Sub-functionalization to ovule development following duplication of a floral organ identity gene.

Abstract Gene duplications result in paralogs that may be maintained due to the gain of novel functions (neo-functionalization) or the partitioning of ancestral function (sub-functionalization). Plant genomes are especially prone to duplication; paralogs are particularly widespread in the floral MADS box transcription factors that control organ identity through the ABC model of flower development. C class genes establish stamen and carpel identity and control floral meristem determinacy, and are largely conserved across the angiosperm phylogeny. Originally, an additional D class had been identified as controlling ovule identity; yet subsequent studies indicated that both C and D lineage genes more commonly control ovule development redundantly. The ranunculid Thalictrum thalictroides has two orthologs of the Arabidopsis thaliana C class gene AGAMOUS (AG) , ThtAG1 and ThtAG2 (Thalictrum thalictroides AGAMOUS1/2) . We previously showed that ThtAG1 exhibits typical C class function; here we examine the role of its paralog, ThtAG2. Our phylogenetic analysis shows that ThtAG2 falls within the C lineage, together with ThtAG1 , and is consistent with previous findings of a Ranunculales-specific duplication in this clade. However, ThtAG2 is not expressed in stamens, but rather solely in carpels and ovules. This female-specific expression pattern is consistent with D lineage genes, and with other C lineage genes known to be involved in ovule identity. Given the divergent expression of ThtAG2 , we tested the hypothesis that it has acquired ovule identity function. Molecular evolution analyses showed evidence of positive selection on ThtAG2 -a pattern that supports divergence of function by sub-functionalization. Down-regulation of ThtAG2 by virus-induced gene silencing resulted in homeotic conversions of ovules into carpel-like structures. Taken together, our results suggest that, although ThtAG2 falls within the C lineage, it has diverged to acquire “D function” as an ovule identity gene, and does not appear to require a direct interaction with the ThtAG1 protein. We therefore present a functional example of ovule identity being specified by either a single gene or a gene pair within the C lineage, with no D lineage contribution. In conclusion, following a Ranunculales-wide duplication in the AG lineage, functional divergence has led to the evolution of ovule identity-specificity in a T. thalictroides C lineage gene.