Incubation of Petunia hybrida cells with H202 leadsto an increase in alternative oxidase activity measured after 24 h. This increased activity is accompanied by an increase in alternative oxidase protein. A model is presented for the regulation of alternative oxidase protein synthesis in which active oxygen species and especially H202 play a crucial role as second messengers in the signal transducing pathway from the mitochondria to the nucleus. It is proposed that also the induction of the alternative oxidase by salicylic acid is mediated via H202.

In the past few years, knowledge of the nature and regulation of the alternative oxidase in plant mitochondria has increased greatly. The protein has been characterized and mechanisms that regulate its activity have been described. The consequences of these regulatory mechanisms are that in vivo the cytochrome pathway and the alternative pathway may compete for electrons. The implications for the interpretation of the ‘Bahr and Bonner’ inhibitor titrations, formerly used to estimate the partitioning of electrons over the two pathways, are discussed. It is proposed that activation and engagement of the alternative oxidase may keep Q reduction levels low in order to prevent harmful high levels of free radical production. A model is presented for the regulation of alternative oxidase protein induction, involving a signalling function of active oxygen species.

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Current understanding of the structure and function of the plant alternative oxidase is reviewed. In particular, the role of the oxidase in the protection of tissues against oxidative stress is developed.

The inflorescences of several members of the Arum lily family warm up during flowering and are able to maintain their temperature at a constant level, relatively independent of the ambient temperature. The heat is generated via a mitochondrial respiratory pathway that is distinct from the cytochrome chain and involves a cyanide-resistant alternative oxidase (AOX). In this paper we have used flux control analysis to investigate the influence of temperature on the rate of respiration through both cytochrome and alternative oxidases in mitochondria isolated from the appendices of intact thermogenic Arum maculatum inflorescences. Results are presented which indicate that at low temperatures, the dehydrogenases are almost in full control of respiration but as the temperature increases flux control shifts to the AOX. On the basis of these results a simple model of thermoregulation is presented that is applicable to all species of thermogenic plants. The model takes into account the temperature characteristics of the separate components of the plant mitochondrial respiratory chain and the control of each process. We propose that 1) in all aroid flowers AOX assumes almost complete control over respiration, 2) the temperature profile of AOX explains the reversed relationship between ambient temperature and respiration in thermoregulating Arum flowers, 3) the thermoregulation process is the same in all species and 4) variations in inflorescence temperatures can easily be explained by variations in AOX protein concentrations.

A possible function for the alternative (nonphosphorylating) pathway is to stabilize the reduction state of the ubiquinone pool (Q r /Q t ), thereby avoiding an increase in free radical production. If the Q r /Q t were stabilized by the alternative pathway, then Q r /Q t should be less stable when the alternative pathway is blocked. Q r /Q t increased when we exposed roots of Poa annua (L.) to increasing concentrations of KCN (an inhibitor of the cytochrome pathway). However, when salicylhydroxamic acid, an inhibitor of the alternative pathway, was added at the same time, Q r /Q t increased significantly more. Therefore, we conclude that the alternative pathway stabilizes Q r /Q t . Salicylhydroxamic acid increasingly inhibited respiration with increasing concentrations of KCN. In the experiments described here the alternative oxidase protein was invariably in its reduced (high-activity) state. Therefore, changes in the reduction state of the alternative oxidase cannot account for an increase in activity of the alternative pathway upon titration with KCN. The pyruvate concentration in intact roots increased only after the alternative pathway was blocked or the cytochrome pathway was severely inhibited. The significance of the pyruvate concentration and Q r /Q t on the activity of the alternative pathway in intact roots is discussed.The Cyt pathway and the alternative pathway constitute the respiratory electron-transport pathways of plant mitochondria. In contrast to the Cyt pathway, beyond the branch point (ubiquinone), the alternative pathway does not contribute to the generation of a proton-motive force. The AOX protein is found in every examined plant species and in every plant organ, and the genes encoding AOX have regions that are very conserved , suggesting that the alternative pathway plays a vital role in plant functioning. However, a clearly identified function for the alternative pathway has been documented only once to our knowledge (in thermogenic flowers; Meeuse, 1975). Purvis and Shewfelt (1993) and speculated that the alternative pathway helps to stabilize Q r /Q t . Q r is a common substrate for both respiratory pathways. It has been suggested that high Q r /Q t levels promote free radical formation when the Cyt pathway is inhibited or restricted; respiration via the alternative pathway might then help to maintain Q r /Q t at a low level.Although there is a linear relationship between the rate of mitochondrial respiration and the rate of radical formation (Puntelarulo et al., 1991; Leprince et al., 1994), radical formation is not directly connected to O 2 consumption, because uncouplers increase radical formation only to a minor extent (Chance et al., 1977; Leprince et al., 1994) and may even decrease it (Liu and Huang, 1996). Rather, radical formation is linked to the relative reduction state of the respiratory chain (Forman and Boveris, 1982). The addition of uncoupler enhances respiration but not Q r /Q t . Radical formation increases if the appropriate inhibitors (Purvis et al., 1995) are...

Mitochondria of the protozoa Acanthamoeba castellanii possess a cyanide-insensitive oxidase cross-reacting with monoclonal antibodies raised against the plant alternative oxidase. Immunoblotting revealed three monomeric forms (38, 35, and 32 kDa) and very low amounts of a single 65 kDa dimeric form. Cross-linking studies suggest that while in plants the alternative oxidase occurs as a dimer, in amoeba it functions as a monomer. Immunologically detectable protein levels change with the age of amoeba cell culture. Increased amounts of the 35 kDa protein are accompanied by an increase in the activity of cyanideresistant respiration.