Monitoring of hypoxic metabolism in superfused plant tissues by in vivo 1H NMR.

We have used a coaxial superfusion system to obtain physiologically interpretable in vivo 1H NMR spectra at 500 MHz of carrot roots, maize roots, and rice shoots in water (no 2H2O). The superfusion system was constructed from common laboratory parts, required no modification of the probe and sample loading procedure, and was inherently leak resistant. The assignment and quantitation of the in vivo 1H NMR resonances were achieved by performing two-dimensional NMR experiments in vivo, and by in vitro analysis including NMR and gas chromatography-mass spectrometry. The in vivo spectra were dominated by resonances arising from sugars, organic acids, amino acids, and ethanol. In vivo measurements of spin-lattice relaxation times and chemical shifts of beta protons of malate in carrot roots suggested that malate was located in a relatively viscous and acidic compartment. In rice shoots, the hypoxic time courses of 9 metabolites were established in vivo, and 23 in vitro. In both cases, accumulation of lactate, ethanol, Ala, and gamma-aminobutyrate as well as a decrease in Gln and Asn concentrations were observed. These findings are consistent with accelerated glycolysis and decreased tricarboxylic acid cycle activity.