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Keywords:

  • apoptosis;
  • Brassica napus;
  • necrosis;
  • programmed cell death;
  • protoplast

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • •  
    The cleavage of nuclear DNA into oligonucleosomal fragments that is the hallmark of apoptosis in animal cells occurs during the culture of Brassica napus leaf protoplasts.
  • •  
    The changes in nuclei of cultured Brassica napus leaf protoplasts were studied by propidium iodide (PI) and 4′, 6-diamino-2-phenylindole, dihydrochloride (DAPI) staining, transmission electron microscopy, flow cytometry analysis, and DNA laddering staining with ethidium bromide and Southern hybridization.
  • •  
    Free 3′-OH termini of nuclear DNA fragments were labelled with DIG-dUTP, catalyzed by terminal deoxynucleotidyl transferase (TdT), and used as probes for Southern hybridization. This method (TUNEL on membrane) allowed visualization of DNA fragments with 3′-OH termini on a nylon membrane.
  • •  
    These results suggest that loss of viability of protoplast with culture time is accompanied by apoptosis-like cell death. However, the forms or processes undergoing to apoptotic cell death in B. napus leaf protoplasts appears to be different in some details to those in animal cells.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plant protoplasts can provide solutions for improving plants by either DNA transfer or by somatic hybridization. In order to attain this goal, a method to regenerate a complete plant from the altered cell must be available to produce transgenic plants by protoplast techniques. Regeneration from protoplasts has been observed in only a limited number of the many plant species which have been studied to date. Leaf mesophyll protoplasts have the advantage that the cells are at the same cell stage, and of a homogenous genetic background. These protoplasts, however, must undergo a process of dedifferentiation before plant regeneration. Events during the process resulting in totipotency remains largely unknown. Protoplasts from many important agricultural crops show recalcitrance to regenerate into whole plants. Protoplast recalcitrance can be expressed at several points: one of which is initiation of cell division. Our previous study showed that Brassica napus leaf protoplasts initiate senescence during the isolation of protoplasts, and senesce thereafter (Watanabe et al., 1998). In view of the previous evidence, and our interest in developing protoplast technology in important agricultural species (many of which we cannot currently regenerate from protoplasts), we have investigated the cause of failure to initiate cell division.

A range of endogenous and exogenous factors effects plant regeneration. The use of different plant species, or even different genotypes within one species, can result in different levels of regeneration (Roest & Gillissen, 1989; Roubelakis, 1993). The conditions of the isolation procedure and the properties of the incubation medium also affect the overall quality of protoplasts and their regeneration potential. Meyer et al. (1993) stated that the procedures used to isolate the protoplasts have more of an effect on their ability to regenerate than the composition of the medium. It is nonetheless inevitable that both the isolation procedure and the medium in which the protoplasts are grown must be determined empirically for any given system. Many papers have described detailed isolation procedures, and the techniques and criteria for specific systems.

Programmed cell death is the active process of cell death which occurs during development and in response to environmental signals (Greenberg, 1996). There are a number of other investigations of plant tissues in which programmed cell death is supposed to occur, for example, in root cap cells (Wang et al., 1996a), in root cortical cells (Liljeroth & Bryngelsson, 2001), in the barley aleurone during germination (Wang et al., 1996b), during tracheary element differentiation (Mittler et al., 1995), in natural leaf senescence (Cheng-Hung & Chang-Hsien, 1998), during salt stress-induced cell death of barley roots (Katsuhara & Kawasaki, 1996), in a resistant cultivar of cowpea after infection with the cowpea rust fungus (Ryerson & Heath, 1996), in ion-beam irradiated maize roots (Kawai et al., 2000), and in ozone-induced cell death of Arabidopsis thaliana (Rao & Davis, 1999). Most of these studies investigate plants either undergoing the hypersensitive response or during abiotic stress. Furthermore, programmed cell death is thought to occur in carrot suspension cells cultured at low cell density (McCabe et al., 1997), in senescence of tobacco suspension cells (O’Brien et al., 1998) and in aluminum and iron-induced tobacco suspension cells (Ikegawa et al., 1998).

Petunia leaf protoplasts can initiate cell division and form calli. Their plating efficiency was 0.7% (Watanabe et al., 1992). The reasons why recalcitrant leaf protoplasts are unable to divide are unknown. We have investigated this phenomenon in leaf protoplasts isolated from a recalcitrant inbred line of Brassica napus, and tried to elucidate the recalcitrance expressed at the point of cell division. We present evidence that B. napus leaf protoplasts die through a process of programmed cell death or apoptosis similar to that observed in animal cells with a few morphological differences. This is the first report that apoptotic-like cell death of leaf protoplasts has been a determining factor which does not carry out cell division.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plant materials

Seeds of B. napus L. cv. Bronowski were obtained from the National Institute of Agrobiological Resources (Tsukuba, Japan). Plants were grown under a 14-h photoperiod, at a photon fluence density of 50 µmol m−2 s−1 from fluorescent tubes and on a temperature day/night cycle of 25/20°C.

Protoplast isolation and culture

Protoplasts were prepared from leaves of 6–8-wk-old plants as previously described (Watanabe et al., 1992). The sterilized leaves were cut into narrow strips and incubated into an enzyme solution containing 1.0% Cellulase Y-C (Kyowa Kasei Co Ltd, Osaka, Japan), 0.1% Pectolyase Y-23 (Kyowa Kasei Co Ltd), 5 mM MES (pH 5.8) and 0.6 M sorbitol. The isolated protoplasts were cultured in 6 well multidishes at 5 × 105 per well in Murashige Skoog (MS) medium (Murashige & Skoog, 1962) supplemented with 4.5 µM 2,4-D, 2.2 µM BA, 2% sucrose, 1% glucose and 0.5 M sorbitol (pH 5.8). 0.8% agar was layered on bottom of each well, which prevented the adhesion of the protoplasts to the bottom. The dishes were maintained in the dark at 25°C.

Protoplast viability assay

FDA-PI stain The viability of protoplasts was monitored by staining with fluorescein diacetate-propidium iodide (FDA-PI) on a Nikon ECLIDSE TE300 inverted microscope (Nikon, Tokyo, Japan). To stain with FDA-PI, stock solutions of FDA and PI were added to the cultured protoplasts at the final concentration of 12 µg and 5 µg per ml of culture medium, respectively. The protoplasts were stained for 5min at room temperature in the dark and viable (green)/non-viable (red) cells were observed with B-2 filter and G filter, respectively.

Evans Blue stain

Protoplasts were incubated with 0.01% (w/v) solution of Evans blue in culture medium for 10 min and viable protoplasts were identified on the basis of stain exclusion (Graff & Okong O-Ogola, 1971). The number of stained and unstained protoplasts was counted with Fuchs-Rosenthal haemocytometer (MINATO MEDICAL Co., Tokyo, Japan).

Adenosine 5′-triphosphate (ATP) determination

Protoplasts were washed and resuspended in 0.6 M sorbitol, and then diluted 10-fold (5 × 104 ml−1). ATP determination was based on the luciferin-luciferase luminescence assay by using CheckLite 250 Plus and Lumitestor C-100 (Kikkoman Co. Noda, Japan). We followed the manufacturer's protocol precisely. Protoplast viability was expressed as relative light units (RLU) depending on the amount of ATP luminescence.

2, 3, 5-Triphenyl tetrazolium chloride (TTC) reduction

The washed protoplasts were resuspended in 0.05 M phosphate-0.6 M sorbitol buffer containing 0.1% TTC. TTC reduction was determined as described previously (Watanabe et al., 1992).

Chromatin condensation and DNA fragmentation

Microscopic analysis Protoplasts were fixed in 2% glutaraldehyde in sorbitol buffer (10 mM Phosphate buffer (pH 7.4), 0.6 M Sorbitol) for 2 h at room temperature. The protoplasts were washed and resuspended in the sorbitol buffer at the final condensation of 1.0 × 106 protoplasts ml−1. 4′, 6-diamino-2-phenylindole, dihydrochloride (DAPI) was added to the suspension at the final concentration of 10 µg ml−1.

Flow cytometry

Before being subjected to flow cytometric analysis, nuclei were released from protoplasts by suspension in solution A of the plant high-resolution DNA kit type P (Partec, Munster, Germany). After incubating the suspension for 5 min at room temperature, 1.5 ml of DAPI staining solution (Mishiba et al., 2001) was added to the suspension and filtered through 30 µm nylon mesh. After 5 min of staining, the suspension of nuclei was run through a Partec PA cytometer (Partec, Munster, Germany) equipped with a mercury lamp. Filter combinations of KG1, BG38, UG1, TK420 and GG435 were used.

Electron microscopy

Leaf protoplasts were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.1, containing 0.6 M sorbitol to sustain the osmotic conditions of the fixation medium, for 1 h at room temperature. They were twice moved into fresh 0.1 M phosphate buffer (pH 7.1) containing 0.6 M sorbitol and left at room temperature for 1 h. The materials were then fixed in 2% osmium tetroxide in the same buffer for 2 h at 4°C and dehydrated through an ethanol series. The specimen were embedded in Spurr's epoxy resin (Spurr, 1969). Ultrathin sections were cut on a Reichert Ultracut E ultramicrotome, and stained with uranyl acetate and lead citrate. The sections were examined with a Hitachi H-7000 electron microscope at 80 kV.

Nuclear DNA extraction and electrophoresis

B. napus leaf protoplasts were collected from multidish wells and centrifuged at 180 g for 10 min. The protoplasts were homogenized in 5 ml of nuclear buffer from FLORACLEAN (BIO 101, Vista, CA, USA), and the nuclear DNA was extracted according to the manufacturer's protocol. 0.5 µg nuclear DNA was loaded per lane and run on a 2% (w/v) agarose gel at constant 100 V. DNA was visualized by staining with 0.1 µg m−1 ethidium bromide.

Southern hybridization

Nuclear DNA was blotted onto Immobilon-Ny + nylon membranes (MILLIPORE, Tokyo, Japan) and hybridized to alkaline phosphatase labelled nuclear DNA probes, following the protocol for the AlkPhos Direct system (Amersham Pharmacia Biotech, Little Chalfont, UK). The resulting signal was detected by the CDP-Star chemiluminescent detection reagent according to manufacturer's protocol (Amersham Pharmacia Biotech, Tokyo, Japan) and visualized with a Lumino Image Analyzer LAS-1000 (Fuji Film, Tokyo, Japan).

TUNEL on membrane detection

To detect nuclear DNA cleavage resulting in free 3′-OH, nuclear DNA was treated with a DIG oligonucleotide 3′-end labelling kit (Roche Diagnostics, Mannheim, Germany), and used as a probe for Southern analysis. This technique is based on TdT-mediated DIG-dUTP 3′-end labelling (TUNEL). The nuclear DNA was blotted onto nylon membranes and hybridized with the DIG labelled probe. Signals were detected by CDP-Star chemiluminescent detection.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Leaf protoplast viability

Protoplasts isolated from B. napus leaves were analyzed for viability by use of microscopic and biochemical assays. As shown in Fig. 1, Evans Blue viability reduced to 80% and 60% over 3 and 5 d after culture. Since Evans Blue stains only dead cells, viability can be determined by measuring the proportion of unstained cells. It was difficult to estimate the percentage of viable cells in a given sample with precision, as the protoplasts aggregated in the liquid culture medium.

image

Figure 1. Changes in viability during the culture of Brassica napus leaf protoplasts. The protoplasts were cultured in Murashige Skoog (MS) medium in the dark. Evans blue dye was added to the protoplast culture medium. Viability was determined by counting the total number of protoplasts, and recording the numbers stained and unstained. Intracellular Adenosine 5′-triphosphate (ATP) was determined by the luciferin-luciferase luminescence assay. 2, 3, 5-Triphenyl tetrazolium chloride (TTC) reduction was recorded at absorbance of 530 nm.

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The presence of ATP is a useful marker for living organisms. Endogenous cellular ATP levels have therefore been used to determine cell proliferation and cytotoxicity in both bacterial (de Rautlin de la Roy et al., 1991) and mammalian cells (Koop & Cobbold, 1993). The ATP levels of the leaf protoplasts decreased with culture time, which concurs with the results of the previous Evans Blue assay (Fig. 1).

Tetrazolium salts are widely used to detect levels of cytotoxicity and to assay for cellular proliferation. TTC reduction is caused by mitochondrial dehydrogenase activity and is used as a biochemical estimation of viability (Watanabe et al., 1992). Figure 1 also shows that the TTC reduction decreases rapidly with time after the initiation of the culture. It reduced to 60%, 30% and 20% over 1, 3 and 5 d after culture. Thus all three approaches to measuring cell viability showed that the viability of the B. napus leaf protoplasts gradually decreased with the period of time after the initiation of the culture.

Morphological description of cell death

Detection of chromatin condensation by microscopic analysis The morphological changes to the protoplasts and nuclei during culture were studied by looking at protoplasts under the microscope and by staining with fluorescein diacetate (FDA)/propidium iodide (PI). When protoplasts were isolated from viable leaves, they gradually vacuolated and swelled during culture (Fig. 2a–c). However, no structures resembling apoptotic bodies in animal cells were observed.

image

Figure 2. Micrographs of Brassica napus leaf protoplasts under light (a–d), and fluorescence after staining with fluorescein diacetate-propidium iodide (FDA-PI) (e) and propidium iodide (PI) (f, g). Freshly prepared leaf protoplasts show a dense cytoplasm (a) and normal cell membrane integrity (e). By contrast, the protoplasts cultured for 2 d are swollen and vacuolated (b) and have lost cell membrane integrity (f). The protoplasts from leaves under nonoptimal physiological conditions have lost integrity of their cell membranes and become degraded (d). Protoplasts after 6 d in culture sometimes show a budding of the cell membrane (c) and an enlarged nucleus within small PI stained granules that represent chromatin condensation (g). bu, budding; cc, chromatin condensation; nu, nucleus.

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While FDA stains only viable cells with intact cell membranes, PI is excluded by dead cells and is therefore an effective stain for identification of living or dead cells. PI stain can also be used to detect chromatin condensation. Freshly prepared living leaf protoplasts emitted green fluorescence throughout the whole cells, which resulted from the intracellular hydrolysis of FDA and not from nuclear binding of PI (Fig. 2e). Figure 2(f) shows that after only 2 d in culture, the swelling leaf protoplasts already retain PI in the nucleus, and several small granules could be detected. After 6 d some of the swollen leaf protoplasts showed morphological changes in the cells similar to yeast budding (Fig. 2c), and the PI binding granules were smaller than those observed earlier in the culture time (Fig. 2g). These PI staining results indicate that cell death of B. napus leaf protoplasts occurs within 2 d of culture and is accompanied by chromatin condensation.

Even though protoplasts were purified at the time of isolation, they contained some corrupted cell corpses that stained with Evans blue and PI. During culture, the number of cell corpses gradually increased, but these were variable between protoplast cultures. When protoplasts were prepared from nonviable leaves that were susceptible to physical stress, these cell corpses accounted for a large percentage of the total number of cells by 2 d after culture (Fig. 2d).

Freshly prepared leaf protoplasts also emit red fluorescence throughout the whole cell, which results from the natural fluorescence of chlorophyll – this sometimes interferes with PI stained nuclear images (Fig. 2f). In order to obtain clearer images of the nucleus, leaf protoplasts were fixed with glutaraldehyde and stained with DAPI. Since the leaf protoplasts were fixed, there is no chlorophyll fluorescence. Figure 3(a,b) show DAPI staining of nuclei from freshly prepared protoplasts and swollen protoplasts after 2 d in culture, respectively. The obervation that DAPI stained the nuclei of the freshly prepared leaf protoplasts evenly indicated that these nuclei maintain normal structures. The nuclei of the protoplasts after 2 d in culture contained smaller granules or had separated into several globules and enlarged in size. These DAPI staining results were consistent with the PI staining, confirming that the morphological changes in the nucleus detected by DAPI are not artifacts of fixation. We could not detect nuclear fragmentation observed in Nicotiana interspecific hybrid calli and seedlings (Yamada et al., 2000).

image

Figure 3. Micrographs of 4′, 6-diamino-2-phenylindole, dihydrochloride (DAPI) stained Brassica napus leaf protoplasts under fluorescent microscopy. Freshly prepared leaf protoplasts have evenly stained nuclei (a), but the swollen and nondividing protoplasts cultured for 2 d have enlarged nuclei with condensed chromatin (b). cc, chromatin condensation; nu, nucleus.

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Ultrastructure of nuclei

The ultrastructure of nuclei obtained from freshly prepared leaf protoplasts, and from protoplasts after 2 and 5 d of culture were examined by transmission electron microscopy (TEM), and typical micrographs are shown (Fig. 4). Sections taken from the freshly prepared protoplasts show normal nuclei with surrounding cytoplasm containing vacuoles, chloroplasts and other organelles (Fig. 4a). High electron density granules that will represent chromatin are dispersed uniformly in a normal nucleus (Fig. 4c). The protoplasts which had been cultured for 2 d were vacuolated, the chloroplasts were swollen, and the chloroplasts and the nucleus were both present peripheral to the central vacuole (Fig. 4b). However, inclusion bodies could not be found in the central vacuole. Nuclei from protoplasts cultured for 2 or 5 d showed gradual condensation of chromatin during culture (Fig. 4d,e).

image

Figure 4. Ultrastructure of Brassica napus leaf protoplasts and nuclei. Examination of whole cells and nuclei with transmission electron microscopy show that the condensed cytoplasm is present around the central vacuole (a), and that the nucleus appears normal in the freshly prepared protoplast (c). The cytoplasm of the protoplast after 2 d of culture has become more vacuolated (b) and the nucleus contains slightly condensed chromatin (d). Chromatin condensation is more evident in the nucleus from the 5 d cultured protoplast (e).

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Detection of DNA fragmentation by flow cytometry (FCM)

Flow cytometry (FCM) is used for the analysis of DNA fragmentation. Figure 5(a) shows that there are two DAPI fluorescence peaks when freshly prepared leaf protoplasts are analyzed by FCM. The first peak is caused by fluorescence of diploid (2n) G1 nuclei (66% of the total area), and the second peak is due to tetraploid (4n) G1 nuclei (8.7% of the total area). When vacuolated protoplasts grown in culture for 5 d were analyzed, the 2n G1 peak decreased to 20% of the total area and additional peaks with smaller fluorescence value than 2n G1 nuclei significantly increased to 43% (Fig. 5b). This fluorescence shift is indicative of nuclear DNA fragmentation. Rapid cell degradation occurred by 2 d after culture (Fig. 2d) and was associated with a fluorescence pattern without discrete peaks (Fig. 5c), indicative of a style of fragmentation resulting from random degradation of nuclear DNA and a necrosis-like death process.

image

Figure 5. Flow cytometric analysis of Brassica napus leaf protoplasts. Histograms indicating increased fragmentation of 4′, 6-diamino-2-phenylindole, dihydrochloride (DAPI) fluorescence nuclear DNA from the cultured protoplasts are shown. The total number of nuclei from freshly prepared protoplasts (a), swollen and nondividing protoplasts (b) and corrupted protoplasts with membrane damage after 5 d of culture (c) were 11528, 23125 and 55407, respectively. Peak 1, 2n G1 nuclei; Peak 2, 4n G1nuclei. Striped area, fragmented nuclear DNA.

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Detection of DNA fragmentation by agarose gel electrophoresis

A typical feature of apoptosis is the cleavage of DNA at specific chromosomal sites by DNA endonucleases (Eastman et al., 1994). To examine whether DNA fragmentation occurred during culture, nuclear DNA from vacuolated protoplasts was isolated and analyzed. Figure 6(a) shows ladders comprising DNA fragments of multiples of c. 180 bp that are diagnostic of some, but not all, forms of apoptosis in animals. Nuclear DNA from freshly prepared leaf protoplasts retained a single high molecular weight band, and there was no DNA laddering. Figure 6(a) also shows that the DNA laddering increased after 4 d in culture, and then gradually decreased, while the high molecular DNA band is narrow in cells taken from the culture at the time of initiation, but gradually broadens in cells cultured for increasing periods of time. These results show that the nuclear DNA is cleaved into nucleosomal fragments during the time of culture. Nuclear DNA from corrupted cell corpses that had accumulated by 2 days after culture (Fig. 2d) was analyzed. Figure 6(b) shows that there is no DNA ladder, but only a smear.

image

Figure 6. Agarose gel analysis of Brassica napus leaf protoplasts for nuclear DNA fragmentation. (a) Nuclear DNA from swollen and nondividing protoplasts was separated on 2% agarose gel electrophoresis, stained with ethidium bromide and photographed under UV illumination. The numbers 0, 2, 4 and 6 on the photograph indicate the number of days after initiation of the culture. (b) Nuclear DNA from corrupted protoplasts with membrane damage after 2 d of culture was separated on 2% agarose gel electrophoresis, stained with ethidium bromide and photographed under UV illumination. (c) Nuclear DNA from protoplasts grown in culture for various periods of time was separated by electrophoresis on a 2% agarose gel, transferred onto a nylon membrane, hybridized with a B. napus genomic DNA probe and detected as described in methods section. (d) TUNEL on membrane detection of nuclear DNA cleavage with free 3′-OH termini. Nuclear DNA from swollen and nondividing protoplasts cultured for 4 d was separated on 2% agarose gel electrophoresis, transferred, hybridized with 3′-OH DIG labelled probe and detected as described in Materials and Methods.

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The DNA ladders observed on the agarose gels were confirmed by Southern analysis as shown in Fig. 6(c). As before, the DNA ladders were only detected in cultured leaf protoplasts, and were absent from freshly prepared leaf protoplasts. The DNA laddering structures that appear during culture suggest that the swollen B. napus leaf protoplasts die through an apoptotic-like process.

Detection of DNA fragmentation by TUNEL on membrane

In order to relate the DNA fragmentation to apoptosis through specific endonuclease cleavages of nuclear DNA, DNA cleavages were detected by TUNEL on membrane. DNA breakage by endonucleases generates free-3′-OH termini which can be labelled with DIG-dUTP catalyzed by TdT, and used as probes for Southern hybridization (see Material and Methods section). This method can visualize the presence of DNA fragments with 3′-OH termini on a nylon membrane. Figure 6(d) shows that the oligonucleosomal DNA ladders of multiples of c. 180 bp are detected in samples of nuclear DNA from the swollen leaf protoplasts cultured for 4 d. These results provide further evidence for the apoptotic-like cell death of B. napus leaf protoplasts.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The cell death occurring in B. napus leaf protoplasts shows many of the characteristics of apoptosis in animals, which suggests that the cell death is occurring through apoptotic-like pathways. Our previous report indicated that protoplast isolation stress triggered the initiation of senescence (Watanabe et al., 1998), while the cell death caused by senescence of leaves from five different plant species has been shown to be a type of programmed cell death (apoptosis) (Cheng-Hung & Chang-Hsien, 1998). TTC viability was significantly different from that revealed by the other two methods (Fig. 1). It may be due to decrease in redox potential of cells by interruption of photosynthesis during culture in the dark. As three different estimations of protoplast viability indicated that B. napus leaf protoplasts gradually died after culture, we speculated that the senescence of B. napus leaf protoplasts might be a form of programmed cell death or apoptosis. Detection of nuclear changes such as chromatin condensation and nuclear pycnosis by use of fluorescence microscopic analysis (Bhattacharya et al., 1996) would support the hypothesis. Microscopic characteristics of apoptosis in mammalian cells are cytoplasmic budding, plasma membrane blebbing, decreases in cell size and the presence of apoptotic bodies. By contrast with mammalian cells, the protoplasts became vacuolated, and their size increased (Fig. 2a–c). Thus apoptotic-like cell death in B. napus leaf protoplasts appears to be different in some details from apoptosis in animal cells.

Mammalian cells undergoing necrosis are characterized by cell swelling, cell lysis and by the leakage of cell contents (Cohen, 1993). In case of B. napus leaf protoplasts, cell corpses with the plasma membrane damage and the leakage of cell contents (Fig. 2d) display microscopic characteristics of necrotic death. The number of these cell corpses that appeared by the time of protoplast isolation was variable between leaves used for preparation. This is thought to be due to the fact that the plasma membrane of the cells had been physically damaged by the protoplast isolation procedures. Except for swelling, morphological characteristics of necrotic death of B. napus leaf protoplasts appears to be identical to necrosis in animal cells. This necrotic death of B. napus was confirmed by FCM and agarose gel analysis (Figs 5 and 6). Expression of recalcitrance at the point of cell division could be explained by necrotic and apoptotic-like cell death. Rapid degradation of protoplasts by 2 d after culture results from necrotic cell death, whereas senescence symptoms appearing during the culture could be due to apoptotic-like cell death.

Chromatin condensation and membrane integrity were assayed by staining with FDA/PI and DAPI (Figs 2 and 3). These observations clearly visualized chromatin condensation in protoplasts after 2 d in culture, by which time cell death is inevitable.

The ultrastructural examination of nuclei by TEM revealed increased chromatin condensation during protoplast culture (Fig. 4). There were no inclusion bodies in the central vacuoles in rice senescence leaves (Lee & Chen, 2002), nuclear lobes that were formed by apoptosis in the root cortex cells of barley (Liljeroth & Bryngelsson, 2001) and in a soybean root necrosis mutant (Kosslak et al., 1997).

The appearance of oligonucleosome-sized DNA fragments is characteristic of mammalian apoptosis and of programmed cell death in some plants. Oligonucleosomal DNA fragmentation indicates that a form of programmed cell death with some mechanistic similarities to apoptosis has taken place. Despite the similarities between programmed cell death in B. napus leaf protoplasts and in animal cells, it is likely that some aspects of the function and mechanism of programmed cell death in the protoplasts will differ from that seen in animals. For example, the central vacuole and chloroplasts became swollen during the process of dying, which results in an increase in size of the protoplasts. This suggests that vacuoles and chloroplasts, which are plant cell specific organelles, probably play a role in plant programmed cell death.

The TUNEL in situ assay has been reported to give variable results due to fixation methods, reaction times, reagent concentrations, etc. False positives can occur as a result of, for example, DNase activity in necrotic cells, or as a result of different washing procedures during the assay. The absolute values should therefore be interpreted with care. It is also difficult to detect TUNEL positive protoplasts in the suspension medium, because the cells aggregate in the liquid medium during culture. Accordingly, in the course of this study, a new TUNEL assay has been developed in which detection occurs on a membrane. Thus we can detect the presence of both 3′-OH termini of oligonucleosomal DNA and ladders, although the assay cannot localize an individual apoptotic cell to a specific tissue. As expected, oligonucleosomal DNA ladders with 3′-OH termini were detected in nuclear DNA from leaf protoplasts after 4 d in culture (Fig. 6d).

Plants contain several endonucleases that do not cleave the nuclear DNA at regular sites (c. 180 bp), and that do not therefore deliver discrete DNA fragments by electrophoresis (Mittler & Lam, 1995). McCabe et al. (1997) demonstrated that endonucleases that do not cleave at oligonucleosome linker sites could operate in carrot suspension cultures. Our experiments revealed that when B. napus leaf protoplasts were dying, endonucleases cleaved nuclear DNA with 3′-OH termini at nucleosomal sites (Fig. 6).

To date, there have been no experimental data to support a model explaining why protoplasts from some species are recalcitrant, while others regenerate. Our study has demonstrated that B. napus leaf protoplasts should progress to programmed cell death during culture, and therefore suggest that its progression is highly controlled at the genetic level.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We gratefully appreciate Dr Masahiro Mii and Dr Kei-ichiro Mishiba for the use of a Partec PA cytometer.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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