Gen-ichiro Arimura

Herbivory-induced volatiles elicit defence genes in lima bean leaves

In response to herbivore damage, several plant species emit volatiles that attract natural predators of the attacking herbivores. Using spider mites (Tetranychus urticae) and predatory mites (Phytoseiulus persimilis), it has been shown that not only the attacked plant but also neighbouring plants are affected, becoming more attractive to predatory mites and less susceptible to spider mites. The mechanism involved in such interactions, however, remains elusive. Here we show that uninfested lima bean leaves activate five separate defence genes when exposed to volatiles from conspecific leaves infested with T. urticae, but not when exposed to volatiles from artificially wounded leaves. The expression pattern of these genes is similar to that produced by exposure to jasmonic acid. At least three terpenoids in the volatiles are responsible for this gene activation; they are released in response to herbivory but not artificial wounding. Expression of these genes requires calcium influx and protein phosphorylation/dephosphorylation.

Chemical and molecular ecology of herbivore-induced plant volatiles: proximate factors and their ultimate functions.

In response to herbivory, plants emit specific blends of herbivore-induced plant volatiles (HIPVs). HIPVs mediate sizable arrays of interactions between plants and arthropods, microorganisms, undamaged neighboring plants or undamaged sites within the plant in various ecosystems. HIPV profiles vary according to the plant and herbivore species, and the developmental stages and conditions of the live plants and herbivores. To understand the regulatory mechanisms underling HIPV biosynthesis, the following issues are reviewed here: (i) herbivore-induced formation of plant volatile terpenoids and green leaf volatiles; (ii) initial activation of plant responses by feeding herbivores; and (iii) the downstream network of the signal transduction. To understand the ecological significance of HIPVs, we also review case studies of insect-plant and inter-/intraplant interactions mediated by HIPVs that have been documented in the field and laboratory in recent years.

Changing green leaf volatile biosynthesis in plants: An approach for improving plant resistance against both herbivores and pathogens

Green leaf volatiles (GLVs) are commonly emitted by green plants, and their production is drastically enhanced when they are under biotic stress. To clarify the ecological function of naturally emitted GLVs, we studied the response of Arabidopsis, whose GLV biosynthesis had been modified, when subjected to herbivory or a pathogenic infection. There was a significant increase in GLV production after herbivory by cabbage white butterfly larvae and pathogen (gray mold) infection in hydroperoxide lyase (HPL) sense Arabidopsis compared with WT controls. The HPL sense modification resulted in the plant being more attractive to the parasitic wasp Cotesia glomerata, leading to higher mortality of the herbivores. The HPL sense modification also resulted in greater inhibition of growth of the fungus. By contrast, HPL antisense Arabidopsis produced fewer GLVs, attracted fewer parasitoids, and was more susceptible to the pathogens than the WT control. These data show that (i) one of the ecological functions of GLV biosynthesis related to resistance against both herbivores and pathogens, and (ii) the genetic modification of GLV biosynthesis could be a unique approach for improving plant resistance against such biotic stresses.

Effects of Feeding Spodoptera littoralis on Lima Bean Leaves. III. Membrane Depolarization and Involvement of Hydrogen Peroxide

In response to herbivore (Spodoptera littoralis) attack, lima bean (Phaseolus lunatus) leaves produced hydrogen peroxide (H2O2) in concentrations that were higher when compared to mechanically damaged (MD) leaves. Cellular and subcellular localization analyses revealed that H2O2 was mainly localized in MD and herbivore-wounded (HW) zones and spread throughout the veins and tissues. Preferentially, H2O2 was found in cell walls of spongy and mesophyll cells facing intercellular spaces, even though confocal laser scanning microscopy analyses also revealed the presence of H2O2 in mitochondria/peroxisomes. Increased gene and enzyme activations of superoxide dismutase after HW were in agreement with confocal laser scanning microscopy data. After MD, additional application of H2O2 prompted a transient transmembrane potential (Vm) depolarization, with a Vm depolarization rate that was higher when compared to HW leaves. In transgenic soybean (Glycine max) suspension cells expressing the Ca2+-sensing aequorin system, increasing amounts of added H2O2 correlated with a higher cytosolic calcium ([Ca2+]cyt) concentration. In MD and HW leaves, H2O2 also triggered the increase of [Ca2+]cyt, but MD-elicited [Ca2+]cyt increase was more pronounced when compared to HW leaves after addition of exogenous H2O2. The results clearly indicate that Vm depolarization caused by HW makes the membrane potential more positive and reduces the ability of lima bean leaves to react to signaling molecules.

Loblolly pine abietadienol/abietadienal oxidase PtAO (CYP720B1) is a multifunctional, multisubstrate cytochrome P450 monooxygenase

Cytochrome P450 monooxygenases (P450s) are important enzymes for generating some of the enormous structural diversity of plant terpenoid secondary metabolites. In conifers, P450s are involved in the formation of a suite of diterpene resin acids (DRAs). Despite their important role in constitutive and induced oleoresin defense, a P450 gene of DRA formation has not yet been identified. By using phylogenetic cluster analysis of P450-like ESTs from loblolly pine (Pinus taeda), functional cDNA screening in yeast (Saccharomyces cerevisiae), and in vitro enzyme characterization, we cloned and identified a multifunctional and multisubstrate cytochrome P450 enzyme, CYP720B1 [abietadienol/abietadienal oxidase (PtAO)]. PtAO catalyzes an array of consecutive oxidation steps with several different diterpenol and diterpenal intermediates in loblolly pine DRA biosynthesis. Recombinant PtAO oxidized the respective carbon 18 of abietadienol, abietadienal, levopimaradienol, isopimara-7,15-dienol, isopimara-7,15-dienal, dehydroabietadienol, and dehydroabietadienal with apparent Michaelis–Menten (Km) values of 0.5–5.3 μM. PtAO expressed in yeast also catalyzed in vivo oxidation of abietadiene to abietic acid, but with activity much lower than with abietadienol or abietadienal. Consistent with a role of DRAs in conifer defense, PtAO transcript levels increased upon simulated insect attack using methyl jasmonate treatment of loblolly pine. The multisubstrate, multifunctional P450 diterpene oxidase PtAO, in concert with expression of a family of single-product and multiproduct diterpene synthases, allows for formation of a diverse suite of DRA defense metabolites in long-lived conifers.

Plant–plant interactions mediated by volatiles emitted from plants infested by spider mites

In an earlier study, we demonstrated plant–plant interactions mediated by volatiles released from lima bean leaves infested by spider mites (Tetranychus urticae) (Nature 406 (2000a) 512, Biochem. Biophys. Res. Commun. 277 (2000b) 305). In the present study, we further show that, under laboratory conditions, volatiles emitted from T. urticae-infested lima bean plants activate transcription of genes encoding pathogenesis-related proteins and phenylalanine ammonia-lyase in leaves of intact neighboring plants. This finding indicates that intact lima bean plants may be responsive to volatile signals. Further, as green leaf volatiles (GLVs) are released from green plants in response to mechanical damage caused by herbivores, we studied possible involvement of GLVs in plant–plant interaction. We found that (Z)-3-hexenol, (E)-2-hexenal, and (Z)-3-hexenyl acetate, induced the expression of defense genes in uninfested leaves. This finding suggests that GLVs may act as signal compounds in plant–plant interactions.

Effects of Feeding Spodoptera littoralis on Lima Bean Leaves: IV. Diurnal and Nocturnal Damage Differentially Initiate Plant Volatile Emission

Continuous mechanical damage initiates the rhythmic emission of volatiles in lima bean (Phaseolus lunatus) leaves; the emission resembles that induced by herbivore damage. The effect of diurnal versus nocturnal damage on the initiation of plant defense responses was investigated using MecWorm, a robotic device designed to reproduce tissue damage caused by herbivore attack. Lima bean leaves that were damaged by MecWorm during the photophase emitted maximal levels of β-ocimene and (Z)-3-hexenyl acetate in the late photophase. Leaves damaged during the dark phase responded with the nocturnal emission of (Z)-3-hexenyl acetate, but with only low amounts of β-ocimene; this emission was followed by an emission burst directly after the onset of light. In the presence of 13CO2, this light-dependent synthesis of β-ocimene resulted in incorporation of 75% to 85% of 13C, demonstrating that biosynthesis of β-ocimene is almost exclusively fueled by the photosynthetic fixation of CO2 along the plastidial 2-C-methyl-d-erythritol 4-P pathway. Jasmonic acid (JA) accumulated locally in direct response to the damage and led to immediate up-regulation of the P. lunatus β-ocimene synthase gene (PlOS) independent of the phase, that is, light or dark. Nocturnal damage caused significantly higher concentrations of JA (approximately 2–3 times) along with enhanced expression levels of PlOS. Transgenic Arabidopsis thaliana transformed with PlOS promoter∷β-glucuronidase fusion constructs confirmed expression of the enzyme at the wounded sites. In summary, damage-dependent JA levels directly control the expression level of PlOS, regardless of light or dark conditions, and photosynthesis is the major source for the early precursors of the 2-C-methyl-d-erythritol 4-P pathway.

Herbivore-induced defense response in a model legume. Two-spotted spider mites induce emission of (E)-beta-ocimene and transcript accumulation of (E)-beta-ocimene synthase in Lotus japonicus

Indirect defense of plants against herbivores often involves the induced emission of volatile infochemicals including terpenoids that attract natural enemies of the herbivores. We report the isolation and characterization of a terpene synthase cDNA (LjEβOS) from a model legume, Lotus japonicus. Recombinant LjEβOS enzyme produced (E)-β-ocimene (98%) and its Z-isomer (2%). Transcripts of LjEβOS were induced in L. japonicus plants infested with two-spotted spider mites (Tetranychus urticae), coinciding with increasing emissions of (E)-β-ocimene as well as other volatiles, (Z)-3-hexenyl acetate and (E)-4,8-dimethyl-1,3,7-nonatriene, by the infested plants. We suggest that LjEβOS is involved in the herbivore-induced indirect defense response of spider mite-infested L. japonicus via de novo formation and emission (E)-β-ocimene. Mechanical wounding of the leaves or application of alamethicin (ALA), a potent fungal elicitor of plant volatile emission, also induced transiently increased levels of LjEβOS transcripts in L. japonicus. However, wounding or ALA did not result in elevated release of (E)-β-ocimene. Differences in volatile emissions after herbivory, mechanical wounding, or treatment with ALA suggest that neither a single mechanical wounding event nor ALA simulate the effect of herbivore activity and indicate that herbivore-induced emission of (E)-β-ocimene in L. japonicus involves control mechanisms in addition to up-regulation of LjEβOS transcripts.

Functional identification of AtTPS03 as (E)-beta-ocimene synthase: a monoterpene synthase catalyzing jasmonate- and wound-induced volatile formation in Arabidopsis thaliana

Abstract. (E)-β-Ocimene is one of the most commonly found monoterpenes of the volatile blends that are emitted from leaves in response to damage by herbivores or mechanical wounding. (E)-β-Ocimene is also a component of many floral scents. Airborne (E)-β-ocimene emitted from plants can serve as a chemical cue for the attraction of parasitoids or predators of plant herbivores and also as an attractant for pollinating insects. Furthermore, exposure of plants to (E)-β-ocimene can activate defense gene expression. In this paper, we describe cDNA cloning and functional characterization of a gene encoding a highly specialized (E)-β-ocimene synthase, AtTPS03, from Arabidopsis thaliana (L.) Heynh. AtTPS03 was identified as a member of the large AtTPS gene family of putative terpene synthases. A cDNA for AtTPS03 was expressed in Escherichia coli and the enzyme function identified in vitro. The A. thaliana (E)-β-ocimene synthase produces almost exclusively (E)-β-ocimene (94%) with minor amounts of the related acyclic monoterpenes (Z)-β-ocimene (4%) and myrcene (2%). Transcripts for AtTPS03 were up-regulated in leaves of Arabidopsis in response to mechanical wounding and treatment with jasmonic acid, concurrent with induced emission of (E)-β-ocimene. AtTPS03 provides an important gene for probing plant–insect and possibly plant–plant interactions mediated by terpenoid volatiles.

Herbivore-induced terpenoid emission in Medicago truncatula: concerted action of jasmonate, ethylene and calcium signaling

Plant volatiles emitted by Medicago truncatula in response to feeding larvae of Spodoptera exigua are composed of a complex blend of terpenoids. The cDNAs of three terpene synthases (TPSs), which contribute to the blend of terpenoids, were cloned from M. truncatula. Their functional characterization proved MtTPS1 to be a β-caryophyllene synthase and MtTPS5 to be a multi-product sesquiterpene synthase. MtTPS3 encodes a bifunctional enzyme producing (E)-nerolidol and geranyllinalool (precursors of C11 and C16 homoterpenes) from different prenyl diphosphates serving as substrates. The addition of jasmonic acid (JA) induced expression of the TPS genes, but terpenoid emission was higher from plants treated with JA and the ethylene precursor 1-amino-cyclopropyl-1-carboxylic acid. Compared to infested wild-type M. truncatula plants, lower amounts of various sesquiterpenes and a C11–homoterpene were released from an ethylene-insensitive mutant skl. This difference coincided with lower transcript levels of MtTPS5 and of 1-deoxy-d-xylulose-5-phosphate synthase (MtDXS2) in the damaged skl leaves. Moreover, ethephon, an ethylene-releasing compound, modified the extent and mode of the herbivore-stimulated Ca2+ variations in the cytoplasm that is necessary for both JA and terpene biosynthesis. Thus, ethylene contributes to the herbivory-induced terpenoid biosynthesis at least twice: by modulating both early signaling events such as cytoplasmic Ca2+-influx and the downstream JA-dependent biosynthesis of terpenoids.