Gene description, activity quantification and physiological responses of mitochondrial alternative NAD(P)H dehydrogenases

Sammanfattning: In addition to proton-pumping complex I, the plant respiratory chain contains type II NAD(P)H dehydrogenases. These extra enzymes do not pump protons and consequently do not contribute to the electrochemical proton gradient. Database screening of the Arabidopsis genome identified seven type II NAD(P)H dehydrogenase homologues of which two and four genes were closely related to potato (Solanum tuberosum) genes nda1 and ndb1, respectively. A novel homologue, ndc1 was also identified, and concluded to be of cyanobacterial origin. The nda1, but not the nda2, was shown to be dependent on light and circadian regulation. In addition nda1 was not expressed in roots of which nda2 was. Taken together, this indicates a role for nda1 and nda2 in photosynthetic and heterotrophic metabolism, respectively. Gene products of all three families were found to be targeted to the mitochondria. The potato type II dehydrogenases, NDA1 and NDB1, have been shown to have an internal and external location, respectively. The functional role of these enzymes is still to a large extent unknown. Cold treatment caused a decrease of NDA1 transcript, consistent with effects at the protein and activity levels. Treatment with antimycin A caused a decrease in internal rotenone-insensitive capacity. The role of NDA1 in response to these stress treatments is discussed in the context of photorespiration (cold stress) and oxidative stress (cold stress and antimycin A). The channel-forming peptide alamethicin was investigated as a potentially less invasive method to permeabilise plant mitochondria and study the internal dehydrogenases of the plant ETC. Alamethicin was shown to permeabilise the inner mitochondrial membrane to NAD(P)H allowing quantification of matrix enzymes as well as the internal NAD(P)H dehydrogenases. Complex I-mediated NADH oxidation in alamethicin-treated mitochondria was much higher than what has been previously measured with other techniques. In addition, differences in substrate specificities were shown for complex I in mitochondria as compared to inside-out submitochondrial particles.