Conserved intragenic elements were critical for the evolution of the floral C-function.

The floral C-function, which specifies stamen and carpel development, played a pivotal role in the evolution of flowers. An important aspect of this was the establishment of mechanisms regulating the temporal and spatial expression domain of the C-function genes. Transcription of the Arabidopsis C-function gene AGAMOUS (AG) is tightly controlled by factors that interact with cis-elements within its large second intron. Little is known about the regulatory role of intragenic elements in C-function genes from species other than Arabidopsis. We show that a binding site for the LEAFY (LFY) transcription factor, present in the AG intron, is conserved in the introns of diverse C-function genes and is positioned close to other conserved motifs. Using an in planta mutagenesis approach, we targeted evolutionarily conserved sequences in the intron of the Antirrhinum PLENA (PLE) gene to establish whether they regulate PLE expression. Small sequence deletions resulted in a novel class of heterochronic C-function mutants with delayed onset of PLE expression and loss of stamen identity. These phenotypes differ significantly from weak C-function mutant alleles in Antirrhinum and Arabidopsis. Our findings demonstrate that the PLE intron contains regulatory cis-elements, including a LFY-binding site, critical for establishing the correct C-function expression domain. We show that the LFY site, and other conserved intron elements, pre-date the divergence of the monocot and dicot lineages, suggesting that they were a determinant in the evolution of the C-function, and propose a threshold model to explain phenotypic divergence observed between C-function mutants.