Postharvest Melatonin Application Preserves Quality and Imparts Chilling Tolerance in Peaches

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Introduction
Peach (Prunus persica L.), which belongs to the Rosaceae family, originated in China.Annual peach fruit production accounts for 15.02 million tons in China, which is nearly 61.12% of the total world's production, thus making it the world's leading producer.After China, Spain secures second position, followed by Italy, Turkey, Iran, the USA, Egypt, Chile, and India [1].Peach is a temperate fruit that has gained worldwide attention due to its excellent commercial value, pleasing aroma, juicy texture, bright attractive colour, and abundant health benefts [2].Peach fruit is considered a good source of proteins, minerals, soluble sugars, and dietary fbers, making it capable of providing immunity against chronic diseases such as cancer and cardiovascular diseases.Furthermore, the peach's nutritional quality is an important indicator factor that determines fruit quality [3].However, peach fruit inevitably experiences rapid ripening, senescence, and deterioration at ambient temperature.Lowtemperature storage is one of the most efective technologies for preserving fruits [4].Unfortunately, the primary limitation of keeping peaches at low temperatures leads to the development of chilling injury (CI).CI, a physiological disorder, mainly occurs in various cultivars of peach fruit stored at a temperature between 2.2 and 7.6 °C.As soon as fruits are harvested, they are kept in cold stores.Since the fresh horticultural produce is gradually subjected to cold storage, continuous cold temperatures above their freezing point degrade the quality of the product within the course of time, and these symptoms can be clearly observed once the horticultural produce is transferred from cold storage condition to ambient conditions [5].
Te development of CI symptoms in the postharvest produce is considered a complex process involving various mechanisms such as degradation of the cell wall, oxidative stress, membrane damage, and hormonal imbalance, where cell wall alteration due to a low temperature is considered the primary physiological response [6,7].A hypothesis was proposed in relation to the accumulation of certain toxic compounds, increased cell wall permeability, decreased state of water dynamics, metabolism imbalance, and other plant mechanisms afected due to CI [6][7][8].
Melatonin (MT), a ubiquitous indoleamine, chemically known as N-acetyl-5-methoxytryptamine, was frst discovered in 1958 in the bovine human pineal gland [17].It has been reported to play its part in important physiological processes including fruit maturation, photoprotection, and fruit ripening.Various reports have stated the efects of MT on CI alleviation in several fruits, such as kiwifruit [18], apricot [19], tomato [20], nectarine [21], bitter lemon [22], sapota [23], bell pepper [24], mango [6-8, 11, 25], and banana [26].MT is the most preferred signaling molecule nowadays for alleviating CI, reducing postharvest decay, maintaining quality, and extending the shelf life of fruits and vegetables.However, the literature in this context is scanty and not conclusive.To the best of our knowledge, no study reported in peach fruit on the mechanism of CI alleviation by exogenous melatonin treatment in relation to proline metabolism.
Tus, in this study, we intend (a) to analyze the efects which MT has on the concentration and metabolism of endogenous proline and (b) to understand what efect and relation endogenous proline has on chilling tolerance in peach fruit stored at 2 ± 1 °C of chilling temperature.Te aim of the study is to build up a sound knowledge of various underlying physiological mechanisms in peach fruit through which a defense action is induced by MT in alleviating chilling tolerance.

Fruit Selection and Treatment Application.
Peach fruit variety "Silver King" was procured from the farmer's feld of Rajgarh, Sirmour, Himachal Pradesh, India."Silver King" is a chilling-sensitive variety, and chilling symptoms are showing easily.Peaches were harvested at physiological maturity but before ripening with yellow colour and just at the start of red blush tinge on the fruit skin.Fruit were transported to the laboratory within 10 h maintaining a temperature of 20 °C throughout the transportation.Fruit were inspected for any bruises, blemishes, injury, disease, etc., and 400 fruit were selected with uniform maturity, colour, size, and free from any disease symptom or disorder.Fruit were divided into two groups (n � 200/group).Sampling was followed by disinfecting the fruit for 2 min with 1% (v/v) PVPP.Te frst group of peaches was again divided into fve lots of 40 fruit each, and separately immersed in distilled water, used as control replicates, whereas the second group also divided into fve lots comprising 40 fruit in each lot, and separately immersed in MT solution of 100 µM, was considered as treated.Te immersion duration of peaches in water, as well as MT, was 2 h at 25 ± 2 °C in dark conditions, to prevent the degradation of MT [2].Te MT concentration (100 µM) and dipping duration (2 h) were standardized in preliminary trials.Later, the fruit were air-dried and kept at 2 °C ± 1 °C with 85-90% relative humidity for 21 d.Observations were recorded by removing fruit from each replication after 0, 7, 14, or 21 d of storage, in addition to a 3 d simulation period under ambient conditions (dry, clean, ventilated area at 25 ± 2 °C, 70-75% relative humidity).All parameters were assessed in fve replicates.In addition, 10 fruit were kept separately to observe changes in colour and weight loss.After analyzing the fruit respiration and ethylene production rates and physicochemical parameters, the tissues of peach fruit were homogenized in liquid nitrogen and preserved at a freezing temperature of −80 °C for further work.

Chilling Injury Index.
Te fruit of both groups were evaluated every 7 d interval of cold storage, plus 3 d at 25 °C, 70-75% relative humidity after removal from cold storage.Te chilling injury index (CII) was then assessed on the basis of the appearance of the fruit.A numerical scale ranging from 0 to 5 was used to analyze CI severity.CI severity including the intensity of the CI symptoms in fruit including reddening, mealiness, fesh browning, leatheriness, and fesh bleeding was recorded.Te 0 score denotes no CI severity, 1 for 1-20% CI severity, 2 for 21-40% CI severity, 3 for 41-60% CI severity, 4 for 61-80% CI severity, and 5 for 81-100% CI severity on the fruit surface area showing CI symptoms [27].Readings were taken in fve replications, and CII was calculated by the following formula: 2 Journal of Food Biochemistry CII �  rank score * number of fruit receiving the rank of chilling symptoms total number of fruits . (1)

Respiration and Ethylene Production Rates.
Measurement of ethylene production and the respiration rate was performed in fve replicates as described by [28].For measuring the rate of respiration, we used an 800 mL airtight glass jar.Te ethylene analyzer (Bioconservacion, Spain) was calibrated.Te peach fruit were weighed and placed inside the glass jar and closed with a rubber septum over its cap.Te flled jar was kept at 20 °C for the entire duration of measurement.An initial reading was taken by piercing the probe of the ethylene analyzer into the jar headspace through the septum.Te fnal reading was taken after a duration of 2 h.After the measurement of 2 h, calculations of the time taken for ethylene production were performed and were expressed in nmol kg −1 •s −1 .
A similar type of experiment was conducted for measuring the respiration rate.An autogas analyzer (PBI Dansensor, Denmark) at 20 °C was used for the estimate of the respiration rate (CO 2 production rate).By inserting the probe of the autogas analyzer into the glass jars, the analyzer gave the reading of available oxygen and carbon dioxide in the gas jars.Te respiration rate was measured in nmol kg −1 (3) 2.5.3.Firmness.For determining the frmness of the fruit, we used a texture analyzer (TA.HD Plus, Stable Microsystems, UK).Te peach fruit was kept on the stationary platform of the frmness analyzer.We used a 3 mm diameter stainless steel probe at a 1.5 mm•s −1 pretest speed, 0.5 mm•s −1 test speed, and 10 mm•s −1 posttest speed, which pierced the fruit and determined its frmness.Te measurements were recorded in fve replicates and taken along the equatorial plane at two opposite points, the penetration depth being 5 mm.Te readings were expressed in Newton (N).

Total Soluble Solids.
For calculating total soluble solids (TSSs), we used the pulp of the fruit.A muslin cloth was used to squeeze the pulp out of the fruit and was followed by fltration.A fltered drop of juice was placed in a hand-held refractometer (Atago, Japan), and the readings were noted in fve replicates and represented as a percentage.

Titratable Acidity, TSS: Acid Ratio, and pH.
Determination of titratable acidity (TA) was performed according to the previously described method [29].Te fruit was squeezed using a muslin cloth to squeeze out its pulp.Its pulp (20 g) was taken, which was then homogenized in 100 mL distilled water and fltered.Titration with 0.1 N NaOH was performed on the above fltrate.Phenolphthalein (1%) was added to the above solution.Titration was performed until the endpoint was attained.Te endpoint was attained by a light pink shade which resulted due to phenolphthalein.Readings were noted in fve replicates, and results were expressed as a percentage.Te TSS: TA ratio was measured by dividing the measurement of TSS by that of TA.
For measuring the pH of the fruit juice, a calibrated pH meter (Cyberscan, Eutech Instruments, Canada) was used.
2.6.Malondialdehyde.Te concentration of malondialdehyde (MDA) was determined by a procedure described earlier with slight modifcations [30].Te fruit was squeezed by hand and crushed in a mortar and pestle.Liquid nitrogen was poured onto the crushed fruit and further ground in the pestle.Te fnely ground tissue was stored at −80 °C for subsequent analysis.For measuring the MDA content, 2 g of fesh tissue was homogenized with 10% TCA having 0.5% (w/v) thiobarbituric acid (TBA).Te mixture was incubated at 100 °C for 10 min, followed by immediate cooling.Te supernatant was collected after centrifugation (3−18 KS, Sigma, Germany) (5,000 × g, 20 min, 4 °C), and the absorbance was read at 450, 532, and 600 nm using a spectrophotometer (Specord200plus, Analytik Jena, Germany).Analysis was performed in fve replicates.Te concentration of MDA present in 2 g tissue was calculated in mol as converted into a per kg basis of fruit.Te results were expressed in mol•kg −1 .

Proline Measurement.
Te concentration of proline in fruit tissues was measured with some modifcations [31].Te fruit was squeezed by hand and crushed in a mortar and pestle.Liquid nitrogen was poured onto the crushed fruit and was further ground in the pestle.Te fnely ground tissue was stored at −80 °C for subsequent analysis.Peach fruit tissue (1 g) was used for proline extraction.Te fruit tissue was dissolved in 10 ml 3% (v/v) sulfosalicylic acid and subjected to centrifugation (3−18 KS, Sigma, Germany) (12,000 × g, 20 min, 4 °C).Te assay mixture contained 1 mL ninhydrin reagent and 2 mL glacial acetic acid.Tis mixture was subjected to 30 min boiling followed by the addition of 2 mL toluene.Its absorbance was analyzed at 520 nm using a spectrophotometer (Specord200plus, Analytik Jena, Germany).Te concentration of proline present in 1 g fruit tissue was calculated in mg with respect to the standard curve of proline.Te proline concentration in 1 g fruit tissue was converted into a per kg basis of fruit.Te results were expressed in mg•kg −1 .

Δ 1 -Pyrroline-5-carboxylate Synthetase (P5CS).
Δ 1 -Pyrroline-5-carboxylate synthetase (P5CS, EC 2.7.2.11/ 1.2.1.41)activity was measured in fruit tissues in fve replicates, according to the method described with some modifcations [32].Te fruit was squeezed by hand and crushed in a mortar and pestle.Liquid nitrogen was poured onto the crushed fruit and further ground in the pestle.Te fnely ground tissue was stored at −80 °C for subsequent analysis.1 g of tissue was used for the analysis.Te tissue was homogenized in a refrigerated mixture of 500 mM Tris-HCl bufer having pH 7.6, 2 mM PMSF, 100 mM β-mercaptoethanol, and 10 mM magnesium chloride, followed by centrifugation (3−18 KS, Sigma, Germany) of the homogenate at 12,000 × g for 20 min.Te supernatant was used as the crude extract.A 0.8 mL reaction mixture was taken, which contained 0.2 mL of crude enzyme extract, 4 mM of ATP, 76 mM sodium glutamate, and 100 mM Tris-HCl having a pH of 7.2.After adding 0.5 mM NADPH, the absorbance was recorded at 340 nm•min −1 using a spectrophotometer (Specord200plus, Analytik Jena, Germany).
Results were defned as the decrease in absorbance by 0.001.
As the above P5CS activity determined was of 1 g fruit tissue, the above value was converted into a per kg basis of fruit.Te results were expressed in U•kg −1 •s −1 .

Ornithine-δ-aminotransferase (OAT).
Activity of ornithine-δ-aminotransferase (OAT, EC 2.6.1.13) was measured by homogenization of 1 g of fruit tissue, performed in a mixture of ice-cooled 50 mM potassium phosphate bufer having a pH of 7.8, consisting of 2 mM DTT, 0.2 mM pyridoxal phosphate, and 2 mM EDTA.Centrifugation (3−18 KS, Sigma, Germany) of the homogenate was performed at 12,000 × g for 20 min.Te supernatant from the described process was used as the crude enzyme extract.Phosphopyridoxal (50 mM), 20 mM α-ketoglutarate, and 35 mM L-ornithine were mixed to form the reaction mixture, which was then incubated at 37 °C for 15 min.After the addition of 2% ninhydrin and 3 M perchloric acid, the mixture was subjected to boiling for 20 min, centrifuged at 12,000 × g for 15 min, the precipitate obtained was dissolved in toluene, and readings were taken in fve replicates, where the absorbance was read using a spectrophotometer (Specord200plus, Analytik Jena, Germany) at 510 nm.As the above OAT activity determined was of 1 g fruit tissue, the above value was converted into a per kg basis of fruit.Te results were expressed in U•kg −1 •s −1 [33].

Proline Dehydrogenase (PDH).
Proline dehydrogenase (PDH, EC 1.5.5.2) activity was measured according to the method described by Lopez-Carrion [32] with slight modifcations.Te liquid nitrogen ground fruit tissue was used for estimating the PDH activity.To determine, 1 g fruit tissue was taken and homogenized in a mixture of ice-cooled 0.1 M potassium phosphate bufer (pH 7.8), containing 10 mM β-mercaptoethanol and 2 mM EDTA.Te above mixture was centrifuged (3−18 KS, Sigma, Germany) (12,000 × g for 20 min).Te supernatant obtained after centrifugation was used as the crude enzyme extract.Te reaction mixture contained 10 mM NAD + , 0.15 M sodium carbonatebicarbonate bufer (pH 10.3), and L-proline.Te crude enzyme extract (0.2 mL) was mixed with 0.9 mL reaction mixture to initiate the reaction.Results were measured in fve replicates.Te above PDH activity determined was of 1 g fruit tissue, and the above value was converted into a per kg basis of fruit.Te results were expressed in U•kg −1 •s −1 .

Principal Component Analysis (PCA) and Comprehensive
Evaluation.Te efect of MT on the storage life of peach fruit was evaluated using the principal component coefcient using R package software.It featured the eigenvalue, variance %, and cumulative variance % (Table 1).Using the values of Table 1, a scree plot was made to determine the principal component factors (Figure 1).After identifying the principal component factors, a variable correlation plot was developed (Figure 2).Furthermore, Figure 3 is developed to interpret which variables contributed the most in the extension of shelf life of MT-treated peaches.

Chilling Injury Index.
Te results revealed a signifcant diference between the CII of control and treated fruit.A higher percentage of CI severity was recorded for the control fruit from day 7 onwards on the numerical scale having ranking from 0 to 5. Mealiness, wooliness, reddening, and peel wrinkling were the main CI symptoms observed in the peach fruit related to CI. Te MT-treated peach fruit started showing symptoms of CI only after 14 d of storage (Figure 4).According to the results, CII in control fruit was 66.66, 78.8, and 66.66% higher than in MT-treated on days 7, 14, and 21, respectively (Figure 5).Hence, the shelf life of the MT-treated fruit was extended and maintained up to 21 d (Figure 5).On the other hand, control peaches showed deteriorating symptoms from day 7, which may reduce their appeal for acceptability by target consumers.

Respiration and Ethylene Production
Rates.One of the most important parameters for determining the metabolic activity of a fruit is its rate of respiration and ethylene production.From the results, it can be estimated that the rate of respiration and ethylene production follow a dependent pattern where no signifcant diference can be seen in both groups until day 7.However, the rate of respiration and ethylene production experienced a climacteric rise which was 60 and 28.57% higher in control fruit when compared to the MT-treated fruit (Figures 6(a) and 6(b)).Te metabolic and other ongoing processes lead to the loss of surface water of a fruit.On weighing the control and treated fruit every 7 d, the percentage of weight loss was more in the control fruit than in the treated one.However, throughout the storage period, no signifcant diference was observed among the groups (Figure 7(a)).Te maturity of the fruit is determined by the frmness of the fruit.Te frmness of both groups showed a decreasing trend as fruit softened with the advancement of storage duration.However, MT-treated peaches exerted less changes (Figure 7(b)).Results revealed signifcant diferences in fruit frmness from 7 d onwards.Te frmness of MT-treated fruit was found to be 8.57, 25, and 150% higher than that of the nontreated fruit.In addition to frmness, an equally important characteristic determining the quality of the fruit is its colour.Te fruit colour is expressed via L * , a * , b * , hue angle (h °), and chroma (C).L * denotes the lightness in fruit, where the negative value means the fruit has lightness in fruit and the positive value depicts darkness.Te L * value decreased in storage and showed an irregular pattern, whereas it is worth mentioning that MT-treated fruit showed delayed darkening as the values moved towards positive at a slower rate.a * depicts redness and greenness of fruit, where the positive value depicts redness and the negative value depicts greenness.At 0 th day of storage, both groups show negative values of a * denoting greenness.With the commencement of cold storage, peaches of both groups start becoming red; however, it is to be noted that MT-treated peaches show lower a * values than the control groups on 14 th and 21 st day, denoting lower fruit redness.b * denotes yellowness and blueness of fruit, where the positive b * value means yellowness and the negative value means blueness.MT-treated peaches showed a more positive value than the control group, depicting more fruit yellowness.Te C value tells us whether the fruit is bright or dull by having a positive and negative value, respectively.h °showed an increasing pattern in both groups, its value reaching a maximum on day 7 (Table 2).

Total Soluble Solids, Titratable
Acidity, TSS: Acid Ratio, and pH.An increasing trend was recorded for TSS with progression in storage duration in both control and treated fruit (Figure 8(a)).TSS was 1.9, 17.19, and 14.67% higher in the nontreated fruit than in the MT-treated one.A decreasing trend in TA was observed in both treated and control fruit.MT-treated fruit showed signifcantly 25.71%, 75%, and 75.2% higher TA on days 7, 14, and 21, respectively (Figure 8(b)).Te TSS: TA ratio was found to be increased in both control and treated fruit in storage, denoting the increase in maturity (Figure 8(c)).Signifcant diferences in the TSS: TA ratio could be observed only after 7 d of storage, where the ratio was 23.08, 88.89, and 11.91% lower in treated fruit on days 7, 14, and 21, respectively.MT treatment was successful in reducing the TSS: TA ratio, thus delaying maturity and enhancing shelf life.Organic acids are constantly utilized in various other internal processes, leading to a decrease in the pH of the fruit juice.pH showed an increasing trend from day 7 onwards, being 21.43, 26.01, and 20% higher in control than in the treated fruit on 7, 14, and 21 days of storage, respectively (Figure 8(d)).

Malondialdehyde.
Results showed a signifcant diference between the control and treated groups from day 14 onwards.MDA content was 26.32% and 17.39% lower in the MT-treated fruit on days 14 and 21, respectively, when compared to the control (Figure 9).

Proline Content, P5CS, OAT, and PDH Enzyme Activity.
An increase in proline concentration was observed both in the control as well as treated fruit with the advancement of storage duration.However, MT application resulted in 13.27, 31.43, and 17.5% higher proline accumulation on days 7, 14, and 21, respectively.Tis trend is related to CII.A signifcant difference was observed on days 7, 14, and 21 between control and MT-treated fruit (Figure 10    Journal of Food Biochemistry 3.6.Determining Principal Components.Te scree plot (Figure 1) shows that approximately 98.06% of the information (variances) contained in the data is retained by the frst fve principal components.So we might want to stop at the ffth principal component.

Correlation Analysis.
Te correlation coefcient values between all the physiological parameters are shown in Figure 2.
It is known as the correlation plot which shows the relation between all variables.Positively correlated variables are grouped together.Negatively correlated variables are positioned on opposite sides of the plot origin (opposed quadrants).
From the plot, it was concluded that the respiration rate, ethylene production, pH, CII, TSS, the TSS/TA ratio, MDA, and PDH are positively correlated, whereas frmness, P5CS, OAT, proline, and mass loss are negatively correlated.

Contribution of Variables.
Te contributions of variables in accounting for the variability in a given principal component are expressed in percentage.Variables that are correlated with PC1 (i.e., Dim. 1) and PC2 (i.e., Dim. 2) are most important in explaining the variability in the dataset.Variables that do not correlate with any PC or correlated with the last dimensions are variables with low contribution and might be removed to simplify the overall analysis.Te larger the value of the contribution, the more the variable contributes to the component.Figure 3 reveals that proline, MDA, TSS, frmness, the respiration rate, ethylene production, CII, PDH, and L * contribute greatly to the extended shelf life of MT-treated peaches.

Principal Component Analysis.
Loading values and scores in PCA enabled visualization of similarities and diferences in physicochemical parameters of the studied peach samples.PCA allowed understanding which parameters contributed the most.Te two components accounted for 83.70% of the variation in the dataset.Te most infuential parameters on frst principal component 1 (PC1) accounted for 66.20% of variance (Figure 3).Tese parameters were MDA, CII, frmness, TSS, TA, P5CS, mass loss, and the TSS/TA ratio.Te main contributions to second principal component 2 (PC2), which accounted for 17.50% of variance, were PDH, OAT, proline, the respiration rate, ethylene production, and L * .

Discussion
Peach being a climacteric fruit has a limited shelf life due to which it is advisable to keep it at a cold temperature.Continuous chilling temperatures above the freezing point of the fruit cause CI, which is the main hindrance responsible for decreasing its postharvest life and quality.A decreasing trend in CI has been reported in various fruit when treated with exogenous MT.Hundred μM MT-treated pomegranate [34] and 100 or 200 μM MT-treated tomato [4] experienced alleviation in CI due to prestorage MT application.MT treatment was studied in relation to CI reduction in mango fruit and established the relationship with proline metabolism, c-aminobutyric acid shunt pathway, fatty acid composition, and internal cell energy status [6-8, 11, 25].Sevillano et al. [35] stated the contribution of various molecular, physiological, and hormonal mechanisms to this discrepancy.Tere are several factors that are responsible for chilling tolerance regulation in peach fruit, among which CO 2 is the most common.Carbon dioxide gives rise to ethylene which is an essential plant hormone responsible for ripening and senescence.Carbon dioxide acts as a cofactor for 1-aminocyclopropane-1-carboxylic (ACC) oxidase [15,27].1-Aminocyclopropane-1-carboxylic (ACC) is the key enzyme for ethylene production and suppressed at cold 10 Journal of Food Biochemistry temperatures.Keeping the fruit at room temperature reconditions it and results in a rapid ethylene increase due to ACC production (Figure 6(b)).Tis, in turn, enhances the occurrence of CI [35].Here, MT is found to delay the climacteric rise in the fruit.Tus, exogenous MT application was capable of shifting the climacteric rise.Te synergistic efect of ripening and catalytic cell wall enzymes such as endo-1,4-β-D-glucanase, polygalacturonase, and pectin esterase contributes to fruit softening.Te cell wall mainly comprises glycoproteins, cellulose, hemicellulose, and calcium pectate.Fruit frmness depends on wall-to-wall adhesion of cells.As ripening progresses, dissolution of pectin starts taking place.Enzyme pectinase breaks down pectin present in the middle lamella of the fruit cell wall and results in softening of the fruit.Tus, the signifcant diference in frmness between both groups contributed to the delayed respiration rate.Te L * value greatly afects the acceptability of the fruit in the market.With the commencement of storage, the ongoing biochemical processes of fruit lead to the production of enzymes, which turns the shade of fruit from light to dark.A lighter shade in MT-treated peaches denotes that MT successfully slowed down biochemical processes leading to a reduction in enzymatic production.Te a * and b * values show that MT-treated fruits experienced lower ripening stress and remained greener/yellower when compared to the control.Troughout cold storage, MT-treated peaches experienced a higher C value than the control, denoting that  MTsuccessfully maintained the brightness of the fruit.h °was higher in MT-treated peaches, denoting MT was successful in maintaining the yellow colour of peaches and delayed its change from yellow to green.A lower value means the colour of the fruit is close to dark, and a higher value represents its lightness.Due to various ongoing processes, the storage of fruit leads to the development of various monosaccharides, mainly glucose, and fructose.Being a climacteric fruit, peach experiences rapid ripening and respiration, leading to the metabolization of complex carbohydrates to simple sugars.Tese simple sugars mainly include glucose and fructose, which contribute to an increase in TSS for both groups [6][7][8]25].Ongoing respiration leads to the depletion of organic acids, leading to the reduction in the pH of fruit juice.Te decreasing trend in TA of both groups can be attributed to several metabolic processes in the fruit.As respiration occurs, it utilizes available organic acids as its substrate and contributes to TA reduction [25].MDA is known to be the fnal product of lipid peroxidation and hence acts as a reliable indicator for the same [11].Progressive fruit ripening leads to reactive oxygen species (ROS) formation, which when produced in excess enhances oxidative stress.In order to counter this deleterious process, enzymatic and nonenzymatic antioxidant systems are ramped up.ROS species are highly active, which initiate the process of lipid peroxidation, leading to the formation of various end products, such as MDA, alkanes, alcohols, epoxides, and lipid alkoxyl [8].Lipoxygenase triggers the activity of lipid peroxidation resulting in cell membrane destabilization.Tus, a decrement in the membrane integrity of the control fruit is linked to an increment in the MDA content.Tese results agree with previous studies which reported positive results in CI alleviation due to low MDA activity in litchi fruit treated with 100 μM MT [36].
Proline is an amino acid that is nonpolar in nature and acts as an osmotic adjustment substance against unfavorable conditions [11].It plays an essential role as a nonenzymatic antioxidant, molecular chaperone, energy supplier, and signaling molecule in plants.Te results obtained favor previous studies which reported better stress tolerance and alleviation of CI by proline accumulation [2,11,37,38].A recent study by Aghdam et al. [39] has documented MT treatment of 100 μM for 5 min and 400 μM MT for 15 min to mitigate CI by initiating proline accumulation in tomato and litchi, respectively.Bhardwaj et al. [11] noticed 1.39 times increased proline content in 100 μM MT-treated "Langra" mangoes (for 2 h) on 28 d of storage, and it is correlated with the decrease in CI incidence.In agreement with the above studies, the current work confrms that administering peaches to 100 μM MT for 2 h results in a signifcant increase in proline levels.Te observed CII pattern is in accordance with the proline accumulation trend.Furthermore, the evident reduction in MDA concentration, weight loss, and chilling symptoms followed by increased levels of proline in MT-treated peaches confrms the antioxidative and protective nature of amino acid, proline.P5CS is the key enzyme that plays a major role in the synthesis of proline through the glutamate pathway [11].It is a dual function and rate-limiting enzyme, resulting in the synthesis of glutamatederived proline.It acts as a catalyst in the formation of Δ 1pyrroline-5-carboxylate (P5C) from the cyclization of glutamate-c-semialdehyde (GSA) which is followed by the production of NADP + .NADP + produced is reduced to NADPH by its coupling with the pentose phosphate pathway (PPP), thus ensuring an uninterrupted reducing power for various cell processes [11].Te biosynthesis pathways of phenylalanine and tryptophan are also supplied, which later leads to an increment in MT and phenol accumulation.Tus, these molecules form an important part of the plant's antioxidant system [8].OAT through the ornithine pathway contributes to proline synthesis.In accordance with the previous studies on litchi [5], this present study confrms that exogenous MT application to fruit signifcantly enhances the activity of P5CS and OAT, contributing to the accumulation of proline.PDH is known as the rate-limiting enzyme as it forms glutamic acid by catalyzing proline [40].It causes hindrance in protein accumulation.In accordance with previous studies in tomatoes [39], litchi [5], and mangoes [11], MT was successful in lowering PDH activity.Terefore, MT-treated peaches increased the P5CS and OAT activity, which in turn decreased the PDH activity, thus adding to their increased chilling tolerance.
Proline being a non-enzymatic antioxidant was found to play an important role in the mitigation of CI in peaches.MT was found to activate P5CS and OAT, initiating glutathione and ornithine pathways, which in turn suppressed the formation of PDH, which led to the increment in proline levels.Te biosynthesis pathways of tryptophan and phenylalanine also contributed to MT accumulation, thus enhancing its performance.A parallel trend was obtained between the proline levels and the occurrence of CII.Proline was found to decrease the MDA content as it lowered excess ROS production, thus preventing lipid peroxidation and electrolyte leakage of the fruit cell wall.In a nutshell, MT application to fruit activated its antioxidant system, where many antioxidants including proline came into action and aided in mitigating the symptoms of chilling injury and increased chilling tolerance.
MT in peaches has been exogenously applied to elucidate many pathways, including phenolic pathways, membrane fatty acid regulation, oxidative damage pathway, sucrose metabolism, cell wall disassembly, and GABA shunt pathway; however, the proline pathway still remains unexplored.Further studies are required at the transcriptomic and genomic levels to elucidate the complex molecular network and proposed biochemical pathways of melatonin in peaches in response to chilling injury.

Conclusion
Te present study focused on the efect of exogenous MT application on the alleviation of CI in the "Silver King" peach cultivar stored at 2 ± 1 °C and 90-95% relative humidity for 21 d.Melatonin-treated fruit experienced a 78.8% lower CI than the control fruit; moreover, they were recorded to experience a 60% lower respiration rate, 1.5 times higher frmness, 17.19% lower TSS, 75.2% higher TA, 26.01%lower pH, 26.32% reduction in MDA levels, and 31.43% higher proline accumulation.P5CS, OAT, and PDH were found to be the key enzymes for proline synthesis.Te partial CI tolerance efcacy of MT treatment shown in "Silver King" peach fruit might be due to proline accumulation in the fruit.

Figure 1 :
Figure 1: A scree plot of the factors studied for shelf-life extension of peaches.It shows variance shown by each factor.Te horizontal axis represents dimensions, and the vertical axis represents the percentage of the explained variances.

Figure 3 :Figure 2 : 1 .
Figure 3: Contribution % of each factor to the frst and second principal components, where the horizontal axis represents the parameters and the vertical axis represents the % contribution.

Figure 5 :Figure 4 :
Figure 5: Efect of 0 μM (control) or 100 μM (treated) melatonin on the chilling injury index in peach fruit stored at 2 ± 1 °C for 21 days followed by a simulation period of 3 days at ambient temperature.Te letters a, b, and c indicated signifcant diferences following the application of MT with 100 µM as based on Tukey's test (p < 0.05).
(a)).Te activity of P5CS expressed in U•kg −1 •s −1 was enhanced by MT treatment.A signifcant change was observed in the fruit on days 14 and 21 when MT-treated fruit experienced 26% and 37.93% higher P5CS enzymatic activity (Figure10(b)).OAT expressed in U•kg −1 •s −1 was observed to increase in control as well as in MT-treated groups; however, MT-treated fruit recorded 31.03,51.67, and 50% higher OAT enzymatic activity on days 7, 14, and 21, respectively (Figure10(c)).MT-treated fruit showed signifcant changes in the PDH enzymatic activity expressed in U•kg −1 •s −1 , which was 59.25, 59.37, and 31.43%lower in the treated fruit than in the control.Tus, MT was capable of decreasing the PDH activity in the peach fruit (Figure10(d)).

Figure 7 :Figure 6 :
Figure 7: Efect of 0 μM (control) or 100 μM (treated) melatonin on the (a) weight loss and (b) frmness of peach fruit stored at 2 ± 1 °C for 21 days followed by a simulation period of 3 days at ambient temperature.Te letters a, b, and c indicated signifcant diferences following the application of MT with 100 µM as based on Tukey's test (p < 0.05).

Figure 8 :
Figure 8: Efect of 0 μM (control) or 100 μM (treated) melatonin on (a) total soluble solids, (b) titratable acidity, (c) TSS/TA ratio, and (d) pH in peach fruit stored at 2 ± 1 °C for 21 days followed by a simulation period of 3 days at ambient temperature.Te letters a, b, and c indicated signifcant diferences following the application of MT with 100 µM as based on Tukey's test (p < 0.05).

Figure 9 :
Figure9: Efect of 0 μM (control) or 100 μM (treated) melatonin on the malondialdehyde concentration in peach fruit stored at 2 ± 1 °C for 21 days followed by a simulation period of 3 days at ambient temperature.Te letters a, b, and c indicated signifcant diferences following the application of MT with 100 µM as based on Tukey's test (p < 0.05).
•s −1 .Te formulas used were as follows:

Table 2 :
Efect of 0 μM (control) or 100 μM (treated) melatonin on the colour parameters in peach fruit during storage at 2 ± 1 °C for 21 days followed by a simulation period of 3 days at ambient temperature.denotes lightness/darkness, a * denotes redness/greenness, b * denotes yellowness/blueness, h °denotes the hue angle, and, C denotes chroma; letters a, b, and c indicate signifcant diferences as based on Tukey's test (p < 0.05). *