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Polypeptide Hormones1 Polypeptide Hormones1 Clarence A. Ryan* and Gregory Pearce Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164–6340 Polypeptide signaling is an emerging field in plant developing assays for biological activity and physical biology, particularly in areas of defense, fertilization, detection. The isolations of systemin and phytosulfo- Downloaded from https://academic.oup.com/plphys/article/125/1/65/6098950 by guest on 30 September 2021 growth, and development. Until 1991, polypeptide kines did not result from direct searches for polypep- hormones and pheromones were thought to be only tide hormones, but from the use of the scientific found in animals and yeast, and it was thought that method in seeking the causes of specific biological plants had evolved signaling systems that did not effects, including the systemic signal(s) for plant de- include polypeptide signals. Following the initial dis- fense (systemin) and the cause of the conditioned covery in 1991 of the 18-amino acid polypeptide de- medium response (phytosulfokines). It would be fu- fense hormone systemin and its precursor prosyste- tile to directly seek polypeptide hormones in plants min in tomato leaves (6, 9), several plant polypeptide without some indication that the particular process signals have been isolated and characterized or else involved a polypeptide ligand. Mutational analyses identified by gene tagging (3, 5, 13, 15). In addition, and gene isolation have been powerful tools in iden- several genes have been identified in plants that code tifying regulatory genes that code for polypeptide for proteins having extracellular Leu-rich domains ligands, and for genes that code for LRR receptors to (LRRs) (1, 4, 7, 12, 14) that are typical of polypeptide- effect biological function. However, these approaches binding motifs. Polypeptides are now considered to are also very difficult and time consuming. The genes be a new class of plant hormones, adding to the list of coding for CLAVATA 1, ENOD40, and SCR are the known plant hormones that includes auxins, gibber- initial examples of the power of these approaches. illins, cytokinins, ethylene, abscisic acid, jasmonic We anticipate that many more polypeptide hormones acid, and brassinolides. In addition to polypeptide will be identified in the near future using biochemi- signals originating within plants, polypeptides that cal and genetic approaches. are generated by pathogens can also activate plant defenses through receptor-mediated signaling, and POLYPEPTIDES ISOLATED BY FUNCTION can play important roles as signals that activate re- sistance responses (8). Tomato Systemin Processing from larger precursors is a characteristic of most animal and yeast polypeptide hormones The initial polypeptide signal that was identified in where prepro-hormones are synthesized through the plants, systemin, was found in our laboratory during secretory system. Insulin is a classic example of a a search for the chemical agent that was responsible hormone that is processed and stored within secre- for the systemic induction of proteinase inhibitors in tory vesicles and released in response to physiologi- tomato leaves. We had found that fractions from crude cal signals. Others, such as growth factors and cyto- extracts obtained from tomato leaves activated pro- kines, are not processed within the vesicles to the teinase inhibitor genes when supplied to young ex- mature form but are anchored in the vesicle mem- cised tomato plants through their cut stems. We iden- branes prior to processing. The vesicle membranes tified the active components as oligogalacturonide fuse with the cell membrane to present the hormone fragments derived from plant cell walls. At the same domain to the extracellular space, where they are time the laboratories of Peter Albersheim (University cleaved and released by membrane proteinases in of Colorado) and Charles West (University of Califor- response to specific signals. nia, Los Angeles) found that oligogalacturonides Because of the low abundance of polypeptide hor- could activate the synthesis of phytoalexins, which are mones in tissues and organs of animals and plants, defense chemicals in soybeans and castor beans. Fur- their isolations have been typically time consuming ther research revealed that the oligogalacturonides and difficult. The barriers to isolations have been in were not mobile in tomato plants and were therefore not likely candidates for systemic signaling, but are 1 localized signals to help defend against pest and This work was supported in part by Project 1791, by the pathogen attacks. College of Agriculture and Home Economics, by Washington State University, by the National Science Foundation (grant no. IBN Our further efforts to identify and purify the sys- 9601099), and by the U.S. Department of Agriculture National temic signal in tomato plants resulted in a long and Research Initiative (grant no. 9801502). tedious search involving over 40 thousand assays * Corresponding author; e-mail [email protected]; fax using young tomato plants. The assay consisted of 509–335–7643. supplying young plants through their cut stems with Plant Physiology, January 2001, Vol. 125, pp. 65–68, www.plantphysiol.org © 2001 American Society of Plant Physiologists 65 Ryan and Pearce fractions purified from columns. The induction of toses attached, but the carbohydrate structures have accumulation of proteinase inhibitors was deter- not been established. The two sequences exhibit lim- mined 24 h later using an immunoradial diffusion ited homology with each other, and they may interact assay, which took another 24 h to run. It was usual with the same receptor. The finding that the tobacco that duplicate assays were performed. Each purifica- systemins are not homologous with tomato, potato, tion step required scaling up the starting tomato leaf pepper, or nightshade systemins raises questions material, with the final purification requiring over 60 concerning the possible universality of systemins and pounds of tomato leaves. The purification resulted in their structural variability among species. Despite just a few micrograms of a pure material that had the structural differences among the polypeptide defense properties of a small polypeptide. Amino acid and signals, we propose that plant-derived polypeptides sequence analyses in the laboratory of B. Vallee (Har- that signal defense genes, locally or systemically, be Downloaded from https://academic.oup.com/plphys/article/125/1/65/6098950 by guest on 30 September 2021 vard Medical School) indicated that it was an 18- called systemins. The data so far indicate that syste- amino acid polypeptide, and we named it systemin mins and their receptors may be a common feature of (9). Systemin was active in the biological assays at plants, but that structurally different systemin levels of femtomoles per plant, and was phloem mo- polypeptides may serve the same functions in differ- bile when labeled with 14C and placed on wounds on ent plant species. Systemins homologous to tomato tomato leaves. Using the sequence of systemin to or tobacco systemins have not been found in species synthesize nucleotide probes to identify the mRNA, outside the Solanaceae family, but searches for their systemin was found to be processed from the C ter- presence in other species continue. The presence of minus of a 200-amino acid precursor, prosystemin systemic wound-inducible defense genes have been (6), a processing scenario common to animal and demonstrated in numerous species in several families, yeast polypeptide hormones. Proof for the defensive and it is likely that polypeptide hormones will be signaling role of systemin was demonstrated by commonly found as wound signals. The identification transforming tomato plants with a gene containing and characterization of additional systemins should an antisense prosystemin cDNA under the control of help establish the fundamental biochemical, physio- the cauliflower mosaic virus 35S promoter. The logical, and evolutionary principles that govern their plants produced large amounts of antisense prosys- existence and functions in plants and their possible temin mRNA, which resulted in abolishing the sys- relationships to other plant and animal polypeptide temic wound response (6) and allowed Manduca sexta hormones and their receptors. larvae to rapidly consume the plants that were nor- mally resistant. A 160-kD high-affinity receptor for systemin was recently identified in plasma mem- Phytosulfokines branes of tomato cells (11), and purified (J. Scheer and C.A. Ryan, unpublished data). Systemin is the A novel class of polypeptide hormones that regu- only polypeptide ligand in plants for which a recep- late cell division was purified and characterized by tor has been identified and isolated, for which the Y. Matsubayashi and Y. Sakagami (Nagoya, Japan) elements of a signal transduction pathway are from conditioned medium of asparagus suspension- known, and for which several genes regulated by the cultured cells (5). The researchers were not seeking a polypeptide have been identified. polypeptide hormone, but the causal factor for con- ditioned media. The factors were isolated using a cell culture assay in which the purified fractions were Systemins in Other Plant Species added to cells and the mitogenic activity was re- corded. Using HPLC, ion exchange, and gel perme- Genes coding for systemins have been identified in ation the factors were purified and found to be small, potato, pepper, and nightshade,
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