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First published online February 25, 2005; 10.1104/pp.104.058974 Plant Physiology 137:1037-1048 (2005) © 2005 American Society of Plant Biologists Differential Regulation of FLOWERING LOCUS C Expression by Vernalization in Cabbage and Arabidopsis1Institute of BioAgricultural Sciences, Academia Sinica, Taipei 115, Taiwan R.O.C. (S.-I.L., J.-G.W., S.-Y.P., C.-l.S., T.-J.C.); and Tainan District Agricultural Research and Extension Station, Tainan 701, Taiwan R.O.C. (S.-S.W.)
Vernalization is required to induce flowering in cabbage (Brassica oleracea var Capitata L.). Since FLOWERING LOCUS C (FLC) was identified as a major repressor of flowering in the vernalization pathway in Arabidopsis (Arabidopsis thaliana), two homologs of AtFLC, BoFLC3-2 and BoFLC4-1, were isolated from cabbage to investigate the molecular mechanism of vernalization in cabbage flowering. In addition to the sequence homology, the genomic organization of cabbage FLC is similar to that of AtFLC, except that BoFLC has a relatively smaller intron 1 compared to that of AtFLC. A vernalization-mediated decrease in FLC transcript level was correlated with an increase in FT transcript level in the apex of cabbage. This observation is in agreement with the down-regulation of FT by FLC in Arabidopsis. Yet, unlike that in Arabidopsis, the accumulation of cabbage FLC transcript decreased after cold treatment of leafy plants but not imbibed seeds, which is consistent with the promotion of cabbage flowering by vernalizing adult plants rather than seeds. To further dissect the different regulation of FLC expression between seed-vernalization-responsive species (e.g. Arabidopsis) and plant-vernalization-responsive species (e.g. cabbage), the pBoFLC4-1::BoFLC4-1::GUS construct was introduced into Arabidopsis to examine its vernalization response. Down-regulation of the BoFLC4-1::GUS construct by seed vernalization was unstable and incomplete; in addition, the expression of BoFLC4-1::GUS was not suppressed by vernalization of transgenic rosette-stage Arabidopsis plants. We propose a hypothesis to illustrate the distinct mechanism by which vernalization regulates the expression of FLC in cabbage and Arabidopsis.
The transition from the vegetative to reproductive phase is essential for completion of the life cycle of flowering plants. This transition is particularly important in agriculture in terms of productivity. The timing of the reproductive transition is determined by developmental status and environmental conditions. A combination of these two factors ensures that flowering occurs at appropriate times, with an accumulation of sufficient nutrients and favorable environmental conditions (Levy and Dean, 1998  ; Reeves and Coupland, 2000; Mouradov et al., 2002; Simpson and Dean, 2002). Genetic analysis has revealed that multiple pathways, such as the photoperiod and autonomous pathways, are involved in regulating this transition (Koornneef et al., 1998; Levy and Dean, 1998; Simpson et al., 1999). In addition, exposure to an extended period of low temperature is needed to promote flowering in many species (Chourad, 1960). This process is known as vernalization (Napp-Zinn, 1987). Vernalization is a natural adaptation ensuring that flowering occurs only after winter, in order for flowers and seeds to develop under favorable conditions.
During the past few years, two major loci, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC), have established a vernalization requirement in Arabidopsis (Arabidopsis thaliana; Burn et al., 1993
FLC encodes a MADS-box transcription factor that functions as a repressor of the floral transition (Michaels and Amasino, 1999
The repression of AtFLC by vernalization is mitotically stable, which means that the effect caused by cold temperature can be maintained even if treated plants are returned to a warm temperature, which suggests an epigenetic repression (Wellensiek, 1964
In addition to Arabidopsis, other species in the family of Brassicaceae rely on vernalization to promote flowering (Friend, 1985
Vernalization can be classified into two types according to the age of the plant that senses low temperature (Friend, 1985
Cabbage (B. oleracea var Capitata L.) is one of the most important and popular vegetable crops in the Brassicaceae family and is a plant-vernalization-responsive type (Ito et al., 1966
Molecular Cloning and Characterization of Cabbage FLC Genes
Cabbage, like Arabidopsis, is a vernalization-dependent plant in the family Brassicaceae. Since AtFLC was identified as a major flowering repressor in the vernalization pathway in Arabidopsis (Michaels and Amasino, 1999
Repression of BoFLC Expression by Vernalization
To determine whether cabbage FLCs are involved in controlling flowering via the vernalization process, RNA gel-blot analysis was carried out to investigate the FLC expression pattern in response to vernalization. Eight-week-old cabbage plants from 3 different varieties, TNSS42-12, Yehsen, and YSL-0, were vernalized at 4°C for 2 to 6 weeks. Vernalization requirements for these three varieties are different. TNSS42-12 requires at least 45 d at 5°C and Yehsen needs about 30 d at 5°C, whereas YSL-0 can flower after the local winter season in the lowlands of Taiwan, where the average winter temperature is 17°C to 20°C from December to February (http://www.cwb.gov.tw/V4; S.-S. Wang, unpublished data). BoFLC transcripts were analyzed in different tissues (apex, young leaf, mature leaf, stem, and root) before and after cold treatment with use of a BoFLC4-1 cDNA probe lacking the MADS box (Fig. 2). In all three varieties, FLC transcripts were most abundant in the apex before cold treatment. The FLC transcript level was decreased mainly in the apex, young and mature leaves, and to a lesser extent in the stem and root during cold treatment. This observation is consistent with the decrease in AtFLC level in all tissues after vernalization in Arabidopsis (Sheldon et al., 2000
The initial transcript levels of FLC in the apex, young leaf, and mature leaf before cold treatment are similar in these three varieties (Fig. 2D); however, the down-regulation of FLC in YSL-0 was more rapid than in TNSS42-12 and Yehsen after cold treatment. The FLC transcript level was significantly reduced in YSL-0 after 2-week treatment and barely detectable after 4-week treatment but remained substantial in the other 2 varieties during the same period of cold treatment (Fig. 2, A–C), which suggests that the repression of FLC in YSL-0 is more sensitive to cold temperature compared to the other 2 varieties. This observation may explain the shorter vernalization requirement for YSL-0 to flower.
Because in Arabidopsis FT is down-regulated by FLC (Samach et al., 2000
The expression of BoFLC4-1 in TNSS42-12 was monitored in different developmental stages by RNA gel-blot analysis (Fig. 3A). Since the apex was shown previously to have the highest expression of BoFLC4-1, apex tissue was harvested from 1- to 8-week-old cabbage plants for further examination. The entire aboveground tissue, including apex and cotyledon, was collected and indicated as "shoot" for the first week of sampling. Cotyledons were collected separately from the apex of 2-week-old seedlings but not at the 4- and 8-week-old stages since they were senescent. The expression of BoFLC4-1 was very weak in the shoot at the 1-week-old stage but significantly increased at the 2-week-old stage and had a dramatic boost at 4 to 8 weeks (Fig. 3A). Thus, FLC expression strongly increases with age. The FT homolog was highly expressed in the shoot of 1-week-old seedlings, whereas FLC expression was barely detectable at the same stage. However, in older plants, the expression of FT in the apex gradually decreased to an almost undetectable level at the 8-week-old stage, whereas the expression of FLC increased. This result again supports the down-regulation of FT by FLC (Samach et al., 2000
Previous studies had shown that FLC expression was down-regulated and flowering promoted after vernalizing Arabidopsis seeds (Michaels and Amasino, 1999 ; Sheldon et al., 2000). Unlike Arabidopsis, in cabbage seed vernalization is not able to induce flowering. Vernalization is effective only when cold treatment is applied to mature cabbage plants (Ito et al., 1966; Friend, 1985). As shown in Figure 2, a decrease in expression of BoFLC was observed in vernalized 8-week-old cabbage plants. However, BoFLC does not respond to seed vernalization: cold treatment of cabbage seeds (4°C for 4 weeks) did not alter the expression pattern of FLC or FT (compare Fig. 3, A and B). This observation is in agreement with seed vernalization not being effective in promoting flowering in cabbage, which is in contrast with the down-regulation of AtFLC in Arabidopsis when seeds are vernalized.
To determine whether the change of signal intensity shown in these RNA gel blots was attributable to BoFLC4-1 or BoFLC3-2, a 3'-untranslated region (UTR) specific sequence of BoFLC3-2 was used as a probe for hybridization. No BoFLC3-2 specific signal was detected in all tissues examined (data not shown). This result indicated that the expression level of BoFLC3-2 was very low, and signals observed in the RNA gel-blot analysis were mainly derived from BoFLC4-1, although we cannot exclude cross-hybridization to other unidentified BoFLCs. DNA gel-blot analysis revealed that both BoFLC4-1 and BoFLC3-2 exist as single-copy genes in cabbage (data not shown). Nevertheless, more cabbage FLC genes are likely to exist based on the pattern of multiple bands from low stringency hybridization. Previously, Schranz et al. cloned three FLC genes (BoFLC1, BoFLC3, and BoFLC5) from a doubled haploid line of B. oleracea (Schranz et al., 2002
To further understand the biological function of cabbage FLC in regulating flowering, heterologous expression of BoFLC4-1 was conducted in Arabidopsis and flowering time was monitored. We took a dominant-negative approach to inactivate the function of the endogenous AtFLC gene. Since the FLC protein is a MADS transcription factor and MADS proteins usually function as a multimer (Pellegrini et al., 1995
Comparison of Promoter and Intron 1 Sequences of BoFLCs and AtFLC To further study the regulation of BoFLC gene expression in response to vernalization, the promoters of both BoFLC4-1 and BoFLC3-2 were isolated by genome walking. Genomic fragments 2.0 kb and 1.5 kb upstream from the translational start sites of BoFLC4-1 and BoFLC3-2, respectively, were cloned. Comparing promoter sequences to that of AtFLC identified three conserved regions (Fig. 5A). The first conserved sequence (P1) includes the 5' UTR and a sequence up to –200 bp upstream from the translational start site. The second conserved sequence (P2), consisting of 51 bp, was found at –219 to –269 bp of AtFLC. The third conserved sequence (P3) was located at about –2.4 kb and –2.0 kb of AtFLC and BoFLC4-1, respectively, but at about –0.4 kb upstream of BoFLC3-2 from the translational start site. A partial open reading frame encoding a 3-keto-acyl-ACP dehydratase was identified in this 1.5-kb upstream sequence of the BoFLC3-2 gene (Fig. 5A), which indicates that the promoter of BoFLC3-2 could be truncated and this may account for the lack of detectable BoFLC3-2 mRNA.
As mentioned earlier, the introns 1 of BoFLC4-1 and BoFLC 3-2 (approximately 1.1 kb) are relatively smaller than that of AtFLC (approximately 3.5 kb), and the intron 1 sequence of AtFLC was identified to be important in the epigenetic repression of AtFLC through chromatin modification (Sheldon et al., 2002
Since the promoter and intragenic sequences were reported to be crucial for the regulation of AtFLC in response to vernalization (Sheldon et al., 2002
The transgenic T2 lines were examined for the response to seed or plant vernalization. Seeds or 10-d-old rosette plants were treated at 4°C for 2, 4, 6, and 8 weeks. Vernalized seedlings were returned to the normal growth temperature for another 10 d before harvest. Vernalized rosette plants were harvested immediately after treatment. Total RNAs from seedlings without vernalization or with seed-vernalization (Fig. 7A) or plant-vernalization treatment (Fig. 7B) were isolated, and the transcript levels of the endogenous AtFLC and BoFLC4-1::GUS transgenes were inspected.
Upon seed vernalization, the expression of endogenous AtFLC deceased significantly and was not detectable after a 4-week cold treatment (Fig. 7A). Endogenous AtFLC in plant-vernalized seedlings were also down-regulated during cold treatment. Thus, both seed- and plant-vernalization treatments were effective, although the repression during plant vernalization was not as complete as that during seed vernalization (Fig. 7B). However, the down-regulation of the BoFLC4-1::GUS transcript by seed vernalization showed heterogeneity among the 11 transgenic T2 lines examined. Three groups of T2 lines were classified, and one independent line from each group was selected for representation in Figure 7. In the 4 independent lines in group 1, the transcript levels of BoFLC4-1::GUS did not change significantly during seed vernalization (line 1 in Fig. 7A). In the 3 independent lines of group 2, transcripts of BoFLC4-1::GUS were undetectable after 4- and 6-week treatments but reappeared after 8-week treatment of seed vernalization (line 2 in Fig. 7A). In the remaining 4 independent lines in group 3, transcripts of BoFLC4-1::GUS were undetectable at the 4- or 6-week time points, but the expression of BoFLC4-1::GUS was restored after that (line 3 in Fig. 7A). The heterogeneity among several transgenic lines and fluctuation of the BoFLC4-1::GUS transcript during vernalization indicate that the suppression of BoFLC4-1::GUS was incomplete and unstable when transgenic Arabidopsis seeds were vernalized. By contrast, none of transgenic lines exhibited reduced BoFLC::GUS expression after plant-vernalization treatment, even though the level of endogenous AtFLC transcript was gradually reduced during the treatment (Fig. 7B). The instability of repression by seed vernalization and lack of repression of BoFLC::GUS after plant vernalization in transgenic Arabidopsis demonstrate that the repression of BoFLC4-1 by vernalization cannot be properly regulated in Arabidopsis. This result suggests that the regulatory machinery controlling FLC expression by vernalization in cabbage is different from that in Arabidopsis.
In this study, two FLC homologs, BoFLC4-1 and BoFLC3-2, were isolated from cabbage (Fig. 1). The expression of BoFLC4-1 and its regulation by vernalization were analyzed. The FLC transcripts were highest in the apex, as was observed in Arabidopsis (Michaels and Amasino, 1999 ). Steady-state levels of the BoFLC transcript decreased during vernalization of cabbage plants, mainly in the apex and leaf (Fig. 2, A–C). It is of interest to note that the three cabbage varieties (TNSS42-12, Yehsen, and YSL-0) with different vernalization requirements showed similar transcript levels of FLC before vernalization (Fig. 2D) but a different degree of reduction of FLC expression after vernalization (Fig. 2, A–C). Suppression of FLC in YSL-0, a short-vernalization requirement variety, occurred much more rapidly than that of the other two varieties, which require longer exposure to cold.
FT and SOC1 are recognized as key integrators of multiple floral pathways in Arabidopsis (Simpson and Dean, 2002
MADS-box proteins are thought to form dimers through the interaction of the K domain at the center and bind to DNA through the MADS-box domain at the N terminus (Schwarz-Sommer et al., 1992
Comparison of promoter sequences upstream of BoFLC4-1, BoFLC3-2, and AtFLC revealed three conserved regions (P1, P2, and P3; Fig. 5A). The alignment of these conserved regions suggests that a large sequence deletion between the P2 and P3 regions occurred in the promoter of BoFLC3-2. The truncated promoter of BoFLC3-2 may explain its low level of transcripts in cabbage. Positive and negative regulatory elements between the P2 and P3 regions were identified in the promoter of AtFLC (Sheldon et al., 2002
Cabbage flowering can be promoted by vernalizing adult plants but not seeds (Friend, 1985
When transgenic Arabidopsis seeds were subjected to vernalization, the BoFLC4-1::GUS transcript did not show significant changes in some of the transgenic lines (e.g. line 1 in Fig. 7A), but its transcript was undetectable at one or two time points of the treatment in other lines (e.g. lines 2 and 3 in Fig. 7A). However, the down-regulation of the BoFLC4-1::GUS transcript was not sustained after prolonged cold treatment, which indicated that down-regulation was not stable. This observation is inconsistent with the quantitative relationship between the duration of cold treatment and the extent of down-regulation of AtFLC, as observed in Figure 7 and in a previous report (Sheldon et al., 2000
The mechanism involved in inactivation of AtFLC during vernalization recently was demonstrated to be associated with chromatin modification, such as histone deacetylation and methylation (Bastow et al., 2004
The expression of BoFLC4-1::GUS was not inactivated when transgenic rosette plants were subjected to vernalization treatment. We postulate that certain cabbage-specific trans-regulatory factors required for the suppression of BoFLC4-1 during cabbage vernalization do not exist in Arabidopsis. The different responses in terms of seed or plant vernalization are likely due to the distinct interactions between specific cis- and trans-regulatory elements in these two different vernalization-responsive types. Nevertheless, it is also possible that the homologs of upstream regulators of AtFLC such as VIN3 (Sung and Amasino, 2004
In Figure 8, a working hypothesis is proposed for the different mechanisms in controlling cabbage and Arabidopsis flowering by vernalization. The BoFLC transcript gradually accumulates along with a decreased level of BoFT in the apex during cabbage growth. Vernalization of cabbage seeds does not alter the expression of FLC and FT. The presence of vernalization repressors or the absence of vernalization activators could be responsible for the ineffectiveness of seed vernalization in cabbage. The expression of repressors or activators may depend on age and be regulated coordinately in an opposite manner. During development, the loss of repressors or accumulation of activators may be associated with increased competence to sense cold temperature. The accumulated vernalization activators, together with cold temperature, in mature cabbage plants can efficiently down-regulate the expression of FLC (Fig. 8A). Without vernalization, cabbage remains in vegetative growth. The expression of FT shows an opposite pattern to that of FLC, and increased FT expression in the apex after plant vernalization may be critical to induce cabbage flowering (Fig. 2A). However, seed vernalization is more effective than plant vernalization in down-regulating FLC expression in Arabidopsis (Fig. 7). This observation is consistent with a previous report that cold treatment at the rosette stage is less effective in promoting flowering than seed cold treatment in Arabidopsis (Nordborg and Bergelson, 1999
In summary, we show that cabbage BoFLC4-1 plays a similar role to Arabidopsis FLC in controlling flowering. However, AtFLC responds to seed vernalization but BoFLC4-1 does not. Comparison of promoter and intragenic sequences between BoFLC4-1 and AtFLC revealed differences that may be specifically involved in either the seed or plant vernalization response. Further characterization of these sequences may provide an opportunity to dissect different regulatory mechanisms of the vernalization response in seed- versus plant-responsive species.
Plant Material and Treatment
Cabbage (Brassica oleracea var Capitata L.) was grown in growth chambers with a 16-h photoperiod (with cool fluorescence light at 150–200 µE m–2 s–1) and a 24°C/20°C light/dark temperature cycle. Three cabbage varieties, TNSS42-12 and YSL-0, two inbred lines, and Yehsen, an open-pollinated local variety, provided by the Tainan District Agricultural Research and Extension Station, were used for vernalization treatment (Wang et al., 2000
The cDNA of BoFLC4-1, lacking the MADS-box domain, was cloned by reverse transcription-PCR using primers designed from the AtFLC sequence (forward primer 5'-ggcgataacctggtcaagat-3' and reverse primer 5'-tacaaacgctcgcccttatc-3'). The full-length BoFLC4-1 gene was subsequently cloned after screening a cabbage cDNA library constructed by the SMART cDNA library construction kit (CLONTECH, Palo Alto, CA). PCR and genomic walking (GenomeWalker kit; CLONTECH) were carried out to obtain the genomic and cDNA sequences of BoFLC4-1 and BoFLC3-2 genes. The promoters of both BoFLC4-1 and BoFLC3-2 also were cloned with use of the same genomic walking approach. The phylogenetic relationship of the partial amino acid sequences of BoFLC3-2 and BoFLC4-1 and other FLC proteins from Arabidopsis, Brassica napus, Brassica rapa, and Brassica oleracea was analyzed by the Phylip program (http://evolution.genetics.washington.edu/phylip.html). Partial amino acid sequences were used in this analysis because the exon 1 sequences of several BoFLC and BrFLC genes are unavailable (Schranz et al., 2002
Total RNA was isolated from various tissues using TRIzol reagent (Invitrogen, Carlsbad, CA). An amount of 15 µg of total RNA was run on a 2.2 M formaldehyde-agarose gel and blotted onto a nylon membrane (Roche, Mannheim, Germany). The membrane was stained with reversible methylene blue (Sigma B-1177; St. Louis) to check the loading and transfer of RNA in each sample. AtFLC and BoFLC4-1 cDNA, lacking the MADS box, and AtFT cDNA (AV563203, a EST clone from Kazusa DNA Research Institute) were used to generate antisense DIG-UTP-labeled riboprobes (Roche). Membranes were hybridized at 62°C in hybridization solution (50% [v/v] formamide, 5x SSC, 0.1% [w/v] sodium lauroyl sarcosine, 0.02% [w/v] SDS, 2% [w/v] blocking reagent) overnight, and then washed in 2x SSC/0.1% (w/v) SDS twice for 5 min at room temperature, and in 0.1x SSC/0.1% (w/v) SDS twice for 15 min at 68°C. Detection was performed by use of the Detection Starter Kit II, following the manufacturer's instructions (Roche). The signal was analyzed with use of a luminescent image analyzer (LAS-1000 plus; Fujifilm, Tokyo) combined with Image Gauge version 3.12 software (Fujifilm).
The sequence containing the 2.0-kb promoter and complete 5' UTR, exons, and introns of BoFLC4-1 (5.5 kb in total) was placed upstream of the GUS reporter gene in the pCambia 1381Z vector (Cambia, Canberra, Australia). The stop codon of BoFLC4-1 was deleted and immediately fused to the start codon of the GUS gene in the vector. A dominant-negative clone was generated by overexpressing N-terminal or C-terminal truncated BoFLC4-1 in the pCambia 2300 (Cambia) driven by a cauliflower mosaic virus 35S promoter. The N-terminal truncated protein lacked the N-terminal MADS-box domain of 59 amino acids (DN1), whereas the C-terminal truncated protein lacked the C domain of 49 amino acids (DN2). These constructs were sequence verified to rule out any mistakes during cloning. They were subsequently transformed into Agrobacterium tumefaciens GV3101pMP90. Arabidopsis transformation was carried out by use of the floral dip method (Clough and Bent, 1998
Flowering time was determined by the number of rosette leaves formed when the inflorescent stem reached approximately 1 to 3 cm long. The flowering time of 19 and 13 independent T2 lines of DN1 and DN2 dominant-negative constructs, respectively, were assessed. Three replicate plants of each line were analyzed.
GUS staining was performed according to Jefferson et al. (1987)
We thank Drs. Kenrick Jaichard and Ning-Sun Yang and Miss Laura Heraty for critically reading the manuscript, Dr. Pei-Ing Hwang and Miss Ho-Ming Chen for sequence analysis, and Dr. Yee-Yung Charng for valuable discussion during the process of this study. Received December 26, 2004; returned for revision December 27, 2004; accepted December 27, 2004.
1 This work was supported by grants from Academia Sinica and the National Science Council of the Republic of China (grant nos. NSC–90–2313–B–001–022 and NSC–91–2313–B–001–032 to T.-J.C.). 
2 Present address: Vita Genomics, Inc., Taipei 248, Taiwan R.O.C. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.058974. * Corresponding author; e-mail tjchiou{at}gate.sinica.edu.tw; fax 886–2–26515600.
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ABSTRACT
RESULTS
-glucuronidase (GUS) reporter gene (pBoFLC4-1::BoFLC4-1::GUS) and transformed into Arabidopsis to examine its expression pattern and vernalization responses in Arabidopsis. GUS staining was observed in the apex, young leaves, and roots of transgenic Arabidopsis seedlings (
