CUL3BPM E3 ubiquitin ligases regulate MYC2, MYC3,and MYC4 stability and JA responsesJose Manuel Chicoa,1, Esther Lechnerb,1, Gemma Fernandez-Barberoa, Esther Canibanoa, Gloria García-Casadoc,Jose Manuel Franco-Zorrillac, Philippe Hammannd, Angel M. Zamarreñoe, Jose M. García-Minae, Vicente Rubioa

,Pascal Genschikb, and Roberto Solanoa,2

aDepartamento de Genética Molecular de Plantas, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain;bInstitut de Biologie Moléculaire des Plantes, CNRS, Université de Strasbourg, 67084 Strasbourg, France; cGenomics Unit, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain; dInstitut de Biologie Moléculaire et Cellulaire, CNRS FR1589, Plateforme ProtéomiqueStrasbourg-Esplanade, Université de Strasbourg, 67084 Strasbourg, France; and eDepartment of Environmental Biology, University of Navarra, 31008Navarra, Spain

Edited by Natasha V. Raikhel, Center for Plant Cell Biology, University of California, Riverside, CA, and approved January 30, 2020 (received for review July16, 2019)

The jasmonate (JA)-pathway regulators MYC2, MYC3, and MYC4are central nodes in plant signaling networks integrating environ-mental and developmental signals to fine-tune JA defenses andplant growth. Continuous activation of MYC activity is potentiallylethal. Hence, MYCs need to be tightly regulated in order to optimizeplant fitness. Among the increasing number of mechanisms regulat-ing MYC activity, protein stability is arising as a major player.However, how the levels of MYC proteins are modulated is stillpoorly understood. Here, we report that MYC2, MYC3, and MYC4are targets of BPM (BTB/POZ-MATH) proteins, which act as substrateadaptors of CUL3-based E3 ubiquitin ligases. Reduction of functionof CUL3BPM in amiR-bpm lines, bpm235 triple mutants, and cul3abdouble mutants enhances MYC2 and MYC3 stability and accumula-tion and potentiates plant responses to JA such as root-growth in-hibition and MYC-regulated gene expression. Moreover, MYC3polyubiquitination levels are reduced in amiR-bpm lines. BPM3 pro-tein is stabilized by JA, suggesting a negative feedback regulatorymechanism to control MYC activity, avoiding harmful runaway re-sponses. Our results uncover a layer for JA-pathway regulation byCUL3BPM-mediated degradation of MYC transcription factors.

phytohormone | proteasome | jasmonate signaling | MYC2 |Cullin ring ligases

Jasmonates (JAs) are oxygenated lipid derivatives (oxylipins)synthesized through the octadecanoid and hexadecatrienoic

pathways and chemically similar to prostaglandins in animals (1, 2).JAs are essential phytohormones for plant development and en-vironmental adaptation, since they 1) are key regulators of re-sponses to biotic and abiotic stresses, 2) coordinate severaldevelopmental processes such as root growth and fertility, and3) fine-tune competitive growth–defense tradeoffs that optimizeplant fitness in response to resource limitations (1, 3).Upon stress or endogenous stimuli, plants accumulate the bio-

active JA (+)-7-iso-JA-Ile (JA-Ile) (4, 5), which is perceived by acoreceptor complex formed by the F-box protein CORONATINEINSENSITIVE 1 (COI1) and a JASMONATE-ZIM (JAZ) do-main protein (6–8). COI1 is the F-box subunit of the SCFCOI1

(Skip1-Cullin1-F box) E3 ubiquitin ligase (9, 10). JAZs act asnuclear repressors of an increasing number of transcription factors(TFs) belonging to different families such as bHLH, MYB,YABBY, and EIN3/EIL (1, 11–15). The JAZ proteins exert theirrepressor activity by a double mechanism involving the recruit-ment of the general corepressor TOPLESS (TPL) by the adaptorprotein NOVEL INTERACTOR OF JAZ (NINJA) (16) andthrough competition with the MEDIATOR25 (MED25) compo-nent of the general transcriptional activation machinery for in-teraction with MYCs (17). Hormone-triggered interaction ofCOI1 and JAZs leads to JAZ ubiquitination and degradation bythe ubiquitin-proteasome system (UPS) (6, 7, 18, 19). Degradation

of JAZs releases their TF targets that, in turn, induce a substantialtranscriptional reprogramming (20–24).Among JAZ targets, MYC2, MYC3, and MYC4 are key tran-

scriptional regulators of JA-mediated gene expression that belongto the bHLH family of TFs (11, 25). JA-dependent transcriptionalactivation by MYCs requires recruitment of MED25, a subunit ofthe MEDIATOR transcriptional coactivator complex (26). BothMYC2 and MED25 regulate a dynamic chromatin looping be-tween JA enhancers and their promoters (27). Continuous acti-vation of JA responses by MYCs inhibits growth and is potentiallyharmful or even lethal for the cell. Thus, many mechanisms forresetting JA signaling and terminating MYC activity have beendescribed, including the MYC-dependent expression of JAZ andbHLH repressors, alternative splicing forms of JAZ, etc. (28, 29).Recently, even a function of MYC2 in regulating the termination

Significance

Jasmonates (JAs) are essential phytohormones regulating amyriad of developmental and defensive responses in plants.MYC2, MYC3, and MYC4 are key transcriptional activators ofJA-mediated gene expression and have become relevant sig-naling hubs. Repression of MYC activity is necessary for re-setting JA signaling and to avoid harmful runaway responses,which contribute to plant fitness. Here, we identified a mech-anism to reduce MYC protein levels by E3 ubiquitin ligases basedon Cullin3 and BPM proteins as substrate adaptors (CUL3BPM).BPM3 stability is enhanced by JA, establishing a negative feed-back regulatory loop to control MYC levels and activity. Ourresults uncover a new layer of JA-pathway regulation that ter-minates MYC activity by CUL3BPM-mediated degradation ofMYC TFs.

Author contributions: P.G. and R.S. designed research; J.M.C., E.L., G.F.-B., E.C., G.G.-C.,J.M.F.-Z., P.H., and A.M.Z. performed research; V.R. supervised ubiquitin experiments;J.M.C., E.L., G.F.-B., G.G.-C., J.M.F.-Z., P.H., A.M.Z., J.M.G.-M., V.R., P.G., and R.S. analyzeddata; and R.S. wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.

Data deposition: Transcriptomic data reported in this paper have been deposited in theGene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo/ (accessionnos. GSE131024 [“Transcriptomic profile of bpm2,3,5 triple mutant and Col-0”] andGSE131037 [“Transcriptomic profile of amiR-bpm and Col-0”]). The mass spectrometry proteo-mics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifierPXD013906.1J.M.C. and E.L. contributed equally to this work.2To whom correspondence may be addressed. Email: rsolano@cnb.csic.es.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1912199117/-/DCSupplemental.

First published March 2, 2020.

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of JA signaling through activation of a small group of bHLH re-pressors that impair the formation of the MYC2–MED25 complexhas been reported (30). Another repression mechanism includesJAV1 [JASMONATE-ASSOCIATED VQ-MOTIF GENE1(14)], a repressor of JA-mediated defenses that is degraded afterherbivory by JAV1-ASSOCIATED UBIQUITIN LIGASE1[JUL1 (31)].In addition to JA signaling and defense, MYCs have been

involved in the regulation of other processes such as responses toabscisic acid (ABA), ethylene, or blue light (32–35). Therefore,MYCs are currently considered a node of convergence of severalpathways, whose activity needs to be tightly regulated to optimizeplant fitness. MYCs are short-lived proteins and their tran-scriptional activity requires phosphorylation, which also triggersMYC proteolysis (36, 37). Besides phosphorylation, MYC pro-tein stability is regulated by JA, light quality, and the circadianclock, suggesting that modulation of protein levels is a majorregulatory mechanism of MYC protein activity (36–39).Several regulators of MYC protein stability have been described.

For instance, the E3 ubiquitin ligase PUB10 targets MYC2 forproteasomal degradation (40). PUB10 affects JA-related geneexpression and phenotypic responses, but its involvement inmodulation of MYC2 protein levels by environmental cues has notbeen reported so far. Similarly, COP1 promotes MYC degrada-tion in the dark, whereas photoreceptors counteract this activity inlight (37). However, the importance of MYCs as nodes of con-vergence of signaling networks and activators of responses that arepotentially harmful for the cell suggests that MYC activity has tobe tightly regulated and that other regulatory mechanisms of MYCstability could be expected.The BPM (BTB/POZ-MATH) proteins are adaptors of Cullin3-

based E3 ubiquitin ligases in animals and plants, and form a smallfamily of six members in Arabidopsis [AtBPM1 (At5g19000),AtBPM2 (At3g06190), AtBPM3 (At2g39760), AtBPM4(At3g03740), AtBPM5 (At5g21010), and AtBPM6 (At3g43700)(41–45)]. They are characterized by the presence of two protein–protein interaction domains: BTB/POZ (broad complex, tram-track, bric-a-brac/POX virus, and zinc finger domain) and MATH(Meprin and TRAF homology domain). The BTB/POZ domainmediates assembly of BTB/POZ proteins with CUL3a and CUL3bin plants and animals. The MATH domain determines the in-teraction of BTB/POZ-MATH proteins with their substrates (10,41, 46–52).CUL3BPM E3 ligases are involved in the regulation of several

physiological processes such as plant growth, fertility, stomataldynamics, fatty acid metabolism, and ABA signaling, but theirtargets are still scarce (43, 44, 53).Here, we identified MYC2, MYC3, and MYC4 as interactors

of BPM proteins in vivo and show that CUL3BPM E3 ligasestarget MYC2 and MYC3 for UPS-mediated degradation. Indeed,reduction of function of CUL3BPM (amiR-bpm) reduces ubiquiti-nation levels of MYC3, enhances MYC accumulation, and rendersthe plants more responsive to JA. Similarly, triple bpm235mutantsshow JA-related phenotypes and a constitutive JA-regulated geneexpression. Reduction of CUL3 function in the double mutantcul3ab also enhances JA responses, altogether indicating thatCUL3BPM regulates MYC activity. Interestingly, BPM3 protein isstabilized by JA, suggesting a negative feedback regulatory mech-anism to control MYC levels and activity. Our results uncover anew tier of JA-pathway regulation by CUL3BPM-mediated degra-dation of MYC TFs.

ResultsMYC2, MYC3, and MYC4 Physically Interact with BPMs. In order toidentify new targets of BPMs, we performed a yeast two-hybrid(Y2H) screen using BPM3 as bait and an Arabidopsis comple-mentary (c)DNA library prepared from Arabidopsis inflores-cences. From over 2 million clones screened, 80 were subsequently

confirmed by retransformation into yeast (43). Among theseclones, we identified two independent prey clones corresponding toMYC2 (MYC228–292 and MYC2187–509). These two clones delimita small MYC2 region of interaction with BPM3 (MYC2187–292),which is highly conserved within the MYC2 partners MYC3 andMYC4 (SI Appendix, Fig. S1). Y2H assays using all six BPMs asbait and the three MYCs as prey showed that MYC2 and MYC3interact strongly with BPM1, BPM2, BPM3, and BPM4 and veryweakly with BPM5 and BPM6 (Fig. 1A and SI Appendix, Fig.S2). In the case of MYC4, only a strong interaction with BPM2could be observed. However, due to the toxicity of MYC ex-pression in yeast, these interactions likely represent only a subsetof all possible interactions in vivo.To further test BPM–MYC interactions, we conducted pull-

down experiments using maltose-binding protein (MBP)–MYCprotein fusions bound to amylose resin and protein extracts fromtransgenic plants expressing hemagglutinin (HA)-BPM3 or myc-BPM6 from the 35S CaMV promoter (SI Appendix, Fig. S3). Asshown in Fig. 1 B and C, similar to the positive control MBP-BPM3 that homodimerizes with HA-BPM3, all three MBP–MYC fusions pulled down both HA-BPM3 and myc-BPM6from these extracts.To find out if BPM–MYC interaction occurs in vivo, we

attempted to identify proteins bound to BPM6 after immuno-precipitation of green fluorescent protein (GFP)-BPM6 fromtransgenic 35S:GFP-BPM6 lines (Materials and Methods) treatedor not with MeJA by mass spectrometry analysis. As shown inFig. 1D, SI Appendix, Table S1, and Dataset S1, several BPMs(BPM1, BPM4, and BPM5) were coimmunopurified by GFP-BPM6, both in the presence and absence of MeJA treatment,supporting the previous observation that BPMs can form heter-odimers in vivo (41, 43). More strikingly, these assays identifiedMYC2, MYC3, MYC4, and JAZ1 both in untreated and JA-treatedplants and NINJA in untreated plants, but not in 35S:GFP controls(Fig. 1D, SI Appendix, Table S1, and Dataset S1). These data sug-gested that MYCs are direct interactors of BPMs in vivo. AlthoughMeJA might potentiate the interaction, particularly in the case ofMYC2 andMYC4, it also occurs in the absence of MeJA treatment.

BPMs Regulate MYC Stability. Protein–protein interaction resultsdescribed above suggested that MYC2, MYC3, and MYC4 couldbe targets of CUL3BPM E3 ubiquitin ligases. Therefore, we nextintrogressed MYC2-GFP from two independent 35S:MYC2-GFP lines into a previously described amiRNA knockdown(KD) BPM line (amiR-bpm), where transcripts for several BPMfamily members are significantly reduced (43). After confirma-tion that, similar to the original lines, the crossed lines main-tained a lower expression level of BPM1, BPM4, BPM5, andBPM6 (SI Appendix, Fig. S4), we analyzed the effect of BPMreduction on MYC2 protein levels. As shown in Fig. 2A, MYC2accumulated more in the mutant amiR-bpm than in wild-type(WT) plants, despite MYC2-GFP transgenic expression beingsimilar or even slightly lower in amiR-bpm (Fig. 2A and SI Ap-pendix, Fig. S5). Since MYC2 is constitutively expressed from the35S promoter in the transgenic lines, we also analyzed its deg-radation rate after inhibition of protein synthesis by cyclohexi-mide (CHX) (Fig. 2B). In WT plants, MYC2 is a short-livedprotein with a rapid turnover that almost disappeared after 3 h.In contrast, MYC2 degradation was much slower in amiR-bpm,with higher levels remaining at all time points.To confirm these results, we analyzed MYC2-GFP levels in a

“recombineering” line in which MYC2-GFP is expressed underits natural promoter in its natural chromosomal environment[recombineering line MYC2 C5025 (54, 55)]. These lines wereobtained by introducing the GFP marker by homologous re-combination in bacteria using big chromosomal pieces containingthe gene of interest and transforming the recombineered chro-mosomal piece back into transgenic plants (54). Similar to the

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35S:MYC2-GFP line, the recombineering MYC2-GFP showed anenhanced stability after CHX treatment (Fig. 2C).Finally, we also analyzed the effect of BPM reduction on

MYC3-HA protein levels using the amiR-bpm background. Asshown in Fig. 2D, results of MYC3 were similar to those ofMYC2, indicating that MYC3 is also a target of CUL3BPMs.We have previously shown that MYC2 protein stability is re-

duced in the dark through COP1 activity (37). To test if BPMscould also have a role in this process, we analyzed MYC2 levelsin WT and amiR-bpm plants under white light or after transfer todarkness for 24 h. Consistent with the regulation of this processby COP1, destabilization of MYC2 was similar in both lines,indicating that BPMs are not involved in light/dark regulation ofMYC2 (SI Appendix, Fig. S6).

CUL3BPMs Ubiquitinate MYC3. To further confirm that elevatedMYC protein levels in the amiR-bpm background correlate withprotein ubiquitination, we analyzed whether a representativemember of the family (MYC3) is ubiquitinated in vivo and whetherubiquitination levels are dependent on BPMs. We chose MYC3,since this protein accumulates to higher levels than other MYCsand is easier to detect. We used p62 resin columns to purifyubiquitinated proteins in WT and amiR-bpm lines expressingMYC3-HA. As shown in Fig. 3A, the MYC3 lanes showed ahigher-size smear of polyubiquitinated bands when the plants wereincubated with proteasome inhibitors overnight, demonstratingthat MYC3 is ubiquitinated in vivo. Remarkably, after purificationof ubiquitinated proteins with the p62 resin, the polyubiquitinated-protein smear was lower in the amiR-bpm background comparedwith the WT. Quantification of ubiquitinated levels of MYC3-HAin WT and amiR-bpm lines in three independent experimentsshowed a consistent reduction of over 40% in all three experi-ments (Fig. 3B). Considering that amiR-bpm lines are only a re-duction of BPM function, these results strongly support a majorrole of CUL3BPMs in ubiquitination of MYC3.

Knocking Down BPM Function Increases JA Responses. Results de-scribed above suggest that BPMs could redundantly regulateMYC levels and, therefore, activity. To test this hypothesis, weanalyzed typical JA responses in amiR-bpm and bpm mutants. Wefirst attempted to obtain and characterize individual bpm mutantsand combinations. We confirmed that available lines in stockcenters for bpm2 (GK_391EO4), bpm4 (Salk_082761), and bpm5(Salk_038471) were loss-of-function mutations. Line Salk_72848,however, seemed to be a reduction of function of bpm3 (SI Ap-pendix, Fig. S7). Phenotypic analyses suggested functional re-dundancy among BPMs due to the lack of JA-related phenotypesin single mutants and some double combinations (bpm23, bpm25,bpm34, bpm35). However, triple bpm235 (SI Appendix, Fig. S8)showed a root-growth phenotype similar to that of amiR-bpm and35S:MYC2 overexpressing lines, both in control conditions andafter treatments with JA or COR (Fig. 4) (25). In control conditions,

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Fig. 1. MYC2, MYC3, and MYC4 interact with several BPM proteins. (A)Yeast cells cotransformed with MYC2, MYC3, and MYC4 fused to the GAL4activation domain (prey) and with BPM1 to BPM6 fused to the GAL4–DNA-binding domain (bait) were selected and subsequently grown on yeast syn-thetic dropout lacking Leu and Trp (−2) as a transformation control (shown inSI Appendix, Fig. S2), or on selective media lacking Ade, His, Leu, and Trp (−4)to test protein interactions. One-third and 1/10 dilutions were included.pGADT7-MYC2, pGADT7-MYC3, and pGADT7-MYC4 cotransformations withpGBKT7 empty vectors were included as controls. (B and C) Immunoblots withanti-HA antibody of recovered HA-BPM3 (B) or with anti-myc antibody of re-covered myc-BPM6 (C) after pull-down reactions using crude protein extractsfrom 35S:HA-BPM3 (+; B), 35S:myc-BPM6 (+; C), or Col-0 (−) Arabidopsis plantsand resin-bound MBP or MBP-fused MYC and BPM3 proteins. Coomassie blue

staining shows the amount of recombinant proteins used (Bottom). Asterisksshow an unspecific band in MYC3 (B) and MYC4 (C). (D) Graphical repre-sentation obtained from the proteomic analysis, depicting the GFP-BPM6–enriched proteins. Proteins are ranked in a volcano plot according to theirrelative abundance ratio (BPM6/control) and the statistical analysis wasperformed by a negative binomial regression (adjusted P values). For eachprotein, the average number of spectra from the control samples (n = 3) wascompared with the average number of spectra from the GFP-BPM6 samples(n = 3). The vertical red dashed lines display large-magnitude fold changes (xaxis, FC < 0.5, Left; FC > 2, Right) whereas the horizontal red dashed linesdisplay high statistical significance (y axis, adjusted P < 0.05 above the line).Green dots represent enriched BPM proteins exhibiting significant foldchanges, whereas red dots represent enriched proteins of interest, amongwhich are MYC proteins. Proteins associated with the volcano plot repre-sentation are listed in SI Appendix, Table S1 and Dataset S1.

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all three lines (35S:MYC2, bmp235, and amiR-bpm) have a slightlyshorter root than the WT, indicative of a mild constitutive JAresponse. Quantification of basal JA levels in these plants showedthat, similar to 35S:MYC2, amiR-bpm lines had increased levels ofJA in control conditions, whereas bpm235 mutants had a highermean JA level but not statistically different from the WT (SIAppendix, Fig. S9). This suggests that the root-growth phenotypein amiR-bpmmay be the consequence of enhanced basal JA levels.However, this is unlikely the case with bpm235. Treatments withJA or coronatine (a JA-Ile mimic of bacterial origin) inhibitedroot growth in WT plants (Fig. 4 A–C) in a percentage similar tothat of 35S:MYC2, bpm235, and amiR-bpm lines, whose final rootlength was shorter thanWT plants. The shorter final root length in35S:MYC2 lines is the result of a stronger response to JA due toelevated MYC2 levels. The similar behavior of bpm235 and amiR-bpm lines to that of 35S:MYC2 transgenics suggests a stronger JAresponse in these lines and is consistent with elevated levels ofMYCs in bpm235 and amiR-bpm lines. Importantly, the effect ofamiR-bpm on root-growth inhibition was partially suppressed injin1-2, a MYC2 loss-of-function allele (25), which confirms thatthe shorter root in basal conditions is due to a constitutive re-sponse to JA of the mutant lines and is consistent with an effect ofBPMs on the activity of several MYCs (Fig. 4D). Altogether, theseresults show that BPMs and MYC2 have opposite effects on rootgrowth and suggest that MYCs are targets of CUL3BPMs in vivo.To further support this hypothesis, we also analyzed JA re-

sponses in cul3 mutants. Since a complete loss of function of thetwo CUL3 genes (CUL3a and CUL3b) is lethal, we used a weakcul3ab allele (56). Additionally, CUL3 also participates in eth-ylene (ET) biosynthesis assembling CUL3ETO1 E3 ligases thattarget ACS5 (48, 56, 57). Thus, cul3ab mutants accumulateethylene and have a strong phenotype. To reduce this ETphenotype, we used a triple cul3ab,ein3 mutant and comparedit with its ET-insensitive ein3 background. As shown in Fig. 5, thecul3ab,ein3 mutant had a stronger response to all JA or CORconcentrations, compared with its ein3 background, indicatingthat the reduction of CUL3 activity in cul3 mutants also leads toenhanced JA response and behaves as the KD mutations inBPMs or overexpression of MYC2.

Knocking Down BPM Function Increases JA-Dependent Gene Expression.Transcriptomic analyses of the amiR-bpm line and bpm235 mu-tants further supported the role of BPMs regulating MYC stability

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Fig. 2. MYC2 and MYC3 accumulate in the amiR-bpm background. (A and B) Immunoblot analyses of MYC2-GFP and actin protein levels in 35S:MYC2-GFPlines in WT or amiR-bpm (ami) backgrounds. “#1” and “#2” indicate two independent 35S:MYC2-GFP transgenic lines. Loading of total protein extracts in A isindicated by 1 (18 μL) and 1/3 (6 μL). To monitor MYC2-GFP protein decay in B, seedlings were treated with 50 μM CHX and protein levels were analyzed at theindicated times (h). (C) Immunoblot analysis of MYC2-GFP and actin protein levels in wild-type and amiR-bpm backgrounds. To check MYC2-GFP proteindecline, seedlings were treated with 50 μM CHX and protein levels were analyzed at the indicated times (h). Expression of MYC2-GFP under its naturalpromoter was assessed using the MYC2 C5025 recombineering line. (D) Immunoblot analysis of MYC3-HA and actin protein levels in 35S:MYC3-HA lines in WTand amiR-bpm backgrounds. #1 and #2 indicate two independent 35S:MYC3-HA transgenic lines. To check MYC3-HA protein levels, seedlings were treatedwith 50 μM CHX and protein levels were analyzed at the indicated times (h). Protein molecular mass is shown (Right).

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Fig. 3. MYC3 polyubiquitination levels are dependent on BPMs. (A) Affinitypurification of polyubiquitinated MYC3-HA was carried out from total proteinextracts obtained fromMYC3-HA seedlings inWT (M3wt) and amiR-bpm (M3ami)backgrounds by incubation with Ub-binding p62 resin (+) or with empty agaroseresin (negative control; −). Anti-Ub was used to detect total ubiquitinated pro-teins. Anti-HA allowed detection of MYC3-HA and its ubiquitinated forms (shownby brackets). To facilitate visualization and quantification of results, the amountsof M3ami andM3wt loaded into the gel were adjusted to obtain similar amountsof the main MYC3-HA band. PD, pull-down. (B) Protein-level analysis of samplesdescribed in A was carried out using ImageJ software. The ratio of poly-ubiquitinated MYC3-HA [Ub(n)-MYC3-HA] compared with unmodified MYC3-HA is shown for M3wt and M3ami samples. Results correspond to three in-dependent experiments.

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(Fig. 6). COR-treated amiR-bpm showed 121 genes differentiallyup-regulated and 77 genes differentially down-regulated comparedwith COR-treated wild-type plants (false discovery rate [FDR] <0.01; fold change [FC] > 2; ref. 58). Among the differentially up-regulated genes, more than 27% (considering FDR < 0.01) or 43%(considering FDR < 0.001) are JA-regulated genes (up-regulatedby MeJA after 0.5, 1, or 3 h according to the BAR database [http://bar.utoronto.ca]; Fig. 6 A and B). These observed values contrastwith the expected number of JA-regulated genes by chance (1.3%considering FDR < 0.01 of BAR data; Fig. 6B). Moreover, geneontology (GO) analysis of up-regulated genes in amiR-bpm showeda statistically significant overrepresentation of JA-related GOterms, such as JA response and wounding (Fig. 6C). These resultsindicate that reduction of BPM activity has a deep impact en-hancing JA-regulated gene expression and support that amiR-bpmplants have a stronger response to JA. Under basal conditions,amiR-bpm plants differentially expressed 172 genes (112 up-regulated and 60 down-regulated) compared with WT (FDR <0.01; FC > 2; SI Appendix, Fig. S10; ref. 58). In this case, thenumber of JA-regulated genes among those differentially regulated

genes was lower but still significant (about 13.3% consideringFDR < 0.01).Results were even clearer in the case of the bpm235 mutant

(Fig. 6 D–F). Under basal conditions, 531 genes were differentiallyexpressed in bpm235 compared with the WT (500 up-regulatedand 31 down-regulated; FDR < 0.01; FC > 2 or <−2; ref. 59).Among up-regulated genes, more than 44% (considering FDR <0.01) or 50% (considering FDR < 0.001) are JA-response genes(induced by MeJA after 0.5, 1, or 3 h according to the BAR da-tabase; Fig. 6 D and E). Moreover, GO analysis of up-regulatedgenes in bpm235 showed a statistically significant over-representation of JA-related GO terms, such as JA response, JAsignaling, wounding, and defense (Fig. 6F). Besides JA, other GOterms such as water transport, water deficit, or ABA were over-represented in our GO analysis. This alteration in basal gene ex-pression is consistent with BPMs regulating other pathways (inaddition to JA), as previously reported (43, 45).

MYC-Dependent Gene Expression Is Up-Regulated in KD bpm Lines.To further contrast if MYCs are targets of CUL3BPMs, we nextchecked if genes up-regulated in amiR-bpm were targets ofMYCs. Toward this, we obtained transcriptomic profiles of triplemyc2myc3myc4 mutants compared with WT Col-0 plants afterJA treatment for 6 h using Agilent microarrays. As shown in Fig.7A, clustering analysis showed that genes differentially down-regulated in myc2myc3myc4 in response to JA are mostly up-regulated in the bmp235 and amiR-bpm backgrounds. Interest-ingly, the overlap in misregulated genes in bmp235 and amiR-bpmwas relatively low (Fig. 7A and SI Appendix, Fig. S11), indicatingthat they affect complementary JA-regulated gene sets. This isconsistent with the different genes knocked out (or down) in thetwo different mutants (bpm235 is a knockout mutant for BPM2and 5 and a knockdown for BPM3, whereas amiR-bpm is aknockdown for BPM4 and 5 and a weak knockdown for BPM1 and6). Therefore, these results suggest that different BPMs may havea complementary function in the regulation of particular JAresponses.

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Fig. 4. amiR-bpm and bpm235 show increased response to JA and COR. (A)Root-growth inhibition assay of 10-d-old Arabidopsis wild-type Col-0 seed-lings, amiR-bpm line, bpm235 triple mutant, and 35S:MYC2 transgenic plants(OEMYC2) grown on Johnson’s media supplemented with JA or coronatineat the indicated concentrations. C, control. (Scale bar, 10 mm.) (B) Quanti-fication of the root length of plants described in A treated with JA. (C)Quantification of the root length of plants described in A treated with COR.(D) Quantification of the root length of 8-d-old Arabidopsis wild-type Col-0seedlings, amiR-bpm line, jin1-2, and two amiR-bpm lines (7.2 and 7.4) inthe jin1-2 mutant background grown on Johnson’s media supplementedwith 50 μM JA or 0.5 μM COR. Results shown in the graphs are the mean ±SD of measurements of 15 to 20 seedlings. Asterisks indicate statisticallysignificant differences compared with WT (Student’s t test, *P < 0.01) andletters indicate statistically significant differences compared with WT andbetween different genotypes (Student’s t test, *P < 0.01).

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Fig. 5. CUL3 knockdown increases responses to JA and COR. Root-growthinhibition assay of 10-d-old Arabidopsis wild-type Col-0 seedlings and ein3-1and cul3ab ein3 mutants grown on Johnson’s media supplemented with JA(A) or COR (B) at the indicated concentrations. Results shown are the mean ±SD of measurements of 15 to 20 seedlings. Asterisks indicate statisticallysignificant differences compared with ein3-1 (Student’s t test, *P < 0.01).

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At1g14120 DAO2 At1g43160 RAP2.6 At1g52430 At1g61110 ANAC025 At1g61120 TPS4 At1g62760 ATPMEI10 At1g64160 DIR5 At1g66280 BGLU22 At1g68620 At1g73260 KTI1 At2g21640 At2g22760 At2g29440 GST24/GSTU6 At2g29460 GST22 At2g30830 At2g36590 PROT3 At2g37770 AKR4C9 At2g47950 At3g09940 MDAR3 At3g11480 BSMT1 At3g24982 RLP40 At3g25180 CYTOCHROME P450 At3g25780 AOC3 At3g46660 UGT76E12 At3g49620 DIN11 At4g15230 ABCG30 At4g21830 MSRB7 At4g23420 At4g27140 SESA1 At4g29570 At5g12020 HSP17 At5g53710 At5g63450 CYP94B1

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Fig. 6. BPMs repress JA-mediated gene expression. (A) BAR data (FC ≥ 2 and FDR < 0.01) showing MeJA regulation of representative genes up-regulated inthe transcriptomic profiling of amiR-bpm vs. Col-0 plants both treated with 0.5 μM COR for 6 h. (B) Percentage of expected (MeJA BAR) or observed (amiR-bpm) JA–up-regulated genes among those differentially expressed in the same transcriptomic analysis. MeJA BAR: % of genes expected to be induced by JAconsidering the whole-genome data of JA treatments (0.5, 1, and 3 h) in the BAR database using FC ≥2 and FDR (rank product) <0.01, <0.005, or 0.001. amiR-bpm: % of genes induced or repressed by MeJA treatment within a list of genes up-regulated by coronatine treatment. (C) GO enrichment analysis of up-regulated genes in amiR-bpm vs. WT using DAVID bioinformatics resources. P values were adjusted by the Benjamini–Hochberg method. JA-dependentbiological processes are highlighted in red. (D) BAR data (FC ≥ 2 and FDR < 0.01) showing MeJA regulation of the 20% most up-regulated genes in thetranscriptomic profiling of bpm235 vs. Columbia plants in basal conditions (untreated). (E) Percentage of expected (MeJA BAR) or observed (bpm235) JA–up-regulated genes among those differentially expressed in the same transcriptomic analysis. MeJA BAR: % of genes expected to be induced by JA consideringthe whole-genome data of JA treatments (0.5, 1, and 3 h) in the BAR database using FC ≥2 and FDR (rank product) <0.01, <0.005, or <0.001. bpm235: % ofgenes induced by MeJA treatment within the list of genes up-regulated in basal conditions in bpm235. (F) GO enrichment analysis of up-regulated genes inthe triple mutant bpm235 vs. WT using DAVID bioinformatics resources. P values were adjusted by the Benjamini–Hochberg method. JA-dependent biologicalprocesses are highlighted in red. The columns in A and D correspond to the 0.5, 1, and 3 h time points of BAR data.

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MYC TFs recognize the G box (5′-CACGTG-3′) or G-box var-iants (5′-AACGTG-3′; 5′-CATGTG-3′) in their cognate promoters(60, 61). As shown in Fig. 7 B and C, analyses of the promoters (0.5or 1 kb upstream of the transcriptional start site) of genes up-regulated in the amiR-bpm or bmp235 lines showed a significantoverrepresentation of these motifs (G box and 5′-AACGTG-3′ inboth lines and 5′-CATGTG-3′ only in bpm235). These resultsfurther support that up-regulation of these genes is mediated byan enhanced activity of MYCs in the KD bpm backgrounds.

JA Stabilizes BPM3. Results described above suggest a mechanismto terminate MYC activity and reset JA signaling by CUL3BPMs.Therefore, we next investigated if JA signaling regulates BPMactivity. BPM genes do not seem transcriptionally regulated by JA(according to our own transcriptomic data, the BAR database, andthe Genevestigator database, as shown in SI Appendix, Fig. S12).To check if JA could regulate BPMs posttranscriptionally, weanalyzed myc-BPM3 and myc-BPM6 stability after JA treatmentin time-course experiments. As shown in Fig. 8A, the amount ofmyc-BPM3 protein was very low in untreated conditions, despitethe myc-BPM3 transgene being expressed constitutively under thestrong 35S promoter. Interestingly, JA treatment greatly stabilizedmyc-BPM3 protein (Fig. 8A). Accumulation was visible after 1 hof treatment and increased to very high levels after 3 h. Sub-cellular fractionation of a different fusion protein (HA-BPM3) inthe absence or presence of JA confirmed these results (JA stabi-lization) and showed that, consistent with previous reports (43),BPM3 is a nuclear protein (Fig. 8B). Simultaneous treatment ofHA-BPM3 transgenic plants with JA and the COI1 specific in-hibitor coronatine-O-methyloxime (COR-MO) (62) prevented thestabilizing effect of JA on HA-BPM3, indicating that this effect of

JA is dependent on the COI1 receptor and, therefore, on the JA-signaling pathway (Fig. 8C).Finally, inhibition of translation by cycloheximide or protea-

some activity by MG132, bortezomib, or epoxomicin showed thatBPM3 is a short-lived protein, degraded by the proteasome, aspreviously described (Fig. 8D and SI Appendix, Fig. S13A) (43).In contrast to BPM3, BPM6 accumulation is not altered by JA

(SI Appendix, Fig. S13B). Moreover, BPM6 seems to be a morestable protein than BPM3, whose levels do not decay after 4 h ofCHX treatment (SI Appendix, Fig. S13C).

DiscussionMYC2, MYC3, and MYC4 are central nodes in plant signalingnetworks integrating environmental and developmental signalsand balancing multiple physiological responses for plant fitness(28, 32–35). Their role in activating plant defenses has thetradeoff of inhibiting growth. Runaway activation of these TFs

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Fig. 7. MYC-dependent gene expression is up-regulated in KD bpm lines. (A)Hierarchical clustering analysis of genes down-regulated (log ratio <−1; FDR <0.05) in the triple myc2,3,4 mutant in response to JA (myc234) and their ex-pression in knockdown bpm lines (amiR-bpm) after COR treatment and in un-treated triple bpm2,3,5 mutant plants. (B and C) The MYC binding sites (G boxand/or G-box variants) are overrepresented in the promoters of genes up-regulated in amiR-bpm and bpm235. Percentage of expected (genome) or ob-served (amiR-bpm or bpm235) boxes (G box or variants) in differentially up-regulated genes in amiR-bpm or bpm235. Genome: % of genes expected tocontain the G box or G-box variants (1 or 0.5 kb upstream of the TSS) consideringthewhole genome obtainedwith the dna-pattern tool in RSAT (http://www.rsat.eu).amiR-bpm and bpm235: % of genes containing the G box or G-box variants withinthe list of genes up-regulated in both genotypes. Asterisks indicate statistically sig-nificant differences compared with the genome (binomial, *P < 0.01).

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Fig. 8. JA induces BPM3 accumulation. (A) Immunoblot analysis (anti-mycantibody) of myc-BPM3 protein levels in 7-d-old 35S:myc-BPM3 transgenicplants untreated or treated with 100 μM JA at the indicated times. Ponceaustaining was used as protein loading control. (B) Immunoblot analysis of HA-BPM3 and histone 2B protein levels in 9-d-old 35S:HA-BPM3 transgenic plantsuntreated (control; C), treated for 4 h (4 h), or grown for 9 d (9 d) in 50 μM JA.Cytoplasmic (S) and nuclear (N) proteins were isolated and tested with anti-HAand histone 2B antibodies, respectively. Histone 2B was used as a control ofcytoplasmic and nuclear fraction isolation. (C) Immunoblot analysis of HA-BPM3 and histone 3 protein levels in 7-d-old 35S:HA-BPM3 transgenic plantsuntreated, treated for 3 h with 100 μM JA, or treated with 100 μM JA plus10 μM COR-MO. Nuclear proteins were isolated and tested with anti-HA andhistone 3 antibodies, respectively. (D) Immunoblot analysis of myc-BPM3 andactin protein levels in 35S:myc-BPM3 transgenic plants. Seven-day-old seed-lings were treated overnight with 50 μM MG132 and then untreated ortreated with 50 μM CHX, harvested after 0.5 or 1 h, and tested with anti-mycor anti-actin antibodies, respectively. Protein molecular mass is shown (Right).

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would be very harmful or even lethal for the cell and thereforeneeds to be tightly regulated spatially and temporally.Many mechanisms to ensure a timely repression of these TFs

have been uncovered. For instance, phosphorylation, which isrequired for MYC2 activation, also triggers MYC2 degradation,ensuring a rapid elimination of active protein (36). Additionally,competition for cis-regulatory elements with bHLH repressorsbalances MYC activity (15, 30, 63–65). Rapid transcriptionalinduction of their JAZ repressors, which are direct transcrip-tional targets of MYCs, and splicing variants (or atypical JAZs)resistant to degradation ensures a pulsed, narrow activation ofMYCs and a quick rerepression (6, 7, 66–69). Daily variation inMYC protein levels fine-tunes MYC activity during the day/nightcycles to optimize the growth–defense balance (38). This dailyvariation is regulated by circadian clock components [i.e., TIC2(38) and light quality (37)]. Photoreceptors are required for MYCstability, and conditions that inactivate photoreceptors (dark orshade) reduce MYC protein levels by favoring COP1-mediateddegradation (37).Altogether, these examples underscore how different envi-

ronmental cues influence plant physiology by modulating theactivity of these TFs. They also show that regulation of proteinlevels is a major way of regulating MYC activity. So far, twoubiquitin ligases have been involved in the regulation of MYCstability, COP1 and PUB10 (37, 40). COP1 reduces MYC levels inthe dark and canopy shade, and photoreceptors counteract itsactivity in the light (37). PUB10 regulates MYC accumulationand, therefore, JA responses, but has not been linked to envi-ronmental regulation so far.Based on the importance of MYC TFs as signaling hubs, ad-

ditional regulators of their stability should exist and remain to beuncovered. In this work, we identified a small family of E3 ubiq-uitin ligases that regulate MYC protein stability. This E3 family isbased on CUL3 and uses BPM proteins as substrate adaptors(CUL3BPM). Several members of the BPM family interact directlywith MYC2, MYC3, and MYC4 in vivo and in vitro. Pull-downand mass spectrometry results of BPM6-coimmunoprecipitatedproteins suggest that MYCs can interact with BPM3 and BPM6directly and with similar affinity. Y2H assays confirmed a directinteraction. The differences observed in Y2H assays among dif-ferent combinations of proteins are likely a consequence of thetoxicity of BPM and MYC proteins in yeast. However, we cannotexclude that BPM6 may recognize MYCs through dimeriza-tion with other BPMs in vivo. This would be consistent withthe identification of BPM1, BPM4, and BPM5 in the BPM6immunoprecipitation purifications.The JA-related phenotypes (root growth and gene expression)

of the reduction-of-function mutants cul3ab, bpm235, and amiR-bpm confirmed that the CUL3BPM complexes regulate MYCprotein stability. This conclusion is also supported by theCUL3BPM-dependent ubiquitination of MYC3, with the higherlevels of MYC2 and MYC3 in amiR-bpm, and by the partialsuppression of the root-growth phenotype of amiR-bpm in thejin1-2 background. This partial suppression is consistent withBPMs regulating several MYCs and not only MYC2, the onlyMYC mutated in jin1-2. It is intriguing, however, that BPMs donot seem to affect all JA-related MYC2-regulated phenotypes.For instance, amiR-bpm and bpm235 do not overaccumulateanthocyanins, in contrast to 35S:MYC2 transgenics. This suggeststhat BPMs may be more active in specific tissues (i.e., roots), orthat different CUL3BPM complexes may have functional differ-ences in the regulation of particular JA responses. Gene expres-sion results in amiR-bpm and bpm235 support this hypothesis byshowing that individual BPMs have a complementary functionregulating specific JA-regulated gene sets (as shown by themoderate overlap in misregulated genes between amiR-bpm andbpm235 shown in Fig. 7A and SI Appendix, Fig. S11). Alterna-tively, BPMs may activate additional pathways that counteract

some of the MYC-dependent effects. Indeed, previous studieshave identified other CUL3BPM targets such as AtHB6 andPP2CA, a negative regulator of ABA signaling, and several ERF/AP2 TFs involved in fatty acid metabolism (43, 45, 70). Consistentwith these reports, GO terms related to ABA physiology, such asABA, water deficit, and water transport, are overrepresented inour transcriptomic analyses. Besides ABA, other hormone-relatedGO terms (i.e., salicylic acid [SA] and ET) are also over-represented, indicating that, in addition to JA and ABA, BPMsmay be involved in the regulation of multiple hormonal signalingpathways. Finally, the amiR-bpm plants might retain residual butsufficient accumulation of BPMs in the aerial part of the plants.Results on BPM3 stability using the protein synthesis inhibitor

cycloheximide and the proteasome inhibitors MG132, bortezo-mid, or epoxomicin indicate that this protein is unstable andsubject to a quick turnover by the proteasome. It is noteworthythat BPM proteins are prone to autoubiquitination in the ab-sence of their substrate, as seen for other Cullin-based ligases(71, 72). Stabilization of BPM3 by JA is dependent on COI1,which parallels the JA-mediated stabilization of MYC proteins(37). Therefore, BPM3 stabilization by JA may be a direct con-sequence of JA-mediated stabilization of its MYC substrates.This hypothesis also suggests a mechanism for activation ofthe CUL3BPM3 complex and its involvement in a negative feed-back regulatory loop to reduce protein levels after MYC activation(Fig. 9). This repressor mechanism of MYCs adds to the manydescribed so far, such as the transcriptional induction of JAZrepressors, degradation-resistant JAZs, and splicing variants (6,7, 66–69), and further underscores the importance of repressingMYC activity to avoid runaway harmful effects. In contrast toBPM3, JA does not regulate BPM6 stability. Stabilization ofdifferent BPMs by different signals would provide specificityamong BPMs and deserves further research. In any case, BPMsmay share overlapping but not fully redundant functions. This isparticularly clear from gene expression results shown in Fig. 7,where knockdown lines amiR-bpm and bpm235 share many up-regulated genes but also show evident specificities. Thus, theclustering results suggest that many BPM genes regulate JA re-sponses complementarily and with some specificity that may comefrom their differential patterns of tissue or temporal expression orrelative effect on different MYCs.Besides BPM3 stabilization, how JA regulates the activity of

other BPMs is currently unknown. Our results using the COI1specific inhibitor COR-MO (62) show that this JA effect is de-pendent on its COI1 receptor; however, additional signals maybe required for BPM–MYC interaction. In this regard, phos-phorylation of Thr328 in the transactivation domain is a pre-requisite for MYC2 activation (36). Interestingly, phosphorylationof this residue also leads to MYC2 degradation, which ensures ashort pulse of MYC2 activity. Although phosphorylation could bethe obvious signal inducing BPM–MYC interaction, available 3Dstructures of MYC proteins do not expand the region containingThr328 (17, 73). Therefore, the role of Thr328 phosphorylation inthe interaction with BPMs remains to be addressed. Clarificationof this issue or the identification of additional phosphorylatedresidues or other amino acid modifications seems essential tounderstand how BPMs recognize their targets and how specificityis achieved.The fact that MYC function is regulated by several simulta-

neous mechanisms, including several E3 ligases (COP1, PUB10,and CUL3BPM), indicates that MYCs have a broad function,which needs to be tightly controlled. Indeed, besides regulatingstress responses, MYCs also inhibit growth by promoting pho-tomorphogenesis, among other mechanisms (74). Thus, thatseveral ubiquitin ligases regulate MYC protein stability is con-sistent with the idea that MYCs should be controlled not only inresponse to stress but also temporally and spatially.

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In summary, we provide a new framework to understand thefine-tuned regulation of MYC activity. We uncovered a feedbackregulatory loop of MYC protein levels mediated by the E3 ligaseCUL3BPM (Fig. 9) that facilitates termination of MYC activityand resetting of the JA pathway.

Materials and MethodsPlant Material and Growth Conditions. Arabidopsis thaliana Columbia-0 eco-type is the genetic background of the wild-type, mutant, and transgeniclines used throughout the work. Seeds were vernalized at 4 °C for 3 d in thedark and grown in 0.5× Murashige and Skoog (MS) medium with 1% sucroseand 0.7% agar at 21 °C under a 16-h light/8-h dark cycle in a growth chamber.

Two 35S:MYC2-GFP and 1 35S:MYC3-HA transgenic lines previously reported(75) were crossed by Col-0 or introgressed by direct crossing into the amiR-bpmbackground (43), and F3 homozygous segregating plants were selected in40 μM hygromycin plates for the wild-type background or in 40 μMhygromycin and 10 μM BASTA plates for the amiR-bpm background, re-spectively.

amiR-bpm was also introgressed by direct crossing into the MYC2 C5025recombineering line in which MYC2-GFP expression is under its naturalpromoter (54, 55). F1 segregating progenies were analyzed by Western blotassays. The MYC2 C5025 line was also crossed by Col-0, and F1 segregatingprogenies were used as controls in the same experiment. Likewise, the amiR-bpm line (43) was introgressed by direct crossing into the jin1-2 mutantbackground (25), and F2 segregating progenies were selected in 10 μMBASTA and 50 μM JA plates.

T-DNA insertion lines for bpm2 (GK_391E04), bpm3 (Salk_72848), bpm4(Salk_082761), and bpm5 (Salk_038471) were obtained from the NottinghamArabidopsis Stock Centre. The bpm2,bpm3,bpm5 (bpm235) triple mutant wasgenerated by crossing the corresponding double-mutant homozygous lines. F2segregating progenies of these crosses were genotyped to obtain homozygousplants for each T-DNA insertion line.

Light/dark experiments were performed as described by Chico et al. (37).

Plasmid Constructs. Full-length BPM1, BPM2, BPM3, BPM4, BPM5, and BPM6coding sequences carrying a stop codon were PCR-amplified with Phusionpolymerase (Thermo Fisher) and cloned by restriction into the pENTR1A

(carrying a gentamicin resistance gene; modified from pENTRY1A fromInvitrogen) vector for BPM3 and into pENTR3C (carrying a gentamicin re-sistance gene; modified from pENTRY3C from Invitrogen) for BPM1, BPM2,BPM4, BPM5, and BPM6. PCR primers and restriction enzymes used for am-plification and cloning of the BPM genes into pENTRY vectors are listed in SIAppendix, Table S2.

Recombinant Proteins. For pull-down assays, MBP–MYC proteins were gen-erated as previously reported (6, 11). Full-length BPM3 and BPM6 codingsequences carrying a stop codon were PCR-amplified with Expand HighFidelity polymerase (Roche) or Phusion polymerase (Thermo Fisher) usingGateway-compatible primers (SI Appendix, Table S2). PCR products werecloned into pDONR207 using the Gateway BP II Kit (Invitrogen) to obtainpENTRY-BPM3 and pENTRY-BPM6 and sequence-verified. pENTRY-BPM3 wasused in Gateway LR reactions (Invitrogen) and recombined in pDEST-H1 (76)and pEarley201 to obtain N-terminal MBP and HA fusions, respectively. Al-ternatively, pENTRY-BPM6 was used in Gateway LR reactions (Invitrogen)and recombined in pGWB21 to obtain N-terminal 10×Myc fusions, and inpMDC43 in order to obtain N-terminal GFP fusions.

RecombinantMBP fusions were expressed in Escherichia coli BL21 cells andpurified on amylose resin columns (New England Biolabs) following the methodpreviously described (6).

To generate transgenic plants expressing 35S:HA-BPM3 and 35S:myc-BPM6,respectively, the corresponding constructs were transferred to Agrobacteriumtumefaciens GV3101 by heat shock and Arabidopsis Col-0 plants were thentransformed by floral dipping (77).

Similar to generating plants expressing 35S:GFP-BPM6, transgenicArabidopsisthat already expressed an artificial microRNA directed to BPM2 and BPM3were transformed by floral dipping using A. tumefaciens GV3101 pMP90expressing the 35S:GFP-BPM6 construct.

Yeast Two-Hybrid Assays. Full-length MYC2, MYC3, and MYC4 coding se-quences cloned into pGADT7gateway (Gal4 AD) were used as previouslydescribed (6, 11). Likewise, full-length BPM1, BPM2, BPM3, and BPM6 con-structs were cloned into pGBKT7gateway (GAL4 BD) and BPM4 and BPM5into pGBT9 (GAL4 BD). To test protein interactions, the correspondingplasmids were cotransformed into Saccharomyces cerevisiae AH 109 cellsfollowing standard heat-shock protocols (75). Successfully transformed col-onies were identified on yeast synthetic dropout lacking Leu and Trp. At 3 dafter transformation, yeast colonies were suspended in water and celldensity was adjusted to 3 × 107 cells mL−1 (OD600 1). A 10-μL sample of cellsuspensions and 1/3 and 1/10 dilutions were plated out on yeast syntheticdropout lacking Ade, His, Leu, and Trp supplemented or not with 5 mM3-aminotriazole to test protein interactions. Plates were incubated at 28 °Cfor 2 to 4 d. Yeast cotransformed with MYC2, MYC3, and MYC4 constructsinto the pGADT7 vector together with a pGBKT7 empty vector were used asnegative controls.

Protein Extracts, Pull-Down Assays, and Western Blots. Ten-day-old Arabidopsiswild-type seedlings and lines expressing 35S:HA-BPM3 or 35S:myc-BPM6 wereground in liquid nitrogen and homogenized in extraction buffer containing50 mM Tris·HCl (pH 7.4), 80 mM NaCl, 10% glycerol, 0.1% Tween 20, 1 mMDTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), 50 μM MG132 (Sigma-Aldrich), and complete protease inhibitor (Roche). After two rounds of 15 minof centrifugation at 13,000 rpm at 4 °C, the supernatant was collected. Forpull-down experiments, 6 mg of resin-bound MBP fusion protein was added to1 mg of total protein extract and incubated for 2 h at 4 °C with rotation. Afterwashing, samples were denatured, loaded on 8% sodium dodecyl sulfate (SDS)polyacrylamide gels, transferred to nitrocellulose membranes, and incubatedwith anti-HA (Roche) or anti–Myc-horseradish peroxidase antibody (Santa CruzBiotechnology). An 8-μL aliquot of MBP-fused protein of each sample was runon SDS polyacrylamide gels and stained with Coomassie brilliant blue toconfirm equal protein loading.

For immunoblotting analysis, 8 to 10 seedlings were harvested per sample,frozen in liquid nitrogen, and homogenized in 200 μL of 2× Laemmli SDSpolyacrylamide gel electrophoresis (PAGE) protein loading buffer. The ex-tracts were boiled at 95 °C for 5 min and kept on ice. After centrifugation(5 min, 13,000 rpm at room temperature) the supernatant was collected. A20- to 40-μL volume of each sample was run on 8% SDS polyacrylamide gels,transferred to nitrocellulose membrane (Bio-Rad), and incubated with anti-GFP(Miltenyi Biotec), anti-HA, anti-Myc, or monoclonal anti-ACTIN (produced inmice; Sigma-Aldrich) antibodies. Blots were developed using ECL (Pierce).

Immunoprecipitation. A. thaliana 35S:GFP-BPM6 seedlings were ground inliquid nitrogen. Seedling powder was transferred in a cold mortar and lysed

Fig. 9. Model of CUL3BPM-dependent regulation of MYCs. Activation of theCOI1-dependent pathway by JA-Ile activates JA-dependent gene expressionthrough the transcription factors MYC2, MYC3, and MYC4. JA-pathway ac-tivation stabilizes BPM3, likely by repressing a negative regulator of BPM3stability, therefore activating a negative feedback regulatory loop to reduceMYC protein levels and their activity. BPMs are substrate adaptors of Cullin3-based E3 ubiquitin ligases that target MYC proteins for proteasome degra-dation in response to JA (i.e., CUL3BPM3) or other unknown signals (i.e., otherCULBPMs).

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with a ratio of weight/lysis (g/mL) buffer volume of 1/3 during 15 min (lysisbuffer containing 50 mM Tris·HCl, pH 8, 50 mM NaCl, 1% Triton, andcOmplete EDTA-free protease inhibitors; Roche). The protein extracts wereclarified by centrifugation and GFP-BPM6 complexes were immunoprecipi-tated using magnetic microparticles (MACS Purification System; MiltenyiBiotec) according to the manufacturer’s instructions and as previously de-scribed (78). μMACS magnetic microbeads were coated with a monoclonalanti-GFP antibody (Miltenyi Biotec). Beads coated with anti-HA antibodieswere used for negative controls. Coimmunoprecipitation experiments werecarried out in independent biological duplicates; each was divided into threeaffinity-purification replicates. Proteins were eluted out of the magneticstand with SDS loading buffer from the kit.

Mass Spectrometry Analysis and Data Processing. Eluted proteins weredigested with sequencing-grade trypsin (Promega) and analyzed by nanoLC-MS/MS (liquid chromatography-tandem mass spectrometry) as describedpreviously (79). Two instruments were used: a QExactive+mass spectrometercoupled to an EASY-nanoLC 1000 (Thermo Fisher Scientific) and a TripleTOF5600 mass spectrometer coupled to a nanoLC Ultra 2D Plus (AB Sciex). Datawere searched against the TAIR 10 database with a decoy strategy. Peptideswere identified with the Mascot algorithm (version 2.5; Matrix Science) anddata were imported into Proline 1.4 software (http://proline.profiproteomics.fr/).Proteins were validated on Mascot pretty rank equal to 1, and 1% FDR on bothpeptide spectrum matches (PSM score) and protein sets (protein set score). Thetotal number of MS/MS fragmentation spectra was used to quantify each pro-tein. This spectral count was submitted to a negative-binomial test using anedgeR GLM regression through the R package (v3.5.0). For each identifiedprotein, an adjusted P value corrected by Benjamini–Hochberg was calcu-lated, as well as a protein fold change. The results are presented in a volcanoplot using protein log2 fold changes and their corresponding adjusted log10P values to highlight enriched proteins. Mass spectrometry data are de-posited in the PRIDE database.

Affinity Purification of Ubiquitinated Proteins. Soluble proteins of MYC3-HAseedlings in WT or amiR-bpm backgrounds were treated overnight with50 μM bortezomib or alternatively with 50 μM MG132 and extracted inbuffer BI (50 mM Tris·HCl, pH 7.5, 20 mM NaCl, 0.1% Nonidet P-40, 5 mMATP, 1 mM PMSF, 50 mM MG132, 10 nM ubiquitin (Ub)-aldehyde, 10 mM N-ethylmaleimide, and plant protease inhibitor mixture; Sigma-Aldrich) beforeincubation with prewashed p62 agarose (Enzo Life Sciences) or the agarosealone at 4 °C for 4 h. Beads were washed twice in BI buffer and once with BIbuffer supplemented with 200 mM NaCl. Proteins were eluted in SDSloading buffer at 100 °C for 5 min. The eluted proteins were separated bySDS/PAGE and analyzed by immunoblotting using anti-Ub (Enzo Life Sci-ences) or anti-HA antibodies (Roche).

Root Measurements. For root-growth inhibition assays, the root length of 15to 20 seedlings was measured 8 to 10 d after germination in the presence orabsence of JA (Sigma-Aldrich) or coronatine (Sigma-Aldrich) at the concen-trations indicated in each experiment. Three independent replicates (15 to 20seedlings each) were measured for each sample. Values represent mean ± SD.Comparisons between different lines and the wild type (Col-0) were done byStudent’s t test.

Microarray Assays. Leaves of 3-wk-old wild-type, amiR-bpm, or bpm235plants treated with 0.5 μM coronatine or mock were harvested and frozen.Three biological replicates were independently hybridized for each tran-scriptomic comparison. RNA was extracted using TRIzol reagent (Invitrogen)

and amplification and labeling were performed basically as previously de-scribed (62) starting from 200 ng of total RNA. Genes with a reportedFDR <0.05 and a fold change ≥2 and ≤−2 were selected for further inves-tigation. Two different microarray designs were used, the Arabidopsis V4Oligo Microarray 4 × 44 (Agilent; 021169) for the analysis of amiR-bpmplants and a custom oligo microarray 8 × 60 K (Agilent; GPL22511) inbpm235 that contains the same oligonucleotide probes as V4 and a newbatch of probes covering a total of two probes per gene. Transcriptomicprofiles of the triple myc234 mutant were previously obtained (74) andcorrespond to Agilent’s V4 design. For comparative analysis of the differentexperiments, common oligonucleotide probes were considered.

Chemical Treatments and Hormone Quantification. Arabidopsis seedlings weregerminated and grown on 0.5× MS plates. Seven-day-old seedlings weretreated with sterile water containing 50 μM CHX, 50 μM MG132, and 50 or100 μM JA and harvested at the indicated times. CHX was dissolved in 100%ethanol, MG132 in dimethyl sulfoxide, and JA in dimethylformamide. Thechemicals were provided by Sigma-Aldrich.

JA levels were quantified as previously described in ref. 2.

Quantitative RT-PCR. Quantitative RT-PCR experiments were performed withRNA extracted from 10-d-old seedlings expressing 35S:MYC2-GFP or 35S:MYC3-HA in wild-type and amiR-bpm backgrounds, respectively. RNA extractionand cleanup were done using TRIzol reagent (Invitrogen) followed by theHigh Pure RNA Isolation Kit (Roche) to remove genomic DNA contamina-tion. cDNA was synthesized from 1 μg of total RNA with the High-CapacitycDNA Reverse Transcription Kit (Applied Biosystems). Four microliters from1/10-diluted cDNA was used to amplify GFP and the housekeeping ACTIN8gene using Power SYBR Green (Applied Biosystems). Primer sequenceswere GFP-F (forward) (5′-TATATCATGGCCGACAAGCA-3′), GFP-R (reverse)(5′-ACTGGGTGCTCAGGTAGTGG-3′), HA-F (5′-ACAAAGTGGTTGATAACAGC-3′), HA-R (5′-GAGCTCTAAGCGCTGCAC-3′), ACT8-F (5′-CCAGTGGTCG-TACAAC-CGGTAT-3′), and ACT8-R (5′-TAGTTCTTTTCGATGGAGGAGCTG-3′).

Quantitative PCR was performed in 96-well optical plates in a 7300 Real-Time PCR System (Applied Biosystems). The PCR conditions were as follows:2 min at 50 °C, 10 min at 95 °C, and 40 cycles of 15 s at 95 °C and 60 s at 60 °C.Data analysis shown was done using three technical replicates from onebiological sample. Similar results were obtained with two other biologicalreplicates.

Data Availability Statement. Microarray datasets have been deposited in theGene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo/ under accession nos. GSE131024 (“Transcriptomic profile of bpm2,3,5triple mutant and Col-0”) and GSE131037 (“Transcriptomic profile of amiR-bpm and Col-0”). These data will be publicly available at the time ofpublication. Besides this, all materials, data, and associated protocols willbe available to readers upon request from the corresponding author.

ACKNOWLEDGMENTS. We thank Marta Godoy, who kindly performedmicroarray assays in the Genomics Unit (Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas), and members of the R.S. labfor critical reading of the manuscript. This work was funded by SpanishMinistry for Science and Innovation Grants BIO2016-77216-R (Ministerio deEconomia [MINECO]/Fondos Europeos de Desarrollo Regional [FEDER]) (toR.S.) and BIO2016-80551-R (MINECO/FEDER) (to V.R.). E.C. was the recipientof a Formación de Personal Investigador grant from MINECO (ReferenceBES-2017-081147). The mass spectrometry instrumentation was funded bythe University of Strasbourg (IdEx “Equipement mi-Lourd” 2015) and by“Laboratoires d’Excellence” Grant ANR-10-LABX-0036 (NETRNA).

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