Sequential action of FRUITFULL as a modulator of the activity of the floral regulators SVP and SOC1

Abstract

The role in flowering time of the MADS-box transcription factor fruitfulL (FUL) has been proposed in many works. FUL has been connected to several 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 these 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 signalling is suppressed by short-day conditions or by high levels of FLC expression, as in non-vernalized winter ecotypes. A model is proposed where the sequential formation of FUL-SVP and FUL-SOC1 heterodimers may mediate the vegetative and meristem identity transitions, counteracting the repressive effect of FLC and SVP on flowering.

Keywords: FLC; FUL; Flowering; MADS-box factors.; SOC1; SVP.

Fig. 1.

Interaction of FUL with SOC1. (A, B) Phenotypes of 35S::FUL 35S::SOC1 double over-expression lines. Only two rosette 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 expressed 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 sector where chlorophyll is shown in red. Negative controls for BiFC experiments are shown in Supplementary Fig. S3 at JXB online. Scale bars: 500mm (A, B), 40 µm (C).

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 analysed by qRT-PCR in WT, ful-2, and 35S::FUL plants at 7, 10, and 12 d after germination. The error bars depict the s.e. based 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 represented by a black box, while the upstream genomic region is represented by a black line. The red stars indicate the sites containing either single mismatch or perfect match with the consensus binding sequence (CArG box) of MADS-domain proteins. Amplicons spanning these sites used in the ChIP analyses are represented by grey lines and marked by roman numbers. (Bottom) ChIP enrichment tests showing the binding of FUL-GFP to the LFY-I region. Bars represent 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 represented by black boxes, upstream genomic region and intron by a black line. The red stars mark CArG boxes. Amplicons spanning these sites used in the ChIP analyses are represented by grey lines and marked by roman numbers. (Bottom) ChIP enrichment tests showing the binding of FUL-GFP to the SOC1-III region. Bars represent 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).

Fig. 3.

Interaction of FUL with SVP. (A) BiFC experiments in tobacco leaf cells between transiently expressed 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 sector 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.

Fig. 4.

FUL over-expression suppresses the effects of high levels of FLC. (A) Vernalization response of FRI;FLC and FRI;FLC ful-2 in LD. The ful-2 mutation greatly enhances the late flowering phenotype of FRI;FLC unvernalized plants (left), while a vernalization treatment causes both genotypes to flower similarly earlier (right). (B–G) Histochemical detection of FLC::GUS activity in FRI;FLC (B–D) and FRI;FLC 35S::FUL (E–G) plants. Apices of 10-d-old plants are compared in (B) and (E), the first rosette leaf in (C) and (F), and inflorescence apices of plants at bolting in (D) and (G). All plants were heterozygous for the FLC::GUS reporter and for the wild-type dominant alleles of FRI or FLC. 35S::FUL in (E–G) was also heterozygous. Scale bars: 500 µm (B, C, E, F) or 100 µm (D, G). (H) Relative expression of FLC analysed by qRT-PCR in FRI;FLC and FRI;FLC 35S::FUL plants 10 d after germination. The error bars depict the s.e. based on two biological replicates. An asterisk (*) indicates a significant difference (P <0.05) from the WT control according to Student’s t-test.

Fig. 5.

A proposed mechanistic model for the role of FUL during floral transition through interaction with SVP and SOC1 factors. During vegetative growth FLC and SVP repress the expression of SOC1 and other flowering promoting factors. Upon FUL accumulation, probably mediated by the age SPL-dependent pathway, FUL–SVP dimerization occurs. The FUL–SVP dimer could compete with the FLC–SVP dimer for binding sites in the SOC1 promoter and/or directly interfering with the FLC–SVP dimer formation. Lower repressive activity of the FLC-SVP dimer on SOC1 or even direct activation of SOC1 by FUL-SVP would lead to SOC1 accumulation, the dimerization of FUL-SOC1 and the activation of both SOC1 and LFY promoters, thus triggering flower initiation.