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ISSN 1021�4437, Russian Journal of Plant Physiology, 2014, Vol. 61, No. 3, pp. 390–396. © Pleiades Publishing, Ltd., 2014.
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1 INTRODUCTION
The plant hormone ethylene plays an importantregulatory role in plant growth and development.Although the role of ethylene on stomatal behavior hasbeen suggested, its effect on this process seems rathercontradictory [1]. In some species, ethylene inducesstomatal opening or inhibits ABA�induced stomatalclosure [2–4], whereas in other species ethyleneinduces stomatal closure [5]. Using Vicia faba leaf epi�dermal tissues, our previous works proved that ethyl�ene probably induces hydrogen peroxide (H2O2)removal, reduces H2O2 levels in guard cells, and finallyinhibits stomatal closure induced by darkness [6]. Thediscrepancy of ethylene effects on stomatal behavior isprobably associated with one or several factors: plantspecies, organ or tissue types, manners of ethylene
1 This text was submitted by the authors in English.
treatment, physiological states of the tissue, and theconcentrations of ethylene gas, ACC, or ethephon(ETH), which can be decomposed into ethylene, butthere is no final conclusion, and the detailed mecha�nism is still under investigation.
Hydrogen peroxide (H2O2), a form of reactive oxy�gen species, is one of the common components ofplant development processes and defense responses.The roles of H2O2 in stomatal closure and plantresponse to a wide variety of abiotic and biotic stressesare at present widely accepted [5, 7, 8]. It has been wellknown that H2O2 production in plants is achieved byseveral enzymatic systems, including amine oxidases[9]. Amine oxidases are represented by a heteroge�neous group of enzymes, such as copper�containingdiamine oxidase (CuAO; EC 1.4.3.6) and flavin�con�taining polyamine oxidase (EC 1.5.3.3). CuAO gener�ally catalyzes the oxidation of aliphatic diaminesputrescine (Put) and cadaverine at the primary aminogroups [10]. The products from Put oxidation byCuAO are H2O2, NH3, and Δ1�pyrroline. Δ1�pyrrolineis further catabolized to γ�aminobutyric acid (GABA),which is subsequently transaminated and oxidized tosuccinic acid (Succ) [10, 11]. Increasing evidence sug�
Involvement of Copper Amine Oxidase (CuAO)�Dependent Hydrogen Peroxide Synthesis in Ethylene�Induced Stomatal
Closure in Vicia faba1 X. G. Songa, b, X. P. Shea, M. Yuec, Y. E. Liub, Y. X. Wanga, X. Zhua, and A. X. Huanga
a School of Life Sciences, Shaanxi Normal University, Xi`an 710062, P.R. China;fax: +86�(0)29 8531�0546; e�mail: [email protected]
b The High School affiliated to Shaanxi Normal University, Xi`an 710061, P.R. Chinac Opening Foundation of Key Laboratory of Resource Biology and Biotechnology in Western China,
The College of Life Sciences, Northwest University, Xi’an 710069, P.R. ChinaReceived January 18, 2013
Abstract—Ethylene promotes stomatal closure via inducing hydrogen peroxide (H2O2) generation. H2O2can be catalytically synthesized by several enzymes in plants. Here, by means of stomatal bioassay, the analysisof enzyme activity and using laser�scanning confocal microscopy based on the H2O2�sensitive probe2',7'�dichlorodihydrofluorescein diacetate (H2DCF�DA), the roles of copper amine oxidase (CuAO) in eth�ylene�induced H2O2 production in guard cells and stomatal closure in Vicia faba L. were investigated. 1�ami�nocyclopropane�1�carboxylic acid (ACC), an immediate precursor of ethylene synthesis, and ethylene gassignificantly activated CuAO in intercellular washing fluid from leaves, the production of H2O2 in guard cells,and stomatal closure. These effects of ACC and ethylene gas were largely prevented by both aminoguanidineand 2�bromoethylamine, which are irreversible inhibitors of CuAO. Among major catalyzed and metabolizedproducts of CuAO, only H2O2 could markedly promote stomatal closure and evidently reversed the effect ofCuAO inhibitor on stomatal closure by ACC and ethylene gas. The data described above show that CuAO�mediated H2O2 production is involved in ethylene�induced stomatal closure.
Keywords: Vicia faba, copper amine oxidase, ethylene, hydrogen peroxide, stomatal closure
DOI: 10.1134/S1021443714020150
Abbreviations: ACC—1�aminocyclopropane�1�carboxylic acid;AG—aminoguanidine; BEA—2�bromoethylamine; CuAO—copper amino oxidase; DCF—dichlorofluorescein; DMSO—dimethyl sulfoxide; ETH—ethephon; GABA—γ�aminobutiricacid; HRP—horseradish peroxidase; LSCM—laser scanningconfocal microscopy; Put—putrescine; Succ—succinic acid.
RESEARCHPAPERS
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INVOLVEMENT OF COPPER AMINE OXIDASE (CuAO)�DEPENDENT 391
gests that H2O2 generated by CuAO participates inplant development and defense responses [10, 12]. Itwas reported that CuAO regulates lateral root develop�ment in soybean via its product H2O2 [13]. Recently,H2O2 generated by CuAO has been shown to mediateABA�induced stomatal closure [14]. However, untilnow, it is unclear whether CuAO and its product H2O2are involved in ethylene�regulated stomatal move�ments. In the present study, we investigated the role ofH2O2 generated by CuAO during ethylene�inducedstomatal closure in V. faba by means of stomatal bio�assay, the analysis of enzyme activity, and using laser�scanning confocal microscopy (LSCM) based on themolecular probe 2',7'�dichlorodihydrofluoresceindiacetate (H2DCF�DA).
MATERIALS AND METHODS
Plant materials. Broad bean (Vicia faba L.) plantswere grown in controlled�environment plant growthchamber with a humidity of 70%, a photon flux den�sity of 300 μmol/(m2 s) PAR generated by cool whitefluorescent tubes (Philips, United States), a 14�h pho�toperiod, and an ambient temperature of 25 ± 2°C.The epidermis was peeled carefully from the abaxialsurface of the youngest, fully expanded leaves of 4�week�old seedlings and cut into pieces about 5 mm widthand 5 mm length.
Stomatal bioassay. Stomatal bioassay was per�formed as described in [6] with slight modifications.Freshly prepared epidermal strips were floated in CO2�free Mes/KCl buffer (10 mM Mes/KOH, 50 mM KCl,100 μM CaCl2, pH 6.15) in the light (300 μmol/(m2 s)at 25 ± 2°C for 3 h. Once the stomata were fully open,the strips were treated with Mes/KCl buffer containingvarious compounds for further 3 h. Besides ACC andethylene gas, the concentrations of other compoundstested were based on our preliminary experiments orother studies where the compounds were used [7, 14].Control treatments involved the addition of appropri�ate solvents used with the compounds.
For treatment with ethylene gas, detached strips,on which stomata were fully open, were incubated inCO2�free Mes/KCl buffer in open Petri dishes in a gas�impermeable sealed Kilner jars, which were injectedwith air or ethylene gas at various concentrations,under light (300 μmol/(m2 s) at 25 ± 2°C for 3 h. Afterthese steps, stomatal aperture was recorded with a lightmicroscope and an eyepiece graticule previously cali�brated with a stage micrometer. To avoid any potentialrhythmic effects on stomatal aperture, experimentswere always started at the same time of the day. In eachtreatment, we scored randomly 30 apertures, and everytreatment was repeated three times. The data pre�sented are the means of 90 measurements ± standarderrors.
Extraction and CuAO activity determination in theintercellular washing fluid (IWF) from leaves. Thetreatment procedure of detached leaves was the same
as that for detached strips, just as described in stomatalbioassay section. IWF was extracted using the methoddescribed in [15]. The leaves were weighted and vac�uum infiltrated for 10 min at 1.0 kPa and 4°C in50 mM K�phosphate buffer (pH 6.5) with 0.2 M NaCland 0.1 mM CaCl2. Leaves were then quickly driedand centrifuged at 1000 g for 5 min at 4°C in a 10�mLsyringe barrel placed in a 50�mL tube. After centrifu�gation, IWF collected from the tube bottom was usedto analyze CuAO activity.
The activity of CuAO in the IWF (IWF�CuAO) wasdetermined according to the method described in [11]with minor modifications. Use of Put, as a substratefor IWF�CuAO activity determination, was based on astudy with the same species [14]. Three milliliters ofthe reaction solution containing 1.9 mL of phosphatebuffer (100 mM, pH 6.5) with 25 U/mL of horseradishperoxidase (HRP), 35 μM 4�aminoantipyrine, and1 mM 3,5�dichloro�2�hydroxybenzenesulfonic acid(DCHBS), 0.1 mL IWF, and 1 mL of 3 mM Put. Thereaction was initiated by the addition of Put. After30 min at 25°C, the changes in absorbance at 515 nmwere recorded with a spectrophotometer. One unit ofenzyme represents the amount of enzyme that cata�lyzes the oxidation of 1 μmol Put/min. The activitywas expressed as enzyme unit per gram of protein.Protein content was determined according to themethod described in [16] with BSA as a standard. Thedeterminations of CuAO activity and protein contentwere repeated three times. The data presented are themeans of three measurements ± standard errors.
Measurement of H2O2 production. H2O2 produc�tion was monitored with H2DCF�DA, as described in[6] with minor modifications. To study the effect ofCuAO inhibitor on ACC� or ethylene gas�inducedH2O2 production, the epidermal strips were treated asdescribed in stomatal bioassay section and then loadedwith 50 μM H2DCF�DA (10 min) in Tris–KCl loadingbuffer (Tris 10 mM, KCl 50 mM, pH 7.2) in darknessat 25 ± 2°C. After excess dye was washed off with freshTris–KCl buffer in darkness, the strips were immedi�ately examined by TCS SP5 laser�scanning confocalmicroscopy (Leica Lasertechnik, Germany) with thefollowing settings: excitation 488 nm, emission530 nm, power 10%, zoom about 4, normal scanningspeed and frame 512 × 512 pixel. Images acquired fromthe confocal microscope were analyzed with Leica imagesoftware and processed with Photoshop 7.0. To enablethe comparison of changes in signal intensity, confocalimages were taken under identical conditions (in man�ual setup) for all samples, and in each treatment wemeasured three epidermal strips, and the treatmentwas repeated at least three times. The selected confo�cal images represented the same results from threereplications.
Chemicals. 1�aminocyclopropane�1�carboxylicacid (ACC), aminoguanidine (AG), 2�bromoethyl�amine (BEA), γ�aminobutyric acid (GABA), succinicacid (Succ), putrescine (Put), horseradish peroxidase
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(HRP), 2�(N�morpholino)ethanesulfonic acid(Mes), and dimethyl sulfoxide (DMSO) wereobtained from Sigma�Aldrich (United States). Themolecular probe 2',7'�dichlorodihydrofluoresceindiacetate (H2DCF�DA, Biotium, United States) wasdissolved in DMSO to produce a 10 mM stock solu�tion, which was aliquoted. Unless stated otherwise,the remaining chemicals were of the highest analyticalgrade available from various suppliers of the Chinesecompanies.
Statistical analysis. All data were treated with SPSS16.0 software (SPSS Inc., United States). Statisticalanalyses were performed by using a one�way ANOVAfollowed by the least significant difference test.
RESULTS
ACC and Ethylene Gas Induce Stomatal Closurein the Epidermal Strips of V. faba
Ethylene is synthesized from methionine via S�ade�nosyl�L�methionine (AdoMet) and the cyclic non�protein amino acid 1�aminocyclopropane�1�carboxy�lic acid (ACC) [17], and the conversion from AdoMetto ACC, which is catalyzed by ACC synthase (ACS), isgenerally considered as the rate�limiting step of ethyl�ene biosynthesis. The enzyme catalyzing the conver�sion of ACC to ethylene is ACC oxidase. To gaininsights into the effect of ACC or ethylene gas on sto�matal pore changes, a system using isolated V. fabaepidermal strips, in which stomatal apertures could bemeasured, was employed. As shown in Fig. 1, bothACC and ethylene gas induced stomatal closure in adose�dependent manner, and the effects of both ACCat the concentration of 150 μM and ethylene gas at theconcentration of 10 μL/L are highly significant (P <0.01, Figs. 1a, 1b). Thus, ethylene does induce sto�matal closure in detached epidermal strips of V. fabaleaves, which is consistent with the previous results[5]. Based on the results shown in Fig. 1, 150 μM ACCand 10 μL/L ethylene gas were respectively used in fol�lowing experiments.
Effects of Inhibitors and Products of CuAOon ACC� and Ethylene Gas�Induced Stomatal Closure
To seek for whether H2O2 generated by CuAOmediates ethylene�induced stomatal closure, weobserved the effects of aminoguanidine (AG) and2�bromoethylamine (BEA), two irreversible inhibitorsof CuAO [14], and H2O2, NH3, GABA, and Succ, sev�eral major catalyzed and metabolized products ofCuAO [10, 14], on ACC� and ethylene gas�inducedstomatal closure. As shown in Fig. 2, ACC and ethyl�ene gas obviously induced stomatal closure (P < 0.05),AG or BEA alone had no effect on stomatal apertures(P < 0.05). However, AG and BEA significantly pre�vented ACC� and ethylene gas�induced stomatal clo�sure (P < 0.05). In addition, among major catalyzedand metabolized products of CuAO, only H2O2 couldmarkedly promote stomatal closure (P < 0.05) and evi�dently reversed the inhibitory effects of AG andBEA on stomatal closure by ACC and ethylene gas(P < 0.05). The data suggest that H2O2 generated by CuAOis implicated in ethylene�induced stomatal closure.
ACC and Ethylene Gas Stimulate CuAO Activity in the Intercellular Washing Fluid (IWF) from Leaves
The data from our preliminary experiments (datanot shown) and previous study [14] show that 70% oftotal CuAO activity was detected in the IWF of V. fabaleaves. Thus, the effects of ACC and ethylene gas onactivity of IWF�CuAO were studied. As shown inFig. 3, the treatment with AG or BEA did not influence
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Fig. 1. Effects of ACC and ethylene gas on stomatal aper�ture.Isolated epidermal strips of V. faba were incubated in CO2�free Mes/KCl buffer only or containing different concen�trations of ACC (25, 50, 100, 150 μM) (a) or in open Petridishes in a gas�impermeable sealed Kilner jars, which wereinjected into with air or ethylene gas at various concentra�tions ( 2.5, 5, 10 μL/L) (b), under light (300 μmol/(m2 s)at 25 ± 2°C for 3 h. Stomatal apertures were determined.Data are the means of 90 measurements ± standard errorsof three independent experiments. Means with differentletters are significantly different at P < 0.05.
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IWF�CuAO activity (P < 0.05), ACC and ethylene gasnotably activated IWF�CuAO (Fig. 3d) (P < 0.05). WhenAG or BEA was applied with ACC or ethylene gas, thisactivation was significantly restricted (Fig. 3) (P < 0.05).The data suggest that ethylene does stimulate theIWF�CuAO activity. The result is consistent with anattenuating effect of AG or BEA on stomatal closureinduced by ACC and ethylene gas (Figs. 2a, 2b).
Inhibitor of CuAO Prevents ACC� and EthyleneGas�Induced H2O2 Production
To investigate whether ethylene�induced H2O2generation is CuAO�dependent, H2O2 production inguard cells of epidermal strips was measured by meansof LSCM based on the molecular probe H2DCF�DA,which can report the content changes of ROS, includ�ing H2O2, in guard cells [7, 8]. As shown in Fig. 4,compared with the control (Figs. 4A, 4J), the treat�ment with ACC or ethylene gas produced an increasein H2DCF fluorescence in guard cells (Figs. 4B, 4C, 4J)(P < 0.05). The increase in H2DCF fluorescence wasmainly found in chloroplasts and cytoplasm (Figs. 4B,4C). When the epidermal peels were treated with AGor BEA in the presence of ACC or ethylene gas, ACCor ethylene gas�induced H2DCF fluorescence waslargely reduced (Figs. 4E, 4F, 4H, 4I, 4J) (P < 0.05).Alone, AG or BEA had no effect on H2DCF fluores�cence (Figs. 4D, 4G, 4J) (P < 0.05). It has beenreported that the H2DCF�DA dye is not specific forH2O2 [7]. In our preliminary experiments, the epider�mal peels were pretreated with 100 U/mL catalase(CAT) or 100 μM ascorbate (ASA), ethylene�inducedH2DCF fluorescence elevation was abolished (datanot shown), which suggests that the increase ofH2DCF fluorescence is derived from H2O2 induced byethylene, and this results is consistent with the previ�ous report [5]. The fact that AG or BEA inhibition ofH2O2 production by ACC or ethylene gas indicatesthat ethylene�induced H2O2 synthesis is dependent onCuAO, which is in accordance with an inhibitoryeffect of AG or BEA on stomatal closure (Figs. 2a, 2b)and IWF–CuAO activation (Fig. 4) induced by ACCor ethylene gas.
DISCUSSION
The effect of phytohormone ethylene on stomatalbehavior has been known to be rather contradictory[2–5]. The present study showed clearly that ethyleneinduced stomatal closure in V. faba, irrespective ofwhether ethylene is supplied directly as a gas, or inplanta via biosynthesis from ACC (Fig. 1). The resultis contrary to our previous study with the same plantspecies, where ethylene was proved to prevent dark�ness� and ABA�induced stomatal closing [4, 19]. Whydoes ethylene have different effects on the stomatalbehavior described in the literature? It is generallyconsidered that the mechanism may be related to one
or other factors, such as one or several factors amongplant species, organ or tissue types, manners of ethyl�ene treatment, physiological states of the tissue, andthe concentrations of ethylene [1, 5, 19]. We proposethat the effect of ethylene on stomatal behavior isrelated to the presence or absence of ABA. In theabsence of ABA, ethylene alone did lead to stomatalclosure in V. faba and this effect of ethylene on sto�matal behavior was also observed in other plant species[5]. However, such as in our previous results and thoseof other researchers [1, 4, 6, 19], in the presence ofABA, ethylene antagonizes ABA and promotes sto�matal opening.
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Fig. 2. Effects of inhibitors and products of CuAO on sto�matal closure induced by ACC (a) and ethylene gas (b).Detached epidermal strips were incubated in CO2�freeMes/KCl buffer only or containing different compoundsunder light conditions (300 μmol/(m2 s) at 25 ± 2°C. After3 h, stomatal apertures were determined. In (a) and (b), thecompounds tested include ACC (150 μM), ethylene gas(ethy, 10 μL/L), AG (10 μM), BEA (10 μM), H2O2 (H,100 μM), γ�GABA (G, 100 μM), Succ (S, 100 μM), andNH3 (N, 100 μM). Other explanations are the same as inFig.1.
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Fig.3. Effects of ACC and ethylene gas on IWF–CuAO activity in V. faba leaves.Detached leaves were treated with CO2�free Mes/KCl buffer only or containing different compounds under light (300 μmol/(m2 s)conditions at 25 ± 2°C. After 3 h, IWF was isolated, and IWF–CuAO activity was determined. Data are the means of 9 measure�ments ± standard errors of three independent experiments. Here, the compounds tested include ACC (150 μM), ethylene (ethy,10 μL/L), AG (10 μM), and BEA (10 μM). Other explanations are the same as in Fig.1.
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Fig. 4. Effect of CuAO inhibitor on ethylene�induced H2O2 production.Guard cells of V. faba shown in image were treated with CO2�free Mes/KCl buffer alone (A), containing 150 μM ACC (B),10 μL/L ethylene gas (C), 10 μM AG (D), 150 μM ACC with 10 μM AG (E), 10 μL/L ethylene gas with 10 μM AG (F), 10 μMBEA (G), 150 μM ACC with 10 μM BEA (H), and 10 μL/L ethylene gas with 10 μM BEA (I) for 3 h in light (300 μmol/(m2 s)at 25 ± 2°C. Herein, treatment with ethylene gas was performed in a sealed environment. Epidermal strips were immediatelyloaded with H2DCF�DA for 10 min in darkness at 25 ± 2°C, then excess dye was removed and the strips were examined by laser�scanning confocal microscopy. (J) The average fluorescence intensity of guard cells in image (A–I), data are means ± standarderrora. Values in (J) with different letters are significantly different at P < 0.05. The guard cells shown in image (a�i) are the rep�resentative of guard cells shown in image (A�I). The insets show the bright�field image corresponding to the fluorescence image(a�i). Scale bars in image (I) and (i) represent 40 and 25 μm for images (A�I) and (a�i), respectively. The bar in the inset of image(i) represents 15 μm for all insets. Each experiment was repeated at least three times and the selected confocal image representedthe same results from approximately nine times measurements.
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INVOLVEMENT OF COPPER AMINE OXIDASE (CuAO)�DEPENDENT 395
H2O2 is the signaling molecule of widespreadimportance in plants, its role in stomatal movementhas been proved [4, 5, 7, 8, 19]. The enzymatic sourcesof H2O2 production in plants include plasma mem�brane�associated NADPH oxidase, cell wall peroxi�dases, and amine oxidases, CuAO in particular [9].Previous studies showed that NADPH oxidases Atr�bohD� and AtrbohF� and CuAO�dependent produc�tion of H2O2 are involved in ABA�induced stomatalclosure [14, 20]. The role of cell wall peroxidase�mediated H2O2 generation in ethylene�induced sto�matal closure has also been accepted [8]. Using Arabi�dopsis mutants and physiological approach, Desikanet al. [5] provided evidence that NADPH oxidase Atr�bohF�dependent H2O2 production is implicated instomatal closure induced by ethylene, and ethyleneand H2O2 signaling are mediated by ethylene receptorETR1 via a nature resistance�associated macrophage(N�RAMP)�like protein (EIN2)� and two�compo�nent response regulator protein (ARR2)�dependentpathways. However, whether CuAO�mediated H2O2production is involved in ethylene�induced stomatalclosure remains unknown. The present study showedthat the role of CuAO�dependent H2O2 productionwas crucial in ethylene�induced stomatal closure in V.faba. The following data support this conclusion: (1)ethylene stimulated CuAO activity, increased H2O2production, and induced stomatal closure; theseeffects of ethylene were prevented by CuAO inhibitors(Figs. 1, 2, 4B, 4C, 4E, 4F, 4H, 4I); (2) among majorcatalyzed and metabolized products (H2O2, NH3,GABA, and Succ) of CuAO, only H2O2 could inducestomatal closure and reverse the effect of CuAO inhib�itor on ethylene�induced stomatal closure (Fig. 2). Itis worthy to notice that in the present work, based onthe fact that 77.9% of total CuAO activity was detectedin IWF from V. faba leaves, only CuAO activity in theIWF was measured, whereas ethylene�induced H2O2production was located in the cytoplasm and chloro�plasts according to the H2DCE fluorescence (Figs. 4B,4C). As plasma membrane has been proposed to bepermeable for H2O2 [21], and the specific aquaporinsof Arabidopsis facilitate the diffusion of H2O2 acrossmembranes in a yeast system [22], we presume thatH2O2 generated by IWF�CuAO rapidly diffuses acrossthe plasma membrane into the cytoplasm and thenregulates stomatal movements in V. faba. According toour knowledge, this is the first report that CuAO andCuAO�mediated H2O2 production function in ethyl�ene�induced stomatal closure.
In summary, an ethylene signaling process in guardcell of V. faba is proposed. CuAO is activated inresponse to ethylene, which produces H2O2. Theincreased H2O2 production finally promotes stomatalclosure. However, the mechanism, by which CuAO isactivated by ethylene, remains unknown. Of course,the underlying mechanism, by which H2O2 generatedby CuAO induces stomatal closure, needs to be furtherinvestigated.
ACKNOWLEDGMENTS
Research is supported by Shaanxi Provincial Natu�ral Science Foundation for the Youth of China(2009JM3001), Fundamental Research Funds for theCentral Universities (GK200902037), and OpeningFoundation of Key Laboratory of Resource Biologyand Biotechnology in Western China (Northwest Uni�versity), Ministry of Education (ZS12006).
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