NAC transcription factors regulate Ca2+ -dependent and Zn2+ -dependent nucleases to cooperatively participate in nuclear DNA degradation during programmed cell death in secretory cavity cells of Citrus fruits

• Three NAC transcription factors CrNAC2, CrNAC87-like and CrNAC100 participate in the development of secretory cavities in Citrus reticulata ‘Chachiensis’ fruits. • Three NAC TFs may regulate two nucleases to co-participate in nuclear DNA degradation during PCD. CrNAC2 negatively regulated the expression of Ca 2+ -dependent nuclease CrCAN , whereas CrNAC87-like and CrNAC100 positively regulated the expression of Zn 2+ -dependent nuclease CrENDO1 . • A model of NAC-CrCAN/CrENDO1 co-participates in nuclear DNA degradation during PCD. Plant transcription factors play a crucial role in plant growth, development, and stress resistance. Among them, the NAC transcription factor family is involved in the process of programmed cell death (PCD) during plant growth and development. PCD is involved in the development process of the secretory cavities in the fruit of Citrus reticulata ‘Chachiensis’. During PCD in secretory cavity cells, Ca 2+ -dependent and Zn 2+ -dependent nucleases collaboratively participate in the degradation of the cell nucleus. However, there are still few studies on the transcription factors that regulate the synergistic action of these two types of nucleases in secretory cavity cells PCD. Therefore, this study investigated the transcription factors involved in the regulation of the synergistic action of Ca²⁺-dependent and Zn²⁺-dependent nucleases which participate in nuclear degradation during the development of secretory cavities in Citrus reticulata ‘Chachiensis’ fruits. In this study, three NAC transcription factors CrNAC2, CrNAC87-like and CrNAC100, were obtained and identified. We found that CrNAC2 had a specific expression peak in the middle initial cell stage of the secretory cavity development. While CrNAC87-like and CrNAC100 were mainly specifically expressed in the late initial cell stage and the lumen-forming stage of the secretory cavity development. These results indicated that three transcription factors were involved in the forming of secretory cavity. Through yeast one-hybrid and dual-luciferase assays, it was found that CrNAC2 negatively regulated the expression of Ca 2+ -dependent nuclease CrCAN , whereas CrNAC87-like and CrNAC100 positively regulated the expression of Zn 2+ -dependent nuclease CrENDO1 . Therefore, we hypothesize that during the development of secretory cavities in Citrus fruits, CrNAC2 negatively regulates the Ca²⁺-dependent nuclease, which mediates limited fragmentation of nuclear DNA in the middle initial cell stage. Subsequently, during the late initial cell stage and the lumen-forming stage, CrNAC87-like and CrNAC100 positively regulate the Zn²⁺-dependent nuclease to completely degrade the nuclear DNA. This study provides additional experimental data for understanding the regulation of nucleases by NAC transcription factors in plant programmed cell death.