The floral regulator LEAFY evolves by substitutions in the DNA binding domain.

The plant-specific transcription factor LEAFY controls general aspects of the life cycle in a basal plant, the moss Physcomitrella patens. In contrast, LEAFY has more specialized functions in angiosperms, where it specifically induces floral fate during the reproductive phase. This raises the question of a concomitant change in the biochemical function of LEAFY during the evolution of land plants. We report that the DNA binding domain of LEAFY, although largely conserved, has diverged in activity. On the contrary, other, more rapidly evolving portions of the protein have few effects on LEAFY activity.

Regulatory elements of the floral homeotic gene AGAMOUS identified by phylogenetic footprinting and shadowing.

In Arabidopsis thaliana, cis-regulatory sequences of the floral homeotic gene AGAMOUS (AG) are located in the second intron. This 3-kb intron contains binding sites for two direct activators of AG, LEAFY (LFY) and WUSCHEL (WUS), along with other putative regulatory elements. We have used phylogenetic footprinting and the related technique of phylogenetic shadowing to identify putative cis-regulatory elements in this intron. Among 29 Brassicaceae species, several other motifs, but not the LFY and WUS binding sites identified previously, are largely invariant. Using reporter gene analyses, we tested six of these motifs and found that they are all functionally important for the activity of AG regulatory sequences in A. thaliana. Although there is little obvious sequence similarity outside the Brassicaceae, the intron from cucumber AG has at least partial activity in A. thaliana. Our studies underscore the value of the comparative approach as a tool that complements gene-by-gene promoter dissection but also demonstrate that sequence-based studies alone are insufficient for a complete identification of cis-regulatory sites.