# The role of low oxygen concentrations on ripening and cell wall-related gene expression in different tomato genotypes

Hayri Ustun, Selman Uluisik, Adem Dogan, Yunus Ozyigit, Mustafa Cetin, Cansu Ural, Mustafa Erkan

<figure><img src="image_1.png" /></figure>

## To cite this version:

Hayri Ustun, Selman Uluisik, Adem Dogan, Yunus Ozyigit, Mustafa Cetin, et al.. The role of low oxygen concentrations on ripening and cell wall-related gene expression in different tomato genotypes. Horticulture, Environment and Biotechnology, 2025, 66 (6), pp.1573 - 1589. 10.1007/s13580-025-00740-9. hal-05507021

HAL Id: hal-05507021

https://ut3-toulouseinp.hal.science/hal-05507021v1

Submitted on 12 Feb 2026

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
# The role of low oxygen concentrations on ripening and cell wall-related gene expression in different tomato genotypes

Hayri Ustun<fnref n="1" />,<fnref n="2" /> ID · Selman Uluisik<fnref n="3" /> ID · Adem Dogan<fnref n="1" /> ID · Yunus Ozyigit<fnref n="1" /> ID · Mustafa Cetin<fnref n="1" /> ID · Cansu Ural<fnref n="1" /> ID · Mustafa Erkan<fnref n="1" /> ID

Received: 16 December 2024 / Revised: 12 June 2025 / Accepted: 15 June 2025 / Published online: 9 July 2025
© The Author(s) 2025

## Abstract

The increasing global demand for fresh produce, coupled with the necessity to reduce postharvest losses, has driven research into alternative storage methods that extend fruit shelf life while maintaining quality. However, concerns about the environmental and human health effects of chemical treatments are driving the shift to nonchemical preservation methods. The aim of this study was to evaluate the effectiveness of different low oxygen concentrations during storage and ripening of different tomato genotypes in comparison with 1-methylcyclopropene (1-MCP), which is widely used as a ripening control agent, and to contribute to a deeper understanding of the molecular mechanisms regulating cell wall degradation. For this purpose, the effect of, 1%, 3%, and 5% O₂, 21% O₂ (control) concentrations and 1-MCP (0.5 µL L⁻<fnref n="1" />) were tested in six tomato genotypes including Micro-Tom and *rin* (ripening inhibitor mutant). The fruit samples were stored in different controlled atmosphere chambers at 20 °C and 85–90% relative humidity. The relationships between the gene expression levels with fruit ripening, firmness, ethylene, and respiration in different tomato genotypes and different O₂ concentrations were discussed. 1% and 3% O₂ inhibited the ripening of tomato fruit. However, there are indications that while ethylene production continues at 3% O₂, ethylene production is blocked at 1% O₂. Cell wall disruption and fruit softening continued under low O₂ conditions with little or no ethylene. 5% O₂ atmospheric composition and 1-MCP treatment provided similar or close results depending on the genotypes, therefore, storage of fruit at 5% O₂ atmospheric composition could be an alternative to 1-MCP application in terms of maintaining tomato fruit firmness, prolonged shelf-life and quality.

**Keywords** Solanum lycopersicum L. · Respiration rate · Cell wall destruction · Hypoxia · Ethylene production

Communicated by Choon-Tak Kwon

<figure><img src="image_3.png" /></figure>

Mustafa Erkan
erkan@akdeniz.edu.tr

Hayri Ustun
hayri.ustun@toulouse-inp.fr

Selman Uluisik
suluisik@mehmetakif.edu.tr

Adem Dogan
ademdogan@akdeniz.edu.tr

Yunus Ozyigit
yunusozyigit09@gmail.com

Mustafa Cetin
mcetin975@gmail.com

Cansu Ural
cansu.url@hotmail.com

<footnote n="1">¹ Department of Horticulture, Faculty of Agriculture, Akdeniz University, Antalya 07070, Türkiye</footnote>

<footnote n="2">² Laboratoire de Recherche en Sciences Végétales-Génomique et Biotechnologie des Fruits-UMR5546, Université de Toulouse, CNRS, Université Paul Sabatier, Institut Polytechnique de Toulouse, Auzeville Tolosan 31326, France</footnote>

<footnote n="3">³ Department of Food Agriculture and Livestock, Burdur Vocational High School, Burdur Mehmet Akif Ersoy University, Burdur 15030, Türkiye</footnote>
# 1 Introduction

Tomato (Solanum lycopersicum L.) is grown worldwide and consumed as fresh or processed product with a global production of around 186 million tons in 2022 (FAOSTAT 2024). The tomato is a climacteric fruit, where ripening is accompanied by a burst in respiration known as the climacteric rise and a large increase in ethylene production (Brumos 2021). The ripening process is irreversible and involves a complex and highly coordinated set of biochemical changes that include those involved in altering the fruit texture, color and taste (Wang et al. 2023). The management of the ripening process is critical for effective transport and storage of fruit without excessive postharvest losses. Over the years, numerous techniques have been developed to effectively manage the ripening of various fruit crops, and these have included modulation of the storage environment and removal or dosing fruit with ethylene to control the onset and progression of ripening (Yang et al. 2007; Dias et al. 2021; Islam et al. 2022). Although it is possible to control the overall rate of ripening with reduced concentration of O₂ or by control of ethylene (Alonso-Salinas et al. 2024) from the surrounding atmosphere of fruit in storage, the effects of altered O₂ or ethylene concentrations on specific ripening processes have received less attention.

1-MCP, a well-known and widely used inhibitor of ethylene action, binds to ethylene receptors and blocks ethylene sensing, thereby delaying ripening in a wide range of fruits, including tomatoes (Dias et al. 2021). The effectiveness of 1-MCP applications varies depending on several factors, including species and cultivar, application dose, fruit maturity stage, duration of exposure, temperature, and application method (Blankenship and Dole 2003). Although 1-MCP is classified as safe by the U.S. Environmental Protection Agency (EPA) and European Food Safe Authority (EFSA), research on the potential health and environmental impacts of long-term exposure remains limited. Due to growing concerns about environmental safety and human health, there is an increasing need to explore alternative methods for postharvest chemical control. As a result, there is a shift away from chemical treatments for plant products, with a focus on developing more environmentally friendly approaches to extending shelf life. One such method is controlled atmosphere (CA) storage, which involves modifying the storage environment by adjusting O₂ or ethylene concentrations. There is a move away from chemical treatments of plant products and a need to develop more environmentally friendly methods to extend shelf life. Indeed, controlling the storage atmosphere by changing the O₂ or ethylene concentrations in storage has proven to be very effective to extend the shelf life of fruit (Fagundes et al. 2015). This approach is well known and termed CA storage. CA systems are

generally used to extend postharvest storage, maintenance of sensory and nutritional quality and reduction of microbial growth in various fruits by industry and researchers (Klein et al. 2020). Several studies have demonstrated that the CA system can extend the shelf life of fruit, such as fig (Dogan 2022), jujube (Islam et al. 2022), litchi (Ali et al. 2019), apple (Wendt et al. 2023) and tomato (Olveira-Bouzas et al. 2021). Although, CA achieves significant advantages when products are preserved in appropriate temperature, humidity, and environment composition, the benefits of CA storage is also dependent on the variety, ripening stage, temperature, and duration of storage (Zhuang et al. 2014).

The recommended range for the storage of most horticultural products, excluding those that respond negatively to low oxygen and those that respond positively to high carbon dioxide, is typically between 2–5% O₂ and 3–8% CO₂. CO₂ concentration is higher than 3–5% and O₂ concentration lower than 1% resulted in physiological disorders such as taste defects, alcohol formation, and internal darkening in fruit (Beaudry 2000). The recommended concentrations of O₂ and CO₂ for optimal storage for tomato are typically around 3–5% O₂ and 0% CO₂ (Sandhya 2010). The storage and shelf life of pink ‘Buffalo’ tomatoes stored at 12 °C in 4% O₂ + 2% CO₂ was increased (Nunes et al. 1996). In contrast, Ratanachinakorn et al. (1997) showed that pink ‘Bermuda’ tomatoes did not suffer any negative effects from being exposed to 0.5% O₂ for 1 d or 80% CO₂ for 2 d at 22 °C. In a more recent study, modified atmosphere packaging (MAP) (10% O₂–10% CO₂) delayed color evolution, firmness loss, and decay of tomato throughout storage (Olveira-Bouzas et al. 2021).

Fruit texture characteristics are determined by a variety of factors including cell turgor, cell size, thickness of the primary cell wall, intercellular adhesion involving the middle lamella region and the organization of the cell wall at the molecular level (Posé et al. 2019). The primary cell walls of dicots such as tomato are composed primarily of three main classes of polysaccharide namely pectic polysaccharides, hemicelluloses and cellulose with a small component of structural proteins, in which alterations in pectin structure are consistently reported as being closely linked to softening (Wang and Seymour 2022). During ripening, these polysaccharide domains are disassembled, and these structural changes within the primary cell wall and middle lamella are the major reason why tissues soften, and cells separate (Brummell 2006). Tomato softening is known to be involved in degradation of the pectic component through the action of enzymes such as pectate lyase (PL) (Uluisik et al. 2016; Yang et al. 2017), polygalacturonase (PG) and a range of other pectin degrading enzymes and proteins such as expansins (Exps) (Wang et al. 2018; Ortega-Salazar et al. 2023). Although these genes showed different responses
to ethylene at biochemical and molecular levels, ethylene would seem to play a significant role in the induction of cell wall enzyme gene expression in many fruits of both the climacteric and non-climacteric types (Tucker et al. 2017). Although the link between ethylene in fruit softening and expression of cell wall-modifying enzymes has been studied (Li et al. 2019; Wang and Seymour 2022), there is no information on the response to ethylene absence under hypoxic conditions in any fruit.

Controlled atmosphere storage, which modifies O₂ and CO₂ concentration, has emerged as a promising technique to delay ripening and maintain fruit firmness. However, the specific effects of reduced O₂ concentration on key aspects of fruit ripening, particularly cell wall degradation and softening, remain underexplored in different tomato genotypes. This study investigates the impact of varying oxygen concentrations on the ripening process of different tomato genotypes, with a particular focus on the expression of four

key cell wall-related genes known to significantly influence tomato texture.

## 2 Materials and methods

### 2.1 Fruit material

In this study, four tomato (Solanum lycopersicum L.) genotypes ('Torry', 'Elips', 'Verty', 'Yeniceri') commercially grown in Türkiye, and two more genotypes, the ripening inhibitor mutant (rin) and the wild type 'Micro-Tom' were used (Li et al., 2020b) (Table 1). These genotypes were selected based on our previous studies evaluating the post-harvest performance of the most widely cultivated tomato genotypes in Türkiye (Üstün 2024). All tomato genotypes were cultivated within a 1000 m² greenhouse at Akdeniz University, Türkiye. The planting arrangement involved double rows with inter-rows and intra-rows distances were

<table><caption>**Table 1** Tomato cultivars and their visual properties</caption><thead><tr><th>Cultivars</th><th>Picture*</th><th>Types</th><th>Color</th><th>Shape</th><th>Average Weight (g)</th></tr></thead><tbody><tr><td>Torry</td><td></td><td>Beefsteak</td><td>Red</td><td>Round oblate</td><td>195.32 ±33.26</td></tr><tr><td>Elips</td><td></td><td>Oval</td><td>Red</td><td>Elongated oval</td><td>134.08 ±14.08</td></tr><tr><td>Verty</td><td></td><td>Cocktail</td><td>Red</td><td>Oval</td><td>35.96 ±3.77</td></tr><tr><td>Yeniceri</td><td></td><td>Cherry</td><td>Red</td><td>Round</td><td>22.30 ±1.68</td></tr><tr><td>Micro-Tom</td><td></td><td>Cherry</td><td>Red</td><td>Round</td><td>11.27 ±1.05</td></tr><tr><td>Rin</td><td></td><td>Cherry</td><td>Yellow</td><td>Round</td><td>9.22 ±1.33</td></tr></tbody></table>

*Pictures are of different maturity stages of tomato cultivars (For red cultivars; immature green, mature green, breaker, turning, pink, light red and red, respectively. Maturity stages for yellow colored genotype were determined according to the scale used for red-colored genotypes). Data are mean (n = 60), and “±” represent the standard deviation.
0.6 m and 0.4 m, respectively. Throughout the cultivation period, identical cultural practices were implemented for all plants, and they were pruned as single stem. Climate data belonging to the cultivation period provided in our previous study (Ustun et al. 2023). Tomatoes were harvested from the 3rd or 4th cluster of plants for each commercial genotype at mature green (MG) stage. For ‘Micro-Tom’ and ‘rin’, the harvest was done at the MG stage as well.

## 2.2 Experimental design, treatments, and sampling

Harvested tomato fruit were stored at 20 °C, optimal ripening temperature (Sharma et al. 2020), at different O₂ concentrations of 21% O₂ as a control, 1% O₂, 3% O₂, and 5% O₂ (Gómez and Camelo 2002). O₂ concentration belonging to the controlled atmosphere environment was adjusted to 0.01% CO₂ for the prevention of CO₂ accumulation and stabilized during the storage period via using palistore storage system. In addition, to determine the effectiveness of low O₂ doses against 1-MCP was applied at a dose of 0.5 µL L⁻¹ for 12 h at 20 °C as chemical control (Guillén et al. 2004). Tomatoes subjected with 1-MCP were stored at 20 °C at a concentration of 21% O₂ similar to the control group, sampling at 5 d intervals.

## 2.3 Ripening indicator [hue angle (h°)]

Color in tomatoes is a crucial external characteristic for assessing both ripeness and postharvest life. It plays a significant role in consumer purchasing decisions and is a key factor in determining tomato quality. It's a non-destructive ripening determination method for tomato and this also allows monitoring of ripening in the same biological samples. The hue angle was measured using a chromometer (Minolta CR-400, Camera Co., Ramsey, NJ) at three different positions around the equator at each time point to ensure that the fruit were in the same state of maturity (Tao et al. 2021). Subsequently, measurements were taken at 5 d intervals on the same fruit using the same method, allowing for the tracking of fruit ripening.

## 2.4 Ethylene production and respiration rate

In order to determine the respiration rates and ethylene production of tomatoes, fruit samples with known weight and volume were introduced into gas-tight plastic containers with a capacity of 2 L. Tomatoes stored at low O₂ were left for 1 h for reoxygenation to stabilize and then the containers were closed. After the fruit were placed into containers and they were kept at 20 °C for 1 h, the gas sample was taken from the enclosed space using a gas-tight injector. After that gas samples were injected into the TCD detector

to determine the respiration rate and into the FID detector to measure ethylene production by gas chromatography (Thermo Finnigan Trace GC Ultra, Thermo Electron SpA Strada Rivoltana, Milan, Italy) device. Respiration rates of fruit samples were calculated in units of mL CO₂ kg⁻¹ h⁻¹, while ethylene production was determined in units of µL C₂H₄ kg⁻¹ h⁻¹ (Ustun et al. 2023).

## 2.5 Fruit firmness

Fruit firmness was measured using a hand penetrometer (Effigies, model FT 011), equipped with a 3 mm cylindrical probe. The probe was inserted into the fruit to a depth of 0.5 cm for accurate assessment. The measurements were taken from the equatorial region of the fruits at equal intervals, and the results are presented as N (Ali et al. 2021).

## 2.6 RNA extraction and quantitative real-time PCR

Frozen tomato fruit pericarp was ground into fine powder in liquid nitrogen using a coffee grinder. Then 0.5 g of powdered tissue was placed in a 1.5 mL centrifuge tube with a mixture of 500 µL of extraction buffer (CTAB (Cetyl trimethylammonium bromide) method) and 500 µL of PCI (Phenol-Chloroform-Isoamyl alcohol, ROTI® Aqua Phenol). The mixture was incubated at 60 °C for 15 min. Samples were then centrifuged at 10,000 g for 15 min at 4 °C and the supernatant was transferred to the new Eppendorf tube. The same volume of chloroform: Iso-amyl (1:1) alcohol was added and centrifuged again as done before. The supernatant was collected in a new Eppendorf and then 10 M of chilled lithium chloride was added and placed at -20 °C for four hours. After samples were centrifuged 10,000 g for 30 min at 4 °C, the supernatant was discarded, and the pellet was washed with 70% ethanol and dried for half an hour. The pellet was dissolved in 50 µL RNA-DNA free water. Quality determination was performed using a NanoDrop (Epoch Microplate, Biotek), with sample readings assessing at 260/280 nm and 260/230 nm ratios, and values between 1.8 and 2.0 were considered suitable for subsequent steps. RNA samples stored at -80 °C for further experiments.

cDNA synthesis was carried out using VitaScript™ First-Strand cDNA Synthesis Kit (Procomcure Biotech, Thalgau, Austria), using 300 ng of total RNA and 1 µL of VitaScript™ enzyme mix. RT-qPCR experiments were conducted in Bio-Rad CFX96 Real-Time PCR System (Bio-Rad, USA). The *EF-1α* (GenBank accession no. X144491) was used as the reference gene, and primer sequences used for RT-qPCR are listed (Table S1). Every reaction was conducted in three separate biological replicates, each with two technical replicates. The relative expression level of the genes was calculated using the 2⁻⁰⁻⁰ΔΔCt method (Livak and Schmittgen 2001).
## 2.7 Statistical analyses

The experimental design of the study comprised a randomized plot design, with three replications and five fruits per replicate. The measurements of the huge angle, fruit firmness, ethylene production, respiration rate and gene expressions were examined in different genotypes using a general linear model (two-way ANOVA). Pairwise comparisons were executed using the Tukey (HSD) test in significant cases ($p<0.05$). Heatmaps for relative gene expressions were prepared using RStudio.

# 3 Results

## 3.1 Color stage as a ripening indicator (h °)

Color stages (h °) of the fruit are shown in Fig. 1 and this parameter is used to track the progress of ripening. After the tomatoes were harvested at the MG stage, their color was measured to ensure that they were at the same maturity stage and a change of 3 ° is considered normal. The colors of the tomatoes at the MG stage differed according to the genotypes. At the beginning, the highest mean h ° was detected in the ‘Torry’ genotype with 113.67 °, and the lowest mean h ° was detected in the ‘Yeniceri’ genotype with 107.34 °. The h° of other genotypes were between these two values. With the onset of ripening, h ° began to decrease. An average value of 60 ° and below indicated fully ripe fruit.

The effects of all treatments on ripening were found to be statistically significant ($p<0.05$). In commercial genotypes (‘Torry’, ‘Elips’ and ‘Verty’; except ‘Yeniceri’), the effectiveness of 1-MCP started to decrease from the 15th d ($p<0.05$). In ‘Yeniceri’, 1-MCP started to lose its effectiveness from the 5th d. Among low oxygen applications, the effect of 1% and 3% O₂ on ripening was generally found to be statistically similar. In ‘Torry’, ‘Elips’, ‘Verty’ genotypes, these two treatments differed statistically ($p<0.05$) only on the 20th d. In the ‘Yeniceri’ variety, this differentiation emerged from the 5th d. There is a statistical ($p<0.05$) difference in 5% O₂ compared to 1% and 3%. In general treatments involving 1-MCP and low O₂ concentrations reduced the rate of ripening. However, 1% and 3% O₂ concentrations did not allow ripening to continue for 20 d. Although 1-MCP and 5% O₂ concentration allow the fruit to ripen, different responses occur according to the genotypes. This phenomenon is discussed and explained in detail below.

## 3.2 Ethylene production and respiration rate

The lowest ethylene production (Fig. 2) at harvest was determined in ‘Elips’ with 0.61 µL C₂H₄ kg⁻¹ h⁻¹ and the highest

ethylene production in ‘rin’ with 3.28 µL C₂H₄ kg⁻¹ h⁻¹. On the 5th d, ethylene production increased dramatically in the control groups except ‘Elips’. In fact, there was an increase in this genotype on the 5th d, but the increase continued until the 15th d. In addition, 1-MCP treatment significantly ($p<0.05$) delayed this increase in all genotypes. After a peak in the production of ethylene in tomatoes, the production of ethylene began to decrease. In general, an increase in ethylene production was observed in control fruits, and treated with 1-MCP and 5% O₂, depending on ripening. (Fig. 2). However, ethylene production in fruit kept at 1% and 3% O₂ conditions remained lower than the riper fruit in general after of reoxygenation. Contrary to expectations, ethylene production did not follow a linear trend from low O₂ to high O₂ concentrations. The ethylene production of the analyzed tomato genotypes was found to be statistically similar ($p<0.05$) at both 1% and 3% O₂ concentrations, except for ‘Yeniceri’. In the ‘Yeniceri’ genotype, 3% O₂ showed higher ethylene production at 10th, 15th and 20th d.

Respiration rate of different tomato genotypes stored for 20 d in 1%, 3% and 5% O₂ atmosphere compositions and 1-MCP treatment are given in Fig. 3. The lowest respiration rate at harvest time was determined in ‘Elips’ with 0.46 mL CO₂ kg⁻¹ h⁻¹ and the highest respiration rate in ‘rin’ mutant with a value of 27.93 mL CO₂ kg⁻¹ h⁻¹. Although, respiration rate generally increased in all genotypes during the storage period, a slower increase trend was observed in fruit treated with 1-MCP and 5% O₂. In addition, this increase was much less in tomatoes stored at 1% and 3% O₂ than those stored at 1-MCP and 5% O₂, although some of these changes are statistically ($p>0.05$) similar to each other.

## 3.3 Fruit firmness

Fruit firmness of different tomato genotypes stored for 20 d in 1%, 3% and 5% O₂ atmosphere compositions or 1-MCP treatment are given in Fig. 4. At the beginning of storage, fruit firmness of tomatoes at the MG stage varies between 16.24 and 30.26 N. The highest firmness value was determined as ‘Yeniceri’, and the softest genotype was determined as ‘Micro-Tom’. During storage, tomatoes started to lose their firmness, and this loss was less in 1-MCP and low O₂ applications compared to the control group. On the 5th d of storage, fruit firmness was statistically ($p>0.05$) similar in ‘Verty’, ‘Elips’ and ‘Yeniceri’ genotypes for all atmosphere compositions, 1-MCP treatment and control group. On the 10th d of storage, the control group fruit of ‘Elips’ were statistically ($p<0.05$) softer compared to other atmospheric compositions and 1-MCP treatment. Additionally, in the control group and 1-MCP treatment of ‘Torry’ and ‘rin’ genotypes; in the control group and 5% O₂ atmosphere composition of ‘Verty’, ‘Yeniceri’ and ‘Micro-Tom’ genotypes
<figure><img src="image_4.png" /><figcaption>**Fig. 1** Hue angles (h°) of tomatoes stored in a 1-MCP-treated and under low-oxygen concentrations (1%, 3% and 5% O₂) during a 20 d storage period. Data are mean (n=3), and “±” represents the standard deviation</figcaption></figure>

were found to be statistically (p<0.05) similar. On the 15th d of storage, ‘Torry’ and ‘Elips’, genotypes preserved fruit firmness better than the control group in all treatments. However, fruit firmness of ‘Verty’ and ‘Yeniceri’ in control group was statistically (p>0.05) similar to firmness values of fruit treated with 1-MCP and stored in 5% O₂ atmosphere

composition. Moreover, the firmness of fruit in control group and 5% O₂ atmosphere composition of ‘Micro-Tom’ and ‘rin’ genotypes was statistically similar (p>0.05). On the 20th d, the fruit stored in 5% O₂ atmosphere composition and 1-MCP treatment displayed fruit firmness values statistically (p>0.05) similar to each other except ‘Verty’
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>**Fig. 2** Ethylene production of tomatoes stored in a 1-MCP-treated and under low-oxygen concentrations (1%, 3% and 5% O₂) during a 20 d storage period. Data are mean (n=3), and “±” represents the standard deviation</figcaption></figure>

and ‘Micro-Tom’. In ‘Torry’ and ‘Elips’, there was not sta-
tistically significant (p>0.05) loss of firmness until the end
of the storage in 1% O₂ atmosphere composition. In general,
fruit firmness loss was better preserved at 1% and 3% O₂
atmosphere composition compared to other applications at
the end of the storage, and fruit firmness was statistically
(p<0.05) similar in 1% and 3% O₂ conditions. However,

in 1% and 3% O₂ storage conditions, tomato fruit did not
continue ripening, therefore there is no need to be stored
for more than 20 d. On the other hand, 5% O₂ atmosphere
composition and 1-MCP treatment were found to be gen-
erally successful in maintaining fruit firmness throughout
the storage period compared to the control group. Because
tomatoes stored at 5% O₂ continue ripening during storage,
<figure><img src="image_11.png" /><figcaption>**Fig. 3** Respiration rates of tomatoes stored in a 1-MCP-treated and under low-oxygen concentrations (1%, 3% and 5% O₂) during a 20 d storage period. Data are mean (n=3), and “±” represents the standard deviation</figcaption></figure>

like 1-MCP and control groups. For this reason, in the atmospheric compositions that were evaluated in the study, only the 5% O₂ is the most suitable composition for the storage of MG tomatoes.

At the end of 20 d, it was determined that small-fruited tomatoes with high respiration rate lost fruit firmness faster

than large-fruited tomatoes with low respiration rate, under low O₂ conditions. Fruit firmness loss was between 28.11% and 66.57% in the 1-MCP group; 23.76% and 68.81% in 5% O₂ group; 16.32% and 55.61% in 3% O₂ group. The fruit stored at 1% O₂, the firmness loss varied between 7.23% and 55.0%. Interestingly, while the loss of fruit firmness is
<figure><img src="image_12.png" /><figcaption>**Fig. 4** Fruit firmness of tomatoes stored in a 1-MCP-treated and under low-O₂ concentrations (1%, 3% and 5% O₂) during a 20 d storage period. Data are mean (n=3), and “±” represents the standard deviation</figcaption></figure>

close in 1% and 3% O₂ in genotypes with high respiration rate ('Yeniceri', 'Verty', 'Micro-Tom', 'rin'); there was a 5% firmness loss difference due to ripening. On the contrary, in genotypes with low respiration rate ('Elips' and 'Torry'), the loss of fruit firmness in 5%, 3% and 1% O₂ (30.75%, 18.30% and 7.78% in 'Elips'; 23.76%, 16.32% and 7.23% in 'Torry'), respectively (Figs. 3 and 4). In general, 5% O₂

atmospheric composition and 1-MCP gave similar or close results depending on the genotypes, which makes 5% O₂ environment a possible alternative to 1-MCP treatments in terms of maintaining tomato fruit firmness in postharvest storage conditions.
## 3.4 Expression of genes involved in cell wall modification

We investigated the expression of cell wall related genes, *SIPL*, *SIPG2*, *SIPME1*, and *SIExp1*, in response to 1-MCP treatment and 1, 3, 5% O₂ storage conditions. Pectate lyase (PL), randomly cleaves β-(1-4) linkages between galacturonosyl residues by β-elimination generating 4,5-unsaturated double bonds oligogalacturonates. In the case of *SIPL*, on the 10th d of storage in control fruit, the gene expression increased dramatically only in ‘Yeniceri’, while no sharp increase was observed in other genotypes (Fig. 5). In low O₂ environment, 1% O₂ storage blocked nearly all *SIPL* gene expression in all genotypes analyzed. However, in fruit stored in 3% and 5% O₂ environments, *SIPL* expression level varied on different d and genotypes as in ‘Torry’ on the 20th d. As expected, ‘rin’ mutant tomato fruit had the lowest *SIPL* expression in all conditions throughout the storage, but with a peak in the 10th d of 5% O₂ condition. Although it was not as suppressed as in fruit stored in 1% O₂ environment, the expression level of *SIPL* in fruit treated with 1-MCP showed a similar pattern to the expression levels in fruit stored at 3–5% O₂ concentrations. Overall,

the expression pattern of *SIPL* in fruit stored at 3–5% O₂ was quite variable, whereas it was much more stable under 1% O₂ conditions and showed the lowest expression levels. These results for *SIPL* might suggest that *SIPL* gene expression might be highly correlated with O₂ present in the environment.

Polygalacturonases (PGs) catalyze the hydrolysis of pectin and are involved in numerous developmental processes including the fruit ripening / softening process. In our analysis, the transcript levels of *SIPG2* were also determined in six different tomato genotypes stored under different O₂ conditions (Fig. 6). The *SIPG2*, known to have very high activity during tomato ripening, expectedly showed higher activity in control fruit in the first period of storage. Especially on the 10th d of storage, *SIPG2* gene expression peaked in ‘Torry’, ‘Yeniceri’ ‘Elips’ and ‘rin’ fruit. In 1-MCP treated fruit, the expression level of *SIPG2* was up-regulated after 15th or 20th d of storage, including ‘rin’. The expression pattern of *SIPG2* transcripts from fruit stored at 3–5% O₂ showed similarities to 1-MCP treated fruit but within different genotypes. The fruit stored at 1% O₂ conditions showed the greatest reduction in the expression level of *SIPG2*. The expression of *SIPG2* in fruit kept at 1% O₂ was greatly

<figure><img src="image_13.png" /><figcaption>**Fig. 5** Relative expression levels (as fold change) of *SIPL* in different tomato cultivars stored at 1-MCP-treated and under low-oxygen concentrations (1%, 3% and 5% O₂) during 20 d of storage. Effect</figcaption></figure>

of storage conditions on selected cell-wall modification related genes represented heat maps. (n=3)
<figure><img src="image_14.png" /><figcaption>**Fig. 6** Relative expression levels (as fold change) of *SIPG2* in different tomato cultivars stored at 1-MCP-treated and under low-oxygen concentrations (1%, 3% and 5% O₂) during 20 d of storage. Effect</figcaption></figure>

of storage conditions on selected cell-wall modification related genes represented heat maps. (n=3)

suppressed compared to the other two low O₂ storage conditions and when compared with 1-MCP treated fruit.

Pectin methyl esterase (PME) is an enzyme that catalyzes the removal of methyl esterified galacturonic acid residues from pectin. PME is one of the key enzymes that hydrolyze methyl esters to yield HG (homogalacturonan) with a low degree of methylation, which can then be cleaved by PL or PG. In this study, *SIPME1* expression was also quantified by RT-qPCR (Fig. 7). *SIPME1* expression showed a similar trend to *SIPG* and *SIPL*, as *SIPME1* expression was more drastically suppressed in fruit stored under low O₂ and 1-MCP conditions compared to control fruit. For example, while *SIPME1* expression was the highest control fruit of ‘Yeniceri’ stored for 10 d, similar expression pattern was observed on the 20th d in fruit stored in 5% O₂ storage condition. The activation of *SIPME1* was greatly down-regulated in all genotypes stored in 1% O₂, similar to *SIPL* and *SIPG2*.

Expansins (Exp) are localized in the cell wall might also indirectly contribute to pectin degradation during fruit ripening. They cause non-enzymatic cell wall relaxation by binding cellulose / hemicellulose and disrupting non-covalent

cross-linking interactions (Cosgrove 2016). Therefore, we also assessed the expression pattern of *SIEXP1* in fruit stored in different storage conditions. Based on the results, generally, the expression of *SIEXP1* in fruit stored in 1-MCP and all low O₂ concentrations were effectively suppressed compared to controls (Fig. 8). ‘Verty’ showed a different *SIEXP1* compared to other genotypes, as *SIEXP1* expression appeared active in 1% O₂ conditions on the 15th d of storage. Moreover, a steady slowly increased *SIEXP1* transcript accumulation was detected in 1-MCP-treated ‘Torry’ fruit compared to all low O₂ storage conditions. Unexpectedly, *rin*’ mutant fruit resulted in a marginal up-regulation of *SIEXP1* on day 10 in 3–5% O₂ storage conditions. Overall, it can be concluded that expression patterns of cell wall modifying genes are quite variable in fruit subjected to 1-MCP and 1–3–5% O₂, with a higher down-regulation in the genes stored in 1% O₂ conditions.
<figure><img src="image_15.png" /><figcaption>**Fig. 7** Relative expression levels (as fold change) of *SIPME1* in different tomato cultivars stored at 1-MCP-treated and under low-oxygen concentrations (1%, 3% and 5% O₂) during 20-days of storage. Effect</figcaption></figure>

of storage conditions on selected cell-wall modification related genes represented heat maps. (n=3)

## 4 Discussion

It is well established that the reduction in O₂ concentration used in CA systems results in decreased respiration and ethylene production, delayed ripening, and extended shelf life in many fruit species, including tomato (Park et al. 2018). Moreover, the response of tomato to hypoxic stress during fruit ripening is complex and can vary according to genotype depending on their respiration rate (Ustun et al. 2023). However, the effect of low O₂ conditions on specific aspects of tomato ripening are often less well understood and especially the effects on softening-related gene expression. Accordingly, we utilized an integrated morphological and molecular approach to characterize the responses of the six tomato genotypes (Table 1) to the different low O₂ regimes adopted during tomato storage looking at their changes in ethylene production and respiration along with cell wall-related gene expression. In our study, although fruit firmness values were higher in all tomato genotypes stored at 1% O₂, compared to other storage conditions including 1-MCP treatment, some of the genotypes kept in 3% or 5% O₂ environment have a firmer or softer texture than the fruit

treated with 1-MCP. The reason for this could be depending on the genotype, cell wall-degrading enzymes are more active, such as ‘Yeniceri’ in 3–5% O₂ environment than 1% O₂ storage conditions. There was not statistically significant (*p*>0.05) loss of firmness in ‘Torry’ and ‘Elips’ until the end of storage at 1% O₂ atmosphere composition and there was no clear expression of the four-cell wall related genes analysed at this storage condition, supporting previous studies that low O₂ prevented the loss of firmness in tomato (Gómes and Camelo 2002). Furthermore, the study conducted by Sozzi et al. (1999) proposed that the potential influence of O₂ and/or CO₂ on cell wall enzyme activities should not be disregarded.

Previous studies have reported that *PG* and *PL* gene expression are related to ethylene production in melon, apple and banana development (Nishiyama et al. 2007; Tatsuki et al. 2007; Payasi et al. 2004). However, in tomato, silencing of *SIPL* resulted in no changes on ethylene biosynthesis in *Ailsa Craig* tomato genotype (Uluisik et al. 2016), but silencing of *SIPL* in Micro-Tom down-regulated the expression of ethylene biosynthetic genes (Yang et al. 2017). In a very recent study, fruit respiration and ethylene
<figure><img src="image_16.png" /><figcaption>**Fig. 8** Relative expression levels (as fold change) of *SIExp1* in different tomato cultivars stored at 1-MCP-treated and under low-oxygen concentrations (1%, 3% and 5% O₂) during 20 d of storage. Effect</figcaption></figure>

of storage conditions on selected cell-wall modification related genes represented heat maps. (n=3)

production were higher in double CRISPR knockout of SIPG and SIPL lines compared to WT fruits. This finding defies logic because fruits with higher respiration and ethylene production rates also typically have higher metabolic rates and transpiration, which leads to senescence or physiological deterioration (Ortega-Salazar et al. 2023). Our findings show that *SIPG2* gene expression increases with increasing ethylene content or at the next sampling time. (Figs 2 and 6). However, *SIPG2* gene expression is absent or negligible under 1% O₂ conditions. This is another indication that there is little or no ethylene production under 1% O₂ conditions as discuss and explain below. The 1-MCP treatment could potentially suppress ethylene signaling and control the pathways responsible for cell wall metabolism, thereby impeding cell wall degradation (Li et al. 2020a, b). In our study, *SIPG2* expression increased after the effect of 1-MCP decreased, suggesting that new ethylene receptors formed (Chang and Brecht 2023) and *SIPG2* expression begins with the resumption of ripening.

It is known that the ripening of tomatoes stored in low O₂ for a short time ripen more slowly (Klieber et al. 1996). In this study, tomatoes stored in low O₂ atmosphere

environment showed different ripening behaviors based on genotypes (Fig. 1). The most striking point is that tomatoes with small fruit size tend to ripen faster than larger tomatoes, except ‘Micro-Tom’ (Table 1; Fig. 1). These results support our previous findings that respiration rate is related to fruit size (Ustun et al. 2023). In other words, tomatoes with small fruit size respire faster than larger ones and thus they benefit more effectively from the small amount of O₂ in the atmosphere composition. In this way, their ripening demands can be met, and they can ripen faster under low O₂. Oxygen availability is an important factor for ethylene formation to continue the ripening process (Johansson et al. 2013). In this study, it has been determined that ethylene production is generally higher in fruit stored at 5% O₂ than in fruit stored in 1% and 3% O₂ concentrations (Fig. 2). However, higher ethylene production was detected in fruit stored at 1% O₂ compared to 3% and 5% O₂ concentrations on some d of storage, as observed in previous studies in apple fruit (Lee et al. 2023) tomato leaf (Wang and Arteca 1992) and tomato fruit (Klieber et al. 1996). The most plausible explanation might be that the ethylene production pathway is interrupted by O₂ deprivation (Yip et al. 1988),
and that 1-aminocyclopropane-1-carboxylic acid (ACC) proteins accumulated in very low O₂ state increased ethylene production due to reoxygenation and recovery of ACO enzymes (Wang and Arteca 1992; Tian et al. 1997). Zhen-guo et al. (1983) reported that low O₂ inhibits ethylene production and therefore causes ACC accumulation. They also reported that ACC synthesis was delayed but eventually accumulated in huge amounts. Blankenship and Richardson (1986) found that the levels of naturally occurring ACC in fruit stored in 1% or 3% O₂ were 20 times higher compared to fruit stored in normal atmospheric conditions. Also, ethylene production rises well-known fact during and after abiotic stress factors (Chen et al. 2021). Therefore, another explanation could be that increased ethylene production is the result of response after low O₂ stress. Although there is detailed information in literature about how ethylene performs a pivotal function in anticipating hypoxia and metabolic acclimation when plants are submergence (Hartman et al. 2019), there is no morpho/physiological and molecular mechanism-based information together proving how ethylene plays a role during fruit ripening in low O₂ stress under postharvest storage conditions. If there is an increase in ethylene production due to accumulation of ACC proteins, we can assume that there is little or no ethylene production in the 1% O₂ storage condition, or that ethylene production might only increase in response to low O₂ stress. In either case, another question arises as to whether ethylene is the main regulator of cell wall degradation because, fruit softening continues even in 1% O₂ environment depending on the genotype.

In this study, 5% O₂ storage condition provided the optimum environment maintaining fruit firmness throughout the storage period compared to the control group and 1-MCP treatment while ripening continued. Interestingly, as it has been mentioned above, although the ripening of tomato fruit stored at 1% O₂ environment was blocked and there was almost no ethylene production, the fruit continued to soften in MG state. The rate of fruit softening was accelerated, particularly in smaller genotypes with higher respiration rates such as ‘Yeniceri’ and ‘Verty’ in 1% O₂ environment. In the absence of elevated concentrations of ethylene production, the fruit continues to soften. What are the genes or mechanisms that soften fruit independent of ethylene? In our study, the expression of genes related to cell wall degradation increased with ripening. However, high gene expression levels were also observed under 1% and 3% O₂ conditions even in the absence of ripening, as well as under 1% O₂ without ethylene production. These findings suggest that certain aspects of fruit softening occur independently of autocatalytic ethylene production.

Respiration rates are reduced at low temperatures. Therefore, the delay in fruit softening observed during cold storage

may be attributed to the suppression of respiration rate under low-temperature conditions. Further evidence supporting this argument comes from the studies that the activity of cell wall degrading enzymes, including PME and PG activity, in chilling effect and in cold storage was reduced (Wei et al. 2010; Chen et al. 2015). Moreover, Sozzi et al. (1999) found no difference in fruit firmness when they applied ethylene at 3% O₂ and kept the tomatoes at only 3% O₂ environment. Based on these discoveries, it can be hypothesized that respiration might be more effective than ethylene and ethylene may not play as important a role in cell wall disruption as previously thought. However, these results need to be supported by a more detailed study to understand the response mechanism of cell wall degrading enzymes to low O₂.

In general, the expression of genes related to the cell wall has been decreased or increased due to changes in ethylene production. However, the applied treatments also affect respiration rates, either decreasing or increasing. Our study argues that the main mechanism regulating cell wall enzymes is related to respiration rather than ethylene, and it presents strong arguments to support this hypothesis.

## 5 Conclusion

Although it is known that most of the cell wall-degrading genes depend on ethylene, in this study, tomato fruit with high respiration stored in environment with 1% O₂ continued to soften rapidly without ripening unlike genotypes with low respiration. Surprisingly, these cell wall-modifying genes were down-regulated in 1% O₂ environment, meaning the fruit continued to soften in the absence of ethylene. Our data show that although ethylene is effective in cell wall catabolism, it is not entirely responsible. This study proposes a new perspective for cell wall studies and is a milestone in this context. However, further studies should explore respiration-linked metabolic pathways that contribute to fruit softening under low O₂ conditions. Investigating energy metabolism and ATP production under low O₂ may help explain why fruit continue to soften in the absence of ethylene. Moreover, testing similar storage conditions in other climacteric fruits (e.g., banana, apple, pear) could determine whether the ethylene-independent softening mechanism is a common phenomenon. Overall, this study provided a new hypothesis for fruit softening, seeking to answer the question of whether respiration, independent of ethylene, plays a greater role in fruit softening than previously thought.

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s13580-0-25-00740-9.
**Acknowledgements** Financial support for this study was provided by The Scientific and Technological Research Council of Türkiye (TUBITAK) under project number 2190042. The authors also express their sincere gratitude to Emeritus Professor Graham Seymour (The University of Nottingham) for his critical review and contributions to the final stages of the manuscript.

**Author contributions** Hayri Ustun: Investigation, Data curation, Formal analysis, Methodology, Writing – original draft preparation. Selman Uluisik: Investigation, Formal analysis, Data curation, Writing – original draft preparation. Adem Dogan: Methodology, Resources, Visualization, Investigation. Yunus Ozyigit: Investigation. Mustafa Cetin: Investigation. Cansu Ural: Investigation. Mustafa Erkan: Writing – review & editing, Methodology, Supervision, Project administration, Funding acquisition.

**Funding** Open access funding provided by the Scientific and Technological Research Council of Türkiye (TÜBİTAK). This declaration is not applicable.

**Data availability** Data will be made available on request.

## Declarations

**Ethical approval** This declaration is not applicable.

**Conflict of interest** The authors declare that they have no conflict of interest.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

## References

Ali S, Khan AS, Malik AU, Anjum MA, Nawaz A, Shah HMS (2019) Modified atmosphere packaging delays enzymatic Browning and maintains quality of harvested litchi fruit during low temperature storage. Sci Hortic 254:14–20. https://doi.org/10.1016/j.scienta.2019.04.065

Ali Q, Kurubaş MS, Erkan M (2021) Comparison of ethylene sensitivity of three tomato cultivars from different tomato types and effects of ethylene on postharvest performance. J Agric Sci 27(4):476–483. https://doi.org/10.15832/ankutbd.715171

Alonso-Salinas R, López-Miranda S, Pérez-López AJ, Acosta-Motos JR (2024) Strategies to delay ethylene-mediated ripening in climacteric fruits: implications for shelf life extension and postharvest quality. Horticulturae 10(8):840. https://doi.org/10.3390/horticulturae10080840

Beaudry RM (2000) Responses of horticultural commodities to low oxygen: limits to the expanded use of modified atmosphere

packaging. HortTechnology 10:491–500. https://doi.org/10.2127/3/HORTTECH.10.3.491

Blankenship SM, Dole JM (2003) 1-Methylcyclopropene: a review. Postharvest Biol Technol 28(1):1–25. https://doi.org/10.1016/S0925-5214(02)00246-6

Blankenship SM, Richardson DG (1986) ACC and ethylene levels in ‘d’anjou’ Pears in air and controlled-atmosphere storage. HortScience 21(4):1020–1022. https://doi.org/10.21273/HORTSCI.21.4.1020

Brummell DA (2006) Cell wall disassembly in ripening fruit. Funct Plant Biol 33(2):103–119. https://doi.org/10.1071/FP05234

Brumos J (2021) Gene regulation in climacteric fruit ripening. Curr Opin Plant Biol 63:102042. https://doi.org/10.1016/j.pbi.2021.102042

Chang LY, Brecht JK (2023) Responses of 1-methylcyclopropene (1-MCP)-treated banana fruit to pre- and post-treatment ethylene exposure. Sci Hortic 309:111636. https://doi.org/10.1016/j.scienta.2022.111636

Chen H, Cao S, Fang X, Mu H, Yang H, Wang X, Xu Q, Gao H (2015) Changes in fruit firmness, cell wall composition and cell wall degrading enzymes in postharvest blueberries during storage. Sci Hortic 188:44–48. https://doi.org/10.1016/j.scienta.2015.03.018

Chen H, Bullock DA Jr, Alonso JM, Stepanova AN (2021) To fight or to grow: the balancing role of ethylene in plant abiotic stress responses. Plants 11(1):33. https://doi.org/10.3390/plants11010033

Cosgrove DJ (2016) Plant cell wall extensibility: connecting plant cell growth with cell wall structure, mechanics, and the action of wall-modifying enzymes. J Exp Bot 67(2):463–476. https://doi.org/10.1093/jxb/erv511

Dias C, Ribeiro T, Rodrigues AC, Ferrante A, Vasconcelos MW, Pintado M (2021) Improving the ripening process after 1-MCP application: implications and strategies. Trends Food Sci Technol 113:382–396. https://doi.org/10.1016/j.tifs.2021.05.012

Dogan A (2022) Effects of different oxygen levels with high-carbon dioxide atmosphere on postharvest quality of fresh Fig under pal-liflex storage systems. Horticulturae 8(5):353. https://doi.org/10.3390/horticulturae8050353

Fagundes C, Moraes K, Pérez-Gago MB, Palou L, Maraschin M, Monteiro AR (2015) Effect of active modified atmosphere and cold storage on the postharvest quality of Cherry tomatoes. Postharvest Biol Technol 109:73–81. https://doi.org/10.1016/j.postharvbio.2015.05.017

FAOSTAT (2024) http://www.fao.org/faostat/en/#data/QCL Accessed 15 Dec 2024

Gómez PA, Camelo AF (2002) Postharvest quality of tomato fruits stored under controlled atmospheres. Hortic Bras 20:38–43. https://doi.org/10.1590/S0102-05362002000100007

Guillén F, Valverde JM, Martinez-Romero D, Castillo S, Valero D, Serrano M (2004) Tomato fruit quality retention during storage by 1-MCP treatment as affected by cultivar and ripening stage at harvest. Acta Hortic 682:1069–1076. https://doi.org/10.17660/ActaHortic.2005.682.140

Hartman S, Liu Z, Van Veen H, Vicente J, Reinen E, Martopawiro S, Zhang H, Van Dongen N, Bosman F, Bassel GW, Visser EJ, Theodoulou FL, Hebelstrup KH, Gibbs DJ, Holdsworth MJ, Sasidharan R, Voesenek LACJ (2019) Ethylene-mediated nitric oxide depletion pre-adapts plants to hypoxia stress. Nat Commun 10(1):4020. https://doi.org/10.1038/s41467-019-12045-4

Islam A, Acıkalın R, Ozturk B, Aglar E, Kaiser C (2022) Combined effects of Aloe vera gel and modified atmosphere packaging treatments on fruit quality traits and bioactive compounds of jujube (Ziziphus Jujuba Mill.) fruit during cold storage and shelf life. Postharvest Biol Technol 187:111855. https://doi.org/10.1016/j.postharvbio.2022.111855
Johansson N, Quehl P, Norbeck J, Larsson C (2013) Identification of factors for improved ethylene production via the ethylene forming enzyme in Chemostat cultures of Saccharomyces cerevisiae. *Microb Cell Factories* 12:1–7. https://doi.org/10.1186/1475-2859-12-89

Klein B, Falk RB, Thewes FR, de Oliveira Anese R, dos Santos ID, Ribeiro SR, Donadel JZ, Brackmann A, Barin JS, Cichoski AJ, Wagner R (2020) Dynamic controlled atmosphere: effects on the chemical composition of cuticular wax of ‘cripps pink’ apples after long-term storage. *Postharvest Biol Technol* 164:111170. https://doi.org/10.1016/j.postharvbio.2020.111170

Klieber A, Ratanachinakorn B, Simons DH (1996) Effects of low oxygen and high carbon dioxide on tomato cultivar ‘bermuda’ fruit physiology and composition. *Sci Hortic* 65(4):251–261. https://doi.org/10.1016/0304-4238(96)00881-3

Lee YC, Chang CW, Hsu MC, Chung HY, Liang YS (2023) Effects of different concentrations of oxygen [blume]sed for storage on the postharvest physiology and quality of wax Apple (Syzygium Samarangense [Blume] merr. & LM Perry cv. pink). *Sci Hortic* 313:111906. https://doi.org/10.1016/j.scienta.2023.111906

Li P, Zhang B, Dhital S (2019) Starch digestion in intact pulse cells depends on the processing induced permeability of cell walls. *Carbohydr Polym* 225:115204. https://doi.org/10.1016/j.carbpol.2019.115204

Li C, Xin M, Li L, He X, Liu G, Li J, Sheng J, Sun J (2020a) Transcriptome profiling helps to elucidate the mechanisms of ripening and epidermal senescence in passion fruit (*Passiflora Edulia* Sims). *PLoS ONE* 15(9):e0236535. https://doi.org/10.1371/journal.pone.0236535

Li S, Zhu B, Pirrello J, Xu C, Zhang B, Bouzayen M, Chen K, Grierson D (2020b) Roles of RIN and ethylene in tomato fruit ripening and ripening-associated traits. *New Phytol* 226(2):460–475. https://doi.org/10.1111/nph.16362

Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2⁻^Δ^ΔCT method. *Methods* 25(4):402–408. https://doi.org/10.1006/meth.2001.1262

Nishiyama K, Guis M, Rose JK, Kubo Y, Bennett KA, Wangjin L, Kato K, Ushijima K, Nakano R, Inaba A, Bouzayen M, Latche A, Pech JC, Bennett AB (2007) Ethylene regulation of fruit softening and cell wall disassembly in Charentais melon. *J Exp Bot* 58(6):1281–1290. https://doi.org/10.1093/jxb/erl283

Nunes MCN, Morais AMMB, Brecht JK, Sargent SA (1996) Quality of Pink tomatoes (cv. Buffalo) after storage under controlled atmosphere at chilling and nonchilling temperatures. *J Food Qual* 19(5):363–374. https://doi.org/10.1111/j.1745-4557.1996.tb00431.x

Oliveira-Bouzas V, Pita-Calvo C, Vázquez-Odériz ML, Romero-Rodríguez MÁ (2021) Evaluation of a modified atmosphere packaging system in pallets to extend the shelf-life of the stored tomato at cooling temperature. *Food Chem* 364:130309. https://doi.org/10.1016/j.foodchem.2021.130309

Ortega-Salazar I, Crum D, Sbodio AO, Sugiyama Y, Adaskaveg A, Wang D, Seymour GB, Li X, Wang SC, Blanco-Ulate B (2023) Double CRISPR knockout of pectin degrading enzymes improves tomato shelf-life while ensuring fruit quality. *Plants People Planet* 6(2):330–340. https://doi.org/10.1002/ppp3.10445

Park MH, Sangwanangkul P, Choi JW (2018) Reduced chilling injury and delayed fruit ripening in tomatoes with modified atmosphere and humidity packaging. *Sci Hortic* 231:66–72. https://doi.org/10.1016/j.scienta.2017.12.021

Payasi A, Misra PC, Sanwal GG (2004) Effect of phytohormones on pectate lyase activity in ripening Musa acuminata. *Plant Physiol Biochem* 42(11):861–865. https://doi.org/10.1016/j.plaphy.2004.10.011

Posé S, Paniagua C, Matas AJ, Gunning AP, Morris VJ, Quesada MA, Mercado JA (2019) A nanostructural view of the cell wall disassembly process during fruit ripening and postharvest storage by atomic force microscopy. *Trends Food Sci Technol* 87:47–58. https://doi.org/10.1016/j.tifs.2018.02.011

Ratanachinakorn B, Klieber A, Simons DH (1997) Effect of short-term controlled atmospheres and maturity on ripening and eating quality of tomatoes. *Postharvest Biol Technol* 11(3):149–154. https://doi.org/10.1016/S0925-5214(97)00021-5

Sandhya (2010) Modified atmosphere packaging of fresh produce: current status and future needs. *LWT-Food Sci Technol* 43(3):381–392. https://doi.org/10.1016/j.lwt.2009.05.018

Sharma L, Ramesh KV, Paul V, Pandey R (2020) Ripening index: a better parameter for colour based assessment of ripening behaviour of tomato fruits. *Plant Physiol Rep* 25:171–177. https://doi.org/10.1007/s40502-020-00499-4

Sozzi GO, Trinchero GD, Fraschina AA (1999) Controlled-atmosphere storage of tomato fruit: low oxygen or elevated carbon dioxide levels alter galactosidase activity and inhibit exogenous ethylene action. *J Sci Food Agric* 79(8):1065–1070. https://doi.org/10.1002/(SICI)1097-0010;79:8%3C1065::AID-JSFA319%3E3.0.CO;2-U

Tao X, Wu Q, Li J, Cai L, Mao L, Luo Z, Li L, Ying T (2021) Exogenous Methyl jasmonate regulates sucrose metabolism in tomato during postharvest ripening. *Postharvest Biol Technol* 181:111639. https://doi.org/10.1016/j.postharvbio.2021.111639

Tatsuki M, Endo A, Ohkawa H (2007) Influence of time from harvest to 1-MCP treatment on Apple fruit quality and expression of genes for ethylene biosynthesis enzymes and ethylene receptors. *Postharvest Biol Technol* 43(1):28–35. https://doi.org/10.1016/j.postharvbio.2006.08.010

Tian MS, Bowen JH, Bauchot AD, Gong YP, Lallu N (1997) Recovery of ethylene biosynthesis in Diazocyclopentadiene (DACP)-treated tomato fruit. *Plant Growth Regul* 22:73–78. https://doi.org/10.1023/A:1005835115937

Tucker G, Yin X, Zhang A, Wang M, Zhu Q, Liu X, Xie X, Chen K, Grierson D (2017) Ethylene and fruit softening. *Food Qual Saf* 1(4):253–267. https://doi.org/10.1093/fqsafe/fyx024

Uluisik S, Chapman NH, Smith R, Poole M, Adams G, Gillis RB, Besong TM, Sheldon J, Stiegelmeyer S, Perez L, Samsulrizal N, Wang D, Fisk ID, Yang N, Baxter C, Rickett D, Fray R, Powell AL, Harding SE, Craigon J, Rose JKC, Fich EA, Sun L, Domozych DS, Fraser PD, Tucker GA, Grierson D, Seymour GB (2016) Genetic improvement of tomato by targeted control of fruit softening. *Nat Biotechnol* 34(9):950–952. https://doi.org/10.1038/nbt.3602

Ustun H, Dogan A, Peker B, Ural C, Cetin M, Ozyigit Y, Erkan M (2023) Determination of the relationship between respiration rate and ethylene production by fruit sizes of different tomato types. *J Sci Food Agric* 103(1):176–184. https://doi.org/10.1002/jsfa.12129

Üstün H (2024) Investigation of the sensitivity of different tomato types to postharvest low oxygen stress. *Dissertation, University of Akdeniz*

Wang TW, Arteca RN (1992) Effects of low O₂ root stress on ethylene biosynthesis in tomato plants (*Lycopersicon esculentum* mill Cv heinz 1350). *Plant Physiol* 98(1):97–100. https://doi.org/10.1104/pp.98.1.97

Wang D, Seymour GB (2022) Molecular and biochemical basis of softening in tomato. *Mol Hortic* 2(1):5. https://doi.org/10.1186/s43897-022-00026-z

Wang D, Yeats TH, Uluisik S, Rose JK, Seymour GB (2018) Fruit softening: revisiting the role of pectin. *Trends Plant Sci* 23(4):302–310. https://doi.org/10.1016/j.tplants.2018.01.006

Wang W, Wang Y, Chen T, Qin G, Tian S (2023) Current insights into posttranscriptional regulation of fleshy fruit ripening. *Plant*
Physiol 192(3):1785–1798. https://doi.org/10.1093/plphys/kiac483

Wei J, Ma F, Shi S, Qi X, Zhu X, Yuan J (2010) Changes and postharvest regulation of activity and gene expression of enzymes related to cell wall degradation in ripening Apple fruit. Postharvest Biol Technol 56(2):147–154. https://doi.org/10.1016/j.postharvbio.2009.12.003

Wendt LM, Soldateli FJ, Both V, Thewes FR, Ludwig V, Berghetti MRP, Ferrão TS, Wagner R, Brackmann A (2023) Effects of ethylene control and dynamic controlled atmosphere storage on ‘galaxy’ Apple quality. J Plant Growth Regul 42(2):701–718. https://doi.org/10.1007/s00344-022-10578-5

Yang Z, Zheng Y, Cao S, Tang S, Ma S, Li NA (2007) Effects of storage temperature on textural properties of Chinese bayberry fruit. J Texture Stud 38(1):166–177. https://doi.org/10.1111/j.1745-4603.2007.00092.x

Yang L, Huang W, Xiong F, Xian Z, Su D, Ren M, Li Z (2017) Silencing of Sl PL, which encodes a pectate lyase in tomato, confers enhanced fruit firmness, prolonged shelf-life and reduced

susceptibility to grey mould. Plant Biotechnol J 15(12):1544–1555. https://doi.org/10.1111/pbi.12737

Yip WK, Jiao XZ, Yang SF (1988) Dependence of in vivo ethylene production rate on 1-aminocyclopropane-1-carboxylic acid content and oxygen concentrations. Plant Physiol 88(3):553–558. https://doi.org/10.1104/pp.88.3.553

Zhen-guo L, Yu L, Jian-guo D, Rong-jiang X, Mei-zhen Z (1983) Effect of low oxygen and high carbon dioxide on the levels of ethylene and 1-aminocyclopropane-1-carboxylic acid in ripening Apple fruits. J Plant Growth Regul 2:81–87. https://doi.org/10.1007/BF02042236

Zhuang H, Barth MM, Cisneros-Zevallos L (2014) Modified atmosphere packaging for fresh fruits and vegetables. In: Han JH (ed) Innovations in food packaging. Academic, UK, pp 445–473. http://doi.org/10.1016/B978-0-12-394601-0.00018-7

**Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.