Effects of Gaseous Chlorine Dioxide on Reactive Nitrogen species metabolism of postharvest ‘Daw’ longan fruit in relation to pericarp browning

Abstract

Reactive nitrogen species (RNS) are generated in plant cells either in healthy or senescent cell. Various environmental stresses lead to excessive production of RNS causing progressive nitrosative damage to cellular constituents (nitrosative stress) and senescence or physiological disorders in the higher plant. Pericarp browning, the major postharvest problem of longan (Dimocarpus longan Lour.), have been attributed to excessive production of reactive oxygen species (ROS) causing oxidative damage and ultimately cell death, leading to the reduction in storage life and market value. Previous studies demonstrated that application of gaseous chlorine dioxide (ClO2) inhibit ROS and oxidative stress which reduce pericarp browning of longan after harvesting. However, there was no investigation on the role of ClO2 on RNS metabolism and nitrosative stress associated with the longan pericarp browning. This research demonstrates the effectiveness of ClO2 on RNS generation and nitrosative damage as well as the protective role of ClO2 against pericarp browning of harvested ‘Daw’ longan through antinitrosative defense mechanism during storage at 25±1 °C and 82±5% RH for 7 d. The effects of gaseous ClO2 on RNS generation and the nitrosative stress of ‘Daw’ longan fruit during storage were first examined. Longan fruits were fumigated with ClO2 (0 and 10 mg L-1) for 10 min before being kept in cardboard boxes and stored at 25±1 °C and 82±5 % RH for 7 d. It was revealed that continuously pericarp browning was evident in the control at 6 h and the intensity increased steadily during storage. Browning developed in tandem with a significant rise in RNS levels (nitric oxide, nitrogen dioxide, peroxynitrite and S-nitrosoglutathione) and nitrosative stress (S-nitrosothiol, 3-nitrotyrosine, 8-nitroguanine and nitro fatty acid). Moreover, nitric oxide producing enzyme activities, including nitric oxide synthase and nitrate reductase, increased with a decline in their substrate contents (L-arginine and nitrate) while their products (L-citrulline and nitrite) and pH were also raised during severe pericarp browning. However, ClO2 fumigation reduced the activities of RNS producing enzymes and maintained their substrates for up to 5-6 d of storage. The reduction of these enzyme activities correlated with lower RNS levels, nitrosative stress and pericarp browning, indicating that ClO2 treatment could diminish postharvest browning of ‘Daw' longan pericarp by lowering the activity of the RNS-producing enzyme and causes a reduction in RNS to counteract nitrosative stress. The next experiment was to study the involvement of RNS on ROS, oxidative stress, antioxidant potential and pericarp browning development of harvested ‘Daw’ longan fruit during storage with the aid of exogenous sodium nitroprusside (SNP; nitric oxide donor) or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO; nitric oxide scavenger). Longan fruit were dipped in distilled water (control), SNP (50 and 100 mM) or cPTIO (100 and 500 µM) for 10 min, then stored at 25±1 °C and 82±5 % RH for 7 d. SNP-treated fruit exhibited higher ROS contents (superoxide radical, hydrogen peroxide and hydroxyl radical), oxidative membrane damage (malondialdehyde, protein carbonyl and electrolyte leakage) and pericarp browning (browning index and polyphenol oxidase activity), compared with control fruits. In contrast, exposure to cPTIO attenuated ROS content, oxidative membrane damage and pericarp browning. In addition, the activity of antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione peroxidase) and the total antioxidant capacity were reduced after SNP treatment but were increased after treatment with cPTIO, corresponding to the reduction in pericarp browning. The severity of pericarp browning, ROS production, oxidative damage and antioxidant potential depended on SNP or cPTIO concentrations. The experiment clearly demonstrated that exposure to excessive RNS (nitric oxide) resulted in the reduction of antioxidant potential and the increment of ROS accumulation, oxidative damage and pericarp browning of harvested ‘Daw' longan fruit. The effects of ClO2 on antinitrosative defense system of ‘Daw’ longan fruit during storage were then investigated. Longan fruit were fumigated with ClO2 (0 and 10 mg L-1) for 10 min and then stored in cardboard boxes as previously described. The glutathione and thioredoxin pathways were activated in ClO2 treated fruit within 6-24 h of storage, illustrated by the increasing activities and gene expression of thioredoxin reductase, thioredoxin and glutathione reductase, glutathione content and nicotinamide adenine dinucleotide phosphate redox state 6-24 h after fumigation. The activity of enzymatic RNS scavenger (glutathione peroxidase, S-nitrosoglutathione reductase, glutaredoxin and peroxiredoxin) and accumulation of non-enzymatic RNS scavenger (ascorbic acid, melatonin, phenolic compounds and α-tocopherol) also increased after the fumigation. Moreover, ClO2 fumigation stimulated the activity of nitric oxide and peroxynitrite scavenging. ClO2-induced alteration in the antinitrosative system was highly correlated with pericarp browning reduction. This experiment indicated that ClO2 fumigation triggers antinitrosative systems, with consequent lessening of the pericarp browning of longan fruit. These results indicated that fumigation with ClO2 can regulate RNS metabolism by alleviating RNS production as well as enhancing antinitrosative defense system which leads to a reduction in oxido-nitrosative stress and delay pericarp browning of postharvest ‘Daw' longan fruit during storage at 25±1 °C and 82±5 % RH for 7 d.