Evidence supporting nitrite-dependent NO release by the green microalga Scenedesmus obliquus

J Plant Physiol. Vol. 157. pp. 40-46 (2000) http://www.urbanfischer.de/journals/j pp

JOURNAL OF PLANT PHYSIOLOGY © 2000 URBAN & FISCHER Verlag

Evidence supporting nitrite-dependent NO release by the green microalga Scenedesmus ob/iquus Nirupama Mallick, Friedrich Helmuth Mohn, Carl J. Soeder Forschungszentrum Jiilich, leG-6, D-52425 Jiilich, Germany Received October 10, 1999 . Accepted December 15, 1999

Summary

Studies conducted with respiratory electron transport chain inhibitors and uncouplers such as antimycin A, rotenone, 2,4-dinitrophenol, pentachlorophenol and carbonyl cyanide m-chlorophenylhydrazone resulted in sudden NO bursts in Scenedesmus suspensions incubated under dark-aerobic conditions with supplemented glucose. Anaerobiosis was also found to increase NO production significantly in dark-incubated cells of Scenedesmus. These NO bursts, quite comparable to the usual
Key words: Scenedesmus, nitric oxide, MSX glyphosate, cycloheximide, pentachlorophenol, anaerobiosis. Abbreviations: NO = nitric oxide; NOS = nitric oxide synthase; MSX = methionine sulfoximine; NR = nitrate reductase; NiR = nitrite reductase, m-CCCP = carbonyl cyanide m-chlorophenylhydrazone. Pisum sativum (Leshem, 1996), Lupinus albus (Cueto et al., 1996), Neurospora crassa (Ninnemann and Maier, 1996), soyThe diatomic gas nitric oxide (NO) is synthesized in bio- bean (Delledone et al., 1998), Nicotiana tabacum (Durner et logical systems by a complex enzyme known as nitric oxide al., 1998; Huang and Knopp, 1998) and maize (Ribeiro et al., synthase (NOS) and is believed to be involved in a number of 1999). Furthermore, tobacco plants resistant to infection by physiological processes (see Fukuto and Chaudhuri, 1995). Ralstonia solanacearum exhibited elevated levels of NOS acAnalysis of the data from the literature shows that studies on tivity (Huang and Knopp, 1998). With immunoblot analysis, medico-biological aspects of NO embrace an unpreceden- antibodies made against rabbit brain NOS or mouse macrotedly wide region at the boundary between biochemistry and phase NOS were also found to react with tobacco and maize molecular biology, physiology and cellular biology as well as extracts that had high levels of NOS activity (Huang and pharmacology and roxicology. Recently, a novel pathway for Knopp, 1998; Ribeiro et al., 1999). In contrast, in our test NO production in humans was discovered that involves the alga, Scenedesmus obliquus, we failed to find a role for NOS in chemical reduction of inorganic nitrite, a reaction that takes nitric oxide biosynthesis (Mallick et al., 2000) when conductplace predominantly during acidic and reducing conditions ing experiments with the widely used potent inhibitors and (Weitzberg and Lundberg, 1998). substrates of NOS. The first report on nitric oxide research in plant sciences Taking recourse to the plant kingdom, production of NO by involvement of the enzyme nitric oxide synthase (NOS, was made by Klepper (1979), where he demonstrated the proEC 1.14.13.39) has been found in some plant genera such as duction of NO from herbicide-treated soybeans grown on Introduction

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41

Nitrite-dependent NO release by Scenedesmus

nitrate as the nitrogen source. He also pointed out that nitrite in NO formation in Scenedesmus obliquus (Mallick et a!., accumulation did occur in the leaf tissues prior to this gase- 1999). Nitrite was, however, found to be essential for NO ous emission. A subsequent study by Dean and Harper production by a hitherto unknown mechanism in the organ(1988) reported the role of constitutive NAD(P)H-nitrate ism under study. This study, therefore, provides some further reductase in converting nitrite to NO in soybean leaf extracts. evidence of nitrite-dependent NO production in the chloroYamasaki et a!. (1999) in a recent report also supported phycean microalga ScenedesmuJ obliquus, nitrate reductase (NR)-dependent NO production in maize plants. In sharp contrast to this, in their experimental system Materials and Methods with reversible conversion with the tungstate (W) of active Mo-NR into inactive W-NR, Mallick et al. (1999) failed to Experiments were conducted with axenic cultures of Scenedesmus produce NO in light or the most common
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Nirupama Mallick, Friedrich Helmuth Mohn, Carl J. Soeder

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(5 L stirred liquid volume) were continuously illuminated with two high-pressure mercury vapour lamps each (Osram HQI, 400 W), yielding 2,200 I1mol m- 2 sec- l in the centre of the empty culture vessel. Photolysis of nitrite as occurring in unfiltered light was reduced by 90 % by placing two 3 mm polymethacrylate sheets (Type GS 303 DIN 4102, Riihm GmbH, Darmstadt) as cut-off filters (transmission >410 nm) between the lamps and the illuminated algal cultures. To avoid carryover of nitrate or nitrite from previous incubation media to newly started experimental batches, the algal suspensions were always precipitated by vacuum on 0.2 11m Millipore filters, washed with sterile Ringer's solution under cleanbench conditions and re-incubated in fresh culture solution at an initial optical density (O.D.) of 0.14 ± 0.01 at 540 nm. Under standard conditions, the O.D. increased to about 0.56 within 24 h. Antimycin A and rotenone were selected as respiratory electron transport chain inhibitors. To inhibit protein synthesis cycloheximide was used. 2-4,-dinitrophenol, pentachlorophenol and carbonyl cyanide m-chlorophenylhydrazone were used as uncouplers. Anaerobiosis was per-

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Fig. 2: (a) Effect of 2,4-dinitrophenol (2mmoIL-1) on NO production by dark-grown glucose-supplemented cultures of Scenedesmus obliquus. (b) Same under illuminated conditions.

formed with nitrogen purging. Methionine sulfoximine, Basta, glutarate and glyphosate were used to inhibit various steps of the amino acid biosynthetic pathway. For measuring gaseous NO, ambient air was purified by charcoal and KMn04 (Purafil) and analysed for NO and N0 2 at the gas inlet and outlet of the algal cultures by a chemoluminiscence detection instrument (Ecophysics, Tecan CLD 770 AL PT) as described in Neubert et al. (1993). All experiments were repeated at least three times. The data presented were well reproducible qualitatively.

Results and Discussion As reported by Rai et al. (1999) and Mallick et al. (1999), the appearance of
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Nitrite-dependent NO release by Scenedesmus 14

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Since we also never observed this phenomenon when the alga was grown in ammonium- or urea-rich medium, we assumed a link between nitrite and NO production. A possible explanation for this could be that as soon as the algal cells are darkened, the sudden imbalance between nitrate reduction and nitrite reduction (as nitrite reduction comes to drastic reduction in the chloroplast due to lack of photosynthetic products in darkness) could be the cause of the instantaneous NO peak. Keeping in mind that the green algae are capable of respiration-linked nitrate reduction Gin et al., 1998) and continuing our earlier work where we observed a complete suppression of NO release in Scenedesmus cells incubated in the dark

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for 15 h, addition of glucose to these dark-incubated cells to boost respiration as well as the respiration-linked nitrate reduction resulted in a gradual rise in NO production concomitant with increased nitrite accumulation (Mallick et ai., 1999). We, therefore, further attempted to boost the cellular nitrite pool by indirect means. It is well known from the report ofYoneyama (1981) that anaerobic incubation (under an atmosphere of N 2 gas) enhanced accumulation of nitrite significantly under dark conditions. Inhibitors of the respiratory electron transport chain, i.e. antimycin A and rotenone, also stimulated nitrite accumulation, but under dark-aerobic conditions (Gray and Cresswell, 1984). These workers further observed an enhanced nitrate reduction and nitrite accumula-

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Nirupama Mallick. Friedrich Helmuth Mohn. Carl J. Soeder Basta 15

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tion in Zea mays following supplementation of respiratory uncouplers under dark-aerobic conditions. Interestingly. in our experimental system supplementation of respiratory electon transport chain inhibitors such as antimycin A and rotenone to the dark-grown glucose-supplemented suspension cultures of Scenedesmus resulted in instantaneous NO peaks

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Fig. 4: Basta-. MSX- and glyphosate-induced NO release by S. obliquus.

(see Fig. 1a. b). The Nypurged Scenedesmus cultures also showed an instant rise in NO production (data not shown). Furthermore, supplementation of uncouplers such as 2-4,dinitrophenol. pentachlorophenol and carbonyl cyanide m-chlorophenylhydrazone also resulted in instantaneous NO peaks under dark-aerobic conditions (data for DNP only

Nitrite-dependent NO release by Scenedesmus shown; Fig. 2a). No such effect was, however, observed when 2,4-dinitrophenol was supplemented in light (Fig. 2 b). Klepper (1976) in his experimental systems, i.e. with wheat and soybean leaves, also failed to observe accumulation of nitrite following DNP treatment in light. This could be due to the fact that the apparent stimulation of nitrate reduction by respiratory uncouplers occurs both in light and dark conditions, but it is not observed in the light where nitrite reductase is fully functional. This, therefore, supports our view of nitrite-mediated NO biosynthesis in the microalga Scenedes-

mus.

Supplementation of cycloheximide to inhibit protein synthesis vis-a-vis nitrite consumption (thereby resulting in nitrite accumulation) also gave rise to an instantaneous NO peak (Fig. 3). Furthermore, such NO peaks were also observed following treatment with methionine sulphoximine (MSX) and Basta (specific inhibitors of glutamine synthetase), glutarate (an inhibitor of glutamate dehydrogenase) and glyphosate (an inhibitor of EPSP synthetase of shikimate pathway) (see Figs. 3 and 4). Accumulation of nitrite following supplementation of glutarate and glyphosate to Scenedesmus cell cultures was also observed by Soeder et al. (unpublished data). Although we do not know the exact cause of nitrite accumulation under such conditions a feedback inhibition of ammonia utilization and, hence, of nitrite reduction and a build-up of nitrite seems more likely. These findings, therefore, again give support to our hypothesis that accumulation of nitrite is essential for NO production in the case of the chlorophycean microalga Scenedesmus. Based on our study we proposed a hypothetical model demonstrating the pathway for nitric oxide production in the case of the test alga Scenedesmus (Fig. 5 a, b). We suppose that in light only a small fraction of nitrite entering the chloroplast stroma (after nitrate reduction in the cytoplasm) is dissociated to NO + OH- by a reductant (Fig. 5 a). A large fraction of nitrite from the cytoplasm, however, can be released into the medium, thereby dissociating to NO by photolysis under wavelengths <410 nm (Fig. 5 a). In the case of sudden darkening, which promptly reduces nitrite reduction by NiR in chloroplast because of a lack of reduced ferredoxin, a fast rise in the intraplastidary nitrite pool is quite likely. This could cause an instantaneous «light-off" peak with a similar reaction with ,RH, (Fig. 5 b). We suspect the ,RH, could be ascorbate and/or glutathione. This is, of course, supported by the fact that both of these substances when added to nitrite solution separately in darkness (to avoid photolysis of nitrite in light) interacted chemically to produce NO (Table O. However, glutathione (GSH) showed far greater efficiency (10 times) than ascorbate in converting nitrite to NO. Though it is evident from the earlier reports of Foyer et al. (1983) and Bielawski and Joy (1986) that ascorbate and glutathione can accumulate to millimolar concentrations in photosynthetic tissues, it should be kept in mind that the intracellularlintraplastidary ascorbate and glutathione pools of the test organism were not measured in this study. According to the observations of Riens and Heldt (1992) in spinach leaves, a strong rise in nitrite concentration was observed immediately after darkening, which of course started decreasing after 2 min, showing a similarity to the normal

45

[AJ NO

NO

NOa- ...~. .

[B] NO NO

Fig. 5: Hypothetical scheme of reactions leading to nitrite-dependent formation of NO in a photosynthesizing algal cell. A: Main route of nitrogen assimilation from nitrate in the light, not showing photo respiration; the rounded compartment to the right represents the chloroplast. B: Assumed situation after sudden darkening. Ms = amino acids, Fd = ferredoxin (Fd- reduced, Fd+ oxidized), Prot. = protein, RH = unknown nitrite reductant, NR = nitrate reductase, NiR =nitrite reductase, circles = pools, squares =enzymes. Table 1: Induction of NO release from nitrite by ascorbate and glutathione (aerated medium NIl, 25·C, darkness). Trearment

NO release (ppb)

Nutrient solution N I I O.09±O.OI (6.4j.lmol L-I) O.l2±O.O] N 11 + nitrire (6.4 j.lmol L-I) + ascorbate (I mmol L-I) 'IO.5±O.51 N I I + nitrite (6.4j.lmoIL -I) + glutathione (I mmol L-I) *I07.3 ± 1.25 N I I + nitrite

, Maximal peak height. peak of nitrite disappeared in the absence of the functional NiR under dark conditions. Reaction with ,RH> to produce NO could be involved. In conclusion, nitrite accumulation is prerequisite for NO production in the case of the chloropycean microalga Scenedesmus.

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Nirupama Mallick, Friedrich Helmuth Mohn, Carl J. Soeder

Acknowledgements

We are grateful to the Alexander von Humboldt Foundation for granting a Fellowship to Nirupama Mallick.

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