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Accumulation of Phosphorylated Repressor for Gibberellin Signaling in an F-box Mutant

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Science  21 Mar 2003:
Vol. 299, Issue 5614, pp. 1896-1898
DOI: 10.1126/science.1081077

Abstract

Gibberellin (GA) regulates growth and development in plants. We isolated and characterized a rice GA-insensitive dwarf mutant,gid2. The GID2 gene encodes a putative F-box protein, which interacted with the rice Skp1 homolog in a yeast two-hybrid assay. In gid2, a repressor for GA signaling, SLR1, was highly accumulated in a phosphorylated form and GA increased its concentration, whereas SLR1 was rapidly degraded by GA through ubiquitination in the wild type. We conclude that GID2 is a positive regulator of GA signaling and that regulated degradation of SLR1 is initiated through GA-dependent phosphorylation and finalized by an SCFGID2-proteasome pathway.

Gibberellins (GAs) are essential regulators of diverse growth and developmental processes in plants (1). Recently, a constitutive GA response mutant,slender rice1 (slr1) (Fig. 1A), was isolated and molecular biological analyses revealed that the SLR1 gene encodes a protein orthologous to Arabidopsis GAI and RGA, wheat Rht, maize d8, and barley SLN1 (2–6). Using the Arabidopsis gai mutant, Peng et al. (2) proposed that GA signaling is governed by derepression of a repressor protein, GAI. This idea has been substantiated by recent studies on RGA, SLR1, and SLN1; that is, these proteins function in nuclei and degrade rapidly when exposed to GA, enabling the GA signal to be transmitted downstream (7–10). More recently, Fuet al. (11) indicated that proteasome-dependent degradation is necessary for GA-mediated destabilization of SLN1. How GA triggers the proteasome-mediated degradation of these repressor proteins remains unknown.

Figure 1

Phenotype of the gid2mutant. (A) Gross morphologies of wild type,gid2-1, gid2-1/slr1-1, andslr1-1 plants (left to right) in 2-week-old seedlings. Bar = 10 cm. (B) Elongation of the second leaf sheath in response to GA3 treatment in gid2-1 (•),gid2-2 (Δ), d18-dy (⊚), and wild-type (⧫) plants. Error bars represent standard deviation from the mean (n = 5). (C) α-Amylase induction in embryoless half seeds of the wild type and gid2-1.Embryoless half seeds from wild-type or gid2-1 heterozygous plants were placed on starch plates with (+) or without (−) 1 μM GA3. Starch was detected by staining with iodine. (D) Effect of GA3 on expression of α-amylase (RAmy1A) and OsGA20ox2 in wild type,d18-dy, and gid2-1. The gel was stained with ethidium bromide as a control (rRNA) (13).

To investigate the molecular mechanism of GA signaling, we isolated several candidates showing a severe dwarf phenotype (12). Genetic analyses of these candidates revealed that the dwarf phenotype was inherited in a recessive manner and could be categorized in two groups: GA-insensitive dwarf 1 and 2(gid1 and -2). Here, we report that the GA-insensitive phenotype of gid2 is caused by the loss of function of a putative F-box protein.

The gid2 mutant showed a severe dwarf phenotype with wide leaf blades and dark green leaves (Fig. 1A), features typical of GA-related mutants such as d1 and d18(13, 14). Thegid2-1/slr1-1 double mutants showed a slender phenotype identical to that of the slr1-1 single mutant (Fig. 1A), indicating that GID2 functions upstream of SLR1. To confirm that gid2 was a GA-insensitive mutant, we compared the GA responsiveness of gid2-1 and gid2-2 with that of the wild type and a GA-deficient mutant, d18-dy. When we treated the second leaf sheaths of these plants with various concentrations of GA3, the wild type and d18-dyelongated at GA3 concentrations of 10 nM or higher, whereas we observed very little response in both gid2 mutants (Fig. 1B). We also examined the induction of α-amylase by GA3in gid2-1 endosperm (Fig. 1C). α-Amylase activity was induced in all wild-type embryoless half seeds by GA treatment. However, GA did not induce α-amylase activity in 25% of the embryoless half seeds derived from gid2-1 heterozygous plants. We observed no GA-dependent induction of α-amylase ingid2-1 at the transcriptional level (Fig. 1D).

We also examined GA biosynthesis in gid2-1. First, we analyzed the expression of the GA20-oxidase gene, OsGA20ox2, which is highly expressed in vegetative organs (15). In the wild type and in d18-dy, OsGA20ox2 expression was suppressed by GA3 treatment, corresponding to feedback regulation of GA biosynthetic genes by bioactive GAs (Fig. 1D). However, GA3 treatment did not suppressOsGA20ox2 expression in gid2-1. We also directly measured the GAs in gid2-1 and wild type. The amount of GA1, the dominant bioactive GA in rice vegetative organs, in gid2-1 was more than 100 times higher than that detected in wild type (Table 1). Taken together, these results demonstrate that gid2 is a GA-insensitive mutant and the GID2 gene encodes a positive regulator of GA signaling.

Table 1

Amounts of endogenous GAs in wild type andgid2 (ng per g of fresh weight).

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To elucidate the molecular function of GID2, we isolated the gene by positional cloning. Because gid2 was sterile, we crossed a japonica variety of GID2/gid2 rice with an indica rice variety (Kasalath) and selected F1plants with a GID2/gid2 genotype. About 2500 F2plants derived from these heterozygous F1 plants were used in this study. The candidate region was narrowed down to a 13-kb sequence containing three putative genes. We compared this 13-kb sequence in the wild type with the corresponding region in the three gid2 mutants and found nucleotide substitution or deletions in one predicted gene (Fig. 2A). The deletions or substitution of nucleotide in all three gid2 alleles introduced a novel stop codon, suggesting that these are null alleles. Introduction of a 4.2-kbSac I fragment covering the entire region of theGID2 gene in the gid2-1 plants recovered the normal phenotype, confirming that the gid2 mutation is caused by a defect in GID2 (16).

Figure 2

Sequence analyses and molecular characterization of GID2. (A) TheGID2 gene consists of one exon encoding an F-box motif (thick line). Nucleotide deletions (thin lines) and substitution (at the site of 618) in the three gid2 alleles are represented. Black and shadow boxes indicate the coding and 5′- or 3′-flanking regions, respectively. (B) Alignment of the F-box motif of GID2 with other Fbox proteins (26): GID2 (rice, accession number AB100246), SLY1 (Arabidopsis, accession number CAB45056), MET30 (yeast, accession number P39014), POF1 (Schizosaccharomyces, accession number P87053), SCON2 (mold, accession number Q01277), 1FQV A (human, accession number 1FQVA), FBX8 (human, accession number AAF04517), FBX22 (human, accession number AAF045223), and FBX29 (human, accession number AAF03129). Dark and light shading indicate identical and similar residues, respectively. (C) Interaction between GID2 and OsSkps in a yeast two-hybrid assay. For bait construct (BD-GID2), full-length GID2 was fused with the GAL4 DNA binding domain. For the prey constructs (AD-fusion), full-length OsSkp2 or OsSkp5 was fused with the GAL4 transcriptional activation domain (13).

The GID2 gene encoded a 639-base-pair open reading frame capable of producing a polypeptide of 212 amino acid residues (Fig. 2A). The deduced amino acid sequence of GID2 contained an F-box domain, which is a conserved motif of F-box proteins that form part of an E3 ubiquitin-ligase complex. The motif found inGID2 is well conserved in other F-box proteins, with the greatest similarity to Arabidopsis SLY1, which is a positive regulator of GA signaling (Fig. 2B) (17).

To confirm that GID2 actually encodes an F-box protein, we examined whether GID2 interacts with rice orthologs of Skp1 by a yeast two-hybrid analysis. F-box protein functions as a component of the SCF complex through interaction with Skp1 (18,19). Thus, if GID2 functions as an F-box protein, it will interact with a suitable Skp1 protein. We isolated 14 homologous genes from rice, designated OsSkp1-14 (Oryza sativa Skp1 homolog 1-14). We tested the interaction between GID2 and two rice Skp1 candidates (OsSkp2 and OsSkp5) because these two proteins showed high similarity to ArabidopsisASK1 (fig. S1) (20). GID2 interacted with OsSkp2 to produce a blue colony, whereas a combination of GID2 and OsSkp5 did not induce lacZ activity (Fig. 2C). This indicates that GID2 preferentially interacts with one or more members of Skp1 proteins.

As already noted, the SLR1 protein functions as a repressor of GA signaling in rice and its degradation may be essential for downstream action of GA. We examined whether this is also the case in the gid2 mutant by probing immunoblots with an antibody to SLR1 (Fig. 3A). The SLR1 protein was abundant in gid2-1 but was detected at a very low level in the wild type (compare lanes 1 and 3). Further, GA-dependent degradation of the SLR1 protein occurred in the wild type but was not observed in gid2-1 (Fig. 3A). In addition, the immunoreactive protein in the gid2 mutant was split into two bands, but only one band was detected in the wild type (Fig. 3A). The band in gid2-1 with higher mobility corresponded to the protein synthesized in Escherichia coli—namely, the nascent protein (Fig. 3A); the band with lower mobility may be an intermediate in the SLR1 degradation process. The high level accumulation of SLR1 and the appearance of a new band with lower mobility in gid2suggest that GA-dependent degradation of SLR1 is deficient ingid2.

Figure 3

Accumulation of the phosphorylated SLR1 protein in gid2. (A) Immunoblotting of SLR1 was done with 10 μg of protein of crude extract from wild type (lanes 1 and 2) and gid2-1(lanes 3 and 4) treated with (+) or without (−) 100 μM GA3. Recombinant SLR1 produced in E. coli was used as a size control (lane 5), and the Coomassie brilliant blue–stained RbcS protein was used as a loading control (CBB). (B) A crude extract of gid2-1 was incubated with (+) or without (−) CIP. Boiled CIP was used as a negative control (boil). (C and D) In vivo phosphorylation of the SLR1 protein. Seedlings of wild type or gid2-1 pretreated with 1 μM uniconazol were incubated with 32PO4 and then treated with (+) or without (−) 100 μM GA3 for 2 hours in (C) or collected with a time course following the GA3 treatment in (D). Proteins immunoprecipitated with the antibody to SLR1 were analyzed by autoradiography [lanes 1 to 4 in (C) and (D)] or immunoblotting with the antibody to SLR1 [lanes 5 and 6 in (C)]. (E) Detection of the polyubiquitinated SLR1 in wild type andgid2-1. Seedlings were pretreated with the proteasome inhibitor MG132 before GA3 treatment. One milligram of total protein extract was immunoprecipitated with antibody to SLR1 (IP: α-SLR1) and analyzed by immunoblotting with antibody to Ub (α-Ub) (13).

To investigate the nature of the modification in the SLR1 protein, we treated a crude extract of gid2-1 with calf intestine alkaline phosphatase (CIP) before immunoblotting (Fig. 3B). This treatment caused the band with lower mobility in gid2-1to disappear, suggesting that this band comprises a phosphorylated form of the SLR1 protein. We also examined phosphorylation of the SLR1 protein by in vivo labeling with 32PO4 . Because we predicted that GA would enhance the degradation of the phosphorylated SLR1 protein, we pretreated the wild type and gid2-1 with uniconazol, an inhibitor of GA biosynthesis. We detected one faint radioactive band in uniconazol-pretreated wild type and this band disappeared after treatment with GA3 (Fig. 3C, lanes 1 and 2). This supports our theory that the phosphorylated SLR1 protein is destabilized by bioactive GA. In contrast, we observed one strong radioactive band in gid2-1 and GA3treatment increased its intensity (Fig. 3C, lanes 3 and 4). The mobility of the radioactive band corresponded to the upper band ingid2-1, and the intensity of the upper band observed by immunoblotting increased after treatment with GA (Fig. 3C, lanes 5 and 6). This GA-induced phosphorylation of SLR1 protein ingid2-1 was gradually increased after GA3treatment (Fig. 3D). These results indicate that GA increases SLR1 phosphorylation and may lead to degradation of phosphorylated SLR1 in wild type but that degradation of the phosphorylated SLR1 in gid2 is inhibited and consequently the protein is accumulated.

The fact that a loss of function in an F-box protein, GID2, causes accumulation of the SLR1 protein leads us to speculate that GA-dependent degradation of SLR1 protein is caused by the ubiquitin/26S proteasome pathway. To test this possibility, we examined the polyubiquitination of SLR1 protein in vivo by immunoblotting with antibody to ubiquitin (Ub). In wild type treated with a proteasome inhibitor, MG132, a low level of polyubiquitinated SLR1 was observed without GA treatment (Fig. 3E, lane 1), and GA treatment induced the accumulation of polyubiquitinated SLR1 protein (Fig 3E, lane2). In contrast, in gid2-1, we observed no ubiquitinated SLR1 with or without GA treatment (Fig. 3E, lanes 3 and 4). These results suggest that the SLR1 protein is degraded via the ubiquitin/26S proteasome pathway mediated by the SCFGID2 complex.

The F-box protein in the SCF complex functions as a receptor that selectively recruits target proteins into the complex to degrade these proteins through ubiquitination. This SCF-mediated signaling pathway is well conserved in yeast, mammals, and higher plants (21–25). According to recent advances in understanding SCF-mediated pathways in yeast and animals (21–23), modification of the target protein is a prerequisite for interaction between the target and F-box proteins, and phosphorylation is one of the most common types of modification of target proteins. Although there are no previous reports that phosphorylation of target proteins triggers SCF-mediated degradation in plants, our results indicate that GA-dependent phosphorylation of SLR1 triggers the ubiquitin-mediated degradation in a manner similar to the SCF-mediated pathway in yeast and animals.

Supporting Online Material

www.sciencemag.org/cgi/content/full/299/5614/1896/DC1

Materials and Methods

Fig. S1

References

  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: makoto{at}agr.nagoya-u.ac.jp

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