Journal of Experimental Botany, Vol. 57, No. 12, pp. 3043–3055, 2006
doi:10.1093/jxb/erl070 Advance Access publication 7 August, 2006
RESEARCH PAPER
Nitric oxide (NO) detection by DAF fluorescence and
chemiluminescence: a comparison using abiotic and
biotic NO sources
Elisabeth Planchet and Werner M. Kaiser*
Julius-von-Sachs Institute for Biosciences, University of Würzburg, Julius-von-Sachs-Platz 2,
D-97082 Würzburg, Germany
Received 20 March 2006; Accepted 30 May 2006
Abstract
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.
Introduction
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 NOspecific 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 (DAF2T). 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
* To whom correspondence should be addressed. E-mail: [email protected]
Abbreviations: AOX, alternative oxidase; cPTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; DAF-2, 4,5-diamino-fluorescein; DAF2DA; 4,5-diamino-fluorescein diacetate; DAF-FM DA, 3-amino,4-aminoethyl-29,79-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.
ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
3044 Planchet and Kaiser
autoxidation of NO in air according to reaction (1), and in
aqueous solution will result in nitrite formation (reaction 2)
(Espey et al., 2001)
4NO + O2 ! 2N2 O3
ð1Þ
N2 O3 + H2 O ! 2NO2 + 2H +
ð2Þ
In that case, fluorescence intensity should not only
depend on NO production, but also on the rate of NO
oxidation which requires oxygen, and which should
strongly depend on the NO concentration. Accordingly, it
was suggested that DAF cannot be used under anoxia.
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 NOproducing enzyme, or intact cell suspensions. Further,
NO scavenging was examined by cPTIO [carboxy-PTIO,
2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl3-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.
Materials and methods
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 8C 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 nitrategrown 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 ml1. Before any treatment with myxothiazol (10 lM)+
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 lM), 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 min1 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 8C in the dark or at room light.
For fluorometric NO determination, the fluorophore 4,5-diaminofluorescein (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 lM DAF-2DA for 15 min
at 24 8C in darkness on a rotary shaker (100 rpm) and then rinsed
with fresh suspension buffer to remove excess fluorophore. Alternatively, DAF-2 (10 lM) 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-500 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 8C), and fluorescence was again measured in the clear
supernatant.
NO detection by DAF and chemiluminescence
Similarly, formation of fluorescing DAF derivates by compounds
released from the cells was measured by adding DAF-2 (10 lM)
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 lm, Roth, Karlsruhe,
Germany).
Nitrite determination
Separate aliquots (400 ll) of the above cell suspensions or solutions
were sampled and quickly mixed with a reaction mixture containing:
600 ll sulphanilamide (1%), 600 ll of N-(1)-(naphthyl)ethylenediaminedihydrochloride (0.02%), and 300 ll of zinc acetate (0.5 M).
After 25 min of incubation at 24 8C, 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 8C 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 ml1. 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 lM 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-3oxide-1-yloxy chloride) were purchased from Alexis Biochemicals
(Gruenberg, Germany). Myxothiazol and SHAM were from SigmaAldrich (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).
Results
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
min1 through the reaction chamber was used at 20 s
measuring intervals. Under these conditions, the detection
limit (23 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
3045
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 8C (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.
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 lM. NO emission from SNP,
measured by chemiluminescence, rapidly reached a low
rate of 5.5 pmol ml1 min1 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 min1 ml1 measured by chemiluminescence corresponded to an increase of fluorescence of
0.286 AU min1. Apparently the donor solution produced
NO and oxidized DAF-reactive derivatives at the same time.
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.
3046 Planchet and Kaiser
Fig. 1. NO release from an aqueous solution of dissolved NO. (A) Chemiluminescence NO detection. A 10 ml aliquot of a buffer solution (100 mM
HEPES, pH 7.5) was pre-flushed with 100 ppm NO in nitrogen for at least 15 min, resulting in a solution that contained 190 pmol NO ml1. Under a gas
stream of air or nitrogen, aliquots of that solution (0.1–1 ml) were injected into 500 ll of buffer solution in a small Petri dish placed in a glass cuvette on
a rotary shaker. Where indicated, cPTIO (200 lM final concentration) was added to the buffer solution 5 min before adding NO. The inset shows that the
integrals of the chemiluminescence signal in air were linear over the whole range. (B) The same experiment with DAF fluorescence detection. Here,
different volumes of the pre-flushed NO solution were added to the buffer solution containing DAF-2 (10 lM), under nitrogen or in air, to give a final
volume of 1 ml. Fluorescence values were read when the signal was stable, usually after 30 min. Each value represents the mean 6SE of three
independent experiments. Note that the fluorescence with NO gas was extremely low compared with the subsequent experiments (Figs 2–8).
Fig. 2. NO production from SNP. The donor (10 lM final concentration) was added at T=0 to 2 ml of buffer (MES 5 mM, pH 6.5) for (A)
chemiluminescence or (B) DAF-2 fluorescence (10 lM). Where indicated, the NO scavenger cPTIO (500 lM) was added 10 min before SNP. The nitrite
content in aliquots of the medium containing SNP was measured at T=0 and at T=120 (columns in B). Note that a constant chemiluminescence signal
means that the NO emission rate is constant. In that case, the fluorescence signal will increase with constant slope, because the concentration of the highly
fluorescing reaction product, DAF-2T, is steadily increasing. Each datum point is the mean of three independent experiments 6SE. In (A), SE is only
indicated for a few points for reasons of clarity.
NO + cPTIO ! NO2 + cPTI
ð3Þ
NO2 + NO ! N2 O3
ð4Þ
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 lM
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 ml1 min1.
Consistent with that, after an initial lag phase, DAF
fluorescence increased steadily (Fig. 3B), confirming a
NO detection by DAF and chemiluminescence
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 min1 ml1 from NR measured
by chemiluminescence corresponded to a fluorescence
increase of only 0.033 AU min1, suggesting that only a
small fraction of NR-derived NO was oxidized to the DAFreactive 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).
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
3047
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 NRfree 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,
Fig. 3. NO generation by purified NR. (A) NO detected by chemiluminescence: 1 ml of a buffer solution (HEPES 100 mM, pH 7.6) was incubated in
a 5 ml glass beaker mounted in the headspace cuvette at 24 8C in air, under vigorous stirring. Purified NR (25 mU) and nitrite (500 lM) were added
5 min before starting the reaction by adding NADH (500 lM) at T=0. After 40 min, the NO scavenger tmaPTIO (200 lM), or KCN (1 mM) was added
to the solution where indicated. Each datum point is the mean of three independent experiments. (B) NO as detected by DAF-2 fluorescence. Conditions as before. Five minutes before starting the reaction by NADH (T=0), DAF-2 (10 lM), NR, and nitrite were already present. NO scavenger and
cyanide were added as indicated. Means are from three independent experiments (6SE).
3048 Planchet and Kaiser
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).
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 longlasting second phase of NO emission observed with WT
cells was completely lacking with Nia30 (Fig. 5B).
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. 4. Cryptogein-mediated NO production by cell suspensions. (A)
Chemiluminescence NO detection from nitrate-grown WT tobacco cell
suspensions after cryptogein treatment. The elicitor cryptogein (20 nM)
was added to the cells at T=0. Each datum point is the mean of three
independent experiments. (B) Intracellular DAF fluorescence from a cell
suspension (10 ml) pre-loaded with DAF-2DA (10 lM, 15 min). After
removal of excess dye (see Materials and methods), cryptogein (20 nM)
was injected in the presence or absence of the NO scavenger cPTIO
(500 lM), which was added 10 min before treatment. At the indicated
times, aliquots (1 ml) were removed for fluorescence measurement
(n=4 6SE). At T=0 and T=120, aliquots from nitrate-grown cell
suspensions (medium+cells) treated with cryptogein were taken for nitrite
determination (n=4 6SE, columns). In controls, nitrite remained at the
level at T=0 (not shown). (C) The same experiment as in (B) with DAF2DA. However, here fluorescence was measured after lysing 1 ml of the
cells by a freeze/thaw cycle and subsequent removal of insoluble material
by centrifugation. Thereby, optical conditions were improved and DAF
fluorescence was measured under well-defined ionic conditions in the
medium. After pre-loading cells with DAF-2DA, the cells were incubated
with cryptogein (20 nM)6cPTIO (500 lM). Values are means (n=3) 6SE.
NO detection by DAF and chemiluminescence
3049
Fig. 5. Inhibitors of mitochondrial electron transport induce low and transient chemiluminescence but high DAF fluorescence. Chemiluminescence NO
detection from (A) nitrate-grown tobacco WT cells and from (B) NR-free tobacco cells (Nia30 cells). At T=0, both inhibitors (myxothiazol 10 lM plus
SHAM 2.5 mM) were injected into the cell suspension. The NO scavenger cPTIO (500 lM) was added 10 min before the inhibitors (n=3–6 6SE).
Fluorescence kinetics of DAF-2DA-pre-loaded (C) nitrate-grown WT cells and (D) cells from the NR-free Nia double mutant. After pre-loading cells
with 10 lM DAF-2DA and subsequent removal of excess dye, myxothiazol+SHAM (6cPTIO 500 lM) were added. At the indicated time points,
aliquots (1 ml) were removed for fluorescence determination of the complete cell suspension (n=4 6SE). Aliquots for nitrite determination from nitrategrown cell suspensions treated with myxothiazol+SHAM were taken at T=0 and at T=120 (n=4 6SE, columns). Note the different scales in (C) and (D).
(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 cryptogeintreated 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).
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 preincubated 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).
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).
3050 Planchet and Kaiser
Fig. 6. Cryptogein treatment induces release of DAF-reactive compounds into the cell medium. DAF-2 (10 lM) fluorescence in the supernatant of
nitrate-grown WT cells (A) or Nia30 cells (E) incubated with cryptogein (20 nM) in the presence or absence of cPTIO (500 lM, added 10 min before
treatment). At the indicated time points, aliquots (1 ml) were removed. The cells were gently centrifuged, the supernatant was cleared through additional
filtration, and fluorescence was immediately measured (n=3 6SE). Alternatively, following a 2 h incubation without DAF, but with cryptogein6cPTIO
(500 lM) in NO-free air, nitrate-grown WT (B–D) or Nia30 cells (F–H) were gently centrifuged and the supernatant was cleared by filtration as before.
To 1 ml of the supernatant, DAF-2 (10 lM)6cPTIO (500 lM) was added and fluorescence was measured during the subsequent 2 h (n=4 6SE).
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
NO detection by DAF and chemiluminescence
3051
Fig. 7. Inhibitors of mitochondrial electron transport trigger the release of DAF-reactive compounds into the medium (cell supernatant). DAF-2 kinetics
from the supernatant of nitrate-grown WT cells (A) or Nia30 cells (E) incubated directly with DAF-2 (10 lM) and treated with myxothiazol (10 lM) and
SHAM (2.5 mM) in the presence or absence of cPTIO (500 lM, added 10 min before treatment). Conditions are as in Fig. 6. Alternatively, after a 2 h
incubation with myxothiazol and SHAM6cPTIO (500 lM) under NO-free air, nitrate-grown (B–D) or Nia30 cells (F–H) were gently centrifuged and
the supernatant was cleared by filtration. A 1 ml aliquot of the supernatant was immediately incubated with DAF-2 (10 lM)6cPTIO (500 lM) and
fluorescence was followed during the subsequent 2 h (n=4 6SE).
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 pretreated 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).
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
As shown above, DAF fluorescence and chemiluminescence may give very different results, depending on
3052 Planchet and Kaiser
Fig. 8. Anoxia triggers the release of DAF-reactive compounds into the medium (cell supernatant). DAF-2 fluorescence in the supernatant of nitrategrown WT cells (A) or Nia30 cells (E) incubated with DAF-2 (10 lM) under nitrogen or in air (control), in the presence or absence of cPTIO (500 lM,
added 10 min before treatment). Alternatively, after a 2 h incubation under nitrogen or NO-free air6cPTIO (500 lM), the nitrate-grown (B–D) or Nia30
cells (F–H) were separated from the medium as before and, after addition of DAF-2 (10 lM)6cPTIO (500 lM), fluorescence was measured. Other
conditions are as in Fig. 6 (n=4 6SE).
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
NO detection by DAF and chemiluminescence
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-2DApre-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
3053
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.
Acknowledgements
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
3054 Planchet and Kaiser
Barbara Dierich and Eva Wirth for maintenance of tobacco cell
suspensions.
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