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Apple and pear peel and pulp and their influence on plasma lipids and antioxidant potentials in rats fed cholesterol-containing diets.

The aim of this study was to assess the bioactive compounds of apple and pear peel and pulp in vitro and their influence on plasma lipids and antioxidant potentials in vivo. The antioxidant potentials measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH), beta-carotene bleaching (beta-carotene), and nitric oxide inhibition radical scavenging (NO) tests in apple peel and pulp were significantly higher than in pear peel and pulp, respectively. The ethanol extract of apple peels showed the strongest inhibition of lipid peroxidation as a function of its concentration and was comparable to the antioxidant activity of butylated hydroxyanisole. The pear pulp extract had the weakest antioxidant ability, whereas other extracts such as apple pulp and pear peel were nearly equal. The antioxidant activities comprised contributions from polyphenols, phenolic acids, and flavonoids and correlated well with polyphenols and flavonoids. The correlation coefficients between polyphenols and antioxidant activities by DPPH, beta-carotene, and NO were as follows: 0.9207, 0.9350, and 0.9453. Contrarily, the correlation coefficient between the content of dietary fiber and the antioxidant activities test was low. The content of all studied indices in apple and pear peel was significantly higher than in peeled fruits (p < 0.05). Diets supplemented with fruit peels exercised a significantly higher positive influence on plasma lipid levels and on plasma antioxidant capacity of rats than diets with fruit pulps.

Antioxidants↗

Inhibition of apple polyphenol oxidase activity by procyanidins and polyphenol oxidation products.

The rate of consumption of dissolved oxygen by apple polyphenol oxidase in cider apple juices did not correlate with polyphenol oxidase activity in the fruits and decreased faster than could be explained by the decrease of its polyphenolic substrates. The kinetics parameters of a crude polyphenol oxidase extract, prepared from apple (Braeburn cultivar), were determined using caffeoylquinic acid as a substrate. Three apple procyanidin fractions of n 80, 10.5, and 4 were purified from the parenchyma of cider apples of various cultivars. Procyanidins, caffeoylquinic acid, (-)-epicatechin, and a mixture of caffeoylquinic acid and (-)-epicatechin were oxidized by reaction with caffeoylquinic acid o-quinone in order to form oxidation products. All the fractions were evaluated for their inhibitory effect on PPO activity. Native procyanidins inhibited polyphenol oxidase activity, the inhibition intensity increasing with n. The polyphenol oxidase activity decreased by 50% for 0.026 g/L of the fraction of n 80, 0.17 g/L of the fraction of n 10.5, and 1 g/L of the fraction of n 4. The inhibitory effect of oxidized procyanidins was twice that of native procyanidins. Oxidation products of caffeoylquinic acid and (-)-epicatechin also inhibited polyphenol oxidase.

Beverages↗

Major phytochemicals in apple cultivars: contribution to peroxyl radical trapping efficiency.

Forty-one samples of apples (peel plus pulp), obtained from eight cultivars, were examined for concentration of some important phytochemicals and for antioxidant activity expressed as peroxyl radical trapping efficiency. Five major polyphenolic groups plus ascorbate were identified and quantified by HPLC in the apple varieties. Oligomeric and polymeric proanthocyanidins were found to be about two-thirds of total polyphenols. The antioxidant efficiency of the apple extracts and of representative pure compounds for each group of phytochemicals was measured in a micellar system mimicking lipid peroxidation in human plasma. Although the amount of polyphenols measured by HPLC is similar to that measured by standard methods, the antioxidant efficiency calculated on the basis of the contribution of the pure compounds was lower than the antioxidant efficiency of the apple extracts. The higher efficiency of apples appears to be strictly related to the overwhelming presence of oligomeric proanthocyanidins.

Antioxidants↗

Determination of pesticides in apple-based infant foods using liquid chromatography electrospray ionization tandem mass spectrometry.

A liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method was developed and validated to quantify and confirm trace levels of 13 pesticides including aldicarb sulfoxide, aldicarb sulfone, oxamyl, methomyl, formetanate, 3-hydroxycarbofuran, carbendazim, thiabendazole, aldicarb, propoxur, carbofuran, carbaryl, and methiocarb in apple-based infant foods such as apple sauces, apples and strawberries, apples and blueberries, and apples and plums. Data acquisition under MS/MS was achieved by applying multiple reaction monitoring of two fragment ion transitions to provide a high degree of sensitivity and selectivity for both quantification and confirmation. LC/ESI-MS/MS quantitative results were significantly affected by matrices, and thus, the standard addition was employed to compensate for the matrix effects to achieve the best accuracy of the method. Recoveries of 13 pesticides, spiked at 5.0, 25.0, and 45.0 microg/kg, were around 100% using the LC/ESI-MS/MS standard addition. The method detection limits (S/N > or = 3:1) of 13 pesticides were less than 0.2 microg/kg.

Chromatography, Liquid↗

Preliminary studies for the differentiation of apple juice samples by chemometric analysis of solid-phase microextraction-gas chromatographic data.

A combination of gas chromatography (GC) and chemometrics was evaluated for its ability to differentiate between apple juice samples on the basis of apple variety and applied heat treatment. The heat treatment involved exposure of 15 mL juice samples for 30 s in a 900 W domestic microwave oven. The chromatographic results were subjected to two chemometric procedures: (1) partial least squares (PLS) regression and (2) linear discriminant analysis (LDA) applied to principal component (PC) scores. The percent correct classification of samples were obtained from PLS and LDA in terms of separation on the basis of apple variety and applied heat treatment. PLS gave the highest level of correct classification of the apple juice samples according to both variety and heat treatment, 92.5% correct classification in each case. When LDA was performed on the PC scores obtained from GC analysis, 87.5% and 80% of samples were correctly classified according to apple variety used and applied heat treatment, respectively.

Beverages↗

Development of liquid chromatography-electrospray mass spectrometry for the determination of patulin in apple juice: investigation of its contamination levels in Japan.

Patulin is a mycotoxin produced by mainly Penicillium species, for example, P. expansum, and Aspergillus species. There are several reports of patulin contamination in apple juice. Last year, the Ministry of Health, Labour and Welfare of Japan set the maximum allowable level of patulin in apple juice at 50 ppb and decided that the measurement of patulin levels in apple juice products should be conducted. To this end, a simple, accurate, and selective analytical method for the detection of patulin at levels lower than 5 ppb, the detection limit, is desired. This paper reports the development of an analytical method that employs solid-phase extraction-liquid chromatography-mass spectrometry (SPE-LC-MS). When MS measurements were conducted with the selected ion monitoring (SIM) mode, the pseudomolecular ions at m/z 153 and 156 were used to monitor patulin and (13)C(3)-labeled patulin, respectively. The detection limit (S/N = 3) and the quantification limit (S/N = 10) of patulin at injection levels into LC-MS were 12.5 and 25 pg, respectively. However, when the actual sample was applied for the analysis based on the developed method including the sample preparation, the detection limit (S/N = 3) and quantification limit (S/N = 10) were 2.5 and 5 pg in sample, respectively. The calibration curve obtained for concentrations ranging from 5 to 500 ppb showed good linearity with a coefficient of determination (r (2)) of 0.999. In addition, the recovery was >95% when an internal standard was used. The method was applied to the analysis of 76 apple juice samples from Japan, and as a result, patulin levels ranging from <1.0 to 45 ppb (detection frequency = 15/76) were detected. In this study, it was found that patulin was a greater contaminant in concentration/reduction than in "not from concentrate" apple juice.

Beverages↗

Effect of gas environment and sorbate addition on flavor characteristics of irradiated apple cider during storage.

Apple cider, with (0.1%) and without potassium sorbate, was packaged in polystyrene containers and exposed to three different gas environments: oxygen flush, nitrogen flush, and atmospheric air. To evaluate the effects of irradiation (2 kGy) and storage on flavor and microbial quality, these irradiated apple cider samples were compared to a control, unirradiated sample exposed to atmospheric air. Volatile compounds, soluble solids, titratable acidity, and microbiological counts were determined weekly throughout 7 weeks of refrigerated (4 degrees C) storage. Cider irradiated and stored in atmospheric air or nitrogen-flush environments had lower rates of loss for characteristic flavor volatiles compared to unirradiated apple cider and cider irradiated and stored in an oxygen-flush environment. The addition of potassium sorbate to the apple cider resulted in lower counts of yeasts and aerobic microorganisms, reduced fermentation of sugars to organic acids, and improved retention of volatile compounds characteristic of apple cider.

Beverages↗

Phenolic extraction from apple peel by cellulases from Thermobifida fusca.

With the optimization of the pretreatment conditions for the crude Thermobifida fusca cellulase activity and phenolic release from apple peel, we focused on the activity of individual purified cellulase related to the antioxidant activity. The overall phenolic release was significantly increased in a synergistic manner with combined pretreatment, not with individual pretreatment such as boiling, acid, and pectinase treatment. Approximately 60 mg of reducing sugar equivalent were produced per g of apple peel by treatment with T. fusca crude extract, and up to 3 times more reducing sugars were released when the apple peel was boiled and then treated with acid and pectinase. There was good correlation between the release of phenolics and reducing sugar by cellulase treatment and also between the amount of total phenolics and antioxidant capacity by each enzyme treatment (r2> 0.95). Among the tested enzymes purified from T. fusca cell extract, cellulase activity on apple peel was the highest with cellulase 6A (Cel 6A; 43% digestion), and the highest antioxidant capacity was obtained by incubation with Cel 6B (16 mg vitamin C equiv/g). Synergism in the activity was found from the combined treatment with Cel 6A and 6B in both cellulase activity and antioxidant capacity after 20 h of incubation. Cel 9A (progressive endocellulase) exhibited greater cellulase activity and antioxidant capacity than Cel 9A cd which lacks in cellulose-binding module, indicating that the cellulose-binding domain might play important roles in cellulolysis of apple peel. This study could provide some insights into the action mechanism of various cellulases on the digestion of cellulose-containing byproducts and expand the opportunity for cellulase utilization in the extraction of functional ingredients from the plant-derived byproducts.

Actinomycetales↗

Polyphenol composition and antioxidant activity of Kei-apple (Dovyalis caffra) juice.

The polyphenolic and ascorbate (ASC) components as well as the antioxidant capacity of Kei-apple (Dovyalis caffra) juice were analyzed and compared to three other fruit juices. The Kei-apple juice had significantly the highest total polyphenolic concentrations (1013 mg gallic acid equivalent/L), and solid phase (C(18)) fractionation identified the majority of these polyphenols to be phenolic acids. The Kei-apple juice also had significantly the highest ASC concentrations (658 mg/L), which showed exceptional heat stability with very little conversion to dehydroascorbate (DHA). Antioxidant capacities of both the unfractionated fruit juices and their solid phase-extracted fractions, as determined by oxygen radical absorbance capacity and ferric reducing antioxidant power analyses, correlated well to the polyphenol concentrations. Gas chromatography-mass spectrometry analyses showed caffeic acid as the most abundant polyphenol present (128.7 mg/L) in the Kei-apple juice; it contributed to 63% of the total antioxidant capacity (of all of the individual compounds identified). Other notable polyphenols identified in higher concentrations included p-coumaric acid, p-hydroxyphenylacetic acid, and protocatechuic acid. Our results therefore support the putative high antioxidant value linked to this fruit and better define this potential in terms of the major antioxidants that exist in the Kei-apple.

Antioxidants↗

Piceid (resveratrol glucoside) synthesis in stilbene synthase transgenic apple fruit.

A stilbene synthase gene along with the selectable marker gene bar for herbicide resistance was transferred via Agrobacterium tumefaciens mediated transformation into apple (Malus domesticaBorkh.) cvs. 'Elstar' and 'Holsteiner Cox'. The stilbene synthase catalyzes the conversion of 1 molecule of p-coumaroyl-CoA and 3 molecules of malonyl-CoA into 3,4',5-trihydroxystilbene, commonly known as resveratrol. This phytoalexin has implications in both phytopathology and human health. Greenhouse-grown transgenic and nontransformed control plants were grafted onto dwarfing rootstock M27. Flowering and fruiting occurred within the following years, offering the opportunity to analyze transgenic apple fruit and fertility of transgenic plants as well as inheritance of the transgenes into the seedling progeny. Molecular analysis revealed that the stilbene synthase is expressed in transgenic plants and in the skin and flesh of transgenic apple fruit. After formation, resveratrol is modified by the addition of a hexose sugar. The resulting component was characterized as piceid. With the aim of characterizing the influence of the novel biosynthetic pathway on the accumulation of other phenolic compounds naturally present in apple fruit, the amounts of flavanols, flavonols, phloretin derivatives and hydroxycinnamic acids in wild type and transgenic fruit were determined by HPLC. In all investigated transformed lines that accumulated piceid, no negative correlation between levels of piceid and the above-mentioned compounds was observed, except for the flavonol contents, which slightly decreased. Inheritance of the transgenes was confirmed in the seedling progeny, which were obtained after pollination of transgenic plants with nontransgenic pollen and vice versa after pollination of nontransgenic plants with pollen obtained from transgenic plants. The fertility of stilbene synthase transgenic plants was demonstrated. To the authors' knowledge this is the first time that data are available on piceid synthesis in transgenic apple fruit and the effects of its accumulation on levels of other phenolic compounds present in the fruit.

Acyltransferases↗

Adulteration of apple with pear juice: emphasis on major carbohydrates, proline, and arbutin.

Detection of juice-to-juice adulteration based on chemical composition studies is a common method used by government regulatory agencies and food companies. This study investigated the use of major carbohydrate (fructose, glucose and sucrose), polyol (sorbitol), proline, and phenolic profiles as indicators of pear adulteration of apple juice (PAAJ). For this work, a total of 105 authentic apple juice samples from 13 countries and 27 authentic pear juice samples from 5 countries were analyzed. Because the major carbohydrate ranges for these juices showed significant overlap their use as markers for PAAJ detection would be very limited. It was found that sorbitol and proline means for apple and pear were significantly different; however, their broad natural ranges would afford PAAJ at levels up to 30% without detection. In addition, careful selection of the pear juice used as the adulterant would further limit the usefulness of these markers for PAAJ detection. Arbutin was conclusively identified as a marker for pear juice on the basis of its presence in all 27 authentic pear samples and its absence (<0.5 microg/mL) in all 105 apple juice samples analyzed in this study. The application of the developed HPLC-PDA method for arbutin analysis to detect PAAJ at levels as low as 2% (v/v) was demonstrated. A confirmation method for the presence of arbutin in pure pear juice and apple adulterated with pear juice was introduced on the basis of the hydrolysis of arbutin to hydroquinone employing beta-glucosidase, with reactant and product monitoring by HPLC-PDA.

Arbutin↗

Odor thresholds of microbially induced off-flavor compounds in apple juice.

Microbially derived off-flavor is a major problem in apple juice production as it diminishes the sensory quality of the juice significantly. Fifteen relevant off-flavor compounds that are formed in apple juice, for example, by the strains Alicyclobacillus acidoterrestris and Actinomycetes (Streptomyces ssp.) were investigated with respect to their sensory relevance. The odor threshold values (i.e., detection and recognition values) were determined for all compounds in the matrix apple juice. Odor threshold values for fenchyl alcohol are reported here for the first time. The obtained values were set in relation to the limits of detection and quantification of a previously published GC-MS method. Eight tainted apple juice samples were analyzed for the presence of the 2 strains and the 15 off-flavor compounds. Both strains could be found in the samples; the presence of Streptomyces ssp. as spoilage bacteria of apple juice is reported for the first time. In samples with distinct off-flavor, 2-isopropyl-3-methoxypyrazine, 2-isobutyl-3-methoxypyrazine, 2-methylisoborneol, 1-octen-3-ol, fenchyl alcohol, geosmin, and guaiacol as well as 2,6-dibromophenol were determined in concentrations higher than the detection threshold.

Bacillaceae↗

Physiological effects of extraction juices from apple, grape, and red beet pomaces in rats.

In comparison to classical fruit juice processing, polyphenols and dietary fiber can be extracted from pomace by means of pectinases and cellulases. In the present study, rats were fed with such produced extraction juices from apples, grapes, and red beets as drinking fluids instead of water for 4 weeks to evaluate their physiological effects. In all test groups, the intake of extraction juices was greater as compared to control (water intake), resulting in a higher urine excretion. In the apple and grape group, pH values in feces was lower than control. Administration of extraction juices from apples increased fecal counts of Lactobacillus and Bifidobacterium. More acetate and total short-chain fatty acids appeared in intestinal contents of the apple and red beet group. Furthermore, the intestinal contents of test groups contained higher concentrations of primary bile acids, cholesterol, and cholesterol metabolites but lower concentrations of secondary bile acids. The total amount of steroids excreted by these groups was also greater than control. Quercetin and isorhamnetin appeared in urine of rats fed extraction juices from apples and grapes; in urine of the former group, phloretin was found also. Administration of the extraction juices, enriched in secondary plant metabolites and dietary fiber, resulted in beneficial nutritional effects in rats.

Animal Nutritional Physiological Phenomena↗

Novel 1,3-dioxanes from apple juice and cider.

Extracts obtained by XAD solid-phase extraction of apple juice and cider were separated by liquid chromatography on silica gel. Several new 1,3-dioxanes including the known 2-methyl-4-pentyl-1,3-dioxane and 2-methyl-4-[2'(Z)-pentenyl]-1,3-dioxane, were identified in the nonpolar fractions by GC/MS analysis and confirmed by chemical synthesis. The enantioselective synthesis of the stereoisomers of the 1,3-dioxanes was performed using (R)- and (R,S)-octane-1,3-diol and (R)- and (R,S)-5(Z)-octene-1,3-diol as starting material. Comparison with the isolated products indicated that the natural products consisted of a mixture of (2S,4R) and (2R,4R) stereoisomers in the ratio of approximately 10:1, except for 1,3-dioxanes generated from acetone and 2-butanone. It is assumed that the 1, 3-dioxanes are chemically formed in the apples and cider from the natural apple ingredients (R)-octane-1,3-diol, (R)-5(Z)-octene-1, 3-diol, (3R,7R)- and (3R,7S)-octane-1,3,7-triol, and the appropriate aldehydes and ketones, which are produced either by the apples or by yeast during fermentation of the apple juice.

Beverages↗

Reduction of azinphos-methyl, chlorpyrifos, esfenvalerate, and methomyl residues in processed apples.

McIntosh, Red Delicious, and Golden Delicious from two years of experimental spray programs using azinphos-methyl, chlorpyrifos, esfenvalerate, and methomyl were processed into frozen apple slices, applesauce, single-strength juice, and juice concentrate. Residue levels were expressed as micrograms per 150 g of apple or the equivalent amount of apple product to calculate the percentage change in these pesticides brought about by processing. Producing single-strength apple juice reduced azinphos-methyl, chlorpyrifos, esfenvalerate, and methomyl residues by 97.6, 100, 97.8, and 78.1%, respectively. Production of applesauce reduced all four compounds by >/=95%. Azinphos-methyl, chlorpyrifos, esfenvalerate, and methomyl residues were reduced in apple slices by 94.1, 85.7, 98.6, and 94.7%, respectively. Processing is shown to be very effective in reducing the levels of these pesticides.

Azinphosmethyl↗

Distribution and fractionation of copper in soils of apple orchards.

BACKGROUND: Frequent application of Bordeaux mixture, which includes copper, as a fungicide in fruit and grape orchards may lead to copper accumulation in the soil, especially when orchard age and application times increase. The objectives of this study were: (i) to investigate the copper content and its spatial distribution in orchard soils; (ii) to identify the copper fractionation in soil and its relationship with plant uptake; (iii) to understand the characteristics of copper contamination in orchard soils. MATERIALS AND METHODS: Soil profile samples were taken in apple orchards with ages of 0, 5, 10, 20, 30 years and pot experiments were also carried out to study the effects of external copper input on copper fractionation. All soil samples were air-dried, ground and extracted with 0.43 mol L(-1) HNO3 for the total absorbed copper. Fractionation determination was conducted following Tessier and Shuman sequential extraction methods, and copper was measured with AAS. Plant samples were first dry ashed, dissolved with 6 mol L(-1) HCI and then copper and other elements were measured with ICP-MS. RESULTS AND DISCUSSION: Soil total Cu was higher in the apple orchards than that in non-orchard fields and was seen to have increased with orchard age. Soil Cu increased substantially with the average annual copper increase, ranging from 2.5 to 9 mg Cu kg(-1). The distribution of copper in the soil profile was uneven, decreasing from surface to deeper layers, and the differences were significant, but the contents in every layer were also significantly correlated with those in the next layers. For all copper fractions, the organically bound, crystalline Mn oxide bound, and amorphous Fe bound fractions extracted with the Shuman method were much higher than the exchangeable and residual fractions. Using the Tessier method, organically bound, carbonate bound and Fe-Mn oxide bound fractions were much higher. With an increase in external copper input, the organically bound, crystalline Mn oxide bound and amorphous Fe bound fractions in the Shuman method and organically bound, carbonate bound and Fe-Mn oxide bound fractions in the Tessier method all increased significantly, while the changes in other fractions were not significant. Soil total copper and copper fractions were found to have good correlations with apple tree uptake. Copper in fruit flesh had significant correlations with soil total content in the 0-10 cm layer, all the copper fractions in the 0-5 cm layer, and some fractions in the deeper layers. CONCLUSION: Copper content in orchard soils increased significantly with intensive application of Bordeaux mixtures and orchard age. Copper content decreased sharply from the topsoil to deeper soil layers. The copper contents in different layers also significantly correlated with those in the next layers. Dominant fractions of the copper in soil were mainly associated with organic matter, iron and manganese oxides and carbonates. A close relationship was found between the copper content in soils and in apple tree organs (which contained 8.9 to 66 mg kg(-1) Cu). RECOMMENDATION AND PERSPECTIVE: Though most copper in the soil was specifically adsorbed or immobilized, and copper was mainly distributed in topsoil, which was essentially devoid of roots, the copper concentration of fruit still had significantly positive correlations with soil copper and most copper fractions. Therefore, measures must be taken to control copper accumulation in orchard soils and to make the apple fruit production sustainable.

Agriculture↗

Food (miles) for thought--energy balance for locally-grown versus imported apple fruit.

This commentary compares the primary energy requirement for apples (cultivar 'Braeburn'), which were either imported or locally-grown in Meckenheim, Germany. Imported apples of the same cultivar were grown in a Southern hemisphere winter in Nelson, Southland, New Zealand, and were picked at the end of March with subsequent 28 d transport by sea for sale in April in Germany. Locally-grown apples (cultivar 'Braeburn') were picked in mid-October and required a primary energy of nearly 6 MJ/kg of fruit including 0.8 MJoule/kg for five months CA storage at 1 degrees C during a Northern hemisphere winter until mid-March. This compared favourably with 7.5 MJoule/kg for overseas shipment from New Zealand, i.e. a ca. 27% greater energy requirement for these imported fruits. Overall, the primary energy requirement of regional produce, stored several months on-site, partially compensated for the larger energy required to import fresh fruit from overseas. This result is in marked contrast to reported overestimates of a reported up to 8-fold energy requirement for domestic versus imported apple juice concentrate. Our own findings of less primary energy required for domestic apple fruit is discussed with respect to providing local employment, fruit orchards preserving the countryside, quality assurance systems for local fruit such as QS and EUREP-GAP, networking and other factors favouring regional production.

Agriculture↗

Parthenocarpic apple fruit production conferred by transposon insertion mutations in a MADS-box transcription factor.

Fruit development in higher plants normally requires pollination and fertilization to stimulate cell division of specific floral tissues. In some cases, parthenocarpic fruit development proceeds without either pollination or fertilization. Parthenocarpic fruit without seed has higher commercial value than seeded fruit. Several apple (Malus domestica) mutants (Rae Ime, Spencer Seedless and Wellington Bloomless) are known to produce only apetalous flowers that readily go on to develop into parthenocarpic fruit. Through genetics, a single recessive gene has been identified to control this trait in apple. Flower phenotypes of these apple mutants are strikingly similar to those of the Arabidopsis mutant pistillata (pi), which produces flowers where petals are transformed to sepals and stamens to carpels. In this study, we have cloned the apple PI homolog (MdPI) that shows 64% amino acid sequence identity and closely conserved intron positions and mRNA expression patterns to the Arabidopsis PI. We have identified that in the apetalous mutants MdPI has been mutated by a retrotransposon insertion in intron 4 in the case of Rae Ime and in intron 6 in the case of Spencer Seedless and Wellington Bloomless. The insertion apparently abolishes the normal expression of the MdPI gene. We conclude that the loss of function mutation in the MdPI MADS-box transcription factor confers parthenocarpic fruit development in these apple varieties and demonstrates another function for the MADS- box gene family. The knowledge generated here could be used to produce parthenocarpic fruit cultivars through genetic engineering.

Amino Acid Sequence↗