Alleviation of cold stress impacts on grapes by the chitosan-salicylic acid nanocomposite (CS-SA NCs) application

Plant material and growing conditions

In accordance with relevant institutional and national guidelines and legislation, uniform-sized cuttings of Vitis vinifera ‘Flame seedless’ cultivar were obtained from a local nursery. The cuttings were rooted in perlite and cultivated in a greenhouse maintained at 22–25 °C during the day and 18–20 °C at night. This experiment was conducted on biennial plants (2 years old). During acclimation, the plants were nourished using the hydroponic nutrient solution described by Cramer & Läuchli (1986). A two-branch pruning method was applied tothe young plants.

Foliar treatment with chitosan-salicylic acid nanocomposite (CS-SA NCs) (2 liters) was applied when the plants had developed 10–12 fully expanded leaves. Treatments were applied on plants 1, 6, and 12 h prior to cold stress exposure, while control plants were sprayed with distilled water (0.5 liter). Following each foliar treatment, the plants were transferred to cold storage for 1, 4, 8, and 16 h at +1 ± 0.5 °C; control plants were maintained at 22 °C. The experimental design included two factors, the first factor was CS-SA NCs application at four intervals (control, 1, 6, and 12 h before cold stress); the second factor was cold stress duration (1, 4, 8, and 16 h at +1 ± 0.5 °C, and control at 22 °C), in four replications. After cold treatment, leaf samples (from fully expanded leaves) were collected. Some were immediately transferred to the laboratory for analysis, while others were frozen in liquid nitrogen and stored at –80 °C until further evaluation.

Synthesis of chitosan-salicylic acid nanocomposites (CS-SA NCs)

Low molecular weight chitosan (Mw = 100 kDa, DD = 85%, Purity = 97 wt%) was obtained from Dr. Mahdavinia company, Maragheh, Iran. Tripolyphosphate (TPP; Merck company, Germany) and used as a crosslinking agent to form chitosan nanoparticles. The synthesis method followed the gelation procedure described by Ahmadi et al. (2018). To load salicylic acid (SA), 0.0138 g (0.1 mmol) of SA powder was dissolved in 1,000 mL of distilled water. Then, 1 mL (0.1 wt%) of acetic acid was added to the SA solution tofacilitate chitosan dissolution. To reach (SA)-loaded chitosan nanocomposites, 0.1 wt% of chitosan solution was prepared by adding 1 g of chitosan powder to the SA solution. After complete dissolution, 0.4 g of TPP was dissolved in 20 mL of distilled water and slowly added to the chitosan solution under continuous stirring (700 rpm). The resulting homogeneous SA-loaded chitosan nanocomposites were used for foliar applications without further modification.

Chitosan-salicylic acid nanocomposite (CS-SA NCs) characterization

Transmission Electron Microscopy (TEM) analysis of the fabricated CS-SA NCs clearly illustrates the successful formation of well-defined octahedral structures, with particle sizes ranging from approximately 70 to 100 nm, indicating a uniform and controlled synthesis process (Fig. 1A). These findings were further supported by Dynamic Light Scattering (DLS) analysis, which validated the consistency of particle size distribution (Fig. 1B).

Measurement of chlorophyll fluorescence

Chlorophyll fluorescence was measured in the most recently developed leaves under light-mode conditions using a portable fluorometer (PAM 2500-WALZ, Germany). These measurements were used to assess photosynthetic efficiency and the physiological response of plants to environmental conditions or treatments. The fluorometer provided precise readings of key parameters, including the maximum quantum yield of Photosystem II (PSII) and other fluorescence-related indicators.

Electrolyte leakage

Electrolyte leakage was assessed by measuring the electrical conductivity (EC) of leaf samples, following the method described by Nayyar (2003). Ten uniform leaves were placed in 20 ml of distilled water andshaken for 24 h at room temperature. The initial electrical conductivity 1 (EC1) was determined with an electrical conductivity (EC) meter. Then, the samples were autoclaved for 2 h at 120 °C, and the final electrical conductivity 2 (EC2) was recorded. Electrolyte leakage was calculated using the formula (Nayyar, 2003):

EC (%) = (EC1/EC2)×100.

Photosynthetic pigments

Chlorophylls (Chl a and Chl b), and carotenoids (CARs) were determined spectrophotometrically. Fresh leaf tissue (0.5 g) was ground using liquid nitrogen to preserve pigments and prevent degradation during the extraction process. The ground material was then suspended in 10 ml of 80% acetone as the extraction solvent, ensuring efficient solubilization of chlorophylls and carotenoids. The resulting mixture was thoroughly homogenized and centrifuged to separate the supernatant containing pigments. The concentrations of Chl a, Chl b, and CARs were accurately quantified by measuring the absorbance of the pigment extract at 663 nm, 645 nm, and 470 nm and expressed as mg g⁻¹ fresh weight (FW) by the below equations (Arnon, 1949).

(1) $Chlorophylla=\left[\left(12.7\left(A663\right)\right)-\left(2.69\left(A645\right)\right)\right]\times v/1000w$

(2) $Chlorophyllb=\left[\left(22.9\left(A645\right)\right)-\left(2.69\left(A663\right)\right)\right]\times v/1000w$

(3) $Carotenoids=\left[100\left(A470\right)+3.27\left(Chla\right)-104\left(Chlb\right)\right]/227$.

Malondialdehyde content

Grape leaf samples (0.2 g) were thoroughly homogenized in 5 mL of 1% trichloroacetic acid (TCA) and centrifuged at 12,000 g for 15 min to separate the solid and liquid phases. 1 mL of the resulting supernatant was mixed with 4 mL of a solution containing 20% TCA and 0.5% thiobarbituric acid (TBA), then heated for 30 min. Samples were immediately cooled on ice. Absorbance of the samples was subsequently measured using a T80+ spectrophotometer (China) at two specific wavelengths: 532 and 600 nm. MDA content was calculated and expressed as nmol g⁻¹ FW using the following formula (Heath & Packer, 1968): ${\displaystyle \mathrm{MDA}=\left[\left(\mathrm{A}532\mathrm{nm}-\mathrm{A}600\mathrm{nm}\right)\times 20\right]/155\times 100.}$

Hydrogen peroxide content

Frozen grape leaf tissue (0.2 g) was homogenized in two mL of 0.1% trichloroacetic acid (TCA) and centrifuged at 12,000 g for 15 min. The supernatant (0.5 mL) was mixed with 0.5 mL of phosphate buffer (10 mL, pH 7.0) and 1 mL of potassium iodide solution (1 M). The reaction produced a colored complex. The absorbance of the resulting solution was measured at 390 nm using a spectrophotometer. Hydrogen peroxide content was quantified using a standard H₂O₂ curve and expressed as mg g⁻¹ FW (Velikova, Yordanov & Edreva, 2000).

Enzymatic extraction

To assess the enzymatic activities of catalase and guaiacol peroxidase, 0.5 g of grape leaf tissue was homogenized in liquid nitrogen. The homogenate was then mixed with 5 mL of cold phosphate buffer (pH = 7.5) containing 0.5 mL ethylenediaminetetraacetic acid (EDTA), which stabilizes enzymes and prevents metal ion interference. The mixture was centrifuged at 15,000 rpm for 15 min at 4 °C. The resulting supernatant, containing proteins and enzymes, was carefully collected and stored at −20 °C until further analysis (Sairam, Rao & Srivastava, 2002).

Total soluble protein content

The reaction mixture for determining total soluble protein content consisted of three components: 100 µL of enzyme solution, 200 µL of Bradford reagent, and 700 µL of deionized water. The mixture was allowed to react for 2 min to ensure optimal complex formation between the reagent and amino acids, resulting in a color change proportional to protein concentration. Absorbance was measured at 535 nm using a spectrophotometer. Protein concentration was quantified using a standard bovine serum albumin (BSA) and expressed as mg g⁻¹ FW (Bradford, 1976).

Catalase activity

To assess catalase activity, the enzyme extract was mixed with cold buffer and EDTA. The reaction was initiated by adding 0.5 mL of 75 mM hydrogen peroxide. Enzymatic activity was monitored by measuring the decrease in absorbance at 240 nm over 1 min, which corresponds to the breakdown of hydrogen peroxide into water and oxygen. The rate of absorbance change was used to calculate catalase activity, expressedas µmol min⁻¹ mg⁻¹ protein (Dezar et al., 2005).

Ascorbate peroxidase (APX) activity

Grape leaf tissue (0.5 g) was homogenized in liquid nitrogen and suspended in 2 ml of 0.1 M phosphate buffer containing 0.5 mM EDTA, 5% polyvinyl pyrrolidone, and 2 mM ascorbate. After centrifugation, the supernatant was used for the enzymatic assay. The reaction mixture consisted of 250 µL of phosphate buffer (25 mM, pH 7.0), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 10 µL of hydrogen peroxide (1 mM), 250 µL of reduced ascorbate (0.25 mM), 50 µL of the enzyme extract, and 190 mL of distilled water. The reaction was monitored spectrophotometrically at 290 nm over 1 min. APX activity was calculated using the extinction coefficient of 2.8 mM⁻¹ cm⁻¹, and expressed as µmol min⁻¹ mg⁻¹ protein) (Stewart & Bewley, 1980).

Guaiacol peroxidase (GPX) activity

The GPX assay was performed using a reaction mixture containing 1 mL of phosphate buffer (100 mM, pH 7.0), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1 mL of guaiacol (15 mM), 1 mL of hydrogen peroxide (3 mM), and 50 µL of enzyme extract. The reaction was initiated by adding the enzyme solution, and the increase in absorbance at 470 nm was monitored spectrophotometrically for 1 min. GPX activity was calculatedusing an extinction coefficient of 26.6 mM⁻¹ cm⁻¹. The enzyme activity was expressed as µmol min⁻¹ mg⁻¹ protein (Yoshimura et al., 2000).

Superoxide dismutase (SOD) activity

Grape leaf tissue (0.5 g) was homogenized in liquid nitrogen and mixed with 50 mM sodium phosphate buffer, 1 mM EDTA, and 2% polyvinylpyrrolidone (PVPP). After centrifugation, the supernatant was used for the enzymatic assay. SOD activity was assessed by its ability to inhibit the photoreduction of nitroblutetrazolium (NBT). The reaction mixture included 50 mL of 50 mM potassium phosphate buffer (pH 7.5) 75 μM nitroblutetrazolium, 13 μM methionine, 0.1 μM EDTA, and 4 μM riboflavin. This process was monitored spectrophotometrically at 560 nm, and SOD activity was expressed as µmol min⁻¹ mg⁻¹ protein (Giannopolitis & Ries, 1977).

Glutathione activity

Grape leaf tissue (0.2 g) was homogenized in 2 mL of 5% sulfosalicylic acid to extract cellular components, including glutathione. The homogenate was centrifuged at 15,000 rpm for 10 min. From the supernatant, 300 μL was collected and neutralized with 18 μL of 7.5 M triethanolamine. For quantification, 50 μL of the neutralized supernatant was mixed with 700 μL of 0.3 mM nicotinamide adenine dinucleotide phosphate (NADPH), 100 μL of 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), and 150 μL of 125 mM phosphate buffer (pH 6.5) containing 6.3 mM EDTA. The reaction was initiated by adding, 0.1 unit of glutathione reductase, which reduces oxidized glutathione (GSSG) to its reduced form (GSH), while oxidizing NADPH to NADP⁺. Absorbance was measured at 412 nm. A standard curve using known concentrations of both reduced (GSH) and oxidized (GSSG) glutathione was used to quantify glutathione levels,. expressed as µmol min⁻¹ mg⁻¹ protein (Griffith, 1980).

Ascorbate activity

A 0.2 g portion of the grape leaf sample was homogenized in 1 metaphosphoric acid. The homogenate was centrifuged at 22,000 rpm for 15 min at 25 °C. To the supernatant, 150 μM phosphate buffer (pH 7.4) and 200 μL of distilled water were added. Subsequently, 400 μL of 10% trichloroacetic acid (TCA), followed by 400 μL of 44% phosphoric acid, 400 μL of 4% bipyridyl in 70% ethanol to form a colored complex, and 200 μL of 3% ferric chloride (FeCl₃) to catalyze the reaction. After vortexing, the samples were incubated at 37 °C for 1 hour to allow full color development. Absorbance was measured spectrophotometrically at 525 nm, corresponding to the absorption maximum of the ascorbate-bipyridyl complex. To determine total ascorbate (including both reduced ascorbate and oxidized ascorbate, dehydroascorbate, DHA), 100 μL of 10 mM dithiothreitol (DTT) was added to the reaction mixture. A standard curve was constructed using known concentrations of reduced ascorbate to ensure accurate quantification. Final results were expressed as µmol g⁻¹ FW (Law, Charles & Halliwell, 1983).

Experimental design and data analysis

The experiment was conducted as a factorial based on a randomized complete block design (RCBD) with three replications. Analysis of variance (ANOVA) was performed using MSTAT-C version 6.2.1 (https://www.canr.msu.edu/afre/projects/microcomputer_statistical_package_mstat._1983_1985). Significant differences among means were evaluated with Duncan’s multiple range test at P < 0.05 and P < 0.01. Standard deviations (n = 3) were calculated for all traits. Pearson’s correlation and cluster dendrogram heat maps were generated using R software (version 4.1.1; June 2024), (URL https://cran.r-project.org/bin/windows/base/old/4.1.1/).

Chlorophyll florescence

Variance analysis revealed that the interaction between cold stress duration and CS-SA nanocomposite treatment significantly affected F0, Fm, Fv, Fv/F0, Fv/Fm and Y(II) (P ≤ 0.01). As cold stress duration increased, F0 and Y(II) values rose in the ‘Flame Seedless‘ cultivar, while Fm, Fv, Fv/F0, and Fv/Fm declined compared to the control. Application of CS-SA NCs under cold stress conditions longer than 4 h led to an increase in the Fv/Fm index, indicating reduced damage to the photosynthetic apparatus. 16 hours of cold stress increased F0 by 1.66 times compared to the temperature control. Pretreatment with CS-SA NCs 12 h before cold exposure under the same conditions. Cold stress for 16 h caused reductions of 28.59, 47.75, 68.5 and 27.71% in Fm, Fv, Fv/F0 and Fv/Fm, respectively, compared to the control. Pretreatment with CS-SA NCs, especially 12-hours prior to cold stress, improved Fm, Fv, Fv/F0 and Fv/Fm under 16-hour cold stress (Table 1).

Photosynthetic pigments content

The interaction between cold stress duration and chitosan-salicylic acid nanocomposite treatment had no significant effect on chlorophyll a, chlorophyll b, or total chlorophyll content. However, the main effect of temperature was significant (P ≤ 0.01). Increasing exposure time at +1 °C significantly reduced chlorophyll a, chlorophyll b, and total chlorophyll content. Foliar application of CS-SA NCs up to 12 h before cold stress improved chlorophyll content. Total chlorophyll content decreased by 69.86% after 16 h of cold stress (+1 °C) compared to the control. Application of CS-SA NCs 12 h before cold treatment increased total chlorophyll content by 14.56% compared to the control. The highest carotenoids content was observed with foliar application of CS-SA NCs applied 12 h before cold stress under 8-hour exposure. The lowest carotenoid content was recorded in the 16-hour cold stress treatment without foliar application (Figs. 2A–2G).

Membrane stability and signaling responses

Electrolyte leakage increased with longer cold stress exposure, while foliar application of CS-SA NCs significantly reduced leakage (P ≤ 0.05). The highest electrolyte leakage (58.15%) occurred in the 16-hour cold stress treatment without foliar application, while the lowest (14.82%) was observed in the temperature control (22 °C). Total soluble protein content increased with cold stress up to 4 h, then significantly decreased At the level of p ≤ 0.05 at 16 h. The highest total soluble protein content was observed in plants treated with CS-SA NCs 12 h before cold stress and exposed for 4 h. The lowest total soluble protein content was observed in the 16-hour cold stress treatment without foliar application. Malondialdehyde content increased with longer cold stress exposure. At 16 h, treatments with CS-SA NCs applied 1, 6, and 12 h before cold stress showed 2.44-, 3.23-, 4.33-, and 3.97- fold increases in MDA compared to the control, respectively. Hydrogen peroxide content also increased with cold stress duration. The highest hydrogen peroxide level was observed in the 16-hour cold stress treatment without foliar application, while the lowest was observed in plants treated with CS-SA NCs 12 h before stress and not exposed to cold (Figs. 3A–3D).

Antioxidant enzymes activity

Activities of catalase (CAT), gluthatione reductase (GR), ascorbate peroxidase (APX), and guaiacol peroxidase (GPX) increased with cold stress up to 4 h, then declined with longer exposure (16 h). The highest CAT, GR, and GPX enzyme activities were observed in plants treated with CS-SA NCs 6 h before cold stress and exposed for 4 h, showing 7.55-, 4.8-, and 6.39-fold increases compared to the control, respectively. The lowest activities of CAT, GR, and GPX enzymes were recorded in the control group. APX activity peaked in plants treated with CS-SA NCs 1 h before cold stress and exposed for 1 and 4 h. The lowest APX activity was recorded in the 16-hour cold stress treatment without foliar application. The highest SOD activity was was recorded in plants treated with CS-SA NCs 6 h before cold stress and exposed for 4 h, while the lowest was found in the 16-hour cold stress treatment without foliar application (Figs. 4A–4E).

Non-enzymatic antioxidant activity

Ascorbate (AsA) levels and AsA/Dehydroascorbate (DHA) ratio decreased with increasing cold stress duration, while DHA activity significantly increased. The highest activity of ascorbate (AsA) was observed in plants treated with CS-SA NCs 12 h before cold exposure and not subjected to cold stress, showing a 62% increase compared to the control. The lowest ascorbate activity was recorded in the 16-hour cold stress treatment without foliar application, which was 58% lower than the control. Similarly, reduced glutathione (GSH) levels and GSH/GSSG (oxidized glutathione) ratio declined with cold stress, while GSSG activity increased significantly with prolonged exposure. The highest glutathione (GSH) activity was observed in plants treated with CS-SA NCs 12 h before cold stress and not exposed to cold, showing a 61% increase compared to the control. The lowest glutathione activity was recorded in the 16-hour cold stress treatment without foliar application, showing a 55% decrease compared to the control (Figs. 5A–5F).

Multivariate analysis of cold stress duration and CS-SA NCs foliar application in ‘Flame seedless’ grapevine

Pearson’s correlation analysis of biochemical, enzymatic, and non-enzymatic antioxidant characteristics are shown in Fig. 5. Positive correlations were observed among Chl a, Chl b, total chl, carotenoids (CARs), enzymatic antioxidants, AsA, AsA/DHA, GSH, and GSH/GSSG. In contrast, photosynthetic pigments were negatively associated with MDA, GSSG, DHA, H₂O_2, and electrolyte leakage (EL). A significant positive correlation (P ≤ 0.05) was observed among EL, GSSG, MDA, H₂O_2, and DHA. Electrolyte leakage was negatively correlated with enzymatic antioxidants, AsA, AsA/DHA, GSH, and GSH/GSSG (Fig. 6).

Heat map analysis of ‘Flame seedless’ grapevine plants responses to cold stress duration and CS-SA NCs treatment revealed that traits such as H₂O₂, MDA, and GSSG were positive associated with cold stress. Conversely, traits including photosynthetic pigments, protein content, and non-enzymatic antioxidants (AsA, GSH, and GSH/GSSG) showed negative associations. The heat map indicated that CS-SA NCs application mitigated the effects of cold stress (Fig. 7A).

Cluster analysis and dendrograms identified three distinct groups under cold stress conditions. Group I contained: H₂O₂, GSSG, EL and MDA; group II included: CARs, DHA, CAT, AsA/DHA, AsA, GSH and GSH/GSSG, and group III consisted of Chl a, Chl b, total Chl, GPX, APX, GR, SOD and proteins content (Fig. 7A). Principal Component Analysis (PCA) confirmed the clustering observed in the heap map (Fig. 7B). Overall, cluster analysis revealed three treatment classes. Class I contained the plants treated with CS-SA NCs for 6 and 12 h and exposed to cold stress for 1 h, as well as untreated plants not exposed to cold stress. Class II contained plants exposed to cold stress for 4, 8, and 16 h and treated with CS-SA NCs for 6 or 12 h. Finally, class III included plants exposed to cold stress treated with CS-SA NCs for 1 h, and untreated plsants under cold stress (Figs. 7A–7B).