Joseph T. Wiskich

Organic acid activation of the alterNatlve oxidase of plant mitochondria

Alternative oxidase activity (oxygen uptake in the presence of KCN, antimycin or myxothiazol) in mitochondria isolated from the roots of soybean seedlings was very slow, even with succinate as substrate. This activity was stimulated substantially (100–400%) by the addition of pyruvate, with half maximal stimulation occurring at 0.1 mM pyruvate. Mitochondria from soybean shoots displayed high alternative oxidase activity with succinate and malate as substrates but lower activity with exogenous NADH; addition of pyruvate stimulated the activity with NADH up to that seen with succinate. This stimulation of cyanide‐insensitive NADH oxidation was seen also with mitochondria from other species. Hydroxypyruvate and oxoglutarate could substitute for pyruvate, although higher concentrations were required to achieve maximum stimulation. Pyruvate stimulation of cyanide‐insensitive oxygen uptake was observed with exogenous quinols as substrates, with sub‐mitochondrial particles, and in the presence of the pyruvate transport inhibitor, cyanohydroxycinnamic acid, but was not observed with detergent‐solubilised mitochondria. It is suggested that pyruvate acts allosterically on the alternatlve oxidase to stimulate its activity. The implications of these findings for respiration in vivo are discussed.

Interdependence between chloroplasts and mitochondria in the light and the dark

2. Interactions between organelles depends on metabolite exchange . . . . . . . . . . . . . . . . . . . . 236 2.1. ATP exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236 2.2. Transport of reducing equivalents across membranes . . . . . . . . . . . . . . . . . . . . . . . . 237 2.3. Exchange of carbon compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

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.

Regulation of alternative oxidase kinetics by pyruvate and intermolecular disulfide bond redox status in soybean seedling mitochondria

Two factors known to regulate plant mitochondrial cyanide‐resistant alternative oxidase activity, pyruvate and the redox status of the enzymes intermolecular disulfide bond, were shown to differently affect activity in isolated soybean seedling mitochondria. Pyruvate stimulated 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.

Differential expression of the multigene family encoding the soybean mitochondrial alternative oxidase.

The alternative oxidase (AOX) of the soybean (Glycine max L.) 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 at a characteristic site and that the AOX2 and AOX3 proteins are more similar to one another than to other AOX proteins, including soybean AOX1. 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.

Regulation of alternative pathway activity in plant mitochondria: Nonlinear relationship between electron flux and the redox poise of the quinone pool

The dependence of respiratory flux via the alternative pathway on the redox poise of the ubiquinone (Q) pool was investigated in soybean cotyledon mitochondria. A marked nonlinear relationship was observed between Q-pool reduction level and O2 uptake via the alternative oxidase. Significant engagement of the alternative pathway was not apparent until Q-pool reduction level reached 35-40% but increased disproportionately on further reduction. Similar results were obtained with electron donation from either Complex 1 or Complex 2. Close agreement was obtained over a range of experimental conditions between the estimated contribution of the alternative pathway to total respiratory flux, as measured with salicylhydroxamic acid, and that predicted from the redox poise of the Q-pool. These results are discussed in terms of existing models of the regulation of respiratory flux via the alternative pathway.

Regulation of alternative oxidase activity in higher plants

Plant mitochondria contain two terminal oxidases: cytochrome oxidase and the cyanideinsensitive alternative oxidase. Electron partioning between the two pathways is regulated by the redox poise of the ubiquinone pool and the activation state of the alternative oxidase. The alternative oxidase appears to exist as a dimer which is active in the reduced, noncovalently linked form and inactive when in the oxidized, covalently linked form. Reduction of the oxidase in isolated tobacco mitochondria occurs upon oxidation of isocitrate or malate and may be mediated by matrix NAD(P)H. The activity of the reduced oxidase is governed by certain other organic acids, notably pyruvate, which appear to interact directly with the enzyme. Pyruvate alters the interaction between the alternative oxidase and ubiquinol so that the oxidase becomes active at much lower levels of ubiquinol and competes with the cytochrome pathway for electrons. These requirements for activation of the alternative oxidase constitute a sophisticated feed-forward control mechanism which determines the extent to which electrons are directed away from the energy-conserving cytochrome pathway to the non-energy conserving alternative oxidase. Such a mechanism fits well with the proposed role of the alternative oxidase as a protective enzyme which prevents over-reduction of the cytochrome chain and fermentation of accumulated pyruvate.

Specificity of the Organic Acid Activation of Alternative Oxidase in Plant Mitochondria.

The claim that succinate and malate can directly stimulate the activity of the alternative oxidase in plant mitochondria (A.M. Wagner, C.W.M. van den Bergen, H. Wincencjusz [1995] Plant Physiol 108: 1035–1042) was reinvestigated using sweet potato (Ipomoea batatas L.) mitochondria. In whole mitochondria, succinate (in the presence of malonate) and both L- and D-malate stimulated respiration via alternative oxidase in a pH- (and NAD+)-dependent manner. Solubilized malic enzyme catalyzed the oxidation of both L- and D-malate, although the latter at only a low rate and only at acid pH. In submitochondrial particle preparations with negligible malic enzyme activity, neither L- nor D-malate stimulated alternative oxidase activity. However, even in the presence of high malonate concentrations, some succinate oxidation was observed via the alternative oxidase, giving the impression of stimulation of the oxidase. Neither L-malate nor succinate (in the presence of malonate) changed the dependence of alternative oxidase activity on ubiquinone reduction state in submitochondrial particles. In 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 [mu]M 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 intra-mitochondrial generation of pyruvate via malic enzyme.

Regulation of alternative oxidase activity by pyruvate in soybean mitochondria

The regulation of alternative oxidase activity by the effector pyruvate was investigated in soybean (Glycine max L.) mitochondria using developmental changes in roots and cotyledons to vary the respiratory capacity of the mitochondria. Rates of cyanide-insensitive oxygen uptake by soybean root mitochondria declined with seedling age. Immunologically detectable protein levels increased slightly with age, and mitochondria from younger, more active roots had less of the protein in the reduced form. Addition of pyruvate stimulated cyanide-insensitive respiration in root mitochondria, up to the same rate, regardless of seedling age. This stimulation was reversed rapidly upon removal of pyruvate, either by pelleting mitochondria (with succinate as substrate) or by adding lactate dehydrogenase with NADH as substrate. In mitochondria from cotyledons of the same seedlings, cyanide-insensitive NADH oxidation was less dependent on added pyruvate, partly due to intramitochondrial generation of pyruvate from endogenous substrates. Cyanide-insensitive oxygen uptake with succinate as substrate was greater than that with NADH, in both root and cotyledon mitochondria, but this difference became much less when an increase in external pH was used to inhibit intramitochondrial pyruvate production via malic enzyme. Malic enzyme activity in root mitochondria declined with seedling age. The results indicate that the activity of the alternative oxidase in soybean mitochondria is very dependent on the presence of pyruvate: differences in the generation of intramitochondrial pyruvate can explain differences in alternative oxidase activity between tissues and substrates, and some of the changes that occur during seedling development.

Measurement of the redox state of the ubiquinone pool in plant mitochondria

We have investigated the dependence of the respiratory rate on the redox poise of the quinone pool in isolated turnip and pea leaf mitochondria. A linear relationship has been found between these two parameters during succinate oxidation under both state 3 and 4 conditions. When succinate is oxidised by the alternative oxidase the dependence of oxygen uptake on the steady‐state reduction level of quinone is markedly non‐linear. These results are discussed within the frame‐work of a homogeneous quinone pool.