Y2SK2- and SK3-type dehydrins from Agapanthus praecox act as protectants to improve plant cell viability during cryopreservation

The cryopreservation of plants through vitrification prevents ice damage by bringing the plants to a glassy state for cryogenic storage. Most angiosperm seeds are dehydrated to extremely low levels of their original water content, accumulate late embryogenesis abundant (LEA) proteins, and greatly increase their cytoplasmic viscosity, thereby reaching a glassy state during maturation. In this study, two LEA family recombinant dehydrin (DHN) proteins (Y2SK2 and SK3) from Agapanthus praecox, when added to the plant vitrification solution, were found to double the survival rate of Arabidopsis thaliana seedlings after cryopreservation. Evans blue staining revealed that the Y2SK2 and SK3 proteins decreased the damage to the plasma membranes of plant cells during cryopreservation. Furthermore, the Y2SK2 and SK3 proteins significantly decreased the malondialdehyde and H2O2 contents, increased the glutathione (GSH) content, and downregulated NADPH oxidase (RbohA) in A. thaliana seedlings. Thus, these DHNs may reduce excessive reactive oxygen species production and membrane lipid peroxidation damage under the complex stresses of cryopreservation. Additionally, Y2SK2 can effectively enhance peroxidase activity and catalase2 (CAT2) expression levels; SK3 obviously improved ascorbic acid (AsA) content, as well as Cu/Zn superoxide dismutase (Cu/Zn-SOD) and ascorbate peroxidase 5 (APX5) expression in plant cells and enhanced H2O2 scavenging capacity by promoting the activity of the AsA-GSH cycle. These findings suggested that Y2SK2 and SK3, the DHNs added at the dehydration step, can relieve cell cryoinjury by inducing high antioxidant levels and positive oxidative stress responses and act as protectants, improving plant cell viability after cryopreservation. Application of dehydrins optimization plant cryopreservation protocol, dehydrins can significantly improve complex stress damage and enhance the cell viability from cryopreservation.