The development of angiosperm flowers is regulated by homeotic MIKC-type MADS-domain transcription factors that activate or repress target genes via the formation of DNA-bound, organ-specific tetrameric complexes. The protein–protein interaction (PPI) capabilities differ considerably between different MIKC-type proteins. In Arabidopsis thaliana the floral homeotic protein SEPALLATA3 (SEP3) acts as a hub that incorporates numerous other MADS-domain proteins into tetrameric complexes that would otherwise not form. However, the molecular mechanisms that underlie these promiscuous interactions remain largely unknown. In this study, we created a collection of amino acid substitution mutants of SEP3 to quantify the contribution of individual residues on protein tetramerization during DNA-binding, employing methods of molecular biophysics. We show that leucine residues at certain key positions form a leucine-zipper structure that is essential for tetramerization of SEP3, whereas the introduction of physicochemically very similar residues at respective sites impedes the formation of DNA-bound tetramers. Comprehensive molecular evolutionary analyses of MADS-domain proteins from a diverse set of flowering plants revealed exceedingly high conservation of the identified leucine residues within SEP3-subfamily proteins throughout angiosperm evolution. In contrast, MADS-domain proteins that are unable to tetramerize among themselves exhibit preferences for other amino acids at homologous sites. Our findings indicate that the subfamily-specific conservation of amino acid residues at just a few key positions accounts for subfamily-specific interaction capabilities of MADS-domain transcription factors and this has shaped the present-day structure of the PPI network controlling flower development.

Phytoplasmas are intracellular bacterial plant pathogens that cause devastating diseases in crops and ornamental plants by the secretion of effector proteins. One of these effector proteins, termed SECRETED ASTER YELLOWS WITCHES’ BROOM PROTEIN 54 (SAP54), leads to the degradation of a specific subset of floral homeotic proteins of the MIKC-type MADS-domain family via the ubiquitin-proteasome pathway. In consequence, the developing flowers show the homeotic transformation of floral organs into vegetative leaf-like structures. The molecular mechanism of SAP54 action involves binding to the keratin-like domain of MIKC-type proteins and to some RAD23 proteins, which translocate ubiquitylated substrates to the proteasome. The structural requirements and specificity of SAP54 function are poorly understood, however. Here, we report, based on biophysical and molecular biological analyses, that SAP54 folds into an α-helical structure. Insertion of helix-breaking mutations disrupts correct folding of SAP54 and compromises SAP54 binding to its target proteins and, concomitantly, its ability to evoke disease phenotypes in vivo. Interestingly, dynamic light scattering data together with electrophoretic mobility shift assays suggest that SAP54 preferentially binds to multimeric complexes of MIKC-type proteins rather than to dimers or monomers of these proteins. Together with data from literature, this finding suggests that MIKC-type proteins and SAP54 constitute multimeric α-helical coiled coils. Our investigations clarify the structure-function relationship of an important phytoplasma effector protein and may thus ultimately help to develop treatments against some devastating plant diseases.

MADS-domain transcription factors (MTFs) are involved in the control of many important processes in eukaryotes. They are defined by the presence of a unique and highly conserved DNA-binding domain, the MADS domain. MTFs bind to double-stranded DNA as dimers and recognize specific sequences termed CArG boxes (such as 5′-CC(A/T)₆GG-3′) and similar sequences that occur hundreds of thousands of times in a typical flowering plant genome. The number of MTF-encoding genes increased by around two orders of magnitude during land plant evolution, resulting in roughly 100 genes in flowering plant genomes. This raises the question as to how dozens of different but highly similar MTFs accurately recognize the cis-regulatory elements of diverse target genes when the core binding sequence (CArG box) occurs at such a high frequency. Besides the usual processes, such as the base and shape readout of individual DNA sequences by dimers of MTFs, an important sublineage of MTFs in plants, termed MIKC^C-type MTFs (M^C-MTFs), has evolved an additional mechanism to increase the accurate recognition of target genes: the formation of heterotetramers of closely related proteins that bind to two CArG boxes on the same DNA strand involving DNA looping. M^C-MTFs control important developmental processes in flowering plants, ranging from root and shoot to flower, fruit and seed development. The way in which M^C-MTFs bind to DNA and select their target genes is hence not only of high biological interest, but also of great agronomic and economic importance. In this article, we review the interplay of the different mechanisms of target gene recognition, from the ordinary (base readout) via the extravagant (shape readout) to the idiosyncratic (recognition of the distance and orientation of two CArG boxes by heterotetramers of M^C-MTFs). A special focus of our review is on the structural prerequisites of M^C-MTFs that enable the specific recognition of target genes.

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 MIKC^C-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 MIKC^C-type proteins. In the stem group of the sister lineage of the MIKC^C-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 MIKC^C-type and MIKC*-type proteins. This way, two largely independent gene regulatory networks could be established, featuring MIKC^C-type or MIKC*-type proteins, respectively, that control different aspects of plant development.

Key message: We studied the DNA-binding profile of the MADS-domain transcription factor SEPALLATA3 and mutant variants by SELEX-seq. DNA-binding characteristics of SEPALLATA3 mutant proteins lead us to propose a novel DNA-binding mode. MIKC-type MADS-domain proteins, which function as essential transcription factors in plant development, bind as dimers to a 10-base-pair AT-rich motif termed CArG-box. However, this consensus motif cannot fully explain how the abundant family members in flowering plants can bind different target genes in specific ways. The aim of this study was to better understand the DNA-binding specificity of MADS-domain transcription factors. Also, we wanted to understand the role of a highly conserved arginine residue for binding specificity of the MADS-domain transcription factor family. Here, we studied the DNA-binding profile of the floral homeotic MADS-domain protein SEPALLATA3 by performing SELEX followed by high-throughput sequencing (SELEX-seq). We found a diverse set of bound sequences and could estimate the in vitro binding affinities of SEPALLATA3 to a huge number of different sequences. We found evidence for the preference of AT-rich motifs as flanking sequences. Whereas different CArG-boxes can act as SEPALLATA3 binding sites, our findings suggest that the preferred flanking motifs are almost always the same and thus mostly independent of the identity of the central CArG-box motif. Analysis of SEPALLATA3 proteins with a single amino acid substitution at position 3 of the DNA-binding MADS-domain further revealed that the conserved arginine residue, which has been shown to be involved in a shape readout mechanism, is especially important for the recognition of nucleotides at positions 3 and 8 of the CArG-box motif. This leads us to propose a novel DNA-binding mode for SEPALLATA3, which is different from that of other MADS-domain proteins known.

The filamentous and unicellular algae of the class Zygnematophyceae are the closest algal relatives of land plants. Inferring the properties of the last common ancestor shared by these algae and land plants allows us to identify decisive traits that enabled the conquest of land by plants. We sequenced four genomes of filamentous Zygnematophyceae (three strains of Zygnema circumcarinatum and one strain of Z. cylindricum ) and generated chromosome-scale assemblies for all strains of the emerging model system Z. circumcarinatum . Comparative genomic analyses reveal expanded genes for signaling cascades, environmental response, and intracellular trafficking that we associate with multicellularity. Gene family analyses suggest that Zygnematophyceae share all the major enzymes with land plants for cell wall polysaccharide synthesis, degradation, and modifications; most of the enzymes for cell wall innovations, especially for polysaccharide backbone synthesis, were gained more than 700 million years ago. In Zygnematophyceae, these enzyme families expanded, forming co-expressed modules. Transcriptomic profiling of over 19 growth conditions combined with co-expression network analyses uncover cohorts of genes that unite environmental signaling with multicellular developmental programs. Our data shed light on a molecular chassis that balances environmental response and growth modulation across more than 600 million years of streptophyte evolution. HIGHLIGHTS: Genomes of four filamentous algae ( Zygnema ) sisters to land plants Zygnema are rich in genes for multicellular growth and environmental acclimation: signaling, lipid modification, and transport Cell wall innovations: diversification of hexameric rosette cellulose synthase in Zygnematophyceae Co-expression networks reveal conserved modules for balancing growth and acclimation