Characterization of proteases and nucleases associated with ethylene-induced programmed cell death in immature cucumber fruit

Abstract

Previous studies demonstrated that continuous 10μLL−1 ethylene exposure induces acute watersoaking of immature beit-alpha cucumber fruit (Cucumis sativus L. cv. Manar). This phenomenon includes increases in nuclease activities and loss of total protein, supporting the idea that watersoaking is a form of programmed cell death (PCD). The present study was designed to further characterize ethylene-induced, PCD-associated nucleases and proteases in immature cucumber fruit. DNases were found to possess bifunctional (DNA/RNA) activities. In addition, four RNA-specific nucleases (17.5, 22.3, 24.4, and 26.1kDa) were detected by in-gel assays, all of which were ethylene inducible. All nucleases were inhibited by β-mercaptoethanol, indicating a requirement for disulfide bond(s) for activity. Both the bifunctional nucleases and the 17.5kDa RNase exhibited Ca2+ dependency. Bifunctional nuclease activities were detected above pH 7, but strongly inhibited at weakly acidic pH (6). By contrast, RNA-specific nuclease activity was optimal at acidic pH and strongly inhibited above pH 8.Ethylene induced significant protein degradation after only 2d of exposure, with total protein levels declining by about 60% after 6d. The decline in total protein was accompanied by significant increases in protease activity. Increased activity was also observed after 2d, reaching levels 9.3-fold higher than initial values after 6d. Total protease activity was greatest at pH 5 and declined sharply through pH 8. In-gel assays revealed three ethylene-responsive proteases with apparent molecular masses of 56, 106, and 180kDa. Levels of the 106 and 180kDa proteases increased after 2d of ethylene exposure, reaching maxima at 4–5d coincident with symptoms of watersoaking. A 56kDa protease was observed only in ethylene-treated fruit and was first detected after 4d of ethylene exposure. Together with up-regulated nuclease activities and reactive oxygen species (ROS)-generating capacity, the early appearance of proteases in response to ethylene provides additional evidence that watersoaking in immature cucumber fruit reflects the execution phase of a PCD pathway.