Essay 11.8

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

Jodi A. Swidzinski, Christopher J. Leaver, and Janneke Balk, Department of Plant Sciences, University of Oxford, UK

May, 2006

Programmed Cell Death

Mitochondria are major sites of energy conversion and carbon metabolism in the cell. In recent years, they have also been shown to play a major role in regulating cell death. This is not surprising given that inherited mitochondrial defects leading to respiratory dysfunction will ultimately lead to cell death, but this can be viewed as a passive physiological process. Conversely, programmed cell death (PCD) is an active form of cellular suicide controlled by a network of genes, and the controlled initiation and execution of PCD is essential for the normal development of both plants and animals. In plants, PCD plays a role during xylem formation, the deletion of the embryonic suspensor and aleurone cells in seeds, and leaf and organ sculpting, as the endpoint of senescence, in response to various forms of abiotic stress, and in response to pathogen attacks as part of the hypersensitive response (for an excellent review, see Lam 2004).

Our knowledge of the molecular processes underlying PCD has dramatically increased with the discovery of genes controlling the initiation and execution of cell death in animals. In particular a morphologically defined form of PCD, called apoptosis, is well-studied. It can be triggered by a receptor-mediated pathway responding to extracellular stimuli. In addition, apoptosis can be initiated by the release of mitochondrial proteins normally resident in the mitochondrial intermembrane space (IMS). In fact, the mitochondrial cell death pathways are often recruited by receptor-mediated apoptosis, probably to speed up the process of cell demise and removal. What is intriguing, however, is that the proteins released are not solely involved in the cell death pathway, but also have an important role in mitochondrial metabolism.

Cell Death Pathways Involving Mitochondria

Three, largely independent, pathways leading to programmed cell death involving the release of mitochondrial proteins to the cytosol have been described in animals (Figure 1; reviewed by Newmeyer & Ferguson-Miller 2003):

Figure 1 Mammalian cell death pathways involving mitochondria. Three pathways leading to programmed cell death in mammals are initiated by signals received by the mitochondrion and the release of proteins from the mitochondrial intermembrane space. The release of cytochrome c to the cytosol triggers the proteolytic activation of a caspase cascade. Conversely, the release of apoptosis inducing factor (AIF) or the mitochondrial endonuclease G and their translocation to the nucleus results in caspase-independent cell death. All three pathways result in the fragmentation of nuclear DNA. Members of the pro-apoptotic Bax family of proteins are stimulatory to all three pathways, while Bcl-2 family members act to inhibit programmed cell death. (Click image to enlarge.)

1. The release of cytochrome c, a component of the electron transport chain, from the IMS to the cytosol is often recognized in the early stages of cell death. In the cytosol, 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 cell death signal, and ultimately bringing about cell death. One of the caspases activates a DNA nuclease, resulting in the cleavage of chromosomal DNA into nucleosomal fragments (180–200 bp). This pattern of DNA "laddering," visualized by gel electrophoresis (Figure 2) is often used as a diagnostic marker for the occurrence of PCD. The potentially lethal activity of caspases is held in check by inhibitor of apoptosis (IAP) proteins in the healthy cell. However, among the proteins released from the IMS are two proteins that inactivate these IAPs. These are a serine protease known as HtrA, or Omi, and a protein that binds directly to IAP, called SMAC, or DIABLO.

Figure 2 Nuclear DNA fragmentation pattern characteristic of programmed cell death. Pea plumules were treated for ten minutes at 55°C to induce cell death or left untreated and incubated at room temperature for the indicated length of time. Total DNA was isolated and separated by agarose gel-electrophoresis. DNA laddering is evident as multimers of approximately 200 bp-sized fragments due to cleavage of the DNA between nucleosomes. The left lane represents molecular weight markers. (Click image to enlarge.)

The release of cytochrome c and other IMS proteins is regulated by a family of Bcl-2-like proteins that bind to the outer mitochondrial membrane and either have anti-apoptotic (Bcl-2) or pro-apoptotic activity (Bax).

The cytochrome c-dependent activation of Apaf-1 is only found in mammals. There is no evidence for the involvement of cytochrome c in nematodes, even though the Bcl‑2/Apaf‑1/caspase pathway plays a conserved role in apoptosis, and tethering of the Bcl-2 homologue Ced-9 is important for its function (Table 1).

Table 1 Evolutionary conservation of components of the programmed cell death pathway (Click image to enlarge.)

2. A protein referred to as apoptosis inducing factor (AIF) is released from the IMS and subsequently translocated to the nucleus. While nuclear DNA cleavage is observed during this process, this is not nucleosomal fragmentation but large, 50 kb fragments that are visualized by gel electrophoresis. The release of AIF and the resulting cell death occurs in a caspase-independent fashion.

3. Another caspase-independent pathway leading to PCD is mediated by endonuclease G, a nuclease that is released from the IMS, moves to the nucleus, and cleaves the chromosomal DNA generating nucleosome-sized fragments.

From Animals to Plants

Over the last 5 years, considerable progress has been made in unravelling the biochemical mechanisms of PCD in plants. Interesting differences with animals have come to the fore, which is not surprising given the different life strategies of plants and animals. Nevertheless, certain mechanisms turn out to be conserved, providing clues about the evolution of PCD.

For a long time, the existence of caspases in plants was hotly debated. Although caspase substrates were cleaved in cell extracts from tissues undergoing PCD, no caspase genes were found in the Arabidopsis genome sequence. Several enzymes have now been identified that possess "caspase activity" and play a role in PCD, for instance, in the defence against pathogen infection or during embryogenesis (see update by Woltering 2004).

Several groups have demonstrated that expression of pro-apoptotic genes such as Bax in transgenic plants leads to cell death, which mimics the hypersensitive response. Bax was found associated with plant mitochondria and appeared to disrupt mitochondrial integrity. Conversely, expression of members of the Bcl-2 family of proteins resulted in increased resistance to irradiation, paraquat treatment, and HR-associated cell death. Despite these results, Bax and Bcl-2 homologues have not been identified in Arabidopsis. In contrast, Bax inhibitor proteins are present in plants and are important in regulating PCD (Lam 2004).

Transgenic studies with Bax show that the release of mitochondrial proteins might activate the cell death program, as in animals. This is supported by research in our laboratory on cytoplasmic male sterility (CMS) in sunflower. CMS is associated with mutations in the mitochondrial genome. In sunflower, PCD is prematurely triggered in anther tissues, resulting in a late arrest of pollen development. Importantly, cell death in the anther tissues is associated with the hallmark production of DNA-laddering and the release of cytochrome c from the mitochondria prior to DNA cleavage (Balk and Leaver 2001). Several other studies involving the use of PCD-inducing toxins and abiotic stresses have also demonstrated a translocation of cytochrome c, but this is not universal in plant PCD. Thus far, there is no evidence that cytochrome c plays an active role in plant PCD. Instead, its redistribution in the cell might be a convenient assay to visualize the loss of outer mitochondrial membrane integrity, for example due to a rapid drop in the mitochondrial transmembrane potential, ΔΨ, which commonly precedes plant PCD (Curtis & Wolpert 2004; Yao et al. 2004).

The mammalian AIF protein bears similarity to plant monodehydroascorbate reductases (MDHAR), enzymes involved in the glutathione-ascorbate cycle in the cell. While some of the MDHARs may be targeted to the mitochondria and/or chloroplast, a secondary role for any of these proteins as components of the cell-death pathway has not been demonstrated (summarized above in Table 1).

Of the three mitochondrial pathways causing apoptosis in animals, the endonuclease-G pathway is the one that is most conserved, and is likely also to exist in plants. The protein has not yet been identified in plants, but then its abundance is thought to be extremely low, as shown in yeast. However, the IMS does contain a strong nuclease activity that is able to induce chromatin condensation and DNA fragmentation in a cell-free system (Balk et al. 2003).

To summarize, there is substantial evidence that mitochondria do play a role in PCD in plants. However, the molecular mechanisms are not yet clear but seem to be far less sophisticated than in mammals.

References

Balk, J., and Leaver, C. J. (2001) The PET1-CMS mitochondrial mutation in sunflower is associated with premature programmed cell death and cytochrome c release. Plant Cell 13: 1803–1818.

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. Plant J. 34: 573–583.

Curtis, M. J., and Wolpert, T. J. (2004) The victorin-induced mitochondrial permeability transition precedes cell shrinkage and biochemical markers of cell death, and shrinkage occurs without loss of membrane integrity. Plant J. 38: 244–259.

Lam, E. (2004) Controlled cell death, plant survival and development. Nature Reviews Mol. Cell Biol. 5: 305–315.

Newmeyer, D. D., and Ferguson-Miller, S. (2003) Mitochondria: Releasing power for life and unleashing the machineries of death. Cell 112: 481–490.

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

Yao, N., Eisfelder, B. J., Marvin, J., and Greenberg, J. T. (2004) The mitochondrion—An organelle commonly involved in programmed cell death in Arabidopsis thaliana. Plant J. 40: 596–610.