RESUMO
Cytotoxic stress activates stress-activated kinases, initiates adaptive mechanisms, including the unfolded protein response (UPR) and autophagy, and induces programmed cell death. Fatty acid unsaturation, controlled by stearoyl-CoA desaturase (SCD)1, prevents cytotoxic stress but the mechanisms are diffuse. Here, we show that 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol) [PI(18:1/18:1)] is a SCD1-derived signaling lipid, which inhibits p38 mitogen-activated protein kinase activation, counteracts UPR, endoplasmic reticulum-associated protein degradation, and apoptosis, regulates autophagy, and maintains cell morphology and proliferation. SCD1 expression and the cellular PI(18:1/18:1) proportion decrease during the onset of cell death, thereby repressing protein phosphatase 2 A and enhancing stress signaling. This counter-regulation applies to mechanistically diverse death-inducing conditions and is found in multiple human and mouse cell lines and tissues of Scd1-defective mice. PI(18:1/18:1) ratios reflect stress tolerance in tumorigenesis, chemoresistance, infection, high-fat diet, and immune aging. Together, PI(18:1/18:1) is a lipokine that links fatty acid unsaturation with stress responses, and its depletion evokes stress signaling.
Assuntos
Transdução de Sinais , Estearoil-CoA Dessaturase , Animais , Apoptose , Ácidos Graxos , Camundongos , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Resposta a Proteínas não DobradasRESUMO
The acylphloroglucinol myrtucommulone A (MC) causes mitochondrial dysfunctions by direct interference leading to apoptosis in cancer cells, but the molecular targets involved are unknown. Here, we reveal the chaperonin heat-shock protein 60 (HSP60) as a molecular target of MC that seemingly modulates HSP60-mediated mitochondrial functions. Exploiting an unbiased, discriminative protein fishing approach using MC as bait and mitochondrial lysates from leukemic HL-60 cells as target source identified HSP60 as an MC-binding protein. MC prevented HSP60-mediated reactivation of denatured malate dehydrogenase in a protein refolding assay. Interference of MC with HSP60 was accompanied by aggregation of two proteins in isolated mitochondria under heat shock that were identified as Lon protease-like protein (LONP) and leucine-rich PPR motif-containing protein (LRP130). Together, our results reveal HSP60 as a direct target of MC, proposing MC as a valuable tool for studying HSP60 biology and for evaluating its value as a target in related diseases, such as cancer.
Assuntos
Apoptose/efeitos dos fármacos , Chaperonina 60/antagonistas & inibidores , Proteínas Mitocondriais/antagonistas & inibidores , Floroglucinol/análogos & derivados , Chaperonina 60/química , Chaperonina 60/metabolismo , Citocromos c/metabolismo , Desenho de Fármacos , Células HL-60 , Resposta ao Choque Térmico/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Terapia de Alvo Molecular , Floroglucinol/farmacologia , Agregados Proteicos/efeitos dos fármacosRESUMO
The defensive chemistry and persistence of plant tissues determine their suitability and apparency - the likelihood of being discovered - to insect herbivores. As consumers of plant tissues with transient apparency, florivores and seed-feeders must frequently migrate between host plants to synchronize colonization with plant phenology. Aggregation pheromones could provide information-based solutions to finding ephemeral hosts, but little is known about plant-influenced variation in this form of chemical communication. Combining analytical chemistry, de novo synthesis and field ecology, we investigated the change in colonization of two sympatric host plants, Nicotiana attenuata and Nicotiana obtusifolia, which differ in apparency-related life history traits, by a heteropteran seed-feeder, Corimelaena extensa. We identified a novel pheromone released by C. extensa males - (5Z,8Z)-tetradeca-5,8-dienal - and performed field assays with the synthetic pheromone, showing that it stimulates the formation of feeding aggregations on the post-fire annual N. attenuata. Corimelaena extensa pheromone emission was 40-fold higher when feeding on N. attenuata compared with the perennial N. obtusifolia, as were adult fecundity and seed capsule content of the putative biosynthetic precursor, linoleic acid. Higher pheromone emission increases the apparency and colonization of the ephemeral, high-quality host N. attenuata. This plant-specific variation in insect signaling could facilitate host-finding by seed-feeders migrating between plant patches.
Assuntos
Heterópteros/fisiologia , Nicotiana/fisiologia , Feromônios/fisiologia , Sementes/fisiologia , Aldeídos/química , Animais , Herbivoria , Ácido Linoleico/análise , Masculino , Feromônios/química , Especificidade da Espécie , Compostos Orgânicos Voláteis/análiseRESUMO
Diatoms are unicellular algae of crucial importance as they belong to the main primary producers in aquatic ecosystems. Several diatom species produce polyunsaturated aldehydes (PUAs) that have been made responsible for chemically mediated interactions in the plankton. PUA-effects include chemical defense by reducing the reproductive success of grazing copepods, allelochemical activity by interfering with the growth of competing phytoplankton and cell to cell signaling. We applied a PUA-derived molecular probe, based on the biologically highly active 2,4-decadienal, with the aim to reveal protein targets of PUAs and affected metabolic pathways. By using fluorescence microscopy, we observed a substantial uptake of the PUA probe into cells of the diatom Phaeodactylum tricornutum in comparison to the uptake of a structurally closely related control probe based on a saturated aldehyde. The specific uptake motivated a chemoproteomic approach to generate a qualitative inventory of proteins covalently targeted by the α,ß,γ,δ-unsaturated aldehyde structure element. Activity-based protein profiling revealed selective covalent modification of target proteins by the PUA probe. Analysis of the labeled proteins gave insights into putative affected molecular functions and biological processes such as photosynthesis including ATP generation and catalytic activity in the Calvin cycle or the pentose phosphate pathway. The mechanism of action of PUAs involves covalent reactions with proteins that may result in protein dysfunction and interference of involved pathways.
Assuntos
Aldeídos/química , Proteínas de Algas/análise , Diatomáceas/química , Sondas Moleculares/química , Proteoma/análise , Trifosfato de Adenosina/biossíntese , Proteínas de Algas/química , Proteínas de Algas/metabolismo , Animais , Cromatografia Líquida , Copépodes/fisiologia , Diatomáceas/metabolismo , Eletroforese em Gel Bidimensional , Cadeia Alimentar , Anotação de Sequência Molecular , Via de Pentose Fosfato/fisiologia , Fotossíntese/fisiologia , Fitoplâncton/fisiologia , Proteoma/química , Proteoma/metabolismo , Coloração e Rotulagem/métodos , Espectrometria de Massas em TandemRESUMO
Nuptial gifts produced by males and transferred to females during copulation are common in insects. Yet, their precise composition and subsequent physiological effects on the female recipient remain unresolved. Male decorated crickets Gryllodes sigillatus transfer a spermatophore to the female during copulation that is composed of an edible gift, the spermatophylax, and the ampulla that contains the ejaculate. After transfer of the spermatophore, the female detaches the spermatophylax and starts to eat it while sperm from the ampulla are evacuated into the female reproductive tract. When the female has finished consuming the spermatophylax, she detaches the ampulla and terminates sperm transfer. Hence, one simple function of the spermatophylax is to ensure complete sperm transfer by distracting the female from prematurely removing the ampulla. However, the majority of orally active components of the spermatophylax itself and their subsequent effects on female behavior have not been identified. Here, we report the first analysis of the proteome of the G. sigillatus spermatophylax and the transcriptome of the male accessory glands that make these proteins. The accessory gland transcriptome was assembled into 17,691 transcripts whilst about 30 proteins were detected within the mature spermatophylax itself. Of these 30 proteins, 18 were encoded by accessory gland encoded messages. Most spermatophylax proteins show no similarity to proteins with known biological functions and are therefore largely novel. A spermatophylax protein shows similarity to protease inhibitors suggesting that it may protect the biologically active components from digestion within the gut of the female recipient. Another protein shares similarity with previously characterized insect polypeptide growth factors suggesting that it may play a role in altering female reproductive physiology concurrent with fertilization. Characterization of the spermatophylax proteome provides the first step in identifying the genes encoding these proteins in males and in understanding their biological functions in the female recipient.
Assuntos
Comportamento Alimentar , Gryllidae/fisiologia , Proteínas de Insetos/genética , Comportamento Sexual Animal , Sequência de Aminoácidos , Animais , Anidrases Carbônicas/análise , Anidrases Carbônicas/genética , Copulação , Feminino , Doações , Gryllidae/química , Gryllidae/genética , Proteínas de Insetos/análise , Peptídeos e Proteínas de Sinalização Intercelular/análise , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Dados de Sequência Molecular , Reprodução , Alinhamento de Sequência , Espermatogônias/química , Espermatogônias/fisiologia , TranscriptomaRESUMO
For sexual communication, moths primarily use blends of fatty acid derivatives containing one or more double bonds in various positions and configurations, called sex pheromones (SPs). To study the molecular basis of novel SP component (SPC) acquisition, we used the tobacco hornworm (Manduca sexta), which uses a blend of mono-, di-, and uncommon triunsaturated fatty acid (3UFA) derivatives as SP. We identified pheromone-biosynthetic fatty acid desaturases (FADs) MsexD3, MsexD5, and MsexD6 abundantly expressed in the M. sexta female pheromone gland. Their functional characterization and in vivo application of FAD substrates indicated that MsexD3 and MsexD5 biosynthesize 3UFAs via E/Z14 desaturation from diunsaturated fatty acids produced by previously characterized Z11-desaturase/conjugase MsexD2. Site-directed mutagenesis of sequentially highly similar MsexD3 and MsexD2 demonstrated that swapping of a single amino acid in the fatty acyl substrate binding tunnel introduces E/Z14-desaturase specificity to mutated MsexD2. Reconstruction of FAD gene phylogeny indicates that MsexD3 was recruited for biosynthesis of 3UFA SPCs in M. sexta lineage via gene duplication and neofunctionalization, whereas MsexD5 representing an alternative 3UFA-producing FAD has been acquired via activation of a presumably inactive ancestral MsexD5. Our results demonstrate that a change as small as a single amino acid substitution in a FAD enzyme might result in the acquisition of new SP compounds.
Assuntos
Substituição de Aminoácidos , Evolução Molecular , Ácidos Graxos Dessaturases/metabolismo , Proteínas de Insetos/metabolismo , Manduca/metabolismo , Atrativos Sexuais/biossíntese , Sequência de Aminoácidos , Animais , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Insaturados/genética , Ácidos Graxos Insaturados/metabolismo , Feminino , Proteínas de Insetos/genética , Manduca/genética , Dados de Sequência Molecular , Filogenia , Atrativos Sexuais/genéticaRESUMO
BACKGROUND: Although leaf herbivory-induced changes in allocation of recently assimilated carbon between the shoot and below-ground tissues have been described in several species, it is still unclear which part of the root system is affected by resource allocation changes and which signalling pathways are involved. We investigated carbon partitioning in root tissues following wounding and simulated leaf herbivory in young Nicotiana attenuata plants. RESULTS: Using 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG), which was incorporated into disaccharides in planta, we found that simulated herbivory reduced carbon partitioning specifically to the root tips in wild type plants. In jasmonate (JA) signalling-deficient COI1 plants, the wound-induced allocation of [(18)F]FDG to the roots was decreased, while more [(18)F]FDG was transported to young leaves, demonstrating an important role of the JA pathway in regulating the wound-induced carbon partitioning between shoots and roots. CONCLUSIONS: Our data highlight the use of [(18)F]FDG to study stress-induced carbon allocation responses in plants and indicate an important role of the JA pathway in regulating wound-induced shoot to root signalling.
Assuntos
Carbono/metabolismo , Fluordesoxiglucose F18/metabolismo , Nicotiana/metabolismo , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Animais , Autorradiografia , Ciclopentanos/metabolismo , Dissacarídeos/biossíntese , Herbivoria , Especificidade de Órgãos , Oxilipinas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Compostos Radiofarmacêuticos/metabolismo , Transdução de Sinais , Estresse FisiológicoRESUMO
BACKGROUND: Overexpressing novel antimicrobial peptides (AMPs) in plants is a promising approach for crop disease resistance engineering. However, the in planta stability and subcellular localization of each AMP should be validated for the respective plant species, which can be challenging due to the small sizes and extreme pI ranges of AMPs which limits the utility of standard proteomic gel-based methods. Despite recent advances in quantitative shotgun proteomics, its potential for AMP analysis has not been utilized and high throughput methods are still lacking. RESULTS: We created transgenic Nicotiana attenuata plants that independently express 10 different AMPs under a constitutive 35S promoter and compared the extracellular accumulation of each AMP using a universal and versatile protein quantification method. We coupled a rapid apoplastic peptide extraction with label-free protein quantification by nanoUPLC-MSE analysis using Hi3 method and identified/quantified 7 of 10 expressed AMPs in the transgenic plants ranging from 37 to 91 amino acids in length. The quantitative comparison among the transgenic plant lines showed that three particular peptides, belonging to the defensin, knottin and lipid-transfer protein families, attained the highest concentrations of 91 to 254 pmol per g leaf fresh mass, which identified them as best suited for ectopic expression in N. attenuata. The chosen mass spectrometric approach proved to be highly sensitive in the detection of different AMP types and exhibited the high level of analytical reproducibility required for label-free quantitative measurements along with a simple protocol required for the sample preparation. CONCLUSIONS: Heterologous expression of AMPs in plants can result in highly variable and non-predictable peptide amounts and we present a universal quantitative method to confirm peptide stability and extracellular deposition. The method allows for the rapid quantification of apoplastic peptides without cumbersome and time-consuming purification or chromatographic steps and can be easily adapted to other plant species.
Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Técnicas de Química Analítica/métodos , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Nicotiana/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Estrutura Quaternária de Proteína , Nicotiana/metabolismoRESUMO
Metabolic profiling is commonly employed to investigate the global metabolic alterations of malignant cells or tissues. In the latter setting, neoplastic lesions are separated from adjacent, healthy tissues and their metabolites are quantified upon a chromatographic run coupled to mass spectrometry. Changes in the abundance of specific metabolites are then mapped on metabolic networks and the underlying metabolic circuitries are investigated as potential targets for the development of novel anticancer drugs. This approach, however, does not take into account the intrinsic heterogeneity of neoplastic lesions, which contain a large amount of non-transformed cells. To circumvent this issue, techniques have been developed that allow for the imaging of metabolites at the single-cell level. Here, we summarize established protocols that are suitable for imaging metabolites in animal cells (be them malignant or not) as well as in plant and prokaryotic cells. These methods are relevant for the study of the metabolic alterations that accompany oncogenesis and tumor progression.
Assuntos
Neoplasias/metabolismo , Análise de Célula Única , Células HeLa , Humanos , Neoplasias/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND: Floral nectar (FN) contains not only energy-rich compounds to attract pollinators, but also defense chemicals and several proteins. However, proteomic analysis of FN has been hampered by the lack of publically available sequence information from nectar-producing plants. Here we used next-generation sequencing and advanced proteomics to profile FN proteins in the opportunistic outcrossing wild tobacco, Nicotiana attenuata. RESULTS: We constructed a transcriptome database of N. attenuata and characterized its nectar proteome using LC-MS/MS. The FN proteins of N. attenuata included nectarins, sugar-cleaving enzymes (glucosidase, galactosidase, and xylosidase), RNases, pathogen-related proteins, and lipid transfer proteins. Natural variation in FN proteins of eleven N. attenuata accessions revealed a negative relationship between the accumulation of two abundant proteins, nectarin1b and nectarin5. In addition, microarray analysis of nectary tissues revealed that protein accumulation in FN is not simply correlated with the accumulation of transcripts encoding FN proteins and identified a group of genes that were specifically expressed in the nectary. CONCLUSIONS: Natural variation of identified FN proteins in the ecological model plant N. attenuata suggests that nectar chemistry may have a complex function in plant-pollinator-microbe interactions.
Assuntos
Flores/metabolismo , Variação Genética , Nicotiana/metabolismo , Néctar de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Eletroforese em Gel Bidimensional , Flores/química , Flores/genética , Flores/crescimento & desenvolvimento , Espectrometria de Massas , Filogenia , Néctar de Plantas/química , Néctar de Plantas/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Nicotiana/classificação , Nicotiana/genética , Nicotiana/crescimento & desenvolvimentoRESUMO
The formation of 4-deoxyaurones, which serve as UV nectar guides in Bidens ferulifolia (Jacq.) DC., was established by combination of UV photography, mass spectrometry, and biochemical assays and the key step in aurone formation was studied. The yellow flowering ornamental plant accumulates deoxy type anthochlor pigments (6'-deoxychalcones and the corresponding 4-deoxyaurones) in the basal part of the flower surface whilst the apex contains only yellow carotenoids. For UV sensitive pollinating insects, this appears as a bicoloured floral pattern which can be visualized in situ by specific ammonia staining of the anthochlor pigments. The petal back side, in contrast, shows a faintly UV absorbing centre and UV absorbing rays along the otherwise UV reflecting petal apex. Matrix-free UV laser desorption/ionisation mass spectrometric imaging (LDI-MSI) indicated the presence of 9 anthochlors in the UV absorbing areas. The prevalent pigments were derivatives of okanin and maritimetin. Enzyme preparations from flowers, leaves, stems and roots of B. ferulifolia and from plants, which do not accumulate aurones e.g. Arabidopsis thaliana, were able to convert chalcones to aurones. Thus, aurone formation could be catalyzed by a widespread enzyme and seems to depend mainly on a specific biochemical background, which favours the formation of aurones at the expense of flavonoids. In contrast to 4-hydroxyaurone formation, hydroxylation and oxidative cyclization to the 4-deoxyaurones does not occur in one single step but is catalyzed by two separate enzymes, chalcone 3-hydroxylase and aurone synthase (catechol oxidase reaction). Aurone formation shows an optimum at pH 7.5 or above, which is another striking contrast to 4-hydroxyaurone formation in Antirrhinum majus L. This is the first example of a plant catechol oxidase type enzyme being involved in the flavonoid pathway and in an anabolic reaction in general.
Assuntos
Benzofuranos/análise , Benzofuranos/metabolismo , Bidens/química , Flores/química , Pigmentos Biológicos/metabolismo , Carotenoides/química , Catecol Oxidase/metabolismo , Cromatografia Líquida de Alta Pressão , Flavonoides/metabolismo , Concentração de Íons de Hidrogênio , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Raios UltravioletaRESUMO
Induced defenses are thought to be economical: growth and fitness-limiting resources are only invested into defenses when needed. To date, this putative growth-defense trade-off has not been quantified in a common currency at the level of individual compounds. Here, a quantification method for ¹5N-labeled proteins enabled a direct comparison of nitrogen (N) allocation to proteins, specifically, ribulose-1,5-bisposphate carboxylase/oxygenase (RuBisCO), as proxy for growth, with that to small N-containing defense metabolites (nicotine and phenolamides), as proxies for defense after herbivory. After repeated simulated herbivory, total N decreased in the shoots of wild-type (WT) Nicotiana attenuata plants, but not in two transgenic lines impaired in jasmonate defense signaling (irLOX3) and phenolamide biosynthesis (irMYB8). N was reallocated among different compounds within elicited rosette leaves: in the WT, a strong decrease in total soluble protein (TSP) and RuBisCO was accompanied by an increase in defense metabolites, irLOX3 showed a similar, albeit attenuated, pattern, whereas irMYB8 rosette leaves were the least responsive to elicitation, with overall higher levels of RuBisCO. Induced defenses were higher in the older compared with the younger rosette leaves, supporting the hypothesis that tissue developmental stage influences defense investments. We propose that MYB8, probably by regulating the production of phenolamides, indirectly mediates protein pool sizes after herbivory. Although the decrease in absolute N invested in TSP and RuBisCO elicited by simulated herbivory was much larger than the N-requirements of nicotine and phenolamide biosynthesis, ¹5N flux studies revealed that N for phenolamide synthesis originates from recently assimilated N, rather than from RuBisCO turnover.
Assuntos
Nicotiana/fisiologia , Nitrogênio/metabolismo , Proteínas de Plantas/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Animais , Ciclopentanos/metabolismo , Herbivoria , Manduca , Nicotina/metabolismo , Oxilipinas/metabolismo , Pentoses , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Nicotiana/crescimento & desenvolvimentoRESUMO
Ribulose-1,5-bisphosphate carboxylase/ oxygenase (RuBisCO) is the most abundant protein on the planet and in addition to its central role in photosynthesis it is thought to function as a nitrogen (N)-storage protein and a potential source of N for defense biosynthesis in plants. In a recent study in the wild tobacco Nicotiana attenuata, we showed that the decrease in absolute N invested in soluble proteins and RuBisCO elicited by simulated herbivory was much larger than the N-requirements of nicotine and phenolamide biosynthesis; (15)N flux studies revealed that N for defensive phenolamide synthesis originates from recently assimilated N rather than from RuBisCO turnover. Here we show that a transgenic line of N. attenuata silenced in the expression of RuBisCO (asRUB) invests similar or even larger amounts of N into phenolamide biosynthesis compared with wild type plants, consistent with our previous conclusion that recently assimilated N is channeled into phenolamide synthesis after elicitation. We suggest that the decrease in leaf proteins after simulated herbivory is a tolerance mechanism, rather than a consequence of N-demand for defense biosynthesis.
Assuntos
Inativação Gênica , Herbivoria/fisiologia , Manduca/fisiologia , Nicotiana/enzimologia , Nicotiana/imunologia , Nitrogênio/metabolismo , Ribulose-Bifosfato Carboxilase/genética , Animais , Modelos Biológicos , Nicotina/metabolismo , Isótopos de Nitrogênio , Subunidades Proteicas/metabolismo , Putrescina/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Espermidina/metabolismoRESUMO
RATIONALE: There is a need to find new antibiotic agents to fight resistant pathogenic bacteria. To search successfully for novel antibiotics from bacteria cultivated under diverse conditions, we need a fast and cost-effective screening method. METHODS: A combination of Liquid Extraction Surface Analysis (LESA), automated chip-based nanoelectrospray ionization, and high-resolution mass or tandem mass spectrometry using an Orbitrap XL was tested as the screening platform. Actinobacteria, known to produce well-recognized thiazolyl peptide antibiotics, were cultivated on a plate of solid medium and the antibiotics were extracted by organic solvent mixtures from the surface of colonies grown on the plate and analyzed using mass spectrometry (MS). RESULTS: LESA combined with high-resolution MS is a powerful tool with which to extract and detect thiazolyl peptide antibiotics from different Actinobacteria. Known antibiotics were correctly detected with high mass accuracy (<4 ppm) and structurally characterized using tandem mass spectra. Our method is the first step toward the development of a novel high-throughput extraction and identification tool for antibiotics in particular and natural products in general. CONCLUSIONS: The method described in this paper is suitable for (1) screening the natural products produced by bacterial colonies on cultivation plates within the first 2 min following extraction and (2) detecting antibiotics at high mass accuracy; the cost is around 2 Euro per sample.
Assuntos
Actinobacteria/metabolismo , Antibacterianos/isolamento & purificação , Extração Líquido-Líquido/métodos , Peptídeos/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodos , Tiazóis/isolamento & purificação , Actinobacteria/citologia , Antibacterianos/química , Antibacterianos/metabolismo , Aderência Bacteriana , Humanos , Peptídeos/química , Peptídeos/metabolismo , Tiazóis/química , Tiazóis/metabolismoRESUMO
Herbivory leads to changes in the allocation of nitrogen among different pools and tissues; however, a detailed quantitative analysis of these changes has been lacking. Here, we demonstrate that a mass spectrometric data-independent acquisition approach known as LC-MS(E), combined with a novel algorithm to quantify heavy atom enrichment in peptides, is able to quantify elicited changes in protein amounts and (15)N flux in a high throughput manner. The reliable identification/quantitation of rabbit phosphorylase b protein spiked into leaf protein extract was achieved. The linear dynamic range, reproducibility of technical and biological replicates, and differences between measured and expected (15)N-incorporation into the small (SSU) and large (LSU) subunits of ribulose-1,5-bisphosphate-carboxylase/oxygenase (RuBisCO) and RuBisCO activase 2 (RCA2) of Nicotiana attenuata plants grown in hydroponic culture at different known concentrations of (15)N-labeled nitrate were used to further evaluate the procedure. The utility of the method for whole-plant studies in ecologically realistic contexts was demonstrated by using (15)N-pulse protocols on plants growing in soil under unknown (15)N-incorporation levels. Additionally, we quantified the amounts of lipoxygenase 2 (LOX2) protein, an enzyme important in antiherbivore defense responses, demonstrating that the approach allows for in-depth quantitative proteomics and (15)N flux analyses of the metabolic dynamics elicited during plant-herbivore interactions.
Assuntos
Nicotiana/metabolismo , Nitrogênio/metabolismo , Folhas de Planta/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Algoritmos , Sequência de Aminoácidos , Animais , Teorema de Bayes , Cromatografia Líquida/normas , Herbivoria , Funções Verossimilhança , Lipoxigenase/química , Lipoxigenase/isolamento & purificação , Lipoxigenase/metabolismo , Dados de Sequência Molecular , Isótopos de Nitrogênio/metabolismo , Fragmentos de Peptídeos/química , Mapeamento de Peptídeos/normas , Fosforilase b/química , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Folhas de Planta/química , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Coelhos , Padrões de Referência , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/normas , Espectrometria de Massas em Tandem/normas , Nicotiana/químicaRESUMO
Recent advances in instrumentation and sample preparation have facilitated the mass spectrometric (MS) imaging of a large variety of biological molecules from small metabolites to large proteins. The technique can be applied at both the tissue and the single-cell level, and provides information regarding the spatial distribution of specific molecules. Nevertheless, the use of MS imaging in plant science remains far from routine, and there is still a need to adapt protocols to suit specific tissues. We present an overview of MALDI-imaging MS (MSI) technology and its use for the analysis of plant tissue. Recent methodological developments have been summarized, and the major challenges involved in using MALDI-MSI, including sample preparation, the analysis of metabolites and peptides, and strategies for data evaluation are all discussed. Some attention is given to the identification of differentially distributed compounds. To date, the use of MALDI-MSI in plant research has been limited. Examples include leaf surface metabolite maps, the characterization of soluble metabolite translocation in planta, and the profiling of protein/metabolite patterns in cereal grain cross-sections. Improvements to both sample preparation strategies and analytical platforms (aimed at both spectrum acquisition and post-acquisition analysis) will enhance the relevance of MALDI-MSI technology in plant research.
Assuntos
Peptídeos/análise , Proteínas de Plantas/análise , Plantas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Metabolômica/métodos , Peptídeos/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/análise , Proteoma/metabolismo , Proteômica/métodos , Reprodutibilidade dos TestesRESUMO
Insect cuticular hydrocarbons (CHCs) are not only essential for desiccation resistance, they also play an important role as chemical signals and cues in social as well as solitary insects. The identification of CHCs is, therefore, crucial to an understanding of the chemical communication within and between insect species. We describe a method for rapid, simple, and unambiguous identification of CHCs using gas chromatography-ion-trap mass spectrometry. External ionization configuration in combination with a low ion-trap temperature resulted in dramatically increased intensities of molecular ions for alkanes, alkenes and alkadienes, and in high-mass fragmentation patterns with intense ions characteristic for methyl-branched hydrocarbons comparable to those obtained with quadrupole instruments. Additionally, we present an external chemical ionization-tandem mass-spectrometric method that allows for the determination of double-bond positions in alkenes and alkadienes without the need for derivatization prior to analysis.
Assuntos
Drosophila melanogaster/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Hidrocarbonetos , Vespas/química , Alcadienos/análise , Alcanos/análise , Alcenos/análise , Animais , Drosophila melanogaster/anatomia & histologia , Feminino , Hidrocarbonetos/análise , Hidrocarbonetos/química , Vespas/anatomia & histologiaRESUMO
Nectars are rich in primary metabolites and attract mutualistic animals, which serve as pollinators or as an indirect defense against herbivores. Their chemical composition makes nectars prone to microbial infestation. As protective strategy, floral nectar of ornamental tobacco (Nicotiana langsdorffii x Nicotiana sanderae) contains "nectarins," proteins producing reactive oxygen species such as hydrogen peroxide. By contrast, pathogenesis-related (PR) proteins were detected in Acacia extrafloral nectar (EFN), which is secreted in the context of defensive ant-plant mutualisms. We investigated whether these PR proteins protect EFN from phytopathogens. Five sympatric species (Acacia cornigera, A. hindsii, A. collinsii, A. farnesiana, and Prosopis juliflora) were compared that differ in their ant-plant mutualism. EFN of myrmecophytes, which are obligate ant-plants that secrete EFN constitutively to nourish specialized ant inhabitants, significantly inhibited the growth of four out of six tested phytopathogenic microorganisms. By contrast, EFN of nonmyrmecophytes, which is secreted only transiently in response to herbivory, did not exhibit a detectable inhibitory activity. Combining two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with nanoflow liquid chromatography-tandem mass spectrometry analysis confirmed that PR proteins represented over 90% of all proteins in myrmecophyte EFN. The inhibition of microbial growth was exerted by the protein fraction, but not the small metabolites of this EFN, and disappeared when nectar was heated. In-gel assays demonstrated the activity of acidic and basic chitinases in all EFNs, whereas glucanases were detected only in EFN of myrmecophytes. Our results demonstrate that PR proteins causally underlie the protection of Acacia EFN from microorganisms and that acidic and basic glucanases likely represent the most important prerequisite in this defensive function.
Assuntos
Acacia/enzimologia , Quitinases/química , Glicosídeo Hidrolases/química , Néctar de Plantas/química , Proteínas de Plantas/química , Acacia/química , Acacia/microbiologia , Animais , Anti-Infecciosos/farmacologia , Formigas/fisiologia , Quitinases/isolamento & purificação , Glicosídeo Hidrolases/isolamento & purificação , Testes de Sensibilidade Microbiana , Fungos Mitospóricos/efeitos dos fármacos , Doenças das Plantas/microbiologia , Proteínas de Plantas/isolamento & purificação , Especificidade da EspécieRESUMO
Here, we show the usefulness of a strong base, 1,8-bis(dimethyl-amino)naphthalene (DMAN; proton sponge), as a novel matrix for MALDI-TOF/MS analysis of anions. Several strong and weakly acidic low-molecular-weight analytes (fatty acids, amino acids, fatty acid-amino acid conjugates, plant and animal hormones, vitamins, and short peptides) were measured at physiologically relevant concentrations. Clear negative-mode MALDI-TOF/MS spectra of all analytes using DMAN as the matrix show only deprotonated analyte signals at a low picomole/femtomole limit-of-detection. Moreover, the spectra were totally devoid of any matrix-related signals. Standard calibration curves gave good linearity over the entire picomole range: over two concentration orders in most cases and over three orders for peptides. Using this method, the crude regurgitate of the tobacco hornworm caterpillars (Manduca sexta, Lepidoptera, Sphingidae) was analyzed. As many as 11 different components were identified from a single spot, including 16:0, 18:2, 18:3, and 21:0 free acids and 5:0-Glu, 6:0-Glu, 18:2-Glu, 18:3-Glu, 16:0-Glu, and 16:3-Glu fatty acid-amino acid conjugates (FACs) in complete qualitative agreement with previously reported anion exchange-HPLC analyses. The identity of these components was confirmed by negative ion collision-induced dissociation (CID) MS2 spectra.
Assuntos
1-Naftilamina/análogos & derivados , Aminoácidos/análise , Cromatografia Líquida de Alta Pressão/métodos , Ácidos Graxos/análise , Manduca/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , 1-Naftilamina/química , Aminoácidos/química , Animais , Ácidos Graxos/químicaRESUMO
Herbivore- and jasmonate-induced volatile organic compounds (VOCs), which mediate indirect defense, must provide reliable information for predators that frequently learn to associate their release with feeding herbivores. Yet little is known about variation of these cues within populations of native plants, on a scale encountered by predators. We examined variation in herbivore-elicited VOC emissions and patterns of herbivore-induced jasmonate signaling from accessions of Nicotiana attenuata co-occurring in a native population. VOC emissions elicited by herbivore oral secretions (OS) and by methyl jasmonate (MJ) were characterized using gas chromatography-mass spectrometry (GC-MS), high-resolution two-dimensional gas chromatography-time-of-flight mass spectrometry (GCxGC-ToF-MS) and micro-hydrolysis and micro-hydrogenation reactions. Accessions varied in emissions of abundant (trans-alpha-bergamotene, alpha-duprezianene, trans-beta-ocimene, and cis-3-hexenol) and total detectable VOCs, as well as the accumulation of jasmonates, the jasmonate antagonist salicylic acid (SA), abscisic acid (ABA) and jasmonate signaling-related transcripts after OS elicitation. Yet MJ treatment exacerbated differences in VOC emission, suggesting that much variation in VOC emission is caused by processes downstream of jasmonate signaling. Co-occurring N. attenuata plants emit different VOCs following simulated herbivore elicitation as a result in part of differences in jasmonate production and responsiveness, which could reduce the effectiveness of induced indirect defense.