RESEARCH PAPER |
Flavin-containing polyamine oxidase is a hydrogen peroxide source in the oxidative response to the protein phosphatase inhibitor cantharidin in Zea mays L.
1Dipartimento di Biologia, Università degli Studi ‘Roma Tre’, Viale Guglielmo Marconi 446, 00146 Rome, Italy
2Istituto di Cristallografia-Consiglio Nazionale delle Ricerche, Monterotondo, 00016 Monterotondo Stazione, Rome, Italy
3Dipartimento Farmaco Chimico Tecnologico, Università degli Studi di Siena, Via A. Moro, 53100 Siena, Italy
*To whom correspondence should be addressed. E-mail: angelini{at}bio.uniroma3.it
Received 4 November 2005; Accepted 13 March 2006
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In this study, the specific contribution of polyamine oxidase (PAO), a hydrogen peroxide (H2O2)-producing enzyme, to the oxidative burst induced in maize mesocotyl by the phosphatase inhibitor cantharidin was examined. For this purpose, a pharmacological approach was applied using, either in vitro or in vivo, two strong inhibitors of maize PAO (MPAO), N-prenylagmatine (G3) and its structural analogue Ro5, as well as diphenyleneiodonium (DPI), an inhibitor of the phagocyte NAD(P)H oxidase. DPI was shown to be a good MPAO inhibitor in vitro. G3, Ro5, and DPI were very effective in inhibiting in vivo the extracellular accumulation of H2O2 that is released by mesocotyl segments upon spermidine supply. G3 and Ro5 did not show any inhibition in vitro of either horseradish peroxidase or barley oxalate oxidase. Moreover, G3 and Ro5 did not inhibit the extracellular accumulation of superoxide radical that is released in vivo upon NADH supply. G3, Ro5, and DPI strongly affected H2O2 production induced in maize mesocotyl by cantharidin. Histochemical localization of H2O2 in cantharidin-treated mesocotyl cross-sections revealed an increase of H2O2-specific staining in the epidermal and subepidermal tissues. The effect was also inhibited by G3 and DPI. Moreover, an increase in MPAO activity was observed in the same tissues upon cantharidin treatment. All these data suggest that G3 and Ro5 behave as powerful and selective inhibitors of MPAO activity either in vitro or in vivo and that MPAO activity contributes to a major part of the cantharidin-induced H2O2 synthesis in the apoplastic milieu of maize mesocotyl.
Key words: Cantharidin, cell wall, polyamines, polyamine oxidase, reactive oxygen species
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Reactive oxygen species (ROS) play basic roles in the regulation of biological processes and behave as signalling molecules in mediating responses to various stimuli (Overmyer et al., 2003; Laloi et al., 2004). In plants, hydrogen peroxide (H2O2) has a key role in the modulation of extension growth by decreasing cell wall extensibility through peroxidase-mediated oxidative cross-linking of cell wall glycoproteins and polysaccharides (Fry, 1986; Hohl et al., 1995; Schopfer, 1996). On the other hand, H2O2 synthesis in the secretory pathway could promote the formation of large hemicellulose coagula that will result in a wall-loosening effect (Fry et al., 2000). Moreover, a role in promoting wall loosening has recently been demonstrated for hydroxyl radicals (·OH) that can be physiologically produced in the cell wall by peroxidase (Liszkay et al., 2003, 2004) or an ascorbate/Cu+-mediated reaction (Fry, 1998). H2O2 and the superoxide radical (
) have both been involved in defence responses against pathogens (Levine et al., 1994; Delledonne et al., 1998, 2001), with H2O2 acting either as a direct toxic molecule or as a cell wall strengthening agent (Rea et al., 2002). Furthermore, H2O2 behaves as a second messenger in the activation of defence genes (Orozco-Cárdenas et al., 2001) and interacts synergistically with nitric oxide (NO) in inducing programmed cell death (PCD) during the hypersensitive response (Delledonne et al., 1998). H2O2 has also been implicated in the auxin-dependent gravitropic stimulus (Joo et al., 2001) and abscisic acid (ABA)-induced stomatal closure (Pei et al., 2000). H2O2 production is induced by a number of elicitors in plant cells, primarily cell wall-derived oligosaccharides, and pathogen avirulence proteins, which are recognized by specific receptors, in many cases represented by leucine-rich repeat (LRR) kinases (Dievart and Clark, 2004). In this regard, it is worth recalling that in soybean suspension cell cultures, different elicitors share a common mitogen-activated protein kinase (MAPK) intermediate in the signal transduction pathway leading to the oxidative burst (Taylor et al., 2001). The activities of specific phosphatases and kinases are also needed in the ABA signalling cascade responsible for stomatal closure (Murata et al., 2001; Mustilli et al., 2002). The activation of a MAPK cascade and transcription factors acting downstream of the LRR receptor has recently been proposed as a conserved mechanism involved in resistance responses to bacterial and fungal pathogens (Asai et al., 2002). The role of the protein phosphorylation status in the regulation of the burst activity has also been demonstrated by pharmacological studies, where the kinase inhibitors staurosporine and K-252a have been shown to block progression of the oxidative burst (Chandra and Low, 1995). Accordingly, protein phosphatase inhibitors such as calyculin A, cantharidin, and okadaic acid can stimulate H2O2 synthesis in the absence of elicitors (Chandra and Low, 1995).
The ROS machinery in plants is made up of specific enzymatic sources, whose activity needs to be strictly regulated by the cell. Indeed. the physiological effects as well as the chemical identity, intensity, duration, and cellular targets of the generated secondary signals depend on the exact spatial and temporal pattern of ROS production. The main putative ROS-delivering systems in the cell wall are represented by membrane-bound NAD(P)H oxidases and apoplastic oxalate oxidases, peroxidases, and amine oxidases. In recent years, a great deal of attention has been devoted to NAD(P)H oxidases, the plant homologues of the catalytic subunit of the activated mammalian phagocytes and neutrophils (gp91phox). These enzymes, whose encoding genes have been isolated from rice (Groom et al., 1996), tobacco (Plas et al., 2002), and Arabidopsis thaliana (Torres et al., 1998), have been shown by genetic and biochemical approaches to be a key ROS-delivering system either in response to pathogen infection (Torres et al., 2002; Plas et al., 2002; Yoshioka et al., 2003) or in physiological processes such as cell growth regulation (Foreman et al., 2003) and stomatal closure (Pei et al., 2000; Kwak et al., 2003). Nevertheless, the role played by the alternative ROS-generating enzyme systems deserves attention, as a growing body of data strongly suggests their involvement in developmental and pathological events. Indeed, exocellular peroxidases have been implicated either in the oxidative stress response to pathogen infections in several species (reviewed in Bolwell et al., 2002), or in the modulation of cell wall extensibility properties in maize (Schopfer, 1996; Liszkay et al., 2004). Several lines of evidence indicate oxalate oxidases as agents of plant defence in cereals as well as in transgenic dicotyledons in which an oxalate oxidase-encoding gene from cereals has been inserted (Lane, 2002). Moreover, amine oxidases have been suggested to be involved in defence responses to wounding and pathogen invasion in chickpea (Rea et al., 2002) and in cell wall stiffening events in maize (Cona et al., 2003). However, the specific contribution of each source to the oxidative burst in different tissues or plants and/or in response to different stimuli is still a matter of debate.
Maize PAO (MPAO) is a secretory flavoprotein of 53 kDa (Tavladoraki et al., 1998; 
ebela et al., 2001), which catalyses the oxidative deamination of secondary amino groups of spermidine (spd) and spermine, producing the corresponding aldehyde, H2O2, and 1,3-diaminopropane. Light and electron microscopic analysis demonstrated its abundance in the cell walls of several maize tissues, especially the xylem, endodermis, and epidermis (Cona et al., 2005). In this context, MPAO has been proposed to be involved in lignification as well as in cell wall stiffening events occurring in the outer tissues during the light-induced inhibition of growth (Cona et al., 2003).
The resolution of the crystal structure of MPAO (Binda et al., 1999) allowed the identification of powerful inhibitors of MPAO enzyme activity. Indeed, N-prenylagmatine (G3), a natural compound initially isolated from the Venezuelan plant Verbesina caracasana Fries (Delle Monache et al., 1999), showed high binding affinity for MPAO with an inhibitory activity value (Ki) of 1.5x10–8 M (Federico et al., 2001). Therefore, considering the chemical structure of G3, a new class of MPAO inhibitors has been designed and synthesized, and their inhibition properties have been analysed (Corelli et al., 2002; Cona et al., 2004). Owing to their high affinity for MPAO, this family of inhibitors potentially represents a very powerful tool in the diagnostic determination of MPAO's contribution to the oxidative burst and/or physiological H2O2 production. In order to verify the binding specificity of these inhibitors, in the present work the effect of G3 and Ro5 (a G3 analogue) on the other putative ROS-generating enzyme systems was analysed. Moreover, as several pharmacological studies indicating NAD(P)H oxidase involvement in a given physiological event have been based on the diphenyleneiodonium (DPI)-mediated inhibition of ROS production, the effect of DPI on MPAO activity was tested. Indeed, considering that iodonium compounds are known to inhibit a variety of flavoproteins as well as to interact with haemoproteins (Riganti et al., 2004), the use of DPI as an unequivocal diagnostic tool in discriminating NAD(P)H oxidase involvement in biological processes often reported in the literature deserves a critical revision. Besides this, the main scope of this study was the understanding of the specific contribution of MPAO to the cantharidin-induced oxidative burst, with the aim of either establishing the reliability of our inhibitors in discriminating PAO-mediated H2O2 production in an in vivo multicomponent ROS production system, or of revealing a possible direct or indirect role of phosphatase activity in modulation of PAO activity. For this purpose, the effect of G3, Ro5, and DPI on this pharmacologically induced production of H2O2 was explored through biochemical and histochemical approaches.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Chemicals
N-prenylagmatine [G3; N-(4-aminobutyl)-N'-(3-methyl-2-butenyl)guanidine] and its analogue Ro5 [N-(6-aminohexyl)-N'-(3-methyl-2-butenyl)guanidine] were prepared as previously described (Corelli et al., 1996, 2002). Na, 4-methoxy-5-[2-(2-methoxy-4-nitro-5-sulpho-phenyl)-5-(phenylcarbamoyl)-1,3,4-triaza-2-azoniacyclopenta-1,4-dien-3-yl]-2-nitro-benzenesulphonate (XTT) was from Polysciences (Germany). DPI, barley oxalate oxidase, horseradish peroxidase, superoxide dismutase (SOD), 3,3'-diaminobenzidine (DAB), 4-aminoantipyrine (AAP), 3,5-dichloro-2-hydroxybenzenesulphonic acid (DCHBS), sodium azide (NaN3), and spermidine (spd) were from Sigma-Aldrich (Italy). Cantharidin was from Alexis (Switzerland) and Etravon was from Novartis (Italy).
Enzyme activity and inhibition assays
The reported data are the average of the values obtained in three different experiments, each with two replicates. The standard error for each point was <8%. All data were obtained at 25 °C.
MPAO activity and inhibition assays:
PAO was purified from maize shoots as previously described (Federico et al., 1989) and then used for inhibition assays. For the determination of Ki values, MPAO activity was measured spectrophotometrically by following the formation of a pink adduct (
515 nm= 2.6x104 M–1 cm–1) as a result of the H2O2-dependent oxidation of AAP catalysed by horseradish peroxidase and the subsequent condensation of the oxidized AAP with DCHBS (Artiss and Entwistle, 1981). Inhibition assays were carried out in 0.2 M sodium phosphate buffer pH 6.5 or 5.5, containing 60 µg horseradish peroxidase, 0.1 mM AAP, and 1 mM DCHBS with spd as a substrate, in the presence or absence of G3, Ro5, and DPI as previously described (Cona et al., 2004). Ki values (where Ki is the apparent dissociation equilibrium constant for the formation of the reversible enzyme–inhibitor complex) were determined according to the Dixon graphical method (Dixon, 1953). Pre-incubation of the enzyme with the inhibitors did not affect the results obtained. G3, Ro5, or DPI did not inhibit the activity of peroxidase in the standard assay reaction (i.e. the oxidation of a phenolic substrate by H2O2) in agreement with what was previously reported for DPI by Frahry and Schopfer (1998). Enzyme activities were expressed in International Units (1 U is the amount of enzyme that catalyses the oxidation of 1 µmol substrate per min).
Peroxidase activity and inhibition assays:
The NADH-oxidizing activity and the NADH-dependent -producing activity of horseradish peroxidase were measured in the same reaction mixture. Reactions were carried out in 0.2 M sodium phosphate buffer pH 4.5, containing 2 µg ml–1 horseradish peroxidase and 0.2 mM NADH, in the presence or absence of 100 µM G3, Ro5, or DPI. NADH oxidation was determined by following the decrease in absorbance at 340 nm (Frahry and Schopfer, 1998). The reducing activity of peroxidase was measured by determinating the H2O2 derived from the dismutation. Five minutes after NADH addition, aliquots of 500 µl were removed from the reaction mixture. After eliminating NADH with 0.1 M HCl (Frahry and Schopfer, 1998), H2O2 was assayed in the samples as described by Bellincampi et al. (2000), by the method based on the H2O2-mediated oxidation of Fe2+ and the subsequent reaction of Fe3+ with xylenol orange (Jiang et al., 1990; Wolff, 1994). The calibration curve was linear in the range of 0.5–3 µM H2O2, and the apparent extinction coefficient obtained in the standard assay reaction was 1.2x104 M–1 cm–1. Preincubation of the enzyme with the inhibitors did not affect the results obtained.
Oxalate oxidase activity and inhibition assays:
Oxalate oxidase activity was measured spectrophotometrically by following the formation of a pink adduct (515 nm= 2.6x104 M–1 cm–1) as a result of the H2O2-dependent oxidation of AAP and subsequent condensation with DCHBS catalysed by horseradish peroxidase (Artiss and Entwistle, 1981). The reaction was performed in 50 mM sodium succinate buffer, pH 3.8 containing 3 mM EDTA, in the presence or absence of 100 µM G3 or Ro5, using 0.01 U of oxalate oxidase and 2 mM oxalic acid (Requena and Bornemann, 1999). Preincubation of the enzyme with the inhibitors did not affect the results obtained.
Plant material and growth conditions
Maize seeds (Zea mays L. cv. Corona; from Monsanto Agricoltura, Italy) were soaked for 12 h in running water and germinated on paper, at 22 °C in a growth chamber in the dark for 5 d. Segments 1 cm long were dissected from the non-elongating zone of the mesocotyl after eliminating the 1 cm long subnodal segment, washed for 2 h in distilled water and then utilized for oxidative burst studies, as described below. Experiments were performed in the dark. Etravon (0.1% v/v) was added to all solutions as wetting agent.
In vivo determination of H2O2 released by maize mesocotyl segments
Procedure 1:
After washing in distilled water, segments were incubated for 30 min (10 segments in 10 ml of buffer; average fresh weight 0.25 g) in buffer A alone [10 mM sodium phosphate buffer, pH 5.5, plus Etravon 0.1% (v/v), 0.3 mg ml–1 horseradish peroxidase, 0.2 mM AAP, and 2 mM DCHBS] or containing 100 µM G3, Ro5, or DPI. Afterwards, spd or cantharidin at the indicated concentrations was added to the incubation medium, either in the presence or in the absence of inhibitors. H2O2 accumulation was monitored up to 5 h, following the formation of a pink adduct (515 nm= 2.6x104 M–1 cm–1) as a result of the H2O2-dependent oxidation of AAP catalysed by horseradish peroxidase and subsequent condensation of the oxidized AAP with DCHBS (Artiss and Entwistle, 1981), as described above. Data reported are the mean of five independent experiments, each performed with two replicates. The 0 time point represents the value obtained immediately after the addition of spd or cantharidin to the incubation medium. Control represents estimation of H2O2 accumulation by mesocotyl segments in the absence of the substrate spd or in the absence of the elicitor cantharidin. In our experimental conditions, no formation of background colour occurred. The percentage increase in H2O2 accumulation after spd or cantharidin addition, as well as the percentage inhibition of this increment due to inhibitor treatments, were calculated comparing the values of the treated samples with respect to the control at the same time, after subtracting from both values the value of the time 0 control.
Procedure 2:
Washed segments were incubated for 30 min in buffer B alone [10 segments in 10 ml of 10 mM sodium phosphate buffer, pH 5.5, plus Etravon 0.1% (v/v)] or containing 100 µM G3, Ro5, or DPI. Afterwards, 100 µM spd was added to the incubation medium, either in the presence or in the absence of inhibitors. H2O2 accumulation was measured at time 0 and after 1 h. In particular, aliquots of 500 µl were removed from the reaction mixture at the indicated time and H2O2 was assayed in the samples as described by Bellincampi et al. (2000), by the method based on the H2O2-mediated oxidation of Fe2+ and the subsequent reaction of Fe3+ with xylenol orange (Jiang et al., 1990; Wolff, 1994). Data reported are the mean of five independent experiments, each performed with two replicates. The 0 time point represents the value obtained immediately after the addition of spd to the incubation medium. Control represents estimation of H2O2 accumulation by mesocotyl segments in the absence of the substrate spd. In our experimental conditions, no formation of background colour occurred. The percentage increase in H2O2 accumulation following spd supply, as well as the percentage inhibition of this increment due to inhibitor treatments, were calculated comparing the values of the treated samples with respect to the control at the same time, after subtracting from both values the value of the time 0 control.
In vivo determination of O2–· released from maize mesocotyl segments
After washing in distilled water, segments were incubated for 30 min (10 segments in 3 ml of buffer; average fresh weight 0.25 g) in buffer C alone [10 mM sodium citrate buffer, pH 6 plus Etravon 0.1% (v/v) and 500 µM XTT] or containing 1 mM NaN3, 100 µM G3, Ro5, or DPI, or 50 µg ml–1 SOD. Afterwards, 200 µM NADH or 100 µM cantharidin were added to the incubation medium, either in the presence or in the absence of inhibitors. accumulation was measured using the method based on the reduction of XTT (Sutherland and Learmonth, 1997) as described by Frahry and Schopfer (2001). The absorbance at 470 nm was monitored up to 15 min and transformed into molar concentration using an extinction coefficient of 2.16x104 M–1 cm–1 (Sutherland and Learmonth, 1997). Data reported are the mean of five independent experiments, each performed with two replicates. The 0 time point represents the value obtained immediately after the addition of NADH or cantharidin to the incubation medium. Control represents estimation of accumulation by mesocotyl segments in the absence of NADH or cantharidin. In our experimental conditions, no formation of background colour occurred. The percentage increase in the level after NADH or cantharidin addition, as well as the percentage inhibition of this increment due to inhibitor treatments, were calculated comparing the values of the treated samples with respect to the control at the same time, after subtracting from both values the value of the time 0 control.
Histochemical visualization of H2O2:
Hand-cut cross-sections (
150 µm thick) were obtained from the non-elongating zone of the mesocotyl of etiolated maize seedlings, after eliminating the 1 cm long subnodal segment. After washing for 1.5 h in distilled water, some sections were preincubated in 10 mM sodium citrate buffer, pH 6 containing 100 µM DPI or G3, for 1.5 h. Other sections were transferred in citrate buffer without inhibitors. Afterwards, sections were incubated with 100 µM cantharidin either in the presence or in the absence of inhibitors for 3 h. H2O2 production was visualized by adding to the incubation medium 2.5 mM DAB (Angelini and Federico, 1989) as the chromogen. Reactions were blocked after 15 min, by washing sections in distilled water. At the end of the treatment, all the sections were mounted on glass slides and observed in an Axioplan 2 Zeiss microscope equipped with a video camera (Delta Sistemi, Rome, Italy). Digitized images were acquired by IAS 2001 software (Delta Sistemi). Experiments were performed independently five times, and a minimum of 10 sections for each segment were observed, yielding reproducible results. The reported micrographs are representative of single experiments.
Determination of MPAO activity in crude extract
Outer (cortical plus epidermal) tissues were obtained by drawing out the stele from segments previously incubated in buffer A containing 100 µM cantharidin for 0, 1, 3, and 5 h. Plant material was ground at 4 °C in 0.2 M sodium phosphate buffer, pH 6.5 [tissue to buffer ratio 1:3 (w/v)]. The suspension was used for total MPAO activity determination. Alternatively, homogenates were centrifuged at 12 000 g for 20 min at 4 °C. Supernatants constituted the extractable MPAO fraction. Pellets were resuspended in the appropriate volume of 0.2 M sodium phosphate buffer, pH 6.5 containing 0.01% Triton X-100 and then filtered onto Miracloth. This step was repeated three times. The washed pellets were resuspended in 0.2 M sodium phosphate buffer, pH 6.5 (3 ml g–1 fresh weight) and the suspension utilized for determination of tightly wall-bound MPAO activity. Total, extractable, and tightly wall-bound MPAO fractions were used for the polarographic measurements of enzyme activity, by measuring oxygen consumption in an oxygraph (Hansatech, Norfolk, UK) equipped with a Clarke electrode, at 30 °C in 0.2 M sodium phosphate buffer, pH 6.5, as described by Augeri et al. (1990). Outer tissues from 10 segments were used for each homogenate. Data reported are the mean of five independent experiments, each performed with two replicates.
Western immunoblotting
Protein content was evaluated by the method of Bradford (1976). Western blot analyses were performed after protein deglycosylation (Woodward et al., 1985), in order to eliminate cross-reactivity due to glycan moieties, using a rabbit polyclonal anti-MPAO antiserum (Federico et al., 1988). Experiments were repeated at least three times, yielding reproducible results.
Statistics
All statistical tests were performed using analysis of variance (ANOVA). The statistical significance of differences was evaluated by P-level. P-values have been calculated comparing ROS levels in stimulated samples with respect to control at the same time, or alternatively comparing ROS levels in inhibited samples with respect to samples under stimulation at the same time.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In vitro and in vivo inhibition of MPAO enzyme activity
In order to investigate the MPAO-specific contribution to H2O2 production in the apoplastic milieu, a pharmacological approach was exploited using two potent inhibitors of MPAO enzyme activity, G3 and Ro5, and the well-characterized inhibitor of the mammalian phagocyte NADPH oxidase, DPI (Ki=1–0.5x10–5 M; O'Donnell et al., 1993). For this purpose, a comprehensive analysis of the in vitro and in vivo inhibitory effects of these three compounds on MPAO enzyme activity was performed. The in vitro inhibition by DPI of MPAO enzyme activity was measured at pH 6.5, the optimum pH value for the enzyme activity, and pH 5.5, the typical pH value occurring in the apoplast, the compartment where MPAO is localized in abundance. DPI competitively inhibited spd oxidation catalysed by purified MPAO at both pH values, with a Ki value similar to that reported for the inhibition of the phagocyte NAD(P)H oxidase activity (O'Donnell et al., 1993). Furthermore, G3 and Ro5 exhibited very high affinity for MPAO at both pH values, with Ki values at pH 6.5 identical to those previously reported by Federico et al. (2001). Results are summarized in Table 1.
|
To determine the in vivo inhibition effectiveness of G3, Ro5, and DPI on MPAO activity, the effect of these compounds on the production of H2O2 that is released in the external medium by subapical maize mesocotyl segments upon supply of spd (Figs 1, 2), the preferred substrate of MPAO (Km=1.0x10–5 M; Cervelli et al., 2001), was analysed. In this assay, the H2O2 colorimetrically revealed in the incubation medium derives from the MPAO activity occurring in the mesocotyl apoplast. In particular, H2O2 determination was achieved by peroxidase-mediated oxidation and subsequent condensation of AAP with DCHBS to a pink-coloured stable compound. In this assay, peroxidase works as a high affinity trap which reduces H2O2 released by mesocotyl segments. Organ segments were washed extensively before adding spd, in order to decrease tissue responses to cutting injury. The addition of 5 µM spd resulted in a time-dependent increase in the accumulation of H2O2 compared with the control. The effect was detectable soon after the addition of the substrate (time 1; P <0.001) and persisted up to the fifth hour (P <0.001), the last time point checked after spd addition. As shown in Fig. 1, the addition of 100 µM G3 (P <0.001 at the fifth hour), 100 µM Ro5 (P <0.001 at the fifth hour), or 100 µM DPI (P <0.001 at the fifth hour) was very effective in inhibiting in vivo H2O2 production due to 5 µM spd oxidation over 5 h. Interestingly, after 1 h incubation in 100 µM spd, that results in a 2.5-fold increase of H2O2 accumulation in the incubation medium, DPI exhibited a lower inhibitory effect on H2O2 production (15% inhibition; P <0.05) compared with G3 and Ro5 (90% inhibition; P <0.001), reflecting the difference in their respective Ki values for MPAO (Fig. 2A). Furthermore, all three compounds also caused a lower accumulation of H2O2 still occurring in untreated segments as a consequence of excision wounding, even after thorough washing (Fig. 1). As expected, when H2O2 production was monitored using xylenol orange (Fig. 2B; see Materials and methods), although similar percentages of increased H2O2 levels under cantharidin stimulation and inhibition with DPI, G3, or Ro5 were revealed, much lower H2O2 accumulation levels were detected in the incubation medium in all the samples. This phenomenon is probably due to the fast degradation of H2O2 by scavenger enzymatic systems which results in a lower accumulation level detectable at a fixed time in the external medium as compared with the peroxidase-mediated determination of H2O2 described above.
|
0.05, 0.01, and 0.001, respectively.
|
In vivo and in vitro specificity of G3, Ro5, and DPI
To verify the reliability of G3 and Ro5 as MPAO selective inhibitors in the study of the MPAO-specific contribution to H2O2 synthesis, the inhibitory activity of these compounds on the other putative ROS-generating enzyme systems in the cell wall was analysed. The effect of G3 and Ro5 on the extracellular accumulation of
that is released in vivo upon NADH supply was tested. was revealed by the accumulation of water-soluble, coloured XTT formazan in the incubation medium, according to Frahry and Schopfer (2001). The effect of DPI was also tested. Washed mesocotyl segments released with a 6-fold concentration increment in 15 min, upon NADH supply (Fig. 3), according to the results reported for maize coleoptiles (Frahry and Schopfer, 2001). G3 and Ro5 did not exert any inhibition of accumulation in vivo from maize mesocotyl segments (P >0.05), while DPI inhibited it by 85% (P <0.001; Fig. 3), in agreement with what has already been demonstrated for the DPI-mediated inhibition of production in maize coleoptiles (Frahry and Schopfer, 2001). Furthermore, as expected, almost no formation of XTT formazan occurred in the presence of SOD (P > 0.05). Since it has been demonstrated previously that XTT, at the concentration used in the assay mixture, inhibits the NADH-dependent, peroxidase-mediated production (Frahry and Schopfer, 2001), it can be assumed that in this condition the formation of XTT formazan depends entirely on the NAD(P)H oxidase-mediated production. Accordingly, sodium azide (NaN3; P >0.05), an inhibitor of the peroxidase-mediated production, did not exert any inhibitory effect on the production of XTT formazan (Fig. 3).
|
Moreover, to study the effect of G3 and Ro5 on other ROS-synthesizing systems, their effect in vitro on peroxidase and oxalate oxidase enzyme activity was investigated. Both the MPAO inhibitors, at the same concentration as used in the in vivo assays, did not show any effect in vitro either on the NADH oxidation or the NADH-dependent
production activity of horseradish peroxidase (Table 2), or on the barley oxalate oxidase enzyme activity (Table 2). DPI, tested in order to validate the peroxidase inhibition assay in our conditions, completely inhibited NADH-dependent H2O2 production without inhibiting the NADH-oxidizing activity of this enzyme (not shown), according to what was previously reported by Frahry and Schopfer (1998).
|
Effect of G3, Ro5, and DPI on the oxidative burst induced by cantharidin in maize mesocotyl
From studies on plant cell suspension cultures and whole plants, it has been established that ROS production can be stimulated by protein phosphatase inhibitors such as calyculin A, cantharidin, and okadaic acid (Chandra and Low, 1995; Taylor et al., 2001). In the present study, an inhibitor-based pharmacological approach was applied to the study of H2O2 and
production stimulated by cantharidin in the maize mesocotyl, with the aim of ascertaining MPAO-specific involvement in ROS production. For this purpose, the levels of extracellular ROS released by washed etiolated mesocotyl segments, the organ in which the physiological roles of MPAO have been studied the most (Laurenzi et al., 1999; Cona et al., 2003), were analysed. The analyses of the dose–response curve for the oxidative burst induced by cantharidin revealed that the increase in H2O2 levels is detectable in the assay system after 3 h in the presence of 50 µM cantharidin, while the maximum response is obtained with cantharidin concentrations of 100 µM (Fig. 4; P <0.001 at the fifth hour after cantharidin addition). Cantharidin did not induce any increment in the level of released by mesocotyl segments as detected with the XTT assay described above (data not shown). G3, Ro5, and DPI strongly affected cantharidin-induced H2O2 accumulation (Fig. 5). Indeed, cantharidin-treated segments incubated in the presence of DPI (P <0.001 at the fifth hour) did not show any increase in H2O2 accumulation over 5 h as compared with the control at time 0, while G3 (P <0.001 at the fifth hour) or Ro5 (P <0.001 at the fifth hour) reversed the cantharidin-induced H2O2 level increase as compared with the control at the fifth hour (Fig. 5). These results demonstrate that the inhibitory activity of these compounds affect, even if to a different extent, either the constitutive or the cantharidin-induced accumulation of H2O2, released in the external medium by segments.
|
|
Histochemical visualization of the H2O2 accumulation induced by cantharidin: effect of G3 and DPI
Histochemical assays exploiting DAB (Angelini and Federico, 1989) were used for localizing H2O2 accumulation, in hand-cut cross-sections of maize mesocotyl of control, cantharidin-treated, and cantharidin plus inhibitors-treated sections. A stronger DAB staining was detected in epidermal and subepidermal tissues of cantharidin-treated versus control sections, while in cantharidin/DPI-treated sections the intensity of DAB staining in the outer tissues appeared to be much lower with respect to the control (Fig. 6). H2O2 accumulation was also inhibited in the outer tissues of cantharidin plus G3-treated sections, which showed a DAB staining level similar to that of the control sections. The high levels of MPAO activity in stelar tissues (Laurenzi et al., 1999) caused a strong DAB staining in all the samples without any detectable difference among different treatments.
|
Effect of cantharidin on MPAO activity in the outer tissues of maize mesocotyls
In order to obtain more direct evidence of MPAO involvement in the cantharidin-induced oxidative burst, the effect of cantharidin supply on the MPAO enzyme activity level was analysed. Considering the tissue specificity of the increase in the H2O2 level evidenced by DAB staining, the effect of cantharidin on MPAO expression was investigated in outer tissues from mesocotyl segments previously incubated in the presence or absence of 100 µM cantharidin for 0, 1, 3, and 5 h. To this end, tissues from the samples were homogenized and utilized for total, extractable, and tightly wall-bound MPAO activity measurements. An increase of total MPAO activity was already evident 1 h after the supply of cantharidin, while after 5 h total MPAO activity levels were 2.5-fold higher in cantharidin-treated versus control tissues (Fig. 7A). The observed increase is entirely due to the tightly wall-bound MPAO activity, while any activity increase is detectable in the extractable fraction (Fig. 7A). Western blot analysis of extractable MPAO fractions showed band intensities that essentially reflect the corresponding MPAO activity values (Fig. 7B).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Pharmacological, molecular, and reverse genetic data in Arabidopsis thaliana and other species suggest that an NAD(P)H oxidase, similarly to mammalian phagocyte oxidase, is mainly responsible for the oxidative burst that leads to rapid H2O2 production in the apoplast as a consequence of biotic and abiotic stresses. Recently, this system has also been suggested to be involved in physiological responses, such as extension growth, stomatal opening, root gravitropism, and PCD events, in which ROS play a key role. On the other hand, circumstantial evidence fosters the view that other enzymatic systems, such as peroxidases, oxalate oxidases, and amine oxidases, are involved in ROS generation in this compartment. Studies on signal transduction leading to the oxidative burst have demonstrated that protein phosphatase inhibitors, such as cantharidin, calyculin, and okadaic acid, are able to induce ROS production in cells or tissues independently from exogenous stimuli or stress (Chandra and Low, 1995).
It is reported here that flavin-containing PAO is a significative source of apoplastic H2O2 produced in maize mesocotyl during the cantharidin-induced oxidative burst. Supply of cantharidin to mesocotyl segments triggered an increase of both tightly wall-bound MPAO activity and the level of H2O2 released in the incubation medium. The absence of an increase in the level of extractable MPAO activity could be accounted for by proteins becoming rapidly insoluble as a consequence of the oxidative burst (Bradley et al., 1992). DAB/peroxidase-based histochemical determination of H2O2 revealed that the cantharidin-induced increase in H2O2 level is mainly localized in the mesocotyl epidermal tissues, while the levels of this ROS are similarly high in the stelar tissues of control and cantharidin-treated sections. This suggests that the cantharidin-induced regulation of MPAO is a tissue-specific event and is likely to be linked to H2O2-consuming wall stiffening reactions, and/or ROS-mediated defence mechanisms, occurring in the epidermal tissues.
Useful tools in this study were the MPAO substrate analogues, agmatine-related compounds G3 and Ro5, that were shown to behave as powerful and selective inhibitors of MPAO activity either in vitro or in vivo at pH values typical of the apoplastic milieu. In particular, they were able efficiently to inhibit the production of H2O2 deriving from mesocotyl segments treated with cantharidin. Interestingly, these compounds did not inhibit the NADH-stimulated synthesis in vivo in mesocotyl segments. Moreover, G3 and Ro5 did not inhibit the activity of many other enzymes such as lentil copper amine oxidase, swine kidney copper amine oxidase, inducible nitric oxide synthase (Federico et al., 2001), papaine (Ki >10–2 M; P Ascenzi and A Bocedi, personal communication), barley oxalate oxidase (this work), or horseradish peroxidase (this work). From these results, it is reasonable to hypothesize that G3 and Ro5 constitute unequivocal diagnostic tools for discriminating in vivo the PAO-catalysed H2O2 production from that arising by the other main H2O2-apoplastic sources, and therefore they could be considered very useful drugs for the in vivo inhibition of PAO activity in plant tissues. In contrast, DPI was shown to be a non-specific inhibitor in our study and to be of no use in discriminating between MPAO- and NAD(P)H oxidase-mediated H2O2 production in maize mesocotyl tissues. Indeed, this compound has recently been demonstrated to behave as an unspecific inhibitor of a number of flavin-containing enzymes and haemoproteins (Riganti et al., 2004), including horseradish peroxidase (Frahry and Schopfer, 1998).
Taken together, these data suggest that MPAO activity, at least in maize epidermal tissues, may significantly account for cantharidin-induced H2O2 synthesis in the apoplastic milieu, although a role for peroxidase in producing this compound, either through an ‘oxidase’ cycle or through compound III decay, cannot be excluded. Indeed, XTT used to determine levels has been demonstrated to be a strong inhibitor of peroxidase-catalysed reactions leading to its synthesis (Frahry and Schopfer, 2001). The absence of an increase in production by maize mesocotyl segments upon cantharidin supply indicates that NAD(P)H oxidase could not be involved in this event or that a rapid dismutation of the is operating in this system.
In addressing the physiological meaning of the cantharidin-mediated PAO regulation leading to increased H2O2 production in plant tissues, the following considerations can be made. As the H2O2 which is detected is released in the segment incubation medium, it is reasonable to hypothesize that the system(s) operating in cantharidin-induced H2O2 production is located in the apoplast or in a vesicular system prepared for rapid exocytic secretion. In this context, it has been reported that in barley leaves infected with Blumeria graminis f.sp. hordei, unsuccessful penetration of the pathogen was associated with the formation of 2 µm large cytosolic vesicles accumulating H2O2 (Hückelhoven et al., 1999). A network of MAPKs are involved either in transduction of the oxidative burst signal (Taylor et al., 2001) or in sensing the oxidative stress signal in plants. Indeed, Arabidopsis MPK3 and MPK6 are both induced by H2O2 either directly, through H2O2-mediated oxidation (Jonack et al., 2002), or via MAPKKK ANP1 (Kovtun et al., 2000). Moreover, it is well known that phosphatases can be inhibited by H2O2 by means of oxidation of key cysteinyl residues (van Monfort et al., 2003). PTP1, an Arabidopsis phosphatase that can inactivate the Arabidopsis MPK6 (Gupta and Luan, 2003), is indeed inactivated by H2O2. Interestingly, the phosphatase inhibitor cantharidin (as well as calyculin A and okadaic acid) is autologously able to stimulate H2O2 synthesis in soybean cells (Chandra and Low, 1995). Thus the cantharidin-mediated regulation of PAO activity could represent a key point in a metabolic loop leading to the oxidative burst that could have important roles in ROS-mediated events. Indeed, PAO has been reported to have a role in the production of H2O2 in defence mechanisms towards fungal and viral pathogens (Cowley and Walters, 2002; Yoda et al., 2003; Takahashi et al., 2004), wall stiffening and lignification (Cona et al., 2003, 2005; Paschalidis and Roubelakis-Angelakis, 2005a), and PCD (Cona et al., 2005).
The cantharidin-induced PAO regulation could be the result of a complex array of signal transduction pathways that could be linked to both development and defence events. Indeed, the cantharidin-induced PAO activity increase might be directly regulated by the phosphorylation status through specific kinase and cognate phosphatase. In this connection, a prediction analysis performed using the NetPhos 2.0 server (http://www.cbs.dtu.dk/services/NetPhos/; Blom et al., 1999) revealed that indeed MPAO shows many potential phosphorylation sites. Analysis of the three-dimensional structure of MPAO highlighted that several of these potential phosphorylation sites are located on the external surface of the protein, therefore being easily accessible to eventual kinase or phosphatase activities (F Polticelli, personal communication). The cantharidin-induced PAO activity increase could also be explained by cantharidin-mediated regulation of PAO gene expression. The above-mentioned oxidative loop could also operate at this level as it is known that H2O2 is active in regulating MAPK/phosphatase involved in modulating the activity of specific transcription factors that induce the expression of an array of genes involved in the oxidative stress response (Desikan et al., 2001).
It is worth recalling here that polyamine catabolism and anabolism may also be involved in phenomena other than the oxidative response. The reported PAO regulation could be part of complex signal transduction pathways also linked to cell division/expansion and cell cycle progression (Paschalidis and Roubelakis-Angelakis, 2005a, b).
Finally, the undetectable amount of freely available polyamines in the extracellular spaces under physiological conditions (Yoda et al., 2003; Rea et al., 2004) implicate PAO substrate delivery in the cell wall as a part of a signal transduction pathway influenced by cantharidin.
|
Acknowledgements |
|---|
This work was supported by the Italian Ministry for University and Research (COFIN-PRIN 2002 and 2005, p.c. 2002073257_002 and p.c. 2005052297_002 to RA, and COFIN-PRIN 2004, p.c. 2004059221_004 to MB and FC). We wish to thank Paraskevi Tavladoraki and Fabio Polticelli (Biology Department, University ‘Roma Tre’, Italy) for critical reading of the manuscript and analysis of the three-dimensional structure of MPAO. We are grateful to Paolo Ascenzi and Alessio Bocedi (Biology Department, University ‘Roma Tre’, Italy) for papaine inhibition studies in the presence of G3.
|
Abbreviations |
|---|
AAP, 4-aminoantipyrine; ABA, abscisic acid; DAB, 3,3'-diaminobenzidine; DCHBS, 3,5-dichloro-2-hydroxybenzenesulphonic acid; DPI, diphenyleneiodonium; G3, N-prenylagmatine [N-(4-aminobutyl)-N'-(3-methyl-2-butenyl)guanidine]; LRR, leucine-rich repeat; MAPK, mitogen-activated protein kinase; MPAO, maize polyamine oxidase; NaN3, sodium azide; PAO, polyamine oxidase; PCD, programmed cell death; Ro5, a G3 analogue [N-(6-aminohexyl)-N'-(3-methyl-2-butenyl)guanidine]; ROS, reactive oxygen species; SOD, superoxide dismutase; spd, spermidine; XTT, Na,4-methoxy-5-[2-(2-methoxy-4-nitro-5-sulpho-phenyl)-5-(phenylcarbamoyl)-1,3,4-triaza-2-azoniacyclopenta-1,4-dien-3-yl]-2-nitro-benzenesulphonate.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Angelini R and Federico R. (1989) Histochemical evidence of polyamine oxidation and hydrogen peroxide generation in the cell wall. Journal of Plant Physiology 135:212–217.
Artiss JD and Entwistle WM. (1981) The application of a sensitive uricase–peroxidase couple reaction to a centrifugal fast analyser for the determination of uric acid. Clinica Chimica Acta 116:301–309.[CrossRef][Web of Science][Medline]
Asai T, Tena G, Plotnikova J, Willmann MR, Chiu W-L, Gomez-Gomez L, Boller T, Ausubel FM, Sheen J. (2002) MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415:977–983.[CrossRef][Medline]
Augeri M, Angelini R, Federico R. (1990) Sub-cellular localization and tissue distribution of polyamine oxidase in maize (Zea mays L.) seedlings. Journal of Plant Physiology 136:690–695.
Bellincampi D, Dipierro N, Salvi G, Cervone F, De Lorenzo G. (2000) Extracellular H2O2 induced by oligogalacturonides is not involved in the inhibition of the auxin-regulated rolB gene expression in tobacco leaf explants. Plant Physiology 122:1379–1385.
Binda C, Coda A, Angelini R, Federico R, Ascenzi P, Mattevi A. (1999) A 30 Å long U-shaped catalytic tunnel in the crystal structure of polyamine oxidase. Structure 7:265–276.[Medline]
Blom N, Gammeltoft S, Brunak S. (1999) Sequence- and structure-based prediction of eukaryotic protein phosphorylation sites. Journal of Molecular Biology 294:1351–1362.[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.
Bradford MM. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein–dye binding. Analytical Biochemistry 72:248–254.[CrossRef][Web of Science][Medline]
Bradley DJ, Kjellbom P, Lamb CJ. (1992) Elicitor- and wound-induced oxidative cross-linking of a proline-rich plant cell wall protein: a novel, rapid defense response. Cell 70:21–30.[CrossRef][Web of Science][Medline]
Chandra S and Low PS. (1995) Role of phosphorylation in elicitation of the oxidative burst in cultured soybean cells. Proceedings of the National Academy of Sciences, USA 92:4120–4123.
Cervelli M, Cona A, Angelini R, Polticelli F, Federico R, Mariottini P. (2001) A barley polyamine oxidase isoform with distinct structural features and subcellular localization. European Journal of Biochemistry 268:3816–3830.[Web of Science][Medline]
Cona A, Cenci F, Cervelli M, Federico R, Mariottini P, Moreno S, Angelini R. (2003) Polyamine oxidase, a hydrogen peroxide-producing enzyme, is up-regulated by light and down-regulated by auxin in the outer tissues of the maize mesocotyl. Plant Physiology 131:803–813.
Cona A, Manetti F, Leone R, Corelli F, Tavladoraki P, Ponticelli F, Botta M. (2004) Molecular basis for the binding of competitive inhibitors of maize polyamine oxidase. Biochemistry 43:3426–3435.[CrossRef][Medline]
Cona A, Moreno S, Cenci F, Federico R, Angelini R. (2005) Cellular re-distribution of flavin-containing polyamine oxidase in differentiating root and mesocotyl of Zea mays L. seedlings. Planta 221:265–276.[CrossRef][Web of Science][Medline]
Corelli F, Dei D, Delle Monache G, Botta B, De Luca C, Carmignani M, Volpe AR, Botta M. (1996) Synthesis and preliminary pharmacological evaluation of analogues of caracasanamide, a hypotensive natural product. Bioorganic and Medicinal Chemistry Letters 6:653–658.[CrossRef]
Corelli F, Federico R, Cona A, Venturini G, Schenone S, Botta M. (2002) Solution and solid-phase synthesis of aminoalkylguanidines inhibiting polyamine oxidase and nitric oxide synthase. Medicinal Chemistry Research 11:309–321.
Cowley T and Walters DR. (2002) Polyamine metabolism in an incompatible interaction between barley and the powdery mildew fungus, Blumeria graminis f.sp. horde. Journal of Phytopathology 150:1–7.[CrossRef]
Delledonne M, Xia Y, Dixon RA, Lamb C. (1998) Nitric oxide functions as a signal in plant disease resistance. Nature 394:585–588.[CrossRef][Medline]
Delledonne M, Zeier J, Marocco A, Lamb C. (2001) Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive disease resistance response. Proceedings of the National Academy of Sciences, USA 98:13454–13459.
Delle Monache G, Volpe AR, Delle Monache F, et al. (1999) Novel hypotensive agents from Verbesina caracasana. 2. Synthesis and pharmacology of caracasanamide. Bioorganic and Medicinal Chemistry Letters 9:3249–3254.[CrossRef]
Desikan R, A-H-Mackerness S, Hancock JT, Neill SJ. (2001) Regulation of the Arabidopsis transcriptome by oxidative stress. Plant Physiology 127:159–172.
Dievart A and Clark SE. (2004) LRR-containing receptors regulating plant development and defense. Development 131:251–261.
Dixon M. (1953) The determination of enzyme inhibitor constants. Biochemical Journal 55:170–171.[Web of Science][Medline]
Federico R, Alisi C, Cona A, Angelini R. (1988) Purification of polyamine oxidase from maize seedlings by immunoadsorbent column. In Zappia V and Pegg AE (Eds.). Advances in experimental medicine and biology, progress in polyamine research (Plenum Press, New York) pp. 617–623.
Federico R, Alisi C, Forlani F. (1989) Properties of the polyamine oxidase from the cell wall of maize seedlings. Phytochemistry 28:45–46.[CrossRef]
Federico R, Leone L, Botta M, Binda C, Angelini R, Venturini G, Ascenzi P. (2001) Inhibition of pig liver and Zea mays L. polyamine oxidase: a comparative study. Journal of Enzyme Inhibition 13:465–471.
Foreman J, Demidchik V, Bothwell JHF, et al. (2003) Reactive oxygen species produced by NADPH oxidase regulate plant cell growth. Nature 422:442–446.[CrossRef][Medline]
Frahry G and Schopfer P. (1998) Inhibition of O2-reducing activity of horseradish peroxidase by diphenyleneiodonium. Phytochemistry 48:223–227.[CrossRef][Web of Science][Medline]
Frahry G and Schopfer P. (2001) NADH-stimulated, cyanide-resistant superoxide production in maize coleoptiles analyzed with a tetrazolium-based assay. Planta 212:175–183.[CrossRef][Web of Science][Medline]
Fry SC. (1986) Cross-linking of matrix polymers in the growing cell walls of angiosperms. Annual Review of Plant Physiology 37:165–186.[CrossRef][Web of Science]
Fry SC. (1998) Oxidative scission of plant cell wall polysaccharides by ascorbate-induced hydroxyl radicals. Biochemical Journal 332:507–515.[Web of Science][Medline]
Fry SC, Willis SC, Paterson AEJ. (2000) Intraprotoplasmic and wall-localised formation of arabinoxylan-bound diferulates and larger ferulate coupling-products in maize cell-suspension cultures. Planta 211:679–692.[CrossRef][Web of Science][Medline]
Groom QJ, Torres MA, Fordham-Sketon AP, Hammond-Kosack KE, Robinsion NJ, Jones JDG. (1996) rbohA, a rice homologue of the mammalian gp91phox respiratory burst oxidase gene. The Plant Journal 10:515–522.[CrossRef][Web of Science][Medline]
Gupta R and Luan S. (2003) Redox control of protein tyrosine phosphatases and mitogen-activated protein kinases in plants. Plant Physiology 132:1149–1152.
Hohl M, Greiner H, Schopfer P. (1995) The cryptic growth response of maize coleoptiles and its relationship to H2O2-dependent cell-wall stiffening. Physiologia Plantarum 94:491–498.
Huckelhoven R, Fodor J, Preis C, Kogel KH. (1999) Hypersensitive cell death and papilla formation in barley attacked by the powdery mildew fungus are associated with hydrogen peroxide but not with salicylic acid accumulation. Plant Physiology 119:1251–1260.
Jiang Z-Y, Woollard ACS, Wolff SP. (1990) Hydrogen peroxide production during experimental protein glycation. FEBS Letters 268:69–71.[CrossRef][Web of Science][Medline]
Jonak C, Okresz L, Bogre L, Hirt H. (2002) Complexity, cross talk and integration of plant MAP kinase signalling. Current Opinion in Plant Biology 5:415–424.[CrossRef][Web of Science][Medline]
Joo JH, Bae YS, Lee JS. (2001) Role of auxin-induced reactive oxygen species in root gravitropism. Plant Physiology 126:1055–1060.
Kovtun Y, Chiu WL, Tena G, Sheen J. (2000) Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proceedings of the National Academy of Sciences, USA 97:2940–2945.
Kwak JM, Mori IC, Pei ZM, Leonhardt N, Torres MA, Dangl JL, Bloom RE, Bodde S, Jones JD, Schroeder JI. (2003) NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signalling in Arabidopsis. EMBO Journal 22:2623–2633.[CrossRef][Web of Science][Medline]
Laloi C, Apel K, Danon A. (2004) Reactive oxygen signalling: the latest new. Current Opinion in Plant Biology 7:323–328.[CrossRef][Web of Science][Medline]
Lane BG. (2002) Oxalate, germins, and higher-plant pathogens. IUBMB Life 53:67–75.[Web of Science][Medline]
Laurenzi M, Rea G, Federico R, Tavladoraki P, Angelini R. (1999) De-etiolation causes a phytochrome-mediated increase of polyamine oxidase expression in outer tissues of the maize mesocotyl: a role in the photomodulation of growth and cell wall differentiation. Planta 208:146–154.[CrossRef]
Levine A, Tenhaken R, Dixon R, Lamb C. (1994) H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79:583–593.[CrossRef][Web of Science][Medline]
Liszkay A, Kenk B, Schopfer P. (2003) Evidence for the involvement of cell wall peroxidase in the generation of hydroxyl radicals mediating extension growth. Planta 217:658–667.[CrossRef][Web of Science][Medline]
Liszkay A, van der Zalm E, Schopfer P. (2004) Production of reactive oxygen intermediates (, H2O2, and ·OH) by maize roots and their role in wall loosening and elongation growth. Plant Physiology 136:3114–3123.
Murata Y, Pei ZM, Mori IC, Schroeder J. (2001) Abscisic acid activation of plasma membrane Ca2+ channels in guard cells requires cytosolic NAD(P)H and is differentially disrupted upstream and downstream of reactive oxygen species production in abi1-1 and abi2-1 protein phosphatase 2C mutants. The Plant Cell 13:2513–2523.
Mustilli AC, Merlot S, Vavasseur A, Fenzi F, Giraudat J. (2002) Arabidopsis OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen production. Plant Cell 14:3089–3099.
O'Donnell VB, Tew DG, Jones OTG, England PJ. (1993) Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase. Biochemical Journal 290:41–49.[Web of Science][Medline]
Orozco-Cárdenas ML, Narváez-Vásquez J, Ryan CA. (2001) Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate. Plant Cell 13:179–191.
Overmyer K, Broschè M, Kangasjärvi J. (2003) Reactive oxygen species and hormonal control of cell death. Trends in Plant Science 8:335–342.[CrossRef][Web of Science][Medline]
Paschalidis KA and Roubelakis-Angelakis KA. (2005a) Sites and regulation of polyamine catabolism in the tobacco plant. Correlations with cell division/expansion, cell cycle progression, and vascular development. Plant Physiology 138:2174–2184.
Paschalidis KA and Roubelakis-Angelakis KA. (2005b) Spatial and temporal distribution of polyamine levels and polyamine anabolism in different organs/tissues of the tobacco plant. Correlation with age, cell division/expansion, and differentiation. Plant Physiology 138:142–152.
Pei ZM, Murata Y, Benning G, Thomine S, Klusener B, Allen GJ, Grill E, Schroeder JI. (2000) Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells. Nature 406:731–734.[CrossRef][Medline]
Plas FS, Elmayan T, Blein JP. (2002) The plasma membrane oxidase NtrbohD is responsible for AOS production in elicited tobacco cells. The Plant Journal 31:137–147.[CrossRef][Web of Science][Medline]
Rea G, de Pinto MC, Tavazza R, Biondi S, Gobbi V, Ferrante P, De Gara L, Federico R, Angelini R, Tavladoraki P. (2004) Ectopic expression of maize polyamine oxidase and pea copper amine oxidase in the cell wall of tobacco plants. Plant Physiology 134:1414–1426.
Rea G, Metoui O, Infantino A, Federico R, Angelini R. (2002) Copper amine oxidase expression in defense responses to wounding and Ascochyta rabiei invasion. Plant Physiology 128:865–875.
Requena L and Bornemann S. (1999) Barley (Hordeum vulgare) oxalate oxidase is a manganese-containing enzyme. Biochemical Journal 343:185–190.[CrossRef][Web of Science][Medline]
Riganti C, Gazzano E, Polimeni M, Costamagna C, Bosia A, Ghigo D. (2004) Diphenyleneiodonium inhibits the cell redox metabolism and induces oxidative stress. Journal of Biological Chemistry 279:47726–47731.
Schopfer P. (1996) Hydrogen peroxide-mediated cell-wall stiffening in vitro in maize coleoptiles. Planta 199:43–49.
ebela M, Radovà A, Angelini R, Tavladoraki P, Frébort I, P
c P. (2001) FAD-containing polyamine oxidases: a timely challenge for researchers in biochemistry and physiology of plants. Plant Science 160:197–207.[Medline]
Sutherland MW and Learmonth BA. (1997) The tetrazolium dyes MTS and XTT provide new quantitative assays for superoxide and superoxide dismutase. Free Radical Research 27:283–289.[Web of Science][Medline]
Takahashi Y, Uehara Y, Berberich T, Ito A, Saitoh H, Miyazaki A, Terauchi R, Kusano T. (2004) A subset of hypersensitive response marker genes, including HSR203J, is the downstream target of a spermine signal transduction pathway in tobacco. The Plant Journal 40:586–595.[CrossRef][Web of Science][Medline]
Tavladoraki P, Shininà ME, Cecconi F, Di Agostino S, Manera F, Rea G, Federico R, Mariottini P, Angelini R. (1998) Maize polyamine oxidase: primary structure from protein and cDNA sequencing. FEBS Letters 426:62–66.[CrossRef][Web of Science][Medline]
Taylor ATS, Kim J, Low PS. (2001) Involvement of mitogen-activated protein kinase activation in the signal-transduction pathways of the soya bean oxidative burst. Biochemical Journal 355:795–803.[CrossRef][Web of Science][Medline]
Torres MA, Onuchi H, Hamada S, Machida G, Hammond-Kosak KE, Robinsion NJ, Jones JDG. (1998) Six Arabidopsis thaliana homologues of the human respiratory burst oxidase (gp91phox). The Plant Journal 14:365–370.[CrossRef][Web of Science][Medline]
Torres MA, Dangl JL, Jones JDG. (2002) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defence response. Proceedings of the National Academy of Sciences, USA 99:517–522.
van Montfort RL, Congreve M, Tisi D, Carr R, Jhoti H. (2003) Oxidation state of the active-site cysteine in protein tyrosine phosphatase 1B. Nature 423:773–777.[CrossRef][Medline]
Wolff SP. (1994) Ferrous ion oxidation in presence of ferric ion indicator xylenol orange for measurement of hydroperoxides. Methods in Enzymology 233:182–189.
Woodward MP, Young WW, Bloodgood RA. (1985) Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation. Journal of Immunological Methods 78:143–153.[CrossRef][Web of Science][Medline]
Yoda H, Yamaguchi Y, Sano H. (2003) Induction of hypersensitive cell death by hydrogen peroxide produced through polyamine degradation in tobacco plants. Plant Physiology 132:1973–1981.
Yoshioka H, Numata N, Nakajima K, Katou S, Kawakita K, Rowland O, Jones JDG, Doke N. (2003) Nicotiana benthamiana gp91(phox) 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:
|
|
 |
|
  A. A. Rodriguez, S. J. Maiale, A. B. Menendez, and O. A. Ruiz Polyamine oxidase activity contributes to sustain maize leaf elongation under saline stress J. Exp. Bot., November 1, 2009; 60(15): 4249 - 4262. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Marina, S. J. Maiale, F. R. Rossi, M. F. Romero, E. I. Rivas, A. Garriz, O. A. Ruiz, and F. L. Pieckenstain Apoplastic Polyamine Oxidation Plays Different Roles in Local Responses of Tobacco to Infection by the Necrotrophic Fungus Sclerotinia sclerotiorum and the Biotrophic Bacterium Pseudomonas viridiflava Plant Physiology, August 1, 2008; 147(4): 2164 - 2178. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Angelini, A. Tisi, G. Rea, M. M. Chen, M. Botta, R. Federico, and A. Cona Involvement of Polyamine Oxidase in Wound Healing Plant Physiology, January 1, 2008; 146(1): 162 - 177. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Beffagna and I. Lutzu Inhibition of catalase activity as an early response of Arabidopsis thaliana cultured cells to the phytotoxin fusicoccin J. Exp. Bot., December 1, 2007; 58(15-16): 4183 - 4194. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||