Biology subjects

Theissen, G.

Publications and source records attributed to Theissen, G..

15 recordsLinked to original sources

bioRxiv2026

Strong correlation between amino acid frequency and codon degeneracy in genetic codes across all domains of life

Since the discovery of the genetic code, a frequently discussed question is whether the numbers of synonymous codons for the various amino acids are randomly distributed or were shaped by evolutionary constraints. In this study, we analyze for the standard as well as alternative genetic codes, the correlations between codon degeneracy and amino acid frequencies in proteins (neglecting differences in gene expression). To taking into account the effect of GC content, expected codon frequencies rather than codon multiplicity need to be considered. A strong correlation of these frequencies with amino acid abundance is revealed. Furthermore, we identify consistent patterns of over- and underrepresentation of amino acids across domains of cellular life as well as viruses. For example, the codons for glutamate, aspartate, lysine, and methionine are consistently overrepresented across domains, while cysteine, arginine, histidine, and proline are underrepresented. Subsequently, we discuss the role of biosynthesis costs of amino acids and other factors such as the order of amino acid recruitment in early evolution, exposure to oxidative stress, and sulphur availability. We hypothesize that in a first phase of evolution, the genetic code evolved in a way so as to comply with the different demands for amino acids. In a second phase, after the code was frozen, changes in demand led to deviations from the strong correlation between codon multiplicity and amino acid frequency by natural selection. This comprehensive analysis offers new insights into the interplay between genetic code structure and amino acid usage across all domains of life.

genomics↗

bioRxiv2025

Overexpression of the MADS-box gene CRM3 in the fern Ceratopteris richardii affects sporophyte development

MADS-box genes encode MADS-domain transcription factors that play important roles in the development and evolution of land plants. Especially well-characterized is their role as organ identity genes during flower development of angiosperms. Even though the first MADS-box genes have been molecularly cloned from ferns almost 30 years ago, little is known about their function, mainly due to a lack of mutants. Also their quite broad expression domain, often comprising both the gametophytic and the sporophytic phase of the life cycle of ferns, provide few specific clues concerning gene function. However, as sister group of seed plants monilophytes (ferns and their allies such as horsetails) are of great interest in many attempts to understand both MADS-box gene and land plant evolution. Here we report, to the best of our knowledge, the first mutant phenotype based on transgenic overexpression of a fern MADS-box gene. Transgenic plants of the fern model system Ceratopteris richardii overexpressing the MIKCC-type MADS-box gene CRM3 under control of the Cauliflower Mosaic Virus (CaMV) 35S promoter show a significant reduction of the length of sporophylls. This finding reveals that overexpression of CRM3 affects sporophyll development in C. richardii, possibly by inhibiting cell proliferation or elongation.

plant biology↗

bioRxiv2025

Ceratopteris richardii U6 promoters and plants expressing Cas9 endonuclease as tools for efficient genome editing in a fern

Ferns and their allies (monilophytes) represent the second most species-rich group of land plants and are of considerable ecological importance. As a sister group of seed plants (including flowering plants) they are also of great evolutionary interest. Compared to flowering plants, however, much less is known about the developmental and molecular biology of ferns. Among the most important reasons have been the huge genome sizes of ferns and technical obstacles such as the lack of an efficient transformation system. In recent years the situation has improved considerably, however. For the fern model system Ceratopteris richardii a whole genome sequence has been published, and an efficient transformation system has been developed. To further facilitate studies on fern biology we aim at simplifying genome engineering of C. richardii with the CRISPR-Cas9 system. We report C. richardii plants that express Cas9 nuclease under control of the strong CaMV 35S promoter. For efficient expression of single guide RNA (sgRNA) by RNA polymerase III we identified C. richardii U6 promoters. These technical improvements may foster many fields of fern physiology, development and evolution.

plant biology↗

bioRxiv2023

The dimorphic diaspore model Aethionema arabicum (Brassicaceae): Distinct molecular and morphological control of responses to parental and germination temperatures

Plants in habitats with unpredictable conditions are often characterized by diversifying their bet-hedging strategies that ensure fitness over a wider range of variable environmental factors. A striking example is the diaspore (seed and fruit) heteromorphism that evolved to maximize species survival in Aethionema arabicum (Brassicaceae) in which external and endogenous triggers allow the production of two distinct diaspores on the same plant. Using this dimorphic diaspore model, we identified contrasting molecular, biophysical, and ecophysiological mechanisms in the germination responses to different temperatures of the mucilaginous seeds (M+ seed morphs), the dispersed indehiscent fruits (IND fruit morphs), and the bare non-mucilaginous M- seeds obtained by pericarp (fruit coat) removal from IND fruits. Large-scale comparative transcriptome and hormone analyses of M+ seeds, IND fruits, and M- seeds provided comprehensive datasets for their distinct thermal responses. Morph-specific differences in co-expressed gene modules in seeds, as well as seed and pericarp hormone contents identified a role of the IND pericarp in imposing coat dormancy by generating hypoxia affecting ABA sensitivity. This involved expression of morph-specific transcription factors, hypoxia response and cell wall-remodeling genes, as well as altered abscisic acid (ABA) metabolism, transport, and signaling. Parental temperature affected ABA contents and ABA-related gene expression and altered IND pericarp biomechanical properties. Elucidating the molecular framework underlying the diaspore heteromorphism can provide insight into developmental responses to globally changing temperatures. IN A NUTSHELL BackgroundHeteromorphic diaspores (fruits and seeds) are an adaptive bet-hedging strategy to ensure survival in spatiotemporally variable environments. The stone cress Aethionema arabicum, an annual plant native to semi-arid habitats in Anatolia (Turkey), one of the worlds hotspots of biodiversity. It is a close relative of Arabidopsis, rapeseed, cabbage and other Brassica crops, but in contrast to these Ae. arabicum disperses two distinct diaspores from the same plant. These dimorphic diaspores are the mucilaginous seeds (dispersed by pod shatter) and indehiscent fruits (dispersed by abscission). The wing-like pericarp (fruit coat) of the single-seeded indehiscent fruit allows wind dispersal over large distances. The amounts and ratios of the dimorphic diaspores are variable and depend on the environmental conditions. The dimorphic diaspores differ in morphology, dormancy and germination properties and thereby make Ae. arabicum an excellent model for the comparative investigation of the underpinning molecular mechanisms. QuestionWe asked how temperature during fruit and seed formation and during seed germination affect dormancy release and germination speed, and how the morphology, hormonal regulation, and the expression of genes differ between the dimorphic diaspores. FindingsLarge-scale comparative transcriptome and hormone analyses of the mucilaginous seeds and the indehiscent fruits, as well as the seeds artificially extracted from indehiscent fruits by pericarp (fruit coat) removal, provided comprehensive datasets for their distinct thermal responses. Material obtained from plants grown at different temperatures during reproduction was imbibed at different temperatures for germination. This altered the abscisic acid (ABA) metabolism and the pericarp biomechanical properties. Diaspore-specific differences in response to distinct imbibition temperatures identified distinct gene expression patterns in seeds, distinct seed and pericarp hormone contents, and a role of the pericarp in generating hypoxia inside the fruit and imposing coat dormancy. This revealed distinct combinations of specific transcription factors, hypoxia responses and cell wall-remodeling genes, as well as altered signaling pathway genes. Next stepsOur large-scale comparative transcriptome datasets are easily and publicly accessible via the Aethionema arabicum web portal (https://plantcode.cup.uni-freiburg.de/aetar_db/index.php). We plan to expand this by future work on seedlings derived from the dimorphic diaspores, by comparing different Ae. arabicum genotypes, and by studying responses to specific stresses. Understanding the molecular basis of this fascinating example of developmental diversity and plasticity and its regulation by temperature is expected to add insight how plants respond to changing environmental conditions.

plant biology↗

bioRxiv2023

Cracking the floral quartet code: How do multimers of MIKC C-type MADS-domain transcription factors recognize their target genes?

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 thousand times in a typical flowering plant genome. The number of MTF-encoding genes increased by about two orders of magnitude during land plant evolution, resulting in roughly about 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 base and shape readout of individual DNA sequences by dimers of MTFs, an important sublineage of MTFs in plants, termed MIKCC-type MTFs (MC-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. MC-MTFs control important developmental processes in flowering plants, ranging from root and shoot to flower, fruit and seed development. The way MC-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 MC-MTFs). A special focus of our treatment is on the structural prerequisites of MC-MTFs that enable the specific recognition of target genes.

genetics↗

bioRxiv2023

Deep evolution of MADS-box genes in Archaeplastida

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.

evolutionary biology↗

bioRxiv2023

Chromosome-level genomes of multicellular algal sisters to land plants illuminate signaling network evolution

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. HIGHLIGHTSO_LIGenomes of four filamentous algae (Zygnema) sisters to land plants C_LIO_LIZygnema are rich in genes for multicellular growth and environmental acclimation: signaling, lipid modification, and transport C_LIO_LICell wall innovations: diversification of hexameric rosette cellulose synthase in Zygnematophyceae C_LIO_LICo-expression networks reveal conserved modules for balancing growth and acclimation C_LI

evolutionary biology↗

bioRxiv2022

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. 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.

genetics↗

bioRxiv2021

Comparative transcriptomics identifies the transcription factors BRANCHED1 and TCP4, as well as the microRNA miR166 as candidate genes involved in the evolutionary transition from dehiscent to indehiscent fruits in Lepidium (Brassicaceae)

BackgroundFruits are the seed-bearing structures of flowering plants and are highly diverse in terms of morphology, texture and maturation. Dehiscent fruits split open upon maturation to discharge their seeds while indehiscent fruits are dispersed as a whole. Indehiscent fruits evolved from dehiscent fruits several times independently in the crucifer family (Brassicaceae). The fruits of Lepidium appelianum, for example, are indehiscent while the fruits of the closely related L. campestre are dehiscent. Here, we investigate the molecular and genetic mechanisms underlying the evolutionary transition from dehiscent to indehiscent fruits using these two Lepidium species as model system. ResultsWe have sequenced the transcriptomes and small RNAs of floral buds, flowers and fruits of L. appelianum and L. campestre and analyzed differentially expressed genes (DEGs) and differently differentially expressed genes (DDEGs). DEGs are genes that show significantly different transcript levels in the same structures (buds, flowers and fruits) in different species, or in different structures in the same species. DDEGs are genes for which the change in expression level between two structures is significantly different in one species than in the other. Comparing the two species, the highest number of DEGs was found in flowers, followed by fruits and floral buds while the highest number of DDEGs was found in fruits versus flowers followed by flowers versus floral buds. Several gene ontology terms related to cell wall synthesis and degradation were overrepresented in different sets of DEGs highlighting the importance of these processes for fruit opening. Furthermore, the fruit valve identity genes FRUITFUL and YABBY3 were among the DEGs identified. Finally, the microRNA miR166 as well as the TCP transcription factors BRANCHED1 (BRC1) and TCP FAMILY TRANSCRIPTION FACTOR 4 (TCP4) were found to be DDEGs. ConclusionsOur study reveals differences in gene expression between dehiscent and indehiscent fruits and uncovers miR166, BRC1 and TCP4 as possible causes for the evolutionary transition from dehiscent to indehiscent fruits in Lepidium.

evolutionary biology↗

bioRxiv2019

Morphologically and physiologically diverse fruits of two Lepidium species differ in allocation of glucosinolates into immature and mature seed and pericarp

The morphology and physiology of diaspores play crucial roles in determining the fate of seeds in unpredictable habitats. In some genera of the Brassicaceae different types of diaspores can be found. Lepidium appelianum produces non-dormant seeds within indehiscent fruits while in L. campestre dormant seeds are released from dehiscent fruits. These different diaspore types offer an excellent model system to analyse the allocation of relevant defence compounds into different tissues, which may maximise diaspore fitness. Total glucosinolate concentration and composition were analysed in immature and mature seeds and pericarps of L. appelianum and L. campestre using high-performance liquid chromatography. Moreover, transgenic RNAi L. campestre lines were used for comparison that produce indehiscent fruits due to silencing of LcINDEHISCENCE, the INDEHISCENCE ortholog of L. campestre. Total glucosinolate concentrations were lower in green compared to mature seeds in all studied Lepidium species and transgenic lines. In contrast, indehiscent fruits of L. appelianum maintained their total glucosinolate concentration in mature pericarps compared to green ones, while in dehiscent L. campestre and in indehiscent RNAi-LcIND L. campestre a significant decrease in total glucosinolate concentrations from green to mature pericarps could be detected. Regarding the distribution of glucosinolate classes, high concentrations of 4-methoxyindol-3-ylmethyl glucosinolate were found in mature seeds of L. appelianum, while no indole glucosinolates were detected in mature diaspores of L. campestre. The diaspores of the latter species may rather depend on aliphatic and aromatic glucosinolates for long-term protection. The allocation patterns of glucosinolates correlate with the morpho-physiologically distinct fruits of L. appelianum and L. campestre and may be explained by the distinct dispersal strategies and the dormancy status of both species.

plant biology↗

bioRxiv2019

Structural requirements of the phytoplasma effector protein SAP54 for causing homeotic transformation of floral organs

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 physical binding to the keratin-like K-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 -helical structures. We also show that the insertion of helix-breaking mutations disrupts correct folding of SAP54, which interferes with the ability of SAP54 to bind to its target proteins and to cause disease phenotypes in vivo. Surprisingly, 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 literature data this finding suggests that MIKC-type proteins and SAP54 constitute multimeric -helical coiled-coils, possibly also involving other partners such as RAD23 proteins. Our investigations clarify the structure-function relationship of an important phytoplasma effector protein and thus may ultimately help to develop treatments against some devastating plant diseases.\n\nSIGNIFICANCE STATEMENTPhytoplasmas are bacterial plant pathogens that cause devastating diseases in crops and ornamental plants by the secretion of effector proteins such as SAP54, which leads to the degradation of some floral homeotic proteins. Our study clarifies the structural requirements of SAP54 function and illuminates the molecular mode of interaction and thus may ultimately help to develop treatments against some devastating plant diseases.

plant biology↗

bioRxiv2019

Independent origin of MIRNA genes controlling homologous target genes by partial inverted duplication of antisense-transcribed sequences

Some microRNAs (miRNAs) are key regulators of developmental processes, mainly by controlling the accumulation of transcripts encoding transcription factors that are important for morphogenesis. MADS-box genes encode a family of transcription factors which control diverse developmental processes in flowering plants. Here we study the convergent evolution of two MIRNA (MIR) gene families, named MIR444 and MIR824, targeting members of the same clade of MIKCC-group MADS-box genes. We show that these two MIR genes most likely originated independently in monocots (MIR444) and in Brassicales (eudicots, MIR824). We provide evidence that in both cases the future target gene was transcribed in antisense prior to the evolution of the MIR genes. Both MIR genes then likely originated by a partial inverted duplication of their target genes, resulting in natural antisense organization of the newly evolved MIR gene and its target gene at birth. We thus propose a new model for the origin of MIR genes, MEPIDAS (MicroRNA Evolution by Partial Inverted Duplication of Antisense-transcribed Sequences). MEPIDAS is a refinement of the inverted duplication hypothesis. According to MEPIDAS, a MIR gene evolves at a genomic locus at which the future target gene is also transcribed in the antisense direction. A partial inverted duplication at this locus causes the antisense transcript to fold into a stem-loop structure that is recognized by the miRNA biogenesis machinery to produce a miRNA that regulates the gene at this locus. Our analyses exemplify how to elucidate the origin of conserved miRNAs by comparative genomics and will guide future studies.

evolutionary biology↗

bioRxiv2017

A dead gene walking: convergent degeneration of a clade of MADS-box genes in Brassicaceae

Genes are born, and eventually they die. In contrast to gene birth, however, gene death has found only limited scientific interest, even though it is of considerable evolutionary importance. Here we use Bsister genes, a subfamily of MIKC-type MADS-box genes, as a model to investigate gene death in unprecedented detail. Typical MIKC-type genes encode conserved transcription factors controlling plant development. We show that ABS-like genes, a clade of Bsister genes, are indeed highly conserved in Brassicaceae maintaining the ancestral function of Bsister genes in ovule and seed development. In contrast, their closest paralogs, the GOA-like genes, have been undergoing convergent gene death in Brassicaceae. Intriguingly, erosion of GOA-like genes occurred after millions of years of co-existence with ABS-like genes. We thus describe Delayed Convergent Asymmetric Degeneration (DCAD), a so far neglected but possibly frequent pattern of duplicate gene evolution that does not fit classical scenarios. DCAD of GOA-like genes may have been initiated by a reduction in the expression of an ancestral GOA-like gene in the stem group of Brassicaceae and driven by dosage subfunctionalization. Our findings have profound implications for gene annotations in genomics, interpreting patterns of gene evolution and using genes in phylogeny reconstructions of species.

evolutionary biology↗

bioRxiv2017

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 and a central player in flower development. MADS-domain proteins bind as dimers to AT-rich sequences termed CArG-boxes which share the consensus 5-CC(A/T)6GG-3. Since only a fraction of the abundant CArG-boxes in the Arabidopsis genome are bound by SEPALLATA3, more elaborate principles have to be discovered to better understand which features turn CArG-box sequences into genuine recognition sites. Here, we investigated to which extent the shape of the DNA contributes to the DNA-binding specificity of SEPALLATA3. We determined in vitro binding affinities of SEPALLATA3 to a variety of DNA probes which all contain the CArG-box motif, but differ in their DNA shape characteristics. We found that binding affinity correlates well with certain DNA shape features associated with A-tracts. Analysis of SEPALLATA3 proteins with single amino acid substitutions in the DNA-binding MADS-domain 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 plant MADS-domain transcription factors heavily depend on shape readout mechanisms and that the absence of a critical arginine residue in the MADS-domain impairs binding specificity.

plant biology↗

bioRxiv2017

Sequence Features Of MADS-Domain Proteins That Act As Hubs In The Protein-Protein Interaction Network Controlling Flower Development

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. 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 account for subfamily-specific interaction capabilities of MADS-domain transcription factors and shaped the present-day structure of the PPI network controlling flower development.

plant biology↗