Light-regulated large-scale reorganization of chromatin during the floral transition in Arabidopsis

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Light-regulated large-scale reorganization of chromatin during the floral transition in Arabidopsis Federico Tessadori, Roeland Kees Schulkes, Roel van Driel and Paul Fransz* Nuclear Organization Group, Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, University of Amsterdam, Kruislaan 318, 1098 SM, Amsterdam, The Netherlands Received 8 November 2006; revised 30 January 2007; accepted 9 February 2007. *For correspondence (fax + 31205257935; e-mail [email protected]). Summary The floral transition marks the switch from vegetative to reproductive growth, and is controlled by different pathways responsive to endogenous and exogenous cues. The developmental switch is accompanied by local changes in chromatin such as histone modifications. In this study we demonstrate large-scale reorganization of chromatin in rosette leaves during the floral transition. An extensive reduction in chromocenters prior to bolting is followed by a recovery of the heterochromatin domains after elongation of the floral stem. The transient reduction in chromocenters is a result of relocation away from chromocenters of methylated DNA sequences, 5S rDNA and interspersed pericentromeric repeats, but not of 45S rDNA or the 180-bp centromere tandem repeats. Moreover, fluorescence in situ hybridization analysis revealed decondensation of chromatin in gene-rich regions. A mutant analysis indicated that the blue-light photoreceptor CRYPTOCHROME 2 is involved in triggering chromatin decondensation, suggesting a light-signaling pathway towards large-scale chromatin modulation. Keywords: Arabidopsis, floral transition, nucleus, heterochromatin. Introduction Floral transition is a fundamental stage in plant develop- ment, during which the shoot apical meristem switches from generating leaf primordia to production of reproductive organs. A pivotal factor for successful plant reproduction is the timing of the floral transition, as favorable environ- mental conditions are essential for fertilization and optimal seed production. Plants have developed various strategies to comply with this aim. For example, although facultative long-day plants, such as Arabidopsis, are induced to flower by a rise in temperature and increasing day length, flowering in maize and rice is promoted by shorter days. In the dicot- yledonous short-day plant Pharbitis nil, even a single exposure to a 14-h night is floral inductive (Liu et al., 2001; Vince-Prue and Gressel, 1985). A number of different pathways have been described in Arabidopsis that induce the floral transition (Boss et al., 2004; He and Amasino, 2005). Two of these, the vernalization and photoperiod pathways, are dependent on environmen- tal factors and promote flowering after a prolonged cold period and a long-day regime, respectively. In contrast, intrinsic control mechanisms, such as the gibberellin path- way and the autonomous pathway, are largely dependent on endogenous developmental signals. The external and endogenous cues converge into a complex interaction between flower-inhibiting and -promoting signals, the bal- ance of which determines the timing of the floral transition. Flower-inducing signals are integrated at the downstream end of the flower-promoting pathways by the MADS-box protein SUPPRESSOR OF OVEREXPRESSION OF CON- STANS 1 (SOC1) and FLOWERING LOCUS T (FT), of which the biochemical function is unknown. These proteins control the expression of floral meristem identity genes such as LEAFY (LFY) and APETALA 1 (AP1; reviewed in Boss et al., 2004; Putterill et al., 2004). SOC1 and FT themselves are under direct control of CONSTANS (CO), a zinc-finger transcription factor, and FLOWERING LOCUS C (FLC), a MADS-box transcription factor. Flowering time is also controlled by the photoreceptors PHYTOCHROME A (PHYA) and CRYPTOCHROME 2 (CRY2) through the photoperiod pathway, by positively regulating CO activity (Valverde et al., 2004; Yanovsky and Kay, 2002). Under long-day light conditions CO exerts a positive control on floral meristem 848 ª 2007 The Authors Journal compilation ª 2007 Blackwell Publishing Ltd The Plant Journal (2007) 50, 848–857 doi: 10.1111/j.1365-313X.2007.03093.x

Transcript of Light-regulated large-scale reorganization of chromatin during the floral transition in Arabidopsis

Light-regulated large-scale reorganization of chromatinduring the floral transition in Arabidopsis

Federico Tessadori, Roeland Kees Schulkes, Roel van Driel and Paul Fransz*

Nuclear Organization Group, Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, University of Amsterdam,

Kruislaan 318, 1098 SM, Amsterdam, The Netherlands

Received 8 November 2006; revised 30 January 2007; accepted 9 February 2007.

*For correspondence (fax + 31205257935; e-mail [email protected]).

Summary

The floral transition marks the switch from vegetative to reproductive growth, and is controlled by different

pathways responsive to endogenous and exogenous cues. The developmental switch is accompanied by local

changes in chromatin such as histone modifications. In this study we demonstrate large-scale reorganization

of chromatin in rosette leaves during the floral transition. An extensive reduction in chromocenters prior to

bolting is followed by a recovery of the heterochromatin domains after elongation of the floral stem. The

transient reduction in chromocenters is a result of relocation away from chromocenters of methylated DNA

sequences, 5S rDNA and interspersed pericentromeric repeats, but not of 45S rDNA or the 180-bp centromere

tandem repeats. Moreover, fluorescence in situ hybridization analysis revealed decondensation of chromatin

in gene-rich regions. A mutant analysis indicated that the blue-light photoreceptor CRYPTOCHROME 2 is

involved in triggering chromatin decondensation, suggesting a light-signaling pathway towards large-scale

chromatin modulation.

Keywords: Arabidopsis, floral transition, nucleus, heterochromatin.

Introduction

Floral transition is a fundamental stage in plant develop-

ment, during which the shoot apical meristem switches from

generating leaf primordia to production of reproductive

organs. A pivotal factor for successful plant reproduction is

the timing of the floral transition, as favorable environ-

mental conditions are essential for fertilization and optimal

seed production. Plants have developed various strategies

to comply with this aim. For example, although facultative

long-day plants, such as Arabidopsis, are induced to flower

by a rise in temperature and increasing day length, flowering

in maize and rice is promoted by shorter days. In the dicot-

yledonous short-day plant Pharbitis nil, even a single

exposure to a 14-h night is floral inductive (Liu et al., 2001;

Vince-Prue and Gressel, 1985).

A number of different pathways have been described in

Arabidopsis that induce the floral transition (Boss et al.,

2004; He and Amasino, 2005). Two of these, the vernalization

and photoperiod pathways, are dependent on environmen-

tal factors and promote flowering after a prolonged cold

period and a long-day regime, respectively. In contrast,

intrinsic control mechanisms, such as the gibberellin path-

way and the autonomous pathway, are largely dependent on

endogenous developmental signals. The external and

endogenous cues converge into a complex interaction

between flower-inhibiting and -promoting signals, the bal-

ance of which determines the timing of the floral transition.

Flower-inducing signals are integrated at the downstream

end of the flower-promoting pathways by the MADS-box

protein SUPPRESSOR OF OVEREXPRESSION OF CON-

STANS 1 (SOC1) and FLOWERING LOCUS T (FT), of which

the biochemical function is unknown. These proteins control

the expression of floral meristem identity genes such as

LEAFY (LFY) and APETALA 1 (AP1; reviewed in Boss et al.,

2004; Putterill et al., 2004). SOC1 and FT themselves are

under direct control of CONSTANS (CO), a zinc-finger

transcription factor, and FLOWERING LOCUS C (FLC), a

MADS-box transcription factor. Flowering time is also

controlled by the photoreceptors PHYTOCHROME A (PHYA)

and CRYPTOCHROME 2 (CRY2) through the photoperiod

pathway, by positively regulating CO activity (Valverde

et al., 2004; Yanovsky and Kay, 2002). Under long-day light

conditions CO exerts a positive control on floral meristem

848 ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd

The Plant Journal (2007) 50, 848–857 doi: 10.1111/j.1365-313X.2007.03093.x

identity genes, among others through the production of a

long-distance systemic florigen signal in the leaves (An

et al., 2004; Huang et al., 2005; Martinez-Garcia et al., 2002;

Zeevaart, 1976). It was recently demonstrated that the FT

gene product is an important component of this signal

(Huang et al., 2005). In contrast to CO, FLC has an inhibiting

action on the expression of the SOC1 and FT genes

(Hepworth et al., 2002). FLC is under the control of several

regulators, including the FRIGIDA gene (FRI), which upreg-

ulates FLC expression (Johanson et al., 2000; Sheldon et al.,

1999). The active FLC locus is associated with transcriptional

activation hallmarks, such as methylation at lysine 4 of

histone 3 and acetylation of histones (He et al., 2003, 2004).

FLC repression is achieved by dimethylation of histone H3 at

lysine 9 (H3 K9me2), mediated by VERNALIZATION INSEN-

SITIVE 1 (VIN1), VERNALIZATION 1 (VRN1) and a Polycomb-

like protein VERNALIZATION 2 (VRN2; Bastow et al., 2004;

Gendall et al., 2001; Levy et al., 2002; Sung and Amasino,

2004). Another protein that epigenetically regulates flower-

ing time is the Arabidopsis HP1 homologue LIKE HETERO-

CHROMATIN PROTEIN 1 (LHP1), also called TERMINAL

FLOWER 2 (TFL2) (Gaudin et al., 2001; Kotake et al., 2003;

Larsson et al., 1998). LHP1 may participate in repression of

the FT gene by counteracting CO (Takada and Goto, 2003).

Consequently, lhp1 mutants are early flowering. The mutant

appears to be impaired in FLC repression, possibly through

interaction with VRN1 (Mylne et al., 2006). VRN1 binds to

chromatin, but the binding site is not restricted to the FLC

locus. Similar to LHP1, the VRN1 protein decorates euchro-

matin regions. Whether the function of VRN1 is part of a

machinery that affects more genes apart from FLC, like LHP1,

(Gaudin et al., 2001; Kotake et al., 2003; Libault et al., 2005) is

not known.

Floral transition is a major developmental switch that

involves physiological processes in the apical meristem, but

also in the rosette leaves, which sense the changing

environmental conditions such as the photoperiod. The

accumulation of light signals triggers a response in the

leaves that is transduced to the shoot apical meristem.

Considering that several components of the light-signa-

ling pathway operate in the nucleus, we addressed the

question of whether the floral transition affects nuclear

processes such as chromatin compaction, involving large

regions of the Arabidopsis genome. In the Arabidopsis

nucleus, the pericentric heterochromatin and the inactive

ribosomal genes are generally clustered into compact

chromatin domains, so-called chromocenters (Fransz et al.,

2002), whereas gene-rich regions, including active and

inactive genes, are largely outside the chromocenters

(Fransz et al., 2006). Unless stated otherwise, in this study

we use the term heterochromatin to indicate compact

chromatin or chromocenters. Previous studies have repor-

ted changes in the appearance of chromocenters under

certain physiological, developmental conditions. For exam-

ple, changes in heterochromatin organization have been

observed during seedling differentiation (Mathieu et al.,

2003), ageing of rosette leaves (Tessadori et al., 2004), de-

differentiation to protoplasts (Tessadori et al., 2007) and

pathogen infection (Pavet et al., 2006). In this study we

demonstrate a transient large-scale reduction of compact

chromatin over a short period (2–4 days) that corresponds

specifically with the floral transition. Mutant analysis of

photoperiod pathway components revealed no typical

reduction in compact chromatin in plants lacking the blue-

light photoreceptor CRY2, suggesting that CRY2 mediates

light-signaling to chromatin organization during the floral

transition.

Results

Mesophyll cells of rosette leaves display large-scale reor-

ganization of heterochromatin domains prior to bolting

The floral transition is marked by changes in the expression

of hundreds of genes (Schmid et al., 2003). We addressed the

question of whether this developmental switch involves

major changes in large-scale chromatin organization.

Parenchyma nuclei from inflorescences display 6–10 con-

spicuous chromocenters (Fransz et al., 2002). We observed a

similar phenotype in nuclei from rosette leaves (Figure 1a).

Up to 20% of the nuclei, however, displayed a phenotype

with smaller or elongated chromocenters and more hetero-

geneous euchromatin (Figure 1b). Remarkably, the fraction

of nuclei with this aberrant phenotype increased to 50%

when visible flower buds appeared at the center of the ro-

sette, which corresponds to stage 5.10 (Boyes et al., 2001).

We therefore carried out a quantitative analysis of

heterochromatin at different developmental stages around

the floral transition. We measured the mean relative hetero-

chromatin fraction (RHF), which is determined by the area

and fluorescence intensity of all chromocenters in relation to

the area and fluorescence intensity of the entire nucleus

(Soppe et al., 2002; Tessadori et al., 2004). Four days before

the appearance of the flower buds (stage 1.09) the RHF

showed a sharp reduction in compact chromatin content

(Figure 1c). We then established the percentage of nuclei

with normal chromocenter appearance (i.e. number, shape

and size), here denoted as the heterochromatin index (HX).

The HX decreased from 70% to about 50% during the same

period before bolting (Figure 1d). Apparently, the appear-

ance of flower buds is accompanied by a large-scale

decondensation of chromocenters. Moreover, within

3 days after bolting the RHF and the HX increased to normal

values, indicating that the reduction of heterochromatin is

transient.

We addressed the question if the change in nuclear

organization is dependent on the flowering time, and

examined two accessions that differ from Col-0 in flowering

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time. Ler flowers earlier (day 17), whereas Cvi-0 flowers

more than 30 days after germination. We monitored the RHF

value of Col-0, Ler and Cvi-0 plants before and during the

floral transition (Figure 2). The results indicate that the

decrease in chromocenters corresponds with the transition

to flowering irrespective of the flowering time. Strikingly,

although Cvi-0 constitutively shows a lower content of

condensed chromatin compared to Col-0 or Ler, it still

Figure 1. Morphological changes of mesophyll

nuclei and reduction in compact chromatin dur-

ing the floral transition.

(a) and (b) 4¢,6-Diamidino-2-phenylindole stained

nuclei before the floral transition (a) and 2-4 days

prior to bolting (b). Note the difference in chro-

mocenter morphology. Scale bar = 5 lm.

(c) and (d) Quantification of compact chromatin

content by means of relative heterochromatin

fraction (c) and heterochromatin index (d). Bars

represent stages before (white) or after (gray)

bolting. For each bar the data are mean � SEM

of more than 30 nuclei (c) or more than 68 nuclei

(d).

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ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 50, 848–857

displays a decrease in heterochromatin during flower trans-

ition (Figure 2).

To investigate if heterochromatin decondensation is cor-

related with the transition to flowering, we delayed the floral

transition in Col-0 plants by growing them under a shorter

day regime (SD, 12-h light/12-h dark). These plants bolted

19 days later than plants grown under long-day conditions

(LD, 16-h light/ 8-h dark).

In leaves of SD-grown plants the RHF value decreased

after 38 days, which is just before bolting, and recovered

after the appearance of the first flower buds (stage 5.10;

Figure 3a). The simultaneous delay in both bolting and

heterochromatin reduction further indicates a strong corre-

lation between heterochromatin reorganization and floral

transition.

We finally tested if chromatin reorganization can be

promoted by transferring plants from a short-day to a

long-day light regime. Such a transfer will induce the

transition from a vegetative state to a reproductive state.

Col-0 plants were grown under short-day conditions for

37 days and subsequently transferred to a long-day regime.

Only 24 h after the transfer the mesophyll nuclei showed a

sharp decrease in RHF value (Figure 3b). By 4 days after the

transfer the plants produced visible flower buds and the

mesophyll nuclei displayed a normal appearance. These

observations confirm that the decrease in heterochromatin

coincides with the period during which bolting is triggered.

Both pericentromeric heterochromatin and gene-rich chro-

matin show large-scale decondensation during the floral

transition

The reduction of chromocenters indicates that certain

chromosomal regions are no longer in heterochromatin

domains. To determine which DNA sequences are involved

in the reduction of heterochromatin, we applied fluores-

cence in situ hybridization (FISH) with probes of dispersed

and tandemly arranged repeat sequences, known to localize

in chromocenters (Fransz et al., 2002): (i) the 180-bp cen-

tromeric tandem repeat, (ii) the pericentromeric BAC F28D6,

which contains numerous interspersed repeats, such as

transposable elements present on all chromosomes, and (iii)

tandemly arranged 5S and 45S rRNA sequences. All repeats

are condensed and reside in chromocenters of mesophyll

nuclei before the floral transition. During the floral transition

the 180-bp tandem repeat and 45S rDNA sequences re-

mained condensed in chromocenters (Figure 4a). In con-

trast, pericentromeric sequences, targeted by a 5S rDNA

probe and BAC F28D6, were decondensed and dispersed.

This decondensation is likely to be responsible for the

reduction in heterochromatin and the decreased RHF.

To find out whether euchromatic regions are also affected

during the floral transition, we monitored the compaction

state of an approximately 0.3-Mb segment in gene-rich chro-

matin. We applied FISH with the BAC contig F28A21, F13C5

and T18B16, which map to the long arm of chromosome 4,

Figure 2. The reduction of heterochromatin in three accessions.

The relative heterochromatin fraction was measured in Col-0, Ler and Cvi-0

plants grown under long-day conditions before (white bars) and during the

floral transition (gray bars). For each bar the data are mean � SEM of more

than 15 nuclei.

Figure 3. Heterochromatin fraction under short-day (SD) conditions.

(a) Relative heterochromatin fraction (RHF) of plants grown in SD conditions

(12-h light/12-h dark). FS: plants have developed a floral stem, but no flower is

yet open.

(b) RHF of plants grown in SD conditions for 37 days and then transferred to

long-day (LD) conditions. Note the decrease in RHF only 24 h after transfer

and the bolting after 96 h. Bars represent stages before (white) or after (gray)

bolting. For each bar the data are mean � SEM of more than 30 nuclei.

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Figure 4. Cytological localization of repetitive sequences, gene-rich regions and DNA methylation.

(a) Fluorescence in situ hybridization (FISH) localization of repetitive sequences (right) and 4¢,6-diamidino-2-phenylindole (DAPI) counterstaining (left) in mesophyll

nuclei during the floral transition. Note that although the pericentromeric repeat-rich F28D6 BAC (I, green) and 5S rDNA (II, green) are dispersed in nuclei during

floral transition, the centromeric 180-bp (I, II and III, red) and the subtelomeric 45S rDNA (III, green) regions remain compact and colocalize with chromocenters.

(b) FISH localization of the F28A21-F13C5-T18B16 BAC contig, covering approximately 0.3 Mb. F13C5 appears in red; T18B16 and F28A21 appear in green. The high

compaction state prior to floral transition (I; arrows) is lost in nuclei during the 2–4-day period before bolting (II; arrows). Nuclei are counterstained with DAPI.

(c) Fraction of nuclei with normal levels of heterochromatin (gray), and condensed FISH signals of BAC F13C5 (red) and T18B16-F28A21 (green) before and during

the floral transition. Data are mean � SEM of 29 nuclei.

(d) Immunolocalization of 5-methylcytosine (5-mC, right) counterstained with DAPI (left). (I) Prior to floral transition 5-mC signals are clustered at chromocenters. (II)

A dispersed pattern of 5-mC is observed in nuclei with low chromocenter content during floral transition. Scale bars = 5 lm.

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ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 50, 848–857

and measured the frequency of relatively compact FISH

signals before and during the floral transition (Figure 4b).

Concomitant to the decrease in chromocenters, the occur-

rence of condensed BAC signals decreased by about 33%

(Figure 4c), indicating that the decompaction of chromatin is

not restricted to the heterochromatic sections of the genome.

As chromocenters are rich in methylated DNA (Fransz et al.,

2002) and because a 5-methyl-cytosine (5-mC) imprint is

required to maintain a chromocenter state (Soppe et al.,

2002), we examined leaf nuclei for changes in nuclear

distribution of 5-mC. Col-0 nuclei displayed a dispersed

pattern of 5-mC during the floral transition (Figure 4d),

similar to the dispersion of the 5S rDNA and the transpo-

son-rich BAC F28D6. This suggests that heavily methylated

DNA sequences are responsible for the decrease of the

chromocenters. Furthermore, we found no reduction in DNA

methylation at centromeric repeats, indicating that no large-

scale decrease of DNA methylation has occurred during the

floral transition (Figure S1).

CO and FT do not affect heterochromatin reorganization,

whereas the photoreceptor CRY2 does

As the decrease in heterochromatin coincides with the floral

transition and its timing is affected by the day length, we

inferred that the reorganization of heterochromatin is under

control of components of the photoperiod pathway. We

therefore examined the RHF in mutants of CO, FT and CRY2.

In the photoperiod pathway, the upregulation of CO in

rosette leaves stimulates the production of an FT transcript

that moves through the phloem to the shoot apex triggering

flower development (Abe et al., 2005; Wigge et al., 2005).

Both co-2 and ft-1 mutants are in the Ler background and

have a late-flowering phenotype in long-day conditions

(Bradley et al., 1997; Putterill et al., 1995). They bolt after

22 days when there are 11 rosette leaves (Figure 5b,c). This

is 6 days later than in wild-type plants, which start flowering

with six rosette leaves (Figure 5a). The RHF in wild-type Ler

decreased sharply at stage 1.05 (Boyes et al., 2001), just prior

to bolting, and increased during bolting (Figure 5a). Both the

co-2 and the ft-1 mutants (Figure 5c) showed a similar sharp

reduction in RHF, but 8 days later than in wild type, corres-

ponding with the delay in flowering time. Apparently, CO

and FT do not affect the reduction in heterochromatin during

the floral transition. However, in comparison with the wild

type, a shorter period of the transient heterochromatin

reduction (2 days) prior to bolting was observed in the mu-

tants. The delayed decrease of RHF in ft-1 and co-2 further

underlines that heterochromatin reduction is related to the

floral transition rather than to the age of the plant or the

number of rosette leaves.

CRY2, a component of the light-signaling pathway, is

located in the nucleus (Mas et al., 2000) and controls floral

transition by stabilizing CO (Valverde et al., 2004). We

examined the nuclear phenotype of the cry2 mutant in a

Col-0 (cry2-1) and a Ler (fha-1) background. The two wild-

type accessions flowered after 22 and 17 days, respectively,

and showed a decrease in RHF during the floral transition.

Flowering time for cry2-1 and fha-1 is delayed compared

with their wild-type background accession (after 31 and

22 days, respectively). However, in contrast to the wild-type

plants, both cry2 mutants displayed no decrease in the HX

during floral transition (Figure 6a,b). We conclude that CRY2

is a major component of the light-signaling pathway leading

to chromatin decondensation during the floral transition.

Taken together with the results obtained in co-2 and ft-1, the

data suggest that the heterochromatin reduction is down-

stream of CRY2, but does not depend on CO and FT activity.

Figure 5. Heterochromatin fraction in co-2 and ft-1 mutants.

Relative heterochromatin fraction (RHF) of wild-type Ler (a), co-2 (b) and ft-1

(c) grown in long-day (LD) conditions. Developmental stages (Boyes et al.,

2001) and age in days (in brackets) are indicated. Flowering is delayed in the

mutants, but the decrease in RHF is still observed 2–4 days before bolting.

Bars represent stages before (white) or after (gray) bolting. For each bar the

data are mean � SEM of more than 30 nuclei.

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ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 50, 848–857

Discussion

The floral transition is one of the major developmental

switches in plants. Hundreds of genes are up- or downreg-

ulated when Arabidopsis is induced to flower (Schmid et al.,

2003). Such an extensive change in the gene expression

pattern is likely to involve major changes in chromatin state.

Our study demonstrates a dramatic decondensation of

pericentromeric and gene-rich chromatin in leaf mesophyll

nuclei during the floral transition. The decrease in compact

chromatin content is under control of a light-signaling

pathway that involves the blue-light photoreceptor CRY2.

Considering that, for an annual plant such as Arabidopsis,

the floral transition is a fundamental and irreversible

developmental event, a correctly established transition is

crucial. Hence, all processes during the floral transition

should be properly timed in order to initiate floral meristem

development. The decondensation of large chromatin

regions including gene-rich and pericentric regions may

facilitate this important developmental switch.

Floral transition is referred to as the switch from veget-

ative to reproductive growth. However, although this devel-

opmental switch results in distinctive morphological

features, such as the formation of flower primordia and the

elongation of internodes (Hempel and Feldman, 1994;

Koornneef and Peeters, 1997), its precise temporal bound-

aries have not been clearly defined. During vegetative

growth the shoot apical meristem produces leaf primordia

and associated axillary buds. Under certain environmental

conditions that induce flowering, such as extended photo-

periods, the shoot apical meristem produces floral primor-

dia upon reception of a florigen signal. This signal is

generated in the leaves and transported to the apical

meristem. It is reasonable to define floral transition under

long-day conditions as the lapse of time between the

initiation of the florigen production in the leaves and the

bolting of the immature flower buds. Of all possible com-

ponents that make up the florigen signal so far only the FT

gene product (mRNA and/or protein) has been identified

(Huang et al., 2005). FT transcription is activated in the

vascular tissue of leaves under long-day conditions by the

flowering activator CO (An et al., 2004; Samach et al., 2000;

Suarez-Lopez et al., 2001; Takada and Goto, 2003). The FT

mRNA and/or protein must be transported to induce

flowering. Within 6 h of induction in the leaves, significant

FT mRNA levels are detectable in the shoot meristem

(Huang et al., 2005). The expression of FT mRNA in the

leaves is maintained for up to 3 days and leads to upregu-

lation of floral identity genes such as AP1 and LFY. This time

frame, between initiation of FT transcript production and the

induction of floral identity genes, corresponds remarkably

well with the period in which we observe the reduction of

pericentric heterochromatin. It suggests that the transient

state of reduced heterochromatin may be mechanistically

related to the emission of FT gene product. In this study we

report decondensation of pericentromeric heterochromatin

and gene-rich chromatin during the floral transition. There-

fore, we propose that decondensation of chromatin in

flowering-induced plants may enhance the accessibility to

DNA for the transcription machinery on a genome-wide

scale.

High CO levels necessary for FT stimulation are estab-

lished by the coincidence of clock-driven high CO expression

in conjunction with CO-stabilizing activities of the photore-

ceptors CRY2 and PHYA during the late afternoon (Valverde

et al., 2004). Both PHYA and CRY2 are involved in the

perception of the photoperiod. CRY2 is a nuclear protein that

has been shown to associate with chromosomes (Cutler

et al., 2000). CRY2 shows homology with DNA photolyase,

but does not have photolyase activity. Virtually nothing is

known about a direct functional interaction between CRY2

and chromatin. The cry2 mutant shows delayed flowering in

LD conditions, whereas overexpression of CRY2 accelerates

flowering under SD conditions (Guo et al., 1998). Our study

Figure 6. Nuclear fraction with normal heterochromatin levels in null mutants

of CRY2 during the floral transition.

Heterochromatin index in cry2 mutant plants in a Col-0 background (a) and a

Ler background, fha-1 (b). Plants were grown in long-day (LD) conditions. Bars

represent stages before (white) or after (gray) bolting. For each bar the data

are mean � SEM of more than 75 nuclei. The age of the plant in days is shown

in brackets.

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ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 50, 848–857

has demonstrated that CRY2 is required for chromatin

decondensation during the floral transition. The transient

reduction of heterochromatin coincides with the floral

transition except in the cry2 mutant, where heterochromatin

levels remain stable. Notably, the correlation between

absence of CRY2 and stable levels of heterochromatin

(Fig. 6) is not only observed during the floral transition.

We have found a dramatic reduction of heterochromatin in

low-light treated wild-type plants, whereas cry2 mutants did

not show this reduction (van Zanten et al., unpublished data

). Low light levels trigger a shade-avoidance response,

which activates a number of processes, such as hyponastic

growth (Ballare, 1999; Millenaar et al., 2005), leading to

morphological changes. Apparently, under certain light

conditions that promote changes of the nuclear program,

CRY2 triggers reorganization of chromatin. The deconden-

sation event is not a prerequisite for flowering, as the cry2

mutant shows high levels of heterochromatin during the

floral transition, yet it produces flowerbuds, albeit delayed

compared with the wild type. We therefore propose that

chromatin decondensation is under control of a light-sign-

aling mechanism involving CRY2, and may facilitate CRY2-

mediated responses to environmental changes.

Heterochromatin reduction coincides with the floral trans-

ition in both LD and SD conditions. The perception of LD or

SD involves the activity of the photoreceptor CRY2 (Guo

et al., 1998). CRY2 stabilizes CO under LD, as its level

fluctuates in SD (El-Din El-Assal et al., 2001). Consequently,

the floral transition is delayed and under control of the

gibberellin pathway (Reeves and Coupland, 2001). Coinci-

dently, the heterochromatin level decreases prior to bolting.

The same happens to co and ft plants grown in LD. However,

in the absence of CRY2, the floral transition is delayed and

under control of the gibberellin pathway, but reduction of

heterochromatin does not take place. This implies that

chromatin decondensation depends on CRY2 action, but is

not affected by CO or FT activity. Conversely, the activities of

CO and FT are unlikely to take place downstream of

heterochromatin decondensation, as they are expressed

before the floral transition. Therefore, we propose that CRY2

promotes two independent processes: CO-mediated flower-

ing and reorganization of chromatin. The two events coin-

cide at the transition from vegetative to reproductive

growth. This explains why the reduction in heterochromatin

occurs prior to bolting and is absent in the cry2 mutant. How

CRY2 may regulate these processes is not clear. It is likely

that E3 ligase complexes, such as COP1, are involved. A

reciprocal effect has been reported between CRY2 and

COP1. CRY2 is unstable and rapidly degraded in blue light

by COP1, whereas COP1 activity in turn is negatively

regulated by CRY2 (Shalitin et al., 2002; Wang et al., 2001).

The latter is carried out via interaction between the WD40

domain of COP1 and the C-terminal domain of CRY2 (Wang

et al., 2001). How CO is stabilized by CRY2 is not yet clear.

CO is degraded via a mechanism that involves SPA proteins

(Laubinger et al., 2006). Proteins of the SPA family are

known to act with COP1 in the degradation of transcription

factors of photomorphogenesis. Accordingly, CRY2 might

stabilize CO via negatively regulating its degradation by a

COP1-like complex. Whether COP1 is required to degrade

CO and if the same mechanism controls chromatin organ-

ization remains to be elucidated.

Experimental procedures

Plant material

Arabidopsis accessions Col-0 (N1092); Ler (NW20) and Cvi-0 (N902)were used for this study. The co-2, ft-1, cry2-1 and fha-1 mutantswere kindly provided by M. Koornneef (MPIZ, Cologne, Germany).The co-2, ft-1 and fha-1 mutants are in the Ler background. The cry2-1 mutant is in the Col-0 background. Plants were grown under whitefluorescent light (70–90 lmol m)2 sec)1) in photoperiods of 16-hlight/8-h dark or 12-h light/12-h dark. Temperature (22�C) andhumidity (70%) remained constant. For experiments, tissue fromyoung rosette leaves, 5–15 mm in size, was used. The first twojuvenile leaves were excluded. The developmental stage of theplants was assessed according to the method described by Boyeset al., 2001.

Fluorescence in situ hybridization (FISH)

The DNA fragments used as FISH probes were labeled with eitherbiotin- or digoxigenin-Nick Translation Mixes (Roche, http://www.roche.com). Plasmid pAL1 (Martinez-Zapater et al., 1986) wasused to detect the 180-bp centromeric tandem repeat. BAC F28D6(DDBJ/EMBL/GenBank accession No. AF147262) in pBeloBAC-Kanvector was obtained from the Nottingham Arabidopsis Stock Centre(NASC, http://nasc.nott.ac.uk) . The AGAMOUS locus was localizedusing BAC F13C5 (DDBJ/EMBL/GenBank accession No. AL021711;119.1 kb) and the two adjacent BACs T18B16 (DDBJ/EMBL/GenBankaccession No. AL021687; 96.5 kb) and F28A21 (DDBJ/EMBL/Gen-Bank accession No. AL025526; 94.3 kb).

FISH experiments were carried out as described in Schubert et al.,(2001) and detected with antibodies Avidin-Texas Red (VectorLaboratories, http://www.vectorlabs.com) and goat anti-Avidin(Vector Laboratories) for biotin-labeled probes; mouse anti-digo-xigenin (Roche), rabbit anti-mouse�FITC (Sigma-Aldrich, http://www.sigmaaldrich.com) and goat anti-rabbit�Alexa488 (MolecularProbes, http://www.invitrogen.com) for the detection of digoxige-nin-labeled probes. Prior to observation, slides were counterstainedwith 4¢,6-diamidino-2-phenylindole (DAPI, 2 lg ml)1; Roche) inVectashield (Vector Laboratories).

5-Methylcytosine detection

Preparations were dried at 60�C for 30 min, treated with RNAse(10 lg ml)1 in 2X SSC ; Roche) for 60 min at 37�C, rinsed twice for5 min in 2X SSC and 5 min in PBS (10 mM sodium phosphate,pH 7.0, 143 mM NaCl), fixed for 10 min in 1% formaldehyde in PBSat room temperature, rinsed twice for 5 min in PBS, dehydrated bysuccessive 1-min baths in 70, 90 and 100% ethanol and then airdried. Denaturation was carried out by adding 50 ll HB50 (50%formamide in 1X SSC) and heating at 80�C for 2 min. The slides

Chromatin reorganization during floral transition 855

ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 50, 848–857

where then washed in 70% ice-cold ethanol and dehydrated bysuccessive ethanol baths. Blocking of the slides was carried out for1 h in 1% BSA in PBS and, after three 5-min washes in TNT (1 M Tris/HCl, pH 8.0, 1 M NaCl, 0.5% Tween 20), incubation with themouse antibody against 5-methylcytosine (Eurogentec, http://www.eurogentec.be) was carried out at room temperature overnight.Detection of the antibody was carried out with the same antibodiesused for FISH with digoxigenin-labeled probes.

Image acquisition and processing

Image acquisition was carried out on an Olympus BX60 microscope(Olympus, http://www.olympus-global.com) with filters for DAPI,Texas Red and fluorescein isothiocyanate (FITC). Pictures werecaptured with a charge-coupled device (CCD) camera (Coolsnap FX;Photometrics, http://www.photomet.com). The images were digit-ally processed using ADOBE PHOTOSHOP (Adobe, http://www.adobe.-com).

Measurement of RHF and HX

Greyscale images were analyzed with OBJECTIMAGE freeware (http://simon.bio.uva.nl/object-image.html). The RHF was calculated frommesophyll nuclei as described by Soppe et al., 2002; Tessadoriet al., 2004; and is defined as the area and fluorescence intensity ofchromocenters in relation to the area and fluorescence intensity ofthe entire nucleus. The HX is defined as the percentage of normalnuclei showing a relatively high content of compact chromatin,represented by 6–10 round or oval conspicuous chromocenters(Fransz et al., 2002, 2003). Nuclei containing fewer chromocenters,smaller chromocenters or chromocenters with vague contours areconsidered abnormal. Only mesophyll nuclei are used to measureRHF or HX. Nuclei from epidermis, vascular tissue or endoredupli-cated cells are not included in the measurements.

Acknowledgements

We thank Maarten Koornneef (MPIZ, Cologne, Germany) forvaluable discussions and Corrie Hanhart (WVR, Wageningen,Netherlands) for the seeds of the co-2, ft-1, cry2-1 and fha-1 mutantsused in this work.

Supplementary Material

The following supplementary material is available for this articleonline:Figure S1. Southern blot analysis of DNA methylation.

This material is available as part of the online article from http://www.blackwell-synergy.com

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