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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]

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 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.

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.