Pepper pathogenesis‐related protein 4c is a plasma membrane‐localized cysteine protease inhibitor that is required for plant cell death and defense signaling

Xanthomonas campestris pv. vesicatoria (Xcv) type III effector AvrBsT triggers programmed cell death (PCD) and activates the hypersensitive response (HR) in plants. Here, we isolated and identified the plasma membrane localized pathogenesis‐related (PR) protein 4c gene (CaPR4c) from pepper (Capsicum annuum) leaves undergoing AvrBsT‐triggered HR cell death. CaPR4c encodes a protein with a signal peptide and a Barwin domain. Recombinant CaPR4c protein expressed in Escherichia coli exhibited cysteine protease‐inhibitor activity and ribonuclease (RNase) activity. Subcellular localization analyses revealed that CaPR4c localized to the plasma membrane in plant cells. CaPR4c expression was rapidly and specifically induced by avirulent Xcv (avrBsT) infection. Transient expression of CaPR4c caused HR cell death in pepper leaves, which was accompanied by enhanced accumulation of H₂O₂ and significant induction of some defense‐response genes. Deletion of the signal peptide from CaPR4c abolished the induction of HR cell death, indicating a requirement for plasma membrane localization of CaPR4c for HR cell death. CaPR4c silencing in pepper disrupted both basal and AvrBsT‐triggered resistance responses, and enabled Xcv proliferation in infected leaves. H₂O₂ accumulation, cell‐death induction, and defense‐response gene expression were distinctly reduced in CaPR4c‐silenced pepper. CaPR4c overexpression in transgenic Arabidopsis plants conferred greater resistance against infection by Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis. These results collectively suggest that CaPR4c plays an important role in plant cell death and defense signaling.     

Overexpression of the pepper antimicrobial protein <Emphasis Type="Italic">CaAMP1</Emphasis> gene regulates the oxidative stress- and disease-related proteome in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Proteomics facilitates our understanding of cellular processes and network functions in the plant defense response during abiotic and biotic stresses. Here, we demonstrate that the ectopic expression of the Capsicum annuum antimicrobial protein CaAMP1 gene in Arabidopsis thaliana confers enhanced tolerance to methyl viologen (MV)-induced oxidative stress, which is accompanied by lower levels of lipid peroxidation. Quantitative comparative proteome analyses using two-dimensional gel electrophoresis coupled with mass spectrometry identified some of the oxidative stress- and disease-related proteins that are differentially regulated by CaAMP1 overexpression in Arabidopsis leaves. Antioxidant- and defense-related proteins, such as 2-cys peroxiredoxin, l-ascorbate peroxidase, peroxiredoxin, glutathione S-transferase and copper homeostasis factor, were up-regulated in the CaAMP1 transgenic leaf tissues. In contrast, GSH-dependent dehydroascorbate reductase and WD-40 repeat family protein were down-regulated by CaAMP1 overexpression. In addition, CaAMP1 overexpression enhanced resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 infection and also H₂O₂ accumulation in Arabidopsis. The identified antioxidant- and defense-related genes were differentially expressed during MV-induced oxidative stress and Pst DC3000 infection. Taken together, we conclude that CaAMP1 overexpression can regulate the differential expression of defense-related proteins in response to environmental stresses to maintain reactive oxygen species (ROS) homeostasis.     

Histochemical Studies of the Non‐Host Resistance and the Role of Actin Cytoskeleton in Pepper Against Colletotrichum orbiculare

The penetration process and defence reactions (hypersensitive response, oxidative burst and cell wall fortification) of Colletotrichum orbiculare were studied histochemically on pepper cultivar ‘A11’ (non‐host) and susceptible cucumber cultivar ‘Changchun Thorn’ (host). The results indicate that C. orbiculare could hardly penetrate the non‐host pepper leaves. It was papillae rather than hypersensitive response and H₂O₂ that played an important role in resisting the colonization and development of C. orbiculare on the non‐host pepper. The depolymerization of the actin microfilament weakened the papilla deposition of pepper and allowed successful penetration of the non‐adapted C. orbiculare, suggesting that the actin cytoskeleton of pepper is significant in preventing the invasion of the non‐host pathogen C. orbiculare.     

Hydrogen peroxide is involved in nitric oxide‐induced cell death in maize leaves

Recent evidence indicates that nitric oxide (NO) plays an important role in plant hypersensitive cell death. Here, we report that NO treatment led to rapid cell death and induced hydrogen peroxide (H₂O₂) accumulation in maize leaves. We also show that NO induced the expression of Zmrboh genes. Pharmacological study suggests that NO‐induced cell death is in part mediated via H₂O₂. In addition, semi‐quantitative RT‐PCR revealed that NO induced expression of the systemic acquired resistance (SAR) genes, ZmPR1 and ZmPR5.     

Induction of alkalinization and an oxidative burst by low doses of cycloheximide in soybean cells

Soybean (Glycine max L. Merr.) Cell-suspension cultures inoculated with avirulent Pseudomonas syringae pv. glycinea bacteria generated a sustained oxidative burst 3–6 h after the infection. The H₂O₂ production was not dependent on protein biosynthesis but, surprisingly, cycloheximide itself was a very strong inducer of the oxidative burst and of the alkalinization measured in the cell culture medium. Both responses were activated in a very similar manner by inhibitors of protein phosphatases, implicating a phosphorylation change evoked by cycloheximide as a trigger for the elicitation. The activation of the oxidative burst was totally blocked by the kinase inhibitor K252a. The alkalinization response preceded the oxidative burst. The generation of H₂O₂ depleted the medium of H⁺ but the expected alkalinization of about one pH-unit did not occur. The H₂O₂ production by the plasma membrane oxidase must therefore be charge-compensated, likely via H⁺-channel activity. Received: 4 October 1997 / Accepted: 12 May 1998     

The elicitor-induced oxidative burst in cultured chickpea cells drives the rapid insolubilization of two cell wall structural proteins

Elicitation of cultured chickpea cells caused rapid insolubilization of two cell wall structural proteins, p190, a putative hydroxyproline-rich glycoprotein and p80, a putative proline-rich protein. This process appeared to result from an H₂O₂-mediated oxidative cross-linking mechanism and was initiated within 5 min and complete within 20 min. Further, elicitation of cells induced a rapid, transient generation of H₂O₂ (oxidative burst), with an onset after 5 min and a maximum H₂O₂-release after 20 min, as measured by a luminol-dependent chemiluminescence assay. Both chemiluminescence and protein insolubilization were suppressed by exogenous application of catalase or diphenylene iodonium, an inhibitor of plasma-membrane NADPH oxidase, respectively. In contrast, exogenous H₂O₂ mimicked the effect of the elicitor, suggesting that the putative oxidative crosslinking of the proteins depends directly on H₂O₂ from the oxidative burst. The peroxidase inhibitor salicylhydroxamic acid blocked both the elicitor- and the exogenous-H₂O₂-stimulated insolubilization, indicating that a peroxidase activity downstream of H₂O₂-supply is required. The protein kinase inhibitor staurosporine blocked the elicitation of the oxidative burst and protein insolubilization. In contrast, the protein phosphatase 2A inhibitor cantharidin accelerated, potentiated and extended the elicited oxidative burst. Cantharidin even stimulated the responses in the absence of the elicitor. The competitive effect of both inhibitors confirms that a coordinated activation of (i) protein kinase(s) and (ii) counteracting protein phosphates(s) is a poised signal transduction step for the induction of an NADPH-oxidase-dependent oxidative burst, which drives the putative peroxidase-catalyzed cross-linking of the cell wall proteins.Abbreviations DPI diphenylene iodonium - Ext-1 extensin-1 - gE1 anti-glycosylated extensin-1 antibodies - HRGP hydroxyp-roline-rich glycoprotein - LDC luminol-dependent chemiluminescence - POD peroxidase - PA polyacrylamide - PRP proline-rich proteins - SHAM salicylhydroxamic acid Financial support by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie is gratefully acknowledged. We thank Dr. C.J. Lamb (Salk Institute, La Jolla, Calif., USA) and Dr. L.A. Staehelin (University of Colorado, Boulder, Colo., USA) for their kind gifts of antibodies.     

The oxidative burst in plant defense: Function and signal transduction

The rapid production and accumulation of active oxygen species (AOS), the oxidative burst, has been shown to occur in a variety of plant/pathogen systems. In particular, two species, hydrogen peroxide (H₂O₂) and the superoxide radical anion O₂^? have received considerable attention. H₂O₂ and O₂^?, while acting directly as antimicrobial agents, may also serve as second messengers or catalysts in plants to activate a more diverse set of defense responses. Some of the better studied downstream responses promoted by AOS are (1) the cross-linking of cell wall proteins, (2) the induction of defense-related genes, (3) the stimulation of phytoalexin biosynthesis and (4) promotion of the hypersensitive response (HR). A useful model for studying the oxidative burst in plants is the neutrophil NADPH ox-idase complex, the primary source of AOS production in mammals. Several of the subunits of the neutrophil NADPH oxidase complex have been immunologically identified in plants. Furthermore, many of the components known to be involved in the signal transduction pathway in neutrophils have also been found to play a role in the oxidative burst in plants. Just as various ligands activate the oxidase complex in neutrophils, several ligands (elicitors or pathogens) also lead to induction of the oxidative burst in plant cells. The similarities between the neutrophil and plant oxidative bursts will be elaborated in this review. Following stimulation with elicitors, different signal transduction pathways are activated in plants, depending on the source of elicitor used. While the identities and chronologies of the major intermediates in these pathways remain largely unknown, there is strong evidence at least for participation of phospholipases, H⁺/K⁺ exchange, Ca²⁺ influxes, protein kinases and phosphatases, and GTP binding proteins. In an effort to integrate these various signaling events into a single scheme, we have constructed a hypothetical model that proposes how different elicitors might induce the oxidative burst in the same cell by different pathways.     

Orchestration of hydrogen peroxide and nitric oxide in brassinosteroid‐mediated systemic virus resistance in Nicotiana benthamiana

Brassinosteroids (BRs) play essential roles in modulating plant growth, development and stress responses. Here, involvement of BRs in plant systemic resistance to virus was studied. Treatment of local leaves in Nicotiana benthamiana with BRs induced virus resistance in upper untreated leaves, accompanied by accumulations of H₂O₂ and NO. Scavenging of H₂O₂ or NO in upper leaves blocked BR‐induced systemic virus resistance. BR‐induced systemic H₂O₂ accumulation was blocked by local pharmacological inhibition of NADPH oxidase or silencing of respiratory burst oxidase homolog gene NbRBOHB, but not by systemic NADPH oxidase inhibition or NbRBOHA silencing. Silencing of the nitrite‐dependent nitrate reductase gene NbNR or systemic pharmacological inhibition of NR compromised BR‐triggered systemic NO accumulation, while local inhibition of NR, silencing of NbNOA1 and inhibition of NOS had little effect. Moreover, we provide evidence that BR‐activated H₂O₂ is required for NO synthesis. Pharmacological scavenging or genetic inhibiting of H₂O₂ generation blocked BR‐induced systemic NO production, but BR‐induced H₂O₂ production was not sensitive to NO scavengers or silencing of NbNR. Systemically applied sodium nitroprusside rescued BR‐induced systemic virus defense in NbRBOHB‐silenced plants, but H₂O₂ did not reverse the effect of NbNR silencing on BR‐induced systemic virus resistance. Finally, we demonstrate that the receptor kinase BRI1(BR insensitive 1) is an upstream component in BR‐mediated systemic defense signaling, as silencing of NbBRI1 compromised the BR‐induced H₂O₂ and NO production associated with systemic virus resistance. Together, our pharmacological and genetic data suggest the existence of a signaling pathway leading to BR‐mediated systemic virus resistance that involves local Respiratory Burst Oxidase Homolog B (RBOHB)‐dependent H₂O₂ production and subsequent systemic NR‐dependent NO generation.     

Oligogalacturonide signal transduction,induction of defense-related responses and protection of grapevine against<Emphasis Type="Italic"> Botrytis cinerea</Emphasis>

Grapevine (Vitis vinifera L.) is vulnerable to a variety of pathogenic fungi, among them Botrytis cinerea, the causal agent of grey mould, is responsible for worldwide yield losses that would be even more important without a successful control that relies mainly on fungicides. In the present work we investigated an alternative way of using oligogalacturonides (OGA) to induce defense responses in grapevine and protection against B. cinerea. Kinetic experiments with grapevine cells showed that OGA induced a rapid and transient generation of H₂O₂, followed by differential expression of nine defense-related genes and stimulation of chitinase and -1,3-glucanase activities. Inhibition of OGA-induced oxidative burst by diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, lowered induction levels of six genes and chitinase activity. Interestingly, the induction of three other genes and -1,3-glucanase activity were inhibited by K252a, a protein kinase inhibitor, but not by DPI. Treatment of grapevine leaves with OGA also reduced infection by B. cinerea by about 55–65%. Accordingly, DPI or K252a with or without OGA increased the susceptibility of grapevine leaves to B. cinerea. We suggest that treatment of grapevine with OGA elicits different signalling pathways, which might act in tandem with the oxidative burst to increase grapevine defense responses required for protection against B. cinerea.Abbreviations AOS Active oxygen species - Chit Chitinase - DPI Diphenylene iodonium - -Glu -1,3-Glucanase - GST Glutathione-S-transferase - MAP Mitogen-activated protein - OGA Oligogalacturonides - PAL Phenylalanine ammonia-lyase - PR Pathogenesis-related - PGIP Polygalacturonase inhibiting protein - PIN Serine-proteinase inhibitor - STS Stilbene synthase     

In planta measurements of oxidative bursts elicited by avirulent and virulent bacterial pathogens suggests that H2O2 is insufficient to elicit cell death in tobacco

A rapid and localized programmed cell death – the hypersensitive response (HR) – is a widely utilized plant resistance mechanism against pathogens. Studies have implicated H₂O₂ generation as a key elicitory mechanism in the HR. The causal relationship between the kinetics of the in planta oxidative burst, the HR and certain defence gene expression was examined. H₂O₂ generation following challenge with avirulent strains of Pseudomonas syringae pv. (P. s. pv.) syringae occurred in two phases. The effects of ROS generation were investigated using the H₂O₂-responsive transgene AoPR10-GUS, the dually responsive (H₂O₂ and salicylic acid) PR1a-GUS as well as measures of cell death. Co-application of catalase with P. s. pv. syringae into tobacco leaf panels suppressed AoPR10- and PR1a-GUS expression and cell death. Conversely, varying H₂O₂ generation with glucose: glucose oxidase influenced both defence gene expression and cell death. AoPR10-GUS proved to be primarily responsive to apoplastic not intracellular oxidative stress, suggesting that the apoplasm was a distinctive source of oxidative signals. A biphasic oxidative burst was also observed with virulent P. s. pv. tabaci, which, although delayed compared to that observed during HR, persisted at equivalent levels for a longer period. Taking all these data together we suggest that either (1) additional factors to the apoplastic oxidative burst are required to explain the rapid kinetics of defence signalling and cell death associated with the HR or (2) P. s. pv. tabaci successfully suppresses the effects of H₂O₂ generation by an unknown mechanism.     

Variations of vinblastine accumulation and redox state affected by exogenous H<Subscript>2</Subscript>O<Subscript>2</Subscript> in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don

Effects of exogenous H₂O₂ application on vinblastine (VBL) and its precursors, vindoline (VIN), catharanthine (CAT) and α-3′,4′-anhydrovinblastine (AVBL), were measured in Catharanthus roseus seedlings in order to explore possible correlation of VBL formation with oxidative stress. VBL accumulation has previously been shown to be regulated by an in vitro H₂O₂-dependent peroxidase (POD)-like synthase. Experimental exposure of plants to different concentrations of H₂O₂ showed that endogenous H₂O₂ and alkaloid concentrations in leaves were positively elevated. The time-course variations of alkaloid concentrations and redox state, reflected by the concentrations of H₂O₂, ascorbic acid (AA), oxidative product of glutathione (GSSG) and POD activity, were significantly altered due to H₂O₂ application. The further correlation analysis between alkaloids and redox status indicated that VBL production was tightly correlated with redox status. These results provide a new link between VBL metabolisms and redox state in C. roseus.     

Activation of an oxidative burst is a general feature of sensitive plants exposed to the air pollutant ozone

Ozone exposure stimulates an oxidative burst in leaves of sensitive plants, resulting in the generation and accumulation of hydrogen peroxide (H₂O₂) in tobacco and tomato, and superoxide (O₂^–?) together with H₂O₂ in Arabidopsis accessions. Accumulation of these reactive oxygen species (ROS) preceded the induction of cell death, and both responses co‐occurred spatially in the periveinal regions of the leaves. Re‐current ozone exposure of the sensitive tobacco cv. Bel W3 in closed chambers or in the field led to an enlargement of existing lesions by priming the border cells for H₂O₂ accumulation. Open top chamber experiments with native herbaceous plants in the field showed that Malva sylvestris L. accumulates O₂^–? at those sites that later exhibit plant cell death. Blocking of ROS accumulation markedly reduced ozone‐induced cell death in tomato, Arabidopsis and M. sylvestris. It is concluded that ozone triggers an in planta generation and accumulation of H₂O₂ and/or O₂^–? depending on the species, accession and cultivar, and that both these reactive oxygen species are involved in the induction of cell death in sensitive crop and native plants.     

Effects of salicylic acid on paraquat tolerance in<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

Foliar spraying ofArabidopsis thaliana (Columbia ecotype) plants with a 1.0-mM salicylic acid (SA) solution significantly improved their tolerance to subsequent paraquat (PQ)-induced oxidative damage. Leaf injuries, including losses of chlorophyll, protein, and fresh weight, were reduced. Our analysis of antioxidant enzymes in the leaves showed that SA pre-treatment effectively retarded rapid decreases in the activities of Superoxide dismutase (SOD), catalase, and ascorbate peroxidase that are normally associated with PQ exposure. In addition, guaiacol peroxidase activity was remarkably increased. In a native gel assay of peroxidase (POD) isozymes, staining activity of the POD1 isozyme, which disappeared in plants exposed only to 10 μM PQ, was significantly recovered by the 1.0-mM SA pre-treatment POD2 isozyme activity was also pronounced in all SA-treated plants compared with the control. A 12-h SA pre-treatment, without subsequent PQ stress, also caused a small increase in the endogenous H₂O₂ content that accompanies the symptoms of mild leaf injuries. This enhanced level occurred in parallel with a slight SOD increase and a catalase decrease. From our results, it can be assumed that, due to the small increase in SOD as well as catalase inactivation via SA pre-treatment, a moderate increase in H₂O₂ levels may occur. In turn, a large induction of guaiacol peroxidase leads to enhanced PQ tolerance inA. thaliana plants.     

Oxidative burst and electrolyte leakage induced by sulfhydryl blockers and by membrane permeabilizing reagents in different organs ofEgeria densa

Summary Sulfhydryl blockers, such as N-ethylmaleimide, iodoacetate and heavy metals induce a transitory stimulation of O₂ consumption and H₂O₂ production (oxidative burst) and a rapid release of electrolytes in leaves of various aquatic plants. The correlation between these two responses to N-ethylmaleimide or to Ag⁺ in separate organs and stages of leaf development was investigated inEgeria densa. Only adult leaves were able to respond to the sulfhydryl blockers with an oxidative burst, whereas this response was absent in immature growing leaves and in stem and root segments. In N-ethyl-maleimide- as well as in Ag⁺-treated adult leaves the oxidative burst was constantly associated with a relevant electrolyte leakage. These data are consistent with a model in which the SH reagent would first interact with a plasmalemma protein, leading to an increase in passive permeability to ions and to the activation of an oxidative enzyme of the type of the superoxide synthase described for granulocytes. In its turn, active-oxygen species produced by the activated oxidase might further damage the plasma membrane, increasing its passive permeability. Digitonin and nystatin, two reagents known to cause a permeabilization of lipid membranes, induced in adultE. densa leaves a transient increase in the rate of O₂ consumption and H₂O₂ production and an electrolyte leakage very similar to those induced by sulfhydryl blockers. These effects, however, were not influenced by the flavin analogues diphenylene iodonium and quinacrine, and were partially inhibited by the presence of CN^– and salicylhydroxamic acid, thus suggesting the involvement of a different oxidase in the oxidative burst elicited by these reagents.Abbreviations BTP 1,3-bis-tris(hydroxymethyl)methylaminopropane - CCCP carbonylcyanide-chlorophenylhydrazone - DCMU 3-(3,4 dichloropheny 1)-1,1-dimethylurea - DPI diphenylene iodonium - NEM N-ethylmaleimide - QO₂ O₂ uptake     

Exogenous Salicylic Acid Alleviates Growth Inhibition and Oxidative Stress Induced by Hypoxia Stress in <Emphasis Type="Italic">Malus robusta</Emphasis> Rehd

Salicylic acid (SA) as a signal molecule mediates many biotic and environmental stress-induced physiologic responses in plants. In this study we investigated the role of SA in regulating growth and oxidative stress in Malus robusta Rehd under both normoxic and hypoxic conditions. Hypoxia stress inhibited plant growth and dramatically reduced biomass. Addition of SA significantly alleviated the plant growth inhibition. The amounts of superoxide radicals (O₂ ⁻) and hydrogen peroxide (H₂O₂) significantly increased in leaves of the plants exposed to hypoxia stress and resulted in oxidative stress, which was indicated by accumulated concentration of malondialdehyde (MDA) and electrolyte leakage. Addition of SA significantly decreased the level of O₂ ⁻, electrolyte leakage, and lipid peroxidation and enhanced the activities of superoxide dismutase (SOD), peroxidase (POD), and ascorbate peroxidase (APX) under hypoxia stress. As important antioxidants, ascorbate (AsA) and glutathione (GSH) contents in the plant leaves were slightly increased by SA treatment compared to hypoxia stress treatment alone. It was concluded that SA could alleviate the detrimental effects of hypoxia stress on plant growth and of oxidative stress by enhancing the antioxidant defense system in leaves of M. robusta Rehd.