A modified chemiluminescence method for hydrogen peroxide determination in apple fruit tissues

Hydrogen peroxide (H2O2) is one of the important by-products produced by plant and fruit tissues during normal metabolism as well as under stress conditions. Evidence suggests that it is actively involved in many physiological activities in plants, including ripening, senescence and the development of disorders. Quantitative measurement of H2O2 in fruit has been a challenge due to variations in methodologies, and their sensitivities and interferences present in plant samples. Among the currently used methodologies, chemiluminescence (CL) is one of the most promising, due to its high specificity and sensitivity. However, direct application of CL methods developed for leaf analysis is not suitable for fruit, especially fruit peel tissues, possibly due to interfering compounds in fruit tissues. In this study, evaluation of the efficiency of removal of interfering compounds by PVP, PVPP and activated charcoal revealed that the PVPP is the most effective compound to remove the interference. This modified protocol can measure H2O2 content in apple peel and flesh tissues. ‘Red Delicious’ apple peel and flesh tissues were measured with amount of 1.48 and 1.03 μmol/g FW, respectively. The established protocol can also be used for a wide variety of tissues in addition to apple fruit, including strawberry tissues (fruit, calyx and leaves) and spinach leaves. This protocol was applied to determine the H2O2 concentration in 1-MCP and DPA treated apples after 5 months of storage, but no significant difference in H2O2 in those samples was found. Direct comparison of CL with a commercial hydrogen peroxide measurement OXIS kit was also made. The challenges to accurately assay H2O2 in fruit/plant tissue were discussed.