Abstract
Reliable measurements of intracellular metabolites are useful for effective plant metabolic engineering. This study explored the application ofin situ31P and13C NMR spectroscopy for long-term measurements of intracellular pH and concentrations of several metabolites in glycolysis, glucan synthesis, and central carbon metabolic pathways in plant tissues. An NMR perfusion reactor system was designed to allowCatharanthus roseushairy root cultures to grow for 3–6 weeks, during which time NMR spectroscopy was performed. Constant cytoplasmic pH (7.40±0.06), observed during the entire experiment, indicated adequate oxygenation.13C NMR spectroscopy was performed on hairy root cultures grown in solutions containing 1-13C-, 2-13C-, and 3-13C-labeled glucose in separate experiments and the flow of label was monitored. Activities of pentose phosphate pathways, nonphotosynthetic CO2fixation, and glucan synthesis pathways were evident from the experimental results. Scrambling of label in glucans also indicated recycling of triose phosphate and their subsequent conversion to hexose phosphates.
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