Characterization of browning during CO 2 deastringency treatment in astringent persimmon fruit

Deastringency treatment with CO 2 is an effective and convenient method for improving the marketability of persimmon fruit. However, the main persimmon cultivars in China turn brown very quickly following exposure to a high-CO 2 atmosphere, causing significant economic loss. However, the mechanisms of persimmon browning under CO 2 treatment remain largely unknown. In this study, we evaluated components and enzymes related to persimmon fruit browning. The results revealed that astringency was alleviated by simultaneous reduction of soluble tannin content and accumulation of insoluble tannin. During persimmon browning, fruit firmness, total phenolic content, and phenylalanine ammonia lyase (PAL) activity decreased significantly, whereas malondialdehyde (MDA), superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and polyphenol oxidase (PPO) activity increased significantly, suggesting that reactive oxygen species (ROS)-scavenging enzymes and non-enzymatic antioxidants cannot effectively maintain redox reaction balance to protect cell membranes from oxidative damage during CO 2 treatment. An untargeted metabolomics analysis identified 19 polyhydroxyphenols that were downregulated in CO 2 -treated fruit, suggesting that phenolics may act as a substrate for persimmon browning. We also identified 11 metabolites associated with abiotic stress. Together, these results study provide valuable information on the mechanism of persimmon fruit browning induced by CO 2 treatment and will contribute to the ongoing development of the persimmon fruit industry.


Introduction
Persimmon (Diospyros kaki Thunb.: Ebenaceae) is a deciduous plant that is well adapted to tropical, subtropical, and temperate areas. As a food source and an alternative to the predominant fruit crops, persimmon is one of the most important fruit crops with high commercial value in Asian countries [1]. In pomology, persimmon cultivars are classified into four types: pollination-variant and astringent, pollination-variant and non-astringent, pollination-constant and astringent, pollination-constant and non-astringent [2]. Persimmon fruit are believed to have originated in China [3], and approximately 1,000 varieties of D. kaki have been found in China thus far. Interestingly, besides a few PCNA germplasms found in Luotian County, Hubei Province, the rest of the varieties in China are considered the PCA type [4]. In addition, astringent persimmon is the largest cultivated species of persimmon in the world because of its high yield, strong resistance, large fruit, and smooth flesh.
According to the Food and Agriculture Organization of the United Nations, the fruit production of persimmon in China was approximately 4,216,400 tons in 2018, accounting for 73.32% of total production worldwide. The majority of persimmon production is PCA cultivars, which 1 3 are usually harvested for the fresh-fruit market when the fruit are still astringent [5]. It is necessary to remove the astringency of these fruit to make them palatable before consumption. Traditional methods to remove astringency at harvest involve over-ripening by treating the fruit with ethephon or ethylene, which is usually accompanied by a dramatic loss of fruit firmness. This handling limitation shortens the postharvest life of the fruit [6]. Thus, it is necessary and urgent to explore a novel and effective method to overcome the deficiencies of the traditional deastringency treatment in persimmon fruit.
Compared to traditional methods, CO 2 treatment is the most widely used deastringency technology due to its efficiency and convenience when preserving high levels of fruit firmness [7,8]. The effectiveness of this treatment is based on its ability to insolubilize soluble tannins through the mediation of acetaldehyde generated during anaerobic respiration, which is triggered when fruit are exposed to a high-CO 2 atmosphere [9,10]. However, our previous studies showed that most main persimmon cultivars in China, especially those with good flavor, such as 'Mopan', 'Huojing', 'Zhongshi No. 1', and 'Gongcheng', were susceptible and turned brown very easily after CO 2 -deastringency treatment. The browning rate reached more than 50% when 100 astringent persimmon cultivars were treated under the optimal CO 2 deastringent conditions. Fruit browning is a physiological disorder that occurs in the fruit of many species such as apple, pear, pineapple, lychee, and persimmon [11][12][13][14][15]; it is characterized by darkened tissues near the fruit surface [13]. Browning significantly affects the economic value of the fruit and greatly limits the application of CO 2 deastringency technology in China.
Fruit and vegetable browning can be divided mainly into two categories: non-enzymatic and enzymatic browning. Non-enzymatic browning is caused by various nonenzymatic chemical reactions, such as auto-oxidation and polymerization of polyphenols that produce brown tissues. Enzymatic browning is caused by the oxidation and polymerization of phenolic compounds produced by the catalysis of enzymes such as phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), and peroxidase (POD) [16,17]. Although the effects of fresh-cut processing, mechanical damage, and hot water treatment have been well studied in persimmon fruit [5,15,18], the mechanisms of browning during CO 2 deastringency treatment have not yet been reported.
In this study, we evaluated the browning-related components and enzymes of the cultivar 'Zhongshi No. 1', which was found to be extremely susceptible to browning in our previous study. The results will provide valuable information on the mechanism of browning during CO 2 deastringency treatment.

Plant materials
The persimmon cultivar 'Zhongshi No. 1' was harvested at the commercial maturity stage on October 8, 2018, in Mengzhou County (34°51′38″N, 112°42′58″E), Henan Province, China. We randomly collected 40 fruits from six healthy 10-year-old persimmon trees, with three biological replicates per sample. After harvest, each replicate was transported to the laboratory, and the samples were divided into control (CK) and CO 2 -deastringency treatment groups. Fruit in the control group were stored at 25 °C without treatment for 48 h. Fruit in the CO 2 treatment group were placed in sealed containers containing 99.9999% food-grade CO 2 at 25℃ and 90% relative humidity for 24 h, subjected to deastringency treatment, and then maintained at 25 °C for another 24 h. Both the control and treated fruit were then peeled after fruit firmness measurement, and the flesh was immediately frozen in liquid nitrogen and stored at -80 °C until further analysis.

Determination of firmness and ascorbic acid, total phenolic, total flavonoid, tannin, and malondialdehyde (MDA) content
Before the fruit were peeled, sample firmness was measured using a firmness tester (GY-4; Zhejiang Tuopu, Ningbo, China) with a 8 mm plunger; the results are expressed as pressure (N). Ascorbic acid content was determined using high-performance liquid chromatography (HPLC) [19,20]. Total phenolic content was determined according to the Folin-Ciocalteu method of Li et al. [21]. Total flavonoid content was measured using the AlCl 3 (HAc-NaAc) method according to Fan [22]. Soluble and insoluble tannin content was measured using the Folin-Ciocalteu method, as described by Oshida et al. [23]. Malondialdehyde (MDA) content was measured using the thiobarbituric acid method, as described by Cao et al. [24].
PPO activity was monitored as previously described [24,25], with slight modifications. The reaction solution consisted of a mixture of 4.0 mL sodium acetate buffer (50 mmol/L, pH 5.5) and 1.0 mL 4-methyl-catechol (50 mmol/L), and was incubated at 25 °C for 5 min. Then, 0.1 mL of the extracted enzyme was added to the reaction system, and the absorbance at 420 nm was measured. The results are expressed as U/g, where 1 U was defined as the amount of enzyme activity required to cause an absorbance change per 1 g of sample under assay conditions. POD activity was monitored according to Cao et al. [24], with a slight modification. The POD extract (0.5 mL) was mixed with 3 mL of 25 mmol/L guaiacol solution. Then, 0.2 mL H 2 O 2 (0.5 mol/L) was added to the reaction system and the absorbance at 470 nm was measured. The results are expressed as U/g. SOD activity was assayed according to Kumar et al. [26], with some modifications. The reaction mixture consisted of 1.2 mL sodium phosphate buffer (50 mmol/L, pH 7.8), 0.3 mL MET (0.13 mol/L), 0.3 mL EDTA (0.1 mmol/L), 0.3 mL nitroblue tetrazolium (NBT; 0.75 mmol/L), 0.3 mL riboflavin (0.02 mmol/L), and 0.5 mL of the SOD extract. After exposure to white light for 15 min at 30 °C, the absorbance of the reaction mixture at 560 nm was measured. The results are expressed as U/g.
CAT activity was determined as the decrease in absorbance at 240 nm for 1 min following the decomposition of H 2 O 2 , in accordance with Duan et al. [27], with some modifications. The reaction mixture consisted of 2.5 mL of 50 mmol/L phosphate buffer (pH 7.0) containing 15 mmol/L H 2 O 2 and 0.5 mL of CAT extract. The results are expressed as U/g. PAL activity was determined as described previously [24,28]. Briefly, 3 mL of 0.1 mol/L boric acid-borax buffer (pH 8.8), 500 µL of 20 mol/L L-phenylalanine, and 500 µL of extracted enzyme were incubated at 37 °C for 60 min. The reaction was terminated by the addition of 0.1 mL of 6 mol/L HCl, and the absorbance of the reaction mixture at 290 nm was measured. The results are expressed as U/g.

Extraction parameter settings for untargeted metabolomic analysis
A portion (100 mg) of each collected sample was extracted with 120 µL of precooled 50% methanol, vortexed for 1 min, and incubated at room temperature for 10 min. The extraction mixture was then stored overnight at − 20 °C. After centrifugation at 4000×g for 20 min, the supernatants were transferred into new 96-well plates. The samples were stored at − 80 °C prior to liquid chromatography-mass spectrometry (LC-MS) analysis. In addition, pooled quality control (QC) samples were prepared by combining 10 µL of each extraction mixture.
A high-resolution tandem mass spectrometer TripleTOF-5600plus (SCIEX) was used to detect metabolites in both positive and negative ion modes. The QC sample was used to evaluate the stability of the LC-MS system throughout data acquisition.

Data preprocessing and pretreatment
LC-MS raw data files were converted into mzXML format after the pretreatments using XCMS software and then processed by the XCMS, CAMERA and metaX toolbox implemented with the R software [29][30][31]. Each ion was identified by combining retention time (RT) and m/z data. The online KEGG and HMDB databases were used to annotate the metabolites. An in-house fragment spectrum library of metabolites was used to validate the metabolite identification.
The pre-processed dataset was used to perform principal component analysis (PCA) for outlier detection and batch effects evaluation. Supervised PLSDA was conducted to discriminate the different variables between groups through metaX, and the VIP value was calculated. Student's t-tests were conducted to detect differences in metabolite concentrations between two phenotypes, adjusting P for multiple tests according to the false discovery rate, as determined by the Benjamini-Hochberg test.

Statistical analyses
All experiments were performed using an absolutely randomized design and the results are expressed on a fresh weight basis. All data were analyzed using the SPSS v23.0 software (SPSS Inc., Chicago, IL, USA). Duncan's multiple comparisons test was used to test for significant differences. Significance was evaluated at a level of P < 0.05.

Physiological parameters related to browning caused by CO 2 deastringency treatment
The phenotype of CO 2 -treated persimmon fruit is shown in Fig. 1. Visible browning on the fruit surface demonstrated that 'Zhongshi No. 1' was susceptible to browning due to CO 2 deastringency treatment. Fruit firmness was also affected by CO 2 treatment ( Fig. 2A), with significant differences detected between the control (74.14 ± 5.88 N) and treated (11.77 ± 2.60 N) fruit.
Tannins are substrates in enzymatic browning reactions with many biological activities for fruit and vegetable quality, and they are closely associated with astringency and enzymatic activity [32][33][34][35]. Soluble tannin content decreased from 14.31 ± 0.06 to 1.19 ± 0.02 g/ kg after deastringency treatment (Fig. 2B, C), perhaps because soluble tannins were accumulated and converted to insoluble tannins, followed by acetaldehyde generation within the fruit during CO 2 deastringency treatment [10,36]. These results also demonstrate that the CO 2 deastringency treatment used in this study was effective, as the threshold soluble tannin content value for astringency is 2 g/kg [37]. The insoluble tannin contents of the control and treated samples were 1.64 ± 0.13 and 9.22 ± 0.22 g/  Phenolics are substrates of enzymatic browning reactions; PAL is involved in the biosynthetic pathway of phenolic compounds and may accelerate browning [16,38,39]. In this study, the total phenolic content showed a downward trend after CO 2 treatment, from 13.19 ± 0.90 to 7.24 ± 0.047 g/kg (Fig. 2D), and PAL activity decreased significantly from 175.33 ± 4.03 U/g in the control group to 136.83 ± 5.06 U/g in the treatment group fruit (Fig. 2E).
In normal cell tissues, PPO is located in the organelles and polyphenol compounds are located in vacuoles; decompartmentalization of the intracellular membrane plays an important role in the browning process. PPO activity was 3.94 ± 0.33 U/g in the control group and 22.22 ± 0.14 U/g in the treatment group (Fig. 2F). MDA is an indicator of cell membrane integrity [40]. In this study, MDA content increased from 3.08 ± 0.38 umol/kg in the control group to 6.09 ± 0.41 umol/kg in the treatment group (Fig. 2G), suggesting that browning caused by CO 2 deastringency treatment damaged cell membrane structure and intracellular membrane decompartmentalization, leading to the reaction of phenolic substrates and PPO. CO 2 deastringency treatment affects oxidative stress in fruit, and has been reported to rapidly increase fruit ROS levels [41]. Effective ROS destruction requires the participation of several ROS-scavenging enzymes, such as SOD, POD, and CAT [14], as well as non-enzymatic antioxidants such as ascorbic acid and flavonoids [41]. In this study, CO 2 deastringency treatment induced significant increases in persimmon fruit SOD (1.12 ± 0.024 to 2.91 ± 0.074 U/g), POD (0.49 ± 0.011 to 22.79 ± 0.91 U/g), and CAT (1.33 ± 0.05 to 53.00 ± 0.99 U/g) activity (Fig. 2H, I, J). Ascorbic acid and flavonoid levels were significantly increased following CO 2 deastringency treatment, by approximately 1.93-and 1.28-fold, respectively (Fig. 2K, L).

Metabolite identification
To explore the changes in metabolites after browning due to CO 2 treatment, we performed untargeted metabolomic analysis in both positive (ESI + ) and negative (ESI − ) modes. PCA was performed to evaluate the repeatability and stability of the metabolic profiles obtained by UPLC-MS, and to detect differences among samples. Both positive and negative electron spray ionization (ESI) models clearly divided the samples into two groups, and grouped samples from the same treatment together, demonstrating the high quality of the data (Fig. 3). This result indicates that CO 2 deastringency treatment had a significant effect on metabolite variation.
Partial least-squares discriminant analysis (PLSDA) clearly distinguished between the control and treatment groups (Fig. 4). No significant outliers were found according to the PLSDA scores, suggesting that the samples were of high quality. The R 2 Y and Q 2 Y data supported the predictive accuracy of the model and reliability of the data.
We obtained 12,009 and 8,012 ion features using the positive and negative models, respectively; among these, 8271 and 4205 were annotated. The important metabolites were identified based on thresholds of the variable importance in projection score (VIP > 1.0), fold change (FC > 2.0 or FC < 0.5), and P < 0.05. A total of 689 and 1573 metabolites were upregulated and downregulated, respectively, in positive mode, and 588 and 1,182 metabolites were upregulated and downregulated, respectively, in  (Table 1). Moreover, 30 metabolites were found to be associated with abiotic stress and phenolics ( Table 2). Browning is caused by the enzymatic oxidation of phenolic compounds by PPO to o-quinones, which are very reactive, forming brown polymers [12]. The levels of 19 polyhydroxyphenols including manniflavanone, quercitrin, procyanidin_B4, proanthocyanidin_A2, and sciadopitysin, are listed in Table 2. Polyhydroxyphenols clearly decreased following treatment; these results were consistent with those for total phenolics (Fig. 2D); enzymatic reaction of phenolic substances produce substrates with PPO, which were significantly more abundant in the treatment group. We also identified 11 compounds associated with abiotic stress: valine, tyrosine, lysine, succinic acid, phenylalanine, trehalose, allose, D-glucose, mannitol, D-sorbitol, and spermidine.

Discussion
When persimmon fruit were exposed to an atmosphere with high CO 2 and low O 2 concentrations, the local anoxic conditions caused oxidative stress and changes in normal cellular metabolism, impairing fruit quality and leading to browning [6,41,42]. Browning induced by CO 2 deastringency treatment of 'Zhongshi No. 1' fruit is a physiological disorder of the internal flesh that is visible at the fruit surface, significantly impacting the economic value of the fruit. SOD, POD, and CAT are ROS-scavenging enzymes in plants. SOD constitutes the first enzymatic line of defense against ROS, catalyzing the dismutation of O 2 − to non-toxic O 2 and less toxic H 2 O 2 , whereas POD and CAT remove H 2 O 2 . Ascorbic acid and flavonoids are potent and versatile antioxidants in plants, effectively quenching free radicals and protecting cell membranes and intracellular proteins from oxidative damage [43,44]. In this study, persimmon fruit SOD, POD, and CAT activity rapidly increased following CO 2 treatment. CO 2 deastringency treatment increased SOD activity by 2.60-fold compared with the control; a similar study reported higher SOD activity in the 'Rojo Brillante' persimmon cultivar following CO 2 deastringency treatment [42]. Under oxidative stress conditions, the contents of ascorbic acid and flavonoids, which function as antioxidants in persimmon fruit, increase to maintain the redox reaction balance within the fruit.
MDA is the main product of membrane lipid peroxidation; its accumulation level can be used as an indicator of tissue cell damage and physiological aging [40]. In this study, the MDA content of browning fruit increased following treatment with high concentrations of CO 2 , enhancing membrane lipid peroxidation. PPO is found in most higher plants and plays a central role in fruit browning [45]; the levels of total phenolics and 19 phenolic compounds decreased significantly as those of PPO activity significantly increased in treated fruit, suggesting that browning damages cell membrane structure and of intracellular membrane decompartmentalization, leading to the reaction of phenolic substrates and PPO. These results suggest that ROS-scavenging enzymes and non-enzymatic antioxidants in 'Zhongshi No. 1' fruit could not effectively maintain the redox reaction balance to protect cell membranes and intracellular proteins from oxidative damage during CO 2 deastringency treatment. Plant metabolomics examines the role of metabolite change in various tissues [46]. As metabolites are the final products of cell biological regulation process, metabolite changes and their levels are regarded as the ultimate response of plants to environmental stresses [47]. Phenolics act as substrates of enzymatic browning reactions; in this study, the levels of total phenolics and 19 phenolic compounds decreased significantly in the treated samples, suggesting that phenolics similarly acted as a substrate for browning in 'Zhongshi No. 1' persimmon fruit.
Many amino acids also play significant roles in plant growth and various stress responses, including as regulatory and signaling molecules [48,49]. Several studies have demonstrated that amino acids accumulate to high levels under abiotic stress [50][51][52][53]. In this study, the levels of valine, tyrosine, lysine, succinic acid, and phenylalanine were increased under high concentration CO 2 ; a possible reason for this is that high-concentration CO 2 could induce the synthesis of amino acids and the degradation of proteins in persimmon fruit. In addition, the results also indicated that soluble sugars such as trehalose, allose, and D-glucose, which accumulate under stress responses caused by abiotic stress, may also contribute to sustaining osmotic pressure and protecting the plasma membrane and proteins, as previously reported [53][54][55]. Polyamines belonging to the aliphatic series can also protect plant cell membranes and reduce oxidative damage of plant cells [56]. Sugar alcohols have been reported to sustain the stability of macromolecules, scavenge ROS in cells, and prevent peroxidation of fatty acids in cell membranes [57]. In this study, the levels of spermidine, mannitol, and D-sorbitol were decreased in CO 2 -treated 'Zhongshi No. 1'fruit, which may have led to browning.

Conclusions
High CO 2 concentrations induced oxidative stress, and damaged cell membrane structure and intracellular membrane decompartmentalization, leading to reactions among phenolic substrates and PPO. ROS scavenging enzymes (SOD, POD, and CAT) and non-enzymatic antioxidants (ascorbic acid and flavonoids) were unable to effectively maintain redox reaction balance and protect cell membranes from oxidative damage during CO 2 treatment. We identified 19 polyhydroxyphenols that showed decreased content in treated fruit, suggesting that phenolics act as a substrate in the enzymatic browning reaction of 'Zhongshi No. 1' persimmon fruit. We also identified 11 metabolites associated with abiotic stress. The findings of this study provide valuable information on the mechanism of browning under CO 2 deastringency treatment, and will contribute to the ongoing development of the persimmon fruit industry.
Funding This work was supported by the Fundamental Research Funds for the Central Non-profit Research Institution of CAF in China (CAFYBB2019MA005), and Scientific and technological project of Henan province, (182102110076).