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The four phosphoenolpyruvate carboxylase encoding genes (1–4) of Arabidopsis thaliana were found to be expressed in rosette leaves, with PEPC2 mRNA being the major species. The enzyme activity was sensitive to feedback inhibition by malate, aspartate and glutamate and this effect was antagonized by glucose-6-phosphate and modulated after a dark–light transition. Taken together, the light/dark differences detected in pH response, malate sensitivity, phosphoenolpyruvate carboxylase kinase1 (PEPCk1) transcript content, and in vivo [ 32 P]-phosphate labelling indicates that PEPC is subjected to reversible, light-dependent phosphorylation on its N-terminal regulatory serine. The light-transduction pathway in isolated mesophyll cell protoplasts involves a PI-dependent phospholipase C (PI-PLC), the second messenger inositol 1,4,5 trisphosphate (IP 3 ), calcium fluxes via IP 3 -dependent tonoplast calcium channels, photosynthetic electron transport and PEPCk1 synthesis.

Two phosphoenolpyruvate carboxylase (PEPC) kinase genes (PPCk1 and PPCk2) are present in the Arabidopsis genome; only PPCk1 is expressed in rosette leaves. Homozygous lines of two independent PPCk1 T-DNA-insertional mutants showed very little (dln1), or no (csi8) light-induced PEPC phosphorylation and a clear retard in growth under our greenhouse conditions. A mass-spectrometry-based analysis revealed significant changes in metabolite profiles. However, the anaplerotic pathway initiated by PEPC was only moderately altered. These data establish the PPCk1 gene product as responsible for leaf PEPC phosphorylation in planta and show that the absence of PEPC phosphorylation has pleiotropic consequences on plant metabolism. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Metabolic profiling by 1-dimensional (1-D)

Metabolic profiling by 1-dimensional (1-D) 1H-nuclear magnetic resonance (NMR) was tested for absolute quantification of soluble sugars, organic acids, amino acids and some secondary metabolites in fruit, roots and leaves. The metabolite responsible for each peak of the 1H-NMR spectra was identified from spectra of pure compounds. Peak identity was confirmed by the addition of a small amount of commercially-available pure substance. 1H-NMR spectra acquisition was automated. 1H-NMR absolute quantification was performed with a synthesised electronic reference signal and validated by comparison with enzymatic or HPLC analyses; the correlation coefficients between 1H-NMR data and enzymatic or HPLC data were highly significant. Depending on the species and tissues, 14–17 metabolites could be quantified with 15–25 min acquisition time. The detection limit was approximately 1–9 µg in the NMR tube, depending on the compound. Quantitative data were used for (1) a genetic study of strawberry fruit quality, (2) a functional study of tomato transformants overexpressing hexokinase and (3) a study of Arabidopsis phosphoenolpyruvate carboxylase transformants with several lines showing decreased activity of the enzyme. Biochemical phenotyping of the fruits of a strawberry offspring allowed the detection of quantitative trait loci (QTL) controlling fruit quality. Comparison of the roots of wild types and hexokinase tomato transformants using principal component analysis of metabolic profiles revealed that environmental factors, i.e. culture conditions, can significantly modify the metabolic status of plants and thus hide or emphasise the expression of a given genetic background. The decrease in phosphoenolpyruvate carboxylase activity (up to 75%) in Arabidopsis transformants impacted on the metabolic profiles without compromising plant growth, thus supporting the idea that the enzyme has a low influence on the carbon flux through the anaplerotic pathway.