Strigolactone Preserves Fresh-Cut Apple Quality during Shelf Life

Abstract

Strigolactone (SL) is a signal factor that plays a vital role in plants. The application of SL for the storability of horticultural products has recently received attention. In this experiment, fresh-cut apples were treated with SL at diverse concentrations and stored at 4 °C for 10 days, and the changes in quality characteristics, antioxidant system, hydrogen sulfide metabolism, and nitric oxide metabolism were determined. Compared with other treatments, the results showed that SL treatment at 0.50 µmol L⁻¹ had excellent effects on maintaining fruit surface color, weight, firmness, reduced respiration rate, soluble solids content, and electrolyte leakage. SL treatment increased antioxidant enzyme activities, reduced reactive oxygen species (ROS) accumulation, altered the nitric oxide synthase (NOS)-like pathway to promote endogenous NO production in the fruit, and facilitated the L-cysteine-catalyzed process to increase the endogenous hydrogen sulfide (H₂S) content. In addition, SL treatment affected the mRNA transcription levels of several genes related to the antioxidant system, H₂S metabolism, and NO synthesis, including MdSOD, MdCAT, MdPOD, and MdSAT. Taken together, the results indicated that 0.50 µmol L⁻¹ SL treatment improves the endogenous synthesis of NO and H₂S, enhances the antioxidative system, and maintains the quality of fresh-cut apples during their shelf life. Therefore, the present study opens up the possibility of using the exogenous application of strigolactone in the fresh-cut processing industry.

1. Introduction

Fresh-cut apples, like whole fruits, provide the nutrients people need and have the characteristics of convenient consumption, so consumers love them. However, apples suffer mechanical damage after cutting, and the flesh is exposed to the atmosphere. Mechanical damage generates excessive reactive oxygen species (ROS), further aggravating oxidative damage [1]. Water loss is also an essential phenomenon of fresh-cut apples, causing wilting and the breakdown of cell walls [2]. The phenolics in the flesh are oxidized to quinones catalyzed by oxygen, which causes the cut surface browning [3]. Oxidative damage, water loss, and surface browning cause a decrease in the economic values and quality of fresh-cut apples.

Apples accumulate ROS after mechanical damage, which can harm plants by producing oxidative damage and promoting polyphenol oxidase (PPO) to oxidize phenolics for browning [4]. In plants, the antioxidant systems play a notable role in protecting cells from oxidative stress caused by ROS. NO and H₂S signaling and function allow plants to regulate the overproduction of ROS under abiotic stresses [5]. H₂S is formed in the plant, and the balance of endogenous H₂S slows the senescence of the product after harvest [6]. It has been found that both the physicochemical and microbiological properties of fresh-cut apples were improved by using the emulsion of carvacrol [7]. In addition, citric acid and cysteine can also lower the pH of an anti-browning solution, react with quinone, and interact with copper ions in the active center of PPO to inhibit browning [8]. However, there are certain safety risks associated with these substances, which is why natural, safe browning inhibitors are preferred.

Diverse methods, including those from the physical, chemical, and biological domains, are utilized to keep the storage quality and prolong the preservation period of fresh-cut apples. Among them, low-temperature storage in conjunction with preservatives is the common preservation method of fresh-cut apples. Strigolactones (SLs) are a distinctive group of carotenoid-derived terpenoid lactones and have a conserved methylbutenolide ring (D-ring), which is pivotal for their bioactivity [9]. As promising signaling molecules, SLs play a notable role in regulating plant growth, development, and stress resistance [10]. Additionally, SL is a novel phytohormone commonly found in plants and plays a vital role in stress tolerance, including promoting stomatal closure and attenuating transpiration, alleviating the damage caused by drought stress, and improving a plant’s adaptation to drought [11]. The application of SL in the storage resistance of horticultural products has recently attracted attention. For example, 200 µmol L⁻¹ GR24 (an artificial analog of SLs) increased the antioxidant capacity of sweet oranges during storage, and GR24 significantly maintained the nutritional quality of sweet oranges [12]. The peak values of peroxidase (POD) and PPO activities in postharvest strawberries were significantly increased by SL treatment, and the possibility of browning was reduced [13].

Safety, effectiveness, and environmental protection are the primary criteria for selecting preservatives. Research on SLs has revealed their potential application as consumer-safe preservatives. Due to their biocompatibility and well-characterized functions, phytohormones like SLs can be applied for cancer treatment for their therapeutic activity, and anti-inflammatory and antiviral activity. The distinctive chemical skeleton of SLs and their action mode in plants have inspired potential applications in biomedical fields. SLs, as common and valuable terpenoids, are widely used due to their various pharmacological activities, such as anti-inflammatory, hepatoprotective, and detoxification properties [14]. Studies have reported that SLs exhibit antiproliferative activity and can induce the apoptosis of breast cancer cells [15]. As a result, SLs could provide a potential perseverative treatment strategy suitable to marketable applications due to their therapeutic character.

However, few studies have investigated the use of SL to restrain deterioration and maintain the storage quality of fresh-cut products. In this study, different concentrations of SL were applied to fresh-cut apples, and the effects of SLs on storage quality, the antioxidant system, NO and H₂S metabolism, and essential genes involved in metabolism were investigated to explore SL’s potential in maintaining the storage quality of fresh-cut apples.

2. Materials and Methods

2.1. Experimental Design

‘Yantai Fuji’ apples at physiological maturity (180 days after full bloom; mean soluble solids content, 11 °Brix) were obtained from local orchards in Taian City, Shandong Province. Fifty-five apples of similar size and color with no apparent damage were selected as the material. Each apple was peeled, cored, and sliced using a sterile slicing knife to apple slices with a thickness of 5 millimeters, and then they were uniformly cut into slices with 50 mm in outer diameter. Eight slices of one apple were randomly collected and gathered. Twenty pieces were randomly selected as untreated at day 0. Four hundred of the rest of the slices were divided into 4 groups, with 100 pieces in each group. They were then soaked in solutions of SL (CAS #76974-79-3, Shanghai Yuanye Bio-Technology Co. Ltd., Shanghai, China) with concentrations of 0.25 µmol L⁻¹, 0.50 µmol L⁻¹, and 1.00 µmol L⁻¹ for 3 min, respectively. The fresh-cut apples soaked in clean water for 3 min were the control. After drying the water, the fresh-cut apples in sterilized polypropylene containers were stored for 10 days at 4 °C and a relative humidity of 95%. Apples were sampled every two days to determine the browning index, firmness, soluble solids content, electrolyte leakage, weight loss, and respiration rate. Fresh-cut apples were ground into powders by a basic analytical mill (A11; IKA, Staufen, Hessen, Germany), and then the apple powders were collected and frozen at −80 °C for subsequent experiments.

2.2. Measurement of Primary Indexes

Fresh-cut apples’ firmness was determined using a GY-4 firmness tester (Yueqing Aidebao Instrument Co., Ltd., Yueqing, China) and expressed in N. The L*, a*, and b* values of the cutting surfaces were measured using a CR-10 color difference meter (Konica Minolta, Tokyo, Japan). The browning index (BI) was calculated with the method of Ruangchakpet and Sajjaanantakul [16] as $\mathrm{B}\mathrm{I}=\left[100\left(x-0.31\right)\right]/0.172,\mathrm{where}x=(a^{*}+1.75L^{*})/(5.645L^{*}+a^{*}-0.3012b^{*}).$ The weight loss was determined using an electronic analytical balance and caculated as weight (W) loss (%) = (W_initial − W_storage) × 100/W_initial. The DDS-307 conductivity meter (Shanghai Jingke Leici Company, Shanghai, China) was used to measure the relative conductivity, and the electrolyte leakage was calculated following Wang et al.’s method [17]. The respiration rate was determined using the SY-1022 fruit respiration measuring instrument (Shijiazhuang, China) and expressed as mg CO₂ Kg⁻¹ h⁻¹. The soluble solids content (SSC) was determined using a hand-held refractometer (WY015R, Abes Ltd., Zurich, Switzerland) and expressed as Brix. A sensory evaluation was performed by 20 trained team members (evenly divided between male and female members) in a sensory testing laboratory. The specific scoring criteria are shown in

Table 1. Sensory evaluation criteria of fresh-cut apples.

Sensory Attributes	Score Values
	9–10	6–8	1–5
Color	No browning, high color degree	Browning slightly, but acceptable, color degree in general	Browning is more serious, unacceptable, and has poor glossiness
Texture	High crispness, high flesh firmness	Firmness slightly decreased, but acceptable	Soft texture, water loss is serious
Taste	Sweet and sour moderate, delicious taste	Slightly uneven in sweet and sour, but acceptable	Odor, acidity imbalance, loss of edible value
Flavor	It has the unique sweet smell of apples	It still has the unique aroma of apple, slightly sour, but acceptable	There is a sour smell
Wholistic evaluation	Fresh appearance, very edible desire	It is acceptable to eat	Loss of edible value

. Evaluation parameters less than 6.0 were considered the end of the shelf life. The final sensory score values were averaged over data from both men and women.

2.3. Determination of Antioxidant System in Fresh-Cut Apples

In this work, an ultraviolet-visible (UV–Vis) spectrophotometer (UV-6100S; Metash Instruments Co., Ltd., Shanghai, China) was used to record the absorbance of the reaction solution at the maximum wavelength (λ_max) of various analytes. Then, the content of the metabolites and the activities of enzymes could be analyzed. Data of content and activity were expressed with units on the fresh weight basis (FW) and the protein content basis, respectively. The total protein content in apples with different treatments was measured following the Bradford method [18].

The content of hydrogen peroxide (H₂O₂) was analyzed following the procedure outlined by Ma et al. [19]. The absorbance at λ_max = 415 nm was recorded, and the content of H₂O₂ was expressed as µmol g⁻¹ FW.

The content of superoxide radicals (O₂˙⁻) was determined by the method of Zhang et al. [20]. The absorbance at λ_max = 525 nm was recorded, and the content of O₂˙⁻ was expressed as µmol g⁻¹ FW.

The determination of hydroxyl radicals (˙OH) content was carried out according to Zhu et al. [21]. The absorbance at λ_max = 532 nm was recorded, and the ˙OH content was expressed as nmol g⁻¹ FW.

Apple powders (3 g) were homogenized in 10 mL phosphate buffer (pH 7.8, 0.05 mol L⁻¹). After 20-min centrifugation (4 °C, 15,000× g), the supernatant was collected to measure the activities of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD). The change in absorbance at λ_max = 240 nm, 560 nm, and 580 nm was recorded to assess the activities of CAT, SOD, and POD, respectively [13,19]. The enzyme activity was expressed as U mg⁻¹ protein. The unit of enzyme activity (U) was defined as the amount of enzyme required to change the absorbance at λ_max by 0.01 in one minute.

Apple powders (3 g) were homogenized in 10 mL phosphate buffer (pH 7.8, 0.05 mol L⁻¹, containing 2 mmol L⁻¹ ethylenediaminetetraacetic acid (EDTA), 2 mmol L⁻¹ ascorbic acid, and 2% (w/v) polyvinylpyrrolidone (PVP)). After 20-min centrifugation (4 °C, 15,000× g), the supernatant was collected to measure the activities of dehydroascorbic acid reductase (DHAR), glutathione reductase (GR), ascorbic acid peroxidase (APX), and monodehydroascorbic acid reductase (MDHAR) in the ascorbate–glutathione (AsA-GSH) cycle [22]. The change in absorbance at λ_max = 290 nm, 340 nm, 265 nm, and 265 nm was recorded to assess the activities of APX, MDHAR, DHAR, and GR, respectively. The enzyme activity was expressed as U mg⁻¹ protein.

Apple powders (3 g) were homogenized in 10 mL of 5% (v/v) metaphosphoric acid. After 20-min centrifugation (4 °C, 15,000× g), the supernatant was collected to measure the contents of reduced ascorbate (AsA), dehydroascorbate (DHA), reduced glutathione (GSH), and oxidized glutathione (GSSG). The contents of AsA, DHA, GSH, and GSSG were expressed as µmol g⁻¹ FW [13,23].

2.4. Determination of Changes in NO Metabolism in Fresh-Cut Apples

The endogenous levels of NO were measured using the NO kit from Beijing Solarbio Technology Co., Ltd., Beijing, China, and the NO content was expressed as µmol g⁻¹ FW.

For nitrate reductase (NR) activity, a Hepes–KOH buffer (pH 7.5, containing 1 mmol L⁻¹ EDTA, 3% (w/v) PVP, 7 mmol L⁻¹ cysteine) was used to homogenize apple powders. The change in absorbance at λ_max = 540 nm was recorded, and the NR activity was expressed as µmol NO h⁻¹ mg⁻¹ protein [24].

Nitric oxide synthase (NOS)-like activity was determined using the method of Dong et al. [25]. The change in absorbance at λ_max = 540 nm was recorded, and the NOS-like activity was expressed as µmol NO h⁻¹ mg⁻¹ protein.

Nitrite and L-arginine contents were determined using the method of Tian et al. [26]. The absorbance at λ_max = 530 nm and 540 nm was recorded to assess the contents of L-arginine and nitrite, respectively. The contents of nitrite and L-arginine were expressed as µmol g⁻¹ FW.

2.5. Determination of Changes in H₂S Metabolism in Fresh-Cut Apples

Apple powders (3 g) were homogenized in 10 mL phosphate buffer (pH 6.8, 0.05 mol L⁻¹, containing 100 mmol L⁻¹ EDTA, 200 mmol L⁻¹ AsA). After 20 min centrifugation (4 °C, 15,000× g), the supernatant was collected to measure the content of endogenous H₂S. The absorbance at λ_max = 667 nm was recorded, and the endogenous H₂S content was expressed as µmol g⁻¹ FW [27].

For the measurement of L-cysteine desulfhydrase (L-CD) activity, the Tris-HCl buffer (pH 8.0, 20 mmol L⁻¹) was used to homogenize apple powders [28]. The change in absorbance at λ_max = 670 nm was recorded, and L-CD activity was expressed as U mg⁻¹ protein.

The enzyme activities of serine acetyltransferase (SAT) and O-acetyl-1-serine (thiol) lyase (OAS-TL) were measured following the protocol described by Geng et al. [28]. The change in absorbance at λ_max = 560 nm and 232 nm was recorded to assess the activities of OAS-TL and SAT, respectively. The enzyme activity was expressed as U mg⁻¹ protein.

2.6. qRT-PCR Analysis

The purity and concentration of RNA were determined using a Q5000 microspectrophotometer (Quawell, San Jose, CA, USA). The total RNA was reverse transcribed using the cDNA PrimeScript^TM II1st Strand cDNA Synthesis Kit (Takara, Dalian, China). qRT-PCR was performed with ACTIN as the housekeeping gene using the method of He et al. [29]. The qRT-PCR primers are shown in Table S1. The relative gene expression scale of untreated fresh-cut apples on day 0 was set to 1. The formula for the PCR exponential amplification was R_n = R₀ (1 + E_r) ⁿ. E_r was the amplification efficiency, n was the number of cycles, R_n was the number of PCR products after n cycles, and R₀ was the number of original templates. Gene expression was calculated by the 2 ^(−ΔΔt) formula.

2.7. Statistical Analysis

Each measurement was operated with three biological replicates. Statistical analyses were performed using the SPSS software (v19.0, Chicago, IL, USA). The data are presented as mean ± standard error (SE) and were processed by one-way ANOVA as significant differences (LSD test, p < 0.05).

3. Results

3.1. Changes of Primary Indexes of Fresh-Cut Apple after Treatment with SL

Figure 1a shows the apple slices with different treatments on the 10th day of storage. Compared with other treatments, 0.50 µmol L⁻¹ SL retained a better appearance and storage quality of the fresh-cut apples during the storage. The apples decayed after they were cut, including an increase in weight loss, electrolyte leakage, browning index, soluble solids content, and the decrease in firmness. Compared with the control, SL treatment delayed the deterioration of fresh-cut apples (Figure 1). On day 10, weight loss, electrolyte leakage, browning index, and the soluble solids content of fresh-cut apples treated with 0.50 µmol L⁻¹ SL were 59.4% (Figure 1b), 73.8% (Figure 1d), 70.7% (Figure 1e), and 88.9% (Figure 1g) of the control, respectively. The firmness of fresh-cut apples decreased continuously during storage, and SL retarded the decrease in the firmness of fresh-cut apples. On day 10, the firmness of fresh-cut apples treated with 0.25, 0.50, and 1.00 µmol L⁻¹ SL were 1.37, 1.53, and 1.32 times that of the control, respectively (Figure 1c). The respiration rate increased in the first 6 days and then decreased. And it had a peak value on day 6. The peak respiration rate of apples with 0.25, 0.50, and 1.00 µmol L⁻¹ SL treatment was 45.2%, 41.7%, and 73.9% of the control, respectively (Figure 1f). Among the different concentrations, 0.50 µmol L⁻¹ SL exhibited a significant effect.

The sensory evaluation scores with a downward trend were observed in fresh-cut apples with different treatments during storage (

Table 2. Sensory evaluation of fresh-cut apples during cold storage.

Sensory Attributes	Treatment	Storage Time (Days)
		0	2	4	6	8	10
Color	Control	9.54 ± 0.46	7.81 ± 0.33b	6.26 ± 0.53b	4.78 ± 0.47b	3.98 ± 0.28b	3.46 ± 0.14c
	0.25 µmol L−1	9.54 ± 0.46	7.93 ± 0.29b	6.73 ± 0.43ab	5.44 ± 0.19ab	5.04 ± 0.29ab	4.52 ± 0.23b
	0.50 µmol L−1	9.54 ± 0.46	8.89 ± 0.21a	7.32 ± 0.27a	6.53 ± 0.22a	6.13 ± 0.37a	5.95 ± 0.32a
	1.00 μmol L−1	9.54 ± 0.46	7.52 ± 0.43b	6.34 ± 0.32b	5.29 ± 0.42b	4.44 ± 0.34b	3.57 ± 0.23c
Texture	Control	9.69 ± 0.29	8.22 ± 0.19b	6.76 ± 0.25c	5.58 ± 0.26c	4.87 ± 0.22b	3.73 ± 0.27c
	0.25 µmol L−1	9.69 ± 0.29	8.47 ± 0.33ab	7.61 ± 0.42a	6.36 ± 0.37b	5.76 ± 0.44a	4.81 ± 0.42b
	0.50 µmol L−1	9.69 ± 0.29	8.84 ± 0.27a	7.92 ± 0.32ab	7.21 ± 0.24a	6.53 ± 0.29a	6.23 ± 0.39a
	1.00 μmol L−1	9.69 ± 0.29	8.56 ± 0.38ab	7.34 ± 0.17b	6.18 ± 0.31bc	5.42 ± 0.41ab	3.87 ± 0.25c
Taste	Control	9.63 ± 0.29	7.99 ± 0.26ab	6.27 ± 0.23b	5.89 ± 0.43b	4.27 ± 0.32c	2.99 ± 0.23c
	0.25 µmol L−1	9.63 ± 0.29	8.21 ± 0.33a	6.92 ± 0.25ab	6.24 ± 0.35ab	5.03 ± 0.27b	3.84 ± 0.21b
	0.50 µmol L−1	9.63 ± 0.29	8.64 ± 0.24ab	7.42 ± 0.14a	6.94 ± 0.26a	5.96 ± 0.21a	5.78 ± 0.27a
	1.00 μmol L−1	9.63 ± 0.29	7.89 ± 0.39b	6.41 ± 0.24ab	6.03 ± 0.32b	4.48 ± 0.14c	3.24 ± 0.25c
Flavor	Control	9.71 ± 0.33	8.11 ± 0.26b	7.06 ± 0.34b	6.64 ± 0.26b	5.27 ± 0.25c	3.94 ± 0.42c
	0.25 µmol L−1	9.71 ± 0.33	8.24 ± 0.33b	7.36 ± 0.25b	6.77 ± 0.31b	5.94 ± 0.32b	4.53 ± 0.24b
	0.50 µmol L−1	9.71 ± 0.33	8.52 ± 0.24a	7.98 ± 0.37a	7.34 ± 0.43a	6.62 ± 0.24a	6.12 ± 0.54a
	1.00 μmol L−1	9.71 ± 0.33	8.06 ± 0.17b	7.41 ± 0.23b	6.80 ± 0.13b	5.51 ± 0.10c	4.23 ± 0.32bc
Holistic evaluation	Control	9.63 ± 0.45	8.02 ± 0.4b	6.36 ± 0.29c	5.12 ± 0.23c	4.03 ± 0.24c	3.54 ± 0.13c
	0.25 µmol L−1	9.63 ± 0.45	8.21 ± 0.1b	7.12 ± 0.17b	6.04 ± 0.11b	5.32 ± 0.34b	4.16 ± 0.29b
	0.50 µmol L−1	9.63 ± 0.45	8.64 ± 0.3a	7.61 ± 0.38a	6.95 ± 0.34a	6.27 ± 0.23a	6.01 ± 0.17a
	1.00 μmol L−1	9.63 ± 0.45	7.97 ± 0.28b	6.95 ± 0.30bc	6.02 ± 0.23b	5.20 ± 0.42b	3.77 ± 0.39bc

Note: The score is an average of 20 ratings. Values within a column labeled with the different letters are significantly different (LSD test, p < 0.05).

), and 0.50 μmol L⁻¹ SL-treated apples always had a higher score than others, demonstrating a positive effect of SL on the storage quality of fresh-cut apples, which was consistent with the results of the primary indexes. In addition, fresh-cut apples lost their commercial value after treatment with 0, 0.25, 0.50, and 1.00 μmol L⁻¹ SL on the 6th, 8th, 10th, and 8th day, respectively, and the results of the sensory evaluation indicated that SL treatment could prolong the shelf life of fresh-cut apples.

3.2. Changes in the Antioxidant System of Fresh-Cut Apple after Treatment with SL

The contents of H₂O₂, ˙OH, and O₂˙⁻ in fresh-cut apples increased continuously during storage (Figure 2a–c). The contents of H₂O₂, O₂˙⁻, and ˙OH were lower in 0.25, 0.50, and 1.00 µmol L⁻¹ SL-treated fresh-cut apples than that in the control. It is noticeable that the contents of H₂O₂, ˙OH, and O₂˙⁻ of fresh-cut apples treated with 0.50 µmol L⁻¹ SL decreased much more significantly than other concentrations, which were 36.8%, 64.8%, and 48.1% of the control on day 10, respectively.

The activities of SOD, CAT, and POD in fresh-cut apples increased in the first 6 days and then decreased (Figure 2d,f). The activity of each enzyme in fresh-cut apple fruit with 0.50 µmol L⁻¹ SL treatment consistently exceeded that of the control and the other two concentrations. On day 6, the activities of SOD, POD, and CAT in fresh-cut apples treated with 0.50 µmol L⁻¹ SL were 1.20, 1.35, and 1.41 times that of the control, respectively. The 0.50 µmol L⁻¹ SL treatment effectively enhanced the enzyme activities in the enzymatic antioxidant system of fresh-cut apples, thereby boosting the scavenging ability of ROS and maintaining a better quality of the fruits.

During the first 6 days of storage, the DHAR activity in fresh-cut apples increased, peaked on day 6, and then decreased (Figure 3a). On day 6, the DHAR activity in fresh-cut apples with 0.50 µmol L⁻¹ SL treatment was 1.38 times higher than the control; that is to say, 0.50 µmol L⁻¹ SL treatment effectively boosted the DHAR activity in fresh-cut apple fruit, promoted the formation of AsA, and strengthened the ability of the fruit to resist oxidative damage.

The MDHAR activity in fresh-cut apples showed an initial increase followed by a decrease as the storage days increased, as depicted in Figure 3b. The application of 0.50 µmol L⁻¹ SL significantly boosted MDHAR activity in fresh-cut apples. Notably, on day 6, fresh-cut apples with 0.50 µmol L⁻¹ SL treatment exhibited an MDHAR activity 1.29 times higher than the control. The results indicate that the 0.50 µmol L⁻¹ SL treatment notably enhanced the MDHAR activity in fresh-cut apples during the whole storage period. This treatment effectively elevated MDHAR activity in the fruit, facilitated AsA regeneration, and bolstered its resistance to oxidative stress.

SL treatments enhanced the APX activity in fresh-cut apples throughout the storage (Figure 3c). Among all treatments, fresh-cut apples with 0.50 µmol L⁻¹ SL treatment consistently exhibited the highest APX activity. On day 4, the APX activity in fresh-cut apples with 0.50 µmol L⁻¹ SL treatment was 1.49 times greater than that of the control. A significant increase in APX activity in fresh-cut apples as a result of SL treatment improved their ability to eliminate reactive oxygen species, thereby contributing to the maintenance of the fruit’s quality.

The GR activity of fresh-cut apples increased during the first 4 days of storage and then gradually decreased (Figure 3d). The GR activity of fresh-cut apples with 0.50 µmol L⁻¹ SL treatment consistently exceeded that of the control. On day 6, the GR activity of fresh-cut apples with 0.50 µmol L⁻¹ SL treatment was 1.71 times higher than the control. Treatment with 0.50 µmol L⁻¹ SL effectively improved the activity of GR in fresh-cut apples, promoted the conversion of GSSG to GSH, and enhanced the ability of the fruits to resist oxidative damage.

During storage, the GSH content in fresh-cut apples initially rose before eventually declining (Figure 3e). The GSH content in fresh-cut apples with 0.50 µmol L⁻¹ SL treatment was consistently higher than that of other treatments. On day 8, the content of GSH in the fresh-cut apple with 0.50 µmol L⁻¹ SL treatment was 2.03 times higher than that of the control apple, and it was always higher than that of other treatments throughout the storage period, which resulted in an increased GSH content in the fruit.

During the whole storage period, the GSSG content in fresh-cut apples shrank gradually (Figure 3f). And the GSSG content of 0.50 µmol L⁻¹ SL treatment remained consistently lower than that of the control. Furthermore, 0.50 µmol L⁻¹ SL treatment significantly affected the GSSG content compared with other treatments. On day 4, the GSSG content in fresh-cut apple fruits with 0.50 µmol L⁻¹ SL treatment was 77.2% of that in the control apple.

During storage, the changes in AsA content in fresh-cut apples gradually decreased, and the AsA content in fresh-cut apples with 0.50 µmol L⁻¹ SL treatment was always higher than the other treatments (Figure 3g). On day 8, the AsA content of fresh-cut apples with 0.50 µmol L⁻¹ SL treatment was 1.75 times higher than the control. The SL treatment consistently increased the AsA content. With the increase in ASA content, the antioxidant damage ability of the fresh-cut apples was enhanced.

During the whole storage period, there was a steady decline in the DHA content of fresh-cut apples. SL treatment significantly decreased the DHA content, and the 0.50 µmol L⁻¹ SL treatment was always lower than the control during the storage period, and the DHA content of 0.50 µmol L⁻¹ SL-treated fresh-cut apple had the most prominent effect on day 4 (Figure 3h), which was 62.1% of that of the control.

3.3. Effect of NO Metabolism in Fresh-Cut Apple after SL Treatment

The endogenous NO content in fresh-cut apples increased during storage (Figure 4a). Throughout the storage period, the NO content in fresh-cut apples with 0.50 µmol L⁻¹ SL treatment consistently exceeded that of the control. On day 10, the NO content in the 0.50 µmol L⁻¹ SL-treated apples was 2.35 times higher than the control, maintaining its lead as the highest NO content observed.

During storage, the NOS-like activity in SL-treated fresh-cut apples was significantly higher than that of the control apples (Figure 4b). The NOS-like activity within fresh-cut apples showed a first increasing and then decreasing trend. Compared with the control, different concentrations of SL treatments increased the NOS-like activity in fresh-cut apples, with the NOS-like activity in fresh-cut apples with 0.50 µmol L⁻¹ SL treatment being the highest. On day 6, the NOS-like activity in 0.50 µmol L⁻¹ SL-treated fresh-cut apples was 1.55 times higher than that of the control.

The nitrite content in fresh-cut apples displayed a pattern of firstly rising and then falling (Figure 4c). Various concentrations of SL treatments all made the nitrite content within fresh-cut apples lower compared with the control, and the content of nitrite in 0.50 µmol L⁻¹ SL-treated fresh-cut apples on day 10 was 1.68 times higher than the control. Therefore, the use of SL treatment suppressed the formation of nitrite in fresh-cut apples.

The L-arginine content within fresh-cut apples increased continuously over the storage time (Figure 4d). The L-arginine content of the 0.50 µmol L⁻¹ SL-treated fresh-cut apples was consistently higher than that of the control. During the whole storage period, the highest L-arginine content was found in the 0.50 µmol L⁻¹ SL-treated fresh-cut apples, which was 2.35 times higher than the control treatment on day 10.

The NR activity irregularly changed over the storage time (Figure 4e). The NR activity in 0.50 µmol L⁻¹ SL-treated fresh-cut apples peaked on day 8, which was 1.79 times that of the control.

3.4. Effect of H₂S Metabolism in Fresh-Cut Apple after SL Treatment

Throughout the storage period, the content of endogenous H₂S in fresh-cut apples increased rapidly in the first 4 days and then peaked on day 6 (Figure 5a). At the peak on day 6, the endogenous H₂S content of fresh-cut apple was improved by different concentrations of SL treatments, in which the endogenous H₂S content of 0.50 µmol L⁻¹ SL-treated fresh-cut apple was 1.62 times that of the control, indicating a significant effect of 0.50 µmol L⁻¹ SL treatment on the H₂S production.

During the 10-day storage period, the L-CD activity and OAS-TL activity in fresh-cut apples rose to a peak in the first 6 days and then declined (Figure 5b,c). It is noticeable that the SL treatment at various concentrations promoted the L-CD activity and OAS-TL activity, compared with the control. In particular, the 0.50 µmol L⁻¹ SL treatment showed a significant effect on these enzyme activities. The L-CD activity and OAS-TL activity of 0.50 µmol L⁻¹ SL-treated apples were higher than others, which were 1.29 and 1.37 times that of the control on day 6, respectively.

The activity of SAT in fresh-cut apples gradually rose over the whole storage period (Figure 5d). Different concentrations of SL treatments resulted in increased SAT activity in fresh-cut apples compared with the control. On day 10, the fresh-cut apples with 0.50 µmol L⁻¹ SL treatment showed the highest SAT activity, which was 1.38 times greater than the control.

3.5. Effect of SL Treatment on the Gene Expression of Essential Enzymes in Fresh-Cut Apples

Fresh-cut apples under SL treatments exhibited different ROS production and scavenging capacities, which were associated with the various transcriptional regulations of essential genes directly or indirectly involved in ROS metabolism. Therefore, this study analyzed the relative expression levels of several genes involved in producing and scavenging ROS (Figure 6). MdSOD and MdCAT play vital roles in scavenging reactive oxygen species. SL treatment resulted in the up-regulation of MdSOD and MdCAT expression in fresh-cut apples compared with the control. On day 6, the relative expression level of MdCAT in 0.50 µmol L⁻¹ SL-treated fresh-cut apples was 20.6 times that of the control (Figure 6a), and the relative expression level of MdPOD was 42.5 times that of the control (Figure 6b). SL treatment at both 0.25 and 0.50 µmol L⁻¹ significantly up-regulated the relative expression level of MdSOD. But on day 4, the relative expression level of MdSOD in fresh-cut apples treated with 0.50 µmol L⁻¹ SL was higher than that in 0.25 µmol L⁻¹ SL-treated apples, which is 25.4-fold higher than that of the control (Figure 6c).

In addition, SL treatment at 0.50 µmol L⁻¹ increased the expression levels of MdDHAR, MdMDHAR, MdGR, and MdAPX. On day 6, the relative expression levels of MdDHAR and MdMDHAR were significantly up-regulated in 0.50 µmol L⁻¹ SL-treated fresh-cut apples, which were 60.0 times (Figure 6d) and 28.8 times (Figure 6e) that of the control, respectively. On day 4, the relative expression level of MdGR in 0.25 µmol L⁻¹ SL-treated fresh-cut apples was the highest, being 12.5 times that of the control (Figure 6f), and that of MdAPX in 0.50 µmol L⁻¹ SL-treated fresh-cut apples was 33.7 times that of the control (Figure 6g).

MdL-CD and MdSAT are some of the critical genes involved in H₂S metabolism, and their expression levels were significantly increased under SL treatment, especially at 0.50 µmol L⁻¹. Fresh-cut apples with SL treatment at 0.50 µmol L⁻¹ exhibited elevated levels of MdSAT and MdL-CD expression. On day 4, the relative expression level of MdL-CD was 39.4 times that of the control (Figure 6h). The relative gene expression of MdSAT was 242 times that of the control (Figure 6i).

4. Discussion

After fresh-cut processing, apple flesh browning occurs rapidly. Its storage life is shortened, and its economic value is reduced [30]. Diverse chemical preservatives are utilized to keep the storage quality and prolong the preservation period of fresh-cut apples. Arginine (50 mM) retarded the browning of fresh-cut apples and extended their postharvest life [31]. The effectiveness of 1-MCP (1 µL L⁻¹) mainly focuses on reducing ethylene and carbon dioxide to maintain the storage quality of fresh-cut apples [32]. Citric acid (0.5%) combined with UV, as an effective preserver, promoted the activity of POD in fresh-cut apples [33]. Fresh-cut apples treated with calcium ascorbate (6 or 12%) and stored in modified atmosphere packaging had a longer shelf life with improved antioxidant activities [34]. NaHS (0.7 mmol L⁻¹) had a surface browning inhibition effect on fresh-cut apples by reducing the prenol lipids content [35]. Strigolactone, which was as effective as these chemical preservatives, also exhibits its positive role in maintaining the storage qualities of strawberries [13], celery [36], sweet orange fruit [12], and fresh-cut apples in this work. Not affordable like other chemical preservatives, natural SLs have not yet been commercially broadly applied, considering their unfeasible high-level production by chemical synthesis [37]. Economically viable synthesis, such as microbial biosynthetic platforms [38], would enable the mass production of SLs and reduce the cost of SLs for preservative application.

Applying SL treatment restrained the respiratory activity of fresh-cut apples over time, prevented weight loss and color alteration, upheld the balance of antioxidants by controlling the activity and content of antioxidant enzymes, enhanced antioxidant metabolism, and notably boosted the levels of endogenous NO and H₂S of fresh-cut apples during the storage period (Figure 7).

As plant-specific organs, apples produce ROS endogenously and maintain ROS homeostasis through ROS scavenging pathways. SL can maintain ROS homeostasis and prolong its storage time by modulating non-enzymatic and enzymatic antioxidant systems. This study investigated the preservation effect of SL analogs on asparagus tubers, and found that the antioxidant enzyme activity of alfalfa was increased after treatment with SL analogs, suggesting that SL has a more substantial preservation effect [30]. In this work, the genes corresponding to the key enzymes of the antioxidant system of fresh-cut apples were activated with 0.50 µmol L⁻¹ SL, which regulated NO metabolism and H₂S metabolism to increase the content of NO and H₂S, and thus were regulated to increase the antioxidant enzyme activity and antioxidant content of the fresh-cut apples. It was demonstrated that SL could reduce the oxidative damage resulting from an excess of ROS by boosting both enzymatic and non-enzymatic mechanisms for scavenging ROS during storage.

In the fruit antioxidant system, SOD, CAT, and POD are the key enzymes to reduce the attack of ROS. In a study on seedless grapes [39], it was found that when SOD, POD, and CAT are kept at high activities, the O₂˙⁻ and H₂O₂ contents in grapes are effectively controlled, which is favorable for long-term storage. Zhao et al. [40] found the GSNO-CS-treated fresh-cut apple slices had high antioxidant enzyme activities and a low ROS content. Altaf et al. [41] report that exogenous melatonin attenuates oxidative damage in tomatoes by modulating the enzyme activities and relative gene expression level of SOD, CAT, APX, GR, and POD, and the trend of their findings is in line with the present study. SL at 0.50 µmol L⁻¹ significantly increased the relative expression levels of MdSOD, MdCAT, MdPOD, MdMDHAR, MdDHAR, MdAPX, and MdGR and the corresponding enzyme activities, thereby increasing the content of antioxidants (GSH and ASA) and decreasing the content of ROS (H₂O₂, ˙OH, O₂˙⁻), which suggests that SL treatment could improve the antioxidant capacity by activating the antioxidant system, thus maintaining the quality of fresh-cut apples.

NO is a bioactive molecule that acts as a second messenger in various life activities, such as plant growth, development, and stress tolerance [42]. NO is endogenously produced in fruits and positively affects their storage and freshness preservation [43]. It has been shown that NO treatment at 10 µmol L⁻¹ can inhibit the increase in the sugar-acid ratio in peaches and prolong their storage time [44]. NO can be generated through nitrite reduction and NOS-like pathways. In plants, there is an interaction between SL and NO, where the addition of external SL can enhance NO levels, while NO can control the synthesis and signaling of SL in plants [45]. In this experiment, 0.50 µmol L⁻¹ SL treatment increased NOS-like activity, L-arginine, and NO content. NR activity also increased significantly after being treated with SL. This suggests that SL can increase endogenous NO content through both the nitrite reduction pathway and the NOS-catalyzed pathway.

H₂S is a gaseous signaling molecule that improves plant stress tolerance and simultaneously reduces the adverse effects of abiotic stresses [46]. Low concentrations of H₂S can act as a source of sulfur in the plant body, and high concentrations of H₂S can be toxic to the plant. The treatment of strawberries with the exogenous H₂S donor NaHS reveals a correlation between the concentration of H₂S produced in fruit, the activities of APX, CAT, POD, and SOD, and the concentration of ROS, which reduces the consumption of nutrients in the fruit and enables the fruits to maintain a high nutritional value [47]. H₂S also maintains brightness, peels firmness, reduces respiration and ethylene release rates, and extends the fruit’s shelf life by delaying ripening [48]. In the present study, 0.50 µmol L⁻¹ SL up-regulated MdSAT and SAT activity, causing the reaction of serine and acetyl coenzyme A to form O-acetylserine which was cleaved to L-serine in the presence of OAS-TL, in turn, catalyzing the cleavage of L-cysteine to increase the endogenous H₂S content. At a concentration of 0.50 µmol L⁻¹, SL enhanced the activity of enzymes and antioxidant levels in fresh-cut apples, resulting in a better preservation of their postharvest quality. Similar results are also found in goji berry [49].

There is a complex interrelationship between the antioxidant system and those metabolisms [50]. H₂S metabolism and NO metabolism in SL application improved the antioxidant system of fresh-cut apples during cold storage and preservation to balance the accumulation of reactive oxygen species and increased endogenous NO through the NOS-like pathway and endogenous H₂S through the modulation of H₂S metabolism. The enhancement of NO and H₂S metabolism also positively affected the antioxidant system, thereby maintaining storage quality.

It is noticeable that SL at 1.00 µmol L⁻¹ did not have a better effect than SL at 0.50 µmol L⁻¹ on the preservation of fresh-cut fruits, although SL at different concentrations showed varying degrees of enhancement effects on the antioxidant system and metabolism of H₂S and NO in this work. In our previous study [13], SL treatment at 1 µmol L⁻¹, but not at higher concentrations, maintained the quality of postharvest strawberries by significantly improving the antioxidant system and metabolism of H₂S and NO. This enhancement effect appears to be related to the concentration of SL. These bell-shaped concentration-response results, which usually exist in pharmacodynamic research on drugs in cells [51], were found in the research on preventing the browning of lettuce [31]. The postharvest life of lettuce treated with arginine significantly increased at the concentrations from 1 mM to 100 mM, but a lower increase was detected at a higher concentration of 250 mM. Aharoni [52] studied the effects of growth regulators on the senescence of lettuce leaves. It showed that the retarding effect of gibberellic acid and kinetin on chlorophyll loss increased at the concentration from 10⁻⁹ M to 10⁻⁶ M and 10⁻⁷ M, respectively. A weakened retarding effect on chlorophyll loss appeared above the optimal concentration. Toxicity was considered to make a probable contribution to reducing the effectiveness of hormones at a supra-optimal applying concentration. Additionally, changes in POD and SOD activity in rice seedlings were proved to be associated with SL concentration, and the optimal concentration of SL could enhance the salt tolerance of plants, while the effect was impaired with a higher SL concentration [53]. A high concentration of SL may result in general toxicity and harm to the appearance and fitness of entire plants [54]. It remains unexplained whether the weakened improvement in the antioxidant system of fruit resulted from the toxicity. Further work is required to explore the interaction between preservation methods and SL concentration. Additional studies are also needed to determine whether SL has potential commercial applications with other fresh-cut foods beyond fresh-cut apples.

5. Conclusions

SL at 0.50 µmol L⁻¹ effectively alleviated oxidative damage in fresh-cut apples. The alleviation of oxidative damage by SL might be associated with the maintenance of high activated levels of non-enzymatic and enzymatic antioxidants system and the endogenous NO and H₂S, which have the potential to prevent ROS from accumulating and thus protect cells from oxidative damage, thus maintaining the quality of fresh-cut fruits. Consequently, the current research suggests the potential application of exogenous strigolactone in the cut-fruit processing industry.