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An apple polyphenol oxidase cDNA is up-regulated in wounded tissues.

A full-length cDNA clone encoding apple (Malus domesticus) polyphenol oxidase (PPO) was isolated from a fruit peel cDNA library. Southern analysis indicated that apple PPO is encoded by a divergent multigene family. By northern analysis, PPO mRNA was only detected in a fruit sample taken one week after full bloom. PPO mRNA accumulated in wounded tissues, and also in peel tissue showing the symptoms of superficial scald, a post-harvest disorder. The induction of PPO mRNA provides the first evidence for transcriptional control of PPO expression after wounding or the manifestation of a physiological disorder.

Catechol Oxidase↗

Purification, properties and partial amino-acid sequence of 1-aminocyclopropane-1-carboxylic acid oxidase from apple fruits.

The enzyme which converts 1-aminocyclopropane-1-carboxylic acid (ACC) into ethylene, ACC oxidase, has been isolated from apple fruits (Malus x domestica Borkh. cv. Golden Delicious), and for the first time stabilized in vitro by 1,10-phenanthroline and purified 170-fold to homogeneity in a five-step procedure. The sodium dodecyl sulfate-denatured and native proteins have similar molecular weights (approx. 40 kDa) indicating that the enzyme is active in its monomeric form. Antibodies raised against a recombinant ACC oxidase over-produced in Escherichia coli from a tomato cDNA recognise the apple-fruit enzyme with high specificity in both crude extracts and purified form. Glycosylation appears to be absent because of (i) the lack of reactivity towards a mixture of seven different biotinylated lectins and (ii) the absence of N-linked substitution at a potential glycosylation site, in a sequenced peptide. Phenylhydrazine and 2-methyl-1-2-dipyridyl propane do not inhibit activity, indicating that ACC oxidase is not a prosthetic-heme iron protein. The partial amino-acid sequence of the native protein has strong homology to the predicted protein of a tomato fruit cDNA demonstrated to encode ACC oxidase.

Amino Acid Oxidoreductases↗

Criteria for keeping quality in Golden Delicious apples.

Apples harvested in October 1975, from several regions of the Netherlands, were stored in air or under a controlled atmosphere (CA), at 3--4 degrees C in 11 batches. They were analysed for L-malate and sucrose, and their acceptability was assessed by pilot test. The decrease of L-malate during storage (0.1 g in 37.5 days in air and in 75 days under CA) was confirmed. Thus one can predict (at the start of the trial) the L-malate content at a later stage of the storage. It had been proposed previously that 0.39--0.45% of L-malate corresponds to similar proportions of acceptable and unacceptable fruits, as determined by a laboratory panel. This hypothesis was not corroborated by the pilot test. Based on the data of this test, the original criteria were adapted as follows. In most batches, the percentage of unacceptable apples is low (0--15%) as long as the content of L-malate is greater than or equal to 0.55%. If L-malate has already decreased to 0.54--0.50%, then sucrose should be greater than or equal to 2%.

Food Preservation↗

[Pesticide residues in dried apples (author's transl)].

60 samples of dried apple cubes, flakes and powder imported from april 1975 to may 1975 were analyzed for organochlorine and organophoshorus pesticides. All values lay well below the maximum limits for pesticide residues allowed in the Fed. Rep. of Germany. The level of total DDT in apple powder was significantly higher than in cubes and flakes.

Aldrin↗

[On the metabolism of sulphur dioxide in apples (author's transl)].

Apples of the two varieties, Boskoop and Brettacher, were exposed to 35S labeled sulphur dioxide. After storage at several conditions the distribution of 35S-activity among the three fractions, sulphite, sulphate and sulphonate was examined. The major portion of the radioactivity was found in the sulphate fraction. The activity decreased remarkably from the peel towards the core. Appreciable differences in distribution patterns were found between the two apples varieties.

Food Preservation↗

Changes in starch content and amylase zymograms during storage of Golden Delicious and Cox's Orange Pippin apples.

Changes in starch content and amylase zymograms were followed during storage of Golden Delicious and Cox's Orange Pippin apples. Although the former was stored at 3-4 degrees C under controlled atmosphere (3--4% O2; 7--8% CO2 by volume) and the latter in air at 17 degrees C, in both, the multiple forms of amylases remained active, even after the starch content decreased to zero. It is the lack of starch substrate, therefore, rather than of enzymes that limits the amylase action in the stored apple.

Amylases↗

[Determination of patulin in foodstuffs. Part I: Determination of patulin in apple juice (author's transl)].

For the determination of Patulin in apple juice a method of analysis was developed permitting the detection of 40 microng Patulin/kg apple juice. With additions between 120--200 microng Patulin/kg the recovery rates lie between 82 and 90%. After a pre-purification of the crude extract by liquid-liquid extraction and column-chromatography the Patulin is determined thinlayer chromatographically by reflectance measurement at 273 nm.

Beverages↗

Dislodgeable azinphosmethyl residues from air blast spraying of apple foliage in Ohio.

The distribution and decay rates of dislodgeable residues of azinphosmethyl applied by two types of air blast sprayers on apple foliage in Ohio were investigated. Leaf discs were taken from nine sites located on the periphery of Cortland apple trees at five dates after spraying. The sprayer delivering the higher airflow rate, but lower velocity, deposited the pesticide much more uniformly over the trees and applied more in the top of the tree. The other sprayer deposited the greatest proportion of pesticide on the site nearest the sprayer. The residue decreased at all sites and ranged from 42 to near 100% decrease at 14 days after spraying.

Agriculture↗

Measurement of sugar content in Fuji apples by FT-NIR spectroscopy.

To evaluate the potential of FT-NIR spectroscopy and the influence of the distance between the light source/detection probe and the fruit for measuring the sugar content (SC) of Fuji apples, diffuse reflectance spectra were measured in the spectral range from 12500 to 4000 cm(-1) at 0 mm, 2 mm, 4 mm and 6 mm distances. Four calibration models at four distances were established between diffused reflectance spectra and sugar content by partial least squares (PLS) analysis. The correlation coefficients (R) of calibrations ranged from 0.982 to 0.997 with SEC values from 0.138 to 0.453 and the SECV values from 0.74 to 1.58. The best model of original spectra at 0 mm distance yielded high correlation de-termination of 0.918, a SEC of 0.092, and a SEP of 0.773. The results showed that different light/detection probe-fruit distances influence the apple reflective spectra-and SC predictions.

Calibration↗

Identification of SCAR markers linked to Pl-w mildew resistance in apple.

Resistance to powdery mildew is an important objective for cultivar improvement programmes of apple and several different major genes for resistance to mildew are available. Molecular markers linked to such key traits can be used to screen progenies for resistant individuals. A progeny derived from the crab apple 'White Angel' (the source of Pl-w) was screened for resistance to mildew for two seasons in the glasshouse and four seasons in the field. DNA bulks of resistant and susceptible seedlings were screened with 176 AFLP primer combinations. Seven AFLP markers were identified that differentiated the bulks, and two of these markers were developed into SCARs, EM M01 and EM M02, mapping at 4.6 and 6.4 recombination units from Pl-w.

Base Sequence↗

Creating a saturated reference map for the apple (Malus x domestica Borkh.) genome.

The availability of a high quality linkage map is essential for the detection and the analysis of quantitative traits. Such a map should cover a significant part of the genome, should be densely populated with markers, and in order to gain the maximum advantage should be transferable to populations or cultivars other than the ones on which it has been constructed. An apple genetic linkage map has been constructed on the basis of a segregating population of the cross between the cultivars Fiesta and Discovery. A total of 840 molecular markers, 475 AFLPs, 235 RAPDs, 129 SSRs and 1 SCAR, were used for the two parental maps constructed with JoinMap and spanning 1,140 cM and 1,450 cM, respectively. Large numbers of codominant markers, like SSRs, enable a rapid transfer of the map to other populations or cultivars, allowing the investigation of any chosen trait in another genetic background. This map is currently the most advanced linkage map in apple with regard to genome coverage and marker density. It represents an ideal starting point for future mapping projects in Malus since the stable and transferable SSR frame of the map can be saturated quickly with dominant AFLP markers.

Automation↗

Narrowing down the region of the Vf locus for scab resistance in apple using AFLP-derived SCARs.

A narrow-down strategy to restrict the Vf region, which controls resistance to the fungal disease apple scab in apple, to a genetic distance of 0.4 cM is presented. Using 11 AFLP-derived SCARs and three RAPD-derived SCARs, all linked to the Vf gene, we subjected 1,412 scab-resistant individuals from 16 mapping populations to genotype analysis. Eleven recombinant individuals were identified within a genetic distance of 0.9 cM around the Vf gene. Using these 11 recombinants, we achieved fine-resolution of several AFLP-derived SCAR markers surrounding the Vf gene, resulting in the following genetic linkage map: ACS-6 and ACS are located left of the Vf gene at genetic distances of 0.2 cM and 0.1 cM, respectively; ACS-7 and ACS-9 are inseparable from the Vf gene; ACS-8, ACS-10, and ACS-4 are located to the right of the Vf gene at genetic distances of 0.1 cM, 0.4 cM, and 0.5 cM, respectively; the remaining five SCARs-ACS-11, ACS-5, ACS-2, ACS-1, and AL07-are inseparable and are located right of the Vf gene at a genetic distance of 0.7 cM. By integrating this linkage data with our previous physical map, we generated a revised map of the narrowed-down region of Vf.

Ascomycota↗

Characterisation and transferability of apple SSRs to two European pear F1 populations.

European pear (Pyrus communis L.) is among the important fruit species for which only few genetic studies have been carried out. Available evidence indicates that simple sequence repeats (SSR) are very useful as molecular markers because they are codominant, highly polymorphic, abundant and reproducible. The present paper reports more than 100 apple SSR markers in two populations of European pear; a total of 41 SSR markers were then positioned on a genetic linkage map of the cross 'Passe Crassane' x 'Harrow Sweet' and 31 in the map 'Abbe Fetel' x 'Max Red Bartlett'. Syntenic relationships between pear and apple maps have been considered for the chromosomes carrying two or more SSR markers. The alignment among the two maps supports the colinearity of the two genomes with respect both to identification and to orientation of the linkage groups.

Chromosome Mapping↗

Identification of molecular markers linked to the mildew resistance gene Pl-d in apple.

Powdery mildew poses a serious problem for apple growers, and resistance to the disease is a major objective in breeding programmes for cultivar improvement. As selective pressure allows pathogens to overcome previously reliable resistances, there is a need for the introduction of novel resistance genes into new breeding lines. This investigation is concerned with the identification of the first set of molecular markers linked to the gene for mildew resistance, Pl-d, from the accession 'D12'. As no prior information on the map position or markers for Pl-d were available, a bulked-segregant approach was used to test 49 microsatellite primers, 176 amplified fragment length polymorphism (AFLP) primers and 80 random amplified polymorphic DNA (RAPD) primers in a progeny segregating for Pl-d resistance, 'Fiesta' (susceptible) x A871-14 ('Worcester Pearmain' x 'D12'). The segregations of the markers identified in the resistant and susceptible bulks were scored in the progeny, then the recombination fractions between Pl-d and the most tightly linked markers were calculated and a map prepared. Three AFLP, one RAPD and two microsatellite markers were identified. One AFLP was developed into a sequence-characterised amplified region marker, while the microsatellites CH03C02 and CH01D03 were flanking markers, 7 and 11 recombination units, respectively, from Pl-d. Two more distant microsatellites on the same linkage group, CH01D09 and CH01G12, confirmed the orientation of the markers on the linkage group. These microsatellites place Pl-d on the bottom of linkage group 12 in published apple maps, a region where a number of other disease resistance genes have been identified.

Ascomycota↗

Retrotransposon characterisation and fingerprinting of apple clones by S-SAP markers.

Retrotransposons have been found to comprise the most common class of transposable elements in eukaryotes and to occur in high copy number in plant genomes. Several of these elements have been sequenced and were found to display a high degree of heterogeneity and insertional polymorphism, both within and between species. The dispersion, ubiquity and prevalence of retrotransposons in plant genomes provide an excellent basis for the development of marker systems and, hence, may be good molecular candidates in distinguishing among apple clones, when they represent bud mutations of the original variety, considering that the random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) used thus far in fingerprinting analyses have failed to meet discrimination expectations. The technique called sequence-specific amplified polymorphism (S-SAP), which makes it possible to identify dominant markers for the detection of variation in the DNA flanking the retrotransposon insertion site, was used in the present study to distinguish several clones of the cultivars 'Gala' and 'Braeburn' in apple fingerprinting. Moreover, our results suggest that the bud mutations, which have generated new patented varieties of 'Gala' and 'Braeburn', appear to derive from retrotransposon insertion.

Alleles↗

Process control of apple winemaking by low-resolution gas-phase Fourier-transform infrared spectroscopy.

Four apple wine fermentation processes have been observed by means of direct-inlet gas-phase FTIR spectroscopy. The apple juice concentrates were each fermented by two species of Saccharomyces cerevisiae starters, and the experiment was repeated. The development of the concentrations of 1-propanol, 4-methylpyridine, acetaldehyde, acetic acid, and ethyl acetate was monitored. Two different sampling methods were used--static headspace and direct injection of the must. The performance of the FTIR method is limited by the high ethanol concentration. It can be mathematically proven that the amount of sample can be selected so that any distortion due to ethanol is minimized. Headspace GC-MS was used for preliminary compound identification.

1-Propanol↗

Antisense inhibition of sorbitol synthesis leads to up-regulation of starch synthesis without altering CO2 assimilation in apple leaves.

Sorbitol is a primary end-product of photosynthesis in apple (Malus domestica Borkh.) and many other tree fruit species of the Rosaceae family. Sorbitol synthesis shares a common hexose phosphate pool with sucrose synthesis in the cytosol. In this study, 'Greensleeves' apple was transformed with a cDNA encoding aldose 6-phosphate reductase (A6PR, EC 1.1.1.200) in the antisense orientation. Antisense expression of A6PR decreased A6PR activity in mature leaves to approximately 15-30% of the untransformed control. The antisense plants had lower concentrations of sorbitol but higher concentrations of sucrose and starch in mature leaves at both dusk and predawn. (14)CO(2) pulse-chase labeling at ambient CO(2) demonstrated that partitioning of the newly fixed carbon to starch was significantly increased, whereas that to sucrose remained unchanged in the antisense lines with decreased sorbitol synthesis. Total activities of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), sucrose-phosphate synthase (EC 2.4.1.14), and ADP-glucose pyrophosphorylase (EC 2.7.7.27) were not significantly altered in the antisense lines, whereas both stromal and cytosolic fructose-1,6-bisphosphatase (EC 3.1.3.11) activities were higher in the antisense lines with 15% of the control A6PR activity. Concentrations of glucose 6-phosphate and fructose 6-phosphate (F6P) were higher in the antisense plants than in the control, but the 3-phosphoglycerate concentration was lower in the antisense plants with 15% of the control A6PR activity. Fructose 2, 6-bisphosphate concentration increased in the antisense plants, but not to the extent expected from the increase in F6P, comparing sucrose-synthesizing species. There was no significant difference in CO(2) assimilation in response to photon flux density or intercellular CO(2) concentration. We concluded that cytosolic FBPase activity in vivo was down-regulated and starch synthesis was up-regulated in response to decreased sorbitol synthesis. As a result, CO(2) assimilation in source leaves was sustained at both ambient CO(2) and saturating CO(2).

Carbon Dioxide↗

Structure and expression of spermidine synthase genes in apple: two cDNAs are spatially and developmentally regulated through alternative splicing.

Three cDNAs (MdSPDS1, 2a and 2b) encoding spermidine synthase (SPDS), a key enzyme in the polyamine biosynthesis, have been cloned from apple [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.]. The deduced amino acid sequences of their protein products share 76-83% identity with SPDSs of other higher plants. A comparison of the sequences of the three cDNAs and of the two corresponding genomic DNA fragments (SPDS1 and SPDS2) indicated that MdSPDS1 was transcribed from the SPDS1 sequence, whereas MdSPDS2a and MdSPDS2b were both derived from SPDS2 by alternative splicing. To learn more about the physiological roles of MdSPDS1, MdSPDS2a and MdSPDS2b, Northern analyses were carried out, together with measurements of polyamine content. Levels of both MdSPDS1 and MdSPD2a were higher in young leaves than in mature leaves and shoots. In fruits, mRNA levels were nearly as high as in young leaves and remained high during fruit development. By RT-PCR, MdSPDS2b transcripts were detected in mature leaves and shoots, but not in young leaves and fruits. These results indicate that MdSPDS2a and MdSPDS2b are differentially regulated in a tissue- and developmentally specific manner. The content of free polyamines in mesocarp tissues was measured at five stages of fruit development. At all stages, spermidine (Spd) was the predominant form of polyamine. The level of Spd was high at the early growth stage and declined to about 90% during later developmental stages. The possible regulation of SPDS expression during apple fruit development is discussed.

Alternative Splicing↗