Loss of deeply conserved C-class floral homeotic gene function and C- and E-class protein interaction in a double-flowered ranunculid mutant.

In the model plant Arabidopsis thaliana, a core eudicot, the floral homeotic C-class gene AGAMOUS (AG) has a dual role 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 all other eudicots. Down-regulation of ThtAG1 by virus-induced gene silencing resulted in homeotic conversion of stamens and carpels into sepaloid organs and loss of flower determinacy. Moreover, flowers exhibiting strong silencing of ThtAG1 phenocopied the double-flower ornamental cultivar T. thalictroides 'Double White.' Molecular analysis of 'Double White' ThtAG1 alleles revealed the insertion of a retrotransposon causing either nonsense-mediated decay of transcripts or alternative splicing that results in mutant proteins with K-domain deletions. Biochemical analysis demonstrated that the mutation abolishes protein-protein interactions with the putative E-class protein ThtSEP3. C- and E-class protein heterodimerization is predicted by the floral quartet model, but evidence for the functional importance of this interaction is scarce outside the core eudicots. Our findings therefore corroborate the importance and conservation of the interactions between C- and E-class proteins. This study provides a functional description of a full C-class mutant in a noncore ("basal") eudicot, an ornamental double flower, affecting both organ identity and meristem determinacy. Using complementary forward and reverse genetic approaches, this study demonstrates deep conservation of the dual C-class gene function and of the interactions between C- and E-class proteins predicted by the floral quartet model.

Virus-induced gene silencing as a tool for comparative functional studies in Thalictrum.

Perennial woodland herbs in the genus Thalictrum exhibit high diversity of floral morphology, including four breeding and two pollination systems. Their phylogenetic position, in the early-diverging eudicots, makes them especially suitable for exploring the evolution of floral traits and the fate of gene paralogs that may have shaped the radiation of the eudicots. A current limitation in evolution of plant development studies is the lack of genetic tools for conducting functional assays in key taxa spanning the angiosperm phylogeny. We first show that virus-induced gene silencing (VIGS) of a PHYTOENE DESATURASE ortholog (TdPDS) can be achieved in Thalictrum dioicum with an efficiency of 42% and a survival rate of 97%, using tobacco rattle virus (TRV) vectors. The photobleached leaf phenotype of silenced plants significantly correlates with the down-regulation of endogenous TdPDS (P<0.05), as compared to controls. Floral silencing of PDS was achieved in the faster flowering spring ephemeral T. thalictroides. In its close relative, T. clavatum, silencing of the floral MADS box gene AGAMOUS (AG) resulted in strong homeotic conversions of floral organs. In conclusion, we set forth our optimized protocol for VIGS by vacuum-infiltration of Thalictrum seedlings or dormant tubers as a reference for the research community. The three species reported here span the range of floral morphologies and pollination syndromes present in Thalictrum. The evidence presented on floral silencing of orthologs of the marker gene PDS and the floral homeotic gene AG will enable a comparative approach to the study of the evolution of flower development in this group.