The role of two f-box proteins, SLEEPY1 and SNEEZY, in Arabidopsis gibberellin signaling

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.

Figure 1.

Domain structure and homology of the SLY1 and SNE genes and predicted protein sequences. A, Gene structure of two F-box proteins, SLY1 and SNE, consisting of N-terminal (N), F-box (F), and C-terminal (C) domains. The number below the SLY1 and above the SNE gene shows the amino acid (a.a.) number, where the first amino acid is +1. B, DNA and amino acid homology between SLY1 and SNE in the N-terminal, F-box, and C-terminal domains.

Figure 2.

The effect of N-terminal, C-terminal, and F-box domain deletions on the ability of HA-SLY1 and HA-SNE to rescue sly1-10. A, Shown are 30-d-old wild-type (WT) Ler, sly1-10, and sly1-10 transformed with the indicated HA fusion constructs. B, Shown are 45-d-old sly1-10 and sly1-10 HA-SNE. The HA-SNE construct partially rescued the dwarfism. C, DELLA RGA and GAI protein accumulation in wild-type Ler, ga1-3, sly1-10, and sly1-10 transformed with constructs described above was determined by anti-RGA immunoblot analysis of 40 μg of total protein extracted from 21-d-old rosette leaves.

Figure 3.

Final plant height and fertility in the wild-type (WT) Ler, sly1-10, and sly1-10 transformed with the indicated constructs. A, Final plant height (cm) was determined after 120 d of incubation in a growth chamber. B, Fertility was determined based on the number of seeds per silique. Wild-type Ler and sly1-10 plants transformed with the HA vector were used as controls.

Figure 4.

SNE overexpression partial rescue of the sly1-10 seed germination phenotype is associated with decreased DELLA RGA but not RGL2 protein accumulation. A, Germination of sly1-10 and two independent sly1-10 lines transformed with HA-SLY1 (#27-2 and #27-3) and HA-SNE (#7 and #12). Seeds were incubated on MS agar plates at 4°C for 3 d, followed by 22°C for 4 d. WT, Wild type. B, Protein-blot analysis of RGA and RGL2 in sly1-10 HA-SLY1 and sly1-10 HA-SNE seeds imbibed on MS agar plates at 4°C for 3 d, followed by 22°C for 24 h. Protein was detected with anti-RGA and anti-RGL2. Sixty micrograms of total protein was loaded. [See online article for color version of this figure.]

Figure 5.

HA-SLY1 and HA-SNE fusion proteins interact with DELLA RGA and CUL1 protein in planta. Protein was extracted from 12-d-old sly1-10 plants transformed with the indicated constructs and incubated with HA matrix agarose in the presence of 0.1% ethanol (mock) or 100 μm GA₃. Immunoprecipitated protein was loaded on an SDS-PAGE gel and detected with anti-HA antibody. Coimmunoprecipitated protein was detected with anti-RGA and anti-CUL1.

Figure 6.

Interaction of SLY1 and GID1 in coimmunoprecipitation (Co-IP) assays. The coimmunoprecipitation experiment was performed using protein extracted from 12-d-old sly1-10 plants transformed with HA-GID1a and/or FLAG-SLY1. The protein extract was incubated with FLAG matrix agarose in the presence of 0.1% ethanol (mock) or 10 μm GA₃ and loaded on an SDS-PAGE gel. Protein-blot analysis was performed using anti-FLAG and anti-HA antibodies. The asterisk indicates a background band. Forty micrograms of total protein was loaded (input). [See online article for color version of this figure.]