JXB Advance Access originally published online on March 21, 2006
Journal of Experimental Botany 2006 57(6):1323-1332; doi:10.1093/jxb/erj107
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Published by Oxford University Press [2006] on behalf of the Society for Experimental Biology.
RESEARCH PAPER |
Characterization of active oxygen-producing proteins in response to hypo-osmolarity in tobacco and Arabidopsis cell suspensions: identification of a cell wall peroxidase
Institut des Sciences du Végétal, UPR 2355, CNRS, 1 av. de la terrasse, 91198 Gif s/Yvette Cedex, France
To whom correspondence should be addressed. E-mail: Christiane.Lauriere{at}isv.cnrs-gif.fr
Received 18 July 2005; Accepted 22 December 2005
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The oxidative response induced by hypo-osmolarity is characterized in tobacco and Arabidopsis cells in order to identify the corresponding active oxygen-producing proteins. The pharmacological profiles of the oxidative responses were clearly different in the two plant materials, leading to the identification of distinct active oxygen producers in tobacco and Arabidopsis cells. In tobacco cells, a 100 kDa protein, localized in the plasma membrane, was demonstrated to produce active oxygen in the presence of NADPH. This production can be activated by fatty acids and is strongly depressed by diphenylene iodonium, as measured by an in vivo response. In Arabidopsis, 30 kDa and 34 kDa proteins localized in the cell wall were shown to be able to produce active oxygen in the presence of cofactors and the production is prevented by peroxidase inhibitors, as is the in vivo response. The two purified proteins were identified by mass spectrometry and both correspond to the peroxidase gene At5g64120.
Key words: Active oxygen, cell wall, NADPH oxidase, osmotic stress, oxidative burst, peroxidase, plasma membrane
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Active oxygen species (AOS) are partially reduced forms of atmospheric oxygen which can lead to the destruction of plant cells. Besides this toxicity, AOS were identified in recent studies as signalling molecules controlling several processes like programmed cell death and pathogen defence. Changes in the AOS concentration are sensed and the signal is transferred, leading to changes in the transcription of genes. Plant AOS signalling appears to be a particularly complex process, since both the chemical identity of a given AOS and the intracellular site of its production affect the specificity of its biological activity (for a review, see Laloi et al., 2004
).
In plant cells, many potential sources of AOS were reviewed, including reactions in normal metabolism and reactions to stress. AOS producers are localized in almost every cell compartment, including chloroplasts, mitochondria, peroxisomes, apoplasm, plasma membranes, and cell walls (Mittler, 2002). Pathogen-induced AOS production is well characterized and involves plasma membrane-bound NADPH oxidase, cell wall-bound peroxidases, oxalate and amine oxidases in the apoplast (Bolwell et al., 2001). Peroxidase isoforms have been shown to produce H2O2 in vitro with a maximum production at neutral to alkaline pH. This H2O2 generation was reported to be highly dependent upon extracellular alkalinization which is classically observed in the apoplast following pathogen recognition (Bolwell and Wojtaszek, 1997). At the same time, secretion of peroxidase directed to sites of infection has been demonstrated (McLusky et al., 1999) as well as a highly localized increase in peroxidase activity at the sites of bacterial attachment (Bestwick et al., 1998). The plant NADPH oxidase, which is homologous to gp91phox the transmembrane subunit of the mammalian oxidase, appears to play various roles, including regulation of plant cell growth (Foreman et al., 2003), mediation of ABA-dependent stomatal closure (Kwak et al., 2003), as well as plant defence and disease resistance (Torres et al., 2002; Yoshioka et al., 2003).
Induction of oxidative burst by physical stimuli like mechanical/hypo-osmotic stresses is still poorly characterized. Mechanically induced oxidative response was demonstrated in soybean (Yahraus et al., 1995) and tobacco cells (Cazalé et al., 1998). In comparison with the elicitor-induced oxidative response, mechanical stimulation of the burst appears to be regulated by a different set of constraints (Chandra et al., 2000) and through differing pathways. In tobacco, the hypo-osmotically induced oxidative response appears to require phospholipase A activation, contrary to the burst induced by oligogalacturonides (Mathieu et al., 2002). It also needs Ca2+ channels to be opened, anion effluxes, and phosphorylation events (Cazalé et al., 1998) which probably involve MAP kinase activity (Cazalé et al., 1999). AOS production was highly depressed by iodonium compounds (Cazalé et al., 1998), but no information is available on the molecular identification of the enzymes producing AOS in response to hypo-osmolarity. Fatty acids like arachidonate and linolenate were shown to be able to stimulate the oxidative response induced by hypo-osmotic stress, and a NADPH-dependent producing system was characterized in plasma membrane vesicles (Mathieu et al., 2002), displaying properties similar to the oxidative burst assayed in vivo.
In this study, the oxidative burst induced by hypo-osmolarity was characterized in Arabidopsis cells and compared with that measured in tobacco cells. Based on the different pharmacological profiles of the two responses assayed in vivo, a search was carried out to find the corresponding AOS producers using purified cellular fractions. Further purification of the proteins of interest led to the characterization of a 100 kDa protein in tobacco which is likely to correspond to NADPH oxidase, and to the molecular identification by mass spectrometry of an Arabidopsis cell wall-bound peroxidase able to produce superoxide anions.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant material
Tobacco cells (Nicotiana tabacum cv. Xanthi) were cultured in Murashige and Skoog medium pH 5.2 with 0.91 µM 2,4-D, 60 nM kinetin, 555 µM myo-inositol, 1.19 µM thiamine-HCl, and 3% (w/v) sucrose. Arabidopsis thaliana cells (Columbia ecotype) were cultured in liquid medium as previously described (Droillard et al., 2002
). The two cell suspensions were grown at 23 °C, in constant light, and used after 4–5 d subculturing with 100 mg FW ml–1 cell density.
Chemicals
All chemicals were purchased from Sigma-Aldrich. Stock solutions of diphenylene iodonium (DPI; up to 20 mM), 2,6 dichlorophenol (DCP; 300 mM), p-coumaric acid (300 mM), and 3,3',5,5'-tetramethylbenzidine (TMBZ; 30 mM) were prepared in DMSO. A stock solution of epinephrine (20 mM) was prepared in water acidified with H2SO4 (6.35 mM final concentration). Stock solutions of horseradish peroxidase (type VI-A, 2.6 U µl–1), superoxide dismutase (SOD) from bovine erythrocytes (100 U µl–1), and guaiacol (80 mM) were prepared in water. The linolenic acid (35.9 mM) was solubilized in absolute ethanol, then diluted in water and finally clarified by addition of 1% (v/v) 1 N NaOH.
Osmotic stresses
Osmolarity was monitored using a freezing point osmometer (Roebling) on 100 µl aliquots. Osmotic stresses were applied essentially as previously described for tobacco (Mathieu et al., 2002) and Arabidopsis (Droillard et al., 2002) cells. Briefly, tobacco cell suspensions were adjusted to 180 mOsm with sucrose and supplemented with 1 mM CaSO4, 10 mM MES-TRIS pH 5.2. After 3–4 h equilibration of aliquots of 8 ml of cell suspension, 6 ml of extracellular medium were replaced by the same volume of either hypo-osmotic medium (10 mM MES-TRIS pH 5.2, 1 mM CaSO4, sucrose-free) or iso-osmotic medium (10 mM MES-TRIS pH 5.2, 1 mM CaSO4, 180 mM sucrose). Arabidopsis cells were treated in a similar way except for the use of MES-TRIS buffer pH 6.25 during equilibration (10 mM) and stress (30 mM).
Oxidative burst assay
The formation of activated oxygen was determined using oxidation of epinephrine to the red product adrenochrome (Mathieu et al., 2002). Epinephrine was added to the cells at zero time (final concentration 750 µM). For Arabidopsis cells, horseradish peroxidase (6.5 U ml–1) was also added. After different times, 350 µl samples of extracellular medium, supplemented with 10 µl of 0.5 N HCl, were transferred to a titration microplate and absorbance was monitored (Dynatech). Absorbance at 490 nm was measured and a control value (600 nm) was subtracted to remove absorbance due to the eventual presence of cells. Adrenochrome formation was quantified, using a calibration curve established in the presence of horseradish peroxidase (13 U ml–1) and H2O2 (0–400 µM).
Production of AOS by model systems
Glucose oxidase:
The production of hydrogen peroxide by the glucose–glucose oxidase system was assayed in a 1 ml reaction mixture containing 30 mM MES-TRIS pH 6.2, 1.2 mM glucose, 0.75 mM epinephrine, and 6.5 U horseradish peroxidase. The reaction was initiated by addition of glucose oxidase (0.35 U ml–1), and the production of hydrogen peroxide was followed by monitoring the changes in absorption at 490 nm due to the oxidation of epinephrine to the red product adrenochrome.
Horseradish peroxidase:
The production of activated oxygen by horseradish peroxidase was measured using either the oxidation of epinephrine or the reduction by NBT (Van Gestelen et al., 1997). Assay mixtures (350 µl) contained 60 mM MES-TRIS pH 6.2, 1 mM MnSO4, 90 µM DCP, 0.75 mM NADPH, and either 0.75 mM epinephrine or 1 mM NBT. Production of AOS was initiated by the addition of horseradish peroxidase (13 U ml–1) and the changes followed by measuring absorbance at 490 nm (epinephrine oxidation) or 530 nm (NBT reduction). Adrenochrome formation was quantified using a calibration curve established in the presence of H2O2 (0–400 µM) and an absorption coefficient of 12.8 mmol–1 cm–1 for the production of formazan.
Preparation of plasma membrane-enriched fractions
Plasma membrane was purified by two-phase partitioning using PEG-dextran essentially as described previously (Mathieu et al., 2002) but with the following modifications. Arabidopsis and tobacco cells were ground twice for 30 s in an electric grinder (D56; Moulinex). The resulting pellet after centrifugation (2000 g, 30 min) was again homogenized 4-fold for 30 s and the two supernatants were mixed to prepare the microsomal fraction. For the two-phase partitioning, 6.4% (w/v) of each polymer was used, with 5 mM KCl for tobacco and 3 mM KCl for Arabidopsis cells.
Preparation of cell-wall fractions
Arabidopsis cells (200 g) were washed in 250 ml of 2 mM MES-TRIS pH 6.2 and then extracted by stirring for 20 min in 100 ml fresh Arabidopsis culture medium adjusted with 0.2 M KCl, 2.2 mM phenylmethylsulphonylfluoride (PMSF), 0.75 µg ml–1 antipain, and 0.75 µg ml–1 leupeptin. After filtration on fritted glass, solubilized proteins were precipitated by (NH4)2SO4 (45–85%). The final pellet was suspended in 20 mM MES-TRIS pH 7, dialysed overnight with the same buffer, and then centrifuged to remove insoluble material. For tobacco cells, cell wall proteins were prepared in a similar way. Proteins were extracted in fresh tobacco culture medium adjusted with 0.2 M KCl, 2.2 mM PMSF, 0.75 µg ml–1 antipain, and 0.75 µg ml–1 leupeptin. After filtration on fritted glass, 0.6% (w/v) polyvinylpyrrolidone and 5 mM ascorbate were added before precipitation with (NH4)2SO4. For both materials, the supernatants were concentrated if necessary by use of a centricon 30 (Amicon concentrator) to get a 5 µg µl–1 protein concentration.
Enzyme assays in plasma membrane and cell-wall fractions
Peroxidase activity:
Enzyme activity was assayed using TMBZ as a substrate (Imberty et al., 1984). The assay contained 700 µM TMBZ and 3 mM H2O2 in 100 mM MES-TRIS pH 6.25. The reaction was initiated by addition of the cellular fraction. Absorbance at 640 nm was measured and the product formation was quantified, using a calibration curve established in the presence of horseradish peroxidase (1 U ml–1) and H2O2 (0–400 µM).
Superoxide production:
pH 7.8. The SOD-dependent NBT reduction was assayed to evaluate superoxide production in the cell fractions. The assay mixture contained 100 mM TRIS-HCl pH 7.8, 1 mM NBT, 1 mM NADPH, and 120 µM C 18:3, and was with or without SOD (200 U ml–1). The reaction was initiated by addition of the cell fraction. Absorbance at 530 nm was measured and the value obtained for the assay containing SOD was subtracted. Activity was calculated using a formazan absorption coefficient of 12.8 mmol–1 cm–1.
Superoxide production:
pH 6.25 and cofactors. Active oxygen production by peroxidases was evaluated by NBT reduction in the presence of NADPH and cofactors. The assay mixture contained 50 mM MES-TRIS pH 6.25, 0.8 mM NBT, and 1 mM NADPH, and was with or without cofactors (0.3 mM DCP, 3.5 mM MnSO4). The reaction was initiated by addition of the cell fraction. Absorbance at 530 nm was measured and the value obtained for the assay deprived of cofactors was subtracted. Activity was calculated using a formazan absorption coefficient of 12.8 mmol–1 cm–1.
H2O2 production:
Production of active oxygen by peroxidases in the presence of NADPH and cofactors was also verified by demonstrating the formation of H2O2 which is able to oxidize guaiacol (Mader and Amberg-Fisher, 1982). The assay mixture contained 50 mM MES-TRIS pH 6.25, 0.3 mM NADPH, 0.3 mM DCP, and 3.5 mM MnSO4. The reaction was initiated by the addition of the Arabidopsis cell wall fraction (3 µg of proteins). When NADPH (absorbance measured at 340 nm) was fully oxidized (
12 min), 5 mM guaiacol were added and the browning was evaluated by measuring the absorbance at 470 nm.
HPLC of plasma membrane and cell wall fractions
Proteins of the tobacco plasma membrane and the Arabidopsis cell wall fractions were separated by HPLC (Beckman) using a cation exchange column (SP 5 PW, 7.5–75 mm; Waters). For the plasma membrane fraction, proteins were solubilized by addition of 0.5% (v/v) Triton X-100 to reach a 1:1 (w/w) protein:detergent ratio and gentle stirring for 2 h on ice. After centrifugation (18 000 g, 30 min, 4 °C), the protein sample (10 mg) was loaded on the column previously equilibrated in 20 mM acetate pH 4.8, 10% (v/v) glycerol, 200 mM saccharose, and 0.5% (v/v) Triton X-100. Proteins were eluted from the column in 2 ml fractions, at a flow rate of 1 ml min–1 with a continuous salt gradient of 0–1 M NaCl in the same buffer. Fractions were then concentrated using a centricon 30, with two successive washings with 500 µl of 20 mM TRIS-HCl pH 6.8, 10% (v/v) glycerol, 200 mM saccharose, and 0.5% (v/v) Triton X-100. For the cell-wall fraction, the sample (9 mg) was loaded on the column previously equilibrated in 50 mM acetate pH 5.25. Proteins were eluted from the column in 2 ml fractions at a flow rate of 1 ml min–1 with a continuous salt gradient of 0–1 M NaCl in the same buffer. Peroxidase activity was assayed in each fraction and the fractions containing peroxidase were then concentrated using a centricon 30, with two successive washings with 500 µl of 20 mM acetate pH 5.2.
Protein assays
Protein concentrations were determined by the Bradford method (Bradford, 1976) or by the bicinchoninic acid (BC assay kit; Uptima-Interchim), when 0.5% (v/v) Triton X-100 is present. In both cases, bovine serum albumin was used as a standard.
Sample preparation and electrophoresis
For SDS-PAGE, cell-fraction proteins were diluted immediately before loading in Laemmli buffer deprived of dithiothreitol and containing only 0.3% (w/v) SDS (cell wall) or 2% (w/v) SDS (plasma membrane). Samples were electrophoresed at 4 °C on 10% (cell wall) or 7.5% (plasma membrane) SDS-polyacrylamide gels. For two-dimensional (2D) electrophoresis, cell fraction proteins were diluted in 2% (w/v) CHAPS, 0.4% (v/v) Triton X-100, and 1% (v/v) Pharmalytes pH 3–10 (Amersham Biosciences). Protein extracts were then loaded at the cathodic side of a 7 cm ReadyStrip-immobilized pH gradient pH 3–10 (Bio-Rad) previously rehydrated at room temperature with a solution containing 0.5% (w/v) CHAPS and 0.8% (v/v) Pharmalytes pH 3–10. Isoelectric focusing was completed overnight, running for a total of 27 kVh, at 6 °C. Gel strips were equilibrated twice for 10 min in 80 mM TRIS-HCl pH 6.8, 20% (v/v) glycerol, and 2% (w/v) SDS (plasma membrane) or without SDS (cell wall) at room temperature before being placed on top of the SDS-polyacrylamide gel as described previously. One- or two-dimensional gels were then washed for SDS removal and renaturation as described below.
Western blot
Protein extracts were separated on 7.5% SDS-polyacrylamide gels and immunoblotted onto polyvinylidene difluoride membranes (Millipore). Blots were blocked with 5% (w/v) defatted milk in TBS-Tween [10 mM TRIS-HCl pH 7.5, 154 mM NaCl, 0.1% (v/v) Tween 20] and probed with 1:250 polyclonal antibodies (Doussiere et al., 1995) directed against a synthetic peptide corresponding to residues 552–559 of the gp91 C-ter sequence. Horseradish peroxidase-conjugated anti-rabbit IgG (Sigma) was used as secondary antibody and the reaction was visualized using the NBT/BCIP kit (BioRad).
In gel enzyme assays
Peroxidase activity:
Peroxidase activity was assayed in gels after renaturation (Schmidt and Trojanowski, 1986). Gels were washed extensively by shaking at 4 °C in water for 20 min and 2 mM MES-TRIS pH 5.2 for 20 min, and then 10 min at room temperature in the same buffer. The gels were then incubated in 100 mM MES-TRIS pH 5.2 containing 480 µM TMBZ and 11% (v/v) DMSO for 5 min before addition of 13 mM H2O2.
Nitro blue tetrazolium (NBT) reduction at pH 7.8:
Gels were washed by shaking for 30 min at 4 °C in 10% (v/v) glycerol and 0.5% (v/v) Triton X-100, and 10 min at room temperature in 10% (v/v) glycerol and 10 mM TRIS-HCl pH 7.8. Then, they were incubated for 5 min in 100 mM TRIS-HCl pH 7.8 and the reaction was initiated by addition of 1 mM NADPH and 120 µM C 18:3.
Production of AOS by peroxidases at pH 6.25 in the presence of cofactors:
Gels were washed as described above for peroxidase activity. The active oxygen production by peroxidases in the presence of NADPH and cofactors (Mn, phenol) was visualized using either NBT or TMBZ. With NBT, gels were incubated in 0.1 M MES-TRIS pH 6.25 containing 1.25 mM NBT with or without cofactors (0.3 mM DCP or p-coumaric acid, 3 mM MnSO4), for 5 min before addition of 1.2 mM NADPH. With TMBZ, gels were incubated in 0.1 M MES-TRIS pH 6.25 with or without the cofactors (0.3 mM DCP or p-coumaric acid, 3 mM MnSO4) for 5 min before addition of 1.2 mM NADPH. After 3 min, H2O2 production was made evident by adding 400 µM TMBZ.
Mass spectrometry analysis
Peptide samples resulting from trypsin digestion were analysed by ion-trap LC-MS/MS for identification by cross-referencing databases (plateforme de protéomique végétale, UMR de génétique végétale and IFR 87, Ferme du Moulon, 91190 Gif sur Yvette, France).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Formation of AOS in response to hypo-osmotic stress is due to distinct producing systems in tobacco and Arabidopsis cells
The production of AOS in response to hypo-osmolarity was described previously in tobacco cell suspensions as highly depressed by the inhibitor DPI (Cazalé et al., 1998
). This hypo-osmotically induced oxidative burst measured by epinephrine oxidation in Arabidopsis cell suspensions (0.37 ±0.02 µmol produced adrenochrome g–1 FW 15 min–1) is low in comparison to the response of tobacco cells (1.44 ±0.06 µmol produced adrenochrome g–1 FW 15 min–1). The Arabidopsis response is also clearly more resistant to 2 µM DPI, which has only a slight inhibitory effect at this concentration (Fig. 1A, B). To test the possibility that a peroxidase may be involved in the oxidative burst induced by hypo-osmolarity, peroxidase inhibitors [KCN, salicyl hydroxamic acid (SHAM)] were assayed, leading to the same result, namely the high susceptibility of the response of Arabidopsis cells to peroxidase inhibitors, contrary to the response of tobacco cells. The effect of 2 mM SHAM on the hypo-osmotically induced oxidative response is illustrated in Fig 1A, B for both tobacco and Arabidopsis cells. Because the AOS assay using epinephrine needs the presence of peroxidase, it was verified that the effect of SHAM on the oxidative response was not due to a direct effect of SHAM on the AOS assay. AOS were produced by a model system using glucose oxidase and glucose and assayed by epinephrine oxidation in the presence of peroxidase. No significant effect of 2 mM SHAM was observed in this assay (Fig. 1C). Thus, responses of Arabidopsis and tobacco cells to the same stimulus, hypo-osmotic stress, display clear pharmacological differences, suggesting that distinct producing systems may be involved.
|
Superoxide production and peroxidase activity are present in plasma membrane and cell wall fractions
One AOS-producing system previously characterized in the plasma membrane of tobacco cells was NADPH dependent, highly depressed by diphenyl iodonium, and capable of being activated by fatty acids (Mathieu et al., 2002
). As these pharmacological properties corresponded to the tobacco oxidative burst induced by hypo-osmolarity, it was interesting to investigate further the tobacco plasma membrane enzyme. For Arabidopsis, the hypothesis that a peroxidase located either in the cell wall or the plasma membrane was involved in the generation of extracellular AOS was formulated. As a first step, superoxide production, evaluated by the SOD-dependent formation of formazan induced by NADPH at pH 7.8 (Fig. 2A) and peroxidase activity (Fig. 2B) were evaluated both in plasma membrane and cell wall fractions to search for a possible different pattern in the two plant species. Unexpectedly, quite similar results were obtained for Arabidopsis and tobacco cells, with higher activities in cell wall fractions compared with plasma membrane fractions for both plant species (Fig. 2). However, it was noticeable that peroxidase activity was clearly higher in tobacco than in the Arabidopsis cell wall (Fig. 2B). In any case, it was only possible to detect peroxidase activity in the cell wall in Arabidopsis, leading to further investigation of this compartment in order to identify the putative peroxidase of interest.
|
Peroxidases are able to produce AOS in the presence of Mn and phenols
The conditions allowing the production of AOS by peroxidases were first investigated using horseradish peroxidase as a model. The production of AOS was evaluated using the formation of both adrenochrome (Fig. 3A) and formazan (Fig. 3B). A mix of two cofactors, DCP and Mn2+ (Halliwell, 1978
), was observed to be highly efficient in inducing the formation of AOS catalysed by the peroxidase, whatever the probe used for the assay. This AOS production, dependent on the presence of cofactors, was strongly reduced by both SOD and catalase, confirming the validity of the assay (Fig. 3C). Additionally, formation of formazan was clearly depressed by the peroxidase inhibitor SHAM but was almost unaffected by DPI. These two properties, in good agreement with the pharmacological profile of the oxidative burst induced by hypo-osmolarity in Arabidopsis, reinforced the hypothesis of a peroxidase acting to produce AOS. The ability of the plant cell fractions to produce AOS when the cofactors Mn2+ and DCP are added was then evaluated (Fig. 4A), using the NBT reduction assay. This cofactor-induced formazan formation was mainly observed in cell wall fractions from both tobacco and Arabidopsis cells. In Arabidopsis, AOS production in the presence of cofactors was not observed in the plasma membrane (Fig. 4A) in accordance with the absence of peroxidase in this cellular compartment (Fig. 2B). The pharmacological profile of AOS generation by the Arabidopsis cell-wall proteins (Fig. 4B) showed similar characteristics to those displayed by horseradish peroxidase (Fig. 3C). However, a significant inhibition of AOS generation (around 20%) by DPI was observed only for the cell wall. This result is in good agreement with the slight inhibition by DPI observed in vivo (Fig. 1B). It suggests that, in addition to peroxidase, a second way of producing AOS that is depressed by DPI, may only contribute in a minor way to AOS generation in the plant cell wall. The formation of AOS by Arabidopsis cell wall peroxidase(s) was further documented, following oxidation of guaiacol by the hydrogen peroxide produced (Fig. 4C). NADPH added to the cell wall was rapidly oxidized only if the cofactors Mn2+ and DCP were present. The production of H2O2 by the reaction may thus be visualized by the browning of gaiacol, one peroxidase substrate.
|
|
Characterization of AOS-producing systems in tobacco PM and Arabidopsis cell wall
To characterize the AOS generators involved in tobacco and Arabidopsis hypo-osmotic signalling, cell fraction proteins were separated by electrophoresis. In tobacco, a search was carried out for an AOS-producing system displaying pharmacological properties corresponding to the hypo-osmotically induced oxidative burst, which was NADPH dependent, highly depressed by iodonium diphenyl, and capable of being activated by fatty acids (Mathieu et al., 2002
) (Fig. 5). Renaturation of the plasma-membrane proteins separated by SDS-PAGE allowed the visualization of three proteins, 165, 100, and 40 kDa, able to reduce NBT in an NADPH-dependent reaction (Fig. 5A). Using an antibody directed against the neutrophile NADPH oxidase (Doussiere et al., 1995), a signal was visualized around 100 kDa, strongly suggesting that the 100 kDa protein, able to produce AOS, corresponds to tobacco NADPH oxidase. Its enzyme activity was dependent on NADPH, capable of being activated by fatty acids, and inhibited by DPI (Fig. 5B). In the presence of NADH instead of NADPH, three activity bands, 165, 85, and 40 kDa, were also visualized, suggesting that the 165 kDa and 40 kDa bands may use both NADPH and NADH contrary to the 100 kDa NADPH oxidase.
|
Electrophoretic analysis was also performed on the Arabidopsis cell wall fraction (Fig. 6). Enzymes able to produce AOS in the presence of the cofactors Mn2+ and DCP (Mader and Amberg-Fisher, 1982
) were identified by NBT staining after renaturation of the proteins. First, the effect of adding cofactors was compared at pH 7.8 and pH 6.2 using NBT reduction (Fig. 6A). Cofactors were efficient in inducing the formation of AOS only at pH 6.2, with the appearance of several activity bands, notably two major bands (100 kDa and 34 kDa) and a minor band (180 kDa) which correspond to classical peroxidase activity, stained using H2O2 and TMBZ as substrates (Fig. 6A). The pharmacological profile of the three proteins corresponded with the oxidative response characterized in vivo: inhibition by SHAM and resistance to the action of DPI (Fig. 6B). Finally, the effect of a phenol naturally present in plants, p-coumaric acid, was compared with the effect of DCP (Fig. 6C). Using the NBT reduction assay, similar profiles were observed with the two phenols (Fig. 6C, left). This result was also confirmed, using another staining assay, TMBZ oxidation (Fig. 6C, right). TMBZ was used in this case to assay the production of AOS in the presence of NADPH and cofactors. All together, these results indicated the presence of enzymes capable of producing AOS and displaying peroxidase characteristics in the Arabidopsis cell wall. The genome of Arabidopsis contains a high number of peroxidase genes containing N-terminal signal peptides so the molecular identification of the enzyme(s) of interest was undertaken.
|
Molecular identification of Arabidopsis AOS-producing peroxidase
The cell-wall proteins were separated by cation exchange chromatography and analysed for peroxidase activity. The active fractions were then separated by SDS-PAGE and the gels stained for peroxidase activity after renaturation of the proteins. A fraction containing a high level of activity was selected, displaying two main active proteins of 34 kDa and 30 kDa (Fig. 7A, left). The fraction was further purified by 2D electrophoresis (Fig. 7A) and the two resulting activity bands were analysed by mass spectrometry. Based on four peptides for the 34 kDa band and three peptides for the 30 kDa band, the same peroxidase protein was identified (Fig. 7B), corresponding to the At5g64120 gene. Thus, the 30 kDa and 34 kDa proteins appear as two forms of the same peroxidase.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Production of AOS in response to hypo-osmolarity was demonstrated in both tobacco and Arabidopsis cell suspensions. The pharmacological profiles of the Arabidopsis and tobacco responses were shown to be different, with a high inhibition of the tobacco burst by DPI (Fig. 1A) and the Arabidopsis burst by peroxidase inhibitors like SHAM (Fig. 1B). Starting from these different in vivo pharmacological profiles, a search was carried out for the corresponding AOS producers, using in vitro AOS assays on purified cell fractions or directly on electrophoresis gels. The SOD-dependent reduction of NBT was used to assay AOS production both on cell fractions (Figs 2, 4) and gels (Figs 5, 6), and the similarity between the results obtained by adrenochrome or formazan formations is illustrated (Fig. 3).
In the first step, NADPH-dependent AOS formation was assayed in purified cell wall and plasma membrane fractions for both Arabidopsis and tobacco cells, and similar levels of activity were observed in the two plants (Fig. 2A). Because SHAM is efficient in preventing the hypo-osmotically induced oxidative burst in Arabidopsis (Fig. 1), suggesting a putative AOS-producing peroxidase, the classical scavenging H2O2 peroxidase activity was also evaluated in the different cell fractions (Fig. 2B). The activities were much higher in tobacco than in Arabidopsis cells, with a main localization of the activity in the cell wall, leading to further characterization of the cell-wall peroxidases in Arabidopsis.
An AOS producer was characterized previously in tobacco plasma membrane vesicles as NADPH-dependent, strongly depressed by iodonium diphenyl, and activated by fatty acids like the oxidative response assayed in vivo (Mathieu et al., 2002). It can be noted that 20 µM iodonium diphenyl used in this previous study and 2 µM DPI used here have similar inhibitory effects. It is known that DPI is a more efficient inhibitor of the flavoproteins than the closely related molecule iodonium diphenyl (Jabs et al., 1997) and DPI was thus preferred here. The producing activity previously described was further clarified and a 100 kDa protein was shown to produce superoxide anions (Fig. 5B), with stimulation of the activity by fatty acids and strong inhibition by 5 µM DPI. This 100 kDa protein is likely to correspond to NADPH oxidase, since an anti-gp91phox antibody also recognizes a 100 kDa protein in the same purified fraction of the plasma membrane (Fig. 5A). An attempt was also made to purify further the enzyme activity in order to allow mass spectrometry analysis. However, the 100 kDa activity was labile and could not be detected any more after further purification. This lability has already been reported in a study aimed at determining the molecular nature of the formazan-generating polypeptides observed in SDS gels using an anti-tomato gp91phox homologue antibody (Sagi and Fluhr, 2001). A second formazan-producing protein was also observed around 40 kDa (Fig. 5). This band was not immunolabelled by the anti-gp91phox antibody, was able to strongly produce AOS using both NADH and NADPH, and retains a part of its activity in the presence of a strong concentration of DPI (Fig. 5B). Although these properties of the 40 kDa protein did not match those of the AOS producer induced by hypo-osmolarity, it will be interesting to identify this second plasma membrane formazan-generating protein.
The Arabidopsis oxidative burst induced by hypo-osmolarity was inhibited by SHAM and was relatively insensitive to DPI (Fig. 1), suggesting the generation of AOS by a peroxidase. Using horseradish peroxidase as a model, conditions allowing the production of AOS were determined first (Fig. 3). In the presence of NADPH, Mn ions, and dichlorophenol, the peroxidase was able to produce AOS at pH 6.2. These conditions of assay were successful in detecting AOS production in the Arabidopsis cell wall, which was again strongly inhibited by SHAM and not very sensitive to DPI (Fig. 4B). The producing activity at pH 6.2 was then analysed in gel after denaturing electrophoresis and observed to correspond to several bands notably around 180, 100, 34, and 30 kDa (Fig. 6). The protein molecular masses predicted from the full-length peroxidase genes identified in the Arabidopsis genome were below 50 kDa, and an attempt was made to purify the active 34 kDa and 30 kDa proteins. The two proteins were recovered in the same HPLC fraction which was further purified by 2D electrophoresis (Fig. 7). Mass spectrometry analysis of the 34 kDa and 30 kDa peroxidases excised from the 2D gel led to the molecular identification of only one peroxidase gene, At5g64120. The corresponding protein, called peroxidase 71 or AtP15 in the databases, comprises 328 amino acids, with predicted molecular mass and isoelectric points of 34.89 and 8.6, respectively. These characteristics match the apparent molecular masses and basic properties observed after 2D electrophoresis and it may be assumed that the 34 kDa and 30 kDa proteins correspond to close forms of the peroxidase, differing in glycosylation status or other post-translational modification. It can be noted that the high molecular weight forms of peroxidases were active in producing AOS and probably correspond to polymers of the same peroxidase. It can also be noted that the Arabidopsis peroxidase identified here differs from the anionic enzyme previously purified as the predominant peroxidase present in cell suspension cultures (Ostergaard et al., 1996).
There are 73 peroxidase genes in Arabidopsis thaliana and the complete sequencing, mRNA expression analyses, and predicted protein structures were recently analysed (Welinder et al., 2002). No unusual feature could be found for this peroxidase, as was also the case for the French bean peroxidase isoform believed to be responsible for the apoplastic oxidative burst induced by fungal elicitor (Blee et al., 2001). The 46 kDa exocellular peroxidase purified from walls of suspension-cultured French bean cells is cationic and differs from other peroxidases in its ability to generate H2O2 at relatively neutral pH. In this apoplastic burst induced by biotic stress, an extracellular alkalinization is absolutely necessary and the French bean oxidative burst does not occur if the pH of the medium is held at 6.0 (Bolwell et al., 2002). A different mechanism of oxidative burst induction has thus to be envisaged for the anionic peroxidase AtP15 which is able to produce AOS at pH 6.2 (Fig. 6). This result suggests that biotic and abiotic stresses might use different pathways to induce AOS production by cell wall peroxidases. In each case, the question of the electron donor/substrate in AOS production by peroxidases remains to be resolved. NADPH is localized in the cytoplasm, suggesting another extracellular donor, and cysteine was used successfully in the French bean peroxidase experiments (Bolwell et al., 2002). Concerning the phenol requirement for the reaction, it is interesting to note that p-coumaric acid is highly efficient in promoting the AOS reaction (Fig. 6). Other naturally occurring phenolic compounds like ferulic acid, which is believed to be an in vivo substrate for plant peroxidases, are found in the cell wall (Gajhede, 2001).
Although plasma membrane NADPH oxidase and one cell wall peroxidase appear to play major roles in oxidative bursts induced by hypo-osmolarity in tobacco and Arabidopsis cells, respectively, other AOS generators may also co-function with these main producers. For example, the oxidative response assayed here in vivo in Arabidopsis cells is slightly inhibited by a low concentration of DPI (Fig. 1B), although the peroxidase characterized is clearly more resistant to DPI action (Fig. 4B). This result suggests that a second AOS generator, highly sensitive to DPI, may account for a minor part of the oxidative response induced by hypo-osmolarity in Arabidopsis. Similarly, at least two distinct sources were shown to contribute to the oxidative burst induced by infection of Arabidopsis by Pseudomonas syringae (Grant et al., 2000). More generally, it is clear that other enzymes able to produce AOS in the apoplast are present in the tobacco cell wall and the Arabidopsis plasma membrane, but they are likely to be involved in other signalling pathways. These different data underline the complexity of the signalling pathways leading to extracellular AOS production. However, the molecular identification of functional AOS producers presented here will support the genetic studies that must now be undertaken.
|
Acknowledgements |
|---|
We thank Luc Negroni (UMR de Génétique Végétale, CNRS/INRA/INAPG and IFR 87 la Plante et son environnement) for performing the mass spectrometry analysis.
|
Footnotes |
|---|
* These authors contributed equally to this work.
Abbreviations: AOS, active oxygen species; DCP, 2,6 dichlorophenol; DPI, diphenylene iodonium; NBT, nitro blue tetrazolium; SHAM, salicyl hydroxamic acid; SOD, superoxide dismutase; TMBZ, 3,3',5,5'-tetramethylbenzidine.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Bestwick CS, Brown IR, Mansfield JW. 1998. Localized changes in peroxidase activity accompany hydrogen peroxide generation during the development of a nonhost hypersensitive reaction in lettuce. Plant Physiology 118, 1067–1078.
Blee KA, Jupe SC, Richard G, Zimmerlin A, Davies DR, Bolwell GP. 2001. Molecular identification and expression of the peroxidase responsible for the oxidative burst in French bean (Phaseolus vulgaris L.) and related members of the gene family. Plant Molecular Biology 47, 607–620.[CrossRef][Web of Science][Medline]
Bolwell GP, Bindschedler LV, Blee KA, Butt VS, Davies DR, Gardner SL, Gerrish C, Minibayeva F. 2002. The apoplastic oxidative burst in response to biotic stress in plants: a three-component system. Journal of Experimental Botany 53, 1367–1376.
Bolwell GP, Page A, Pislewska M, Wojtaszek P. 2001. Pathogenic infection and the oxidative defences in plant apoplast. Protoplasma 217, 20–32.[CrossRef][Web of Science][Medline]
Bolwell GP, Wojtaszek P. 1997. Mechanisms for the generation of reactive oxygen species in plant defence – a broad perspective. Physiological and Molecular Plant Pathology 51, 347–366.
Bradford M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254.[CrossRef][Web of Science][Medline]
Cazalé A-C, Droillard M, Wilson C, Barbier-Brygoo H, Laurière C. 1999. MBP kinase activation by hypoosmotic stress of tobacco cell suspensions: towards the oxidative burst response? The Plant Journal 19, 297–307.[CrossRef][Web of Science][Medline]
Cazalé A-C, Rouet-Mayer MA, Mathieu Y, Barbier-Brygoo H, Laurière C. 1998. Oxidative burst and hypoosmotic stress in tobacco cell suspensions. Plant Physiology 116, 659–669.
Chandra S, Cessna SG, Yahraus T, Devine R, Low PS. 2000. Homologous and heterologous desensitization and synergy in pathways leading to the soybean oxidative burst. Planta 211, 736–742.[CrossRef][Web of Science][Medline]
Doussiere J, Buzenet G, Vignais PV. 1995. Photoaffinity labeling and photoinactivation of the O2(-)-generating oxidase of neutrophils by an azido derivative of FAD. Biochemistry 34, 1760–1770.[CrossRef][Medline]
Droillard MJ, Boudsocq M, Barbier-Brygoo H, Laurière C. 2002. Different protein kinase families are activated by osmotic stresses in Arabidopsis thaliana cell suspensions: involvement of the MAP kinases AtMPK3 and AtMPK6. FEBS Letters 527, 43–50.[CrossRef][Web of Science][Medline]
Foreman J, Demidchik V, Bothwell JH, et al. 2003. Reactive oxygen species produced by NADPH oxidase regulate plant cell growth. Nature 422, 442–446.[CrossRef][Medline]
Gajhede M. 2001. Plant peroxidases: substrate complexes with mechanistic implications. Biochemical Society Transactions 29, 91–98.[CrossRef][Web of Science][Medline]
Grant JJ, Yun BW, Loake GJ. 2000. Oxidative burst and cognate redox signalling reported by luciferase imaging: identification of a signal network that functions independently of ethylene, SA and Me-JA but is dependent on MAPKK activity. The Plant Journal 24, 569–582.[CrossRef][Web of Science][Medline]
Halliwell B. 1978. Lignin synthesis: the generation of hydrogen peroxide and superoxide by HRP and its stimulation by manganese (II) and phenols. Planta 140, 81–88.[CrossRef]
Imberty A, Goldberg R, Catesson A-M. 1984. Tetramethylbenzidine and p-phenylenediamine-pyrocatechol for peroxidase histochemistry and biochemistry: two new, non-carcinogenic chromogens for investigating lignification process. Plant Science Letters 35, 103–108.[CrossRef]
Jabs T, Tschöpe M, Colling C, Hahlbrock K, Scheel D. 1997. Elicitor-stimulated ion fluxes and 
from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley. Proceedings of the National Academy of Sciences, USA 94, 4800–4805.
Kwak JM, Mori IC, Pei ZM, Leonhardt N, Torres MA, Dangl JL, Bloom RE, BoddeS, Jones JD, Schroeder JI. 2003. NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis. EMBO Journal 22, 2623–2633.[CrossRef][Web of Science][Medline]
Laloi C, Apel K, Danon A. 2004. Reactive oxygen signalling: the latest news. Current Opinion in Plant Biology 7, 323–328.[CrossRef][Web of Science][Medline]
Mader M, Amberg-Fisher V. 1982. Role of peroxidase in lignification of tobacco cells. I. Oxidation of nicotinamide adenine dinucleotide and formation of hydrogen peroxide by cell wall peroxidases in Nicotiana tabacum. Plant Physiology 70, 1128–1131.
Mathieu Y, Rouet-Mayer MA, Barbier-Brygoo H, Laurière C. 2002. Activation by fatty acids of the production of active oxygen species by tobacco cells. Plant Physiology and Biochemistry 40, 313–324.[CrossRef]
McLusky S, Bennett M, Beale M, Lewis M, Gaskin P, Mansfield J. 1999. Cell wall alterations and localized accumulation of feruloyl-3'-methoxytyramine in onion epidermis at sites of attempted penetration by Botrytis allii are associated with actin polarisation, peroxidase activity and suppression of flavonoid biosynthesis. The Plant Journal 17, 523–534.[CrossRef]
Mittler R. 2002. Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science 7, 405–410.[CrossRef][Web of Science][Medline]
Ostergaard L, Abelskov AK, Mattsson O, Welinder KG. 1996. Structure and organ specificity of an anionic peroxidase from Arabidopsis thaliana cell suspension culture. FEBS Letters 398, 243–247.[CrossRef][Web of Science][Medline]
Sagi M, Fluhr R. 2001. Superoxide production by plant homologues of the gp91(phox) NADPH oxidase: modulation of activity by calcium and by tobacco mosaic virus infection. Plant Physiology 126, 1281–1290.
Schmidt ML, Trojanowski JQ. 1986. Enzymatic detection of native and derivatized horseradish peroxidase in sodium dodecyl sulfate polyacrylamide gels. Analytical Biochemistry 155, 371–375.[CrossRef][Web of Science][Medline]
Torres MA, Dangl JL, Jones JD. 2002. Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proceedings of the National Academy of Sciences, USA 99, 517–522.
Van Gestelen P, Asard H, Caubergs RJ. 1997. Solubilization and separation of a plant plasma membrane NADPH-O2-synthase from other NAD(P)H oxidoreductases. Plant Physiology 115, 543–550.[Abstract]
Welinder KG, Justesen AF, Kjaersgard IV, Jensen RB, Rasmussen SK, Jespersen HM, Duroux L. 2002. Structural diversity and transcription of class III peroxidases from Arabidopsis thaliana. European Journal of Biochemistry 269, 6063–6081.[Web of Science][Medline]
Yahraus T, Chandra S, Legendre L, Low PS. 1995. Evidence for mechanically induced oxidative burst. Plant Physiology 109, 1259–1266.[Abstract]
Yoshioka H, Numata N, Nakajima K, Katou S, Kawakita K, Rowland O, Jones JD, Doke N. 2003. Nicotiana benthamiana gp91phox homologs NbrbohA and NbrbohB participate in H2O2 accumulation and resistance to Phytophthora infestans. The Plant Cell 15, 706–718.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C. Cosio and C. Dunand Specific functions of individual class III peroxidase genes J. Exp. Bot., February 1, 2009; 60(2): 391 - 408. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Bustos, R. Lascano, A. L. Villasuso, E. Machado, M. E. Senn, A. Cordoba, and E. Taleisnik Reductions in Maize Root-tip Elongation by Salt and Osmotic Stress do not Correlate with Apoplastic O2*- Levels Ann. Bot., October 1, 2008; 102(4): 551 - 559. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Vellosillo, M. Martinez, M. A. Lopez, J. Vicente, T. Cascon, L. Dolan, M. Hamberg, and C. Castresana Oxylipins Produced by the 9-Lipoxygenase Pathway in Arabidopsis Regulate Lateral Root Development and Defense Responses through a Specific Signaling Cascade PLANT CELL, March 1, 2007; 19(3): 831 - 846. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||