ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 4, с. 580-584
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
УДК 581.1
AZIDE-DEPENDENT NITRIC OXIDE EMISSION FROM THE WATER FERN Azolla pinnata1 © 2014 S. Gurung*, M. F. Cohen**, H. Yamasaki*
*Faculty of Science, University of the Ryukyus, Nishihara, Japan **Department of Biology, Sonoma State University, Rohnert Park, USA Received April 4, 2013
Nitric oxide (NO) is involved in versatile functions in plant growth and development as a signaling molecule. To date, plants have been reported to produce NO following exposure to nitrite (NO2 ), the amino acid
L-arginine, hydroxylamine, or polyamines. Here we demonstrate azide-dependent NO production in plants. The water fern Azolla pinnata emitted NO into air upon exposure to sodium azide (NaN3). The NO production was dependent on azide concentration and was strongly inhibited by potassium cyanide (KCN). Incubation of A. pinnata with the catalase inhibitor 3-aminotriazole (3-AT) abolished the azide-dependent NO production. Although nitrite-dependent NO production was inhibited by sodium azide, azide-dependent NO production was not affected by nitrite. These results indicate that A. pinnata enzymatically produces NO using azide as a substrate. We suggest that plants are also capable of producing NO from azide by the action of catalase as previously reported in animals.
Keywords: Azolla pinnata - nitric oxide - nitrite - azide - catalase - ROS
DOI: 10.7868/S0015330314040083
INTRODUCTION
Nitric oxide (NO) is a gaseous signaling molecule that is involved in a multitude of physiological processes in plants [1] and animals [2]. In the late 1980s, it was discovered that NO is synthesized in animal cells by the enzyme nitric oxide synthase (NOS) using the amino acid L-arginine as the substrate (arginine pathway). Later, NO was found to also be produced from nitrite in animals by distinctive mechanisms (nitrite pathway) [3]. Since the regulation of NO production is important in physiological responses, the sources of NO and its production mechanisms are of particular interest in biology and medicine.
In plants, it has been shown that NO is produced from nitrite through enzymatic as well as non-enzymatic mechanisms, whereas the arginine pathway has yet to be elucidated due to the lack of a NOS homolog in plants [1]. Nitrate reductase (NR) is the first enzyme whose NO producing activity was confirmed in plants by both in vitro [4] and in vivo [5] studies. In contrast to the arginine pathway that is catalyzed by
1 The article is published in the original.
Abbreviations: 3-AT - 3-aminotriazole; NOS - nitric oxide synthase; NR - nitrate reductase.
Corresponding author: Hideo Yamasaki. Faculty of Science, University of the Ryukyus, Nishihara 903-0213, Japan. Fax: +81-98895-8576; e-mail: yamasaki@sci.u-ryukyu.ac.jp
NOS enzymes, the nitrite pathway involves multiple routes and mechanisms. More recently, many plant enzymes other than NR have been reported to produce NO from nitrite: peroxisomal xanthine oxidase
[6], plasma membrane bound nitrite:NO reductase
[7], and nonsymbiotic hemoglobin [8]. One electron reduction of nitrite by electron transport systems also produces NO in chloroplasts [9] and mitochondria [10]. In addition to these enzymatic mechanisms, non-enzymatic NO production in acidic and reducing environments, that may occur in the apoplast [11] and plastids [12], has physiological relevance.
Compounds other than L-arginine and nitrite have been shown to induce NO production in plants: the polyamines spermine and spermidine in Arabidopsis [13] and hydroxylamine in NR-free plant cells [14]. These studies imply that plants may have the potential to utilize a variety of chemicals to produce NO.
Sodium azide (NaN3) has been applied for research purposes as a vasodilator [15, 16], but it is cytotoxic because it inhibits a range of metal-containing enzyme activities [17]. It is now evident that the vasodilative activity of azide is due to its function as a precursor of NO in animals [16, 18]. However, there is no literature available to confirm the presence of azide-dependent NO production in plants.
In this study we used the floating fern Azolla pinnata as a plant model to investigate azide-dependent NO
AZIDE-DEPENDENT NITRIC OXIDE EMISSION FROM THE WATER FERN
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production. A. pinnata is a freshwater fern in symbiotic relationship with the nitrogen-fixing cyanobacterium Nostoc (Anabaena) azollae. Its small size and aquatic habitat made A. pinnata ideally suited for this study, facilitating the delivery of exogenous chemicals to the plants in solution. Employing the water fern, we show here the first experimental evidence to verify that plants are capable of producing NO from azide.
MATERIALS AND METHODS
Azolla pinnata was collected from a local taro field in Okinawa, Japan and surface-disinfected in a solution containing 0.12% (v/v) sodium hypochlorite and 0.01% (v/v) Triton X-100. The plants were then cultured in autoclaved cobalt-supplemented 40% nitrogen-free Hoagland E-medium under laboratory conditions (27 ± 1°C, a 16 h photoperiod, and light intensity of 50 ^mol/(m2 s)). For experiments, manually de-rooted fronds were cultured in nutrient medium that was replaced with a fresh-autoclaved one every four days. Details of the method of disinfection, composition of the culture medium, and plant growth conditions have been previously described [19].
A confluent layer of water ferns (10-13-day-old) enough to cover the surface area was placed in a 10-cm in diameter plastic Petri dish that contained 20 mL of 10 mM potassium phosphate buffer (pH 7.0). Two pinholes were made on the Petri dish cover: one at the side was for inserting the outlet pipe to the nitric oxide analyzer and the other on the top cover served as the inlet of air and various chemical solutions. To facilitate mixing of the medium, the Petri dish apparatus was rotated on a shaker.
NO was measured with a chemiluminescence technique that can monitor real-time emission of gaseous NO [20]. The measurement was carried out at room temperature (23 ± 1°C) with a Sievers Nitric Oxide Analyzer (NOA) 280i having a 50 mL/s intake flow rate and the data collected by NO Analysis software ("GE Analytical Instruments", United States). The sampling frequency was 8/s.
RESULTS
Figure 1 shows time courses of NO production in A. pinnata monitored by the chemiluminescence technique. Real-time measurements, as well as high specificity for NO, are advantages of the chemiluminescence technique [20]. In control experiments, the fronds of A. pinnata incubated only with phosphate buffer emitted negligible basal amounts of NO (<0.5 ppb). The signal of NO increased rapidly when azide (NaN3) was supplied into the medium (fig. 1). The initial rate of NO production and the extent of apparent steady-state level strongly depended on the concentrations of azide added (fig. 1, inset).
0 2 4 6 8 10 12 Time, min
Fig. 1. Time courses of azide-induced NO emission from A. pinnata.
Fronds of A. pinnata were placed onto a plastic Petri dish that contained 10 mM potassium phosphate buffer (pH 7.0). NO concentration of the headspace was monitored with a chemiluminescence technique. Sodium azide (NaN3) at various concentrations was added at the arrow indicated. The inset figure shows azide concentration dependence of NO emission from A. pinnata. The correlation and regression coefficients were R = 0.95 and R2 = 0.91, respectively.
We next examined whether azide-dependent NO production in A. pinnata arises from enzymatic or non-enzymatic (chemical) reactions. The effect ofen-zyme inhibitors was assessed to test for the involvement of enzymatic activity in the NO production (fig. 2). Cyanide is a strong inhibitor that binds to many metal-containing enzymes [21]. Figure 2a shows the effects of potassium cyanide (KCN) on azide-dependent NO production in A. pinnata. When cyanide was added to plants maintaining apparent steady-state production of NO, the NO production rapidly declined to the basal level. 5 mM KCN completely abolished the 1 mM azide-induced NO production (fig. 2a, trace 1). When the fronds were pre-treated with cyanide (5 mM) before the addition of azide (1 mM), we observed negligible NO production, which was identical to the basal level (fig. 2a, trace 2). The addition of cyanide alone did not induce NO production by A. pinnata (data not shown).
Figure 2b demonstrates effects of the catalase inhibitor 3-aminotriazole (3-AT) on azide-dependent NO production. When 3-AT was added to plants that had reached an apparent steady-state azide-depen-dent NO production, there was only a little inhibitory effect of the catalase inhibitor on NO production (fig. 2b, trace 1). Previous studies reported that the inhibitory effect of 3-AT on catalase activity in plants becomes apparent only after a long incubation time of
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GURUNG и др.
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Fig. 2. Effects of enzyme inhibitors on azide-dependent NO production in A. pinnata.
a - the effects of potassium cyanide (KCN) on the NO production. Trace 1 — time course of NO production induced by 1 mM NaN3. At the arrow indicated, 5 mM KCN was added. Trace 2 — NO production of the KCN-pretreated sample. Prior to the NO measurement, A. pinnata had been incubated with 5 mM KCN for 15 min; b — the effects of the catalase inhibitor 3-AT on azide-dependent NO production. Trace 1 — a condition similar to those in fig. 2a trace 1 except that 3-AT (10 mM) was added instead of KCN. Trace 2 — a condition similar to those in fig. 2a trace 2 except that the 15 min pre-incubation was made with 3-AT (10 mM) instead of KCN. Trace 3 — time course of NO production in A. pinnata pre-incubated with 3-AT (10 mM) for 3 h. Experimental conditions were similar to those in fig. 1.
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several hours [22]. Therefore, we incuba
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