Roles and evolution of four LEAFY homologs in floral patterning and leaf development in woodland strawberry

The plant-specific transcription factor LEAFY (LFY), generally maintained as a single-copy gene in most angiosperm species, plays critical roles in flower development. The woodland strawberry (Fragaria vesca) possesses four LFY homologs in the genome; however, their respective functions and evolution remain unknown. Here, we identified and validated that mutations in one of the four LFY homologs, FveLFYa, cause homeotic conversion of floral organs and reiterative outgrowth of ectopic flowers. In contrast to FveLFYa, FveLFYb/c/d appear dispensable under normal growth conditions, as fvelfyc mutants are indistinguishable from wild type and FveLFYb and FveLFYd are barely expressed. Transgenic analysis and yeast one-hybrid assay showed that FveLFYa and FveLFYb, but not FveLFYc and FveLFYd, are functionally conserved with AtLFY in Arabidopsis (Arabidopsis thaliana). Unexpectedly, LFY-binding site prediction and yeast one-hybrid assay revealed that the transcriptional links between LFY and the APETALA1 (AP1) promoter/the large AGAMOUS (AG) intron are missing in F. vesca, which is due to the loss of LFY-binding sites. The data indicate that mutations in cis-regulatory elements could contribute to LFY evolution. Moreover, we showed that FveLFYa is involved in leaf development, as approximately 30% of mature leaves have smaller or fewer leaflets in fvelfya. Phylogenetic analysis indicated that LFY homologs in Fragaria species may arise from recent duplication events in their common ancestor and are undergoing convergent gene loss. Together, these results provide insight into the role of LFY in flower and leaf development in strawberry and have important implications for the evolution of LFY.