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Programmed cell death (PCD) occurs during plant development and in response to various stimuli, including biotic and abiotic stresses. Characteristics of PCD were detected in cucumber fruit after 9 days of cold storage at 2C, including chromatin condensation and DNA fragmentation. PCD development was accompanied by ethylene emission and cytosolic free calcium ([Ca 2+ ]cyt) release. The role of ethylene production and [Ca 2+ ]cyt in chill-induced PCD were studied through the application of 1-methylcyclopropene (1-MCP; an ethylene perception inhibitor) and ethylene glycol bis (2-aminoethyl) tetraacetic acid (EGTA; a divalent cation chelator). Results indicated that hallmarks of PCD were identified in cucumber fruit suffering from chilling stress and diagnostic events of PCD were alleviated and/or delayed by 1-MCP or EGTA applications. Therefore, ethylene and calcium could play significant roles in the initiation and execution progress of coldinduced PCD. PRACTICAL APPLICATIONSCucumber fruit is chilling sensitive at temperatures below 10C. It has also been showed that exogenous ethylene could induce PCD features in cucumber fruits and [Ca 2+ ]cyt plays a key role in regulating apoptosis. However, information about roles of endogenous ethylene and calcium in the PCD process of cucumber under cold stress has been rarely reported. The data in this study provided a theoretical basis for understanding the mechanism of chilling injury in cucumber fruit and benefited exploration of strategies to prevent it.