SummaryMembers of the MADS-box transcription factor family play essential roles in almost every developmental process in plants. Many MADS-box genes have conserved functions across the flowering plants, but some have acquired novel functions in specific species during evolution. The analyses of MADS-domain protein interactions and target genes have provided new insights into their molecular functions. Here, we review recent findings on MADS-box gene functions in Arabidopsis and discuss the evolutionary history and functional diversification of this gene family in plants. We also discuss possible mechanisms of action of MADS-domain proteins based on their interactions with chromatin-associated factors and other transcriptional regulators. Key words: MADS-box genes, Plant development, Evolution, Transcriptional regulationIntroduction MADS-domain transcription factors comprise one of the beststudied gene families in plants and members of this family play prominent roles in plant development. Two decades ago, the first MADS-box genes AGAMOUS (AG) from Arabidopsis thaliana (Yanofsky et al., 1990) and DEFICIENS (DEF) from Antirrhinum majus (Schwarz-Sommer et al., 1990) were discovered as regulators of floral organ identity. The sequence of the ~60 amino acid DNA-binding domains within these proteins showed striking similarities to that of the previously characterized proteins serum response factor (SRF) in Homo sapiens (Norman et al., 1988) and Minichromosome maintenance 1 (Mcm1) in Saccharomyces cerevisiae (Passmore et al., 1988). This shared and conserved domain was named the MADS domain (for MCM1, AG, DEF and SRF) and is present in all MADS-domain transcription factor family members (Schwarz-Sommer et al., 1990). Structural analysis of animal and yeast MADS domains showed that the N-terminal and central parts of the MADS domain make contacts with the DNA, while the C-terminal part of this domain contributes mainly to protein dimerization, resulting in a DNA-binding protein dimer consisting of two interacting MADS monomers (e.g. Pellegrini et al., 1995;Huang et al., 2000). Over the past 22 years, many MADS-box gene functions were uncovered in the model species Arabidopsis thaliana and in other flowering plants. Important model plant species for MADS-box gene research include snapdragon (Antirrhinum majus) (reviewed by Schwarz-Sommer et al., 2003), tomato (Solanum lycopersicum), petunia (Petunia hybrida) (Gerats and Vandenbussche, 2005), gerbera (Gerbera hybrida) (Teeri et al., 2006) and rice (Oryza sativa) (reviewed by Yoshida and Nagato, 2011).Initially, MADS-box genes were found to be major players in floral organ specification, but more recent studies revealed functions for MADS-box genes in the morphogenesis of almost all organs and throughout the plant life cycle, from embryo to gametophyte development. The MADS-box gene family in higher plants is significantly larger than that found in animals or fungi, with more than 100 genes in representative flowering plant Development 139, 3081-3098 (2012) REVIEW Box 1...

Floral organs are specified by the combinatorial action of MADSdomain transcription factors, yet the mechanisms by which MADSdomain 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 MADSdomain 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.protein complex isolation | transcriptional regulation | chromatin activation | histone marks F lower development is one of the best understood developmental processes in plants. According to the classic ABC model (1), floral organs in the model plant species Arabidopsis are specified by the combinatorial activity of three functional gene classes. The A class genes represented by APETALA1 (AP1) and APETALA2 (AP2) specify sepal identity, and together with B class genes APETALA3 (AP3) and PISTILLATA (PI), they determine the identity of petals. The C class gene AGA-MOUS (AG) alone determines carpel identity and, together with B class genes, it specifies stamen identity. The ABC model was extended to the ABCE model, in which E class genes [SEPAL-LATA1-4 (SEP1-4)] are required for the specification of all four types of floral organs (2, 3). Based on genetic and yeast n-hybrid protein interaction data, it was later proposed in the "floral quartet model" that floral organs are specified by combinatorial protein interactions of ABCE-class MADS-domain transcription factors, which are thought to assemble into organ-specific quaternary protein complexes that bind to two CArG boxes, DNA consensus sequence CC[A/T] 6 GG, in regulatory regions of target genes (4, 5). E-class proteins have a special role in this model as major mediators of higher-order complex formation. Although interactions that were predicted in this model were further supported by additional in vitro DNA-binding assays and protoplast , formation and composition of these complexes in endogenous tissues remained unknown.Heterologous interaction studies in yeast and genetic data suggest recruitment of transcriptional coregulators such as SEUSS (SEU) and LEUNIG (LUG) by floral MADS-domain proteins (9). Ovule-specific MADS-domain protein complexes were found to form higher-order interactions with BELL1 (BEL1), a mem...

The molecular mechanisms by which floral homeotic genes act as major developmental switches to specify the identity of floral organs are still largely unknown. Floral homeotic genes encode transcription factors of the MADS-box family, which are supposed to assemble in a combinatorial fashion into organ-specific multimeric protein complexes. Major mediators of protein interactions are MADS-domain proteins of the SEPALLATA subfamily, which play a crucial role in the development of all types of floral organs. In order to characterize the roles of the SEPALLATA3 transcription factor complexes at the molecular level, we analyzed genome-wide the direct targets of SEPALLATA3. We used chromatin immunoprecipitation followed by ultrahigh-throughput sequencing or hybridization to whole-genome tiling arrays to obtain genome-wide DNA-binding patterns of SEPALLATA3. The results demonstrate that SEPALLATA3 binds to thousands of sites in the genome. Most potential target sites that were strongly bound in wild-type inflorescences are also bound in the floral homeotic agamous mutant, which displays only the perianth organs, sepals, and petals. Characterization of the target genes shows that SEPALLATA3 integrates and modulates different growth-related and hormonal pathways in a combinatorial fashion with other MADS-box proteins and possibly with non-MADS transcription factors. In particular, the results suggest multiple links between SEPALLATA3 and auxin signaling pathways. Our gene expression analyses link the genomic binding site data with the phenotype of plants expressing a dominant repressor version of SEPALLATA3, suggesting that it modulates auxin response to facilitate floral organ outgrowth and morphogenesis. Furthermore, the binding of the SEPALLATA3 protein to cis-regulatory elements of other MADS-box genes and expression analyses reveal that this protein is a key component in the regulatory transcriptional network underlying the formation of floral organs.