Nitric Oxide in Controlled Atmosphere Storage of ‘Fuji Mishima’ Apples

Abstract

‘Fuji’ apples require long-term storage to ensure year-round supply, and controlled-atmosphere (CA) technology is widely used to preserve their quality and reduce postharvest losses. Nitric oxide (NO), a natural signaling molecule in plants, has shown potential to delay ripening and reduce physiological and pathological disorders during fruit storage. This study evaluated the effect of nitric oxide (NO) treatment during controlled-atmosphere (CA) storage on the postharvest quality of ‘Fuji Mishima’ apples. Apples were stored for 8 months at 1 kPa O₂ +< 0.5 kPa CO₂, 1.0 ± 0.2 °C, and 94 ± 2% RH. The treatments consisted of a control (without NO) and five NO application regimes: 5 µL L⁻¹ applied at the beginning of storage; 5 µL L⁻¹ applied both at the beginning and end; 5 µL L⁻¹ applied every 30 days; 10 µL L⁻¹ applied at the beginning; and 10 µL L⁻¹ applied both at the beginning and end of storage. All NO treatments delayed ethylene production and reduced its levels after 4 days under ambient conditions compared to the control. However, NO had no effect on flesh firmness, soluble solids content, titratable acidity, peel color, or flesh browning. Repeated NO applications (5 or 10 µL L⁻¹) increased peel yellowing. Treatment with 5 µL L⁻¹ applied every 30 days increased decay incidence. Phenolic compounds in the flesh were unaffected, while in the peel, they decreased with 10 µL L⁻¹. Overall, NO application in CA storage of ‘Fuji Mishima’ apples did not maintain fruit quality and, in some cases, increased peel yellowing and decay.

1. Introduction

The Brazilian apple harvest occurs over a short period, mainly from February to April, resulting in a large portion of the yield needing to be stored in order to regulate the supply of the fruit throughout the year. Controlled-atmosphere (CA) storage is the leading technology for preserving apple during long-term storage [1].

Despite the advantages of CA, apples from the ‘Fuji’ group are susceptible to damage caused by high CO₂ partial pressure (>0.5 kPa) during CA storage. Therefore, it is recommended to adopt <0.5 kPa CO₂ during CA storage to avoid browning damage [2]. Additionally, the incidence of decay, especially in prolonged storage, can account for up to 80% of postharvest losses of ‘Fuji’ apples [3]. Therefore, additional technologies are required to complement CA storage and maintain fruit quality.

Nitric oxide (NO) is a gaseous molecule naturally produced by plants which participates in various physiological processes, including fruit ripening, with positive effects on postharvest quality regarding flavor preservation, reduction of disease development, and physiological disorders [4]. In the postharvest period, NO has an antagonistic effect on ethylene production by interfering with the production of its precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), which results in lower activity of the enzymes ACC synthase and ACC oxidase, and consequently inhibits the activity of enzymes responsible for fruit softening [5]. NO also increases the ability to metabolize reactive oxygen species (ROS) through the induction of antioxidant metabolism enzymes, such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and polyphenol oxidase (PPO), resulting in the maintenance of overall antioxidant capacity [4].

The postharvest application of NO, associated with CA storage, has already shown positive results in various fruits, including ‘Baigent’ [6] and ‘Cripps Pink’ apples [7,8]. NO application has also proven effective in reducing surface browning of minimally processed ‘Granny Smith’ apples [9], ‘Royal Gala’, ‘Golden Delicious’, ‘Sundowner’, ‘Fuji’, and ‘Red Delicious’ apples [10].

The postharvest metabolic pathways influenced by NO remain only partially understood. It is known that the effectiveness of using NO may change depending on factors such as cultivar, epigenetic modifications, storage conditions, and others, such as concentration, time, and moments of application, influencing its action [11,12,13]. Additionally, NO also has the ability to interact with other molecules, resulting in the alteration of fruit responses [11]. During postharvest storage, nitric oxide may interact with a variety of molecules, including ethylene, reactive oxygen species (ROS), hydrogen sulfide (H₂S), melatonin, and antioxidant-related enzymes. Interaction with ethylene leads to inhibition of its biosynthesis, thereby delaying ripening and mitigating physiological disorders. NO modulates the antioxidant system by inducing enzymes such as catalase, peroxidase and superoxide dismutase, thus enhancing the fruit’s tolerance to oxidative stress. Additionally, NO’s interplay with signaling molecules like H₂S and melatonin further influences stress response and maintenance of fruit quality during storage [14,15]. Currently, there is no information of NO application in ‘Fuji’ apples group during CA storage. In this context, the objective of this study was to evaluate the effect of two doses of NO, applied at different moments during CA storage, on ‘Fuji Mishima’ apples quality maintenance.

2. Materials and Methods

2.1. Plant Material and Experimental Details

The experiment was conducted with ‘Fuji Mishima’ apples harvested during the 2022/2023 season from a commercial orchard located in the municipality of São Joaquim, Santa Catarina, Brazil (28°16′01.7′′ S; 49°54′17′′ W; 1400 m altitude, Cfb climate according to the Köppen classification). The ‘Fuji Mishima’ apple trees used in this study were 12 years old and grafted on the vigorous rootstock ‘Marubakaido’ with an M.9 interstem (Marubakaido/M.9 combination). This rootstock combination is commonly used in commercial orchards in southern Brazil, particularly in São Joaquim (SC). The orchard was established with a planting spacing of 4.0 m between rows and 1.2 m between trees within the row. The canopy training system used was the central leader, which is commonly adopted in commercial apple orchards in southern Brazil. After harvest, apples were transported to the laboratory, sorted for uniform size and maturity, and fruit showing any mechanical or pathogen-related damage were discarded.

The fruit initially presented the following ripening attributes: background peel color with lightness (L*), chromaticity (C*), and hue angle of 70.5, 42.1, and 100.8°, respectively; flesh firmness of 74.2 N, soluble solids content of 15.8 °Brix, and titratable acidity of 0.38% malic acid, with an average iodine-starch index of 4.5 (scale 1–5).

For NO treatment, the fruit were placed in experimental micro-chambers (80 L) under CA conditions of 1 kPa O₂ + < 0.5 kPa CO₂, at a temperature of 1.0 ± 0.2 °C and relative humidity of 94 ± 2%, for 8 months. The evaluated treatments were as follows: control (without NO); 5 µL L⁻¹ NO applied at the beginning of storage; 5 µL L⁻¹ NO applied at the beginning and end of storage; 5 µL L⁻¹ NO applied at the beginning and every 30 days during storage; 10 µL L⁻¹ NO applied at the beginning of storage; and 10 µL L⁻¹ NO applied at the beginning and end of storage. NO concentrations (5 and 10 µL L⁻¹) were selected according to previous studies conducted with other apple cultivars such as Baigent and Cripps Pink [6,7].

The application of NO in the micro-chambers was performed using a mixture of NO + N₂ gas (standard mixture containing 1000 μL L⁻¹ of NO + N₂ in balance, White Martins^®, Brazil) from high-pressure cylinders. The initial application was carried out when microchambers O₂ partial pressure reached 1 kPa (4 days after storage), every 30 days from the first application, or at the end of storage, according to the treatment. The last NO application, for treatments with reapplication every 30 days and at the end of storage, occurred 5 days before the finished of the storage period.

The O₂ and CO₂ partial pressure were monitored daily using an automated system for monitoring and controlling CA (Multiplex Isosoft, Isolcell, Laives, Italy). The oxygen consumption due to fruit respiration were corrected by injecting O₂ from high-pressure cylinders. For CO₂ absorption, hydrated lime was placed inside the micro-chamber in the proportion of 1 kg of hydrated lime per 20 kg of fruit. The storage temperature was monitored daily using mercury thermometers (5100, Incoterm^®, Porto Alegre, Brazil).

2.2. Evaluation of Fruit Quality Attributes

The fruits were evaluated upon opening the chambers for ethylene production rate, background peel color, and incidence of decay. After 7 days of shelf life (20 ± 1 °C; RH of 65 ± 5%) flesh firmness, soluble solids content (SSC), titratable acidity (TA), incidence of flesh browning, and decay were also performed. The ethylene production rate was evaluated every 2 days during shelf life, from the exit of storage (D0), until 6 days of shelf life (D2, D4, and D6, respectively).

After 7 days of shelf life, samples of flesh and peel were separated to determine phenolic compounds and antioxidant activity using the ABTS (2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging methods. The samples were frozen in liquid nitrogen immediately after fruit cutting. The skin samples were processed and kept fresh in an ultrafreezer (Thermo Fischer Scientific, 910, Waltham, MA, USA) at −50 °C until analysis, while the flesh samples were freeze-dried in a freeze dryer (LH, Terroni^®, São Carlos, Brazil). The freeze-dried samples were processed in a vibratory ball mill (BM 500, Anton Paar^®, Graz, Austria) and stored in a desiccator until analysis. The analyses were carried out within 30 days after freeze-drying (lyophilization) of the samples.

To determine ethylene production (ηmol C₂H₄ kg⁻¹ s⁻¹), approximately 1 kg of fruit per replicate were placed in hermetically sealed glass jars (6.1 L), for 120 min. Then, air samples were withdrawn from the headspace of the jars, using plastic syringes of 1 mL, and injected into a gas chromatograph with flame ionization detection (GC-FID) (Clarus 580, PerkinElmer^®, Shelton, CT, USA), equipped with a 3 m Porapak N column (mesh 80–100). The temperatures of the column, detector, and injector were 70, 250, and 130 °C, respectively. The gas flow rates were 70, 30, and 300 mL min⁻¹ for nitrogen, hydrogen, and synthetic air, respectively. Nitrogen gas was used as the mobile phase.

Skin background color was measured on the greenest side of the fruit peel using a CR-400 colorimeter (Konica, Minolta^®, Tokyo, Japan), on the L* (lightness), C* (chromaticity), and h° (hue) scale.

Flesh firmness was determined on the equatorial region of each fruit after removing a small portion of the epidermis, at two opposite points on each fruit, using an electronic penetrometer (Güss Manufacturing Ltd., Cape Town, South Africa), equipped with an 11 mm diameter probe, and the results were expressed in Newtons (N).

For the SSC and TA, samples of flesh and skin were taken from transverse slices cut from the equatorial region of the fruits, which were then crushed in an electric centrifuge (PG 710/G5, Champion Juicer^®, Lodi, CA, USA). The SSC was evaluated using a digital precision refractometer with automatic temperature measurement and compensation to 20 °C (PR201α, Atago^®, Tokyo, Japan) using 0.5 mL of juice, with the results expressed in °Brix. The TA was determined using an automatic titrator (TitroLine Easy, Schott Instruments^®, Weilheim, Germany) using a 5 mL juice sample diluted in 45 mL of distilled water, which was titrated with a 0.1 N NaOH solution until pH 8.1, with the results expressed as % malic acid.

To evaluate flesh browning (results expressed as %), the fruits were cut in a transverse section at the equatorial region and observed for the presence of browning (i.e., visually presented any type of browning). The incidence of decay was evaluated by counting the number of apples exhibiting fungal infection symptoms with lesions larger than 5 mm.

To obtain the extracts for the analysis of total phenolic compounds and antioxidant activity, 0.5 g of freeze-dried flesh and 1 g of fresh skin were added to 10 mL of 80% methanol, followed by homogenization in an Ultra-Turrax (SilentCruscher M, Heidolph, Schwabach, Germany), and left to rest protected from light for 60 min at room temperature. The extracts were then centrifuged at 15,000 rpm for 15 min at 4 °C in a centrifuge (CR22N, Hitachi, Tokyo, Japan), subsequently filtered, and the supernatant collected.

The determination of phenolic compound content was performed using the Folin-Ciocalteau reagent (Sigma Aldrich, Darmstadt, Germany) adapted from Roesler et al. [16]. The standard curve was obtained with gallic acid. For analysis, 0.25 µL of extract, 200 µL of distilled water, and 25 µL of 2 N Folin-Ciocalteau reagent were added to microplates. The mixture was allowed to stand for 5 min, and then 25 µL of 10% sodium carbonate was added. Readings were taken after 60 min using a microplate reader (EnSpire, PerkinElmer^®, Shelton, CT, USA) at 725 nm. The results were expressed in milligrams of gallic acid equivalent per gram of fresh (FM) or dry mass (DM) (mg GAE g⁻¹ FM for peel or mg GAE g⁻¹ DM for flesh).

The determination of antioxidant capacity by the ABTS free radical scavenging method was performed according to Rufino et al. [17], with modifications. In microplates, 10 μL of extract and 290 μL of ABTS radical were added and kept in the dark for 6 min, and then the absorbance was read at 734 nm using a microplate reader (EnSpire, PerkinElmer^®, Shelton, CT, USA). The calibration curve was constructed with standard Trolox solutions, and the results were expressed in mg TEAC g⁻¹ FM for skin or mg TEAC g⁻¹ DM for flesh.

The determination of antioxidant capacity by the DPPH free radical scavenging method was conducted according to the method described by Rufino et al. [18], with modifications. In microplates, 40 µL of extract from the samples were added, followed by 260 µL of DPPH solution, and allowed to react for 30 min in the dark. Reading were then taken at a wavelength of 525 nm using a microplate reader (EnSpire, PerkinElmer^®, Shelton, CT, USA). The results were expressed as a percentage of DPPH radical scavenging (% DPPH inhibition).

2.3. Experimental Design and Statistical Analysis

The experimental design was completely randomized, with four replicates, each consisting of 30 fruits. The data were analyzed using RStudio 3.5.0 software (Posit, Boston, MA, USA). Shapiro–Wilk normality tests of residuals (p < 0.05) and Bartlett’s homogeneity of variance tests (p < 0.05) were first employed. Normal data were then subjected to analysis of variance (ANOVA), and the means were compared using the Scott–Knott test (p < 0.05). Data expressed as percentages were previously transformed into arcsin√(x + 0.1)/100.

3. Results and Discussion

After 8 months of storage, the ethylene production rate of ‘Fuji Mishima’ apples showed no differences between treatments upon chamber opening, as well as at 2 and 6 days of shelf life (Figure 1). After 4 days of shelf life, control fruit reached the ethylene production peak, a rate about 1.5 times higher than those treated with different NO doses and frequencies. However, this effect of reduced ethylene production with NO application did not persist until 6 days under ambient conditions.

Similar results were observed for ‘Cripps Pink’ apples, where the application of NO at the beginning of CA storage was effective in reducing ethylene production at 4 days of shelf life [8]. However, at 6 and 7 days of shelf life, respectively, for ‘Galaxy’ and ‘Cripps Pink’, there was no effect from NO. In ‘Galaxy’ apples, the effect of NO on ethylene production was, at least in part, due to the lower activity of the ACC oxidase enzyme, a key enzyme in ethylene synthesis. NO acts by inhibiting ethylene synthesis through the formation of the ACC-oxidase-NO-ACC complex. Additionally, NO induces the reduction of ACC to 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), decreases the activity of ACC synthase and ACC oxidase enzymes, and negatively regulates the expression of genes involved in the ethylene signaling pathway [11,19]. The results of this study demonstrate that NO application delayed the peak of ethylene production, corroborating the results obtained by Coser et al. [6] in ‘Baigent’ apples.

For the background peel color, differences between treatments were observed for the hue angle attribute upon exiting the chamber, and for L* after 7 days of shelf life (

Table 1. Skin background color attributes of ‘Fuji Mishima’ apples after 8 months of storage in a controlled atmosphere (1.0 kPa O₂ + < 0.5 kPa CO₂; 1.0 ± 0.2 °C; RH of 94 ± 2%), upon chamber opening, and after 7 days of shelf life (20 ± 1 °C; RH of 65 ± 5%), as a function of nitric oxide (NO) application in two doses and at different times during storage. NO: nitric oxide.

Treatments		Skin Background Color
NO Doses	Period of Application	L*	C*	h°
		Exit of the Chamber
0 µL L−1	No application	67.9 ns	37.0 ns	98.8 a
5 µL L−1	Beginning	69.1	35.3	98.2 a
5 µL L−1	Beginning and end	68.4	36.6	96.3 b
5 µL L−1	Beginning + every 30 days	69.4	38.2	97.0 b
10 µL L−1	Beginning	69.9	37.8	99.4 a
10 µL L−1	Beginning and end	69.3	37.0	96.6 b
CV (%)		2.1	3.9	1.5
		7 days of shelf life
0 µL L−1	No application	69.0 b	42.1 ns	97.0 ns
5 µL L−1	Beginning	70.8 a	39.8	96.0
5 µL L−1	Beginning and end	67.7 b	40.2	95.0
5 µL L−1	Beginning + every 30 days	69.7 b	41.9	96.5
10 µL L−1	Beginning	71.7 a	39.8	98.4
10 µL L−1	Beginning and end	71.9 a	40.8	95.3
CV (%)		2.2	3.2	2.4

Means followed by the same letter in the column do not differ by the Scott–Knott test (p < 0.05). ns: not significant by ANOVA.

). Higher hue angle were observed in fruits not treated with NO, and in those treated with 5 and 10 µL L⁻¹ NO at the beginning of storage, indicating a greener background peel color and, therefore, less mature fruits. However, this result did not persist after 7 days of shelf life, where the treatments did not differ from each other. After 7 days of shelf-life, the treatments with 5 and 10 µL L⁻¹ NO at the beginning of storage and 10 µL L⁻¹ NO applied at the beginning and end of storage, showed fruit with higher L* values than the other treatments. The NO application did not show benefits in maintaining the background color of ‘Fuji Mishima’ apples.

Contrary to the present study, ‘Baigent’ apples, regardless of the dose and application time, after 7 days of shelf life, showed higher hue angle for fruit treated with NO compared to the control, while no difference was observed in terms of L* [6]. A similar result was observed for ‘Cripps Pink’ apples, where the weekly application of a NO dose between 5.3 and 5.9 μL L⁻¹ kept the fruits greener after 7 days of shelf life [7]. However, in another study with ‘Cripps Pink’ apples, pre-storage treatments with NO application, regardless of the dose, the fruit did not differ from control (1-MCP) [9]. The results obtained in the present study and in the literature indicate that the effect of NO on maintaining skin background color can be variable among cultivars, as well as between different harvests.

NO acts as an anti-senescence agent, correlating negatively with the process in different plant species, as endogenous NO deficiency accelerates chlorophyll degradation [20]. The application of NO can delay chlorophyll breakdown by regulating the activity of chlorophyllase and Mg-dechelatase enzymes, reducing carotenoid accumulation, which is responsible for the yellow background color of apples, and by promoting the antioxidant system during ripening [19,21]. However, based on the skin background color data observed in this study, it is possible that the higher frequencies of NO application (at the beginning and end and at the beginning and every 30 days of storage) were detrimental or even cumulative, accelerating chlorophyll loss in the fruit, since the greener fruits were those treated only at the beginning of storage or not treated with NO. These findings suggest a negative impact of excessive NO application.

Changes in peel color indicate that frequent or excessive NO applications accelerated yellowing rather than preserving fruit appearance, which contradicts the expected senescence-delaying effect of NO. Zhu et al. [22], when treating ‘Feicheng’ peaches with 5, 10, and 15 µL L⁻¹ of NO gas, observed toxicity from the highest dose of NO in the fruit, while 5 and 10 µL L⁻¹ were beneficial. This contrary effect of NO, when in high doses, may be related to the formation of reactive nitrogen species (RNS) and reactive oxygen species (ROS), which can be harmful to metabolism [22,23]. In this case, NO, instead of inducing antioxidant activity, acts as a facilitator of cellular disorder [24]. ‘Bartlett’ pears treated with 50 and 10 µL L⁻¹ of NO for 12 h showed accelerated ripening, unlike those treated with 10 µL L⁻¹ of NO for 2 h, which showed a delay in this process [25], indicating that in addition to high doses, longer exposure periods can also have a negative effect. However, the mode of action of NO on chlorophyll degradation and/or carotenoid biosynthesis and accumulation in fruit still needs to be further investigated [21].

After 8 months in CA storage followed by 7 days on the shelf, no differences were observed for flesh firmness, SSC, and TA (

Table 2. Flesh firmness, soluble solids content (SSC), titratable acidity (TA), flesh browning, and decay in ‘Fuji Mishima’ apples after 8 months of storage in a controlled atmosphere (1.0 kPa O₂ + < 0.5 kPa CO₂; 1.0 ± 0.2 °C; RH of 94 ± 2%) and 7 days of shelf life (20 ± 1 °C; RH of 65 ± 5%), as a function of nitric oxide (NO) application in two doses and at different times during storage. NO: nitric oxide.

Treatments		Flesh Firmness (N)	SSC (°Brix)	TA (% of Malic Acid)	Flesh Browning (%)	Decay (%)
NO Doses	Period of Application
0 µL L−1	No application	68.4 ns	15.6 ns	14.0 ns	14.0 ns	23.8 b
5 µL L−1	Beginning	65.7	15.1	17.6	17.6	27.3 b
5 µL L−1	Beginning and end	66.2	15.2	15.9	15.9	26.4 b
5 µL L−1	Beginning + every 30 days	65.0	15.2	16.3	16.3	44.5 a
10 µL L−1	Beginning	65.8	14.8	16.7	16.7	33.8 b
10 µL L−1	Beginning and end	66.3	14.9	15.0	15.0	26.4 b
CV (%)		2.8	2.5	5.6	14.9	14.9

Means followed by the same letter in the column do not differ by the Scott–Knott test (p < 0.05). ns: not significant by ANOVA.

). Similarly, no differences in flesh firmness were found with NO application for ‘Cripps Pink’, and ‘Baigent’ apples during storage [6,8]. However, ‘Cripps Pink’ apples stored in ultra-low oxygen (ULO-CA) for 8 months showed higher flesh firmness when treated weekly with NO at doses of 5.9 and 6.7 μL L⁻¹ for less and more mature fruits, respectively [7]. The loss of flesh firmness is one of the main changes that occur during ripening, making fruits more susceptible to physical damage and fungal infections during handling. During ripening, several cell wall-modifying enzymes can cause structural modifications to pectin polymers, reducing cohesion between cells and leading to fruit softening [26]. Although no significant difference in flesh firmness were observed between control and NO-treated apples, all maintained their average values between 66 and 69 N, which is suitable for the commercialization of ‘Fuji Mishima’ apples [27]. This stability may indicate a positive effect of CA on firmness maintenance that overlapped the NO applications. The lower O₂ partial pressure during CA storage, may itself reduce various oxidative reactions such as respiration and ethylene biosynthesis, lowering the activity of cell wall breakdown enzymes responsible for softening [26].

The SSC in all treatments was above 14.8 °Brix, which is also suitable for the fresh consumption of ‘Fuji Mishima’ apples [27] and was not influenced by the doses and frequencies of NO application. The same result was verified for ‘Cripps Pink’ apples, where SSC values were not affected by NO application, regardless of the maturity stage and duration of ULO-CA storage [7]. For ‘Baigent’ apples, the highest SSC values were observed for the treatment of 2 μL L⁻¹ applied at the beginning, every 30 days, and at the end of the storage period, but not differing from control [6]. Under refrigerated storage, ‘Cripps Pink’ apples only showed differences in SSC between the control and the treatment with 20 μL L⁻¹ NO applied for 2 h [8].

No differences were also observed regarding TA, with values remaining between 0.21 and 0.23% malic acid. In ‘Cripps Pink’ apples, the acid content was not affected by postharvest NO application [8]. On the other hand, in ‘Baigent’ apples, there was a decrease in TA with the use of higher NO doses, 10 μL L⁻¹ at the beginning, and higher application frequencies, 5 μL L⁻¹ every 30 days or at the beginning and end of the storage period [6]. For ‘Cripps Pink’ apples stored in ULO-CA, there was a linear increasing response, where 10 μL L⁻¹ NO provided higher TA values for less mature apples, while for more mature apples, there was no difference between treatments [7]. This result highlights the importance of the effect of the maturity stage at harvest on the effect of NO on TA. The ‘Fuji Mishima’ apples used in this study were already mature at harvest, according to the quality parameters described by Fioravanço et al. [27] for the cultivar, which may have led to the lack of response to NO.

The treatments with NO also did not show an effect on flesh browning (Table 2). Temperature is the main environmental factor influencing the conservation of fruit postharvest. In general, apples are non-chilling sensitive fruits and can be stored at temperatures above the freezing point [21], yet they are susceptible to cold damage such as flesh browning. The postharvest application of NO can reduce cold injuries in fruit by decreasing membrane permeability, malondialdehyde content, lipid peroxidation, and ion leakage, effects associated with a reduction in ROS generation, ROS detoxification, and an increase in antioxidant enzyme activity or expression [28,29]. Flesh browning is attributed to the oxidation of phenolic compounds, while NO may inhibit the activity of enzymes such as phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), and peroxidase (POD), thereby maintaining fruit quality [11]. For ‘Baigent’ apples, treatment with 5 μL L⁻¹ NO at the beginning and end of storage in CA reduced the incidence of flesh browning [6].

The incidence of decay in ‘Fuji Mishima’ apples after 7 days of shelf life was highest in the treatment with application of 5 µL L⁻¹ NO every 30 days of storage, while all other strategies of NO treatment did not differ from control (Table 2). For ‘Baigent’ apples, the application of NO, regardless of dose and application period, showed favorable results in reducing the incidence of decay [6]. NO has the potential to improve postharvest quality by acting in pest and disease control by inducing defense responses at the genetic and molecular level, depending on the dose and exposure time [5]. It also acts in the activation of enzymes related to defense metabolism, such as cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase, in addition to the accumulation of antifungal compounds, phenolic compounds, and antioxidant activity [11,21].

The NO application did not alter the phenolic compound content in the flesh or the antioxidant capacity quantified by the DPPH method (

Table 3. Total phenolic compound content and total antioxidant activity by ABTS and DPPH methods in the flesh and skin of ‘Fuji Mishima’ apples after 8 months of storage in a controlled atmosphere (1.0 kPa O₂ + < 0.5 kPa CO₂; 1.0 ± 0.2 °C; RH of 94 ± 2%) and after 7 days of shelf life (20 ± 1 °C; RH of 65 ± 5%), as a function of nitric oxide (NO) application in two doses and at different times during storage. TPC: total phenolic compound; GAE: gallic acid equivalent; TEAC: Trolox equivalent; DM: dry mass; FM: fresh mass; NO: nitric oxide.

Treatments		TPC Flesh	TPC Peel	ABTS Flesh	ABTS Peel	DPPH Flesh	DPPH Peel
NO Dose	Period of Application	(mg GAE g−1DM)	(mg GAE g−1FM)	(mg TEAC g−1DM)	(mg TEAC g−1FM)	(% DPPH Inhibition)	(% DPPH Inhibition)
0 µL L−1	No application	2.14 ns	3.30 a	2.56 b	4.67 b	90.1 ns	84.3 ns
5 µL L−1	Beginning	2.25	3.61 a	4.57 a	4.80 b	89.3	84.1
5 µL L−1	Beginning and end	2.16	3.31 a	5.34 a	4.95 b	89.6	84.8
5 µL L−1	Beginning + every 30 days	2.49	2.90 a	5.83 a	6.16 a	82.9	83.2
10 µL L−1	Beginning	2.33	2.36 b	5.21 a	5.56 a	89.8	83.5
10 µL L−1	Beginning and end	2.50	2.09 b	5.68 a	4.90 b	89.1	85.0
CV (%)		11.0	12.9	12.2	9.8	5.6	1.9

Means followed by the same letter in the column do not differ by the Scott–Knott test (p < 0.05). ns: not significant by ANOVA.

). The control and all frequencies of NO application at a dose of 5 μL L⁻¹ showed fruits with higher phenolic compound content in the skin compared to treatments with 10 μL L⁻¹ NO, indicating a possible negative effect of higher NO doses. On the other hand, the antioxidant capacity of the flesh, as measured by the ABTS method, was lower in fruits not treated with NO, while in the skin it was lower in untreated fruits, as well as in those treated with 5 μL L⁻¹ NO only at the beginning and beginning + end of the storage period, and 10 μL L⁻¹ NO applied at the beginning and end of CA storage. NO may end up inducing oxidative stress instead of suppressing it. In ‘Micro-Tom’ tomatoes, NO repressed the activity of H₂O₂ scavenging enzymes, contributing to increased ROS and RNS production [30].

Gupta et al. [31] emphasize that NO has an ambiguous effect, acting as an antioxidant at low concentrations and as a pro-oxidant at high doses. In this context, the application of 5 μL L⁻¹ NO, regardless of frequency, seems to have been more effective in increasing the phenolic compound content and antioxidant capacity in the skin and flesh of ‘Fuji Mishima’ apples. However, when 5 μL L⁻¹ NO was applied every 30 days, it may have led to negative effects on fruit decay due to frequent exposure to the gas, again pointing to the negative effect of NO when applied in higher doses or frequencies. In parallel, these fruits showed both higher phenolic compound content and antioxidant capacity, mainly in the skin, the primary barrier against pathogens. Frequent NO application may have caused structural damage to the fruit and compromised the barriers, leading to oxidative stress [21]. This contradictory effect, between higher decay incidence and the antioxidant system, may have been caused as a response to stress induced by frequent NO treatment, which led to an increase in decay occurrence. The metabolic response of the fruit to stress may have led to an increase in the synthesis of antioxidant metabolism compounds as a defense mechanism [31,32]. Despite minor variations in phenolic content and antioxidant activity, NO treatment every 30 days increased decay, indicating that higher application frequency may compromise fruit integrity during long-term storage.

It is well established in the literature that the effect of NO is dependent on the applied dose, exposure time, frequency of application during storage, and the fruit species used [6,8,21]. Although in the present study NO reduced ethylene production and suppressed the climacteric peak during the evaluation period, this did not result into delayed ripening and quality maintenance after storage. Other studies conducted with different cultivars, such as ‘Baigent’ and ‘Cripps Pink’, observed positive results of NO on ethylene production and fruit quality maintenance after storage [6,7]. This differentiated response among different cultivars raises the hypothesis that the effect of NO may not only be species-dependent but also cultivar-dependent. However, different clones of the ‘Gala’ cultivar also showed a differentiated effect to NO application during storage. As mentioned earlier, ‘Baigent’ apples showed positive results with NO application (5 µL L⁻¹) at the beginning and end of storage in CA (1.2 kPa O₂ + 2.0 kPa CO₂).

In ‘Maxi Gala’ apples, the application of NO (10 µL L⁻¹) at the beginning and end of storage did not suppress ethylene production in CA (1.2 kPa O₂ + 2.0 kPa CO₂) and increased phytohormone synthesis in a CA with extremely low O₂ partial pressure (pO₂ varying between 0.1 and 0.7 kPa) and under dynamic controlled-atmosphere conditions (minimum pO₂ of 0.08 kPa; maximum of 0.5 kPa; average of 0.18 kPa) [33]. Furthermore, the authors did not observe a positive effect of NO, regardless of storage conditions, on maintaining fruit quality. These results contrast with each other even when conducted with clones of apples from the same cultivar. Therefore, in addition to raising the hypothesis of a differential effect based on cultivar, it is possible that the effect of NO is dependent on storage conditions, maturity stage, and even the year of production. Future studies considering these aspects need to be conducted for a better understanding of the effects of NO during apple storage. The evaluation period of eight months corresponds to the long-term storage duration used commercially for Fuji group apples under controlled atmosphere conditions. This storage length was intentionally adopted to replicate realistic postharvest scenarios and to assess the persistence of NO effects over the actual marketing period. Shorter storage durations might reveal transient physiological changes, but they would not represent the fruit quality observed under commercial conditions.

Overall, the findings demonstrate that while NO influenced some metabolic processes, it was not effective in maintaining the quality parameters of ‘Fuji Mishima’ apples throughout prolonged controlled-atmosphere storage. These outcomes indicate that NO, under the tested conditions, does not extend storage duration or preserve marketable quality, reinforcing the cultivar’s sensitivity and the need for complementary preservation methods.

4. Conclusions

The application of NO, in the evaluated doses and frequencies and under CA conditions of 1 kPa O₂ + < 0.5 kPa CO₂, 1.0 ± 0.2 °C, and 94 ± 2% RH, did not show a positive effect on maintaining the quality of ‘Fuji Mishima’ apples. The dose of 5 µL L⁻¹ NO, applied every 30 days during storage, despite reducing ethylene synthesis, resulted in more mature fruit and increased the incidence of decay in ‘Fuji Mishima’ apples. The results provide practical insight into the limited benefit of NO application for maintaining the postharvest quality of ‘Fuji’ apples and highlight the importance of adjusting storage strategies according to cultivar-specific responses. However, it is important to recognize that the effectiveness of NO may vary depending on cultivar characteristics, maturity stage at harvest, storage conditions, and gas concentration or exposure frequency. These factors can influence the metabolic and physiological responses of fruit to NO, which may explain the contrasting results reported among different studies and apple cultivars. Future research should focus on optimizing dose and application timing for diverse genotypes to clarify the mechanisms underlying these responses and enhance the practical use of NO in commercial storage systems.