Journal of Experimental Botany, Vol. 51, No. 347, pp. 1027–1036, June 2000
A nitric oxide burst precedes apoptosis in angiosperm
and gymnosperm callus cells and foliar tissues
M. Cristina Pedroso1,4, J.R. Magalhaes2 and D. Durzan3
1 Centro de Biotecnologia Vegetal, Departamento Biologia Vegetal, Faculdade de Ciências de Lisboa, Bloco
C2, Piso 1, Campo Grande, P-1749–016 Lisboa, Portugal
2 Centro Nacional de Recursos Genéticos e Biotecnologia—EMBRAPA-70770–900 Brası́lia, DF-Brazil
3 Department of Environmental Horticulture, University of California, Davis, CA 95616–8587, USA
Received 26 November 1999; Accepted 3 February 2000
Abstract
Leaves and callus of Kalanchoë daigremontiana and
Taxus brevifolia were used to investigate nitric oxideinduced apoptosis in plant cells. The effect of nitric
oxide (NO) was studied by using a NO donor, sodium
nitroprusside (SNP), a nitric oxide-synthase (NOS)
inhibitor, NG-monomethyl-L-arginine (NMMA), and centrifugation (an apoptosis-inducing treatment in these
species). NO production was visualized in cells and
tissues with a specific probe, diaminofluorescein
diacetate (DAF-2 DA). DNA fragmentation was detected
in situ by the terminal deoxynucleotidyl transferasemediated dUTP nick end labelling (TUNEL) method. In
both species, NO was detected diffused in the cytosol
of epidermal cells and in chloroplasts of guard cells
and leaf parenchyma cells. Centrifugation increased
NO production, DNA fragmentation and subsequent
cell death by apoptosis. SNP mimicked centrifugation
results. NMMA significantly decreased NO production
and apoptosis in both species. The inhibitory effect of
NMMA on NO production suggests that a putative NOS
is present in Kalanchoë and Taxus cells. The present
results demonstrated the involvement of NO on DNA
damage leading to cell death, and point to a potential
role of NO as a signal molecule in these plants.
Key words: Apoptosis, cell death, centrifugation,
Kalanchoë daigremontiana, nitric oxide, Taxus brevifolia.
Introduction
Nitric oxide (NO) is involved in ethylene emission
(Leshem and Haramaty, 1996), plant response to drought
stress (Leshem, 1996; Haramaty and Leshem, 1997),
disease resistance (Delledonne et al., 1998; Durner et al.,
1998; Van Camp et al., 1998), growth and proliferation,
senescence (Leshem et al., 1998), and apoptosis
(Magalhaes et al., 1999). In animals, NO is a biologically
active messenger molecule that acts as a physiological
mediator and as a patho-physiological entity, being
involved in several processes including apoptosis and
cancer (Schmidt et al., 1992, 1998; MeBmer et al., 1994;
Dawson, 1998; Kojima et al., 1998b; Moncada, 1998).
Evidence suggests that NO signal transduction may also
be operating in plants as in animals (Haramaty and
Leshem, 1997; Delledonne et al., 1998; Durner et al.,
1998; Kim et al., 1998; Klessig et al, 1999).
NO is endogenously produced from -arginine,
NADPH, and molecular oxygen, by constitutive and
inducible forms of nitric oxide synthase (NOS; EC
1.14.13.39). This oxidative reaction requires NADPH and
the cofactor tetrahydrobiopterin to form -citrulline, NO,
and NADP+ (Leshem, 1996). Recent studies have indicated that NOS is also present in plants ( Yamasaki et al.,
1999; Wambutt et al., unpublished results). Cyclic and
acyclic amidines, guanidines and isothioureas are known
inhibitors of the active site of this enzyme (Schmidt et al.,
1998). In plants, NO can also be produced as a by-product
of the activity of constitutive nitrate reductase (Dean and
Harper, 1988; Yamasaki et al., 1999). NO can also be
generated non-enzymatically, by chemical breakdown of
NO donor molecules, such as sodium nitroprusside
(SNP), S-nitroso-N-acetylpenicillamine, and 3-morpholinosydnomine (Rucki, 1977; Leshem, 1996).
Because NO is a free radical, it reacts rapidly with
4 To whom correspondence should be addressed. Fax: +351 21 75 000 48. E-mail: [email protected]
Abbreviations: DAF-2 DA, 4,5-diaminofluorescein diacetate; DAPI, 4∞-6-diamino-2-phenylindole dihydrochloride; NMMA, NG-monomethyl-l-arginine; NO,
nitric oxide; NOS, nitric oxide-synthase; RT, room temperature; SNP, sodium nitroprusside; TUNEL, terminal deoxynucleotidyl transferase-mediated
dUTP nick end labelling; UV, ultraviolet;×g, times unit gravity.
© Oxford University Press 2000
1028 Pedroso et al.
atoms or molecules with unpaired electrons, such as
molecular oxygen (O ), superoxide (O−), water (H O),
2
2
2
and metals (iron, copper or manganese) in metalloproteins
(Lancaster Jr, 1992). In animal systems, NO can cause
genomic alterations ( Wink et al., 1991; Tannenbaum,
1998), mutation ( Tannenbaum, 1998), necrosis or
apoptosis, through p53-dependent (MeBmer et al., 1994)
or p53-independent pathways (Brüne et al., 1998). NO
production, p53 expression and apoptosis are prevented
or attenuated by NOS-inhibitors such as NG-monomethyl-arginine (NMMA) (Brüne et al., 1998).
The fluorescent probe, DAF-2 DA, has recently been
developed for direct detection of NO in live animal cells
( Kojima et al., 1998b). It is specific, highly sensitive, and
it does not react with stable oxidized forms of N, such as
nitrite and nitrate, nor with other reactive oxygen species
such as molecular oxygen, superoxide anion, hydrogen
peroxide and peroxynitrite ( Kojima et al., 1998a, b;
Nakatsubo et al., 1998).
In the present work, the hypothesis was tested that
DAF-2 DA can be used to visualize NO in plant cells,
and that NO is involved in irreversible DNA damage
leading to apoptosis in explants exposed to centrifugation
(mechanical stress). Treatments with a non-enzymatic NO
donor (SNP) and centrifugation, with or without
NMMA, a competitive substrate inhibitor of NOS, were
used to induce or prevent NO formation and apoptosis.
In Kalanchoë and Taxus, apoptosis (for review, see Bell,
1994, 1996; Greenberg, 1996; Havel and Durzan, 1996a;
Pennel and Lamb, 1997; Gilchrist, 1998) occurs at different stages of plant development (Havel and Durzan,
1996a, b; 1999; Pedroso and Durzan, 1998; Durzan,
1999), or as a response to stressful environments (Pedroso
and Durzan, 1998; Durzan, 1999). In Kalanchoë, leaf and
plantlet exposure to hypergravity (centrifugation;
mechanical stress) led to a significant increase in chloroplastidial and nuclear DNA fragmentation, and in cell
death by apoptosis (Pedroso and Durzan, 1999b). Bursts
in reactive oxygen species are frequently correlated with
mechanical stress ( Yahraus et al., 1995) and subsequent
cell death (Allan and Fluhr, 1997). The reaction of NO
with superoxide (O−) produces the toxic compound per2
oxynitrite (OONO−) more damaging than NO itself,
exerting deleterious effects on DNA, lipids and proteins,
similar to that observed with oxygen-derived species
(Stamler et al., 1992; Pryor and Squadrito, 1995;
Yamasaki et al., 1999). The fact that a stressful treatment,
such as centrifugation, results in an increase in apoptosis
in both Kalanchoë and Taxus (angiosperm and gymnosperm, respectively) (Durzan, 1999; Pedroso and Durzan,
1999b), makes them ideal explants to show the effect of
mechanical stress on NO production, as well as NO
involvement on DNA fragmentation leading to cell death.
In this study, the terminal deoxynucleotidyl transferasemediated dUTP nick end labelling ( TUNEL) method was
used for in situ detection of DNA fragmentation following
exposure to SNP or to centrifugation in the presence or
absence of NMMA. To determine if DNA fragmentation
occurred in the cells producing NO, a protocol
DAF-2DA-TUNEL was developed for simultaneous
detection of NO and DNA fragmentation.
Materials and methods
Plant materials
Leaves and stem-derived callus of Kalanchoë daigremontiana
were used for the angiosperm trials. Shoot cultures, used as a
source of leaves and stems for callus production, were
maintained on modified half-strength Murashige and Skoog
basal medium (Murashige and Skoog, 1962) (MS) supplemented with 25 g l−1 -glucose (Pedroso, 1998), and subcultured
every month. Callus induction from stem segments was
performed on MS13 medium consisting of MS basal medium
with 5 mg l−1 dithiothreitol, 25 g l−1 -glucose, 1 mg l−1 N6benzylaminopurine and 0.5 mg l−1 2,4-dichlorophenoxyacetic
acid. Cultures were kept at 25 °C, under a 16 h photoperiod
provided by Phillips F40 AGRO AGROLITE fluorescent lamps
(maxinum radiation at 350–450 nm (blue) and 650 nm (red )).
Leaves, isolated from 4-week-old shoots, and 3-week-old callus,
were harvested immediately before the assays.
Leaves from a 30-year-old female Taxus brevifolia plant, and
egg cell-derived callus cultures, derived from the same plant,
cultured in darkness in modified B5 medium (Durzan and
Ventimiglia, 1994), were used for the gymnosperm trials.
Leaves, isolated from young branches, and 3-week-old callus,
were harvested immediately before the assays.
Nitric oxide donor assays
NO was generated non-enzymatically by chemical breakdown
of sodium nitroprusside (SNP). Friable callus cells and leaves,
isolated and hand-sectioned under aseptic conditions, were
incubated for 3 h in 10−6, 10−4 and 10−2 M SNP in filtersterilized modified B5 media (Durzan and Ventimiglia, 1994).
Incubation without SNP were used as a control. Samples, kept
in a shaker at 60 rpm, at 23±2 °C, were then washed in sterile
fresh medium and immediately assayed for NO visualization or
kept in culture overnight for the detection of apoptosis. On a
separate set of experiments, live cells and leaf sections, collected
immediately after the treatments and after overnight incubation,
were doubled stained for the simultaneous detection of NO and
DNA fragmentation using DAF-2 DA followed by fixation and
TUNEL as described below.
Inhibition of NO production
Friable callus cells and leaf sections were centrifuged for 3 h in
liquid culture medium, at 150×g, with or without 0.5 mM NGmonomethyl--arginine, NMMA (NO-synthase inhibitor). The
NMMA enantiomer, NG-monomethyl--arginine, which does
not have any significant effect on NO-synthase, was used as
negative control to investigate non-specific -NMMA activity.
Samples kept for 3 h, in 1×g, under agitation, were used as
additional controls.
Visualization of NO
Cells and leaf sections (SNP treated, centrifuged, and controls)
were incubated for 1 h, at 25 °C, with 10 mM 4,5diaminofluorescein diacetate (DAF-2 DA; Calbiochem, La
Nitric oxide and apoptosis
Jolla, CA, USA) ( Kojima et al., 1998b). Samples incubated at
1×g without SNP, boiled in water for 1 min (dead cells) and
incubated in DAF-2 DA, incubated in the absence of DAF-2
DA, or incubated in medium supplemented with NO− or
3
NO− (10 mg ml−1) and then incubated in DAF-2 DA, were
2
used as controls. After incubation, the samples were washed in
water and mounted in water or Vectashield ( Vector
Laboratories, Inc., Burlingame, CA, USA). DAF-2 DA fluoresces bright green (excitation 495 nm; emission 515 nm).
In situ detection of apoptosis
For detection of nuclear DNA fragmentation, all samples were
fixed in a 4% (v/v) formaldehyde solution for 24 h, at 4 °C, and
labelled using the one-step modified TUNEL method
(Boehringer Mannheim, Germany) (Havel and Durzan, 1996b).
In negative controls, only TUNEL label (nucleotide mix,
containing fluorescein-dUTP and dNTP without terminal
deoxynucleotidyl transferase) was added. In positive controls,
cells were incubated with DNAse I (1 mg ml−1) (Boehringer
Mannheim, Germany) for 10 min, at 25 °C, prior to labelling.
Counterstaining for 15 min, at RT, with 1 mg ml−1 4∞-6-diamino2-phenylindole dihydrochloride (DAPI ), which binds to adenine-thiamine-rich deoxyribonucleic acids, was performed to
distinguish non-apoptotic nuclei (blue fluorescence at 360 nm)
from apoptotic ones (bright green fluorescence at 450–490 nm).
‘Apoptotic nuclei’ were considered to be those nuclei that were
only TUNEL-positive, indicated irreversible DNA fragmentation, and exhibited cytological features characteristics of plant
apoptosis ( Wang et al., 1996; Yeng and Yang, 1998; Pedroso
and Durzan, 1999b).
Fluorescence and confocal microscopy
Leaf sections and cells stained with DAF-2 DA and TUNEL
were observed with an epi-fluorescence microscope, equipped
with DAPI (excitation at 360 nm; emission ≥420 nm) and
FITC filters (excitation at 450–490 nm; emission ≥520 nm),
and a Nikon camera with 400 ASA Elite Chromo Kodak film.
Confocal images were obtained using a Leitz UV 40× 1NA oil
PL FLUOTA objective lense under a Leica TCS-NT confocal
laser scanning microscope (Leica Lasertechnik GmbH,
Heidelberg, Germany), equipped with Argon/Kripton and UV
lasers (ex. spliter DD488/586) and Leica TCS-NT software
(TCS-NT version 1.5.451). Serial confocal optical sections were
taken at a step size of 1 mm. Molecular Dynamics ImageSpace
software (Molecular Dynamics, Inc., Sunnyvale, CA, USA)
was used in confocal image processing. Images in Fig. 5 were
desaturated using image processing software (Photoshop; Adobe
Systems, Inc., Mountain View, CA, USA). Plates were printed
on a Kodak ds 8650P Color Printer (Eastman Kodak Company,
Rochester, NY, USA).
Data analysis
The mean (±SD) for DAF-2 DA-positive and DAF-2
DA-negative cells and the percent of NO-stained cells were
determined. Apoptosis was quantified by counting the number
of TUNEL-positive nuclei (apoptotic) versus the total number
of DAPI-positive nuclei (non-apoptotic). The number of
TUNEL-positive nuclei in each of 10 microscope fields randomly
selected was also determined. Statistical analysis using ANOVA
at P<0.05 (n at least 8) was performed and results are presented
in the text. Experiments were repeated at least three times.
1029
Results
Visualization of NO
Fluorescence microscopy examination showed that autofluorescence was present in both plants. In Kalanchoë
leaves, autofluorescence was red in chloroplasts (chlorophyll ), yellow in tracheids ( lignin), and greenish in some
vacuoles and for cuticle (phenolics). In Taxus callus,
autofluorescence (yellow) was observed in cell walls and
some vacuoles; in leaves, autofluorescence was also
observed in tracheids ( lignin) and cuticle (phenolics).
Taxus callus cells stained uniformly for NO (green
fluorescence; Fig. 1A, B). Kalanchoë stem-derived callus
cells were preferentially stained at the chloroplast level
( Fig. 1D). In leaves of both species (Fig. 1E–H and I–L),
NO-positive cells were preferentially observed at the
epidermis (including guard cells), and in subepidermal
parenchyma cells. Independently of the species and cell
types, NO fluorescence was visibly brighter at cell walls
( Fig. 1A, J ) and in dense cytoplasmic regions (Fig. 1J,
L). Vacuoles did not stain for NO ( Fig. 1J–L). DAF-2
DA reaction was negative for stained dead cells (boiled
samples).
The pattern of staining for NO in foliar tissues was
variable in the two species, for both control and treated
samples. In Taxus leaves (Fig. 1E–H ), NO was mainly
detected diffused in the cytosol of epidermal and subepidermal cells. Bright fluorescence of the cytosol, with or
without chloroplast staining, was commonly observed
in parenchyma cells, after centrifugation (Fig. 1F ).
Chloroplast staining without cytosol staining was not
observed. In contrast, in Kalanchoë leaves ( Fig. 1I–L),
NO was detected only in chloroplasts ( Fig. 1C versus
Fig. 1D) and/or diffused in the cytosol of epidermal cells.
NO staining of both chloroplast and cytosol was common
in guard cells (Fig. 1I, J ).
In both species, an average of 13–28% of control cells
(non-treated ) were stained for NO ( Fig. 1A, E, I ).
Although cell walls were stained for NO (Fig. 1A), less
than 5% of those cells showed a bright NO staining in
the cytoplasm (as in Fig. 1B).
The number of NO-positive cells increased with increasing concentrations of SNP ( Fig. 2). Since these results
were highly reproducible, 10−4 M SNP treatment was
selected for the remaining assays.
The effect of centrifugation on NO production was
shown to be similar to incubation in medium with SNP,
for callus cells of Kalanchoë and Taxus ( Fig. 3). For both
species, the number of NO-positive cells was significantly
higher (P<0.05, n=10) in SNP-treated and centrifuged
cells compared to stained but untreated controls (Fig. 3A,
B). DAF-2 DA green fluorescence was also brighter
( Fig. 1B, F, H, J, L) than in controls ( Fig. 1A, E, I ). For
callus and foliar tissues, no significant differences were
detected between 10−4 M SNP-treated and centrifuged
1030 Pedroso et al.
Fig. 1. Visualization of NO and apoptosis in Taxus brevifolia and Kalanchoë daigremontiana by fluorescence microscopy using DAF-2 DA and the
TUNEL method (see text for details). (A, B) Cells from egg-derived callus of T. brevifolia. (A) NO-positive control cells (green) cultured at 1 g
without any further treatment. Note the presence of cells with low or null NO production (arrows) (×250). (B) NO-positive cell after 3 h
centrifugation at 150×g. (C, D) Cells from stem-derived callus of K. daigremontiana (×400). (C ) NO-negative control cells. Red autofluorescence
was due to chorophyll in chloroplasts, (×600). (D) Centrifuged callus cells (3 h, at 150×g) stained for NO. Note NO-positive chloroplasts (green)
(×400). (E–H ) Leaf sections of T. brevifolia. ( E ) NO (green fluorescence) in epidermal and some parenchyma cells, in control leaf (×250). (F )
Increase of NO (green) after induction of apoptosis by centrifugation at 150×g (×250). (G) Absence of NO in leaf sections after centrifugation in
the presence of 0.5 mM NG-monomethyl--arginine, NMMA, a NO-synthase inhibitor (×400). (H ), NO-positive epidermal cells (arrows) after
incubation in 10−4 M SNP ( longitudinal section; top view), (×400). (I–M ) Leaf sections of K. daigremontiana. (I ) NO-positive guard cell (green;
arrows) in control (×250). (J ) NO in epidermal cells (green) after induction of apoptosis by centrifugation at 150×g (×250). ( K ) NO in epidermal
cells after centrifugation in the presence of 0.5 mM NMMA. Note the presence of few dimly stained cells (arrows) (×250). (L) NO-positive
epidermal cells (green) after incubation in 10−4 M sodium nitroprusside (SNP) (×400). (M ) Apoptotic nuclei (green) in epidermis and subepidermis
after a NO burst (×250) (see text and Table 1 for details).
Nitric oxide and apoptosis
1031
Fig. 2. Effect of increasing concentrations (10−6, 10−4 and 10−2 M ) of
sodium nitroprusside (SNP; nitric oxide donor), on the number of
nitric oxide (NO) positive cells of Taxus brevifolia, stained with DAF-2
DA (see text for details). Live cells mounted in water or dead cells
stained with DAF-2 DA (C; negative controls); cells stained with
DAF-2 DA (C+DAF ). Cells incubated for 3 h with different concentrations of sodium nitroprusside (10−6, 10−4 and 10−2 M SNP). Values
are the mean±SD of at least three independent experiments.
cells, in terms of number of NO-positive cells (Fig. 3A,
B) or brightness of fluorescence ( Fig. 1F, H, J, L), except
for guard cells (Fig. 4). In centrifuged Kalanchoë leaves,
the number of guard cells stained for NO was significantly
higher (P<0.05, n=10) than in 10−4 M SNP treated
leaves ( Fig. 4).
For both species, the number of DAF-2 DA-stained
cells in medium supplemented with NO− or NO− was
3
2
not significantly different (P<0.05) from the values
obtained for samples incubated in medium without nitrogen supplementation (controls).
NMMA significantly decreased (P<0.05; n=10) NO
production in both centrifuged plant materials ( Figs 1G,
K; 3), including in guard cells of Kalanchoë leaves
(Fig. 4). NMMA reduced NO production to levels below
those detected in controls, in Kalanchoë stem-derived
callus ( Fig. 3A) and leaves (Fig. 1K ), and in Taxus leaves
(Fig. 1G). NMMA was more effective in decreasing or
preventing NO production in parenchyma cells and guard
cells (chloroplasts) than in epidermal cells (cytosol ).
Centrifugation in medium containing -NMMA (negative
control; -NMMA enantiomer), indicated that the NO
detected in at least 14% and 26% of the stained cells, in
Kalanchoë and Taxus, respectively, was not produced
through NO-synthase ( Fig. 3).
Detection of apoptosis
No apoptosis was observed in negative controls ( Table 1).
Positive controls, DNase-I treated samples, showed
DAPI-positive non-apoptotic nuclei under UV light, and
some TUNEL-positive nuclei (DNase-I-induced DNA
fragmentation) under blue light (bright green) ( Table 1).
In leaves, apoptotic nuclei were mainly detected in epidermal cells (Fig. 1M ).
Apoptotic cells were rarely (1–10%) observed in con-
Fig. 3. Effects of sodium nitroprusside (SNP; nitric oxide donor) and
centrifugation, with and without 0.5 mM NG-monomethyl--arginine
(-NMMA; nitric oxide-synthase inhibitor) or NG-monomethyl-arginine (-NMMA; no effect on NO-synthase), on the percentage of
nitric oxide-4,5-diaminofluorescein diacetate (DAF-2 DA)-stained cells
from stem-derived callus of Kalanchoë daigremontiana (A) and eggderived callus of Taxus brevifolia (B) (see text for details). Live cells
mounted in water or dead cells stained with DAF-2 DA (C; negative
controls). Cells stained with DAF-2 DA (C+DAF ). No significant
differences were recorded between cells incubated in medium with and
without NO− or NO−, so those samples are represented here as the
3
2
same population (C+DAF ). Cells incubated for 3 h with 10−4 M
sodium nitroprusside (10−4 SNP). Cells centrifuged for 3 h at 150×g
for the induction of apoptosis (150×g). Cells centrifuged with NMMA (150×g+-NMMA) or with -NMMA (150×g+NMMA). Values are the mean of at least three independent experiments.
trols ( Table 1). In both species, addition of SNP or
centrifugation significantly increased (P<0.05, n=8) the
percentage of apoptotic cells up to 82% ( Table 1).
In Taxus, low levels of SNP (10−6 M ) increased
apoptosis from 4% (controls) to 17%. Apoptosis was
highest (up to 75%) for 10−2 M SNP-treated cells and
leaves ( Table 1). Results were identical for Kalanchoë
with the exception that apoptotic bodies and drastic DNA
fragmentation were detected in leaf sections incubated
with 10−4 and 10−2 M SNP.
In both species, centrifugation increased (3–5-fold ) the
percentage of apoptotic cells ( Table 1). No significant
1032 Pedroso et al.
( Fig. 5A) or chloroplasts ( Fig. 5C ) was generally
observed.
The sequential detection of NO-producing cells, and of
apoptotic nuclei ( Fig. 5D), showed that all apoptotic cells
were NO-positive, but not all NO-positive cells presented
apoptotic nuclei.
Discussion
Fig. 4. Specific effect of sodium nitroprusside (SNP; nitric oxide donor)
and centrifugation, with and without 0.5 mM NG-monomethyl-arginine (-NMMA; nitric oxide-synthase inhibitor) or NG-monomethyl-arginine (-NMMA; no effect on NO-synthase), on the percentage
of nitric oxide-4,5-diaminofluorescein diacetate (DAF-2 DA)-stained
guard cells in Kalanchoë daigremontiana leaves. Procedure and controls
were identical to Fig. 3. Live cells mounted in water or dead cells
stained with DAF-2 DA (C; negative controls). Cells stained with
DAF-2 DA (C+DAF ). No significant differences were recorded
between cells incubated in medium with and without NO− or NO−, so
3
2
those samples are represented here as the same population (C+DAF ).
Cells incubated for 3 h in 10−4 M sodium nitroprusside (10−4 SNP).
Cells centrifuged for 3 h at 150×g for the induction of apoptosis
(150×g). Cells centrifuged with -NMMA (150×g+-NMMA) or
with -NMMA (150×g+-NMMA). Values are the mean of at least
three independent experiments.
differences (P<0.05, n=8) were recorded between
10−4 M SNP-treated and centrifuged samples, for the
number of apoptotic cells. Apoptotic bodies were not
observed in centrifuged samples. NMMA significantly
decreased apoptosis in centrifuged cells and leaves, compared to centrifugation without NMMA ( Table 1). In
both plants, the increase in the number of cells stained
for NO coincided with an increase in apoptotic nuclei.
Confocal microscopy and double staining
Confocal microscopy (Fig. 5A–D) confirmed the observations with fluorescence microscopy. Nuclei and vacuoles
(Fig. 5C ) were not stained with DAF-2 DA. NO was
diffused in the cytosol. A bright staining of amyloplasts
DAF-2 DA enabled the direct visualization of NO in
Kalanchoë and in Taxus cells. The use of controls
(unstained samples and stained-untreated ones) and the
use of chemical agents to increase or prevent NO generation (SNP and NMMA, respectively), confirmed the
absence of artefacts and showed that DAF-2 DA efficiently entered plant cells in both species. The negative
reaction obtained with boiled samples reaffirms the need
for the presence of intracellular esterases to hydrolyse
DAF-2 DA to DAF-2, which upon reaction with NO
becomes fluorescent ( Kojima et al., 1998a, b). The
absence of significant differences between samples incubated in medium with and without NO− and NO−
3
2
supplementation, confirmed that nitrates and nitrites do
not react with DAF-2. As reported for animal cells
( Kojima et al., 1998b), the fluorescence in plant cells was
also observed diffused in the cytosol. However, in both
Kalanchoë and in Taxus, positive staining of chloroplasts
was also detected, with and without positive-staining of
the cytosol. These results and the preferential staining of
stomata, point clearly to the differences existing between
animals and plants, concerning N metabolism (Bidwell
and Durzan, 1975; Durzan and Steward, 1983) and
defence mechanisms against oxidative stress (Inzé and
Montagu, 1995).
The simultaneous visualization of NO-producing cells
(green) and nuclear DNA fragmentation (green) was
possible because nuclei did not stain for NO, and DAF-2
was not washed out during TUNEL. In cells without
chloroplasts, the visualization process can be further
improved by using the two-step TUNEL method (Havel
and Durzan, 1996b) with a red fluorochrome.
Table 1. Apoptosis (expressed as percentage of apoptotic cells) in leaves and callus of Kalanchoë daigremontiana and Taxus brevifolia
incubated for 3 h in media with 10−6 M, 10−4 M and 10−2 M sodium nitroprusside (SNP) or centrifuged for 3 h at 150×g, with or
without 0.5 mM NG-monomethyl--arginine (NMMA; NOS inhibitor)
In situ detection of irreversible DNA fragmentation was performed using the TUNEL method (see text for details). Negative controls, no TUNEL
enzyme; positive controls, incubated in DNase-I, for 10 min, without SNP; Control, incubation without SNP. 10−6 M, 10−4 M and 10−2 M,
concentrations of SNP treatments. Centrif., centrifuged. Centrif.+NMMA, centrifuged with a NOS inhibitor.
Kalanchoë leaves
Kalanchoë callus cells
Taxus leaves
Taxus callus cells
Negative
controls
(0%)
Positive
controls
(5–14%)
Control
(no SNP)
(1–10%)
10−6 M
SNP
(15–25%)
10−4 M
SNP
(30–50%)
10−2 M
SNP
(>70%)
Centrif.
(30–50%)
Centrif.
+NMMA
(1–10%)
–
–
–
–
+
+
+
+
±
±
±
±
++
++
++
++
+++
+++
+++
+++
++++
++++
++++
++++
+++
+++
+++
+++
±
±
±
±
Nitric oxide and apoptosis
1033
Fig. 5. Confocal microphotographs of Taxus brevifolia cells (A) and of Kalanchoë daigremontiana leaf sections (B, C, D) centrifuged for 3 h at
150×g (see text for details). Nitric oxide (NO) was visualized using the 4,5-diaminofluorescein diacetate (DAF-2 DA) probe. DNA fragmentation
was detected in situ using the TUNEL method (see text for details). (A) NO in two ( light gray) out of three Taxus cells centrifuged for 3 h at
150×g, showing stained amyloplasts (arrow); Bar=18 mm. (B) NO formation in guard cells; (v) unstained vacuoles. Bar=8 mm. (C ) NO in
epidermal cells and in chloroplasts of subepidermal parenchyma cells, in a Kalanchoë leaf section. Vacuoles (v) from epidermal cells and stoma
guard cells (arrow) do not stain; Bar=17 mm. (D) Stoma in a Kalanchoë leaf section double stained for detection of NO and DNA fragmentation;
note, in the guard cell, left side, the TUNEL-positive nucleus (arrow) and the NO-positive cytosol; (v) vacuoles. Bar=6 mm.
The addition of 10−6, 10−4 and 10−2 M SNP, as well
as centrifugation, significantly increased the number of
cells showing NO and apoptosis. As predicted by Kojima
et al. ( Kojima et al., 1998b), a NO concentrationdependent enhancement of fluorescence was detected with
increasing SNP concentrations. These results show that
centrifugation (mechanical stress) induced a burst in NO
which correlated with DNA fragmentation, an effect that
was mimicked by the addition of SNP. This extends the
results with pea and carnation plants which released NO
to the atmosphere in response to drought stress
(Leshem, 1996).
NMMA, a NOS inhibitor, significantly decreased NO
production and apoptosis in centrifuged cells and tissues.
This implies that a putative NOS is operating in plants,
as earlier suggested (Delledonne et al., 1998; Durner
et al., 1998; Van Camp et al., 1998; Magalhaes et al.,
1999; Pedroso and Durzan, 1999b). The recent discovery
1034 Pedroso et al.
in Arabidopsis thaliana of a gene sequence with similarity
with the NOS from Rattus norvegicus ( Wambutt et al.,
unpublished results) reinforce this assumption. NO production was not, however, completely prevented by
NMMA. Similar results were reported for rat aortic
smooth muscle cells with 10 times the concentration used
in the present study ( Kojima et al., 1998b). In this work,
the fluorescence detected might have resulted from NO
generated before the inhibition of NOS, low levels of the
inhibitor, possible differences in enzyme activity
(Marletta, 1999), different affinity of plant NOS to
NMMA, or the presence of an alternative pathway for
NO formation in plants ( Yamasaki et al., 1999). This
prediction is supported by the results obtained with the
NMMA enantiomer, -NMMA, which showed nonspecific (-) NMMA activity in 14–26% of the
NO-stained cells.
In both species, there was a differential staining of
tissues and cells in all treatments. In SNP-treated samples,
staining was preferentially detected in the cytosol of
epidermal cells, whereas in centrifuged samples staining
was preferentially detected in parenchyma and guard
cells. NMMA significantly reduced, or totally eliminated,
the number of NO-stained chloroplasts in parenchyma
cells, but was less effective in reducing NO-staining in the
cytosol of epidermal cells. This difference can be partially
explained by how NO was generated in each case. In the
samples incubated in medium with SNP, NO was generated non-enzymatically, while in centrifuged samples NO
production was induced by centrifugation and prevented
with NMMA. Differences between guard and epidermal
cells in response to active oxygen species and cell death
were reported for tobacco (Allan and Fluhr, 1997).
Presence versus absence of chloroplasts, NO production
by nitrate reductase in the cytosol ( Yamasaki et al.,
1999), and the presence of NOS isoforms in different
tissues (Marletta, 1999) are factors to consider.
Previous studies had shown that centrifugation of plant
materials at 150×g induced a stress response, cell damage,
cell death, and cell proliferation (Pedroso and Durzan,
1998, 1999a, b). Cell damage caused by active oxygen
species (AOS) generated as a consequence of abiotic and
biotic stresses is widely documented ( Inzé and Montagu,
1995). The results of this study, using a probe that
specifically reacts with NO but not with AOS, revealed
the involvement of NO in stress responses and DNA
damage. Although a senescence-like process is induced in
leaves and leaf-plantlets cultured in 150×g, for 15 d
(Pedroso and Durzan, 1998, 1999a, b), until now the
results have pointed to NO as a cause (trigger) and not
an effect of senescence-associated proteolysis (Pedroso
and Durzan, 1999b). Besides, the decrease of apoptosis
in cells and foliar tissues centrifuged in the presence of
NMMA, demonstrated that NO preceded and was directly, or indirectly, responsible for nuclear DNA frag-
mentation leading to apoptosis, in both Kalanchoë and
Taxus species. These results parallel previous reports in
animals, where the increase in NO production, either
produced enzymatically or generated by SNP, led to
DNA damage ( Wink et al., 1991; MeBmer et al., 1994;
Tannenbaum, 1998) and to apoptosis (Dimmerler and
Zeiher, 1997). The present results suggest that a
NO-signalling pathway might have emerged early in
evolution and become highly conserved.
The reaction of NO with the superoxide anion is the
fastest biochemical rate constant currently known,
resulting in the formation of the potent oxidant, peroxynitrite (ONOO−) (Dawson, 1998). Peroxynitrite is a lipid
permeable molecule, with a wider range of chemical
targets than NO, which can oxidize proteins, lipids, RNA,
and DNA. As a consequence, alteration of receptor
functionality, activation/deactivation of enzyme systems
and depletion of ATP and NAD(P)H can occur and lead
to cell injury and cell death ( Kehrer, 1993). According
to Brüne et al. (Brüne et al., 1998), the toxicity of NO is
influenced by the relative rates of NO formation, oxidation and reduction, combination with oxygen, superoxide,
and other biomolecules. One predicted reaction site of
NO is the tyrosine residue in proteins, which would be
converted to 3-nitrotyrosine ( Eiserich et al., 1996). The
post-translational modification of tyrosine residues by
NO might affect processes dependent on tyrosine residue
phosphorylation as the cell cycle and apoptosis.
The biochemical, ecophysiological and evolutionary
characteristics of these taxonomically distant species,
could account for particular differences in NO-mediated
apoptosis. Both Kalanchoë ( Yamagishi et al., 1981) and
Taxus (Bidwell and Durzan, 1975; Durzan and Steward,
1983; Durzan, 1999) produce secondary metabolites with
anti-cancer activities which interfere with apoptosis (Oishi
and Yamaguchi, 1994; Suffness, 1995; Tepper et al., 1995;
Lanni et al., 1997; Al-alami et al., 1998). Kim et al.
suggest that the cancer-preventive activity of some plant
secondary metabolites may be related to their effect on
NO production and NOS activity ( Kim et al., 1998). In
Taxus, the action of NO may be complicated by the
formation of paclitaxel ( TaxolA), other taxanes
(Appendino, 1995; Platel, 1998) and monosubstituted
guanidines (Bidwell and Durzan, 1975; Durzan and
Steward, 1983) that are known inhibitors of NOS in
animal cells (Marletta, 1999). Paclitaxel increases protein–tyrosine phosphorylation (Manthey et al., 1992) and
induces p53-independent apoptosis (Lanni et al., 1997;
Al-alami et al., 1998; Durzan, 1999). Considering that
NO plays a vital role in mediating paclitaxel-induced
apoptosis (Al-alami et al., 1998), it is possible that
NO-mediated apoptosis in Taxus brevifolia might be
operative through a similar signalling pathway. Phorbol
esters produced by Kalanchoë, promote the expression of
the inducible NOS in cultured hepatocytes, inhibit
Nitric oxide and apoptosis
apoptosis induced by the Fas antigen, and may induce
apoptosis in HL-60 promyelocytic leukemia cells (Oishi
and Yamaguchi, 1994; Tepper et al., 1995). Interactions
between these anti-cancer metabolites and NO-mediated
apoptosis will require further study.
In summary, NO in plant cells can be visualized by
DAF-2 DA. Centrifugation with and without NMMA, a
NO-synthase inhibitor, showed that centrifugation
induced a burst in NO which preceded DNA fragmentation and subsequent cell death by apoptosis in Kalanchoë
(angiosperm) and Taxus (gymnosperm) cells. The effect
of centrifugation was mimicked by the addition of SNP.
These results support the role of NO as a stress signal
molecule and its involvement in plant apoptosis.
Acknowledgements
This work was partially supported by INVOTAN (fellowship
grant 3/B/96/PO), PRAXIS XXI (3/3.1/CTAE/1930/95),
EMBRAPA, and by NASA grant NAG 9–825.
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