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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.
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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.