Protein-protein interactions between jasmonate-related master regulator MYC and transcriptional mediator MED25 depend on a short binding domain.

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 JAZJas < MED25CMIDM < JAZCMID, 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.

Immune gene activation by NPR and TGA transcriptional regulators in the model monocot Brachypodium distachyon.

The nonexpressor of pathogenesis-related (NPR) gene family is well known to play a crucial role in transactivation of TGA transcription factors for salicylic acid (SA)-responsive genes, including pathogenesis-related protein 1 (PR1), during plants' immune response after pathogen attack in the model dicot Arabidopsis thaliana. However, little is known about NPR gene functions in monocots. We therefore explored the functions of NPRs in SA signaling in the model monocot Brachypodium distachyon. BdNPR1 and BdNPR2/3 share structural similarities with A. thaliana AtNPR1/2 and AtNPR3/4 subfamilies, respectively. The transcript level of BdNPR2 but not BdNPR1/3 appeared to be positively regulated in leaves in response to methyl salicylate. Reporter assays in protoplasts showed that BdNPR2 positively regulated BdTGA1-mediated activation of PR1. This transactivation occurred in an SA-dependent manner through SA binding at Arg468 of BdNPR2. In contrast, BdNPR1 functioned as a suppressor of BdNPR2/BdTGA1-mediated transcription of PR1. Collectively, our findings reveal that the TGA-promoted transcription of SA-inducible PR1 is orchestrated by the activator BdNPR2 and the repressor BdNPR1, which function competitively in B. distachyon.

JUL1, Ring-Type E3 Ubiquitin Ligase, Is Involved in Transcriptional Reprogramming for ERF15-Mediated Gene Regulation.

JAV1-associated ubiquitin ligase 1 (JUL1) is a RING-type E3 ubiquitin ligase that catalyzes ubiquitination of JAV1, a jasmonate signaling repressor, in Arabidopsis thaliana in response to herbivore attack. Here we present a new insight into the nature of JUL1 as a multi-targeting enzyme for not only JAV1 but also transcription factors (TFs) screened using in vitro and in vivo protein interaction assays. Reporter assays using protoplasts showed that the JUL1-interacting TFs (JiTFs), including ERF15, bZIP53 and ORA59, were involved in transcriptional activation of jasmonate-responsive PDF1.2 and abscisic acid-responsive GEA6. Likewise, assays using mutant plants suggested that the 3 JiTFs were indeed responsible for transcriptional regulation of PDF1.2 and/or GEA6, and ERF15 and ORA59 were substantially responsible for the anti-herbivore trait. In vitro protein ubiqutination assays showed that JUL1 catalyzed ubiqutination of JAV1 but not any of the TFs. This was in accord with the finding that JUL1 abolished JAV1's interference with ERF15 function, according to the reporter assay. Moreover, of great interest is our finding that ERF15 but not bZIP53 or ORA59 serves as a scaffold for the JAV1/JUL1 system, indicating that there is narrow selectivity of the transcriptional reprogramming by the JAV1/JUL1 system.

The rice wound-inducible transcription factor RERJ1 sharing same signal transduction pathway with OsMYC2 is necessary for defense response to herbivory and bacterial blight.

KEY MESSAGE: This study describes biological functions of the bHLH transcription factor RERJ1 involved in the jasmonate response and the related defense-associated metabolic pathways in rice, with particular focus on deciphering the regulatory mechanisms underlying stress-induced volatile emission and herbivory resistance. RERJ1 is rapidly and drastically induced by wounding and jasmonate treatment but its biological function remains unknown as yet. Here we provide evidence of the biological function of RERJ1 in plant defense, specifically in response to herbivory and pathogen attack, and offer insights into the RERJ1-mediated regulation of metabolic pathways of specialized defense compounds, such as monoterpene linalool, in possible collaboration with OsMYC2-a well-known master regulator in jasmonate signaling. In rice (Oryza sativa L.), the basic helix-loop-helix (bHLH) family transcription factor RERJ1 is induced under environmental stresses, such as wounding and drought, which are closely linked to jasmonate (JA) accumulation. Here, we investigated the biological function of RERJ1 in response to biotic stresses, such as herbivory and pathogen infection, using an RERJ1-defective mutant. Transcriptome analysis of the rerj1-Tos17 mutant revealed that RERJ1 regulated the expression of a typical family of conserved JA-responsive genes (e.g., terpene synthases, proteinase inhibitors, and jasmonate ZIM domain proteins). Upon exposure to armyworm attack, the rerj1-Tos17 mutant exhibited more severe damage than the wildtype, and significant weight gain of the larvae fed on the mutant was observed. Upon Xanthomonas oryzae infection, the rerj1-Tos17 mutant developed more severe symptoms than the wildtype. Among RERJ1-regulated terpene synthases, linalool synthase expression was markedly disrupted and linalool emission after wounding was significantly decreased in the rerj1-Tos17 mutant. RERJ1 appears to interact with OsMYC2-a master regulator of JA signaling-and many OsJAZ proteins, although no obvious epistatic interaction was detected between them at the transcriptional level. These results indicate that RERJ1 is involved in the transcriptional induction of JA-mediated stress-responsive genes via physical association with OsMYC2 and mediates defense against herbivory and bacterial infection through JA signaling.

A simple method for labeling proteins and antibodies with biotin using the proximity biotinylation enzyme TurboID.

Proteins and antibodies labeled with biotin have been widely used for protein analysis, enzyme immunoassays, and diagnoses. Presently, they are prepared using either a chemical reaction involving a biotin N-hydroxysuccinimide (NHS) ester compound or by enzymatic biotin ligation using a combination of a biotinylation-peptide tag and Escherichia coli BirA. However, these methods are relatively complicated. Recently BirA was improved to TurboID, a highly active enzyme for proximity labeling with biotin. Here, we demonstrate a novel simple biotin labeling method for proteins and antibodies using TurboID. Purified TurboID was mixed with a protein or an antibody in the presence of biotin and ATP in the general biochemical buffer condition, followed by biotin labeling. Biotin labeling sites by TurboID were found on the surface of green fluorescent protein. Biotin labeling of IκBα by TurboID indicated its binding to RelA. Furthermore, TurboID-dependent biotin labeling of monoclonal antibodies from rabbits and mice could be directly used for immunoblotting detection of specific proteins without the purification step. These results indicate that TurboID provides a very useful and simple method for biotin labeling of functional proteins.

The Ring-Type E3 Ubiquitin Ligase JUL1 Targets the VQ-Motif Protein JAV1 to Coordinate Jasmonate Signaling.

Jasmonates regulate plant defense and development. In Arabidopsis (Arabidopsis thaliana), JASMONATE-ASSOCIATED VQ-MOTIF GENE1 (JAV1/VQ22) is a repressor of jasmonate-mediated defense responses and is degraded through the ubiquitin-26S proteasome system after herbivory. We found that JAV1-ASSOCIATED UBIQUITIN LIGASE1 (JUL1), a RING-type E3 ubiquitin ligase, interacted with JAV1. JUL1 interacted with JAV1 in the nucleus to ubiquitinate JAV1, leading to proteasomal degradation of JAV1. The transcript levels of JUL1 and JAV1 were coordinately and positively regulated by the CORONATINE INSENSITIVE1-dependent signaling pathway in the jasmonate signaling network, but in a manner that was not dependent on CORONATINE INSENSITIVE1-mediated signaling upon herbivory by Spodoptera litura Gain or loss of function of JUL1 modulated the expression levels of the defensin gene PDF1.2 in leaves, conferring on the plants various defense properties against the generalist herbivore S. litura Because neither the JUL1 mutant nor overexpression lines showed any obvious developmental defects, we concluded that the JAV1/JUL1 system functions as a specific coordinator of reprogramming of plant defense responses. Altogether, our findings offer insight into the mechanisms by which the JAV1/JUL1 system acts specifically to coordinate plant defense responses without interfering with plant development or growth.

Dysregulation of a potassium channel, THIK-1, targeted by caspase-8 accelerates cell shrinkage.

Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the physiological role of CASP8 is not fully understood. Here, we identified a two-pore domain potassium channel, tandem-pore domain halothane-inhibited K+ channel 1 (THIK-1), as a novel CASP8 substrate. The intracellular region of THIK-1 was cleaved by CASP8 in apoptotic cells. Overexpression of THIK-1, but not its mutant lacking the CASP8-target sequence in the intracellular portion, accelerated cell shrinkage in response to apoptotic stimuli. In contrast, knockdown of endogenous THIK-1 by RNA interference resulted in delayed shrinkage and potassium efflux. Furthermore, a truncated THIK-1 mutant lacking the intracellular region, which mimics the form cleaved by CASP8, led to a decrease of cell volume of cultured cells without apoptotic stimulation and excessively promoted irregular development of Xenopus embryos. Taken together, these results indicate that THIK-1 is involved in the acceleration of cell shrinkage. Thus, we have demonstrated a novel physiological role of CASP8: creating a cascade that advances the cell to the next stage in the apoptotic process.

Evidence that the Entamoeba histolytica Mitochondrial Carrier Family Links Mitosomal and Cytosolic Pathways through Exchange of 3'-Phosphoadenosine 5'-Phosphosulfate and ATP.

Entamoeba histolytica, a microaerophilic protozoan parasite, possesses mitosomes. Mitosomes are mitochondrion-related organelles that have largely lost typical mitochondrial functions, such as those involved in the tricarboxylic acid cycle and oxidative phosphorylation. The biological roles of Entamoeba mitosomes have been a long-standing enigma. We previously demonstrated that sulfate activation, which is not generally compartmentalized to mitochondria, is a major function of E. histolytica mitosomes. Sulfate activation cooperates with cytosolic enzymes, i.e., sulfotransferases (SULTs), for the synthesis of sulfolipids, one of which is cholesteryl sulfate. Notably, cholesteryl sulfate plays an important role in encystation, an essential process in the Entamoeba life cycle. These findings identified a biological role for Entamoeba mitosomes; however, they simultaneously raised a new issue concerning how the reactions of the pathway, separated by the mitosomal membranes, cooperate. Here, we demonstrated that the E. histolytica mitochondrial carrier family (EhMCF) has the capacity to exchange 3'-phosphoadenosine 5'-phosphosulfate (PAPS) with ATP. We also confirmed the cytosolic localization of all the E. histolytica SULTs, suggesting that in Entamoeba, PAPS, which is produced through mitosomal sulfate activation, is translocated to the cytosol and becomes a substrate for SULTs. In contrast, ATP, which is produced through cytosolic pathways, is translocated into the mitosomes and is a necessary substrate for sulfate activation. Taking our findings collectively, we suggest that EhMCF functions as a PAPS/ATP antiporter and plays a crucial role in linking the mitosomal sulfate activation pathway to cytosolic SULTs for the production of sulfolipids.

Diversity in guanosine 3',5'-bisdiphosphate (ppGpp) sensitivity among guanylate kinases of bacteria and plants.

The guanosine 3',5'-bisdiphosphate (ppGpp) signaling system is shared by bacteria and plant chloroplasts, but its role in plants has remained unclear. Here we show that guanylate kinase (GK), a key enzyme in guanine nucleotide biosynthesis that catalyzes the conversion of GMP to GDP, is a target of regulation by ppGpp in chloroplasts of rice, pea, and Arabidopsis. Plants have two distinct types of GK that are localized to organelles (GKpm) or to the cytosol (GKc), with both enzymes being essential for growth and development. We found that the activity of rice GKpm in vitro was inhibited by ppGpp with a Ki of 2.8 µM relative to the substrate GMP, whereas the Km of this enzyme for GMP was 73 µM. The IC50 of ppGpp for GKpm was ∼10 µM. In contrast, the activity of rice GKc was insensitive to ppGpp, as was that of GK from bakers' yeast, which is also a cytosolic enzyme. These observations suggest that ppGpp plays a pivotal role in the regulation of GTP biosynthesis in chloroplasts through specific inhibition of GKpm activity, with the regulation of GTP biosynthesis in chloroplasts thus being independent of that in the cytosol. We also found that GKs of Escherichia coli and Synechococcus elongatus PCC 7942 are insensitive to ppGpp, in contrast to the ppGpp sensitivity of the Bacillus subtilis enzyme. Our biochemical characterization of GK enzymes has thus revealed a novel target of ppGpp in chloroplasts and has uncovered diversity among bacterial GKs with regard to regulation by ppGpp.

Biochemical analyses of ppGpp effect on adenylosuccinate synthetases, key enzymes in purine biosynthesis in rice.

The ppGpp-signaling system functions in plant chloroplasts. In bacteria, a negative effect of ppGpp on adenylosuccinate synthetase (AdSS) has been suggested. Our biochemical analysis also revealed rice AdSS homologs are apparently sensitive to ppGpp. However, further investigation clarified that this phenomenon is cancelled by the high substrate affinity to the enzymes, leading to a limited effect of ppGpp on adenylosuccinate synthesis.

Characterization of the plastidic phosphate translocators in the inducible crassulacean acid metabolism plant Mesembryanthemum crystallinum.

In plant Mesembryanthemum crystallinum, which has the inducible crassulacean acid metabolism (CAM), isoforms of plastidic phosphate translocators (pPTs) are categorized into three subfamilies: the triose phosphate/phosphate translocator (McTPT1), the phosphoenolpyruvate/phosphate translocator (McPPT1), and the glucose 6-phosphate/phosphate translocator (McGPT1 and McGPT2). In order to elucidate the physiological roles of these pPTs in M. crystallinum, we determined the substrate specificity of each pPT isoform. The substrate specificities of McTPT1, McPPT1, and McGPT1 showed overall similarities to those of orthologs that have been characterized. In contrast, for glucose 6-phosphate, McGPT2 showed higher selectivity than McGPT1 and other GPT orthologs. Because the expression of McGTP2 is specific to CAM while that of McGTP1 is constitutively expressed in both the C3- and the CAM-state in M. crystallinum, we propose that McGPT2 functions as a CAM system-specific GPT in this plant.

Effect of Thickening Agents on the In Vitro Antibacterial Activity of Vancomycin Hydrochloride Powder.

When vancomycin hydrochloride (VCM) powder mixes with xanthan gum-based thickening agents in food, lumps or other property-related changes may occur. Previous studies have reported delayed disintegration and elution of the drug and its adsorption on to xanthan gum, which is the main ingredient of thickened food products. If the addition of thickening agents can affect the antimicrobial activity of VCM powder as previously reported, it might interfere with the treatment of Clostridioides difficile infection (CDI). In this study, we investigated the effect of the addition of xanthan gum-based thickening agents on the antibacterial activity of VCM against Clostridioides difficile in vitro. The VCM concentration at 0 min after adding 3% Tsururinko Quickly (Clinico, Tokyo) to VCM powders (Shionogi, Osaka and Meiji Seika Pharma, Tokyo) was lower than that of the control [Shionogi: 65.15±35.57%, Meiji Seika Pharma: 77.00±15.81% (mean±standard deviation), ** p<0.01, Dunnet's test]. However, the VCM concentration at 30 min after the addition recovered to the control level. The drug susceptibility tests for C. difficile and Staphylococcus aureus using the disk diffusion method showed no effect of addition of 3% Tsururinko Quickly. Our in vitro evaluations showed that the addition of xanthan gum-based thickeners to VCM powders had a negligible effect on the treatment of CDI.

Cytoplasmic Kinase Network Mediates Defense Response to Spodoptera litura in Arabidopsis.

Plants defend against folivores by responding to folivore-derived elicitors following activation of signaling cascade networks. In Arabidopsis, HAK1, a receptor-like kinase, responds to polysaccharide elicitors (Frα) that are present in oral secretions of Spodoptera litura larvae to upregulate defense genes (e.g., PDF1.2) mediated through downstream cytoplasmic kinase PBL27. Here, we explored whether other protein kinases, including CPKs and CRKs, function with PBL27 in the intracellular signaling network for anti-herbivore responses. We showed that CRK2 and CRK3 were found to interact with PBL27, but CPKs did not. Although transcripts of PDF1.2 were upregulated in leaves of wild-type Arabidopsis plants in response to mechanical damage with Frα, this failed in CRK2- and PBL27-deficient mutant plants, indicating that the CRK2/PBL27 system is predominantly responsible for the Frα-responsive transcription of PDF1.2 in S. litura-damaged plants. In addition to CRK2-phosphorylated ERF13, as shown previously, ethylene signaling in connection to CRK2-phosphorylated PBL27 was predicted to be responsible for transcriptional regulation of a gene for ethylene response factor 13 (ERF13). Taken together, these findings show that CRK2 regulates not only ERF13 phosphorylation but also PBL27-dependent de novo synthesis of ERF13, thus determining active defense traits against S. litura larvae via transcriptional regulation of PDF1.2.

Identification of a new gibberellin receptor agonist, diphegaractin, by a cell-free chemical screening system.

Gibberellin (GA) is a phytohormone that regulates various developmental processes during the plant life cycle. In this study, we identify a new GA agonist, diphegaractin, using a wheat cell-free based drug screening system with grape GA receptor. A GA-dependent interaction assay system using GA receptors and DELLA proteins from Vitis vinifera was constructed using AlphaScreen technology and cell-free produced proteins. From the chemical compound library, diphegaractin was found to enhance the interactions between GA receptors and DELLA proteins from grape in vitro. In grapes, we found that diphegaractin induces elongation of the bunch and increases the sugar concentration of grape berries. Furthermore, diphegaractin shows GA-like activity, including promotion of root elongation in lettuce and Arabidopsis, as well as reducing peel pigmentation and suppressing peel puffing in citrus fruit. To the best of our knowledge, this study is the first to successfully identify a GA receptor agonist showing GA-like activity in agricultural plants using an in vitro molecular-targeted drug screening system.

ppGpp inhibits peptide elongation cycle of chloroplast translation system in vitro.

Chloroplasts possess common biosynthetic pathways for generating guanosine 3',5'-(bis)pyrophosphate (ppGpp) from GDP and ATP by RelA-SpoT homolog enzymes. To date, several hypothetical targets of ppGpp in chloroplasts have been suggested, but they remain largely unverified. In this study, we have investigated effects of ppGpp on translation apparatus in chloroplasts by developing in vitro protein synthesis system based on an extract of chloroplasts isolated from pea (Pisum sativum). The chloroplast extracts showed stable protein synthesis activity in vitro, and the activity was sensitive to various types of antibiotics. We have demonstrated that ppGpp inhibits the activity of chloroplast translation in dose-effective manner, as does the toxic nonhydrolyzable GTP analog guanosine 5'-(ß,γ-imido)triphosphate (GDPNP). We further examined polyuridylic acid-directed polyphenylalanine synthesis as a measure of peptide elongation activity in the pea chloroplast extract. Both ppGpp and GDPNP as well as antibiotics, fusidic acid and thiostrepton, inhibited the peptide elongation cycle of the translation system, but GDP in the similar range of the tested ppGpp concentration did not affect the activity. Our results thus show that ppGpp directly affect the translation system of chloroplasts, as they do that of bacteria. We suggest that the role of the ppGpp signaling system in translation in bacteria is conserved in the translation system of chloroplasts.

Cell-free synthesis, reconstitution, and characterization of a mitochondrial dicarboxylate-tricarboxylate carrier of Plasmodium falciparum.

The malaria parasite, Plasmodium falciparum, was recently shown to operate a branched pathway of tricarboxylic acid (TCA) metabolism. To identify and characterize membrane transporters required for such TCA metabolism in the parasite, we isolated a cDNA for a dicarboxylate-tricarboxylate carrier homolog (PfDTC), synthesized the encoded protein with the use of a cell-free translation system, and determined the substrate specificity of its transport activity with a proteoliposome reconstitution system. PfDTC was found to mediate efficient oxoglutarate-malate, oxoglutarate-oxaloacetate, or oxoglutarate-oxoglutarate exchange across the liposome membrane. Our results suggest that PfDTC may mediate the oxoglutarate-malate exchange across the inner mitochondrial membrane required for the branched pathway of TCA metabolism in the malaria parasite.

Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system.

BACKGROUND: Recently, some groups have reported on cell-free synthesis of functional membrane proteins (MPs) in the presence of exogenous liposomes (liposomes). Previously, we reported synthesis of a functional AtPPT1 plant phosphate transporter that was associated with liposomes during translation. However, it is unclear whether or not lipid/MP complex formation is common to all types of MPs in the wheat cell-free system. RESULTS: AtPPT1 was synthesized using a wheat cell-free system with or without liposomes. AtPPT1 synthesized with liposomes showed high transport activity, but the activity of AtPPT1 synthesized without liposomes was less than 10% activity of that with liposomes. To test whether co-translational association with liposomes is observed in the synthesis of other MPs, we used 40 mammalian MPs having one to 14 transmembrane domains (TMDs) and five soluble proteins as a control. The association rate of all 40 MPs into liposomes was more than 40% (mean value: 59%), while that of the five soluble proteins was less than 20% (mean value: 12%). There were no significant differences in association rate among MPs regardless of the number of TMDs and synthesis yield. These results indicate that the wheat cell-free system is a highly productive method for lipid/MP complex formation and is suitable for large-scale preparation. The liposome association of green fluorescent protein (GFP)-fusion MPs were also tested and recovered as lipid/MP complex after floatation by Accudenz density gradient ultracentrifugation (DGU). Employment of GFP-MPs revealed optimal condition for Accudenz floatation. Using the optimized Accudenz DGU condition, P2RX4/lipid complexes were partially purified and detected as a major band by Coomassie Brilliant Blue (CBB)-staining after SDS-PAGE. CONCLUSION: Formation of lipid/AtPPT1 complex during the cell-free synthesis reaction is critical for synthesis of a functional MP. The lipid/MP complex during the translation was observed in all 40 MPs tested. At least 29 MPs, as judged by their higher productivity compared to GFP, might be suitable for a large-scale preparation. MPs synthesized by this method form lipid/MP complexes, which could be readily partially purified by Accudenz DGU. Wheat cell-free protein synthesis in the presence of liposomes will be a useful method for preparation of variety type of MPs.

In vitro protein import of a putative amino acid transporter from Arabidopsis thaliana into chloroplasts and its suborganellar localization.

We identified a gene product of At5g19500 (At5g19500p) from Arabidopsis thaliana that is homologous to EcTyrP, a tyrosine-specific transporter from Escherichia coli. Computational analyses of the amino acid sequence of At5g19500p predicted 11 transmembrane domains (TMDs) and a potential plastid targeting signal at its amino terminus. As a first step toward understanding the possible role of At5g19500p in plant cells, we attempted to determine the localization of At5g19500p by an in vitro chloroplastic import assay using At5g19500p translated in a cell-free wheat germ system (Madin et al., Proc. Natl. Acad. Sci. USA, 97, 559-564 (2000)), followed by subfractionation of the chloroplasts. At5g19500p was successfully imported into chloroplasts, and the newly transported mature form of At5g19500p was recovered from the inner envelope membrane.

Deciphering OPDA Signaling Components in the Momilactone-Producing Moss Calohypnum plumiforme.

Jasmonic acid (JA) and its biologically active form jasmonoyl-L-isoleucine (JA-Ile) regulate defense responses to various environmental stresses and developmental processes in plants. JA and JA-Ile are synthesized from α-linolenic acids derived from membrane lipids via 12-oxo-phytodienoic acid (OPDA). In the presence of JA-Ile, the COI1 receptor physically interacts with JAZ repressors, leading to their degradation, resulting in the transcription of JA-responsive genes by MYC transcription factors. Although the biosynthesis of JA-Ile is conserved in vascular plants, it is not recognized by COI1 in bryophytes and is not biologically active. In the liverwort Marchantia polymorpha, dinor-OPDA (dn-OPDA), a homolog of OPDA with two fewer carbons, and its isomer dn-iso-OPDA accumulate after wounding and are recognized by COI1 to activate downstream signaling. The moss Calohypnum plumiforme produces the antimicrobial-specialized metabolites, momilactones. It has been reported that JA and JA-Ile are not detected in C. plumiforme and that OPDA, but not JA, can induce momilactone accumulation and the expression of these biosynthetic genes, suggesting that OPDA or its derivative is a biologically active molecule in C. plumiforme that induces chemical defense. In the present study, we investigated the biological functions of OPDA and its derivatives in C. plumiforme. Searching for the components potentially involving oxylipin signaling from transcriptomic and genomic data revealed that two COI1, three JAZ, and two MYC genes were present. Quantification analyses revealed that OPDA and its isomer iso-OPDA accumulated in larger amounts than dn-OPDA and dn-iso-OPDA after wounding. Moreover, exogenously applied OPDA, dn-OPDA, or dn-iso-OPDA induced the transcription of JAZ genes. These results imply that OPDA, dn-OPDA, and/or their isomers potentially act as biologically active molecules to induce the signaling downstream of COI1-JAZ. Furthermore, co-immunoprecipitation analysis showed the physical interaction between JAZs and MYCs, indicating the functional conservation of JAZs in C. plumiforme with other plants. These results suggest that COI1-JAZ-MYC mediated signaling is conserved and functional in C. plumiforme.

Efficient production and purification of functional bacteriorhodopsin with a wheat-germ cell-free system and a combination of Fos-choline and CHAPS detergents.

Cell-free translation is one potential approach to the production of functional transmembrane proteins. We have now examined various detergents as supplements to a wheat-germ cell-free system in order to optimize the production and subsequent purification of a functional model transmembrane protein, bacteriorhodopsin. We found that Fos-choline and CHAPS detergents counteracted each other's inhibitory effects on cell-free translation activity and thereby allowed the efficient production and subsequent purification of functional bacteriorhodopsin in high yield.