Derepression of ethylene-stabilized transcription factors (EIN3/EIL1 ...

Derepression of ethylene-stabilized transcriptionfactors (EIN3/EIL1) mediates jasmonate andethylene signaling synergy in ArabidopsisZiqiang Zhu a,1 , Fengying An a,1 , Ying Feng a , Pengpeng Li a,2 , Li Xue b ,MuA a , Zhiqiang Jiang a , Jong-Myong Kim c ,Taiko Kim To c , Wei Li b , Xinyan Zhang a , Qiang Yu a , Zhi Dong a , Wen-Qian Chen a , Motoaki Seki c , Jian-Min Zhou b ,and Hongwei Guo a,3a State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, China; b National Institute of BiologicalSciences, Beijing 102206, China; and c RIKEN Plant Science Center, Yokohama 2300045, JapanEdited by Mark Estelle, University of California at San Diego, La Jolla, CA, and approved June 9, 2011 (received for review March 11, 2011)Jasmonate (JA) and ethylene (ET) are two major plant hormonesthat synergistically regulate plant development and tolerance tonecrotrophic fungi. Both JA and ET induce the expression ofseveral pathogenesis-related genes, while blocking either signalingpathway abolishes the induction of these genes by JA and ETalone or in combination. However, the molecular basis of JA/ETcoaction and signaling interdependency is largely unknown. Here,we report that two Arabidopsis ET-stabilized transcription factors(EIN3 and EIL1) integrate ET and JA signaling in the regulation ofgene expression, root development, and necrotrophic pathogendefense. Further studies reveal that JA enhances the transcriptionalactivity of EIN3/EIL1 by removal of JA-Zim domain (JAZ)proteins, which physically interact with and repress EIN3/EIL1. Inaddition, we find that JAZ proteins recruit an RPD3-type histonedeacetylase (HDA6) as a corepressor that modulates histone acetylation,represses EIN3/EIL1-dependent transcription, and inhibitsJA signaling. Our studies identify EIN3/EIL1 as a key integrationnode whose activation requires both JA and ET signaling, andillustrate transcriptional derepression as a common mechanismto integrate diverse signaling pathways in the regulation of plantdevelopment and defense.root hair | Botrytis cinereaPlants are sessile organisms and face different environmentalchanges during their lifespan. To survive various abiotic andbiotic stresses, plants synthesize a number of small moleculesfunctioning as phytohormones to elaborately regulate their growth,development, and defense. Two types of phytohormones—ethylene (ET) and jasmonate (JA)—are crucial for plant developmentand defense against necrotrophic fungi infections (1–3). Complicated modes of interaction between ET and JA havebeen documented in different processes. For example, ETstrongly suppresses JA-induced wounding-responsive gene expression,but JA suppresses ET-induced apical hook formation(4, 5), indicative of their antagonisms. Upon necrotrophic fungiinfections, plants can quickly produce ET and JA and induce theexpression of downstream defense genes (like ERF1, ORA59,and PDF1.2) that help plants tolerate or fight against the fungalpathogens (1). Plants treated with exogenous JA or ET expresshigh levels of defense genes (6, 7), and simultaneous treatmentwith JA and ET results in the highest expression (8). Nevertheless,in the ET or JA insensitive mutant (ein2 or coi1, respectively),JA and ET alone or in combination fail to induce theexpression of those defense genes (8, 9), indicating that the twohormone-signaling pathways are required concomitantly for theactivation of plant-defense response. These results suggest thatJA and ET act synergistically and mutually dependently in regulatingnecrotrophic pathogen responses. However, the moleculardetails underlying such hormone synergy and signalinginterdependency are currently unknown.ET is a gaseous hormone, which is perceived by its receptorsand represses a Raf-like kinase CONSTITUTIVE ETHYL-ENE RESPONSE 1 (CTR1) (10, 11). Downstream of CTR1 isETHYLENE INSENSITIVE 2 (EIN2), which is an essentialpositive regulator in ET signaling (12). ETHYLENE IN-SENSITIVE 3 (EIN3) and its closest homolog EIN3-LIKE 1(EIL1) are two primary transcription factors downstream ofEIN2 (6, 7, 13), which are necessary and sufficient for the inductionof the vast majority of ethylene response genes (13–15).In the absence of ET, EIN3/EIL1 are quickly degraded throughthe action of two EIN3-binding F-box proteins 1 and 2 (EBF1and EBF2), and ET enhances EIN3/EIL1 accumulation, probablythrough the repression of EBF1/2 (14, 16–18).JA is synthesized and conjugated with isoleucine to form theactive hormone JA-Ile (19, 20), which is perceived by its receptorCORONATINE INSENSITIVE 1 (COI1), an F-box protein(21–25). JASMONATE ZIM-DOMAIN (JAZ) proteins aretranscriptional repressors, which interact with MYC2 transcriptionfactor and repress its function (23, 26, 27). Recently, onepossible mechanism underlying JAZ repression on MYC2 hasbeen demonstrated in that JAZ proteins associate with NOVELINTERACTOR of JAZ (NINJA) to recruit TOPLESS as a corepressorto fulfill this function (28). The perception of JA-Ileinduces the interaction between COI1 and JAZs, which bringsJAZs for degradation and relieves their repression on MYC2(23, 26). Two MYC2-like bHLH proteins (MYC3 and MYC4)were also able to interact with JAZs and act addictively withMYC2 in mediating a subset of JA-regulated responses, includinginhibition of root elongation, wound response, and metabolism(29–31). Besides MYC2/MYC3/MYC4, two MYB transcriptionfactors (MYB21 and MYB24) were recently identified as JAZtargets in regulating plant stamen development (32).Histone deacetylation, which is catalyzed by histone deacetylases(HDAs), is a type of chromatin modification commonlyassociated with transcriptional repression (33). It has beenreported that histone deacetylation affects many growth anddevelopmental events in plants, such as flowering time, coldtolerance, embryogenesis, root hair development, abscisic acid,and salt responses (34–38). A molecular link between this epigeneticcontrol and JA signaling has been implied, as an RPD3-type histone deacetylase 6 (HDA6) was reported to associatewith COI1 in a coimmunoprecipitation (co-IP) assay (39). It wasalso found that the transcript levels of some JA-responsive genesare altered in HDA6 or HDA19 loss-of-function mutants andAuthor contributions: Z.Z. and H.G. designed research; Z.Z., F.A., Y.F., P.L., L.X., M.A., W.L.,X.Z., Q.Y., and Z.D. performed research; J.-M.K., T.K.T., W.-Q.C., and M.S. contributed newreagents/analytic tools; Z.Z., Z.J., J.-M.Z., and H.G. analyzed data; and Z.Z. and H.G. wrotethe paper.The authors declare no conflict of interest.This article is a PNAS Direct Submission.1 Z.Z. and F.A. contributed equally to this work.2 Present address: Department of Biology, Stanford University, Stanford, CA 94305.3 To whom correspondence should be addressed. E-mail: hongweig@pku.edu.cn.This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1103959108/-/DCSupplemental.PLANT BIOLOGYwww.pnas.org/cgi/doi/10.1073/pnas.1103959108 PNAS Early Edition | 1of6

transgenic overexpression plants (40, 41). However, the action ofthese HDAs in JA signaling is not clear.Here, we report that EIN3 and EIL1 integrate ET and JAsignaling in plant development and defense against necrotrophicpathogens. JAZ proteins directly interact with EIN3/EIL1 andrecruit HDA6 as a corepressor to repress the transcriptional activityof EIN3/EIL1. Our work defines EIN3/EIL1 as a previouslyunexplored class of JAZ-associated transcription factors that aresubject to JAZ repression via the action of histone deacetylation,and provides unique insights into how plant hormones coordinatelyregulate plant development and defense response.ResultsEIN3/EIL1 Are Positive Regulators Mediating a Subset of JA Responses.Previous studies have revealed that JA alone or in combinationwith ET fails to induce pathogenesis-related genes (such asERF1, ORA59, PDF1.2) inein2 background (8, 9). Because ein3eil1 is phenotypically similar to ein2 and one of ET/JA-regulatedgenes (ERF1) is a direct target of EIN3 (7, 13), we hypothesizedthat EIN3 and EIL1 may function as key transcriptional regulatorsof JA/ET-induced gene expression. We first examined theinduction of JA-responsive genes in ein3 eil1 upon JA treatment.The JA induction of pathogenesis-related genes (ERF1, ORA59,and PDF1.2) was abolished in ein3 eil1, but the expression ofwound-response genes (e.g., VSP2) that are regulated by MYC2was still induced (Fig. 1A). ein3 eil1 was also insensitive to thecombination of JA and ET treatments in terms of ERF1 induction(Fig. S1A). Because the expression of pathogenesisrelatedgenes is correlated with plant tolerance to necrotrophicfungi (42), we tested plant response to Botrytis cinerea infection.Our result showed that plants lacking EIN3, EIN3/EIL1, orEIN2 were more susceptible to B. cinerea, whereas myc2 wasmore tolerant to the fungus (Fig. 1B and Fig. S1 B and C) (13).Next, we sought to determine whether loss of EIN3/EIL1affects other JA-regulated responses. JA has been reported toinduce root hair development (43). However, such induction inein3 eil1 was largely abolished (Fig. 1 C and D). We also foundthat ein3 eil1 was partially insensitive in JA-induced inhibition ofroot elongation (Fig. 1E). Conversely, transgenic plants overexpressingEIN3 or EIL1 (EIN3ox or EIL1ox) (6) were hypersensitiveto JA in root-growth inhibition (Fig. 1F). On the otherhand, ein3 eil1 was indistinguishable from WT in terms of fertilityand JA-induced anthocyanin biosynthesis (Fig. S2). Collectively,we conclude that EIN3 and EIL1 are positive regulators ofa subset of JA responses, including pathogenesis-related geneexpression, plant resistance to necrotrophic fungi, and root development,but not fertility and pigment metabolism.JAZ Proteins Interact with EIN3/EIL1. To understand how EIN3/EIL1 mediate JA signaling, we speculated that EIN3 and EIL1might be targeted by JAZ proteins, and thus tested the interactionbetween JAZ proteins and EIN3/EIL1. We found thatJAZ1 interacted with EIN3 in yeast cells, and an EIN3 fragmentcontaining amino acid residues 200 to 500 was sufficient forJAZ1 binding (Fig. 2A). The interaction between JAZ1 and anEIL1 fragment (amino acids 201–501) was also observed (Fig.S3A). To test if EIN3/EIL1 interacts with other JAZ proteins, wechose JAZ3 and JAZ9 and found that both JAZ3 and JAZ9were able to interact with EIN3 (amino acids 200–500) and EIL1(amino acids 201–501) in yeast cells (Fig. S3B). A pull-downassay also verified JAZ1-EIN3 interaction in which Escherichiacoli-expressed GST-JAZ1 successfully pulled down EIN3 fromplant total protein extracts (Fig. 2B). To confirm JAZ1-EIN3interaction in vivo, we performed a co-IP assay in the 35S:JAZ1-GUS transgenic plants (23) and found that the anti-GUS antibodywas able to precipitate EIN3 along with JAZ1-GUS, indicatingtheir association in planta (Fig. 2C). A bimolecularfluorescence complementation (BiFC) assay in onion epidermiscells also confirmed JAZ1-EIN3 interaction in vivo (Fig. 2D). Tofurther characterize the interaction domain in JAZ1, we used anFig. 1. EIN3 and EIL1 are positive regulators of JA signaling.(A) Analysis of JA-responsive gene expression by quantitativeRT-PCR (qRT-PCR) in 7-d-old light-grown seedlings with orwithout 100 μM JA treatment for 4 h. Mean ± SEM, n =3.(B)B. cinerea infection analysis. The percentages of differentinfection ratios in each genetic background were scored. (Cand D) Root-hair phenotypes. (C) Representative picture ofroots from seedlings grown on MS medium for 4 d and thentransferred onto MS (Mock) or 25 μM JA medium for additional2 d. Arrows indicate root hairs. (Scale bars, 50 μm.) (D)Root hair densities of 7-d-old seedlings grown on MS mediumsupplemented with indicated concentrations of JA werescored by counting the numbers of entire root hairs. Mean ±SEM, n = 10. (E and F) Relative root growth of 7-d-oldseedlings grown on indicated concentrations of JA medium.Mean ± SEM, n = 10. **P ≤ 0.01, significant differences betweenCol-0 and mutants.2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1103959108 Zhu et al.

E. coli-expressed GST-fused N terminus of JAZ1 (containing theZIM domain) or a GST-fused C terminus of JAZ1 (containingthe Jas domain) to pull down EIN3 protein from extracted planttotal proteins. Our result revealed that the C terminus of JAZ1was mainly responsible for EIN3 binding (Fig. S3C). It has beendemonstrated that the C terminus of JAZs (Jas domain) is theinteraction domain for JAZs-MYC2 binding (26), therefore JAZproteins seem to use the same Jas domains to interact with varioustranscription factors. Together, these results indicate EIN3and EIL1 as a previously unexplored class of JAZs-associatedtranscription factors.JAZ Proteins Repress the Function of EIN3/EIL1. We next determinedwhether JAZ proteins repress the function of EIN3/EIL1. First,we carried out a transient transcriptional activity assay in Arabidopsisprotoplasts using ERF1:LUC (firefly luciferase-codinggene driven by the ERF1 promoter) as a reporter. Overexpressionof EIN3 (35S:EIN3) induced ERF1:LUC expression, butoverexpression of JAZ1 (35S:JAZ1) suppressed EIN3-inducedexpression of ERF1:LUC (Fig. 3A), indicating that JAZ1 somehowrepresses EIN3 function. Second, we introduced 35S:JAZ1-D3A (a stable form of JAZ1) (23) into EIN3ox plants and foundthat overaccumulation of JAZ1 dramatically reduced the sensitivityof EIN3ox to JA (Fig. 3 B and C). Third, we found that thecoi1 mutation also reduced the sensitivity of EIN3ox to JA in termsof ERF1 gene expression and root hair development (Fig. 3D).Finally, we introduced 5×EBS::GUS (the glucuronidase gene isdriven by a promoter containing five tandem repeats of the EIN3-binding sites) (44) into Col-0, ein3-1, orein3 eil1 backgrounds.Upon ET or JA treatment, the transcriptional activity of EIN3indicated by the GUS level was augmented in Col-0 but diminishedor abolished in ein3-1 or ein3 eil1, respectively (Fig. 3E).Together, these results demonstrate that JAZs repress the functionof EIN3/EIL1 and suggest that JA might relieve such repressionby removing JAZ proteins.Next, we investigated how JAZ proteins repress EIN3/EIL1.Because JAZ1 interacts with the EIN3 (amino acids 200–500) andEIL1 (amino acids 201–501) fragments that overlap with theirDNA binding domains (amino acids 59–359 in EIN3) (7), we determinedwhether JAZ1 interferes with EIN3 binding to DNA.ChIP-PCR assay showed that the in vivo association of EIN3 to theERF1 promoter was increased by JA or ET (Fig. 3F). However, anin vitro binding experiment using electrophoretic mobility shiftassay failed to detect the interference of JAZ1 on EIN3 binding tothe ERF1 promoter element. One explanation for this discrepancyis that JAZ repressors possibly need additional components (corepressors)to suppress the DNA binding of EIN3 in vivo.JAZ Proteins Recruit HDA6 to Associate with EIN3/EIL1. We thentested the possibility whereby corepressors are required for JAZrepression of EIN3 function. It was reported that HDA6 iscoimmunoprecipitated with COI1 and its expression is upregulatedby both JA and ET (39, 41). In addition, histonedeacetylation is generally associated with transcriptional repression(33). These features make HDA6 a good candidate tobe a corepressor of JAZ proteins. We thus tested the physicalinteractions among JAZs, HDA6, and EIN3/EIL1. We foundthat HDA6 interacted with EIN3 (amino acids 200–500) andEIL1 (amino acids 201–501) in yeast cells (Fig. 4A). We alsocarried out a co-IP assay using an inducible EIN3-3×FLAG inein3 eil1 ebf1 ebf2 background, in which the induced FLAGtaggedEIN3 becomes more stabilized because of the lack ofEBF1 and EBF2 and thus easier to detect (14). Our resultsshowed that the anti-FLAG antibody efficiently coprecipitatedHDA6 together with FLAG-tagged EIN3, suggesting their associationin vivo (Fig. 4B).Meanwhile, we found that HDA6 also interacted with JAZ1 inyeast cells (Fig. 4C). To verify JAZ1-HDA6 interaction, we dida pull-down assay and found that His-JAZ1 could pull downHDA6 purified from plant extracts (Fig. 4D). To further confirmHDA6-JAZ1 association, we performed a BiFC assay anddemonstrated their in vivo interaction (Fig. S4B). We also testedthe interactions between HDA6 and other JAZ proteins (e.g.,JAZ3 and JAZ9) and found that HDA6 interacted with bothJAZ3 and JAZ9 (Fig. S4A). To define the interaction domain ofJAZ1 for HDA6 binding, we used GST-JAZ1 ΔN or GST-JAZ1ΔC to do a pull-down assay and found that GST-JAZ1 ΔC wassufficient for HDA6 binding (Fig. 4E), suggesting that JAZ1 usesdistinctive interaction modules for EIN3- and HDA6-binding.Because both JAZ1 and HDA6 interact with EIN3 (amino acids200–500), we further performed yeast two-hybrid analysis withEIN3 deletion fragements and found that EIN3 (amino acids300–500) was sufficient for JAZ1-EIN3 interaction but not forHDA6-EIN3 interaction (Fig. S4C), suggesting that JAZ1 andHDA6 interact with different domains of EIN3. To investigatethe biological consequence of JAZ interactions with EIN3 andHDA6, we used anti-FLAG antibody to coprecipitate HDA6 intransgenic plants expressing FLAG-tagged EIN3 with or withoutJA treatment. We found that JA treatment reduced the in vivoHDA6-EIN3 association (Fig. 4 F and G), suggesting that JAZproteins or other components are necessary for maximal interactionbetween HDA6 and EIN3 in vivo.HDA6 Is a Repressor for EIN3-Mediated Transcription and JASignaling. Histone deacetylases usually associate with transcriptionalrepression. To investigate whether HDA6 is involved inthe repression of EIN3-mediated transcription and JA signaling,we studied JA responses in HDA6 loss-of-function mutant (axe1-4)and transgenic overexpressing plant (HDA6ox) (Fig. S5A). TheJA-induced ERF1 gene expression, root hair development, androot growth inhibition were enhanced in axe1-4 (Fig. 5 A and Band Fig. S5B) but were attenuated in HDA6ox (Fig. 5A and Fig.S5 B and C). Meanwhile, we used a specific histone deacetylaseinhibitor, trichostatin A (TSA) (37) to mimic the loss of HDAPLANT BIOLOGYFig. 2. JAZ proteins physically interact with EIN3. (A) Yeasttwo-hybrid assays showing the interaction between EIN3 andJAZ1. White colony indicates protein interaction in yeast cellsgrown on selective medium. (B) Pull-down assay. E. coliexpressedGST or GST-JAZ1 proteins were incubated withprotein extracts from Col-0 and further immobilized by glutathioneSepharose 4B. Half of the pull-down products wereprobed with anti-EIN3 antibody to test interaction (Upper)and the other half of pull-down products were subjected toCoomassie blue staining as loading controls (Lower). (C) EIN3was coimmunoprecipitated with JAZ1. The 35S:JAZ1-GUSseedlings were treated with 100 μM MG132 for 4 h to stabilizeJAZ1-GUS and EIN3 proteins. Protein extracts from JAZ1-GUSwere immunoprecipitated with anti-GUS antibody (+) or noantibody (−). Precipitated products were probed with anti-GUS or anti-EIN3 antibody to detect the interaction. (D) BiFCassay in onion epidermis cells. YFP fluorescence indicatesprotein interaction. (Upper) Two arbitrary independent fields showing JAZ1-EIN3 interaction. (Lower) Negative controls. (Scale bars, 50 μm.)Zhu et al. PNAS Early Edition | 3of6

Fig. 3. JAZ proteins repress the transcriptional activity ofEIN3. (A) Transient assay of ERF1:LUC expression. ERF1:LUC-35S:REN reporter constructs were transiently expressed inArabidopsis protoplasts together with control vector, 35S:EIN3, or 35S:EIN3 and 35S:JAZ1 effectors, respectively. Theexpression of REN was used as an internal control. LUC/RENratio represents the relative activity of the ERF1 promoter.Results are repeated three times with the same pattern.Mean ± SEM, n =3.(t test: ** P ≤ 0.01). (B and C) Rootelongation phenotypes of 7-d-old seedlings grown on 20μM JA medium. (Scale bar in B, 1 mm.) Mean ± SEM, n =10.(D) Root hair densities of 7-d-old seedlings grown on MS(Mock) or 10 μM JA medium (Upper, mean ± SEM, n ≥ 5),and qRT-PCR results showing relative expression of ERF1from 7-d-old seedlings treated without (Mock) or with 100μM JAfor4h(Lower, mean ± SEM, n = 3). (E) GUS staining.5×EBS::GUS expression in 7-d-old seedlings treated withMock solution, 100 μM ACC (ET) or 100 μM JA (JA) for 4 h.ACC (1-aminocyclopropane-1-carboxylic acid) is the ethylenebiosynthetic precursor. (Scale bar, 1 mm.) (F) ChIP-PCRresult showing that JA enhances the DNA binding ability ofEIN3 in vivo. Chromatin was extracted from 12-d-old seedlingsafter either 100 μM JAor100μM ACC treatment for3.5 h and was precipitated using anti-EIN3 antibody. PrecipitatedDNA was amplified by primers corresponding tosequences neighboring the EIN3 binding sites in the promoterof ERF1. The ein3 eil1 samples and no-antibodypanels were negative controls showing the specificity ofprecipitation. Input indicates samples before immunoprecipitation.activities. TSA treatment was sufficient to induce root hair formationand ERF1 expression in Col-0, similar to the effect of JA(Fig. S6 A–C). Interestingly, TSA induced root hair formation inthe coi1-2 mutant but not in the ein3 eil1 mutant (Fig. S6A).Consistent with this observation, ein3 eil1 completely suppressedthe JA-hypersensitivity phenotypes of axe1-4 (Fig. 5 A and B).These results suggest that HDA6 (and probably other histonedeacetylases) is a negative regulator of the JA-regulated processes(e.g., ERF1 gene expression and root hair development)acting in an EIN3/EIL1-dependent manner.To further explore the role of histone deacetylation in EIN3-mediated transcription, we analyzed the histone acetylationlevels in the promoter region of the EIN3 target gene, ERF1.Upon JA treatment, the levels of both histone H3 and H4acetylation were enhanced, but in axe1-4, this enhancement wasmore pronounced, particularly for histone H4 acetylation (Fig.5C), which agrees with JA induction of ERF1 expression in axe1-4(Fig. 5A). Meanwhile, the levels of histone H4 acetylation incoi1-2 and ein3 eil1 were greatly reduced compared with that ofCol-0 upon JA treatment (Fig. 5D), which again was in agreementwith the levels of ERF1 expression and JA sensitivity inthese mutants (Fig. 1A and Fig. S1A). Taking these data together,we conclude that JAZ proteins recruit HDA6 (andprobably other HDAs as well) to deacetylate histones (especiallyH4) and obstruct the chromatin binding of EIN3/EIL1, but JAtreatment removes JAZs-HDA6 corepressors away from EIN3/EIL1 by degrading JAZs.DiscussionEmerging evidences show that the action of JA and ET issynergistic and mutually dependent in regulating tolerance tonecrotrophic fungi (8, 9), although not much is known about themolecular details of this hormonal synergy and signaling interdependency.In this study, we report EIN3 and EIL1 as twoFig. 4. JAZ proteins recruit HDA6 to associate with EIN3. (A)Yeast two-hybrid assays indicating that HDA6 interacts withEIN3 and EIL1. (B) HDA6 was coimmunoprecipitated with EIN3-3×FLAG. EIN3-3×FLAG is preinduced in pER8-EIN3-3×FLAG/ein3eil1 ebf1 ebf2 transgenic plants by 10 μM β-estrogen for 4 hand then immunoprecipitated with anti-FLAG antibody (+Ab)or no antibody (−Ab), followed by immunoblots with anti-FLAG or anti-HDA6 antibody. (C) Yeast two-hybrid assays indicatingthat HDA6 interacts with JAZ1. (D and E) Pull-downassays. His-JAZ1, His-APRT or His-APT3 proteins (D) and GST-JAZ1ΔC or GST-JA1ΔN proteins (E) were incubated with Col-0 protein extracts and further immobilized by Ni-NTA Agarose(D) or glutathione Sepharose 4B (E). Half of the pull-downproducts were probed with anti-HDA6 antibody to test interaction(D and E) and the other half of pull-down products were probed with anti-His (D) or anti-GST (E) as loading controls. His-APRT and His-APT3 areproteins as negative controls. (F and G) JA treatment reduces EIN3-HDA6 association. (F) pER8-EIN3-3×FLAG/ein3 eil1 ebf1 ebf2 seedlings were initially treatedwith 10 μM β-estrogen for 4 h and then treated with 100 μM JA (+JA) or mock (−JA) for additional 1 h. Extracted protein was immunoprecipitated with anti-FLAG antibody (+Ab) or no antibody (−Ab), followed by immunoblots with anti-FLAG or anti-HDA6 antibody. Ten percent of protein extracts were used asinput controls. Protein extracts from Col-0 and axe1-4 were used as controls showing the specificity of anti-HDA6 antibody. (G) The relative intensities ofHDA6 and EIN3 were calculated with Image J software (National Institutes of Health) from input or precipitated products (+Ab) to show the association ofHDA6 and EIN3. Results from JA-treated samples (+JA) were normalized to samples without JA treatment (−JA). Similar results were obtained from twoindependent experiments and error bar represents SEM. (t test: *P = 0.026.)4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1103959108 Zhu et al.

JAZ-associated transcription factors integrating JA and ETsignaling to regulate gene expression, pathogen defense, androot development. Under normal conditions, EIN3/EIL1 arerepressed by JAZs, which recruit HDA6 as a corepressor todecrease EIN3/EIL1 transcriptional ability. ET treatmentenhances EIN3/EIL1 protein stability (14, 16–18), while JAtreatment leads to JAZs degradation and transcriptional derepression,providing a nice explanation for the hormonal synergyin ET/JA-regulated gene expression and developmentalprocesses (Fig. S7). When ET signaling is blocked (e.g., in ein2),EIN3/EIL1 proteins are rapidly degraded (14, 18); therefore, theabsence of EIN3/EIL1 accumulation fails to induce downstreamgene expression and leads to ET/JA insensitivity. When JA signalingis blocked (e.g., in coi1), JAZs are highly accumulated(23, 26), which strongly repress EIN3/EIL1 function even in thepresence of ET and JA.The discovery of JAZ proteins has successfully linked JAreceptors to downstream transcription factors. bHLH transcriptionfactors (MYC2/MYC3/MYC4) and MYB transcriptionfactors (MYB21/MYB24) are reported primary transcriptionfactors associated with and repressed by JAZ protein (23, 30–32,45). Our study has identified EIN3/EIL1 as a unique class ofJAZ-targeted transcription factors. EIN3 and EIL1 physicallyinteract with a number of JAZ proteins including JAZ1, JAZ3,and JAZ9, and JAZ proteins use the same module containingthe Jas domain to interact with EIN3/EIL1 and MYC2 (26).Unlike MYC2/3/4 that substantially contribute to JA-mediatedwound response and metabolism (30, 46), EIN3/EIL1 largelyregulate JA-induced root hair development and resistance tonecrotrophic fungi. We also examined the phehotypes of ein3 eil1for other types of JA responses, such as fertility and anthocyaninbiosynthesis, and found that EIN3/EIL1 have little role in theseprocesses, which are more likely regulated by MYC2 or MYBtranscription factors (32, 45–47). Consistently, JA was still ableto robustly induce the expression of VSP2 in ein3 eil1, a MYC2-regulated gene invloved in wounding response (45). Interestingly,both EIN3/EIL1 and MYC2-mediated pathways participatein JA inhibition of root elongation, although EIN3/EIL1seem to play a minor role in this growth response. Therefore,EIN3/EIL1 work separately and cooperatively with other JAZsassociatedtranscription factors to mediate a diverse range of JAresponses. EIN3/EIL1 are predominant in JA regulation offungal defense and root hair development, whereas MYC2-liketranscription factors are indispensible for JA-mediated woundingresponse and metabolism, and MYB21/MYB24 function mainlyin stamen development (32).JAZ proteins have been identified as a group of respressors inJA signaling (23, 26, 27). We have presented several lines ofevidence to demonstrate that JAZ proteins act to inhibit thefunction of EIN3/EIL1, and JA initiates a transcriptional derepressionevent by degrading JAZ proteins (Fig. 3). HDA6/19had been implicated in the regulation of JA-responsive genes(40, 41), although it was not clear how histone deacetylationmodulates JA signaling. Here, we reveal a previously unexploredmode of action for JAZ repressors, in which JAZ proteins recruitHDA6 (and probably other HDAs) to switch chromatin structureinto a less accessible state. Treatment of a specific HDA inhibitor,TSA, was sufficient to drastically induce ERF1 expressionand root hair formation, suggesting that histone deacetylation isa major repression mechanism of these JA-regulated processes.Consistent with this notion, the acetylation levels of histones(particularly H4) in the ERF1 promoter region were significantlyincreased by JA treatment, and such enhancement was morepronounced in the hda6 mutant, which were in good correlationwith the expression levels of ERF1. Interestingly, we noted thatthe H4 acetylation level of the ERF1 promoter was greatly reducedin ein3 eil1, which could hardly be explained by the actionof HDAs, implying the involvement of histone acetyltransferasesin EIN3/EIL1-mediated gene activation. It thus remains to beinvestigated whether and how histone acetylation and deacetylationcoordinately modulate JA/ET-regulated processes.It was recently reported that JAZs interact with an adaptorprotein NINJA that recruits Groucho/Tup1-type corepressorTOPLESS and its related proteins to suppress MYC2-dependenttranscription (28). As TOPLESS-mediated repression has beenimplicated to link with histone deacetylation (33), it would beinteresting to ascertain whether JAZs-NINJA-TOPLESS andJAZs-HDA6 repressors work as a large repressive module inmodulating JA signaling. Alternatively, JAZ proteins might adoptdistinct repression mechanisms to attenuate the function oftheir associated transcription factors in different JA responses. Insupport of this view, HDAs seem not involved in repressingMYC2 as TSA treatment does not induce VSP2 expression,which is a MYC2 responsive gene.Transcriptional derepression has also been implicated in othersignaling integration. Eliminating repressors (DELLA proteins)from transcriptional regulators (PIFs) has been reported in thecoordinated regulation of plant growth and development bygibberellins and light (48, 49). Our work provides further evidenceto highlight transcriptional derepression as a possiblycommon mechanism for the integration of multiple signalingpathways in plants. Given the existence of a number of repressorsin plant hormone signaling (DELLA proteins for gibberellin,AUX/IAA proteins for auxin, JAZ proteins for JA,A-type Arabidopsis-response regulators for cytokinin) (50), furtherresearch should focus on the identification of repressorassociatedtranscription factors that might be integration pointsPLANT BIOLOGYFig. 5. HDA6 is a repressor for EIN3-mediatedtranscription and JA signaling. (A) qRT-PCR resultsshowing relative expression of ERF1 from 7-d-oldseedlings treated without (Mock) or with 100 μMJAfor 4 h. Mean ± SEM, n = 3. Significant differencesbetween Col-0 and axe1-4 or HDA6ox were indicated:**P ≤ 0.01. (B) Root-hair numbers. Fourday-oldseedlings grown on MS were transferred toMS or 25 μM JA medium for 2 d. Root-hair numberswere counted from the 2-mL region of root tipunder an Olympus microscope. Mean ± SEM, n ≥ 5.Significant differences between Col-0 and axe1-4are indicated: **P ≤ 0.01. (C and D) Histone acetylationanalysis by ChIP-PCR. Before ChIP analysis,seedlings were treated with or without 100 μM JAfor 4 h. Antibodies specifically recognizing eitheracetylated histone H3 (AcH3) or H4 (AcH4) wereused to precipitate chromatins. Precipitated DNAwas amplified by primers corresponding to sequencesneighboring the EIN3 binding sites in the promoter of ERF1. qPCR (C) or PCR product separated on ethidium bromide stained gel (D) shows thehistone acetylation levels of the ERF1 promoter region. Mean ± SD, n =3(t test: *P ≤ 0.05, **P ≤ 0.01). Actin was used as a negative control. Input indicatessamples before immunoprecipitation.Zhu et al. PNAS Early Edition | 5of6

of plant hormones and other environmental or developmentalsignals.Experimental ProceduresRoot Hair Density. We counted the entire root hair numbers from the root tipto the root-hypocotyl junction under an Olympus dissecting microscope, anddivided this number by root length to obtain the value of root hair density.Each root hair observation was carried out for at least three times with thesame result.Yeast Two-Hybrid Assays. The coding sequences of COI1, JAZ1, and HDA6were amplified from wild-type cDNA and cloned into pGBKT7 vectors, andthe coding sequences of EBF2, MYC2, HDA6, EIN3, EIN3 (200–500), EIN3 (1–300), EIN3 (200–400), EIN3 (300–500), and EIL1 (201–501) were cloned intopGADT7 vectors and transformed into yeast strain AH109 following theMatchmaker user’s manual protocol (Clontech). Yeast transformants werestreaked onto SD (-Leu/-His/-Ade/-Trp) medium (Clontech) and grown at 30 °Cfor 4 d. The white colonies represented interactions. Each yeast two-hybridassay has been independently repeated at least twice with the same results.All primers used in this study are summarized in Table S1.Protein Expression and Purification. The coding sequences of JAZ1, JAZ1ΔN,and JAZ1ΔC were cloned into pGEX 5×-1 vectors (GE Healthcare) for GSTfusion or pET-16b vectors (Novagen) for His fusion and transformed into BL21(DE3)-competent cells. Protein expression was induced by 0.1 mM isopropylbeta-D-thiogalactopyranosideand proteins were purified by glutathioneSepharose 4B (GE Healthcare) or Ni-NTA Agarose (Qiagen) following themanufacturer’s instructions.ACKNOWLEDGMENTS. We thank Drs. Yan Guo, Lijia Qu, Joseph R. Ecker,Yajie Niu, and John Browse for sharing research materials; and Drs. DaoxinXie and Ian Wilson and colleagues in the H.G. laboratory for stimulatingdiscussions and critical reading of this manuscript. 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