THIRD WORLD NETWORK BIOSAFETY INFORMATION SERVICE
Dear Friends and Colleagues
Glyphosate-based Herbicides Cause Unintended Effects Even in Glyphosate-Tolerant Soy
Researchers from GenØk – Centre for Biosafety and the Federal University of Santa Catarina in Brazil, have identified that glyphosate-based herbicides can trigger unintended effects even in genetically modified (GM) varieties resistant to them (Item 1).
Glyphosate kills undesired plants by inhibiting the EPSPS synthase enzyme, thus blocking the synthesis of aromatic amino acids. This study compared the effect of glyphosate-based herbicide (GBH) spraying on single-transgene and stacked, herbicide-resistant soybean varieties on various biological processes, metabolic pathways, and key shikimic enzymes. The varieties tested were genetically engineered for glyphosate tolerance and widely grown by famers in Brazil.
The scientists used a molecular analytical method known as transcriptomics, which allows the total profile of gene expression of an organism to be determined. Their analysis revealed that GBH caused adverse effects on carbon metabolism and flow, redox metabolism, photosynthesis, plant defense responses as well as oxidative stress. Plants with two inserted transgenes showed a more intense stress response. Redox imbalance is known to severely damage the cell integrity and can negatively interfere with photosynthetic processes, leading to reduction in plant growth.
An earlier study had also found that a GM Roundup-tolerant maize (NK603) showed increased oxidative stress, which was believed to be caused by expression of the glyphosate-tolerance transgene. Both studies confirm not only that GM plants are not substantially equivalent to their non-GM counterparts, but also that they are especially vulnerable to environmental stresses. Thus, the use of GM crops can undermine the resilience of farming systems. (Item 2)
The researchers recommend that an assessment of the risks of the actual GMO to be released in the environment should consider combinatorial and cumulative effects derived from stacking transgenes into single organisms using omics profiling, which can also support risk assessment in detecting unintended effects due to the GBH application.
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Item 1
STACKED GENETICALLY MODIFIED SOYBEAN HARBORING HERBICIDE RESISTANCE AND INSECTICIDE RCRY1AC SHOWS STRONG DEFENSE AND REDOX HOMEOSTASIS DISTURBANCE AFTER GLYPHOSATE-BASED HERBICIDE APPLICATION
by Zanatta, C.B., Benevenuto, R.F., Nodari, R.O. et al.
Environ Sci Eur 32, 104 (2020)
25 July 2020
https://doi.org/10.1186/s12302-020-00379-6
https://enveurope.springeropen.com/articles/10.1186/s12302-020-00379-6
[EXCERPTS ONLY]
ABSTRACT
Background
World agricultural production of genetically modified (GM) products, in particular, the combination of different traits/genes in the same plant has been a trend over the last decade. There have been concerns raised over stacking multiple herbicide and insect-resistant transgenes that could result in fitness costs depending on the type and strength of selection pressures exerted by the environment. Here, we report the results of transcriptomic analysis comparing the effect of glyphosate-based herbicide (GBH) in the single-transgene versus stacked, herbicide-resistant soybean varieties on various biological processes, metabolic pathways, and key shikimic enzymes.
Results
Gene expression data showed that defense metabolism and redox homeostasis were equally modulated in single-transgene and stacked-variety samples. Carbon accumulation and energy metabolisms were distinct between the varieties and photosynthesis metabolism was found negatively affected in the single-transgene variety only. In the stacked variety, the shikimate pathway was modulated by the accumulation of transcripts from phenylalanine gene and other cascade genes. As expected, the expression of native EPSPS was upregulated in both varieties when herbicide was applied. On the other hand, transgenic EPSPS expression was down-regulated in both GM varieties upon herbicide application which cannot be explained.
Conclusion
Glyphosate-based herbicides toxicity suggests its effects on carbon central metabolism and flux, redox metabolism, photosynthesis, and to hormone and defense response in plants. The observed unintended effects in GM herbicide-tolerant varieties unravel the deleterious effects previously observed on GM-tolerant varieties growth and production. The impact of GBH on shikimate and cascade pathways was observed in terms of both native and transgenic insensitive EPSPS modulation, alteration of jasmonic acid and lignin metabolism in both single-transgene and stacked variety. The energy metabolism and carbon flux were differently affected in these varieties. Oxidative stress, more specifically glutathione metabolism, induced by GBH, was also observed in this study. The stacked variety showed a more pronounced stress response (activation of specific stress defense proteins, Rboh, WRKY) and secondary compounds (β-glucosidase, isoflavone 7-O-methyltransferase). Omics profiling techniques, such as transcriptomics, can be considered tools to support risk assessment in detecting unintended effects due to the GBH application.
RELEVANCE TO RISK ASSESSMENT OF STACKED GM CROPS
Worldwide, a growing number of GM crops with stacked transgenic traits are being developed to confer resistance to herbicide active ingredients and some insect species. For most varieties, the single-transgene events might never reach market and pre-market risk assessment. Therefore, an assessment of the risks of the actual GMO to be released in the environment should consider combinatorial and cumulative effects derived from stacking transgenes into single organism.
Omics profiling analysis can contribute to the identification of combinatorial effects that may occur due to interactions among the proteins and metabolites produced by the transgenes or endogenous genes of a stacked GM plant. In addition, interactions between the stacked transgenes or their products, or interactions among the physiological pathways in which the transgenes are involved, taking into account the possibility that these interactions could result in potentially harmful substances, such as anti-nutritional factors, some of which may persist or accumulate in the environment.
Stacked GM plants can be produced through different approaches. In addition to the cross-breeding of two GM plants, multiple traits can be also achieved by the natural cross of transgenic lines that have been found in crop field boundaries [96, 97], such as feral transgenic canola outside of cultivation [98, 99].
Accordingly, it is reasonable to anticipate future occurrence of stacked traits within ruderal and wild populations. Despite the potential for the formation of feral populations with multiple transgenes, we have little understanding of how these traits could migrate, evolve or influence native and naturalized plant communities. Thus, such profiling studies could generate useful information to assist risk assessment of stacked GM crops and potential feral populations.
Our study on stacked-transgene soybean variety showed GBH effects on shikimate genes, carbon metabolism and flux, photosynthesis, oxidative events and defense response. Whereas GBH effects in single-transgene plants have been reported [100,101,102] our data suggest that GBH affects stacked-transgene plants at a higher extend than its single-transgene near-isogenic comparator. In addition, GBH adverse effects in GM tolerant plants are widely variable depending on species and cultivar and herbicide regime. Therefore, it is intrinsically important to elucidate the GBH effects on physiological processes related to metabolic disturbances in order to better understand the glyphosate-herbicidal mechanism and its possible unintended effects on commercialized transgenic varieties.
CONCLUSIONS
It is well known that glyphosate kills undesired plants by inhibiting EPSPS synthase enzyme, thus blocking the synthesis of aromatic amino acids. However, glyphosate-based herbicides have shown to promote several indirect effects on plant physiology which may also explain its herbicidal effects. Glyphosate-based herbicides toxicity suggests its effects on carbon central metabolism and flux, redox metabolism, photosynthesis, and to the plant’s hormone and defense response. Most relevant, such unintended effects are also present in GM herbicide-tolerant varieties even when they do not lead to plant’s death. The alteration of these cellular processes unravels the deleterious effects previously observed on GM tolerant varieties growth and production. The impact of GBH on shikimate and cascade pathways was observed in terms of both native and transgenic insensitive EPSPS modulation, alteration of jasmonic acid and lignin metabolism in both single-transgene and stacked variety. Whereas the energy metabolism and carbon flux were differently affected in these varieties. In the stacked variety, trehalose levels were altered up to sevenfold increase. Oxidative stress, more specifically glutathione metabolism, induced by GBH, was observed in this study. Redox imbalance is known to severely damage the cell integrity and can negatively interfere with photosynthetic processes, for example by decreasing the chlorophyll content, photochemical efficiency, and C metabolism, leading to reduction in plant growth. We found Ca2+ signaling responses and several up-regulated molecular chaperones in both varieties. However, distinct stress responses were also observed. The stacked variety showed a more pronounced stress response (activation of specific stress defense proteins (Rboh, WRKY) and secondary compounds (β-glucosidase, isoflavone 7-O-methyltransferase). Unintended effects of GBH applications in GM tolerant varieties as well as the differences in the variety’s response show the relevance in elucidating the GBH effects on physiological processes as means to establish its safety.
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Item 2
GLYPHOSATE HERBICIDE HARMS EVEN GM GLYPHOSATE-TOLERANT SOYBEANS
Claire Robinson
GM Watch
25 August 2020
https://www.gmwatch.org/en/news/latest-news/19515
Scientists have found that spraying with glyphosate-based herbicide triggers unintended effects even in genetically modified soybean varieties resistant to this type of pesticide. They report their findings in a new study published in Environmental Sciences Europe.
The researchers, from GenØk–Centre for Biosafety in Norway and the Federal University of Santa Catarina (UFSC) in Brazil, found severe metabolic disturbances in both stacked (multiple transgene) and single transgene trait GM soybeans caused by exposure to a glyphosate-based herbicide.
The scientists used a molecular analytical method known as transcriptomics, which allows the total profile of gene expression of an organism to be determined. The authors of this new study used transcriptomics to investigate if there were alterations in gene expression patterns following spraying with a glyphosate-based herbicide (Roundup Transorb®) on the stacked-trait GM soybean Intacta Roundup Ready 2 Pro MON-877Ø1-2×MON-89788-1, engineered for glyphosate tolerance and to express a Bt toxin insecticide, and on the single-trait GM soybean Roundup Ready MON-Ø4Ø32-6, genetically engineered for glyphosate tolerance.
Both GM soybean varieties are approved in the EU for food and feed use but not for cultivation. They are widely grown by famers in Brazil.
The researchers looked at the gene expression levels of the stacked and single-trait soy varieties 8 hours after application of the dose of Roundup Transorb® legally permitted in Brazil. The results showed that spraying with the glyphosate-based herbicide caused adverse effects on carbon metabolism and flow, energy metabolism, and photosynthesis, as well as plant defence responses. Oxidative stress (an imbalance in the production of reactive oxygen-based molecules that can lead to DNA, RNA, cell and tissue damage) was found to be induced by herbicide application, as suggested by increased activity of the plants’ detoxification system via the important antioxidant molecule glutathione.
The stacked variety had a more intense response to the stress of herbicide application, in that more biochemical pathways were affected.
The results build on previous field evidence of GM soybeans’ increased susceptibility to other environmental stressors, including drought and diseases – a susceptibility that appears to be caused by the combination of the GM genes and the herbicide application. This previous evidence is detailed in the new paper.
Reflective of real-farm conditions
The authors of the new study say that their research, which was conducted in the controlled environment of a greenhouse, reflects real-farm conditions for GM soybean production.
Dr Sarah Agapito-Tenfen, a scientist from GenØk and the lead author of the new publication, added that the new study contains important lessons for improving risk assessment: “Since we have found differences between the stacked varieties (which combine two or more transgenes) and with the herbicide exposure, we recommend the regulatory agencies to include these same [molecular analysis] techniques that we have used in this study as a risk assessment criterion.”
The authors state in their paper that scientists need to look more closely at the effects of herbicide applications on genetically modified plants in order to ensure their safety for consumption and for the environment.
On the question of food safety, Dr Agapito-Tenfen told GMWatch, “Anti-nutrients found in plant species are usually associated with some kind of stress response to an abiotic [non-living] or biotic [living] stressor. Since we detected a disturbance in the biochemical pathways related to stress response, such as glutathione and redox [balance in oxygen production and utilisation] metabolism, we suggest that a new risk hypothesis should be drawn from the altered pathways. For instance, a quantitative investigation of anti-nutrients and the presence of new anti-nutrients should be performed.”
GM maize similarly affected
An earlier study, which includes Dr Agapito-Tenfen among its authors, similarly found that a GM Roundup-tolerant maize (NK603) showed increased oxidative stress, which was believed to be caused by expression of the glyphosate-tolerance transgene. The maize also showed altered glutathione metabolism when glyphosate-based herbicide was sprayed during cultivation. The authors commented that the increased levels of antioxidant enzymes in the GM plants were likely to be a response to oxidative stress “in order to maintain proper physiological function”.
For GMWatch, these studies show that the GM transformation process results in unpredictable and undesirable alterations in the function of many genes. The studies confirm not only that GM plants are not substantially equivalent to their non-GM counterparts, but also that they are especially vulnerable to environmental stresses. The first law of thermodynamics tells us that energy cannot be created or destroyed, but only changed from one form to another. Accordingly, the genetic manipulation of plants to confer certain traits such as herbicide tolerance redirects energy that the plant would normally use to adapt to environmental stresses. Thus the use of genetically manipulated crops can undermine the resilience of farming systems.