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Mild reductions in cytosolic NADP-dependent isocitrate dehydrogenase activity result in lower amino acid contents and pigmentation without impacting growth.

ABSTRACT: Transgenic tomato (Solanum lycopersicum) plants were generated targeting the cytosolic NADP-dependent isocitrate dehydrogenase gene (SlICDH1) via the RNA interference approach. The resultant transformants displayed a relatively mild reduction in the expression and activity of the target enzyme in the leaves. However, biochemical analyses revealed that the transgenic lines displayed a considerable shift in metabolism, being characterized by decreases in the levels of the TCA cycle intermediates, total amino acids, photosynthetic pigments, starch and NAD(P)H. The plants showed little change in photosynthesis with the exception of a minor decrease in maximum photosynthetic efficiency (F (v)/F (m)), and a small decrease in growth compared to the wild type. These results reveal that even small changes in cytosolic NADP-dependent isocitrate dehydrogenase activity lead to noticeable alterations in the activities of enzymes involved in primary nitrate assimilation and in the synthesis of 2-oxoglutarate derived amino acids. These data are discussed within the context of current models for the role of the various isoforms of isocitrate dehydrogenase within plant amino acid metabolism.

SUBMITTER: Sulpice R

PROVIDER: S-EPMC2945463 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Mild reductions in cytosolic NADP-dependent isocitrate dehydrogenase activity result in lower amino acid contents and pigmentation without impacting growth.

Sulpice Ronan R Sienkiewicz-Porzucek Agata A Osorio Sonia S Krahnert Ina I Stitt Mark M Fernie Alisdair R AR Nunes-Nesi Adriano A

Amino acids 20100516 4

Transgenic tomato (Solanum lycopersicum) plants were generated targeting the cytosolic NADP-dependent isocitrate dehydrogenase gene (SlICDH1) via the RNA interference approach. The resultant transformants displayed a relatively mild reduction in the expression and activity of the target enzyme in the leaves. However, biochemical analyses revealed that the transgenic lines displayed a considerable shift in metabolism, being characterized by decreases in the levels of the TCA cycle intermediates, ...[more]

PMID: 20473773

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Project description:Cytoplasmic NADP(+)-isocitrate dehydrogenase (NADP(+)-IDH) has been purified and characterized, and its gene sequenced in many animal, plant, and yeast species. However, much less information is available on this enzyme-gene in insects. As a first step in investigating the biochemical and molecular mechanisms by which NADP(+)-IDH contributes to adaptations for flight vs. reproduction in insects, the enzyme was purified to homogeneity in the wing-dimorphic cricket, Gryllus firmus, characterized, and its corresponding gene sequenced. Using a combination of polyethylene glycol precipitation, Cibacron-Blue affinity chromatography, and hydrophobic interaction chromatography the enzyme was purified 291-fold (7% yield; specific activity = 15.8 µmol NADPH/min/mg protein). The purified enzyme exhibited a single band on SDS PAGE (46.3 kD), but consisted of two N-terminal amino acid sequences that differed in the first two amino acids. Purified enzyme exhibited standard Michaelis-Menten kinetics at pH 8.0 and 28° C (K(M(NADP+)) = 2.3 ± 0.4 µM; K(M(Na+-Isocitrate)) = 14.7 + 1.8 µM). Subunit molecular mass and K(M)S were similar to published values for NADP(+)-IDHs from a variety of vertebrate and two insect species. PCR amplification of an internal sequence using genomic DNA followed by 3' and 5' RACE yielded a nucleotide sequence of the mature protein and translated amino-acid sequences that exhibited high similarity (40-50% and 70-80%, respectively) to sequences from insect and vertebrate NADP(+)-IDHs. Two potential ATG start codons were identified. Both Nterminal amino-acid sequences matched the nucleotide sequence, consistent with both enzyme forms being transcribed from the same gene, although these variants could also be encoded by different genes. Bioinformatic analyses and differential centrifugation indicated that the majority, if not all, of the enzyme is cytoplasmic. The enzyme exhibited high specific activity in fat body, head and gut, and a single band on native PAGE.

Project description:The effect of inhibition of NADP-specific isocitrate dehydrogenase (EC 1.1.1.42) by DL-threo-alpha-methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylase) on urea synthesis was studied in isolated rat hepatocytes. alpha-Methylisocitrate substantially inhibited the rate of urea synthesis (35--84%) with substrates requiring net reductive amination of 2-oxoglutarate to glutamate for aspartate synthesis (i.e., L-serine, D-alanine, or NH4Cl + L-lactate). alpha-Methylisocitrate did not inhibit synthesis of urea from substrates not requiring reductive formation of glutamate (i.e. L-alanine, L-glutamine, L-asparagine, or NH4Cl + L-ornithine). The rate-limiting role of NADPH in urea synthesis was correlated with the decrease in NADPH content that occurred upon addition of NH4Cl or of alpha-methylisocitrate to hepatocytes incubated with lactate and pyruvate, indicating utilization of NADPH for reductive amination of 2-oxoglutarate and inhibition of NADPH generation via NADP-isocitrate dehydrogenase, respectively. Similar results were obtained with D-alanine and L-serine; however, alpha-methylisocitrate or NH4Cl did not substantially decrease NADPH content when L-alanine was the substrate. Inhibitors or ornithine--2-oxo acid transaminase (L-canaline or gabaculine) decreased the uptake of ornithine by hepatocytes and inhibited the alpha-methylisocitrate insensitive urea synthesis from ornithine and NH4Cl. Canaline did not inhibit urea synthesis from lactate, ornithine, and NH4Cl but the inhibition by alpha-methylisocitrate of urea formation from this combination was appreciably larger with canaline (approx. 82%) than without canaline (approx. 48%). Inhibition of urea synthesis from NH4Cl + lactate by alpha-methylisocitrate was partially prevented by oleate, octanoate, or 3-hydroxybutyrate. When the NADH content of hepatocytes was increased by 3-hydroxybutyrate, the addition of NH4Cl and/or alpha-methylisocitrate caused a decline in NADH (and NADPH) content, suggesting that reducing equivalents from NADH as well as from NADPH can support net reductive amination of 2-oxoglutarate when required for urea synthesis.

Dataset Information

Mild reductions in cytosolic NADP-dependent isocitrate dehydrogenase activity result in lower amino acid contents and pigmentation without impacting growth.

Publications

Mild reductions in cytosolic NADP-dependent isocitrate dehydrogenase activity result in lower amino acid contents and pigmentation without impacting growth.

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