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Antimycin A treatment decreases respiratory internal rotenone-insensitive NADH oxidation capacity in potato leaves.

Geisler DA, Johansson FI, Svensson AS, Rasmusson AG - BMC Plant Biol. (2004)

Bottom Line: The internal rotenone-insensitive NADH oxidation decreases after antimycin A treatment of potato leaves.However, the decrease is not due to changes in expression of known nda genes.One consequence of the lower NADH dehydrogenase capacity may be a stabilisation of the respiratory chain reduction level, should the overall capacity of the cytochrome and the alternative pathway be restricted.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept of Cell and Organism Biology, Lund University, Sölvegatan 35B, Lund, (SE-223 62), Sweden. daniela.geisler@cob.lu.se

ABSTRACT

Background: The plant respiratory chain contains several energy-dissipating enzymes, these being type II NAD(P)H dehydrogenases and the alternative oxidase, not present in mammals. The physiological functions of type II NAD(P)H dehydrogenases are largely unclear and little is known about their responses to stress. In this investigation, potato plants (Solanum tuberosum L., cv. Desiree) were sprayed with antimycin A, an inhibitor of the cytochrome pathway. Enzyme capacities of NAD(P)H dehydrogenases (EC 1.6.5.3) and the alternative oxidase were then analysed in isolated leaf mitochondria.

Results: We report a specific decrease in internal rotenone-insensitive NADH dehydrogenase capacity in mitochondria from antimycin A-treated leaves. External NADPH dehydrogenase and alternative oxidase capacities remained unaffected by the treatment. Western blotting revealed no change in protein abundance for two characterised NAD(P)H dehydrogenase homologues, NDA1 and NDB1, nor for two subunits of complex I. The alternative oxidase was at most only slightly increased. Transcript levels of nda1, as well as an expressed sequence tag derived from a previously uninvestigated closely related potato homologue, remained unchanged by the treatment. As compared to the daily rhythm-regulated nda1, the novel homologue displayed steady transcript levels over the time investigated.

Conclusions: The internal rotenone-insensitive NADH oxidation decreases after antimycin A treatment of potato leaves. However, the decrease is not due to changes in expression of known nda genes. One consequence of the lower NADH dehydrogenase capacity may be a stabilisation of the respiratory chain reduction level, should the overall capacity of the cytochrome and the alternative pathway be restricted.

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Alternative pathway activity. Antimycin A-insensitive oxidation of NADPH, NADH and succinate was measured to oxygen in the presence of DTT and pyruvate to assure maximum rates. Respiratory chain activities are displayed as percentage of control activity in each experiment. Control activities were: 12, 52 and 59 nmol NADPH min-1 mg-1; 25, 40 and 57 nmol NADH min-1 mg-1; or 60 and 52 nmol succinate min-1 mg-1 in the independent experiments. Rates of succinate oxidation were corrected for a small rate remaining after addition of antimycin A and salicylhydroxamic acid; 3–6 and 7–10 nmol O2 min-1 mg-1 for control/A3, and A10/A30, respectively. Samples and error bars are denoted as for Fig. 1, where the corresponding rates for total respiration (cytochrome path plus alternative path) are presented.
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Figure 3: Alternative pathway activity. Antimycin A-insensitive oxidation of NADPH, NADH and succinate was measured to oxygen in the presence of DTT and pyruvate to assure maximum rates. Respiratory chain activities are displayed as percentage of control activity in each experiment. Control activities were: 12, 52 and 59 nmol NADPH min-1 mg-1; 25, 40 and 57 nmol NADH min-1 mg-1; or 60 and 52 nmol succinate min-1 mg-1 in the independent experiments. Rates of succinate oxidation were corrected for a small rate remaining after addition of antimycin A and salicylhydroxamic acid; 3–6 and 7–10 nmol O2 min-1 mg-1 for control/A3, and A10/A30, respectively. Samples and error bars are denoted as for Fig. 1, where the corresponding rates for total respiration (cytochrome path plus alternative path) are presented.

Mentions: To determine the capacity of the AOX, the cytochrome pathway activity was completely inhibited by adding antimycin A to the reaction medium. Fig. 3 summarises the rates after antimycin A addition with succinate and external NAD(P)H as substrates. No difference in AOX capacity is seen between mitochondria from antimycin A-treated leaves and control leaves. With the 30 μM antimycin A treatment, the rates were completely insensitive to in vitro addition of antimycin A (data not shown), indicating a high degree of in vivo bc1 complex inhibition at this concentration.


Antimycin A treatment decreases respiratory internal rotenone-insensitive NADH oxidation capacity in potato leaves.

Geisler DA, Johansson FI, Svensson AS, Rasmusson AG - BMC Plant Biol. (2004)

Alternative pathway activity. Antimycin A-insensitive oxidation of NADPH, NADH and succinate was measured to oxygen in the presence of DTT and pyruvate to assure maximum rates. Respiratory chain activities are displayed as percentage of control activity in each experiment. Control activities were: 12, 52 and 59 nmol NADPH min-1 mg-1; 25, 40 and 57 nmol NADH min-1 mg-1; or 60 and 52 nmol succinate min-1 mg-1 in the independent experiments. Rates of succinate oxidation were corrected for a small rate remaining after addition of antimycin A and salicylhydroxamic acid; 3–6 and 7–10 nmol O2 min-1 mg-1 for control/A3, and A10/A30, respectively. Samples and error bars are denoted as for Fig. 1, where the corresponding rates for total respiration (cytochrome path plus alternative path) are presented.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC424582&req=5

Figure 3: Alternative pathway activity. Antimycin A-insensitive oxidation of NADPH, NADH and succinate was measured to oxygen in the presence of DTT and pyruvate to assure maximum rates. Respiratory chain activities are displayed as percentage of control activity in each experiment. Control activities were: 12, 52 and 59 nmol NADPH min-1 mg-1; 25, 40 and 57 nmol NADH min-1 mg-1; or 60 and 52 nmol succinate min-1 mg-1 in the independent experiments. Rates of succinate oxidation were corrected for a small rate remaining after addition of antimycin A and salicylhydroxamic acid; 3–6 and 7–10 nmol O2 min-1 mg-1 for control/A3, and A10/A30, respectively. Samples and error bars are denoted as for Fig. 1, where the corresponding rates for total respiration (cytochrome path plus alternative path) are presented.
Mentions: To determine the capacity of the AOX, the cytochrome pathway activity was completely inhibited by adding antimycin A to the reaction medium. Fig. 3 summarises the rates after antimycin A addition with succinate and external NAD(P)H as substrates. No difference in AOX capacity is seen between mitochondria from antimycin A-treated leaves and control leaves. With the 30 μM antimycin A treatment, the rates were completely insensitive to in vitro addition of antimycin A (data not shown), indicating a high degree of in vivo bc1 complex inhibition at this concentration.

Bottom Line: The internal rotenone-insensitive NADH oxidation decreases after antimycin A treatment of potato leaves.However, the decrease is not due to changes in expression of known nda genes.One consequence of the lower NADH dehydrogenase capacity may be a stabilisation of the respiratory chain reduction level, should the overall capacity of the cytochrome and the alternative pathway be restricted.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept of Cell and Organism Biology, Lund University, Sölvegatan 35B, Lund, (SE-223 62), Sweden. daniela.geisler@cob.lu.se

ABSTRACT

Background: The plant respiratory chain contains several energy-dissipating enzymes, these being type II NAD(P)H dehydrogenases and the alternative oxidase, not present in mammals. The physiological functions of type II NAD(P)H dehydrogenases are largely unclear and little is known about their responses to stress. In this investigation, potato plants (Solanum tuberosum L., cv. Desiree) were sprayed with antimycin A, an inhibitor of the cytochrome pathway. Enzyme capacities of NAD(P)H dehydrogenases (EC 1.6.5.3) and the alternative oxidase were then analysed in isolated leaf mitochondria.

Results: We report a specific decrease in internal rotenone-insensitive NADH dehydrogenase capacity in mitochondria from antimycin A-treated leaves. External NADPH dehydrogenase and alternative oxidase capacities remained unaffected by the treatment. Western blotting revealed no change in protein abundance for two characterised NAD(P)H dehydrogenase homologues, NDA1 and NDB1, nor for two subunits of complex I. The alternative oxidase was at most only slightly increased. Transcript levels of nda1, as well as an expressed sequence tag derived from a previously uninvestigated closely related potato homologue, remained unchanged by the treatment. As compared to the daily rhythm-regulated nda1, the novel homologue displayed steady transcript levels over the time investigated.

Conclusions: The internal rotenone-insensitive NADH oxidation decreases after antimycin A treatment of potato leaves. However, the decrease is not due to changes in expression of known nda genes. One consequence of the lower NADH dehydrogenase capacity may be a stabilisation of the respiratory chain reduction level, should the overall capacity of the cytochrome and the alternative pathway be restricted.

Show MeSH