TY - JOUR
AU - Marubashi, Wataru
AB - Abstract Hybrid cells from the cross Nicotiana suaveolens × N. tabacum expressed hybrid lethality at 28°C in a thin layer cell culture system. Features characteristic of apoptosis, such as DNA fragmentation, chromatin condensation and nuclear fragmentation, were detected during expression of hybrid lethality. Actinomycin D (ActD) or cycloheximide (CHX) added to the medium suppressed apoptotic cell death during hybrid lethality. This indicates that hybrid lethality requires de novo transcription and translation, and is thus under genetic control. To estimate the time course of apoptotic cell death during the expression of hybrid lethality, we determined when factors controlling hybrid lethality were expressed by observing the point of no return. When cells were exposed to 28°C for 2 h or less in inhibitor-free medium before addition of ActD or CHX, the percentage of dead cells did not increase. However, when cells were exposed to 28°C for 4 h before the addition of inhibitor, the percentage of dead cells increased. When cells were exposed to 28°C for 3 h before the addition of inhibitor, the percentage of dead cells varied from experiment to experiment. These data indicate that the factors controlling hybrid lethality are expressed 3 h after induction of hybrid lethality. In addition, we found a time difference between the expression of cell death and nuclear fragmentation. This suggests that the factor controlling cell death is different from the one controlling nuclear fragmentation. (Received September 11, 2002; Accepted February 8, 2003) Introduction Hybrid lethality is a kind of reproductive isolation of species (Stebbins 1966) and has been observed in interspecific hybrids of several genera, including crop plants (Phillips and Reid 1975, Zeven 1981, De Verna et al. 1987). Genetic research of hybrid lethality has revealed a few genes involved in this process in interspecific crosses of Gossypium (Samora et al. 1994) and of Oryza (Chu and Oka 1972). In the genus Nicotiana, lethality expressed in two interspecific crosses has been studied genetically. Using monosomic analysis, Gerstel et al. (1979) suggested that in the cross N. africana × N. tabacum, one or more genes on the H chromosome of N. tabacum are involved in the expression of lethality in hybrid seedlings. In the cross N. suaveolens × N. tabacum, Marubashi and Onosato (2002) reported that the Q chromosome of N.tabacum is responsible for lethality. It has been reported that the expression of hybrid lethality is influenced by temperature. In the cross N. suaveolens × N. tabacum, Manabe et al. (1989) showed that lethality is expressed by culturing the hybrid seedlings at 28°C, and is suppressed at high temperatures (30–36°C). Similarly, in some other crosses in the genus Nicotiana, lethality is suppressed under high temperature conditions (Yamada et al. 1999). Temperature sensitivity of hybrid lethality was confirmed in several crosses of other genera, such as Gossypium (Lee 1981) and Triticum (Dhaliwal et al. 1986), but the physiological and molecular mechanisms of induction and suppression of hybrid lethality have not been explained. Apoptosis is a distinct type of cell death characterized by condensation of chromatin, fragmentation of nuclei, cell membrane shrinkage, and fragmentation of DNA (Kerr et al. 1972, Wylie et al. 1984). Apoptosis plays major roles during animal development, homeostasis and disease (Afford and Randhawa 2000). This type of cell death is performed through a conserved process under genetic control, and many molecules associated with this process have been reported in animal cells (Aravind et al. 1999). Apoptosis can be divided into three distinct phases: initiation, commitment and execution. The initiation phase is the first stage of the apoptotic time course, characterized by the cell’s recognition and response to an apoptotic signal. This phase is defined as the stage wherein the signal from any of a variety of inducers of apoptosis is transduced to the commitment phase but is reversible. In the commitment phase, the process of apoptosis becomes irreversible and cell death becomes inevitable. The execution phase is characterized by numerous pathways, such as activation of caspases, and nuclear changes that result in the apoptotic morphology of cell death (Mills 2001). In plants, distinct features of apoptosis have been reported during cell death associated with development (Wang et al. 1999, Lindholm et al. 2000), senescence (Yen and Yang 1998, LoSchiavo et al. 2000), and in response to phytotoxins and pathogens (Navarre and Wolpert 1999, Mittler et al. 1997, Che et al. 1999), and environmental stress (McCabe et al. 1997, Danon and Gallois 1998). Several reports have described hybrid lethality as necrosis (Nishikawa et al. 1974, Phillips and Reid 1975, Zeven 1981, De Verna et al. 1987) without any cytological or biochemical observations. However, Marubashi et al. (1999) reported that characteristic features of apoptosis, such as chromatin condensation, nuclear fragmentation and DNA fragmentation, were observed in hybrid seedlings of N. glutinosa × N. repanda. Moreover, in the cross N. suaveolens × N. tabacum, Yamada et al. (2000) reported that hybrid seedlings expressed lethality with features characteristic of apoptosis within 2 weeks after germination, and calli derived from this cross also expressed lethality with apoptotic features. The fact that apoptosis during expression of hybrid lethality was detected in suspension-cultured cells derived from N. suaveolens × N. tabacum indicates that the same phenomenon occurs in seedlings, calli and suspension-cultured cells (Yamada et al. 2001). These studies also revealed that when hybrid cells from the cross Nicotiana suaveolens × N. tabacum are exposed to 28°C for 2 h or less and then cultured at 36°C, hybrid lethality is suppressed. On the other hand, when the cells are exposed for more than 3 h and then cultured at 36°C, hybrid lethality is not suppressed. These data indicate that cells exposed to 28°C for more than 3 h become irreversibly committed to death, so that the point of no return in the hybrid cells is 3 h after exposure to 28°C. In this study, we developed a thin layer cell culture (TLCC) system that enabled reducing the scale of the experiments and that was useful for analysis of apoptotic cell death during expression of hybrid lethality. Using this system, we tried to suppress hybrid lethality by actinomycin D (ActD) (a transcriptional inhibitor) or cycloheximide (CHX) (a translation inhibitor) to determine the time course of apoptotic cell death during expression of hybrid lethality in tobacco hybrid cells (Nicotiana suaveolens × N. tabacum). Results Features characteristic of apoptotic cell death in a thin layer cell culture system A TLCC system was developed to observe particular cells serially and to enable reduction of the scale of the experiments. Using this system, the amount of medium is reduced by 90% compared with a suspension culture system; this also simplifies investigation of the effects of inhibitors. Using this system, we tried to detect the apoptotic features, such as chromatin condensation, nuclear fragmentation, cell membrane shrinkage, and fragmentation of DNA into multimers of about 180 base pairs (bp), which had been detected in a suspension culture system in hybrid cells from the cross Nicotiana suaveolens × N. tabacum (Yamada et al. 2001). Particular cells were observed serially by phase-contrast microscopy (Fig. 1). Normal cells before exposure to 28°C had normal nuclei with a nucleolus and mesh-like structural chromatin, and exhibited protoplasmic streaming (Fig. 1A). In cells exposed to 28°C for 3–6 h, chromatin condensation was observed and protoplasmic streaming ceased (Fig. 1B). In cells exposed to 28°C for 6–9 h, plasma membrane shrinkage had started (Fig. 1C). Fluorescence microscopy of 4′-6′-diamino-2-phenylindole (DAPI)-stained cells showed structural changes of the nucleus in the TLCC system. Thousands of cells were observed at different times after 28°C treatment. All the cells had normal nuclei maintaining a mesh-like structure 0–3 h after exposure to 28°C (Fig. 1D). The nuclei of half of the cells lost their mesh-like structure and had condensed chromatin 3–6 h after exposure to 28°C (Fig. 1E). A few cells exposed to 28°C for 15 h showed nuclear fragmentation (Fig. 1F). Nuclear fragmentation was further evaluated by analysis of the DNA content of nuclei isolated from the hybrid cells using flow cytometry. The DNA histogram of the cells 0 h after exposure to 28°C showed two peaks, which probably correspond to the G1 and G2 phases of the cell cycle (Fig. 2A). In cells exposed to 28°C for 24 h, the G1 peak decreased and additional peaks with lower fluorescence values appeared. The percentage of these additional peaks out of the total was about 12% in cells before exposure to 28°C, and increased gradually over time at 28°C. In cells exposed to 28°C for 24 h, the percentage of these additional peaks became about 60% (Fig. 2B). A ladder-like DNA banding pattern (indicating DNA fragmentation) was detected in hybrid cells using 2% agarose gel electrophoresis. Total DNA isolated from cells exposed to 28°C for 12 and 24 h showed a ladder-like DNA banding pattern (Fig. 2C, lanes 5, 6). This pattern was not detectable in total DNA isolated from cells exposed to 28°C for 0–6 h (Fig. 2C, lanes 2–4). These data suggest that apoptotic cell death during expression of hybrid lethality is readily detected in the TLCC system. Suppression of apoptotic cell death by actinomycin D or cycloheximide during expression of hybrid lethality We attempted to demonstrate whether hybrid lethality in the cross N. suaveolens × N. tabacum is under genetic control. The percentage of cell death was detected using Evans Blue staining in medium with ActD or CHX. In cells exposed to 28°C for 24 h in culture medium with 100 µg ml–1 ActD or CHX, the percentage of dead cells was about 30%. This percentage was the same as that of negative controls, which were cells maintained at 36°C, which suppresses hybrid lethality (Fig. 3). In contrast, the percentage of dead cells after exposure to 28°C without these inhibitors was about 70%. These data indicate that ActD and CHX suppress cell death associated with hybrid lethality. Nuclear fragmentation in cells from the cross N. suaveolens × N. tabacum was detected in media with ActD or CHX using a flow cytometer (Fig. 4). The histogram of the cells exposed to 28°C for 24 h in the presence of ActD or CHX showed the normal two peaks, which presumably correspond to the G1 and G2 phases of the cell cycle, and the percentage of additional peaks, which are evidence of nuclear fragmentation, did not increase (Fig. 4C, D). These data indicate that ActD and CHX suppress nuclear fragmentation. These data therefore suggest that expression of hybrid lethality requires de novo transcription and translation. Estimation of the commitment phase (point of no return) Apoptosis can be divided into three distinct phases, initiation, commitment and execution, in animal cells (Mills 2001). In the commitment phase, the cells undergoing apoptosis become irreversibly committed to death. We tried to estimate the duration of the commitment phase for hybrid lethality after exposure to 28°C in the cross N. suaveolens × N. tabacum. In this experiment, according to the timetable illustrated in Fig. 5A, cells cultured at 36°C were exposed to 28°C in medium without inhibitor for 0–5 h, and then in medium containing the inhibitor. After the cells had been incubated at 28°C for a total of 12 h, the percentage of dead cells was measured. When an inhibitor is added to the medium before the commitment phase, hybrid lethality will be suppressed and the percentage of dead cells will not increase. On the other hand, when an inhibitor is added to the medium after the commitment phase, it will have no effect on the percentage of dead cells. We tried to estimate the duration of the commitment phase during induction of hybrid lethality by determining when the percentage of dead cells was no longer suppressed by addition of ActD or CHX. As shown in Fig. 5B, when inhibitor was added to cells exposed to 28°C for 0–2 h in inhibitor-free medium, the percentage of dead cells did not increase and was the same as for cells exposed to 36°C, in which hybrid lethality is suppressed. When inhibitor was added to cells exposed to 28°C for 4 h, hybrid lethality was not suppressed. When inhibitor was added to cells exposed to 28°C for 3 h, the percentage of dead cells varied from experiment to experiment and showed a large standard deviation. These data suggest that the commitment phase in our experimental model was 3 h after exposure to 28°C. Nuclear fragmentation was analyzed by flow cytometry. As shown in Fig. 6, when cells were exposed to 28°C for 3 h or less without inhibitor and then an inhibitor was added, the resulting histogram had two approximately normal peaks and no additional peaks with a lower fluorescence value. This indicates that nuclear fragmentation was suppressed by the inhibitor (Fig. 6A, D). In contrast, when inhibitor was added to cells exposed to 28°C for 5 h, the histogram showed peaks indicating that nuclear fragmentation had taken place (Fig. 6C, F). In spite of cell death occurring (Fig. 5), when cells were exposed to 28°C for 4 h before addition of inhibitor, the histogram showed the normal two peaks. This means that cell death took place but nuclear fragmentation did not (Fig. 6B, E). These data suggest that the timing of expression of the factor controlling nuclear fragmentation lags behind the factor controlling cell death. Discussion Using a TLCC system, we observed morphological and biochemical changes characteristic of apoptosis, such as chromatin condensation (Fig. 1B, E), cell membrane shrinkage (Fig. 1C), nuclear fragmentation (Fig. 1F, 2B), and DNA fragmentation (Fig. 2C). These results demonstrate that apoptotic cell death is accompanied by hybrid lethality in hybrid tobacco cells (N. suaveolens × N. tabacum) in this TLCC system. Rhoads and McIntosh (1993) used ActD and CHX to inhibit transcription and translation, respectively, in tobacco BY-2 suspension cells. In our experiments, ActD or CHX was added to the medium to confirm that hybrid lethality is controlled genetically in the TLCC system. Hybrid lethality (Fig. 3) and nuclear fragmentation (Fig. 4) in cells exposed to 28°C, which is the lethal temperature, were suppressed by ActD or CHX. These data indicate that hybrid lethality requires de novo transcription and translation. This suggests that one or more genes are responsible for hybrid lethality. De Verna et al. (1987) proposed that the cause of hybrid lethality is interspecific incongruity. Some parts of this concept may be explained by the analysis of genes responsible for hybrid lethality. In animal cells, ActD and CHX are common inducers of apoptosis, and in some cases, the mechanism of induction of apoptosis by ActD or CHX has been reported (Kimura and Yamamoto 1997, Kleeff et al. 2000). However, in our experiment, these inhibitors suppressed apoptotic cell death. This suggests that the mechanism of inducing apoptosis in plant cells, as it is involved in hybrid lethality, is different from that in animal cells. In animal cells, apoptosis is divided into three distinct phases, initiation, commitment and execution. The mechanisms of the commitment and execution phases are evolutionarily conserved, but that of the initiation phase depends on the cell type (Mills 2001). We estimated the point of no return, at which the factor controlling hybrid lethality was expressed. Hybrid lethality was divided into the process responsible before the point of no return and the process after the point of no return. The process responsible requires expression of hybrid lethality-related gene(s), and is reversible and temperature sensitive. The process following the point of no return does not require gene expression, and uses the existing mechanism of the apoptotic execution phase. When cells were exposed to 28°C in inhibitor-free medium for up to 2 h and then exposed to inhibitor, hybrid lethality was suppressed (Fig. 5). On the other hand, when cells were exposed to 28°C for 4 h and then exposed to inhibitor, hybrid lethality was not suppressed (Fig. 5). When cells were exposed to 28°C for 3 h, the percentage of dead cells differed (Fig. 5). These data indicate that the factors controlling hybrid lethality are expressed 3 h after induction of hybrid lethality, which might correspond to the commitment phase of apoptosis in animal cells. This suggests that the process 0–3 h after transfer to 28°C is characteristic of hybrid lethality and is responsible for temperature sensitivity and hybrid lethality. Yamada et al. (2001) revealed that when hybrid cells from the cross Nicotiana suaveolens × N. tabacum are exposed to 28°C for more than 3 h and then cultured at 36°C, hybrid lethality is not suppressed. This suggests that the point of no return in hybrid cells is 3 h after exposure to 28°C. The observation that 3 h after induction of hybrid lethality was also the point of no return in our experiments shows that a process critical to expression of hybrid lethality exists 3 h after induction of apoptotic cell death. Despite the fact that nuclear fragmentation was suppressed by addition of inhibitor 4 h after exposure to 28°C (Fig. 6B, E), the percentage of dead cells rose (Fig. 5). The percentage of dead cells was counted by Evans Blue staining, which is an indicator of cell death. Nuclear fragmentation was measured by flow cytometry. The time difference between the expression of cell death and nuclear fragmentation suggests that the factor controlling cell death is different from the one controlling nuclear fragmentation. A TLCC system is suited to serial observation. We observed identical cells undergoing various stages of apoptotic cell death by inverted phase contrast microscopy; they showed typical morphological changes such as chromatin condensation and cell membrane shrinkage (Fig. 1). At the final stages of apoptosis in animal cells, the cells form an apoptotic body and are phagocytosed, while apoptosis or programmed cell death in plants might involve autolysis (Danon et al. 2000). During differentiation of tracheary elements in Zinnia, the programmed cell death process was observed serially (Obara et al. 2001); however, the final stages of apoptotic cell death during expression of hybrid lethality are unknown. Using the TLCC system, vital staining and serial observation of hybrid cells undergoing apoptotic cell death may reveal the final stages of apoptotic cell death in hybrid lethality. Materials and Methods Cell culture Hybrid cells were cultured at a high temperature (36°C), which suppresses lethality in hybrid seedlings from the cross N. suaveolens × N. tabacum (Manabe et al. 1989), and under continuous illumination (ca. 3,000 lux). To establish cultures, hybrid calli were first induced from cotyledonary segments (Yamada et al. 2000). The segments were excised from hybrid seedlings 4 d after germination. Next, hybrid calli prepared from cotyledon-segment cultures 30 d after initiation were transferred into liquid MS medium (pH 5.8) supplemented with 50 µM α-naphthalenacetic acid, 0.04 µM 6-benzylaminopurine, and 3% sucrose, pH 5.8, and grown in conical flasks with constant shaking (130 rpm). Cell suspensions were maintained under the same conditions on a 7-d subculture cycle and used for experiments 3 d after subculturing. Thin layer cell culture system To remove old medium from cells maintained in suspension culture, hybrid cells were sieved through a 200-µm nylon mesh and about 0.5 g (FW) of cells was transferred to culture dishes (ϕ90 mm) at a high temperature (36°C). Three ml of fresh medium was added and cells were placed in order to form a single layer of cells. Then about 2 ml of the extra culture medium was removed to expose the cells to air in order to keep the dish under observation. Induction of lethality and measurement of cell death Hybrid cells cultured at 36°C were transferred to 28°C, which is the lethal temperature for hybrid seedlings (Manabe et al. 1989), and maintained at 28°C in the TLCC system. Hybrid cells exposed to 28°C were sieved through a 200-µm nylon mesh to remove the clustered cells and were resuspended in 50 µl fresh medium after centrifugation (2,000 rpm, 10 min). Ten µl of this cell suspension was dropped on a glass slide and observed by light or fluorescence microscopy. The progression of lethality in hybrid cells was estimated from the percentage of dead cells after 28°C treatment for different intervals over 24 h. Dead cells were scored under a light microscope after staining with 2.5% (w/v) Evans Blue. Detection of apoptotic cell death According to Yamada et al. (2001), apoptotic features are detectable in hybrid cells that express lethality after an inducing temperature treatment (transfer from 36°C to 28°C). Hybrid cells were fixed with 5% paraformaldehyde in PBS (137 mM NaCl, 2.68 mM KCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.4). Morphological changes were observed using fluorescence microscopy under U excitation (330–380 nm) and photographed by a cooled CCD camera (Quantix 1401E; Roper Scientific, Trenton, NJ, U.S.A.) after staining with 0.5% (w/v) DAPI. For serial observation, a particular cell in the culture dish on a plate heater (stage warmer MP10DM; Kitazato, Shizuoka, Japan) adjusted to 28°C was observed using an inverted phase contrast microscope (IX70; Olympus, Tokyo, Japan) and photographed at hly intervals for 24 h. For detection of nuclear fragmentation, nuclei were isolated from harvested hybrid cells chopped in ice-cold buffer (Michaelson et al. 1991) and filtered through a 59- and a 20-µm nylon mesh. The nuclei were collected from the filtrate by centrifugation for 5 min at 700×g, suspended in ice-cold buffer supplemented with 5 µg ml–1 PI and 10 µg ml–1 RNase, and incubated for 15 min at 37°C. The DNA content of the isolated nuclei was analyzed using a flow cytometer (FACS Calibur; Becton Dickinson, San Jose, CA, U.S.A.). DNA fragmentation, a biochemical change that occurs during apoptotic cell death, was detected by electrophoretic analysis on an agarose gel. Total DNA was extracted from hybrid cells using a modified CTAB method (Yamada et al. 2000) and then electrophoresed in a 2% agarose gel and stained with SYBR Gold (Molecular Probes, Eugene, OR, U.S.A.). Suppression of apoptotic cell death by ActD (a transcriptional inhibitor) and CHX (a translation inhibitor) To confirm the influence of these inhibitors on hybrid lethality, ActD or CHX was added to culture medium to a final concentration of 100 µg ml–1 from a 5 mg ml–1 stock solution prepared in 80% ethanol. Medium with the inhibitor was added to the TLCC system instead of 3 ml fresh medium. Cells in the TLCC system were suspended 10 min before removing the extra culture medium. Estimation of the commitment phase (point of no return) in induction of hybrid lethality Cells cultured at 36°C were exposed to 28°C in medium without inhibitor for 0–5 h, and then the culture medium was exchanged for medium with inhibitor, as illustrated by the timetable in Fig. 5A. After cells had been exposed to 28°C for a total of 12 h, the percentage of dead cells and the extent of nuclear fragmentation were determined to confirm that the cells were undergoing apoptotic cell death. Acknowledgments This work was partly supported by Grant-in-Aid for Exploratory Research and Grant-in-Aid for Scientific Research (A) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. 3 Corresponding author: E-mail, marubasi@mx.ibaraki.ac.jp; Fax, +81-298-88-8644. View largeDownload slide Fig. 1 Apoptotic changes in hybrid cells from the cross Nicotiana suaveolens × N. tabacum expressing lethality at 28°C; serial observation of an individual cell by phase-contrast microscopy (A–C). Arrowheads indicate nuclei. Fluorescence microscopic observation of individual cells stained with DAPI (D–F). (A) Cells cultured at 36°C had normal structure of nuclei and exhibited protoplasmic streaming. (B) Cells exposed to 28°C for 6 h showed chromatin condensation. (C) Cells exposed to 28°C for 8 h showed plasma membrane shrinkage. (D) Cells cultured at 36°C had normal nuclei with a nucleolus and mesh-like structural chromatin. (E) Cells exposed to 28°C for 6 h had condensed chromatin. (F) Cells exposed to 28°C for 15 h had fragmented nuclei. View largeDownload slide Fig. 1 Apoptotic changes in hybrid cells from the cross Nicotiana suaveolens × N. tabacum expressing lethality at 28°C; serial observation of an individual cell by phase-contrast microscopy (A–C). Arrowheads indicate nuclei. Fluorescence microscopic observation of individual cells stained with DAPI (D–F). (A) Cells cultured at 36°C had normal structure of nuclei and exhibited protoplasmic streaming. (B) Cells exposed to 28°C for 6 h showed chromatin condensation. (C) Cells exposed to 28°C for 8 h showed plasma membrane shrinkage. (D) Cells cultured at 36°C had normal nuclei with a nucleolus and mesh-like structural chromatin. (E) Cells exposed to 28°C for 6 h had condensed chromatin. (F) Cells exposed to 28°C for 15 h had fragmented nuclei. View largeDownload slide Fig. 2 Detection of apoptotic features in hybrid cells from the cross Nicotiana suaveolens × N. tabacum expressing lethality at 28°C; nuclear fragmentation was detected by flow cytometry of PI-treated nuclei (A, B). The vertical line in each figure divides the area containing additional peaks with lower fluorescence values from the G1+G2 peaks. The upper-left number in each figure is the percentage of additional peaks with lower fluorescence values out of the total. These indicate that the status of nuclear fragmentation. DNA fragmentation was detected using 2% agarose gel electrophoresis (C). (A) Cells cultured at 36°C showing the normal 2 peaks of G1 and G2 nuclei. (B) Cells exposed to 28°C for 24 h showing additional peaks with lower fluorescence values. (C) DNA solution was electrophoresed in a 2% agarose gel and stained with SYBR Gold (Molecular Probes); M, 123 bp ladder marker (BRL); lane 1, cells cultured at 28°C for 0 h; lane 2, cells cultured at 28°C for 3 h; lane 3, cells cultured at 28°C for 6 h; lane 4, cells cultured at 28°C for 12 h; lane 5, cells cultured at 28°C for 24 h. View largeDownload slide Fig. 2 Detection of apoptotic features in hybrid cells from the cross Nicotiana suaveolens × N. tabacum expressing lethality at 28°C; nuclear fragmentation was detected by flow cytometry of PI-treated nuclei (A, B). The vertical line in each figure divides the area containing additional peaks with lower fluorescence values from the G1+G2 peaks. The upper-left number in each figure is the percentage of additional peaks with lower fluorescence values out of the total. These indicate that the status of nuclear fragmentation. DNA fragmentation was detected using 2% agarose gel electrophoresis (C). (A) Cells cultured at 36°C showing the normal 2 peaks of G1 and G2 nuclei. (B) Cells exposed to 28°C for 24 h showing additional peaks with lower fluorescence values. (C) DNA solution was electrophoresed in a 2% agarose gel and stained with SYBR Gold (Molecular Probes); M, 123 bp ladder marker (BRL); lane 1, cells cultured at 28°C for 0 h; lane 2, cells cultured at 28°C for 3 h; lane 3, cells cultured at 28°C for 6 h; lane 4, cells cultured at 28°C for 12 h; lane 5, cells cultured at 28°C for 24 h. View largeDownload slide Fig. 3 Changes in the percentage of dead hybrid cells from the cross Nicotiana suaveolens × N. tabacum in culture medium with ActD (A) or CHX (B) incubated at 28°C. Cells that were stained with Evans Blue after initiation of the culture at 28°C were scored as dead. Values are means with SD (vertical bars) of results from five independent determinations. View largeDownload slide Fig. 3 Changes in the percentage of dead hybrid cells from the cross Nicotiana suaveolens × N. tabacum in culture medium with ActD (A) or CHX (B) incubated at 28°C. Cells that were stained with Evans Blue after initiation of the culture at 28°C were scored as dead. Values are means with SD (vertical bars) of results from five independent determinations. View largeDownload slide Fig. 4 Nuclear fragmentation in hybrid cells from the cross N. suaveolens × N. tabacum in culture medium with or without ActD or CHX, with hybrid lethality induced by treatment at 28°C; histograms indicate the extent of fragmentation of nuclei in the cells as measured by flow cytometry of PI-treated nuclei. The vertical line in each figure divides the area containing additional peaks with lower fluorescence values from the G1+G2 peaks. The upper-left number in each figure is the percentage of additional peaks with lower fluorescence values out of the total. These indicate that the status of nuclear fragmentation. (A) Cells cultured at 36°C showing the normal two peaks of G1 and G2 nuclei. (B) Cells exposed to 28°C for 24 h without inhibitor showing additional peaks with lower fluorescence values. (C) Cells exposed to 28°C for 24 h with ActD showing the normal two peaks of G1 and G2 nuclei. (D) Cells exposed to 28°C for 24 h with CHX showing the normal two peaks of G1 and G2 nuclei. View largeDownload slide Fig. 4 Nuclear fragmentation in hybrid cells from the cross N. suaveolens × N. tabacum in culture medium with or without ActD or CHX, with hybrid lethality induced by treatment at 28°C; histograms indicate the extent of fragmentation of nuclei in the cells as measured by flow cytometry of PI-treated nuclei. The vertical line in each figure divides the area containing additional peaks with lower fluorescence values from the G1+G2 peaks. The upper-left number in each figure is the percentage of additional peaks with lower fluorescence values out of the total. These indicate that the status of nuclear fragmentation. (A) Cells cultured at 36°C showing the normal two peaks of G1 and G2 nuclei. (B) Cells exposed to 28°C for 24 h without inhibitor showing additional peaks with lower fluorescence values. (C) Cells exposed to 28°C for 24 h with ActD showing the normal two peaks of G1 and G2 nuclei. (D) Cells exposed to 28°C for 24 h with CHX showing the normal two peaks of G1 and G2 nuclei. View largeDownload slide Fig. 5 Estimation of the commitment phase in hybrid cells from the cross Nicotiana suaveolens × N. tabacum incubated at 28°C. (A) Timetable of ActD or CHX treatment of hybrid cells to estimate the point of no return for the lethality induced by incubation at 28°C. Cells cultured at 36°C were exposed to 28°C for 0–5 h in the medium without inhibitor, and then the culture medium was either exchanged with medium containing ActD or CHX, or left as a control. After the cells were exposed to 28°C for a total of 12 h, the percentage of dead cells was measured. (B) Changes in the percentage of dead cells in hybrid cells from the cross N. suaveolens × N. tabacum cultured as illustrated in (A). Cells that were stained with Evans Blue were scored as dead. Values are means with SD (vertical bars) of results from five independent determinations. View largeDownload slide Fig. 5 Estimation of the commitment phase in hybrid cells from the cross Nicotiana suaveolens × N. tabacum incubated at 28°C. (A) Timetable of ActD or CHX treatment of hybrid cells to estimate the point of no return for the lethality induced by incubation at 28°C. Cells cultured at 36°C were exposed to 28°C for 0–5 h in the medium without inhibitor, and then the culture medium was either exchanged with medium containing ActD or CHX, or left as a control. After the cells were exposed to 28°C for a total of 12 h, the percentage of dead cells was measured. (B) Changes in the percentage of dead cells in hybrid cells from the cross N. suaveolens × N. tabacum cultured as illustrated in (A). Cells that were stained with Evans Blue were scored as dead. Values are means with SD (vertical bars) of results from five independent determinations. View largeDownload slide Fig. 6 Nuclear fragmentation in hybrid cells from the cross N. suaveolens × N. tabacum incubated at 28°C without inhibitor for 3–5 h, then with fresh medium containing inhibitor as shown in Fig. 5A; histograms indicate the extent of fragmentation of nuclei in the cells, as measured by flow cytometry of PI-treated nuclei. The vertical line in each figure divides the area containing additional peaks with lower fluorescence values from the G1+G2 peaks. The upper-left number in each figure is the percentage of additional peaks with lower fluorescence values out of the total. These indicate that the status of nuclear fragmentation. (A) 3 h ActD (–), 9 h ActD (+), (B) 4 h ActD (–), 8 h ActD (+), (C) 5 h ActD (–), 7 h ActD (+), (D) 3 h CHX (–), 9 h CHX (+), (E) 4 h CHX (–), 8 h CHX (+), (F) 5 h CHX (–), 7 h CHX (+). View largeDownload slide Fig. 6 Nuclear fragmentation in hybrid cells from the cross N. suaveolens × N. tabacum incubated at 28°C without inhibitor for 3–5 h, then with fresh medium containing inhibitor as shown in Fig. 5A; histograms indicate the extent of fragmentation of nuclei in the cells, as measured by flow cytometry of PI-treated nuclei. The vertical line in each figure divides the area containing additional peaks with lower fluorescence values from the G1+G2 peaks. The upper-left number in each figure is the percentage of additional peaks with lower fluorescence values out of the total. These indicate that the status of nuclear fragmentation. (A) 3 h ActD (–), 9 h ActD (+), (B) 4 h ActD (–), 8 h ActD (+), (C) 5 h ActD (–), 7 h ActD (+), (D) 3 h CHX (–), 9 h CHX (+), (E) 4 h CHX (–), 8 h CHX (+), (F) 5 h CHX (–), 7 h CHX (+). 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TI - Time Course Analysis of Apoptotic Cell Death during Expression of Hybrid Lethality in Hybrid Tobacco Cells (Nicotiana suaveolens × N. tabacum)
JO - Plant and Cell Physiology
DO - 10.1093/pcp/pcg055
DA - 2003-04-15
UR - https://www.deepdyve.com/lp/oxford-university-press/time-course-analysis-of-apoptotic-cell-death-during-expression-of-v6jVMFhsWC
SP - 420
EP - 427
VL - 44
IS - 4
DP - DeepDyve
ER -