Optimal Preharvest Melatonin Applications to Enhance Endogenous Melatonin Content, Harvest and Postharvest Quality of Japanese Plum

Abstract

Plum is one of the most produced stone fruits worldwide. Melatonin is an environmentally eco-friendly substance that, in low concentrations, activates defence systems against biotic and abiotic stresses. This substance is considered a tool that could increase fruit quality. This study aimed to evaluate the impact of different preharvest foliar applications with different melatonin concentrations (0.1, 0.3, 0.5 mmol L⁻¹) to enhance melatonin content and shelf life of ‘Primetime’ plum. To this purpose, two and three applications were carried out at different critical stages of fruit growth. Different quality characteristics such as size, colour, titratable acidity, total soluble solids, ripening index, respiration rate, ethylene production rate, anthocyanins and total antioxidant activity, as well as endogenous melatonin content, were tested at harvest and after 40 days of cold storage. Results showed that ‘Primetime’ plums that received 3 applications of 0.5 mmol L⁻¹ enhanced endogenous melatonin content at harvest and showed less softening, delayed darkening, higher anthocyanin concentration and total antioxidant activity after 40 days of storage. Therefore, the concentration of 0.5 mmol L⁻¹ melatonin in 3 applications was effective in improving the quality of ‘Primetime’ plums.

1. Introduction

Japanese plum (Prunus salicina L.) is among the world’s most widely produced stone fruits with a relatively short postharvest life. In addition to its organoleptic characteristics, it presents several beneficial health properties for consumers due to its fibre, sorbitol and phenolic compounds content [1,2]. Intake of Japanese plums increases urinary levels of 6-sulfatoxymelatonin and total antioxidant capacity in humans [3]. This is why plum consumers demand fruit of high overall quality. The plum ripening process is characterised by a double sigmoid curve, where there are four key moments such as cell elongation (S1), pit hardening (S2), colour change (S3) and consumption maturity (S4). During this process, modulated by different phytohormones, there is an increase in fruit size, acids, sugars and pigment changes, and an accumulation of volatile compounds [4,5]. Notwithstanding, plums should be harvested before they reach the S4 stage to ensure good postharvest handling and shelf life [6]. It is also known that fruit quality is defined by genotype and growing conditions [4]. However, in recent years, the environmentally friendly application of natural substances or biostimulants, such as melatonin, can play a fundamental role in modulating the biosynthetic pathways of the different compounds that determine fruit quality during ripening process on the tree and subsequent postharvest life [7,8,9,10,11,12,13]. Melatonin is an indolamine, structurally analogous to auxins phytohormones that regulate stress response processes throughout a plant’s growth and development cycle [14]. Thus, the use of exogenous melatonin has improved photosynthesis and crop yield in grape berries [15]. In cherries, a delay in the flowering and ripening process was also observed [16]. In addition, melatonin shows high antioxidant activity, which allows to inhibit harmful substances [17] and thereby resist abiotic and biotic stresses [18,19,20], directly enhancing postharvest life and bioactive compounds in fruits and vegetables [21,22]. Exogenous melatonin concentration usually ranges from 150 nmol L⁻¹ to 1 mmol L⁻¹ [17], with 10⁻⁴ mol L⁻¹ concentrations being the most commonly used [11,12,23]. Postharvest application is usually performed by immersion in aqueous solutions of this substance before cold storage. In preharvest, foliar spraying is generally chosen and applied at critical fruit growth stages. However, the concentration and number of preharvest applications require further studies, as they may vary depending on the species and varieties. Thus, several authors have proposed a single application [15,24], while others have suggested several applications during the fruit growth cycle [11,12,25,26]. To the best of our knowledge, no studies comparing the effect of different numbers of melatonin applications on fruit quality and postharvest life of Japanese plums have yet been found.

On the other hand, melatonin also plays an essential role in regulating circadian rhythms in mammals. Because of its antioxidant capacity, melatonin helps prevent degenerative and cardiovascular diseases, allergies, cancer and infections such as COVID-19 [27,28]. In this sense, medical drugs that enhance endogenous melatonin levels have been developed, but this is not the only alternative. Consuming melatonin-enriched foods may be a natural approach to help prevent and combat diseases caused by oxidative stress [29]. Vegetable- and fruit-based nutraceuticals have been developed to improve sleep and antioxidant status in humans [30] and can modulate serum markers of inflammation in rats and wood pigeons [31]. The melatonin content of some fruits depends on species, cultivar, fruit tissue and ripening stage [21,32]. Plums have moderate melatonin content (around 14 and 20 ng g⁻¹ DW) [7], so it would be interesting to increase their endogenous content, thus enhancing the health benefits of consuming of this fruit [8].

Therefore, this study aimed to evaluate the impact of different preharvest foliar applications using different melatonin concentrations to enhance endogenous melatonin levels and the postharvest potential of ‘Primetime’ Japanese plum.

2. Materials and Methods

2.1. Plant Material and Experimental Design

Experiments were conducted in a commercial orchard located at Entrerríos (Badajoz, Spain, 38°59′49.6” N 5°40′17.0” W; 279 m altitude) on Japanese plum trees (Prunus salicina Lindl.) of the cultivar ‘Primetime’ in the 2019 and 2021 seasons, planted in 2013 in an open vase training system with a spacing of 5.5 m × 3 m and grafted onto Mariana rootstock. Plum trees were subjected to agronomic practices specific to the crop in both study years. Climate data from May to July in each year were obtained from the closest weather station (Supplementary Table S1), showing mean temperatures of 23.78 ± 3.55 °C and 23.61 ± 4.07 °C for 2019 and 2021, respectively, with no significant differences between the 2 study years. Three concentrations of melatonin (M), 0.1, 0.3 and 0.5 mmol L⁻¹ plus a control treatment (CO) were evaluated, in which trees were sprayed with an aqueous solution, containing 0.5 mL L⁻¹ of Tween-20 as a surfactant, 2 (2APP) or 3 times (3APP). Three trees per treatment were randomly selected in 2APP and 3APP (n = 24). Applications were carried out at sunset, as melatonin is a photosensitive molecule, by hand spraying and applying 2 L of product per tree. The application moments were based on those described by Martínez-Esplá et al. [6], with 2APP being applied at the stage of pit hardening (S2) and colour change (S3). For 3APP, a 3rd application was applied 7 days before harvesting (before S4) in addition to the 2 moments mentioned above. All plums from each treatment/tree were hand-picked at the commercial ripening stage (before S4) according to commercial practices. They were then transported to the fruit packing house for further processing. A total of 22 kg of plums from each treatment at 2APP and 3APP were automatically sorted by maximum diameter using a sorting machine. The ranges used to sort the plums were more than 60 mm, between 60 and 50 mm and less than 50 mm. After sorting, the plums were packed in 30-fruit boxes and transferred to the CICYTEX facilities, where postharvest storage was carried out under controlled conditions of temperature (1 ± 0.8 °C) and relative humidity (92.4 ± 3.2% RH) for 40 days in darkness.

2.2. Physicochemical Parameters

To evaluate physicochemical parameters, 90 fruits (30 fruits per tree) of each 2APP and 3APP treatment were used at harvest (Day 0), and 30 fruits (10 from 3 independent boxes) were used for postharvest storage. A CR-400 reflectance colourimeter (Minolta Camera Co, Osaka, Japan) was used to measure exocarp and mesocarp colour at two opposite sites. The results were expressed as CIRG index (colour index of red grapes) [33]. Firmness was determined using a Stable Micro Systems TAXT2i texturometer. The penetration test was performed on 2 equatorials opposite faces of the mesocarp with an 8 mm diameter probe, and the maximum force expressed as Newtons (N) was recorded. Subsequently, 3 homogenates (n = 3) were performed for each treatment. Total soluble solids (TSS) were quantified using a portable digital refractometer PR-01 (°Brix). Titratable acidity (TA) was measured on 3 g of each homogenate made up to a final volume of 60 mL using distilled water. Titration was carried out using 0.1 mol L⁻¹ NaOH solution up to pH 8.1 on an automatic T-50 Graphix titrator. TA was expressed as g of malic acid per 100 g of fresh weight (FW). Ripening index (RI) was calculated as TSS/TA. Weight losses were only calculated in stored fruits by comparing the initial weight of 10 independent fruits per replicate (n = 30) and treatment with the weight at the end of cold storage.

2.3. Determination of Respiration Rate and Ethylene Production Rate

Respiration rate and ethylene production rate measurements were performed in triplicate (n = 3). At room temperature (20 °C), 5 fruits from each replicate were placed in a 1.7 L sealed glass jar. Respiratory rate was quantified by static method [34] using a PBI-Dansensor CheckMate 3 gas analyser (DK-4100 Ringsted, Denmark) and results were expressed as mL CO₂ kg⁻¹ h⁻¹. Ethylene production rate was quantified using the static method [35] with slight modifications. Briefly, 1 mL of gas sample was syringe-collected from each jar 1.5 h after sealing and measured using an Agilent Technologies 7890A (G3440A) Network GC system gas chromatograph (Santa Clara, CA, USA) equipped with Agilent 113-4332 Gaspro column (30 m, 0.32 mm, 0 mm), maintained at 50 °C and a detection temperature of 280 °C. The N₂ carrier gas was used at a flow rate of 8.6 mL min⁻¹ and a pressure of 180 kPa. Air and H₂ had a 350 and 35 mL min⁻¹ flow rate in the flame ionization detector (FID). Results were expressed as µL C₂H₄ kg⁻¹ h⁻¹.

2.4. Bioactive Parameters

For each 2APP and 3APP treatment and sampling date, 15 plums were frozen at −80 °C until analysis. The analysis of bioactive compounds was performed independently for the exocarp and mesocarp. Three homogenates were prepared from 5 frozen plums to extract all bioactive compounds (n = 3). Phenolic compounds were quantified using HPLC-DAD (Agilent Technologies 1100, Santa Clara, CA, USA) according to the method described by Manzano Durán et al. [36]. Briefly, 2 g of exocarp or 2 g of mesocarp were extracted with 10 mL MeOH (HCl 0.1%), containing 2 mmol L⁻¹ NaF. Samples were sonicated at 4 °C for 30 min at dark. Then, a chromatographic method with photometric detection (HPLC-DAD) was employed for the separation and quantification of the extract. A Gemini-NX C18 column (150 mm × 4.6 mm; 3 µm) and a mixture of water and acetonitrile, both acidified (0.1% formic acid), were used as stationary and mobile phase, respectively. Flow and temperature were set at 1 mL min⁻¹ and 40 °C, respectively; injection volume was 10 µL. The detection of the anthocyanin was performed at 515 nm and expressed as mg cyanidin 3-O-glucoside 100 g⁻¹ of FW. Total antioxidant activity (TAA) was quantified using the DPPH method as described by Pérez-Jiménez et al. [37], with little modifications. Concretely, frozen fruit was used instead of freeze-dried fruit. Briefly, 2 g of exocarp or 2 g of mesocarp was extracted using 20 mL of MeOH:H₂O (50:50) acidified with HCl to pH 2.0 and shaken for 1 h in an orbital shaker covered by ice and protected from light. Then, they were centrifuged, and the supernatant was separated. The residue was extracted again under the same conditions, but using an acetone:H₂O mixture (70:30). Finally, both supernatants were pooled in a flask and diluted up to 50 mL using MilliQ. The extracts were mixed with a methanolic solution of DDPH: The absorbance was measured at 515 nm at minute 0 and after 120 min, when the reaction with the plum extract had terminated. Results were expressed as mg Trolox 100 g⁻¹ of FW.

2.5. Quantification of Melatonin

Melatonin extraction and quantification were performed from freeze-dried plum samples, as described by González-Gómez et al. [38], with a minor modification to improve melatonin quantification, using deuterated melatonin as an internal standard. The limit of quantification (LQ) of the calibration line was 3.9 µg L⁻¹ melatonin. The LQ considering the extraction process was 197 ng 100 g⁻¹ DW. Results were expressed as ng 100 g⁻¹ DW.

2.6. Statistical Analysis

The statistical analysis of the results was performed using SPSS 25 (IBM). Results were subjected to a one-way analysis of variance (ANOVA). Tukey’s test was performed to evaluate the treatment effect. Differences were considered significant at p < 0.05.

3. Results

3.1. Quality at the Time of Harvest

3.1.1. Physicochemical Parameters

Figure 1 shows the fruit size distribution for each treatment and number of applications. It was observed that M0.3 and M0.5 fruits were larger than M0.1 and the control in 3APP, and most plums were larger than 60 mm (Figure 1a). However, in 2APP, most plums were in the 50-60 mm size range (approximately 60%) without differences among treatments (Figure 1b).

The effect of treatments on plum exocarp and mesocarp colour was studied, and the CIRG index for each treatment is shown in Figure 2. This index is commonly used to measure the red-purple colour of plums [39]. There were slight differences in exocarp colour between 3APP and 2APP, with 3APP plums being more purplish than 2APP plums (Figure 2a). Control plums were generally darker than treated plums. In 3APP, M0.3 plums were observed to be more reddish, although there were no significant differences between treatments. However, the mesocarp CIRG index was similar in both cases studied (Figure 2b). Significant differences were found between M0.3 and control in 2APP, with CO slightly darker and more reddish.

Table 1. Physicochemical traits of ‘Primetime’ Japanese plums at time of harvest.

		CO	M0.1	M0.3	M0.5
TSS	3APP	12.77 ± 0.20 a 1	10.60 ± 0.21 b	10.37 ± 0.69 b	13.20 ± 0.15 a
	2APP	11.07 ± 0.27	10.67 ± 0.53	10.70 ± 0.23	10.73 ± 0.37
TA	3APP	1.35 ± 0.09	1.36 ± 0.03	1.31 ± 0.04	1.41 ± 0.05
	2APP	1.44 ± 0.04	1.36 ± 0.02	1.35 ± 0.02	1.43 ± 0.02
RI	3APP	9.59 ± 0.73	7.79 ± 0.26	7.92 ± 0.33	9.40 ± 0.38
	2APP	7.71 ± 0.18	7.83 ± 0.30	7.90 ± 0.11	7.50 ± 0.24
Firmness	3APP	28.46 ± 1.28 ab	23.79 ± 0.13 b	25.67 ± 0.72 ab	30.39 ± 1.70 a
	2APP	23.71 ± 0.87	22.39 ± 0.30	22.47 ± 0.91	21.93 ± 1.17

¹ In each row, different lowercase letters indicate a significant difference at p < 0.05 (Tukey’s test). Mean values ± SE. TSS (°Brix), TA (g malic acid 100 g⁻¹ FW), RI (TSS/TA) and firmness (N). 3APP (three applications) and 2APP (two applications).

shows TSS, TA, RI and firmness mean values of control and melatonin-treated plums. No significant differences were found among treatments in 2APP. However, differences were found in TSS and firmness for 3APP; M0.1 and M0.3 led to plums with lower TSS content and, thus, a lower TSS/TA ratio than the M0.5 and control treatments. There were no differences between the melatonin-treated fruits and control regarding firmness. Nevertheless, in 3APP, M0.5 fruits were firmer than the M0.1 treatment.

3.1.2. Determination of Respiration Rate and Ethylene Production Rate

Respiration rate and ethylene production rate mean values are shown in Figure 3. No significant differences in respiration rate were found between treatments and the number of applications. The mean values for 3App and 2App were 16.94 and 21.45 mL CO₂ kg⁻¹ h⁻¹, respectively (Figure 3a). There were significant differences in ethylene production rate between the control and M0.3 in 2APP, with M0.3 plums producing more ethylene (Figure 3b). On the other hand, fruit treated in 3APP showed no significant differences among treatments.

3.1.3. Bioactive Parameters

The most important family of phenolic compounds in the ‘Primetime’ plum was anthocyanins, especially cyanidin 3-O-glucoside (C3OG) in both the exocarp and the endocarp (Figure 4). In the plums of 2APP, no significant differences were found between treated fruit and the control. Nevertheless, differences were found in plums that received three preharvest applications (3APP); M0.1 and M0.3 fruit showed significantly lower C3OG content in the exocarp than the control (Figure 4a). On the other hand, only M0.1 treatment produced this reduction in the mesocarp (Figure 4b).

The total antioxidant activity (TAA) of the exocarp and mesocarp was quantified using the DPPH method, and the mean values obtained are shown in Figure 5. No significant differences were found between the control and melatonin treatments in 2APP. However, a significant TAA reduction in the exocarp was observed in M0.1 plums after 3APP (Figure 5a).

3.1.4. Quantification of Melatonin

Melatonin content was quantified to confirm that melatonin was absorbed and retained in the fruit (Figure 6). Using two applications (2APP), the melatonin content of treated and untreated plums was very similar; there was a slight increase in melatonin content using the concentration applied, but no significant difference. However, using three applications (3APP), plums showed a significant increase in this compound, especially M0.5 fruits, in which the increase in melatonin levels was almost fivefold.

3.2. Postharvest Quality

As plums of 3APP had higher endogenous melatonin content at harvest, the impact of this enhancement on the postharvest life of ‘Primetime’ plums was evaluated.

Table 2. Postharvest quality of ‘Primetime’ plums with 3APP (three applications) after 40 days of cold storage.

3APP	CO	M0.1	M0.3	M0.5
CIRG exocarp	4.49 ± 0.14 a1	4.03 ± 0.18 ab	3.81 ± 0.15 b	3.70 ± 0.14 b
CIRG mesocarp	2.55 ± 0.07	2.52 ± 0.10	2.37 ± 0.08	2.43 ± 0.11
TSS	12.33 ± 0.03 a	10.67 ± 0.09 ab	11.83 ± 0.98 a	9.40 ± 0.32 b
TA	1.10 ± 0.06	1.14 ± 0.06	1.02 ± 0.08	1.11 ± 0.08
RI	11.31 ± 0.52	9.46 ± 0.56	11.87 ± 1.90	8.52 ± 0.33
Firmness	3.29 ± 0.25	3.59 ± 0.29	4.12 ± 0.30	4.36 ± 0.47
Weight loss	3.03 ± 0.27	3.00 ± 0.25	3.57 ± 0.34	2.61 ± 0.29
Respiration rate	29.17 ± 1.56	32.22 ± 2.85	32.04 ± 2.08	33.14 ± 1.23
Ethylene production	1.34 ± 0.35	1.04 ± 0.12	0.52 ± 0.04	0.93 ± 0.13
C3OG exocarp	115.49 ± 15.74 ab	114.72 ± 8.90 ab	77.96 ± 0.38 b	147.93 ± 9.01 a
C3OG mesocarp	8.07 ± 2.64	8.93 ± 1.02	9.24 ± 0.69	7.84 ± 0.13
TAA exocarp	508.25 ± 7.74 b	504.77 ± 0.41 b	375.52 ± 7.72 c	656.67 ± 17.03 a
TAA mesocarp	141.32 ± 5.50 a	136.71 ± 4.91 a	99.01 ± 1.27 c	117.41 ± 0.30 b

Mean values ± SE. Colour (CIRG index), TSS (°Brix), TA (g malic acid 100 g⁻¹ FW), RI (TSS/TA), firmness (N), weight loss (%), respiration rate (mL CO₂ kg⁻¹ h⁻¹), ethylene production (µL C₂H₄ kg⁻¹ h⁻¹), anthocyanins (mg cyanidin 3-O-glucoside 100 g⁻¹ FW) and TAA (mg Trolox 100 g⁻¹ FW). ¹ For each row, different lowercase letters indicate a significant difference at p < 0.05 (Tukey’s test).

shows the quality traits of ‘Primetime’ plums after 40 days of cold storage.

The plums treated using melatonin were visually less dark than the control after 40 days of cold storage. This difference was evident in the CIRG index of mesocarp and exocarp, the latter being significantly lower than CO in M0.3 and M0.5 plums. M0.5 fruit using 3APP showed better overall quality than control plums. Additionally, M0.5 mM plums showed slightly higher firmness, lower TSS and weight loss after cold storage.

On the other hand, it could be observed that treated fruits showed a slight increase in respiration rate and a decrease in ethylene production rate in comparison to the control. However, no significant differences were found (Table 2).

Regarding bioactive compounds, the exocarp of M0.5 plums showed the highest levels of C3OG (147.93 ± 9.01 mg cyanidin 3-O-glucoside 100 g⁻¹ FW) and TAA (656.67 ± 17.03 mg Trolox 100 g⁻¹ FW).

4. Discussion

Melatonin is an important signalling molecule in different plant organs that stimulates various physiological processes during plant growth and development [8,32]. In addition, it has also been shown to be an effective molecule in minimising biotic and abiotic stress, ROS accumulation, delaying postharvest diseases and the senescence process [18,21,40,41]. In recent years, several studies have reported on the effect of exogenous preharvest melatonin on vegetables and fruits, demonstrating that it is a safe and nontoxic substance for humans and has a positive effect on shelf life and food quality [17,21,22,25,42].

One of the objectives of this study was to determine the number of preharvest melatonin applications necessary to enhance the overall quality and endogenous melatonin content of ‘Primetime’ plums. The results obtained in our study (Table 1 and Table 2, Figure 4, Figure 5 and Figure 6) showed that three (3APP) vs. two (2APP) preharvest applications of melatonin, independent concentration, was able to enhance quality and endogenous melatonin content in ‘Primetime’ plums. In this sense, Carrión-Antolí et al. [11,12] proposed that three preharvest melatonin applications (at stone hardening, at the onset of colour change, and days before harvest) in cherry improved fruit quality traits. In contrast, others such as Meng et al. [15] and Xia et al. [24] proposed that a unique preharvest melatonin application improved grape berry quality in terms of fruit colour change. Considering the results obtained in the present study, it can be inferred that the endogenous melatonin content depends on the concentration, number and moment of applications during fruit growth, which have an impact on plum quality traits.

In addition to yield, fruit size is a critical parameter that determines fruit quality and commercial value. Several authors have shown that the exogenous melatonin application in preharvest can be essential for both parameters. Previous studies [11,15,41] have reported improved yield and size in treated fruits. However, in our study, this effect was not so noticeable (Figure 1a), although it can be observed that M0.3 and M0.5 fruits treated using 3APP showed the lowest percentage of small fruits.

Skin colour and firmness define fruit harvesting time in plums [7]. Firmness is considered a key parameter for fruit shelf life [43]. At harvest time, only M0.5 plums were firmer than the control after three applications, although no significant differences were observed (Table 2). Regarding skin colour, no significant impact of melatonin treatments was observed. Another critical parameter is the ripening index (RI = TSS/TA), widely used to establish consumer acceptance [44]. It has been shown that using biostimulants by foliar spraying on fruit can delay the increase in TSS content and maintain TA [45]. In this study, melatonin showed no impact on TA of ‘Primetime’ plums. By contrast, lower TSS content was found at harvest, which is more noticeable in plums treated with 0.1 mmol L⁻¹ and 0.3 mmol L⁻¹ (Table 1), indicating delayed ripening. These results are consistent with those found in the available literature. Tijero et al. [16] reported that tree melatonin treatment delayed TSS accumulation and, thus, delayed ripening. Yan et al. [46] also reported that treatment using 0.05 mmol L⁻¹ and 0.1 mmol L⁻¹ melatonin delayed ‘Taoxingli’ plum ripening.

In exogenous melatonin applications, much controversy exists regarding its effects on respiration rate. Wang et al. [47] concluded that exogenous melatonin applications caused a delayed respiration rate in sweet cherries. Moreover, Bal et al. [13] reported that melatonin treatments delayed respiration and the climacteric peak of nectarines. Conversely, Michailidis et al. [48] and Fan et al. [49] reported an increase in the respiration rate of melatonin-treated fruit. Our study showed this decrease in respiration rate and a slight increase in ethylene production rate at harvest time. However, no significant differences were observed among treatments when three applications were performed (Figure 3).

Anthocyanins are mainly responsible for the skin colour of ‘Primetime’ plums. The synthesis and conversion of anthocyanins take place during veraison [7]. It was observed that a 0.1 mmol L⁻¹ concentration of melatonin led to a delay in the ripening process of ‘Primetime’ plums. The anthocyanin content and, consequently, the total antioxidant activity of these plums were lower than that of M0.5 and the control (Figure 4 and Figure 5). Tijero et al. [16] reported that melatonin treatment at 10⁻⁴ and 10⁻⁵ mol L⁻¹ delayed anthocyanin synthesis and ripening.

Environmental factors, technology and the lifestyles of today’s society have significant repercussions on public health [50]. In the literature, melatonin has been shown to reduce disorders caused by infectious organisms, cardiovascular and inflammatory diseases, sleep disorders or cancer in mammals [27,28,29,50,51,52]. Melatonin levels in fruit vary according to species and variety [23]. In the available literature, we found that melatonin content in plums ranges between 0 and 20 ng g⁻¹ DW [3,7] (Figure 6). In ‘Primetime’ plums, the mean melatonin content for the control was 4.12 ng g⁻¹ DW (3APP) and 3.29 ng g⁻¹ DW (2APP). Authors such as González-Flores et al. [3] found no melatonin in Japanese ‘Crimson Globe’ plums, and only serotonin was detected. On the other hand, Arabia et al. [7] determined melatonin contents of 14 ng g⁻¹ DW (pulp) and 20 ng g⁻¹ DW (skin) in the plum cultivar ‘Angeleno’. In our study, all the melatonin treatments tested provided plums with higher endogenous melatonin levels than the control. This effect was remarkably significant when three preharvest applications (3APP) were performed, and the melatonin content in M0.5 plums was more than fivefold higher than the control. Meng et al. [15] increased the endogenous melatonin content in grape berries more discretely using a unique preharvest application of 100 mg L⁻¹ of melatonin.

Another objective of our study was to enhance the endogenous melatonin levels in plums. The increase in endogenous melatonin has a positive effect on all stakeholders in the fruit supply chain. Consumers benefit from healthy foods with nutraceutical functions. In addition, producing high-quality fruit at harvest ensures better postharvest storage. Melatonin has been shown to have positive effects in delaying senescence and preserving the sensory and nutritional quality of different types of fruits during postharvest [53]. This is of considerable economic importance worldwide, as it could contribute to reducing food waste [17].

Plums undergo many biochemical changes during ripening and postharvest life. Throughout these processes, there is softening of the cell wall, a decrease in total acidity, production of aromatic compounds and anthocyanin accumulation [54]. Plums can be preserved for one to six weeks after harvest if suitable handling and storage conditions are provided. In our study, ‘Primetime’ plums were stored under refrigeration for 40 days in order to test the effect of the 3 preharvest melatonin applications (3APP) on postharvest quality.

Plums, as a climacteric fruit, continued with the postharvest ripening process during cold storage, showing skin darkening and an increase in softening and weight loss, as well as in respiration rate and ethylene production rate. However, a slower evolution of these parameters was observed in M0.5 plums. These fruits not only resulted in higher endogenous melatonin content but also led to plums of better quality and shelf life. After 40 days of postharvest storage, M0.5 ‘Primetime’ plums had a less dark exocarp colour; lower RI, weight loss and ethylene production rate; and, in addition, higher firmness, anthocyanin content and TAA (Table 2), showing better fruit acceptance potential and senescence delay. These results agree with Agham et al. [17], Arabia et al. [7,23], Xia et al. [24,42], Carrión-Antolí et al. [11,12], Yan et al. [46] and Cortés-Montaña et al. [53], who proposed melatonin treatment as a potential strategy to maintain fruit quality, due to its implication in the reduction of biotic and abiotic stress, weight loss, incidence of chilling injury and activation of bioactive systems that promote free radical scavenging, which allows for increasing the shelf life of the fruit and reducing food waste.

5. Conclusions

The results obtained in this study corroborate the effect of exogenous melatonin in low doses to regulate specific physiological processes in fruit and, thus, to enhance their quality and directly extend their shelf life. Three applications of melatonin at the highest concentration studied (0.5 mmol L⁻¹) were shown to be effective in increasing the endogenous content of this substance in the ‘Primetime’ plum. In addition, this endogenous increase in melatonin improved the shelf life of these plums, delaying darkening and softening after 40 days of cold storage. Further studies are needed to clarify the different pathways that the exogenous melatonin application regulates.