Evolution of the isoprene biosynthetic pathway in kudzu

Isoprene synthase converts dimethylallyl diphosphate, derived from the methylerythritol 4-phosphate (MEP) pathway, to isoprene. Isoprene is made by some plants in substantial amounts, which affects atmospheric chemistry, while other plants make no isoprene. As part of our long-term study of isoprene synthesis, the genetics of the isoprene biosynthetic pathway of the isoprene emitter, kudzu (Pueraria montana), was compared with similar genes in Arabidopsis (Arabidopsis thaliana), which does not make isoprene. The MEP pathway genes in kudzu were similar to the corresponding Arabidopsis genes. Isoprene synthase genes of kudzu and aspen (Populus tremuloides) were cloned to compare their divergence with the divergence seen in MEP pathway genes. Phylogenetic analysis of the terpene synthase gene family indicated that isoprene synthases are either within the monoterpene synthase clade or sister to it. In Arabidopsis, the gene most similar to isoprene synthase is a myrcene/ocimene (acyclic monoterpenes) synthase. Two phenylalanine residues found exclusively in isoprene synthases make the active site smaller than other terpene synthase enzymes, possibly conferring specificity for the five-carbon substrate rather than precursors of the larger isoprenoids. Expression of the kudzu isoprene synthase gene in Arabidopsis caused Arabidopsis to emit isoprene, indicating that whether or not a plant emits isoprene depends on whether or not it has a terpene synthase capable of using dimethylallyl diphosphate.     

(E)-beta-ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: function and expression of three terpene synthase genes of a new terpene synthase subfamily

Snapdragon flowers emit two monoterpene olefins, myrcene and (E)-beta-ocimene, derived from geranyl diphosphate, in addition to a major phenylpropanoid floral scent component, methylbenzoate. Emission of these monoterpenes is regulated developmentally and follows diurnal rhythms controlled by a circadian clock. Using a functional genomics approach, we have isolated and characterized three closely related cDNAs from a snapdragon petal-specific library that encode two myrcene synthases (ama1e20 and ama0c15) and an (E)-beta-ocimene synthase (ama0a23). Although the two myrcene synthases are almost identical (98%), except for the N-terminal 13 amino acids, and are catalytically active, yielding a single monoterpene product, myrcene, only ama0c15 is expressed at a high level in flowers and contributes to floral myrcene emission. (E)-beta-Ocimene synthase is highly similar to snapdragon myrcene synthases (92% amino acid identity) and produces predominantly (E)-beta-ocimene (97% of total monoterpene olefin product) with small amounts of (Z)-beta-ocimene and myrcene. These newly isolated snapdragon monoterpene synthases, together with Arabidopsis AtTPS14 (At1g61680), define a new subfamily of the terpene synthase (TPS) family designated the Tps-g group. Members of this new Tps-g group lack the RRx(8)W motif, which is a characteristic feature of the Tps-d and Tps-b monoterpene synthases, suggesting that the reaction mechanism of Tps-g monoterpene synthase product formation does not proceed via an RR-dependent isomerization of geranyl diphosphate to 3S-linalyl diphosphate, as shown previously for limonene cyclase. Analyses of tissue-specific, developmental, and rhythmic expression of these monoterpene synthase genes in snapdragon flowers revealed coordinated regulation of phenylpropanoid and isoprenoid scent production.     

Volatile C6-aldehydes and Allo-ocimene activate defense genes and induce resistance against Botrytis cinerea in Arabidopsis thaliana

Green leafy volatiles or isoprenoids are produced after mechanical wounding or pathogen/herbivore attacks in higher plants. We monitored expression profiles of the genes involved in defense responses upon exposing Arabidopsis thaliana to the volatiles. Among the genes investigated, those known to be induced by mechanical wounding and/or jasmonate application, such as chalcone synthase (CHS), caffeic acid-O-methyltransferase (COMT), diacylglycerol kinase1 (DGK1), glutathione-S-transferase1 (GST1) and lipoxygenase2 (LOX2), were shown to be induced with (E)-2-hexenal, (Z)-3-hexenal, (Z)-3-hexenol or allo-ocimene (2,6-dimethyl-2,4,6-octatriene). A salicylic acid-responsive gene, pathogenesis-related protein2 (PR2), was not induced by the volatiles. Detailed analyses of the expression profiles showed that the manner of induction varied depending on either the gene monitored or the volatile used. A chemically inert compound, (Z)-3-hexenol, was also potent, which suggested that chemical reactivity was not the sole requisite for the inducing activity. With a jasmonate-insensitive mutant (jar1), the induction by the volatiles was mostly suppressed, however, that of LOX2 was unaltered. An ethylene-insensitive mutant (etr1) showed responses almost identical to the wild type, with minor exceptions. From these observations, it was suggested that both the jasmonate-dependent and -independent pathways were operative upon perception of the volatiles, while the ETR1-dependent pathway was not directly involved. When Botrytis cinerea was inoculated after the volatile treatment, retardation of disease development could be seen. It appears that volatile treatment could make the plants more resistant against the fungal disease.     

UV-C-irradiated Arabidopsis and tobacco emit volatiles that trigger genomic instability in neighboring plants

We have previously shown that local exposure of plants to stress results in a systemic increase in genome instability. Here, we show that UV-C-irradiated plants produce a volatile signal that triggers an increase in genome instability in neighboring nonirradiated Arabidopsis thaliana plants. This volatile signal is interspecific, as UV-C-irradiated Arabidopsis plants transmit genome destabilization to naive tobacco (Nicotiana tabacum) plants and vice versa. We report that plants exposed to the volatile hormones methyl salicylate (MeSA) or methyl jasmonate (MeJA) exhibit a similar level of genome destabilization as UV-C-irradiated plants. We also found that irradiated Arabidopsis plants produce MeSA and MeJA. The analysis of mutants impaired in the synthesis and/or response to salicylic acid (SA) and/or jasmonic acid showed that at least one other volatile compound besides MeSA and MeJA can communicate interplant genome instability. The NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (npr1) mutant, defective in SA signaling, is impaired in both the production and the perception of the volatile signals, demonstrating a key role for NPR1 as a central regulator of genome stability. Finally, various forms of stress resulting in the formation of necrotic lesions also generate a volatile signal that leads to genomic instability.     

茉莉酸甲酯喷施和光肩星天牛（Anoplophora glabripennis(Motschulsky)咬食后五角枫释放的挥发物

茉莉酸甲酯喷施和光肩星天牛(Anoplophora glabripennis(Motschulsky)咬食后五角枫(Acer mono Maxim.)植株均诱导产生了乙酸丁酯、2-壬醇、乙酸己酯、3-甲基-2-丁醇、1-辛烯、里那醇、3-蒈烯、1-丁醇、(Z)-2-己烯醛9种正常植株中没有检测到的挥发物。两种处理均诱导或促进了萜烯类、醇类和醛类挥发物的大量释放,且乙酸-3-己烯酯、乙酸乙酯、己醇、反-2-己烯醇、乙基己醇、1-辛烯、石竹烯、法尼烯等挥发物的时序变化趋势也非常相似。因而推测光肩星天牛咬食五角枫后在植物体内诱导产生了茉莉酸信号传导途径。     

C6-volatiles derived from the lipoxygenase pathway induce a subset of defense-related genes

Six-Carbon (C6-) volatiles, including the aldehydes trans-2-hexenal, hexanal and cis-3-hexenal, as well as their corresponding alcohols, are produced from damaged or wounded plant tissue as a product of the enzymatic activity of hydroperoxide lyase (HPL), a component of the lipoxygenase (LOX) pathway. Aerial treatment of Arabidopsis seedlings with 10 microM concentrations of trans-2-hexenal induces several genes known to be involved in the plant's defense response, including phenylpropanoid-related genes as well as genes of the LOX pathway. Genes encoding the pathogenesis-related proteins PR-1 or PR-2, however, were not induced. Trans-2-hexenal induction thus closely mimics the group of genes induced by methyl jasmonate (MeJA), also a LOX-derived volatile. However, unlike MeJA, trans-2-hexenal did not induce hydroxymethylglutaryl-coenzyme A reductase (HMGR) or thionin2-1. The inductive effect seemed to be limited to C6-related volatiles, as C8-, C9- and other related volatiles did not induce LOX mRNA levels. As has been demonstrated for MeJA, trans-2-hexenal quantitatively reduced wild-type seed germination. Trans-2-hexenal also reduced the germination frequency of the MeJA resistant Arabidopsis mutant, jar1-1, supporting the notion that trans-2-hexenal and MeJA are recognized via different mechanisms. In addition, trans-2-hexenal had a moderate inhibitory effect on root length relative to similar concentrations of MeJA and was approximately 10-fold less effective than MeJA at inducing anthocyanin accumulation in Arabidopsis seedlings. These results suggest that C6-volatiles of the LOX pathway act as a wound signal in plants, but result in a moderate plant response relative to MeJA at both the physiological and molecular level.     

Two Methyl Jasmonate-Insensitive Mutants Show Altered Expression of AtVsp in Response to Methyl Jasmonate and Wounding

Jasmonates are plant signal molecules that are derived from lipids through the action of lipoxygenase. Jasmonates regulate gene expression during plant development and in response to water deficit, wounding, and pathogen elicitors. The signal transduction chain that mediates jasmonate action was investigated by isolating and studying two methyl jasmonate (MeJA)-insensitive mutants of Arabidopsis thaliana. The recessive mutants, jin1 and jin4, are nonallelic and neither corresponds to coi1, a previously identified MeJA-insensitive mutant. Both mutants showed reduced sensitivity to MeJA-mediated root growth inhibition as well as reduced MeJA induction of AtVsp in leaves. Expression of AtVsp in flowers was not altered in the mutants. Furthermore, MeJA modulation of the jasmonate-responsive lipoxygenase and phenylalanine ammonia lyase genes was not altered in the mutants. jin4 plants exhibited increased sensitivity to abscisic acid in seed germination assays, whereas jin1 plants showed wild-type sensitivity. Neither mutant showed altered sensitivity to ethylene in hypocotyl growth inhibition assays. jin1 and jin4 identify genes that modulate the response of AtVsp to MeJA in leaves of A. thaliana.     

Investigations into plant biochemical wound-response pathways involved in the production of aphid-induced plant volatiles

Feeding damage to plants by insect herbivores induces the production of plant volatiles, which are attractive to the herbivores natural enemies. Little is understood about the plant biochemical pathways involved in aphid-induced plant volatile production. The aphid parasitoid Diaeretiella rapae can detect and respond to aphid-induced volatiles produced by Arabidopsis thaliana. When given experience of those volatiles, it can learn those cues and can therefore be used as a novel biosensor to detect them. The pathways involved in aphid-induced volatile production were investigated by comparing the responses of D. rapae to volatiles from a number of different transgenic mutants of A. thaliana, mutated in their octadecanoid, ethylene or salicylic acid wound-response pathways and also from wild-type plants. Plants were either undamaged or infested by the peach-potato aphid, Myzus persicae. It is demonstrated that the octadecanoid pathway and specifically the COI1 gene are required for aphid-induced volatile production. The presence of salicylic acid is also involved in volatile production. Using this model system, in combination with A. thaliana plants with single point gene mutations, has potential for the precise dissection of biochemical pathways involved in the production of aphid-induced volatiles.     

The social wasp Polybia fastidiosuscula Saussure (Hymenoptera: Vespidae) uses herbivore‐induced maize plant volatiles to locate its prey

Social wasps in the Polybia genus are important for use as pest‐control agents in agricultural systems. The objective of this study was to investigate the behavioural responses of Polybia fastidiosuscula Saussure (Hymenoptera: Vespidae) to volatiles from maize, both constitutive volatiles and those induced by the herbivory of Spodoptera frugiperda (JE Smith) (Lepidoptera: Noctuidae). To assess the behavioural response of P. fastidiosuscula to S. frugiperda larvae, undamaged plants, S. frugiperda‐damaged plants, mechanically damaged plants, mechanically damaged plants plus regurgitant from larvae and extracts from various treatments, bioassays were conducted in a Y‐olfactometer. In addition, the volatiles from plants subjected to different treatments were collected via aeration, and they were quantified and identified. The wasps showed a greater preference for plants with damage induced either by larval feeding or by being mechanically damaged plus regurgitant than for undamaged plants or either larvae alone or mechanically damaged plants. Wasps were more attracted to extracts from plants + S. frugiperda larvae and to an extract from mechanically damaged plants + the regurgitant of larvae compared to hexane. The primary compounds induced by herbivory for 5–6 h after the beginning of the damage or regurgitant treatment were identified as α‐pinene, β‐myrcene, (Z)‐3‐hexenyl acetate, limonene, (E)‐ocimene, linalool, DMNT, (E)‐β‐farnesene, TMTT and indole. The results presented here show that the social wasp P. fastidiosuscula uses herbivore‐induced plant volatiles from maize to locate its prey.     

Induced parasitoid attraction by Arabidopsis thaliana: involvement of the octadecanoid and the salicylic acid pathway

Plants can use indirect defence mechanisms to protect themselves against herbivorous insects. An example of such an indirect defence mechanism is the emission of volatiles by plants induced by herbivore feeding. These volatiles can attract the natural enemies of these herbivores, for example, parasitoid wasps. Here, it is shown that the octadecanoid and the salicylic acid pathways are involved in the induced attraction of the parasitoid wasp Cotesia rubecula by Arabidopsis thaliana infested with the herbivore Pieris rapae. Besides exogenous application of jasmonic acid or salicylic acid, use is also made of transgenic Arabidopsis that do not show induced jasmonic acid levels after wounding (S-12) and transgenic Arabidopsis that do not accumulate salicylic acid (NahG). Treatment of Arabidopsis with jasmonic acid resulted in an increased attraction of parasitoid wasps compared with untreated plants, whereas treatment with salicylic acid did not. Transgenic plants impaired in the octadecanoid or the salicylic acid pathway were less attractive than wild-type plants.     

Terpenoid secondary metabolism in Arabidopsis thaliana: cDNA cloning, characterization, and functional expression of a myrcene/(E)-beta-ocimene synthase

The Arabidopsis genome project has recently reported sequences with similarity to members of the terpene synthase (TPS) gene family of higher plants. Surprisingly, several Arabidopsis terpene synthase-like sequences (AtTPS) share the most identity with TPS genes that participate in secondary metabolism in terpenoid-accumulating plant species. Expression of a putative Arabidopsis terpene synthase gene, designated AtTPS03, was demonstrated by amplification of a 392-bp cDNA fragment using primers designed to conserved regions of plant terpene synthases. Using the AtTPS03 fragment as a hybridization probe, a second AtTPS cDNA, designated AtTPS10, was isolated from a jasmonate-induced cDNA library. The partial AtTPS10 cDNA clone contained an open reading frame of 1665 bp encoding a protein of 555 amino acids. Functional expression of AtTPS10 in Escherichia coli yielded an active monoterpene synthase enzyme, which converted geranyl diphosphate (C(10)) into the acyclic monoterpenes beta-myrcene and (E)-beta-ocimene and small amounts of cyclic monoterpenes. Based on sequence relatedness, AtTPS10 was classified as a member of the TPSb subfamily of angiosperm monoterpene synthases. Sequence comparison of AtTPS10 with previously cloned monoterpene synthases suggests independent events of functional specialization of terpene synthases during the evolution of terpenoid secondary metabolism in gymnosperms and angiosperms. Functional characterization of the AtTPS10 gene was prompted by the availability of Arabidopsis genome sequences. Although Arabidoposis has not been reported to form terpenoid secondary metabolites, the unexpected expression of TPS genes belonging to the TPSb subfamily in this species strongly suggests that terpenoid secondary metabolism is active in the model system Arabidopsis.