E3 SUMO ligase AtSIZ1 regulates FLC-mediated flowering repression

Abstract

Flowering Locus C (FLC), a floral repressor, is antagonistic regulator for transition from vegetative to the reproductive phase. FLC represses expression of several genes involved in floral induction and its transcription is positively or negatively regulated by FRIGIDA or by vernalization, respectively. Although several factors affecting the transcription of FLC have been described, the post-translational regulation of FLC stability and function has not been clearly characterized. Here, I investigated the mechanisms regulating the activity and stability of the FLC protein. Sumoylation, a post translational process of attaching small ubiquitin-related modifier (SUMO) to a lysine residue in proteins through an enzyme cascade catalyzed by E1, E2 and E3 enzymes, plays crucial role in protein stability and biological processes. Bimolecular fluorescence complementation, co-immunoprecipitation and in vitro pull down analysis showed that FLC interacts with the E3 small ubiquitin-like modifier (SUMO) ligase AtSIZ1, suggesting that AtSIZ1 is an E3 SUMO ligase for FLC. In vitro sumoylation assays showed that FLC is modified by SUMO in presence of SUMO-activating enzyme E1 and conjugating enzyme E2 but its sumoylation is inhibited by AtSIZ1. In transgenic plants, inducible AtSIZ1 overexpression led to an increase in the concentration of FLC and delayed the post-translational decay of FLC, indicating that AtSIZ1 stabilizes FLC through direct binding. Also, the flowering time in mutant FLC (K154R, a mutation of sumoylation site)-overexpressing plants was comparable to that in wild type, whereas flowering was considerably delayed in FLC-overexpressing plants, supporting the notion that sumoylation is an important mechanism for FLC function. These results indicate that the sumoylation of FLC is critical for its role in the control flowering time and AtSIZ1 positively regulates FLC-mediated floral suppression.