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

Ethylene induced significant protein degradation after only 2 d of exposure, with total protein levels declining by about 60% after 6 d. The decline in total protein was accompanied by significant increases in protease activity. Increased activity was also observed after 2 d, reaching levels 9.3-fold higher than initial values after 6 d. 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 180 kDa. Levels of the 106 and 180 kDa proteases increased after 2 d of ethylene exposure, reaching maxima at 4-5 d coincident with symptoms of watersoaking. A 56 kDa protease was observed only in ethylene-treated fruit and was first detected after 4 d 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.