Investigation of carbohydrate metabolism and transport in castor bean seedlings by cyclicjcross polarization imaging and spectroscopy

Abstract

NMR experiments using¹³C-labeled compounds offer the possibility of noninvasive monitoring of carbohydrate transport and metabolism in living plants, but are usually hampered by the low sensitivity of the¹³C nucleus. The problem of low sensitivity can be overcome by using the cyclicJcross polarization (CYCLCROP) technique, which allows the indirect detection of¹³C nuclei coupled to¹H nuclei with the high NMR sensitivity of protons. We report here on methods for imaging and spectroscopy based on the CYCLCROP technique, and their use in the firstin vivoNMR study of carbohydrate transport and metabolism in castor bean seedlings (Ricinus communis L.). Comprehensive acquisition strategies for the various NMR methods are given, including the procedure for setting up the experiments. In addition, a full analysis of the effect of relaxation on the signals generated from smallJ-coupled spin systems by the CYCLCROP sequence is given, and the high sensitivity of the sequence is demonstrated. In thein vivostudy of six-day-old castor bean seedlings, we were able to measure the uptake of labeled hexoses, supplied in solution to the cotyledons, and their conversion to sucrose, as well as the transport of this sucrose in the vascular bundles. Images of the actual distribution of labeled sucrose in the hypocotyl of the seedling have also been obtained. The resulting data show some evidence for a preferential incorporation of labeled fructose in the process of sucrose synthesis, which decreases with the time of incubation.