Pseudomonas syringae elicits emission of the terpenoid (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene in Arabidopsis leaves via jasmonate signaling and expression of the terpene synthase TPS4

Volatile, low-molecular weight terpenoids have been implicated in plant defenses, but their direct role in resistance against microbial pathogens is not clearly defined. We have examined a possible role of terpenoid metabolism in the induced defense of Arabidopsis thaliana plants against leaf infection with the bacterial pathogen Pseudomonas syringae. Inoculation of plants with virulent or avirulent P. syringae strains induces the emission of the terpenoids (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT), beta-ionone and alpha-farnesene. While the most abundant volatile, the C16-homoterpene TMTT, is produced relatively early in compatible and incompatible interactions, emission of both beta-ionone and alpha-farnesene only increases in later stages of the compatible interaction. Pathogen-induced synthesis of TMTT is controlled through jasmonic acid (JA)-dependent signaling but is independent of a functional salicylic acid (SA) pathway. We have identified Arabidopsis T-DNA insertion lines with defects in the terpene synthase gene TPS4, which is expressed in response to P. syringae inoculation. The tps4 knockout mutant completely lacks induced emission of TMTT but is capable of beta-ionone and alpha-farnesene production, demonstrating that TPS4 is specifically involved in TMTT formation. The tps4 plants display at least wild type-like resistance against P. syringae, indicating that TMTT per se does not protect against the bacterial pathogen in Arabidopsis leaves. Similarly, the ability to mount SA-dependent defenses and systemic acquired resistance (SAR) is barely affected in tps4, which excludes a signaling function of TMTT during SAR. Besides P. syringae challenge, intoxication of Arabidopsis leaves with copper sulfate, a treatment that strongly activates JA biosynthesis, triggers production of TMTT, beta-ionone, and alpha-farnesene. Taken together, our data suggest that induced TMTT production in Arabidopsis is a by-product of activated JA signaling, rather than an effective defense response that contributes to resistance against P. syringae.     

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.     

Trehalose metabolism in Arabidopsis: occurrence of trehalose and molecular cloning and characterization of trehalose-6-phosphate synthase homologues

Axenically grown Arabidopsis thaliana plants were analysed for the occurrence of trehalose. Using gas chromatography-mass spectrometry (GC-MS) analysis, trehalose was unambiguously identified in extracts from Arabidopsis inflorescences. In a variety of organisms, the synthesis of trehalose is catalysed by trehalose-6-phosphate synthase (TPS; EC 2.4.1.15) and trehalose-6-phosphate phosphatase (TPP; EC 3.1.3.12). Based on EST (expressed sequence tag) sequences, three full-length Arabidopsis cDNAs whose predicted protein sequences show extensive homologies to known TPS and TPP proteins were amplified by RACE-PCR. The expression of the corresponding genes, AtTPSA, AtTPSB and AtTPSC, and of the previously described TPS gene, AtTPS1, was analysed by quantitative RT-PCR. All of the genes were expressed in the rosette leaves, stems and flowers of Arabidopsis plants and, to a lower extent, in the roots. To study the role of the Arabidopsis genes, the AtTPSA and AtTPSC cDNAs were expressed in Saccharomyces cerevisiae mutants deficient in trehalose synthesis. In contrast to AtTPS1, expression of AtTPSA and AtTPSC in the tps1 mutant lacking TPS activity did not complement trehalose formation after heat shock or growth on glucose. In addition, no TPP function could be identified for AtTPSA and AtTPSC in complementation studies with the S. cerevisiae tps2 mutant lacking TPP activity. The results indicate that while AtTPS1 is involved in the formation of trehalose in Arabidopsis, some of the Arabidopsis genes with homologies to known TPS/TPP genes encode proteins lacking catalytic activity in trehalose synthesis.     

Feeding of pea leafminer larvae simultaneously activates jasmonic and salicylic acid pathways in plants to release a terpenoid for indirect defense

The pea leafminer, Liriomyza huidobrensis, is an important pest species affecting ornamental crops worldwide. Plant damage consists of oviposition and feeding punctures created by female adult flies as well as larva-bored mines in leaf mesophyll tissues. How plants indirectly defend themselves from these two types of leafminer damage has not been sufficiently investigated. In this study, we compared the indirect defense responses of bean plants infested by either female adults or larvae. Puncturing of leaves by adults released green leaf volatiles and terpenoids, while larval feeding caused plants to additionally emit methyl salicylate and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT). Puncturing of plants by female adults induced increases in jasmonic acid (JA) and JA-related gene expressions but reduced the expressions of salicylic acid (SA)-related genes. In contrast, JA and SA and their-related gene expression levels were increased significantly by larval feeding. The exogenous application of JA+SA significantly triggered TMTT emission, thereby significantly inducing the orientation behavior of parasitoids. Our study has confirmed that larval feeding can trigger TMTT emission through the activation of both JA and SA pathways to attract parasitoids; however, TMTT alone is less attractive than the complete blend of volatiles released by infested plants.     

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.     

Inhibition of polyunsaturated fatty acid accumulation in plants expressing a fatty acid epoxygenase

Earlier, we described the isolation of a Crepis palaestina cDNA (Cpal2) which encoded a Delta12-epoxygenase that could catalyse the synthesis of 12,13-epoxy-cis-9-octadecenoic acid (18:1E) from linoleic acid (18:2). When the Cpal2 gene was expressed under the control of a seed-specific promoter in Arabidopsis, plants were able to accumulate small amounts 18:1E and 12,13-epoxy-cis-9,15-octadec-2-enoic acid in their seed lipids. In this report we give results obtained from a detailed analysis of transgenic Arabidopsis plants containing the Cpal2 gene. The seeds from these plants accumulate varying levels of 18:1E, but show a marked increase in 18:1 and equivalent decrease in 18:2 and 18:3. We further observed that the co-expression of a C. palaestina Delta12-desaturase in Arabidopsis appears to return the relative proportions of the C(18) seed fatty acids to normal levels and results in a 2-fold increase in total epoxy fatty acids.     

Immunogold localization of trehalose-6-phosphate synthase in leaf segments of wild-type and transgenic tobacco plants expressing the AtTPS1 gene from Arabidopsis thaliana

Summary. Following the establishment of a transgenic line of tobacco (B5H) expressing the trehalose-6-phosphate synthase (TPS) gene from Arabidopsis thaliana, a preliminary immunolocalization study was conducted using leaves of adequately watered B5H and wild-type plants. Immunocytochemical staining, followed by electron microscopy showed that the enzyme could be detected in both B5H and wild-type plants at two different levels. Quantification showed the signal to be two to three times higher in transgenic plants than in the wild type. This enzyme was markedly present in the vacuoles and the cell wall, and to a lesser extent in the cytosol. Moreover, a high profusion of gold particles was detected in adjacent cells and in the sieve elements. Occasional spots were also detected in chloroplasts and the nucleus, especially in the transgenic B5H line. No labeling signal was detected in mitochondria. Protein localization seems to confirm the important role of TPS in sugar metabolism and transport through the plant, which could explain its role in plant stress tolerance. Finally, it can be expected that TPS from tobacco has a relatively high similarity to the TPS of Arabidopsis thaliana. Correspondence and reprints: Laboratório de Biotecnologia de Células Vegetais, ITQB, Apartado 127, Avenida da República (E.A.N.), 2781-901 Oeiras, Portugal.     

干旱诱导性启动子驱动的海藻糖－6－磷酸合酶基因载体的构建及转基因烟草的耐旱性

The trehalose-6-phosphate synthase gene (TPS) from Saccharomyces cerevisiae Hansen and the drought-responsive promoter from Arabidopsis thaliana (L.) Heynh. have been cloned by PCR procedure. A plant expression vector with TPS under control of Prd29A has been constructed and used for the genetic transformation of tobacco. The transgenic tobacco with Prd29A/TSP demonstrated TPS expression with increased drought tolerance under drought stress. Some obvious morphological changes including dwarf and fine shoot, lancet-shaped leaves and vigorous auxiliary buds have been observed in a few transformed plants.     

Genetic Transformation of Tobacco with the Trehalose Synthase Gene from Grifola frondosa Fr. Enhances the Resistance to Drought and Salt in Tobacco

Trehalose is a non-reducing disaccharide of glucose that functions as a protectant in the stabilization of biological structures and enhances the tolerance of organisms to abiotic stress. In the present study, we report on the expression of the Grifolafrondosa Fr. trehalose synthase (TSase) gene for manipulating abiotic stress tolerance in tobacco (Nicotiana tabaccum L.). The expression of the transgene was under the control of two tandem copies of the CaMV35S promoter and was transferred into tobacco by Agrobacterium tumefaciens EHA105. Compared with non-transgenic plants, transgenic plants were able to accumulate high levels of products of trehalose, which were increased up to 2.126-2.556 mg/g FW, although levels were undetectable in non-transgenic plants. This level of trehalose in transgenic plants was 400-fold higher than that of transgenic tobacco plants cotransformed with Escherichia coli TPS and TPP on independent expression cassettes, twofold higher than that of transgenic rice plants transformed with a bifunctional fusion gene (TPSP) of the trehalose-6-phosphate (T-6-P) synthase (TPS) and T-6-P phosphatase (TPP) of E. coli, and 12-fold higher than that of transgenic tobacco plants transformed the yeast TPS1 gene.It has been reported that transgenic plants with E. coli TPS and/or TPP were severely stunted and had morphological alterations of their roots. Interestingly, our transgenic plants have obvious morphological changes, including thick and deep-coloured leaves, but show no growth inhibition; moreover, these morphological changes can restore to normal type in T2 progenies. Trehalose accumulation in 35S-35S:TSase plants resulted in increased tolerance to drought and salt, as shown by the results of tests on drought, salt tolerance, and drought physiological indices, such as water content in excised leaves, malondialdehyde content, chlorophyll a and b contents, and the activity of superoxide dismutase and peroxidase in excised leaves. These results suggest that transgenic plants transformed with the TSase gene can accumulate high levels of trehalose and have enhanced tolerance to drought and salt.     

Trehalose-6-phosphate synthase 1, which catalyses the first step in trehalose synthesis, is essential for Arabidopsis embryo maturation

Despite the recent discovery that trehalose synthesis is widespread in higher plants very little is known about its physiological significance. Here we report on an Arabidopsis mutant (tps1), disrupted in a gene encoding the first enzyme of trehalose biosynthesis (trehalose-6-phosphate synthase). The tps1 mutant is a recessive embryo lethal. Embryo morphogenesis is normal but development is retarded and stalls early in the phase of cell expansion and storage reserve accumulation. TPS1 is transiently up-regulated at this same developmental stage and is required for the full expression of seed maturation marker genes (2S2 and OLEOSN2). Sucrose levels also increase rapidly in seeds during the onset of cell expansion. In Saccharomyces cerevisiae trehalose-6-phosphate (T-6-P) is required to regulate sugar influx into glycolysis via the inhibition of hexokinase and a deficiency in TPS1 prevents growth on sugars (Thevelein and Hohmann, 1995). The growth of Arabidopsis tps1-1 embryos can be partially rescued in vitro by reducing the sucrose level. However, T-6-P is not an inhibitor of AtHXK1 or AtHXK2. Nor does reducing hexokinase activity rescue tps1-1 embryo growth. Our data establish for the first time that an enzyme of trehalose metabolism is essential in plants and is implicated in the regulation of sugar metabolism/embryo development via a different mechanism to that reported in S. cerevisiae.     

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.     

Expression of lima bean terpene synthases in rice enhances recruitment of a beneficial enemy of a major rice pest

Volatile terpenoids play a key role in plant defence against herbivory by attracting parasitic wasps. We identified seven terpene synthase genes from lima bean, Phaseolus lunatus L. following treatment with either the elicitor alamethicin or spider mites, Tetranychus cinnabarinus. Four of the genes (Pltps2, Pltps3, Pltps4 and Pltps5) were up‐regulated with their derived proteins phylogenetically clustered in the TPS‐g subfamily and PlTPS3 positioned at the base of this cluster. Recombinant PlTPS3 was able to convert geranyl diphosphate and farnesyl diphosphate to linalool and (E)‐nerolidol, the latter being precursor of the homoterpene (E)‐4,8‐dimethyl‐1,3,7‐nonatriene (DMNT). Recombinant PlTPS4 showed a different substrate specificity and produced linalool and (E)‐nerolidol, as well as (E,E)‐geranyllinalool from geranylgeranyl diphosphate. Transgenic rice expressing Pltps3 emitted significantly more (S)‐linalool and DMNT than wild‐type plants, whereas transgenic rice expressing Pltps4 produced (S)‐linalool, DMNT and (E,E)‐4,8,12‐trimethyl‐1,3,7,11‐tridecatetraene (TMTT). In laboratory bioassays, female Cotesia chilonis, the natural enemy of the striped rice stemborer, Chilo suppressalis, were significantly attracted to the transgenic plants and their volatiles. We further confirmed this with synthetic blends mimicking natural rice volatile composition. Our study demonstrates that the transformation of rice to produce volatile terpenoids has the potential to enhance plant indirect defence through natural enemy recruitment.     

Aphid antixenosis in cotton is activated by the natural plant defence elicitor cis-jasmone

Upon insect herbivory, plants can release blends of volatile organic compounds (VOCs) that modify herbivore and natural enemy behaviour. We have shown recently that cotton, Gossypium hirsutum, emits a blend of defence VOCs that repels the cotton aphid, Aphis gossypii, upon herbivory by this notorious crop pest, including (Z)-3-hexenyl acetate, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), methyl salicylate and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT). In this study, we investigated changes in the defence VOC profile of G. hirsutum induced by the naturally-occurring plant elicitor cis-jasmone (CJ) and whether these changes modify the behaviour of A. gossypii. In four-arm olfactometer assays, VOCs from untreated plants were significantly attractive (P < 0.05), whilst VOCs from CJ-treated plants were significantly repellent (P < 0.05). The VOCs induced by CJ appeared to comprise (Z)-3-hexenyl acetate, DMNT, methyl salicylate and TMTT. In quantitative VOC collection studies, sustained release of DMNT and TMTT was observed in CJ-treated plants over a period of five days, with levels becoming statistically significantly higher than for control treated plants on the fifth day in most cases. Despite earlier indications, no statistically significant differences were observed in levels of (Z)-3-hexenyl acetate or methyl salicylate between CJ and control treatments on any day. Furthermore, DMNT and TMTT emissions from CJ-treated plants were further enhanced by subsequent addition of A. gossypii. CJ treatment induced statistically significantly higher DMNT and TMTT expression levels as early as day three, when A. gossypii was present. The results in this study show that CJ can induce the production of A. gossypii-induced VOCs from G. hirsutum, with potential for deployment in novel crop protection strategies.