Postharvest Application of Abscisic Acid and Methyl Jasmonate on Fruit Quality of ‘Red Zaosu’ Pear

Abstract

Subsequent to the harvesting of ‘Red Zaosu’ pears, a swift decline in quality becomes evident. This is characterized by the discoloration of the peel, the softening of the flesh and metabolic alterations during storage. To elucidate the regulatory roles of phytohormone in fruit preservation, postharvest pears were treated with 100 μmol/L abscisic acid (ABA), 100 μmol/L methyl jasmonate (MeJA) or their combination (ABA + MeJA). The results indicated that the phytohormone treatment groups exhibited varying degrees of efficacy in improving the postharvest quality of pear fruits. The combined treatments did not show synergistic effects, but rather inhibited anthocyanin accumulation and antioxidant enzyme (POD, CAT, APX, POD) activities and significantly reduced soluble solids, acidity and flavonoids, although peel brightness was maintained. ABA alone treatment promoted anthocyanin accumulation and peel coloring, but reduced fruit firmness, crispness, chewiness and soluble solids, enhanced total flavonoids and CAT activity and reduced malondialdehyde accumulation, while MeJA alone treatment inhibited anthocyanin synthesis and coloring, but also reduced firmness and soluble solids, and enhanced total flavonoids and CAT activity. The results indicate that ABA and MeJA exhibit differential regulatory effects on fruit quality when applied individually, and their combined application showed inferior effects compared to individual treatments. This finding provides a theoretical basis for optimizing combined phytohormone-preservation techniques.

1. Introduction

Pear (Pyrus spp.) is a perennial deciduous fruit tree of the genus Pyrus in the family Rosaceae, widely cultivated in China [1]. China’s pear production reached 19 million metric tons in 2022 [2]. However, pear peel has the problem of unstable red coloring and pears also face the problem of yellowing peel, softening flesh and decreasing fruit quality during the shelf life [3]. Many physical and chemical methods have been applied to fruit postharvest treatments to improve storage quality [4].

Phytohormones play an important role in the regulation of postharvest fruit quality. Depending on the rate of respiration and the capacity to produce and respond to ethylene during ripening, fruits can be classified into two groups: climacteric and non-climacteric fruits [5]. While pears belong to the climacteric fruits, more and more studies have shown that abscisic acid (ABA) can directly regulate the ripening of climacteric fruits, such as pigment synthesis, sugar accumulation and fruit softening [6,7]. It has been shown that ABA is closely associated with the process of ripening and anthocyanin synthesis in fruits such as pears [8], apples [9] and strawberries [10]. In strawberries, ABA may play a role in strawberry fruit ripening and coloration processes by upregulating ethylene production and phenylalanine ammonia lyase (PAL) activity [11]. However, studies have shown that 6 μmol/L ABA treatment significantly inhibited anthocyanin biosynthesis in callus cultures of red-fleshed apples [12]. Furthermore, the application of ABA on jujube increased the malondialdehyde (MDA) content in the fruit peel, resulting in elevated free radical-induced cytoplasmic membrane damage, which adversely impacted the quality preservation of jujube fruits [13]. These differences may be related to factors such as fruit type, ABA concentration and treatment duration. Therefore, when applying ABA for postharvest fruit treatment, a variety of factors need to be considered comprehensively to achieve the best preservation effect.

Methyl jasmonate (MeJA), a derivative of jasmonic acid (JA), is an important plant signaling compound and growth regulator. It plays significant roles in plant growth and development, stress responses and the enhancement of fruit internal and external quality [14]. MeJA treatment significantly promoted red coloration in ‘Danxiahong’ pears and increased the fruit soluble solids, improved the total sugar content, decreased the fruit acid content and significantly increased the total sugar/total acid ratio [15]. In addition to color improvement, the application of exogenous MeJA significantly increased bioactive compounds and antioxidant levels in a wide range of fruits. Postharvest treatment with MeJA at 500 μmol/L and 1500 μmol/L significantly increased total flavonoid content in blood oranges [16]. Preharvest spraying of MeJA promotes the accumulation of phenolics, flavonoids and carotenoids in ‘Yoho’ persimmon [17]. Postharvest spraying of 100 μmol/L MeJA and storing Rosa roxburghii fruits at low temperature could effectively reduce decay, maintain fruit firmness and brightness, inhibit respiration and reduce MDA content. Further analyses revealed that MeJA enhanced antioxidant capacity by upregulating both the activity and gene expression of superoxide dismutase (POD), catalase (CAT) and peroxidase (POD) [18].

An increasing number of studies have demonstrated that the composite postharvest preservation method exhibits a significant synergistic effect and provides better results in maintaining fruit postharvest quality compared to single treatments [19,20,21]. Postharvest application of MeJA not only improves fruit quality in plums [22] and kiwifruit [23], but also boosts antioxidant enzyme activity, increases total antioxidant capacity, delays senescence and extends shelf life. Additionally, studies have indicated that MeJA can alleviate the inhibitory effect of ABA on anthocyanin biosynthesis in red-fleshed apple callus [12]. However, there are fewer studies on the synergistic effects of ABA and MeJA in regulating fruit quality and extending shelf life, particularly regarding their combined application in pears. The promotive or inhibitory mechanisms underlying the combined ABA and MeJA treatment on postharvest fruit quality preservation in ‘Red Zaosu’ pears remain unclear. Therefore, investigating the synergistic mechanisms between ABA and MeJA is critical to advance understanding of fruit-ripening and senescence processes, enhance fruit quality and prolong shelf life. This study aims to elucidate how ABA and MeJA synergistically regulate postharvest pear fruit quality, thereby providing both theoretical insights and practical strategies for fruit quality improvement and shelf life extension.

2. Materials and Methods

2.1. Materials

‘Red Zaosu’ pear trees were planted in the Pear Germplasm and Innovative Resources Nursery at the Chongzhou Modern Agriculture R&D Base of Sichuan Agricultural University, located at 103°64′52″ E and 30°55′67″ N. Three ‘Red Zaosu’ trees of similar age (≥5 years old), vigor and fruit load were selected as experimental trees. Young fruits were chosen for bagging and shading 15 days after flowering. The fruits remained bagged and were transported to the laboratory with shock-absorbing packaging after harvesting at 100 days post-bagging. Fruits of uniform size and free from mechanical damage were selected under light-restricted conditions.

2.2. Treatments

In our preliminary experiment, ‘Red Zaosu’ apples were treated with 50 μmol/L and 100 μmol/L ABA, respectively. The data indicated that 100 μmol/L ABA treatment was more effective in promoting pear anthocyanin accumulation (Figure S1). In addition, the data indicated that 100 μmol/L MeJA treatment was effective in maintaining the postharvest quality of Rosa roxburghii fruit [18]. Therefore, 100 μmol/L ABA and 100 μmol/L MeJA were selected in this study.

The experiment consisted of four treatments, each containing 45 pear fruits, with three replicates. The solution was prepared by dissolving ABA powder (Solarbio, Beijing, China) and 98% MeJA (Macklin, Shanghai, China) in 1% (v/v) aqueous ethanol solution containing 0.02% (v/v) Tween-20 (this solvent was chosen because MeJA requires dissolution in 1% ethanol). For the combined treatment (ABA + MeJA), 100 μmol/L ABA and 100 μmol/L MeJA solutions were mixed in a 1:1 (v/v) ratio. Fruits were soaked in ABA, MeJA or ABA + MeJA solutions for 30 min (obtained through pre-experimental screening), while the control group (CK) was treated with 1% aqueous ethanol solution containing 0.02% Tween-20 for the same duration. All fruits were then air-dried under light-restricted conditions. The treated fruits were placed in a controlled environment with a temperature of 23 ± 2 °C, relative humidity of 80–90% and a light intensity of 220 μmol/(m²·s). Fruits were turned over every 2 days. Physiological indices (color, texture, soluble solid content, titratable acid content, etc.) were measured on days 0, 3, 6, 9 and 18. After each measurement, samples were rapidly collected. The peel and pulp from the light-exposed surface of each fruit were scraped to a depth of approximately 1 cm at the equatorial region under liquid nitrogen immersion, then stored in an ultra-low-temperature freezer at −80 °C for subsequent analysis. At each sampling time point, three fruits were randomly selected from each treatment group to form composite samples for the determination of relevant indices, repeated three times.

2.3. Color Properties and Texture

Peel color was determined using the method of McGuire [24] with slight modifications. A colorimeter (CR-400, Minolta, Tokyo, Japan) was used to determine the color parameters (L*, a* and b*) of each fruit. Measurements were taken three times at five randomly selected positions on the light-exposed surface of each fruit. The values of chroma C (Chroma) and h° (hue angle) were calculated from the a* and b* values: C = $\sqrt{{a\ast }^2+{b\ast }^2}$; h° = tan(b*/a*) when both a* and b* are greater than 0 and h° = 180 + tan⁻¹(b*/a*) when a* < 0 and b* > 0.

Fruit texture was analyzed with a TA-XTplus texture analyzer (Bosin Tech, Shanghai, China) referring to the method of Ding, X. et al. [25] with minor modifications. Instrument settings were as follows: target displacement 8.000 mm, test duration 3.00 s, pre-test speed 5.00 mm/s, test speed 3.00 mm/s, post-test speed 3.00 mm/s, trigger force 10.000 gf and probe contact area 2.000 mm². For each treatment, five evenly distributed points on the equatorial plane of each fruit were tested for texture parameters.

2.4. Total Soluble Solids (TSSs), Titratable Acid (TA) and Sugar Contents

TSSs (Brix %) and TA (%) were measured using a Pocket Brix-Acidity Meter Master Kit (ATAGO, Tokyo, Japan). The undiluted fruit juice was used to measure TSS contents. To measure TA contents, about 100 μL of fruit juice was collected and diluted to 5 mL (1:50 v/v) using redistilled water.

The extraction, detection and quantification of sugar and acid components were conducted following the method of Ma, Y. et al. [26] with slight modifications. Fresh samples were completely freeze-dried in a vacuum environment of −80 °C using a freeze dryer (FDU-2110,EYELA, Tokyo, Japan). The dry samples were ground into powder in liquid nitrogen. Approximately 0.2 g of powder were mixed with 10 mL of redistilled water and heated in an 80 °C water bath for 15 min. The suspension was then centrifuged at 10,000× g for 15 min at 4 °C (Sorvall ST 16R centrifuge, Thermo Fisher Scientific, San Jose, CA, USA) and the supernatant was collected. The sample was extracted twice and the combined supernatants were diluted to 10 mL with ultrapure water. The supernatant was filtered through a 0.22 μm nylon membrane to remove large particles. Sugar components were determined using an HPLC method, employing an Agilent 1260 II high-performance liquid chromatography system (Agilent Technologies Co., Ltd., Santa Clara, CA, USA) as follows: chromatographic column Athema (reversed-phase), 5 μmL, NH2-RP column, detector RID, detector temperature 30 °C; mobile phase: ethanol∶ultrapure water = 80∶20 (v/v), flow rate 1 mL/min, isocratic elution. Three biological replicates of each sample were used for extraction and HPLC analysis.

The peak areas of glucose, fructose, sucrose and sorbitol standards were used to draw a standard curve to calculate the content of each sugar component in the samples. The soluble sugar contents were expressed as mg/g dry weight (DW).

2.5. Total Anthocyanin and Total Chlorophyll Contents

The total anthocyanin content was determined by referring to the method of Honda, C. et al. [27] with slight modifications. The sample weighed 0.5 g, was ground with liquid nitrogen and was homogenized with 5 mL of pre-cooled 1% (v/v) hydrochloric acid–methanol solution. The mixture was then extracted in the dark at 4 °C for 24 h and centrifuged at 12,000 rpm for 20 min (4 °C). The absorbances of the extracts at 530, 620 and 650 nm were measured using a spectrophotometer (UV-2450, Shimadzu, Kyoto, Japan). Anthocyanin content was calculated as normalized OD530/g fresh weight (FW). Normalized OD530 = [(OD530 − OD650) − 0.2(OD650 − OD620)].

The total chlorophyll content was determined by the method of Kou, X. et al. [13] with slight modification. The sample weighed 0.5 g, was ground with liquid nitrogen and was extracted with 5 mL of 80% (v/v) acetone. The extract was kept in the dark at 4 °C for 24 h until the precipitate turned white. Absorbance values at 663 nm and 645 nm were recorded using a spectrophotometer. The chlorophyll concentration (mg/g FW) of the extract was calculated: chlorophyll a = 12.7 OD663 − 2.69 OD645, chlorophyll b = 22.9 OD645 − 4.86 OD663, total chlorophyll = chlorophyll a + chlorophyll b. Mixed sample analysis was conducted on samples consisting of three fruits each, with three replicates.

2.6. Total Phenolic and Total Flavonoid Contents

The total phenol content of the pears was determined according to the method of Xu, F. [28] with slight modifications. A 0.2 g mixed sample of pear peel and pulp was homogenized in 4 mL of 2% (v/v) hydrochloric acid–methanol solution on ice. The homogenate underwent ultrasonic extraction at 30 °C for 30 min, followed by centrifugation at 9000× g for 10 min at 4 °C. The supernatant was collected and the residue was re-extracted once under the same conditions. The combined supernatants were diluted to a final volume of 10 mL with the extraction solvent. For analysis, 200 μL of the extract was mixed with 1.8 mL distilled water and 0.5 mL Folin-Ciocalteu reagent. After 5 min of incubation, 4 mL of 15% (w/v) sodium carbonate (Na₂CO₃) solution was added. The mixture was allowed to react for 1.5 h in the dark and absorbance was measured at 765 nm. Total phenol content was quantified using a gallic acid standard curve and expressed as micrograms of gallic acid equivalents per gram of fresh weight (μg GAE/g FW).

Total flavonoid content of pears was determined according to the method of Men, C. et al. [29] with slight modifications. The extraction procedure was identical to the phenol method. Subsequently, 1 mL of the extract was mixed with 4.8 mL distilled water and 600 μL of 5% (w/v) sodium nitrite (NaNO₂) solution. The mixture was incubated for 5 min, followed by the addition of 600 μL of 10% (w/v) aluminum nitrate (Al(NO₃)₃) solution and 6 min incubation. Finally, 3 mL of 1 mol/L sodium hydroxide (NaOH) was added and the solution was incubated for 15 min. Absorbance was recorded at 510 nm. The total flavonoid content of the pear sample was calculated against a rutin standard curve and expressed as micrograms of rutin equivalent per gram of fresh weight (μg rutin/g FW).

2.7. MDA Content and the Activities of SOD, POD, CAT and APX

The MDA content was determined using the method of Kou, X. et al. [13] with slight modifications. A 1.0 g mixed sample of peel and pulp (pooled from three pears) was homogenized in 5 mL of 5% (w/v) trichloroacetic acid (TCA) and centrifuged at 10,000× g for 20 min. Then, 2 mL of supernatant was mixed with 2 mL of 0.67% (w/v) thiobarbituric acid (TBA) solution and incubated in boiling water for 30 min, rapidly cooled on ice and centrifuged again at 10,000× g for 20 min. Absorbance of the supernatant was measured at 450, 532 and 600 nm. Total MDA (μmol/g FW) = amount of extraction (mL) × [6.45(OD532 − OD600) − 0.56 × OD450]/[amount of supernatant (mL) × amount of sample (g)].

The activities of SOD, POD, CAT and APX enzymes were assayed using commercial kits (Jiangsu Aidisheng Biological Technology Co., Ltd., Yancheng, China) according to the manufacturer’s instructions.

2.8. Statistical Analysis

Statistical analyses were performed using Microsoft Excel 2016, combined with IBM SPSS Statistics 27.0 for one-way analysis of variance (ANOVA) and Waller–Duncan multiple comparison significance tests (p < 0.05, significant difference) and Origin 2021 for charting.

3. Results

3.1. Effect of Different Treatments on Coloration of ‘Red Zaosu’ Pears

Fruits in the control (CK), ABA and MeJA alone treatment groups began coloration at 6 days post-treatment. The ABA alone treatment induced significantly larger red areas compared to CK. However, the ABA + MeJA combination treatment significantly inhibited skin coloration in ‘Red Zaosu’ pears. Peel pigmentation progressively shifted from yellow to green with prolonged treatment duration (Figure 1A). The L* values indicated progressive darkening of peel coloration with extended treatment time. Notably, MeJA and the combined treatment maintained higher L* values, showing increases of 17.52% and 30.40%, respectively, compared to the CK after 18 days of treatment (Figure 1B). On the other hand, the a* value dynamics correlated with observed phenotypic alterations, where combination treatment caused values to decline below zero, indicating that the skin became green (Figure 1C). Both b* values and chroma displayed initial decreases followed by increases in each treatment. All treatments enhanced b* and chroma values compared to CK, with the combination treatment showing the most significant efficacy (Figure 1D,E). Comprehensive analysis of color parameters in each group demonstrated distinct coloring conditions: fruit of CK and single-treatment groups changed from yellow to yellow-red color, where MeJA treatment alone inhibited peel reddening compared to CK and ABA treatment had a similar effect to CK; while the fruit of the combined treatment gradually turned yellow-green and the ABA + MeJA treatment significantly inhibited peel reddening and promoted peel greening.

3.2. Effects of Different Treatments on Total Anthocyanin and Chlorophyll Contents of ‘Red Zaosu’ Pears

With prolonged treatment time, the anthocyanin content in the peels of both the CK group and phytohormone alone treatment groups gradually increased. Compared to CK, ABA treatment significantly promoted anthocyanin accumulation, whereas MeJA and ABA + MeJA treatments markedly inhibited this process. At 18 days post-treatment, anthocyanin content in the ABA-treated group increased by 12.34% compared to the CK, while MeJA and ABA + MeJA treatments reduced anthocyanin levels by 59.31% and 81.42%, respectively (Figure 2A,B). Chlorophyll content also affects the color of the fruit; the chlorophyll content of the CK group exhibited a gradual increase over time. In contrast, the ABA and MeJA alone treatment groups showed no significant chlorophyll accumulation from 0 to 9 days, but displayed rapid accumulation from 9 to 18 days, ultimately exceeding CK levels. Conversely, the ABA + MeJA treatment group showed a decline in chlorophyll content from 0 to 9 days, followed by accumulation from 9 to 18 days, though its overall levels remained lower than those of the CK (Figure 2C,D). Overall, ABA treatment promoted the accumulation of both anthocyanins and chlorophyll in pear fruits, while MeJA treatment inhibited anthocyanin accumulation with minimal impact on chlorophyll content. In contrast, the ABA + MeJA combined treatment exhibited inhibitory effects on both pigments.

3.3. Effects of Different Treatments on Fruit Texture of ‘Red Zaosu’ Pears

Fruit firmness decreased gradually with increasing treatment time. Compared to the CK, ABA and MeJA alone treatments significantly reduced fruit firmness, whereas the combined ABA + MeJA treatment exhibited no significant effect on fruit firmness (Figure 3A). As shown in Figure 3B, the trend of fracturability is similar to that of firmness. All three phytohormone treatments significantly reduced fruit fracturability compared to the CK, and ABA or MeJA alone treatments exhibited stronger effects than the combined treatment. Adhesiveness reflects the tendency of fruit particles to adhere to oral surfaces. The trend of adhesiveness showed similar gradual increases in both the CK and ABA alone treatment. In contrast, MeJA alone and ABA + MeJA treatments first increased then decreased adhesiveness. On day 18 after treatment, the MeJA treatment alone increased total adhesiveness of pear fruits by 17.68% compared to the CK, while the ABA and ABA + MeJA combined treatments decreased it by 22.42% and 23.47%, respectively. Thus, MeJA alone enhanced adhesiveness, while ABA alone and the combination reduced it (Figure 3C). Resilience reflects the ability to recover from deformation. Under CK and ABA treatments, it fluctuated, while under MeJA and ABA + MeJA treatments, it initially decreased and then increased. On the 18th day post-treatment, resilience values reached 5.52 and 4.81, respectively, significantly higher than those observed in CK and ABA alone treatments (Figure 3D). Cohesiveness, which reflects the ability of fruit tissues to resist damage and maintain structural integrity during mastication, can indicate intercellular bonding strength. Chewiness is the product of firmness, cohesiveness and springiness, which represents the fruit’s sustained resistance during mastication. The results indicated that adhesiveness and chewiness exhibited similar trends in their changes, with an overall pattern of initially decreasing and then increasing. On day 18 after treatment, the cohesiveness and chewiness of pear fruit ranked from highest to lowest as ABA, MeJA treatment group, CK and ABA + MeJA combined treatment group. In addition, MeJA has little effect on cohesiveness and chewiness. This suggests that the combined application of ABA + MeJA resulted in looser flesh tissue and diminished fruit flavor, while the use of ABA alone could enhance the flavor profile (Figure 3E,F).

3.4. Effects of Different Treatments on TSS, TA and Sugar Compositions of ‘Red Zaosu’ Pears

The TSS content in the CK, ABA alone treatment group and ABA + MeJA treatment groups exhibited a trend of initial decrease followed by increase, whereas the MeJA alone treatment group showed an opposite trend, with TSS accumulation peaking on day 6. On day 18 after treatment, the TSS content in the ABA, MeJA and ABA + MeJA treatment groups decreased by 10.79%, 12.96% and 14.85%, respectively, compared to the CK. Therefore, all three phytohormone treatments significantly inhibited TSS accumulation (Figure 4A). The TA content in pear fruits peaked on day 3, then gradually decreased. Compared to the CK, both ABA and ABA + MeJA treatments significantly accelerated TA reduction, whereas MeJA alone showed minimal impact on TA degradation (Figure 4B).

As shown in Figure 4C–F, fructose was the most abundant sugar component in ‘Red Zaosu’ pear fruit, followed by glucose, sorbitol and sucrose. During the 0–18 d post-treatment period, the fructose content in CK showed an initial increase followed by a decrease, while treated groups exhibited a fluctuating pattern of increase–decrease–increase. On day 18 after treatment, the ABA + MeJA combined treatment increased fructose content by 6.68% compared to the CK, while ABA treatment alone significantly decreased it by 5.74%. In contrast, the MeJA treatment showed no significant effect (Figure 4C). The glucose content in both CK and ABA + MeJA groups displayed an initial increase followed by decline. However, the ABA + MeJA group maintained significantly lower levels than CK throughout. Notably, individual ABA or MeJA treatments resulted in gradual glucose accumulation, surpassing the CK group’s level by day 9 post-treatment. At day 18 after treatment, glucose content in these treatments increased by 38.46% and 28.14%, respectively, compared to the CK. These results suggest that phytohormone treatment alone enhances glucose biosynthesis, whereas combined treatment has an inhibitory effect (Figure 4D). The sorbitol content of the CK, MeJA and ABA + MeJA treatment groups fluctuated within a certain range in the post-treatment period. The ABA alone treatment group exhibited a gradual decline, consistently below CK levels. On day 18 after treatment, the sorbitol contents in the ABA, MeJA and combined treatments were 110.69, 169.24 and 188.58 mg/g, respectively, compared to 197.56 mg/g in the CK. These results indicate that all three treatments inhibited sorbitol metabolism, with the ABA treatment alone exhibiting the strongest inhibitory effect (Figure 4E). In contrast to the CK, which exhibited an initial increase followed by gradual decline in sucrose content, the content of sucrose in the treatment group gradually accumulated over the treatment period, ultimately exceeding CK levels and reaching statistical significance by day 18. This suggests phytohormone treatments enhanced sucrose biosynthesis, with ABA alone treatment showing the greatest efficacy, followed by MeJA and ABA + MeJA treatments (Figure 4F).

3.5. Effects of Different Treatments on Total Phenol and Flavonoid Contents of ‘Red Zaosu’ Pears

The total phenolic content of pear fruits gradually accumulated from 0 to 6 days after treatment, followed by a gradual decline. The ABA alone treatment group exhibited a significantly higher total phenol content than the CK from 3 to 9 days, indicating that ABA alone could markedly promote total phenol accumulation in pear fruits. The promotion effect of MeJA treatment alone is relatively weak. In contrast, the ABA + MeJA combination treatment significantly suppressed total phenol accumulation, maintaining levels below the CK throughout the post-treatment period (Figure 5A). The total flavonoid content in pear fruits from both the CK and single phytohormone treatment groups exhibited an initial increase followed by a decrease during the post-treatment period. On day 18 after treatment, the total flavonoid content in the ABA and MeJA treatment groups increased by 10.22% and 29.93%, respectively, compared to the CK, indicating that individual treatments with these phytohormones promoted total flavonoid accumulation. In contrast, the ABA + MeJA combined treatment group showed significantly lower flavonoid levels from 9 to 18 days compared to the CK, suggesting that the combination treatment suppressed flavonoid accumulation (Figure 5B). The results that individual treatments of ABA and MeJA promote flavonoid accumulation align with findings by Mattus-Araya, Elena et al. [30] and Wang, K. et al. [31], whereas the combined ABA + MeJA treatment induced an antagonistic effect, indicating that this treatment impedes changes in secondary metabolites during postharvest fruit ripening.

3.6. Effects of Different Treatments on MDA Content and Activities of SOD, CAT, APX, POD of ‘Red Zaosu’ Pears

MDA content of pear fruit exhibited a gradual increase during storage. Compared to the CK, both ABA and ABA + MeJA combined treatments significantly inhibited MDA accumulation. However, the inhibitory effect of MeJA treatment on MDA was not observed until the ninth day. On day 18 after treatment, ABA, MeJA and ABA + MeJA combined treatment reduced values by 39.24%, 79.66% and 60.43%, respectively, compared to the CK. These results indicate that all three phytohormone treatments (ABA, MeJA and ABA + MeJA combined) can alleviate membrane lipid peroxidation damage in pear fruit cells, with the ABA + MeJA combined treatment demonstrating the most pronounced effect (Figure 6A).

The antioxidant enzyme system in plants includes SOD, which acts as the initial defense mechanism. SOD rapidly converts O₂⁻ into H₂O₂ and O₂, effectively eliminating O₂⁻ and protecting oxygen-metabolizing cells from its harm. With prolonged post-treatment time, the SOD activity in pear fruit gradually increased. On day 18, the CK exhibited the highest SOD enzyme activity (0.9427 U/g), while the treatment groups ranked in descending order as follows: ABA + MeJA combined treatment (0.8676 U/g), MeJA treatment (0.5728 U/g) and ABA treatment (0.373 U/g) (Figure 6B). The CAT activity exhibited a trend similar to that of superoxide dismutase (SOD). Both ABA and MeJA alone treatments enhanced CAT activity, showing significant increases of 27.36% and 19.22%, respectively, compared to the CK at 18 days post-treatment. However, in both the CK and ABA + MeJA combined treatment groups, CAT activity declined after 9 days, with the combined treatment group ultimately demonstrating lower enzyme activity than CK (Figure 6C). POD activity fluctuated within a certain range in MeJA and ABA + MeJA treatment groups. ABA treatment caused an initial increase followed by a decrease in POD activity. All three phytohormone treatment groups ultimately maintained significantly lower POD activity compared to the CK (Figure 6D). The APX activity in both the CK and MeJA alone treatment groups fluctuated during the post-treatment period. In contrast, ABA alone treatment and the ABA + MeJA combined treatment induced a progressive decrease in APX activity. Compared to CK, the ABA and combined treatment groups exhibited significantly lower APX activity (Figure 6E).

3.7. Correlation Analysis of Different Treatments

The correlation analysis of the different treatment groups is shown in Figure 7. Certain variables consistently exhibited significant correlations in the CK and individual phytohormone treatment groups, whereas opposite or non-significant correlations were observed in the combined phytohormone treatment groups. For example, L* value showed positive correlations with h°, firmness, fracturability and total flavonoid content, but negative correlations with a* value, adhesiveness, MDA content and SOD enzyme activity in CK and individual treatments. However, in combined treatments, L* value was positively correlated with MDA content and SOD enzyme activity. a* value displayed negative correlations with h° and firmness and positive correlations with MDA content and SOD enzyme activity in CK and individual treatments, but exhibited reversed trends in combined treatments. Notably, significant correlations were identified in CK and individual phytohormone treatments between b* value and total flavonoid content, h° and firmness/fracturability, anthocyanin content and firmness, MDA content and SOD enzyme activity, TSS and total phenol content. These relationships became non-significant in the combined phytohormone treatment groups.

4. Discussion

The fruit-ripening process involves physiological and biochemical changes such as pigment accumulation, softening and flavor formation. During the postharvest shelf life of pear fruit, endogenous ethylene biosynthesis accelerates fruit ripening and senescence, leading to discoloration, textural changes, flavor loss and reduced nutritional value, thereby significantly compromising fruit quality and consumer acceptance [32,33,34,35]. ABA and MeJA have been demonstrated to maintain fruit quality in a variety of fruits [36,37]. However, the combined treatment of ABA + MeJA failed to show superiority over individual treatments in improving pear fruit postharvest quality, but exhibited distinct effects compared to individual treatments on specific physiological indicators.

Color is one of the most important indicators of the external quality of fruits. It is widely believed that better fruit peel coloration indicates riper fruit with higher nutritional value, thereby attracting more customers to purchases [38]. The coloring of the fruit skin is the result of the combined action of various pigments (e.g., chlorophyll, carotenoids and anthocyanins) [39]. The formation of red color in pear skin mainly depends on the composition of the peel and anthocyanin concentration [40]. Additionally, anthocyanins in the fruit peel can help prevent cardiovascular and cerebrovascular diseases [41]. The ripening process of pear fruit is typically characterized by the accumulation of anthocyanins and the degradation of chlorophyll [42]. In this experiment, ABA treatment alone promoted peel coloring and also promoted anthocyanin accumulation. Similar findings have also been observed in apples [43], strawberries [44] and grapes [45]. Previous studies suggested that ABA, as a phytohormone promoting fruit senescence and ripening, enhances chlorophyll degradation in postharvest banana fruit [46]. Li et al. [47] demonstrated through transcriptomic analysis that ABA upregulates the expression of chlorophyll degradation-related genes, thereby accelerating chlorophyll degradation, whereas in this experiment, ABA promoted chlorophyll accumulation. This may be attributed to the removal of bagging, which exposes the fruit to light and thereby promotes chlorophyll accumulation. Huang, Chunhui et al. [48] have also reported similar findings in their study. Post-bloom spraying of MeJA was shown to promote red color development in the peel of apple [49] and pear [15], significantly increasing anthocyanin content. However, in this experiment, MeJA did not significantly affect fruit color but inhibited anthocyanin accumulation, possibly due to concentration-dependent effects. Similarly, chlorophyll content changes were MeJA concentration-dependent: 10 μmol/L MeJA inhibited chlorophyll degradation in apples, while 1500 μmol/L MeJA promoted it [50]. In addition, the chlorophyll content of grape berries increased when treated with 10 mmol/L MeJA [51]. In contrast, 100 μmol/L MeJA in this experiment promoted chlorophyll degradation. Unlike single phytohormone treatments, the ABA + MeJA combination significantly inhibited red coloration in ‘Red Zaosu’ pears, with a decrease in h° values indicating a shift toward yellowish-green. The anthocyanin and chlorophyll contents in ABA + MeJA-treated fruits were significantly lower than those in the CK and single phytohormone treatments, suggesting that the combined treatment adversely affects fruit appearance quality.

In this experiment, the combined treatment of ABA and MeJA significantly inhibited the accumulation of anthocyanins in the fruit peel, suggesting an antagonistic relationship between ABA and MeJA in anthocyanin metabolism. Additionally, studies on strawberry treated with MeJA revealed a decrease in ABA levels and downregulation of FaNCED1, indicating that the JA pathway may antagonize the ABA pathway to promote anthocyanin accumulation in strawberry fruits [52]. Further research has demonstrated that the interaction between ABA and MeJA mediates anthocyanin enrichment in Lycium plants [53]. However, further investigations are required to elucidate the synergistic mechanisms underlying the combined treatment in regulating postharvest quality of pear fruits. The regulatory network of signal transduction between ABA and MeJA also needs to be clarified. Future studies could employ transcriptomics and other molecular approaches to investigate the precise regulatory mechanisms of phytohormone signaling involved in the flavonoid biosynthetic pathway via the phenylpropanoid pathway leading to anthocyanin synthesis.

Texture is the result of complex interactions between food components [54] and is an important component of the intrinsic quality of fruits. Texture profile analysis (TPA), a mechanical process simulating human mastication through dual compression of specimens, enables measurement of probe pressure and other texture parameters [55]. Fruit ripening has been shown to correlate with progressive softening [56]. Substantial evidence indicates that ABA acts as a pivotal regulator in the ripening of numerous fruits [57] and that it significantly accelerates the softening process during strawberry ripening [58]. Specifically in sweet cherry, ABA reduces fruit firmness by regulating the expression of cell wall modification genes PavPL18, PavPME44 and PavXTH26/31 [59]. In this experiment, ABA reduced fruit firmness, fracturability and adhesiveness and enhanced fruit cohesiveness, chewiness and resilience, indicating that ABA did promote fruit softening and that it enhanced fruit flavor. Previous studies have found contradictory effects of MeJA on fruit ripening, with exogenous MeJA promoting ripening and softening in apples [60] and kiwifruit [61], whereas the application of 10 μmol/L MeJA to aubergines delayed fruit ripening and improved fruit firmness [62]. In this experiment, MeJA treatment reduced fruit firmness, fracturability, cohesiveness, adhesiveness and improved chewiness and resilience. Moreover, ABA + MeJA combined treatment showed different changes in fruit texture characteristics compared to phytohormone alone treatment. The combined treatment improved fruit firmness, adhesiveness, fracturability, cohesiveness, chewiness and resilience. Changes in fruit texture are closely related to pectic acid, hemicellulose, cellulose, cell expansibility, cell cohesion and assimilation [63]. Therefore, the effect of phytohormones on fruit texture still needs to be further explored.

Soluble sugars and acids are the core edible qualities of fruits [64]. Soluble solids, which are mainly composed of soluble sugars, serve as an important indicator for evaluating fruit quality [65]. In this experiment, ABA, MeJA and their combination treatments reduced TSS content. Previous studies showed that applying 150 μL/L and 300 μL/L ABA reduced TSS content in grapes after storage [66], with similar results observed in jujube. This phenomenon could be attributed to ABA treatment potentially increasing endogenous ABA concentrations and accelerating fruit metabolism, leading to the lowest TSS content at the end of storage [13]. Therefore, ABA treatment is detrimental to the preservation of pear fruit flavor. Different concentrations of MeJA exhibit distinct effects on regulating apple fruit physiological processes [50,67]. For example, treatment of peaches at 102 DAF with 0.44 mmol/L MeJA delayed ripening and reduced soluble solid accumulation [68], whereas 8 mmol/L MeJA promoted both peach fruit ripening and leaf senescence [69]. However, in this experiment, MeJA did not effectively enhance TSS accumulation in pear fruit. Additionally, ABA and combined treatments reduced TA content, while MeJA exerted minimal impact on TA levels. Therefore, the optimal MeJA concentration for regulating quality in ‘Red Zaosu’ pear remains to be determined. In additional, studies have shown that sucrose can promote the accumulation of anthocyanins [70] and there is a positive correlation between sucrose concentration and anthocyanin accumulation [48]. However, no significant correlation between the two was observed in this experiment.

Total phenols and total flavonoids, which are beneficial to human health, serve as important secondary metabolites that help plants adapt quickly to environmental changes through their antioxidant properties. Their levels correlate with the strength of the antioxidant system [71]. In this experiment, the accumulation of total phenol content peaked at 6 days before declining, while both ABA and MeJA significantly increased this peak level and promoted the accumulation of total phenolic compounds. The application of ABA to grapes also enhanced total phenolic content, particularly through a marked increase in total flavonol levels [72]. Although ABA slightly contributed to total flavonoid accumulation, its effect was less pronounced compared to MeJA. The ability of ABA to induce flavonoids has been demonstrated in strawberry [44]. Notably, while the ABA + MeJA combination treatment showed minimal impact on total phenol accumulation in this study, it exhibited a significant inhibitory effect on total flavonoid accumulation.

Fruits gradually senesce and produce reactive oxygen species (ROSs) after harvesting. ROSs oxidize and damage cytoplasmic membranes, accelerating fruit senescence. The damage caused by ROSs can be counteracted by the SOD, CAT, APX and POD defense enzyme systems [72]. However, the postharvest activity trends of antioxidant enzymes such as SOD and CAT exhibit considerable variability across studies, attributable to differences in cultivars, harvest maturity and environmental conditions during experimentation [73,74,75,76]. For instance, in metabolism fruit, the activities of SOD and CAT progressively decreased during fruit ripening, while POD activity exhibited a gradual increase [77]. In contrast, peach fruits demonstrated a distinct pattern: SOD activity initially rose then declined, CAT levels remained relatively stable and POD activity showed a consistent downward trend throughout maturation [78]. In this experiment, compared to the control group, the ABA + MeJA combined treatment maintained lower activities of SOD, POD, CAT and APX. Combined with the observed lower MDA content, higher fruit firmness in ABA + MeJA-treated fruits, we speculate that the combined treatment of ABA and MeJA may inhibit the senescence of pear fruits, thereby slowing down the physiological changes in postharvest fruits. This could result in lower TSS and total flavonoid content, while maintaining higher peel brightness.

5. Conclusions

In this study, ABA alone treatment exhibited superior efficacy compared to MeJA alone treatment in enhancing postharvest pear fruit coloration and quality. However, although the combined ABA + MeJA treatment maintained peel brightness and fruit firmness, it exhibited a significant antagonistic effect on peel pigmentation promotion, thereby adversely impacting the postharvest appearance quality enhancement of ‘Red Zaosu’ pears. Furthermore, the combined treatment failed to outperform individual treatments in preserving intrinsic postharvest fruit quality parameters such as TSS and total flavonoid content. Furthermore, the results revealed that MDA content in the ABA + MeJA treatment group was significantly reduced compared to the CK, while also exhibiting lower activities of SOD, CAT, APX and POD. This phenomenon is likely attributed to the ABA + MeJA treatment delaying fruit senescence. Therefore, the combined ABA + MeJA treatment exerted an inhibitory effect on the regulation of postharvest senescence in pear fruits, thereby impeding the development of postharvest fruit quality. Our findings establish a scientific basis for utilizing ABA and MeJA in postharvest fruit quality management. The observed antagonisms between these phytohormones offer critical guidance for optimizing combined treatment protocols, paving the way for innovative approaches to preserve the postharvest quality of ‘Red Zaosu’ pears.