Hydrogen peroxide and nitric oxide are involved in programmed cell death induced by cryopreservation in Dendrobium protocorm-like bodies

Abstract

Programmed cell death (PCD) plays a key role in animal tissue cell death following cryopreservation. However, there are few studies evaluating the plant PCD characteristics induced by cryopreservation and the possible role of signal molecules in PCD. Here, we performed cell ultrastructural observation and evaluation of PCD-regulating gene expression, caspase-3-like activity, and hydrogen peroxide (H2O2) and nitric oxide (NO) contents occurring in Dendrobium protocorm-like bodies (PLBs) cryopreserved by vitrification. The main changes in cell ultrastructure were noted at the plant vitrification solution 2 (PVS2) dehydration stage with severe plasmolysis, chromatin condensation and vesicle formation. Liquid nitrogen exposure increased autophagy activity with the appearance of many autophagic vesicles. Expression of the autophagy-related protein 8C gene (Atg 8C) and reticulon-like protein B8 gene (Rtnl B8) was increased at the preculture and cooling-rewarming stages, while bax inhibitor 1-like gene (BIL 1) expression was decreased at the preculture and PVS2 dehydration stages compared with expression levels in fresh PLBs (CK). Caspase-3-like activity was low at the preculture stage but increased significantly with the treatment of loading and PVS2 dehydration and finally reached a peak at the stage of cooling-rewarming. Both H2O2 and NO began to increase at the preculture stage, reached the highest value at the loading stage, and then decreased at the late stage of cryopreservation. Catalase (CAT) and 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) addition at the preculture stage significantly increased survival and decreased caspase-3-like activity in cryopreserved PLBs. These results indicated that the cryopreservation protocol, especially PVS2 dehydration and cooling-rewarming, induced PCD in D. nobile PLBs. H2O2 and NO produced at the preculture stage may also be involved. Cryopreservation protocol by vitrification, especially the PVS2 dehydration and cooling-rewarming, induced programmed cell death in PLBs. Inhibition of H2O2 and NO produced at the preculture stage can decrease caspase-3-like activity and improve PLBs viability.