Zhang Di, Ren Li, Chen Guan-Qun, Zhang Jie, Reed Barbara M, Shen Xiao-Hui
Key Laboratory of Urban Agriculture (South) Ministry of Agriculture, School of Agriculture and Biology, Shanghai Jiao Tong University, No. 800, Rd. Dong Chuan, Shanghai, People's Republic of China.
Plant Cell Rep. 2015 Sep;34(9):1499-513. doi: 10.1007/s00299-015-1802-0. Epub 2015 Jun 24.
Oxidative stress and apoptosis-like programmed cell death, induced in part by H 2 O 2 , are two key factors that damage cells during plant cryopreservation. Their inhibition can improve cell viability. We hypothesized that oxidative stress and apoptosis-like event induced by ROS seriously impact plant cell viability during cryopreservation. This study documented changes in cell morphology and ultrastructure, and detected dynamic changes in ROS components (O 2 (·-) , H2O2 and OH·), antioxidant systems, and programmed cell death (PCD) events during embryonic callus cryopreservation of Agapanthus praecox. Plasmolysis, organelle ultrastructure changes, and increases in malondialdehyde (a membrane lipid peroxidation product) suggested that oxidative damage and PCD events occurred at several early cryopreservation steps. PCD events including autophagy, apoptosis-like, and necrosis also occurred at later stages of cryopreservation, and most were apoptosis. H2O2 is the most important ROS molecule mediating oxidative damage and affecting cell viability, and catalase and AsA-GSH cycle are involved in scavenging the intracellular H2O2 and protecting the cells against stress damage in the whole process. Gene expression studies verified changes of antioxidant system and PCD-related genes at the main steps of the cryopreservation process that correlated with improved cell viability. Reducing oxidative stress or inhibition of apoptosis-like event by deactivating proteases improved cryopreserved cell viability from 49.14 to 86.85 % and 89.91 %, respectively. These results verify our model of ROS-induced oxidative stress and apoptosis-like event in plant cryopreservation. This study provided a novel insight into cell stress response mechanisms in cryopreservation.
氧化应激和部分由H₂O₂诱导的凋亡样程序性细胞死亡是植物冷冻保存过程中损害细胞的两个关键因素。对它们的抑制可以提高细胞活力。我们推测，冷冻保存过程中由活性氧诱导的氧化应激和凋亡样事件会严重影响植物细胞活力。本研究记录了百子莲胚性愈伤组织冷冻保存过程中细胞形态和超微结构的变化，并检测了活性氧成分（O₂(·-)、H₂O₂和OH·）、抗氧化系统以及程序性细胞死亡（PCD）事件的动态变化。质壁分离、细胞器超微结构变化以及丙二醛（一种膜脂过氧化产物）增加表明，在冷冻保存的几个早期步骤中发生了氧化损伤和PCD事件。包括自噬、凋亡样和坏死在内的PCD事件在冷冻保存后期也会发生，且大多数为凋亡。H₂O₂是介导氧化损伤并影响细胞活力的最重要的活性氧分子，过氧化氢酶和AsA-GSH循环在整个过程中参与清除细胞内H₂O₂并保护细胞免受应激损伤。基因表达研究证实了冷冻保存过程主要步骤中抗氧化系统和PCD相关基因的变化，这些变化与提高细胞活力相关。通过使蛋白酶失活来降低氧化应激或抑制凋亡样事件，分别将冷冻保存细胞的活力从49.14%提高到86.85%和89.91%。这些结果验证了我们关于植物冷冻保存中活性氧诱导氧化应激和凋亡样事件的模型。本研究为冷冻保存中的细胞应激反应机制提供了新的见解。
