Nitric Oxide Interacts with Salicylate to Regulate Biphasic Ethylene Production during the Hypersensitive Response

C₂H₄ is associated with plant defense, but its role during the hypersensitive response (HR) remains largely uncharacterized. C₂H₄ production in tobacco (Nicotiana tabacum) following inoculation with HR-eliciting Pseudomonas syringae pathovars measured by laser photoacoustic detection was biphasic. A first transient rise (C₂H₄-I) occurred 1 to 4 h following inoculation with HR-eliciting, disease-forming, and nonpathogenic strains and also with flagellin (flg22). A second (avirulence-dependent) rise, at approximately 6 h (C₂H₄-II), was only seen with HR-eliciting strains. Tobacco leaves treated with the C₂H₄ biosynthesis inhibitor, aminoethoxyvinylglycine, suggested that C₂H₄ influenced the kinetics of a HR. Challenging salicylate hydroxylase-expressing tobacco lines and tissues exhibiting systemic acquired resistance suggested that C₂H₄ production was influenced by salicylic acid (SA). Disrupted expression of a C₂H₄ biosynthesis gene in salicylate hydroxylase tobacco plants implicated transcriptional control as a mechanism through which SA regulates C₂H₄ production. Treating leaves to increase oxidative stress or injecting with SA initiated monophasic C₂H₄ generation, but the nitric oxide (NO) donor sodium nitroprusside initiated biphasic rises. To test whether NO influenced biphasic C₂H₄ production during the HR, the NO synthase inhibitor NG-nitro-L-arginine methyl ester was coinoculated with the avirulent strain of P. syringae pv phaseolicola into tobacco leaves. The first transient C₂H₄ rise appeared to be unaffected by NG-nitro-L-arginine methyl ester, but the second rise was reduced. These data suggest that NO and SA are required to generate the biphasic pattern of C₂H₄ production during the HR and may influence the kinetics of HR formation.