Post-translational modifications of the endogenous and transgenic FLC protein in Arabidopsis thaliana.

FLC is a MADS box transcription factor that acts as a dosage-dependent repressor of flowering. We carried out a 2D gel analysis and showed that the majority of endogenous FLC and overexpressed FLC-FLAG proteins are post-translationally modified. The endogenous and transgenic proteins have different floral repressor activities; however, they have similar, if not the same, profiles of post-translational modifications. The protein modification profile was also not changed by vernalization treatment. The activities of other MADS box proteins have been shown to be affected by phosphorylation and we found that both the endogenous FLC and the transgenic FLC-FLAG protein are phosphorylated. When eight potential serine kinase target sites in FLC were changed to mimic phosphorylated residues, expression of the mutant FLC-FLAG protein led to early flowering, suggesting that the repressive function was abolished. When the same eight serine residues were changed to non-phosphorylatable residues, expression of the resulting protein gave the same weak flowering repression as overexpressed unmodified FLC-FLAG. The non-phosphorylatable variant of FLC-FLAG showed a similar spectrum of post-translational modifications to unmodified FLC-FLAG, indicating that modifications other than the predicted phosphorylations occur. Our data provide evidence for a post-translational regulation of FLC function.