Involvement of Reactive Nitrogen and Oxygen Species (RNS and ROS) in SunflowerMildew Interaction

Nitric oxide (NO) has been shown to participate in plant response against pathogen infection; however, less is known of the participation of other NO-derived molecules designated as reactive nitrogen species (RNS). Using two sunflower (Helianthus annuus L.) cultivars with different sensitivity to infection by the pathogen Plasmopara halstedii, we studied key components involved in RNS and ROS metabolism. We analyzed the superoxide radical production, hydrogen peroxide content, l-arginine-dependent nitric oxide synthase (NOS) and S-nitrosoglutathione reductase (GSNOR) activities. Furthermore, we examined the location and contents of NO, S-nitrosothiols (RSNOs), S-nitrosoglutathione (GSNO) and protein 3-nitrotyrosine (NO2-Tyr) by confocal laser scanning microscopy (CLSM) and biochemical analyses. In the susceptible cultivar, the pathogen induces an increase in proteins that undergo tyrosine nitration accompanied by an augmentation in RSNOs. This rise of RSNOs seems to be independent of the enzymatic generation of NO because the l-arginine-dependent NOS activity is reduced after infection. These results suggest that pathogens induce nitrosative stress in susceptible cultivars. In contrast, in the resistant cultivar, no increase of RSNOs or tyrosine nitration of proteins was observed, implying an absence of nitrosative stress. Therefore, it is proposed that the increase of tyrosine nitration of proteins can be considered a general biological marker of nitrosative stress in plants under biotic conditions.