JXB Advance Access originally published online on August 7, 2006
Journal of Experimental Botany 2006 57(12):3043-3055; doi:10.1093/jxb/erl070
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RESEARCH PAPER |
Nitric oxide (NO) detection by DAF fluorescence and chemiluminescence: a comparison using abiotic and biotic NO sources
Julius-von-Sachs Institute for Biosciences, University of Würzburg, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany
*To whom correspondence should be addressed. E-mail: kaiser{at}botanik.uni-wuerzburg.de
Received 20 March 2006; Accepted 30 May 2006
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Abstract |
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Because of controversies in the literature on nitric oxide (NO) production by plants, NO detection by the frequently used diaminofluorescein (DAF-2 and DAF-2DA) and by chemiluminescence were compared using the following systems of increasing complexity: (i) dissolved NO gas; (ii) the NO donor sodium nitroprusside (SNP); (iii) purified nitrate reductase (NR); and (iv) tobacco cell suspensions. Low (physiological) concentrations (
1 nM) of dissolved NO could be precisely quantified by chemiluminescence, but caused no DAF-2 fluorescence. In contrast to NO gas, SNP, NR, or cell suspensions produced both good DAF fluorescence and chemiluminescence signals which were completely (chemiluminescence) or partly (DAF fluorescence) prevented by NO scavengers. Signal strength ratios between the two methods were variable depending on the NO source, and eventually reflect variable NO oxidation. DAF fluorescence in cell suspension cultures was also increased by an as yet unidentified compound(s) released from cells into the medium. These compounds gave no chemiluminescence signal and were not produced by NR-free mutants. Their production was stimulated by anoxia, by inhibitors of mitochondrial electron transport, and by the fungal elicitor cryptogein. Thus, changes in DAF fluorescence are not necessarily indicative for NO production, but may also reflect NO oxidation and/or production of other DAF-reactive compounds. Key words: Anoxia, chemiluminescence, cryptogein, DAF fluorescence, mitochondria, nitrate reductase, nitric oxide, NO oxidation
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Introduction |
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Various methodological approaches have been used to analyse nitric oxide (NO) production in biological samples and also in plants. Most of them detect NO only after it has diffused out of the cell or tissues, or they require cell extraction, such as the haemoglobin method (Delledonne et al., 1998; Clarke et al., 2000; Orozco-Cárdenas and Ryan, 2002), electron spin resonance based on non-permeating spin traps (Pagnussat et al., 2002; Huang et al., 2004; Modolo et al., 2005), chemiluminescence (gas phase) (Rockel et al., 2002; Planchet et al., 2005), mass spectrometry (Conrath et al., 2004), amperometric methods with NO-specific electrodes (Yamasaki and Sakihama, 2000), or laser-photoacoustics (Leshem and Pinchasov, 2000; Mur et al., 2005). Only the frequently used fluorophore diaminofluorescein (DAF) in its cell-permeable forms (DAF-2DA or DAF-FM DA) (Kojima et al., 1998a, b; Itoh et al., 2000) is thought to indicate NO production inside cells (Guo et al., 2003; Zeidler et al., 2004), where DAF-2 is N-nitrosated forming the highly fluorescing triazolofluorescein (DAF-2T). It has been suggested that DAF-2 does not react directly with the NO free radical, but rather with N2O3 (nitrous anhydride) (Kojima et al., 1998a; Nakatsubo et al., 1998; Espey et al., 2001). The latter may be formed by autoxidation of NO in air according to reaction (1), and in aqueous solution will result in nitrite formation (reaction 2) (Espey et al., 2001)
 | (1) |
 | (2) |
It was of interest to identify sources of NO and to quantify NO production in whole plants, organs, and cell suspensions, specifically under anoxia and during plant–pathogen interactions (Tischner et al., 2004; Gupta et al., 2005; Planchet et al., 2005, 2006). For these investigations, chemiluminescence detection of NO emitted into the gas phase was largely used. However, the data obtained so far with chemiluminescence and data published by many others based on DAF fluorescence (Foissner et al., 2000; Krause and Durner, 2004; Lamotte et al., 2004; Yamamoto et al., 2004; Prats et al., 2005), but also on laser-photoacoustics (Mur et al., 2005), mass spectrometry (Conrath et al., 2004), or haemoglobin (Delledonne et al., 1998; Clarke et al., 2000; Xu et al., 2005), were partly contradictory. Briefly, literature reports show that NO is produced in response to elicitors or pathogens as a transient burst, but also continuously in later phases, and that this NO triggers a sequence of events leading ultimately to defence including programmed cell death. However, using chemiluminescence detection, no or very little NO emission from tobacco leaves (Nicotiana tabacum cv. Xanthi) elicited with the fungal peptide cryptogein (Planchet et al., 2006) or with avirulent bacteria (Pseudomonas syringae pv. tomato, unpublished results) was found. Accordingly, the importance of NO as a second messenger in plant–pathogen interactions has been questioned (Planchet et al., 2006).
Here, in order to solve the contradictions, DAF fluorescence and chemiluminescence were systematically compared using NO gas, a chemical NO donor, an NO-producing enzyme, or intact cell suspensions. Further, NO scavenging was examined by cPTIO [carboxy-PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide]. It is shown that chemiluminescence detects dissolved NO in low concentrations not registered by DAF fluorescence. It is also shown that DAF reacts with as yet unidentified stable compounds produced and released by cells under certain conditions.
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Materials and methods |
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Cell cultures and treatments
The cell suspension derived from tobacco (Nicotiana tabacum cv. Xanthi) was cultured in 300 ml Erlenmeyer flasks containing 100 ml of LS medium pH 5.8 (Linsmaier and Skoog, 1965) at a constant temperature of 24 °C and a continuous illumination with fluorescent tubes, on a rotary shaker (New Brunswick Scientific, NJ, USA) at 100 rpm. Subcultures were made weekly by transferring 20 ml of the cell suspension into 80 ml of fresh growth medium. Three days after subculturing, cells were used for the experiments. In specific cases, NR-free tobacco cells, Nia30 cells, which are totally devoid of NR activity, were grown in an MS medium with small modifications, i.e. 3 mM NH4Cl instead of 20 mM NH4NO3, 10 mM KNO3, 3 mM (NH4)2SO4, and the MES concentration was 10 mM in order to maintain the pH at
6.2.
Cells from cultures in the exponential growth phase (subcultured after 3 d) were washed and resuspended with a medium containing only the macro-elements (5 mM KNO3 or 3 mM NH4Cl for nitrate-grown or Nia30 cells, respectively, 2 mM CaCl2, 1.5 mM MgSO4, 1.25 mM KH2PO4), sucrose, and 2.35 mM MES in order to maintain the pH at 6.5. The cells were adjusted to a cell density of 0.1 g FW ml–1. Before any treatment with myxothiazol (10 µM)+salicylhydroxamic acid (SHAM; 2.5 mM), with cryptogein (20 nM), or incubation under nitrogen, in the presence or absence of the NO scavenger (cPTIO; 500 µM), cells were allowed to adjust to the new conditions for a period of 30 min to 1 h.
NO measurements
For experiments with cell suspensions or solutions, a defined liquid volume (1–10 ml) was placed in a Petri dish in a transparent glas cuvette (1.0 l) mounted on a shaker. A constant flow of measuring gas (pressurized air or nitrogen) of 1.3 l min–1 was pulled through the cuvette and subsequently through the chemiluminescence detector (CLD 770 AL ppt, Eco-Physics, Dürnten, Switzerland) by a vacuum pump connected to an ozone destroyer. The ozone generator of the chemiluminescence detector was supplied with dry oxygen (99%). The measuring gas (air or nitrogen) was made NO free by conducting it through a custom-made charcoal column (1 m long, 3 cm internal diameter, particle size 2 mm). The detector was adjusted to 20 s time resolution. Calibration was routinely carried out with NO-free air and with various concentrations of NO (1–35 ppb) adjusted by mixing the calibration gas (500 ppb NO in nitrogen, Messer Griesheim, Darmstadt, Germany) with NO-free air. Flow controllers (FC-260, Tylan General, Eching, Germany) were used to adjust all gas flows. The air temperature in the cuvette was continuously monitored, and was usually 24 °C in the dark or at room light.
For fluorometric NO determination, the fluorophore 4,5-diamino-fluorescein (DAF-2), the cell-permeable diacetate (DAF-2DA), or, occasionally, diaminorhodamine-4M (DAR-4M) was used (Alexis Biochemicals, Gruenberg, Germany). Filtrated and washed cell suspensions were pre-incubated with 10 µM DAF-2DA for 15 min at 24 °C in darkness on a rotary shaker (100 rpm) and then rinsed with fresh suspension buffer to remove excess fluorophore. Alternatively, DAF-2 (10 µM) was added to the solutions or suspensions. Aliquots (1 ml) of the solutions or suspensions were sampled at different times after incubation, and DAF-2T fluorescence was measured using a spectrofluorometer (SPF-500TM Ratio, American Instrument Company, MD, USA) with 495 nm excitation and 515 nm emission wavelength (2 nm band width). Fluorescence was expressed as arbitrary fluorescence units (AU), and was measured at the same instrument settings in all experiments.
Where indicated, DAF fluorescence from cells pre-loaded with DAF-2DA was also measured in cell extracts. For that purpose, the previously measured 1 ml aliquots were gently centrifuged and the supernatant was removed. A 1 ml aliquot of 100 mM HEPES pH 7.5 was added to the cells, which were then frozen in liquid nitrogen and thawed. The resulting cell homogenate was centrifuged (10 min at 16 000 g, 4 °C), and fluorescence was again measured in the clear supernatant.
Similarly, formation of fluorescing DAF derivates by compounds released from the cells was measured by adding DAF-2 (10 µM) to the supernatant of intact cell suspensions previously incubated under the conditions indicated in the figure legends. The supernatant was collected by gentle centrifugation (500 g) of an aliquot of the cell suspension and was additionally cleared by filtration through PET-membrane filters (15 mm diameter, 0.45 µm, Roth, Karlsruhe, Germany).
Nitrite determination
Separate aliquots (400 µl) of the above cell suspensions or solutions were sampled and quickly mixed with a reaction mixture containing: 600 µl sulphanilamide (1%), 600 µl of N-(1)-(naphthyl)ethylene-diaminedihydrochloride (0.02%), and 300 µl of zinc acetate (0.5 M). After 25 min of incubation at 24 °C, the mixture was cleared by centrifugation (16 000 g, 5 min), and the nitrite content from the supernatant was determined photometrically (Hageman and Reed, 1980).
Generation of NO and NO titration experiments
Stock solutions of the NO donor SNP (sodium nitroprusside, 10 mM) or GSNO (S-nitrosoglutathione, 200 mM) were freshly prepared for each experiment and stored on ice until use. NO gas (100 ppm in nitrogen; Messer-Griesheim, Darmstadt, Germany) was flushed for 15 min at 24 °C through a glass vial containing buffer (HEPES-KOH 100 mM, pH 7.5). According to the solubility of pure NO in water at atmospheric pressure (1.9 mM), the NO equilibrium concentration of the solution flushed with 100 ppm NO is 190 pmol ml–1. For the chemiluminescence measurement, aliquots of the freshly prepared solution were sampled with a syringe (1 ml) fitted with a stainless steel needle, and immediately injected into a small beaker mounted in the NO cuvette, through a gas-tight serum stopper, without interrupting the NO measurement. For DAF fluorescence, the same amount of the NO solution was added to 10 µM DAF-2 and fluorescence was measured after 30 min, as described above.
Chemicals
The cell-permeating NO scavenger cPTIO and the non-cell-permeating water-soluble NO scavenger tmaPTIO (trimethylammonio-PTIO,3,3,4,4-tetramethyl-2-trimethylammonio-phenyl-2-imidazoline-3-oxide-1-yloxy chloride) were purchased from Alexis Biochemicals (Gruenberg, Germany). Myxothiazol and SHAM were from Sigma-Aldrich (Taufkirchen, Germany). The NO-donor SNP and potassium cyanide (KCN) were from Merck (Darmstadt, Germany), and GSNO from Calbiochem (Schwalbach/Ts, Germany). Purified maize NR was from The Nitrate Elimination Company Inc. (Lake Linden, MI, USA).
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Results |
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Dissolved NO gas
Chemiluminescence measures light emitted by the reaction of NO with ozone in a given gas volume. In the instrumental set up used here, a constant gas flow of 1.3 l min–1 through the reaction chamber was used at 20 s measuring intervals. Under these conditions, the detection limit (2x above the mean of the noise) of NO in air was at or below 10 ppt. In contrast to chemiluminescence, DAF-2 at constant NO concentration in the reaction medium should give a constant increase (slope) of fluorescence over time, reflecting a constant rate of the formation of the highly fluorescing reaction product, DAF-2T. Changes in the rate of NO production result in changes in the slope of the fluorescence increase.
First, the response of both methods to dissolved NO gas was compared. In order quickly to add a defined amount of NO to the reaction mixture in the chemiluminescence cuvette, a buffer solution was flushed for 15 min with NO gas [100 ppm (or occasionally 1% in nitrogen)]. Aliquots (0.1–1 ml) of the equilibrium solutions were rapidly injected into a small beaker with buffer solution mounted in the headspace cuvette on a magnetic stirrer. Under these conditions, NO was quickly and transiently released into purified air or under nitrogen and was detected by chemiluminescence (Fig. 1A). The NO scavenger cPTIO prevented 95% of the chemiluminescence signal (Fig. 1A). The sum of all measuring points of a single emission curve gives the total amount of NO released into NO-free air. These integrated amounts were practically identical to the theoretical NO content of the equilibrium solution calculated from the solubility of pure NO in water [1.9 mM at atmospheric pressure and 22 °C (inset in Fig. 1A)], indicating that (i) NO detection by chemiluminescence was quantitative already at NO concentrations in the pM range and (ii) NO oxidation was negligible.
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When similar concentrations of NO (
1 nM) were injected into a solution of DAF-2, and allowed to react for 30 min in a closed cuvette, in air or under nitrogen, DAF fluorescence was hardly detectable (Fig. 1B). Thus, chemiluminescence generally appeared much more sensitive than DAF fluorescence. Only solutions flushed with much higher NO concentrations (1%) gave good DAF fluorescence (not shown). These solutions (after 10–15 min flushing) also accumulated nitrite (not shown), indicating that under these conditions NO was oxidized according to equations (1) and (2).
NO donor (SNP)
Next, the two methods were compared by following continuous NO production in solutions of a frequently used NO donor, SNP (Stamler et al., 1992, and references therein). Freshly prepared SNP solutions were rapidly injected (at room light) into a buffer solution to give a final concentration of 10 µM. NO emission from SNP, measured by chemiluminescence, rapidly reached a low rate of 5.5 pmol ml–1 min–1 which was constant over a time span of several hours (only 1 h is shown in Fig. 2A). While the chemiluminescence signal was rather low, DAF-2 fluorescence increased linearly with time (Fig. 2B). A rate of 1 nmol NO min–1 ml–1 measured by chemiluminescence corresponded to an increase of fluorescence of 0.286 AU min–1. Apparently the donor solution produced NO and oxidized DAF-reactive derivatives at the same time.
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In this experiment, the effect of the widely used NO scavenger cPTIO, which reacts with NO by converting it to NO2 (3), was also examined.
 | (3) |
 | (4) |
NO2 may react with NO to give N2O3 (4), which, as discussed above, is the substrate that nitrosates DAF-2 to DAF-2T. When measured by chemiluminescence, 500 µM cPTIO completely prevented NO production by SNP (Fig. 2A), whereas SNP-dependent DAF fluorescence was scavenged by only 85% (Fig. 2B). SNP produced not only NO, but also nitrite (Fig. 2B, columns), and nitrite production was higher when cPTIO was present (not shown), as expected from reactions (1–3) (compare Pfeiffer et al., 1997).
The NO donor GSNO was also examined and it gave ratios of fluorescence versus chemiluminescence similar to SNP (not shown).
NO from purified NR
NR is probably the prevailing NO source in higher plants. Normally, it reduces nitrate to nitrite via a two-electron transfer, and with a much lower capacity it may also catalyse a one-electron transfer from nitrite to NO, at the expense of NADH (Rockel et al., 2002; Planchet et al., 2005). Similarly, oxygen is reduced to superoxide anion which may rapidly react with NO to yield peroxynitrite (Yamasaki and Sakihama, 2000).
When measured by chemiluminescence, the NO concentration in the gas stream above a solution of highly purified maize NR containing NADH and nitrite increased continuously until a constant rate was achieved, which in Fig. 3A corresponded to 0.293 nmol NO ml–1 min–1. Consistent with that, after an initial lag phase, DAF fluorescence increased steadily (Fig. 3B), confirming a constant rate of NO and N2O3 formation. In contrast to what was observed with the NO donor SNP, an NO production rate of 1 nmol min–1 ml–1 from NR measured by chemiluminescence corresponded to a fluorescence increase of only 0.033 AU min–1, suggesting that only a small fraction of NR-derived NO was oxidized to the DAF-reactive N2O3. When NR was blocked with cyanide, both methods indicated a rapid and complete cessation of NO production (Fig. 3A, B). Addition of the NO scavenger tmaPTIO completely prevented NO detection by both methods (Fig. 3A, B).
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Tobacco cell suspensions
In contrast to the above simple systems, intact cells are far more complex because they may produce NO by different sources and in different subcellular compartments, and because NO or its congenitors produced inside cells may partly react with haem groups, thiols, and lipids, or with reactive oxygen species, such as superoxide or hydrogen peroxide (Stamler et al., 1992; Pryor and Squadrito, 1995; Koppenol, 1998; Wink and Mitchell, 1998; Henry and Guissani, 1999). Thus, only a small part of the NO produced might actually diffuse out of cells into the medium and into the gas phase of the chemiluminescence apparatus (or other detection systems; see Introduction). The cell-permeable fluorescence indicator DAF-2DA should be much closer to the sites of NO production and oxidation. As DAF may be differentially distributed between subcellular compartments, and because the ionic composition in these compartments may affect fluorescence (Kojima et al., 1998a; Nagata et al., 1999; Zhang et al., 2002; Rodriguez et al., 2005), DAF-2T fluorescence from cell suspensions was measured in two different ways: first, the fluorescence was determined in a suspension of the DAF-2DA-pre-loaded intact cells. Then, the cells were ruptured by a freeze/thaw cycle and the insoluble material was removed by centrifugation. DAF-2T fluorescence could then be measured in the clear (buffered) supernatant at almost constant ionic composition and pH and at better optical conditions.
As shown previously by chemiluminescence measurements, tobacco cell suspensions emit very little NO under normal aerated conditions, even when supplied with nitrite as substrate for NO production. Using chemiluminescence detection, conditions provoking high NO production have previously been defined, which were (i) a chemical inhibition of respiration in air or (ii) inhibition of respiration by anoxia (Gupta et al., 2005; Planchet et al., 2005). These conditions have now been used to compare the two methods for quantitative NO detection. The fungal elicitor cryptogein, which caused only a minor and delayed NO production from cell suspensions, as measured previously by chemiluminescence (Planchet et al., 2006), was also used. In order to evaluate the contribution of nitrate and nitrate reduction to NO production, experiments were also carried out with cell suspensions from a completely NR-free Nia double mutant.
Induction of NO production by elicitors or by pathogens has been well established using various NO detection methods (Delledonne et al., 1998; Clarke et al., 2000; Lamotte et al., 2004; Yamamoto et al., 2004; Mur et al., 2005; Prats et al., 2005). However, as mentioned above, in previous experiments the fungal elicitor cryptogein caused no or only a small NO emission which appeared only several hours after elicitor addition, whereas DAF-2DA fluorescence increased almost immediately (Planchet et al., 2006). These different kinetic responses of the two methods were now examined in more detail (Fig. 4). As expected, in the first 2 h following elicitor addition, chemiluminescence gave no signal (Fig. 4A). In contrast, an immediate increase of DAF fluorescence took place with DAF-2DA-pre-loaded cells, and this fluorescence was prevented by cPTIO (Fig. 4B). In NR-free Nia cells treated with cryptogein, no increase of fluorescence could be observed (not shown), indicating that fluorescence obtained with wild-type (WT) cells was somehow related to nitrate reduction (also compare Planchet et al., 2006). Importantly, there was no basic difference when fluorescence was measured in vivo (Fig. 4B) or after extracting the pre-incubated cells (Fig. 4C), except that the cell-free extract always gave higher fluorescence levels, probably due to the better optical conditions. This also shows that the fluorescence increase was not due to artefacts of the ionic environment (pH) of the cell cytoplasm. Nitrite concentrations in the cell suspension increased significantly 2 h after cryptogein addition (columns in Fig. 4B).
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It has been shown previously that reduction of nitrite to NO by mitochondrial electron transport is almost completely blocked by the inhibitors myxothiazol and SHAM, which act on complex III and on alternative oxidase (AOX), respectively. For a complete inhibition of respiratory electron transport (not shown), it was necessary to add both inhibitors together (compare Gupta et al., 2005). Addition of the inhibitors produced a transient peak of the chemiluminescence signal, followed by low and constant NO emission (Fig. 5A). The inhibitors also induced the transient NO emission in Nia30 cells, whereas the long-lasting second phase of NO emission observed with WT cells was completely lacking with Nia30 (Fig. 5B).
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With DAF-2DA-pre-incubated WT cells, a strong linear increase in fluorescence was triggered by the inhibitors, but this increase was only weakly responsive to cPTIO (Fig. 5C). Nitrite concentrations in the cell suspension were almost undectable at T=0, but increased drastically when the inhibitors were added (columns in Fig. 5C). DAF-2-DA fluorescence from Nia30 cells was very low (Fig. 5D). Thus, DAF fluorescence appeared consistent with the continuous part of the chemiluminescence signal obtained with WT cells, but it did not reflect the transient NO emission peak. It should be noted that even a direct continuous recording of DAF fluorescence (not shown) gave no indication of the rapid and transient NO burst observed with chemiluminescence detection.
As before, there was no basic difference when fluorescence was measured in extracts from pre-incubated cells, except that the fluorescence intensity was higher than in the intact cell suspensions (not shown).
Cells release stable DAF-reactive compounds not related to NO
The observation that DAF fluorescence was occasionally not or only partly prevented by the NO scavenger cPTIO (Fig. 5) prompted us to check whether DAF fluorescence could also be caused by compounds or reactions not directly related to NO. First, cell suspensions containing DAF-2 together with the elicitor cryptogein were incubated for 2 h. Subsequently, cells and supernatant were quickly separated by centrifugation, and fluorescence was determined in the clear supernatant. Under these conditions, DAF fluorescence in the supernatant of the cryptogein-treated cells remained low at control levels (Fig. 6A). With NR-free Nia30 cells, the fluorescence level was even lower and cryptogein had no effect (Fig. 6E).
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Next, cell suspensions were pre-treated with cryptogein as before, but without DAF. After 2 h, cells were again separated from the medium, DAF-2 was added to the supernatant, and fluorescence was registered as indicated in Fig. 6B. The cell-free supernatant obtained from cells pre-incubated for 2 h with cryptogein gave a steep fluorescence increase, whereas the supernatant from control cells produced only a very low fluorescence signal (Fig. 6B).
The strong increase of DAF fluorescence in the supernatant from cryptogein-treated cells was largely prevented when cPTIO was present during the pre-incubation with cryptogein (Fig. 6C). In contrast, when cPTIO and DAF-2 were added together to the supernatant following 2 h of pre-incubation of cells with cryptogein, the fluorescence increase was insensitive to the NO scavenger (Fig. 6D). With NR-deficient cells (Nia30) treated in the same way as WT cells, DAF fluorescence was generally very low, almost at the control levels (Fig. 6F–H).
Next, DAF fluorescence was followed in the supernatant of cells pre-treated with myxothiazol and SHAM (Fig. 7). With nitrate-grown WT cells, the two inhibitors caused a strong and linear increase of DAF-2 fluorescence with time, which was completely insensitive to cPTIO (Fig. 7A). In contrast, NR-free Nia30 cells showed hardly any DAF-2 fluorescence (Fig. 7E). The fluorescence increase with WT cells was even higher when DAF-2 was added to the supernatant of pre-treated (2 h) cells (Fig. 7B). Here, cPTIO had no effect at all, whether it was added together with the inhibitors or after 2 h pre-incubation (Fig. 7C, D). As with cryptogein, Nia30 cells gave only low DAF fluorescence under any of the conditions (Fig. 7F–H).
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The experiments with supernatant from myxothiazol+SHAM-pre-treated cell suspensions were also carried out with the new fluorescent NO indicator DAR-4M. The results, including the absolute fluorescence intensity (560 nm excitation, 575 nm emission, 2 nm bandwidth), were very similar to those recorded with DAF-2 (data not shown).
In order to give a preliminary characterization of the DAF- (or DAR-4M-) reactive compounds released, the supernatant in experiment Fig. 7B was subjected to a number of treatments before adding DAF. Desalting the supernatant on Sephadex G25 columns, or brief boiling before addition of the fluorophore strongly reduced fluorescence (not shown). Further, in order to check if the putative DAF-reacting compounds in the supernatant were related to reactive oxygen species, superoxide dismutase and catalase were also added to the supernatant obtained from cells treated with myxothiazol and SHAM. However, the presence of these enzymes did not interfere at all with DAF fluorescence (not shown).
As shown previously, NO emission from cell suspensions (measured by chemiluminescence) was orders of magnitude higher under nitrogen than in air (Planchet et al., 2005). If DAF did not react with NO, but with N2O3, as mentioned above, oxygen should always be required for DAF fluorescence. It was therefore unexpected that WT cells incubated under nitrogen gave an almost linear and strong increase in DAF-2 fluorescence, which was, however, insensitive to cPTIO (Fig. 8A). Similarly, addition of DAF-2 to the supernatant of anaerobically pre-treated cells also gave an increase in DAF fluorescence, which was largely insensitive to cPTIO (Fig. 8B–D). As for the respiratory inhibitors, NR-free Nia30 cells produced hardly any DAF fluorescence (Fig. 8E–H).
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It is important to note that the cell supernatant, in spite of producing a strong increase in DAF-2 fluorescence by the treatments used in Figs 6–8
, never triggered a chemiluminescence signal characteristic for NO production. |
Discussion |
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As shown above, DAF fluorescence and chemiluminescence may give very different results, depending on conditions and on the systems under investigation. With dissolved NO at the low concentrations thought to exist in plants (compare Planchet et al., 2005), only chemiluminescence was sensitive enough to detect NO. In spite of that, in a number of systems (NO donor, NR, cell suspensions) both methods gave reasonable signal strength. Thus, either part of the NO that was formed as the primary product must have been oxidized to NO2 or N2O3 (Kojima et al., 1998a; Nakatsubo et al., 1998; Espey et al., 2001), or these oxidized and DAF-reactive forms were congenitors of NO. Thus, the occasionally large differences in signal strength of chemiluminescence and DAF fluorescence (as, for example, with purified NR or with SNP) may reflect the relative rates of the production of NO and oxidized congenitors, or of NO oxidation, or, as will be discussed later, of the production of other DAF-reactive compounds.
Most important for biological research is the response of the two methods to NO produced by living cells. As already shown previously, NO production by cells as detected by chemiluminescence could only be weakly induced by pathogen-derived elicitors (Planchet et al., 2006), but strongly by anoxia (Planchet et al., 2005) or by inhibitors of mitochondrial electron transport (Tischner et al., 2004; Gupta et al., 2005; Planchet et al., 2005). All these three conditions have been applied here. Consistent with previous results (Planchet et al., 2006), cryptogein induced a low but immediate increase of fluorescence in DAF-2DA-pre-loaded cells, but without any detectable chemiluminescence signal (Fig. 4). With the non-cell-permeable DAF-2, the fluorescence increase was much lower, which may indicate that NO or other DAF-reactive compounds were initially formed inside cells. After prolonged pre-incubation (2 h) of cells with any of the three conditions (elicitor, myxothiazol+SHAM, or anoxia), compounds were released to the medium which gave a strong reaction with DAF-2 (Figs 6–8), and also with the new NO indicator DAR-4M (not shown), but without producing a chemiluminescence signal. Thus, the DAF-2- or DAR-4M -reactive compound was not NO itself. Its production, however, must have been related to nitrate reduction, because Nia cells never showed this response. The chemical nature of the compound(s) has not been identified yet. However, these compounds were (i) removable by gel filtration, i.e. of low molecular weight; (ii) destroyable by brief boiling; (iii) largely non-reactive with cPTIO; and (iv) formed even in the presence of excess catalase or superoxide dismutase.
If NO was rapidly released from ‘NO storage pools’, like S-nitrosothiols, nitrated aromatic amino acids, proteins, lipids, or from iron-nitrosyl-haemoglobins, this NO should still be scavenged by cPTIO or tmaPTIO (for reviews, see O'Donnell and Freeman, 2001; Zhang and Hogg, 2004; Gladwin et al., 2005). However, this was not the case. Thus, it appears presently that either these ‘NO storage compounds’ can donate NO directly to DAF (but why not to cPTIO?) without liberating free NO, or, nitrate (or nitrite) reduction led to as yet unknown meta-stable intermediates that react with both DAF-2 and DAR-4M. It has been shown previously that DAF can also react with dehydroascorbic acid and ascorbic acid, forming complexes that fluoresce with characteristics similar to those of DAF-2T (Zhang et al., 2002). DAF nitrosation can be enhanced in the presence of peroxynitrite or peroxidase (Jourd'heuil, 2002), and nitroxyl-compounds can elicit similar effects (Espey et al., 2002). In our experiments, however, very high ascorbate concentrations (
5 mM) were required to provoke DAF-2 fluorescence (data not shown), which are probably not reached in the cell supernatant.
The inhibitors of mitochondrial complex III (myxothiazol) and AOX (SHAM) specifically provoked a rapid and transient NO burst, which was detected by chemiluminescence, but not by DAF fluorescence. In WT cells, this rapid burst was followed by a lower but continuous NO emission, which was lacking in Nia cells. Thus, the NO burst was independent of nitrate reduction, whereas the continuous NO emission required NR.
The above NO burst might have been due to a nitric oxide synthase (NOS) activity. In experiments not shown here, it was indeed found that commercially available recombinant iNOS (inducible NOS), like NR, gave rise to both a DAF-2 and a chemiluminescence signal. However, in contrast to NR-dependent NO production, the iNOS reaction was absent under anoxia, as should be expected. Consistent with that, the above-mentioned NO burst was not observed under nitrogen (not shown). The recent suggestion that plant NOS (AtNOS1) is located in the mitochondria (Guo and Crawford, 2005) might render the above response to mitochondrial inhibitors more plausible. On the other hand, mitochondria purified from tobacco cell suspensions did not show the transient NO burst in response to myxothiazol and SHAM (not shown). Thus, either the NO burst is not caused by NOS, or NOS is not a mitochondrial enzyme, contrary to what has been suggested recently (Guo and Crawford, 2005). However, without further experimental data, the interpretation remains speculative at this point.
In summary, the data suggest that DAF fluorescence is not a good indicator of NO itself, and indeed its specificity for NO has been questioned (Jourd'heuil, 2002; Roychowdhury et al., 2002; Balcerczyk et al., 2005). Increased DAF fluorescence and/or chemiluminescence may actually be traced back to three different reactions: (i) to NO production without oxidation (resulting in chemiluminescence but no DAF fluorescence); (ii) to NO production with slow oxidation or co-production of oxidized NO derivatives (resulting in variable ratios of chemiluminescence and DAF fluorescence); and (iii) to production of nitrate-dependent, as yet unidentified meta-stable compounds resulting in cPTIO-insensitive DAF fluorescence but no chemiluminescence.
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Acknowledgements |
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This work was supported by the DFG, SFB 567, Ka 456-15/1-3. We gratefully acknowledge the technical assistance of Maria Lesch. Cryptogein was kindly provided by Dr Michel Ponchet (INRA, Unité Mixte de Recherches, Interactions Plantes-Microorganismes et Santé Végétale, Sophia-Antipolis, France). We wish also thank Barbara Dierich and Eva Wirth for maintenance of tobacco cell suspensions.
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Abbreviations |
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AOX, alternative oxidase; cPTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; DAF-2, 4,5-diamino-fluorescein; DAF-2DA, 4,5-diamino-fluorescein diacetate; DAF-FM DA, 3-amino,4-aminoethyl-2',7'-difluorofluorescein diacetate; DAF-2T, 4,5-diamino-fluorescein triazole; DAR-4M, diaminorhodamine-4M; GSNO, S-nitrosoglutathione; Nia, nitrate reductase gene; NO, nitric oxide; NOS, nitric oxide synthase; NR, nitrate reductase; SHAM, salicylhydroxamic acid; SNP, sodium nitroprusside; tmaPTIO, 3,3,4,4-tetramethyl-2-trimethylammonio-phenyl-2-imidazoline-3-oxide-1-yloxy chloride; WT, wild type.
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