Metabolomics should be deployed in the identification and characterization of gene‐edited crops

Gene‐editing techniques are currently revolutionizing biology, allowing far greater precision than previous mutagenic and transgenic approaches. They are becoming applicable to a wide range of plant species and biological processes. Gene editing can rapidly improve a range of crop traits, including disease resistance, abiotic stress tolerance, yield, nutritional quality and additional consumer traits. Unlike transgenic approaches, however, it is not facile to forensically detect gene‐editing events at the molecular level, as no foreign DNA exists in the elite line. These limitations in molecular detection approaches are likely to focus more attention on the products generated from the technology than on the process in itself. Rapid advances in sequencing and genome assembly increasingly facilitate genome sequencing as a means of characterizing new varieties generated by gene‐editing techniques. Nevertheless, subtle edits such as single base changes or small deletions may be difficult to distinguish from normal variation within a genotype. Given these emerging scenarios, downstream ‘omics’ technologies reflective of edited affects, such as metabolomics, need to be used in a more prominent manner to fully assess compositional changes in novel foodstuffs. To achieve this goal, metabolomics or ‘non‐targeted metabolite analysis’ needs to make significant advances to deliver greater representation across the metabolome. With the emergence of new edited crop varieties, we advocate: (i) concerted efforts in the advancement of ‘omics’ technologies, such as metabolomics, and (ii) an effort to redress the use of the technology in the regulatory assessment for metabolically engineered biotech crops.     

Opportunities and surprises in crops modified by transgenic technology: metabolic engineering of benzylisoquinoline alkaloid, gossypol and lysine biosynthetic pathways

The development of new or improved traits in plants, whether that is through traditional genetic modification and selection or through transgenic technologies, is associated with the potential risk of unintended changes with harmful or unacceptable consequences. The greater definition and precision of transgenic modification and the regulatory oversight of such technology may, however, confer advantages in safety and efficacy. This bears considerable relevance to the use of transgenic-based metabolic engineering in agricultural trait development. Metabolic engineering seeks to modify the amounts or chemical structures within selected biosynthetic routes without introducing inadvertent effects on other metabolic pathways. Examples discussed here include attempts to; (i) modify benzylisoquinoline alkaloid biosynthesis in poppy, (ii) improve the nutritional value of maize by increasing levels of free lysine, and (iii) increase the nutritional value of cottonseed by eliminating gossypol production. Clearly, evaluation of the efficacy (and unintended consequences) of such approaches is vital. A role for metabolomics in the compositional and metabolite analyses of new plant varieties derived from transgenic-based metabolic engineering is discussed. Major themes discussed in this review include; (i) the heightened level of scrutiny associated with genetically modified (GM) crop evaluations has markedly contributed to the safety in the adoption of transgenic technology, and (ii) the nature of any introduced trait may prove more relevant to safety assessments than the means by which the trait is introduced.     

The impact of biomass crop cultivation on temperate biodiversity

The urgency for mitigation actions in response to climate change has stimulated policy makers to encourage the rapid expansion of bioenergy, resulting in major land‐use changes over short timescales. Despite the potential impacts on biodiversity and the environment, scientific concerns about large‐scale bioenergy production have only recently been given adequate attention. Environmental standards or legislative provisions in the majority of countries are still lagging behind the rapid development of energy crops. Ranging from the field to the regional scale, this review (i) summarizes the current knowledge about the impact of biomass crops on biodiversity in temperate regions, (ii) identifies knowledge gaps and (iii) drafts guidelines for a sustainable biomass crop production with respect to biodiversity conservation. The majority of studies report positive effects on biodiversity at the field scale but impacts strongly depend on the management, age, size and heterogeneity of the biomass plantations. At the regional scale, significant uncertainties exist and there is a major concern that extensive commercial production could have negative effects on biodiversity, in particular in areas of high nature‐conservation value. However, integration of biomass crops into agricultural landscapes could stimulate rural economy, thus counteracting negative impacts of farm abandonment or supporting restoration of degraded land, resulting in improved biodiversity values. Given the extent of landconversion necessary to reach the bioenergy targets, the spatial layout and distribution of biomass plantations will determine impacts. To ensure sustainable biomass crop production, biodiversity would therefore have to become an essential part of risk assessment measures in all those countries which have not yet committed to making it an obligatory part of strategic landscape planning. Integrated environmental and economic research is necessary to formulate standards that help support long‐term economic and ecological sustainability of biomass production and avoid costly mistakes in our attempts to mitigate climate change.     

Current progress in computational metabolomics

Being a relatively new addition to the 'omics' field, metabolomics is still evolving its own computational infrastructure and assessing its own computational needs. Due to its strong emphasis on chemical information and because of the importance of linking that chemical data to biological consequences, metabolomics must combine elements of traditional bioinformatics with traditional cheminformatics. This is a significant challenge as these two fields have evolved quite separately and require very different computational tools and skill sets. This review is intended to familiarize readers with the field of metabolomics and to outline the needs, the challenges and the recent progress being made in four areas of computational metabolomics: (i) metabolomics databases; (ii) metabolomics LIMS; (iii) spectral analysis tools for metabolomics and (iv) metabolic modeling.     

Tracking genes from seed to supermarket: techniques and trends

Analytical techniques to track plant genes in the environment and the food chain are essential for environmental risk assessment, government regulation and production and trade of genetically modified (GM) crops. Here, I review laboratory techniques to track plant genes during pre-commercialization research on gene flow and post-commercialization detection, identification and quantification of GM crops from seed to supermarket. At present, DNA- and protein-based assays support both activities but the demand for fast, inexpensive, sensitive methods is increasing. Part of the demand has been generated by stringent food labeling and traceability regulations for GM crops. The increase in GM crops, changes in GM crop design, evolution of government regulations and adoption of risk-assessment frameworks will continue to drive development of analytical techniques.     

A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using quantitative nuclear magnetic resonance

Absolute analyte quantification by nuclear magnetic resonance (NMR) spectroscopy is rarely pursued in metabolomics, even though this would allow researchers to compare results obtained using different techniques. Here we report on a new protocol that permits, after pH-controlled serum protein removal, the sensitive quantification (limit of detection [LOD] = 5−25 μM) of hydrophilic nutrients and metabolites in the extracellular medium of cells in cultures. The method does not require the use of databases and uses PULCON (pulse length-based concentration determination) quantitative NMR to obtain results that are significantly more accurate and reproducible than those obtained by CPMG (Carr–Purcell–Meiboom–Gill) sequence or post-processing filtering approaches. Three practical applications of the method highlight its flexibility under different cell culture conditions. We identified and quantified (i) metabolic differences between genetically engineered human cell lines, (ii) alterations in cellular metabolism induced by differentiation of mouse myoblasts into myotubes, and (iii) metabolic changes caused by activation of neurotransmitter receptors in mouse myoblasts. Thus, the new protocol offers an easily implementable, efficient, and versatile tool for the investigation of cellular metabolism and signal transduction.     

Anticipating potential biodiversity conflicts for future biofuel crops in South Africa: incorporating spatial filters with species distribution models

Liquid biofuel production will likely have its greatest impact through the large‐scale changes in land use that will be required to meet the production of this energy source. In this study, we develop a framework which integrates species distribution models, land cover, land capability and various biodiversity conservation data to identify natural areas with (i) a potentially high risk of transformation for biofuel production and (ii) potential impact to biodiversity conservation areas. The framework was tested in the Eastern Cape of South Africa, a region which has been earmarked for the cultivation of biofuels. We expressly highlight the importance of biodiversity conservation data that enhance the protected area network to limit potential losses by comparing the overlap of areas likely to become cultivated with (i) protected areas; (ii) biodiversity hot spots not currently protected; and (iii) ‘ecological corridors’ (areas deemed important for the migration of species and linkages between important biodiversity areas). Results indicate that the introduction of spatial filters reduced available land from 54% to 45%. Including all biodiversity scenarios reduced available land to 15% of the Eastern Cape should avoiding conflict with biodiversity conservation areas be prioritized. The assumption that agriculturally marginal land offers a unique opportunity to be converted to biofuel crops does not consider the biodiversity value attached to these areas. We highlight that decisions relating to large‐scale transformation and changes in land cover need to take account of broader ecological processes. Determining the spatial extent of threats to biodiversity facilitates the analysis of spatial conflict. This article demonstrates a proactive approach for anticipating likely habitat transformation and provides an objective means of mitigating potential conflict with existing land use and biodiversity.     

Compositional differences between near‐isogenic GM and conventional maize hybrids are associated with backcrossing practices in conventional breeding

Here, we show that differences between genetically modified (GM) and non‐GM comparators cannot be attributed unequivocally to the GM trait, but arise because of minor genomic differences in near‐isogenic lines. Specifically, this study contrasted the effect of three GM traits (drought tolerance, MON 87460; herbicide resistance, NK603; insect protection, MON 89034) on maize grain composition relative to the effects of residual genetic variation from backcrossing. Important features of the study included (i) marker‐assisted backcrossing to generate genetically similar inbred variants for each GM line, (ii) high‐resolution genotyping to evaluate the genetic similarity of GM lines to the corresponding recurrent parents and (iii) introgression of the different GM traits separately into a wide range of genetically distinct conventional inbred lines. The F1 hybrids of all lines were grown concurrently at three replicated field sites in the United States during the 2012 growing season, and harvested grain was subjected to compositional analysis. Proximates (protein, starch and oil), amino acids, fatty acids, tocopherols and minerals were measured. The number of statistically significant differences (α = 0.05), as well as magnitudes of difference, in mean levels of these components between corresponding GM variants was essentially identical to that between GM and non‐GM controls. The largest sources of compositional variation were the genetic background of the different conventional inbred lines (males and females) used to generate the maize hybrids and location. The lack of any compositional effect attributable to GM suggests the development of modern agricultural biotechnology has been accompanied by a lack of any safety or nutritional concerns.     

Current knowledge of gene flow in plants: implications for transgene flow

Plant evolutionary biologists' view of gene flow and hybridization has undergone a revolution. Twenty-five years ago, both were considered rare and largely inconsequential. Now gene flow and hybridization are known to be idiosyncratic, varying with the specific populations involved. Gene flow typically occurs at evolutionarily significant rates and at significant distances. Spontaneous hybridization occasionally has important applied consequences, such as stimulating the evolution of more aggressive invasives and increasing the extinction risk for rare species. The same problems have occurred for spontaneous hybridization between crops and their wild relatives. These new data have implications for transgenic crops: (i) for most crops, gene flow can act to introduce engineered genes into wild populations; (ii) depending on the specific engineered gene(s) and populations involved, gene flow may have the same negative impacts as those observed for traditionally improved crops; (iii) gene flow's idiosyncratic nature may frustrate management and monitoring attempts; and (iv) intercrop transgene flow, although rarely discussed, is equally worthy of study.     

Observation conflict resolution in steady-state metabolic network dynamics analysis

Steady state metabolic network dynamics analysis (SMDA) is a recently proposed computational metabolomics tool that (i) captures a metabolic network and its rules via a metabolic network database, (ii) mimics the reasoning of a biochemist, given a set of metabolic observations, and (iii) locates efficiently all possible metabolic activation/inactivation (flux) alternatives. However, a number of factors may cause the SMDA algorithm to eliminate feasible flux scenarios. These factors include (i) inherent error margins in observations (measurements), (ii) lack of knowledge to classify measurements as normal versus abnormal, and (iii) choosing a highly constrained metabolic subnetwork to query against. In this work, we first present and formalize these obstacles. Then, we propose techniques to eliminate them and present an experimental evaluation of our proposed techniques.     

Land Use Implications of Increased Biomass Production Identified by GIS-Based Suitability and Yield Mapping for Miscanthus in England

The need for climate change mitigation and to meet increasing energy demands has led to a rise in the land area under bioenergy crops in many countries. There are concerns that such large-scale land conversion will conflict with food production and impact on the environment. Perennial biomass crops could be grown on more marginal agricultural land. However, for sustainable solutions, biomass yields will need to be sufficient and the wider implications of land-use changes considered. Here, focusing on Miscanthus in England as an example, we combined an empirical model with GIS to produce a yield map and estimated regional energy generation potentials after masking out areas covered by environmental and socio-economic factors which could preclude the planting of energy crops. Agricultural land quality and the distributions of currently grown food crops were then taken into account. Results showed that: (i) regional contrasts occur in the importance of different factors affecting biomass planting; (ii) areas with the highest biomass yields co-locate with food producing areas on high grade land, and; (iii) when such high grade land and unsuitable areas are excluded, a policy-related scenario for increased planting on 350,000 ha utilised 4–28% (depending on the region) of lower grade land and would not necessarily greatly impact on UK food security. We conclude that the GIS-based yield and suitability mapping described here can help identify important issues in bioenergy generation potentials and land use implications at regional or finer spatial scales that would be missed in analyses at the national level.     

组学技术评价转基因农作物的非预期效应

安全性评价是转基因农作物商品化应用的必要环节。组学技术能在转录物、蛋白质、代谢物水平上对转基因农作物进行无偏倚的安全性评价。文章综述了近10年来应用转录组学、蛋白质组学和代谢物组学技术评价转基因农作物非预期效应的研究进展, 结果表明在转基因农作物非预期变异中, 环境因素(种植地点和季节)和基因型差异比转基因本身的影响更大。     

Protection goals in environmental risk assessment: a practical approach

Policy protection goals are set up in most countries to minimise harm to the environment, humans and animals caused by human activities. Decisions on whether to approve new agricultural products, like pesticides or genetically modified (GM) crops, take into account these policy protection goals. To support decision-making, applications for approval of commercial uses of GM crops usually comprise an environmental risk assessment (ERA). These risk assessments are analytical tools, based on science, that follow a conceptual model that includes a problem formulation step where policy protection goals are considered. However, in most countries, risk assessors face major problems in that policy protection goals set in the legislation are stated in very broad terms and are too ambiguous to be directly applicable in ERAs. This means that risk assessors often have to interpret policy protection goals without clear guidance on what effects would be considered harmful. In this paper we propose a practical approach that may help risk assessors to translate policy protection goals into unambiguous (i.e., operational) protection goals and to establish relevant assessment endpoints and risk hypotheses that can be used in ERAs. Examples are provided to show how this approach can be applied to two areas of environmental concern relevant to the ERAs of GM crops.     

Metabolomics in pesticide research and development: review and future perspectives

The emerge of metabolomics within functional genomics has provided a new dimension in the study of biological systems. In regards to the study of agroecosystems, metabolomics enables monitoring of metabolic changes in association with biotic or abiotic agents such as agrochemicals. Focusing on crop protection chemicals, a great effort has been given towards the development of crop protection agents safer for consumers and the environment and more efficient than the existing ones. Within this framework, metabolomics has so far been a valuable tool for high-throughput screening of bioactive substances in order to discover those with high selectivity, unique modes-of-action, and acceptable eco-toxicological/toxicological profiles. Here, applications of metabolomics in the investigation of the modes-of-action and ecotoxicological–toxicological risk assessment of bioactive compounds, mining of biological systems for the discovery of bioactive metabolites, and the risk assessment of genetic modified crops are discussed.     

Metabolic adaptations of Leishmania donovani in relation to differentiation,drug resistance,and drug pressure

Antimonial (sodium stibogluconate, SSG) resistance and differentiation have been shown to be closely linked in Leishmania donovani, with SSG‐resistant strains showing an increased capacity to generate infectious (metacyclic) forms. This is the first untargeted LC‐MS metabolomics study which integrated both phenomena in one experimental design and provided insights into metabolic differences between three clinical L. donovani strains with a similar genetic background but different SSG‐susceptibilities. We performed this analysis at different stages during promastigote growth and in the absence or presence of drug pressure. When comparing SSG‐resistant and SSG‐sensitive strains, a number of metabolic changes appeared to be constitutively present in all growth stages, pointing towards a clear link with SSG‐resistance, whereas most metabolic changes were only detected in the stationary stage. These changes reflect the close intertwinement between SSG‐resistance and an increased metacyclogenesis in resistant parasites. The metabolic changes suggest that SSG‐resistant parasites have (i) an increased capacity for protection against oxidative stress; (ii) a higher fluidity of the plasma membrane; and (iii) a metabolic survival kit to better endure infection. These changes were even more pronounced in a resistant strain kept under Sb^III drug pressure.     

Large‐scale,long‐term trends in British river macroinvertebrates

Rivers are among the world's most modified ecosystems, with poor water quality representing a prominent problem for over 200 years, especially in urban areas. In Western Europe, however, industrial decline, tighter regulation and improved wastewater treatment have combined over recent decades to create conditions conducive to extensive restoration and positive biological change. Here, we evaluate the river macroinvertebrate fauna of England and Wales in relation to water quality, physical habitat and climate over almost two decades. We predicted that biological recovery would be characterized by: (i) greater taxon richness and prevalence of pollution‐sensitive taxa, (ii) larger changes in more heavily urbanized catchments, and (iii) temporal trends in assemblage structure that correlated with improving water quality. Family level richness increased on average by nearly 20% during 1991–2008, accompanied by a widespread shift towards taxa characteristic of well‐oxygenated and less polluted waters. Changes were largest in the most urbanized catchments. A combination of natural gradients and anthropogenic pressures explained the variation among sites, whereas temporal changes correlated with improving water quality and variations in discharge. Positive trends were not universal, however, and there was localized deterioration in some streams draining upland areas and in the lowland south east. Our results are consistent with a large‐scale ecological recovery of English and Welsh rivers since 1990, probably continuing a trend from the mid‐20th century. Based on these results, we suggest: (i) freshwater communities are resilient to long‐term anthropogenic pressures, (ii) biodiversity benefits can arise from investment and long‐term restoration intended largely to enhance ecosystem services such as drinking water and sanitary concerns, and (iii) long‐term monitoring data collected for statutory purposes–based in this case on nearly 50 000 samples–can address scientific questions at spatial and temporal extents seldom achieved in research programmes.     

Safety assessment of genetically modified plants with deliberately altered composition

The development and marketing of ‘novel’ genetically modified (GM) crops in which composition has been deliberately altered poses a challenge to the European Union (EU)'s risk assessment processes, which are based on the concept of substantial equivalence with a non‐GM comparator. This article gives some examples of these novel GM crops and summarizes the conclusions of a report that was commissioned by the European Food Safety Authority on how the EU's risk assessment processes could be adapted to enable their safety to be assessed.     

Risk assessment of gene flow from genetically engineered virus resistant cassava to wild relatives in Africa: an expert panel report

The probability and consequences of gene flow to wild relatives is typically considered in the environmental risk assessment of genetically engineered crops. This is a report from a discussion by a group of experts who used a problem formulation approach to consider existing information for risk assessment of gene flow from cassava (Manihot esculenta) genetically engineered for virus resistance to the ‘wild’ (naturalized) relative M. glaziovii in East Africa. Two environmental harms were considered in this case: (1) loss of genetic diversity in the germplasm pool, and (2) loss of valued species, ecosystem resources, or crop yield and quality due to weediness or invasiveness of wild relatives. Based on existing information, it was concluded that gene flow will occur, but it is not likely that this will reduce the genetic diversity in the germplasm pool. There is little existing information about the impact of the virus in natural populations that could be used to inform a prediction about whether virus resistance would lead to an increase in reproduction or survival, hence abundance of M. glaziovii. However, an increase in the abundance of M. glaziovii should be manageable, and would not necessarily lead to the identified environmental harms.