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.     

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.     

Blue light-dependent interactions of CRY1 with GID1 and DELLA proteins regulate gibberellin signaling and photomorphogenesis in Arabidopsis

Cryptochromes are blue light photoreceptors that mediate various light responses in plants and mammals. In Arabidopsis (Arabidopsis thaliana), cryptochrome 1 (CRY1) mediates blue light-induced photomorphogenesis, which is characterized by reduced hypocotyl elongation and enhanced anthocyanin production, whereas gibberellin (GA) signaling mediated by the GA receptor GA-INSENSITIVE DWARF1 (GID1) and DELLA proteins promotes hypocotyl elongation and inhibits anthocyanin accumulation. Whether CRY1 control of photomorphogenesis involves regulation of GA signaling is largely unknown. Here, we show that CRY1 signaling involves the inhibition of GA signaling through repression of GA-induced degradation of DELLA proteins. CRY1 physically interacts with DELLA proteins in a blue light-dependent manner, leading to their dissociation from SLEEPY1 (SLY1) and the inhibition of their ubiquitination. Moreover, CRY1 interacts directly with GID1 in a blue light-dependent but GA-independent manner, leading to the inhibition of the interaction between GID1 with DELLA proteins. These findings suggest that CRY1 controls photomorphogenesis through inhibition of GA-induced degradation of DELLA proteins and GA signaling, which is mediated by CRY1 inhibition of the interactions of DELLA proteins with GID1 and SCF^SLY1, respectively.

Blue light-dependent interactions of CRY1 with GID1 and DELLA proteins inhibit gibberellin (GA)-induced degradation of DELLA proteins to regulate GA signaling and photomorphogenesis.     

Functional analysis of SPINDLY in gibberellin signaling in Arabidopsis

The Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) protein negatively regulates the gibberellin (GA) signaling pathway. SPY is an O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) with a protein-protein interaction domain consisting of 10 tetratricopeptide repeats (TPR). OGTs add a GlcNAc monosaccharide to serine/threonine residues of nuclear and cytosolic proteins. Determination of the molecular defects in 14 new spy alleles reveals that these mutations cluster in three TPRs and the C-terminal catalytic region. Phenotypic characterization of 12 spy alleles indicates that TPRs 6, 8, and 9 and the catalytic domain are crucial for GA-regulated stem elongation, floral induction, and fertility. TPRs 8 and 9 and the catalytic region are also important for modulating trichome morphology and inflorescence phyllotaxy. Consistent with a role for SPY in embryo development, several alleles affect seedling cotyledon number. These results suggest that three of the TPRs and the OGT activity in SPY are required for its function in GA signal transduction. We also examined the effect of spy mutations on another negative regulator of GA signaling, REPRESSOR OF ga1-3 (RGA). The DELLA motif in RGA is essential for GA-induced proteolysis of RGA, and deletion of this motif (as in rga-delta17) causes a GA-insensitive dwarf phenotype. Here, we demonstrate that spy partially suppresses the rga-delta17 phenotype but does not reduce rga-delta17 or RGA protein levels or alter RGA nuclear localization. We propose that SPY may function as a negative regulator of GA response by increasing the activity of RGA, and presumably other DELLA proteins, by GlcNAc modification.     

New insights into gibberellin signaling in regulating flowering in Arabidopsis

In angiosperms,floral transition is a key developmental transition from the vegetative to reproductive growth,and requires precise regulation to maximize the reproductive success.A complex regulatory network governs this transition through integrating flowering pathways in response to multiple exogenous and endogenous cues.Phytohormones are essential for proper plant developmental regulation and have been extensively studied for their involvement in the floral transition.Among various phytohormones,gibberellin(GA)plays a major role in affecting flowering in the model plant Arabidopsis thaliana.The GA pathway interact with other flowering genetic pathways and phytohormone signaling pathways through either DELLA proteins or mediating GA homeostasis.In this review,we summarize the recent advances in understanding the mechanisms of DELLA-mediated GA pathway in flowering time control in Arabidopsis,and discuss its possible link with other phytohormone pathways during the floral transition.     

油菜素内酯、赤霉素与光共同调控拟南芥的细胞伸长和光形态建成

在植物的生长发育过程中,植物激素发挥着重要的作用. 最新研究对油菜素内酯、赤霉素两类植物激素与光的信号通路共同调控植物的细胞伸长和光形态建成的分子机制给予了精确的阐述,这也为提高农作物产量提拱了理论基础.     

Transcriptional regulation of gibberellin metabolism genes by auxin signaling in Arabidopsis

Auxin and gibberellins (GAs) overlap in the regulation of multiple aspects of plant development, such as root growth and organ expansion. This coincidence raises questions about whether these two hormones interact to regulate common targets and what type of interaction occurs in each case. Auxins induce GA biosynthesis in a range of plant species. We have undertaken a detailed analysis of the auxin regulation of expression of Arabidopsis (Arabidopsis thaliana) genes encoding GA 20-oxidases and GA 3-oxidases involved in GA biosynthesis, and GA 2-oxidases involved in GA inactivation. Our results show that auxin differentially up-regulates the expression of various genes involved in GA metabolism, in particular several AtGA20ox and AtGA2ox genes. Up-regulation occurred very quickly after auxin application; the response was mimicked by incubations with the protein synthesis inhibitor cycloheximide and was blocked by treatments with the proteasome inhibitor MG132. The effects of auxin treatment reflect endogenous regulation because equivalent changes in gene expression were observed in the auxin overproducer mutant yucca. The results suggest direct regulation of the expression of GA metabolism genes by Aux/IAA and ARF proteins. The physiological relevance of this regulation is supported by the observation that the phenotype of certain gain-of-function Aux/IAA alleles could be alleviated by GA application, which suggests that changes in GA metabolism mediate part of auxin action during development.     

Plant ubiquitin-proteasome pathway and its role in gibberellin signaling

The ubiquitin-proteasome system (UPS) in plants, like in other eukaryotes, targets numerous intracellular regulators and thus modulates almost every aspect of growth and development. The well-known and best-characterized outcome of ubiquitination is mediating target protein degradation via the 26S proteasome, which represents the major selective protein degradation pathway conserved among eukaryotes. In this review, we will discuss the molecular composition, regulation and function of plant UPS, with a major focus on how DELLA protein degradation acts as a key in gibberellin signal transduction and its implication in the regulation of plant growth.     

GID1-mediated gibberellin signaling in plants

Gibberellin (GA) perception is mediated by GID1 (GA-INSENSITIVE DWARF1), a receptor that shows similarity to hormone-sensitive lipases. A key event in GA signaling is the degradation of DELLA proteins, which are negative regulators of GA response that interact with GID1 in a GA-dependent manner. This GID1-DELLA GA-perception system is conserved among vascular plants but is not found in the moss Physcomitrella patens. The identification of factors in GA signaling downstream of DELLA and the development of a new concept of DELLA function beyond its role as a repressor of GA signaling are important advances. DELLA proteins appear to have at least two other distinct roles: maintaining GA homeostasis and regulating cross-talk between GA and other plant hormones.     

Distinct and overlapping roles of two gibberellin 3-oxidases in Arabidopsis development

Gibberellin (GA) 3-oxidase, a class of 2-oxoglutarate-dependent dioxygenases, catalyzes the conversion of precursor GAs to their bioactive forms, thereby playing a direct role in determining the levels of bioactive GAs in plants. Gibberellin 3-oxidase in Arabidopsis is encoded by a multigene family consisting of at least four members, designated AtGA3ox1 to AtGA3ox4. It has yet to be investigated how each AtGA3ox gene contributes to optimizing bioactive GA levels during growth and development. Using quantitative real-time PCR analysis, we have shown that each AtGA3ox gene exhibits a unique organ-specific expression pattern, suggesting distinct developmental roles played by individual AtGA3ox members. To investigate the sites of synthesis of bioactive GA in plants, we generated transgenic Arabidopsis that carried AtGA3ox1-GUS and AtGA3ox2-GUS fusions. Comparisons of the GUS staining patterns of these plants with that of AtCPS-GUS from previous studies revealed the possible physical separation of the early and late stages of the GA pathway in roots. Phenotypic characterization and quantitative analysis of the endogenous GA content of ga3ox1 and ga3ox2 single and ga3ox1/ga3ox2 double mutants revealed distinct as well as overlapping roles of AtGA3ox1 and AtGA3ox2 in Arabidopsis development. Our results show that AtGA3ox1 and AtGA3ox2 are responsible for the synthesis of bioactive GAs during vegetative growth, but that they are dispensable for reproductive development. The stage-specific severe GA-deficient phenotypes of the ga3ox1/ga3ox2 mutant suggest that AtGA3ox3 and AtGA3ox4 are tightly regulated by developmental cues; AtGA3ox3 and AtGA3ox4 are not upregulated to compensate for GA deficiency during vegetative growth of the double mutant.     

The Arabidopsis SLEEPY1 gene encodes a putative F-box subunit of an SCF E3 ubiquitin ligase

The Arabidopsis SLY1 (SLEEPY1) gene positively regulates gibberellin (GA) signaling. Positional cloning of SLY1 revealed that it encodes a putative F-box protein. This result suggests that SLY1 is the F-box subunit of an SCF E3 ubiquitin ligase that regulates GA responses. The DELLA domain protein RGA (repressor of ga1-3) is a repressor of GA response that appears to undergo GA-stimulated protein degradation. RGA is a potential substrate of SLY1, because sly1 mutations cause a significant increase in RGA protein accumulation even after GA treatment. This result suggests SCF(SLY1)-targeted degradation of RGA through the 26S proteasome pathway. Further support for this model is provided by the observation that an rga null allele partially suppresses the sly1-10 mutant phenotype. The predicted SLY1 amino acid sequence is highly conserved among plants, indicating a key role in GA response.     

Synergistic derepression of gibberellin signaling by removing RGA and GAI function in Arabidopsis thaliana

RGA and GAI are negative regulators of the gibberellin (GA) signal transduction pathway in Arabidopsis thaliana. These genes may have partially redundant functions because they are highly homologous, and plants containing single null mutations at these loci are phenotypically similar to wild type. Previously, rga loss-of-function mutations were shown to partially suppress defects of the GA-deficient ga1-3 mutant. Phenotypes rescued include abaxial trichome initiation, rosette radius, flowering time, stem elongation, and apical dominance. Here we present work showing that the rga-24 and gai-t6 null mutations have a synergistic effect on plant growth. Although gai-t6 alone has little effect, when combined with rga-24, they completely rescued the above defects of ga1-3 to wild-type or GA-overdose phenotype. However, seed germination and flower development defects were not restored. Additionally, rga-24 and rga-24/gai-t6 but not gai-t6 alone caused increased feedback inhibition of expression of a GA biosynthetic gene in both the ga1-3 and wild-type backgrounds. These results demonstrate that RGA and GAI have partially redundant functions in maintaining the repressive state of the GA-signaling pathway, but RGA plays a more dominant role than GAI. Removing both RGA and GAI function allows for complete derepression of many aspects of GA signaling.     

Cross talk between gibberellin and cytokinin: the Arabidopsis GA response inhibitor SPINDLY plays a positive role in cytokinin signaling

SPINDLY (SPY) is a negative regulator of gibberellin (GA) responses; however, spy mutants exhibit various phenotypic alterations not found in GA-treated plants. Assaying for additional roles for SPY revealed that spy mutants are resistant to exogenously applied cytokinin. GA also repressed the effects of cytokinin, suggesting that there is cross talk between the two hormone-response pathways, which may involve SPY function. Two spy alleles showing severe (spy-4) and mild (spy-3) GA-associated phenotypes exhibited similar resistance to cytokinin, suggesting that SPY enhances cytokinin responses and inhibits GA signaling through distinct mechanisms. GA and spy repressed numerous cytokinin responses, from seedling development to senescence, indicating that cross talk occurs early in the cytokinin-signaling pathway. Because GA3 and spy-4 inhibited induction of the cytokinin primary-response gene, type-A Arabidopsis response regulator 5, SPY may interact with and modify elements from the phosphorelay cascade of the cytokinin signal transduction pathway. Cytokinin, on the other hand, had no effect on GA biosynthesis or responses. Our results demonstrate that SPY acts as both a repressor of GA responses and a positive regulator of cytokinin signaling. Hence, SPY may play a central role in the regulation of GA/cytokinin cross talk during plant development.     

Direct interaction between the Arabidopsis disease resistance signaling proteins, EDS1 and PAD4

The Arabidopsis EDS1 and PAD4 genes encode lipase-like proteins that function in resistance (R) gene-mediated and basal plant disease resistance. Phenotypic analysis of eds1 and pad4 null mutants shows that EDS1 and PAD4 are required for resistance conditioned by the same spectrum of R genes but fulfil distinct roles within the defence pathway. EDS1 is essential for elaboration of the plant hypersensitive response, whereas EDS1 and PAD4 are both required for accumulation of the plant defence-potentiating molecule, salicylic acid. EDS1 is necessary for pathogen-induced PAD4 mRNA accumulation, whereas mutations in PAD4 or depletion of salicylic acid only partially compromise EDS1 expression. Yeast two-hybrid analysis reveals that EDS1 can dimerize and interact with PAD4. However, EDS1 dimerization is mediated by different domains to those involved in EDS1-PAD4 association. Co-immunoprecipitation experiments show that EDS1 and PAD4 proteins interact in healthy and pathogen-challenged plant cells. We propose two functions for EDS1. The first is required early in plant defence, independently of PAD4. The second recruits PAD4 in the amplification of defences, possibly by direct EDS1-PAD4 association.     

Role of Arabidopsis BBX proteins in light signaling

Journal of Plant Biochemistry and Biotechnology - Light regulates numerous aspects of plant growth and development like seed germination, seedling de-etiolation, pigment accumulation, cotyledon...