Proteases Associated with Programmed Cell Death and Watersoaking in Ethylene-treated Immature Cucumber Fruit

Continuous ethylene exposure induces acute tissue watersoaking of immature, commercial-harvest maturity beit-alpha cucumber fruit (Cucumis sativus L., cv. Manar). Ethylene-induced watersoaking is preceded by hallmark features of programmed cell death (PCD) including loss of cell viability, increased nuclease activity, and DNA laddering. Our previous studies demonstrated that enhanced reactive oxygen species (ROS)-generating capacity and increases in specific and bifunctional nucleases represent early events in ethylene-induced PCD in cucumber fruit. The present study examined the participation of proteases in ethylene-induced PCD. Immature cucumber fruit (avg. length 12.7 cm, avg. mass 69.4 g) were treated continuously with air or 10 µL L^-1 ethylene for up to 6 d at 15 °C. Ethylene-treated fruit showed incipient watersoaking in hypodermal tissue at 4 d that intensified and progressed into mesocarp and endocarp at 6 d. Protease activity and total protein content in air-stored fruit remained unchanged through 6 d.. In ethylene-treated fruit, significant protein degradation was observed at 2 d, with total protein levels declining about 60% through 6 d. The decline in total protein in response to ethylene was accompanied by significant increases in protease activity. Increased activity was noted after 2 d, reaching levels 9.3-fold higher than initial values after 6 d. Total protease activity was highest at pH 5 and declined sharply through pH 8. Acidic pH preference suggests vacuolar localization, or activation in the cytoplasm under acidic conditions upon tonoplast rupture. In-gel assays revealed three ethylene-responsive proteases with apparent molecular weights of 56, 106, and 180 kDa. The 106 and 180 kDa proteins increased after 2 d of ethylene exposure, reaching maxima at 4 to 5 d coincident with symptoms of watersoaking. The 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 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.