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

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.     

Proteolysis-independent downregulation of DELLA repression in Arabidopsis by the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1

This article presents evidence that DELLA repression of gibberellin (GA) signaling is relieved both by proteolysis-dependent and -independent pathways in Arabidopsis thaliana. DELLA proteins are negative regulators of GA responses, including seed germination, stem elongation, and fertility. GA stimulates GA responses by causing DELLA repressor degradation via the ubiquitin-proteasome pathway. DELLA degradation requires GA biosynthesis, three functionally redundant GA receptors GIBBERELLIN INSENSITIVE DWARF1 (GID1a, b, and c), and the SLEEPY1 (SLY1) F-box subunit of an SCF E3 ubiquitin ligase. The sly1 mutants accumulate more DELLA proteins but display less severe dwarf and germination phenotypes than the GA biosynthesis mutant ga1-3 or the gid1abc triple mutant. Interestingly, GID1 overexpression rescued the sly1 dwarf and infertility phenotypes without decreasing the accumulation of the DELLA protein REPRESSOR OF ga1-3. GID1 rescue of sly1 mutants was dependent on the level of GID1 protein, GA, and the presence of a functional DELLA motif. Since DELLA shows increasing interaction with GID1 with increasing GA levels, it appears that GA-bound GID1 can block DELLA repressor activity by direct protein-protein interaction with the DELLA domain. Thus, a SLY1-independent mechanism for GA signaling may function without DELLA degradation.     

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

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.     

Seed germination of GA-insensitive sleepy1 mutants does not require RGL2 protein disappearance in Arabidopsis

We explore the roles of gibberellin (GA) signaling genes SLEEPY1 (SLY1) and RGA-LIKE2 (RGL2) in regulation of seed germination in Arabidopsis thaliana, a plant in which the hormone GA is required for seed germination. Seed germination failure in the GA biosynthesis mutant ga1-3 is rescued by GA and by mutations in the DELLA gene RGL2, suggesting that RGL2 represses seed germination. RGL2 protein disappears before wild-type seed germination, consistent with the model that GA stimulates germination by causing the SCF(SLY1) E3 ubiquitin ligase complex to trigger ubiquitination and destruction of RGL2. Unlike ga1-3, the GA-insensitive sly1 mutants show variable seed dormancy. Seed lots with high seed dormancy after-ripened slowly, with stronger alleles requiring more time. We expected that if RGL2 negatively controls seed germination, sly1 mutant seeds that germinate well should accumulate lower RGL2 levels than those failing to germinate. Surprisingly, RGL2 accumulated at high levels even in after-ripened sly1 mutant seeds with 100% germination, suggesting that RGL2 disappearance is not a prerequisite for seed germination in the sly1 background. Without GA, several GA-induced genes show increased accumulation in sly1 seeds compared with ga1-3. It is possible that the RGL2 repressor of seed germination is inactivated by after-ripening of sly1 mutant seeds.     

Mutations in the F-box gene SNEEZY result in decreased Arabidopsis GA signaling

We previously reported that the SLEEPY1 (SLY1) homolog, F-box gene SNEEZY/SLEEPY2 (SNE/SLY2), can partly replace SLY1 in gibberellin (GA) hormone signaling through interaction with DELLAs RGA and GAI. To determine whether SNE normally functions in GA signaling, we characterized the phenotypes of two T-DNA alleles, sne-t2 and sne-t3. These mutations result in no apparent vegetative phenotypes, but do result in increased ABA sensitivity in seed germination. Double mutants sly1-t2 sne-t2 and sly1-t2 sne-t3 result in a significant decrease in plant fertility and final plant height compared to sly1-t2. The fact that sne mutations have an additive effect with sly1 suggests that SNE normally functions as a redundant positive regulator of GA signaling.Key words: gibberellin signaling, GA, SLEEPY1, SNEEZY, DELLA, F-box proteinThis paper describes genetic evidence that the SLEEPY1 (SLY1) homolog SNEEZY/SLEEPY2 (SNE/SLY2) functions redundantly with SLY1 to stimulate gibberellin signaling. GA responses such as seed germination, stem elongation and fertility are promoted by proteolysis of DELLA proteins, negative regulators of the GA signaling.¹ In the classic GA signaling model, GA binding to the GA receptor GID1 increases GID1 affinity for DELLA protein. This GID1-GA binding to DELLA causes SLY1, the F-box subunit of an SCF E3 ubiquitin ligase complex, to recognize, bind and ubiquitinate DELLA proteins thereby targeting them for destruction by the 26S proteasome. Thus, loss of SLY1 function results in decreased GA responses, causing dwarfism, delayed flowering, infertility and seed dormancy. The sly1 mutants over-accumulate DELLA proteins due to failure to destroy them through the ubiquitin-proteasome pathway.Overexpression of the SLY1 homolog, SNE, partially rescues the germination, dwarfism and infertility of the sly1-10 mutant.^2–4 SNE overexpression in the sly1-10 background is associated with reduced accumulation of DELLA proteins RGA and GAI, but not of DELLA RGL2. Co-immunoprecipitation assays demonstrated that SNE directly binds RGA protein as well as the cullin subunit of the SCF E3 complex. These recently published data suggest that SNE forms a functional SCF E3 ubiquitin ligase complex that negatively regulates a subset of the DELLA proteins regulated by SLY1.⁴The finding that SNE overexpression rescues sly1-10 phenotypes through down-regulation of DELLA RGA and GAI suggests that SNE is normally a positive regulator of GA signaling. If this is true, then we expect sne mutations to cause phenotypes resulting from reduced GA response including reduced germination, stature and fertility. To examine this hypothesis, three sne T-DNA mutants were identified: sne-t1, sne-t2 and sne-t3. The sne-t1 allele is a SALK line containing a T-DNA insertion 183-bp upstream of the coding region.⁵ This line showed no apparent phenotype and was not further characterized. The sne-t2 allele is a Sussman T-DNA line^4,6 containing a T-DNA insertion immediately before the ATG that is the SNE translational start codon (Fig. 1A). While this insertion does not disrupt the coding region, it likely disrupts SNE protein translation as the T-DNA contains multiple stop codons. The sne-t3 allele contains a T-DNA insertion within the SNE ORF before amino acid 146 of the 157 amino acid predicted protein (FLAG_461E03).^7,8 This allele should result in loss of the last 11 SNE amino acids. We know that loss of the last 8 SLY1 amino acids in sly1-10 results in dwarfism, suggesting that loss of the last 11 SNE amino acids may also cause some loss of function in the small F-box protein. When the homozygous sne-t2 and sne-t3 lines were compared to wild-type Ws, no change was observed either in final plant height or fertility measured in seeds/silique (Fig. 1B). An ABA dose-response curve in seed germination detected a small but reproducible increase in ABA sensitivity during seed germination of sne-t2 and sne-t3 (Fig. 1C). The fact that the sne-t2 and sne-t3 mutants, like sly1-2 and sly1-10, show increased ABA sensitivity suggests that SNE and SLY1 may have similar functions in GA signaling during seed germination.²Open in a separate windowFigure 1The phenotypes of sne-t2 and sne-t3 T-DNA mutants. (A) Schematic diagram of the sne-t2 T-DNA insertion at position −1 bp and of sne-t3 at position +435 bp with respect to the translation start site. (B) Final plant height (upper) and fertility (lower) of indicated genotypes. Letters indicate statistically different classes as determine by t-test. Bars represent standard error. (C) sne mutants show increase in ABA sensitivity. Seeds of wild-type Ws, sne-t2 and sne-t3 were after-ripened for 2 weeks then sown on MS-agar containing indicated concentrations of ABA as described by Steber et al.¹¹ Germination was scored based on radical emergence after incubating 3 days at 4°C followed by 14 days at 22°C. (D) Mutations in SNE cause no significant effect on DELLA RGA, GAI and RGL2 protein accumulation. Total protein was extracted from leaves of 12-d-old seedlings (Top) or flower buds (FB, bottom) and detected as described in Ariizumi et al.⁴One possible explanation for the lack of apparent GA-insensitive phenotypes in sne T-DNA insertion lines, is that SNE function is redundant with SLY1 in GA signaling.⁹ If so, we would expect sly1 sne double mutants to show stronger GA-insensitive phenotypes than the sly1 single mutation. Double mutants were constructed containing either the sne-t2 or sne-t3 mutation in the sly-t2 null background. The sly1-t2 allele was chosen because sly1-t2, sne-t2 and sne-t3 are all in the Ws ecotype. The sly1-t2 allele contains a T-DNA insertion within the F-box domain resulting in severe GA-insensitive phenotypes including failure to germinate, reduced stature and infertility.¹⁰ The sly1-t2 sne-t2 and sly1-t2 sne-t3 double mutants showed a small but significant decrease in final plant height and fertility (seeds/silique) compared to sly1-t2 (Fig. 1B). This increase in phenotype severity was not associated with an apparent increase in DELLA RGA, GAI or RGL2 protein accumulation (Fig. 1D). It could be that DELLA protein levels in sly1-t2 are so high that any slight increase due to sne mutations is undetectable. Our previous study of SNE overexpression lines showed that SNE has the ability to downregulate RGA and GAI protein accumulation. Figure 1 shows that the chromosomal SNE gene contributes to GA signaling presumably through ubiquitination of DELLA protein.Taken together, the fact that sne mutants show only mild GA-insensitive phenotypes and that the natural SNE expression cannot compensate for lack of SLY1, indicate that SLY1 is the main E3 ubiquitin ligase stimulating GA signaling (this study, reviewed in ref. ⁴). We cannot rule out the possibility that stronger SNE alleles would show either stronger GA response phenotypes or phenotypes that are unrelated to GA signaling. Indeed, there is evidence to suggest that SNE may have unique functions. The sne-t3 (sne-1) allele results in a shortened root phenotype.⁸ That SNE is expressed in the endodermis and quiescent center of the root whereas SLY1 is expressed in the stele, suggests that SNE may function independently in the root.⁸ Moreover, SNE overexpression, but not SLY1 overexpression, results in decreased apical dominance and a prone growth habit suggesting that SNE may play a unique role in development.^2,4 Our model is that in addition to regulating DELLA proteins RGA and GAI, SNE may also regulate a yet unidentified target involved in apical dominance (Fig. 2). Future research will need to elucidate the role of SNE in Arabidopsis growth and development.Open in a separate windowFigure 2Model for SNE function in Arabidopsis. Both SLY1 and SNE act as positive regulators of GA responses via DELLA protein destruction. SNE may negatively regulate an unknown protein that maintains apical dominance.     

Understanding gibberellic acid signaling--are we there yet?

The phytohormone gibberellic acid (GA) controls important aspects of plant growth such as seed germination, elongation growth, and flowering. The key components of the GA signaling pathway have been identified over the past 10 years. The current view is that GA binds to a soluble GID1 receptor, which interacts with the DELLA repressor proteins in a GA-dependent manner and thereby induces DELLA protein degradation via the E3 ubiquitin ligase SCF(GID2/SLY1). GA-dependent growth responses can generally be correlated with and be explained by changes in DELLA repressor abundance, where the DELLA repressor exerts a growth restraint that is relieved upon its degradation. However, it is obvious that other mechanisms must exist that control the activity of this pathway. This review discusses recent advances in the understanding of GA signaling, of its homeostasis, and of its cross-talk with other signaling pathways.     

The Arabidopsis mutant sleepy1gar2-1 protein promotes plant growth by increasing the affinity of the SCFSLY1 E3 ubiquitin ligase for DELLA protein substrates

DELLA proteins restrain the cell proliferation and enlargement that characterizes the growth of plant organs. Gibberellin stimulates growth via 26S proteasome-dependent destruction of DELLAs, thus relieving DELLA-mediated growth restraint. Here, we show that the Arabidopsis thaliana sleepy1gar2-1 (sly1gar2-1) mutant allele encodes a mutant subunit (sly1gar2-1) of an SCF(SLY1) E3 ubiquitin ligase complex. SLY1 (the wild-type form) and sly1gar2-1 both confer substrate specificity on this complex via specific binding to the DELLA proteins. However, sly1gar2-1 interacts more strongly with the DELLA target than does SLY1. In addition, the strength of the SCFSLY1-DELLA interaction is increased by target phosphorylation. Growth-promoting DELLA destruction is dependent on SLY1 availability, on the strength of the interaction between SLY1 and the DELLA target, and on promotion of the SCFSLY1-DELLA interaction by DELLA phosphorylation.     

Geminivirus C4 proteins inhibit GA signaling via prevention of NbGAI degradation,to promote viral infection and symptom development in N. benthamiana

The phytohormone gibberellin (GA) is a vital plant signaling molecule that regulates plant growth and defense against abiotic and biotic stresses. To date, the molecular mechanism of the plant responses to viral infection mediated by GA is still undetermined. DELLA is a repressor of GA signaling and is recognized by the F-box protein, a component of the SCF^SLY1/GID2 complex. The recognized DELLA is degraded by the ubiquitin-26S proteasome, leading to the activation of GA signaling. Here, we report that ageratum leaf curl Sichuan virus (ALCScV)-infected N. benthamiana plants showed dwarfing symptoms and abnormal flower development. The infection by ALCScV significantly altered the expression of GA pathway-related genes and decreased the content of endogenous GA in N. benthamiana. Furthermore, ALCScV-encoded C4 protein interacts with the DELLA protein NbGAI and interferes with the interaction between NbGAI and NbGID2 to prevent the degradation of NbGAI, leading to inhibition of the GA signaling pathway. Silencing of NbGAI or exogenous GA₃ treatment significantly reduces viral accumulation and disease symptoms in N. benthamiana plants. The same results were obtained from experiments with the C4 protein encoded by tobacco curly shoot virus (TbCSV). Therefore, we propose a novel mechanism by which geminivirus C4 proteins control viral infection and disease symptom development by interfering with the GA signaling pathway.     

Inter- and intra-molecular interactions of Arabidopsis thaliana DELLA protein RGL1

The phytohormone gibberellin and the DELLA proteins act together to control key aspects of plant development. Gibberellin induces degradation of DELLA proteins by recruitment of an F-box protein using a molecular switch: a gibberellin-bound nuclear receptor interacts with the N-terminal domain of DELLA proteins, and this event primes the DELLA C-terminal domain for interaction with the F-box protein. However, the mechanism of signalling between the N- and C-terminal domains of DELLA proteins is unresolved. In the present study, we used in vivo and in vitro approaches to characterize di- and tri-partite interactions of the DELLA protein RGL1 (REPRESSOR OF GA1-3-LIKE 1) of Arabidopsis thaliana with the gibberellin receptor GID1A (GIBBERELLIC ACID-INSENSITIVE DWARF-1A) and the F-box protein SLY1 (SLEEPY1). Deuterium-exchange MS unequivocally showed that the entire N-terminal domain of RGL1 is disordered prior to interaction with the GID1A; furthermore, association/dissociation kinetics, determined by surface plasmon resonance, predicts a two-state conformational change of the RGL1 N-terminal domain upon interaction with GID1A. Additionally, competition assays with monoclonal antibodies revealed that contacts mediated by the short helix Asp-Glu-Leu-Leu of the hallmark DELLA motif are not essential for the GID1A-RGL1 N-terminal domain interaction. Finally, yeast two- and three-hybrid experiments determined that unabated communication between N- and C-terminal domains of RGL1 is required for recruitment of the F-box protein SLY1.     

猪链球菌2型溶血素基因的表达及其免疫原性

【背景】猪链球菌(Streptococcus suis,SS)是一种重要的人兽共患病原菌,有35种血清型,其中以猪链球菌2型(SS2)危害最为严重。研究发现,各种血清型SS分泌的溶血素(Suilysin,SLY)可能具有较好的免疫保护作用,因此,SLY作为SS基因工程亚单位疫苗的成分具有较大优势。【目的】获得SS2安徽强毒株(AH10-8株)溶血素基因(sly)的表达产物,并对其免疫原性进行测定分析。【方法】根据GenBank数据库中登录号为DQ443533.1的全长sly序列,设计合成一对分别带有Bam HI、XhoI酶切位点的特异性引物,利用PCR从AH10-8株中扩增sly基因,构建pET-30a-sly原核表达重组质粒,将重组质粒转化大肠杆菌BL21(DE3)中,进行IPTG诱导表达和His-Tag镍柱纯化蛋白,SDS-PAGE检测SLY,Western blotting鉴定SLY的反应原性;利用昆明鼠和斑马鱼进行SLY免疫攻毒保护试验,间接ELISA方法检测昆明鼠血清IgG抗体效价,观察比较病理组织变化及其免疫保护率。【结果】重组质粒pET-30a-sly在大肠杆菌中实现高效表达,获得大小为60kD的目的蛋白,与预期大小的SLY分子质量一致,能与SS2阳性血清发生特异性反应。SLY和AH10-8株灭活全菌体3次免疫昆明鼠后的血清IgG抗体效价分别为1:6 400、1:204 800 (以AH10-8株全菌体的超声裂解物为包被抗原)和1:102 400、1:51 200 (以SLY为包被抗原);SLY和AH10-8株灭活全菌体对昆明鼠和斑马鱼的免疫保护率分别为40%、80%和84%、92%;病理组织变化与攻毒对照组之间差异明显。【结论】成功表达的SLY具有良好的反应原性和免疫原性,可诱导机体产生保护性免疫反应,有望成为研制SS2新型疫苗的候选成分。     

The yeast SLY gene products, suppressors of defects in the essential GTP-binding Ypt1 protein, may act in endoplasmic reticulum-to-Golgi transport.

It has been shown previously that defects in the essential GTP-binding protein, Ypt1p, lead to a block in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus in the yeast Saccharomyces cerevisiae. Here we report that four newly discovered suppressors of YPT1 deletion (SLY1-20, SLY2, SLY12, and SLY41) to a varying degree restore ER-to-Golgi transport defects in cells lacking Ypt1p. These suppressors also partially complement the sec21-1 and sec22-3 mutants which lead to a defect early in the secretory pathway. Sly1p-depleted cells, as well as a conditional lethal sly2 null mutant at nonpermissive temperatures, accumulate ER membranes and core-glycosylated invertase and carboxypeptidase Y. The sly2 null mutant under restrictive conditions (37 degrees C) can be rescued by the multicopy suppressor SLY12 and the single-copy suppressor SLY1-20, indicating that these three SLY genes functionally interact. Sly2p is shown to be an integral membrane protein.     

Release of the repressive activity of rice DELLA protein SLR1 by gibberellin does not require SLR1 degradation in the gid2 mutant

The rice (Oryza sativa) DELLA protein SLR1 acts as a repressor of gibberellin (GA) signaling. GA perception by GID1 causes SLR1 protein degradation involving the F-box protein GID2; this triggers GA-associated responses such as shoot elongation and seed germination. In GA-insensitive and GA biosynthesis mutants, SLENDER RICE1 (SLR1) accumulates to high levels, and the severity of dwarfism is usually correlated with the level of SLR1 accumulation. An exception is the GA-insensitive F-box mutant gid2, which shows milder dwarfism than mutants such as gid1 and cps even though it accumulates higher levels of SLR1. The level of SLR1 protein in gid2 was decreased by loss of GID1 function or treatment with a GA biosynthesis inhibitor, and dwarfism was enhanced. Conversely, overproduction of GID1 or treatment with GA(3) increased the SLR1 level in gid2 and reduced dwarfism. These results indicate that derepression of SLR1 repressive activity can be accomplished by GA and GID1 alone and does not require F-box (GID2) function. Evidence for GA signaling without GID2 was also provided by the expression behavior of GA-regulated genes such as GA-20oxidase1, GID1, and SLR1 in the gid2 mutant. Based on these observations, we propose a model for the release of GA suppression that does not require DELLA protein degradation.     

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.