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First published online April 10, 2003; 10.1105/tpc.010207 American Society of Plant Biologists The COP9 Signalosome Interacts Physically with SCFCOI1 and Modulates Jasmonate Responses
a Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520-8104 2 To whom correspondence should be addressed. E-mail xingwang.deng{at}yale.edu; fax 203-432-5726
The COP9 signalosome (CSN) is an evolutionarily conserved, nucleus-enriched multiprotein complex. CSN plays roles in photomorphogenesis, auxin response, and floral organ formation, possibly via the regulation of ubiquitin-proteasome–mediated protein degradation. COI1 encodes an F-box protein, which is a subunit of SCFCOI1 E3 ubiquitin ligase, and is required for jasmonate (JA) responses. Here, we demonstrate using coimmunoprecipitation and gel-filtration analyses that endogenous as well as epitope-tagged COI1 forms SCFCOI1 and associates directly with CSN in vivo. Like the coi1-1 mutant, CSN reduction-of-function plants exhibited a JA-insensitive root elongation phenotype and an absence of JA-induced–specific gene expression. Genome expression profile analyses indicated that JA-triggered genome expression is critically dependent on COI1 dosage. More importantly, most of the COI1-dependent JA-responsive genes also required CSN function, and CSN abundance was shown to be important for JA responses. Furthermore, we showed that both COI1 and CSN are essential for modulating the expression of genes in most cellular pathways responsive to JA. Thus, CSN and SCFCOI1 work together to control genome expression and promote JA responses.
The COP9 signalosome (CSN) was characterized genetically as a repressor of photomorphogenesis in darkness. Ten pleiotropic COP/DET/FUS loci were identified by screening for mutants that showed light-grown phenotypes when grown in complete darkness (Chory, 1993  ; Deng, 1994; Kwok et al., 1996) and for mutants with purple cotyledons (high level of anthocyanin) in young seedlings or mature seeds (Miséra et al., 1994). To date, molecular and genetic studies have shown that 6 of the 10 loci—COP9, FUS6/COP11, FUS5, FUS4/COP8, FUS11, and FUS12—encode subunits of the COP9 signalosome, which are CSN8, CSN1, CSN7, CSN4, CSN3, and CSN2, respectively (Chamovitz et al., 1996; Staub et al., 1996; Karniol et al., 1999; Serino et al., 1999, 2003; Peng et al., 2001b).
CSN is conserved in diverse organisms, including mammals, flies, and fission yeast (Seeger et al., 1998
The 26S proteasome is a proteolytic machine that degrades proteins conjugated with a multiubiquitin chain and plays a central role in selective proteolysis and the regulation of a wide variety of biological processes (Baumeister et al., 1998
CSN has been shown to associate with multiple SCF-type E3 ubiquitin ligases in vivo (Lyapina et al., 2001
Jasmonates (JAs) not only mediate plant defense responses to both mechanical trauma and pathogenesis but also regulate stamen and pollen development (Liechti and Farmer, 2002 Here, we report that CSN and SCFCOI1 interact physically and are capable of forming a large complex in vivo. We also show that Arabidopsis lines partially deficient in CSN exhibit JA-insensitive phenotypes that are analogous to those of coi1-1 mutants. Using an EST microarray with 6126 genes, we demonstrate that both COI1 and CSN are required for JA-responsive genome expression in Arabidopsis and that the regulation of gene expression is critically dependent on COI1 and CSN dosage. Moreover, we reveal that the COI1 and CSN requirement for expression changes in genes that encode several distinctive cellular pathway components. Our results strongly suggest that CSN and SCFCOI1 associate with each other in vivo and mediate JA responses collaboratively.
Construction and Expression of the COI1-Flag Fusion Protein in Wild-Type and coi1-1 Mutant Arabidopsis Plants As a first step in the biochemical analysis of COI1, we constructed a chimeric gene using the 35S promoter of Cauliflower mosaic virus to drive the expression of the full-length COI1 protein with three copies of the flag epitope tag at the C terminus (Figure 1A). The chimeric gene was first transformed into wild-type Arabidopsis plants, and T2 progeny carrying a single-locus transgene were selected. The transgene was introduced subsequently into the coi1-1 mutant (Feys et al., 1994 ) by genetic crossing. Protein gel blot analysis using polyclonal antibodies against COI1 resulted in the identification of the 66-kD COI1 protein as well as the slightly low-mobility tagged COI1 protein, COI1-flag, in wild-type or coi1-1 mutant plants carrying the COI1-flag transgene (Figure 1B).
Interestingly, the level of the COI1-flag protein was lower in the coi1-1 mutant background than in the wild-type background, which makes the CF/coi1 line a reduction-of-function strain for COI1 (Figure 1B). However, the male-sterile phenotype of the coi1-1 mutant was rescued completely in the CF/coi1 line (data not shown), suggesting that the low COI1-flag level in the CF/coi1 line is sufficient to properly regulate stamen and pollen development. Thus, this CF/coi1 line provides a viable and low-level COI1 strain with which to analyze the COI1 protein dosage effect on JA responses (see below).
The COP9 Signalosome Associates Physically with SCFCOI1 in Vivo
The COI1-Flag Fusion Protein Also Forms SCFCOI1-flag and Associates Physically with the COP9 Signalosome in Vivo We next examined whether the COI1-flag fusion protein can act properly in forming the SCF complex and its association with CSN. We used monoclonal anti-flag antibody to precipitate the COI1-flag fusion protein from JA-untreated CF flower extract and then examined the pull-down of the SCF and CSN components. As shown in Figure 2B, immunoprecipitation of COI1-flag readily pulls down the SCF complex scaffold component CUL1, implying the formation of an SCF complex with the COI1-flag fusion protein. Furthermore, five representative CSN subunits also were detected clearly in the immunoprecipitate from the CF flower extract. This pull down was specific, because the same flag antibodies did not reduce CSN subunits or CUL1 from the wild-type flower extract, and the endogenous COI1 and TBP were not reduced in either wild-type or transgenic COI1-flag flower extracts (Figure 2B). These data demonstrate that the COI1-flag fusion protein also forms SCFCOI1-flag and associates physically with the COP9 signalosome in vivo.
We further analyzed the association of SCFCOI1-flag and CSN by comparing the gel-filtration profiles of the COI1-flag fusion protein, CUL1, and CSN5 in JA-untreated CF flower extract. As shown in Figure 3, the COI1-flag fusion protein eluted from fractions 2 to 16, with a minor peak centered on fractions 3 and 4 and a major peak centered on fractions 10 to 12. CUL1 exhibited an elution profile over a similar range of fractions. Interestingly, we observed three peaks of CSN5 in the gel-filtration profiles. Two of the peaks are similar to the previously reported observations for CSN5 (Kwok et al., 1998
JA Responses Are Dependent on COI1 To test a possible role of CSN in JA responses, we first set out to establish the effect of exogenously applied JA on Arabidopsis seedling root growth and gene expression under our experimental conditions. JA-conferred root growth inhibition in wild-type Arabidopsis seedlings was dose dependent, with 60% root length reduction on 10 µM JA, 70% on 50 µM JA, and 90% on 100 µM JA (Figures 4A and 4B). The coi1-1 mutants showed insensitivity to root growth inhibition by JA, with only  10% length reduction on 100 µM JA (Figures 4A and 4B). Consistently, the VSP2 (Vegetative Storage Protein2) gene that is induced normally by JA in wild-type Arabidopsis seedlings was not induced at all in coi1-1 mutants (Figure 4C). Similarly, we found that PDF1.2, a plant defensin gene, can be induced by JA in wild-type Arabidopsis seedlings but not in coi1-1 mutants (Figure 4C), as reported previously (Penninckx et al., 1998). By contrast, the expression level of BGL2 ( -1,3-glucanase), which is inducible by salicylic acid but not by JA (Cao et al., 1994), was the same in wild-type and coi1-1 seedlings treated or untreated with JA (Figure 4C). These data indicate that our experimental conditions can adequately reproduce the previously reported JA responses and the effect of the coi1-1 mutation. Thus, we examined the effect of low COI1 level and the role of CSN on JA responses.
The coi1-1 mutant with the COI1-flag transgene, CF/coi1, which contains only a low level of the functional COI1-flag protein, was used to analyze the JA responses. Unlike coi1-1, CF/coi1 seedlings showed root growth inhibition by JA. However, compared with wild-type seedlings, they were less sensitive to JA, with 75% (instead of 90%) root length reduction on 100 µM JA (Figures 4A and 4B). In addition, VSP2 transcript induction upon JA treatment was reduced significantly in CF/coi1 seedlings compared with wild-type seedlings (Figure 4C), suggesting that a sufficient protein level of COI1 is important to maintain proper JA responses. Surprisingly, PDF1.2 was induced by JA to an even greater level in CF/coi1 than in the wild type (Figure 4C). It seems that PDF1.2 induction by JA is optimal at lower COI1 levels and decreases in magnitude at higher COI1 levels. Those observations imply that distinct JA responses have different sensitivities to COI1 abundance. In an independent experiment, a transgenic line expressing wild-type levels of a flag-COI1-hemagglutinin fusion protein in the coi1-1 mutant background was generated. In contrast to CF/coi1, the coi1-1 mutant phenotypes, including JA insensitivity and sterility, were rescued fully in this line (D. Xie, unpublished data). This finding suggests that the partial rescue of the coi1-1 mutant phenotype in CF/coi1 is attributable to the low COI1-flag protein level.
Proper Responses to JA Require CSN Function
However, there was no detectable change in VSP2 transcript induction by JA in FL seedlings (Figure 4C). It is interesting that both CSN5 (Schwechheimer et al., 2002
Expression Profile Analyses Show That JA-Responsive Gene Expression Is Largely COI1 Dependent Next, we compared the genome expression profiles of JA-treated wild-type seedlings with those of JA-treated coi1-1 mutant seedlings. This time, 290 (4.7%) of the 6126 genes were upregulated or downregulated by at least twofold. Of these genes, 130 had higher expression in JA-treated wild-type seedlings, and 160 had higher expression in JA-treated coi1-1 seedlings (Table 1; see also supplemental data at http://plantgenomics.biology.yale.edu). This group of genes was designated COI1-dependent JA-responsive (or COI1-regulated) genes, because their responsiveness to JA required COI1 function.
Cluster analysis showed a significant overlap between JA-responsive and COI1-regulated genes. Indeed, most genes showed similar regulation patterns in the two clusters, although differences in magnitude often were observed (Figure 5A, compare lanes 1 and 2). Our data confirmed the previous observation that most JA-induced responses require COI1 function.
JA Control of Genome Expression Is Critically Dependent on COI1 Dosage To further analyze the role of COI1 in JA responses, we examined the effect of reduced COI1 abundance on JA-triggered genome expression profiles. To this end, the genome expression profiles of CF/coi1 (low COI1 activity) with or without JA treatment (Figure 5A, lane 3) were obtained. A careful comparison with the JA-responsive and COI1-regulated genome expression profiles (Figure 5A, lanes 1 and 2) revealed that most of the JA-responsive and COI1-regulated genes displayed reduced responses to JA in the CF/coi1 line (see Table 1 for statistics). These genes can be categorized further into two groups. First, for the majority of JA-responsive and COI1-regulated genes, their responses to JA in CF/coi1 were compromised partially to varying degrees (Figure 5A), indicating that a reduced level of COI1 is able to regulate their responses to JA but a high level of COI1 is required for optimal responses. Second, the rest of the JA-responsive and COI1-regulated genes no longer respond to JA in the low COI1 abundance line (Figure 5A), suggesting that their responses to JA require normal high levels of COI1. Selected genes from the two groups are represented in Figures 5B and 5C, respectively. Our results indicate that most COI1-dependent JA-responsive genes require COI1 in a dose-dependent manner and that different genes exhibit distinct sensitivities to COI1 abundance.
The COI1-Dependent JA-Responsive Genes Require Fully Functional CSN
A closer examination of gene regulation patterns reveals clear differential sensitivities of different COI1-dependent JA-responsive genes to CSN abundance. One group of genes is very sensitive to CSN level, because the genes do not respond to JA in the FL line, which has a low CSN level (Figure 6B, lane 2). On the contrary, JA still can regulate some other genes in the FL line, although the magnitude is reduced to variable extents (Figure 6C, lane 2). Furthermore, genes in both clusters are no longer responsive to JA in cop9 and fus11 mutants (Figures 6B, lanes 3 and 4, and 6C, lanes 3 and 4). Together, the microarray data and the physiological studies (Figure 4) demonstrate that, as with COI1, the requirement for CSN in JA responses also is dose dependent.
A Small Group of COI1-Dependent JA-Responsive Genes Do Not Depend on CSN and Are Insensitive to COI1 Dosage
COI1- and CSN-Dependent JA-Responsive Genes Are Involved in Specific Cellular Processes Various genes involved in JA responses and COI1 regulation had been identified previously using arrays containing selected groups of genes (Reymond et al., 2000 ; Schenk et al., 2000; Sasaki et al., 2001). Those studies involved arrays with 150, 2375, and 2880 genes, respectively. Our data agree well with the previous results (see supplemental data at http://plantgenomics.biology.yale.edu) but provide a more comprehensive analysis as a result of our large gene coverage (6126 genes). We looked for functional groups in the COI1- and CSN-regulated JA-responsive genes, and those genes with an assigned functional category are summarized in Table 2. Ten putative stress-related genes were found in the genes that were upregulated, which suggests that COI1 and CSN work together to promote stress responses triggered by JA (Table 2). Another upregulated pathway is polysaccharide metabolism, in which six genes were identified (Table 2). Both COI1 and CSN are required for the JA-responsive expression of genes involved in amino acid metabolism, signal transduction, and transcription. For different genes in these pathways, the regulation could be either stimulatory or inhibitory (Table 2). Photosynthesis, energy generation, and resistance to certain diseases seem to be downregulated by COI1 and CSN in response to JA (Table 2).
The COP9 Signalosome Associates Physically with SCFCOI1 in Vivo and Is Essential for Its Function In this study, we used coimmunoprecipitation experiments to show that CSN is associated with CUL1 and COI1 or COI1-flag in wild-type and COI1-flag transgenic Arabidopsis plants (Figure 2), respectively. Moreover, COI1-flag, CUL1, and CSN5 were found to cofractionate in a large complex (>700 kD) in gel-filtration analysis (Figure 3). The size of this large complex is greater than that of CSN and SCFCOI1 combined, which indicates that it contains other components. It has been reported that neddylated CUL1 was enriched in cellular SCF complexes immunoprecipitated by antibodies against SCF substrates (Read et al., 2000 ; Kawakami et al., 2001). Here, we observed that most of the neddylated CUL1 exists in higher molecular mass fractions corresponding to the large complex. It is reasonable to speculate that this large complex, with SCF and CSN, may be the active form and that it contains SCF substrates. Phenotypic and gene expression studies support the idea that the observed CSN and SCFCOI1 physical association is functionally significant. The CSN partial loss-of-function line, fus6/CSN1-11 (FL), was found to be partially defective in JA responses as well (Figure 4). Furthermore, microarray analyses confirm that most of the COI1-regulated JA-responsive genes are controlled by CSN (Figure 6). Thus, our data suggest that CSN associates physically with SCFCOI1 in vivo and plays an essential regulatory role in its function.
In an exhaustive genome-wide search, 694 potential F-box proteins were identified in the Arabidopsis genome, constituting the largest gene superfamily currently known in plants (Gagne et al., 2002
With this study, three Arabidopsis SCF complexes, SCFTIR1, SCFUFO, and SCFCOI1, have been demonstrated to associate with CSN in vivo. In all cases, CSN has been found not only to interact with but also to directly modulate the activities of SCFTIR1, SCFUFO, and SCFCOI1 and the developmental responses they regulate (Schwechheimer et al., 2001
The Dosage Effect of SCFCOI1 and the COP9 Signalosome in JA Responses Like the COI1 dosage effect, we found that the abundance of CSN also is important for JA responses. The FL line, with a low CSN level, displayed reduced responsiveness to JA (Figure 4). It turns out that the expression of a majority of the COI1-regulated genes in response to JA was affected in the FL line, whereas almost all of these genes were affected in severe csn mutants (Figure 6, Table 1). Furthermore, COI1-regulated JA-responsive genes showed different sensitivities to CSN level (Figure 6). Based on these findings, we hypothesize that protein ubiquitination and degradation mediated by SCFCOI1 is a process that affects most aspects of the JA pathway and that regulation by CSN is essential for SCFCOI1 to perform its function. Thus, adequate COI1 and CSN protein levels are required for proper JA responses. In some rare cases, in which minimal COI1 activity is sufficient for their JA-responsive gene expression, CSN function is not essential (Figure 7).
Specific JA-Triggered Pathways That Are Regulated by Both COI1 and CSN Among these genes, we found several interesting functional groups. Clearly, stress responses were increased by COI1 and CSN when plants encountered exogenous JA (Table 2). By contrast, photosynthesis and energy-generating processes were suppressed (Table 2). These findings are consistent with the current understanding of JA-responsive pathways. JA is produced when plants suffer from mechanical trauma and pathogenesis. Under such circumstances, stress responses are of high priority to plant cells, whereas photosynthesis and energy metabolism should be limited. Because signal transduction and transcription processes are involved in various aspects of cellular activities, it is not surprising that the regulation of different genes in these pathways by COI1 and CSN could be either stimulatory or inhibitory (Table 2). Interestingly, COI1 and CSN can downregulate certain disease resistance genes (Table 2). This finding implies that JA pathways and other defense-related pathways might have antagonistic effects to each other. Overall, EST microarray analyses provide an overview of how plant cells, using COI1 and CSN, respond to JA, adjust genome expression patterns, and affect necessary cellular pathways.
Plant Materials and Growth Conditions The cop9-1, fus11-U203, coi1-1, and fus6/CSN1-11 mutants were described previously (Feys et al., 1994 ; Wei et al., 1994; Kwok et al., 1996; Wang et al., 2003). When not specified, we use cop9 to indicate cop9-1, fus11 to indicate fus11-U203, coi1 to indicate coi1-1, and FL to indicate fus6/CSN1-11 throughout the text. The wild-type Arabidopsis thaliana used in this study was the Columbia ecotype. Arabidopsis seeds were surface-sterilized, plated on Murashige and Skoog (1962) (MS) medium (GIBCO) containing 1% sucrose, and vernalized by incubating at 4°C for 3 to 5 days before being placed in a standard long-day growth chamber at 22°C. After 7 to 9 days of incubation, seedlings were transferred to soil and grown to adult plants in a standard long-day growth room. For exogenous jasmonate treatment experiments, Arabidopsis seedlings were grown on MS medium containing 50 µM methyl jasmonate (Bedoukian, Danbury, CT) for 7 days. For root growth inhibition experiments, Arabidopsis seedlings were grown on normal MS medium for 4 days and then transferred to MS medium containing 10, 50, or 100 µM methyl jasmonate. Photographs of the seedlings were taken 3 days after transfer, as were root length measurements.
Plasmid Construction, Arabidopsis Transformation, and Antibodies
Full-length cDNA of COI1 was amplified by reverse transcriptase–mediated PCR with the forward primer COI1-5NKS (5'-ATAAGAATGCGGCCGCAAGGTACCATTCCCGGGATGGAGGATCCTGATATCAAG-3') and the reverse primer COI1-3SSS (5'-ACGCGTCGACGAGCTCTCACC-CGGGTATTGGCTCCTTCAGGACTC-3'), both of which contain a SmaI site. The resulting PCR product was cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA). The SmaI fragment of the PCR product then was subcloned into pF3PZPY122, which, besides three copies of flag tag, also carries a gentamicin-resistant marker and a 35S promoter of Cauliflower mosaic virus to drive the expression of the transgene (Yamamoto et al., 1998
Arabidopsis plants of Columbia ecotype were used in the transformation of the COI1-flag transgene. The methods of plant transformation were described separately (Wang et al., 2002
The primary antibodies used in this study include anti-CSN3 (Peng et al., 2001b
Immunoblot, Immunoprecipitation, and Gel-Filtration Analyses
Experimental conditions for the immunoprecipitation experiments were as described previously (Staub et al., 1996
Gel-filtration chromatography was performed as described previously (Peng et al., 2001b All protein samples were run on SDS-PAGE (8 to 12%), transferred to nylon Immobilon membranes (Millipore, Bedford, MA), and subjected to immunoblot analysis.
RNA Gel Blot Analysis PDF1.2 and BGL2 probes were prepared by PCR amplification from Columbia wild-type genomic DNA. For PDF1.2, the forward primer was 5'-ACGCACCGGCAATGGTGGAAGCAC-3' and the reverse primer was 5'-CGGGAAAATAAACATTAAAACAG-3'. For BGL2, the forward primer was 5'-TCAGAGCTACAGAGATGGTGTCAG-3' and the reverse primer was 5'-AACAGTGGACTGGGCGGTGAACAG-3'. The SalI-NotI fragment from a VSP2 EST clone was used as the VSP2 probe. All of the probes were radiolabeled using the Rediprime kit (Amersham).
Microarray Analysis Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes.
Accession Number
We thank Elizabeth Strickland and Vicente Rubio for reading and commenting on the manuscript, the members of our laboratory for technical assistance and helpful discussion, and J.G. Turner for coi1-1 mutants. This research was supported by grants from the U.S. Department of Agriculture (2001-35304-10852) and the National Science Foundation (MCB-0077217 and MCB-0115870) to X.W.D. and by grants from the National Science Foundation of China (30170489 and 39725002).
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010207.
1 These two authors contributed equally to this work. Received December 22, 2002; accepted February 21, 2003.
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INTRODUCTION
-CSN4) or anti-COI1 antibody–conjugated beads (
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