Abstract Programmed cell death (PCD) plays a crucial role in plant development (dPCD) and is also activated under the influence of environmental factors (ePCD). As the mechanism of PCD under heavy metal stress is poorly understood, a suspension culture of cells from the facultative metallophyte Viola tricolor was used as a model to study the role of caspase-like proteases in PCD in plant cells under heavy metal stress. Cell suspension cultures treated with Zn or Pb (2000 μM for 72 h) were subjected to TUNEL assay and ligand blotting using a biotinylated inhibitor of caspase-like proteases (biotin-xVAD-fmk). The results from the TUNEL assay showed that the frequency of PCD events was significantly higher in the metal-treated suspended cells (6.8–19.6 % vs 21.8–30.1 %, Zn or Pb, respectively) than it was in the control cells (7.1–9.1 %). The results from the biotinylated inhibitor blot analysis revealed that papain-like cysteine proteases (PLCPs) were involved in heavy metal-induced PCD. Exogenous application of the PLCPs inhibitor E-64-d at a concentration of 50 μM together with Zn or Pb remarkably increased cell viability. This is the first report on the role of the PLCPs in ePCD in plant cells under heavy metal stress.

中文翻译：

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木瓜蛋白酶样半胱氨酸蛋白酶参与重金属胁迫下植物的程序性细胞死亡

摘要 程序性细胞死亡 (PCD) 在植物发育 (dPCD) 中起着至关重要的作用，并且在环境因素 (ePCD) 的影响下也会被激活。由于对重金属胁迫下 PCD 的机制知之甚少，以兼性金属植物三色堇细胞的悬浮培养物为模型，研究了重金属胁迫下植物细胞 PCD 中半胱天冬酶样蛋白酶的作用。用 Zn 或 Pb（2000 μM，72 小时）处理的细胞悬浮培养物进行 TUNEL 测定和配体印迹，使用半胱天冬酶样蛋白酶的生物素化抑制剂（生物素-xVAD-fmk）。TUNEL 分析的结果表明，金属处理的悬浮细胞中 PCD 事件的频率（分别为 6.8-19.6 % 与 21.8-30.1 %，Zn 或 Pb）显着高于对照细胞（7.1- 9.1%)。生物素化抑制剂印迹分析的结果表明，木瓜蛋白酶样半胱氨酸蛋白酶 (PLCP) 参与重金属诱导的 PCD。外源性应用浓度为 50 μM 的 PLCPs 抑制剂 E-64-d 与 Zn 或 Pb 可显着提高细胞活力。这是关于 PLCPs 在重金属胁迫下植物细胞 ePCD 中的作用的第一份报告。