RESUMEN
Expression 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, specific for nda1, is seen at different times of the day with highest expression in the morning. This variation also influences the apparent developmental induction. Further, the nda1 mRNA in leaves specifically and completely disappears during dark treatment, with a rapid re-induction when plants are returned to light. Corresponding immunodetected NDA protein is specifically 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 specific 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 specific role of the encoded enzyme in the photosynthetically associated mitochondrial metabolism.
Asunto(s)
Transporte de Electrón/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Luz , NADH Deshidrogenasa/genética , Hojas de la Planta/efectos de la radiación , Solanum tuberosum/efectos de la radiación , Envejecimiento/fisiología , Western Blotting , Ritmo Circadiano/genética , Oscuridad , Regulación del Desarrollo de la Expresión Génica , Genes de Plantas/genética , Mitocondrias/enzimología , NAD/metabolismo , NADH Deshidrogenasa/metabolismo , NADP/metabolismo , Oxidación-Reducción/efectos de los fármacos , Hojas de la Planta/citología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rotenona/farmacología , Solanum tuberosum/citología , Solanum tuberosum/genética , Solanum tuberosum/metabolismoRESUMEN
Two 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 dehydro- genases, the NDB amino-acid sequence contains a non-conserved 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.