Programmed cell death (PCD) is a major cause of reduced cell viability following cryopreservation, yet the underlying mechanism remains unclear. In this study, pollen from Paeonia lactiflora was used as the experimental material to investigate the role of the microtubule cytoskeleton in PCD during pollen cryopreservation, which exhibits significant viability decline after cryopreservation. The results showed that post-cryopreservation addition of the microtubule-depolymerizing agent oryzalin significantly decreased pollen viability. This effect was accompanied by the activation of caspase-like proteases, reduced mitochondrial membrane potential, elevated intracellular cytochrome C levels, accumulation of PCD signaling molecules, and ultimately increased apoptosis rates. In contrast, treatment with the microtubule-stabilizing agent paclitaxel exerted the opposite effect. At the transcriptional level, paclitaxel treatment induced 754 differentially expressed genes (DEGs); oryzalin treatment resulted in 575 DEGs, a total of 63 DEGs were shared between the two treatments. At the protein level, paclitaxel treatment yielded 262 differentially expressed proteins (DEPs), while oryzalin treatment led to 270 DEPs, with 100 DEPs overlapping between the two groups. Integrated transcriptomic and proteomic analyses revealed that these DEGs and DEPs were significantly enriched in two key pathways: cysteine and methionine metabolism, and protein processing in the endoplasmic reticulum. Notably, heat shock proteins were prominently expressed at both the transcriptional and protein levels in the endoplasmic reticulum protein processing pathway, while malate dehydrogenase played an extremely critical role in cysteine and methionine metabolism pathway. Collectively, these findings indicate that the microtubule cytoskeleton is involved in regulating PCD during pollen cryopreservation, with cysteine and methionine metabolism and endoplasmic reticulum protein processing serving as the core pathways. • Added microtubule cytoskeleton regulators affeced cryopreserved pollen viability. • Also Microtubule cytoskeleton regulators affected the PCD in cryopreserved pollen. • Endoplasmic reticulum protein processing pathway involved in the regulating effect. • Cysteine and methionine metabolism pathway played a role in the regulatory process.
Ruifen et al. (Sun,) studied this question.