Postharvest methyl jasmonate dipping modulates lipid composition and volatile profiles to alleviate chilling injury in yellow-fleshed peach fruit
Po-Kai Huang, Diane M. Beckles, Pedro J. Martínez-García, Carlos H. Crisosto
TL;DR
This study shows how methyl jasmonate treatment helps yellow-fleshed peaches resist cold storage damage by changing their lipid and aroma profiles.
Contribution
The study reveals new insights into how MeJA protects yellow-fleshed peaches from chilling injury through lipid and volatile compound changes.
Findings
MeJA-treated peaches had higher phospholipid levels and more unsaturated fatty acids, which may preserve membrane integrity.
MeJA increased levels of γ-decalactone and δ-decalactone, important for peach flavor, and reduced chilling injury biomarkers.
Autophagy-related genes were upregulated in MeJA-treated peaches, suggesting a role in CI resistance.
Abstract
Methyl jasmonate (MeJA) has been widely shown to mitigate chilling injury (CI) but the underlying mechanisms in peach remain largely unknown. Further, most MeJA studies focus on white-fleshed, rather than the more CI-resistant, yellow-fleshed peaches. To address this knowledge gap, we integrated lipidomic, volatile organic compound (VOC), and transcriptomic analyses to elucidate the mechanisms underlying MeJA protection in yellow-fleshed peaches. We identified ethyl acetate and methyl cinnamate as potential early biomarkers of CI right after cold storage. Our lipidomic characterization revealed that after 21 days of cold storage at 5 °C, MeJA-treated peaches showed increased phospholipid levels and reduced triacylglycerols and diacylglycerols compared to control fruit. Additionally, membrane lipids in MeJA-treated peaches exhibited greater unsaturation in fatty acids, particularly an…
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Taxonomy
TopicsPostharvest Quality and Shelf Life Management · Plant Physiology and Cultivation Studies · Ion Channels and Receptors
Introduction
After harvest, peach (Prunus persica (L.) Batsch) fruit undergoes rapid ripening and senescence. Low temperatures slow these processes, but fruit stored between 2.2–7.6 °C (“the killing zone”) can lead to chilling injury (CI) [1, 2]. Fruit mealiness, ‘off flavor’, and internal flesh browning are among the symptoms of CI, each associated with different and sometimes overlapping underlying physiological mechanisms [2, 3]. CI may arise from unsuccessful or insufficient responses that fail to protect the fruit from cold stress [3]. While several physiological changes associated with CI have been linked to disruptions in cold response pathways [4], the precise underlying mechanisms remain under investigation. Moreover, the influence of CI-alleviating treatments on these pathways also remains largely unclear.
Lipid remodeling is one of the earliest cellular responses to cold stress in plants [5]. In peach, an increased unsaturated-to-saturated fatty acid ratio and double bond index were associated with reduced CI symptoms [6–9]. Furthermore, elevated levels of linolenic acid (18:3) were linked to enhanced CI tolerance and corresponded with differences in fatty acid desaturase (FAD) gene expression [9]. Cold stress also alters lipid class composition [5], and changes in phospholipid content were associated with CI symptoms in peach [6, 7, 10, 10–14]. Although phospholipid biosynthesis pathways are well characterized [15], their regulation in peach remains unexplored.
Low temperatures also reduce fruit volatile organic compounds (VOCs), several of which are derived from lipid precursors [16, 17]. In peach, aldehydes, alcohols, esters, lactones, and terpenoids are the primary contributors to peach aroma and are affected by CI [18]. Current molecular studies in peach primarily focused on six-carbon (C6) fatty acid-derived VOCs and lactones [19–21]. C6 VOCs are associated with “green” or “grassy” aromas, while lactones, particularly γ-decalactone and δ-decalactone, are major contributors to the characteristic “peachy” aroma. The green and glassy aromas were also the primary driver of liking determined for seven peach and nectarine California cultivars using sensory analysis [22]. These VOCs are derived from unsaturated fatty acids synthesized by FADs [18]. C6 VOCs are produced via the lipoxygenase (LOX) pathway, which involves LOX and hydroperoxide lyase (HPL) [23], whereas lactone biosynthesis involves the β-oxidation initiated by acyl-CoA oxidase (ACX) [24, 25]. Alcohol acyltransferase 1 (AAT1), regulated by NAC transcription factors, catalyzes the final step of lactone and ester biosynthesis [26–30]. However, most genes involved in the peach VOC synthesis pathways remain unknown, and their association with CI is still under investigation.
Exogenous application of the plant hormone methyl jasmonate (MeJA) alleviates CI across various peach cultivars through a broad range of regulatory mechanisms. MeJA treatment reduced membrane leakage [31, 32] and increased the unsaturated fatty acid ratio during and after cold storage [33]. Lipidomic analysis further demonstrated that MeJA treatment induces phospholipid remodeling [12]. In addition to regulating lipid metabolism, MeJA mitigated VOC loss associated with CI [34, 35]. Despite these advances, several knowledge gaps remain in understanding how MeJA modulates cold responses, particularly given the variation among peach cultivars, the intrinsic complexity of CI syndromes, and their dynamic progression throughout storage [4, 36, 37].
MeJA application mode and concentration may influence the effective cellular concentration, which in turn differentially affects fruit biological pathways. The most common application is exposing peach fruit to MeJA vapors at 10 µmol L^−1^ for 24 h [12, 33, 34], although Jin et al. [32] used a milder treatment of 1 µmol L^−1^. In contrast, Duan et al. (2022) is the only report where peach fruit (white-fleshed) were dipped in liquid MeJA for 10 min. After testing lower concentrations (0.01 mM and 0.1 mM), they found that 1 mM MeJA was optimal for fruit stored at 0 °C for up to 28 days [35]. Examining fruit response to immersion in other MeJA concentration–time combinations would inform on MeJA effectiveness over a broader range of treatments, which is important for understanding its biological action in vivo and for its practical use.
Most CI studies focus on white-fleshed peaches, and it is unknown if the same regulatory mechanisms occur in yellow-fleshed cultivars, which differ in key traits such as biochemical composition, including volatile characteristics, and importantly for this work, in CI susceptibility [38–40]. Although a few studies characterized CI responses in yellow-fleshed peaches [41, 42], none have examined the effects of MeJA on CI in these cultivars. Furthermore, many previous studies evaluated fruit at 0 °C storage rather than within the “killing zone” (2.2–7.6 °C) and examined secondary responses that occur during rewarming when CI symptoms are pronounced. Thus, the molecular regulation occurring immediately after cold storage, a critical time point for identifying early CI biomarkers, remains understudied.
Here, we used the yellow-fleshed cultivar ‘August Flame’ to investigate the molecular mechanisms by which MeJA alleviates CI. First, we confirmed that MeJA application, by immersing fruit at a concentration of 0.3 mM for 1 min, was effective in mitigating poor flavor attributes associated with chilling. These results were reproducible in fruit harvested in the 2022 and 2023 seasons. We then focused on changes in the lipidomic and VOC profiles measured immediately after cold storage within the “killing zone”. We tested the following hypotheses: (1) Does MeJA induce cold-responsive lipid modifications, including changes in lipid classes and double bond levels? (2) Are flavor-associated VOCs maintained at higher levels in MeJA-treated peaches compared to controls? (3) Do differentially expressed VOCs correspond with changes in their lipid-derived precursors? (4) Do these regulatory effects involve changes at the transcriptional level?
Materials and methods
Plant material
Peach (Prunus persica L. Batsch cv. ‘August Flame’) fruits with flesh firmness of 12–16 lb were harvested from Reedley, California, during the 2022 and 2023 seasons. ‘August Flame’ is a late-season, yellow-fleshed freestone cultivar that is industry-favored and exhibits susceptibility to CI. Additional details on plant materials are provided in Supplementary Text S1. Lipidomic and RNA-seq analyses were conducted using fruit harvested in 2022. A portion of the fruit was sampled immediately after harvest without any treatment (Ctrl_H), while the remaining fruit was stored at 5 °C for 21 d without (Ctrl_CS) or after dipping in 0.3 mmol/L MeJA (VBC-30530; Valent BioSciences Corporation, Libertyville, IL, USA) solution for 1 min (MJ_CS). A 21-day storage period at 5 °C was selected based on prior evaluations of this cultivar and industry-established market life ranges for chilling injury-susceptible peaches [1, 2, 43, 44]. Samples were collected, and CI symptoms were assessed immediately after cold storage. For lipidomic analysis, an additional treatment group was included in which cold-stored peaches were ripened at room temperature (Ctrl_CS_R). VOC analysis was performed using fruit from the 2023 harvest. In addition to Ctrl_CS and MJ_CS, a higher MeJA concentration treatment (3 mmol/L, MJ_CS-Hi) was included before cold storage. CI phenotypes were analyzed using four biological replicates, with each replicate consisting of 25 peaches. Flesh tissue slices were immediately frozen in liquid nitrogen and stored at −80 °C for subsequent analyses. RNA-seq and VOC analyses were conducted using three randomly selected replicates, while lipidomic analysis was performed using eight replicates. Please refer to Fig. 1 for a schematic summary of the experimental design.Fig. 1. Schematic summary of the experimental design. Sample names: Ctrl, control fruit (untreated with methyl jasmonate, MeJA); MJ, fruit dipped in MeJA solution; CS, fruit kept in cold storage; R, fruit rewarmed to room temperature after cold storage; Hi, samples dipped in 3 mmol/L MeJA
CI evaluation
During two seasons, in 2022 and 2023, CI was evaluated after cold storage. “Sound” fruits were identified by excluding those exhibiting fungal decay caused by brown rot (Monilinia fructicola). Sound fruits were cut into halves, and CI symptoms were assessed as previously described [1, 2, 44–46]. In brief, fruits exhibiting a dry appearance with little or no juice upon hand squeezing were classified as mealy. Flesh browning was defined as uniform brown areas extending from the pit cavity into at least 25% of the flesh. ‘Off flavor’ was defined as the absence of the typical peach flavor after informal tasting.
Lipidome analysis
Lipidomic profiling was performed using the Complex Lipids Targeted Panel by Metabolon, Inc. (Morrisville, NC, USA). Briefly, homogenized samples were soaked overnight in dichloromethane/methanol, followed by a modified Bligh-Dyer extraction with internal standards [47]. Lipid extracts were concentrated under nitrogen and reconstituted in dichloromethane:methanol (1:1, v/v) containing 10 mM ammonium acetate. Reconstituted samples were analyzed by infusion-mass spectrometry using a Shimadzu liquid chromatography with nano PEEK tubing and a Sciex SelexION-5500 QTRAP. Data acquisition was performed in multiple reaction monitoring (MRM) mode with more than 1,100 transitions. Individual lipid species were quantified by calculating the intensity ratio of each target compound to the internal standard, and multiplying by the concentration of the internal standard added to the sample. The raw data were natural log-transformed for analysis.
Univariate analyses were conducted by fitting linear models using the lm function in the stats package, and the results were processed using the emmeans function from the emmeans package [48]. Multiple comparison corrections, either Tukey’s or Dunnett’s method, were applied using the contrast function in the emmeans package. For screening the differentially abundant lipid species, false discovery rate (FDR) adjustment was performed using the q-value function from the qvalue package, with an adjusted p-value cutoff of < 0.05.
Multivariate analyses were performed using the mixOmics package following the official tutorial [49]. Briefly, variables with more than 20% missing data were excluded, and missing values were imputed using the Nonlinear Iterative Partial Least Squares (NIPALS) method via the impute.nipals function. Partial Least-Squares Discriminant Analysis (PLS-DA) [50] was then conducted using the plsda function. Variable Importance in Projection (VIP) scores were calculated with the vip function, using a cutoff value of > 1.0.
For examining lipid unsaturation levels, the unsaturated/saturated fatty acid ratio was calculated as the total concentration of unsaturated fatty acids divided by that of saturated fatty acids. The double bond index (DBI), which weights unsaturated fatty acids by the number of double bonds, was calculated as follows:
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathrm{DBI}}=\frac{\sum \left(\text{double bonds}\times \left[\text{Unsaturated FA}\right]\right)}{\sum \left[\text{Saturated FA}\right]}$$\end{document}VOC analysis
Frozen peach samples were ground into powder using a Retsch Mixer Mill MM 300 (Retsch GmbH, Haan, Germany) with liquid nitrogen. Five grams of powder were dissolved in 40 mL of sodium citrate buffer (1 M, pH 6.0) containing 400 µL of 1.14 M ascorbic acid. The mixture was centrifuged at 3,800 g for 20 min at 4 °C, and 5 mL of the supernatant was transferred into a 20 mL headspace vial with 1.5 g of NaCl. The sample was spiked with 20 µL of 10 ppm 2-undecanone as an internal standard and sealed.
Volatile profiles were analyzed using headspace solid-phase microextraction gas chromatography-mass spectrometry (HS–SPME–GC–MS), automated with a GERSTEL MultiPurpose Sampler MPS2 (GERSTEL GmbH & Co. KG, Mülheim an der Ruhr, Germany) coupled to an Agilent 6890 N gas chromatograph and a 5975 mass spectrometer (Agilent Technologies, Wilmington, DE, USA). VOCs were extracted using Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS) SPME fiber (Supelco, Bellefonte, PA, USA). After a 5-min incubation with agitation, the fiber needle was inserted 22 mm into the vial headspace and exposed for 30 min at 40 °C. After extraction, the fiber was desorbed in the GC injection port at 250 °C. The injection was performed in split mode (10:1) at 250 °C. VOCs were separated using a DB-Wax column (30 m × 0.25 mm i.d., 0.25 µm film thickness; J&W Scientific, Folsom, CA, USA), with helium as the carrier gas at a constant flow rate of 1.0 mL/min. The oven temperature was initially held at 40 °C for 5 min, then ramped at 6 °C/min to 250 °C, and held for 5 min. The mass spectrometer was operated in electron ionization (EI) mode at 70 eV, scanning from m/z 30 to 300. The MS source temperature was set to 230 °C and the quadrupole to 150 °C. Each sample was analyzed in technical triplicate.
Compounds were identified using linear retention indices (RIs), calculated from a series of n-alkanes (C8–C26) using MassHunter Unknowns Analysis (version 10.0, Agilent), with the NIST Mass Spectral Library (NIST-23) and the setting of SNR threshold = 5 and Min match factor = 80. Target VOCs were confirmed based on both RI values and spectral matching. Quantification was performed using MassHunter Quantitative Analysis (version 10.0, Agilent), with an internal standard applied for relative quantification. The univariate and multivariate analyses were performed using the same pipeline described in lipidome analysis. Aroma descriptions were obtained from the FlavorDB2 database [51].
RNA-seq analysis
RNA-seq library preparation and alignment were performed by Novogene (Sacramento, CA, USA). Total RNA was extracted using a column-based purification system established by Novogene. RNA integrity was assessed using the RNA Nano 6000 Assay Kit on the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Libraries were constructed using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs, USA), and sequenced on an Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA) to generate 150 bp paired-end reads. Reads were aligned to the Prunus_persica_NCBIv2 reference genome obtained from Ensembl Plants [52] using HISAT2 v2.0.5 [53]. Gene-level count tables were then generated using featureCounts v1.5.0-p3 [54].
The count table was analyzed using an independently developed workflow. For differential gene expression (DGE) analysis, model fitting was performed using the voom method in limma package (v3.60.2) [55]. Gene Ontology (GO) annotations were queried from Dicots PLAZA 5.0 [56], and GO enrichment analysis was performed using the goseq package (v1.56.0), with correction for gene length bias [57]. A comprehensive list of genes within families of interest was identified by integrating annotations from Phytozome v13 [58] and Dicots PLAZA 5.0, as previously described (Table S1) [59]. Unless otherwise specified, candidate genes were selected based on an FDR threshold of 5%, using a Benjamini–Hochberg adjusted p-value cutoff of 0.05 [60].
Statistical analysis
All analyses were conducted using R (version 4.4.0), except for CI phenotyping, which was performed using Minitab. Data visualization was performed using the ggplot2 package [61].
Results
MeJA alleviates CI symptoms in ‘August Flame’
CI symptoms were evaluated using industry standards after 21 d of storage at 5 °C. They were as follows: the percentage of peaches that (a) were ‘sound’ (i.e., free of fungal decay), (b) had mealy flesh (i.e., not juicy), (c) exhibited internal browning, and (d) had an ‘off flavor’ when ingested. These fruits were either without (Ctrl_CS) or with (MJ_CS) postharvest MeJA dipping treatment (0.3 mmol/L for 1 min).
As shown in Table 1, MeJA treatment significantly reduced the percentage of fruit exhibiting flesh mealiness, browning, and ‘off flavor’, while increasing the proportion of sound peaches. This data was reproducible over two seasons, supporting the role of MeJA in alleviating CI symptoms.Table 1. Differences in CI phenotypes between MeJA-treated and control ‘August Flame’ peach. Fruit were stored at 5 °C for three weeks in the 2022 and 2023 season. Different letters within a column indicate statistically significant differences within each season using Fisher’s least significant difference (LSD) at p < 0.05TreatmentsYearSound (%)Flesh Mealiness (%)Flesh Browning (%)‘Off Flavor’ (%)Ctrl_CS202227.7^b^69.3^b^65.6^b^70.3^b^MJ_CS80.2^a^14.1^a^3.4^a^15.9^a^Ctrl_CS202338.0^b^60.1^b^58.6^b^60.3^b^MJ_CS84.8^a^13.2^a^4.5^a^15.2^a^
Changes in lipidomic profiles during cold storage and post-storage ripening in peaches without MeJA treatment
To better understand the lipidomic changes associated with chilling, profiles were assessed in peaches without MeJA treatment at three stages: immediately after harvest (Ctrl_H), after cold storage (Ctrl_CS), and after ripening following cold storage (Ctrl_CS-R). Major classes of plant lipids include phospholipids, galactolipids, sphingolipids, and neutral lipids.
The percentages of triacylglycerols (TAG) and diacylglycerols (DAG), both neutral lipids, were higher in Ctrl_CS. In contrast, the levels of membrane lipids such as phosphatidylcholine (PC) and phosphatidylinositol (PI) were lower compared to both Ctrl_H and Ctrl_CS-R. Phosphatidylethanolamine (PE) was also significantly reduced in Ctrl_CS relative to Ctrl_CS-R. Similar patterns were observed in the total concentrations of each lipid class (Fig. 2, Table S2).Fig. 2. Differences in percentage distribution and concentrations of lipid classes across peach treatments. In a each stacked bar represents a treatment group: untreated peaches after harvest (Ctrl_H), after cold storage (Ctrl_CS), after ripening following cold storage (Ctrl_CS-R), and postharvest MeJA-treated peaches (MJ_CS). In b treatments are indicated by color, and lipid classes are grouped along the x-axis. A zoom-in of CER is shown in the upper left corner, and enlarged views of the other lipid classes are provided in Fig. S1. Error bars represent standard deviation. Asterisks indicate significant differences between Ctrl_CS and MJ_CS (p < 0.05) based on Tukey’s test for multiple comparisons within each lipid class. The full Tukey’s test results are presented in Table S2. CE: Cholesteryl ester; CER: Ceramide; DCER: Dihydroceramide; HCER: Hexosylceramide; LPC: Lysophosphatidylcholine; LPE: Lysophosphatidylethanolamine; PC: Phosphatidylcholine; PE: Phosphatidylethanolamine; PI: Phosphatidylinositol; MAG: Monoacylglycerol; DAG: Diacylglycerol; TAG: Triacylglycerol
Among membrane lipids, the double bond index (DBI) significantly increased after cold storage and continued to rise during ripening following cold storage (Fig. 3b). A similar increasing trend was observed for linolenic acid (18:3), whereas other fatty acid species generally remained stable or showed decreasing trends after cold storage (Fig. 4).Fig. 3. Assessment of fatty acid saturation in peach fruit. a Ratio of unsaturated to saturated fatty acids and b the double bond index (DBI) across untreated peaches immediately after harvest (Ctrl_H), after cold storage (Ctrl_CS), after ripening following cold storage (Ctrl_CS-R), and postharvest MeJA-treated peaches (MJ_CS). Boxplots show the median (center line), interquartile range (25th-75th percentiles; boxes), and whiskers represent the minimum and maximum values within 1.5 times the interquartile range. Numbers above the boxes were generated using Tukey’s method for multiple pairwise comparisons, with different numbers indicating statistically significant differences (p < 0.05)Fig. 4. Dynamics of fatty acid (FA) concentrations across peach fruit. Included are untreated peaches immediately after harvest (Ctrl_H), after cold storage (Ctrl_CS), after ripening following cold storage (Ctrl_CS-R), and postharvest MeJA-treated peaches (MJ_CS). Each panel represents a different FA. Numbers above the boxes were generated using Tukey’s method for multiple pairwise comparisons, with different numbers indicating statistically significant differences (p < 0.05). Panels without numbers indicate no significant differences were detected. Except for FA 22:0 and FA 26:1, which are low in abundance but show differences in at least one pairwise comparison, all significant differences were observed among the most abundant FAs. Boxplots show the median (center line), interquartile range (25th-75th percentiles; boxes), and whiskers represent the minimum and maximum values within 1.5 times the interquartile range
MeJA alters the lipid class composition and concentration in cold-stored fruit
A higher proportion of TAGs and DAGs together with lower percentages of PEs and PCs (all p < 0.001, Fig. 2a) were observed in non-MeJA-treated fruits (Ctrl_CS) compared to MeJA-treated fruits (MJ_CS). Interestingly, the distribution of lipid classes in MJ_CS was more similar to those in Ctrl_H and Ctrl_CS-R, suggesting that MeJA treatment partially reverses the cold-induced lipid profile shifts observed in Ctrl_CS.
A similar trend was observed when the concentration of each lipid class was considered (Fig. 2b). Ctrl_CS had higher concentrations of TAGs (difference = 61.9 nmol/g, p < 0.01), DAGs (difference = 31.2 nmol/g, p = 0.01), and CERs (difference = 0.04 nmol/g, p = 0.01) compared to MJ_CS. The concentration of PCs and PEs trended lower in Ctrl_CS by 37.6 and 35.2 nmol/g (p = 0.07 and p = 0.10, respectively). MeJA treatment therefore alters the composition and concentration of several neutral and membrane lipid classes after cold storage.
MJ_CS exhibits a higher DBI driven by 18:3-containing membrane lipids
Two indices were used to assess plant cold stress response, i.e., the unsaturated/saturated fatty acid ratio, and DBI (Fig. 3). The unsaturated/saturated ratio was similar between Ctrl_CS and MJ_CS; however, the DBI was higher in MJ_CS. Since the DBI weighs unsaturated fatty acids by their number of double bonds, an elevated DBI in MJ_CS indicates increased abundance of highly unsaturated fatty acids.
To test this assumption, we compared the concentrations of fatty acids in membrane lipids. The five most abundant fatty acids were palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), and linolenic acid (18:3) (Fig. 4), with 18:3 significantly higher (p < 0.05) in MJ_CS compared to Ctrl_CS. We then determined if increased 18:3 fatty acid was linked to specific lipid species. Eight species were more abundant in MJ_CS, all of which contained 18:3 (Fig. 5). These included four PCs: PC (16:0/18:3), PC (18:2/18:3), PC (18:0/18:3), and PC (18:1/18:3), as well as four PEs: PE(18:0/18:3), PE(16:0/18:3), PE(18:1/18:3), and PE(18:2/18:3). Therefore, higher membrane DBI in MJ_CS is driven by increased levels of 18:3-containing phospholipids.Fig. 5. Phospholipids that differ between fruit with (MJ_CS) and without (Ctrl_CS) MeJA treatment after cold storage. A total of 214 lipid species differed after adjusting for multiple comparisons using FDR (q-values), including four PCs and four PEs. Boxplots show the median (center line), interquartile range (25th-75th percentiles; boxes), and whiskers represent the minimum and maximum values within 1.5 times the interquartile range. Asterisks indicate lipid species with q-values < 0.05
Multivariate analysis distinguishes lipid profiles of MeJA-treated from untreated cold-stored fruit
Global lipidomic profiles of MJ_CS and Ctrl_CS fruit showed substantial differences using partial least squares discriminant analysis (PLS-DA) along the first two components. Except for Ctrl_CS-7 and Ctrl_CS-8, most Ctrl_CS samples were distinctly separated from MJ_CS on the first PLS component (Fig. 6a). Variable Importance in Projection (VIP) scores > 1 from the first PLS component, showed that, in addition to TAGs and DAGs, several PCs and PEs contributed substantially to the group separation (Table S3). These included PC(16:0/18:3), PC(18:0/18:3), PC(18:1/18:3), PC(18:2/18:3), PE(16:0/18:3), PE(18:0/18:3), PE(18:1/18:3), and PE(18:2/18:3). This pattern is consistent with univariate results above and further supports the role of 18:3-enriched membrane lipids in MeJA-mediated lipid remodeling after cold storage.Fig. 6. Multivariate analysis of a lipidome and b VOC profiles using PLS-DA. Peaches treated with MeJA (MJ_CS and MJ_CS-Hi) and untreated controls (Ctrl_CS) after cold storage are separated along the first two components. MJ_CS-Hi is a higher-concentration MeJA treatment compared to MJ_CS. Each dot represents a biological replicate for the corresponding treatment group
MeJA alleviates CI-induced defects in peach aroma compounds
VOCs such as lactones and esters, which contribute to peach flavor, are closely linked to lipid metabolism and are affected by CI. To assess if MeJA influences fruit aroma profiles, we analyzed fruit collected immediately after cold storage (Ctrl_CS and MJ_CS) and included a high-concentration MeJA treatment group (MJ_CS-Hi).
We identified 20 VOCs, comprising three lactones, six esters, five aldehydes, two alcohols, and four ketones (Table S4). Among these, 11 differed (p < 0.05) in at least one pairwise comparison across treatments (Fig. 7). The greatest variation was observed for ethyl acetate and methyl cinnamate between control and MeJA-treated peaches. Except for ethyl acetate, all VOCs were elevated in at least one MeJA-treated group. In particular, γ-decalactone and δ-decalactone were higher in MeJA-treated fruit, suggesting partial restoration of key aroma compounds suppressed by CI. Notably, (E)−2-hexenal and 3-hexenal, which are derived from 18:3 FA in peach, were also higher in MeJA-treated fruit, consistent with the increased 18:3 observed in the lipidomic analysis.Fig. 7VOC differences among 20 detected compounds in response to MeJA treatment after cold storage. Treatments included control (Ctrl_CS) and MeJA-treated peaches (MJ_CS and MJ_CS-Hi), with MJ_CS-Hi representing 3 mM MeJA treatment. Boxplots show the median (center line), interquartile range (25th-75th percentiles; boxes), and whiskers represent the minimum and maximum values within 1.5 times the interquartile range. VOC category abbreviations follow each compound name: ALC (alcohol), ALD (aldehyde), EST (ester), KET (ketone), and LAC (lactone). Asterisks indicate significant differences from Ctrl_CS using Dunnett’s test (p < 0.05) following ANOVA with FDR correction. Panels without asterisks indicate no significant differences after ANOVA with FDR correction
To assess global differences in VOC profiles, we performed PLS-DA. Ctrl_CS was separated from the MeJA-treated groups along the first PLS component, while MJ_CS and MJ_CS-Hi were further distinguished along the second component (Fig. 6b). Twelve VOCs had VIP > 1 for Component 1 and another twelve for Component 2, with substantial overlap (Table S5). Hexanal and δ-decalactone contributed more to Component 1 separation, whereas (Z)−3-hexenyl acetate and benzaldehyde were more influential along Component 2. These multivariate findings were consistent with the univariate results and further demonstrated the impact of MeJA on VOC profiles associated with CI.
Most lipid and VOC metabolism genes are not differentially expressed, except FADs
MeJA may affect the lipidome and VOC profiles through transcriptional regulation. To test this, we compared the expression of key genes involved in peach lipid metabolism and VOC biosynthesis between Ctrl_CS and MJ_CS using RNA-seq. To reduce cultivar-related variation and potential bias, we comprehensively examined all gene families encoding enzymes with the same predicted activity.
Because MeJA increased the levels of both PC and PE, we examined the expression of phosphocholine cytidylyltransferase (CCT) and ethanolamine-phosphate cytidylyltransferase (ECT), which catalyze the rate-limiting steps in the respective biosynthetic pathways [15]. However, no significant differences in expression were observed among CCT and ECT genes between control and MeJA-treated peaches (Fig. S2). The expression of glycerol-3-phosphate acyltransferase (GPAT), which catalyzes the rate-limiting step of TAG biosynthesis, was also examined [62]. Five GPATs are predicted in peach, of which three were detected in our samples. Among these, one was upregulated in MJ_CS compared to Ctrl_CS, while the others showed no significant change (Fig. S3). Similar patterns were observed for diacylglycerol acyltransferase (DGAT), an important enzyme that catalyzes the final step of TAG biosynthesis [63]. Of the eight expressed DGATs, four were upregulated in MJ_CS, while the others were not (Fig. S3). These findings are inconsistent with the observed decrease in TAG levels in MJ_CS.
For VOC regulation, we analyzed 13-LOXs, HPL1, ACXs, and AATs, which are involved in the biosynthesis of C6 volatiles and/or lactones. We also included CXEs, known to degrade esters in peach [64], and NAC1, a transcription factor implicated in broad ripening-related regulatory processes linked to CI. Similar to lipid-related genes, most of these VOC-associated genes did not show significant expression differences between Ctrl_CS and MJ_CS (Fig. S4-8). Interestingly, NAC1 expression was significantly higher in MJ_CS (difference = 512 CPM, p = 0.047) (Fig. S8), indicating that transcriptional regulation via this transcription factor began during cold storage.
FADs are involved in both lipid modification and VOC biosynthesis. In peach, ω−6 FADs (FAD2 and FAD6) catalyze the conversion of 18:1 to 18:2, while ω−3 FADs (FAD3-1, FAD3-2, FAD7, and FAD8) convert 18:2 to 18:3 [65]. Although expression levels of FAD genes varied between Ctrl_CS and MJ_CS, the two most highly expressed isoforms, FAD2 (ω−6) and FAD3-1 (ω−3), had higher expression in MJ_CS, with FAD2 showing a statistically significant difference (Fig. 8). This expression pattern is consistent with the elevated 18:3 content observed in MeJA-treated fruit. Taken together, except for NAC1 and FADs, the expression of most lipid- and VOC-related genes did not align with the observed trends in lipid and VOC levels.Fig. 8. Expression of FAD genes in control (Ctrl_CS) and MeJA-treated (MJ_CS) peaches after cold storage. FAD2 and FAD6 are ω−6 FADs, while FAD3-1, FAD3-2, FAD7, and FAD8 are ω−3 FADs. Boxplots show the median (center line), interquartile range (25th-75th percentiles; boxes), and whiskers represent the minimum and maximum values within 1.5 times the interquartile range. Asterisks indicate significant differences from Ctrl_CS using Student’s t-test (p < 0.05)
GO analysis of differentially expressed genes reveals enrichment of autophagy-related annotations
Despite the pronounced differences in lipidomic and VOC profiles between control and MeJA-treated peaches after cold storage, we did not observe corresponding changes in the expression of targeted lipid- and VOC-related genes. To assess broader transcriptional responses, we performed a global differential expression analysis. Among the 17,572 expressed genes, 628 were upregulated and 1,120 were downregulated in MJ_CS compared to Ctrl_CS.
Gene Ontology (GO) enrichment analysis revealed 13 significantly enriched terms in the Biological Process (BP) domain among the downregulated genes, including those related to peptide metabolism (GO:0043043, GO:0006518) and amide metabolism (GO:0043604, GO:0043603), which may be associated with protein turnover (Fig. 9b). In contrast, 11 BP terms were enriched among upregulated genes, five of which were related to autophagy (Fig. 9a). Given the prominence of autophagy-related GO terms, we examined standardized expression across all genes annotated with autophagy-related functions. As shown in Fig. 9c, MJ_CS exhibited significantly higher expression of autophagy-associated genes. These results suggest that autophagy plays an important role in the MeJA-mediated response following cold storage.Fig. 9. Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEG) in MeJA-treated peaches (MJ_CS) compared to the control (Ctrl_CS) after cold storage. DEG that are a upregulated and b downregulated are shown, with the circle size indicating the number of DEG associated with each GO term. Color represents p-values on a log scale, with pink indicating lower p-values and green indicating higher p-values. c Standardized expression of all genes annotated with autophagy-related terms in Ctrl_CS and MJ_CS. Boxplots show the median (center line), interquartile range (25th-75th percentiles; boxes), and whiskers represent the minimum and maximum values within 1.5 times the interquartile range. Asterisks indicate significant differences from Ctrl_CS using Student’s t-test (p < 0.05)
Discussion
A comprehensive integration of lipidomic, VOC, and transcriptomics analyses was performed to understand MeJA-mediated CI mitigation on a yellow-fleshed cultivar ‘August Flame’. Historically, MeJA has been reported to alleviate CI in a wide range of fruits and vegetables [66]. However, in peach, due to initial inconsistencies in findings, the understanding of the underlying mechanisms remains in its early stages. This arises because CI comprises multiple physiological syndromes, each potentially involving distinct pathways [4, 36], variation across cultivars [67], and further, individual studies often vary in their experimental design and research focus. In this work, we formulated several hypotheses to dissect the molecular mechanisms by which MeJA alleviates CI. In the following sections, we revisit these hypotheses presented in the Introduction section and integrate insights from our findings with previous research.
Does MeJA induce cold-responsive lipid modifications, including changes in lipid classes and double bond levels?
Lipidomic responses to cold storage and MeJA treatment in ‘August Flame’
Most studies used only a limited set of lipid metrics, whereas we used a wide range to assess lipid classes, degree of unsaturation, fatty acid composition, and individual lipid species, providing a deep analysis of underlying biological pathways. Our study is among the first to comprehensively profile the lipidome of yellow peach fruit stored at “killing zone” temperatures. In contrast, prior studies in peach primarily examined lipid dynamics during cold storage or CI-alleviation treatments at 0 °C, rather than within the “killing zone” [3, 36].
Increased double bonds in FAs is a well-established cold response in plants, including CI tolerance in peach [6–9]. Greater unsaturation enhances membrane fluidity, which maintains membrane integrity under cold stress [5]. We observed significantly higher DBI and total 18:3 levels in the membrane lipids in MJ_CS. Additionally, both univariate and multivariate analyses revealed differences in 18:3-containing PEs and PCs between Ctrl_CS and MJ_CS, providing a more detailed view of lipid remodeling. Finally, elevated expression of FAD2 and FAD3-1 may underline the increased 18:3 content and higher DBI in MeJA-treated fruit. Taken together, our multi-omic results underscore the importance of double bond content in MeJA-mediated CI alleviation.
Another well-established cold response in plants is the accumulation of TAGs [5]. TAG levels were highest in Ctrl_CS and lower in Ctrl_H and Ctrl_CS-R, consistent with this trend. Interestingly, TAG levels in MJ_CS were lower than in Ctrl_CS, and more similar to those in Ctrl_H and Ctrl_CS-R. A similar decrease in TAG content occurred with peaches stored under 0 °C to mitigate CI, suggesting a potential association between TAG accumulation and CI development [13].
The reduced TAG content observed in MJ_CS raises intriguing questions. TAG accumulation is thought to promote cold tolerance [5] by functioning as an energy store, by regulating redox reactions, or by removing toxic free fatty acids from membrane lipid metabolism [68]. However, excessive TAG accumulation can be harmful by disrupting lipid homeostasis [69], and in the leaves of some species, by causing lipotoxicity and cell death [70, 71]. Reduced TAG content in MJ_CS was associated with a better response to chilling stress compared to Ctrl_CS, suggesting that harmful pathways promoting lipotoxins and/or lipid homeostasis loss were suppressed; however, the underlying mechanism requires further investigation. Similarly, determining whether MeJA treatment reduces TAG levels, by accelerating TAG degradation, or inhibiting TAG accumulation during cold storage would require time-course experiments tracking TAG dynamics and related pathways in MeJA-treated and untreated peach fruit.
MeJA-induced lipid changes may involve autophagy
Despite substantial differences in the lipidome, there were few significant differences in the transcripts of enzymes involved in lipid metabolism. MeJA may regulate lipid remodeling through translational or epigenetic regulation rather than at the transcriptional level [35, 72]. Another potential regulatory mechanism is autophagy, as indicated by the upregulation of autophagy-related pathways and the generally higher expression of autophagy-associated genes in MJ_CS (Fig. 9).
Autophagy is a membrane-mediated process essential for maintaining intracellular homeostasis through nutrient recycling. In plants, it plays key roles in leaf senescence and responses to starvation and stress [73–75]. Fruit ripening is a senescent process, and autophagy is important to fleshy fruit ripening through metabolic and developmental regulation in tomato and strawberry [75–77], as well as to postharvest resistance against Botrytis cinerea in tomato [78, 79]. However, lipid degradation mediated by autophagy (lipophagy) remains less studied in plants, and to date, no studies have been conducted in fruit [80, 81]. Therefore, further research is needed to determine whether autophagy contributes to TAG regulation in MeJA-treated peach fruit.
Are flavor-associated VOCs maintained at higher levels in MeJA-treated peaches compared to controls?
Ethyl acetate and methyl cinnamate are potential early VOC biomarkers of CI following cold storage
Over 100 VOCs have been identified in studies of various peach cultivars [18]. However, similar to lipidomic studies, most investigations of VOCs focused on white-fleshed peaches. A few studies acknowledge the potential of early-stage VOC biomarkers for earlier CI detection [42], but most research investigated VOC changes during shelf life after cold storage. We observed that, even immediately after cold storage, there were substantial differences in VOC profiles between MeJA-treated and control peaches. Particularly, γ-decalactone and δ-decalactone levels differed between treatments, which may contribute to the off-flavor defect [82]. We did not detect any jasmonate-derived VOCs, even with lenient screening criteria (i.e., SNR threshold = 0 and minimum match factor = 50), suggesting that the floral-like aroma associated with jasmonates is unlikely to be a relevant concern under practical MeJA application conditions.
Although VOC concentrations differed significantly after cold storage, most compounds were low and may not be robust biomarkers, especially taking into account the inherent high variability among fruits. Based on fold-change magnitude, we propose that ethyl acetate and methyl cinnamate are more suitable early VOC markers for CI in our system, as they exhibited fold changes greater than two in at least one comparison. Both compounds differed significantly between control and MeJA-treated peaches (Fig. 7) and had VIP > 1 in our PLS-DA analysis (Table S5). In particular, ethyl acetate levels were more than tenfold higher in control fruit than in MeJA-treated fruit. Consistent with this, ethyl acetate and methyl cinnamate exhibited significant differences (p < 0.05) between healthy and CI peaches after cold storage within the control group (Table S6). Further testing using authentic reference standards is required for definitive GC–MS identification and absolute quantification. In addition, it will be important to evaluate the reliability and practical applicability of these candidate VOC biomarkers.
Direct comparisons between healthy and CI peaches are needed in future studies
To allow comparability with previous peach CI studies, we examined control and treatment groups, rather than between healthy and CI fruit, as done in prior studies [9, 12–14]. However, the inherent variation in the proportion of healthy and CI fruits within each treatment can introduce additional variability across biological replicates.
A direct comparison can reduce this variation and provide clearer insight into CI mechanisms. We conducted such a comparison by analyzing healthy and CI peaches in the group without MeJA treatment after cold storage (Table S6). However, due to the small sample size, only four VOC was identified with a p-value < 0.05 after FDR adjustment (2,4-hexadienal, hexanal, ethyl acetate, methyl cinnamate). Thus, larger and more systematic healthy-CI comparisons are required in future studies to characterize CI-associated metabolic changes.
Do differentially expressed VOCs correspond with changes in their lipid-derived precursors and do these regulatory effects involve changes at the transcriptional level?
Limitations in the current understanding of lipid-derived VOC biosynthesis in peaches
Several peach VOCs are predicted to be synthesized from lipid-derived precursors [16, 23]. Consistent with previous studies [83], we found that 18:3 levels were higher in MeJA-treated peaches, which may be linked to higher levels of C6 VOCs such as (E)−2-hexenal and 3-hexenal. However, because most precursors and genes involved in VOC biosynthesis in peach remain poorly understood and experimental evidence is limited, it is difficult to attribute the broad VOC improvements observed in MeJA-treated fruit to lipidomic changes alone.
This limited understanding of the peach VOC biosynthetic pathway may partly explain why we did not detect significant differences in the known VOC gene expression. For example, we did not observe differential expression of AAT1 or other AATs, which play important roles in ester and lactone biosynthesis [26–29]. It is possible that other gene families or regulatory steps specific to yellow-fleshed peaches play a more prominent role in regulating these VOCs. Another possibility is that key enzymes involved in VOC biosynthesis are not transcriptionally regulated or are expressed at low levels during cold storage, reducing the power to detect changes. Interestingly, the transcription factor NAC1 was differentially expressed, suggesting a potential explanation for the delayed but stronger VOC-related CI defects during the shelf-life stages. However, further studies are needed to clarify these mechanisms.
Conclusion
By integrating lipidomic, VOC profiling, and transcriptomic data, we gained insights into the molecular mechanisms underlying MeJA’s alleviation of CI in the yellow-flesh peach ‘August Flame’ after 5 °C storage. We identified shifts in the proportions of phospholipids and neutral lipids, as well as an increase in the degree of unsaturation in fatty acids in membrane lipids, primarily driven by the elevated 18:3 content in fruit dipped in MeJA postharvest. This increase in polyunsaturated fatty acids may not only contribute to membrane integrity but also alleviate VOC loss in MeJA-treated fruit, supporting the hypothesis that CI results from inadequate cold responses, which fail to protect the fruit from cold stress. Our VOC assay suggests that ethyl acetate and methyl cinnamate are potential early VOC biomarkers of CI following cold storage. Interestingly, the RNA-seq results indicate that transcriptional regulation plays a limited role in the expression of genes involved in lipid and VOC metabolism. However, autophagy-related genes were significantly enriched among the differentially expressed genes in MeJA-treated peaches, providing a future direction for investigating the effects of MeJA in CI alleviation and its role in fruit senescence and ripening.
Supplementary Information
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