Effects of chlorine dioxide fumigation on redox balancing potential of antioxidative ascorbate-glutathione cycle in ‘Daw’ longan fruit during storage

Scientia Horticulturae 222 (2017) 76–83

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Research Paper

Effects of chlorine dioxide fumigation on redox balancing potential of antioxidative ascorbate-glutathione cycle in ‘Daw’ longan fruit during storage

MARK

Athiwat Chumyama, Lalida Shankb, Bualuang Faiyuec, Jamnong Uthaibutraa,d, ⁎ Kobkiat Saengnila,d, a

Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand Department of Biology, Mahidol Wittayanusorn School, Salaya, Phuttamonthon, Nakhon Pathom 73170, Thailand d Postharvest Technology Research Institute, Chiang Mai University/Postharvest Technology Innovation Center, Commission on Higher Education, Bangkok 10140, Thailand b c

A R T I C L E I N F O

A B S T R A C T

Keywords: Redox status Ascorbate Glutathione NADPH Chlorine dioxide Dimocarpus longan

Redox status in ascorbate-glutathione (ASA-GSH) cycle plays a pivotal role in plant responses to physiological and environmental changes. The change of ASA-GSH redox potential is associate with oxidative stress leading to plant senescence. The present study was aimed to investigate the effects of gaseous ClO2 on the redox status involved in delaying fruit senescence during storage of harvested longan fruit (Dimocarpus longan Lour. cv. Daw). Fresh longan fruit were fumigated with 10 mg/L ClO2 for 10 min, packed into cardboard boxes and stored at room temperature for 7 d. The results show that redox status of the fruit determined by ASA/DHA, GSH/GSSG and NADPH/NADP ratios decreased steadily in ClO2 untreated control group during storage. On the contrary, those ratios increased immediately after ClO2 treatment before declining steadily afterwards. The activities of NADPH-generating dehydrogenases including glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, and ASA-GSH cycle enzymes including ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase were upregulated upon the ClO2 treatment and remained stable for a few days. ClO2 treatment decreased hydrogen peroxide content and fruit senescence index, including browning and disease symptoms in pericarp during storage. It was concluded that the imbalance of redox status of ASA-GSH cascade involved senescence in ‘Daw’ longan, and ClO2 could maintain the redox balance, leading to the alleviation and delay fruit senescence.

1. Introduction ‘Daw’ longan is an important economic fruit in Thailand which is exported to the international markets more than 400,000 t with a value of $270 million per year (Chanrittisen and Chomsri, 2010). However, longan fruit undergo rapid pericarp browning, a symptom of fruit senescence, and develop disease after a few days at room temperature, resulting in a short storage life and reducing market value, as pericarp appearance is a key factor determining consumer selection (Jiang et al., 2002; Saengnil et al., 2014). Pericarp browning is caused by the oxidation of phenols to quinones, which are then polymerized to form brown pigments (Jiang et al., 2002). Moreover, longan fruit are susceptible to postharvest pathogens. High sugar and moisture induce microorganisms to rot the fruit rapidly, which also causes severe

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browning (Apai, 2010). Consequently, prevention of senescence including pericarp browning and fruit decay is important for improving fruit marketing for longan. Saengnil et al. (2014) reported that applications of gaseous chlorine dioxide (ClO2) at 10 mg/L for 10 min on longan fruit before storage at room temperature significantly reduced pericarp browning and prolonged shelf life by reducing the oxidation of phenolic compound, delaying the occurrence of disease and maintaining higher fruit quality. Similarly, ClO2 treatment of longan fruit reduced pericarp browning, enhanced the antioxidant defense system and reduced ROS and oxidative damage (Chomkitichai et al., 2014a, 2014b). Fruit senescence involved the production and accumulation of reactive oxygen species (ROS) which cause detrimental effects through oxidative damage of macromolecules especially lipids, proteins and nucleic acids (Mittler, 2002; Chomkitichai et al., 2014a). However,

Corresponding author at: Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand. E-mail address: [email protected] (K. Saengnil).

http://dx.doi.org/10.1016/j.scienta.2017.05.022 Received 21 February 2017; Received in revised form 2 May 2017; Accepted 6 May 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.

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2.2. Determination of ASA and DHA contents

plants have evolved free radical scavenging systems to minimize the accumulation of ROS and repair oxidative damage. The ascorbateglutathione (ASA-GSH) cycle is one of those scavenging systems (Foyer and Noctor, 2011). Four antioxidative enzymes in ASA-GSH cycle including ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR), interplay with ascorbate (ASA) and glutathione (GSH) to dissipate hydrogen peroxide (H2O2) or other ROS (Noctor and Foyer, 1998). The ASA-GSH cycle controlled by ASA/dehydroascorbate (DHA) and GSH/glutathione disulfide (GSSG) ratios, is related to senescence in some horticultural crops (Potters et al., 2010). For example, the decrease in ASA/DHA and GSH/GSSG ratios increased senescence of broccoli during storage at 20 °C (Mori et al., 2009). In loquat fruit, decreased ASA/DHA and GSH/GSSG ratios were related to decreased activities of APX, MDHAR, DHAR and GR, while browning was found to increase during cold storage (Cai et al., 2011). In ASA-GSH cycle, there is a complex relationship between ASA, GSH and reduced nicotinamide adenine dinucleotide phosphate (NADPH) which is regenerated from its oxidized form (oxidized nicotinamide adenine dinucleotide phosphate, NADP) by a group of NADPH-generating dehydrogenases such as glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) (Corpas and Barroso, 2014; Manai et al., 2014). The redox status of NADPH indicated by NADPH/NADP ratio, and activities of G6PDH and 6PGDH are affected by stresses. For example, NADPH redox balance in bean seedlings was declined during sulphur-deficient stress, while the decrease in G6PDH has been reported in rice under salt stress (Juszczuk and Ostaszewska, 2011; Zhang et al., 2013). Similarly, salt stress was shown to induce the decrease in NADPH/NADP ratio, G6PDH and 6PGDH activities in roots of tomato which coincided with a decrease in the growth of seedlings (Manai et al., 2014). In order to delay or reduce senescence by mean of maintaining the redox status balance, various chemical methods have been employed on harvested fruit. For example, Mori et al. (2009) reported that an application of ethanol vapor on broccoli before storage at 20 °C delayed senescence by increasing ASA/DHA and GSH/GSSG ratios and activities of APX, MDHAR, DHAR and GR. The objectives of this research were to investigate how ClO2 involves in the antioxidative redox homeostasis, especially the ASA-GSH cycle during storage longan fruit and to evaluate the effects of ClO2 on the status of ASA, GSH and NADPH in the ASA-GSH cycle, as a possible mechanism of delaying senescence in ‘Daw’ longan.

ASA and DHA contents were assayed by the procedure of Kampfenkel et al. (1995) with some modifications. Pericarp of 30 fruit in each treatment was cut into small pieces and mixed. Then 1 g was sampled and homogenized in 5 mL of 6% (w/v) trichloroacetic acid (TCA) at 4 °C for 1 min. The homogenate was then centrifuged at 15,600g and 4 °C for 5 min and the supernatant was immediately assayed for ASA and DHA contents. For ASA determination, 0.2 mL of the supernatant was added into 3.8 mL of reaction mixture containing 0.2 M potassium phosphate buffer (pH 7.4), 10% (w/v) TCA, 42% (v/v) ortho-phosphoric acid, 4% (w/v) 2,2′-dipyridyl and 3% (w/v) FeCl3. The mixed solution was placed at 42 °C for 40 min before measuring the absorbance with a spectrophotometer (Thermo Spectronic model Helios Epsilon, USA) at 525 nm. Total ASA was determined by the reduction of DHA into ASA using dithiothreitol (DTT). Briefly, 0.2 mL of the supernatant was added to 0.8 mL of 10 mM DTT. The reaction was incubated at 42 °C for 15 min and stopped by adding 0.2 mL of 0.5% (w/v) n-ethylmaleimide. The reduced samples were then assayed for ASA as described above. ASA and total ASA contents were determined from the linear equation of a standard curve prepared with ASA. The concentration of DHA was determined by subtracting ASA from the total ASA. The concentrations of ASA and DHA were expressed on a fresh weight basis (g/kg). 2.3. Determination of GSH and GSSG contents GSH and GSSG contents were assayed by the 5,5′-dithiobis-(2nitrobenzoic acid) (DTNB)-glutathione reductase (GR) recycling method as described by Hodges and Forney (2000) with some modifications. One gram of pericarp (from 30 fruit) was sampled and homogenized in 15 mL of 5% (w/v) TCA at 4 °C for 1 min. The homogenate was then centrifuged at 12,000g and 4 °C for 15 min and the supernatant was immediately assayed for GSH and GSSG content. For total glutathione (GSH + GSSG) determination, the supernatant (0.2 mL) was neutralized with 0.5 M potassium phosphate buffer (pH 7.0) with the ratio of 1:25 (supernatant per buffer). The reaction medium consisted of 2.2 mL of 0.1 M potassium phosphate buffer (pH 7.5) containing 5 mM ethylenediaminetetraacetic acid (EDTA) and 0.2 mL of 1 mM NADPH. The reaction was initiated by adding 0.2 mL of 6 mM DTNB prepared in 0.1 M potassium phosphate buffer (pH 7.5) and 0.2 mL of supernatant to the reaction medium. The change in absorbance of the mixture was measured with a spectrophotometer at 412 nm for 5 min. For determination of GSSG, 0.2 mL of the supernatant was neutralized with 0.5 M potassium phosphate buffer (pH 6.5) at the ratio of 1:25. GSH was first sequestered by incubating 5 mL of the supernatant with 0.1 mL of 2-vinylpyridine at 25 °C for 1 h, and then subjected to a similar reaction as described above for total glutathione. Total glutathione and GSSG contents were determined from the linear equation of a standard curve prepared with GSH. For each sample, GSH concentration was obtained by subtracting GSSG from total glutathione. The concentrations of GSH and GSSG were expressed on a fresh weight basis (μg/kg).

2. Materials and methods 2.1. Plant materials and ClO2 treatments Mature longan fruit were harvested from a commercial orchard in Lamphun province, Thailand in June to December 2016 (rainy and winter seasons of Thailand) and transported to the Postharvest Physiology and Technology Research Laboratory at Chiang Mai University within 2 h. Fruit were selected for uniformity in shape, color, size and lack of defects and divided into 2 groups of 720 fruits each. ClO2 gas was prepared according to Saengnil et al. (2014). A 10 min fumigation with ClO2 (0 and 10 mg/L) at 25 ± 1 °C in a chamber (75 L capacity) was done in triplicate. After fumigation, the chamber was ventilated for 30 min to remove any residual ClO2. The fumigated fruit in each group were placed in a cardboard box (25 cm (L) × 17 cm (W) × 9 cm (H)) for 30 fruit per box. All boxes were stored in a storage room at 25 ± 1 °C with a relative humidity of 82 ± 5% for 7 d. A single cardboard box (30 fruit) of each replication was randomly taken every day to determine redox status of ASA, GSH and NADPH, ASA-GSH cycle enzyme activities, NADPH regenerating enzyme activities, H2O2 level and senescence index.

2.4. Determination of NADP and NADPH contents NADP and NADPH contents were assayed by the procedure of Brugidou et al. (1991) with some modifications. One gram of pericarp (from 30 fruit) was sampled and ground in liquid nitrogen and the powder was then homogenized in 10 mL of 0.5 M perchloric acid in 10% (v/v) methanol for NADP extraction, or 0.5 M NaOH in 10% (v/v) methanol for NADPH extraction at 4 °C for 1 min. The homogenate was then centrifuged at 5000g and 4 °C for 15 min. The supernatant was adjusted to either pH 5.0 with 6 M KOH for the NADP extraction or pH 77

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6PGDH activity was assayed in a 1.9 mL of reaction mixture containing 50 mM HEPES (pH 7.6), 2 mM MgCl2, 0.8 mM NADP and 5 mM 6-phosphogluconate and 0.1 mL of supernatant. Subsequent increase in NADPH was observed at 340 nm (E = 6.2 mM/cm). The 6PGDH activity was expressed as mmol/(kg min).

8.0 with 1 M HCl for the NADPH extraction. After centrifugation (5000g) at 4 °C for 10 min, the supernatant was filtered through a 0.45 μm filter (MS® Nylon Syringe Filter, USA). Twenty microliter of the supernatant was used for high performance liquid chromatography (HPLC) (Agilent Corporation, USA) analysis. NADP and NADPH were eluted with 0.1 M KH2PO4, adjusted to pH 6.0 with 0.1 M Tris-base as a mobile phase with a flow rate of 1.3 mL/ min and detected at 254 nm. NADP and NADPH in the samples were identified by comparison with retention time of standards, while the concentrations of NADP and NADPH were determined using the external standard method. The concentrations of NADP and NADPH were expressed on a fresh weight basis (mg/kg).

2.7. Determination of hydrogen peroxide content H2O2 content was determined according to the method of Velikova et al. (2000) with slight modifications. One gram of pericarp (from 30 fruit) was sampled and homogenized in 10 mL of 1% (w/v) TCA at 4 °C for 1 min. The homogenate was centrifuged at 20,000g and 4 °C for 20 min. Then 0.5 mL of the supernatant was added to 2.4 mL of 10 mM potassium phosphate buffer (pH 7.0) and 0.1 mL of 1 M KI. The absorbance of the mixture was read at 390 nm (distilled water was used as blank) and the standard curve was constructed with H2O2·H2O2 content was expressed on a fresh weight basis (mmol/kg).

2.5. Extraction and assay of ASA-GSH cycle enzymes activities Activities of APX, DHAR, MDHAR and GR were assayed by the procedure of Hodges and Forney (2000) with some modifications. One gram of pericarp (from 30 fruit) was sampled and ground in liquid nitrogen and the powder was then homogenized in 10 mL of extraction buffer containing 100 mM potassium phosphate buffer (pH 7.0), 1 mM ASA, 1 mM EDTA and 2.5% (w/v) polyvinylpolypyrrolidone at 4 °C for 1 min. The homogenate was then centrifuged at 15,000g and 4 °C for 25 min and the supernatant was filtered with Whatman® No.1 filter paper (Whatman, England) immediately before being assayed for APX, DHAR, MDHAR and GR activities. APX activity was assayed in 1.9 mL of a reaction mixture containing 100 mM potassium phosphate buffer (pH 7.0), 1 mM ASA, 0.1 mM EDTA and 1 mM H2O2 and 0.1 mL of supernatant. The subsequent decrease in ascorbic acid was observed at 290 nm (E = 2.8 mM/cm). The APX activity was expressed as mmol/(kg min). DHAR activity was assayed in 1.9 mL of a reaction mixture containing 100 mM potassium phosphate buffer (pH 7.0), 5 mM GSH, 0.1 mM EDTA and 0.2 mM DHA and 0.1 mL of supernatant. The subsequent reduction of DHA was observed at 265 nm (E = 14.7 mM/cm). The DHAR activity was expressed as mmol/(kg min). MDHAR activity was assayed in 1.9 mL of a reaction mixture containing 100 mM potassium phosphate buffer (pH 7.0), 0.25 unit ascorbate oxidase, 0.1 mM EDTA, 0.2 mM NADH and 2.5 mM ASA and 0.1 mL of supernatant. The subsequent decrease in NADH was observed at 340 nm (E = 6.2 mM/cm). The MDHAR activity was expressed as mmol/(kg min). GR activity was assayed in 1.9 mL of a reaction mixture containing 100 mM potassium phosphate buffer (pH 7.0), 2.5 mM GSSG, 0.1 mM EDTA and 0.5 mM NADPH in 1% (w/v) NaHCO3 and 0.1 mL of supernatant. The subsequent decrease in NADPH was observed at 340 nm (E = 6.2 mM/cm). The GR activity was expressed as mmol/ (kg min). Protein content was determined according to Lowry et al. (1951) using bovine serum albumin as standard.

2.8. Browning index Pericarp browning was estimated using the browning index (BI) (Jiang and Li, 2001). Browning was estimated visually by measuring the extent of the total brown area on each fruit surface using the following scale: 1 = no browning (excellent quality), 2 = slight browning, 3 = less than 25% browning, 4 = 25-50% browning and 5 = > 50% browning (poor quality). BI was calculated using the following formula: ∑ (browning scale × percentage of fruit in each class). Fruit having BI above 3.0 were considered as unacceptable for marketing quality. 2.9. Disease index Disease index (DI) was visually assessed by the lesions or rot on the fruit surface. The severity of fungal development on the surface was given a score from 0 to 4 with 0 = no visual evidence of fungi, 1 = less than 10%, 2 = 10–30%, 3 = 31–70% and 4 = more than 70% of the surface affected with fungus. DI was calculated using the following formula: ∑ (disease scale × percentage of fruit in each class) (Thavong et al., 2010). 2.10. Statistical analysis The data collected from each experiment were statistically analyzed by using analysis of variance (ANOVA) and the Duncan’s multiple range tests at a significant level of 0.05. All data are presented as mean ± standard deviation. All graphs were created by the Microsoft® Excel for Windows®. 3. Results and discussion

2.6. Extraction and assay of NADPH regenerating enzymes activities

3.1. Associations of redox balancing potential of antioxidative ASA-GSH cycle with longan senescence

Activities of G6PDH and 6PGDH were assayed by the procedure of Valderrama et al. (2006) with some modifications. One gram of pericarp (from 30 fruit) was sampled and ground in liquid nitrogen and the powder was then homogenized in 10 mL of extraction buffer containing 50 mM Tris-HCl buffer (pH 7.8), 0.1 mM EDTA, 0.2% (v/v) TritonX-100 and 10% (v/v) glycerol at 4 °C for 1 min. The homogenate was then centrifuged at 20,000g and 4 °C for 20 min and the supernatant was immediately assayed for G6PDH and 6PGDH activities. G6PDH activity was assayed in 1.9 mL of a reaction mixture containing 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.6), 2 mM MgCl2, 0.8 mM NADP and 5 mM glucose-6phosphate and 0.1 mL of supernatant. Subsequent increase in NADPH was observed at 340 nm (E = 6.2 mM/cm). The G6PDH activity was expressed as mmol/(kg min).

The ratios of ASA/DHA, GSH/GSSG or NADPH/NADP are considered as markers for cellular redox balance and oxidative stress in plants (Noctor et al., 2006; Kumari et al., 2010). In the present study, ASA, GSH and NADPH contents in longan pericarp during storage at room temperature were investigated in order to prove the possibility that a disturbance in the redox balance affected the quality of longan fruit. As shown in Figs. 1–3, the results clearly demonstrated that ASA/DHA, GSH/GSSG and NADPH/NADP ratios of non-ClO2 fumigated (control) fruit markedly decreased during storage for 7 days caused by the decrease in their reduced forms and the increase in oxidized forms. The decrease in these redox ratios (Figs. 1C, 2C and 3C) coincided with an increase in H2O2 content (Fig. 6) and fruit senescence indicated by BI and DI (Fig. 7). It is possible that the reduction in these ratios down78

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Fig. 1. Effects of ClO2 fumigation on ASA content (A), DHA content (B) and ASA/DHA ratio (C) of ‘Daw’ longan pericarp during storage. BF: before fumigation. AF: after fumigation. Bars with the different letters between the control and ClO2 treatment in each storage time indicate significance at P < 0.05. Mean ± standard deviation (n = 3) is presented.

Fig. 2. Effects of ClO2 fumigation on GSH content (A), GSSG content (B) and GSH/GSSG ratio (C) of ‘Daw’ longan pericarp during storage. BF: before fumigation. AF: after fumigation. Bars with the different letters between the control and ClO2 treatment in each storage time indicate significance at P < 0.05. Mean ± standard deviation (n = 3) is presented.

regulates reactive oxygen inactivating enzymes in the ASA-GSH pathway such as APX, MDHAR, DHAR and GR, leading to the accumulation of H2O2 accelerating oxidative damage and cellular senescence (Ramakrishna and Rao, 2013; Chomkitichai et al., 2014a, 2014b). In the control fruit, APX activity increased slightly during the first two days whereas MDHAR activity markedly increased in the first day of storage and decreased thereafter (Fig. 4). On the other hand, DHAR and GR activities decreased over time (Fig. 4). Water and nutrition

stresses upon picking longan fruit might trigger the production and accumulation of ROS. The increase in ROS after harvest has also been reported in loquat (Cai et al., 2006) and litchi (Yang et al., 2009; Sun et al., 2010). It is possible that H2O2 produced at an early stage of the stress acts as an activation signal for APX to scavenge H2O2 and for MDHAR to regenerate ASA from MDHA (Gill and Tuteja, 2010; Sharma et al., 2012; Chomkitichai et al., 2014b). Hence, as the activities of these enzymes generally decreased during the storage of the control 79

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was induced by oxidative stress in the early storage time. Induction of antioxidant defense system by elevated activity of enzymes in the ASA-GSH cycle in association with the redox status of ASA and GSH was observed in longan fruit during storage. In nonfumigated longan fruit, ASA was oxidized to MDHA and then converted to DHA (Fig. 1A and B). This caused a marked decrease in ASA/DHA ratio (Fig. 1C). A marked decline in ASA/DHA ratio and ASA content coincided with the reduction of APX and DHAR activities. When ASA/ DHA ratio was lower than 0.25 on Day 3, the APX and DHAR activities sharply dropped and continuously declined during storage time (Figs. 1C, 4A and B) indicating that lower ASA/DHA ratio down regulated the promotion of APX and DHAR activities. The decrease in ASA/DHA ratio associated with activity loss of ASA-DHA pathway enzymes was also reported in broccoli (Mori et al., 2009), loquat (Cai et al., 2011) and plum (Singh and Singh, 2013). GSH redox state has an important function in maintaining ASA-GSH cycle capacity (Noctor and Foyer, 1998). In addition, GSH/GSSG ratio and GSH content also play an indispensable role in detoxification of ROS (Anjum et al., 2012). In the present study, GSH/GSSG ratio and GSH level of non-fumigated longan fruit continuously decreased during storage (Fig. 2A and C). It is possible that GSH was used in ROS detoxification by antioxidant enzymes such as DHAR, glutathione peroxidase (GPX) and glutathione-S-transferase (GST) (Ramakrishna and Rao, 2013), resulting in a decrease in GSH and increase in GSSG. Moreover, GR activities, the GSH regenerating enzymes, also decreased (Fig. 4D), causing a marked decrease in GSH/GSSG ratio. The decrease coincided with a decrease in DHAR activity (Figs. 2C and 4B) indicating that imbalance of GSH redox state is a cause of ASA redox state imbalance through the reduction of DHAR and GR activities. The decrease in GSH/GSSG ratio and GSH content associated with activity loss of GSH-GSSG cycle enzymes, leading to the alteration of ASA redox state was also reported in broccoli (Mori et al., 2009), loquat (Cai et al., 2011) and plum (Singh and Singh, 2013). NADPH is the primary source of reducing equivalents for the ASAGSH cycle. The NADPH redox state (NADPH/NADP ratio) plays a significant role in the regeneration of GSH by GR and the regeneration of ASA by MDHAR (Foyer and Noctor, 2011; Manai et al., 2014). In the present study, NADPH redox state declined gradually while the NADP pool increased (Fig. 3). It is possible that stresses during longan fruit storage such as water and nutrition deficit, wound or aging elevated NADP biosynthesis to provide sufficient reducing power for ROS detoxification (Ying, 2008; Juszczuk and Ostaszewska, 2011; Manai et al., 2014). However, the activity of NADPH-generating dehydrogenases (G6PDH and 6PGDH) was down regulated during storage time (Fig. 5). This caused a marked increase in NADP level with a decrease in NADPH/NADP ratio. It was found that a decrease in NADPH/NADP ratio closely related to downregulation of MDHAR and GR activities after Day 3 (Figs. 3C, 4C and D) indicating that NADPH redox state directly affected GR. The changes in MDHAR may be broadly regulated by NADPH redox state and other unknown cellular components such as ROS, since its activity increased before gradually decreased thereafter. 3.2. Effects of ClO2 on redox balancing potential of antioxidative ASA-GSH cycle in relation with longan senescence

Fig. 3. Effects of ClO2 fumigation on NADPH content (A), NADP content (B) and NADPH/ NADP ratio (C) of ‘Daw’ longan pericarp during storage. BF: before fumigation. AF: after fumigation. Bars with the different letters between the control and ClO2 treatment in each storage time indicate significance at P < 0.05. Mean ± standard deviation (n = 3) is presented.

ClO2 fumigation significantly increased both ASA/DHA and GSH/ GSSG ratios immediately after fumigation and maintained higher redox level than those of the control (Figs. 1 and 2). The increase in redox ratios by ClO2 coincided with the significant increase in ASA and GSH contents and the significant decrease in DHA and GSSG contents (Figs. 1 and 2) indicating that the ClO2-related mechanism of ASA and GSH is enhanced. The finding that ClO2 maintained ASA/DHA ratios of longan during storage might be explained by increased activities of DHAR and MDHAR (Fig. 4B and C). Moreover, ClO2 maintained GSH/GSSG ratio could be also explained by increased activities of GR enzymes (Fig. 4D). Consequently, higher GSH/GSSG ratio upregulated DHAR activity for

fruit (Fig. 4), the ability to scavenge H2O2 was diminished resulting in perceivable damage to the fruit. Although APX and MDHAR activities were stimulated, the effective time was short (Fig. 4A and C). Moreover, DHAR and GR activities were relatively low (Fig. 4B and D). These might cause the reduction of ASA-GSH cycle potential for the detoxification of H2O2, resulting in the cellular oxidative damage (Sharma et al., 2012; Chomkitichai et al., 2014a,2014b) indicating that APX and MDHAR in longan were mainly important in scavenging H2O2, which 80

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Fig. 4. Effects of ClO2 fumigation on APX (A), DHAR (B), MDHAR (C) and GR (D) activities of ‘Daw’ longan pericarp during storage. BF: before fumigation. AF: after fumigation. Bars with the different letters between the control and ClO2 treatment in each storage time indicate significance at P < 0.05. Mean ± standard deviation (n = 3) is presented.

maintaining the reduced state of ascorbate. This indicated that enhanced enzyme activity in ASA and GSH metabolism (DHAR, MDHAR and GR) by ClO2 played an important role in controlling the redox states of ASA and GSH in longan fruit. The increase in ASA/DHA and GSH/GSSG redox status by ClO2 fumigation coincided with low H2O2 content and fruit senescence index including browning and disease (Figs. 6 and 7). It is possible that ClO2-

induced alteration of redox status due to higher ASA/DHA ratio upregulated APX activity to scavenge H2O2. APX stimulation occurred only in the first few days after which its activity declined coinciding with the increase in H2O2. These results were in line with the work of Ramakrishna and Rao (2013) describing that the higher GSH redox status enhanced H2O2 scavenging capacity through GPX activity. The redox status of ASA and GSH was higher in the stress-tolerant plant than

Fig. 5. Effects of ClO2 fumigation on G6PDH (A) and 6PGDH (B) activities of ‘Daw’ longan pericarp during storage. BF: before fumigation. AF: after fumigation. Bars with the different letters between the control and ClO2 treatment in each storage time indicate significance at P < 0.05. Mean ± standard deviation (n = 3) is presented.

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capacity of APX, DHAR and MDHAR as well as decrease the level of H2O2 via a high GR activity in longan fruit. ClO2 fumigation maintained NADPH redox equilibrium of ‘Daw’ longan which was indicated by higher NADPH/NADP ratio than that of the control (Fig. 3). It is possible that ClO2 modulated NADPH redox potential by the enhancement of NADPH-regenerating enzymes (G6PDH and 6PGDH), resulting in increased thiol and NADPH (Figs. 3 and 5). G6PDH and 6PGDH play a vital role in tolerance to stresses (Hou et al., 2007; Liu et al., 2013). Enhanced expression of the genes of these enzymes in maintaining NADPH redox homeostasis might be a mechanism of response against oxidative stress (Hou et al., 2007; Corpas and Barroso, 2014; Yang et al., 2014). Moreover, G6PDH activities are also activated by phosphorylation through glycogen synthase kinase 3 (GSK3) such as ASKα which had been reported in Arabidopsis (Santo et al., 2012). Thus, ClO2 might upregulate G6PDH and 6PGDH activities through gene expression and/or phosphorylation kinase pathway. Future studies are needed to evaluate the effects of ClO2 on the regulation of NADPH-generating dehydrogenase (G6PDH and 6PGDH) activity of ‘Daw’ longan fruit. ClO2 has been reported in enhancing antioxidant defense system to reduce oxidative stress in ‘Daw’ longan (Chomkitichai et al., 2014a, 2014b). In the present study, reduction in ROS such as H2O2 through ASA-GSH cycle by ClO2 treatment could lessen oxidative damage and retard cell senescence, leading to a higher longan fruit quality. The enhancing effects of ClO2 may be due to increased H2O2 scavenging capacity though maintaining redox homeostasis of ASA-GSH. From the results of this study, a model by which ClO2 modulates the redox state of NADPH and the ASA and GSH redox balance was proposed (Fig. 8). ClO2 may govern the redox state of NADPH by enhancing G6PDH and 6PGDH activities and may administer the ASA and GSH redox balance by stimulating MDHAR, DHAR and GR. Higher ASA redox ratio promoted APX activity to remove excess H2O2 leading to the reduction of oxidative damage and delay senescence of ‘Daw’ longan fruit during storage.

Fig. 6. Effects of ClO2 fumigation on H2O2 content of ‘Daw’ longan pericarp during storage. BF: before fumigation. AF: after fumigation. Bars with the different letters between the control and ClO2 treatment in each storage time indicate significance at P < 0.05. Mean ± standard deviation (n = 3) is presented.

the sensitive ones (Zagorchev et al., 2013) indicating that high ASA/ DHA and GSH/GSSG ratios may play a role in protection against oxidative stress in longan fruit. Similar results were found in chickpea and maize induced by NO or H2S (Kumari et al., 2010; Shan et al., 2014). In the present study, the effects of ClO2 on ASA and GSH redox balancing potential of longan was associated with increased NADPH redox state (Figs. 1–3). A higher NADPH/NADP ratio in ClO2 treated fruit coincided with the higher MDHAR and GR activities, resulting in a higher ASA/DHA and GSH/GSSG ratios (Figs. 1–4). It is possible that the higher NADPH/NADP ratio showed the capacity to upregulate the ASA and GSH recycling enzyme activity including MDHAR and GR. Moreover, MDHAR was also activated by thioredoxin through NADPHdependent thioredoxin reductase (NTR) system which has been reported in sweet potato (Huang et al., 2008). ClO2 might induce MDHAR as well as GR through the NADPH-dependent NTR system for DHA and GSSG reduction. However, the relationship between NADPH redox potential and MDHAR and GR activities in harvested horticultural crops is not clear. The data presented here provide an evidence of this relationship activated by ClO2 in ‘Daw’ longan fruit. Interestingly, the effects of ClO2 on GR activity was greatly induced (3–4 folds after fumigation) and maintained a higher value than that of the control over storage time (Fig. 4D). This indicated that ClO2 fumigation might enhance the capacity of antioxidative ASA-GSH cycle and the increased

4. Conclusions The imbalance of ASA, GSH and NADPH redox states directly affected the senescence of ‘Daw’ longan fruit during storage. ClO2 treatment delayed senescence of the fruit by maintaining redox balance of ASA, GSH and NADPH through ASA-GSH cycle and NADPHregeneration. Acknowledgements This study was financially supported by a grant from The Science

Fig. 7. Effects of ClO2 fumigation on browning index (A) and disease index (B) of ‘Daw’ longan pericarp during storage. BF: before fumigation. AF: after fumigation. Bars with the different letters between the control and ClO2 treatment in each storage time indicate significance at P < 0.05. Mean ± standard deviation (n = 3) is presented.

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Fig. 8. Probable mechanism for reduction of longan senescence by ClO2 via enhancing ASA-GSH cycle through maintaining redox homeostasis during storage at 25 ± 1 °C. = inhibitory effect). ( = activation effect,

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