Postharvest 2,4-Epibrassinolide Treatment Delays Senescence and Increases Chilling Tolerance in Flat Peach

Abstract

Chilling injury (CI) frequently occurs in postharvest flat peach fruit during cold storage, leading to quality deterioration and a reduced shelf life. Therefore, investigating the key factors involved in alleviating CI and developing effective preservatives are vital scientific issues for the industry. 2,4-Epibrassinolide (EBR) is a crucial endogenous hormone involved in plant response to both biological and environmental stressors. At present, most studies focus on the mechanisms of mitigating CI using a single concentration of EBR treatment, while few studies focus on the effects varying EBR concentrations have on CI. The purpose of this research is to explore the effects of varying concentrations of EBR on the postharvest quality and cold resistance of peach fruit, thereby establishing a basis for refining a technical framework of environmentally sustainable strategies to mitigate postharvest CI. The results show that EBR treatment effectively inhibits the generation of reactive oxygen species (ROS) and malondialdehyde (MDA) by maintaining the activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), thereby delaying the internal browning process of postharvest peaches. In addition, EBR treatment reduced the consumption of total phenolics by inhibiting the activities of polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL). Experimental results identify that 5 μmol L⁻¹ EBR treatment emerged as the most effective concentration for maintaining core postharvest quality attributes. It significantly delayed the decrease in firmness, reduced weight loss, effectively inhibited the production of H₂O₂ and O₂⁻·, particularly during the early storage period, strongly restrained the activity of PAL, and maintained lower rot rates and internal browning indexes. While the 15 μmol L⁻¹ EBR treatment enhanced antioxidant activity, increased total phenolic content at certain stages, and maintained higher soluble solids and acid content, its effects on key physical quality parameters, like firmness and weight loss, were less pronounced compared to the 5 μmol L⁻¹ treatment.

1. Introduction

Peach (Prunus persica (L.) Batsch) is a horticulturally significant fruit crop with substantial economic value, renowned for its high nutritional content and bright color. The global annual production of peaches is approximately 25 million tons, of which China accounts for over half [1]. However, as a climacteric fruit, peaches have a limited shelf life at room temperature and are prone to rapid softening and decay during postharvest storage [2]. Currently, cold storage is widely utilized to prolong the availability of fresh produce. However, peaches are highly susceptible to cold stress during storage, and temperatures below 8 °C can lead to metabolic disorders, often resulting in chilling injury (CI). Symptoms of CI include woolliness, flesh browning, loss of flavor, and diminished ripening capacity, which reduce the commercial value and limit the development of the peach industry [3,4,5].

CI is especially common in low-temperature-sensitive fruits, vegetables, and flowers [6]. The cell membrane acts as a protective barrier, safeguarding against damage and physiological disorders while maintaining a stable intracellular environment essential for diverse biochemical processes. Nevertheless, it is highly vulnerable to abiotic stresses, including cold stress, which can compromise its integrity and disrupt cellular functions [7]. Numerous studies have demonstrated that preserving cell membrane integrity in horticultural crops plays a crucial role in alleviating CI during cold storage [8]. Cold stress induces an increase in membrane lipid peroxidation, resulting in decreased membrane fluidity. This is evidenced by elevated levels of malondialdehyde (MDA), which is widely recognized as a reliable indicator of semipermeable membrane loss due to membrane fatty acid peroxidation and is commonly used to assess membrane integrity [9,10]. CI also leads to the overproduction of reactive oxygen species (ROS). This overproduction disrupts the balance between ROS generation and ROS scavenging systems, thereby stimulating lipid peroxidation of cell membranes and resulting in the loss of membrane integrity [11]. Thus, maintaining the dynamic equilibrium of ROS within cells is crucial for preserving the shelf life of postharvest fruit and vegetables during storage [12]. To ensure survival, plants have developed sophisticated antioxidant systems comprising both enzymatic and non-enzymatic mechanisms to regulate ROS homeostasis. Recent studies have revealed that enhancing the activity of antioxidant enzymes, such as catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), and ascorbate peroxidase (APX), can effectively suppress ROS accumulation. This, in turn, helps preserve mitochondrial structure and alleviates the development of CI in postharvest mangoes [12].

Various physical and chemical pre-storage treatments have been utilized to reduce the occurrence of CI. Widely used physical approaches include edible coatings [11], hypobaric treatment [13], and modified atmosphere packaging (MAP) treatment [14]. Chemical treatments primarily involve the application of plant hormones and natural elicitors, including nitric oxide, 1-MCP, melatonin, methyl jasmonate, and brassinosteroids [15,16,17]. Among them, 2,4-Epibrassinolide (EBR), a synthetic analog of brassinosteroids, is notable for its demonstrated safety and efficacy [16].

Brassinosteroids (BRs) are a class of naturally occurring, polyhydroxylated steroidal phytohormones. Extensive research has demonstrated that BRs are integral to plant growth and development, while also improving resilience to a range of environmental stressors, such as drought, cold, salinity, and heavy metal toxicity [18]. EBR has been shown to effectively preserve the postharvest quality of horticultural products and prevent the occurrence of CI [16]. Hu et al. [19] have demonstrated that postharvest treatment with 2,4-Epibrassinolide enhances the chilling resistance of tomatoes by modulating the antioxidant system and the expression of SlCBF1 through mediating SlCYP90B3. Furthermore, treatment with EBR has been demonstrated to improve the cold tolerance of green bell peppers and loquat [20,21]. Gao et al. [22] demonstrated the effect of EBR on reducing CI in peaches via modulation of phenolic and proline metabolism. Although extensive research has confirmed the pivotal role of EBR in cold resistance, there is limited research on the role of EBR in the postharvest cold control of peach fruits and its latent mechanisms.

Hence, this research aims to evaluate the impact of varying EBR concentrations on the storage quality and cold resistance of peaches, and identify the optimal EBR treatment for improving peach cold resistance. Furthermore, we assess the activity of key enzymes in the antioxidant system to investigate the mechanisms by which EBR treatment alleviates CI in postharvest peach fruit, thereby providing theoretical support for the study of peach fruit chilling injuries.

2. Materials and Methods

2.1. Fruit Materials and Sampling

Flat peach (Prunus persica (L.) Batsch ‘Zaolu’) fruit were harvested at commercial maturity stage with total soluble solid of 11–13% and firmness of 14–16 N from an orchard located in Urumchi, Xinjiang Uyghur Autonomous Region, China, in mid-July. Approximately 1080 peaches of uniform size, free from disease, and without mechanical damage were selected and partitioned into four groups, with each group consisting of 270 fruit with three replicates. The fruit were immersed in 2,4-Epibrassinolide solutions (0, 5, 15, and 25 μmol L⁻¹; Shanghai yuanye Bio-Technology Co., Ltd., Shanghai, China) for 10 min at ambient temperature. After air-drying, the fruit were stored at 4 °C with a relative humidity of 80–85% for 35 days. Every 7 days, 18 fruit from each treatment were chosen for the determination of quality and biochemical indexes. After measurement of firmness and total soluble solids (TSS) content, the fruit samples were chopped, frozen with liquid nitrogen, and stored at −80 °C. To evaluate the effect of transferring fruit to shelf temperature, 45 peach fruit stored at 4 °C were selected for each treatment at 14, 21, 28, and 35 d and transferred to ambient temperature for an additional 3 d.

2.2. Measurement of Internal Browning Index

The internal browning (IB) index was employed to evaluate the severity of internal browning [23]. According to the different degrees of internal browning, the peaches were divided into the following 5 grades: 0 = no browning, 1 = 1–25% browning area, 2 = 26–50% browning area, 3 = 51–75% browning area, 4 = >76% browning area. The IB index was calculated as follows:

IB Index = 100% × [(IB score) × (number of fruit at the IB score)]/(4 × total number of fruit).

2.3. Measurement of L*, a*, b*

The color change of peach peel during ripening was evaluated by measuring the parameters of lightness (L*), red–green chromaticity (a*), and yellow–blue chromaticity (a*). In total, 12 peaches from each group were selected arbitrarily, and 4 symmetrical points near the equator were gauged with a handheld colorimeter (model CR 310, Minolta, Tokyo, Japan).

2.4. Measurement of Rot Rate, Weight Loss and Firmness

When the fruit surface shows obvious mold spots, softening, water-soaked disease spots or a rotten area exceeding 5%, it should be classified as rotten [24]. During each inspection, record the number of rotten fruit and remove them to prevent cross-infection. The rot rate was calculated by summing the number of rotten fruit detected in each inspection. The formula is as follows:

Rot rate (%) = (Number of rotten fruit/Total number of fruit) × 100.

Weight loss rate of fruit was calculated using the weighing method with 6 biological replicates per group. The results were averaged. Calculation formula: Weight loss rate (%) = [(initial weight − weight after storage)/initial weight] × 100%.

Firmness was measured using a hand-held firmness tester GY-4 (Zhejiang TOP Instrument Co., Ltd., Hangzhou, China), provided with a 5 mm probe. Six peach fruit were picked randomly from each treatment, and each fruit was gauged four times symmetrical positions near the equator. Firmness results were expressed as N.

2.5. Measurement of Respiratory Rate and Ethylene Production

Peaches were placed in a well-sealed box at room temperature and were allowed to stand for 2 h before measurement. The respiratory rate and ethylene production were determined using an F-950 C₂H₄/O₂/CO₂ portable gas analyzer (Felix Instrument, Washington, DC, USA). The respiratory rate was expressed as CO₂ production (mL kg⁻¹ h⁻¹), and the ethylene production rate was expressed as µL kg⁻¹ h⁻¹.

2.6. Measurement of TSS, Titratable Acid (TA) and Sucrose Content

The TSS content and TA were determined using a handheld refractometer (Model: PAL-BXIACID F5, ATAGO Scientific Instruments Co., Ltd., Tokyo, Japan). Four pieces of peach fruit tissue from different parts were homogenized, and the juice was extracted. In total, 1 mL of the mixed juice was analyzed, and the results were expressed as %.

Sucrose content was determined using a commercial assay kit (BC2465-100T/96S, Solarbio Inc., Beijing, China) by strictly according to the manufacturer’s protocol. Each experimental group consisted of three biological replicates, and the final results are expressed as mg g⁻¹.

2.7. Measurement of MDA Content and Total Phenol Content (TPC)

The malondialdehyde (MDA) content in peach fruit was assayed according to the MDA content detection kit (BC0025-100T/96S, Solarbio, Beijing, China). The MDA content was expressed as nmol g⁻¹.

Approximately 3 g of fully ground peach tissue was weighed and mixed well with 30 mL 70% ethanol solution, followed by ultrasonic extraction for 90 min. Then, the mixture was centrifuged at 8000× g for 5 min at 4 °C so as to obtain the supernatant. The TPC in peach tissue was measured using the Folin–Ciocalteu colorimetric method [25]. A standard curve was constructed using gallic acid as the reference standard. The reaction system included 1 mL of ten-fold diluted supernatant, 19 mL distilled water, 1 mL Folin–Ciocalteu reagent (1 mol L⁻¹, Shifeng Biotechnology Co., Ltd., Shanghai, China), and 4 mL of Na₂CO₃ (10.6%), which were fully mixed and incubated at 25 °C in the dark for 1 h. The optical density (OD) value was measured at 765 nm. Total phenol content was expressed as gallic acid equivalents (mg g⁻¹).

2.8. Measurement of O₂⁻ Production Rate and H₂O₂ Content

Approximately 0.1 g fully ground peach tissue was weighed and thoroughly mixed with 0.5 mL extract solution. Then, the mixture was centrifuged at 8000× g for 10 min at 4 °C so as to obtain the supernatant. The H₂O₂ content and O₂⁻ production rate were determined using an assay kit (BC3595-100T/96S, BC1295-100T/96S, Solarbio, Beijing, China) according to the instructions. The H₂O₂ content and O₂⁻ production rate were expressed as μmol g⁻¹.

2.9. Measurement of Antioxidant Enzyme Activities

Approximately 0.1 g fully ground peach tissue was weighed and mixed well with 0.5 mL extract solution. The mixture was then centrifuged at 8000× g for 10 min at 4 °C to obtain the supernatant. The POD, SOD, and CAT activity test kit (BC0090-50T/48S; BC0175-100T/48S, BC0200-50T/48S, Solarbio, Beijing, China) instructions were followed. Enzyme activity was characterized as the change in absorbance of 0.01 per minute for every gram of tissue within each milliliter of reaction system. The activity is presented as U g⁻¹.

2.10. Measurement of PPO and PAL Activity

The PPO and PAL activities in peach fruit were quantified utilizing the PPO and PAL activity detection kit (BC5165-100T/48; BC0205-100T/96S, Solarbio, Beijing, China). Enzyme activity was defined as the alteration in absorbance at a rate of 0.01 per minute per gram of tissue in each milliliter of reaction system. The activity is expressed as U g⁻¹.

2.11. Statistical Analysis

The mean value and standard deviation (SD) of the experimental data were computed using Excel 2022 software. The results are presented as mean ± SD. Statistical analysis was conducted using SPSS 26.0 software. One-way ANOVA was employed to compare different treatment groups at the same storage time. Significant differences were identified using Duncan’s multiple comparison test (p < 0.05). The experimental results were visualized using Origin 2021 software.

3. Results

3.1. Effect of Different-Concentration EBR Treatments on Internal Browning Index

The presence of slight internal browning can be observed after 21 days of storage at 4 °C (Figure 1a,b). After being refrigerated for 14 days, the fruit were transferred to room temperature to simulate a three-day shelf life. As a result, distinct water-soaked spots appeared inside the fruits, while EBR-treated fruit exhibited no signs of browning or only mild browning symptoms (Figure 1a). Throughout the storage period, the IB index of the control group demonstrated a significantly higher level compared to that of the 5 μmol L⁻¹ and 15 μmol L⁻¹ EBR treatment groups (p < 0.05). The IB index of the control group at 21 + 3 d was 11.67%, which was seven times than of the 15 μmol L⁻¹ EBR treatment group. The results demonstrate that EBR significantly mitigated the browning induced by low temperature; however, its inhibitory effect on internal browning decreased with increasing EBR concentration.

3.2. Effect of Different-Concentration EBR Treatments on L*, a*, b*

The color of the fruit’s surface is one of the key indicators to evaluate the quality of the fruit. The skin color of peach fruit changes from green to yellow and then to red during ripening. As shown in Figure 2, during storage, the L* and a* values of peach fruit gradually increased, whereas the b* value showed a trend of first increasing and then decreasing. As shown in Figure 2a, there was no significant difference in L* value among the four groups (p ≥ 0.05). Meanwhile, Figure 2b demonstrates that, throughout the storage period, the a* value of peach fruit in EBR treatment groups were significantly lower than those in the control group (p < 0.05). Although the inhibitory effect on a* value increased with increasing EBR concentration, no significant differences were observed among the different EBR concentrations. As shown in Figure 2c, EBR treatment effectively maintained the b* value of peach fruit, with the 15 μmol L⁻¹ EBR treatment consistently showing a higher b* value of than the other groups (p < 0.05). These results suggest that EBR treatment effectively preserves the apparent color of peach fruits during storage, as higher b* values indicate a brighter yellow tone, associated with freshness.

3.3. Effect of Different-Concentration EBR Treatments on Rot Rate, Weight Loss and Firmness

As shown in Figure 3a, compared to the control group, EBR treatment significantly inhibited the increase in the rot rate of postharvest peaches. The peaches of the control group began to rot from the 14th day, whereas peaches treated with 5, 15, and 25 μmol·L⁻¹ EBR exhibited only slight decay symptoms from day 21 onwards. By day 35, the rot rate reached 42.22% in the control group. In contrast, the rot rate of peaches treated with 15 μmol·L⁻¹ EBR was 20%, which was only half that of the control group.

The weight loss rate of peach fruit stored at 4 °C increased almost linearly after 35 days of storage. Additionally, the application of EBR treatment could inhibit the water loss in peach fruit during storage. Although there was no significant difference between the control and 5 μmol·L⁻¹ EBR treatment, it was significantly lower compared to 15 μmol·L⁻¹ EBR treatment. (Figure 3b).

The firmness of peach fruit decreased rapidly during the initial 14 days of storage, followed by a gradual decline thereafter (Figure 3c). It can be seen from the figure that EBR treatment effectively retards the decline in postharvest fruit firmness. Furthermore, peach fruit treated with 5 μmol L⁻¹ and 25 μmol L⁻¹ EBR exhibited significantly higher firmness compared to the control at most time points (p < 0.05). There was no significant difference in firmness between the 15 μmol L⁻¹ EBR-treated fruit and the control group.

3.4. Effect of Different-Concentration EBR Treatments on TSS, Titratable Acid (TA) and Sucrose Content

The TSS of peach fruit in the control group exhibited a slight fluctuation from 12.12% to 12.78% during the initial 14 d of storage, indicating a tendency rather than a significant increase, followed by a subsequent decline (Figure 4a). By the 14th day, the TSS reached 13.64% and 13.24% in the 15 μmol L⁻¹ and 25 μmol L⁻¹ EBR-treated groups, respectively, representing a 1.07-fold and 1.04-fold increase compared to the control group. The TSS of peach fruit treated with 5 μmol L⁻¹ EBR had no significant change during early storage, but showed a significant increase (p < 0.05) in later stages, ultimately exceeding all other treatment groups.

Figure 4b demonstrates that the TA content in postharvest peach fruit generally decreased, with a gradual initial decline followed by a more pronounced reduction in later stages. Throughout the middle storage period, EBR-treated peaches maintained relatively higher TA levels compared to the control group. The effects of different EBR concentrations on TA content varied significantly, as the 25 μmol·L⁻¹ treatment showed no significant difference from the control (p ≥ 0.05), but the 15 μmol·L⁻¹ treatment maintained significantly higher TA content than the 5 μmol·L⁻¹ treatment (p < 0.05). These results suggest that 15 μmol·L⁻¹ EBR treatment effectively retards TA consumption during peach storage.

As shown in Figure 4c, the sucrose content of peach fruits showed a trend of increasing first and then decreasing during storage. Peaches treated with 15 μmol·L⁻¹ EBR showed a rapid 61.9% increase in sucrose content during the first week of storage, rising from 76.54 to 123.92 mg·g⁻¹. The sucrose content in peaches treated with 25 μmol·L⁻¹ EBR and 15 μmol·L⁻¹ EBR followed a similar trend. However, the decrease in sucrose content was more significant from the 7th to the 21st day, dropping sharply from a peak of 111.24 mg·g⁻¹ to 74.60 mg·g⁻¹, a reduction of 32.93%. The control group exhibited a gentler increase in sucrose content during the early storage period. Over the first 7 days, sucrose levels rose from 76.54 mg·g⁻¹ to 96.68 mg·g⁻¹, an increase of only 26.3%, which was significantly lower than that observed in the 15 μmol·L⁻¹ EBR treatment group (p < 0.05). Additionally, the 5 μmol·L⁻¹ EBR treatment group showed a sucrose content trend nearly identical to the control, with no statistically significant difference between them (p > 0.05).

3.5. Effect of Different-Concentration EBR Treatments on Respiratory Rate and Ethylene Production

Figure 5a illustrates the dynamic changes in respiratory rate across all treatment groups during storage. The respiratory rate in each group exhibited an initial decline, reaching its lowest point at approximately day 14, followed by an increase to peak levels around day 21, before subsequently decreasing. Treatment concentration significantly influenced respiratory rate. On day 21, the respiratory rates of the control group and the 25 μmol·L⁻¹ EBR treatment group were 20.82 mL kg⁻¹ h⁻¹ and 19.85 mL kg⁻¹ h⁻¹, respectively. In contrast, respiratory rates in the 5 μmol·L⁻¹ and 15 μmol·L⁻¹ EBR treatment groups were significantly lower at 16.24 mL kg⁻¹ h⁻¹ and 12.56 mL kg⁻¹ h⁻¹, respectively (p < 0.05).

As shown in Figure 5b, ethylene production rate in all groups exhibited an increasing trend throughout the storage period. During the initial storage phase, the production rate of ethylene was relatively slow, followed by a substantial increase in the later stage. EBR treatment significantly suppressed ethylene release in peaches compared to the control group (p < 0.05). Notably, the 5 μmol·L⁻¹ and 15 μmol·L⁻¹ EBR treatments demonstrated superior inhibitory effects on ethylene production relative to the 25 μmol·L⁻¹ treatment.

3.6. Effect of Different-Concentration EBR Treatments on MDA Content and TPC

A trend of MDA content in peach fruit first increasing and then decreasing was shown over time (Figure 6a). The MDA content in peach fruits treated with 15 μmol L⁻¹ EBR peaked at 21 d, whereas the other groups reached their maximum values at 14 d. The results suggest that EBR treatment can slow down the increase in MDA content, with a higher efficacy observed at 15 μmol L⁻¹.

As depicted in Figure 6b, the TPC in peach fruit from the control and 15 μmol L⁻¹ EBR-treated groups showed a declining trend. The total phenol content in the control group decreased rapidly during the initial 7 days of storage, reaching only 56.20% of the initial value at 7 days. However, the total phenol content in the 15 μmol L⁻¹ EBR-treated group decreased more slowly. In addition, the total phenol content in the 5 μmol L⁻¹ and 25 μmol L⁻¹ EBR-treated groups reached minimum values of 0.59 mg g⁻¹ and 1.10 mg g⁻¹ on the 21st and 14th days of storage, respectively, after which they increased rapidly. Compared to the control group, the 15 μmol L⁻¹ and 25 μmol L⁻¹ EBR treatments significantly maintained total phenol content in postharvest peach fruit (p < 0.05).

3.7. Effect of Different-Concentration EBR Treatments on H₂O₂ Content and O₂⁻ Production Rate

The content of O₂⁻ in peach fruit of the control group and the EBR-treated groups showed an increasing trend (Figure 7a). However, during early storage stage, the O₂⁻content in the EBR-treated group was consistently lower than that in the control group, with a significant difference noted between the 5 μmol L⁻¹ EBR-treated group and the control group (p < 0.05).

As shown in Figure 7b, the H₂O₂ content in peach fruit from both the control group and the EBR-treated groups exhibited a pattern of ascending initially and then decreasing. The H₂O₂ content in the control group peaked at 4.51 μmol g⁻¹ on the 14th day, which was significantly higher than that in the EBR-treated groups (p < 0.05), and then decreased rapidly. In the 5 μmol L⁻¹ EBR-treated group, the H₂O₂ content remained at a low level and peaked at 2.95 μmol g⁻¹ on the 35th day, demonstrating particularly effective suppression during the critical early and middle storage periods (14–21 d) compared to other EBR concentrations.

3.8. Effect of Different-Concentration EBR Treatments on the Activities of SOD, POD, and CAT

As shown in Figure 8a, the SOD activity in the peach fruit from both the control and EBR-treated groups increased during storage. The SOD activity in the control group decreased rapidly within 21 days of storage. In contrast, the SOD activity in EBR-treated groups remained relatively higher or more stable at different stages. At the beginning of storage, the SOD activity in the 15 μmol L⁻¹ EBR-treated group was consistently higher than that in the control and the 5 μmol L⁻¹ EBR-treated groups (p < 0.05). Additionally, the SOD activity in the 25 μmol L⁻¹ EBR-treated group was relatively high during both the initial and late storage periods.

POD activity fluctuated during storage, but generally followed an upward trend (Figure 8b). In the early stages of storage, no significant differences in POD activity were detected between the EBR-treated and control groups. However, after 21 days of storage, the POD activity in EBR-treated peach fruit increased by 46.68%, 30.71%, and 46.78% compared to the control group, respectively (p < 0.05). During the later stages of storage, the POD activity in 15 μmol L⁻¹ EBR-treated group remained high.

As illustrated in Figure 8c, the CAT activity in flat peach fruit displayed an initial rapid increase, peaking on the 14th and 21st days of storage, followed by a subsequent rapid decline. Throughout the 21-day storage period, the CAT activity in the 15 μmol L⁻¹ EBR-treated group was significantly higher compared to the other groups (p < 0.05). No significant differences in CAT activity were noted among the 5 μmol L⁻¹ EBR-treated, 25 μmol L⁻¹ EBR-treated, and control groups.

3.9. Effects of EBR Treatment on the Activities of PPO and PAL

PAL activity showed an increasing trend during storage. However, the 5 μmol L⁻¹ and 25 μmol L⁻¹ EBR treatments inhibited the increase compared to the control group (Figure 9a). Throughout the storage period, PAL activity in the control group was significantly higher than that in the 5 μmol L⁻¹ EBR-treated group (p < 0.05). By the 35th day of storage, PAL activity in the control group was 1.72-fold higher than that in the 5 μmol L⁻¹ EBR-treated group. No significant difference in PAL activity was observed between 15 μmol L⁻¹ EBR-treated and control groups.

As shown in Figure 9b, PPO activity in peach fruit from both the control and EBR-treated groups increased steadily with storage time. In the control group, PPO activity increased from 84.43 U g⁻¹ on day 0 to 138.87 U g⁻¹ on day 35, representing a 0.65-fold increase. In the 15 μmol L⁻¹ EBR-treated group, PPO activity reached 107.11 U g⁻¹ on 35 d, representing only a 0.27-fold increase compared to day 0, and it was significantly lower than that in the control group (p < 0.05). Furthermore, compared to the control group, the 5 μmol L⁻¹ and 25 μmol L⁻¹ EBR-treated groups also inhibited PPO activity, but no significant differences were observed.

3.10. Cluster Heatmap-Based Analysis of EBR Treatment Effects on Flat Peach Fruit Quality and Cold Resistance Indicators

The samples were analyzed using cluster heat maps, which included a total of 21 physicochemical indicators (Figure 10). The 15 μmol L⁻¹ EBR treatment group exhibited elevated levels (shown in red) of quality-related indicators, including weight loss rate, TA, sucrose content, TSS, TPC, and antioxidant enzyme activities. Conversely, the control group showed higher expression of cold-damage-associated indicators, specifically IB index, ROS, and MDA content. Cluster analysis demonstrated that samples from both the 5 μmol L⁻¹ and 25 μmol L⁻¹ EBR treatments grouped together, while those from the 15 μmol L⁻¹ EBR treatment formed a distinct cluster, clearly separated from other groups. These findings suggested that both 5 μmol L⁻¹ and 15 μmol L⁻¹ EBR treatments can maintain postharvest peach quality by enhancing antioxidant capacity while reducing oxidative damage, thereby mitigating cold injury symptoms during storage, with the 5 μmol L⁻¹ treatment showing particular strength in maintaining core quality parameters like firmness and weight loss.

4. Discussion

Low-temperature storage is the most common way to prolong the postharvest shelf life of fruit and vegetables after harvest. However, prolonged cold storage under inappropriate conditions can result in CI, thereby impacting the quality and commercial value of postharvest peach fruit [26]. This study investigated the efficacy of EBR treatment in preserving postharvest peach quality and mitigating CI symptoms. EBR treatment significantly suppressed low-temperature-induced browning and preserved fruit visual quality. However, its inhibitory effect on internal browning decreased with increasing EBR concentration. This finding echoes the results of EBR in other fruits such as bananas [27], loquats [21], and citrus fruits [28], and aligns with previous findings from peaches [22], further confirming the extensive role of EBR as a plant hormone modulating postharvest stress responses. In terms of quality indicators, EBR treatment delayed a* value increment (Figure 2b), stabilized b* value (Figure 2c), and reduced fruit weight loss (Figure 3b). This suggests that EBR may mitigate water loss by regulating epidermal structure or wax synthesis, which is of great significance for maintaining the postharvest quality of peach fruits [28,29].

Further analysis demonstrated that EBR alleviates CI through coordinated regulation of oxidative stress and phenolic metabolism. Low-temperature stress can lead to excessive accumulation of ROS, causing damage to the membrane system and resulting in a sharp increase in MDA content in the control group within 14 days of storage (Figure 6a) [17,26]. The exogenous EBR treatment effectively suppressed H₂O₂ and O₂⁻·levels (Figure 7a,b) and MDA accumulation, with the 5 μmol·L⁻¹ treatment showing particularly effective suppression of H₂O₂ during the critical early storage period. This might be attributed to the activation of the antioxidant enzyme system in fruit by EBR. Our research shows that EBR treatment significantly enhances the activities of antioxidant enzymes such as SOD, CAT, and POD, which is a result that is consistent with the research results from plants such as star fruit and basil, indicating that EBR may maintain the stability of cell membranes by enhancing the scavenging ability of ROS [30,31]. The phenolic compounds exhibit strong antioxidant activity and play a crucial role in combating both biological and abiotic stresses [32]. PPO, an important biocatalyst, facilitates the oxidation of phenolic compounds to quinones, which leads to enzymatic browning of agricultural products [33]. The results of this study indicate that EBR treatment also influences the metabolic process of phenolic substances. On the one hand, it delays the decrease in total phenolic content, and on the other hand, it inhibits PPO activity, thereby mitigating enzymatic browning by reducing phenolic oxidation. This is consistent with previous studies [34].

Some studies have shown that the accumulation of lignin is directly related to the pulp floc and cotton in peach fruit cold injury symptoms [35]. In this study, EBR treatment significantly suppressed PAL activity, a key enzyme in lignin biosynthesis, particularly at the 5 μmol·L⁻¹ concentration. This is consistent with the research findings of EBR treatment of asparagus [36]. As lignin accumulation directly correlates with pulp flocculation in CI peaches, EBR likely mitigates chilling-induced texture deterioration through PAL regulation. Notably, the effects of EBR on various physiological parameters exhibit concentration-dependent differences. For instance, the 5 μmol·L⁻¹ treatment most effectively inhibits PAL activity and reduces weight loss, whereas the 15 μmol·L⁻¹ treatment shows strong potential in mitigating ROS accumulation, although lower H₂O₂ levels were also observed with 5 μmol·L⁻¹ EBR at 14 and 21 days. These variations suggest that EBR may act through distinct signaling pathways, warranting further investigation into its specific mechanisms.

This study demonstrates that EBR treatment significantly suppressed respiration intensity and ethylene production in postharvest peach fruit. Additionally, EBR maintained the nutritional quality of the fruit, with soluble solids content in the treatment group consistently sustained at elevated levels. These findings suggest that EBR mitigates nutrient depletion by modulating respiratory metabolism [37]. In conclusion, EBR effectively alleviates low-temperature-induced CI in peach fruit through multiple mechanisms, including stabilizing cell membrane integrity, enhancing antioxidant system activity, and inhibiting phenolic oxidation and lignification processes. These results provide novel experimental evidence for clarifying the mechanism of EBR and establish a theoretical foundation for the development of innovative postharvest preservation technologies for peach fruit.

5. Conclusions

In conclusion, the application of EBR markedly mitigated CI in peach fruit during storage. Different concentrations of EBR may delay the chilling damage of peach fruit after harvest by acting on different pathways and substances. The 5 μmol L⁻¹ EBR treatment emerged as the most effective concentration for maintaining core postharvest quality attributes. It significantly delayed the decrease in firmness, reduced weight loss, effectively inhibited the production of H₂O₂ and O₂⁻, particularly during the early storage period, strongly restrained the activity of PAL, and maintained lower rot rates and internal browning indexes. While the 15 μmol L⁻¹ EBR treatment enhanced antioxidant activity, increased total phenolic content at certain stages, and maintained higher soluble solids and acid content, its effects on key physical quality parameters like firmness and weight loss were less pronounced compared to the 5 μmol L⁻¹ treatment. This study not only provides a new idea for developing new preservatives and improving the quality of peach fruit after harvest, but also provides an important theoretical basis for optimizing the preservation process of peach fruit after harvest. However, the detailed regulatory mechanism of EBR-alleviated CI of peach fruit observed in these results needs to be further revealed with more systematic molecular evidence in the future.