The powerful potential of amino acid menthyl esters for anti-inflammatory and anti-obesity therapies.

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.

Development of Menthyl Esters of Valine for Pest Control in Tomato and Lettuce Crops.

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.

Novel Potential of Rose Essential Oil as a Powerful Plant Defense Potentiator.

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 ß-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.

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.

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.

Metabolic engineering of betacyanin in vegetables for anti-inflammatory therapy.

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.

Sustained defense response via volatile signaling and its epigenetic transcriptional regulation.

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 ß-ocimene. Defense traits of Arabidopsis plants toward larvae of Spodoptera litura were induced in response to ß-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 ß-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.

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.

An amino acid ester of menthol elicits defense responses in plants.

KEY MESSAGE: Valine menthyl ester (ment-Val) acts as a plant defense potentiator for several crop species including soybean. Terpenoids, including menthol, exhibit potent abilities as plant defense potentiators in agriculture and horticulture. In the current study, we developed new terpene derivatives that consisted of menthol and various amino acids and that were expected to act as powerful plant defense potentiators. We used 6 amino acids possessing low-reactive sidechains to synthesize an array of amino acid ester of menthol (ment-aa) compounds. Transcript levels of two defense genes (pathogenesis-related protein 1 [PR1] and trypsin inhibitor [TI]) were evaluated in leaves of soybean plants 24 h after application of aquatic solution of menthol or menthol-aa, and revealed that the valine menthyl ester (ment-Val) alone elevated the transcript level of defense genes, and it did so only at the low dose of 1 µM, not at higher or lower doses tested. Moreover, it appeared that histone acetylation was involved in this effect. Application of ment-Val enabled soybean plants to sustain the increased transcript levels in their leaves for up to 3 days. Moreover, when ment-Val was additionally applied at day 4, at which time the transcript level had declined to the basal level, the transcript level was re-elevated, indicating the possibility that ment-Val could be repeatedly used to sustain pest control. Ment-Val was found to be chemically stable and effective for defense of several crop species. Collectively, these data show that terpenoid conjugates are useful for pest control instead of or in addition to pesticides.

Phytohormone-dependent plant defense signaling orchestrated by oral bacteria of the herbivore Spodoptera litura.

A vast array of herbivorous arthropods live with symbiotic microorganisms. However, little is known about the nature and functional mechanism of bacterial effects on plant defense responses towards herbivores. We explored the role of microbes present in extracts of oral secretion (OS) isolated from larvae of Spodoptera litura, a generalist herbivore, in phytohormone signaling-dependent defense responses in Arabidopsis thaliana (Arabidopsis). In response to mechanical damage (MD) with application of bacteria-free OS (OS- ) prepared by sterilization or filtration of OS, Arabidopsis leaves exhibited enhanced de novo synthesis of oxylipins, and induction of transcript abundance of the responsible genes, in comparison to those in leaves with MD + nonsterilized OS (OS+ ), indicating that OS bacteria serve as suppressors of these genes. By contrast, de novo synthesis/signaling of salicylic acid and signaling of abscisic acid were enhanced by OS bacteria. These signaling networks were cross-regulated by each other. Meta-analysis of OS bacteria identified 70 bacterial strains. Among them was Staphylococcus epidermidis, an anaerobic staphylococcus that was shown to contribute to the suppression/manipulation of phytohormone-dependent plant defense signaling. The presence of OS bacteria was consequently beneficial for S. litura larvae hosted by Brassicaceae.

Making Sense of the Way Plants Sense Herbivores.

Plants are constantly threatened by herbivore attacks and must devise survival strategies. Some plants sense and respond to elicitors including specific molecules secreted by herbivores and molecules that are innate to plants. Elicitors activate diverse arrays of plant defense mechanisms that confer resistance to the predator. Recent new insights into the cellular pathways by which plants sense elicitors and elicit defense responses against herbivores are opening doors to a myriad of agricultural applications. This review focuses on the machinery of herbivory-sensing and on cellular and systemic/airborne signaling via elicitors, exemplified by the model case of interactions between Arabidopsis hosts and moths of the genus Spodoptera.

Soy and Arabidopsis receptor-like kinases respond to polysaccharide signals from Spodoptera species and mediate herbivore resistance.

Plants respond to herbivory by perceiving herbivore danger signal(s) (HDS(s)), including "elicitors", that are present in herbivores' oral secretions (OS) and act to induce defense responses. However, little is known about HDS-specific molecules and intracellular signaling. Here we explored soybean receptor-like kinases (RLKs) as candidates that might mediate HDS-associated RLKs' (HAKs') actions in leaves in response to OS extracted from larvae of a generalist herbivore, Spodoptera litura. Fractionation of OS yielded Frα, which consisted of polysaccharides. The GmHAKs composed of their respective homomultimers scarcely interacted with Frα. Moreover, Arabidopsis HAK1 homomultimers interacted with cytoplasmic signaling molecule PBL27, resulting in herbivory resistance, in an ethylene-dependent manner. Altogether, our findings suggest that HAKs are herbivore-specific RLKs mediating HDS-transmitting, intracellular signaling through interaction with PBL27 and the subsequent ethylene signaling for plant defense responses in host plants.

Evidence that ERF transcriptional regulators serve as possible key molecules for natural variation in defense against herbivores in tall goldenrod.

We collected Solidago altissima clones to explore their leaf damage resistance, and as a result identified five accessions that exhibited variable defense abilities against the generalist herbivore Spodoptera litura. In order to characterize molecules involved in such natural variation, we focused on ethylene response factors (ERFs) that exhibited distinct transcription patterns in the leaves of the five accessions (e.g., S1 and S2) after wounding: the transcript of SaERF1 and SaERF2 was induced in wounded S1 and S2 leaves, respectively. Although transcription levels of SaERFs in leaves of the five accessions did not correlate with the accessions' phytohormone levels, these transcription levels accorded with the possibility that ethylene and jasmonate signaling play crucial roles in wound-induced transcription of SaERF1 in S1 leaves, and SaERF2 in S2 leaves, respectively. SaERF1 was found to be a positive regulator of the GCC box and DRE element in the upstream regions of promoters of defense genes, whereas SaERF2 served as a negative regulator of genes controlled through the GCC box. Transgenic Arabidopsis plants expressing SaERF1 or SaERF2 showed enhanced and suppressed transcript levels, respectively, of a defensin gene, indicating that ERFs may be partly responsible for herbivore resistance properties of S. altissima accessions.

Mint companion plants enhance the attraction of the generalist predator Nesidiocoris tenuis according to its experiences of conspecific mint volatiles.

Mint plants enable improvement of pest management by attracting herbivore enemies to constitutively released mint volatiles. The generalist predator Nesidiocoris tenuis is used worldwide to control agricultural pests, but little is known about whether mint can serve as a companion plant that attracts this predator. To examine this, olfactory responses of N. tenuis were assessed using apple mint, candy mint, and spearmint as odor sources. Of the volatiles released by these mint species, candy mint volatiles alone were more attractive than those from undamaged eggplant, and were as attractive as volatiles from eggplant damaged with Spodoptera litura larvae. However, no prominent preference for particular volatile(s) among the mint volatiles was shown by O. strigicollis. When N. tenuis had been previously exposed to candy mint, the predator showed a stronger preference for candy mint volatiles than damaged eggplant volatiles. It was, however, irrelevant whether the predator received benefit or not by predating animal prey during the mint-experience period. In contrast, spearmint-experience increased the preference for spearmint volatiles only when the predator acquired prey during the mint-experience period. These findings suggest that previous exposure of N. tenuis to some particular mint species can increase its preference for volatiles from the conspecific mints.

Current opinions about herbivore-associated molecular patterns and plant intracellular signaling.

Elicitor-associated compounds included in oral secretions of herbivorous arthropods, defined as herbivore-associated molecular patterns (HAMPs), induce defense responses in plants. Recognition of HAMPs by the host plants triggers the activation of downstream intracellular and intercellular signaling, resulting in the production of defensive secondary metabolites and volatile emissions to defend against herbivore attack. Thus far, several chemical classes of HAMPs, e.g., fatty acid-amino acid conjugates, peptides, enzymes, and oligosaccharides, have been characterized from not only plant-chewing arthropod herbivores but also plant-sucking arthropod herbivores. Here, we introduce the latest insights about HAMPs and the HAMPs-induced defense signaling network in host plants.

Tyrosine Kinase-Dependent Defense Responses Against Herbivory in Arabidopsis.

Tyrosine (Tyr) phosphorylation (TP) is important for promotion of plants' signaling. Arabidopsis calcium-dependent protein kinase related protein kinases (CRK2 and CRK3) phosphorylate Tyr residues of a subset of transcription factors (TFs), including herbivory-responsive ethylene response factor 13 (ERF13), but the in vivo functions of these kinases in plant defense responses and development remain to be clarified. We show that when CRKs were coexpressed with ERF13 in Arabidopsis leaf protoplasts, the transcription activity regulated via ERF13 was elevated by CRK2 but not CRK3 or their kinase-dead form mutants. Moreover, this elevation was abolished when a Tyr-phosphorylation mutant of ERF was coexpressed with CRK2, indicating that CRK2 serves as an effector of ERF13 mediated by Tyr-phosphorylation. Moreover, CRK2 and CRK3 acted as effectors of RAP2.6 and WRKY14, respectively. CRK-overexpressing lines and knockout mutants of Arabidopsis plants showed increased and decreased expression levels of the defensin gene PDF1.2 in leaves, respectively, conferring on the plants modulated defense properties against the generalist herbivore Spodoptera litura. However, these lines did not show any obvious developmental defects, indicating that CRKs play a role in defense responses but not in the ordinary growth or development of plants. Transcription of both CRK2 and CRK3 was positively regulated by jasmonate signaling and abscisic acid (ABA) signaling upon herbivory. Our findings suggest that these phytohormone-responsive CRKs work coordinately for plant defense responses via Tyr phosphorylation of herbivory-responsive regulators.

Tetranins: new putative spider mite elicitors of host plant defense.

The two-spotted spider mite (Tetranychus urticae) is a plant-sucking arthropod herbivore that feeds on a wide array of cultivated plants. In contrast to the well-characterized classical chewing herbivore salivary elicitors that promote plant defense responses, little is known about sucking herbivores' elicitors. To characterize the sucking herbivore elicitors, we explored putative salivary gland proteins of spider mites by using an Agrobacterium-mediated transient expression system or protein infiltration in damaged bean leaves. Two candidate elicitors (designated as tetranin1 (Tet1) and tetranin2 (Tet2)) triggered early leaf responses (cytosolic calcium influx and membrane depolarization) and increased the transcript abundances of defense genes in the leaves, eventually resulting in reduced survivability of T. urticae on the host leaves as well as induction of indirect plant defenses by attracting predatory mites. Tet1 and/or Tet2 also induced jasmonate, salicylate and abscisic acid biosynthesis. Notably, Tet2-induced signaling cascades were also activated via the generation of reactive oxygen species. The signaling cascades of these two structurally dissimilar elicitors are mostly overlapping but partially distinct and thus they would coordinate the direct and indirect defense responses in host plants under spider mite attack in both shared and distinct manners.

Dual nitrogen species involved in foliar uptake of nitrogen dioxide in Arabidopsis thaliana.

Foliar uptake of nitrogen dioxide (NO2) is governed by its reactive absorption mechanism, by which NO2 molecules diffuse through cell wall layers and simultaneously react with apoplastic ascorbate to form nitrous acid, which freely diffuses across plasmalemma. However, whether free diffusion of nitrous acid is the sole mechanism of foliar uptake of NO2 remains unknown. The involvement of ammonia-inhibitable nitrite transporters in the foliar uptake of NO2, as reported in nitrite transport in Arabidopsis roots, is also unknown. In this study, we treated Arabidopsis thaliana leaves with methionine sulfoximine (MSX) to inhibit incorporation of ammonia into glutamate and exposed them to 4 ppm 15N-labeled NO2 for 4 h in light followed by quantification of total nitrogen, reduced nitrogen, and ammonia nitrogen derived from NO2 using mass spectrometry and capillary electrophoresis. The total nitrogen derived from NO2 in leaves without MSX treatment was 587.0 nmol NO2/g fresh weight, of which more than 65% was recovered as reduced nitrogen. In comparison, MSX treatment decreased the total nitrogen and reduced nitrogen derived from NO2 by half. Thus, half of the foliar uptake of NO2 is not attributable to passive diffusion of nitrous acid but to ammonia-inhibitable nitrite transport. Foliar uptake of NO2 is mediated by a dual mechanism in A. thaliana: nitrous acid-free diffusion and nitrite transporter-mediated transport.