School of Design, Shanghai Jiao Tong University, Shanghai, 200240, China.
School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, China.
Plant Sci. 2021 Jul;308:110928. doi: 10.1016/j.plantsci.2021.110928. Epub 2021 Apr 29.
Cell death is an inevitably cryo-injury in cell and tissue cryopreservation. The research on programmed cell death (PCD) in plant cryopreservation is still in its infancy. In this study, the survival rate of Agapanthus praecox embryogenic callus was significantly improved when the vitrification solution was added with 20 μM E-64, which is an inhibitor of cathepsin B. For further investigating the relation between cathepsin B and cryo-injury, the coding gene of cathepsin B, ApCathB was isolated and characterized. A subcellular localization assay showed that ApCathB was located in cytomembrane. Heterologous overexpression of ApCathB reduced the recovery rate during Arabidopsis seedlings cryopreservation from 29.56 % to 16.46 %. Transgenic seedlings lost most of cell viability in hypocotyl after dehydration and lead to aggravated cryo-injury. The reduced survival rate of ApCathB-overexpressing embryogenic callus of A. praecox further confirmed its negatively function in cryo-injury tolerance. In addition, the survival of ApCathB-overexpressing lines was almost rescued by E-64. TUNEL detection showed intensified signal and ROS was burst, especially for HO. Furthermore, VPE, Metacaspase 1, Cyp15a and AIF genes related to cell death regulation were remarkably up-regulated in ApCathB-overexpressing embryogenic callus during cryopreservation. Additionally, the expression level of genes regulating cell degradation was also elevated, indicating accelerated cell death caused by ApCathB-overexpressing. Taken together, this work verified that ApCathB negatively regulated the cryo-injury tolerance and cell viability through mediating the PCD event in plant cryopreservation. Significantly, cathepsin B has potential to be a target to improve survival rate after cryopreservation.
细胞死亡是细胞和组织冷冻保存中不可避免的冷冻损伤。植物冷冻保存中程序性细胞死亡（PCD）的研究仍处于起步阶段。在这项研究中，当玻璃化溶液中添加 20μM 的 E-64（一种组织蛋白酶 B 的抑制剂）时，Agapanthus praecox 胚性愈伤组织的存活率显著提高。为了进一步研究组织蛋白酶 B 与冷冻损伤之间的关系，分离并鉴定了组织蛋白酶 B 的编码基因 ApCathB。亚细胞定位实验表明，ApCathB 位于细胞质膜上。异源过表达 ApCathB 导致拟南芥幼苗冷冻保存后的恢复率从 29.56%降至 16.46%。转基因幼苗在脱水后失去大部分下胚轴细胞活力，导致冷冻损伤加重。Agapanthus praecox 胚性愈伤组织中 ApCathB 过表达导致的存活率降低进一步证实了其在抗冷冻损伤方面的负功能。此外，E-64 几乎可以挽救 ApCathB 过表达系的存活率。TUNEL 检测显示信号增强，ROS 爆发，尤其是 HO。此外，在冷冻保存过程中，与细胞死亡调控相关的 VPE、Metacaspase 1、Cyp15a 和 AIF 基因在 ApCathB 过表达的胚性愈伤组织中显著上调。此外，调节细胞降解的基因的表达水平也升高，表明 ApCathB 过表达导致细胞死亡加速。总之，这项工作验证了 ApCathB 通过介导植物冷冻保存中的 PCD 事件，负调控冷冻损伤耐受性和细胞活力。重要的是，组织蛋白酶 B 有可能成为提高冷冻保存后存活率的靶标。
