Introduction

Tangelo tangerines hybrids result from crosses between Dancy tangerine species (tangerine groups) and Duncan grapefruit (C. reticulate × C. paradise). Two well-known varieties of this group are Minneola and Orlando tangelo (Lim and Lim 2012). Citrus fruit is a highly preferred fruit worldwide due to its delightful taste, appealing appearance, aroma, and beneficial components such as vitamins and phenolic compounds (Shorbagi et al. 2022). The major reason for economic losses in citrus postharvest is the natural senescence process of the fruits, which indicates a decline in their quality. This deterioration encompasses various factors such as dehydration and reduction in weight (Lufu 2020), shrinkage and peel damage (Zhou et al. 2021), changes in flesh texture and granulation (Chen et al. 2021), as well as the degradation of sugar and organic acids, resulting in a reduction in overall flavor (Tang et al. 2016).

The utilization of cold storage is crucial for fruit producers and distributors to guarantee that their products are delivered to consumers in their best possible condition. However, in several tropical and subtropical varieties, such as citrus fruits, long-term cold storage might result in physiological irregularities, making them susceptible to chilling damage during storage. This could ultimately lead to decreased quality and increase in postharvest losses (Strano et al. 2022). The proposed minimum temperature for safe storage of mandarin varies from 5 to 8 °C (Kader and Rolle 2004). Due to the increasing demand from consumers for fruit that is free from harmful chemical fungicides and strict regulations on the amount of chemical residues permitted, exporters are now more worried about decay development and are opting to transport mandarins at lower temperatures of about 3–4 °C to prevent losses (Tietel et al. 2012). The chilling effect on citrus fruit is marked by the appearance of rind discoloration, pitting, reddish patches, scorching, and softening of the flavedo (Strano et al. 2022).

Currently, there is an increasing focus on the use of GRAS (Generally Recognized as Safe) materials in the postharvest production of fruits as evidenced by the growing number of research studies in this area (Burdock and Carabin 2004). Melatonin (N-acetyl-5-methoxytryptamine) and GABA (γ-aminobutyric acid) are two endogenous compounds that participate in regulating plant responses to alterations in their environment (Zeng et al. 2022; Kaspal et al. 2021). GABA functions as a suppressor of malondialdehyde production during lipid peroxidation, maintains the integrity of membrane, enhances the activity of antioxidants and GABA shunt pathway, and helps to regulate osmotic balance (Hayat et al. 2023). Furthermore, γ-aminobutyric acid (GABA) has been acknowledged as a crucial signaling molecule with a diverse array of functions. It regulates ROS signaling and the carbon–nitrogen balance in plants encountering different stress conditions (Seifikalhor et al. 2019).

The secure and beneficial compound melatonin, which is an indole amine molecule, acts as an endogenous stimulant and signaling molecule in mitigating both biotic and abiotic stress (Zeng et al. 2022). Melatonin is considered a potential contender for improving the quality of crops due to its substantial contribution to different physiological processes of plants, particularly its ability to alleviate stress (Arnao and Hernández‐Ruiz 2015). Numerous earlier studies have indicated that administering melatonin externally enhances the shelf life of vegetables and fruits, modifies their sugar and acid levels, and influences the taste and aroma of various fresh produce (Xu et al. 2019)(Rastegar et al. 2020).

No documented investigation has been conducted regarding the effects of utilizing melatonin and GABA to prolong the postharvest life, diminish chilling damage and maintain the quality of Orlando tangelo during cold storage. The present study pursued to investigate the effects of melatonin and GABA, either in combination or separately, in mitigating chilling injury and preserving the quality of Orlando tangerine stored at a temperature of 3 ± 0.5 °C for 90 days, followed by 5 days at 20 °C. Internal composition such as phenol, flavonoid, antioxidants systems, and TSS/TA and external qualities like color and chilling injury were evaluated.

Materials and methods

Fruit materials and experimental design

Mandarin fruit (Orlando tangelo) was harvested from a commercial orchard located in Ziyarat Ali village, Rodan City (57°29ˊE and 27°59ˊN), within the Hormozgan province of Iran. The region is characterized by alkaline soil with a pH of approximately 8–9, and an average annual temperature of 27 ℃. In addition, the average rainfall in the region is around 250 mm and it is situated at an elevation of approximately 190 m above sea level. The fruit was transported to the laboratory in an artificial container within a period of 2 h after being packed. For the following analysis, selected fruits were uniform in size and color and free from any visible damage or disease. The fruits were sterilized with sodium hypochlorite (0.05%) and then immersed in melatonin (500 µM), GABA (5 mM) and melatonin (500 µM) + GABA (5 mM) for 10 min. The control fruits were soaked in distilled water for a duration of 10 min. The choice of concentrations was made after conducting a preliminary test, in which various concentrations and durations of melatonin and GABA were evaluated. The optimal concentration and duration were chosen for carrying out this experimental. Once air-dried, the fruits were arranged in plastic containers and kept in an environment at a temperature of 3 ± 0.5 °C and a relative humidity of 80–90% for a duration of 90 days. To assess quality and biochemical composition, samples were collected following 30, 60, and 90 days of storage, as well as 5 days at room temperature. Furthermore, a fraction of the samples was frozen in liquid nitrogen and kept at − 80 °C.

Weight loss

The rate of reduction in weight was determined every week throughout the period of storage. The initial weight was used as a reference point, and the reduction in weight was expressed as a percentage (Lo’Ay and Taher 2018).

Chilling injury evaluation

During the storage, the fruit was examined for any signs of chilling indications. In mandarin fruit, the symptoms of chilling injury were commonly identified as minor brown pit-shaped dents in the flavedo. This condition is known as 'pitting' and can progress into deeper and darker collapses, spreading across the fruit surface as it is exposed to lower temperatures. To evaluate the seriousness and range of chilling symptoms, fruits were visually examined and categorized using the following grading system: 0 = no pitting observed; 1 = slight depressions covering less than 25% of the fruit surface; 2 = deeper indentations covering 25–50% of the surface; and 3 = severe damage covering 50% or more of the fruit surface. The outcome was presented as the chilling indicator, which was computed by summing up the outcome of the number of fruits in each classification multiplied by the rating for each classification. This sum was then divided by the overall number of fruits assessed (Rey et al. 2020).

Malondialdehyde (MDA)

The samples of Orlando tangelo were ground utilizing 5 ml of trichloroacetic acid (1%). Subsequently, they were rotated in a centrifuge (10 min at 10,000 rpm), and 500 µL of the resultant extract was taken out and rendered acidic by adding 2 trichloroacetic acid (20%, which had 0.5% thiobarbituric acid). Afterward, the blend was heated at 95 °C for 30 min. Upon cooling, the mixture was once more centrifuged at 10,000×g for 10 min. The samples were then examined for absorbance at a wavelength of 532 nm. By utilizing the MDA extinction coefficient (0.155 mM−1 cm−1), the quantity of malondialdehyde (MDA) present in each gram of fresh weight was calculated and expressed as μmol MDA g−1 fresh weight.

Total phenolic and total flavonoids content

The process of extraction involved blending 500 µL of fruit juice with 1500 µL of methanol (85%). The resultant mixture was subjected to centrifugation at a rate of 11,000×g for 20 min at ambient temperature. The resulting supernatant was used to assess the antioxidant activity, total flavonoids content and total phenol.

The determination of total phenolic concentration was carried out through the Folin–Ciocalteu spectrophotometric technique, as delineated by Singleton et al. (1999), with certain modifications. Specifically, 150 µL of methanol extract was combined with 600 µL of a 7% sodium carbonate solution and 750 µL of diluted Folin–Ciocalteu reagent. After a 90-min incubation period at room temperature, the mixture was analyzed at 750 nm using a UV–Vis spectrophotometer (CECIL-2501, England). The findings were presented as mg of gallic acid equivalents per g of fresh weight.

The total flavonoids content was determined following the protocol outlined by Chang et al. (2002). Briefly, 200 μL of the methanol extract sample was mixed with 40 μL of 10% AlCl3 and 40 μL of 1 mM acetate potassium solution. The mixture was allowed to react for 30 min, and the absorbance was measured at 415 nm using a UV–visible spectrophotometer (CECIL-2501, England). Quercetin was used as a reference substance to formulate the calibration graph. The results were reported as mg of quercetin equivalents per g of FW.

Activity of antioxidants

To explore the activity of antioxidants in the juice samples, the method of DPPH (2,2-diphenyl-1-picrylhydrazyl) was employed (Brand-Williams et al. 1995). In this method, 0.025 g of DPPH was dissolved in 100 ml of 85% methanol and subsequently mixed well with 30 μL of methanol extract using a vortex. The mixture was placed in a dimly lit space for half an hour, and the absorbance was measured at a wavelength of 517 nm. The percentage inhibition of the DPPH radical was considered as the measure of radical scavenging potential (RSA).

CAT and POD enzyme activity

To extract the enzyme, samples were homogenized with potassium phosphate buffer (50 mM, pH 7.4, 1.5 mL) containing 1 mM EDTA and 1% PVP (weight/volume) at 4 °C. The resulting mixture was subjected to centrifugation at 13,000 × g and 4 °C for 20 min. The resulting supernatant was used to measure the activity of CAT and POD with a spectrophotometer (CECIL Model 3000, Cambridge, England).

Peroxidase (POD, EC: 1.11.1.7) activity was determined using guaiacol as the substrate. Sample extracts were mixed with 50 μL of guaiacol and 150 μL of H2O2. The absorbance was measured for 1 min at a wavelength of 460 nm. POD activity was expressed as the change in OD 460 per gram of FW per min. The measurements were carried out in triplicate (Chance and Maehly 1955).

The enzyme catalase (CAT, EC: 1.11.1.6) was assessed using the technique established by Chance and Maehly 1955). Sample extracts were mixed with 1000 μL of 50 mM phosphate buffer (KH2PO4 pH 7.0) and 20 μL of H2O2. The absorbance was measured at a wavelength of 470 nm using a spectrophotometer (Cecil 2501, England). Each evaluation was carried out in triplicate.

TSS, TA, and pH

The TSS concentration of the fruits was evaluated using a digital refractometer (DBR 95) at a temperature of 25 °C and expressed as a percentage. Titratable acidity concentration was determined by titrating 5 mL of juice with 0.1 N NaOH and phenolphthalein until the solution turned light pink (pH 8.1), and expressed as a percentage of citric acid. Fruit juice pH was measured using a pH meter.

External peel color evaluation

The color of the peel of the fruit was evaluated at two points located on opposite sides of the fruit using a Minolta Chroma meter CR-400 (EC Minolta, Japan). The recorded values represented the color attributes: b* for blue/yellow, a* for green/red, and L* for darkness/whiteness (McGuire 1992).

Statistical assessment

Analysis of variance (ANOVA) was used to statistically analyze the treatment-related data. Clustering was performed using the SAS program (Version 9.4). Mean values were compared using the LSD test at a significance level of 5% (P < 0.05).

Result

Weight loss

The percentage of fruit weight loss gradually increased during storage, but the treated fruit effectively prevented weight loss. Therefore, 90 days after storage, the control group exhibited the highest weight loss.

(Fig. 1a). A high correlation between chilling and weight loss was observed in different treatments (Fig. 5a).

Fig. 1
figure 1

The effect of postharvest treatment of melatonin, GABA, and melatonin + GABA on a weight loss and b weight loss of Orlando tangelo fruits stored at 3 ± 0.5 °C and 80–90% RH for 90 days plus 5 days at 20 °C. Data represent mean values of n = 3 and the error bars represent standard errors (SD) of the means. Statistical analysis was performed using LSD test at P ≤ 0.05 level

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Chilling injury

As shown in Fig. 1b, chilling damage gradually increased during storage, but it was noticeably more severe in the untreated fruits compared to the treated ones. The melatonin + GABA treatment significantly controlled chilling, and no signs of chilling were observed in the treated fruits for up to 90 days of storage.

MDA

As shown in Fig. 2a, b, the MDA present in both the peel and juice of the fruit steadily rose throughout the storage period. The increase was lower in the treated fruits than in the control group. At the end of the storage period, the degree of chilling damage in the peel and juice of the control group was 43% and 50% greater, respectively, than that in the treated fruits. In different treatments, a high correlation was observed between chilling and MDA of peel and juice (Fig. 5b, c).

Fig. 2
figure 2

The effect of postharvest treatment of melatonin, GABA, and melatonin + GABA on a water MDA b peel MDA of Orlando tangelo fruits stored at 3 ± 0.5 °C and 80–90% RH for 90 days plus 5 days at 20 °C. Data represent mean values of n = 3, and the error bars represent standard errors (SD) of the means. Statistical analysis was performed using LSD test at P ≤ 0.05 level

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Total phenol and flavonoid and antioxidant capacity

As shown in Fig. 3a, b, the total phenol and flavonoid content of the fruit gradually decreased during storage, regardless of treatment. Although some treatments showed higher phenol and flavonoid contents than those of the control group, this increase was not statistically significant at the 0.05 level.

Fig. 3
figure 3

The effect of postharvest treatment of melatonin, GABA, and melatonin + GABA on a phenol, b flavonoid, c) antioxidant, and correlation between antioxidant and phenol (d) and correlation between antioxidant and flavonoid of fruit juice of Orlando tangelo fruits stored at 3 ± 0.5 °C and 80–90% RH for 90 days plus 5 days at 20 °C. Data represent mean values of n = 3 and the error bars represent standard errors (SD) of the means. Statistical analysis was performed using LSD test at P ≤ 0.05 level

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During storage, the levels of antioxidants in the control and treated fruits also reduced. During the first 60 days of storage, there were no observable differences between the control group and the treatments. However, by the end of storage, the treated fruits showed a significant increase compared to the control group (Fig. 3c). As shown in Fig. 3a, d, e high correlation was observed between antioxidant with phenol and flavonoids. Except GABA melatonin treatment, a high correlation between frostbite and reduction of antioxidant compounds was observed in other treatments (Fig. 5d, e, f).

Enzyme activity

As shown in Fig. 4a, b, the POD of the peel and juice of the fruit increased slightly during the first 60 days of storage and then decreased slightly on the 90th day of storage. The treated fruit showed higher POD enzyme activity than the control group during storage. Therefore, the control group exhibited the lowest enzyme activity on the final day of storage.

Fig. 4
figure 4

The effect of postharvest treatment of melatonin, GABA, and melatonin + GABA on a peel POD, b water POD, c peel CAT, and d water POD of Orlando tangelo fruits stored at 3 ± 0.5 °C and 80–90% RH for 90 days plus 5 days at 20 °C. Data represent mean values of n = 3 and the error bars represent standard errors (SD) of the means. Statistical analysis was performed using LSD test at P ≤ 0.05 level

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During the initial 60 days of storage, the CAT enzyme activity remained relatively stable across most treatments, except for GABA + melatonin treatment. This treatment exhibited a statistically significant variation compared to the control group and other treatments on both the 60th and 90th day of storage, demonstrating a substantial increase of 192% and 163% respectively in comparison to the enzyme activity observed on the first day. In the fruit juice, CAT activity remained consistent until the 30th day of storage, after which a significant increase was detected in the GABA + melatonin treatment. By the 90th day of storage, all treatments showed progressive activity levels than the control group. Among the treatments, the melatonin treatment exhibited the highest enzyme activity. In different treatments, different correlations were observed between enzymes and the chilling (Fig. 5g–j).

Fig. 5
figure 5

Correlation between chilling injury with different traits in different treatments

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TSS, TA and pH

As indicated in Table 1, the TSS and pH levels of the fruit showed a gradual increase over the storage period, with no significant differences observed between the control and treated fruits at different stages. The level of fruit TA decreased significantly during storage, regardless of the treatment. At the end of the storage period, the fruits treated with the different treatments displayed significantly elevated levels of TA (total acidity) in comparison to the control group. Except for the GABA + melatonin treatment, other treatments displayed a strong correlation between chilling and both TA and TSS. (Fig. 5k, l).

Table 1 The effect of postharvest treatment of melatonin, GABA, and melatonin + GABA on TSS, pH, and TA of Orlando tangelo fruits stored at 3 ± 0.5 °C and 80–90% RH for 90 days plus 5 days at 20 °C
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Color

The color indices of L* and b* gradually decreased during storage (Fig. 6 a,b). During different storage periods, no significant variations were observed between the control group and the treatments, except for the melatonin + GABA treatment. On the 30th day of storage, the melatonin + GABA treatment showed significantly higher levels of L* and b* than the other groups and the control group. The a* level remained relatively constant throughout the storage period, with a slight increase detected on the 30th day (Fig. 6c). On the 30th day, the GABA and GABA + melatonin treatments showed lower values than the control group, However, there were no notable distinctions observed between the control group and treatments during other stages. Regardless of the treatment, no significant change in Hue angle was observed up to 30 days of storage, but a noticeable increase was observed on day 60 of storage, and it remained relatively stable until day 90 of storage. At the end of the storage period, both the highest (74.7) and lowest (68.9) levels of Hue were observed in GABA + melatonin treatment and the control group (Fig. 6d).

Fig. 6
figure 6

The effect of postharvest treatment of melatonin, GABA and melatonin + GABA on color measurement (a) L*, (b) b*, (c) a*, and (d) hue angle of Orlando tangelo fruits stored at 3 ± 0.5 °C and 80—90% RH for 90 days plus 5 days at 20 °C. Data represent mean values of n = 3 and the error bars represent standard errors (SD) of the means. Statistical analysis was performed using LSD test at P ≤ 0.05 level

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Correlation analysis

A normalized heatmap matrix was created to assess the overall reactions of fruit biochemical parameters to treatments throughout the storage. In Fig. 7, it can be seen the changes in the physicochemical properties of Orlando tangelo fruits during storage periods. Two significant clusters were identified based on the relative responses of parameters to treatment and storage duration. The first cluster (I), which included control and treated fruit at 0 and 30 days of storage, was characterized by fruits with high to very high antioxidant and TA, low weight loss, and low chilling. The second group (II), which included fruit at 60 and 90 days of storage, was characterized by fruit with low to very low phenol and flavonoid. Melatonin + chilling treatment showed a lower chilling, higher POD peel, and TA than other treatments.

Fig. 7
figure 7

Hierarchical clustering analysis (HCA) of the melatonin and GABA treatments and variable trait relationships in Orlando tangelo fruit, including a heatmap of Pearson correlation coefficients (r values) for variable traits. The colored scale shows the r coefficient values (r = − 1 to + 1.5) indicating positive (red) and negative (blue) correlations, respectively

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Discussion

Weight loss during storage is an important factor to consider in maintaining the quality and shelf life of fruits. Weight loss can also affect the appearance and marketability of fruits. Fruit weight loss can occur due to the dehydration of the fruit's surface, which is caused by metabolic processes such as respiration and transpiration. This event is influenced by the difference in humidity levels between the product and its surrounding atmosphere, as noted by Bovi et al. (2018). In both blueberry and fresh-cut pitaya fruit, the use of melatonin as a treatment has been discovered to enhance the fruit's firmness and slow down the rate at which it loses weight (Magri and Petriccione 2022; Ba et al. 2022). In the current study, all treatments reduced weight loss compared to the control fruit during storage. The beneficial effect of melatonin in reducing weight loss may be attributed to the fruit's exceptional peel resilience properties, which played a vital role in preventing weight reduction (Liu et al. 2018a, b). According to the findings of Palma et al. (2019), the application of GABA improved the quality of zucchini crops stored at 4 °C, leading to a decrease in the occurrence of cold damage and weight loss.

Exposure of citrus fruits to cold temperatures can cause chilling symptoms, leading to a decrease in appearance quality and an increase in postharvest losses. The chilling index is a commonly used method for assessing chilling injury and determining the cold tolerance of fruit during cold storage (Rey et al. 2020).

The results obtained from our study showed that GABA and melatonin had similar effects in reducing chilling injury. When fruit was exposed to both GABA and melatonin, the chilling injury was further reduced compared to the fruit treated with only GABA or melatonin. This indicates a synergistic effect between these two substances in minimizing chilling injury. Previous studies have reported that activating the GABA shunt pathway can enhance the impact of melatonin on GABA homeostasis (Sharafi et al. 2019).

Melatonin treatment can upregulate early hydrogen peroxide signaling and activities of enzymatic and non-enzymatic antioxidants, leading to the mitigation of chilling injury and maintenance of quality in cold-stored horticultural produce (Shah et al. 2023). According to Cao et al. (2016), administering melatonin helped to reduce the effects of chilling injury on peach fruits. This was achieved by enhancing the expression of arginine decarboxylase and ornithine decarboxylase genes, leading to an increase in endogenous polyamine accumulation. It has also been reported that the application of GABA at both pre and postharvest stages leads to an increase in proline content. Proline acts as both an osmotic protector and a membrane stabilizer, helping to stabilize and integrate cell membranes during low-temperature storage of horticultural crops (Aghdam et al. 2015).

External application of GABA has proven effective in reducing chilling injury of horticultural produce during cold storage. This is achieved through various mechanisms, including activation of the GABA shunt pathway, enhanced prevention of ROS, increased levels of endogenous proline, inhibition of phospholipase D (PLD) and lipoxygenase (LOX) activities, promotion of PAL activity, suppression of PPO activity, elevated levels of anthocyanins, phenols, and flavonoids, improved scavenging activity of DPPH, and maintenance of firmness by inhibiting pectin methylesterase (PME) and polygalacturonase (PG) activities.

(Asgarian et al. 2022; Ali et al. 2022). The application of GABA has been used in various types of fruits to prolong their postharvest shelf life (Sheng et al. 2017) and enhance their ability to resist chilling (Malekzadeh et al. 2017), yielding comparable outcomes to those observed in this study on mandarin fruit. A different research study demonstrated that the application of GABA treatment improved the quality of stored fruit and decreased the occurrence of chilling injury in Chinese olive fruit (Fan et al. 2022).

During cold storage, the loss of membrane semi permeability may lead to a decrease in intracellular ATP levels and an increase in ROS accumulation in persimmon fruit, as indicated by the increase in electrolyte leakage and MDA accumulation (Niazi et al. 2021). It has been proposed that GABA acts as a suppressor of MDA generation during lipid peroxidation, which may explain the fruit's increased ability to tolerate cold stress (Wang et al. 2014). Various studies have been conducted on zucchini fruit (Palma et al. 2019), cornelian cherry fruit (Rabiei et al. 2019), and pear fruit (Li et al. 2019). These studies have demonstrated that the application of GABA lead to in reduced electrolyte leakage and MDA accumulation. Application of melatonin can also delay the occurrence of oxidative stress and minimize the extent of membrane lipid peroxidation in the fruits of 'Newhall' navel oranges (Ma et al. 2021). Adverse impacts of melatonin on oxidative pressure and lipid oxidation have been noted in horticultural crops such as peaches (Gao et al. 2016) and kiwifruit (Wang et al. 2019). The use of GABA may offer benefits in protecting the membrane integrity of fruit against oxidative damage.

In the current study, the fruit's phenolic and flavonoid levels decreased during storage, and the treatments significantly reduced the changes in the fruit phenolic and flavonoid content. Earlier studies have established that melatonin is effective in preserving fruit phenolic compounds (Rastegar et al. 2020). Melatonin treatment has been found to increase the polyphenol accumulation and antioxidant activity in blueberry fruit during cold storage (Magri and Petriccione 2022). The study conducted by Xu et al. (2017) found that melatonin enhanced the antioxidant potential of grapefruit by facilitating the buildup of polyphenols. Treating various fruit species with melatonin has been found to increase the concentrations of phenolic and anthocyanin compounds during storage (Wang et al. 2019; Xu et al. (2017); . This effect is believed to result from the stimulation of the phenylpropanoid pathway by melatonin, which enhances the activities of phenylalanine ammonolysis and chalcone synthase enzymes (Sharafi et al. 2019; Liu et al. 2018b). In addition, it has been reported that the use of GABA increased the phenol and flavonoid content of ‘Sahebi’ grape during cold storage (Asgarian et al. 2022). GABA preserved the levels of phenolic compound, ascorbic acid and other compounds in peach fruit, as well as its capacity to remove free radical (Zhou et al. 2022). The increased antioxidant capacity observed in fruit under GABA treatment may be due to the accumulation of endogenous GABA (Ngaffo Mekontso et al. 2021). Research has shown that the application of melatonin to kiwifruit resulted in an increase in the overall phenolic content and antioxidant capacity, compared to untreated fruits, throughout the storage period (Wang et al. 2019).

Although the use of GABA or melatonin alone did not significantly affect peel CAT, their combined use resulted in the highest degree of enzymatic action. Consistent with our results, the activity of POD in banana fruit peel stored at low temperature was significantly enhanced by GABA (Wang et al. 2014). Our findings also align with earlier studies on the impact of GABA in enhancing the levels of CAT and POD activity in pear fruit (Li et al. 2019). These results are consistent with previous research that observed increased POD activity in fruit treated with GABA during storage. It has been reported that persimmon fruit treated with GABA showed increased levels of CAT, APX, and SOD activities throughout cold storage (Niazi et al. 2021). Melatonin administration enhances the AsA-GSH pathway and other antioxidants, such as CAT, SOD and total phenols, which efficiently eliminate dangerous ROS. As a result, this mitigates lipid peroxidation and delays fruit senescence (Ma et al. 2021).

In citrus crops, organic acids, sugars, and amino acids are the most significant elements for the fruit's internal quality, with organic acids being the most crucial (Hussain et al. 2017). Any changes in organic acids can significantly affect the fruit's flavor, as well as its senescence process and storage capabilities. Typically, an increased concentration of organic acids or a reduced pH level indicates delayed fruit senescence (Batista-Silva et al. 2018). In the present study, the properties of the fruit, including TSS levels, TA, and pH, underwent changes during the storage period. The use of GABA and melatonin significantly contributed to the preservation of the fruit's acidity. The application of GABA treatment has been shown to significantly maintain crop quality by increasing the accumulation of citrate and ATP (Pott et al. 2020). Previous investigations have also indicated that GABA plays a key role in regulating the metabolism of natural acids in harvested citrus fruit (Badiche et al. 2023). Administering exogenous GABA has been demonstrated to impact the GABA shunt, which regulates the production of endogenous GABA, the tricarboxylic acid (TCA) cycle, citric acid degradation, and mitigates cellular damage caused by various stresses in fruit (Ansari et al. 2021). According to Yang et al. (2022), the fruit flavor of 'Olinda' oranges can be maintained by reducing respiration intensity with the use of melatonin. This may also result in a delay in the consumption of the sugar and acid nutrients present in the fruit. Mature non-climacteric citrus fruit evolves very low amounts of ethylene, but ethylene sharply increases in response to stress (Lafuente et al. 2001). (Han et al. (2018) confirmed that GABA treatment prevented the reduction of malic acid levels during the fruit ripening process by inhibiting malate breakdown and stimulating malate production. The treatment also resulted in higher levels of oxalate and succinate. Previous research has indicated that melatonin has a favorable effect on total soluble solids (TSS) and fruit firmness. However, its impact on titratable acidity (TA) varies depending on the type and cultivar of the fruit (Yang et al. 2022). It has been suggested that the delayed taste deterioration in 'Newhall' oranges after harvest, induced by external melatonin, may have resulted from a reduction in the respiration rate (Ma et al. 2021).

In our study, GABA and melatonin did not have a significant impact on the indicators of fruit color index. However, Ma et al. (2021) revealed that the use of melatonin considerably accelerated and improved the modification of color in orange fruit, as demonstrated by raised measurements of a* and b* starting from 28 days after harvesting. Researchers demonstrated that melatonin has the potential to improve the chromaticity features of L*, C*, and H◦ in cherries and strawberries, as shown by analysis of chromaticity index (Wang et al. 2019). However, in a different study, the application of melatonin to mango fruit resulted in a contrary effect, delaying the yellowing of the fruit and reducing the amount of beta-carotene produced (Liu et al. 2020).

Conclusion

The results of the current research suggest a synergistic impact of GABA and melatonin in improving the cold tolerance of Orlando tangelo fruit during cold storage. Additionally, the treatments used in the study were effective in preventing weight loss of the fruit during storage. All treatments were found to have the highest antioxidant capacity at the end of the experiment. The improvement of cold resistance in the fruit occurred due to increased antioxidant enzymes (CAT and POD) and non-enzymatic antioxidant compounds such as phenol and flavonoid. These findings could potentially be useful for managing fruit quality during low-temperature storage. It is recommended to conduct further comprehensive investigation on the molecular mechanisms by which melatonin and GABA improve chilling tolerance in Orlando tangelo fruit.