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Antagonistic regulation of flowering-time gene SOC1 by CONSTANS and FLC via separate promoter motifs

2002; Springer Nature; Volume: 21; Issue: 16 Linguagem: Inglês

10.1093/emboj/cdf432

1460-2075

Shelley R. Hepworth, Federico Valverde, Dean Ravenscroft, Aidyn Mouradov, George Coupland,

Plant Reproductive Biology

Article15 August 2002free access Antagonistic regulation of flowering-time gene SOC1 by CONSTANS and FLC via separate promoter motifs Shelley R. Hepworth Shelley R. Hepworth John Innes Centre, Colney Lane, Norwich, NR4 7UH UK Present address: Department of Botany, University of British Columbia, Vancouver, BC V6T 1Z4 Canada Search for more papers by this author Federico Valverde Federico Valverde John Innes Centre, Colney Lane, Norwich, NR4 7UH UK Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany Search for more papers by this author Dean Ravenscroft Dean Ravenscroft John Innes Centre, Colney Lane, Norwich, NR4 7UH UK Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany Search for more papers by this author Aidyn Mouradov Aidyn Mouradov Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany Search for more papers by this author George Coupland Corresponding Author George Coupland John Innes Centre, Colney Lane, Norwich, NR4 7UH UK Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany Search for more papers by this author Shelley R. Hepworth Shelley R. Hepworth John Innes Centre, Colney Lane, Norwich, NR4 7UH UK Present address: Department of Botany, University of British Columbia, Vancouver, BC V6T 1Z4 Canada Search for more papers by this author Federico Valverde Federico Valverde John Innes Centre, Colney Lane, Norwich, NR4 7UH UK Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany Search for more papers by this author Dean Ravenscroft Dean Ravenscroft John Innes Centre, Colney Lane, Norwich, NR4 7UH UK Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany Search for more papers by this author Aidyn Mouradov Aidyn Mouradov Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany Search for more papers by this author George Coupland Corresponding Author George Coupland John Innes Centre, Colney Lane, Norwich, NR4 7UH UK Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany Search for more papers by this author Author Information Shelley R. Hepworth1,2, Federico Valverde1,3, Dean Ravenscroft1,3, Aidyn Mouradov3 and George Coupland 1,3 1John Innes Centre, Colney Lane, Norwich, NR4 7UH UK 2Present address: Department of Botany, University of British Columbia, Vancouver, BC V6T 1Z4 Canada 3Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:4327-4337https://doi.org/10.1093/emboj/cdf432 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Flowering in Arabidopsis is controlled by endogenous and environmental signals relayed by distinct genetic pathways. The MADS-box flowering-time gene SOC1 is regulated by several pathways and is proposed to co-ordinate responses to environmental signals. SOC1 is directly activated by CONSTANS (CO) in long photoperiods and is repressed by FLC, a component of the vernalization (low-temperature) pathway. We show that in transgenic plants overexpressing CO and FLC, these proteins regulate flowering time antagonistically and FLC blocks transcriptional activation of SOC1 by CO. A series of SOC1::GUS reporter genes identified a 351 bp promoter sequence that mediates activation by CO and repression by FLC. A CArG box (MADS-domain protein binding element) within this sequence was recognized specifically by FLC in vitro and mediated repression by FLC in vivo, suggesting that FLC binds directly to the SOC1 promoter. We propose that CO is recruited to a separate promoter element by a DNA-binding factor and that activation by CO is impaired when FLC is bound to an adjacent CArG motif. Introduction In plants, the transition from vegetative growth to flowering occurs in response to both environmental stimuli and endogenous signals. Genetic analyses of the control of flowering in Arabidopsis thaliana identified four major floral promotion pathways (reviewed in Simpson et al., 1999; Reeves and Coupland, 2000; Araki, 2001). The photoperiod and vernalization pathways mediate the response to environmental signals, whereas the autonomous and gibberellin (GA) pathways appear to act independently of these signals (Koornneef et al., 1991). The photoperiod pathway mediates the promotion of flowering by daylength. Arabidopsis is a facultative long-day plant, flowering more rapidly under long-day (LD) conditions of 16 h of light than in short days (SDs) of 10 h light. CONSTANS (CO) and FT were placed in this pathway because mutations in these genes delay flowering in LDs, but not in SDs, and thereby modulate the response to photoperiod (reviewed in Reeves and Coupland, 2000; Araki, 2001). CO encodes a putative transcription factor (Putterill et al., 1995; Robson et al., 2001), whilst FT encodes a protein with similarity to RKIP proteins (Kardailsky et al., 1999; Kobayashi et al., 1999; Pnueli et al., 2001). The vernalization pathway promotes flowering in response to extended exposures to low temperature. This pathway acts redundantly with the autonomous pathway. Both of these pathways promote flowering by preventing accumulation of the FLOWERING LOCUS C (FLC) mRNA (Michaels and Amasino, 1999, 2001; Sheldon et al., 1999, 2000). FLC acts synergistically with FRI to repress flowering in late-flowering accessions (Koornneef et al., 1994; Sanda and Amasino, 1996; Johanson et al., 2000; Sheldon et al., 2000), and encodes a MADS-domain transcription factor that represses flowering when overexpressed in transgenic plants (Michaels and Amasino, 1999; Sheldon et al., 1999). Mutation of FLC accelerates flowering in LDs and SDs, and is epistatic to mutations in the autonomous pathway and to dominant alleles of FRI (Michaels and Amasino, 2001). The abundance of FLC mRNA and protein is elevated by mutations in the autonomous pathway and is reduced by vernalization, suggesting that modulation of FLC expression is central to the control of flowering time (Michaels and Amasino, 1999; 2001; Sheldon et al., 2000), but it is not essential for a vernalization response (Michaels and Amasino, 2001). The floral promotion pathways ultimately converge to regulate the expression and function of the floral meristem identity genes that control flower development (Blázquez and Weigel, 2000; Borner et al., 2000; Lee et al., 2000; Samach et al., 2000; Rouse et al., 2002). For example, the floral meristem identity gene LEAFY (LFY) is regulated both by CO, a component of the photoperiod pathway, and GA (Blázquez and Weigel, 2000). These act through different motifs within the LFY promoter, although CO probably does not directly activate LFY, and the transcription factor that regulates LFY in response to GA is not yet known (Blázquez and Weigel, 2000; Samach et al., 2000). The flowering-time genes FT and SOC1 (or AGL20) are also common targets of distinct pathways and are proposed to function upstream of the floral meristem identity genes. SOC1 and FT were shown to be direct targets of CO by using plants that overexpressed a translational fusion of CO to the ligand-binding domain of the glucocorticoid receptor (35S::CO:GR) (Samach et al., 2000). In agreement with this, FT expression is reduced in co mutants (Kardailsky et al., 1999; Kobayashi et al., 1999; Samach et al., 2000; Suárez-López et al., 2001), whilst SOC1 expression responds to photoperiod and is slightly reduced in co mutants (Borner et al., 2000; Lee et al., 2000; Samach et al., 2000). In addition, ft or soc1 mutations partially suppress the early flowering of 35S::CO plants (Onouchi et al., 2000). Thus, FT and SOC1 act downstream of CO in the photoperiod pathway and CO promotes their expression. However, FT and SOC1 also act downstream of the floral inhibitor FLC, which is a component of the autonomous/vernalization pathway and does not affect CO expression. For example, SOC1 mRNA abundance is reduced in genotypes with high levels of FLC and is increased in flc mutants (Borner et al., 2000; Lee et al., 2000; Samach et al., 2000; Michaels and Amasino, 2001). These observations are consistent with the proposal that FLC represses SOC1. Thus, the antagonistic effect of transcription factors CO and FLC on the expression of downstream genes FT and SOC1 may represent a direct convergence of signalling pathways and provide a means of co-ordinating the control of flowering by daylength and temperature. Here we further characterize how CO and FLC interact to generate antagonistic effects on SOC1 expression. Results Phenotypes associated with overexpression of FLC are suppressed by overexpression of CONSTANS To examine the antagonistic effect of CO and FLC on flowering, the phenotypes of plants overexpressing both genes from the strong CaMV 35S promoter were examined. A 35S::CO 35S::FLC line was generated by crossing plants carrying 35S::CO (Onouchi et al., 2000) with those containing 35S::FLC (Michaels and Amasino, 1999). The flowering times of wild-type (Ler), 35S::CO, 35S::CO 35S::FLC, and 35S::FLC plants were scored in LDs. 35S::CO plants flowered earlier than wild type, whilst 35S::FLC plants flowered much later (Table I; Figure 1A). The 35S::CO 35S::FLC plants flowered much earlier than 35S::FLC and at a time between that of 35S::CO and wild-type plants (Table I). CO and FLC, therefore, have antagonistic effects on flowering time and overexpression of CO can largely overcome the delay in flowering caused by overexpression of FLC. Figure 1.Antagonistic effect of 35S::CO and 35S::FLC on flowering time, floral morphology, and expression of SOC1 and FT. (A) Phenotype of wild-type (WT), 35S::CO, 35S::CO 35S::FLC, and 35S::FLC plants. (a) Thirty-two-day-old plants grown in LDs; (b–f) morphology of siliques; (g) dissection of silique in (f) to show an ectopic inflorescence. (B) Northern analysis of SOC1 and FT mRNA in WT, 35S::CO, 35S::CO 35S::FLC, and 35S::FLC plants. One filter made with RNA harvested 8 h after dawn was hybridized with probes for SOC1 and UBQ10 (upper rows). A second filter made with RNA harvested 16 h after dawn was sequentially hybridized with probes for FT and UBQ10 (lower rows). Download figure Download PowerPoint Table 1. Effect of overexpression of CO and FLC on flowering time in LDs Genotype No. of rosette leaves No. of cauline leaves Total No. of leaves Ler 5.5 ± 0.5 3.1 ± 0.3 8.6 ± 0.5 35S::CO 3.0 ± 0.0 1.9 ± 0.5 4.9 ± 0.5 35S::CO 35S::FLC 4.4 ± 0.5 2.5 ± 0.5 6.9 ± 0.6 35S::FLC 33.3 ± 3.4 6.9 ± 0.7 40.2 ± 3.9 Overexpression of FLC also caused defects in floral morphogenesis. A proportion of flowers produced anthers with little or no pollen (data not shown), the petioles were often retained at the base of mature siliques (Figure 1A) and floral reversion caused development of an inflorescence inside some siliques (Figure 1A). These defects were absent in 35S::CO 35S::FLC lines (Figure 1A), although the mRNA expressed from 35S::FLC was still present in these plants. Similarly, defects associated with overexpression of CO, such as the presence of extra carpels and the short club-like appearance of siliques, were absent in 35S::CO 35S::FLC lines (see Onouchi et al., 2000; Figure 1), although 35S::CO mRNA was present. This genetic analysis supports the notion that CO and FLC interact antagonistically to regulate flowering time, and indicates that this antagonism persists throughout development when these genes are overexpressed. Antagonistic effect of CONSTANS and FLC on expression of target genes Previously, SOC1 and FT were shown to be immediate targets of CO, and their mRNA levels correlate with the level of CO expression (Samach et al., 2000). SOC1 mRNA levels also correlate with the level of FLC expression (Lee et al., 2000; Michaels and Amasino, 2001). We compared the level of SOC1 and FT mRNAs in 35S::CO, 35S::CO 35S::FLC, 35S::FLC and wild-type plants to determine whether they correlate with flowering time. RNA was extracted from 10-day-old seedlings and subjected to northern analysis. As expected, SOC1 and FT mRNAs accumulated to a higher level in 35S::CO plants than in wild type and were not detected in 35S::FLC plants (Figure 1B). In 35S::CO 35S::FLC plants, the levels of SOC1 and FT mRNAs were dramatically reduced compared with those in 35S::CO plants. Thus, 8.3-fold less SOC1 mRNA and 11.3-fold less FT mRNA was detected in 35S::CO 35S::FLC plants compared with 35S::CO plants (Figure 1B). Nevertheless, 35S::CO 35S::FLC plants flowered only slightly later than 35S::CO plants (Figure 1A; Table I). Furthermore, SOC1 mRNA in 35S::CO 35S::FLC plants was 3-fold less abundant in comparison to wild-type plants, although 35S::CO 35S::FLC plants flowered earlier than wild type. Despite the lack of correlation between SOC1 mRNA levels and flowering time, our analysis confirmed that CO and FLC have antagonistic effects on SOC1 and FT expression. Expression of SOC1 in cauline leaves and flowers and accumulation of transcript with age require promoter sequences between nucleotides −4105 and −1911 Previously, SOC1 mRNA was shown to accumulate early in development and to be present in most tissues of mature plants (Borner et al., 2000; Lee et al., 2000; Samach et al., 2000). To identify SOC1 promoter sequences, expression of a SOC1::GUS reporter gene containing ∼4 kb of sequence upstream of the SOC1 transcriptional start site was monitored (Materials and methods). This was assumed to contain the full-length promoter since a genomic DNA fragment containing 1.4 kb of upstream sequence complemented the soc1 mutation (Samach et al., 2000). Twenty primary transformants carrying SOC1::GUS were analysed by β-glucuronidase (GUS) staining. Staining was first visible in the germinating seed after 1 day of growth in LDs, and was subsequently detected in the roots, apex and cotyledons of seedlings. GUS expression was observed in mature plant tissues such as rosette leaves, cauline leaves, inflorescences and flowers, but not in mature siliques or in seeds (Figure 2B). The pattern of expression of the 4 kb SOC1::GUS reporter gene was, therefore, similar to that of the endogenous SOC1 gene as monitored by RT–PCR (Borner et al., 2000; Lee et al., 2000). Figure 2.Analysis of expression of SOC1::GUS reporter genes in wild-type plants. (A) Diagram of SOC1::GUS reporter genes with full-length (4 kb) or truncated promoters (2, 1 and 0 kb). The 5′ endpoints of constructs are numbered relative to the transcription start site (+1). (B) Expression of SOC1::GUS reporter genes in seedlings, cauline leaves, flowers and siliques as monitored by GUS staining. Days of growth are in the lower right of panels. Four kilobase SOC1::GUS reporter gene expression in (a–g) seedlings, (h) cauline leaves, (i) flowers and (j) siliques; 2 kb SOC1::GUS reporter gene expression in (k and l) seedlings, (m) cauline leaves, (n) flowers and (o) siliques; 1 kb SOC1::GUS expression in (p and q) seedlings, (r) cauline leaves, (s) flowers and (t) siliques; 0 kb SOC1::GUS expression in (u and v) seedlings, (w) cauline leaves, (x) flowers and (y) siliques. (C) Time course of 4, 2 and 1 kb SOC1::GUS reporter gene expression in wild-type seedlings as determined by GUS activity assays (see Materials and methods). RFU, relative fluorescence units. Download figure Download PowerPoint To identify a minimal promoter sequence that would still mediate activation by CO and repression by FLC, the effects of sequential 5′ deletions of the 4 kb promoter on expression of SOC1::GUS were monitored in wild-type plants. SOC1::GUS reporter genes with deletion endpoints at nucleotide (nt) −1911 (2 kb SOC1::GUS), nt −966 (1 kb SOC1::GUS) and nt −89 (0 kb SOC1::GUS) were introduced into wild-type plants (Figure 2A; Materials and methods). Twenty primary transformants were obtained for each construct and analysed for activity by GUS staining. The patterns of expression for the 1 and 2 kb SOC1::GUS reporter genes were similar (Figure 2B). Staining was detected in seedlings and rosette leaves, but was reduced or absent in cauline leaves and was not detected in inflorescences or flowers. GUS staining was not detected for plants transformed with a 0 kb SOC1::GUS reporter gene (Figure 2B). Therefore, the 5′ boundary of promoter sequences required for SOC1 expression in cauline leaves and in flowers was located upstream of nt −1911, but sequences between nt −966 and −89 were sufficient for SOC1 expression in seedlings. Previously, SOC1 mRNA was detected at a low level early in development and slowly accumulated over a 12 day period (Lee et al., 2000). To determine whether the 4 kb SOC1::GUS reporter gene was similarly expressed in developing seedlings and to monitor activity of the truncated 2 and 1 kb promoters, seedlings with 4, 2 or 1 kb SOC1::GUS reporter genes were monitored for GUS activity over 21 days (Figure 2C). Expression of 4 kb SOC1::GUS was detected early in development (day 3) and gradually increased until about day 12. For the 1 and 2 kb SOC1::GUS reporter genes, activity was also detected early in development (day 3), but at ∼10-fold lower levels than for 4 kb SOC1::GUS. Expression reached a maximum at about day 5 and remained constant until day 21. Therefore, truncation of the SOC1 promoter resulted in an overall decrease in the level of reporter gene expression and abolished the age-dependent increase in expression that is observed for the 4 kb promoter. The 5′ boundary of sequences required for maximal accumulation of SOC1 mRNA in seedlings must be located upstream of nt −1911. The 1 kb SOC1::GUS reporter is activated by CO and repressed by FLC To test whether the 1 kb SOC1::GUS reporter retained the ability to be activated by CO and/or repressed by FLC, 1 kb SOC1::GUS was introduced into 35S::CO, 35S::CO 35S::FLC, and 35S::FLC plants by crossing. Expression of 1 kb SOC1::GUS in these lines was then monitored by GUS staining and northern blotting (Figure 3). Figure 3.One kilobase SOC1::GUS reporter gene expression is activated in 35S::CO lines and repressed by FLC. (A) Analysis of 1 kb SOC1::GUS expression in wild-type (WT), 35S::CO and 35S::FLC plants by GUS staining: (a) 10-day-old seedlings, 1 kb SOC1::GUS WT (left) and 1 kb SOC1::GUS 35S::CO (right); (b) 14-day-old seedlings, 1 kb SOC1::GUS WT (left) and 1 kb SOC1::GUS 35S::FLC (right); 1 kb SOC1::GUS 35S::CO in (c) cauline leaves and (d) flower. (B) Northern analysis of 1 and 0 kb SOC1::GUS expression. RNA was purified from 1 kb SOC1::GUS or 0 kb SOC1::GUS seedlings: wild type (WT), 35S::CO (CO), 35S::CO 35S::FLC (CO FLC), 35S::FLC (FLC) and 35S::CO:GR. Dexamethasone (Dex) treatment, (+) or (−). The filter was sequentially hybridized with probes for GUS, SOC1 and β-TUB (loading control). (C) Transient expression assays of 1 kb SOC1::LUC. Leaves of Columbia (Col), flc-1 (in Col background), Landsberg erecta (Ler) and fca-1 (in Ler) plants were bombarded with beads coated with DNA of plasmids carrying 1 kb SOC1::LUC and 35S::GFP (Materials and methods). The ratio of luciferase to GFP expression is shown for each genotype (Materials and methods). In each case, the column represents the mean value, with the standard error. Download figure Download PowerPoint GUS activity was higher in all tissues of 35S::CO seedlings in comparison to wild type (Figure 3A). An increase in GUS activity was also detected in cauline leaves and in flowers relative to that of wild-type plants (Figures 2B and 3A). In contrast, GUS activity was dramatically reduced in 35S::FLC seedlings in comparison with wild-type seedlings. Repression was strongest in the leaves; a residual amount of expression was retained in the roots, at the apex and in the veins of the leaves (Figure 3A). GUS expression in 35S::CO 35S::FLC seedlings was similar to that in 35S::FLC seedlings (data not shown). The accumulation of GUS mRNA in whole seedlings was also measured by northern blotting (Figure 3B). The 1 kb SOC1::GUS transcript was upregulated 3.1-fold in 35S::CO seedlings and repressed 5.1-fold in 35S::FLC seedlings in comparison with wild type (Figure 3B). In addition, a transient assay was developed to determine whether the 1 kb fragment of the SOC1 promoter responded to FLC in wild-type plants and in fca mutants, which contain elevated levels of FLC (Michaels and Amasino, 1999, 2001; Sheldon et al., 2000). A 1 kb SOC1::LUCIFERASE fusion was introduced into wild-type plants, flc loss-of-function mutants and fca mutants by microprojectile bombardment. Levels of luciferase expression were compared with those of co-bombarded 35S::GFP (Materials and methods). Luciferase expression was ∼1.75-fold higher in flc loss-of-function mutants than in wild-type plants, and ∼0.6-fold wild-type levels in fca mutants (Figure 3C). The differences between the mutants and wild-type plants were confirmed as significantly different (P < 0.001) using the Mann–Whitney rank sum test. The 1 kb SOC1 promoter fragment therefore also confers responses to FLC at levels of expression found in wild-type and fca mutant plants. In 35S::CO 35S::FLC seedlings carrying 1 kb SOC1::GUS, GUS mRNA was 4.1-fold less abundant than in wild type, and was most similar to that in 35S::FLC (Figure 4B). Analysis of SOC1 mRNA demonstrated that the endogenous gene was upregulated in 35S::CO plants and repressed in 35S::FLC plants to a similar extent to 1 kb SOC1::GUS (Figure 3B). Figure 4.Identification of a 234 bp region of the SOC1 promoter that mediates activation by CO and repression by FLC. (A) Summary of expression of SOC1::GUS reporter genes in wild-type (WT), 35S::CO and 35S::FLC lines. Relative activities were determined by GUS staining. Top line, 1 kb SOC1::GUS reporter gene; second to fifth lines, 300 bp SOC1::GUS reporter genes containing overlapping fragments A, B, C or D from the SOC1 promoter. Fragments of 300 bp were each inserted upstream of the minimal 0 kb reporter gene at a unique BamHI site (see Materials and methods). Asterisk denotes that fragment D contains two copies of the minimal promoter region between −89 and +5. Bottom line, minimal 0 kb SOC1::GUS reporter gene that contains the TATA box and transcription start site (+1) for SOC1. (B) Promoter fragment C mediates activation in 35S::CO plants and repression in 35S::FLC plants. Expression of 1 kb SOC1::GUS (1 kb) and 300 bp SOC1::GUS (A, B, C, D) reporter genes was monitored in WT, 35S::CO and 35S::FLC seedlings by northern blotting using probes for GUS and UBQ10 (loading control). These data were quantified and are presented in histogram format. The amount of GUS/UBQ transcript in WT plants containing the 1 kb SOC1::GUS reporter gene was given an arbitrary value of 1. The relative level of transcript for each reporter gene construct in each background is presented as an average. Download figure Download PowerPoint We also tested whether upregulation of 1 kb SOC1::GUS expression in 35S::CO:GR lines was likely to be mediated directly by CO (Samach et al., 2000). Expression of 1 kb SOC1::GUS was monitored in 35S::CO:GR plants by northern blotting (Figure 3B). A 3.5-fold increase in GUS mRNA abundance was detected after 4 h of dexamethasone (Dex) treatment in comparison to untreated control plants (Figure 3B), whereas no GUS mRNA was detected for the minimal 0 kb SOC1::GUS reporter gene in 35S::CO:GR plants (Figure 3B). The endogenous SOC1 gene was also analysed, demonstrating that treatment with Dex was sufficient for upregulation of endogenous SOC1 mRNA. These experiments indicated that sequences between nt −966 and −89 in the SOC1 promoter mediate activation by CO and repression by FLC, and that 1 kb SOC1::GUS is regulated by CO and FLC in a similar manner to the endogenous gene. Overlapping 300 bp fragments in the 1 kb promoter of SOC1 mediate activation by CO and repression by FLC To further define the sequences that mediate activation by CO and repression by FLC, the 1 kb promoter was subdivided into four overlapping fragments of ∼300 bp each. These fragments were cloned upstream of the minimal 0 kb SOC1::GUS reporter gene to generate the 300 bp A, B, C and D SOC1::GUS reporter genes (Figure 4A; Materials and methods). Each of these constructs was used to transform wild-type plants. Twenty primary transformants for each construct were obtained. Reporter genes present in these lines were introduced into 35S::CO and 35S::FLC lines by crossing. The expression pattern of each construct was first analysed qualitatively by staining seedlings for GUS activity in the T2 generation (Figure 4A). In wild-type plants, constructs containing fragments B and C supported expression of the minimal 0 kb SOC1::GUS gene. In general, constructs containing fragment B supported higher levels of GUS activity than those containing fragment C. However, the GUS activity mediated by fragment C was strongly increased in 35S::CO lines, whereas that mediated by fragment B was not. The GUS expression caused both by fragment B and C was significantly repressed by 35S::FLC, indicating that the overlapping region between these two sequences was likely to contain sequences that mediate repression by FLC. Little or no GUS activity for constructs containing fragments A and D was detected in wild-type, 35S::CO or 35S::FLC lines. The accumulation of GUS transcript in homozygous T3 lines was also monitored in whole seedlings by northern blotting. Figure 4B shows that for constructs containing fragment C, SOC1::GUS mRNA abundance increased by an average of 2.7-fold in 35S::CO seedlings. This increase is similar to that observed for 1 kb SOC1::GUS mRNA, which was increased 3.1-fold in 35S::CO seedlings in comparison to wild type. SOC1::GUS mRNA expressed from fragments A, B or D was not significantly increased in abundance in 35S::CO seedlings compared with wild type. Figure 4B also shows that constructs containing fragment B or C were repressed by an average of 5.6- or 2.1-fold, respectively, in 35S::FLC seedlings compared with wild type. The level of repression of fragment B is similar to that observed for the 1 kb SOC1::GUS reporter gene in 35S::FLC seedlings (Figure 4). The mRNA of SOC1::GUS constructs containing fragments A and D was not significantly reduced in abundance in 35S::FLC seedlings in comparison to wild type. This analysis showed that overlapping fragments B and C support activity of a minimal 0 kb SOC1::GUS reporter gene in wild-type plants, and that this activity is repressed by 35S::FLC. Therefore, sequences between nt −482 and −372 of the SOC1 promoter mediate repression by FLC. Furthermore, only fragment C could support efficient activation of the minimal 0 kb SOC1::GUS reporter in 35S::CO lines, suggesting that the sequences required for this response are located between nt −372 and −248. Specific in vitro binding of FLC to DNA containing the CArG box at nt −400 of the SOC1 promoter To test whether CO and/or FLC bind directly to the SOC1 promoter, gel retardation experiments were performed using fragments B and C as probe. A protein–DNA complex was detected by gel retardation after incubation of recombinant FLC protein with labelled fragments B and C (Figure 5A). Formation of a similar complex was observed when FLC and CO proteins were present together in the reaction mixture, and no complex was observed when recombinant CO protein alone was incubated with the probes (Figure 5A). Figure 5.Specific binding of FLC protein to a CArG box at nt −400 of the SOC1 promoter. (A) FLC protein forms a gel retardation complex (arrow) with 300 bp fragments B and C. FLC and/or CO protein were incubated with promoter fragments B or C as probes. The protein contained in each reaction is indicated above the panel. f.p., free probe. The asterisk indicates an apparent non-specific protein–DNA complex that was not consistently observed and was competed with non-specific competitor. (B) Comparison of wild-type (WT) and mutant MADS-box protein binding sites (CArG boxes): includes CArG box at nt −400 of the SOC1 promoter (CCAAAATAAG), as well as mutant versions of SOC1 CArG box. (C) Specific binding of FLC protein to fragment C probe. The presence of FLC protein is indicated above the panels and competitor DNAs are described in the text. Lane 1, no protein and no competitor DNA; lanes 3–5, SOC1 WT fragment as competitor DNA; lanes 6–8, SOC1 M1 fragment as competitor DNA. Non-labelled DNA in molar excess was used as competitor in lanes 3 and 6 (10-fold), lanes 4 and 7 (100-fold), and lanes 5 and 8 (1000-fold). (D) Specific binding of FLC protein to a 30 bp fragment containing the CArG sequence at nt −400. FLC protein was incubated with 30 bp fragment probes as indicated below the panel. The presence of FLC protein is indicated above the panels and competitor DNAs are described in the text. Lanes 1, 3, 5, no protein and no competitor DNA; lanes 2, 4, 6, FLC and no competitor DNA; lanes 7–9, SOC1 WT fragment as competitor DNA; lanes 10–12, SOC1 M1 fragment as competitor DNA; lanes 13–15, SOC1 M2 fragment as competitor DNA. Non-labelled DNA in molar excess was used as competitor in lanes 7, 10 and 13 (10-fold), lanes 8, 11 and 14 (100-fold), and lanes 9, 12 and 15 (1000-fold). Download figure Download PowerPoint Inspection of the overlapping region between fragments B and C revealed a sequence motif centred at nt −400 that resembled a CArG box, the site to which MADS-domain proteins bind (Figure 5B; reviewed in Shore and Sharrocks, 1995). This CArG sequence in the