From Decision to Commitment: The Molecular Memory of Flowering

Molecular Plant

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Volume 2

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Number 4

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Pages 628–642

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July 2009

REVIEW ARTICLE

From Decision to Commitment: The Molecular Memory of Flowering Jessika Adrian, Stefano Torti and Franziska Turck1 Max Planck Institute for Plant Breeding Research, Carl von Linne´ Weg 10, 50829 Ko¨ln, Germany

Key words: Memory; floral commitment; floral transition; chromatin; Polycomb group; transcription regulatory network motif.

INTRODUCTION During the floral transition the identity of the shoot apical meristem (SAM) changes from a vegetative meristem (VM) to an inflorescence meristem (IM). The VM maintains a pool of self-renewing stem cells and generates leaves and side shoot meristems at its flanks, whereas the IM generates floral meristems (FMs). In Arabidopsis thaliana (Arabidopsis), the IM is indeterminate; however, FMs are determinate structures that eventually consume their self-renewing stem cells during the formation of floral organs. If the IM is transformed to a FM, as is the case in some plant species and Arabidopsis mutants, a terminal flower is formed. The transition to flowering is controlled by genetic pathways that integrate environmental cues like temperature and day length, and the developmental state of the plant (Amasino, 2005; Quesada et al., 2005; Baurle and Dean, 2006; Kobayashi and Weigel, 2007; Turck et al., 2008). In many species, including Arabidopsis, an external floral inductive stimulus is required only transiently to cause a stable transition from vegetative to reproductive development (Corbesier et al., 2007). This ability to continue flowering when inductive conditions no longer exist is called floral commitment. Nevertheless, in some other species and in some Arabidopsis mutants, a floral inductive stimulus is required continuously and its absence causes reversion (Tooke et al., 2005). This indicates that flowering needs to be not only established, but also maintained. In a possible molecular scenario that explains floral commitment, a key gene that is induced (or repressed) by the floral stimulus maintains its novel expression state in absence of

the primary signal. Many examples from animals and yeast indicate that epigenetic mechanisms can be the instrument to switch the default expression state of a gene in a stable fashion (Klar, 2007; Schwartz and Pirrotta, 2007; Kohler and Villar, 2008; Schatlowski et al., 2008). In the context of this review, an epigenetic mechanism is defined as a change in the chromatin at the regulated locus that persists after the disappearance of a transient primary stimulus. In particular, this encompasses stable changes in histone- or DNA-methylation. It is important to keep in mind that not every stable chromatin change is involved in expression maintenance (Farrona et al., 2008). Furthermore, bistable expression modules have been modeled in theoretical and experimental approaches without the causal implication of chromatin modifications. Such bistable modules are capable to define a cellular memory even in organisms that do not possess a structured chromatin such as bacteria (Ferrell, 2002; Smits et al., 2006; Alon, 2007). One simple example is the ‘genetic toggle switch’ that is formed by two mutually repressing transcription factors. Toggle switches have two stable and distinct states (either one gene is expressed or the other) that can be switched through the transient action of either an inducer of the down-regulated gene or a repressor of

1 To whom correspondence should be addressed. E-mail [email protected].

ª The Author 2009. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: 10.1093/mp/ssp031 Received 16 February 2009; accepted 13 April 2009

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ABSTRACT During the floral transition the shoot apical meristem changes its identity from a vegetative to an inflorescence state. This change in identity can be promoted by external signals, such as inductive photoperiod conditions or vernalization, and is accompanied by changes in expression of key developmental genes. The change in meristem identity is usually not reversible, even if the inductive signal occurs only transiently. This implies that at least some of the key genes must possess an intrinsic memory of the newly acquired expression state that ensures irreversibility of the process. In this review, we discuss different molecular scenarios that may underlie a molecular memory of gene expression.

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REPRESSION OF FLC: EPIGENETIC REGULATION AS IN TEXTBOOKS The MADS-box transcription factor FLC is a key floral repressor in Arabidopsis (Michaels and Amasino, 1999). High levels of FLC render the plant incapable of initiating flowering, even under inductive long-day (LD) conditions, which gives a good reason why transcriptional repression of FLC is the main object of floral enabling pathways (Sheldon et al., 2000; Ausin et al., 2004; Lim et al., 2004). Components of the vernalization pathway repress FLC during prolonged exposure to cold temperatures and also maintain this repression after a subsequent increase in ambient temperature (Michaels and Amasino, 1999; Sheldon et al., 1999). In contrast, the autonomous pathway results in FLC repression in response to internal cues at late developmental stages. The autonomous pathway is not a linear genetic pathway, but rather a collection of genetic components targeting FLC regulation at different levels (Koornneef et al., 1998). We will not discuss FLC down-regulation through the autonomous pathway here, but refer to other recent reviews (He and Amasino, 2005; Baurle and Dean, 2006; Marquardt et al., 2006; Farrona et al., 2008). FLC is expressed in the vasculature of leaves and the SAM and acts as a transcriptional repressor. In leaves direct targets of FLC are the floral integrator genes FT and SOC1, while FD and SOC1 are repressed in the SAM (Helliwell et al., 2006; Searle et al., 2006). It has been shown that FLC acts in both tissues to prevent the response to floral inductive signals (Searle et al., 2006). The impact of FLC on plant development is the subject of natural variation in Arabidopsis. Winter annual Arabidopsis accessions have high levels of FLC and therefore require vernalization, while summer annuals have only low levels and flower without exposure to cold temperatures. This natural variation depends on the FLC locus itself and on its upstream activator FRIGIDA (FRI), which is non-functional in many summer annuals (Johanson et al., 2000; Shindo et al., 2005; Werner et al., 2005).

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The transcriptional repression of FLC during vernalization is one of the best studied epigenetic mechanisms in plants (Figure 1). In a first phase, starting from a steady state of high FLC expression such as observed in winter annual accessions or mutants of the autonomous pathway, transcriptional repression of FLC is induced as an acute response to cold. During this phase, the plant homeodomain (PHD) finger proteins VERNALIZATION INSENSITIVE 3 (VIN3) and VERNALIZATION 5 (VRN5)/ VIN3-like 1 (VIL1) associate with a region near the 5#-end of intron 1 of FLC (De Lucia et al., 2008). Plants lacking one of these genes fail to down-regulate FLC in response to cold treatment (Sung and Amasino, 2004; Sung et al., 2006a; Greb et al., 2007). VIN3 expression is up-regulated after extended exposure to cold and, therefore, VIN3 is one of the early components of the vernalization pathway (Sung and Amasino, 2004). VRN5/VIL1 association with FLC is dependent on the presence of VIN3 (De Lucia et al., 2008). During the first FLC-repressive phase, chromatin modifications observed at the FLC locus change, so that those generally associated with highly expressed genes decrease, whereas others that are correlated with low gene expression levels increase. For instance, VIN3 is required for

Figure 1. FLC States during a Lifecycle in Arabidopsis. High levels of FLC expression render the plant unable to respond to floral inductive conditions. In winter annual accessions, high FLC levels require the presence of an active allele of FRI. The VRN2– PRC2 complex is associated with highly expressed FLC but does not repress transcription. Upon experience of cold temperature, expression of FLC is repressed. This acute response to cold is accompanied by a loss of histone acetylation and a gradual increase in H3K27 tri-methylation. During this phase, the PHD finger proteins VIN3 and VRN5/VIL1 associate with the FLC locus as part of a PHD– VRN2–PRC2 super-complex. The presence of VIN3 and VRN5/VIL1 is required to down-regulate FLC. Maintenance of the repressed state requires the function of the PRC2 component VRN2 and the PRC1related protein LHP1. It is associated with a further increase and spreading of the H3K27me3 mark at FLC. The silent state needs to be maintained and cell division may be necessary to maintain FLC repression. In older tissues that do not divide, H3K27me3 does not spread and repression of FLC is leaky. The default expression state of FLC is reset during early embryo development by a mechanism that does not involve FRI. In contrast, FRI is important to increase FLC levels in late embryo development.

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the expressed gene. In an even simpler device, a self-activating transcription factor stabilizes its own expression after an initial activation event. In the following, we will discuss physiological and molecular examples of the genetic flowering pathways in the context of these principal mechanisms. In Arabidopsis, floral promotion pathways such as the photoperiod and gibberellin (GA) pathway ultimately increase the expression levels of genes called the floral integrators, such as FLOWERING LOCUS T (FT), TWIN SISTER OF FT (TSF), SUPPRESSOR OF CONSTANS 1 (SOC1)/AGAMOUS-LIKE 20 (AGL20), AGAMOUS-LIKE 24 (AGL24), and LEAFY (LFY), whereas enabling pathways regulate the expression of floral repressors, such as FLOWERING LOCUS C (FLC), TERMINAL FLOWER 1 (TFL1), and SHORT VEGETATIVE PHASE (SVP) and thus define the competence of the plant to flower under inductive conditions (Boss et al., 2004; Kobayashi and Weigel, 2007; Turck et al., 2008).

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et al., 2006; De Lucia et al., 2008). The evolutionary conserved PRC2 is capable of tri-methylating lysine 27 of H3 in animals and plants (Schwartz and Pirrotta, 2007; Farrona et al., 2008; Schatlowski et al., 2008). In Arabidopsis most components of the PRC2 are encoded by small gene families and the composition of the complex might differ, depending on developmental states and tissues (see Figure 2 for details). It has been shown that a PRC2 that contains VERNALIZATON 2 (VRN2) is constitutively associated with the FLC locus at sites where it correlates with the H3K27me3 chromatin mark (De Lucia et al., 2008). The current model proposes that, upon cold perception, association of the PHD finger proteins VRN5/VIL1– VIN3–VEL1/VIL2 with VRN2–PRC2 increases H3K27tri-methylation activity of the resulting PHD–VRN2–PRC2 super-complex. The effect of this stimulation is an increase in H3K27me3 levels and a broader spreading of the histone mark over the FLC locus that largely takes place after an increase in ambient temperature (Finnegan and Dennis, 2007; De Lucia et al., 2008). Accordingly, loss-of-function of either VRN2, VRN5/VIL1, or VIN3 inhibits accumulation of tri-methylated H3K27 in the cold and attenuates the down-regulation of FLC during vernalization. In addition, FLC repression is unstable after vernalization in vrn5/vil1, vin3, and vrn2 mutant plants, although a local increase in H3K27me3 is still observed (but it is reduced in comparison to wild-type plants) (Greb et al., 2007; De Lucia et al., 2008). The increased and expanded H3K27me3 mark is thought to recruit repressors that assist in the maintenance of a stable FLC repression. The increase in H3K27me3 at the FLC locus during vernalization correlates with increased LIKE HETERCHROMATIN PROTEIN 1 (LHP1)/TERMINAL FLOWER 2 (TFL2) association and lhp1 mutants fail to maintain FLC repression (see Figure 1 and 2) (Mylne et al., 2006; Sung et al., 2006b). Interestingly, LHP1 physically interacts with INCURVATA 2 (ICU2), the catalytic subunit of DNA-polymerase alpha, which, again, links the maintenance of FLC repression with DNA replication and mitosis (Barrero et al., 2007). An elegant model has been proposed to accommodate the role of cell division in repression maintenance (Finnegan and Dennis, 2007). It is based on the observation that the existing variants of H3 have a differential propensity to become tri-methylated at K27 (Johnson et al., 2004). Variants that are predominantly expressed during replication are easily modified, whereas variants that are preferentially utilized during nucleosome replacement in arrested cells are rather recalcitrant to the modification. Thus, increased need to replace nucleosomes in aging cells may lead to a loss of H3K27me3 and, as a consequence, re-activation of FLC. Interestingly, in the Arabidopsis relative, Arabis alpina, non-maintained repression of the FLC ortholog PERPETUAL FLOWERING 1 (PEP1) is the rule and not the exception (Wang et al., 2009). In contrast to Arabidopsis, A. alpina has a perennial, polycarpic life strategy and, for most accessions, vernalization is an obligate requirement for flowering. In contrast to annual plants, in perennial plants, the memory of vernalization has to be reset during each annual cycle, allowing repeated response to seasonal cues. In A. alpina carrying the

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vernalization-induced deacetylation of lysine 9 of histone 3 (H3K9) at a region of intron 1 and a region upstream of the transcription site of FLC (Sung and Amasino, 2004). Tri-methylation of lysine 4 (H3K4me3) decreases during vernalization and the presence of di-methylation of lysine 36 of histone 3 (H3K36me2) has also been correlated with high FLC expression (Zhao et al., 2005; Finnegan and Dennis, 2007; Xu et al., 2008). Although the impact of chromatin modifications on gene expression cannot be deduced from correlated changes in histone marks and transcription levels, functional analysis of Arabidopsis mutants agrees with the notion that histone modifications indeed play a functional role in FLC regulation. For example, mutants of components of the Arabidopsis PAF1 complex, such as EARLY FLOWERING 7 and EARLY FLOWERING 8 (ELF7 and ELF8), fail to express high levels of FLC even in winter annual accessions or autonomous pathway mutant backgrounds and are therefore early flowering (He et al., 2004; Oh et al., 2004). Studies of the yeast PAF1 complex have established its involvement in initiation and elongation of transcripts in connection with chromatin (Belotserkovskaya and Reinberg, 2004). In yeast, the PAF1 complex interacts with the histone methyltransferases SET1 and SET2. Recruitment of SET1 and SET2 to the chromatin leads to increased methylation of H3K4 and H3K36, respectively. In Arabidopsis, EARLY FLOWERING IN SHORT DAYS (EFS)/SET DOMAIN GROUP 8 (SDG 8) encodes a homolog of SET2 that is involved in di-methylation of H3K36 in the promoter region and first intron of FLC (Zhao et al., 2005; Xu et al., 2008). Loss-of-function of EFS causes reduced FLC expression and early flowering in short days (SD) (Kim et al., 2005; Zhao et al., 2005). The ARABIDOPSIS TRITHORAX (ATX) 1–6 proteins belong to the same clade as the histone methyltransferase SET1. Mutations in ATX1 result in reduced FLC expression concomitant with reduced H3K4me3 levels at the 5#-end of FLC (Pien et al., 2008). Interestingly, the closest relative of ATX1, ATX2, affects H3K4 di-methylation more strongly than tri-methylation and may therefore possess divergent catalytic activity (Saleh et al., 2008b). After initial FLC repression during cold exposure, a second phase of vernalization is marked by a gradual increase in repressive chromatin modifications such as di-methylation and tri-methylation of lysine 27 of histone 3 (H3K27me2 and H3K27me3) and di-methylation of H3 lysine 9 (H3K9me2) at the FLC locus (Bastow et al., 2004; Sung and Amasino, 2004; De Lucia et al., 2008). During this phase, FLC repression becomes irreversible so that it persists after a return to warmer ambient temperatures. However, there appears to be a requirement for active maintenance of FLC repression as well. Cell division seems instrumental to maintain FLC repression, which is stable in young leaves that still undergo cell divisions but becomes leaky in older leaves that have terminated their development (Finnegan and Dennis, 2007). Recently, it has been reported that VRN5/VIL1 and VIN3 together with their homolog VERNALIZATION5-like 1 (VEL1)/ VIN3-like 2 (VIL2) form a complex with proteins of the Polycomb Repressive Complex 2 (PRC2) (Sung et al., 2006a; Wood

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The mechanism by which PRC1 and PRC2 repress target loci is indicated on the left. The PRC2 is recruited to target loci where it tri-methylates H3K27. The catalytic core of the PRC2 is the SET domain protein E(Z). The PRC2 is well conserved between animals and plants (compare middle and left panels, domain relatives are marked in identical color, conserved domains are indicated at the right). The plant PRC2 components are usually encoded by small gene families and form sub-complexes with distinct and overlapping functions. The PRC1 recognizes the H3K27me3 mark via the chromodomain of PC. Presumably, LHP1 fulfills an analogous function in plants although the protein is more related to HP1 than PC. The PRC1 component SCE possesses catalytic activity to ubiquitylate lysine 119 of H2A. AtRING1a and AtRING1b are the closest relatives of SCE in Arabidopsis and a link to PcG proteins has been recently reported although ubiquitylation of H2AK119 was not demonstrated (Sanchez-Pulido et al., 2008; Xu and Shen, 2008). Other PRC1 components are not identified in plants. For comprehensive recent review, see also Schwartz and Pirrotta (2007), Farrona et al. (2008), and Schatlowski et al. (2008).

pep1 mutation, the requirement for vernalization and the seasonal behavior is lost. Similar to FLC, PEP1 expression is down-regulated during prolonged exposure to cold that is accompanied by a marked increase in H3K27me3 over the PEP1 locus. However, PEP1 down-regulation and the increase in H3K27me3 are not maintained after a return to warmer ambient temperature, regardless of whether the tissue originated from inflorescent apices or from those that were preserved in a vegetative state (Wang et al., 2009). Despite the observation that different modifications of histone tails are correlated with specific transcription states in general, it is impossible to predict from the pattern of histone modification at a locus whether the gene is expressed or not. As example, ‘active’ H3K4me3 and ‘repressive’ H3K27me3 marks co-occur at the FLC locus in summer annual accessions with very low levels of FLC but also in winter annuals that express FLC highly (Figure 3C) (Jiang et al., 2008; Oh et al., 2008;

Pien et al., 2008; Saleh et al., 2008a). Such bivalent distribution of H3K4me3 and H3K27me3 modifications was first observed in mammalian embryonic stem (ES) cells, where many bivalent target genes are important for cell differentiation (Bernstein et al., 2006; Lee et al., 2006). In ES cells, the bivalent genes produce RNA, but at barely detectable levels. Upon ES cell differentiation, the ratio between the histone marks changes, leading to a complete loss of one mark that is paralleled by either expression or repression of the target genes (Schwartz and Pirrotta, 2008). At FLC, stable repression may depend on the disappearance of the H3K4me3 signal from the 5#-end of the transcribed region upon vernalization. The effect of H3K4me3 seems to be region-specific, since both H3K27me3 and H3K4me3 remain in the middle part of the FLC transcribed region throughout development (Saleh et al., 2008a). Since H3K27me3 increases upon vernalization, a quantitative aspect of ratios between

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Figure 2. Polycomb Repressive Complexes in Animals and Plants.

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Figure 3. Active and Repressive Chromatin Marks at Gene Loci Regulating Floral Transition. Cis-genome browser view of genomic ChIP-chip data for H3K27me3 (red) and H3K4me3 (green) from the van Nocker group (Ji et al., 2008; Oh et al., 2008). The material analyzed was generated from soil-grown seedlings and enrichment was calculated as the log ratio of ChIP with histone methylation-specific antibodies versus ChIP with antibodies recognizing H3 in general. (A) The genomic locus of CO illustrates that tissue-specific expression does not depend on H3K27me3, which is absent from the locus. (B) FT, LFY, and AG show only H3K27me3, whereas H3K4me3 is not enriched. Interestingly, transcription factor-associated regions that were identified by ChIP experiments for FT and LFY correlate to locally H3K27me3 depleted regions (Searle et al., 2006; Lee et al., 2008; Li et al., 2008; Liu et al., 2008). At the AG locus, the large second intron has been shown to confer transcriptional regulation by LFY and WUS (Lohmann et al., 2001). (C) FLC, AGL24, and SOC1 are bivalent H3K27me3 target genes, since they show also a clear peak of H3K4me3 at the 5#-end of the transcribed region. Transcription factor binding sites that have been mapped by ChIP experiments correlate to locally H3K27me3 depleted regions (Searle et al., 2006; Liu et al., 2007; Li et al., 2008; Liu et al., 2008).

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FLORAL PROMOTION PATHWAYS: EPIGENETICS WITHOUT MEMORY? Repression of FLC is not sufficient to induce flowering, but rather confers competence to respond to a flower promoting signal such as photoperiod. Differences in day length are perceived in the leaves that communicate via a mobile signal with the apical meristem. In the facultative LD plant Arabidopsis, flowering is promoted under long periods of light while, under SD conditions, flower initiation is delayed. Transcriptional and post-transcriptional regulation of CONSTANS (CO) is crucial for measurement of day length. CO expression is circadian-controlled and rises around ten to twelve hours after dawn but drops rapidly at the beginning of the day. Only under LD conditions does CO mRNA expression coincide with light, which allows accumulation of CO protein. CRYPTOCHROM 1 (CRY1), CRY2, and PHYTOCHROME A (PhyA) stabilize CO protein and prevent its proteasome-dependent degradation at the end of LDs (Valverde et al., 2004). During the night, CO degradation is dependent on SUPRESSOR OF PHYA-105-1 (SPA1), SPA3, and SPA4, which act in concert with the ubiquitin-ligase CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1) (Laubinger et al., 2006; Jang et al., 2008). In the morning negative regulation of CO protein abundance is mediated by PHYTOCHROME B (PhyB) (Valverde et al., 2004). Experiments with Arabidopsis plants that express chemically inducible CO indicate that CO acts as a direct activator of FT (Samach et al., 2000). CO mRNA is expressed in the vasculature and in the shoot apical region above the protophloem, but CO protein seems to be restricted to the phloem companion cells (An et al., 2004). As a result, FT mRNA accumulation occurs only in the veins of leaves (Takada and Goto, 2003). In contrast to its place of expression, FT protein acts in the meristem, where it induces the expression of the inflorescence identity genes (Abe et al., 2005; Wigge et al., 2005). FT is a small protein that migrates through the phloem into the apex and this movement is required for flowering promotion (Abe et al., 2005; Wigge et al., 2005; Corbesier et al., 2007).

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When Arabidopsis plants are grown in SD, a transient shift for three days to LD growth conditions is sufficient to irreversibly commit the plants to flower (Corbesier et al., 2007). FT expression is induced immediately upon experience of LD conditions. FT mRNA follows a diurnal pattern that is driven by CO, which means that FT expression rises towards the end of the day when CO protein accumulates. If plants are shifted from inductive LD conditions to SD, FT mRNA levels decrease to pre-induced levels during the first SD (Corbesier et al., 2007). Interestingly, although FT does not show a memory of its expression state, the FT locus is targeted by epigenetic mechanisms. The repressor LHP1 and the H3K27me3 histone mark distribute widely over the FT locus (Turck et al., 2007; Zhang et al., 2007) (Figure 3B). Lack of LHP1 and the PRC2 components EMBRYONIC FLOWER 2 (EMF2), CURLY LEAF (CLF), and FERTILIZATION INDEPENDENT ENDOSPERM (FIE) cause high expression of FT and, therefore, early flowering (Kotake et al., 2003; Moon et al., 2003b; Barrero et al., 2007; Jiang et al., 2008). This raises the question of what other functions the Polycomb group (PcG) protein-mediated regulation fulfils at FT. It has been observed that, on the genomic level, H3K27me3 target genes are more tissue-specific in their expression than the average Arabidopsis gene (Turck et al., 2007; Zhang et al., 2007). However, analysis of FT expression in the lhp1 mutant background revealed that FT expression was increased but still restricted to the CO-activity domain (Takada and Goto, 2003). CO expression, in turn, is tissue-specific, despite the absence of H3K27me3 at the CO locus (Figure 3A). Possibly, PcG proteins help to create a threshold for activation that ensures that FT is not induced under low inductive conditions. The chromatin repression could also play a role in shaping the diurnal peak of FT expression by creating an element of hysteresis. Hysteresis is a term used to describe memory effects of materials in experimental physics. For example, a piece of iron that is brought into a magnetic field retains some magnetization, even after the external magnetic field is removed. To demagnetize the iron, it would be necessary to apply a magnetic field in the opposite direction. Hysteresis of the locus response would signify that higher levels of CO are required to induce than to uphold FT expression, thereby allowing FT mRNA to accumulate throughout the first half of the night, although CO protein is rapidly degraded. Such a more dynamic role for PcG protein-mediated repression has recently also been discussed in animals where targets of H3K27me3 and PcG complexes have also been identified at a genome-wide level (Bernstein et al., 2006; Schwartz et al., 2006). Many genomic PcG targets in animals show a more dynamic regulation throughout development than that originally observed for homeotic target genes. The closest relative of FT, TSF, is induced in parallel to FT and probably is also a direct target of CO. Compared to FT, expression of TSF is very low. The tsf mutation enhances the flowering phenotype of ft in LD conditions and overexpression of TSF causes early flowering independently from the day length (Michaels et al., 2005; Yamaguchi et al., 2005).

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activating and repressing chromatin marks could also be important. Another factor to consider is the impact of other chromatin marks like di-methylated H3K9. Failure to maintain the repressed FLC state and an absence of increase in H3K9me2 upon vernalization have been reported in plants lacking the DNA binding protein VRN1 (Levy et al., 2002; Bastow et al., 2004; Sung and Amasino, 2004). The memory of stable FLC repression must be reset during the change to the next generation. FLC is re-activated after fertilization in early embryos but reports are conflicting on whether activation also occurs during male gametogenesis (Sheldon et al., 2008; Choi et al., 2009). Although FRI is expressed throughout all stages of embryo development, it is not implicated in the early activation phase. However, presence of FRI has an impact on the level of FLC expression during late embryogenesis (Choi et al., 2009).

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In conclusion, the commitment of flower development in Arabidopsis is unlikely to involve the leaf-born signal or at least the FT/TSF loci per se. This may be different in other species in which the signals coming from the leaves are more persistent. In Perilla crispa, grafting of an induced leaf to a non-induced shoot was able to induce flowering up to several times over a period of 3 months (King and Zeevaart, 1973).

THE COMMITTED NETWORK: TRANSITION FROM VEGETATIVE TO INFLORESCENCE MERISTEM

Figure 4. Meristem Identity Changes upon Floral Induction. (A) The identity of the SAM changes during floral transition from a VM to an IM. Floral pathways promoting flowering increase transcription of floral integrator genes (blue). Transition to FM identity is mediated by the so-called meristem identity genes (yellow), which are activated by the floral integrators. (B) Floral enabling pathways regulate the expression of floral repressors (red) that target the floral transition pathway at different stages and tissues.

The floral integrator gene LFY, a plant-specific single copy gene transcription factor, is already expressed in leaf primordia, but, upon floral transition, its abundance in the apex rises (Blazquez et al., 1997; Hempel et al., 1997). Inductive photoperiods and GA lead to this increased expression (Blazquez and Weigel, 2000). SOC1 and AGL24 are responsible for the regulation of LFY in response to the photoperiod (Yu et al., 2002). A recent report suggests that cytosolic SOC1 protein is transferred to the nucleus upon interaction with AGL24, and that the complex of the two proteins directly activates LFY (Lee et al., 2008). It is still not clear whether FT regulates LFY also directly or only indirectly through SOC1 (Schmid et al., 2003).

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Since the leaf-derived floral signal composed of FT and TSF appears to be required only transiently, changes that mark floral commitment are likely to be observed in the SAM (Figure 4). The earliest molecular marker for the VM-to-IM transition is the expression of the floral integrator SOC1. The expression of this MADS-box transcription factor shows a sharp increase in the apex upon floral induction, even before any physiological changes in the meristem architecture can be observed (Borner et al., 2000; Lee et al., 2000; Samach et al., 2000). The early activation of SOC1 by inductive LD conditions is dependent on FT and FD (Searle et al., 2006). Several reports suggest that FT and the bZIP transcription factor FD interact in the SAM, and directly regulate gene expression, such as activating the meristem identity gene, APETALA1 (AP1) (Abe et al., 2005; Wigge et al., 2005). Whether the activation of SOC1 by FT/FD is direct has to be investigated. Later in development activation of SOC1 in SD conditions is mediated through the GA pathway (Moon et al., 2003a). SOC1 and the floral integrator AGL24 mutually activate each other by direct binding to their respective promoters (Liu et al., 2008). Similar to SOC1, the expression of the MADS-box transcription factor AGL24 is up-regulated by inductive photoperiods, but also by the autonomous pathway and vernalization, even though the latter is mediated in a FLC-independent manner (Yu et al., 2002; Michaels et al., 2003). Together, AGL24 and SOC1 form parallel entry points to a self-maintained loop that, as such, should be independent of primary inductive signals (Figures 4 and 5A). However, at least SOC1 up-regulation is a steep uphill race against repression mediated by the MADS-box transcription factor SVP (Li et al., 2008). SVP directly binds to SOC1 at regions that are also targeted by FLC (Searle et al., 2006; Li et al., 2008). Since SVP and FLC interact, these transcription factors may bind as a complex and, therefore, residual levels of FLC that are expressed in non-vernalized summer accessions may also play a role in antagonizing the SOC1/AGL24 loop (Figures 4B and 5A) (Li et al., 2008). AGL24 and SOC1 are partially redundant, since double mutants flower later than single mutants and the effect of single mutants on flowering is mild. However, the impact of mutations in SOC1 and AGL24, either single or double, is more pronounced in SD than LD, which can be explained by the direct induction of AP1 by FT/FD (Figure 4A).

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Four main mechanisms ensure a linear progression from vegetative to inflorescence meristems and further to floral meristems in Arabidopsis. These mechanisms also permit inflorescence and floral meristems to co-exist. (A) Back-locked feed-forward loop by AGL24/SOC1, LFY, and AP1. Mutual activation of SOC1 and AGL24 stabilizes the flower transition signal and SOC1 directly activates LFY (Lee et al., 2008; Liu et al., 2008). LFY feeds forward to activate AP1. Repression of SOC1/AGL24 in the emerging floral meristems is a prerequisite to define floral identity and seems to be mediated by direct interaction of AP1 with SOC1 and AGL24 regulatory regions (Liu et al., 2007). (B) Positive loop between the genes LFY and AP1/CAL. Upon floral transition, AP1 is directly up-regulated by the FT/FD dimer and LFY (Figure 4A). LFY induces CAL transcription directly and through a feed-forward motif that includes LMI1 (Wagner et al., 1999; Schmid et al., 2003; Wagner et al., 2004; Saddic et al., 2006). Increased abundance of AP1 and CAL leads back to increased LFY expression, which might be a direct effect or in part be mediated by antagonizing TFL1 (Liljegren et al., 1999). Thus, LFY and AP1/CAL reinforce each other’s expression and stabilize the floral development program. (C) Toggle-switch between the floral identity genes LFY and AP1/CAL and the shoot identity gene TFL1. LFY and AP1/CAL are only activated in floral primordia but not in inflorescence meristems, which therefore remain indeterminate (Shannon and Meekswagner, 1991; Liljegren et al., 1999). The floral repressor TFL1 prevents expression of LFY and AP1/CAL in inflorescence meristems (Bradley et al., 1997). Since LFY represses TFL1, and AP1 and CAL seem also to negatively regulate TFL1, the expression domains of these groups of genes do not overlap (Liljegren et al., 1999; Ratcliffe et al., 1999; Parcy et al., 2002). (D) Back-locked feed-forward loop created by LFY/WUS and AG. This regulatory loop determinates the floral meristems. LFY and WUS bind to regulatory regions in the second AG intron and are both required to activate AG expression in the early floral meristem. AG maintains its own expression by direct positive feed-back (Gomez-Mena et al., 2005). Increased expression of AG represses WUS and LFY from the initiation of carpel primordia onwards by a not yet identified mechanism (Mayer et al., 1998).

Interestingly, the SOC1, AGL24, and LFY loci are also widely associated with the repressive H3K27me3 mark and therefore potentially regulated by an epigenetic mechanism (Oh et al., 2008). In addition, SOC1 and AGL24 show a strong accumulation of H3K4me3 at the 5#-end of the transcribed region and therefore fall into the class of bivalent PcG targets (Figure 3C). However, as for FT, the function of chromatin modifications at these loci is not known and it is unclear whether the mark is involved in memorizing an expression state. In fact, since cells in the apex change their position with growth and considering the temporal and spatial expression pattern of SOC1, AGL24, and LFY, these genes are only transiently expressed in a given cell, which argues strongly against a long-term epigenetic fixation of the expression state. Strikingly, the regions within the LFY, SOC1, and AGL24 loci that were shown to be direct targets

of these and other transcription factors coincide with local gaps in H3K27me3 regions (Figure 3) (Gregis et al., 2008; Lee et al., 2008; Liu et al., 2008; Oh et al., 2008). This is even more remarkable if one considers that the histone mark profiles were carried out with chromatin from entire seedlings, whereas the expression domain of the meristem identity genes is tissue-restricted. However, similar observations have been made in Drosophila melanogaster (Drosophila), where it has been shown that Polycomb responsive elements (PREs) are rather depleted in H3K27me3 content, although they represent the peak of binding for PRC2 proteins (Schwartz et al., 2006). Typically, PREs are up to several thousand base-pair in sequence and contain elements recruiting proteins of the Trithorax group (TrxG) in addition to those that cause PRC2 association (Schwartz and Pirrotta, 2008). Besides SET-domain

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Figure 5. Transcriptional Loops Mediating Floral Commitment.

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proteins related to Trithorax, the TrxG proteins include transcription factors that oppose the effect of PcG proteins on the transcription of their shared targets. In Drosophila, the PREs are sites of increased nucleosome turn-over, which results in a general depletion in histone content and a higher ratio of the replacement form of H3 (Mito et al., 2007). Although, in Arabidopsis nucleosome-depleted regions were not overrepresented within or adjacent to H3K27me3 regions, there may be a hidden function worthwhile pursuing in the gaps observed within H3K27me3 regions (Zhang et al., 2007; Oh et al., 2008).

The transition from an inflorescence to a floral meristem requires the function of a set of genes, called floral meristem identity genes. The generation of FMs is not only irreversible, but it also needs to be locally restricted so that inflorescence and FMs can co-exist in close proximity. In Arabidopsis LFY, AP1, CAULIFLOWER (CAL), and possibly also FRUITFULL (FUL) have been identified as genes that confer floral identity to the arising meristems (Figure 4) (Blazquez et al., 2006). The closely related MADS-box transcription factors AP1, CAL, and FUL are bivalent H3K27me3/H3K4me3 target genes (Oh et al., 2008). LFY acts as a key regulator in the meristem. Besides its floral integrator function, LFY is essential to confer determinacy to the FM and determines floral organ patterning (Weigel et al., 1992; Parcy et al., 1998; Wagner et al., 1999). Mutations of LFY in Arabidopsis result in conversion of the FMs into inflorescence shoots in SD conditions (Schultz and Haughn, 1991; Weigel et al., 1992). Conversely, ectopic LFY expression results in a conversion of the IM into a terminal flower (Weigel and Nilsson, 1995). Through induction of AP1 and other floral homeotic genes, LFY determines floral organ patterning and meristem growth (Parcy et al., 1998; Wagner et al., 1999). Together with the homeodomain transcription factor WUSCHEL (WUS), LFY activates the floral homeotic gene AGAMOUS (AG) that plays an important role in terminating meristem growth (Figure 5D). Induction of the direct LFY target genes AP1 and CAL defines FM identity (Mandel et al., 1992; Mandel and Yanofsky, 1995; Wagner et al., 1999; William et al., 2004). LFY and CAL are also indirectly connected by a feed-forward loop transcriptional network that includes LATE MERISTEM IDENTITY 1 (LMI1) (Figure 5B) (Saddic et al., 2006). LFY directly activates the homeodomain-zipper transcription factor LMI1 and both LFY and LMI1 directly bind to proximal promoter regions of CAL. Combination of LMI1 loss-of-function with weak lfy alleles leads to a reduced induction of CAL and enhances the lfy phenotype. In general, feed-forward motifs stabilize the expression of the output (in this case CAL) if the strength of the input signal (in this case LFY) fluctuates (Alon, 2007).

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REMEMBER THE NEXT STEP: THE TRANSITION FROM INFLORESCENCE TO FLORAL MERISTEM

AP1 and CAL are expressed in young flower primordia that rise from the inflorescence meristems and act in a redundant way to specify floral meristem identity (Mandel et al., 1992). While loss of AP1 results in moderate defects in floral meristem identity and floral organ identity and loss of function of CAL does not show any visible phenotype, floral meristems of ap1 cal double mutants undergo a complete transformation into inflorescence meristems (Bowman et al., 1993). The primordia produced on the flanks of the IM cannot develop flowers, but produce new meristems that reiterate this pattern indefinitely, forming structures similar to cauliflower heads (Bowman et al., 1993). However, late in development, even these structures eventually gain floral identity and produce stamens and carpels. Conversely, overexpression of AP1 or CAL results in the conversion of the IM into a terminal flower (Mandel and Yanofsky, 1995). Once AP1 and CAL are activated by LFY, they are able to induce expression of LFY (Bowman et al., 1993). This creates a positive loop that is important to establish a stable floral induction, regardless of the environmental conditions (Figures 4A and 5B). FUL is involved in carpel and fruit development (Gu et al., 1998). Mutation of FUL does not affect FM identity, but enhances the ap1 cal double mutant phenotype so that the meristems of ap1 cal ful triple mutants fail to acquire floral identity but generate cauline leaf-like structures (Gu et al., 1998; Ferrandiz et al., 2000). FUL is strongly up-regulated in the IM during the floral transition but absent from young developing flowers until its up-regulation in carpel primordia; therefore, FUL’s precise role in flower development is still unclear. Its expression pattern is negatively correlated with that of AP1 but the mechanism of this regulation has not yet been elucidated (Ferrandiz et al., 2000). The floral repressor TFL1 confers shoot identity to the meristem and therefore has an antagonistic role to the FM identity genes LFY, AP1, CAL, and possibly FUL (Figure 5C). In tfl1 mutants, the meristems of the inflorescence shoots are converted into FMs and form terminal flowers (Shannon and Meekswagner, 1991). The transformation of the IM into a FM in tfl1 mutants is due to ectopic expression of AP1 and LFY (Weigel et al., 1992; Bowman et al., 1993; Gustafsonbrown et al., 1994; Bradley et al., 1997). In wild-type plants the expression of TFL1 occurs in the center of the SAM, just below the IM, where it prevents LFY and AP1 expression and the consequent termination of the IM (Bradley et al., 1997). In contrast, LFY represses TFL1, and AP1 and CAL seem also to negatively regulate TFL1 (Liljegren et al., 1999; Ratcliffe et al., 1999; Parcy et al., 2002). In consequence, TFL1 on the one side and LFY, AP1/CAL on the other side form a toggle-switch transcriptional network motif that results in mutually exclusive expression domains (Figure 5C). However, TFL1 protein is mobile and spreads through the meristem beyond the region where TFL1 mRNA is transcribed. In addition, it seems that a signal from LFY feeds back to promote this protein movement, adding even more complexity to the picture (Conti and Bradley, 2007).

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WHAT CAN BE LEARNED IF THINGS GO WRONG? REVERSION IN ARABIDOPSIS Failure to maintain the floral commitment in the absence of inductive conditions leads to different developmental changes that have been subsumed under the term ‘floral reversion’.

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A switch from floral development back to more vegetative development can occur at different stages. Inflorescence reversion takes place during or after inflorescence development and leads back to formation of leaves and vegetative shoots. Flower reversion that occurs later in floral development results in alteration of the flower. Incomplete flowers or replacement of some flower parts by leaves are characteristic for this type of reversion but also reversion from determinate to indeterminate meristem growth (Tooke et al., 2005). Floral reversion phenomena are closely linked to failure of Arabidopsis plants to make floral conversion and it is not always possible to distinguish between those two observations. Reversion in wildtype Arabidopsis occurs partially, only at low frequency and mainly from floral to inflorescence development. For example, the first flowers formed in the Arabidopsis accession Landsberg erecta under SD conditions can occasionally revert to indeterminate inflorescence-type development (Okamuro et al., 1996). Consistent with their role to confer floral identity, lack of meristem identity genes enhances the frequency of flower reversion in Arabidopsis. Nevertheless, heterozygous lfy and homozygous ap1 or ag mutants only revert under non-inductive SD conditions (Okamuro et al., 1996, 1997). A combination of ag mutations with mutations in the photoperiod pathway can mimic low inductive conditions, which again causes floral reversion (Mizukami and Ma, 1997). In contrast, increased gibberellin concentrations suppress SD-induced reversion in heterozygous lfy and homozygous ag mutants (Okamuro et al., 1996). As discussed previously, LFY and AP1 are connected by a mutually positive feed-back loop that is important to establish floral development (Figure 5B). AP1 activation by the FT/TSF systemic signal occurs either directly or indirectly through LFY. It is possible that the mis-functioning of the AP1/LFY loop in ap1 mutant or lfy heterozygous plants needs to be compensated by a continued input from the photoperiod-induced signal. GA induces SOC1 and LFY expression independently of photoperiod and may thereby substitute the photoperiod signal. A special case of reversion in Arabidopsis is represented by the late flowering accession Sy-0, in which an alteration of the body plan causes aerial rosette production in the axils of cauline leaves, and inflorescence and floral reversion. The consequence of this morphological change is reflected in a longer vegetative phase, increasing the life of the plant to at least 1.5 years. This phenotype might be due to a lower level of floral signals or a lower competence to respond to them. Two genes, an active FRI allele and the gene AERIAL ROSETTE 1 (ART1), synergistically activate the floral repressor FLC and are responsible for the Sy-0 phenotype (Poduska et al., 2003). The expression levels of SOC1 and FUL are strongly reduced in Sy-0, which could partially explain similarities of this accession with soc1 ful double mutants in the Columbia and Landsberg erecta background. Loss of function of SOC1 and FUL leads to an unexpected inflorescence reversion phenotype (Melzer et al., 2008). In these plants, some apical inflorescence

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Since tfl1 mutants flower early, TFL1 is also a repressor of the floral transition (Shannon and Meekswagner, 1991; Bradley et al., 1997). These two roles of TFL1 in shoot identity and flowering seem not to be separate, but rather based on a general mechanism to govern the transition at the SAM by TFL1 (Ratcliffe et al., 1998). Like FT, TFL1 is not a transcription factor and belongs to a family of proteins with homology to phosphatidyl ethanolamine-binding proteins (Ahn et al., 2006). Similar to the FT/FD module, TFL1 might interact with another bZIP transcription factor or compete with FT for FD binding (Abe et al., 2005; Wigge et al., 2005). The activation of AG by LFY and WUS and the repression of WUS by AG create a back-locked feed-forward loop motif that ensures the relationships between determinate and indeterminate meristems (Figure 5D). In a back-locked feed-forward loop, element A (here, WUS and LFY) induces element B (here, AG) and both A and B induce element C (in this case, also AG, see below). Last, element C negatively feeds back on A, thereby becoming independent from the primary inducer. The meristem maintenance factor WUS is expressed in the floral meristem in early phase, but decreases when AG is activated and disappears when carpel primordia initiate (Mayer et al., 1998). LFY and WUS bind to regulatory regions in the second AG intron and are both required to activate AG expression in the early FM (Busch et al., 1999; Lohmann et al., 2001). In addition, AG maintains its own expression by direct positive feedback (Gomez-Mena et al., 2005). It is not yet clear whether the repression of WUS by AG is direct or mediated by another transcription factor. The AG locus is widely decorated with the H3K27me3 mark and AG expression is up-regulated in Arabidopsis plants that carry mutations in PcG-proteins and lhp1 (Goodrich et al., 1997; Hennig et al., 2003; Nakahigashi et al., 2005; Schubert et al., 2006). In contrast to the situation observed for FT, AG expression in lhp1 and clf mutant background is ectopic. In these mutants, AG is highly expressed in leaves, where it activates the floral homeotic gene, AP3 (also a H3K27me3 positive locus). The ectopic expression of AP3 is mainly responsible for the curled leaf phenotype of the clf and lhp1 mutant (Goodrich et al., 1997; Kotake et al., 2003). In conclusion, one role of the epigenetic mechanism observed at AG could be to limit the access to cis-regulatory elements at the locus and thereby prevent out-of-domain expression. Interestingly, AG transcription is also dependent on ATX1 and AG has been identified as bivalent H3K4me3/H3K27me3 target (Saleh et al., 2007). Double atx1 clf mutants lose the curled leaf phenotype observed in clf mutant plants concomitant with a reduction in ectopic expression of AG (Saleh et al., 2007).

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meristems revert to a vegetative state, leading to the formation of small true leaves with axillary meristems in apical rosettes. Bud-type shoots and aerial rosettes form new inflorescences, which repeatedly start a new growth cycle. The phenotype, in addition to the markedly increased lifespan of these plants and extensive secondary growth, strongly resembles the characteristics of perennial plants.

REVERSION AS RULE

CONCLUSIONS Floral commitment and its maintenance clearly require a molecular memory of gene expression. Sometimes, this memory is mediated by epigenetic mechanisms that implicate chromatinbased gene regulation. A beautiful example is the stable repression of FLC by vernalization, although, also in this case, repression seems to require active maintenance. In contrast, the sole presence of histone marks such as H3K27me3 that have the potential to ‘encode’ epigenetic memory does not allow the conclusion that a target gene relies on epigenetic mechanisms to achieve bistable regulation. Epigenetic mechanisms may also be the basis of threshold sensitivity, response hysteresis, and tissue specificity of target loci. In contrast, bistable gene switches such as required for flower development can be built by transcriptional network motifs that are shared between eukaryotes and non-chromatin organisms such as bacteria. Intriguingly, however, the genes that built these regulatory loops also show a propensity to be regulated by epigenetic mechanisms. Clearly, future work needs to consider how to combine these two models of molecular memory into one function.

FUNDING Jessika Adrian was supported by an International Max Planck Research School (IMPRS) studentship. Stefano Torti was supported by an EC funded Marie Curie studentship through the training network TRANSISTOR. The work in Franziska Turck’s laboratory is supported by a core grant from the Max Planck Society. No conflict of interest declared.

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