An Investigation of N Me[tacute]abolism and pH Regulation in Sphagnum Using In Vivo Nuclear Magnetic Resonance and Stable Isotope Mass Spectrometry

Abstract

Glutamine synthetase (GS) and glutamate dehydrogenase (GDH) are both present in Sphagnum and in principle both enzymes could be involved in the assimilation of ammonium. 15NH4* application led to the formation γ- 15N-glutamine, followed by α-amino glutamate/glutamine as observed by in vivo 15N NMR. No assimilation was detected in the presence of the GS-specific inhibitor methionine sulfoximine. This labelling pattern suggests, that ammonium assimilation in Sphagnum is mediated solely by the GS/glutamate synthase pathway. The application of 15N-labelled glutamate led to the liberation of 15NH4*, indicating that GDH mainly catalyses the deamination of glutamate. It was possible to record good-quality in vivo 31P NMR spectra of Sphagnum flexuosum tissue, and the chemical shift of the orthophosphate resonances indicated that cytoplasmic pH was not affected during the ammonium application, whereas the vacuolar pH was slightly reduced.