Default Activation and Nuclear Translocation of the Plant Cellular Energy Sensor SnRK1 Regulate Metabolic Stress Responses and Development.

Energy homeostasis is vital to all living organisms. In eukaryotes, this process is controlled by fuel gauging protein kinases: AMP-activated kinase in mammals, Sucrose Non-Fermenting1 (SNF1) in yeast (Saccharomyces cerevisiae), and SNF1-related kinase1 (SnRK1) in plants. These kinases are highly conserved in structure and function and (according to this paradigm) operate as heterotrimeric complexes of catalytic-α and regulatory ß- and γ-subunits, responding to low cellular nucleotide charge. Here, we determined that the Arabidopsis (Arabidopsis thaliana) SnRK1 catalytic α-subunit has regulatory subunit-independent activity, which is consistent with default activation (and thus controlled repression), a strategy more generally used by plants. Low energy stress (caused by darkness, inhibited photosynthesis, or hypoxia) also triggers SnRK1α nuclear translocation, thereby controlling induced but not repressed target gene expression to replenish cellular energy for plant survival. The myristoylated and membrane-associated regulatory ß-subunits restrict nuclear localization and inhibit target gene induction. Transgenic plants with forced SnRK1α-subunit localization consistently were affected in metabolic stress responses, but their analysis also revealed key roles for nuclear SnRK1 in leaf and root growth and development. Our findings suggest that plants have modified the ancient, highly conserved eukaryotic energy sensor to better fit their unique lifestyle and to more effectively cope with changing environmental conditions.

The hybrid four-CBS-domain KINßγ subunit functions as the canonical γ subunit of the plant energy sensor SnRK1.

The AMPK/SNF1/SnRK1 protein kinases are a family of ancient and highly conserved eukaryotic energy sensors that function as heterotrimeric complexes. These typically comprise catalytic α subunits and regulatory ß and γ subunits, the latter function as the energy-sensing modules of animal AMPK through adenosine nucleotide binding. The ability to monitor accurately and adapt to changing environmental conditions and energy supply is essential for optimal plant growth and survival, but mechanistic insight in the plant SnRK1 function is still limited. In addition to a family of γ-like proteins, plants also encode a hybrid ßγ protein that combines the Four-Cystathionine ß-synthase (CBS)-domain (FCD) structure in γ subunits with a glycogen-binding domain (GBD), typically found in ß subunits. We used integrated functional analyses by ectopic SnRK1 complex reconstitution, yeast mutant complementation, in-depth phylogenetic reconstruction, and a seedling starvation assay to show that only the hybrid KINßγ protein that recruited the GBD around the emergence of the green chloroplast-containing plants, acts as the canonical γ subunit required for heterotrimeric complex formation. Mutagenesis and truncation analysis further show that complex interaction in plant cells and γ subunit function in yeast depend on both a highly conserved FCD and a pre-CBS domain, but not the GBD. In addition to novel insight into canonical AMPK/SNF/SnRK1 γ subunit function, regulation and evolution, we provide a new classification of plant FCD genes as a convenient and reliable tool to predict regulatory partners for the SnRK1 energy sensor and novel FCD gene functions.

RNAi-based GM plants: food for thought for risk assessors.

RNA interference (RNAi) is an emerging technology that offers new opportunities for the generation of new traits in genetically modified (GM) plants. Potential risks associated with RNAi-based GM plants and issues specific to their risk assessment were discussed during an international scientific workshop (June 2014) organized by the European Food Safety Authority (EFSA). Selected key outcomes of the workshop are reported here.

Extensive expression regulation and lack of heterologous enzymatic activity of the Class II trehalose metabolism proteins from Arabidopsis thaliana.

Trehalose metabolism has profound effects on plant growth and metabolism, but the mechanisms involved are unclear. In Arabidopsis, 21 putative trehalose biosynthesis genes are classified in three subfamilies based on their similarity with yeast TPS1 (encoding a trehalose-6-phosphate synthase, TPS) or TPS2 (encoding a trehalose-6-phosphate phosphatase, TPP). Although TPS1 (Class I) and TPPA and TPPB (Class III) proteins have established TPS and TPP activity, respectively, the function of the Class II proteins (AtTPS5-AtTPS11) remains elusive. A complete set of promoter-beta-glucurinidase/green fluorescent protein reporters demonstrates their remarkably differential tissue-specific expression and responsiveness to carbon availability and hormones. Heterologous expression in yeast furthermore suggests that none of the encoded enzymes displays significant TPS or TPP activity, consistent with a regulatory rather than metabolic function for this remarkable class of proteins.

Plant development: introducing trehalose metabolism.

Trehalose metabolism, a short side-branch of primary carbon metabolism that is controlled by a surprisingly large gene family, is emerging as an important new regulatory pathway in plants. Two recent studies by Namiko Satoh-Nagasawa et al. and Leonardo Gomez et al. have highlighted its novel and possibly pivotal role in coordinating carbon supply with plant growth and development.

Assessment of genetically modified maize MON 89034 × 1507 × NK603 × DAS-40278-9 and subcombinations independently of their origin for food and feed uses, import and processing, under Regulation (EC) No 1829-2003 (application EFSA-GMO-NL-2013-112).

Maize MON 89034 × 1507 × NK603 × DAS-40278-9 (four-event stack maize) was produced by conventional crossing to combine four single events: MON 89034, 1507, NK603 and DAS-40278-9. The GMO Panel previously assessed the four single events and four of their subcombinations and did not identify safety concerns. No new data on the maize single events or their four subcombinations that could lead to modification of the original conclusions on their safety have been identified. The molecular characterisation, comparative analysis (agronomic, phenotypic and compositional characteristics) and the outcome of the toxicological, allergenicity and nutritional assessment indicates that the combination of the single maize events and of the newly expressed proteins in the four-event stack maize does not give rise to food/feed safety and nutritional concerns. The GMO Panel concludes that the four-event stack maize, as described in this application, is as safe as and nutritionally equivalent to its non-GM comparator and the non-GM reference varieties tested. In the case of accidental release of viable grains of the four-event stack maize into the environment, this would not raise environmental safety concerns. The GMO Panel assessed the likelihood of interactions among the single events in the six maize subcombinations for which no experimental data were provided, and concludes that these are expected to be as safe as and nutritionally equivalent to the single events, the previously assessed subcombinations and the four-event stack maize. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of the four-event stack maize. No post-market monitoring for food/feed is necessary. The GMO Panel concludes that the four-event stack maize and its subcombinations are as safe as its non-GM comparator and the tested non-GM reference varieties with respect to potential effects on human and animal health and the environment.

Assessment of genetically modified maize MON 89034 × 1507 × MON 88017 × 59122 × DAS-40278-9 and subcombinations independently of their origin for food and feed uses, import and processing under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2013-113).

Maize MON 89034 × 1507 × MON 88017 × 59122 × DAS-40278-9 (five-event stack maize) was produced by conventional crossing to combine five single events: MON 89034, 1507, MON 88017, 59122 and DAS-40278-9. The GMO Panel previously assessed the 5 single maize events and 11 of their subcombinations and did not identify safety concerns. No new data on the single maize events or their 11 subcombinations that could modify the original conclusions on their safety were identified. The molecular characterisation, comparative analysis (agronomic, phenotypic and compositional characteristics) and the outcome of the toxicological, allergenicity and nutritional assessment indicates that the combination of the single maize events and of the newly expressed proteins in the five-event stack maize does not give rise to food and feed safety and nutritional concerns. The GMO Panel concludes that the five-event stack maize, as described in this application, is as safe as and nutritionally equivalent to its non-GM comparator and the non-GM reference varieties tested. In the case of accidental release of the five-event stack maize into the environment, this would not raise environmental safety concerns. The GMO Panel assessed the likelihood of interactions among the single events in the 14 maize subcombinations for which no experimental data were provided, and concludes that they are expected to be as safe as and nutritionally equivalent to the single events, the previously assessed subcombinations and the five-event stack maize. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of the five-event stack maize. No post-market monitoring of food/feed is considered necessary. The GMO Panel concludes that the five-event stack maize and its subcombinations are as safe as its non-GM comparator and the tested non-GM reference varieties with respect to potential effects on human and animal health and the environment.

Risk assessment of new sequencing information for genetically modified soybean BPS-CV127-9.

The GMO Panel has previously assessed genetically modified (GM) soybean BPS-CV127-9. This soybean was found to be as safe and nutritious as its conventional counterpart and commercial soybean varieties with respect to potential effects on human and animal health and the environment in the context of its intended uses. On 16 February 2018, European Commission requested EFSA to analyse new nucleic acid sequencing data and updated bioinformatics data for GM soybean BPS-CV127-9 and to indicate whether the previous conclusions of the GMO Panel on safety of GM soybean BPS-CV127-9 remain valid. The new sequencing data indicated a two nucleotide difference in the unannotated Arabidopsis genomic DNA sequence downstream of the 3' untranslated region of the ahasl (also referred to as csr1-2) gene as compared to the sequencing data originally provided. One of these nucleotide differences reported in the new nucleic acid sequencing data from GM soybean event BPS-CV127-9 was shown to be already present in the original plant material used for the risk assessment. However, for the other nucleotide difference reported, no evidence could be provided to differentiate between a sequencing error and point-mutation. With the exception of bioinformatics analyses, the studies performed for the risk assessment of the single event soybean BPS-CV127-9 remain valid. The new sequencing data and the bioinformatics analyses performed on the new sequence did not give rise to safety issues. Therefore, EFSA concludes that the original risk assessment of soybean BPS-CV127-9 remains valid.

Risk assessment of new sequencing information on genetically modified carnation FLO-40685-2.

The GMO Panel has previously assessed genetically modified (GM) carnation FLO-40685-2 and concluded that there is no scientific reason to consider that the import, distribution and retailing in the EU of carnation FLO-40685-2 cut flowers for ornamental use will cause any adverse effects on human health or the environment. On 7 November 2017, European Commission requested EFSA to analyse new nucleic acid sequencing data and updated bioinformatics data for carnation FLO-40685-2 and to indicate whether the conclusions of the GMO Panel on the previously assessed GM carnation FLO-40685-2 remain valid. The new sequencing data indicated an additional three base pairs compared to the sequencing data originally provided: one base pair addition to the polyA tail of each of the two inserted flavonoid 3',5'-hydroxylase elements and one base pair addition to the sequence of one of the two D8 promoters in locus 1. These sequence differences are located outside the coding sequence for the newly expressed proteins and the base pairs described as differences in the new nucleic acid sequencing data for carnation FLO-40685-2 were reported to have been already present in the original plant material used for the risk assessment. Thus, with the exception of bioinformatics analyses, the studies performed for the risk assessment of GM carnation FLO-40685-2 remain valid. The new sequencing data and the bioinformatics analyses performed on the new sequence did not give rise to safety issues. Therefore, EFSA concludes that the original risk assessment of carnation FLO-40685-2 remains valid.

Risk assessment of new sequencing information on genetically modified carnation FLO-40689-6.

The GMO Panel has previously assessed genetically modified (GM) carnation FLO-40689-6 and concluded that there is no scientific reason to consider that the import, distribution and retailing in the EU of carnation FLO-40689-6 cut flowers for ornamental use will cause any adverse effects on human health or the environment. On 27 October 2017, the European Commission requested EFSA to analyse new nucleic acid sequencing data and updated bioinformatics data for carnation FLO-40689-6 and to indicate whether the conclusions of the GMO Panel on the previously assessed GM carnation FLO-40689-6 remain valid. The new sequencing data indicated the correction of one nucleotide compared to the sequencing data originally provided. The new sequence was corrected by removal of one nucleotide from the polylinker region in locus 1. The removal of this base pair reported in the new nucleic acid sequencing data for carnation FLO-40689-6 has been already present in the original plant material used for the risk assessment. Thus, with the exception of bioinformatics analyses, the studies performed for the risk assessment of GM carnation FLO-40689-6 remain valid. The new sequencing data and the bioinformatic analyses performed on the new sequence, did not give rise to safety issues. Therefore, EFSA concludes that the original risk assessment of carnation FLO-40689-6 remains valid.

Assessment of genetically modified maize MON 87403 for food and feed uses, import and processing, under Regulation (EC) No 1829/2003 (application EFSA-GMO-BE-2015-125).

Maize MON 87403 was developed to increase ear biomass at early reproductive phase through the expression of a modified AtHB17 gene from Arabidopsis thaliana, encoding a plant transcription factor of the HD-Zip II family. The molecular characterisation data and bioinformatic analyses did not identify issues requiring assessment for food and feed safety. No statistically significant differences in the agronomic and phenotypic characteristics tested between maize MON 87403 and its conventional counterpart were identified. The compositional analysis of maize MON 87403 did not identify differences that require further assessment. The GMO Panel did not identify safety concerns regarding the toxicity and allergenicity of the AtHB17∆113 protein, as expressed in maize MON 87403. The nutritional value of food and feed derived from maize MON 87403 is not expected to differ from that of food and feed derived from non-genetically modified (GM) maize varieties. Based on the outcome of the studies considered in the comparative analysis and molecular characterisation, the GMO Panel concludes that maize MON 87403 is as safe and nutritious as the conventional counterpart and the non-GM maize reference varieties tested. In the case of accidental release of viable maize MON 87403 grains into the environment, maize MON 87403 would not raise environmental safety concerns. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of maize MON 87403. In conclusion, the GMO Panel considers that maize MON 87403, as described in this application, is as safe as its conventional counterpart and the tested non-GM maize reference varieties with respect to potential effects on human and animal health and the environment.

Technical Note on the quality of DNA sequencing for the molecular characterisation of genetically modified plants.

As part of the risk assessment (RA) requirements for genetically modified (GM) plants, according to Regulation (EU) No 503/2013 and the EFSA guidance on the RA of food and feed from GM plants (EFSA GMO Panel, 2011), applicants need to perform a molecular characterisation of the DNA sequences inserted in the GM plant genome. The European Commission has mandated EFSA to develop a technical note to the applicants on, and checking of, the quality of the methodology, analysis and reporting covering complete sequencing of the insert and flanking regions, insertion site analysis of the GM event, and generational stability and integrity. This Technical Note puts together requirements and recommendations for when DNA sequencing is part of the molecular characterisation of GM plants, in particular for the characterisation of the inserted genetic material at each insertion site and flanking regions, the determination of the copy number of all detectable inserts, and the analysis of the genetic stability of the inserts, when addressed by Sanger sequencing or NGS. This document reflects the current knowledge in scientific-technical methods for generating and verifying, in a standardised manner, DNA sequencing data in the context of RA of GM plants. From 1 October 2018, this Technical Note will replace the JRC guideline of 2016 (updated April 2017) related to the verification and quality assessment of the sequencing of the insert(s) and flanking regions. It does not take into consideration the verification and validation of the detection method which remains under the remit of the JRC.

Assessment of genetically modified maize MON 87411 for food and feed uses, import and processing, under Regulation (EC) No 1829/2003 (application EFSA-GMO-NL-2015-124).

Maize MON 87411 was developed to confer resistance to corn rootworms (Diabrotica spp.) by the expression of a modified version of the Bacillus thuringiensis cry3Bb1 gene and a DvSnf7 dsRNA expression cassette, and tolerance to glyphosate-containing herbicides by the expression of a CP4 5-enolpyruvylshikimate-3-phosphate synthase (cp4 epsps) gene. The molecular characterisation data and bioinformatics analyses did not identify issues requiring assessment for food and feed safety. No statistically significant differences in the agronomic and phenotypic characteristics tested between maize MON 87411 and its conventional counterpart were identified. The compositional analysis of maize MON 87411 did not identify differences that required further assessment except for palmitic acid levels in grains from not treated maize MON 87411. The GMO Panel did not identify safety concerns regarding the toxicity and allergenicity of the Cry3Bb1 and CP4 EPSPS proteins, as expressed in maize MON 87411 and found no evidence that the genetic modification might significantly change the overall allergenicity of maize MON 87411. The nutritional impact of maize MON 87411-derived food and feed is expected to be the same as those derived from the conventional counterpart and non-GM commercial reference varieties. The GMO Panel concludes that maize MON 87411, as described in this application, is nutritionally equivalent to and as safe as the conventional counterpart and the non-GM maize reference varieties tested, and no post-market monitoring of food/feed is considered necessary. In the case of accidental release of viable maize MON 87411 grains into the environment, maize MON 87411 would not raise environmental safety concerns. The post-market environmental monitoring plan and reporting intervals are in line with the intended uses of maize MON 87411. The GMO Panel concludes that maize MON 87411, as described in this application, is as safe as its conventional counterpart and the tested non-GM maize reference varieties with respect to potential effects on human and animal health and the environment.

Chemical Intervention: New Tools to Dissect Metabolic Signaling.

The metabolic intermediate trehalose-6-P (T6P) has emerged as a key regulator of plant growth and development, but the underlying mechanisms remain largely elusive. A recent publication reported a new chemical intervention strategy, providing a powerful tool to dissect T6P-mediated metabolic signaling.

Risk assessment of new sequencing information on genetically modified soybean event 40-3-2.

The GMO Panel has previously assessed genetically modified (GM) soybean 40-3-2 as a single event and as part of a two-event stack, 305423 × 40-3-2. These soybean events were found to be as safe as their conventional counterparts and other appropriate comparators with respect to potential effects on human and animal health and the environment. On 4 April 2017, European Commission requested EFSA to analyse new nucleic acid sequencing and updated bioinformatics data for soybean event 40-3-2 and to indicate whether the conclusions of the GMO Panel on the previously assessed GM soybeans remain valid. The new sequencing data indicated that, the sequence of soybean event 40-3-2 as present in the stacked soybean 305423 × 40-3-2 contains an additional nucleotide in the 5' flanking region of a 72 bp additional insert of CP4 EPSPS present in soybean 40-3-2. Re-examination of the original sequencing data of the single soybean event 40-3-2 by the applicant, indicated that this additional nucleotide was already present in the original plant material used for the risk assessment of soybean event 40-3-2. Thus, with the exception of bioinformatics analyses, the studies performed for the risk assessment of the single event soybean 40-3-2 and the two-event stack soybean 305423 × 40-3-2 remain valid. The updated bioinformatic analyses performed on the corrected sequence did not give rise to safety issues. Therefore, EFSA concludes, based on the information provided, that the original risk assessment of the soybean event 40-3-2 as a single and the stacked soybean 305423 × 40-3-2 remains valid.

Predictive Protein Toxicity and Its Use in Risk Assessment.

In the EU novel proteins used in food or feed are assessed for their potential toxic effects in humans and livestock animals. The discovery of clear molecular features linked to the toxicity of a protein may be an important step towards the use of predictive protein toxicity in risk assessment.

Assessment of the potential integration of the DNA plasmid vaccine CLYNAV into the salmon genome.

The European Commission mandated EFSA to review a new data package provided by the company Elanco, for the possible integration/non-integration of the DNA plasmid vaccine CLYNAV into the genome of Atlantic salmon (Salmo salar) and to indicate whether EFSA agrees with the conclusions drawn by Elanco. The vaccine is injected into fish to confer protection against pancreas disease caused by the salmonid alphavirus. The majority of the experimental data provided by the company was for muscle tissue close to the injection site and for gonadal tissue. EFSA considers that the long persistence of DNA plasmid in muscle tissue close to the injection site and the potential heritability of an integration event in gonad cells support the focus of the assessment on both these tissues. The experimental data did not provide scientifically robust evidence for a true integration event. The company overall concluded that the likelihood of integration is negligible, based on considerations in the context of the company's environmental risk assessment, but did not provide a quantitative value for the rate of integration linked to the term 'negligible'. It is therefore not possible to evaluate this statement specifically with regard to integration rates. EFSA notes that knowledge about homologous and non-homologous integration predicts that integration could occur with certain frequency. Therefore, EFSA has constructed worst-case scenarios leading to upper estimates for possible integration rates of the DNA plasmid vaccine into the Atlantic salmon genome. EFSA concludes that, based on the worst-case scenarios described here and taking into account additional factors decreasing the likelihood of integration, the actual integration rate is likely to be orders of magnitude lower than the upper estimated integration rate calculated in the context of the worst-case scenarios. With the available evidence, the actual integration rate cannot be estimated with more precision.

Risk assessment of new sequencing information on genetically modified soybean event 305423.

The GMO Panel has previously assessed genetically modified (GM) soybean 305423 as a single event and as part of a two-event stack, 305423 × 40-3-2. These soybean events were found to be as safe as their conventional counterparts and other appropriate comparators with respect to potential effects on human and animal health and the environment. On 23 February 2017, European Commission requested EFSA to analyse new nucleic acid sequencing data and updated bioinformatics data for soybean event 305423 and to indicate whether the previous conclusions of the GMO Panel on the previously assessed GM soybeans remain valid. The new sequencing data indicated a four base pair (bp) difference compared to the sequencing data originally provided: one bp located in the genomic 3' flanking region, two bp located in a gene silencing cassette and one bp in a partial promoter. These bp reported as differences in the new nucleic acid sequencing data on soybean event 305423 were already present in the original plant material used for the risk assessment. Thus, with the exception of bioinformatics analyses, including an off-target search with the dsRNA expression cassette, the studies performed for the risk assessment of the single event soybean 305423 and the two-event stack soybean 305423 × 40-3-2 remain valid. The new sequencing data and the bioinformatic analyses performed on the new sequence including the RNAi off-target search, did not give rise to safety issues. Therefore, EFSA concludes that the original risk assessment of the single soybean event 305423 and the two-event stack soybean 305423 × 40-3-2 remains valid.

Scientific opinion on an application for renewal of authorisation for continued marketing of maize 1507 and derived food and feed submitted under Articles 11 and 23 of Regulation (EC) No 1829/2003 by Pioneer Overseas Corporation and Dow AgroSciences LLC.

Following the submission of application EFSA-GMO-RX-001 under Regulation (EC) No 1829/2003 from Pioneer Overseas Corporation and Dow Agrosciences LLC, the Panel on Genetically Modified Organisms of the European Food Safety Authority (GMO Panel) was asked to deliver a scientific risk assessment on the data submitted in the frame of a renewal of authorisation application of the insect-resistant and herbicide-tolerant genetically modified (GM) maize 1507. The data package received in the frame of this renewal application contained post-market environmental monitoring reports, a systematic search and evaluation of literature, an updated bioinformatics analysis and additional documents or studies performed by or on behalf of the applicant. The GMO Panel assessed this data package for possible new hazards, modified exposure or new scientific uncertainties identified during the authorisation period and not previously assessed in the frame of the original application. Under the assumption that the DNA sequence of the event in maize 1507 considered for renewal is identical to the corrected sequence of the originally assessed event, the GMO Panel concludes that no new hazards or modified exposure and no new scientific uncertainties were identified for the application for renewal that would change the conclusions of the original risk assessment on maize 1507 (EFSA, 2005b, 2009).

Assessment of genetically modified sugar beet H7-1 for renewal of authorisation under Regulation (EC) No 1829/2003 (application EFSA-GMO-RX-006).

Following the submission of application EFSA-GMO-RX-006 under Regulation (EC) No 1829/2003 from KWS SAAT SE and Monsanto Company, the Panel on Genetically Modified Organisms of the European Food Safety Authority (GMO Panel) was asked to deliver a scientific risk assessment on the data submitted in the context of the renewal of authorisation application for the herbicide-tolerant genetically modified sugar beet H7-1. The data received in the context of this renewal application contained a systematic search and evaluation of literature, updated bioinformatics analyses and additional documents or studies performed by or on behalf of the applicant. The GMO Panel assessed these data for possible new hazards, modified exposure or new scientific uncertainties identified during the authorisation period and not previously assessed in the context of the original application. Under the assumption that the DNA sequence of the event in sugar beet H7-1 considered for renewal is identical to the originally assessed event, the GMO Panel concludes that there is no evidence in the context of this renewal application for new hazards, modified exposure or scientific uncertainties that would change the conclusions of the original risk assessment on sugar beet H7-1.