Abstract
Flowering locus C (FLC), a floral repressor, is a critical factor for the transition from the vegetative to the reproductive phase. Here, the mechanisms regulating the activity and stability of the FLC protein were investigated. Bimolecular fluorescence complementation and in vitro pull-down analyses 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 the 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 the sumoylation site)-overexpressing plants was comparable with that in the wild type, whereas flowering was considerably delayed in FLC-overexpressing plants, supporting the notion that sumoylation is an important mechanism for FLC function. The data indicate that the sumoylation of FLC is critical for its role in the control of flowering time and that AtSIZ1 positively regulates FLC-mediated floral suppression.
Highlights
In eukaryotic cells, protein function and stability are posttranslationally regulated by small and large molecules such as phosphates, carbohydrates, lipids, and small proteins (Castro et al, 2012)
Overexpression of mFLC, a sumoylation site mutant gene, had no effect on flowering time. These findings indicate that Flowering locus C (FLC) is stabilized by the E3 small ubiquitinlike modifier (SUMO) ligase AtSIZ1, and FLC-mediated flowering repression is stimulated by sumoylation
Arabidopsis FLC tagged with the C-terminus of enhanced yellow fluorescent protein (EYFP) and AtSIZ1 tagged with the N-terminus of EYFP were transiently expressed in onion epidermal cells
Summary
Protein function and stability are posttranslationally regulated by small and large molecules such as phosphates, carbohydrates, lipids, and small proteins (Castro et al, 2012). SUMO modification in plants has been implicated in numerous basic cellular processes, such as stress and defence responses, nitrogen metabolism, and the regulation of flowering (Hotson et al, 2003; Kurepa et al, 2003; Lois et al, 2003; Murtas et al, 2003; Miura et al, 2005, 2007; Catala et al, 2007; Lee et al, 2007; Conti et al, 2008; Yoo et al, 2006; Park et al, 2011). A few Arabidopsis proteins, such as the nitrate reductases NIA1 and NIA2, inducer of CBF expression 1 (ICE1), the R2R3-type transcription factor MYB30, and the SUMO machinery proteins AtSIZ1 and AtSCE1, have been experimentally demonstrated to be sumoylated (Miura and Hasegawa, 2010; Park et al, 2011; Zheng et al, 2012)
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