ISSN 10214437, Russian Journal of Plant Physiology, 2014, Vol. 61, No. 4, pp. 543–547. © Pleiades Publishing, Ltd., 2014. RESEARCH PAPERS AzideDependent Nitric Oxide Emission from the Water Fern Azolla pinnata1 S. Gurunga, M. F. Cohenb, and H. Yamasakia a Faculty of Science, University of the Ryukyus, Nishihara 9030213, Japan; fax: +81988958576; email: [email protected] b Department of Biology, Sonoma State University, Rohnert Park, CA 94928, USA Received April 4, 2013 Abstract—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 Larginine, hydroxylamine, or polyamines. Here we demonstrate azidedependent 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 3aminotriazole (3AT) abolished the azidedependent NO production. Although nitritedependent NO production was inhibited by sodium azide, azidedepen dent 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: 10.1134/S1021443714040086 1 INTRODUCTION duce NO from nitrite: peroxisomal xanthine oxidase [6], plasma membrane bound nitrite:NO reductase Nitric oxide (NO) is a gaseous signaling molecule [7], and nonsymbiotic hemoglobin [8]. One electron that is involved in a multitude of physiological pro reduction of nitrite by electron transport systems also cesses in plants [1] and animals [2]. In the late 1980s, produces NO in chloroplasts [9] and mitochondria it was discovered that NO is synthesized in animal cells [10]. In addition to these enzymatic mechanisms, by the enzyme nitric oxide synthase (NOS) using the nonenzymatic NO production in acidic and reducing amino acid Larginine as the substrate (arginine path environments, that may occur in the apoplast [11] and way). Later, NO was found to also be produced from plastids [12], has physiological relevance. nitrite in animals by distinctive mechanisms (nitrite Compounds other than Larginine and nitrite have pathway) [3]. Since the regulation of NO production is been shown to induce NO production in plants: the important in physiological responses, the sources of polyamines spermine and spermidine in Arabidopsis NO and its production mechanisms are of particular [13] and hydroxylamine in NRfree plant cells [14]. interest in biology and medicine. These studies imply that plants may have the potential In plants, it has been shown that NO is produced to utilize a variety of chemicals to produce NO. from nitrite through enzymatic as well as nonenzy Sodium azide (NaN3) has been applied for research matic mechanisms, whereas the arginine pathway has purposes as a vasodilator [15, 16], but it is cytotoxic yet to be elucidated due to the lack of a NOS homolog because it inhibits a range of metalcontaining enzyme in plants [1]. Nitrate reductase (NR) is the first activities [17]. It is now evident that the vasodilative enzyme, whose NO producing activity was confirmed activity of azide is due to its function as a precursor of in plants by both in vitro [4] and in vivo [5] studies. In NO in animals [16, 18]. However, there is no literature contrast to the arginine pathway that is catalyzed by available to confirm the presence of azidedependent NOS enzymes, the nitrite pathway involves multiple NO production in plants. routes and mechanisms. More recently, many plant In this study we used the floating fern Azolla pin enzymes other than NR have been reported to pro nata as a plant model to investigate azidedependent 1 The article is published in original. NO production. A. pinnata is a fresh water fern in sym biotic relationship with the nitrogenfixing cyanobac Abbreviations: 3AT—3aminotriazole; NOS—nitric oxide syn terium Nostoc (Anabaena) azollae. Its small size and thase; NR—nitrate reductase. aquatic habitat made A. pinnata ideally suited for this 543 544 GURUNG et al. mixing of the medium, the Petri dish apparatus was 200 rotated on a shaker. 150 200 NO was measured with a chemiluminescence tech 100 nique that can monitor realtime emission of gaseous 50 NO emission, NO [20]. The measurement was carried out at room 0 ± nmol/(g dry wt min) temperature (23 1°C) with a Sievers Nitric Oxide 160 10–5 10–3 10–1 Concentration 10 mM Analayzer (NOA) 280i having a 50 mL/s intake flow of NaN3, M rate and the data collected by NO Analysis software 120 (GE Analytical Instruments, United States). The 1 mM sampling frequency was 8/s. 80 NO emission, ppb 0.05 mM RESULTS 40 0 mM Figure 1 shows time courses of NO production in A. pinnata monitored by the chemiluminescence tech NaN3 nique. Realtime measurements, as well as high spec ificity for NO, are advantages of the chemilumines 0 2 4 6 8 10 12 Time, min cence 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 Fig. 1. Time courses of azideinduced NO emission from A. pinnata. signal of NO increased rapidly when azide (NaN3) was Fronds of A. pinnata were placed onto a plastic Petri dish supplied into the medium (Fig. 1). The initial rate of that contained 10 mM potassium phosphate buffer NO production and the extent of apparent steady (pH 7.0). NO concentration of the headspace was moni state level strongly depended on the concentrations of tored with a chemiluminescence technique. Sodium azide azide added (Fig. 1, inset). (NaN3) at various concentrations was added at the arrow indicated. The inset figure shows azide concentration We next examined whether azidedependent NO dependence of NO emission from A. pinnata. The correla production in A. pinnata arises from enzymatic or tion and regression coefficients were R = 0.95 and R2 = nonenzymatic (chemical) reactions. The effect of 0.91, respectively. enzyme inhibitors was assessed to test for the involve ment of enzymatic activity in the NO production (Fig. 2). Cyanide is a strong inhibitor that binds to many metal study, facilitating the delivery of exogenous chemicals containing enzymes [21]. Figure 2a shows the effects to the plants in solution. Employing the water fern, we of potassium cyanide (KCN) on azidedependent NO show here the first experimental evidence to verify that production in A. pinnata. When cyanide was added to plants are capable of producing NO from azide. plants maintaining apparent steadystate production of NO, the NO production rapidly declined to the basal level. 5 mM KCN completely abolished the MATERIALS AND METHODS 1 mM azideinduced NO production (Fig. 2a, trace 1). Azolla pinnata was collected from a local taro field When the fronds were pretreated with cyanide (5 mM) in Okinawa, Japan and surfacedisinfected in a solu before the addition of azide (1 mM), we observed neg tion containing 0.12% (v/v) sodium hypochlorite and ligible NO production, which was identical to the 0.01% (v/v) Triton X100. The plants were then cul basal level (Fig. 2a, trace 2). The addition of cyanide tured in autoclaved cobaltsupplemented 40% nitro alone did not induce NO production by A. pinnata genfree Hoagland Emedium under laboratory con (data not shown). ditions (27 ± 1°C, a 16 h photoperiod, and light inten Figure 2b demonstrates effects of the catalase sity of 50 μmol/(m2 s). For experiments, manually de inhibitor 3aminotriazole (3AT) on azidedependent rooted fronds were cultured in nutrient medium that NO production. When 3AT was added to plants that was replaced with a freshautoclaved one every four had reached an apparent steadystate azidedepen days. Details of the method of disinfection, composi dent NO production, there was only a little inhibitory tion of the culture medium, and plant growth condi effect of the catalase inhibitor on NO production tions have been previously described [19]. (Fig. 2b, trace 1). Previous studies reported that the A confluent layer of water ferns (10–13dayold) inhibitory effect of 3AT on catalase activity in plants enough to cover the surface area was placed in a 10cm becomes apparent only after a long incubation time of in diameter plastic Petri dish that contained 20 mL of several hours [22]. Therefore, we incubated the plants 10 mM potassium phosphate buffer (pH 7.0). Two with 3AT (10 mM) for 15 min, 1, 3, and 5 h to evalu pinholes were made on the Petri dish cover: one at the ate its effect. In good agreement with previous reports side was for inserting the outlet pipe to the nitric oxide on the inhibition of catalase, azidedependent NO analyzer and the other on the top cover served as the production was strongly suppressed depending on the inlet of air and various chemical solutions. To facilitate incubation time with 3AT (Fig. 2b, traces 2 and 3). RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 61 No. 4 2014 AZIDEDEPENDENT NITRIC OXIDE EMISSION 545 (a) KCN (b) 3AT 1 1 NaN3 NaN3 80 2 60 2 40 NaN3 NaN3 20 3 0 0 5 10 15 20 NO emission, ppb Time, min NaN3 Fig. 2. Effects of enzyme inhibitors on azidedependent 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 KCNpretreated sample.