13C labelling reveals details of the soybean (Glycine max (L.) Merrill) seedling metabolic network

Seedlings depend upon seed reserves for the prevision of carbon skeletons for growth and energy production. Post-germinative growth of soybean is therefore marked by the breakdown of carbohydrates, proteins, and lipids and the interconversion of the products of these catabolic processes. Here, we refined a method for ¹³C isotope labelling of heterotrophic soybean seedlings and used it to probe metabolism during this critical phase of plant development. We anticipated that ¹³C labelling would reveal differences in metabolism between the cotyledons (COT) and hypocotyl-root axis (HRA). Feeding with U-¹³C glucose followed by analysis of isotope incorporation indicated uptake and metabolism of this labelled precursor by both COT and HRA. Fractional enrichments were generally greater in the HRA reflecting the catabolism of unlabelled reserves of lipids and proteins in COT. Mass isotopomer distributions confirmed operation of the TCA cycle and glycolysis along with hexose-phosphate cycling in both organs, whilst amino acid synthesis was limited, as expected, given the significant protein reserves. COT differed from HRA in TCA cycle citrate and anapleurotic metabolism. Experiments with ¹³C glycine indicated that glycine decarboxylase and serine hydroxymethyltransferase enzymes may function in heterotrophic tissues as well as in photorespiration. Labelling of the majority of metabolites was constant over time, suggesting that the experimental system could be used for metabolic flux analysis. Overall stable isotope labelling provided significant insight into metabolism of soybean seedlings and could be used to investigate seedling metabolism in other genotypes or species.