RESUMO
Secondary metabolites produced in glandular trichomes of tomato are involved in interactions with herbivores. In cultivated tomato (Solanum lycopersicum) glandular trichomes accumulate a blend of abundant monoterpenes and smaller amounts of a few sesquiterpenes. These mono- and sesquiterpenes are synthesized by three terpene synthases, TPS20 as well as TPS9 and TPS12, respectively. To study effects of these terpenes on performance and choice behavior of potato aphid (Macrosiphum euphorbiae), we utilized two tomato trichome mutants, hairless and odorless-2, that are differently affected in mono- and sesquiterpene production. Non-choice assays demonstrated that longevity and fecundity of M. euphorbiae were increased when kept on the trichome mutants. A principal component analysis of these aphid performance parameters and terpene production in the trichome mutants indicated that longevity and fecundity of M. euphorbiae were negatively correlated with production of the TPS12-derived sesquiterpenes ß-caryophyllene and α-humulene. While we had previously shown that addition of pure ß-caryophyllene/α-humulene to an artificial feeding diet affected M. euphorbiae apterae survivorship and feeding behavior, no such effects were observed here upon addition of a mixture of pure TPS20-derived monoterpenes. In olfactometer assays M. euphorbiae alates displayed differential choice behaviors towards the hairless and odorless-2 mutants suggesting a role of TPS20-derived monoterpenes in aphid attraction, which was further confirmed using a mixture of pure monoterpenes. Our analyses revealed contrasting roles of glandular trichome-derived terpenes in S. lycopersicum. While TPS12-derived sesquiterpenes contribute to host plant resistance against M. euphorbiae, TPS20-derived monoterpenes appear to be exploited as cue for host plant orientation by aphids.
Assuntos
Afídeos/fisiologia , Monoterpenos/metabolismo , Sesquiterpenos Policíclicos/metabolismo , Solanum lycopersicum/metabolismo , Tricomas/metabolismo , Animais , Fertilidade , Longevidade , Solanum lycopersicum/genética , Sesquiterpenos Monocíclicos/metabolismo , OlfatometriaRESUMO
In plants, geranylgeranyl diphosphate (GGPP) is produced by plastidic GGPP synthase (GGPPS) and serves as a precursor for vital metabolic branches, including chlorophyll, carotenoid, and gibberellin biosynthesis. However, molecular mechanisms regulating GGPP allocation among these biosynthetic pathways localized in the same subcellular compartment are largely unknown. We found that rice contains only one functionally active GGPPS, OsGGPPS1, in chloroplasts. A functionally active homodimeric enzyme composed of two OsGGPPS1 subunits is located in the stroma. In thylakoid membranes, however, the GGPPS activity resides in a heterodimeric enzyme composed of one OsGGPPS1 subunit and GGPPS recruiting protein (OsGRP). OsGRP is structurally most similar to members of the geranyl diphosphate synthase small subunit type II subfamily. In contrast to members of this subfamily, OsGRP enhances OsGGPPS1 catalytic efficiency and specificity of GGPP production on interaction with OsGGPPS1. Structural biology and protein interaction analyses demonstrate that affinity between OsGRP and OsGGPPS1 is stronger than between two OsGGPPS1 molecules in homodimers. OsGRP determines OsGGPPS1 suborganellar localization and directs it to a large protein complex in thylakoid membranes, consisting of geranylgeranyl reductase (OsGGR), light-harvesting-like protein 3 (OsLIL3), protochlorophyllide oxidoreductase (OsPORB), and chlorophyll synthase (OsCHLG). Taken together, genetic and biochemical analyses suggest OsGRP functions in recruiting OsGGPPS1 from the stroma toward thylakoid membranes, thus providing a mechanism to control GGPP flux toward chlorophyll biosynthesis.
Assuntos
Clorofila/biossíntese , Geraniltranstransferase/metabolismo , Complexos Multiproteicos/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Tilacoides/metabolismo , Clorofila/genética , Geraniltranstransferase/genética , Complexos Multiproteicos/genética , Oryza/genética , Proteínas de Plantas/genética , Tilacoides/genéticaRESUMO
Terpenoids, compounds found in all domains of life, represent the largest class of natural products with essential roles in their hosts. All terpenoids originate from the five-carbon building blocks, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), which can be derived from the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways. The absence of two components of the MVA pathway from archaeal genomes led to the discovery of an alternative MVA pathway with isopentenyl phosphate kinase (IPK) catalyzing the final step, the formation of IPP. Despite the fact that plants contain the complete classical MVA pathway, IPK homologs were identified in every sequenced green plant genome. Here, we show that IPK is indeed a member of the plant terpenoid metabolic network. It is localized in the cytosol and is coexpressed with MVA pathway and downstream terpenoid network genes. In planta, IPK acts in parallel with the MVA pathway and plays an important role in regulating the formation of both MVA and MEP pathway-derived terpenoid compounds by controlling the ratio of IP/DMAP to IPP/DMAPP. IP and DMAP can also competitively inhibit farnesyl diphosphate synthase. Moreover, we discovered a metabolically available carbon source for terpenoid formation in plants that is accessible via IPK overexpression. This metabolite reactivation approach offers new strategies for metabolic engineering of terpenoid production.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Archaea/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Terpenos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Citosol/metabolismo , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Genes de Plantas , Hemiterpenos/metabolismo , Cinética , Redes e Vias Metabólicas/genética , Ácido Mevalônico/metabolismo , Compostos Organofosforados/metabolismo , Plantas Geneticamente Modificadas , Plastídeos/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Homologia de Sequência de Aminoácidos , Sesquiterpenos/metabolismo , Nicotiana/genéticaRESUMO
Synthetic plant volatile lures attract natural enemies, but may have non-target effects due to the multifunctional nature of volatile signals. For example, methyl salicylate (MeSA) is used to attract predators, yet also serves as a signaling hormone involved in plant pathogen defense. We investigated the consequences of deploying MeSA lures to attract predators for tomato (Solanum lycopersicum) defense against herbivores. To understand the spatial distribution of the lure's effect, we exposed tomatoes in the field to MeSA along a linear distance gradient and induced defenses by simulating feeding by hornworm caterpillars in a fully crossed factorial design (+/- MeSA, +/- herbivory). Subsequently, we analyzed activity of several defensive proteins (protease inhibitors, polyphenol oxidase, peroxidase), development of hornworm larvae (Manduca sexta), growth of fungal pathogens (Cladosporium and Alternaria), and attractiveness to herbivores and predators. Overall, MeSA-exposed plants were more resistant to both insects and pathogens. Secondary pathogen infection was reduced by 25% in MeSA exposed plants, possibly due to elevated polyphenol oxidase activity. Interestingly, we found that lures affected plant pathogen defenses equivalently across all distances (up to 4 m away) indicating that horizontal diffusion of a synthetic volatile may be greater than previously assumed. While thrips avoided colonizing hornworm- damaged tomato plants, this induced resistance was not observed upon pre-exposure to MeSA, suggesting that MeSA suppresses the repellant effect induced by herbivory. Thus, using MeSA lures in biological control may inadvertently protect crops from pathogens, but has mixed effects on plant resistance to insect herbivores.
Assuntos
Herbivoria , Manduca/fisiologia , Salicilatos/química , Salicilatos/farmacologia , Solanum lycopersicum , Alternaria/crescimento & desenvolvimento , Animais , Catecol Oxidase/metabolismo , Cladosporium/crescimento & desenvolvimento , Larva/fisiologia , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiologia , Peroxidase/metabolismo , Controle Biológico de Vetores , Folhas de Planta/microbiologia , Inibidores de Proteases/metabolismo , Espectrometria de Massas em Tandem , Compostos Orgânicos Voláteis/química , Compostos Orgânicos Voláteis/metabolismoRESUMO
Geranyl diphosphate (GPP), the precursor of most monoterpenes, is synthesized in plastids from dimethylallyl diphosphate and isopentenyl diphosphate by GPP synthases (GPPSs). In heterodimeric GPPSs, a non-catalytic small subunit (GPPS-SSU) interacts with a catalytic large subunit, such as geranylgeranyl diphosphate synthase, and determines its product specificity. Here, snapdragon (Antirrhinum majus) GPPS-SSU was over-expressed in tomato fruits under the control of the fruit ripening-specific polygalacturonase promoter to divert the metabolic flux from carotenoid formation towards GPP and monoterpene biosynthesis. Transgenic tomato fruits produced monoterpenes, including geraniol, geranial, neral, citronellol and citronellal, while exhibiting reduced carotenoid content. Co-expression of the Ocimum basilicum geraniol synthase (GES) gene with snapdragon GPPS-SSU led to a more than threefold increase in monoterpene formation in tomato fruits relative to the parental GES line, indicating that the produced GPP can be used by plastidic monoterpene synthases. Co-expression of snapdragon GPPS-SSU with the O. basilicum α-zingiberene synthase (ZIS) gene encoding a cytosolic terpene synthase that has been shown to possess both sesqui- and monoterpene synthase activities resulted in increased levels of ZIS-derived monoterpene products compared to fruits expressing ZIS alone. These results suggest that re-direction of the metabolic flux towards GPP in plastids also increases the cytosolic pool of GPP available for monoterpene synthesis in this compartment via GPP export from plastids.
Assuntos
Alquil e Aril Transferases/genética , Antirrhinum/genética , Difosfatos/metabolismo , Diterpenos/metabolismo , Monoterpenos/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Alquil e Aril Transferases/metabolismo , Citosol/metabolismo , Frutas/genética , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Geraniltranstransferase/genética , Geraniltranstransferase/metabolismo , Sesquiterpenos Monocíclicos , Ocimum basilicum/genética , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Plantas Geneticamente Modificadas , Plastídeos/genética , Plastídeos/metabolismo , Poligalacturonase/genética , Regiões Promotoras Genéticas , Sesquiterpenos/metabolismoRESUMO
Recently it was shown that monoterpenes in tomato trichomes (Solanum lycopersicum) are synthesized by phellandrene synthase 1 (PHS1) from the non-canonical substrate neryl diphosphate (NPP), the cis-isomer of geranyl diphosphate (GPP). As PHS1 accepts both NPP and GPP substrates forming different monoterpenes, it was overexpressed in tomato fruits to test if NPP is also available in a tissue highly active in carotenoid production. However, transgenic fruits overexpressing PHS1 produced only small amounts of GPP-derived PHS1 monoterpene products, indicating the absence of endogenous NPP. Therefore, NPP formation was achieved by diverting the metabolic flux from carotenoids via expression of tomato neryl diphosphate synthase 1 (NDPS1). NDPS1 transgenic fruits produced NPP-derived monoterpenes, including nerol, neral and geranial, while displaying reduced lycopene content. NDPS1 co-expression with PHS1 resulted in a monoterpene blend, including ß-phellandrene, similar to that produced from NPP by PHS1 in vitro and in trichomes. Unexpectedly, PHS1×NDPS1 fruits showed recovery of lycopene levels compared to NDPS1 fruits, suggesting that redirection of metabolic flux is only partially responsible for the reduction in carotenoids. In vitro assays demonstrated that NPP serves as an inhibitor of geranylgeranyl diphosphate synthase, thus its consumption by PHS1 leads to recovery of lycopene levels. Monoterpenes produced in PHS1×NDPS1 fruits contributed to direct plant defense negatively affecting feeding behavior of the herbivore Helicoverpa zea and displaying antifungal activity against Botrytis cinerea. These results show that NPP-derived terpenoids can be produced in plant tissues; however, NPP has to be consumed to avoid negative impacts on plant metabolism.
Assuntos
Frutas , Engenharia Metabólica , Monoterpenos/metabolismo , Plantas Geneticamente Modificadas , Fosfatos de Poli-Isoprenil/metabolismo , Solanum lycopersicum , Carotenoides/biossíntese , Carotenoides/genética , Frutas/genética , Frutas/metabolismo , Licopeno , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismoRESUMO
Terpenoids constitute one of the largest and most chemically diverse classes of primary and secondary metabolites in nature with an exceptional breadth of functional roles in plants. Biosynthesis of all terpenoids begins with the universal fivecarbon building blocks, isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP), which in plants are derived from two compartmentally separated but metabolically crosstalking routes, the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways. Here, we review the current knowledge on the terpenoid precursor pathways and highlight the critical hidden constraints as well as multiple regulatory mechanisms that coordinate and homeostatically govern carbon flux through the terpenoid biosynthetic network in plants.
Assuntos
Plantas , Terpenos , Terpenos/metabolismo , Terpenos/química , Plantas/metabolismo , Vias Biossintéticas , Ácido Mevalônico/metabolismo , Compostos Organofosforados , HemiterpenosRESUMO
The invasive shrub glossy buckthorn (Frangula alnus) has been progressively colonizing the Northeastern United States and Southeastern Canada for more than a century. To determine the dominant arthropod orders and species associated with F. alnus, field surveys were conducted for two years across 16 plots within the Allegheny National Forest, Pennsylvania, USA. Statistical analyses were employed to assess the impact of seasonal variation on insect order richness and diversity. The comprehensive arthropod collection yielded 2845 insects and arachnids, with hemipterans comprising the majority (39.8%), followed by dipterans (22.3%) and arachnids (15.5%). Notably, 16.2% of the hemipterans collected were in the immature stages, indicating F. alnus as a host for development. The two dominant insect species of F. alnus were Psylla carpinicola (Hemiptera: Psyllidae) and Drosophila suzukii (Diptera: Drosophilidae); D. suzukii utilized F. alnus fruits for reproduction. Species richness and diversity exhibited significant variations depending on the phenology of F. alnus. The profiles of volatile compounds emitted from the leaves and flowers of F. alnus were analyzed to identify factors that potentially contribute to the attraction of herbivores and pollinators. The results of our study will advance the development of novel F. alnus management strategies leveraging the insects associated with this invasive species.
RESUMO
Aromatic L-amino acid decarboxylases (AADCs) are key enzymes operating at the interface between primary and secondary metabolism. The Arabidopsis thaliana genome contains two genes, At2g20340 and At4g28680, encoding pyridoxal 5'-phosphate-dependent AADCs with high homology to the recently identified Petunia hybrida phenylacetaldehyde synthase involved in floral scent production. The At4g28680 gene product was recently biochemically characterized as an L-tyrosine decarboxylase (AtTYDC), whereas the function of the other gene product remains unknown. The biochemical and functional characterization of the At2g20340 gene product revealed that it is an aromatic aldehyde synthase (AtAAS), which catalyzes the conversion of phenylalanine and 3,4-dihydroxy-L-phenylalanine to phenylacetaldehyde and dopaldehyde, respectively. AtAAS knock-down and transgenic AtAAS RNA interference (RNAi) lines show significant reduction in phenylacetaldehyde levels and an increase in phenylalanine, indicating that AtAAS is responsible for phenylacetaldehyde formation in planta. In A. thaliana ecotype Columbia (Col-0), AtAAS expression was highest in leaves, and was induced by methyl jasmonate treatment and wounding. Pieris rapae larvae feeding on Col-0 leaves resulted in increased phenylacetaldehyde emission, suggesting that the emitted aldehyde has a defensive activity against attacking herbivores. In the ecotypes Sei-0 and Di-G, which emit phenylacetaldehyde as a predominant flower volatile, the highest expression of AtAAS was found in flowers and RNAi AtAAS silencing led to a reduction of phenylacetaldehyde formation in this organ. In contrast to ecotype Col-0, no phenylacetaldehyde accumulation was observed in Sei-0 upon wounding, suggesting that AtAAS and subsequently phenylacetaldehyde contribute to pollinator attraction in this ecotype.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Descarboxilases de Aminoácido-L-Aromático/metabolismo , Folhas de Planta/metabolismo , Tirosina Descarboxilase/metabolismo , Acetaldeído/análogos & derivados , Acetaldeído/metabolismo , Acetatos/farmacologia , Animais , Arabidopsis/efeitos dos fármacos , Arabidopsis/parasitologia , Proteínas de Arabidopsis/genética , Descarboxilases de Aminoácido-L-Aromático/genética , Ciclopentanos/farmacologia , Comportamento Alimentar , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Insetos/patogenicidade , Larva/patogenicidade , Odorantes , Oxilipinas/farmacologia , Filogenia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/parasitologia , Pólen/genética , Pólen/metabolismo , Interferência de RNA , Homologia de Sequência de Aminoácidos , Tirosina Descarboxilase/genética , Compostos Orgânicos Voláteis/metabolismo , VolatilizaçãoRESUMO
Native polyacrylamide gel electrophoresis (PAGE) is an important technique for the analysis of membrane protein complexes. A major breakthrough was the development of blue native (BN-) and high resolution clear native (hrCN-) PAGE techniques. Although these techniques are very powerful, they could not be applied to all systems with the same resolution. We have developed an alternative protocol for the analysis of membrane protein complexes of plant chloroplasts and cyanobacteria, which we termed histidine- and deoxycholate-based native (HDN-) PAGE. We compared the capacity of HDN-, BN- and hrCN-PAGE to resolve the well-studied respiratory chain complexes in mitochondria of bovine heart muscle and Yarrowia lipolytica, as well as thylakoid localized complexes of Medicago sativa, Pisum sativum and Anabaena sp. PCC7120. Moreover, we determined the assembly/composition of the Anabaena sp. PCC7120 thylakoids and envelope membranes by HDN-PAGE. The analysis of isolated chloroplast envelope complexes by HDN-PAGE permitted us to resolve complexes such as the translocon of the outer envelope migrating at approximately 700 kDa or of the inner envelope of about 230 and 400 kDa with high resolution. By immunodecoration and mass spectrometry of these complexes we present new insights into the assembly/composition of these translocation machineries. The HDN-PAGE technique thus provides an important tool for future analyses of membrane complexes such as protein translocons.
Assuntos
Ácido Desoxicólico/química , Eletroforese em Gel de Poliacrilamida/métodos , Histidina/química , Proteínas de Membrana/análise , Complexos Multiproteicos/análise , Anabaena/química , Animais , Bovinos , Parede Celular/química , Cloroplastos/química , Cianobactérias/química , Coração , Espectrometria de Massas , Medicago sativa/química , Proteínas de Membrana/isolamento & purificação , Mitocôndrias/química , Complexos Multiproteicos/isolamento & purificação , Músculos/química , Pisum sativum/química , Transporte Proteico , Tilacoides/química , Yarrowia/químicaRESUMO
Geranyl diphosphate (GPP), the precursor of many monoterpene end products, is synthesized in plastids by a condensation of dimethylallyl diphosphate and isopentenyl diphosphate (IPP) in a reaction catalyzed by homodimeric or heterodimeric GPP synthase (GPPS). In the heterodimeric enzymes, a noncatalytic small subunit (GPPS.SSU) determines the product specificity of the catalytic large subunit, which may be either an active geranylgeranyl diphosphate synthase (GGPPS) or an inactive GGPPS-like protein. Here, we show that expression of snapdragon (Antirrhinum majus) GPPS.SSU in tobacco (Nicotiana tabacum) plants increased the total GPPS activity and monoterpene emission from leaves and flowers, indicating that the introduced catalytically inactive GPPS.SSU found endogenous large subunit partner(s) and formed an active snapdragon/tobacco GPPS in planta. Bimolecular fluorescence complementation and in vitro enzyme analysis of individual and hybrid proteins revealed that two of four GGPPS-like candidates from tobacco EST databases encode bona fide GGPPS that can interact with snapdragon GPPS.SSU and form a functional GPPS enzyme in plastids. The formation of chimeric GPPS in transgenic plants also resulted in leaf chlorosis, increased light sensitivity, and dwarfism due to decreased levels of chlorophylls, carotenoids, and gibberellins. In addition, these transgenic plants had reduced levels of sesquiterpene emission, suggesting that the export of isoprenoid intermediates from the plastids into the cytosol was decreased. These results provide genetic evidence that GPPS.SSU modifies the chain length specificity of phylogenetically distant GGPPS and can modulate IPP flux distribution between GPP and GGPP synthesis in planta.
Assuntos
Antirrhinum/enzimologia , Farnesiltranstransferase/metabolismo , Nicotiana/enzimologia , Sesquiterpenos/metabolismo , Antirrhinum/genética , Clonagem Molecular , Difosfatos/metabolismo , Diterpenos/metabolismo , Farnesiltranstransferase/genética , Flores/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Monoterpenos/metabolismo , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , RNA de Plantas/genética , Nicotiana/genéticaRESUMO
Terpenoids constitute one of the largest and most diverse classes of plant metabolites. While some terpenoids are involved in essential plant processes such as photosynthesis, respiration, growth, and development, others are specialized metabolites playing roles in the interaction of plants with their biotic and abiotic environment. Due to the distinct functions and properties of specific terpenoid compounds, there is a growing interest to introduce or modify their production in plants by metabolic engineering for agricultural, pharmaceutical, or industrial applications. The MVA and MEP pathways and the prenyltransferases providing the general precursors for terpenoid formation, as well as the enzymes of the various downstream metabolic pathways leading to the formation of different groups of terpenoid compounds have been characterized in detail in plants. In contrast, the molecular mechanisms directing the metabolic flux of precursors specifically toward one of several potentially competing terpenoid biosynthetic pathways are still not well understood. The formation of metabolons, multi-protein complexes composed of enzymes catalyzing sequential reactions of a metabolic pathway, provides a promising concept to explain the metabolic channeling that appears to occur in the complex terpenoid biosynthetic network of plants. Here we provide an overview about examples of potential metabolons involved in plant terpenoid metabolism that have been recently characterized and the first attempts to utilize metabolic channeling in terpenoid metabolic engineering. In addition, we discuss the gaps in our current knowledge and in consequence the need for future basic and applied research.
RESUMO
Over the last decade, the Allegheny National Forest (ANF) in the USA has experienced issues with the regeneration of black cherry (Prunus serotina). This study was conducted to investigate the effects of silvicultural treatment on the insect communities that may affect black cherry pollination and regeneration. We conducted a 2-year study to compare the abundance, richness, and diversity of insects in unmanaged, shelterwood seed-tree, and shelterwood clear-cut stands. Using pan traps, we sampled insects at the ground level and in the canopies of flowering mature black cherry trees. The results of this study showed significant increases in the abundance of insects captured in shelterwood seed-tree stands and in species richness and diversity of insects captured in the canopy of black cherry in shelterwood removal stands, indicating that silvicultural treatment affected the insect community significantly. The dominant insect order was Diptera (true flies, 72.91%, n = 12,668), and Anthalia bulbosa (Diptera: Hybotidae) was the dominant species comprising 33% of all insects found in the canopy of flowering black cherry. The findings in this study could help land managers in managing black cherry for its pollination and natural regeneration.
RESUMO
Tomato produces a number of terpenes in their glandular trichomes that contribute to host plant resistance against pests. While glandular trichomes of cultivated tomato Solanum lycopersicum primarily accumulate a blend of monoterpenes, those of the wild tomato species Solanum habrochaites produce various sesquiterpenes. Recently, we have identified two groups of sesquiterpenes in S. habrochaites accessions that negatively affect the performance and choice behavior of the potato aphid (Macrosiphum euphorbiae). Aphids are piercing-sucking herbivores that use their mouthpart to penetrate and probe plant tissues in order to ultimately access vascular tissue and ingest phloem sap. Because secondary metabolites produced in glandular trichomes can affect the initial steps of the aphid feeding behavior, introducing the formation of defensive terpenes into additional plant tissues via metabolic engineering has the potential to reduce tissue penetration by aphids and in consequence virus transmission. Here, we have developed two multicistronic expression constructs based on the two sesquiterpene traits with activity toward M. euphorbiae previously identified in S. habrochaites. Both constructs are composed of sequences encoding a prenyl transferase and a respective S. habrochaites terpene synthase, as well as enhanced green fluorescent protein as a visible marker. All three coding sequences were linked by short nucleotide sequences encoding the foot-and-mouth disease virus 2A self-processing oligopeptide which allows their co-expression under the control of one promoter. Transient expression of both constructs under the epidermis-specific Arabidopsis CER5-promoter in tomato leaves demonstrated that formation of the two sets of defensive sesquiterpenes, ß-caryophyllene/α-humulene and (-)-endo-α-bergamotene/(+)-α-santalene/(+)-endo-ß-bergamotene, can be introduced into new tissues in tomato. The epidermis-specific transgene expression and terpene formation were verified by fluorescence microscopy and tissue fractionation with subsequent analysis of terpene profiles, respectively. In addition, the longevity and fecundity of M. euphorbiae feeding on these engineered tomato leaves were significantly reduced, demonstrating the efficacy of this novel aphid control strategy.
RESUMO
Black cherry is an ecologically important high-value wood. A decline of its regeneration has been reported in the USA, which could be associated with a lack of pollination. This study was conducted to identify insects visiting black cherry flowers, to determine whether insects captured on the flowers carry black cherry pollen and to identify the volatile organic compounds (VOCs) emitted by flowers of black cherry. A two-year insect survey was conducted before, during and after the black cherry bloom. A total of 9533 insects were captured in traps and Diptera was the most abundant (64.1%). Significantly more insects in Diptera, Lepidoptera and Thysanoptera were captured in the traps installed in the canopy than those on the ground, and Anthalia bulbosa (Diptera: Hybotidae) was the dominant species. Electron microscopy analyses demonstrated that insects captured in the canopy indeed carried black cherry pollen. Black cherry flowers emitted a VOC blend that is composed of 34 compounds and dominated by ß-ocimene and several phenylpropanoids/benzenoids. This floral VOC profile is similar to that of other pollinator-dependent Prunus species. This study reports pollinator insects and associated VOCs, for the first time, that could play a significant role in the pollination and regeneration of black cherry.
RESUMO
Terpenoids are a large and diverse class of plant metabolites that also includes volatile mono- and sesquiterpenes which are involved in biotic interactions of plants. Due to the limited natural availability of these terpenes and the tight regulation of their biosynthesis, there is strong interest to introduce or enhance their production in crop plants by metabolic engineering for agricultural, pharmaceutical and industrial applications. While engineering of monoterpenes has been quite successful, expression of sesquiterpene synthases in engineered plants frequently resulted in production of only minor amounts of sesquiterpenes. To identify bottlenecks for sesquiterpene engineering in plants, we have used two nearly identical terpene synthases, snapdragon (Antirrhinum majus) nerolidol/linalool synthase-1 and -2 (AmNES/LIS-1/-2), that are localized in the cytosol and plastids, respectively. Since these two bifunctional terpene synthases have very similar catalytic properties with geranyl diphosphate (GPP) and farnesyl diphosphate (FPP), their expression in target tissues allows indirect determination of the availability of these substrates in both subcellular compartments. Both terpene synthases were expressed under control of the ripening specific PG promoter in tomato fruits, which are characterized by a highly active terpenoid metabolism providing precursors for carotenoid biosynthesis. As AmNES/LIS-2 fruits produced the monoterpene linalool, AmNES/LIS-1 fruits were found to exclusively produce the sesquiterpene nerolidol. While nerolidol emission in AmNES/LIS-1 fruits was 60- to 584-fold lower compared to linalool emission in AmNES/LIS-2 fruits, accumulation of nerolidol-glucosides in AmNES/LIS-1 fruits was 4- to 14-fold lower than that of linalool-glucosides in AmNES/LIS-2 fruits. These results suggest that only a relatively small pool of FPP is available for sesquiterpene formation in the cytosol. To potentially overcome limitations in sesquiterpene production, we transiently co-expressed the key pathway-enzymes hydroxymethylglutaryl-CoA reductase (HMGR) and 1-deoxy-D-xylulose 5-phosphate synthase (DXS), as well as the regulator isopentenyl phosphate kinase (IPK). While HMGR and IPK expression increased metabolic flux toward nerolidol formation 5.7- and 2.9-fold, respectively, DXS expression only resulted in a 2.5-fold increase.
RESUMO
The twin arginine translocation (Tat) machinery which is capable of transporting folded proteins across lipid bilayers operates in the thylakoid membrane of plant chloroplasts as well as in the cytoplasmic membrane of bacteria. It is composed of three integral membrane proteins (TatA, TatB, and TatC) which form heteromeric complexes of high molecular weight that accomplish binding and transport of substrates carrying Tat pathway-specific signal peptides. Western analyses using affinity purified antibodies showed in both, juvenile and adult tissue from Arabidopsis thaliana, an approximately equimolar ratio of the TatB and TatC components, whereas TatA was detectable only in minor amounts. Upon Blue Native-PAGE, TatB and TatC were found in four heteromeric TatB/C complexes possessing molecular weights of approximately 310, 370, 560 and 620 kDa, respectively, while TatA was detected only in a molecular weight range below 200 kDa. The implications of these findings on the currently existing models explaining the mechanism of Tat transport are discussed.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Subunidades Proteicas/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/isolamento & purificação , Membranas Intracelulares/metabolismo , Pisum sativum/metabolismo , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação , Transporte Proteico , Solubilidade , Especificidade da Espécie , Tilacoides/metabolismoRESUMO
Glandular trichomes of tomato produce a number of secondary metabolites including terpenes that contribute to host plant resistance against pests. While glandular trichomes of cultivated tomato Solanum lycopersicum primarily accumulate a monoterpene blend, those of wild tomato species like Solanum habrochaites produce various sesquiterpenes. Previous studies have shown that glandular trichome derived terpenes in cultivated and wild tomato species have repellent and toxic activity against multiple biting-chewing herbivores. In contrast, considerably less is known about the effect of these glandular trichome derived terpenes on piercing-sucking herbivores such as aphids. Here, we have screened a collection of S. habrochaites accessions representing five chemotypes that produce distinct sets of sesquiterpenes to identify those affecting the potato aphid (Macrosiphum euphorbiae). Non-choice assays demonstrated that the longevity and fecundity of M. euphorbiae was significantly reduced when kept on the leaf surface of S. habrochaites accessions producing ß-caryophyllene and α-humulene, or α-santalene, α-bergamotene, and ß-bergamotene, respectively. When M. euphorbiae apterae were feeding on artificial diets with added terpene containing leaf dip extracts, the same ß-caryophyllene/α-humulene and α-santalene/α-bergamotene/ß-bergamotene producing S. habrochaites accessions were found to affect aphid survivorship and feeding behavior as indicated by gel saliva investment and honeydew production. Olfactometer assays revealed that the sesquiterpenes emitted from these S. habrochaites accessions also have repellent activity against M. euphorbiae alatae affecting their choice behavior prior to landing on host plants. Assays performed with pure sesquiterpene compounds and an introgression line carrying respective S. habrochaites terpene biosynthetic genes in the S. lycopersicum background confirmed that ß-caryophyllene/α-humulene and α-santalene/α-bergamotene/ß-bergamotene were responsible for the observed effects on performance, feeding and choice behavior of M. euphorbiae.
Assuntos
Afídeos , Sesquiterpenos , Solanum lycopersicum , Solanum , Animais , Comportamento Alimentar , TricomasRESUMO
Terpenoids emitted from snapdragon flowers include three monoterpenes derived from geranyl diphosphate (GPP), myrcene, (E)-beta-ocimene and linalool, and a sesquiterpene, nerolidol, derived from farnesyl diphosphate (FPP). Using a functional genomics approach, we have isolated and biochemically characterized two nearly identical nerolidol/linalool synthases, AmNES/LIS-1 and AmNES/LIS-2, two enzymes responsible for the terpenoid profile of snapdragon scent remaining to be characterized. The AmNES/LIS-2 protein has an additional 30 amino acids in the N-terminus, and shares 95% amino acid sequence identity with AmNES/LIS-1, with only 23 amino acid substitutions distributed across the homologous regions of the proteins. Although these two terpene synthases have very similar catalytic properties, and synthesize linalool and nerolidol as specific products from GPP and FPP, respectively, they are compartmentally segregated. GFP localization studies and analysis of enzyme activities in purified leucoplasts, together with our previous feeding experiments, revealed that AmNES/LIS-1 is localized in cytosol, and is responsible for nerolidol biosynthesis, whereas AmNES/LIS-2 is located in plastids, and accounts for linalool formation. Our results show that subcellular localization of bifunctional enzymes, in addition to the availability of substrate, controls the type of product formed. By directing nearly identical bifunctional enzymes to more than one cellular compartment, plants extend the range of available substrates for enzyme utilization, thus increasing the diversity of the metabolites produced.
Assuntos
Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Antirrhinum/enzimologia , Flores/enzimologia , Monoterpenos/metabolismo , Sesquiterpenos/metabolismo , Monoterpenos Acíclicos , Alquil e Aril Transferases/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
The chloroplast is an organelle of prokaryotic origin that is situated in an eukaryotic cellular environment. As a result of this formerly endosymbiotic situation, the chloroplast houses a unique set of protein transport machineries. Among those are evolutionarily young transport pathways which are responsible for the import of the nuclear-encoded proteins into the organelle as well as ancient pathways operating in the 'export' of proteins from the stroma (the former cyanobacterial cytosol) across the thylakoid membrane into the thylakoid lumen. In this review, we have tried to address the main features of these various transport pathways.