Nitric Oxide Modulates Postharvest Physiology to Maintain Abelmoschus esculentus Quality Under Cold Storage

Abstract

Cold storage is widely used for the postharvest preservation of fruits and vegetables; however, okra, as a tropical vegetable, is susceptible to chilling injury under low-temperature storage conditions, leading to quality deterioration, reduced nutritional value, and significant economic losses. Nitric oxide (NO), as an important signaling molecule, plays a crucial role in the postharvest preservation of fruits and vegetables. To investigate the effects of different concentrations of nitric oxide on the postharvest quality of okra under cold storage, fresh okra pods were treated with sodium nitroprusside (SNP), a commonly used NO donor, at concentrations of 0 (control), 0.5 (T1), 1.0 (T2), 1.5 (T3), and 2.0 mmol·L⁻¹ (T4). The results showed that low-concentration NO treatment (T1) significantly reduced weight loss, improved texture attributes including hardness, springiness, chewiness, resilience, and cohesiveness, and suppressed the increase in adhesiveness. T1 treatment also effectively inhibited excessive accumulation of cellulose and lignin, thereby maintaining tissue palatability and structural integrity. Additionally, T1 significantly delayed chlorophyll degradation, preserved higher levels of soluble sugars and proteins, and enhanced the activities of key antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), contributing to improved oxidative stress resistance and membrane stability. In contrast, high-concentration NO treatments (T3 and T4) led to pronounced quality deterioration, characterized by accelerated membrane lipid peroxidation as evidenced by increased malondialdehyde (MDA) content and relative conductivity, and impaired antioxidant defense, resulting in rapid texture degradation, chlorophyll loss, nutrient depletion, and oxidative damage. These findings provide theoretical insights and practical guidance for the precise application of NO in extending shelf life and maintaining the postharvest quality of okra fruits.

1. Introduction

Okra (Abelmoschus esculentus L.), an annual herbaceous plant of the Malvaceae family, first grew in tropical climates. Having been transported to Southern China via India, it has been widely cultivated throughout provinces, including Guangxi, Hainan, Taiwan, and Fujian [1,2]. The immature pods—the major edible component—are rich in dietary fiber, proteins, pectin, flavonoids, calcium, and vitamin C. Because of its nutritional and therapeutic properties, okra pods are becoming a more well-known functional vegetable with great health-promoting power [3,4]. Directly connected to its economic worth is the postharvest pod quality of okra, which influences the quality of associated processed goods. China’s harvest season runs from June to September, at which time the temperature increases greatly, compounding the physiological deterioration that follows harvest. Fast moisture loss, high respiration rates, and mechanical damage incurred during harvesting and transportation produce accelerating senescence and increased sensitivity to microbial deterioration. Typical symptoms in storage and distribution include pod dehydration, surface browning, tissue softness and degradation, and the loss of both nutritional and visual quality [5,6]. Low-temperature storage is being employed more and more (usually at 4 °C) to slow down such degradation by controlling metabolic activity and rotting [7]. On the other hand, okra is very sensitive to cold; hence, prolonged low-temperature exposure may produce symptoms like membrane disruption, decreased cellular activity, and accelerated senescence [8]. These metabolic issues affect not only postharvest quality but also limit the effectiveness of cold storage as a preservation technique. Given increasing consumer demand for premium fresh products and the increasing economic value of okra farming, effective postharvest treatments are thus urgently needed to limit cold-induced damage and maintain storage quality.

Recent developments in postharvest biology have shown how well exogenous therapies might increase the storability of horticulture crops. Glycine betaine (GB), for example, has been shown to improve cold tolerance in peach (Prunus persica L.) by encouraging membrane fatty acid production and thereby maintaining membrane integrity [9]. The analogous preservation of postharvest quality of chili pepper (Capsicum annuum L.) during cold storage has been shown by exogenous melatonin (MT [10]). Among many exogenous regulators, nitric oxide (NO)—a tiny, gaseous signaling molecule—has become more important in postharvest preservation, especially for the modulation of plant stress responses. Usually administered by fumigation or immersion in NO-donor solutions, NO has been shown to be successful in lowering oxidative stress, thereby prolonging the shelf life of many fruits and vegetables and preventing chilling harm. Sodium nitroprusside (SNP) is one of the most widely used NO donors and has been extensively applied to explore NO-mediated physiological processes in plants. Recent research has underscored the diverse roles of NO in regulating postharvest physiology and prolonging the shelf life of various horticultural commodities. For instance, the exogenous application of SNP has been shown to enhance disease resistance and mitigate fruit softening in bitter melon by upregulating the expression and activity of key enzymes in the phenylpropanoid biosynthetic pathway [11]. In bananas, NO treatment has been shown to inhibit ripening by delaying chlorophyll degradation and reducing the production rates of ethylene and CO₂ during storage [12]. Moreover, in peaches, exogenous NO improves antioxidant capacity and enhances cold tolerance, thereby mitigating chilling injury [13,14]. Similar effects have been observed in mangoes, where NO application under cold storage conditions delays fruit ripening and helps maintain commercial quality for a longer period [15,16]. In pepper fruits, NO modulates H₂O₂ levels via differential regulation of ascorbate peroxidase (APX) isozymes, which in turn influences both shelf life and nutritional quality [17]. Additionally, exogenous NO has shown great promise in reducing postharvest browning and preserving the quality of mushrooms [18,19]. The protective effects of NO in postharvest preservation are largely attributed to its ability to enhance the antioxidant defense system. This includes the upregulation of key enzymes such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and phenylalanine ammonia-lyase (PAL), which collectively mitigate oxidative stress, preserve cellular homeostasis, and reduce membrane lipid peroxidation under low-temperature storage conditions [20,21].

To sum up, NO treatment combined with low-temperature storage has proven to be an effective strategy for preserving the postharvest quality of fruits and vegetables. However, the specific effects of NO on okra during cold storage remain insufficiently explored. To address this gap, the present study aims to systematically investigate the effects of exogenous SNP at varying concentrations on the postharvest quality of okra stored at 4 ± 0.5 °C. Key quality-related parameters were examined, including weight loss, texture properties, chlorophyll content, nutritional composition (soluble sugars and proteins), and the activities of antioxidant enzymes (SOD, POD, and CAT). By elucidating the concentration-dependent physiological and biochemical responses of okra to NO treatment, this study provides both theoretical insights and practical guidance for the precise application of NO in the postharvest preservation of okra.

2. Materials and Methods

2.1. Plant Materials and Experimental Treatments

The experiment was conducted using the “Lvba” cultivar of okra (Abelmoschus esculentus L.), grown at the Excellence Training Base for Agricultural and Forestry Talents of Kaili University (26°31′14″ N, 107°53′24″ E, altitude: 685 m), located in Kaili City, Guizhou Province, China. The region experiences a subtropical humid monsoon climate, with average temperatures ranging from 21.9 °C during the growing season (March to October), annual extremes between 4 °C and 36 °C, and summer rainfall between 400 and 700 mm. The experimental field soil is classified as yellow loam with a pH of approximately 6.2 and moderate fertility. Fresh okra pods were harvested on the 5th day after anthesis, ensuring consistent developmental stage. Uniformly sized and mature pods free from visible mechanical damage, disease, or insect infestation were selected. For each treatment, 15 pods were placed into black polyvinyl chloride (PVC) bags (thickness: 0.03 mm), with three replicates per treatment. There were five treatment groups: control—distilled water (ddH₂O); T1—0.5 mmol·L⁻¹ sodium nitroprusside (SNP, a nitric oxide donor); T2—1.0 mmol·L⁻¹ SNP; T3—1.5 mmol·L⁻¹ SNP; T4—2.0 mmol·L⁻¹ SNP. Sodium nitroprusside (SNP) was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Pods were immersed in their respective treatment solutions for 2 min and then gently blotted dry with paper towels to remove surface moisture. All treated samples were stored at 4 ± 0.5 °C to simulate cold storage conditions. Sampling for physiological and biochemical analyses was conducted on days 0, 3, 6, and 9 of storage.

2.2. Weight Loss Measurement

Weight loss was determined using an electronic analytical balance with a precision of 0.01 g. For each treatment, the fresh weight of okra pods was recorded at the beginning of storage (denoted as W₀) and after 3, 6, and 9 days of cold storage (denoted as W_t). The percentage of weight loss was calculated using the following formula:

$$\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{L}\mathrm{o}\mathrm{s}\mathrm{s}\left(\mathrm{\%}\right)=({\mathrm{W}}_{\mathrm{t}}-{\mathrm{W}}_0)/{\mathrm{W}}_0\times 100$$

where W₀ represents the initial weight (g), and W_t represents the weight after t days of storage.

2.3. Texture Quality Assessment

The texture quality of okra pods was evaluated using a texture analyzer (TA.XT Plus, Stable Micro Systems Ltd., Surrey, Godalming, UK). Six parameters—hardness, springiness, chewiness, adhesiveness, cohesiveness, and resilience—were assessed to comprehensively reflect textural characteristics. Each okra pod was longitudinally sectioned into three equal segments (apical, median, and basal), and 5 mm thick samples were collected from each portion. The samples were positioned on the analyzer’s platform and tested using a cylindrical probe (P/75, 75 mm diameter). The test conditions were set as follows: a pre-test speed of 5.0 mm/s, test speed of 2.0 mm/s, post-test speed of 2.0 mm/s, deformation distance of 25% of sample height, trigger force of 5 g, and two consecutive compression cycles with a 5 s interval. The texture profile analysis (TPA) mode was applied, and parameters were automatically calculated based on force–time curves using the Texture Exponent 32 software. For each treatment, five pods were randomly selected, and measurements were performed in triplicate. Mean values were used for statistical analysis.

2.4. Determination of Cellulose and Lignin Contents

Cellulose content was determined following the method described by Ren [22], while lignin content was measured according to the protocol reported by Cai [23].

2.5. Determination of Chlorophyll Content

The contents of chlorophyll a, chlorophyll b, and total chlorophyll in the okra pod epidermis were determined based on the method described by Kotiková et al. [24], with slight modifications. Specifically, 0.25 g of okra pod tissue was homogenized with 15 mL of 100% acetone and extracted in the dark at 4 °C for 48 h. The absorbance (A) of the supernatant was then measured at wavelengths of 662 nm, 645 nm, and 470 nm using a spectrophotometer. Chlorophyll concentrations were calculated using the following equations:

Chlorophyll a (mg/g FW) = 11.75 × A₆₆₂ − 2.35 × A₆₄₅

Chlorophyll b (mg/g FW) = 18.61 × A₆₄₅ − 3.96 × A₆₆₂

Total chlorophyll (mg/g FW) = Chlorophyll a + Chlorophyll b

All extraction and measurement procedures were conducted under dim light to minimize chlorophyll degradation.

2.6. Determination of Soluble Protein Content

The soluble protein content in okra pods was determined using the Coomassie Brilliant Blue G-250 staining method, as described by Qi et al. [21], with bovine serum albumin (BSA) as the standard. Briefly, a series of BSA standard solutions were prepared and mixed with distilled water and Coomassie Brilliant Blue G-250 reagent. After incubation for 5 min at room temperature, the absorbance was measured at 595 nm (A₅₉₅), and a standard curve was generated. For sample analysis, 0.2 mL of okra pod extract was mixed with the dye reagent, incubated for 2 min, and the absorbance was recorded at 595 nm. The soluble protein concentration in each sample was calculated based on the standard curve.

2.7. Determination of Soluble Sugar Content

The soluble sugar content was measured following the method outlined by Li et al. [25], with slight modifications. Glucose was used to prepare the standard solutions. Approximately 100 mg of okra pod tissue was homogenized with 1 mL of 80% ethanol and incubated in a water bath at 80 °C for 30 min. The homogenate was centrifuged at 2600× g for 10 min, and the supernatant was collected. The extraction was repeated once, and the supernatants were combined. A 20 μL aliquot of the pooled extract was added to a 96-well polystyrene microplate and incubated at 4 °C for 15 min. Then, 100 μL of anthrone reagent (0.2% anthrone dissolved in 72% sulfuric acid) was added to each well. After vortexing, the mixture was incubated in a 92 °C water bath for 3 min, cooled at room temperature for 5 min, and further incubated at 45 °C for 15 min. The absorbance was measured at 630 nm (A₆₃₀), and soluble sugar concentration was determined using the standard curve.

2.8. Determination of MDA Content and Relative Conductivity

Fresh okra samples (1 g) were accurately weighed and homogenized with 2 mL of 10% trichloroacetic acid (TCA) and quartz sand. The mixture was ground into a homogeneous paste, followed by the addition of 8 mL of 10% TCA and further grinding. The homogenate was centrifuged at 4000 r/min for 10 min, and the supernatant was collected as the MDA extract. An aliquot of 2 mL MDA extract was transferred to a 10 mL stoppered test tube, with distilled water serving as the control. Subsequently, 2 mL of 0.6% thiobarbituric acid (TBA) solution was added to each tube. The mixture was shaken thoroughly and incubated in a boiling water bath for 15 min, followed by rapid cooling and centrifugation. The supernatant was collected, and the absorbance was measured at wavelengths of 532, 600, and 450 nm using a spectrophotometer (UV-1900i, Shimadzu Corporation, Kyoto, Japan).

For relative conductivity measurement, okra peel samples were punched into uniform circular discs using a blade puncher (Huaou, Jiangsu, China). Ten discs were placed in a conical flask with 30 mL of distilled water and shaken at constant speed on a shaker for 30 min. The initial conductivity (P₁) was then measured. Subsequently, the flask was heated in a boiling water bath for 10 min, cooled to room temperature, and the final conductivity (P₂) was measured. The measurement was repeated three times, and the average value was calculated. The relative conductivity was calculated using the following formula:

$$\mathrm{r}\mathrm{e}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}(\mathrm{\%})={\displaystyle \frac{{\mathrm{P}}_1-{\mathrm{P}}_0}{{\mathrm{P}}_2-{\mathrm{P}}_0}}\times 100$$

where P₁ represents the initial conductivity, P₂ represents the final conductivity after heating, and P₀ represents the conductivity of distilled water.

2.9. Determination of Antioxidant Enzyme Activities

The activities of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), were determined using commercial assay kits (Suzhou Keming Biotechnology Co., Ltd., Suzhou, China), following the manufacturer’s instructions.

2.10. Data Analysis

The mean ± standard error (SE) is used to represent all experimental results. Microsoft Excel 2019 was used to process the data. SPSS 19.0 software was used to perform a one-way analysis of variance (ANOVA), and Duncan’s multiple range test was used to identify significant differences between treatments at a significance level of p < 0.05. Software called Origin 2021 was used to create the graphs. At least three biological replicates were used in every experiment to guarantee the precision and dependability of the findings.

3. Results

3.1. Effects of Different Concentrations of NO on the Weight Loss Rate of Okra Under Low-Temperature Storage

One important physiological metric for assessing moisture loss and fruit and vegetable shelf-life quality during storage is weight loss. Greater water loss and decreased commercial quality are usually reflected in higher weight loss. Over the course of the storage period, the weight loss rate of okra rose steadily in all treatment groups, as seen in Figure 1. Weight loss for the whole storage period was greatly inhibited by treatment with 0.5 mmol·L⁻¹ sodium nitroprusside (SNP), denoted as T1, in comparison to the control, suggesting a strong water-retention effect. Only on days 3 and 6 did the 1.0 mmol·L⁻¹ SNP treatment (T2) result in a substantial decrease in weight loss; nevertheless, by day 9, this group’s weight loss was noticeably more than the control’s. On the other hand, during the course of the storage period, weight loss was continuously considerably larger for higher concentrations of SNP (T3 and T4) than for the control. Notably, by day 9, the T4 group had lost 34.68% of their body weight, indicating that okra pod water loss may be accelerated by heavy NO application.

3.2. Effects of Different NO Concentrations on the Texture Profile Analysis (TPA) of Okra Fruit Under Low-Temperature Storage

Figure 2 illustrates how the texture profile analysis (TPA) characteristics of okra, including cohesiveness, resilience, chewiness, hardness, and springiness, gradually decreased while adhesiveness rose during the course of cold storage. These patterns suggest that okra pods’ textural quality is negatively impacted by cold stress. Increased hardness, springiness, chewiness, resilience, and cohesiveness were among the texture-related measures that were markedly enhanced by treatment with 0.5 mmol·L⁻¹ sodium nitroprusside (T1) in comparison to the control. Furthermore, T1 treatment demonstrated excellent texture retention under cold storage conditions by successfully suppressing the rise in adhesiveness. However, the decline in fruit texture quality, which was shown by hardness, springiness, and chewiness, was made worse by higher-concentration NO treatment (T2, T3, and T4). To differing degrees, the cohesiveness and resilience decline. With longer storage times, the adhesiveness of the T3 and T4 treatments increased gradually and was significantly higher than that of the control.

3.3. Effects of Different NO Concentrations on Lignin and Cellulose Contents of Okra Fruit Under Low-Temperature Storage

Important structural elements of plant cell walls, lignin, and cellulose play a crucial role in the lignification and fiber buildup of fruits and vegetables. All treatment groups’ lignin and cellulose concentrations gradually rose over storage, as shown in Figure 3. Treatment with 0.5 mmol·L⁻¹ SNP (T1) considerably decreased the buildup of lignin and cellulose on days 3, 6, and 9 of storage compared to the control group (control). In particular, the content of cellulose decreased by 15.3%, 18.9%, and 18.3%, while the amount of lignin decreased by 11.3%, 14.7%, and 10.8%. Additionally, the T2 treatment dramatically lowered the amount of lignin on days three and six, as well as the amount of cellulose for the whole storage period. But when the NO concentration increased further, treatments T3 and T4 dramatically increased the amounts of lignin and cellulose throughout the course of the storage period when compared to the control.

3.4. Effects of Different NO Concentrations on Chlorophyll Content in Okra Fruit During Low-Temperature Storage

One of the key physiological markers used to evaluate fresh vegetable storage quality is the chlorophyll concentration. Usually, it reflects the degree of nutrition and preservation, as well as variations in fruits and vegetables’ quality of appearance throughout storage. As shown in Figure 4, the concentrations of chlorophyll a, chlorophyll b, and total chlorophyll progressively dropped with the storage period, suggesting that under refrigeration, okra had varied degrees of chlorophyll degradation. The 0.5 mmol·L⁻¹ SNP (T1) treatment notably slowed down the breakdown of chlorophyll a and b compared to the control. Showing excellent chlorophyll preservation, the chlorophyll concentration across the storage procedure was much greater than that of the control group. Although it exhibited a significantly reduced impact compared to the T1 treatment, the 1.0 mmol·L⁻¹ SNP (T2) treatment also had a slight green retention effect. On the other hand, chlorophyll breakdown was accelerated by treatments (T3 and T4) that had greater NO contents. Compared to the control, the levels of chlorophyll a, b, and total chlorophyll were noticeably lower. Furthermore, the degradation in T4 treatment was the most severe and much more severe than in T3 treatment.

3.5. Effects of Different NO Concentrations on Soluble Protein Content in Okra Fruit During Low-Temperature Storage

When evaluating the nutritional value and storage stability of fruits and vegetables, one of the key metrics is their soluble protein concentration. Under low-temperature storage circumstances, the soluble protein content of okra fruits exhibits a highly steady trend with a relatively narrow overall fluctuation range, as seen in Figure 5. A suitable NO concentration helps preserve the protein stability of okra during storage, as evidenced by the fact that T1 treatment considerably raised the soluble protein content of okra fruits over the course of the storage period after treatment with varying NO concentrations. Only the third day saw a substantial decrease in protein concentration due to the T2 treatment; at other times, there was no discernible change from the control. However, treatment with greater NO concentrations (T3 and T4) markedly reduced the amount of soluble protein that accumulated in okra fruits.

3.6. Effects of Different NO Concentrations on Soluble Sugar Content in Okra Fruit During Low-Temperature Storage

In addition to influencing the taste and texture of fruits and vegetables, the soluble sugar concentration is a crucial determinant of the fruits’ capacity to be stored over time. As indicated by Figure 6, the soluble sugar content of okra fruits steadily drops when stored at low temperatures, suggesting that as storage time increases, the sugar in okra gradually breaks down or changes. Its composition was significantly affected by treatment with varying NO concentrations. Among them, the administration of 0.5 mmol·L⁻¹ SNP (T1) and 1.0 mmol·L⁻¹ SNP (T2) markedly reduced the breakdown of soluble sugar, demonstrating a favorable impact on glucose retention. On the other hand, throughout the storage period, the okra fruits’ soluble sugar content was considerably decreased by high-concentration NO treatments (T3 and T4), which also accelerated sugar loss. The T3 treatment’s range of decrease was 4.5% to 13.6%, but the T4 treatment’s range was 10.4% to 33.1%.

3.7. Effects of Different NO Concentrations on MDA Content and Relative Conductivity in Okra Fruit Under Low-Temperature Storage

The malondialdehyde (MDA) content and relative conductivity are important indicators of membrane lipid peroxidation and membrane integrity, respectively. As shown in Figure 7, both MDA content and relative conductivity in okra fruit increased progressively during cold storage, indicating a gradual decline in membrane structural integrity over time. Treatment with 0.5 mmol·L⁻¹ SNP (T1) effectively suppressed the accumulation of MDA and limited the increase in relative conductivity compared with the control, suggesting that low-dose NO could reduce oxidative stress and maintain membrane stability. Similarly, 1.0 mmol·L⁻¹ SNP (T2) showed protective effects, although slightly less pronounced than T1. In contrast, higher concentrations of NO (1.5 and 2.0 mmol·L⁻¹, T3 and T4) accelerated membrane lipid peroxidation, as reflected by significantly elevated MDA levels and relative conductivity throughout the storage period. In particular, the T4 treatment group showed the most severe membrane damage, with MDA levels increasing by 28.0% and relative conductivity rising by 27.9% at day 9 compared to the control.

3.8. Effects of Different NO Concentrations on Antioxidant Enzyme Activities in Okra Fruit Under Low-Temperature Storage

The three most important enzymatic indicators for removing reactive oxygen species (ROS) from fruits and vegetables’ antioxidant defense systems are superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). These indicators are often used to determine how resistant a fruit is to stress. Figure 8 illustrates that the CAT activity of okra fruits initially increased and subsequently decreased when stored at low temperatures, possibly due to the accumulation of reactive oxygen species (ROS) exceeding the detoxification capacity of CAT at later stages, leading to enzyme inactivation or degradation. On the third and sixth days of treatment, it climbed progressively, but on the ninth day, it drastically reduced. The activity of CAT was significantly impacted by the treatment with varying NO concentrations. Out of all of them, the T1 treatment markedly raised CAT activity throughout the course of the storage period and had a pattern of gradually increasing activity with an increase in storage duration. Like the control, the T2 treatment had a fluctuating tendency, initially rising and then falling. The activity of CAT was markedly suppressed by the high-concentration treatment (T3 and T4), which also showed a continuous declining trend.

Additionally, during the low-temperature storage phase, POD and SOD activities demonstrated a generally steadily rising tendency. The two enzymes’ improved activity was markedly aided by the T1 treatment, suggesting that it successfully increased the antioxidant potential of okra fruits. POD activity was not significantly affected by the T2 treatment on days three or six, although it did significantly decline on day nine. SOD activity was not significantly impacted over the storage period. A persistent downward trend and considerable inhibition of the rise in CAT POD and SOD activities were seen in T3 and T4 treatments.

4. Discussion

The tender pods of okra are rich in mucilaginous polysaccharides, flavonoids, vitamins, and mineral elements, indicating its high potential for commercial and nutritional exploitation. This makes it a popular ingredient in the food processing, traditional medicine, and functional food industries [26]. But because of its high moisture content, quick respiration rate, and thin skin, okra fruit is very prone to drying, softening, and rotting, which reduces its shelf life and severely restricts its postharvest handling and commercialization [27]. The most widely used technique for postharvest okra preservation at the moment is cold storage because of its technical viability, low cost, and capacity to suppress microbial growth and metabolic activity [28]. However, cellular homeostasis may also be upset by low-temperature storage, which often leads to an excessive buildup of reactive oxygen species (ROS). This, in turn, impairs membrane integrity, accelerates the symptoms of chilling injury and senescence, and promotes lipid peroxidation [8]. An essential signaling molecule, nitric oxide (NO), has strong antioxidant and stress-resilient properties in the physiological control of fruits and vegetables after harvest. By scavenging ROS, stabilizing membrane systems, and modifying gene expression, it can delay senescence [29]. Okra weight loss during storage was significantly decreased in the current study when treated with a low concentration of NO (T1, 0.5 mmol·L⁻¹ SNP) (Figure 1). This reduction may have been caused by decreased membrane permeability and the suppression of excessive water evaporation, which improved water retention and freshness [30]. As shown in apricot fruits (Prunus armeniaca L. cv. Amal) [31], Lycium barbarum [32], and pointed gourd (Trichosanthes dioica) [33], NO may control the expression of plasma membrane aquaporins, helping to maintain cellular water balance. In contrast, higher concentrations of NO (T3 and T4) resulted in significantly increased weight loss, possibly due to intensified membrane lipid peroxidation, leading to membrane disruption and accelerated water loss.

The Texture Profile Analysis (TPA) may emulate the motions of a human chew. Fruits get compressed twice to produce various textural characteristics, including hardness, springiness, cohesiveness, resilience, and chewiness [34]. The maintenance of fruit texture is highly dependent on the integrity of the cell wall, which is primarily composed of polysaccharide components such as cellulose, hemicellulose, pectin, and lignin [35]. During storage, the degradation of cell wall polysaccharides is one of the main causes of texture deterioration and tissue softening in fruits. Research results indicated that low-concentration NO treatment (T1) significantly improved the hardness, springiness, chewiness, resilience, and cohesiveness of okra fruits during cold storage while inhibiting the increase in adhesiveness (Figure 2). This indicates that NO exerts a favorable effect on maintaining the structural stability of fruit tissues. This might be attributed to the regulatory effects of NO on the activity of cell wall-degrading enzymes, including pectin lyase (PG), pectin methylesterase (PME), and cellulase (Cx). Inhibiting the activity of these enzymes can retard the degradation of cell wall components, thereby preserving fruit texture characteristics [36]. Further analysis revealed that T1 treatment significantly maintained the cellulose and lignin contents in okra fruits (Figure 3). As the most abundant structural polysaccharide in the cell wall, cellulose’s crystalline microfibrils play a critical role in maintaining cell morphology and fruit mechanical strength; lignin, on the other hand, enhances the hydrophobicity and compressive resistance of the cell wall, further improving the stability and stress resistance of the tissue structure [37]. SNP treatment increased the endogenous NO content in melons and elevated lignin content by enhancing the activity of lignin biosynthesis-related enzymes (phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, cinnamyl alcohol dehydrogenase, and peroxidase) [38]. NO retarded tomato fruit softening by inhibiting the transcriptional activation of SlCCEL2, SlPG2a, and SlPL8 via SlNAP2, thereby reducing the activities of cellulase, PG, and PME [39]. In contrast, higher-concentration NO treatments (T3, T4) failed to exert these positive effects; instead, they caused rapid declines in texture parameters and significant reductions in cellulose and lignin contents. It is speculated that this may be related to excessive oxidative stress induced by high-concentration NO, which damages cell membranes and wall structures. Previous studies have indicated that NO exhibits bidirectional regulatory effects within a certain concentration range: low concentrations contribute to stress resistance and homeostatic regulation, whereas high concentrations may act as pro-oxidant factors, inducing programmed cell death or membrane lipid peroxidation [40]. Therefore, the regulatory effects of NO treatment for storage are significantly concentration-dependent, and reasonable control of its application concentration is a key factor in achieving high-efficiency preservation of okra fruits.

The color of vegetables, particularly their green appearance, serves as a critical quality attribute determining consumer acceptance. Generally, the reduction in chlorophyll content is recognized as the primary cause of yellowing or chlorophyll loss in green vegetables [41]. Chlorophyll degradation is typically mediated by a series of enzymatic reactions involving chlorophyllase, Mg-dechelatase, Mg-protoporphyrin IX monomethyl ester oxidase, and red chlorophyll catabolite reductase, whose activities are regulated by various environmental factors and signaling molecules [42]. Previous studies have demonstrated that nitric oxide (NO) can effectively delay chlorophyll breakdown by inhibiting the activities of these enzymes, thereby mitigating fruit yellowing and extending postharvest visual quality and marketability [12]. Our results revealed that low-concentration NO treatment (T1) significantly slowed the degradation rates of chlorophyll a and chlorophyll b, maintaining a higher greenness index in okra fruits, indicating its remarkable role in preserving color stability (Figure 4). This phenomenon may be attributed to NO-mediated regulation of the expression of key genes involved in chlorophyll metabolism or its enhancement of the stability of chlorophyll-binding proteins [43]. In contrast, higher-concentration treatments (T3, T4) exhibited an accelerated decline in chlorophyll content, which might be due to enhanced oxidative stress leading to membrane system damage and subsequent upregulation of the expression and activity of pigment-degrading enzymes. Beyond visual quality, soluble sugars and proteins are essential indicators for assessing the nutritional value and sensory quality of okra. Their contents are largely influenced by metabolic intensity, respiration rate, and cellular integrity [44]. The findings showed that T1 treatment significantly increased the levels of soluble sugars and proteins during the late storage period (Figure 5 and Figure 6), which could be attributed to NO-mediated inhibition of respiration rate and the activities of proteolytic enzymes, thereby slowing nutrient consumption while enhancing cellular vitality during storage [45]. Furthermore, NO was found to regulate the expression of genes encoding key enzymes in sugar metabolism (e.g., sucrose synthase and amylase), thereby influencing sugar accumulation and transformation [46]. In contrast, the decline in soluble nutrient contents under high-concentration NO treatments might result from oxidative stress-induced protein degradation and sugar metabolism disorders, coupled with increased material loss due to membrane damage.

Malondialdehyde (MDA), a major end-product of membrane lipid peroxidation in plant tissues, serves as a reliable biomarker for oxidative damage and senescence progression [47]. Relative conductivity reflects membrane permeability changes and is widely employed as an indicator of cellular membrane integrity [48]. In the present study, both MDA content and relative conductivity exhibited progressive increases during cold storage across all treatments, indicating enhanced membrane permeability and accelerated cellular senescence (Figure 7). These observations align with previous findings for chili pepper [47] and mushroom [49]. Among all treatments, the 0.5 mmol·L⁻¹ SNP group (T1) consistently maintained the lowest MDA content and relative conductivity values throughout the storage period, suggesting that low-concentration exogenous NO effectively mitigates membrane lipid peroxidation and preserves membrane structural integrity. Conversely, higher SNP concentrations (T3 and T4) resulted in substantial increases in both parameters, particularly during the later storage phases, reflecting intensified oxidative damage and compromised membrane stability. The antioxidant defense system plays a pivotal role in plant responses to abiotic stress. Superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) constitute the first line of defense against reactive oxygen species (ROS) by collaboratively catalyzing the conversion of ROS intermediates, effectively preventing the accumulation of oxidative damage [50]. In this study, T1 treatment significantly elevated the enzymatic activities of SOD, POD, and CAT (Figure 8), indicating that NO enhances the antioxidant capacity of okra fruits, maintains redox homeostasis, reduces membrane lipid peroxidation, and preserves membrane system integrity. In contrast, high-concentration NO treatments suppressed the activities of the antioxidant enzyme system, leading to decreased oxidative scavenging capacity and further promoting ROS accumulation, forming a vicious cycle that results in membrane lipid peroxidation and cellular structural damage.

5. Conclusions

This study systematically evaluated the physiological and biochemical responses of okra pods to different concentrations of sodium nitroprusside (SNP), a nitric oxide (NO) donor, under cold storage (4 ± 0.5 °C). The results demonstrate that an appropriate concentration of exogenous NO, particularly 0.5 mmol·L⁻¹ SNP (T1), significantly delayed postharvest senescence and maintained okra quality. This was achieved by reducing weight loss, preserving textural attributes via the inhibition of cellulose and lignin accumulation, delaying chlorophyll degradation, retaining higher levels of soluble sugars and proteins, and enhancing the activities of antioxidant enzymes (SOD, POD, and CAT). Conversely, higher SNP concentrations (1.5 and 2.0 mmol·L⁻¹) induced excessive oxidative stress, as indicated by increased MDA content and electrolyte leakage, ultimately accelerating quality deterioration. These findings offer physiological evidence supporting the precise use of SNP as a postharvest treatment strategy to mitigate chilling injury and extend the shelf life of okra during cold storage.