The Systemin Signaling Cascade As Derived from Time Course Analyses of the Systemin-responsive Phosphoproteome

The early phosphorylation signaling cascade of the peptide hormone systemin was defined through comparing systemin-induced responses with non-specific responses induced by the inactive analogon systemin-A17 or mock treatment. Systemin-responsive phosphorylation of kinases and phosphatases was identified over a time course of 2 to 45 minutes, as well as typical substrate phosphorylation sites. We identified the C-terminal threonine of H+-ATPase LHA1 as a substrate of phosphatase PLL5 and MAP-Kinase MPK2, thereby defining a signaling circuit of rapid dephosphorylation and re-phosphorylation after 15 minutes. Systemin is a small peptide with important functions in plant wound response signaling. Although the transcriptional responses of systemin action are well described, the signaling cascades involved in systemin perception and signal transduction at the protein level are poorly understood. Here we used a tomato cell suspension culture system to profile phosphoproteomic responses induced by systemin and its inactive Thr17Ala analog, allowing us to reconstruct a systemin-specific kinase/phosphatase signaling network. Our time-course analysis revealed early phosphorylation events at the plasma membrane, such as dephosphorylation of H+-ATPase, rapid phosphorylation of NADPH-oxidase and Ca2+-ATPase. Later responses involved transient phosphorylation of small GTPases, vesicle trafficking proteins and transcription factors. Based on a correlation analysis of systemin-induced phosphorylation profiles, we predicted substrate candidates for 44 early systemin-responsive kinases, which includes receptor kinases and downstream kinases such as MAP kinases, as well as nine phosphatases. We propose a regulatory module in which H+-ATPase LHA1 is rapidly de-phosphorylated at its C-terminal regulatory residue T955 by phosphatase PLL5, resulting in the alkalization of the growth medium within 2 mins of systemin treatment. We found the MAP kinase MPK2 to have increased phosphorylation level at its activating TEY-motif at 15 min post-treatment. The predicted interaction of MPK2 with LHA1 was confirmed by in vitro kinase assays, suggesting that the H+-ATPase LHA1 is re-activated by MPK2 later in the systemin response. Our data set provides a resource of proteomic events involved in systemin signaling that will be valuable for further in-depth functional studies in elucidation of systemin signaling cascades.