Following Plant Metabolismin Vivoand in Extracts with Heteronuclear Two-Dimensional Nuclear Magnetic Resonance Spectroscopy

Abstract

Limits of sensitivity and spectral resolution currently restrict the application of nuclear magnetic resonance (NMR) spectroscopy in plant metabolism. This study shows that these limits can be substantially expanded through the application of heteronuclear single- and multiple-quantum two-dimensional (2D) spectroscopic methods using pulsed field gradients bothin vivoand in extracts. The course of metabolism in approximately 0.2 g of maize (Zea maysL.) root tips labeled with [1-13C]glucose was followed with 1 min time resolution using heteronuclear multiple quantum coherence (HMQC)13C–1H spectroscopyin vivo.The timing of alanine, lactate, and ethanol synthesis was followed during the transition from normal to hypoxic conditions. In extracts of labeled maize root tips,13C–1H heteronuclear single quantum coherence and heteronuclear multiple quantum coherence (HMBC) spectra acquired in 2–3 h allowed the detection and assignment of resonances that are not seen in one-dimensional (1D)13C NMR spectra of the same samples taken in 12 h. In root tips labeled with15NH+415N–1H HMQC spectrain vivoshowed labeling in the amide of glutamine. In extracts,15N labeling in amines and amides was detected using15N–1H HMBC spectra that is not seen in 1D15N spectra of the same sample.