Journal of Experimental Botany, Vol. 65, No. 4, pp. 1193–1203, 2014
doi:10.1093/jxb/ert482 Advance Access publication 24 January, 2014
This paper is available online free of all access charges (fordjournals/open_access.html for further details)
ReaRch papeR
Sequential action of FRUITFULL as a modulator of the activity of the floral regulators SVP and SOC1
Vicente Balanzà, Irene Martínez-Fernández and Cristina Ferrándiz*
Instituto de Biología Molecular y Celular de Plantas, Conjo Superior de Investigaciones Científicas–Universidad Politécnica de Valencia, Avenida de los Naranjos s/n, 46022 Valencia, Spain
*To whom correspondence should be addresd. E-mail: cferrandiz@ibmcp.upv.es
新居乔迁邀请函
Received 18 July 2013; Revid 11 November 2013; Accepted 12 December 2013
Abstract
The role in flowering time of the MADS-box transcription factor FRUITFULL (FUL) has been propod in many works. FUL has been connected to veral flowering pathways as a target of the photoperiod, ambient temperature, and age pathways and it is has been shown to promote flowering in a partially redundant manner with SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1). However, the position of FUL in the genetic networks, as well as the functional output of FUL activity during floral transition, remains unclear. In this work, a genetic approach has been undertaken to understand better the functional hierarchies involving FUL and other MADS-box factors with well established roles as floral integrators such as SOC1, SHORT VEGETATIVE PHASE (SVP) or FLOWERING LOCUS C (FLC). Our results suggest a prominent role of FUL in promoting reproductive transition when photoinductive signal-ling is suppresd by short-day conditions or by high levels of FLC expression, as in non-vernalized winter ecotypes.
A model is propod where the quential formation of FUL–SVP and FUL–SOC1 heterodimers may mediate the veg-etative and meristem identity transitions, counteracting the repressive effect of FLC and SVP on flowering.
Key words:Flowering, FUL, SVP, SOC1, FLC, MADS-box factors.
Introduction
Arabidopsis thaliana adult life cycle compris three major pha transitions that are mainly characterized by the identity of the lateral structures produced by the shoot apical meris-tem (SAM). The vegetative pha transition marks the change from the production of juvenile leaves to the production of adult leaves. Both types of leaves form a rotte through the period of vegetative growth of the plant and, then, trig-gered by both environmental and endogenous cues, the SAM undergoes two subquent pha transitions leading to repro-ductive development: the reproductive transition that caus bolting of the primary inflorescence and the production of cauline leaves subtending condary inflorescences, and the meristem identity transition, after which the SAM will pro-duce floral meristems directly (Araki, 2001; Yamaguchi et al., 2009; Huijr and Schmid, 2011).
Both reproductive and meristem identity transitions, that are collectively named as floral transition, are highly controlled by developmental and environmental signals. Six promoting pathways have been propod to regulate this pro-cess (reviewed in Fornara et al., 2010; Srikanth and Schmid, 2011): the photoperiod, vernalization, ambient temperature, age, autonomous, and gibberellin pathways. The first three pathways respond to environmental signals such as daylength and asonal or day growth temperature, while the age and autonomous patways respond to endogenous signals, and the gibberellin pathway responds to both environmental and endogenous clues. All the pathways converge at the level of a few genes, named floral transition integrators.
Within this group of floral transition integrators, veral members of the MADS-box family have major roles: the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) is activated by the photoperiod, age and gibberellin pathways to promote floral transition (Borner et al., 2000; Lee et al., 2000; Samach et al., 2000; Lee and Lee,
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2010) which is, in part, mediated by the activation of the floral identity gene LEAFY (LFY) (Lee et al., 2008; Liu et al., 2008). Converly, FLOWERING LOCUS C (FLC) and SHORT VEGETATIVE PHASE (SVP) act as floral transition repres-sors (Hartmann et al., 2000; Michaels and Amasino, 1999; Sheldon et al., 1999). High levels of FLC expression compete the inductive floral signals at the
SAM, and thus, flowering is promoted when the vernalization and autonomous path-ways repress FLC expression (Michaels and Amasino, 1999; Lee et al., 2000; Sheldon et al., 1999, 2000; Hepworth et al., 2002; Michaels et al., 2004; Kim et al., 2009). Likewi, the expression of the flowering repressor SVP is controlled by the autonomous, thermonsory, and gibberellin pathways (Lee et al., 2007; Li et al., 2008). FLC and SVP are able to form heterodimers that directly bind to the SOC1 promoter to down-regulate SOC1 expression, as well as to other floral transition integrators such as FLOWERING LOCUS T (FT) (Lee et al., 2007; Fujiwara et al., 2008; Li et al., 2008).
The MADS-box transcription factor FRUITFULL (FUL), a cloly related gene to the flower meristem identity genes APETALA1 (AP1) and CAULIFLOWER, has been associ-ated with veral developmental process. In addition to its well-known function during fruit development, FUL roles in floral meristem identity specification, shoot maturation, and the control of floral transition have also been described (Hempel et al., 1997; Gu et al., 1998; Ferrándiz et al., 2000a, b; Melzer et al., 2008; Shikata et al., 2009; Wang et al., 2009). FUL is partially redundant with SOC1 in flowering pro-motion. Although the ful mutants are only slightly late flow-ering under long-day growth conditions (Ferrándiz et al., 2000a), the double ful soc1 mutants show a strong delay in floral transition (Melzer et al., 2008). As SOC1, FUL is one of the earliest responsive genes to photoinductive signals (Hemp
el et al., 1997; Schmid et al., 2003) being a target of the FT–FD dimer (Schmid et al., 2003; Teper-Bamnolker and Samach, 2005; Torti et al., 2012). FUL also responds to signals derived from the age pathway, being one of the most responsive genes to the SQUAMOSA PROMOTER BINDING LIKE (SPL) proteins (Shikata et al., 2009; Wang et al., 2009; Yamaguchi et al., 2009). A recent study also places FUL in the promotion of flowering in respon to ambient temperature through the action of miR156/SPL3 and FT (Kim et al., 2012).中国面积最小的五个省
In spite of mounting evidence linking FUL to the main flowering pathways, the importance of FUL in controlling the process, as well as its position, downstream effectors, and mode of action in the pathways are still unclear. In this study, genetic analys have been ud to understand better the regulatory hierarchies involving FUL and other floral integrators of the MADS-box family such as SOC1, SVP, and FLC in the control of floral transition in Arabidopsis. Our results show that FUL is able to act both upstream and co-operatively with SOC1, forming a heterodimer and bind-ing directly to the LFY promoter. In addition, it is shown that the promotive effect of FUL on floral transition depends of the prence of a functional allele of SVP and that FUL is able to counteract the repressive effect of FLC on flowering both affecting FLC expression levels and probably competing with FLC for common targets. Taking all the data together,
a dynamic model is propod for the role of FUL during flo-
ral transition, where the progressive formation of different heterodimers of FUL and other MADS transcription fac-
tors may act as a molecular switch between the vegetative and reproductive states.
Materials and methods
Plant material and growth conditions
Arabidopsis thaliana plants were grown in cabinets at 21 °C under
LD (16 h light) or SD (8 h light) conditions, illuminated by cool-
white fluorescent lamps (150 µE m–2 s–1), in a 1:1:1 by vol. mixture
of sphagnum:perlite:vermiculite. To promote germination, eds
were stratified on soil at 4 °C for 3 d in the dark. The Arabidopsis
plants ud in this work were in the Col-0 background, except ful-1
and 35S::SOC1, that were in L er. Mutant alleles and transgenic lines
have been previously described: soc1-2 (Lee et al., 2000), ful-1 (Gu穷途未路
et al., 1998), ful-2 (Ferrándiz et al., 2000a), svp-32 (Lee et al., 2007),
FRI FLC (Lee and Amasino, 1995), 35S::SOC1, (Lee et al., 2000),
35S::FUL (Ferrándiz et al., 2000b), 35S::SVP (Masiero et al., 2004),
35S::FLC (Michaels and Amasino, 1999), LFY:GUS (Blázquez
et al., 1997) and FLC:GUS (Sheldon et al., 2002).
35S::FUL::GFP was generated by cloning the FUL CDS into the pEarley103 vector (Earley et al., 2006). Agrobacterium strain C58
pM090 was ud to transform Arabidopsis using the floral dip pro-
tocol (Clough and Bent, 1998), and transgenic lines carrying a sin-
gle transgene inrtion and with similar phenotypes to the reference
35S::FUL line were lected.
Flowering time measurements
Flowering time was scored as number of leaves at bolting. The num-
ber of rotte and cauline leaves was counted when the bolting shoot
had produced the first open flower. At least 15 genetically identi-
cal plants were ud to score flowering time of each genotype. The Student’s t-test was ud to test the significance of flowering time differences.
Chromatin immunoprecipitation (ChIP)
35S::FUL and 35S::FUL::GFP eds were grown for 15 d in soil
and inflorescences were collected for analysis. The ChIP experiments
were performed as previously described by Sorefan et al. (2009) with
minor modifications using an anti-GFP antibody (Abcam, Ab290).
Q-PCR was performed using the SYBR®Green PCR Master Mix (Applied Biosystems) in a ABIPRISM 7700 quence detection system (Applied Biosystems). The values correspond to the ratios between the pull-down DNA with the GFP antibody from 35S::FUL
and 35S::FUL:GFP lines and between a 10% fraction of the input genomic DNA from both samples, all of them initially normalized
颈动脉斑块形成
by ACT7 or UBQ10 genomic region. The primers ud for this study
are described in Supplementary Table S1 at JXB online.
Quantitative RT-PCR (qRT-PCR)
Total RNA was extracted from whole plants with the RNeasy Plant
Mini kit (Qiagen). 2 µg of total RNA were ud for cDNA synthesis performed with the First-Strand cDNA Synthesis kit (Invitrogen)
and the qPCR master mix was prepared using the iQTM SYBR Green Supermix (Bio-Rad). Results were normalized to the expres-
sion of the TIP41-like reference gene. The PCR reactions were run
and analyd using the ABI PRISM 7700 Sequence detection system (Applied Biosystems). Three technical and two biological replicates
were performed for each sample. See Supplementary Table S1 at
JXB online for the primer quences.
at Institute of Botany, CAS on May 4, 2014
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FUL modulates SVP and SOC1 activities |1195
β-Glucuronida (GUS) staining and activity measurements
For GUS histochemical detection, samples were treated for 15 min in 90% ice-cold acetone and then washed for 5 min with washing buffer (25 mM sodium phosphate, 5 mM ferrocyanide, 5 mM ferri-cya
nide, and 1% Triton X-100) and incubated from 4–16 h at 37 °C with staining buffer (washing buffer+1 mM X-Gluc). Following staining, plant material was fixed, cleared in chloral hydrate, and mounted to be viewed under bright-field microscopy.
For quantitative measurements, the protocol described in Blazquez et al. (1997) was followed. Briefly, apices were incubated at 37 °C for 16 h in 1 mM MUG assay solution (1 mM 4-methyl umbelliferyl glucuronide, 50 mM sodium phosphate buffer pH 7, 10 mM EDTA, 0.1% SDS, 0.1% Triton X-100), in individual wells of a microtitre plate. After the reaction had been stopped by the addition of 0.3 M Na2CO3, fluorescence at 430 nm was measured on a luminescence spectrophotometer equipped with an ELISA plate reader (Perkin Elmer, model LS50B).
Bimolecular Fluorescence Complementation (BiFC)
Open reading frames of full-length FUL, SOC1, and SVP CDS were cloned into vectors pYFPN43 and pYFPC43 (www. ibmcp.upv.es/FerrandoLabVectors.php), and BiFC was performed as previously described by Belda-Palazon et al. (2012).
Confocal microscopy
Confocal microscopy was performed using a Leica TCS SL (Leica Microsystems GmbH, Heidelberg, Germany) equipped with an Argon krypton lar (Leica).
Accession numbers
Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databas under the follow-ing accession numbers: FUL (AT5G60910), SOC1 (AT2G45660), SVP (AT2G22540), FLC (AT5G10140), FRI (AT4G00650), LFY (AT5G61850), UBQ10 (AT4G05320), act7 (AT5G09810), and tip41-like (AT4G34270).
Results
Genetic interactions of FUL and SOC1
The timing of both reproductive and meristem pha tran-sitions were compared by the quantification of rotte and cauline leaves of wild-type, ful, and 35S::FUL plants. As previously reported, it was obrved that the loss of FUL function caud a small delay in flowering time both in long-day (LD) and short-day (SD) conditions, while the over-expression of FUL caud a strong early flowering phenotype (Table 1) (Ferrándiz et al., 2000a; Melzer et al., 2008). The late flowering pheno
type of ful mutants mainly affected the ont of the meristem identity transition, since the number of rotte leaves did not significantly differ from the wild type, while the number of cauline leaves was incread in both LD and SD conditions (Table 1). In addition, when grown in SD, the axillary meristems of cauline leaves of single ful-2 mutants formed aerial rottes (e Supplementary Fig. S1 at JXB online), and flowers were subtended by bracts (e Supplementary Fig. S1 at JXB online).
It has been described that FUL and SOC1 have similar roles and probably promote flowering redundantly (Melzer et al., 2008). However, it is still unclear how precily the two fac-tors interact genetically and how each of them contributes to the reproductive or the meristem identity transitions. To understand better the genetic relationship of FUL and SOC1, the effect on flowering time of different combinations of FUL and SOC1 loss- and gain-of-function alleles was compared. In LD conditions, the ful-2 soc1-2 double mutant showed a synergistic late-flowering phenotype, in agreement with pre-viously reported data (Melzer et al., 2008), producing more rotte leaves than the soc1-2 single mutant and more cauline leaves than both ful-2 and soc1-2 single mutants (Table 1). Additional phenotypes were obrved such as the production of small leaves subtending flowers, the development of aerial rottes at the cauline leaf axils, and frequent SAM rever-sion (e Supple
mentary Fig. S1B at JXB online), similar to what was obrved in ful-2 single mutants grown in SD and in other studies (Torti et al., 2012).
The soc1-2 mutant grown in SD showed a dramatic increa in rotte leaf number, and also a delay in meris-tem identity transition, although not as important as the delay produced by ful-2 (Table 1). The ful-2 soc1-2 double mutants grown in SD produced a similar number of rotte
Table 1. Genetic interaction of FUL and SOC1: effect on flowering
Long day Short day
Rotte leaves Cauline leaves Rotte leaves Cauline leaves
Columbia-010.2 ± 1.0 3.2 ± 0.455.1 ± 3.49.3 ± 0.7
ful-210.7 ± 0.8 4.4 ± 0.5a59.9 ± 3.8a23.7 ± 3.2a
soc1-219.3 ± 0.9a 4.2 ± 0.5a75.0 ± 4.2a15.2 ± 0.5a
ful-2 soc1-224.5 ± 0.8a,b,c9.7 ± 1.9a.b,c75.1 ± 3.5a,b,28.1 ± 1.7a,b,c
35S::FUL 3.5 ± 0.5a 1.7 ± 0.7a10.6 ± 0.9a 3.6 ± 0.7a
35S::FUL soc1-29.0 ± 1.1d 2.2 ± 0.7d44.6 ± 12.8d7.2 ± 4.5d Landsberg er7.3 ± 0.5 1.8 ± 0.4nd nd
ful-18.4 ± 0.5e 2.5 ± 0.5e nd nd
35S::SOC1 4.0 ± 0.0e0.4 ± 0.5e nd nd
35S::SOC1 ful-1 4.0 ± 0.0f0.7 ± 0.5f,g nd nd
35S::FUL 35S::SOC1 2.0 ± 0.0g0.2 ± 0.4g nd nd Flowering time is expresd as the mean of rotte and cauline leaves produced in long- and short-day conditions. Errors are reprented as the standard deviation. Superscript letters indicate a significant difference (P <0.05) from (a) Col, (b) ful-2, (c) soc1-2, (d) 35S::FUL, (e) L er, (f) ful-1, and (g) 35S::SOC1 controls, respectively, according to Student’s t-test; nd=not determined. at Institute of Botany, CAS on May 4, fordjournals/ Downloaded from
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leaves than the soc1-2 mutant, indicating that, in the abnce of photoperiodic stimulus, the promoti
ng role of FUL on the reproductive transition could depend on the prence of SOC1. On the other hand, the number of cauline leaves produced by ful-2 soc1-2 was only moderately higher than in ful-2 single mutants, suggesting that FUL would have a predominant effect in the control of meristem identity tran-sition (Table 1).
35S::FUL soc1-2 plants flowered earlier than the wild type, but significantly later than 35S::FUL lines (Table 1) sup-porting the idea that the flowering-promoting role of FUL was partially dependent on the prence of an active allele of SOC1. In contrast, 35S::SOC1 ful-1 plants were iden-tical to 35S::SOC1 plants in rotte leaf number, while the abnce of FUL only slightly incread the number of caul-ine leaves produced in the 35S::SOC1 background (Table 1). Finally, lines that over-expresd both genes simultaneously flowered extremely early, producing only two rotte leaves before the SAM directly differentiated into one or two flow-ers, although occasionally one cauline leaf with an axillary flower was formed (Table 1;Fig. 1A, B). Moreover, the axil-lary meristems from rotte leaves were also converted into flowers (Fig. 1A). This strong synergistic effect, together with the partial dependence of FUL on the prence of SOC1 to promote flowering, was compatible with FUL acting in part as an upstream regulator of SOC1, together with a sub-quent co-operative action of both proteins in the regulation of putative common targets, although it did not exclude other possible scenarios.
SOC1 and LFY are FUL direct targets
It has been described that FUL and SOC1 are able to inter-act in yeast two-hybrid experiments as homo- and heter-odimers (de Folter et al., 2005; Immink et al., 2012). To confirm this interaction in planta, a Bimolecular Fluorescence Complementation (BiFC) experiment was performed through transient expression on Nicotiana benthamiana leaves,
obrving FUL-SOC1 dimerization in the nuclei of the cells (Fig. 1C).
The floral identity gene LFY has been identified as a bona fide SOC1 direct target (Lee et al., 2008). In addition, FUL has been also suggested to up-regulate LFY (Ferrándiz et al., 2000a). To confirm this suggestion, the expression of a LFY::GUS reporter line was analyd in the ful-2 and 35S::FUL backgrounds, and it was obrved that the level of LFY expression was dependent on FUL, being lower in the ful-2 mutant and higher in the 35S::FUL line than in WT plants (Fig. 2A–C). The relative levels of expression were also confirmed by quantitative RT-PCR of LFY expression in apices at 7, 10, and 12 d after germination (Fig. 2D). In addition, GUS activity was also quantitatively determined in individual discted apices, using the substrate 4-methyl umbelliferyl glucuronide (MUG), which is converted by GUS into the fluorescent product 4-MU. A time-cour per-apex quanti
fication was performed on the three genetic back-grounds, obrving that LFY::GUS activity was consistently higher in 35S::FUL plants and lower in ful-2 plants than in the WT (Fig 2E). Chromatin immunoprecipitations (ChIP) experiments using a 35S::FUL::GFP line (e Supplementary Fig. S2 at JXB online) revealed that FUL was able to bind a region 2.2 kb upstream to the ATG codon of the LFY gene (Fig. 2F), overlapping with a previously identified region also bound by SOC1 (Lee et al., 2008).
Moreover, FUL–GFP was also found to bind the SOC1 promoter, around 800 bp upstream of the ATG codon (Fig. 2G). Again, this region bound by FUL overlaps with
a region bound by SOC1 itlf, which confirms in planta the
Y1H experiment reported previously, which shows a FUL–
SOC1 heterodimer binding to this fragment of the SOC1 promoter (Immink et al., 2012). Taken together, the results strongly support the hypothesis of SOC1 and FUL binding
as heterodimers to the promoters of their target genes and could explain the genetic interactions obrved.
Genetic interactions of FUL and SVP
SVP has been shown to repress SOC1 directly, in part by binding to the SOC1 promoter as a heterodimer with FLC,
a potent repressor of flowering involved in the vernalization
Fig. 1. Interaction of FUL with SOC1. (A, B) Phenotypes of 35S::FUL
35S::SOC1 double over-expression lines. Only two rotte leaves are
produced (arrows in A) and occasionally one cauline leaf (arrowhead in
B). All axillary meristems are determinate, directly producing flowers.
Asterisks mark the cotyledons in (A). (C) Bimolecular Fluorescence Complementation in tobacco epidermal leaf cells between transiently expresd FUL and SOC1 fusions to the C- and N-terminal fragments of
YFP, respectively. The left panel shows reconstituted YFP fluorescence (green) and the right panel is an overlay with a bright field image of the
same ctor where chlorophyll is shown in red. Negative controls for BiFC experiments are shown in Supplementary Fig. S3 at JXB online. Scale bars: 500 mm (A, B), 40 µm (C). at Institute of Botany, CAS on May 4, 2014
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FUL modulates SVP and SOC1 activities | 1197
and autonomous pathways (Michaels and Amasino, 1999; Sheldon et al., 2002; Helliwell et al., 2006). Our results indicated that FUL could also act as an upstream regu-lator of SOC1, binding directly the SOC1 promoter. To explore whether FUL could interact with SVP to regulate SOC1, the effect on flowering time of different combina-tions of FUL and SVP loss- and gain-of-function alleles
was characterized.
Fig. 2. FUL regulates key genes in the floral transition process binding directly to SOC1 and LFY promoters. (A–C) Histochemical detection of LFY::GUS activity in the apices of 6-d-old wild type (A), ful-2 (B) or 35S::FUL (C) plants. Scale bars, 250 µm. (D) Relative expression of LFY analyd by qRT -PCR in WT, ful-2, and 35S::FUL plants at 7, 10, and 12 d after germination. The error bars depict bad on two biological replicates. Asterisks (*) indicate a significant difference (P <0.05) from the WT control according to Student’s t -test. (E) Quantification of LFY:GUS activity in WT, ful-2, and 35S::FUL
backgrounds. Plants were grown on plates under long days (LD). At each time point, GUS activity was measured in at least 12 individual apices, and the means ±s.e are given. (F) (Top) Schematic diagram of the LFY upstream promoter region. First exon is reprented by a black box, while the u
pstream genomic region is reprented by a black line. The red stars indicate the sites containing either single mismatch or perfect match with the connsus binding quence (CArG box) of MADS-domain proteins. Amplicons spanning the sites ud in the ChIP analys are reprented by grey lines and marked by roman numbers. (Bottom) ChIP enrichment tests showing the binding of FUL-GFP to the LFY-I region. Bars reprent the ratio of amplified DNA (35S::FUL:GFP/35S::FUL) in the starting genomic DNA (input) or in the immunoprecipitated DNA with the GFP antibody (Ab). (G) (Top) Schematic diagram of the SOC1 genomic region, including upstream promoter, exons 1 and 2 and the first intron. Exons are reprented by black boxes, upstream genomic region and intron by a black line. The red stars mark CArG boxes. Amplicons spanning the sites ud in the ChIP analys are reprented by grey lines and marked by roman numbers. (Bottom) ChIP enrichment tests showing the binding of FUL-GFP to the SOC1-III region. Bars reprent the ratio of amplified DNA (35S::FUL:GFP/35S::FUL) in the starting genomic DNA (input) or in the immunoprecipitated DNA with the GFP antibody (Ab).
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1198 | Balanzà et al .
The svp-32 mutant showed a clear early-flowering pheno-type both in LD and SD conditions, reducing the number of rotte leaves produced when compared with the WT control, as previously described by Lee et al. (2007) (Table 2). ful-2 svp-32 flowered with a similar number of leaves as the svp-32 single mutant (Table 2) (Torti et al., 2012), suggesting that SVP repress additional targets that can promote flower-ing in the abnce of FUL , as has already been propod by Torti et al. (2012). If this was true, we could expect plants over-expressing FUL in a svp background to flower earlier or at least like 35S::FUL plants. However, 35S::FUL svp-32 plants also flowered similarly to svp-32, both in LD and SD, (Table 2) suggesting an alternative scenario where FUL over-expression was not able to promote flower transition in the abnce of an active SVP protein. Thus, the epistatic effect of svp mutation on both FUL loss- or gain-of-function may sug-gest that FUL required SVP to regulate its targets, and this could be mediated by the physical interaction of both factors.Interaction of FUL and SVP proteins has already been reported in yeast-two-hybrid experiments (de Folter et al., 2005; Immink et al., 2012). To test if this heterodimer also occurred in planta , a BiFC experiment was performed that confirmed such interaction (Fig 3A ). If FUL required inter-action with SVP to promote floral transition, it could be expected that simultaneous over-expression of FUL and SVP would result in early flowering, overcoming the late-flower-ing phenotype caud by SVP over-expression. A 35S::SVP 35S::FUL line was then generated and flowering time quan-tified in this dou
ble transgenic line. As described above, 35S::FUL flowered early, while 35S::SVP flowered very late, as expected for a potent repressor of flowering transition (Table 2; Fig. 3B ). The line harbouring both the 35S::FUL and the 35S::SVP transgenes flowered early, similarly to 35S::FUL or 35S::FUL svp plants (Fig. 3B ; Table 2). This phenotype indicated that SVP was not able to repress floral transition when both high levels of SVP and FUL were pre-nt, suggesting that the FUL–SVP dimer could suppress the repressor effect of SVP on flowering or even act as a flowering promoting factor.
Genetic interactions of FUL and FLC
Becau the repressor effect of SVP in flowering transition is partially mediated by the formation of a heterodimer with FLC (Lee et al., 2007; Fujiwara et al., 2008; Li et al., 2008), the genetic relationship of FUL and FLC was studied.
Much of the natural variation in flowering time in Arabidopsis depends on the allelic variation of FLC and
Table 2. Genetic interaction of FUL and SVP: effect on flowering
Long day Short day
Rotte leaves
Cauline leaves
Rotte leaves
Cauline leaves元宵节的图片
Columbia-012.4 ± 1.7 2.5 ± 0.464.4 ± 6.08.6 ± 0.8ful-212.9 ± 0.9 3.8 ± 0.6a 70.2 ± 7.0a 20.8 ± 3.8a svp-32
5.6 ± 0.5a 2.8 ± 0.41
6.4 ± 2.1 4.6 ± 1.0ful-2 svp-32 5.3 ± 0.5b 3.3 ± 0.516.1 ± 2.5
7.1 ± 1.635S::FUL
4.0 ± 0.0a 1.4 ± 0.5a 8.3 ± 1.8a 3.5 ± 0.8a 35S::FUL svp-32
5.8 ± 0.4 2.5 ± 0.514.9 ± 2.1c,d 3.4 ± 1.2c 35S::SVP
27.5 ± 1.7a 7.3 ± 1.0a nd nd 35S::FUL 35S::SVP
5.8 ± 1.2e
2.7 ± 0.8d,e
nd
nd
Flowering time is expresd as the mean of rotte and cauline leaves produced in long- and short-day conditions. Errors are reprented as the standard deviation. Superscript letters indicate a significant difference (P <0.05) from (a) Col, (b) ful-2, (c) svp-32, (d) 35S::FUL, and (e) 35S::SVP controls, respectively, according to Student’s t
-test; nd=not determined.
Fig. 3. Interaction of FUL with SVP . (A) BiFC experiments in tobacco leaf cells between transiently expresd FUL and SOC1 fusions to the C- and N-terminal fragments of YFP , respectively. The left panel shows YFP reconstituted fluorescence (green) and the right panel is an overlay with a bright field image of the same ctor where chlorophyll is shown in red. Negative controls for BiFC experiments are shown in Supplementary Fig. S3 at JXB online. Scale bars: 40 µm. (B) Phenotypes
of the 35S::FUL, 35S::SVP , and 35S::FUL 35S::SVP double over-expression lines. FUL
over-expression reverts the late flowering phenotype of 35S::SVP , although inflorescence development is partially restored respect to the 35S::FUL plants.
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