In addition to proton-pumping complex I, plant mitochondria contain several type II NAD(P)H dehydrogenases in the electron transport chain. The extra enzymes allow the nonenergy-conserving electron transfer from cytoplasmic and matrix NAD(P)H to ubiquinone. We have investigated the type II NAD(P)H dehydrogenase gene families in Arabidopsis. This model plant contains two and four genes closely related to potato (Solanum tuberosum) genes nda1 and ndb1, respectively. A novel homolog, termed ndc1, with a lower but significant similarity to potato nda1 and ndb1, is also present. All genes are expressed in several organs of the plant. Among the nda genes, expression of nda1, but not nda2, is dependent on light and circadian regulation, suggesting separate roles in photosynthesis-associated and other respiratory NADH oxidation. Genes from all three gene families encode proteins exclusively targeted to mitochondria, as revealed by expression of green fluorescent fusion proteins and by western blotting of fractionated cells. Phylogenetic analysis indicates that ndc1 affiliates with cyanobacterial type II NADH dehydrogenase genes, suggesting that this gene entered the eukaryotic cell via the chloroplast progenitor. The ndc1 should then have been transferred to the nucleus and acquired a signal for mitochondrial targeting of the protein product. Although they are of different origin, the nda, ndb, and ndc genes carry an identical intron position.Plant and fungal mitochondria have highly branched electron transport chains. The protonpumping respiratory complexes I, III, and IV work to a varying extent in parallel with non-protonpumping enzymes, i.e. type II NAD(P)H dehydrogenases and alternative oxidase. Thus, the coupling of electron transport to ATP formation varies depending on the electron path (Siedow and Umbach, 1995;Joseph-Horne et al., 2001).Complex I, the proton-pumping (type I) NAD(P)H dehydrogenase, is a multisubunit enzyme that is inhibited by rotenone in most organisms. It is present in ␣-proteobacteria and mitochondria of all eukaryotes except fermenting yeasts e.g. Saccharomyces cerevisiae. A homologous complex with unclear enzymatic properties is also present in chloroplasts (Yagi, 1991;Friedrich et al., 1995;Rasmusson et al., 1998).Type II, or rotenone-insensitive, NAD(P)H dehydrogenases have been found in several bacterial species and in plant and fungal mitochondria. The most studied enzymes, Escherichia coli NDH and S. cerevisiae NDI1, are FAD-containing single-polypeptide enzymes of 45 to 50 kD de Vries and Grivell, 1988). The S. cerevisiae NDI1 is located on the inner surface of the inner mitochondrial membrane, where it catalyzes the oxidation of matrix NADH (de Vries et al., 1992). Two homologous S. cerevisiae proteins, NDE1 and NDE2, are located on the external side of the inner membrane, where they oxidize cytoplasmic NADH (Luttik et al., 1998;Small and McAlister-Henn, 1998). An NADPHspecific type II dehydrogenase, NDE1, is present on the external surface of the inner membrane of Neurospora crassa ...

SummaryExpression of genes for respiratory chain dehydrogenases was investigated in potato (Solanum tuberosum L. cv. Desiree) leaves. The recently characterized nda1 and ndb1 genes, homologues to genes encoding the non-proton pumping respiratory chain NADH-dehydrogenases of Escherichia coli and yeast, were compared to genes encoding catalytic subunits of the proton-pumping NADH dehydrogenase (complex I). As leaves develop from young to mature, the nda1 transcript level increases, accompanied by an elevation in immunodetected NDA protein and internal rotenone-insensitive NADH oxidation. The other investigated transcripts, proteins and NAD(P)H oxidation activities were essentially unchanged. A variation in transcript level, speci®c for nda1, is seen at different times of the day with highest expression in the morning. This variation also in¯uences the apparent developmental induction. Further, the nda1 mRNA in leaves speci®cally and completely disappears during dark treatment, with a rapid reinduction when plants are returned to light. Corresponding immunodetected NDA protein is speci®cally decreased in mitochondria isolated from dark-treated plants, accompanied by a lower capacity for internal rotenone-insensitive NADH oxidation. Complete light dependence and diurnal changes in expression have previously not been reported for genes encoding respiratory chain proteins. Qualitatively similar to NDA, the alternative oxidase showed developmental induction and light dependence. In addition to the speci®c change in nda1, a general, slower down-regulation in darkness was seen for the other NAD(P)H dehydrogenase genes. The nda1 expression during development, and in response to light, indicates a speci®c role of the encoded enzyme in the photosynthetically associated mitochondrial metabolism.

SummaryTwo different cDNAs, homologous to genes for rotenone-insensitive NADH dehydrogenases of bacteria and yeast, were isolated from potato. The encoded proteins, called NDA and NDB, have calculated molecular masses of 55 and 65 kDa, respectively. The N-terminal parts show similarity to mitochondrial targeting peptides and the polypeptides are in vitro imported into potato mitochondria. Import processing to a smaller polypeptide is seen for the NDA but not the NDB protein. After import, NDA is intramitochondrially sorted to the matrix side of the inner membrane, whereas NDB becomes exposed to the intermembrane space. Imported proteins are associated to membranes upon digitonin permeabilization. On expression in Escherichia coli, NDB is released from the bacterial membrane in the absence of divalent cations whereas detergents are necessary for solubilization of NDA. Both deduced amino-acid sequences contain the dual motifs for nucleotide binding with the characteristics of the core criteria, similar to the bacterial homologues. Unique among NADH dehydrogenases, the NDB amino-acid sequence contains a nonconserved insert, which is similar to EF-hand motifs for calcium binding. Phylogenetic analyses group the rotenone-insensitive NADH dehydrogenases largely by species, but suggest ancient gene duplications.

Controlled oxidation reactions catalyzed by the large, proton-pumping complexes of the respiratory chain generate an electrochemical gradient across the mitochondrial inner membrane that is harnessed for ATP production. However, several alternative respiratory pathways in plants allow the maintenance of substrate oxidation while minimizing the production of ATP. We have investigated the role of light in the regulation of these energy-dissipating pathways by transcriptional profiling of the alternative oxidase, uncoupling protein, and type II NAD(P)H dehydrogenase gene families in etiolated Arabidopsis seedlings. Expression of the nda1 and ndc1 NAD(P)H dehydrogenase genes was rapidly up-regulated by a broad range of light intensities and qualities. For both genes, light induction appears to be a direct transcriptional effect that is independent of carbon status. Mutant analyses demonstrated the involvement of two separate photoreceptor families in nda1 and ndc1 light regulation: the phytochromes (phyA and phyB) and an undetermined blue light photoreceptor. In the case of the nda1 gene, the different photoreceptor systems generate distinct kinetic induction profiles that are integrated in white light response. Primary transcriptional control of light response was localized to a 99-bp region of the nda1 promoter, which contains an I-box flanked by two GT-1 elements, an arrangement prevalent in the promoters of photosynthesis-associated genes. Light induction was specific to nda1 and ndc1. The only other substantial light effect observed was a decrease in aox2 expression. Overall, these results suggest that light directly influences the respiratory electron transport chain via photoreceptor-mediated transcriptional control, likely for supporting photosynthetic metabolism.The electron transport chain (ETC) and ATP synthase catalyze the final steps of aerobic respiration, whereby reduced organic compounds are converted into chemical energy in the form of ATP. The ETC is located in the inner mitochondrial membrane and is composed of four large, multiprotein complexes common to both plants and animals. Complexes I and II catalyze the oxidation of matrix NADH and succinate, respectively, transferring electrons to lipid-soluble ubiquinone. Reduced ubiquinone is then oxidized via complex III, which donates electrons to the cytochrome c protein. Complex IV then transfers electrons from cytochrome c to the terminal electron acceptor, O 2 , generating water. The oxidation reactions mediated by complexes I, III, and IV are coupled to the pumping of protons across the inner membrane, generating an electrochemical gradient. This membrane gradient is harnessed by the F o F 1 -ATP synthase for the production of ATP (Siedow and Day, 2000).In addition to the basal ETC described above, plants possess several alternative respiratory pathways that bypass energy conservation by circumventing the formation or utilization of the electrochemical proton gradient. These energy-dissipating pathways are formed by several simple proteins: type ...