Combined transcript and metabolite analysis reveals genes involved in spider mite induced volatile formation in cucumber plants

Many plants have an indirect defense against herbivores by emitting volatiles that attract carnivorous enemies of the herbivores. In cucumber (Cucumis sativus) the production of carnivore attractants can be induced by herbivory or jasmonic acid spraying. From the leaves of cucumber plants with and without spider mite infestation, two subtractive cDNA libraries were made that were enriched in cDNA fragments up- or down-regulated by spider mite infestation. A total of 713 randomly selected clones from these libraries were used to make a cDNA microarray. Subsequently, cucumber plants were sprayed with jasmonic acid, mechanically damaged, infested with spider mites, or left untreated (control). Leaf samples were taken at a range of different time points, and induced volatile compounds and mRNA (from the same leaves) were collected. cDNAs prepared from the mRNA were hybridized to the clones on the microarray. The resulting gene expression profiles were analyzed in combination with volatile production data in order to gain insight in the possible involvement of the studied genes in the synthesis of those volatiles. The clones on the microarray and the induced cucumber volatiles could be grouped into a number of clusters in which specific biosynthetic genes clustered with the product of that pathway. For example, lipoxygenase cDNA clones clustered with the volatile (Z)-3-hexenyl acetate and the volatile sesquiterpene (E,E)- alpha-farnesene clustered with an up-regulated sesquiterpene synthase fragment. This fragment was used to screen a cDNA library which resulted in the cloning of the cucumber (E,E)-alpha-farnesene and (E)-beta-caryophyllene synthases. The use of combined global gene expression analysis and metabolite analysis for the discovery of genes involved in specific biosynthetic processes is discussed.     

Overexpression of the trehalose-6-phosphate synthase gene <Emphasis Type="Italic">OsTPS1</Emphasis> enhances abiotic stress tolerance in rice

Trehalose plays an important role in metabolic regulation and abiotic stress tolerance in a variety of organisms. In plants, its biosynthesis is catalyzed by two key enzymes: trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). The genome of rice (Oryza sativa) contains 11 OsTPS genes, and only OsTPS1 shows TPS activity. To demonstrate the physiological function of OsTPS1, we introduced it into rice and found that OsTPS1 overexpression improved the tolerance of rice seedling to cold, high salinity and drought treatments without other significant phenotypic changes. In transgenic lines overexpressing OsTPS1, trehalose and proline concentrations were higher than in the wild type and some stress-related genes were up-regulated, including WSI18, RAB16C, HSP70, and ELIP. These results demonstrate that OsTPS1 may enhance the abiotic stress tolerance of plants by increasing the amount of trehalose and proline, and regulating the expression of stress-related genes. Furthermore, we found that overexpression of some Class II TPSs also enhanced plant tolerance of abiotic stress. This work will help to clarify the role of trehalose metabolism in abiotic stress response in higher plants.     

Expression of a Flax Allene Oxide Synthase cDNA Leads to Increased Endogenous Jasmonic Acid (JA) Levels in Transgenic Potato Plants but Not to a Corresponding Activation of JA-Responding Genes

Both jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are thought to be significant components of the signaling pathway regulating the expression of plant defense genes in response to various stresses. JA and MeJA are plant lipid derivatives synthesized from [alpha]-linolenic acid by a lipoxygenase-mediated oxygenation leading to 13-hydroperoxylinolenic acid, which is subsequently transformed by the action of allene oxide synthase (AOS) and additional modification steps. AOS converts lipoxygenase-derived fatty acid hydroperoxide to allene epoxide, which is the precursor for JA formation. Overexpression of flax AOS cDNA under the regulation of the cauliflower mosaic virus 35S promoter in transgenic potato plants led to an increase in the endogenous level of JA. Transgenic plants had six- to 12-fold higher levels of JA than the nontransformed plants. Increased levels of JA have been observed when potato and tomato plants are mechanically wounded. Under these conditions, the proteinase inhibitor II (pin2) genes are expressed in the leaves. Despite the fact that the transgenic plants had levels of JA similar to those found in nontransgenic wounded plants, pin2 genes were not constitutively expressed in the leaves of these plants. Transgenic plants with increased levels of JA did not show changes in water state or in the expression of water stress-responsive genes. Furthermore, the transgenic plants overexpressing the flax AOS gene, and containing elevated levels of JA, responded to wounding or water stress by a further increase in JA and by activating the expression of either wound- or water stress-inducible genes. Protein gel blot analysis demonstrated that the flax-derived AOS protein accumulated in the chloroplasts of the transgenic plants.     

Analysis of trehalose-6-phosphate synthase (TPS) gene family suggests the formation of TPS complexes in rice

Trehalose-6-phosphate (T6P), an intermediate in the trehalose biosynthesis pathway, is emerging as an important regulator of plant metabolism and development. T6P levels are potentially modulated by a group of trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) homologues. In this study, we have isolated 11 TPS genes encoding proteins with both TPS and TPP domains, from rice. Functional complement assays performed in yeast tps1 and tps2 mutants, revealed that only OsTPS1 encodes an active TPS enzyme and no OsTPS protein possesses TPP activity. By using a yeast two-hybrid analysis, a complicated interaction network occurred among OsTPS proteins, and the TPS domain might be essential for this interaction to occur. The interaction between OsTPS1 and OsTPS8 in vivo was confirmed by bimolecular fluorescence complementation and coimmunoprecipitation assays. Furthermore, our gel filtration assay showed that there may exist two forms of OsTPS1 (OsTPS1a and OsTPS1b) with different elution profiles in rice. OsTPS1b was particularly cofractionated with OsTPS5 and OsTPS8 in the 360 kDa complex, while OsTPS1a was predominantly incorporated into the complexes larger than 360 kDa. Collectively, these results suggest that OsTPS family members may form trehalose-6-phosphate synthase complexes and therefore potentially modify T6P levels to regulate plant development.     

Identification of a Novel <i>OsCYP2</i> Allele that Was Involved in Rice Response to Low Temperature Stress

Cyclophilin (CYP) plays an important role in plant response to stress, and OsCYP2, one gene of cyclophlilin family, is involved in auxin signal transduction and stress signaling in rice. However, the mechanism that OsCYP2 is involved in rice response to low temperature is still unclear. We identified a new OsCYP2 allelic mutant, lrl3, with fewer lateral roots, and the differences in shoot height, primary root length and adventitious root length increased with the growth process compared to the wild-type plant. Auxin signaling pathway was also affected and became insensitive to gravity. The transgenic rice plants with over-expression of OsCYP2 were more tolerant to low temperature than the wild-type plants, suggesting that OsCYP2 was involved in the low temperature response in rice. In addition, OsCYP2 negatively regulated the expression of OsTPS38, a terpene synthase gene, and was dependent on the OsCDPK7-mediated pathway in response to low temperature stress. OsTPS38- overexpressed transgenic line ox-2 was more sensitive to low temperature. Therefore, OsCYP2 may negatively regulate OsTPS38 through an OsCDPK7-dependent pathway to mediate the response to low temperature in rice. These results provide a new basis for auxin signaling genes to regulate rice response to low temperature stress.     

Induction of volatile terpene biosynthesis and diurnal emission by methyl jasmonate in foliage of Norway spruce

Terpenoids are characteristic constitutive and inducible defense chemicals of conifers. The biochemical regulation of terpene formation, accumulation, and release from conifer needles was studied in Norway spruce [Picea abies L. (Karst)] saplings using methyl jasmonate (MeJA) to induce defensive responses without inflicting physical damage to terpene storage structures. MeJA treatment caused a 2-fold increase in monoterpene and sesquiterpene accumulation in needles without changes in terpene composition, much less than the 10- and 40-fold increases in monoterpenes and diterpenes, respectively, observed in wood tissue after MeJA treatment (D. Martin, D. Tholl, J. Gershenzon, J. Bohlmann [2002] Plant Physiol 129: 1003-1018). At the same time, MeJA triggered a 5-fold increase in total terpene emission from foliage, with a shift in composition to a blend dominated by oxygenated monoterpenes (e.g. linalool) and sesquiterpenes [e.g. (E)-beta-farnesene] that also included methyl salicylate. The rate of linalool emission increased more than 100-fold and that of sesquiterpenes increased more than 30-fold. Emission of these compounds followed a pronounced diurnal rhythm with the maximum amount released during the light period. The major MeJA-induced volatile terpenes appear to be synthesized de novo after treatment, rather than being released from stored terpene pools, because they are almost completely absent from needle oleoresin and are the major products of terpene synthase activity measured after MeJA treatment. Based on precedents in other species, the induced emission of terpenes from Norway spruce foliage may have ecological and physiological significance.     

Molecular and genomic basis of volatile-mediated indirect defense against insects in rice

Rice plants fed on by fall armyworm ( Spodoptera frugiperda , FAW) caterpillars emit a blend of volatiles dominated by terpenoids. These volatiles were highly attractive to females of the parasitoid Cotesia marginiventris . Microarray analysis identified 196 rice genes whose expression was significantly upregulated by FAW feeding, 18 of which encode metabolic enzymes potentially involved in volatile biosynthesis. Significant induction of expression of seven of the 11 terpene synthase ( TPS ) genes identified through the microarray experiments was confirmd using real-time RT-PCR. Enzymes encoded by three TPS genes, Os02g02930, Os08g07100 and Os08g04500, were biochemically characterized. Os02g02930 was found to encode a monoterpene synthase producing the single product S- linalool, which is the most abundant volatile emitted from FAW-damaged rice plants. Both Os08g07100 and Os08g04500 were found to encode sesquiterpene synthases, each producing multiple products. These three enzymes are responsible for production of the majority of the terpenes released from FAW-damaged rice plants. In addition to TPS genes, several key genes in the upstream terpenoid pathways were also found to be upregulated by FAW feeding. This paper provides a comprehensive analysis of FAW-induced volatiles and the corresponding volatile biosynthetic genes potentially involved in indirect defense in rice. Evolution of the genetic basis governing volatile terpenoid biosynthesis for indirect defense is discussed.     

Two sesquiterpene synthases are responsible for the complex mixture of sesquiterpenes emitted from Arabidopsis flowers

Despite the fact that Arabidopsis is largely self-pollinating, its flowers emit a complex mixture of terpene volatiles consisting predominantly of a large group of over 20 sesquiterpenes. Here we report that only two terpene synthases, encoded by the florally expressed genes At5g23960 and At5g44630, are responsible for the formation of virtually all sesquiterpenes found in the Arabidopsis floral volatile blend. Two independent mutant lines with T-DNA insertions in the previously identified At5g23960 gene lacked the emission of three sesquiterpenes, including the main sesquiterpene volatile (E)-beta-caryophyllene, confirming the previous in vitro functional assignment for this gene. Flowers of a mutant line carrying a T-DNA insertion in gene At5g44630 emitted these three sesquiterpenes, but did not emit any of the remaining sesquiterpene volatiles. An At5g44630 cDNA was expressed in Escherichia coli and the produced protein catalyzed the conversion of farnesyl diphosphate into over 15 sesquiterpenes in similar proportions to those found in the floral volatile blend. At5g23960 and At5g44630 promoter-beta-glucuronidase (GUS) fusion experiments demonstrated that both genes are expressed in several parts of the Arabidopsis flower, with strong At5g23960 promoter-GUS activity in the stigma and strong expression of At5g44630 in intrafloral nectaries. Given the previously reported antimicrobial activity of terpenes, their production in stigmas and nectaries may serve to inhibit microbial infection at these vulnerable sites. A survey of 37 Arabidopsis thaliana ecotypes revealed quantitative, but almost no qualitative, variations of floral monoterpene and sesquiterpene emissions suggesting that floral terpene volatiles must play some significant role in the life of the Arabidopsis plant.     

Isolation and characterization of terpene synthases in cotton (Gossypium hirsutum)

Cotton plants accumulate gossypol and related sesquiterpene aldehydes, which function as phytoalexins against pathogens and feeding deterrents to herbivorous insects. However, to date little is known about the biosynthesis of volatile terpenes in this crop. Herein is reported that 5 monoterpenes and 11 sesquiterpenes from extracts of a glanded cotton cultivar, Gossypium hirsutum cv. CCRI12, were detected by gas chromatography–mass spectrometry (GC–MS). By EST data mining combined with Rapid Amplification of cDNA Ends (RACE), full-length cDNAs of three terpene synthases (TPSs), GhTPS1, GhTPS2 and GhTPS3 were isolated. By in vitro assays of the recombinant proteins, it was found that GhTPS1 and GhTPS2 are sesquiterpene synthases: the former converted farnesyl pyrophosphate (FPP) into β-caryophyllene and α-humulene in a ratio of 2:1, whereas the latter produced several sesquiterpenes with guaia-1(10),11-diene as the major product. By contrast, GhTPS3 is a monoterpene synthase, which produced α-pinene, β-pinene, β-phellandrene and trace amounts of other monoterpenes from geranyl pyrophosphate (GPP). The TPS activities were also supported by Virus Induced Gene Silencing (VIGS) in the cotton plant. GhTPS1 and GhTPS3 were highly expressed in the cotton plant overall, whereas GhTPS2 was expressed only in leaves. When stimulated by mechanical wounding, Verticillium dahliae (Vde) elicitor or methyl jasmonate (MeJA), production of terpenes and expression of the corresponding synthase genes were induced. These data demonstrate that the three genes account for the biosynthesis of volatile terpenes of cotton, at least of this Upland cotton.     

内源茉莉酸甲酯的积累改变了大豆叶片与根的发育

茉莉酸甲酯是一种调节植物形态发生、诱导防御相关基因的植物信号转导分子。为了解内源茉莉酸甲酯在植物发育中的作用,将编码茉莉酸甲基转移酶的NTR1基因与CaMV 35S启动子连接并导入大豆植株。PCR及Northern杂交结果表明,NTR1基因稳定整合在大豆基因组并得到过量表达。与野生型植株相比,转基因大豆叶片与根的形态发生了显著的变化。大部分转基因大豆叶片变得细长,初生根生长受到抑制而侧根的生长却受到促进。定量分析结果表明,转基因大豆植株叶片中茉莉酸甲酯的含量比对照高出 2～2.5 倍。这些结果表明,内源茉莉酸甲酯的积累参与了大豆形态发生的调控。     

Overexpression of (At)NPR1 in rice leads to a BTH- and environment-induced lesion-mimic/cell death phenotype

Systemic acquired resistance (SAR) is an inducible defense response that protects plants against a broad spectrum of pathogens. A central regulator of SAR in Arabidopsis is NPR1 (nonexpresser of pathogenesis-related genes). In rice, overexpression of Arabidopsis NPR1 enhances plant resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae. This report demonstrates that overexpression of (At)NPR1 in rice also triggers a lesion-mimic/cell death (LMD) phenotype. The LMD phenotype is environmentally regulated and heritable. In addition, the development of lesions and death correlates with the expression of rice defense genes and the accumulation of hydrogen peroxide. Application of the salicylic acid (SA) analog, benzo(1,2,3) thiadiazole-7-carbothioc acid S-methyl ester (BTH), potentiates this phenotype Endogenous SA levels are reduced in rice overexpressing (At)NPR1 when compared with wildtype plants, supporting the idea that (At)NPR1 may perceive and modulate the accumulation of SA. The association of (At)NPR1 expression in rice with the development of an LMD phenotype suggests that (At)NPR1 has multiple roles in plant stress responses that may affect its efficacy as a transgenic tool for engineering broad-spectrum resistance.     

The rice terpene synthase gene OsTPS19 functions as an (S)‐limonene synthase in planta,and its overexpression leads to enhanced resistance to the blast fungus Magnaporthe oryzae

Rice blast disease, caused by the fungus Magnaporthe oryzae, is the most devastating disease of rice. In our ongoing characterization of the defence mechanisms of rice plants against M. oryzae, a terpene synthase gene OsTPS19 was identified as a candidate defence gene. Here, we report the functional characterization of OsTPS19, which is up‐regulated by M. oryzae infection. Overexpression of OsTPS19 in rice plants enhanced resistance against M. oryzae, while OsTPS19 RNAi lines were more susceptible to the pathogen. Metabolic analysis revealed that the production of a monoterpene (S)‐limonene was increased and decreased in OsTPS19 overexpression and RNAi lines, respectively, suggesting that OsTPS19 functions as a limonene synthase in planta. This notion was further supported by in vitro enzyme assays with recombinant OsTPS19, in which OsTPS19 had both sesquiterpene activity and monoterpene synthase activity, with limonene as a major product. Furthermore, in a subcellular localization experiment, OsTPS19 was localized in plastids. OsTPS19 has a highly homologous paralog, OsTPS20, which likely resulted from a recent gene duplication event. We found that the variation in OsTPS19 and OsTPS20 enzyme activities was determined by a single amino acid in the active site cavity. The expression of OsTPS20 was not affected by M. oryzae infection. This indicates functional divergence of OsTPS19 and OsTPS20. Lastly, (S)‐limonene inhibited the germination of M. oryzae spores in vitro. OsTPS19 was determined to function as an (S)‐limonene synthase in rice and plays a role in defence against M. oryzae, at least partly, by inhibiting spore germination.     

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.     

Manipulation of VOC emissions with methyl jasmonate and carrageenan in the evergreen conifer Pinus sylvestris and evergreen broadleaf Quercus ilex

Plant defence can be induced by exposing plants to the plant hormone jasmonic acid (JA) or its volatile ester, methyl jasmonate (MeJA). Carrageenans (Carr) - sulphated D-galactans extracted from red algae - can also induce plant defences. In this study, the effects of exogenous MeJA and Carr application (concentration 300 and 12.7 μmol, respectively) on volatile emissions from two widespread evergreen woody species, Pinus sylvestris (nine Turkish and one Finnish provenance) and Quercus ilex (Italian provenance) were investigated. We collected headspace samples from seedlings and analysed the quality and quantity of volatile compounds emitted by treated and control plants. In total, 19 monoterpenes, 10 sesquiterpenes, 10 green leaf volatiles (GLVs) and two aromatic compounds were emitted by P. sylvestris from all the provenances studied. Foliar MeJA application clearly affected the volatile profiles of trees from all the provenances. Effects of Carr were genotype specific. In Q. ilex, emissions of sesquiterpenes, GLVs and the homoterpene (E)-DMNT were all induced by MeJA application. However, emissions of most constitutively emitted monoterpenes were significantly reduced. Carr application also led to a significant reduction in monoterpene emissions, but without corresponding increases in other emissions. Our results indicate that exogenously applied MeJA and Carr can both significantly modify the volatile profiles of P. sylvestris and Q. ilex, but also that there are important provenance- and species-specific differences in the overall degree of elicitation and compositions of elicited compounds.