Effects of Gaseous Chlorine Dioxide and Sulfur Dioxide on Hydrogen Peroxide Production and Cellular Activation of Oxidative Stress Responses of ‘Daw’ Longan Fruit During Storage

Abstract

During oxidative stress, hydrogen peroxide (H2O2) is a strong toxic oxidant causing cell damage or even cell death. At the same time it serves conversely as a signaling molecule to activate defense system against stresses. Pericarp browning, the major postharvest problem of longan (Dimocarpus longan Lour.), have been attributed to the stresses leading to reduced storage life and market value. Fumigation with either chlorine dioxide (ClO2) and sulfur dioxide (SO2) has been reported to inhibit longan browning effectively by reducing reactive oxygen species (ROS) accumulation and oxidative membrane damage which consequently alleviate pericarp browning. However, their ability to counteract the disorder through the H2O2 signaling pathway to stimulate the antioxidant defense system, has not been studied. Therefore, the aims of this study were to demonstrate that fumigation induces the transient H2O2 production, which is believed to act as the upstream signaling molecule in the antioxidant defense responses to oxidative stress involved in pericarp browning of longans. The effects of gaseous SO2 and ClO2 on the fruit quality of ‘Daw’ longan fruit during storage were first examined. Freshly harvested longan fruits were fumigated with either SO2 (500, 1,000 and 2,500 mg L-1) or ClO2 (10 mg L-1) or in combination and stored at 251 °C with 82% relative humidity for 8 days. The fruit were randomly sampled to determine fruit quality. The combined treatment based on 1,000 mg L−1 SO2 with 10 mg L−1 ClO2 gave the best results for limiting the browning development, preserving the visual quality and maintaining overall consumer acceptance. Fumigation with either 1,000 mg L−1 SO2 or 10 mg L−1 ClO2 or in combination, reduced pericarp browning and maintained fruit quality for up to 3, 5 or 7 d, respectively, comparing with a 2 d shelf life for the non-fumigated control fruit. Fumigation with ClO2 and SO2 also reduced disease development effectively and delayed the decrease in overall quality acceptance. Moreover, the levels of SO2 and ClO2 residues on pericarp and aril of the treated fruits were low and below the prescribed limit. The effects of gaseous SO2 and ClO2 on H2O2 production and cellular activation of oxidative stress responses of ‘Daw’ longan fruit were then investigated. H2O2 content increased rapidly after fumigation in the treated fruit, reaching the maximum within 6 to 12 h. Treatments increased expression of the plasma membrane nicotinamide adenine dinucleotide phosphate oxidase (RbohD) and superoxide dismutase (SOD) genes. Subsequent increase in the activities of nicotinamide adenine dinucleotide phosphate oxidase (NOX) and superoxide dismutase coincided with the rise of H2O2. Compared with the fumigated samples, H2O2 concentrations did not increase in the non-fumigated control until Day 1, but increased rapidly thereafter reaching the concentrations that were about three times higher than those of the fumigated samples at the end of the experiment, while that of the fumigated fruit remained lower. Mitogen-activated protein kinase (MAPK, MPK3 and MPK6) gene expression was activated during the first 3 hours to 2 days after the fumigation in the treated fruit. Compared with the fumigated samples, the MAPK activation was barely detectable and remained unchanged throughout the storage time in non-fumigated control fruit. Adenosine triphosphate (ATP) also increased rapidly after fumigation, while that of the control fruit was reduced throughout the storage. In addition, MPK3 and MPK6 expression were activated by exogenous ATP (eATP) in a time-dependent manner similar to the treated fruit. The antioxidant genes, such as catalase (CAT), ascorbate peroxidase (APX) and glutathione peroxidase (GPX), and the activities of antioxidant enzymes CAT, APX, and GPX were stimulated in the treated fruit, reaching the maximum at 12-24 and 6-24 h of treatment, respectively. While the non-fumigated control fruit, had low gene expression and the antioxidant enzyme activity remained low throughout the storage time. 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assays indicated that the total antioxidant capacity were enhanced in the treated fruit. The ascorbate (ASA)/dehydroascorbate (DHA), reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios were also enhanced. These results indicated that fumigation with either SO2 or ClO2 or in combination triggers H2O2 signaling pathway by inducing the NOX-dependent H2O2 generation and MAPK activation as well as enhanced the efficiency of antioxidant defense system in longan fruit. These aimed to overcome the subsequent H2O2 production, thereby reducing the pericarp browning and maintaining fruit quality during storage.