Efeitos de Roundup Transorb no perfil transcriptômico de folhas e na composição de grãos de Glycine max (L.) Merr.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Programa de Pós-Graduação em Recursos Genéticos Vegetais, Florianópolis, 2020.A abordagem atual de avaliação com base somente na equivalência substancial entre Organismos Modificados comparados com sua linhagem isogênica mais próxima, não é considerada suficiente, por muitos cientistas, como método seguro de análise de risco. Assim, o presente trabalho no seu primeiro capítulo apresenta uma análise de expressão de EPSPS transgênica e nativa de soja (Glycine max), com o uso da técnica RT-PCR. As análises dos resultados indicaram que a expressão gênica foi regulada igualmente para eventos únicos e estaqueados (stacking). Com a interpretação do RNA-seq verificou-se que apesar das variedades compartilharem respostas semelhantes, a variedade simples exibe um comportamento fisiológico esperado como resposta ao estresse, aumento do teor de açúcar, diminuição da taxa de captação de clorofila A e B e comprometimento do balanço celular redox. Por sua vez, a variedade estaqueada apresentou forte regulação nas rotas relacionadas a metabólitos secundários (ß-glucosidase, isoflavona 7-O-metiltransferase, bem como, componentes de defesa e mecanismos de sinalização de Ca2+, chaperonas moleculares, proteínas Rboh e WRKY, o que sugere uma resposta ao estresse mais pronunciada. No segundo capítulo, foi obsevado que aplicação no estádio fenológico V2 produziu resíduos de glifosato e AMPA no grão colhido pronto para consumo. A análise dos resíduos permite refletir sobre o atual cenário alimentar, considerando que apenas um herbicida e em dosagens recomendas pela bula foram adotadas. O efeito do HBG sobre alguns componentes também foi testado e foi observado que as amostras não-GM e GM tenderam ao agrupamento independente do tratamento com herbicida nas duas dosagens. É imprescindível conhecer o organismo biológico GM de maneira mais complexa, neste sentido, a utilização de transcriptômica permitiu observar alterações metabólicas oriundas do efeito de HBG. Por exemplo, foram descritas alterações em uma série de genes relacionados ao metabolismo defesa, carbono, homeostase redox e fotossíntese e na variedade estaqueada, chama a atenção a alteração da rota do chiquimato, constatada devido a presença do aminoácido fenilalanina e subprodutos do mesmo.Abstract: The current assessment approach based only on the substantial equivalence between Modified Organisms compared to their closest isogenic lineage, is not considered sufficient, by many scientists, as a safe method of risk analysis. Thus, the present work in its first chapter analyzes and presents an analysis of the expression of transgenic and native EPSPS, from soy (Glycine max), using the RT-PCR technique. Through the analysis of the results, it was observed that the gene expression was regulated equally for single and stacked events (staking). Stacking analysis. With the interpretation of RNA-seq it was demonstrated that despite the varieties sharing similar responses, the simple variety exhibits an expected physiological behavior as a response to stress (, increase in sugar content, decrease in the rate of uptake of chlorophyll A and B harvest and impairment of the redox cell balance). On the other hand, the stacked variety, on the other hand, showed a high regulation in the pathways related to secondary metabolites (ß-glucosidase, isoflavone 7-O-methyltransferase), as well as defense components and signaling mechanisms of Ca2 +, molecular chaperones, Rboh and WRKY proteins, which suggests a more pronounced stress response. In the second chapter, it was observed that application in the phenological stage V2 produced residues of glyphosate and AMPA in the harvested grain ready for consumption. The analysis of residues allows us to reflect on the current food scenario, considering that only one herbicide and in dosages recommended by the package insert were adopted. The effect of HBG on some components was also tested and it was observed that the non-GM and GM samples tended to group independently of the herbicide treatment at the two dosages. It is essential to know the biological organism GM in a more complex way, in this sense, the use of transcriptomics allowed us to observe metabolic changes arising from the effect of HBG. For example, changes have been described in a series of genes related to defense metabolism, carbon, redox homeostasis, and photosynthesis, and in the staked variety, attention is drawn to the alteration in the route of the shikimate, found due to the presence of the amino acid phenylalanine and its by-product

Integration of omics analyses into GMO risk assessment in Europe: a case study from soybean field trials

In Europe, genetically modified organisms (GMOs) are subject to an authorization process including a mandatory risk assessment. According to the respective guidance by the European Food Safety Authority (EFSA), one of the pillars of this GMO risk assessment is a comparative analysis of the compositional and agronomic characteristics. This targeted approach has been criticized for its limitations, as it only considers pre-determined compounds, being insufficient to assess a comprehensive range of relevant compounds, including toxins and anti-nutrients, on a case-specific basis. Strategies based on advanced untargeted omics technologies have been proposed as a potential broader approach to be implemented into the initial step of the risk assessment framework. Here, we provide an example of a step-by-step omics analysis based on systems biology approach to fit into the context of European GMO regulation. We have performed field trial experiments with genetically modified (GM) Intacta™ Roundup Ready™ 2 Pro soybean containing both cry1Ac and cp4epsps transgenic inserts and analyzed its proteomic profile against the non-GM counterpart and reference varieties. Based on EFSA’s comparative endpoint-by-endpoint approach, the proteomics analysis revealed six proteins from the GMO outside the 99% tolerance intervals of reference varieties (RVs) in the equivalence test. Interestingly, from the near-isogenic (non-GM) comparator we found as many as ten proteins to be outside of the said RVs’ equivalence limits. According to EFSA’s statistical guidelines, differences found in metabolite abundance between a GMO and its non-GM comparator would not be considered biologically relevant as all compounds of concern remained within the equivalence limits of commercial RVs. By assessing the proteomic and metabolomic data through our proposed systems biology approach, we found 70 proteins, and the metabolite xylobiose as differentially expressed between the GMO and its non-GM comparator. Biological relevance of such results was revealed through a functional biological network analysis, where we found alterations in several metabolic pathways related to protein synthesis and protein processing. Moreover, the allergenicity analysis identified 43 proteins with allergenic potential being differentially expressed in the GM soybean variety. Our results demonstrate that implementation of advanced untargeted omics technologies in the risk assessment of GMOs will enable early and holistic assessment of possible adverse effects. The proposed approach can provide a better understanding of the specific unintended effects of the genetic modification on the plant’s metabolism, the involved biological networks, and their interactions, and allows to formulate and investigate dedicated risk hypotheses in the first place. We draw conclusions on a detailed comparison with the comparative assessment according to EFSA and provide scientific arguments and examples on how the current comparative approach is not fit for purpose.publishedVersio

A DNA-Free Editing Platform for Genetic Screens in Soybean via CRISPR/Cas9 Ribonucleoprotein Delivery

CRISPR/Cas9-based ribonucleoprotein (RNP)-mediated system has the property of minimizing the effects related to the unwanted introduction of vector DNA and random integration of recombinant DNA. Here, we describe a platform based on the direct delivery of Cas9 RNPs to soybean protoplasts for genetic screens in knockout gene-edited soybean lines without the transfection of DNA vectors. The platform is based on the isolation of soybean protoplasts and delivery of Cas RNP complex. To empirically test our platform, we have chosen a model gene from the soybean genetic toolbox. We have used five different guide RNA (gRNA) sequences that targeted the constitutive pathogen response 5 (CPR5) gene associated with the growth of trichomes in soybean. In addition, efficient protoplast transformation, concentration, and ratio of Cas9 and gRNAs were optimized for soybean for the first time. Targeted mutagenesis insertion and deletion frequency and sequences were analyzed using both Sanger and targeted deep sequencing strategies. We were able to identify different mutation patterns within insertions and deletions (InDels) between + 5 nt and –30 bp and mutation frequency ranging from 4.2 to 18.1% in the GmCPR5 locus. Our results showed that DNA-free delivery of Cas9 complexes to protoplasts is a useful approach to perform early-stage genetic screens and anticipated analysis of Cas9 activity in soybeans.publishedVersio

Alterations in genetically modified crops assessed by omics studies: Systematic review and meta-analysis

Background International agreements and domestic legislation regulate genetically modified (GM) crops for environmental release, recognizing that genetic engineering could result in unintended genotypic and phenotypic effects. In that context, omics technologies, which allow comprehensive characterization of the molecular profile of GM crops at all levels, may be used to assess alterations or effects of genetic engineering. Objective To determine whether omics techniques are suitable tools to comprehensively screen for metabolic changes due to genetic modification in plants. Approaches A literature search was conducted in four online scientific databases for relevant publications. After removal of duplicates, we retained only studies that included cry, epsps and pat/bar transgenes. We evaluated the full texts of the remaining papers and performed data extraction. We placed the extracted outcomes into an evidence table, which comprised six major categories, including an analysis of altered metabolic pathways based on the KEGG pathway database. Main findings Sixty articles were included in this review. We found a high proportion of publicly funded studies (86.7%) compared to just three with industry financial support. We found that 40% of the plant material analyzed was produced in the field, 26.7% in growth chambers, and 18.3% in greenhouse experiments, although this information could not be extracted from all studies. More than one third (38.4%) of the studies did not use a non-GM near-isogenic line as a comparator, and half did not specify the number of plants used per sample in their reports. All the studies (except three that did not perform a comparative analysis) reported statistical differences in GM versus non-GM omic profiles. A heatmap analysis showed that the most frequently affected metabolic pathways were related to metabolism of carbohydrates, energy, lipids, and amino acids, as well as genetic information processing and environmental information processing. Conclusion This review shows that omics techniques can profile different levels of genetic information and metabolism and can be useful tools in assessing alterations in genetically modified plants. In recent years, there have been intensive efforts to harmonize omics methods. Consistent guidelines with standardized frameworks are needed to capitalize on the unquestionable potential of implementing untargeted omics analyses in the risk assessment process. Finally, there is a need to build an assessment framework connecting omics results to biologically relevant changes in the GM organism, and this framework to be operable for the risk assessment process

Proteomic Profile of Glyphosate-Resistant Soybean under Combined Herbicide and Drought Stress Conditions

While some genetically modified (GM) plants have been targeted to confer tolerance to abiotic stressors, transgenes are impacted by abiotic stressors, causing adverse effects on plant physiology and yield. However, routine safety analyses do not assess the response of GM plants under different environmental stress conditions. In the context of climate change, the combination of abiotic stressors is a reality in agroecosystems. Therefore, the aim of this study was to analyze the metabolic cost by assessing the proteomic profiles of GM soybean varieties under glyphosate spraying and water deficit conditions compared to their non-transgenic conventional counterparts. We found evidence of cumulative adverse effects that resulted in the reduction of enzymes involved in carbohydrate metabolism, along with the expression of amino acids and nitrogen metabolic enzymes. Ribosomal metabolism was significantly enriched, particularly the protein families associated with ribosomal complexes L5 and L18. The interaction network map showed that the affected module representing the ribosome pathway interacts strongly with other important proteins, such as the chloro-plastic gamma ATP synthase subunit. Combined, these findings provide clear evidence for increasing the metabolic costs of GM soybean plants in response to the accumulation of stress factors. First, alterations in the ribosome pathway indicate that the GM plant itself carries a metabolic burden associated with the biosynthesis of proteins as effects of genetic transformation. GM plants also showed an imbalance in energy demand and production under controlled conditions, which was increased under drought conditions. Identifying the consequences of altered metabolism related to the interaction between plant transgene stress responses allows us to understand the possible effects on the ecology and evolution of plants in the medium and long term and the potential interactions with other organisms when these organisms are released in the environment

Proteomic Profile of Glyphosate-Resistant Soybean under Combined Herbicide and Drought Stress Conditions

While some genetically modified (GM) plants have been targeted to confer tolerance to abiotic stressors, transgenes are impacted by abiotic stressors, causing adverse effects on plant physiology and yield. However, routine safety analyses do not assess the response of GM plants under different environmental stress conditions. In the context of climate change, the combination of abiotic stressors is a reality in agroecosystems. Therefore, the aim of this study was to analyze the metabolic cost by assessing the proteomic profiles of GM soybean varieties under glyphosate spraying and water deficit conditions compared to their non-transgenic conventional counterparts. We found evidence of cumulative adverse effects that resulted in the reduction of enzymes involved in carbohydrate metabolism, along with the expression of amino acids and nitrogen metabolic enzymes. Ribosomal metabolism was significantly enriched, particularly the protein families associated with ribosomal complexes L5 and L18. The interaction network map showed that the affected module representing the ribosome pathway interacts strongly with other important proteins, such as the chloro-plastic gamma ATP synthase subunit. Combined, these findings provide clear evidence for increasing the metabolic costs of GM soybean plants in response to the accumulation of stress factors. First, alterations in the ribosome pathway indicate that the GM plant itself carries a metabolic burden associated with the biosynthesis of proteins as effects of genetic transformation. GM plants also showed an imbalance in energy demand and production under controlled conditions, which was increased under drought conditions. Identifying the consequences of altered metabolism related to the interaction between plant transgene stress responses allows us to understand the possible effects on the ecology and evolution of plants in the medium and long term and the potential interactions with other organisms when these organisms are released in the environment

Alterations in genetically modified crops assessed by omics studies: Systematic review and meta-analysis

Background International agreements and domestic legislation regulate genetically modified (GM) crops for environmental release, recognizing that genetic engineering could result in unintended genotypic and phenotypic effects. In that context, omics technologies, which allow comprehensive characterization of the molecular profile of GM crops at all levels, may be used to assess alterations or effects of genetic engineering. Objective To determine whether omics techniques are suitable tools to comprehensively screen for metabolic changes due to genetic modification in plants. Approaches A literature search was conducted in four online scientific databases for relevant publications. After removal of duplicates, we retained only studies that included cry, epsps and pat/bar transgenes. We evaluated the full texts of the remaining papers and performed data extraction. We placed the extracted outcomes into an evidence table, which comprised six major categories, including an analysis of altered metabolic pathways based on the KEGG pathway database. Main findings Sixty articles were included in this review. We found a high proportion of publicly funded studies (86.7%) compared to just three with industry financial support. We found that 40% of the plant material analyzed was produced in the field, 26.7% in growth chambers, and 18.3% in greenhouse experiments, although this information could not be extracted from all studies. More than one third (38.4%) of the studies did not use a non-GM near-isogenic line as a comparator, and half did not specify the number of plants used per sample in their reports. All the studies (except three that did not perform a comparative analysis) reported statistical differences in GM versus non-GM omic profiles. A heatmap analysis showed that the most frequently affected metabolic pathways were related to metabolism of carbohydrates, energy, lipids, and amino acids, as well as genetic information processing and environmental information processing. Conclusion This review shows that omics techniques can profile different levels of genetic information and metabolism and can be useful tools in assessing alterations in genetically modified plants. In recent years, there have been intensive efforts to harmonize omics methods. Consistent guidelines with standardized frameworks are needed to capitalize on the unquestionable potential of implementing untargeted omics analyses in the risk assessment process. Finally, there is a need to build an assessment framework connecting omics results to biologically relevant changes in the GM organism, and this framework to be operable for the risk assessment process.publishedVersio