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This commentary focuses on the potential added value of and need for (sub)-chronic testing of whole genetically modified (GM) foods in rodents to assess their safety. Such routine testing should not be required since, due to apparent weaknesses in the approach, it does not add to current risk assessment of GM foods. Moreover, the demand for routine testing using animals is in conflict with the European Union (EU) Commission's efforts to reduce animal experimentation. Regulating agencies in the EU are invited to respect the sound scientific principles applied to the risk assessment of foods derived from GM plants and not to interfere in the risk assessment by introducing extra requirements based on pseudo-scientific or political considerations., (© 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

This manuscript provides insights into ongoing debates on the regulatory issues surrounding groups of biotechnology-driven 'New Plant Breeding Techniques' (NPBTs). It presents the outcomes of preliminary discussions and in some cases the initial decisions taken by regulators in the following countries: Argentina, Australia, Canada, EU, Japan, South Africa and USA. In the light of these discussions we suggest in this manuscript a structured approach to make the evaluation more consistent and efficient. The issue appears to be complex as these groups of new technologies vary widely in both the technologies deployed and their impact on heritable changes in the plant genome. An added complication is that the legislation, definitions and regulatory approaches for biotechnology-derived crops differ significantly between these countries. There are therefore concerns that this situation will lead to non-harmonised regulatory approaches and asynchronous development and marketing of such crops resulting in trade disruptions., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Conventional plant breeding exploits existing genetic variability and introduces new variability by mutagenesis. This has proven highly successful in securing food supplies for an ever-growing human population. The use of genetically modified plants is a complementary approach but all plant breeding techniques have limitations. Here, we discuss how the recent evolution of targeted mutagenesis and DNA insertion techniques based on tailor-made site-directed nucleases (SDNs) provides opportunities to overcome such limitations. Plant breeding companies are exploiting SDNs to develop a new generation of crops with new and improved traits. Nevertheless, some technical limitations as well as significant uncertainties on the regulatory status of SDNs may challenge their use for commercial plant breeding., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A metabolite profiling approach based on gas chromatography-mass spectrometry (GC-MS) was applied to investigate the metabolite profiles of genetically modified (GM) Bt-maize (DKC78-15B, TXP 138F) and Roundup Ready-maize (DKC78-35R). For the comparative investigation of the impact of genetic modification versus environmental influence on the metabolite profiles, GM maize was grown together with the non-GM near-isogenic comparators under different environmental conditions, including several growing locations and seasons in Germany and South Africa. Analyses of variance (ANOVA) revealed significant differences between GM and non-GM maize grown in Germany and South Africa. For the factor genotype, 4 and 3%, respectively, of the total number of peaks detected by GC-MS showed statistically significant differences (p < 0.01) in peak heights as compared to the respective isogenic lines. However, ANOVA for the factor environment (growing location, season) revealed higher numbers of significant differences (p < 0.01) between the GM and the non-GM maize grown in Germany (42%) and South Africa (10%), respectively. This indicates that the majority of differences observed are related to natural variability rather than to the genetic modifications. In addition, multivariate data assessment by means of principal component analysis revealed that environmental factors, that is, growing locations and seasons, were dominant parameters driving the variability of the maize metabolite profiles.

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.

"Omics" technologies provide coverage of gene, protein and metabolite analysis that is unsurpassed compared with traditional targeted approaches. There are a growing number of examples indicating that profiling approaches can be used to expose significant sources of variation in the composition of crop and model plants caused by genetic background, breeding method, growing environment (site, season), genotype × environment interactions and crop cultural practices to name but a few. Whilst breeders have long been aware of such variation from tried and tested targeted analytical approaches, the broad-scale, so called "unbiased" analysis of the metabolome now possible, offers a major upside to our understanding of the true extent of variation in a plethora of metabolites relevant to human and animal health and nutrition. Metabolomics is helping to provide targets for plant breeding by linking gene expression, and allelic variation to variation in metabolite complement (functional genomics), and is also being deployed to better assess the potential impacts of climate change and reduced input agricultural systems on crop composition. This review will provide examples of the factors driving variation in the metabolomes of crop species., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Phytochemical diversity was examined by gas chromatography-mass spectrometry in tubers of genotypes belonging to groups Andigena, Phureja, Stenotomum, and Tuberosum of the potato, Solanum tuberosum. Polar extracts (mainly amino acids, organic acids, sugars, and sugar alcohols) and nonpolar extracts (mainly fatty acids, fatty alcohols, and sterols) were examined. There was a large range in levels of metabolites, including those such as asparagine, fructose, and glucose, that are important to tuber quality, offering considerable scope for selecting germplasm for breeding programmes. There were significant differences in the levels of many metabolites among the groups. The metabolite profiles of genotypes belonging to Phureja and Stenotomum were similar and different from those of Tuberosum and the majority of Andigena genotypes. There was some agreement with the phylogeny of the groups in that Stenotomum is believed to be the ancestor of Phureja and they are both distinct from Tuberosum. Andigena genotypes could be partially distinguished according to geographical origin, Bolivian genotypes being particularly distinct from those from Ecuador. Biosynthetic links between metabolites were explored by performing pairwise correlations of all metabolites. The significance of some expected and unexpected strong correlations between many amino acids (e.g., between isoleucine, lysine, valine, and other amino acids) and between several nonpolar metabolites (e.g., between many fatty acids) is discussed. For polar metabolites, correlation analysis gave essentially similar results irrespective of whether the whole data set, only Andigena genotypes, or only Phureja genotypes were used. In contrast, for the nonpolar metabolites, Andigena only and Phureja only data sets resulted in weaker and stronger correlations, respectively, compared to the whole data set, and may suggest differences in the biochemistry of the two groups, although the interpretation should be viewed with some caution.

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.

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.

The novel intragenic approach to genetic engineering improves existing varieties by eliminating undesirable features and activating dormant traits. It transforms plants with native expression cassettes to fine-tune the activity and/or tissue specificity of target genes. Any intragenic modification of traits could, at least in theory, also be accomplished by traditional breeding and transgenic modification. However, the new approach is unique in avoiding the transfer of unknown or foreign DNA. By consequently eliminating various potential risk factors, this method represents a relatively safe approach to crop improvement. Therefore, we argue that intragenic crops should be cleared through the regulatory process in a timely and cost-effective manner.

Steroidal glycoalkaloids (SGAs) are potentially harmful specialty metabolites found in Solanaceous plants. Two tri-glycosylated alkaloids, alpha-chaconine and alpha-solanine accumulate in potato tubers. Expressed sequence tags (ESTs) were identified in the available database by searching for protein homology to the Sgt1 (SOLtu:Sgt1) steriodalalkaloid galactosyltransferase. The EST sequence data was used to isolate Sgt3 cDNA sequences by polymerase chain reaction (PCR) from a wounded potato tuber cDNA library. The resulting 1515bp open reading frame of Sgt3, encodes a predicted SGT3 amino acid sequence that is 18 residues longer than, 45% identical to, and 58% homologous to the SGT1 protein. The amino-terminal region of the Sgt3 cDNA was used to create an antisense transgene under control of the granule bound starch synthase, GBSS6, promoter and the ubiquitin, Ubi3, polyadenylation signal. Analysis of SGA metabolites in selected transgenic tubers revealed a dramatic decrease in the accumulation of alpha-chaconine and alpha-solanine. This decrease was compensated by an increase in beta-solanine and beta-chaconine with minor accumulation of alpha-SGAs. These results allowed the identification of the function for SGT3 as the beta-solanine/beta-chaconine rhamnosyl transferase, the terminal step in formation of the potato glycoalkaloid triose side chains.

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.

The tuber of potato (Solanum tuberosum) is commonly used as a model for underground storage organs. In this study, changes in the proteome were followed from tuberization, through tuber development and storage into the sprouting phase. Data interrogation using principal component analysis was able to clearly discriminate between the various stages of the tuber life cycle. Moreover, five well-defined protein expression patterns were found by hierarchical clustering. Altogether 150 proteins showing highly significant differences in abundance between specific stages in the life cycle were highlighted; 59 of these were identified. In addition, 50 proteins with smaller changes in abundance were identified, including several novel proteins. Most noticeably, the development process was characterized by the accumulation of the major storage protein patatin isoforms and enzymes involved in disease and defense reactions. Furthermore, enzymes involved in carbohydrate and energy metabolism and protein processing were associated with development but decreased during tuber maturation. These results represent the first comprehensive picture of many proteins involved in the tuber development and physiology.

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.

To provide tools for breeders to control the steroidal glycoalkaloid (SGA) pathway in potato, we have investigated the steroidal alkaloid glycosyltransferase (Sgt) gene family. The committed step in the SGA pathway is the glycosylation of solanidine by either UDP-glucose or UDP-galactose leading to alpha-chaconine or alpha-solanine, respectively. The Sgt2 gene was identified by deduced protein sequence homology to the previously identified Sgt1 gene. SGT1 has glucosyltransferase activity in vitro, but in vivo serves as the UDP-galactose:solanidine galactosyltransferase. Two alleles of the Sgt2 gene were isolated and its function was established with antisense transgenic lines and in vitro assays of recombinant protein. In tubers of transgenic potato (Solanum tuberosum) cvs. Lenape and Desirée expressing an antisense Sgt2 gene construct, accumulation of alpha-solanine was increased and alpha-chaconine was reduced. Studies with recombinant SGT2 protein purified from yeast show that SGT2 glycosylation activity is highly specific for UDP-glucose as a sugar donor. This data establishes the function of the gene product (SGT2), as the primary UDP-glucose:solanidine glucosyltransferase in vivo.

The European Food Safety Authority, following a request from the European Commission, has published a guidance document for the risk assessment of GM plants and derived food and feed to assist in the implementation of provisions of Regulation (EC) 1829/2003 of the European Parliament and Council on GM food and feed. This regulation has applied since 18 April 2004. In principle, hazard identification and characterisation of GM crops is conducted in four steps: characterisation of the parent crop and any hazards associated with it; characterisation of the transformation process and of the inserted recombinant DNA, including an assessment of the possible production of new fusion proteins or allergens; assessment of the introduced proteins (toxicity, allergenicity) and metabolites; identification of any other targetted and unexpected alterations in the GM crop, including changes in the plant metabolism resulting in compositional changes and assessment of their toxicological, allergenic or nutritional impact. In relation to allergenicity specifically, it is clear that this property of a given protein is not intrinsic and fully predictable but is a biological activity requiring an interaction with individuals with a predisposed genetic background. Allergenicity, therefore, depends on the genetic diversity and variability in atopic human subjects. Given this lack of complete predictability it is necessary to obtain, from several steps in the risk-assessment process, a cumulative body of evidence that minimises any uncertainty about the protein(s) in question.

The antioxidant capacity of red cloudberry (Rubus chamaemorus) juice correlates well with its phenolic content. The red berries have a markedly higher content of anthocyanins, particularly cyanidin and pelargonidin derivatives, than that found in the more common yellow fruit. Conversely, the yellow juice has higher ellagitannin content. A feeding study was conducted to show the in vivo effects of the juices on lipid peroxidation in a sensitive Drosophila melanogaster stock. In young female flies there were significant (P < 0.01) effects of cloudberry juice on lipid peroxidation. In young male flies significant (P < 0.05) effects were found on primary products (hydroxyperoxides) with yellow juice and on secondary products (ketodienes) with red juice. With the red juice, a significant (P < 0.05) decrease in ketodienes was found in both young and old males. This study demonstrates that the effects of berry antioxidants on lipid peroxidation are easily and rapidly tested in vivo with the sensitive Drosophila model.

Potato plants (Solanum tuberosum L. cvs Desiree and Record) transformed with sense and antisense constructs of a cDNA encoding the potato fructokinase StFK1 exhibited altered transcription of this gene, altered amount of protein and altered enzyme activities. Measurement of the maximal catalytic activity of fructokinase revealed a 2-fold variation in leaf (from 90 to 180% of wild type activity) and either a 10- or 30-fold variation in tuber (from 10 or 30% to 300% in Record and Desiree, respectively) activity. The comparative effect of the antisense construct in leaf and tuber tissue suggests that this isoform is only a minor contributor to the total fructokinase activity in the leaf but the predominant isoform in the tuber. Antisense inhibition of the fructokinase resulted in a reduced tuber yield; however, its overexpression had no impact on this parameter. The modulation of fructokinase activity had few, consistent effects on carbohydrate levels, with the exception of a general increase in glucose content in the antisense lines, suggesting that this enzyme is not important for the control of starch synthesis. However, when metabolic fluxes were estimated, it became apparent that the transgenic lines display a marked shift in metabolism, with the rate of redistribution of radiolabel to sucrose markedly affected by the activity of fructokinase. These data suggest an important role for fructokinase, acting in concert with sucrose synthase, in maintaining a balance between sucrose synthesis and degradation by a mechanism independent of that controlled by the hexose phosphate-mediated activation of sucrose phosphate synthase.

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.

Four related phenolic amides previously undescribed from the species were revealed during metabolic profiling of potato (Solanum tuberosum) tubers. N(1),N(12)-Bis(dihydrocaffeoyl)spermine (kukoamine A) and N(1),N(8)-bis(dihydrocaffeoyl)spermidine were positively identified by comparison with authentic standards, while the structures N(1),N(4),N(12)-tris(dihydrocaffeoyl)spermine and N(1),N(4),N(8)-tris(dihydrocaffeoyl)spermidine are proposed for the other two metabolites. Each amide was present at several tens of micrograms per gram of dry matter. Several of these compounds were subsequently detected in other solanaceous species, such as tomato (Lycopersicon esculentum) and Nicotiana sylvestris. They appeared not to be present in Arabidopsis thaliana or Beta vulgaris. Bis(dihydrocaffeoyl)spermine isomers have previously been identified in only a single plant, the Chinese medicinal species Lycium chinense (Solanaceae), where they may account for some of the described biological activity. The other compounds have not until now been reported in vivo, though some of the equivalent hydroxycinnamoyl derivatives are known. The surprising discovery of kukoamine and allies in a range of solanaceous species including potato, a common food crop that has a long history of scientific investigation, provides exemplary evidence for the potential of the nontargeted techniques of metabolomics in studying plant metabolites.

Globally, urea is the most widely used nitrogen fertilizer and is made accessible to plants via the urease reaction. However, sequence information for the plant enzyme is scarce. A cDNA encoding urease from soybean (Glycine max) has been cloned and sequence information has been obtained for two alleles (11 and 19 kbp, respectively) of the potato (Solanum tuberosum, cv. Desiree) urease gene and the corresponding cDNAs. It was found that urease is encoded by a single copy gene in several solanaceous species, and maps to chromosome V in potato. By contrast, the presence of two urease genes was reported for soybean. Comparative analysis of 11 kbp overlapping allelic DNA allowed the quantification of single nucleotide polymorphisms and revealed the presence of a truncated Ty1-copia retrotransposon in one of the alleles. Both alleles contained a copy of a terminal-repeat retrotransposon in miniature (TRIM). 25-50% of urease pre-mRNAs from both alleles were alternatively spliced in a variety of different ways. The retrotransposons had no effect on splicing. While urease is expressed in all tissues tested, its mRNA and protein are of low abundance. The TATA-less urease promoter appears to drive low-level housekeeping transcription. An in silico analysis showed that eukaryotic urease protein sequences are very similar to sequences from prokaryotic enzymes, conserving all residues of known functional importance. It is therefore likely that all known ureases are structurally similar, employing the same catalytic mechanism.

In order to enhance the carotenoid content of potato tubers, transgenic potato plants have been produced expressing an Erwinia uredovora crtB gene encoding phytoene synthase, specifically in the tuber of Solanum tuberosum L. cultivar Desiree which normally produces tubers containing c. 5.6 microg carotenoid g(-1) DW and also in Solanum phureja L. cv. Mayan Gold which has a tuber carotenoid content of typically 20 microg carotenoid g(-1) DW. In developing tubers of transgenic crtB Desiree lines, carotenoid levels reached 35 microg carotenoid g(-1) DW and the balance of carotenoids changed radically compared with controls: beta-carotene levels in the transgenic tubers reached c. 11 microg g(-1) DW, whereas control tubers contained negligible amounts and lutein accumulated to a level 19-fold higher than empty-vector transformed controls. The crtB gene was also transformed into S. phureja (cv. Mayan Gold), again resulting in an increase in total carotenoid content to 78 microg carotenoid g(-1) DW in the most affected transgenic line. In these tubers, the major carotenoids were violaxanthin, lutein, antheraxanthin, and beta-carotene. No increases in expression levels of the major carotenoid biosynthetic genes could be detected in the transgenic tubers, despite the large increase in carotenoid accumulation. Microarray analysis was used to identify a number of genes that were consistently up- or down-regulated in transgenic crtB tubers compared with empty vector controls. The implications of these data from a nutritional standpoint and for further modifications of tuber carotenoid content are discussed.

Total and individual fatty acid contents were determined in raw tubers of four genotypes from each of the two species Solanum phureja and S. tuberosum. The four S. phureja genotypes contained statistically significantly higher concentrations of total fatty acids, with their average value being 37% greater than that for the mean of the four S. tuberosum cultivars. In both species, a total of 17 fatty acids were detected in quantifiable amounts, and in all genotypes the predominant fatty acid was linoleic followed by alpha-linolenic and palmitic acids. Unusually, 15-methyl hexadecanoate was present as a minor acid in both species. Although a number of statistically significant differences in the fatty acid percentage compositions were found between and within the two species, these were generally small. Averaged over all species and genotypes, tuber storage resulted in an initial small but statistically significant increase in total fatty acid content, but prolonged storage resulted in a fall to the initial values detected close to harvest. The same trend was evident for S. phureja alone (for mean values of all genotypes), but for S. tuberosum the total fatty acid content remained constant over the whole storage period. For both species, the contents (both as absolute levels and as percent compositions) of linoleic acid decreased and alpha-linolenic acid increased in tubers over the whole storage period, and possible mechanisms are discussed. Also, the absolute levels of these two acids were greater in S. phureja than in S. tuberosum, and this is discussed in relation to the development of flavor-related compounds during cooking. The polar lipids of one representative of S. tuberosum and of S. phureja were qualitatively similar. There were only minor differences in the polar lipid percentage compositions and in the corresponding fatty acid compositions of the individual polar lipids between the two species, although the absolute levels of the total, and of some individual, polar lipids were higher in S. phureja.

Metabolite profiling has been carried out to assess the compositional changes occurring in potato tubers after genetic modifications have been made to different metabolic pathways. Most major features in the (1)H NMR and HPLC-UV profiles of tuber extracts have been assigned. About 40 GM lines and controls belonging to 4 groups of samples (derived from cv. Record or cv. Desirée and modified in primary carbon metabolism, starch synthesis, glycoprotein processing, or polyamine/ethylene metabolism) were analyzed. Differences were assessed at the level of whole profiles (by PCA) or individual compounds (by ANOVA). The most obvious differences seen in both NMR and HPLC-UV profiles were between the two varieties. There were also significant differences between two of the four Desirée GM lines with modified polyamine metabolism and their controls. Compounds notably affected were proline, trigonelline, and numerous phenolics. However, that modification gave rise to a very abnormal phenotype. Certain lines from the other groups had several compounds present in significantly higher or lower amounts compared to the control, but the differences in mean values amounted to no more than a 2-3-fold change: in the context of variability in the whole data set, such changes did not appear to be important.

Salt-extractable proteins from the cell walls of immature and ripe strawberry (Fragaria x ananassa Duch. cv. Elsanta) fruit were separated using two-dimensional polyacrylamide gel electrophoresis. Seven polypeptides (enzymes) were characterized from their N-terminal sequences: (1) glyceraldhyde-3-phosphate dehydrogenase (EC 1.2.1.12); (2) triose phosphate isomerase (TPI; EC 5.3.1.1); (3) mitochondrial malate dehydrogenase (mMDH; EC 1.1.1.37); (4) NADH glutamate dehydrogenase (EC 1.4.1.3); (5) chalcone synthase (ChS; EC 2.3.1.74); (6) mitochondrial citrate synthase (mCS; EC 4.1.3.7); and (7) UDP glucose:flavonoid 3-O-glucosyltransferase (UDPG:FGT; EC 2.4.1.91). The sequenced polypeptides identified only cytosolic proteins, two of which (ChS and UDPG:FGT) had already been identified as being up-regulated in ripening (strawberry) fruit and important contributors to ripe fruit character. Our focus was therefore diverted to the enzymes mMDH and mCS for further molecular characterization as potentially important determinants of fruit flavour via regulation of the sugar : acid balance. Citrate synthase (CS) and malate dehydrogenase (MDH) enzyme activities increased substantially during ripening, as did citrate and malate contents. The increase in CS activity is supported by western blot analysis. One strawberry mCS (Fa-mCS-I) and two mMDH (Fa-mMDH-I and -II) cDNAs were cloned that were 77, 82 and 53% identical (respectively) to sequences from other plant sources. Northern analysis showed that CS and MDH expression did not correlate with enzyme activities and these findings are discussed.

Limit dextrinase (EC 3.2.1.41) from germinating barley (Hordeum vulgare L) can be activated by millimolar concentrations of linear maltodextrins with a degree of polymerisation > or = 2. The activation was assay-dependent; it was detected using assays based on the solubilisation of cross-linked dyed pullulan but not in assays that directly measured cleavage events such as the formation of new reducing termini. This strongly suggested that maltodextrins did not increase the catalytic rate of limit dextrinase i.e. this is not a true activation. On the other hand, considerable activation was noted in assays that measured pullulan degradation by reduction in viscosity. Taken together, this suggested that maltodextrins altered the mode of action of limit dextrinase, causing more rapid decreases in viscosity or greater solubilisation of dye-linked pullulan fragments per cleavage event. The proposed mechanism of activation by alteration in action pattern was reminiscent of initial work in the discovery of xyloglucan endotransglycosylase. Therefore, the ability of limit dextrinase to catalyse transglycosylation reactions into pullulan was tested and confirmed by an assay based on the incorporation of a fluorescently labelled maltotriose derivative into higher-molecular-weight products. The transglycosylation reaction was dependent on limit dextrinase activity and was enhanced in more highly purified preparations of limit dextrinase. Transglycosylation was inhibited by unlabelled maltotriose. How transglycosylation accounts for the apparent activation of limit dextrinase by maltodextrins and the physiological relevance of this novel reaction are discussed.

The gene coding for agglutinin from Galanthus nivalis (GNA) was expressed in, and secreted by, the methylotrophic yeast, Pichia pastoris. Transformants of P. pastoris were selected and a process to produce and purify gram quantities of recombinant GNA was developed. GNA was secreted at approximately 80 mg l(-1) at the 200 1 scale and was purified to 95% homogeneity using hydrophobic interaction chromatography. The recombinant protein was similar to the protein synthesised in plant with respect to structure and biological activity.

The influence of urease activity on N distribution and losses after foliar urea application was investigated using wild-type and transgenic potato (Solanum tuberosum cv Désirée) plants in which urease activity was down-regulated. A good correlation between urease activity and (15)N urea metabolism (NH(3) accumulation) was found. The general accumulation of ammonium in leaves treated with urea indicated that urease activity is not rate limiting, at least initially, for the assimilation of urea N by the plant. It is surprising that there was no effect of urease activity on either N losses or (15)N distribution in the plants after foliar urea application. Experiments with wild-type plants in the field using foliar-applied (15)N urea demonstrated an initial rapid export of N from urea-treated leaves to the tubers within 48 h, followed by a more gradual redistribution during the subsequent days. Only 10% to 18% of urea N applied was lost (presumably because of NH(3) volatilization) in contrast to far greater losses reported in several other studies. The pattern of urease activity in the canopy was investigated during plant development. The activity per unit protein increased up to 10-fold with leaf and plant age, suggesting a correlation with increased N recycling in senescing tissues. Whereas several reports have claimed that plant urease is inducible by urea, no evidence for urease induction could be found in potato.