Interaction between reactive oxygen species and nitric oxide in programmed cell death of Paeonia lactiflora seeds...

In PCD of Paeonia lactiflora seeds during cryopreservation, the nitrate reductase pathway is the critical path of ROS-induced nitric oxide (NO) biosynthesis. The AsA-GSH antioxidant cycle, catalase and superoxide dismutase, are the action sites of NO-mediated ROS regulation. Programmed cell death (PCD) is one of the main reasons affecting the changes in cell viability after cryopreservation. Reactive oxygen species (ROS) and nitric oxide (NO) are key signaling molecules in PCD. However, the relationship between these two signaling molecules in the occurrence of PCD during plant cryopreservation remains unclear. Therefore, in this study, Paeonia lactiflora seeds with varying moisture contents were used as the material. This is based on the changes in seed viability, ROS, and NO before and after cryopreservation. The relationship between ROS and NO and their interaction mechanism during the occurrence of PCD in seed cryopreservation under the action of exogenous ROS and NO regulators are studies. The results showed that after cryopreservation, the viability with high-moisture-content seeds significantly decreased, and the contents of ROS and NO significantly increased. Conversely, the trend was the opposite for seeds with low moisture content. Correlation analysis indicated a significant correlation between seeds viability and the levels of hydroxyl radical (·OH), hydrogen peroxide (H₂O₂), and NO during cryopreservation. Exogenous application of ROS and NO scavengers at optimal concentrations resulted in a significant reduction in the activities of caspase-3-like and caspase-9-like proteases. This also decreased the number of apoptotic and necrotic cells. Exogenous supplementation of H₂O₂ and NO carrier at optimal concentrations elicited opposing trends in two key PCD-related indicators, with the exception of caspase-9-like protease activity. Exogenous application of ROS scavengers at optimal concentrations led to a significant reduction in NO content, whereas exogenous H₂O₂ supplementation exerted an opposite effect on NO levels. Exogenous application of ROS modulators resulted in a consistent variation trend between nitrate reductase (NR) activity and endogenous NO content. Exogenous application of NO scavenger resulted in a marked elevation in ·OH generation and H₂O₂ content, while supplementation with NO carrier significantly decreased in both of these ROS species. Concurrently, the activity profiles of antioxidant enzymes, including ascorbic acid (AsA), glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX), exhibited concordant variation trends with those of ·OH and H₂O₂. These results indicate that ROS and NO have an interactive relationship in the PCD occurrence of P. lactiflora seeds during cryopreservation.