ApSerpin-ZX from Agapanthus praecox, is a potential cryoprotective agent to plant cryopreservation

Abstract

Cryopreservation-induced cell death is regarded as an important problem faced by cryobiologists. Oxidative stress and programmed cell death are detrimental to cell survival. Serine protease inhibitors (serpins) inhibit pro-cell-death proteases and play a pro-survival role in excessive cell death induced by abiotic stress. In this study, ApSerpin-ZX was isolated from Agapanthus praecox and characterized as a protective protein in plant cryopreservation. The mRNA level of ApSerpin-ZX was elevated under abiotic stress, such as salt, osmosis, oxidative, cold, and cryoinjury. The purified recombinant protein expressed in E. coli was added to the plant vitrification solution and used for A. praecox embryogenic callus cryopreservation. The concentration of 0.6-4.8 mg∙L^-1 of ApSerpin-ZX protein was beneficial to the survival of cryopreserved embryogenic callus of A. praecox. The most effective concentration was 1.2 mg∙L^-1, which elevated the survival by 37.15%. Subsequently, the cryopreservation procedure with 1.2 mg∙L^-1 of ApSerpin-ZX protein was regarded as the treated group, compared to standard procedure, to determine the physiological mechanism of ApSerpin-ZX protein on cryopreserved cell. The MDA and H₂O₂ contents were significantly decreased in the treated group, along with reduced OH· generation activity in the recovery stage. After the addition of ApSerpin-ZX, the POD and CAT activities keep increased, while SOD activity increased only after dehydration. Besides, the caspase-1-like and caspase-3-like activities were lower than the standard procedure. This study indicated that ApSerpin-ZX was a potential cryoprotective agent that alleviated oxidative stress and cell death induced by cryopreservation.

Keywords: Agapanthus praecox; Cryopreservation; Plant serpin; Programmed cell death; cell viability.