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Systemin, the first peptide hormone identified in plants, was initially isolated from tomato (Solanum lycopersicum) leaves. Systemin mediates local and systemic wound-induced defense responses in plants, conferring resistance to necrotrophic fungi and herbivorous insects. Systemin is recognized by the leucine-rich-repeat receptor-like kinase (LRR-RLK) receptor SYSTEMIN RECEPTOR1 (SYR1), but how the systemin recognition signal is transduced to intracellular signaling pathways to trigger defense responses is poorly understood. Here, we demonstrate that SERK family LRR-RLKs function as co-receptors for SYR1 to mediate systemin signal transduction in tomato. By using chemical genetic approaches coupled with engineered receptors, we revealed that the association of the cytoplasmic kinase domains of SYR1 with SERKs leads to their mutual trans-phosphorylation and the activation of SYR1, which in turn induces a wide range of defense responses. Systemin stimulates the association between SYR1 and all tomato SERKs (SlSERK1, SlSERK3A, and SlSERK3B). The resulting SYR1-SlSERK heteromeric complexes trigger the phosphorylation of TOMATO PROTEIN KINASE 1B (TPK1b), a receptor-like cytoplasmic kinase that positively regulates systemin responses. Additionally, upon association with SYR1, SlSERKs are cleaved by the Pseudomonas syringae effector HopB1, further supporting the finding that SlSERKs are activated by systemin-bound SYR1. Finally, genetic analysis using Slserk mutants showed that SlSERKs are essential for systemin-mediated defense responses. Collectively, these findings demonstrate that the systemin-mediated association of SYR1 and SlSERKs activates defense responses against herbivorous insects.
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[This corrects the article DOI: 10.3389/fpls.2020.00529.].
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Plant defence peptides are paramount endogenous danger signals secreted after a challenge, intensifying the plant immune response. The peptidic hormone Systemin (Sys) was shown to participate in resistance in several plant pathosystems, although the mechanisms behind Sys-induced resistance when exogenously applied remain elusive. We performed proteomic, metabolomic, and enzymatic studies to decipher the Sys-induced changes in tomato plants in either the absence or the presence of Botrytis cinerea infection. Sys treatments triggered direct proteomic rearrangement mostly involved in carbon metabolism and photosynthesis. However, the final induction of defence proteins required concurrent challenge, triggering priming of pathogen-targeted proteins. Conversely, at the metabolomic level, Sys-treated plants showed an alternative behaviour following a general priming profile. Of the primed metabolites, the flavonoids rutin and isorhamnetin and two alkaloids correlated with the proteins 4-coumarate-CoA-ligase and chalcone-flavanone-isomerase triggered by Sys treatment. In addition, proteomic and enzymatic analyses revealed that Sys conditioned the primary metabolism towards the production of available sugars that could be fuelling the priming of callose deposition in Sys-treated plants; furthermore, PR1 appeared as a key element in Sys-induced resistance. Collectively, the direct induction of proteins and priming of specific secondary metabolites in Sys-treated plants indicated that post-translational protein regulation is an additional component of priming against necrotrophic fungi.
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Botrytis , Resistencia a la Enfermedad , Enfermedades de las Plantas , Solanum lycopersicum , Solanum lycopersicum/microbiología , Solanum lycopersicum/inmunología , Solanum lycopersicum/metabolismo , Solanum lycopersicum/genética , Botrytis/fisiología , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/inmunología , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteómica , PéptidosRESUMEN
The green leaf volatiles (GLVs) Z-3-hexen-1-ol (Z3-HOL) and Z-3-hexenyl acetate (Z3-HAC) are airborne infochemicals released from damaged plant tissues that induce defenses and developmental responses in receiver plants, but little is known about their mechanism of action. We found that Z3-HOL and Z3-HAC induce similar but distinctive physiological and signaling responses in tomato seedlings and cell cultures. In seedlings, Z3-HAC showed a stronger root growth inhibition effect than Z3-HOL. In cell cultures, the two GLVs induced distinct changes in MAP kinase (MAPK) activity and proton fluxes as well as rapid and massive changes in the phosphorylation status of proteins within 5 min. Many of these phosphoproteins are involved in reprogramming the proteome from cellular homoeostasis to stress and include pattern recognition receptors, a receptor-like cytoplasmic kinase, MAPK cascade components, calcium signaling proteins and transcriptional regulators. These are well-known components of damage-associated molecular pattern (DAMP) signaling pathways. These rapid changes in the phosphoproteome may underly the activation of defense and developmental responses to GLVs. Our data provide further evidence that GLVs function like DAMPs and indicate that GLVs coopt DAMP signaling pathways.
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Células Vegetales , Compuestos Orgánicos Volátiles , Células Vegetales/metabolismo , Plantones/metabolismo , Plantas/metabolismo , Transducción de Señal , Hojas de la Planta/metabolismo , Compuestos Orgánicos Volátiles/metabolismoRESUMEN
Cellular damage inflicted by wounding, pathogen infection, and herbivory releases a variety of host-derived metabolites, degraded structural components, and peptides into the extracellular space that act as alarm signals when perceived by adjacent cells. These so-called damage-associated molecular patterns (DAMPs) function through plasma membrane localized pattern recognition receptors to regulate wound and immune responses. In plants, DAMPs act as elicitors themselves, often inducing immune outputs such as calcium influx, reactive oxygen species generation, defense gene expression, and phytohormone signaling. Consequently, DAMP perception results in a priming effect that enhances resistance against subsequent pathogen infections. Alongside their established function in local tissues, recent evidence supports a critical role of DAMP signaling in generation and/or amplification of mobile signals that induce systemic immune priming. Here, we summarize the identity, signaling, and synergy of proposed and established plant DAMPs, with a focus on those with published roles in systemic signaling.
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Enfermedades de las Plantas , Transducción de Señal , Reguladores del Crecimiento de las PlantasRESUMEN
Phytocytokines are endogenous danger peptides that are actively released after a pest or pathogen attack, triggering an amplification of plant immune responses. Here, we found that Systemin, a peptide from tomato, has a substantial impact at the molecular level in Arabidopsis plants that leads to induced resistance against Plectosphaerella cucumerina. Using transcriptional and metabolomics approaches, and loss-of-function mutants to analyse the molecular mechanisms underlying induced resistance against the necrotroph, we decipher the enhanced molecular responses in Systemin-treated plants following infection. Some protein complexes involved in the response to other damage signals, including the BAK1-BIK1 protein complex and heterotrimeric G proteins, as well as MPK activation, were among the early signalling events triggered by Systemin in Arabidopsis upon infection. Non-targeted analysis of the late responses underlying Systemin-Induced Resistance1 (Sys-IR) showed that phenolic and indolic compounds were the most representative groups in the Systemin metabolic fingerprint. Lack of flavonoids resulted in the impairment of Sys-IR. On the other hand, some indolic compounds showed a priming profile and were also essential for functional Sys-IR. Evidence presented here shows that plants can sense heterologous peptides from other species as danger signals driving the participation of common protein cascades activated in the PTI and promoting enhanced resistance against necrotrophic fungus.
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Proteínas de Arabidopsis , Arabidopsis , Solanum lycopersicum , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ascomicetos , Péptidos/metabolismo , Enfermedades de las Plantas/microbiología , Proteínas Serina-Treonina Quinasas , Triptófano/metabolismoRESUMEN
Systemin (Sys) is an octadecapeptide, which upon wounding, is released from the carboxy terminus of its precursor, Prosystemin (ProSys), to promote plant defenses. Recent findings on the disordered structure of ProSys prompted us to investigate a putative biological role of the whole precursor deprived of the Sys peptide. We produced transgenic tomato plants expressing a truncated ProSys gene in which the exon coding for Sys was removed and compared their defense response with that induced by the exogenous application of the recombinant truncated ProSys (ProSys(1-178), the Prosystemin sequence devoid of Sys region). By combining protein structure analyses, transcriptomic analysis, gene expression profiling and bioassays with different pests, we demonstrate that truncated ProSys promotes defense barriers in tomato plants through a hormone-independent defense pathway, likely associated with the production of oligogalacturonides (OGs). Both transgenic and plants treated with the recombinant protein showed the modulation of the expression of genes linked with defense responses and resulted in protection against the lepidopteran pest Spodoptera littoralis and the fungus Botrytis cinerea. Our results suggest that the overall function of the wild-type ProSys is more complex than previously shown, as it might activate at least two tomato defense pathways: the well-known Sys-dependent pathway connected with the induction of jasmonic acid biosynthesis and the successive activation of a set of defense-related genes, and the ProSys(1-178)-dependent pathway associated with OGs production leading to the OGs mediate plant immunity.
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Peptides are important regulators that participate in the modulation of almost every physiological event in plants, including defense. Recently, many of these peptides have been described as defense elicitors, termed phytocytokines, that are released upon pest or pathogen attack, triggering an amplification of plant defenses. However, little is known about peptides sensing and inducing resistance activities in heterologous plants. In the present study, exogenous peptides from solanaceous species, Systemins and HypSys, are sensed and induce resistance to the necrotrophic fungus Plectosphaerella cucumerina in the taxonomically distant species Arabidopsis thaliana. Surprisingly, other peptides from closer taxonomic clades have very little or no effect on plant protection. In vitro bioassays showed that the studied peptides do not have direct antifungal activities, suggesting that they protect the plant through the promotion of the plant immune system. Interestingly, tomato Systemin was able to induce resistance at very low concentrations (0.1 and 1 nM) and displays a maximum threshold being ineffective above at higher concentrations. Here, we show evidence of the possible involvement of the JA-signaling pathway in the Systemin-Induced Resistance (Sys-IR) in Arabidopsis. Additionally, Systemin treated plants display enhanced BAK1 and BIK1 gene expression following infection as well as increased production of ROS after PAMP treatment suggesting that Systemin sensitizes Arabidopsis perception to pathogens and PAMPs.
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Systemin, a peptide plant hormone of 18 amino acids, coordinates local and systemic immune responses. The activation of the canonical systemin-mediated systemic signaling pathway involves systemin release from its precursor prosystemin, systemin binding to its membrane receptor SYSTEMIN RECEPTOR1 (SYR1), and the transport of long-distance signaling molecules, including jasmonic acid, the prosystemin mRNA, volatile organic compounds and possibly systemin itself. Here, we review emerging evidence that the disordered structure and unconventional processing and secretion of systemin contribute to the regulation of systemin-mediated signaling during plant defense. We highlight recent advances in systemin research, which elucidated how cells integrate multiple long-distance signals into the systemic defense response. In addition, we discuss the perception of systemin by SYR1 and its mediation of downstream defense responses.
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Solanum lycopersicum , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/metabolismo , Péptidos/metabolismo , Proteínas de Plantas/metabolismoRESUMEN
Systemin (Sys) is an 18-aa plant peptide hormone involved in the regulation of plant's defensive response. Sys is considered as a fast-spreading systemic wound signal. We developed a simple and rapid CE method to monitor the spreading of Sys peptides through tomato plant. A 1,2,3-triazole-linked AZT-systemin conjugate was designed as a model to study the possibility of translocating small cargo molecules 3'-Azido-2',3'-dideoxythymidine by systemin. The Sys peptides (Sys, N-propiolyl Sys, and AZT-systemin conjugate) were injected into the stem and leaves of mature tomato plant. Its transportation throughout the plant tissue was traced by CE. The peptides were clearly visible in the crude tomato exudates and an optimum separation was achieved in 25 mM phosphate "buffer" at pH 2.5 and a voltage of 20 kV using uncoated fused silica capillary. CE analysis showed that Sys peptides are well separated from tomato plant exudates ingredients and are stable in tomato stem and leaf exudates for up to 24 h. CE study revealed that the Sys peptides are effectively spreading throughout tomato stem and leaves and the peptides could be directly detected in the crude plant matrixes. The translocation was strongly inhibited by sodium azide. The results showed that the established CE method can be used to characterize plant peptides spreading under plant physiological conditions.
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Electroforesis Capilar/métodos , Péptidos , Solanum lycopersicum , Solanum lycopersicum/química , Solanum lycopersicum/metabolismo , Solanum lycopersicum/fisiología , Péptidos/análisis , Péptidos/metabolismo , Péptidos/fisiología , Hojas de la Planta/química , Hojas de la Planta/metabolismo , Hojas de la Planta/fisiología , Tallos de la Planta/química , Tallos de la Planta/metabolismo , Tallos de la Planta/fisiología , Zidovudina/análisis , Zidovudina/metabolismo , Zidovudina/farmacocinéticaRESUMEN
Plants need to respond appropriately to wounding and herbivorous insects. Peptide signals have been implicated in local and systemic induction of appropriate plant defense responses. To study these peptide signals and their perception in host plants, it is important to have reproducible bioassays. Several assays, such as treatment of peptide solution via pressure infiltration, have been developed. Here, we provide detailed protocols for peptide feeding and mechanical wounding for tomato seedlings. To directly introduce peptides into tomato seedlings, peptide solution is fed through the excised stem via the transpiration stream. To mimic the wounding caused by insect feeding, leaflets of tomato seedlings are mechanically damaged with a hemostat; and wounded and systemic unwounded leaves are harvested and analyzed separately. Samples from both assays may be further assessed by examining the transcript level of marker genes by quantitative real-time PCR (qRT-PCR).
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Jasmonic acid (JA) is a phytohormone involved in plant development and defense. A major role of JA is the enhancement of secondary metabolite production, such as response to herbivory. Systemin is a bioactive plant peptide of 18 amino acids that contributes to the induction of local and systemic defense responses in tomato (Solanum lycopersicum) through JA biosynthesis. The overexpression of systemin (PS-OE) results in constitutive JA accumulation and enhances pest resistance in plants. Conversely, mutant plants affected in linolenic acid synthesis (spr2) are negatively compromised in the production of JA which favors damage and oviposition by insect herbivores. With undirected mass fingerprinting analyses, we found global metabolic differences between genotypes with modified jasmonic acid production. The spr2 mutants were enriched in di-unsaturated fatty acids and generally showed more changes. The PS-OE genotype produced an unidentified compound with a mass-to-charge ratio of 695 (MZ695). Most strikingly, the steroidal glycoalkaloid biosynthesis was negatively affected in the spr2 genotype. Complementation with jasmonic acid could restore the tomatine pathway, which strongly suggests the control of steroidal glycoalkaloid biosynthesis by jasmonic acid. spr2 plants were more susceptible to fungal infection with Fusarium oxysporum f.sp. ciceris, but not to bacterial infection with Clavibacter michiganensis subsp. michiganensis which supports the involvement of steroidal glycoalkaloids in the plant response against fungi.
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Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Solanum lycopersicum/metabolismo , Fusarium/patogenicidad , Genotipo , Solanum lycopersicum/genética , Solanum lycopersicum/microbiología , Metabolómica , Péptidos/genética , Péptidos/metabolismoRESUMEN
Systemin (SYS), an octadecapeptide hormone processed from a 200-amino-acid precursor (prosystemin, PS), plays a central role in the systemic activation of defense genes in tomato in response to herbivore and pathogen attacks. However, whether PS mRNA is transferable and its role in systemic defense responses remain unknown. We created the transgenic tomato PS gene tagged with the green fluorescent protein (PS-GFP) using a shoot- or root-specific promoter, and the constitutive 35S promoter in Arabidopsis. Subcellular localization of PS-/SYS-GFP was observed using confocal laser scanning microscopy and gene transcripts were determined using quantitative real-time PCR. In Arabidopsis, PS protein can be processed and SYS is secreted. Shoot-/root-specific expression of PS-GFP in Arabidopsis, and grafting experiments, revealed that the PS mRNA moves in a bi-directional manner. We also found that ectopic expression of PS improves Arabidopsis resistance to the necrotrophic fungus Botrytis cinerea, consistent with substantial upregulation of the transcript levels of specific pathogen-responsive genes. Our results provide novel insights into the multifaceted mechanism of SYS signaling transport and its potential application in genetic engineering for increasing pathogen resistance across diverse plant families.
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Arabidopsis/genética , Arabidopsis/microbiología , Botrytis/fisiología , Resistencia a la Enfermedad/genética , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/genética , Transporte de ARN/genética , Solanum lycopersicum/microbiología , Arabidopsis/efectos de los fármacos , Botrytis/efectos de los fármacos , Resistencia a la Enfermedad/efectos de los fármacos , Fluorescencia , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , Péptidos/farmacología , Enfermedades de las Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/genética , Plantas Modificadas Genéticamente , Proteolisis/efectos de los fármacos , Transporte de ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Plantones/efectos de los fármacos , Plantones/crecimiento & desarrollo , Plantones/fisiología , Fracciones Subcelulares/metabolismoRESUMEN
Main conclusion: The transcripts of transgenic prosystemin (PS) gene are mobile and the PS mRNA can be translated into protein in tomato and tobacco plants. Systemin (SYS) and its precursor protein, prosystemin (PS), are upstream components of the wound-induced signaling pathway in tomato. Although the mobile signal(s) for wound responses has been the subject of considerable research, its identity remains controversial. Intensive studies have revealed the essential role of mRNA on plant systemic signaling. We hypothesize that PS mRNA can act as a transmissible signal in tomato. Herein, we demonstrated that transgenic PS mRNA occurs in leaves located at considerable distances from the initial site of its generation by a transient Agrobacterium-infiltration assay system. We also showed that PS protein is present in the vascular bundle of the distant leaves. Our results indicate that transgenic PS mRNA may be functional as a long-distance signal to modulate systemic defense responses in tomato, providing novel insights into the multifaceted systems by which SYS signaling transports.
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Subtilisin-like proteases (SBTs) constitute a large family of extracellular plant proteases, the function of which is still largely unknown. In tomato plants, the expression of SBT3 was found to be induced in response to wounding and insect attack in injured leaves but not in healthy systemic tissues. The time course of SBT3 induction resembled that of proteinase inhibitor II and other late wound response genes suggesting a role for SBT3 in herbivore defense. Consistent with such a role, larvae of the specialist herbivore Manduca sexta performed better on transgenic plants silenced for SBT3 expression (SBT3-SI). Supporting a contribution of SBT3 to systemic wound signaling, systemic induction of late wound response genes was attenuated in SBT3-SI plants. The partial loss of insect resistance may thus be explained by a reduction in systemic defense gene expression. Alternatively, SBT3 may play a post-ingestive role in plant defense. Similar to other anti-nutritive proteins, SBT3 was found to be stable and active in the insect's digestive system, where it may act on unidentified proteins of insect or plant origin. Finally, a reduction in the level of pectin methylesterification that was observed in transgenic plants with altered levels of SBT3 expression suggested an involvement of SBT3 in the regulation of pectin methylesterases (PMEs). While such a role has been described in other systems, PME activity and the degree of pectin methylesterification did not correlate with the level of insect resistance in SBT3-SI and SBT3 overexpressing plants and are thus unrelated to the observed resistance phenotype.
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Proteínas de Plantas/fisiología , Solanum lycopersicum/fisiología , Subtilisinas/fisiología , Animales , Herbivoria , Solanum lycopersicum/enzimología , Manduca , Péptido Hidrolasas/fisiología , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Systemin is a signal peptide that promotes the response to wounding and herbivore attack in tomato. This 18-amino acid peptide is released from a larger precursor, prosystemin. To study the role of systemin as a modulator of defense signaling, we generated tomato (Solanum lycopersicum) transgenic plants that overexpress the prosystemin cDNA. We carried out a transcriptomic analysis comparing two different transgenic events with the untransformed control. The Gene Ontology categories of the 503 differentially expressed genes indicated that several biological functions were affected. Systemin promotes the expression of an array of defense genes that are dependent on different signaling pathways and it downregulates genes connected with carbon fixation and carbohydrate metabolism. These alterations present a degree of overlap with the response programs that are classically associated to pathogen defense or abiotic stress protection, implying that end products of the systemin signaling pathway may be more diverse than expected. We show also that the observed transcriptional modifications have a relevant functional outcome, since transgenic lines were more resistant against very different biotic stressors such as aphids (Macrosiphum euphorbiae), phytopathogenic fungi (Botrytis cinerea and Alternaria alternata) and phytophagous larvae (Spodoptera littoralis). Our work demonstrated that in tomato the modulation of a single gene is sufficient to provide a wide resistance against stress by boosting endogenous defense pathways. Overall, the data provided evidence that the systemin peptide might serve as DAMP signal in tomato, acting as a broad indicator of tissue integrity.
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Methanol is a byproduct of cell wall modification, released through the action of pectin methylesterases (PMEs), which demethylesterify cell wall pectins. Plant PMEs play not only a role in developmental processes but also in responses to herbivory and infection by fungal or bacterial pathogens. Molecular mechanisms that explain how methanol affects plant defenses are poorly understood. Here we show that exogenously supplied methanol alone has weak effects on defense signaling in three dicot species, however, it profoundly alters signaling responses to danger- and microbe-associated molecular patterns (DAMPs, MAMPs) such as the alarm hormone systemin, the bacterial flagellum-derived flg22 peptide, and the fungal cell wall-derived oligosaccharide chitosan. In the presence of methanol the kinetics and amplitudes of DAMP/MAMP-induced MAP kinase (MAPK) activity and oxidative burst are altered in tobacco and tomato suspension-cultured cells, in Arabidopsis seedlings and tomato leaf tissue. As a possible consequence of altered DAMP/MAMP signaling, methanol suppressed the expression of the defense genes PR-1 and PI-1 in tomato. In cell cultures of the grass tall fescue (Festuca arundinacea, Poaceae, Monocots), methanol alone activates MAPKs and increases chitosan-induced MAPK activity, and in the darnel grass Lolium temulentum (Poaceae), it alters wound-induced MAPK signaling. We propose that methanol can be recognized by plants as a sign of the damaged self. In dicots, methanol functions as a DAMP-like alarm signal with little elicitor activity on its own, whereas it appears to function as an elicitor-active DAMP in monocot grasses. Ethanol had been implicated in plant stress responses, although the source of ethanol in plants is not well established. We found that it has a similar effect as methanol on responses to MAMPs and DAMPs.
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Plants are continuously exposed to agents such as herbivores and environmental mechanical stresses that cause wounding and open the way to the invasion by microbial pathogens. Wounding provides nutrients to pathogens and facilitates their entry into the tissue and subsequent infection. Plants have evolved constitutive and induced defense mechanisms to properly respond to wounding and prevent infection. The constitutive defenses are represented by physical barriers, i.e., the presence of cuticle or lignin, or by metabolites that act as toxins or deterrents for herbivores. Plants are also able to sense the injured tissue as an altered self and induce responses similar to those activated by pathogen infection. Endogenous molecules released from wounded tissue may act as Damage-Associated Molecular Patterns (DAMPs) that activate the plant innate immunity. Wound-induced responses are both rapid, such as the oxidative burst and the expression of defense-related genes, and late, such as the callose deposition, the accumulation of proteinase inhibitors and of hydrolytic enzymes (i.e., chitinases and gluganases). Typical examples of DAMPs involved in the response to wounding are the peptide systemin, and the oligogalacturonides, which are oligosaccharides released from the pectic component of the cell wall. Responses to wounding take place both at the site of damage (local response) and systemically (systemic response) and are mediated by hormones such as jasmonic acid, ethylene, salicylic acid, and abscisic acid.
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The Cu(I) catalyzed Huisgen 1,3-dipolar azide-alkyne cycloaddition (CuAAC) was applied for a nucleoside-peptide bioconjugation. Systemin (Sys), an 18-aa plant signaling peptide naturally produced in response to wounding or pathogen attack, was chemically synthesized as its N-propynoic acid functionalized analog (Prp-Sys) using the SPPS. Next, CuAAC was applied to conjugate Prp-Sys with 3'-azido-2',3'-dideoxythymidine (AZT), a model cargo molecule. 1,4-Linked 1,2,3-triazole AZT-Sys conjugate was designed to characterize the spreading properties and ability to translocate of cargo molecules of systemin. CuAAC allowed the synthesis of the conjugate in a chemoselective and regioselective manner, with high purity and yield. The presence of Cu(I) ions generated in situ drove the CuAAC reaction to completion within a few minutes without any by-products. Under typical separation conditions of phosphate 'buffer' at low pH and uncoated fused bare-silica capillary, an increasing peak intensity assigned to triazole-linked AZT-Sys conjugate was observed using capillary electrophoresis (CE) during CuAAC. CE analysis showed that systemin peptides are stable in tomato leaf extract for up to a few hours. CE-ESI-MS revealed that the native Sys and its conjugate with AZT are translocated through the tomato stem and can be directly detected in stem exudates. The results show potential application of systemin as a transporter of low molecular weight cargo molecules in tomato plant and of CE method to characterize a behavior of plant peptides and its analogs.
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Fármacos Anti-VIH/química , Química Clic/métodos , Portadores de Fármacos/síntesis química , Péptidos/síntesis química , Técnicas de Síntesis en Fase Sólida , Zidovudina/química , Portadores de Fármacos/química , Electroforesis Capilar , Péptidos/químicaRESUMEN
Systemin is an important group of plant peptide hormones participating in the regulation of plant defensive responses. An improved method, based on dynamic pH junction and capillary electrophoresis-quadrupole time-of-flight mass spectrometry, was developed for online enrichment and sensitive determination of trace systemins in plants. After optimization, the online enrichment factors for six target systemins ranged from 90- to 127-fold. The detection limits reached lower than 0.5 nM, which were comparable with the sensitivity of LC-MS method. Satisfactory quantitative results were obtained in terms of linearity (R(2) ≥ 0.993), dynamic range (3-120 ng/mL), and reproducibility (≤6.7%). For the analysis of real plant samples, a rapid sample preparation method was developed, using two steps of SPE purification with different retention and separation mechanisms. Finally, this method realized the successful detection of tomato systemin and tobacco hydroxyproline-rich systemin I from plant leaves with shorter analysis time.