F-box Protein CORONATINE INSENSITIVE1 Research Articles

JAZ4 is involved in plant defense, growth, and development in Arabidopsis

Jasmonate zim-domain (JAZ) proteins comprise a family of transcriptional repressors that modulate jasmonate (JA) responses. JAZ proteins form a co-receptor complex with the F-box protein coronatine insensitive1 (COI1) that recognizes both jasmonoyl-l-isoleucine (JA-Ile) and the bacterial-produced phytotoxin coronatine (COR). Although several JAZ family members have been placed in this pathway, the role of JAZ4 in this model remains elusive. In this study, we observed that the jaz4-1 mutant of Arabidopsis is hyper-susceptible to Pseudomonas syringae pv. tomato (Pst) DC3000, while Arabidopsis lines overexpressing a JAZ4 protein lacking the Jas domain (JAZ4∆Jas) have enhanced resistance to this bacterium. Our results show that the Jas domain of JAZ4 is required for its physical interaction with COI1, MYC2 or MYC3, but not with the repressor complex adaptor protein NINJA. Furthermore, JAZ4 degradation is induced by COR in a proteasome- and Jas domain-dependent manner. Phenotypic evaluations revealed that expression of JAZ4∆Jas results in early flowering and increased length of root, hypocotyl, and petiole when compared with Col-0 and jaz4-1 plants, although JAZ4∆Jas lines remain sensitive to MeJA- and COR-induced root and hypocotyl growth inhibition. Additionally, jaz4-1 mutant plants have increased anthocyanin accumulation and late flowering compared with Col-0, while JAZ4∆Jas lines showed no alteration in anthocyanin production. These findings suggest that JAZ4 participates in the canonical JA signaling pathway leading to plant defense response in addition to COI1/MYC-independent functions in plant growth and development, supporting the notion that JAZ4-mediated signaling may have distinct branches.

The Pseudomonas syringae type III effectors AvrRpm1 and AvrRpt2 promote virulence dependent on the F-box protein COI1.

Type III effectors AvrRpm1 and AvrRpt2 promote bacterial growth dependent on a COI1-mediated pathway in the absence of the RPM1 and RPS2 resistance proteins. The type III effectors, AvrRpm1 and AvrRpt2, promote bacterial virulence by suppressing host defense responses. The defense suppressing activities of AvrRpm1 and AvrRpt2 are best studied in the absence of the resistance proteins RPM1 and RPS2, which induce defense responses to them. We tested whether the type III effectors could modulate a CORONATINE INSENSITIVE1 (COI1)-mediated hormone signaling pathway to promote virulence. COI1 has been demonstrated to contribute in the induction of chlorosis during Pseudomonas syringae infection. By comparing the activity of inducibly expressed AvrRpm1-HA or AvrRpt2-HA in rpm1rps2 and rpm1rps2coi1 backgrounds, we demonstrate that both effectors promote bacterial growth dependent on a COI1-mediated pathway and additively with the action of coronatine (COR) and that AvrRpt2-HA induces COI1-dependent chlorosis. Further, PATHOGENESISRELATED1 (PR-1) expression resulting from inducible expression of AvrRpm1-HA or AvrRpt2-HA is elevated in coi1 plants consistent with the effectors activating JA-signaling to antagonize SA-signaling. In addition, we found that AvrRpm1-HA or AvrRpt2-HA requires COI1 to promote bacterial growth through suppression of both SA-dependent and SA-independent defense responses. Collectively, these results indicate that type III effectors AvrRpm1 and AvrRpt2 promote bacterial virulence by targeting a COI1-dependent signaling pathway.

The RING E3 Ligase KEEP ON GOING Modulates JASMONATE ZIM-DOMAIN12 Stability.

Jasmonate (JA) signaling in plants is mediated by the JASMONATE ZIM-DOMAIN (JAZ) proteins that repress the activity of several transcription factors regulating JA-inducible gene expression. The hormone JA-isoleucine triggers the interaction of JAZ repressor proteins with the F-box protein CORONATINE INSENSITIVE1 (COI1), part of an S-phase kinase-associated protein1/Cullin1/F-box protein COI1 (SCF(COI1)) E3 ubiquitin ligase complex, and their degradation by the 26S proteasome. In Arabidopsis (Arabidopsis thaliana), the JAZ family consists of 13 members. The level of redundancy or specificity among these members is currently not well understood. Here, we characterized JAZ12, encoded by a highly expressed JAZ gene. JAZ12 interacted with the transcription factors MYC2, MYC3, and MYC4 in vivo and repressed MYC2 activity. Using tandem affinity purification, we found JAZ12 to interact with SCF(COI1) components, matching with observed in vivo ubiquitination and with rapid degradation after treatment with JA. In contrast to the other JAZ proteins, JAZ12 also interacted directly with the E3 RING ligase KEEP ON GOING (KEG), a known repressor of the ABSCISIC ACID INSENSITIVE5 transcription factor in abscisic acid signaling. To study the functional role of this interaction, we circumvented the lethality of keg loss-of-function mutants by silencing KEG using an artificial microRNA approach. Abscisic acid treatment promoted JAZ12 degradation, and KEG knockdown led to a decrease in JAZ12 protein levels. Correspondingly, KEG overexpression was capable of partially inhibiting COI1-mediated JAZ12 degradation. Our results provide additional evidence for KEG as an important factor in plant hormone signaling and a positive regulator of JAZ12 stability.

The F-box protein COI1 functions upstream of MYB305 to regulate primary carbohydrate metabolism in tobacco (Nicotiana tabacum L. cv. TN90).

Jasmonate (JA) plays an important role in regulating plant male fertility and secondary metabolism, but its role in regulating primary metabolism remains unclear. The F-box protein CORONATINE INSENSITIVE 1 (COI1) is a critical component of the JA receptor, and mediates JA-signalling by targeting JASMONATE ZIM-domain (JAZ) proteins for proteasomal degradation in response to JA perception. Here, we found that RNA interference-mediated knockdown of NtCOI1 in tobacco (Nicotiana tabacum L. cv. TN90) recapitulated many previously observed phenotypes in coi1 mutants, including male sterility, JA insensitivity, and loss of floral anthocyanin production. It also affected starch metabolism in the pollen, anther wall, and floral nectary, leading to pollen abortion and loss of floral nectar. Transcript levels of genes encoding starch metabolism enzymes were significantly altered in the pollen, anther wall, and floral nectary of NtCOI1-silenced tobacco. Changes in leaf primary metabolism were also observed in the NtCOI1-silenced tobacco. The expression of NtMYB305, an orthologue of MYB305 previously identified as a flavonoid metabolic regulator in Antirrhinum majus flowers and as a floral-nectar regulator mediating starch synthesis in ornamental tobacco, was extremely downregulated in NtCOI1-silenced tobacco. These findings suggest that NtCOI1 functions upstream of NtMYB305 and plays a fundamental role in coordinating plant primary carbohydrate metabolism and correlative physiological processes.

TheArabidopsisF-Box Protein CORONATINE INSENSITIVE1 Is Stabilized by SCFCOI1 and Degraded via the 26S Proteasome Pathway

Jasmonate regulates critical aspects of plant development and defense. The F-box protein CORONATINE INSENSITIVE1 (COI1) functions as a jasmonate receptor and forms Skp1/Cullin1/F-box protein COI1 (SCF(COI1)) complexes with Arabidopsis thaliana Cullin1 and Arabidopsis Skp1-like1 (ASK1) to recruit its substrate jasmonate ZIM-domain proteins for ubiquitination and degradation. Here, we reveal a mechanism regulating COI1 protein levels in Arabidopsis. Genetic and biochemical analysis and in vitro degradation assays demonstrated that the COI1 protein was initially stabilized by interacting with ASK1 and further secured by assembly into SCF(COI1) complexes, suggesting a function for SCF(COI1) in the stabilization of COI1 in Arabidopsis. Furthermore, we show that dissociated COI1 is degraded through the 26S proteasome pathway, and we identified the 297th Lys residue as an active ubiquitination site in COI1. Our data suggest that the COI1 protein is strictly regulated by a dynamic balance of SCF(COI1)-mediated stabilization and 26S proteasome-mediated degradation and thus maintained at a protein level essential for proper biological functions in Arabidopsis development and defense responses.

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.

Design and synthesis of biotin-tagged photoaffinity probes of jasmonates

Jasmonates (JAs) are a class of oxylipin compounds that play diverse roles in plant defense and development. The F-box protein coronatine insensitive1 (COI1) plays a crucial role in the JA signaling pathway. To determine whether COI1 binds directly to jasmonates, three biotin-tagged photoaffinity probes for JAs, a jasmonic acid photoaffinity probe (PAJA), a JAIle photoaffinity probe (PAJAIle), and a coronatine photoaffinity probe (PACOR), were designed and synthesized based on analysis of JA structure–activity relationships and molecular modeling of the interaction between COI1 and JAs. Among them, PACOR exhibited the most significant biological activity in inhibiting root growth, promoting accumulation of JA-responsive proteins, and triggering COI1–JAZ1 interaction in Arabidopsis seedlings. PACOR is an effective tool for elucidating the interaction between COI1 and JA.

A Leaky Mutation in DWARF4 Reveals an Antagonistic Role of Brassinosteroid in the Inhibition of Root Growth by Jasmonate in Arabidopsis

The F-box protein CORONATINE INSENSITIVE1 (COI1) plays a central role in jasmonate (JA) signaling and is required for all JA responses in Arabidopsis (Arabidopsis thaliana). To dissect JA signal transduction, we isolated the partially suppressing coi1 (psc1) mutant, which partially suppressed coi1 insensitivity to JA inhibition of root growth. The psc1 mutant partially restored JA sensitivity in coi1-2 background and displayed JA hypersensitivity in wild-type COI1 background. Genetic mapping, sequence analysis, and complementation tests revealed that psc1 is a leaky mutation of DWARF4 (DWF4) that encodes a key enzyme in brassinosteroid (BR) biosynthesis. Physiological analysis showed that an application of exogenous BR eliminated the partial restoration of JA sensitivity by psc1 in coi1-2 background and the JA hypersensitivity of psc1 in wild-type COI1 background. Exogenous BR also attenuated JA inhibition of root growth in the wild type. In addition, the expression of DWF4 was inhibited by JA, and this inhibition was dependent on COI1. These results indicate that (1) BR is involved in JA signaling and negatively regulates JA inhibition of root growth, and (2) the DWF4 is down-regulated by JA and is located downstream of COI1 in the JA-signaling pathway.

The Jasmonate Receptor: Protein Modeling and Photoaffinity Labeling Reveal That the CORONATINE INSENSITIVE1 Protein Binds Jasmonoyl-Isoleucine and Coronatine

The plant defense hormone jasmonate (JA) functions through a multiprotein complex containing the F-box protein CORONATINE INSENSITIVE1 (COI1) ([reviewed in Katsir et al., 2008][1]; [Browse, 2009][2]). This complex forms an E3 ubiquitin ligase, which binds to downstream negative transcriptional

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.