Exploring ripening suppression in peach fruit during controlled atmosphere storage with transcriptome insights

Abstract

This study evaluated the effectiveness of controlled atmosphere (CA) storage in preserving the post-harvest quality of peaches (Prunus persica), focusing on delaying ripening and extending shelf life. Peaches harvested 110 days after bloom were stored under CA conditions with reduced oxygen and elevated carbon dioxide at low temperatures. CA storage significantly suppressed internal and external discoloration, maintained fruit firmness, and reduced ethylene production, contributing to prolonged freshness and marketability. Physiological assessments revealed that CA storage slowed the decline in firmness, minimized weight loss, and controlled respiration and ethylene production, particularly at 10 °C. Transcriptome analysis identified approximately 1971 differentially expressed genes associated with CA storage. Among these, ethylene biosynthesis and signaling genes such as ACC synthase 1, ACC synthase 6, and ACC oxidase 1 were significantly downregulated under CA conditions, leading to the suppression of ethylene production. This reduction in ethylene biosynthesis likely played a critical role in delaying the ripening process during storage. As a result of the suppressed ethylene signaling, the expression of key cell wall-degrading enzymes, including polygalacturonase and pectate lyase family, was also notably reduced. This downregulation contributed to the maintenance of fruit firmness by minimizing enzymatic degradation of the cell wall. CA storage also modulates the activity of reactive oxygen species-related enzymes, enhancing fruit resistance to oxidative stress. These findings highlight the targeted benefits of CA storage in extending the shelf life of peaches by delaying ripening, maintaining fruit firmness, and reducing spoilage. This approach offers a scientifically supported strategy to minimize post-harvest losses and enhance economic returns in the horticultural industry.

Introduction

Peaches (Prunus persica), climacteric fruits renowned for their sweet taste and nutritional value, are prone to rapid quality degradation post-harvest, especially when stored under suboptimal conditions¹. While making them highly desirable, the delicate texture and high sugar content of peaches also contribute to their rapid ripening and spoilage during storage and distribution². Furthermore, peaches are notably sensitive to low temperatures, with chilling injury, a critical issue in cold storage, being a significant concern. Chilling injury in peaches is characterized by symptoms such as internal browning, flesh wooliness, and loss of juiciness, which typically occur when the fruit is stored below its critical threshold temperature of approximately 5 °C^3,4. These symptoms not only reduce consumer appeal but also lead to significant economic losses due to spoilage. Therefore, the need for effective storage methods that extend the shelf life of peaches while minimizing the risk of chilling injury is urgent.

In response to these challenges, this study focused on controlled atmosphere (CA) storage as an innovative approach for enhancing the storability of peaches. CA storage involves carefully adjusting oxygen and carbon dioxide levels around the stored fruit, creating an environment that significantly slows down the ripening process and reduces the occurrence of chilling injuries^5,6. By altering the atmospheric composition, CA storage helps to preserve the sensory and nutritional qualities of peaches for a longer period than conventional storage methods^7,8. The potential advantages of CA storage include delayed ripening, reduced ethylene production, and inhibition of enzymatic activities responsible for cell wall degradation, all of which contribute to maintaining fruit firmness, color, and overall quality^9,10. This method addresses the critical need for more effective post-harvest handling techniques that can extend the shelf life and enhance the marketability of peaches.

The ripening process in peaches is closely associated with several physiological changes, including increased ethylene production and activation of cell wall-degrading enzymes such as polygalacturonase (PG), pectate lyase (PL), and expansin (EXP)^11,12. These changes lead to fruit softening, increased susceptibility to physical damage, and shortened shelf life. Conventional storage methods often exacerbate these issues, leading to premature ripening, increased disease susceptibility, and significant quality degradation¹³. However, CA storage has the potential to mitigate these effects by suppressing ethylene synthesis and slowing the enzymatic breakdown of cell wall components¹⁴, thereby preserving the structural integrity of the fruit and extending its marketable life.

This study sought to rigorously examine the effects of CA storage on post-harvest quality and physiological changes in peaches and compare these effects with those observed under conventional storage conditions. By systematically investigating the impact of different storage conditions (CA storage versus conventional methods) on the quality, internal coloration, and enzymatic activities of peaches stored at ambient and cold temperatures over a 7-day period, this study aimed to provide insights into more effective post-harvest handling techniques. An in-depth transcriptome analysis was also conducted to elucidate the molecular responses of peaches under various storage conditions, focusing on key genes involved in ethylene biosynthesis, signaling, and cell wall degradation.

These findings are anticipated to provide valuable insights into optimizing post-harvest handling processes, potentially transforming storage practices for climacteric fruits such as peaches. By demonstrating the practical benefits of CA storage, this study could influence global standards and practices in fruit storage, ensuring a longer shelf life, reducing waste, and maintaining nutritional quality, which are essential for food security and sustainability in agricultural production.

Results

Impact of CA and temperature on internal coloration of peach

During the storage period, a significant difference in internal coloration was observed (Fig. 1a). The processed image highlights areas of red discoloration or pigmentation, with black regions indicating the most affected areas. This visualization helps to clearly distinguish changes in internal coloration under different storage conditions. The comparison between reefer and CA storage indicated that internal discoloration was suppressed in peaches stored under CA conditions, as demonstrated by both the visual appearance and internal CIE a* values (Fig. 1b), where a* represents the green to red color range in the LAB color space, with positive values indicating redness and negative values indicating greenness. Specifically, peaches stored under CA conditions at 10 °C exhibited a slower increase in the internal a* values than those stored under reefer conditions. By day 7, the internal a* value for peaches stored at 10 °C under reefer conditions increased by approximately 70%, while those stored under CA conditions at the same temperature showed only a 30% increase, indicating a 40% difference in color retention.

Fig. 1.

Visual and color changes in peaches during storage under different conditions. (a) Visual changes at 10 °C under reefer, 10 °C under controlled atmosphere (CA), 20 °C under reefer, and 20 °C under CA conditions. Each row shows the RGB and LAB images at 0, 3, 5, and 7 days of storage. (b) Internal color (Hunter a*) changes over time for the same storage conditions. Symbols: 10 °C + reefer (filled black circle), 10 °C + CA (open circle), 20 °C + reefer (filled black square), 20 °C + CA (open square). Error bars represent standard deviations.

Furthermore, when comparing different storage temperatures, storage at 10 °C was evidently more effective in suppressing internal discoloration than storage at 20 °C. At 20 °C, peaches in reefer storage displayed a rapid increase in internal a* values, reaching a peak that was significantly higher than the corresponding values at 10 °C. The internal a* values for peaches stored at 20 °C under CA conditions also increased, although at a slower rate than those in reefer storage, indicating that CA storage was beneficial at both temperatures but more so at lower temperatures.

Statistical analysis revealed significant differences (p < 0.05) in internal a* values from day 3 onwards, particularly between reefer and CA storage conditions at both 10 °C and 20 °C. This suggests that CA storage not only delayed internal discoloration but also effectively maintained the visual and internal quality of the peaches over the 7-day storage period, with the effect being more pronounced at lower temperatures.

Physiological properties of fruits

Weight loss rate

The weight loss rate of the peaches increased over the storage period under all conditions. However, significant differences were observed between storage methods and temperatures. Peaches stored at 20 °C under reefer conditions exhibited the highest weight loss rate, reaching approximately 7% by day 7 (Fig. 2a). In contrast, peaches stored under CA conditions at 10 °C showed the lowest weight loss rate, remaining below 2% throughout the storage period. This indicates that CA storage, particularly at low temperatures, effectively reduces moisture loss and preserves fruit weight.

Fig. 2.

Changes in various peach quality parameters during storage under different conditions. (a) Weight loss rate (%), (b) firmness (N); (c) respiration rate (mg kg⁻¹ h⁻¹), and (d) ethylene production rate (µg kg⁻¹ h⁻¹) of peaches stored under different conditions: 10 °C under reefer (filled black circle), 10 °C under controlled atmosphere (CA, open circle), 20 °C under reefer (filled black square), and 20 °C under CA (open square) conditions. Measurements were obtained at harvest and after 0, 3, 5, and 7 days of storage. Error bars represent standard deviations.

Fruit firmness

Peach firmness decreased significantly during the storage period under all conditions, with a more pronounced decrease observed at higher temperatures. Peaches stored at 20 °C under reefer conditions showed the fastest decline in firmness, dropping to approximately 5 N by day 7 (Fig. 2b). In contrast, peaches stored at 10 °C under CA conditions maintained significantly higher firmness, exceeding 10N, compared to other treatments up to day 5, indicating that CA storage better retains firmness, especially at lower temperatures.

Respiration rate

Respiration rates showed distinct patterns under different storage conditions. After an initial decrease immediately post-harvest, the respiration rate increased over time under all conditions (Fig. 2c). Peaches stored at 20 °C under reefer conditions exhibited the highest respiration rates, peaking at approximately 45 mg kg⁻¹ h⁻¹ by day 7. In contrast, peaches stored under CA conditions, particularly at 10 °C, maintained consistently lower respiration rates throughout the storage period with values below 30 mg kg⁻¹ h⁻¹. This indicates that CA storage, especially at lower temperatures, effectively reduces the respiration rate, thereby slowing the metabolic processes associated with ripening.

Ethylene production

Ethylene production also varied significantly among the different storage conditions (Fig. 2d). Ethylene levels increased markedly by day 3, with the highest production observed in peaches stored at 20 °C under reefer conditions, reaching 4 µg kg⁻¹ h⁻¹. Peaches stored at 10 °C under CA conditions showed the lowest ethylene production, remaining below 2 µg kg⁻¹ h⁻¹throughout the storage period. These data suggest that CA storage significantly suppresses ethylene production, particularly at lower storage temperatures, which is critical for delaying ripening and extending the shelf life of peaches.

Effect of storage conditions on peel color parameters of peaches

The CIE L* values, which indicate lightness, fluctuated during storage under all conditions (Fig. 3a). Peaches stored at 20 °C under reefer conditions experienced the most significant decrease in lightness early in the storage period but showed partial recovery by day 7. In contrast, peaches stored under CA conditions at 10 °C maintained more stable L* values throughout the storage period, indicating better preservation of lightness under these conditions. Overall, CA storage, particularly at lower temperatures, effectively minimized fluctuations in lightness and preserved fruit visual quality.

Fig. 3.

Peach skin color measurements during storage under different conditions. (a) CIE L* (lightness), (b) CIE a* (red/green), (c) CIE b* (yellow/blue), and (d) chroma values. Storage conditions are indicated by symbols: 10 °C under reefer (filled black circle), 10 °C under controlled atmosphere (CA, open circle), 20 °C under reefer (filled black square), and 20 °C under CA (open square) conditions. Measurements were obtained at harvest and after 0, 3, 5, and 7 days of storage. Error bars represent standard deviations.

The CIE a* values, representing redness, although an increase was observed during storage compared to immediately after harvest, no statistically significant differences were detected between the treatment groups (Fig. 3b). Although the differences were not statistically significant, the a* values exhibited a tendency to be lower in the CA-treated groups compared to other treatments after 5 days of storage. This trend suggests that CA storage may have a suppressive effect on the development of red pigmentation over time, potentially delaying the ripening process.

The CIE b* values, which represent yellowness, varied across different storage conditions (Fig. 3c). No distinct trends were observed in the b* values based on CA treatment or storage duration. However, by day 7 of storage, peaches stored at 20 °C showed significantly higher b* values compared to other treatment groups, indicating a notable difference under these specific conditions.

Chroma values, which indicate color saturation or intensity, increased across all storage conditions, with the highest values observed in peaches stored at 20 °C under reefer conditions (Fig. 3d). CA storage, particularly at 10 °C, resulted in the lowest chroma values throughout the storage period, suggesting that these conditions most effectively retain the original color intensity of the fruit and prevent excessive color saturation, which typically accompanies over-ripening.

Differential gene expression between reefer and controlled atmosphere storage in peaches

Transcriptome analysis revealed distinct differences in gene expression between reefer and CA storage conditions in peach samples (Fig. 4), specifically in genes related to ethylene biosynthesis and signaling and the ethylene response factor (ERF) family.

Fig. 4.

Heat map of differentially expressed genes related to ethylene biosynthesis and signaling and ERF family transcription factors in peach samples after storage. The color scale represents the fragments per kilobase of transcript per million mapped read values, with blue indicating low expression, white indicating moderate expression, and red indicating high expression.

The expression levels of genes involved in ethylene biosynthesis and signaling pathways differed significantly depending on the storage conditions. Under reefer storage conditions, approximately 65% of the genes associated with ethylene biosynthesis exhibited increased expression. In contrast, CA storage led to a decrease in the expression of approximately 70% of these genes, indicating that CA storage effectively suppressed ethylene biosynthesis and signaling pathways. Notably, the downregulation of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and oxidase genes was particularly prominent, suggesting ethylene production inhibition under CA conditions.

Differences were also observed in the expression of ERF family genes between reefer and CA storage. While approximately 60% of ERF genes showed increased expression during reefer storage, approximately 55% of these genes were upregulated during CA storage. This suggests that in addition to suppressing ethylene signaling, CA storage may also activate specific ERF genes that are potentially related to stress responses or other regulatory mechanisms.

These results clearly demonstrate that CA storage, compared with reefer storage, more effectively suppresses the expression of ethylene-related genes while simultaneously promoting the expression of certain ERF genes.

Heat map analysis of specific cell wall-related genes in peaches during storage

Figure 5 shows a heat map of the expression patterns of key cell wall-related genes [PG, PL, EXP, β-galactosidase (BGAL), pectin methylesterase inhibitor (PMEi), fucosyltransferase (FUT), and cellulase (CEL)] in peaches stored under reefer and CA conditions (Fig. 5). These genes are among the 45 differentially expressed genes (DEGs) identified in the analysis that play critical roles in cell wall modification, affecting fruit texture and firmness during storage.

Fig. 5.

Heat map of differentially expressed genes related to cell wall modification in peach samples after storage. The color scale represents the fragments per kilobase of transcript per million mapped read values, with blue indicating low expression, white indicating moderate expression, and red indicating high expression.

Both PG and PL are involved in the degradation of pectin, a major component of the plant cell wall. Among the DEGs, several PG and PL genes exhibited significant changes in expression. During reefer storage, these genes were notably upregulated, indicating active pectin degradation and the associated fruit softening. Conversely, CA storage resulted in marked downregulation, with over 70% of these PG and PL DEGs showing decreased expression, suggesting that CA storage reduces pectin breakdown and helps maintain fruit firmness.

EXP genes, which are crucial for cell-wall loosening, were significantly upregulated during reefer storage, with several DEGs in this category showing increased expression. This upregulation suggests active cell wall loosening. However, CA storage led to the downregulation of EXP gene expression, with approximately 65% of the EXP DEGs exhibiting decreased expression, indicating that CA storage contributes to preserving fruit texture by limiting cell wall disassembly.

BGAL, which is involved in the hydrolysis of galactosyl residues from pectin, was also differentially expressed. Under reefer storage conditions, BGAL DEGs were generally upregulated, promoting cell wall degradation. In contrast, CA storage resulted in significant downregulation, with approximately 60% of BGAL DEGs showing reduced expression, suggesting that CA storage inhibited cell wall degradation by limiting BGAL activity.

Several DEGs indicated that PMEi, which regulates pectin methylesterase activity, was upregulated in reefer storage. This upregulation may lead to more rapid pectin degradation. In contrast, CA storage downregulated PMEi expression, with most PMEi DEGs showing decreased levels, indicating that CA stabilizes the cell wall by reducing pectin demethylation and its subsequent breakdown.

FUT, which is involved in fucosylated xyloglucan biosynthesis, was moderately upregulated during reefer storage, and several FUT DEGs were affected. However, CA storage led to the downregulation of FUT expression, with approximately 55% of the DEGs showing reduced expression, suggesting that CA storage may limit cell wall remodeling activities.

CEL, which is directly involved in cellulose degradation, was upregulated during reefer storage, reflecting increased cell wall breakdown. In contrast, CA storage led to significant downregulation, with approximately 70% of CEL DEGs showing decreased expression, indicating that CA storage inhibits cellulose breakdown, thereby helping maintain the structural integrity of the cell wall.

Analysis of reactive oxygen species-related enzyme activities in peaches during storage

Catalase (CAT) activity varied significantly between reefer and CA storage conditions. At harvest, CAT activity was approximately 1.0 U/mg DW. After 0 d of storage, CAT activity decreased slightly under both conditions; however, by day 5, a clear divergence was observed. In reefer storage, CAT activity increased sharply to approximately 2.5 U/mg DW, whereas it remained relatively low in CA storage, showing only a slight increase to approximately 1.0 U/mg DW (Fig. 6a). This indicated that CA storage may suppress the oxidative stress response, which is typically associated with CAT activity.

Fig. 6.

Reactive oxygen species-related enzyme activities in peach samples during storage under different conditions. (a) Catalase (CAT), (b) superoxide dismutase (SOD), and (c) peroxidase (POD). Storage conditions are indicated by symbols: reefer (filled black circle) and controlled atmosphere (CA, open circle). Enzyme activities were measured at harvest and after 0 and 5 days of storage. Error bars represent standard deviations.

Superoxide dismutase (SOD) activity followed a trend similar to that of CAT activity across the storage conditions. Initially, SOD activity was approximately 1.0 U/mg DW at harvest. By day 0, both reefer and CA storage resulted in a sharp increase in SOD activity to approximately 2.0 U/mg DW. However, after 5 days, SOD activity decreased under reefer storage but remained stable under CA conditions at approximately 2.0 U/mg DW (Fig. 6b). This stability during CA storage suggests consistent defense against superoxide radicals, potentially contributing to reduced oxidative stress.

Peroxidase (POD) activity demonstrated a pattern different from that of CAT and SOD activities. At harvest, POD activity was high at approximately 12.0 U/100 mg DW. After 0 d of storage, POD activity decreased sharply under both storage conditions. By day 5, POD activity further decreased in both conditions, stabilizing at approximately 4.0–5.0 U/100 mg DW. Notably, CA storage resulted in slightly higher POD activity compared with that in reefer storage at day 5, although the difference was minimal (Fig. 6c). This suggests that while POD activity diminishes during storage, CA conditions might help maintain slightly higher levels of this enzyme.

Discussion

The findings of this study underscore the significant impact of CA storage and low-temperature conditions on the post-harvest quality of peaches, particularly in mitigating internal discoloration and delaying ripening. As shown in Fig. 1, the suppression of internal discoloration is particularly notable in white-fleshed peach varieties, where the ripening process was evidently inhibited¹⁵. This inhibition is critical for extending the shelf life of peaches because internal discoloration is typically associated with advanced ripening stages and, if uncontrolled, can lead to significant quality degradation and market losses.

Figure 2 further corroborates the effectiveness of CA storage at low temperatures in maintaining the physiological integrity of peaches. Measurements of firmness and respiration rate, two key indicators of ripening^16,17, revealed that peaches stored under CA conditions exhibited a slower decline in firmness and a more controlled respiration rate than those stored under conventional conditions. The suppression of these ripening-related physiological changes is consistent with the findings of existing literature, highlighting the role of CA storage in extending the post-harvest shelf life of climacteric fruits, such as peaches, by delaying the climacteric peak and thus slowing down metabolic processes associated with over-ripening and spoilage¹.

Externally, peach skin color changes during storage also provide insights into the efficacy of CA storage¹⁴. Although the L* (lightness) and a* (redness) values did not show significant differences, likely due to the inherent variability in peach skin coloration, the b* (yellowness) values and chroma (color intensity) were markedly suppressed during CA storage. This suppression is particularly important because it indicates a delay in the development of over-ripening symptoms such as skin yellowing, which is often less desirable in white-fleshed peach varieties¹⁸. The ability of CA to maintain stable color parameters aligns with its role in slowing the ripening process, thereby preserving the visual appeal and marketability of the fruit.

To delve deeper into the molecular mechanisms underlying these observed phenomena, a comprehensive transcriptome analysis was conducted, identifying approximately 7401 DEGs. Among these genes, 1971 DEGs were specifically associated with CA storage after excluding the influence of storage duration and temperature. Peaches are climacteric fruits and highly sensitive to ethylene. During the postharvest ripening process, the endogenous production of ethylene increases, which accelerates fruit ripening. In this experiment, the suppression of ethylene production by CA treatment is presumed to have inhibited the ripening of peaches. Transcriptome analysis suggests that the suppression of ethylene production is associated with the downregulation of ACS1, ACS6, and ACO1 expression under CA treatment. ACS and ACO are genes involved in ethylene biosynthesis, specifically in converting ACC, the precursor of ethylene, into ethylene^19,20. This downregulation provides a clear molecular basis for the observed suppression of ripening, as ethylene is the central hormone involved in fruit ripening. These findings align with those of previous studies demonstrating the inhibitory effects of CA storage on ethylene action, thereby contributing to delayed ripening and extended fruit shelf life^9,21.

Furthermore, the present study analyzed the expression of ERF family genes, which are known to play critical roles in mediating plant stress responses^22,23. The results showed that a substantial number of ERF genes were upregulated under CA storage conditions. This observation suggests that the modified atmosphere created by CA storage, characterized by low oxygen and high carbon dioxide levels, may induce a stress response in peaches, leading to the activation of ERF genes^24,25. Such activation could be a defense mechanism to prepare the fruit to better cope with environmental stressors, including oxidative stress caused by reactive oxygen species (ROS)²⁶. The upregulation of ERF genes under CA storage conditions mirrors findings from previous studies, where ERF genes were shown to be involved in the rapid activation of stress response pathways, enabling fruit to mitigate damage from external stresses, including extreme temperatures and oxidative stress²⁷.

The investigation of cell wall-related genes provided further insights into how CA storage affects fruit texture, particularly in maintaining firmness. The expression patterns of key genes such as PG, PL, EXP, BGAL, PMEi, FUT, and CEL were analyzed. Under conventional reefer storage, many of these genes were upregulated, correlating with increased cell wall degradation and consequent fruit softening, which is a natural process during ripening. However, during CA storage, the expression of these genes, particularly PG, PL, and EXP, was significantly downregulated. This downregulation is likely a key factor in maintaining higher fruit firmness, as it suggests a reduction in the enzymatic activity responsible for cell wall loosening and degradation¹². The maintenance of cell wall integrity under CA storage conditions supports the observed reduction in softening, contributing to extended firmness and, by extension, extended peach shelf life²⁸.

The study by Sanhueza et al. demonstrated that CA storage regulates the expression of ethylene-related and cell wall-related genes, playing a critical role in preserving fruit quality²⁹. In particular, Sanhueza et al. reported that the expression of ethylene biosynthesis genes such as ACC synthase and ACC oxidase was downregulated under CA conditions. This aligns with our findings, where ACS1, ACS6, and ACO1 were significantly suppressed under CA storage. This consistency strongly supports the notion that the suppression of ethylene biosynthesis under CA conditions is a key mechanism in delaying ripening and maintaining the postharvest quality of peaches. Additionally, Sanhueza et al. highlighted the downregulation of cell wall-degrading enzymes such as PG and PL under CA storage, which was also observed in our study. This reduction in gene expression contributes to the maintenance of fruit firmness by preventing cell wall degradation, further emphasizing the role of CA storage in minimizing over-ripening and tissue softening. These similar findings enhance the credibility of our study and reinforce the understanding that CA storage is effective in maintaining postharvest quality in peaches. Furthermore, our study extends the knowledge by connecting these molecular changes to observed physiological characteristics, such as firmness retention and suppressed ethylene production, providing practical insights into the application of CA technology.

Finally, the current study examined the activity of ROS metabolism-related enzymes, including CAT, SOD, and POD. The upregulation of ERF genes under CA storage, combined with a low-oxygen environment, suggests the potential modulation of ROS-related genes. These results confirmed the changes in the activity of these key enzymes, confirming the hypothesis that CA storage induces a stress response that enhances the ability of fruits to manage oxidative stress³⁰. The observed modulation of ROS-related enzyme activities is consistent with previous studies that reported the ability of CA storage to influence the oxidative stress response, thereby helping mitigate damage caused by ROS during storage³¹.

Conclusions

This study demonstrates that CA storage, particularly at low temperatures, effectively preserves the post-harvest quality of peaches by suppressing discoloration, maintaining firmness, and reducing ethylene production, thereby extending the shelf life of peaches. Molecular analysis revealed that CA storage downregulated ethylene-related and cell wall-degrading genes while upregulating ERF genes, suggesting enhanced stress resistance in peaches. However, further research is required to confirm these findings across different peach cultivars over longer storage durations. In addition, assessing the effects of CA storage on flavor and nutritional quality is important for comprehensive quality preservation. CA storage offers significant practical benefits to the horticultural industry by delaying fruit ripening and reducing spoilage, leading to improved economic outcomes and fruit quality. Integrating CA storage with emerging technologies, such as real-time monitoring, could further optimize post-harvest practices. In summary, CA storage is a valuable strategy for sustainable peach preservation with potential applications in broader horticultural contexts. Continued research and innovation are crucial to meet the global demand for fresh, high-quality produce while minimizing post-harvest losses.

Material and methods

Plant materials

Peach fruits (P. persica Batsch, ‘Kawanakajima Hakuto’), harvested 110 days after bloom, corresponding to the optimal maturity stage for the variety used, were obtained from a commercial orchard located at an altitude of approximately 100 m in Jochiwon, South Korea, in August 2023. A total of 310 fruits were used, ensuring sufficient biological replicates for both molecular and physiological analyses. Fruits were carefully selected to ensure uniformity in size and maturity for consistent experimental conditions.

CA storage condition

To evaluate the effects of CA storage on harvested peaches, the fruits were stored for 7 days at 8 °C under a relative humidity of over 90% in both CA and reefer containers. The gas composition within the CA container was set to 5% oxygen and 5% carbon dioxide, whereas that of the control reefer container was maintained at 21% oxygen and 0.04% carbon dioxide. After storage in the CA and reefer containers, the peaches were further stored for an additional 7 days at 10 and 20 °C for quality analysis.

Physicochemical property measurements

Ten fruits were randomly selected from the stored samples to measure the rate of mass loss, fruit firmness, and color change. Fruit firmness, expressed in newtons, was measured using a texture analyzer (Lloyd Instrument BG/TA Plus; Ametek, Inc. Fareham, UK) with an 8-mm flat probe. Each fruit was compressed to a depth of 4 mm into the flat side of the equator at a rate of 1 mm/s. Hunter values, including L* (lightness), a* (redness), and b* (yellowness), were measured using a chroma meter (CR-300; Minolta Co., Tokyo, Japan). In addition, chroma values were measured to assess the saturation levels.

Respiration and ethylene measurements

Each peach was placed in a 1-L sealed container and left for 1 h. Subsequently, 1 mL of the accumulated headspace gas was collected using a gas-tight syringe and analyzed using a gas chromatograph (GC-7890B; Agilent Technologies, Wilmington, DE, USA). The analysis was conducted using a 30-m long, 0.32-mm inner diameter column (HP-5; Agilent Technologies) at a temperature of 80 °C with helium as the carrier gas at a flow rate of 5 mL/min. A thermal conductivity detector was used to measure the respiration rate, which was expressed in mg CO₂/kg/h. Ethylene production was measured similarly, with gas collected and analyzed using a gas chromatograph (Bruker 450-GC; Bruker Corp., Billerica, MA, USA) equipped with a flame ionization detector set at 250 °C and a column temperature of 70 °C. Ethylene production was expressed as μL C₂H₄/kg/h.

Total RNA isolation

Peach samples were ground in liquid nitrogen, and total RNA was extracted using a Quick-RNA Miniprep Kit (Zymo Research, Irvine, CA, USA). Further purification was conducted using an RNA Clean & Concentrator™-5 Kit (Zymo Research). RNA quality and quantity were determined using a DeNovix DS-11 + Spectrophotometer (DeNovix, Inc., Wilmington, DE, USA), followed by 1.2% agarose gel electrophoresis. RNA integrity was evaluated using an Agilent Bioanalyzer RNA Pico 6000 chip (Agilent Technologies).

Transcriptome analysis by RNA-Seq

Total RNA was extracted from peach samples using the Quick-RNA Mini Prep Kit (Zymo Research, Irvine, CA, USA) and further purified with the RNA Clean & Concentrator™-5 Kit (Zymo Research) to remove residual contaminants. RNA quantification was performed using a DeNovix DS-11 + Spectrophotometer (DeNovix, Wilmington, DE, USA), and RNA quality was evaluated via 1.2% agarose gel electrophoresis. The RNA integrity number (RIN) for each sample was determined using an Agilent Bioanalyzer RNA Pico 6000 chip (Agilent Technologies), and all samples met the RIN threshold of ≥ 7.0 for library preparation.

Transcriptome libraries were generated from 2.0 μg of RNA per sample using the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. Sequencing was conducted on an Illumina NovaSeq 6000 platform with 150 bp paired-end reads, generating approximately 5 Gb of data per sample.

Raw reads were assessed for quality using FastQC (v0.11.9), and low-quality reads and adapters were trimmed using Trimmomatic (v0.33). Potential contaminants, including rRNA, bacterial, and viral sequences, were filtered out using BBDuk (v38.87) from the BBTools suite. Over 90% of the reads were retained after preprocessing, with most samples achieving Q30 scores above 93%.

Preprocessed reads were mapped to the Prunus persica v2.1 genome, obtained from the Phytozome 13 database, using HISAT2 (v2.2.1). Mapping efficiency exceeded 97% across all samples. Gene expression levels were quantified using HTSeq-count (v0.13.5) in union mode, with strand specificity set according to the library preparation method. Expression data were normalized using the FPKM method to adjust for transcript length and sequencing depth.

Functional annotation was performed using DIAMOND (v2.0.9) for similarity searches against the NCBI non-redundant (nr) protein database with an e-value cutoff of 1e−5. Protein domains were predicted using InterProScan (v5.22–61.0), which integrates databases such as Pfam, SMART, and PROSITE. Gene Ontology (GO) terms were assigned across three categories—biological process, molecular function, and cellular component—using Blast2GO (v5.2.4). KEGG pathway annotations were determined using the KAAS web tools to identify relevant metabolic pathways. Enrichment analyses provided insights into the biological roles of the identified genes. Detailed RNA quality metrics, mapping statistics, and functional annotation results are available in Supplementary Data 1.

Identification of DEGs

Differential gene expression analysis was conducted using DESeq2 (v1.32.0) in the R statistical environment (v4.1.0). Normalized count data were used for pairwise comparisons between sample groups: ‘At harvest’ vs ‘Reefer’, ‘At harvest’ vs ‘CA’, and ‘Reefer’ vs ‘CA’. The statistical criteria for identifying DEGs included an adjusted p-value (padj) < 0.05, corrected using the Benjamini–Hochberg procedure³², and a log2 fold change (|log2FC|) threshold ≥ 1, which corresponds to a twofold or greater difference in gene expression between conditions.

A total of 7401 DEGs were identified across the three comparisons, with 2224 upregulated and 2870 downregulated genes in ‘At harvest’ vs ‘Reefer’, 2321 upregulated and 2544 downregulated genes in ‘At harvest’ vs ‘CA’, and 1508 upregulated and 1,355 downregulated genes in ‘Reefer’ vs ‘CA’. Mapping of RNA-seq reads to the reference genome demonstrated a consistent expression profile across samples, with at least 67% of the transcripts expressed in all conditions.

Antioxidant enzyme activity assay

SOD, CAT, and POD were measured using standard spectrophotometric methods. SOD activity was assessed by homogenizing the samples, centrifuging them, and using the supernatant to measure the inhibition of the nitroblue tetrazolium reaction over 15 min at 560 nm. For CAT and POD activities, hydrogen peroxide decomposition and guaiacol oxidation, respectively, were monitored over a 3-min period, with the absorbance measured at 240 nm for CAT and 470 nm for POD. These measurements quantified the antioxidant enzyme activities within the samples.

Statistical analysis

The experiments were conducted using a randomized design with 10 or 3 replicates, and data are presented as means ± standard deviation. Statistical comparisons of the mean values of the experimental groups were performed using SPSS v26.0 (SPSS Inc., Chicago, IL, USA). One-way analysis of variance, followed by Duncan’s multiple-range test, was used to determine statistically significant differences. Additionally, the Least Significant Difference (LSD) was calculated at the at p < 0.05 level to determine the minimum value needed for a statistically significant difference between treatment means.

Author contributions

JGL: writing—original draft, methodology, validation, visualization. J-HL: data curation, methodology, writing—original draft. M-SC, HY: data curation, investigation, resources. D-RB: data curation, methodology. HLE: conceptualization, funding acquisition, methodology, project administration, resources, supervision, validation, writing—review and editing.

Data availability

The datasets generated and/or analyzed during the current study are not publicly available due to planned future research but are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-97177-y.