Expression of a yeast acetyl CoA hydrolase in the mitochondrion

Abstract

Acetyl Coenzyme A (acetyl CoA) is required in the mitochondria to fuel the operation of the Krebs cycle and within the cytosolic, peroxisomal and plastidial compartments wherein it acts as the immediate precursor for a wide range of anabolic functions. Since this metabolite is impermeable to membranes it follows that discrete pathways both for its synthesis and for its utilization must be present in each of these organelles and that the size of the various compartmented pools are independently regulated. To determine the specific role of acetyl CoA in the mitochondria we exploited a transgenic approach to introduce a yeast acetyl CoA hydrolase (EC 3.1.2.1.) into this compartment in tobacco plants. Despite the facts that the introduced enzyme was correctly targeted and that there were marked reductions in the levels of citrate and malate and an increase in the acetate content of the transformants, the transgenic plants surprisingly exhibited increased acetyl CoA levels. The lines were further characterised by a severe growth retardation, abnormal leaf colouration and a dramatic reduction in photosynthetic activity correlated with a marked reduction in the levels of transcripts of photosynthesis and in the content of photosynthetic pigments. The altered rate of photosynthesis in the transgenics was also reflected by a modified carbon partitioning in leaves of these lines, however, further studies revealed that this was most likely caused by a decreased source to sink transport of carbohydrate. In summary these results suggest that the content of acetyl CoA is under tight control and that alterations in the level of this central metabolite have severe metabolic and developmental consequences in tobacco.

Mild Reductions in Mitochondrial NAD-Dependent Isocitrate Dehydrogenase Activity Result in Altered Nitrate Assimilation and Pigmentation But Do Not Impact Growth

“…The procedure of extraction and assay of NAD, NADH, NADP, and NAPH was performed according to the method described by Gibon and Larher (1997). Photosynthetic pigments were determined exactly as described in Bender-Machado et al (2004).…”

Arabidopsis Seed Development and Germination Is Associated with Temporally Distinct Metabolic Switches

“…A retention time and mass spectral library for automatic peak quantification of metabolite derivatives was implemented within the MassLab method. Relative and absolute metabolite contents were determined exactly as previously described (Roessner-Tunali et al, 2003) with the exception that quantitative analysis was expanded to include novel metabolites as previously described (Bender-Machado et al, 2004;Schauer et al, 2005). The absolute concentrations of most metabolites were determined by comparison with standard calibration curve response ratios of various concentrations of standard substance solutions, including the internal standard ribitol, which was derivatized concomitantly with tissue samples.…”

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

“…The procedure of extraction and assay of NAD, NADH, NADP and NAPH was performed according to the method described by Gibon and Larher (1997). Photosynthetic pigments were determined exactly as described in Bender-Machado et al (2004) . …”