Properties of Nucleases Associated with Watersoaking and Programmed Cell Death in Ethylene-treated Cucumber Fruit, Poster Board #015

Our previous studies have demonstrated that continuous ethylene exposure predictably induces acute tissue watersoaking of immature mini-cucumber fruit (Cucumis sativus L. cv. Manar) (Postharvest Biol. Technol. 2010, 58:13-20). The attendant changes, including loss of cell viability, increased total nuclease activity, and DNA fragmentation support the idea that watersoaking represents the culmination of stress-mediated programmed cell death (PCD) triggered by ethylene. Prior to the onset of watersoaking, increases in activity of two ethylene-responsive nucleases, with apparent MWs of 34.5 and 37 kDa, were detected by in-gel nuclease assay. The 37 kDa protein was ethylene induced and the 34.5 kDa was ethylene enhanced. The objective of the present study was to further characterize the ethylene-responsive nucleases. Mini-cucumber fruit at immature stage were provided with continuous air or 10 µL·L^-1 ethylene for up to 6 d at 15 °C. Fruit provided with ethylene exhibited watersoaking at 6 d of treatment. Protein from epidermis-associated tissue was subjected to in-gel nuclease assay under different conditions or in-gel substrates to characterize the PCD-related nucleases. Activities of both nucleases were eliminated by 2-β-mercaptoethanol, indicating that disulfide bonds are required for activity. Both nucleases were bifunctional, degrading both DNA and RNA, and the calcium-specific- chelator EGTA (1 mM) inhibited activity which was partially restored by addition of 1 mM Ca²⁺ but not Mg²⁺. Nuclease activities were strongly inhibited at pH below 6. However, they were detected at pH 7 to 9. Therefore, these nucleases might function in the nucleus or cytoplasm prior to tonoplast rupture during PCD. These results indicate that both nucleases exhibit DNase and RNase activities, and require Ca²⁺ and neutral/weak basic conditions for activity. In addition to the ethylene-responsive, bifunctional nucleases, two RNA-specific nucleases (17.5 and 22.3 kDa) were detected using RNA as in-gel substrate. The 17.5 kDa protein was ethylene induced and 22.3 kDa was ethylene enhanced. Contrary to the bifunctional nucleases, RNA-specific nucleases were calcium-independent and were not inhibited by 1 mM EGTA. In addition, RNase activity was detected below pH 7 and strongly inhibited above pH 8. Therefore, even after vacuole collapse during PCD, RNA-specific nucleases might maintain activity. The early appearance of bifunctional and RNA-specific nucleases in the development of ethylene-mediated PCD in mini-cucumber fruit parallels events occurring in several types of PCD in plants. We propose that PCD-like events may explain other types of postharvest disorders mediated by ethylene.