NAD malic enzyme and the control of carbohydrate metabolism in potato tubers.

Potato (Solanum tuberosum) plants were transformed with a cDNA encoding the 59-kD subunit of the potato tuber NAD-dependent malic enzyme (NADME) in the antisense orientation. Measurements of the maximum catalytic activity of NADME in tubers revealed a range of reductions in the activity of this enzyme down to 40% of wild-type activity. There were no detrimental effects on plant growth or tuber yield. Biochemical analyses of developing tubers indicated that a reduction in NADME activity had no detectable effects on flux through the tricarboxylic acid cycle. However, there was an effect on glycolytic metabolism with significant increases in the concentration of 3-phosphoglycerate and phosphoenolpyruvate. These results suggest that alterations in the levels of intermediates toward the end of the glycolytic pathway may allow respiratory flux to continue at wild-type rates despite the reduction in NADME. There was also a statistically significant negative correlation between NADME activity and tuber starch content, with tubers containing reduced NADME having an increased starch content. The effect on plastid metabolism may result from the observed glycolytic perturbations.

Mitochondrial pyruvate dehydrogenase. Molecular cloning of the E1 alpha subunit and expression analysis.

A polymerase chain reaction-based approach was used to isolate cDNA clones encoding the E1 alpha subunit of the mitochondrial pyruvate dehydrogenase from higher plants. Putative full-length clones were identified on the basis of similarity to E1 alpha sequences from nonplant sources. Southern blot analysis revealed a small family of genes in potato (Solanum tuberosum L.), whereas in cucumber (Cucumis sativus) there are only one or two genes. Tissue-specific variation in the relative amounts of E1 alpha mRNA was observed in northern blot analysis of different potato tissues, with the highest steady-state transcript levels found in floral tissue. Measurement of pyruvate dehydrogenase activity in cucumber cotyledons showed that there is a transient increase to a maximum at 4 to 5 d postimbibition. Western blot analysis revealed that the amount of E1 alpha protein also peaks at this time. Steady-state transcript levels in germinating cucumber cotyledons also show transient accumulation, peaking 2 d postimbibition. These data are consistent with regulation of E1 alpha at the level of transcription and/or mRNA stability in postgerminative cucumber cotyledons.

Plant mitochondrial NAD+-dependent malic enzyme. cDNA cloning, deduced primary structure of the 59- and 62-kDa subunits, import, gene complexity and expression analysis.

The 59- and 62-kDa subunits of the mitochondrial NAD+-dependent malic enzyme (EC 1.1.1.39) were purified from Solanum tuberosum L. (potato). NH2-terminal and internal amino acid sequence information was used to identify cDNAs encoding the two subunits. Comparison of the nucleotide sequences revealed that the subunits have 60% identity at the DNA level and 65% identity at the deduced amino acid level, implying that they are derived from a common ancestral gene. The plant NAD+-dependent malic enzymes belong to a family of related enzymes, including cytosolic and chloroplastic NADP+-dependent malic enzymes (EC 1.1.1.40) and bacterial NAD+-dependent malic enzymes (EC 1.1.1.38). The cDNAs were transcribed and translated in vitro and the resultant polypeptides imported into isolated mitochondria and shown to be processed. Southern blot analysis of potato genomic DNA revealed a simple pattern of hybridization for both subunits, indicating a simple gene structure or small number of genes encoding the two subunits. Northern blot analysis of RNA from a range of potato tissues has shown that the steady state levels for the two subunits are equivalent, suggesting that they are coordinately expressed.

The adenine nucleotide translocator of higher plants is synthesized as a large precursor that is processed upon import into mitochondria.

Two maize genes and cDNAs encoding the mitochondrial adenine nucleotide translocator (ANT), a nuclear-encoded inner mitochondrial membrane carrier protein, have previously been isolated in this laboratory. Sequence analysis revealed the existence of much longer open reading frames than the corresponding fungal and mammalian ANT genes. Potato ANT cDNAs have subsequently been isolated and sequenced and alignment of the deduced plant amino acid sequences with the equivalent fungal and mammalian polypeptides indicated that the plant proteins contain N-terminal extensions. When the plant cDNA clones are expressed in vitro they direct the synthesis of precursor proteins that are specifically processed at the N-terminus upon import into isolated mitochondria. N-terminal amino acid sequence data obtained from the native proteins purified from both maize and potato mitochondria has allowed identification of the putative processing sites. Further import analysis has shown that two distinct regions of the maize precursor protein contain targeting information, the 97 amino acids at the N-terminus and the 267 C-terminal amino acids. This is the first report that provides experimental evidence that the adenine nucleotide translocator of higher plants is synthesized as a large precursor protein that is specifically cleaved upon import into mitochondria. Import of ANT into higher plant mitochondria therefore appears to be different to the corresponding process in fungal and mammalian systems where targeting of ANT to mitochondria is mediated by internal signals and there is no N-terminal processing.