Cathepsin B-like cysteine protease ApCathB negatively regulates cryo-injury tolerance in transgenic Arabidopsis and Agapanthus praecox
Cathepsin B-like cysteine protease ApCathB negatively regulates cryo-injury tolerance in transgenic Arabidopsis and Agapanthus praecox
复制标题
组织蛋白酶 B 样半胱氨酸蛋白酶 ApCathB 负向调节转基因拟南芥和百子莲的低温损伤耐受性
DOI：
10.1016/j.plantsci.2021.110928
复制
发表时间：
2021-05-06
期刊：
影响因子：
5.2
通讯作者：
Shen, Xiaohui
中科院分区：
文献类型：
--
作者：
Chen, Guanqun;Zhang, Di;Shen, Xiaohui
细胞死亡是细胞和组织低温保存中不可避免的低温损伤。植物超低温保存过程中细胞程序性死亡的研究尚处于起步阶段。在本研究中，百子莲胚性愈伤组织的玻璃化液中加入20 μ M的组织蛋白酶B抑制剂E-64，可显著提高其存活率。为了进一步研究组织蛋白酶B与冷冻损伤的关系，本研究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定位于细胞膜。ApCathB的过表达使拟南芥幼苗超低温保存的复苏率从29.56%降低到16.46%。转基因幼苗在脱水后下胚轴丧失了大部分细胞活力，导致低温伤害加重。过量表达ApCathB的拟南芥胚性愈伤组织存活率降低。Praecox进一步证实了其在低温损伤耐受中的负作用。此外，ApCathB过表达株系的存活几乎被E-64挽救。TUNEL检测显示信号增强，ROS大量释放，尤其是H2 O2。此外，在超表达ApCathB的胚性愈伤组织中，与细胞死亡调控相关的VPE、Metacaspase 1、Cyp 15 a和AIF基因在超低温保存过程中表达显著上调。此外，调节细胞降解的基因的表达水平也升高，表明ApCathB过表达引起的加速细胞死亡。综上所述，本工作证实了ApCathB通过介导植物低温保存中的PCD事件负调控植物的低温损伤耐受性和细胞活力。值得注意的是，组织蛋白酶B有可能成为提高冻存后存活率的靶点。
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 mu M 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 H2O2. 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.