The Arabidopsis F-Box Protein CORONATINE INSENSITIVE1Is Stabilized by SCFCOI1 and Degraded via the 26SProteasome PathwayC W

Jianbin Yan,a,1 Haiou Li,a,1 Shuhua Li,a Ruifeng Yao,a Haiteng Deng,a Qi Xie,b and Daoxin Xiea,2

a The Tsinghua University-Peking University Center for Life Sciences, MOE Key Laboratory of Bioinformatics, School of Life Sciences,Tsinghua University, Beijing 100084, Chinab State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Genetics and Developmental Biology,Chinese Academy of Sciences, Beijing 100101, China

Jasmonate regulates critical aspects of plant development and defense. The F-box protein CORONATINE INSENSITIVE1(COI1) functions as a jasmonate receptor and forms Skp1/Cullin1/F-box protein COI1 (SCFCOI1) complexes with Arabidopsisthaliana Cullin1 and Arabidopsis Skp1-like1 (ASK1) to recruit its substrate jasmonate ZIM-domain proteins for ubiquitinationand degradation. Here, we reveal a mechanism regulating COI1 protein levels in Arabidopsis. Genetic and biochemicalanalysis and in vitro degradation assays demonstrated that the COI1 protein was initially stabilized by interacting with ASK1and further secured by assembly into SCFCOI1 complexes, suggesting a function for SCFCOI1 in the stabilization of COI1 inArabidopsis. Furthermore, we show that dissociated COI1 is degraded through the 26S proteasome pathway, and we identifiedthe 297th Lys residue as an active ubiquitination site in COI1. Our data suggest that the COI1 protein is strictly regulated bya dynamic balance of SCFCOI1-mediated stabilization and 26S proteasome–mediated degradation and thus maintained ata protein level essential for proper biological functions in Arabidopsis development and defense responses.

INTRODUCTION

In all eukaryotes, the ubiquitin/26S proteasome pathway playsa key role in regulating diverse aspects of developmental andphysiological responses by selectively removing intracellularproteins (Moon et al., 2004; Smalle and Vierstra, 2004; Ho et al.,2008). These intracellular proteins are recognized and modifiedby the covalent attachment of polymeric ubiquitin chains via anATP-dependent reaction cascade involving the sequential andcoordinated action of three enzyme families, including the E1ubiquitin-activating enzymes, the E2 ubiquitin-conjugating en-zymes, and the E3 ubiquitin ligases (Hershko and Ciechanover,1998). The best characterized class of E3 ubiquitin ligases is theSCF (SKP1/CUL1/F-box protein) E3 ligases, which contain coreconstant subunits, including Cullin1 (CUL1) and SKP1, and onevariable F-box protein (Moon et al., 2004). SKP1 serves as abridge between CUL1 and the F-box protein (Zheng et al., 2002).The N-terminal F-box motif of the F-box protein is responsiblefor interaction with SKP1, while its C-terminal WD40 motif orLeucine-rich repeat region binds targeted substrates to providereaction specificity (Hua and Vierstra, 2011).

In Arabidopsis thaliana, there are ;700 F-box proteins (Xuet al., 2009). These F-box proteins, which form distinct SCFcomplexes with Arabidopsis SKP-like proteins (ASK) and Arabi-dopsis CUL1, specifically recognize substrate proteins and dy-namically regulate their abundance to modulate a diverse range ofbiological processes (Hellmann and Estelle, 2002; Stone andCallis, 2007). During the past two decades, tremendous progresshas been achieved in understanding how F-box proteins controlthe abundance of short-lived regulatory proteins that mediate keyplant functions, including hormonal signaling, light signaling, cir-cadian clock control, organ development, self-incompatibility, cellcycle, and defense responses (Gray et al., 2001; del Pozo et al.,2002; Stirnberg et al., 2002; Más et al., 2003; Potuschak et al.,2003; Sasaki et al., 2003; Qiao et al., 2004; Imaizumi et al., 2005;Lechner et al., 2006; Santner and Estelle, 2010).Recent studies have shown that regulating the abundance of

F-box proteins themselves is essential to exert their proper bi-ological functions in Arabidopsis. For example, the F-box proteinZEITLUPE (ZTL) functions as a circadian photoreceptor and iscritical to the maintenance of circadian rhythmicity (Somerset al., 2004). Rhythmic changes in ZTL protein levels are nec-essary to sustain a normal circadian period in Arabidopsis (Kimet al., 2003). Further studies revealed that the GIGANTEA (GI)protein interacts with ZTL in a blue light–dependent manner (Kimet al., 2007), to stabilize the ZTL protein, which mediates thedegradation of TIMING OF CAB EXPRESSION1 (TOC1), a cen-tral transcriptional repressor of core circadian genes (Más et al.,2003). The F-box proteins EBF1/2 modulate ethylene responsesvia regulating the abundance of their substrate protein, the EIN3/EIL1 transcription factor (Guo and Ecker, 2003; Potuschak et al.,2003). A recent study revealed that abundance of the EBF1/2

1 These authors contributed equally to this work.2 Address correspondences to daoxinlab@tsinghua.edu.cn.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Daoxin Xie (daoxinlab@tsinghua.edu.cn).C Some figures in this article are displayed in color online but in black andwhite in the print edition.W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.112.105486

This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been

edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online

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proteins adjusts in response to ethylene, which is vital for reg-ulating EIN3/EIL1 degradation (An et al., 2010).

The F-box protein CORONATINE INSENSITIVE1 (COI1) (Xieet al., 1998) functions as a receptor for jasmonate (Katsir et al.,2008; Yan et al., 2009; Sheard et al., 2010), an important planthormone essential for plant defense and development (Farmerand Ryan, 1992; Staswick et al., 1992; Howe et al., 1996; McConnet al., 1997; Cheong and Choi, 2003; Farmer et al., 2003;Wasternack, 2007). COI1 forms SCFCOI1 complexes with CUL1and ASK1/2 (Xu et al., 2002) and recruits its substrates, thejasmonate ZIM-domain proteins (JAZs) (Chini et al., 2007; Thineset al., 2007; Yan et al., 2007), for ubiquitination and degradationvia the 26S proteasome pathway. Degradation of JAZs activatestheir downstream transcription factors or complexes, includingMYC2/3/4 (Cheng et al., 2011; Fernández-Calvo et al., 2011; Niuet al., 2011), MYB21/24 (Song et al., 2011), andWD-repeat/bHLH/MYB (Qi et al., 2011), to mediate various jasmonate responses.Although understanding the molecular basis for COI1 function inregulation of jasmonate responses has received significant at-tention, the mechanism by which cells regulate and maintain therequired level of the F-box protein COI1 itself to properly executeits biological function in Arabidopsis remains unclear.

In this study, we revealed a mechanism regulating the abun-dance of COI1 in Arabidopsis. We demonstrate that the COI1protein is stabilized by the integrity of the SCFCOI1 complex.Furthermore, we show that disassociated COI1 is degradedthrough the 26S proteasome pathway. Our data suggest that theCOI1 protein is regulated by a dynamic balance of SCFCOI1-mediated stabilization and 26S proteasome–mediated degra-dation and maintained at a proper protein level essential forexerting its biological functions in Arabidopsis. It will be in-teresting to determine whether this mechanism is more widelyused for regulation of other Arabidopsis F-box proteins bySCF complexes, in addition to their regulatory role in substratedegradation.

RESULTS

ASK1 Is Required to Maintain COI1 Stability

Previous studies have shown that the abundance of some F-boxproteins is dynamically regulated by hormones in Arabidopsis(An et al., 2010; Liu and Stone, 2010; Jurado et al., 2008, 2010).To examine the effect of plant hormones on abundance of theF-box protein COI1, we treated Arabidopsis wild-type seedlingswith various plant hormones in the presence of the proteinsynthesis inhibitor cycloheximide (CHX). Immunoblot analysisshowed that treatments with plant hormones, including abscisicacid, 1-aminocyclopropane-1-carboxylic acid, 6-benzylaminopurine,gibberellin, salicylate, jasmonic acid, jasmonic acid methyl ester(MeJA), jasmonoyl-Ile, cis(+)-12-oxophytodienoic acid, and co-ronatine (a molecular mimic of jasmonoyl-Ile), had no obviouseffect on COI1 abundance. The COI1 protein level was un-changed over 24 h in hormone-treated Arabidopsis seedlingsin which protein synthesis was inhibited by CHX (Figure 1A).These results indicate that COI1 experiences a high degree ofstability in Arabidopsis.

Stabilities of F-box proteins ZTL and Homolog of Slimb (HOS)are maintained by the ZTL-interacting protein GI and the HOSsubstrate IkBa, respectively (Li et al., 2004; Kim et al., 2007). Toinvestigate the molecular basis for COI1 stability, we examinedwhether the COI1 protein was stabilized by COI1-interactingprotein ASK1 or its substrate protein JAZ. When the COI1 genewas coexpressed with ASK1 in a yeast pBridge expressionsystem, high levels of the COI1 protein were detected in thesupernatant of lysed yeast cells. However, under equivalentgrowth conditions, the COI1 protein was hardly detected whenCOI1 was coexpressed with JAZ1 or expressed alone (Figure 1B).These results clearly indicate that ASK1, but not JAZ1, played animportant role in COI1 stability in a heterologous system.To verify the role for ASK1 in COI1 stability in planta, we in-

vestigated whether disruption of ASK1 results in reduced COI1levels by comparing the COI1 level between the wild type andask1-1, an Arabidopsis mutant with a Ds insertion in the ASK1gene (Yang et al., 1999). As shown in Figure 1C, the COI1protein level was severely reduced in ask1-1 compared with thewild type, whereas the COI1 mRNA level was equivalent in bothgenotypes, as revealed by RT-PCR (Figure 1D) and real-timequantitative PCR analysis (Figure 1E). Consistent with the re-duction of COI1 protein in ask1-1, expression of VSP1, a typicaljasmonate-responsive gene (Anderson, 1991; Staswick, 1994),was also reduced in ask1-1 (Figure 1F). These results demon-strate that ASK1 is required to maintain stability of the COI1protein for eliciting jasmonate responses.

Mutation in CUL1 Results in a Reductionof COI1 Protein in Arabidopsis

ASK1 interacts directly with COI1 (Xu et al., 2002) and stabilizesthe COI1 protein (Figure 1). Here, we investigated whether thestability of COI1 is also controlled by another COI1-associatedprotein, CUL1, which forms an SCF complex with COI1 viaASK1 (Xu et al., 2002; Ren et al., 2005; Hua and Vierstra, 2011).As the axr6-1 and axr6-2 homozygous mutants with missensemutations of Phe-111 to Val and Ile residues, respectively, inCUL1 are seedling lethal (Hobbie et al., 2000; Hellmann et al.,2003), axr6-1/+ and axr6-2/+ heterozygous mutants were usedfor immunoblot analysis of COI1 protein.Compared with the wild type, COI1 protein levels were clearly

reduced in axr6-1/+ and axr6-2/+ adult plants (Figure 2A), whereasthe COI1mRNA levels were unchanged (Figure 2C). Similar resultswere obtained for 10-d-old seedlings, which exhibited normalmorphological phenotypes (Figure 2B), and minimized potentialeffect of the severe morphological defects on COI1 stability inaxr6-1/+ and axr6-2/+ adult plants (Figure 2A). Consistent withreduction of the COI1 protein, jasmonate induction of VSP1 ex-pression and anthocyanin accumulation were also reduced inaxr6-1/+ and axr6-2/+ compared with the wild type (Figures 2Dand 2E). These results suggest a role for CUL1 in the regulation ofCOI1 stability.Missense mutations of Phe-111 to Val and Ile residues in CUL1

do not reduce the CUL1 level (Hellmann et al., 2003) but com-promise the ability of CUL1 to interact with ASK1 and attenuateassembly of the ASK1-COI1 into SCF complexes (Ren et al.,2005). Attenuation of the COI1-ASK1 assembly into SCFCOI1 by

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Figure 1. ASK1 Regulates COI1 Stability.

(A) Ten-day-old Arabidopsis seedlings were treated with CHX (1 mM) for 2 h, followed by additional 24-h treatment with 20 mM cis(+)-12-oxophytodienoic acid (OPDA), (6)-jasmonic acid (JA), MeJA, (6)-jasmonoyl-Ile (JA-Ile), (6)-abscisic acid (ABA), 1-aminocyclopropane-1-carboxylic acid(ACC), 6-benzylaminopurine (6-BA), gibberellic acid GA3 (GA), indole-3-acetic acid (IAA), salicylic acid (SA), or coronatine (COR). Total protein extractedfrom the treated seedlings was separated by SDS-PAGE and immunoblotted with anti-COI1 antibody (a-COI1). The PVDF membrane was stained withMemstain to visualize the large subunit of ribulose-1,5-bisphosphate as a control for equal loading (Loading Control). CK1 and CK2 refer to thetreatment with CHX for 0 h (CK1) and 26 h (CK2).(B) Immunoblot analysis of COI1 expression in yeast cells. Yeast cells transfected with constructs pBridge-COI1 (COI1), pBridge-JAZ1-COI1 (COI1+JAZ1), and pBridge-ASK1-COI1 (COI1+ASK1) were lysed in detergent. The supernatants of whole-cell lysates were subjected to immunoblot analysisusing COI1 antiserum (a-COI1). The PVDF membrane was stained with Memstain to serve as loading control.(C) Leaves from 4-week-old Arabidopsis Landsberg erecta (the wild type [WT]) and ask1-1 (ask1) plants were harvested for protein extraction andsubjected to immunoblot analysis using the COI1 antibody (a-COI1) or the ASK1 antibody (a-ASK1).(D) RT-PCR analysis of the COI1 transcript levels (COI1) in the wild type and ask1-1 described in (C). ACTIN1 was used as an internal control.(E) Real-time quantitative PCR analysis of COI1 transcript levels in the wild type and ask1-1 described in (C). Error bars represent SD (n = 6).(F) Real-time quantitative PCR analysis of the VEGETATIVE STORAGE PROTEIN1 (VSP1) gene in 2-week-old seedlings of Landsberg erecta (WT) andask1-1 (ask1) treated with or without MeJA at the indicated concentrations. Error bars represent SD (n = 6).

Regulating Mechanism in COI1 Level 3 of 13

the CUL1 mutations of Phe-111 to Val and Ile residues causesdecreased COI1 protein levels in axr6-1/+ and axr6-2/+ (Figures2A and 2B), suggesting that integrity of SCFCOI1 is essential forthe COI1 stability in Arabidopsis.

The COI1 Protein Is Regulated at the PosttranslationalLevel in Arabidopsis

We next generated transgenic lines constitutively overexpressingCOI1 to test whether high and stable COI1 transcript levels would

affect COI1 protein abundance in Arabidopsis. The COI1 gene,tagged with hemagglutinin (HA) at its C terminus and Flag at its Nterminus, was driven by the cauliflower mosaic virus (CaMV) 35Spromoter and introduced into the coi1-1 mutant.The transgenic line, referred as to coi1:HiCOI1 (coi1-1 highly

expressing COI1), had an;10-fold increase inCOI1 transcripts asdetermined by RT-PCR and real-time quantitative PCR analysis(Figure 3A). Immunoblot analysis showed that coi1:HiCOI1 re-tained the normal COI1 protein level, similar to that in the wild type(Figure 3B), suggesting that the high level of COI1 transcripts was

Figure 2. Mutations in CUL1 Affect COI1 Stability.

(A) and (B) Proteins were extracted from adult plants (A) and seedlings (B) of Arabidopsis Columbia (the wild type [WT]), axr6-1/AXR6 (axr6-1/+), andaxr6-2/AXR6 (axr6-2/+) heterozygous mutant plants and subjected to immunoblot using the COI1 antiserum (a-COI1). Morphological phenotypes of5-week-old adult plants (top panel in [A]) and 7-d-old seedlings (top panel in [B]) are also shown.(C) RT-PCR analysis of the COI1 transcript levels (COI1) in the Arabidopsis plants, as indicated.(D) Real-time quantitative PCR analysis of the VSP1 (VSP) transcripts in the wild type, axr6-1/+, and axr6-2/+ treated with MeJA (25 mM) or solvent. Errorbars represent SD (n = 6).(E) Anthocyanin accumulation in the wild type, axr6-1/+, and axr6-2/+ treated with MeJA (25 mM) or solvent. The o.d. (535 nm–650 nm)/g fresh weightwas regarded as the Y value. Error bars represent SD (n = 6).[See online article for color version of this figure.]

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Figure 3. The Significantly Increased COI1 Transcripts Were Unable to Elevate the COI1 Protein Level.

(A) RT-PCR (top panel) and real-time quantitative PCR (bottom panel) of the COI1 transcript level in the leaves of 4-week-old Arabidopsis Columbia (thewild type [WT]), coi1-1 (coi1), and coi1:HiCOI1 plants. Error bars represent SD (n = 4).(B) Immunoblot analysis of the COI1 protein level in the plants described in (A) using the COI1 antibody (a-COI1).(C) Phenotype of 10-d-old seedlings grown on Murashige and Skoog (MS) medium without (top panel) or with (middle panel) MeJA (25 mM). The bottompanel shows average root lengths of seedlings pictured in the middle panel. Error bars represent SD (n > 100). JA, jasmonate.(D) Analysis of stamen length (top panel), anther dehiscence (second panel), and the pollen viability in the wild type, coi1-1, and coi1:HiCOI1. Fluo-rescein diacetate and propidium iodide were used to stain pollen grains with viable pollen staining green, while inviable pollen stains red (third panel).Pollen grains of the wild type and coi1:HiCOI1 germinate normally (bottom panel).(E) The coi1:HiCOI1 and wild-type plants similarly exhibited normal seed production. The top panel shows representative primary inflorescences. Themiddle panel shows representative siliques. Seed numbers counted from mature siliques are shown in the bottom panel. Error bars represent SD (n = 10).(F) Anthocyanin accumulation in Col-0, coi1-1, and coi1:HiCOI1 treated with MeJA (25 mM). Error bars represent SD (n = 6).

unable to further elevate COI1 protein levels in coi1:HiCOI1.Consistent with the COI1 level, the coi1:HiCOI1 plants exhibitedfull complementation with restoration of normal jasmonate re-sponses, as indicated by jasmonate-mediated root inhibition(Figure 3C), anthocyanin accumulation (Figure 3F), and plantfertility (stamen length, anther dehiscence, pollen viability, andseed production; Figures 3D and 3E). Similar observations wereobtained when other independent transgenic lines were analyzed.These data suggest that overexpression of COI1 transcript level isunable to further increase the COI1 protein abundance in Arabi-dopsis, indicating that the COI1 protein level was regulated at aposttranslational level.

We further conducted genetic crosses of coi1:HiCOI1 withask1-1 and axr6-1/+ mutants to generate plants that constantlyoverexpress the COI1 gene in a background with compromisedASK1 or CUL1 function (ask1:HiCOI1 or axr6:HiCOI1). Indeed, theCOI1 transcripts in ask1:HiCOI1 and axr6:HiCOI1were maintainedat a high level similar to coi1:HiCOI1 (Figures 4A and 4B). How-ever, immunoblot analysis showed that the levels of COI1 proteinwere greatly reduced in the ask1:HiCOI1 and axr6:HiCOI1 lines(Figures 4A and 4B). These data further suggested that COI1 wasregulated at a posttranslational level and that COI1 is stabilized bythe SCFCOI1 core subunits ASK1 and CUL1 in Arabidopsis.

COI1 Stability Is Regulated by SCFCOI1 in Vitro

Having shown that SCFCOI1 is essential for COI1 stability inArabidopsis (Figures 1 to 4), we used in vitro assays to further

verify that COI1 stability is dependent on the SCFCOI1 complex.Myc-tagged COI1 (Myc-COI1) was expressed in a Nicotianabenthamiana transient expression system, purified with anti-Mycaffinity beads and evaluated for its bioactivity as assayed for itsinteraction with JAZ in a coronatine-dependent manner (Figure5A). Myc-COI1 protein was then mixed with total protein ex-tracts prepared from ask1-1, axr6-1/+, or wild-type plants, andMyc-COI1 stability was assessed by immunoblot analysis afterthe treatment at 22°C for various periods of time up to 8 h.As shown in Figures 5B and 5C, the Myc-COI1 protein de-

graded more rapidly in either ask1-1 and axr6-1/+ extracts com-pared with the wild type. These data demonstrated that both SCFcore components ASK1 and CUL1 are necessary for Myc-COI1stability, implying that an intact SCF complex is required formaintaining COI1 levels. Furthermore, as the Myc-COI1 proteinwas used for the in vitro assays, these data confirmed that Myc-COI1 stability is regulated at the posttranslational level and not atthe level of transcription or translation.All data, obtained from our in vivo and in vitro assays (Figures

1 to 5), collectively point to the COI1 protein being stabilized bythe SCFCOI1 complex.

COI1 Is Degraded via the 26S Proteasome Pathwayin an SCFCOl1-Independent Manner

Recent studies reported that degradation of the ArabidopsisF-box proteins TIR1, EBF1/2, and ZTL are mediated by

Figure 4. Stable and High COI1 Transcription Failed to Increase COI1 abundance in ask1-1 and axr6-1/+.

The protein level of HA-tagged COI1 was determined for the leaves of 4-week-old ask1:HiCOI1 (A) and axr6:HiCOI1 (B) by immunoblot analysis usinganti-HA antibody (top panel). The COI1 transcript level was determined by RT-PCR (middle panel) and real-time quantitative PCR analysis (bottompanel) using the leaves of 4-week-old plants. Error bars represent SD (n = 3). coi1:HiCOI1, as described in Figure 3, served as reference sample.

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proteasome-dependent proteolysis (Stuttmann et al., 2009; Anet al., 2010; Ito et al., 2012); we further investigated the mech-anism involved in COI1 protein degradation. Total protein ex-tracts from 4-week-old coi1:HiCOI1 plants were used to assessCOI1 stability in presence of the specific proteasome inhibitorsMG132 and PS341 and a mixture of inhibitors against a broadrange of proteases. COI1 degradation was attenuated byMG132 and PS341 (Figures 6A and 6B) but not by the non-specific protease inhibitors (Figure 6A), suggesting that COI1may be degraded via the 26S proteasome pathway. As ubiq-uitin/proteasome degradation is an ATP-dependent process(Etlinger and Goldberg, 1977), we were not surprised to find thatATP treatment significantly enhanced the COI1 degradation rate(Figure 6C).

Mutations in RPT5a, an important component of the 26S pro-teasome (Rubin et al., 1998), attenuated the degradation of manyproteins (Sakamoto et al., 2011). Here, we found that the level ofCOI1 protein was moderately increased in the rpt5a-4 mutantcompared with wild-type plants (Figure 7A). The rpt5a-4 mutantwas moderately but clearly more sensitive to jasmonate as in-dicated by enhanced expression of the typical jasmonate-responsivegene VSP1 (Figure 7B) and accumulation of anthocyanin (Figure 7C).Taken together, these results further suggest that the COI1degradation was mediated by the 26S proteasome pathway.

Figure 5. In Vitro Analysis of the Regulatory Role for SCFCOI1 in the COI1Stability.

(A)Myc-tagged COI1 transiently expressed in N. benthamianawas extractedfrom tobacco leaves (Myc-COI1 ext), purified with Anti-c-Myc AgaroseAffinity Gel (Purified Myc-COI1), and subsequently assessed for its ability tointeract with recombinant JAZ1-His protein in presence of coronatine atvarious concentrations (0, 0.01, 0.1, 1, and 10 mM) in a pull-down assay.Total protein extracts from tobacco leaves infiltrated with empty expressionvector is shown as the control. The samples were purified by SDS-PAGE andimmunoblotted with the anti-Myc antibody (top panel). The PVDF membranewas stained with Memstain to visualize the recombinant JAZ1-His protein inthe nickel-nitrilotriacetic acid (Ni-NTA) affinity resin (bottom panel).(B) The purified Myc-COI1 protein was added to total crude proteinextracts from ask1-1 (ask1 Crude) and Arabidopsis Landsberg erectaplants (wild type [WT] Crude), incubated at 22°C for the indicated timeperiods, and subjected to immunoblot analysis with anti-Myc antibody(a-Myc). The PVDF membrane was stained with Memstain to visualizethe large subunit of ribulose-1,5-bisphosphate as a control for equalloading (Loading Control).(C) The purified Myc-COI1 protein was added to total crude proteinextracts from axr6-1/+ plants (axr6 Crude) or Columbia wild-type controlplants (WT Crude), incubated at 22°C for the indicated time periods, andsubjected to immunoblot analysis with anti-Myc antibody (a-Myc).

Figure 6. COI1 Is Degraded via the 26S Proteasome Pathway.

(A) Total protein extracts from the wild-type Arabidopsis Columbia wereincubated at 22°C with DMSO (Mock), MG132 (50 mM), PS341 (50 mM),or protease inhibitor mixture (13 protease inhibitor cocktail from Roche)for 0 (start), 4, and 8 h, separated by SDS-PAGE, and immunoblottedwith COI1 antibody (top panel). The PVDF membrane was stained withMemstain to visualize the large subunit of ribulose-1,5-bisphosphate asa loading control (bottom panel).(B) Quantitative analysis of the relative abundance of COI1 in the pres-ence of proteasome inhibitors. Total protein extracts from wild-typeplants were incubated at 22°C with DMSO (Mock), MG132 (50 mM), orPS341 (50 mM) for the time periods indicated. The abundance of COI1 atthe start point (0-h incubation) was set to 100 as a reference for calcu-lating its relative abundance after various incubation periods. Error barsrepresent SD. The experiment was repeated three times.(C) The total protein extracts were incubated at 22°C with 5 mM ATP orsolvent (Mock) for the indicated time periods and immunoblotted withCOI1 antiserum (a-COI1).[See online article for color version of this figure.]

Regulating Mechanism in COI1 Level 7 of 13

Many F-box proteins are recruited by their respective SCFcomplexes for ubiquitination and degradation via the 26S pro-teasome pathway (Galan and Peter, 1999; He et al., 2005). Toexamine whether the COl1 degradation is dependent on SCFCOI1,we used the in vitro assay to evaluate stability of the truncatedMyc-tagged COI1 lacking the F-box motif (Myc-COI1DF). Wefound that Myc-COI1DF was degraded very rapidly and abolishedcompletely after 4-h incubation with the total protein extractsfrom the wild type (Figures 8A and 8B) and coi1-1 where theSCFCOl1 complexes are absent (Figure 8B), suggesting that COI1was not recruited by SCFCOI1 for degradation. Together with the

observation that the presence of the proteasome inhibitor MG132significantly attenuated degradation of Myc-COI1DF (Figure 8C),our data collectively demonstrated that degradation of COI1is mediated by the 26S proteasome pathway in an SCFCOl1-independent manner.

The 297th Lys Residue of COI1 Is a Ubiquitination Site

Having shown that COI1 is degraded through the ubiquitin/26Sproteasome pathway, we sought to identify possible ubiquiti-nation sites in COI1. Isolated Flag-tagged COI1 was enrichedand purified using anti-Flag agarose affinity gel from the totalprotein extracts of the MG132-treated coi1:HiCOI1 seedlings.The purified COI1 protein was separated on SDS-PAGE; the gelband of interest was excised, alkylated, in-gel digested withtrypsin, and subjected to liquid chromatography–tandem massspectrometry (LC-MS/MS) analysis.By database searching, we identified a peptide ion with the

mass corresponding to the modified peptide (297-KLDLLYAL-LETEDHCTLIQK-316) with an addition of 114 D (mass of Gly-Gly),suggesting that the 297th Lys residue of COI1 is the ubiquitination

Figure 7. The COI1 Protein Level Is Elevated in the 26S ProteasomeMutant rpt5a-4.

(A) Immunoblot analysis of COI1 with COI1 antiserum (a-COI1) in therpt5a-4 (rpt5a) mutant and wild-type Arabidopsis Columbia plants (thewild type [WT]).(B) Real-time quantitative PCR analysis of VSP1 (VSP) transcripts in8-d-old seedlings grown on Murashige and Skoog medium (0) or Murashigeand Skoog medium supplemented with 25 mM MeJA (25). Error barsrepresent SD (n = 6).(C) Anthocyanin accumulation in 8-d-old seedlings grown onMurashige andSkoog medium (0) or Murashige and Skoog medium supplemented withMeJA (5 and 25 mM). The o.d. of (535 to 650 nm)/g fresh weight was re-garded as the Y value (error bars indicate SE). Error bars represent SD (n = 6).

Figure 8. COI1 Is Degraded via the 26S Proteasome Pathway in anSCFCOl1-Independent Manner.

(A) Purified Myc-COI1 and Myc-COI1 without the F-box motif (Myc-COI1DF) were incubated with total crude protein extracts prepared fromthe wild-type Arabidopsis Columbia, incubated at 22°C for the indicatedtime periods, and subjected to immunoblot analysis with anti-Myc anti-body (a-Myc). The PVDF membrane was stained with Memstain to vi-sualize the large subunit of ribulose-1,5-bisphosphate as a control forequal loading (Loading Control).(B) The purified Myc-COI1DF protein were incubated with total crudeprotein extracts prepared from the wild type (wild type [WT] Crude) or thecoi1-1 plants (coi1 Crude) at 22°C and subjected to immunoblot analysiswith anti-Myc antibody (a-Myc).(C) Purified Myc-COI1 lacking the F-box motif (Myc-COI1DF) was addedto total protein extracts from wild-type plants, incubated at 22°C with 50mMMG132 (MG132) or solvent (Mock) for the indicated time periods, andimmunoblotted with anti-Myc antibody.

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site. This has been confirmed by the MS/MS spectrum (Figure9A), in which masses of fragment ions matched to predictedb and y ions of the modified peptides with ubiquitination at the297th Lys residue. Based on the COI1 crystal structure, the 297thLys residue is localized within the 9th tandem Leucine-rich repeat inthe COI1 C-terminal horseshoe-shaped solenoid domain (Figure 9B)(Sheard et al., 2010).

To experimentally verify that the 297th Lys residue of COI1 isa ubiquitination site, we investigated whether changing the 297thCOI1 residue from Lys to Ala, by generating Myc-COI1 (Myc-

COI1K297A), could attenuate COI1 degradation. The Myc-COI1K297A

protein was transiently expressed in tobacco plants, purified withanti-Myc affinity beads, and added to the total wild-type proteinextracts. COI1 degradation was subsequently assessed by im-munoblot analysis with anti-Myc antibody. Myc-COI1K297A wasmarginally more stable compared with the Myc-COI1 protein when

Figure 9. The 297th Lys Residue of COI1 Is a Ubiquitination Site.

(A) The MS/MS spectrum of a doubly charged ion at mass-to-chargeratio (m/z) 844.1127 for MH22+ corresponding to the mass of theubiquitination peptide (297-KLDLLYALLETEDHCTLIQK-316) with thefirst Lys residue was modified. The labeled peaks correspond to massesof b and y ions of the modified peptides. Observation of the addition of114 D (mass of Gly-Gly) for all b ions confirms that 297th Lys residue ofCOI1 is the ubiquitination site.(B) A diagrammatic representation of the mutant site of K297A in theCOI1 protein. The structure of the COI1 protein is shown as a ribbon andthe 297th Lys residue of COI1 is labeled.(C) and (D) The effect of the K297A mutation on COI1 stability by in vitroassays. Myc-COI1 or Myc-COI1K297A was transiently expressed in N.benthamiana leaves, purified with anti-c-Myc agarose affinity gel, addedto total protein extracts from the wild-type Arabidopsis Columbia plants,incubated at 22°C (C) or 27°C (D) for the indicated time periods, andsubjected to immunoblot analysis with anti-Myc antibody. The PVDFmembrane was stained with Memstain to visualize the large subunit ofribulose-1,5-bisphosphate as a loading control.[See online article for color version of this figure.]

Figure 10. A Simplified Model for Regulation of the Dynamic Balancebetween COI1 Stabilization and Destabilization in Arabidopsis.

The COI1 protein is stabilized by interaction with ASK to form the COI1-ASK heterodimers (such as COI1-ASK1), which are further stabilized bythe assembly of SCFCOI1 complexes (such as ASK1-based SCFCOI1) viainteraction with CUL1. The mutation in CUL1 in axr6-1/+, or dissociationof CUL1 from the SCF complex (Hua and Vierstra, 2011), releases theCOI1-ASK heterodimer and might also affect the interaction betweenCOI1 and ASK proteins. The dissociated COI1 protein in the absence ofinteraction with ASK proteins, including the transgenically overexpressedCOI1 proteins and the mutated COI1 proteins that are unable to interactwith ASKs, is recruited by an unidentified E3 ligase for ubiquitination andsubsequent degradation via the 26S proteasome pathway. COI1 proteinis maintained at a proper and stable level in Arabidopsis througha combination of SCFCOI1-mediated stabilization and the 26S protea-some–mediated degradation.

Regulating Mechanism in COI1 Level 9 of 13

incubated at 22°C (Figure 9). When the incubation temperature wasincreased to 27°C, the degradation of Myc-COI1 protein acceler-ated and was not detectable by 8 h of incubation, whereas Myc-COI1K297A was still observed. A higher incubation temperaturemore clearly exposed the differences in protein stability (Figure9D), which further demonstrated that the K297A mutation im-proved the stability of Myc-COI1, suggesting that the 297th Lysresidue is an active ubiquitination site.

DISCUSSION

SCF complexes, an important class of ubiquitin E3 ligases, havebeen extensively studied with respect to their roles in substraterecognition for ubiquitin-mediated proteolysis (Petroski and De-shaies, 2005; Hua and Vierstra, 2011). In plants, the SCF complexesSCFCOI1, SCFTIR1, SCFEBF1/2, SCFSLEEPY (and SCFGID2), SCFFKF1,SCFSKP2A, and SCFZTL are responsible for ubiquitination and sub-sequent degradation of their respective substrate JAZ proteins:auxin/indole-3-acetic acids, EIN3/EIL1, DELLAs, CDF1, E2FC/DPB,and TOC1 (Lechner et al., 2006; Santner and Estelle, 2010; Shanet al., 2012). In this study, we revealed a function of SCFCOI1 byshowing that it is responsible for stabilization of the F-box proteinCOI1 in Arabidopsis.

We found that COI1 abundance is strictly maintained at a properand stable level in Arabidopsis (Figure 1A). Mutations in eitherASK1 or CUL1 clearly reduced COI1 protein stability in Arabi-dopsis (Figures 1C, 2A, and 2B), and when COI1 transcripts wereincreased 10-fold in transgenic Arabidopsis lines, they were unableto significantly elevate the COI1 protein level (Figures 3 and 4). Thein vitro assays showed that purified COI1 protein was unstable andquickly degraded in total protein extracts from the mutant plantswhere either of the core SCFCOI1 subunits ASK1 or CUL1 wascompromised (Figures 5B and 5C). These data collectively sug-gest that integrity of SCFCOI1 plays an essential role in maintainingCOI1 stability in Arabidopsis. Consistent with this, a gel filtrationassay showed that that the majority of COI1 is assembled into thecomplex in Arabidopsis (Feng et al., 2003; data not shown). Itwould be interesting to determine whether other SCF complexes,such as SCFTIR1 involved in auxin response, have a similar novelfunction to stabilize their F-box proteins in Arabidopsis.

We observed that the ASK1 mutation severely decreased, butdid not entirely abolish, the COI1 protein in the ask1-1 mutantplants. The small amount of COI1 protein that remained in theask1-1mutant plants was possibly stabilized by other ASKs-basedSCFCOI1 complexes, as other ASK proteins were also reported tointeract with COI1 (Xu et al., 2002; Takahashi et al., 2004). In ouryeast assays when ASK1 was coexpressed with COI1 (Figure 1B),ASK1 preferentially interacted with COI1, leading to an increasedCOI1 protein level. However, in Arabidopsis, it may not necessarilyfollow that overexpression of ASK1 would obviously increase theCOI1 protein abundance, as is known to interact with many otherArabidopsis F-box proteins (Risseeuw et al., 2003;Lechner et al.,2006), thereby limiting its availability for interaction with COI1.

Degradation of many F-box proteins is dynamically regulatedby an autocatalytic mechanism. Such F-box proteins, includingGrr1, Cdc4, Met30, and FWD1, are recruited by their respectiveSCF complexes, including SCFGrr1, SCFCdc4, SCFMet30, andSCFFWD1, for ubiquitination and subsequent degradation in a

proteasome-dependent manner (Galan and Peter, 1999; Heet al., 2005). Mutations occurring in their F-box motifs have beenshown to attenuate ubiquitination and degradation of theseF-box proteins (Galan and Peter, 1999; He et al., 2005). In thisstudy, we demonstrated that deleting the F-box motif did notattenuate, but on the contrary accelerated the degradation ofCOI1 (Figure 8A). This implies that COI1 degradation is notsubjected to an autocatalytic mechanism. Furthermore, wefound that the 26S proteasome inhibitors MG132 and PS341effectively stabilized COI1 protein (Figure 6) and that COI1accumulated in the 26S proteasome mutant rpt5a-4 (Figure 7).In addition, we showed that the 297th Lys residue of COI1 isone of the active ubiquitination sites (Figure 9). These resultssuggest that COI1 is recruited by another, unidentified E3 li-gase, but not by SCFCOI1 itself, for ubiquitination and sub-sequent degradation via 26S proteasome pathway. Previousstudies showed that the F-box proteins TIR1, EBF1/2, and ZTLwere degraded via the ubiquitin-proteasome pathway in Arab-idopsis (Kim et al., 2007; Stuttmann et al., 2009; An et al., 2010). Itremains to be elucidated whether the degradation of these Arabi-dopsis F-box proteins shares this mechanism with COI1 oremploys the more common autocatalytic mechanism.Our results provide new mechanistic insights into how plant

cells regulate COI1 abundance. We propose that COI1 proteinis strictly regulated by a dynamic balance between SCFCOI1-mediated stabilization and 26S proteasome–mediated degra-dation and maintained at a protein level essential to exert itsproper functions in Arabidopsis (Figure 10). In the wild type, theCOI1 protein is stabilized by interacting with ASK1 and furtherstabilized by assembly into the SCFCOI1 complex. COI1 levelswere lower in the cul1 mutants axr6-1 and axr6-2, where re-duced CUL1 activity potentially limited the assembly of SCFCOI1.The most rapid degradation of COI1 occurred in ask1-1 mutantswhere the COI1-ASK interaction was completely abolished. FreeCOI1 proteins without interaction with ASKs, including the mu-tated COI1 proteins that are unable to associate with ASKs andthe overexpressed COI1 proteins in the transgenic lines, arepredicted to be recruited by a yet unidentified E3 ligase, butnot by SCFCOI1, for targeted ubiquitination and subsequentdegradation via the 26S proteasome. Characterization of thisunidentified E3 ligase would provide a new insight into molecularmechanism regulating the COI1 protein to satisfy the cellular de-mands for the SCFCOI1 activity essential for jasmonate-mediatedplant defense and development.

METHODS

Plant Materials and Growth Conditions

The Arabidopsis thaliana mutants coi1-1, axr6-1 and axr6-2, rpt5a-4, andask1-1 were gifts from J. Turner, M. Estelle, H. Huang, and H. Ma,respectively. Arabidopsis seeds were surface sterilized and plated onMurashige and Skoog medium with 2% Suc and 7 g/L agar, chilled at 4°Cin the dark for 3 d, and then placed in a growth room under a 16-h-light(21 to 23°C)/8-h-dark (16 to 19°C) photoperiod for 10 d. For adult plants,seedlings from plates were transferred to soil and grown to maturity at22°C under a 16-h-light/8-h-dark cycle.

Nicotiana benthamiana seeds were directly sown into soil and grownunder similar growth conditions.

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Plasmid Constructs

The yeast expression construct pBridge-COI1was generated by cloning theCOI1 cDNA into multiple cloning site 2 of the pBridge vector (Clontech). Theyeast coexpression constructs pBridge-ASK1-COI1 and pBridge-JAZ1-COI1were generated by cloning theASK1 or JAZ1 cDNA, respectively, intothe multiple cloning site 1 of the construct pBridge-COI1.

For the constructs used in the N. benthamiana transient expressionsystem (Liu et al., 2010), the COI1 cDNA fragment was in-frame taggedwith the Myc epitope at the 59 terminus and driven by the CaMV 35Spromoter in the plant binary vector (Xu et al., 2002) to generate the Myc-COI1 construct. The Myc-COI1DF and Myc-COI1K297A constructs weresimilarly generated. The point mutation of COI1K297A was introduced bysite-directed mutagenesis using a fast mutagenesis kit (Transgen).

In Figures 3 and 4, the COI1 cDNA fragment was in-frame tagged withthe Flag epitope at the 59 terminus and the HA epitope at the 39 terminusand driven by the CaMV 35S promoter in the plant binary vector (Xu et al.,2002) to generate the Flag-COI1-HA construct. The Arabidopsis-expressed Flag-COI1-HA fusion proteins were detected with anti-HAantibody (Sigma-Aldrich; H6908; Figures 3 and 4).

All the primers using in generation of the plasmid constructs are shownin Supplemental Table 1 online.

Expression of COI1 Protein in Yeast

The constructs pBridge-ASK1-COI1 and pBridge-JAZ1-COI1 weretransformed into the yeast strain AH109. The yeast cultures were grownon SD synthetic medium lacking Trp and Met at 30°C for 16 h. Whole-celllysates were prepared using Yeast Protein Extraction Reagent (Y-PER;Thermo), followed by centrifugation at 14,000g for 15 min. The super-natants of whole-cell lysates were separated by SDS-PAGE, transferredto polyvinylidene fluoride (PVDF) membrane, and detected with anti-COI1antiserum (Xu et al., 2002).

Transient Expression of COI1 in N. benthamiana

Leaves of 4-week-old N. benthamiana were inoculated with Agro-bacterium tumefaciens GV3101 strain carrying the Myc-tagged COI1,Myc-COI1K297A, orMyc-tagged COI1DF constructs following a previouslydescribedmethod (Liu et al., 2010). Two days after infiltration, total proteinwas extracted from infiltrated leaves with extraction buffer (50 mM Tris-Cl,pH 7.8, 100 mMNaCl, 10% [v/v] glycerol, and 20 mM b-mercaptoethanol)and incubated with Anti-c-Myc Agarose Affinity Gel (Sigma-Aldrich) at 4°Cfor 3 to 4 h. After washing, the Myc-tagged proteins were eluted with 50mg/mL Myc peptide (Sigma-Aldrich).

A pull-down assay was used to determine the activity of purified Myc-COI1 as described previously (Yan et al., 2009).

In Vitro Protein Degradation Assay

For the in vitro degradation assay of Myc-COI1, Myc-COI1DF, and Myc-COI1K297A described in Figures 5B, 5C, 8, 9C, and 9D, 5 mg of purifiedprotein was added to 900 mL of total crude protein extracts (1 mg/mL)prepared from young rosette leaves of 4-week-old plants, as indicated.The reaction mixture was incubated at 22°C for the time periods indicatedin the figures, separated by SDS-PAGE, transferred to PVDF membrane,and detected with anti-Myc antibody (Roche).

In Figures 6A to 6C, the total protein extracts (1 mg/mL) from 4-week-old wild-type young rosette leaves were incubated at 22°C for indicatedtime periods with or without MG132 (50 mM), PS341 (50 mM), 13 proteaseinhibitor cocktail (Roche), or ATP (5 mM), separated by SDS-PAGE,transferred to PVDF membrane, and detected with the anti-COI1 anti-serum (Xu et al., 2002).

Anthocyanin Measurement, Pollen Viability, and Pollen Germination

Eight-day-old Arabidopsis seedlings grown on Murashige and Skoogmedium supplemented with MeJA at the indicated concentrations wereused for anthocyanin measurements following the method describedpreviously (Shan et al., 2009). The anthocyanin content is presented as(A535–A650)/ g fresh weight. The experiment was repeated three times.

Pollen grains were resuspended in 6 mg/mL fluorescein diacetate and0.5 mg/mL propidium iodide stain for microscopy to assess pollen viability(Regan and Moffatt, 1990).

The germination medium (10% [w/v] Suc, 0.01% boric acid, 1 mMMgSO4, 5 mMCaCl2, and 5mMKCl, pH 7.5, with 1.5% agar) was used forthe pollen germination assay (Song et al., 2011).

RT-PCR and Real-Time Quantitative PCR

The total RNA extracts were treated with DNase I (NEB) according to themanufacturer’s instructions of the Reverse Transcriptase M-MLV (RNaseH; TaKaRa). RT-PCR amplifications were performed with a limited numberof cycles, ensuring the amplifications were within the linear range, andamplified transcripts were detected by ethidium bromide staining on 2%(w/v) agarose gels. ACTIN1 was used as an internal control.

Real-time quantitative PCR analysis was performed using the ABI 7500real-time PCR system and the RealMasterMix (SYBR Green I; TaKaRa).ACTIN8 was used as the internal control.

Primer pairs used for RT-PCR and real-time quantitative PCR analysisare listed in Supplemental Table 1 online.

MS Analysis

Total protein was extracted from 30 g of young rosette leaves of 4-week-old plants transgenic for the Flag-COI1-HA construct (coi1:Hi-COI1) withextraction buffer (50 mM Tris-Cl, pH 7.8, 100 mMNaCl, 25 mM imidazole,10% [v/v] glycerol, 0.1% [v/v] Tween 20, and 20 mM b-mercaptoethanol)in the presence of MG132. The total protein extracts were concentratedand incubated with anti-Flag agarose affinity gel (Sigma-Aldrich) for 8 h at4°C with gentle rocking. After washing, the purified Flag-tagged COI1proteins were denatured at 98°C and separated by SDS-PAGE. The gelbands at the approximate size of Flag-tagged COI1 were excised, re-duced with 10 mM DTT, and alkylated with 55 mM iodoacetamide(Calbiochem). Then the in-gel digestion was performed with trypsin(Promega) in 50 mM ammonium bicarbonate at 37°C overnight. Thepeptides were extracted twice with 1% (v/v) trifluoroacetic acid in 50%(v/v) acetonitrile aqueous solution for 30 min.

For LC-MS/MS analysis, the trypsin-digested sample was separatedby a 65-min gradient elution at a flow rate of 0.25 µL/min with the EASY-nLCII integrated nano-HPLC system (Proxeon), which directly interfacedwith the Thermo LTQ-Orbitrap Velos mass spectrometer. The analyticalcolumn was a homemade fused silica capillary column (75 µm i.d., 150-mm length; Upchurch) packed with C-18 resin (300 Å, 5 µm; Varian).Mobile phase A consisted of 0.1% (v/v) formic acid, and mobile phase Bconsisted of 100% acetonitrile and 0.1% formic acid. The LTQ-Orbitrapmass spectrometer was operated in the data-dependent acquisitionmode using Xcalibur 2.0.7 software with a single full-scan mass spectrumin the Orbitrap (400 to 1800 mass-to-charge ratio; 30,000 resolution)followed by six data-dependent MS/MS scans in the ion trap at 35%normalized collision energy. The MS/MS spectra from each LC-MS/MSrun were searched against the selected databases using an in-houseMascot or Proteome Discovery searching algorithm.

Accession Numbers

The Arabidopsis Genome Initiative accession numbers for the genes andgene products mentioned in this article are as follows: COI1 (At2g39940),

Regulating Mechanism in COI1 Level 11 of 13

ASK1 (At1g75950), AtVSP1 (AT5G24780), AtCUL1 (At4g02570), RPT5a(At3g05530), ACTIN1 (AT2G37620), and ACTIN8 (AT1G49240).

Supplemental Data

The following material is available in the online version of this article.

Supplemental Table 1. List of Constructs and Primers Used in This Study.

ACKNOWLEDGMENTS

This work was financially supported by the grants from the NationalScience Foundation of China (91017012, 31230008 and 31200214) andthe Ministry of Agriculture (2011ZX08011-006).

AUTHOR CONTRIBUTIONS

J.Y. and H.L. performed all the experiments. S.L. and R.Y. performeddegradation in vitro and LC-MS/MS assay, respectively. H.D. and Q.X.provided useful advice in mass spectrometry analysis. J.Y. and D.X.designed the experiments and wrote the article.

Received September 26, 2012; revised December 16, 2012; acceptedJanuary 12, 2013; published February 5, 2013.

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Regulating Mechanism in COI1 Level 13 of 13

DOI 10.1105/tpc.112.105486; originally published online February 5, 2013;Plant Cell

Jianbin Yan, Haiou Li, Shuhua Li, Ruifeng Yao, Haiteng Deng, Qi Xie and Daoxin XieDegraded via the 26S Proteasome Pathway

andCOI1 F-Box Protein CORONATINE INSENSITIVE1 Is Stabilized by SCFArabidopsisThe

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