Treatment of tobacco (Nicotiana tabacum L. cv Petit Havana SR1) cells with cysteine (Cys) triggers a signal pathway culminating in a large loss of mitochondrial cytochrome (cyt) pathway capacity. This down-regulation of the cyt path likely requires events outside the mitochondrion and is effectively blocked by cantharidin or endothall, indicating that protein dephosphorylation is one critical process involved. Generation of reactive oxygen species, cytosolic protein synthesis, and Ca 2ϩ flux from organelles also appear to be involved. Accompanying the loss of cyt path is a large induction of alternative oxidase (AOX) protein and capacity. Induction of AOX allows the cells to maintain high rates of respiration, indicating that the lesion triggered by Cys is in the cyt path downstream of ubiquinone. Consistent with this, transgenic (AS8) cells unable to induce AOX (due to the presence of an antisense transgene) lose all respiratory capacity upon Cys treatment. This initiates in AS8 a programmed cell death pathway, as evidenced by the accumulation of oligonucleosomal fragments of DNA as the culture dies. Alternatively, wild-type cells remain viable and eventually recover their cyt path. Induction of AOX in response to a chemical inhibition of the cyt path (by antimycin A) is also dependent upon protein dephosphorylation and the generation of reactive oxygen species. Common events required for both down-regulation of the cyt path and induction of AOX may represent a mechanism to coordinate the biogenesis of these two electron transport paths. Such coordinate regulation may be necessary, not only to satisfy metabolic demands, but also to modulate the initiation of a programmed cell death pathway responsive to mitochondrial respiratory status.Mitochondria play a central role in energy and carbon metabolism of eukaryotic cells, being the site of both the tricarboxylic acid cycle and oxidative phosphorylation pathways (Siedow and Day, 2000). Mitochondria have other important functions, such as taking an active role in programmed cell death (PCD) pathways of animals (Green and Reed, 1998) and possibly plants (Jones, 2000; Lam et al., 2001).In plant mitochondria, the electron transport chain (ETC) supporting oxidative phosphorylation branches at ubiquinone (Siedow and Day, 2000; Vanlerberghe and Ordog, 2002). Electrons flow from ubiquinone through the cytochrome (cyt) pathway (including ubiquinol:cyt c oxidoreductase [Complex III], cyt c, and cyt oxidase) or to alternative oxidase (AOX). Electron flow from ubiquinone to AOX is not coupled to the generation of proton motive force. Thus, this pathway bypasses two of the three sites of energy conservation that otherwise support oxidative phosphorylation. Study of transgenic plant cells with altered levels of AOX supports the hypothesis that this protein dampens the mitochondrial generation of reactive oxygen species (ROS), presumably by preventing overreduction of ETC components such as ubiquinone (Maxwell et al., 1999; Parsons et al., 1999; Yip and Vanlerberghe, 2001).M...

Transgenic tobacco (Nicotiana tabacum) lacking mitochondrial alternative oxidase (AOX) have been compared with wild-type (Wt) tobacco using two different systems, either suspension cell cultures or leaves. In both systems, a lack of AOX was accompanied by an increase in some anti-oxidant defenses, consistent with the hypothesis that a lack of AOX increases the mitochondrial generation of reactive oxygen species (ROS). In most cases, this increase in anti-oxidant defenses could more than offset the presumed increased rate of ROS generation, resulting paradoxically in a lower steady-state level of ROS than was found in Wt leaves or suspension cells. We also found that the amount of cell death induced by salicylic acid or nitric oxide correlated strongly with the level of ROS (irrespective of the level of AOX), while death induced by azide was dependent upon the presence or absence of AOX. These results suggest that susceptibility to cell death by signaling molecules (salicylic acid and nitric oxide) is dependent upon the steady-state cellular level of ROS and that AOX levels clearly contribute to this steady state, perhaps by influencing the rate of mitochondrial-generated ROS and hence the cellular level of anti-oxidant defenses.

When wild-type (wt) tobacco (Nicotiana tabacum cv. Petit Havana SR1) cells are grown under macronutrient (P or N) limitation, they induce large amounts of alternative oxidase (AOX), which constitutes a non-energy-conserving branch of the respiratory electron transport chain. To investigate the significance of AOX induction, wt cells were compared with transgenic (AS8) cells lacking AOX. Under nutrient limitation, growth of wt cell cultures was dramatically reduced and carbon use efficiency (g cell dry weight gain g(-1) sugar consumed) decreased by 42-63%. However, the growth of AS8 was only moderately reduced by the nutrient deficiencies and carbon use efficiency values remained the same as under nutrient-sufficient conditions. As a result, the nutrient limitations more severely compromised the tissue nutrient status (P or N) of AS8 than wt cells. Northern analyses and a comparison of the mitochondrial protein profiles of wt and AS8 cells indicated that the lack of AOX in AS8 under P limitation was associated with increased levels of proteins commonly associated with oxidative stress and/or stress injury. Also, the level of electron transport chain components was consistently reduced in AS8 while tricarboxylic acid cycle enzymes did not show a universal trend in abundance in comparison to the wt. Alternatively, the lack of AOX in AS8 cells under N limitation resulted in enhanced carbohydrate accumulation. It is concluded that AOX respiration provides an important general mechanism by which plant cells can modulate their growth in response to nutrient availability and that AOX also has nutrient-specific roles in maintaining cellular redox and carbon balance.

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 (H 2 O 2 , 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 H 2 O 2 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 cantharidininduced 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). An example is the hypersensitive response (HR), a rapid, localized cell death that occurs at sites of invasion by an incompatible pathogen and that acts to restrict the pathogen to the immediate area (Alvarez, 2000).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;Adams and Corey, 2001;Bratton and Cohen, 2001;Kaufmann and Hengartner, 2001). Animal PCD is primarily achieved by the activation of the Asp-specific Cys protease (caspase) cascade (Bratton and Cohen, 2001). Two types of pathways can lead to activation of this cascade. One pathway depends upon the participation of the mitochondrion, whereas the other pathway involves the interaction of a death receptor and ligand. A key event in the mitochondrial pathway (and in some cases the receptor pathway as well) is release of the mitochondrial electron transport chain (ETC) protein cytochrome (cyt) c from the mitochondrion to the cytosol. Cyt c in the cytosol catalyzes the oligomerization of apoptotic protease activating factor-1. This promotes the activation o...