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Grapevine is one of the most relevant crops in the world being used for economically important products such as wine. Yet, relevant grapevine cultivars are heavily affected by diseases such as the downy mildew disease caused by Plasmopara viticola. Improvements on grapevine resistance are made mainly by breeding techniques where resistance traits are introgressed into cultivars with desired grape characteristics. However, there is still a lack of knowledge on how resistant or tolerant cultivars tackle the P. viticola pathogen. In this study, using a shotgun proteomics LC-MS/MS approach, we unravel the protein modulation of a highly tolerant grapevine cultivar, V. vinifera ‘Regent’, in the first hours post inoculation (hpi) with P. viticola. At 6 hpi, proteins related to defence and to response to stimuli are negatively modulated while at 12 hpi there is an accumulation of proteins belonging to both categories. The co-occurrence of effector-triggered susceptibility (ETS) and effector-triggered immunity (ETI) is detected at both time-points, showing that these defence processes present high plasticity. The results obtained in this study unravel the tolerant grapevine defence strategy towards P. viticola and may provide valuable insights on resistance associated candidates and mechanisms, which may play an important role in the definition of new strategies on breeding approaches.

Grapevine downy mildew, caused by the fungus Plasmopara viticola (Berk. & Curt.) Berl. & de Toni was introduced in European vineyards in the 1870s and quickly spread to all major grape-producing regions worldwide. The early events of grapevine resistance to P. viticola were accessed using transcriptomic, metabolomic and proteomics approaches in the cultivars, ‘Regent' and ‘Trincadeira', resistant and susceptible to downy mildew, respectively. At the transcript level the resistant genotype ‘Regent' presents signalling and defence-related transcripts up-regulated as soon as 6hpi. At the metabolome level, our results show that ‘Trincadeira' and ‘Regent' are intrinsically different as ‘Regent', the resistant cultivar, is able to activate the phenylpropanoid pathway and to accumulate linolenic acid, the precursor of jasmonate in the octadecanoid cascade. At the proteome level ‘Regent' presents upregulated proteins mainly belonging to the functional categories of photosynthesis, carbohydrate metabolism and defence, indicating that this cultivar makes a more efficient use of light, in CO2 assimilation and obtaining intermediate metabolites from photoassimilates for the biosynthesis of secondary metabolites and subsequent defence responses. Our data brings new insights on the molecular processes underlying resistance to Plasmopara viticola, which may eventually enable the design of novel strategies for pathogen control., Revista de Ciências Agrárias, vol. 38 n.º 2 (2015)

“NOTICE: this is the author’s version of a work that was accepted for publication in Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. Volume 1834, Issue 6, June 2013, Pages 1010–1022, DOI 10.1016/j.bbapap.2012.12.001" "Protein glycation induces structural and stability changes that impair protein function, and is associated with several human neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease and Familial Amyloidotic Polyneuropathy. Recently we have shown that methylglyoxal induces and stabilizes the formation of small native-like aggregates in the amyloidogenic protein insulin and the same was previously shown for α-synuclein. However, the fundamental biophysical mechanism underlying such methylglyoxal-induced protein aggregation is not yet fully understood. In this study, we used the model protein cytochrome c to characterize the specific glycation targets and to investigate the glycation effects on protein structure, stability and aggregation. Methylglyoxal was found modify cytochrome c in a single residue and to induce the formation of cytochrome c native-like aggregates. Additionally, it is shown that methylglyoxal glycation of cytochrome c also results in the formation of a partially unfolded species. Interestingly, the formation of this partially unfolded species is not implicated in the aggregation process, a clear difference from amyloid fibril mechanisms that involve partially or totally unfolded intermediates. Equilibrium-unfolding experiments using guanidinium hydrochloride shows that glycation strongly reduces cytochrome c conformational stability. This reduction is balanced by aggregation that increases conformational stability. The data collected from analytical and spectroscopic techniques along with kinetic analysis based on least-squares parameter fitting and statistical model discrimination permitted the proposal of a comprehensive thermodynamic and kinetic model for native-like aggregation of methylglyoxal glycated cytochrome c."

Articles in International Journals BACKGROUND: Shellfish farming is an important economic activity that provides society with a valuable source of food. Analyses of the protein content and metabolism of shellfish are therefore of utmost importance to monitor the presence and effects of environmental contaminants in these organisms and also to assess food quality and authenticity. The aim of the present study was to compare different protein extraction protocols commonly used in two-dimensional gel electrophoresis (2DE) research and select the most suitable for the analysis of gill and digestive gland proteomes from the marine mussel Mytilus galloprovincialis. RESULTS: High-resolution protein separation was achieved by direct solubilisation of proteins from M. galloprovincialis tissues with urea (7 mol L−1), thiourea (2 mol L−1), CHAPS (40 g L−1), DTT (65 mmol L−1) and ampholytes (pH 4–7, 8mLL−1). Subsequent protein identification from 2DEgels by MALDI-TOF/TOFmass spectrometry revealed a high number of proteinswith functions in cytoskeleton structure, dynamics andmaintenance. Other proteins identified in the 2DE gels are involved in energy production and carbohydratemetabolism, metal transport, chaperones and stress response, cell signalling and regulation, proteolysis and protein transduction. CONCLUSION: Important protein markers for contaminant and quality assessment of shellfish food products can be analysed using 2DE.

Sheep and goats differ in diet selection, which may reflect different abilities to deal with the ingestion of plant secondary metabolites. Although saliva provides a basis for immediate oral information via sensory cues and also a mechanism for detoxification, our understanding of the role of saliva in the pre-gastric control of the intake of herbivores is rudimentary. Salivary proteins have important biological functions, but despite their significance, their expression patterns in sheep and goats have been little studied. Protein separation techniques coupled to mass spectrometry based techniques have been used to obtain an extensive comprehension of human saliva protein composition but far fewer studies have been undertaken on animals' saliva. We used two-dimensional electrophoresis gel analysis to compare sheep and goats parotid saliva proteome. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) and liquid chromatography tandem mass spectrometry (LC-MS/MS) were used to identify proteins. From a total of 260 sheep and 205 goat saliva protein spots, 117 and 106 were identified, respectively. A high proportion of serum proteins were found in both salivary protein profiles. Major differences between the two species were detected for proteins within the range of 25–35 kDa. This study presents the parotid saliva proteome of sheep and goat and highlights the potential of proteomics for investigation relating to intake behavior research.

The recovery of high added-value products from waste plant material has been an important issue with economic relevance for the pharmaceutical and food industries. The recovery of antioxidants from wine industry by-products is of great importance in the nutraceutical field. The aim of this study was to identify the main flavonol glycosides, present in winemaking by-products from two white grape varieties: ‘Arinto’ and ‘Moscatel’ using liquid chromatography with diode array and electrochemical detection. Liquid chromatography with electrospray and atmospheric pressure chemical ionization, in negative and positive modes, were also used to characterize the structures of the compounds detected by means of MS²and MS ³. In the by-products analysed quercetin-3-glucoside and quercetin-3-glucuronide were identified as well as kaempferol-3-glucoside. By-products from ‘Arinto’ variety showed higher levels of quercetin glycosides and kaempferol-3-glucoside than the by-products of ‘Moscatel variety’. Uvas, vinho e subprodutos da vinificação contêm diversos compostos fenólicos pertencentes ao grupo dos flavonóides, em elevadas concentrações. Os glicósidos de flavonóis estão entre os compostos fenólicos presentes nas uvas. A sua actividade biológica é de grande interesse para as indústrias farmacêutica, cosmética e alimentar, não só pela actividade antioxidante, mas também pelas suas propriedades anti-inflamatórias e anti-cancerígenas. O objectivo deste estudo foi identificar glicósidos de flavonóis maioritários em subprodutos da vinificação em branco das variedades ‘Arinto’ e ‘Moscatel’. Foram utilizadas metodologias analíticas que incluiram a cromatografia líquida acoplada a detectores de díodos e electroquímico. Cromatografia líquida com espectrometria de massa foi ainda utilizada com duas fontes de ionização à pressão atmosférica: electrospray (ESI) e ionização química (APCI), nas polaridades positiva e negativa no sentido de comparar resultados. Foram também efectuados ensaios MS²e MS³ para caracterizar as estruturas químicas dos glicósidos de flavonóis detectados. Nas amostras analisadas foi identificada a quercetina-3-glucósido e a quercetina-3-glucuronido e também o quempferol-3-glucósido. Os subprodutos da variedade ‘Arinto’ mostraram, proporcionalmente, maior teor de glicósidos de quercetina que quempferol-3-glucósido, quando comparados com os resíduos da variedade ‘Moscatel’. Le rétablissement de produits de haute valeur obtenue de la matière végétale est une question importante avec la pertinence économique pour les industries pharmaceutique, cosmétique et alimentaire, à cause de son activité antioxydant mais aussi pour ces propriétés antiinflammatoire et anti-cancérigène. Le rétablissement des antioxydants des sous-produits d’industrie vinicole est de grande importance dans le domaine nutraceutique. Le but de cette étude était d’identifier les principaux glycosides de flavonol dans les sousproduits de la vinification de deux variétés blanches de raisin: ‘Arinto’ et ‘ Moscatel’. La chromatographie liquide avec la detection par diode et électrochimique on été employées en tandem. La chromatographie liquide était également employée pour caractériser les structures des composés détectés par MS²et MS³en utilisant electrospray et l’ionisation chimique de pression atmosphérique, en modes négative et positive. Dans les sous-produits analysés quercétine-3-glucoside, quercétine-3-glucuronide et kaempférol-3-glucoside ont été identifiés. Les sous-produits de la variété d’ ‘Arinto’ ont des proportions plus élevée des glycosides de quercétine et kaempférol-3-glucoside en comparaison avec les sous-produits de variété ‘Moscatel ‘.