Derepression of Ethylene-Stabilized Transcription Factors (EIN3/EIL1) Mediates Jasmonate and Ethylene Signaling Synergy in Arabidopsis

Derepression of ethylene-stabilized transcription factors (EIN3/EIL1) mediates jasmonate and ethylene signaling synergy in Arabidopsis

Ziqiang Zhua,1, Fengying Ana,1, Ying Fenga, Pengpeng Lia,2, Li Xueb,MuAa, Zhiqiang Jianga, Jong-Myong Kimc, Taiko Kim Toc, Wei Lib, Xinyan Zhanga, Qiang Yua, Zhi Donga, Wen-Qian Chena, Motoaki Sekic, Jian-Min Zhoub, and Hongwei Guoa,3

aState Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, China; bNational Institute of Biological Sciences, Beijing 102206, China; and cRIKEN Plant Science Center, Yokohama 2300045, Japan

Edited 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 hormones ETHYLENE INSENSITIVE 2 (EIN2), which is an essential that synergistically regulate plant development and tolerance to positive regulator in ET signaling (12). ETHYLENE IN- necrotrophic fungi. Both JA and ET induce the expression of SENSITIVE 3 (EIN3) and its closest homolog EIN3-LIKE 1 several pathogenesis-related genes, while blocking either signal- (EIL1) are two primary transcription factors downstream of ing pathway abolishes the induction of these genes by JA and ET EIN2 (6, 7, 13), which are necessary and sufficient for the in- – alone or in combination. However, the molecular basis of JA/ET duction of the vast majority of ethylene response genes (13 15). coaction and signaling interdependency is largely unknown. Here, In the absence of ET, EIN3/EIL1 are quickly degraded through we report that two Arabidopsis ET-stabilized transcription factors the action of two EIN3-binding F-box proteins 1 and 2 (EBF1 and EBF2), and ET enhances EIN3/EIL1 accumulation, prob- (EIN3 and EIL1) integrate ET and JA signaling in the regulation of – gene expression, root development, and necrotrophic pathogen ably through the repression of EBF1/2 (14, 16 18). JA is synthesized and conjugated with isoleucine to form the defense. Further studies reveal that JA enhances the transcrip- active hormone JA-Ile (19, 20), which is perceived by its receptor tional activity of EIN3/EIL1 by removal of JA-Zim domain (JAZ) CORONATINE INSENSITIVE 1 (COI1), an F-box protein PLANT BIOLOGY proteins, which physically interact with and repress EIN3/EIL1. In – fi (21 25). JASMONATE ZIM-DOMAIN (JAZ) proteins are addition, we nd that JAZ proteins recruit an RPD3-type histone transcriptional repressors, which interact with MYC2 transcrip- deacetylase (HDA6) as a corepressor that modulates histone acet- tion factor and repress its function (23, 26, 27). Recently, one ylation, represses EIN3/EIL1-dependent transcription, and inhibits possible mechanism underlying JAZ repression on MYC2 has JA signaling. Our studies identify EIN3/EIL1 as a key integration been demonstrated in that JAZ proteins associate with NOVEL node whose activation requires both JA and ET signaling, and INTERACTOR of JAZ (NINJA) to recruit TOPLESS as a co- illustrate transcriptional derepression as a common mechanism repressor to fulfill this function (28). The perception of JA-Ile to integrate diverse signaling pathways in the regulation of plant induces the interaction between COI1 and JAZs, which brings development and defense. JAZs for degradation and relieves their repression on MYC2 (23, 26). Two MYC2-like bHLH proteins (MYC3 and MYC4) root hair | Botrytis cinerea were also able to interact with JAZs and act addictively with MYC2 in mediating a subset of JA-regulated responses, including lants are sessile organisms and face different environmental inhibition of root elongation, wound response, and metabolism Pchanges during their lifespan. To survive various abiotic and (29–31). Besides MYC2/MYC3/MYC4, two MYB transcription biotic stresses, plants synthesize a number of small molecules factors (MYB21 and MYB24) were recently identified as JAZ functioning as phytohormones to elaborately regulate their growth, targets in regulating plant stamen development (32). development, and defense. Two types of phytohormones— Histone deacetylation, which is catalyzed by histone deacety- ethylene (ET) and jasmonate (JA)—are crucial for plant de- lases (HDAs), is a type of chromatin modification commonly velopment and defense against necrotrophic fungi infections (1– associated with transcriptional repression (33). It has been 3). Complicated modes of interaction between ET and JA have reported that histone deacetylation affects many growth and been documented in different processes. For example, ET developmental events in plants, such as flowering time, cold strongly suppresses JA-induced wounding-responsive gene ex- tolerance, embryogenesis, root hair development, abscisic acid, pression, but JA suppresses ET-induced apical hook formation and salt responses (34–38). A molecular link between this epi- (4, 5), indicative of their antagonisms. Upon necrotrophic fungi genetic control and JA signaling has been implied, as an RPD3- infections, plants can quickly produce ET and JA and induce the type histone deacetylase 6 (HDA6) was reported to associate expression of downstream defense genes (like ERF1, ORA59, with COI1 in a coimmunoprecipitation (co-IP) assay (39). It was and PDF1.2) that help plants tolerate or fight against the fungal also found that the transcript levels of some JA-responsive genes pathogens (1). Plants treated with exogenous JA or ET express are altered in HDA6 or HDA19 loss-of-function mutants and high levels of defense genes (6, 7), and simultaneous treatment with JA and ET results in the highest expression (8). Neverthe- less, in the ET or JA insensitive mutant (ein2 or coi1, re- Author contributions: Z.Z. and H.G. designed research; Z.Z., F.A., Y.F., P.L., L.X., M.A., W.L., spectively), JA and ET alone or in combination fail to induce the X.Z., Q.Y., and Z.D. performed research; J.-M.K., T.K.T., W.-Q.C., and M.S. contributed new expression of those defense genes (8, 9), indicating that the two reagents/analytic tools; Z.Z., Z.J., J.-M.Z., and H.G. analyzed data; and Z.Z. and H.G. wrote hormone-signaling pathways are required concomitantly for the the paper. activation of plant-defense response. These results suggest that The authors declare no conflict of interest. JA and ET act synergistically and mutually dependently in reg- This article is a PNAS Direct Submission. ulating necrotrophic pathogen responses. However, the molec- 1 ular details underlying such hormone synergy and signaling Z.Z. and F.A. contributed equally to this work. interdependency are currently unknown. 2Present address: Department of Biology, Stanford University, Stanford, CA 94305. ET is a gaseous hormone, which is perceived by its receptors 3To whom correspondence should be addressed. E-mail: [email protected]. and represses a Raf-like kinase CONSTITUTIVE ETHYL- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ENE RESPONSE 1 (CTR1) (10, 11). Downstream of CTR1 is 1073/pnas.1103959108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1103959108 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 transgenic overexpression plants (40, 41). However, the action of induce root hair development (43). However, such induction in these HDAs in JA signaling is not clear. ein3 eil1 was largely abolished (Fig. 1 C and D). We also found Here, we report that EIN3 and EIL1 integrate ET and JA that ein3 eil1 was partially insensitive in JA-induced inhibition of signaling in plant development and defense against necrotrophic root elongation (Fig. 1E). Conversely, transgenic plants over- pathogens. JAZ proteins directly interact with EIN3/EIL1 and expressing EIN3 or EIL1 (EIN3ox or EIL1ox) (6) were hyper- recruit HDA6 as a corepressor to repress the transcriptional ac- sensitive to JA in root-growth inhibition (Fig. 1F). On the other tivity of EIN3/EIL1. Our work defines EIN3/EIL1 as a previously hand, ein3 eil1 was indistinguishable from WT in terms of fertility unexplored class of JAZ-associated transcription factors that are and JA-induced anthocyanin biosynthesis (Fig. S2). Collectively, subject to JAZ repression via the action of histone deacetylation, we conclude that EIN3 and EIL1 are positive regulators of and provides unique insights into how plant hormones co- a subset of JA responses, including pathogenesis-related gene ordinately regulate plant development and defense response. expression, plant resistance to necrotrophic fungi, and root development, but not fertility and pigment metabolism. Results EIN3/EIL1 Are Positive Regulators Mediating a Subset of JA Responses. JAZ Proteins Interact with EIN3/EIL1. To understand how EIN3/ Previous studies have revealed that JA alone or in combination EIL1 mediate JA signaling, we speculated that EIN3 and EIL1 with ET fails to induce pathogenesis-related genes (such as might be targeted by JAZ proteins, and thus tested the in- ERF1, ORA59, PDF1.2)inein2 background (8, 9). Because ein3 teraction between JAZ proteins and EIN3/EIL1. We found that eil1 is phenotypically similar to ein2 and one of ET/JA-regulated JAZ1 interacted with EIN3 in yeast cells, and an EIN3 fragment genes (ERF1) is a direct target of EIN3 (7, 13), we hypothesized containing amino acid residues 200 to 500 was sufficient for that EIN3 and EIL1 may function as key transcriptional regu- JAZ1 binding (Fig. 2A). The interaction between JAZ1 and an lators of JA/ET-induced gene expression. We first examined the EIL1 fragment (amino acids 201–501) was also observed (Fig. induction of JA-responsive genes in ein3 eil1 upon JA treatment. S3A). To test if EIN3/EIL1 interacts with other JAZ proteins, we The JA induction of pathogenesis-related genes (ERF1, ORA59, chose JAZ3 and JAZ9 and found that both JAZ3 and JAZ9 and PDF1.2) was abolished in ein3 eil1, but the expression of were able to interact with EIN3 (amino acids 200–500) and EIL1 wound-response genes (e.g., VSP2) that are regulated by MYC2 (amino acids 201–501) in yeast cells (Fig. S3B). A pull-down was still induced (Fig. 1A). ein3 eil1 was also insensitive to the assay also verified JAZ1-EIN3 interaction in which Escherichia combination of JA and ET treatments in terms of ERF1 in- coli-expressed GST-JAZ1 successfully pulled down EIN3 from duction (Fig. S1A). Because the expression of pathogenesis- plant total protein extracts (Fig. 2B). To confirm JAZ1-EIN3 related genes is correlated with plant tolerance to necrotrophic interaction in vivo, we performed a co-IP assay in the 35S:JAZ1- fungi (42), we tested plant response to Botrytis cinerea infection. GUS transgenic plants (23) and found that the anti-GUS anti- Our result showed that plants lacking EIN3, EIN3/EIL1, or body was able to precipitate EIN3 along with JAZ1-GUS, in- EIN2 were more susceptible to B. cinerea, whereas myc2 was dicating their association in planta (Fig. 2C). A bimolecular more tolerant to the fungus (Fig. 1B and Fig. S1 B and C) (13). fluorescence complementation (BiFC) assay in onion epidermis Next, we sought to determine whether loss of EIN3/EIL1 cells also confirmed JAZ1-EIN3 interaction in vivo (Fig. 2D). To affects other JA-regulated responses. JA has been reported to further characterize the interaction domain in JAZ1, we used an

Fig. 1. EIN3 and EIL1 are positive regulators of JA signaling. (A) Analysis of JA-responsive gene expression by quantitative RT-PCR (qRT-PCR) in 7-d-old light-grown seedlings with or without 100 μM JA treatment for 4 h. Mean ± SEM, n =3.(B) B. cinerea infection analysis. The percentages of different infection ratios in each genetic background were scored. (C and D) Root-hair phenotypes. (C) Representative picture of roots from seedlings grown on MS medium for 4 d and then transferred onto MS (Mock) or 25 μM JA medium for additional 2 d. Arrows indicate root hairs. (Scale bars, 50 μm.) (D) Root hair densities of 7-d-old seedlings grown on MS medium supplemented with indicated concentrations of JA were scored by counting the numbers of entire root hairs. Mean ± SEM, n = 10. (E and F) Relative root growth of 7-d-old seedlings grown on indicated concentrations of JA medium. Mean ± SEM, n = 10. **P ≤ 0.01, signiﬁcant differences between Col-0 and mutants.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1103959108 Zhu et al. Downloaded by guest on September 30, 2021 E. coli-expressed GST-fused N terminus of JAZ1 (containing the regulated by both JA and ET (39, 41). In addition, histone ZIM domain) or a GST-fused C terminus of JAZ1 (containing deacetylation is generally associated with transcriptional re- the Jas domain) to pull down EIN3 protein from extracted plant pression (33). These features make HDA6 a good candidate to total proteins. Our result revealed that the C terminus of JAZ1 be a corepressor of JAZ proteins. We thus tested the physical was mainly responsible for EIN3 binding (Fig. S3C). It has been interactions among JAZs, HDA6, and EIN3/EIL1. We found demonstrated that the C terminus of JAZs (Jas domain) is the that HDA6 interacted with EIN3 (amino acids 200–500) and interaction domain for JAZs-MYC2 binding (26), therefore JAZ EIL1 (amino acids 201–501) in yeast cells (Fig. 4A). We also proteins seem to use the same Jas domains to interact with var- carried out a co-IP assay using an inducible EIN3-3×FLAG in ious transcription factors. Together, these results indicate EIN3 ein3 eil1 ebf1 ebf2 background, in which the induced FLAG- and EIL1 as a previously unexplored class of JAZs-associated tagged EIN3 becomes more stabilized because of the lack of transcription factors. EBF1 and EBF2 and thus easier to detect (14). Our results showed that the anti-FLAG antibody efficiently coprecipitated JAZ Proteins Repress the Function of EIN3/EIL1. We next determined HDA6 together with FLAG-tagged EIN3, suggesting their as- whether JAZ proteins repress the function of EIN3/EIL1. First, sociation in vivo (Fig. 4B). we carried out a transient transcriptional activity assay in Ara- Meanwhile, we found that HDA6 also interacted with JAZ1 in bidopsis protoplasts using ERF1:LUC (firefly luciferase-coding yeast cells (Fig. 4C). To verify JAZ1-HDA6 interaction, we did gene driven by the ERF1 promoter) as a reporter. Overex- a pull-down assay and found that His-JAZ1 could pull down pression of EIN3 (35S:EIN3) induced ERF1:LUC expression, but HDA6 purified from plant extracts (Fig. 4D). To further con- overexpression of JAZ1 (35S:JAZ1) suppressed EIN3-induced firm HDA6-JAZ1 association, we performed a BiFC assay and expression of ERF1:LUC (Fig. 3A), indicating that JAZ1 some- demonstrated their in vivo interaction (Fig. S4B). We also tested how represses EIN3 function. Second, we introduced 35S:JAZ1- the interactions between HDA6 and other JAZ proteins (e.g., D3A (a stable form of JAZ1) (23) into EIN3ox plants and found JAZ3 and JAZ9) and found that HDA6 interacted with both that overaccumulation of JAZ1 dramatically reduced the sensi- JAZ3 and JAZ9 (Fig. S4A). To define the interaction domain of tivity of EIN3ox to JA (Fig. 3 B and C). Third, we found that the JAZ1 for HDA6 binding, we used GST-JAZ1 ΔN or GST-JAZ1 coi1 mutation also reduced the sensitivity of EIN3ox to JA in terms ΔC to do a pull-down assay and found that GST-JAZ1 ΔC was of ERF1 gene expression and root hair development (Fig. 3D). sufficient for HDA6 binding (Fig. 4E), suggesting that JAZ1 uses Finally, we introduced 5×EBS::GUS (the glucuronidase gene is distinctive interaction modules for EIN3- and HDA6-binding. driven by a promoter containing five tandem repeats of the EIN3- Because both JAZ1 and HDA6 interact with EIN3 (amino acids binding sites) (44) into Col-0, ein3-1,orein3 eil1 backgrounds. 200–500), we further performed yeast two-hybrid analysis with Upon ET or JA treatment, the transcriptional activity of EIN3 EIN3 deletion fragements and found that EIN3 (amino acids PLANT BIOLOGY indicated by the GUS level was augmented in Col-0 but di- 300–500) was sufficient for JAZ1-EIN3 interaction but not for minished or abolished in ein3-1 or ein3 eil1, respectively (Fig. 3E). HDA6-EIN3 interaction (Fig. S4C), suggesting that JAZ1 and Together, these results demonstrate that JAZs repress the func- HDA6 interact with different domains of EIN3. To investigate tion of EIN3/EIL1 and suggest that JA might relieve such re- the biological consequence of JAZ interactions with EIN3 and pression by removing JAZ proteins. HDA6, we used anti-FLAG antibody to coprecipitate HDA6 in Next, we investigated how JAZ proteins repress EIN3/EIL1. transgenic plants expressing FLAG-tagged EIN3 with or without Because JAZ1 interacts with the EIN3 (amino acids 200–500) and JA treatment. We found that JA treatment reduced the in vivo EIL1 (amino acids 201–501) fragments that overlap with their HDA6-EIN3 association (Fig. 4 F and G), suggesting that JAZ DNA binding domains (amino acids 59–359 in EIN3) (7), we de- proteins or other components are necessary for maximal in- termined whether JAZ1 interferes with EIN3 binding to DNA. teraction between HDA6 and EIN3 in vivo. ChIP-PCR assay showed that the in vivo association of EIN3 to the ERF1 promoter was increased by JA or ET (Fig. 3F). However, an HDA6 Is a Repressor for EIN3-Mediated Transcription and JA in vitro binding experiment using electrophoretic mobility shift Signaling. Histone deacetylases usually associate with transcrip- assay failed to detect the interference of JAZ1 on EIN3 binding to tional repression. To investigate whether HDA6 is involved in the ERF1 promoter element. One explanation for this discrepancy the repression of EIN3-mediated transcription and JA signaling, is that JAZ repressors possibly need additional components (co- we studied JA responses in HDA6 loss-of-function mutant (axe1-4) repressors) to suppress the DNA binding of EIN3 in vivo. and transgenic overexpressing plant (HDA6ox)(Fig. S5A). The JA-induced ERF1 gene expression, root hair development, and JAZ Proteins Recruit HDA6 to Associate with EIN3/EIL1. We then root growth inhibition were enhanced in axe1-4 (Fig. 5 A and B tested the possibility whereby corepressors are required for JAZ and Fig. S5B) but were attenuated in HDA6ox (Fig. 5A and Fig. repression of EIN3 function. It was reported that HDA6 is S5 B and C). Meanwhile, we used a specific histone deacetylase coimmunoprecipitated with COI1 and its expression is up- inhibitor, trichostatin A (TSA) (37) to mimic the loss of HDA

Fig. 2. JAZ proteins physically interact with EIN3. (A) Yeast two-hybrid assays showing the interaction between EIN3 and JAZ1. White colony indicates protein interaction in yeast cells grown on selective medium. (B) Pull-down assay. E. coli- expressed GST or GST-JAZ1 proteins were incubated with protein extracts from Col-0 and further immobilized by glutathione Sepharose 4B. Half of the pull-down products were probed with anti-EIN3 antibody to test interaction (Upper) and the other half of pull-down products were subjected to Coomassie blue staining as loading controls (Lower). (C) EIN3 was coimmunoprecipitated with JAZ1. The 35S:JAZ1-GUS seedlings were treated with 100 μM MG132 for 4 h to stabilize JAZ1-GUS and EIN3 proteins. Protein extracts from JAZ1-GUS were immunoprecipitated with anti-GUS antibody (+) or no antibody (−). Precipitated products were probed with anti- GUS or anti-EIN3 antibody to detect the interaction. (D) BiFC assay in onion epidermis cells. YFP ﬂuorescence indicates protein interaction. (Upper) Two arbitrary independent ﬁelds showing JAZ1-EIN3 interaction. (Lower) Negative controls. (Scale bars, 50 μm.)

Zhu et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 Fig. 3. JAZ proteins repress the transcriptional activity of EIN3. (A) Transient assay of ERF1:LUC expression. ERF1:LUC- 35S:REN reporter constructs were transiently expressed in Arabidopsis protoplasts together with control vector, 35S: EIN3, or 35S:EIN3 and 35S:JAZ1 effectors, respectively. The expression of REN was used as an internal control. LUC/REN ratio 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) Root elongation phenotypes of 7-d-old seedlings grown on 20 μM 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 ERF1 from 7-d-old seedlings treated without (Mock) or with 100 μMJAfor4h(Lower, mean ± SEM, n = 3). (E) GUS staining. 5×EBS::GUS expression in 7-d-old seedlings treated with Mock solution, 100 μM ACC (ET) or 100 μM JA (JA) for 4 h. ACC (1-aminocyclopropane-1-carboxylic acid) is the ethylene biosynthetic precursor. (Scale bar, 1 mm.) (F) ChIP-PCR result showing that JA enhances the DNA binding ability of EIN3 in vivo. Chromatin was extracted from 12-d-old seedlings after either 100 μMJAor100μM ACC treatment for 3.5 h and was precipitated using anti-EIN3 antibody. Pre- cipitated DNA was ampliﬁed by primers corresponding to sequences neighboring the EIN3 binding sites in the promoter of ERF1.Theein3 eil1 samples and no-antibody panels were negative controls showing the speciﬁcity of precipitation. Input indicates samples before immunoprecipitation.

activities. TSA treatment was sufﬁcient to induce root hair for- (Fig. 5A). Meanwhile, the levels of histone H4 acetylation in mation and ERF1 expression in Col-0, similar to the effect of JA coi1-2 and ein3 eil1 were greatly reduced compared with that of (Fig. S6 A–C). Interestingly, TSA induced root hair formation in Col-0 upon JA treatment (Fig. 5D), which again was in agree- the coi1-2 mutant but not in the ein3 eil1 mutant (Fig. S6A). ment with the levels of ERF1 expression and JA sensitivity in Consistent with this observation, ein3 eil1 completely suppressed these mutants (Fig. 1A and Fig. S1A). Taking these data to- the JA-hypersensitivity phenotypes of axe1-4 (Fig. 5 A and B). gether, we conclude that JAZ proteins recruit HDA6 (and These results suggest that HDA6 (and probably other histone probably other HDAs as well) to deacetylate histones (especially deacetylases) is a negative regulator of the JA-regulated pro- H4) and obstruct the chromatin binding of EIN3/EIL1, but JA cesses (e.g., ERF1 gene expression and root hair development) treatment removes JAZs-HDA6 corepressors away from EIN3/ acting in an EIN3/EIL1-dependent manner. EIL1 by degrading JAZs. To further explore the role of histone deacetylation in EIN3- mediated transcription, we analyzed the histone acetylation Discussion levels in the promoter region of the EIN3 target gene, ERF1. Emerging evidences show that the action of JA and ET is Upon JA treatment, the levels of both histone H3 and H4 synergistic and mutually dependent in regulating tolerance to acetylation were enhanced, but in axe1-4, this enhancement was necrotrophic fungi (8, 9), although not much is known about the more pronounced, particularly for histone H4 acetylation (Fig. molecular details of this hormonal synergy and signaling in- 5C), which agrees with JA induction of ERF1 expression in axe1-4 terdependency. In this study, we report EIN3 and EIL1 as two

Fig. 4. JAZ proteins recruit HDA6 to associate with EIN3. (A) Yeast two-hybrid assays indicating that HDA6 interacts with EIN3 and EIL1. (B) HDA6 was coimmunoprecipitated with EIN3- 3×FLAG. EIN3-3×FLAG is preinduced in pER8-EIN3-3×FLAG/ein3 eil1 ebf1 ebf2 transgenic plants by 10 μM β-estrogen for 4 h and 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 indicating that HDA6 interacts with JAZ1. (D and E) Pull-down assays. 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-down products 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 are proteins as negative controls. (F and G) JA treatment reduces EIN3-HDA6 association. (F) pER8-EIN3-3×FLAG/ein3 eil1 ebf1 ebf2 seedlings were initially treated with 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 as input controls. Protein extracts from Col-0 and axe1-4 were used as controls showing the speciﬁcity of anti-HDA6 antibody. (G) The relative intensities of HDA6 and EIN3 were calculated with Image J software (National Institutes of Health) from input or precipitated products (+Ab) to show the association of HDA6 and EIN3. Results from JA-treated samples (+JA) were normalized to samples without JA treatment (−JA). Similar results were obtained from two independent experiments and error bar represents SEM. (t test: *P = 0.026.)

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1103959108 Zhu et al. Downloaded by guest on September 30, 2021 JAZ-associated transcription factors integrating JA and ET function of EIN3/EIL1, and JA initiates a transcriptional de- signaling to regulate gene expression, pathogen defense, and repression event by degrading JAZ proteins (Fig. 3). HDA6/19 root development. Under normal conditions, EIN3/EIL1 are had been implicated in the regulation of JA-responsive genes repressed by JAZs, which recruit HDA6 as a corepressor to (40, 41), although it was not clear how histone deacetylation decrease EIN3/EIL1 transcriptional ability. ET treatment modulates JA signaling. Here, we reveal a previously unexplored enhances EIN3/EIL1 protein stability (14, 16–18), while JA mode of action for JAZ repressors, in which JAZ proteins recruit treatment leads to JAZs degradation and transcriptional de- HDA6 (and probably other HDAs) to switch chromatin structure repression, providing a nice explanation for the hormonal syn- into a less accessible state. Treatment of a specific HDA in- ergy in ET/JA-regulated gene expression and developmental hibitor, TSA, was sufficient to drastically induce ERF1 expression processes (Fig. S7). When ET signaling is blocked (e.g., in ein2), and root hair formation, suggesting that histone deacetylation is EIN3/EIL1 proteins are rapidly degraded (14, 18); therefore, the a major repression mechanism of these JA-regulated processes. absence of EIN3/EIL1 accumulation fails to induce downstream Consistent with this notion, the acetylation levels of histones gene expression and leads to ET/JA insensitivity. When JA sig- (particularly H4) in the ERF1 promoter region were significantly coi1 naling is blocked (e.g., in ), JAZs are highly accumulated increased by JA treatment, and such enhancement was more (23, 26), which strongly repress EIN3/EIL1 function even in the pronounced in the hda6 mutant, which were in good correlation presence of ET and JA. with the expression levels of ERF1. Interestingly, we noted that The discovery of JAZ proteins has successfully linked JA the H4 acetylation level of the ERF1 promoter was greatly re- receptors to downstream transcription factors. bHLH transcrip- duced in ein3 eil1, which could hardly be explained by the action tion factors (MYC2/MYC3/MYC4) and MYB transcription of HDAs, implying the involvement of histone acetyltransferases factors (MYB21/MYB24) are reported primary transcription in EIN3/EIL1-mediated gene activation. It thus remains to be factors associated with and repressed by JAZ protein (23, 30–32, fi investigated whether and how histone acetylation and deacety- 45). Our study has identi ed EIN3/EIL1 as a unique class of lation coordinately modulate JA/ET-regulated processes. JAZ-targeted transcription factors. EIN3 and EIL1 physically It was recently reported that JAZs interact with an adaptor interact with a number of JAZ proteins including JAZ1, JAZ3, protein NINJA that recruits Groucho/Tup1-type corepressor and JAZ9, and JAZ proteins use the same module containing TOPLESS and its related proteins to suppress MYC2-dependent the Jas domain to interact with EIN3/EIL1 and MYC2 (26). Unlike MYC2/3/4 that substantially contribute to JA-mediated transcription (28). As TOPLESS-mediated repression has been wound response and metabolism (30, 46), EIN3/EIL1 largely implicated to link with histone deacetylation (33), it would be regulate JA-induced root hair development and resistance to interesting to ascertain whether JAZs-NINJA-TOPLESS and ein3 eil1 JAZs-HDA6 repressors work as a large repressive module in necrotrophic fungi. We also examined the phehotypes of PLANT BIOLOGY for other types of JA responses, such as fertility and anthocyanin modulating JA signaling. Alternatively, JAZ proteins might adopt biosynthesis, and found that EIN3/EIL1 have little role in these distinct repression mechanisms to attenuate the function of processes, which are more likely regulated by MYC2 or MYB their associated transcription factors in different JA responses. In – support of this view, HDAs seem not involved in repressing transcription factors (32, 45 47). Consistently, JA was still able VSP2 to robustly induce the expression of VSP2 in ein3 eil1, a MYC2- MYC2 as TSA treatment does not induce expression, regulated gene invloved in wounding response (45). Inter- which is a MYC2 responsive gene. estingly, both EIN3/EIL1 and MYC2-mediated pathways par- Transcriptional derepression has also been implicated in other ticipate in JA inhibition of root elongation, although EIN3/EIL1 signaling integration. Eliminating repressors (DELLA proteins) seem to play a minor role in this growth response. Therefore, from transcriptional regulators (PIFs) has been reported in the EIN3/EIL1 work separately and cooperatively with other JAZs- coordinated regulation of plant growth and development by associated transcription factors to mediate a diverse range of JA gibberellins and light (48, 49). Our work provides further evi- responses. EIN3/EIL1 are predominant in JA regulation of dence to highlight transcriptional derepression as a possibly fungal defense and root hair development, whereas MYC2-like common mechanism for the integration of multiple signaling transcription factors are indispensible for JA-mediated wounding pathways in plants. Given the existence of a number of re- response and metabolism, and MYB21/MYB24 function mainly pressors in plant hormone signaling (DELLA proteins for gib- in stamen development (32). berellin, AUX/IAA proteins for auxin, JAZ proteins for JA, JAZ proteins have been identified as a group of respressors in A-type Arabidopsis-response regulators for cytokinin) (50), fur- JA signaling (23, 26, 27). We have presented several lines of ther research should focus on the identification of repressor- evidence to demonstrate that JAZ proteins act to inhibit the associated transcription factors that might be integration points

Fig. 5. HDA6 is a repressor for EIN3-mediated transcription and JA signaling. (A) qRT-PCR results showing relative expression of ERF1 from 7-d-old seedlings treated without (Mock) or with 100 μMJA for 4 h. Mean ± SEM, n = 3. Significant differences between Col-0 and axe1-4 or HDA6ox were indicated: **P ≤ 0.01. (B) Root-hair numbers. Four- day-old seedlings grown on MS were transferred to MS or 25 μM JA medium for 2 d. Root-hair numbers were counted from the 2-mL region of root tip under an Olympus microscope. Mean ± SEM, n ≥ 5. Significant differences between Col-0 and axe1-4 are indicated: **P ≤ 0.01. (C and D) Histone acetylation analysis by ChIP-PCR. Before ChIP analysis, seedlings were treated with or without 100 μMJA for 4 h. Antibodies specifically recognizing either acetylated histone H3 (AcH3) or H4 (AcH4) were used to precipitate chromatins. Precipitated DNA was amplified by primers corresponding to sequences neighboring the EIN3 binding sites in the promoter of ERF1. qPCR (C) or PCR product separated on ethidium bromide stained gel (D) shows the histone 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 indicates samples before immunoprecipitation.

Zhu et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 of plant hormones and other environmental or developmen- assay has been independently repeated at least twice with the same results. tal signals. All primers used in this study are summarized in Table S1.

Experimental Procedures Protein Expression and Purification. The coding sequences of JAZ1, JAZ1ΔN, Δ × Root Hair Density. We counted the entire root hair numbers from the root tip and JAZ1 C were cloned into pGEX 5 -1 vectors (GE Healthcare) for GST BL21 to the root-hypocotyl junction under an Olympus dissecting microscope, and fusion or pET-16b vectors (Novagen) for His fusion and transformed into divided this number by root length to obtain the value of root hair density. (DE3)-competent cells. Protein expression was induced by 0.1 mM isopropyl- fi Each root hair observation was carried out for at least three times with the beta-D-thiogalactopyranoside and proteins were puri ed by glutathione same result. Sepharose 4B (GE Healthcare) or Ni-NTA Agarose (Qiagen) following the manufacturer’s instructions. Yeast Two-Hybrid Assays. The coding sequences of COI1, JAZ1, and HDA6 were amplified from wild-type cDNA and cloned into pGBKT7 vectors, and ACKNOWLEDGMENTS. We thank Drs. Yan Guo, Lijia Qu, Joseph R. Ecker, the coding sequences of EBF2, MYC2, HDA6, EIN3, EIN3 (200–500), EIN3 (1– Yajie Niu, and John Browse for sharing research materials; and Drs. Daoxin Xie and Ian Wilson and colleagues in the H.G. laboratory for stimulating 300), EIN3 (200–400), EIN3 (300–500), and EIL1 (201–501) were cloned into discussions and critical reading of this manuscript. This work was supported pGADT7 vectors and transformed into yeast strain AH109 following the by National Natural Science Foundation of China Grants 91017010, ’ Matchmaker user s manual protocol (Clontech). Yeast transformants were 30625003, and 30730011 (to H.G.), and Ministry of Science and Technology streaked onto SD (-Leu/-His/-Ade/-Trp) medium (Clontech) and grown at 30 °C of China Grant 2009CB119101 (to H.G.); the publication fee is covered by the for 4 d. The white colonies represented interactions. Each yeast two-hybrid 111 project of Peking University.

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