Phytaspase‐mediated precursor processing and maturation of the wound hormone systemin

Abstract

Peptide hormones are implicated in many important aspects of plant life and are usually synthesized as precursor proteins. In contrast to animals, data for plant peptide hormone maturation are scarce and the specificity of processing enzyme(s) is largely unknown. Here we tested a hypothesis that processing of prosystemin, a precursor of tomato (Solanum lycopersicum) wound hormone systemin, is performed by phytaspases, aspartate-specific proteases of the subtilase family. Following the purification of phytaspase from tomato leaves, two tomato phytaspase genes were identified, the cDNAs were cloned and the recombinant enzymes were obtained after transient expression in Nicotiana benthamiana. The newly identified tomato phytaspases hydrolyzed prosystemin at two aspartate residues flanking the systemin sequence. Site-directed mutagenesis of the phytaspase cleavage sites in prosystemin abrogated not only the phytaspase-mediated processing of the prohormone in vitro, but also the ability of prosystemin to trigger the systemic wound response in vivo. The data show that the prohormone prosystemin requires processing for signal biogenesis and biological activity. The identification of phytaspases as the proteases involved in prosystemin maturation provides insight into the mechanisms of wound signaling in tomato. Our data also suggest a novel role for cell death-related proteases in mediating defense signaling in plants.

From structure to function – a family portrait of plant subtilases

“…Prosystemin is cleaved by phytaspases purified from tomato leaves at two sites flanking the systemin sequence, thus excising the peptide hormone from its precursor. In agreement with this in vitro evidence for the involvement of phytaspases in prohormone processing, mutating the phytaspase cleavage sites in prosystemin abolished its defense signaling activity in tomato plants (Beloshistov et al ., ). Most recently, phytaspase was found to hydrolyze and inactivate the human gastrointestinal peptide hormones gastrin and cholecystokinin in vitro (Galiullina et al ., ).…”

“…Signaling by small secreted peptides is, in fact, emerging as an important regulatory phenomenon in plant development (Matsubayashi, ; Tavormina et al ., ), and in some cases SBTs were shown to be involved. As mentioned earlier, the formation of the wound signal systemin involves phytaspases in tomato (Beloshistov et al ., ), while in Arabidopsis, AtSBT6.1 contributes to the formation of growth‐regulating peptides, including RALFs and GOLVEN1 (Srivastava et al ., ; Ghorbani et al ., ; Stegmann et al ., ). However, the cleavage products generated by AtSBT6.1 from the respective precursor proteins are not the mature and bioactive peptides.…”

Regulation of plant peptide hormones and growth factors by post‐translational modification

“…Substitution of phytaspase cleavage sites precluded PS processing in vitro and inhibited systemin signalling in vivo , indicating that Asp‐specific cleavage is required for signal biogenesis (Beloshistov et al . ). However, which of the 12 tomato phytaspases (Reichardt et al .…”

“…Asp-specific proteases (phytaspases) had previously been identified within the subtilase family, and tobacco phytaspase was shown to be instrumental for the induction of programmed cell death in response to biotic and abiotic stress stimuli (Chichkova et al 2004(Chichkova et al , 2010Galiullina et al 2015). Recombinant tomato phytaspases were shown to cleave prosystemin in an Asp-specific manner, producing Lsystemin (Beloshistov et al 2017). Substitution of phytaspase cleavage sites precluded PS processing in vitro and inhibited systemin signalling in vivo, indicating that Asp-specific cleavage is required for signal biogenesis (Beloshistov et al 2017).…”

Unravelling the mode of action of plant proteases

“…In addition, SBT proteins are also involved in the regulation of biotic and abiotic stress responses. Beloshistov et al . (in this issue, pp.…”