vernalization-induced repression of flowering locus c stimulates flowering in sinapis alba and...

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Vernalization-induced repression of FLOWERING LOCUS C stimulates flowering in Sinapis alba and enhances plant responsiveness to photoperiod

Maria D’Aloia, Pierre Tocquin and Claire PérilleuxLaboratory of Plant Physiology, Department of Life Sciences, University of Liège, Bât. B22 Sart Tilman, Boulevard de Colonster 27, B-4000 Liège, Belgium

Summary

• Of the Brassicaceae, Sinapis alba has been intensively studied as a physiological modelof induction of flowering by a single long day (LD), while molecular-genetic analysesof Arabidopsis thaliana have disclosed complex interactions between pathwayscontrolling flowering in response to different environmental cues, such as photoperiodand vernalization. The vernalization process in S. alba was therefore analysed here.• The coding sequence of S. alba SaFLC, which is orthologous to the A. thaliana floralrepressor FLOWERING LOCUS C, was isolated and the transcript levels quantifiedin different conditions.• Two-week-old seedlings grown in noninductive short days (SDs) were vernalizedfor 1–6 wk. Down-regulation of SaFLC was already marked after 1 wk of cold but2 wk was needed for a significant acceleration of flowering. Flower buds wereinitiated during vernalization. When vernalization was stopped after 1 wk, repressionof SaFLC was not stable but a significant increase in plant responsiveness to 16-h LDswas observed when LDs followed immediately after the cold treatment.• These results suggest that vernalization does not only work when plants expe-rience long exposure to cold during the winter: shorter cold periods might stimulateflowering of LD plants if they occur when photoperiod is increasing, such as in spring.

Key words: Brassicaceae, flowering, mustard, photoperiod, Sinapis alba, vernalization.

New Phytologist (2008) 178: 755–765

© The Authors (2008). Journal compilation © New Phytologist (2008) doi: 10.1111/j.1469-8137.2008.02404.x

Author for correspondence:Claire PérilleuxTel: +32 4366 38 33Fax: +32 4366 38 31Email: [email protected]

Received: 28 November 2007Accepted: 10 January 2008

Introduction

In the natural conditions of temperate areas, many factors ofthe environment influence plant reproduction, and thoseexhibiting predictable seasonal variation were selected forreliable timing of floral transition (Bernier & Périlleux, 2005).Primary factors controlling flowering time include winter cold(vernalization) and photoperiod, on which most experimentalwork was focused. Early physiological studies showed thatthese environmental factors are mainly perceived by differentorgans: vernalization is mostly sensed by mitotically activetissues – including the shoot apical meristem (SAM) itself –while photoperiod is perceived by expanded leaves and requiressystemic signalling towards the SAM in order to induceflowering (Bernier et al., 1981; Metzger, 1988). Moreover, a

majority of cold-requiring plants require long days (LDs) aftervernalization, suggesting that vernalization acts as a first stepin bringing about the competence to flower (Chouard, 1960).Spatial and temporal separation thus explained why themechanisms of vernalization and photoperiodic induction offlowering were studied separately and physiological modelswere selected for their strong requirement for one or the otherenvironmental factor. Among photoperiodic species, physio-logists favoured those that flower in response to a singleinductive cycle because of the high synchrony that can beachieved during the transition. For example, Sinapis alba(white mustard) plants, when grown in controlled conditionsin phytotronic cabinets, remain vegetative in 8-h short days(SDs) and can be induced to flower by a single LD when2 months old (Bernier, 1969). This experimental system has

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been used extensively to analyse physiological signals leadingto flowering, and a detailed picture of sequential events offloral transition has been described (reviewed in Bernier et al.,1993; Bernier & Périlleux, 2005). In contrast, much less isknown about vernalization in S. alba although, in naturalconditions, this species behaves as a quantitative LD- andcold-requiring plant (Bernier, 1969; Bodson, 1985).

Most recent studies of the molecular-genetic mechanismsthat control flowering have focused on Arabidopsis thaliana, amodel plant of geneticists. At the physiological level, A. thaliana,like S. alba, is a quantitative LD- and cold-requiring species,and hence the first screenings and characterizations of mutantswere based on flowering response to the primary factorsphotoperiod and vernalization (Koornneef et al., 1991;Martínez-Zapater et al., 1994). Mutants and genes weretherefore classified into three main classes which, after furtherepistasis and molecular analyses, defined three genetic pathwayscontrolling flowering time in A. thaliana: the LD-promotingpathway, the vernalization-promoting pathway, and the ‘auton-omous’ pathway which is defective in mutants that are lateflowering but still sensitive to photoperiod and vernalization.A fourth route – the gibberellin (GA) pathway – was added onthe basis of the observation that flowering of GA-deficient mutantsis much delayed, mainly in SDs. These flowering pathwayscontrol a set of ‘integrator’ genes, which include FLOWER-ING LOCUS T (FT) and SUPPRESSOR OF OVER-EXPRES-SION OF CO 1 (SOC1, previously named AGL20) that actupstream of the genes involved in the flower initiation processsuch as LEAFY (LFY ) and APETALA1 (AP1) (reviewed inBoss et al., 2004; Bernier & Périlleux, 2005; Corbesier &Coupland, 2005).

The photoperiodic pathway involves interactions betweenlight signalling and the circadian clock. LDs promote floweringin A. thaliana by the activation of the transcription factorCONSTANS (CO): the abundance of CO exhibits circadianoscillations (Suárez-López et al., 2001) and only in LDs doesa peak in the amount of protein coincide with the presence oflight. Far-red and blue lights perceived by phytochrome A andcryptochrome 2 stabilize the protein (Valverde et al., 2004).This ‘external coincidence’ allows CO to activate its targetsFT and SOC1 (Onouchi et al., 2000; Samach et al., 2000). Ofthe utmost importance, the FT protein was recently shown toparticipate in the systemic signalling that induces floweringin A. thaliana (Corbesier et al., 2007; Jaeger & Wigge, 2007;Mathieu et al., 2007).

Both the vernalization and autonomous pathways act byrepressing a flowering inhibitor: the MADS box gene FLOW-ERING LOCUS C (FLC ) (Michaels & Amasino, 2000).Vernalization down-regulates the FLC mRNA level, withlonger periods of exposure to cold leading to less FLC mRNA(Sheldon et al., 2000). The process is saturated after severalweeks of cold treatment; the vernalized state then remains stablefor the rest of the plant life cycle and is reset at meiosis. Thisepigenetic silencing of FLC is mediated by histone modifica-

tions in the promoter and intron 1 regions: an early step inthis process is H3 deacetylation, and the stable maintenanceof FLC repression involves H3K9 and H3K27 methylation.These modifications create a ‘histone code’ associated withFLC repression (reviewed in Sung & Amasino, 2006).

The identification of distinct genetic pathways controllingflowering time in A. thaliana was consistent with the spatialand temporal separation of vernalization and LD inductiondiscussed above. However, the rapid progress in the field andthe fact that experimental research extended to the analysis of‘secondary’ environmental factors revealed more intricategenetic networks. For example, FLC has been found to beinvolved in the control of flowering by ambient temperature(Blázquez et al., 2003; Balasubramanian et al., 2006) and inthe regulation of the circadian clock (Edwards et al., 2006;Salathia et al., 2006).

We were therefore interested in studying vernalization andthe participation of FLC in the control of flowering in the LDplant S. alba. This strategy was greatly encouraged by the taxo-nomic proximity of Sinapis and Arabidopsis, both members ofthe Brassicaceae. A number of FLC orthologues have beencloned in Brassica species, using genomics information onA. thaliana (Tadege et al., 2001; Schranz et al., 2002; Martynov& Khavkin, 2004; Lin et al., 2005; Razi et al., 2008). A highrate of conservation between A. thaliana and S. alba sequenceswas also reported. For example, SaMADS A, which is orthol-ogous to SOC1, was cloned from S. alba as the earliest MADSbox gene expressed in the shoot apical meristem at floraltransition (Menzel et al., 1996). Overexpression of SaMADSA in A. thaliana caused precocious flowering (Bonhommeet al., 2000), as also found for other SOC1 orthologues clonedfrom other Brassicaceae species (Kim et al., 2003). We reporthere isolation of SaFLC and physiological analyses of its functionin flowering response to vernalization and photoperiod.

Materials and Methods

Plant growth conditions

The seeds of Sinapis alba L. cv. Carla were purchased fromJob-semences S.A.R.L. (Nancy, France). Plants were grown in8-h SDs, as described by Lejeune et al. (1988). The fluencerate at the top of the plants was 150 µmol m−2 s−1 over thewaveband 300–700 nm and was provided by very-high-outputSylvania fluorescent white tubes (Zaventem, Belgium). Thetemperature was kept constant at 20°C, except during thevernalization treatments, where 2-wk-old plants were trans-ferred to 7°C and 85 µmol m−2 s−1 for 1–6 wk before beingreturned to standard conditions. Flowering time was scored as‘days to macroscopic appearance of floral buds’.

In experiments where plants were transferred transiently to16-h LDs, flowering was measured by dissection of the apicalbuds under the binocular microscope 2 wk after the start ofexposure to LDs and was expressed as a percentage of floral


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plants. A plant was classified as floral when at least one flowerprimordium was present within the apical bud. Each experi-mental batch numbered 15–20 individual plants.

Isolation of SaFLC cDNA and sequence analysis

A λgt10 cDNA library made from S. alba leaf mRNA (Menzelet al., 1996) was screened with a cDNA probe of A. thalianaFLC (AtFLC). For probe synthesis, cDNA was prepared fromfca mutant mRNA. A fragment of AtFLC cDNA was amplifiedwith the following primers: AtFLC-fwd: 5′-TCATCATGTG-GGAGCAGAAG-3′ and AtFLC-rev: 5′-TACAAACGCTC-GCCCTTATC-3′.

Approximately 150 000 clones from the library werescreened and one positive colony was isolated, subclonedinto pGEM®-T Vector (Promega, Madison, WI, USA; http://www.promega.com/) and sequenced. Sequence alignmentshowed a high level of identity with Brassica napus BnFLC1and AtFLC; thereafter we called the isolated sequence SaFLC.In order to isolate a full-length cDNA, we designed a pair ofprimers based on the nucleotide sequence of BnFLC1 (GenBankaccession no. AY036888) for the 5′ region (BnFLC1-fwd:5′-AGGGCGCAAAGCACTGTTGGAGAC) and on thepartial SaFLC clone for the 3′ region (SaFLC-rev: 5′-TTACAAG-GGGATAAATACACATCTTG-3′). Reverse transcriptionpolymerase chain reaction (RT-PCR) was performed on RNAextracted from aerial parts of 2-wk-old S. alba plants. A fragmentof 702 bp was obtained, cloned into pGEM®-T Vector andsequenced (GenBank accession no. EF542803).

Amino acid sequence alignment of SaFLC and other FLCproteins from A. thaliana, Arabidopsis arenosa, Arabidopsissuecicea, Arabidopsis lyrata, B. napus, Brassica oleracea, Brassicarapa, Brassica juncea and Raphanus sativus were generatedusing the ClustalW program (http://www.infobiogen.fr/services/analyseq/cgi-bin/clustalw_in.pl). Partial amino acidsequences were used for alignment because the sequence ofthe MADS box is unavailable for some FLC genes (BoFLC,BrFLC and BjFLC) (Schranz et al., 2002; Martynov &Khavkin, 2004). Aligned sequences were analysed using thepaup* program (Phylogenetic Analysis Using Parsimony;Sinauer Associates, Sunderland, MA, USA).

Genomic Southern blot hybridization

Genomic DNA was isolated from leaves as described before(Dellaporta et al., 1983) and modified by Saumitou-Lapradeet al. (1999). Digestion was performed with the restrictionenzyme EcoRI, HindIII or NcoI (5 U µg–1) overnight at 37°C.Twenty-µg samples were separated on 1% (w/v) agarose geland blotted onto positively charged nylon membrane (Roche,Mannheim, Germany). The probe was synthesized fromtruncated SaFLC cDNA lacking the MADS box (337 pb)and cloned in pGEM®-T. Radiolabelling was performedby random priming using 32P-ATP according to the manu-

facturer’s instructions (RadPrime DNA labelling system;Invitrogen, Carlsbad, CA, USA). Blotted DNA fragmentswere hybridized overnight at 55°C in Herby buffer: 250 mMsodium phosphate, 7% (w/v) sodium dodecyl sulphate (SDS),1 mM ethylenediaminetetraacetic acid (EDTA), and 1%(w/v) bovine serum albumin, pH 7.2. Blots were washed twicein 2 × saline sodium citrate (SSC) containing 0.1% (w/v) SDSfor 10 min, once in 2 × SSC containing 0.1% (w/v) SDS at60°C and finally in 1 × SSC containing 0.1% (w/v) SDS for5 min at 60°C. X-ray film (Kodak BioMax MS film) wasexposed for 3 d.

RNA extraction and quantification

Total RNA was extracted from tissues using the TRIzolmethod (Invitrogen; http://www.invitrogen.com/). DNA-freeRNA was obtained by DNase treatment (0.2 U DNase µg−1)according to the manufacturer’s instructions (Promega). Thefirst strand of cDNA was synthesized from 5 µg of total RNAusing the Moloney murine leukaemia virus reverse transcriptaseand an Oligo(dT)15 primer according to the manufacturer’sprotocol (Promega) in a reaction volume of 40 µl. The cDNAwas diluted 2.5-fold with water and 5 µl of diluted cDNA wasused for quantification.

Quantitative real-time RT-PCR for SaFLC mRNA quantifi-cation For quantification of SaFLC transcripts by quantitativereal-time RT-PCR (qRT-PCR), cDNA was prepared from3-mm-high shoot apices (at least 15 apices per sample, harvestedin the plant growing chambers, at either 7 or 20°C). Serialdilutions of a concentrated first-strand cDNA stock were usedas relative standards. The PCR reaction mix was preparedusing a commercially available master mix containing Taq DNApolymerase, SYBR-Green I, deoxyribonucleoside triphosphatesand MgCl2 (iQ SYBR Green Supermix; Bio-Rad, Hercules,CA, USA). Primers were used in a final concentration of0.5 µM. The PCR programme was: 95°C for 5 min; 40 ×(95°C for 30 s, 57°C for 30 s and 72°C for 1 min); 72°Cfor 10 min. PCR reactions were performed in triplicate oneach cDNA sample, using the iCycler iQ real-time PCRdetection system from Bio-Rad. Transcript levels were normalizedwith the amount of transcripts from a β-TUBULIN gene(SaTUB). The specificity of the amplification was confirmedby melting curve analysis and agarose gel electrophoresis. Theprimers used (SaFLC-fwd, SaFLC-rev, SaTUB-fwd andSaTUB-rev) are listed below.

Semi-quantitative RT-PCR for SaMADS A mRNA quanti-fication For SaMADS A expression analyses, cDNA wasprepared from 3-mm-high shoot apices (at least 15 apices persample). The PCR programme was: 95°C for 5 min;25 × (95°C for 30 s, 55°C for 30 s and 72°C for 1 min); 72°Cfor 10 min SaTUB was used as a control gene (same PCRprogramme, 20 cycles). The PCR products were transferred

http://www.promega.com

http://www.promega.com

http://www.infobiogen.fr/services/analyseq/cgi-bin/clustalw_in.pl

http://www.infobiogen.fr/services/analyseq/cgi-bin/clustalw_in.pl

http://www.invitrogen.com



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onto a nylon Hybond-N membrane (GE Healthcare, Diegem,Belgium) and were hybridized using a digoxigenin-labelledprobe prepared according to the manufacturer’s instructions(Roche). The SaMADS A probe was a fragment amplifiedby PCR from a full-length cDNA clone (Bonhomme et al.,2000). The primers used (SaMADSA-fwd, SaMADSA-rev,SaTUB-fwd and SaTUB-rev) are given below.

The sequences of primers were:SaFLC-fwd: 5′-GAAAAGGAGAAATTGCTGGAAGAGGA-3′SaFLC-rev: 5′-GGAGCGTTACCGGAAGATTGATGT-3′SaTUB-fwd: 5′-CGAAAACGCTGACGAGTGTATG-3′SaTUB-rev: 5′-TTAAGCTGGCCAGGGAAACGAA-3′SaMADSA-fwd: 5′-TAGCTGCAGAAAACGAGAAG-3′SaMADSA-rev: 5′-ACTTTCTGGAAGAACAAGGTAAC-3′

In situ hybridization

Shoot apices of S. alba plants were fixed in 2% formaldehyde,100 mM phosphate buffer, pH 7.2 (16 h at 4°C). Fixedtissues were dehydrated, embedded in paraffin accordingto standard procedures and cut with a rotary microtome.Longitudinal sections (8 µm) were mounted onto poly L-lysine-coated slides and were pretreated for hybridizationaccording to the method of Angerer & Angerer (1992).35S-UTP-labelled antisense riboprobes were synthesized usingT7 and T3 RNA polymerases according to the manufacturer’sinstructions (Promega). The SaFLC probe was obtained fromthe same truncated cDNA clone (337 bp) as used for Southernblot hybridization; the SaLFY probe was obtained from acomplete cDNA (1374 bp) cloned in pBluescript II SK+(Melzer et al., 1999). The hybridization mix was prepared as

described by Bonhomme et al. (1997). Incubation, RNasetreatment, washing steps, coating with Kodak NTB-2 nuclearTrack emulsion, and development of slides were performedaccording to Angerer & Angerer (1992). Slides were exposedfor 2 wk and were stained with 0.1% calcofluor. Autora-diographs were observed with dark-field illumination and theunderlying tissue visualized using UV fluorescence.

Results

Effect of vernalization on flowering time in short days

In order to analyse the process of vernalization in S. alba, wefirst decided to grow the plants in noninductive 8-h SDs andto start the vernalization treatments when they were 2 wk old.At that stage, plants had their cotyledons plus two leavesexpanded and shoot apices could easily be harvested. Verna-lization was always carried out at 7°C (day and night).

Different durations of vernalization (1–6 wk) were testedin three independent experiments, where flowering time wasrecorded as ‘days to macroscopic appearance of floral buds’. Asshown in Fig. 1, the effect of vernalization was clearly duration-dependent: while 1 wk had no effect on flowering time in SDs,2-wk and longer treatments clearly accelerated flowering.After 6 wk of vernalization, flowering of S. alba in SDs occurredtwice as fast as in nonvernalized plants. The vernalizationresponse was saturated after 6 wk: plants actually initiatedflowers during their growth in cold conditions and floral budsbecame macroscopically visible ∼70 d after sowing, that is,∼55 d after start of the cold treatment.

Isolation and characterization of SaFLC

An FLC-like cDNA from S. alba (hereafter referred to asSaFLC) was obtained in two steps: screening of a leaf cDNAlibrary with an AtFLC cDNA probe, followed by full-lengthcDNA cloning by RT-PCR (see the Materials and Methods).SaFLC (702 bp; GenBank accession no. EF542803) encodesa MADS-box protein with predicted amino acid sequenceshowing 95% identity with BnFLC1 and 85% identity withAtFLC. Phylogenetic analyses performed with Brassicaceaesequences indicated that the predicted SaFLC protein fell intoone well-segregated clade with the BnFLC1, BoFLC1 andBrFLC1 proteins (Fig. 2). This group was the closest to theArabidopsis genus FLC proteins.

We have estimated the number of FLC copies in the S. albagenome by Southern blot analysis (Fig. 3). Genomic DNAwas digested and hybridized with a SaFLC cDNA probe lackingthe MADS-box region. We detected one band in all digestionreactions, suggesting that SaFLC is present as a single-copygene in the S. alba genome.

This result was confirmed by an alternative PCR approachdeveloped in B. rapa (Schranz et al., 2002). The methodologywas based on the fact that the structure of the AtFLC gene –

Fig. 1 Effect of vernalization duration on flowering time of Sinapis alba grown in 8-h short days (SDs). Vernalization treatments started when plants were 2 wk old, and were given at 7°C (day and night) for 1–6 wk. Flowering time is expressed as ‘days to macroscopic appearance of floral buds’. Results of three independent experiments are shown. Data are means ± standard deviation for a minimum of 15 plants. *Statistically different from nonvernalized plants (Student’s t-test).



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containing seven exons and six introns – is conserved amongBrassica species, while the length of introns is highly variable.In B. rapa, four different PCR products were amplified withconserved primers of exons 2 and 7, indicating the existenceof four BrFLC copies (Schranz et al., 2002). Using the samestrategy, we obtained a single 1693-bp amplicon in S. alba,giving further support to our inference that SaFLC is a uniquegene. The 1693-bp PCR fragment was sequenced and showed87% identity with the genomic region from exon 2 to exon 7of BrFLC1 (AY115678) (results not shown).

Expression pattern of SaFLC and downstream genes during vernalization

Expression of SaFLC was detected by in situ hybridization inthe shoot apex of 8-wk-old nonvernalized plants (Fig. 4a), butnot in plants that had been vernalized for 6 wk (Fig. 4b). Notethat the apex shown in Fig. 4(b) was initiating flower primordiaduring the vernalization treatment.

Real-time RT-PCR was carried out for a time-course analysisof SaFLC expression during vernalization. Shoot apices wereharvested weekly; four independent experiments were con-ducted. As can be seen in Fig. 4(c), SaFLC transcript levels

Fig. 2 FLOWERING LOCUS C (FLC) proteins in Brassicaceae species. (a) Alignment of the deduced amino acid sequences of SaFLC from Sinapis alba, BnFLC1 from Brassica napus, and AtFLC from Arabidopsis thaliana. (b) Maximum parsimony tree based on amino acid sequences excluding the MADS-box domain. The tree includes FLC proteins from A. thaliana (AtFLC, NP_196576), Arabidopsis arenosa (AaFLC1, AAZ92552 and AaFLC2, AAZ92550), Arabidopsis suecicea (AsFLC, AAZ92553), Arabidopsis lyrata (AlFLC, AAV51231), S. alba (SaFLC, ABP96967), B. napus (BnFLC1, AAK70215; BnFLC2, AAK70216; BnFLC3, AAK70217; BnFLC4, AAK70218 and BnFLC5, AAK70219), Brassica oleracea (BoFLC1, AAN87902; BoFLC3, AAN87901; BoFLC5, AAN87900; BoFLC3-2, AAQ76274 and BoFLC4-1, AAQ76275), Brassica rapa (BrFLC1, AAO13159; BrFLC2, AAO86066/AAO86067; BrFLC3, AAO13158 and BrFLC5, AAO13157), Raphanus sativus (RsFLC, AAP31676) and Brassica juncea (BjFLC3, AAP42143 and BjFLCx, AAP31243). Symbols I and II indicate the groups formed by FLC1 proteins from S. alba and other Brassica species (I) and by FLC proteins from the Arabidopsis genus (II).

Fig. 3 Southern blot analysis of Sinapis alba genomic DNA. Genomic DNA was digested with EcoRI, HindIII or NcoI, blotted after electrophoresis onto a nylon membrane and probed with a 32P-labelled S. alba FLOWERING LOCUS C (SaFLC) probe.



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Fig. 4 Sinapis alba FLOWERING LOCUS C (SaFLC) expression patterns. (a, b) In situ hybridization pattern of SaFLC at the shoot apex of 8-wk-old plants of S. alba grown in short days (SDs), either nonvernalized (a) or vernalized for 6 wk (b). Bar, 100 µm. fm, flower meristem; im, inflorescence meristem; l, leaf; vm, vegetative meristem. (c) Time-course analysis of SaFLC transcripts in shoot apices of plants of S. alba grown in 8-h SDs. Plants were either vernalized at 7°C from 2 wk after sowing (V; open bars) or continuously grown at 20°C (nonvernalized controls; NV; closed bars). Shoot apices were harvested from 15 plants for each sample. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was used on total RNA and expression of SaTUBULIN was used for data normalization. Results shown are means ± standard deviation of four independent experiments. *Statistically different from nonvernalized plants (Student’s t-test).

Fig. 5 Expression of Sinapis alba SaMADS A and LEAFY (SaLFY) during vernalization. (a) Time-course analysis of SaMADS A expression duringvernalization in 8-h short days (SDs) at 7°C. Shoot apices were harvested from 15 plants for each sample. Semi-quantitative reverse transcriptionpolymerase chain reaction (RT-PCR) was used on total RNA and expression of SaTUBULIN was used as a control. (b–d) In situ hybridization pattern of SaLFY at the shoot apex of plants of S. alba grown in SDs and either nonvernalized (NV; 8-wk-old plants shown in b) or vernalized for 4 wk (4-w V; 6-wk-old plants shown in c) or vernalized for 6 wk (6-w V; 8-wk-old plants shown in d). Bar, 100 µm. fm, flower meristem; im, inflorescence meristem; l, leaf; vm, vegetative meristem.

remained quite high and constant in nonvernalized controlsduring the course of the experiment. In contrast, SaFLCdecreased sharply in apices of vernalized plants, and the tran-script level was already very low after 1 wk at 7°C. Furtherexposure to cold correlated with a further decrease in the SaFLCtranscript level.

AtFLC was shown to repress SOC1 in the SAM (Searleet al., 2006). We therefore examined, using semi-quantitativeRT-PCR, the expression pattern of the orthologous geneSaMADS A (Bonhomme et al., 2000) during the vernalizationtreatment. As shown in Fig. 5(a), expression of SaMADS Awas not detected in nonvernalized apices, as already shownby Bonhomme et al. (2000). The SaMADS A transcript levelincreased in the shoot apex from the 3rd week of vernalizationand was highest after 6 wk of vernalization. Because, asmentioned above, floral buds were initiated during long vernal-ization treatments, we also performed in situ hybridizationswith an SaLFY probe. In nonvernalized apices, expression ofSaLFY was detected at the tip of leaf primordia, but not in theSAM (Fig. 5b). After 4 wk of vernalization, up-regulationof the gene was observed in the whole SAM, which hadincreased in size and was more domed (Fig. 5c). After6 wk of vernalization, SaFLY was highly expressed in flowerprimordia and in the flanks of the SAM (Fig. 5d).

Expression pattern of SaFLC postvernalization

The effect of vernalization appeared to be much greater onSaFLC repression (Fig. 4) than on flowering time in SDs(Fig. 1). We therefore quantified SaFLC transcripts aftervernalization. In two independent experiments, plants werevernalized for 1 or 3 wk and were harvested either immediatelyafter the vernalization treatment (post vernalization time (PVT)0) or 2 wk later (PVT2) (Fig. 6a). Nonvernalized controls



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were harvested at each sampling time. As shown in Fig. 6(b),it was observed that the level of SaFLC transcript, althoughvery low at the end of the vernalization treatment, recoveredwhen plants were returned to 20°C. When plants had beenvernalized for only 1 wk, the level reached at PVT2 was notdifferent from that observed in nonvernalized controls. Whenplants had been vernalized for 3 wk, the SaFLC transcriptlevel remained lower than in nonvernalized controls.

Effect of vernalization and SaFLC on plant sensitivity to photoperiod

In order to examine whether 1 wk of vernalization, althoughinsufficient for stable repression of SaFLC, could have aphysiological effect on flowering, we studied plant respon-siveness to LDs. Plants were exposed to one or two 16-h LDsjust after the vernalization treatment (PVT0), or after having

been returned to 20°C for 2 wk (PVT2) (Fig. 7a). Nonver-nalized controls were also exposed to the LD(s) but, becauseplant growth was slowed down during the cold treatment,vernalized plants exposed to LDs at PVT0 were comparedwith nonvernalized controls that received the LD(s) 4 dbefore, when they had approximately the same mean leafarea (10.4 ± 2.1 cm2 and 9.3 ± 1.4 cm2 for vernalized andnonvernalized plants, respectively). The flowering responsewas evaluated 2 wk after the LD(s) by dissecting apical budsand calculating the ‘percentage of floral plants’. Threeindependent experiments were carried out. As can be seen inFig. 7(b), the 1-wk vernalization treatment had a strongpromotive effect on the floral response to LDs when the LDsimmediately followed vernalization: we indeed observed a3-fold increase in the flowering response of vernalized plantsto two 16-h LDs given at PVT0, as compared with nonverna-lized controls. When 2 wk at 20°C were interpolated betweenthe vernalization treatment and LD exposure (PVT2), theshort vernalization treatment appeared to be insufficient topromote the flowering response to one or two 16-h LDs, aswas found previously in continuous SDs (Fig. 1). Figure 7(b)

Fig. 6 Effect of vernalization duration on stability of Sinapis alba FLOWERING LOCUS C (SaFLC) down-regulation. (a) Experimental set-up. Two-week-old plants of S. alba were vernalized for 1 (1-w V) or 3 (3-w V) wk and compared with nonvernalized (NV) controls. Shoot apices were harvested at the end of vernalization (PVT0) or 2 wk later (PVT2) for SaFLC transcript quantification in short days (SDs). NV controls were sampled at PVT2. (b) SaFLC transcript level in shoot apices. SaFLC transcript levels were quantified by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) on total RNA and the expression of SaTUBULIN was used for data normalization. PVT0, open bars; PVT2, hatched bars; NV, cross-hatched bars. Data are means ± standard deviation of two independent experiments. *PVT2 statistically different from NV controls; °PVT2 statistically different from PVT0 (Student’s t-test).

Fig. 7 Effect of 1-wk vernalization on flowering response to 16-h long days (LDs). (a) Experimental set-up. Two-week-old plants of Sinapis alba were vernalized for 1 wk (1-w V) and exposed to one or two 16-h LDs at the end of vernalization (PVT0) or 2 wk later (PVT2) and compared with nonvernalized (NV) controls. (b) Flowering response was evaluated as ‘percentage of floral plants’ as observed by dissecting the shoot apices of 15 plants 2 wk after the LD(s). V, open bars; NV, closed bars. Data are means ± standard deviation of three independent experiments. *Statistically different from nonvernalized plants (Student’s t-test).



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also illustrates – by the comparison of PVT0 with PVT2 –that the flowering response to LDs increases with plant age.

Discussion

The vernalization response of S. alba is quantitative

The aim of the research was to investigate the vernalizationpathway of flowering time control in S. alba. This species hasbeen previously shown to be sensitive to vernalization at theseed and adult stages (Bernier, 1969; Bodson, 1985). Weshow here that vernalization of 2-wk-old seedlings couldaccelerate flowering in noninductive SDs, and that floraltransition occurred during the vernalization period when itwas extended over ∼4 wk. However, plants of S. alba do notrequire vernalization, as nonvernalized plants do flower –albeit very late – even in SDs (Fig. 1). Sinapis alba is indeed afacultative vernalization- and LD-responsive species.

The flowering response to vernalization in SDs wasobserved only in plants that had been exposed to cold for atleast 2 wk (Fig. 1). When macroscopic appearance of floralbuds was used as a criterion for flowering, the positive effectof increasing vernalization duration was observed for up to5–6 wk of cold treatment. However, the use of SaMADS Aand SaLFY as earlier markers of the floral shift showed thatfloral transition actually started after 3–4 wk at 7°C (Fig. 5).

SaFLC is orthologous to AtFLC

SaFLC was cloned in order to investigate the molecularmechanisms of the S. alba response to vernalization. Thesequence that was isolated showed a high identity withAtFLC1. Phylogenetic data presented herein demonstrate thatthe predicted SaFLC protein clusters with the FLC1 sequencesfound in different members of the Brassicaceae family(BnFLC1, BoFLC1 and BrFLC1). This is consistent with thefact that S. alba is called Brassica hirta by some authors(Primard et al., 1988). Among all the amino acid sequencesfound in the phylogenetic tree, the group made up of FLC1is the closest to FLC proteins from the Arabidopsis genus,suggesting that SaFLC is orthologous to AtFLC.

SaFLC was found here as a single-copy gene (Fig. 3) althoughthe S. alba genome is assumed to have been triplicated duringevolution, containing three Arabidopsis genome equivalents(Nelson & Lydiate, 2006). A high rate of chromosomal rear-rangement (fusion and fission) following polyploidy eventsmight explain discrepancies between expected and actual genecopy numbers (Lysak et al., 2005; Town et al., 2006). For exam-ple, in B. napus, which originated from an interspecific hybridi-zation between B. rapa and B. oleracea, Tadege et al. (2001)found five FLC genes instead of six. We therefore hypothesizethat S. alba has lost two copies of FLC from its ancestor.

Expression of SaFLC was detected in the shoot apex ofnonvernalized plants and its level of expression seemed to be

independent of plant age, at least for up to 8 wk of growth inSDs at 20°C (Fig. 4), as previously reported in A. thaliana(Sheldon et al., 1999; Michaels & Amasino, 2000; Rouseet al., 2002; Sheldon et al., 2006). It is well known, however,that plants of increasing age are more and more sensitive toflower-inducing signals (Bernier et al., 1981): 8-wk-old non-vernalized plants of S. alba are fully responsive to a single LD(Bernier, 1969) although SaFLC is highly expressed in theSAM. We can thus infer that this increased sensitivity is notcaused by a developmental decrease in FLC activity in the SAM.As it was recently reported that the repression of flowering byFLC is also caused by FLC activity in the leaves (Searle et al.,2006), we analysed SaFLC transcript levels by semiquantitativeRT-PCR in the leaves and observed no developmental decreasein these tissues either (results not shown).

We observed a clear down-regulation of SaFLC by vernali-zation, which is in agreement with results obtained not onlyin A. thaliana, but also in other Brassicaceae species such asB. napus, B. oleracea, B. rapa and Thellungiella halophila (Tadegeet al., 2001; Li et al., 2005; Lin et al., 2005; Fang et al., 2006;Kim et al., 2006) and, more recently, outside this plant family(Reeves et al., 2007). We showed here that, in S. alba, theamount of SaFLC transcripts was already greatly decreased bya short cold treatment of 1 wk (Fig. 4c), but stabilization ofthe repression required longer exposure to cold (Fig. 6b). Thelonger the vernalization treatment, the lower the remainingSaFLC transcript level, and the stronger the effect on floweringtime (Fig. 1). This correlation between FLC repressionand the quantitative effect of vernalization was described inA. thaliana by Sheldon et al. (2000).

AtFLC targets include SOC1 in the SAM and FT in theleaves (Searle et al., 2006), which are repressed unless FLC isdown-regulated by vernalization. We followed expression ofSaMADS A, which is orthologous to SOC1 (Bonhommeet al., 2000), during vernalization and we observed that itincreased in the shoot apex after ∼3 wk of exposure to cold(Fig. 5a), that is, after down-regulation of SaFLC. It is stillpossible, however, that SaMADS A was also up-regulated bycold independently of SaFLC, as – in A. thaliana – vernalizationmay cause induction of SOC1 in an flc mutant (Moon et al.,2003). SaLFY appeared in the SAM approx. 1 wk afterSaMADS A (Fig. 5b), which is consistent with the idea thatLFY is activated by SOC1 (Moon et al., 2005). Although ourtiming needs to be refined by more frequent sampling, arather long lapse of time between up-regulation of SaMADSA and up-regulation of SaLFY could be expected, as thisoccurred at 7°C.

Stabilization of SaFLC down-regulation requires exposure to cold for longer than 1 wk

Although the expression pattern of SaFLC (Fig. 4) fits nicelywith the quantitative effect of vernalization on the floweringresponse of S. alba (Fig. 1), a major discrepancy appeared:



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1 wk of vernalization was enough for SaFLC down-regulation(Fig. 4b), but not for flowering stimulation in SDs (Fig. 1).This suggests that the down-regulation of SaFLC by 1 wk ofvernalization was transient. Indeed, quantification of SaFLCtranscript postvernalization showed that, when plants weretransferred back to 20°C, its level returned to that ofnonvernalized plants when vernalization was short (1 wk) butremained lower if vernalization was longer (Fig. 6b) This resultclearly indicates that 1 wk of vernalization was not sufficientfor stable repression of SaFLC, while 3 wk of vernalizationwas; this difference may explain the ineffectiveness of theshortest treatment for accelerating flowering in SDs.

In A. thaliana, maintenance of AtFLC repression afterexposure to cold requires the functions of the VERNALIZA-TION 1 (VRN1), VRN2 and LIKE HETEROCHROMATINPROTEIN 1 genes (Gendall et al., 2001; Levy et al., 2002;Bastow et al., 2004; Mylne et al., 2006; Sung et al., 2006).Stabilization of the AtFLC silenced state also depends on cisregulatory sequences, as shown by the fact that the AtFLCpromoter and intron 1 are sufficient for down-regulation andmaintenance of repression by cold (Sheldon et al., 2002,2006; Sung et al., 2006). Recently, natural variation in vernal-ization response has been correlated with the stability ofAtFLC repression and with the rate of accumulation of AtFLChistone H3K27 trimethylation (Shindo et al., 2006).

Maintenance of SaFLC down-regulation might be controlledby mechanisms in S. alba similar to those in A. thaliana, andhence regulatory sequences may account for the degree ofstability of the vernalized state. Variations in AtFLC intron 1sequences have already been described in the literature (Lempeet al., 2005). For instance, a mutator-like sequence found atthe 3′ end of intron 1 in the Ler AtFLC allele seems to limitAtFLC RNA accumulation (Gazzani et al., 2003; Michaelset al., 2003). Also, A. thaliana transgenic lines containing theBoFLC transgene (from B. oleracea var. capitata) have beenshown to display variable repression of the transgene, whichcould be a result of a 2.6-kb deletion in BoFLC intron 1, ascompared to AtFLC (Lin et al., 2005). The regulatory functionof SaFLC intron 1 remains to be investigated.

Down-regulation of SaFLC enhances sensitivity to photoperiod

We observed that 1 wk of vernalization – which was sufficientfor SaFLC down-regulation but insufficient for stabilizationof the repressed state – did not stimulate flowering in SDs(Fig. 1), but had a clear promotive effect when followedimmediately by inductive LDs: the percentage of plants thatflowered in response to two 16-h LDs was tripled when theyhad been vernalized for 1 wk, as compared with nonvernalizedplants (Fig. 7b). However, the vernalizing effect of the 1-wktreatment did not persist: when the LDs were given after 2 wkof growth postvernalization, vernalized and nonvernalizedplants showed a similar flowering response to LDs. Extrapo-

lation of these results to natural conditions means that therequirement for vernalization is longer when experienced inwinter – because LDs do not immediately follow and SaFLCrepression requires stabilization – while short periods of coldoccurring in spring could enhance plant response to increasingphotoperiod. Such environmental conditions are far from rarein temperate regions.

Down-regulation of FLC might stimulate the photoperiodicpathway directly through FT and SOC1, as recent literaturehas provided evidence that these integrator genes are repressedby AtFLC (Searle et al., 2006), and hence vernalization acts inboth the leaf and the SAM. Cold could also have a less directeffect on the floral response to LDs. Indeed, Salathia et al.(2006) reported that vernalization of A. thaliana seedlings ledto a shortening of the circadian period of leaf movements, andperiod shortening can be associated with earlier flowering undershorter photoperiods (Yanovsky & Kay, 2002). Whether theeffect of short vernalization treatments is produced via suchcomplex networks warrants further investigation and is ofgreat ecological relevance.

Acknowledgements

We would like to thank Prof G. Bernier and Dr L. Corbesierfor their critical reading of the manuscript. We are muchindebted to A. Pieltain and N. Detry for their excellent technicalassistance and to A. Havelange, P. Perruzza and D. Libion fortaking care of the plants. We acknowledge Dr S. Melzer(Department of Plant Systems Biology, VIB/Ghent University,Belgium) for the opportunity to screen the S. alba cDNAlibrary he had constructed and for providing us with theSaLFY probe. MD is grateful to the FRIA for the award of aPhD fellowship. The research was funded by InteruniversityAttraction Poles Programme, Belgian State, Belgian SciencePolicy, P5/13 and by the National Fund for Scientific Research(Grant 9.4547.03).

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