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.

Arabidopsis thaliana was transformed with a Sorghum C4 PEPC cDNA under the control of the 35S promoter. Primary transformants were isolated and classified on a seed PEPC activity basis: A lines (two-fold increase) and B lines (four-fold increase). Primary transformants also expressed the C4 PEPC in leaves (up to a 10-fold increase in activity). In seeds, ectopically expressed PEPC impacted on dry weight and total protein content that were increased by up to 30%. In the stable progeny of A and B lines, the increase in PEPC activity was amplified in seeds; however, a decrease in PEPC content and activity (inversely proportional to seed levels) was observed in maternal tissues (leaves and siliques). This was due to the absence of the exogenous C4 PEPC and a partial decrease in the endogenous C3 PEPC forms (mRNA, protein and activity). There was no apparent compensation by a PEPC hyperphosphorylation. In these plants, the anaplerotic pathway flux was altered (notably, a decrease in organic acids and glutamine levels) as judged by 1H NMR metabolite profiling. Whereas in the corresponding seeds (four- to eight-fold increase in PEPC activity) there was no increase in protein and seed mass, but a moderate decrease in lipid content was observed (compared to control plants). Under normal culture conditions, the transformants (primary and progeny) did not show any apparent growth phenotype or modification in seed production per plant. However, the A and B lines exhibited severe growth defects when salt stressed by LiCl.