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. 2004 Jun;16(6):1392-405.
doi: 10.1105/tpc.020958. Epub 2004 May 21.

The Arabidopsis F-box protein SLEEPY1 targets gibberellin signaling repressors for gibberellin-induced degradation

Alyssa Dill  1 , Stephen G Thomas, Jianhong Hu, Camille M Steber, Tai-Ping Sun
Affiliations

The Arabidopsis F-box protein SLEEPY1 targets gibberellin signaling repressors for gibberellin-induced degradation

Alyssa Dill et al. Plant Cell. 2004 Jun.

Abstract

The nuclear DELLA proteins are highly conserved repressors of hormone gibberellin (GA) signaling in plants. In Arabidopsis thaliana, GA derepresses its signaling pathway by inducing proteolysis of the DELLA protein REPRESSOR OF ga1-3 (RGA). SLEEPY1 (SLY1) encodes an F-box-containing protein, and the loss-of-function sly1 mutant has a GA-insensitive dwarf phenotype and accumulates a high level of RGA. These findings suggested that SLY1 recruits RGA to the SCFSLY1 E3 ligase complex for ubiquitination and subsequent degradation by the 26S proteasome. In this report, we provide new insight into the molecular mechanism of how SLY1 interacts with the DELLA proteins for controlling GA response. By yeast two-hybrid and in vitro pull-down assays, we demonstrated that SLY1 interacts directly with RGA and GA INSENSITIVE (GAI, a closely related DELLA protein) via their C-terminal GRAS domain. The rga and gai null mutations additively suppressed the recessive sly1 mutant phenotype, further supporting the model that SCFSLY1 targets both RGA and GAI for degradation. The N-terminal DELLA domain of RGA previously was shown to be essential for GA-induced degradation. However, we found that this DELLA domain is not required for protein-protein interaction with SLY1 in yeast (Saccharomyces cerevisiae), suggesting that its role is in a GA-triggered conformational change of the DELLA proteins. We also identified a novel gain-of-function sly1-d mutation that increased GA signaling by reducing the levels of the DELLA protein in plants. This effect of sly1-d appears to be caused by an enhanced interaction between sly1-d and the DELLA proteins.

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Figures

Figure 1.
Figure 1.
Suppression of sly1-10 by rga-24 and gai-t6. (A) Representative 42-d-old homozygous plants. a, sly1-10; b, gai-t6 sly1-10; c, rga-24 sly1-10; d, rga-24 gai-t6 sly1-10; e, the wild type. The rga-24 and gai-t6 single mutants were not shown because their phenotypes are similar to the wild type (Silverstone et al., 1997; Dill and Sun, 2001; King et al., 2001). (B) to (E) Twenty-four plants per line were characterized except for days to flower in sly1-10 gai-t6 rga-24, for which 13 plants were studied. Shown are means ± se. (B) Flowering time in leaf number. Black bars indicate rosette leaves; gray bars show cauline leaves. (C) Final height. (D) Branch number. (E) Days to flower and fertility. Many siliques on sly1-10–containing plants were infertile. “All” indicates the average fertility of all siliques on the primary inflorescence; “fertile” indicates the average fertility of only those siliques that contained seeds.
Figure 2.
Figure 2.
SLY1 Regulates GA-Induced GAI Degradation. The blots contain 20 μg of total proteins from rosette leaves of 24-d-old wild-type and homozygous mutant plants treated with 100 μM GA3 (+) or water (−) for 2 h. The protein blots were probed with anti-RGA antibodies from rabbit. Protein bands indicated with an asterisk represent nonspecific immunoreactive proteins. Blot (B) was exposed ∼10 times longer than blot (A).
Figure 3.
Figure 3.
gar2 Is a Gain-of-Function sly1 Allele That Partially Suppresses gai-1 and rga-Δ17 Dwarf Phenotypes. (A) gar2 partially suppresses rga-Δ17 but does not restore GA responsiveness. Representative 36-d-old homozygous plants grown on soil are shown. Plants treated with GA (+) were sprayed with 100 μM GA3 weekly, beginning at 18 d after sowing. (B) Sequence alignment of the LSL motif in SLY1 and other F-box proteins that contain both GGF and LSL motifs. Identical residues are shown in black boxes, and similar residues are in gray boxes. The asterisk indicates the residue mutated in sly1-d (gar2). (C) to (H) Phenotype of 42-d-old control and T1 transgenic plants. Shown are gai-1 (C), gai-1∷pSLY1 number 4-1 (D), gai-1∷psly1-d number 6-1 (E), GFP-(rga-Δ17) (F), GFP-(rga-Δ17)∷pSLY1 number 4-1 (G), and GFP-(rga-Δ17)∷psly1-d number 5-1 (H). (I) The average final height of parental control lines and transgenic lines containing pSLY1 or psly1-d transgene. a, The total number of T1 transgenic or control plants measured.
Figure 4.
Figure 4.
sly1-d Reduces RGA and rga-Δ17 Protein Levels but Not the RGA mRNA. (A) and (B) Proteins or mRNA were extracted from homozygous lines as labeled, except that the sample for the rga-Δ17 line was extracted from a mix of hemizygous and homozygous plants. (A) The blots contain 25 μg of total proteins from 8-d-old seedlings after 2 h of treatment with water (−) or 100 μM GA3 (+) as labeled. Affinity-purified rabbit anti-RGA antibodies and a peroxidase-conjugated goat anti-rabbit IgG were used to detect the RGA (64-kD) and rga-Δ17 (62-kD) proteins (indicated by arrows). The blot at right was exposed for a shorter time than the blot at left because rga-Δ17 accumulates to a higher level than RGA in plants. (B) Relative RGA and SLY1 mRNA levels determined by quantitative RT-PCR. Total RNA was isolated from the wild type and various mutants after 100 μM GA3 or water treatment. The relative RGA and SLY1 mRNA levels were determined by running three quantitative RT-PCR reactions for each sample and normalized using the housekeeping gene GAPC. The value of water-treated wild type was arbitrarily set to 1.0. Bars = means ± se.
Figure 5.
Figure 5.
Interaction of DELLA Proteins with SLY1 and sly1-d through the GRAS Domain. (A) A schematic diagram showing the full-length RGA and GAI and the AD deletion constructs of GAI used in the yeast two-hybrid assay. The positions of conserved motifs (DELLA and VHYNP motif, poly Ser/Thr [polyS/T] sequence, nuclear localization signal [NL], and Leu heptad repeats [LR]) within the RGA and GAI full-length sequence are indicated. The numbers indicate the amino acid position at which the deletions start with regard to the full-length GAI sequence. (B) DELLA proteins interact with SLY1 and sly1-d in yeast two-hybrid assays. Interactions of DB and AD fusion proteins in the L40 yeast cells were scored for the relative growth on His− plates containing 3-AT (0 to 60 mM) and β-gal activity (means ± se). A dash indicates no growth on His− plates at 0 mM 3-AT. The fold change indicates the relative β-gal activity with the activity of the DB-SLY1/AD-RGA L40 strain arbitrarily set as 1.0. (C) Recombinant GST-SLY1, GST-(sly1-d), or GST was used in pull-down assays with lysates from sly1-10 and sly1-10 rga-24 leaves. The blot was probed with a rat anti-RGA antibody. The sly1-10 protein extract (2 μg) indicates the position of endogenous RGA on the blot. The asterisk indicates a nonspecific protein copurified from E. coli with GST-SLY and GST-(sly1-d), which is recognized by the RGA antibody. (D) rga-1 protein is insensitive to GA-induced destabilization in the rga-1 ga1-3 mutant, whereas RGA and rga-22 proteins are degraded after GA treatment. Immunoblots contain 50 μg of total protein extracted from tissues of 8-d-old plants after a 30 min treatment with water (−) or 0.5 μM GA4 (+) as labeled. Blots were probed with a rabbit anti-RGA antibody. The asterisk represents a nonspecific immunoreactive protein.
Figure 6.
Figure 6.
Nuclear Localization of the SLY1- and (sly1-d)-CFP Fusion Proteins in Onion Cells. Individual cells were imaged by differential interference contrast (DIC) and epifluorescence (fluor) microscopy. The CFP protein (control) is present in both the cytoplasm and the nucleus because of its small size.
Figure 7.
Figure 7.
An Updated Model of GA Signaling in Plants. GA induced phosphorylation (P) of DELLA proteins via an unidentified kinase and the SCFSLY1 complex interacts with the GRAS domain of DELLA proteins and targets their polyubiquitination ([Ub]n) and degradation via the ubiquitin-26S proteasome pathway. The novel sly1-d mutation results in an elevated affinity of SCFsly1-d with both wild-type and GA-resistant mutant DELLA proteins.
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