- DAF-FM DA
nitric oxide synthase
programmed cell death
reactive oxygen species.
Cell death can be induced by cytokinin 6-benzylaminopurine (BA) at high dosage in suspension-cultured Arabidopsis cells. Herein, we provide evidence that BA induces nitric oxide (NO) synthesis in a dose-dependent manner. A reduction in cell death can be observed when the cytokinin is supplemented with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or the nitric oxide synthase (NOS) inhibitors: 2-aminoethyl-isothiourea (AET) and NG.-monomethyl- l-arginine ( l-NMMA), which suggests that NO is produced via a NOS and is a signalling component of this form of programmed cell death. In BA-treated cells, mitochondrial functionality is altered via inhibition of respiration. This inhibition can be prevented by addition of either cPTIO or AET implying that NO acts at the mitochondrial level.
In both animals and plants, programmed cell death (PCD) is a genetically determined process leading to cell suicide (Havel & Durzan 1996). In plants, PCD occurs in many forms and plays an important role in cellular defence and development; during this process specific groups of cells are induced to die. The hypersensitive response and senescence are two of the different forms of PCD in plants. Different types of molecules, such as endogenous plant growth regulators (Hoeberichts & Woltering 2003), fungal elicitors (Hammond-Kosack & Jones 1996), reactive oxygen species (Levine et al. 1994), nitric oxide (NO) (Delledonne et al. 1998), salicylic acid (Dempsey, Shah & Klessig 1999) and others have been proposed to be involved in either induction or signal transduction of PCD. Recently, cytokinins have been demonstrated to induce programmed cell death, both in animals and plants (Ishii et al. 2002; Mlejnek & Prochazka 2002; Carimi et al. 2003). The cytokinin 6-benzylaminopurine (BA), if added at high dosage to cultivated cell suspensions of Arabidopsis, induces programmed cell death by accelerating senescence. In fact, cell death and oligonucleosomal DNA fragmentation, due to high levels of the cytokinin, is preceded by the early expression of SAG12, a cysteine protease that is a typical senescence-associated marker not found in other forms of PCD (Carimi et al. 2004).
The signalling intermediates of cytokinin-induced PCD have not yet been identified. In animal systems, reactive oxygen species (ROS) have been demonstrated to be involved in BA-induced PCD. In fact, Ishii et al. (2002) reported that apoptosis was significantly reduced when human myeloid leukemia HL-60 cells were incubated with cytokinin ribosides in the presence of a superoxide scavenger or antioxidant. In plants, Mlejnek, Dolezel & Prochazka (2003) reported that the cytokinin benzyladenosine induced oxidative stress in tobacco suspension cultures. However, the authors concluded that the formation of ROS did not appear to be the event governing the cellular decision for PCD.
In recent years, NO has emerged as a key signalling molecule in plants. This small water and lipid soluble gas is involved in several biological processes, including stomatal closure, seed germination, root development, expression of defence-related genes and PCD (Zottini et al. 2002; Neill, Desikan & Hancock 2003; Lamotte et al. 2004). It has been reported that in some cases NO mediates the biological effects of primary signalling molecules such as hormones (Neill et al. 2002). Its synthesis has also been shown to be induced by cytokinins in Arabidopsis, tobacco and parsley cultured cells (Tun, Holk & Scherer 2001).
These results prompted us to analyse the role of NO in cytokinin-induced PCD in Arabidopsis suspension cells. In this report we provide data suggesting that NO signalling is involved in BA-induced PCD, most likely via inhibition of mitochondrial respiration.
MATERIALS AND METHODS
Cell cultures and treatments
The Arabidopsis cell line was generated from hypocotyls dissected from young plantlets of Arabidopsis thaliana (L) Heynh. ecotype Landsberg erecta (Ler), and subcultured in AT3 medium (Desikan et al. 1996). Culture conditions and treatments have previously been described in detail (Carimi et al. 2003).
Two different compounds were utilized to inhibit nitric oxide synthases (NOS). The animal non-aminoacid based-inhibitor 2-aminoethyl-isothiourea (AET; Southan, Szabo & Thiemermann 1995) was from Sigma Aldrich; the animal (Southan & Szabo 1996) and plant (Foissner et al. 2000; Garces, Durzan & Pedroso 2001) NOS l-arginine-based inhibitor NG-monomethyl- l-arginine ( l-NMMA) as well as the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO; Pfeiffer et al. 1997) were from Alexis Biochemicals, Vinci, Italy. Cytokinin was added to 3-day-old cells; AET (1 m m), l-NMMA (1 m m) or cPTIO (0.2 m m) were added 1 h before cytokinin treatment. In the experiments regarding ‘spontaneous’ senescence, l-NMMA or cPTIO were added to the medium 3 d after culture initiation.
Imaging of intracellular nitric oxide production
The cell-permeable diacetate derivative diaminofluorescein-FM (DAF-FM DA, Alexis Biochemicals, Cod 620–071-M001) was used as a fluorescent probe for the detection of NO in cells (Kojima et al. 1998). In the presence of NO and O2, DAF probes are converted to the fluorescent triazole derivative thereby increasing the quantum yield of fluorescence more than 180-fold (Nakatsubo et al. 1998). A 3-day-old suspension culture, corresponding to 50 µL of packed cells, was added to a solution containing 0.5 µm DAF-FM DA in a final volume of 1 mL of AT3 medium. After 15 min incubation at 25 °C on a rotating plate agitator, cells were washed with 1.5 mL AT3 medium for 20 min. For microscopic analysis cells were layered onto a coverslip.
To determine the effect of BA in planta, seedlings of A. thaliana ecotype Wassileskija were used. Five-day-old seedlings were incubated for 15 min in loading buffer (5 m m MES-KOH, pH 5.7, 0.25 m m KCl, 1 m m CaCl2), with or without NOS inhibitors (1 mm l-NMMA, 1 m m AET) or the NO scavenger (0.2 m m cPTIO). The seedlings were then incubated for 15 min in the same loading buffer containing 15 µm DAF-FM DA, followed by 20-min of washing in order to remove excess fluorescent probe. The seedlings were subsequently incubated in buffer with or without BA for 2 h in the presence or absence of the NOS inhibitors or the NO scavenger.
DAF-FM DA fluorescence was estimated using confocal laser scanning microscopy (excitation 488 nm, emission 515–530 nm; Nikon PCM2000). The images acquired from the confocal microscope were processed using the software Corel Photo-Paint (Corel Corporation, Dallas, TX, USA) and relative pixel intensities determined using Scion Image software (Scion Corporation, Frederick, MD, USA).
Extracellular NO quantification by fluorimetric analysis with DAF-2
NO was determined by binding to 4,5-diaminofluorescein (DAF-2; Alexis Biochemicals, Cod 620–052-M001) in a fluorimetric assay (Nakatsubo et al. 1998). Fluorescence measurements were performed with a Perkin Elmer LS-55 Luminescence Spectrometer with an excitation wavelength of 495 nm and an emission wavelength of 515 nm, with a slit width of 3 nm.
We followed the procedure of Tun et al. (2001) with slight modifications: 25.5 mg of cells were transferred into 2 mL buffered medium consisting of 50 µL of 0.5 m sodium phosphate buffer (pH 7.2), 1 mL of H2O, 2.5 µm DAF-2 and AT3 medium to the final volume. After 15 min incubation at 25 °C on a rotating plate agitator, the DAF-2-loaded cell suspension was transferred to a 10-mm quartz cuvette and the fluorescence emission was determined at 24 °C with slow stirring. All reactions were carried out at least in duplicate and their reproducibility was checked. Each experiment was repeated three times. Relative fluorescence is expressed as the ratio to the control values obtained with untreated cells.
Cell viability and measurement of oxygen consumption
Cell death was determined by spectrophotometric measurements of the uptake of Evan's blue, as described by Gaff & Okong'o-Ogola (1971).
Respiratory activity was measured as previously described (Zottini et al. 2002). Briefly, 100 mg of cells were re-suspended in 1.3 mL of culture medium and analysed for O2 consumption in a glass chamber, maintained at 25 °C, equipped with a Clark-type oxygen electrode (Rank Brothers, Bottisham, UK) connected to a chart recorder. The oxygen electrode was calibrated with air-saturated medium kept at 25 °C, assuming an oxygen concentration of 240 µm. Then 13 µm BA was added to 3-day-old cells; AET (1 m m) or cPTIO (0.2 m m) were added 1 h before cytokinin treatment.
For DNA extraction, cells were ground using a mortar and pestle with liquid N2. Genomic DNA was isolated as described by Doyle & Doyle (1987) and quantified by measurement of the OD260 as described by Sambrook, Fritsch & Maniatis (1989). For DNA fragmentation analysis, 10 µg of each sample was electrophoresed on 1% (w/v) agarose gels containing 1 × TAE (40 m m Tris-acetate, 1 m m EDTA) and stained with ethidium bromide.
BA induces NO synthesis in Arabidopsis cells
The effect of BA on NO accumulation was determined by using the cell-permeable fluorescent probe DAF-FM DA, which allows the direct detection of NO at the cellular level. NO production was induced in BA-treated cells in comparison with control cells (Fig. 1a & b). In control, untreated cells low-levels of fluorescence were observed that were mainly associated with chloroplasts (Fig. 1a), whereas in 27 µm BA-treated cells the fluorescence was higher and more diffuse in the cytosol (Fig. 1b). Two hours after BA treatment, the average fluorescence intensity increased by 76% (Fig. 1c).
Effects of different levels of BA on Arabidopsis cells
One of the simplest and most sensitive probes for the quantification of NO is DAF-2. Accordingly, we therefore used this probe in order to measure the release of NO from cells treated with different levels of BA.
Figure 2a shows the release of NO 1 h after the addition of 4, 13 and 27 µm BA to 3-day-old cells. At these concentrations, fluorescence levels increased by 1, 22 and 34%, respectively, demonstrating the dose-dependent nature of NO production. Moreover, both the fresh weight and cell death were dependent on the concentration of BA (Fig. 2a & c). The presence of 4 µm BA in the medium had only a moderate effect on cell growth and no effect on cell viability (12.5 versus 10.4% of control). The other two concentrations of BA used (13 and 27 µm) reduced cell growth to about 50 and 20% of control, and induced 29 and 41% cell death, respectively, after 4 d.
BA-induced PCD requires NO synthesis
To test the effect of BA after longer incubation periods, we measured the NO release from cells treated for 1, 2 and 3 d. The results show that at all these time points, the amount of NO released, remained higher with respect to untreated, control cells. In order to test the hypothesis that BA-induced PCD involves the synthesis of NO we measured the effect of the NO scavenger cPTIO (0.2 m m) on cell death and cell growth induced by BA. Pre-treatment with cPTIO reduced the accumulation of NO (Fig. 3a), increased cell growth (360 versus 230 mg mL−1 of BA-treated cells), and more than halved cell death (Fig. 3c). We next pretreated cells with the NOS inhibitor AET (1 m m) for 1 h. Tun et al. (2001) have previously shown that the animal NOS inhibitor AET inhibits the release of NO in tobacco cells treated with cytokinins. Likewise, in our experiment the production of NO was prevented and the NO level became lower than in untreated cultures (Fig. 3a).
The reduced cell growth and the increased cell death induced by BA were both inhibited by pretreatment with AET (cell growth 400 versus 230 mg mL−1 of BA-treated cells, cell death 17 versus 29% of BA-treated cells; Fig. 3b & c). These results suggest that NO is a signalling component of this form of programmed cell death induced by BA and that NO production probably occurs via a NOS enzyme. When added alone, neither the NOS inhibitor nor the NO scavenger altered cell viability (data not shown).
Mitochondrial functionality in BA-treated cells
NO is able to inhibit cell respiration in plants by interacting with components of the respiratory chain (Zottini et al. 2002). Since we measured NO production in Arabidopsis cells after treatment with BA, we wanted to investigate whether mitochondrial functionality was impaired. For this purpose, 3-day-old Arabidopsis cells were incubated with 13 µm BA and, after 24 h, respiratory activity was measured using 100 mg of treated and untreated cells (Fig. 4). It was found that the rate of O2 consumption was inhibited by 36% with respect to controls. This inhibition was prevented by the NO scavenger pretreatment: in this case respiration was nearly similar to control cells (90% O2 consumption). When cells were pretreated for 1 h with the NOS inhibitor, the inhibition of O2 consumption was 14%.
BA induces NO synthesis in planta
In order to verify the NO production induced by BA in plant tissues we treated young Arabidopsis seedlings with BA (27 µm) for 2 h in presence or absence of the NO scavenger cPTIO and the NOS inhibitors AET or l-NMMA. The latter has been shown by Garces et al. (2001) to inhibit a NOS enzyme cloned and characterized from Arabidopsis. Endogenous NO production was examined in roots by using the cell-permeable fluorescent probe DAF-FM DA.
When seedlings were treated with BA, the increase of fluorescence signal was clearly observed in roots (Fig. 5b). In addition, BA-induced NO production was inhibited by the NOS inhibitors (Fig. 5c & d) and was further reduced by cPTIO (Fig. 5e).
Senescence in Arabidopsis suspension cultured cells
In a previous report, we have shown that high levels of BA were able to induce PCD by accelerating a senescence process in cell culture (Carimi et al. 2003, 2004). We thus wanted to investigate if production of NO is also associated with the ‘spontaneous’ senescence that normally occurs at the end of the subculture cycle if the medium is not renewed.
Arabidopsis cultures are subcultured with a cycle of approximately 10 d (Fig. 6a): this period comprises the initial adaptation phase, followed by the exponential growth phase up to the stationary phase. If the medium is not changed at this time (10–13 d) a rapid decrease in cell fresh weight is observed (decline phase).
The examination of the survival curves suggests that this decrease is due to the rapid increase of cell death (Fig. 6b). Agarose gel analysis of DNA isolated from suspension cultures collected at different times after culture initiation showed that DNA laddering (a characteristic feature of apoptosis) occurred in a time-dependent fashion (Fig. 6c). If the medium was not changed, DNA laddering was observed 14 d after culture initiation (when cell death reaches 40%) and was more pronounced afterwards.
The results presented in Fig. 6d clearly indicate that NO production increased during the progression of ‘spontaneous’ senescence. In fact, in suspension cultures, the amount of NO released from cells (measured as described in the Materials and Methods), began to rise at the end of the exponential growth phase and continued to increase during the stationary and decline phases (Fig. 6d). Cell death and DNA fragmentation during the ‘spontaneous’ senescence are preceded by an increase in NO. This result leads to the question of whether NO is consequence or cause of ‘spontaneous’ senescence observed in suspension culture. To address this question, we measured the effect of the NO scavenger cPTIO and NOS inhibitor l-NMMA on senescence-associated cell death.
In the presence of NO scavenger or NOS inhibitor (added 3 d after culture initiation), the accumulation of NO was prevented and the NO levels became lower than untreated cultures (Fig. 7a). Cell death (Fig. 7b), as compared to control cells, was reduced: at T17 cell death reaches 30% and DNA laddering was not observed, which indicates that NO affects the triggering of cell senescence.
In the presence of the NOS inhibitor, or the NO scavenger, cell growth was comparable with the control untreated cells until the end of the exponential growth phase (11 d after subculture initiation). Instead after 11 d untreated cultures started to senesce (measured as decline in fresh weight) whereas the NOS inhibitor and the NO scavenger allowed the continued growth of the cultures (Fig. 7c).
Recently, the role of cytokinins as a signalling molecule in plants and animals during cell death has received much consideration (Ishii et al. 2002; Mlejnek & Prochazka 2002; Carimi et al. 2003). The fact that this death is programmed is shown by the characteristic oligonucleosomal degradation of DNA. However, the precise role of the cytokinin in PCD and its relation with other signalling molecules still need clarification.
While it has been shown that benzyladenosine positively affects the level of ROS in tobacco cells (Mlejnek et al. 2003), the same authors also reported that the selective inhibition of ROS production did not provide any protection to cells. This result was interpreted in terms of reduced endogenous levels of ATP. In experiments on Arabidopsis cell cultures, we observed that ROS level was increased in cells incubated with cytokinin, although the presence of the superoxide scavenger was not able to reduce cell death (data not shown). A recent paper reported that the PCD induced by cryptogein required NO, but was independent of H2O2 (Lamotte et al. 2004).
In this report we show that NO is produced in suspension cell cultures treated with BA in a dose-dependent manner. Neither the release of NO nor cell death was influenced by low levels of BA (4 µm). Higher levels of the cytokinin (13–27 µm) had effects on both NO production and cell death.
NO appears to be produced via a NOS enzyme since both its level and cellular effects, namely the inhibition of cell growth and cell death, were strongly reduced by pretreatment with the NOS inhibitor AET and not only with the NO scavenger cPTIO. A key experiment to investigate the role of NO in BA-induced PCD showed that cell death was significantly reduced and cell growth inhibition was attenuated in cells incubated with BA in the presence of the NOS inhibitor.
Interestingly, we observed for the first time that BA also induces NO production in plants at the level of root tissues, and that this production can be reduced by pretreatment with a NO scavenger (cPTIO) and NOS inhibitors (AET and l-NMMA).
It is known that NO can also be produced by nitrate reductase, which can be induced in environmental conditions that are unfavorable to photosynthesis (i.e. dark, photosynthesis inhibitors) (Rockel et al. 2002). However the inhibitory effect of AET or l-NMMA shows that in the present case at least a significant proportion of NO is synthesized by NOS.
In a previous report we showed that NO affects mitochondrial functionality in plant cells and reduces total cell respiration due to strong inhibition of the cytochrome pathway (Zottini et al. 2002). The results presented here show that treatment with 13 µm BA reduced total cell respiration by approximately 36% within 24 h. In addition, we observed that the reduction of respiration is lower if cells were pretreated with the NO scavenger (reduced by 10%) or the NOS inhibitor (reduced by 14%). These results demonstrate that the reduction of cell respiration in cytokinin-treated cells is mediated by NO.
It should also be noted that the inhibition of mitochondrial respiration occurs as an early event in the cell death programme, since mitochondrial functionality is impaired even when cell viability is still not affected. The observation that the ATP content is depleted when cells are treated with cytokinins (Mlejnek, Dolezel & Prochazka 2003), another indication of mitochondrial involvement in cytokinin action, is in agreement with both the present results and the previous observation of the early release of cytochrome c from mitochondria in BA-treated cells (Carimi et al. 2003).
In the literature we find contrasting reports on the effect on senescence of NO and, for that matter of BA [see, e.g. Leshem & Haramaty (1996); Leshem, Willis & Ku (1998); Magalhares, Monte & Durzan (2000) or the editorial comment of Eckardt (2003) in a recent issue of The Plant Cell]. In a previous paper we demonstrated that high levels of BA are able to induce PCD by accelerating senescence in cell culture (Carimi et al. 2004). In the present report we demonstrate that BA treatment induces NO production, and that NO levels increase during the progression of ‘spontaneous’ senescence. In addition, either a NOS inhibitor or NO scavenger prevents the increase of NO levels during the progression of senescence, and, moreover, cell death is reduced (Fig. 7) and DNA laddering was not detected (not shown). In our cell culture conditions (Fig. 6a), DNA laddering becomes evident only when cell death approaches 40% (Fig. 6b & c). These results are indicative of the involvement of endogenous NO metabolism in the progression of ‘spontaneous’ senescence. Taken together, these data suggest that NO is a key player both in spontaneous and in BA-induced PCD/senescence and that NO synthesis proceeds, at least partially, via a NOS-dependent route.
This work was supported by the PRIN Program of the Italian Ministry of Scientific Research. Michela Zottini was supported by ‘Progetto giovani ricercatori’ grant of the University of Padova. We are indebted to Elisabetta Barizza for technical assistance.