Effects of Programmed Cell Death Induced by NO on Pollen Viability After Cryopreservation

Abstract Low activity level of biomaterials after cryopreservation is a bottleneck problem that limited 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 promoting PCD (pro-PCD) gene was up-regulated, while the expression level of inhibiting PCD (anti-PCD) gene was down-regulated after preserved in liquid nitrogen (LN); the NO content in pollen cells increased significantly after LN preserved. The results of correlation analysis showed that NO was significantly correlated with pollen viability and all indicators of PCD. The addition of 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, and 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; and the addition of NO scavenger c-PTIO produced the opposite effect of SNP. It was suggested that NO induced the PCD during the cryopreservation of pollen, which was one of the reasons for the significant decrease of pollen viability after cryopreservation.