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

Document Type : Research Article


Shahid Bahonar University


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.


1- Aghdam M.S., and Bodbodak S. 2013. Physiological and biochemical mechanisms regulating chilling tolerance in fruits and vegetables under postharvest salicylates and jasmonates treatments. Scientia Horticulturae. 156: 73-85.
2-Asghari M.R., and Abdollahi R. 2012. Changes in quality of strawberries during cold storage in response to postharvest nitric oxide and putrescine treatments. Acta Alimentaria 12:1-13.
3-Basiouny F.‌‌‌M. 1996.‌ Blueberry fruit quality and storability influenced by postharvest application of polyamines and heat treatments. Proceeding Fland State Horticultural Society, 109, 269-272.
4-Beligni M.V., and Lamatina L .1999. Nitric oxide counteract cytotoxic processes mediated by reactive oxygen species in plant tissues. Planta 208: 337-344.
5-Bradford M.‌M. 1976.‌A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248- 254.
6-Burdurlu H.‌S., Nuray K., and Feryal K. 2006.‌ Degradation of vitamin C in citrus juice concentrates during storage. Journal of Food Engineering, 74, 211-216.
7-Chang K. 1992. The evaluation of citrus demand and supply.Proceeding of International Society Citric, Italy. 3: 1153-1155.
8-Cioroi M. 2007. Study on L-ascorbic acid contents from exotic fruits. Cercetari Agronomicin Moldova.1:23-27.
9-Dhindsa R.S., Dhindsa P., and Thorpe A.T. 1981.‌ Leaf senescence correlated with increased levels of membrane permeability and lipid peroxidation and decrease levels of superoxide dismutase and catalase. Journal Experimental Botany, 32, 93-101.
10-FAO. 2014.‌ Food and Agriculture Organization of the United Nations Website. in: http:// www.faosat.org.
11-Forney C.‌F., and Peterson S.‌J. 1990.‌Chilling induced potassium leakage of cultured Citrus cells. Physiologia Plantarum, 78, 193-196.
12-Fotouhi-Ghazvini R., and Fattahi-Moghadam J. 2006. Citrus Growth in Iran. University of Guilan. Rasht. Iran. 305p. (in Persian with English abstract)
13-Heath R.‌L., and ‌Packer L.‌1969.‌ Photoperoxidation in isolated chloroplast, kinetics and stoichiometry of fatty acid peroxidation. Archive of Biochemistry and Biophysiology, 125,189-198.
14-Kelebek H., Selli S., Canbas A., and Cabaroglu T. 2009. HPLC determination of organic acids, sugars, phenolic composition and antioxidant capacity of orange wine made from a Turkish cv. Kozan. Microchemical Journal. 91:187-192.
15-Kochba J., Lavee S., and Spiegel-Roy P. 1977. Differences in peroxidase activity and isoenzymes in embryogenic and non-embryogenic ‘Shamouti’ orange ovular callus lines. Plant and Cell Physiology, 18, 463-497.
16-Leshem Y.Y., and Haramaty E. 1996. Nitric oxide in biological systems. Plant Growth Regulator. 18: 155-159.
17-Leshem Y.Y., and Haramaty E.1998.The characterization and contrasting effects of the nitric oxide free radical in vegetative stress and senescence of Pisum sativum foliage. Journal of Plant Physiology. 148: 258-263.
18-Nilprapruck P., Authanithee F., and Keebjan P. 2008. Effect of exogenous methyljasmonate on chilling injury and quality of pineapple. Silpakorn University Science and Techology. 2: 33-42.
19-Pantastico E.‌B., Soule J., and Grierson W. 1968. Chilling injury in tropical and subtropical fruits: II.limes and grapefruit. Proceeding of Tropical Region American Society. Horticultural Science, 12, 171-183.
20-Purvis A.‌C. 1985. Relationship between chilling injury of grape fruit and moisture loss during storage. Amelioration by polyethylene shrink film. Journal of American Society Horticultural Science, 110, 385-388.
21-Sairam R.K., Deshmukh P.S., and Shukla, D.S. 1997. Tolerance to drought and temperature stress is relation to increased antioxidant enzyme activity in Wheat. Journal of Agronomy Crop Science, 178, 171-177.
22-Schirra M., and D’hallewin G.1997. Storage performance of “Fortune” mandarins following hot water dips. Postharvest Biology and Technology, 10: 229-238.
23-Schirra M., Mulas M., Fadda A., Mignani I., and Lurie S. 2005. Chemical and quality traits of ‘Olinda’ and ‘Campbell’ oranges after heat treatment at 44 or 46_C for fruit fly disinfestations. Lebenson. Wiss. Technology. 38:519-527.
24-Shahbaz M., and Ashraf M. 2007. Influence of exogenous application of nitric oxide on growth and mineral nutrients of wheat under saline conditions. Plant Physiology, 143, 513-522.
25-Singh S.P., Singh Z., and Swinny E.E. 2009. Postharvest nitric oxide fumigation delay fruit ripening and alleviates chilling injury during cold storage of Japanese plums (Prunus salicina L.). Postharvest Biology and and Technology. 53: 101-108.
26-Velikova V., Yordanov I., and Edreva A. 2000. Oxidaive stress and some antioxidant systems in acid rain-treated bean plants. Protective role of exogenous polyamines. Plant Science, 151, 59-66.
27-Wills R.B.H., and Bowyer M.C. 2000. Use of nitric oxide to extend the postharvest life of horticultural produce. Acta Horticulturael. 217:141-147.
28-Wu B., Guo Q., Li Q., and Ha Y. 2014. Impact of postharvest nitric oxide treatment on antioxidant enzymes and related genes in banana fruit in response to chilling tolerance. Postharvest Biology and Technology.92:157-163.
29-Xu M., Dong J., Zhang M., Xu X., and Sun L. 2012. Cold-induced endogenous nitric oxide Generation plays a role in chilling tolerance of loquat fruit during postharvest
Storage. Postharvest Biology and Technology.65:5-12.
30- Zaharah S.S., and Singh Z. 2011. Postharvest nitric oxide fumigation alleviates chilling injury, delays fruit ripening and maintains quality in cold-stored ‘Kensington Pride’ mango. . Postharvest Biology and Technology.60:202-210.
31- Zhu S., Sun L., Liu M., and Zhou J. 2008. Effect of nitric oxide on reactive oxygen species and antioxidant enzymes in kiwifruit during storage. Journal of the Science of Food and Agriculture. 88: 2324-2331.
32 -Zhu L. Q., Zhou J ., and Zhu S.H. 2010. Effect of a combination of nitric oxide treatment and intermittent warming on prevention of chilling injury of ’Feicheng‘peach fruit during storage. Food Chemistry. 121: 165-170.