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1.
J Sci Food Agric ; 98(2): 737-750, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28675480

RESUMO

BACKGROUND: Lettuce is a leafy vegetable that is extensively commercialized as a ready-to-eat product because of its widespread use in human nutrition as salad. It is well known that washing treatments can severely affect the quality and shelf-life of ready-to-eat vegetables. The study presented here evaluated the effect of two washing procedures on fresh-cut lettuce during storage. RESULTS: An omics approach was applied to reveal global changes at molecular level induced by peracetic acid washing in comparison with sodium hypochlorite treatment. Microbiological analyses were also performed to quantify total bacterial abundance and composition. The study revealed wide metabolic alterations induced by the two sanitizers. In particular, transcriptomic and proteomic analyses pointed out a number of transcripts and proteins differentially accumulated in response to peracetic acid washing, mainly occurring on the first day of storage. In parallel, different microbiota composition and significant reduction in total bacterial load following washing were also observed. CONCLUSION: The results provide useful information for the fresh-cut industry to select an appropriate washing procedure preserving fresh-like attributes as much as possible during storage of the end product. Molecular evidence indicated peracetic acid to be a valid alternative to sodium hypochlorite as sanitizer solution. © 2017 Society of Chemical Industry.


Assuntos
Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Lactuca/metabolismo , Ácido Peracético/farmacologia , Hipoclorito de Sódio/farmacologia , Eletroforese em Gel Bidimensional/métodos , Lactuca/efeitos dos fármacos , Espectrometria de Massas/métodos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polimorfismo de Fragmento de Restrição , Proteômica/métodos , Transcriptoma
2.
FEMS Microbiol Lett ; 363(1): fnv209, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26511951

RESUMO

With the growing demand of fresh-cut vegetables, a variety of packaging films are produced specifically to improve safety and quality of the fresh vegetables over the storage period. The aim of our work was to evaluate the influence of different packaging films on the quality of fresh-cut lettuce analyzing changes in bacterial community composition and modifications at the proteome level, by means of culture-dependent/culture-independent methods and differential gel electrophoresis combined with mass spectrometry analysis. Total viable counts indicated the presence of a highly variable and complex microbial flora, around a mean value of 6.26 log10 CFU g(-1). Analysis of terminal-restriction fragment length polymorphism data indicated that bacterial communities changed with packaging films and time, showing differences in community composition and diversity indices between the commercially available package (F) and the new packages (A and C), in the first days after packaging. Also proteomic analysis revealed significant changes, involving proteins related to energy metabolism, photosynthesis, plant defense and oxidative stress processes, between F and A/C packages. In conclusion, microbiological and proteomic analysis have proved to be powerful tools to provide new insights into both the composition of leaf-associated bacterial communities and protein content of fresh-cut lettuce during the shelf-life storage process.


Assuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Biota , Embalagem de Alimentos , Lactuca/química , Lactuca/microbiologia , Proteoma/análise , Contagem de Colônia Microbiana , Eletroforese , Genômica , Espectrometria de Massas , Técnicas Microbiológicas , Polimorfismo de Fragmento de Restrição , Proteômica
3.
Proteomics ; 15(23-24): 4159-74, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26444578

RESUMO

Posidonia oceanica is a marine angiosperm, or seagrass, adapted to grow to the underwater life from shallow waters to 50 m depth. This raises questions of how their photosynthesis adapted to the attenuation of light through the water column and leads to the assumption that biochemistry and metabolism of the chloroplast are the basis of adaptive capacity. In the present study, we described a protocol that was adapted from those optimized for terrestrial plants, to extract chloroplasts from as minimal tissue as possible. We obtained the best balance between tissue amount/intact chloroplasts yield using one leaf from one plant. After isopynic separations, the chloroplasts purity and integrity were evaluated by biochemical assay and using a proteomic approach. Chloroplast proteins were extracted from highly purified organelles and resolved by 1DE SDS-PAGE. Proteins were sequenced by nLC-ESI-IT-MS/MS of 1DE gel bands and identified against NCBInr green plant databases, Dr. Zompo database for seagrasses in a local customized dataset. The curated localization of proteins in sub-plastidial compartments (i.e. envelope, stroma and thylakoids) was retrieved in the AT_CHLORO database. This purification protocol and the validation of compartment markers may serve as basis for sub-cellular proteomics in P. oceanica and other seagrasses.


Assuntos
Cloroplastos/metabolismo , Proteômica/métodos , Alismatales/metabolismo , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas em Tandem
4.
Int J Mol Sci ; 16(3): 5803-29, 2015 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-25775160

RESUMO

Filamentous fungi possess the extraordinary ability to digest complex biomasses and mineralize numerous xenobiotics, as consequence of their aptitude to sensing the environment and regulating their intra and extra cellular proteins, producing drastic changes in proteome and secretome composition. Recent advancement in proteomic technologies offers an exciting opportunity to reveal the fluctuations of fungal proteins and enzymes, responsible for their metabolic adaptation to a large variety of environmental conditions. Here, an overview of the most commonly used proteomic strategies will be provided; this paper will range from sample preparation to gel-free and gel-based proteomics, discussing pros and cons of each mentioned state-of-the-art technique. The main focus will be kept on filamentous fungi. Due to the biotechnological relevance of lignocellulose degrading fungi, special attention will be finally given to their extracellular proteome, or secretome. Secreted proteins and enzymes will be discussed in relation to their involvement in bio-based processes, such as biomass deconstruction and mycoremediation.


Assuntos
Fungos/metabolismo , Proteômica , Eletroforese em Gel de Poliacrilamida , Enzimas/metabolismo , Lignina/análise , Lignina/metabolismo
5.
PLoS One ; 8(1): e53563, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23349718

RESUMO

BACKGROUND: Widespread in the Mediterranean basin, Olea europaea trees are gaining worldwide popularity for the nutritional and cancer-protective properties of the oil, mechanically extracted from ripe fruits. Fruit development is a physiological process with remarkable impact on the modulation of the biosynthesis of compounds affecting the quality of the drupes as well as the final composition of the olive oil. Proteomics offers the possibility to dig deeper into the major changes during fruit development, including the important phase of ripening, and to classify temporal patterns of protein accumulation occurring during these complex physiological processes. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we started monitoring the proteome variations associated with olive fruit development by using comparative proteomics coupled to mass spectrometry. Proteins extracted from drupes at three different developmental stages were separated on 2-DE and subjected to image analysis. 247 protein spots were revealed as differentially accumulated. Proteins were identified from a total of 121 spots and discussed in relation to olive drupe metabolic changes occurring during fruit development. In order to evaluate if changes observed at the protein level were consistent with changes of mRNAs, proteomic data produced in the present work were compared with transcriptomic data elaborated during previous studies. CONCLUSIONS/SIGNIFICANCE: This study identifies a number of proteins responsible for quality traits of cv. Coratina, with particular regard to proteins associated to the metabolism of fatty acids, phenolic and aroma compounds. Proteins involved in fruit photosynthesis have been also identified and their pivotal contribution in oleogenesis has been discussed. To date, this study represents the first characterization of the olive fruit proteome during development, providing new insights into fruit metabolism and oil accumulation process.


Assuntos
Frutas/crescimento & desenvolvimento , Frutas/metabolismo , Olea/crescimento & desenvolvimento , Olea/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Frutas/genética , Olea/genética , Azeite de Oliva , Óleos de Plantas/metabolismo , Proteínas de Plantas/genética , Análise de Componente Principal , Proteoma/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo
6.
Plant Cell Environ ; 35(5): 994-1012, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22082487

RESUMO

In order to sense and respond to the fluctuating light conditions, higher plants possess several families of photoreceptors, such as phytochromes (PHYs), cryptochromes (CRYs) and phototropins. CRYs are responsible for photomorphogenesis and play a role in circadian, developmental and adaptive growth regulation of plants. In tomato (Solanum lycopersicum), CRY2 controls vegetative development, flowering time, fruit antioxidant content as well as the diurnal transcription of several other photoreceptor genes. We applied large-scale molecular approaches to identify altered transcripts and proteins in tomato wild-type (WT) versus a CRY2 overexpressing transgenic genotype, under a diurnal rhythm. Our results showed that tomato CRY2 profoundly affects both gene and protein expression in response to daily light cycle. Particularly altered molecular pathways are related to biotic/abiotic stress, photosynthesis, including components of the light and dark reactions and of starch and sucrose biosynthesis, as well as to secondary metabolism, such as phenylpropanoid, phenolic and flavonoid/anthocyanin biosynthesis pathways. One of the most interesting results is the coordinated up-regulation, in the transgenic genotype, of a consistent number of transcripts and proteins involved in photorespiration and photosynthesis. It is conceivable that light modulates the energetic metabolism of tomato through a fine CRY2-mediated transcriptional control.


Assuntos
Ritmo Circadiano , Criptocromos/genética , Regulação da Expressão Gênica de Plantas/genética , Solanum lycopersicum/genética , Estresse Fisiológico/genética , Respiração Celular , Cloroplastos/genética , Criptocromos/metabolismo , Regulação para Baixo/genética , Regulação para Baixo/efeitos da radiação , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Solanum lycopersicum/fisiologia , Solanum lycopersicum/efeitos da radiação , Mitocôndrias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Peroxissomos/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Proteômica , RNA de Plantas/genética , Transdução de Sinais , Estresse Fisiológico/efeitos da radiação , Regulação para Cima/genética , Regulação para Cima/efeitos da radiação
7.
Microb Cell Fact ; 9: 95, 2010 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-21110849

RESUMO

BACKGROUND: Proteomics was recently used to reveal enzymes whose expression is associated with the production of the glycopeptide antibiotic balhimycin in Amycolatopsis balhimycina batch cultivations. Combining chemostat fermentation technology, where cells proliferate with constant parameters in a highly reproducible steady-state, and differential proteomics, the relationships between physiological status and metabolic pathways during antibiotic producing and non-producing conditions could be highlighted. RESULTS: Two minimal defined media, one with low Pi (0.6 mM; LP) and proficient glucose (12 g/l) concentrations and the other one with high Pi (1.8 mM) and limiting (6 g/l; LG) glucose concentrations, were developed to promote and repress antibiotic production, respectively, in A. balhimycina chemostat cultivations. Applying the same dilution rate (0.03 h-1), both LG and LP chemostat cultivations showed a stable steady-state where biomass production yield coefficients, calculated on glucose consumption, were 0.38 ± 0.02 and 0.33 ± 0.02 g/g (biomass dry weight/glucose), respectively. Notably, balhimycin was detected only in LP, where quantitative RT-PCR revealed upregulation of selected bal genes, devoted to balhimycin biosynthesis, and of phoP, phoR, pstS and phoD, known to be associated to Pi limitation stress response. 2D-Differential Gel Electrophoresis (DIGE) and protein identification, performed by mass spectrometry and computer-assisted 2 D reference-map http://www.unipa.it/ampuglia/Abal-proteome-maps matching, demonstrated a differential expression for proteins involved in many metabolic pathways or cellular processes, including central carbon and phosphate metabolism. Interestingly, proteins playing a key role in generation of primary metabolism intermediates and cofactors required for balhimycin biosynthesis were upregulated in LP. Finally, a bioinformatic approach showed PHO box-like regulatory elements in the upstream regions of nine differentially expressed genes, among which two were tested by electrophoresis mobility shift assays (EMSA). CONCLUSION: In the two chemostat conditions, used to generate biomass for proteomic analysis, mycelia grew with the same rate and with similar glucose-biomass conversion efficiencies. Global gene expression analysis revealed a differential metabolic adaptation, highlighting strategies for energetic supply and biosynthesis of metabolic intermediates required for biomass production and, in LP, for balhimycin biosynthesis. These data, confirming a relationship between primary metabolism and antibiotic production, could be used to increase antibiotic yield both by rational genetic engineering and fermentation processes improvement.


Assuntos
Actinomycetales/metabolismo , Antibacterianos/biossíntese , Proteoma/análise , Vancomicina/análogos & derivados , Actinomycetales/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Eletroforese em Gel Bidimensional , Ácidos Graxos/metabolismo , Glucose/farmacologia , Biossíntese de Proteínas , Vancomicina/biossíntese
8.
J Proteome Res ; 9(11): 5684-97, 2010 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-20815412

RESUMO

Cucumber mosaic virus (CMV), a member of the Cucumovirus genus, is the causal agent of several plant diseases in a wide range of host species, causing important economic losses in agriculture. Because of the lack of natural resistance genes in most crops, different genetic engineering strategies have been adopted to obtain virus-resistant plants. In a previous study, we described the engineering of transgenic tomato plants expressing a single-chain variable fragment antibody (scFv G4) that are specifically protected from CMV infection. In this work, we characterized the leaf proteome expressed during compatible plant-virus interaction in wild type and transgenic tomato. Protein changes in both inoculated and apical leaves were revealed using two-dimensional gel electrophoresis (2-DE) coupled to differential in gel electrophoresis (DIGE) technology. A total of 2084 spots were detected, and 50 differentially expressed proteins were identified by nanoscale liquid chromatographic-electrospray ionization-ion trap-tandem mass spectrometry (nLC-ESI-IT-MS/MS). The majority of these proteins were related to photosynthesis (38%), primary metabolism (18%), and defense activity (14%) and demonstrated to be actively down regulated by CMV in infected leaves. Moreover, our analysis revealed that asymptomatic apical leaves of transgenic inoculated plants had no protein profile alteration as compared to control wild type uninfected plants demonstrating that virus infection is confined to the inoculated leaves and systemic spread is hindered by the CMV coat protein (CP)-specific scFv G4 molecules. Our work is the first comparative study on compatible plant-virus interactions between engineered immunoprotected and susceptible wild type tomato plants, contributing to the understanding of antibody-mediated disease resistance mechanisms.


Assuntos
Cucumovirus/imunologia , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/imunologia , Plantas Geneticamente Modificadas/química , Solanum lycopersicum/genética , Solanum lycopersicum/virologia , Engenharia Genética , Interações Hospedeiro-Patógeno/imunologia , Imunidade/genética , Fragmentos de Imunoglobulinas/genética , Solanum lycopersicum/imunologia , Doenças das Plantas/imunologia , Doenças das Plantas/virologia , Folhas de Planta , Proteínas de Plantas/análise , Vírus de Plantas , Proteômica/métodos
9.
BMC Biotechnol ; 9: 96, 2009 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-19930574

RESUMO

BACKGROUND: In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. RESULTS: The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. CONCLUSION: We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant-derived Nef was purified, with enhanced yield, exploiting a two-step purification protocol. These results represent a first step towards the development of a plant-derived HIV vaccine.


Assuntos
Nicotiana/genética , Interferência de RNA , Tombusvirus/genética , Proteínas do Core Viral/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/biossíntese , Cromatografia de Afinidade , Homólogo 5 da Proteína Cromobox , Regulação da Expressão Gênica de Plantas , Engenharia Genética/métodos , Espectrometria de Massas , RNA Interferente Pequeno/genética , Nicotiana/virologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/isolamento & purificação
10.
J Proteomics ; 72(4): 586-607, 2009 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-19135558

RESUMO

Strawberry is worldwide appreciated for its unique flavour and as a source of macronutrients and high levels of antioxidants which are closely related to fruit ripening. We report the investigation of the complex physiological processes of strawberry fruit ripening at proteomic level. Multiple approaches were used to investigate strawberry fruit proteome. In particular, a proteome reference map of strawberry fruit from Queen Elisa élite genotype was achieved by 2-D analyses of proteins extracted from berries at immature, turning and red stages to isolate a set of proteins commonly present in fruit during ripening. In addition, several hundreds of proteins were identified by a combination of multidimensional liquid chromatography/tandem mass spectrometry and one dimensional SDS-PAGE coupled with nano-liquid chromatography/tandem mass spectrometry. DIGE technology was also used to identify differentially accumulated proteins during ripening and to correlate fruit protein expression with quality traits of the reference variety Queen Elisa and its parental genotypes. A number of constitutive or differentially accumulated proteins were found. Generally, the pattern of protein expression as well as the putative function of identified proteins argues for a role in major fruit physiological developmental and ripening processes. The role of some of the identified proteins is discussed in relation to strawberry fruit ripening and to quality traits. Consequently, this study provides the first characterization of the strawberry fruit proteome and the time course of variation during maturation by using multiple approaches.


Assuntos
Fragaria/metabolismo , Frutas/metabolismo , Proteínas de Plantas/análise , Proteoma/metabolismo , Cromatografia Líquida/métodos , Eletroforese em Gel Bidimensional/métodos , Regulação da Expressão Gênica de Plantas , Genótipo , Espectrometria de Massas em Tandem/métodos
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