Market introduction of genetically modified (GM) food crops in Europe has given rise to broad public concern, which of a great deal is based on uncertainties related to safety for humans, animals and environment. One of the key issues in safety evaluation of GM plants is whether unexpected or unintended changes may have taken place in the organism due to genetic modification, that could affect its safety or nutritional status. The project was designed to answer the question: Does genetic modification induce (un-)intended effects in the Genetically Modified (GM) crop plant, that may affect levels of nutrients, natural toxins or other compounds if compared to the parental line? The answer addresses the issue of 'Substantial Equivalence' and therefore, the main thrust of the research programme targets on exploiting 'omics' techniques like genomics (DNA chip technology), proteomics (2D-PAGE/MALDI-TOF) and metabolomics (LC-NMR/GC-MS) for a 'holistic' compositional analysis of plant tissues. The overall objective is to adapt these new technologies to identify and/or eliminate potential hazardous metabolic perturbations in whole complex GM crop plants at the earliest step in the food chain.
The overall first year objective was to imply the 'omics' technologies, which should be of sufficient sensitivity and specificity, to assure a screen for identifying possible unintended effects (i.e. metabolic perturbations) in GM crop plants. Implicit in this objective was the development of new knowledge intended to understand the implications of genetic modification processes, such as the use of sense/antisense constructs, on metabolic pathways in crop plants. The 'omics' tools were tested through the evaluation of transgenic potato lines modified in, for example, starch, polyamine, glycoalkaloid and amino acid metabolism and transgenic tomato lines with modified carotenoids content. Other GM-lines like Arabidopsis and Nicotiana were used for academic and control purposes in order to determine what genes are involved in the process of flavonoid and lysine metabolism, growth and development.
The wealth of techniques and methods developed and evaluated during the first year of the project provided a firm basis for identification of possible hazardous metabolic perturbations in whole complex GM crop plants. In the first year crop plant production (i.e. glasshouse, polytunnel and field environments) has been arranged to supply sufficient transgenic plants for experimental usage in both 'targeted (i.e. selected critical nutrients and key natural toxins analysis)' as well as in 'non-targeted (i.e. non-specific broad-spectrum profiling analysis)' compositional studies. Transgenic plants have been selected as follows: 5 types of GM potato lines i.e. Mal1 (defective glycoprotein processing), SAM35S (polyamine metabolism), W2GBSS (starch composition), FK (modified sugar metabolism) and DHDPS (lysine content); 5 types of GM tomato lines i.e. 35S crtl (2-fold increase of b-carotene and lutein), crtB (increased carotenoids), anti-Psy (dramatically reduced carotenoids), beta-Lcy (2.5-fold increase of b-carotene) and HMGR (increased end-product phytosterols). The GM crop plants have been characterised on sizes of T-DNA, vector backbone integration, copy number and expression levels. These transgenic plants have been screened for phenotypes with metabolic and/or developmental disturbations, which would be of particular interest. For academic purposes Arabidopsis cv. Wassilewskija has been selected and characterised further i.e. one parental line, 3 CHS (modified flavonoid content) antisense (single copy) and 3 CHS (multi copy) transformants. The construction of Arabidopsis lines with antisense/sense constructs of DFR under control of an ethanol inducible promoter is under way. In the case of Nicotiana sylvestris 4 types of mutants i.e. RAEC-1 (mutated dhdps-r1), RLT-70 (mutated aklys 1), hybrid RLT-70/RAEC-1 and parental line have been chosen and studied in more detail.
Different methodologies as tools for a targeted analytical comparison (e.g. sugars, fatty acids, glycoalkaloids, caretonoids, amino acids, organic acids, aromatic compounds, flavonoids etc.) have been evaluated for the compositional analysis of the selected transgenic and parental plant material (e.g. potato, tomato, Arabidopsis and tobacco), which were bred under identical conditions. Technologically, these tools are operational and reproducibility tests including data analysis by chemometrics are under way.
Genomics: Technologically, the DNA array technology (i.e. genomics) is operational. With respect to the gene expression profiling experiments, two tomato cDNA libraries have been constructed and part of these libraries have been spotted in array formats. These microarrays consist of ESTs (i.e. expressed sequence tags = fragments of genes that are expressed under specific conditions) derived from red and green tomato fruit as well as cDNAs corresponding to genes with known function. Hybridisation experiments have taken place in order to determine levels of gene expression in subsequent ripening stages of tomato (i.e. natural variation).
Proteomics: The evaluation of sample preparation and extraction procedures for the proteome analysis of plant material (i.e. protein expression profiling or proteomics) has been completed and optimised to find a practical approach for sample delivery and compatibility with other analyses done within the project. Protein (2D-PAGE) patterns of various potato, tomato and Arabidopsis GM lines have been analysed and the identification of landmark proteins by ESI-MS in GM and non-GM material is under way. A strategy for glycome analysis (i.e. post-translation modifications in protein-linked carbohydrates) of minor amounts of glycoprotein post-PAGE and in whole plant tissue has been developed and its application to the selected GM and non-GM materials is in progress. For the development of tools to profile (possible) alterations in endogenous allergens, human sera containing specific IgE antibodies directed to rape seed has been collected and analysed for specific subclasses between allergic patients. A strategy to prepare western blots based on minimal amounts of human sera has been evaluated and demonstrated for plant material. The collection of pollen from (wild type) Arabidopsis and subsequent selection of Arabidopsis-specific human IgE is under way.
Metabolomics: The utilisation of special growth chambers for potato, tomato, Arabidopsis and tobacco pot plants has been evaluated for the metabolite profiling (metabolomics) experiments in vivo (during growth). Specific adaptations have been made. At present biomass is fixed for 13C-NMR analysis and other measurements. For the metabolomics experiments post harvest sample preparation and extraction procedures for proton-NMR, HPLC and GC-MS have been established. The fractionation procedure for tomato and potato has been tested and modified to reduce running time. The automated acquisition and processing of NMR spectra has been evaluated, whereas NMR, HPLC and GC-MS reproducibility check is in progress including the chemometrics treatment of the large potato and tomato batches.