Plant Physiol, January 2001, Vol. 125, pp. 65-68

Polypeptide Hormones¹

Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340

	INTRODUCTION

TOP INTRODUCTION POLYPEPTIDES ISOLATED BY... POLYPEPTIDE HORMONES IDENTIFIED... SUMMARY LITERATURE CITED

Polypeptide signaling is an emerging field in plant biology, particularly in areas of defense, fertilization, growth, and development. Until 1991, polypeptide hormones and pheromones were thought to be only found in animals and yeast, and it was thought that plants had evolved signaling systems that did not include polypeptide signals. Following the initial discovery in 1991 of the 18-amino acid polypeptide defense hormone systemin and its precursor prosystemin in tomato leaves (6, 9), several plant polypeptide signals have been isolated and characterized or else identified by gene tagging (3, 5, 13, 15). In addition, several genes have been identified in plants that code for proteins having extracellular Leu-rich domains (LRRs) (1, 4, 7, 12, 14) that are typical of polypeptide-binding motifs. Polypeptides are now considered to be a new class of plant hormones, adding to the list of known plant hormones that includes auxins, gibberillins, cytokinins, ethylene, abscisic acid, jasmonic acid, and brassinolides. In addition to polypeptide signals originating within plants, polypeptides that are generated by pathogens can also activate plant defenses through receptor-mediated signaling, and can play important roles as signals that activate resistance responses (8).

Processing from larger precursors is a characteristic of most animal and yeast polypeptide hormones where prepro-hormones are synthesized through the secretory system. Insulin is a classic example of a hormone that is processed and stored within secretory vesicles and released in response to physiological signals. Others, such as growth factors and cytokines, are not processed within the vesicles to the mature form but are anchored in the vesicle membranes prior to processing. The vesicle membranes fuse with the cell membrane to present the hormone domain to the extracellular space, where they are cleaved and released by membrane proteinases in response to specific signals.

Because of the low abundance of polypeptide hormones in tissues and organs of animals and plants, their isolations have been typically time consuming and difficult. The barriers to isolations have been in developing assays for biological activity and physical detection. The isolations of systemin and phytosulfokines did not result from direct searches for polypeptide hormones, but from the use of the scientific method in seeking the causes of specific biological effects, including the systemic signal(s) for plant defense (systemin) and the cause of the conditioned medium response (phytosulfokines). It would be futile to directly seek polypeptide hormones in plants without some indication that the particular process involved a polypeptide ligand. Mutational analyses and gene isolation have been powerful tools in identifying regulatory genes that code for polypeptide ligands, and for genes that code for LRR receptors to effect biological function. However, these approaches are also very difficult and time consuming. The genes coding for CLAVATA 1, ENOD40, and SCR are the initial examples of the power of these approaches. We anticipate that many more polypeptide hormones will be identified in the near future using biochemical and genetic approaches.

	POLYPEPTIDES ISOLATED BY FUNCTION

TOP INTRODUCTION POLYPEPTIDES ISOLATED BY... POLYPEPTIDE HORMONES IDENTIFIED... SUMMARY LITERATURE CITED

Tomato Systemin

The initial polypeptide signal that was identified in plants, systemin, was found in our laboratory during a search for the chemical agent that was responsible for the systemic induction of proteinase inhibitors in tomato leaves. We had found that fractions from crude extracts obtained from tomato leaves activated proteinase inhibitor genes when supplied to young excised tomato plants through their cut stems. We identified the active components as oligogalacturonide fragments derived from plant cell walls. At the same time the laboratories of Peter Albersheim (University of Colorado) and Charles West (University of California, Los Angeles) found that oligogalacturonides could activate the synthesis of phytoalexins, which are defense chemicals in soybeans and castor beans. Further research revealed that the oligogalacturonides were not mobile in tomato plants and were therefore not likely candidates for systemic signaling, but are localized signals to help defend against pest and pathogen attacks.

Our further efforts to identify and purify the systemic signal in tomato plants resulted in a long and tedious search involving over 40 thousand assays using young tomato plants. The assay consisted of supplying young plants through their cut stems with fractions purified from columns. The induction of accumulation of proteinase inhibitors was determined 24 h later using an immunoradial diffusion assay, which took another 24 h to run. It was usual that duplicate assays were performed. Each purification step required scaling up the starting tomato leaf material, with the final purification requiring over 60 pounds of tomato leaves. The purification resulted in just a few micrograms of a pure material that had the properties of a small polypeptide. Amino acid and sequence analyses in the laboratory of B. Vallee (Harvard Medical School) indicated that it was an 18-amino acid polypeptide, and we named it systemin (9). Systemin was active in the biological assays at levels of femtomoles per plant, and was phloem mobile when labeled with ¹⁴C and placed on wounds on tomato leaves. Using the sequence of systemin to synthesize nucleotide probes to identify the mRNA, systemin was found to be processed from the C terminus of a 200-amino acid precursor, prosystemin (6), a processing scenario common to animal and yeast polypeptide hormones. Proof for the defensive signaling role of systemin was demonstrated by transforming tomato plants with a gene containing an antisense prosystemin cDNA under the control of the cauliflower mosaic virus 35S promoter. The plants produced large amounts of antisense prosystemin mRNA, which resulted in abolishing the systemic wound response (6) and allowed Manduca sexta larvae to rapidly consume the plants that were normally resistant. A 160-kD high-affinity receptor for systemin was recently identified in plasma membranes of tomato cells (11), and purified (J. Scheer and C.A. Ryan, unpublished data). Systemin is the only polypeptide ligand in plants for which a receptor has been identified and isolated, for which the elements of a signal transduction pathway are known, and for which several genes regulated by the polypeptide have been identified.

Systemins in Other Plant Species

Genes coding for systemins have been identified in potato, pepper, and nightshade, but not in tobacco, a more distantly related solanaceous species (2). Tobacco plants also did not respond to tomato systemin, although wounding caused a systemic activation of the synthesis of proteinase inhibitors in tobacco leaves. This wound response utilizes the octadecanoid pathway, similar to wound signaling in tomato plants, with methyl jasmonate being a potent inducer of the defense genes. In searching for the systemic signal in tobacco leaves two 18-amino acid tobacco systemins were recently isolated in our laboratory (G. Pearce, D. Moura, J. Stratmann, and C.A. Ryan, submitted for publication) that exhibit no homology to tomato systemin. In both processed systemins, several prolines have been modified to hydroxy-Pros. Some of the hydroxy-Pros have pentoses attached, but the carbohydrate structures have not been established. The two sequences exhibit limited homology with each other, and they may interact with the same receptor. The finding that the tobacco systemins are not homologous with tomato, potato, pepper, or nightshade systemins raises questions concerning the possible universality of systemins and their structural variability among species. Despite structural differences among the polypeptide defense signals, we propose that plant-derived polypeptides that signal defense genes, locally or systemically, be called systemins. The data so far indicate that systemins and their receptors may be a common feature of plants, but that structurally different systemin polypeptides may serve the same functions in different plant species. Systemins homologous to tomato or tobacco systemins have not been found in species outside the Solanaceae family, but searches for their presence in other species continue. The presence of systemic wound-inducible defense genes have been demonstrated in numerous species in several families, and it is likely that polypeptide hormones will be commonly found as wound signals. The identification and characterization of additional systemins should help establish the fundamental biochemical, physiological, and evolutionary principles that govern their existence and functions in plants and their possible relationships to other plant and animal polypeptide hormones and their receptors.

Phytosulfokines

A novel class of polypeptide hormones that regulate cell division was purified and characterized by Y. Matsubayashi and Y. Sakagami (Nagoya, Japan) from conditioned medium of asparagus suspension-cultured cells (5). The researchers were not seeking a polypeptide hormone, but the causal factor for conditioned media. The factors were isolated using a cell culture assay in which the purified fractions were added to cells and the mitogenic activity was recorded. Using HPLC, ion exchange, and gel permeation the factors were purified and found to be small, four- to five-amino acid polypeptides that were sulfated on Tyr residues. The polypeptides, called phytosulfokines, when added to the medium of unconditioned cell cultures, caused cell proliferation as if the cultures were conditioned. Synthetic sulfated peptides were as fully active as the native compounds. The phytosulfokines were later found to promote organogenesis in roots, buds, and embryos in various plant species. A cDNA encoding a precursor of the pentapeptide was isolated from rice, revealing that the polypeptide is processed from the C terminus of the prohormone. Specific, low, and high affinity binding sites have been identified in plasma membranes of rice, indicating that the phytosulfokines are receptor mediated. Cumulative evidence indicates that phytosulfokines are widespread in the plant kingdom and are important signals involved in growth processes.

RALF

A 50-amino acid polypeptide called RALF (rapid alkalinization factor) that causes a rapid alkalinization of suspension cultured cells has been isolated recently from tomato, tobacco, and alfalfa leaves (G. Pearce, D. Moura, J. Stratmann, and C.A. Ryan, unpublished data). It also causes a rapid activation of a mitogen-activated protein kinase in the cells. However, no function has been found for the polypeptide. Expressed sequence tags coding for RALF have been identified in 10 species of plants from eight plant families. The precursor cDNAs contain signal sequences, indicating that they are synthesized through the secretory pathway, and then further processed. The role of the polypeptide in plants is not known, but it is found in a variety of tissues and it is not wound inducible.

	POLYPEPTIDE HORMONES IDENTIFIED FROM CLONED GENES

TOP INTRODUCTION POLYPEPTIDES ISOLATED BY... POLYPEPTIDE HORMONES IDENTIFIED... SUMMARY LITERATURE CITED

ENOD40

The signaling polypeptide called ENOD40 (early nodulation; 15) was the second polypeptide identified in plants and the first to be deduced using gene analysis. The ENOD40 polypeptide was initially identified by the concerted efforts of several scientists in laboratories of J. Schell of the Max Planck Institute (Koln) and T. Bisseling of Wageningen (The Netherlands). ENOD40 is the small translated product of the Enod40 mRNA that plays an important early role in the establishment of root nodule primordia during Rhizobium infection. The soybean ENOD40 polypeptide is coded by a small open reading frame and is expressed in the root pericycle just opposite to the nodule primordium, where its expression precedes the induction of cortical cell divisions. The soybean polypeptide is composed of 12 amino acids, and homologs have been identified in tobacco, pea, and alfalfa. In legume and non-legume species the polypeptide is thought to play a specific role in cell division. The Enod40 gene is expressed in a temporally similar manner as 1-aminocyclopropane-1-carboxylic acid oxidase during the early events of nodulation and the polypeptide may counteract the effects of ethylene in cortical cell division during Rhizobium infection leading to nitrogen fixation. Antibodies prepared against soybean ENOD40 identified the polypeptide in tobacco protoplasts and soybean root nodules, where it is synthesized without a pre- or pro-sequence and therefore does not involve the secretory pathway. However, no biological activity has been directly associated with the polypeptide in vivo.

CLAVATA3

The CLAVATA3 gene in Arabidopsis (3) codes for a polypeptide that appears to be the ligand for the CLAVATA1 receptor that balances cell proliferation and cell differentiation in flower meristems. The CLAVATA1 gene was first isolated in the laboratory of E. Meyerowitz (California Institute of Technology) and was shown to be a regulator of shoot and floral meristem size in Arabidopsis. CLAVATA1, a Ser-Thr transmembrane receptor with an N-terminal LRR repeat, is associated with the meristematic regions of flower primordia. Genetic analyses in the Meyerowitz laboratory demonstrated that the CLAVATA1 and the CLAVATA3 gene together control the balance between meristem cell proliferation and meristem development. CLAVATA3 codes for a polypeptide that is secreted from an adjacent meristematic region, and it appears to be the ligand for CLAVATA1. The expression of the two genes together results in the coordination of growth between the adjacent meristematic regions. This example of communication between interconnecting cells may be a prototype of what is occurring in other developmental processes that involve LRR receptors and their ligands in cells that are in close proximity.

SCR

An example of polypeptide signaling between different organs involves the polypeptide ligand, SCR, a family of small secreted Cys-rich proteins that are essential for S-locus control of incompatibility in Brassica (13). Recognition of the plants own pollen at the surface of the stigma epidermal cells leads to the inhibition of pollen growth. The laboratory of J. Nasrallah (Cornell University) discovered a small, anther-specific gene that was a consistent feature of the S haplotype, which controls pollen function in self incompatibility (SI). The SCR gene was shown to be anther-specific and was the male determinant of SI. The newly translated, small SCR protein exhibits a signal sequence, and is secreted from the developing microspores where it subsequently interacts with a receptor (SRK) to activate a signal transduction system leading to the inhibition of pollen development. Whether SCR is further processed to a smaller polypeptide signal before or after secretion is not known.

	SUMMARY

TOP INTRODUCTION POLYPEPTIDES ISOLATED BY... POLYPEPTIDE HORMONES IDENTIFIED... SUMMARY LITERATURE CITED

The polypeptide hormones that have now been identified in plants are presented in Table I. The various scenarios in plants for synthesis, storage, processing, and release of polypeptide hormones are not yet known, and from the limited data available it is apparent that no consistent patterns can yet be deduced. Prosystemin and ENOD40 lack signal sequences that would target them through the secretory system, and they may be synthesized in the cytoplasm. PSKs, CLAVATA3, and SCR pre-proteins all exhibit signal peptides, as does RALF, and are likely synthesized through the secretory system. Signal peptidase sites, where known, appear to have similar specificity requirements as the proteinases that cleave animal, yeast, and bacterial pre-proteins. Only systemin and phytosulfokines are known to be internally processed to produce smaller signaling polypeptides, although others may be. No pro-protein processing enzymes or their possible processing sites have yet been identified for any of the plant polypeptide hormones.

In addition to the polypeptide receptor proteins and genes mentioned above, several other receptor genes have been identified through mutagenesis and cloning, including Erecta (1), Crinkly4 (14), PRK1 (7), SLRK (12), and BRl1 (4). All exhibit Leu repeat domains that are typical of extracellular LRR polypeptide-binding motifs. LRRs are associated with protein-protein interactions in animals and plants, which are often found associated with polypeptide hormone receptors. Over 100 LRR-containing genes have been identified in Arabidopsis alone and it is possible that many of these proteins are receptors for polypeptide hormones.

The signaling pathway for systemin is a complex cascade that bears striking similarities to the inflammatory response of animals (10) that has raised interesting questions concerning the ancestral origins of both signaling systems. If the two pathways are found to share a common ancestral origin, then it must be established whether other polypeptide signals and signaling pathways in plants also track back to ancestral origins common to plants and animals. It will be important not only to investigate the occurrence of polypeptide hormones, their receptors, and their signaling pathways throughout the plant kingdom, but also to begin to understand how various stimuli cause the release of these signals and orchestrate their activities within the overall environmental and developmental status of the plants. The understanding of the scope and roles of polypeptide hormones in plants and their relationships with other plant hormones and signals should provide new insights into many of the complex signaling networks that orchestrate plant growth and development, and the responses of plants to biotic and abiotic stresses.

	FOOTNOTES

¹ This work was supported in part by Project 1791, by the College of Agriculture and Home Economics, by Washington State University, by the National Science Foundation (grant no. IBN 9601099), and by the U.S. Department of Agriculture National Research Initiative (grant no. 9801502).

^* Corresponding author; e-mail cabudryan{at}hotmail.com; fax 509-335-7643.

	LITERATURE CITED

TOP INTRODUCTION POLYPEPTIDES ISOLATED BY... POLYPEPTIDE HORMONES IDENTIFIED... SUMMARY LITERATURE CITED

• Becraft PW, Stinard PO, McCarty DR (1996) Science 273: 1406-1409 [Abstract]
• Constabel CP, Yip L, Ryan CA (1998) Plant Mol Biol 36: 55-62 [CrossRef][Web of Science][Medline]
• Fletcher JC, Brandu U, Running MP, Simon R, Meyerowitz EM (2000) Science 283: 1911-1914 [Abstract/Free Full Text]
• Li J, Chory J (1997) Cell 90: 929-938 [CrossRef][Web of Science][Medline]
• Matsubayashi Y, Sakagami Y (1996) Proc Natl Acad Sci USA 93: 7623-7627 [Abstract/Free Full Text]
• McGurl B, Pearce G, Orozco-Cardenas M, Ryan CA (1992) Science 255: 1570-1573 [Abstract/Free Full Text]
• Mu J-H, Lee H-S, Kao T-H (1994) Plant Cell 6: 709-721 [Abstract/Free Full Text]
• Nurnberger T, Nennsties K, Jabs T, Sacks WR, Hahlbrock K, Scheel D (1994) Cell 78: 449-460 [CrossRef][Web of Science][Medline]
• Pearce G, Strydom D, Johnson S, Ryan CA (1991) Science 253: 895-898 [Abstract/Free Full Text]
• Ryan CA (2000) Biochim Biophys Acta 1477: 112-121 [CrossRef][Medline]
• Scheer JM, Ryan CA (1999) Plant Cell 11: 1525-1535 [Abstract/Free Full Text]
• Schmidt EDL, Guzzo F, Toonen MAJ, De Bries SC (1997) Development 124: 2049-2062 [Abstract]
• Schopfer CR, Nasrallah ME, Nasrallah JB (1999) Science 286: 1697-1700 [Abstract/Free Full Text]
• Torii KU, Mitsukawa N, Oosumi T, Matsuura Y, Yokoyama R, Whittier RF, Komeda Y (1996) Plant Cell 8: 735-746 [Abstract]
• van de Sande D, Pawlowski K, Czaja I, Wieneke J, Schell J, Schmidt J, Walden R, Matvienko M, Wellink J, Van Kammen A, Franssen J, Bisseling T (1996) Science 273: 370-373 [Abstract]