The mitochondrial external NADPH dehydrogenase modulates the leaf NADPH/NADP+ ratio in transgenic Nicotiana sylvestris

Plant mitochondria contain alternative external NAD(P)H dehydrogenases, which oxidise cytosolic NADH or NADPH and reduce ubiquinone without inherent linkage to proton pumping and ATP production. In potato, St-NDB1 is an external Ca2+-dependent NADPH dehydrogenase. The physiological function of this enzyme was investigated in homozygous Nicotiana sylvestris lines overexpressing St-ndb1 and co-suppressing St-ndb1 and an N. sylvestris ndb1. In leaf mitochondria isolated from the overexpressor lines, higher activity of alternative oxidase (AOX) was detected. However, the AOX induction was substantially weaker than in the complex I deficient CMSII mutant, previously shown to contain elevated amounts of NAD(P)H dehydrogenases and AOX. An aox1b and an aox2 gene were up-regulated in CMSII, but only aox1b showed a response, albeit smaller, in the transgenic lines, indicating differences in AOX activation between the genotypes. As in CMSII, the increase of AOX in the overexpressing lines was not due to a general oxidative stress. The lines overexpressing St-ndb1 had consistently lowered leaf NADPH/NADP+ ratios in the light and variably decreased levels in darkness, but unchanged NADH/NAD+ ratios. CMSII instead had similar NADPH/NADP+ and lower NADH/NAD+ ratios than wildtype. These results demonstrate that St-NDB1 is able to modulate the cellular balance of NADPH and NADP+ at least in the day and that reduction of NADP(H) and NAD(H) is independently controlled. Similar growth rates, chloroplast malate dehydrogenase activation and xanthophyll ratios indicate that the change in reduction does not communicate to the chloroplast, and that the cell tolerates significant changes in NADP(H) reduction without deleterious effects.