Ailantoin and Allantoic Acid in the Nitrogen Economy of the

The ureides, allantoin and allantoic acid, represented major fractions of the soluble nitrogen pool of nodulated plants of cowpea ( Vigna unuiulata [L.] Walp. cv. Caloona) throughout vegetative and reproductive growth. Stem and petioles were the principal sites of ureide accumulation, especialy in early fruiting. Labeling studies using 14C02 and 15N2 and incubation periods of 25 to 245 minutes indicated that synthesis of aDantoin and allantoic acid in root nodules involved currentiy delivered photosynthate and recentiy fixed N, and that the ureides were exported from nodule to shoot via the xylem. From 60 to 80% of xylem-borne N consisted of ureides; the remainder was glutamine, asparagine, and amino acids. ADlantoin predominated in the soluble N fraction of nodules and fruits, allantoin and allantoic acid were present in approximately equal proportions in xylem exudate, stems, and petioles. Extracts of the plant tissue fraction of nitrogen-fixing cowpea nodules contained glutamate synthase (EC 2.6.1.53) and glutamine synthetase (EC 63.1.2), but little activity of glutamate dehydrogenase (EC 1A.13). Hih levels of uricase (EC 1.733) and allantoinase (EC 3.5.2.5) were also detected. Aflantoinase but little uricase was found in extracts of leaflets, pods, and seeds. Balance sheets were constructed for production, storage, and utilization of ureide N during growth. VirtuaDy anl (average 92%) of the ureides exported from roots was metabolzed on entering the shoot, the compounds being presumably used as N sources for protein synthesis. The ureides, allantoin and allantoic acid, are major solutes of N in a wide variety of plants (5, 6, 21). In grain legumes they feature prominently in seeds and seedlings (e.g. Phaseolus mungo L., Dolichos spp., Glycine max [L.] Merr. [6]), in the soluble N of vegetative and reproductive organs of nodulated plants (G. max [10, 12]) and in xylem sap of nodulated plants (Pisum spp., Phaseolus vulgaris L. [16]; Vigna unguiculata [L.] Walp. [7]), or of plants utilizing NH4', N03, or urea (Pisum arvense L. [19]). In soybean (G. max) nodules are the presumed site of synthesis of ureides (11, 22). This study examines the significance of ureides in the nitrogen economy of nodulated cowpea (V. unguiculata), describing labeling studies of ureide synthesis in root nodules and patterns of transport, accumulation, and utilization of the compounds during plant growth. MATERIALS AND METHODS Growth, Harvesting, and Extraction of Plant Material. Effectively nodulated plants of cowpea (V. unguiculata [L.] Walp. cv. 1 Supported by funds from the Australian Research Grants Committee and the Wheat Industry Research Council. Caloona) were grown during summer on N-free culture solution in sand culture in a naturally lighted glasshouse. Samples ofplants were harvested and samples of xylem bleeding sap collected from decapitated plants at regular intervals during growth as described previously (17). Xylem sap was collected from freshly detached nodules of flowering and fruiting plants as detailed by Pate et al. (17). Plant parts were weighed and extracted at 0 C in 80o (v/v) ethanol. Nitrogen of the ethanol-soluble and ethanol-insoluble fractions was estimated by Kjeldahl analysis. Aliquots of ethanolsoluble extracts were evaporated to dryness, partitioned between petroleum ether and water, and the water-soluble fraction assayed for ureides and amino acids. Determination of Nitrogenous Solutes in Xylem Sap and Tissue Extracts. Allantoin and allantoic acid were separated on cellulose thin layers and estimated by densitometry after treatment with Ehrlich's reagent (7). Using this method the lower limit of detection for both compounds was 2.5 nmol. Between 2.5 and 100 nmol of both ureides applied to the plates were quantitated with a standard error of ±5% (N = 12). Amino acids and amides were estimated on a Beckman 118 C amino acid analyzer (4). 15N2and 14C02-Feeding Studies. Nodulated plants in late vegetative growth were used with their root systems enclosed as described earlier for root respiration measurements (3, 7). The shoots of a series of replicate groups of five plants were enclosed in water-cooled feeding chambers and exposed to 14CO2 (10 ,uCi/plant) for 10 min in full sunlight. At the end of the "GO2feeding period the gas space around the intact root systems was replaced by a mixture of A/02/N2 (56:14:30%o v/v) containing 18 atom % excess "5N. This gas mixture remained around the roots until the plants were harvested. Five plants were decapitated at successive intervals after the commencement of feeding with 14CO2 and xylem bleeding sap collected from their root stumps for a 10or 15-min period. Roots were then excavated and the nodules harvested. Assay of 14C and "N in Xylem Sap and "N in the Ethanolsoluble Fraction of Nodules. Basic compounds were removed from the samples using a column (1 x 6 cm) of Dowex 50-X1O (H+ form, 200-400 mesh) ion exchange resin. The eluate, containing the labeled allantoin and allantoic acid, was passed through a column (1 x 15 cm) of Dowex l-XIO (formate form, 200-400 mesh) and allantoin collected in the column wash. The adsorbed allantoic acid was then resolved from other acidic compounds with a linear gradient of formic acid (2). The allantoin eluted from the Dowex columns was hydrolyzed to allantoic acid by boiling with 1 mm NaOH for 5 min, and the neutralized solution applied to the formate column to resolve the allantoic acid. Allantoic acid was detected colorimetrically in the column eluates as the phenyl hydrazone of glyoxylate (24). The compound identified as allantoic acid in labeled xylem sap and tissue extracts cochromatographed with authentic allantoic acid (Sigma Chemical Co.) when separated on the formate gradients, on the cellulose thin layer plates, or on the lithium buffer system used in the amino acid analyzer. 495 www.plantphysiol.org on January 6, 2018 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 62, 1978 Determination of "4C was by liquid scintillation counting with appropriate correction for quenching. Ratios of 15N/"4N were determined by optical emission spectroscopy following Dumas combustion of the separated nitrogenous solutes (12-pig N samples) in quartz discharge tubes (20). Enzyme Assays. Nitrogenase activity was estimated by acetylene reduction assays of freshly harvested whole nodulated roots. Other enzymes were extracted from freshly harvested nodules or other plant tissues by thorough homogenization in a mortar and pestle at 4 C with 2 volumes of 0.1 M (pH 7.5) Tris-HCl, containing 5 to 10 mM DTT and I mm disodium EDTA. Sucrose (0.5 M) was added to the extracting medium for nodules to minimize disruption of bacteroids. Extracts were centrifuged at 30,000g (30 min, 4 C) and the supernatant passed through a Sephadex G-25 column before assay. The pellet from nodule extracts was resuspended in extracting medium and the bacteroids disrupted by four passages through a chilled French pressure cell (1). The bacteroid extract was then assayed for enzyme activity. Protein was estimated following precipitation with 20% (w/v) trichloroacetic acid using BSA as standard (9). Enzyme assays were carried out at 30 C. NADH-glutamate dehydrogenase (EC 1.4.1.4) and NADH-glutamate synthase (EC 2.6.1.53) were assayed as described previously (4), glutamine synthetase (EC 6.3.1.2) by the formation of glutamyl hydroxamate (14), uricase (EC 1.7.3.3) by the conversion of uric acid to allantoin (23), and allantoinase (EC 3.5.2.5) by measuring the glyoxylate freed from enzymically derived allantoic acid (15).