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Clarence A. Ryan - One of the best experts on this subject based on the ideXlab platform.
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CHAPTER 10 – Systemins
Handbook of Biologically Active Peptides, 2020Co-Authors: Gregory Pearce, Javier Narváez-vásquez, Clarence A. RyanAbstract:Systemins are a family of functionally related peptide signals in plants of 15 to 20 amino acids in length, derived from precursors that activate defensive genes in response to herbivore and pathogen attacks [25]. Two subfamilies of nonhomologous peptides are included in the Systemin superfamily, based on several structural and functional properties. Members of one subfamily are homologous to tomato Systemin [21], now called Le Sys (the name derived from Lycopersicon esculentum Systemin), with members in potato, pepper, and nightshade [ 3]. The precursors of this subfamily lack leader sequences and are synthesized in the cytosol and are not posttranslationally modified. The second subfamily members are hydroxyproline-rich glycopeptides (called HypSys peptides) that are derived from precursor proteins that have homologs in tobacco [19], tomato [20], petunia, nightshade, and potato [G. Pearce and C. A. Ryan, unpublished data). Members of each subfamily exhibit the following characteristics: They are all small peptides of about 15–20 amino acids each, are derived from larger precursor proteins, contain multiple proline and/or hydroxyproline residues, are active at low nM concentrations, appear to be receptor mediated, and the precursor genes are all activated by methyl jasmonate.
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Systemic wound signaling in tomato leaves is cooperatively regulated by Systemin and hydroxyproline-rich glycopeptide signals
Plant Molecular Biology, 2007Co-Authors: Javier Narváez-vásquez, Martha L. Orozco-cárdenas, Clarence A. RyanAbstract: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.
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Systemins: a functionally defined family of peptide signals that regulate defensive genes in Solanaceae species.
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Clarence A. Ryan, Gregory PearceAbstract:Numerous plant species have been known for decades that respond to herbivore attacks by systemically synthesizing defensive chemicals to protect themselves from predators. The nature of systemic wound signals remained obscure until 1991, when an 18-aa peptide called Systemin was isolated from tomato leaves and shown to be a primary signal for systemic defense. More recently, two new hydroxyproline-rich, glycosylated peptide defense signals have been isolated from tobacco leaves, and three from tomato leaves. Because of their origins in plants, small sizes, hydroxyproline contents (tomato Systemin is proline-rich), and defense-signaling activities, the new peptides are included in a functionally defined family of signals collectively called Systemins. Here, we review structural and biological properties of the Systemin family, and discuss their possible roles in systemic wound signaling.
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generation of Systemin signaling in tobacco by transformation with the tomato Systemin receptor kinase gene
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Justin M Scheer, Gregory Pearce, Clarence A. RyanAbstract:Abstract The tomato Systemin receptor, SR160, a plasma membrane-bound, leucine-rich repeat receptor kinase that signals systemic plant defense, and the brassinolide (BL) receptor, BRI1, that regulates developmental processes, have been shown recently to have identical amino acid sequences. We report herein that tobacco, a solanaceous species that does not express a Systemin precursor gene nor responds to Systemin, when transformed with the SR160 receptor gene, expresses the gene in suspension-cultured cells, evidenced by mRNA and protein analyses and photoaffinity-labeling experiments. Additionally, Systemin induced an alkalinization response in the transgenic tobacco cells similar to that found in tomato cells, but not in WT cells. The gain in function in tobacco cells indicates that early steps of the Systemin signaling pathway found in tomato are present in tobacco cells. A tomato line, cu-3, in which a mutation in the BRI1 gene has rendered the plant nonfunctional in BL signaling, exhibits a severely reduced response to Systemin. In leaves of WT tomato plants, BL strongly and reversibly antagonized systemic signaling by Systemin. The results suggest that the Systemin-mediated systemic defense response may have evolved in some solanaceous species by co-opting the BRI1 receptor and associated components for defense signaling.
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hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding Systemin and methyl jasmonate
The Plant Cell, 2001Co-Authors: Martha L Orozcocardenas, Javier Narvaezvasquez, Clarence A. RyanAbstract:The systemic accumulation of both hydrogen peroxide (H2O2) and proteinase inhibitor proteins in tomato leaves in response to wounding was inhibited by the NADPH oxidase inhibitors diphenylene iodonium (DPI), imidazole, and pyridine. The expression of several defense genes in response to wounding, Systemin, oligosaccharides, and methyl jasmonate also was inhibited by DPI. These genes, including those of four proteinase inhibitors and polyphenol oxidase, are expressed within 4 to 12 hr after wounding. However, DPI did not inhibit the wound-inducible expression of genes encoding proSystemin, lipoxygenase, and allene oxide synthase, which are associated with the octadecanoid signaling pathway and are expressed 0.5 to 2 hr after wounding. Accordingly, treatment of plants with the H2O2-generating enzyme glucose oxidase plus glucose resulted in the induction of only the later-expressed defensive genes and not the early-expressed signaling-related genes. H2O2 was cytochemically detected in the cell walls of vascular parenchyma cells and spongy mesophyll cells within 4 hr after wounding of wild-type tomato leaves, but not earlier. The cumulative results suggest that active oxygen species are generated near cell walls of vascular bundle cells by oligogalacturonide fragments produced by wound-inducible polygalacturonase and that the resulting H2O2 acts as a second messenger for the activation of defense genes in mesophyll cells. These data provide a rationale for the sequential, coordinated, and functional roles of Systemin, jasmonic acid, oligogalacturonides, and H2O2 signals for systemic signaling in tomato plants in response to wounding.
Johannes W Stratmann - One of the best experts on this subject based on the ideXlab platform.
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Systemin and jasmonic acid regulate constitutive and herbivore induced systemic volatile emissions in tomato solanum lycopersicum
Phytochemistry, 2010Co-Authors: David C Degenhardt, Johannes W Stratmann, Sarah Refihind, David E LincolnAbstract:Abstract Transgenic tomato ( Solanum lycopersicum ) plants that overexpress the ProSystemin gene ( 35S::PS ) and plants with a mutation in the JA biosynthetic pathway ( def1 ) are known to exhibit a constitutive or reduced wound response, respectively. Here it is demonstrated that several independent 35S::PS lines emit high levels of specific volatiles in addition to increased accumulation of proteinase inhibitors (PIs). Furthermore, the temporal dynamics of systemically induced volatile compounds including green-leaf volatiles, terpenes, and shikimic acid-derivatives from 35S::PS and def1 plants in response to herbivore wounding and treatment with jasmonic acid (JA) are described. Application of JA induced defense protein accumulation and volatile emissions in wild type plants, but did not further increase systemic volatile emissions from 35S::PS plants. Wounding by Manduca sexta larvae induced synthesis of defense proteins and emission of volatiles in wild type plants, but not in def1 plants. Application of jasmonic acid restored the local and systemic accumulation of defense proteins in def1 , as well as enhanced herbivore-induced volatile emissions. These results provide strong support for the role of proSystemin- and JA-signaling in the regulation of volatile emissions in tomato plants.
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tissue type specific Systemin perception and the elusive Systemin receptor
Plant Signaling & Behavior, 2010Co-Authors: Sarah R Hind, Robert Malinowski, Roopa Yalamanchili, Johannes W StratmannAbstract:Systemin is a wound signaling peptide from tomato that is important for plant defenses against herbivory. The Systemin receptor was initially identified as the tomato homolog of the brassinosteroid receptor BRI1, but genetic evidence argued against this finding. However, we found that BRI1 may function as an inappropriate Systemin binding protein that does not activate the Systemin signaling pathway. Here we provide evidence that Systemin perception is localized in a tissue-type specific manner. Mesophyll protoplasts were not sensitive to Systemin, while they responded to other elicitors. We hypothesize that the elusive Systemin receptor is a protein with high similarity to BRI1 which is specifically localized in vascular tissue like the Systemin precursor proSystemin. Binding of Systemin to BRI1 may be an artifact of transgenic BRI1-overexpressing plants, but does not take place in wild type tomato cells.
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the tomato brassinosteroid receptor bri1 increases binding of Systemin to tobacco plasma membranes but is not involved in Systemin signaling
Plant Molecular Biology, 2009Co-Authors: Robert Malinowski, Rebecca Higgins, Laverne Piper, Azka Nazir, Vikramjit S Bajwa, Steven D Clouse, Paul R Thompson, Johannes W StratmannAbstract:The tomato wound signal Systemin is perceived by a specific high-affinity, saturable, and reversible cell surface receptor. This receptor was identified as the receptor-like kinase SR160, which turned out to be identical to the brassinosteroid receptor BRI1. Recently, it has been shown that the tomato bri1 null mutant cu3 is as sensitive to Systemin as wild type plants. Here we explored these contradictory findings by studying the responses of tobacco plants (Nicotiana tabacum) to Systemin. A fluorescently-labeled Systemin analog bound specifically to plasma membranes of tobacco suspension-cultured cells that expressed the tomato BRI1-FLAG transgene, but not to wild type tobacco cells. On the other hand, signaling responses to Systemin, such as activation of mitogen-activated protein kinases and medium alkalinization, were neither increased in BRI1-FLAG-overexpressing tobacco cells nor decreased in BRI1-silenced cells as compared to levels in untransformed control cells. Furthermore, in transgenic tobacco plants BRI1-FLAG became phosphorylated on threonine residues in response to brassinolide application, but not in response to Systemin. When BRI1 transcript levels were reduced by virus-induced gene silencing in tomato plants, the silenced plants displayed a phenotype characteristic of bri1 mutants. However, their response to overexpression of the ProSystemin transgene was the same as in control plants. Taken together, our data suggest that BRI1 can function as a Systemin binding protein, but that binding of the ligand does not transduce the signal into the cell. This unusual behavior and the nature of the elusive Systemin receptor will be discussed.
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micro electrode flux estimation confirms that the solanum pimpinellifolium cu3 mutant still responds to Systemin
Plant Physiology, 2007Co-Authors: Frank C Lanfermeijer, Johannes W Stratmann, Robert Malinowski, Marten Staal, Theo J M ElzengaAbstract:In this study, we introduce the Micro-Electrode Ion Flux Estimation technique as a sensitive and accurate technique to study Systemin-induced changes in ion fluxes from isolated nearly intact plant tissues. Our results demonstrate the effectiveness and value of the Micro-Electrode Ion Flux Estimation technique to monitor and characterize those elicitor-induced ion flux changes from intact tissues. We used the method to monitor the Systemin-induced changes in ion fluxes from leaf tissue of various plant species, including wild-type and cu3 mutant tomato (Solanum pimpinellifolium) plants, and confirm previous observations, but now in intact leaf tissue. Upon exposure of leaf tissue of plant species from the subtribe solaneae to Systemin, the H+ influx and K+ efflux were transiently strongly increased. Plant species of other clades did not show a response upon Systemin exposure. Although it has been reported that the gene containing the cu3 null mutation is identical to the SR160/tBRI1 gene, which encodes the Systemin/brassinosteroid receptor and is essential in Systemin and brassinosteroid perception, we observed no differences in the response of H+ and K+ fluxes from both wild-type and mutant leaf tissue to Systemin. Also, the effects of various pharmacological effectors on Systemin-induced flux changes were similar. Moreover, a SR160/tBRI1 transgene-containing tobacco (Nicotiana tabacum) line was insensitive to Systemin, whereas both this line and its wild-type predecessor were responsive to the elicitor flg22. Our results support the conclusion that the Cu3 receptor of tomato is not the Systemin receptor, and, hence, another receptor is the principal Systemin receptor.
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tomato mapks lempk1 lempk2 and lempk3 function in the Systemin mediated defense response against herbivorous insects
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Pramod K Kandoth, Gregg A Howe, David E Lincoln, Stefanie Ranf, Suchita S Pancholi, Sastry S Jayanty, Michael D Walla, Wayne Miller, Johannes W StratmannAbstract:Systemin is a wound-signaling peptide that mediates defenses of tomato plants against herbivorous insects. Perception of Systemin by the membrane-bound receptor SR160 results in activation of MAPKs, synthesis of jasmonic acid (JA), and expression of defense genes. To test the function of MAPKs in the response to Systemin, we used virus-induced gene silencing (VIGS) in plants that overexpress the Systemin precursor proSystemin (35S::prosys plants). These transgenic plants accumulate high levels of defense proteins and exhibit increased resistance to herbivorous insects. Cosilencing of the MAPKs MPK1 and MPK2 reduced MPK1/2 kinase activity, JA biosynthesis, and expression of JA-dependent defense genes. Application of methyl-JA restored the full defense response. These data show that MPK1 and MPK2 are essential components of the Systemin signaling pathway and most likely function upstream of JA biosynthesis. MPK1 and MPK2 are 95% identical at the amino acid level. Specific VIGS of only MPK1 or MPK2 resulted in the same reduction of defense gene expression as cosilencing of MPK1 and MPK2, indicating that gene dosage effects may be important for MPK signaling. In addition, VIGS of the closely related MPK3 also reduced Systemin-induced defense responses. The function of MPK1/2 and orthologs in pathogen-induced defenses is well established. Here we show that cosilencing of MPK1 and MPK2 compromised proSystemin-mediated resistance to Manduca sexta (Lepidoptera) herbivory, demonstrating that MPK1 and MPK2 are also required for successful defenses against herbivorous insects.
Gerard J Bishop - One of the best experts on this subject based on the ideXlab platform.
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tomato bri1 and Systemin wound signalling
Plant Signaling & Behavior, 2008Co-Authors: Nicholas Holton, Kate Harrison, Takao Yokota, Gerard J BishopAbstract:Brassinosteroids (BRs) are perceived by Brassinosteroid Insensitive 1 (BRI1), that encodes a leucine-rich repeat receptor kinase. Tomato BRI1 has previously been implicated in both Systemin and BR signalling. The role of tomato BRI1 in BR signalling was confirmed, however it was found not to be essential for Systemin/wound signalling. Tomato roots were shown to respond to Systemin but this response varied according to the species and growth conditions. Overall the data indicates that mutants defective in tomato BRI1 are not defective in Systemin-induced wound signalling and that Systemin perception can occur via a non-BRI1 mechanism.
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tomato brassinosteroid insensitive1 is required for Systemin induced root elongation in solanum pimpinellifolium but is not essential for wound signaling
The Plant Cell, 2007Co-Authors: Nicholas Holton, Kate Harrison, Gerard J Bishop, Ana I Canodelgado, Teresa Montoya, Joanne ChoryAbstract:The tomato Leu-rich repeat receptor kinase BRASSINOSTEROID INSENSITIVE1 (BRI1) has been implicated in both peptide (Systemin) and steroid (brassinosteroid [BR]) hormone perception. In an attempt to dissect these signaling pathways, we show that transgenic expression of BRI1 can restore the dwarf phenotype of the tomato curl3 (cu3) mutation. Confirmation that BRI1 is involved in BR signaling is highlighted by the lack of BR binding to microsomal fractions made from cu3 mutants and the restoration of BR responsiveness following transformation with BRI1. In addition, wound and Systemin responses in the cu3 mutants are functional, as assayed by proteinase inhibitor gene induction and rapid alkalinization of culture medium. However, we observed BRI1-dependent root elongation in response to Systemin in Solanum pimpinellifolium. In addition, ethylene perception is required for normal Systemin responses in roots. These data taken together suggest that cu3 is not defective in Systemin-induced wound signaling and that Systemin perception can occur via a non-BRI1 mechanism.
Gregory Pearce - One of the best experts on this subject based on the ideXlab platform.
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CHAPTER 10 – Systemins
Handbook of Biologically Active Peptides, 2020Co-Authors: Gregory Pearce, Javier Narváez-vásquez, Clarence A. RyanAbstract:Systemins are a family of functionally related peptide signals in plants of 15 to 20 amino acids in length, derived from precursors that activate defensive genes in response to herbivore and pathogen attacks [25]. Two subfamilies of nonhomologous peptides are included in the Systemin superfamily, based on several structural and functional properties. Members of one subfamily are homologous to tomato Systemin [21], now called Le Sys (the name derived from Lycopersicon esculentum Systemin), with members in potato, pepper, and nightshade [ 3]. The precursors of this subfamily lack leader sequences and are synthesized in the cytosol and are not posttranslationally modified. The second subfamily members are hydroxyproline-rich glycopeptides (called HypSys peptides) that are derived from precursor proteins that have homologs in tobacco [19], tomato [20], petunia, nightshade, and potato [G. Pearce and C. A. Ryan, unpublished data). Members of each subfamily exhibit the following characteristics: They are all small peptides of about 15–20 amino acids each, are derived from larger precursor proteins, contain multiple proline and/or hydroxyproline residues, are active at low nM concentrations, appear to be receptor mediated, and the precursor genes are all activated by methyl jasmonate.
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Systemin hydroxyproline rich Systemin and the induction of protease inhibitors
Current Protein & Peptide Science, 2011Co-Authors: Gregory PearceAbstract:: 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.
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Systemins: a functionally defined family of peptide signals that regulate defensive genes in Solanaceae species.
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Clarence A. Ryan, Gregory PearceAbstract:Numerous plant species have been known for decades that respond to herbivore attacks by systemically synthesizing defensive chemicals to protect themselves from predators. The nature of systemic wound signals remained obscure until 1991, when an 18-aa peptide called Systemin was isolated from tomato leaves and shown to be a primary signal for systemic defense. More recently, two new hydroxyproline-rich, glycosylated peptide defense signals have been isolated from tobacco leaves, and three from tomato leaves. Because of their origins in plants, small sizes, hydroxyproline contents (tomato Systemin is proline-rich), and defense-signaling activities, the new peptides are included in a functionally defined family of signals collectively called Systemins. Here, we review structural and biological properties of the Systemin family, and discuss their possible roles in systemic wound signaling.
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generation of Systemin signaling in tobacco by transformation with the tomato Systemin receptor kinase gene
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Justin M Scheer, Gregory Pearce, Clarence A. RyanAbstract:Abstract The tomato Systemin receptor, SR160, a plasma membrane-bound, leucine-rich repeat receptor kinase that signals systemic plant defense, and the brassinolide (BL) receptor, BRI1, that regulates developmental processes, have been shown recently to have identical amino acid sequences. We report herein that tobacco, a solanaceous species that does not express a Systemin precursor gene nor responds to Systemin, when transformed with the SR160 receptor gene, expresses the gene in suspension-cultured cells, evidenced by mRNA and protein analyses and photoaffinity-labeling experiments. Additionally, Systemin induced an alkalinization response in the transgenic tobacco cells similar to that found in tomato cells, but not in WT cells. The gain in function in tobacco cells indicates that early steps of the Systemin signaling pathway found in tomato are present in tobacco cells. A tomato line, cu-3, in which a mutation in the BRI1 gene has rendered the plant nonfunctional in BL signaling, exhibits a severely reduced response to Systemin. In leaves of WT tomato plants, BL strongly and reversibly antagonized systemic signaling by Systemin. The results suggest that the Systemin-mediated systemic defense response may have evolved in some solanaceous species by co-opting the BRI1 receptor and associated components for defense signaling.
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proteinase inhibitor inducing activity of the prohormone proSystemin resides exclusively in the c terminal Systemin domain
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: James E Dombrowski, Gregory Pearce, Clarence A. RyanAbstract:ProSystemin is the 200-amino acid precursor of the 18-amino acid polypeptide defense hormone, Systemin. Herein, we report that proSystemin was found to be as biologically active as Systemin when assayed for proteinase inhibitor induction in young tomato plants and nearly as active in the alkalinization response in Lycopersicon esculentum suspension-cultured cells. Similar to many animal prohormones that harbor multiple signals, the Systemin precursor contains five imperfect repetitive domains N-terminal to a single Systemin domain. Whether the five repetitive domains contain defense signals has not been established. N-terminal deletions of proSystemin had little effect on its activity in tomato plants or suspension-cultured cells. Deletion of the C-terminal region of proSystemin containing the 18-amino acid Systemin domain completely abolished its proteinase inhibitor induction and alkalinization activities. The apoplastic fluid from tomato leaves and the medium of cultured cells were analyzed for proteolytic activity that could process proSystemin to Systemin. These experiments showed that proteolytic enzymes present in the apoplasm and medium could cleave proSystemin into large fragments, but the enzymes did not produce detectable levels of Systemin. Additionally, inhibitors of these proteolytic enzymes did not affect the biological activity of proSystemin. The cumulative data indicated that proSystemin and/or large fragments of proSystemin can be active inducers of defense responses in both tomato leaves and suspension-cultured cells and that the only region of proSystemin that is responsible for activating the defense response resides in the Systemin domain.
Andreas Schaller - One of the best experts on this subject based on the ideXlab platform.
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the Systemin signaling cascade as derived from time course analyses of the Systemin responsive phosphoproteome
Molecular & Cellular Proteomics, 2019Co-Authors: Fatima Haj Ahmad, Andreas Schaller, Xu Na Wu, Annick Stintzi, Waltraud X SchulzeAbstract:Systemin is a small peptide with important functions in plant wound response signaling. Although the transcriptional responses of Systemin action are well described, the signaling cascades involved in Systemin perception and signal transduction at the protein level are poorly understood. Here we used a tomato cell suspension culture system to profile phosphoproteomic responses induced by Systemin and its inactive Thr17Ala analog, allowing us to reconstruct a Systemin-specific kinase/phosphatase signaling network. Our time-course analysis revealed early phosphorylation events at the plasma membrane, such as dephosphorylation of H+-ATPase, rapid phosphorylation of NADPH-oxidase and Ca2+-ATPase. Later responses involved transient phosphorylation of small GTPases, vesicle trafficking proteins and transcription factors. Based on a correlation analysis of Systemin-induced phosphorylation profiles, we predicted substrate candidates for 44 early Systemin-responsive kinases, which includes receptor kinases and downstream kinases such as MAP kinases, as well as nine phosphatases. We propose a regulatory module in which H+-ATPase LHA1 is rapidly de-phosphorylated at its C-terminal regulatory residue T955 by phosphatase PLL5, resulting in the alkalization of the growth medium within 2 mins of Systemin treatment. We found the MAP kinase MPK2 to have increased phosphorylation level at its activating TEY-motif at 15 min post-treatment. The predicted interaction of MPK2 with LHA1 was confirmed by in vitro kinase assays, suggesting that the H+-ATPase LHA1 is re-activated by MPK2 later in the Systemin response. Our data set provides a resource of proteomic events involved in Systemin signaling that will be valuable for further in-depth functional studies in elucidation of Systemin signaling cascades.
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the Systemin signaling cascade as derived from phosphorylation time courses under stimulation by Systemin and its inactive thr17ala a17 analog
bioRxiv, 2019Co-Authors: Fatima Hai Ahmad, Andreas Schaller, Xu Na Wu, Annick Stintzi, Waltraud X SchulzeAbstract:Systemin is a small peptide with important functions in plant wound response signaling. Although transcriptional responses of Systemin action are well described, the precise signaling cascades involved in its perception and signal transduction are poorly understood at the protein level. Here we use a phosphoproteomic profiling study involving stimulation time courses with Systemin and its inactive analogon A17 to reconstruct a Systemin-specific kinase/phosphatase signaling network. The time course analysis of Systemin-induced phosphorylation patterns revealed early events at the plasma membrane, such dephosphorylation of H+-ATPase, rapid phosphorylation of NADPH-oxidase and Ca2+-ATPase. Later responses involved transient phosphorylation of small GTPases and vesicle trafficking proteins, as well as transcription factors. Based on a correlation analysis of Systemin-specific phosphorylation profiles, we predict substrate candidates for 56 Systemin specific kinases and 18 phosphatases. Among the kinases are several Systemin-specific receptor kinases as well as kinases with downstream signaling functions, such as MAP-kinases. A regulatory circuit for plasma membrane H+-ATPase was predicted and confirmed by in-vitro activity assays. In this regulatory model we propose that upon Systemin treatment, H+-ATPase LHA1 is rapidly de-phosphorylated at its C-terminal regulatory residue T955 by phosphatase PLL5, resulting in the alkalization of the growth medium within 2 minutes of Systemin treatment. We further propose that the H+-ATPase LHA1 is re-activated by MAP-Kinase MPK2 later in the Systemin response. MPK2 was identified with increased phosphorylation at its activating TEY-motif at 15 minutes of treatment and the predicted interaction with LHA1 was confirmed by in-vitro kinase assays. Our data set provides a valuable resource of proteomic events involved in the Systemin signaling cascade with a focus on predictions of substrates to Systemin-specific kinases and phosphatases.
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Characterization of proSystemin expressed in the baculovirus/insect cell system reveals biological activity of the Systemin precursor.
Planta, 2000Co-Authors: Michael Vetsch, Ingar Janzik, Andreas SchallerAbstract:Tomato (Lycopersicon esculentum Mill.) proSystemin in fusion with a viral signal peptide was expressed in Sf21 insect cell cultures after infection with recombinant baculoviruses. ProSystemin was purified from culture supernatants and its identity was confirmed by N-terminal sequence and mass-spectral analyses. Recombinant proSystemin was found to be equally active as compared to Systemin in inducing the expression of wound-response genes in tomato plants. In cultured cells of L. peruvianum, proSystemin elicited a rapid alkalinization of the growth medium. The timing and dose-dependence of the alkalinization response were found to be identical for proSystemin and Systemin, respectively. ProSystemin-triggered defense responses were inhibited by a competitive antagonist of Systemin activity, indicating that the Systemin sequence within the primary structure of proSystemin determines its activity.
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characterization of proSystemin expressed in the baculovirus insect cell system reveals biological activity of the Systemin precursor
Planta, 2000Co-Authors: Michael Vetsch, Ingar Janzik, Andreas SchallerAbstract:Tomato (Lycopersicon esculentum Mill.) proSystemin in fusion with a viral signal peptide was expressed in Sf21 insect cell cultures after infection with recombinant baculoviruses. ProSystemin was purified from culture supernatants and its identity was confirmed by N-terminal sequence and mass-spectral analyses. Recombinant proSystemin was found to be equally active as compared to Systemin in inducing the expression of wound-response genes in tomato plants. In cultured cells of L. peruvianum, proSystemin elicited a rapid alkalinization of the growth medium. The timing and dose-dependence of the alkalinization response were found to be identical for proSystemin and Systemin, respectively. ProSystemin-triggered defense responses were inhibited by a competitive antagonist of Systemin activity, indicating that the Systemin sequence within the primary structure of proSystemin determines its activity.
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action of proteolysis resistant Systemin analogues in wound signalling
Phytochemistry, 1998Co-Authors: Andreas SchallerAbstract:Abstract In cultured cells of Lycopersicon peruvianum , the oligopeptide Systemin which mediates systemic signalling in the tomato wound response is rapidly inactivated by proteolytic cleavage of the bond carboxyterminal to Lys 14 . A Systemin derivative in which this peptide bond had been modified by N -methylation was resistant to proteolytic inactivation. Systemin elicits a rapid, transient alkalinization of the growth medium in L. peruvianum cells. Consistent with its metabolic stability, the response elicited by the N -methylated peptide was found to be more sustained than that caused by Systemin. In differentiated tomato plants, the stabilized peptide was found to be 3 times more active than Systemin with respect to the induction of proteinase inhibitors I and II. This result indicates the possible physiological significance of the observed proteolytic degradation for Systemin inactivation in planta . The activity of a protease capable of processing Systemin carboxy-terminal of Lys 14 was detected in tomato plasma membranes and may be responsible for the inactivation process. Two further peptides, N -methylated at the bonds carboxy-terminal of Gln 3 and Arg 10 had proteinase inhibitor inducing activities lower by a factor of 8 and 80, respectively, as compared to Systemin. Correspondingly, the alkalinization response elicited by these two peptides in cultured cells was found to be more transient than the Systemin response. The correlation between the duration of the alkalinization response and the proteinase inhibitor inducing activities of Systemin analogues may be indicative of a casual relationship between ion fluxes and defense gene induction.