Tissue-Specific Recovery Capability of Aroma Biosynthesis in ‘Golden Delicious’ Apple Fruit after Low Oxygen Storage

Abstract

The impact of low-oxygen (2 kPa) controlled atmosphere storage on the recovery of aroma biosynthesis in ‘Golden Delicious’ (GD) apple tissues during their subsequent shelf life was investigated. The results showed that the highest ester content was found in skin tissue, followed by Flesh 2 and Flesh 1. The 2 kPa O₂ storage of GD apples resulted in a decrease in the emission of volatile esters and alcohols, but an increase in aldehyde emission. Notably, compared with skin tissue, the flesh tissue of 2 kPa O₂-stored GD apples had a relatively high recovery capacity of ester biosynthesis but a low recovery capacity of alcohol synthesis for its shelf life. The impact was associated with increased levels in the MdLOX1a and MdAATs (MdAAT1 and MdAAT2) transcripts, as well as a decreased level in the MdHPL transcript in the flesh tissue. In addition, a complex regulatory network of ethylene on fruit aroma biosynthesis in response to low-oxygen conditions was also indicated. Collectively, there was a tissue-specific recovery capability of aroma biosynthesis in GD apples after low-oxygen storage.

1. Introduction

The apple has a complex aroma composed of a wide variety of volatile compounds including alcohols, aldehydes, and esters, which are the basic components involved in fruit flavor formation [1]. Over 300 types of volatile components were identified in apple fruit; however, only 20–30 volatiles are directly related to the formation of the characteristic apple aroma [2,3]. Esters, including butyl acetate, butyl butyrate, butyl caproate, hexyl butyrate, hexyl propanoate, and hexyl 2-methylbutyrate, account for more than 50% of total aromatic compounds in most apple cultivars [3,4]. Notably, hexyl butyrate was also reported as a major contributor to apple sweetness, which is known as an odor-induced enhancement of taste perception [5]. The dual role of aromatic components emphasizes their great significance to the overall sensory quality of apple fruit. However, the production of aromatic volatiles in apple fruit was largely suppressed during cold or controlled atmosphere (CA) storage, especially for long-term periods [4,6,7]. Most importantly, the negative impact of commercial storage conditions on aroma emission in fruit persists throughout the retail chain, resulting in poor consumer acceptability [8].

Oxygen partial pressure is considered an important factor for the CA storage of most apple cultivars, being applied as follows: 2.0–5.0 kPa for conventional CA, 1.5–2.0 kPa for low oxygen, and 0.8–1.2 kPa for ultra-low oxygen [9]. Generally, compared with single cold storage, the application of low-oxygen partial pressure could result in better fruit quality, especially in skin color and flesh firmness, thereby preventing physiological alterations [4]. Notably, the storage of apples under low oxygen may effectively inhibit the production of straight-chain esters due to the reduced availability of corresponding acid and alcohol precursors [1,2,6]. Although the GD apple has a strong tolerance in terms of its physiological response to low-oxygen partial pressure, its emission of straight-chain esters can still be suppressed under hypoxic conditions during long-term storage in a CA [10,11,12]. Brackmann et al. hypothesized that the suppression of aroma production in GD apples might be related to retarded synthesis or degradation of fatty acids [10]. However, the underlying response mechanism of aroma production to low oxygen in GD apples is still unclear.

Additionally, the existence of the oxygen gradient in plant tissue is a common phenomenon: the area with low-oxygen perfusion expands when surrounding oxygen availability decreases [13]. In apple fruit, the local oxygen gradient in the tissue was found to be higher than the surrounding environment [14]. The hypoxia caused by the oxygen gradient may cause negative metabolic adaptation in apple tissues [15]. Therefore, the oxygen gradient might, to some extent, be an influencing factor in the spatiotemporal pattern of aroma biosynthesis in fruit. However, limited data about the spatial response of aroma biosynthesis to low oxygen in GD apple tissues are available so far.

Since the early 20th century, the Golden Delicious apple was cultivated around the world, and its high yield and sweet flavor make it a favorite amongst apple growers and customers. The study aimed to investigate the spatial effect of low oxygen on the emission of volatile compounds in GD apples under CA storage and for their subsequent shelf life, and the mechanism underlying the impact of low-oxygen storage on the pathways involved in aroma biosynthesis of GD apples. This work provides insights into the tissue-specific recovery capability of aroma biosynthesis in GD apples after storage under low-oxygen conditions.

2. Materials and Methods

2.1. Apple Fruit Handling and Treatment

Apples (Malus × domestica Borkh. cv. Golden Delicious) were harvested from a commercial orchard in Jinzhou, China. At harvest, fruit firmness was 100.0 ± 4.2 N and soluble solid content was 13.5 ± 0.57%. Four hundred harvested apples of uniform color and size, and free of disease and insect infestation, were selected and transported to a Bohai University laboratory within two hours. Among the selected apples, two hundred apples were placed in plastic trays and transferred to a controlled atmosphere chamber (YS-XCAB/G4, YISHIKEJI Co., Ltd., Hangzhou, China) under 2 kPa O₂ and 98 kPa N₂ at 4 °C and 90–95% relative humidity (2 kPa O₂ group); the remaining two hundred apples were air-stored at the same temperature and humidity as the control group. The apples were stored for 56 days, and then transferred to retail-mimicking conditions at 20 °C for 44 days. Samplings were performed on days 14, 28, 42, and 56 during the storage period, and on days 56 + 9, 56 + 23, and 56 + 44 of the shelf-life period. Apple-tissue samples were collected from fifteen fruit, immediately frozen in liquid nitrogen, and then stored at −80 °C until further analysis. As shown in Figure S1, 0.1 cm thickness of fruit skin was peeled with a sterile peeler; and flesh tissues located at 0.1–1.0 cm (Flesh1) and 2.0–3.0 cm (Flesh2) below the exocarp were sampled with a sterile blade. The entire experiment was repeated three times.

2.2. Determination of Firmness and Soluble Solid Content

Flesh firmness was measured on the opposite side of the peeled equatorial region of fruit using a handheld fruit firmness tester (GY-4 digital fruit penetrometer; HANDPI Instrument Co., Ltd., Yueqing, China) equipped with an 11-mm diameter probe. The measurement was performed in five replicates, and the results were expressed in Newtons (N) [16].

For the determination of soluble solid content (SSC), 15 g of flesh tissue from five fruit were homogenized and then centrifuged at 10,000× g for 10 min using a 5417R Eppendorf centrifuge (Merck KGaA, Darmstadt, Germany). The obtained supernatants were used to measure SSC with a 3810 PAL-1 digital handheld pocket refractometry (Atago Co., Ltd., Tokyo, Japan). SSC was measured in triplicate for each sample and expressed in percentage [16,17].

2.3. Determination of Ethylene Production and Respiration Rates

To determine ethylene production, approximately 1.5 kg of fruit was placed into a 5-L sealed hermetical container at 20 °C for 1 h. One milliliter of the headspace air was collected and injected into a gas chromatograph (GC-2014C, Shimadzu Corporation, Kyoto, Japan) equipped with a flame ionization detector (FID-2014, Shimadzu Corporation, Kyoto, Japan). Simultaneously, the level of CO₂ was determined with a GC equipped with a thermal conductivity detector (TCD-2014, Shimadzu Corporation, Kyoto, Japan). Ethylene production was expressed in kg⁻¹ s⁻¹ and the respiration rate was in CO₂ kg⁻¹ s⁻¹ [16,17].

2.4. Volatile Compound Analysis

Volatile compounds in apple fruit were analyzed according to the method proposed by Both et al. with some modifications [18]. Specifically, fruit samples were ground into a fine powder in liquid nitrogen with a mortar and pestle. An aliquot of 5.0 g powder for each sample was mixed with 5 mL of saturated sodium chloride solution in a 20-mL vial sealed with a polytetrafluoroethylene-coated silicone lid. Cyclohexanone was used as an internal standard. The mixture was equilibrated for 5 min, and the volatile compounds in the headspace were then extracted at 60 °C for 1 h by solid-phase microextraction (SPME) using a fiber assembly 50/30 μm divinylbenzene/carbon/polydimethylsiloxane (DVB/CAR/PDMS) (Supelco^®, Bellefonte, PA, USA), preconditioned following the manufacturer’s protocol.

Volatile compounds were quantified in a gas chromatography-mass spectrometer (GC-MS) with a flame ionization detector (Agilent 7890N/5975C GC-MSD, Agilent Technologies, Inc., Santa Clara, CA, USA). The fiber was thermally desorbed into the injection port at 250 °C for 5 min. Ultra-pure helium (99.99%) was used as carrier gas at constant pressure (15 psi) and flow rate (0.02 mL s⁻¹). Heating conditions were as follows: the initial temperature was set at 40 °C for 5 min, then increased to 120 °C at a rate of 0.05 °C s⁻¹ and held for 2 min, followed by an increase at a rate of 0.15 °C s⁻¹ to reach 230 °C and held for 6 min. MS was conducted in the electron-impact ionization mode with ionization energy of +70 eV and a scanning mass range of m/z from 30 to 550 anu. The temperature of the quadrupole and MS ion source were held at 150 °C and 230 °C, respectively. Analytes were identified using the database of the National Institute of Standards and Technology (U.S. Department of Commerce, Gaithersburg, MD, USA). The emission of volatile compounds was expressed in mg kg⁻¹ fresh weight.

2.5. Gene Expression Analysis

The total RNA of the apple fruit tissue was extracted by the cetyltrimethyl ammonium bromide (CTAB) method [19]. DNase was used to remove genomic DNA. The first-strand cDNA from the total RNA was reverse-transcribed using a FastKing RT Kit (Beijing Tiangen Biochemical Technology Co., Ltd., Beijing, China). Real-time quantitative PCR (RT-qPCR) was performed in a StepOne™ Plus Real-Time PCR System (Applied Biosystems Inc., Carlsbad, CA, USA) using a SuperReal PreMix Plus kit (Beijing Tiangen Biochemical Technology Co., Ltd., China). Primers for RT-qPCR in Supplementary Table S1 were designed using Primer Premier 5.0 software (PREMIER Biosoft International, San Francisco, CA, USA). The gene-encoding fatty acid metabolism enzymes, MdLOX1a, MdHPL, MdADHs (MdADH1 and MdADH2), MdAATs (MdAAT1 and MdAAT2) were selected. Additionally, the genes related to ethylene biosynthesis (MdACS and MdACO1) and signaling (MdERFa-f) were also analyzed. MdGAPDH (Locus ID: MDP0000757565) was used as the housekeeping gene. Relative mRNA expression was calculated using the 2^−ΔΔCT method [20]. RT-qPCR amplifications were performed in triplicate for each sample.

2.6. Statistical Analysis

Data are presented as mean ± standard deviation. One-way analysis of variance (ANOVA) and Duncan’s multiple range test were conducted using IBM SPSS Statistics software v.19.0 (IBM Corporation, Armonk, NY, USA). The difference between treatments was considered to be significant if p < 0.05.

3. Results

3.1. Firmness, Ethylene Production, Respiration Rate, and SSC

Apple flesh firmness was monitored during storage and the subsequent shelf-life period (Figure 1A). Firmness of air-stored fruit progressively decreased during the 56-day storage, especially within the initial 28 days of storage, as well as the 44-day shelf life. Compared with the air-stored group, apples treated with 2 kPa O₂ had a higher firmness throughout the experimental period, indicating that storage under low oxygen retarded the softening of the GD apples. Interestingly, no difference in SSC was observed between the 2 kPa O₂ and air groups during the storage and shelf-life periods (Figure 1B).

Ethylene production in both groups increased in the early days of storage, then decreased during the late stage, which was consistent with the typical characteristics of climacteric fruit (Figure 1C). The apples in the 2 kPa O₂ group had a lower ethylene production than those in the air group during the storage and the shelf-life periods (Figure 1C). Ethylene production in the air-stored fruit peaked on day 56, while the peak of ethylene production in the fruit stored at 2 kPa O₂ was observed on day 56 + 23. Low-oxygen partial pressure, therefore, not only inhibited the production of ethylene during storage, but also delayed the ethylene peak during the subsequent shelf-life period. Similarly, the respiration rate in the apples gradually increased during the 56 days of storage and reached its peak on day 56 + 9 and day 56 + 23 for the air and 2 kPa O₂ groups, respectively (Figure 1D). The respiration rate in the fruit stored under 2 kPa O₂ was always lower than that of the air group (Figure 1D).

3.2. Emission of Key Esters

Aroma emission in skin and flesh tissues of GD apples during the storage and shelf-life periods were comprehensively investigated. In total, 38 aroma-related volatiles were detected in the GD apples, including five alcohols, five aldehydes, and twenty-eight esters (Supplementary Tables S2–S4). Of these, twenty-one volatiles were commonly found in all tissues, whereas six, two, and four volatiles were exclusively detected in Skin, Flesh1, and Flesh2, respectively.

During the storage period, the total ester content of fruit in the 2 kPa O₂ group was lower than that in the air group as the content of butyl acetate, hexyl acetate, and butyl butyrate were dramatically reduced in the 2 kPa O₂-stored apples (Figure 2). During the shelf-life period, the total ester content of the 2 kPa O₂-stored fruit increased, but still had lower levels than that of the air-stored fruit (Figure 2A). A similar situation was also observed in the levels of butyl acetate, hexyl acetate, and butyl butyrate (Figure 2B–E). The results suggest that the negative effect of low-oxygen storage on ester accumulation continued when the apples were transferred to ambient temperature during the shelf-life period. However, for 2-methybutyl acetate, no difference was found in content between fruit in the 2 kPa O₂ and air groups at the end of the shelf-life period (Figure 2E). On day 56 + 9 of the shelf-life period, the total ester content in the apples under low oxygen increased to 29.7%, 48.7%, and 48.0% for Skin, Flesh1, and Flesh2, respectively, compared with those in the air-stored fruit (Figure 2A), indicating a high recovery capability of ester biosynthesis in flesh tissue under low-oxygen storage.

3.3. Emission of Key Alcohols and Aldehydes

Alcohols in the GD apples were mainly 1-hexanol, 1-butanol, 2-methyl-1-butanol, and 1-octanol (Supplementary Tables S2–S4). The content of alcohols in both the 2 kPa O₂ and air groups initially increased and then gradually decreased during storage (Figure 3). During early storage, the apples stored under 2 kPa O₂ had a lower alcohol content than the air-stored fruit (Figure 3). However, on day 56, for both Flesh1 and Flesh2, the alcohol content in the 2 kPa O₂-stored fruit increased and was even higher than that in the air-stored fruit (Figure 3A). Interestingly, on day 56 + 23 of the shelf-life period, the total alcohol content of both Flesh1 and Flesh2 in the 2 kPa O₂ group became lower than that in the air group (Figure 3A), suggesting a low recovery capability of alcohols in apple flesh tissue after 2 kPa O₂ storage. The flesh tissue generally had higher alcohol levels than did skin tissue in the apples (Figure 3). On day 56 + 44 of the shelf-life period, higher alcohol levels were still observed in Flesh2 compared with Skin and Flesh1 (Figure 3A). As an abundant alcohol in the GD apple, 1-hexanol showed a similar trend in total alcohol content (Figure 3B, Supplementary Tables S2–S4). In addition, during the shelf-life period, the 1-butanol produced in Flesh1 of the 2 kPa O₂-stored fruit was also found to be at a lower level compared with that of the air group (Supplementary Tables S2–S4).

The aldehydes (Z)-2-heptenal, nonanal, (E)-2-octenal, hexanal, and (E)-2-hexenal, were detected in GD apple tissues (Supplementary Tables S2–S4). The hexanal and (E)-2-hexenal, related to the ‘green apple’ flavor, accounted for more than 95% of aldehydes in the GD apples (Supplementary Tables S2–S4). Total aldehyde had relatively higher levels in Skin but were lower in Flesh2 (Figure 3C). During early storage, the hexanal content in the skin of 2 kPa O₂-stored apples was higher, but the (E)-2-hexenal content was lower than that in the air group (Figure 3D,E). However, for Flesh1 on day 14 of storage, the levels of both hexanal and (E)-2-hexenal in apples stored under low oxygen were higher than those in air-stored fruit (Figure 3D,E), which agreed with previous research on ‘Fuji’ and ‘Gala’ apples [21]. Moreover, on day 56 + 23 of the shelf-life period, the aldehyde content in Flesh1 of the 2 kPa O₂-stored apples was also higher than that in the air group (Figure 3C).

3.4. Expressions of Genes Involved in Fatty Acid Metabolism

The expression level of MdLOX1a in both the 2 kPa O₂ and air groups increased along with storage time (Figure 4A). Specifically, in Skin, MdLOX1a expression was inhibited in apples under low oxygen during early storage. Meanwhile, for flesh tissues, especially Flesh1, MdLOX1a expression was inhibited throughout the storage period (Figure 4A). During the shelf-life period, the expression of MdLOX1a in Skin and Flesh2 of 2 kPa O₂-stored fruit was upregulated. The results indicate that MdLOX1a expression was sensitive to the change in oxygen partial pressure in the surrounding environment, which is similar to the discussion by Espino-Díaz et al. [2].

The expression of MdHPL in all apples continuously decreased during storage but increased during the shelf-life period (Figure 4B). In the 2 kPa O₂-stored fruit skin, the MdHPL had a higher expression than that in the air group, being consistent with the content of hexanal and (E)-2-hexenal (Figure 3E). In contrast, Flesh2 in the 2 kPa O₂ group had lower levels of MdHPL expression than did the air-stored apples throughout their storage and shelf life.

Alcohol dehydrogenase is an enzyme involved in fermentation and is highly sensitive to changes in oxygen level [22]. On day 14 of storage, the expression of MdADH1 and MdADH2 in 2 kPa O₂-stored apples was dramatically upregulated, especially in flesh tissues (Figure 4C,D), which could explain the decrease in alcohol levels and increase in aldehyde levels (Figure 3). However, the levels of MdADH1 and MdADH2 expression in all tissues decreased after being transferred to the shelf-life stage (Figure 4D).

For MdAATs (MdAAT1 and MdAAT2), the expression levels in all apples increased during storage, and their peaks occurred on day 56 + 9 of the shelf-life period, decreasing, thereafter, during the late stage of shelf life (Figure 4E,F). During storage, the expression of MdAATs, especially MdAAT2, in 2 kPa O₂-stored fruit were at lower levels compared with that in air-stored fruit, suggesting that low-oxygen conditions could downregulate ester biosynthesis by reducing the expression of MdAATs. On day 56 + 9 of the shelf-life period, the expression of MdAAT2 in the 2 kPa O₂-stored fruit increased (Figure 4F), although its levels were still lower than those of the air group (Figure 3A). At the end of the shelf-life period, the level of MdAAT1 expression decreased but was higher in the 2 kPa O₂ group than in the air group, especially for Flesh2 tissue, confirming the better recovery of ester components in apple flesh tissue.

3.5. Expressions of Genes Involved in Ethylene Production

The genes MdACS and MdACO1 encode the key enzymes for ethylene biosynthesis in GD apples. Over the storage period, the expression of MdACS and MdACO1 in all tissues showed an upward trend (Figure 5A,B). However, compared with the air group, the 2 kPa O₂-stored apples had lower levels of MdACS and MdACO1 expression. During the shelf-life period, the expression of MdACS and MdACO1 in 2 kPa O₂-stored fruit peaked on day 56 + 23, while the peaks mostly occurred on day 56 + 9 in the air group (Figure 5A,B). Additionally, higher transcript levels of MdACS and MdACO1 were observed in Flesh1 compared with Skin and Flesh2 (Figure 5A,B).

The expression patterns of six ethylene responsive factor (ERF) VII genes in GD apples are shown in Figure 5. Compared with the air group, the expression of MdERFa in 2 kPa O₂-stored fruit was upregulated during early storage, while the upregulations of MdERFb and MdERFc expressions were observed in late storage (Figure 5C–E). However, during storage, the expressions of MdERFd, MdERFe, and MdERFf were downregulated in 2 kPa O₂-stored fruit, especially in flesh tissue (Figure 5F–H). During the shelf-life period, the transcript levels of MdERFs in apple fruit peaked on day 56 + 9 or day 56 + 23 (Figure 5C–F), which is consistent with ethylene production in apples (Figure 1C).

4. Discussion

Deliberate storage under low-oxygen partial pressure in CA was widely applied to reduce the respiration rate and ethylene biosynthesis in fruits, which would consequently delay ripening, and thereby prolong fruit storage life, especially for apples and pears [22,23]. As expected, GD apples stored under 2 kPaO₂ had reduced ethylene production and respiration rates; the progression of softening was also retarded (Figure 1). Notably, the residual effect of storage under hypoxic conditions on aroma emission depended upon aroma components and fruit tissues, and was associated with the aroma biosynthetic pathways that directly influenced the recovery ability of aroma during the shelf-life period (Figure 2, Figure 3 and Figure 4). The flesh tissue of GD apples demonstrated a high recovery capability of ester biosynthesis, which was confirmed by the relatively high levels of expression of MdLOX1a and MdATTs during the shelf-life period (Figure 2 and Figure 4).

In terms of fruit quality, the results presented herein reveal that lowering the partial oxygen pressure could benefit retarding fruit softening, whereas there was no impact on soluble solid content (Figure 1A,B), thus confirming the results reported by Lopez et al. [12]. After fruit harvest, sugars are the secondary energy source during early storage as there are sufficient organic acids participating in the tricarboxylic acid cycle during respiration [24]. Many apple cultivars are suitable for long-term cold storage partly because the high content of organic acids in fruit at harvest can provide a sufficient primary energy source at low temperatures for a long period [25].

Generally, most apple cultivars have more abundant aroma volatiles in skin tissue compared with flesh tissue [26,27]. In GD apples, the amount of aroma volatiles, especially esters, in the skin was over two-fold higher than in the flesh tissues (Figure 3 and Tables S2–S4). Volatile esters are critical compounds for aroma development in apple fruit and are responsible for the characteristic fruity or floral flavors [28]. Under 2 kPa O₂ hypoxic conditions, the emissions of esters were dramatically suppressed in both the skin and flesh of GD apples (Figure 3). The flesh tissue of apples under low-oxygen conditions is likely to suffer an oxygen shortage due to a relatively low diffusion rate of oxygen in the apple tissue [29,30], which further affects aroma biosynthesis in the apples. Therefore, considering the oxygen gradient in fruit tissue, the availability of oxygen may be a critical factor that affects aroma biosynthesis in apples [14,15,29].

During the shelf-life period, the residual impact of storage under 2 kPaO₂ low oxygen was more pronounced on the straight-chain esters in GD apples, such as butyl acetate, hexyl acetate, and butyl butyrate, rather than the branched-chain ester 2-methylbutyl acetate (Figure 2). In apples, straight-chain esters are synthesized via the lipoxygenase pathway, while branched-chain esters are synthesized via amino acid metabolic pathways [4]. Previous research revealed that low-oxygen storage led to decreasing the production of fatty acids, which are the precursors of corresponding alcohols and straight-chain esters in apples [31]. In the present study, the reduced transcripts of genes encoding lipoxygenase and alcohol acetyltransferase in GD apples suggest that storage under 2 kPa O₂ repressed the lipoxygenase pathway (Figure 2 and Figure 4). Even so, the increase in MdLOX1a and MdAATs expression during the subsequent shelf-life stage did not promote the production of esters in the apples (Figure 2, Figure 4 and Figure 6). Similarly, Villatoro et al. found that the differential production of volatile esters in ‘Pink Lady’ apples could be attributed to biochemical modifications rather than the recovery of ester-synthesizing capability upon being transferred to air conditions [32]. Additionally, apple flesh tissue had a better capability of ester biosynthesis recovery than the skin tissue after low-oxygen storage (Figure 2). Thus, the production of volatile esters after storage in CA is more likely subjected to the limited availability of precursors such as free fatty acids in apples [4,26,33].

Straight-chain alcohols are the metabolic intermediates in the biosynthesis of linear esters from fatty acids [21]. In 2 kPa O₂-stored GD apples, hexyl acetate content decreased with the decreasing content of its precursor, 1-hexanol (Figure 2 and Figure 3). Furthermore, the increase in MdADH transcripts was observed in flesh tissue, which might contribute to the reduction in alcohol production (Figure 4 and Figure 6). The findings can also be confirmed by the high levels of aldehydes in apple flesh tissue under low-oxygen conditions during early storage (Figure 2) as MdADHs encode alcohol dehydrogenases that facilitate the interconversion between alcohols and aldehydes or ketones [34]. In addition, low-oxygen storage increased the transcription of MdHPL (Figure 4 and Figure 6) which encodes hydroperoxide lyase and is responsible for the oxidative cleavage of hydroperoxyl fatty acids to form C6 or C9 aldehydes and the corresponding C12 or C9 ω-fatty acids [1]. Nevertheless, the increased aldehydes and other volatile compounds related to the ‘green apple’ flavor were indicated as a cause of the reduction in flavor quality in apples during CA storage [28].

Ethylene is an important regulator of the biosynthesis of aroma volatiles, such as esters and alcohols [26]. Storage under 2 kPa O₂ negatively affected ethylene production by repressing the transcript accumulation of two rate-limited enzymes related to ethylene biosynthesis, namely l-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase (Figure 1C and Figure 5A,B). Previous literature indicated that continuous ethylene activity was required to maximize the production of volatile compounds in apples [7,35,36]. Thus, the reduction in aroma compounds during storage under low-oxygen partial pressure may be related to low ethylene production. However, the transcript levels of MdACS and MdACO1 did not show a positive correlation with ester emission in the tissues of apples (Figure 2, Figure 4 and Figure 6). The findings suggest that regulatory processes other than ethylene might also be involved in the production of fruit aroma, as suggested by Defilippi et al. [26]. Additionally, the VII ERFs in Arabidopsis were reported to participate in the fine-tuning regulation of hypoxia-responsive genes [37]. The VII ERF genes in GD apples had differential expression patterns in their response to low-oxygen conditions, indicating the multiple roles of ethylene (Figure 5 and Figure 6).

5. Conclusions

In conclusion, (1) for GD apples, 2 kPa O₂ storage influenced the abundance and spatiotemporal pattern of aroma emissions remarkably, mainly through the fatty acid lipoxygenase pathway. (2) Notably, the residual effect of storage under hypoxic conditions on apple aroma partly depended on aroma components and fruit tissues. Compared with skin tissue, the flesh tissue of GD apples was more resilient in ester biosynthetic activity after low-oxygen storage, with a recovery of about 48% of total esters in the flesh tissue compared with about 30% in skin tissue on day 56 + 9 of shelf life, indicating the differential recovery ability of aroma biosynthesis in tissues. However, there was a low recovery capability of alcohol in apple flesh tissue after 2 kPa O₂ storage as its total alcohol recovery was below 72%, although above 82% recovery occurred in skin tissue on day 56 + 23 of shelf life. For aldehydes, Flesh 1 had the highest recovery of 158% on day 56 + 23 of shelf life after 2 kPa O₂ storage. (3) Moreover, low-oxygen conditions during storage reduced ethylene production by 62.8% in peak values, which was associated with ester biosynthesis in GD apples. However, the differential expression patterns of genes associated with ethylene biosynthesis and signaling suggest more complex regulatory mechanisms of ethylene on aroma biosynthesis of GD apples under low-oxygen conditions.