Characterization of MADS-domain transcription factor complexes in Arabidopsis flower development

Abstract

Floral organs are specified by the combinatorial action of MADS-domain transcription factors, yet the mechanisms by which MADS-domain proteins activate or repress the expression of their target genes and the nature of their cofactors are still largely unknown. Here, we show using affinity purification and mass spectrometry that five major floral homeotic MADS-domain proteins (AP1, AP3, PI, AG, and SEP3) interact in floral tissues as proposed in the "floral quartet" model. In vitro studies confirmed a flexible composition of MADS-domain protein complexes depending on relative protein concentrations and DNA sequence. In situ bimolecular fluorescent complementation assays demonstrate that MADS-domain proteins interact during meristematic stages of flower development. By applying a targeted proteomics approach we were able to establish a MADS-domain protein interactome that strongly supports a mechanistic link between MADS-domain proteins and chromatin remodeling factors. Furthermore, members of other transcription factor families were identified as interaction partners of floral MADS-domain proteins suggesting various specific combinatorial modes of action.

Fig. 1.

In planta MADS-domain protein interactions. (A) Average MADS-domain protein abundance ratios between the IP samples and the control samples scaled to the ratio of the bait protein. Ratios calculated based on 4–5 most abundant and unique peptides of a particular protein identified by LC-MS/MS are marked with an asterisk. Ratios calculated based on three or fewer identified peptides were not marked. (B) 3D maximum projections of in situ BiFC data using MADS-domain proteins expressed from their own promoters, confirming the interactions between MADS-domain proteins in floral meristems. Left, pAG:AG-eYFP/N + pSEP3:SEP3-eYFP/C. The yellow spots are characteristic of the nuclear localized interaction signal. The signal is positioned in the FM center where stamens and carpels will arise. Center, pSEP3:SEP3-eYFP/N + pAP1:AP1-eYFP/C. Most YFP signal is located in sepal tips and at the edges of the FM from where petals will be formed. Right, pAP3:AP3-eYFP/N + pPI:PI-eYFP/C. Weak YFP signal is found in the meristematic domain giving rise to petal and stamens, which is characteristic for PI and AP3 protein expression patterns (Fig. S1 J and K). 1–6, flower bud stages; FM, flower meristem; IM, inflorescence meristem; P, petal initiation site; Sp, sepal; St, stamen initiation site. (Scale bars, 25 μm.)

Fig. 2.

Assembly of the MADS-domain protein complexes in a distal region of the SEP3 promoter. (A) Graphic representation of the SEP3 locus with a 4.1-kb promoter region and the SEP3 and AP1 ChIP-SEQ profiles. Fragments used in the EMSA experiments were flanked with the biotin primers used for amplification and detection. Vertical lines in the sequence map indicate position of the CArG boxes. (B) EMSA of the different MADS-domain protein complexes with the SEP3 wild-type promoter fragment and possible model representations of formed protein–DNA complexes. (C) Left, EMSA of the SEP3/AG/AP3/PI protein mix with the “SEP3 wt” DNA fragment containing two CArG boxes. Center, EMSAs where the concentration of a single protein component was gradually reduced from approximately equimolar amounts to 0. Only the part of the gel containing the slow migrating complexes (rectangle in the left EMSA) is shown. Right, Model representation of the higher-order protein complexes formed in the presence of SEP3, AG, AP3, and PI binding to the SEP3 promoter fragment in vitro. (D) EMSA of the SEP3/AG protein mix with the truncated versions of the SEP3 wild-type DNA fragments. The SEP3 wt fragment was shortened from both 3′ and 5′ ends and contains either a single or double binding site. CArG3 (96 bp) – A, CArG3 (96 bp) – B, and CArG3 (96 bp) – C are different, randomized versions of the 3′-end flanking region of the CArG3 fragment.

Fig. 3.

Interactions between MADS-domain transcription factors and other transcriptional regulators. (A) Gel filtration reveals that SEP3 is present in large nuclear complexes. (B) SEP3 promoter and genomic locus representation with the quantitative PCR fragments in the distal enhancer site (e) and weaker proximal promoter site (p). Fragments were designed according to ChIP-SEQ profiles of AP1 and SEP3 (see Fig. 2A). Vertical bars indicate CArG box sequences. (C) Enrichment analysis of H3K27me3 at the MADS binding site in the distal SEP3 enhancer (e). ChIP was analyzed by quantitative PCR; material was obtained from inflorescence tissue of 35S:AP1-GR ap1 cal before (0 h) or 48 h after dexamethasone treatment and then subjected to ChIP with antibodies specific to H3K27me3. Results are presented as fold enrichment of input chromatin. Graphs represent average values from triplicates. Error bars represent SE of the mean. Asterisks indicate values that are significantly different from wild-type leaves (*) or from untreated 35S:AP1-GR ap1 cal plants (**) (P < 0.05 using Student t test). (D) Enrichment analysis of H3K27me3 in the proximal SEP3 promoter (p). For both C and D, H3K27me3 signal is reduced 48 h after AP1 induction compared with signal in 35S:AP1-GR ap1 cal uninduced tissues. (E–H) Scanning electron microscopy (SEM) pictures of chr11 chr17 double mutant inflorescences. (E) Overview of an inflorescence showing aberrations in floral organ development. (F) Close-up of a dissected chr11 chr17 flower (sepal in front was removed) with malformed stamens and petals replaced by pin-like structures (see arrow). (G) Close-up of a developing chr11 chr17 flower showing outgrowth of pin-like structures that replace the petals. (H) Incompletely closed carpel.