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l h e claim that succinate and malate can directly stimulate the activity of the alternative oxidase i n plant mitochondria (A.M. Wagner, C.W.M. van den Bergen, H. Wincencjusz [1995] Plant Physiol 108: 1035-1 042) was reinvestigated using sweet potato (Ipomoea batatas 1.) mitochondria. I n whole mitochondria, succinate (in the presence of malonate) and both i-and D-malate stimulated respiration via alternative oxidase in a pH-(and NAD+)-dependent manner. Solubilized malic enzyme catalyzed the oxidation of both i-and D-malate, although the latter at only a low rate and only at acid pH. I n submitochondrial particle preparations with negligible malic enzyme activity, neither i-nor o-malate stimulated alternative oxidase activity. However, even i n the presence of high malonate concentrations, some succinate oxidation was observed via the alternative oxidase, giving the impression of stimulation of the oxidase. Neither i-malate nor succinate (in the presence of malonate) changed the dependence of alternative oxidase activity on ubiquinone reduction state in submitochondrial particles. I n contrast, a large change in this dependence was observed upon addition of pyruvate. Half-maximal stimulation of alternative oxidase by pyruvate occurred at less than 5 p~ in submitochondrial particles, one-twentieth of that reported for whole mitochondria, suggesting that pyruvate acts on the inside of the mitochondrion.We suggest that malate and succinate do not directly stimulate alternative oxidase, and that reports to the contrary reflect intramitochondrial generation of pyruvate via malic enzyme.

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.

Two factors known to regulate plant mitochondrial cyanide-resistant alternative oxidase activity, pyruvate and the redox status of the enzyme's intermolecular disulfide bond, were shown to differently affect activity in isolated soybean seedling mitochond~a. Pyruvate st~ulated alternative oxidase activity at low levels of reduced ubiquinone, shifting the threshold level of ubiquinone reduction for enzyme activity to a lower value. The disulfide bond redox status determined the maximum enzyme activity obtainable in the presence of pyruvate, with the highest rates occurring when the bond was reduced. With variations in cellular pyruvate levels and in the proportion of reduced alternative oxidase protein, a wide range of enzyme activity is possible in vivo.

The alternative oxidase (AOX) of the soybean (Glycine max 1.) inner mitochondrial membrane is encoded by a multigene family (Aox) with three known members. Here, the Aox2 and Aox3 primary translation products, deduced from cDNA analysis, were found to be 38.1 and 36.4 kD, respectively. Direct N-terminal sequencing of partially purified AOX from cotyledons demonstrates that the mature proteins are 31.8 and 31.6 kD, respectively, implying that processing occurs upon import of these proteins into the mitochondrion. Sequence comparisons show that the processing of plant AOX proteins occurs ata characteristic site and that the AOX2 and AOX3 proteins are more similar to one another than to other AOX proteins, including soybean AOXl . Transcript analysis using a polymerase chain reaction-based assay in conjunction with immunoblot experiments indicates that soybean Aox genes are differentially expressed in a tissue-dependent manner. Moreover, the relative abundance of both Aox2 transcripts and protein in cotyledons increase upon greening of dark-grown seedlings. These results comprehensively explain the multiple AOX-banding patterns observed on immunoblots of mitochondrial proteins isolated from various soybean tissues by matching protein bands with gene products.