Role of mitochondrial membrane homeostasis in the occurrence of programmed cell death during pollen cryopreservation: Mitochondrial ROS eruption and bioenergetic deficiency as key contributors

Cryopreservation is an ideal approach for the long-term storage of pollen, but the decline in pollen viability caused by necrosis and programmed cell death (PCD) after cryopreservation remains a challenge. Mitochondrial homeostasis is considered to play a crucial role in plant PCD. However, there is no further explanation on how it specifically alters. To investigate changes in mitochondrial reactive oxygen species (ROS) signaling, membrane homeostasis, and bioenergetics during PCD occurrence triggered by cryopreservation, pollen from three Paeonia lactiflora cultivars with decreased, stable, and increased PCD (i.e., 'Yan Zi Xiang Yang', DEC-PCD, 'Fen Yu Nu', STABLE-PCD, 'Zi Feng Chao Yang', INC-PCD) after cryopreservation was used. The results revealed that: (1) Mitochondrial ROS acts as a signaling molecule induced PCD during pollen cryopreservation, and its content was associated with changes in the activity of mitochondrial respiratory chain (MRC) complex I, II and III. (2) After cryopreservation, the extent of mitochondrial permeability transition pore opening, the reduction in mitochondrial membrane potential, and the oxidation of mitochondrial inner membrane in INC-PCD were 16.57 %,50.91 %, and 615.32 % higher, respectively, than those in STABLE-PCD, indicating more severe mitochondrial membrane damage in INC-PCD. (3) During pollen cryopreservation, mitochondrial bioenergetic supply gradually declined as PCD intensified. These results demonstrated that cryopreservation induces excessive mitochondrial ROS production in certain cultivars, leading to the imbalance in mitochondrial membrane homeostasis and impaired bioenergetic supply, ultimately resulting in the occurrence of PCD during pollen cryopreservation of P. lactiflora.