Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 10 (2), R24

SEPALLATA3: The 'Glue' for MADS Box Transcription Factor Complex Formation

Affiliations

SEPALLATA3: The 'Glue' for MADS Box Transcription Factor Complex Formation

Richard G H Immink et al. Genome Biol.

Abstract

Background: Plant MADS box proteins play important roles in a plethora of developmental processes. In order to regulate specific sets of target genes, MADS box proteins dimerize and are thought to assemble into multimeric complexes. In this study a large-scale yeast three-hybrid screen is utilized to provide insight into the higher-order complex formation capacity of the Arabidopsis MADS box family. SEPALLATA3 (SEP3) has been shown to mediate complex formation and, therefore, special attention is paid to this factor in this study.

Results: In total, 106 multimeric complexes were identified; in more than half of these at least one SEP protein was present. Besides the known complexes involved in determining floral organ identity, various complexes consisting of combinations of proteins known to play a role in floral organ identity specification, and flowering time determination were discovered. The capacity to form this latter type of complex suggests that homeotic factors play essential roles in down-regulation of the MADS box genes involved in floral timing in the flower via negative auto-regulatory loops. Furthermore, various novel complexes were identified that may be important for the direct regulation of the floral transition process. A subsequent detailed analysis of the APETALA3, PISTILLATA, and SEP3 proteins in living plant cells suggests the formation of a multimeric complex in vivo.

Conclusions: Overall, these results provide strong indications that higher-order complex formation is a general and essential molecular mechanism for plant MADS box protein functioning and attribute a pivotal role to the SEP3 'glue' protein in mediating multimerization.

Figures

Figure 1
Figure 1
SEP3 protein sequence, domains and motifs. Predicted alpha helices are outlined and numbered (I-III) and the K-box (AA75-177, PFAM [84]) is shaded. Motifs predicted to be involved in transcriptional activation are underlined (NxNQ, HQxQ, QxQH, and MGxxxxxN). The arrow indicates the position at which SEP3ΔC1 stops (after amino acid 171) and the end of SEP3ΔC2 is indicated by an arrowhead (after amino acid 184).
Figure 2
Figure 2
MADS box transcription factor interaction networks. (a) Visualization of a sub-network representing all SEP3 interactions and (b) the network representing all identified higher-order complexes. Proteins are indicated by ovals and interactions by lines. Purple lines indicate dimer formation and blue lines indicate ternary interactions. Ternary complexes are graphically represented in the network as a line between the protein that is expressed from the pAD-GAL4 vector and the protein expressed from the pARC352 vector (the dimer combination), and a line between the protein in the pARC352 vector and the pBD-GAL4 vector. Layout computed using the Pathway Studio 4.0 software (Ariadne Genomics, Inc., Rockville, MD, USA). Type I and type II MADS box protein sub-networks are indicated.
Figure 3
Figure 3
Localization of MADS box proteins in living cells. The MADS box proteins under study were fused to CFP or YFP and transiently expressed in Arabidopsis protoplasts. (a) PI-CFP; (b) SEP3-YFP; (c) AP3-YFP; (d) YFP-AP3; (e) YFP-AP3 + PI-CFP; (f) AP3-YFP + PI-CFP. Note that the proteins accumulate in a ring-like pattern at the position of the nucleolus. Scale bar = 10 μm.
Figure 4
Figure 4
Analyses of MADS box protein interactions in protoplasts by FRET. Arabidopsis leaf protoplasts, co-expressing MADS box proteins fused to either CFP or YFP, were analyzed by FLIM, in order to detect FRET. One representative protoplast is shown for each analyzed combination. The left panels display the intensity channel, the middle panels show the fluorescence lifetime image of the same nucleus in a false color code, and the right panels depict histograms representing the distribution of fluorescence lifetime values over the nucleus. FLIM analysis on a protoplast transiently expressing (a) pECFP + PI-YFP (negative control); (b) SEP3-CFP + SEP3-YFP; (c) AP3-CFP + AP3-YFP; (d) PI-CFP + PI-YFP; (e) AP1-CFP + SEP3-YFP; (f) PI-CFP + AP3-YFP; (g) PI-CFP + AP3-YFP + SEP3. Scale bars = 10 μm.
Figure 5
Figure 5
SEP3 ternary complexes that, based on expression patterns of the genes encoding the involved proteins, might be formed in the shoot apical meristem (SAM) at the moment of the phase switch between vegetative and generative development. Taking into account known functions for some of these proteins, the complexes have been categorized in two classes; one for complexes supposed to be involved in regulating the timing of flowering, and one for complexes that might function in negative auto-regulatory loops. Our hypothesis is that complexes from this latter group are essential for the repression of the genes involved in timing of flowering in the floral organs.
Figure 6
Figure 6
Putative function of SEP3 complexes during plant development. Some of the identified higher-order SEP3 complexes have been placed in the Arabidopsis life cycle at the stage in which they are supposed to be functional. For all the indicated complexes the genes encoding the proteins are co-expressed in a particular tissue or developmental stage (Tables S3 and S7 and Figure S1 in Additional data file 1). Note, that the graphical representation probably does not reflect the real stoichiometry of the complexes. It is possible, for example, that proteins are present as homodimers in the complexes.

Similar articles

See all similar articles

Cited by 84 PubMed Central articles

See all "Cited by" articles

References

    1. Ng M, Yanofsky MF. Function and evolution of the plant MADS box gene family. Nat Rev Genet. 2001;2:186–195. - PubMed
    1. Parenicova L, de Folter S, Kieffer M, Horner DS, Favalli C, Busscher J, Cook HE, Ingram RM, Kater MM, Davies B, Angenent GC, Columbo L. Molecular and phylogenetic analyses of the complete MADS box transcription factor family in Arabidopsis: New openings to the MADS world. Plant Cell. 2003;15:1538–1551. - PMC - PubMed
    1. Portereiko MF, Lloyd A, Steffen JG, Punwani JA, Otsuga D, Drews GN. AGL80 is required for central cell and endosperm development in Arabidopsis. Plant Cell. 2006;18:1862–1872. - PMC - PubMed
    1. Coen ES, Meyerowitz EM. The war of the whorls: genetic interactions controlling flower development. Nature. 1991;353:31–37. - PubMed
    1. Mizukami Y, Ma H. Ectopic expression of the floral homeotic gene agamous in transgenic Arabidopsis plants alters floral organ identity. Cell. 1992;71:119–131. - PubMed

Publication types

MeSH terms

LinkOut - more resources

Feedback