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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.

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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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Western analyses of respiratory chain proteins. Potato leaf mitochondria were purified from control and antimycin A-sprayed plants. Twenty μg of mitochondrial protein was loaded in each lane. The proteins analysed for are indicated to the right. For NDA, NDB and complex I subunits, blots from one representative experiment out of 3 are shown. For AOX, the experiment with highest induction is depicted. Samples are denoted as for Fig. 1.
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Figure 4: Western analyses of respiratory chain proteins. Potato leaf mitochondria were purified from control and antimycin A-sprayed plants. Twenty μg of mitochondrial protein was loaded in each lane. The proteins analysed for are indicated to the right. For NDA, NDB and complex I subunits, blots from one representative experiment out of 3 are shown. For AOX, the experiment with highest induction is depicted. Samples are denoted as for Fig. 1.

Mentions: The isolated leaf mitochondria were analysed by western blotting to investigate if antimycin A leaf treatment had any effects on the protein abundance of respiratory chain enzymes. The antisera against NDA1 and NDB1 specifically recognised proteins of 48 kDa and 61 kDa, respectively, as previously seen for isolated potato leaf mitochondria [27]. However, the immunosignals for NDA, NDB, as well as the NAD9 and 76 kDa subunits of complex I showed no significant changes between the treatments and the control (Fig. 4). In two experiments, AOX was seen to increase slightly after leaf treatment with 3 and 10 μM antimycin A. The blot with the clearest increase is shown in Fig. 4. In a third experiment, this increase could not be seen. These results suggest that the AOX protein may be slightly increased by treatment of potato leaves with antimycin A.


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)

Western analyses of respiratory chain proteins. Potato leaf mitochondria were purified from control and antimycin A-sprayed plants. Twenty μg of mitochondrial protein was loaded in each lane. The proteins analysed for are indicated to the right. For NDA, NDB and complex I subunits, blots from one representative experiment out of 3 are shown. For AOX, the experiment with highest induction is depicted. Samples are denoted as for Fig. 1.
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Related In: Results  -  Collection

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Figure 4: Western analyses of respiratory chain proteins. Potato leaf mitochondria were purified from control and antimycin A-sprayed plants. Twenty μg of mitochondrial protein was loaded in each lane. The proteins analysed for are indicated to the right. For NDA, NDB and complex I subunits, blots from one representative experiment out of 3 are shown. For AOX, the experiment with highest induction is depicted. Samples are denoted as for Fig. 1.
Mentions: The isolated leaf mitochondria were analysed by western blotting to investigate if antimycin A leaf treatment had any effects on the protein abundance of respiratory chain enzymes. The antisera against NDA1 and NDB1 specifically recognised proteins of 48 kDa and 61 kDa, respectively, as previously seen for isolated potato leaf mitochondria [27]. However, the immunosignals for NDA, NDB, as well as the NAD9 and 76 kDa subunits of complex I showed no significant changes between the treatments and the control (Fig. 4). In two experiments, AOX was seen to increase slightly after leaf treatment with 3 and 10 μM antimycin A. The blot with the clearest increase is shown in Fig. 4. In a third experiment, this increase could not be seen. These results suggest that the AOX protein may be slightly increased by treatment of potato leaves with antimycin A.

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