Molecular Basis of Late-Flowering Phenotype Caused by Dominant Epi-Alleles of the FWA Locus in Arabidopsis

The late-flowering phenotype of dominant fwa mutants is caused by hypomethylation in the FWA locus leading to ectopic expression of a homeodomain leucine zipper (HD-ZIP) protein. However, little is known about whether FWA has any role in regulation of flowering and how ectopically expressed FWA delays flowering. Through analysis of FWA expression in wild-type seedlings, it was shown that FWA is not expressed during the vegetative phase. This suggests that FWA has no role in flowering. The previous reports that fwa suppressed the precocious-flowering phenotype of plants overexpressing FLOWERING LOCUS T (FT) suggest that the flowering pathway(s) either at and/or downstream of FT is blocked by FWA. Comparison of gene expression profiles in three genetic backgrounds ectopically expressing FWA and their respective wild types failed to detect common changes, ruling out the possibility that FWA acts through transcriptional misregulation. Yeast two-hybrid analysis and in vitro pull-down assay showed that FWA protein can specifically interact with FT protein. The importance of protein interaction with FT in delaying flowering was supported by studies involving N-terminal and C-terminal truncations of FWA. The C-terminal truncation with abolished interaction did not delay flowering when overexpressed, while the N-terminal truncation, which retains interaction, did. Specific interaction of FWA with FT enabled us to use FWA protein as a specific inhibitor of FT protein function. Through tissue-specific ectopic expression of FWA, further support for the shoot apex being the site of action of FT protein was provided.