Involvement of Reactive Oxygen Species, Glutathione Metabolism, and Lipid Peroxidation in the Cf-Gene-Dependent Defense Response of Tomato Cotyledons Induced by Race-Specific Elicitors of Cladosporium fulvum

The chronological order of responses to Cladosporium fulvum (Cooke) (Cf) race-specific elicitors was assessed in cotyledons of three near-isogenic tomato (Lycopersicon esculentum Mill.) lines carrying either Cf-9 or Cf-2 or no Cf gene. The responses observed were dependent on the presence of a Cf gene, Avr-gene product dose injected, and the relative humidity (RH) of the growth chamber. At ambient RH, superoxide formation and lipid peroxidation occurred after 2 h (Cf9) and 4 h (Cf2). At elevated RH (98%) and at lower avirulence elicitor dose, Cf-Avr-dependent lipid peroxidation was considerably attenuated. Significant electrolyte leakage occurred by 18 h but only at the lower RH. Total glutathione levels began to increase 2 to 4 h and 4 to 8 h after challenge of Cf9 and Cf2 cells, respectively, and by 48 h reached 665 and 570% of initial levels. A large proportion of this accumulation (87%) was as oxidized glutathione. When the RH was increased to 98%, increases in glutathione levels were strongly attenuated. Increased lipoxygenase enzyme activity was detected 8 h postchallenge in either incompatible interaction. These results indicate that the activation of the Cf-Avr-mediated defense response results in severe oxidative stress.     

Electrolyte Leakage, Lipoxygenase, and Lipid Peroxidation Induced in Tomato Leaf Tissue by Specific and Nonspecific Elicitors from Cladosporium fulvum

Glycoprotein nonspecific elicitor (NSE) and a specific elicitor preparation from intercellular fluids (SE) of tomato (Lycopersicon esculentum Mill. cv Bonny Best or Potentate) infected with race 2.4.5 of Cladosporium fulvum Cooke [syn. Fulvia fulva (Cooke) Ciferri] were injected into cv Sonatine (resistant to race 2.4.5) to compare electrolyte leakage, lipoxygenase activity, and lipid peroxidation induced in response to these elicitors. Increased electrolyte leakage was induced by NSE or SE; the leakage due to NSE but not to SE was inhibited by the nonsteroidal antiinflammatory drug (NSAID) piroxicam. Under normal photoperiod conditions, higher levels of lipoxygenase activity were detected 6 hours after injection with either elicitor. This activity peaked by 12 hours with both elicitors and declined to control levels by 24 hours when visible necrosis could be detected. Both NSE and SE-induced lipoxygenase was inhibited by piroxicam in vitro. Lipid peroxidation in elicitor-treated tissue was also assayed at 6, 12, and 24 hours after injection using the TBA test for malonaldehyde. Increased peroxidation was detected in response to NSE or SE at 12 hours with similar values obtained at 24 hours. With plants incubated in the dark, lipoxygenase, and lipid peroxidation were similarly induced in SE-injected tissue whereas necrosis induction by SE was light dependent.     

Race-Specific Elicitors of Cladosporium fulvum Induce Changes in Cell Morphology and the Synthesis of Ethylene and Salicylic Acid in Tomato Plants Carrying the Corresponding Cf Disease Resistance Gene

Defense responses mediated by the genetically unlinked Cf-9 and Cf-2 genes were compared with those involving no Cf gene (Cf0). Compatible tomato (Lycopersicon esculentum)-Cladosporium fulvum intercellular washing fluids were injected into tomato cotyledons, and the kinetics of responses was monitored under conditions of 70 and 98% relative humidity. The latter conditions suppressed the normal macroscopic responses. For the Cf-9-Avr9 interaction, stomatal opening was induced within 3 to 4 h and after 9 h mesophyll cell death commenced. A burst of ethylene production occurred between 9 and 12.5 h and remained elevated. Free salicylic acid levels increased after 12 h, peaked at 24 h, and thereafter declined. For the Cf-2-Avr2 interaction, stomata became plugged after 8 h, and salicylic acid and ethylene levels increased by 12 and 18 h, respectively, and thereafter declined. Host cell death commenced around vascular tissue by 24 h. Cell death in both incompatible interactions was frequently preceded by cell enlargement. For Cf0-injected plants, no significant responses were detected. High humidity delayed and reduced the Cf-Avr-gene-dependent cell death and ethylene synthesis, whereas induced salicylic acid levels were unaffected for Cf-2-Avr2 and reduced in magnitude only for Cf-9-Avr9.     

Identification of Two Genes Required in Tomato for Full Cf-9-Dependent Resistance to Cladosporium fulvum

Mutagenesis was used to identify and characterize plant genes required for fungal disease resistance gene function in tomato. Seed of a stock homozygous for the Cf-9 gene for resistance to Cladosporium fulvum were treated with ethyl methanesulfonate, and 568 M2 families were screened for mutations to C. fulvum sensitivity. Eight mutants with reduced resistance were isolated. Four mutations, all of which mapped to the Cf-9 gene, lost both resistance and response to the race-specific AVR9 elicitor. The other four mutations partially lost resistance and response to the AVR9 elicitor. Cytological analysis revealed that a unique host cell staining pattern accompanied the reduced-resistance phenotype in three mutants. Two of the mutants with reduced resistance mapped to Cf-9, and two mapped to two distinct loci designated Rcr-1 and Rcr-2 (Required for Cladosporium resistance) that are unlinked to Cf-9.     

Involvement of Reactive Oxygen Species in Capsaicinoid-induced Apoptosis in Transformed Cells

Some varieties of sweet pepper accumulate non-pungent isosters of capsaicin, a type of compounds exemplified by capsiate. The only structural difference between capsaicin and capsiate is the link between the vanillyl and the acyl moieties, via an amide bond in the former and via an ester bond in the latter. By flow cytometry analyses we have determined that nor-dihydrocapsiate, a simplified analogue of capsiate, is a pro-oxidant compound that induces apoptosis in the Jurkat tumor cell line. The nuclear DNA fragmentation induced by nor-dihydrocapsiate is preceded by an increase in the production of reactive oxygen species and by a subsequent disruption of mitochondria transmembrane potential. Capsiate-induced apoptosis is initiated at the S phase of the cell cycle and is mediated by a caspase-3-dependent pathway. The accumulation of intracellular reactive oxygen species in capsiate-treated cells is greatly prevented by the presence of ferricyanide, suggesting that capsiates target a cellular redox system distinct from the one involved in the mitochondrial electron-chain transport. Methylation of the phenolic hydroxyl of nor-dihydrocapsiate completely abrogated the ability to induce reactive oxygen species and apoptosis, highlighting the relevance of the presence of a free phenolic hydroxyl for the pro-oxidant properties of capsaicinoids.     

A High-Affinity Binding Site for the AVR9 Peptide Elicitor of Cladosporium fulvum Is Present on Plasma Membranes of Tomato and Other Solanaceous Plants

The race-specific Cladosporium fulvum peptide elicitor AVR9, which specifically induces a hypersensitive response in tomato genotypes carrying the Cf-9 resistance gene, was labeled with iodine-125 at the N-terminal tyrosine residue and used in binding studies. 125I-AVR9 showed specific, saturable, and reversible binding to plasma membranes isolated from leaves of tomato cultivar Moneymaker without Cf resistance genes (MM-Cf0) or from a near-isogenic genotype with the Cf-9 resistance gene (MM-Cf9). The dissociation constant was found to be 0.07 nM, and the receptor concentration was 0.8 pmol/mg microsomal protein. Binding was highly influenced by pH and the ionic strength of the binding buffer and by temperature, indicating the involvement of both electrostatic and hydrophobic interactions. Binding kinetics and binding capacity were similar for membranes of the MM-Cf0 and MM-Cf9 genotypes. In all solanaceous plant species tested, an AVR9 binding site was present, whereas in the nonsolanaceous species that were analyzed, such a binding site could not be identified. The ability of membranes isolated from different solanaceous plant species to bind AVR9 seems to correlate with the presence of members of the Cf-9 gene family, but whether this correlation is functional remains to be determined.     

Induction of Defense-Related Responses in Cf9 Tomato Cells by the AVR9 Elicitor Peptide of Cladosporium fulvum Is Developmentally Regulated

The AVR9 elicitor from the fungal pathogen Cladosporium fulvum induces defense-related responses, including cell death, specifically in tomato (Lycopersicon esculentum Mill.) plants that carry the Cf-9 resistance gene. To study biochemical mechanisms of resistance in detail, suspension cultures of tomato cells that carry the Cf-9 resistance gene were initiated. Treatment of cells with various elicitors, except AVR9, induced an oxidative burst, ion fluxes, and expression of defense-related genes. Agrobacterium tumefaciens-mediated transformation of Cf9 tomato leaf discs with Avr9-containing constructs resulted efficiently in transgenic callus formation. Although transgenic callus tissue showed normal regeneration capacity, transgenic plants expressing both the Cf-9 and the Avr9 genes were never obtained. Transgenic F₁ seedlings that were generated from crosses between tomato plants expressing the Avr9 gene and wild-type Cf9 plants died within a few weeks. However, callus cultures that were initiated on cotyledons from these seedlings could be maintained for at least 3 months and developed similarly to callus cultures that contained only the Cf-9 or the Avr9 gene. It is concluded, therefore, that induction of defense responses in Cf9 tomato cells by the AVR9 elicitor is developmentally regulated and is absent in callus tissue and cell-suspension cultures, which consists of undifferentiated cells. These results are significant for the use of suspension-cultured cells to investigate signal transduction cascades.     

Adriamycin Affects Glomerular Renal Function: Evidence for the Involvement of Oxygen Radicals

The early nephrotoxic effect of the antitumor drug adriamycin (ADR) is suggested to be related to the generation of oxygen free radicals. Therefore the O₂ -dependence and the influence of free radical scavengers were studied in the model of the isolated perfused single glomerulus of Myxine glutinosa and by histochemi-cal demonstration of the glomerular ATP-ase. In Myxine, the glomerular ATP-ase activity was decreased after injection of ADR (5 mg/kg, i.v.).

Both ADR-treated Myxine and controls were exposed for 48 h to an artificial atmosphere of 20% O₂/80% N₂ or 80% O₂/20% N₂, respectively. After 10 days a significant decrease of the hydraulic conductivity (k) was measured in the experimental group exposed to 80% O₂ (k-values expressed as nl/s.mmHg.mm²: controls (7): 0.059 ± 0.017; ADR (7): 0.033 ± 0.026). The reduction of k following the administration of ADR (20 mg/kg) could be prevented by the sulphydryl donor N-acetylcysteine (NAC). The sieving coefficient for albumin (μ) was significantly increased in ADR-treated animals, showing no O₂-d

ependence (μ × 10^?2: controls (7) 1.3 ± 0.2; ADR 20% O₂ (8): 8.1 ± 9.6; ADR 80% O₂ (7): 6.9 ± 6.7). ω was not affected by NAC.

The lipid peroxide levels in liver, kidney and heart of Myxine increased after the administration of ADR, peaking by day 2 to 5. The circulation disorders of ADR-treated Myxine were not due to an accumulation of the drug in the heart, but rather to a lack of the intracellular antioxidant glutathione.

It is concluded that the early nephrotoxic effect of ADR, as reflected by a decreased glomerular ATP-ase activity, is mediated by free radical formation. Oxidative stress on membrane compounds seems to reduce the water permeability of the glomerular barrier, while the ADR-induced sieving defect may be due to oxygen independent pathological mechanisms.     

Comparison of the Effect of Different Fungal Elicitors on Rubia Tinctorum L. Suspension Culture

The effect of two elicitor types prepared from three different fungi on the alizarin content and the ultrastructure of Rubia tinctorum cells was studied. The de novo alizarin synthesis took place predominantly during the first day of treatment then it was followed by a constant release of alizarin into the medium. The alkalinization of the medium was similar in every treatment. The number of the living cells did not changed during 24-h elicitor treatments, but it decreased significantly after 96 h. The appearance of vacuolar bodies, and the change of the plasmalemmasome structure from vesiculo-reticular to reticulo-lamellar were the most typical morphological syndromes due to elicitation. Phytium elicitor proved to be the least effective, and Botrytis elicitor seemed to be the most effective.     

Development of a real-time PCR assay for the detection of Cladosporium fulvum in tomato leaves

Aims: The aim of this study was to develop a sensitive real-time polymerase chain reaction (PCR) assay for the rapid detection of Cladosporium fulvum in tomato leaves. Methods and Results: Three PCR primer pairs were designed based on the nucleotide sequences of: (i) the internal transcribed spacer regions of ribosomal RNA; (ii) a microsatellite region amplified by the microsatellite primer M13; and (iii) the β-tubulin gene of C. fulvum. Each primer pair amplified the expected target DNA fragment from geographically diverse isolates of C. fulvum. No PCR products were amplified with these primer pairs from DNA of other fungal species. Among the three pairs of primers, the primer pair CfF1/CfR1 developed based on the microsatellite region was the most sensitive. Using this sensitive primer pair, a real-time PCR assay was developed to detect early infection of C. fulvum in tomato leaves. Significance and Impact of the Study: DNA regions amplified by the microsatellite primer M13 have a high potential for developing highly sensitive species-specific PCR primers for the detection of phytopathogenic fungi. The real-time PCR assay developed in this study is useful in monitoring early infection of C. fulvum, and can help growers make timely decisions on fungicide application.     

Overexpression of the Tomato 13-Lipoxygenase Gene TomloxD Increases Generation of Endogenous Jasmonic Acid and Resistance to Cladosporium fulvum and High Temperature

Expression of the tomato gene encoding 13-lipoxygenase,TomloxD, is stimulated by wounding, pathogen infection, jasmonate, and systemin, but its role during growth and development of tomato (Lycopersicon Spp.) remains unclear. To assess the physiological role of TomloxD, we produced transgenic tomato plants with greatly increased TomloxD content using sense constructs under the control of the CaMV 35S promoter. Overexpression of TomloxD in transgenic tomatoes led to a marked increase in the levels of lipoxygenase activity and content of endogenous jasmonic acid (JA), which suggested that TomloxD can use α-linolenic acid as a substrate to produce (13S)-hydroperoxyoctadecatrienoic acid (13-HPOT); the 13-HPOT produced appears to be metabolized further to synthesize JA. Real-time RT-PCR revealed that the expression levels of defense genes LeHSP90, LePR1, LePR6 and LeZAT in the transformants were higher than those in non-transformed plants. Assay for resistance to pathogenic fungus and high temperature stresses suggested that transgenic plants harboring TomloxD were more tolerant to Cladosporium fulvum and high temperature stress than non-transformed tomato plants. The data presented here indicate clearly that TomloxD is involved in endogenous JA synthesis and tolerance to biotic and abiotic stress. The tomloxD gene has potential applications in engineering cropping plants that are resistant to biotic and/or abiotic stress factors.     

活性氧簇对蛋白尿的影响

蛋白尿不仅反映肾小球损伤,而且是一个独立的导致肾脏病变进展的主要因素,任何能够使蛋白尿减少的治疗干预都有利于减慢肾脏疾病的进展,遗传性蛋白尿性肾病是由于基因突变所致,获得性肾病大量蛋白尿成因目前尚未阐明。免疫异常,炎症介质及氧化应激反应均可导致肾损伤。氧自由基是肾损伤的主要介质,它作为强氧化剂是造成蛋白尿的重要原因之一。活性氧分子(ROS)可以通过降解肾小球乙酰肝素硫酸盐、肾小球基底膜Ⅳ型胶原富含赖氨酸的NCl区域发生交联、损伤足细胞破坏肾小球滤过屏障及与其他活性因子作用增强血清蛋白的渗透性等作用,造成蛋白尿。本文就近年来人们对活性氧造成蛋白尿的机制的研究做一综述,便于帮助医务工作者更好的了解和治疗蛋白尿性肾病。     

Down‐regulation of cladofulvin biosynthesis is required for biotrophic growth of Cladosporium fulvum on tomato

Fungal biotrophy is associated with a reduced capacity to produce potentially toxic secondary metabolites (SMs). Yet, the genome of the biotrophic plant pathogen Cladosporium fulvum contains many SM biosynthetic gene clusters, with several related to toxin production. These gene clusters are, however, poorly expressed during the colonization of tomato. The sole detectable SM produced by C. fulvum during in vitro growth is the anthraquinone cladofulvin. Although this pigment is not detected in infected leaves, cladofulvin biosynthetic genes are expressed throughout the pre‐penetration phase and during conidiation at the end of the infection cycle, but are repressed during the biotrophic phase of tomato colonization. It has been suggested that the tight regulation of SM gene clusters is required for C. fulvum to behave as a biotrophic pathogen, whilst retaining potential fitness determinants for growth and survival outside its host. To address this hypothesis, we analysed the disease symptoms caused by mutant C. fulvum strains that do not produce or over‐produce cladofulvin during the biotrophic growth phase. Non‐producers infected tomato in a similar manner to the wild‐type, suggesting that cladofulvin is not a virulence factor. In contrast, the cladofulvin over‐producers caused strong necrosis and desiccation of tomato leaves, which, in turn, arrested conidiation. Consistent with the role of pigments in survival against abiotic stresses, cladofulvin protects conidia against UV light and low‐temperature stress. Overall, this study demonstrates that the repression of cladofulvin production is required for C. fulvum to sustain its biotrophic lifestyle in tomato, whereas its production is important for survival outside its host.     

Disulfide bond structure of the AVR9 elicitor of the fungal tomato pathogen Cladosporium fulvum: evidence for a cystine knot

Disease resistance in plants is commonly activated by the product of an avirulence (Avr) gene of a pathogen after interaction with the product of a matching resistance (R) gene in the host. In susceptible plants, Avr products might function as virulence or pathogenicity factors. The AVR9 elicitor from the fungus Cladosporium fulvum induces defense responses in tomato plants carrying the Cf-9 resistance gene. This 28-residue beta-sheet AVR9 peptide contains three disulfide bridges, which were identified in this study as Cys2-Cys16, Cys6-Cys19, and Cys12-Cys26. For this purpose, AVR9 was partially reduced, and the thiol groups of newly formed cysteines were modified to prevent reactions with disulfides. After HPLC purification, the partially reduced peptides were sequenced to determine the positions of the modified cysteines, which originated from the reduced disulfide bridge(s). All steps involving molecules with free thiol groups were performed at low pH to suppress disulfide scrambling. For that reason, cysteine modification by N-ethylmaleimide was preferred over modification by iodoacetamide. Upon (partial) reduction of native AVR9, the Cys2-Cys16 bridge opened selectively. The resulting molecule was further reduced to two one-bridge intermediates, which were subsequently completely reduced. The (partially) reduced cysteine-modified AVR9 species showed little or no necrosis-inducing activity, demonstrating the importance of the disulfide bridges for biological activity. Based on peptide length and cysteine spacing, it was previously suggested that AVR9 isa cystine-knotted peptide. Now, we have proven that the bridging pattern of AVR9 is indeed identical to that of cystine-knotted peptides. Moreover, NMR data obtained for AVR9 show that it is structurally closely related to the cystine-knotted carboxypeptidase inhibitor. However, AVR9 does not show any carboxypeptidase inhibiting activity, indicating that the cystine-knot fold is a commonly occurring motif with varying biological functions.     

Harpin,An Elicitor of the Hypersensitive Response in Tobacco Caused by Erwinia amylovora,Elicits Active Oxygen Production in Suspension Cells

Active oxygen (AO) production and a K+/H+ exchange response (XR) are two concurrent early events associated with incompatible plant-bacteria interactions that result in a hypersensitive response (HR). Recently, a protein, termed harpin, produced by Erwinia amylovora has been reported to be the elicitor responsible for the HR caused by this pathogen. Although both the bacterium and harpin are reported to induce XR in tobacco (Nicotiana tabacum) cell suspensions, there have been no reports regarding the concurrent production of AO in this system. Here we report that E. amylovora stimulates the AO response, whereas an E. amylovora mutant that does not produce harpin does not elicit the AO response. In addition, a cell-free preparation of harpin induces AO production. This study indicates that harpin may be the bacterial elicitor of the XR and AO responses during the development of E. amylovora-induced HR.     

用自旋捕集技术研究硒化合物对活性氧自由基的清除作用

亚硒酸钠,硒代甲硫氨酸和硒代硫酸软骨素对黄嘌呤氧化酶体系和人多形核白细胞（ＰＭＮ）呼吸暴发时产生的超氧阴离子（Ｏ）自由基;和Ｆｅｎｔｏｎ反应生成的羟自由基（·ＯＨ）有明显的清除作用,并抑制ＰＭＮ呼吸暴发时对氧的消耗。实验结果说明不同硒化合物不仅能够清除细胞体系产生的活性氧自由基,而且对化学反应生成的活性氧自由基也有清除作用。     

Reactive Oxygen Species in Unstimulated Hemocytes of the Pacific Oyster Crassostrea gigas: A Mitochondrial Involvement

The Pacific oyster Crassostrea gigas is a sessile bivalve mollusc whose homeostasis relies, at least partially, upon cells circulating in hemolymph and referred to as hemocytes. Oyster’s hemocytes have been reported to produce reactive oxygen species (ROS), even in absence of stimulation. Although ROS production in bivalve molluscs is mostly studied for its defence involvement, ROS may also be involved in cellular and tissue homeostasis. ROS sources have not yet been described in oyster hemocytes. The objective of the present work was to characterize the ROS sources in unstimulated hemocytes. We studied the effects of chemical inhibitors on the ROS production and the mitochondrial membrane potential (Δψ_m) of hemocytes. First, this work confirmed the specificity of JC-10 probe to measure Δψ_m in oyster hemocytes, without being affected by ΔpH, as reported in mammalian cells. Second, results show that ROS production in unstimulated hemocytes does not originate from cytoplasmic NADPH-oxidase, nitric oxide synthase or myeloperoxidase, but from mitochondria. In contrast to mammalian cells, incubation of hemocytes with rotenone (complex I inhibitor) had no effect on ROS production. Incubation with antimycin A (complex III inhibitor) resulted in a dose-dependent ROS production decrease while an over-production is usually reported in vertebrates. In hemocytes of C. gigas, the production of ROS seems similarly dependent on both Δψ_m and ΔpH. These findings point out differences between mammalian models and bivalve cells, which warrant further investigation about the fine characterization of the electron transfer chain and the respective involvement of mitochondrial complexes in ROS production in hemocytes of bivalve molluscs.