Heat shock proteins (HSPs) have many positive stabilizing effects on the proteins of stressed cells. This study investigated the effect of HSP70 on cryopreserved pollen. HSP70 was differentially expressed in Paeonia lactiflora and Magnolia denudate pollen before and after exposure to liquid nitrogen (LN). In this study, cryopreserved pollen samples of P. lactiflora ‘Fen Yu Nu’, ‘Zi Feng Chao Yang’ and ‘Hong Pan Tuo Jing’ were incubated with HSP70 at six concentrations, and the pollen germination, oxidative stress and apoptosis-like programmed cell death indicators were measured after LN storage. HSP70 significantly improved pollen viability after LN storage, and the concentration of HSP70 that was required varied from cultivar to cultivar, ranging from 0.5 to 10 μg/mL. Compared to the activity of the control treatment without HSP70, the activity of superoxide dismutase (SOD) increased, the contents of reactive oxygen species (ROS) and malondialdehyde (MDA) decreased, and the Ca2+ level, apoptosis rate and caspase-3-like activity decreased depending on the appropriate concentration of HSP70 added. Pollen germination was negatively correlated with MDA content, Ca2+ level, and caspase-3-like activity and positively correlated with SOD activity. In conclusion, HSP70 improved the viability of pollen after cryopreservation by improving the activity of antioxidant enzymes, reducing the contents of ROS and MDA, and affecting the Ca2+ signal to inhibit apoptosis-like programmed cell death events. This report is the first time to describe the application of HSP70 for improving cryopreserved pollen viability by regulating oxidative and apoptosis-like programmed cell death events, and provide a novel insight into the mechanisms of HSP70 action in cryopreservation.
After the pollen was rewarmed and then incubated with appropriate concentrations of exogenous HSP70, it significantly increasing the post-freezing germination of the pollen. Which by affecting oxidative stress and apoptosis-like events, the ROS level, MDA content, intracellular Ca2+ concentration, apoptosis rate and caspase-3-like enzyme activity were reduced, and SOD activity was increased, thereby significantly increasing the post-freezing germination of the pollen.
Cryopreservation Pollen HSP70 Oxidative stress Apoptosis-like programmed cell death events
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This research was supported by the National Natural Science Foundation of China (Nos. 31370693 and 31770741). We thank Prof. Barbara M. Reed for editing the manuscript.
RR designed and conducted the research, analyzed the data and drafted the manuscript. XJ and WD edited the manuscript and offered some help on experiment technical. ZL offered some help on material collection. BL and JX conceived the research and provided technical assistance. YL conceived the project, supervised the analysis and critically revised the manuscript. All authors read and approved the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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1.Beijing Laboratory of Urban and Rural Ecological Environment, Beijing Municipal Education Commission, Beijing Key Laboratory of Ornamental Plants Germplasm Innovation & Molecular Breeding, National Engineering Research Center for Floriculture, College of Landscape ArchitectureBeijing Forestry UniversityBeijingChina
2.College of Architecture and Urban PlanningGuangzhou UniversityGuangzhouChina