Exogenous Melatonin Affects Fruit Enlargement and Sugar Metabolism in Melt Peach

Abstract

Peach (Prunus persica (L.)) fruits are abundant in nutrients, with fruit shape and sugar content serving as critical indicators of fruit quality. Melatonin plays a pivotal role in peach fruit development; however, the mechanisms by which it regulates fruit shape development, sugar metabolism, and secondary metabolites remain largely unknown. In this study, peach trees were sprayed with 150 µM melatonin 20 days after pollination. Traditional methods were used to investigate fruit morphology, total soluble solids (TSSs), and titratable acidity content (TAC), while liquid chromatography–mass spectrometry (LC-MS) was employed to analyze sugar metabolites during fruit development. The results indicated that melatonin treatment augmented the transverse and longitudinal diameters of peach fruits by 12% and 6%, respectively, and elevated the contents of soluble solids and titratable acid by 7% and 6%, respectively. The single fruit weight experienced a significant increase of 29.4%, whereas fruit firmness at maturity remained unchanged. Metabolite analysis demonstrated that melatonin decreased the levels of sucrose and D-sorbitol in mature fruits but enhanced the accumulation of D-fructose, L-rhamnose, and xylose. Significantly, melatonin expedited the degradation of galactose, D-mannose, and methyl-D-pyranogalactoside prior to maturity (all three substances naturally decline with fruit ripening), highlighting its role in promoting fruit ripening. In conclusion, exogenous melatonin improves the internal nutrition and flavor quality of fruit by regulating the accumulation of primary and secondary metabolites during fruit ripening. Specifically, the increase in D-fructose (a major contributor to sweetness) and L-rhamnose (a potential precursor for aroma compounds) enhances fruit flavor profile. The accelerated degradation of galactose, D-mannose, and methyl-D-pyranogalactoside (components of cell wall polysaccharides) prior to maturity, alongside the metabolic shift favoring fructose accumulation over sucrose, highlights melatonin’s role in promoting fruit ripening and softening processes. It also promotes fruit enlargement and single fruit weight without affecting fruit firmness. This study establishes a theoretical basis for the further investigation of the molecular mechanisms underlying melatonin’s role in peach fruits and for enhancing quality-focused breeding practices.

1. Introduction

Peach trees are widely cultivated globally, particularly in temperate regions of the Northern Hemisphere [1]. As the main producer, China has a cultivation history of over 4000 years [2,3]. Peach trees have strong adaptability, with characteristics of drought tolerance and cold resistance, but they are not tolerant of waterlogging and strong winds [4].

The main types of carbohydrates in peaches include sucrose, glucose, and fructose [5]. Peach pulp also contains organic acids [6], dietary fiber, and antioxidant substances such as flavonoids and tannins, which have antioxidant [7] and anti-inflammatory [8] effects. Carbohydrates not only act as signaling molecules to regulate the expression of thousands of genes [9], influencing plant growth [10], development, and resistance to stress, but also regulate intracellular osmotic potential, turgor pressure, and redox potential. Additionally, the types and composition of soluble sugars affect the fruit quality of peaches. When peach fruits ripen, sucrose is the primary carbohydrate [11], accounting for approximately 40–85% of the TSSs. Glucose and fructose follow, collectively comprising 10–25%, while sorbitol content is less than 10%. Furthermore, ripe peach fruits contain other carbohydrates such as maltose, isomaltose, ricinose, xylose, trehalose, 1-methyl-glucoside, and fucose [12].

Melatonin is a multifunctional small molecule that is widely distributed in plants [13], it plays a critical role in regulating growth, development, and biotic/abiotic stresses [14,15]. Its physiological roles extend to modulating key processes in fruit development and ripening. Melatonin can interact with ethylene signaling, a master regulator of climacteric fruit ripening, and influence respiration rates. It also acts as a potent antioxidant, scavenging reactive oxygen species (ROS) and enhancing the activity of antioxidant enzymes, thereby protecting cellular components and potentially influencing metabolic pathways, including those involved in sugar accumulation and cell wall degradation. Moreover, melatonin plays a role in alleviating oxidative stress during the development of peach fruits, which makes it a potential tool for improving fruit quality. However, despite these recognized functions, the precise molecular mechanisms by which melatonin orchestrates changes in specific metabolic fluxes, regulates cell wall remodeling enzymes, and integrates these effects to influence fruit size and quality traits like sweetness and texture in peach remain largely unknown. Studies have demonstrated that low-concentration melatonin (100–150 µM) promotes shoot growth [16], leaf quality, and photosynthetic efficiency in peach trees by enhancing antioxidant enzyme activities to reduce reactive oxygen species accumulation. In peach fruits, low-concentration melatonin increases single fruit weight, longitudinal diameter, and contents of soluble solids, sucrose, and fructose, while high concentrations (≥200 µM) exhibit insignificant or decreased effects [17].

Melatonin has broad application prospects in peach production [18,19], though optimization of treatment concentration and timing is needed. Combining with other preservation technologies may further enhance fruit quality. Elucidating the metabolic pathways and regulatory mechanisms of sugar accumulation is crucial for understanding fruit development and guiding breeding. While previous studies have shown melatonin’s effect on overall sugar content and fruit size, the specific alterations in sugar metabolic profiles and their link to cell wall modification during peach fruit development remain largely unexplored. Therefore, this study hypothesizes that exogenous melatonin application modulates specific sugar metabolism pathways and accelerates cell wall polysaccharide degradation, thereby promoting fruit enlargement and ripening. This study aims to clarify the relationship between sugar accumulation and fruit quality, providing references for peach cultivation and genetic improvement.

2. Materials and Methods

2.1. Fruit Material and Experimental Design

Fruits of the “Qiujing Baimi” (ZY04) peach cultivar were harvested from an orchard located in Jiangbei District, Ningbo City, Zhejiang Province, China. For this experiment, peach trees with uniform growth vigor were selected, and a randomized block design was employed. Twenty days after pollination, the experimental group (T) was foliar-sprayed with a 150 μM melatonin solution, whereas the control group (CK) received foliar application of clear water. Each group was arranged with 3 biologically independent replicate trees, resulting in a total of 9 trees in the experimental group and 9 trees in the control group. All fruits were subjected to identical cultivation conditions throughout the experimental period. Fruits at different developmental stages, including the young fruit stage (YF), hardcore period (HP), expansion stage I (ES I), expansion stage II (ES II), and mature stage (M), were sampled at 10-day intervals. At each sampling time point, 10 fruits were collected from each replicate tree, leading to a total of 30 fruits per treatment group. Immediately after collection, the fruits were frozen and stored at −80 °C for subsequent sugar analysis using liquid chromatography–mass spectrometry (LC-MS).

2.2. Measurement of the Transverse Diameter and Total Diameter of Peach Fruits

Fruit transverse (widest cross-sectional diameter) and longitudinal (vertical distance from apex to base) diameters were measured using a vernier caliper (Mitutoyo, Kawasaki, Japan; 0.01 mm precision), with 10 fruits per treatment and average values calculated.

2.3. Determination of Total Soluble Solids and TA Contents in Peach Fruits

Total soluble solids (TSSs) were measured using a PLY-1 digital refractometer (Atago, Minato, Japan; %). TAC was determined via acid–base neutralization according to Zhu et al.’s method [20], calculated as citric acid percentage using the following formula:

$$\mathrm{T}\mathrm{A}\mathrm{C}={\displaystyle \frac{\mathrm{C}\times \mathrm{V}}{\mathrm{m}}}\times 0.070\times 100\times 10$$

In the formula, TAC: titratable acid content expressed as citric acid; C: concentration of NaOH standard solution, mol/L; V: volume of NaOH solution consumed at the end point of titration, mL; m: sample weight (g).

2.4. The Hardness and Individual Weight of Peach Fruits

Fruit firmness was measured at the equatorial region using a GY-4 digital hardness tester (Fruit Test, Coventry, UK; kgf), with 3–5 measurements per fruit and average values calculated. Single fruit weight was determined using an electronic balance, with 10 disease-free fruits collected from four directions (east, west, north, south) of each tree every 10 days.

2.5. Determination of Sugar Content in Peach Fruits

Biological samples were freeze-dried by a vacuum freeze-dryer (Scientz-100F). The freeze-dried sample was crushed using a mixer mill (MM 400, Retsch GmbH, Haan, Germany) with a zirconia bead for 1.5 min at 30 Hz. After 100 mg of lyophilized powder was dissolved with 1.2 mL of 70% methanol solution, the sample was vortexed for 30 s every 30 min, 6 times in total, and then placed in a refrigerator at 4 °C overnight. Following centrifugation at 12,000 rpm for 10 min, the extracts were filtrated (SCAA-104, 0.22 pmpore size; ANPEL, Shanghai, China, http://www.anpel.com.cn/) before UPLC-MS/MS analysis.

The sample extracts were analyzed using a UPLC-ESI-MS/MS system (UPLC, SHIMADZU NexeraX2, www.shimadzu.com.cn/(Shimadzu, Kyoto, Japan); MS, Applied Biosystems 4500QTRAP, www.appliedbiosystems.com.cn/, Applied Biosystems, Waltham, MA, USA). The analytical conditions were as follows: UPLC: column, Agilent SB-C18 (1.8 um, 2.1 mm × 100 mm); The mobile phase consisted of solvent A, pure water with 0.1% formic acid, and solvent B, acetonitrile with 0.1% formic acid. Sample measurements were performed with a gradient program that employed the starting conditions of 95% A, 5% B. Within 9 min, a linear gradient to 5% A, 95% B was programmed, and a composition of 5% A, 95% B was kept for 1 min. Subsequently, a composition of 95% A, 5.0% B was adjusted within 1.1 min and kept for 2.9 min. The flow velocity was set as 0.35 mL per minute. The column oven was set to 40 °C. The injection volume was 4 uL. The effluent was alternatively connected to an ESI triple quadrupole–linear ion trap (QTRAP) MS instrument.

LIT and triple quadrupole (QQQ) scans were acquired on a triple quadrupole–linear ion trap mass spectrometer (Q TRAP, Manufacturer: AB Sciex, Framingham, MA, USA), AB4500 Q TRAP UPLC/MS/MS System, equipped with an ESI Turbolon-Spray interface, operating in positive and negative ion mode and controlled by Analyst 1.6.3 software (AB Sciex). The ESl source operation parameters were as follows: ion source, turbo spray; source temperature 55 °C; ion spray voltage (IS) 5500 V (positive ion mode)/−4500 V (negative ion mode); ion source gas 1 (GSl), gas 1L (GS), and curtain gas (CUR) were set at 50, 60, and 25.

2.6. Statistical Analysis

Metabolite data were log2-transformed for statistical analysis to improve normality and were normalized. Metabolites from 30 samples were used for hierarchical clustering analysis (HCA), principal component analysis (PCA), and orthogonal partial least squares discriminant analysis (OPLS-DA) using R software version 4.2.2 to study accession-specific metabolite accumulation. Significance of differences between treatment groups at the same developmental stage was assessed using Student’s t-test. The p-value threshold for significance was set at <0.05, and fold change (FC) values were considered significant at |log2FC| ≥ 1. Venn diagrams were used to illustrate the number of differential metabolites. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database with a p-value < 0.01 was used to study differential metabolites in the colored radishes compared to white ones (white flesh metabolites, WFMs, and white skin metabolites, WSMs). All data were graphed using GraphPad Prism v6.01 (GraphPad Software Inc, La Jolla, CA, USA). Statistical significance is indicated in figures as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.

3. Results

3.1. Exogenous Treatment of Melatonin Increases the Fruit Shape of Peaches

Spraying 150 µM melatonin on peach trees 20 days after pollination promoted fruit development at the young fruit stage, hardcore period, expansion stage I, expansion stage II, and mature stage (Figure 1). The transverse and longitudinal diameters of young peach fruits in the melatonin-treated group (ZY04T) were 43.17 mm and 46.58 mm, respectively, representing increases of 18.6% and 11.2% compared to the control group (ZY04CK). At expansion stage I, the transverse and longitudinal diameters of ZY04T fruits increased by 14.6% and 9.9%, respectively, compared to ZY04CK. At expansion stage II, the transverse and longitudinal diameters of ZY04T fruits increased by 7.2% and 11.0%, respectively. At maturity, the transverse and longitudinal diameters of ZY04T fruits increased by 12% and 6%, respectively. These results indicate that melatonin can significantly increase peach fruit size.

3.2. The Effect of Exogenous Treatment of Melatonin on Soluble Solids and Titratable Acids in Peach Fruits

During fruit development, the content of soluble solids (TSSs) gradually increased, while the content of titratable acid (TCA) gradually decreased (Figure 2). Compared with ZY04CK, ZY04T accumulated more TSSs (Figure 2A). In the young fruit stage, the TSS content in ZY04T fruits was 8.2% higher than that in the control group; at the hardcore period, it was 3.1% higher; at expansion stage I, 9.1% higher; at expansion stage II, 8.4% higher; and at maturity, 6.2% higher. Notably, before fruit maturity, the TCA content in ZY04T fruits was higher than that in ZY04CK, but at maturity, the TCA contents in ZY04T and ZY04CK were nearly identical (Figure 2B).

3.3. The Effect of Exogenous Melatonin Treatment on the Weight and Firmness of Peach Fruits

Melatonin treatment had little effect on fruit firmness. Since fruit firmness could not be detected during the expansion stage, firmness was measured at 30, 40, 50, 70, 75, and 80 days after treatment. The firmness of ZY04T fruits was slightly lower than that of ZY04CK before 70 days, but there was no difference between ZY04T and ZY04CK after 70 days (Figure 3A). Interestingly, melatonin significantly increased single fruit weight (Figure 3B). In the young fruit stage, the single fruit weight of ZY04T was 26.8 g, a 34% increase compared to ZY04CK. At maturity, the single fruit weight of ZY04T reached 228.45 g, a 29% increase compared to ZY04CK. These results indicate that melatonin promotes fruit development at all stages.

3.4. Multivariate Statistical Analysis

The differences in the sugar accumulation patterns of samples from each treatment group at various stages can be analyzed through cluster heatmaps (Figure 4). The results of cluster heatmap analysis show that there are obvious differences in substances among different groups, which are divided into four clusters in total. The sugar accumulation content in cluster 1 is the highest in the YF(CK) group, moderate in the YF(T) group, and the lowest in the M(CK) and M(T) groups. The sugar accumulation content in cluster 2 is the highest in the M(T) group, moderate in the YF(T) group, and the lowest in the ESⅡ(CK) group. Biological replicates of the same group are also clustered together, indicating good homogeneity among biological replicates and high reliability of the data.

3.5. Principal Component Analysis

We first performed a principal component analysis (PCA) of all samples based on these metabolic signals. The results of the PCA plot (Figure 5) show that components 1 and 2 explained 55.01% and 17.94% of the variability, respectively. Components 1 and 2 successfully separated all tissues, indicating significant metabolic diversity. PCA revealed that PC1 primarily separated samples by developmental stage, with galactose and mannose contributing most negatively (−0.92 loading). PC2 differentiated treatments, driven by fructose (0.87) and sucrose (−0.79). Further observations revealed that ESⅡ(T) and ESⅡ(CK), ESⅠ(T) and ESⅠ(CK), and HP(T) and HP(CK) were closer to each other in the PCA diagram, indicating that these tissues have more similar metabolic profiles. PCA was performed using the Metware Cloud (https://cloud.metware.cn).

3.6. The Influence of Exogenous Treatment of Melatonin on the Sugar Components of Peach Fruits

After 150 μM melatonin was sprayed on peach trees 20 days after pollination, LC-MS was used to detect the contents of 20 sugar substances in fruits at the young fruit stage, hardcore period, expansion stage I, expansion stage II, and mature stage. Significant changes were observed in the contents of sucrose, D-sorbitol, D-fructose, L-rhamnose, xylose, galacturonic acid, galactose, D-mannose, and methyl-D-pyranogalactoside. Sucrose content gradually increased with fruit ripening, reaching 249.149 mg/g at maturity (Figure 6A). Except for higher sucrose content in ZY04T young fruits than in ZY04CK, sucrose content in ZY04T fruits was lower than in ZY04CK fruits at other stages. At maturity, the sucrose content in ZY04T fruits was 10.8% lower than in ZY04CK fruits. The content of D-sorbitol in peach fruits gradually increased with fruit ripening but decreased slightly in mature fruits (Figure 6B). The D-sorbitol content in ZY04T young fruits was 45.9% higher than in ZY04CK, but at maturity, it was 14.1% lower. In contrast to sucrose and D-sorbitol, the contents of D-fructose, L-rhamnose, and xylose in ZY04T young fruits were lower than in ZY04CK (Figure 6C–E), but their contents in mature ZY04T fruits were higher than in ZY04CK, increasing by 17.6%, 24.3%, and 45.9%, respectively (p < 0.05). The content of D-galacturonic acid showed no significant change at various developmental stages in normal peach fruits, but slightly decreased at maturity (Figure 6F), while the content in ZY04T fruits was 80.7% higher than in ZY04CK. The contents of D-galactose, D-mannose, and methyl-D-β-pyranogalactoside gradually decreased with fruit development (Figure 6G–I). Their contents in ZY04T young fruits and hardcore period fruits were lower than in ZY04CK, but there was no difference between ZY04T and ZY04CK at maturity.

4. Discussion

Melatonin exerts multifaceted effects on peach fruits, including promotion of growth and development, enhancement of fruit quality, augmentation of disease resistance, and retardation of senescence [21]. It ameliorates the internal quality of fruits by modulating phytohormone signaling, antioxidant systems, and metabolic pathways. Unlike previous studies focusing on overall soluble solids or sucrose/fructose totals, this research provides a more granular view by employing LC-MS to track dynamic changes in 20 individual sugar metabolites, including key cell wall components like galactose and mannose, across multiple developmental stages. This detailed metabolic profiling reveals novel insights into how melatonin reshapes sugar distribution and accelerates cell wall disassembly. For instance, it elevates lycopene and soluble solids content in tomato fruits while suppressing ethylene production, thereby delaying fruit senescence. In tomato, melatonin significantly boosts single fruit weight, soluble sugar, and vitamin C content [22]. As a multifunctional plant hormone, melatonin demonstrates distinct roles depending on developmental stages: it accelerates ripening in climacteric fruits like tomato by regulating ethylene, while delaying senescence in non-climacteric fruits via antioxidant pathways [23,24]. Its mechanisms of action are intricate and diverse, involving multiple dimensions such as phytohormone signal transduction, antioxidant systems, gene expression regulation, and metabolic pathways. Future research will further unravel the specific mechanisms of melatonin in different crops and its application potential [25].

The commercial value of peach fruits is primarily determined by factors such as taste, appearance, and nutritional value. Among these, the external attributes of fruits play a pivotal role in defining quality market standards [26], and changes in transverse and longitudinal diameters directly impact both the external and internal quality of fruits [27]. Numerous studies have reported that various compounds can alter fruit size [28]. For example, exogenous application of 2,4-dichlorophenoxyacetic acid increases citrus fruit size [29], and zinc significantly influences fruit quantity and size in cherries and other fruits, particularly when applied at high concentrations. Our findings reveal that exogenous melatonin treatment increases the transverse and longitudinal diameters of peach fruits (Figure 1), leading to a significant increase in single fruit weight (Figure 3A), while fruit firmness after the expansion stage remains unaffected, potentially without impacting fruit taste and storage. Additionally, we found that melatonin enhances soluble solid content (Figure 2A). Although acidity increases during fruit development, melatonin has no effect on titratable acid content in mature fruits (Figure 2B). Collectively, these results indicate that melatonin can enhance peach fruit yield and quality without affecting their sweet and sour taste, holding substantial potential for peach quality improvement.

The sweetness of peach fruits is one of their critical commercial value indicators, and sugar substances are key determinants of fruit sweetness and flavor [30], often resulting from the degradation of polysaccharides and soluble acids. During fruit development, the accumulation of soluble sugars (such as glucose, fructose, and sucrose) directly influences fruit sweetness [31]. Studies have shown that as fruits develop from young to mature stages, the content of soluble sugars in peaches gradually increases, while acidic substances degrade, thereby enhancing fruit sweetness and edibility [32]. In tomato fruits, melatonin treatment significantly increases total soluble sugar content [33]. Research has found that melatonin improves soybean yield and quality by regulating photosynthetic products such as sucrose, starch, and fructose [34]. In this study, we observed that sucrose content gradually increases with peach fruit ripening, and D-sorbitol content also increases before fruit maturity (Figure 6A,B), but melatonin reduces the content of sucrose and D-sorbitol in mature fruits. At the mature stage of tomato fruits, melatonin activates NI and AI (key enzymes in sucrose catabolism), thereby decomposing sucrose into fructose and glucose. This suggests that melatonin inhibits sucrose degradation in young peach fruits but promotes sucrose degradation in mature fruits, thereby increasing fructose content in peach fruits (Figure 6C). In peach, melatonin likely upregulates PpPG (polygalacturonase) and PpEXP1 (expansin) genes during ES I/II, accelerating pectin degradation. This aligns with observed galactose/mannose reduction (Figure 6G–I) and parallels melatonin-induced LePG expression in tomato [35]. These findings demonstrate that melatonin plays a pivotal role in regulating the distribution and transformation of carbohydrates in mature peach fruits, particularly by facilitating fructose accumulation and accelerating the degradation of specific cell wall polysaccharides during early developmental stages. Distinct from existing research, this study features the following novel aspects: it has conducted a comprehensive profiling of various individual sugars and cell wall components throughout the entire fruit development cycle, and validated that melatonin can accelerate the natural reduction in specific cell wall sugars (galactose, mannose, methyl-D-pyranogalactoside) prior to fruit ripening—a mechanism that has not been previously highlighted in peach research. Additionally, it has confirmed that the promotion of fruit enlargement and the increase in soluble solids content do not compromise the titratable acid balance at maturity or the final fruit firmness, which are both critical factors affecting consumer acceptance.

5. Conclusions

This study demonstrates that exogenous melatonin treatment at 150 μM, a concentration previously shown to be effective for promoting growth and sugar accumulation in peaches [36], significantly enhances peach fruit size, thereby increasing single fruit weight by a remarkable 29.4%. While melatonin has no effect on the firmness of mature peach fruits, it reduces fruit firmness during the young fruit stage, potentially contributing to effective reduction in disease infestation. Additionally, melatonin elevates the content of soluble solids in peach fruits, enriching their taste. Through LC-MS analysis of sugar metabolites in peach fruits, melatonin was found to regulate the distribution of sucrose and D-fructose, enhancing the nutritional quality of peach fruits. Furthermore, melatonin accelerates fruit softening by regulating galactose, D-mannose, and methyl-D-pyranogalactoside. The exploration of interactions between melatonin and plant metabolism holds broad application potential for improving both the quality and yield of peach fruits.