Prohormone processing and regulation of the wound response by plant death proteases

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While the importance of peptide hormone signaling is well recognized in animal systems, peptides as signaling molecules have long been neglected in plants. In recent years, however, about a dozen of secreted plant signaling peptides have been identified and recognized to play important roles in cellular communication. In addition, hundreds of potential signaling peptides have been predicted by bioinformatics. Most of these signaling peptides require post-translational modifications for maturation including proteolytic processing to release the active peptides from their larger precursor proteins. This also applies to systemin, which was identified in 1991 as the first hormone-like signaling peptide in plants, and as an essential component of the wound signaling pathway for the induction of defense responses against herbivorous insects in plants of the nightshade family. However, for systemin and most other signaling peptides alike, the question of how they are released from their precursor proteins and the identity of the processing proteases remain unresolved. In an attempt to identify the prosystemin-processing enzyme, we purified an aspartate (Asp)-specific protease from tomato. The protease was found to cleave prosystemin at two Asp residues flanking the systemin peptide. The cleavage product was active in bioassays for the induction of wound response markers. Cleavage at these sites was also found to be necessary for activation, since proteolytic processing and bioactivity were lost in a site-directed prosystemin double mutant, in which the two Asp residues had been substituted by Ala. Using mass spectrometry we could identify the protease as either SlPhyt-1 or SlPhyt-2, two homologs of phytaspase, a cell death-related Asp-specific protease previously characterized in tobacco and rice. The data suggest that after gene duplication, phytaspase homologs in tomato may have acquired novel roles in prosystemin processing and the regulation of the wound response, in addition to the recognized function of phytaspase in programmed cell death. This hypothesis will be addressed in the proposed research project. Using gain- and loss-of-function analyses we will characterize the function of SlPhyt-1 and SlPhyt-2 in vivo and identify their specific contributions to cell death on the one hand, and prosystemin processing, wound signaling and insect resistance on the other. With the proposed research we address the long-standing question of how wounding leads to the formation of the signaling peptide systemin as part of the systemic defense response against insect herbivores. On a more general note, the study will provide insight into the mechanisms of peptide hormone maturation and the function of proteases as regulators of cell death and plant defense.