Introduction

Strawberry (Fragaria × ananassa Duch.) is a globally valued fruit crop prized for its sensory qualities, high nutritional content, and significant economic contribution to horticulture. However, its inherently soft texture, high respiration rate, and susceptibility to microbial decay contribute to a short postharvest life1. Rapid deterioration is primarily driven by cell wall disassembly—mediated by enzymes such as polygalacturonase (PG), pectin methylesterase (PME), and cellulase—and oxidative stress arising from reactive oxygen species (ROS) accumulation during ripening and storage. Conventional postharvest preservation relies heavily on synthetic chemical preservatives, but increasing safety, regulatory, and environmental concerns have intensified the search for eco-friendly alternatives2. Preharvest application of natural or low-toxicity plant regulators offers a sustainable strategy to improve fruit resilience before harvest. Among such agents, salicylic acid (SA)—a naturally occurring phenolic phytohormone—has been widely reported to modulate stress signaling pathways, activate the phenylpropanoid pathway, and enhance antioxidant enzyme systems, including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD)3,4. In strawberries, SA delays ripening, suppresses oxidative stress, and maintains firmness by regulating ROS-scavenging mechanisms and secondary metabolite biosynthesis5,6. Maleic hydrazide (MH) is a plant growth regulator with anti-senescent properties that inhibits cell division and slows metabolic processes. It delays ripening by suppressing ethylene biosynthesis and reducing the activity of cell wall-degrading enzymes7. MH improves firmness retention and delays senescence8,9, though its role in strawberries remains limited.Postharvest ethylene action inhibition is a proven approach for slowing fruit senescence. 1-Methylcyclopropene (1-MCP) binds irreversibly to ethylene receptors, thereby blocking ethylene perception and delaying senescence10. Although strawberries are non-climacteric, ethylene influences softening, color development, aroma formation, and decay susceptibility11,12. Application of 1-MCP reduces respiration, slows softening, maintains ascorbic acid, and enhances antioxidant activity during cold storage13,14,15, partly by suppressing cell wall-related gene expression16. Despite evidence supporting SA, MH, and 1-MCP individually, no study has integrated preharvest SA and MH priming with postharvest ethylene inhibition using 1-MCP in strawberries. The present two-season study evaluates the interactive effects of these treatments on postharvest physiology, biochemical attributes, antioxidant status, and enzyme activities of ‘Nabila’ strawberries.

Materials and methods

Experimental site and plant material

A two-season field experiment (2023–2024) was conducted at the experimental orchard of the Division of Fruit Science, Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu (SKUAST-J), Chatha, India (32.63°N, 74.85°E; 332 m above sea level). The site experiences a subtropical climate with mild winters and warm summers. Uniform, healthy, and disease-free plants of Fragaria × ananassa Duch. cv. ‘Nabila’ grown under open-field conditions in raised beds were selected. Standard agronomic practices were maintained throughout the crop cycle.

Experimental design and treatment application

The trial was arranged in a randomized complete block design (RCBD) with three replications per treatment, each replication consisting of 10 plants. Treatments comprised:

T₁: Control (distilled water spray).

T₂: Salicylic acid (SA, 1 mM).

T₃: Maleic hydrazide (MH, 250 ppm).

T₄: SA (1 mM) + MH (250 ppm).

T₅: SA (1 mM) + MH (250 ppm) + postharvest 1-methylcyclopropene (1-MCP, 1 µL L⁻¹ for 12 h at 20 °C).

Preharvest sprays(approximately 20–25 mL solution per plant) were applied at three phenological stages—10%, 50%, and 100% flowering—using a hand-held pressure sprayer (Solo 408, Germany) in the early morning to ensure uniform coverage of foliage and developing fruits. A surfactant (0.05% Tween-20) was included in all spray solutions to enhance penetration.

Postharvest 1-MCP treatment

For treatments involving 1-MCP, freshly harvested fruits were placed in an airtight chamber and exposed to 1-methylcyclopropene gas (1 µL L⁻¹) for 12 h at 20 ± 1 °C. This was carried out immediately after sanitization and prior to cold storage.

Harvesting and storage

Fruits were harvested at commercial ripeness (75–100% red surface coloration), sanitized in 0.01% sodium hypochlorite, rinsed with distilled water, and air-dried. Samples were packed in perforated PET clamshell containers (EcoFresh, India) and stored at 4 ± 1 °C and 85–90% relative humidity (RH) in a cold chamber (Remi CI-10 Plus, India) for 6 days. Quality assessments were performed on day 0 (immediately after harvest) and day 6 of storage.

Physiological quality parameters

Fruit firmness

Firmness was assessed using a digital texture analyzer (TA.XT2i, Stable Micro Systems, UK) equipped with a 2 mm cylindrical stainless-steel probe. Fruits were placed on the flat platform, and the probe was driven perpendicularly into the equatorial zone at a constant speed of 1 mm/s. The maximum force (in Newtons) required to penetrate the fruit was recorded.

Weight loss

Fruits were individually weighed at the beginning and at designated intervals during storage using a precision digital balance (Mettler Toledo ME204, Switzerland; ±0.01 g accuracy). Weight loss (%) was calculated as: (Initial weight − Final weight)/Initial weight] × 100.

Total soluble solids (TSS)

TSS content was determined in °Brix using a digital refractometer (Atago PAL-1, Japan). Juice was extracted from pooled fruits, filtered, and 2–3 drops were placed on the refractometer prism.

Titratable acidity (TA)

TA was measured by titrating 10 mL of clarified juice with 0.1 N NaOH to an endpoint of pH 8.1 using phenolphthalein as an indicator. The results were expressed as a percentage of citric acid equivalents.

TSS: TA ratio

This ratio was calculated by dividing the TSS by TA, providing an index of fruit palatability and sensory balance.

Respiration rate

Respiration rate was measured using a static closed system with approximately 200 g of fruit in a 2 L airtight jar incubated for 2 h at 20 ± 1 °C. The incubation time and fruit load were deliberately optimized based on preliminary trials to ensure that CO2 accumulation remained within the linear response range of the infrared gas analyzer and below levels known to suppress strawberry respiration. Given the relatively short incubation period and the moderate respiration rate of strawberry at 20 °C, the estimated CO2concentration in the headspace did not exceed 0.5–1%, a threshold widely considered non-inhibitory for respiratory activity.

Cell wall-degrading enzyme activities

Crude enzyme extracts were obtained by homogenizing 5 g of fruit tissue in 10 mL of cold 50 mM sodium acetate buffer (pH 5.0) containing 1 mM EDTA and 1% PVP to inhibit phenolic oxidation. The homogenate was centrifuged at 10,000 rpm for 15 min at 4 °C using a refrigerated centrifuge (Eppendorf 5810R, Germany), and the supernatant was used for enzyme assays. All enzymatic activities were expressed as units per g fresh weight (U/g FW), and as percentage changes relative to the control.

Polygalacturonase (PG)

PG activity was assayed by incubating the extract with 1% polygalacturonic acid in 50 mM acetate buffer (pH 5.0) at 37 °C for 1 h. Reducing sugars released were quantified using the DNS method, and absorbance was measured at 540 nm using a UV–Vis spectrophotometer (Shimadzu UV-1900i, Japan).

Pectin methylesterase (PME)

PME activity was estimated by mixing the extract with 1% citrus pectin in 0.1 M NaCl (pH 7.5), and titrating released carboxyl groups with 0.01 N NaOH to pH 8.0 using a pH-stat titration system (MetrohmTitrando 888, Switzerland).

Cellulase

Cellulase activity was determined by incubating the enzyme extract with 2% carboxymethyl cellulose (CMC) in 50 mM acetate buffer at 37 °C. The released glucose was measured using the DNS method at 540 nm on the Shimadzu UV-1900i spectrophotometer.

Biochemical and antioxidant analyses

Total phenolic content (TPC)

TPC was measured by adding 0.5 mL of extract to 2.5 mL of 10% Folin–Ciocalteu reagent and 2 mL of 7.5% Na₂CO₃. After incubation at 25 °C for 30 min, absorbance was measured at 765 nm using a UV–Vis spectrophotometer (Shimadzu UV-1900i, Japan). Results were expressed as mg gallic acid equivalents (GAE)/100 g FW. Phenolics contribute to antioxidant activity and disease resistance17.

Total flavonoid content (TFC)

TFC was estimated by mixing 1 mL of extract with 4 mL distilled water, 0.3 mL 5% NaNO₂, 0.3 mL 10% AlCl₃, and 2 mL 1 M NaOH. Absorbance was read at 510 nm using the Shimadzu UV-1900i spectrophotometer (Japan), and results expressed as mg quercetin equivalents (QE)/100 g FW. Flavonoids possess strong antioxidant and anti-inflammatory properties18.

Anthocyanin content

Anthocyanin levels were determined by the pH differential method. Absorbance at 520 and 700 nm was measured using buffers at pH 1.0 and pH 4.5 on a Shimadzu UV-1900i spectrophotometer (Japan). Results were expressed as mg cyanidin-3-glucoside equivalents/100 g FW. Anthocyanins are key contributors to color and antioxidant functionality19.

Ascorbic acid content

Ascorbic acid was extracted with 3% metaphosphoric acid and titrated against 2,6-dichlorophenolindophenol (DCPIP) using a manual burette system (Borosil, India) until a persistent pink color appeared. Results were expressed as mg/100 g FW. Ascorbate plays a central role in ROS scavenging and enzyme cofactor activity20.

Lipid peroxidation (MDA)

Malondialdehyde (MDA) was measured using the thiobarbituric acid reactive substances (TBARS) assay. Extracts were reacted with 0.5% TBA in 20% TCA and heated at 95 °C for 30 min. Absorbance was read at 532 and 600 nm using the Shimadzu UV-1900i spectrophotometer (Japan). Results were expressed as nmol MDA/g FW. Elevated MDA indicates oxidative membrane damage21.

Antioxidant enzyme assays

Enzyme extracts were prepared by homogenizing 5 g of fresh fruit tissue in 10 mL of ice-cold 50 mM phosphate buffer (pH 7.0) with 1% PVP and 0.1 mM EDTA. The homogenate was centrifuged at 12,000 rpm for 15 min at 4 °C using a refrigerated centrifuge (Eppendorf 5810R, Germany). The clear supernatant was used for enzyme activity assays.

Superoxide dismutase (SOD)

SOD activity was determined by monitoring the inhibition of NBT photoreduction at 560 nm using the Shimadzu UV-1900i spectrophotometer (Japan). One unit of SOD was defined as the amount of enzyme required to cause 50% inhibition22.

Catalase (CAT)

CAT activity was measured by monitoring the decomposition of H₂O₂ at 240 nm for 1 min using the Shimadzu UV-1900i spectrophotometer. Results were expressed as µmol H2O2decomposed/min/mg protein23.

Ascorbate peroxidase (APX)

APX activity was assayed by measuring the decline in absorbance at 290 nm due to ascorbate oxidation. The reaction mixture contained 50 mM phosphate buffer, 0.5 mM ascorbate, and 0.1 mM H2O2. Absorbance was read on the Shimadzu UV-1900i spectrophotometer24.

Guaiacol peroxidase (POD)

POD activity was estimated using guaiacol as a substrate in the presence of H2O2. The increase in absorbance at 470 nm due to tetraguaiacol formation was recorded using the Shimadzu UV-1900i spectrophotometer (Japan)25.

Statistical analysis

The experimental data collected for the years 2023 and 2024 were subjected to multivariate statistical analysis using R version 4.3.3. Mean differences among treatments were compared using Tukey’s HSD test at p < 0.05. Principal Component Analysis (PCA) was applied to reduce dimensionality and identify major contributing variables. A scree plot was used to determine the number of significant principal components, while a biplot provided insight into the correlation and distribution of variables. All values are reported as the mean ± standard error (SE) of three biological replicates.

Results

Fruit firmness and enzyme activity

Across both seasons (2023 and 2024), preharvest applications of salicylic acid (SA) and maleic hydrazide (MH), either alone or in combination, significantly enhanced firmness retention during cold storage compared to the untreated control (T₁). The effect was most pronounced when the combined preharvest application was supplemented with a postharvest 1-methylcyclopropene (1-MCP) treatment (T₅) (Fig. 1). On day 6 of cold storage, fruits from T₅ retained firmness values of 3.05 N in 2023 and 3.12 N in 2024, representing 64% and 67% higher firmness, respectively, than T₁ (1.86 N in 2023; 1.87 N in 2024). The combined preharvest SA + MH treatment (T₄) also showed notable firmness preservation—2.85 N in 2023 and 2.91 N in 2024—corresponding to 52% and 54% higher values than T₁. SA alone (T₂) and MH alone (T₃) maintained intermediate firmness levels, indicating partial but less pronounced protection against softening. Enzymatic profiling revealed a treatment-dependent suppression of cell wall-degrading enzymes. In T₅, polygalacturonase (PG) activity was reduced by 53.4% (2023) and 51.8% (2024) compared with T₁, while pectin methylesterase (PME) activity declined by 48.6% and 46.9%, and cellulase activity dropped by 55.2% and 56.4%, respectively. T₄ also showed substantial enzyme suppression—PG activity decreased by 41.2% (2023) and 39.5% (2024), PME by 37.8% and 35.6%, and cellulase by 43.1% and 45.0%. The inhibitory effect was consistently lower in T₂ and T₃. The dual-action preharvest SA + MH approach (T₄) clearly delayed the onset of pectin demethylation, pectin depolymerization, and cellulose hydrolysis, thereby reducing cell wall disassembly. However, the integration of postharvest 1-MCP (T₅) further amplified these effects, suggesting a combined interplay between ethylene action inhibition (via 1-MCP) and preharvest elicitor-induced fortification of cell wall structure.

Fig. 1
figure 1

Effect of preharvest SA, MH, SA + MH, and postharvest 1-MCP treatments on firmness and cell wall-degrading enzyme activities in strawberry fruit (2023 and 2024). Each bar represents the mean value ± standard error (SE) of different parameters. (a) Fruit firmness expressed as Newtons (N) measured on day 6 of cold storage. (b) Polygalacturonase (PG) activity expressed as U mg⁻¹ protein. (c) Pectin methylesterase (PME) activity expressed as U mg⁻¹ protein. (d) Cellulase activity expressed as U mg⁻¹ protein.

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The combined preharvest application of salicylic acid (SA) and maleic hydrazide (MH) (T₄), together with the postharvest treatment of 1-methylcyclopropene (1-MCP; T₅), markedly enhanced key biochemical quality parameters of strawberry fruits during postharvest storage in both 2023 and 2024 (Fig. 2). Total soluble solids (TSS) exhibited significant elevation under T₅, reaching 13.8 °Brix in 2023 and 14.2 °Brix in 2024—representing a 23.3% and 22.4% increase, respectively, relative to the untreated control (T₁). Likewise, T₄-treated fruits recorded TSS values of 9.8 °Brix (2023) and 10.1 °Brix (2024), corresponding to 19.5% and 21.2% increases over the control. The enhancement in TSS under these treatments can be attributed to improved carbohydrate metabolism and reduced respiratory consumption of soluble sugars during storage.

Titratable acidity (TA) was also maintained at comparatively higher levels in treated fruits, particularly under T₄, which showed 13.8% and 12.6% higher TA than the control in 2023 and 2024, respectively, while T₅ treatment further elevated TA by 18.8% and 20.6% across the two seasons. This maintenance of organic acid pools indicates a delay in acid degradation, contributing to sustained flavor and metabolic stability during storage.

In contrast, the respiration rate exhibited a pronounced decline under both treatments. Fruits treated with T₅ showed 34.3% and 36.4% lower CO₂ evolution in 2023 and 2024, respectively, while those under T₄ recorded reductions of 24.6% and 26.9% relative to the control. The suppression of respiration reflects reduced metabolic turnover, lower energy dissipation, and delayed senescence—key indicators of improved storage performance.

Ascorbic acid content, a critical determinant of antioxidant capacity and nutritional quality, increased substantially under these treatments. T₅-treated fruits exhibited 44.4% and 47.6% higher ascorbic acid levels in 2023 and 2024, respectively, compared to the control, whereas T₄-treated fruits showed moderate but consistent increases of 34.2% and 36.7%. These enhancements suggest improved preservation of vitamin C, likely due to reduced oxidative degradation and the synergistic antioxidative effects induced by SA and MH priming.

Collectively, these results demonstrate that the integration of preharvest SA + MH and postharvest 1-MCP treatments effectively modulates fruit metabolism by minimizing respiration-driven senescence while sustaining sugar–acid balance and enhancing antioxidant retention. Such biochemical optimization underpins extended shelf life, superior postharvest stability, and improved marketable quality in strawberries across both growing seasons.

Fig. 2
figure 2

Effect of Preharvest SA, MH, SA + MH, and Postharvest 1-MCP Treatments on Total Soluble Solids, Titratable Acidity, Respiration Rate, and Ascorbic Acid Content in Strawberry Fruit During Cold Storage (2023 and 2024). Each bar represents the mean value ± standard error (SE) of different parametersa) Total soluble solids (TSS) expressed as °Brix, measured on day 6 of cold storage. (b) Titratable acidity (TA) expressed as percentage (% citric acid equivalents). (c) Respiration rate expressed as mg CO2 kg⁻¹ h⁻¹. (d) Ascorbic acid content expressed as mg/ 100 g⁻¹ fresh weight (FW).

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The combined preharvest treatment of salicylic acid (SA) and maleic hydrazide (MH) (T₄), together with the postharvest application of 1-methylcyclopropene (1-MCP) (T₅), markedly enhanced the accumulation of key bioactive compounds in strawberry fruits during both 2023 and 2024 (Fig. 3). Total phenolic content (TPC) under T₅ reached 260.6 mg GAE/100 g FW in 2023 and 264.2 mg GAE/100 g FW in 2024, corresponding to 50.6% and 51.7% higher values, respectively, compared with the untreated control (T₁). Similarly, T₄-treated fruits recorded TPC values of 239.8 mg GAE/100 g FW (2023) and 242.1 mg GAE/100 g FW (2024), reflecting 41.7% and 42.9% increases over the control. This marked elevation in phenolic content indicates activation of the phenylpropanoid pathway—a well-established response to SA-mediated defense signaling. Total flavonoid content (TFC) also increased significantly under elicitor and 1-MCP treatments. T₅-treated fruits exhibited TFC values of 88.1 mg QE/100 g FW (2023) and 89.2 mg QE/100 g FW (2024), representing 45.6% and 47.2% higher levels, respectively, than the control, whereas T₄ fruits recorded 78.5 mg QE/100 g FW (2023) and 79.3 mg QE/100 g FW (2024), corresponding to 37.3% and 39.1% increases. Flavonoids, as potent antioxidants, contribute to enhanced oxidative stability and nutraceutical quality, suggesting that preharvest elicitation effectively stimulated secondary metabolite synthesis. Anthocyanin accumulation followed a similar trend. Fruits under T₅ recorded 50.4 mg and 48.7 mg cyanidin-3-glucoside equivalents/100 g FW in 2023 and 2024, showing 42.6% and 43.2% increases relative to the control, while T₄ fruits recorded 40.2 mg and 40.8 mg equivalents/100 g FW, reflecting 34.4% and 36.5% higher levels, respectively. The pronounced enhancement in anthocyanin biosynthesis under T₅ is likely mediated through coordinated regulation of key transcription factors such as MYB and bHLH, which govern the flavonoid biosynthetic pathway. Collectively, these findings demonstrate that the integration of preharvest SA + MH with postharvest 1-MCP application exerts a synergistic effect on secondary metabolism, leading to significant enrichment of phenolics, flavonoids, and anthocyanins. The consistent responses across both seasons highlight the stability, reproducibility, and practical potential of this integrated strategy for improving the phytochemical richness and antioxidant capacity of strawberries.

Fig. 3
figure 3

Impact of Preharvest SA, MH, SA + MH, and Postharvest 1-MCP Treatments on Strawberry Phytochemical Profiles (2023–2024). Each bar represents the mean value ± standard error (SE) of different parameters (a) Total phenolic content (TPC; mg GAE/100 g FW). (b) Total flavonoid content (TFC; mg QE/100 g FW). (c) Total anthocyanin content (mg cyanidin-3-glucoside equiv./100 g FW).

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The combined preharvest treatment of salicylic acid (SA) and maleic hydrazide (MH) (T₄), together with the postharvest application of 1-methylcyclopropene (1-MCP) (T₅), significantly mitigated oxidative damage and enhanced the antioxidant defense system in strawberry fruits across both 2023 and 2024 seasons. As shown in Fig. 4, malondialdehyde (MDA) content—a reliable indicator of lipid peroxidation and membrane injury—was markedly reduced in T₅ fruits (1.11 nmol g⁻¹ FW in 2023; 1.16 nmol g⁻¹ FW in 2024), representing 48.1% and 47.2% decreases, respectively, compared with the untreated control (T₁). Similarly, T₄-treated fruits recorded MDA levels of 1.38 nmol g⁻¹ FW (2023) and 1.36 nmol g⁻¹ FW (2024), corresponding to 41.2% and 42.0% reductions, indicating substantial suppression of oxidative stress and improved membrane stability. In parallel, antioxidant enzymes exhibited significant upregulation under elicitor and 1-MCP treatments. Superoxide dismutase (SOD) activity increased by 45.6% (2023) and 44.1% (2024) under T₅, compared to the control, whereas T₄ fruits recorded respective increases of 38.7% and 39.5%. Catalase (CAT) activity under T₅ rose by 42.2% in 2023 and 40.8% in 2024, while ascorbate peroxidase (APX) and guaiacol peroxidase (POD) showed respective enhancements of 44.5–45.0% and 41.0–43.2% across both seasons. These pronounced increases in enzymatic antioxidant activities reflect the synergistic interaction between SA and MH-induced defense priming and the ethylene action inhibition conferred by 1-MCP, resulting in superior reactive oxygen species (ROS) detoxification efficiency. Collectively, the concurrent decline in MDA content and elevation of antioxidant enzyme activities under T₅ and T₄ treatments demonstrate their efficacy in reinforcing oxidative stress tolerance, maintaining membrane integrity, and extending postharvest fruit longevity. The reproducibility of these responses across two consecutive seasons underscores the robustness, consistency, and practical potential of this integrated preharvest–postharvest strategy for improving the oxidative stability and storage quality of strawberries.

Fig. 4
figure 4

Effect of Preharvest SA, MH, SA + MH, and Postharvest 1-MCP Treatments on Lipid Peroxidation and Antioxidant Enzyme Activities in Strawberry Fruit (2023 & 2024). Each bar represents the mean value ± standard error (SE) of different parameters.(A) Malondialdehyde (MDA) content (nmol g⁻¹ FW). (B) Superoxide dismutase (SOD) (U mg⁻¹ protein). (C) Catalase (CAT) (µmol H₂O₂ min⁻¹ mg⁻¹ protein). (D) Ascorbate peroxidase (APX) (µmol ascorbate min⁻¹ mg⁻¹ protein). (E) Guaiacol peroxidase (POD) (µmol tetraguaiacol min⁻¹ mg⁻¹ protein).

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The PCA biplot Fig. 5 illustrates the relationships among physiological and biochemical attributes of strawberry fruit. The first principal component (PC1) explains 79.9% of the total variance, primarily associated with positive loadings for firmness, TSS, ascorbic acid, total phenolics, and antioxidant enzymes (SOD, CAT), indicating strong intercorrelations among these traits. The second component (PC2), accounting for 9.1% variance, is influenced by anthocyanins and titratable acidity. Negative loadings for PG, PME, cellulase, MDA, and respiration indicate their inverse relationship with fruit quality traits. Overall, PC1 effectively distinguishes quality-enhancing parameters from those linked to senescence.

Fig. 5
figure 5

PCA Biplot of Biochemical and Physiological Traits (2023–2024).

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The scree plot Fig. 6 shows the percentage of variance explained by each principal component (PC) in the dataset. PC1 accounts for the largest share of variance (79.9%), indicating it captures most of the variability among strawberry fruit attributes. PC2 contributes an additional 9.1%, while PC3 explains only 4.0%. The sharp decline after PC1 suggests that the first two components are sufficient to represent the majority of data variability, making them the most significant for interpretation.

Fig. 6
figure 6

Scree plot of principal components.

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The correlation matrix Fig. 7 illustrates the interrelationships among physiological and biochemical attributes of strawberry fruit. Firmness, TSS, ascorbic acid, TPC, flavonoids, and antioxidant enzymes (SOD and CAT) exhibited strong positive correlations with each other, indicating their collective role in maintaining fruit quality. Negative correlations were observed between these quality traits and enzymes associated with fruit softening and senescence (PG, PME, cellulase) as well as MDA and respiration rate. Titratable acidity (TA) also showed a strong positive association with firmness and TSS. Conversely, weight loss demonstrated a negative correlation with most biochemical quality parameters, highlighting its inverse relationship with fruit stability and shelf life.

Fig. 7
figure 7

Correlation matrix depicting relationships among physiological and biochemical attributes of strawberry fruit.

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Discussion

The present study demonstrated that preharvest application of salicylic acid (SA) and maleic hydrazide (MH), individually or in combination, significantly enhanced the postharvest firmness, biochemical composition, and antioxidant capacity of strawberry fruits, with the most pronounced effects observed when combined with postharvest treatment of 1-methylcyclopropene (1-MCP).Maleic hydrazide (MH) is an established plant growth regulator whose dietary safety has been evaluated by international regulatory authorities. The FAO/WHO Joint Meeting on Pesticide Residues (JMPR) has assessed MH toxicology and established an Acceptable Daily Intake (ADI) of 0–0.3 mg kg⁻¹ body weight day⁻¹, indicating low chronic toxicity when used according to good agricultural practices26. In addition, regulatory evaluations by the United States Environmental Protection Agency have concluded that MH residues do not pose a significant risk to consumers at recommended application rates, supporting its safe use across food commodities27. These assessments collectively support the consumer safety of preharvest MH application in soft fruits when applied within approved limits. These findings are consistent across two consecutive seasons (2023 and 2024), indicating a robust and reproducible response.Strawberries are highly perishable fruits with a relatively short postharvest shelf life, even under refrigerated conditions; typical shelf life at cold storage (≈ 4 °C) is generally in the range of 5–7 days, after which quality parameters such as firmness, soluble solids, and decay change rapidly and marketability declines sharply28. Quantitative studies have shown that significant declines in textural and biochemical attributes frequently occur within the first week of cold storage, with many quality indicators exhibiting substantial variation by Day 629]– [30. Therefore, Day 6 was selected as the terminal point in the present study to capture this critical transition from acceptable to deteriorated quality in the untreated control group and assess treatment efficacy up to the practical limit of commercial storage life. We acknowledge that the absence of intermediate sampling limits the temporal resolution of physiological changes between Days 0 and 6; however, the chosen interval aligns with the typical timeframe wherein strawberry quality undergoes its most pronounced deterioration under cold storage and is commonly used in postharvest research evaluating shelf life and treatment effects. Future work may integrate additional intermediate time points (e.g., Day 2 or Day 4) to better detail the kinetics of quality changes.

Firmness retention and cell wall enzyme modulation

Firmness retention is a key indicator of postharvest quality and consumer acceptability. In the present study, the combined preharvest application of salicylic acid (SA) and maleic hydrazide (MH) with postharvest 1-methylcyclopropene (1-MCP) (T₅) resulted in the highest firmness retention, approximately 64–67% higher than the untreated control. This improvement was associated with strong suppression of cell wall-degrading enzymes such as polygalacturonase (PG), pectin methylesterase (PME), and cellulase. Coordinated downregulation of these enzymes is critical for maintaining the integrity of the pectocellulosic matrix and reducing middle lamella polysaccharide disassembly during cold storage31.

The observed reductions in PG (~ 53%), PME (~ 48%), and cellulase (~ 55%) activities under T₅ indicate that the combined preharvest–postharvest strategy effectively delayed pectin solubilization and cellulose hydrolysis. These results are consistent with32, who reported that 1-MCP inhibited PG, β-galactosidase, and α-arabinofuranosidase activity in strawberries, resulting in improved textural stability. Ethylene inhibition by 1-MCP likely suppressed the transcription of ripening-related genes involved in cell wall disassembly, while SA and MH elicitation may have induced endogenous enzyme inhibitors such as polygalacturonase-inhibiting proteins (PGIPs) or PME inhibitors (PMEIs), further reinforcing cell wall structure33,34.

SA is known to modulate fruit softening through signaling cross-talk with ethylene and abscisic acid (ABA) pathways, leading to reduced PG and PME activity in several fruits35. MH, in contrast, functions as a growth regulator that can reduce ethylene biosynthesis and delay cell wall loosening, likely through interference with auxin- and ethylene-mediated transcription of cell wall hydrolases36. In non-climacteric fruits such as strawberry, softening is primarily governed by the transcriptional regulation of cell wall-modifying enzymes rather than by ethylene bursts alone. MH is a well-characterized plant growth regulator that interferes with cell division and phytohormone signaling, particularly by inhibiting auxin-related responses and downstream gene expression involved in cell wall metabolism37. Studies in other plant systems have shown that MH treatment leads to downregulation of auxin-responsive genes and alteration of hormone balance, including enhanced ABA signaling, which influences ripening-related metabolic pathways and cell wall dynamics38.Although direct transcriptomic studies in strawberry are limited, the established inhibitory effect of MH on auxin signaling supports the hypothesis that MH can reduce the expression of cell wall–degrading enzymes by modulating hormone-regulated transcriptional networks underlying softening. By restricting the activation of genes encoding pectinolytic and cellulolytic enzymes through repression of auxin-mediated pathways, MH contributes to slower pectin depolymerization and maintenance of cell wall integrity. This, together with postharvest ethylene inhibition via 1-MCP, explains the strong firmness retention observed in T₅-treated fruit.

Maintenance of biochemical quality and respiration suppression

The marked enhancement of total soluble solids (TSS) and titratable acidity (TA) under SA + MH and 1-MCP treatments suggests improved carbohydrate retention and sustained metabolic stability during cold storage. Fruits treated with T₅ exhibited 22–23% higher TSS and up to 20% higher TA compared with controls, accompanied by a 34–36% reduction in respiration rate. The suppression of CO₂ evolution indicates reduced metabolic turnover and delayed senescence, a hallmark of prolonged storability39.

The elevated TSS could be attributed to improved carbohydrate metabolism and decreased consumption of soluble sugars as respiratory substrates. Similarly, the retention of TA implies slower degradation of organic acids such as citric and malic acid—key intermediates in the tricarboxylic acid (TCA) cycle—under reduced respiratory activity. This pattern is consistent with previous reports in strawberries and apples, where 1-MCP application suppressed respiration and delayed acid catabolism32,34. Preharvest SA treatment has also been shown to reduce respiration rate by downregulating glycolytic enzymes, thus conserving soluble sugars35. The concurrent suppression of respiration and ethylene signaling in T₅ likely minimized carbon loss and maintained the sugar–acid equilibrium that defines sensory quality and storage potential.

Enhancement of antioxidant system and phenolic metabolism

The combined treatments significantly elevated antioxidant potential, as indicated by higher levels of ascorbic acid, total phenolics, flavonoids, and anthocyanins, along with marked reductions in malondialdehyde (MDA) accumulation. The increase in ascorbic acid content (up to 47.6% higher under T₅) underscores the capacity of SA- and MH-primed fruits to preserve redox balance by maintaining the ascorbate–glutathione cycle. The elevation of enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxidase (POD) under both T₄ and T₅ further supports the activation of ROS-scavenging mechanisms40.

The accumulation of phenolic and flavonoid compounds (up to 42–43% and 39% increases under T₅, respectively) indicates stimulation of the phenylpropanoid pathway. SA acts as a signaling molecule that enhances phenylalanine ammonia-lyase (PAL) activity and the expression of downstream biosynthetic genes involved in phenolic and anthocyanin synthesis41. The synergistic enhancement observed under 1-MCP application suggests that reduced ethylene signaling may further redirect metabolic flux towards secondary metabolite accumulation rather than catabolic oxidation. Anthocyanin enrichment under T₅ (34–36% higher than control) aligns with previous studies linking SA treatment to increased expression of MYB and bHLH transcription factors regulating flavonoid biosynthesis36.

In parallel, the reduction in MDA levels (up to 43% lower in T₅) reflects decreased lipid peroxidation and improved membrane integrity. This decrease can be directly linked to enhanced antioxidant enzyme activities, which efficiently detoxify hydrogen peroxide and superoxide radicals. The coordinated activation of both enzymatic and non-enzymatic antioxidants demonstrates a priming effect of SA + MH, reinforced by ethylene inhibition through 1-MCP. Such reinforcement of antioxidant metabolism is consistent with observations by36, who noted that combined elicitor and ethylene inhibitor treatments minimize oxidative stress and senescence in perishable fruits.

Integrated mechanism and practical implications

Taken together, the results suggest a dual-mechanism model: (i) preharvest SA + MH priming strengthens cell wall structure and enhances the antioxidant system, and (ii) postharvest 1-MCPprevents ethylene-induced degradation and metabolic overactivation. The resulting synergy yields superior firmness retention, delayed respiration-driven senescence, and improved nutraceutical quality during cold storage. The reproducibility of these effects over two seasons confirms the robustness of this integrated approach under field-to-storage transition conditions.From an applied standpoint, this combined strategy offers a sustainable method to improve strawberry shelf life and marketable quality without resorting to synthetic preservatives or high-energy storage interventions. By modulating both structural (cell wall) and metabolic (antioxidant) processes, the preharvest SA + MH and postharvest 1-MCP treatments represent a holistic solution for extending strawberry storability while retaining its sensory and nutritional attributes.

Conclusion

The integrated preharvest application of salicylic acid (SA) and maleic hydrazide (MH), combined with postharvest treatment using 1-methylcyclopropene (1-MCP), markedly enhanced strawberry fruit quality and storage performance. This synergistic strategy effectively preserved fruit firmness by suppressing the activities of key cell wall–degrading enzymes, including polygalacturonase, pectin methylesterase and cellulase, thereby delaying pectin depolymerization and maintaining cell wall integrity. The structural stability was further associated with a significant reduction in respiration rate, reflecting lowered metabolic activity and delayed senescence during cold storage. Improved retention of total soluble solids and titratable acidity indicated sustained carbohydrate reserves and moderated organic acid degradation, collectively contributing to better flavor preservation and metabolic balance. Concurrently, the combined treatments reinforced the antioxidant defense system through enhanced activities of superoxide dismutase, catalase, ascorbate peroxidase, and peroxidase, resulting in reduced malondialdehyde accumulation and improved membrane stability. Elevated concentrations of phenolics, flavonoids, and anthocyanins further augmented antioxidant capacity and supported color retention, underscoring the role of elicitor-induced metabolic priming alongside ethylene inhibition in preserving nutritional and sensory attributes. Future investigations should incorporate direct measurements of rotting incidence and pathogen dynamics across extended storage durations to more comprehensively validate the commercial applicability of this integrated preharvest–postharvest strategy.