Toward the mechanism of NH4+ sensitivity mediated by Arabidopsis GDPamannose pyrophosphorylase

The ascorbic acid (AA)-deficient Arabidopsis thaliana mutant vtc1-1, which is defective in GDP-mannose pyrophosphorylase (GMPase), exhibits conditional hypersensitivity to ammonium (NH4+), a phenomenon that is independent of AA deficiency. As GMPase is important for GDP-mannose biosynthesis, a nucleotide sugar necessary for protein N-glycosylation, it has been thought that GDP-mannose deficiency is responsible for the growth defect in vtc1-1 in the presence of NH4+. Therefore, the motivation for this work was to elucidate the growth and developmental processes that are affected in vtc1-1 in the presence of NH4+ and to determine whether GDP-mannose deficiency generally causes NH4+ sensitivity. Furthermore, as NH4+ may alter cytosolic pH, we investigated the responses of vtc1-1 to pH changes in the presence and absence of NH4+. Using qRT-PCR and staining procedures, we demonstrate that defective N-glycosylation in vtc1-1 contributes to cell wall, membrane and cell cycle defects, resulting in root growth inhibition in the presence of NH4+. However, by using mutants acting upstream of vtc1-1 and contributing to GDP-mannose biosynthesis, we show that GDP-mannose deficiency does not generally lead to and is not the primary cause of NH4+ sensitivity. Instead, our data suggest that GMPase responds to pH alterations in the presence of NH4+.