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First published online November 20, 2002; 10.1105/tpc.006197 American Society of Plant Biologists Gibberellin-Mediated Proteasome-Dependent Degradation of the Barley DELLA Protein SLN1 Repressor
a John Innes Centre, Colney Lane, Norwich NR4 7UJ, United Kingdom 3 To whom correspondence should be addressed. E-mail nicholas.harberd{at}bbsrc.ac.uk; fax 44-1603-450025
DELLA proteins are nuclear repressors of plant gibberellin (GA) responses. Here, we investigate the properties of SLN1, a DELLA protein from barley that is destabilized by GA treatment. Using specific inhibitors of proteasome function, we show that proteasome-mediated protein degradation is necessary for GA-mediated destabilization of SLN1. We also show that GA responses, such as the aleurone  -amylase response and seedling leaf extension growth, require proteasome-dependent GA-mediated SLN1 destabilization. In further experiments with protein kinase and protein phosphatase inhibitors, we identify two additional signaling steps that are necessary for GA response and for GA-mediated destabilization of SLN1. Thus, GA signaling involves protein phosphorylation and dephosphorylation steps and promotes the derepression of GA responses via proteasome-dependent destabilization of DELLA repressors.
Bioactive gibberellins (GAs) are essential regulators of plant growth and development (Hooley, 1994  ). For example, during the germination of cereal grains, GA is synthesized by the embryo and secreted into the aleurone. In this situation, GA regulates the synthesis and secretion of hydrolyzing enzymes (such as -amylase) into the endosperm. The hydrolyzing enzymes then catalyze the breakdown of endosperm storage macromolecules, releasing nutrients that are used by the establishing seedling (Bethke et al., 1997; Ritchie and Gilroy, 1998; Lovegrove and Hooley, 2000).
GA is thought to elicit GA responses in the following manner. First, GA appears to be perceived on the surface of plant cells by an unidentified outward-facing plasma membrane–associated GA receptor (Hooley et al., 1991
Mutants of wheat, barley, and rice that are affected in GA signaling display an altered aleurone
Wheat Rht-B1a and Rht-D1a, rice SLR1, and barley SLN1 encode proteins orthologous with Arabidopsis GAI, a member of the GRAS family of putative transcriptional regulators (Peng et al., 1997
Recent studies using DELLA proteins fused to the green fluorescent protein have shown that RGA, SLR1, and SLN1 accumulate in the nucleus of plant cells and that treatment with exogenous GA causes the disappearance of these proteins from the nucleus (Silverstone et al., 2001
Here, we describe the molecular analysis of the barley SLN1 gene and the mechanism by which its product (SLN1) mediates barley GA responses. We investigated the mechanism of GA-induced SLN1 destabilization by studying the effects of a number of different inhibitory compounds on this process. In particular, we show that specific inhibitors of 26S proteasome function block both the GA-mediated destabilization of SLN1 and GA responses (the aleurone
Molecular Characterization of the Barley sln1-1 Mutant Allele As shown in Figure 1A , recessive mutations at SLN1 (e.g., sln1-1) confer exaggerated elongation growth of barley seedlings. This phenotype persists throughout the development of the plant, resulting in adult plants that are taller than wild-type plants, with thin, pale green leaves and sterile flowers (the "slender" phenotype) (Foster, 1977 ; Chandler, 1988; Lanahan and Ho, 1988). In addition, the growth of sln1-1 mutants is resistant to the growth-inhibitory effects of the GA biosynthesis inhibitor paclobutrazol, suggesting that SLN1 encodes a repressor of GA responses and that loss-of-function mutations at SLN1 confer a constitutive GA response (Chandler, 1988; Lanahan and Ho, 1988).
Because mutations at SLN1 confer altered GA responses, we reasoned that SLN1 might be a barley ortholog of the GAI/RGA/d8/Rht-B1a/Rht-D1a/SLR1 genes (genes that encode DELLA proteins from a variety of species) (Chandler et al., 2002 ; Gubler et al., 2002). Therefore, we amplified SLN1 using PCR primers derived from the wheat Rht-D1a sequence (see Methods). The predicted amino acid sequence of the protein encoded by the amplified SLN1 was identical to that described previously (Chandler et al., 2002; Gubler et al., 2002; X. Fu and D.E. Richards, unpublished data). We also amplified SLN1 from the sln1-1 mutant (see Methods) (Figure 1A). DNA sequencing showed that the sln1-1 mutation is a single nucleotide substitution (GAG to TAG) that converts the codon encoding Glu-250 to a stop codon (Figure 1B).
Immunoblot analysis showed that sln1-1 plants lack detectable SLN1 protein. As shown in Figure 1C, the shoots of SLN1 seedlings germinated and grown in the presence of exogenous GA3 were longer than those germinated in water, whereas there was no effect of GA on sln1-1 seedlings. Total proteins extracted from these seedlings were electrophoretically fractionated and analyzed using anti-GAI antibodies. These experiments identified an
GA-Induced Disappearance of SLN1 Is Unaffected by the Protease Inhibitors Pefabloc SC, Aprotinin, and Phenylmethylsulfonyl Fluoride
GA-Induced Disappearance of SLN1 Is Affected by Proteasome Inhibitors In contrast with the results reported above, the GA-induced disappearance of SLN1 was affected by the addition of five different cell-permeable proteasome-specific inhibitors: MG115, MG132, proteasome inhibitor I, proteasome inhibitor II, and lactacystin (for details of inhibitors, see Methods). As shown in Figure 2, extracts from SLN1 seedlings treated with both GA and MG115, MG132, or proteasome inhibitor I contained detectable levels of SLN1. Similar results were obtained with proteasome inhibitor II and lactacystin and from samples treated with GA and proteasome inhibitors for 30 min or 24 h (data not shown). These results show that proteasome inhibitors can prevent the GA-induced disappearance of SLN1, suggesting that this disappearance might be attributable to proteasome-mediated degradation.
GA-Regulated Seedling Leaf Extension Growth Is Dependent on Proteasome-Mediated SLN1 Degradation
-Amylase Induction in Aleurone Cells Is Dependent on Proteasome-Mediated SLN1 DegradationGA-mediated destruction of SLN1 in aleurone cells is associated with the GA induction of -amylase (Gubler et al., 2002). Therefore, we examined the effects of a range of inhibitors on the -amylase responses of de-embryonated SLN1 and sln1-1 half-grains (see Methods). As described previously, sln1-1 half-grains produce comparable amounts of -amylase activity in the presence or absence of GA (Chandler, 1988; Lanahan and Ho, 1988) (Table 1). None of the inhibitors tested (Pefabloc SC, MG115, MG132, and proteasome inhibitor I) affected the production of -amylase by sln1-1 half-grains in the presence or absence of GA (Table 1). By contrast, the proteasome inhibitors MG115 and MG132 largely blocked the GA induction of -amylase activity from SLN1 half-grains (Table 1). The fact that MG115 and MG132 blocked the -amylase response in SLN1 half-grains but did not inhibit the -amylase production of sln1-1 half-grains shows that the observed effects of these inhibitors on GA responses is not attributable to nonspecific effects or the poisoning of cellular metabolism. Rather, MG115 and MG132 block the -amylase response of SLN1 half-grains by inhibiting proteasome activity, and proteasome-dependent degradation of SLN1 is necessary for the induction of -amylase activity.
Protein Kinase and Phosphatase Inhibitors Block GA-Induced SLN1 Protein Degradation and -Amylase ProductionTo identify additional steps in the GA signal transduction pathway, we tested the effects of various protein phosphorylation and dephosphorylation inhibitors on GA-induced SLN1 degradation. Previous experiments have shown that the Ser/Thr protein phosphatase inhibitor okadaic acid (OA) is effective at blocking the GA-induced production of -amy-lase by wheat aleurone cells (Kuo et al., 1996). We examined the effects of OA and sodium vanadate (SV), a widely used general inhibitor of protein phosphatases (for details of these inhibitors, see Methods), on the GA-induced degradation of SLN1. SLN1 was detected in extracts from SLN1 seedlings treated with water or with GA and either OA or SV for 2 h (Figure 4A)
or 24 h (data not shown) and was not detected in GA-only controls. Thus, treatment with OA or SV blocked the GA-induced degradation of SLN1. We also showed that both OA and SV blocked the GA-mediated induction of -amylase activity in SLN1 half-grains but did not block the constitutive production of -amylase by sln1-1 half-grains (Table 1). These results show that OA and SV affect GA responses by perturbing the signaling chain associated with SLN1 (or they affect SLN1 itself), making SLN1 resistant to GA-mediated destabilization.
Staurosporine is a broad-range inhibitor of Ser/Thr protein kinases, whereas protein phosphatase 2 (PP2) is a selective protein kinase inhibitor (see Methods). Treatment with either staurosporine or PP2 failed to block the GA-induced degradation of SLN1 or the production of -amylase activity (Figure 4B, Table1). By contrast, treatment with two protein Tyr kinase inhibitors, genistein (a broad-range protein kinase inhibitor) and Tyrophostin B46 (AG555; for details, see Methods), blocked the GA-induced degradation of SLN1 (Figure 4B). Figure 4 shows extracts from seedlings treated for 2 h; similar results were obtained for each inhibitor after treatments for 30 min and 24 h (data not shown). Genistein and Tyrophostin B46 also blocked GA-induced -amylase production in SLN1 half-grains but did not block constitutive -amylase production in sln1-1 half-grains (Table 1). Together, the results described here suggest that protein kinases and protein phosphatases mediate GA-induced degradation of SLN1, thus eliciting GA responses.
Here, we show that several well-defined, cell-permeable inhibitors of proteasome function can block the GA-induced disappearance of SLN1. Thus, proteasome function is necessary for the destabilization of SLN1 in response to the GA signal. Furthermore, we show that two GA responses, leaf extension growth and the aleurone -amylase response, are blocked by proteasome inhibitors. Our results suggest that GA induces GA responses in barley via proteasome-dependent degradation of SLN1.
To identify additional steps in the GA signaling pathway, we tested the effects of a range of protease, kinase, and phosphatase inhibitors on GA responses and on the GA-mediated destabilization of SLN1. None of these inhibitors (and none of the proteasome inhibitors described above) inhibited the production of
We identified two additional steps in GA signaling. First, we showed that protein phosphorylation inhibitors can block GA responses and the GA-mediated destabilization of SLN1, implying a protein phosphorylation step in GA signaling. The inhibitors that were effective at blocking GA responses and SLN1 destabilization are Tyr kinase inhibitors, making it possible that phosphotyrosine is involved in GA signaling. It has been suggested previously that the DELLA proteins are structurally, and perhaps functionally, related to the STAT proteins (which signal via Tyr phosphorylation) (Peng et al., 1999
We propose the following model to explain the phenomena reported in this article (Figure 5)
. First, GA interacts with an unknown plasma membrane–associated specific receptor (Hooley et al., 1991
It has become apparent that targeted proteasome-mediated protein degradation is crucial to many signaling pathways in plants. For example, the accumulation of AUX/IAA proteins is key to auxin signaling (Rouse et al., 1998 ). AUX/IAA accumulation is regulated via auxin-mediated targeting of AUX/IAAs for destruction via the proteasome (Gray et al., 2001). Other signaling pathways are regulated at the level of targeted protein destruction, either via the proteasome or the related COP9 signalosome complex, including jasmonic acid signaling (Xie et al., 1998), photomorphogenesis (Osterlund et al., 2000), floral development pathways (Samach et al., 1999), and disease resistance pathways (Austin et al., 2002; Azevedo et al., 2002). Here, we have shown that GA signaling in barley operates via proteasome-dependent, GA-elicited destabilization of SLN1. The fact that targeted protein destruction is key to so many different plant signaling pathways may explain why they often operate negatively, via derepression.
Plant Material and Growth Conditions The experiments described here used the barley (Hordeum vulgare) cv Herta as the source of the wild-type SLN1 allele. The sln1-1 mutant allele was induced by diethyl sulfate treatment of Herta (Foster, 1977 ), and the segregating material used was the selfed progeny of a line backcrossed multiple times onto the Herta genetic background. Seeds were surface-sterilized by washing first with 70% ethanol for 2 min, then with sodium hypochlorite for 30 min, and finally with sterile distilled water. Sterilized seeds then were grown at 20°C (16-h photoperiod) on moistened filter paper. In tests of the seedling growth response to gibberellin (GA), seeds were germinated in 100 µM GA3 (Sigma). In further tests of GA-promoted leaf extension growth, 3-day-old seedlings were incubated with water or 100 µM GA3 in the presence or absence of 100 µM MG132. In tests of GA-induced SLN1 protein degradation, 5-day-old seedlings were treated with 100 µM GA3 (Sigma) or with water in the presence or absence of different pharmacological agents in the presence of 1% DMSO.
Cloning and Sequence Analysis of SLN1 Alleles
Production of Anti-GAI Polyclonal Antibodies The antisera obtained, although prepared against E. coli–expressed Arabidopsis GAI, were capable of detecting the SLN1 protein. A band that approximated the size expected for SLN1 was detected in extracts from SLN1 plants but not in extracts from sln1-1 plants.
Protein Gel Blot Analysis
Inhibitor Studies
MG115 is a potent, reversible proteasome inhibitor that specifically inhibits the chymotrypsin-like activity of the proteasome (Peptides International, Louisville, KY). MG132 (also from Peptides International) is a tripeptide aldehyde, a potent, reversible proteasome inhibitor (Callis and Vierstra, 2000
Okadaic acid (Calbiochem) is an inhibitor of the Ser/Thr protein phosphatases PP1 and PP2B (Bialojan and Takai, 1988 For the inhibitor analyses, 5-day-old seedlings were transferred to 100 µM GA3 (Sigma) for 30 min, 2 h, or 24 h in the presence of 1% DMSO (control) or 1% DMSO with inhibitor at the following concentrations: MG115 (100 µM), MG132 (100 µM), Pefabloc SC (0.5 mg/mL), okadaic acid (1 µM), sodium vanadate (3 mM), AG555 (10 µg/mL), staurosporine (50 µM), genistein (50 µg/mL), PP2 (10 µg/mL), and proteasome inhibitor I (100 µM). After treatment, the seedlings were harvested and extracted for immunoblot analysis as described above. Data shown are representative of the results of three independent experiments.
Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes.
We thank David Laurie for helpful discussion, advice on the growth of barley, and provision of plant material, and Patrick Achard and Kathryn King for discussion of the manuscript. This work was supported by a Core Strategic Grant from the Biotechnology and Biological Science Research Council to the John Innes Centre and by Biotechnology and Biological Science Research Council Grant 208/P15108 to N.P.H.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.006197.
1 These authors contributed equally to this work.
2 Current address: Functional Genomics Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609. Received July 9, 2002; accepted September 13, 2002.
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Abstract
INTRODUCTION
65-kD immunoreactive protein that was detectable in water-treated SLN1 seedlings but not in water-treated sln1-1 seedlings, indicating that the protein identified is SLN1 (the predicted molecular mass of SLN1 is 65.2 kD) (
-galactoside for 90 min, and then harvested. Cells were resuspended in a buffer containing 50 mM Tris-HCl, pH 8.0, 0.2 mM EDTA, 100 mg/mL lysozyme, and 0.1% Triton X-100 and sonicated on ice. After centrifugation at 10,000 rpm for 20 min at 4°C, inclusion bodies were resuspended in the binding buffer solution containing 6 M urea, 5 mM imidazole, 50 mM NaCl, and 20 mM Tris-HCl, pH 8.0. The GAI recombinant protein was affinity-purified with nickel–nitrilotriacetic acid agarose beads (Qiagen). The purified GAI recombinant protein solution then was dialyzed against PBS solution overnight at 4°C before being used to raise antibodies in a rabbit. 
