HypSys peptides are 18-20 amino acids glycopeptide defense signal first discovered in tobacco and tomato that activate expression of defensive genes against insect-herbivores. Discovery of their orthologs in other Solanaceaous and nonsolanaceous plants demonstrated their possible ubiquitous nature and species specific functional diversity. In our continued search to establish the paradigm of defense signalling by HypSys peptides, we isolated a cDNA from potato leaves encoding putative analogs of tomato HypSys peptides flanked by conserved proteolytic cleavage sites. The gene encoding the cDNA was a member of a gene family in the tetraploid genome of potato and its expression was transcriptionally activated by wounding and methyl jasmonate. The deduced precursor protein contained a leader peptidase splice site and three putative HypSys peptides with conserved N- and C-termini along with central proline-rich motifs. In defense signalling, the three HypSys peptides elicit H₂O₂ generation in vivo and activate several antioxidant defensive enzymes in young potato leaves. Similar to potato systemin, the HypSys peptides activate the expression of octadecanoid pathway genes and protease inhibitors for insect defense. In addition, the HypSys peptides also activate the essential genes of the innate pathogen defense response in young potato leaves, acting as common elicitors of signalling associated with anti-herbivore and anti-pathogen defense in potato.

A 16-amino-acid peptide was isolated from the leaves of sweet potato. The peptide caused a rapid alkalinization response in tomato suspension culture media, a characteristic of defense peptides in plants. No post-translational modification was observed on the peptide according to MALDI-MS analysis. We have named the peptide Ipomoea batatas anti-cancer peptide (IbACP). IbACP also was shown with the ability to dose-dependently inhibit Panc-1, a pancreatic cancer line, cell proliferation. The morphological observations of the Panc-1 cells by phase contrast microscopy showed significant changes after treatment with IbACP. Moreover, caspase-3 and PARP [poly(ADP-ribose) polymerase] were activated by IbACP treatment, followed by cell death. An increase in the levels of cleaved caspase-3 and -9 was also detected by an immunoblot assay after treatment with IbACP. In addition, genomic DNA fragmentation and decreased cellular proliferation were induced when IbACP was supplied to the Panc-1 cells, further demonstrating its biological relevance. The combined data indicates that IbACP peptide may have an important role in the regulation of cellular proliferation by inducing and promoting apoptosis through the mitochondrial apoptotic pathway. This report also showed that IbACP peptide contains potent anti-cancer effects and may play an important role in herbal medicine development.

Ever since the seminal discovery of systemic wound signaling in tomato and potato plants by Green and Ryan (Science 1972), a number of candidate systemic wound signals have been proposed. These can be classified into three groups: (1) Chemical signals, including the alarm hormone systemin and other peptide hormones, jasmonic acid is a phytohormone, as well as reactive oxygen species (ROS); (2) physical signals, including electrical and hydraulic signals; and (3) herbivore-induced volatile compounds, including green leafy volatiles and terpenes. These signals are generated at or close to the site of herbivore-inflicted injury and systemically move to target tissues where they induce defense responses. Chemical and physical signals depend on the connectivity of the plant body, whereas volatile compounds are released into the airspace. Different plants with different morphologies and ecological niches employ different modes of systemic signaling.

Insect-induced defenses occur in nearly all plants and are regulated by conserved signaling pathways. As the first described plant peptide signal, systemin regulates antiherbivore defenses in the Solanaceae, but in other plant families, peptides with analogous activity have remained elusive. In the current study, we demonstrate that a member of the maize (Zea mays) plant elicitor peptide (Pep) family, ZmPep3, regulates responses against herbivores. Consistent with being a signal, expression of the ZmPROPEP3 precursor gene is rapidly induced by Spodoptera exigua oral secretions. At concentrations starting at 5 pmol per leaf, ZmPep3 stimulates production of jasmonic acid, ethylene, and increased expression of genes encoding proteins associated with herbivory defense. These include proteinase inhibitors and biosynthetic enzymes for production of volatile terpenes and benzoxazinoids. In accordance with gene expression data, plants treated with ZmPep3 emit volatiles similar to those from plants subjected to herbivory. ZmPep3-treated plants also exhibit induced accumulation of the benzoxazinoid phytoalexin 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside. Direct and indirect defenses induced by ZmPep3 contribute to resistance against S. exigua through significant reduction of larval growth and attraction of Cotesia marginiventris parasitoids. ZmPep3 activity is specific to Poaceous species; however, peptides derived from PROPEP orthologs identified in Solanaceous and Fabaceous plants also induce herbivory-associated volatiles in their respective species. These studies demonstrate that Peps are conserved signals across diverse plant families regulating antiherbivore defenses and are likely to be the missing functional homologs of systemin outside of the Solanaceae.

Only a handful of endogenous peptide defense signals have been isolated from plants. Herein, we report a novel peptide from soybean (Glycine max) leaves that is capable of alkalinizing the media of soybean suspension cells, a response that is generally associated with defense peptides. The peptide, DHPRGGNY, was synthesized and found to be active at 0.25 nM and requiring only 5 to 10 min to obtain a maximal pH change. The peptide is located on the carboxy-terminal end of a 52-amino acid precursor protein (Glyma12g00990) deduced from the soybean genome project. A search of the soybean databank revealed a homolog (Glyma09g36370) that contained a similar peptide, DLPRGGNY, which was synthesized and shown to have identical activity. The peptides, designated GmPep914 (DHPRGGNY) and GmPep890 (DLPRGGNY), were capable of inducing the expression of both Glyma12g00990 (GmPROPEP914) and Glyma09g36370 (GmPROPEP890) in cultured soybean suspension cells within 1 h. Both peptides induced the expression of defense genes, including CYP93A1, a cytochrome P450 gene involved in phytoalexin synthesis, chitinaseb1-1, a chitinase involved in pathogen defense, and Glycine max chalcone synthase1 (Gmachs1), chalcone synthase, involved in phytoalexin production. Both GmPROPEP914 and GmPROPEP890 were highly expressed in the roots, relative to the aerial portions of the plant. However, treatment of the aerial portion of soybean plants with hormones involved in elicitation of defense responses revealed a significant increase in expression levels of GmPROPEP914 and GmPROPEP890. A search of gene databases revealed homologous sequences in other members of the Fabales and also in the closely related Cucurbitales but not in any other order of plants.

Systemin, an 18-amino acid signaling peptide isolated from tomato leaves, has been found to be an integral component of the jasmine acid signaling pathway, leading to the synthesis of protease inhibitors (PIs). The discovery of systemin has led to a search for other peptide signals involved in defense in the Solanaceae and in other plant families. A new class of peptides having similar signaling properties but little sequence homology to systemin have been found and termed hydroxyproline-rich glycopeptide systemins (HypSys). These small (18-20 amino acids) glycopeptides, like systemin, are derived from larger precursor proteins (proHypSys) and until recently were thought to function only in protection from herbivore attack. However, HypSys peptides isolated from petunia induced the defensin gene, known for its involvement in pathogen defense. More recently, a HypSys glycopeptide was isolated from sweet potato, a member of the Convolvulaceae family and found to induce the sporamin gene which codes for the major storage protein in tubers with trypsin inhibitor activity. These recent discoveries expand the function and range of the HypSys family of glycopeptides and establish these unique inducible signaling molecules as potential components of defense pathways throughout the Eudicots. Herein we review the signaling and structural properties of systemin and the HypSys glycopeptides and their roles in the induction of PIs.

GmSubPep, a 12-amino acid peptide isolated from soybean leaves, induces the expression of genes in soybean suspension-cultured cells that encode proteins involved in defense against pathogens. The peptide is derived from an extracellular subtilisin-like protease (subtilase) and binds a putative cell-surface receptor that initiates a defense signaling cascade. Interaction of the peptide with its receptor results in alkalinization of soybean suspension cell media which can be utilized to analyze the kinetics of receptor binding. Substitutions of alanine at each of the 12 amino acid positions revealed that the amino acids at positions 10 (arginine) and 12 (histidine) were essential for activity. Both analogs were able to reduce the physiological effects of GmSubPep associated with receptor binding. Deletion of the C-terminal histidine [GmSubPep(1-11)] abolished the alkalinizing activity and this peptide was also a strong competitor for receptor binding. Deletion of N-terminal amino acids from GmSubPep caused a sequential loss of activity with no alkalinizing activity for GmSubPep(4-12). However, the N-terminal deleted peptides did not compete with GmSubPep for receptor binding. Further modifications at the arginine-10 position indicated that an ionizable proton was not essential for activity as an attenuated response was found for a citrulline substitution. Substitution of the histidine-12 with methylated histidine at position N-1 of the imidazole group abolished activity, whereas substitution at N-3 was completely active, indicating that the N-3 analog retains important receptor binding properties. This study indicates that the extreme C-terminal of GmSubPep has important signal transduction properties while the C-terminal is essential for receptor interaction.

Among the arsenal of plant-derived compounds activated upon attack by herbivores and pathogens are small peptides that initiate and amplify defense responses. However, only a handful of plant signaling peptides have been reported. Here, we have isolated a 12-aa peptide from soybean (Glycine max) leaves that causes a pH increase of soybean suspension-cultured cell media within 10 min at low nanomolar concentrations, a response that is typical of other endogenous peptide elicitors and pathogen-derived elicitors. The amino acid sequence was determined and was found to be derived from a member of the subtilisin-like protease (subtilase) family. The sequence of the peptide was located within a region of the protein that is unique to subtilases in legume plants and not found within any other plant subtilases thus far identified. We have named this peptide signal Glycine max Subtilase Peptide (GmSubPep). The gene (Glyma18g48580) was expressed in all actively growing tissues of the soybean plant. Although transcription of Glyma18g48580 was not induced by wounding, methyl jasmonate, methyl salicylate, or ethephon, synthetic GmSubPep peptide, when supplied to soybean cultures, induced the expression of known defense-related genes, such as Cyp93A1, Chib-1b, PDR12, and achs. GmSubPep is a unique plant defense peptide signal, cryptically embedded within a plant protein with an independent metabolic role, providing insights into plant defense mechanisms.

Rapid Alkalinization Factors (RALFs) are plant peptides that rapidly increase the pH of plant suspension cell culture medium and inhibit root growth. A pollen-specific tomato (Solanum lycopersicum) RALF (SlPRALF) has been identified. The SlPRALF gene encodes a preproprotein that appears to be processed and released from the pollen tube as an active peptide. A synthetic SlPRALF peptide based on the putative active peptide did not affect pollen hydration or viability but inhibited the elongation of normal pollen tubes in an in vitro growth system. Inhibitory effects of SlPRALF were detectable at concentrations as low as 10 nm, and complete inhibition was observed at 1 mum peptide. At least 10-fold higher levels of alkSlPRALF, which lacks disulfide bonds, were required to see similar effects. A greater effect of peptide was observed in low-pH-buffered medium. Inhibition of pollen tube elongation was reversible if peptide was removed within 15 min of exposure. Addition of 100 nm SlPRALF to actively growing pollen tubes inhibited further elongation until tubes were 40 to 60 mum in length, after which pollen tubes became resistant to the peptide. The onset of resistance correlated with the timing of the exit of the male germ unit from the pollen grain into the tube. Thus, exogenous SlPRALF acts as a negative regulator of pollen tube elongation within a specific developmental window.

Rapid Alkalinization Factor (RALF) is a 49-amino acid peptide initially isolated from tobacco leaves that is capable of arresting both root and pollen tube growth With suspension cells addition of RALF causes an elevation of the pH of the extracellular media caused by the blockage of a proton pump RALF associates with a putative receptor(s) on the surface of the plant cell initiating a signal transduction pathway Although the exact function(s) of RALFs are unknown its presence throughout the plant kingdom attests to its importance in some type of basic regulatory role In the present study deletion and substitution analyses of RALF reveal a specific - YISY - motif located at positions 5 through 8 from the N-terminus highly conserved within the plant kingdom which is a requirement for productive binding of RALF to its putative receptor Replacement of isoleucine with alanine in the - YISY - motif caused a severe reduction in alkalinization of suspension cell media and a loss of root growth inhibition with tomato seedlings (C) 2010 Elsevier Inc All rights reserved

Peptide signaling regulates a variety of developmental processes and environmental responses in plants. For example, the peptide systemin induces the systemic defense response in tomato and defensins are small cysteine-rich proteins that are involved in the innate immune system of plants. The CLAVATA3 peptide regulates meristem size and the SCR peptide is the pollen self-incompatibility recognition factor in the Brassicaceae. LURE peptides produced by synergid cells attract pollen tubes to the embryo sac. RALFs are a recently discovered family of plant peptides that play a role in plant cell growth.

Peptide hormones are extracellular signaling molecules that are commonly found in animals and plants (38). A broad spectrum of physiological processes are regulated by peptide signals including metabolism, cell division, growth, pain, well being, reproduction and immunity, as examples. The first peptide hormone, insulin, was isolated in 1921 (1), which led to the isolation of hundreds of peptide signals from animals and, more recently, to their isolation from plants (34). The majority of our fundamental knowledge about peptide signals has been from studies in animals, where peptide hormones are synthesized through the secretory pathway and are derived from larger precursor proteins by proteolytic processing enzymes. Two general classes of peptide hormones are found in animals, endocrine hormones (10) and membrane-anchored cytokines and growth factors (21).

This chapter contains sections titled: Introduction Defense-Related Peptides Peptides Involved in Growth and Development Peptides Involved in Self-Recognition Methods in Plant Regulatory Peptide Research Conclusions References

A gene encoding a preprohydroxyproline-rich systemin, SnpreproHypSys, was identified from the leaves of black nightshade (Solanum nigrum), which is a member of a small gene family of at least three genes that have orthologs in tobacco (Nicotiana tabacum; NtpreproHypSys), tomato (Solanum lycopersicum; SlpreproHypSys), petunia (Petunia hybrida; PhpreproHypSys), potato (Solanum tuberosum; PhpreproHypSys), and sweet potato (Ipomoea batatas; IbpreproHypSys). SnpreproHypSys was induced by wounding and by treatment with methyl jasmonate. The encoded precursor protein contained a signal sequence and was posttranslationally modified to produce three hydroxyproline-rich glycopeptide signals (HypSys peptides). The three HypSys peptides isolated from nightshade leaf extracts were called SnHypSys I (19 amino acids with six pentoses), SnHypSys II (20 amino acids with six pentoses), and SnHypSys III (20 amino acids with either six or nine pentoses) by their sequential appearance in SnpreproHypSys. The three SnHypSys peptides were synthesized and tested for their abilities to alkalinize suspension culture medium, with synthetic SnHypSys I demonstrating the highest activity. Synthetic SnHypSys I was capable of inducing alkalinization in other Solanaceae cell types (or species), indicating that structural conformations within the peptides are recognized by the different cells/species to initiate signal transduction pathways, apparently through recognition by homologous receptor(s). To further demonstrate the biological relevance of the SnHypSys peptides, the early defense gene lipoxygenase D was shown to be induced by all three synthetic peptides when supplied to excised nightshade plants.

AtPep1, a 23-amino acid peptide recently isolated from Arabidopsis leaves, induces the expression of the genes encoding defense proteins against pathogens. We investigated the structure–activity relationship of AtPep1 with its receptor, a 170kDa leucine-rich repeat receptor kinase (AtPEPR1) by utilizing a suspension cell assay (the alkalinization assay). Binding of AtPep1 to AtPEPR1 on the cell surface is accompanied by an increase in the pH of Arabidopsis suspension cell media by 1 pH unit in 15min with a half-maximal response of 0.25nM. Sequential removal of N-terminal amino acids had little effect on activity until the peptide was reduced to 15 amino acids [AtPep1(9–23)], which decreased the activity by less than one order of magnitude. Activity was completely abolished when nine C-terminal amino acids remained. Removal of the C-terminal asparagine from AtPep1(9–23), resulted in a decrease in activity (12 max∼100nM). AtPep1(9–23) was used for alanine-substitution analysis and revealed two important residues for activity, a serine, [A15]AtPep1(9–23) (12 max∼10nM), and a glycine, [A17]AtPep1(9–23) (12 max∼1000nM). Neither [A17]AtPep1(9–23) nor the C-terminal truncated AtPep1, AtPep1(9–22), were able to compete with AtPep1(9–23) in the alkalinization assay. The importance of the glycine residue for binding to the AtPep receptor was also confirmed by competition assays using radiolabeled AtPep1. d-Alanine or 2-methylalanine substituted at the glycine position displayed only a slight decrease in activity whereas l- and d-proline substitution caused a loss of activity. Homologs of AtPep1 identified in Arabidopsis and other species revealed a strict conservation of the glycine residue.

Hydroxyproline-rich systemins (HypSys) are small defense signaling glycopeptides found within the Solanaceae family that until recently were thought to only induce defense genes to herbivore attack. The glycopeptides are processed from larger proproteins with up to 3 different glycopeptides being processed out of a single precursor protein. A conserved central hydroxyproline motif within each HypSys is the site of pentose sugar attachment. Recently, it was found that in Petunia hybrida, these defense signaling glycopeptides did not induce protease inhibitor but instead, increased levels of defensin, a gene that is involved in pathogen attack. More recently, a HypSys peptide was isolated from Ipomoea batatas (sweet potato) of the Convolvulaceae family and found to induce sporamin. The proprotein precursor contained six putative peptide signals and had a propeptidase processing region with homology to solanaceous proHypSys. Thus, the HypSys defense peptides are no longer confined to defense against herbivory or exclusivity to the Solanaceae family, redefining both function and dispersion.