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

Authors

  • Maria D’Aloia,

    1. Laboratory of Plant Physiology, Department of Life Sciences, University of Liège, Bât. B22 Sart Tilman, Boulevard de Colonster 27, B-4000 Liège, Belgium
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  • Pierre Tocquin,

    1. Laboratory of Plant Physiology, Department of Life Sciences, University of Liège, Bât. B22 Sart Tilman, Boulevard de Colonster 27, B-4000 Liège, Belgium
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  • Claire Périlleux

    1. Laboratory of Plant Physiology, Department of Life Sciences, University of Liège, Bât. B22 Sart Tilman, Boulevard de Colonster 27, B-4000 Liège, Belgium
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Author for correspondence:
Claire Périlleux
Tel:+32 4366 38 33
Fax:+32 4366 38 31
Email: cperilleux@ulg.ac.be

Summary

  • • Of the Brassicaceae, Sinapis alba has been intensively studied as a physiological model of induction of flowering by a single long day (LD), while molecular-genetic analyses of Arabidopsis thaliana have disclosed complex interactions between pathways controlling flowering in response to different environmental cues, such as photoperiod and 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 floral repressor FLOWERING LOCUS C, was isolated and the transcript levels quantified in different conditions.
  • • Two-week-old seedlings grown in noninductive short days (SDs) were vernalized for 1–6 wk. Down-regulation of SaFLC was already marked after 1 wk of cold but 2 wk was needed for a significant acceleration of flowering. Flower buds were initiated during vernalization. When vernalization was stopped after 1 wk, repression of SaFLC was not stable but a significant increase in plant responsiveness to 16-h LDs was observed when LDs followed immediately after the cold treatment.
  • • These results suggest that vernalization does not only work when plants experience long exposure to cold during the winter: shorter cold periods might stimulate flowering of LD plants if they occur when photoperiod is increasing, such as in spring.

Introduction

In the natural conditions of temperate areas, many factors of the environment influence plant reproduction, and those exhibiting predictable seasonal variation were selected for reliable timing of floral transition (Bernier & Périlleux, 2005). Primary factors controlling flowering time include winter cold (vernalization) and photoperiod, on which most experimental work was focused. Early physiological studies showed that these environmental factors are mainly perceived by different organs: vernalization is mostly sensed by mitotically active tissues – including the shoot apical meristem (SAM) itself – while photoperiod is perceived by expanded leaves and requires systemic signalling towards the SAM in order to induce flowering (Bernier et al., 1981; Metzger, 1988). Moreover, a majority of cold-requiring plants require long days (LDs) after vernalization, suggesting that vernalization acts as a first step in bringing about the competence to flower (Chouard, 1960). Spatial and temporal separation thus explained why the mechanisms of vernalization and photoperiodic induction of flowering were studied separately and physiological models were selected for their strong requirement for one or the other environmental factor. Among photoperiodic species, physiologists favoured those that flower in response to a single inductive cycle because of the high synchrony that can be achieved during the transition. For example, Sinapis alba (white mustard) plants, when grown in controlled conditions in phytotronic cabinets, remain vegetative in 8-h short days (SDs) and can be induced to flower by a single LD when 2 months old (Bernier, 1969). This experimental system has been used extensively to analyse physiological signals leading to flowering, and a detailed picture of sequential events of floral transition has been described (reviewed in Bernier et al., 1993; Bernier & Périlleux, 2005). In contrast, much less is known about vernalization in S. alba although, in natural conditions, this species behaves as a quantitative LD- and cold-requiring plant (Bernier, 1969; Bodson, 1985).

Most recent studies of the molecular-genetic mechanisms that control flowering have focused on Arabidopsis thaliana, a model 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 mutants were based on flowering response to the primary factors photoperiod and vernalization (Koornneef et al., 1991; Martínez-Zapater et al., 1994). Mutants and genes were therefore classified into three main classes which, after further epistasis and molecular analyses, defined three genetic pathways controlling flowering time in A. thaliana: the LD-promoting pathway, the vernalization-promoting pathway, and the ‘autonomous’ pathway which is defective in mutants that are late flowering but still sensitive to photoperiod and vernalization. A fourth route – the gibberellin (GA) pathway – was added on the basis of the observation that flowering of GA-deficient mutants is much delayed, mainly in SDs. These flowering pathways control a set of ‘integrator’ genes, which include FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVER-EXPRESSION OF CO 1 (SOC1, previously named AGL20) that act upstream of the genes involved in the flower initiation process such as LEAFY (LFY) and APETALA1 (AP1) (reviewed in Boss et al., 2004; Bernier & Périlleux, 2005; Corbesier & Coupland, 2005).

The photoperiodic pathway involves interactions between light signalling and the circadian clock. LDs promote flowering in A. thaliana by the activation of the transcription factor CONSTANS (CO): the abundance of CO exhibits circadian oscillations (Suárez-López et al., 2001) and only in LDs does a peak in the amount of protein coincide with the presence of light. Far-red and blue lights perceived by phytochrome A and cryptochrome 2 stabilize the protein (Valverde et al., 2004). This ‘external coincidence’ allows CO to activate its targets FT and SOC1 (Onouchi et al., 2000; Samach et al., 2000). Of the utmost importance, the FT protein was recently shown to participate in the systemic signalling that induces flowering in A. thaliana (Corbesier et al., 2007; Jaeger & Wigge, 2007; Mathieu et al., 2007).

Both the vernalization and autonomous pathways act by repressing a flowering inhibitor: the MADS box gene FLOWERING LOCUS C (FLC) (Michaels & Amasino, 2000). Vernalization down-regulates the FLC mRNA level, with longer periods of exposure to cold leading to less FLC mRNA (Sheldon et al., 2000). The process is saturated after several weeks of cold treatment; the vernalized state then remains stable for the rest of the plant life cycle and is reset at meiosis. This epigenetic silencing of FLC is mediated by histone modifications in the promoter and intron 1 regions: an early step in this process is H3 deacetylation, and the stable maintenance of FLC repression involves H3K9 and H3K27 methylation. These modifications create a ‘histone code’ associated with FLC repression (reviewed in Sung & Amasino, 2006).

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

We were therefore interested in studying vernalization and the participation of FLC in the control of flowering in the LD plant S. alba. This strategy was greatly encouraged by the taxonomic proximity of Sinapis and Arabidopsis, both members of the Brassicaceae. A number of FLC orthologues have been cloned in Brassica species, using genomics information on A. thaliana (Tadege et al., 2001; Schranz et al., 2002; Martynov & Khavkin, 2004; Lin et al., 2005; Razi et al., 2008). A high rate of conservation between A. thaliana and S. alba sequences was also reported. For example, SaMADS A, which is orthologous to SOC1, was cloned from S. alba as the earliest MADS box gene expressed in the shoot apical meristem at floral transition (Menzel et al., 1996). Overexpression of SaMADS A in A. thaliana caused precocious flowering (Bonhomme et al., 2000), as also found for other SOC1 orthologues cloned from other Brassicaceae species (Kim et al., 2003). We report here isolation of SaFLC and physiological analyses of its function in flowering response to vernalization and photoperiod.

Materials and Methods

Plant growth conditions

The seeds of Sinapis alba L. cv. Carla were purchased from Job-semences S.A.R.L. (Nancy, France). Plants were grown in 8-h SDs, as described by Lejeune et al. (1988). The fluence rate at the top of the plants was 150 µmol m−2 s−1 over the waveband 300–700 nm and was provided by very-high-output Sylvania fluorescent white tubes (Zaventem, Belgium). The temperature was kept constant at 20°C, except during the vernalization treatments, where 2-wk-old plants were transferred to 7°C and 85 µmol m−2 s−1 for 1–6 wk before being returned to standard conditions. Flowering time was scored as ‘days to macroscopic appearance of floral buds’.

In experiments where plants were transferred transiently to 16-h LDs, flowering was measured by dissection of the apical buds under the binocular microscope 2 wk after the start of exposure to LDs and was expressed as a percentage of floral plants. A plant was classified as floral when at least one flower primordium was present within the apical bud. Each experimental batch numbered 15–20 individual plants.

Isolation of SaFLC cDNA and sequence analysis

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

Approximately 150 000 clones from the library were screened and one positive colony was isolated, subcloned into pGEM®-T Vector (Promega, Madison, WI, USA; http://www.promega.com/) and sequenced. Sequence alignment showed a high level of identity with Brassica napus BnFLC1 and AtFLC; thereafter we called the isolated sequence SaFLC. In order to isolate a full-length cDNA, we designed a pair of primers based on the nucleotide sequence of BnFLC1 (GenBank accession no. AY036888) for the 5′ region (BnFLC1-fwd: 5′-AGGGCGCAAAGCACTGTTGGAGAC) and on the partial SaFLC clone for the 3′ region (SaFLC-rev: 5′-TTACAAGGGGATAAATACACATCTTG-3′). Reverse transcription polymerase chain reaction (RT-PCR) was performed on RNA extracted from aerial parts of 2-wk-old S. alba plants. A fragment of 702 bp was obtained, cloned into pGEM®-T Vector and sequenced (GenBank accession no. EF542803).

Amino acid sequence alignment of SaFLC and other FLC proteins from A. thaliana, Arabidopsis arenosa, Arabidopsis suecicea, Arabidopsis lyrata, B. napus, Brassica oleracea, Brassica rapa, Brassica juncea and Raphanus sativus were generated using the ClustalW program (http://www.infobiogen.fr/services/analyseq/cgi-bin/clustalw_in.pl). Partial amino acid sequences were used for alignment because the sequence of the MADS box is unavailable for some FLC genes (BoFLC, BrFLC and BjFLC) (Schranz et al., 2002; Martynov & Khavkin, 2004). Aligned sequences were analysed using the paup* 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-Laprade et al. (1999). Digestion was performed with the restriction enzyme EcoRI, HindIII or NcoI (5 U µg–1) overnight at 37°C. Twenty-µg samples were separated on 1% (w/v) agarose gel and blotted onto positively charged nylon membrane (Roche, Mannheim, Germany). The probe was synthesized from truncated SaFLC cDNA lacking the MADS box (337 pb) and cloned in pGEM®-T. Radiolabelling was performed by random priming using 32P-ATP according to the manufacturer's instructions (RadPrime DNA labelling system; Invitrogen, Carlsbad, CA, USA). Blotted DNA fragments were hybridized overnight at 55°C in Herby buffer: 250 mM sodium 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 twice in 2 × saline sodium citrate (SSC) containing 0.1% (w/v) SDS for 10 min, once in 2 × SSC containing 0.1% (w/v) SDS at 60°C and finally in 1 × SSC containing 0.1% (w/v) SDS for 5 min at 60°C. X-ray film (Kodak BioMax MS film) was exposed for 3 d.

RNA extraction and quantification

Total RNA was extracted from tissues using the TRIzol method (Invitrogen; http://www.invitrogen.com/). DNA-free RNA was obtained by DNase treatment (0.2 U DNase µg−1) according to the manufacturer's instructions (Promega). The first strand of cDNA was synthesized from 5 µg of total RNA using the Moloney murine leukaemia virus reverse transcriptase and an Oligo(dT)15 primer according to the manufacturer's protocol (Promega) in a reaction volume of 40 µl. The cDNA was diluted 2.5-fold with water and 5 µl of diluted cDNA was used for quantification.

Quantitative real-time RT-PCR for SaFLC mRNA quantifi-cation  For quantification of SaFLC transcripts by quantitative real-time RT-PCR (qRT-PCR), cDNA was prepared from 3-mm-high shoot apices (at least 15 apices per sample, harvested in the plant growing chambers, at either 7 or 20°C). Serial dilutions of a concentrated first-strand cDNA stock were used as relative standards. The PCR reaction mix was prepared using a commercially available master mix containing Taq DNA polymerase, SYBR-Green I, deoxyribonucleoside triphosphates and MgCl2 (iQ SYBR Green Supermix; Bio-Rad, Hercules, CA, USA). Primers were used in a final concentration of 0.5 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°C for 10 min. PCR reactions were performed in triplicate on each cDNA sample, using the iCycler iQ real-time PCR detection system from Bio-Rad. Transcript levels were normalized with the amount of transcripts from a β-TUBULIN gene (SaTUB). The specificity of the amplification was confirmed by melting curve analysis and agarose gel electrophoresis. The primers used (SaFLC-fwd, SaFLC-rev, SaTUB-fwd and SaTUB-rev) are listed below.

Semi-quantitative RT-PCR for SaMADS A mRNA quantification  For SaMADS A expression analyses, cDNA was prepared from 3-mm-high shoot apices (at least 15 apices per sample). 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°C for 10 min SaTUB was used as a control gene (same PCR programme, 20 cycles). The PCR products were transferred onto a nylon Hybond-N membrane (GE Healthcare, Diegem, Belgium) and were hybridized using a digoxigenin-labelled probe prepared according to the manufacturer's instructions (Roche). The SaMADS A probe was a fragment amplified by 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). Fixed tissues were dehydrated, embedded in paraffin according to standard procedures and cut with a rotary microtome. Longitudinal sections (8 µm) were mounted onto poly L-lysine-coated slides and were pretreated for hybridization according to the method of Angerer & Angerer (1992). 35S-UTP-labelled antisense riboprobes were synthesized using T7 and T3 RNA polymerases according to the manufacturer's instructions (Promega). The SaFLC probe was obtained from the same truncated cDNA clone (337 bp) as used for Southern blot hybridization; the SaLFY probe was obtained from a complete 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, RNase treatment, washing steps, coating with Kodak NTB-2 nuclear Track emulsion, and development of slides were performed according to Angerer & Angerer (1992). Slides were exposed for 2 wk and were stained with 0.1% calcofluor. Autoradiographs were observed with dark-field illumination and the underlying 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, we first decided to grow the plants in noninductive 8-h SDs and to start the vernalization treatments when they were 2 wk old. At that stage, plants had their cotyledons plus two leaves expanded and shoot apices could easily be harvested. Vernalization was always carried out at 7°C (day and night).

Different durations of vernalization (1–6 wk) were tested in three independent experiments, where flowering time was recorded as ‘days to macroscopic appearance of floral buds’. As shown 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 occurred twice as fast as in nonvernalized plants. The vernalization response was saturated after 6 wk: plants actually initiated flowers during their growth in cold conditions and floral buds became macroscopically visible ∼70 d after sowing, that is, ∼55 d after start of the cold treatment.

Figure 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).

Isolation and characterization of SaFLC

An FLC-like cDNA from S. alba (hereafter referred to as SaFLC) was obtained in two steps: screening of a leaf cDNA library with an AtFLC cDNA probe, followed by full-length cDNA cloning by RT-PCR (see the Materials and Methods). SaFLC (702 bp; GenBank accession no. EF542803) encodes a MADS-box protein with predicted amino acid sequence showing 95% identity with BnFLC1 and 85% identity with AtFLC. Phylogenetic analyses performed with Brassicaceae sequences indicated that the predicted SaFLC protein fell into one well-segregated clade with the BnFLC1, BoFLC1 and BrFLC1 proteins (Fig. 2). This group was the closest to the Arabidopsis genus FLC proteins.

Figure 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).

We have estimated the number of FLC copies in the S. alba genome by Southern blot analysis (Fig. 3). Genomic DNA was digested and hybridized with a SaFLC cDNA probe lacking the MADS-box region. We detected one band in all digestion reactions, suggesting that SaFLC is present as a single-copy gene in the S. alba genome.

Figure 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.

This result was confirmed by an alternative PCR approach developed in B. rapa (Schranz et al., 2002). The methodology was based on the fact that the structure of the AtFLC gene – containing seven exons and six introns – is conserved among Brassica species, while the length of introns is highly variable. In B. rapa, four different PCR products were amplified with conserved primers of exons 2 and 7, indicating the existence of four BrFLC copies (Schranz et al., 2002). Using the same strategy, we obtained a single 1693-bp amplicon in S. alba, giving further support to our inference that SaFLC is a unique gene. The 1693-bp PCR fragment was sequenced and showed 87% identity with the genomic region from exon 2 to exon 7 of BrFLC1 (AY115678) (results not shown).

Expression pattern of SaFLC and downstream genes during vernalization

Expression of SaFLC was detected by in situ hybridization in the shoot apex of 8-wk-old nonvernalized plants (Fig. 4a), but not in plants that had been vernalized for 6 wk (Fig. 4b). Note that the apex shown in Fig. 4(b) was initiating flower primordia during the vernalization treatment.

Figure 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).

Real-time RT-PCR was carried out for a time-course analysis of SaFLC expression during vernalization. Shoot apices were harvested weekly; four independent experiments were conducted. As can be seen in Fig. 4(c), SaFLC transcript levels remained quite high and constant in nonvernalized controls during the course of the experiment. In contrast, SaFLC decreased sharply in apices of vernalized plants, and the transcript level was already very low after 1 wk at 7°C. Further exposure to cold correlated with a further decrease in the SaFLC transcript level.

AtFLC was shown to repress SOC1 in the SAM (Searle et al., 2006). We therefore examined, using semi-quantitative RT-PCR, the expression pattern of the orthologous gene SaMADS A (Bonhomme et al., 2000) during the vernalization treatment. As shown in Fig. 5(a), expression of SaMADS A was not detected in nonvernalized apices, as already shown by Bonhomme et al. (2000). The SaMADS A transcript level increased in the shoot apex from the 3rd week of vernalization and was highest after 6 wk of vernalization. Because, as mentioned above, floral buds were initiated during long vernalization treatments, we also performed in situ hybridizations with an SaLFY probe. In nonvernalized apices, expression of SaLFY was detected at the tip of leaf primordia, but not in the SAM (Fig. 5b). After 4 wk of vernalization, up-regulation of the gene was observed in the whole SAM, which had increased in size and was more domed (Fig. 5c). After 6 wk of vernalization, SaFLY was highly expressed in flower primordia and in the flanks of the SAM (Fig. 5d).

Figure 5.

Expression of Sinapis alba SaMADS A and LEAFY (SaLFY) during vernalization. (a) Time-course analysis of SaMADS A expression during vernalization in 8-h short days (SDs) at 7°C. Shoot apices were harvested from 15 plants for each sample. Semi-quantitative reverse transcription polymerase 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.

Expression pattern of SaFLC postvernalization

The effect of vernalization appeared to be much greater on SaFLC repression (Fig. 4) than on flowering time in SDs (Fig. 1). We therefore quantified SaFLC transcripts after vernalization. In two independent experiments, plants were vernalized for 1 or 3 wk and were harvested either immediately after the vernalization treatment (post vernalization time (PVT) 0) or 2 wk later (PVT2) (Fig. 6a). Nonvernalized controls were harvested at each sampling time. As shown in Fig. 6(b), it was observed that the level of SaFLC transcript, although very low at the end of the vernalization treatment, recovered when plants were returned to 20°C. When plants had been vernalized for only 1 wk, the level reached at PVT2 was not different from that observed in nonvernalized controls. When plants had been vernalized for 3 wk, the SaFLC transcript level remained lower than in nonvernalized controls.

Figure 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).

Effect of vernalization and SaFLC on plant sensitivity to photoperiod

In order to examine whether 1 wk of vernalization, although insufficient for stable repression of SaFLC, could have a physiological effect on flowering, we studied plant responsiveness to LDs. Plants were exposed to one or two 16-h LDs just after the vernalization treatment (PVT0), or after having been returned to 20°C for 2 wk (PVT2) (Fig. 7a). Nonvernalized controls were also exposed to the LD(s) but, because plant growth was slowed down during the cold treatment, vernalized plants exposed to LDs at PVT0 were compared with nonvernalized controls that received the LD(s) 4 d before, when they had approximately the same mean leaf area (10.4 ± 2.1 cm2 and 9.3 ± 1.4 cm2 for vernalized and nonvernalized plants, respectively). The flowering response was evaluated 2 wk after the LD(s) by dissecting apical buds and calculating the ‘percentage of floral plants’. Three independent experiments were carried out. As can be seen in Fig. 7(b), the 1-wk vernalization treatment had a strong promotive effect on the floral response to LDs when the LDs immediately followed vernalization: we indeed observed a 3-fold increase in the flowering response of vernalized plants to two 16-h LDs given at PVT0, as compared with nonvernalized controls. When 2 wk at 20°C were interpolated between the vernalization treatment and LD exposure (PVT2), the short vernalization treatment appeared to be insufficient to promote the flowering response to one or two 16-h LDs, as was found previously in continuous SDs (Fig. 1). Figure 7(b) also illustrates – by the comparison of PVT0 with PVT2 – that the flowering response to LDs increases with plant age.

Figure 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).

Discussion

The vernalization response of S. alba is quantitative

The aim of the research was to investigate the vernalization pathway of flowering time control in S. alba. This species has been previously shown to be sensitive to vernalization at the seed and adult stages (Bernier, 1969; Bodson, 1985). We show here that vernalization of 2-wk-old seedlings could accelerate flowering in noninductive SDs, and that floral transition occurred during the vernalization period when it was extended over ∼4 wk. However, plants of S. alba do not require vernalization, as nonvernalized plants do flower – albeit very late – even in SDs (Fig. 1). Sinapis alba is indeed a facultative vernalization- and LD-responsive species.

The flowering response to vernalization in SDs was observed only in plants that had been exposed to cold for at least 2 wk (Fig. 1). When macroscopic appearance of floral buds was used as a criterion for flowering, the positive effect of increasing vernalization duration was observed for up to 5–6 wk of cold treatment. However, the use of SaMADS A and SaLFY as earlier markers of the floral shift showed that floral 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 molecular mechanisms of the S. alba response to vernalization. The sequence that was isolated showed a high identity with AtFLC1. Phylogenetic data presented herein demonstrate that the predicted SaFLC protein clusters with the FLC1 sequences found in different members of the Brassicaceae family (BnFLC1, BoFLC1 and BrFLC1). This is consistent with the fact that S. alba is called Brassica hirta by some authors (Primard et al., 1988). Among all the amino acid sequences found in the phylogenetic tree, the group made up of FLC1 is 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) although the S. alba genome is assumed to have been triplicated during evolution, containing three Arabidopsis genome equivalents (Nelson & Lydiate, 2006). A high rate of chromosomal rearrangement (fusion and fission) following polyploidy events might explain discrepancies between expected and actual gene copy numbers (Lysak et al., 2005; Town et al., 2006). For example, in B. napus, which originated from an interspecific hybridization between B. rapa and B. oleracea, Tadege et al. (2001) found five FLC genes instead of six. We therefore hypothesize that S. alba has lost two copies of FLC from its ancestor.

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

We observed a clear down-regulation of SaFLC by vernalization, which is in agreement with results obtained not only in A. thaliana, but also in other Brassicaceae species such as B. napus, B. oleracea, B. rapa and Thellungiella halophila (Tadege et 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, the amount of SaFLC transcripts was already greatly decreased by a short cold treatment of 1 wk (Fig. 4c), but stabilization of the repression required longer exposure to cold (Fig. 6b). The longer the vernalization treatment, the lower the remaining SaFLC transcript level, and the stronger the effect on flowering time (Fig. 1). This correlation between FLC repression and the quantitative effect of vernalization was described in A. thaliana by Sheldon et al. (2000).

AtFLC targets include SOC1 in the SAM and FT in the leaves (Searle et al., 2006), which are repressed unless FLC is down-regulated by vernalization. We followed expression of SaMADS A, which is orthologous to SOC1 (Bonhomme et al., 2000), during vernalization and we observed that it increased in the shoot apex after ∼3 wk of exposure to cold (Fig. 5a), that is, after down-regulation of SaFLC. It is still possible, however, that SaMADS A was also up-regulated by cold independently of SaFLC, as – in A. thaliana– vernalization may cause induction of SOC1 in an flc mutant (Moon et al., 2003). SaLFY appeared in the SAM approx. 1 wk after SaMADS A (Fig. 5b), which is consistent with the idea that LFY is activated by SOC1 (Moon et al., 2005). Although our timing needs to be refined by more frequent sampling, a rather long lapse of time between up-regulation of SaMADS A and up-regulation of SaLFY could be expected, as this occurred 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 nicely with the quantitative effect of vernalization on the flowering response of S. alba (Fig. 1), a major discrepancy appeared: 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 of vernalization was transient. Indeed, quantification of SaFLC transcript postvernalization showed that, when plants were transferred back to 20°C, its level returned to that of nonvernalized plants when vernalization was short (1 wk) but remained lower if vernalization was longer (Fig. 6b) This result clearly indicates that 1 wk of vernalization was not sufficient for stable repression of SaFLC, while 3 wk of vernalization was; this difference may explain the ineffectiveness of the shortest treatment for accelerating flowering in SDs.

In A. thaliana, maintenance of AtFLC repression after exposure to cold requires the functions of the VERNALIZATION 1 (VRN1), VRN2 and LIKE HETEROCHROMATIN PROTEIN 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 cis regulatory sequences, as shown by the fact that the AtFLC promoter and intron 1 are sufficient for down-regulation and maintenance of repression by cold (Sheldon et al., 2002, 2006; Sung et al., 2006). Recently, natural variation in vernalization response has been correlated with the stability of AtFLC repression and with the rate of accumulation of AtFLC histone H3K27 trimethylation (Shindo et al., 2006).

Maintenance of SaFLC down-regulation might be controlled by mechanisms in S. alba similar to those in A. thaliana, and hence regulatory sequences may account for the degree of stability of the vernalized state. Variations in AtFLC intron 1 sequences have already been described in the literature (Lempe et al., 2005). For instance, a mutator-like sequence found at the 3′ end of intron 1 in the Ler AtFLC allele seems to limit AtFLC RNA accumulation (Gazzani et al., 2003; Michaels et al., 2003). Also, A. thaliana transgenic lines containing the BoFLC transgene (from B. oleracea var. capitata) have been shown to display variable repression of the transgene, which could be a result of a 2.6-kb deletion in BoFLC intron 1, as compared to AtFLC (Lin et al., 2005). The regulatory function of SaFLC intron 1 remains to be investigated.

Down-regulation of SaFLC enhances sensitivity to photoperiod

We observed that 1 wk of vernalization – which was sufficient for SaFLC down-regulation but insufficient for stabilization of the repressed state – did not stimulate flowering in SDs (Fig. 1), but had a clear promotive effect when followed immediately by inductive LDs: the percentage of plants that flowered in response to two 16-h LDs was tripled when they had been vernalized for 1 wk, as compared with nonvernalized plants (Fig. 7b). However, the vernalizing effect of the 1-wk treatment did not persist: when the LDs were given after 2 wk of growth postvernalization, vernalized and nonvernalized plants showed a similar flowering response to LDs. Extrapolation of these results to natural conditions means that the requirement for vernalization is longer when experienced in winter – because LDs do not immediately follow and SaFLC repression requires stabilization – while short periods of cold occurring in spring could enhance plant response to increasing photoperiod. Such environmental conditions are far from rare in temperate regions.

Down-regulation of FLC might stimulate the photoperiodic pathway directly through FT and SOC1, as recent literature has provided evidence that these integrator genes are repressed by AtFLC (Searle et al., 2006), and hence vernalization acts in both the leaf and the SAM. Cold could also have a less direct effect on the floral response to LDs. Indeed, Salathia et al. (2006) reported that vernalization of A. thaliana seedlings led to a shortening of the circadian period of leaf movements, and period shortening can be associated with earlier flowering under shorter photoperiods (Yanovsky & Kay, 2002). Whether the effect of short vernalization treatments is produced via such complex networks warrants further investigation and is of great ecological relevance.

Acknowledgements

We would like to thank Prof G. Bernier and Dr L. Corbesier for their critical reading of the manuscript. We are much indebted to A. Pieltain and N. Detry for their excellent technical assistance and to A. Havelange, P. Perruzza and D. Libion for taking 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 cDNA library he had constructed and for providing us with the SaLFY probe. MD is grateful to the FRIA for the award of a PhD fellowship. The research was funded by Interuniversity Attraction Poles Programme, Belgian State, Belgian Science Policy, P5/13 and by the National Fund for Scientific Research (Grant 9.4547.03).

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