Salicylic acid induces H2O2 production and endochitinase gene expression but not ethylene biosynthesis in Castanea sativa in vitro model system

Salicylic acid (SA), ethylene (ET), and wounding are all known to influence plant defense response. Experiments attempting to determine SAʹs relation to ET biosynthesis and defense gene expression have shown conflicting results. To confront this, we developed an in vitro model system to investigate how SA affects ET biosynthesis, hydrogen peroxide (H2O2) production and endochitinase gene expression in the European chestnut. ET measurements of in vitro shoots indicated a critical time point for SA exogenous application, enabling us to study its effects independent of ET. In addition, ET measurements demonstrated that its own increased biosynthesis was a response to wounding but not to SA treatment. Application of the ET biosynthesis inhibitor, aminoethoxyvinylglycine (AVG), on wounded and SA-treated shoots blocked wounding-induced ET production. Interestingly, SA inhibited ET production, but to a lesser extent than AVG. Additionally, SA also induced the accumulation of endochitinase transcript level. Likewise, a sensitive tissue-print assay showed that SA further increased the level of H2O2. Yet, SA-induced endochitinase gene expression and SA-enhanced H2O2 production levels were independent of ET. The cumulative results indicate that SA acts as an inducer of endochitinase PR gene expression and of H2O2 oxidative burst. This suggests that SA is a component of the signal transduction pathway leading to defense against pathogens in chestnut. Further, the model system developed for this experiment should facilitate the deciphering of defense signaling pathways and their cross-talk. Moreover, it should also benefit the study of trees of long generation time that are known to be recalcitrant to in vitro studies.