Deep conservation of class C floral homeotic gene function in a ranunculid

In the model plant Arabidopsis thaliana, a core eudicot, the C-class gene AGAMOUS (AG) has a dual role in specifying reproductive organ identity and floral meristem determinacy. We conduct a functional analysis of the putative AG ortholog ThtAG1 from the ranunculid Thalictrum thalictroides, a representative of the sister lineage to eudicots. Virus Induced Gene Silencing (VIGS) of ThtAG1 resulted in homeotic conversion of stamens and carpels into sepaloid organs and loss of flower determinacy. Moreover, it was sufficient to phenocopy the “double flower” ornamental cultivar T. thalictroides ‘Double White’. Molecular analysis of  ‘Double White’ ThtAG1 alleles revealed the insertion of an LTR retrotransposon, which induces alternative splicing that results in mutant proteins with K domain deletions. Biochemical analysis demonstrated that the mutation abolishes protein-protein interactions with the E class protein ThtSEP3. C- and E- class protein heterodimerization is predicted by the floral quartet model, yet evidence for the functional importance of this interaction is scarce outside of the core eudicots. Our finding, that a class C mutant with a defective K domain of an AG protein hampers the interaction with a SEP protein, corroborates the importance and conservation of this interaction. This constitutes the first functional description of a full C class mutant in a non-core eudicot, an ornamental double flower, affecting both organ identity and meristem determinacy. Using complementary forward and reverse genetic approaches this study reveals deep conservation of the dual C class gene function, and of the C-E protein interactions predicted by the floral quartet model.