A large decrease of cytosolic triosephosphate isomerase in transgenic potato roots affects the distribution of carbon in primary metabolism

Abstract

Triosephosphate isomerase (TPI, EC 5.3.1.1) catalyzes the interconversion of dihydroxyacetone-P and glyceraldehyde 3-P in the glycolytic pathway. A constitutively expressed antisense construct for cytosolic TPI was introduced into potato (Solanum tuberosum) using Agrobacterium rhizogenes to examine the metabolic effects of a reduction in cytosolic TPI in roots. We obtained a population of transgenic root clones displaying ~36 to 100 % of the TPI activity found in control clones carrying an empty binary vector. Ion exchange chromatography and immunoblot analysis showed that the antisense strategy significantly decreased the cytosolic TPI isoform, while levels of plastidial TPI activity remained apparently unaffected. Transgenic roots were characterized with respect to the activity of glycolytic enzymes, their metabolite contents and carbon fluxes. Metabolite profiling of sugars, organic acids, amino acids and lipids showed elevated levels of sucrose, glucose, fructose, fumarate, isocitrate, 4-aminobutyrate, alanine, glycine, aromatic amino acids and saturated long chain fatty acids in roots containing the lowest TPI activity. Labelings with (14)C-glucose, (14)C-sucrose and (14)C-acetate indicated that a reduction of cytosolic TPI activity in roots increased carbon metabolism through the pentose phosphate pathway, O(2) uptake and catabolism of sucrose to CO(2), and capacity for lipid synthesis. These results demonstrate that a large reduction of cytosolic TPI alters the distribution of carbon in plant primary metabolism.

Proteomic Analysis of Embryogenesis and the Acquisition of Seed Dormancy in Norway Maple (Acer platanoides L.)

“…DHAP is a precursor for the glycerol moiety in glycerolipids; GAP and/or DHAP are also precursors for a number of secondary metabolites [36]. Dorion et al [37] demonstrated that a large reduction of cytosolic TPI alters the distribution of carbon in plant primary metabolism. In our study, the abundance of TPIs increased as seed development progressed, most likely due to the demand for carbon skeletons during the growth of embryo tissues.…”

Cytosolic Triosephosphate Isomerase from Arabidopsis thaliana Is Reversibly Modified by Glutathione on Cysteines 127 and 218

“…It has been demonstrated that cells with reduced TPI activity have a higher NADPH/NADP + ratio due to an increase in carbon flux through the PPP, thus providing more reducing power within the cytosol (Ralser et al, 2007). In plants, transgenic potato roots with reduced cTPI activity also showed a greater carbon flux through the PPP compared to controls (Dorion et al, 2012). Metabolic modeling simulations also suggested a rerouting of carbon flux through PPP in plant cells with low cTPI activity (Valancin et al, 2013).…”

The Dinoflagellate Lingulodinium polyedrum Responds to N Depletion by a Polarized Deposition of Starch and Lipid Bodies

“…To test if the changes in protein levels observed for f/2-N cultures were also reflected in the free amino acid (FAA) pools, we next characterized the FAA profile of N-depleted cultures using a previously described HPLC-based protocol [13]. Of the 14 FAAs that were quantified by this method, only Gln and Arg levels were found to decrease after N deprivation consistent with the requirement of NH 3 + for their biosynthesis.…”

“…Amino acid quantification was performed by HPLC using a protocol previously optimized for plant tissues [13]. Samples were lysed mechanically in 80% (v/v) ethanol for 2 min at 4°C in a bead beater (BioSpec products).…”