Characterization of mitochondrial alternative NAD(P)H dehydrogenases in Arabidopsis: Intraorganelle location and expression

• Published in: Plant and cell physiology Vol. 47; no. 1; pp. 43 - 54
• Main Authors: Elhafez, D.(University of Western Australia, Crawley), Murcha, M.W, Clifton, R, Soole, K.L, Day, D.A, Whelan, J
• Format: Journal Article
• Language: English
• Published: Japan Oxford University Press 01.01.2006; Oxford Publishing Limited (England)
• Subjects: 51 kDa subunit of complex I; alternative NAD(P)H dehydrogenase; Alternative NAD(P)H dehydrogenase(s); Alternative oxidase; Aox; ARABIDOPSIS; Arabidopsis - enzymology; Arabidopsis - genetics; Base Sequence; CI51; Circadian Rhythm; cytosolic ribosomal protein of the large subunit; DNA, Plant - genetics; EXPRESION GENICA; EXPRESSION DES GENES; GENE; GENE EXPRESSION; Gene Expression Profiling; GENES; Genes, Plant; GFP; green fluorescent protein; MITOCHONDRIA; Mitochondria - enzymology; MITOCHONDRIE; MITOCONDRIA; Multienzyme Complexes - genetics; Multienzyme Complexes - metabolism; NADH, NADPH Oxidoreductases - genetics; NADH, NADPH Oxidoreductases - metabolism; NADPH Oxidases - genetics; NADPH Oxidases - metabolism; nuclear-encoded mitochondrial ribosomal proteins of the small subunit; Organelles - enzymology; P-subunit of glycine decarboxylase; PGDC; Photoperiod; Protein import; proteinase K; RESPIRACION; RESPIRATION; Reverse Transcriptase Polymerase Chain Reaction; reverse transcription–PCR; RPL19; RPS1 and RPS13; RT–PCR; TIM; translocase of the inner mitochondrial membrane; Ucp; uncoupling proteins
• ISSN: 0032-0781; 1471-9053
• DOI: 10.1093/pcp/pci221

The intramitochondrial location of putative typeII NAD(P)H dehydrogenases (NDs) in Arabidopsis was investigated by measuring the ability of isolated mitochondria to take up precursor proteins generated from cDNAs using an in vitro translation system. The mature proteins of NDA1, NDA2 and NDC1 were judged to be located on the inside of the inner membrane because they were protected from protease added after the mitochondrial outer membrane had been ruptured. In contrast, NDB1, NDB2 and NDB4 were not protected from protease digestion in mitochondria with ruptured outer membranes and were deemed to be located on the outside of the inner membrane. Expression of all ND genes was measured using quantitative reverse transcription-PCR (RT-PCR) to determine transcript abundance, and compared with expression of alternative oxidase, uncoupler proteins and selected components of the oxidative phosphorylation complexes. NDA1 and NDB2 were the most prominently expressed members in a variety of tissues, and were up-regulated in the early daytime in a diurnal manner. Analysis of array data suggested that NDA1 clustered closest to the gene encoding the P-subunit of glycine decarboxylase. Taken together with the diurnal regulation of NDA1 observed here and in other studies, this suggests that NDA1 plays a role in integrating metabolic activities of chloroplasts and mitochondria. NDA2, NDB2 and Aox1a were up-regulated in a coordinated manner under various treatments, potentially forming a complete respiratory chain capable of oxidizing matrix and cytosolic NAD(P)H. NDB1 and NDC1 were down-regulated under the same conditions and may be regarded as housekeeping genes.