JXB Advance Access originally published online on February 13, 2008
Journal of Experimental Botany 2008 59(4):815-825; doi:10.1093/jxb/erm370
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RESEARCH PAPER |
Hydrogen peroxide generated by copper amine oxidase is involved in abscisic acid-induced stomatal closure in Vicia faba
College of Life Sciences, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, PR China
* To whom correspondence should be addressed. E-mail: whzhang{at}njau.edu.cn
Received 13 October 2007; Revised 16 December 2007 Accepted 20 December 2007
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Abstract |
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H2O2 is an essential signal in absicic acid (ABA)-induced stomatal closure. It can be synthesized by several enzymes in plants. In this study, the roles of copper amine oxidase (CuAO) in H2O2 production and stomatal closure were investigated. Exogenous ABA stimulated apoplast CuAO activity, increased H2O2 production and [Ca2+]cyt levels in Vicia faba guard cells, and induced stomatal closure. These processes were impaired by CuAO inhibitor(s). In the metabolized products of CuAO, only H2O2 could induce stomatal closure. By the analysis of enzyme kinetics and polyamine contents in leaves, putrescine was regarded as a substrate of CuAO. Putrescine showed similar effects with ABA on the regulation of H2O2 production, [Ca2+]cyt levels, as well as stomatal closure. The results suggest that CuAO in V. faba guard cells is an essential enzymatic source for H2O2 production in ABA-induced stomatal closure via the degradation of putrescine. Calcium messenger is an important intermediate in this process.
Key words: Abscisic acid, calcium, copper amine oxidase, hydrogen peroxide, putrescine, stomatal closure, Vicia faba
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Introduction |
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The plant hormone abscisic acid (ABA) is a vital signal that modulates a variety of growth and developmental processes and responses to environmental stresses such as drought, high salt, and cold (Grill and Christmann, 2007). When plants are drought-stressed, ABA acts as a key regulator of stomatal apertures to restrict transpiration and reduce water loss. Much progress has been made in the identification of components in the ABA signal network, from perception to nuclear events. They include ABA receptors, heterotrimeric G-proteins, lipid signalling, protein kinases and protein phosphatases, transcription factors, proteins involved in post-translational protein modification, and RNA processing (Li et al., 2000; Hugouvieux et al., 2001; Merlot et al., 2001; Sanchez and Chua, 2001; Wang et al., 2001; Xiong et al., 2001a, b; Finkelstein et al., 2002; Mishra et al., 2006). Three ABA receptors have been identified so far, including the flowering control protein FCA, the Mg-chelatase H subunit (ABAR/CHLH), and the G-protein coupled receptor (GCR2) (Razem et al., 2006; Shen et al., 2006; Liu et al., 2007). ABAR/CHLH and GCR2 are involved in ABA-induced stomatal movement (Shen et al., 2006; Liu et al., 2007), while the latter has not been confirmed for its function in the ABA response according to a more recent report (Gao et al., 2007). On the other hand, G-protein activation upon ABA binding leads to the activation of PLD
1, producing a head group and phosphatidic acid, which anchors ABI1, a negative ABA response regulator, to the plasma membrane (Zhang et al., 2004; Grill and Christmann, 2007). The PA-ABI1 binding impairs ABI1 PP2C activity, thereby transducing ABA signals (Zhang et al., 2004; Mishra et al., 2006). The increased phosphatidic acid may, in turn, regulate GPA1 (heterotrimeric G-protein 1 subunit) through sphingosine kinase that acts upsteam of GPA1 (Coursol et al., 2003), because phosphatidic acid has been implicated in binding sphingosine kinase in animal cells (Delon et al., 2004). The second messengers, such as Ca2+, reactive oxygen species (ROS), and nitric oxide are also important in ABA-induced stomatal closure. ABA-induced H2O2 production and the H2O2-activated Ca2+ channels in the plasma membrane are important mechanisms for ABA-induced stomatal closing (Pei et al., 2000; Sokolovski et al., 2005). Molecular genetic evidence shows that, in Arabidopsis, the plasma membrane-associated NADPH oxidase catalytic subunits AtrbohD and AtrbohF are implicated in the ROS-dependent activation of Ca2+ channels and cytosolic Ca2+ increase (Kwak et al., 2003).
Besides NADPH oxidases, apoplast amine oxidases and oxalate oxidase are enzymatic sources of ROS production (Grant and Loake, 2000; Mittler, 2002; Vranová et al., 2002; Cona et al., 2006). Amine oxidases include copper-containing diamine oxidases (CuAO; EC 1.4.3.6) and FAD-containing polyamine oxidases (PAO; EC 1.5.3.3
[EC]
) (Cona et al., 2006). Plant CuAO generally catalyse the oxidation of the aliphatic diamines putrescine (Put) and cadaverine (Cad) at the primary animo groups (Cona et al., 2006). The products from Put oxidation by CuAO include 
1-pyrroline, H2O2, and ammonia. 1-pyrroline is further catabolized to 
-aminobutyric acid (GABA), which is subsequently transaminated and oxidized to succinic acid (Succ) (Rea et al., 2004; Cona et al., 2006). Whereas, PAO catalyse the oxidation of spermine (Spm) and spermidine (Spd) and their acetylated derivates at the secondary amino groups (Cona et al., 2006). Increasing evidence suggests that CuAO and PAO participate in plant development and defence responses via their reaction products (Rea et al., 2004; Cona et al., 2006). These H2O2-generated enzymes have been proposed to mediate lignification and cross-linking of extension, programmed cell death during both normal growth and response to biotic and abiotic stresses (Pennell and Lamb, 1997; Laurenzi et al., 1999; Rea et al., 2002; Walters, 2003; Yoda et al., 2003; Su et al., 2005; Cona et al., 2006). However, it is still unclear whether H2O2 from amine oxidation participates in ABA-mediated stomatal closure. On the other hand, polyamines have been implicated in inhibiting the inward K+ channels across the plasma membrane and inducing stomatal closure through unknown factors (Liu et al., 2000). Moreover, ABA stimulates CuAO activity and increases the H2O2 level in rice roots (Lin and Kao, 2001). Altogether, these findings prompted us to investigate whether and how amine oxidation is involved in ABA regulation of stomatal closure.
In this paper, the evidence that CuAO in Vicia faba guard cells is involved in ABA-induced stomatal closure via the degradation of putrescine to generate H2O2 is provided. Calcium messenger is an important intermediate in this process.
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Materials and methods |
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Plant material
Vicia faba plants were grown in a plant growth chamber under a 12/12 h, light/dark and 25/20 °C cycles. The light intensity was 0.18 mmol m–2 s–1. 4-week-old seedlings were used for following determination.
Stomatal aperture measurement
Stomatal aperture was measured according to the procedure of Zhang et al. (2004) with minor modifications. Epidermal peels were stripped from V. faba leaves, and floated in CO2-free solution containing 10 mM KCl, 0.2 mM CaCl2, 0.1 mM EGTA, and 10 mM MES–KOH (pH 6.15). After the incubation in white light (0.18 mmol m–2 s–1) at 25 °C for 2 h to induce stomata opening, the inhibitors of CuAO, aminoguanidine (AG) and 2-bromoethylamine (BEA), were added 30 min prior to the 2 h treatment with ABA or polyamines. Stomatal apertures were measured under a microscope. Each assay was repeated three times.
Extraction of enzymes in intercellular washing fluid, cytoplasm, and cell wall
The intercellular washing fluid (IWF) was extracted using the method of Hernández et al. (2001). The V. faba leaves were weighted and washed in deionized water, and vacuum infiltrated for 5 min at 1.0 kPa and 4 °C in 50 mM K-phosphate buffer (pH 6.5) with 0.2 M KCl and 0.1 mM CaCl2. Leaves were then quickly dried and centrifuged at 1000 g for 5 min at 4 °C in a 25 ml syringe barrel placed in a 50 ml tube. After the centrifugation, IWF collected from the tube bottom was used to analyse CuAO activity.
The soluble cytoplasmic CuAO, strong ionically-bound cell wall CuAO, and total CuAO in leaves were extracted by the method of Li and McClure (1989). Briefly, after IWF was extracted, the rest of the leaves were homogenized in 3-fold volume of 50 mM K-phosphate buffer (pH 6.5), centrifuged at 27 000 g for 15 min, and the supernatant was used as a source of soluble cytoplasmic CuAO. The cell wall pellets were washed twice by centrifuging in 50 mM K-phosphate buffer (pH 6.5) plus 1% (v/v) Triton X-100 and three times with the buffer alone to remove traces of contaminating cytoplasmic CuAO. The washed pellets were resuspended in 3-fold volume of 50 mM K-phosphate buffer with 1 M NaCl, stirred on ice for 30 min, centrifuged at 27 000 g for 15 min, and the supernatant was used as a source of CuAO strongly ionically bound to the cell wall. To extract the total CuAO in V. faba leaves, the leaves were ground in 50 mM K-phosphate buffer with 1 M NaCl (3 ml of the buffer per gram of fresh weight, pH 6.5) at 4 °C. Homogenates were centrifuged at 10 000 g for 20 min at 4 °C. Supernatants were used for the determination of protein content and amine oxidase activities.
Determination of the effects of exogenous ABA, polyamines and AG on CuAO activity
Fully expanded leaves of 4-week-old seedlings were sprayed with 100 µM ABA or 1 mM Put, Spd or Spm with or without AG. To promote the absorption of these regulators by leaves, 0.01% (v/v) Triton X-100 was used as a detergent, which was set as the control. The leaves were cut at the indicated time and used for enzyme activity assay.
CuAO activity was determined according to Rea et al. (2004) with minor modifications. Three millilitres of reaction solution contained sodium phosphate buffer (100 mM, pH 6.5), horseradish peroxidase (25 U), 35 µM 4-aminoantipyrine, 1 mM 3, 5-dichloro-2-hydroxybenzenesulphonic acid (DCHBS), and 0.1 ml crude enzyme extracts. The reaction was initiated by the addition of Put (or Cad) as a substrate. The changes in absorbance resulting from CuAO enzyme activity were recorded in a spectrometer (Shimadzu UV-1700). One unit of enzyme represents the amount of enzyme that catalyses the oxidation of 1 µmol of substrate min–1. The activity was expressed as enzyme unit per gram of the protein. Protein content was determined according to the method of Bradford (1976) with bovine serum albumin as a standard.
Isolation of plasma membrane vesicles and determination of NADPH oxidase activity
Plasma membrane vesicles were isolated according to the procedure described by Linnemeyer et al. (1990) with minor modifications. The leaves were ground with a chilled mortar and pestle in 4 vols of the buffer containing 0.25 M sucrose, 2 mM EGTA, 2 mM MgSO4, 2 mM ATP, 10% (v/v) glycerol, 1 mM PMSF, 2 mM DTT, 0.5% BSA, and 25 mM TRIS-MES (pH 7.6). The homogenate was filtered through four layers of cheesecloth, and the resulting filtrate was centrifuged at 10 000 g for 20 min. The supernatant was centrifuged at 100 000 g for 20 min to obtain the microsomal pellets which were then resuspended in a buffer (250 mM sucrose, 5 mM potassium phosphate pH 7.8, and 14 mM KCl). The resuspended pellets were added to a phase-partitioning system of a weight equal to the fresh weight of the leaves from which the pellet had been obtained. This system contained 5.8% (w/w) dextran T500, 5.8% (w/w) PEG 3350, 250 mM sucrose, 5 mM potassium phosphate (pH 7.8), and 14 mM KCl. After three partitions (at 1000 g, 10 min), the resulting upper phase was diluted 5-fold in TRIS-HCl buffer (pH 7.5) containing 250 mM sucrose and 1 mM EDTA, centrifuged for 30 min at 100 000 g. The resulting pellet was resuspended in a solution containing 25 mM TRIS-MES (pH 7.0), 1 mM EDTA, 1 mM DTT, and 20% glycerol at approximately 1 mg ml–1 protein and stored in liquid N2.
The NADPH oxidase activity in the plasma membrane was assayed by measuring the superoxide dismutase (SOD)-inhibitable and NADPH-dependent reduction of sodium, 3'-[1-[phenylamino-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulphonic acid hydrate (XTT) by O
(Sagi and Fluhr, 2001). The assay mixture of 1 ml contained 50 mM TRIS-HCl buffer (pH 7.5), 0.5 mM XTT, 100 µM NADPH and 15–20 µg of membrane proteins. The reaction was initiated with the addition of NADPH, and XTT reduction was determined at 470 nm. Corrections were done for background production in the presence of 50 units of SOD.
H2O2- and Ca2+-sensitive fluorescent dye loading
H2O2- and Ca2+-sensitive fluorescent dye loading were according to the procedure described by Chen et al. (2004). For H2O2-sensitive fluorescent dye loading, the abaxial epidermal strips from V. faba were incubated in 50 µM H2DCF-DA loading buffer (10 mM MES-TRIS, pH 6.1) for 15 min at room temperature (25 °C), following by washing three times with the MES-TRIS buffer (pH 6.1) before ABA (or polyamines) addition.
For Ca2+-sensitive fluorescent dye loading, the abaxial epidermal strips were incubated in 10 µM 1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl) phenoxy]-2-(2-amino-5-methylphenoxy) ethane-N,N,N’,N’-tetraacetic acid, pentaacetoxymethyl ester (fluo-3 AM) loading buffer (10 mM MES-TRIS, pH 6.1) for 2 h at 4 °C in darkness. After washing with MES-TRIS buffer (pH 6.1) for three times, strips were kept at room temperature for 1 h before ABA (or polyamines) addition.
Confocal microscopy
The fluorescence of H2O2 or Ca2+ dye was observed using a TCS-SP2 confocal system with an inverted confocal laser scanning microscope (CLSM, Leica Microsystems, Wetzlar, Germany). Excitation and emission was at 488 nm and 535 nm, respectively. The fluorescence intensity was measured by Leica confocal software (version 2.5).
Polyamine measurement
Polyamine extraction and analysis were done according to the method of Kotzabasis et al. (1993).
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Results |
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CuAO is involved in the regulation of stomatal closure in V. faba leaves
ABA significantly induced stomatal closing in V. faba leaves (Fig. 1A). It has been well known that ABA-induced H2O2 production is an essential effector in stomatal closure (Pei et al., 2000; Zhang et al., 2001; Bright et al., 2006). In addition, amine oxidation is one of sources for the production of H2O2 (Cona et al., 2006). To investigate whether amine oxidation by CuAO in guard cells is involved in ABA-mediated stomatal closure, aminoguanidine (AG) and 2-bromoethylamine (BEA), which are irreversible inhibitors of the copper-containing amine oxidases (CuAO) (Medda et al., 1997; Rea et al., 2002), were used. 0.2 mM AG or BEA alone did not affect stomatal apertures (Fig. 1A). However, after epidermal peels were pretreated with 0.1 mM AG or BEA, the effect of ABA on stomatal closure was obviously attenuated (Fig. 1A). The results indicate that the CuAO might be involved in ABA-induced stomatal closure.
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To characterize the CuAO effect on ABA response further, its products were tested. The major catalysed and metabolized products of CuAO are H2O2, ammonia,
-aminobutyric acid (GABA), and succinate (Succ) (Cona et al., 2006). Therefore, the effects of these compounds on stomatal closure were determined. Figure 1B showed that only H2O2 could restore the ABA effect reduced by the CuAO inhibitor AG, indicating that H2O2 is an effective product of CuAO in ABA-induced stomatal closure. The effects of the substrate(s) of CuAO or PAO on stomatal closure were then investigated. At first, the content of diamines (or polyamines) was measured in V. faba leaves. The content of Cad was much lower than that of Put, Spm, and Spd respectively (Fig. 1C, inset). Therefore, exogenous Put, Spm, and Spd were then used to examine whether they have effects on stomatal closure. The data showed that all these polyamines (or diamine) could induce stomatal closure (Fig. 1C, D), and Put showed most effectively, especially at an early time (0.5–1 h) (Fig. 1D). When epidermal peels were pretreated with AG before polyamines (or diamine) addition, only the effect of Put on stomatal closure was inhibited (Fig. 1C). The results suggest (i) Put may be used mainly by CuAO to catalyse the oxidation reaction in the ABA response; and (ii) Put, Spd, and Spm participate in the ABA-induced stomatal closure with different mechanisms.
ABA stimulates CuAO activity in V. faba leaves
To determine directly whether CuAO activity was activated by ABA, CuAO activity was measured. First, CuAO activity was determined in crude leaf extracts using Put or Cad as a substrate. The enzyme kinetics showed that Vmax of CuAO using Put as a substrate was 1.3-fold of that using Cad. Considering (i) Cad concentration in V. faba leaf cells was about 4 µM (calculated from the contents in Fig. 1C inset), much less than the 60 µM at which the CuAO activity could be tested (Fig. 2A); (ii) CuAO activity catalysing Put was 40-fold higher than that catalysing Spd or Spm (data not shown), so it is proposed that CuAO may use Put as its substrate in V. faba leaves. This notion also agrees with the observation that the inhibitory effect of CuAO inhibitor on polyamine-induced stomatal closure was only found in the Put treatment (Fig. 1C). Therefore, Put was used as a substrate for CuAO in the following experiments.
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As shown in Fig. 2B, both CuAO inhibitors (AG and BEA) could inhibit CuAO activity significantly. The inhibitory effect of AG was stronger than that of BEA when their concentrations were below 0.1 mM. The CuAO activity was then detected in the different subcellular compartments, expressed as intercellular washing fluid (IWF), ionically-bound cell wall, and soluble cytoplasm. As shown in Fig. 2C, 77.9% of total CuAO activity was detected in IWF. On the contrary, 10.7% and 5.8% activity was found in ionically-bound cell wall and soluble cytoplasm, respectively. The data were consistent with the previous study showing that CuAO exists at a high level mainly in leguminous plants, loosely associated to cell walls (Cona et al., 2006). Taken together, the data described so far suggest that CuAO existing in IWF mainly catalyses Put to produce H2O2 in V. faba leaves.
ABA sprayed onto V. faba leaves evoked a biphasic activation of IWF–CuAO activity in leaves (Fig. 3A). The activity increased rapidly after the onset of ABA treatment, reaching a first peak after 5 min. It then declined to a minimum at 10 min, and rose to a second lower peak at 30 min (Fig. 3A). The ABA-stimulated CuAO activity was also found in the epidermal tissues (Fig. 3A, inset). The activation of IWF–CuAO by ABA was impaired by AG with dose dependence. When 0.2 mM AG was applied, this activation was abolished (Fig. 3B). The data suggest that AG did inhibit the IWF–CuAO activity. The result is consistent with an attenuated effect of the inhibitor on stomatal closure induced by ABA (Fig. 1A).
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IWF–CuAO activity was also stimulated significantly by applied Put, although this stimulation appeared 5 min later than that by ABA. Neither Spd nor Spm could activate IWF–CuAO activity during the experiments (Fig. 3C). Taken together, the results described so far suggest that Put oxidation by IWF–CuAO is involved in ABA response.
Oxidation of putrescine by CuAO is involved in H2O2 generation induced by ABA in V. faba guard cells
To examine whether CuAO and its substrate, Put, are involved in H2O2 production induced by ABA, ROS generation was measured in guard cells in epidermal strips using a fluorescent dye, H2DCF-DA, which reports the changes in ROS in guard cells (Pei et al., 2000). Treatment of epidermal strips with 10 µM ABA produced a rapid increase in H2O2 indicated by the increase in fluorescence intensity as compared with the control tissue (Fig. 4A, B). Significant H2O2 production was observed as early as 3 min after ABA was added, and maximum fluorescence intensity was recorded in 4–20 min (Fig. 4B). The increase of H2O2 generation was found mainly in chloroplasts, cytoplasm, as well as in the apoplast (and/or plasma membrane) (Fig. 4A). At the maximum fluorescent intensity, ABA-mediated H2O2 generation was about 3.5-fold of the control in V. faba guard cells (Fig. 4B), while 1.4-fold of the control in Arabidopsis (data not shown). Put also induced guard cell H2DCF-fluorescence increase as did ABA treatment (Fig. 4A, B). As compared with ABA, Put induced the increase in H2DCF-fluorescence more slowly. Maximum fluorescence intensity recorded in Put-treated cells was 3 min later and 33% lower in degree than that in ABA-treated cells (Fig. 4B). When the epidermal peels were pretreated with CuAO inhibitor, AG or BEA, the increase in fluorescence intensity induced by ABA or Put was reduced. Alone, AG or BEA had no effect on fluorescence intensity (Fig. 4A, C, D).
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It has been reported that, the H2DCF-DA dye is not specific for H2O2, and also reacts with nitric oxide (Bright et al., 2006). To determine whether the increase of H2DCF-fluorescence is derived from H2O2 production by ABA or Put, the following experiments were designed. As shown in Fig. 4C and D, when the epidermal peels were pretreated with 100 U ml–1 catalase (CAT) or 1 mM ascorbate (ASC), ABA- or Put-induced H2DCF-fluorescence elevation was abolished, suggesting that the elevation of H2DCF-fluorescence by ABA (or Put) was due to H2O2 generation.
To determine whether other polyamines could mediate H2O2 production in V. faba guard cells, Spd and Spm were also tested. Neither of them could induce H2O2 production in 20 min after treatment (data not shown). Taken together, these data indicate that CuAO catalysing Put contributes to ABA-induced H2O2 generation.
CuAO and NADPH oxidase contribute to ABA-induced H2O2 production independently
In plants, both plasma membrane NADPH oxidase and CuAO are sources for H2O2 production (Cona et al., 2006). In order to investigate whether the inhibition of H2O2 generation by CuAO inhibitor(s) was related to its inhibitory effect on NADPH oxidase, the NADPH oxidase activity was assayed using plasma membrane vesicles. As shown in Fig. 5A, treatment of leaves with ABA resulted in a rapid activation of NADPH oxidase. The activation by ABA was not affected by AG up to a concentration of 0.2 mM (Fig. 5B), which significantly inhibited CuAO activity (Fig. 2B). The results suggest that CuAO inhibitor does not inhibit NADPH oxidase activity. Whereas NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI), impaired the ABA-mediated H2O2 generation (Fig. 4C). DPI did not affect CuAO activity (data not shown). When DPI and AG (or BEA) were used together, ABA-induced H2O2 generation was almost abolished (Fig. 4C), indicating H2O2 generation induced by ABA is mainly attributed by CuAO and NADPH oxidase activity. CuAO or NADPH oxidase inhibitor significantly inhibited ABA-induced stomatal closure (Fig. 5C). The above results suggest that CuAO and NADPH oxidase are involved in ABA-mediated H2O2 generation and stomatal closure independently.
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CuAO activation is necessary to increase cytosolic calcium level in ABA response in V. faba guard cells
To investigate the cellular mechanisms of CuAO in the regulation of H2O2 and stomatal closure further, we next determined cytosolic calcium ([Ca2+]cyt) changes in V. faba guard cells, which has been proved to be regulated by H2O2 in Arabidopsis and V. faba guard cells (Pei et al., 2000; Kohler et al., 2003). Confocal laser scanning microscopy (CLSM) was used to visualize calcium-sensitive fluorescent dye fluo-3, which was loaded into guard cells to monitor [Ca2+]cyt. Dramatic [Ca2+]cyt elevation was found when epidermal strips were incubated with 10 µM ABA for 9 min (Fig. 6A, B). Put could increase the [Ca2+]cyt, although this increase was little later as compared with that by ABA (Fig. 6A, B).
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It was then asked whether CuAO activation is crucial for ABA- or Put-mediated [Ca2+]cyt elevation. To test the hypothesis, CuAO inhibitor, AG, was incubated with epidermal strips for 30 min before ABA or Put added. The data showed that AG significantly reduced both ABA- and/or Put-induced [Ca2+]cyt increase (Fig. 6A, C). The results suggest that the CuAO activity, which uses Put as a substrate, is essential to ABA-induced [Ca2+]cyt increase.
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Discussion |
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Despite the importance of signalling via H2O2 in plants, little is known about the sources for H2O2 production and their physiological roles. Under normal conditions, H2O2 can be generated in normal metabolism via the Mehler reaction in chloroplasts, electron transport in mitochondria, and photorespiration in peroxisomes (Neill et al., 2002). Under biotic or abiotic stresses, H2O2 generation increases through these pathways, and other enzymatic routes, including plasma membrane-associated NADPH oxidase, cell wall peroxidase, apoplast amine oxidase (Mittler, 2002; Neill et al., 2002). Different stimuli activate specific H2O2-generating pathways, and produce the signal leading to specific physiological processes (Neill et al., 2002). With the challenge with cryptogein, nuclei act as a potential active source of H2O2 production (Ashtamker et al., 2007). As to the ABA signal, it mediates H2O2 generation via the plasma membrane NADPH oxidase (AtrbohD and AtrbohF catalytic subunits) in Arabidopsis (Kwak et al., 2003), or in the chloroplasts in V. faba (Zhang et al., 2001).
In this work, new evidence is provided that H2O2 coming from the degradation of putrescine by CuAO is involved in ABA-induced stomatal closure in V. faba leaves. The following results support this conclusion: (i) The inhibitors of CuAO impaired the ABA-induced stomatal closure; (ii) exogenous ABA stimulated CuAO activity; (iii) the inhibition of CuAO activity resulted in the attenuation of the elevation of H2O2 level in guard cells induced by ABA; (iv) putrescine, a substrate of CuAO, could induce H2O2 increase and stomatal closure, which was impaired by CuAO inhibitors.
H2O2 production regulated by CuAO was parallel to that by plasma membrane NADPH oxidase in the ABA response, because the inhibitor of CuAO had no effect on the activation of NADPH oxidase by ABA (Fig. 5B). When both CuAO and NADPH oxidase inhibitors were used together, they had an additive inhibition on ABA-induced H2O2 production (Fig. 4C). For ABA-mediated stomatal closure, such an additive inhibition was not as obvious as in H2O2 production (Fig. 5C), suggesting that there are other signal pathways regulating ABA-mediated stomatal closure besides H2O2 signalling.
It should be noticed that both ABA and putrescine-induced H2O2 increase was located in the cytoplasm, chloroplasts, and the apoplast (and/or plasma membrane) according to the H2DCF fluorescence (Fig. 4A). Whereas, CuAO in V. faba was located mainly in apoplasts, because the major activity was checked in intercellular washing fluids from leaf tissues (Fig. 2C). The explanation for this conflict is as follows: (i) the apoplast has only a small proportion of the cell's antioxidant capacity (Neill et al., 2002), which results in less H2O2 degraded; (ii) H2O2 rapidly diffuses across the plasma membrane. H2O2 has been proposed to be permeable across the plasma membrane (Sagi and Fluhr, 2006). Recently, the role of such an aquaporin family of Arabidopsis was studied in a yeast system (Bienert et al., 2007). CuAO activity was also found in the epidermis, and was activated by ABA (Fig. 3A, inset). In addition, polyamines were measured in the epidermis, in which the content of putrescine was higher than other polyamines (see Supplementary data at JXB online). Therefore, the production of H2O2 from the degradation of putrescine in the apoplast and the following permeance into guard cells may exist in both the leaf tissues and the detached epidermal strips of V. faba.
Some components downsteam of H2O2 production in the ABA signal have been characterized in guard cells, such as Ca2+, nitric oxide, inward and outward K+ channels, protein phosphatase 2C, ABI1, and ABI2 (Pei et al., 2000; Bright et al., 2006; Kwak et al., 2006, and references within). In this study, it was found that ABA and putrescine enhanced the Ca2+ concentration in guard cells in V. faba (Fig. 6A, B). The increase in Ca2+ level induced by ABA or putrescine was impaired by CuAO inhibitors (Fig. 6C), suggesting that H2O2 from CuAO-catalysed putrescine oxidation may contribute to the Ca2+ increase in the guard cells in response to ABA. In Arabidopsis guard cells, the increased Ca2+ level is due to the activation of plasma membrane Ca2+-permeable channels by NADPH oxidases-derived H2O2 (Kwak et al., 2003). It still remains unsolved whether H2O2 from diverse sources stimulates different kinds of Ca2+ channels.
The role of polyamines in ABA signalling is complex. Besides being a substrate of CuAO, putrescine could inhibit the K+ inward current in V. faba guard cells (Liu et al., 2000). Compared with spermine and spermidine, putrescine inhibits the K+ current less effectively (Liu et al., 2000). On the other hand, spermine and spermidine did not contribute to ABA-promoted H2O2 generation in V. faba guard cells (data not shown). However, these three polyamines promoted stomatal closure (Fig. 1C, D). Therefore, in vivo, polyamines might regulate stomatal closure through different signal routes.
Based on these results, a model of H2O2 generation by CuAO and its role in the ABA signaling process is proposed (Fig. 7). CuAO is activated in response to ABA, which produces H2O2 by catalysing putrescine oxidation. The increased H2O2 production may activate the Ca2+ channel, resulting in an increase in Ca2+ level in guard cells. Therefore, a Ca2+ messenger mediates ABA signalling processes and induces stomatal closure.
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Acknowledgements |
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This work was supported by the National Science Foundation of China Research Grants (30470162, 30625027), the Chinese National Key Basic Research Project (2006CB100100), and grants from the Ministry of Education of China (NCET-04-0504,20060307019, 111 project) to W Zhang.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Ashtamker C, Kiss V, Sagi M, Davydov O, Fluhr R. Diverse subcellular locations of cryptogein-induced reactive oxygen species production in tobacco bright yellow-2 cells. Plant Physiology (2007) 143:1817–1826.
Bienert GP, Møller AB, Kristiansen KA, Schulz A, Møller IM, Schjoerring JK, Jahn TP. Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes. Journal of Biological Chemistry (2007) 282:1183–1192.
Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry (1976) 72:248–254.[CrossRef][Web of Science][Medline]
Bright J, Desikan R, Hancock JT, Weir LS, Neill SJ. ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis. The Plant Journal (2006) 45:113–122.[CrossRef][Web of Science][Medline]
Chen YL, Huang RF, Xiao YM, Lu P, Chen J, Wang XC. Extracellular calmodulin-induced stomatal closure is mediated by heterotrimeric G protein and H2O2. Plant Physiology (2004) 136:4096–4103.
Cona A, Rea G, Angelini R, Federico R, Tavladoraki P. Function of amine oxidases in plant development and defence. Trends in Plant Science (2006) 11:80–88.[CrossRef][Web of Science][Medline]
Coursol S, Fan LM, Le Stunff H, Spiegel S, Gilroy S, Assmann SM. Sphingolipid signaling in Arabidopsis guard cells involves heterotrimeric G proteins. Nature (2003) 423:651–654.[CrossRef][Medline]
Delon C, Manifava M, Wood E, Thompson D, Krugmann S, Pyne S, Ktistakis NT. Sphingosine kinase 1 is an intracellular effector of phosphatidic acid. Journal of Biological Chemistry (2004) 279:44763–44774.
Finkelstein RR, Gampala SSL, Rock CR. Abscisic acid signaling in seeds and seedlings. The Plant Cell (2002) 14:S15–S45.
Gao Y, Zeng Q, Guo J, Cheng J, Ellis BE, Chen J-G. Genetic characterization reveals no role for the reported ABA receptor, GCR2, in ABA control of seed germination and early seedling development in Arabidopsis. The Plant Journal (2007) 52:1001–1013.[Web of Science][Medline]
Grant JJ, Loake GJ. Role of reactive oxygen intermediates and cognate redox signaling in disease resistance. Plant Physiology (2000) 124:21–29.
Grill E, Christmann A. A plant receptor with a big family. Science (2007) 315:1676–1677.
Hernández JA, Ferrer MA, Jiménez A, Barceló AR, Sevilla F. Antioxidant systems and O/H2O2 production in the apoplast of pea leaves: its relation with salt-induced necrotic lesions in minor veins. Plant Physiology (2001) 127:817–831.
Hugouvieux V, Kwak JM, Schroeder JI. An mRNA cap binding protein, ABH1, modulates early abscisic acid signal transduction in Arabidopsis. Cell (2001) 106:477–487.[CrossRef][Web of Science][Medline]
Kohler B, Hills A, Blatt MR. Control of guard cell ion channels by hydrogen peroxide and abscisic acid indicates their action through alternate signaling pathways. Plant Physiology (2003) 131:385–388.
Kotzabasis K, Christakis-Hampsas MD, Roubelakis-Angelakis KA. A narrow-bore HPLC method for the identification and quantitation of free, conjugated and bound polyamines. Analytical Biochemistry (1993) 214:484–489.[CrossRef][Web of Science][Medline]
Kwak JM, Mori IC, Pei ZM, Leonhardt N, Torres MA, Dangl JL, Bloom RE, Bodde S, Jones JD, Schroeder JI. NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis. EMBO Journal (2003) 22:2623–2633.[CrossRef][Web of Science][Medline]
Kwak JM, Nguyen V, Schroeder JI. The role or reactive oxygen species in hormonal responses. Plant Physiology (2006) 141:323–329.
Laurenzi M, Rea G, Federico R, Tavladoraki P, Angelini R. 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 (1999) 208:146–154.[CrossRef][Web of Science]
Li J, Wang XQ, Watson MB, Assmann SM. Regulation of abscisic acid-induced stomatal closure and anion channels by guard cell AAPK kinase. Science (2000) 287:300–303.
Li ZC, McClure JW. polyamine oxidase of primary leaves is apoplastic in oats but symplastic in barley. Phytochemistry (1989) 28:2255–2259.[CrossRef][Web of Science]
Lin CC, Kao CH. Abscisic acid induced changes in cell wall peroxidase activity and hydrogen level in roots of rice seedlings. Plant Science (2001) 160:323–329.[Medline]
Linnemeyer PA, Volkenburgh EV, Cleland RE. Characterization and effect of light on the plasma membrane H+-ATPase of bean leaves. Plant Physiology (1990) 94:1671–1676.
Liu K, Fu HH, Bei QX, Luan S. Inward potassium channel in guard cell as a target for polyamine regulation of stomatal movements. Plant Physiology (2000) 124:1315–1325.
Liu XG, Yue YL, Li B, Nie YL, Li W, Wu WH, Ma LG. A G protein-coupled receptor is a plasma membrane receptor for the plant hormone abscisic acid. Science (2007) 315:1712–1716.
Medda R, Padiglia A, Pedersen JZ, Agrò AF, Rotilio G, Floris G. Inhibition of copper amine oxidase by haloamines: a killer product mechanism. Biochemistry (1997) 36:2595–2602.[CrossRef][Web of Science][Medline]
Merlot S, Gosti F, Guerrier D, Vavasseur A, Giraudat J. The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway. The Plant Journal (2001) 25:295–303.[CrossRef][Web of Science][Medline]
Mishra G, Zhang W, Deng F, Zhao J, Wang X. A bifurcating pathway directs abscisic acid effects on stomatal closure and opening in Arabidopsis. Science (2006) 312:264–266.
Mittler R. Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science (2002) 7:405–410.[CrossRef][Web of Science][Medline]
Neill SJ, Desikan R, Hancock JT. Hydrogen peroxide signalling. Current Opinion in Plant Biology (2002) 5:388–395.[CrossRef][Web of Science][Medline]
Pei ZM, Murata Y, Benning G, Thomine S, Klusener B, Allen GJ, Grill E, Schroeder JI. Calcium channels activated by hydrogen peroxide mediate abscisic acid signaling in guard cells. Nature (2000) 406:731–734.[CrossRef][Medline]
Pennell RI, Lamb C. Programmed cell death in plants. The Plant Cell (1997) 9:1157–1168.[CrossRef][Web of Science][Medline]
Razem FA, El-Kereamy A, Abrams SR, Hill RD. The RNA-binding protein FCA is an abscisic acid receptor. Nature (2006) 439:290–294.[CrossRef][Medline]
Rea G, De Pinto MC, Tavazza R, Biondi S, Gobbi V, Ferrante P, De Gara L, Federico R, Angelini R, Tavladoraki P. Ectopic expression of maize polyamine oxidase and pea copper amine oxidase in the cell wall of tobacco plants. Plant Physiology (2004) 134:1414–1426.
Rea G, Metoui O, Infantino A, Federico R, Angelini R. Copper amine oxidase expression in defence response to wounding and Ascochyta rabiei invasion. Plant Physiology (2002) 128:865–875.
Sagi M, Fluhr R. Superoxide production by plant homologues of the gp91phox NADPH oxidase. Modulation of activity by calcium and by tobacco mosaic virus infection. Plant Physiology (2001) 126:1281–1290.
Sagi M, Fluhr R. Production of reactive oxygen species by plant NADPH oxidases. Plant Physiology (2006) 141:336–340.
Sanchez JP, Chua NH. Arabidopsis PLC1 is required for secondary responses to abscisic acid signals. The Plant Cell (2001) 13:1143–1154.
Shen YY, Wang XF, Wu FQ, et al. The Mg-chelatase H subunit is an abscisic acid receptor. Nature (2006) 443:823–826.[CrossRef][Medline]
Sokolovski S, Hills A, Gay R, Garcia-Mata C, Lamattina L, Blatt MR. Protein phosphorylation is a prerequisite for intracellular Ca2+ release and ion channel control by nitric oxide and abscisic acid in guard cells. The Plant Journal (2005) 43:520–529.[CrossRef][Web of Science][Medline]
Su GX, An ZF, Zhang W, Liu YL. Light promotes the synthesis of lignin through the production of H2O2 mediated by diamine oxidase in soybean hypocotyls. Journal of Plant Physiology (2005) 162:1297–1303.[CrossRef][Web of Science][Medline]
Vranová E, Inzé D, van Breusegem F. Signal transduction during oxidative stress. Journal of Experimental Botany (2002) 53:1227–1236.
Walters DR. Resistance to plant pathogens: possible roles for free polyamines and polyamine catabolism. New Phytologist (2003) 159:109–115.[CrossRef][Web of Science]
Wang XQ, Ullah H, Jones AM, Assmann SM. G protein regulation of ion channels and abscisic acid signaling in Arabidopsis guard cells. Science (2001) 292:2070–2072.
Xiong L, Gong Z, Rock C, Subramanian S, Guo Y, Xu W, Galbraith D, Zhu JK. Modulation of abscisic acid signal transduction and biosynthesis by an Sm-like protein in Arabidopsis. Developmental Cell (2001b) 1:771–781.[CrossRef][Web of Science][Medline]
Xiong L, Lee B, Ishitani M, Lee H, Zhang C, Zhu JK. FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signaling in Arabidopsis. Genes and Development (2001a) 15:1971–1984.
Yoda H, Yamaguchi Y, Sano H. Induction of hypersensitive cell death by hydrogen peroxide produced through polyamine degradation in tobacco plants. Plant Physiology (2003) 132:1973–1981.
Zhang W, Qin C, Zhao J, Wang X. Phospholipase D1-derived phosphatidic acid interacts with ABI1 phosphatase 2C and regulates abscisic acid signaling. Proceedings of the National Academy of Sciences, USA (2004) 101:9508–9513.
Zhang X, Zhang L, Dong F, Gao J, Galbraith DW, Song CP. Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba. Plant Physiology (2001) 126:1438–1448.
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