Journal of Experimental Botany, Vol. 51, No. 347, pp. 1027-1036,
June 2000
© 2000 Oxford University Press
A nitric oxide burst precedes apoptosis in angiosperm and gymnosperm callus cells and foliar tissues
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 Brasília, DF-Brazil
3 Department of Environmental Horticulture, University of California, Davis, CA 95616–8587, USA
Received 26 November 1999; Accepted 3 February 2000
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Abstract |
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Leaves and callus of Kalanchoë daigremontiana and Taxus brevifolia were used to investigate nitric oxide-induced 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 transferase-mediated 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.
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Introduction |
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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 L-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 L-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 atoms or molecules with unpaired electrons, such as molecular oxygen (O2), superoxide (
), water (H2O), 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-L-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 () produces the toxic compound peroxynitrite (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 transferase-mediated 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.
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Materials and methods |
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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 D-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 D-glucose, 1 mg l-1 N6-benzylaminopurine 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 filter-sterilized 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 150xg, with or without 0.5 mM NG-monomethyl-L-arginine, NMMA (NO-synthase inhibitor). The NMMA enantiomer, NG-monomethyl-D-arginine, which does not have any significant effect on NO-synthase, was used as negative control to investigate non-specific L-NMMA activity. Samples kept for 3 h, in 1xg, 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 µM 4,5- diaminofluorescein diacetate (DAF-2 DA; Calbiochem, La Jolla, CA, USA) (Kojima et al., 1998b). Samples incubated at 1xg 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 
or 
(10 mg ml-1) and then incubated in DAF-2 DA, were 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 µg ml-1 4'-6-diamino-2-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 40x 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 µm. 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).
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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.
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Results |
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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).
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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.
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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 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).
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For both species, the number of DAF-2 DA-stained cells in medium supplemented with
or was 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 D-NMMA (negative control; L-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).
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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 controls (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 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 (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.
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Discussion |
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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 and 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.
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 concentration-dependent 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 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, D-NMMA, which showed non-specific (L-) 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 150xg 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 150xg, 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 fragmentation 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 (Taxol®), 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 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.
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Acknowledgments |
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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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Notes |
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4 To whom correspondence should be addressed. Fax: +351 21 75 000 48. E-mail: nop25892{at}mail.telepac.pt
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Abbreviations |
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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; xg, times unit gravity..
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References |
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Al-alami O, Sammons J, Martin JH, Hassan HT.1998. Divergent effect of taxol on proliferation, apoptosis and nitric oxide production in MHH 225 CD 34 positive and U 937 CD 34 negative human leukaemia cells. Leukaemia Research 22, 939–945.
Allan AC, Fluhr R.1997. Two distinct sources of elicited reactive oxygen species in tobacco epidermal cells. The Plant Cell 9, 1559–1572.[Abstract]
Appendino G.1995. Phytochemistry of the yew tree. Natural Products Reports 12, 349–360.
Bell PR.1994. Commentary. Apomictic features revealed in a conifer. International Journal of Plant Sciences 155, 621–622.
Bell PR.1996. Megaspore abortion: a consequence of selective apoptosis? International Journal of Plant Sciences 157, 1–7.
Bidwell RGS, Durzan DJ.1975. Some recent aspects of nitrogen metabolism. In: Davies, PJ, ed. Historical and recent aspects of plant physiology. Ithaca, NY: Cornell University Press, 152–225.
Brüne B, Knethen AV, Sandau K.1998. Nitric oxide and its role in apoptosis. European Journal of Pharmacology 351, 261–272.[Web of Science][Medline]
Dawson TM.1998. Nitric oxide, PARP and other perpetrators relevant to stroke and neurodegeneration. Progress in Brain Research ‘1998’. http://www.pasteur.fr/Conf/nitric-abstracts.html.
Dean JV, Harper JE.1988. The conversion of nitrite to nitrogen oxide(s) by the constitutive NAD(P)H-nitrate reductase enzyme from soybean. Plant Physiology 88, 389–395.
Delledonne M, Xia Y, Dixon RA, Lamb C.1998. Nitric oxide functions as a signal in plant disease resistance. Nature 394, 585–588.[Medline]
Dimmerler S, Zeiher AM.1997. Nitric oxide and apoptosis: another paradigm for the doubled edged role of nitric oxide. Nitric Oxide 1, 75–281.
Durner J, Wendehenne D, Klessig F.1998. Defense gene induction in tobacco by nitric oxide, cyclic GMP, and cyclic ADP-ribose. Proceedings of the National Academy of Sciences, USA 95, 10328–10333.
Durzan DJ.1999. Gravisensing, apoptosis, and drug recovery in Taxus cell suspensions. American Society for Gravitational and Space Biology Bulletin 12, 47–55.
Durzan D, Steward FC.1983. Nitrogen metabolism. In: Steward FC, ed. Plant physiology. an advanced treatise, Vol. VIII. New York: Academic Press, 55–265.
Durzan D, Ventimiglia F.1994. Free taxanes and the release of bound compounds having taxane antibody reactivity by xylanase in female. Haploid-derived cell suspension cultures of Taxus brevifolia. In vitro Cellular and Developmental Biology 30P, 219–227.
Eiserich JP, Cross CE, Jones AD, Haliwell B, Vliet AVD.1996. Formation of nitrating and chlorinating species by reaction of nitrite with hypochlorous acid. The Journal of Biological Chemistry 271, 19199–19208.
Gilchrist DG.1998. Programmed cell death in plant disease: the purpose and promise of cellular suicide. Annual Review of Phytopathology 36, 393–414.[Web of Science][Medline]
Greenberg JT.1996. Programmed cell death: a way of life for plants. Proceedings of the National Academy of Sciences, USA 93, 12094–12097.
Haramaty E, Leshem YY.1997. Ethylene regulation by the nitric oxide (NO) free radical: a possible mode of action of endogenous NO. In: Kanellis et al., eds. Biology and biotechnology of the plant hormone ethylene. The Netherlands: Kluwer Academic Publishers, 253–258.
Havel L, Durzan DJ.1996a. Apoptosis in plants. Botanica Acta 109, 1–10.
Havel L, Durzan DJ.1996b. Apoptosis during diploid parthenogenesis and early somatic embryogenesis of Norway Spruce. International Journal of Plant Sciences 157, 8–16.
Havel L, Durzan DJ.1999. Apoptosis during early somatic embryogenesis in Picea sp. In: Jain SM, Gupta PK, Newton RJ, eds. Somatic embryogenesis in woody plants. Dordrecht: Kluwer Academic (in press).
Inzé D, Montagu M.1995. Oxidative stress in plants. Current Opinion in Biotechnology 6, 153–158.
Kehrer J.1993. Free radicals as mediators of tissue injury and disease. Critical Reviews in Toxicology 23, 21–48.[Web of Science][Medline]
Kim OK, Murakami A, Nakamura Y, Ohigashi H.1998. Screening of edible Japanese plants for nitric oxide generation inhibitory activities in RAW 264.7 cells. Cancer Letters 125, 199–207.[Web of Science][Medline]
Klessig D, Durner J, Zang S, Wendehenne D, Du H, Chen Z, Anderson M.1999. Salicylic acid-mediated signal transduction in disease resistance. In: 17th Annual Missouri Symposium. Current topics in plant biochemistry, physiology and molecular biology. plant hormone: signalling gene expression. Columbia: University of Missouri, April 14–17.
Kojima H, Nakatsubo N, Kikuchi K, Kawahara S, Kirino Y, Nagoshi H, Hirata Y, Nagano T.1998b. Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins. Analytical Chemistry 70, 2446–2453.[Medline]
Kojima H, Sakurai K, Kikuchi K, Kawahara S, Kirino Y, Nagoshi H, Hirata Y, Nagano T.1998a. Development of a fluorescent indicator for nitric oxide based on the fluorescence chromophore. Chemical and Pharmaceutical Bulletin 46, 373–375.
Lancaster Jr JR.1992. Nitric oxide in cells. American Scientist 80, 248–259.
Lanni SS, Lowe SW, Lictria EJ, Liu JO, Jacks T.1997. p-53-independent apoptosis induced by paclitaxel through an indirect mechanism. Proceedings of the National Academy of Sciences, USA 94, 9679–9683.
Leshem YY.1996. Nitric oxide in biological systems. Plant Growth Regulators 18, 155–159.
Leshem YY, Haramaty E.1996. The characterization and contrasting effects of the nitric oxide free radical in vegetative stress and senescence of Pisum sativum Linn. foliage. Journal Plant Physiology 148, 258–263.
Leshem YY, Wills RBH, Ku V-VV.1998. Evidence for function of the free radical gas–nitric oxide (NO·)—as an endogenous maturation and senescence regulating factor in higher plants. Plant Physiology and Biochemistry 36, 825–833.[Web of Science]
Magalhaes JR, Pedroso MC, Durzan D.1999. Nitric oxide, apoptosis and plant stresses. Physiology and Molecular Biology of Plants 5, 115–125.
Manthey CL, Brandes ME, Perera PY, Vogel SN.1992. Taxol increases steady-state levels of lipopolysaccharide-inducible genes and protein-tyrosine phosphorylation in murine macrophages. Journal of Immunology 149, 2459–2465.[Abstract]
Marletta MA.1999. Cellular signalling with nitirc oxide. In: Calbiochem signal transduction catalog and technical resource. San Diego, CA: Calbiochem-Novabiochem, Co., 419–422.
MeBmer UK, Ankarcrona M, Nicotera P, Brüne B.1994. p53 expression in nitric oxide-induced apoptosis. FEBS Letters 355, 23–26.[Web of Science][Medline]
Moncada S.1998. Nitric oxide as a biological mediator—a ten year perspective. Nitric oxide: basic research and clinic application-extensive abstracts 1, 6–11.
Murashige T, Skoog F.1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15, 473–497.
Nakatsubo N, Kojima H, Kikuchi K, Nagoshi H, Hirata Y, Maeda D, Imai Y, Irimura T, Nagano T.1998. Direct evidence of nitric oxide production from bovine aortic endothelial cells using new fluorescence indicators: diaminofluoresceins. FEBS Letters 427, 263–266.[Web of Science][Medline]
Oishi K, Yamaguchi M.1994. Effect of phorbol 12-myristate 13-acetate on Ca2+-ATPase activity in rat liver nuclei. Journal of Cellular Biochemistry 55, 168–172.[Web of Science][Medline]
Pedroso MC.1998. Report 3/B/96/PO. Instituto de Cooperacao Cientifica e Tecnologica Internacional, Lisboa: INVOTAN, 15–71.
Pedroso MC, Durzan D.1998. Effect of hypergravity and simulated hypogravity on leaf-plantlet development and asexual reproduction of Kalanchoë daigremontiana. Gravitational and Space Biology Bulletin 12, 74 (146).
Pedroso MC, Durzan D.1999a. Effect of gravitational stimuli on leaf-plantlet formation and asexual reproduction of Kalanchoë daigremontiana. Life Odyssey Symposium-7th European Symposium on Life Sciences Research in Space, Maastricht, the Netherlands, 29 May-2 June (in press).
Pedroso MC, Durzan D.1999b. Detection of apoptosis in chloroplasts and nuclei in different gravitational environments. 20th Annual International Gravitational Physiology Meeting, 6–11 June, Orlando, Florida (in press).
Pennel RI, Lamb C.1997. Programmed cell death in plants. The Plant Cell 9, 1157–1168.[Web of Science][Medline]
Platel RN.1998. Tour de paclitaxel. Annual Review of Microbiology 98, 361–395.
Pryor WA, Squadrito GL.1995. The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide? American Journal of Physiology 268, L699-L700.
Rucki R.1977. Sodium nitroprusside. In: Florey K, ed. Analytical profiles of drug substances, Vol. 6. New York: Academic Press, 781–792.
Schmidt HHW, Warner TD, Ishii K, Sheng H, Murad F.1992. Insulin secretion from pancreatic B cells caused by L-arginine-derived nitrogen oxides. Science 255, 721–724.
Schmidt HHW, Frohlich L, Pfleiderer W, Kotsonis P.1998. Pterins and anti-pterins in NO synthesis. Nitric oxide research and clinical applications. extensive abstracts. http://www.pasteur.fr/Conf/nitric-abstracts.html.
Stamler JS, Singel DJ, Loscalzo J.1992. Biochemistry of nitric oxide and its redox-activated forms. Science 258, 1898–1902.
Suffness M.1995. Overview of taxol research: progress on many fronts. In: Georg G, Chen TT, Ojima I, Vyas DM, eds. Taxane anticancer agents: basic science and current status. American Chemical Society Symposium Series 83, 1–17.
Tannenbaum SR.1998. Effects of NO on DNA damage and DNA synthesis. Nitric oxide:basic research and clinical applications—extensive abstracts. http://www.pasteur.fr/Conf/nitric-abstracts.html.
Tepper CG et al. 1995. Role of ceramide as an endogenous mediator of FAS-induced cytotoxicity. Proceedings of the National Academy of Sciences of the United States of America 92, 8443–8447.
Van Camp W, Van Montagu M, Inzé D.1998. H2O2 and NO: redox signals in disease resistance. Trends in Plant Science 3, 330–334.
Wang H, Li J, Bostock RM, Gilchrist DG.1996. Apoptosis: a functional paradigm for programmed cell death induced by a host-selective phytotoxin and invoked during development. The Plant Cell 8, 375–391.[Abstract]
Wink DA, Kasprzak SK, Maragos CM, Elespuru RK, Misra M, Dunams TM, Cebula TA, Koch WH, Andrews AW, Allen JS, Keefer LK.1991. DNA deaminating ability and genotoxicity of nitric oxide and its progenitors. Science 254, 1001–1003.
Yahraus T, Chandra S, Legendre L, Low PS.1995. Evidence for a mechanically induced oxidative burst. Plant Physiology 109, 1259–1266.[Abstract]
Yamagishi T, Haruna M, Yan X-Z, Chang J-J, Lee K-H.1981. Antitumor agents, 110. Bryophyllin B, A novel potent cytotoxic bufadienolide from Bryophyllym pinnatum. Journal of Natural Products 52, 1071–1979.
Yamasaki H, Sakihama Y, Takahashi S.1999. An alternative pathway for nitric oxide production in plants: new feature of an old enzyme. Trends in Plant Science 4, 128–129.[Web of Science][Medline]
Yeng C-H, Yang C-H.1998. Evidence for programmed cell death during leaf senescence in plants. Plant Cell Physiology 39, 922–927.
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