The evolutionary origin of "floral quartets" : clues from molecular interactions of orthologues of floral homeotic proteins from the gymnosperm Gnetum gnemon

The identity of floral organs in angiosperms is specified by multimeric transcription factor complexes composed of floral homeotic MADS-domain proteins that bind to specific cis-regulatory DNA-elements (‘CArG-boxes’) of their target genes, thus constituting floral quartets. Gymnosperms possess orthologues of floral homeotic genes enconding MIKC-type MADS-domain proteins, but when and how the interactions constituting floral quartets were established during evolution has remained unknown. To better understand the ‘abominable mystery’ of flower origin, in this project a comprehensive study was carried out to detect the dimerization and DNA-binding of several classes of MADS-domain proteins from a gymnosperm, Gnetum gnemon of the Gnetales. Determination of protein-protein interactions by pull-down assays revealed complex patterns of heterodimerization among orthologues of class B, class C and class E floral homeotic proteins and Bsister proteins, while homodimerization was not observed. In contrast, electrophoretic mobility shift assays (EMSAs) revealed that all proteins tested except one bind to CArG-boxes also as homodimers, suggesting that homodimerization is relatively weak, but facilitated by DNA-binding. Proteins able of DNA-based homodimerization include orthologues of class B and C proteins; B and C proteins also form heterodimers in vitro and in yeast, which is in sharp contrast to their orthologues from angiosperms, which require class E floral proteins to ‘glue’ them together in multimeric complexes. Remarkably, the heterodimers of B and C proteins from G. gnemon are not capable of binding to CArG-boxes, suggesting that DNA-binding in vivo is based on homodimers, while heterodimerization of B and C proteins may constitute multimeric, DNA-bound complexes by mediating the interaction between two DNA-bound homodimers. EMSAs and DNase I footprint assays indicated that both B with C proteins and C proteins alone but not B proteins alone can induce DNA-looping to form tetrameric protein-DNA complexes similar to floral quartets. These data suggest that at least some of the gymnosperm orthologues of floral homeotic proteins may have the capability of forming higher-order complexes and that gymnosperm B and C proteins control male organ identity and C proteins controls female organ identity, respectively, by forming quartet-like complexes composed of two homodimers, each bound to a CArG-box.


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