Ca2+- and Zn2+-dependent nucleases co-participate in nuclear DNA degradation during programmed cell death in secretory cavity development in Citrus fruits

Abstract Calcium (Ca2+)- and zinc 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 in PCD-associated nuclear DNA degradation remain unclear. Here, the spatiotemporal expression patterns of two nucleases (CrCAN and CrENDO1) were analyzed 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. Quantitative real-time reverse-transcription polymerase chain reaction, in situ hybridization assays, the subcellular localization 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.