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.

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.

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.

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.

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.

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.

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.

Pest management using mint volatiles to elicit resistance in soy: mechanism and application potential.

Plants can eavesdrop on volatile cues emitted from neighboring plants to boost their defense responses. When 10 categories of mints were tested for their effects on Glycine max (soybean) plants cultivated nearby, candy mint (Mentha × piperita cv. Candy) and peppermint (Mentha × piperita L.) induced the strongest enhancement in RNA levels of defense genes in the soybean leaves. The mechanism by which the mint volatiles enhanced these transcript levels was based on histone acetylation within the promoter regions of defense genes. These increases in transcript levels were induced when receiver plants were cultivated near to candy mint, but the priming of the defense responses was instead induced when receiver plants were cultivated at mid-length intervals. Field assays revealed that anti-herbivore ability of soy was strengthened both by co-cultivation and by pre-incubation of receiver plants with candy mint. The same held true for another receiver, Brassica rapa, when the receiver was co-cultivated or pre-incubated with peppermint. Exposure to mint volatiles resulted in lower damage to receiver plants, although ecological effects on the herbivores and predators probably also contributed. Together, our findings indicate that pest management systems relying on mint as companion plants might be commercially useful for reducing herbivore damage in crops.

JRE4 is a master transcriptional regulator of defense-related steroidal glycoalkaloids in tomato.

Steroidal glycoalkaloids (SGAs) are specialized anti-nutritional metabolites that accumulate in Solanum lycopersicum (tomato) and Solanum tuberosum (potato). A series of SGA biosynthetic genes is known to be upregulated in Solanaceae species by jasmonate-responsive Ethylene Response Factor transcription factors, including JRE4 (otherwise known as GAME9), but the exact regulatory significance in planta of each factor has remained unaddressed. Here, via TILLING-based screening of an EMS-mutagenized tomato population, we isolated a JRE4 loss-of-function line that carries an amino acid residue missense change in a region of the protein important for DNA binding. In this jre4 mutant, we observed downregulated expression of SGA biosynthetic genes and decreased SGA accumulation. Moreover, JRE4 overexpression stimulated SGA production. Further characterization of jre4 plants revealed their increased susceptibility to the generalist herbivore Spodoptera litura larvae. This susceptibility illustrates that herbivory resistance is dependent on JRE4-mediated defense responses, which include SGA accumulation. Ethylene treatment attenuated the jasmonate-mediated JRE4 expression induction and downstream SGA biosynthesis in tomato leaves and hairy roots. Overall, this study indicated that JRE4 functions as a primary master regulator of SGA biosynthesis, and thereby contributes toward plant defense against chewing insects.

Overexpression of geraniol synthase induces heat stress susceptibility in Nicotiana tabacum.

MAIN CONCLUSION: Transgenic tobacco plants overexpressing the monoterpene alcohol geraniol synthase exhibit hypersensitivity to thermal stress, possibly due to suppressed sugar metabolism and transcriptional regulation of genes involved in thermal stress tolerance. Monoterpene alcohols function in plant survival strategies, but they may cause self-toxicity to plants due to their hydrophobic and highly reactive properties. To explore the role of these compounds in plant stress responses, we assessed transgenic tobacco plants overexpressing the monoterpene alcohol geraniol synthase (GES plants). Growth, morphology and photosynthetic efficiency of GES plants were not significantly different from those of control plants (wild-type and GUS-transformed plants). While GES plants' direct defenses against herbivores or pathogens were similar to those of control plants, their indirect defense (i.e., attracting herbivore enemy Nesidiocoris tenuis) was stronger compared to that of control plants. However, GES plants were susceptible to cold stress and even more susceptible to extreme heat stress (50 °C), as shown by decreased levels of sugar metabolites, invertase activity and its products (Glc and Fru), and leaf starch granules. Moreover, GES plants showed decreased transcription levels of the WRKY33 transcription factor gene and an aquaporin gene (PIP2). The results of this study show that GES plants exhibit enhanced indirect defense ability against herbivores, but conversely, GES plants exhibit hypersensitivity to heat stress due to suppressed sugar metabolism and gene regulation for thermal stress tolerance.

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.

'Hidden' Terpenoids in Plants: Their Biosynthesis, Localization and Ecological Roles.

Terpenoids are the largest group of plant specialized (secondary) metabolites. These naturally occurring chemical compounds are highly diverse in chemical structure. Although there have been many excellent studies of terpenoids, most have focused on compounds built solely of isoprene units. Plants, however, also contain many 'atypical' terpenoids, such as glycosylated volatile terpenes and composite-type terpenoids, the latter of which are synthesized by the coupling of isoprene units on aromatic compounds. This mini review describes these 'hidden' terpenoids, providing an overview of their biosynthesis, localization, and biological and ecological activities.

Jasmonates induce both defense responses and communication in monocotyledonous and dicotyledonous plants.

Jasmonic acid (JA) and its derivatives (jasmonates, JAs) are phytohormones with essential roles in plant defense against pathogenesis and herbivorous arthropods. Both the up- and down-regulation of defense responses are dependent on signaling pathways mediated by JAs as well as other stress hormones (e.g. salicylic acid), generally those involving the transcriptional and post-transcriptional regulation of transcription factors via protein modification and epigenetic regulation. In addition to the typical model plant Arabidopsis (a dicotyledon), advances in genetics research have made rice a model monocot in which innovative pest control traits can be introduced and whose JA signaling pathway can be studied. In this review, we introduce the dynamic functions of JAs in plant defense strategy using defensive substances (e.g. indole alkaloids and terpenoid phytoalexins) and airborne signals (e.g. green leaf volatiles and volatile terpenes) in response to biotrophic and necrotrophic pathogens as well as above-ground and below-ground herbivores. We then discuss the important issue of how the mutualism of herbivorous arthropods with viruses or bacteria can cause cross-talk between JA and other phytohormones to counter the defense systems.

Herbivore-induced indirect defense across bean cultivars is independent of their degree of direct resistance.

We tested the extent to which resistance of common bean (Phaseolus vulgaris) cultivars to the spider mite Tetranychus urticae parallels the extent to which these plants display indirect defenses via the induced attraction of the predatory mite Phytoseiulus persimilis. First, via field and greenhouse trials on 19 commercial bean cultivars, we selected two spider mite-resistant (Naz and Ks41128) and two susceptible (Akthar and G11867) cultivars and measured the spider mite-induced volatiles and the subsequently induced attraction of predatory mites via olfactory choice assays. The two major volatiles, 4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) and (Z)-3-hexenyl-acetate, were induced in the resistant but not in the susceptible cultivars. However, uninfested susceptible cultivars emitted these volatiles at levels similar to those of mite-infested resistant cultivars. Significant induction of several minor components was observed for all four cultivars except for the infested-susceptible cultivar G11867. Both, the spider mite-resistant cultivar Naz and the susceptible cultivar G11867, attracted more predatory mites when they were infested. In contrast, spider mites induced increased emission of two major and five minor volatiles in Ks41128, but predatory mites did not discriminate between infested and uninfested plants. Overall, the attraction of predatory mites appeared to correlate positively with the presence of TMTT and (Z)-3-hexenyl acetate and negatively with ß-caryophyllene and α-pinene in the bean headspace. Taken together, our data suggest that resistance and attraction of natural enemies via induced volatiles are independent traits. We argue that it should be possible to cross predator-attraction promoting traits into resistant cultivars that lack sufficiently inducible indirect defenses.

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.

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.

Metabolic engineering of the C16 homoterpene TMTT in Lotus japonicus through overexpression of (E,E)-geranyllinalool synthase attracts generalist and specialist predators in different manners.

Plant defenses against herbivores include the emission of specific blends of volatiles, which enable plants to attract natural enemies of herbivores. We characterized a plastidial terpene synthase gene, PlTPS2, from lima bean (Phaseolus lunatus). The recombinant PlTPS2 protein was multifunctional, producing linalool, (E)-nerolidol and (E,E)-geranyllinalool, precursors of (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene [TMTT]. Transgenic Lotus japonicus and Nicotiana tabacum plants, expressing PlTPS2 or its homolog Medicago truncatula TPS3 (MtTPS3), were produced and used for bioassays with herbivorous and predatory mites. Transgenic L. japonicus plants expressing PlTPS2 produced (E,E)-geranyllinalool and TMTT, whereas wild-type plants and transgenic plants expressing MtTPS3 did not. Transgenic N. tabacum expressing PlTPS2 produced (E,E)-geranyllinalool but not TMTT. Moreover, in olfactory assays, the generalist predatory mite Neoseiulus californicus but not the specialist Phytoseiulus persimilis was attracted to uninfested, transgenic L. japonicus plants expressing PlTPS2 over wild-type plants. The specialist P. persimilis was more strongly attracted by the transgenic plants infested with spider mites than by infested wild-type plants. Predator responses to transgenic plant volatile TMTT depend on various background volatiles endogenously produced by the transgenic plants. Therefore, the manipulation of TMTT is an ideal platform for pest control via the attraction of generalist and specialist predators in different manners.