The Origin of Floral Quartet Formation-Ancient Exon Duplications Shaped the Evolution of MIKC-type MADS-domain Transcription Factor Interactions.

During development of flowering plants, some MIKC-type MADS-domain transcription factors (MTFs) exert their regulatory function as heterotetrameric complexes bound to two sites on the DNA of target genes. This way they constitute "floral quartets" or related "floral quartet-like complexes" (FQCs), involving a unique multimeric system of paralogous protein interactions. Tetramerization of MTFs is brought about mainly by interactions of keratin-like (K) domains. The K-domain associated with the more ancient DNA-binding MADS-domain during evolution in the stem group of extant streptophytes (charophyte green algae + land plants). However, whether this was sufficient for MTF tetramerization and FQC formation to occur, remains unknown. Here, we provide biophysical and bioinformatic data indicating that FQC formation likely originated in the stem group of land plants in a sublineage of MIKC-type genes termed MIKCC-type genes. In the stem group of this gene lineage, the duplication of the most downstream exon encoding the K-domain led to a C-terminal elongation of the second K-domain helix, thus, generating the tetramerization interface found in extant MIKCC-type proteins. In the stem group of the sister lineage of the MIKCC-type genes, termed MIKC*-type genes, the duplication of two other K-domain exons occurred, extending the K-domain at its N-terminal end. Our data indicate that this structural change prevents heterodimerization between MIKCC-type and MIKC*-type proteins. This way, two largely independent gene regulatory networks could be established, featuring MIKCC-type or MIKC*-type proteins, respectively, that control different aspects of plant development.

The floral homeotic protein SEPALLATA3 recognizes target DNA sequences by shape readout involving a conserved arginine residue in the MADS-domain.

SEPALLATA3 of Arabidopsis thaliana is a MADS-domain transcription factor (TF) and a key regulator of flower development. MADS-domain proteins bind to sequences termed 'CArG-boxes' [consensus 5'-CC(A/T)6 GG-3']. Because only a fraction of the CArG-boxes in the Arabidopsis genome are bound by SEPALLATA3, more elaborate principles have to be discovered to better understand which features turn CArG-boxes into genuine recognition sites. Here, we investigate to what extent the shape of the DNA is involved in a 'shape readout' that contributes to the binding of SEPALLATA3. We determined in vitro binding affinities of SEPALLATA3 to DNA probes that all contain the CArG-box motif, but differ in their predicted DNA shape. We found that binding affinity correlates well with a narrow minor groove of the DNA. Substitution of canonical bases with non-standard bases supports the hypothesis of minor groove shape readout by SEPALLATA3. Analysis of mutant SEPALLATA3 proteins further revealed that a highly conserved arginine residue, which is expected to contact the DNA minor groove, contributes significantly to the shape readout. Our studies show that the specific recognition of cis-regulatory elements by a plant MADS-domain TF, and by inference probably also of other TFs of this type, heavily depends on shape readout mechanisms.