Abstract
The SLEEPY1 (SLY1) F-box gene is a positive regulator of gibberellin (GA) signaling in Arabidopsis (Arabidopsis thaliana). Loss of SLY1 results in GA-insensitive phenotypes including dwarfism, reduced fertility, delayed flowering, and increased seed dormancy. These sly1 phenotypes are partially rescued by overexpression of the SLY1 homolog SNEEZY (SNE)/SLY2, suggesting that SNE can functionally replace SLY1. GA responses are repressed by DELLA family proteins. GA relieves DELLA repression when the SCF(SLY1) (for Skp1, Cullin, F-box) E3 ubiquitin ligase ubiquitinates DELLA protein, thereby targeting it for proteolysis. Coimmunoprecipitation experiments using constitutively expressed 35S:hemagglutinin (HA)-SLY1 and 35S:HA-SNE translational fusions in the sly1-10 background suggest that SNE can function similarly to SLY1 in GA signaling. Like HA-SLY1, HA-SNE interacted with the CULLIN1 subunit of the SCF complex, and this interaction required the F-box domain. Like HA-SLY1, HA-SNE coimmunoprecipitated with the DELLA REPRESSOR OF GA1-3 (RGA), and this interaction required the SLY1 or SNE carboxyl-terminal domain. Whereas HA-SLY1 overexpression resulted in a decrease in both DELLA RGA and RGA-LIKE2 (RGL2) protein levels, HA-SNE caused a decrease in DELLA RGA but not in RGL2 levels. This suggests that one reason HA-SLY1 is able to effect a stronger rescue of sly1-10 phenotypes than HA-SNE is because SLY1 regulates a broader spectrum of DELLA proteins. The FLAG-SLY1 fusion protein was found to coimmunoprecipitate with the GA receptor HA-GA-INSENSITIVE DWARF1b (GID1b), supporting the model that SLY1 regulates DELLA through interaction with the DELLA-GA-GID1 complex.
Highlights
The SLEEPY1 (SLY1) F-box gene is a positive regulator of gibberellin (GA) signaling in Arabidopsis (Arabidopsis thaliana)
This study examines the relative roles of the two F-box proteins, SLY1 and SNE/SLY2, first through a careful analysis of their ability to complement the sly1-10 mutation, by determining which domains are required for function, and by examining the ability of these proteins to bind to other proteins involved in GA signaling, including the CUL1 subunit of the SCF complex and the DELLA protein REPRESSOR OF GA1-3 (RGA) in Arabidopsis
Previous work suggested that the SLY1 homolog SNE/SLY2 may encode a second F-box protein functioning in GA signaling, since overexpression of SNE
Summary
The SLEEPY1 (SLY1) F-box gene is a positive regulator of gibberellin (GA) signaling in Arabidopsis (Arabidopsis thaliana). Loss of SLY1 results in GA-insensitive phenotypes including dwarfism, reduced fertility, delayed flowering, and increased seed dormancy These sly phenotypes are partially rescued by overexpression of the SLY1 homolog SNEEZY (SNE)/SLY2, suggesting that SNE can functionally replace SLY1. Based on yeast three-hybrid data, Arabidopsis GID1-GA binding to DELLA appears to increase the affinity of the F-box protein SLY1 for DELLA (Griffiths et al, 2006). Addition of four ubiquitin moieties to a target protein triggers its recognition and proteolysis by the 26S proteasome (Smalle and Vierstra, 2004) It appears that DELLA is destroyed via the ubiquitin-proteasome pathway, because both mutations in SLY1 and 26S proteasome inhibitors result in stabilization and increased accumulation of DELLA protein in the presence of GA (McGinnis et al, 2003; Dill et al, 2004; Fu et al, 2004)
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