Evolution of the interaction of floral homeotic proteins
Final Report Abstract
During flower development the identity of each floral organ is controlled by a set of homeotic MIKCC-type MADS-domain transcription factors (MADS-TFs) that act in a combinatorial manner. As proposed by the "floral quartet model" floral organ-specific complexes of four MADS-TFs bind to two adjacent DNA-binding sites via looping the DNA between both binding sites. Although all floral homeotic MADS-TFs can be incorporated into floral quartets, their individual abilities to form DNA-bound homo- and hetero-tetramers differ considerably. One extreme is represented by many APETALA3 (AP3)- and PISTILLATA (PI)-like proteins that only form obligate heterodimers and that require other MADS-TFs to be incorporated into floral quartets. The other extreme are SEPALLATA (SEP)-like proteins that are capable of forming homo-tetramers and that mediate the tetramerization of numerous other MADS-TFs. In addition to a central role in floral organ identity determination some SEP proteins are also involved in controlling flowering time, floral transition and ovule development. SEP proteins thus constitute a major hub in the protein-protein interaction (PPI) network underlying reproductive organ development. When this DFG project was initiated very little was known about sequence determinants that mediate the different PPIs of MADS-TFs belonging to different subfamilies. Furthermore, the conservation and the evolutionary trajectory of the complex PPI network of MADS-TFs was largely unexplored. During this project we investigated the PPI capabilities of MADS-TFs from different early diverging angiosperms such as Amborella, Nuphar and Liriodendron. Our analysis revealed that all homo- and heterodimeric interactions that are important for floral organ development in monocotyledonous and eudicotyledonous plants are also present in early diverging angiosperms, suggesting a high conservation of these interactions during angiosperm evolution. Beside these highly conserved interactions many MADS-TFs from early diverging angiosperms displayed additional PPIs and an overall more promiscuous interaction behavior. These observations suggest a trend from labile to more constraint complex formation capabilities of MADS-TFs during angiosperm evolution. We hypothesize that this increase in interaction constraints constituted an important precondition for the development of an exact merosity of the flower, which in turn is believed to have contributed to the impressive radiation of derived angiosperms. Furthermore, we could demonstrate that also MADS-TFs from extant gymnosperms, angiosperms closest living relatives, form different floral quartet-like complexes (FQCs) that are probably involved in female and male organ identity determination. It appears likely, therefore, that FQC formation predated the split of the lineages that led to extant angiosperms and gymnosperms. To further trace back the evolutionary origin of FQCs we thus tested the tetramerization capabilities of MADS-TFs of representatives of lycophytes, ferns and mosses. Our results suggest that FQC formation is an ancestral feature of MADS-TFs that had probably already been established in the stem group of extant land plants (embryophytes) more than 400 million years ago. To investigate which molecular determinants mediate tetramerization capabilities of MADS-TFs we investigated FQC formation of SEP3 of Arabidopsis thaliana in a combined in vitro and in silico approach. Our results reveal that leucine residues at interacting sites within the protein-protein interacting keratine-like domain (K-domain) of SEP3 are essential mediators of tetramerization. Our data also indicate that preferences for amino acids other than leucine at homologous sites may account for the less promiscuous interactions of floral homeotic proteins belonging to other subfamilies such as AP3- and PI-like proteins. In a side project of this DFG grant we investigated the molecular mechanism by which phytopathogenic bacteria, so called phytoplasmas, manipulate the meristem identity determination of their host plants. Based on in silico analyses we proposed that the phytoplasma effector protein SAP54 binds to a specific subset of MADS-TFs via molecular mimicry of the K-domain, and that the interaction is probably mediated by mechanisms similar to that mediating PPIs among MADS-TFs. We hypothesize that the high structural similarity evolved via convergent structural and sequence evolution. The data and insights gathered during this DFG project resulted in about ten publications in international peer reviewed journals in the fields of botany, molecular genetics and developmental biology. This DFG project provided full support for three Ph.D. students and partial support for one more, which all successfully acquired their doctoral degrees. Furthermore, several Diploma, Bachelor and Master Students prepared their theses in the context of this project and successfully completed their studies. One of the co-applicants and co-supervisors obtained a permanent academic position during the second period of the project and the respective grant and the results obtained during this project may well have helped him to secure this position.
Publications
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(2014). DEF- and GLO-like proteins may have lost most of their interaction partners during angiosperm evolution. Ann. Bot. 114, 1431-1443
Melzer, R., Härter, A., Rümpler, F., Kim, S., Soltis, P. S., Soltis, D. E., Theißen, G.
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(2015). Character-state reconstruction to infer ancestral protein-protein interaction patterns. Bio-protocol 5(16): e1566
Rümpler, F., Theißen, G., Melzer, R.
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(2015). Did convergent protein evolution enable Phytoplasmas to generate 'zombie plants'? Trends Plant Sci. 20, 798-806
Rümpler, F., Gramzow, L., Theißen, G., Melzer, R.
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(2016). MADS-domain transcription factors and the floral quartet model of flower development: linking plant development and evolution. Development 143, 3259-3271
Theißen, G., Melzer, R., Rümpler, F.
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(2017). The floral homeotic protein SEPALLATA3 recognizes target DNA sequences by shape readout involving a conserved arginine residue in the MADS-domain. bioRxiv
Gusewski, S., Melzer R., Rümpler, F., Gafert, C., Theißen, G.
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(2018). A conserved leucine zipper-like motif accounts for strong tetramerization capabilities of SEPALLATA-like MADS-domain transcription factors. J. Exp. Bot.
Rümpler, F., Theißen, G., Melzer, R.
