Abstract
The present study was conducted to investigate the effects of pre-harvest methyl jasmonate treatments on peel color formation, total anthocyanin, total phenolics, total antioxidant capacity, and other selected fruit quality parameters of “Fuji” apples. Results revealed significant decreases in color parameters (L*, chroma, and hue values) with methyl jasmonate treatments. Total anthocyanin, total phenolics, and total antioxidant capacity of methyl jasmonate-treated fruits were significantly higher than control fruits. Methyl jasmonate concentrations increase linearly with increase in total anthocyanin, total phenolics, and total antioxidant capacity with high correlations. Ethylene synthesis-promoting impact of methyl jasmonate increased with increasing implementation doses. Methyl jasmonate treatments also increased fruit flesh firmness. While 1120 and 2240 mg/L methyl jasmonate treatments did not cause any significant changes in the starch degradation, 4480 mg/L methyl jasmonate treatment delayed the starch degradation. All methyl jasmonate treatments did not cause any or much significant changes in fruit mass, geometric mean diameter and soluble solids content of the fruits but yielded significant increase in titratable acidity values.
INTRODUCTION
Erratic and poor red color formation decreases the market values of red apples and causes significant economic losses for producers. Anthocyanin is a primary compound responsible for red color formation in apples, while flavonols and proanthocyanidins are the phenolic compounds contributing to the red color formation.[Citation1] Besides improving fruit color, anthocyanin and other phenolics are also significant compounds with regard to their antioxidant characteristics and nutritional values.[Citation2−Citation4]
Beside the environmental factors (solar radiation, temperature), cultural practices (pruning, training, bagging, reflective mulch) and nutrient treatments (phostrade Ca, seniphos), growth regulators methyl jasmonate (MeJA; ethephon) are also effective in phenolic compounds synthesis and red color formation in apples.[Citation5−Citation7] It was reported in previous researches carried out with different apple cultivars that ethylene increased red color formation and anthocyanin accumulations.[Citation8] Mattheis et al.[Citation9] reported that ethylene alone was not significantly effective in “Fuji” apples but increased anthocyanin levels significantly when used together with MeJA.
MeJA is a member of jasmonate group and increases ethylene and anthocyanin biosynthesis in various fruits including apples.[Citation10,Citation11] While some researchers indicated that promoting effects of MeJA on color formation and anthocyanin synthesis were because of ethylene,[Citation12,Citation13] others indicated that such effects were independent of ethylene.[Citation14,Citation15]
The present study was conducted to investigate the effects of increasing MeJA doses on color formation, internal ethylene concentration, total anthocyanin and total phenolics, total antioxidant capacity, and other quality parameters of “Fuji” apples. Improved color development and bioactive compounds of “Fuji” apples through MeJA treatments will definitely provide significant contributions to market and nutritional values of the fruits.
MATERIALS AND METHODS
Plant Material
The study was carried out at the Horticultural Research Center of Gaziosmanpaşa University (40° 20ʹ 02.19”N latitude, 36° 28ʹ 30.11”E longitude and 623 m above sea level) of Tokat, in middle Black Sea region of Turkey, during the years 2010 and 2011. Thirty-two five-year-old “Fuji” apples (Malus x domestica Borkh)/M9 were selected and grouped (randomized block design) into four blocks of eight trees based on proximity in orchard and crop load. The trees were spaced at 1.0 × 3.0 m and trained to “slender spindle system.”
The trees were hand-thinned to one fruit per cluster at 42 days after full bloom. Trees were sprayed with 1120, 2240, and 4480 mg/L MeJA (Sigma-Aldrich, Germany) at 1-week intervals from 147th day to 175th day after full bloom. MeJA solutions were composed of 0.077% (v/v) Triton X-100 (Sigma-Aldrich, Germany). Treatments were applied to the fruits using hand-pump-actuated spray bottles. Each fruit was sprayed to drip. For each treatment, one pair of trees was used in each block. Two trees in each block were only sprayed with 0.077% Triton X-100 and served as control (containing 0 mg/L MeJA). Beginning at 168th day after full bloom, 40 fruits from two trees of each treatment in each block were randomly harvested at 1-week intervals from the whole canopy until normal harvest time (182 DAFB), and instantly transported to the laboratory for the determination of the color and other quality properties.
Of these fruits, 20 samples were used for the fruit quality parameters (color characteristics [L*, chroma, and hue angle], fruit mass, geometric mean diameter, fruit firmness). A ten-fruit sub-sample was used for bioactive compounds (total anthocyanin, total phenolics, and total antioxidant capacity). The other sub-samples of ten fruits were used for the determination of soluble solids content (SSC), titratable acidity, and starch degradation. To evaluate internal ethylene concentration, ten fruits were also randomly harvested from two trees in each block for each treatment in 2011.
The color characteristics (L*, chroma, and hue angle), total anthocyanin, total phenolics, total antioxidant capacity were determined in each analysis within the period of 2010 and 2011. The internal ethylene concentration was measured only between October 4 and 25, 2011 at 7-day intervals. The fruit mass, geometric mean diameter, fruit firmness, SSC, titratable acidity, and starch degradation were determined only at normal harvest dates of 2010 and 2011.
Color Characteristics
Peel color of the sun-exposed and shade-exposed sides of each fruit was analyzed using a colorimeter (Minolta, model CR–400, Tokyo, Japan), and expressed as the average. Measurements were obtained using the CIE L* (light to dark), a* (green to red), b* (blue to yellow), color space, then a* and b* values were converted to chroma and hue angle. Chroma was calculated by (a*2+b*2)1/2 and hue angle by [hº = tan−1 × b*/a*] equations.[Citation16]
Bioactive Compounds
Total phenolics and total antioxidant were only measured in the flesh of the fruits. Total anthocyanin was only measured in the peel of fruit. A total of ten fruits were homogenized and placed into four different tubes and measurements were taken from each tube in each replication. The fruit samples were kept in 50 mL tubes at –20°C for bioactive analysis. Samples were thawed at room temperature (≈21°C) and homogenized in a food grade blender. The resultant slurry was centrifuged (12,000 g) for 30 min at 4°C to separate the juice from the pulp. The freshly obtained juice materials were diluted with distilled water, divided into multiple sample aliquots, and refrozen at –20°C until used in phenolics, antioxidant, and anthocyanin assay procedures.
Total phenolics were measured according to the Singleton and Rossi[Citation17] procedure. Briefly, fruit slurries (only cortex) were extracted with buffer containing acetone, water, and acetic acid (70:29.5:0.5 v/v) for 2 h in darkness. Samples were replicated three times. Extracts were combined with Folin-Ciocalteus phenol reagent and water, and incubated for 8 min followed by the addition of 7% sodium carbonate. After 2 h, the absorbance at 750 nm was measured in an automated UV-Vis spectrophotometer (Model T60U, PG Instruments, USA). Gallic acid was used as the standard. The results were expressed as μg gallic acid equivalents (GAE)/g flesh weight (fw).
Total antioxidant activity was estimated using one standard procedure, the TEAC assay, as suggested by Ozgen et al.[Citation18] For the standard TEAC assay, 10 mmol/L ABTS (2.2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) was dissolved in acetate buffer and prepared with potassium persulfate. The mixture was diluted using an acidic medium of 20 mM sodium acetate buffer (pH 4.5) to an absorbance of 0.700 ± 0.01 at 734 nm for longer stability. For the spectrophotometric assay, 2.90 mL of the ABTS+ solution and 100 μL of fruit extract were mixed and incubated for 10 min. The absorbance at 734 nm was then determined. The results were expressed in μmol trolox equivalents (TE)/g fw.
The total anthocyanins of fruit skin were estimated by a pH differential method[Citation19] using a UV–Vis spectrophotometer (Model T60U, PG Instruments, USA). Absorbance was measured at 533 and 700 nm in buffers at pH 1.0 and 4.5 using pH 4.5 with a molar extinction coefficient of 29.600. Results were expressed as μg cyanidin-3-galactoside/g fresh peel weight equivalent.
Internal Ethylene Concentration
To measure internal ethylene concentrations, 1 mL air sample from the core cavity of each fruit was injected into a gas chromatograph equipped with an active alumina column and Flame Ionization Detector (Perkin Elmer-Clarus 500, USA), using the method of Bramlage et al.[Citation20] The resulting peaks were compared to that of 100 μL/L ethylene standard and the internal ethylene concentration was calculated.
Other Quality Parameters
Fruit mass (g) were measured with a digital balance (±0.01 g; Radvag PS 4500/C/1, Poland). Dimensional characteristics (length [L], width [W], and thickness [T]) were measured with a digital caliper (±0.01 mm; Model No: CD-6”CSX, Mitutoyo, Japan) and geometric mean diameter was calculated by using the equation of geometric diameter = (L.W.T).1/3[Citation21] Fruit flesh firmness was measured on three sides of equatorial line of each fruit using a press-mounted Effegi penetrometer (FT 327; McCormick Fruit Tech. Torino, Italy) with an 11.1 mm tip. Fruit firmness was expressed as Newton (N).
A sample of juice was also taken from each piece of the fruits (20 fruits) and the percentage SSC was measured using a digital refractometer (PAL-1, McCormick Fruit Tech., Yakima, WA, USA). A pH meter (Hanna, model HI9321, USA) was used to measure the pH of extracts. For titratable acidity, 10 mL extract was taken from each sample, 10 mL distilled water was added and the value corresponding to consumed sodium hydroxide (NaOH) during the titration with 0.1 N sodium hydroxide to increase the pH of samples to 8.1 was expressed in malic acid (g malic acid/100 mL). Starch-iodine tests of sliced fruits were carried out by using the Cornell Generic Starch-Iodine Index Chart, where 1 = 100, space bar between the numbers and equality sign. The same for 8 = 0% starch in the same line.[Citation22]
Statistical Analysis
Experiments were carried out in randomized complete-block design. All statistical analyses were performed with SAS Version 9.3 (SAS Institute Inc., Cary, NC, USA). Data were analyzed by means of analysis of variance. Where appropriate, means were separated by orthogonal polynomial comparison or Duncan’s multiple range test.
RESULTS AND DISCUSSION
L*, chroma, and hue angle values were determined to evaluate the variations in peel color. L* indicates luminance or lightness of the color and varies between 0 (dark) and 100 (light). Bizjak et al.[Citation6] indicated decreasing L* values with the progress of ripening in “Braeburn” apples. Similarly in the present study, L* values of “Fuji” apples also decreased with the progress of ripening. L* values linearly decreased with increasing MeJA doses in both experimental years. Compared to the control treatment, 1120 and 2240 mg/L MeJA treatments of the first year did not have significant effects on L* value of the first two sampling dates but 4480 mg/L the MeJA treatment significantly decreased L* values. In the last sampling date, on the other hand, the entire MeJA treatments had lower L* values than the control treatment (). Similar results were also observed during the second experimental year. Complying with the findings of the present study, decreased L* values were observed in “Golden Delicious” and “Fuji” apples with post-harvest MeJA treatments,[Citation14] and in “Cripps Pink” apples with pre-harvest MeJA treatments.[Citation7] By definition, chroma indicates the degree of departure from gray toward a pure chromatic color.[Citation16] Contrary to the findings of Fan and Mattheis[Citation14] in “Fuji” and of Shafiq et al.[Citation7] in “Cripps Pink” apples, the chroma values of the present study linearly decreased with increasing MeJA doses. The hue angle is the best indicator of color changes during the ripening of apples.[Citation23] Bizjak et al.[Citation6] reported that hue angle decreased toward the ripening in “Breaburn” apples, indicating a greater intensity of red color. Complying with the findings of Rudell et al.[Citation13] and Rudell and Mattheis,[Citation24] hue angles of almost all MeJA treatments were lower than the control treatment. The hue angles decreased linearly with increasing MeJA doses in both experimental years ().
TABLE 1 Effects of pre-harvest methyl jasmonate treatments on the color characteristics (L*, chroma, and hue angle) of “Fuji” apples.
Similar to the findings of Kondo et al.[Citation15] and Rudell et al.,[Citation25] the MeJA treatments in the present study distinctively increased the anthocyanin contents in both experimental years. A clear linear relationship was observed between the treatment doses and the anthocyanin contents. Similar case was also observed for total phenolics. Increasing MeJA treatments linearly increased the total phenolics contents of apples (). Shafiq et al.[Citation26] also reported increased flavonoid levels with pre-harvest MeJA treatments in “Cripps Pink” apples. Similarly, pre-harvest MeJA treatments increased the flavonoid levels in blueberries,[Citation27] blackberries,[Citation28] and grapes.[Citation29] Phenolics have antioxidant characteristics. There is a linear relationship between total phenolics or anthocyanin and antioxidant capacity of berries.[Citation30] In the present study, antioxidant capacity of the fruits increased with MeJA treatments as a natural outcome of increased total phenolics and anthocyanin production ().
TABLE 2 Effects of pre-harvest methyl jasmonate treatments on the total anthocyanin, total phenolics and total antioxidant capacity of “Fuji” apples.
It is assumed that there was a synergic interaction between MeJA and ethylene to regulate the anthocyanin synthesis in apples.[Citation24] Fan et al.[Citation11] reported that MeJA promoted the ethylene synthesis in pre-climacteric fruits. MeJA-induced increase in ethylene synthesis has also been documented in different fruit species.[Citation10] Complying with all these relevant findings, clear increases were observed in ethylene synthesis of MeJA-treated “Fuji” apples of the present study. Internal ethylene concentrations linearly increased with increasing MeJA doses in all sampling dates ().
TABLE 3 Effects of pre-harvest methyl jasmonate treatments on the internal ethylene concentration of “Fuji” apples in 2011.
Rudell et al.[Citation13] reported that early-season MeJA treatments (48 days after full bloom) decreased fruit weights and diameters, but late-season MeJA treatments (119 days after full bloom) on the other hand did not alter fruit weights and diameters. In the present study, the MeJA treatments did not cause any significant changes in fruit weights and geometric mean diameters. In the first year (2010), all MeJA treatments increased flesh firmness. In the second year (2011), while 1120 mg/L MeJA did not cause a significant change, the fruits subjected to 2240 and 4480 mg/L MeJA treatments had higher flesh firmness values than the control fruits. While there were no significant differences in starch degradation between 1120 or 2240 mg/L MeJA treatments and control treatment in both experimental years, 4480 mg/L MeJA treatment delayed starch degradation (). Rudell et al.[Citation13] reported that although MeJA promoted ethylene synthesis, it also increased flesh firmness and delayed starch degradation, and concluded that MeJA might act independent of ethylene during the ripening process. The results of the present study support this consideration.
TABLE 4 Effects of pre-harvest methyl jasmonate treatments on the fruit mass, geometric diameter, fruit flesh firmness, soluble solids content, titratable acidity, and starch degradation of “Fuji” apples.
A regular sugar production is necessary for anthocyanin production and well-color development.[Citation31] Shafiq et al.[Citation26] reported that weekly MeJA treatments increased SSC/titratable acidity ratios of “Cripps Pink” apples. Similarly, it was reported that MeJA treatments increased sugar content and decreased acid content of raspberry.[Citation32] In the present study, MeJA treatments did not cause significant changes in SSC values of “Fuji” apples but significantly increased acid contents of the apples (). Such a conflicting finding may be as a result of the differences in the fruit cultivars or implementations.
CONCLUSIONS
It was concluded in the present study that MeJA could reliably be used to improve color formation and antioxidant capacity of “Fuji” apples without any significant changes in fruit quality parameters.
ACKNOWLEDGMENTS
The authors are grateful to Zeki Gökalp for his critical reading and thorough syntactic corrections of the manuscript.
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