Ca2+- and Zn2+-dependent nucleases play pivotal roles in plant nuclear DNA degradation in programmed cell death (PCD). However, the mechanisms by which these two nucleases co-participate PCD-associated nuclear DNA degradation remain unclear. Here, the spatiotemporal expression patterns of two nucleases (CrCAN and CrENDO1) were analysed qualitatively and quantitatively during PCD in secretory cavity formation in Citrus reticulata 'Chachi' fruits. Results show that the middle and late initial cell stages and lumen-forming stages are key stages for nuclear degradation during the secretory cavity development. CAN and ENDO1 exhibited potent in vitro DNA degradation activity at pH 8.0 and pH 5.5, respectively. qRT-PCR, in situ hybridisation assays, the subcellular localisation of Ca2+ and Zn2+ and immunocytochemical localization showed that CrCAN was activated at the middle and late initial cell stages, while CrENDO1 was activated at the late initial cell and lumen-forming stages. Furthermore, we used immunocytochemical double labelling to simultaneously locate CrCAN and CrENDO1. The DNA degradation activity of the two nucleases was verified by simulating the change of intracellular pH in vitro. Our results also showed that CrCAN and CrENDO1 worked respectively and co-participated in nuclear DNA degradation during PCD of secretory cavity cells. In conclusion, we propose the model for the synergistic effect of Ca2+- and Zn2+-dependent nucleases (CrCAN and CrENDO1) in co-participating in nuclear DNA degradation during secretory cavity cell PCD in Citrus fruits. Our findings provide direct experimental evidence for exploring different ion-dependent nucleases involved in nuclear degradation during plant PCD.

中文翻译：

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在柑橘类水果分泌腔发育过程中的程序性细胞死亡过程中，Ca2+ 和 Zn2+ 依赖性核酸酶共同参与核 DNA 降解。

Ca2+ 和 Zn2+ 依赖性核酸酶在程序性细胞死亡 (PCD) 中植物核 DNA 降解中发挥关键作用。然而，这两种核酸酶共同参与 PCD 相关核 DNA 降解的机制仍不清楚。在这里，对柑橘'Chachi'果实分泌腔形成过程中PCD过程中两种核酸酶（CrCAN和CrENDO1）的时空表达模式进行了定性和定量分析。结果表明，中晚期初始细胞阶段和管腔形成阶段是分泌腔发育过程中核降解的关键阶段。CAN 和 ENDO1 分别在 pH 8.0 和 pH 5.5 下表现出有效的体外 DNA 降解活性。qRT-PCR、原位杂交、Ca2+和Zn2+的亚细胞定位以及免疫细胞化学定位显示CrCAN在初始细胞中后期被激活，而CrENDO1在初始细胞后期和管腔形成阶段被激活。此外，我们使用免疫细胞化学双标记同时定位 CrCAN 和 CrENDO1。通过体外模拟细胞内pH的变化验证了两种核酸酶的DNA降解活性。我们的结果还表明，CrCAN 和 CrENDO1 在分泌腔细胞 PCD 过程中分别发挥作用并共同参与核 DNA 降解。总之，我们提出了柑橘类水果分泌腔细胞 PCD 过程中 Ca2+ 和 Zn2+ 依赖性核酸酶（CrCAN 和 CrENDO1）共同参与核 DNA 降解的协同效应模型。我们的研究结果为探索植物 PCD 过程中参与核降解的不同离子依赖性核酸酶提供了直接的实验证据。