Systemic induction of NO-, redox-, and cGMP signaling in the pumpkin extrafascicular phloem upon local leaf wounding

Cucurbits developed the unique extrafascicular phloem (EFP) as a defensive structure against herbivorous animals. Mechanical leaf injury was previously shown to induce a systemic wound response in the EFP of pumpkin (Cucurbita maxima). Here, we demonstrate that the phloem antioxidant system and protein modifications by NO are strongly regulated during this process. Activities of the central antioxidant enzymes dehydroascorbate reductase, glutathione reductase and ascorbate reductase were rapidly down-regulated at 30 min with a second minimum at 24 h after wounding. As a consequence levels of total ascorbate and glutathione also decreased with similar bi-phasic kinetics. These results hint toward a wound-induced shift in the redox status of the EFP. Nitric oxide (NO) is another important player in stress-induced redox signaling in plants. Therefore, we analyzed NO-dependent protein modifications in the EFP. Six to forty eight hours after leaf damage total S-nitrosothiol content and protein S-nitrosylation were clearly reduced, which was contrasted by a pronounced increase in protein tyrosine nitration. Collectively, these findings suggest that NO-dependent S-nitrosylation turned into peroxynitrite-mediated protein nitration upon a stress-induced redox shift probably involving the accumulation of reactive oxygen species within the EFP. Using the biotin switch assay and anti-nitrotyrosine antibodies we identified 9 candidate S-nitrosylated and 6 candidate tyrosine-nitrated phloem proteins. The wound-responsive Phloem Protein 16-1 (PP16-1) and Cyclophilin 18 (CYP18) as well as the 26.5 kD isoform of Phloem Protein 2 (PP2) were amenable to both NO modifications and could represent important redox-sensors within the cucurbit EFP. We also found that leaf injury triggered the systemic accumulation of cyclic guanosine monophosphate (cGMP) in the EFP and discuss the possible function of this second messenger in systemic NO and redox signaling within the EFP.