Identification of a mitochondrial external NADPH dehydrogenase by overexpression in transgenic Nicotiana sylvestris.
The plant respiratory chain contains a complex setup of non-energy conserving NAD(P)H dehydrogenases, the physiological consequences of which are highly unclear. An expression construct for the potato (Solanum tuberosum L., cv. Desiree) ndb1 gene, a homologue of bacterial and fungal type II NAD(P)H dehydrogenases, was introduced into Nicotiana sylvestris. Transgenic lines with high transcript and protein levels for St-NDB1 had up to threefold increased activity of external NADPH dehydrogenase in isolated mitochondria as compared to the wild type (WT). In two lines, the external NADPH dehydrogenase activity was instead 10-fold decreased, indicating that the corresponding N. sylvestris gene had been suppressed. Activities of external and internal rotenone-insensitive NADH dehydrogenases were unchanged in the transgenic lines. The results demonstrate that the St-ndb1 encodes an external dehydrogenase specific for NADPH and dependent on calcium for activity. Transgenic lines overexpressing St-ndb1 had specifically increased protein levels for alternative oxidase and uncoupling protein, as compared to the WT and one co-suppressing line. This indicates cross-talk in the expressional control, or metabolic conditions influencing it, for the different categories of energy-dissipating proteins that bypass oxidative phosphorylation. The potential effects of external NADPH oxidation on other cellular processes are discussed.
Michalecka
Agnieszka M
AM
Department of Cell and Organism Biology, Biology Building, Lund University, Sölvegatan 35B, SE-223 62 Lund, Sweden.
Agius
Stephanie C
SC
Møller
Ian M
IM
Rasmusson
Allan G
AG
D000595:Amino Acid Sequence; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009252:NADPH Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics
D012333:RNA, Messenger; D009252:NADPH Dehydrogenase