Jasmonic acid (JA) regulates plant growth, development, and stress responses. Coronatine insensitive 1 (COI1) and jasmonate ZIM-domain (JAZ) proteins form a receptor complex for jasmonoyl-l-isoleucine (JA-Ile), a biologically active form of JA. Three COIs (OsCOI1a, OsCOI1b, and OsCOI2) are encoded in the rice genome. In the present study, we generated mutants for each rice COI gene using genome editing to reveal the physiological functions of the three rice COIs. The oscoi2 mutants, but not the oscoi1a and oscoi1b mutants, exhibited severely low fertility, indicating the crucial role of OsCOI2 in rice fertility. Transcriptomic analysis revealed that the transcriptional changes after methyl jasmonate (MeJA) treatment were moderate in the leaves of oscoi2 mutants compared to those in the wild-type or oscoi1a and oscoi1b mutants. MeJA-induced chlorophyll degradation and accumulation of antimicrobial secondary metabolites were suppressed in oscoi2 mutants. These results indicate that OsCOI2 plays a central role in JA response in rice leaves. In contrast, assessment of growth inhibition upon exogenous application of JA to seedlings of each mutant revealed that rice COIs are redundantly involved in shoot growth, whereas OsCOI2 plays a primary role in root growth. In addition, a co-immunoprecipitation assay showed that OsJAZ2 and OsJAZ5 containing divergent Jas motifs physically interacted only with OsCOI2, whereas OsJAZ4 with a canonical Jas motif interacts with all three rice COIs. The present study demonstrated the functional diversity of rice COIs, thereby providing clues to the mechanisms regulating the various physiological functions of JA.

The phytohormone (+)-7-iso-jasmonoyl-L-isoleucine regulates many developmental and stress responses in plants and induces protein–protein interactions between COI1, the F-box component of E3 ubiquitin ligase, and jasmonate ZIM domain (JAZ) repressors. These interactions cause JAZ degradation and activates jasmonate (JA), leading to plant defense responses, growth inhibition, and senescence. Thirteen JAZ subtypes are encoded in the Arabidopsis thaliana genome, but a detailed understanding of the physiological functions of these JAZ subtypes remains unclear, partially because of the genetic redundancy of JAZ genes. One strategy to elucidate the complex JA signaling pathways is to develop a reliable and comprehensive binding assay system of the ligands with all combinations of the co-receptors. Herein, we report the development of a fluorescent anisotropy-based in vitro binding assay system to screen for the ligands of the COI1/JAZ co-receptors. Our assay enabled the first quantitative analysis of the affinity values and JAZ-subtype selectivity of various endogenous JA derivatives, such as coronatine, jasmonic acid and 12-hydroxy jasmonoyl-L-isoleucine. Because of its high signal-to-noise ratio and convenient mix-and-read assay system, our screening approach can be used in plate reader–based assays of both agonists and antagonists of COI1/JAZ co-receptors.

We demonstrate that ligand-directed tosyl (LDT) chemistry is applicable to off-target identification in live cells.

A network of protein–protein interactions (PPI) is involved in the activation of (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile), a plant hormone that regulates plant defense responses as well as plant growth and development. In the absence of JA-Ile, inhibitory protein jasmonate-ZIM-domain (JAZ) represses JA-related transcription factors, including a master regulator, MYC. In contrast, when JA-Ile accumulates in response to environmental stresses, PPI occurs between JAZ and the F-box protein COI1, which triggers JAZ degradation, resulting in derepressed MYC that can interact with the transcriptional mediator MED25 and upregulate JA-Ile-related gene expression. Activated JA signaling is eventually suppressed through the catabolism of JA-Ile and feedback suppression by JAZ splice variants containing a cryptic MYC-interacting domain (CMID). However, the detailed structural basis of some PPIs involved in JA-Ile signaling remains unclear. Herein, we analyzed PPI between MYC3 and MED25, focusing on the key interactions that activate the JA-Ile signaling pathway. Biochemical assays revealed that a short binding domain of MED25 (CMIDM) is responsible for the interaction with MYC, and that a bipartite interaction is critical for the formation of a stable complex. We also show the mode of interaction between MED25 and MYC is closely related to that of CMID and MYC. In addition, quantitative analyses on the binding of MYC3-JAZs and MYC3-MED25 revealed the order of binding affinity as JAZ^Jas < MED25^CMIDM < JAZ^CMID, suggesting a mechanism for how the transcriptional machinery causes activation and negative feedback regulation during jasmonate signaling. These results further illuminate the transcriptional machinery responsible for JA-Ile signaling.

Ethylene (ET) and jasmonate (JA) are plant hormones that act synergistically to regulate plant development and defense against necrotrophic fungi infections, and antagonistically in response to wounds and apical hook formation. Previous studies revealed that the coordination of these responses is due to dynamic protein-protein interactions (PPI) between their master transcription factors (TFs) EIN3/EIL1 and MYC in ET and JA signaling, respectively. In addition, both TFs are activated via interactions with the same transcriptional mediator MED25, which upregulates downstream gene expression. Herein, we analyzed the PPI between EIN3/EIL1 and MED25, and as with the PPI between MYC3 and MED25, found that the short binding domain of MED25 (CMIDM) is also responsible for the interaction with EIN3/EIL1 − a finding which suggests that both TFs compete for binding with MED25. These results further inform our understanding of the coordination between the ET and JA regulatory systems.