An engineered phosphoenolpyruvate carboxylase redirects carbon and nitrogen flow in transgenic potato plants

Abstract

Phosphoenolpyruvate carboxylase (PEPC) plays a central role in the anaplerotic provision of carbon skeletons for amino acid biosynthesis in leaves of C3 plants. Furthermore, in both C4 and CAM plants photosynthetic isoforms are pivotal for the fixation of atmospheric CO2. Potato PEPC was mutated either by modifications of the N-terminal phosphorylation site or by an exchange of an internal cDNA segment for the homologous sequence of PEPC from the C4 plant Flaveria trinervia. Both modifications resulted in enzymes with lowered sensitivity to malate inhibition and an increased affinity for PEP. These effects were enhanced by a combination of both mutated sequences and pulse labelling with 14CO2 in vivo revealed clearly increased fixation into malate for this genotype. Activity levels correlated well with protein levels of the mutated PEPC. Constitutive overexpression of PEPC carrying both N-terminal and internal modifications strongly diminished plant growth and tuber yield. Metabolite analysis showed that carbon flow was re-directed from soluble sugars and starch to organic acids (malate) and amino acids, which increased four-fold compared with the wild type. The effects on leaf metabolism indicate that the engineered enzyme provides an optimised starting point for the installation of a C4-like photosynthetic pathway in C3 plants.

Individual Maize Chromosomes in the C3 Plant Oat Can Increase Bundle Sheath Cell Size and Vein Density

“…It has been reported that expression of maize PEPC causes a slight reduction in net assimilation and stunting in transgenic rice plants (Taniguchi et al, 2008). Retarded growth that correlated with PEPC activity was also reported for potato (Solanum tuberosum; C 3 ) plants overexpressing a modified C 4 PEPC from Flaveria trinervia, and metabolite analysis of these plants revealed elevated levels of malate and total amino acids (Rademacher et al, 2002). Increases in malate and nitrogen content were also observed in maize PEPC-overexpressing rice plants (Agarie et al, 2002).…”

Nuclear Magnetic Resonance Spectroscopy-Based Metabolite Profiling of Transgenic Tomato Fruit Engineered to Accumulate Spermidine and Spermine Reveals Enhanced Anabolic and Nitrogen-Carbon Interactions

“…of PEPC in transgenic potato (Solanum tuberosum) plants showed that the flux of soluble sugars and starch is directed to organic acids such as malate and the amino acids Glu and Gln (Rademacher et al, 2002). We surmised that a fruit such as tomato may also use a similar mechanism, i.e.…”

“…Integration of glycolysis with N assimilation is likely linked with phosphoenolpyruvate carboxylase (PEPC), which provides C skeletons (Huppe and Turpin, 1994;Foyer and Noctor, 2002;Rademacher et al, 2002), and cytosolic isocitrate dehydrogenase is given as the sum of citrate (citr) and malate to that of Glc plus Fru plus Suc. These results were generated from the data given in Figure 2.…”

Alteration of the Interconversion of Pyruvate and Malate in the Plastid or Cytosol of Ripening Tomato Fruit Invokes Diverse Consequences on Sugar But Similar Effects on Cellular Organic Acid, Metabolism, and Transitory Starch Accumulation

“…Since pyruvate and PEP metabolites are transformed via the gluconeogenic pathway to sugar phosphates, which can subsequently be converted to starch (Lara et al, 2004), and the fact that previous studies demonstrated that phosphoenolpyruvate carboxylase (PEPC) activity plays an important role within potato (Solanum tuberosum) starch metabolism (Rademacher et al, 2002), we next evaluated the starch content of both the wild type and transformants at breaker stage (Fig. 5).…”