JAZ Proteins Research Articles

Oryza sativa COI homologues restore jasmonate signal transduction in Arabidopsis coi1-1 mutants.

CORONATINE INSENSITIVE 1 (COI1) encodes an E3 ubiquitin ligase complex component that interacts with JAZ proteins and targets them for degradation in response to JA signaling. The Arabidopsis genome has a single copy of COI1, but the Oryza sativa genome has three closely related COI homologs. To examine the functions of the three OsCOIs, we used yeast two-hybrid assays to examine their interactions with JAZ proteins and found that OsCOIs interacted with OsJAZs and with JAZs, in a coronatine dependent manner. We also tested whether OsCOI1a and OsCOI1b could complement Arabidopsis coi1-1 mutants and found that overexpression of either gene in the coi1-1 mutant resulted in restoration of JA signal transduction and production of seeds, indicating successful complementation. Although OsCOI2 interacted with a few OsJAZs, we were not able to successfully complement the coi1-1 mutant with OsCOI2. Molecular modeling revealed that the three OsCOIs adopt 3D structures similar to COI1. Structural differences resulting from amino acid variations, especially among amino acid residues involved in the interaction with coronatine and JAZ proteins, were tested by mutation analysis. When His-391 in OsCOI2 was substituted with Tyr-391, OsCOI2 interacted with a wider range of JAZ proteins, including OsJAZ1, 2, 5∼9 and 11, and complemented coi1-1 mutants at a higher frequency than the other OsCOIs and COI1. These results indicate that the three OsCOIs are orthologues of COI1 and play key roles in JA signaling.

A MultiSite GatewayTM vector set for the functional analysis of genes in the model Saccharomyces cerevisiae

BackgroundRecombinatorial cloning using the GatewayTM technology has been the method of choice for high-throughput omics projects, resulting in the availability of entire ORFeomes in GatewayTM compatible vectors. The MultiSite GatewayTM system allows combining multiple genetic fragments such as promoter, ORF and epitope tag in one single reaction. To date, this technology has not been accessible in the yeast Saccharomyces cerevisiae, one of the most widely used experimental systems in molecular biology, due to the lack of appropriate destination vectors.ResultsHere, we present a set of three-fragment MultiSite GatewayTM destination vectors that have been developed for gene expression in S. cerevisiae and that allow the assembly of any promoter, open reading frame, epitope tag arrangement in combination with any of four auxotrophic markers and three distinct replication mechanisms. As an example of its applicability, we used yeast three-hybrid to provide evidence for the assembly of a ternary complex of plant proteins involved in jasmonate signalling and consisting of the JAZ, NINJA and TOPLESS proteins.ConclusionOur vectors make MultiSite GatewayTM cloning accessible in S. cerevisiae and implement a fast and versatile cloning method for the high-throughput functional analysis of (heterologous) proteins in one of the most widely used model organisms for molecular biology research.

Chemical and genetic exploration of jasmonate biosynthesis and signaling paths

Jasmonates are lipid-derived compounds that act as signals in plant stress responses and developmental processes. Enzymes participating in biosynthesis of jasmonic acid (JA) and components of JA signaling have been extensively characterized by biochemical and molecular-genetic tools. Mutants have helped to define the pathway for synthesis of jasmonoyl-L-isoleucine (JA-Ile), the bioactive form of JA, and to identify the F-box protein COI1 as central regulatory unit. Details on the molecular mechanism of JA signaling were recently unraveled by the discovery of JAZ proteins that together with the adaptor protein NINJA and the general co-repressor TOPLESS form a transcriptional repressor complex. The current model of JA perception and signaling implies the SCF(COI1) complex operating as E3 ubiquitin ligase that upon binding of JA-Ile targets JAZ proteins for degradation by the 26S proteasome pathway, thereby allowing MYC2 and other transcription factors to activate gene expression. Chemical strategies, as integral part of jasmonate research, have helped the establishment of structure-activity relationships and the discovery of (+)-7-iso-JA-L-Ile as the major bioactive form of the hormone. The transient nature of its accumulation highlights the need to understand catabolism and inactivation of JA-Ile and recent studies indicate that oxidation of JA-Ile by cytochrome P450 monooxygenase is the major mechanism for turning JA signaling off. Plants contain numerous JA metabolites, which may have pronounced and differential bioactivity. A major challenge in the field of plant lipid signaling is to identify the cognate receptors and modes of action of these bioactive jasmonates/oxylipins.

JAZ8 lacks a canonical degron and has an EAR motif that mediates transcriptional repression of jasmonate responses in Arabidopsis.

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 LxLxL-type 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.

The Arabidopsis JAZ2 Promoter Contains a G-Box and Thymidine-Rich Module that are Necessary and Sufficient for Jasmonate-Dependent Activation by MYC Transcription Factors and Repression by JAZ Proteins

Jasmonate (JA) is a critical hormone for both plant defense and reproductive development. Until now, early JA-responsive promoters have not been well characterized. To identify the cis-acting DNA element involved in the early JA response at the transcriptional level, we analyzed the promoter of the Arabidopsis gene encoding jasmonate-ZIM domain2 protein (JAZ2). The full-length JAZ2 promoter in JAZ2::GUS plants is active in vegetative and reproductive tissue, with expression in stamen filaments being dependent on JA biosynthesis. We identified a G-box element in the JAZ2 promoter that is required for JA-mediated promoter activation in carrot protoplasts and Arabidopsis seedlings. Three copies of a G-box and flanking sequences has autonomous JA-dependent activity and was transactivated by MYC2, MYC3 and MYC4 transcription factors in carrot protoplasts. Expression of MYC2, MYC3 or MYC4 fused to an EAR (ETHYLENE RESPONSE FACTOR-associated amphiphilic repression) motif, or expression of JAZ1 or JAZ6 all repressed JA- and MYC-dependent activation of the JAZ2 promoter. A thymidine-rich sequence 3' to the G-box was required for full JA activation of the JAZ2 promoter. Because the G-box is also present in genes unresponsive to JA, we propose that this thymidine-rich sequence together with the G-box provides JA specificity to promoters induced early in the JA response. Together, these results indicate that an extended G-box located in the promoter region of an early JA-responsive gene is required for gene induction in vivo, probably through selective binding of MYC2, MYC3 and MYC4 transcription factors.

The JAZ Proteins: A Crucial Interface in the Jasmonate Signaling Cascade

Jasmonates are phytohormones that regulate many aspects of plant growth, development, and defense. Within the signaling cascades that are triggered by jasmonates, the JASMONATE-ZIM DOMAIN (JAZ) repressor proteins play a central role. The endogenous bioactive JA-Ile conjugate mediates the binding of JAZ proteins to the F-box protein CORONATINE INSENSITIVE1 (COI1), part of the Skp1/Cullin/F-box SCF(COI1) ubiquitin E3 ligase complex. Upon the subsequent destruction of the JAZ proteins by the 26S proteasome, multiple transcription factors are relieved from JAZ-mediated repression, allowing them to activate their respective downstream responses. However, many questions remain regarding the targets, specificity, function, and regulation of the different JAZ proteins. Here, we review recent studies on the model plant Arabidopsis thaliana that provided essential and novel insights. JAZ proteins have been demonstrated to interact with a broad array of transcription factors that each control specific downstream processes. Recruitment of the corepressor TOPLESS unveiled a mechanism for JAZ-mediated gene repression. Finally, the presence of JAZ proteins was also found to be regulated by alternative splicing and interactions with proteins from other hormonal signaling pathways. Overall, these contemporary findings underscore the value of protein-protein interaction studies to acquire fundamental insight into molecular signaling pathways.

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

Jasmonate (JA) and ethylene (ET) are two major plant hormones that synergistically regulate plant development and tolerance to necrotrophic fungi. Both JA and ET induce the expression of several pathogenesis-related genes, while blocking either signaling pathway abolishes the induction of these genes by JA and ET alone or in combination. However, the molecular basis of JA/ET coaction and signaling interdependency is largely unknown. Here, we report that two Arabidopsis ET-stabilized transcription factors (EIN3 and EIL1) integrate ET and JA signaling in the regulation of gene expression, root development, and necrotrophic pathogen defense. Further studies reveal that JA enhances the transcriptional activity of EIN3/EIL1 by removal of JA-Zim domain (JAZ) proteins, which physically interact with and repress EIN3/EIL1. In addition, we find that JAZ proteins recruit an RPD3-type histone deacetylase (HDA6) as a corepressor that modulates histone acetylation, represses EIN3/EIL1-dependent transcription, and inhibits JA signaling. Our studies identify EIN3/EIL1 as a key integration node whose activation requires both JA and ET signaling, and illustrate transcriptional derepression as a common mechanism to integrate diverse signaling pathways in the regulation of plant development and defense.

Transcriptional regulation of plant inducible defenses against herbivores: a mini-review

Inducible plant defenses against herbivores are controlled by a transient burst of jasmonic acid (JA) and its conversion to the active hormone (3R,7S)-jasmonoyl-L-isoleucine (JA-Ile). JA-Ile shows high affinity for binding to the COI1 protein complex with JAZ repressor protein(s), a multi component JA-Ile receptor, promoting hormone-dependent ubiquitination and degradation of JAZ transcriptional repressors. Degradation of JAZ proteins in Arabidopsis leads to the release of a bHLH transcription factor, MYC2, which functions as a master regulator of JA-dependent defense responses. Because the activity of the MYC2 coincides with the presence of active jasmonate in cells, it is unlikely that MYC2, alone, regulates prolonged transcriptional responses of genes encoding enzymes required for the accumulation of defense metabolites. In this review, we focus on MYC2 and a specific group of MYC2-regulated ‘secondary’ transcription factors as critical components of the JA signal transduction pathway that controls inducible chemical defense responses in plants.

Tobacco MYC2 Regulates Jasmonate-Inducible Nicotine Biosynthesis Genes Directly and By Way of the NIC2-Locus ERF Genes

In Arabidopsis, the MYC2-family basic helix-loop-helix transcription factors mediate transcriptional regulation of jasmonate-responsive genes, and their transcriptional activities are suppressed by physical interactions with jasmonate-ZIM domain (JAZ) proteins. Jasmonate-inducible nicotine formation in Nicotiana plants has been shown to be suppressed by tobacco JAZ proteins, and be regulated by both MYC2-related and NIC2-locus ethylene response factor (ERF) transcription factors. We here show that tobacco MYC2 (NtMYC2) recognizes the G-box sequences, 5'-CAC(G/A)T(G/T)-3', found in the proximal promoter regions of several nicotine biosynthesis genes, including Putrescine N-Methyltransferase 2 (PMT2) and Quinolinate Phosphoribosyltransferase 2 (QPT2). Transient transactivation assays using cultured tobacco cells showed that NtMYC2 and NIC2-locus ERF189 additively activated the PMT2 and QPT2 promoters depending on their cognate binding sites. RNA interference (RNAi) silencing of NtMYC2 in tobacco hairy roots strongly decreased transcript levels of jasmonate-responsive structural genes, including those involved in nicotine biosynthesis, as well as the NIC2-locus ERF genes. Conversely, ERF189 was not required for the expression of NtMYC2. NtMYC2, but not ERF189, interacted with tobacoo JAZs in a yeast two-hybrid assay. These results indicate that NtMYC2 controls nicotine biosynthesis genes in two combinatorial ways, by directly binding the G-box in the target promoters and by up-regulating the NIC2-locus ERF genes.

Jasmonate perception by inositol-phosphate-potentiated COI1–JAZ co-receptor

Jasmonates (JAs) are a family of plant hormones that regulate plant growth, development, and responses to stress. The F-box protein CORONATINE-INSENSITIVE 1 (COI1) mediates JA signaling by promoting hormone-dependent ubiquitination and degradation of transcriptional repressor JAZ proteins. Despite its importance, the mechanism of JA perception remains unclear. Here we present structural and pharmacological data to show that the true JA receptor is a complex of both COI1 and JAZ. COI1 contains an open pocket that recognizes the bioactive hormone, (3R,7S)-jasmonoyl-L-isoleucine (JA-Ile), with high specificity. High-affinity hormone binding requires a bipartite JAZ degron sequence consisting of a conserved α-helix for COI1 docking and a loop region to trap the hormone in its binding pocket. In addition, we identify a third critical component of the JA co-receptor complex, inositol pentakisphosphate, which interacts with both COI1 and JAZ adjacent to the ligand. Our results unravel the mechanism of JA perception and highlight the ability of F-box proteins to evolve as multi-component signaling hubs.

Alternative splicing expands the repertoire of dominant JAZ repressors of jasmonate signaling

Jasmonates (JAs) are fatty acid-derived signaling compounds that control diverse aspects of plant growth, development and immunity. The F-box protein COI1 functions both as a receptor for jasmonoyl-l-isoleucine (JA-Ile) and as the component of an E3-ubiquitin ligase complex (SCF(COI1) ) that targets JAZ transcriptional regulators for degradation. A key feature of JAZ proteins is the C-terminal Jas motif that mediates the JA-Ile-dependent interaction with COI1. Here, we show that most JAZ genes from evolutionarily diverse plants contain a conserved intron that splits the Jas motif into 20 N-terminal and seven C-terminal (X(5) PY) amino acid submotifs. In most members of the Arabidopsis JAZ family, alternative splicing events involving retention of this intron generate proteins that are truncated before the X(5) PY sequence. In vitro pull-down and yeast two-hybrid assays indicate that these splice variants have reduced capacity to form stable complexes with COI1 in the presence of the bioactive stereoisomer of the hormone (3R,7S)-JA-Ile. cDNA overexpression studies showed that some, but not all, truncated splice variants are dominant repressors of JA signaling. We also show that strong constitutive expression of an intron-containing JAZ10 genomic clone is sufficient to repress JA responses. These findings provide evidence for functional differences between JAZ isoforms, and establish a direct link between the alternative splicing of JAZ pre-mRNA and the dominant repression of JA signal output. We propose that production of dominant JAZ repressors by alternative splicing reduces the negative consequences associated with inappropriate or hyperactivation of the JA response pathway.

Dissection of the one-MegaDalton JAZ1 protein complex

Jasmonates (JAs) comprise a class of plant-specific hormones that mediate a large variety of processes involved in plant growth, development and defense. Perception of jasmonoyl-isoleucine (JA-Ile), the bioactive amino acid conjugate of JA, initiates the expression of JA-responsive genes through the degradation of the jasmonate ZIM domain (JAZ) repressor proteins and the subsequent release of the transcriptional activator MYC2. By using a tandem affinity purification based approach, we demonstrated that the Groucho/Tup1-type co-repressor TOPLESS (TPL) and TPL-related proteins are connected to the JAZ proteins via an adaptor protein, designated Novel Interactor of JAZ (NINJA). Both NINJA and TPL were shown to function as negative regulators of JA signaling. Here, we provide additional data, demonstrating that JAZ1 incorporates into high-molecular weight (HMW) protein complexes of >1 MDa and speculate about their composition.

Jasmonates: Structural Requirements for Lipid-Derived Signals Active in Plant Stress Responses and Development

Jasmonates are lipid-derived signals that mediate plant stress responses and development processes. Enzymes participating in biosynthesis of jasmonic acid (JA) (1, 2) and components of JA signaling have been extensively characterized by biochemical and molecular-genetic tools. Mutants of Arabidopsis and tomato have helped to define the pathway for synthesis of jasmonoyl-isoleucine (JA-Ile), the active form of JA, and to identify the F-box protein COI1 as central regulatory unit. However, details of the molecular mechanism of JA signaling have only recently been unraveled by the discovery of JAZ proteins that function in transcriptional repression. The emerging picture of JA perception and signaling cascade implies the SCF(COI1) complex operating as E3 ubiquitin ligase that upon binding of JA-Ile targets JAZ repressors for degradation by the 26S-proteasome pathway, thereby allowing the transcription factor MYC2 to activate gene expression. The fact that only one particular stereoisomer, (+)-7-iso-JA-l-Ile (4), shows high biological activity suggests that epimerization between active and inactive diastereomers could be a mechanism for turning JA signaling on or off. The recent demonstration that COI1 directly binds (+)-7-iso-JA-l-Ile (4) and thus functions as JA receptor revealed that formation of the ternary complex COI1-JA-Ile-JAZ is an ordered process. The pronounced differences in biological activity of JA stereoisomers also imply strict stereospecific control of product formation along the JA biosynthetic pathway. The pathway of JA biosynthesis has been unraveled, and most of the participating enzymes are well-characterized. For key enzymes of JA biosynthesis the crystal structures have been established, allowing insight into the mechanisms of catalysis and modes of substrate binding that lead to formation of stereospecific products.

A Critical Role for the TIFY Motif in Repression of Jasmonate Signaling by a Stabilized Splice Variant of the JASMONATE ZIM-Domain Protein JAZ10 inArabidopsis

JASMONATE ZIM-domain (JAZ) proteins act as repressors of jasmonate (JA) signaling. Perception of bioactive JAs by the F-box protein CORONATINE INSENSITIVE1 (COI1) causes degradation of JAZs via the ubiquitin-proteasome pathway, which in turn activates the expression of genes involved in plant growth, development, and defense. JAZ proteins contain two highly conserved sequence regions: the Jas domain that interacts with COI1 to destabilize the repressor and the ZIM domain of unknown function. Here, we show that the conserved TIFY motif (TIFF/YXG) within the ZIM domain mediates homo- and heteromeric interactions between most Arabidopsis thaliana JAZs. We have also identified an alternatively spliced form (JAZ10.4) of JAZ10 that lacks the Jas domain and, as a consequence, is highly resistant to JA-induced degradation. Strong JA-insensitive phenotypes conferred by overexpression of JAZ10.4 were suppressed by mutations in the TIFY motif that block JAZ10.4-JAZ interactions. We conclude that JAZ10.4 functions to attenuate signal output in the presence of JA and further suggest that the dominant-negative action of this splice variant involves protein-protein interaction through the ZIM/TIFY domain. The ability of JAZ10.4 to interact with MYC2 is consistent with a model in which a JAZ10.4-containing protein complex directly represses the activity of transcription factors that promote expression of JA response genes.

Alternative Splicing Produces a JAZ Protein That Is Not Broken Down in Response to Jasmonic Acid

As the old song goes, “You've got to accentuate the positive, eliminate the negative,” and that is what happens in the plant response to the hormone jasmonic acid (JA). In this case, the task of eliminating the negative falls to the ubiquitin-meditated protein degradation pathway, specifically

A critical role of two positively charged amino acids in the Jas motif of Arabidopsis JAZ proteins in mediating coronatine- and jasmonoyl isoleucine-dependent interactions with the COI1 F-box protein.

Coronatine is an important virulence factor produced by several pathovars of the bacterial pathogen Pseudomonas syringae. The structure of coronatine is similar to that of a class of plant hormones called jasmonates (JAs). An important step in JA signaling is the SCF(COI1) E3 ubiquitin ligase-dependent degradation of JAZ repressor proteins. We have recently shown that jasmonoyl isoleucine (JA-Ile) promotes physical interaction between Arabidopsis JAZ1 and COI1 (the F-box component of SCF(COI1)) proteins, and that the JA-Ile-dependent COI1-JAZ1 interaction could be reconstituted in yeast cells (i.e. in the absence of other plant proteins). Here we show that coronatine, but not its two biosynthetic precursors, also promotes interaction between Arabidopsis COI1 and multiple JAZ proteins. The C-terminal Jas motif, but not the N-terminal (NT) domain or central ZIM domain of JAZ proteins, is critical for JA-Ile/coronatine-dependent interaction with COI1. Two positively charged amino acid residues in the Jas domain were identified as essential for coronatine-dependent COI1-JAZ interactions. Mutations of these two residues did not affect the ability of JAZ1 and JAZ9 to interact with the transcription factor AtMYC2. Importantly, transgenic Arabidopsis plants expressing JAZ1 carrying these two mutations exhibited JA-insensitive phenotypes, including male sterility and enhanced resistance to P. syringae infection. These results not only suggest that coronatine and JA-Ile target the physical interaction between COI1 and the Jas domain of JAZ repressors, but also illustrate the critical role of positively charged amino acids in the Jas domain in mediating the JA-Ile/coronatine-dependent JAZ interaction with COI1.

JAZ repressors set the rhythm in jasmonate signaling

Jasmonates (JAs) are essential hormones for plant defense and development. In spite of their importance, the molecular details of their signaling pathways remain largely unknown. A new family of regulators of JA signaling named JAZ, jasmonate ZIM-domain proteins, has recently been described. JAZ proteins repress of JA signaling and are targeted by the E3-ubiquitin ligase SCF(COI1) for proteasome degradation in response to JA. Hormone binding depends on a functional COI1 protein suggesting that COI1 is the JA receptor. MYC2, a positive regulator of JA-dependent responses, has been identified as a target of JAZ repressors. Interestingly, MYC2 and JAZ proteins are involved in a negative regulatory feedback loop, suggesting a model to explain how transcriptional reprogramming is turned on and off in response to JA. The discovery of JAZ repressors provides a new framework to understand JA-signaling pathways from hormonal perception to transcriptional activation.

The JAZ Proteins Link Jasmonate Perception with Transcriptional Changes

The oxylipin jasmonic acid (JA) and its metabolites, collectively known as jasmonates, are important plant signaling molecules that mediate biotic and abiotic stress responses as well as aspects of growth and development. Although it is well known that JA regulates transcription, the mechanism of

Stress Responses: JAZ Players Deliver Fusion and Rhythm

How do stress-induced jasmonates cause genetic re-programming of plant cells? New research shows that the JAZ proteins provide the missing link.

All That JAZ

The intoxicating scent of jasmine depends in part on jasmonates, fatty acid-derived hormones implicated in plant defense and development. Jasmonate signaling in plants depends on COI1 (CORONATINE INSENSITIVE1), which is part of the Skp-Cullin-F-box complex SCF COI1 , an E 3 ubiquitin ligase that ubiquitinates proteins and targets them for destruction (see Farmer). Two groups, Thines et al . and Chini et al ., have elucidated the pathway linking jasmonates to SCF COI1 and the mechanism through which proteolytic degradation of ubiquitinated proteins leads to activation of jasmonate target genes. Thines et al . found that eight genes of unknown function, each containing a ZIM (ZO-1 interaction motif) domain, were transcriptionally activated in an Arabidopsis jasmonate-synthesis mutant shortly after treatment with exogenous jasmonate. Jasmonate transcriptionally activated these JAZ (for JASMONATE-ZIM-DOMAIN ) genes in wild-type plants, and fluorescently labeled JAZ1 and JAZ6 fusion proteins localized to the nucleus. When expressed in wild-type plants, a C-terminal deletion mutant of JAZ1 (JAZ1Δ3A) elicited a phenotype similar to that of jasmonate-synthesis mutants or plants lacking COI1, suggesting that it blocked jasmonate signaling. Jasmonate stimulated the COI1- and proteasome-dependent degradation of a fusion protein encoding JAZ1 or JAZ6 linked to β-glucuronidase (JAZ1-GUS or JAZ6-GUS) but not that of JAZ1Δ3A-GUS. Yeast two-hybrid and in vitro pull-down assays indicated that jasmonyl-isoleucine stimulated the direct interaction of Arabidopsis JAZ1 and COI1 as well as that of related tomato proteins. Chini et al ., who previously isolated a jasmonate-insensitive Arabidopsis mutant, determined that the mutated gene ( JAI3 , for JASMONATE-INSENSITIVE 3 ) encoded a member of a family of ZIM-domain-containing proteins (JAZ proteins). Like Thines et al ., they showed that constitutive expression of a JAI3 mutant lacking the C-terminal domain ( JAI3ΔC ) rendered transgenic plants insensitive to jasmonate and that JAI3 interacted with COI1. Jasmonate stimulated the COI1- and proteasome-dependent degradation of a JAI3-green-fluorescent-protein (JAI3-GFP)-fusion protein, but not that of JAI3ΔC-GFP, and JAI3 degradation was impaired in a mutant unable to conjugate jasmonate with amino acids. JAI3 bound MYC2, a transcriptional activator downstream of jasmonate, and microarray analyses indicated that expression of MYC2 target genes was decreased in plants with a dominant-negative JAI3 mutant and that MYC2 regulated expression of JAZ genes. Thus, both studies indicate that jasmonyl-isoleucine stimulates the interaction of COI1 with JAZ-family transcriptional repressors, thereby enabling JAZ protein degradation and the transcriptional activation of jasmonate target genes (including the JAZ proteins themselves). A third study by Yan et al . also identified JAZ proteins as repressors of jasmonate signaling. B. Thines, L. Katsir, M. Melotto, Y. Niu, A. Mandaokar, G. Liu, K. Nomura, S. Y. He, G. A. Howe, J. Browse, JAZ repressor proteins are targets of the SCF COI1 complex during jasmonate signalling. Nature 448 , 661-665 (2007). [PubMed] A. Chini, S. Fonseca, G. Fernández, B. Adie, J. M. Chico, O. Lorenzo, G. García-Casado, I. López-Vidriero, F. M. Lozano, M. R. Ponce, J. L. Micol, R. Solano, The JAZ family of repressors is the missing link in jasmonate signalling. Nature 448 , 666-671 (2007). [PubMed] E. E. Farmer, Jasmonate perception machines. Nature 448 , 659-660 (2007). [PubMed] Y. Yan, S. Stolz, A. Chételat, P. Reymond, M. Pagni, L. Dubugnon, E. E. Farmer, A downstream mediator in the growth repression limb of the jasmonate pathway. Plant Cell , E-pub ahead of print, 3 August (2007). [PubMed]