Effects of Sodium Nitroprusside Applications on Antioxidant Defense System and Oxidative Stress in Leaves and Berries of the ‘Horoz Karası’ Grapevine (Vitis vinifera L.)

Effects of Sodium Nitroprusside Applications on Antioxidant Defense System and Oxidative Stress in Leaves and Berries of the ‘Horoz Karası’ Grapevine (Vitis vinifera L.) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of Sodium Nitroprusside Applications on Antioxidant Defense System and Oxidative Stress in Leaves and Berries of the ‘Horoz Karası’ Grapevine (Vitis vinifera L.) İbrahim Samet GÖKÇEN This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8888370/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background Nitric oxide (NO) has been widely recognized as a multifunctional signaling molecule in plants, participating in the regulation of growth, developmental transitions, and stress adaptation processes. Its involvement in modulating cellular redox homeostasis is considered one of the key mechanisms underlying these effects. In experimental studies, sodium nitroprusside (SNP) is frequently employed as an exogenous NO donor, and numerous reports indicate that SNP treatments can activate antioxidant systems in different plant species. However, information regarding the temporal effects of repeated SNP applications under vineyard conditions and their integrated effect on leaf and grape berries antioxidant responses in grapevines remains limited. Results Foliar SNP applications significantly modulated antioxidant enzyme activities and oxidative stress indicators in the Horoz Karası grape variety. Among the tested concentrations, 300 ppm SNP consistently resulted in the highest activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR), while effectively suppressing malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) accumulation in leaf tissues. The effects of SNP were more pronounced during the veraison and harvest periods than in the pre-veraison stage, indicating a stronger regulatory role under elevated metabolic activity. In grape berries tissue, SNP applications, particularly at 300 ppm, reduced MDA levels and promoted higher antioxidant enzyme activities, demonstrating a systemic leaf– grape berries interaction mediated by NO signaling. Conclusions Repeated foliar application of SNP effectively enhances antioxidant defense capacity and limits oxidative damage in Horoz Karası grapevines in a dose- and time-dependent manner. The 300 ppm concentration emerged as the optimal dose for maintaining redox homeostasis in both leaf and grape berries tissues. These findings highlight the potential of NO-based applications as an environmentally friendly strategy to improve oxidative stress management and quality preservation in sustainable viticulture practices. Vitis vinifera L. antioxidant enzymes nitric oxide sodium nitroprusside Horoz Karası Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Grapes ( Vitis vinifera L.), an important member of the Vitaceae family, possess considerable economic and nutritional value worldwide, both as fresh fruit and in processed products. Grape berries are characterized by their attractive aroma and flavor, as well as their rich nutritional composition. Grapes at harvest maturity represent an important source of dietary fiber, organic acids, vitamins, and minerals, and are particularly rich in phenolic compounds and antioxidant capacity. Owing to these bioactive components, grapes provide significant health benefits and are widely accepted as a functional food. Horoz Karası, one of the important grape varieties endemic to Türkiye, stands out especially for its high adaptability and favorable fruit quality. Due to increasing consumer demand and the need to protect local genetic resources, the cultivation of this variety has become increasingly important. In viticulture, increasing both yield and quality is a fundamental goal for producers, and the development of environmentally friendly and sustainable practices plays a crucial role in achieving this goal. [ 1 – 3 ]. Today, various chemical treatments are used in agricultural production to increase yield and improve quality. However, the intensive use of pesticides and chemical fertilizers poses risks not only to the environment but also to product quality and human health. Therefore, there is increasing interest in lower-risk alternative applications that support the physiological and biochemical mechanisms of plants. [ 4 ]. In this context, the use of signaling molecules that regulate growth, development, and stress responses in plants is gaining prominence. Nitric oxide (NO), one of these signaling molecules, is an important compound that plays a role in regulating many physiological processes in plants, such as photosynthesis, respiration, cell division, and aging [ 5 – 7 ]. NO also plays a critical role in the development of tolerance to environmental stress conditions in plants. In particular, it contributes to the maintenance of cellular redox balance through its interaction with reactive oxygen species (ROS) [ 8 , 9 ]. Sodium nitroprusside (SNP) is one of the compounds widely used for the external application of NO in plants, considered an effective donor for controlled NO release. Owing to its light and temperature-sensitive structure, small molecular size, and high diffusion capacity, it can easily cross cell membranes and effectively regulate intracellular signaling mechanisms in plant tissues [ 5 , 10 ]. In plants, NO is synthesized via NO synthase-like enzymes during the conversion of L-arginine to L-citrulline and is widely distributed in different plant tissues. NO is known to exert significant effects on seed germination, stomatal movement, root development, defense responses against pathogens, and leaf physiology [ 11 – 13 ]. It also plays an important role in the regulation of cell division, chlorophyll metabolism, photosynthesis, and respiration processes [ 14 , 15 ]. One of the most important functions of NO in plants is the control of oxidative stress. NO activates the antioxidant defense system, regulating the activities of enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR); conversely, it limits the accumulation of hydrogen peroxide (H 2 O 2 ) and malondialdehyde (MDA) which are indicators of lipid peroxidation. The application of NO donors such as SNP has been reported to protect membrane integrity and reduce cellular damage in different plant and fruit species [ 16 – 18 ]. Recent studies have shown that SNP applications are effective not only in vegetative tissues but also in fruit development and veraison, with significant effects on quality characteristics. Pre- or post-harvest SNP applications have been reported to enhance antioxidant capacity in fruits, regulate phenylpropanoid metabolism, and reduce quality losses [ 17 – 21 ]. These findings demonstrate that SNP has the potential to improve fruit quality by maintaining oxidative balance in fruit tissues. Although studies on NO and SNP applications in grapevines are limited, existing data indicate a regulatory role of SNP on the antioxidant defense system [ 19 , 22 ]. However, studies evaluating the combined effects of repeated foliar SNP applications on antioxidant enzyme responses in leaves and the reflection of these responses in fruit tissue, particularly under vineyard conditions, are quite insufficient. This study aims to determine the effects of repeated applications of different SNP concentrations at specific intervals on antioxidant enzyme activities (GR, SOD, APX and CAT) and oxidative stress indicators (MDA and H 2 O 2 ) in the leaves of the Horoz Karası grape variety, and to evaluate the relationship between these effects and biochemical responses in the fruit tissue following the final application. The originality of this study lies in the consideration of SNP applications in a temporal framework under vineyard conditions and their evaluation within the context of leaf–fruit integrity. It is expected that the findings will contribute to a better understanding of the physiological and biochemical effects of NO-based applications in viticulture. Materials and methods Plant material and experimental design This study was conducted in a commercial vineyard in Kilis province, located in the Southeastern Anatolia Region of Turkey, during the 2025 growing season. The experimental material consisted of vines of the Horoz Karası grape variety ( Vitis vinifera L.), commonly cultivated in the region. All vines in the vineyard were of the same age (15 years), at a similar phenological development stage, and grown under uniform conditions in terms of cultural practices such as irrigation, fertilization, pruning, and disease and pest control. The experiment was designed with three treatment groups, each consisting of ten vines. At the beginning of the experiment, leaf samples were taken for control purposes before the SNP application. Subsequently, SNP solutions were applied to the treatment groups at concentrations of 100, 300, and 500 ppm respectively, using a foliar spray method; the control group was sprayed only with pure water. Leaf samples were taken 15 days after the first SNP application (pre-veraison period), and a second SNP application was performed at the same doses. On the thirtieth day (veraison period), leaf samples were collected again and a third SNP application was performed. On the forty-fifth day (harvest period), both leaf and fruit samples were harvested for analysis. During this period, fruits from vines not treated with SNP were also collected as a control group. All collected leaf and fruit samples were transported to the laboratory under cold chain conditions and stored under appropriate conditions until analysis. Biochemical analyses were performed to determine oxidative stress indicators and enzyme activities of the antioxidant defense system. Biochemical Measurements and Analyses Preparation of Enzyme Extracts Leaf and grape berries tissues collected after SNP treatments were immediately immersed in liquid nitrogen, pulverized to a fine powder, and preserved at − 80°C prior to biochemical analyses. For the preparation of crude enzyme extracts, 1 g of frozen material was thoroughly homogenized in a pre-chilled mortar using 100 mM phosphate buffer (pH 7.0) supplemented with 10 mM Na₂EDTA, 1 mM MgCl₂, 1% (w/v) polyvinylpyrrolidone (PVP), 10 mM KCl, and 2 mM dithiothreitol (DTT). The homogenate was subsequently filtered and centrifuged at 14,000 rpm for 20 min at 4°C. The clear supernatant obtained after centrifugation served as the enzyme source. Determination of Catalase (CAT; EC 1.11.1.6) Enzyme Activity Catalase (CAT) activity was assayed by following the decomposition of hydrogen peroxide spectrophotometrically. The reaction mixture consisted of 30 mM KH₂PO₄/K₂HPO₄ buffer (pH 7.0) containing 10 mM H₂O₂, and the assay was started by the addition of 300 µL enzyme extract, bringing the final reaction volume to 1.4 mL. The decline in absorbance at 240 nm, corresponding to H₂O₂ degradation, was recorded for 3 min at 30 s intervals. Enzyme activity was calculated using an extinction coefficient of 36 mM⁻¹ cm⁻¹ for H₂O₂ and expressed as units per milligram of protein (U mg⁻¹ protein). The procedure followed the method of Aebi (1984) [ 23 ]. Determination of Ascorbate Peroxidase (APX; EC 1.11.1.11) Enzyme Activity APX activity was assayed spectrophotometrically by monitoring the oxidation of ascorbate in the presence of hydrogen peroxide. The reaction mixture contained 0.8 mL of 50 mM potassium phosphate buffer (pH 7.0), 0.1 mL of 0.5 mM ascorbate, and 0.1 mL of enzyme extract. The reaction was started by the addition of 0.1 mL of 100 mM H₂O₂. The decrease in absorbance at 290 nm, reflecting ascorbate oxidation, was recorded over a 3-min period. Enzyme activity was calculated using an extinction coefficient of 2.8 mM⁻¹ cm⁻¹ and expressed on a protein basis (U mg⁻¹ protein). The assay procedure followed the method described by Nakano and Asada (1981) [ 24 ]. Determination of Glutathione Reductase (GR; EC 1.6.4.2) Enzyme Activity GR activity was assayed by following the oxidation of NADPH during the reduction of oxidized glutathione. The reaction mixture (final volume 2.8 mL) consisted of 120 mM phosphate buffer (pH 7.2; 2.5 mL), 0.065 mM GSSG (0.1 mL), 0.015 mM Na₂EDTA (0.1 mL), 9.6 mM NADPH (0.05 mL), and 0.1 mL enzyme extract. The decline in absorbance at 340 nm, corresponding to NADPH consumption, was monitored for 3 min. Enzyme activity was calculated and expressed as U mg⁻¹ protein. The assay was carried out according to the procedure described by Goldberg and Spooner (1983) [ 25 ]. Determination of Superoxide Dismutase (SOD; EC 1.15.1.1) Enzyme Activity SOD activity was determined by a method based on the inhibition of nitroblue tetrazolium (NBT) reduction by superoxide radicals under light. The reaction medium was prepared to contain 50 mM phosphate buffer (pH 7.8), 12 mM methionine, 75 µM NBT, 3 mM Na₂EDTA, and 50 mM Na₂CO₃, with different volumes of enzyme extract added. The assay was initiated by adding 10 µM riboflavin, after which the reaction mixtures were exposed to fluorescent light at 25°C to allow photochemical reduction. The formation of the blue formazan product resulting from NBT reduction was quantified spectrophotometrically at 560 nm. One unit of superoxide dismutase (SOD) activity was defined as the amount of enzyme required to cause 50% inhibition of NBT reduction under the assay conditions. Enzyme activity was expressed as U mg⁻¹ protein. The determination of SOD activity was carried out following the method of Giannopolitis and Ries (1977) [ 26 ]. Determination of Malondialdehyde (MDA) Content MDA content, used as an indicator of lipid peroxidation, was quantified according to the thiobarbituric acid (TBA) assay [ 27 ]. For this purpose, 0.5 g of leaf tissue previously frozen in liquid nitrogen and stored at − 80°C was homogenized in 0.1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 10,000 rpm for 15 min at 4°C, and the supernatant obtained was used for subsequent measurements. For color development, 1 mL of the supernatant was mixed with 4 mL of 0.5% (w/v) TBA prepared in 20% (w/v) TCA. The reaction mixture was heated at 95°C for 30 min, followed by rapid cooling in an ice bath to terminate the reaction. After cooling, samples were centrifuged again at 10,000 rpm for 10 min. The absorbance of the clear supernatant was recorded at 532 nm, and non-specific turbidity was corrected by subtracting the absorbance measured at 600 nm. The MDA amount was calculated using a standard curve created with MDA standard solutions prepared in the range of 0–120 µM (Fig. 1 ). The standard equation is y = 0.0023x + 0.0241, and determination coefficient R² = 0.991. Results are expressed in µmol MDA g⁻¹ fresh weight. Determination of Hydrogen Peroxide (H₂O₂) Content H₂O₂ content in leaf and grape berries samples was determined using a colorimetric method described in the literature, adapted to the conditions of our study [ 28 ]. For analysis, 0.5 g of samples were taken from those powdered with liquid nitrogen and stored at -80°C. The samples were homogenized with a suitable extraction buffer under cold conditions. The resulting homogenates were centrifuged at 14,000 rpm at 4°C for 20 minutes, and the supernatant was used in the analyses. The determination of H₂O₂ content is based on the spectrophotometric measurement of the colored complex formed as a result of the reaction of the sample extract with the relevant reagents. At the completion of the reaction, absorbance was measured at 390 nm against a reagent blank. The concentration of H₂O₂ was quantified based on the calibration curve generated using known H₂O₂ standards (Fig. 2 ). The linear regression equation (y = 0.01x + 0.0359; R² = 0.992) was used to calculate H₂O₂ content, and the results were expressed as mmol g⁻¹ fresh weight. Statistical Analysis All measurement data obtained from the experiments were expressed as mean ± standard deviation for each application. All data were subjected to statistical analysis using SPSS version 24.0. Differences among treatments were evaluated by one-way analysis of variance (ANOVA). When significant effects were detected, mean comparisons were performed using Duncan’s multiple range test to determine the source of variation among groups. In statistical analyses, p-value ≤ 0.05 was accepted as the threshold value at which the differences between applications were significant. Results Effects of Sodium Nitroprusside (SNP) Applications on Antioxidant Defense System and Oxidative Stress İndicators in Grape Leaves The effects of SNP applications on antioxidant defense system and oxidative stress indicators in the leaves of Horoz Karası grape variety showed significant dose-dependent changes throughout the phenological development process (pre-veraison, veraison, and harvest period). Compared to the control group, 100, 300, and 500 ppm SNP applications affected biochemical parameters to varying degrees in all periods. Findings regarding the pre-veraison period are presented in Table 1 . In this period, generally limited differences were observed between the SNP-treated and control groups in terms of GR, SOD, CAT, and APX activities. The one-way analysis of variance indicated that none of the evaluated parameters differed significantly among the treatments. Nevertheless, an examination of the mean values revealed a tendency toward higher GR and SOD activities in the 300 ppm SNP treatment compared with the control. In contrast, MDA and H₂O₂ levels were generally lower in the SNP-treated groups than in the untreated control. These trends point to the potential of SNP to regulate oxidative balance in the early phenological stage. Table 1 Effects of SNP application at pre-veraison on antioxidant enzyme activities and oxidative stress markers in leaves SNP doses GR SOD CAT APX MDA H₂O₂ Control 3,75 ± 0,48ᵇ 75,87 ± 2,48ᵇ 6,77 ± 0,61ᵃᵇ 12,91 ± 1,07ᵇ 5,35 ± 0,01ᵇ 93,61 ± 0,99ᵇ 100 ppm 4,74 ± 1,34ᵃᵇ 81,93 ± 4,59ᵇ 8,24 ± 0,52ᵃ 15,47 ± 0,00ᵃᵇ 5,30 ± 0,05ᵇ 80,61 ± 0,42ᶜ 300 ppm 6,74 ± 1,14ᵃ 95,36 ± 6,78ᵃ 8,40 ± 0,84ᵃ 16,80 ± 1,70ᵃ 5,55 ± 0,05ᵃ 97,71 ± 0,28ᵃ 500 ppm 4,82 ± 0,45ᵃᵇ 86,32 ± 0,31ᵃᵇ 6,02 ± 0,60ᵇ 14,29 ± 0,00ᵃᵇ 5,55 ± 0,09ᵃ 80,51 ± 0,85ᶜ a, b, c ↓: Different lower cases represent statistically significant differences among means Comparisons of pre-veraison period parameters depending on SNP doses are presented in Fig. 3 . Graphical evaluation shows that, in particular, the application of 300 ppm SNP resulted in a numerical increase in antioxidant enzyme activities and had a suppressive effect on oxidative stress indicators. Leaf traits measured at the veraison stage are presented in Table 2 . The effects of SNP applications became more pronounced during this period, with statistically significant differences observed between applications, particularly in terms of GR and SOD activities (p ≤ 0.05). The highest levels of GR and SOD activity were observed in the 300 ppm SNP application, while the 100 and 500 ppm doses exhibited intermediate values. With regard to the CAT and APX activities, the results of the ANOVA indicated no overall significance. However, it was observed that all groups treated with SNP exhibited higher averages in comparison to the control group. When the oxidative stress indicators were evaluated, it was determined that the contents of MDA and H 2 O 2 decreased significantly, especially in the 100 and 300 ppm SNP applications, compared to the control group (p ≤ 0.05). Table 2 Effects of SNP application at veraison on antioxidant enzyme activities and oxidative stress markers in leaves SNP doses GR SOD CAT APX MDA H₂O₂ Control 3,75 ± 0,48ᵃ 75,87 ± 2,48ᵇ 6,77 ± 0,61ᵃ 12,91 ± 1,07ᵃ 5,35 ± 0,01ᵈ 93,61 ± 0,99ᶜ 100 ppm 5,93 ± 0,44ᵇ 56,37 ± 3,29ᵃ 9,85 ± 0,94ᵇ 17,37 ± 0,98ᵇ 4,56 ± 0,06ᵃ 61,26 ± 0,49ᵃ 300 ppm 9,67 ± 0,72ᶜ 110,54 ± 9,39ᶜ 9,62 ± 0,85ᵇ 20,95 ± 0,80ᶜ 4,79 ± 0,04ᵇ 91,96 ± 0,49ᶜ 500 ppm 6,67 ± 0,00ᵇ 91,74 ± 6,29ᵇ 9,17 ± 0,45ᵇ 17,31 ± 1,17ᵇ 5,18 ± 0,02ᶜ 72,06 ± 2,19ᵇ a, b, c ↓: Different lower cases represent statistically significant differences among means The comparative effects of SNP doses during the veraison period are presented in Fig. 4 . The graph shows that antioxidant enzyme activities reach maximum levels during the veraison period and that SNP applications enhance this response. Leaf-related parameters measured at harvest are presented in Table 3 . It was observed that the effects of SNP applications differed depending on the parameter. Statistically significant differences were identified between the applications in terms of GR and CAT activities (p ≤ 0.05), with the highest values determined in the 300 ppm SNP application. Despite the absence of statistically significant findings for SOD and APX activities, it was observed that all SNP-treated groups exhibited higher mean values in comparison to the control group. With regard to the MDA and H 2 O 2 content, the 100 ppm SNP application exhibited the lowest values, thus suggesting that a lower SNP dose may be more effective in limiting oxidative stress during the harvest period. Table 3 Effects of SNP application at harvest on antioxidant enzyme activities and oxidative stress markers in leaves SNP doses GR SOD CAT APX MDA H₂O₂ Control 3,75 ± 0,48ᵃ 75,87 ± 2,48ᵃ 6,77 ± 0,61ᵃ 12,91 ± 1,07ᵃ 5,35 ± 0,01ᶜ 93,61 ± 0,99ᵇ 100 ppm 4,14 ± 0,98ᵃᵇ 89,12 ± 9,60ᵃ 8,47 ± 0,28ᵃ 14,57 ± 1,08ᵃ 4,34 ± 0,04ᵃ 73,66 ± 0,78ᵃ 300 ppm 8,30 ± 1,41ᶜ 82,67 ± 3,22ᵃ 12,40 ± 1,11ᵇ 19,95 ± 5,23ᵃ 5,02 ± 0,04ᵇ 89,16 ± 0,49ᵇ 500 ppm 6,53 ± 0,49ᵇ 81,98 ± 3,77ᵃ 7,38 ± 0,39ᵃ 17,60 ± 1,08ᵃ 5,35 ± 0,05ᶜ 89,86 ± 3,18ᵇ a, b, c ↓: Different lower cases represent statistically significant differences among means Harvest-related parameters were compared across SNP doses (Fig. 5 ). SNP applications suppress oxidative stress indicators and support the antioxidant defense system throughout phenological progression. The results related to grape berries parameters are summarized in Table 4 . Analysis of variance revealed no significant effect of SNP treatments on grape berries GR activity compared with the control (p = 0.408). Although the control group exhibited the lowest GR activity and the 300 ppm SNP treatment showed the highest value, these differences were not statistically meaningful. Similarly, SOD activity displayed some variation among treatments; however, the observed differences did not reach statistical significance (p = 0.053).The highest SOD activity was detected at 300 ppm SNP, while lower activities were observed in the control and 500 ppm treatments. The CAT activity varied among the treatments; however, the difference was not statistically significant (p = 0.052). The highest activity was recorded at 300 ppm SNP, while the control and 500 ppm treatments exhibited lower values. The effect of SNP applications on APX activity was not statistically significant. Although the highest APX value was observed in the 300 ppm application, no significant difference was observed between the applications. MDA content varied significantly depending on the treatments (p < 0.05). The lowest MDA values ​​were determined in the 500 and 300 ppm SNP treatments, while the control group had the highest MDA content. Hydrogen peroxide (H₂O₂) content did not differ significantly between the SNP-treated groups and the control. Nevertheless, the lowest H₂O₂ level was observed in the 300 ppm SNP treatment, whereas the control group exhibited the highest value. Table 4 Effects of SNP application on antioxidant enzyme activities and oxidative stress markers in in grape berries tissue SNP doses GR SOD CAT APX MDA H₂O₂ Control 1,564 ± 0,170ᵃ 17,264 ± 0,480ᵃ 1,874 ± 0,491ᵃ 4,819 ± 0,757ᵃ 2,129 ± 0,037ᶜ 9,860 ± 0,212ᵈ 100 ppm 1,645 ± 0,332ᵃ 18,415 ± 0,815ᵃᵇ 2,237 ± 0,096ᵃᵇ 5,229 ± 0,739ᵃ 1,981 ± 0,025ᵇ 7,510 ± 0,141ᵇ 300 ppm 1,833 ± 0,324ᵃ 19,808 ± 0,929ᵇ 3,237 ± 0,467ᵇ 6,116 ± 0,000ᵃ 1,755 ± 0,049ᵃ 6,710 ± 0,283ᵃ 500 ppm 1,682 ± 0,297ᵃ 17,906 ± 0,338ᵃ 1,759 ± 0,257ᵃ 5,380 ± 0,331ᵃ 1,720 ± 0,049ᵃ 8,610 ± 0,424ᶜ a, b, c ↓: Different lower cases represent statistically significant differences among means A comparison of antioxidant enzyme activities and oxidative stress markers in grape berries among different SNP applications is presented in Fig. 6 . No statistically significant differences were observed among treatments for GR, SOD, CAT, and APX activities; however, 300 ppm SNP consistently exhibited higher enzyme activities compared to the control and 500 ppm treatments. In contrast, MDA content differed significantly among treatments, with markedly lower values in the 300 and 500 ppm SNP applications relative to the control. Although H₂O₂ content did not differ significantly, the lowest levels were observed at 300 ppm SNP, whereas the control showed the highest values. Overall, the comparative graphical analysis indicates that SNP treatment, particularly at 300 ppm, is associated with enhanced antioxidant responses and reduced oxidative stress in grape berries.” To provide an integrated overview of the antioxidant system and oxidative stress responses, a heatmap based on Z-score normalization was constructed (Figure X). The visualization revealed clear stage-dependent differences in the response to SNP applications. At veraison, particularly under 300 ppm SNP, antioxidant enzyme activities (GR, SOD, CAT, and APX) showed pronounced increases compared to the control, as indicated by the intense red clusters. Importantly, this enzymatic activation was accompanied by a marked reduction in oxidative stress markers, with both MDA and H₂O₂ exhibiting lower relative values than the control treatment. This pattern suggests that SNP application, especially at 300 ppm, effectively enhanced antioxidant defense while simultaneously mitigating lipid peroxidation and hydrogen peroxide accumulation. A similar but less pronounced trend was observed at pre-veraison and harvest stages, where elevated enzymatic activities were generally associated with reduced MDA and H₂O₂ levels relative to the control. In contrast, grape berries displayed comparatively lower antioxidant enzyme activities and a distinct oxidative profile, indicating tissue-specific regulation of redox metabolism during fruit development. Overall, the coordinated increase in antioxidant enzymes together with the decrease in MDA and H₂O₂ under 300 ppm SNP highlights this dose as the most effective treatment for improving redox homeostasis across developmental stages. Discussion In this study, foliar SNP applications were found to dynamically modulate the antioxidant defense system throughout the phenological stages and to limit oxidative damage in the Horoz Karası grape variety. In particular, the observation that the 300 ppm SNP dose was the most effective concentration in both leaf and grape berries tissues supports current findings indicating that NO exhibits a hormetic effect in plants. While low doses act as signaling molecules that trigger defense responses, the reduced effectiveness observed at higher doses (such as 500 ppm) can be explained by the tightly regulated and delicate balance of NO metabolism [ 29 ]. The integrative heatmap analysis provides further evidence that SNP-mediated nitric oxide signaling enhances redox regulation in a stage-dependent manner. The coordinated increase in antioxidant enzyme activities (GR, SOD, CAT, and APX), together with the reduction of MDA and H₂O₂ levels compared to the control, indicates that SNP application effectively mitigates oxidative damage while strengthening the antioxidant defense system. Particularly, the 300 ppm dose appears to optimize redox homeostasis across developmental stages. These findings suggest that exogenous SNP plays a regulatory role in maintaining cellular oxidative balance during grapevine development. The results indicated that SNP treatments led to a marked increase in the activities of major antioxidant enzymes, namely SOD, CAT, and GR, with the most pronounced effects observed during the veraison stage. Superoxide dismutase (SOD) acts as the primary enzymatic barrier against oxidative stress by catalyzing the dismutation of superoxide radicals. Subsequently, enzymes such as CAT and APX facilitate the conversion of the generated H₂O₂ into less harmful molecules, thereby contributing to the regulation of cellular redox balance. Consistent with these results, previous studies have reported that NO applications activate the Ascorbate–Glutathione (Asada–Halliwell) cycle in grape tissues, leading to increased activities of SOD, CAT, APX, and GR, while simultaneously suppressing ROS accumulation and lipid peroxidation [ 30 ]. In line with this, a molecular-level study conducted on grape berries in 2023 demonstrated that NO donors have been reported to stimulate phenylpropanoid pathway activity and to enhance the transcription of antioxidant defense–associated genes, including VvSOD and VvCAT [ 31 ]. In line with these findings, earlier studies have shown that the combined treatment of NO and H₂S alleviated oxidative damage by promoting the activities of major antioxidant enzymes such as APX, CAT, and GR. This coordinated upregulation was accompanied by reduced accumulation of MDA and H₂O₂, indicating an improved cellular redox status [ 32 ]. These molecular insights provide a robust mechanistic explanation for the observation that the 300 ppm SNP application was associated with higher antioxidant enzyme activities compared to the other doses evaluated in the present study. When evaluated in terms of oxidative stress indicators, the significant reduction in MDA levels in both leaf and grape berries tissues following SNP applications reflects the protective effect of NO against membrane lipid peroxidation [ 6 ]. NO can stabilize free radicals through direct interactions with reactive oxygen species and can also indirectly limit ROS accumulation by inducing the antioxidant defense system [ 13 ]. Current studies demonstrate that NO applications slow the oxidation of unsaturated fatty acids in cell membranes, preserve fruit tissue integrity, and reduce post-harvest quality losses [ 19 , 33 – 35 ]. For Horoz Karası, which is among thin-skinned and oxidative stress–sensitive grape varieties, this protective effect is particularly important for the sustainability of fruit quality. Another noteworthy finding of the study is that the effects of SNP applications became more pronounced during the veraison and harvesting periods compared to the pre-veraison stage. This suggests that exogenous NO applications act as a “buffer mechanism” against the increased production of ROS associated with elevated metabolic activity during veraison [ 36 , 37 ]. Indeed, recent studies have demonstrated that NO can delay fruit ripening and softening processes. For example, a recent study reported that NO significantly enhanced the activity of key enzymes involved in the phenylpropanoid biosynthesis pathway, thereby increasing disease resistance and mitigating fruit softening in bitter melon [ 38 ]. In another study, the combined application of gibberellic acid and NO was shown to delay fruit ripening and color development in strawberry fruits by regulating chromatin-level processes [ 39 ]. Similarly, in tomato fruits, NO application delayed ripening and softening by inhibiting the expression of cell wall-softening genes [ 40 ]. In addition, S-nitrosoglutathione application has been reported to delay color change and slow the ripening process during post-harvest storage of tomato fruits [ 41 ]. The observation that foliar-applied SNP influenced antioxidant enzyme activities and MDA levels in fruit tissue indicates that the NO signal is systemically transported through the vascular system of the grapevine, thereby proactively regulating fruit quality within the framework of leaf–fruit integrity. Conclusion This study shown that foliar application of SNP effectively regulates the antioxidant defense system and limits oxidative damage throughout the phenological process in the Horoz Karası grape variety. Considering all evaluated physiological and biochemical parameters, the 300 ppm SNP dose emerged as the optimum concentration, maximizing antioxidant enzyme activities while minimizing MDA and H₂O₂ accumulation. The more pronounced effects of SNP applications during the verasion and harvest stages indicate that exogenous NO supplementation acts as an efficient regulator, balancing the oxidative load associated with increased metabolic activity. Moreover, the influence of foliar SNP application on oxidative stress indicators and antioxidant responses in fruit tissue suggests that NO signaling operates systemically, maintaining leaf–fruit functional integrity. Overall, these findings highlight the strong potential of NO-based applications as a sustainable strategy for oxidative stress management and grape quality preservation in viticulture. Abbreviations APX Ascorbate peroxidase AsA–GSH cycle Ascorbate–Glutathione cycle CAT Catalase GA Gibberellic acid GR Glutathione reductase H₂O₂ Hydrogen peroxide MDA Malondialdehyde NO Nitric oxide NO + H₂S Nitric oxide + Hydrogen sulfide ROS Reactive oxygen species SNP Sodium nitroprusside SOD Superoxide dismutase Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests Not applicable. Funding This research received no external funding. Author Contribution İSG was solely responsible for the conceptualization and design of the study, experimental implementation, data acquisition and statistical analysis, interpretation of findings, and preparation and critical revision of the manuscript. The author approved the final version of the manuscript. Acknowledgements Not applicable. Data Availability The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. References Kamiloglu O. Influence of some cultural practices on yield, fruit quality and individual anthocyanins of table grape cv.‘Horoz Karasi’. J Anim Plant Sci. 2011;21(2):240–5. Sucu Dağ S, Gün B, Şekeroğlu N. The Effects of Cluster Thinning Yield and Quality on ‘Horoz Karası’ Cultivar of Grapevine. Black Sea J Agric. 2026;9(1):1–8. https://doi.org/10.47115/bsagriculture.1792498 . Brás LP, Luís Â, Chatel G, Socorro S, Duarte AP. Stilbenes from Vine Extracts: Therapeutic Potential and Mechanisms. 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Anal Biochem. 1979;95(2):351–8. https://doi.org/10.1016/0003-2697(79)90738-3 . Velikova M, Bankova V, Sorkun K, Houcine S, Tsvetkova I, Kujumgiev A. Propolis from the Mediterranean region: chemical composition and antimicrobial activity. Z Naturforsch C J Biosci. 2000;55(9–10):790–3. https://doi.org/10.1515/znc-2000-9-1019 . Calabrese EJ, Agathokleous E. Nitric oxide, hormesis and plant biology. Sci Total Environ. 2023;866:161299. https://www.sciencedirect.com/science/article/pii/S0048969722084030 . Zhang Z, Xu J, Chen Y, Wei J, Wu B. Nitric oxide treatment maintains postharvest quality of table grapes by mitigation of oxidative damage. Postharvest Biol Technol. 2019;152:9–18. https://www.sciencedirect.com/science/article/pii/S0925521418311098 . Shi J, Huang D, Du Y, Zhu S, Hussain Z, Haider MS, et al. Effects of Exogenous Nitric Oxide Treatment on Grape Berries Against Botrytis cinerea and Alternaria alternata Related Enzymes and Metabolites. Plant Dis. 2023;107(5):1510–21. https://doi.org/10.1094/pdis-04-22-0928-re . Liu R, Ji N, Wang R, Li Y, Nie H, Chen C, et al. Transcriptomic and metabolomic analyses insight into the synergistic effects of nitric oxide and hydrogen sulfide fumigation on enhancing postharvest antioxidant defense and phenylpropane metabolism in ‘crystal’ grape. Food Bioscience. 2025;66:106110. https://www.sciencedirect.com/science/article/pii/S221242922500286X . Li C, Yu W, Liao W. Role of Nitric Oxide in Postharvest Senescence of Fruits. Int J Mol Sci. 2022;23(17). https://doi.org/10.3390/ijms231710046 . Ghorbani B, Pakkish Z, Najafzadeh R. Shelf life improvement of grape (Vitis vinifera L. cv. Rish Baba) using nitric oxide (NO) during chilling damage. Int J Food Prop. 2017;20(sup3):S2750–63. https://doi.org/10.1080/10942912.2017.1373663 . Plakunmonthon T, Chutimanukul P, Matsui K, Seraypheap K. Integrated effects of pre-harvest high blue light and postharvest sodium nitroprusside on volatile oil composition and quality of cold-stored holy basil. Food Chem X. 2026;33:103493. https://doi.org/10.1016/j.fochx.2026.103493 . Mishra AK, Gupta S, Tiwari S. Differential antioxidant and metabolic responses to ozone stress in ozone-sensitive and tolerant wheat cultivars treated with nitric oxide donor. Sci Total Environ. 2025;1002:180588. https://doi.org/10.1016/j.scitotenv.2025.180588 . Ahsan M, Tufail A, Jamal A, Al-Yasi HM, Radicetti E, Raza MA, et al. Nitric oxide regulates water status, antioxidant enzymes, nutritional balance, and growth of gazania (Gazania rigens) under drought stress. Funct Plant Biol. 2025;52. https://doi.org/10.1071/fp25092 . Wang H, Li L, Ma L, Fernie AR, Fu A, Bai C, et al. Revealing the specific regulations of nitric oxide on the postharvest ripening and senescence of bitter melon fruit. aBIOTECH. 2024;5(1):29–45. https://doi.org/10.1007/s42994-023-00110-y . Yang M, Hou G, Peng Y, Jiang Y, He C, She M, et al. editors. Exogenous GSNO and CPTIO treatment affected the ripening and glutathione metabolism of strawberry fruit. III International Symposium on Fruit Culture along Silk Road Countries 1401; 2023. https://doi.org/10.17660/ActaHortic.2024.1401.34 Yao Y, Yang Y, Ding Z, Yao K, Zhang J, Liu Z, et al. Nitric oxide delays tomato fruit softening by inhibiting SlNAP2 (NAC-like, activated by apetala3/pistillata2) transcription factor-activated transcription of soften-related genes. Int J Biol Macromol. 2025;309:143148. https://www.sciencedirect.com/science/article/pii/S0141813025037006 . Liu Z, Huang D, Yao Y, Pan X, Zhang Y, Huang Y et al. The Crucial Role of SlGSNOR in Regulating Postharvest Tomato Fruit Ripening. International Journal of Molecular Sciences. 2024;25(5):2729. https://www.mdpi.com/1422-0067/25/5/2729 Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.jpg Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 26 Mar, 2026 Reviews received at journal 24 Mar, 2026 Reviewers agreed at journal 24 Mar, 2026 Reviews received at journal 21 Mar, 2026 Reviewers agreed at journal 15 Mar, 2026 Reviewers agreed at journal 14 Mar, 2026 Reviewers agreed at journal 27 Feb, 2026 Reviewers invited by journal 25 Feb, 2026 Editor assigned by journal 21 Feb, 2026 Submission checks completed at journal 21 Feb, 2026 First submitted to journal 15 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8888370","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":596858306,"identity":"e5c7914e-292a-4e4b-869c-6c466d6b597a","order_by":0,"name":"İbrahim Samet 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20:53:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8888370/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8888370/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103616875,"identity":"b16e2723-6409-49b4-a393-8f1125e1c2cc","added_by":"auto","created_at":"2026-02-27 16:55:11","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":32731,"visible":true,"origin":"","legend":"\u003cp\u003eStandard curve of 1,1,3,3-Tetraethoxypropane (MDA).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8888370/v1/9d39e97a7dfea1d4cc4b5484.jpg"},{"id":103616873,"identity":"5a6c0a11-152b-4a15-8f98-1ddd9e769767","added_by":"auto","created_at":"2026-02-27 16:55:11","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":30678,"visible":true,"origin":"","legend":"\u003cp\u003eStandard curve of Hydrogen Peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2)\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8888370/v1/ae8d5657fd25649b31ab1579.jpg"},{"id":103616893,"identity":"d390efed-cd2f-4f7c-ae1f-cfbd8a546be4","added_by":"auto","created_at":"2026-02-27 16:55:15","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":58281,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in antioxidant enzyme activities and oxidative stress indicators in grape leaves during pre-veraison under different SNP doses\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8888370/v1/4cfaac6332377521009fb129.jpg"},{"id":103616876,"identity":"d5aa7514-58da-451a-ab1c-aacf193a021a","added_by":"auto","created_at":"2026-02-27 16:55:12","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":57765,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in antioxidant enzyme activities and oxidative stress indicators in grape leaves during veraison under different SNP doses\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8888370/v1/efa0175ea52c24c58ae9cc0f.jpg"},{"id":103616878,"identity":"20fb09cc-8c83-4733-972f-434cb45beccf","added_by":"auto","created_at":"2026-02-27 16:55:13","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":56066,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in antioxidant enzyme activities and oxidative stress indicators in grape leaves at harvest under different SNP doses\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8888370/v1/c736c207910b888e84aee926.jpg"},{"id":103616877,"identity":"6f155a84-45fa-4eb3-b817-b282c5ab904f","added_by":"auto","created_at":"2026-02-27 16:55:12","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":53898,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in antioxidant enzyme activities and oxidative stress indicators in grape berries at harvest under different SNP doses\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8888370/v1/e1acf2d40dfd0e59cc950a9a.jpg"},{"id":103616879,"identity":"6ded325f-459a-4c0e-90e1-2a8131f74382","added_by":"auto","created_at":"2026-02-27 16:55:13","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":102950,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap illustrating the relative changes in antioxidant enzyme activities (GR, SOD, CAT, APX) and oxidative stress markers (MDA and H₂O₂) across developmental stages (pre-veraison, veraison, harvest) and grape berries under different SNP doses. Data were standardized using Z-score normalization to allow comparison among variables with different units and scales.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8888370/v1/fa9d460bef5e7e8b2ec3ab61.jpg"},{"id":103616899,"identity":"f79ced92-2a7b-499f-b08a-5785fcc62de4","added_by":"auto","created_at":"2026-02-27 16:55:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1347441,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8888370/v1/ee97801c-2582-4c59-a46c-6e681afafd96.pdf"},{"id":103616892,"identity":"0e5946e9-5d89-46ca-b245-57b264d66753","added_by":"auto","created_at":"2026-02-27 16:55:15","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":223710,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8888370/v1/856d7db9ab66630fe3fcc428.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of Sodium Nitroprusside Applications on Antioxidant Defense System and Oxidative Stress in Leaves and Berries of the ‘Horoz Karası’ Grapevine (Vitis vinifera L.)","fulltext":[{"header":"Background","content":"\u003cp\u003eGrapes (\u003cem\u003eVitis vinifera\u003c/em\u003e L.), an important member of the Vitaceae family, possess considerable economic and nutritional value worldwide, both as fresh fruit and in processed products. Grape berries are characterized by their attractive aroma and flavor, as well as their rich nutritional composition. Grapes at harvest maturity represent an important source of dietary fiber, organic acids, vitamins, and minerals, and are particularly rich in phenolic compounds and antioxidant capacity. Owing to these bioactive components, grapes provide significant health benefits and are widely accepted as a functional food. Horoz Karası, one of the important grape varieties endemic to T\u0026uuml;rkiye, stands out especially for its high adaptability and favorable fruit quality. Due to increasing consumer demand and the need to protect local genetic resources, the cultivation of this variety has become increasingly important. In viticulture, increasing both yield and quality is a fundamental goal for producers, and the development of environmentally friendly and sustainable practices plays a crucial role in achieving this goal. [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eToday, various chemical treatments are used in agricultural production to increase yield and improve quality. However, the intensive use of pesticides and chemical fertilizers poses risks not only to the environment but also to product quality and human health. Therefore, there is increasing interest in lower-risk alternative applications that support the physiological and biochemical mechanisms of plants. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In this context, the use of signaling molecules that regulate growth, development, and stress responses in plants is gaining prominence. Nitric oxide (NO), one of these signaling molecules, is an important compound that plays a role in regulating many physiological processes in plants, such as photosynthesis, respiration, cell division, and aging [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. NO also plays a critical role in the development of tolerance to environmental stress conditions in plants. In particular, it contributes to the maintenance of cellular redox balance through its interaction with reactive oxygen species (ROS) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSodium nitroprusside (SNP) is one of the compounds widely used for the external application of NO in plants, considered an effective donor for controlled NO release. Owing to its light and temperature-sensitive structure, small molecular size, and high diffusion capacity, it can easily cross cell membranes and effectively regulate intracellular signaling mechanisms in plant tissues [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In plants, NO is synthesized via NO synthase-like enzymes during the conversion of L-arginine to L-citrulline and is widely distributed in different plant tissues. NO is known to exert significant effects on seed germination, stomatal movement, root development, defense responses against pathogens, and leaf physiology [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. It also plays an important role in the regulation of cell division, chlorophyll metabolism, photosynthesis, and respiration processes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. One of the most important functions of NO in plants is the control of oxidative stress. NO activates the antioxidant defense system, regulating the activities of enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR); conversely, it limits the accumulation of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) and malondialdehyde (MDA) which are indicators of lipid peroxidation. The application of NO donors such as SNP has been reported to protect membrane integrity and reduce cellular damage in different plant and fruit species [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent studies have shown that SNP applications are effective not only in vegetative tissues but also in fruit development and veraison, with significant effects on quality characteristics. Pre- or post-harvest SNP applications have been reported to enhance antioxidant capacity in fruits, regulate phenylpropanoid metabolism, and reduce quality losses [\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These findings demonstrate that SNP has the potential to improve fruit quality by maintaining oxidative balance in fruit tissues.\u003c/p\u003e \u003cp\u003eAlthough studies on NO and SNP applications in grapevines are limited, existing data indicate a regulatory role of SNP on the antioxidant defense system [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, studies evaluating the combined effects of repeated foliar SNP applications on antioxidant enzyme responses in leaves and the reflection of these responses in fruit tissue, particularly under vineyard conditions, are quite insufficient.\u003c/p\u003e \u003cp\u003eThis study aims to determine the effects of repeated applications of different SNP concentrations at specific intervals on antioxidant enzyme activities (GR, SOD, APX and CAT) and oxidative stress indicators (MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) in the leaves of the Horoz Karası grape variety, and to evaluate the relationship between these effects and biochemical responses in the fruit tissue following the final application. The originality of this study lies in the consideration of SNP applications in a temporal framework under vineyard conditions and their evaluation within the context of leaf\u0026ndash;fruit integrity. It is expected that the findings will contribute to a better understanding of the physiological and biochemical effects of NO-based applications in viticulture.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material and experimental design\u003c/h2\u003e \u003cp\u003eThis study was conducted in a commercial vineyard in Kilis province, located in the Southeastern Anatolia Region of Turkey, during the 2025 growing season. The experimental material consisted of vines of the Horoz Karası grape variety (\u003cem\u003eVitis vinifera\u003c/em\u003e L.), commonly cultivated in the region. All vines in the vineyard were of the same age (15 years), at a similar phenological development stage, and grown under uniform conditions in terms of cultural practices such as irrigation, fertilization, pruning, and disease and pest control.\u003c/p\u003e \u003cp\u003eThe experiment was designed with three treatment groups, each consisting of ten vines. At the beginning of the experiment, leaf samples were taken for control purposes before the SNP application. Subsequently, SNP solutions were applied to the treatment groups at concentrations of 100, 300, and 500 ppm respectively, using a foliar spray method; the control group was sprayed only with pure water.\u003c/p\u003e \u003cp\u003eLeaf samples were taken 15 days after the first SNP application (pre-veraison period), and a second SNP application was performed at the same doses. On the thirtieth day (veraison period), leaf samples were collected again and a third SNP application was performed. On the forty-fifth day (harvest period), both leaf and fruit samples were harvested for analysis. During this period, fruits from vines not treated with SNP were also collected as a control group.\u003c/p\u003e \u003cp\u003eAll collected leaf and fruit samples were transported to the laboratory under cold chain conditions and stored under appropriate conditions until analysis. Biochemical analyses were performed to determine oxidative stress indicators and enzyme activities of the antioxidant defense system.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBiochemical Measurements and Analyses\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of Enzyme Extracts\u003c/h2\u003e \u003cp\u003eLeaf and grape berries tissues collected after SNP treatments were immediately immersed in liquid nitrogen, pulverized to a fine powder, and preserved at \u0026minus;\u0026thinsp;80\u0026deg;C prior to biochemical analyses. For the preparation of crude enzyme extracts, 1 g of frozen material was thoroughly homogenized in a pre-chilled mortar using 100 mM phosphate buffer (pH 7.0) supplemented with 10 mM Na₂EDTA, 1 mM MgCl₂, 1% (w/v) polyvinylpyrrolidone (PVP), 10 mM KCl, and 2 mM dithiothreitol (DTT). The homogenate was subsequently filtered and centrifuged at 14,000 rpm for 20 min at 4\u0026deg;C. The clear supernatant obtained after centrifugation served as the enzyme source.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetermination of Catalase (CAT; EC 1.11.1.6) Enzyme Activity\u003c/h3\u003e\n\u003cp\u003eCatalase (CAT) activity was assayed by following the decomposition of hydrogen peroxide spectrophotometrically. The reaction mixture consisted of 30 mM KH₂PO₄/K₂HPO₄ buffer (pH 7.0) containing 10 mM H₂O₂, and the assay was started by the addition of 300 \u0026micro;L enzyme extract, bringing the final reaction volume to 1.4 mL. The decline in absorbance at 240 nm, corresponding to H₂O₂ degradation, was recorded for 3 min at 30 s intervals. Enzyme activity was calculated using an extinction coefficient of 36 mM⁻\u0026sup1; cm⁻\u0026sup1; for H₂O₂ and expressed as units per milligram of protein (U mg⁻\u0026sup1; protein). The procedure followed the method of Aebi (1984) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eDetermination of Ascorbate Peroxidase (APX; EC 1.11.1.11) Enzyme Activity\u003c/h3\u003e\n\u003cp\u003eAPX activity was assayed spectrophotometrically by monitoring the oxidation of ascorbate in the presence of hydrogen peroxide. The reaction mixture contained 0.8 mL of 50 mM potassium phosphate buffer (pH 7.0), 0.1 mL of 0.5 mM ascorbate, and 0.1 mL of enzyme extract. The reaction was started by the addition of 0.1 mL of 100 mM H₂O₂. The decrease in absorbance at 290 nm, reflecting ascorbate oxidation, was recorded over a 3-min period. Enzyme activity was calculated using an extinction coefficient of 2.8 mM⁻\u0026sup1; cm⁻\u0026sup1; and expressed on a protein basis (U mg⁻\u0026sup1; protein). The assay procedure followed the method described by Nakano and Asada (1981) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Glutathione Reductase (GR; EC 1.6.4.2) Enzyme Activity\u003c/h2\u003e \u003cp\u003eGR activity was assayed by following the oxidation of NADPH during the reduction of oxidized glutathione. The reaction mixture (final volume 2.8 mL) consisted of 120 mM phosphate buffer (pH 7.2; 2.5 mL), 0.065 mM GSSG (0.1 mL), 0.015 mM Na₂EDTA (0.1 mL), 9.6 mM NADPH (0.05 mL), and 0.1 mL enzyme extract. The decline in absorbance at 340 nm, corresponding to NADPH consumption, was monitored for 3 min. Enzyme activity was calculated and expressed as U mg⁻\u0026sup1; protein. The assay was carried out according to the procedure described by Goldberg and Spooner (1983) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetermination of Superoxide Dismutase (SOD; EC 1.15.1.1) Enzyme Activity\u003c/h3\u003e\n\u003cp\u003eSOD activity was determined by a method based on the inhibition of nitroblue tetrazolium (NBT) reduction by superoxide radicals under light. The reaction medium was prepared to contain 50 mM phosphate buffer (pH 7.8), 12 mM methionine, 75 \u0026micro;M NBT, 3 mM Na₂EDTA, and 50 mM Na₂CO₃, with different volumes of enzyme extract added. The assay was initiated by adding 10 \u0026micro;M riboflavin, after which the reaction mixtures were exposed to fluorescent light at 25\u0026deg;C to allow photochemical reduction. The formation of the blue formazan product resulting from NBT reduction was quantified spectrophotometrically at 560 nm. One unit of superoxide dismutase (SOD) activity was defined as the amount of enzyme required to cause 50% inhibition of NBT reduction under the assay conditions. Enzyme activity was expressed as U mg⁻\u0026sup1; protein. The determination of SOD activity was carried out following the method of Giannopolitis and Ries (1977) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eDetermination of Malondialdehyde (MDA) Content\u003c/h3\u003e\n\u003cp\u003eMDA content, used as an indicator of lipid peroxidation, was quantified according to the thiobarbituric acid (TBA) assay [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. For this purpose, 0.5 g of leaf tissue previously frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C was homogenized in 0.1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 10,000 rpm for 15 min at 4\u0026deg;C, and the supernatant obtained was used for subsequent measurements. For color development, 1 mL of the supernatant was mixed with 4 mL of 0.5% (w/v) TBA prepared in 20% (w/v) TCA. The reaction mixture was heated at 95\u0026deg;C for 30 min, followed by rapid cooling in an ice bath to terminate the reaction. After cooling, samples were centrifuged again at 10,000 rpm for 10 min. The absorbance of the clear supernatant was recorded at 532 nm, and non-specific turbidity was corrected by subtracting the absorbance measured at 600 nm.\u003c/p\u003e \u003cp\u003eThe MDA amount was calculated using a standard curve created with MDA standard solutions prepared in the range of 0\u0026ndash;120 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The standard equation is y\u0026thinsp;=\u0026thinsp;0.0023x\u0026thinsp;+\u0026thinsp;0.0241, and determination coefficient R\u0026sup2; = 0.991. Results are expressed in \u0026micro;mol MDA g⁻\u0026sup1; fresh weight.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Hydrogen Peroxide (H₂O₂) Content\u003c/h2\u003e \u003cp\u003eH₂O₂ content in leaf and grape berries samples was determined using a colorimetric method described in the literature, adapted to the conditions of our study [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. For analysis, 0.5 g of samples were taken from those powdered with liquid nitrogen and stored at -80\u0026deg;C. The samples were homogenized with a suitable extraction buffer under cold conditions. The resulting homogenates were centrifuged at 14,000 rpm at 4\u0026deg;C for 20 minutes, and the supernatant was used in the analyses. The determination of H₂O₂ content is based on the spectrophotometric measurement of the colored complex formed as a result of the reaction of the sample extract with the relevant reagents. At the completion of the reaction, absorbance was measured at 390 nm against a reagent blank. The concentration of H₂O₂ was quantified based on the calibration curve generated using known H₂O₂ standards (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The linear regression equation (y\u0026thinsp;=\u0026thinsp;0.01x\u0026thinsp;+\u0026thinsp;0.0359; R\u0026sup2; = 0.992) was used to calculate H₂O₂ content, and the results were expressed as mmol g⁻\u0026sup1; fresh weight.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll measurement data obtained from the experiments were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation for each application. All data were subjected to statistical analysis using SPSS version 24.0. Differences among treatments were evaluated by one-way analysis of variance (ANOVA). When significant effects were detected, mean comparisons were performed using Duncan\u0026rsquo;s multiple range test to determine the source of variation among groups. In statistical analyses, p-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 was accepted as the threshold value at which the differences between applications were significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEffects of Sodium Nitroprusside (SNP) Applications on Antioxidant Defense System and Oxidative Stress İndicators in Grape Leaves\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe effects of SNP applications on antioxidant defense system and oxidative stress indicators in the leaves of Horoz Karası grape variety showed significant dose-dependent changes throughout the phenological development process (pre-veraison, veraison, and harvest period). Compared to the control group, 100, 300, and 500 ppm SNP applications affected biochemical parameters to varying degrees in all periods.\u003c/p\u003e \u003cp\u003eFindings regarding the pre-veraison period are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. In this period, generally limited differences were observed between the SNP-treated and control groups in terms of GR, SOD, CAT, and APX activities. The one-way analysis of variance indicated that none of the evaluated parameters differed significantly among the treatments. Nevertheless, an examination of the mean values revealed a tendency toward higher GR and SOD activities in the 300 ppm SNP treatment compared with the control. In contrast, MDA and H₂O₂ levels were generally lower in the SNP-treated groups than in the untreated control. These trends point to the potential of SNP to regulate oxidative balance in the early phenological stage.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of SNP application at pre-veraison on antioxidant enzyme activities and oxidative stress markers in leaves\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNP doses\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSOD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCAT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAPX\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMDA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eH₂O₂\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3,75\u0026thinsp;\u0026plusmn;\u0026thinsp;0,48ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e75,87\u0026thinsp;\u0026plusmn;\u0026thinsp;2,48ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e6,77\u0026thinsp;\u0026plusmn;\u0026thinsp;0,61ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e12,91\u0026thinsp;\u0026plusmn;\u0026thinsp;1,07ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e5,35\u0026thinsp;\u0026plusmn;\u0026thinsp;0,01ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e93,61\u0026thinsp;\u0026plusmn;\u0026thinsp;0,99ᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100 ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4,74\u0026thinsp;\u0026plusmn;\u0026thinsp;1,34ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e81,93\u0026thinsp;\u0026plusmn;\u0026thinsp;4,59ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e8,24\u0026thinsp;\u0026plusmn;\u0026thinsp;0,52ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e15,47\u0026thinsp;\u0026plusmn;\u0026thinsp;0,00ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e5,30\u0026thinsp;\u0026plusmn;\u0026thinsp;0,05ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e80,61\u0026thinsp;\u0026plusmn;\u0026thinsp;0,42ᶜ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e300 ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6,74\u0026thinsp;\u0026plusmn;\u0026thinsp;1,14ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e95,36\u0026thinsp;\u0026plusmn;\u0026thinsp;6,78ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e8,40\u0026thinsp;\u0026plusmn;\u0026thinsp;0,84ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e16,80\u0026thinsp;\u0026plusmn;\u0026thinsp;1,70ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e5,55\u0026thinsp;\u0026plusmn;\u0026thinsp;0,05ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e97,71\u0026thinsp;\u0026plusmn;\u0026thinsp;0,28ᵃ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e500 ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4,82\u0026thinsp;\u0026plusmn;\u0026thinsp;0,45ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e86,32\u0026thinsp;\u0026plusmn;\u0026thinsp;0,31ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e6,02\u0026thinsp;\u0026plusmn;\u0026thinsp;0,60ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e14,29\u0026thinsp;\u0026plusmn;\u0026thinsp;0,00ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e5,55\u0026thinsp;\u0026plusmn;\u0026thinsp;0,09ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e80,51\u0026thinsp;\u0026plusmn;\u0026thinsp;0,85ᶜ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003ea, b, c \u0026darr;: Different lower cases represent statistically significant differences among means\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eComparisons of pre-veraison period parameters depending on SNP doses are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Graphical evaluation shows that, in particular, the application of 300 ppm SNP resulted in a numerical increase in antioxidant enzyme activities and had a suppressive effect on oxidative stress indicators.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLeaf traits measured at the veraison stage are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The effects of SNP applications became more pronounced during this period, with statistically significant differences observed between applications, particularly in terms of GR and SOD activities (p\u0026thinsp;\u0026le;\u0026thinsp;0.05). The highest levels of GR and SOD activity were observed in the 300 ppm SNP application, while the 100 and 500 ppm doses exhibited intermediate values. With regard to the CAT and APX activities, the results of the ANOVA indicated no overall significance. However, it was observed that all groups treated with SNP exhibited higher averages in comparison to the control group. When the oxidative stress indicators were evaluated, it was determined that the contents of MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decreased significantly, especially in the 100 and 300 ppm SNP applications, compared to the control group (p\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of SNP application at veraison on antioxidant enzyme activities and oxidative stress markers in leaves\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNP doses\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSOD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCAT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAPX\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMDA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eH₂O₂\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3,75\u0026thinsp;\u0026plusmn;\u0026thinsp;0,48ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e75,87\u0026thinsp;\u0026plusmn;\u0026thinsp;2,48ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e6,77\u0026thinsp;\u0026plusmn;\u0026thinsp;0,61ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e12,91\u0026thinsp;\u0026plusmn;\u0026thinsp;1,07ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e5,35\u0026thinsp;\u0026plusmn;\u0026thinsp;0,01ᵈ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e93,61\u0026thinsp;\u0026plusmn;\u0026thinsp;0,99ᶜ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100 ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5,93\u0026thinsp;\u0026plusmn;\u0026thinsp;0,44ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e56,37\u0026thinsp;\u0026plusmn;\u0026thinsp;3,29ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e9,85\u0026thinsp;\u0026plusmn;\u0026thinsp;0,94ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e17,37\u0026thinsp;\u0026plusmn;\u0026thinsp;0,98ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e4,56\u0026thinsp;\u0026plusmn;\u0026thinsp;0,06ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e61,26\u0026thinsp;\u0026plusmn;\u0026thinsp;0,49ᵃ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e300 ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e9,67\u0026thinsp;\u0026plusmn;\u0026thinsp;0,72ᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e110,54\u0026thinsp;\u0026plusmn;\u0026thinsp;9,39ᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e9,62\u0026thinsp;\u0026plusmn;\u0026thinsp;0,85ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e20,95\u0026thinsp;\u0026plusmn;\u0026thinsp;0,80ᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e4,79\u0026thinsp;\u0026plusmn;\u0026thinsp;0,04ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e91,96\u0026thinsp;\u0026plusmn;\u0026thinsp;0,49ᶜ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e500 ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6,67\u0026thinsp;\u0026plusmn;\u0026thinsp;0,00ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e91,74\u0026thinsp;\u0026plusmn;\u0026thinsp;6,29ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e9,17\u0026thinsp;\u0026plusmn;\u0026thinsp;0,45ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e17,31\u0026thinsp;\u0026plusmn;\u0026thinsp;1,17ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e5,18\u0026thinsp;\u0026plusmn;\u0026thinsp;0,02ᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e72,06\u0026thinsp;\u0026plusmn;\u0026thinsp;2,19ᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003ea, b, c \u0026darr;: Different lower cases represent statistically significant differences among means\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe comparative effects of SNP doses during the veraison period are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The graph shows that antioxidant enzyme activities reach maximum levels during the veraison period and that SNP applications enhance this response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLeaf-related parameters measured at harvest are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. It was observed that the effects of SNP applications differed depending on the parameter. Statistically significant differences were identified between the applications in terms of GR and CAT activities (p\u0026thinsp;\u0026le;\u0026thinsp;0.05), with the highest values determined in the 300 ppm SNP application. Despite the absence of statistically significant findings for SOD and APX activities, it was observed that all SNP-treated groups exhibited higher mean values in comparison to the control group. With regard to the MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content, the 100 ppm SNP application exhibited the lowest values, thus suggesting that a lower SNP dose may be more effective in limiting oxidative stress during the harvest period.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of SNP application at harvest on antioxidant enzyme activities and oxidative stress markers in leaves\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNP doses\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSOD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCAT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAPX\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMDA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eH₂O₂\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3,75\u0026thinsp;\u0026plusmn;\u0026thinsp;0,48ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e75,87\u0026thinsp;\u0026plusmn;\u0026thinsp;2,48ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e6,77\u0026thinsp;\u0026plusmn;\u0026thinsp;0,61ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e12,91\u0026thinsp;\u0026plusmn;\u0026thinsp;1,07ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e5,35\u0026thinsp;\u0026plusmn;\u0026thinsp;0,01ᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e93,61\u0026thinsp;\u0026plusmn;\u0026thinsp;0,99ᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100 ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4,14\u0026thinsp;\u0026plusmn;\u0026thinsp;0,98ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e89,12\u0026thinsp;\u0026plusmn;\u0026thinsp;9,60ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e8,47\u0026thinsp;\u0026plusmn;\u0026thinsp;0,28ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e14,57\u0026thinsp;\u0026plusmn;\u0026thinsp;1,08ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e4,34\u0026thinsp;\u0026plusmn;\u0026thinsp;0,04ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e73,66\u0026thinsp;\u0026plusmn;\u0026thinsp;0,78ᵃ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e300 ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8,30\u0026thinsp;\u0026plusmn;\u0026thinsp;1,41ᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e82,67\u0026thinsp;\u0026plusmn;\u0026thinsp;3,22ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e12,40\u0026thinsp;\u0026plusmn;\u0026thinsp;1,11ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e19,95\u0026thinsp;\u0026plusmn;\u0026thinsp;5,23ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e5,02\u0026thinsp;\u0026plusmn;\u0026thinsp;0,04ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e89,16\u0026thinsp;\u0026plusmn;\u0026thinsp;0,49ᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e500 ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6,53\u0026thinsp;\u0026plusmn;\u0026thinsp;0,49ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e81,98\u0026thinsp;\u0026plusmn;\u0026thinsp;3,77ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e7,38\u0026thinsp;\u0026plusmn;\u0026thinsp;0,39ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e17,60\u0026thinsp;\u0026plusmn;\u0026thinsp;1,08ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e5,35\u0026thinsp;\u0026plusmn;\u0026thinsp;0,05ᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e89,86\u0026thinsp;\u0026plusmn;\u0026thinsp;3,18ᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003ea, b, c \u0026darr;: Different lower cases represent statistically significant differences among means\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eHarvest-related parameters were compared across SNP doses (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). SNP applications suppress oxidative stress indicators and support the antioxidant defense system throughout phenological progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results related to grape berries parameters are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Analysis of variance revealed no significant effect of SNP treatments on grape berries GR activity compared with the control (p\u0026thinsp;=\u0026thinsp;0.408). Although the control group exhibited the lowest GR activity and the 300 ppm SNP treatment showed the highest value, these differences were not statistically meaningful. Similarly, SOD activity displayed some variation among treatments; however, the observed differences did not reach statistical significance (p\u0026thinsp;=\u0026thinsp;0.053).The highest SOD activity was detected at 300 ppm SNP, while lower activities were observed in the control and 500 ppm treatments. The CAT activity varied among the treatments; however, the difference was not statistically significant (p\u0026thinsp;=\u0026thinsp;0.052). The highest activity was recorded at 300 ppm SNP, while the control and 500 ppm treatments exhibited lower values. The effect of SNP applications on APX activity was not statistically significant. Although the highest APX value was observed in the 300 ppm application, no significant difference was observed between the applications. MDA content varied significantly depending on the treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The lowest MDA values ​​were determined in the 500 and 300 ppm SNP treatments, while the control group had the highest MDA content. Hydrogen peroxide (H₂O₂) content did not differ significantly between the SNP-treated groups and the control. Nevertheless, the lowest H₂O₂ level was observed in the 300 ppm SNP treatment, whereas the control group exhibited the highest value.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of SNP application on antioxidant enzyme activities and oxidative stress markers in in grape berries tissue\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSNP doses\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSOD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCAT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAPX\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMDA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eH₂O₂\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1,564\u0026thinsp;\u0026plusmn;\u0026thinsp;0,170ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e17,264\u0026thinsp;\u0026plusmn;\u0026thinsp;0,480ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1,874\u0026thinsp;\u0026plusmn;\u0026thinsp;0,491ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4,819\u0026thinsp;\u0026plusmn;\u0026thinsp;0,757ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e2,129\u0026thinsp;\u0026plusmn;\u0026thinsp;0,037ᶜ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e9,860\u0026thinsp;\u0026plusmn;\u0026thinsp;0,212ᵈ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100 ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1,645\u0026thinsp;\u0026plusmn;\u0026thinsp;0,332ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e18,415\u0026thinsp;\u0026plusmn;\u0026thinsp;0,815ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2,237\u0026thinsp;\u0026plusmn;\u0026thinsp;0,096ᵃᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e5,229\u0026thinsp;\u0026plusmn;\u0026thinsp;0,739ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1,981\u0026thinsp;\u0026plusmn;\u0026thinsp;0,025ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e7,510\u0026thinsp;\u0026plusmn;\u0026thinsp;0,141ᵇ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e300 ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1,833\u0026thinsp;\u0026plusmn;\u0026thinsp;0,324ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e19,808\u0026thinsp;\u0026plusmn;\u0026thinsp;0,929ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3,237\u0026thinsp;\u0026plusmn;\u0026thinsp;0,467ᵇ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e6,116\u0026thinsp;\u0026plusmn;\u0026thinsp;0,000ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1,755\u0026thinsp;\u0026plusmn;\u0026thinsp;0,049ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e6,710\u0026thinsp;\u0026plusmn;\u0026thinsp;0,283ᵃ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e500 ppm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1,682\u0026thinsp;\u0026plusmn;\u0026thinsp;0,297ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e17,906\u0026thinsp;\u0026plusmn;\u0026thinsp;0,338ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1,759\u0026thinsp;\u0026plusmn;\u0026thinsp;0,257ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e5,380\u0026thinsp;\u0026plusmn;\u0026thinsp;0,331ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e1,720\u0026thinsp;\u0026plusmn;\u0026thinsp;0,049ᵃ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e8,610\u0026thinsp;\u0026plusmn;\u0026thinsp;0,424ᶜ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003ea, b, c \u0026darr;: Different lower cases represent statistically significant differences among means\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eA comparison of antioxidant enzyme activities and oxidative stress markers in grape berries among different SNP applications is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. No statistically significant differences were observed among treatments for GR, SOD, CAT, and APX activities; however, 300 ppm SNP consistently exhibited higher enzyme activities compared to the control and 500 ppm treatments. In contrast, MDA content differed significantly among treatments, with markedly lower values in the 300 and 500 ppm SNP applications relative to the control. Although H₂O₂ content did not differ significantly, the lowest levels were observed at 300 ppm SNP, whereas the control showed the highest values. Overall, the comparative graphical analysis indicates that SNP treatment, particularly at 300 ppm, is associated with enhanced antioxidant responses and reduced oxidative stress in grape berries.\u0026rdquo;\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo provide an integrated overview of the antioxidant system and oxidative stress responses, a heatmap based on Z-score normalization was constructed (Figure X). The visualization revealed clear stage-dependent differences in the response to SNP applications.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt veraison, particularly under 300 ppm SNP, antioxidant enzyme activities (GR, SOD, CAT, and APX) showed pronounced increases compared to the control, as indicated by the intense red clusters. Importantly, this enzymatic activation was accompanied by a marked reduction in oxidative stress markers, with both MDA and H₂O₂ exhibiting lower relative values than the control treatment. This pattern suggests that SNP application, especially at 300 ppm, effectively enhanced antioxidant defense while simultaneously mitigating lipid peroxidation and hydrogen peroxide accumulation.\u003c/p\u003e \u003cp\u003eA similar but less pronounced trend was observed at pre-veraison and harvest stages, where elevated enzymatic activities were generally associated with reduced MDA and H₂O₂ levels relative to the control. In contrast, grape berries displayed comparatively lower antioxidant enzyme activities and a distinct oxidative profile, indicating tissue-specific regulation of redox metabolism during fruit development.\u003c/p\u003e \u003cp\u003eOverall, the coordinated increase in antioxidant enzymes together with the decrease in MDA and H₂O₂ under 300 ppm SNP highlights this dose as the most effective treatment for improving redox homeostasis across developmental stages.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, foliar SNP applications were found to dynamically modulate the antioxidant defense system throughout the phenological stages and to limit oxidative damage in the Horoz Karası grape variety. In particular, the observation that the 300 ppm SNP dose was the most effective concentration in both leaf and grape berries tissues supports current findings indicating that NO exhibits a hormetic effect in plants. While low doses act as signaling molecules that trigger defense responses, the reduced effectiveness observed at higher doses (such as 500 ppm) can be explained by the tightly regulated and delicate balance of NO metabolism [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The integrative heatmap analysis provides further evidence that SNP-mediated nitric oxide signaling enhances redox regulation in a stage-dependent manner. The coordinated increase in antioxidant enzyme activities (GR, SOD, CAT, and APX), together with the reduction of MDA and H₂O₂ levels compared to the control, indicates that SNP application effectively mitigates oxidative damage while strengthening the antioxidant defense system. Particularly, the 300 ppm dose appears to optimize redox homeostasis across developmental stages. These findings suggest that exogenous SNP plays a regulatory role in maintaining cellular oxidative balance during grapevine development.\u003c/p\u003e \u003cp\u003eThe results indicated that SNP treatments led to a marked increase in the activities of major antioxidant enzymes, namely SOD, CAT, and GR, with the most pronounced effects observed during the veraison stage. Superoxide dismutase (SOD) acts as the primary enzymatic barrier against oxidative stress by catalyzing the dismutation of superoxide radicals. Subsequently, enzymes such as CAT and APX facilitate the conversion of the generated H₂O₂ into less harmful molecules, thereby contributing to the regulation of cellular redox balance. Consistent with these results, previous studies have reported that NO applications activate the Ascorbate\u0026ndash;Glutathione (Asada\u0026ndash;Halliwell) cycle in grape tissues, leading to increased activities of SOD, CAT, APX, and GR, while simultaneously suppressing ROS accumulation and lipid peroxidation [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In line with this, a molecular-level study conducted on grape berries in 2023 demonstrated that NO donors have been reported to stimulate phenylpropanoid pathway activity and to enhance the transcription of antioxidant defense\u0026ndash;associated genes, including VvSOD and VvCAT [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In line with these findings, earlier studies have shown that the combined treatment of NO and H₂S alleviated oxidative damage by promoting the activities of major antioxidant enzymes such as APX, CAT, and GR. This coordinated upregulation was accompanied by reduced accumulation of MDA and H₂O₂, indicating an improved cellular redox status [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. These molecular insights provide a robust mechanistic explanation for the observation that the 300 ppm SNP application was associated with higher antioxidant enzyme activities compared to the other doses evaluated in the present study. When evaluated in terms of oxidative stress indicators, the significant reduction in MDA levels in both leaf and grape berries tissues following SNP applications reflects the protective effect of NO against membrane lipid peroxidation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. NO can stabilize free radicals through direct interactions with reactive oxygen species and can also indirectly limit ROS accumulation by inducing the antioxidant defense system [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Current studies demonstrate that NO applications slow the oxidation of unsaturated fatty acids in cell membranes, preserve fruit tissue integrity, and reduce post-harvest quality losses [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. For Horoz Karası, which is among thin-skinned and oxidative stress\u0026ndash;sensitive grape varieties, this protective effect is particularly important for the sustainability of fruit quality.\u003c/p\u003e \u003cp\u003eAnother noteworthy finding of the study is that the effects of SNP applications became more pronounced during the veraison and harvesting periods compared to the pre-veraison stage. This suggests that exogenous NO applications act as a \u0026ldquo;buffer mechanism\u0026rdquo; against the increased production of ROS associated with elevated metabolic activity during veraison [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Indeed, recent studies have demonstrated that NO can delay fruit ripening and softening processes. For example, a recent study reported that NO significantly enhanced the activity of key enzymes involved in the phenylpropanoid biosynthesis pathway, thereby increasing disease resistance and mitigating fruit softening in bitter melon [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In another study, the combined application of gibberellic acid and NO was shown to delay fruit ripening and color development in strawberry fruits by regulating chromatin-level processes [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Similarly, in tomato fruits, NO application delayed ripening and softening by inhibiting the expression of cell wall-softening genes [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In addition, S-nitrosoglutathione application has been reported to delay color change and slow the ripening process during post-harvest storage of tomato fruits [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe observation that foliar-applied SNP influenced antioxidant enzyme activities and MDA levels in fruit tissue indicates that the NO signal is systemically transported through the vascular system of the grapevine, thereby proactively regulating fruit quality within the framework of leaf\u0026ndash;fruit integrity.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study shown that foliar application of SNP effectively regulates the antioxidant defense system and limits oxidative damage throughout the phenological process in the Horoz Karası grape variety. Considering all evaluated physiological and biochemical parameters, the 300 ppm SNP dose emerged as the optimum concentration, maximizing antioxidant enzyme activities while minimizing MDA and H₂O₂ accumulation.\u003c/p\u003e \u003cp\u003eThe more pronounced effects of SNP applications during the verasion and harvest stages indicate that exogenous NO supplementation acts as an efficient regulator, balancing the oxidative load associated with increased metabolic activity. Moreover, the influence of foliar SNP application on oxidative stress indicators and antioxidant responses in fruit tissue suggests that NO signaling operates systemically, maintaining leaf\u0026ndash;fruit functional integrity.\u003c/p\u003e \u003cp\u003eOverall, these findings highlight the strong potential of NO-based applications as a sustainable strategy for oxidative stress management and grape quality preservation in viticulture.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eAPX\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAscorbate peroxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eAsA\u0026ndash;GSH cycle\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAscorbate\u0026ndash;Glutathione cycle\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCAT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCatalase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGibberellic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGR\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlutathione reductase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eH₂O₂\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHydrogen peroxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMDA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMalondialdehyde\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eNO\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNitric oxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eNO\u0026thinsp;+\u0026thinsp;H₂S\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNitric oxide\u0026thinsp;+\u0026thinsp;Hydrogen sulfide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eROS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSNP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSodium nitroprusside\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSOD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSuperoxide dismutase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research received no external funding.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eİSG was solely responsible for the conceptualization and design of the study, experimental implementation, data acquisition and statistical analysis, interpretation of findings, and preparation and critical revision of the manuscript. The author approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKamiloglu O. Influence of some cultural practices on yield, fruit quality and individual anthocyanins of table grape cv.\u0026lsquo;Horoz Karasi\u0026rsquo;. J Anim Plant Sci. 2011;21(2):240\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSucu Dağ S, G\u0026uuml;n B, Şekeroğlu N. The Effects of Cluster Thinning Yield and Quality on \u0026lsquo;Horoz Karası\u0026rsquo; Cultivar of Grapevine. 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International Journal of Molecular Sciences. 2024;25(5):2729. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.mdpi.com/1422-0067/25/5/2729\u003c/span\u003e\u003cspan address=\"https://www.mdpi.com/1422-0067/25/5/2729\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chemical-and-biological-technologies-in-agriculture","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Chemical and Biological Technologies in Agriculture](https://chembioagro.springeropen.com/)","snPcode":"40538","submissionUrl":"https://submission.nature.com/new-submission/40538/3","title":"Chemical and Biological Technologies in Agriculture","twitterHandle":"@SpringerPlants","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Vitis vinifera L., antioxidant enzymes, nitric oxide, sodium nitroprusside, Horoz Karası","lastPublishedDoi":"10.21203/rs.3.rs-8888370/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8888370/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNitric oxide (NO) has been widely recognized as a multifunctional signaling molecule in plants, participating in the regulation of growth, developmental transitions, and stress adaptation processes. Its involvement in modulating cellular redox homeostasis is considered one of the key mechanisms underlying these effects. In experimental studies, sodium nitroprusside (SNP) is frequently employed as an exogenous NO donor, and numerous reports indicate that SNP treatments can activate antioxidant systems in different plant species. However, information regarding the temporal effects of repeated SNP applications under vineyard conditions and their integrated effect on leaf and grape berries antioxidant responses in grapevines remains limited.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFoliar SNP applications significantly modulated antioxidant enzyme activities and oxidative stress indicators in the Horoz Karası grape variety. Among the tested concentrations, 300 ppm SNP consistently resulted in the highest activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR), while effectively suppressing malondialdehyde (MDA) and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) accumulation in leaf tissues. The effects of SNP were more pronounced during the veraison and harvest periods than in the pre-veraison stage, indicating a stronger regulatory role under elevated metabolic activity. In grape berries tissue, SNP applications, particularly at 300 ppm, reduced MDA levels and promoted higher antioxidant enzyme activities, demonstrating a systemic leaf– grape berries interaction mediated by NO signaling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepeated foliar application of SNP effectively enhances antioxidant defense capacity and limits oxidative damage in Horoz Karası grapevines in a dose- and time-dependent manner. The 300 ppm concentration emerged as the optimal dose for maintaining redox homeostasis in both leaf and grape berries tissues. These findings highlight the potential of NO-based applications as an environmentally friendly strategy to improve oxidative stress management and quality preservation in sustainable viticulture practices.\u003c/p\u003e","manuscriptTitle":"Effects of Sodium Nitroprusside Applications on Antioxidant Defense System and Oxidative Stress in Leaves and Berries of the ‘Horoz Karası’ Grapevine (Vitis vinifera L.)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-27 16:54:32","doi":"10.21203/rs.3.rs-8888370/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-26T07:42:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-24T11:47:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"187059538587266008088965350390791663468","date":"2026-03-24T07:20:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-21T04:19:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"187059538587266008088965350390791663468","date":"2026-03-15T09:34:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"219176297440920862201899942215837133108","date":"2026-03-14T14:11:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"15624711356637364145536537754326638767","date":"2026-02-27T11:37:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-25T08:21:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-21T08:36:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-21T08:33:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chemical and Biological Technologies in Agriculture","date":"2026-02-15T20:48:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chemical-and-biological-technologies-in-agriculture","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Chemical and Biological Technologies in Agriculture](https://chembioagro.springeropen.com/)","snPcode":"40538","submissionUrl":"https://submission.nature.com/new-submission/40538/3","title":"Chemical and Biological Technologies in Agriculture","twitterHandle":"@SpringerPlants","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"78a3a8c5-db0b-44bc-bcf5-ff96c9b4c628","owner":[],"postedDate":"February 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T09:26:01+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-27 16:54:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8888370","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8888370","identity":"rs-8888370","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}