Prohormone processing and regulation of the wound response by plant death proteases
Plant Physiology
Final Report Abstract
While the importance of peptide hormone signaling is well recognized in animal systems, peptides as signaling molecules have long been neglected in plants. Systemin was identified in 1991 as the first hormone-like signaling peptide in tomato plants, and as an essential component of the wound signaling pathway for the induction of defense responses against herbivorous insects. A few years later, phytosulfokine (PSK) was identified as a growth promoting signaling peptide in asparagus and later in Arabidopsis. Systemin and PSK, like most other peptide hormones, are derived from larger precursor proteins by proteolytic processing. The proteases that are involved in the biogenesis of plant peptide hormones, and their relevance for the regulation of peptide hormone activity are largely unknown. Aiming to identify the prosystemin-processing enzyme, we purified an aspartate (Asp)- specific protease activity from tomato. The purified activity was found to cleave prosystemin at two Asp residues flanking the systemin peptide to release the bioactive peptide. Cleavage at these sites was also found to be necessary for activation, since bioactivity of prosystemin was lost in a site-directed double mutant, in which the two Asp residues had been substituted by Ala. Using mass spectrometry, we could identify the protease as a homologue of phytaspase, a cell death-related Asp-specific protease previously characterized in tobacco and rice. Using a bioinformatics approach, 12 potential phytaspases were identified in the tomato genome. Five of these enzymes were expressed and purified to homogeneity. Substrate specificity was analyzed using a proteomics assay and a panel of fluorogenic peptide substrates confirming them as bona fide phytaspases. Several of them also cleaved prosystemin in vitro at the two Asp residues flanking the systemin peptide. However, gainand loss-of-function analyses of these proteases in transgenic tomato plants indicated that they are not involved in prosystemin processing in vivo, or else, that they act redundantly with another member(s) of the phytaspase family. The protease(s) responsible for Aspspecific processing of prosystemin in vivo is thus still elusive. Unexpectedly, we observed premature abscission of flowers in plants overexpressing the phytaspase SlPhyt-2. The flower drop phenotype was enhanced under drought conditions, while silencing of SlPhyt-2 resulted in higher fruit set and seed production. Consistent with a role of SlPhyt-2 in drought-induced flower drop, the protease was found to be expressed in the pedicel of tomato flowers and its expression was induced before the onset of abscission on the proximal side of the abscission zone. Hypothesizing that SlPhyt-2 may be involved in the formation of a peptide as abscission signal, known peptide hormone precursors were scanned for the presence of Asp at the processing site, resulting in the identification of phytosulfokine (PSK) precursors as potential SlPhyt-2 substrates. Cleavage of PSK precursors by SlPhyt-2 was confirmed in vitro, and SlPhyt-2 was found to be required for PSK formation in vivo. Mature PSK induced the expression of cell wall hydrolases in the abscission zone and restored flower abscission in SlPhyt-2-deficient plants. The data indicate that SlPhyt-2 is expressed in the pedicel where it is responsible for the generation of PSK as a peptide signal that travels from the proximal region into the abscission zone for the induction of cell wall hydrolases as executors of the abscission process. Our results provide fundamental insight into the molecular control of abscission, they reveal a novel function for PSK that is known primarily for its growth promoting and immune-modulating activities, and they demonstrate the relevance of precursor processing as a regulatory step in plant peptide hormone biogenesis and signaling.
Publications
- (2018): From structure to function - a family portrait of plant subtilases. New. Phytol. 218: 901-915
Schaller, A., Stintzi, A., Rivas, S., Serrano, I., Chichkova, N.V., Vartapetian, A.B., Martínez, D., Guiamét, J.J., Sueldo, D.J., van der Hoorn, R.A.L., Ramírez, V., Vera P.
(See online at https://doi.org/10.1111/nph.14582) - (2018): Phytaspase-mediated precursor processing and maturation of the wound hormone systemin. New. Phytol. 218: 1167–1178
Beloshistov, R.E., Dreizler, K., Galiullina, R.A., Tuzhikov, A.I., Serebryakova, M.V., Reichardt, S., Shaw, J., Taliansky, M.E., Pfannstiel, J., Chichkova, N.V., Stintzi, A., Schaller, A., Vartapetian, A.B.
(See online at https://doi.org/10.1111/nph.14568) - (2018): The tomato subtilase family includes several cell death-related proteinases with caspase specificity. Sci. Rep. 8:10531
Reichardt, S., Repper, D., Tuzhikov, A.I., Galiullina, R.A., Planas-Marquès, M., Chichkova, N.V., Vartapetian, A.B., Stintzi, A. and Schaller, A.
(See online at https://doi.org/10.1038/s41598-018-28769-0) - (2020) Peptide signaling for tomato flower drop. Science 367: 1482-1485
Reichardt, S., Piepho, H.-P., Stintzi, A., Schaller, A.
(See online at https://doi.org/10.1126/science.aaz5641)
