Molecular Genetic Evidence of the Ability of Alternative Oxidase to Support Respiratory Carbon Metabolism.

With the cytochrome pathway inhibited, AOX was able to support considerable growth of cultured tobacco (Nicotiana tabacum cv Petit Havana SR1) cells but the efficiency of carbon utilization decreased dramatically. Antisense cells with decreased AOX protein did not grow, whereas sense cells with elevated AOX protein had higher growth and respiration rates than the wild type. In antisense cells a large accumulation of pyruvate resulted in aerobic ethanolic fermentation.

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.

Molecular Genetic Alteration of Plant Respiration (Silencing and Overexpression of Alternative Oxidase in Transgenic Tobacco).

The alternative oxidase (AOX) of plant mitochondria is encoded by the nuclear gene Aox1. Sense and antisense DNA constructs of Nicotiana tabacum Aox1 were introduced into tobacco, and transgenic plants with both increased and decreased levels of mitochondrial AOX protein were identified. Suspension cells derived from wild-type and transgenic plants were grown in heterotrophic batch culture. Transgenic cells with increased AOX protein had an increased capacity for cyanide-resistant, salicylhydroxamic acid-sensitive respiration compared to wild-type cells, whereas transgenic cells with decreased AOX protein had a decreased capacity for such respiration. Thus, genetic alteration of the level of AOX protein was sufficient to alter the capacity for electron transport through the alternative pathway. Under our standard growth conditions, "antisense" cells with dramatically reduced levels of AOX protein had growth and respiration rates similar to the wild type. However, whereas wild-type cells were able to grow under conditions that severely suppressed cytochrome pathway activity, antisense cells could not survive this treatment. This suggests that a critical function of AOX may be to support respiration when the cytochrome pathway is impaired. The much higher level of AOX protein in "sense" cells compared to the wild type did not appreciably alter the steady-state partitioning of electrons between the cytochrome path and the alternative pathway in vivo, suggesting that this partitioning may be subject to additional regulatory factors.

[Pulmonary carbon monoxide exchange constants in the "stable state" at rest in healthy non-smokers]. / Constantes d'échange pulmonaire du monoxyde de carbone en "état stable" de repos chez le sujet sain non fumeur.

During simultaneous evaluation of pulmonary O2, CO2, and CO in the stable state (ES) at rest, CO volume per minute (V'CO) standardized (V'COs) for FICO = 0.001 [2, 3] can be expressed relative to simultaneous flow of "respiratory" gases (V'O2 and V'CO2) by the quotient of the gas concentrations concerned, if its is admitted that V'I = V'E. Whether at the "expired"mean E or "alveolar" A levels, these concentrations have identical paired relationships. In the strict stable state, the proposed expressions of V'CO are constants independent of age, sex, stature, and ventilatory regimen in healthy non-smokers [6]; the result is that V'COs in relation to alveolar ventilation, V'COs/V'A, which is related to V'COs/V'CO2 by PaCO2, is also a constant. When the so-called "stable" state is not a strict one, experimental arguments suggest that V'COs/V'CO2 alone is not markedly influenced by ventilatory instability. Measurements conducted by means of different biological modalities and techniques in 3 groups of healthy women of homogeneous age breathing in a semi-open circuit: 1) established the value of the constants V'COs/V'CO2, V'COs/V'O2, V'COs/V'A in the true stable state (ESV) controlled by R and by gasometry on simultaneously collected blood samples; and 2) confirmed the constant character of V'COs/V'CO2 whatever the degree of respiratory stabilization, even when the ventilatory regimen was in reality not stabilised (ENS); V'COs/V'CO2 should therefore be the only value for defining a "specific capacity of pulmonary CO exchange". V'COs/V'A appears to be related to Q'/V'A. As DuCO/(DuCO2.1,21), which measures this ratio, it is independent of age and of ventilatory frequency. Several facts indicate that the "parallelism" of modifications affecting DuCO and DuCO2 [15] is not found in all physiological and pathological circumstances.