Vernalization: Winter and the Timing of Flowering in Plants...cess known as vernalization. A requirement for vernalization is an adaptation to temperate cli-mates that prevents ﬂowering

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

Vernalization: Winter and theTiming of Flowering in PlantsDong-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 andMolecular Biology, University of Texas, Austin, Texas 787122Department 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

First published online as a Review in Advance onAugust 13, 2009

The Annual Review of Cell and DevelopmentalBiology is online at cellbio.annualreviews.org

This article’s doi:10.1146/annurev.cellbio.042308.113411

Copyright c© 2009 by Annual Reviews.All rights reserved

1081-0706/09/1110-0277$20.00

Key Words

Arabidopsis, cereals, histone modifications, epigenetics, Polycomb,Trithorax

AbstractPlants have evolved many systems to sense their environment and tomodify their growth and development accordingly. One example isvernalization, the process by which flowering is promoted as plantssense exposure to the cold temperatures of winter. A requirement forvernalization is an adaptive trait that helps prevent flowering beforewinter and permits flowering in the favorable conditions of spring. InArabidopsis and cereals, vernalization results in the suppression of genesthat repress flowering. We describe recent progress in understandingthe molecular basis of this suppression. In Arabidopsis, vernalization in-volves the recruitment of chromatin-modifying complexes to a clade offlowering repressors that are silenced epigenetically via histone modifi-cations. We also discuss the similarities and differences in vernalizationbetween Arabidopsis and cereals.

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Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . 278Floral Integrators and

Environmental Sensing. . . . . . . . . . 279Vernalization in Arabidopsis . . . . . . . . . 280The Autonomous Pathway . . . . . . . . . 281Vernalization as an Epigenetic

Switch. . . . . . . . . . . . . . . . . . . . . . . . . . 282Genetic Analysis of the

Vernalization Response. . . . . . . . . . 283Vernalization-Mediated Changes

in FLC Chromatin . . . . . . . . . . . . . . 283Polycomb Repression Complexes

and Vernalization-MediatedFLC Repression . . . . . . . . . . . . . . . . . 286

Activation of FLC Is Associatedwith Specific ChromatinModifications . . . . . . . . . . . . . . . . . . . 287

Generational Resetting of FLC . . . . . 289Other Targets of Vernalization

in Arabidopsis . . . . . . . . . . . . . . . . . . . . 289Vernalization in Cereals . . . . . . . . . . . . 290

FUTURE DIRECTIONS . . . . . . . . . . . . 292

INTRODUCTION

Like all organisms, species of flowering plantshave evolved mechanisms to maximize their re-productive success. One component of optimiz-ing this success is the proper timing of the tran-sition from vegetative to reproductive growth.Proper timing is important for many reasons;the following are a few examples. A wide rangeof plants require cross-pollination for success-ful reproduction, and thus flowering must occursynchronously within individuals of the samespecies. For many plants, flowering must alsooccur at a time when pollinators are present.In many parts of the world, flowering must co-incide with weather conditions that will permitflowers, which are relatively delicate structures,to develop properly, followed by conditions thatwill permit seeds to mature properly.

To ensure that flowering occurs within a par-ticular season, plants rely primarily on envi-ronmental cues. One such cue is photoperiod,

the relative change in the length of day andnight that occurs throughout the course of ayear. Plants that respond to lengthening daysand flower in the spring or early summer areknown as long-day (LD) plants. Short-day (SD)plants flower in the late summer or autumn inresponse to shortening days and lengtheningnights (Thomas & Vince-Prue 1997). In tem-perate climates, many winter-annual, biennial,and perennial plants also use cold as an en-vironmental cue to flower at the proper timeof year. Certain plant species need to experi-ence a period of winter cold to overcome ablock to flowering. This occurs through a pro-cess known as vernalization. A requirement forvernalization is an adaptation to temperate cli-mates that prevents flowering prior to winterand permits flowering in the favorable condi-tions of spring. Many vernalization-requiringplants are LD plants. A LD requirement cou-pled to a vernalization requirement further en-sures that precocious flowering does not occurduring the decreasing day lengths of fall andfavors flowering as day length increases duringspring.

The physiology of vernalization in a widerange of species has been intensively studiedfor many decades, and there are several com-prehensive reviews of vernalization physiology(e.g., Bernier et al. 1981, Chouard 1960, Lang1965). The effective temperature and the lengthof cold exposure required to achieve the vernal-ized state vary among different plant species anddifferent varieties within a species. This vari-ation is expected for a response that providesan adaptation to a range of different environ-mental niches. A vernalization response occursonly at temperatures around or above freezing;temperatures below freezing are not effective,which is not surprising because vernalization isan active process that requires changes in geneexpression during cold exposure, as discussedbelow. The focus of this article is to discuss re-cent progress in understanding the molecularbasis of vernalization. The link between classicphysiological studies and more recent molec-ular genetic analyses have been reviewed else-where (Amasino 2004, Sung & Amasino 2005).

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Floral Integrators andEnvironmental Sensing

Flowers result from the expression of regulatorygenes known as floral meristem-identity genes,which specify that certain cells in the growingtips of the plant (the shoot apical meristems) dif-ferentiate into a floral meristem and ultimatelyform a flower (Coen & Meyerowitz 1991).Many of these genes are conserved through-out the plant kingdom including MADS-boxtranscription factors like APETALA1 (AP1),FRUITFUL (FUL), and LEAFY (LFY), whichis a protein unique in the plant lineage (re-viewed in Soltis et al. 2007). Upstream of thefloral meristem-identity genes are a group ofgenes known as floral integrators such as FTand FD (Figure 1); i.e., the floral integrators areinvolved in the regulation of meristem-identitygenes. The floral integrators are so named be-cause their expression is in turn regulated byflowering pathways that sense environmentalcues, such as photoperiod and cold, and/or de-velopmental cues such as the levels of the hor-mone gibberellin; i.e., the regulation of thesegenes serves to integrate a range of environ-mental and developmental cues (Figure 1).

There is not a precise demarcation betweena floral integrator and a meristem-identity gene,and a gene can have properties of both. For ex-ample, LFY is a key meristem-identity gene, butbecause it is activated directly by gibberellin(Figure 1) (Blazquez et al. 1998), it can alsobe considered a floral integrator. In this review,we discuss how the circuitry of photoperiod andvernalization pathways are connected to floralintegrators in the well-studied model Arabidop-sis thaliana.

Studies of the photoperiodic control of flow-ering indicated the existence of a common flo-ral stimulus in plants, sometimes referred to asflorigen (Zeevaart 1976). Physiological studiesrevealed that the floral stimulus was producedin leaves exposed to inductive photoperiods andtraveled to the meristem and caused flower-ing. Recent studies in Arabidopsis and rice havemade a strong case that florigen, or at least acomponent of the floral stimulus, is the floral

Inductivephotoperiod

pathway Autonomous

pathway Vernalization

pathway

CO

FT SOC1

SEP3 AP1FUL

FD

FLC

Floral meristem identity genes

Floral integrator genes

LFY

FLC cladeGibberellins

Flowering

Figure 1Outline of flowering pathways in Arabidopsis. Components and geneticinteractions that promote flowering are shown in blue; those that repressflowering are shown in red. Flowering is repressed in Arabidopsis byFLOWERING LOCUS C (FLC) and FLC relatives designated the FLCclade. FLC represses the expression of the floral integrator genes FD, FT, andSUPPRESSOR OF CONSTANS 1 (SOC1). These floral integrator genes arepromoters of flowering. One of the floral integrator genes, FT, is induced bythe photoperiod pathway through CONSTANS (CO); FT protein is a mobileflowering signal that partners with FD protein in the meristem to activateSOC1 and the floral meristem-identity genes SEPALATA (SEP), FRUITFUL(FUL), and APETALA 1 (AP1) [for review see Turck et al. (2008)]. Thus, FLCand the photoperiod pathway act antagonistically. The floral meristem-identitygenes specify the formation of floral meristems that will develop into flowers.The basal level of FLC expression is set in part by the level of repression thatresults from the action of autonomous pathway genes. In response to exposureto the prolonged cold of winter, the vernalization pathway provides furtherFLC repression by chromatin remodeling. The gibberellin class of planthormones promotes flowering by activating SOC1 and the floralmeristem-identity gene LEAFY (LFY).

integrator FT. The FT gene is expressed inleaves, and the protein travels to the meris-tem where it interacts with another integrator,FD, to initiate the floral transition (reviewed inTurck et al. 2008, Zeevaart 2008). FT-like genesare ubiquitous in plants and have been found toregulate flowering in a variety of species includ-ing wheat and poplar (reviewed in Turck et al.2008). [Note: Several genes discussed in this re-view such as FT, FD, FVE, FCA, FY, and FPAwere given two- or three-letter designations butnot longer names (Koornneef et al. 1991).]

www.annualreviews.org • Vernalization 279

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There is also a significant level of conser-vation in the molecular mechanisms plants useto sense photoperiod. Arabidopsis is a facultativeLD plant; i.e., it flowers most rapidly in LD, butit also flowers eventually in SD. In Arabidop-sis, the perception of day length is a functionof coincidence between light and expression ofthe circadian-regulated gene CONSTANS (CO)(reviewed in Turck et al. 2008). In LD-grownArabidopsis plants, CO transcription extends intothe daylight phase. Light stabilizes CO protein,which in turn leads to increased expression ofFT (Figure 1) (Corbesier et al. 2007, Hepworthet al. 2002, Wenkel et al. 2006). The photope-riodic induction system, in which CO levels areaffected by day length and translated into theregulation of FT (or FT homologs such as Hd3ain rice or VRN3 in cereals), appears to be wellconserved among flowering plants (reviewed inTurck et al. 2008).

Much of what we know about the molecularmechanism of the vernalization response comesfrom studies of Arabidopsis and temperate cere-als. These studies of vernalization indicate thatthis process is not conserved at a biochemicallevel like the photoperiodic flowering pathway.However, as discussed in detail below, the flo-ral integrator FT/VRN3 is one of the targetsof the vernalization pathway in both Arabidopsisand temperate cereals, and vernalization alle-viates the repression of FT/VRN3 expression.Thus, the control of FT/VRN3 expression maybe conserved as an integration point of the pho-toperiod and vernalization pathways.

In Arabidopsis, there are additional floralintegrators including the MADS-box genesSUPPRESSOR OF OVEREXPRESSION OFCONSTANS1 (SOC1), AGAMOUS-LIKE 19(AGL19), and AGL24 (Lee et al. 2000, Liu et al.2008, Michaels et al. 2003, Samach et al. 2000,Schonrock et al. 2006). FT and FD partner toactivate SOC1 in the meristem, and SOC1 ex-pression is also controlled by vernalization, asdiscussed below. Thus, SOC1 regulation inte-grates inputs from multiple flowering pathways(Figure 1). SOC1 also functions with FUL tomaintain the meristem in a floral state (i.e., asan inflorescence meristem) (Melzer et al. 2008),

and therefore, SOC1 can also be considereda meristem-identity gene. SOC1 and AGL24positively regulate each other (Liu et al. 2008,Michaels et al. 2005), interact at the proteinlevel, and are both required for proper activa-tion of LFY (Lee et al. 2008). Indeed, there isan intricate network of positive feedback loopsamong floral integrators and meristem-identitygenes to ensure that once flowering is initiatedin Arabidopsis, it is maintained in the absenceof environmental and developmental cues. It isbeyond the scope of this review to discuss thedetails of these loops. Hereafter we focus onvernalization and note the connections to theintegrators.

Vernalization in Arabidopsis

The identification of genes involved in the ver-nalization response in Arabidopsis began withthe study of natural variation in flowering foundamong different isolates collected from a rangeof locations worldwide. Most commonly usedlab strains of Arabidopsis do not require vernal-ization to flower rapidly; however, many isolatesflower very late unless first vernalized (Burnet al. 1993, Clarke & Dean 1994, Lee et al.1993, Napp-Zinn 1987). The rapid-floweringtypes are sometimes referred to as summer an-nuals, which indicates that their life cycle islikely to be completed in one growing season.Plants with a strong vernalization requirementare often called winter annuals, because theymay not complete their life cycle until the sec-ond growing season after an intervening winter.However, the actual life cycle in nature is clearlyinfluenced by environment as well as genotype;in one environment a genotype might take twoseasons to flower, whereas in a different envi-ronment the same genotype might flower in asingle season (e.g., Wilczek et al. 2009).

In Arabidopsis, studies of natural variationdemonstrated that the vernalization require-ment is conferred by two dominant genes,FRIGIDA (FRI ) and FLOWERING LOCUS C(FLC ) (Koornneef et al. 1994, Lee et al. 1994).FRI encodes a nuclear protein found only inplants ( Johanson et al. 2000) that may interact

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with the mRNA cap-binding protein to reg-ulate the level of FLC mRNA (Geraldo et al.2009). FLC encodes a MADS-box DNA bind-ing protein that functions as a repressor of flow-ering (Michaels & Amasino 1999, Sheldon et al.1999). Genetic studies have shown that in thecontext of vernalization FRI acts solely to up-regulate FLC (Michaels & Amasino 2001).

The level of FLC expression is the primarydeterminant of the vernalization requirementin Arabidopsis. FLC represses flowering, in part,by repressing the expression of floral integra-tors such as FT, FD, and SOC1 (Helliwell et al.2006, Hepworth et al. 2002, Lee et al. 2000,Samach et al. 2000, Searle et al. 2006). FLCbinds to promoter regions of SOC1 and FD andto the first intron of FT (Searle et al. 2006). Thisbinding likely attenuates the ability of the pho-toperiod pathway to activate these integrators.Indeed, the expression of integrators from con-stitutive promoters bypasses the repressive ef-fects of FLC and the vernalization requirement(Lee et al. 2000, Michaels et al. 2005).

There are many examples of MADS-boxproteins acting in multimeric complexes withother MADS-box proteins (de Folter et al.2005, Honma & Goto 2001, Pelaz et al.2000). Recent studies show that FLC inter-acts directly with another MADS-box pro-tein SHORT VEGETATIVE PHASE (SVP)(Fujiwara et al. 2008, Li et al. 2008). Geneticstudies indicate that this interaction is biologi-cally relevant: loss of SVP provides partial sup-pression of the FLC-mediated delay in flow-ering (Li et al. 2008). However, the loss ofSVP cannot fully suppress the FLC-mediateddelay of flowering, which indicates that FLCmay redundantly interact with other MADS-box proteins. Although SVP and other interac-tion partners may be critical for FLC-mediatedrepression of flowering, SVP levels or activitydo not appear to be involved in the regulation offlowering like FLC. For example, SVP mRNAlevels are not affected by vernalization, whereasvernalization results in the stable repression ofFLC (Bastow et al. 2004, Michaels & Amasino1999, Sheldon et al. 1999, Sung & Amasino2004). Moreover, overexpression of FLC alone

is sufficient to cause extremely late flowering(Michaels & Amasino 2001), which indicatesthat the levels of binding partners are not lim-iting in vivo.

The circadian clock has an influence on SVPlevels; SVP protein accumulates to higher levelsin a double mutant of two clock components,LATE ELONGATED HYPOCOTYL (LHY )and CIRCADIAN CLOCK ASSOCIATED 1(CCA1), than in wild type (Fujiwara et al. 2008).Thus, there may be cross talk in the circuitrybetween the photoperiod pathway and FLC-dependent repression in Arabidopsis; however,no effect of different photoperiods on SVP lev-els has been reported.

The Autonomous Pathway

Most commonly used types of Arabidopsisare null mutants for FRI (e.g., Columbia,Landsberg, Wassilewskija) and thus do not re-quire vernalization for rapid flowering. Histori-cally, a group of late-flowering mutants derivedfrom such rapid-flowering accessions have beenreferred to as the autonomous pathway (AP) offloral promotion (Henderson & Dean 2004).AP mutants are characterized by delayed flow-ering in both LD and SD, which is in contrastto photoperiod-pathway mutants that show de-layed flowering in inductive LD photoperiodsonly. The flowering behavior of AP mutants issimilar to FRI-containing winter-annual acces-sions (i.e., both have strong FLC expression,are late flowering, and require vernalization forrapid flowering) (Michaels & Amasino 2001).To date, eight AP genes have been identified:LUMINIDEPENDENS (LD), FCA, FY, FPA,FLOWERING LOCUS D (FLD), FVE, FLK,and REF6 (Noh et al. 2004, Simpson 2004).FCA, FPA, FY, and FLK encode proteins that arepredicted to be involved in RNA metabolism(Lim et al. 2004, Macknight et al. 1997, Schom-burg et al. 2001, Simpson et al. 2003); how-ever, there is no evidence to suggest thatthese components directly interact with FLCmRNA. FVE, FLD, and REF6 have domainscommon to chromatin-modifying components.FVE (also known as MSI4) is a member of the


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MSI1-like protein family (MSI1–MSI5) in Ara-bidopsis (Ausin et al. 2004, Kim et al. 2004);MSI proteins are found in several chromatin-modifying complexes in eukaryotes (Henniget al. 2005). FLD and REF6 are predicted toencode two different types of histone demethy-lases (He et al. 2003, Jiang et al. 2007, Noh et al.2004).

The repression of flowering by AP genesacts primarily through FLC because flc nullmutants completely suppress the delayed-flowering phenotypes of AP mutants (Michaels& Amasino 2001). Recent studies demonstratethat many AP genes are also involved in the reg-ulation of other genes that are not involved inflowering (Baurle et al. 2007, Veley & Michaels2008). Indeed, most of the AP genes do not ap-pear to be specific to flowering, nor do theycomprise a pathway in the typical sense. In-stead, these genes are involved in a range of re-pressive mechanisms that act on FLC and othergenes. For example, the RNA-binding proteinsFPA and FCA are broadly required for RNA-mediated silencing, which raises the possibil-ity that their role in FLC repression involvesRNA-mediated silencing (Baurle et al. 2007).In addition, all tested AP mutants showed dere-pression of certain transposons (Baurle et al.2007, Veley & Michaels 2008), and double mu-tant analysis among the AP mutants revealeda wide range of nonflowering phenotypes(Veley & Michaels 2008). It is interesting tonote that genetic analysis showed FCA requiresFVE for the repression of certain transposons(Baurle & Dean 2008), whereas FLD is neces-sary for FCA to repress FLC (Liu et al. 2007).Thus, there appears to be a range of mecha-nisms and combinatorial interactions by whichAP genes affect gene expression.

Recent work provides additional support forthe involvement of RNA-mediated silencing inFLC repression. Double mutants of dicer-like 1and dicer-like 3 (dcl1/3) have phenotypes sim-ilar to that of AP mutants: elevated levels ofFLC expression and delayed flowering that issuppressed by both flc mutations and vernaliza-tion (Schmitz et al. 2007). Because late flower-ing in dcl1/3 and AP mutants is overcome by

vernalization, FLC repression by these genesoccurs via a vernalization-independent mech-anism. Perhaps RNA-mediated silencing playsa role in setting the prevernalization basal levelof FLC expression.

Notably, there is an intermediate-size, non-coding RNA that arises from beyond 3′UTR ofFLC (Swiezewski et al. 2007). Mutations in theregion of this RNA have a small effect on flow-ering relative to dcl1/3 or other AP mutants, andthe relationship of this RNA to other floweringpathways is not known.

Vernalization as an Epigenetic Switch

Chouard (1960) provided a useful definition ofvernalization as “the acquisition or accelerationof the ability to flower by a chilling treatment.”As defined, vernalization does not necessarilyinduce flowering. Rather, prolonged chillingprovides competence to flower. As noted above,in many vernalization-requiring plant species,other endogenous and/or environmental con-ditions, such as inductive photoperiods, are alsorequired for flowering. A classic experiment il-lustrates that this competence to flower can bestable through mitotic cell divisions. Lang andMelchers used a biennial variety of Hyoscyamusniger (henbane) that requires both vernaliza-tion and inductive photoperiods (LD) to initi-ate flowering (reviewed by Lang 1965). Whenplants are first exposed to cold (vernalized)and then kept in a noninductive photoperiod(SD) in a normal, warm growth temperature,the plants grow vegetatively but do not flower.However, when the previously vernalized plantsare moved to inductive photoperiods, even aftermany months in noninductive conditions, flow-ering occurs. Thus, the vernalization-mediatedchange in competence is stable through a largenumber of mitotic divisions in the apical meris-tem. Mitotic stability in the absence of the in-ducing signal (cold in the case of vernalization)is arguably an epigenetic switch (e.g., Amasino2004, Dennis & Peacock 2007, Wu & Morris2001). The molecular basis of competence inArabidopsis is the mitotically stable repression ofFLC and some FLC relatives discussed below.

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It is worth noting that not all plant speciesexhibit behavior that fits the stable acquisitionof competence model described above; for ex-ample, there are species that need to flower dur-ing cold exposure presumably because compe-tence is not stably maintained through mitoticcell divisions after cold exposure ceases (Bernieret al. 1981). Indeed, there are mutants inArabidopsis in which FLC repression occurs dur-ing cold exposure but it is not stably maintainedupon a return to warm; not surprisingly, thesemutants affect chromatin-modifying proteins(Gendall et al. 2001, Levy et al. 2002, Mylneet al. 2006, Sung et al. 2006a).

Genetic Analysis of theVernalization Response

As discussed above, winter-annual accessionsof Arabidopsis exhibit a delayed-flowering phe-notype if they are not vernalized, and mostof the natural variation in the vernalizationrequirement is due to allelic variation atFRI and FLC. The molecular mechanism bywhich FLC is epigenetically repressed by ver-nalization in Arabidopsis has been addressedby molecular genetic analyses. Using winter-annual accessions as a parental line, one canscreen for mutants that can no longer bevernalized; such vernalization-insensitive mu-tants have lesions in genes that participatein the vernalization pathway. To date, thesescreens have revealed five genes: VERNALIZA-TION 1 (VRN1), VRN2, VERNALIZATIONINSENSITIVE 3 (VIN3), VRN5/ VIN3-LIKE1(VIL1), and atPRMT5 (Bastow et al. 2004,Gendall et al. 2001, Greb et al. 2007, Schmitzet al. 2008, Sung & Amasino 2004, Sung et al.2006b). As expected, given the nature of the ge-netic screen, all mutants fail to stably repressFLC by vernalization. All of these genes areconstitutively expressed except for VIN3. VIN3is expressed specifically during a vernalizingcold treatment, and expression is completelyabolished when plants are returned to a warmtemperature (Sung & Amasino 2004). The coldinduction and transient nature of VIN3 expres-sion indicates that VIN3 may be a part of the

trigger to set in motion the molecular eventsthat stably repress FLC during vernalization.

Vernalization-Mediated Changesin FLC Chromatin

The stable nature of the vernalized state isconsistent with a role for chromatin modifi-cation of target genes. In Arabidopsis, FLC re-pression is the molecular basis of vernalization-induced competence. Thus, FLC is a likelytarget for chromatin modification. The firstmolecular evidence for chromatin modificationin Arabidopsis vernalization came from the iden-tification of VRN2 and VIN3. VRN2 encodesa homolog of Suppressor of zeste (Su(z)12),a component of Polycomb repression com-plex 2 (PRC2) that was first identified in an-imals (Gendall et al. 2001). VIN3 encodes aPHD (plant homeodomain) protein (Sung &Amasino 2004); the PHD domain is commonlyfound in a wide range of complexes that areinvolved in chromatin-level regulation (Mellor2006).

Vernalization results in an increase in tworepressive histone modifications at FLC chro-matin (Figure 2): histone H3 Lys 9 (H3K9)and histone H3 Lys 27 (H3K27) methylation(Bastow et al. 2004, Sung & Amasino 2004).In vin3 and vrn2 mutants, H3K9 and H3K27methylation are not enriched at FLC follow-ing a sufficient cold treatment, and FLC ex-pression is not repressed (Bastow et al. 2004,Sung & Amasino 2004). The loss of VRN1,which encodes a DNA-binding protein con-taining two plant-specific B3 domains, showsdistinctive chromatin modifications from thoseof vin3 and vrn2 mutants. Whereas methylationof both H3K9 and H3K27 are affected in vin3and vrn2 mutants, only H3K9 methylation failsto occur in vrn1 during and after vernalization(Bastow et al. 2004, Sung & Amasino 2004),which suggests the two methylation events canoccur independently and that VRN1 is involvedspecifically in H3K9 methylation (Figure 2).

VIL1/VRN5 was identified independentlyby a yeast two-hybrid screen for VIN3-interacting proteins (Sung et al. 2006b) and by a


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EFS

VIN3

H

2A

H2

AZ

H2

AZ

Winter cold(vernalization)

VIL1/VRN5

FLC repression initiates

FLC active

H2B

H2B

H3

H3

K9

K9

H3K27Me and H3K9Medensity increases

K9

M

PRC2core

FIE1

ARP4

ARP6

PRC2core

Ubiquitinase

Mitotically stable repression

H2

AZ

PRC2core

H2

A

H

2A

H

2A

H

2A

H

2A

M

M

H3K4triMe

H3K26triMe

H2Bub1

H3K9triMe

H3K27triMe

Methyl groupM

K9

Winter cold(vernalization)

PRC2core

PRC2core

VIL1/VRN5 VIL1/VRN5

H

2A

VIL1/VRN5

H2B

H3

PRC2core

H2

A

H

2A

H

2A

H

2A

H

2A

M

VRN1 VRN1 VRN1VRN1

K9 K9

H

2A

H3

H2

A

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genetic screen for mutants with a reduced ver-nalization response (Greb et al. 2007). VIN3and VIL1/VRN5 belong to a small family of pro-teins that contain a PHD domain, a fibronectinIII (FNIII) domain, and a conserved C-terminalregion. Whereas the PHD and FNIII do-mains are found throughout eukaryotes, theC-terminal domain is unique in plants andmediates protein-protein interactions betweenVIN3 and VIL1/VRN5 (Greb et al. 2007, Sunget al. 2006b). Similar to VIN3, VIL1/VRN5 isalso required for vernalization-mediated his-tone modifications at FLC chromatin includingmethylation of both H3K9 and H3K27 (Grebet al. 2007, Sung et al. 2006b). Recent reportsclaim that yet another member of the fam-ily, VIL2/VEL1, is part of a VIL1-containingcomplex that is physically located at FLC aftervernalization-mediated repression (De Luciaet al. 2008). However, this repression is not im-paired when VIL2/VEL1 levels are decreasedeither by mutation (Kim, Schmitz, Amasino,and Sung, unpublished work) or by RNA in-terference (Greb et al. 2007), which indicatespossible functional redundancy of VIL2/VEL1with other members of the VIN3/VEL proteinfamily.

Recent studies demonstrated that specificPHD domains associate with specific histonemodifications, which include methylated hi-stone H3K4 (Mellor 2006), nonmethylated

histone H3 (Lan et al. 2007), methylatedhistone H3K9 (Karagianni et al. 2008), andmethylated histone H3K36 (Shi et al. 2007).The diverse binding activities of PHD domainsmake them excellent candidates for readers ofhistone modifications. As expected, amino acidsequence variation within PHD domains is re-sponsible for variations in their specificity (Liet al. 2007). In the VIN3/VEL family there arevariations in amino acid sequences within thePHD domains, and thus, if VIN3/VEL fam-ily members indeed bind modified histone tails,there may be variation within the family as towhich modifications are bound. One possibil-ity is that VIN3 binds to modifications associ-ated with active FLC chromatin and in doingso initiates the transition to a repressed state.After a certain degree of conversion to the re-pressed state is achieved and modifications asso-ciated with active FLC chromatin are replacedby repressive marks, VIN3 is no longer neces-sary, as in spring, when other positive feedbackloops of histone modification maintain repres-sion (Figure 2). However, the specificity of var-ious members of the VIN3/VEL protein familyfor modified histones and the significance of anyvariability in modifying FLC chromatin remainto be investigated.

The mechanism of how FLC is targeted forrepression remains a major unanswered ques-tion. The induction of VIN3 is a necessary first

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 2Vernalization-mediated changes in FLC chromatin. (a) Prior to cold exposure, FLC is actively expressed. Thecomplexes that maintain this active chromatin conformation include the PAF complex, which methylateshistone 3 tails at lysine 4 and 36 (H3K4triMe and H3K36triMe), a SWR1-like complex, which deposits ahistone 2A variant in the nucleosomes of FLC chromatin, and H2B ubiquitinases like HUB1 and HUB2 thatubiquitinate histone 2B tails (H2Bub1). Although FLC is in an active state, there are repressive complexespresent 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 isinduced, VIN3 and VIL1/VRN5 associate with the Polycomb complex, the density of repressive chromatinmodifications such as lysine 27 methylation of histone 3 increases, and repressors such as LIKEHETEROCHROMATIN 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, amitotically stable state of repression that no longer requires VIN3 is achieved. This mitotically stable state islikely to involve positive feedback loops in which the repressive chromatin modifications serve to recruit thechromatin-modifying complexes including VRN1 to maintain a repressive state. As the FLC locus passes tothe 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 theactivities of chromatin-modifying complexes atthe FLC locus is not at all understood. Evenif the PHD domain of VIN3 binds to specifichistone modifications characteristic of FLC inthe pre-vernalized state (see below), such mod-ifications are not likely to be unique to FLCchromatin. Clearly, there is much to learn abouttargeting specificity.

In addition to vernalization-mediatedmethylation at H3K9 and H3K27 at FLCchromatin, histone arginine methylation alsoplays a role in maintaining stable repressionof FLC (Schmitz et al. 2008). Mutations ina Type II protein arginine methyltransferasegene atPRMT5, reduced the vernalizationresponse. atPRMT5 is required for the sym-metric methylation of Arg 3 of histone H4(sMeH4R3), and the level of this methylationis increased at FLC chromatin by vernalization.Furthermore, sMeH4R3 appears to be requiredfor the vernalization-mediated enrichment ofboth H3K9 and H3K27 methylation (Schmitzet al. 2008).

Polycomb Repression Complexesand Vernalization-MediatedFLC Repression

As discussed above, the identification of anArabidopsis homolog of Su(z)12, VRN2, in avernalization-insensitive mutant screen sug-gested the involvement of a PRC2-like com-plex in FLC repression (Gendall et al. 2001). Inanimals, there are two distinct PRC complexes,PRC2 and PRC1 (reviewed in Schuettengruberet al. 2007). Polycomb group (PcG) proteinswere first identified in Drosophila as essen-tial components for maintaining HOMEOTIC(HOX) genes in a repressed state. The PRC2complex is involved in the initial di- ortrimethylation of H3K27 at target chromatin.Then, in many systems, methylated H3K27 isbound by the PRC1 complex, which acts tomaintain chromatin in a repressed state andto maintain H3K27 methylation through mi-totic cell divisions (Cao et al. 2002, Mulleret al. 2002). Core components of PRC2 are well

conserved from plants to animals (Hsieh et al.2003), but, as discussed below, PRC1 compo-nents are not.

In Drosophila, core components of PRC2consist of Enhancer of zeste [E(z)], ExtraSex Combs (ESC), and Suppressor of zesteSu(z)12 (Cao et al. 2002). Arabidopsis and otherplants contain multiple copies of many PRC2components (Hsieh et al. 2003, Wood et al.2006). For example, in Arabidopsis, there arethree homologs of the histone methyltrans-ferase E(z). These include CURLY LEAF(CLF), SWINGER (SWN), and MEDEA(MEA) (Hsieh et al. 2003). CLF was first iden-tified as a repressor of the floral homeoticgene AGAMOUS (AG) (Goodrich et al. 1997),whereas MEA was identified in a screen for mu-tants that have altered endosperm development(Grossniklaus et al. 1998). SWN is partiallyredundant with CLF (Chanvivattana et al.2004). None of the Arabidopsis E(z) homologswere identified in screens for vernalization-insensitive mutants, which is likely due to thefunctional redundancy among these proteinswith regard to FLC repression. In fact, VRN2 isthe only PRC2 component identified as beinginvolved in vernalization from mutant screens.

Biochemical approaches revealed thatArabidopsis PRC2 complexes include VIN3during cold exposure (De Lucia et al. 2008,Wood et al. 2006). PRC2 complexes inArabidopsis can also include VIL1/VRN5;purification of proteins associated with aVIL1/VRN5-TAP fusion revealed componentsof PRC2, which include VRN2, SWN, MSI1,and FERTILIZATION INDEPENDENTENDOSPERM (FIE; an Arabidopsis ESChomolog) (De Lucia et al. 2008). The theme ofPHD domain-containing proteins associatingwith PRC2 may be widespread in eukaryotes;PHD–domain proteins also associate withPRC2 in Drosophila and humans (Cao et al.2008a, Nekrasov et al. 2007, Sarma et al. 2008).

Analyses of the occupancy of PRC2 com-ponents at FLC chromatin showed that VRN2is already present at FLC chromatin prior tovernalization (De Lucia et al. 2008). In cold,VIN3 is induced and VIN3 protein is present at

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a region encoding the first intron of FLC. As-sociation of VIL1/VRN5 is VIN3-dependent,and thus VIN3 may serve as a guiding factorfor the recruitment of VIL1/VRN5 to FLC(De Lucia et al. 2008). Whereas the enrich-ment of VIL1/VRN5 is restricted to the first in-tron of FLC in cold temperatures, VIL1/VRN5spreads throughout the FLC chromatin whenplants return to warm temperatures. Thisspreading may require warm temperatures, orwarm temperatures may simply increase therate of these chromatin changes. Presumably,the spreading of VIL1/VRN5 across FLC chro-matin maintains the repressed state of FLC(Figure 2) (De Lucia et al. 2008).

Although the presence of PRC2 com-ponents in plants is well established, Ara-bidopsis lacks components with an overallsimilarity to PRC1 components (Hsieh et al.2003, Schubert et al. 2005). Instead, LIKE-HETEROCHROMATIN PROTEIN 1(LHP1) likely binds to and stably maintains thePRC2-mediated modified histones at FLC fol-lowing vernalization (Mylne et al. 2006, Sunget al. 2006a). In lhp1 mutants, the active histonemark, H3K4triMe, is only transiently reducedwhen plants are kept in cold (Sung et al. 2006a).Interestingly, lhp1 mutants were not found inscreens for vernalization-insensitive mutantsbecause the loss of LHP1 also results in thederepression of flowering promoters such asFT, and thus lhp1 mutants are early floweringwithout vernalization (Kotake et al. 2003, Sunget al. 2006a). LHP1 is likely to play a broadrole in gene repression in plants and may bepart of a protein complex that serves a similarrole to that of PRC1 in animals.

Activation of FLC Is Associated withSpecific Chromatin Modifications

As discussed above, FLC repression is associatedwith dynamic changes in histone composition,which are initiated during prolonged cold ex-posure. Conversely, the high levels of FLC thatcreate a vernalization requirement are associ-ated with active histone marks. Mutant screensfor the loss of the vernalization requirement

(i.e., plants that flower early without cold) inwinter-annual Arabidopsis have revealed manyof the components required for FLC activation.

H3K4triMe is associated with active chro-matin in most eukaryotes (Schneider et al.2004). A specific Saccharomyces cerevisiae HM-Tase, Set1, is responsible for mono- totrimethylation of H3K4, and mutations in set1cause diverse phenotypic defects such as slowgrowth and rDNA derepression (Briggs et al.2001, Fingerman et al. 2005). Yeast Set1 isan essential component of a complex calledCOMPASS (complex proteins associated withSet1) (Krogan et al. 2003). Another complex,the RNA polymerase II-Associated Factor 1(PAF1)-containing complex, is necessary forH3K4triMe enrichment at target chromatin.These two complexes, COMPASS and PAF1,physically associate to coordinate the transcrip-tion of target genes (Krogan et al. 2003).

Screens for rapidly flowering mutants iden-tified two Arabidopsis homologs of the yeastPAF1 complex components PAF1 and CTR9:EARLY FLOWERING 7 (ELF7) and ELF8,respectively (He & Amasino 2005). ELF7 andELF8 are required for high FLC expression andfor H3K4triMe enrichment at FLC chromatin(He et al. 2004). Other Arabidopsis genes thatencode relatives of PAF1 and COMPASScomplex components are VERNALIZATIONINDEPENDENCE 3 (VIP3; Arabidopsis ho-molog of human hSki8), VIP4 (Arabidopsishomolog of yeast Leo1), VIP5 (Arabidopsishomolog of yeast Rtf1), ARABIDOPSISTRITHORAX-LIKE 1 (ATX1), and ATX2 (seebelow for roles of ATX1 and ATX2) (Alvarez-Venegas et al. 2003, He et al. 2004, Oh et al.2004, Pien et al. 2008, Saleh et al. 2008, Zhanget al. 2003, Zhang & van Nocker 2002). Mu-tations in these genes typically cause reducedH3K4triMe deposition at FLC chromatin andearly-flowering phenotypes similar to elf7 andelf8 (Alvarez-Venegas et al. 2003, He et al.2004, Pien et al. 2008, Saleh et al. 2008, Zhang& van Nocker 2002). Methylation of H3K4at FLC chromatin appears to be mediatedby ATX1 and ATX2, which are homologousto Drosophila Trithorax (Trx). Trx is a H3K4


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methyltransferase that belongs to the Trithoraxgroup of proteins (TrxG), which are activatorsof Drosophila homeotic genes (a role oppositeto that of PcG proteins) (Pien et al. 2008, Salehet al. 2008). ATX1 mediates trimethylation onH3K4, whereas ATX2 dimethylates H3K4 invitro (Saleh et al. 2008). The involvement ofTrxG- and PcG-like genes in the regulationof FLC indicates that FLC is controlled byan evolutionarily conserved mechanism thatinvolves a dynamic balance between PcG andTrxG. A recent study reveals that this dynamicbalance occurs throughout the genome inplants (Oh et al. 2008).

In Drosophila, TrxG activator proteins aswell as components of PRC2 repressive com-plexes are constitutively bound to HOX targetchromatin; i.e., both repressive and activatingmodifiers can reside at the same locus (Papp& Muller 2006). As discussed above, PRC2 isdetectable at FLC chromatin prior to vernal-ization (De Lucia et al. 2008). Furthermore,H3K27triMe is present at FLC chromatin priorto vernalization when FLC is actively tran-scribed, but the level of H3K27triMe is muchlower than when FLC is repressed (Pien et al.2008). This indicates that a threshold level ofH3K27triMe enrichment is necessary to estab-lish repressed FLC chromatin.

Another Trx-like SET domain protein isalso required for proper FLC activation. EARLYFLOWERING IN SHORT DAYS (EFS) en-codes a SET domain protein, which medi-ates di- and trimethylation of histone H3 Lys36 (H3K36) on target chromatin (Xu et al.2008). Mutants with EFS lesions exhibit re-duced FLC expression and early flowering sim-ilar to mutations in PAF1-COMPASS compo-nents (Kim et al. 2005, Xu et al. 2008, Zhao et al.2005). In winter-annual strains of Arabidopsis,EFS may also be required for the enrichmentof H3K4triMe at FLC chromatin (Kim et al.2005). Thus, the three Arabidopsis HMTases,ATX1, ATX2, and EFS, appear to act coordi-nately to mediate the transcriptional activationof FLC.

Monoubiquitination at lysine 123 of His-tone H2B (H2Bub1), like H3K4, is a histone

modification associated with active transcrip-tion. In yeast, a complex that contains RAD6(with E2-ubiquitin-conjugating activity) andBRE1 (with E3-ubiquitin ligase activity) actsto monoubiquitinate histone H2B at specifictarget chromatin (Tenney & Shilatifard 2005).H2Bub1 is an important prerequisite for theproper enrichment of H3K4triMe and fortranscriptional activity of target genes (Woodet al. 2003). In Arabidopsis, H2B monoubiq-uitination of FLC chromatin is also requiredfor proper activation of FLC. Investigatorsidentified two Arabidopsis BRE1 homologs, HI-STONE MONOUBIQUITINATION 1 (HUB1)and HISTONE MONOUBIQUITINATION 2(HUB2) (Cao et al. 2008b, Gu et al. 2009),and found that mutations in either hub1 orhub2 result in early flowering and the lossof H3K4triMe enrichment at the promoterregion of FLC. Arabidopsis has three RAD6homologs: UBIQUITIN-CONJUGATINGENZYME 1, 2, and 3 (AtUBC1, AtUBC2,and AtUBC3) (Cao et al. 2008b, Gu et al.2009). AtUBC1 and AtUBC2 are involved inflowering and act redundantly for the enrich-ment of H2Bub1 at FLC chromatin, whereasAtUBC3 does not play a role in FLC activation(Xu et al. 2009). Although the enrichment ofH2Bub1 is required for the proper activationof FLC, over-enrichment of H2Bub1 results inthe failure of FLC activation; a mutation in aH2B deubiquitinase, UBIQUITIN-SPECIFICPROTEASE26 (UBP26 ), results in a rapid-flowering phenotype due to the loss of FLCexpression (Schmitz et al. 2009). Interestingly,ubp26 mutants have a reduced level of H3K36trimethylation at FLC chromatin withoutaltered enrichment of H3K4triMe (Schmitzet al. 2009). Thus, altered levels of H2Bub1 atFLC chromatin result in a phenotype similar toatx1, atx2, and efs mutants, which indicates thepresence of an intricate regulatory loop amonghistone modifiers in FLC activation.

In addition to modifications of histone pro-teins, the exchange of certain histone variantsalso plays a role in achieving proper levels ofFLC expression. The replacement of H2A withits variant H2AZ is mediated by the SWR1

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complex in yeast (Kobor et al. 2004, Mizuguchiet al. 2004). H2AZ deposition by the SWR1complex is required for transcriptional regu-lation, maintenance of heterochromatic barri-ers, and genome stability in yeast (Kamakaka &Biggins 2005). Arabidopsis homologs of SWR1components are also involved in the activa-tion of FLC (Reyes 2006). PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1(PIE1) is the Arabidopsis gene most homolo-gous to SWR1; PIE1 is required for FLC ac-tivation and mediates H2AZ deposition ontotarget chromatin including FLC (Deal et al.2007, Noh & Amasino 2003). Other homologsof SWR1 complex components in Arabidop-sis include ACTIN-RELATED PROTEIN 4(AtARP4) and SUPPRESSOR OF FRIGIDA 3(SUF3) (also known as AtARP6 and ESD1)(Choi et al. 2005, Deal et al. 2005, Kandasamyet al. 2005, Martin-Trillo et al. 2006). Muta-tions in any of these homologs cause an early-flowering phenotype and a reduction in FLCexpression. Thus, FLC activation, and the re-sulting creation of a vernalization requirementin Arabidopsis, requires deposition of H2AZvia PIE1, AtARP4, and AtARP6/ESD1/SUF3(Choi et al. 2005; Deal et al. 2005, 2007; Noh& Amasino 2003).

Generational Resetting of FLC

Although FLC repression in Arabidopsis is stablymaintained throughout mitotic cell divisionsfollowing vernalization, FLC is reactivated atsome point as the locus is passed to the nextgeneration. This reactivation re-establishes therequirement for vernalization (Figure 2). Theresetting of FLC expression in each generationdistinguishes this type of epigenetic repressionfrom heritable (i.e., not reset) epigenetic silenc-ing that involves DNA methylation and smallinterfering RNAs (siRNAs) (e.g., Henderson &Jacobsen 2007). Changes in DNA methylationdo not appear to be involved in the resetting ofFLC expression (Finnegan et al. 2005).

Studies of expression of a vernalized FLClocus can determine the time by which FLCresetting must have occurred (Sheldon et al.

2008, Choi et al. 2009). However, it is impor-tant to note that a locus can be reset prior toactual expression; for example, key chromatinchanges can occur before the transcription fac-tors necessary for expression are present. Ex-pression studies reveal that FLC is transientlyexpressed during male gametogenesis (Sheldonet al. 2008), but there is no expression during fe-male gametogenesis (Sheldon et al. 2008, Choiet al. 2009). Whether this transient expressionduring male gametogenesis represents resettingor a transient state in which there is some ex-pression of repressed gene remains to be deter-mined. In the next generation, FLC is clearlyre-expressed in the mid- to late stages of em-bryo development (i.e., in the early stages ofthe next generation) (Sheldon et al. 2008, Choiet al. 2009). Interestingly, the earliest stages ofFLC expression during early embryogenesis aredependent on the presence of PIE1 but not onthe presence of FRI; however, later maintenanceof FLC expression requires both FRI and PIE1(Choi et al. 2009). Perhaps reduced levels ofsome of the key players in FLC repression inpollen, including VRN1 and LHP1, may con-tribute to the reactivation of FLC during game-togenesis (Mylne et al. 2006). Indeed, a majorityof FLC regulators, which includes FLC activa-tors as well as repressors, are poorly expressedin pollen (Choi et al. 2009). Perhaps any FLCreactivation mechanism that occurs in male ga-metogenesis might be expected to apply to thefemale gamete as well. The mechanism of FLCresetting will be an interesting area to explore.

Other Targets of Vernalizationin Arabidopsis

In Arabidopsis there are five paralogs of FLC(often called the FLC clade) FLOWER-ING LOCUS M (FLM)/MADS AFFECT-ING FLOWERING 1 (MAF1), MAF2, MAF3,MAF4, and MAF5. FLM/MAF1, MAF2, andMAF4 act as floral repressors (Ratcliffe et al.2001, 2003; Scortecci et al. 2001). It is likelythat these proteins repress the expression of flo-ral integrators in a manner that is biochemi-cally redundant with that of FLC (Figure 1).


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In the presence of FRI, FLC provides the ma-jority of the repressive activity, but in rapid-flowering lines that lack FRI, other clade mem-bers such as FLM/MAF1 can provide a greateramount of repression than FLC under certainenvironmental conditions (Ratcliffe et al. 2001,Scortecci et al. 2001, Werner et al. 2005). Like-wise, when FRI is present, the repression ofFLC accounts for the majority of the vernaliza-tion response. However, vernalization clearlypromotes flowering in flc null mutants in SD.This vernalization response has been referredto as an FLC-independent vernalization path-way (Michaels & Amasino 2001). The repres-sion of FLC paralogs is likely to account forsome, and possibly all, of the vernalization

Inductive photoperiod (LD)

Non-inductive photoperiod (SD) Vernalization

PPD1

VRN1

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Floral meristem identity genes

Floral integrator genes

Flowering

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b

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Figure 3Outline of flowering pathways in cereals. As in Figure 1, components andgenetic interactions that promote flowering are shown in blue; those thatrepress flowering are shown in red. The photoperiod pathway in cerealsactivates the floral integrator VRN3 (an Arabidopsis FT homolog), which inturn activates the floral meristem-identity gene VRN1 (an ArabidopsisAP1/FUL homolog). However, in non-vernalized plants, VRN2 repressesVRN3 and attenuates photoperiod-pathway activation of VRN3. Vernalizationalleviates this VRN3 repression by suppressing VRN2 expression. VRN2 issuppressed by VRN1, and VRN1 is induced by cold exposure. The dual role ofVRN1 in promoting the floral transition as a meristem-identity gene and inrepressing VRN2 is depicted by placing VRN1 at two different locations in thediagram. Note that the induction of VRN1 by cold initiates a positive feedbackloop: (a) VRN1 represses VRN2, (b) repression of VRN2 allows thephotoperiod pathway to activate VRN3, and (c) VRN3 activates the expressionof VRN1. In addition, non-inductive SD represses VRN2 providing anadditional pathway to downregulate VRN2 during the SD of winter.

response that remains in flc null mutants. Giventhe presumably functionally overlapping FLCparalogs, it is perhaps simpler to think of ver-nalization as repressing FLC and several otherFLC-like genes rather than being comprised ofFLC-dependent and independent pathways. In-deed, at least one other member of the FLCclade, FLM/MAF1, undergoes the same VIN3-dependent, vernalization-mediated chromatinchanges as FLC (Sung et al. 2006b). Whetherthere is an effect of vernalization in Arabidopsisthat is independent of repression of all mem-bers of the FLC clade remains to be determined.For example, it will be interesting to determinewhether the induction of genes such as AGL19(Alexandre & Hennig 2008, Schonrock et al.2006) or AGL24 (Michaels et al. 2003) by ver-nalization is independent of not only FLC butof other FLC clade members as well.

Vernalization in Cereals

The cereals of the grass subfamily Pooideae,particularly wheat and barley, are the only othergroup of plants in which vernalization has beencharacterized molecularly. There are winter va-rieties of wheat and barley that possess a clearvernalization requirement and spring varietiesthat flower without vernalization. As in Ara-bidopsis, the genetic differences between win-ter and spring varieties have been explored, andgenes responsible for the winter/spring differ-ence have been cloned (reviewed in Colasanti &Coneva 2009, Distelfeld et al. 2009, Trevaskiset al. 2007). As discussed below, although someaspects of the circuitry of the vernalizationpathway are similar in Arabidopsis and cereals,the differences indicate that the respective ver-nalization pathways evolved independently.

Genetic studies comparing winter andspring cultivars of wheat and barley have iden-tified three loci that play a role in the ver-nalization response: VRN1, VRN2, and VRN3(Figure 3). VRN1 and VRN2 are not ho-mologous to the Arabidopsis genes with thesame name. VRN1 encodes a MADS-box tran-scription factor that resembles the Arabidopsisfloral meristem-identity genes AP1 and FUL

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(Danyluk et al. 2003, Preston & Kellogg 2006,Trevaskis et al. 2003, Yan et al. 2003) and is re-quired for flowering (Shitsukawa et al. 2007).VRN2 encodes a CCT-domain protein thathas no clear homolog in Arabidopsis (Yan et al.2004). VRN2 acts as a repressor of flowering byblocking the expression of VRN3, which is thewheat/barley homolog of FT (Yan et al. 2006).Constitutive expression of VRN3 from a pro-moter not subject to VRN2-mediated repres-sion bypasses the vernalization requirement(Yan et al. 2006), just as the constitutive expres-sion of FT bypasses FLC repression of floweringand the vernalization requirement in Arabidopsis(Michaels et al. 2005).

In cereals, the LD floral promotion pathwayfunctions similarly to that in Arabidopsis: Uponexposure to LD, a CO-like gene, PPD1, acti-vates VRN3 (Turner et al. 2005). VRN3, in turn(and in conjunction with proteins homologousto the Arabidopsis floral integrator FD), activatesthe floral meristem-identity gene, VRN1 (Li &Dubcovsky 2008). VRN2, as noted above, actsto repress the expression of VRN3 under LDconditions. This repression prevents the acti-vation of VRN3 by PPD1 and establishes a re-quirement for vernalization in LD (Hemminget al. 2008). VRN2 expression, like that of FLC,is repressed by cold (Yan et al. 2004).

As discussed above, the induction of VIN3 bya long exposure to cold is key to FLC repressionin Arabidopsis. In wheat, prolonged cold elevatesthe expression level of VIN3-like genes; how-ever, the change in expression is not as definedas that seen for Arabidopsis VIN3, and none ofthe wheat homologs to date have been linked tothe control of flowering time (Fu et al. 2007). Incereals, VRN1 is also induced by a vernalizingcold exposure, and VRN1 acts to repress VRN2expression (Loukoianov et al. 2005, Trevaskiset al. 2006). Thus, VRN1 serves a similar roleto VIN3 in cereal vernalization, but that doesnot rule out the existence of other componentsthat play a VIN3-like role in cereals.

Thus, there are three general similarities inthe vernalization circuitry between cereals andArabidopsis: (a) a block to flowering results froma repressor (FLC in Arabidopsis or VRN2 in

wheat) that represses a homologous floral in-tegrator in both groups of plants (FT/VRN3),(b) the repressors of flowering (FLC or VRN2)are downregulated in the cold, which permitsFT/VRN3 to be expressed, and (c) downreg-ulation of FLC and VRN2 is mediated by up-regulation in the cold of VIN3 and VRN1 inArabidopsis and cereals, respectively (Figures 1and 3).

There are, however, some distinct differ-ences in both the vernalization circuitry andcomponents between Arabidopsis and cereals.As discussed above, the repressors (FLC andVRN2) are not related proteins. With respectto circuitry, the VRN1 gene in cereals plays adual role in the regulation of flowering. VRN1is both a promoter of flowering downstream ofVRN3 and a cold-activated repressor upstreamof VRN2. This dual role of VRN1 in cerealscreates a situation not found in Arabidopsis: apositive flowering feedback loop that involvesvernalization components (Figure 3). Such apositive feedback loop ensures that once flow-ering initiates, it continues in the absence of en-vironmental cues. The regulation of floweringin Arabidopsis also incorporates positive feed-back loops as discussed above, but these involvecomponents that are downstream of vernaliza-tion such as the reciprocal reinforcement ofLFY and AP1 expression (Figure 1) (Sablowski2007).

Another circuitry difference is the conver-gence of photoperiod and vernalization on thefloral repressor VRN2 in cereals (Figure 3).In addition to being repressed by cold, VRN2expression is also governed by day length. InSD, VRN2 expression is greatly reduced, andthis reduction occurs rapidly upon transfer ofLD-grown plants to SD (Dubcovsky et al. 2006,Trevaskis et al. 2006). Cold and SD appear torepress VRN2 by distinct mechanisms. VRN2repression via cold is mediated by VRN1, butVRN1 is not expressed in SD (Dubcovsky et al.2006, Trevaskis et al. 2006). Wheat and bar-ley are LD plants, so even though SD removesVRN2, LD is still required to activate VRN3.SD and cold are indicators of winter, and cere-als can apparently use both as cues to remove a


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block to flowering, whereas in Arabidopsis onlycold is a winter cue. Grasses such as wheat andbarley may have evolved in a region where win-ters can be too mild to induce floral compe-tence. Thus, there may be adaptive value inlinking a photoperiod response with vernaliza-tion because, regardless of severity, winter al-ways coincides with short days.

That vernalization alleviates repression ofthe homologous genes FT in Arabidopsis andVRN3 in cereals is perhaps not surprising. Asdiscussed above, FT-like genes are ubiquitousin plants and have been found to initiate thetransition to flowering in all angiosperms ex-amined (Turck et al. 2008). Because the roleof FT/VRN3 homologs as a key flowering ini-tiator is conserved among flowering plants, itmakes a logical target for the repression of flow-ering; i.e., selection for this target may be anexample of convergent evolution in cereals andArabidopsis.

It is important to note that vernalizationhas been more thoroughly studied in Arabidop-sis than in cereals. As more of the molecularcomponents of vernalization in grasses are iden-tified, it is possible that additional similaritiesbetween the vernalization processes in thesetwo groups may be revealed.

FUTURE DIRECTIONS

In cereals and Arabidopsis, the only two sys-tems in which vernalization has been studiedat a molecular level, the first known molecu-lar event of vernalization is the induction ofgenes by cold. Upstream of these genes theremust be a biochemical process that serves as acold sensor. The mechanisms of cold sensingand subsequent gene activation are not known.

There are a variety of possibilities for how coldsensing might operate during vernalization [seeSung & Amasino (2005) for a partial list], butin the absence of data these possibilities remainspeculative. Indeed, little is known about themolecular basis of any cold-induced process inplants. A challenge for the future will be to un-derstand how plants sense cold.

Another challenge will be to explore therange of vernalization mechanisms that existin flowering plants. There are some strikingdifferences between the circuitry and compo-nents of vernalization in Arabidopsis and cereals.Perhaps the lack of conservation in vernaliza-tion pathways compared with the photoperiodpathway is not surprising. Flowering plants be-gan to diversify less than 200 mya (Solds et al.2008) when the climate was warmer and the lo-cations of continents were quite different thanat present. The major groups of angiospermsarose before continental drift, and a changingclimate created environments in which a vernal-ization response would have had adaptive value.In contrast, evolving a mechanism to sense pho-toperiod would have had adaptive value muchearlier as seasonal changes in day length oc-cur nearly everywhere. Moreover, the molecu-lar bases of other long-term cold responses suchas bud dormancy remain to be determined. Inmany perennials, buds become dormant in thefall season and, once dormant, buds of manyspecies must be exposed to a long period ofcold before they become competent to exitdormancy and resume growth (e.g., Chouard1960). Exploring the cold response pathways ina range of species will hopefully provide insightinto the molecular mechanisms that plants haveevolved to coordinate their life cycles with thechanging seasons.

DISCLOSURE STATEMENT

The authors are not aware of any biases that might be perceived as affecting the objectivity of thisreview.

ACKNOWLEDGMENTS

R.M.A. is grateful to the National Institutes of Health, the National Science Foundation, theU.S. Department of Agriculture National Research Initiative Competitive Grants Program, the

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College of Agricultural and Life Sciences, and the Graduate School of the University of Wisconsinfor their generous support of our flowering research. S.S. is grateful to the College of NaturalSciences and the Institute for Cellular and Molecular Biology of the University of Texas for theirgenerous start-up support. We apologize to those in the flowering field whose work was not citeddue to length limits.

LITERATURE CITED

Alexandre CM, Hennig L. 2008. FLC or not FLC: the other side of vernalization. J. Exp. Bot. 59:1127–35Alvarez-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. 13:627–37Amasino R. 2004. Vernalization, competence, and the epigenetic memory of winter. Plant Cell 16:2553–59Ausin 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. 36:162–66Bastow R, Mylne JS, Lister C, Lippman Z, Martienssen RA, Dean C. 2004. Vernalization requires epigenetic

silencing of FLC by histone methylation. Nature 427:164–67Baurle I, Dean C. 2008. Differential interactions of the autonomous pathway RRM proteins and chromatin

regulators in the silencing of Arabidopsis targets. PLoS One 3:e2733Baurle I, Smith L, Baulcombe DC, Dean C. 2007. Widespread role for the flowering-time regulators FCA

and FPA in RNA-mediated chromatin silencing. Science 318:109–12Bernier G, Kinet JM, Sachs RM, eds. 1981. The Control of Floral Evocation and Morphogenesis. Boca Raton:

CRCBlazquez MA, Green R, Nilsson O, Sussman MR, Weigel D. 1998. Gibberellins promote flowering of

Arabidopsis by activating the LEAFY promoter. Plant Cell 10:791–800Briggs 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 theinitiation 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 H3K27methylation 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 methylationin Polycomb-group silencing. Science 298:1039–43

Cao Y, Dai Y, Cui S, Ma L. 2008b. Histone H2B monoubiquitination in the chromatin of FLOWERINGLOCUS 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-groupproteins 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 LOCUSC expression during Arabidopsis reproductive development. Plant J. 57:918–31

Choi K, Kim S, Kim SY, Kim M, Hyun Y, et al. 2005. SUPPRESSOR OF FRIGIDA3 encodes a nuclearACTIN-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. 11:191–238Clarke JH, Dean C. 1994. Mapping FRI, a locus controlling flowering time and vernalization response in

Arabidopsis thaliana. Mol. Gen. Genet. 242:81–89Coen ES, Meyerowitz EM. 1991. The war of the whorls: genetic interactions controlling flower development.

Nature 353:31–37Colasanti J, Coneva V. 2009. Mechanisms of floral induction in grasses: something borrowed, something new.

Plant Physiol. 149:56–62Corbesier 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 316:1030–33Danyluk 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


Ann

u. R

ev. C

ell D

ev. B

iol.

2009

.25:

277-

299.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Uni

vers

ity o

f W

isco

nsin

- M

adis

on o

n 08

/09/

12. F

or p

erso

nal u

se o

nly.


de Folter S, Immink RG, Kieffer M, Parenicova L, Henz SR, et al. 2005. Comprehensive interaction map ofthe 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 triggersthe 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 ARP6is a pleiotropic developmental regulator required for the maintenance of FLOWERING LOCUS Cexpression 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 requiresactivation 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. 10:520–27Distelfeld A, Li C, Dubcovsky J. 2009. Regulation of flowering in temperate cereals. Curr. Opin. Plant Biol.

12:178–84Dubcovsky 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. 60:469–80Fingerman 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. 280:28761–65Finnegan 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 byvernalization 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 CCA1regulate 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 epigeneticregulation 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-groupgene 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 inthe epigenetic silencing of Arabidopsis FLC. Curr. Biol. 17:73–78

Grossniklaus U, Vielle-Calzada JP, Hoeppner MA, Gagliano WB. 1998. Maternal control of embryogenesisby 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 H2Bmonoubiquitination. Plant J. 57:422–33

He Y, Amasino RM. 2005. Role of chromatin modification in flowering-time control. Trends Plant Sci. 10:30–35He 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.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 proteininteracts directly in vivo with SOC1 and FT chromatin and is part of a high-molecular-weight proteincomplex. 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. Development131:3829–38

Henderson IR, Jacobsen SE. 2007. Epigenetic inheritance in plants. Nature 447:418–24Hennig 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.

Ann

u. R

ev. C

ell D

ev. B

iol.

2009

.25:

277-

299.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Uni

vers

ity o

f W

isco

nsin

- M

adis

on o

n 08

/09/

12. F

or p

erso

nal u

se o

nly.


Hepworth SR, Valverde F, Ravenscroft D, Mouradov A, Coupland G. 2002. Antagonistic regulation offlowering-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 influencemultiple 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 Demethylase1repress 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, amajor determinant of natural variation in Arabidopsis flowering time. Science 290:344–47

Kamakaka RT, Biggins S. 2005. Histone variants: deviants? Genes Dev. 19:295–310Kandasamy 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 proteinubiquitination 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 floweringtime 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 intoeuchromatin. 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 erectawild-type. Plant J. 6:911–19

Koornneef M, Hanhart CJ, Van Der Veen JH. 1991. A genetic and physiological analysis of late floweringmutants in Arabidopsis thaliana. Mol. Gen. Genet. 229:57–66

Kotake T, Takada S, Nakahigashi K, Ohto M, Goto K. 2003. Arabidopsis TERMINAL FLOWER 2 geneencodes a heterochromatin protein 1 homolog and represses both FLOWERING LOCUS T to regulateflowering 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 histoneH3 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 H3lysine 4 links BHC80 to LSD1-mediated gene repression. Nature 448:718–22

Lang A. 1965. Physiology of flower initiation. In Encyclopedia of Plant Physiology, ed. W Ruhland, pp. 1371–536.Berlin: Springer-Verlag

Lee H, Suh SS, Park E, Cho E, Ahn JH, et al. 2000. The AGAMOUS-LIKE 20 MADS domain proteinintegrates 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 andmutations 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 directlyregulates leafy. Plant J. 55:832–43

Levy YY, Mesnage S, Mylne JS, Gendall AR, Dean C. 2002. Multiple roles of Arabidopsis VRN1 in vernalizationand 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


Ann

u. R

ev. C

ell D

ev. B

iol.

2009

.25:

277-

299.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Uni

vers

ity o

f W

isco

nsin

- M

adis

on o

n 08

/09/

12. F

or p

erso

nal u

se o

nly.


Li D, Liu C, Shen L, Wu Y, Chen H, et al. 2008. A repressor complex governs the integration of floweringsignals 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 methylationstate-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 RNAbinding 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 integratesflowering signals in Arabidopsis. Development 135:1481–91

Liu F, Quesada V, Crevillen P, Baurle I, Swiezewski S, Dean C. 2007. The Arabidopsis RNA-binding proteinFCA 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 innormal 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 inArabidopsis, encodes a protein containing RNA-binding domains. Cell 89:737–45

Martin-Trillo M, Lazaro A, Poethig RS, Gomez-Mena C, Pineiro MA, et al. 2006. EARLY IN SHORT DAYS1 (ESD1) encodes ACTIN-RELATED PROTEIN 6 (AtARP6), a putative component of chromatinremodelling 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 126:22–24Melzer 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. 40:1489–92Michaels SD, Amasino RM. 1999. FLOWERING LOCUS C encodes a novel MADS domain protein that

acts as a repressor of flowering. Plant Cell 11:949–56Michaels 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 apromoter 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 inwinter-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 variantcatalyzed 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 aDrosophila 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 ofHETEROCHROMATIN PROTEIN1, is required for epigenetic silencing of FLC. Proc. Natl. Acad. Sci.USA 103:5012–17

Napp-Zinn K. 1987. Vernalization: environmental and genetic regulation. In Manipulation of Flowering, ed.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 highlevels 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 repressionin 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 geneexpression in Arabidopsis. PLoS Genet. 4:e1000077

Oh S, Zhang H, Ludwig P, van Nocker S. 2004. A mechanism related to the yeast transcriptional regulatorPaf1c is required for expression of the Arabidopsis FLC/MAF MADS box gene family. Plant Cell 16:2940–53

296 Kim et al.

Ann

u. R

ev. C

ell D

ev. B

iol.

2009

.25:

277-

299.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Uni

vers

ity o

f W

isco

nsin

- M

adis

on o

n 08

/09/

12. F

or p

erso

nal u

se o

nly.


Papp B, Muller J. 2006. Histone trimethylation and the maintenance of transcriptional ON and OFF statesby 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 requireSEPALLATA MADS-box genes. Nature 405:200–3

Pien S, Fleury D, Mylne JS, Crevillen P, Inze D, et al. 2008. Arabidopsis TRITHORAX1 dynamically regulatesFLOWERING 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 AFFECTINGFLOWERING 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 FLChomologue. Plant Physiol 126:122–32

Reyes JC. 2006. Chromatin modifiers that control plant development. Curr. Opin. Plant Biol. 9:21–27Sablowski R. 2007. Flowering and determinacy in Arabidopsis. J. Exp. Bot. 58:899–907Saleh 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 20:568–79Samach 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 288:1613–16Sarma 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. 28:2718–31Schmitz 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. Genetics176:1359–62

Schmitz RJ, Sung S, Amasino RM. 2008. Histone arginine methylation is required for vernalization-inducedepigenetic silencing of FLC in winter-annual Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 105:411–16

Schmitz RJ, Tamada Y, Doyle MR, Zhang X, Amasino RM. 2009. Histone H2B deubiquitination is required fortranscriptional 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 H3lysine 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 inArabidopsis, 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 thefloral 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-groupproteins. Curr. Opin. Plant Biol. 8:553–61

Schuettengruber B, Chourrout D, Vervoort M, Leblanc B, Cavalli G. 2007. Genome regulation by Polycomband Trithorax proteins. Cell 128:735–45

Scortecci KC, Michaels SD, Amasino RM. 2001. Identification of a MADS-box gene, FLOWERING LOCUSM, that represses flowering. Plant J. 26:229–36

Searle I, He Y, Turck F, Vincent C, Fornara F, et al. 2006. The transcription factor FLC confers a floweringresponse to vernalization by repressing meristem competence and systemic signaling in Arabidopsis. GenesDev. 20:898–912

Sheldon CC, Burn JE, Perez PP, Metzger J, Edwards JA, et al. 1999. The FLF MADS box gene: a repressorof 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 LOCUSC 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 cerevisiaeidentifies several PHD fingers as novel direct and selective binding modules of histone H3 methylated ateither 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


Ann

u. R

ev. C

ell D

ev. B

iol.

2009

.25:

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299.

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ity o

f W

isco

nsin

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adis

on o

n 08

/09/

12. F

or p

erso

nal u

se o

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Simpson GG. 2004. The autonomous pathway: epigenetic and post-transcriptional gene regulation in thecontrol 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 factorthat 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 EvolutionaryBiology 2008, pp. 3–25. Oxford: Blackwell

Soltis DE, Ma H, Frohlich MW, Soltis PS, Albert VA, et al. 2007. The floral genome: an evolutionary historyof 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 statein 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 andphotoperiod pathways in Arabidopsis. Genes Dev. 20:3244–48

Swiezewski S, Crevillen P, Liu F, Ecker JR, Jerzmanowski A, Dean C. 2007. Small RNA-mediated chromatinsilencing 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-containingcomplexes: 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: AcademicTrevaskis B, Bagnall DJ, Ellis MH, Peacock WJ, Dennis ES. 2003. MADS box genes control vernalization-

induced flowering in cereals. Proc. Natl. Acad. Sci. USA 100:13099–104Trevaskis B, Hemming MN, Dennis ES, Peacock WJ. 2007. The molecular basis of vernalization-induced

flowering in cereals. Trends Plant Sci. 12:352–57Trevaskis B, Hemming MN, Peacock WJ, Dennis ES. 2006. HvVRN2 responds to daylength, whereas

HvVRN1 is regulated by vernalization and developmental status. Plant Physiol. 140:1397–405Turck F, Fornara F, Coupland G. 2008. Regulation and identity of florigen: FLOWERING LOCUS T moves

center stage. Annu. Rev. Plant Biol. 59:573–94Turner A, Beales J, Faure S, Dunford RP, Laurie DA. 2005. The pseudo-response regulator Ppd-H1 provides

adaptation to photoperiod in barley. Science 310:1031–34Veley KM, Michaels SD. 2008. Functional redundancy and new roles for genes of the autonomous floral-

promotion pathway. Plant Physiol. 147:682–95Wenkel 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

Werner JD, Borevitz JO, Warthmann N, Trainer GT, Ecker JR, et al. 2005. Quantitative trait locus mappingand DNA array hybridization identify an FLM deletion as a cause for natural flowering-time variation.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 perturbationon 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 histonemonoubiquitination by the Rad6-Bre1 complex, which signals for histone methylation by COMPASSand Dot1p. J. Biol. Chem. 278:34739–42

Wood CC, Robertson M, Tanner G, Peacock WJ, Dennis ES, Helliwell CA. 2006. The Arabidopsis thalianavernalization response requires a Polycomb-like protein complex that also includes VERNALIZATIONINSENSITIVE 3. Proc. Natl. Acad. Sci. USA 103:14631–36

Wu C, Morris JR. 2001. Genes, genetics, and epigenetics: a correspondence. Science 293:1103–5Xu 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 offlowering in Arabidopsis thaliana. Plant J. 57:279–88

298 Kim et al.

Ann

u. R

ev. C

ell D

ev. B

iol.

2009

.25:

277-

299.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Uni

vers

ity o

f W

isco

nsin

- M

adis

on o

n 08

/09/

12. F

or p

erso

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Xu L, Zhao Z, Dong A, Soubigou-Taconnat L, Renou JP, et al. 2008. Di- and tri- but not monomethylationon histone H3 lysine 36 marks active transcription of genes involved in flowering time regulation andother 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 anorthologue 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 aflowering 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 thewheat vernalization gene VRN1. Proc. Natl. Acad. Sci. USA 100:6263–68

Zeevaart JA. 2008. Leaf-produced floral signals. Curr. Opin. Plant Biol. 11:541–47Zeevaart JAD. 1976. Physiology of flower formation. Annu. Rev. Plant Physiol. 27:321–48Zhang 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 switchflowering locus C. Genetics 164:347–58

Zhang H, van Nocker S. 2002. The vernalization independence 4 gene encodes a novel regulator of floweringlocus C. Plant J. 31:663–73

Zhao Z, Yu Y, Meyer D, Wu C, Shen WH. 2005. Prevention of early flowering by expression of FLOWERINGLOCUS C requires methylation of histone H3 K36. Nat. Cell Biol. 7:1256–60


Ann

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AR389-FM ARI 14 September 2009 14:58

Annual Reviewof Cell andDevelopmentalBiology

Volume 25, 2009

ContentsChromosome Odds and Ends

Joseph G. Gall � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Small RNAs and Their Roles in Plant DevelopmentXuemei Chen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �21

From Progenitors to Differentiated Cells in the Vertebrate RetinaMichalis Agathocleous and William A. Harris � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �45

Mechanisms of Lipid Transport Involved in Organelle Biogenesisin Plant CellsChristoph Benning � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �71

Innovations in Teaching Undergraduate Biologyand Why We Need ThemWilliam B. Wood � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �93

Membrane Traffic within the Golgi ApparatusBenjamin S. Glick and Akihiko Nakano � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 113

Molecular Circuitry of Endocytosis at Nerve TerminalsJeremy Dittman and Timothy A. Ryan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 133

Many Paths to Synaptic SpecificityJoshua R. Sanes and Masahito Yamagata � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 161

Mechanisms of Growth and Homeostasis in the Drosophila WingRicardo M. Neto-Silva, Brent S. Wells, and Laura A. Johnston � � � � � � � � � � � � � � � � � � � � � � � � � 197

Vertebrate Endoderm Development and Organ FormationAaron M. Zorn and James M. Wells � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 221

Signaling in Adult NeurogenesisHoonkyo Suh, Wei Deng, and Fred H. Gage � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 253

Vernalization: Winter and the Timing of Flowering in PlantsDong-Hwan Kim, Mark R. Doyle, Sibum Sung, and Richard M. Amasino � � � � � � � � � � � � 277

Quantitative Time-Lapse Fluorescence Microscopy in Single CellsDale Muzzey and Alexander van Oudenaarden � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 301

Mechanisms Shaping the Membranes of Cellular OrganellesYoko Shibata, Junjie Hu, Michael M. Kozlov, and Tom A. Rapoport � � � � � � � � � � � � � � � � � � � � 329

The Biogenesis and Function of PIWI Proteins and piRNAs: Progressand ProspectTravis Thomson and Haifan Lin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 355

vii

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AR389-FM ARI 14 September 2009 14:58

Mechanisms of Stem Cell Self-RenewalShenghui He, Daisuke Nakada, and Sean J. Morrison � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 377

Collective Cell MigrationPernille Rørth � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 407

Hox Genes and Segmentation of the Hindbrain and Axial SkeletonTara Alexander, Christof Nolte, and Robb Krumlauf � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 431

Gonad Morphogenesis in Vertebrates: Divergent Means to aConvergent EndTony DeFalco and Blanche Capel � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 457

From Mouse Egg to Mouse Embryo: Polarities, Axes, and TissuesMartin H. Johnson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 483

Conflicting Views on the Membrane Fusion Machinery and the FusionPoreJakob B. Sørensen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 513

Coordination of Lipid Metabolism in Membrane BiogenesisAxel Nohturfft and Shao Chong Zhang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 539

Navigating ECM Barriers at the Invasive Front: The CancerCell–Stroma InterfaceR. Grant Rowe and Stephen J. Weiss � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 567

The Molecular Basis of Organ Formation: Insights from theC. elegans ForegutSusan E. Mango � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 597

Genetic Control of Bone FormationGerard Karsenty, Henry M. Kronenberg, and Carmine Settembre � � � � � � � � � � � � � � � � � � � � � � 629

Listeria monocytogenes Membrane Trafficking and Lifestyle:The Exception or the Rule?Javier Pizarro-Cerda and Pascale Cossart � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 649

Asymmetric Cell Divisions and Asymmetric Cell FatesShahragim Tajbakhsh, Pierre Rocheteau, and Isabelle Le Roux � � � � � � � � � � � � � � � � � � � � � � � � � � � 671

Indexes

Cumulative Index of Contributing Authors, Volumes 21–25 � � � � � � � � � � � � � � � � � � � � � � � � � � � 701

Cumulative Index of Chapter Titles, Volumes 21–25 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 704

Errata

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

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Documents

Vernalization: Winter and the Timing of Flowering in Plants...cess known as vernalization. A requirement for vernalization is an adaptation to temperate cli-mates that prevents ﬂowering