The field of nutrition has been shifting away from addressing single nutritional deficiencies or excesses, to aiming towards the overall improvement of individual health and/or performance through dietary manipulations. Metabolomics is a tool that has been applied to different areas of food science and nutrition, including analysis of food components, and evaluation of the physiological impact of nutritional challenges, dietary ingredients and bioactive compounds. The pig is a well characterized animal model of particular importance in nutritional studies given its physiological similarities to humans. The aim of this project is to apply Gas Chromatography/Mass Spectrometry (GC/MS) and proton Nuclear Magnetic Resonance (1H-NMR) spectroscopy metabolomics to investigate changes induced by nutritional deficiencies or diet supplementation in neonatal pigs. Metabolomics results will be followed up by gene and protein expression experiments to validate and characterize the affected metabolic pathways, therefore leading to a better understanding of the relationship between diet and health.
Esca is a grapevine trunk disease with worldwide distribution. The disease is destructive, without effective treatment, leading to high costs in the viticultural sector. Esca disease is complex, being associated with fungal infection of the xylem, as well as with environmental factors. External symptom appearance is inconstant, and esca-related fungi may live within the plant without causing apparent disease. Water stress is thought to be a factor involved in esca symptom appearance. The objective of this project was to better understand the factors involved in the pathogenicity of esca-related fungi, specifically how water stress could facilitate esca disease. Xylem sap from greenhouse plants inoculated with esca-related fungi and concomitantly subjected to drought was analyzed by proton Nuclear Magnetic Resonance (1H-NMR) spectroscopy. The results revealed an overall concentration increase of several amino acids, sugars and other small molecules in xylem sap. Most of these changes were associated with the water stress, but the presence of fungal infection also contributed to the concentration increase of specific metabolites. The results suggest the increase of nutrients in xylem sap associated with drought may be involved in esca disease progression.
Plants and animals require iron as an essential nutrient. Iron deficiency adverse effects on agriculture include reduced yield and quality of crops, and lower nutritional value of plant foods. Besides, iron deficiency is a public health concern causing iron deficiency anemia, which affects over 30% of the world population (those relying on plant-based diets being more affected). In this project, the responses to iron deficiency of plants grown in hydroponics were investigated by analyzing changes in mineral composition of root and shoot tissues, gene expression, enzyme activity, proteome and metabolome. Proton Nuclear Magnetic Resonance (1H-NMR) spectroscopy-based metabolomics experiments showed that several leaf metabolic pathways were affected by iron deficiency, and some metabolites were implicated for the first time in the response to iron deficiency. Gel-based proteomics analysis of roots revealed the existence of a fine tuning of root metabolism associated with ferric iron reduction. Additionally, a human intestinal mucosa cell line model, Caco-2 cells, was used to analyze the bioavailability of iron in common beans. Caco-2 cells showed higher iron concentration and increased iron uptake from cooked beans, suggesting the presence of components that enhance iron intestinal absorption after cooking.
Pine Wilt Disease is caused by Bursaphelenchus xylophilus, a nematode recently introduced to Europe. The disease affects conifer forests with a high mortality rate. The objective of this project was to assess the impact of the nematode in autochthonous plant species of Portuguese forests and to investigate the mechanism(s) of resistance. Although species of the genus Pinus are the primary host of B. xylophilus, the inoculation of some autochthonous non-Pinus species with this nematode showed that non-Pinus species could be compatible with this nematode, thus suggesting that other species could act as a repository of the nematode in an ecosystem. A large-scale susceptibility screening test was developed and used to screen pine trees of different geographic origins. Trees from different locations presented different degrees of resistance to the nematode. Additionally, the pine volatile component of pine trees infected with the nematode was investigated using Solid-Phase MicroExtraction-Gas Chromatography/Mass spectrometry (SPME-GC/MS). The results showed that nematode infection induced differential volatile production within a week after inoculation, suggesting these compounds could be used as disease biomarkers.
Esca is serious disease of grapevine for which no effective treatment exists. The disease is complex; it involves infection by several fungi and is influenced by environmental factors. In this project, a grapevine cell suspension culture challenged with Phaeomoniella chlamydospora was used as a model to study the plant’s molecular and biochemical defense responses. An High-Performance Liquid Chromatography (HPLC) analysis showed that the fungal-challenged cells increased the production of phenolic compounds, some with recognized antifungal activity. The grapevine cells exhibited up-regulation of several defense-related genes in response to fungal elicitation. This in vitro model also suggested a role for methyl jasmonate, and the involvement of reactive oxygen species and calcium signaling in the defense mechanism. Leaves from plants naturally infected with esca were also analyzed by HPLC and proton Nuclear Magnetic Resonance (1H-NMR) spectroscopy. The 1H-NMR spectroscopy-based metabolomics analysis showed that diseased leaves increased phenolic production, confirming the results obtained in vitro, and rerouted carbon and energy from primary to secondary metabolism. The HPLC results showed that phenolic production was altered in different symptomatic stages of esca disease, suggesting that the leaf phenolic compounds could be used to develop an early detection (non-destructive) diagnostic method.