The plant respiratory chain: Redox responses and catalytic definition of alternative pathways

Sammanfattning: The respiratory chain of plants contains both proton-pumping enzymes that conserve energy and non-proton-pumping alternative enzymes that bypass the sites of energy conservation. In addition to the proton-pumping complex I, plant mitochondria contain alternative type II NAD(P)H dehydrogenases, which allow oxidation of NADH and NADPH from the matrix and the cytosol. The seven gene homologues of type II NAD(P)H dehydrogenases in Arabidopsis thaliana group into three families, including two nda homologues, four ndb homologues and one ndc gene. By producing the seven type II NAD(P)H dehydrogenases in Escherichia coli, we could show that NDB1 and NDB2 bind calcium. A single amino acid substitution identified an EF hand motif as the calcium-binding site of NDB1. Furthermore, NDB1 was characterised as a calcium-dependent NADPH-specific dehydrogenase, while NDB4 and NDB2 were found to be NADH-specific dehydrogenases, with only the latter enzyme being stimulated by calcium. NADH and NADPH are major reducing equivalents in the cell. Supplying A. thaliana seedlings with ammonium or nitrate, which have different reductant demands for assimilation, induced opposite effects on transcription of non-proton-pumping respiratory enzymes. Ammonium led to broad inductions in alternative pathways, also reflected at the enzyme level, whereas nitrate suppressed gene expression in the same families. This points to a role of non-proton-pumping respiratory enzymes in cellular redox balancing. Respiratory inhibitors can elevate the reduction state of the inner membrane, thereby inducing oxidative stress. In potato, inhibition of complex III by antimycin A resulted in a decrease of internal NADH oxidation via the alternative dehydrogenase in isolated mitochondria, which could potentially counteract over-reduction. A method for permeabilisation of membranes by alamethicin was adapted for use on intact plant suspension cells. This allows in situ studies on mitochondrial and cytosolic enzymes in their native environment. Furthermore, the mitochondrial purification method for A. thaliana, the major plant model organism, was improved.