Demonstration and characterization of (E)-nerolidol synthase from maize: a herbivore-inducible terpene synthase participating in (3E)-4,8-dimethyl-1,3,7-nonatriene biosynthesis

Upon herbivore attack, maize (Zea mays L.) emits a mixture of volatile compounds that attracts herbivore enemies to the plant. One of the major components of this mixture is an unusual acyclic C₁₁ homoterpene, (3E )-4,8-dimethyl-1,3,7-nonatriene (DMNT), which is also emitted by many other species following herbivore damage. Biosynthesis of DMNT has been previously shown to proceed via the sesquiterpene alcohol, (E )-nerolidol. Here we demonstrate an enzyme activity that converts farnesyl diphosphate, the universal precursor of sesquiterpenes, to (3S)-(E )-nerolidol in cell-free extracts of maize leaves that had been fed upon by Spodoptera littoralis. The properties of this (E )-nerolidol synthase resemble those of other terpene synthases. Evidence for its participation in DMNT biosynthesis includes the direct incorporation of deuterium-labeled (E )-nerolidol into DMNT and the close correlation between increases in (E )-nerolidol synthase activity and DMNT emission after herbivore damage. Since farnesyl diphosphate has many other metabolic fates, (E )-nerolidol synthase may represent the first committed step of DMNT biosynthesis in maize. However, the formation of this unusual acyclic terpenoid appears to be regulated at both the level of (E )-nerolidol synthase and at later steps in the pathway. Received: 20 August 1999 / Accepted: 27 October 1999     

Identification, functional characterization, and regulation of the enzyme responsible for floral (E)-nerolidol biosynthesis in kiwifruit (Actinidia chinensis)

Flowers of the kiwifruit species Actinidia chinensis produce a mixture of sesquiterpenes derived from farnesyl diphosphate (FDP) and monoterpenes derived from geranyl diphosphate (GDP). The tertiary sesquiterpene alcohol (E)-nerolidol was the major emitted volatile detected by headspace analysis. Contrastingly, in solvent extracts of the flowers, unusually high amounts of (E,E)-farnesol were observed, as well as lesser amounts of (E)-nerolidol, various farnesol and farnesal isomers, and linalool. Using a genomics-based approach, a single gene (AcNES1) was identified in an A. chinensis expressed sequence tag library that had significant homology to known floral terpene synthase enzymes. In vitro characterization of recombinant AcNES1 revealed it was an enzyme that could catalyse the conversion of FDP and GDP to the respective (E)-nerolidol and linalool terpene alcohols. Enantiomeric analysis of both AcNES1 products in vitro and floral terpenes in planta showed that (S)-(E)-nerolidol was the predominant enantiomer. Real-time PCR analysis indicated peak expression of AcNES1 correlated with peak (E)-nerolidol, but not linalool accumulation in flowers. This result, together with subcellular protein localization to the cytoplasm, indicated that AcNES1 was acting as a (S)-(E)-nerolidol synthase in A. chinensis flowers. The synthesis of high (E,E)-farnesol levels appears to compete for the available pool of FDP utilized by AcNES1 for sesquiterpene biosynthesis and hence strongly influences the accumulation and emission of (E)-nerolidol in A. chinensis flowers.     

The biochemistry of homoterpenes--common constituents of floral and herbivore-induced plant volatile bouquets

Volatile organic compounds emitted by plants mediate a variety of interactions between plants and other organisms. The irregular acyclic homoterpenes, 4,8-dimethylnona-1,3,7-triene (DMNT) and 4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT), are among the most widespread volatiles produced by angiosperms with emissions from flowers and from vegetative tissues upon herbivore feeding. Special attention has been placed on the role of homoterpenes in attracting parasitoids and predators of herbivores and has sparked interest in engineering homoterpene formation to improve biological pest control. The biosynthesis of DMNT and TMTT proceeds in two enzymatic steps: the formation of the tertiary C₁₅-, and C₂₀-alcohols, (E)-nerolidol and (E,E)-geranyl linalool, respectively, catalyzed by terpene synthases, and the subsequent oxidative degradation of both alcohols by a single cytochrome P450 monooxygenase (P450). In Arabidopsis thaliana, the herbivore-induced biosynthesis of TMTT is catalyzed by the concerted activities of the (E,E)-geranyllinalool synthase, AtGES, and CYP82G1, a P450 of the so far uncharacterized plant CYP82 family. TMTT formation is in part controlled at the level of AtGES expression. Co-expression of AtGES with CYP82G1 at wound sites allows for an efficient conversion of the alcohol intermediate. The identified homoterpene biosynthesis genes in Arabidopsis and related genes from other plant species provide tools to engineer homoterpene formation and to address questions of the regulation and specific activities of homoterpenes in plant-herbivore interactions.     

Three-step pathway engineering results in more incidence rate and higher emission of nerolidol and improved attraction of Diadegma semiclausum

The concentration and ratio of terpenoids in the headspace volatile blend of plants have a fundamental role in the communication of plants and insects. The sesquiterpene (E)-nerolidol is one of the important volatiles with effect on beneficial carnivores for biologic pest management in the field. To optimize de novo biosynthesis and reliable and uniform emission of (E)-nerolidol, we engineered different steps of the (E)-nerolidol biosynthesis pathway in Arabidopsis thaliana.Introduction of a mitochondrial nerolidol synthase gene mediates de novo emission of (E)-nerolidol and linalool. Co-expression of the mitochondrial FPS1 and cytosolic HMGR1 increased the number of emitting transgenic plants (incidence rate) and the emission rate of both volatiles. No association between the emission rate of transgenic volatiles and their growth inhibitory effect could be established. (E)-Nerolidol was to a large extent metabolized to non-volatile conjugates.     

The influence of different nutrient levels on insect-induced plant volatiles in Bt and conventional oilseed rape plants

Transgenic Bt (expressing the cry1Ac endotoxin gene) and conventional oilseed rape plants grown in different soils were used to study nutrient uptake and emission of volatiles after herbivore damage. All plants were greenhouse-grown in soils representing low-, medium- and high-nutrient levels. The concentrations of N, P, K, Mg and Zn were significantly affected by the transgene, while the main effect of soil type appeared in N, P, Ca, Mg, B, Mn and Zn concentrations in the plants. Plants with four to five leaves were infested with the third instar larvae of Bt-susceptible Plutella xylostella for 48 h, and samples of volatiles were collected and analysed. In the first experiment, the soil nutrient level had a significant effect on the emissions of (Z)-3-hexen-1-ol, (Z)-3-hexenyl acetate, hexyl acetate, (E)-4,8-dimethyl-1,3,7-non-atriene (DMNT), beta-elemene, gamma-bisabolene, alpha-bisabolene and (E)-nerolidol. The induction of these volatiles was significantly higher in infested conventional plants grown at a high-soil nutrient level compared to infested conventional plants at a low-soil nutrient level. In the second experiment, the soil nutrient level had a significant effect on the emissions of (Z)-3-hexen-1-ol, (Z)-3-hexenyl acetate and beta-elemene and, again, this was significantly higher in infested conventional plants grown at high-soil nutrient levels in comparison with infested plants at a low-soil nutrient level. In both experiments, the transgene effect was significant on the emissions of DMNT and (E,E)-alpha-farnesene. The differences in emissions between the two separate experiments suggest that growth conditions (particularly daylength) and sampling procedure may affect the ratio of compounds detected in the emission blend, even though the response to herbivory, nutrient availability and the transgene were similar.     

植物的环境信号分子茉莉酸及其生物学功能

茉莉酸信号分子参与植物生长发育众多生理过程的调控,尤其是作为环境信号分子能有效地介导植物对生物及非生物胁迫的防御反应。迄今已知具有信号分子生理功能的至少包括茉莉酸(jasmonic acid,JA)以及茉莉酸甲酯(methyl jasmonate,Me JA)和茉莉酸-异亮氨酸复合物(jasmonoyl-isoleucine,JA-Ile)等茉莉酸衍生物,统称为茉莉酸类化合物(jasmonates,JAs)。从环境信号分子角度介绍了茉莉酸信号的启动(环境信号感知与转导、茉莉酸类化合物合成)、传递(局部传递、维管束传输、空气传播)和生物学功能(茉莉酸信号受体、调控的转录因子、参与的生物学过程)。     

Complex regulation of ABA biosynthesis in plants.

Abscisic acid (ABA) is a plant hormone that plays important roles during many phases of the plant life cycle, including seed development and dormancy, and in plant responses to various environmental stresses. Because many of these physiological processes are correlated with endogenous ABA levels, the regulation of ABA biosynthesis is a key element facilitating the elucidation of these physiological characteristics. Recent studies on the identification of genes encoding enzymes involved in ABA biosynthesis have revealed details of the main ABA biosynthetic pathway. At the same time, the presence of gene families and their respective organ-specific expression are indicative of the complex mechanisms governing the regulation of ABA biosynthesis in response to plant organ and/or environmental conditions. There have been recent advances in the study of ABA biosynthesis and new insights into the regulation of ABA biosynthesis in relation to physiological phenomena.     

Spider Mite-Induced (3S)-(E)-Nerolidol Synthase Activity in Cucumber and Lima Bean. The First Dedicated Step in Acyclic C11-Homoterpene Biosynthesis

Many plant species respond to herbivory with de novo production of a mixture of volatiles that attracts carnivorous enemies of the herbivores. One of the major components in the blend of volatiles produced by many different plant species in response to herbivory by insects and spider mites is the homoterpene 4,8-dimethyl-1,3(E), 7-nonatriene. One study (J. Donath, W. Boland [1995] Phytochemistry 39: 785-790) demonstrated that a number of plant species can convert the acyclic sesquiterpene alcohol (3S)-(E)-nerolidol to this homoterpene. Cucumber (Cucumis sativus L.) and lima bean (Phaseolus lunatus L.) both produce 4,8-dimethyl-1,3(E),7-nonatriene in response to herbivory. We report the presence in cucumber and lima bean of a sesquiterpene synthase catalyzing the formation of (3S)-(E)-nerolidol from farnesyl diphosphate. The enzyme is inactive in uninfested cucumber leaves, slightly active in uninfested lima bean leaves, and strongly induced by feeding of the two-spotted spider mite (Tetranychus urticae Koch) on both plant species, but not by mechanical wounding. The activities of the (3S)-(E)-nerolidol synthase correlated well with the levels of release of 4, 8-dimethyl-1,3(E),7-nonatriene from the leaves of the different treatments. Thus, (3S)-(E)-nerolidol synthase is a good candidate for a regulatory role in the release of the important signaling molecule 4,8-dimethyl-1,3(E),7-nonatriene.     

Identification and functional analysis of two P450 enzymes of Gossypium hirsutum involved in DMNT and TMTT biosynthesis

The homoterpenes (3E)‐4,8‐dimethyl‐1,3,7‐nonatriene (DMNT) and (E,E)‐4,8,12‐trimethyl‐1,3,7,11‐tridecatetraene (TMTT) are major herbivore‐induced plant volatiles that can attract predatory or parasitic arthropods to protect injured plants from herbivore attack. In this study, DMNT and TMTT were confirmed to be emitted from cotton (Gossypium hirsutum) plants infested with chewing caterpillars or sucking bugs. Two CYP genes (GhCYP82L1 and GhCYP82L2) involved in homoterpene biosynthesis in G. hirsutum were newly identified and characterized. Yeast recombinant expression and enzyme assays indicated that the two GhCYP82Ls are both responsible for the conversion of (E)‐nerolidol to DMNT and (E,E)‐geranyllinalool to TMTT. The two heterologously expressed proteins without cytochrome P450 reductase fail to convert the substrates to homoterpenes. Quantitative real‐time PCR (qPCR) analysis suggested that the two GhCYP82L genes were significantly up‐regulated in leaves and stems of G. hirsutum after herbivore attack. Subsequently, electroantennogram recordings showed that electroantennal responses of Microplitis mediator and Peristenus spretus to DMNT and TMTT were both dose dependent. Laboratory behavioural bioassays showed that females of both wasp species responded positively to DMNT and males and females of M. mediator could be attracted by TMTT. The results provide a better understanding of homoterpene biosynthesis in G. hirsutum and of the potential influence of homoterpenes on the behaviour of natural enemies, which lay a foundation to study genetically modified homoterpene biosynthesis and its possible application in agricultural pest control.     

Monitoring codling moth (Lepidoptera: Tortricidae) in sex pheromone-treated orchards with (E)-4,8-dimethyl-1,3,7-nonatriene or pear ester in combination with codlemone and acetic acid

Traps baited with ethyl (E,Z)-2,4-decadienoate (pear ester) or (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) in two- or three-way combinations with the sex pheromone (E,E)-8,10-dodecadien-1-ol (codlemone) and acetic acid (AA) were evaluated for codling moth, Cydia pomonella (L.). All studies were conducted in apple orchards, Malus domestica Borkhausen, treated with sex pheromone dispensers during 2010. Septa were loaded with codlemone, DMNT, and pear ester individually or codlemone with either DMNT or pear ester together (combo lures). Polyethylene vials loaded with AA were added as a co-lure. Residual analyses of field-aged combo lures and weight loss of the AA co-lure were conducted. AA vials lost 50-150 mg wk(-1). Weekly weight loss was not affected by field aging, but was closely correlated with the daily mean temperature. Pear ester was released from septa at a slightly higher but nonsignificant rate than codlemone. DMNT was released at a significantly higher rate than codlemone, and lures were effective for 4 wk. The addition of codlemone to traps baited with either host plant volatile plus AA had either no effect or significantly increased total moth catches. The addition of AA significantly increased the catch of female moths with either combo lure. Total moth catches in traps baited with pear ester or DMNT combo lures and AA did not differ and were either significantly higher or similar to the pear ester combo lure. These data suggest that codling moth may be more effectively monitored in sex pheromone-treated orchards with multi-component lures, including codlemone, AA, and host plant volatiles.     

植物赤霉素生物合成和信号传导的分子生物学

赤霉素 (GAs)在植物的种子萌发、茎的伸长和花的发育等许多方面起着非常重要的作用。最近几年 ,对GA生物合成及其信号传导途径相关基因的研究取得了惊人的进展。这些进展促进了对其生物合成及其信号传导途径的认识。GA生物合成相关基因的表达受到多种内源和外源因子的调控 ,其中研究较多的是发育阶段、激素水平和光信号等内源及环境因子的调控。GA信号传导通常处于抑制状态 ,GA信号通过去抑制作用激活该传导途径而促进GA刺激植物生长和发育。     

Of light and length: regulation of hypocotyl growth in Arabidopsis

At all stages, plant development results from a complex integration of multiple endogenous and environmental signals. The sedentary nature of plants strongly enhances the impact of the environment on plant development as compared to animal development. The embryonic and postembryonic seedling stem, called the hypocotyl, of the model species Arabidopsis (thale cress) has proved to be an excellent system for studying such signal interplay in the regulation of growth and developmental responses. The extension of the hypocotyl, which is regulated by a network of interacting factors, including light and plant hormones, is such a process. These regulatory factors often reciprocally regulate their biosynthesis and/or signalling. Here we present the current state of knowledge about the regulation of hypocotyl growth by a large repertoire of internal and external cues.     

从水分胁迫的识别到ABA积累的细胞信号转导

由于植物在生长和发育过程中不可避免地要遭受各种环境胁迫的影响,植物只有通过对环境胁迫的快速感知和主动反应才得以生存和发展.植物这种对环境胁迫的快速感知和主动反应体现在环境胁迫下植物可以通过一系列基因的表达调控来实现各种抗逆的生理生化反应.虽然得以鉴定的水分胁迫应答基因越来越多,但其中只有极少的基因在抗逆中的基本功能已得到初步认识.从细胞对水分胁迫原初信号的感知到基因表达调控包括了一系列复杂的细胞逆境信息传递过程.脱落酸(abscisic acid, ABA)作为重要的细胞逆境信号物质介导了一系列基因表达,因此从细胞对水分胁迫原初信号的感知到编码ABA生物合成关键酶基因的表达是一条最为关键的细胞逆境信息传递途径.逆境应答基因功能的鉴定以及对整个细胞信号传递过程中详尽的分子机制的了解无疑是今后最有趣的也是最为重要的研究课题.     

从水分胁迫的识别到ABA积累的细胞信号转导

由于植物在生长和发育过程中不可避免地要遭受各种环境胁迫的影响 ,植物只有通过对环境胁迫的快速感知和主动反应才得以生存和发展。植物这种对环境胁迫的快速感知和主动反应体现在环境胁迫下植物可以通过一系列基因的表达调控来实现各种抗逆的生理生化反应。虽然得以鉴定的水分胁迫应答基因越来越多 ,但其中只有极少的基因在抗逆中的基本功能已得到初步认识。从细胞对水分胁迫原初信号的感知到基因表达调控包括了一系列复杂的细胞逆境信息传递过程。脱落酸 (abscisicacid ,ABA)作为重要的细胞逆境信号物质介导了一系列基因表达 ,因此从细胞对水分胁迫原初信号的感知到编码ABA生物合成关键酶基因的表达是一条最为关键的细胞逆境信息传递途径。逆境应答基因功能的鉴定以及对整个细胞信号传递过程中详尽的分子机制的了解无疑是今后最有趣的也是最为重要的研究课题。     

植物赤霉素生物合成和信号传导的分子生物学

赤霉素(GAs)在植物的种子萌发、茎的伸长和花的发育等许多方面起着非常重要的作用。最近几年,对GA生物合成及其信号传导途径相关基因的研究取得了惊人的进展。这些进展促进了对其生物合成及其信号传导途径的认识。GA生物合成相关基因的表达受到多种内源和外源因子的调控, 其中研究较多的是发育阶段、激素水平和光信号等内源及环境因子的调控。GA信号传导通常处于抑制状态, GA信号通过去抑制作用激活该传导途径而促进GA刺激植物生长和发育。     

Integration of C/N-nutrient and multiple environmental signals into the ABA signaling cascade

Due to their immobility, plants have developed sophisticated mechanisms to robustly monitor and appropriately respond to dynamic changes in nutrient availability. Carbon (C) and nitrogen (N) are especially important in regulating plant metabolism and development, thereby affecting crop productivity. In addition to their independent utilization, the ratio of C to N metabolites in the cell, referred to as the “C/N balance”, is important for the regulation of plant growth, although molecular mechanisms mediating C/N signaling remain unclear. Recently ABI1, a protein phosphatase type 2C (PP2C), was shown to be a regulator of C/N response in Arabidopsis plants. ABI1 functions as a negative regulator of abscisic acid (ABA) signal transduction. ABA is versatile phytohormone that regulates multiple aspects of plant growth and adaptation to environmental stress. This review highlights the regulation of the C/N response mediated by a non-canonical ABA signaling pathway that is independent of ABA biosynthesis, as well as recent findings on the direct crosstalk between multiple cellular signals and the ABA signaling cascade.     

油菜素内酯调控作物农艺性状和非生物胁迫响应的研究进展

植物对不利环境的适应依赖于将外部胁迫信号传递到内部信号通路中,在进化过程中形成一系列的胁迫响应机制。其中,油菜素内酯(brassinosteroids, BRs)是一种类固醇激素,广泛参与植物生长发育和逆境响应过程。BRs被包括受体BRI1和共受体BAK1在内的细胞表面受体感知,继而触发信号级联,导致蛋白激酶BIN2的抑制和转录因子BES1/BZR1的激活,BES1/BZR1可直接调控数千个下游响应基因的表达。在模式植物拟南芥中的研究表明,BR的生物合成和信号转导通路成员,特别是BIN2和其下游的转录因子BES1/BZR1,可以被各种环境因子广泛地调节。本文系统总结了BR相关的最新研究进展,对BR的生物合成和信号转导是如何被复杂的环境因子所调节,以及BR与环境因子如何协同调控作物重要农艺性状、冷胁迫和盐胁迫的响应进行了综述。