Plants, some fungi, and protists contain a cyanide-resistant, alternative mitochondrial respiratory pathway. This pathway branches at the ubiquinone pool and consists of an alternative oxidase encoded by the nuclear gene Aox1. Alternative pathway respiration is only linked to proton translocation at Complex 1 (NADH dehydrogenase). Alternative oxidase expression is influenced by stress stimuli-cold, oxidative stress, pathogen attack-and by factors constricting electron flow through the cytochrome pathway of respiration. Control is exerted at the levels of gene expression and in response to the availability of carbon and reducing potential. Posttranslational control involves reversible covalent modification of the alternative oxidase and activation by specific carbon metabolites. This dynamic system of coarse and fine control may function to balance upstream respiratory carbon metabolism and downstream electron transport when these coupled processes become imbalanced as a result of changes in the supply of, or demand for, carbon, reducing power, and ATP.

Alternative oxidase (AOX) is a non-energy conserving terminal oxidase in the plant mitochondrial electron transport chain. While respiratory carbon oxidation pathways, electron transport, and ATP turnover are tightly coupled processes, AOX provides a means to relax this coupling, thus providing a degree of metabolic homeostasis to carbon and energy metabolism. Beside their role in primary metabolism, plant mitochondria also act as “signaling organelles”, able to influence processes such as nuclear gene expression. AOX activity can control the level of potential mitochondrial signaling molecules such as superoxide, nitric oxide and important redox couples. In this way, AOX also provides a degree of signaling homeostasis to the organelle. Evidence suggests that AOX function in metabolic and signaling homeostasis is particularly important during stress. These include abiotic stresses such as low temperature, drought, and nutrient deficiency, as well as biotic stresses such as bacterial infection. This review provides an introduction to the genetic and biochemical control of AOX respiration, as well as providing generalized examples of how AOX activity can provide metabolic and signaling homeostasis. This review also examines abiotic and biotic stresses in which AOX respiration has been critically evaluated, and considers the overall role of AOX in growth and stress tolerance.

Transgenic Nicofiana tabacum (cv Petit Havana SR1) containing high levels of mitochondrial alternative oxidase (AOX) protein due t o the introduction of a sense transgene(s) of Aoxl, the nuclear gene encoding AOX, were used t o investigate mechanisms regulating AOX activity. After purification of leaf mitochondria, a large proportion of the AOX protein was present as the oxidized (covalently associated and less active) dimer. High AOX activity in these mitochondria was dependent on both reduction of the protein by DTT (to the noncovalently associated and more active dimer) and its subsequent activation by certain a-keto acids, particularly pyruvate. Reduction of AOX t o its more active form could also be mediated by intramitochondrial reducing power generated by the oxidation of certain tricarboxylic acid cycle substrates, most notably isocitrate and malate. Our evidence suggests that NADPH may be specifically required for AOX reduction. All of the above regulatory mechanisms applied to AOX in wild-type mitochondria as well. Transgenic leaves lacking AOX due t o the introduction of an Aoxl antisense transgene or multiple sense transgenes were used to investigate the potential physiological significance of the AOXregulatory mechanisms. Under conditions in which respiratory carbon metabolism is restricted by the capacity of mitochondrial electron transport, feed-forward activation of AOX by mitochondrial reducing power and pyruvate may act t o prevent redirection of carbon metabolism, such as to fermentative pathways.

Suspension cells of tobacco (Nicotiana tabacum 1. cv Bright Yellow) were used to investigate signals regulating the expression of the nuclear gene Aoxl encoding the mitochondrial alternative oxidase (AOX) protein responsible for cyanide-resistant respiration in plants. We found that an increase i n the tricarboxylic acid cycle intermediate citrate (either after its exogenous supply to cells or after inhibition of aconitase by monofluoroacetate) caused a rapid and dramatic increase i n the steady-state level of

Summary• The nonenergy-conserving alternative oxidase (AOX) has been hypothesized to modulate the amount of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in plant mitochondria but there is sparse direct in planta evidence to support this.• Laser scanning fluorescent confocal microscopy and biochemical methods were used to directly estimate in planta leaf concentrations of superoxide (O À 2 ), nitric oxide (NO), peroxynitrite (ONOO ) ) and hydrogen peroxide (H 2 O 2 ) in wildtype (Wt) tobacco (Nicotiana tabacum) and transgenic tobacco with altered amounts of AOX.• We found that plants lacking AOX have increased concentrations of leaf mitochondriallocalized O À 2 and leaf NO in comparison to the Wt, while leaf concentrations of H 2 O 2 were similar or lower in the AOX-suppressed plants.• Based on our results, we suggest that AOX respiration acts to reduce the generation of ROS and RNS in plant mitochondria by dampening the leak of single electrons from the electron transport chain to O 2 or nitrite. This may represent a universal role for AOX in plants. More work is now needed to establish the functional implications of this role, such as during abiotic and biotic stress.

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...

Suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow) have been used to study the effect of growth temperature on the CN-resistant, salicylhydroxamic acid-sensitive alternative pathway of respiration. Mitochondria isolated from cells maintained at 30°C had a low capacity to oxidize succinate via the alternative pathway, whereas mitochondria isolated from cells 24 tago (28), and other species. Although it appears there is an increased capacity (or potential) for AP respiration in these tissues, the degree of engagement of this respiratory pathway at lower temperatures is not clear because of the inherent problems in using KCN and SHAM to measure engagement (22). Therefore, a conclusive answer concerning the degree of engagement of the pathway awaits measurements based upon oxygen-isotope discrimination (9, 31).The role of AP respiration in growth at lower temperatures is not known. Growth at lower temperature can result in a substantial redirection of respiratory metabolism, possibly due to the differential effect of temperature on enzymes of metabolism (1,8). Possibly, this altered metabolism places demands upon mET that bring about increased AP respiration. It has been shown that transfer of plants to lower temperatures can result in the accumulation of soluble sugars (8,25,27). In several species, high sugar levels correlate with increased AP respiration (2, 14), which is consistent with the hypothesis that AP respiration acts to remove excess carbohydrate (the 'energy overflow' hypothesis, ref. 14). Changes in AP capacity at different temperatures might also be related to the sensitivity of plant mET pathways to temperature extremes. It has been suggested that the CP is more coldlabile than the AP (16,21), and also that the AP is more heat-labile (3, 18).It is not known what components of the mET chain are responsible for increased AP capacity at low temperature. In maize, plants grown at lower temperatures had more AO protein in some tissues (30), suggesting that increased AP capacity may have been brought about by an increase in the amount of the terminal oxidase of the pathway. We have now investigated this further in tobacco (Nicotiana tabacum L.), a species in which the effect of temperature on AP respiration has not yet been thoroughly investigated. In this paper, we show that when suspension cells of tobacco are transferred to a lower temperature there is a rapid increase in the capacity for AP respiration in whole cells and in isolated mitochondria due, at least in part, to de novo synthesis of the AO. MATERIALS

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.