Integrating multi-omics to reveal the regulatory mechanisms of microfilament cytoskeleton in programmed cell death during seed cryopreservation.

Programmed cell death (PCD), one of the main reasons of cell viability changes after cryopreservation. The microfilament cytoskeleton serves as a core modulator of cold stress responses and PCD signaling cascades, but the molecular mechanism linking microfilament cytoskeletal dynamics to PCD during seed cryopreservation has not yet been elucidated. Therefore, in this study, two types of Paeonia lactiflora seeds with different viability changes after cryopreservation were used as experimental materials. Taking the microfilament cytoskeleton as insertion point, we explored the mechanism of its action during the occurrence of PCD in seed cryopreservation combining the physiological, transcriptome and proteome. The results showed that the application of microfilaments cytoskeletal stabilizer (phalloidin) increased the seed viability after cryopreservation, and significantly inhibited PCD-regulated proteases, cytochrome C and related signaling molecules, and further reduced the cell apoptosis rates. Conversely, the treatment with microfilaments cytoskeletal depolymerizer (chlorpromazine B) exerted an opposite effect. At the transcriptomic level, the DEGs were mainly enriched in the MAPK signaling pathway- plant, the phosphatidylinositol signaling system, and protein processing in the endoplasmic reticulum. Moreover, weighted gene co-expression network analysis of DEGs identified the salmon module centered on HSP90A, TUBA, cynT, thrC, and SNW1, and the turquoise module centered on aroDE, NEIL3, thrC, SNW1, and PCF11. At the protein level, the DEPs in the two comparison groups were mainly enriched in pathways such as protein processing in the endoplasmic reticulum, glycolysis/gluconeogenesis, and glutathione metabolism. Combined transcriptomic and proteomic analyses showed that both DEGs and DEPs were highly enriched in the pathways of protein processing in the endoplasmic reticulum and glycerolipid metabolism. However, members of the HSP and ALDH family exhibited marked differential expression in pathways protein processing in the endoplasmic reticulum pathway and glycerolipid metabolism pathway separately, both at the transcriptional and protein levels. This indicated that the microfilament cytoskeleton participated in the occurrence of PCD, then affecting the seed viability after cryopreservation. Among them, protein processing in the endoplasmic reticulum and glycerolipid metabolism are the key pathways through which microfilament cytoskeleton participating PCD during seed cryopreservation, and HSP and ALDH may serve as the core action targets in this process.