IDENTIFICATION AND CHARACTERIZATION OF ARABIDOPSIS MUTANT PLANTS IMPAIRED IN NO-MEDIATED HR-CELL DEATH

Nitric oxide (NO) is a gaseous free radical and a key signaling molecule involved in the mediation of various developmental and stress-related conditions in plants. In plant-pathogen interaction, NO plays a crucial role in defense against biotrophic microbes, working together with ROS to trigger cell death in the infected area; a mechanism known as hypersensitive response (HR). However, the molecular mechanisms of how NO coordinates this process is still unknown. To identify candidate genes involved in NO signaling during the HR-cell death process, we carried out a forward genetic screen, using a double-selection system to identify Arabidopsis mutant plants compromised in NO-mediated HR-cell death. For the primary screening, an NO fumigation platform was developed and conditions inducing uniform cell death on wild-type Arabidopsis plants identified. Fumigating in total 25,600 M2 ethyl methane sulfonate (EMS) and fast neutron (FN) mutant plants, we identified 19 mutant lines as non-responsive for NO-induced cell death. The second screening step consisted then, in examining the reduction in pathogen induced HR-cell death in these pre-selected mutants. Evaluation of 13 NO resistant mutant lines for decreased cell death upon avirulent pathogen infection, we finally identified 7 mutant lines that were impaired in this process. An extensive characterization of HR-cell death process was performed in three of these selected mutants. Analyzing the antioxidant system and the presence of normal NO and ROS bursts in these lines, was possible to allocate their mutation in different position in NO signaling in plant resistance. Our results demonstrated that all three mutants showed alteration in the antioxidant system, what affected negatively the ROS burst in one of them. The down-regulation of catalase activity contributed for their higher endogenous level of ROS. Moreover, the NO burst was reduced instead in all three lines but, only one presented a fully compromised NO burst. Interestingly, in all three mutants the modulation of GSNOR activity during HR was disturbed. Concluding, our data indicated that not only the rate of NO and ROS production but also the mechanisms for their turnover were determinant for having normal development of HR-cell death; and therefore, the designed selection strategy also could select mutants impaired in these processes. Thus, these findings reinforce the importance of having balanced bioavailability between H2O2 and NO in the execution of cell death program in plant resistance response. Characterization of NO and ROS in other identified mutants will allow to select most interesting mutants to be sequenced. Mapping population obtained from original M3 mutant plants for outcrossing (OC) revealed to be not the best material for our study. The presence of polymorphic genotypes in the hybrid F2 OC population yielded a distorted genetic segregation and could preclude to determine unequivocally, the mutated gene. Therefore additional strategy for identification of the causal mutation, like the use of F2 backcrossed mutants for creating a mapping population, should be considered once best candidates will be finally selected.