Hydroxyproline-rich glycopeptide signals in potato elicit signalling associated with defense against insects and pathogens.

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.

IbACP, a sixteen-amino-acid peptide isolated from Ipomoea batatas leaves, induces carcinoma cell apoptosis.

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.

GmPep914, an eight-amino acid peptide isolated from soybean leaves, activates defense-related genes.

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.

Ca2+ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca2+ channels.

A family of peptide signaling molecules (AtPeps) and their plasma membrane receptor AtPepR1 are known to act in pathogen-defense signaling cascades in plants. Little is currently known about the molecular mechanisms that link these signaling peptides and their receptor, a leucine-rich repeat receptor-like kinase, to downstream pathogen-defense responses. We identify some cellular activities of these molecules that provide the context for a model for their action in signaling cascades. AtPeps activate plasma membrane inwardly conducting Ca(2+) permeable channels in mesophyll cells, resulting in cytosolic Ca(2+) elevation. This activity is dependent on their receptor as well as a cyclic nucleotide-gated channel (CNGC2). We also show that the leucine-rich repeat receptor-like kinase receptor AtPepR1 has guanylyl cyclase activity, generating cGMP from GTP, and that cGMP can activate CNGC2-dependent cytosolic Ca(2+) elevation. AtPep-dependent expression of pathogen-defense genes (PDF1.2, MPK3, and WRKY33) is mediated by the Ca(2+) signaling pathway associated with AtPep peptides and their receptor. The work presented here indicates that extracellular AtPeps, which can act as danger-associated molecular patterns, signal by interaction with their receptor, AtPepR1, a plasma membrane protein that can generate cGMP. Downstream from AtPep and AtPepR1 in a signaling cascade, the cGMP-activated channel CNGC2 is involved in AtPep- and AtPepR1-dependent inward Ca(2+) conductance and resulting cytosolic Ca(2+) elevation. The signaling cascade initiated by AtPeps leads to expression of pathogen-defense genes in a Ca(2+)-dependent manner.

Structure-activity studies of GmSubPep, a soybean peptide defense signal derived from an extracellular protease.

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.

Structure-activity studies of RALF, Rapid Alkalinization Factor, reveal an essential--YISY--motif.

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.

A subtilisin-like protein from soybean contains an embedded, cryptic signal that activates defense-related genes.

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.

A pollen-specific RALF from tomato that regulates pollen tube elongation.

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.

PEPR2 is a second receptor for the Pep1 and Pep2 peptides and contributes to defense responses in Arabidopsis.

Pep1 is a 23-amino acid peptide that enhances resistance to a root pathogen, Pythium irregulare. Pep1 and its homologs (Pep2 to Pep7) are endogenous amplifiers of innate immunity of Arabidopsis thaliana that induce the transcription of defense-related genes and bind to PEPR1, a plasma membrane leucine-rich repeat (LRR) receptor kinase. Here, we identify a plasma membrane LRR receptor kinase, designated PEPR2, that has 76% amino acid similarity to PEPR1, and we characterize its role in the perception of Pep peptides and defense responses. Both PEPR1 and PEPR2 were transcriptionally induced by wounding, treatment with methyl jasmonate, Pep peptides, and pathogen-associated molecular patterns. The effects of Pep1 application on defense-related gene induction and enhancement of resistance to Pseudomonas syringae pv tomato DC3000 were partially reduced in single mutants of PEPR1 and PEPR2 and abolished completely in double mutants. Photoaffinity labeling and binding assays using transgenic tobacco (Nicotiana tabacum) cells expressing PEPR1 and PEPR2 clearly demonstrated that PEPR1 is a receptor for Pep1-6 and that PEPR2 is a receptor for Pep1 and Pep2. Our analysis demonstrates differential binding affinities of two receptors with a family of peptide ligands and the corresponding physiological effects of the specific receptor-ligand interactions. Therefore, we demonstrate that, through perception of Peps, PEPR1 and PEPR2 contribute to defense responses in Arabidopsis.

Isolation and characterization of hydroxyproline-rich glycopeptide signals in black nightshade leaves.

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.

Structure-activity studies of AtPep1, a plant peptide signal involved in the innate immune response.

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 170 kDa 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 15 min with a half-maximal response of 0.25 nM. 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 approximately 100 nM). AtPep1(9-23) was used for alanine-substitution analysis and revealed two important residues for activity, a serine, [A(15)]AtPep1(9-23) (12 max approximately 10nM), and a glycine, [A(17)]AtPep1(9-23) (12 max approximately 1000 nM). Neither [A(17)]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.

Six peptide wound signals derived from a single precursor protein in Ipomoea batatas leaves activate the expression of the defense gene sporamin.

A mixture of three homologous bioactive hydroxyproline-rich glycopeptides (HypSys peptides) of 18 amino acids in length, differing only at two residues, was isolated from leaves of Ipomoea batatas, the common sweet potato. One of the peptides represented over 95% of the isolated isopeptides, which, at 2.5 nm concentration, induced the expression of sporamin, a major defense protein of I. batatas. The sequence of the major isoform was used to synthesize a primer that identified a cDNA encoding a precursor protein. The protein contained six proline-rich regions whose sequences suggested that they might be HypSys defense signals. One of the encoded peptides, called IbHypSys IV, was identical to one of two minor components of the isolated isopeptides, but neither the major isopeptide nor the other minor isoform was found within the precursor. The six peptides encoded by the precursor gene were synthesized but with hydroxyproline residues at positions found in the native isoforms and lacking carbohydrate moieties. All of the peptides were biologically active when supplied to leaves of sweet potato plants. The gene is the first ortholog of the preproHypSys gene family to be found outside of the Solanaceae family, and its encoded peptide precursor is the first example in plants of a precursor protein with six potential peptide defense signals, a scenario only found previously in animals. The data indicate that multiple copies of the HypSys peptides in a single precursor may have an important role in amplifying wound signaling in leaves in response to herbivore attacks.

Systemic wound signaling in tomato leaves is cooperatively regulated by systemin and hydroxyproline-rich glycopeptide signals.

Hydroxyproline-rich glycopeptides (HypSys peptides) have been isolated recently from tobacco and tomato leaves that are powerful activators of protease inhibitor synthesis. The peptides are processed from polyprotein precursors, two from a single tobacco precursor and three from a single tomato precursor. The precursor genes are expressed in response to wounding and methyl jasmonate, similar to the expression of the systemin precursor prosystemin in tomato leaves. Here we investigate the relationships between systemin and the tomato HypSys peptides in regulating wound signaling in tomato plants. Analysis of transgenic tomato plants over-expressing sense and antisense constructs of the tomato HypSys precursor under the 35S CaMV promoter show that the transgenic plants regulate protease inhibitor gene expression in response to wounding in a manner similar to prosystemin. The evidence indicates that the expression of both the tomato HypSys precursor gene and the prosystemin gene in response to wounding are necessary for strong systemic signaling. The data supports a role for both genes in an amplification loop that up-regulates the octadecanoid pathway and the synthesis of jasmonates to effect strong systemic signaling of defense genes. This report provides the first demonstration of the involvement of two plant peptides derived from two unrelated genes in regulating long distance wound signaling in plants.

New insights into innate immunity in Arabidopsis.

The term innate immunity has been described as '. . . the surveillance system that detects the presence and nature of the infection and provides the first line of host defense . . .' (Medzhitov, 2001; Nat Rev Immunol 1: 135-145). The strategy of innate immunity is based on the recognition of constitutive and conserved molecules from pathogens by specific receptors, triggering defence responses (Medzhitov and Janeway, 2002; Science 296: 298-300). It has been only within the past few years that studies of plant innate immunity, especially in Arabidopsis, have provided important insights into molecular details that define innate immunity in plants. Here we review the innate immune response in Arabidopsis, where leucine-rich repeat (LRR) cell surface receptors play central roles in monitoring the presence of pathogen (microbe) associated molecules to initiate the rapid expression of defence genes. The PAMPS also activate the expression of genes encoding a family of endogenous peptides (AtPep1 paralogues) and their receptor (PEPR1) that amplify defence signalling through a feedback loop initiated by PAMPS. The concept of innate immunity has provided a valuable framework for researchers to re-evaluate the roles of exogenous and endogenous signals that regulate the expression of plant defensive genes.

Endogenous peptide defense signals in Arabidopsis differentially amplify signaling for the innate immune response.

AtPep1, a 23-aa peptide encoded by Arabidopsis PROPEP1, a member of a small, six-member gene family, activates expression of the defense gene PDF1.2 (encoding defensin) and its own precursor gene, PROPEP1, through the jasmonate/ethylene signaling pathway, mediated by a cell-surface receptor, PEPR1. Overexpression of two family members, PROPEP1 and PROPEP2, enhances resistance of Arabidopsis plants against the pathogen Pythium irregulare, and PROPEP2 and PROPEP3 are expressed at highly elevated levels in Arabidopsis in response to pathogen infections and to several pathogen-associated molecules (general elicitors). Here, we report that PDF1.2, PR-1 (pathogenesis protein), and PROPEP genes were differentially expressed in the leaves of intact plants sprayed with methyl jasmonate and methyl salicylate and in excised leaves supplied through cut petioles with peptides derived from the C terminus of each of the encoded proteins. The expression of PDF1.2 and PR-1 elicited by the peptides was blocked in mutant plants deficient in the jasmonate/ethylene and salicylate pathways, and in wild-type plants by treatment with diphenylene iodonium chloride, an inhibitor of hydrogen peroxide production. PROPEP1, PROPEP 2, and PROPEP3 genes appear to have roles in a feedback loop that amplifies defense signaling pathways initiated by pathogens.

Three hydroxyproline-rich glycopeptides derived from a single petunia polyprotein precursor activate defensin I, a pathogen defense response gene.

Hydroxyproline-rich glycopeptides (HypSys peptides) are recently discovered 16-20-amino acid defense signals in tobacco and tomato leaves that are derived from cell wall-associated precursors. The peptides are powerful wound signals that activate the expression of defensive genes in tobacco and tomato leaves in response to herbivore attacks. We have isolated a cDNA from petunia (Petunia hybrida) leaves encoding a putative protein of 214 amino acids that is a homolog of tobacco and tomato HypSys peptide precursors and is inducible by wounding and MeJA. The deduced protein contains a leader sequence and four predicted proline-rich peptides of 18-21 amino acids. Three of the four peptides were isolated from leaves, and each peptide contained hydroxylated prolines and glycosyl residues. Each of the peptides has a -GR- motif at its N terminus, indicating that it may be the substrate site for a processing enzyme. The peptides were active in a petunia suspension culture bioassay at nanomolar concentrations, but they did not induce the expression of defense genes that are directed against herbivores, as found in tobacco and tomato leaves. They did, however, activate expression of defensin 1, a gene associated with inducible defense responses against pathogens.

An endogenous peptide signal in Arabidopsis activates components of the innate immune response.

Innate immunity is initiated in animals and plants through the recognition of a variety of pathogen-associated molecules that in animals are called pathogen-associated molecular patterns and in plants are called elicitors. Some plant pathogen-derived elicitors have been identified as peptides, but peptide elicitors derived from the plant itself that activate defensive genes against pathogens have not been previously identified. Here, we report the isolation and characterization of a 23-aa peptide from Arabidopsis, called AtPep1, which activates transcription of the defensive gene defensin (PDF1.2) and activates the synthesis of H(2)O(2), both being components of the innate immune response. The peptide is derived from a 92-aa precursor encoded within a small gene that is inducible by wounding, methyl jasmonate, and ethylene. Constitutive expression of the AtPep1 precursor gene PROPEP1 in transgenic Arabidopsis plants causes a constitutive transcription of PDF1.2. When grown in soil, the transgenic plants exhibited an increased root development compared with WT plants and an enhanced resistance toward the root pathogen Pythium irregulare. Six paralogs of PROPEP1 are present in Arabidopsis, and orthologs have been identified in species of several agriculturally important plant families, where they are of interest for their possible use in crop improvement.

The cell surface leucine-rich repeat receptor for AtPep1, an endogenous peptide elicitor in Arabidopsis, is functional in transgenic tobacco cells.

AtPep1 is a 23-aa endogenous peptide elicitor from Arabidopsis leaves that signals the activation of components of the innate immune response against pathogens. Here, we report the isolation of an AtPep1 receptor from the surface of Arabidopsis suspension-cultured cells. An (125)I-labeled AtPep1 analog interacted with suspension-cultured Arabidopsis with a K(d) of 0.25 nM, and an (125)I-labeled azido-Cys-AtPep1 photoaffinity analog specifically labeled a membrane-associated protein of approximately 170 kDa. The labeled protein was purified to homogeneity, and its tryptic peptides were identified as gene At1g73080, which encodes a leucine-rich repeat receptor kinase, here called PEPR1. Verification of the binding protein as the receptor for AtPep1 was established by demonstrating the loss of function of microsomal membranes of two SALK insertional mutants and by a gain in function of the alkalinization response to AtPep1 by tobacco suspension-cultured cells expressing the At1g73080 transgene. Synthetic homologs of AtPep1, deduced from the C termini of six known paralogs of PROPEP1, were biologically active and were competitors of the interaction of an AtPep1 radiolabeled analog with the receptor. The data are consistent with a role for PEPR1 as the receptor for AtPep1 to amplify innate immunity in response to pathogen attacks.

Isolation and characterization of isoinhibitors of the potato protease inhibitor I family from the latex of the rubber trees, Hevea brasiliensis.

Three isoinhibitors have been isolated to homogeneity from the C-serum of the latex of the rubber tree, Hevea brasiliensis clone RRIM 600, and named HPI-1, HPI-2a and HPI-2b. The three inhibitors share the same amino acid sequence (69 residues) but the masses of the three forms were determined to be 14,893+/-10, 7757+/-5, and 7565+/-5, respectively, indicating that post-translational modifications of the protein have occurred during latex collection. One adduct could be removed by reducing agents, and was determined to be glutathione, while the other adduct could not be removed by reducing agents and has not been identified. The N-termini of the inhibitor proteins were blocked by an acetylated Ala, but the complete amino acid sequence analysis of the deblocked inhibitors by Edman degradation of fragments from endopeptidase C digestion and mass spectrometry confirmed that the three isoinhibitors were derived from a single protein. The amino acid sequence of the protein differed at two positions from the sequence deduced from a cDNA reported in GenBank. The gene coding for the inhibitor is wound-inducible and is a member of the potato inhibitor I family of protease inhibitors. The inhibitor strongly inhibited subtilisin A, weakly inhibited trypsin, and did not inhibit chymotrypsin. The amino acid residues at the reactive site P(1) and P(1)(') were determined to be Gln45 and Asp46, respectively, residues rarely reported at the reactive site in potato inhibitor I family members. Comparison of amino acid sequences revealed that the HPI isoinhibitors shared from 33% to 55% identity (50-74% similarity) to inhibitors of the potato inhibitor I family. The properties of the isoinhibitors suggest that they may play a defensive role in the latex against pathogens and/or herbivores.

The plant cell wall matrix harbors a precursor of defense signaling peptides.

Proteins of plant cell walls serve as structural macromolecules and play important roles in morphogenesis and development but have not been reported to be the origins of peptide signals that activate genes for plant defense. We report here that the mRNA coding the tomato leaf polyprotein precursor of three hydroxyproline-rich glycopeptide defense signals (called LeHypSys I, II, and III) is synthesized in phloem parenchyma cells in response to wounding, systemin, and methyl jasmonate, and the nascent protein is sequestered in the cell wall matrix. These findings indicate that the plant cell wall can play an active role in defense as a source of peptide signals for systemic wound signaling.