ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 3, с. 419-425
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
YM 581.1
INVOLVEMENT OF COPPER AMINE OXIDASE (CuAO)-DEPENDENT HYDROGEN PEROXIDE SYNTHESIS IN ETHYLENE-INDUCED STOMATAL
CLOSURE IN Vicia faba1
© 2014 X. G. Song***, X. P. She*, M. Yue***, Y. E. Liu**, Y. X. Wang*, X. Zhu*, A. X. Huang*
*School of Life Sciences, Shaanxi Normal University, Xian, P.R. China **The High School affiliated to Shaanxi Normal University, Xian, P.R. China ***Opening Foundation of Key Laboratory of Resource Biology and Biotechnology in Western China, The College of Life Sciences, Northwest University, Xian, P.R. China Received January 18, 2013
Ethylene promotes stomatal closure via inducing hydrogen peroxide (H2O2) generation. H2O2 can be cata-lytically synthesized by several enzymes in plants. Here, by means of stomatal bioassay, the analysis of enzyme activity and using laser-scanning confocal microscopy based on the H2O2-sensitive probe 2\7'-dichlorodi-hydrofluorescein diacetate (H2DCF-DA), the roles of copper amine oxidase (CuAO) in ethylene-induced H2O2 production in guard cells and stomatal closure in Vicia faba L. were investigated. 1-aminocyclopro-pane-1-carboxylic acid (ACC), an immediate precursor of ethylene synthesis, and ethylene gas significantly activated CuAO in intercellular washing fluid (IWF) from leaves, the production of H2O2 in guard cells, and stomatal closure. These effects of ACC and ethylene gas were largely prevented by both aminoguanidine (AG) and 2-bromoethylamine (BEA), which are irreversible inhibitors of CuAO. Among major catalyzed and metabolized products of CuAO, only H2O2 could markedly promote stomatal closure and evidently reversed the effect of CuAO 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 (CuAO) - ethylene - hydrogen peroxide - stomatal closure
DOI: 10.7868/S001533031402016X
INTRODUCTION
The plant hormone ethylene plays an important regulatory role in plant growth and development. Although the role of ethylene on stomatal behavior has been suggested, its effect on this process seems rather contradictory [1]. In some species, ethylene induces stomatal opening or inhibits ABA-induced stomatal closure [2-4], whereas in other species ethylene induces stomatal closure [5]. Using Vicia faba leaf epidermal tissues, our previous works proved that ethyl-ene probably induces hydrogen peroxide (H2O2) removal, reduces H2O2 levels in guard cells, and finally inhibits stomatal closure induced by darkness [6]. The
1 This text was submitted by the authors in English.
Abbreviations: ACC - 1-aminocyclopropane-1-carboxylic acid; AG - aminoguanidine; BEA - 2-bromoethylamine; DCF -dichlorofluorescein; DMSO - dimethyl sulfoxide; ETH - ethep-hon; GABA - y-aminobutyric acid; H2DCF-DA - 2',7'-dichlo-rodihydro fluorescein diacetate; HRP - horseradish peroxidase; LSCM - laser scanning confocal microscopy; Put - putrescine; Succ - succinic acid.
Corresponding author: X. P. She. School of Life Sciences, Shaanxi Normal University, Xi'an 710062, P.R. China. Fax: +86-(0)29 8531-0546; e-mail: shexiaoping530@163.com
discrepancy of ethylene effects on stomatal behavior is probably associated with one or several factors: plant species, organ or tissue types, manners of ethylene treatment, physiological states of the tissue, and the concentrations of ethylene gas, ACC, or ethephon (ETH), which can be decomposed into ethylene, but there is no final conclusion, and the detailed mechanism is still under investigation.
Hydrogen peroxide (H2O2), a form of reactive oxygen species, is one of the common components of plant development processes and defense responses. The roles of H2O2 in stomatal closure and plant response to a wide variety of abiotic and biotic stresses are at present widely accepted [5, 7, 8]. It has been well known that H2O2 production in plants is achieved by several enzymatic systems, including amine oxidases [9]. Amine oxidases are represented by a heterogeneous group of enzymes, such as copper-containing diamine oxidase (CuAO; EC 1.4.3.6) and flavin-containing polyamine oxidase (EC 1.5.3.3). CuAO generally catalyzes the oxidation of aliphatic diamines pu-trescine (Put) and cadaverine at the primary amino groups [10]. The products from Put oxidation by
419
8*
420
SONG h gp.
CuAO are H2O2, NH3, and A1-pyrroline. A1-pyrroline is further catabolized to y-aminobutyric acid (GABA), which is subsequently transaminated and oxidized to succinic acid (Succ) [10, 11]. Increasing evidence suggests that H2O2 generated by CuAO participates in plant development and defense responses [10, 12]. It was reported that CuAO regulates lateral root development in soybean via its product H2O2 [13]. Recently, H2O2 generated by CuAO has been shown to mediate ABA-induced stomatal closure [14]. However, until now, it is unclear whether CuAO and its product H2O2 are involved in ethylene-regulated stomatal movements. In the present study, we investigated the role of H2O2 generated by CuAO during ethylene-induced stomatal closure in V. faba by means of stomatal bio-assay, the analysis of enzyme activity, and using laser-scanning confocal microscopy (LSCM) based on the molecular probe 2\7'-dichlorodihydrofluorescein di-acetate (H2DCF-DA).
MATERIALS AND METHODS
Plant materials. Broad bean (Vicia faba L.) plants were grown in controlled-environment plant growth chamber with a humidity of 70%, a photon flux density of 300 ^mol/(m2 s) PAR generated by cool white fluorescent tubes ("Philips", United States), a 14-h photoperiod, and an ambient temperature of 25 ± 2°C. The epidermis was peeled carefully from the abaxial surface of the youngest, fully expanded leaves of 4-week-old seedlings and cut into pieces about 5 mm width and 5 mm length.
Stomatal bioassay. Stomatal bioassay was performed 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 ^M CaCl2, pH 6.15) in the light (300 ^mol/(m2 s)) at 25 ± 2°C for 3 h. Once the stomata were fully open, the strips were treated with Mes/KCl buffer containing various compounds for further 3 h. Besides ACC and ethylene gas, the concentrations of other compounds tested were based on our preliminary experiments or other studies where the compounds were used [7, 14]. Control treatments involved the addition of appropriate solvents used with the compounds.
For treatment with ethylene gas, detached strips, on which stomata were fully open, were incubated in CO2-free Mes/KCl buffer in open Petri dishes in a gasimpermeable sealed Kilner jars, which were injected into with air or ethylene gas at various concentrations, under light (300 ^mol/(m2 s)) at 25 ± 2 °C for 3 h. After these steps, stomatal aperture was recorded with a light microscope and an eyepiece graticule previously calibrated with a stage micrometer. To avoid any potential rhythmic effects on stomatal aperture, experiments were always started at the same time of the day. In each treatment, we scored randomly 30 apertures, and every
treatment was repeated three times. The data presented are the means of 90 measurements ± standard errors.
Extraction and CuAO activity determination in the intercellular washing fluid (IWF) from leaves. The
treatment procedure of detached leaves was the same as that for detached strips, just as described in stomatal bioassay section. IWF was extracted using the method described in [15]. The leaves were weighted and vacuum infiltrated for 10 min at 1.0 kPa and 4°C in 50 mM K-phosphate buffer (pH 6.5) with 0.2 M NaCl and 0.1 mM CaCl2. Leaves were then quickly dried and centrifuged at 1000 g for 5 min at 4°C in a 10-mL syringe barrel placed in a 50-mL tube. After centrifuga-tion, IWF collected from the tube bottom was used to analyze CuAO activity.
The activity of CuAO in the IWF (IWF-CuAO) was determined according to the method described in [11] with minor modifications. Use of Put, as a substrate for IWF-CuAO activity determination, was based on a study with the same species [14]. Three milliliters of the reaction solution containing 1.9 mL of phosphate buffer (100 mM, pH 6.5) with 25 U/mL ofhorseradish peroxidase (HRP), 35 ^M 4-aminoantipyrine, and 1 mM 3,5-dichloro-2-hydroxybenzenesulphonic acid (DCHBS), 0.1 mL IWF, and 1 mL of 3 mM Put. The reaction was initiated by the addition of Put. After 30 min at 25°C, the changes in absorbance at 515 nm were recorded with a spectrophotometer. One unit of enzyme represents the amount of enzyme that catalyzes the oxidation of 1 ^mol Put/min. The activity was expressed as enzyme unit per gram of protein. Protein content was determined according to the method described in [16] with BSA as a standard. The determinations of CuAO activity and protein content were repeated three times. The data presented are the means of three measurements ± standard errors.
Measurement of H2O2 production. H2O2 production was monitored with H2DCF-DA, as described in [6] with minor modications. To study the effect of CuAO inhibitor on ACC- or ethylene gas-induced H2O2 production, the epidermal strips were treated as described in stomatal bioassay section and then loaded with 50 ^M H2DCF-DA (10 min) in Tris-KCl loading buffer (Tris 10 mM, KCl 50 mM, pH 7.2) in darkness at 25 ± 2°C. After excess dye was washed off with fresh Tris-KCl buffer in darkness, the strips were immediately examined by TCS SP5 laser-scanning confocal microscopy ("Leica Lasertechnik", Germany) with the following settings: excitation 488 nm, emission 530 nm, power 10%, zoom about 4, normal scanning speed and frame 512 x 512 pixel. Images acquired from the confocal microscope were analyzed with Leica image software and processed with Photoshop 7.0. To enable the comparison of changes in signal intensity, confocal images were
Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.