BACKGROUND:Chronic inflammation contributes to the development of lifestyle-related diseases, and dietary phytochemicals are recognized as important modulators of inflammatory responses. However, the synergistic anti-inflammatory effects of phytochemical combinations and their underlying mechanisms remain insufficiently understood. METHODS:The anti-inflammatory activities of menthol (ME), 1,8-cineole (CI), β-eudesmol (EU), and capsaicin (CA) were evaluated in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Pro-inflammatory gene expression was quantified by quantitative PCR, intracellular Ca2+ signaling was assessed by calcium imaging, and the involvement of transient receptor potential (TRP) channels was examined using selective inhibitors. Synergistic effects were analyzed based on changes in half-maximal effective concentrations (EC50). RESULTS:All compounds suppressed LPS-induced pro-inflammatory genes, including tumor necrosis factor-alpha (Tnf) and interleukin-6 (Il6), in a dose-dependent manner, with CA showing the lowest EC50 for Tnf expression (0.087 µM). Notably, combinations of CA with ME or CI exhibited strong synergy, reducing their EC50 values by 699-fold and 154-fold, respectively, without cytotoxicity. These effects likely resulted from the synergic interaction between ME/CI-induced TRP-mediated signaling and CA-activated, TRP-independent signaling. CONCLUSIONS:Specific combinations of plant-derived functional components can markedly enhance anti-inflammatory efficacy, supporting dietary strategies that harness multiple phytochemicals for inflammation control and disease prevention.

Ubiquitination regulates pattern-triggered immune responses in plants, but the underlying mechanisms remain incompletely understood. In Arabidopsis, the E3 ubiquitin ligase PUB4 has been implicated in growth, development, and stress responses, and is known to interact with and be phosphorylated by CERK1, resulting in the positive regulation of chitin-triggered immunity. In this study, we identified ubiquitinated proteins enriched from chitin-treated microsomal fractions and discovered the receptor kinase FERONIA as a potential target of PUB4. FERONIA interacted with PUB4 both in vivo and in vitro, as evidenced by yeast two-hybrid, BiFC, and co-immunoprecipitation assays. An in vitro ubiquitination assay demonstrated that PUB4 ubiquitinates the cytoplasmic domain of FERONIA. Moreover, transient expression assays in wild-type and pub4-2 mutant protoplasts revealed that PUB4 mediates chitin-induced FERONIA degradation. Unlike PUB4, which positively regulates chitin-induced defense responses, FERONIA negatively regulates them. For example, fer4 mutants exhibited elevated basal and chitin-induced ROS and callose levels. This suggests that FERONIA suppresses immune signaling under steady-state conditions, and that PUB4-mediated degradation of FERONIA relieves this inhibition. These findings clearly reveal a novel mechanism by which PUB4 regulates chitin-induced immunity driven by CERK1 through targeted degradation of FERONIA. This highlights the balance between positive and negative regulators of plant immune signaling.

While the epidermal cells of terrestrial plants typically lack chloroplasts, this is not the case for aquatic plants, a trait that likely evolved to enhance photosynthetic efficiency underwater. Amphibious plants display various response strategies to survive under both terrestrial and aquatic environments. In this study, we described an environmental response in the amphibious plant Rorippa aquatica that involves chloroplasts differentiating into epidermal cells upon submergence. This phenomenon has not been previously documented and was named environmentally responsive epidermal chloroplast differentiation (ECD). To elucidate the mechanisms underlying ECD, we conducted RNA-sequencing analysis. The results revealed ethylene signaling, hypoxia responses, and light-regulated chloroplast development as key pathways. Physiological experiments showed that ethylene is a central trigger; exogenous ethylene or 1-aminocyclopropane-1-carboxylic acid promote ECD, whereas silver nitrate inhibits it. Submergence alone was insufficient to fully induce ECD, with underwater light exposure accelerating chloroplast maturation. Comparative analysis of phylogenetically distant amphibious species revealed that ECD is not unique to R. aquatica, implying ECD occurs in other species as well, with its extent varying among them. These findings provide new insights on plant acclimation to aquatic environments and highlight the intricate interplay between submergence, ethylene, and light in regulating chloroplast differentiation.

Gold nanoparticles with brush structures of nucleic acid drugs (Nuc-AuNPs) are prepared by mixing thiol-modified nucleic acid drugs and AuNPs due to the strong affinity of the Au-S bond. However, effectively regulating the intracellular kinetics of nucleic acids remains a challenge in achieving highly efficient nucleic-acid delivery. In this study, we designed new DNA-Schiff-AuNPs. The DNA release behaviors of these DNA-modified AuNPs were characterized under acidic conditions analogous to those in intracellular endosomes. Cy5-labeled DNA with an amine terminus (Cy5-DNA-NH2) reacted with 4-mercaptobenzaldehyde in dimethyl sulfoxide to form a Schiff base. The resulting Cy5-DNA-Schiff-SH was added to the AuNP dispersion system to prepare Cy5-DNA-Schiff-AuNPs. These nanoconjugates were characterized by fluorescence spectroscopy. In the appropriate buffer, the fluorescence intensity of AuNPs remained constant at pH 7.4, while exhibiting an approximately four-fold increase at pH 4.0 and 5.0. In addition, a drastic color change from red to blue was observed after 24 h, suggesting that DNA was released from AuNPs at a pH of 5.5. The DNA-Schiff-AuNPs were effectively incorporated into HeLa cells without any toxicity. Furthermore, siRNA-AuNPs incorporating Schiff base linkages demonstrated significantly enhanced suppression of epidermal growth factor receptor gene expression compared to conventional siRNA-AuNPs. These findings highlight the potential of Schiff base-linked Nuc-AuNPs as a promising platform for intracellular nucleic acid delivery.

Spider mites (Tetranychus urticae) are a major threat to economically important crops. Here, we investigated the potential of tetranins, in particular Tet3 and Tet4, as T. urticae protein-type elicitors that stimulate plant defense. Truncated Tet3 and Tet4 proteins showed efficacy in activating the defense gene pathogenesis-related 1 (PR1) and inducing phytohormone production in leaves of Phaseolus vulgaris. In particular, Tet3 caused a drastically higher Ca2+ influx in leaves, but a lower reactive oxygen species (ROS) generation compared to other tetranins, whereas Tet4 caused a low Ca2+ influx and a high ROS generation in the host plants. Such specific and non-specific elicitor activities were examined by knockdown of Tet3 and Tet4 expressions in mites, confirming their respective activities and in particular showing that they function additively or synergistically to induce defense responses. Of great interest is the fact that Tet3 and Tet4 expression levels were higher in mites on their preferred host, P. vulgaris, compared to the levels in mites on the less-preferred host, Cucumis sativus, whereas Tet1 and Tet2 were constitutively expressed regardless of their host. Furthermore, mites that had been hosted on C. sativus induced lower levels of PR1 expression, Ca2+ influx and ROS generation, i.e., Tet3- and Tet4-responsive defense responses, in both P. vulgaris and C. sativus leaves compared to the levels induced by mites that had been hosted on P. vulgaris. Taken together, these findings show that selected tetranins respond to variable host cues that may optimize herbivore fitness by altering the anti-mite response of the host plant.

Interplant interactions mediated by plant-emitted volatile organic compounds (VOCs) occur with heterospecific neighboring plants, making VOC-emitting companion plants a practical tool for pest management. In this study, we investigated the cocultivation of common bean (Phaseolus vulgaris L.) with bush basil (Ocimum basilicum L. var. minimum), which induced the expression of defense-related genes, such as pathogenesis-related protein 1, via salicylate signaling in the leaves. This coculture enhanced the defense capacity of P. vulgaris against spider mites (Tetranychus urticae) and field herbivores. Eugenol, one of the major VOCs emitted by bush basil plants, was likely responsible for conferring the antiherbivore activity on the receiver plants. Furthermore, bush basil VOCs were able to prime P. vulgaris plants to attract Phytoseiulus persimilis, a predatory mite of T. urticae, in response to T. urticae attack. Our results suggest a practical use of bush basil as companion plants in organic crop production.

Our newly developed menthyl esters of valine and isoleucine exhibit anti-inflammatory properties beyond those of the well-known menthol in macrophages stimulated by lipopolysaccharide (LPS) and in a mouse model of colitis induced by sodium dextran sulfate. Unlike menthol, which acts primarily through the cold-sensitive TRPM8 channel, these menthyl esters displayed unique mechanisms that operate independently of this receptor. They readily penetrated target cells and efficiently suppressed LPS-stimulated tumour necrosis factor-alpha (Tnf) expression mediated by liver X receptor (LXR), a key nuclear receptor that regulates intracellular cholesterol and lipid balance. The menthyl esters showed affinity for LXR and enhanced the transcriptional activity through their non-competitive and potentially synergistic agonistic effect. This effect can be attributed to the crucial involvement of SCD1, an enzyme regulated by LXR, which is central to lipid metabolism and plays a key role in the anti-inflammatory response. In addition, we discovered that the menthyl esters showed remarkable efficacy in suppressing adipogenesis in 3T3-L1 adipocytes at the mitotic clonal expansion stage in an LXR-independent manner as well as in mice subjected to diet-induced obesity. These multiple capabilities of our compounds establish them as formidable allies in the fight against inflammation and obesity, paving the way for a range of potential therapeutic applications.

Volatile organic compounds (VOCs) are essential airborne mediators of interactions between plants. These plant-plant interactions require sophisticated VOC-sensing mechanisms that enable plants to regulate their defenses against pests. However, these interactions are not limited to specific plants or even conspecifics, and can function in very flexible interactions between plants. Sensing and responding to VOCs in plants is finely controlled by their uptake and transport systems as well as by cellular signaling via, for example, chromatin remodeling system-based transcriptional regulation for defense gene activation. Based on the accumulated knowledge about the interactions between plants and their major VOCs, companion plants and biostimulants are being developed for practical applications in agricultural and horticultural pest control, providing a sustainable alternative to harmful chemicals.

Terpenoids, natural compounds released by plants, function to enhance plant defense. The aim of this study was to investigate the effects of terpenoid-enriched essential oils (EOs) on tomato plants. From the application of a highly diluted solution of 11 different EOs to potted tomato soil, our study showed that rose essential oil (REO), rich in β-citronellol, played a crucial role in activating defense genes in tomato leaves. As a result, leaf damage caused by herbivores, such as Spodoptera litura and Tetranychus urticae, was significantly reduced. In addition, our results were validated in field trials, providing evidence that REO is an effective biostimulant for enhancing plant defense against pests. Notably, the REO solution also had the added benefit of attracting herbivore predators, such as Phytoseiulus persimilis. Our findings suggest a practical approach to promote organic tomato production that encourages environmentally friendly and sustainable practices.

Plants defend themselves against herbivores by recognizing herbivore-derived elicitors and activating intracellular signaling. In Arabidopsis, the receptor-like kinase HAK1 recognizes the poly-saccharide elicitor (FrA) from Spodoptera litura larvae, leading to the expression of defense-related genes such as PDF1.2. During this process, the cytoplasmic kinase CRK2 phosphorylates PBL27, triggers the ERF13 expression via ethylene signaling and subsequently leads to PDF1.2 expression. Herein, we investigated four cytoplasmic kinases from the same receptor-like cytoplasmic kinase (RLCK) VII family as PBL27 that interacts with CRK2. Among them, PBL11, like PBL27, is phosphorylated by CRK2 and induces PDF1.2 expression but does not affect ERF13 expression. The weight gain of S. litura larvae on PBL11-deficient mutant plants was only slightly higher than that of wild-type plants, suggesting that PBL11 may function as a minor RLCK that supports the defense response.

Histone modification is a cellular process for transcriptional regulation. In herbivore-damaged plants, activation of genes involved in defence responses is required for antiherbivore properties, but little is known about how the chromatin remodelling system is involved. In Arabidopsis (Arabidopsis thaliana) plants responding to Spodoptera litura larvae, HAC1 and HDA6, a histone acetyltransferase and a histone deacetylase, respectively, were found here to be involved in histone H3 (Lys9; H3K9) acetylation/deacetylation at the promoter region of the plant defensin gene PDF1.2 and the gene body of ethylene response factor 13 (ERF13) as early as 2 h after the onset of herbivore attack. The H3K9 acetylation was responsible for the robust upregulation of PDF1.2 later, at 24 h, and ERF13 even earlier, at 1 h. TOPLESS (TPL) and TOPLESS-related (TPR) corepressors interacted with HDA6 to deacetylate H3K9 at PDF1.2 and ERF13, while negatively regulating the expression of PDF1.2 but not ERF13. Furthermore, TPL also interacted with ERF13, resulting in ERF13-mediated regulation of PDF1.2. Taken together, these data suggest a model of promoter-restricted, TPL/TPR-directed histone deacetylation and transcription factor repression in healthy Arabidopsis plants for the feedback regulation of the antiherbivore response.

Menthyl ester of valine (MV) has been developed as a plant defense potentiator to induce pest resistance in crops. In this study, we attempted to establish MV hydrochloride (MV-HCl) in lettuce and tomato crops. When MV-HCl solutions were used to treat soil or leaves of potted tomato and lettuce plants, 1 µM MV-HCl solution applied to potted plant soil was most effective in increasing the transcript level of defense genes such as pathogenesis-related 1 (PR1). As a result, leaf damage caused by Spodoptera litura and oviposition by Tetranychus urticae were significantly reduced. In addition, MV-HCl-treated plants showed an increased ability to attract Phytoseiulus persimilis, a predatory mite of T. urticae, when they were attacked by T. urticae. Overall, our findings showed that MV-HCl is likely to be effective in promoting not only direct defense by activating defense genes, but also indirect defense mediated by herbivore-induced plant volatiles. Moreover, based on the results of the sustainability of PR1 expression in tomato plants treated with MV-HCl every 3 days, field trials were conducted and showed a 70% reduction in natural leaf damage. Our results suggest a practical approach to promoting organic tomato and lettuce production using this new plant defense potentiator.

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.

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.

Biotechnology and BioengineeringVolume 120, Issue 5 p. 1167-1170 ISSUE INFORMATIONFree Access Biotechnology and Bioengineering: Volume 120, Number 5, May 2023 First published: 12 April 2023 https://doi.org/10.1002/bit.28138AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Volume120, Issue5May 2023Pages 1167-1170 RelatedInformation

Betalains, which consist of the subgroups betaxanthins and betacyanins, are hydrophilic pigments that have classically been used for food colorants. Owing to their strong antioxidant property, their usefulness for application for therapeutic use is also expected. In addition, as betalains are mainly naturally available from plants of the order Caryophyllales, including beet (Beta vulgaris), metabolic engineering for betalain production in crops such as vegetables, fruits and cereals may provide new food resources useful for healthcare. Here we conducted metabolic engineering of betacyanins in tomato fruits and potato tubers. The transgenic tomato fruits and potato tubers with coexpression of betacyanin biosynthesis genes, CYP76AD1 from B. vulgaris, DOD (DOPA 4,5-dioxygenase) and 5GT (cyclo-DOPA 5-O-glucosyltransferase) from Mirabilis jalapa, under control of suitable specific promoters, possessed dark red tissues with enriched accumulation of betacyanins (betanin and isobetanin). The anti-inflammatory activity of transgenic tomato fruit extract was superior to that of wild-type fruit extract on macrophage RAW264.7 cells stimulated with lipopolysaccharide (LPS), as a result of decreased LPS-stimulated transcript levels of proinflammatory genes. These findings were in accord with the observation that administration of the transgenic tomato fruits ameliorated dextran sulfate sodium (DSS)-induced colitis as well as body weight loss and disease activity index in mice, via suppression of DSS-stimulated transcript levels of pro-inflammatory genes, including Tnf (encoding TNF-alpha), Il6, and Ptgs2 (encoding cyclooxygenae 2). Intriguingly, given the fact that the transgenic potato tuber extract failed to enrich the anti-inflammatory activity of macrophage cells, it is likely that metabolic engineering of betacyanins will be a powerful way of increasing the anti-inflammatory property of ordinary foods such as tomato.

Plants perceive volatiles emitted from herbivore-damaged neighboring plants to urgently adapt or prime their defense responses to prepare for forthcoming herbivores. Mechanistically, these volatiles can induce epigenetic regulation based on histone modifications that alter the transcriptional status of defense genes, but little is known about the underlying mechanisms. To understand the roles of such epigenetic regulation of plant volatile signaling, we explored the response of Arabidopsis (Arabidopsis thaliana) plants to the volatile β-ocimene. Defense traits of Arabidopsis plants toward larvae of Spodoptera litura were induced in response to β-ocimene, through enriched histone acetylation and elevated transcriptional levels of defense gene regulators, including ethylene response factor genes (ERF8 and ERF104) in leaves. The enhanced defense ability of the plants was maintained for 5 d but not over 10 d after exposure to β-ocimene, and this coincided with elevated expression of those ERFs in their leaves. An array of histone acetyltransferases, including HAC1, HAC5, and HAM1, were responsible for the induction and maintenance of the anti-herbivore property. HDA6, a histone deacetylase, played a role in the reverse histone remodeling. Collectively, our findings illuminate the role of epigenetic regulation in plant volatile signaling.