Effects of Treatment with a Composite Preparation (2-Chloroethylphosphonic Acid and Methacide) or Butylated Hydroxyanisole on Ethylene Release in Apples

We studied the effect of a Russian composite preparation (2-chloroethylphosphonic acid and methacide) and butylated hydroxyanisole on ethylene release in whole fruit and peel disks of two apple cultivars, Antonovka obyknovennaya (Antonovka) and Simirenko's rennet (Simirenko). Treatment with the composite preparation was followed by an increase in ethylene release from both the whole apples and peel disks. The development of microbial infection (fruit rot) in the whole apples became less pronounced after the treatment. Treatment of whole apples with the antioxidant butylated hydroxyanisole (BHA) increased the intensity of ethylene release during the first subsequent days; thereafter, ethylene release decreased and was 10–15% lower than in the control on days 10–12. In model experiments, BHA decreased ethylene release from apple peel disks below control levels as early as on the first day after treatment. Antonovka apples gave quick responses to the treatment. In the late-ripening Simirenko apples, the response persisted for a longer period. Our results suggest that treatment with physiologically active preparations affects the ethylene release, ripening, and preservation of apples in storage.     

Patterns of pigment changes in apple fruits during adaptation to high sunlight and sunscald development

Reflectance spectra of four apple (Malus domestica Borkh.) cultivars were studied and chlorophyll, carotenoid, anthocyanin and flavonoid content in sunlit and shaded peel was determined. In all cases sunlit peel accumulated high amounts of phenolics (flavonoid glycosides). Adaptation to strong sunlight of an apple cultivar with limited potential for anthocyanin biosynthesis (Antonovka) was accompanied by a decrease in chlorophyll and a significant increase in total carotenoid content. The increase in carotenoids also took place in sunlit sides of the Zhigulevskoye fruits, accumulating high amounts of anthocyanins, but chlorophyll content in sunlit peel was higher than that in shaded peel. Significant increases in carotenoids and anthocyanins were detected during fruit ripening when chlorophyll content fell below 1.5–1.8 nmol cm^–2. Chlorophyll in sunlit fruit surfaces of both cultivars was considerably more resistant to photobleaching than in shaded (especially of Zhigulevskoye) sides. Induced by sun irradiation, the photoadaptive responses were cultivar-dependent and expressed at different stages of fruit ripening even after storage in darkness. The development of sunscald symptoms in susceptible apple cultivars (Granny Smith and Renet Simirenko) led to a dramatic loss of chlorophylls and carotenoids, which was similar to that observed during artificial photobleaching. The results suggest that apple fruits exhibit a genetically determined strategy of adaptation of their photoprotective pigments to cope with mediated by reactive oxygen species photodynamic activity of chlorophyll under strong solar irradiation. This includes induction of synthesis and accumulation of flavonoids, anthocyanins and carotenoids that could be expressed, if necessary, at different stages of fruit development     

Effect of a Protein Polygalacturonidase Inhibitor from Apple Fruit Tissue on the Enzyme Released by Fungal Pathogens

A protein polygalacturonidase inhibitor isolated from fruit of the apple cultivars Antonovka and Mantuanskoe differently affects the polygalacturonidases of different phytopathogenic fungi. Three groups of fungi were recognized according to the sensitivity of their polygalacturonidases to the inhibitory effect. Storage of apples after harvesting is accompanied by changes in the inhibitor activity, and the time pattern of these changes depends on the cultivar. An increase in the inhibitor activity occurs concurrently with the elevation in ethylene release characteristic of the stage of elevated respiration (a climacteric increase). The data suggest that a decrease in the apple fruit resistance to microbes at the end of the storage period is related, along with other reasons, to a change in the activity of the protein polygalacturonidase inhibitor.     

Effect of proteinaceous polygalacturonase inhibitors from apple seed tissue on an enzyme isolated from phytopathogenic fungi

A protein polygalacturonidase inhibitor isolated from fruit of the apple varieties Antonovka and Mantuanskoe differently affects the polygalacturonidases of different phytopathogenic fungi. Three groups of fungi were recognized by the sensitivity of their polygalacturonidases to the inhibitory effect. Storage of apples after harvesting is accompanied by changes in the inhibitor activity, and the time pattern of these changes depends on the variety. An increase in the inhibitor activity occurs concurrently with the elevation in ethylene release characteristic of the stage of elevated respiration (a climacteric increase). The data suggest that a decrease in the apple fruit resistance to microbes at the end of the storage period is related, along with other reasons, to a change in the activity of the protein polygalacturonidase inhibitor.     

Limitations in the Use of Cycloheximide in Studies of Apple Ripening

In order to discover whethor the production of aroma volatilesby apple fruits is dependent on the synthesis of appropriateenzymes during ripening, excised peel, excised cortical tissue,and whole apples were treated with cycloheximide (CH). Volatilerelease, ethylene production, respiration, flesh softening,and peel chlorophyll degradation were measured. The ethylene and volatile compounds produced by excised peelapparently resulted from wounding rather than processes analogousto fruit ripening. Excised cortical tissue was capable of autonomousripening with ethylene production, respiration, and softeningcomparable to that in intact fruits. After infiltration withsucrose solution the same changes occurred, but they were delayedby up to 4 d. Cycloheximide inhibited respiration although theextent of this inhibition decreased after 3 d. Cycloheximideprevented the onset of rapid ethylene production but stimulatedproduction of ethanol, ethyl acetate, and other volatiles. Softeningof CH-treated cortical discs was associated with progressivenecrosis. When whole apples were infiltrated with CH through hypodermicneedles inserted into the core, [¹⁴C]valine incorporation wasinhibited from the core to the mid-cortex but not in the peeland outer cortex. Infiltration with sucrose solution delayedmany ripening changes although the time of maximum [¹⁴C]valineincorporation was unaffected. Early effects of CH on respirationwere masked by the effects of infiltration, but after 5 d CH-infiltratedfruit contained higher CO₂ concentrations and respired morerapidly than controls. Internal ethylene concentrations wereusually lower in CH-treated apples than in controls. CH stimulated release of ethanol and ethyl acetate but inhibitedrelease of higher molecular weight esters such as propyl andbutyl acetates. Cycloheximide-treated fruit softened, but thiswas apparently due to internal necrosis. Peel chlorophyll degradationwas inhibited by CH treatment of whole apples although the tissuehad apparently received no inhibitor.     

Heat treatment of ripening apples: Differential effects on physiology and biochemistry

Apples ( Malus domestica Borkh.) were heated for 4 days at 38°C immediately after harvest and then placed at 20°C for 7–10 days. Protein synthesis, ethylene production and fruit softening were reversibly inhibited by the heat treatment. Fruit respiration, membrane permeability and chlorophyll degradation in the fruit peel were enhanced during the treatment. The heat-treated apples ripened normally but more slowly than untreated apple We hypothesize that heat treatment differentially affects processes which normally increase simultaneously during fruit ripening, by inhibiting those processes which require tie novo protein synthesis and enhancing those that do not.     

Foliar application of aminoethoxyvinylglycine (AVG) delays fruit ripening and reduces pre-harvest fruit drop and ethylene production of bagged “Kogetsu” apples

Covering apple fruits with double layer waterproof bags to enhance fruit quality and evenness of blush colour is typical on many cultivars in Korea and Japan. Aminoethoxyvinylglycine (AVG) applied to unbagged apple fruits at 3–4 weeks before commercial harvest reduces ethylene production in the fruit, delays fruit ripening and reduces pre-harvest fruit drop. Spray application of AVG to trees of bagged apples should have no effect on apple ripening as there is␣no direct contact with the fruit and the translocation of AVG in apple trees is regarded as negligible. However, preliminary experiments suggested that AVG applied to trees of bagged apples reduced pre-harvest fruit drop in “Kotgetsu” apples. This study investigated the effect of spray treatments of 125 ppm of AVG on fruit drop, fruit ripening (firmness, starch conversion and soluble solids) and ethylene production to whole trees with bagged or unbagged “Kogetsu” fruit, as well as sprays of only the bagged or unbagged fruit on trees on two orchards. AVG applied to whole trees with unbagged apples reduced fruit drop from an average of 58.9% to 10.4%, delayed starch conversion and decreased ethylene production. AVG applied to whole trees with bagged fruit was equally effective in reducing pre-harvest drop, delaying fruit ripening and reducing ethylene production. Application of AVG to unbagged fruit only was nearly as effective as application to whole trees with unbagged fruit but application to bagged fruit only had no effect on fruit ripening or ethylene production. Application of AVG to bagged fruit only did reduce fruit drop to an average of 42.5% but this was not as effective as spraying unbagged fruit only or whole trees with bagged fruit. Possible mechanisms for this effect are discussed.     

Ethylene-promoted conversion of 1-aminocyclopropane-1-carboxylic Acid to ethylene in peel of apple at various stages of fruit development

Internal ethylene concentration, ability to convert 1-amino-cyclopropane-1-carboxylic acid (ACC) to ethylene (ethylene-forming enzyme [EFE] activity) and ACC content in the peel of apples (Malus domestica Borkh., cv Golden Delicious) increased only slightly during fruit maturation on the tree. Treatment of immature apples with 100 microliters ethylene per liter for 24 hours increased EFE activity in the peel tissue, but did not induce an increase in ethylene production. This ability of apple peel tissue to respond to ethylene with elevated EFE activity increased exponentially during maturation on the tree. After harvest of mature preclimacteric apples previously treated with aminoethoxyvinyl-glycine, 0.05 microliter per liter ethylene did not immediately cause a rapid increase of development in EFE activity in peel tissue. However, 0.5 microliter per liter ethylene and higher concentrations did. The ethylene concentration for half-maximal promotion of EFE development was estimated to be approximately 0.9 microliter per liter. CO₂ partially inhibited the rapid increase of ethylene-promoted development of EFE activity. It is suggested that ethylene-promoted CO₂ production is involved in the regulation of autocatalytic ethylene production in apples.     

Effect of orchard and postharvest application of daminozide on ethylene synthesis by apple fruit

The effects of daminozide (butanedioic acid-2,2-dimethylhydrazide) on ethylene synthesis by apple fruits were investigated. The objective was to determine the effects of postharvest applications as compared to the standard application of diaminozide in the orchard. Immersion in a solution containing 4.25 g L^?1 active ingredient for 5 min delayed the rise in ethylene production in individual “Cox” apples at 15°C by about 2 days, whereas orchard application of 0.85 g L^?1 caused delays of about 3 days. Both modes of application depressed the maximal rate of ethylene production attained by ripe apples by about 30%. Daminozide did not affect the stimulation of respiration by ethylene treatment of “Gloster” apples, but it delayed the increase in ethylene synthesis. Daminozide applied immediately after harvest delayed the rise in ethylene synthesis in “Golden Delicious” held at 15°C, but it was less effective when applied 48 h after harvest or when apples were held at 5°C. Exposure to 1–2 μl L^?1 ethylene for 48 h was less effective in promoting the rise in ethylene in daminozidetreated “Cox” and “Gloster” apples than in untreated fruit. High (100–1000 μl L^?1) concentrations of ethylene more or less overcame the daminozide effect. Apples absorbed about 40% of surface-applied [¹⁴C]daminozide in 48 h, but more than 90% of the radioactivity in the fruit was recovered from the peel and outer 1 cm of the cortex. Daminozide was partly converted to carbon dioxide and other metabolites.     

Apple flavonols during fruit adaptation to solar radiation: spectral features and technique for non-destructive assessment

Spectral properties of flavonols of three varieties (Golden Delicious, Antonovka, and Renet Simirenko) of anthocyanin-free apple fruit were investigated with reflectance spectroscopy. The results of spectral and biochemical analyses suggested that fruit reflectance in a broad spectral range 365-430 nm is strongly dependent on and, in sunlit fruit surfaces, governed by flavonols. The build up of peel flavonols (mainly rutin and other quercetin glycosides) resulted in a dramatic decrease of fruit reflectance in this range, flattening of the spectrum, and extending the region with low reflectance (4-5%) to ca. 410 nm. The spectral features observed suggest that flavonols contribute significantly to screening of excessive radiation, not only UV-A, but in the short-wave bands of chlorophyll and carotenoid absorption in the visible part of the spectrum as well. To retrieve quantitatively flavonol content from reflectance spectra, we tested the applicability of an inversion technique developed for non-destructive leaf pigment assessment. The model for flavonol content assessment was suggested in the form (R(-1)410 - R(-1)460)R800, where Rlambda is reflectance at wavelength lambda. The model was linearly related to flavonol content between 8 and 220nmol/cm2 with the coefficient of determination r2=0.92 and root mean square error of flavonol estimation of 20 nmol/ cm2 regardless of cultivar, chlorophyll, and carotenoid content.     

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.     

The role of anthocyanin in photoprotection and its relationship with the xanthophyll cycle and the antioxidant system in apple peel depends on the light conditions

The synthesis of anthocyanin, the xanthophyll cycle, the antioxidant system and the production of active oxygen species (AOS) were compared between red and non‐red apple cultivars, in response to either long‐term sunlight exposure (high light intensity) during fruit development, or to exposure of bagged fruits to lower light intensity late in fruit development. During fruit development of red and non‐red apples, the xanthophyll cycle pool size decreased much more in red apple peel late in development. With accumulation of AOS induced by long‐term sunlight exposure, enhancement of the antioxidant system was found. However, this change became significantly lower in red apple than non‐red apple as fruit developed, which might serve to accelerate the anthocyanin synthesis in red apple peel. When, late in fruit development, bagged fruits were exposed to sunlight, the accumulation of AOS was lower in red apple peel than in non‐red peel. This could be due to the higher anthocyanin concentration in the red peels. Meanwhile, compared with that in non‐red cultivar, the xanthophyll cycle and the antioxidant system in red apple peel were protected first but then down‐regulated by its higher anthocyanin concentration during sunlight exposure. In conclusions, red and non‐red apples peel possess different photoprotective mechanisms under high light conditions. The relationship between anthocyanin synthesis and the xanthophyll cycle, and the antioxidant system, depends on the light conditions that fruit undergoes.     

Isolation and study of active ATP-dependent phosphofructokinase from apple fruits Pyrus domestica Borkh

The activity of ATP-dependent phosphofructokinase (PFK) from subepidermal tissue of apple fruits was studied. The enzyme extracted under optimal conditions was stable for 14 h at room temperature. The enzyme was partially purified by ammonium sulfate fractionation and dialysis. PFK from apple fruits was found to be ATP-, UTP-, and CTP-specific. It was inhibited by PEP, Gly-2-P, Gly-1,3-DP, and ADP. The effect of the listed inhibitors was diminished by the presence of phosphate. The activity of PFK was stimulated by magnesium cations. The activity of the enzyme in fruits of an Antonovka cultivar was higher than in the Simirenko rennet cultivar by a factor of 1.3.     

1-MCP对‘粉红女士’苹果冷藏期间品质变化和香气形成的影响

以'粉红女士'苹果为试验材料,研究了1 μL/L 1-MCP(1-甲基环丙烯)对苹果冷藏期间乙烯释放速率、呼吸速率、果实硬度以及香气成分和相对含量的影响.结果表明,1-MCP处理可显著抑制'粉红女士'苹果冷藏期间呼吸作用和乙烯释放,有效延缓果实硬度的下降.冷藏期内'粉红女士'苹果香气物质主要有醇类、醛类、酯类、烯类、酸类和烷烃类等,并以酯类香气为主(占46.15%);1-MCP能显著减少果实贮藏期间酯类、醇类和烷烃类香气成分种类和相对含量,处理果中酯类和醇类香气成分种类比同期对照分别减少了50%和78%,主要香气成分丁酸己酯在处理和对照果实的相对含量分别为1.12%～1.73%和1.87%～5.18%.可见,1-MCP处理对'粉红女士'苹果具有良好保鲜效果,也显著地抑制了贮藏期间香气的形成.     

The Production of Ethylene by Whole Apple Fruits and by Tissue 1

It is shown that the production of ethylene by whole apples,discs of peel prepared from them, and extracts prepared fromthe discs and supplied with linolenic acid, increases simultaneouslyduring the development of the respiration climacteric in apples.As the climacteric peak is reached, the ability of the extractsto produce ethylene declines and this is associated with a rapidloss of ethylene-producing activity on short term (up to 24h) ageing of the peel discs from which the extracts were obtained. It is suggested that the gmn.ll ethylene production by extracts(with linolenic acid) from pre-climacteric fruits, which arenot themselves evolving sufficient ethylene for its detectionin the ambient atmosphere, may be explained on the grounds thateven before the climacterio the fruit is producing small amountsof ethylene within the tissue; the tissue, as the climactericphase is approached, becomes more sensitive to ethylene andautocatalysed production of the gas then stimulates the fruitinto the respiration climacteric.     

Ethylene Requirement for Germination of Partly After-ripened Apple Embryo

The effect and involvement of ethylene in germination of partially after-ripened apple (Malus domestica Borkh. cv. Antonovka) embryos were studied. The requirement of ethylene for germination was shown by the use of a rhizobitoxine analog and 8-hydroxy-quinoline sulphate which are inhibitors of ethylene biosynthesis, and by mercuric perchlorate, a trap for endogenously produced ethylene. Excised apple embryos were found to produce progressively greater amounts of ethylene with increasing periods of after-ripening. Inhibition of germination by rhizobitoxine analog or abscisic acid was accompanied by inhibition of ethylene formation in embryos. Thus ethylene appears to be required for the release of dormancy and germination of apple embryos.     

Effects of the butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on the arylamines N-acetyltransferase activity in rat white blood cells

Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were used to determine any effects on the N-acetyltransferase (NAT) activity in rat whole blood and white blood cells as measured by high performance liquid chromatography assay for the amounts of N-acetyl-2-aminofluorene (AAF) and 2-aminofluorene (AF). Two assay systems were performed, one with cellular cytosols, the other with intact white blood cells. The NAT activity in the whole blood and white blood cell cytosols was suppressed by BHA and BHT in a dose-dependent manner, i.e. the higher the concentrations of BHA and BHT, the higher the inhibition of NAT activity. Time-course experiments showed that NAT activity measured from the intact white blood cells was inhibited by BHA and BHT up to 24 h. The results suggest that BHA and BHT suppressed AF acetylation in rat blood with intact white blood cells.     

Isolation and study of active ATP-dependent phosphofructokinase from apple fruits Pyrus domestica Borkh

The activity of ATP-dependent phosphofructokinase (PFK) from subepidermal tissue of apple fruits was studied. The enzyme extracted under optimal conditions was stable for 14 h at room temperature. The enzyme was partially purified by ammonium sulfate fractionation and dialysis. PFK from apple fruits was found to be ATP-, UTP-, and CTP-specific. It was inhibited by PEP, Gly-2-P, Gly-1,3-DP, and ADP. The effect of the listed inhibitors was diminished by the presence of phosphate. The activity of PFK was stimulated by magnesium cations. The activity of the enzyme in fruits of an Antonovka cultivar was higher than in the Simirenko rennet cultivar by a factor of 1.3.     

Butylated hydroxyanisole specifically inhibits tumor necrosis factor-induced cytotoxicity and growth enhancement.

The effect of commonly used food antioxidants on recombinant tumor necrosis factor alpha (rTNF-alpha)-induced cytotoxicity, growth enhancement and adhesion has been evaluated. Butylated hydroxyanisole (BHA) and 4-hydroxymethyl-2,6-di-t-butylphenol (HBP) were the only two of nine antioxidants that completely inhibited rTNF-alpha-induced cytotoxicity in L929 and WEHI 164 fibrosarcoma cells. Ethoxyquin, propyl gallate and butylated hydroquinone only partially inhibited rTNF-alpha-induced cytotoxicity, while the antioxidants butylated hydroxytoluene (BHT), alpha-tocopherol, ascorbic acid and thiodipropionic acid had minimal effects. The only difference between the molecular structure of the efficient HBP and the non-efficient BHT, is a hydroxymethyl group instead of a hydroxyl group on the phenolic ring. Neither BHA nor BHT inhibited the activation of NF kappa B after 10 or 60 min challenge with rTNF-alpha in L929 cells. BHA also inhibited rTNF-alpha-induced, but not rIL-1 beta-induced growth enhancement in FS-4 fibroblasts. Further, BHA blocked both rTNF-alpha-induced and rIL-1 beta-induced prostaglandin E2 synthesis in FS-4 fibroblasts. BHA inhibited the rTNF-alpha-induced release of arachidonic acid in both FS-4 and L929 cells, suggesting that BHA inhibits cellular phospholipase(s). Neither alpha-tocopherol nor BHA inhibited rTNF-alpha-induced adhesiveness of human endothelial cells. The results indicate that BHA is a specific and potent inhibitor of rTNF-alpha- and rTNF-beta-induced cytotoxicity, as well as of rTNF-alpha-induced growth enhancement.