Nitric oxide treatment delays quality deterioration and enzymatic browning of Agaricus bisporus via reactive oxygen metabolism regulation

Abstract

Nitric oxide (NO) is a highly reactive gaseous small molecule that regulates plant growth. Sodium nitroprusside (SNP) was used as an NO donor to study the effects of NO treatment on browning and antioxidant activity of button mushrooms (Agaricus bisporus). The findings revealed that exogenous NO treatment alleviated the browning, decreased hardness, and increased weight loss of mushrooms stored at 4°C for 15 days, and reduced the accumulation of reactive oxygen species and membrane lipid peroxidation. Moreover, exogenous NO treatment dramatically increased the anabolism of endogenous NO, maintained the content of antioxidant substances including flavonoids and total phenols, and kept catalase and superoxide dismutase activities at a high level. Further studies revealed that exogenous NO treatment inhibited the activities of enzymes that are involved in browning, which including polyphenol oxidase (PPO) and tyrosinase (TYR), and increased phenylalanine ammonia-lyase (PAL) activity, with SNP treatment at 0.05 mM being the most effective. Furthermore, SNP treatment at 0.05 mM also inhibited PPO and TYR gene expressions while increasing PAL gene expression. Therefore, our findings suggest that exogenous NO has the potential for development in delaying postharvest browning and maintaining the quality of mushrooms.

INTRODUCTION

The button mushroom (Agaricus bisporus), which is currently one of the most productive mushroom species on the planet (Gholami et al., 2017; Wang et al., 2021a), has high functional ingredients such as protein, amino acids, vitamins, and minerals, and could lower the risk of cancer, improve metabolic syndrome, immune function, and gastrointestinal health (Blumfield et al., 2020). However, because of the extremely high water content and the absence of epidermal protective tissue, harvested button mushrooms show undesirable phenomena such as browning, rotting, softening, opening, and odor, among which browning plays a crucial part in causing the reduction of sensory quality of mushrooms (Qian et al., 2021). After harvesting, the phenolics produced by phenylalanine ammonialyase (PAL) of button mushrooms undergo enzymatic browning by the action of polyphenol oxidase (PPO) and tyrosinase (TYR) to produce a brown-black substance (Shekari et al., 2021; Xu et al., 2022). In addition, respiration during storage of button mushrooms generates reactive oxygen species (ROS), resulting in peroxidation of membrane lipids that disrupt cell membrane integrity and promote exposure to phenolics and enzymes, which in turn aggravate browning occurrence (Chen et al., 2014; Dokhanieh & Aghdam, 2016). Therefore, delaying enzymatic browning and increasing antioxidant activity are effective ways to maintain the storage quality of button mushrooms.

Recently, research has shown that Arginine treatment delays the browning of button mushrooms’ caps by increasing PAL and superoxide dismutase (SOD) enzyme activities (Li et al., 2019). Under high-voltage electrostatic field (HVEF) treatment, the secondary and tertiary structures of PPO are disrupted, causing changes in its properties and loss of enzymatic catalytic activity, which is responsible for the inhibition of enzymatic browning by HVEF (Yan et al., 2020). Ozone fumigation combined with nano-film packaging can reduce the decay rate and weight loss of mushrooms, improve the oxidative stress capacity, and decrease H₂O₂ content and O₂•- production rate to mitigate membrane damage, thus controlling post-harvest browning (Wang et al., 2021a). Besides, ultrasound (Li et al., 2017), UV-C (Lei et al., 2018), plasma-activated water (Zhao et al., 2021), and lemon essential oils (Wang et al., 2021b) have also been shown to inhibit browning in button mushrooms. However, these methods suffer from low safety, high costs, and are challenging to operate, so there are limitations in their practical application.

Nitric Oxide (NO), a freely diffusible membrane permeable signaling molecule, is involved in the growth of fruits and vegetables such as germination, flowering, and senescence (Azizi et al., 2021; Begara-Morales et al., 2018). In plants, arginine is converted to NO by nitric oxide synthase (NOS), and nitrate/nitrite can also be converted to NO by nitrate reductase (NR) (Calabrese & Agathokleous, 2022). Recently, a growing number of reports have shown that changes occurring during the ripening of fruits and vegetables are not only related to ROS and antioxidant metabolism but also to NO (Corpas et al., 2020; Kolbert et al., 2019; Manjunatha et al., 2010). Therefore, the use of exogenous NO to regulate the ripening of fruits and vegetables and its application for preservation has become one of the current research hotspots in postharvest biology. Indeed, 10 μl L⁻¹ NO for 1 h effectively inhibits surface browning in apples (Pristijono et al., 2006). Besides, 10 μl L⁻¹ NO increases the enzymatic activity of the AsA-GSH recycling system and reduces senescence and cold damage in peaches after harvesting (Ma et al., 2019). It has also been shown that sodium nitroprusside (SNP) (donor compound of NO) solution retarded external browning and lignification of bamboo shoots (Yang et al., 2010) and also inhibited enzymatic browning of fresh pistachios (Gheysarbigi et al., 2020). But to our knowledge, limited research has examined the impact of exogenous NO on the storage quality and physiological activity of postharvest button mushrooms.

The purpose of this study was to determine how exogenous NO affected the quality, antioxidant system, browning-related enzymes, and their gene expression during the storage of button mushrooms. This study may provide evidence to support applying NO in the postharvest of button mushrooms.

MATERIALS AND METHODS Treatments

The button mushroom was purchased from Zhejiang Jiashan Ningyuan Agricultural Development Co., Ltd., and transported back to the laboratory quickly under low temperatures. The mushrooms of uniform size, consistent maturity, white color, and no mechanical damage were selected as experimental materials.

The selected mushrooms were randomly divided into four groups and given sodium nitroprusside solution (SNP, donor compound of NO) at the concentration of 0 (control), 0.025, 0.05, and 0.1 mM. Spraying 100 ml of solution per 60 mushrooms. The mushrooms were dried naturally and packed with PE bags of the same thickness (10 pores were evenly left on the surface of the PE bags). All mushrooms were stored at 4°C and 95 ± 5% relative humidity in the dark for 15 days.

Color, firmness, and weight loss

The color of the pileus was determined using a Chroma Meter (CR-400; Konica Minolta, Japan). To record the L*, a*, and b* values, the browning index was calculated using the equation below (Ali et al., 2016): [Image Omitted. See PDF]where x = (a^*+1.75 L^*)/(5.645 L^*+a^*−3.012 b^*).

The firmness was determined with a texture analyzer (TA-XT plus; Lotun Science Co., Ltd., Austria), and all parameters were set as follows: probe preparation speed = 5 mm s⁻¹, determination speed = 1 mm s⁻¹, probe after measurement speed = 2 mm s⁻¹, determination height = 6 mm, and minimum perception = 0.005 kg.

The rate of weight loss of button mushrooms was expressed by weighing mushroom bodies before and after storage, which was calculated according to the following equation: [Image Omitted. See PDF]where m_i is the average value of the weight of mushrooms stored on day i and m₀ is the average value of the initial weight of mushrooms.

Transmission electron microscopy observation

Transmission electron microscopy (TEM) (H7650; Hitachi Co., Ltd, Japan) was used to examine the microstructure of mushrooms by Zhang et al. (2022) with minor modifications. The mushroom tissues smaller than 1 mm³ were taken, fixed with 2.5% glutaraldehyde for more than 2 h, and rinsed with phosphate buffer. The tissues were then fixed with 1% osmium acid for 2–3 h and rinsed with phosphate buffer. The final samples were dehydrated in ethanol, embedded in acetone and stained, and then viewed with TEM.

Malondialdehyde and electrolyte leakage

Malondialdehyde (MDA) content was assessed following the method described by Shang et al. (2021). Mushrooms (0.1 g) were suspended in 100 g L⁻¹ TCA (1 ml) and centrifuged for 20 min at 4°C and 10,000 g. After boiling the supernatant with 0.67% TBA (0.2 ml), absorbance at 450, 532, and 600 nm was recorded after cooling to 25°C. The accumulation of MDA in mushrooms was expressed in nmol g⁻¹.

Based on the method of Wang et al. (2021c), electrolyte leakage was measured. The small pieces of mushroom were placed in distilled water. A conductivity meter was used to determine the electrolyte leakage, which was recorded as P₀. The solutions were left for 10 min and then boiled for 10 min, which were recorded as P₁ and P₂, respectively. The membrane electrolyte leakage was assessed according to the following equation: [Image Omitted. See PDF]

Hydrogen peroxide (H₂O₂) and superoxide anion (O₂•-) generation

The H₂O₂ content and O₂•- generation were measured using the method of Xu et al. (2021) with slight modifications. Mushroom samples (0.3 g) were added into 1.5 ml extraction (containing 0.3% Triton X-100, 1 mM EDTA, and 2% PVPP) and centrifuged for 20 min. The mixture (containing supernatant, 1 mM hydroxylamine hydrochloride, and 50 mM phosphoric acid buffer) was incubated for 1 h at 25°C, and then 7 mM 1-naphthylamine solution and 17 mM p-aminobenzene sulfonic acid solution were added. After incubating at 25°C for 20 min, the absorbance was recorded at 530 nm, indicating O₂•- level.

The mushroom samples (0.1 g) were added into 0.5 ml pre-cooled acetone and centrifuged for 15 min. Afterward, the supernatant was added with 10% titanium tetrachloride-hydrochloric acid and concentrated ammonia, and then centrifuged at 10,000 g. Finally, the residue was rinsed repetitively with ice-cold acetone and dissolved in 2 mol L⁻¹ sulfuric acid. The absorbance was detected at 412 nm.

NO content, inducible nitric oxide synthase activity, and NR activity

The NO content was measured by the method in the instruction of the NO kit (Nanjing Jiancheng Biological Co., Ltd.), in which the absorbance value was recorded at 550 nm. The NO content was indicated as nmol g⁻¹.

The determination of inducible nitric oxide synthase (iNOS) activity was modified as described by Zhang et al. (2011), and the change of absorbance value was detected at 530 nm. The result was expressed as U mg⁻¹. One nanomole NO production per mg histone per minute was an enzyme activity unit.

The NR activity was determined using the approach proposed by Dong et al. (2018). Absorbance of the solution was measured at 540 nm. The result was expressed as U g⁻¹.

Total phenolic and flavonoid

The total phenolic content was carried out by using the method of Louis et al. (2021). Mushroom samples (0.1 g) were placed in 2 ml 60% ethanol and centrifuged at 4°C. The reaction mixture (containing supernatant, folinol, and 12% sodium carbonate) was placed in darkness for 60 min. Absorbance of the solution was recorded at 760 nm.

The flavonoid content was carried out based on Ge et al. (2021). Mushrooms (0.1 g) were placed in 0.5 ml 60% ethanol and centrifuged at 4°C. The supernatant (200 μl) was added to 2 ml 0.05 ml 5% (w/v) Na₂CO₃ liquor and 160 μl 10% (w/v) Al (NO₃)₃ liquor successively and then reacted for 15 min at 25°C. The absorbance value was determined at 510 nm. The canonical plotting was determined as different concentrations of rutin, and the unit of flavonoid was mg g⁻¹.

Superoxide dismutase activity and catalase activity

SOD activity was assessed by the method in the instruction of the SOD kit (Nanjing Jiancheng Biological Co., Ltd.). Based on the inhibitory effect of superoxide dismutase on photoreduction of nitroblue tetrazole (NBT), the inhibition of NBT photochemical reduction to 50% per gram of sample per minute was taken as an SOD activity unit, which was expressed as U g⁻¹.

Catalase (CAT) activity was assessed referring to Valizadeh et al. (2021). The mixed system was composed of 20 mM H₂O₂ solution (2.9 ml) and 100 μl enzyme extracting solution. A 0.01 reduction in the absorbance change value at 240 nm per minute per gram of sample was taken as a CAT activity unit, and the unit was expressed as U g⁻¹.

PPO, TYR, and PAL activities

PPO activity was evaluated based on a method previously suggested by Shekari et al. (2021a). Mushroom samples (0.1 g) were added into 1 ml extraction solution (containing 1% Triton X-100, 1 mM PEG, and 4% PVPP) and centrifuged for 30 min. The mixture contained 50 μl crude enzyme solution, 4 ml acetic acid-sodium acetate (50 mM, pH 5.5), and 1 ml 50 mM catechol. The absorbance was monitored at 420 nm, and the unit was expressed as U mg⁻¹.

TYR activity was evaluated according to the instruction manual of the TYR kit (Beijing Solexpro Technology Co., Ltd.), and the absorbance was monitored at 475 nm. The unit was recorded as U g⁻¹.

PAL activity adopted the method with reference to Shekari et al. (2021b) with a slight modification. Mushroom samples (0.1 g) were added into 1 ml extraction buffer (containing 2 mM EDTA, 40 g L⁻¹ PVPP, and 5 mM β-mercaptoethanol) and centrifuged for 30 min. The mixture contained 3 ml boric acid (50 mM, pH 8.8) and 50 μl supernatant. The change of absorbance was evaluated at 280 nm, and PAL activity was shown as U g⁻¹.

Total RNA extraction, cDNA synthesis, and real-time quantitative PCR

Total RNA was extracted from mushrooms by HiPure Fungal RNA Kits (Magen Biotech Co., China). The cDNA was produced using HiScrip®II Q RT SuperMix for qPCR (+gDNA WIper) (Vazyme Biotech Co., Shanghai, China). The real-time quantitative PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co.). Primers were designed using Primer3 plus software (listed in Table 1) with 18S rRNA as an endogenous gene (Weijn et al., 2013). The expression of each gene was statistically analyzed using the 2^−ΔΔCT method.

TABLE 1 Gene-specific primers used for gene expression analysis in Agaricus bisporus by real-time quantitative PCR (RT-qPCR)

Gene	Gene ID	Encoded proteins	Primer sequence (5′ to 3′)
PPO	18082695	Polyphenoloxidase	F-ACGACTTGGCCTCATTTCAG
			R-GTTACCACCGACGAGGACAT
TYR	18081838	Tyrosinase	F-CGCTCAGATCAACGACGAGA
			R-CCGAGCAGTATCAGCGAGAG
PAL	18082277	Phenylalanine ammonialyase	F-GCTCGGTTATTTAGCGAATCC
			R-TTTGTCGGCCTGAGATCAAAG
18S rRNA	AY787216	18S rRNA	F-TGCTCTGCTGGGTCTTACCT
			R-CGGCGACTCTAGAAACCAAC

Statistical analysis

Each experiment was repeated three times. All data were analyzed by one-way analysis of variance using SPSS (version 25), and significance analysis was performed using Duncan's test with a significance level of p < .05. The correlation analysis was illustrated using Pearson correlation. Origin software (version 2021) was used for mapping.

RESULTS Appearance quality

As shown in Figure 1a, after 6 days of storage, SNP treatments dramatically reduced the browning of the mushroom surface compared to the control. The L* was consistently greater in the treatments (0.025 mM and 0.05 mM SNP) than in the control after 6 days (p < .05), and from 9th day forward, 0.1 mM SNP treatment maintained a larger L* (Figure 1b). In addition, the browning index (BI) showed an increasing trend during 15 days of storage at 4°C, and SNP treatments significantly decreased the BI from 6th day (p < .05). On the last day of storage, the BI of 0.05 mM SNP treated was 24.86, which was 19% lower than that of the control (Figure 1c), and the differences between the 0.025 mM and 0.1 mM SNP treatments were not significant (p > .05). Throughout the storage, firmness showed a decreasing trend, and SNP treatments maintained a slower downward trend (Figure 1e), where firmness in mushrooms treated with SNP at 0.05 mM was noticeably larger than that of the control (p < .05). Besides, firmness of 0.05 mM SNP treatment was 2.77 N, 18.9% higher than that of the control, and 0.025 mM and 0.1 mM SNP treatments were 10.8% and 12.9% higher than that of the control at 15th day of storage, respectively. In contrast to the change in firmness, the weight loss rate increased with prolonged storage period (Figure 1d), and the SNP treatments suppressed the weight loss rate after 6 days of storage, but the differences among the three groups were not significant (p > .05).

FIGURE 1. Effects of nitric oxide (NO) treatment on the appearance (a), L* (b), browning index (c), weight loss (d), and (e) in Agaricus bisporus. Error bars show the sample standard deviations of means.

Cell ultrastructure

The cell membrane of fresh mushroom was intact, the cytoplasm was full and evenly distributed, and there was a clear boundary between the cytoplasm and cell membrane. At 15th day of storage, the cell membrane of the control was destroyed, a large number of vacuole structures appeared, and the boundary between the cytoplasm and cell membrane was blurred, while the SNP treatment maintained a more intact cell membrane, fuller cytoplasm, fewer vacuole structures, and apparent structural partitioning (Figure 2a).

FIGURE 2. Effects of nitric oxide (NO) treatment on the ultrastructure (a), malondialdehyde (MDA) content (b), electrolyte leakage (c), hydrogen peroxide (H2O2) content (d), and superoxide anion (O2•-) generation (e) in Agaricus bisporus. (a1 and b1) Fresh mushroom tissue at day 0; (a2) mushroom tissue without treatment (control) at day 15; (b2) mushroom tissue with NO treatment at day 15. Image magnification is 7000 (bar = 2 μm). Error bars show the sample standard deviations of means. Abbreviations: CW, cell wall; M, mitochondria; V, vacuole

MDA, electrolyte leakage, and ROS

As shown in Figure 2b,c, the MDA content and electrolyte leakage showed an overall increasing trend during storage, and the SNP treatment delayed the increasing trend. Compared to the control, 0.025 mM, 0.05 mM, and 0.1 mM SNP treatments decreased by 25%, 34%, and 15%, respectively. The electrolyte leakage was significantly lower in 0.05 mM-treated mushrooms (p < .05) and only increased by 8.63% throughout the storage time.

The accumulation of ROS is an essential factor contributing to membrane damage. As indicated in Figure 2d,e, the H₂O₂ content and O₂•- generation rate first decreased rapidly and then increased slowly. The H₂O₂ content in the control increased by 8.6% compared with that before storage until 15th day. In comparison, it increased by only 3.2% in the 0.1 mM SNP treatment and decreased by 7.8% and 3.6% in the 0.025 mM and 0.05 mM SNP treatments, and both treatments were remarkably lower than the control (p < .05). The O₂•- generation rate in the control increased by 7.9% compared with that before storage. In comparison, it increased only by 4.6% in the 0.1 mM SNP-treated mushrooms and decreased by 2.3% and 5.6% in the 0.025 mM and 0.05 mM SNP-treated mushrooms, and it was insignificant between the two treatments (p > .05).

NO content, iNOS activity, and NR activity

As shown in Figure 3a, the accumulation of NO had a decreasing trend at the beginning of storage. The NO content of mushrooms treated with 0.05 mM SNP was considerably higher than that of the control from 3 to 15 days (p < .05), then from 9 to 15 days, NO content of the 0.1 mM SNP-treated mushrooms also increased gradually (p < .05). At the same time, there was no remarkable difference between the control and the 0.025 mM SNP-treated mushrooms (p > .05).

FIGURE 3. Effects of nitric oxide (NO) treatment on NO content (a), inducible nitric oxide synthase (iNOS) activity (b), and nitrate reductase (NR) activity (c) in Agaricus bisporus. Error bars show the sample standard deviations of means.

iNOS and NR are the two enzymes that catalyze NO production in mushrooms. Similar to the trend of NO content, iNOS activity showed a decreasing and then increasing trend (Figure 3b). At 15th day of storage, iNOS activity in the control decreased by 3.2% compared with that before storage. In contrast, iNOS activity in SNP treatments increased significantly (p < .05); the 0.025 mM, 0.05 mM, and 0.1 mM SNP treatments increased by 16.5%, 31.6%, and 22.2%, respectively. NR activity continued to increase during storage (Figure 3c). At 6–15 days of storage, NR activity in the 0.05 mM SNP-treated mushrooms was remarkably higher than the other treatments (p < .05).

Antioxidative system

The browning of mushrooms is closely related to the antioxidative system in which some substances play an essential role. The variations in the total phenolic content are shown in Figure 4a, and it decreased during storage. On the last day of the storage, total phenolic content of the control was 0.38 mg g⁻¹, which decreased by 72.5% compared with that before storage. In contrast, the 0.05 mM and 0.1 mM SNP treatments decreased gradually, only 23.2% and 28.3% at 15th day. The total phenolic content of mushrooms treated with SNP was remarkably higher than that of the control until 15th day of storage (p < .05). As shown in Figure 4b, SNP treatment promoted the rise in flavonoid content, with the 0.025 mM and 0.1 mM SNP treatments maintaining higher flavonoid content after 12 days, the flavonoid content of mushrooms with the 0.05 mM SNP treatment was remarkably higher than the control after 9 days (p < .05).

FIGURE 4. Effects of nitric oxide (NO) treatment on total phenolic content (a), flavonoid content (b), superoxide dismutase (SOD) activity (c), and catalase (CAT) activity (d) in Agaricus bisporus. Error bars show the sample standard deviations of means.

In addition to these substances, antioxidant enzymes could also inhibit the degree of browning of mushrooms by mitigating oxidative damage. The change in SOD activity is shown in Figure 4c. During 15 days of storage, SOD activity of all samples reduced in general. The 0.025, 0.05 mM, and 0.1 mM SNP treatments decreased by 20%, 17%, and 18.7%, respectively, and the 0.05 mM and 0.1 mM SNP treatments were remarkably higher than the control (p < .05). As shown in Figure 4d, SNP treatment increased CAT activity to different degrees, with a gentle decreasing trend in CAT activity in the early storage period and a rapid decrease in CAT activity after 6 days of storage. At 15th day of storage, CAT activities of mushrooms treated with 0.025 mM, 0.05 mM, and 0.1 mM SNP were 5.2%, 62.4%, and 46.8%, respectively, higher than those of the control. Moreover, the 0.05 mM and 0.1 mM SNP treatments were remarkably more active than the control (p < .05), while there was a statistically insignificant difference between the other two groups (p > .05).

Browning-related enzymes and gene expression

PPO and TYR are the key enzymatic browning enzymes in mushrooms. PPO activity increased with increasing storage period (Figure 5a). Compared to the control, SNP treatment exerted a highly significant inhibitory effect on PPO activity (p < .01).

FIGURE 5. Effects of nitric oxide (NO) treatment on polyphenol oxidase (PPO) activity (a), tyrosinase (TYR) activity (c) and phenylalanine ammonialyase (PAL) activity (e) and the relative expression of PPO (b), TYR (d) and PAL (f) gene in Agaricus bisporus. Error bars show the sample standard deviations of means. The Student's t-test was used to establish statistical significance (**p [less than] .01; *p [less than] .05).

TYR activity also showed an increasing trend (Figure 5c); within 15 days of storage, TYR activity severally decreased by 31.3%, 42.9%, and 33.3% in the 0.025 mM, 0.05 mM, and 0.1 mM SNP-treated mushrooms, respectively, compared with the control. In particular, the 0.05 mM SNP treatment remarkably inhibited TYR activity until 15 days of storage (p < .01).

The different concentrations of SNP treatments promoted the increase of PAL activity (Figure 5e). After 3 days of storage, PAL activity of 0.05 mM and 0.1 mM SNP-treated mushrooms increased rapidly (p < .01). After 9 days of storage, the PAL activity of 0.025 mM SNP-treated mushrooms dramatically larger than that of the control (p < .01). At 15 day of storage, the PAL activities of the 0.025 mM, 0.05 mM, and 0.1 mM SNP treatments were 39.9%, 73.7%, and 48.9%, respectively, higher than those of the control.

Since the 0.05 mM SNP treatment showed better storage result of mushrooms and maintained the most significant inhibition of browning-related enzyme activity, its browning-related gene expressions were deeper investigated (Figure 5b,d,f). Unlike the trend of enzyme activity, the expression of genes did not show constant changes with the storage period being extended. However, in the late storage period, PPO and TYR gene expressions were remarkably lower (p < .05). Compared to the control, PAL gene expression was remarkably higher (p < .01) in the SNP-treated mushrooms. The PPO gene expression with SNP-treated decreased sharply at 3rd day and continued to increase after 9 days of storge (p < .05). Additionally, SNP could markedly downregulate the TYR gene expression except at 6th day (p < .05). The PAL gene expression in control increased in the early stage, reached a peak at 3rd day, and then showed a slow increase, but overall showed a lower level. The SNP treatment substantially upregulated PAL gene expression (p < .01).

Correlation analysis

As shown in Figure 6a,b, there were significantly positive or negative correlations between most of the several indicators in the SNP treatments and control of mushrooms (p < .05). In control, BI showed a strong positive correlation with weight loss, MDA content, electrolyte leakage, PPO activity, TYR activity, and PAL activity (p < .05), and a strong negative correlation with L*, total phenols content, and antioxidant enzymes (SOD and CAT) activities (p < .05). In addition, BI of mushrooms treated with SNP showed a strong positive correlation with weight loss, electrolyte leakage, iNOS activity, NR activity, PPO activity, TYR activity, and PAL activity, and a strong negative correlation with L* and SOD activity (p < .05).

FIGURE 6. Analysis of the correlation of all the indexes in the control group (a) and nitric oxide (NO) treatment group (b). Positive effects are shown in red, while negative effects are shown in blue (p [less than] .05).

DISCUSSION

After harvesting, button mushroom is highly susceptible to browning, wilting, rotting, and other deteriorations that lower its commercial value and storage quality. NO, an essential redox signaling molecule in plants, has been widely studied for its application in fruit and vegetable preservation (Azizi et al., 2021). In this study, we found that mushrooms stored for 15 days at 4°C showed different degrees of quality deterioration in terms of browning degree, weight loss rate, and hardness. The exogenous NO-treated mushrooms showed a reduced browning index and maintained a lower weight loss rate and higher hardness. Exogenous NO has also been shown to inhibit browning, maintain hardness, and minimize weight loss in dates and grapes (Zhang et al., 2019; Zhao et al., 2020).

Membrane lipid peroxidation is the main cause of postharvest quality deterioration of button mushrooms (Jolivet et al., 1998). The attack of ROS accelerates the lipid oxidation of mushrooms membranes. It causes damage to the cell membrane, manifested by higher MDA content and cell membrane permeability, leading to a weakened enzyme and substrate segregation distribution, thus inducing the browning of mushrooms (Lei et al., 2018; Lin & Sun, 2019). In this study, MDA content and electrolyte leakage were maintained at low levels, and cell membranes remained more intact in NO-treated mushrooms because exogenous NO treatment reduced ROS accumulation. This finding is consistent with the research by Yang et al. (2011) and Li et al. (2014).

The antioxidant mechanism in button mushrooms can defend against the attack of ROS, either through enzymatic or nonenzymatic systems, and reduce the oxidative damage of cell membranes (Liu et al., 2023). The enzymatic antioxidant system is the main method of scavenging ROS, SOD could scavenge O₂•- and generate H₂O₂, CAT could decompose H₂O₂, which plays an essential role in oxidative stress. We found that the antioxidant enzyme activities of exogenous NO-treated mushrooms were maintained at a high level. Additionally, the same results were reported in bananas (Wu et al., 2014), cantaloupe (Zhang et al., 2017), dates (Zhao et al., 2020), and grapes (Zhang et al., 2019). PAL is a crucial enzyme for plant phenolic metabolism, which encourages the buildup of flavonoids and total phenols and are critical antioxidant substances that accelerate the removal of ROS and delay the epidermal browning of vegetables and fruits. In this study, it is found that PAL gene expression was higher in mushrooms treated with exogenous NO, which kept the PAL enzyme activity at a high level and thus resulted in a large accumulation of total phenols and flavonoids. Therefore, this observation could be one of the reasons for the decrease in H₂O₂ content and O₂•- production rate. Furthermore, NO treatment also enhanced PAL activity and maintained a high phenolic and flavonoid content in kiwifruit and Pitaya, as confirmed by other studies (Hu et al., 2019; Zheng et al., 2017).

In addition, endogenous NO plays a supporting role in the ROS scavenging system, where NO undergoes autoxidation and catalyzes the generation of a strong oxidant, ONOO-, from O₂•-, which disrupts the lipid peroxidation process (Simontacchi et al., 2013). In this study, SNP promoted endogenous NO production and increased NO synthase activity. In contrast, another study showed that the addition of the NO inhibitor L-NAME caused a considerable drop in the amount of NO produced by the plants (Ziogas et al., 2013).

After harvesting of button mushrooms, the phenolic substances undergo enzymatic browning, generating brown-black substances. The enzymes that cause browning in mushrooms are mainly PPO and TYR, where PPO is a substrate that catalyzes the formation of brown quinones from bisabolol. At the same time, TYR is an enzyme with a dual function that catalyzes the hydroxylation reaction of monophenols to form bisabolol and then oxidizes bisabolol to form levodopa and melanin, so forth. In our study, the content of phenolic substrates was increased due to the rise in PAL activity, but the expression of PPO and TYR genes was lower in exogenous NO-treated mushrooms, and the low expression of PPO and TYR genes would cause their enzymatic activity to decrease correspondingly, thus delaying the enzymatic browning process. Other studies have demonstrated that exogenous NO has a specific inhibitory effect on PPO activity, thus alleviating the epidermal browning of vegetables and fruits (Gheysarbigi et al., 2020; Yang et al., 2010).

CONCLUSION

Our results reveal the effectiveness of exogenous NO treatment in slowing postharvest browning and maintaining the antioxidant capacity of mushrooms (Figure 7). Exogenous NO could prevent mushrooms from browning, and maintain high hardness and low weight loss, by reducing the gene expression of browning-related enzymes and thus reducing enzyme activity to achieve the purpose of delaying browning. Notably, exogenous NO treatment also improved the scavenging rate of free radicals by increasing the content of antioxidant substances and the activity of antioxidant enzymes, thus maintaining a more intact cell membrane and reducing the degree of membrane lipid damage. In summary, NO can potentially be a practical method to maintain the quality of button mushrooms and extend their shelf life.

FIGURE 7. Schematic diagram of nitric oxide (NO) treatment delaying browning of Agaricus bisporus by regulating reactive oxygen metabolism

ACKNOWLEDGMENT

This work was supported by the National Natural Science Foundation of China (Grant No. 32072294) and Cooperation on Agricultural Science and Technology of Zhejiang Agriculture and Rural Affairs (Grant No. 2022SNJF045).

CONFLICT OF INTEREST

The authors confirm that they have no conflict of interest to declare for this publication.

ETHICS STATEMENT

None declared