Louise V. T. Shepherd

Comparison of Tuber Proteomes of Potato Varieties, Landraces, and Genetically Modified Lines

Crop improvement by genetic modification remains controversial, one of the major issues being the potential for unintended effects. Comparative safety assessment includes targeted analysis of key nutrients and antinutritional factors, but broader scale-profiling or “omics” methods could increase the chances of detecting unintended effects. Comparative assessment should consider the extent of natural variation and not simply compare genetically modified (GM) lines and parental controls. In this study, potato (Solanum tuberosum) proteome diversity has been assessed using a range of diverse non-GM germplasm. In addition, a selection of GM potato lines was compared to assess the potential for unintended differences in protein profiles. Clear qualitative and quantitative differences were found in the protein patterns of the varieties and landraces examined, with 1,077 of 1,111 protein spots analyzed showing statistically significant differences. The diploid species Solanum phureja could be clearly differentiated from tetraploid (Solanum tuberosum) genotypes. Many of the proteins apparently contributing to genotype differentiation are involved in disease and defense responses, the glycolytic pathway, and sugar metabolism or protein targeting/storage. Only nine proteins out of 730 showed significant differences between GM lines and their controls. There was much less variation between GM lines and their non-GM controls compared with that found between different varieties and landraces. A number of proteins were identified by mass spectrometry and added to a potato tuber two-dimensional protein map.

Assessing the potential for unintended effects in genetically modified potatoes perturbed in metabolic and developmental processes. Targeted analysis of key nutrients and anti-nutrients

Targeted compositional analysis was carried out on transgenic potato tubers of either cultivar (cv.) Record or cv. Desirée to assess the potential for unintended effects caused by the genetic modification process. The range of transgenic lines analysed included those modified in primary carbohydrate metabolism, polyamine biosynthesis and glycoprotein processing. Controls included wildtype tubers, tubers produced from plants regenerated through tissue culture (including a callus phase) and tubers derived from transformation with the ‘empty vector’ i.e. no specific target gene included (with the exception of the kanamycin resistance gene as a selectable marker). Metabolite analysis included soluble carbohydrates, glycoalkaloids, vitamin C, total nitrogen and fatty acids. Trypsin inhibitor activity was also assayed. These cover the major compounds recommended by the OECD in their Consensus Document on Compositional Considerations for New Varieties of Potatoes: Key Food and Feed Nutrients, Anti-Nutrients and Toxicants (2002). Data was statistically analysed using analysis of variance (ANOVA) for individual compounds and, where applicable, principal component analysis (PCA). In general, targeted compositional analysis revealed no consistent differences between GM lines and respective controls. No construct specifically induced unintended effects. Statistically significant differences between wildtype controls and specific GM lines did occur but appeared to be random and not associated with any specific construct. Indeed such significant differences were also found between wildtypes and both tissue culture derived tubers and tubers derived from transformation with the empty vector. This raises the possibility that somaclonal variation (known to occur significantly in potato, depending on genotype) may be responsible for an unknown proportion of any differences observed between specific GM lines and the wildtype. The most obvious differences seen in GC-MS profiles were between the two potato varieties used in the study.

Effects of agricultural production systems and their components on protein profiles of potato tubers

A range of studies have compared the level of nutritionally relevant compounds in crops from organic and nonorganic farming systems, but there is very limited information on the effect of farming systems and their key components on the protein composition of plants. We addressed this gap by quantifying the effects of different farming systems and key components of such systems on the protein profiles of potato tubers. Tuber samples were produced in the Nafferton factorial systems study, a group of long‐term, replicated factorial field experiments designed to identify and quantify the effect of fertility management methods, crop protection practices and rotational designs used in organic, low input and conventional production systems. Protein profiles were determined by 2‐DE and subsequent protein identification by HPLC‐ESI‐MS/MS. Principal component analysis of 2‐DE data showed that only fertility management practices (organic matter vs. mineral fertiliser based) had a significant effect on protein composition. Quantitative differences were detected in 160 of the 1100 tuber proteins separated by 2‐DE. Proteins identified by MS are involved in protein synthesis and turnover, carbon and energy metabolism and defence responses, suggesting that organic fertilisation leads to an increased stress response in potato tubers.

Phytochemical Diversity in Tubers of Potato Cultivars and Landraces Using a GC-MS Metabolomics Approach

Phytochemical diversity with respect to a range of polar (including amino acids, organic acids, sugars, and sugar alcohols) and nonpolar (including fatty acids, alkanols, and sterols) metabolites was examined within tubers from a total of 29 genetically diverse potato cultivars and Chilean landraces using a metabolomics approach by gas chromatography-mass spectrometry. From principal component analysis of the polar and nonpolar metabolite data there was insufficient variation to differentiate the majority of cultivars and landraces. Analysis of all polar metabolite profiles revealed separation of two cultivars (Glenna and Morag) from the other cultivars and landraces and a separate cluster of one landrace line, largely due to higher levels of sugars. Pentland Javelin was distinct in containing high levels of many amino acids. The two Solanum tuberosum group phureja cultivars (Inca Sun and Mayan Gold) were not particularly similar and were not separated from the S. tuberosum group tuberosum cultivars. Analysis of the nonpolar metabolite data revealed partial separation of two landrace lines and, on the basis of some minor fatty acids, Mayan Gold was distinct. The differences in metabolite profiles are considered in terms of the taxonomy and breeding history of the cultivars and possible influences from other factors such as developmental stage of the tuber. With a view to exploring biosynthetic links between metabolites, a pairwise correlation analysis was performed on all metabolites. The significance of high correlations between many amino acids and between several nonpolar metabolites is discussed.

Transcriptome analysis of potato tubers: Effects of different agricultural practices

The use of profiling techniques such as transcriptomics, proteomics, and metabolomics has been proposed to improve the detection of side effects of plant breeding processes. This paper describes the construction of a food safety-oriented potato cDNA microarray (FSPM). Microarray analysis was performed on a well-defined set of tuber samples of two different potato varieties, grown under different, well-recorded environmental conditions. Data were analyzed to assess the potential of transcriptomics to detect differences in gene expression due to genetic differences or environmental conditions. The most pronounced differences were found between the varieties Sante and Lady Balfour, whereas differences due to growth conditions were less significant. Transcriptomics results were confirmed by quantitative PCR. Furthermore, the bandwidth of natural variation of gene expression was explored to facilitate biological and/or toxicological evaluation in future assessments.

Metabolomic analysis of the potato tuber life cycle

An analysis of the potato (Solanum tuberosum L.) tuber life cycle has been completed using a range of mass-spectrometry (MS) based approaches. Six stages have been examined which included developing and mature tubers, sprouting mature tubers and mature tubers stored at 5 or 10°C. The impact of excising developing tubers from the mother plant (source-sink manipulation) was also determined. Data was subjected to Principal Components Analysis, Analysis of Variance and Hierarchical Cluster Analysis to assess the potential for separating the life cycle stages, to define the major profiles for metabolite changes during the life cycle stages examined, and to inform on which metabolites underpinned these profiles. We have shown that it is possible to separate all of the stages using combined analytical approaches and that five major profiles can be used to describe the changes in metabolite levels. Data also indicate that, within a relatively short timeframe, manipulation of source-sink relations has a significant impact on metabolite pools beyond what is currently known for sugar–starch metabolism. We have also demonstrated that the metabolomics data can be mined to provide answers to specific questions––in this case to identify temporal changes in metabolites related to acrylamide-forming potential.

The Identification and Interpretation of Differences in the Transcriptomes of Organically and Conventionally Grown Potato Tubers

In the European integrated research project SAFEFOODS, one of the aims was to further establish the potential of transcriptomics for the assessment of differences between plant varieties grown under different environmental conditions. Making use of the knowledge of cellular processes and interactions is one of the ways to obtain a better understanding of the differences found with transcriptomics. For the present study the potato genotype Santé was grown under both organic and conventional fertilizer, and each combined with either organic or conventional crop protection, giving four different treatments. Samples were derived from the European project QualityLowInputFood (QLIF). Microarray data were analyzed using different statistical tools (multivariate, principal components analysis (PCA); univariate, analysis of variance (ANOVA)) and with pathway analysis (hypergeometric distribution (HGD) and gene set enrichment analysis (GSEA)). Several biological processes were implicated as a result of the different treatments of the plants. Most obvious were the lipoxygenase pathway, with higher expression in organic fertilizer and lower expression in organic crop protection; the starch synthase pathway, with higher expression in both organic crop protection and fertilizer; and the biotic stress pathway, with higher expression in organic fertilizer. This study confirmed that gene expression profiling in combination with pathway analysis can identify and characterize differences between plants grown under different environmental conditions.

Modifying glycoalkaloid content in transgenic potato - Metabolome impacts

Metabolite profiling has been used to assess the potential for unintended composition changes in potato (Solanum tuberosum L. cv. Desirée) tubers, which have been genetically modified (GM) to reduce glycoalkaloid content, via the independent down-regulation of three genes SGT1, SGT2 and SGT3 known to be involved in glycoalkaloid biosynthesis. Differences between the three groups of antisense lines and control lines were assessed using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography (GC)-MS, and data analysed using principal component analysis and analysis of variance. Compared with the wild-type (WT) control, LC-MS revealed not only the expected changes in specific glycoalkaloid levels in the GM lines, but also significant changes in several other metabolites, some of which were explicable in terms of known pathways. Analysis of polar and non-polar metabolites by GC-MS revealed other significant (unintended) differences between SGT lines and the WT, but also between the WT control and other control lines used.

Compositional and toxicological analysis of a GM potato line with reduced α-solanine content--a 90-day feeding study in the Syrian Golden hamster.

Steroidal glycoalkaloids (GAs) are toxins, produced by plants of the Solanaceae family. The potato plant (Solanum tuberosum L.) and its tubers predominantly contain the two GAs α-chaconine and α-solanine. These compounds are believed to act in synergy, and the degree of toxicity may therefore depend on their ratio in the potato. To determine the influence of α-solanine: α-chaconine ratio in potatoes on toxicity, a GM potato line (SGT 9-2) with reduced α-solanine content, and the parental control line (Desirée wild-type) having a traditional α-solanine: α-chaconine ratio were (1) studied for compositional similarity by analysing for a range of potato constituents, and (2) used in a 90-day feeding trial with the Syrian Golden hamster to study differential toxicity. The animal feeding study used diets with up to 60% freeze-dried potato powder from either line. Whilst data indicated some compositional differences between the GM line and its wildtype control these did not raise concerns related to nutritional value or safety. Results of the feeding trials showed a low number of significant differences between potato lines with different α-solanine: α-chaconine ratio but none were considered to raise safety concerns with regard to human (or animal) consumption.

Impact of light-exposure on the metabolite balance of transgenic potato tubers with modified glycoalkaloid biosynthesis.

Metabolite profiling (liquid chromatography-mass spectrometry (LC-MS) and gas chromatography (GC-MS)) was used to assess the impact of light on the composition of transgenic potato (Solanum tuberosum L. cv. Desirée) with reduced glycoalkaloid content via the down-regulation of the SGT1 gene. Transgenic tubers exhibited an almost complete knock-out of α-solanine production and light had little impact on its accumulation. Levels of α-chaconine increased significantly in the peel of both the control and transgenic lines when exposed to light, particularly in the transgenic line. Major differences in metabolite profiles existed between outer and inner tuber tissues, and between light and dark-treated tubers. Many of the light-induced changes are explicable in terms of pathways known to be affected by stress responses. The impact of transgenesis on profiles was much less than that of tissue type or light and most differences were explicable in terms of the modification to the glycoalkaloid pathway.