The protective effect of drying on the cryopreservation of Neoporphyra haitanensis

The gametophytic thalli of Porphyra can survive long-term frozen storage under appropriate drying conditions. Herein we explored the effects of different drying and freezing conditions on the resuscitation of Neoporphyra haitanensis. The mechanisms of adaptation of algae to freezing were also investigated by transcriptomic analysis. The results revealed that the suitable relative water content (RWC) of thalli for frozen storage was 5%-10%; this resulted in a high thalli rehydration rate and high survival. Within 30 days of frozen storage at -20 °C, thalli with 5% RWC could be rehydrated effectively, and high percent thalli remained healthy. Desiccation significantly increased the level of protective floridoside, isofloridoside and some phytohormones (trans-zeatin, N6-isopentenyladenosine and indoleacetic acid) in the thalli, which remained stable for the following 30-days of frozen storage. However, frozen storage for one year reduced the percentage of healthy thalli to 68.67 ± 5.77% and led to a reduction in levels of the protective substances. Transcriptome data revealed that the mRNA pool was in an active transcription state after drying and was well protected during 30-days frozen storage. However, long-term frozen storage for one year altered the transcription status, with a large number of differentially expressed genes identified. Of note, genes in the carbon fixation pathway were up-regulated while genes involved in the citrate cycle (TCA cycle) and protein synthesis were down-regulated. In summary, a limited drying regime is key to the survival of N. haitanensis at frozen temperature; drying to 5% RWC provides the metabolic and transcriptional conditions required for cryoprotection and low temperature protects the stability of these metabolites and the mRNA pool.