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Characterization of ionically bound peroxidases from apple (Mallus pumilus) fruits.

Ionically bound peroxidases (POD) were salt extracted from the pulp of four Indian apple varieties, i.e., Golden delicious HP, Golden delicious JK, Red delicious, and Royal delicious. They were precipitated with chilled ethanol. Thermal treatments of partially purified enzymes were given from 40-70 degrees C for 30 minutes. Golden delicious HP peroxidase showed thermostability at 60 degrees C, while three other peroxidases were observed at 50 degrees C. Phenolic compounds (i.e., caffeic acid, ferulic acids, p-coumaric acid, protocatechuic acid) and metal ions (i.e., Cu2+ and Fe2+) activated all apple peroxidases. However, Mn2+ inhibited the peroxidases from Golden delicious HP, Golden delicious JK, and Red delicious, and a substantial increase was observed in Royal delicious peroxidase. Mg2+ inhibited the peroxidases from Golden delicious HP and Red delicious, but marginal activation was reported in peroxidases from Golden delicious JK and Royal delicious. Zn2+ established stimulation in Golden delicious HP and Golden delicious JK peroxidases, but inhibition was observed in peroxidases in Red delicious and Royal delicious.. Methionine, proline, tryptophan, and valine stimulated all four apple peroxidases, but cysteine showed inhibition in Golden delicious JK.

Amino Acids↗

Causes and effects of changes in xylem functionality in apple fruit.

BACKGROUND AND AIMS: The xylem in fruit of a number of species becomes dysfunctional as the fruit develops, resulting in a reduction of xylem inflow to the fruit. Such a reduction may have consequential effects on the mineral balance of the fruit. The aim of this study was to elucidate the dynamics and nature of xylem failure in developing apples (Malus domestica) showing differing susceptibilities to bitter pit, a calcium-related disorder. METHODS: Developmental changes in xylem functionality of the fruit were investigated in "Braeburn" and "Granny Smith" apples by using a dye infusion technique, to stain the vasculature along the path of dye movement. The vascular bundles were clearly visible in transverse section when fruit were sectioned equatorially. The intensity of staining of the vascular bundles in the fruit was recorded at regular intervals throughout the season. Tissue containing dysfunctional bundles was fixed and embedded in wax for subsequent sectioning and examination. KEY RESULTS: As the season progressed, an increasing proportion of vascular bundles failed to show any staining, with the most marked change occurring in the primary bundles, and in nearly all bundles with increasing distance from the stalk end of the fruit. Decreased conductance in the primary bundles of "Braeburn" occurred earlier than in "Granny Smith". Microscopy revealed that the xylem in vascular bundles of the fruit suffered substantial damage, indicating that the mode of dysfunction was via the physical disruption of the xylem caused by expansion of the flesh. CONCLUSIONS: Results support the view that the relative calcium deficiency of apple fruit is due to a progressive breakdown of xylem conductance caused by growth-induced damage to the xylem strand in the bundle. The earlier onset of xylem dysfunction in the cultivar more susceptible to bitter pit suggests that the relative growth dynamics of the fruit may control the occurrence of calcium-related disorders.

Biological Transport↗

Feasibility of thiocarbamate pesticide analysis in apples by supercritical fluid extraction and high-performance liquid chromatography.

Supercritical fluid extraction produced comparable results with liquid-solid extraction for the analysis of several thiocarbamate pesticides from apples at the 2 ppm spike level. These results were achieved with a simple one-step extraction procedure. The use of diatomaceous earth (Celite, Supelco, Inc.; Bellefonte, PA) served to increase thiocarbamate recoveries by aiding in the immobilization of the aqueous component of the apple matrix. High-performance liquid chromatography coupled with ultraviolet absorbance detection (HPLC-UV) had the most viable means of quantitation when compared with micro-HPLC-sulfur chemiluminescence detection (SCD) and gas chromatography-flame-ionization detection (GC-FID). The small injection volumes used with the micro-HPLC-SCD system made thiocarbamate detection at a spiking level of 2 ppm impossible. SCD did provide, however, valuable qualitative information about the nature of the apple coextractants.

Chromatography, High Pressure Liquid↗

Identification and characterisation of proteinase inhibitors and their genes from seeds of apple (Malus domestica).

Trypsin and papain proteinase inhibitors have been identified and purified from aqueous extracts of apple seeds (Malus domestica). Superdex G75 gel filtration chromatography identified a higher molecular weight (HMW) papain inhibitory fraction (22-26 kDa) and a lower molecular weight papain and trypsin inhibitory fraction (6-12 kDa). The lower molecular weight fraction was separated into a trypsin inhibitor (designated Trp1) and early (designated Pap1) and late (designated Pap2) eluting papain inhibitors after anion exchange (Hitrap SP) chromatography. For Pap2, two inhibitory peaks (designated Pap2-1 and Pap2-2) were identified after further anion exchange (Resource S) chromatography. Each of these lower molecular weight inhibitors was purified by reverse phase HPLC to homogeneity as determined by SDS-PAGE and by mass spectrometry. The HMW papain inhibitory fraction was purified further by anion-exchange (Hitrap Q followed by Resource Q) column chromatography where a minor inhibitor (HMWPap1) and major inhibitor (HMWPap2) fraction were identified. The relative abundance in seeds of apple and the spectrum of proteinase inhibition has been determined for all of these inhibitors. Reverse-phase HPLC separated HMWPap2 into a minor (HMWPap2-1) and a major (HMWPap2-2) inhibitory fraction, and SDS-PAGE and mass spectrometry confirmed that HMWPap2-2 was purified to homogeneity. Amino acid composition data were obtained from Trp1, Pap1, Pap2-2, and HMWPap2-2, and N-terminal sequence data from Trp1, Pap2-1, Pap2-2, and HMWPap2-2, with two of these sequences (Pap2-2 and HMWPap2-2) perfectly matching predicted protein sequences based on EST sequences from an apple database. The relationship of these inhibitors with those of other species is discussed.

Amino Acid Sequence↗

Comparison of microcapsule density with various apple tissues and formulations of oriental fruit moth (Lepidoptera: Tortricidae) sprayable pheromone.

The density of microcapsules was compared on 'Golden Delicious' mature and immature foliage, fruit, and 1-yr-old limbs after dip treatments with Phase III oriental fruit moth, Grapholita molesta (Busck), microencapsulated pheromone. Microcapsules were counted with a dissecting microscope at 50x magnification after apple tissues were treated with a two-step dye staining process to increase the visibility of the microcapsules. The number of Phase III microcapsules on 1-yr limbs treated with a field concentration was two- to threefold greater than the number of microcapsules on similarly treated fruit or foliage. Phase I, III, and V OFM MEC and Checkmate OFM-F formulations of microencapsulated pheromone also were compared to determine their abundance on mature apple foliage. The Phase V OFM MEC formulation had the highest density of microcapsules when mature foliage was treated at the field rate. The Phase I treatment had the greatest difference between upper and lower leaf surfaces with 18-fold greater microcapsule density on the upper surface. On mature apple leaves treated with Phase III MEC, the number of microcapsules/cm2 was two- to threefold greater on fields of view without the mid-vein than those that included the mid-vein. The cuticle structure and abundance of trichomes are two factors that may have contributed to differences in microcapsule density among plant tissue types, top and bottom leaf surfaces, and fields of view with and without the mid-vein.

Alkanes↗

Transmission of apple proliferation phytoplasma by Cacopsylla melanoneura (Homoptera: Psyllidae).

Transmission trials of apple proliferation (AP) phytoplasma to healthy apple plants were carried out with Cacopsylla melanoneura (Forster). Both field naturally infected and experimentally infected psyllids were evaluated. The capacity of the different life stages of the insect in transmitting AP was tested. Overwintered adults collected in the orchards were able to transmit AP with a variable efficiency, depending on the infectivity rate of source plants. Experimentally infected nymphs and springtime adults succeeded in the transmission of AP, but the lower number of insect tested and the shorter inoculation period, due to difficulties in rearing the whole cycle of the insect in the laboratory, affected the efficiency. Considering the life history of C. melanoneura, the overwintered adults are the most responsible of the diffusion of AP in apple orchards.

Animals↗

Protection of fruit against infestation by apple maggot and blueberry maggot (Diptera: Tephritidae) using compounds containing spinosad.

Two insecticide formulations containing the naturalyte insecticide spinosad, GF-120 Fruit Fly Bait and SpinTor 2 SC, were compared for control of apple maggot, Rhagoletis pomonella (Walsh), and blueberry maggot, Rhagoletis mendax Curran. In 2002 and 2003, larval infestation in blueberries and apples was significantly lower in plots treated with GF-120 (spinosad bait) or SpinTor than in untreated control plots. Fruit fly infestation in apples was reduced by 67% in 2002 after weekly application of GF-120 for 6 wk. Six weeks of GF-120 treatment reduced infestation in blueberries by 85% in 2002 and 98% in 2003. Plots treated weekly with the bait component of GF-120 for 6 wk had significantly higher infestation of blueberry maggot larvae compared with untreated plots in 2002. Observations of wild R. mendax flies revealed that similar numbers of flies landed on blueberry foliage treated with spinosad bait, the bait component alone, or water droplets. However, flies on spinosad bait and bait treated plants spent significantly more time within 5 cm of the treatment droplets compared with control (water) droplets. Overall, the results demonstrate a high degree of efficacy of baited spinosad formulations against these key pests of temperate fruit and suggest that GF-120 is an arrestant for foraging flies.

Animals↗

Increased root and shoot production during micropropagation of cherry and apple rootstalks: effect of subculture frequency.

Shoot cultures of apple rootstock cv. M9 and cherry rootstock cv. F12/1 were established and then divided into several sub-lines that were subcultured at 28-42-day intervals. Consequently, similarly aged cultures received various numbers of subcultures. Cultures kept at 24 degrees C showed an increase in shoot and root production over time. There were differences in shoot and root production between apple lines, but there were no differences among sub-lines. In cherry, altering the subculture interval affected rooting competence, which increased with time. Cherry cultures maintained at 4 degrees C gave rise to cultures that were as easy to root as cultures kept at 24 degrees C with more frequent subculturing. We conclude that total time in culture is the most important factor bringing about physiological changes in these genotypes of micropropagated apple and cherry.

Journal Article↗

Remobilization and uptake of N by newly planted apple (Malus domestica) trees in response to irrigation method and timing of N application.

Environmentally sound management of N in apple orchards requires that N supply meets demand. In 1997, newly planted apple trees (Malus domestica Borkh. var. Golden Delicious on M.9 rootstock) received daily applications of N for six weeks as Ca(15NO3)(2) through a drip irrigation system at a concentration of 112 mg l(-1) at 2-8, 5-11 or 8-14 weeks after planting. Irrigation water was applied either to meet estimated evaporative demand or at a fixed rate. In 1997, trees were harvested at 5, 8, 11 and 14 weeks after planting; and in 1998 at 3 weeks after full bloom. The amount of fertilizer N recovered was similar in trees in both irrigation treatments, but efficiency of fertilizer use was greater for trees receiving demand-controlled irrigation than fixed-rate irrigation. This was attributed to lower N inputs, greater retention time in the root zone and less N leaching in the demand-controlled irrigation treatments compared with fixed-rate irrigation treatments. Less fertilizer N was recovered by trees receiving an early application of N than a later application of N and this was related to the timing of N supply with respect to tree demand. Demand for root-supplied N was low until 11 weeks after planting, because early shoot and root growth was supported by N remobilized from woody tissue, which involved 55% of the total tree N content at planting. Rapid development of roots > 1 mm in diameter occurred between 11 and 14 weeks after planting, after remobilization ended, and was greater for trees receiving an early application of N than for trees receiving a later application of N. Late-season tree N demand was supplied by native soil N, and uptake and background soil solution N concentrations were higher for trees receiving demand-supplied irrigation compared with fixed-rate irrigation. Total annual N uptake by roots was unaffected by treatments and averaged 6-8 g tree(-1). Nitrogen applications in 1997 affected growth and N partitioning in 1998. Trees receiving early applications of N had more flowers, spur leaves and bourse shoots than trees receiving later applications of N. Consequently, more N was remobilized into fruits in trees receiving early applications of N compared with fruits in trees receiving later applications of N. Demand for N in the young apple trees was low. Early season demand was met by remobilization from woody tissues and the timing of demand for root-supplied N probably depends on whether flowering occurs. Method of N delivery affected the efficiency of N use. We conclude that N demand can be met at soil solution N concentrations of around 20 mg l(-1).

Agriculture↗

Soil temperature and plant growth stage influence nitrogen uptake and amino acid concentration of apple during early spring growth.

In spring, nitrogen (N) uptake by apple roots begins about 3 weeks after bud break. We used 1-year-old 'Fuji' Malus domestica Borkh on M26 bare-root apple trees to determine whether the onset of N uptake in spring is dependent solely on the growth stage of the plant or is a function of soil temperature. Five times during early season growth, N uptake and total amino acid concentration were measured in trees growing at aboveground day/night temperatures of 23/15 degrees C and belowground temperatures of 8, 12, 16 or 20 degrees C. We used (15NH4)(15NO3) to measure total N uptake and rate of uptake and found that both were significantly influenced by both soil temperature and plant growth stage. Rate of uptake of 15N increased with increasing soil temperature and changed with plant growth stage. Before bud break, 15N was not detected in trees growing in the 8 degrees C soil treatment, whereas 15N uptake increased with increasing soil temperatures between 12 and 20 degrees C. Ten days after bud break, 15N was still not detected in trees growing in the 8 degrees C soil treatment, although total 15N uptake and uptake rate continued to increase with increasing soil temperatures between 12 and 20 degrees C. Twenty-one days after bud break, trees in all temperature treatments were able to acquire 15N from the soil, although the amount of uptake increased with increasing soil temperature. Distribution of 15N in trees changed as plants grew. Most of the 15N absorbed by trees before bud break (approximately 5% of 15N supplied per tree) remained in the roots. Forty-six days after bud break, approximately one-third of the 15N absorbed by the trees in the 12-20 degrees C soil temperature treatments remained in the roots, whereas the shank, stem and new growth contained about two-thirds of the 15N taken up by the roots. Total amino acid concentration and distribution of amino acids in trees changed with plant growth stage, but only the amino acid concentration in new growth and roots was affected by soil temperature. We conclude that a combination of low soil temperature and plant developmental stage influences the ability of apple trees to take up and use N from the soil in the spring. Thus, early fertilizer application in the spring when soil temperatures are low or when the aboveground portion of the tree is not actively growing may be ineffective in promoting N uptake.

Amino Acids↗

Low temperature, but not photoperiod, controls growth cessation and dormancy induction and release in apple and pear.

In contrast to most temperate woody species, apple and pear and some other woody species of the Rosaceae family are insensitive to photoperiod, and no alternative environmental seasonal signal is known to control their dormancy. We studied growth and dormancy induction in micropropagated plants of four apple (Malus pumila Mill.) and one pear (Pyrus communis L.) commercial rootstock cultivars in controlled environments. The results confirm that growth cessation and dormancy induction in apple and pear are not influenced by photoperiod, and demonstrate that low temperature (< 12 degrees C) consistently induces both processes, regardless of photoperiodic conditions. Successive stages of the autumn syndrome (growth cessation, formation of bud scales and winter buds, leaf senescence and abscission, and dormancy induction) occurred in response to low temperature. Long days increased internode length at higher temperatures, but had no significant effect on leaf production in any of the cultivars. Chilling at 6 or 9 degrees C for at least 6 weeks (about 1000 h) was required for dormancy release and growth resumption, whereas treatment at 12 degrees C was marginally effective, even after 14 weeks of exposure. We are thus faced with the paradox that the same low temperature conditions that induce dormancy are also required for dormancy release in these species.

Cold Temperature↗

DspA/E, a type III effector essential for Erwinia amylovora pathogenicity and growth in planta, induces cell death in host apple and nonhost tobacco plants.

Erwinia amylovora is responsible for fire blight, a necrotic disease of apples and pears. E. amylovora relies on a type III secretion system (TTSS) to induce disease on hosts and hypersensitive response (HR) on nonhost plants. The DspA/E protein is essential for E. amylovora pathogenicity and is secreted via the TTSS in vitro. DspA/E belongs to a type III effector family that is conserved in several phytopathogenic bacteria. In E. amylovora, DspA/E has been implicated in the generation of an oxidative stress during disease and the suppression of callose deposition. We investigated the fate of DspA/E in planta. DspA/E delivered artificially to apple or tobacco cells by agroinfection induced necrotic symptoms, indicating that DspA/E was probably injected via the TTSS. We confirmed that DspA/E acts as a major cell-death inducer during disease and HR, because the dspA/E mutant is severely impaired in its ability to induce electrolyte leakage in apple and tobacco leaves. Expression of the defense marker gene PR1 was delayed when dspA/E was transiently expressed in tobacco, suggesting that DspA/E-mediated necrosis may be associated with an alteration of defense responses.

Amino Acid Sequence↗

Apple proteins that interact with DspA/E, a pathogenicity effector of Erwinia amylovora, the fire blight pathogen.

The disease-specific (dsp) gene dspA/E of Erwinia amylovora encodes an essential pathogenicity effector of 198 kDa, which is critical to the development of the devastating plant disease fire blight. A yeast two-hybrid assay and in vitro protein pull-down assay demonstrated that DspA/E interacts physically and specifically with four similar putative leucine-rich repeat (LRR) receptor-like serine/threonine kinases (RLK) from apple, an important host of E. amylovora. The genes encoding these four DspA/E-interacting proteins of Malus xdomestica (DIPM1 to 4) are conserved in all genera of hosts of E. amylovora tested. They also are conserved in all cultivars of apple tested that range in susceptibility to fire blight from highly susceptible to highly resistant. The four DIPMs have been characterized, and they are expressed constitutively in host plants. In silico analysis indicated that the DIPMs have similar sequence structure and resemble LRR RLKs from other organisms. Evidence is presented for direct physical interaction between DspA/E and the apple proteins encoded by the four identified clones, which may act as susceptibility factors and be essential to disease development. Knowledge of DIPMs and the interaction with DspA/E thus may facilitate understanding of fire blight development and lead to new approaches to control of disease.

Amino Acid Sequence↗

The complete nucleotide sequence of apple mosaic virus (ApMV) RNA 1 and RNA 2: ApMV is more closely related to alfalfa mosaic virus than to other ilarviruses.

The complete nucleotide sequences of apple mosaic virus RNA 1 and 2 have been characterized. Apple mosaic virus RNA 1 is 3476 nucleotides in length and encodes a single large open reading frame (ORF), whereas apple mosaic virus RNA 2 is 2979 nucleotides in length and also encodes a single ORF. The amino acid sequences encoded by RNA 1 and 2 show similarity to all of the other ilarviruses for which sequence data are available, but both are more closely related to alfalfa mosaic virus (AMV) than to other ilarviruses. Points of similarity include the absence of ORF 2b, present on the RNA 2 of all previously characterized ilarviruses. The close relationship to AMV also occurs in the movement protein, encoded by RNA 3, but not with the coat protein. These data suggest that the present taxonomy should be revised, and that AMV should be considered an aphid-transmissible ilarvirus.

5' Untranslated Regions↗

Endopolygalacturonase in Apples (Malus domestica) and Its Expression during Fruit Ripening.

The activity of polygalacturonase (PG) has been detected in ripe McIntosh apples (Malus domestica Borkh. cv McIntosh) both by enzyme activity measurement and immunoblotting using an anti-tomato-PG antibody preparation. PG activity increased during fruit ripening and remained steady, or decreased slightly, after 5 months of controlled atmospheric storage. The enzyme had a relative molecular weight of 45,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 56,000 to 61,000 when determined by gel filtration. Viscosity and reducing end group measurements with a commercial pectin preparation showed that the enzyme is endo acting. In RNA and DNA blot hybridization experiments, a full-length tomato PG cDNA hybridized with the apple RNA and DNA, showing the identity of genes encoding the activity of the enzyme in tomato and apple.

Journal Article↗

Loss of highly branched arabinans and debranching of rhamnogalacturonan I accompany loss of firm texture and cell separation during prolonged storage of apple.

Growth and maturation of the edible cortical cells of apples (Malus domestica Borkh) are accompanied by a selective loss of pectin-associated (1-->4)-beta-D-galactan from the cell walls, whereas a selective loss of highly branched (1-->5)-alpha-L-arabinans occurs after ripening and in advance of the loss of firm texture. The selective loss of highly branched arabinans occurs during the overripening of apples of four cultivars (Gala, Red Delicious, Firm Gold, and Gold Rush) that varied markedly in storage life, but, in all instances, the loss prestages the loss of firm texture, measured by both breaking strength and compression resistance. The unbranched (1-->5)-linked arabinans remain associated with the major pectic polymer, rhamnogalacturonan I, and their content remains essentially unchanged during overripening. However, the degree of rhamnogalacturonan I branching at the rhamnosyl residues also decreases, but only after extensive loss of the highly branched arabinans. In contrast to the decrease in arabinan content, the loss of the rhamnogalacturonan I branching is tightly correlated with loss of firm texture in all cultivars, regardless of storage time. In vitro cell separation assays show that structural proteins, perhaps via their phenolic residues, and homogalacturonans also contribute to cell adhesion. Implications of these cell wall modifications in the mechanisms of apple cortex textural changes and cell separation are discussed.

Carbohydrates↗

Metabolic Changes in Excised Fruit Tissue. IV. Changes Occurring in Discs of Apple Peel During the Development of the Respiration Climacteric.

It is shown that a sequential development of a series of enzyme systems occurs in the peel of the apple as the respiration climacteric develops in the whole fruit. The sequence of development of these systems, i.e. acetate incorporation into lipid, production of ethylene, incorporation of amino acid into protein and, finally, the decarboxylation of added malate (malate effect) is the same as that shown earlier for the short term (24 hr) aging of peel discs from pre-climacteric apples. As these systems appear in the initial discs from fruit passing through the climacteric they gradually cease to increase during the 24 hour aging period. Uptake studies show that none of the changes in these systems can be due solely to changes in the permeability of the tissue over the climacteric period. On the basis of these results it is tentatively suggested that the aging of discs from pre-climacteric tissue might provide a model system for a detailed study of the physiological and biochemical changes occurring during the climacteric of apple fruits.

Journal Article↗

The occurrence of nitrate reductase in apple leaves.

Nitrate reductase utilizing NADH or reduced flavin mononucleotide (FMNH(2)) as electron donor was extracted from the leaves, stems and petioles, and roots of apple seedlings. Successful extraction was made possible by the use of insoluble polyvinylpyrrolidone (Polyclar AT) which forms insoluble complexes with polyphenols and tannins. The level of nitrate reductase per gram fresh weight was highest in the leaf tissue although the nitrate content of the roots was much higher than that of the leaves. Nitrite reductase activity was detected only in leaf extracts and was 4 times higher than nitrate reductase activity. Nitrate was found in all parts of young apple trees and trace amounts were also detected in mature leaves from mature trees. Nitrate reductase was induced in young leaves of apple seedlings and in mature leaves from 3 fruit-bearing varieties. An inhibitor of polyphenoloxidase, 2-mercaptobenzothiazole was used in both the inducing medium and the extracting medium in concentrations from 10(-3) to 10(-5)m with no effect upon nitrate reductase activity.

Journal Article↗