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Published on October 8, 2008; 10.1104/pp.108.126219

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Received July 11, 2008
Accepted October 1, 2008

Inhibition of 2-oxoglutarate dehydrogenase in potato tuber suggests the enzyme is limiting for respiration and confirms its importance in nitrogen assimilation

Wagner L. Araujo , Adriano Nunes-Nesi , Sandra Trenkamp , Victoria I. Bunik , and Alisdair R. Fernie *

Max-Planck-Institut fur Molekulare Pflanzenphysiologie, Am Muhlenberg 1, 14476 Potsdam-Golm, Germany; A.N. Belozersly Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia

* Corresponding author; email: fernie{at}mpimp-golm.mpg.de.

The 2-oxoglutarate dehydrogenase complex constitutes a mitochondrially localized tricarboxylic acid cycle multienzyme system responsible for the conversion of 2-oxoglutarate to succinyl CoA concomitant with NAD+ reduction. Although regulatory mechanisms of plant enzyme complexes have been characterized in vitro, little is known concerning their role in plant metabolism in situ. This issue has recently been addressed at the cellular level in non-plant systems via the use of specific phosphonate inhibitors of the enzyme. Here we describe the application of these inhibitors for the functional analysis of the potato tuber 2-oxoglutarate dehydrogenase complex. In vitro experiments revealed that succinyl phosphonate (SP) and a carboxy ethyl ester of SP (CESP) are slow-binding inhibitors of the 2-oxoglutarate dehydrogenase complex, displaying greater inhibitory effects than a diethyl ester of SP (DESP), a phosphono ethyl ester of SP (PESP) or a triethyl ester of SP (TESP). Incubation of potato tuber slices with the inhibitors revealed that they were adequately taken up by the tissue and produced the anticipated effects on the in situ enzyme activity. In order to assess the metabolic consequences of the 2-oxoglutarate dehydrogenase complex inhibition, we evaluated the levels of a broad range of primary metabolites using an established gas chromatography-mass spectrometry method. We additionally analysed the rate of respiration in both tuber discs and isolated mitochondria. Finally, we evaluated the metabolic fate of radiolabeled acetate, 2-oxoglutarate or glucose and 13C labeled pyruvate and glutamate following incubation of tuber discs in the presence or absence of either SP or CESP. The data obtained are discussed in the context of the roles of the 2-oxoglutarate dehydrogenase complex in respiration and carbon nitrogen interactions.






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