CO2-treated Fruit Research Articles

Structural Changes Caused by CO2 or Ethanol Deastringency Treatments in Cold-Stored ‘Giombo’ Persimmon

Persimmon cv. Giombo is astringent at harvest and must be subjected to astringency removal treatment. To date, the most widespread treatment for this variety involves applying ethanol instead of high CO2 concentrations, which is the usual treatment with other varieties. This study aims to evaluate the effect of high CO2 or ethanol concentrations as deastringency treatments on the quality and flesh structure of ’Giombo´ persimmon during cold storage. The deastringency process was faster in the fruit treated with CO2 than with ethanol. One day after treatment, the CO2-treated fruit showed lower soluble tannin levels than those detected sensorially for this variety, while with the ethanol-treated fruit, these values were obtained after 25 storage days plus the shelf-life period. The tannin insolubilisation process was observed by light microscopy. Loss of flesh firmness during storage was more pronounced when fruit were previously treated with ethanol than with CO2. This is closely related to greater parenchyma degradation during storage caused by ethanol treatment, which was observed by a microstructural study by cryo-scanning electron microscopy. Therefore, as deastringency treatment for ‘Giombo’, applying CO2 instead of ethanol treatment is recommended for better fruit quality, especially when fruit are to be cold-stored.

Characterization of browning during CO2 deastringency treatment in astringent persimmon fruit

Deastringency treatment with CO2 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-CO2 atmosphere, causing significant economic loss. However, the mechanisms of persimmon browning under CO2 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 CO2 treatment. An untargeted metabolomics analysis identified 19 polyhydroxyphenols that were downregulated in CO2-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 CO2 treatment and will contribute to the ongoing development of the persimmon fruit industry.

Effects of postharvest deastringency and 1-methylcyclopropene treatments on membrane permeability, membrane-degrading enzymes and their encoding genes in persimmon (Diospyros kaki, cv Mopanshi) fruit

Astringent persimmon fruit softens rapidly after harvest deastringency treatment. The purpose of this study was to understand the characteristics of membrane deterioration and its mechanism during rapid softening. The experiment included three treatments: (1) control, (2) CO2 treatment (≈100% CO2, 24 h), and (3) CO2 plus 1-methylcyclopropene (1-MCP, 1.5 μL∙L-1) treatment. CO2 treatment can effectively remove the astringency of persimmon fruit and cause a sharp drop in fruit firmness. The addition of 1-MCP was beneficial for firmness maintenance, but the process of astringency removal was delayed by one day. The fruit respiration rate and ethylene evolution rate were significantly increased by CO2 treatment, regardless of the presence or absence of 1-MCP, but the addition of 1-MCP could reduce the peak value and delay the occurrence of ethylene peaks by one day. The deastringency treatment resulted in a significant increase in membrane permeability and malondialdehyde content, and 1-MCP could significantly reduce membrane damage. CO2 treatment induced an enormous increase in LOX activity and the transcription of DkLOX3; however, 1-MCP significantly inhibited these increases. The PLD activity and DkPLDα1 expression level of the CO2-treated fruit were significantly promoted, and the addition of 1-MCP had less inhibition effect on the PLD activity and no significant inhibition on the DkPLDα1 expression. These results demonstrate that CO2 deastringency treatment could cause severe damage to the membrane integrity by promoting LOX and PLD activities and related gene expression. 1-MCP may ameliorate membrane damage mainly by inhibiting LOX activity and DkLOX3 gene expression and partly by suppressing PLDα1 expression.

Application of a new wax containing ethanol as a method to remove persimmon astringency during cold storage.

Nowadays the treatment based on applying high CO2 concentrations to fruit is the main method used in astringent persimmon prior to being commercialized, but it can cause quality problems for fruit during cold storage. The aim of this study was to evaluate the effectiveness of a recently patented astringency removal method based on applying a new wax whose formulation includes ethanol before commercial packaging. During two seasons, three treatments were evaluated in cv. Rojo Brillante and Triumph: (1) CO2- standard treatment; (2) waxed and packed in plastic film according to the patented method; (3) packed in plastic film without any treatment. During a third season, the new method's effectiveness in removing astringency was evaluated under industrial conditions. After treatments fruit was stored at 0°C for 15, 21 and 30days before being transferred at 20°C to simulate a 5-days shelf-life. All the fruit treated with the new wax completely lost astringency after 30days at 0°C, and commercial firmness was maintained. At the end of the storage, fruit quality was substantially higher in fruit submitted to the new treatment. CO2-treated fruit, manifested internal browning after 30 storage days and shelf-life, while this disorder was not detected in waxed fruit.

High Carbon Dioxide Treatment Modulates Sugar Metabolism and Maintains the Quality of Fresh-Cut Pear Fruit.

The purpose of this study is to explore the effect of 10% carbon dioxide (CO2) on the fruit quality and sugar metabolism of fresh-cut pear during storage. The results indicated that carbon dioxide treatment maintained fruit quality by delaying the decline of firmness and promoting the accumulation of total soluble solids (TSS). Moreover, carbon dioxide enhanced activities of sucrose synthase (SS), and sucrose phosphate synthase (SPS). The activities of amylase, acid invertase (AI), neutral invertase (NI), SS-cleavage, fructokinase (FK), hexokinase (HK), sorbitol oxidase (SOX), NAD-dependent sorbitol dehydrogenase (NAD-SDH), and NADP-SDH in CO2-treated fruit were inhibited. Expression levels of key genes were found to correspond with the related enzyme activities. As a result, the accumulation of glucose, fructose, sorbitol, and sucrose were accelerated by CO2, which were 12.58%, 13.86%, 24.7%, and 13.9% higher than those of the control at the end of storage, respectively. The results showed that CO2 could maintain the quality of fresh-cut pears by regulating the conversion of various sugar components to enhance soluble sugars content.

Delaying the biosynthesis of aromatic secondary metabolites in postharvest strawberry fruit exposed to elevated CO2 atmosphere

Aromatic secondary metabolites are closely related to quality attributes of postharvest fruit. In the present study, 20% CO2 was applied to strawberry fruit to investigate the regulation of elevated CO2 on aromatic secondary metabolites. The results showed that elevated CO2 delayed accumulations of anthocyanins, eugenol and lignin. Phenylalanine and tyrosine, the precursors of the above secondary metabolites, were 18.90% and 35.61%, respectively, lower in CO2-treated fruit compared with the control on day 6. Furthermore, enzyme activities and transcriptional profiles analysis showed pentose phosphate pathway and glycolysis were activated by elevated CO2 whereas the aromatic amino acids (AAAs) pathway was inhibited. These results indicated that elevated CO2 restricted carbon flux into aromatic secondary metabolism by inhibiting the AAAs pathway, leading to the decrease of phenylalanine and tyrosine, and thus, delayed the accumulation of aromatic secondary metabolites. In addition, the effect of elevated CO2 was eliminated after transferred CO2-treated fruit to air.

Effects of elevated CO2 on energy metabolism and γ-aminobutyric acid shunt pathway in postharvest strawberry fruit

The elevated CO2 was applied to the strawberry fruit during storage at 0 °C, in which its effects on energy metabolism and the γ-aminobutyric acid (GABA) shunt pathway were investigated. 10% and 20% CO2 maintained quality of the fruit. The energy charge of 10% and 20% CO2-treated fruit was decreased by 7.58% and 23.93% on day 12, respectively, compared with the control fruit, which was associated with the decline of NADH/NAD+. The GABA in 10% and 20% CO2-treated fruit was increased by 1.2-fold and 1.6-fold compared with the control fruit on day 12, respectively. The accumulation of GABA resulted from the decrease of GABA transaminase activity and gene expression, which may partially contribute to the decrease of NADH at later storage. These results indicated that 20% CO2 is an optimal concentration for strawberry fruit to prolong the storage to 12 days.

Short-term high CO 2 treatment reduces water loss and decay by modulating defense proteins and organic osmolytes in Cardinal table grape after cold storage and shelf-life

Abstract Shelf-life quality was improved when Cardinal table grapes (Vitis vinifera L.) were pretreated with 20 kPa of CO2 for three days at the beginning of a long-term cold storage. This pretreatment was effective in avoiding postharvest losses of cv. Cardinal grapes in terms of water loss, oxidative damage and disease prevention. To elucidate those physiological and biochemical factors involved in preserving the postharvest shelf-life quality of table grapes, we studied the expression pattern of defense proteins such as pathogenesis-related proteins (PRs) and dehydrins, as well as the profile of protective osmolytes. The efficacy of a short-term high CO2 pretreatment in reducing fungal disease could be mediated by the increase in the low molecular mass chitinase isoform of 16 kDa, which is up-regulated in the skin of CO2-treated grapes in parallel with the shelf-life fungal decay control. In addition, the increasing accumulation of a 22 kDa dehydrin isoform and the endogenous levels of organic osmolytes proline and glycine betaine in fruit stored at 20 °C (mainly in CO2-treated fruit) revealed that these protective biomolecules might play a more effective role in maintaining the structural and cellular homeostasis of table grapes after the shelf-life period, helping to reduce water loss and membrane oxidative damage (malondialdehyde accumulation) associated with the senescence-related disorders of postharvest table grapes.

The application of carbon dioxide and 1-methylcyclopropene to maintain fruit quality of ‘Niuxin’ persimmon during storage

Abstract The different sequential combined effect of carbon dioxide (CO2, 90%) and 1-methylcyclopropene (1-MCP, 0.5 μL L−1) on storage quality of persimmon fruit was investigated. In this study, the application of CO2 could induce the decrease of soluble tannin. But, the CO2-treated persimmon fruit soften rapidly, reducing their shelf life. Compared with CO2 treatment before cold storage, the treatment with CO2 deastringency after cold storage (air → CO2), CO2 deastringency followed by 1-MCP treatment before cold storage (CO2 → 1-MCP), and 1-MCP treatment followed cold storage before CO2 deastringency (1-MCP → CO2) were more effective in prolonging the storage time of persimmon fruit, especially the application of CO2 → 1-MCP, as indicated by higher firmness and soluble solids content, and the lower electrical conductivity and MDA content during most of the storage periods. Moreover, the treatment with CO2 → 1-MCP could induce the enhancement of antioxidant enzyme activities including superoxide dismutase, peroxidase and catalase. Meanwhile, the treatment with CO2 → 1-MCP could also maintain the lower lipoxygenase activity. These results suggest that the treatment of CO2 → 1-MCP may be an useful technique to decrease the soluble tannin content, maintain quality and prolong the storage time in postharvest persimmon fruit during storage.

Impact of Precooling and Controlled-Atmosphere Storage on γ-Aminobutyric Acid (GABA) Accumulation in Longan (Dimocarpus longan Lour.) Fruit.

Longan (Dimocarpus longan Lour.) fruit cultivars 'Chuliang' and 'Shixia' were analyzed for γ-aminobutyric acid (GABA) accumulation after precooling and in controlled-atmosphere storage. Fruit were exposed to 5% O2 plus 3%, 5%, or 10% CO2 at 4 °C, and GABA and associated enzymes, aril firmness, and pericarp color were measured. Aril softening and pericarp browning were delayed by 5% CO2 + 5% O2. GABA concentrations and glutamate decarboxylase (GAD; EC 4.1.1.15) activities declined during storage at the higher-CO2 treatments. However, GABA aminotransferase (GABA-T; EC 2.6.1.19) activities in elevated CO2-treated fruit fluctuated during storage. GABA concentrations increased after precooling treatments. GAD activity and GABA-T activity were different between cultivars after precooling. GABA concentrations in fruit increased after 3 days of 10% CO2 + 5% O2 treatment and then declined as storage time increased. GABA accumulation was associated with stimulation of GAD activity rather than inhibition of GABA-T activity.

Differential regulation of dehydrin expression and trehalose levels in Cardinal table grape skin by low temperature and high CO2

Dehydrins and trehalose are multifunctional protective biomolecules that play a role in counteracting cellular damage during dehydrative stresses. In this paper, we studied dehydrin isoform patterns, dehydrin gene expression and trehalose levels in the skin of Cardinal (Vitis vinifera L.) table grapes, along with their regulation by different cold postharvest storage conditions. Immunoanalysis with K-segment antibody recognizes four constitutive dehydrins (from 17 to 44kDa) that are tightly regulated by low temperature and high CO2. Phosphatase treatment showed that DHN44 and DHN22 isoforms are phosphorylated polypeptides, while MALDI-TOF MS and MS/MS analysis suggested that 44kDa polypeptide may be a dehydrin homodimer. At the transcriptional level, dehydrins are also regulated by low temperature and high CO2, showing a fairly good correlation with their mRNA levels. Trehalose was quantified by high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD), revealing a progressive increase of this metabolite throughout storage at 0°C and the sudden transitory increases in short-term high CO2-treated fruit. We propose that the constitutive presence and up-regulation of dehydrins and trehalose during low temperature postharvest storage could be positively correlated with the relative chilling tolerance of table grapes and the adaptive responses activated by high CO2 levels to preserve cell water status and to counteract the disruption of physiological processes during cold storage.

NADP-malic enzyme and glutathione reductase contribute to glutathione regeneration in Fragaria vesca fruit treated with protective high CO2 concentrations

Treatment of harvested strawberries (Fragaria vesca L. Mara des Bois) with high concentrations of CO2 is an effective means of limiting fungal decay and avoiding disorders caused by low temperature storage. In the present study, we investigated the role of NADP-ME gene expression and activity in lowering malic acid contents and in the provision of NADPH required for the regeneration of the reduced form of glutathione (GSH). We also measured glutathione reductase (GR: EC 1.6.4.2) activity in strawberries treated with different high CO2 concentrations (0, 20 and 40% for 3 days) during storage at 0°C. A decrease in malic acid content in fruit exposed to 20% CO2 was primarily mediated by the stimulation of NADP-ME activity, rather than associated with changes in the expression of cytosolic NADP-ME genes. Moreover, malic acid decarboxylation was associated with a marked increase in GR activity, which may account for the increased levels of glutathione in fruit following exposure to 20% CO2. This chain of events was not observed in untreated fruit stored in air or in fruit treated with 40% CO2, suggesting that the unique cellular redox status of 20% CO2-treated fruit plays an important role in detoxification and protection from damage during storage. Based on these findings, we propose that NADP-ME activation in fruit exposed to 20% CO2 provides NADPH for glutathione regeneration by GR, thereby conferring protection against the cellular damage caused by low temperatures or excessive high CO2 levels.

Increasing Catechin and Procyanindin Accumulation in High-CO2-Treated Fragaria vesca Strawberries

This paper deals with the impact of low temperature and high CO2 levels on flavonols, proanthocyanidins, and anthocyanins, synthesized via branched pathways from common precursors, in strawberries (Fragaria vesca L.). Flavonoids were identified with Q-TOF equipment and quantified by HPLC-quadrupole. Proanthocyanins B1 and B3 accumulated in CO2-treated strawberries, whereas in untreated (air) fruit, flavonoid production was redirected toward anthocyanin accumulation with a sharp decrease in catechin and procyanidin B3 levels. Moreover, in CO2-treated fruit, mainly in those with 20% CO2, anthocyanin accumulation did not decline. Due to its antifungal activity, catechin induction in CO2-treated strawberries could explain the capacity of high CO2 treatments to reduce fungal decay. Ascorbic acid content increased in 40% CO2-treated fruits, whereas in those treated with 20% CO2 an increase in flavonol content was observed. Despite these differences, similar antioxidant capacities were found in untreated and CO2-treated Mara de Bois strawberries.

Water distribution and ionic balance in response to high CO2 treatments in strawberries (Fragaria vesca L. cv. Mara de Bois)

The main organic (acids, amino acids and sugars) and inorganic (mono and divalent cations) solutes associated with changes in water status in response to low temperature and high CO2 levels were analyzed in untreated and 20% or 40% CO2 treated strawberries (Fragaria vesca L. cv. Mara de Bois) stored at 0°C. Intercellular water distribution and cellular tissue integrity were visualized using low temperature scanning electron microscopy (LT-SEM). The results indicated that high CO2 treatments prevented the weight loss and cell structure disorganization observed in untreated strawberries. However, there were differences in water loss regulation dependent on CO2 levels. In addition to mediating osmotic adjustment, treatment with 20% CO2 had a protective effect on cellular structure and prevented the movement of water into the intercellular spaces. Specifically, an accumulation in total sugars and proline were detected in 20% CO2-treated fruit. Moreover, water loss was controlled in these fruit and K+/Na+ homeostasis maintained similar to that found in freshly harvested fruit. By contrast, 40% CO2 controlled water loss, but the intercellular spaces were filled with aqueous solution, possibly as a result of a change in water status. Moreover, these changes were associated with an increase in free soluble Ca2+. In view of the opposite patterns of accumulation in malic, succinic acid, γ-aminobutyric acid (GABA) and glutamate found when comparing 20% and 40% CO2 treated strawberries, we suggest that their corresponding metabolic pathways play a regulatory role in CO2 tolerance.

A cryoprotective and cold-adapted 1,3-β-endoglucanase from cherimoya ( Annona cherimola) fruit

A 1,3-β-glucanase with potent cryoprotective activity was purified to homogeneity from the mesocarp of CO 2-treated cherimoya fruit ( Annona cherimola Mill.) stored at low temperature using anion exchange and chromatofocusing chromatography. This protein was characterized as a glycosylated endo-1,3-β-glucanase with a M r of 22.07 kDa and a p I of 5.25. The hydrolase was active and stable in a broad acidic pH range and it exhibited maximum activity at pH 5.0. It had a low optimum temperature of 35 °C and it retained 40% maximum activity at 5 °C. The purified 1,3-β-glucanase was relatively heat unstable and its activity declined progressively at temperatures above 50 °C. Kinetic studies revealed low k cat (3.10 ± 0.04 s −1) and K m (0.32 ± 0.03 mg ml −1) values, reflecting the intermediate efficiency of the protein in hydrolyzing laminarin. Moreover, a thermodynamic characterization revealed that the purified enzyme displayed a high k cat at both 37 and 5 °C, and a low E a (6.99 kJ mol −1) within this range of temperatures. In vitro functional studies indicated that the purified 1,3-β-glucanase had no inhibitory effects on Botrytis cinerea hyphal growth and no antifreeze activity, as determined by thermal hysteresis analysis using differential scanning calorimetry. However, a strong cryoprotective activity was observed against freeze–thaw inactivation of lactate dehydrogenase. Indeed, the PD 50 was 8.7 μg ml −1 (394 nM), 9.2-fold higher (3.1 on a molar basis) than that of the cryoprotective protein BSA. Together with the observed accumulation of glycine-betaine in CO 2-treated cherimoya tissues, these results suggest that 1,3-β-glucanase could be functionally implicated in low temperature-defense mechanism activated by CO 2.

Potent cryoprotective activity of cold and CO 2-regulated cherimoya ( Annona cherimola) endochitinase

A cryoprotective chitinase (BChi14) was isolated and purified from the mesocarp of CO 2-treated cherimoya fruit ( Annona cherimola Mill.) stored at chilling temperature by anion exchange and chromatofocusing chromatography. This hydrolase was characterized as an endochitinase with a M r of 14.31 kDa and a pI of 8.26, belonging to the family 19 of glycosyl hydrolases (GH19). While it was stable over a wide pH range and active in a broad acidic pH range, it had an optimum pH of 7.0. Its optimum temperature was low, 35 °C, and it retained about 30% of its maximum activity at 5 °C. Moreover, BChi14 was relatively heat unstable and its activity was progressively lost at temperatures above 50 °C. Kinetic studies revealed many similarities with other plant endochitinases. However, BChi14 had high k cat (6.93 s −1) value for the fluorogenic substrate 4-MU-(GlcNAc) 3, reflecting its great catalytic efficiency. Moreover, a thermodynamic characterization revealed that the purified enzyme displayed a high k cat at 37 and 5 °C, and a low E a (11.32 kJ mol −1). In vitro functional studies indicated that BChi14 had no effect on the inhibition of Botrytis cinerea hyphal growth and no antifreeze activity, as shown by the thermal hysteresis analysis using differential scanning calorimetry. However, the purified endochitinase showed very strong cryoprotective activity against freeze–thaw inactivation of lactate dehydrogenase. The PD 50 was 12.5 times higher than that of the cryoprotective protein BSA, and 2 or 3 orders of magnitude greater than sucrose, comparable with that of most cryoactive plant dehydrins. These results, together with the consolidated microstructure and the integrity of CO 2-treated mesocarp tissue, indicate that BChi14 is functionally implicated in the mechanisms underlying chilling tolerance activated by high CO 2 concentrations.

Anthocyanin, antioxidant activity and stress-induced gene expression in high CO 2-treated table grapes stored at low temperature

A pretreatment with 20kPa CO2+20 kPa O2+60 kPa N2 for 3 days proved effective in maintaining the fruit quality and controlling decay in table grapes (Vitis vinifera cv. Cardinal) stored at 0 degrees C. In the present work, we analyzed whether total anthocyanin content, the molecular mechanism implicated in their biosynthesis and antioxidant activity is related to the beneficial effect of this gaseous treatment. We isolated partial cDNAs that codified for enzymes implicated in the anthocyanin biosynthesis such as l-phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS), and an antioxidant enzyme such as ascorbate peroxidase (APX). Low temperatures induced an accumulation of total anthocyanin content in the skin of both treated and non-treated grapes, although levels were lower in CO2-treated fruit. By contrast, antioxidant activity decreased during storage at 0 degrees C in non-treated grapes but did not change in CO2-treated grapes. The up-regulation of anthocyanin biosynthesis gene expression and VcAPX mRNA observed in non-treated grape is not enhanced in CO2-treated grapes, which presented low total decay. These results point out the ability of CO2-treated grapes to prevent the generation of reactive oxygen species rather than their inactivation by means of induction of studied defense systems.

Antioxidant enzyme activities in strawberry fruit exposed to high carbon dioxide atmospheres during cold storage

The effects of storage, in air or a 20% CO 2 in air (16.8% O 2) atmosphere for 12 d at 2 °C, on antioxidant enzymes of strawberry fruit ( Fragaria x ananassa Duch. cv. ‘Jewel’) were investigated. The concentrations of acetaldehyde, ethanol and ethyl acetate associated with fermentation were measured, and the activities of peroxidase (POX), catalase (CAT) and superoxide dismutase (SOD) assayed. Ethanol, acetaldehyde and ethyl acetate concentrations increased in CO 2-treated fruit within a day of treatment, but more extensively after four days, while concentrations remained low in air-stored fruit. The total protein content extracted was not affected by CO 2 or storage time. Activities of POX were similar in air- and CO 2-treated fruit, with an increase occurring only in air-treated fruit on day-12. Neither CAT nor SOD activities were affected by CO 2 treatment. In summary, a 20% CO 2 storage treatment induced fermentation but did not significantly affect total antioxidant enzyme activities.

Fruit Quality, Fermentation Products, and Activities of Associated Enzymes During Elevated CO2 Treatment of Strawberry Fruit at High and Low Temperatures

The effects of postharvest treatments of air and 20 kPa CO2 (in air) at 2 or 20 °C on color, firmness, accumulations of acetaldehyde, ethanol, and ethyl acetate, activities of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) activity, and expression of an ADH gene were studied in strawberry fruit (Fragaria ×ananassa Duch. cv. Jewel). CO2 treatment enhanced strawberry fruit firmness at 2 °C but not 20 °C, while the rate of color changes was affected by CO2 treatment at 20 °C but not at 2 °C. Temperature also affected the accumulation of acetaldehyde, ethanol and ethyl acetate in CO2-treated fruit. All three compounds accumulated in fruits at 2 °C. At 20 °C, ethanol accumulated slightly by day 6, although ethyl acetate accumulated in fruit from both atmospheres. PDC enzyme activity was higher in CO2-treated fruit than their air-treated control at 2 °C but not at 20 °C. ADH activity and ADH mRNA accumulation of the CO2-treated berries were higher than in air at 20 °C but not 2 °C. The results, overall, indicate that patterns of change among gene expression, enzyme activities, and fermentation product accumulation were not consistent.

Malate Metabolism and Adaptation to Chilling Temperature Storage by Pretreatment with High CO2 Levels in Annona cherimola Fruit

In this study we focused on the effect of a pretreatment with high (20%) CO2 levels on malic acid metabolism in cherimoya (Annona cherimola Mill) fruit stored at chilling temperature. We analyzed the activity of phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), and the NADP-malic enzyme (NADP-ME), involved in the carboxylation/decarboxylation of malate. Our results show that CO2 treatment, which improves tolerance to prolonged storage at chilling temperature, was closely linked to considerably greater NADP-ME activity. These results, combined with lower PEPC activity, may explain the significantly lower amount of malic acid and titratable acidity quantified in CO2-treated fruit. Moreover, the high cytoplasmic MDH enzyme activity and the strong stimulation of NADP-ME activity exhibited by CO2-treated fruit could be contributing factors in the maintenance of fruit energy metabolism, pH stability, and the promotion of synthesis of defense compounds that prevent or repair damage caused by chilling temperature.