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, but not in tobacco, a four- to five-amino acid polypeptides that were sul- more distantly related solanaceous species (2). To- fated on Tyr residues. The polypeptides, called bacco plants also did not respond to tomato syste- phytosulfokines, when added to the medium of un- min, although wounding caused a systemic activa- conditioned cell cultures, caused cell proliferation as tion of the synthesis of proteinase inhibitors in if the cultures were conditioned. Synthetic sulfated tobacco leaves. This wound response utilizes the oc- peptides were as fully active as the native com- tadecanoid pathway, similar to wound signaling in pounds. The phytosulfokines were later found to tomato plants, with methyl jasmonate being a potent promote organogenesis in roots, buds, and embryos inducer of the defense genes. In searching for the in various plant species. A cDNA encoding a precur- systemic signal in tobacco leaves two 18-amino acid sor of the pentapeptide was isolated from rice, re- tobacco systemins were recently isolated in our lab- vealing that the polypeptide is processed from the C oratory (G. Pearce, D. Moura, J. Stratmann, and C.A. terminus of the prohormone. Specific, low, and high Ryan, submitted for publication) that exhibit no ho- affinity binding sites have been identified in plasma mology to tomato systemin. In both processed sys- membranes of rice, indicating that the phytosulfo- temins, several prolines have been modified to kines are receptor mediated. Cumulative evidence hydroxy-Pros. Some of the hydroxy-Pros have pen- indicates that phytosulfokines are widespread in the

66 Plant Physiol. Vol. 125, 2001 Polypeptide Hormones plant kingdom and are important signals involved in CLAVATA3 growth processes. The CLAVATA3 gene in Arabidopsis (3) codes for a polypeptide that appears to be the ligand for the CLAVATA1 receptor that balances cell prolifera- RALF tion and cell differentiation in flower meristems. The CLAVATA1 gene was first isolated in the laboratory A 50-amino acid polypeptide called RALF (rapid of E. Meyerowitz (California Institute of Technology) alkalinization factor) that causes a rapid alkaliniza- and was shown to be a regulator of shoot and floral tion of suspension cultured cells has been isolated meristem size in Arabidopsis. CLAVATA1, a Ser-Thr recently from tomato, tobacco, and alfalfa leaves (G. transmembrane receptor with an N-terminal LRR re- Pearce, D. Moura, J. Stratmann, and C.A. Ryan, un- peat, is associated with the meristematic regions of Downloaded from https://academic.oup.com/plphys/article/125/1/65/6098950 by guest on 30 September 2021 published data). It also causes a rapid activation of a flower primordia. Genetic analyses in the Meyerow- mitogen-activated protein kinase in the cells. How- itz laboratory demonstrated that the CLAVATA1 and ever, no function has been found for the polypeptide. the CLAVATA3 gene together control the balance Expressed sequence tags coding for RALF have been between meristem cell proliferation and meristem identified in 10 species of plants from eight plant development. CLAVATA3 codes for a polypeptide families. The precursor cDNAs contain signal se- that is secreted from an adjacent meristematic region, quences, indicating that they are synthesized through and it appears to be the ligand for CLAVATA1. The the secretory pathway, and then further processed. expression of the two genes together results in the The role of the polypeptide in plants is not known, coordination of growth between the adjacent meris- but it is found in a variety of tissues and it is not tematic regions. This example of communication be- wound inducible. tween interconnecting cells may be a prototype of what is occurring in other developmental processes that involve LRR receptors and their ligands in cells POLYPEPTIDE HORMONES IDENTIFIED FROM that are in close proximity. CLONED GENES ENOD40 SCR The signaling polypeptide called ENOD40 (early An example of polypeptide signaling between dif- nodulation; 15) was the second polypeptide identified ferent organs involves the polypeptide ligand, SCR, a family of small secreted Cys-rich proteins that are in plants and the first to be deduced using gene anal- essential for S-locus control of incompatibility in ysis. The ENOD40 polypeptide was initially identified Brassica (13). Recognition of the plants own pollen at by the concerted efforts of several scientists in labora- the surface of the stigma epidermal cells leads to the tories of J. Schell of the Max Planck Institute (Koln) inhibition of pollen growth. The laboratory of J. Nas- and T. Bisseling of Wageningen (The Netherlands). rallah (Cornell University) discovered a small, ENOD40 is the small translated product of the Enod40 anther-specific gene that was a consistent feature of mRNA that plays an important early role in the estab- the S haplotype, which controls pollen function in lishment of root nodule primordia during Rhizobium self incompatibility (SI). The SCR gene was shown to infection. The soybean ENOD40 polypeptide is coded be anther-specific and was the male determinant of by a small open reading frame and is expressed in the SI. The newly translated, small SCR protein exhibits root pericycle just opposite to the nodule primordium, a signal sequence, and is secreted from the develop- where its expression precedes the induction of cortical ing microspores where it subsequently interacts with cell divisions. The soybean polypeptide is composed a receptor (SRK) to activate a signal transduction of 12 amino acids, and homologs have been identified system leading to the inhibition of pollen develop- in tobacco, pea, and alfalfa. In legume and non-legume ment. Whether SCR is further processed to a smaller species the polypeptide is thought to play a specific polypeptide signal before or after secretion is not role in cell division. The Enod40 gene is expressed in known. a temporally similar manner as 1-aminocyclopropane- 1-carboxylic acid oxidase during the early events of nodulation and the polypeptide may counteract the SUMMARY effects of ethylene in cortical cell division during Rhi- The polypeptide hormones that have now been zobium infection leading to nitrogen fixation. Antibod- identified in plants are presented in Table I. The var- ies prepared against soybean ENOD40 identified the ious scenarios in plants for synthesis, storage, process- polypeptide in tobacco protoplasts and soybean root ing, and release of polypeptide hormones are not yet nodules, where it is synthesized without a pre- or known, and from the limited data available it is ap- pro-sequence and therefore does not involve the se- parent that no consistent patterns can yet be deduced. cretory pathway. However, no biological activity has Prosystemin and ENOD40 lack signal sequences that been directly associated with the polypeptide in vivo. would target them through the secretory system, and

Plant Physiol. Vol. 125, 2001 67 Ryan and Pearce

Table I. Polypeptide hormones in plants Size Name Origin Role Initial Reference Preprotein Processed Systemin Tomato, potato, pepper, and 200 Amino acids 18 amino acids Defense signaling 9 nightshade leaves Systemins Tobacco leaves Unknown 18 amino acidsa Defense signaling ENOD40 Tobacco, alfalfa, pea soy- 10–13 Amino acids 10–13 amino acids Cell division 15 bean, pea, and vetch PSK Many species 89 Amino acids 4–5 amino acidsb Cell proliferation 5 RALF Many species ϳ120 Amino acids ϳ50 amino acids Unknown

CLAVATA3 Arabidopsis meristems 96 Amino acids 78 amino acids Coordinate growth 13 Downloaded from https://academic.oup.com/plphys/article/125/1/65/6098950 by guest on 30 September 2021 SCR Brassica pollen 74–77 Amino acids 50–53 amino acids Self incompatibility 3 a Two systemins, both glycosylated. b Sulfated. they may be synthesized in the cytoplasm. PSKs, and developmental status of the plants. The under- CLAVATA3, and SCR pre-proteins all exhibit signal standing of the scope and roles of polypeptide hor- peptides, as does RALF, and are likely synthesized mones in plants and their relationships with other through the secretory system. Signal peptidase sites, plant hormones and signals should provide new in- where known, appear to have similar specificity re- sights into many of the complex signaling networks quirements as the proteinases that cleave animal, that orchestrate plant growth and development, and yeast, and bacterial pre-proteins. Only systemin and the responses of plants to biotic and abiotic stresses. phytosulfokines are known to be internally processed to produce smaller signaling polypeptides, although others may be. No pro-protein processing enzymes or LITERATURE CITED their possible processing sites have yet been identified for any of the plant polypeptide hormones. 1. Becraft PW, Stinard PO, McCarty DR (1996) Science In addition to the polypeptide receptor proteins 273: 1406–1409 and genes mentioned above, several other receptor 2. Constabel CP, Yip L, Ryan CA (1998) Plant Mol Biol genes have been identified through mutagenesis and 36: 55–62 cloning, including Erecta (1), Crinkly4 (14), PRK1 (7), 3. Fletcher JC, Brandu U, Running MP, Simon R, Mey- SLRK (12), and BRl1 (4). All exhibit Leu repeat do- erowitz EM (2000) Science 283: 1911–1914 mains that are typical of extracellular LRR 4. Li J, Chory J (1997) Cell 90: 929–938 polypeptide-binding motifs. LRRs are associated 5. Matsubayashi Y, Sakagami Y (1996) Proc Natl Acad with protein-protein interactions in animals and Sci USA 93: 7623–7627 plants, which are often found associated with 6. McGurl B, Pearce G, Orozco-Cardenas M, Ryan CA polypeptide hormone receptors. Over 100 LRR- (1992) Science 255: 1570–1573 containing genes have been identified in Arabidopsis 7. Mu J-H, Lee H-S, Kao T-H (1994) Plant Cell 6: 709–721 alone and it is possible that many of these proteins 8. Nurnberger T, Nennsties K, Jabs T, Sacks WR, Hahl- are receptors for polypeptide hormones. brock K, Scheel D (1994) Cell 78: 449–460 The signaling pathway for systemin is a complex 9. Pearce G, Strydom D, Johnson S, Ryan CA (1991) cascade that bears striking similarities to the inflam- Science 253: 895–898 matory response of animals (10) that has raised in- 10. Ryan CA (2000) Biochim Biophys Acta 1477: 112–121 teresting questions concerning the ancestral origins 11. Scheer JM, Ryan CA (1999) Plant Cell 11: 1525–1535 of both signaling systems. If the two pathways are 12. Schmidt EDL, Guzzo F, Toonen MAJ, De Bries SC found to share a common ancestral origin, then it (1997) Development 124: 2049–2062 must be established whether other polypeptide sig- 13. Schopfer CR, Nasrallah ME, Nasrallah JB (1999) Sci- nals and signaling pathways in plants also track back ence 286: 1697–1700 to ancestral origins common to plants and animals. It 14. Torii KU, Mitsukawa N, Oosumi T, Matsuura Y, will be important not only to investigate the occur- Yokoyama R, Whittier RF, Komeda Y (1996) Plant Cell rence of polypeptide hormones, their receptors, and 8: 735–746 their signaling pathways throughout the plant king- 15. van de Sande D, Pawlowski K, Czaja I, Wieneke J, dom, but also to begin to understand how various Schell J, Schmidt J, Walden R, Matvienko M, Wellink stimuli cause the release of these signals and orches- J, Van Kammen A, Franssen J, Bisseling T (1996) trate their activities within the overall environmental Science 273: 370–373

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