Studies On Endonucleases During The Salt-Induced Programmed Cell Death In Root Tip Cells Of Rice

Salt stressed rice root tips were used to investigate the progress of programmed cell death (PCD). The results indicated that 500 mmol/L NaCl treatment could lead to specific features of PCD in root tip cells, such as DNA ladder, nuclear condensation and deformation, and transferase mediated dUTP nick end labeling positive reaction, which were initiated at 4 h of treatment and progressed thereafter. Cytochrome c release from mitochondria into cytoplasm was also observed, which occurred at 2 h and was earlier than the above nuclear events. All these results above indicated that 500mmol NaCl was capable of inducing PCD in root tip cells of rice.We used root tip cells of rice, which have been induced by NaCl stress to undergo PCD, to analyze the endonucleases activities of cytoplasmic and nuclear extracts. Two endonucleases, a cytoplasmic of 20 kDa (OsCyt20) and a nuclear of 37 kDa (OsNuc37), were identified as PCD-related. Our results indicated that OsCyt20 is a Ca²⁺/Mg²⁺-dependent nuclease that is most active at neutral pH, and that OsNuc37 is Zn²⁺-dependent, with a pH optimum of 4.5-6. Both nucleases were induced at the early stage of PCD (2 h salt treatment) and exhibited the highest activity approximately 4 h after exposure to NaCl, paralleling with the occurrence of DNA laddering. In vitro assays of endonuclease activities further revealed that the OsNuc37, a glycoprotein localized in the nucleus, is the executor for DNA laddering.Based on results above,in the present study we identified a new PCD-associated endonuclease, OsNuc37, which is a glycoprotein and responsible to the DNA laddering in salt-induced PCD in root tip cells of rice .This research made it possible to discover the mechanism of PCD in plants better.