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

Corrigendum: An EU Perspective on Biosafety Considerations for Plants Developed by Genome Editing and Other New Genetic Modification Techniques (nGMs)

The question whether new genetic modification techniques (nGM) in plant development might result in non-negligible negative effects for the environment and/or health is significant for the discussion concerning their regulation. However, current knowledge to address this issue is limited for most nGMs, particularly for recently developed nGMs, like genome editing, and their newly emerging variations, e.g., base editing. This leads to uncertainties regarding the risk/safety-status of plants which are developed with a broad range of different nGMs, especially genome editing, and other nGMs such as cisgenesis, transgrafting, haploid induction or reverse breeding. A literature survey was conducted to identify plants developed by nGMs which are relevant for future agricultural use. Such nGM plants were analyzed for hazards associated either (i) with their developed traits and their use or (ii) with unintended changes resulting from the nGMs or other methods applied during breeding. Several traits are likely to become particularly relevant in the future for nGM plants, namely herbicide resistance (HR), resistance to different plant pathogens as well as modified composition, morphology, fitness (e.g., increased resistance to cold/frost, drought, or salinity) or modified reproductive characteristics. Some traits such as resistance to certain herbicides are already known from existing GM crops and their previous assessments identified issues of concern and/or risks, such as the development of herbicide resistant weeds. Other traits in nGM plants are novel; meaning they are not present in agricultural plants currently cultivated with a history of safe use, and their underlying physiological mechanisms are not yet sufficiently elucidated. Characteristics of some genome editing applications, e.g., the small extent of genomic sequence change and their higher targeting efficiency, i.e., precision, cannot be considered an indication of safety per se, especially in relation to novel traits created by such modifications. All nGMs considered here can result in unintended changes of different types and frequencies. However, the rapid development of nGM plants can compromise the detection and elimination of unintended effects. Thus, a case-specific premarket risk assessment should be conducted for nGM plants, including an appropriate molecular characterization to identify unintended changes and/or confirm the absence of unwanted transgenic sequences

An EU Perspective on Biosafety Considerations for Plants Developed by Genome Editing and Other New Genetic Modification Techniques (nGMs)

The question whether new genetic modification techniques (nGM) in plant development might result in non-negligible negative effects for the environment and/or health is significant for the discussion concerning their regulation. However, current knowledge to address this issue is limited for most nGMs, particularly for recently developed nGMs, like genome editing, and their newly emerging variations, e.g., base editing. This leads to uncertainties regarding the risk/safety-status of plants which are developed with a broad range of different nGMs, especially genome editing, and other nGMs such as cisgenesis, transgrafting, haploid induction or reverse breeding. A literature survey was conducted to identify plants developed by nGMs which are relevant for future agricultural use. Such nGM plants were analyzed for hazards associated either (i) with their developed traits and their use or (ii) with unintended changes resulting from the nGMs or other methods applied during breeding. Several traits are likely to become particularly relevant in the future for nGM plants, namely herbicide resistance (HR), resistance to different plant pathogens as well as modified composition, morphology, fitness (e.g., increased resistance to cold/frost, drought, or salinity) or modified reproductive characteristics. Some traits such as resistance to certain herbicides are already known from existing GM crops and their previous assessments identified issues of concern and/or risks, such as the development of herbicide resistant weeds. Other traits in nGM plants are novel; meaning they are not present in agricultural plants currently cultivated with a history of safe use, and their underlying physiological mechanisms are not yet sufficiently elucidated. Characteristics of some genome editing applications, e.g., the small extent of genomic sequence change and their higher targeting efficiency, i.e., precision, cannot be considered an indication of safety per se, especially in relation to novel traits created by such modifications. All nGMs considered here can result in unintended changes of different types and frequencies. However, the rapid development of nGM plants can compromise the detection and elimination of unintended effects. Thus, a case-specific premarket risk assessment should be conducted for nGM plants, including an appropriate molecular characterization to identify unintended changes and/or confirm the absence of unwanted transgenic sequences

Suberin biosynthesis in <em>O. sativa : </em>characterisation of a cytochrome P450 monooxygenase

In endodermal and exodermal cell walls of roots the polyester suberin forms a radial diffusion barrier for water, solutes and gases. Depolymerization of this otherwise insoluble lipophilic polymer releases its aliphatic monomers, which comprise derivates of very long chain carboxylic acids such as alcohols, fatty acids, ω-hydroxyacids and dicarboxylic acids, as well as the aromatic compounds ferulic and coumaric acid. Based on the chemical composition of suberin some key functions of its biosynthesis have been predicted and first genes could be characterized in the model plant Arabidopsis thaliana in the last decade. In the course of this work, a number of suberin candidate genes could be identified in the globally important crop and model species Oryza sativa based on in silico studies and the analysis of gene expression during physiological conditions promoting suberisation. A reverse genetic approach with focus on cytochrome P450 enzymes led to the discovery of a monooxygenase of very long chain fatty acids in roots. Plants carrying knock out mutations in CYP86B3 were lacking bifunctional aliphatic suberin monomers with carbon chain lengths C24 and higher. Additionally, an accumulation of putative precursors of the affected monomers, the acid methyl esters with corresponding chain lengths, was found in root total lipid extracts of cyp86b3-1. Though no visible phenotype of the mutation was determined after cultivation in hydroponic culture and under osmotic stresses, permeability of diffusion barriers was altered: transport experiments with a photosynthesis inhibitor revealed significantly faster uptake of this apoplastic tracer in roots of cyp86b3-1 impaired in the suberin composition. The transgenic expression of CYP86B3 in A. thaliana could restore the wild type level of ω-hydroxy acids with chain length C22 and C24 in root suberin of the heterologous knock out mutant ralph and ralph/horst

Shoot (A) and root (B) dry weight and root growth rate (C) of rice, maize and onion plants as affected by Si supply.

<p>Data are mean ± s.e., n = 4. Different letters indicate a significant difference between Si treatments of a species; t-test with p < 0.05.</p

Concentration of lignin and lignin-like polymers in the outer cell layers of the root comprising the exodermis and the sclerenchyma in rice, and the exodermis in maize and onion as affected by Si supply.

<p>Amounts were determined photometrically. Root zone B was at 4–6 cm drt in rice and onion and at 6–8 cm drt in maize roots. Data are mean ± s.e., n = 4. Different letters indicate a significant difference between Si treatments of a plant species; <i>t</i>-test with p < 0.05.</p

Distribution of Si in the root zone B of rice (A), maize (B) and onion (C).

<p>The values indicate the relative Si abundance relating to a maximum value of 1.0. Root zone B was at 4–6 cm drt in rice and onion and at 6–8 cm drt in maize roots. Data are mean ± s.e., n = 4. The black bar indicates 100 μm. EX, exodermis; SCL, sclerenchyma; EN, endodermis; CC, central cylinder. The formation of an aerenchyma was observed not only in rice but also in maize roots, which was reported before for well-aerated maize plants [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138555#pone.0138555.ref061" target="_blank">61</a>].</p

Development of Casparian bands (CB) in the exodermis of rice (A), maize (B) and onion (C) roots as affected by Si supply.

<p>Root zone where the formation of CB initiated was defined as root zone A, which was at 0–2 cm drt for rice and onion, and at 2–4 cm drt for maize. Root zone B started 2 cm behind root zone A. Exodermal CB were classified into stages I-IV according to 0, 0–25, 25–50 or > 50% of the length of the anticlinal cell wall with developed CB. n = 4. Different letters indicate significant difference between Si treatments and root sections of a species; cumulative link mixed models with p < 0.05.</p

Silicon concentration in shoot (A) and root (B) of rice, maize and onion plants as affected by Si supply.

<p>Data are mean ± s.e., n = 4. Different letters indicate a significant difference between Si treatments of a species; t-test with p < 0.05.</p