NO way back: nitric oxide and programmed cell death in Arabidopsis thaliana suspension cultures

Recent research has implicated nitric oxide (NO) in the induction of the hy persensitive response (HR) during plant-pathogen interactions. Here we demo nstrate that Arabidopsis suspension cultures generate elevated levels of NO in response to challenge by avirulent bacteria, and, using NO donors, show that these elevated levels of NO are sufficient to induce cell death in Ar abidopsis cells independently of reactive oxygen species (ROS). We also pro vide evidence that NO-induced cell death is a form of programmed cell death (PCD), requiring gene expression, and has a number of characteristics of P CD of mammalian cells: NO induced chromatin condensation and caspase-like a ctivity in Arabidopsis cells, while the caspase-1 inhibitor, Ac-YVAD-CMK, b locked NO-induced cell death. A well-established second messenger mediating NO responses in mammalian cells is cGMP, produced by the enzyme guanylate cyclase. A specific inhibitor of guanylate cyclase blocked NO-induced cell death in Arabidopsis cells, and this inhibition was reversed by the cell-pe rmeable cGMP analogue, 8Br-cGMP, although 8Br-cGMP alone did not induce cel l death or potentiate NO-induced cell death. This suggests that cGMP synthe sis is required but not sufficient for NO-induced cell death in Arabidopsis . In-gel protein kinase assays showed that NO activates a potential mitogen -activated protein kinase (MAPK), although a specific inhibitor of mammalia n MAPK activation, PD98059, which blocked H2O2-induced cell death, did not inhibit the effects of NO.