 21:4327–37 Honma T, Goto K. 2001. Complexes of MADS-box proteins are sufficient to convert leaves into floral organs. Nature 409:525–29 Hsieh TF, Hakim O, Ohad N, Fischer RL. 2003. From flour to flower: how Polycomb group proteins influence multiple aspects of plant development. Trends Plant Sci. 8:439–45 Jiang D, Yang W, He Y, Amasino RM. 2007. 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 19:2975–87 Johanson U, West J, Lister C, Michaels S, Amasino R, Dean C. 2000. Molecular analysis of FRIGIDA, a major determinant of natural variation in Arabidopsis flowering time. Science 290:344–47 Kamakaka RT, Biggins S. 2005. Histone variants: deviants? Genes Dev. 19:295–310 Kandasamy MK, McKinney EC, Deal RB, Meagher RB. 2005. Arabidopsis ARP7 is an essential actin-related protein required for normal embryogenesis, plant architecture, and floral organ abscission. Plant Physiol. 138:2019–32 Karagianni P, Amazit L, Qin J, Wong J. 2008. ICBP90, a novel methyl K9 H3 binding protein linking protein ubiquitination with heterochromatin formation. Mol. Cell. Biol. 28:705–17 Kim HJ, Hyun Y, Park JY, Park MJ, Park MK, et al. 2004. A genetic link between cold responses and flowering time through FVE in Arabidopsis thaliana. Nat. Genet. 36:167–71 Kim SY, He Y, Jacob Y, Noh YS, Michaels S, Amasino R. 2005. Establishment of the vernalization-responsive, winter-annual habit in Arabidopsis requires a putative histone H3 methyl transferase. Plant Cell 17:3301–10 Kobor MS, Venkatasubrahmanyam S, Meneghini MD, Gin JW, Jennings JL, et al. 2004. A protein com- plex 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. 1994. The phenotype of some late- flowering mutants is enhanced by a locus on chromosome 5 that is not effective in the Landsberg erecta wild-type. Plant J. 6:911–19 Koornneef M, Hanhart CJ, Van Der Veen JH. 1991. 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. 2003. 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. 44:555–64 Krogan NJ, Dover J, Wood A, Schneider J, Heidt J, et al. 2003. The Paf1 complex is required for histone H3 methylation by COMPASS and Dot1p: linking transcriptional elongation to histone methylation. Mol. Cell 11:721–29 Lan F, Collins RE, De Cegli R, Alpatov R, Horton JR, et al. 2007. Recognition of unmethylated histone H3 lysine 4 links BHC80 to LSD1-mediated gene repression. Nature 448:718–22 by University of Wisconsin - Madison on 08/09/12. For personal use only. Lang A. 1965. Physiology of flower initiation. In Encyclopedia of Plant Physiology, ed. W Ruhland, pp. 1371–536. Berlin: Springer-Verlag Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org Lee H, Suh SS, Park E, Cho E, Ahn JH, et al. 2000. The AGAMOUS-LIKE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis. Genes Dev. 14:2366–76 Lee I, Bleecker A, Amasino R. 1993. Analysis of naturally occurring late flowering in Arabidopsis thaliana. Mol. Gen. Genet. 237:171–76 Lee I, Michaels SD, Masshardt AS, Amasino RM. 1994. 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. 2008. SOC1 translocated to the nucleus by interaction with AGL24 directly regulates leafy. Plant J. 55:832–43 Levy YY, Mesnage S, Mylne JS, Gendall AR, Dean C. 2002. Multiple roles of Arabidopsis VRN1 in vernalization and flowering time control. Science 297:243–46 Li C, Dubcovsky J. 2008. Wheat FT protein regulates VRN1 transcription through interactions with FDL2. Plant J. 55:543–54

www.annualreviews.org • Vernalization 295 ANRV389-CB25-12 ARI 12 September 2009 8:41

Li D, Liu C, Shen L, Wu Y, Chen H, et al. 2008. A repressor complex governs the integration of flowering signals in Arabidopsis. Dev. Cell 15:110–20 Li H, Fischle W, Wang W, Duncan EM, Liang L, et al. 2007. Structural basis for lower lysine methylation state-specific readout by MBT repeats of L3MBTL1 and an engineered PHD finger. Mol. Cell 28:677–91 Lim MH, Kim J, Kim YS, Chung KS, Seo YH, et al. 2004. A new Arabidopsis gene, FLK, encodes an RNA binding protein with K homology motifs and regulates flowering time via FLOWERING LOCUS C. Plant Cell 16:731–40 Liu C, Chen H, Er HL, Soo HM, Kumar PP, et al. 2008. Direct interaction of AGL24 and SOC1 integrates flowering signals in Arabidopsis. Development 135:1481–91 Liu F, Quesada V, Crevillen P, Baurle I, Swiezewski S, Dean C. 2007. The Arabidopsis RNA-binding protein FCA requires a lysine-specific demethylase 1 homolog to downregulate FLC. Mol. Cell 28:398–407 Loukoianov A, Yan L, Blechl A, Sanchez A, Dubcovsky J. 2005. Regulation of VRN-1 vernalization genes in normal and transgenic polyploid wheat. Plant Physiol. 138:2364–73 Macknight R, Bancroft I, Page T, Lister C, Schmidt R, et al. 1997. FCA, a gene controlling flowering time in Arabidopsis, encodes a protein containing RNA-binding domains. Cell 89:737–45 Martin-TrilloM, Lazaro A, Poethig RS, Gomez-Mena C, Pineiro MA, et al. 2006. 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 133:1241– 52 Mellor J. 2006. 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. 2008. Flowering-time genes modulate meristem determinacy and growth form in Arabidopsis thaliana. Nat. Genet. 40:1489–92 Michaels SD, Amasino RM. 1999. FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering. Plant Cell 11:949–56 Michaels SD, Amasino RM. 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 13:935–41 Michaels SD, Ditta G, Gustafson-Brown C, Pelaz S, Yanofsky M, Amasino RM. 2003. AGL24 acts as a promoter of flowering in Arabidopsis and is positively regulated by vernalization. Plant J. 33:867–74 Michaels SD, Himelblau E, Kim SY, Schomburg FM, Amasino RM. 2005. Integration of flowering signals in winter-annual Arabidopsis. Plant Physiol. 137:149–56 Mizuguchi G, Shen X, Landry J, Wu WH, Sen S, Wu C. 2004. ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex. Science 303:343–48 Muller J, Hart CM, Francis NJ, Vargas ML, Sengupta A, et al. 2002. Histone methyltransferase activity of a Drosophila Polycomb group repressor complex. Cell 111:197–208 Mylne JS, Barrett L, Tessadori F, Mesnage S, Johnson L, et al. 2006. LHP1, the Arabidopsis homologue of HETEROCHROMATIN PROTEIN1, is required for epigenetic silencing of FLC. Proc. Natl. Acad. Sci. by University of Wisconsin - Madison on 08/09/12. For personal use only. USA 103:5012–17 Napp-Zinn K. 1987. Vernalization: environmental and genetic regulation. In Manipulation of Flowering, ed. Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org JG Atherton, pp. 123–32. London: Butterworths Nekrasov M, Klymenko T, Fraterman S, Papp B, Oktaba K, et al. 2007. Pcl-PRC2 is needed to generate high levels of H3-K27 trimethylation at Polycomb target genes. EMBO J. 26:4078–88 Noh B, Lee SH, Kim HJ, Yi G, Shin EA, et al. 2004. Divergent roles of a pair of homologous jumonji/zinc- finger-class transcription factor proteins in the regulation of Arabidopsis flowering time. Plant Cell 16:2601– 13 Noh YS, Amasino RM. 2003. PIE1, an ISWI family gene, is required for FLC activation and floral repression in Arabidopsis. Plant Cell 15:1671–82 Oh S, Park S, van Nocker S. 2008. Genic and global functions for Paf1 C in chromatin modification and gene expression in Arabidopsis. PLoS Genet. 4:e1000077 Oh S, Zhang H, Ludwig P, van Nocker S. 2004. A mechanism related to the yeast transcriptional regulator Paf1c is required for expression of the Arabidopsis FLC/MAF MADS box gene family. Plant Cell 16:2940– 53

296 Kim et al. ANRV389-CB25-12 ARI 12 September 2009 8:41

Papp B, Muller J. 2006. Histone trimethylation and the maintenance of transcriptional ON and OFF states by trxG and PcG proteins. Genes Dev. 20:2041–54 Pelaz S, Ditta GS, Baumann E, Wisman E, Yanofsky MF. 2000. 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. 2008. Arabidopsis TRITHORAX1 dynamically regulates FLOWERING LOCUS C activation via histone 3 lysine 4 trimethylation. Plant Cell 20:580–88 Preston JC, Kellogg EA. 2006. Reconstructing the evolutionary history of paralogous APETALA1/ FRUITFULL-like genes in grasses (Poaceae). Genetics 174:421–37 Ratcliffe OJ, Kumimoto RW, Wong BJ, Riechmann JL. 2003. Analysis of the Arabidopsis MADS AFFECTING FLOWERING gene family: MAF2 prevents vernalization by short periods of cold. Plant Cell 15:1159–69 Ratcliffe OJ, Nadzan GC, Reuber TL, Riechmann JL. 2001. Regulation of flowering in Arabidopsis by an FLC homologue. Plant Physiol 126:122–32 Reyes JC. 2006. Chromatin modifiers that control plant development. Curr. Opin. Plant Biol. 9:21–27 Sablowski R. 2007. Flowering and determinacy in Arabidopsis. J. Exp. Bot. 58:899–907 Saleh A, Alvarez-Venegas R, Yilmaz M, Le O, Hou G, et al. 2008. The highly similar Arabidopsis homologs of trithorax ATX1 and ATX2 encode proteins with divergent biochemical functions. Plant Cell 20:568–79 Samach A, Onouchi H, Gold SE, Ditta GS, Schwarz-Sommer Z, et al. 2000. Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis. Science 288:1613–16 Sarma K, Margueron R, Ivanov A, Pirrotta V, Reinberg D. 2008. Ezh2 requires PHF1 to efficiently catalyze H3 lysine 27 trimethylation in vivo. Mol. Cell. Biol. 28:2718–31 Schmitz RJ, Hong L, Fitzpatrick KE, Amasino RM. 2007. DICER-LIKE 1 and DICER-LIKE 3 redundantly act to promote flowering via repression of FLOWERING LOCUS C in Arabidopsis thaliana. Genetics 176:1359–62 Schmitz RJ, Sung S, Amasino RM. 2008. Histone arginine methylation is required for vernalization-induced epigenetic silencing of FLC in winter-annual Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 105:411–16 Schmitz RJ, TamadaY, Doyle MR, Zhang X, Amasino RM. 2009. Histone H2B deubiquitination is required for transcriptional activation of FLOWERING LOCUS C and for proper control of flowering in Arabidopsis. Plant Physiol. 149:1196–204 Schneider R, Bannister AJ, Myers FA, Thorne AW, Crane-Robinson C, Kouzarides T. 2004. Histone H3 lysine 4 methylation patterns in higher eukaryotic genes. Nat. Cell Biol. 6:73–77 Schomburg FM, Patton DA, Meinke DW, Amasino RM. 2001. FPA, a gene involved in floral induction in Arabidopsis, encodes a protein containing RNA-recognition motifs. Plant Cell 13:1427–36 Schonrock N, Bouveret R, Leroy O, Borghi L, Kohler C, et al. 2006. Polycomb-group proteins repress the floral activator AGL19 in the FLC-independent vernalization pathway. Genes Dev. 20:1667–78 Schubert D, Clarenz O, Goodrich J. 2005. Epigenetic control of plant development by Polycomb-group proteins. Curr. Opin. Plant Biol. 8:553–61 Schuettengruber B, Chourrout D, Vervoort M, Leblanc B, Cavalli G. 2007. Genome regulation by Polycomb

by University of Wisconsin - Madison on 08/09/12. For personal use only. and Trithorax proteins. Cell 128:735–45 Scortecci KC, Michaels SD, Amasino RM. 2001. Identification of a MADS-box gene, FLOWERING LOCUS M, that represses flowering. Plant J. 26:229–36 Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org Searle I, He Y, Turck F, Vincent C, Fornara F, et al. 2006. The transcription factor FLC confers a flowering response to vernalization by repressing meristem competence and systemic signaling in Arabidopsis. Genes Dev. 20:898–912 Sheldon CC, Burn JE, Perez PP, Metzger J, Edwards JA, et al. 1999. The FLF MADS box gene: a repressor of flowering in Arabidopsis regulated by vernalization and methylation. Plant Cell 11:445–58 Sheldon CC, Hills MJ, Lister C, Dean C, Dennis ES, Peacock WJ. 2008. Resetting of FLOWERING LOCUS C expression after epigenetic repression by vernalization. Proc. Natl. Acad. Sci. USA 105:2214–19 Shi X, Kachirskaia I, Walter KL, Kuo JH, Lake A, et al. 2007. 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. 282:2450–55 Shitsukawa N, Ikari C, Shimada S, Kitagawa S, SakaMoto K, et al. 2007. The einkorn wheat (Triticum mono- coccum) mutant, maintained vegetative phase, is caused by a deletion in the VRN1 gene. Genes Genet. Syst. 82:167–70

www.annualreviews.org • Vernalization 297 ANRV389-CB25-12 ARI 12 September 2009 8:41

Simpson GG. 2004. The autonomous pathway: epigenetic and post-transcriptional gene regulation in the control of Arabidopsis flowering time. Curr. Opin. Plant Biol. 7:570–74 Simpson GG, Dijkwel PP, Quesada V, Henderson I, Dean C. 2003. FY is an RNA 3 end-processing factor that interacts with FCA to control the Arabidopsis floral transition. Cell 113:777–87 Solds DE, Bell CD, Kim S, Soltis PS. 2008. Origin and early evolution of angiosperms. In Year in Evolutionary Biology 2008, pp. 3–25. Oxford: Blackwell Soltis DE, Ma H, Frohlich MW, Soltis PS, Albert VA, et al. 2007. The floral genome: an evolutionary history of gene duplication and shifting patterns of gene expression. Trends Plant Sci. 12:358–67 Sung S, Amasino RM. 2004. Vernalization in Arabidopsis thaliana is mediated by the PHD finger protein VIN3. Nature 427:159–64 Sung S, Amasino RM. 2005. Remembering winter: toward a molecular understanding of vernalization. Annu. Rev. Plant Biol. 56:491–508 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. 38:706–10 Sung S, Schmitz RJ, Amasino RM. 2006b. A PHD finger protein involved in both the vernalization and photoperiod pathways in Arabidopsis. Genes Dev. 20:3244–48 Swiezewski S, Crevillen P, Liu F, Ecker JR, Jerzmanowski A, Dean C. 2007. Small RNA-mediated chromatin silencing directed to the 3 region of the Arabidopsis gene encoding the developmental regulator, FLC. Proc. Natl. Acad. Sci. USA 104:3633–38 Tenney K, Shilatifard A. 2005. A COMPASS in the voyage of defining the role of trithorax/MLL-containing complexes: linking leukemogenesis to covalent modifications of chromatin. J. Cell Biochem. 95:429–36 Thomas B, Vince-Prue D, eds. 1997. Photoperiodism in Plants. San Diego: Academic Trevaskis B, Bagnall DJ, Ellis MH, Peacock WJ, Dennis ES. 2003. MADS box genes control vernalization- induced flowering in cereals. Proc. Natl. Acad. Sci. USA 100:13099–104 Trevaskis B, Hemming MN, Dennis ES, Peacock WJ. 2007. The molecular basis of vernalization-induced flowering in cereals. Trends Plant Sci. 12:352–57 Trevaskis B, Hemming MN, Peacock WJ, Dennis ES. 2006. HvVRN2 responds to daylength, whereas HvVRN1 is regulated by vernalization and developmental status. Plant Physiol. 140:1397–405 TurckF, Fornara F, Coupland G. 2008. Regulation and identity of florigen: FLOWERING LOCUS T moves center stage. Annu. Rev. Plant Biol. 59:573–94 Turner A, Beales J, Faure S, Dunford RP, Laurie DA. 2005. The pseudo-response regulator Ppd-H1 provides adaptation to photoperiod in barley. Science 310:1031–34 Veley KM, Michaels SD. 2008. Functional redundancy and new roles for genes of the autonomous floral- promotion pathway. Plant Physiol. 147:682–95 Wenkel S, Turck F, Singer K, Gissot L, Le Gourrierec J, et al. 2006. CONSTANS and the CCAAT box binding complex share a functionally important domain and interact to regulate flowering of Arabidopsis. Plant Cell 18:2971–84 by University of Wisconsin - Madison on 08/09/12. For personal use only. Werner JD, Borevitz JO, Warthmann N, Trainer GT, Ecker JR, et al. 2005. Quantitative trait locus mapping and DNA array hybridization identify an FLM deletion as a cause for natural flowering-time variation. Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org Proc. Natl. Acad. Sci. USA 102:2460–65 Wilczek AM, Roe JL, Knapp MC, Cooper MD, Lopez-Gallego C, et al. 2009. Effects of genetic perturbation on seasonal life history plasticity. Science 323:930–34 Wood A, Schneider J, Dover J, Johnston M, Shilatifard A. 2003. 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. 278:34739–42 Wood CC, Robertson M, Tanner G, Peacock WJ, Dennis ES, Helliwell CA. 2006. The Arabidopsis thaliana vernalization response requires a Polycomb-like protein complex that also includes VERNALIZATION INSENSITIVE 3. Proc. Natl. Acad. Sci. USA 103:14631–36 Wu C, Morris JR. 2001. Genes, genetics, and epigenetics: a correspondence. Science 293:1103–5 Xu L, Menard R, Berr A, Fuchs J, Cognat V, et al. 2009. 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. 57:279–88

298 Kim et al. ANRV389-CB25-12 ARI 12 September 2009 8:41

Xu L, Zhao Z, Dong A, Soubigou-Taconnat L, Renou JP, et al. 2008. 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. 28:1348–60 Yan L, Fu D, Li C, Blechl A, Tranquilli G, et al. 2006. The wheat and barley vernalization gene VRN3 is an orthologue of FT. Proc. Natl. Acad. Sci. USA 103:19581–86 Yan L, Loukoianov A, Blechl A, Tranquilli G, Ramakrishna W, et al. 2004. The wheat VRN2 gene is a flowering repressor down-regulated by vernalization. Science 303:1640–44 Yan L, Loukoianov A, Tranquilli G, Helguera M, Fahima T, Dubcovsky J. 2003. Positional cloning of the wheat vernalization gene VRN1. Proc. Natl. Acad. Sci. USA 100:6263–68 Zeevaart JA. 2008. Leaf-produced floral signals. Curr. Opin. Plant Biol. 11:541–47 Zeevaart JAD. 1976. Physiology of flower formation. Annu. Rev. Plant Physiol. 27:321–48 Zhang H, Ransom C, Ludwig P, 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 164:347–58 Zhang H, van Nocker S. 2002. The vernalization independence 4 gene encodes a novel regulator of flowering locus C. Plant J. 31:663–73 Zhao Z, YuY, Meyer D, Wu C, Shen WH. 2005. Prevention of early flowering by expression of FLOWERING LOCUS C requires methylation of histone H3 K36. Nat. Cell Biol. 7:1256–60 by University of Wisconsin - Madison on 08/09/12. For personal use only. Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org

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Contents Annual Review of Cell and Chromosome Odds and Ends pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp Developmental Joseph G. Gall 1 Biology

Small RNAs and Their Roles in Plant Development Volume 25, 2009 Xuemei Chen pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp21 From Progenitors to Differentiated Cells in the Vertebrate Retina Michalis Agathocleous and William A. Harris pppppppppppppppppppppppppppppppppppppppppppppp45 Mechanisms of Lipid Transport Involved in Organelle Biogenesis in Plant Cells Christoph Benning ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp71 Innovations in Teaching Undergraduate Biology and Why We Need Them William B. Wood pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp93 Membrane Traffic within the Golgi Apparatus Benjamin S. Glick and Akihiko Nakano ppppppppppppppppppppppppppppppppppppppppppppppppppp113 Molecular Circuitry of Endocytosis at Nerve Terminals Jeremy Dittman and Timothy A. Ryan pppppppppppppppppppppppppppppppppppppppppppppppppppp133 Many Paths to Synaptic Specificity Joshua R. Sanes and Masahito Yamagata pppppppppppppppppppppppppppppppppppppppppppppppppp161 Mechanisms of Growth and Homeostasis in the Drosophila Wing Ricardo M. Neto-Silva, Brent S. Wells, and Laura A. Johnston ppppppppppppppppppppppppp197 Vertebrate Endoderm Development and Organ Formation

by University of Wisconsin - Madison on 08/09/12. For personal use only. Aaron M. Zorn and James M. Wells ppppppppppppppppppppppppppppppppppppppppppppppppppppppp221 Signaling in Adult Neurogenesis Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org Hoonkyo Suh, Wei Deng, and Fred H. Gage pppppppppppppppppppppppppppppppppppppppppppppp253 Vernalization: Winter and the Timing of Flowering in Plants Dong-Hwan Kim, Mark R. Doyle, Sibum Sung, and Richard M. Amasino pppppppppppp277 Quantitative Time-Lapse Fluorescence Microscopy in Single Cells Dale Muzzey and Alexander van Oudenaarden ppppppppppppppppppppppppppppppppppppppppppp301 Mechanisms Shaping the Membranes of Cellular Organelles Yoko Shibata, Junjie Hu, Michael M. Kozlov, and Tom A. Rapoport pppppppppppppppppppp329 The Biogenesis and Function of PIWI Proteins and piRNAs: Progress and Prospect Travis Thomson and Haifan Lin ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp355

vii AR389-FM ARI 14 September 2009 14:58

Mechanisms of Stem Cell Self-Renewal Shenghui He, Daisuke Nakada, and Sean J. Morrison ppppppppppppppppppppppppppppppppppp377 Collective Cell Migration Pernille Rørth pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp407 Hox Genes and Segmentation of the Hindbrain and Axial Skeleton Tara Alexander, Christof Nolte, and Robb Krumlauf pppppppppppppppppppppppppppppppppppppp431 Gonad Morphogenesis in Vertebrates: Divergent Means to a Convergent End Tony DeFalco and Blanche Capel ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp457 From Mouse Egg to Mouse Embryo: Polarities, Axes, and Tissues Martin H. Johnson pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp483 Conflicting Views on the Membrane Fusion Machinery and the Fusion Pore Jakob B. Sørensen pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp513 Coordination of Lipid Metabolism in Membrane Biogenesis Axel Nohturfft and Shao Chong Zhang pppppppppppppppppppppppppppppppppppppppppppppppppppp539 Navigating ECM Barriers at the Invasive Front: The Cancer Cell–Stroma Interface R. Grant Rowe and Stephen J. Weiss ppppppppppppppppppppppppppppppppppppppppppppppppppppppp567 The Molecular Basis of Organ Formation: Insights from the C. elegans Foregut Susan E. Mango ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp597 Genetic Control of Bone Formation Gerard Karsenty, Henry M. Kronenberg, and Carmine Settembre pppppppppppppppppppppp629 Listeria monocytogenes Membrane Trafficking and Lifestyle: The Exception or the Rule? by University of Wisconsin - Madison on 08/09/12. For personal use only. Javier Pizarro-Cerd´a and Pascale Cossart ppppppppppppppppppppppppppppppppppppppppppppppppp649

Annu. Rev. Cell Dev. Biol. 2009.25:277-299. Downloaded from www.annualreviews.org Asymmetric Cell Divisions and Asymmetric Cell Fates Shahragim Tajbakhsh, Pierre Rocheteau, and Isabelle Le Roux ppppppppppppppppppppppppppp671

Indexes

Cumulative Index of Contributing Authors, Volumes 21–25 ppppppppppppppppppppppppppp701 Cumulative Index of Chapter Titles, Volumes 21–25 pppppppppppppppppppppppppppppppppppp704

Errata

An online log of corrections to Annual Review of Cell and Developmental Biology articles may be found at http://cellbio.annualreviews.org/errata.shtml

viii Contents