Percorrer por autor "Renaut, Jenny"
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- Characterization of maize allergens - MON810 vs. its non-transgenic counterpart.Publication . Fonseca, Cátia; Planchon, Sébastien; Renaut, Jenny; Oliveira, Maria Margarida; Batista, RitaOne of the main concerns about genetically modified foods and their potential impacts on human health is that the introduction of a new/ altered gene may putatively alter the expression of others, namely endogenous allergens. We intended to evaluate, and to compare, using quantitative real time RT-PCR technique, the expression of 5 already known maize allergens (Zea m14, Zea m25, Zea m27kD, 50kD Zein and trypsin inhibitor) in MON 810 vs. its non-transgenic counterpart, throughout seed development (10, 16 and 23 days after pollination). We have shown that none of the tested allergen genes presented differential expression, with statistic significance, along all tested seed development stages, in MON810 vs. its conventional counterpart. We have also used bidimensional gel electrophoresis followed by Western blotting with plasma from two maize allergic subjects to characterize their immunologic responses against MON 810 vs. its non-transgenic control. Immunoreactive spots were characterized by MS. We have identified fourteen new IgE-binding proteins present in both transgenic and non-transgenic maize.
- A cultura in vitro tem impacto nas diferenças encontradas entre os alimentos transgénicos e seus controlosPublication . Fonseca, Cátia; Planchon, Sébastien; Serra, Tânia; Chander, Subhash; Saibo, Nelson JM; Renaut, Jenny; Oliveira, M Margarida; Batista, Rita
- Environmental stress is the major cause of transcriptomic and proteomic changes in GM and non-GM plantsPublication . Batista, Rita; Fonseca, C.; Planchon, Sébastien; Negrão, Sónia; Renaut, Jenny; Oliveira, MargaridaThe approval of genetically modified (GM) crops is preceded by years of intensive research to demonstrate safety to humans and environment. We recently showed that in vitro culture stress is the major factor influencing proteomic differences of GM vs. non-GM plants. This made us question the number of generations needed to erase such “memory”. We also wondered about the relevance of alterations promoted by transgenesis as compared to environment-induced ones. Here we followed three rice lines (1-control, 1-transgenic and 1-negative segregant) throughout eight generations after transgenesis combining proteomics and transcriptomics, and further analyzed their response to salinity stress on the F6 generation. Our results show that: (a) differences promoted during genetic modification are mainly short-term physiological changes, attenuating throughout generations, and (b) environmental stress may cause far more proteomic/transcriptomic alterations than transgenesis. Based on our data, we question what is really relevant in risk assessment design for GM food crops.
- In vitro culture is the major contributing factor for transgenic vs. non-transgenic proteomic plant differencesPublication . Fonseca, Cátia; Planchon, Sébastien; Serra, Tânia; Chander, Subhash; Saibo, Nelson; Renaut, Jenny; Oliveira, M.Margarida; Batista, RitaThe identification of similarities and differences between genetically modified plants and derived food/feed and their comparators plays a central role in risk assessment strategy. Our main goal was to better understand the relevance of transgene presence, genetic and epigenetic changes promoted by transgene insertion and in vitro culture in the potential unintended effects/differences between transgenic and conventional counterparts. In order to achieve this goal we have used Multiplex fluorescence 2D gel electrophoresis technology coupled with MS to characterize the proteome of three different rice lines (Oryza sativa L. ssp. japonica cv. Nipponbare): a control conventional counterpart, an Agrobacterium-transformed transgenic line (Ta) and a negative segregant (NSb), progeny of a different transgenic line. We have observed that Ta and NSb plant lines grouped together (only 1 differentially regulated spot - fold difference ≥ 1.5), separated from the control (49 spots with fold difference ≥1.5, in both Ta and NSb lines comparing to control line). Since the only feature in common between the tested transgenic and negative segregant lines is that they have both suffered in vitro culture procedures we hypothesised that stress caused by in vitro culture was, in this case, the most relevant factor contributing for the encountered proteomic differences. MS identifications of differentially regulated spots supported our hypothesis, demonstrating that Ta and NSb rice lines present several adjusted metabolic pathways and several proteins with altered expression that were previously associated with plant stress response.
- Maize Allergens during seed development: transgenic versus non-transgenicPublication . Fonseca, Cátia; Oliveira, Maria Margarida; Renaut, Jenny; Planchon, Sébastien; Batista, RitaRecombinant DNA technology, also known as genetic engineering, allows the transfer of genes between unrelated species. As a result, a genetically modified organism (GMO) may contain one or more proteins coming from other organism/s. The application of genetic engineering to plants improvement and food production is becoming a common practice. New and diverse plant varieties have been obtained which are pest and disease resistant, more productive and with improved nutritional quality, flavour and shelf life. In spite of all these potential benefits, some apprehension persist regarding genetically modified organisms putative effects over human health and environment. One of the main concerns regards GMO’s potential allergenicity. One of the possibilities is that the introduction of a new/ altered gene may putatively alter the expression of others, namely endogenous allergens. In maize, there are already some proteins characterized as allergens, namely a lipid transport protein (Pastorello et al. 2000), a tioredoxin Zea m25 (Weichel et al. 2006), two glutelins Zea m27kD (Frisner et al. 2000) and 50kD Zein (Pasini et al. 2002) and one trypsin inhibitor (Pastorello et al. 2000). In this study, we have evaluated the expression of these 5 allergens, throughout MON 810 vs its non-transgenic counterpart seed development (10, 16 and 23 days after pollination). The expression profile of each one of these allergens varies during seed development, although the observed differences between Transgenic and Non-Transgenic maize were not statistical significant (t-test). Since there is a lack of experimental data regarding the correlation of allergen expression with food allergy clinical relevance, we are now comparing the immunologic response of maize allergic individuals against MON 810 maize vs its non transgenic control. We also intend to characterize the encountered potential maize allergens by mass spectrometry.
- Maize IgE binding proteins: each plant a different profile?Publication . Fonseca, Cátia; Planchon, Sébastien; Pinheiro, Carla; Renaut, Jenny; Ricardo, Cândido; Oliveira, M. Margarida; Batista, RitaBackground: Allergies are nearly always triggered by protein molecules and the majority of individuals with documented immunologic reactions to foods exhibit IgE hypersensitivity reactions. In this study we aimed to understand if natural differences, at proteomic level, between maize populations, may induce different IgE binding proteins profiles among maize-allergic individuals. We also intended to deepen our knowledge on maize IgE binding proteins. Results: In order to accomplish this goal we have used proteomic tools (SDS-PAGE and 2-D gel electrophoresis followed by western blot) and tested plasma IgE reactivity from four maize-allergic individuals against four different protein fractions (albumins, globulins, glutelins and prolamins) of three different maize cultivars. We have observed that maize cultivars have different proteomes that result in different IgE binding proteins profiles when tested against plasma from maize-allergic individuals. We could identify 19 different maize IgE binding proteins, 11 of which were unknown to date. Moreover, we found that most (89.5%) of the 19 identified potential maize allergens could be related to plant stress. Conclusions: These results lead us to conclude that, within each species, plant allergenic potential varies with genotype. Moreover, considering the stress-related IgE binding proteins identified, we hypothesise that the environment, particularly stress conditions, may alter IgE binding protein profiles of plant components.
- Maize-induced allergy: each plant a different reaction?Publication . Fonseca, Cátia; Planchon, Sébastien; Renaut, Jenny; Pinheiro, Carla; Oliveira, M.Margarida; Batista, RitaCereals are the most important crops in the world. For the majority of the world’s human population, cereal-based foods constitute the most important source of energy and other nutrients. In the poorest parts of the world starchy foods, including cereals, may supply 70% of total energy. Although a number of cereal species are grown for food worldwide, only three - maize, wheat and rice (respectively, 883, 704 and 722 million tonnes in 2011) - together account for over 85% of the total production (FAOSTAT- http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567#ancor). Maize is present in a wide range of foods (bread, breakfast cereals, corn snacks, corn flour, polenta, popcorn). Maize variation may be categorized based on the quality, quantity and pattern of endosperm composition in the kernel. Maize types are generally divided in: flint, dent, flour, pop, sweet and pop corn. Maize allergy can occur after the ingestion of maize or maize derivatives, or by the inhalation of maize flour or pollen. Recently, some papers were published on maize allergy. However, the factors that may influence allergen production are still unknown. Considering that proteins are the elicitors of the majority of allergic food reactions, it would be expected that different cultivars may induce different allergic reactions. Actually, some authors have already reported an high heterogeneity in the distribution and quantification of several already known plant allergens among different cultivars. In this study we aimed to contribute to the characterisation of maize flour allergens via proteomic tools (SDS-PAGE and 2-D gel electrophoresis followed by Western blot) trying to understand if natural proteomic differences, between maize varieties, may conduct to different allergic reactions among maize-allergic individuals. In order to accomplish this goal we have tested plasma IgE reactivity from four maize-allergic individuals against four different protein fractions (albumin, globulin, glutelin and prolamin) of three different maize cultivars. We have observed that maize cultivars have different proteomic profiles inducing different allergic reactions in the tested individuals. We could identify 19 different maize IgE-binding proteins, 11 of which unknown to date. Five of these 11 proteins, were already identified as potential allergens in other organisms. Moreover, we found that most (89.5%) of the 19 identified potential maize allergens could be related to stress. These results lead us to conclude that, like the proteome, plant allergenic potential varies with genotype and have also the potential to be highly influenced by environmental stress.
- Selection of the best comparator for the risk assessment of GM plants- conventional counterpart vs. negative segregantPublication . Fonseca, Cátia; Planchon, Sébastien; Serra, Tânia; Chandler, Subhash; Saibo, Nelson; Renaut, Jenny; Oliveira, Margarida; Batista, RitaAbstract for 12th international Symposium on biosafety of genetically modified organisms The identification of similarities and differences between GM plants and derived food/feed and their comparators plays a central role in risk assessment strategy. Therefore, selecting the right comparators must be one of the top priorities. The question is which control would allow us to better evaluate the potential unintended effects related directly to the transgene and/or DNA rearrangements, discounting the potential effects caused by in vitro culture procedures; since, these are non-controversial procedures also used in conventional breeding. Aiming to answer the previous question we have used Multiplex fluorescence 2D gel electrophoresis technology (DyeAGNOSTICS Refraction-2D) coupled with MS to characterize the proteome of three different rice lines (Oriza sativa L. ssp. Japonica cv Nipponbare): A control conventional counterpart. An Agrobacterium transformed transgenic line. A negative segregant (homozygous negative progeny) from a different transgenic line. We have observed that transgenic and negative segregant plant lines grouped together (only 1 differentially regulated spot - fold difference > 1.5, ANOVA, P<0.05,) and apart from control (49 spots with fold difference >1.5, ANOVA, P<0.05, in both transgenic and negative segregant lines comparing to control line). Additionally, the 35 proteins identified (using MS) in this study, were already associated with stress response by other authors. The only feature in common between the transgenic and negative segregant lines is that they have both suffered in vitro culture procedures. Hence, the results obtained indicate that, in this study, different gene disruption and/or DNA rearrangements and the presence/absence of transgene were factors with less impact on rice proteome than the proteomic promoted differences caused by in vitro culture, and eventually the stress caused by this process. This work highlights the importance of continuous revision and upgrade of the guidance criteria to be followed for the selection of suitable comparators in GMO risk assessment.
- O stress ambiental pode provocar mais alterações nas plantas do que a engenharia genéticaPublication . Batista, Rita; Fonseca, Cátia; Planchon, Sébastien; Negrão, Sónia; Renaut, Jenny; Oliveira, M. MargaridaA aprovação das plantas geneticamente modificadas é precedida de vários anos de investigação intensiva de forma a garantir a sua segurança para o homem e ambiente. Recentemente demonstrámos que o stress da cultura in vitro parece ser o fator que mais influencia as diferenças proteómicas encontradas entre as plantas geneticamente modificadas (GM) e os seus controlos. Neste trabalho tentámos avaliar quantas gerações são necessárias para os efeitos da “memória” desse stress serem apagados. Tentámos também comparar a relevância das alterações provocadas pelo stress da cultura in vitro quando comparadas com as provocadas por outros stresses ambientais. Para tal seguimos três linhas de arroz durante oito gerações após a transgénese combinando técnicas de análise transcritómica e proteómica. Na geração F6 as plantas foram sujeitas a stress salino. Os resultados deste trabalho demonstraram que: (a) as diferenças promovidas durante a modificação genética são maioritariamente diferenças fisiológicas de curta duração, que atenuam ao longo do tempo; (b) os stresses ambientais podem causar mais alterações proteómicas/trascritómicas que a engenharia genética. Com base nos dados obtidos questionamos quais os ensaios que são realmente relevantes e quais aqueles claramente excessivos na avaliação do risco das plantas GM para a saúde humana e animal, plantas e ambiente.