Arginine Metabolism in Developing Soybean Cotyledons 111 . Utilization 1

Tracerkinetic experiments were performed using L-[guanidino14C]arginine, L-[U-14C]arginine, L-[ureido-14C]citrulline, and L-[114C]omithine to investigate arginine utilization in developing cotyledons of Glycine max (L.) Merrill. Excised cotyledons were injected with carrier-free 14C compounds and incubated in sealed vials containing a CO2 trap. The free and protein amino acids were analyzed using high performance liquid chromatography and arginine-specific enzyme-linked assays. After 4 hours, 75% and 90% of the 14C metabolized from [guanidino-14C]arginine and [U-14C]arginine, respectively, was in protein arginine. The net protein arginine accumulation rate, calculated from the depletion of nitrogenous solutes in the cotyledon during incubation, was 17 nanomoles per cotyledon per hour. The data indicated that arginine was also catabolized by the arginase-urease reactions at a rate of 5.5 nanomoles per cotyledon per hour. Between 2 and 4 hours 14CO2 was also evolved from carbons other than C-6 of arginine at a rate of 11.0 nanomoles per cotyledon per hour. It is suggested that this extra '4CO2 was evolved during the catabolism of omithine-derived glutamate; 14C-omithine was a product of the arginase reaction. A model for the estimated fluxes associated with arginine utilization in developing soybean cotyledons is presented. The maximum specific radioactivity ratios between arginine in newly synthesized protein and total free arginine in the 14Ccitrulline and 14C-ornithine experiments indicated that only 3% of the free arginine was in the protein precursor pool, and that argininosuccinate and citrulline were present in multiple pools. Nitrogen budgets for developing cowpeas ( 11) and soybeans (8) suggest that the legume cotyledon plays a major role in the provision of amino acids for the synthesis of seed protein. A comparison between the N composition of soybean seedcoat exudate and cotyledonary protein suggests that 35% of the N in exudate is directly incorporated into protein, whereas 65% of the N undergoes transformation before incorporation (8). This comparison assumes that amino acids delivered to the cotyledon in deficit amounts are not metabolized. In addition, biochemical pathways not directly related to protein amino acid biosynthesis, and the existence of multiple cellular amino acid pools are not considered. In-depth metabolic ' Supported by an operating grant to B.J.S. from the Natural Sciences and Engineering Research Council of Canada. B.J.M. was the recipient of an Ontario Graduate Scholarship. studies of all seed amino acids are required to understand fully the processing of incoming N. Arginine is the predominant form of amino acid N in soybean cotyledons, constituting 18% of the total protein N and 14% to 62% of the free pool N during seed development (8). Micallef and Shelp (8) estimated that 72% of the cotyledon's Arg requirement for protein synthesis is satisfied by in situ biosynthesis, and, correspondingly, '4C tracer studies have demonstrated that this amino acid is actively synthesized in developing cotyledons (9). The remaining Arg is supplied by the vascular system. In this study, 14C tracer experiments involving L-[guanidino-'4C]Arg and L-[U-'4C]Arg indicated that exogenously supplied Arg was incorporated into seed protein and was metabolized via the arginase reaction and additional degradatory routes. In '4C tracer experiments that used L-[ureido'4C]Cit2 and L-[1-'4C]Orn, which are intermediates of Arg biosynthesis (9), the specific radioactivity of the Arg incorporated into protein was compared to that in the free pool; this information indicated the existence of multiple cellular pools of Arg and other pathway intermediates. In addition, the depletion of free pool N compounds provided an estimate of the rate of Arg incorporated into protein, and, consequently, permitted estimates of the fluxes through the various pathways associated with Arg utilization. MATERIALS AND METHODS Soybean plants were grown, and developing cotyledons were chosen from seeds at 23 DAA as previously described (8). All L-'4C-amino acids were obtained from Du Pont-New England Nuclear. The shipping medium was replaced by 50 mM Hepes (pH 8.0), and the '4C solution was injected into intact excised cotyledons, which were incubated at 30°C in a closed 34-mL serum bottle containing a CO2 trap, as described by Micallef and Shelp (8). Tissue was ground in sulfosalicylic acid (30 mg/mL), and the supernatant was collected; the protein was extracted from the pellet and hydrolyzed in 6 N HCI (8). The composition and 14C content of the amino acids present in the soluble fraction were determined by reverse-phase HPLC after orthophthalaldehyde derivatization (10). The techniques for the determination of the intramolecular position (C1 or C-6) of 2 Abbreviations: Cit, citrulline; Abu, 4-aminobutyric acid; Orn, ornithine; ArgS, argininosuccinate; DAA, days after anthesis.