Search

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. [ABSTRACT FROM AUTHOR]

Laser photoacoustic spectroscopy continuously quantified the ethylene (C2H4) produced by strawberry flowers and fruits developing in planta. C2H4 was first detected as flower buds opened and exhibited diurnal oscillations (to approximately 200 pl flower−1 h−1) before petal abscission. Exogenous application of silver thiosulphate (STS) to detached flowers inhibited petal abscission and flower senescence. In fruit, C2H4 production was maintained at a ‘low level’ (10–60 pl fruit−1 h−1) until fruit expanded when levels increased in a diurnal pattern (to 200 pl fruit−1 h−1). After expansion, C2H4 production declined to a low level until fruit attained the red-ripe stage for at least 24 h. After this time, C2H4 levels increased linearly (no diurnal fluctuation) to approximately 1 nL fruit−1 h−1. Twenty-four hours after the re-initiation of C2H4 production by red fruit, CO2 levels increased approximately three-fold, indicative of a respiratory climacteric. STS applied to fruits developing in planta and dissected fruit parts ex situ established that C2H4 production is regulated by negative feedback until fruits had expanded. The C2H4 produced by red-ripe fruit was regulated by positive feedback. Anti-1-amino-cyclopropane-1-carboxylic acid oxidase IgG localization identified immunoreactive antigens of 40 and 30 kDa (Mr) within the fruit achenes of expanding and red-ripe fruit. Analysis of dissected fruit showed that seed C2H4 accounts for 50% the C2H4 that is detectable from ripe fruit. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Désirée) 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. [ABSTRACT FROM PUBLISHER]

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 Désirée which normally produces tubers containing c. 5.6 μg carotenoid g−1 DW and also in Solanum phureja L. cv. Mayan Gold which has a tuber carotenoid content of typically 20 μg carotenoid g−1 DW. In developing tubers of transgenic crtB Désirée lines, carotenoid levels reached 35 μg carotenoid g−1 DW and the balance of carotenoids changed radically compared with controls: β-carotene levels in the transgenic tubers reached c. 11 μg 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 μg carotenoid g−1 DW in the most affected transgenic line. In these tubers, the major carotenoids were violaxanthin, lutein, antheraxanthin, and β-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. [ABSTRACT FROM PUBLISHER]

Fruit starch reserves can be an important contributor to the sugar content of some ripe fruit, and despite the relatively high financial premiums (compared to other fruit) commanded by ripe strawberries, neither their starch or sugar biochemistry has been examined in detail. This study assessed the rate of starch biosynthesis and breakdown in developing strawberry and sought to determine the temporal changes in the activities of selected enzymes known to be involved in sucrose-starch interconversions. Scanning electron microscopy revealed that starch levels appeared greatest in immature strawberry (Fragaria×ananassa, cv. Elsanta) at 7 days postanthesis, as evidenced by a decrease in the number of cells containing starch granules as ripening progressed. Levels of key enzymes of starch and sugar metabolism estimated using Western blotting and enzyme activity analysis showed that activities did not correlate with antigen levels. In particular, enzyme activity recovery experiments indicated that losses were due to non-proteinaceous inhibitors, and in particular protein binding: highlighting the potential for misinterpretation of enzyme activity data gathered from ripening (strawberry) fruit tissue extracts. Consequently, in vitro experiments using [U-14C] glucose revealed that incorporation to starch is low (11%) at the earliest developmental stages when starch content is greatest. Starch synthesis rate then declines to non-detectable levels as fruit expand and ripen. These results show that starch accumulates extremely early in the fruit formation process and that starch degradation predominates during fruit growth and development. We estimate that breakdown of transient starch can contribute up to 3% of the sugar accumulated in ripe fruit. [ABSTRACT FROM AUTHOR]

Salt-extractable proteins from the cell walls of immature and ripe strawberry (Fragaria × 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Polymerase chain reaction-based methodology was used to obtain a cDNA clone (MAL2) from potato (Solanum tuberosum L.) with the sequence characteristics of an α-glucosidase. Phylogenetic analysis of the deduced polypeptide encoded by this cDNA demonstrated that the most similar sequences were α-glucosidases and α-xylosidases of plant origin. The MAL2 cDNA was expressed in Escherichia coli and the recombinant MAL2 protein was affinity-purified. MAL2 catalysed the hydrolysis of a range of maltooligomers and p-nitrophenyl-α-D-glucopyranoside with a pH optimum of 5.5–5.7. The substrate with the lowest Km value was maltotetraose (3.7 mM). The MAL2 expression product did not catalyse the hydrolysis of xyloglucan oligosaccharides, p-nitrophenyl-α-D-xylopyranoside or gelatinised potato starch. MAL2 was down-regulated in transgenic potato plants using an antisense approach. In several independent transgenic antisense lines, MAL2 expression was severely down-regulated. Despite this, no decrease in total extractable α-glucosidase and α-xylosidase activity could be detected in tissues from the transgenic plants. In glasshouse trials, no visible phenotype, change in tuber yield or carbohydrate content was associated with MAL2 down-regulation. The implications of these results are discussed. [ABSTRACT FROM AUTHOR]

Presents a study in which transgenic plants, in which the potato gene MAL1 expression was down-regulated, were generated. Methods; Results; Conclusion that the results suggest that MAL1 encodes glucosidase II activity.

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. [ABSTRACT FROM AUTHOR]

Tuberising stolon tips of potato (Solonum tuberosum L. cv. Record) accumulate starch and sucrose but the hexose content, particularly fructose, declines rapidly. Similar changes occur in the region 2cm behind the swelling apex but the decline in glucose is far more pronounced than in the developing tuber. Tuberisation is characterised by an apparent switch from an invertase-dominated sucrolytic system (both acid and alkaline invertases [EC 3.2.1.26] are present to one dominated by sucrose synthase (EC 2.4.1.13). Sucrose synthase and fructokinase (EC 2.7.1.4) activities were, at a maximum. ca 10- and 5-fold higher, respectively in the swelling stolon tip compared with the non-tuberising region. At the highest starch contents attained, the starch level in the young developing tuber was approximately double that in the adjacent non-tuberising stolon region. Immunoblots revealed that developmental changes in sucrose synthase, fructokinase and alkaline invertase polypeptides corresponded with enzyme activities. Antibodies raised against the N-terminal amino acid sequence of a soluble invertase purified from mature tubers did not detect significant quantities of a polypeptide in stolons and young, developing tubers. Antibodies raised against an in vitro expression product of an apoplastic invertase cloned from a leaf cDNA library detected a polypeptide in developing tubers but not in mature ones. However, expression of the protein did not correlate well with acid invertase activity during early tuber formation. [ABSTRACT FROM AUTHOR]

The antioxidant status of potato (Solanum tuberosum L.) tubers of two genotypes. cv. Désirée and clone 10337de40 was investigated in relation to susceptibility to internal rust spot (IRS), a Ca2+-related physiological disorder. Concentrations of total calcium within the perimedulla tissue of tubers, grown with a restricted (1 mM CaCI2) Ca2+ supply, were similar in cv. Désirée (IRS resistant) and clone 10337de40 (IRS susceptible). A range of antioxidants was assayed in order to assess antioxidant status in both genotypes under the two Ca2+ treatments. Although no appreciable differences were detected between low Ca2+ and control treatments, certain antioxidants were present at significantly higher levels in the IRS resistant genotype. cv. Désirée. These included dehydroascorbate reductase (EC 1.8.5.1) activity (more than 100% higher), total glutathione content (ca 40% higher), glutathione reductase (EC 1.6.4.2) activity (almost 50% higher). peroxidase (EC 1.11.1.7) activity (ca 60% higher) and superoxide dismutase (EC 1.15.1.1) activity (almost 80% higher). There was no difference in ascorbate content. ascorbate free radical reductase activity (EC 1.6.5.4), α-tocopherol levels and catalase activity (EC 1.11.1.6) between the two genotypes. The possible relationship between resistance to IRS and a superior antioxidant status, found in cv. Désirée. is discussed. [ABSTRACT FROM AUTHOR]

Focuses on the sequence analysis, genomic organization and gene expression of StNR2 and StNR3 cDNA clones representing alleles of the nitrate reductase gene of potato, Solanum tuberosum L. cv. Desiree. Comparison of deduced amino acid sequence encoded by StNR2 and StNR3 cDNA; Distribution of nitrate reductase transcript and activity.

Summary: Using an Arabidopsis thaliana expressed sequence tag with sequence similarity to human lysosomal α-glucosidase as a probe, a potato cDNA was isolated. The cDNA encodes a polypeptide with an Mr value of 105,400 and the most significant matches of the deduced amino acid sequence are with members of family 31 of glucosyl transferase. The potato cDNA was expressed in a strain of Saccharomyces cerevisiae that is deficient in maltase activity and unable to grow using maltose as a carbon source (ABYSMAL81). Expression of the potato cDNA in the mutant yeast strain restores its ability to use maltose as a carbon source for growth. Additionally, α-glucosidase activity could be measured in extracts of the yeast cells following complementation. A range of maltodextrins were substrates for this activity. The steady-state expression level of the potato α-glucosidase gene was low in most tissues examined, the highest levels occurring in sprouting tubers and source leaves. [ABSTRACT FROM AUTHOR]