Carbon-13 Nuclear Magnetic Resonance Analysis of Metabolism in Soybean Labeled by ¹³CO₂

Abstract

Fourier transform 13C nuclear magnetic resonance spectra have been obtained of intact, fresh soybean ovules (Glycine max L. cv. Dare) harvested from pods subtended by a trifoliolate exposed to "3CO2 1 to 3 days earlier. The high resolution spectra are interpreted in terms of the labeled sugars and lipids in the ovule. Comparison of the spectra taken over the 3-day period permits qualitative estimates of sugar metabolism and rates of lipid synthesis. The spectra also contain information about the distribution of labels within the lipid chains. This information leads to a method of estimating the extent to which glucose degradation in the synthesizing soybean ovule is involved in the reactions of the phosphogluconate pathway.production, but also, in part, the distribution of the '2C labels within the lipids.This distribution in the lipids is complicated by the effect of recycling of hexose phosphates in the pentose cycle during synthesis in the ovule. That is, the label, when in the precursor sugar, is subject to a redistribution among unlabeled sugars due to reactions of the complete pentose cycle, as well as by the reversible transaldolase-transketolase reactions which form the nondehydrogenase part of the pentose cycle (13). The oxidation of sugar by the reactions of the pentose phosphate cycle is a source of NADPH for the synthesizing ovule (3). The information concerning pairs of labels available from a '2C labeling and NMR experiment leads to a fast and simple way of estimating the amount of glucose in an intact synthesizing soybean ovule which is involved in the reactions of the pentose cycle, relative to that utilized by other metabolic pathways.Carbon-1 3 labels can be introduced into soybean ovules by feeding`CO,2 to a single soybean trifoliolate in a closed system photosynthesis cell. About 200 mg of '2CO2 are fixed in a 4-hour experiment. The fixed 'C is transported to the ovules in the form of massively labeled sucrose, which is then used in the synthesis of fatty acids, amino acids, and various insoluble materials (3). Analysis of the '3C NMR2 spectra of intact ovules clearly identifies the labeled lipids present, as well as those labeled sugars in solution. The spectra are well resolved, displaying widely spaced, relatively narrow lines, features common to the 22C NMR spectra of many nonrigid systems (7,8).Although the use of 1'C labels, followed by an NMR detection of the label, has been reported before, previous experiments have been, for the most part, restricted to the introduction of labeled intermediates into bacterial or mammalian systems (9). There are at least two important advantages in using '2CO2, rather than labeled intermediates, in the study of certain types of metabolism in soybeans. First MATERIALS AND METHODSA single soybean plant (Glycine max, L. cv. Dare) was labeled with`2CO, between 8 AM and noon on a day in the beginning of its log growth ovule-filling period (6). The labeling was performed using a photosynthesis system in which a single soybean trifoliolate could be ...

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