World agricultural production of genetically modified (GM) products in particular, the combination of different traits/genes in the same plant has been intense over the last decade. The stacking of herbicide and insect-resistant transgenic genes can result in fitness costs that rely on the type and strength of the selection pressure exerted by the environment. Here we report the results of transcriptomic analysis comparing the effect of glyphosate on various biological processes, metabolic

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... ays, and main shikimic enzymes in stacked versus single soybean resistant varieties. Results Gene expression data were grouped according to treatment, ie the herbicidal factor strongly influenced. Common physiological results between the single and established varieties were mainly in Redox metabolism, energy, and metabolism. Photosynthesis was only found negatively affected in the single variety. The defense components, although present in both varieties, show a more intense presence in staked pathways, that demonstrated pathways related to up-regulated secondary metabolites biosynthesis, a known response when plants are under various stress conditions. RT-PCR results confirmed that native EPSPS expression was up-regulated at the same level for single and stacked events. However, metabolic differences in expression were observed, suggesting a distinct cascade effect between simple and stacked, triggered by glyphosate application. Conclusion Changes in plant metabolism by glyphosate application have been observed in several pathways, particularly the shiquimate pathway, suggesting that event staking may promote a more intense defensive genetic response. Omics profiling techniques, such as transcriptome, can be considered tools to support risk assessment based on detecting unwanted effects, both on plant physiological changes and on the safety of foods and products from new genetic editing technologies. Background The combination of different traits or genes in genetically modified (GM) plants has rapidly emerged in worldwide crop production. In recent years, an increasing number of GM plants with stacked traits 3 reached about 81 million hectares equivalent to 42% of the total 191.7 million hectares planted with transgenic crops worldwide in 2018 [1]. The predominant trait, for both single and stacked crop varieties is herbicide resistance and it is estimated to remain so in the near future [2]. According to the current regulatory practice within the European Union (EU), stacked events are considered new GM organisms, requiring similar risk assessment procedures to those from single events [3] . Whereas in other countries, such as Brazil, stacked events are also considered new GMOs but require simplified risk assessments upon approval of single parental events (CTNBio, 2009). Previous studies have shown that stacking herbicide and insect-resistant transgenes can result in fitness costs that are dependent on the type and strength of selection pressure, and could also contribute to changes in plant communities through hitchhiking of unselected traits [4] . In that particular study, one of the tested selective pressure was the spray of glyphosate, which has been shown to adversely affect plant uptake and transport of micronutrients (e.g. Mn, Fe, Cu, and Zn) whose undersupply can reduce disease resistance and plant growth [5] [6]. Glyphosate manufacturers claim that it works by inhibiting the enzyme 5-enolpyruvylshikimate-3phosphate synthase (EPSPS), which catalyzes the penultimate step of the shikimate pathway leading the conversion of shikimic acid to chorismate, the precursor for aromatic amino acids (tyrosine, phenylalanine, and tryptophan) and other secondary plant metabolites. Glyphosate competes with phosphoenolpyruvate (PEP), a substrate for the EPSPS enzyme, to form a very stable enzyme −herbicide complex that inhibits the product-formation reaction [7] . Despite glyphosate widespread use in global crop production, its precise mode(s)-of-action and cascade metabolic effects in plants remain unclear. After inhibition of EPSPS, many physiological processes were observed to be affected by glyphosate and these could also be associated with glyphosate toxicity [8] It has been also shown that carbon and nitrogen metabolism are affected within hours after glyphosate treatment (Gruys and Sikorski, 1999) i.e., total free amino acid content increases, soluble protein content decreases [9] and carbohydrate content accumulates [10]. Application of transcriptomic and proteomic methods have helped to identify the common causes/mechanisms of the effect of glyphosate on the metabolism of resistant plants. The