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- The effect of sprouting in lentil (lens culinaris) nutritional and microbiological profilePublication . Santos, Carla S.; Silva, Beatriz; Valente, Luisa M. P.; Gruber, Sabine; Vasconcelos, Marta W.Biological and vegetarian raw food products, in particular based on legume sprouts, are an increasing food trend, due to their improved nutritional value when compared to seeds. Herein, protein and mineral profiles were studied in 12 lentil varieties, with varieties Du Puy, Kleine Schwarze, Rosana, Flora, Große Rote and Kleine Späths II demonstrating the highest protein percentages. After sprouting, protein percentages increased significantly in 10 of the 12 varieties, with the highest increases ranging between 20–23% in Dunkelgrün Marmorierte, Du Puy, Große Rote and Kleine Späths II varieties. While Fe concentration was significantly decreased in three varieties (Samos, Große Rote and Kleine Späths II), Zn and Mn were positively impacted by sprouting (p ≤ 0.05). Magnesium concentration was not affected by sprouting, while Ca and K had percentage increases between 41% and 58%, and 28% and 30%, respectively, in the best performing varieties (Kleine Schwarze, Dunkelgrün Marmorierte, Samos and Rosana). Regardless of the associated nutritional benefits, issues pertaining to sprouts microbiological safety must be ensured. The best results for the disinfection protocols were obtained when combining the seed treatment with SDS reagent followed by an Amukine application on the sprouts, which did not affect germination rates or sprout length. The increasing levels of sprout consumption throughout the world require efficient implementation of safety measures, as well as a knowledge-based selection for the nutritional quality of the seeds.
- The potencial role of soil application of a tris (3-hydroxy-4-pyridinonate) iron (III) complex in iron deficiency chlorosis treatmentPublication . Rodrigues, Elsa; Ferreira, Sofia; Leite, Andreia; Moniz, Tânia; Rangel, Maria; Vasconcelos, Marta W.; Carvalho, Susana M. P.; Santos, Carla S.
- Unravelling the molecular and physiological components that contribute to iron deficiency chlorosisPublication . Santos, Carla Sofia Sancho dos; Vasconcelos, Marta Wilton Pereira Leite de; Rangel, António Osmaro Santos Silva; Carvalho, Susana Maria Pinto deIron (Fe) deficiency chlorosis (IDC) is a serious condition affecting plants which are grown under calcareous or water logged soils. Under such conditions,Fe forms insoluble oxides and becomes unavailable for plant uptake, leading to stunted growth and severe yield reduction, causing aggravated agricultural losses. In the past years,efforts have been made to increase plant Fe content(so-called plant biofortification), in order to reduce the incidence of iron deficiency anaemia (IDA) prevalent around the world. To this end, legume grains and cereals, due to their rich nutritional profile and high worldwide intake by the population,have gained an important role in biofortification studies, which depend on the available molecular and physiological data for their successful implementation. The aim of this thes is was to contribute to the understanding of the molecular, physiological and biochemical mechanisms associated to Fe uptake and transport in Fe-stressed plants and to test a new class of Fe chelates as an efficient tool to prevent IDC. With the purpose of understanding the transcriptomic response to Fe deficiency in a set of different legume species, a non-targeted analysis was performed using Illumina technology. Transcriptome analysis was performed in the roots of soybean (Glycine max), common bean (Phaseolus vulgaris) and barrel medic (Medicago truncatula) grown in Fe deficiency and Fe sufficiency, and 114,723 annotated genes were obtained for all samples. Four IDC-related gene families were up-regulated in common by the three species and can be considered key players involved in the IDC response, namely, metal ligands, transferases, zinc ion binding and metal ion binding genes. Also, amongst the most highly expressed genes were genes of theisoflavonoid pathway and, on the other hand, oxidoreductases were the most down-regulated genes.Still on the search for IDC molecular players, two targetedgenetic analyses were performed,one on G.maxand M. truncatula and another on rice (Oryza sativa). Both studies involved the growth of plants under Fe sufficiency and Fe deficiency in order to compare the regulation of IDC related genes. Soybean and barrel medicare strategy I-crops,which means that, before uptake, they need to reduce Fe(III) to Fe(II) via an enzyme encoded by the FRO2 gene and, afterwards, Fe(II) is transported to the roots via ametal transporter encoded by the IRT1 gene. The expression of these two genes was analysed and both behaved similarly between species, appearingto be co-regulated.Moreover, the Fe transportersYSL1 andVIT1and the main Fe storage protein-encoding gene –ferritin–were up-regulated in the presence of Fe. The NRAMP3 gene, responsible for Fe remobilization from the vacuoles, was up-regulated under Fe deficiency,as was theGCN2gene, indicating a putative role of the latterin Fe metabolism and homeostasis.The targeted study performed in rice, a strategy II cereal that releases phytosiderophores in order to chelate and absorb Fe, involved the analysis of two rice cultivars with distinct susceptibilities to IDC –cv. Nipponbare and cv. Bico Branco. This different susceptibility was confirmed by their contrasting leaf chlorosis development and tissue nutrient accumulation patterns. Thecv. Nipponbare, that showed lower IDC susceptibility, was able to induce higher levels of the key reduction enzyme activity(Fe reductase)and showed higher levels of expression of the strategy I-OsFRO2 gene in roots.In contrast, cv. Bico Branco induced more genes involved in strategy II, specially, the transcription factor OsIRO2 and the phytosiderophore precursor OsTOM1.The screening for tolerant genotypes to IDC is an important tool in plant breeding programs. The most common IDC indicator is the degree of chlorosis development, which is quantified using a numerical scale. Therefore, after gathering the molecular data, the physiological mechanisms triggered by IDC were studied. The model crop G. max was selected,as it comprises lines well characterized according to their IDC-susceptibilities. To this end, two studies were performed. In the first study we aimed at understanding if the ability to partition Fe could be related to Fe-efficiency. We concluded that IDC susceptible lines, when compared to efficient lines,have lower ability to translocate Fe to the shoots, having about two fold higher Fe content at the root level, and they have lower capacity to induce the ferric reductase enzyme, having about three fold lower enzyme activity. In the second study the regulation of the antioxidant and tetrapyrrole systems under Fe deficiency was analysedfor the first time and we inferred that higher levels of oxidative stress might induce the oxidation of the tetrapyrrole heme into hemin, which leads to the induction of the heme-containing catalase enzyme and the reduction of ferric reductase activity. Taken together, the previous results indicate that low ferric reductase activity and Fe accumulation in the root tissue could be added as new IDC-related physiological markers.The application of fertilizers and Fe chelating agentsis one of the most frequently used tools to manage IDC. However, most of them are ineffective,too expensive or recalcitrant in the environment. Hence, the search for new Fe chelates is of utmost importance. In the last step of this thesis, we investigated the potential of a tris(3-hydroxy-4-pyridinonate) Fe(III) complex(Fe(mpp)3, which has never be enutilized in agricultural context)as na Fe fertilizer. Soybean plants were grown hydroponically under Fe deficiency and with Fe(mpp)3or Fe EDDHA supplementation. Results of both physiological and molecular markers showed that the new Fe complex led to healthier plants with increased growthby 24%,42% higher SPAD units and lower Fe retention in the roots.In general, the results presented in this thesis have contributed to a better understanding of the IDC-associated mechanisms and elucidated the key factors to be considered when analysing Fe deficient plants and their defence responses.
- Legume responses and adaptations to nutrient deficienciesPublication . Duarte, Rafael D. C.; Santos, Carla S.; Vasconcelos, Marta W.Legumes have unique mechanisms to respond to nutrient deficiencies that can be considered as important advantages for agricultural purposes. The preponderance of plant-based protein is on the rise, and the market value of protein crops is expected to be worth billions by 2025. To match the global demand for plant-based products, crops productivity must be ensured; however, this might be impaired either by environmental or anthropogenic pressures that lead to soil nutrient disturbance. The responses activated by legumes to nutrient deficiencies and the mechanisms they utilize to adapt to such conditions will be discussed in this chapter. The study of these factors enables breeding programs specific for legumes and crop improvement. Understanding legumes responses also allows for a better management of agricultural practices and the adoption ofmore sustainablemethods. It is important to reflect on the impact of climate change and intensive farming on food quality and on the future of agriculture, and this chapter contributes with important facts about the role of legumes in our current scenario.
- Identification of genes differentially expressed in P. pinea and P. pinaster after infection with the pine wood nematode (PWD) using the SSH techniquePublication . Santos, Carla Sofia Sancho dos; Vasconcelos, Marta Wilton deO objectivo deste estudo consistiu na comparação da resposta à infecção com o nemátode da madeira do pinheiro (NMP) Bursaphelenchus xylophilus entre Pinus pinaster e Pinus pinea no estádio inicial da doença, três horas após inoculação. O NMP é o agente causal da doença da madeira do pinheiro, tem em Portugal como hospedeiro principal o pinheiro bravo, P. pinaster, e tem como vector Monochamus galloprovincialis. Curiosamente, esta doença parece não afectar a espécie P. pinea e vários factores podem estar na origem desta diferença de susceptibilidade. Nas primeiras horas após uma infecção por patogénios, a árvore desenvolve uma resposta hipersensitiva, que consiste na produção de proteínas de resistência, entre outros compostos cruciais de defesa. Actualmente, não há estudos que descrevam os efeitos da infecção do nemátode nas plantas a um estádio inicial da doença. No que diz respeito aos efeitos fisiológicos e metabólicos nas plantas, verificou-se uma diminuição de 10,9% no teor de água em P. pinaster (por comparação com árvores inoculadas com água) e 6,7% em P. pinea; relativamente à concentração de clorofilas, uma diminuição para cerca de metade do valor controlo foi registado para ambas as espécies, mas mais pronunciadamente em P. pinaster. Um estudo exploratório com GC também foi efectuado, que demonstrou que os compostos voláteis produzidos permitem uma clara diferenciação entre espécies, mas não entre inoculação controlo vs. nemátode. No que diz respeito aos efeitos da doença ao nível da transcrição de genes, utilizou-se a técnica de SSH para identificar ESTs em P. pinaster e P. pinea inoculados com NMP. Os ESTs foram isolados, clonados, sequenciados e identificados usando BlastN e BlastX, e indicam claramente que no estádio inicial da doença existe a activação de uma resposta de defesa ao nível molecular relacionada principalmente com o stress oxidativo, produção de lenhina e de etileno e regulação pós-transcripcional dos ácidos nucleicos. Finalmente, 58% das sequências isoladas não estão ainda descritas, o que mostra a falta de informação genómica actual existente para o pinheiro.
- Comparative life cycle assessment of plant and beef-based patties, including carbon opportunity costsPublication . Saget, Sophie; Costa, Marcela Porto; Santos, Carla Sancho; Vasconcelos, Marta; Styles, David; Williams, MikeLegume-derived foods have been shown to have comparatively low greenhouse gas (GHG) intensities whilst providing high amounts of nutrients. However, processing legumes into meat analogues can incur significant energy costs. Here, we undertake a comprehensive life cycle assessment of plant-based and (Brazilian and Irish) beef burger patties. Sixteen impact categories are supplemented with the carbon opportunity cost of land occupation, and benchmarked against nutrient density units (NDU) to provide holistic evidence on the potential contribution of plant-based patties to environmentally-sustainable nutritional density. Plant-based patties have a smaller environmental footprint across most categories, including a 77% smaller climate change burden, but incur 8% more energy use compared with Brazilian beef patties. Normalised scores (person equivalents) were significantly larger (p < 0.05) for the beef products across key categories including land use, acidification, and marine and terrestrial eutrophication. Sensitivity analyses indicated significant variance across impact categories if beef cattle are reared in South Africa, France or the United States, including a 16-fold difference in land occupation. Biophysical allocation of co-products reduced environmental burdens of beef burgers. However, owing to a 68% higher NDU per serving, reflecting higher fibre and essential fatty acid content, plant-based patties are associated with 81–87% less climate change and 92–95% less marine eutrophication per NDU compared with beef burger patties. Accounting for carbon opportunity cost of land further increased the climate change advantage of plant-based patties by 25–44%. A simple extrapolation indicates that switching from beef to vegetable patties in the UK could save between 9.5 and 11 million tonnes CO2e annually, representing up to 2.4% of territorial GHG emissions.
- Peristomal infection after percutaneous endoscopic gastrostomy: a 7-year surveillance of 297 patients.Publication . Duarte, Helena; Santos, Carla; Capelas, Manuel Luís; Fonseca, JorgeCONTEXT: Healthcare-associated infection represents the most frequent adverse event during care delivery. Medical advances like percutaneous endoscopic gastrostomy have brought improvement on quality of life to patients but an increased risk of healthcare-associated infection. Predictive risk factors for peristomal wound infection are largely unknown but evidence suggests that antibiotic prophylaxis and preventive strategies related to infection control may reduce infection rates. OBJECTIVES: The primary aim was to evaluate the global prevalence rate of peristomal infection. Secondary objectives were to characterise the positive culture results, to evaluate the prophylactic antibiotic protocol and to identify potential risk factors for peristomal infection. METHODS: Retrospective study of 297 patients with percutaneous endoscopic gastrostomy performed at a general hospital between January 2004 and September 2010. Patients received prophylactic cefazolin before the endoscopic gastrostomy procedure. Medical records were reviewed for demographic data, underling disease conditions to percutaneous endoscopic gastrostomy and patient potential intrinsic risk factors. Statistical analysis was made with the statistical program SPSS 17.0. RESULTS: A total of 297 percutaneous endoscopic gastrostomy tubes were inserted. Wound infection occurred in 36 patients (12.1%). Staphylococcus aureus methicillin resistant was the most frequently isolated microorganism (33.3%) followed by Pseudomonas aeruginosa (30.6%). The incidence rate had been rising each year and differ from 4.65% in 2004/2007 to 17.9% in 2008/2010. This finding was consistent with the increasing of prevalence global infection rates of the hospital. Most of the infections (55.6%) were detected in the first 10 days post procedure. There was no significant difference in age, body mass index values, mean survival time and duration of percutaneous endoscopic gastrostomy feeding between patients with and without periostomal infection. Institutional factors, namely global prevalence infection rates and the endemic character of Staphylococcus aureus methicillin resistant, play an important role in peristomal infection rates. Traditional antibiotic prophylaxis with cefazolin is not adequate due to the prevalence of resistant organisms.CONCLUSIONS: Peristomal infection is a frequent problem with clinical impact in percutaneous endoscopic gastrostomy patients and should be considered a healthcare associated infection. The antimicrobial prophylaxis regimens using cephalosporins are not adequate and need to be reviewed due to the high prevalence of Staphylococcus aureus methicillin resistant and other resistant organisms in hospitals and nursing homes.
- Comparative analysis of Iron Deficiency Chlorosis responses in soybean (Glycine max) and barrel medic (Medicago truncatula)Publication . Santos, Carla S.; Serrão, Inês; Vasconcelos, Marta W.Legume grains have an important socio-economical role, being highly utilized in human and animal nutrition. Although iron (Fe) is abundant in the earths crust, its limited solubility makes it poorly bioavailable for plants, contributing to iron deficiency chlorosis (IDC). In this work the physiological and molecular mechanisms associated with IDC were studied, namely, the mechanisms involved on Fe deficiency response, as well as a new Fe metabolism related gene in two important legume crops, Glycine max and Medicago truncatula. Fe deficient plants developed: decreased root and shoot length, increased number of secondary roots and lower chlorophyll levels. Fe shoot content decreased six- and 11-fold for G. max and M truncatula in Fe-deficiency. Whilst in G. max roots no significant differences were detected, in M. truncatula roots Fe decreased nine-fold in Fe-deficiency. Genes involved in Fe uptake (FRO2-like and IRT1-like), were over-expressed in roots of Fe-sufficient G. max and in Fe-deficient M. truncatula. VIT1-like, YSL1-like and ferritin presented higher expression levels in Fe-sufficient shoots and roots, whereas NRAMP3-like and GCN2-like showed higher expression values in Fe-deficiency.
- Impact of iron deficiency in five legume speciesPublication . Carvalho, Ana Lúcia; Serrão, Inês; Santos, Carla; Vasconcelos, Marta W.
- Effect of tris(3-hydroxy-4-pyridinonate) iron(III) complexes on iron uptake and storage in soybean (Glycine max L.)Publication . Santos, Carla S.; Carvalho, Susana M.P.; Leite, Andreia; Moniz, Tânia; Roriz, Mariana; Rangel, António O. S. S.; Rangel, Maria; Vasconcelos, Marta W.Iron deficiency chlorosis (IDC) is a serious environmental problem affecting the growth of several crops in the world. The application of synthetic Fe(III) chelates is still one of the most common measures to correct IDC and the search for more effective Fe chelates remains an important issue. Herein, we propose a tris(3-hydroxy-4-pyridinonate) iron(III) complex, Fe(mpp)3, as an IDC corrector. Different morphological, biochemical and molecular parameters were assessed as a first step towards understanding its mode of action, compared with that of the commercial fertilizer FeEDDHA. Plants treated with the pyridinone iron(III) complexes were significantly greener and had increased biomass. The total Fe content was measured using ICP-OES and plants treated with pyridinone complexes accumulated about 50% more Fe than those treated with the commercial chelate. In particular, plants supplied with compound Fe(mpp)3 were able to translocate iron from the roots to the shoots and did not elicit the expression of the Fe-stress related genes FRO2 and IRT1. These results suggest that 3,4-HPO iron(III) chelates could be a potential new class of plant fertilizing agents.