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1.
Summary The in vivo induction of H2O2 production was tested on tobacco cell suspension cultures (Nicotiana tabacum cv. Bright Yellow-2). The measurement of H2O2 was based on the oxidation of 3,5-dichloro-2-hydroxybenzensulfonic acid by endogenous peroxidases and spectrophotometric detection after reaction with 4-aminoanti-pyrine. The phosphatase inhibitor cantharidin induced a transient increase in H2O2 synthesis. The timing of the H2O2 production, the level of induction by cantharidin and the background H2O2 production were dependent on the tobacco cell concentration used. A concentration curve of cantharidin revealed saturating kinetics for the H2O2 detection (E50=46 to 70 M, Emax=101 to 128 mol/h·g fresh weight). An inhibitor study with the tobacco BY-2 cells showed high inhibitions of the H2O2 induction with the flavin analogues diphenylene iodonium (I50=1.26M) and acridine orange and with membrane-permeative thiol reagents (N-ethyl maleimide, N-pyrene maleimide, iodoacetate); whereas the nonpermeative thiol reagentp-chloromercuribenzoic acid was ineffective. Therefore, the induction of H2O2 production with phosphatase inhibitors (cantharidin) showed comparable properties to the elicitor-induced oxidative-burst response in other plant cells.Abbreviations AcOr acridine orange - AOS active-oxygen species - BY-2 Bright Yellow-2 - pCMBS p-chloromercuribenzoic acid - DHBS 3,5-dichloro-2-hydroxybenzenesulfonic acid - DMSO dimethylsulfoxide - DPI diphenylene iodonium - EtOH ethanol - H2O2 hydrogen peroxide - HRP horseradish peroxidase - MS Murashige and Skoog - NEM N-ethyl maleimide - NPM N-pyrene maleimide - O 2 superoxide - SOD superoxide dismutase  相似文献   

2.
Generation of reactive oxygen species (ROS) constitutes an important first reaction under many stress conditions in plants. We demonstrate that Nicotiana tabacum L. cv. Bright Yellow 2 (TBY-2) cells in suspension cultures, generate superoxide radical and hydrogen peroxide upon treatment with cadmium and zinc. Addition of catalase and N,N-diethyldithiocarbamate (DDC) decreased the level of H2O2, whereas superoxide dismutase (SOD) induced a slight increase of the H2O2 production. The effects of catalase, DDC and SOD on the heavy metal-induced ROS production indicate that it occurs outside of the cells, and that at least part of the hydrogen peroxide is produced by dismutation of the superoxide radical (O2 ·−). The effect of pretreatment of the cell cultures with commonly used mammalian NADPH oxidase inhibitors was also tested. Strong inhibitions of cadmium and zinc-mediated ROS production were obtained with the flavoprotein inhibitors—diphenylene iodonium (DPI) and quinacrine and with an inhibitor of b-type cytochromes—imidazol. Membrane permeable-N-ethyl maleimide (NEM) and iodoacetate, and membrane non-permeable thiol reagents—para-chloromercuribenzoic acid (pCMBS) also inhibited the ROS production. These results suggested that the enzyme responsible for cadmium and zinc-induced ROS production in tobacco cells contains a flavocytochrome. They also show the importance of intra- and extracellular thiol groups in the observed stress reaction. The induction of ROS production with heavy metals showed properties comparable to the elicitor-induced oxidative burst in other plant cells.  相似文献   

3.
Large amounts of the sesquiterpenoid capsidiol accumulated in the media of tobacco (Nicotiana tabacum L. cv KY14) cell suspension cultures upon addition of fungal elicitor. Capsidiol accumulation was proportional to the amount of elicitor added. The accumulation of capsidiol was preceded by a transient increase in the capsidiol de novo synthesis rate as measured by the incorporation of exogenous [14C]acetate. Changes in 3-hydroxy-3-methylglutaryl-CoA reductase activity (HMGR; EC 1.1.1.34), an enzyme of general isoprenoid metabolism, paralleled the changes in [14C]acetate incorporation into capsidiol. Incubation of the cell cultures with mevinolin, a potent in vitro inhibitor of the tobacco HMGR enzyme activity, inhibited the elicitor-induced capsidiol accumulation in a concentration dependent manner. [14C]Acetate incorporation into capsidiol was likewise inhibited by mevinolin treatment. Unexpectedly, [3H] mevalonate incorporation into capsidiol was also partially inhibited by mevinolin, suggesting that mevinolin may effect secondary sites of sesquiterpenoid biosynthesis in vivo beyond HMGR. The data indicated the importance of the induced HMGR activity for capsidiol production in elicitor-treated tobacco cell suspension cultures.  相似文献   

4.
The signal compound ethylene and its relationships with oxidative burst and cell death were analyzed in cultured tobacco cells treated with the proteinaceous elicitor quercinin. Quercinin belongs to the protein family of elicitins and was isolated from the soil-born oak pathogen Phytophthora quercina. It was shown to induce a dose-dependent oxidative burst in tobacco cell culture in concentrations from 0.05 to 0.5 nM, and subsequently, cell death. The characteristics of quercinin-induced cell death included both membrane damage and DNA fragmentation in tobacco cell culture.

At higher quercinin concentrations (2 nM), H2O2 formation and ethylene biosynthesis were inhibited. Ethylene at low concentrations proved to be necessary for induction and maintenance of H2O2 production in tobacco cells treated with quercinin. It was demonstrated that external addition of inhibitors of ethylene biosynthesis such as -amino-oxy-acetic acid (AOA) and CoCl2 also decreased or even inhibited the quercinin-induced oxidative burst, but did not influence cell death induction. These results demonstrate evidence for a requirement of the plant hormone ethylene for the onset of the quercinin-induced oxidative burst.  相似文献   


5.
We investigated the possible existence of superoxide dismutase (SOD; EC 1.15.1.1) isoenzymes in the pollen of Nicotiana tabacum (Petit Havana SR-1 cultivar). To detect SOD activity, crude extracts from tobacco pollen were subjected to native polyacrylamide gel electrophoresis followed by staining with nitroblue tetrazolium (NBT). The presence of six SOD isoenzymes was detected in tobacco pollen. Treatment with SOD inhibitors indicated the presence of one manganese SOD (Mn SOD), five copper-zinc SOD (Cu/Zn SOD) isoenzymes, and the absence of iron SOD (Fe SOD).  相似文献   

6.
We investigated the possible existence of superoxide dismutase (SOD; EC 1.15.1.1) isoenzymes in the pollen of Nicotiana tabacum (Petit Havana SR-1 cultivar). To detect SOD activity, crude extracts from tobacco pollen were subjected to native polyacrylamide gel electrophoresis followed by staining with nitroblue tetra-zolium (NBT). The presence of six SOD isoenzymes was detected in tobacco pollen. Treatment with SOD inhibitors indicated the presence of one manganese SOD (Mn SOD), five copper-zinc SOD (Cu/Zn SOD) isoenzymes, and the absence of iron SOD (Fe SOD).  相似文献   

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10.
Salicylic acid beta-glucoside (SAG) is a storage form of a defense signal against pathogens, releasing free salicylic acid (SA), to meet the requirements in plants. Since excess SA induces locally restricted cell death following oxidative burst and Ca2+ influx in plants, the effects of SAG on cell viability, Ca2+ influx, and generation of superoxide (O2*-) were examined in suspension-cultured tobacco BY-2 cells expressing aequorin. Among SA-related chemicals tested, only SAG induced the slow and long-lasting O2*- generation, although SAG was less active in acute O2*- generation, Ca2+ influx and induction of cell death. The prolonging action of SAG is likely due to gradual release of SA and the data suggested that a peroxidase-dependent reaction is involved. Notably, pretreatment with low-dose SA (50 micromu) enhanced the response to SAG by 2.5-fold. There are four possible secondary messengers in early SA signaling detectable in the BY-2 culture, namely O2*-, H2O2, Ca2+ and protein kinase (PK). If these messengers are involved in the low-dose SA-dependent priming for SAG response, they should be inducible by low-dose SA. Among the four SA-inducible signaling events, PK activation was excluded from the low-dose SA action since a much higher SA dose (> 0.4 mmu) was required for PK activation.  相似文献   

11.
《Phytochemistry》1987,26(8):2259-2260
Addition of fungal elicitor to tobacco cell suspension cultures induced extracellular accumulation of capsidiol.  相似文献   

12.
Summary.  In cell suspension cultures of Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) a rapid and concentration-dependent accumulation of H2O2 is induced by excess concentrations of copper (up to 100 μM). This specific and early response towards copper stress was shown to be extracellular. Addition of 300 U of catalase per ml decreased the level of H2O2. Superoxide dismutase (5 U/ml) induced an increase in H2O2 production by 22.2%. This indicates that at least part of the H2O2 is produced by dismutation of superoxide. Pretreatment of the cell cultures with the NAD(P)H oxidase inhibitors diphenylene iodonium (2 and 10 μM) and quinacrine (1 and 5 mM) prevented the generation of H2O2 under copper stress for 90%. The influence of the pH on the H2O2 production revealed the possible involvement of cell-wall-dependent peroxidases in the generation of reactive oxygen species after copper stress. Received May 20, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Plant Physiology, Department of Biology, University of Antwerp (RUCA), Groenenborgerlaan 171, 2020 Antwerp, Belgium.  相似文献   

13.
In cell suspension cultures of Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) a rapid and concentration-dependent accumulation of H(2)O(2) is induced by excess concentrations of copper (up to 100 microM). This specific and early response towards copper stress was shown to be extracellular. Addition of 300 U of catalase per ml decreased the level of H(2)O(2). Superoxide dismutase (5 U/ml) induced an increase in H(2)O(2) production by 22.2%. This indicates that at least part of the H(2)O(2) is produced by dismutation of superoxide. Pretreatment of the cell cultures with the NAD(P)H oxidase inhibitors diphenylene iodonium (2 and 10 microM) and quinacrine (1 and 5 mM) prevented the generation of H(2)O(2) under copper stress for 90%. The influence of the pH on the H(2)O(2) production revealed the possible involvement of cell-wall-dependent peroxidases in the generation of reactive oxygen species after copper stress.  相似文献   

14.
Zhang Q  Soole KL  Wiskich JT 《Planta》2001,212(5-6):765-773
Cells of Nicotiana tabacum L. suspension cultures were treated with the respiratory inhibitor rotenone, which specifically inhibits complex I activity of mitochondria. Rotenone retarded cell growth, as shown by decreases in fresh weight, dry weight and cell numbers on a suspension-volume basis. However, rates of the coupled respiration were higher in rotenone-treated compared to control cells when expressed on a fresh-weight basis. Rates of the rotenone-insensitive respiration increased substantially on both a fresh-weight and extractable-cellular-protein basis 24 h after rotenone treatment. ATP/ADP ratios were not significantly different between control and rotenone-treated cells. Our results indicated that cells of tobacco suspension cultures were able to maintain a slow rate of growth and adequate ATP/ADP ratios without the operation of complex I. Received: 12 June 2000 / Accepted: 2 August 2000  相似文献   

15.
Summary The effect ofin situ extraction and elicitor treatment on shikonin production was studied with the suspension cultures ofLithospermum erythrorhizon. Shikonin concentration of 60 mg/L was achieved by the use of both techniques which was 24 times higher than that of control culture, and 65 times higher in terms of shikonin productivity. The host-pathogen effect of elicitor treatment andin situ extraction for product removal were effective for shikonin production.  相似文献   

16.
Farmer EE 《Plant physiology》1985,78(2):338-342
Soybean (Glycine max L.) cells cultured in B5 medium produce extremely low amounts of lignin. However, modification in the growth medium, by lowering the concentration of NO3 and PO2−4, results in the lignification of these cells without affecting levels of cell wall-esterified 4-coumaric and ferulic acid. The production of an extracellular, macromolecular complex by the cultured soybean cells (Moore TS Jr 1973 Plant Physiol 51: 529-536) allows a rapid, nondestructive solubilization of the lignin which can be estimated by reaction with phloroglucinol in free solution. This system has been used to study the effects of fungal elicitor on the synthesis of lignin in soybean cells. The inclusion of very low levels of an elicitor fraction from the cell walls of Phytophthora megasperma in the medium in which lignification of the soybean cells occurs suppressed both the accumulation of extracellular lignin and phloroglucinol staining of the cell walls without affecting the levels of bound hydroxycinnamic acids. The activity profiles of phenylalanine ammonia-lyase (EC 4.3.1.5) and isoenzymes of 4-coumarate:CoA ligase (EC 6.2.1.12) were compared in lignifying and elicitor-treated cell cultures as was the activity of chalcone synthase, an enzyme of flavonoid biosynthesis. The measured activities of these enzymes in cell cultures treated with elicitor were considerably lower than in untreated cells.  相似文献   

17.
Plants respond to the attack of pathogens with the oxidative burst, a production of reactive oxygen species (ROS). In this work a cell culture suspension of Phaseolus vulgaris was used to investigate the oxidative burst triggered by a conidia suspension of different races of Colletotrichum lindemuthianum. As a defence response of the cells a two-phase peak was observed with all used races of Colletotrichum lindemuthianum, varying only in the produced amounts of hydrogen peroxide. Findings with additives such as superoxide dismutase (SOD), diphenyleneiodonium (DPI) and catalase gave rise to the conclusion that more superoxide radicals were produced than be detectable with Amplex Red as hydrogen peroxide. It is assumed that the conversion of the superoxide radical is spontaneous and not driven via a cell-derived superoxide dismutase. The addition of low-molecular cell wall components (ergosterol, glucosamine, galactosamine) showed clearly that compounds like this act as elicitors and thus are involved in triggering the burst. Furthermore, an evaluation of the metabolizing capacities of hydrogen peroxide of the suspension culture cells revealed the enormous capacity of the cells to detoxify this ROS.  相似文献   

18.
Meyer A  Pühler A  Niehaus K 《Planta》2001,213(2):214-222
The lipopolysaccharides (LPSXcc) of the phytopathogenic bacteria Xanthomonas campestris pv. campestris (X.c.c.) were purified from an exopolysaccharide-deficient mutant strain. The isolated LPSxcc induced an oxidative burst reaction in cell-suspension cultures of the non-host plant tobacco (Nicotiana tabacum L.) SRI. The oxidative burst elicited by LPSXcc differed from that induced by yeast elicitor (YE), a cell wall preparation of baker's yeast. The LPSXcc-induced oxidative burst was characterised by a slow increase in H2O2 production and an extended decline. Both the LPSXcc-and YE-induced oxidative bursts were completely blocked by the NAD(P)H-oxidase inhibitor diphenylene-iodonium. When LPSXcc and YE were applied in combination, a synergistic effect and the establishment of refractory states in the generation of H2O2 were observed. The amount of cytosolic calcium was measured in transgenic tobacco cell cultures carrying the apoaequorin gene by coelenterazine-derived chemiluminescence. Whereas YE induced a calcium peak within 1 min after application, LPSXcc induced a long-term calcium signal without transients. To our knowledge this is the first report on the elicitation of an oxidative burst in plant cell cultures by isolated LPS of a phytopathogenic bacterium.  相似文献   

19.
《Phytochemistry》1987,26(2):401-405
A carbohydrate fraction isolated from yeast extract by ethanolic precipitation was used as an elicitor to induce secondary product formation in plant cell suspension cultures. The elicitor preparation is effective in inducing glyceollin isomer synthesis (up to 200 μg glyceollin per g dry wt) in cells of Glycine max and enhancing berberine biosynthesis (up to four-fold) in cells of Thalictrum rugosum. The response of the cell cultures to the elicitor treatment is dependent on the amount of carbohydrate per unit of biomass and on the physiological state of the cells. Cells are optimally induced in late exponential or early stationary growth phases.  相似文献   

20.
Uncaria tomentosa cell suspension cultures were grown in a 2-L stirred tank bioreactor operating at a shear rate gamma(.)(avg)=86 s(-1). The cultures showed an early monophasic oxidative burst measured as H2O2 production (2.15 micromol H2O2 g(-1) dw). This response was followed by a transient production of monoterpenoid oxindole alkaloids (178 +/- 40 microg L(-1) at 24 h). At the stationary phase (144 h), the increase of the shear rate gamma(.)(avg) up to 150 s(-1) and/or oxygen tension up to 85% generated H2O2, restoring oxindole alkaloid production. U. tomentosa cells cultured in Erlenmeyer flasks also exhibited the monophasic oxidative burst but the H2O2 production was 16-fold lower and the alkaloids were not detected. These cells exposed to H2O2 generated in situ produced oxindole alkaloids reaching a maximum of 234 +/- 40 microg L(-1). A positive correlation was observed between the oxindole alkaloid production and the endogenous H2O2 level. On the other hand, addition of 1 microM diphenyleneiodonium (NAD(P)H oxidase inhibitor) or 10 microM sodium azide (peroxidases inhibitor) reduced both H2O2 production and oxindole alkaloids build up, suggesting that these enzymes might play a role in the oxidative burst induced by the hydrodynamic stress.  相似文献   

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