Essay 12.5 Balancing Life and Death: The Role of the Mitochondrion in Programmed Cell Death

Essay 12.5 Balancing Life and Death: The Role of the Mitochondrion in Programmed Cell Death

Theresa J. Reape and Paul F. McCabe, School of Biology and Environmental Science, University College Dublin, Ireland


Mitochondria are the major sites of energy generation within the cell, hence being described in most undergraduate textbooks as the “powerhouse” of the cell. Research over the last two decades has established the mitochondrion as a central regulator of apoptosis in animal cells. Mitochondria also play a pivotal role in the life and death decisions of plant cells (Diamond and McCabe 2011). There are, however, major differences between plant and animal programmed cell death (PCD) pathways and, furthermore, various modes of PCD within these kingdoms. Hence, there are differences in the roles mitochondria play in these PCD pathways. PCD is a fundamental process in plants, controlling the elimination of cells during development, defense, and stress responses (Reape and McCabe 2008). In other words, this death process can occur as an integral part of a highly specialized developmental program such as xylogenesis or during the hypersensitive response (HR) following pathogen attack or simply in overstressed, neglected houseplants. It is likely, therefore, that every plant cell is capable of activating an organized series of events that result in its own destruction. Whether or not the plant activates this destructive machinery is determined by information it receives from a number of sources, including its environment, developmental signals, and assessment of its metabolic condition. The cell must assess these different external and internal factors and act upon them by remaining as it is, or by differentiating, dividing or dying.

The Mitochondrion and Mammalian Apoptosis

The most widely studied and understood form of PCD in animal cells is apoptosis. Apoptosis was initially defined, in the seminal paper by Kerr et al. (1972), in very specific morphological terms and is still characterized by cell shrinkage, nuclear condensation and fragmentation, and eventually the break-up of the cells into ‘apoptotic bodies.’ The early events that occur during apoptosis in mammalian cells are centered around the permeability of the mitochondrial outer membrane, which when compromised (following cellular stress), releases proteins normally contained within the intermembrane space, most notably cytochrome c. Released from its normal functional space, where it is a component of the electron transport chain, cytochrome c binds Apaf-1 (apoptotic protease activating factor 1), resulting in the formation of a complex of proteins referred to as the “apoptosome.” This leads to the proteolytic activation of caspases (a family of cysteine proteases responsible for cleaving cellular proteins), amplifying the death signal and resulting in the death of the cell. Several members of the Bcl-2 family of proteins are thought to function by maintaining the integrity of the mitochondrial outer membrane and preventing release of cytochrome c and other pro-apoptotic proteins, including Smac/Diablo, apoptosis inducing factor (AIF), and EndoG. On the other hand, other members of Bcl-2 family, Bax and Bak, are pro-apoptotic in function and are involved in the permeabilization of the mitochondrial outer membrane by formation of a pore or channel. A second way that mitochondria may release apoptotic proteins is through physical rupturing of the outer mitochondrial membrane. This rupture is caused by a rapid influx of water into the mitochondrial matrix, mediated through the formation of a transient pore known as the permeability transition pore (PTP). The PTP is a multiprotein complex formed at contact sites between the inner and outer mitochondrial membrane (IMM/OMM) and thought to act through the interaction between the voltage-dependent anion channel in the OMM, the adenine nucleotide transporter in the IMM, and cyclophylin D in the matrix (Delivani and Martin 2006; Kroemer et al. 2007).

An Apoptotic-like Plant Programmed Cell Death

Plant cells undergo various forms of PCD, one of which has been described as apoptotic-like (AL) or AL-PCD (see review by Reape and McCabe 2008). A study using carrot cells showed that heat treatment up to 55°C, dilution (i.e., removal of social signals) or embryogenic cell proliferation resulted in dead cells with a very specific cellular morphology, the most obvious feature being the retraction of the protoplast away from the cell wall (McCabe et al. 1997). Apart from morphology, other markers of animal apoptosis include caspase activation and DNA cleavage. Although there is no evidence for classic caspases in the Arabidopsis genome, caspase-like molecules do exist in plants, and there is evidence that caspase substrates are cleaved during plant PCD (Woltering 2004). A recurrent feature of apoptosis in animal cells and of AL-PCD in plant cells is cleavage of cellular DNA by PCD-associated endonucleases. In animal cells, DNA cleavage is thought to facilitate the packaging of DNA into apoptotic bodies that are then engulfed by macrophages. In plants, the rationale for DNA degradation is less clear, but several interesting possibilities have been proposed, such as it being a pathogen-defense response that prevents replication of viruses or other pathogens, or a method to recycle DNA phosphorus to other parts of the plant.

The Role of the Mitochondrion in Plant AL-PCD

Release of cytochrome c and other intermembrane space proteins

In animal cells the release of cytochrome c from the mitochondria results in it binding to the cytosolic protein, Apaf-1, that in turn activates procaspase-9. The activation of caspase-9 promotes activation of the other cytosolic caspases. The presence of “caspase-like” activity in plant cells made it tempting to assume a similar pathway of cytochrome c activation of plant PCD and indeed, cytochrome c release has been documented in numerous studies of plant PCD during abiotic stress, developmental PCD and defense response (see review by Reape and McCabe 2008). Yet, despite this strong association of cytochrome c release with plant PCD, a study using a cell-free system containing purified Arabidopsis nuclei indicated that other mitochondrial components are involved (Balk et al. 2003). Addition of broken mitochondria to the purified nuclei resulted in chromatin condensation, high-molecular weight DNA fragmentation, and DNA laddering, but the addition of purified plant cytochrome c had no such effect. Using submitochondrial fractionation and pharmacological studies, these authors were able to show that the generation of the 30 kb DNA fragments was the result of a Mg2+-dependent nuclease that normally resides in the mitochondrial intermembrane space.

The role of mitochondrial redox metabolism in plant PCD

If release of cytochrome c does not result in the direct activation of cytosolic proteases during plant PCD, is the cytochrome c release observed in plants simply a by-product of the release of other cell death molecules? It is possible that cytochrome c activates or amplifies the cell death process in ways other than direct protease activation. Release of cytochrome c from the mitochondria results in disruption of electron transport, leading to a rise in levels of reactive oxygen species (ROS). While originally thought of as toxic by-products of aerobic metabolism, it has become increasingly clear that ROS play important signaling roles in plants in processes such as PCD (Mittler 2002). Maxwell et al. (2002) inhibited mitochondrial electron transport with antimycin A and, using differential display, they showed that seven cDNAs were increased. Treatment with hydrogen peroxide or salicylic acid, which causes a rise in ROS and leads to decreased rates of electron transport, also induced the cDNAs. Interestingly, when the authors pretreated the cells with bongkrekic acid, an inhibitor of the mitochondrial PTP, the increase in gene expression was blocked. This work demonstrates that disruption of electron transport can certainly result in mitochondrial signals being transported to the nucleus, possibly as a result of PTP opening, and result in alteration of gene expression. It may be that ROS act as signaling molecules, which leads to the opening of the PTP, which would lead to release of cytochrome c and the generation of more ROS, causing a feedback loop which amplifies the original PCD-inducing stress signal (Jabs 1999). Further evidence that cytochrome c release is dependent on ROS production is that its release does not occur in the presence of scavengers of ROS (Vacca et al. 2006). Blackstone and Kirkwood (2003) hypothesize that during the evolution of mitochondria, the proto-mitochondrial electron transport chain and ROS may have played important roles in signaling between the mitochondrial symbiont and the host cell. These redox signaling mechanisms may have subsequently been adapted into primordial PCD mechanisms, which were driven by ROS production and release of cytochrome c (causing increased ROS production). Blackstone and Kirkwood further suggest that in animal cells this release of cytochrome c could have been refined over time, leading to the recruitment of caspases as the terminal effectors of apoptosis. As plants and animals share a common unicellular ancestor, it would not be surprising that plant cells also used ROS production and release of cytochrome c in cell death pathways.

How is release of mitochondrial intermembrane space proteins controlled in plant cells?

Given the potential for cellular damage if ROS levels were to rise dramatically, you can imagine that in order for them to be involved in signaling cues for life and death decisions in the plant cell, their production and accumulation must be under tight control. If cytochrome c or other mitochondrial intermembrane space proteins are involved in generation of ROS during PCD, what controls are in place to regulate their release in response to stress? As mentioned earlier, one way that mitochondrial membrane permeability (MMP) is controlled in mammalian cells is via a pore formed by Bax/Bcl-2 (Delivani and Martin 2006; Kroemer 2007). Briefly, the Bcl-2 family consists of proteins that promote (Bid, Bad, Bak, and Bax) or inhibit (Bcl-2 and Bcl-xL) apoptosis. Under normal conditions, OMM integrity is maintained through a fine balance of these proteins, preventing the release of cytochrome c and other intermembrane space proteins. Plants have no known homologs of the Bcl-2 family and, while there is evidence of pro or anti-apoptotic activity when the appropriate mammalian Bcl-2 proteins are expressed in tobacco (Lacomme and Santa Cruz 1999; Mitsuhara et al. 1999), there is no evidence that a pore formed from Bcl-2 family proteins operates during plant PCD. As mentioned earlier, animal mitochondria can also release apoptogenic factors from the mitochondria via the PTP. The PTP can be formed following cellular stress—including ROS production, build-up of Ca2+ or changes in phosphate and/or ATP levels—and this results in loss of the inner mitochondrial membrane potential (∆Ψm), osmotic swelling of the mitochondria, disruption of the OMM, and subsequent release of intermembrane space proteins. There is increasing evidence for involvement of the PTP during plant PCD (see review by Reape and McCabe 2010). But how is this pore controlled? Hexokinase, the enzyme that catalyzes the initial step in intracellular glucose metabolism, is a strong contender for controlling MMP in plants. Mammalian hexokinase can bind with high affinity to mitochondria at sites in the OMM through its interaction with the voltage-dependent anion chanel (VDAC) (Wilson 2003). This interaction is maintained by the serine/threonine kinase Akt and has been shown to play an important role in the control of mammalian apoptosis in the presence or absence of Bax and Bak (Wilson 2003). More recently, studies in plants have shown that mitochondrially associated hexokinases play an important role in the regulation of PCD in Nicotiana benthamiana (Kim et al. 2006). Kim et al. (2006) used virus-induced gene silencing (VIGS) to investigate the function of signaling genes in N. benthamiana. This screen revealed that VIGS of a hexokinase-encoding gene, Hxk1, caused the formation of necrotic lesions in leaves similar to those formed during the HR. When cells in the affected areas were examined, they displayed hallmark features of AL-PCD, including nuclear condensation and DNA fragmentation, and death was associated with loss of ∆Ψm, cytochrome c release, activation of caspase-9-like and caspase-3-like activities, and expression of genes known to be induced during the HR. Interestingly, Kim et al. also found that overexpresson of mitochondrially associated HXK1 and HXK2 in Arabidopsis increased the plants’ resistance to oxidative stress-induced PCD. Another study has shown that mitochondria-bound hexokinase has an antioxidant function in potato tubers (Camocho-Pereira et al. 2009). The potential involvement of mitochondria-associated hexokinase in regulation of both mitochondrial respiration and ROS production in plants has been recently reviewed by Bolouri-Moghaddam et al. (2010). It appears that the association of hexokinase with mitochondria is important in maintaining mitochondrial integrity during plant PCD. Of considerable interest is the fact that dissociation of hexokinase from animal mitochondria in the presence of apoptotic stimuli, but also in the presence of Bax and Bak, still results in cytochrome c release (though not as effective), which is not suppressed by Bcl-2 (Majewski et al. 2004). It is possible that while both animal and plant cells employ hexokinases to control MMP, animals may have evolved a further level of control involving the Bcl-2 family of proteins. Over time plants may have found this pathway sufficient for its cell death needs; subsequently, they may also have recruited caspase-like molecules into PCD or, alternatively, recruited other, as yet unidentified, molecules into plant cell death pathways.

Figure 1 Schematic representation of mitochondrial involvement in plant PCD. It is thought that the mitochondrion plays a role in integrating signals generated through developmental signals or stress, thus determining whether the cell activates its PCD pathway or not. Similar to animal cells, cytochrome c is released rapidly from the mitochondria in the early stages of plant PCD. Unlike in the animal system, however, cytochrome c release in plants does not appear to be directly responsible for activating a caspase-driven cascade of events leading to PCD, but rather may serve to amplify the death process. Cytochrome c release disrupts the electron transport chain, resulting in generation of ROS. As well as being a signal that can lead to opening of the PTP and release of cytochrome c, more ROS can be generated in this way, causing a feedback loop that amplifies the original death signal. The PTP in plants may be regulated through its interaction with mitochondrially associated hexokinase, which also has a role in regulating antioxidant activity, which in turn regulates ROS signaling and release. We also know that a Mg2+-dependent nuclease, which also normally resides in the mitochondrial intermembrane space, can be released from mitochondria, causing DNA degradation. HK = hexokinase; IMS = intermembrane space; PTP = permeability transition pore.


Balk, J., Chew, S. K., Leaver, C. J., and McCabe, P. F. (2003) The intermembrane space of plant mitochondria contains a DNase activity that may be involved in programmed cell death. The Plant Journal 34: 573–583.

Blackstone, N. W., and Kirkwood, T. B. L. (2003) Mitochondria and programmed cell death: “slave revolt” or community homeostasis? In Genetic and Cultural Evolution of Cooperation, P. Hammerstein, ed., The MIT Press, Cambridge, MA, pp. 309–325.

Bolouri-Moghaddam, M. R., Le Roy, K., Xiang, L., Rolland, F., and Van den Ende, W. (2010) Sugar signaling and antioxidant network connections in plant cells. FEBS Journal 277: 2022–2037.

Camocho-Pereira, J., Meyer, L. E., Machado, L. B., Oliveira, M. F., and Galina, A. (2009) Reactive oxygen species production by potato tuber mitochondria is modulated by mitochondrially bound hexokinase activity. Plant Physiology 149: 1099–1110.

Delivani, P., and Martin, S. J. (2006) Mitochondrial membrane remodelling in apoptosis: an inside story. Cell Death and Differentiation 13: 2007–2010.

Diamond, M., and McCabe, P. F. (2011) Mitochondrial regulation of plant programmed cell death. In Plant Mitochondria, Advances in Plant Biology 1, F. Kemden, ed., Verlag:Springer Publishing, pp. 439–465.

Jabs, T. (1999) Reactive oxygen intermediates as mediators of programmed cell death in plants and animals. Biochemical Pharmacology 57: 231–245.

Kerr, J. F., Wyllie, A. H., and Currie, A. R. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer 26: 239–257.

Kim, M., Lim, J. H., Ahn, J-H., Park, K., Kim, G. T., Kim, W. T., and Pai, H. S. (2006) Mitochondria-associated hexokinases play a role in the control of programmed cell death in Nicotiana benthamiana. The Plant Cell 18: 2341–2355.

Kroemer, G., Galluzzi, L., and Brenner, C. (2007) Mitochondiral membrane permeabilization in cell death. Physiological Reviews 87: 99–163.

Lacomme, C., and Santa Cruz, S. (1999) Bax-induced cell death in tobacco is similar to the hypersensitive response. Proceedings of the National Academy of Sciences USA 96: 7956–7961.

Majewski, N., Nogueira, V., Bhaskar, P., Coy, P. E., Skeen, J. E., Gotlob, K., Chandel, N. S., Thompson, C. B., Brooks Robey, R., and Hay, N. (2004) Hexokinase-mitochondria interaction mediated by Akt is required to inhibit apoptosis in the presence of Bax and Bak. Molecular Cell 16: 819–830.

Maxwell, D. P., Nickels, R., and McIntosh, L. (2002) Evidence of mitochondrial involvement in the transduction of signals required for the induction of genes associated with pathogen attack and senescence. The Plant Journal 29: 269–279.

McCabe, P. F., Levine, A., Meijer, P. J., Tapon, N. A., and Pennell, R. I. (1997) A programmed cell death pathway activated in carrot cells cultured at low cell density. The Plant Journal 12: 267–280.

Mitsuhara, I., Malik, K. A., Miura, M., and Ohashi, Y. (1999) Animal cell-death suppressors Bcl-x(L) and Ced-9 inhibit cell death in tobacco plants. Current Biology 15: 775–778.

Mittler, R. (2002) Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science 7: 405–410.

Reape, T. J., and McCabe, P. F. (2008) Apoptotic-like programmed cell death in plants. New Phytologist 180: 13–26.

Reape, T. J., and McCabe, P. F. (2010) Apoptotic-like regulation of programmed cell death in plants. Apoptosis 15: 249–256.

Vacca, R. A., Valenti, D., Bobba, A., Merafina, R. S., Passarella, S., and Marra, E. (2006) Cytochrome c is released in a reactive oxygen species-dependent manner and is degraded via caspase-like proteases in tobacco Bright-Yellow 2 cells en route to heat shock-induced cell death. Plant Physiology 141: 208–219.

Wilson, J. E. (2003) Isozymes of mammalian hexokinase: structure, subcellular localization and metabolic function. Journal of Experimental Biology 206: 2049–2057.

Woltering, E. J. (2004) Death proteases come alive. Trends in Plant Science 9: 469–472.

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