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. Tetramerisation 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 tetramerisation and FQC formation to occur, remains unknown. Here, we provide biophysical and bioinformatic data indicating that the ancestral MTFs were not able to form FQCs. According to our data, FQC formation 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 tetramerisation 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 exons of the K-domain occurred, extending the K-domain at its N-terminal end. Our data indicate that this structural change prevents heterodimerisation 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.

MADS-box genes represent a paneukaryotic gene family encoding transcription factors. Given its importance for essential functions in plants, animals and fungi, such as development of organ identity and mating type determination, the phylogeny of MADS-box genes is of great biological interest. It has been well established that a gene duplication in the stem group of extant eukaryotes generated two clades of MADS-box genes, termed Type I and Type II genes. Almost all Type II genes of land plants contain a keratin-like (K) domain in addition to the family-defining, DNA-binding MADS (M) domain and are also termed MIKC-type genes. Due to a lack of sampling of MADS-box genes in Archaeplastida (rhodophytes, glaucophytes, chlorophytes, and streptophytes) except land plants, the deep evolution of MADS-box genes in plants remains poorly understood, however. Here we use the genomic and transcriptomic ressources that have become available in recent years to answer longstanding questions of MADS-box gene evolution in Archaeplastida. Our results reveal that archaeplastid algae likely do not harbour Type I MADS-box genes. However, rhodophytes, glaucophytes, prasinodermophytes and chlorophytes possess Type II MADS-box genes without a K domain. Type II MADS-box genes with a K domain are found only in streptophytes. This corroborates previous views that some Type II gene acquired a K domain in the stem group of extant streptophytes, generating MIKC-type genes. Interestingly, we found both variants of Type II genes - with (MIKC) and without a K domain - in streptophyte algae, but not in land plants (embryophytes), suggesting that Type II genes without a K domain (ancestral Type II genes) were lost in the stem group of land plants. Our data reveal that the deep evolution of MADS-box genes in "plants" (Archaeplastida) was more complex than has previously been thought.

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. Tetramerisation 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 tetramerisation 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 tetramerisation 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 heterodimerisation 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.