Ca2+ participates in programmed cell death by modulating ROS during pollen cryopreservation.

KEY MESSAGE: After cryopreservation, the Ca2+ content increased, which affected the intracellular ROS content, then participated in the occurrence of programmed cell death in pollen. Programmed cell death (PCD) is one of the reasons for the decline in pollen viability after cryopreservation. However, the role of calcium ions (Ca2+) in PCD during pollen cryopreservation has not been revealed in the existing studies. In this study, Paeonia lactiflora 'Fen Yu Nu' pollen was used as the research material for investigating the effects of Ca2+ changes on PCD indices and reactive oxygen species (ROS) during pollen cryopreservation. The results showed that after cryopreservation, with the decrease of pollen viability, the Ca2+ content significantly increased. The regulation of Ca2+ content had a significant effect on PCD indices, which showed that the Ca2+ carrier A23187 accelerated the decrease of mitochondrial membrane potential level and increased the activity of caspase-3-like and caspase-9-like proteases and the apoptosis rate. The expression levels of partial pro-PCD genes were upregulated, the anti-PCD gene BI-1 was downregulated, and the addition of Ca2+-chelating agent EGTA had the opposite effect. The addition of the Ca2+ carrier A23187 after cryopreservation significantly increased the ROS content of pollen, the addition of the Ca2+-chelating agent EGTA had the opposite effect, and Ca2+ regulators also had significant effects on the contents of ROS production and clearance-related substances. Ca2+ affected intracellular ROS content by acting on the ROS production and clearance system during the cryopreservation of pollen and is thus involved in the occurrence of PCD.