Abstract
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.
Highlights
Almost 30 years ago, the quest for signaling molecules mediating systemic defense responses after local injury by insect herbivores culminated in the discovery of systemin as the first peptide with signaling function in plants [1, 2]
Cellular responses to systemin include a rapid influx of calcium and depolarization of the plasma membrane which are necessary and sufficient for systemin signaling and induction of the wound response [17,18,19, 42, 43]
When A17 was added to the systemin treatment, the alkalization response was reduced with increasing concentrations of A17 (Figure 2B)
Summary
Almost 30 years ago, the quest for signaling molecules mediating systemic defense responses after local injury by insect herbivores culminated in the discovery of systemin as the first peptide with signaling function in plants [1, 2]. Systemin was initially described as the long-sought hormonal signal that is released at the site of wounding, that travels through the vasculature and induces the defense response in distal, unwounded tissues [2, 5]. This model had to be modified when it was shown that systemin rather acts locally at the site of wounding, where it induces and amplifies the production of jasmonates as systemic signals for defense gene activation in distal tissues [6, 7]. If systemin is released passively as a result of tissue disruption, it could be addressed as a damageassociated molecular pattern, a DAMP [8]
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