Alternative NAD(P)H dehydrogenases in plant mitochondria - localisation, catalytic functions and physiological roles

Abstract: In addition to complex I, the plant mitochondrial electron transport chain possesses several alternative NAD(P)H dehydrogenases, not present in animals. These enzymes allow nonenergy-conserving electron transfer from cytoplasmic and matrix NAD(P)H to ubiquinone. The mitochondrial inner membrane was permeabilised with a channel-forming antibiotic, alamethicin, and the activity of internal NADH dehydrogenases was studied in the matrix. This technique revealed that changes occur in substrate specificity of complex I upon isolation of submitochondrial particles. Alamethicin permeabilisation was shown to be a reliable method for measurements of internal NADH dehydrogenases and soluble matrix proteins in their native environment of plant mitochondria. Three gene families encoding alternative NAD(P)H dehydrogenases were detected in Arabidopsis genome. There are two At-nda genes, four At-ndb genes and one At-ndc gene. The nda and ndb families are more closely related to fungal homologues, while the ndc family has its origin in cyanobacteria. Representative genes of all three families were all shown to encode proteins targeted to mitochondria. Expression of nda1 but not nda2 was shown to be light-dependent. Based on the sequence similarity between NDA proteins it is possible that they have the same submitochondrial localisation and enzymatic function. Most likely, NDA proteins are internal alternative NADH dehydrogenases. Expression of the St-ndb1 gene in transgenic Nicotiana sylvestris plants imposed specifically increased and decreased external NADPH oxidation in mitochondria isolated from different transgenic lines. A strict correlation to transcript and protein amounts allowed the assignment of St-NDB1 as an external NADPH dehydrogenase. As a consequence the St-ndb1 overexpressing transgenic plants had specifically increased protein levels for alternative oxidase and uncoupling protein, indicating crosstalk in regulation of the protein amount for the enzymes involved in non-energy-conserving pathways.