Greg C. Vanlerberghe

Alternative Oxidase: A Mitochondrial Respiratory Pathway to Maintain Metabolic and Signaling Homeostasis during Abiotic and Biotic Stress in Plants

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 Plant Cells Lacking Mitochondrial Alternative Oxidase Have Increased Susceptibility to Mitochondria-Dependent and -Independent Pathways of Programmed Cell Death

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.

Induction of Mitochondrial Alternative Oxidase in Response to a Cell Signal Pathway Down-Regulating the Cytochrome Pathway Prevents Programmed Cell Death

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 Ca2+ 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.

Alternative Oxidase Activity in Tobacco Leaf Mitochondria (Dependence on Tricarboxylic Acid Cycle-Mediated Redox Regulation and Pyruvate Activation)

Transgenic Nicotiana tabacum (cv Petit Havana SR1) containing high levels of mitochondrial alternative oxidase (AOX) protein due to the introduction of a sense transgene(s) of Aox1, the nuclear gene encoding AOX, were used to 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 [alpha]-keto acids, particularly pyruvate. Reduction of AOX to 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 to the introduction of an Aox1 antisense transgene or multiple sense transgenes were used to investigate the potential physiological significance of the AOX-regulatory 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 to prevent redirection of carbon metabolism, such as to fermentative pathways.

Is the maintenance of homeostatic mitochondrial signaling during stress a physiological role for alternative oxidase

All plants maintain a non-energy-conserving pathway of mitochondrial electron transport referred to as alternative oxidase (AOX) respiration. Here, we briefly review some of the most prevailing themes for the metabolic and physiological roles of this respiratory pathway. Many of these themes relate to the potential of AOX to provide metabolic homeostasis in response to fluctuating cellular conditions, such as is often seen during stress. We then review reverse genetic experiments that have been used to test these hypotheses. To date, such experiments have been limited to just two dicot species and have only targeted one member (a stress-induced member) of the AOX multigene family. Nonetheless, the experiments to date strongly reinforce the idea that AOX respiration is of particular importance during abiotic and biotic stress. Finally, we propose that another core role of AOX may be to modulate the strength of a stress-signaling pathway from the mitochondrion that controls cellular responses to stress. In this way, AOX could be acting to provide a degree of signaling homeostasis from the mitochondrion. This hypothesis may provide explanation for some of the disparate results seen in reverse genetic experiments regarding the impact of AOX on the reactive oxygen network and oxidative damage.

Alternative oxidase modulates leaf mitochondrial concentrations of superoxide and nitric oxide

• 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 (O2(-)), 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 mitochondrial-localized O2(-) 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.

Molecular Localization of a Redox-Modulated Process Regulating Plant Mitochondrial Electron Transport

Using in organellar assays, we found that significant tobacco alternative oxidase (AOX) activity is dependent on both reduction of a putative regulatory disulfide bond and the presence of pyruvate, which may interact with a Cys sulfhydryl. This redox modulation and pyruvate activation thus may be important in determining the partitioning of electrons to AOX in vivo. To investigate these regulatory mechanisms, we generated tobacco plants expressing mutated AOX proteins. Mutation of the most N-terminal Cys residue (Cys-126) to an Ala residue produced an AOX that could not be converted to the disulfide-linked form, thus identifying this Cys residue as being responsible for redox modulation. Although this mutation might be expected to produce an AOX with constitutive high activity in the presence of pyruvate, we found it to have minimal in organellar activity in the presence of pyruvate. Nonetheless, the Cys-126 mutation did not appear to have compromised the catalytic function of AOX, given that cells expressing the protein displayed high rates of cyanide-resistant respiration in vivo. The striking difference between in vivo and in organellar results suggests that an additional mechanism(s), as yet unidentified by in organellar assays, may promote activity in vivo. Mutation of the Cys residue nearest the presumptive active site (Cys-176) to an Ala residue did not prevent disulfide bond formation or affect the ability of AOX to be stimulated by pyruvate, indicating that this Cys residue is involved in neither redox modulation nor pyruvate activation.

Mitochondrial alternative oxidase is not a critical component of plant viral resistance but may play a role in the hypersensitive response.

Transgenic tobacco (Nicotiana tabacum) with altered levels of mitochondrial alternative oxidase (AOX) were used to examine the potential role of this electron transport chain protein in resistance to tobacco mosaic virus. We examined the effect of AOX expression on the salicylic acid-induced resistance in susceptible plants and the resistance responses of plants harboring the N-gene. A lack of AOX did not compromise the ability of salicylic acid treatment to heighten the resistance of susceptible plants. In plants with the N-gene, a lack of AOX did not compromise the ability of the hypersensitive response to restrict the virus or the ability of the plant to develop systemic acquired resistance. Overexpression of AOX did not heighten the resistance of susceptible plants, but did result in smaller hypersensitive response lesions, suggesting a link between mitochondrial function and this programmed cell death event. We conclude that AOX is not a critical component of the previously characterized salicylhydroxamic acid-sensitive pathway important in viral resistance.

Branched Mitochondrial Electron Transport in the Animalia: Presence of Alternative Oxidase in Several Animal Phyla

The mitochondrion of most eukaryotes has multiple electron transport components that increase the points of entry and/or exit of electrons, thus giving a branched nature to the respiratory chain. In plants and many other organisms, a prominent example is alternative oxidase, a non‐energy conserving branch in the respiratory chain and an additional terminal oxidase for the exit of electrons. Our genome database searches have now revealed the presence of alternative oxidase in four animal species from three different phyla (Mollusca, Nematoda and Chordata), consistent with frequent reports of cyanide‐resistant respiration in the Animalia. In Ciona intestinalis and Crassostrea gigas, alternative oxidase is expressed in several different tissues. Phylogenetic analysis is consistent with the animal proteins having originated by vertical inheritance. We hypothesize that alternative oxidase is likely widespread in the Animalia and discuss some of the potential role(s) for such a branched respiratory chain. IUBMB Life, 56: 333‐341, 2004

Origins, evolutionary history, and taxonomic distribution of alternative oxidase and plastoquinol terminal oxidase

Alternative oxidase (AOX) and plastoquinol terminal oxidase (PTOX) are related quinol oxidases associated with respiratory and photosynthetic electron transport chains, respectively. Contrary to previous belief, AOX is present in numerous animal phyla, as well as heterotrophic and marine phototrophic proteobacteria. PTOX appears limited to organisms capable of oxygenic photosynthesis, including cyanobacteria, algae and plants. We propose that both oxidases originated in prokaryotes from a common ancestral di-iron carboxylate protein that diversified to AOX within ancient proteobacteria and PTOX within ancient cyanobacteria. Each then entered the eukaryotic lineage separately; AOX by the endosymbiotic event that gave rise to mitochondria and later PTOX by the endosymbiotic event that gave rise to chloroplasts. Both oxidases then spread through the eukaryotic domain by vertical inheritance, as well as by secondary and potentially tertiary endosymbiotic events.