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Plant Physiol, August 2002, Vol. 129, pp. 1908-1920 Transgenic Plant Cells Lacking Mitochondrial Alternative Oxidase Have Increased Susceptibility to Mitochondria-Dependent and -Independent Pathways of Programmed Cell Death1Division of Life Sciences and Department of Botany, University of Toronto at Scarborough, 1265 Military Trail, Scarborough, Ontario, Canada M1C 1A4
The plant mitochondrial electron transport chain is branched such that electrons at ubiquinol can be diverted to oxygen via the alternative oxidase (AOX). This pathway does not contribute to ATP synthesis but can dampen the mitochondrial generation of reactive oxygen species. Here, we establish that transgenic tobacco (Nicotiana tabacum L. cv Petit Havana SR1) cells lacking AOX (AS8 cells) show increased susceptibility to three different death-inducing compounds (H2O2, salicylic acid [SA], and the protein phosphatase inhibitor cantharidin) in comparison with wild-type cells. The timing and extent of AS8 cell death are very similar among the three treatments and, in each case, are accompanied by the accumulation of oligonucleosomal fragments of DNA, indicative of programmed cell death. Death induced by H2O2 or SA occurs by a mitochondria-dependent pathway characterized by cytochrome c release from the mitochondrion. Conversely, death induced by cantharidin occurs by a pathway without any obvious mitochondrial involvement. The ability of AOX to attenuate these death pathways may relate to its ability to maintain mitochondrial function after insult with a death-inducing compound or may relate to its ability to prevent chronic oxidative stress within the mitochondrion. In support of the latter, long-term treatment of AS8 cells with an antioxidant compound increased the resistance of AS8 cells to SA- or cantharidin-induced death. The results indicate that plants maintain both mitochondria-dependent and -independent pathways of programmed cell death and that AOX may act as an important mitochondrial "survival protein" against such death.
Programmed cell death (PCD) is an
essential physiological process occurring during plant development and
in response to biotic and abiotic stress (Beers and McDowell, 2001 Although the cellular and molecular events involved in plant PCD are
only beginning to be elucidated, the events involved in animal
PCD (commonly called apoptosis) have been extensively characterized
(for recent reviews, see Desagher and Martinou, 2000 Cyt c release from the mitochondrion is tightly regulated by
the Bcl-2 family of proteins. Anti-apoptotic Bcl-2 family members act
to prevent cyt c release, whereas pro-apoptotic Bcl-2 family members promote such release (Adams and Corey, 2001 There is also debate regarding the relative importance of caspase
activation versus mitochondrial dysfunction in promoting animal PCD.
Although it is apparent that changes in key mitochondrial parameters
(such as membrane potential, rate of electron transport, rate of ATP
synthesis, rate of reactive oxygen species [ROS] generation, and matrix Ca2+ concentration) occur during death
programs, the functional significance of these changes in terms of
promoting or attenuating cell death remains controversial (Green and
Kroemer, 1998 A prevalent theme in animal PCD research is that the intracellular
redox state may play a critical role in the overall process (Voehringer
et al., 2000 It is unclear to what extent PCD events in plants are similar to those
in animals. For example, although there is recent evidence for the
release of cyt c from the mitochondrion during plant PCD (Sun et al., 1999 The plant mitochondrion has unique components that alter mitochondrial
function in comparison with animal cells and, hence, could alter the
specific mechanisms by which the mitochondrion takes part in PCD. One
such component is alternative oxidase (AOX), a mitochondrial inner
membrane protein that functions as a part of the ETC, catalyzing the
O2-dependent oxidation of ubiquinol, producing
ubiquinone and water (Vanlerberghe and Ordog, 2002 A few studies have implicated AOX in plant PCD. In Arabidopsis, a
strategy used to identify genes induced early in the HR identified both
AOX and a mitochondrial anion channel gene (Lacomme and Roby, 1999 We previously generated transgenic tobacco (Nicotiana
tabacum L. cv Petit Havana SR1) cells in which AOX gene expression
was silenced by an antisense transgene (Vanlerberghe et al., 1994
Antisense Cells Lacking Mitochondrial AOX Have Increased Susceptibility to Several Different Death-Inducing Compounds When a low concentration of H2O2 was added to the growth medium of wt tobacco cells (at 2 d after subculture), it resulted in a large (approximately 30%-60%) drop in culture viability within 4 h (Fig. 1). Culture viability dropped somewhat further by 8 and 18 h but then stabilized and began to recover so that by 120 h after treatment, the culture was again near 100% viable cells.
We compared the above wt response with that of transgenic tobacco cells
(AS8) that lack mitochondrial AOX and found that the extent, pattern,
and timing of cell death were much different. In the AS8 culture, there
was no loss of culture viability at 4 h after treatment and only
about a 10% to 30% drop by 8 h (Fig. 1). However,
despite this apparent increased resistance to
H2O2 in the short term
(0-8 h), we consistently found that between 8 and 18 h, AS8
culture viability dropped to at or near 0%. In most cases, there was
no recovery of the culture by 120 h (Fig. 1). These differences in
the pattern and extent of cell death between wt and AS8 cells were not
attributable to differences in the dosage of
H2O2 (millimoles per gram
dry weight of cells) applied (Fig. 1; see "Materials and
Methods" for further explanation). The increased short-term
resistance of AS8 cells to
H2O2 (0-8 h) may relate to
the increased level of expression of antioxidant enzymes previously
observed in these cells (Maxwell et al., 1999 We compared the effects of
H2O2 with those of two
other compounds, the plant phenolic salicylic acid (SA) and
cantharidin, a potent inhibitor of Ser/Thr protein phosphatase types 1 and 2A (Li and Casida, 1992
In the case of cantharidin, both wt and AS8 cultures again showed little loss of culture viability within the first 8 h (Fig. 2C). However, between 8 and 18 h, the AS8 culture again dropped to at or near 0%. In the wt, viability at 18 h was much higher (approximately 65%) and then decreased only gradually over the 120-h period. Accumulation of Oligonucleosomal Fragments of DNA Indicates That AS8 Cells Undergo PCD To determine whether cells were experiencing a necrotic or programmed form of cell death, we examined genomic DNA extracted from cells at different times (8, 24, 48, and 120 h) after addition of a death-inducing compound. For AS8 cells treated with
H2O2, we saw only
high-Mr DNA at 8 h (when viability of
the culture is still high), but by 24 h, we observed the
accumulation of lower Mr fragments of DNA
(Fig. 3B). Comparison with a 100-bp
DNA ladder indicated that the accumulating fragments matched the
expected size of oligonucleosomal fragments (approximately 180-200 bp)
and multiples thereof. This pattern (commonly called DNA laddering and
indicative of PCD; Ryerson and Heath, 1996
In the case of AS8 cells treated with SA, we readily observed DNA laddering at 24, 48, and 120 h (Fig. 3D). Also, interestingly, the loss of high-Mr DNA in this case was much more complete than in H2O2-treated AS8 cells. As an alternative, for wt cells treated with SA, only high-Mr DNA was observed throughout the 120-h period (Fig. 3C). In the case of AS8 cells treated with cantharidin, DNA laddering was once again readily observed at 24, 48, and 120 h (Fig. 3F). For wt cells treated with cantharidin, laddering was only observed in the long term (120 h, Fig. 3E), coincident with the large amount of cell death by this time point (see Fig. 2C). PCD Is Preceded by Changes in Respiration and Mitochondrial Function The respiratory characteristics of wt and AS8 cells were compared at 8 h after treatment with a death-inducing compound (i.e. before the major drops in viability). Also, we compared the respiratory characteristics being measured in whole cells (i.e. in vivo measurements) with those of mitochondria isolated from such cells (i.e. in organello measurements). This comparison was valuable in determining whether changes in whole-cell respiratory characteristics were the result of changes in mitochondrial ETC function specifically or the result of changes in upstream carbon metabolism. We also confirmed cell viability of the cultures for these experiments. In all cases, viability after the 8-h treatments was similar to that seen in Figure 2. That is, viability remained reasonably high (averaging from 82% to 97%, depending on the treatment) with the exception of wt cells treated with H2O2, in which case culture viability had dropped to approximately 59%, as expected (see Figs. 1 and 2). AOX Capacity In vivo and in organello assays were used to estimate the maximum potential rate of electron transport through the AOX pathway (see "Materials and Methods"). In the wt, AOX capacity in untreated (control) cells was high, whether measured in vivo (Fig. 4A) or in organello (Fig. 4B). An 8-h treatment of cells with H2O2 or SA reduced this capacity somewhat (whether measured in vivo or in organello), but significant capacity was still maintained. Cantharidin treatment had no impact on in vivo AOX capacity, although it was reduced somewhat when measured in organello.
As expected, the AOX capacity of untreated (control) AS8 cells was very low in comparison with the wt, whether measured in vivo (Fig. 4A) or in organello (Fig. 4B). This indicates that the antisense transgene was effectively suppressing AOX expression in these cells. With H2O2 or SA treatment, capacity was negligible either in vivo or in organello. As an alternative, in vivo or in organello AOX capacity after cantharidin treatment remained low and similar to untreated AS8 cells. Respiration The respiration rate of whole cells (treated or untreated) was determined simply by monitoring oxygen uptake (in the absence of any inhibitors), whereas the respiration rate of isolated mitochondria was determined by monitoring oxygen uptake in the presence of a suite of respiratory substrates (see "Materials and Methods"). The in vivo data is, thus, a measure of actual cell respiratory activity, whereas the in organello data is a measure of the functional state of the mitochondrion. The respiration rate of wt cells was reduced by approximately 60% at 8 h after treatment with H2O2 or SA (Fig. 5A). In organello respiration was reduced by a similar extent (by approximately 40%) in response to these treatments (Fig. 5C). As an alternative, cantharidin-treated wt cells showed only a small decline in cell respiration and no decline (in fact a small increase) in mitochondrial respiration (Fig. 5, A and C).
The respiration of H2O2-treated AS8 cells was completely abolished, whether measured in vivo (Fig. 5A) or in organello (Fig. 5C). Similarly, SA-treated AS8 cells showed no cell respiration, although a low level of mitochondrial respiration (<10% of control) was still observed. Interestingly, cantharidin reduced in vivo AS8 respiration to less than 30% of control rates (Fig. 5A), but this occurred in the absence of any effect on mitochondrial respiration, which remained at levels slightly greater than control values (Fig. 5C). These data indicate that cantharidin treatment was reducing the respiratory activity of AS8 cells but that this was not attributable to a direct loss of mitochondrial function. Cyt Pathway Capacity In vivo and in organello assays were used to estimate the functional state of the cyt pathway (see "Materials and Methods"). In wt cells, treatment with H2O2 or SA reduced cyt path capacity measured in either cells or mitochondria to a similar extent as respiration was reduced (compare Fig. 5, B and D with A and C). As an alternative, no loss of cyt path (in vivo or in organello) was seen with cantharidin, similar to what had been noted for the respiration of cantharidin-treated wt cells. In fact, the mitochondrial cyt path capacity of cantharidin-treated cells actually increased nearly 1.6-fold in comparison with untreated wt cells (Fig. 5D). In AS8, both the cell and the mitochondrial cyt path were completely abolished after an 8-h treatment with H2O2 or SA, except for a small amount of the mitochondrial cyt path in the SA-treated cells (Fig. 5, B and D). In the case of canthardin, the AS8 cell cyt path was almost abolished, despite no loss of the mitochondrial cyt path (Fig. 5, B and D). In Antisense Cells, the Loss of Mitochondrial Function after H2O2 or SA Treatment and Preceding PCD Involves Cyt c Release from the Mitochondrion We examined the level of ETC proteins in mitochondria isolated after the 8-h treatments with a death-inducing compound. In mitochondria from wt cells, AOX protein was always visible in the mitochondria from both treated and untreated cells, consistent with the presence of AOX capacity in these cells (Fig. 6A). As an alternative, we were unable to detect any AOX protein in mitochondria isolated from AS8 cells, which is consistent, in these cases, with the lack of AOX capacity (Fig. 6A).
The levels of two proteins in the cyt pathway were also examined. In the wt, cyt c was present in mitochondria from both untreated cells and cells treated with H2O2, SA, or cantharidin (Fig. 6A). However, in the case of AS8 cells, we found that both the H2O2 and SA treatments resulted in a massive loss of cyt c protein from the mitochondrion. As an alternative, cantharidin treatment did not generate this loss of cyt c. Cyt oxidase subunit II (cox II) was abundant in mitochondria from both wt and AS8 cells, whether untreated or treated with one of the death-inducing compounds (Fig. 6A). Figure 6B is an example of a Coomassie-stained SDS-PAGE gel similar to that used for the immunoblot analyses in Figure 6A. We consistently found that in the AS8 cells treated for 8 h with either H2O2 or SA (i.e. the two treatments that result in cyt c release from the mitochondrion; Fig. 6A), there were several changes in the mitochondrial protein profile, and these changes always occurred similarly in both of these treatments and in none of the other treatments. One prominent change (marked by the arrow in Fig. 6B) was the increased intensity of an unidentified protein of approximately 60 kD. A time-course experiment examined the effects of SA on AS8 cells in more detail. We found that AS8 cell respiration and cell cyt pathway capacity were severely depressed within 2 h after addition of SA, despite no loss of mitochondrial respiration or mitochondrial cyt path at this time point (Fig. 7). This indicates that a general depression of cell respiration precedes changes in mitochondrial function. By 5 h, cell respiration and cell cyt path were completely lost. By this time as well, mitochondrial respiration and mitochondrial cyt path were each reduced to about 50% of control levels (Fig. 7), and this correlated with some loss of cyt c from the mitochondrion by this time (Fig. 8A). Finally, at 8 h, mitochondrial respiration and cyt path were almost completely lost (Fig. 7), as was the level of cyt c (Fig. 8A). Importantly, all of these events precede any significant loss of culture viability (Fig. 8A).
To examine whether cyt c loss might still be an event preceding the death of cantharidin-treated AS8 cells (despite the lack of cyt c release by 8 h, Fig. 6A), a time-course experiment examined viability and cyt c level in the longer term. By 11 and 14 h after cantharidin treatment, viability dropped to 52% and 17%, respectively (Fig. 8B). Despite these now large drops in viability, cyt c remained abundant in isolated mitochondria (Fig. 8B), and cyt path capacity was still evident (data not shown). Long-Term Pretreatment of Antisense Cells with Antioxidants Attenuates PCD We tested whether the antioxidant compound flavone would delay or prevent PCD in AS8 cells. In one type of experiment, 100 µM flavone was added to the cell culture 15 min before the addition of either SA or cantharidin. This short-term pretreatment with flavone had no significant effect on either the timing or extent of cell death in response to either SA or cantharidin (data not shown). In another type of experiment, cells were grown for 2 d (i.e. from the time of subculture) in the presence of flavone before the addition of a death-inducing compound. This long-term pretreatment was found to significantly delay the death induced by either SA or cantharidin (Fig. 9). The treatments in which death was delayed still displayed DNA laddering (indicative of PCD), except that the laddering was delayed because of the slower death of the culture (data not shown).
Two key findings of the present research are as follows: (a) Transgenic cells lacking AOX are more susceptible than wt cells to PCD. To our knowledge, this is the first example showing that manipulation of a component of plant mitochondrial metabolism can dramatically influence PCD. Our discussion of this comparison between wt and transgenic cells and our hypotheses regarding the increased susceptibility of AS8 cells will be discussed later. (b) Using different death-inducing compounds, we have identified two different pathways of PCD, one pathway with an apparent mitochondrial connection, and a second pathway lacking any obvious mitochondrial involvement. Mitochondria-Dependent and -Independent Pathways of PCD in Tobacco Cells The discussion below pertains to AS8 cells, in which PCD was readily induced. H2O2, SA, and cantharidin each induced PCD in AS8, and the timing and extent of death were remarkably similar between the three treatments (Figs. 1 and 2). In each case, a major drop in culture viability occurred between 8 and 18 h after treatment. DNA laddering became apparent by 24 h after each treatment and then persisted through 120 h (Fig. 3). Despite these similarities between treatments, analyses of cellular events preceding death (i.e. within the first 8 h) indicated that the effects of H2O2 and SA were very similar to one another but that the effects of cantharidin were fundamentally different. Examination of AS8 respiratory characteristics in response to the different death-inducers revealed a striking similarity between the effects of H2O2 and SA. Both compounds abolished the capacity for cyt path respiration by 8 h after treatment. This was confirmed in both cells and mitochondria derived from such cells (Fig. 5, B and D). As a result of this inhibition (and the lack of AOX in AS8), neither cells nor mitochondria displayed any respiratory O2 uptake (Fig. 5, A and C). Importantly, the lack of cyt path at 8 h was accompanied by an almost complete loss of cyt c from the mitochondrion (Fig. 6). To examine the above changes more closely, a time-course experiment was performed using SA. Two important observations arose from these experiments. First, cell respiration was dramatically depressed at 2 h after treatment, but this was clearly not due to an inhibition of mitochondrial ETC function. Mitochondrial ETC function appeared normal, whether evaluated using the mitochondrial respiration or mitochondrial cyt path assay (Fig. 7). Note that the cell cyt path assay did indicate a low cyt path capacity at 2 h. However, this is artifactual, arising from the fact that cell respiration had been dramatically reduced by this time point. These results illustrate the importance of having done measurements of both cell and mitochondrial respiratory characterisitics. Examining both determined whether changes in cell respiratory characteristics were due to changes in mitochondrial ETC function or some other event. The other important observation arising from the time-course experiment was that the change in mitochondrial cyt path capacity approximately correlated with the change in cyt c level in the mitochondrion (Figs. 7B and 8A). At 2 h after treatment, no loss of cyt path capacity or cyt c level was obvious, whereas at 5 h, both parameters began to decline. By 8 h, both cyt path capacity and cyt c level were very low. Importantly, these events precede any significant level of cell death in the culture. In summary, the results show the following order of events in SA-treated AS8 cells: (a) a severe depression of whole-cell respiratory activity before any change in mitochondrial ETC function (between approximately 0 and 2 h after treatment); (b) a rapid decline in mitochondrial ETC function in which a loss of cyt c from the mitochondrion may be a critical event (between approximately 5 and 8 h after treatment); and (c) rapid PCD (between 8 and 18 h after treatment). SA and H2O2 were chosen as
death inducers for our experiments because each has been implicated to
play a role in the HR (Jabs, 1999 We were struck by how similar the response of AS8 cells were to SA and
H2O2. Both compounds
induced almost identical changes in respiratory characteristics and
induced cyt c release. Also, both compounds generated
several changes in the mitochondrial protein profile of AS8 cells, and
these changes were identical between the two treatments (Fig. 6B).
Attempts are under way to identify these proteins, with the hope that
it will provide insight into the plant mitochondrial PCD pathway. The
similarity between the SA and
H2O2 treatments is
consistent with evidence that these compounds may act as complementary
or synergistic signals during the HR and in response to abiotic stress
(Jabs, 1999 Cantharidin treatment had a different effect than H2O2 and SA on AS8 respiratory characteristics. Cell respiration and apparent cell cyt pathway capacity were dramatically reduced at 8 h (just as seen with the other treatments), but these changes in cell respiratory characteristics were not attributable to changes in mitochondrial ETC function. Mitochondrial ETC function at 8 h (whether assessed by the mitochondrial respiration or mitochondrial cyt pathway capacity assay) was similar to that of mitochondria from the untreated cells (Fig. 5). Consistent with this, these mitochondria retained their cyt c (Fig. 6A). A longer term time-course experiment with cantharidin (8, 11, and 14 h) clearly showed that cyt c loss was not an event preceding death of the culture. For example, at 11 h after cantharidin treatment, 48% of the cells were already dead but high levels of cyt c were still evident (Fig. 8B). Some loss of cyt c appeared to occur at the later time points (particularly at 14 h), but this is probably attributable to a general decline in cell organization, because culture viability by this time point had already dropped to only 17%. In summary, the results suggest the following order of events in
cantharidin-treated AS8 cells: (a) a depression of whole-cell respiratory activity (within 8 h), before any change in
mitochondrial ETC function; and (b) rapid PCD (between 8 and 18 h), before any apparent cyt c release from the
mitochondrion. Previous studies also found that addition of Ser/Thr
protein phosphatase inhibitors induced rapid plant cell death,
suggesting that changes in protein phosphorylation may be a common
event in death induction (Suzuki et al., 1999 Despite no indication that the mitochondrion is involved in the
cantharidin-induced pathway (at least with respect to cyt c
release), it is interesting that cantharidin severely depressed whole-cell respiration in AS8 cells, just as was found with
H2O2 and SA. This may be
coincidental or may indicate that a depression of whole-cell
respiration is a common early event among these different PCD pathways.
The ability of SA to dramatically inhibit whole-cell respiration in the
short term (30 min) but without any apparent effect on mitochondrial
ETC function was also seen previously (Xie and Chen, 1999 How Does AOX Influence Susceptibility to PCD? An important challenge in future experiments is to determine why
antisense cells lacking AOX are more susceptible to PCD-inducing compounds than wt cells with abundant AOX. Such a determination could
provide insight into the cellular conditions that promote cell survival
versus those promoting cell death. The usefulness of such an approach
is illustrated by experiments using an apoptosis-sensitive and
-resistant murine B-cell lymphoma model system (Voehringer et al.,
2000 Our work shows that tobacco cells can undergo PCD by at least two different pathways: a mitochondria-dependent pathway involving cyt c release and a pathway without any obvious mitochondrial involvement (Fig. 10B). Nonetheless, the AS8 cells with altered mitochondrial function (i.e. a lack of AOX) have increased susceptibility to compounds inducing either of these PCD pathways. Below, we describe our two broad hypotheses as to why wt and AS8 cells differ in their susceptibility to PCD. Although discussed separately, the two hypotheses are not mutually exclusive, and it seems possible that both may play a role or that their relative importance may depend upon whether the mitochondria-dependent or -independent pathway is considered.
Mitochondrial Function Hypothesis In this case, it is the activity of AOX in wt cells after the insult with a death-inducer that is primarily responsible for the differential sensitivity of wt and AS8 cells to PCD. In other words, after the insult of wt cells with a death-inducing treatment, active participation of AOX in the respiration of these cells acts to attenuate cell death in some fashion. Such a hypothesis is dependent upon wt cells maintaining a functional AOX pathway after the treatments, which is what we observed. After an 8-h treatment with H2O2, SA, or cantharidin, significant levels of AOX capacity were present, whether measured in cells or mitochondria (Fig. 4). A characteristic of AOX activity is that electron flow from ubiquinol to oxygen is uncoupled from the generation of proton motive force (Fig. 10A). This, combined with the ability to biochemically regulate electron flux to AOX in a sophisticated manner (Vanlerberghe et al., 1998 ), provides the mitochondrion with considerable metabolic flexibility. This flexibility could potentially allow the mitochondrion to (a) modulate the rate of ATP production; (b) maintain electron flux
to oxygen when other downstream ETC components or ADP are limiting; or
(c) modulate the reduction state of ETC components, thereby controlling
the rate of generation of ROS by electron transport (Fig. 10A).
Significantly, mitochondrial parameters such as these are commonly
implicated as important during animal PCD, although the role of
individual parameters in promoting or attenuating important
mitochondrial events such as cyt c release remains unclear (see introduction).
If AOX activity in wt cells acts to attenuate PCD in response to SA or
H2O2, it seems probable
that it would have to do so by promoting, either directly of
indirectly, the maintenance of cyt c. In plants, nothing is
known of the mechanisms responsible for cyt c loss, making
it difficult to speculate on a mechanism by which AOX activity might
attenuate this event.
In animals, the Bcl-2 family of proteins tightly regulates the cyt
c release associated with PCD (Adams and Corey, 2001). Such
active regulation is critical so that PCD is not inappropriately triggered or suppressed. As it becomes more firmly established that cyt
c release is also an important event in plant PCD, the question arises as to how the process is actively regulated, because bioinformatics analyses have not revealed the presence of Bcl-2-like proteins in plants. If plants do lack a Bcl-2-like regulatory system,
it seems that some other regulatory mechanism would be required. Our
work here suggests that the abundance and activity of AOX may serve
such a regulatory function.
Chronic Oxidative Stress Hypothesis In this case, it is the absence of AOX in AS8 cells before the insult with a death inducer that is primarily responsible for the differential sensitivity of wt and AS8 cells to PCD. An important consequence of the lack of AOX in AS8 is that these cells experience constitutive higher in vivo levels of ROS than do wt cells (Maxwell et al., 1999; Parsons et al., 1999; Yip and Vanlerberghe, 2001). It has
been elegantly shown that the source of these increased ROS is the
mitochondrion (Maxwell et al., 1999) and that this is accompanied by
changes in the expression of nuclear genes encoding antioxidant enzymes
(such as catalase) and other genes known to respond to increased ROS,
such as PR-1 (Maxwell et al., 1999). Such results clearly indicate that
the increased mitochondrial ROS being produced in AS8 cells is
physiologically relevant, having altered the pattern of gene expression
in these cells.
Mitochondrial oxidative damage and changes in gene expression
attributable to oxidative stress are widely implicated as important contributors to many PCD pathways in animal and plant cells (for review, see Jabs, 1999; see introduction). It seems possible that the
chronic increased level of mitochondrial ROS ensures that AS8 cells are
more "competent to die" than are wt cells. In plants, we are only
beginning to identify genes that may play a role in PCD (Lacomme and
Roby, 1999; Pontier et al., 1999; Hoeberichts et al., 2001). It will be
of interest whether the expression of such candidate genes differs
between wt and AS8 cells. It will also be of interest to determine
whether AS8 mitochondria show signs of increased oxidative damage in
comparison with wt cells.
Results of experiments using the antioxidant flavone are consistent
with the chronic oxidative stress hypothesis. Flavone was used in these
experiments because it has previously been shown to reduce the in vivo
level of ROS in these plant cells (Maxwell et al., 1999). If the
increased susceptibility of AS8 cells to PCD is due to oxidative damage
and/or to changes in gene expression resulting from chronic oxidative
stress, then one might expect that a short-term treatment with flavone
(i.e. the 15-min treatment) would be insufficient time to remedy this
and, hence, insufficient time to alter the cell death pattern. This is
what we observed. This experiment also illustrates that the presence of
flavone during treatment with SA or cantharidin is, in itself,
insufficient to alter the death pattern. As an alternative, a long-term
treatment with flavone (i.e. the 2-d treatment; Fig. 9) would allow
sufficient time for oxidative damage and/or gene expression patterns
(and, hence, the cell death pattern) to be altered. Again, this is what we observed (Fig. 9).
Together, the above results are consistent with the hypothesis that
chronic oxidative stress is "priming" AS8 cells for PCD and that if
this stress is relieved (such as with flavone), the cells become less
susceptible to PCD. With this view, the importance of AOX in
susceptibility to PCD lies in its ability to continually dampen the
mitochondrial generation of ROS, hence preventing oxidative damage,
aberrant gene expression, or some other consequence of increased ROS
that favors PCD (Fig. 10A).
Plant Material, Growth Conditions, and Experimental Treatments The cells used were derived from leaves of wt and transgenic
tobacco (Nicotiana tabacum L. cv Petit Havana SR1) and
were in culture for approximately 8 years before this study
(Vanlerberghe et al., 1994 Cell cultures (200 mL of culture in 500-mL Erlenmeyer flask) were grown
in the dark on a rotary shaker (140 rpm) at 28°C and were subcultured
every 7 d by dilution in fresh growth medium. The growth medium
(Linsmaier and Skoog, 1965 We were careful to ensure that our subculturing routine was generating wt and AS8 cultures that, at 2 d after subculture, were of similar density to one another and of similar density from one experiment to the next. In initial experiments, we both determined culture density just before H2O2 addition and varied the initial dosage of H2O2 applied (i.e. millimoles of H2O2 per gram dry weight of cells; Fig. 1). These analyses showed that the differences seen between wt and AS8 cultures in the extent of cell death were not due to differences in culture density at the time of H2O2 addition. Similar conclusions were drawn for the SA and cantharidin treatments. Respiratory Characteristics of Cells Suspension cells (1-2.5 mg dry weight
mL Respiratory Characteristics of Isolated Mitochondria Washed mitochondria were isolated under cold conditions from 4 × 200 mL of suspension cells. Cells were collected onto qualitative filter paper (VWR Canlab, Mississauga, ON, Canada) by vacuum filtration and washed briefly with ice-cold water. Cells were then scraped into a cooled commercial blender to which 300 mL of ice-cold homogenization medium was added. The homogenization medium consisted of 350 mM mannitol, 30 mM 3-(N-morpholino) propanesulfonic acid (pH 7.5), 1 mM EDTA, 10 mM Cys, 0.6% (w/v) polyvinylpolypyrrolidone, and 0.2% (w/v) bovine serum albumin. Cells were then disrupted by four pulses (of 3-s duration each) of the blender at maximum speed, and the homogenate was immediately filtered through two layers of Miracloth (Calbiochem-Novabiochem, San Diego). The filtrate was centrifuged at low speed (3,850g, 2 min, 4°C), and the supernatant was then centrifuged at higher speed (20,600g, 10 min, 4°C) to pellet the mitochondria. The pellet was then carefully suspended in wash medium consisting of 300 mM mannitol, 20 mM 3-(N-morpholino) propanesulfonic acid (pH 7.2), 1 mM EDTA, and 0.2% (w/v) bovine serum albumin. The sample was then centrifuged at low speed (3,850g, 2 min, 4°C), and the supernatant was centrifuged at high speed (20,600g, 10 min, 4°C) to pellet the mitochondria. The pellet was then suspended in a small volume of wash medium. Immediately after isolation, O2 uptake by mitochondria
(adjusted to 0.1-0.25 mg protein mL Protein Analysis of Mitochondria Reducing SDS-PAGE was performed as previously described
(Vanlerberghe et al., 1998 DNA Isolation and Analysis Isolation of genomic DNA was based on a method described by
Mettler (1987) Other Methods Cell viability was determined by microscopic observation of
cells treated with Evans blue, which accumulates in dead cells as a
blue protoplasmic stain (Baker and Mock, 1994
We thank Dr. Sasan Amirsadeghi, David Couto, Sandi H. Ordog, Mark Sandico, and Justine Y.H. Yip for their contributions to this work.
Received March 1, 2002; returned for revision April 9, 2002; accepted April 26, 2002. 1 This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada and by a Premiers Research Excellence Award of Ontario (both to G.C.V.).
* Corresponding author; e-mail gregv{at}utsc.utoronto.ca; fax 416-287-7642.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.004853.
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TOP
ABSTRACT
INTRODUCTION
) and transgenic (AS8; 
)
tobacco cell cultures at different times after the addition of
H2O2 (3-5 mM)
to the culture medium at time 0. Each symbol represents data from an
independent experiment.
1
dry weight h
); cantharidin only
(
); SA + flavone (
); and cantharidin + flavone (
). Note that
the black triangles are being obscured by the black squares at most
time points. Data are the means ± SE from three
independent experiments. In some cases, error bars are smaller than the
data symbols.
80°C. The O2
concentration in air-saturated water at 28°C was assumed to be 253 µM and protein concentration was determined as
described below.
