THE MOLECULAR MECHANISM OF JAZ PROTEINS IN REGULATING JASMONATE RESPONSES IN ARABIDOPSIS By Christine Shyu A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Plant Biology 2012 ABSTRACT THE MOLECULAR MECHANISM OF JAZ PROTEINS IN REGULATING JASMONATE RESPONSES IN ARABIDOPSIS By Christine Shyu Jasmonate (JA) is a lipid-derived phytohormone that regulates a broad range of physiological processes, including wound responses, defense against insect herbivores and pathogens, and plant growth and development. JASMONATE ZIM-DOMAIN (JAZ) proteins are negative regulators of transcription factors that repress JA-induced gene expression. The current JA signaling model indicates that JAZ proteins require the adaptor protein NOVEL INTERACTOR OF JAZ (NINJA) to recruit the co-repressor TOPLESS (TPL) and repress transcription of JA-response genes. Upon wounding or other biological cues that lead to accumulation of bioactive JA, JAZ proteins are targeted for ubiquitination by the F-box protein CORONATINE INSENSITIVE1 (COI1) and degraded by the 26S proteasome. Degradation of JAZ proteins results in induction of JAresponsive gene expression. There are 12 JAZ proteins in Arabidopsis, and it is unknown how they regulate such a wide range of biological processes. In this study, I first show that JAZ proteins exhibit differential stability upon JA treatment, with JAZ8 being a relatively stable repressor. The increased stability of JAZ8 is a consequence of a unique degron motif that differs from the canonical degron of most others JAZs. Secondly, I report that JAZ8 represses JA-induced gene expression through its N-terminal EAR (for EAR-associated amphiphilic repression) motif, and that the EAR motif is required for direct interaction of JAZ8 with TPL. This finding suggests that JAZ8 does not repress JA responses through NINJA. Finally, I report the identification of a novel JAZ protein (called JAZ13) in Arabidopsis that is most closely related to JAZ7 and JAZ8. I provide genetic and biochemical evidence that JAZ13, like JAZ8, negatively regulates JA responses through a NINJA-independent pathway. Taken together, results from this study elucidate a novel mechanism to recruit TPL and repress JA responses, presumably for the purpose of fine-tuning myriad JA responses that balance plant defense and development. Copyright by CHRISTINE SHYU 2012 ACKNOWLEGDEMENTS It has been an amazing six years here at MSU-PRL, and there are many people I would like to thank. I came out fresh from college when I joined the Department of Plant Biology and PRL at MSU, and people and experiences here really helped train me to become a scientist. Without these people this dissertation would not have been possible. I would like to greatly thank Dr. Gregg Howe for letting me join the lab and be a part of the exciting JAZ research team. Gregg is a wonderful advisor that trained and shaped me to become the scientist I am now. He is very patient and supportive, and always makes sure that I am on track, without making me feel pushed. His inspiring yet critical thinking and high standards for solid and key scientific data really help me set a solid foundation for my dissertation research. I know that this training will also greatly impact my future scientific career. Gregg is truly a role model for me in many aspects. Words cannot express how grateful I am to be a graduate student in the Howe Lab. I am very fortunate to have a group of extremely smart, supportive and passionate professors as my committee members to guide me during my graduate studies. Being one of the “JAZzers”, Dr. Sheng Yang He provided many critical suggestions for the dissertation project. He also inspired me greatly in looking at the “big picture” and future of science. His pure passion for science will be a strong encouragement that I will always remember. Dr. Jianping Hu’s lecture in Plant Growth and Development was one of the most enjoyable courses I have taken. Her deep and comprehensive knowledge on plant hormones and developmental signaling has helped me significantly. I would also like to thank Dr. Michael Thomashow for giving me great comments and suggestions to ! "! improve my research. I will always remember the fun discussions on flowering time and epigenetics we had during the comprehensive exam. Committee meetings and comprehensive exams with this fantastic committee have truly been very enjoyable processes that helped me grow in knowledge, and shaped my academic career. It has been a wonderful experience to work with Howe Lab members. Thanks to all current and past lab members, who provided a stimulating and joyful environment to brainstorm, troubleshoot, conduct research, and discuss science and life as a scientist. Thanks to Abe Koo and Jin Ho Kang for sharing life experiences with me, Javier Moreno for many fun JAZ discussions and vacation suggestions, Yuki Yoshida for exciting scientific discussions on signaling, development, and experimental procedures. Leron Katsir and Hoo Sun Chung are good friends and pioneers in the JAZ project that set high standards and great examples for me to follow as a graduate student. Marcelo Campos and Caitlin Thireault are talented and fun graduate students that will surely JAZ up some exciting stories in the near future. I would like to especially thank Cody DePew and Li (Adam) Deng for friendship and technical assistance. Thanks go to Marco Herde, Lalita Patel, Eliana Gonzales-Vigil, Ian Major and other Howe Lab members that have helped me in multiple aspects throughout my PhD. I learned a lot from each and every one of you. Special thanks go to Yuki Yoshida and Marco Herde, who have been great company in the lab during late night and weekends. PRL and the MSU plant science community provided a friendly and supportive environment for me to learn and grow scientifically. I would like to thank many friends in the PRL, specifically John Withers and Jian Yao for fruitful discussions on the JAZ project, and study group members Kyaw Aung, Jeongwoon Kim, Chin Mei Lee and Yani ! "#! Chen for being great friends throughout the years. Special thanks also go to Day Lab members, specifically Liewei (Leo) Yan, Masaki Shimono and Patricia Santos, who were great company and support during the writing process of this dissertation, which sometimes lasted till 3 or 4 am. Finally, my deepest thanks and gratitude goes to God, for letting me learn and experience so much, and giving me wonderful friends and family to walk with me during this fantastic journey. I would like to dedicate this dissertation to my parents Shin-Guang Shyu and Hue-Min Wu, who are outstanding scientists that gave me their science genes and let me run around in their labs as I was growing up. Thanks to my loving sister Doris, who probably will also become a scientist when she grows up, for being super supportive. I would like to thank my dear friends in Christ, Jih-Hsuan Tammy Lin, Rui Zhang, Kris Wang and Lena Liao, and their beautiful kids Matthew and Josiah for their friendship and love. I also thank Tom and Sharon Snivley for giving me the wonderful Snivley hugs when I needed them the most. Thanks to Chu-Yin Yeh for her support and care as a roommate while I was preparing for comprehensive exams, and for being that wonderful friend that could talk science, basketball and faith. Special thanks go to my cat LaLa, who is the sweetest and most well-behaved cat ever, given that I spend more time in lab than at home. Thank you, who are reading this dissertation right now. It is extremely rewarding for a researcher to know that someone is interested in his or her work. I hope that research described in this dissertation contributes positively to the field, and that you may somehow benefit from it. ! "##! TABLE OF CONTENTS LIST OF TABLES..……………………………………………………………………...x LIST OF FIGURES…..…………………………………………………………………xi CHAPTER ONE – LITERATURE REVIEW…………………………………………1 1. THE BIOLOGY OF JASMONATES……………………………………………….2 2. JA BIOSYNTHESIS AND METABOLISM……………………………………….4 3. JA SIGNALING – BACKGROUND AND HISTORY…………………………….6 4. JASMONATE ZIM-DOMAIN PROTEINS………………………………………...7 4.1 Discovery of JAZ proteins………………………………………………………7 4.2 Domain features of JAZ proteins………………………………………………..9 4.3 JAZ-COI1 interaction – The JA-Ile receptor complex………………………...11 4.4 Alternative splice variants of JAZ proteins……………………………………12 4.5 The JAZ-NINJA-TOPLESS repression complex……………………………...13 4.6 JAZ-JAZ dimerization…………………………………………………………14 4.7 Involvement of transcription factors in jasmonate signaling…………………..15 4.8 Other JAZ-interacting proteins………………………………………………...16 4.9 Physiological role of JAZ proteins…………………………………………….18 5. CURRENT MODEL OF JA SIGNALING………………………………………..18 6. AIM OF RESEARCH……………………………………………………………...19 7. FIGURES AND TABLES…………………………………………………………21 8. REFERENCES…………………………………………………………………….28 CHAPTER TWO – JAZ8 LACKS A CANONICAL DEGRON AND HAS AN EAR MOTIF THAT MEDIATES TRANSCRIPTIONAL REPRESSION OF JASMONATE RESPONSES IN ARABIDOPSIS …………………………...………34 ABSTRACT……………………………………………………………………………35 INTRODUCTION……………………………………………………………………..36 RESULTS……………………………………………………………………………...40 DISCUSSION………………………………………………………………………….48 MATERIAL AND METHODS………………………………………………………..55 FIGURES AND TABLES……………………………………………………………..64 REFERENCES………………………………………………………………………...98 CHAPTER THREE – THE JAZ8 SUB-CLADE OF JAZ PROTEINS REPRESS JA SIGNALING THROUGH A NINJA-INDEPENDENT PATHWAY……………...105 ABSTRACT…………………………………………………………………………..106 INTRODUCTION……………………………………………………………………107 RESULTS…………………………………………………………………………….110 DISCUSSION………………………………………………………………………...115 MATERIAL AND METHODS………………………………………………………120 FIGURES AND TABLES……………………………………………………………125 REFERENCES……………………………………………………………………….142 ! "###! CHAPTER FOUR – SUMMARY AND FUTURE PERSPECTIVES……………..147 SUMMARY OF DISSERTATION…………………………………………………..148 FUTURE PERSPECTIVES…………………………………………………………..150 REFERENCES……………………………………………………………………….156 ! "#! LIST OF TABLES Supplemental Table 2.1. List of oligonucleotide primers used in this study……...….87 Supplemental Table 2.2. JAZ genes used for construction of consensus motifs shown in Figure 2.6……………………………………………………………………………93 Supplemental Table 3.1. List of oligonucleotide primers used in this article……....140 ! "! LIST OF FIGURES Figure 1.1. The Jasmonate Biosynthesis Pathway in Arabidopsis………...…………..21 Figure 1.2. Chemical Structures of Jasmonyl-Isoleucine (JA-Ile) and Coronatine…...23 Figure 1.3. The JAZ Family of Proteins in Arabidopsis…………………………...….24 Figure 1.4. Current Jasmonate Signaling Model………………………………………25 Figure 1.5. Domain Structure of JAZ Proteins…………………………...…………...26 Figure 2.1. JAZ Proteins Differentially Interact with COI1 in the Presence of JA-Ile ………………………………………………………………………………………….64 Figure 2.2. Overexpression of JAZ8 Results in Decreased Sensitivity to JA .……......66 Figure 2.3. JAZ8 is Resistant to JA-Mediated Degradation in Vivo……………...…..68 Figure 2.4. A Jas Domain Swap Converts JAZ8 to a COI1-Interacting Protein That Does Not Exert Dominant Repression of JA Responses………………………………69 Figure 2.5. The Canonical LPIAR Degron Promotes JAZ8 Binding to COI1, Destabilizes JAZ8, and Restores JA Responsiveness………………………………….71 Figure 2.6. The EAR Motif of JAZ8 is Required for Repression of JA Responses ….72 Figure 2.7. The EAR Motif is Required for Transcriptional Repression by JAZ8 …...74 Figure 2.8. Protein-Protein Interaction Domains in JAZ8………………...…………..76 Figure 2.9. The ZIM Domain is not Required for JAZ8-mediated Repression of JAinduced Root Growth Inhibition……………………………………………………….78 Supplemental Figure 2.1. Phylogenetic Tree Constructed from the Jas Motif of Arabidopsis JAZ Proteins…………………………………………………………..….79 Supplemental Figure 2.2. JA-Mediated Root Growth Inhibition in Independent 35S:JAZ8 Lines………………………………………………………………………...80 Supplemental Figure 2.3. 35S:JAZ8 Plants are Compromised in Resistance to Herbivory by S. exigua Larvae………………………………………………………...81 Supplemental Figure 2.4. JA-Mediated Root Growth Inhibition in 35S:JAZ8-YFP Lines……………………………………………………………………………………82 ! "#! Supplemental Figure 2.5. Substitution of PKASM to LPIAR does not Affect the Ability of JAZ8 to Interact with MYC2, JAZ1, or TPL. …………………………..….84 Supplemental Figure 2.6. Nuclear Localization of JAZ8-YFP Fusion Proteins….….85 Supplemental Figure 2.7. Yeast Two-hybrid Analysis of JAZ8 and JAZ8!ZIM Interactions with other Arabidopsis JAZ proteins…...………………………………...86 Figure 3.1. Amino acid sequence alignment of full length JAZ7, JAZ8 and JAZ13 ………………………………………………………………………………………...125 Figure 3.2. Phylogenetic tree of the JAZ family of proteins in Arabidopsis…….......126 Figure 3.3. Induction of JAZ7, JAZ8 and JAZ13 in response to wounding, MeJA and coronatine treatment…………………………………………………………………..127 Figure 3.4. Overexpression of JAZ13 leads to decreased sensitivity to JA-induced root growth inhibition……………………………………………………………………...128 Figure 3.5. The EAR motif of JAZ13 is required for interaction with TPL…..……..130 Figure 3.6. The Jas motif is required for interaction of JAZ7, JAZ8 and JAZ13 with MYC2………………………………………………………………………………...131 Figure 3.7. The Jas domain is required for JAZ-MYC2 interaction and JAZ repression of JA responses……………………………………………………………………….132 Figure 3.8. Characterization of knockout lines of JAZ7, JAZ8 and JAZ13 …...……..134 Figure 3.9. Root growth inhibition assay of jaz7-1, jaz8-1 and jaz13-1 lines. ……...135 Supplemental Figure 3.1. Identification of 35S-JAZ13 transgenic lines overexpressing JAZ13…………………………………………………………………………………136 R117A Supplemental Figure 3.2. Identification of 35S-JAZ8 overexpressing JAZ8 R117A transgenic lines …………………………………..………………………137 R117A Supplemental Figure 3.3. Characterization of 35S-JAZ8 –YFP transgenic lines…………………………………………………………………………………...138 Supplemental Figure 3.4. JAZ8 R117A –YFP localization in N. tabacum…..….…..139 ! ! "##! CHAPTER ONE LITERATURE REVIEW ! "! 1. THE BIOLOGY OF JASMONATES Plants are faced with numerous environmental changes on a daily basis. The sessile nature of plants prevents them from escaping or hiding from environmental stresses such as insect herbivores or extreme weather conditions. Adaptive responses to environmental stimuli are thus important for plants to survive and successfully carry on their next generation. Plant hormones provide an important mechanism for plants to regulate growth and responses to stress. There are multiple plant hormones, each of which has distinct and overlapping roles in regulating plant growth, development and defense (reviewed in Santner et al., 2009). Jasmonates (JA) are a particularly interesting group of phytohormones because of their dual role in inhibiting growth and promoting defense and reproduction. JA was first identified in the form of methyl-JA (MeJA) from jasmine essential oil extracts and later widely used in the perfume industry due to its pleasure scent (Demole and Lederer, 1962). Jasmonic acid was isolated from multiple plant species, and a biological role for JA as a growth inhibitor was described in 1981 (Yamane et al., 1981). Exogenous application of JA inhibits seedling growth, promotes senescence and represses photosynthesis (Ueda and Kato, 1980; Reinbothe et al., 1994; ). JA also has roles in reproductive development in multiple species such as rice, maize, Arabidopsis and tomato. MeJA inhibits spikelet formation in rice(Kim et al., 2009). Mutations in a maize lipoxygenase TASSELSEED1 (TS1) lead to disruption in stamen development that can be restored by applying JA (Acosta et al., 2009). Independent mutations in several steps of the JA biosynthetic pathway result in male sterility, in which Arabidopsis flowers of these mutants have shorter anther filaments and defects in pollen development (McConn and Browse, 1996; Sanders et al., 2000; Stintzi and Browse, 2000; Ishiguro et al., 2001). Exogenous JA restores fertility of these lines. On the other hand, mutations ! "! in a key JA perception component COI1 also leads to male sterility (Feys et al., 1994). Interestingly, mutations in the tomato ortholog of COI1 result in female sterility and defects in male fertility as well (Li et al., 2001). Taken together, research on multiple plant species demonstrate a role for JA in inhibiting plant growth and promoting reproductive development. The discovery of JA as a master regulator of plant defense was a key advance in understanding JA biology. Tomato protease inhibitors (PIs), which are involved in defense against insect herbivores, were shown to be highly expressed upon MeJA treatment (Farmer and Ryan, 1990). Indeed, mutations in genes regulating JA biosynthesis or signaling severely compromise defense against insect feeding (Howe et al., 1996; McConn and Browse, 1996). Many other roles for JA in plant defense have been characterized, including secondary metabolite production, trichome development, and defense against pathogens (Vijayan et al., 1998; Chen et al., 2006; Yoshida et al., 2009). All of these discoveries establish JA as a well known “wound hormone” (Koo and Howe, 2009). In addition to defense against biotic stresses, JA also has multiple roles in plant responses to abiotic stress. These roles include responses to UV light, ozone stress and other abiotic stresses (Conconi et al., 1996; Weiler et al., 1993; Dombrowski et al., 2003). JA has dual roles in regulating growth and defense. It has been increasingly important to understand how multiple signaling pathways in a regulatory network crosstalk with each other to fine-tune signal outputs under different conditions. Understanding how JA regulates different responses to balance growth and defense is an important starting point toward this goal. Understanding of JA biology can be further applied to agricultural and industrial purposes, in which one can manipulate crops to become more resistant to insect herbivores and pathogens, which will increase crop yields. In addition to its role as an essential phytohormone, JA has been ! "! shown to be an effective anti-cancer agent that inhibits tumor cell growth and promotes apoptosis (Xiao et al., 2011; Milrot et al., 2012). These results highlight the importance of JA research in both agricultural and medical research. 2. JA BIOSYNTHESIS AND METABOLISM A detailed understanding of the biosynthesis of JA has been obtained mainly by research initiated from forward and reverse genetic screens in Arabidopsis and tomato. JA is synthesized via an octadecanoid pathway in two cellular components: the chloroplast and peroxisome (Figure 1.1). The first step of JA biosynthesis is in the chloroplast, which is composed of high levels of trienoic fatty acids. A role for chloroplast lipids in JA biosynthesis was demonstrated in experiments utilizing a triple mutant of fatty acid desaturases (fad 3 fad7 fad8). The fad 3 fad7 fad8 mutant, which is defective in trienoic fatty acids, is male sterile, and exogenous application of JA restores fertility (McConn and Browse, 1996). In the second step of JA biosynthesis, !linolenic acid (18:3) is released from membrane lipids by the lipase DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1). Similar to fad 3 fad7 fad8, dad1 shows defects in male fertility that can be restored by exogenous JA (Ishiguro et al., 2001). !-Linolenic acid (18:3) is converted to 13-hydroxyperoxylinolenic acid by 13-lypoxygenases, such as LOX2 (Bell et al., 1995). 13Hydroxyperoxylinolenic acid is further metabolized to 12-oxo-phytodienoic acid (OPDA) by allene oxide synthase (AOS) and allene oxide cyclase (AOC) (Song and Brash, 1991; Ziegler et al., 2000). The production of OPDA completes the chloroplastic steps in JA biosynthesis. Transport of OPDA from the chloroplast to peroxisome requires the ATP-binding cassette transporter protein COMATOSE (CTS) (Theodoulou et al., 2005). Upon transport to the ! "! peroxisome, OPDA is further metabolized to 3-oxo-2-(cis-2’-pentenyl)-cyclopentane-1-octanoic acid (OPC 8:0) and 3-oxo-2-(cis-2’-pentenyl)-cyclopentane-1-octanoyl CoA (OPC 8:0-CoA) by peroxisomal OPDA reductases (OPR) and OPC-8:0 CoA ligases (OPCL) (Stintzi and Browse, 2000; Schneider et al., 2005). The final phase of JA biosynthesis involves three cycles of !oxidation in the peroxisome by oxidases such as acyl CoA oxidases (ACX) and ABNORMAL INFLORESCENCE MERISTEM (AIM1) (Li et al., 2005; Delker et al., 2007; Schilmiller et al., 2007). Following export from the peroxisome, JA is further metabolized to various metabolites in the cytoplasm. Examples include methylation of JA by a methyl transferase to produce MeJA, conjugation of JA to isoleucine (Ile) to yield JA-Ile and hydroxylation of JA to produce 12-OHJA (Seo et al., 2001; Staswick and Tiryaki, 2004; Koo et al., 2011). Although many JA metabolites have been identified in plants, (3R, 7S)-JA-Ile is the only known receptor-active form of the hormone. JA-Ile levels are rapidly induced upon wounding, and JA-Ile is likely the most abundant conjugated form of JA in Arabidopsis (Koo et al., 2009). JA-Ile synthesis from JA and Ile is catalyzed by JASMONATE RESISTANT 1 (JAR1) (Staswick and Tiryaki, 2004). Mutation of JAR1 results in loss of JA-Ile accumulation and decreased sensitivity to JA in root growth inhibition assays. Application of JA-Ile, but not other JA metabolites, complements the jar1 root growth phenotype (Staswick and Tiryaki, 2004). jar1 plants also have defects in other JA responses such as insect feeding (Kang et al., 2006). These results collectively indicate that JA-Ile plays an essential role in JA signaling. JA-Ile can be further metabolized to a less active form, 12-OH-JA-Ile. This involves a subgroup of cytochrome P450 hydroxylases in the CYP94 family (Koo et al., 2011). Mutant lines of CYP94B3 hyperaccumulate JA-Ile and are deficient in 12-OH-JA-Ile in response to wounding. cyp94b3 lines exhibit increased sensitivity to JA-mediated root growth inhibition, and overexpression of ! "! CYP94B3 results in decreased sensitivity to JA ((Kitaoka et al., 2011; Koo et al., 2011; Heitz et al., 2012). In vitro enzymatic assays show that CYP94B3 catalyzes oxidation steps or JA-Ile to produce 12OH-JA-Ile, which is less active than JA-Ile in promoting COI1 interaction with JAZ proteins (Koo et al., 2011; Heitz et al., 2012). These results firmly demonstrate that catabolism of JA-Ile is an essential pathway to deactivate JA-Ile. Interestingly, several strains of the plant pathogen Pseudomonas syringae produce a phytotoxin called coronatine that is structurally similar to JA-Ile (Bender et al., 1999) (Figure 1.2). Indeed, coronatine and JA elicit many of the same responses, including inhibition of seedling growth (Feys et al., 1994). It is generally accepted that coronatine is a molecular mimic of JA-Ile and widely used in biochemical assays to approach questions related to JA signaling. 3. JASMONATE SIGNALING – BACKGROUND AND HISTORY An important breakthrough in jasmonate research was the discovery of CORONATINE INSENSITIVE 1 (COI1). Application of coronatine to Arabidopsis growth medium results in inhibition of seedling growth, similar to effects of MeJA application. Seedlings treated with coronatine have shorter primary roots compared to those grown without coronatine. MeJA and coronatine also elicit chlorosis in vegetative tissues. Feys et al. (2004) reported an ethyl methane sulfonate (EMS) mutant screen to identify mutants that have reduced sensitivity to coronatine. The coi1 mutant was identified as being insensitive to both coronatine and MeJA. In addition to seedling growth phenotypes, coi1 plants also have shorter anther filaments and are male sterile. This is a phenotype commonly observed among mutants deficient in JA biosynthesis. Moreover, coi1 is insensitive to the coronatine-producing bacterial pathogen Pseudomonas syringae. The ! "! COI1 gene was identified by map-based cloning and shown to encode an F-box protein (Xie et al., 1998). Similar findings were reported in tomato; a screen for MeJA-insenstive tomato seedlings identified a mutant called jasmonate insensitive 1 (jai1), that that lacks all known JA responses. Mapping of jai1 revealed that it defines the tomato ortholog of Arabidopsis COI1 (Li et al., 2004). F-box proteins are components of SCF (Skp/Cullin/F-box) E3-type ubiquitin ligases that provide targeting specificity for the SCF complex to recognize and target proteins for ubiquitination and degradation via the 26S proteasome pathway (Hershko and Ciechanover, 1998). Identification of COI1 as a F-box protein suggested that it may target repressors of jasmonate responses for degradation in response to a JA signal. Transcription factors are also important components of the JA signaling pathway. One transcription factor that is involved in this process is JASMONATE INSENSITIVE 1 (JIN1), also known as MYC2. Mutations in the MYC2 gene were identified in mutant screens for seedlings insensitive to jasmonate treatment (Lorenzo et al., 2004). MYC2 belongs to a family of basic helix-loop-helix transcriptional regulators. Unlike coi1 and mutants affected in JA biosynthesis, jin1 mutants are fertile and do not exhibit obvious growth-related phenotypes. This indicates that other transcription factors are likely involved in other jasmonate responses. Knowledge of COI1 and MYC2 led to the following model: in the presence of JA, COI1 targets repressor proteins that work through an unknown mechanism to repress transcription factors such as MYC2. 4. JASMONATE ZIM-DOMAIN PROTEINS 4.1. Discovery of JAZ proteins ! "! The missing link in the JA signaling pathway are the JASMONATE ZIM-DOMAIN (JAZ) proteins, which were identified nearly a decade after the discovery of COI1. Three independent groups identified JASMONATE ZIM-DOMAIN PROTEINS as JA-responsive transcriptional repressors (Chini et al., 2007; Thines et al., 2007; Yan et al., 2007). Thines et al. (2007) identified JAZ genes in a microarray experiment comparing gene expression patterns in wild type (WT) and the JA-biosynthesis mutant opr3, treated with exogenous jasmonate. Seven genes of unknown function were identified in a list of rapidly induced genes in WT but not opr3, 0.5-hours after JA treatment. All seven genes contain a conserved ZIM domain. Homology search of the Arabidopsis genome identified five additional ZIM-domain-containing genes of unknown function. The 12 ZIM-domain containing proteins were named JASMONATE ZIM DOMAIN (JAZ) 1-12 (Figure 1.3). Loss-of-function and gain-of-function analyses of various JAZ family members did not reveal obvious JA-related phenotypes. However, overexpression of a form of JAZ1 lacking the C terminus (JAZ1!3A) resulted in several JA-insensitive phenotypes, including male sterility. This suggests that JAZ proteins are involved in JA signaling. Chini et al. (2007) identified jai3-1 (later renamed jaz3) in a mutant screen screening for seedlings with altered JA sensitivity. Map-based cloning revealed that jai3-1 contains a G to A mutation in the JAZ3 splicing-acceptor site of its fifth intron, resulting in deletion of the JAZ3 Cterminus. The jai3-1 mutant is insensitive to JA-induced root growth inhibition, similar to 35SJAZ1!3A lines. Yan et al. (2007) identified JASMONATE-ASSOCIATED1 (JAS1)/JAZ10 using a transcript profiling approach similar to that described by Thines et al. (2007). Genes differentially expressed between WT and aos upon wounding were overexpressed in WT Arabidopsis plants to screen for JA-related phenotypes. Although overexpression of JAZ10 did ! "! not result in altered JA-responsiveness, overexpression of an alternative-spliced form of JAZ10, JAZ10.3, resulted in decreased sensitivity to JA. JAZ proteins are hypothesized to be repressors that are targeted by COI1 for degradation in response to JA treatment (Chini et al., 2007; Thines et al., 2007). Indeed, JAZ1-GUS is degraded in a JA- and COI1-dependent manner. JAZ1-GUS degradation is also reduced when seedlings overexpressing JAZ1-GUS were treated with the protease inhibitor MG132. This suggests that JAZ1-GUS is degraded via the 26S proteasome pathway, consistent with known SCF ubiquitination-degradation pathways. Similarly, JAZ3-GFP is also rapidly degraded in a COI1-dependent manner upon JA treatment (Chini et al., 2007). Other JAZs, including JAZ6, JAZ9 and JAZ10 are also degraded in responses to JA treatment (Chini et al., 2007; Thines et al., 2007; Chung and Howe, 2009). Moreover, JAZ proteins physically interact with COI1 in the presence of JA-Ile (Thines et al., 2007). These results collectively demonstrate that JAZ proteins are direct targets of SCFCOI1. Another important observation reported in Chini et al. (2007) is that JAZ3 interacts with the JA-responsive transcription factor MYC2 in yeast two-hybrid and in vitro pull-down assays. This discovery, along with other observations reported in these three pioneer papers (Chini et al., 2007; Thines et al., 2007; Yan et al., 2007) leads to the following model: JAZ proteins are repressor proteins that bind to MYC2 in the absence of JA. Upon accumulation of bioactive JA, JAZ repressors are targeted for ubiquitination by COI1 and degraded via the 26S proteasome pathway to activate JA-induced gene expression (Figure 1.4). 4.2. Domain Features of JAZ Proteins ! "! JAZ proteins share three conserved domains: an N-terminal Domain 1 (D1), a ZIM domain and a C-terminal Jas domain (Figure 1.5). The ZIM domain was first identified in the Zinc-finger protein expressed in inflorescence meristem (ZIM) (Nishii et al., 2000). A hallmark of ZIM domains is the TIFY sequence motif, hence the ZIM domain is sometimes referred as the TIFY domain (Vanholme et al., 2007). In silico approaches revealed the presence of TIFY family proteins in all species of land plants, but not the single-cellular green algae Chlamydomonas rainhartii or the multicellular green algae Volvox carteri (Bai et al., 2011). In the case of JAZ proteins, the ZIM domain has important roles in protein-protein interaction, specifically in mediating interaction with a NOVEL INTERACTOR OF JAZ (NINJA) corepressor adapter protein. The ZIM domain is also involved in interaction between JAZ proteins to form homo or heterodimers. Functional roles of JAZ-NINJA and JAZ-JAZ interactions mediated by the ZIM domain are discussed in later sections of this chapter. The Jas domain is a 27-amino-acid motif located at the C-terminus of all 12 JAZ proteins. The Jas domain shares sequence similarity to the N-terminal region of CCT domains reported in CONSTANS (CO) (Strayer et al., 2000; Robson et al., 2001; Chung et al., 2009). Although the CCT domain in CO is reported to be involved in DNA binding, analyses of the Jas domain suggested different functions (Ben-Naim et al., 2006). A key feature of the Jas domain is the degron motif that facilitates JAZ-COI1 interaction (Melotto et al., 2008; Sheard et al., 2010). The Jas domain is also required for interaction with MYC2 and other transcription factors involved in JA signaling (Chini et al., 2007; Fernandez-Calvo et al., 2011; Qi et al., 2011; Song et al., 2011). Moreover, Grunewald et al (2009) suggested a role for the C-terminal region of Jas motif in nuclear localization of JAZ1 (Grunewald et al., 2009). ! "#! The N-terminal D1 is less conserved among JAZ proteins. It has been reported that JAZ1 interacts with DELLA proteins, which are important transcriptional repressors in regulating gibberellin responses (Hou et al., 2010; Yang et al., 2012). The N-terminal motif of JAZ1, which contained D1, is critical for JAZ1-DELLA interaction (Hou et al., 2010). The functional role of D1 in other JAZ proteins is not known. 4.3. JAZ-COI1 interaction – the JA-Ile receptor complex One key feature of JAZ proteins is their role as targets of SCFCOI1. JAZ proteins interact with COI1 in a JA-Ile-dependent manner in yeast two-hybrid and in vitro pull-down assays (Thines et al., 2007; Katsir et al., 2008; Melotto et al., 2008). JAZ-COI1 interaction is also detected in the presence of coronatine, which is a molecular mimic of JA-Ile (Melotto et al., 2008). Coronatine binds specifically to COI1-JAZ complexes. The ability of JA-Ile to compete with COR shows that both are recognized by the same receptor (Katsir et al., 2008). These results suggest an important role for the COI1-JAZ complex in perceiving bioactive ligands. JAZ3-COI1 interaction is dependent on the Jas domain. JAZ3 recombinant proteins lacking the Jas domain fail to interact with COI1 in in vitro pull down assays (Kastir et al., 2008). A JAZ3 C-terminal fragment containing the Jas domain is sufficient to interact with COI1 in a JA-Ile-dependent manner. Indeed, coronatine binding assays using purified COI1 and different fragments of the Jas domain of JAZ1 indicate that the N-terminal motif within the Jas domain is critical for coronatine binding (Sheard et al., 2010). A peptide containing 21 amino acids of the JAZ1 Jas motif, including an N-terminal ELPIAR motif, is sufficient to bind coronatine. ! ""! Mutation or deletion of the ELPIAR motif results in loss of coronatine binding. Sheard et al (2010) hence defined the first 21 amino acids within the Jas domain as the JAZ “degron” motif. Structural analysis of COI1 revealed that the COI1-JAZ complex is the true receptor for JA-Ile (Sheard et al., 2010). COI1 was crystalized along with a JAZ1 degron peptide in the presence of the bioactive (3R, 7S) JA-Ile. The JA-Ile COI1-JAZ receptor complex is very similar to the auxin TIR1-Aux/IAA receptor complex, with the F-box protein COI1 forming a binding pocket for JA-Ile, and the JAZ degron forming a molecular clamp to secure ligand binding in the binding pocket. The LPIAR residues within the JAZ degron are responsible for direct contact with JA-Ile. This explains why the DLPIAR motif is required for COI1-JAZ1 to bind coronatine. Interestingly, crystallization of the COI1-JAZ JA-Ile receptor complex revealed inositol pentakisphosphate (InsP5) as a cofactor in the receptor complex (Sheard et al., 2010). This is supported by the JA hypersensitive phenotype of the Arabidopsis ipk1-1 mutant (Mosblech et al., 2011), which accumulates InsP4 and InsP5 due to a defect in the conversion of InsP5 to InsP6 by the INOSITOL POLYPHOSPHATE KINASE1 (IPK1) (Stevenson-Paulik et al., 2005). It is interesting to speculate that biosynthesis and metabolism of InsP5 may provide a mechanism of regulating JA-Ile perception. In summary, structural and biochemical analyses have demonstrated that the jasmonate receptor is composed of both COI1 and JAZ and a cofactor InsP5. The degron motif that mediates COI1-JAZ1-interaction has been defined. It is yet to be explored whether or not there is a role for the degron motif of JAZ proteins in planta. 4.4. Alternative Splice Variants of JAZ Proteins ! "#! Ectopic expression of artificially truncated JAZ proteins that lack the Jas motif results in decreased sensitivity to JA (Chini et al., 2007; Thines et al., 2007). Interestingly, many plants express natural JAZ splice variants (JAZ!Jas) lacking the Jas motif (Chung et al., 2010). In most Arabidopsis genes, alternative splicing of a conserved intron within the Jas domain coding region prematurely truncates the highly conserved X5PY submotif that defines the C-term end of the Jas domain. The resulting splice variants lacking the C-terminal X5PY sequence are resistant to JAIle-promoted JAZ-COI1 interaction and, as a consequence, exert dominant repression on JA signaling (Chung and Howe, 2009; Chung et al., 2010). For example, overexpression of the JAZ10.3 splice variant results in decreased sensitivity to JA (Yan et al., 2007; Chung and Howe, 2009). Based on these findings, it was proposed that stable JAZ splice variants play a role in preventing plants from hyper-responding to JA. The biological role of stabilized JAZ splice variants and how they are removed from the cell are interesting future questions to investigate. 4.5. The JAZ-NINJA-TOPLESS Repression Complex JAZ proteins repress JA-responsive gene expression by recruiting a Groucho/Tup1 corepressor TOPLESS (TPL) through an adapter protein NOVEL INTERACTOR OF JAZ (NINJA) (Pauwels et al., 2010). Tandem affinity purification (TAP) assays followed by mass spectrometry (MS) were used to identify JAZ1-interacting proteins in suspension-cultured Arabidopsis cells. Among the proteins identified with this approach were COI1, MYC2, JAZ12, and protein of unknown function, which was named NINJA. NINJA interacts with the ZIM domain of JAZ1. Overexpression of NINJA represses jasmonate-induced root growth inhibition, whereas knock-down of NINJA expression by RNAi leads to hypersensitivity to JA. These ! "#! results collectively indicate that NINJA is involved in repression of jasmonate responses, presumably through direct interaction with JAZ proteins. Pauwels et al. (2010) searched for NINJA-interacting proteins using the same TAP-MS method, which resulted in identification of the corepressor TPL. This result is striking because TPL was previously shown to regulate auxin responses by interacting directly with Aux/IAA transcriptional repressors (Szemenyei et al., 2008). Indeed, tpl-1, which has strong temperaturesensitive auxin phenotypes, is hypersensitive to JA (Long et al., 2002; Pauwels et al., 2010). NINJA interacts with TPL through an ETHYLENE RESPONSIVE FACTOR-associated amphiphilic repressor (EAR) motif located near the N-terminal end of NINJA. Based on these findings, it was proposed that JAZ proteins repress JA responses by interacting simultaneously with MYC2 and, via the NINJA adaptor, TPL. The mechanism by which TPL represses expression of JA response genes is currently not known. Interestingly, JAZ7 and JAZ8 do not interact with NINJA (Pauwels et al., 2010). Genome-wide analysis of EAR motif-containing proteins in Arabidopsis identified EAR motif sequences in JAZ5, JAZ6, JAZ7 and JAZ8 (Kagale et al., 2010). Whether or not JAZ proteins can repress through an alternative mechanism that directly recruits EAR-interacting corepressors, similar to the auxin repression pathway, remains an open question. 4.6. JAZ-JAZ Dimerization JAZ proteins homo- and heterodimerize with other members in the JAZ family (Chini et al., 2009; Chung and Howe, 2009). JAZ-JAZ interaction is mediated through the ZIM domain. Ala-scanning of the TIFY motif of JAZ10 revealed that the I residue within the TIFY motif is ! "#! required for JAZ10 dimerization (Chung and Howe, 2009). Ectopic expression of a stabilized form of JAZ10, JAZ10.4, results in decreased sensitivity to JA. Overexpression of JAZ10.4 I!A compromises the JA-insensitive phenotype that was observed in 35S-JAZ10.4 lines. This result suggests that the TIFY motif is required for JAZ repression. Interestingly, the TIFY motif is also involved in JAZ-NINJA interaction (Pauwels et al., 2010). Whether the JAZ10.4 I! A overexpression phenotype is due to lack of JAZ-JAZ interaction, or to perturbation of the JAZNINJA interaction, remains to be elucidated. Detailed analyses of the ZIM domain structure is expected to provide important information about how JAZ proteins repress JA responses. 4.7. Involvement of Transcription Factors in Jasmonate Signaling JAZ proteins regulate gene expression by physically interacting with JA-responsive transcription factors. Prior to JAZ identification, MYC2 was the only transcription factor known to have a direct role in promoting JA responses. Discovery of JAZ proteins led to rapid identification of novel transcription factors that regulate JA responses. MYC3 and MYC4 are two bHLH transcription factors closely related to MYC2 that interact with multiple JAZ proteins (Fernandez-Calvo et al., 2011). Domain mapping experiments indicate that MYC2/3/4 have an N-terminal JAZ-interacting domain (JID). Genetic analyses show that MYC3 and MYC4 play an important role in JA-mediated insect resistance, whereas MYC2 is mainly involved in regulating JA-mediated root growth inhibition. Wounding or application of MeJA induces trichome formation (Yoshida et al., 2009). Trichome development is regulated by WD-repeat/bHLH/MYB complexes that involve a WDrepeat protein TRANSPARENT TESTA 1 (TTG1), three bHLH proteins (GLABRA3, GL3; ! "#! ENHANCER OF GLABRA3, EGL3; TRANSPARENT TESTA 8, TT8) and the MYB transcription factors MYB75 and GLABRA 1 (GL1). All three bHLH proteins contain a JID that interacts with at least eight distinct JAZ proteins (Qi et al., 2011). The two MYB proteins, MYB75 and GL1, interact with JAZ1, JAZ8 and JAZ11. GL1 also interacts with JAZ10. TTG1, on the other hand, does not reported interact with JAZ proteins. Interestingly, overexpression of JAZ1 abolishes formation of the WD-repeat/bHLH/MYB complex. Furthermore, 35S-JAZ1!3 transgenic lines show reduced JA-induced trichome formation and anthocyanin accumulation. These results collectively show that JAZ proteins interact with WD-repeat/bHLH/MYB complexes to disrupt the complex and repress trichome development. In addition to bHLH transcription factors, JAZ proteins also interact with MYB transcription factors. MYB21 and MYB24 were identified as JAZ8-interactors via yeast twohybrid screens (Song et al., 2011).Genetic analyses of MYB21 and MYB24 loss-of-function and gain-of-function lines revealed that MYB21 and MYB24 play a role in JA-regulated male fertility but not other JA responses such as insect resistance or anthocyanin accumulation. To summarize, JAZ proteins interact with a wide array of transcription factors to regulate different JA signaling outputs. Understanding whether or not there are specificities for JAZ-TF interaction, and examining expression patterns of different JAZs and transcription factors will lead to greater understanding of how JAZ proteins regulate different JA responses. 4.8. Other JAZ-Interacting Proteins JAZ proteins are important players in cross-regulation of JA and other signaling pathways. One example is JA and gibberellin (GA) cross-regulation through JAZ-DELLA ! "#! interaction. DELLA proteins function as transcriptional repressors that are rapidly degraded in the presence of GA (Dill et al., 2001). JAZ1 and JAZ9 interact with the DELLA protein REPRESSOR OF GAI-1 (RGA) (Hou et al., 2010; Yang et al., 2012). Interestingly, JA antagonizes the GA signaling pathway by delaying GA-induced DELLA degradation. Moreover, DELLAs compete with MYC2 for binding to JAZ1 to control the expression of MYC2-regulated target genes (Hou et al., 2010). coi1 and JAZ9 overexpression mutant are hypersensitive to GA (Yang et al., 2012). These results collectively demonstrate that JA and GA crosstalk is regulated through JAZ-DELLA interaction, and that this may provide a way for plants to regulate the balance between growth and defense. JAZ1 also interacts with ethylene-responsive transcription factors ETHYLENE INSENSITIVE 3 (EIN3) and its closest homolog EIN3-LIKE 1 (EIL1) (Zhu et al., 2011). EIN3 and EIL1 are transcription factors that are rapidly degraded in the absence of ethylene, and are stabilized in the presence of ethylene to regulate ethylene responses (An et al., 2010). JAZ proteins repress the function of EIN3 and EIL1 by recruiting a histone deacetylase HDA6 to repress EIN3/EIL1 activity (Zhu et al., 2011). These results suggest that EIN3/EIL1 responsive gene expression requires both ethylene and JA. This also indicates that JAZ proteins are important nodes to integrate JA and ethylene responses. In summary, JAZ proteins interact with and modulate transcription factors in other hormone signaling pathways. JAZ binds to DELLA repressors to positively regulate GA responses, and interact with EIN3/EIL1 to repress ethylene responses. JAZ1 interaction with HDA6 also suggests an alternative mechanism for JAZ proteins to repress gene expression. It has become increasingly accepted that response to a particular signal is not the result of a single linear pathway, but integration of multiple signaling pathways. This phenomenon is referred as ! "#! “crosstalk” between signaling pathways (Vert and Chory, 2011). Crosstalk between different signaling pathways can be “direct” or “indirect”, depending on whether or not there are shared components between different pathways that regulate a common signaling output (Vert and Chory, 2011). JAZ proteins have emerged as one of the best examples of direct crosstalk, in which JAZ proteins directly interact with signaling components in other pathways to regulate downstream responses. 4.9. Physiological Role of JAZ proteins Although much has been revealed about the molecular mechanism of JAZ function, very little is known about the physiological role of individual JAZ proteins. Most jaz mutants reported to date have no obvious growth or development phenotypes, presumably due to functional redundancy of other JAZ protiens (Thines et al., 2007). Currently the only two JAZ loss-offunction lines that exhibit detectable phenotypes are described below. jaz10-1 lines are hypersensitive to JA-induced root growth inhibition and JAZ10 RNAi lines are also hypersensitive to JA-inhibited growth responses (Demianski et al., 2010; Yan et al., 2007). JAZ9, on the other hand, is involved in cross-regulating GA responses. jaz9-1 and jaz9-3 lines are lateflowering and have shorter petiole lengths, which are typical GA-insensitive phenotypes (Yang et al., 2012). Characterization of other JAZ loss-of-function mutants and higher-order mutants will provide more insight in how different JAZ proteins regulate diverse JA signaling outputs. 5. CURRENT MODEL FOR JA SIGNALING ! "#! The discovery and characterization of JAZ proteins has significantly sped up our understanding of the JA signaling pathway. Most importantly, JAZ proteins connected prior knowledge of the F-box protein COI1 to the transcription factor MYC2. The current JA signaling pathway is summarized in the following paragraph: (Figure 1.3) In the absence of bioactive JA, JAZ proteins interact with multiple transcriptional regulators, including bHLH, MYB, and EIN3/EIL1, to repress JA-induced gene expression. JAZ proteins repress gene expression by recruiting the TOPLESS corepressor complex through the adapter protein NINJA. Upon wounding or other developmental cues that lead to accumulation of JA-Ile, JAZ proteins form receptor complexes with COI1 to perceive JA-Ile, and are further ubiquitinated and degraded through the 26S proteasome pathway. Degradation of JAZ proteins leads to activation of JA-responsive transcription factors, and induction of gene expression. 6. AIMS OF DISSERTATION The main focus of this dissertation is to determine the molecular mechanism of JAZ function in response to JA. Firstly, I aim to determine whether or not all JAZ proteins interact with COI1 in a JA-Ile-dependent manner. I show that not all JAZ proteins interact with COI1 in the presence of JA-Ile, and JAZ8 is a weak COI1-interactor that is stable upon JA treatment. The results of this aim are described in Chapter 2 (Shyu et al., 2012). Another central aim is to understand the repression mechanism of JAZ8. I report that JAZ8 represses JA responses in a NINJA-independent pathway that is different from the NINJA-dependent pathway utilized by other JAZ proteins. Experimental results of this aim are illustrated in Chapter 2 (Shyu et al., 2012). Finally, I report identification and characterization of a novel JAZ protein, JAZ13, which ! "#! also represses JA responses via a NINJA-independent pathway. JAZ13 shares high sequence similarity to JAZ8, and groups with JAZ8 in the JAZ8 subclade of JAZ repressors. This research is described in Chapter 3. In summary, I show that the JAZ8 subclade of JAZ proteins consists of unique JAZ repressors that are stable in the presence of JA, and repress through NINJAindependent pathways to regulate JA responses. ! "#! FIGURES AND TABLES !"#$%&'"()*+*,!"#$%&'!"#"$%&"'() !!!"#$%&#"'()'"* !"$!"#$%&'()*+(,-!./0 ! !"!!"#$%&"'($%&")*+%)(+*,-.,*#!""#$#%&'()#%*+$,-.*#%/!012 !""#$#%&'()#%*(*"+,#%-!./0 !"!!"!!!"#$%&'()%'*+,*'&+-./012+ !"!!"!!!"#$%&'()%'*+,*'&+-./012+ !"#$%&'()*+,-'%.!"/0 !!!"!!!!!"#$!!!!!"#$"#%&'!!"!#$%&'()'&!!!!"#$%!&"'$"&(')*+,'-./0' !"#$%&'$#()$*+,-./$0!"#12 ! !!!"!!!!!"#$!!!!!"#$"#%&'!!"!#$%&'()'&!!!!"#$%!&'()!*(+,-)(./0!!"#$% !$!"#$%&#!'()*+, !"#$%&'()*+,-(*./01 !"#$%&'()"('* !"#$%&'()"('* !"# !"#$%&'()*+,-)#" !"#$ !"#$%&' !"#$%&'(!!"#$%&'!(% !"!!"!!"#$%&'(!!"#$%&'!(% Figure 1.1. The Jasmonate Biosynthesis Pathway in Arabidopsis ! "#! Figure 1.1. The Jasmonate Biosynthesis Pathway in Arabidopsis – Continued. For interpretation of the references to color in this and all other figures, the reader is referred to the electronic version of this dissertation. ! ""! ! ! ! ! ! ! ! ! ! ! ! !! ! H OOC ! NH !! ! ! ! HOOC ! NH ! ! ! ! O ! ! ! ! O !! ! ! ! !! ! ! ! ! !! ! O ! O coronatine ! JA-Ile ! Figure 1.2. Chemical Structures of Jasmonyl-Isoleucine (JA-Ile) and Coronatine. ! "#! ! JAZ3 ! JAZ4 JAZ9 ! ! ! JAZ12 JAZ10 ! ! ! ! ! JAZ6 ! ! ! !! ! ! JAZ11 ! ! JAZ1 JAZ2 JAZ5 ! 0.1 ! ! JAZ8 JAZ7 Figure 1.3. The JAZ Family of Proteins in Arabidopsis. ! "#! 26S )*.' !"#"$%&' Wounding, Biological cues Prot. )*.' !"#"$%&' /01' JA-Ile )*.' +,"-' +,"-' Figure 1.4. Current Jasmonate Signaling Model. ! (%()*' (%()*' )*.' "#! /01' TIFY motif JAZ ()$ !"#$ %&'$ Jas degron Figure 1.5. Domain Structure of JAZ Proteins. Sequence logos of the conserved ZIM and Jas domains of 12 Arabidopsis JAZ proteins. Sequences used to generate the ZIM domain and Jas domain alignments were 28 and 27 amino acids, respectively, in length. The height of each stack of symbols indicates the sequence conservation at that position, whereas the height of symbols within the stack represents the relative frequency of the corresponding amino or nucleic acid at that position (Crooks et al., 2004). ! "#! REFERENCES ! "#! REFERENCES Acosta IF, Laparra H, Romero SP, Schmelz E, Hamberg M, Mottinger JP, Moreno MA, Dellaporta SL (2009) tasselseed1 is a lipoxygenase affecting jasmonic acid signaling in sex determination of maize. Science 323: 262–265 An F, Zhao Q, Ji Y, Li W, Jiang Z, Yu X, Zhang C, Han Y, He W, Liu Y, et al (2010) Ethylene-induced stabilization of ETHYLENE INSENSITIVE3 and EIN3-LIKE1 is mediated by proteasomal degradation of EIN3 binding F-box 1 and 2 that requires EIN2 in Arabidopsis. 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C.S. and G.A.H. wrote the article with contributions and edits from P.F., L.B.S., N.Z., and J.B. !"# ABSTRACT The lipid-derived hormone jasmonoyl-L-Ile (JA-Ile) initiates large-scale changes in gene expression by stabilizing the interaction of JASMONATE ZIM domain (JAZ) repressors with the F-box protein CORONATINE INSENSITIVE1 (COI1), which results in JAZ degradation by the ubiquitin-proteasome pathway. Recent structural studies show that the JAZ1 degradation signal (degron) includes a short conserved LPIAR motif that seals JA-Ile in its binding pocket at the COI1-JAZ interface. Here, we show that Arabidopsis thaliana JAZ8 lacks this motif and thus is unable to associate strongly with COI1 in the presence of JA-Ile. As a consequence, JAZ8 is stabilized against jasmonate (JA)-mediated degradation and, when ectopically expressed in Arabidopsis, represses JA-regulated growth and defense responses. These findings indicate that sequence variation in a hypervariable region of the degron affects JAZ stability and JA-regulated physiological responses. We also show that JAZ8-mediated repression depends on an LxLxLtype EAR (for ERF-associated amphiphilic repression) motif at the JAZ8 N terminus that binds the corepressor TOPLESS and represses transcriptional activation. JAZ8-mediated repression does not require the ZIM domain, which, in other JAZ proteins, recruits TOPLESS through the EAR motif– containing adaptor protein NINJA. These findings show that EAR repression domains in a subgroup of JAZ proteins repress gene expression through direct recruitment of corepressors to cognate transcription factors. !"# INTRODUCTION The lipid-derived hormone jasmonoyl-L-isoleucine (JA-Ile) and structurally related jasmonates (collectively referred to as JAs) play an essential role in controlling plant growth, development, and responses to environmental stress. Among the major functions ascribed to JAs are activation of defense responses to insect attack and pathogen infection, reproductive development, and growth inhibition (Glazebrook, 2005; Wasternack, 2007; Howe and Jander, 2008; Browse, 2009). These general roles for the hormone suggest that JA signaling evolved as a mechanism to optimize plant fitness in rapidly changing terrestrial environments. This hypothesis is supported by emerging mechanistic evidence for extensive crosstalk between JA and other hormones that mediate developmental plasticity (Dombrecht et al., 2007; Moreno et al., 2009; Pieterse et al., 2009; Hou et al., 2010; Robson et al., 2010; Ballare, 2011; Kazan and Manners, 2011; Zhu et al., 2011). A greater understanding of the molecular mechanism of JA signaling is therefore expected to impact broad areas of plant biology (Howe, 2010; Kazan and Manners, 2012). The discovery of JASMONATE ZIM-domain (JAZ) proteins spurred remarkable progress in understanding how JAs regulate large-scale changes in gene expression (Chini et al., 2007; Thines et al., 2007; Yan et al., 2007; Pauwels and Goossens, 2011). JAZs belong to the plant-specific TIFY family of transcriptional regulators that are defined by the presence of a TIF[F/Y]XG motif within a larger (~28 amino acids) conserved region known as the ZIM (or TIFY) domain (Vanholme et al., 2007; Chung et al., 2009; Bai et al., 2011). JAZs are distinguished from other TIFY proteins by the presence of a multifunctional C-terminal region known as the Jas motif. Both the ZIM domain and Jas motif are required for JAZ-mediated repression of JA responses in cells containing low JA-Ile levels. The Jas motif interacts with !"# members of the bHLH (e.g., MYC2) and R2R3 MYB family of transcription factors that promote the expression of JA-response genes (Chini et al., 2007; Melotto et al., 2008; Cheng et al., 2011; Fernandez-Calvo et al., 2011; Niu et al., 2011; Qi et al., 2011; Song et al., 2011). The ZIM domain and its associated TIFY motif mediate JAZ interaction with an adaptor protein called NOVEL INTERACTOR OF JAZ (NINJA), which functions to recruit the transcription co-repessor TOPLESS (TPL) and TPL-related proteins (TPRs) (Pauwels et al., 2010; Pauwels and Goossens, 2011). As is the case for other proteins that repress transcription through TPL and associated chromatin remodeling enzymes (Ohta et al., 2001; Krogan and Long, 2009; Kagale and Rozwadowski), NINJA contains an EAR (ERF-associated amphiphilic repression) motif that is necessary and sufficient for TPL interaction (Pauwels et al., 2010). The ZIM domain also promotes homo- and heteromeric interactions between JAZ proteins, which in Arabidopsis are encoded by 12 genes (JAZ1 – JAZ12) (Chini et al., 2009; Chung and Howe, 2009; Chung et al., 2009). Alternative splicing of JAZ genes expands the repertoire and potential combinatorial diversity of JAZ-JAZ interactions (Yan et al., 2007; Chung and Howe, 2009; Chung et al., 2010). A current challenge is to determine the contribution of individual JAZ isoforms to the regulation of JA signaling. JAZ-mediated repression is relieved in response to stimuli that activate the production of JA-Ile and subsequent degradation of JAZ proteins via the ubiquitin/26S proteasome pathway. JA-Ile initiates the signaling cascade by promoting interaction of JAZ with the F-box protein CORONATINE INSENSITIVE 1 (COI1). As the specificity determinant of the SCF (SKP1CUL1-F-box)-type ubiquitin ligase SCFCOI1, COI1 binding to many JAZ proteins is stimulated directly by JA-Ile (Xie et al., 1998; Thines et al., 2007; Katsir et al., 2008b; Melotto et al., 2008; Fonseca et al., 2009; Yan et al., 2009). Recent structural studies revealed that the N-terminal 20 !"# amino acids of the Jas motif adopt a bipartite structure consisting of a loop region that interacts with both COI1 and JA-Ile, and an !-helix that contacts the surface of COI1 adjacent to the ligand binding pocket (Sheard et al., 2010). Point mutations affecting conserved basic amino acids in the loop region of JAZ1 and JAZ9 abrogate COI1 binding, and also enhance the repression activity of ectopically expressed JAZ1 (Melotto et al., 2008). High-affinity ligand binding to the COI1-JAZ1 coreceptor complex also requires an inositol pentakisphosphate (IP5) cofactor that interacts with COI1 and JAZ near the hormone-binding pocket (Sheard et al., 2010). Initial insight into the identity of JAZ proteins as repressors came from the observation that truncated JAZs lacking the Jas motif are resistant to JA-mediated degradation and, when expressed in Arabidopsis, confer dominant insensitivity to exogenous JA (Chini et al., 2007; Thines et al., 2007; Yan et al., 2007). A role for JAZ10 as a negative regulator is supported by the phenotype of jaz10 loss-of-function mutants, which exhibit increased sensitivity to JA (Yan et al., 2007; Demianski et al., 2011). The repressive function of JAZ10 appears to result from the action of alternative splice variants that lack either a portion of the Jas motif (JAZ10.3) or the entire Jas motif (JAZ10.4) (Yan et al., 2007; Chung and Howe, 2009; Chung et al., 2010). These naturally occurring truncated JAZs have reduced capacity to interact with COI1 and thus are stabilized against hormone-dependent degradation (Chung and Howe, 2009). Ectopic expression of JAZ10.3 and JAZ10.4, which retain the ability to interact with MYC2, results in reduced sensitivity to JA. Transgenic lines overexpressing a JAZ10 genomic clone also show reduced sensitivity to JA, presumably as a consequence of overproduction of JAZ10.3 and JAZ10.4 (Chung et al., 2010). !"# Although it is established that dominant repression by JAZ10 splice variants and artificially truncated JAZs results from decreased association with COI1, a role for full-length (i.e., Jas motif containing) JAZs as transcriptional repressors remains to be shown. For example, JA-insensitive phenotypes have not been observed in transgenic lines of Arabidopsis that overexpress full-length JAZ1, JAZ2, JAZ3, or JAZ10 (Chini et al., 2007; Thines et al., 2007; Yan et al., 2007; Chung and Howe, 2009; Chung et al., 2010). Other than jaz10, the only jaz mutant reported to exhibit enhanced sensitivity to JA is an RNA interference (RNAi) line of JAZ1 (Grunewald et al., 2009). This phenotype, however, was not observed in a T-DNA insertion mutant (jaz1-1) that was characterized as a likely null mutant (Demianski et al., 2011). The lack of obvious JA-hypersensitive phenotypes among most jaz mutants reported to date may result from functional redundancy between JAZ proteins. Determining the role of specific JAZ isoforms in transcriptional repression is needed to further understand how JAs control myriad responses during the plant life cycle. Here, we establish a role for full-length JAZ8 as a transcriptional repressor, and describe two novel features of the protein that are critical for repression activity. First, we show that JAZ8 lacks the canonical degron that promotes JA-Ile-dependent interaction of other JAZ proteins with COI1. Second, we demonstrate that an LxLxL-type EAR motif near the N-terminus of JAZ8 mediates transcriptional repression. Significantly, JAZ8-mediated repression does not require the ZIM domain, which in other JAZs recruits TPL through the EAR motif of NINJA. Based on these findings, we propose that JAZ8 and other EAR motif-containing JAZ proteins directly recruit corepressors to transcription factors as a mechanism for repressing JA responses. !"# RESULTS JAZ8 Weakly Associates with COI1 in the Presence of Known Receptor Ligands We used in vitro pull-down assays to investigate the relationship between sequence diversity in the Jas motif and hormone-dependent binding of full-length JAZ proteins to COI1. A phylogenetic tree constructed from the 27-amino acid Jas motif of all 12 Arabidopsis JAZs divided the family into six groups: JAZ1/2; JAZ3/4/9; JAZ5/6; JAZ7/8; JAZ10.1; JAZ11/12 (Supplemental Figure 2.1). Representative full-length proteins from each group were expressed in E. coli as MBP-JAZ-6xHis fusions, and tested for their ability to interact with COI1 in the presence or absence of the active form of the hormone, (3R,7S)-JA-Ile. At a concentration of 1 !M, JA-Ile stimulated binding of COI1 to JAZ2.1, JAZ3.1, JAZ6, JAZ10.1, and JAZ12, but not JAZ8 (Figure 2.1A). Higher concentrations of JA-Ile increased the amount of COI1 binding to JAZ10.1, but failed to promote interaction with JAZ8 (Figure 2.1B). The phytotoxin coronatine, which is a potent agonist of the COI1-JAZ receptor system (Katsir et al., 2008b; Melotto et al., 2008; Yan et al., 2009; Sheard et al., 2010), stimulated low-level, dose-dependent binding of COI1 to JAZ8 (Figure 2.1C). Saturation binding assays confirmed that 3H-coronatine binds specifically to COI1-JAZ8 receptor complexes, albeit with much lower affinity (91.4 nM) than that observed for COI1-JAZ10.1 complexes (7.0 nM) (Figure 2.1D). JAZ8 Attenuates JA Responses and is Resistant to JA-mediated Degradation in Vivo Truncated JAZ proteins that fail to interact with COI1 in the presence of JA-Ile can repress JA signaling when overexpressed in Arabidopsis (Thines et al., 2007; Melotto et al., 2008; Chung et al., 2010). To determine whether full-length JAZ8 exerts dominant repression in vivo, we !"# constructed transgenic lines of Arabidopsis that express the JAZ8 cDNA under the control of the Cauliflower Mosaic Virus 35S promoter. T1 plants (24 independent lines) selected for the presence of the 35S:JAZ8 transgene did not exhibit obvious defects in flower development or fertility. Subsequently, homozygous lines (T3 generation) that were selected for high expression of the transgene (Materials and Methods) were tested in root growth inhibition assays. All 35S:JAZ8 lines (n = 5) selected in this manner were insensitive to JA-mediated root growth inhibition, and were significantly more insensitive to JA than the myc2 mutant, jin1-7 (Supplemental Figure 2) (Lorenzo et al., 2004). Dose-response experiments performed with a representative 35S:JAZ8 homozygous line (#24) showed that JAZ8 overexpression confers insensitivity to a broad range of concentrations of exogenous MeJA, but does not affect root growth in seedlings grown on medium lacking the compound (Figure 2.2A). Consistent with this finding, RNA blot analysis demonstrated that 35S:JAZ8 seedlings are impaired in the expression of the JA-responsive genes VSP2 and JAZ7 (Figure 2.2B). To test the effect of JAZ8 overexpression on JA-mediated defense responses, we compared the weight gain of Spodoptera exigua larvae grown on adult WT and 35S:JAZ8 plants. Insects reared on 35S:JAZ8 lines for either 9 or 14 d were significantly heavier (P<0.0001) than insects grown on the WT (Figure 2.2C and D). The increased mass of caterpillars grown on 35S:JAZ8 plants was associated with increased consumption of leaf tissue (Supplemental Figure 2.3). In summary, these results show that overexpression of full-length JAZ8 impairs JA-mediated root growth inhibition and defense responses, as well as JA-dependent gene expression. To determine whether the JA-insensitive phenotype of 35S:JAZ8 plants is associated with increased JAZ8 stability in vivo, we used confocal laser-scanning microscopy to compare JAmediated changes in fluorescence of JAZ8-YFP and JAZ10.1-YFP fusion proteins that were !"# stably expressed in Arabidopsis. Root growth inhibition assays showed that 35S:JAZ8-YFP seedlings, similar to 35S:JAZ8 lines, exhibit reduced sensitivity to exogenous MeJA (Supplemental Figure 4A). In the absence of JA treatment, a nuclear-localized YFP signal was observed in both 35S:JAZ10.1-YFP and 35S:JAZ8-YFP seedlings (Figure 3A). Within 10 min of treatment with 50 !M MeJA, this signal was largely eliminated in 35S:JAZ10.1-YFP seedlings, as previously reported (Chung and Howe, 2009). The nuclear-localized YFP signal in roots of 35S:JAZ8-YFP seedlings persisted up to 30 min after MeJA treatment. Consistent with the ability of coronatine to associate with COI1-JAZ8 complexes in vitro (Figure 2.1), exogenous coronatine stimulated JAZ8-YFP turnover in vivo (Figure 2.3B). This effect was partially inhibited by pre-treatment of seedlings with the 26S proteasome inhibitor MG132. These findings show that JAZ8 is more resistant than JAZ10.1 to JA-mediated degradation in vivo, but can be degraded in response to ligands (i.e., coronatine) that promote COI1-JAZ8 association. JAZ8 Lacks a Canonical JA-Ile Degron We performed domain swap experiments to test whether the observed functional differences between JAZ8 and JAZ10.1 can be attributed to sequence variation in the Jas motif (Figure 2.4A). For this purpose, we generated constructs encoding chimeric proteins in which the Jas motif and remaining C-terminal amino acids (two and five amino acids for JAZ8 and JAZ10.1, respectively) of JAZ8 and JAZ10.1 were reciprocally exchanged as shown schematically in Figure 4B. In vitro pull-down assays performed with the resulting fusion proteins showed that the Jas motif of JAZ10.1 confers on JAZ8 the ability to interact with COI1 in the presence of 2.5 !M JA-Ile (Figure 2.4C). Moreover, binding of COI1 to the JAZ10-Jas8 chimera, in which the C-terminal region of JAZ10.1 is replaced by the Jas motif from JAZ8, was not stimulated by JA- !"# Ile. To determine whether these changes in hormone-dependent COI1 interaction impact JAregulated physiological responses, we constitutively expressed the chimeric proteins in Arabidopsis. Seedlings that express the JAZ8-Jas10 chimera were fully sensitive to JA. Conversely, lines expressing JAZ10-Jas8 exhibited a JA-insensitive root growth phenotype that was indistinguishable from that of 35S:JAZ8 plants (Figure 2.4D). We conclude that the Jas motif of JAZ8 lacks sequence determinants that are required for JA-Ile-dependent binding to COI1. The COI1-interacting degron of JAZ1 includes a six-amino acid (LPIARR) loop region that encloses JA-Ile in its binding pocket (Sheard et al., 2010). This sequence is largely conserved in other JAZs (e.g., JAZ10.1) that strongly interact with COI1 in the presence of JAIle (Figure 4A). The sequence (PKASMK) of the corresponding region in JAZ8 differs markedly from the canonical LPIARR motif. To determine whether this variation is responsible for the weak COI1-JAZ8 association observed in vitro (Figure 2.1), we constructed a modified version (JAZ8LPIAR) of JAZ8 in which five amino acids (PKASM) in the loop region were replaced with the canonical LPIAR. In vitro pull-down assays showed that this substitution is sufficient to confer JA-Ile-dependent interaction of JAZ8 with CO11 (Figure 2.5A). Consistent with this finding, the PKASM!LPIAR substitution completely suppressed the JA-insensitive root growth phenotype of 35S:JAZ8 seedlings (Figure 2.5B). The ability of JAZ8 to interact with MYC2 (Chini et al., 2009) raised the possibility that the PKASM!LPIAR substitution impairs this interaction, which could account for the JA-sensitive phenotype of 35S:JAZ8LPIAR seedlings. Yeast two-hybrid assays showed that JAZ8LPIAR, like JAZ8, interacts with MYC2, thus excluding this possibility (Supplemental Figure 2.5). Fluorescence microscopy showed that a JAZ8LPIAR-YFP fusion protein accumulates in the nucleus (Figure 2.5C), further indicating that !"# the substitution does not impair expression or localization of the protein. Imaging of 35S:JAZ8YFP and 35S:JAZ8LPIAR-YFP seedlings treated with MeJA (for 15 min) demonstrated that JAZ8LPIAR-YFP is more sensitive than JAZ8-YFP to JA-mediated degradation. The ability of MG132 to block JA-mediated turnover of JAZ8LPIAR-YFP showed that degradation of JAZ8LPIAR is dependent on the 26S proteasome. We conclude that the non-canonical degron sequence (PKASM) in JAZ8 does not effectively interact with COI1 in a JA-Ile-dependent manner, and that this sequence is important for JAZ8’s function as a dominant repressor of JA signaling. These results led us to investigate whether JAZ proteins in other plant species have a JAZ8-like Jas motif. Phylogenetic analyses indicate that angiosperm JAZs cluster into five groups, including those represented by JAZ7/8 (group IV) and JAZ10 (group III) (Bai et al., 2011). Alignment of the predicted Jas motif in group IV proteins from diverse plant species showed that Ser and Met residues within the PKASM motif are highly conserved in other members of this group, whereas the PKA sequence is more variable (Figure 2.6A). Comparison of this motif with a consensus Jas motif constructed from JAZ10 orthologs showed that these two groups of JAZs differ markedly in the N-terminal degron sequence, but not in other regions of the Jas motif (Figure 2.6A). An EAR Motif in JAZ8 Mediates Transcriptional Repression Previous genome-wide sequence searches identified an LxLxL-type EAR motif in Arabidopsis JAZ8 and its close relative, JAZ7 (Kagale et al., 2010). This sequence (LELRL) is located close to the N-terminus of JAZ8 and is highly conserved among group IV JAZs from diverse plant species (Figure 6B) (Bai et al., 2011). To investigate the functional relevance of the motif in JAZ8-mediated repression of JA responses, we generated transgenic lines that overexpress a !!" mutant version (JAZ8!EAR) of JAZ8 in which the LELRL sequence was deleted. Root growth assays performed with multiple homozygous 35S:JAZ8!EAR lines showed that deletion of the EAR motif suppresses the JA-insensitive phenotype conferred by JAZ8 overexpression (Figure 2.6C). Similar results were obtained with lines expressing YFP-tagged JAZ8 proteins in which the LELRL motif was deleted (JAZ8!EAR-YFP) or substituted with Ala residues (JAZ8LELRL!5xA-YFP) (Supplemental Figure 2.4B). Fluorescence microscopy showed that these JAZ8-YFP derivatives are expressed in these lines and, like JAZ8-YFP, accumulate in the nucleus (Supplemental Figure 2.6). These results support the hypothesis that the conserved LELRL motif performs a critical role in JAZ8-mediated inhibition of JA responses. We next used a heterologous carrot protoplast transfection system (Tiwari et al., 2004) to test directly whether JAZ8 functions as a transcriptional repressor. This assay relied on the use of a GUS (ß-glucuronidase) reporter gene [35S(-46)LexA(2x)-Gal4(2x):GUS] that is driven by a 35S minimal promoter harboring LexA and Gal4 DNA-binding sites. The reporter was cotransfected with two effector constructs: one encoding a fusion protein (LD-VP16) consisting of the LexA DNA binding domain (LD) fused to the VP16 activation domain, and a second effector encoding the Gal4 DNA binding domain (GD) fused to either full-length JAZ8 (GDJAZ8) or the N-terminal region of JAZ8 (GD-JAZ8-N). Cotransfection of the GUS reporter with LD-VP16 and a construct encoding only GD resulted in strong activation (~13-fold) of the reporter in comparison to the GD construct alone (Figure 2.7A). The EAR motif repression domain of IAA17 was used as a positive control for transcriptional repression; cotransfection of this effector (GD-IAA17[I]) resulted in strong reduction in reporter gene expression, as previously reported (Tiwari et al., 2004). GD-JAZ8 repressed LD-VP16-mediated transcription of the reporter to a level similar to that obtained with GD-IAA17[I]. Cotransfection with GD- !"# JAZ-N, which encodes the EAR motif-containing N-terminal 44 amino acids of JAZ8, also resulted in strong repression (Figure 2.7A). These findings show that JAZ8 directly represses a transcriptional activator, and that the repression activity resides within the N-terminal 44 amino acids of JAZ8. Ala-scanning mutagenesis was used to determine whether the LELRL motif of JAZ8 is required for transcriptional repression. Individual Ala substitutions within the LELRL motif resulted in partial or complete loss of repression by GD-JAZ-N in the protoplast transfection assay (Figure 2.7B). The effect of mutating any one of the three Leu residues was stronger than that of mutating the intervening hydrophilic residues. The Leu!Ala substitution completely impaired GD-JAZ8-N repression activity, suggesting that this residue is important for recruitment of co-repressors. Similar findings were reported for the characterization of EAR motifs in SUPERMAN and IAA17 repressors (Hiratsu et al., 2003; Tiwari et al., 2004). Mechanism of Repression by JAZ8 We used the yeast two-hybrid (Y2H) system to investigate how interaction of JAZ8 with other components of the JA signaling module may contribute to the mechanism of JAZ8-mediated repression. We first investigated the interaction of JAZ8 with the bHLH transcription factor MYC2, which plays a major role in controlling JA-dependent root growth inhibition (Lorenzo et al., 2004; Fernandez-Calvo et al., 2011). Full-length JAZ8 interacted with MYC2 (Figure 2.8A), as previously reported (Chini et al., 2009). A JAZ8!Jas construct lacking the Jas motif did not bind MYC2 but maintained the ability to heterodimerize with JAZs. Moreover, a 36-amino acid C-terminal fragment containing the Jas motif was sufficient for binding of MYC2. !"# Consistent with previous Y2H studies (Pauwels et al., 2010; Arabidopsis Interactome Mapping Consortium, 2011), full-length JAZ8 interacted with TPL but not with NINJA (Figure 8B). Deletion of the ZIM domain abolished JAZ8 heterodimerization with JAZ1 and all other JAZs tested (Supplemental Figure 2.7), but did not affect the interaction with MYC2 or TPL. Removal of the LELRL motif (JAZ8!EAR construct), or substitution of this sequence with five Ala residues (JAZ8LELRL!5xA), eliminated the interaction with TPL but did not affect binding to JAZ1 or MYC2 (Figure 2.8B). A construct (JAZ8!EZ) that lacks both the EAR and ZIM domains interacted with MYC2 but not with TPL or JAZ1. These collective results show that the EAR, ZIM, and Jas motifs mediate JAZ8 interaction with TPL, other JAZ proteins, and MYC2, respectively. Pauwels et al (2010) showed that although JAZ8 does not interact with NINJA in the Y2H system, the two proteins do interact in pull-down assays performed with GFP-tagged NINJA expressed in planta. We used a yeast-expressed, HA-tagged NINJA derivative to reexamine this question. Whereas JAZ8-His and JAZ10.1-His recovered comparable amounts of HA-JAZ1, the amount of HA-NINJA recovered by JAZ8-His was much less than that associated with JAZ10.1-His (Figure 2.8C). The amount of HA-NINJA bound by JAZ8-His, however, was consistently greater than that recovered by MBP-His alone. This finding, together with the results of Pauwels et al (2010), raised the possibility that JAZ8-mediated repression may depend in part on recruitment of TPL via JAZ8’s ZIM domain. To test this hypothesis, we constructed transgenic lines (35S:JAZ8!ZIM) that overexpress JAZ8!ZIM. Multiple independent T1 lines showing high transgene expression were selected for use in root growth inhibition assays. The mean root length ratio (0.72 ± 0.01; n=127) exhibited by seedlings from eight independent homozygous 35S:JAZ8!ZIM lines was not significantly different from that of 35S:JAZ8 !"# seedlings (0.70 ± 0.04; n=23) (Figure 2.9A). As an additional control, we constructed transgenic lines that express the JAZ8!EZ construct (Figure 2.8B) in which both the EAR and ZIM motifs of JAZ8 are deleted. Eleven independent lines that express the 35S:JAZ8!EZ transgene to high levels were selected for root growth inhibition assays. The mean root length ratio of seedlings from all lines was not different (P-value = 0.62, Student’s t-test) from that of WT seedlings (Figure 2.9B), indicating that overexpression of JAZ8!EZ does not significantly repress JA signaling. These results show that the ZIM domain is not required for repression of JA-mediated root growth inhibition by ectopically expressed JAZ8. DISCUSSION Sequence Diversity in the JAZ Degron Modulates JA Responsiveness Our results provide new insight into sequence determinants within the Jas motif that promote hormone-dependent interaction of JAZ proteins with COI1. Recent structural studies showed that the JAZ1 degron is located in the N-terminal region of the Jas motif, and includes a hexapeptide motif (LPIARR) that seals JA-Ile at the COI1-JAZ1 interface (Sheard et al., 2010). The Cterminal end (ARR) of the motif makes direct contact with JA-Ile, whereas N-terminal residues (LPI) clamp down on the COI1 surface to trap the hormone in place. These structural studies are supported by genetic evidence showing that mutations in the C-terminal basic residues (RR) result in dominant repression of JA responses by JAZ1 (Melotto et al., 2008). Here, we show that the LPIARR sequence is largely conserved in JAZ proteins (including JAZ2, JAZ3, JAZ9, JAZ10.1, and JAZ12) that strongly interact with COI1 in the presence of JA-Ile. This finding is !"# consistent with the rapid JA-mediated turnover of this group of JAZs (Chini et al., 2007; Thines et al., 2007; Chung and Howe, 2009; Grunewald et al., 2009; Pauwels et al., 2010). The importance of the LPIARR motif as a JAZ degradation signal is highlighted by the finding that JAZ8, which lacks this motif, does not readily associate with COI1 in the presence of JA-Ile. The weak COI1-JAZ8 interaction was associated with increased stability of JAZ8 in JA-treated cells and the ability of JAZ8 to repress JA responses in 35S:JAZ8 plants. Domain swap and site-directed mutagenesis experiments demonstrated that these unique features of JAZ8 are attributed to sequence variation in the degron region; replacement of PKASM in the Jas motif of JAZ8 with LPIAR conferred JA-Ile-dependent interaction with COI1, and also decreased the stability of JAZ8. The PKASM!LPIAR substitution also suppressed the JA-insensitive phenotype of 35S:JAZ8 plants, thus establishing a direct link between sequence variation in the ligand-contacting region of the degron and JA signal output. These findings show that the LPIARR motif functions in vivo as a critical part of the JAZ degron, thus confirming and extending our previous structural studies (Sheard et al., 2010). The central (SLX2FX2KRX2R) and C-terminal (X5PY) regions of the Jas motif are highly conserved among all JAZs, whereas the N-terminal amino acids corresponding to the degron loop are much more variable (Figure 2.6A) (Chung et al., 2009; Sheard et al, 2010). Our work on JAZ8 indicates that sequence variation in the N-terminus reflects the existence of functionally distinct JAZ subgroups that differ in their stability. These subgroups include the canonical LPIAR(R/K)-containing JAZs (e.g., JAZ1) that interact robustly with COI1 in the presence of JA-Ile, and X3SMK-containing JAZs such as JAZ8 that do not strongly associate with COI1 under these conditions. The degron loop region of JAZ5 and JAZ6 (Figure 2.4A) is unusual in that it contains the canonical C-terminal residues (IARR) that contact JA-Ile (Sheard et al., !"# 2010), but lacks the N-terminal residues (LP) that clamp the hormone in the binding pocket. Interestingly, this sequence variation correlates with the reduced capacity of JAZ6 to recover COI1 in pull-down assays (Figure 2.1A). Differential association of JAZ isoforms with COI1, mediated by sequence variation in the degron, may enable plants to perceive and respond appropriately to a wide dynamic range of JA-Ile. Our results support a scenario in which the intracellular level of JA-Ile dictates which JAZs in a given cell associate with COI1. It is likely, for example, that some JAZs are recruited to COI1 in response to low JA-Ile concentrations, whereas other JAZ associate with COI1 only in the presence of high JA-Ile levels. Sequence variation in the auxin degron has also been shown to contribute to the diversification of Aux/IAA proteins (Dreher et al., 2006; Sato and Yamamoto, 2008), further highlighting the conserved nature of the COI1-JAZ and TIR1-Aux/IAA receptor systems (Katsir et al., 2008a). The mechanisms by which JAZ8 and other stable JAZs are removed from cells remain unknown. One hypothesis is that stable JAZs are eliminated via COI1-independent proteolytic pathways, perhaps involving other F-box proteins. However, the ability of coronatine to destabilize JAZ8 in vivo, together with the inhibitory effect of MG132 on this process, suggests that JAZ8 can be degraded by a pathway involving COI1 and the 26S proteasome. Given that coronatine functions as a potent agonist of the JA-Ile receptor, it is possible that JAZ8 is slowly degraded by a COI1-dependent route in the presence of very high intracellular levels of JA-Ile. Our results also raise the possibility that recruitment of JAZ8 to COI1 is mediated by a small molecule other than (3R,7S)-JA-Ile. The ability of JAZ8 to associate with COI1 in the presence of coronatine demonstrates that JAZ8 is capable of interacting with COI1 in a ligand-dependent manner. Although we cannot exclude the possibility that this interaction is mediated by a cryptic COI1-binding site located outside the Jas motif, the most straightforward interpretation is that the !"# non-canonical loop region of JAZ8 is a weak substrate for coronatine-triggered COI1 association. The putative degron loop of JAZ8 contains a KK motif (Figure 4A) that may partially fulfill the function of the critical dibasic motif (R205R206) in JAZ1, which interacts directly with the carboxyl group of the ligand at the bottom of the binding pocket (Sheard et al., 2010). Among the small molecules that could potentially promote COI1-JAZ8 association are metabolic precursors or derivatives of JA-Ile. Several studies have provided evidence that the JA-Ile precursors 12-oxo-phytodienoic acid and jasmonic acid elicit JA-related responses without their prior conversion to JA-Ile (Hopke et al., 1994; Blechert et al., 1999; Miersch et al., 1999; Stintzi et al., 2001; Ribot et al., 2008; Wang et al., 2008). To date, however, the only naturally occurring JA derivatives known to promote COI1-JAZ binding are JA-Ile, structurally related JA-amino acid conjugates (e.g., JA-Leu), and 12-hydroxy-JA-Ile (Thines et al., 2007; Katsir et al., 2008b; Melotto et al., 2008; Chini et al., 2009; Koo et al., 2011). The expression pattern of JAZ8 in WT plants provides insight into the physiological role of JAZ8 as a repressor of JA responses. The most striking aspect of JAZ8 expression is its rapid and strong induction in response to exogenous JA and other stress-related cues that activate the pathway (Figure 2B; Thines et al., 2007; Chini et al., 2007; Chung et al., 2008). In mature Arabidopsis leaves, for example, JAZ8 and its paralog, JAZ7, are highly expressed in response to mechanical wounding and infection with coronatine-producing strains of P. syringae (Chung et al., 2008; Koo et al., 2009; Demianski et al., 2011). The enhanced expression of JAZ8 in wounded leaves of a cyp94b3 mutant that hyperaccumulates JA-Ile further suggests that expression of JAZ8 is promoted by high endogenous levels of JA-Ile (Koo et al., 2011). Given that the repressive activity of JAZ8 is dominant and persists in the presence of JA-Ile, stressinduced expression of JAZ8 is expected to result in attenuation of JA responses, consistent with !"# the JA-insensitive phenotype exhibited by 35S:JAZ8 plants. We therefore propose that JAZ8, like the alternative splice variants of JAZ10 (Yan et al., 2007; Chung and Howe, 2009; Chung et al., 2010), is a component of a JA-triggered negative feedback loop to prevent runaway activation of JA responses that could lead to inhibition of plant growth and reduced fitness (Zhang and Turner, 2008). Repression of JA Responses by EAR Motif-Containing JAZ Proteins The recent identification of NINJA provided an important advance in understanding how JAZ proteins repress JA-dependent transcriptional responses (Pauwels et al., 2010). NINJA negatively regulates the expression of JA response genes by bridging the ZIM domain of target JAZs to members of the TPL family of corepressors. As is the case for other TPL-interacting proteins (Szemenyei et al., 2008; Krogan and Long, 2009), NINJA contains an EAR motif that functions to recruit TPL. Our results indicate that JAZ8 employs a distinct and more direct mechanism to repress JA-dependent transcription; through its N-terminal EAR and C-terminal Jas motifs, we propose that JAZ8 provides a direct link between TPL and cognate transcription factors. Three independent lines of evidence demonstrate a key role for the EAR motif in JAZ8mediated repression. First, we show that JAZ8 interacts with TPL in yeast and that a canonical LxLxL-type EAR motif at the N-terminus of JAZ8 is required for this interaction. These findings are in agreement with a recent large-scale Y2H study in which TPL was shown to interact with JAZ8 (Arabidopsis Interactome Mapping Consortium, 2011). Second, we demonstrate that the EAR motif is required for JAZ8-mediated repression of JA-dependent root growth inhibition in 35S:JAZ8 plants. Finally, we show that JAZ8 represses VP16-mediated transcriptional activation in a protoplast transfection assay and, moreover, that the EAR motif is important for the !"# mechanism of repression. To our knowledge, this is the first direct demonstration that a fulllength JAZ protein functions as a transcriptional repressor. A key role for the EAR motif in JAZ8-mediated repression is supported by our finding that 35S:JAZ8 and 35S:JAZ8!ZIM lines exhibit a similar level of insensitivity to JA. This finding shows that the ZIM domain is not strictly required for repression by ectopically expressed JAZ8, thus suggesting that JAZ8 action under these conditions depends neither on NINJA nor on interaction with other JAZs. Such a ZIM/TIFY-independent mechanism of repression differs from that of JAZ10.4, whose function as a dominant repressor is blocked by point mutations within the ZIM domain (i.e., TIFY motif) that disrupt JAZ10.4 homo- and heterodimerization (Chung and Howe, 2009). In this context, JAZ8!ZIM appears to define the minimal domain architecture of a JAZ repressor, which in this case consists only of an EAR repression domain and a transcription factor-binding Jas motif. Given the ability of JAZ8 to interact with NINJA in pull-down assays (Pauwels et al., 2010; this study), we cannot exclude the hypothesis that JAZ8 is capable of repressing gene expression through both a NINJA-independent pathway involving direct binding of TPL/TPR to JAZ8’s EAR motif, as well as a NINJA-dependent pathway in which the ZIM domain of JAZ8 recruits TPL/TPR via NINJA. Our collective results, however, favor the hypothesis that JAZ8 represses gene expression mainly via a direct, NINJAindependent mechanism. The bHLH transcription factor MYC2 plays a major role in JA-mediated root growth inhibition (Fernandez-Calvo et al., 2011). We found that the JA-insensitive root growth phenotype of JAZ8-overexpressing plants is stronger than that of a myc2 mutant (jin1-7). This finding, together with the fact that JAZ8 physically associates with MYC2 (Cheng et al., 2011; Fernandez-Calvo et al., 2011) (this study), suggests that JAZ8 targets MYC2 for repression. !"# JAZ8 also interacts with the MYC2-related proteins MYC3 and MYC4, which serve a minor but significant role in activating JA responses in the root (Cheng et al., 2011; Fernandez-Calvo et al., 2011; Niu et al., 2011), as well as MYB-type transcription factors (MYB21 and MYB24) implicated in JA-regulated male fertility (Song et al., 2011). In contrast to male sterile phenotype of transgenic lines that overexpress various truncated JAZ proteins (Thines et al., 2007; Melotto et al., 2008; Chung and Howe, 2009), we did not observe reproductive defects associated with overexpression of JAZ8. Identification of transcription factors and physiological processes that are regulated by endogenous JAZ8 is an important question for future studies. Genome-wide sequence surveys showed that JAZ5, JAZ6, and JAZ7 also contain predicted EAR motifs (Kagale et al., 2010). Moreover, proteome-wide mapping of the Arabidopsis interactome identified JAZ5 and JAZ6 as TPL-binding proteins in yeast (Arabidopsis Interactome Mapping Consortium, 2011; Causier et al., 2012). It thus seems likely that repression of JA-responsive genes through direct JAZ-TPL coupling is not unique to JAZ8. Several reported features of JAZ7 are reminiscent of JAZ8, including the N-terminal location of the EAR motif and the ability to interact with MYC-related transcription factors but not NINJA (Pauwels et al., 2010; Cheng et al., 2011; Fernandez-Calvo et al., 2011). JAZ5 and JAZ6 both interact with NINJA but are distinct in that they each contain two predicted EAR motifs in the central and C-terminal regions of the protein (Kagale et al., 2010; Pauwels et al., 2010; Fernandez-Calvo et al., 2011). These observations support our general conclusion that different subgroups of JAZ employ distinct domain architectures to recruit corepressors to cognate transcription factors. Future work aimed at delineating the mechanisms and evolutionary origins of NINJA-dependent and NINJA-independent JAZ repression pathways should help to unravel the complexities of JA biology. !"# MATERIAL AND METHODS Plant Material & Growth Conditions Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used as the wild-type (WT) parent for all experiments. Plants were grown in soil under controlled conditions and were transformed with Agrobacterium tumefaciens as previously described (Chung et al., 2008; Chung and Howe, 2009). 35S:JAZ10.1 and 35S:JAZ10.1-YFP transgenic lines were described previously (Chung and Howe, 2009). The jin1-7 mutant (SALK_040500) was previously described (Lorenzo et al., 2004). Lines that express transgenes to high levels were identified as follows. Seedlings (T1 generation) of transformed lines were screened on MS agar medium containing sucrose (0.8%), kanamycin (50 µg/mL), and vancomycin (100 µg/mL). For each construct, 32 independent T1 plants were transferred to soil for evaluation of transgene expression level by Northern blot analysis. RNA isolated from untreated leaf tissue was hybridized with a JAZ8 cDNA as a probe as previously described (Chung et al., 2008). Selected high-expressing lines (typically five per construct) were further propagated for identification of homozygous T3 lines. Lines expressing JAZ8-YFP fusion proteins were screened at the seedling stage by fluorescence microscopy (Axio Scope, Carl Zeiss). Plant Treatments The effect of exogenous JA on root growth inhibition was determined as described previously (Chung and Howe, 2009), with minor modifications. Surface-sterilized and stratified seeds were sown on square petri plates containing MS agar medium (0.8% agar, Caisson Labs) supplemented with 0.8% sucrose (w/v) and the indicated concentration of MeJA (SigmaAldrich). WT and transgenic lines were grown on the same plate to control for plate-to-plate !!" variation. Petri plates were incubated vertically in a growth chamber maintained at 21°C and continuous light, unless otherwise indicated. Following eight d of growth, the length of primary roots was measured using Image J software (http://rsbweb.nih.gov/ij/index.html). Root length ratios (% inhibition by MeJA) were calculated by dividing the average the root length of seedlings grown on MeJA-containing medium by the average root length of seedlings of the same genotype grown in the absence of MeJA. Spodoptera exigua eggs were obtained from Benzon Research and hatched at 30°C. Newly hatched larvae were transferred to fully expanded rosette leaves of 5-week-old WT and 35S:JAZ8 transgenic (line 24) plants. Eight larvae were reared on each of 12 plants per genotype. Insect-challenged and unchallenged control plants were maintained under standard growth conditions (Chung et al., 2008). Larval weights were measured at various times after challenge, and weighed larvae were returned to their plant of origin. The experiment was independently repeated four times with similar results. Northern blot analysis of RNA isolated from liquidgrown seedlings was performed as previously described (Chung et al., 2008). Transgene Constructs and Site-Directed Mutagenesis A complete list of constructs and primers used to generate transgenic lines is provided in Supplemental Table 1 online. Three PCR reactions were used to generate constructs encoding the JAZ8-JAZ10 chimeric proteins. In the case of the JAZ8-Jas10 chimera, one reaction used the JAZ8 cDNA as a template with primer set JAZ8-XbaI-FP and JAZ8-Jas10-RP to amplify a portion (amino acids 1-100) of the JAZ8 coding sequence that lacks the Jas motif. A second PCR reaction used the JAZ10 cDNA as a template with a primer set (JAZ8-Jas10-FP and JAZ10KpnI-RP) to amplify the Jas motif-containing region of JAZ10 (amino acids 166-205). The PCR !"# products from these two reactions contain complementary sequences (built into the primers) that allow the products to anneal as template in a third PCR reaction with primer set JAZ8-XbaI-FP and JAZ10-KpnI-RP. The resulting chimeric product was cloned into pGEM-T Easy (Promega) for sequencing and subcloning into the final destination vector. A similar approach was used to construct the JAZ10-Jas8 chimera, using the primer sets listed in Supplemental Table 1. The JAZ8!ZIM construct was generated using three PCR reactions. The first PCR reaction used JAZ8 cDNA as a template with a primer set (JAZ8-NcoI-FP and JAZ8-deltaZIMRP) to amplify the N-terminal region (amino acids 1-44) of JAZ8. A second PCR was used to amplify the C-terminal region of JAZ8 (amino acids 72-131) with primers JAZ8-deltaZIM-FP and JAZ8-XhoI-RP. The two PCR products contain complementary sequences built into the primers (JAZ8-deltaZIM-FP and JAZ8-deltaZIM-RP). In a third PCR reaction (primers JAZ8NcoI-FP and JAZ8-XhoI-RP), the two PCR products were annealed as a template to generate JAZ8!ZIM (i.e., JAZ8 lacking amino acids 45-71). The resulting product was cloned into pGEM-T Easy for sequencing and subcloning into Y2H and overexpression vectors. The JAZ8!EAR construct was generated in two PCR reactions. The first reaction used the JAZ8 cDNA as a template with a primer set (JAZ8-deltaEAR-FP and JAZ8-pENTR-RP) that amplifies a portion of the JAZ8 coding region lacking the EAR motif. This PCR product was then used as template in the second PCR (primers JAZ8-pENTR-FP and JAZ8-pENTR-RP) to generate the JAZ8!EAR product, which was subsequently cloned into pENTR/D-TOPO (Invitrogen) and pGEM-T Easy for sequencing and subcloning. Site-directed mutagenesis of the Jas and EAR motif of JAZ8 was performed with Pfu Turbo DNA Polymerase (Stratagene) as previously described (Chung and Howe, 2009). PCR reactions were performed with JAZ8 cDNA in the vector pGEM-T Easy. PCR-amplified !"# products were treated with restriction enzyme DpnI to remove the parental plasmid. The presence of the desired mutation was verified by DNA sequencing. In Vivo Degradation of JAZ-YFP Fusion Proteins Transgenic seedlings expressing JAZ-YFP fusion proteins were grown on MS plates (containing kanamycin) and then transferred to water (0.6 ml) 48-well microtiter plates. Seedlings were incubated in the presence or absence of MeJA or coronatine at room temperature on an orbital shaker (70 rpm). YFP fluorescence was analyzed on an Olympus Fluoview confocal microscope, with imaging software provided by the manufacturer. In some experiments, seedlings were pretreated with water or 100 !M MG132 (Sigma-Aldrich) for ~75 min prior to the addition of MeJA. Images shown in a single panel were taken at the same exposure time and microscope parameters, unless otherwise indicated in the figure legend. Protein-Protein Interaction Assays JAZ cDNAs were cloned into pRMG-nMAL to produce plasmids that encode maltose binding protein (MBP) and hexa-histidine (His6)-tagged fusion proteins, referred to as JAZ-His (Thines et al., 2007). Primers used for amplifying JAZ cDNAs are listed in Supplemental Table 1. JAZHis fusion proteins were expressed in Escherichia coli and purified by Ni-affinity chromatography as previously described (Katsir et al., 2008b; Chung et al., 2010). Leaf extracts prepared from a transgenic line of Arabidopsis that expresses Myc-tagged AtCOI1 were used as the source of COI1 (Melotto et al., 2008; Chung et al., 2010). In vitro COI1-JAZ pull-down assays were performed with chemically synthesized (3R,7S)-JA-Ile or coronatine as previously described (Chung et al., 2010). Purified JAZ-COI1 complexes were separated by SDS-PAGE on !"# a 10% acrylamide gel, transferred to polyvinylidene fluoride (PVDF) membrane, and probed with an anti-c-Myc antibody (Roche). In vitro JAZ8-NINJA and JAZ8-JAZ1 interaction assays were performed with purified JAZ-His protein (25 µg) and crude extract (600 µg) from yeast cells expressing N-terminal HA-tagged derivatives of NINJA or JAZ1, which were cloned into the yeast expression vector pB42AD (Clontech) using primers listed in Supplemental Table 1. JAZ-His complexes were recovered by Ni-affinity chromatography, protein-protein interactions were detected with an anti-HA antibody (Covance), as described above. Yeast two-hybrid (Y2H) assays were performed with the Matchmaker LexA system (Clontech) as previously described (Melotto et al., 2008; Chung and Howe, 2009). JAZ8 and JAZ8-deletion constructs were subcloned into the pGILDA “bait” vector to generate translational fusions with the LexA DNA-binding domain. Full-length cDNAs encoding MYC2, other JAZs, NINJA, and TPL were subcloned into the pB42AD “prey” vector to generate fusions with B42 activation domain (AD). Bait and prey vectors were co-transformed into yeast (Saccharomyces cerevisae) strain EGY48 using the frozen-EZ yeast transformation II kit (Zymo Research). Transformants were selected for colorimetric detection (!-galactosidase) of protein-protein interaction as described by Chung and Howe (2009). Photographic images of Y2H plates were taken after 48 h of incubation at 30°C. Coronatine Binding Assays Full-length Arabidopsis COI1 and ASK1 were co-expressed as a glutathione S-transferase (GST)-fusion protein and an untagged protein, respectively, in Hi5 suspension insect cells. The COI1-ASK1 complex was isolated from the soluble cell lysate by glutathione affinity chromatography. Full-length JAZ substrate proteins were expressed as 6xHis-fusion proteins in !"# Escherichia coli and purified on Ni-NTA resin with subsequent dialysis into 20 mM Tris-HCl, pH 8.0, 200 mM NaCl, and 10% glycerol. Radioligand binding was assayed with purified COI1ASK1 complex (2 mg) and JAZ proteins at a 1:3 molar ratio. Reactions were prepared in 100 mL final volume and in a binding buffer containing 20 mM Tris-HCl, 200 mM NaCl, and 10% glycerol. Saturation binding experiments were conducted with serial dilutions of 3H-coronatine in binding buffer. Non-specific binding was determined in the presence of 300 mM coronatine (Sigma-Aldrich). Specific binding was calculated by subtracting non-specific binding from total binding. Following incubation with mixing at 4°C, all samples were collected with a cell harvester (Brandel, Gaithersburg, MD) on polyethyleneimine (Sigma)-treated filters. Samples were incubated in liquid scintillation fluid for >1 h before counting with a Packard Tri-Carb 2200 CA liquid scintillation analyzer (Packard Instrument Co. Inc., Rockville, MD). Saturation binding experiments were analyzed by non-linear regression using GraphPad Prism version 5.00 for MacOSX. 3H-coronatine was synthesized commercially as previously described (Katsir et al., 2008b). Carrot Protoplasts Transactivation Assay The effector constructs 35S:GD (GD: Gal4 DNA binding domain), 35S:GD-IAA17[I] (IAA17/AXR3 domain I, amino acids 1 to 29), and 35S:LD-VP16 (LD: LexA DNA binding domain; VP16: Herpes simplex virus VP16 activation domain) were all driven by the CaMV 35S promoter and have been described previously (Tiwari et al., 2004). The 35S:GD vector was converted into a Gateway destination version (pGD-RfA) by inserting the reading frame A cassette (RfA, Invitrogen) downstream of the sequence encoding the Gal4 DNA binding domain. JAZ8 (amino acids 1 to 131) and JAZ8-N (N-terminus; amino acids 1 to 44) coding regions were !"# amplified by RT-PCR using RNA from Arabidopsis seedlings as template and the primer pairs listed in Supplementary Table S1. cDNAs were cloned into pDONR/Zeo via Gateway BP cloning reactions (Invitrogen). GD-JAZ8 and GD-JAZ8-N effector constructs were generated by recombination between pDNOR clones described above and pGD-RfA via Gateway LR cloning reactions. The reporter constructs were 35S:LexA(2x)-Gal4(2x):GUS, which is driven by CaMV 35S -46 minimal promoter containing LexA and Gal4 DNA binding sites, and 35S:LUC have been described previously (Tiwari et al., 2004). To generate JAZ8 effector constructs encoding mutated proteins (L9A, E10A, L11A, R12A, L13A and F14A), the JAZ8-N coding region was mutagenized with a Quickchange II site-directed mutagenesis kit (Stratagene) and a complementary pair of primers (Supplementary Table S1) containing the relevant mutation. All mutations were confirmed by DNA sequencing. Reporter and effector plasmids were isolated from the E. coli strain ER2925 (New England Biolabs) using Wizard midiprep DNA purification kit (Promega). Isolation of protoplasts from carrot (Daucus carota) suspension culture cells, transfections, luciferase (LUC), and GUS assays have been described (Niu et al., 2011). GUS activities for each measurement were normalized by cotransfection with a 35S:LUC reporter gene (Liu et al., 1994). GUS activity measurements were performed with 4-methylumbelliferyl!-glucuronide (Sigma-Aldrich) as a substrate, and excitation and emission wavelengths of 365 and 455 nm, respectively. LUC activity was determined with a Luciferase Assay System (Promega), using an emission wavelength of 550 nm and a photomultiplier gain of 775 V. Fluorescence and luminescence were measured with a 96-well luminescence spectrometer LS50B (Perkin Elmer). Three transfections were performed for each construct and the measurements were averaged. All transfections were repeated two or three times using !"# independent protoplast preparations. Results from representative experiments are reported. Significant differences were determined by Student´s t-test using Graphpad Prism version 4.00 for Windows software (GraphPad Software). Differences between mean values are reported as significant at P<0.01. Multiple Sequence Alignments JAZ8-related sequences were identified using Basic Local Alignment Search Tools (BLASTP and TBLASTN) and the Phytozome database (http://www.phytozome.net/), with JAZ8 as the query sequence. Of the resulting 43 sequences identified from 18 monocot and dicot species, 35 proteins contained a complete ZIM domain and Jas motif and were used to generate the Weblogo (Crooks et al., 2004) shown in Figure 6. Manual annotation identified 30 JAZ8-like proteins that contain a canonical an EAR motif at the N-terminus, and these sequences were used to generate the Weblogo shown in Figure 6. A similar approach was used to identify 97 JAZ10-like proteins for construction of the Weblogo. Accession Numbers Arabidopsis Genome Initiative numbers described in this article are listed below: ACT8 (At1g49240), COI1 (At2g39940), IAA17 (At1g04250), JAZ1 (At1g19180), JAZ2 (At1g74950), JAZ3 (At3g17880), JAZ4 (At1g48500), JAZ5 (At1g17380), JAZ6 (At1g72450), JAZ7 (At2g34600), JAZ8 (At1g30135), JAZ9 (At1g70700), JAZ10.1 (At5g13220.1), JAZ11 (At3g43440), JAZ12 (At5g20900), MYC2 (At1g15750), and VSP2 (At5g24770). !"# (At1g32640), NINJA (At4g28910), TPL ACKNOWLEDGMENTS We acknowledge Chad Seippel and Li Deng (Michigan State University) for technical assistance throughout the project. We thank Kyaw Aung and Jiangping Hu (Michigan State University) for providing Gateway-compatible Y2H vectors, and Rob Larkin (Michigan State University) for providing seed for the jin1-7 mutant. We also thank Tom Guilfoyle and Gretchen Hagen (University of Missouri) for carrot cell cultures and plasmid constructs used in transfection assays. Shiv Tiwari (Mendel Biotechnology Inc.) provided assistance with protoplasts isolation and transfection, and Yajie Niu (Washington State University) provided the 35S:GD vector. This research was supported by the National Institutes of Health (grants T32 GM07270 to L.B.S., R01 CA107134 to N.Z., and R01 GM57795 to G.A.H.,), the National Science Foundation (0929100 to N.Z.), the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy (grants DE-FG02-99ER20323 to J.B. and DE-FG02-91ER20021 to G.A.H.), the Agricultural Research Center at Washington State University, and the Howard Hughes Medical Institute (N.Z.). We dedicate this work to the memory of Laura Sheard, who was an extraordinarily talented and energetic young scientist. As a cherished colleague, she made key discoveries that paved the way for this study. We unfortunately lost her to a car accident while this manuscript was under review. !"# FIGURE AND TABLES [3H-COR], µM Figure 2.1. JAZ Proteins Differentially Interact with COI1 in the Presence of JA-Ile. !"# Figure 2.1. JAZ Proteins Differentially Interact with COI1 in the Presence of JA-Ile. – Continued. (A) JA-Ile differentially promotes COI1 interaction with various JAZ proteins. Pull-down assays were performed using crude leaf extracts from 35S:COI1-Myc transgenic plants and purified recombinant JAZ-His proteins. Reactions were supplemented (+) with 1 µM (3R,7S)-JA-Ile or an equivalent volume of assay buffer (-). Protein bound to JAZ-His was separated by SDS-PAGE and analyzed by immunoblotting with anti-Myc antibody for the presence of COI1-Myc. The blotted membrane was stained with Coomassie blue to visualize the amount of JAZ-His loaded. (B) JAZ8 does not associate with COI1 in the presence of JA-Ile. Pull-down assays with purified JAZ8-His and JAZ10.1-His were performed as described in (A). Reaction mixtures were supplemented with the indicated concentration of (3R,7S)-JA-Ile. (C) JAZ8 weakly associates with COI1 in the presence of coronatine. Pull-down assays were performed as described in (A). Reactions were supplemented with the indicated concentration of coronatine (COR). 3 (D) Saturation binding of H-labeled coronatine to COI1-JAZ8 (circle) and COI1-JAZ10.1 (triangle) complexes. Data show the mean ± SE of 2 replicates performed in duplicate. !"# Figure 2.2. Overexpression of JAZ8 Results in Decreased Sensitivity to JA. !!" Figure 2.2. Overexpression of JAZ8 Results in Decreased Sensitivity to JA. – Continued. (A) Root growth inhibition assay of wild-type (WT; open bars) and 35S:JAZ8 (filled bars) seedlings grown on MS media containing the indicated concentration of MeJA. Root length measurements were made 6 d after seed germination. Data show the mean ± SE (n > 20 seedlings per genotype for each concentration of MeJA except 100 µM, in which n = 6). Asterisks denote statistically significant differences (P<0.05, Student’s t-test) between the two plant genotypes at the indicated concentration of MeJA. (B) JA-responsive gene expression in wild-type (WT) and 35S:JAZ8 (JAZ8) seedlings. Seedlings were grown in liquid MS medium for nine d and then treated for two hr with the indicated concentration of MeJA or a mock control (“0”). Total RNA isolated from the treated seedlings was subjected to Northern blot analysis with the indicated probes. A JAZ8 probe was used to verify overexpression of JAZ8 in the transgenic line, and an ACTIN8 (ACT8) probe was used as a loading control. (C) Weight of S. exigua larvae reared on WT (open bars) or 35S:JAZ8 (filled bars) plants for 9 or 14 d. Data show the mean ± SE (n = 56 to 65 larvae per plant genotype). The experiment was repeated four times with similar results. Data from a representative experiment are shown. (D) Photograph of representative S. exigua larvae recovered from WT and 35S:JAZ8 (JAZ8) plants after 14 d of feeding. !"# Figure 2.3. JAZ8 is Resistant to JA-Mediated Degradation in Vivo. (A) Differential stability of JAZ8 and JAZ10.1 in the presence of JA. Transgenic seedlings (seven-d-old) expressing JAZ8-YFP or JAZ10.1-YFP fusion proteins were treated either with water (Mock) or with 50 !M MeJA for the indicated amount of time prior to imaging of roots by confocal fluorescence microscopy. Image settings used for seedlings of the same genotype at different time points were identical. (B) Coronatine stimulates JAZ8 turnover in a 26S proteasome-dependent manner. Transgenic seedlings (four-d-old) expressing JAZ8-YFP were pretreated with water or the 26S proteasome inhibitor MG132 (100 !M) for 80 min, at which time seedlings were treated with coronatine (1 !M) for an additional 10 or 30 min. Seedlings treated with both coronatine and MG132 were imaged at the 30 min time point. YFP signal in root tissue was visualized by confocal microscopy. Microscope settings were identical for all images. !"# Figure 2.4. A Jas Domain Swap Converts JAZ8 to a COI1-Interacting Protein That Does Not Exert Dominant Repression of JA Responses. ! ! !"# Figure 2.4. A Jas Domain Swap Converts JAZ8 to a COI1-Interacting Protein That Does Not Exert Dominant Repression of JA Responses. – Continued. (A) Amino acid alignment of the Jas motif in 12 Arabidopsis full-length JAZ proteins. Structural features within the Jas motif that physically associate with COI1 and JA-Ile (Sheard et al., 2010) are indicated. (B) Schematic diagram of chimeric proteins constructed by swapping of the Jas motif. Sequence regions derived from JAZ8 and JAZ10.1 are shown in grey and white, respectively. (C) Pull-down assays performed with parental (JAZ8 and JAZ10.1) and chimeric (JAZ10-Jas8, J10-J8; JAZ8-Jas10, J8-J10) proteins in the presence (+) or absence (-) of 2.5 !M JA-Ile. (D) JA-mediated root growth inhibition in WT and transgenic seedlings (35S:JAZ8, JAZ8; 35S:JAZ10.1, JAZ10.1; 35S:JAZ8-Jas10, J8-J10; and 35S:JAZ10-Jas8, J10-J8). Seedlings were grown for 6 d on MS medium supplemented or not supplemented with 20 µM MeJA. The root length ratio was calculated by dividing the average the root length of seedlings grown on MeJAcontaining medium by the average root length of seedlings of the same genotype grown in the absence of MeJA. Data points show the mean ± SE (n = 18 to 24 seedlings per data point). Asterisks denote significant differences (P<0.05, Student’s t-test) in comparisons between the indicated transgenic line and WT. !"# A JAZ10 - JA-Ile: JAZ8 + - + JAZ8LPIAR - Mock control JAZ-His Root Length Ratio JAZ8-YFP JAZ8LPIAR -YFP + COI1-Myc B C 0.8 + MeJA 0.6 0.4 + MG132 + MeJA 0.2 0 WT JAZ10 JAZ8 JAZ8LPIAR Figure 2.5. The Canonical LPIAR Degron Promotes JAZ8 Binding to COI1, Destabilizes JAZ8, and Restores JA Responsiveness. LPIAR (A) JAZ8 interacts with COI1 in a JA-Ile-dependent manner. Pull-down reactions were performed in the presence (+) or absence (-) of 2.5 !M JA-Ile. LPIAR (B) Transgenic lines overexpressing JAZ8 are sensitive to JA. Root growth assays were LPIAR LPIAR performed with WT, 35S:JAZ10 (JAZ10.1), 35S:JAZ8 (JAZ8), and 35S:JAZ8 (JAZ8 ) seedlings. Data show the mean ± SE for each genotype (n = 20). Asterisk denotes significant differences (P<0.05, Student’s t-test) in comparisons between the indicated transgenic line and WT. LPIAR (C) JAZ8 -YFP is degraded in vivo in response to JA treatment. Six-d-old transgenic LPIAR seedlings expressing JAZ8-YFP (left) or JAZ8 -YFP (right) were pretreated with water or the 26S proteasome inhibitor MG132 (100 !M) for 75 min, at which time seedlings were treated with either MeJA (“+MeJA”, 50 !M) or water (Mock control) for an additional 15 min. YFP signal in root tissue was visualized by fluorescence microscopy. Microscope settings were identical for all images. Scale bars = 50 µm. !"# Figure 2.6. The EAR Motif of JAZ8 is Required for Repression of JA Responses. !"# Figure 2.6. The EAR Motif of JAZ8 is Required for Repression of JA Responses. – Continued. (A) Consensus sequence of the Jas motif in JAZ8 (upper) and JAZ10 (lower) orthologs from diverse plant species. The height of each stack of symbols indicates the sequence conservation at that position, whereas the height of symbols within the stack represents the relative frequency of the corresponding amino or nucleic acid at that position (Crooks et al., 2004). (B) Consensus sequence of the N-terminal 15-amino acids from JAZ8-like proteins from diverse plant species. (C) Comparison of JA-mediated root growth inhibition in 35S:JAZ8 and 35S:JAZ8!EAR seedlings. Wild-type (WT), 35S:JAZ8 (line #24), and 35S:JAZ8!EAR (five independent homozygous lines) were grown for 8 d on medium supplemented or not supplemented with 20 µM MeJA. Root length ratios were calculated as described in the legend of Figure 4D. Data show the mean ± SE for each genotype (n >14 seedlings per genotype). Asterisks denote a significant difference (P<0.05, Student’s t-test) in comparisons between transgenic and WT seedlings. The mean root length ratio calculated for seedlings (n = 92) from all five independent 35S:JAZ8!EAR lines was not significantly different (P-value = 0.068, Student’s t-test) from that of WT. !"# Figure 2.7. The EAR Motif is Required for Transcriptional Repression by JAZ8. !"# Figure 2.7. The EAR Motif is Required for Transcriptional Repression by JAZ8. – Continued. (A) Carrot protoplasts were transfected with the GUS reporter [35S(-46)LexA(2x)Gal4(2x):GUS] alone or in combination with the indicated effector and LD-VP16 constructs as described in the text. GUS activities are expressed relative to the sample in which LD-VP16 was cotransfected with the GD effector (Gal4 DNA binding domain alone; set to 100%). GUS activities were normalized by cotransfection with a 35S:LUC construct. Asterisks denote statistically significant differences (P<0.01, Student’s t-test) in comparisons to the LD-VP16 + GD activation control. Data show the mean ± SD of three replicate assays. (B) Carrot protoplasts were cotransfected with the GUS reporter gene described in (A), LD-VP16, and the indicated effector construct. L9A, E10A, L11A, R12A and L13A correspond to single Ala-substitution mutations in the EAR motif of JAZ8-N (black boxes). The F14A mutation is located C-terminal to the EAR motif and serves as a control. GUS activities are expressed as described in (A). Asterisks denote statistically significant differences (P<0.01, Student’s t-test) in comparisons to the unmodified GD-JAZ8-N construct. Data points show the mean ± SD of three replicate assays. !"# 9-13 45-72 101-127 9-13 45-72 101-127 LELRL ! 5xA Figure 2.8. Protein-Protein Interaction Domains in JAZ8. !"# Figure 2.8. Protein-Protein Interaction Domains in JAZ8. – Continued. (A) Yeast two-hybrid (Y2H) assay of JAZ8 and JAZ8 deletion derivatives with MYC2 and JAZ3. Yeast strains expressing both the bait (JAZ8) and prey (MYC2 or JAZ3) proteins were plated on media containing X-gal. LacZ-mediated blue-color formation is indicative of protein-protein interaction. JAZ8 proteins were co-expressed with an empty prey vector (pB42AD) as a negative control. Photographic images of yeast cells were taken after 48 h of incubation at 30°C. (B) Y2H analysis of JAZ8 deletion proteins with MYC2, JAZ1, NINJA and TPL. Yeast strains expressing both the bait (JAZ8 or JAZ8 deletion) and prey (MYC2, JAZ1, NINJA, or TPL) proteins were tested as described in (A). Empty bait (pGILDA) and prey (pB42AD) vectors were used as negative controls. As a positive control for NINJA interaction, yeast cells were cotransformed with pB42AD-NINJA and pGILDA-JAZ1 (lower right). (C) JAZ8 interacts weakly with NINJA in vitro. Purified JAZ-His proteins (fused to MBP) were incubated with crude extract from yeast cells expressing HA-tagged derivatives of NINJA (N), JAZ1 (J), or an empty vector control (E). MBP-His was used as a control for specificity. Purified protein complexes were separated by SDS-PAGE and probed with an anti-HA antibody for the presence of HA-tagged NINJA (HA-NINJA) or JAZ1 (HA-JAZ1). Lower panel shows a Coomassie blue (CB)-stained gel to visualize the amount of protein loaded. CE, crude yeast extracts which were used as an input control. Protein molecular weight markers were run in the fourth lane from the left. !!" Figure 2.9. The ZIM Domain is not Required for JAZ8-mediated Repression of JA-induced Root Growth Inhibition. (A) 35S:JAZ8!ZIM plants are insensitive to JA-mediated root growth inhibition. Assays were performed with WT, 35S:JAZ8 (JAZ8; line #24), and eight independent 35S:JAZ8!ZIM (JAZ8!ZIM; gray bars) lines that are homozygous for the transgene. Root length ratios were calculated as described in the legend of Figure 4D. Data show the mean ± SE for each genotype (n = 12 to 24 seedlings per genotype). The mean root length ratio of seedlings (n = 127) from all eight 35S:JAZ8!ZIM lines was not significantly different than that of 35S:JAZ8 seedlings (Pvalue = 0.69, Student’s t-test). Means with different italicized letters are significantly different at P<0.001. (B) JA-mediated root growth phenotype of 35S:JAZ8!EZ lines expressing a JAZ8 derivative lacking both the EAR and ZIM motifs. Assays were performed as described in panel (A), and included eleven independent 35S:JAZ8!EZ T2 lines (JAZ8!EZ; gray bars). Data show the mean ± SE for each genotype (n = 13 to 22 seedlings per genotype). The mean root length ratio of seedlings (n = 212) from all eleven 35S:JAZ8!EZ lines was not significantly different than that of WT seedlings (P-value = 0.62, Student’s t-test). Means with different italicized letters are significantly different at P<0.001. !"# Supplemental Figure 2.1. Phylogenetic Tree Constructed from the Jas Motif of Arabidopsis JAZ Proteins. The 27-amino-acid Jas motif in all 12 Arabidopsis JAZ proteins was aligned with MUSCLE (Edgar, 2004). The phylogenetic tree was generated with the Neighbor-joining method using MEGA5 software (http://www.megasoftware.net). Red circles indicate JAZ proteins that have a predicted EAR motif. Reference Edgar, R.C. (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32:1792-1797. !"# Supplemental Figure 2.2. JA-Mediated Root Growth Inhibition in Independent 35S:JAZ8 Lines. (A) Five independent homozygous 35S:JAZ8 lines, which were selected for high expression of the transgene, were grown together with WT and jin1-7 seedlings on MS medium supplemented (filled bars) or not supplemented (open bars) with 20 µM MeJA. Plants were grown for 8 d on a 16-h light and 8-h dark cycle. Data show the mean ± SE for each genotype (n >18 seedlings per genotype). The mean root length of each 35S:JAZ8 line was significantly longer (P<0.05, Student’s t-test) than that of the jin1-7 mutant in the presence of MeJA. (B) Data shown in (A) were plotted as root length ratio, which was calculated by dividing the average the root length of seedlings grown on MeJA-containing medium by the mean root length of seedlings of the same genotype grown in the absence of MeJA. Means with different italicized letters are significantly different at P<0.05. !"# Supplemental Figure 2.3. 35S:JAZ8 Plants are Compromised in Resistance to Herbivory by S. exigua Larvae. Six-week-old 35S:JAZ8 (line #24) and wild-type (WT) plants were challenged (+) for 14 d with Spodoptera exigua larva. A separate set of control plants grown side-by-side with the challenged plants were not infested (-) with larvae. The photograph was taken at the end of the feeding trial. !"# Supplemental Figure 2.4. JA-Mediated Root Growth Inhibition in 35S:JAZ8-YFP Lines. !"# Supplemental Figure 2.4. JA-Mediated Root Growth Inhibition in 35S:JAZ8-YFP Lines. – Continued. (A) 35S:JAZ8-YFP plants are insensitive to MeJA. Wild-type (WT), 35S:JAZ8 (line #24; JAZ8), 35S:YFP (YFP), and 35S:JAZ8-YFP (JAZ8-YFP) seedlings were grown on MS medium supplemented with or without 25 µM MeJA for 8 d under continuous light. Root length ratios were calculated as described in the legend of Supplemental Figure 2B. Data show the mean ± SE for each genotype (n > 20 seedlings per genotype). Asterisks indicate significant differences (P<0.05, Student’s t-test) in comparisons between the indicated transgenic line and WT. (B) Mutation of the EAR motif suppresses the JA-insensitive root growth phenotype of 35S:JAZYFP seedlings. Root growth inhibition assays were performed with 35S:YFP (YFP), 35S:JAZ8YFP (JAZ8-YFP), 35S:JAZ8!EAR-YFP (JAZ8!EAR-YFP; gray bars), and four independent LELRL!5xA LELRL!5xA 35S:JAZ8 -YFP (JAZ8 -YFP; gray bars) homozygous lines (T3 generation). Seedlings were grown for 8 d on MS medium containing or lacking 20 µM MeJA. Data were plotted as root length ratio as described in panel (A). Data show the mean ± SE for each genotype (n > 23 seedlings per genotype). Means with different italicized letters are significantly different at P<0.05. The root length ratio of 35S:JAZ8 was calculated as the mean of all four independent 35S:JAZ8 !"# LELRL!5xA LELRL!5xA -YFP seedlings -YFP lines. JAZ8 LPIAR Supplemental Figure 2.5. Substitution of PKASM to LPIAR does not Affect the Ability of JAZ8 to Interact with MYC2, JAZ1, or TPL. LPIAR Yeast strains expressing both the bait (JAZ8 or JAZ8 ) and prey (MYC2, JAZ1, NINJA or TPL) were plated on SD-Gal medium containing X-gal. Blue color indicates protein-protein interaction. Photographic image was taken 48 h after incubation of yeast cells at 30°C. As a positive control for NINJA interaction, yeast cells were cotransformed with pB42AD-NINJA and pGILDA-JAZ1 (lower right). !"# YFP JAZ8-YFP JAZ8!EAR-YFP JAZ8 LELRL ! 5XA -YFP Supplemental Figure 2.6. Nuclear Localization of JAZ8-YFP Fusion Proteins. Root tips of 9-day-old seedlings expressing the indicated JAZ-YFP reporter were imaged with an Olympus Fluoview confocal microscope. Transgenic lines were 35S:YFP (YFP), 35S:JAZ8-YFP (JAZ8-YFP), 35S:JAZ8!EAR-YFP #1-1 (JAZ8!EAR-YFP), and 35S:JAZ8LELRL!5xA-YFP #5-1 (JAZ8LELRL!5xA-YFP). Scale bar = 50 µm. !"# Supplemental Figure 2.7. Yeast Two-hybrid Analysis of JAZ8 and JAZ8!ZIM Interactions with other Arabidopsis JAZ proteins. Yeast strains expressing both the bait (JAZ8 or JAZ8!ZIM) and prey (JAZ1 – JAZ12) were plated on plates containing X-gal. Blue colors indicate protein-protein interaction. Photographic images were taken 48 h after incubation of yeast cells at 30°C. !"# Supplemental Table 2.1 List of oligonucleotide primers used in this study. Primers in this section used for Y2H and overexpression constructs INSERT VECTOR pGILDA & pB42AD pBI-TONY JAZ8 pBI-EYFP pENTR-TOPO pGILDA & pB42AD JAZ8Jas10 pBITONY pBI-EYFP pGILDA & pB42AD JAZ10Jas8 pBITONY pBI-EYFP JAZ8!ZIM ! pGILDA & pB42AD pBI-TONY PRIMER NAME JAZ8_NcoI_FP JAZ8_XhoI_RP JAZ8_XbaI_FP JAZ8_KpnI_RP JAZ8_XbaI_FP JAZ8ns_XhoI_RP JAZ8_pENTR_FP JAZ8_pENTR_RP JAZ8_XbaI_FP JAZ10_NcoI_RP JAZ8_XbaI_FP JAZ10_KpnI_RP JAZ8_XbaI_FP JAZ10ns_KpnI_RP JAZ10_NcoI_FP JAZ8_XhoI_RP JAZ10_BamHI_FP JAZ8_XhoI_RP JAZ10_BamHI_FP JAZ8ns_XhoI_RP JAZ8_NcoI_FP JAZ8_XhoI_RP JAZ8_XbaI_FP SEQUENCE 5'-CCATGGATGAAGCTACAGCAAAATTGTG-3' 5'-CTCGAGTTATCGTCGTGAATGGTACG-3' 5'-ATATTCTAGAATGAAGCTACAGCAAAATTGTG-3' 5'-ATATGGTACCTTATCGTCGTGAATGGTACG-3' 5'-ATATTCTAGAATGAAGCTACAGCAAAATTGTG-3' 5'-CTCGAGTCGTCGTGAATGGTACGGTG-3' 5'-CACCATGAAGCTACAGCAAAATTGTG-3' 5'-TTATCGTCGTGAATGGTACGGTG-3' 5'-ATATTCTAGAATGAAGCTACAGCAAAATTGTG-3' 5'-CCATGGTTAGGCCGATGTCGGATAGTAAG-3' 5'-ATATTCTAGAATGAAGCTACAGCAAAATTGTG-3' 5'-GGTACCTTAGGCCGATGTCGGATAGTAAG-3' 5'-ATATTCTAGAATGAAGCTACAGCAAAATTGTG-3' 5'-GGTACCGGCCGATGTCGGATAGTAAGGAG-3' 5'-CCATGGATGTCGAAAGCTACCATAGAAC-3' 5'-CTCGAGTTATCGTCGTGAATGGTACG-3' 5'-GGATCCATGTCGAAAGCTACCATAGAAC-3' 5'-CTCGAGTTATCGTCGTGAATGGTACG-3' 5'-GGATCCATGTCGAAAGCTACCATAGAAC-3' 5'-CTCGAGTCGTCGTGAATGGTACGGTG-3' 5'-CCATGGATGAAGCTACAGCAAAATTGTG-3' 5'-CTCGAGTTATCGTCGTGAATGGTACG-3' 5'-ATATTCTAGAATGAAGCTACAGCAAAATTGTG-3' !"# Supplemental Table 2.1 - Continued. ! JAZ8_KpnI_RP JAZ8_XbaI_FP pBI-EYFP JAZ8ns_XhoI_RP JAZ8_XbaI_FP pBI-TONY JAZ8_XhoI_RP JAZ!EAR JAZ8_XbaI_FP pBI-EYFP JAZ8ns_XhoI_RP JAZ8_XbaI_FP pBI-TONY JAZ8_XhoI_RP JAZ8!EZ JAZ8_XbaI_FP pBI-EYFP JAZ8ns_XhoI_RP 5'-ATATGGTACCTTATCGTCGTGAATGGTACG-3' 5'-ATATTCTAGAATGAAGCTACAGCAAAATTGTG-3' 5'-CTCGAGTCGTCGTGAATGGTACGGTG-3' 5'-ATATTCTAGAATGAAGCTACAGCAAAATTGTG-3' 5'-CTCGAGTTATCGTCGTGAATGGTACG-3' 5'-ATATTCTAGAATGAAGCTACAGCAAAATTGTG-3' 5'-CTCGAGTCGTCGTGAATGGTACGGTG-3' 5'-ATATTCTAGAATGAAGCTACAGCAAAATTGTG-3' 5'-CTCGAGTTATCGTCGTGAATGGTACG-3' 5'-ATATTCTAGAATGAAGCTACAGCAAAATTGTG-3' 5'-CTCGAGTCGTCGTGAATGGTACGGTG-3' Primers in this section used for MBP-JAZ-His constructs INSERT JAZ6 pRMG-nMAL JAZ7 pRMG-nMAL JAZ8 pRMG-nMAL JAZ12 pRMG-nMAL JAZ8-Jas10 ! VECTOR pRMG-nMAL PRIMER NAME AtJAZ6-5'NotI AtJAZ6-3'XhoI NotI-C-JAZ7-FP JAZ7_NotI_FP JAZ7_XhoI_RP JAZ8_NotI_FP JAZ8 XhoI RP AtJAZ12-NotI F AtJAZ12-SalI R JAZ8_NotI_FP SEQUENCE 5'-GCGCGGCCGCCATGTCAACGGGACAAGCGC-3' 5'-CGGCTCGAGAAGCTTGAGTTCAAGGTTTTTGG-3' 5'-GCGCGGCCGCCATCATCATCAAAAACTGCGACAAGCC-3' 5'-GCGGCCGCATGATCATCATC-3' 5'-CTCGAGCTATCGGTAACGGTGGTAAG-3' 5'-GCGGCCGCATGAAGCTACAGC-3' 5'-CTCGAGTTATCGTCGTGAATGGTACG 5'-GCGCGGCCGCCACTAAGGTGAAAGATGAG-3' 5'-TCGGTCGACAGCAGTTGGAAATTCCTCC-3' 5'-GCGGCCGCATGAAGCTACAGC-3' !!" Supplemental Table 2.1 - Continued. ! AtJAZ10-SalI AtJAZ10-NotI JAZ10-Jas8 pRMG-nMAL JAZ8 XhoI RP JAZ8_NotI_FP JAZ8LPIAR pRMG-nMAL JAZ8 XhoI RP 5'-TCGGTCGACGGCCGATGTCGGATAG-3' 5'-GCGCGGCCGCCTCGAAAGCTACCATAGAACTCG-3' 5'-CTCGAGTTATCGTCGTGAATGGTACG 5'-GCGGCCGCATGAAGCTACAGC-3' 5'-CTCGAGTTATCGTCGTGAATGGTACG Primers in this section used for chimeric proteins and deletion constructs CONSTRUCT JAZ8-Jas10 JAZ10-Jas8 JAZ8!ZIM ! PRIMER NAME JAZ8_XbaI_FP JAZ8_Jas10_RP JAZ8_Jas10_FP JAZ10_KpnI_RP JAZ8_XbaI_FP JAZ10_KpnI_RP JAZ10_BamHI_FP JAZ10_Jas8_RP JAZ10_Jas8_FP JAZ8_XhoI_RP JAZ10_BamHI_FP JAZ8_XhoI_RP JAZ8_NcoI_FP JAZ8_deltaZIM_RP JAZ8_deltaZIM_FP JAZ8_XhoI_RP SEQUENCE 5'-ATATTCTAGAATGAAGCTACAGCAAAATTGTG-3' 5'-GCGATGGGAAGATCTGGAAGCTGATTATGATGAAATG-3' 5'-CATAATCAGCTTCCAGATCTTCCCATCGCAAGGAG-3' 5'-GGTACCTTAGGCCGATGTCGGATAGTAAG-3' 5'-ATATTCTAGAATGAAGCTACAGCAAAATTGTG-3' 5'-GGTACCTTAGGCCGATGTCGGATAGTAAG-3' 5'-GGATCCATGTCGAAAGCTACCATAGAAC-3' 5'-GATGCTTTTGGATTTCCTTCTAGATTCTGGCCAAAG-3' 5'-GAATCTAGAAGGAAATCCAAAAGCATCAATGAAAAAATC-3' 5'-CTCGAGTTATCGTCGTGAATGGTACG-3' 5'-GGATCCATGTCGAAAGCTACCATAGAAC-3' 5'-CTCGAGTTATCGTCGTGAATGGTACG-3' 5'-CCATGGATGAAGCTACAGCAAAATTGTG-3' 5'-GGTTTTCATTTCTCTGCTTTGAGATTCTTCATTTGG-3' 5'-CCAAATGAAGAATCTCAAAGCAGAGAAATGAAAACC-3' 5'-CTCGAGTTATCGTCGTGAATGGTACG-3' !"# Supplemental Table 2.1 - Continued. ! JAZ8_NcoI_FP JAZ8_XhoI_RP JAZ8_deltaEAR_FP JAZ8_pENTR_RP JAZ8!EAR JAZ8_pENTR_FP JAZ8_pENTR_RP JAZ8_deltaEAR_FP JAZ8_pENTR_RP JAZ8!EZ JAZ8_pENTR_FP JAZ8_pENTR_RP JAZ8_NcoI_FP JAZ8!Jas JAZ8_XhoI_306R JAZ8_ATG133_NcoI_FP JAZ8-Jas JAZ8_XhoI_RP 5'-CCATGGATGAAGCTACAGCAAAATTGTG-3' 5'-CTCGAGTTATCGTCGTGAATGGTACG-3' 5'-GCTACAGCAAAATTGTGAC TTTCCCACTTCTTATGATTC-3' 5'-TTATCGTCGTGAATGGTACGGTG-3' 5'-CACCATGAAGCTACAGCAAAATTGTG-3' 5'-TTATCGTCGTGAATGGTACGGTG-3' 5'-GCTACAGCAAAATTGTGAC TTTCCCACTTCTTATGATTC-3' 5'-TTATCGTCGTGAATGGTACGGTG-3' 5'-CACCATGAAGCTACAGCAAAATTGTG-3' 5'-TTATCGTCGTGAATGGTACGGTG-3' 5'-CCATGGATGAAGCTACAGCAAAATTGTG-3' 5'-CTCGAGTTATGGATTTGGAAGCTGATTATG-3' 5'-CCATGGATGCATAATCAGCTTCCAAATCC-3' 5'-CTCGAGTTATCGTCGTGAATGGTACG-3' Primers in this section used for site-directed mutagenesis CONSTRUCT JAZ8LPIAR"AAAAA ! PRIMER NAME JAZ8_AR_FP JAZ8_AR_RP JAZ8_IAR_FP JAZ8_IAR_RP JAZ8_PIAR_FP JAZ8_PIAR_RP SEQUENCE 5'-CCAAATCCAAAAGCAGCAAGGAAAAAATCTCTCC-3' 5'-GGAGAGATTTTTTCCTTGCTGCTTTTGGATTTGG-3' 5'-CCAAATCCAAATCCAAAAATCGCAAGGAAAAAATCTCTC-3' 5'-GAGAGATTTTTTCCTTGCGATTTTTGGATTTGGATTTGG-3' 5'-CTTCCAAATCCAAATCCACCCATCGCAAGGAAAAAATC-3' 5'-GATTTTTTCCTTGCGATGGGTGGATTTGGATTTGGAAG-3' !"# Supplemental Table 2.1 - Continued. ! 5'-CATAATCAGCTTCCA AATCTTCCCATCGCAAGGAAAAAATC-3' 5'-GATTTTTTCCTTGCGATGGGAAGATTTGGAAGCTGATTATGJAZ8_LPIAR_RP 3' 5'JAZ8_LEtoAA_FP GCTACAGCAAAATTGTGACGCGGCACTTCGTCTTTTTCCCAC-3' 5'GTGGGAAAAAGACGAAGTGCCGCGTCACAATTTTGCTGTAGCJAZ8_LEtoAA_FP 3' 5'JAZ8_LELRLtoAAAAA_ GCAAAATTGTGACGCGGCAGCTGCTGCTTTTCCCACTTCTTAT FP GATTC-3' 5'JAZ8_LELRLtoAAAAA_ GAATCATAAGAAGTGGGAAAAGCAGCAGCTGCCGCGTCACAA RP TTTTGC-3' JAZ8_LPIAR_FP JAZ8LELRL!AAAAA Primers in this section used in carrot protoplast assay CONSTRUCT PRIMER NAME JAZ8-F 35S::GD-JAZ8 SEQUENCE 5´GGGGACAAGTTTGTACAAAAAAGCAGGCTCAATGAAGCTACAGCAAAATTGTG ACTTGG-3´ 5´GGGGACCACTTTGTACAAGAAAGCTGGGTCTCATCGTCGTGAATGGTACGGTG AAGTAG-3´ ! ! !"# Supplemental Table 2.1 - Continued. ! 5´JAZ8-F GGGGACAAGTTTGTACAAAAAAGCAGGCTCAATGAAGCTACAGCAAAATTGTG 35S::GDACTTGG-3´ JAZ8!Jas 5´JAZ8-Jas-R GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAATTTGGAAGCTGATTATGATG AAATGAGG-3´ 5´JAZ8-F GGGGACAAGTTTGTACAAAAAAGCAGGCTCAATGAAGCTACAGCAAAATTGTG 35S::GDACTTGG-3´ JAZ8-N 5´JAZ8[I]-R GGGGACCACTTTGTACAAGAAAGCTGGGTCCTATTGAGATTCTTCATTTGGTTG TGGATTT-3 JAZ8-L9A-F 5´-AAGCTACAGCAAAATTGTGACGCGGAACTTCGTCTTTTTCCCAC-3´ JAZ8-L9A JAZ8-L9A-R 5´-GTGGGAAAAAGACGAAGTTCCGCGTCACAATTTTGCTGTAGCTT-3´ JAZ8-L11A-F 5´-GCTACAGCAAAATTGTGACTTGGAAGCTCGTCTTTTTCCCACT-3´ JAZ8-L11A JAZ8-L11A-R 5´-AGTGGGAAAAAGACGAGCTTCCAAGTCACAATTTTGCTGTAGC-3´ JAZ8-L13A-F 5´-CAAAATTGTGACTTGGAACTTCGTGCTTTTCCCACTTCTTATGATTCTGA-3´ JAZ8-L13A JAZ8-L13A-R 5´-TCAGAATCATAAGAAGTGGGAAAAGCACGAAGTTCCAAGTCACAATTTTG-3´ JAZ8-F14A-F 5´-ACAGCAAAATTGTGACTTGGAACTTCGTCTTGCTCCCACTTCTTATGATT-3´ JAZ8-F14A JAZ8-F14A-R 5´-AATCATAAGAAGTGGGAGCAAGACGAAGTTCCAAGTCACAATTTTGCTGT-3´ 5´JAZ8-AAA-F CGCATATGAAGCTACAGCAAAATTGTGACGCGGCCGCTCGTCTTTTTCCCACTT CTTATGATTCTGATTCTT-3´ JAZ8-AAA 5´JAZ8-AAA-R AAGAATCAGAATCATAAGAAGTGGGAAAAAGACGAGCGGCCGCGTCACAATTT TGCTGTAGCTTCATATGCG-3´ ! !"# Supplemental Table 2.2. JAZ genes used for construction of consensus motifs shown in Figure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upplemental Table 2.2. Continued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upplemental Table 2.2. Continued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upplemental Table 2.2. Continued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ai, Y., Meng, Y., Huang, D., Qi, Y., and Chen, M. 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Derepression of ethylene-stabilized transcription factors (EIN3/EIL1) mediates jasmonate and ethylene signaling synergy in Arabidopsis. Proc. Natl. Acad. Sci. USA. 108: 1253912544. !"#$ CHAPTER THREE THE JAZ8 SUBCLADE OF JAZ PROTEINS REPRESS JA SIGNALING THOUGH A NINJA-INDEPENDENT PATHWAY "#$! ! ! ABSTRACT Jasmonoyl-L-Ile (JA-Ile) is a lipid-derived hormone that regulates a broad range of gene expression in response to wounding, insect herbivores and developmental cues. The JASMONATE ZIM-DOMAIN (JAZ) proteins are repressors of JA-responsive gene expression. Twelve JAZ proteins were identified in Arabidopsis. The current JA signaling model suggests that JAZ proteins interact with an adapter protein NOVEL INTERACTOR OF JAZ (NINJA) through the JAZ ZIM domain to recruit the corepressor TOPLESS and repress JA responses. In contrast to the current JAZ repression model, JAZ8 represses JA responses through an Nterminal EAR motif, in a NINJA-independent manner. JAZ8 does not interact with NINJA in yeast, but directly interacts with TOPLESS in the absence of NINJA. Here, we report the identification and characterization of a thirteenth member (JAZ13) in the Arabidopsis JAZ family of proteins. JAZ13 shares high sequence similarity with JAZ8. JAZ13 contains a conserved EAR motif, as well as non-canonical ZIM and Jas motifs that are present in all JAZ members. JAZ13 negatively regulates jasmonate responses. Overexpression of JAZ13 results in decreased sensitivity to JA. JAZ13 does not interact with NINJA but does interact with TOPLESS through the EAR motif, suggesting that JAZ13 also represses JA responses through a NINJA-independent mechanism. The JAZ8 subclade of JAZ proteins, which includes JAZ7, JAZ8 and JAZ13, all interact with MYC2 strongly in yeast. We show that this interaction is through the Jas motif and we identify a critical Arg residue in the Jas motif of JAZ8 that is required for JAZ8-MYC2 interaction and the repressive activity of JAZ8. Collectively, these results suggest that the JAZ8 subfamily of JAZ proteins work through a common NINJAindependent pathway to repress the JA-mediated transcriptional activity of MYC2. Our results "#$! ! ! strengthen the importance of NINJA-independent repression mechanisms in regulating defense responses in Arabidopsis. INTRODUCTION Jasmonates, collectively known as JAs, are lipid-derived phytohormones involved in multiple aspects of plant growth, as well as responses to the environment. JAs were first identified as growth inhibitors (Aldridge et al., 1971), and reported to have multiple roles in regulating growth and development, including growth inhibition of seedlings (Yamane et al., 1981), male fertility in Arabidopsis, female fertility in tomato, and trichome development (Feys et al., 1994; Li et al., 2004; Yoshida et al., 2009). JAs also have important roles in regulating plant defense (Farmer et al., 1992). JAs are important activators of defense against insects, pathogen infection, UV light and other abiotic stresses (Weiler et al., 1993; Conconi et al., 1996; Dombrowski, 2003; Browse, 2009). These reports collectively suggest that JAs have a role in regulating a broad range of responses balancing plant growth and defense (Kazan and Manners, 2012; Ballaré, 2011). Understanding the molecular mechanism of this process is expected to expand our current knowledge in JA biology. JASMONATE ZIM-DOMAIN (JAZ) proteins are key players in the JA signaling pathway that repress JA responses. In the absence of JA-Ile, JAZs interact with transcription factors to repress JA-induced gene expression. Upon accumulation of JA-Ile, JAZ proteins interact with the F-box protein CORONATINE INSENSITIVE 1 (COI1) to form a JA-Ile receptor complex (Katsir et al., 2008; Sheard et al., 2010). Interaction of JAZ-COI1 leads to ubiquitination and degradation of JAZ proteins via the 26S proteasome pathway. Turnover of "#$! ! ! JAZ repressors result in activation of JAZ-interacting transcriptional activators to induce JAresponsive gene expression In Arabidopsis, the 12-member JAZ family of proteins belongs to the larger plant specific TIFY family of transcriptional regulators. TIFY proteins are named for their conserved TIF[F/Y]XG motif at the N terminus of a larger conserved motif known as the ZIM domain (Vanholme et al., 2007; Bai et al., 2011). The ZIM domain is important for JAZ repression and protein-protein interaction (Chung and Howe, 2009; Pauwels et al., 2010). In addition to the ZIM domain, JAZ proteins also share a conserved Jas motif at the C terminus, and a less conserved Domain 1 at the N terminus (Thines et al., 2007). The Jas motif contains the degron sequence that promotes JAZ-COI1 interaction and instability of JAZ substrates of SCF COI1 complex (Sheard et al., 2010; Shyu et al., 2012). The Jas motif is also required for interaction with basic helix-loop-helix (bHLH) and R2R3 MYB transcription factors such as MYC2 (Chini et al., 2007; Chini et al., 2009; Qi et al., 2011; Song et al., 2011; Shyu et al., 2012), and regulation of JAZ nuclear localization (Grunewald et al., 2009). The N-terminal Domain 1, on the other hand, is involved in JAZ1-DELLA interactions that connect jasmonate signaling with the gibberellin signaling pathway (Hou et al., 2010). The current JAZ repression model suggests that JAZ proteins interact with NOVEL INTERACTOR OF JAZ (NINJA) to recruit a Groucho/Tup1-type corepressor TOPLESS (TPL) to represses gene expression (Pauwels et al., 2010). NINJA was identified as a JAZ-interacting protein in a tandem-affinity purification assay followed by mass spectrometry with JAZ1 as bait. The TIFY motif of JAZ1 is important for interaction with NINJA. TPL was then identified using the same assay with NINJA as bait. The EAR motif of NINJA is required for NINJA-TPL "#$! ! ! interaction. Loss-of-function mutants of NINJA and TPL are hypersensitive to JA-induced root growth inhibition. These results collectively suggest that JAZ proteins repress jasmonate responses through a NINJA-TOPLESS corepressor complex. Recent studies suggest an alternative model for JAZ repression independent of NINJA (Shyu et al., 2012). The non-NINJA-interacting protein JAZ8 contains an N-terminal EAR motif that is required for repression of JA responses in transgenic plants that overexpress JAZ8 (Shyu et al., 2012). JAZ8 interacts with TPL in in the absence of NINJA. This interaction requires the EAR motif of JAZ8. Furthermore, although the ZIM domain was shown to be important for NINJA interaction with JAZ1, deletion of the ZIM domain of JAZ8 had no effect in JAZ8mediated repression of jasmonate responses (Shyu et al., 2012). These results lead to an alternative repression model in which JAZ8 directly recruits corepressors through its EAR motif to repress expression of JA-response genes. JAZ repression of JA responses also depends on direct interaction with transcription factors. JAZ proteins interact with various transcription factors in the MYB R2R3 and basic helix-loop-helix (bHLH) family to regulate different JA responses (Chini et al., 2007; FernandezCalvo et al., 2011; Niu et al., 2011; Qi et al., 2011; Song et al., 2011; Shyu et al., 2012). JAZ8 interacts with many of these transcription factors, suggesting an important role for JAZ8 in regulating diverse JA responses. Most JAZ proteins, including JAZ8, interact with transcription factors through the C-terminal Jas motif (Chini et al., 2009; Qi et al., 2011; Song et al., 2011). In addition, the Jas motif is also involved in JAZ-COI1 interaction (Sheard et al., 2010; Shyu et al., 2012) and localization of JAZ proteins (Grunewald et al., 2009). The N-terminal motif of Jas domains is critical for JAZ-COI1 interaction, and the C-terminal region of Jas domains is "#$! ! ! important for JAZ1 localization (Sheard et al., 2010; Shyu et al., 2012). Sequence determinants within the Jas motif that mediate JAZ-MYC2 interaction remains to be determined. Here, we report identification of a novel JAZ protein, JAZ13, which shares high sequence similarity with JAZ8. JAZ13 interacts with MYC2 and TPL, but not NINJA. We also show that members in the NINJA-independent JAZ8 subfamily require their Jas motif to interact with MYC2. We identify critical amino acids responsible for JAZ8-MYC2 interaction and show that this interaction is required for JAZ repressive activity. These results suggest that the JAZ8 subclade of JAZ repressors use a common NINJA-independent pathway to repress JA responses. Based on these findings, we propose that NINJA-independent repression mechanisms are important for JAZ proteins to regulate defense responses in Arabidopsis. RESULTS Identification of a novel JAZ protein, JAZ13 We asked the question whether or not there are other genes in the Arabidopsis genome that share similar functions with NINJA-independent JAZ repressors. A BLAST search performed with JAZ8 as a query sequence identified a previously uncharacterized protein (At3g22275) of unknown function that shared 21% and 23% amino acid identity with JAZ7 and JAZ8, respectively. A multiple sequence alignment of JAZ7, JAZ8 and At3g22275 revealed that At3g22275 contains: (i) an LxLxL-type EAR motif at the N-terminus; (ii) a central ZIM-like domain having a non-canonical TIFYXG motif (NAFYXG), and (iii) a Jas-like motif at the Cterminus (Figure 3.1). There are two conserved submotifs within the Jas motif of JAZ proteins: the Jas degron and a C-terminal PY motif. At3g22275 shares a similar Jas degron submotif as ""#! ! ! JAZ7 and JAZ8, but does not have a conserved PY submotif at the C-terminus. Phylogenetic analysis showed that At3g22275, which we tentatively designated as JAZ13, groups closely with JAZ7 and JAZ8 as a distinct subclade (Figure 3.2). JAZ13 Expression is Induced by JA Genes involved in jasmonate responses are typically induced by wounding or other stress-related cues that activate JA synthesis (Chung et al., 2008). To understand whether or not JAZ13 expression is regulated by wounding and JA treatment, we measured gene expression patterns of JAZ7, JAZ8 and JAZ13 in wounded wild-type plants. Consistent with previous results (Chung et al., 2008), JAZ7 and JAZ8 expression was not detectable in unwound plants and rapidly induced 30 minutes after mechanical wounding. JAZ7 and JAZ8 expression peaked at 1 hour after wounding and then decreased at time points thereafter (Figure 3.3A). Similar to JAZ7 and JAZ8, JAZ13 transcript levels were very low in unwound plants but rapidly induced 1 hour after wounding. JAZ13 expression was also strongly induced in seedlings treated with 20 !M MeJA or 0.1 !M coronatine, resembling the expression pattern as JAZ7 and JAZ8 (Figure 3.3B). These results show that JAZ13 is highly responsive to activation of the JA signaling pathway and is tightly co-expressed with JAZ7 and JAZ8. JAZ13 functions to repress jasmonate responses To determine whether JAZ13 has a functional role in regulating JA responses, we generated stable transgenic lines expressing a full-length JAZ13 cDNA driven by the cauliflower """! ! ! mosaic virus (CaMV) 35S promoter. We selected 32 independent kanamycin-resistant T1 lines and transferred them to soil. We used RNA gel-blot analysis to select 10 lines showing high levels of transgene expression (Supplemental Figure 3.1). None of these lines exhibited obvious defects in flower morphology or fertility. However, all ten lines (T2 generation) showed decreased sensitivity to MeJA-induced root growth inhibition in comparison with wild type (WT) (Figure 3.4). This finding demonstrates that ectopic expression of JAZ13 negatively regulates the JA response pathway. JAZ8 is known to repress jasmonate responses through its N-terminal EAR motif, presumably through interaction with TPL (Shyu et al., 2012). We therefore tested whether the Nterminal EAR motif of JAZ13 interacts with TPL. In yeast two-hybrid assays, JAZ13 interacted with TPL but not NINJA (Figure 3.5). We generated JAZ13 constructs lacking the entire EAR motif, or substituted the conserved Leucine (L) residues in the EAR motif with Alanine (A), to test the dependence of the TPL interaction on the EAR motif. The constructs were named LDLHL!ADAHA JAZ13!EAR and JAZ13 LDLHL!ADAHA JAZ13!EAR nor JAZ13 , respectively. The results showed that neither interact with TPL (Figure 3.5), indicating that the EAR motif is required for JAZ13-TPL interaction. The Jas motif is critical for JAZ7/8/13 interaction with MYC2 and this interaction is required for JAZ8 to repress JA responses JAZ proteins interact with JA-inducible transcription factors, such as MYC2, to regulate JA responses. Yeast two-hybrid analysis of JAZ13 shows that JAZ13 strongly interacts with ""#! ! ! MYC2 (Figure 3.5). This strengthens the hypothesis of JAZ13 being involved in regulation of JA signaling. JAZ8 interacts through its Jas motif with MYC2 and other bHLH and MYB family transcription factors (Qi et al., 2011; Song et al., 2011; Shyu et al., 2012). To determine whether or not the Jas motif is required for other members in the JAZ8 subgroup to interact with transcription factors, we generated deletion constructs of JAZ7 and JAZ13 lacking the Jas motif, and tested their interaction with MYC2 in yeast. Indeed, JAZ7!Jas and JAZ13!Jas failed to interact with MYC2 in this assay (Figure 3.6). To further define the MYC2-interacting region of the JAZ8 subclade of proteins, we generated nested deletions at the C-terminal end of the JAZ8 Jas motif and tested these mutants for interaction with MYC2. Yeast two-hybrid assays showed that removal of 9, 11, or 13 amino acids from the JAZ8 C-terminus (constructs JAZ8!C122, JAZ8!C120 and JAZ8!C118, respectively) do not significantly affect interaction with MYC2 (Figure 3.7A). Strikingly, however, further deletion of three amino acids (KRK; JAZ8!C115) abolished the JAZ8-MYC2 interaction. This finding, together with the highly conserved nature of the KRK motif in all JAZ proteins, suggested that these residues perform a critical role of TF binding. Indeed, site-directed mutagenesis showed that Ala substitution of the central R residue (mutant JAZ8 R117A ) is sufficient to abrogate JAZ8 binding to MYC2 (Figure 3.7A). We next tested whether JAZ8 Among 32 independent 35S- JAZ8 R117A R117A retains the ability to repress JA responses in vivo. transgenic lines identified, RNA-blot analysis showed that seven lines the transgene to high levels (Supplemental Figure 3.2). Root growth assays showed that all seven lines (T2 generation) were as sensitive as WT seedlings to MeJA-induced root growth inhibition (Figure 3.7B). Similar results were obtained with transgenic lines R117A overexpressing JAZ8 -YFP (Supplemental Figure 3.3). Interestingly, confocal microscopy ""#! ! ! R117A showed that although JAZ8-YFP is exclusively localized in the nucleus, JAZ8 -YFP is localized in both the nucleus and cytoplasm, similar to the YFP only control (Supplemental Figure 3.4). We conclude that the R117A mutation affects both interaction with MYC2 and localization of the protein, which correlates with in the inability of JAZ8 R117A to repress JA. T-DNA Knockout lines of JAZ7, JAZ8 and JAZ13 are sensitive to JA To begin to investigate the physiological role of the JAZ7/8/13 clade, we obtained TDNA insertion lines that are defective in each of the corresponding genes (Figure 3.8A). jaz7-1 (WiscDsLox7H11) contains a T-DNA insertion in the second exon of JAZ7 and has been previously reported to be a null mutant (Thines et al., 2007). The WiscDsLox255G12 line, which we designated jaz8-1, harbors the T-DNA insertion 134 bp upstream of the translational start site. The SALK_149961 line (designated jaz13-1) carries a T-DNA insertion in the promoter region of JAZ13, 203 bp upstream of the translational start site (Figure 3.8A). We used RNA blot analysis to assess the effect of each T-DNA insertion on the wound-induced expression of the corresponding transcript. Consistent with previous reports (Thines et al., 2007), JAZ7 transcripts were undetectable in wounded leaves of jaz7-1. Likewise, JAZ8 and JAZ13 mRNAs also failed to accumulate in wounded leaves of the jaz8-1 and jaz13-1 mutants, respectively (Figure 3.8B). These results show that jaz7-1, jaz8-1 and jaz13-1 are null or severe loss of function alleles for the respective genes, and thus are suitable for use in physiological assays. Obvious morphological phenotypes related to growth and development were not observed in either of the three mutant lines. We also tested the effect of jaz7-1, jaz8-1 and jaz13- ""#! ! ! 1 on JA-induced root growth inhibition in MS medium containing 20 uM MeJA. The sensitivity of all three mutants was not significantly different from that of wild-type seedlings (Figure 3.9). Interestingly, JAZ7 expression levels were higher in jaz8-1 and jaz13-1 (Figure 3.8B). JAZ8 expression is also slightly higher in jaz7-1 and jaz13-1. This supports the hypothesis of negative feedback of JAZ7/8 expression by JAZ7/8/13 proteins (Chung et al., 2008). DISCUSSION JAZ proteins are key components in the JA signaling pathway regulating multiple biological processes balancing growth and defense. There are 12 JAZ members in Arabidopsis, and the current model suggests two independent models for JAZ proteins to repress JA-induced gene expression: a NINJA-dependent pathway in which JAZ proteins recruit corepressor TPL through an adaptor protein NINJA; and a NINJA-independent pathway in which JAZ proteins directly repress transcriptional responses through a TPL-interacting EAR motif, in the absence of NINJA (Pauwels et al., 2010; Shyu et al., 2012). Here, we report the identification of a thirteenth member in the JAZ family, and show that it represses JA responses and recruits TPL through a NINJA-independent pathway. JAZ13, A New Member in the Arabidopsis JAZ Family Key features of JAZ proteins include their function to repress JA-induced gene expression, their conserved ZIM domain and Jas motif, and their ability to interact with transcription factors (TFs) involved in JA signaling. JAZ13 shares all of these features (further ""#! ! ! discussed below), thus we designate it as a novel JAZ family member. Another important feature is that JAZ proteins are targeted for ubiquitination by COI1, and degraded through the 26S proteasome pathway. This was shown not to be the case in some JAZ proteins, in which JAZ8 and some JAZ alternative splice variants lack the canonical degron motif that mediates JAZCOI1 interaction. These JAZ variants are more stable upon JA treatment, and overexpression of these noncanonical JAZ proteins lead to decreased sensitivity to JA (Yan et al., 2007; Chung and Howe, 2009; Chung et al., 2010; Shyu et al., 2012). Similar observations were made in 35SJAZ13 lines, in which 35S-JAZ13 seedlings were less sensitive to JA-induced root growth inhibition, compared to WT seedlings. These results demonstrate a role for JAZ13 as a negative regulator in JA signaling, that is presumably stable upon JA treatment. JAZ13 shares high sequence similarity with JAZ7 and JAZ8. JAZ13 contains an LxLxL EAR motif at the N-terminus, which represents a key feature of NINJA-independent JAZ repressors (Shyu et al., 2012). JAZ13 interacts with TPL but not NINJA, and JAZ13 interaction with TPL is dependent on its EAR motif. These results demonstrate that domain functions are similar between JAZ13 and JAZ8. Overexpression of JAZ13 leads to repression of JA responses, presumably due to direct recruitment of TPL. Whether or not JAZ13 repression requires its EAR motif remains to be examined. The ZIM domain and Jas domain sequences of JAZ13 are less conserved among other JAZ family members. This presumably explains why JAZ13 was not identified previously. JAZ proteins share a very conserved TIFY motif at the N-terminus of ZIM domains, which is also a key feature in the TIFY family of proteins (Vanholme et al., 2007). The ZIM domain, specifically the TIFY motif within the ZIM domain, is important for JAZ homo- and heterodimerization (Chini et al., 2009; Chung and Howe, 2009). Site-directed mutagenesis of the I ""#! ! ! residue within the TIFY motif of JAZ10 resulted in abolishment of JAZ10 homo- and heterodimerization with other JAZ proteins (Chung and Howe, 2009). JAZ13 contains a NAFY motif instead of a TIFY motif (Figure 3.1). Blast search of JAZ13 ZIM-like sequences reveals that this NAFY-containing ZIM domain is not found in other proteins in Arabidopsis. Yeast twohybrid analysis shows that JAZ13 does not interact with other JAZ proteins, presumably due to the diversified NAFY motif. In addition to its role in JAZ dimerization, the ZIM domain in most JAZ proteins plays an important role to mediate JAZ-NINJA interaction (Pauwels et al., 2010). The only two JAZ proteins that do not interact with NINJA are JAZ7 and JAZ8 (Pauwels et al., 2010). JAZ13 also does not interact with NINJA, supporting the hypothesis of JAZ13 repressing through a NINJA-independent pathway. The Jas motif of canonical JAZ proteins is composed of three conserved sub-regions: a N-terminal LPIAR(R/K) motif, a central (SLX2FX2KRX2R) and a C-terminal (X5PY) region. The first two sub-regions compose the Jas degron, which mediates JAZ-COI1 interaction (Sheard et al., 2010). Interestingly, the LPIAR(R/K) is highly diversified among JAZ proteins, in which stable JAZs such as JAZ8 contain a X3S(M/V)K motif instead (Shyu et al., 2012). JAZ13 shares a similar degron motif as JAZ8, with X3S(M/V)K and SLX2FX2KRX2R regions. This suggests that JAZ13 may be a weak COI1-interactor in the presence of JA-Ile, thus stable upon JA treatment. Indeed, overexpression of JAZ13 leads to decreased sensitivity, similar to what has been observed for overexpression of stable JAZ variants such as JAZ8 and JAZ splice variants (Chung and Howe, 2009; Chung et al., 2010; Shyu et al., 2012). The C-terminal (X5PY) region is the least conserved domain in JAZ13, when comparing with other JAZ proteins. Instead of the highly conserved X5PY motif, JAZ13 contains a long stretch of Serine (S) and Threonine (T) ""#! ! ! residues at the C-terminus of its Jas motif. Phosphorylation of serine and threonine residues is an important posttranslational modification pathway that regulates many aspects in plant signaling (Nakagami et al., 2012). Whether phosphorylation plays a role in regulation of JAZ13 function is an exciting area to explore. The NINJA-Independent JAZ8 Subclade Identification of JAZ13 expands the repertoire of JAZ proteins and strengthens the importance of NINJA-independent JAZ repressors. NINJA-independent JAZ repressors have important features that are distinct from other JAZ proteins: an N-terminus EAR motif that is required for TPL-interaction and JAZ repression, and a diversified ZIM domain that does not mediate JAZ-NINJA interaction. They also contain a diversified degron motif that causes low affinity of JAZ-COI1 interaction in the presence of JA-Ile (Shyu et al., 2012). Currently three JAZ members belong to the NINJA-independent subclade in Arabidopsis: JAZ7, JAZ8 and the newly identified JAZ13. Blast search of JAZ8-like sequences reveals that NINJA-independent JAZ proteins are also present in other species (Shyu et al., 2012). All of these JAZ8-like sequences contain an EAR motif at the N-terminus and a diversified degron motif with conserved X3S(M/V)K sequences. Although the EAR motif of NINJA-independent JAZ repressors was shown to be required for both TPL-interaction and JAZ repression, we would like to point out that our results do not exclude the possibility of NINJA-independent JAZ proteins repressing though other EAR motif-interacting corepressors, such as mSin3-associated polypeptide 18 (SAP18) (Song and Galbraith, 2006; Kagale and Rozwadowski, 2011). Tandem affinity purification (TAP) assays followed by mass spectrometry (MS) would be helpful to ""#! ! ! determine JAZ8- or JAZ13-interacting proteins in vivo to determine the composition of NINJAindependent repression complexes. Although much has been reported on the molecular mechanism of JAZ action, little is known about the physiological role of JAZ proteins. Currently knockout or knockdown lines of JAZ1 and JAZ10 have been reported to be hypersensitive to JA treatment in growth inhibition assays (Yan et al., 2007; Grunewald et al., 2009; Demianski et al., 2012). jaz10-1, a null allele for JAZ10, has also been reported to have enhanced susceptibility to Pseudomonas syringae DC3000 (Demianski et al., 2012). All of the knockout or knockdown lines characterized so far are all JAZ proteins that undergo NINJA-dependent pathways to repress jasmonate responses. It would be interesting to determine whether NINJA-dependent and NINJA-independent repression mechanisms result in different signaling outputs. Here we report identification of knockout lines of all members in the NINJA-independent clade of JAZ proteins. Root growth inhibition assays do not indicate any difference in wild type and jaz7-1, jaz8-1 and jaz13-1 lines possibly due to redundancy. Other jasmonate-related bioassays would help gain more insight in unveiling the physiological role of JAZ7, JAZ8 and JAZ13. Furthermore, generation of high-order mutants within subclades of JAZ family members will strongly benefit understanding of physiological roles of different JAZ proteins. Role of JAZ-TF Interaction in Regulating JA Responses JAZ proteins interact with many transcription factors that are involved in regulating JA responses, but sequence determinants that mediate JAZ-TF interaction were not known. Furthermore, whether or not JAZ-TF interaction is required for JAZ repression has not been ""#! ! ! reported previously. Our results demonstrate that the R residue within the SLX2FX2KRX2R region of the Jas motif is critical for JAZ8-MYC2 interaction, and also JAZ8 repression. Although the interaction pattern is different, all JAZ proteins interact with JA-related transcription factors, such as MYC2 (reviewed in Pauwels and Goossens, 2011). It has been shown in the case of multiple JAZ proteins (JAZ3, JAZ8 and JAZ11) that JAZ-TF interaction is mediated through the Jas domain (Chini et al., 2009; Qi et al., 2011; Song et al., 2011; Shyu et al., 2012). Furthermore, the SLX2FX2KRX2R region, especially the KRK submotif, is extremely conserved among the JAZ family members. We suggest that the importance of the R residue within the KRK submotif to mediate JAZ-MYC2 interaction can be widely applied to other JAZ members. Interestingly, mutation of the R117 residue in JAZ8 not only led to abolishment of JAZ8-MYC2 interaction, but also mislocalization of JAZ8. Although most of JAZ8 was detected in the nucleus, JAZ8 R117A of JAZ8 R117A R117A -YFP -YFP was also detected in the cytosol. Overexpression results in restoration of JA sensitivity. Whether this is due to loss-of-interaction with MYC2, or mislocalization of JAZ8 remains to be examined. MATERIALS AND METHODS Plant Materials Arabidopsis thaliana ecotype Colombia-0 (Col-0) was used as wild type for all experiments. All mutants and transgenic lines reported in this article are in the Col-0 background. T-DNA "#$! ! ! insertion lines WiscDsLox255G12 (jaz8-1) and SALK_149961 (jaz13-1) were obtained from the Arabidopsis Biological Resource Center (ABRC) (Alonso et al., 2003). jaz7-1 (WiscDsLox7H11) was previously described (Thines et al., 2007). Molecular Cloning and Generation of Constructs JAZ13 was cloned from a PCR reaction with JAZ13 sequence-specific primers (JAZ13_pENTR_FP and JAZ13_XhoI_RP) using wild type wounded mature leaf RNA as template. PCR reactions were performed using Pfu Turbo DNA polymerase (Stratagene). JAZ13 was cloned and sequenced in the pENTR-TOPO vector (Invitrogen) and further cloned into pGILDA and pB42AD vectors for further yeast two-hybrid assays using the Gateway system (Invitrogen). JAZ8-related constructs were cloned into pGEMT-Easy (Promega) for sequencing and subcloning into pGILDA and pB42AD. A complete list of constructs and primers used in this article is provided in Supplemental Table 1. Site-Directed Mutagenesis All site-directed mutagenesis assays were performed using Pfu Turbo DNA polymerase (Stratagene) as previously described (Chung and Howe, 2009). PCR reactions were performed using templates in pGILDA vectors. PCR-amplified products were treated with the restriction enzyme DpnI to remove the parental plasmid, and transformed into E. coli strain DH5! for further amplification. Presence of the desired mutation was verified by DNA sequencing. "#"! ! ! Protein-Protein Interaction assays Yeast two-hybrid assays were performed with the Matchmaker LexA system (Clontech). Full length cDNA of JAZ10 and JAZ8-related sequences were cloned into the pGILDA bait vector to generate fusion proteins with the LexA DNA binding domain. MYC2, other JAZs, NINJA and TPL full length cDNA were cloned into the pB42AD prey vector to generate fusion proteins with the B42 activation domain (AD). Bait and prey vectors were cotransformed into yeast strain EGY48 using the Frozen-EZ yeast Transformation II Kit (Zymo Research). Transformants were selected on SD-glucose plates containing amino acid supplements lacking uracil (U), tryptophan (T) and histidine (H) after 48 hours of incubation at 30°C. Protein-protein interaction was detected by adding X-gal and selection for blue colors (!-galactosidase) on SD-galactose plates with proper amino acid supplements. Photographic images were taken after 48 hours of incubation at 30°C. Generation of Transgenic Lines All constructs for generating transgenic lines were made in the pBI-AS vector. The pBI-AS vector is a modified pBI121 vector, as described previously (Schilmiller et al., 2007). Sequenced overexpression constructs were transformed into Agrobacterium tumefaciens strain C58C1, and further transformed into wild type plants using the floral dip method (Clough and Bent, 1998). T1 seedlings were screened on Murashige and Skoog (MS) agar medium containing sucrose (0.8%) and kanamycin (50 µM/mL) and vancomycin (100 µM/mL). For each construct, 32 kanamycin-resistant lines were selected for RNA gel-blot analysis to screen for lines expressing high levels of the transgene. RNA extracted from untreated leaf tissue was hybridized with the "##! ! ! transgene cDNA in RNA gel-blot assays as previously described (Shyu et al., 2012). Highexpression lines were further used for root growth inhibition assays and propagated for identification of homozygous T3 lines. YFP-tagged lines were identified by fluorescence microscopy at the T1 seedling stage (Axio Scope; Carl Zeiss). Root Growth Inhibition Assays Seedlings were plated on MS agar medium containing sucrose (0.8%) with or without 20 µM MeJA for 8 days under continuous light unless otherwise noted. Primary root lengths were measured using Image J (http://rsbweb.nih.gov/ij/indext.html). Root growth ratios indicate primary root lengths of seedlings grown on MeJA-containing plates, divided by the average of primary root lengths of seedlings of the same genotype grown on MS only plates. RNA Gel-Blot Analysis Primers used to amplify cDNA probes are described in Supplemental Table 1. PCR reactions were performed using Pfu Turbo DNA polymerase (Stratagene) and amplified products were cloned and sequenced in pGEMT-Easy (Promega). RNA extraction was performed using RNeast Plant Mini Kit (Qiagen) or the phenol/chloroform extraction method as described in Li et al., 2002. Gel-blot analyses were performed as described previously (Li et al., 2002). "#$! ! ! Multiple Sequence Alignment and Phylogenetic Analysis All sequences were obtained from The Arabidopsis Information Resource (TAIR) (http://www.arabidopsis.org). Multiple sequence alignments were performed using the MUSCLE method (Edgar, 2004) in software BioEdit (http://www.mbio.ncsu.edu/bioedit/bioedit.html ). Phylogenetic trees were generated with the Neighbor-joining Method (Saitou and Nei, 1987) (Tamura et al., 2011). Accession Numbers Arabidopsis Genome Initiative numbers described in this article are listed below: ACT8 (At1g49240), COI1 (At2g39940), JAZ1 (At1g19180), JAZ2 (At1g74950), JAZ3 (At3g17880), JAZ4 (At1g48500), JAZ5 (At1g17380), JAZ6 (At1g72450), JAZ7 (At2g34600), JAZ8 (At1g30135), JAZ9 (At1g70700), JAZ10 (At5g13220.1), JAZ11 (At3g43440), JAZ12 (At5g20900), JAZ13 (At3g22275), MYC2 (At1g32640), NINJA (At4g28910) and TPL (At1g15750). ACKNOWLEDGEMENTS We would like to thank Li Deng (Michigan State University) for technical assistance. We thank John Browse (Washington State University) for providing the jaz7-1 mutant. This research was supported by the National Institute of Health Grant (Grant R01 GM57795) and the Chemical Sciences, Geosciences, and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy (Grant DE-FG02-91ER20021). "#$! ! ! FIGURES AND TABLES Figure 3.1. Amino acid sequence alignment of full length JAZ7, JAZ8 and JAZ13. Full length amino acid sequences of JAZ7, JAZ8 and JAZ13 were aligned by MUSCLE using the BioEdit software. Conserved EAR, ZIM and Jas motifs are underlined. "#$! ! ! Figure 3.2. Phylogenetic tree of the JAZ family of proteins in Arabidopsis. Full-length amino acid sequences were used to generate phylogenetic tree by the NeighborJoining method. Evolutionary analyses were conducted in MEGA5. "#$! ! ! Figure 3.3. Induction of JAZ7, JAZ8 and JAZ13 in response to wounding, MeJA and coronatine treatment. (A) RNA gel blot analysis of RNA extracted from 4-week-old mature leaf tissue of WT plants untreated or treated with mechanical wounding at the indicated time after wounding (hours). (B) RNA gel-blot analysis of 9-day-old WT seedling RNA grown in liquid MS medium and treated with the indicated concentration of MeJA, coronatine or a mock control. An ACTIN8 (ACT8) probe was used as a control. "#$! ! ! Figure 3.4. Overexpression of JAZ13 leads to decreased sensitivity to JA-induced root growth inhibition.! ! !"#$ Figure 3.4. Overexpression of JAZ13 leads to decreased sensitivity to JA-induced root growth inhibition. –Continued. T2 seedlings were grown under continuous light on MS plates containing ± 20 !M MeJA for 8 days. The root length ratio was calculated by dividing the average root length of seedlings grown on MeJA-containing medium by the average root length of seedlings of the same genotype grown in the absence of MeJA. Data points show the mean ± SE (n = 17-22 per data point). Asterisks indicate significant differences (p value < 0.05, Student’s t-test) in comparisons between the indicated transgenic line and WT. "#$! ! ! Figure 3.5. The EAR motif of JAZ13 is required for interaction with TPL. Yeast two-hybrid analysis of JAZ13 deletion proteins with TPL, NINJA and MYC2. Yeast strains expressing both the bait (JAZ13 or JAZ13 deletions) and prey (TPL, NINJA or MYC2) were tested on media containing X-Gal. LacZ-mediated blue-color formation is indicative of protein-protein interaction. Images were taken 48 hours after incubation at 30 °C. DB indicates empty bait vector. AD indicates empty prey vector. "#$! ! ! MYC2 TPL AD JAZ7 ZIM JAZ7!Jas ZIM JAZ8 ZIM JAZ8!Jas Jas ZIM Jas Jas JAZ8-Jas JAZ13 ZIM JAZ13!Jas Jas ZIM DB Figure 3.6. The Jas motif is required for interaction of JAZ7, JAZ8 and JAZ13 with MYC2. Yeast two-hybrid analysis of yeast strains coexpressed with JAZ related sequences fused with DB and MYC2 or TPL fused with AD. Experiments were performed as described in Material & Methods and Figure 3.5. "#"! ! ! Figure 3.7. The Jas domain is required for JAZ-MYC2 interaction and JAZ repression of JA responses. "#$! ! ! Figure 3.7. The Jas domain is required for JAZ-MYC2 interaction and JAZ repression of JA responses. –Continued. (A) The R117 residue is critical for JAZ8-MYC2 interaction. (B) Transgenic lines R117A overexpressing JAZ8 are sensitive to JA-induced root growth inhibition. Seedlings were grown on ± 20 !M MeJA containing MS plates for 9 days under continuous light. The root length ratio was calculated by dividing the average root length of seedlings grown on MeJAcontaining medium by the average root length of seedlings of the same genotype grown in the absence of MeJA. Data points show the mean ± SE (n = 7-20 per data point). Asterisks indicate significant differences (p value < 0.05, Student’s t-test) in comparisons between the indicated transgenic line and WT. "##! ! ! A jaz7-1 WiscDsLox7H11 JAZ7 jaz8-1 WiscDsLox255G12 JAZ8 jaz13-1 SALK_149961 JAZ13 100 bp B jaz7-1 WT - + - jaz8-1 + - + jaz13-1 - + JAZ7 JAZ8 JAZ13 ACT8 Figure 3.8. Characterization of knockout lines of JAZ7, JAZ8 and JAZ13. (A) T-DNA insertion lines of the JAZ8 subclade of JAZ proteins. Black boxes indicate exons, gray boxes indicate 5’ or 3’ UTR. Black lines indicate introns or untranslated genomic sequences. Black diamond indicates a G-box cis-regulatory element. Scale bar = 100 bp. (B) RNA gel blot analysis of jaz7-1, jaz8-1 and JAZ13-1 upon wounding. Fully expanded leaves of 4-week-old mature plants were used for RNA extraction and RNA gel blot analysis. Samples were collected prior to wounding (-) or 1 hour after wounding (+). Probes containing full-length cDNA of JAZ7, JAZ8 and JAZ13 were used to detect target gene expression. ACTIN8 (ACT8) was used as a loading control. "#$! ! ! Figure 3.9. Root growth inhibition assay of jaz7-1, jaz8-1 and jaz13-1 lines. (A) Primary root length of jaz7-1, jaz8-1 and jaz13-1 lines grown on ± 20 !M MeJA containing MS plates. WT and 35S-JAZ8 were used as controls. Seedlings were grown on ± 20 !M MeJA containing MS plates for 8 days under continuous light. Data points show the mean ± SE (n = 17-20 per data point). (B) Root length ratio of the identical experiment described in (A). The root length ratio was calculated by dividing the average root length of seedlings grown on MeJAcontaining medium by the average root length of seedlings of the same genotype grown in the absence of MeJA. Asterisks indicate significant differences (p value < 0.05, Student’s t-test) in comparisons between the indicated transgenic line and WT. "#$! ! ! Supplemental Figure 3.1. Identification of 35S-JAZ13 transgenic lines overexpressing JAZ13. RNA gel blot analysis of RNA extracted from T1 35S-JAZ13 mature leaf tissues. Each number represents independent lines resistant to kanamycin. WT plants grown side by side to the transgenic lines were used as a control. No wound or JA-related treatment was done before harvesting samples. "#$! ! ! Supplemental Figure 3.2. Identification of 35S-JAZ8 JAZ8 R117A R117A transgenic lines overexpressing . R117A RNA gel blot analysis of RNA extracted from T2 35S-JAZ8 2-week-old seedlings. Each number represents independent lines resistant to kanamycin. WT plants grown side by side to the transgenic lines were used as a control. No wound or JA-related treatment was done before harvesting samples. Lines #1, #4, #14, #15, #19, #20 and #23 were selected for further analyses. "#$! ! ! Supplemental Figure 3.3. Characterization of 35S-JAZ8 R117A –YFP transgenic lines. Transgenic lines overexpressing JAZ8R117A – YFP are sensitive to JA-induced root growth inhibition. Primary root length of 35S-JAZ8R117A-YFP lines grown on 20 !M MeJA containing MS plates. 35S-YFP and 35S-JAZ8-YFP were used as controls. Seedlings were grown on plates for 10 days under continuous light. N = 15-28. b was significantly different than a (p value < 0.0001). c was significantly different than b (p = 0.02). "#$! ! ! Supplemental Figure 3.4. JAZ8 R117A –YFP localization in N. tabacum. R117A JAZ8 –YFP transiently expressed in N. tabacum leaves. YFP and JAZ8-YFP was used as controls. Confocal images were taken 48 hours after infiltration. DAPI (4',6-diamidino-2phenylindole) was applied 2 hours before imaging to locate the nucleus. "#$! ! ! Supplemental Table 3.1. List of oligonucleotide primers used in this study.!! INSERT JAZ14 JAZ14 JAZ14!EAR JAZ14 ADAHA JAZ7!Jas JAZ7-Jas JAZ14!Jas JAZ8!C122 JAZ8!C120 JAZ8!C118 JAZ8!C115 ! PRIMER NAME PRIMER SEQUENCE JAZ14_pENTR_FP JAZ14_KpnI_RP JAZ14_XhoI_FP JAZ14_KpnI_RP JAZ14dEAR_pENTR_FP JAZ14_KpnI_RP 5'-CACCATGAAGGGTTGCAGCTTAG-3' 5'-GGTACCTTAGAAATTATGAAGAGAGGAGG-3' 5'-CTCGAGATGAAGGGTTGCAGCTTAG-3' 5'-GGTACCTTAGAAATTATGAAGAGAGGAGG-3' 5'-CACCATGAAGGGTTGCAGCTCTCCAATGGCCTCTACG-3' 5'-GGTACCTTAGAAATTATGAAGAGAGGAGG-3' 5'CACCATGAAGGGTTGCAGCGCAGATGCTCACGCATCTCCAATGGCCTCT JAZ14_ADAHA_FP ACGC-3' 5'GCGTAGAGGCCATTGGAGATGCGTGAGCATCTGCGCTGCAACCCTTCAT JAZ14_ADAHA_RP GGTG-3' JAZ7_NcoI_FP 5'-CCATGGATGATCATCATCATCAAAAAC-3' JAZ7dJas_XhoI_RP 5'-CTCGAGCTATTGATAATGAAATCGAGTCG-3' JAZ7Jas_NcoI_FP 5'-CCATGGATGTCGACTCGATTTCATTATC-3' JAZ7_XhoI_RP 5'-CGAGCTATCGGTAACGGTGGTAAG-3' JAZ14_pENTR_FP 5'-CACCATGAAGGGTTGCAGCTTAG-3' JAZ14ns_XhoI_249R_stop 5'-CTCGAGTTATAACGGTGATTCCAGTCTCACCG-3' JAZ8_NcoI_FP 5'-CCATGGATGAAGCTACAGCAAAATTGTG-3' JAZ8dC122_s_XhoI_RP 5'-CTCGAGTTAAATTCGAATTTTCCGTTTCTG-3' JAZ8_NcoI_FP 5'-CCATGGATGAAGCTACAGCAAAATTGTG-3' JAZ8dC120_s_XhoI_RP 5'-CTCGAGTTAAATTTTCCGTTTCTCAAG-3' JAZ8_NcoI_FP 5'-CCATGGATGAAGCTACAGCAAAATTGTG-3' JAZ8dC118_s_XhoI_RP 5'-CTCGAGTTACCGTTTCTGAAGAAAAC-3' JAZ8_NcoI_FP 5'-CCATGGATGAAGCTACAGCAAAATTGTG-3' !"#$ Supplemental Table 3.1. – Continued.! JAZ8 R117A JAZ8 R117A JAZ8 R117A JAZ8dC115_s_XhoI_RP JAZ8_R117A_FP JAZ8_R117A_RP JAZ8_XhoI_FP JAZ8_KpnI_RP JAZ8_XhoI_FP JAZ8ns_KpnI_RP 5'-CTCGAGTTAAAGAAAACTTTGGAG-3' 5'-CCAAAGTTTTCTTCAGAAAGCGAAAATTCGAATTCAAGC-3' 5'-GCTTGAATTCGAATTTTCGCTTTCTGAAGAAAACTTTGG-3' 5'-CTCGAGATGAAGCTACAGCAAAATTGTG-3' 5'-ATATGGTACCTTATCGTCGTGAATGGTACG-3' 5'-CTCGAGATGAAGCTACAGCAAAATTGTG-3' 5'-ATATGGTACCTCGTCGTGAATGGTACG-3' ! ! ! !"!# ! REFERENCES ! !"#$ ! ! REFERENCES Aldridge DC, Galt S, Giles D, Turner WB (1971) Metabolites of Lasiodiplodia theobromae. J Chem Soc, C 1623–1627 Bai Y, Meng Y, Huang D, Qi Y (2011) Origin and evolutionary analysis of the plant-specific TIFY transcription factor family. Genomics 98: 128–136 Ballaré CL (2011) Jasmonate-induced defenses: a tale of intelligence, collaborators and rascals. Trends Plant Sci 16: 249–257 Browse J (2009) Jasmonate passes muster: a receptor and targets for the defense hormone. 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CHAPTER FOUR SUMMARY AND FUTURE PERSPECTIVES ! !"#$ SUMMARY OF DISSERTATION Identification of the JASMONATE ZIM DOMAIN (JAZ) family of proteins has spurred remarkable progress towards understanding of jasmonate (JA) signaling. At the time this dissertation research was initiated, little was known about the molecular mechanism of JAZ function. CORONATINE INSENSITIVE 1 (COI1)-JAZ interaction was shown to require JA-Ile as a molecular ligand, but sequence determinants of COI1-JAZ interaction were not identified. Moreover, 12 JAZ proteins were hypothesized to have similar mechanisms in repressing JA responses. In this dissertation research, I characterize JAZ8 as a weak interactor of COI1, and I determine the sequence motifs that are responsible for COI1-JAZ interaction and JAZ stability in vivo (Shyu et al., 2012). I report that JAZ8 represses JA responses through a NOVEL INTERACTOR OF JAZ (NINJA)-independent pathway, which is unique from the known NINJA-dependent repression mechanism (Shyu et al., 2012). I also report the identification of a previously unidentified JAZ protein, JAZ13. JAZ13 shares high sequence similarity with JAZ8, and represses JA responses through the NINJA-independent repression pathway. My findings suggest that JAZ proteins regulate JA-induced gene expression through diverse mechanisms to generate different signaling outputs. I demonstrate that JAZ8 is a stable JAZ repressor in Chapter Two of this dissertation (Shyu et al., 2012). In vitro pull down assays show that different JAZ proteins have different interaction affinities to COI1 in the presence of JA-Ile. JAZ8 does not interact with COI1 in the presence of JA-Ile. JAZ8-YFP was stable upon MeJA treatment compared to JAZ10.1-YFP. Moreover, overexpression of JAZ8 leads to decreased sensitivity to JA, indicating that JAZ8 is a stable repressor of JA responses. I further show that JAZ8 stability is due to it’s diversified ! !"#$ degron motif. Domain swap of the N-terminal Jas degron of JAZ10.1 into JAZ8 results in promotion of JAZ8LPIAR-COI1 interaction and rapid degradation of JAZ8LPIAR-YFP in the presence of MeJA. These results collectively demonstrate that JAZ proteins have different stabilities in response to JA, due to their diversified degron. I report that JAZ8 represses JA-induced gene expression through a previous uncharacterized NINJA-independent pathway. Experiments demonstrating this finding are recorded in Chapter Two. JAZ8 contains a EAR motif at the N-terminus, in which the EAR motif is required for JAZ8’s repressive activity (Kagale et al., 2010; Shyu et al., 2012). JAZ8 interacts with corepressor TOPLESS in the absence of NINJA (Arabidopsis Interactome Mapping Consortium, 2011; Shyu et al., 2012). Furthermore, the ZIM domain, which is important for JAZ-NINJA interaction in other JAZ members, is not required for JAZ8 to repress JA responses. These findings illustrate that JAZ8 recruits TOPLESS and represses JA responses through a NINJA-independent pathway, different from the previous demonstrated NINJAdependent repression mechanism. This is significant because it indicates that JAZ proteins repress JA-induced gene expression through alternative mechanisms. Finally, I identify and characterize a previously undiscovered JAZ member, JAZ13. Research related to JAZ13 is recorded in Chapter Three of this dissertation. JAZ13 was identified from a BLAST search using JAZ8 amino acid sequences as query. JAZ13 has high sequence similarities with JAZ8, and shares a conserved EAR motif at the N-terminus. JAZ13 also has sequence motifs similar to the ZIM domain and Jas motif of JAZ8. Overexpression of JAZ13 leads to decreased sensitivity to JA, indicating JAZ13 has a negative role in regulating JA responses. Interestingly, JAZ13 interacts with TOPLESS in a NINJA-independent manner, suggesting that JAZ13 represses JA responses through a NINJA-independent pathway. I also ! !"#$ report that the JAZ8 subclade of JAZ repressors requires interaction with MYC2 to repress JA responses. I identified sequence determinants for JAZ8-MYC2 interaction, and show that mutations in the JAZ8-MYC2 interaction motif abolish JAZ8 repression. Findings in this chapter strengthen the importance of NINJA-independent repression mechanisms, and expand the repertoire of JAZ proteins. To summarize, research in this dissertation demonstrates an alternative mechanism for JAZ repression. I show that JAZ proteins differ in stability and repression mechanisms to regulate JA responses. JAZ8 is a stabilized JAZ protein, and JAZ8 represses JA responses through a NINJA-independent pathway. Identification and characterization of JAZ13 reveals that the JAZ8 subclade of JAZ proteins represses JA responses in a NINJA-independent manner. Taken together, I report the identification of a novel repression mechanism, presumably for the purpose of fine-tuning myriad JA responses to balance growth and defense. FUTURE PERSPECTIVES In the past five years, many exciting discoveries have drastically advanced our understanding towards JA biology. JAZ proteins were identified as substrates of COI1 (Chini et al., 2007; Thines et al., 2007; Yan et al., 2007). (3R, 7S) JA-Ile was identified as the bioactive form of JA (Fonseca et al., 2009). Crystal structure of the COI1-JAZ JA-Ile receptor complex was resolved (Sheard et al., 2010). Domain analyses of JAZs were done to understand the molecular mechanism of JAZ function, and NINJA and TOPLESS were identified as important components in the JAZ repression complex (Melotto et al., 2008; Chini et al., 2009; Chung and ! !"#$ Howe, 2009; Pauwels et al., 2010; Shyu et al., 2012). Furthermore, many transcription factors were identified as JAZ-interactors, connecting JA perception with different transcriptional signaling outputs (Hou et al., 2010; Cheng et al., 2011; Fernandez-Calvo et al., 2011; Qi et al., 2011; Song et al., 2011; Zhu et al., 2011; Yang et al., 2012). These findings collectively place JAZ proteins as core internodes that link JA-Ile perception to diverse JA signaling outputs. However, as all exciting discoveries do, advancement in JAZ research raises many questions that must now be answered. It is not known how stable JAZ repressors are turned over. In Chapter Two of the dissertation I demonstrated that not all JAZ proteins interact with COI1 in a JA-Ile-dependent manner, and JAZ8 is a weak COI1-interactor (Shyu et al., 2012). JAZ8-YFP is stable upon MeJA treatment. In addition to JAZ8, alternative splice variants of JAZ10 (JAZ10.3 and JAZ10.4) are stable upon MeJA treatment compared to JAZ10.1 (Chung and Howe, 2009). Other JAZ proteins such as JAZ2 and JAZ6 also produce alternative splice variants that interact weakly with COI1 (Chung et al., 2010). Although JA-Ile is characterized as the bioactive ligand that can promote JAZ-COI1 interaction, it is interesting to point out that JA-amino acid conjugates that are structurally similar to JA-Ile also promote JAZ-COI1 interaction in in vitro pull down experiments (Katsir et al., 2008). A variety of JA derivatives are present endogenously, and many of which are wound-inducible (Koo et al., 2009). It would be interesting to examine whether or not other JA derivatives can promote JAZ-COI1 interaction and JAZ degradation. JAZ8 has a diversified degron motif that prevents JAZ8-COI1 interaction in the presence of JAIle (Shyu et al., 2012). Therefore it is reasonable to propose that JAZ8 may form a binding pocket that does not recognize JA-Ile, but interacts with other ligands. Indeed, JAZ8 interacts with COI1 in the presence of high concentrations of coronatine (Shyu et al., 2012). Whether or ! !"!# not endogenous ligands other than JA-Ile can promote JAZ8 degradation is not known. Some alternative splice variants, on the other hand, lack the entire Jas degron. How these stable splice variants are turned over still remains to be elucidated. In the case of Aux/IAA repressors in auxin signaling, it is known that different Aux/IAA proteins have different half-lives (Dreher et al., 2006). Examining the half-life of JAZ proteins may also provide alternative insights to JAZ stability. Taken together, whether or not other endogenous ligands are able to promote JAZ degradation, and examining the half-life of JAZ proteins are expected to advance our knowledge in JAZ signaling. Composition of the JAZ repression complex still remains to be elucidated. The current JA signaling model suggests that the JAZ repression complex involves MYC2, NINJA and TPL (Pauwels et al., 2010). However, this model is mainly based on protein-protein interaction assays that restricts to examining one protein-protein pair at a time. Very little is known about the endogenous JAZ repression complex. Currently two repression models are proposed for JAZ proteins: a NINJA-dependent pathway that JAZ proteins recruit TPL via NINJA, and a NINJAindependent pathway in which JAZ repressors directly interact with TPL through a EAR motif (Pauwels et al., 2010; Shyu et al., 2012). Interestingly, JAZ5 and JAZ6 interact with NINJA, but also contain TPL-interacting EAR motifs (Kagale et al., 2010; Pauwels et al., 2010). Whether or not JAZ5/6 repression requires NINJA or TPL is not known. Understanding the composition of JAZ repression complexes involving JAZ5 and JAZ6 will advance understanding towards the JAZ repression machinery. On the other hand, JAZ proteins form homo- and heterodimers (Chini et al., 2009; Chung and Howe, 2009). Transcription factors and TPL also homodimerize (Szemenyei et al., 2008; Fernandez-Calvo et al., 2011). The role of dimerization and how dimerization contributes to JAZ repression is not known. Geerinck et al. (2010) isolated the ! !"#$ JAZ1 complex by tandem affinity purification (TAP) of JAZ1-TAP overexpressing Arabidopsis cell lines and showed that the JAZ1-TAP complex assembles into protein complexes that are up to 1 MDa (Geerinck et al., 2010). Mass spectrometry analyses of these protein complexes are expected to provide more insight to composition of the JAZ repression complex. How JAZ proteins control the specificity of JA signaling outputs is largely unknown. Although overexpression of stable JAZ proteins result in decreased sensitivity to JA, 35SJAZ10.4 lines are male sterile, whereas 35S-JAZ8 are fertile (Chung and Howe, 2009; Shyu et al., 2012). This suggests that different JAZ proteins may repress gene expression of different target genes. One hypothesis is that JAZ repressors are expressed in various tissues at different developmental stages to regulate a myriad of responses. Little is known about expression patterns of Arabidopsis JAZ repressors. Most JAZs in Arabidopsis have a very low basal expression level, and are rapidly induced in mature leaf tissues or whole seedlings upon wounding or JA treatment (Chung et al., 2008). Microarray analyses of JAZ genes in Nicotiana attenuata showed that different JAZ genes have different expression patterns (Oh et al., 2012). Interestingly, NaJAZg, which is an ortholog of Arabidopsis JAZ3 and JAZ4, is highly expressed in root tissues, but not leaf tissues, whereas other NaJAZ genes are present in leaf tissues. These findings support the hypothesis in which regulation of JA signaling outputs is due to the presence of different JAZ proteins at different tissues and/or developmental stages. Another hypothesis is that JAZ proteins interact with different transcription factors (TFs) to regulate different signaling outputs. Multiple transcription factors interact with JAZ proteins. Interestingly not all JAZ proteins have similar interaction patterns with these transcription factors (Fernandez-Calvo et al., 2011; Qi et al., 2011; Song et al., 2011). Sequence determinants of specificity for JAZ-TF interaction are largely unknown. Examination of JAZ and TF expression patterns and chromatin ! !"#$ immunoprecipitation (ChIP) analyses of JAZ-associated DNA motifs will greatly accelerate understanding towards JAZ target specificity. Moreover, characterization of jaz mutants will also advance understanding towards the biological role of different JAZ proteins. Multiple stable JAZ isomers are characterized, but little is known about the physiological relevance of stable JAZ proteins. One proposed hypothesis is that stable JAZ proteins function to attenuate JA responses to prevent plants from over-responding to JA-Ile. Upon wounding or other biological cues that cause high levels of JA-Ile to accumulate, liable JAZ proteins are rapidly degraded, resulting in accumulation of stable JAZ proteins such as JAZ8. Stable JAZ proteins then interact with transcription factors to turn of JA responses. Another hypothesis is that stable JAZ proteins are only expressed in certain tissues to provide specificity in signaling outputs. Results supporting this hypothesis are that NaJAZ genes have differential expression patterns upon wounding (Oh et al., 2012). It is also possible that a combination of the two models exist simultaneously, to provide a complex regulation of JA signaling outputs. Live cell imaging of native promoter driven JAZ-reporter lines in combination with characterization of JAZ targets (as mentioned in the previous chapter) are expected to provide more understanding to the physiological relevance of stable JAZ proteins. Taken together, characterization of JAZ proteins has introduced us into a new era of understanding the molecular mechanism of JA signaling. Large-scale gene expression analysis and protein-protein interaction techniques played a significant role in identification of new members in the JA signaling pathway and revealing the molecular mechanism of JAZ function. Continued work that expands our knowledge in JA perception, composition of the JAZ repressor complex, and target specificity of different JAZ proteins, is required to answer the big question ! !"#$ of how plants utilize JA to balance growth and defense in response to myriad environmental conditions. ! !""# REFERENCES ! !"#$ REFERENCES Arabidopsis Interactome Mapping Consortium (2011) Evidence for network evolution in an Arabidopsis interactome map. Science 333: 601–607 Cheng Z, Sun L, Qi T, Zhang B, Peng W, Liu Y, Xie D (2011) The bHLH Transcription Factor MYC3 Interacts with the Jasmonate ZIM-Domain Proteins to Mediate Jasmonate Response in Arabidopsis. 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