Effect of Nitric Oxide Application on Reduction of Undesirable Effects of Chilling on Washington Navel orange (Citrus sinensis L.) Fruit During Storage

Introduction: Chilling injury (CI) is the primary postharvest problem of orange (Citrus sinensis L.) and many other horticultural crops during storage. Washington Navel orange fruits are susceptible to CI during storage below 5°C, and the main CI symptoms are surface pitting, browning, discoloration and decay. Several promising methods have been developed to alleviate CI symptoms of orange fruit. These include postharvest physical treatments with UV-C, modified atmosphere packaging, temperature conditioning, and chemical treatments with plant growth regulators. Oxidative stress from excessive reactive oxygen species (ROS) has been associated with appearance of chilling damage in fruits. The oxidation of ROS is due to their reaction with numerous cell components coursing a cascade of oxidative reactions and consequent inactivation of enzymes, lipid peroxidation, protein degradation, and DNA damage. Aerobic organisms have evolved well-developed defense systems to establish a fine-tuned balance between ROS production and removal plants are protected against ROS effects by a complex antioxidant system. This involved both lipid soluble antioxidant (α- tocopherol and carotenoids) and water soluble reductants (glutathion and ascorbate) and enzymes, such as catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD) and peroxidase (POD). Previous studies have shown that there is a positive relationship between the antioxidant enzymes activity and the chilling tolerance in harvested fruits. Nitric oxide (NO) is an important signaling molecule involved in many plant physiological processes. It has also been indicated that NO protects plant cells against oxidative stress by reducing ROS accumulation. When exogenously applied, NO has been shown to result in an improved chilling tolerance and reduced incidence of chilling injury in several fruits. The objectives of this study were to evaluate the effects of NO on chilling injury, lipid peroxidation content, peroxide hydrogen content, and the induction of antioxidant enzymes in Washington Navel orange (Citrus sinensis L.) fruit during storage at 5±1°C. Materials and Methods: Washington Navel orange (Citrus sinensis L.) fruits were harvested at commercial maturity from a commercial orchard in Kerman, Iran, and transported to the laboratory on the same day. Orange fruits were treated with 0.25 and 0.5 mM nitric oxide for 5 min and then stored at 5±1°C and relative humidity of 85-90 % for 5 months. No nitric oxide use was considered as control. The experiment was arranged in completely randomized design (CRD) with three replicates. Characteristics such as chilling injury, total soluble solids, titratable acidity, pH, ascorbic acid, and activity of antioxidant enzymes (peroxidase and catalase) were evaluated in the present experiment. Results and Discussion: The results showed that use of nitric oxide in fruits reduced significantly chilling injury, ion leakage, lipid peroxidation and hydrogen peroxide compared to control, though it increased activity of antioxidant enzymes. According to these results, unlike organic acids which decreased in treated and non-treated fruits, total soluble solids, ascorbic acid and pH of the fruits increased during storage, however, nitric oxide treatment reduced the rate of changes, be either reducing or increasing, in the mentioned parameters compared to control. So, fruits treated with 0.5 mMol nitric oxide showed the highest effect on the reduction of chilling injury. In the present study, the results indicated that NO significantly reduced CI of orange fruits during storage at 5±1 °C. NO has been applied to reduce the development of chilling injury symptoms in a number of horticultural crops. Thus NO has the potential of application in postharvest treatment by alleviating chilling injury and maintaining quality, and the aim of this study was to determine how NO alleviates the anti-oxidative systems, probably one of the mechanisms of improved chilling tolerance, of orange fruit during chilling stress. This indicates that the chilling tolerance of orange fruit was also enhanced by postharvest treatment with NO. Lipid peroxidation and protective enzyme systems are often evaluated in studies of plant mechanisms under various stresses. Low temperature disrupts the balance of active oxygen species metabolism, leading to their accumulation and destruction of scavenging enzymes such as catalase and peroxidase. In the present study, exogenous per-treatment with nitric oxide at 0.25 and 0.5 mM significantly decreased the lipid peroxidation content and electrolyte leakage of cold stored orange fruit compared to untreated fruits. The level of H2O2 was maintained by NO treatment, which led to an increase in chilling tolerance. It has been reported that the improvement of chilling tolerance in harvested horticultural crops is related to the enhancement in activates of antioxidant enzyme. Researchers found that chilling-tolerant mandarins have a higher antioxidant enzyme activity than the chilling-sensitive ones. A number of postharvest treatments that induce chilling tolerance and alleviate chilling injury also enhanced antioxidant enzyme activity. However, to the best of our knowledge, this is the first paper reporting the beneficial effects of NO on CI of postharvest orange fruits. In this study, there was a continuous increase in peel and pulp lipid peroxidation content in all fruits, but the application of NO significantly delayed the increase of lipid peroxidation. Moreover, the change in membrane permeability (revealed by H2O2 content) showed trends similar to lipid peroxidation content; in other words, peel and pulp H2O2 content increased with storage duration, but NO markedly delayed the increase. NO has been considered to be involved in a network of interacting signal transduction pathways, which regulate defense responses to abiotic stress. The detoxification of ROS is dependent on antioxidant enzymes such as CAT and POD. The increase in these enzymes’ activity contributes to the adaptation of plants to cold stress and ameliorates oxidative damage such as lipid peroxidation (lipid peroxidation increase as indicator) and H2O2 content. Conclusion: In conclusion, application of NO reduced CI of oranges stored at 5±1°C and maintained oranges quality as well. The chilling injury, lipid peroxidation, and peroxide hydrogen were significantly reduced by NO treatment especially at 0.5 mM. Induced cold resistance by NO treatment may be due to the stimulation of antioxidant enzymes, and protection against membrane oxidative damage, decreased lipid peroxidation and H2O2 content in orange fruits. These results may have implications for the use of NO in managing postharvest CI of other subtropical fruits stored at low temperatures