تأثیر تیمار تریپتوفان بر حفظ بازارپسندی و افزایش عمر انبارمانی میوه گیلاس

Introduction
Sweet cherry (Prunus avium L.) is a popular stone fruit globally, prized for its desirable flavor, high nutritional value, and significant antioxidant properties (Nava-Ochoa et al., 2025). As a non-climacteric fruit with low ethylene production during ripening, it is highly perishable and has a short postharvest life (Blando and Oomah, 2019). This fruit faces substantial postharvest challenges due to its high respiration rate and susceptibility to fungal decay, leading to rapid quality losses such as reduced firmness, stem browning, and undesirable changes in color, flavor, and nutritional value during storage (Mujtaba et al., 2023). In recent years, the use of bioactive compounds to elicit defense responses has emerged as a sustainable postharvest strategy. Amino acids (AAs), as organic compounds and protein precursors, act as biostimulants that enhance plant metabolic efficiency and growth (Matysiak et al., 2020; Trovato et al., 2021). By scavenging reactive oxygen species (ROS) and maintaining cellular integrity, they can reinforce plant defense systems against environmental stresses. Among them, tryptophan (β-indolyl alanine), an aromatic amino acid biosynthesized via the shikimate pathway, has found extensive applications in food, medical, and agricultural industries (Xiao et al., 2023). In the postharvest context, exogenous application or endogenous accumulation of tryptophan has been linked to reduced chilling injury and fungal decay, delayed senescence, and quality preservation in fruits. These beneficial effects are largely attributed to its role as a precursor for key bioactive molecules such as melatonin, serotonin, auxin, and nicotinamide (NAD+), which are involved in signaling regulation and strengthening plant defense mechanisms (Yuxiao et al., 2023; Aghdam and Arnao, 2024). Supporting evidence highlights the efficacy of tryptophan in various fruits. For example, a 100 µM tryptophan treatment in strawberries and blueberries increased endogenous melatonin and salicylic acid accumulation while reducing ethylene and abscisic acid production, thereby extending storability. Furthermore, by supplying necessary nitrogen for cell wall synthesis, tryptophan enhanced firmness in cherries and raspberries by reinforcing pectin, hemicellulose, and lignin structures (Arabia et al., 2025). Previous studies indicate that postharvest tryptophan application can preserve fruit quality by upregulating phenolic biosynthesis genes (e.g., CHS and PAL), increasing NAD+/NADH and NADPH/NADP+ ratios, and activating the antioxidant system (e.g., enhancing SOD, CAT, and APX gene expression), thereby reducing ROS accumulation, as demonstrated in strawberry (Zhou et al., 2024). In ‘Le-Conte’ pear, preharvest tryptophan treatment (100 ppm) improved fruit set, yield, weight, skin color (L* and a*), total soluble solids (TSS), total soluble carbohydrates, total phenols, and total amino acid content (Khedr, 2018), and subsequently helped maintain TSS and reduce quality loss during 12 weeks of cold storage (Khedr, 2019). Despite promising results in various fruits, information regarding the impact of tryptophan on the physicochemical and marketability attributes of sweet cherry remains limited. Consequently, this study was designed to evaluate the effect of different concentrations of postharvest tryptophan application on maintaining quality, delaying senescence, extending shelf life, and enhancing the antioxidant system of ‘Tak Daneh’ sweet cherries during cold storage.

Materials and Methods
Sweet cherry fruits (Prunus avium L. cv. ‘Tak Daneh’) were harvested at commercial maturity on June 21, 2023, from an orchard in Saqqez, Kurdistan Province, Iran, and promptly transported to the Postharvest Physiology Laboratory, Department of Horticultural Sciences, University of Zanjan. Fruits with uniform size and color and free of visible defects were immersed for 20 minutes in tryptophan solutions at 0 (control), 0.5, 1, and 2 mM (Sigma Chemical Co.) containing 0.01% Tween-20 as a surfactant. The experiment was conducted as a factorial arrangement based on a completely randomized design (CRD) with three replications per treatment combination. After air-drying, fruits were placed in polyethylene containers and stored at 1 ± 0.5 °C and 85-90% relative humidity. A comprehensive set of postharvest quality and biochemical attributes was evaluated after 0, 7, 14, 21, and 28 days of storage. For assessments on days 7-28, fruits were equilibrated at room temperature (20±1 °C) for 24 h before analysis to simulate market conditions. Day-0 measurements were performed on three freshly harvested, untreated fruits. The key quality parameters were assessed using established methods as follows: the stem browning index (SBI) was determined using a visual scoring method (Yang et al., 2019); stem chlorophyll content was measured spectrophotometrically according to Arnon (1967); total anthocyanin content (TAC) was quantified using the pH-differential method (Giusti and Wrolstad, 2001). Fruit color coordinates (L*, a*, b*, C*, h°, ΔE) were obtained using a digital colorimeter (Lutron RGB-1002) with calculations based on standard formulas (Shahabi-Ghahafarrokhi et al., 2015; Mujtaba et al., 2023). Titratable acidity (TA), total soluble solids (TSS), and pH were analyzed from fruit juice, and the flavor index was calculated as the TSS/TA ratio (Naser et al., 2018). Total soluble carbohydrates (TSC) were assayed using the anthrone method (Irigoyen et al., 1992). Proline content was determined via the ninhydrin assay (Sánchez et al., 2001). Total soluble protein (TSP) was quantified using the Bradford method (Bradford, 1976). Peroxidase (POD) activity was measured by monitoring guaiacol oxidation (Zhang et al., 2013). Total antioxidant capacity was evaluated based on DPPH radical scavenging activity (Dehghan and Khoshkam, 2012). Data were analyzed using SPSS (version 26) software. Analysis of variance (ANOVA) was performed, and mean separation was carried out using Duncan’s multiple range test at a significance level of p < 0.05. Graphs were prepared with Microsoft Excel 2019.

Results and Discussion
Analysis of variance (ANOVA) indicated that tryptophan treatment, storage duration, and their interaction significantly affected the stem browning index and most colorimetric and biochemical traits, including L*, a*, C*, hue angle, ΔE, anthocyanin content (TAC), stem chlorophyll (TChl), pH, titratable acidity (TA), total soluble solids (TSS), total soluble carbohydrates (TSC), proline (Prol), total soluble protein (TSP), peroxidase (POD) activity, and total antioxidant capacity (p < 0.01). For the b* coordinate, the main effect of tryptophan treatment was not significant, whereas both storage time and the treatment × storage interaction had a highly significant effect (p < 0.01). Moreover, the flavor index was significantly influenced by the interaction between tryptophan application and storage duration (p < 0.05). Consistent with these statistical findings, postharvest application of tryptophan, particularly at 2 mM, markedly strengthened the antioxidant defense system and alleviated oxidative damage in ‘Tak Daneh’ sweet cherries during cold storage. Fruits treated with 2 mM tryptophan exhibited higher levels of proline, total soluble protein, peroxidase activity, total antioxidant capacity, and total soluble anthocyanins, indicating improved metabolic adaptation to storage-induced stress. These biochemical adjustments contributed to the preservation of key physicochemical and sensory attributes, including pH, titratable acidity, total soluble solids, flavor index, total soluble carbohydrates, and a significantly reduced incidence of stem browning. From a mechanistic perspective, the observed enhancement in antioxidant capacity and anthocyanin content can be attributed to tryptophan's role as a precursor for key signaling molecules. Tryptophan can enhance endogenous melatonin synthesis by upregulating genes such as TDC, T5H, SNAT, and ASMT (Madebo et al., 2021; Sharafi et al., 2021). Melatonin, a potent indoleamine with anti-senescence properties, subsequently stimulates the accumulation of phenolics, flavonoids, and anthocyanins by activating phenylalanine ammonia-lyase and chalcone synthase while suppressing polyphenol oxidase (Sharafi et al., 2021; Magri and Petriccione, 2022). Furthermore, tryptophan may elevate anthocyanin levels by upregulating key biosynthesis genes like DFR and UFGT (Miranda et al., 2020; Zhou and Zhang, 2024). Concurrently, tryptophan is implicated in stimulating endogenous salicylic acid (SA) production (Arabia et al., 2025). SA enhances plant tissue resistance by altering fruit physiology and boosting secondary metabolites, which likely contributed to delayed senescence and reduced stem browning in treated fruits. Thus, the combined action of tryptophan-derived melatonin and SA, along with a direct boost to the enzymatic antioxidant system (e.g., POD activity), effectively mitigated oxidative stress, preserving the overall quality and extending the marketable life of sweet cherries during cold storage. Therefore, the superior preservation of quality attributes in tryptophan-treated 'Tak Daneh' cherries, as evidenced by the biochemical and physical data, can be mechanistically linked to this multifaceted, elicitor-induced defense response.

Conclusion
Postharvest application of tryptophan at 2 mM is an effective, natural strategy for maintaining quality, extending shelf life, and enhancing the antioxidant defense system in sweet cherry during cold storage. This treatment mitigates oxidative damage by elevating proline, soluble proteins, peroxidase activity, and antioxidant and anthocyanin contents, thereby improving biochemical resilience and marketability of ‘Tak Daneh’ sweet cherry fruit.