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Potato (Solanum tuberosum L.) cv. Sante was grown over 2 years under both conventional and organic fertiliser and crop protection regimes. The tuber metabolome was analysed using mass-spectrometry (MS) based approaches, principally liquid chromatography (LC)–MS and gas chromatography (GC)–MS. Data were analysed using Principal Components Analysis (PCA) and Analysis of Variance (ANOVA) to assess any differences between production practices. GC–MS analysis of non-polar metabolites did not detect any statistically significant differences, but GC–MS analysis of polar compounds identified 83 metabolites showing significant differences in the metabolome between the fertiliser treatments. Of the 62 metabolites that were less abundant in tuber samples from organic compared with conventionally fertilised crops, consistent year on year differences were dominated by free amino acids. The effect on free amino acids is associated with the lower nitrogen (N) content of the organically grown potatoes in this instance (50 % lower than for conventional production). LC–MS provided indications that levels of certain glycoalkaloids may be lower under the organic fertiliser regime in one growing season. Differences associated with the crop protection measures used were much less consistent, and relatively small, compared with the fertiliser effects found.

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.

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.

New plant breeding techniques challenge the current regulation of genetically modified crops. Scientists and regulators must find new ways to guarantee safety whilst supporting innovation and agriculture.

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.

Acrylamide is a neurotoxin and possible carcinogen, and concern has been voiced over human exposure to acrylamide in cooked foods. Processed potato products such as crisps and French fries are often cited as being particularly high in acrylamide. In this manuscript a sub-set of clonal progeny from a specific tetraploid potato breeding population has been assessed for acrylamide forming potential in stored tubers processed into crisps. The clone with the lowest acrylamide content in crisps had both low reducing sugars and asparagine contents. Our data show that, in the segregating breeding population used, both asparagine and reducing sugars levels needed to be taken into account to explain most of the variation in acrylamide and that selection for low levels of both metabolites should be targeted for crop improvement.

Risk assessment frameworks, such as those used for GM crops, have detailed comparative analysis with appropriate non-GM counterparts as their cornerstone. Opinions have been voiced that current analytical approaches are too specific and need to be complemented by more unbiased, larger scale analysis of gene expression and protein expression using transcriptomics and proteomics, respectively. In parallel, the use of metabolomics has been advocated as an approach to expand significantly the range of metabolites that can be measured to assess more stringently the potential for any unintended effects. Transcriptomics, proteomics and metabolomics have been termed collectively “omics” technologies. This review assesses the potential for using “omics” techniques in risk assessment. Importantly, the review provides information on sources of natural variation which can result from crop management practices, from interactions between genotype and growing environment and from non-GM breeding systems. This provides an important benchmark for risk assessors and risk managers.

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.

Three main changes to current risk analysis processes are proposed to improve their transparency, openness, and accountability. First, the addition of a formal framing stage would allow interested parties, experts and officials to work together as needed to gain an initial shared understanding of the issue, the objectives of regulatory action, and alternative risk management measures. Second, the scope of the risk assessment is expanded to include the assessment of health and environmental benefits as well as risks, and the explicit consideration of economic- and social-impacts of risk management action and their distribution. Moreover approaches were developed for deriving improved information from genomic, proteomic and metabolomic profiling methods and for probabilistic modelling of health impacts for risk assessment purposes. Third, in an added evaluation stage, interested parties, experts, and officials may compare and weigh the risks, costs, and benefits and their distribution. As part of a set of recommendations on risk communication, we propose that reports on each stage should be made public.

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 challenges facing potato breeding have actually changed very little over the years with resistance to pests and pathogens remaining high on the agenda together with improvements in storability, reduction in blemishes, and novelty and consistency in cooking/processing qualities. The need to expand the range of targets for potato improvement is being driven by requirements for reduced agrochemical usage and by predictions of the effects of changing climates. Thus fertiliser and water use efficiency are moving up the political agenda. Genetic variation present in germplasm collections needs to be harnessed to provide the genes and alleles required. This paper provides examples of the functional genomics tools and approaches being developed and deployed to provide new options for advancing the breeding of next generation crops. Whilst genetic modification (GM) approaches remain contentious in Europe, this paper will also provide some recent examples of the range of potential impacts that GM approaches could make. It will also consider the value of so-called intragenic or cisgenic approaches to potato genetic engineering.

Sprouted, stressed, or spoiled potato tubers have reportedly led to human acute intoxication, coma, and death when consumed in high amounts. These effects have been attributed to glycoalkaloids (GAs), primarily alpha-solanine and alpha-chaconine, naturally present in all potatoes. The level of GAs in potato tubers has previously been shown to increase substantially as a result of improper handling and postharvest storage. A short-term study was performed to investigate the dose-response profile of alpha-solanine and alpha-chaconine alone or in combination, administered daily by oral gavage to Syrian Golden hamsters. Daily doses of 100 mg of alpha-solanine [kg body weight (BW)] (-1) induced death in two of four hamsters within 4 days, when administered by gavage to female Syrian hamsters. Doses of 100 mg of alpha-chaconine alone or alpha-solanine and alpha-chaconine combined in a ratio of 1:2.5, in doses of 75 or 100 mg (kg BW) (-1), induced death in one of four hamsters within the same period. Animals dosed with alpha-solanine alone or in combination with alpha-chaconine suffered from fluid-filled and dilated small intestines. The GA administration had no effect on acetyl cholinesterase (AChE) or butyryl cholinesterase (BuChE) activity in plasma or brain. Liquid chromatography-mass spectrometry-based metabolomics showed that there was a specific accumulation of alpha-chaconine in the liver tissues. In addition, metabolomics gave direct evidence of glycolytic metabolism of the GA with the beta 1, beta 2, and gamma-GAs detected in the urine and, to a lesser extent, the feces. Doses from 75 mg (kg BW) (-1) of alpha-chaconine, alpha-solanine, or the two compounds combined were potentially lethal within 4-5 days in the Syrian Golden hamster. However, the cause of death in these studies could not be established. No synergistic effects of alpha-solanine combined with alpha-chaconine were evident.

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.

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. Desiree) 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.

Metabolite profiling has been used to assess the potential for unintended composition changes in potato (Solanum tuberosum L. cv. Desiree) 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.

International audience; 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 dearly 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

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 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.

Steroidal glycoalkaloids (SGAs) are secondary metabolites of Solanaceous plants. Two predominant glycoalkaloids, a-chaconine and asolanine are produced in potatoes. An antisense transgene was constructed to down-regulate glycoalkaloid biosynthesis using a potato cDNA encoding a sterol alkaloid glycosyltransferase (Sgt1). Introduction of this construct into potatoes resulted in some lines with an almost complete inhibition of a-solanine accumulation. This inhibition was compensated by elevated levels of a-chaconine and resulted in wild type total SGA levels in the transgenic lines. In vitro assays with the recombinant SGT1 isolated from yeast demonstrated that Sgt1 encodes an enzyme capable of both glucosyltransferase and galactosyltransferase activity with a preference for UDP-galactose as the sugar donor. Together this data confirms SGT1’s role in vivo as the solanidine:UDP-galactose galactosyltransferase. # 2004 Elsevier Ireland Ltd. All rights reserved.

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.

Twelve potato cultivars were grown as microtubers in constant environment chambers at two temperatures in total darkness for 28 days. For starch extracted from microtubers grown at 12 or 22 °C, the amylose content ranged from 25.0 to 29.0% (average 27.3 ± 1.4%) or 29.5 to 32.7% (average 30.9 ± 1.4%), the amylose/amylopectin ratio from 1:2.5 to 1:3.0 (average 1:2.7) or 1:2.1 to 1:2.4 (average 1:2.2) and the phosphorus content from 0.41 to 0.93 g kg−1 (average 0.72 g kg−1) or 0.38 to 1.67 g kg−1 (average 0.97 g kg−1) respectively. Two major fractions (F1 and F2) were obtained for isoamylase-debranched starch (amylopectin), with the chain length (CL) averaging 56 ± 3 or 55 ± 1 for F1 and 20 ± 1 or 21 ± 1 for F2 from 12 or 22 °C growth temperature respectively. Peak gelatinisation temperature (Tp) and enthalpy (ΔH) were influenced strongly by growth temperature, with Tp ranging from 60.8 to 64.5 °C (average 62.1 ± 1.0 °C) or 66.9 to 69.6 °C (average 68.2 ± 0.9 °C) and ΔH from 13.3 to 16.9 J g−1 (average 14.8 ± 1.0 J g−1) or 11.3 to 19.5 J g−1 (average 16.3 ± 2.4 J g−1) from 12 or 22 °C growth temperature respectively. The collective data generated at 12 °C were generally more comparable to data published elsewhere (but discussed in the text) for the same potato cultivars grown in field plots (Perthshire). However, there were cultivar specific responses to field or constant environment chambers which make direct comparisons between starches produced from the two systems subject to some variation. Copyright © 2004 Society of Chemical Industry

Germplasm of Solanum tuberosum and Solanum phureja exhibit a wide (over 20-fold) variation in tuber carotenoid content. The levels of carotenoids during tuber development and storage were compared in a high carotenoid-accumulating S. phureja accession (DB375\1) with two S. tuberosum cultivars (Pentland Javelin and Desiree) that accumulate lower levels of tuber carotenoid. On a dry weight basis, total carotenoid levels were at a maximum early in tuber development. However, in the S. phureja accession, carotenoid levels remained at a high level throughout tuber development, whereas in the S. tuberosum accessions, carotenoid content decreased as dry weight increased. The carotenoid profiles of tissues during tuber development were analysed in greater detail by reverse phase HPLC. In S. phureja tubers at maturity the major carotenoids were zeaxanthin, antheraxanthin, and violaxanthin. Following 9 months storage at 4 degrees C the levels of zeaxanthin and antheraxanthin decreased, whereas the level of lutein increased; overall, however, there was only a small decrease in total carotenoid content. In order to explore the reasons for the wide variation in tuber carotenoid content, the expression patterns of the major genes encoding the enzymes of the carotenoid biosynthetic pathway were compared. Significant differences in the profiles were detected, suggesting that transcriptional control or mRNA stability gives rise to the large differences in tuber carotenoid content. In particular, there was an inverse trend between the level of zeaxanthin epoxidase transcript level and tuber carotenoid content in a range of potato germplasm, giving rise to an hypothesis for the regulation of carotenogenesis in potato tubers.

The levels of xylanolytic activities ((1→4)-β-endoxylanases, β- d -xylosidases and α- l -arabinofuranosidases) and the levels of water extractable arabinoxylans (WEAX) in hot water extracts of malts were compared during malting of four barley cultivars of differing quality. Endoxylanase activity increased from day 1 after steeping showing a slight lag compared with the development of (1→3,1→4)-β-glucanase activity. The levels of endoxylanase activity were different between the cultivars. For example, at day 4, which would be a suitable endpoint for malting of good quality cultivars, total endoxylanase activities of Static and Chariot malts were twice that of Hart and Optic. This variation in level of endoxylanase between cultivars did not correlate with the content of water extractable arabinoxylans in hot water extracts of the malts. However, those cultivars that accumulated most water extractable arabinoxylan had a different set of endoxylanase isoforms. This suggests that expression of specific endoxylanase isoforms may influence the extent of arabinoxylan solubilisation and/or degradation. The β-xylosidases and α-arabinosidases increased from low levels after steep to a plateau of activity at day 6 or 7 of germination. Total xylosidase was always higher than the arabinosidase activity. There were differences between cultivars but these were small compared with the variation in endoxylanase activity. (1→4)-β- d -Endomannanase activity was detected in all four cultivars at 2 days germination then rose to a maximum around 5 days.

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 polymerisationor = 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.

Free and total limit dextrinase (LD) activity was measured in four barley varieties of differing malting quality. LD activity was not detected until 2 days after completion of steeping, at which point, total LD activity was 4–5 fold higher in Static and Chariot than in Optic or Hart. Free LD rose to higher levels in Static and Chariot than in Optic and Hart during malting. In addition, the proportion of free to total LD approached 35% in Static and Chariot whereas it did not reach 15% for Optic and Hart. Lower free LD activity was not reflected in a higher total content of branched dextrins in hot water extracts of the different varieties, but may have contributed to the persistence of branched dextrins of higher degree of polymerisation persisting in the hot water extracts of Hart and Optic. The variation in the proportion of free to total LD activity between varieties was not explained by differences in total LD activity and may be related to the presence of inhibitors of LD. Protein extracts made from the malts following steeping inhibited exogenous partially purified LD activity by >80%. After 3 days germination, Chariot, Optic and Static lost much of their inhibitory activity whereas it was retained in Hart. The increase in free LD activities during malting appeared to mirror the disappearance in inhibitory activity. Inhibitors of LD activity were also present in hot water extracts of Chariot and Hart malts. Chariot lost inhibitory activity after 3 days malting and hot water extracts from Chariot after 4 days malting caused apparent activation of LD. By contrast, hot water extracts from Hart retained inhibitory activity up to 5 days malting. These findings are discussed with reference to the availability and effectiveness of LD during malting.

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 l 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 15N urea metabolism (NH3accumulation) 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 15N distribution in the plants after foliar urea application. Experiments with wild-type plants in the field using foliar-applied 15N 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 NH3 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.

Tubers of potato (Solanum tuberosum L. cv. Estima) genetically modified to reduce polyphenol oxidase (PPO) activity and enzymatic discolouration were assessed for changes in the metabolome using Liquid Chromatography–Mass Spectrometry (LC–MS) and Gas Chromatography (GC)–MS. Metabolome changes induced over a 48 hour (h) period by tuber wounding (sliced transverse sections) were also assessed using two PPO antisense lines (asPPO) and a wild-type (WT) control. Data were analysed using Principal Components Analysis and Analysis of Variance to assess differences between genotypes and temporal changes post-tuber wounding (by slicing). The levels of 15 metabolites (out of a total of 134 that were detected) differed between the WT and asPPO lines in mature tubers at harvest. A considerably higher number (63) of these metabolites changed significantly over a 48 h period following tuber wounding. For individual metabolites the magnitude of the differences between the WT and asPPO lines at harvest were small compared with the impacts of tuber wounding on metabolite levels. Some of the observed metabolite changes are explicable in terms of pathways known to be affected by wound responses. Whilst some statistically significant interactions (11 metabolites) were observed between line and time after wounding, very few profiles were consistent when comparing the WT with both asPPO lines, and the underlying metabolites appeared to be random in terms of the pathways they occupy. Overall, mechanical damage to tubers has a considerably greater impact on the metabolite profile than any potential unintended effects resulting from the down-regulation of PPO gene expression.

The activation of the nickel metalloenzyme urease is a complex process. In bacteria, several urease accessory proteins are essential for incorporation of nickel into the active centre of urease. Comparatively little is known about the activation process and the proteins involved in plants. We cloned five different cDNAs encoding isoforms of urease accessory protein G (ureG) in potato. The 5′-coding region of these cDNAs is highly polymorphic within Solanum tuberosum ssp. tuberosum, containing mainly a simple sequence repeat encoding histidine and aspartate. Mapping on an ultrahigh-density map of the potato genome and Southern blot analysis showed that the isoforms arise from allelic differences of a single-copy gene which was located on chromosome 2. Expression analysis at the mRNA and protein levels indicated the presence of ureG in almost all tissues examined, consistent with the ubiquitous expression of urease. An attempt to correlate urease activity with ureG expression levels in different tissues was made. Allelic copies of ureG were expressed in a tissue-specific manner. UreG from potato and the Klebsiella aerogenes urease operon defective in bacterial ureG were co-expressed in Escherichia coli. The plant gene complements the K. aerogenes ureG mutation, demonstrating that it encodes a urease accessory protein and indicating a structural conservation between the plant and the bacterial urease activation complexes.

During raspberry (Rubus ideaus L. cv Glen Clova) fruit ripening, endo-β-1,4-glucanase (EGase; EC 3.2.1.4) specific activity (per g fresh weight) increases approximately 15-fold. Highest activity was associated with the surfaces of the receptacles where the weakening abscission zones are located. Immunoblotting using antibodies raised against a bean abscission-EGase (ab.-EGase) identified a single protein of M r 52 kDa that was present only in ripe fruit and was most prominent in the receptacle. Using reverse transcription-polymerase chain reaction (RT-PCR), two 497-bp partial putative EGase clones were obtained from ripe receptacle mRNA (termed RI-EGL1 and 2) which share 53% amino acid identity. The more abundant RI-EGL1 was used to obtain its full length clone from a ripe receptacle cDNA library. The deduced amino acid sequence of RI-EGL1 was similar to other ab.-EGase sequences (ca 67%) and contained the conserved motifs present in all E2-class EGases. Northern analysis revealed that RI-EGL1 expression was limited to ripe-fruit receptacles. Application of the ethylene antagonist 1-methylcyclopropene (1-MCP) to green fruits indicated that endogenous ethylene accelerates raspberry abscission and increases both EGase activity and RI-EGL1 expression.

In order to elucidate more fully the function of a potato gene (MAL1) encoding alpha-glucosidase activity, transgenic plants in which MAL1 expression was down-regulated were generated using antisense technology. In transgenic lines severely down-regulated in the expression of MAL1, total alpha-glucosidase activity was not decreased in leaves and tubers, and the contents of starch, glucose, fructose and sucrose remained unchanged in tubers. Phylogenetic analysis indicated that the MAL1 gene product was more similar to the glycoprotein-processing alpha-glucosidase II of mammalian and yeast origin than to other plant alpha-glucosidases. Using [14C-Glc]-labelled Glc2Man9GlcNAc2 as a substrate, it was demonstrated that glucosidase II activity was markedly down-regulated in microsomes isolated from tubers of four independent antisense lines studied in detail, strongly suggesting that MAL1 encodes glucosidase II activity. In field trials (but not in the glasshouse), MAL1 down-regulation produced an extremely stunted phenotype - the leaves were curled and tuber yield was decreased by 90% compared to control values. Microscopic analysis of leaves revealed significant differences between the antisense and control samples. Plants with down-regulated glucosidase II activity showed a greater degree of plasmolysis, and an increase in the size of mesophyll intracellular spaces. Analysis of cell walls also indicated changes in structure as a result of MAL1 down-regulation. In leaves from four antisense lines, the steady-state transcript level corresponding to the endoplasmic reticulum chaperone, BiP, was enhanced. This is diagnostic of stress in the endoplasmic reticulum.

Processes that contribute to the overall phenomenon of fruit ripening are not well understood for many soft fruit species, including raspberry (Rubus idaeus L.) Recent biochemical data implicate ethylene and a range of cell wall hydrolases in ripening processes. However, the genes encoding these activities, and others related to ripening, remain to be characterised. With the advent of high-throughput RNA-fingerprinting techniques it is possible to characterise rapidly the changes in gene expression during developmental processes. This paper describes the application of two RNA-fingerprinting techniques (cDNA amplified fragment length polymorphism and differential display reverse transcription-polymerase chain reaction) to the ripening fruit of Rubus idaeus. Copy-DNA tags were isolated representing 34 genes, up-regulated during fruit ripening. The expression profiles of these genes were determined by RNA-blot analysis and their sequences were compared with those in public databases. Potential roles for some of these gene products are considered, providing valuable insights into the processes that underpin fruit ripening. Many of the cDNAs isolated in this study provide tools for the biotechnological improvement of fruit quality.

Antibodies have been expressed in plants to confer novel traits such as virus resistance or altered phenotype. However, not every antibody is suitable for plant expression, and successful intracellular expression of antibody fragments depends primarily on their amino acid sequence in a way that is as yet unpredictable. Therefore it is desirable to assess different constructs before embarking on the production of transgenic plants. We have used a transient expression system based on potato virus X to compare different cDNA constructs for expression and stability of antibody variable gene fragments in plants. Constructs contained an anti-plant enzyme (granule-bound starch synthase I) scFv sequence derived from a naive phage display library together with different combinations of sequences encoding the human IgG constant domain, a murine IgG secretory signal sequence, or the endoplasmic reticulum retention signal peptide KDEL. The results obtained with the potato virus X vector correlated with those from Agrobacterium-mediated stable transformation of potato. The best expression levels were obtained by incorporating sequences that target scFv to the lumen of the endoplasmic reticulum and the secretory pathway. The anti-enzyme scFv retained activity during storage of potato tubers for more than five months. The results demonstrate the utility of the potato virus X vector for the analysis and comparison of many scFv with different epitope specificities or sequence modifications. Evaluation of scFv by transient expression from the PVX vector should aid progress in selection of functional scFv for applications in plant biotechnology.

The antioxidative capacities of a number of Rubus species of varied pigmentation have been investigated. In addition, total phenol, anthocyanin and ascorbic acid contents have been determined. Two methods to assess the antioxidant potential of fruit juices have been used. The antioxidant capacities of the fruit ranged from 0 to 25.3 mmol Trolox equivalents g ˇ1 (TEAC) or from 190 to 66000 mmol l ˇ1 ferric reducing antioxidant power (FRAP). Ascorbic acid contributes only minimally to the antioxidant potential of Rubus juices (

SummaryThe L-ascorbic acid (AsA) content of six genotypes of blackcurrant (Ribes nigrum L.) representative of the available genetic resources for this crop, was measured in relation to berry development. The main period of AsA accumulation coincided with the berry expansion phase, soon after fertilization, and genotypic rankings of AsA content established in this early stage remained constant thereafter. No correlation was found between AsA content, soluble solids content or sugar levels.

Potato tubers (cv Maris Piper) were grown at 10, 16, 20 and 25 °C in constant-environment chambers until maturity, whereupon the starches were extracted and subjected to rigorous chemical and physical analysis. The structure of the amylopectin molecules from the different starches post-debranching with isoamylase showed very little variation. The amylose and phosphorus content of the starches did show some variability while granule size tended to decrease as growth temperature was increased. There was, however, a marked increase in gelatinisation temperatures with a roughly constant enthalpy of gelatinisation as a function of growth temperature. The number of amylopectin double helices was determined by 13C CP/MAS-NMR and crystallinity by wide-angle X-ray diffraction and in common with the enthalpy of gelatinisation found to be almost constant. It is proposed that the differences in the gelatinisation temperatures reflect enhanced registration of the amylopectin double helices in crystallites which restricts hydration and hence elevates gelatinisation temperatures. This is probably associated with enhanced rigidity of amorphous regions. The consequence of these ordering effects is that swelling is restricted, even if there is no detectable order by DSC (post-Tc), because of steric hindrance to hydration exerted by the closer proximity/improved registration of the amylopectin chains. © 1999 Society of Chemical Industry

Red algae (Rhodophyceae) are photosynthetic eukaryotes that accumulate starch granules in the cytosol. Starch synthase activity in crude extracts of Gracilaria tenuistipitata Chang et Xia was almost 9-fold higher with UDP[U-14C]glucose than with ADP[U-14C]glucose. The activity with UDP[U-14C]glucose was sensitive to proteolytic and oxidative inhibition during extraction whilst the activity with ADP[U-14C]glucose appeared unaffected. This indicates the presence of separate starch synthases with different substrate specificities in G. tenuistipitata. The UDPglucose: starch synthase was purified and characterised. The enzyme appears to be a homotetramer with a native M(r) of 580 kDa and displays kinetic properties similar to other alpha-glucan synthases such as stimulation by citrate, product (UDP) inhibition and broad primer specificity. We propose that this enzyme is involved in cytosolic starch synthesis in red algae and thus is the first starch synthase described that utilises UDPglucose in vivo. The biochemical implications of the different compartmentalisation of starch synthesis in red algae and green algae/plants are also discussed.

S-Adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) is a key enzyme in the biosynthesis of the polyamines spermidine and spermine from putrescine and its activity is rate limiting in this pathway. Transgenic potato (Solanum tuberosum L. cv. Desiree) plants containing both sense and antisense SAMDC constructs driven by the tuber-specific patatin promoter have been generated and analysed. In sense transformants, developing tubers expressed higher steady-state levels of the SAMDC-specific transcript, had higher levels of SAMDC activity and contained significantly higher levels of spermidine than vector-transformed controls. Additionally, there was a significant shift in tuber size distribution with larger numbers of smaller tubers but no overall change in tuber yield. In developing tubers from the antisense transformed lines, there was a decrease in SAMDC transcript level, SAMDC activity and total polyamine levels. However, no obvious phenotypic effect was detected in the tuberisation physiology of the antisense lines.

In screening to isolate a full-length copy of a previously isolated cDNA clone, a further three cDNAs were also isolated from a library prepared from sub-apical swelling-stolon tissue of potato (Solanum tuberosum L.). Sequence analysis showed these clones to be similar to extensin-like protein genes, acyl carrier protein thioesterase genes and high mobility group protein genes, respectively. A further cDNA, isolated by subtractive hybridisation, was similar to a tomato cDNA previously isolated on the basis of its down-regulation following nematode infection. While all the newly isolated genes were expressed in swelling stolons, for most, maximal expression was seen to be in stem tissue. Possible roles for these genes in the development of potato plants are discussed, as is the significance of gene expression in stems and stolons to the process of tuberisation.

This study focuses on four raspberry (Rubus idaeus) genotypes from two different genetic backgrounds: cvs Glen Prosen and Glen Clova, bred at the Scottish Crop Research Institute (SCRI) and genotypes bred at Horticulture Research International (HRI), East Malling (EM), EM 4997 and EM 5007. The ripe fruit of each genotype pair were characterised subjectively by raspberry breeders as relatively firm or soft, respectively. Different stages of fruit development from each genotype were used to quantify fruit firmness, rates of ethylene evolution and ripening rate. Penetrometry data confirmed suspected firmness differences. Firmness correlated with rates of ethylene evolution. Rates of ethylene production also correlated with receptacle size. Storage of green fruits in 20 μl l -1 ethylene reduced fruit firmness, enhanced respiration rate and colour (anthocyanin) development and stimulated the development of cell wall hydrolase activities. However, during natural ripening in the field, fruit respiration rate declined, which indicates a non-climacteric ripening pattern. In drupelets, the activities of polygalacturonase (PG), pectin methylesterase (PME), Cx-cellulase (Cx) and β-galactosidase (β-gal.) increased substantially as ripening progressed. More detailed studies with ripe fruit of cv. Glen Clova indicated major isoforms of PG at pIs 3.3, 8.6 and 10.1; of PME at pIs 7.2, 8.5, 8.7, 8.8; of Cx at pI 2.4; and of β-gal. at pIs 6.3 and 6.7.

Potato microtubers (cv Maris Piper) were grown at 10, 16 and 24°C in total darkness for 28 days. Soluble and insoluble starch synthase, ADPglucose pyrophosphorylase, sucrose synthase and fructokinase were assayed in extracts of the microtubers and, in the case of soluble and insoluble starch synthase, activity was found to be particularly sensitive to increasing growth temperature. The starch content of the microtubers increased slightly with increasing growth temperature, but with little effect on the number of granules per microtuber and a small increase in the average granule size. The microtuber starch granules were much smaller than those found in commercial potato starch (c 8–9 μm modal diameter compared to c 21 μm). Although the amylose content of the microtuber starches tended to increase with increasing growth temperature, the phosphorus content was variable. Gel permeation chromatographic elution profiles of native and debranched microtuber and a commercial potato starch showed that no differences could be detected in either amylose or amylopectin molecular size, polydispersity or unit chain distribution of amylopectin (which contained two major unit chain fractions at DP 21 and 56). The onset, peak and conclusion temperatures of the DSC gelatinisation endotherm increased linearly as a function of growth temperature whilst the enthalpy of gelatinisation decreased. It is suggested that elevated temperature during starch biosynthesis facilitates ordering of amylopectin double helices into crystalline domains. © 1998 SCI.

Summary Measurements of respiration rate show that blackcurrant ( Ribes nigrum cv. Ben Alder) fruit do not exhibit a respiratory climacteric during ripening, and that ripe fruit produce only very low levels of ethylene. Differential screening of a cDNA library constructed from RNA extracted from blackcurrant fruit enabled the isolation of the cDNA clones of five genes that showed gready enhanced steady-state transcript levels in fully ripe fruit compared with green fruit. The expression patterns of the corresponding genes were also determined in other tissues of the blackcurrant plant. The sequences of the cDNA clones were compared with known sequences in databases. Four of the clones were identified tentatively on the basis of sequence similarity. pRIB 1 was similar to a protein found in the fruit of kiwifruit, pRIB3 was similar to other plant metaUothionein-like proteins, pRIB6 to members of the cysteine proteinase family and pRIB7 to a mitochondrial RNA splicing protein. It was not possible to identify pRIB5 on the basis of sequence similarity. Southern analysis indicated that all of these genes were present in the blackcurrant genome at low copy number. Sequences similar to RIB 3 and RIB 5 are not present in the genomes of red raspberry ( Rubus idaeus ) or Tayberry ( Rubus loganobaccus ). The nucleotide sequence data reported appears in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the following accession numbers: AJ007576 (pRIBl), AJ007577 (pRIB3), AJ007578 (pRIB5), AJ007579 (pRIB6) & AJ007580 (pRIB7).

Summary cDNA clones of three genes, up-regulated as raspberry ( R. idaeus cv. Glen Clova) fruit ripen, were isolated by differential screening of a cDNA library constructed from RNA extracted from ripe fruit. The expression patterns of these genes were determined in ripening fruit and in other tissues of the raspberry plant. When compared with sequences in databases, the genes were identified on the basis of sequence similarity. The predicted polypeptides coded for by the three clones show similarity to major latex proteins, a class of fruit-specific metallothionein-like proteins, and fruit-ripening associated endo-polygalactu-ronases, respectively. The potential roles of these gene products in fruit-ripening is discussed.

Physiological disorders such as Brown Center and Internal Brown Spot affect the quality of potato tubers destined for fresh and processing markets. Cultural and environmental factors giving rise to such disorders have been studied extensively. Considerably less information is available on the underpinning physiological, molecular and biochemical mechanisms. However, the cell’s calcium status clearly plays an important role in resistance responses. Whilst extreme genotypic susceptibility to these disorders exist such variation has not been exploited fully to dissect the genetics and biochemistry of the key processes. This is central to the selection of resistant germplasm or to the manipulation of existing genotypes for improved tolerance

Calibrations are provided to relate near infrared reflectance (NIR) data to total N concentration in potato leaves, stems, and tubers, estimated by Dumas combustion. Separate calibrations are necessary for each tissue-type. the NIR models simulate Dumas [N] estimates accurately, quickly and cheaply and have been shown to be stable. The methods of deriving the calibrations, correcting them and then providing independent tests are described.

Extraction of high-quality RNA from blackcurrant fruit has hitherto proved difficult, probably owing to high levels of phenolic and polysaccharide components in the berries. The procedure described here is a modification of one described for grape berries, and yields RNA suitable for in vitro translations, RNA blot analysis, and cDNA library construction.

Summary In Douglas fir seeds ( Pseudotsuga menziesii (Mirb.) Franco), dormancy-breakage is associated with quantitative and qualitative changes in gene expression. Specific examples of genes that are up-regulated during dormancy-breakage include those encoding LEA proteins (DF6, DF65 and DF77) (Jarvis et al., J. Plant Physiol. 147: 559–566, 1996). In particular, DF65, a dehydrin, was specifically expressed during stratification and DF6 and DF77 were significantly up-regulated. On examination of the tissue distribution of the LEA gene transcripts during chilling (which breaks dormancy) and warm-incubation, LEA transcripts were detected in both embryo and megagametophyte tissues. Expression of DF65 was lower, and the transcript appeared later, in the embryo than in the megagametophyte. With DF6 and DF77, transcripts reached maximal levels in the megagametophyte earlier than in the embryo. While de novo synthesis of ABA was important for the expression of dormancy, four weeks of chilling, only led to a 36% reduction in endogenous ABA and did not change its distribution between the embryo and megagametophyte, compared with warm-incubated seeds. However, following chilling, seeds showed a reduced sensitivity to applied ABA. To determine if there was a link between the expression pattern of LEA genes and the dormancy status of the seeds and/or abiotic stresses, seeds were treated with the plant growth regulators ABA and Me-JA, or subjected to osmotic, salt, and heat stresses. ABA treatment prevented germination of non dormant seeds, and did not induce expression of LEA genes. Me-JA stimulated dormant seeds to germinate and induced low level expression of all three LEA genes after 24 hours, but not after one week. Expression of all three LEA genes was detected in seeds which had been subjected to osmotic and salt stresses expression. However, under heat stress only DF6 and DF77 were expressed. The degree of induction by these treatments was much less than that induced by chilling. We conclude that the primary role of these gene products is connected with changes that occur during the cold, moist treatment that leads to dormancy-breakage in seeds.

Risk assessment of genetically modified organisms (GMOs) remains a contentious area and a major factor influencing the adoption of agricultural biotech. Methodologically, in many countries, risk assessment is conducted by expert committees with little or no recourse to databases and expert systems that can facilitate the risk assessment process. In this paper we describe DTREEv2, a computer-based decision support system for the identification of hazards related to the introduction of GM-crops into the environment. DTREEv2 structures hazard identification and evaluation by means of an Event-Tree type of analysis. The system produces an output flagging identified hazards and potential risks. It is intended to be used for the preparation and evaluation of biosafety dossiers and, as such, its usefulness extends to researchers, risk assessors and regulators in government and industry.