Cryopreserved-pollen viability is regulated by NO-induced programmed cell death

After cryopreservation, the NO content in pollen increased, inducing programmed cell death as a key reason for reduced viability. Low recovery of biomaterials after cryopreservation is a bottleneck that limits the application of this technology. At present, the mechanism of viability decline after cryopreservation is not fully understood. In this study, the effects of nitric oxide (NO) on programmed cell death (PCD) and its relationship with viability were investigated, using Paeonia lactiflora 'Fen Yu Nu' pollen with significantly decreased viability after cryopreservation. The results showed that: the activity of caspase-3-like and caspase-9-like protease and the apoptosis rate of pollen cells were significantly increased, the expression level of the promoting PCD (pro-PCD) genes was up-regulated, while the expression level of the inhibiting PCD (anti-PCD) genes was down-regulated after preservation in liquid nitrogen (LN); the NO content in pollen cells increased significantly after LN exposure. The correlation analysis showed that NO was significantly correlated with pollen viability and all indicators of PCD. The addition of a NO carrier SNP after LN storage reduced pollen viability, increased endogenous NO content, decreased mitochondrial membrane potential level, activated caspase-3-like and caspase-9-like protease in pollen cells, and increased cell apoptosis rate. The expression levels of pro-PCD genes PDCD2 and ATG8CL were significantly up-regulated, while the expression levels of anti-PCD genes DAD1, BI-1 and LSD1 were significantly down-regulated. The addition of NO scavenger c-PTIO improved pollen viability, and produced the opposite effect of sodium nitroferricyanide (III) dihydrate (SNP), but did not change the mitochondrial membrane potential. These results suggest that NO induced PCD during the cryopreservation of pollen, which was one of the reasons for the significant decrease of pollen viability after cryopreservation.