ABSTRACrThe various nitrogenous solutes important to embryo development in symbiotic soybean plants were determined during the midpodfilling stage. Glutamine was the principal form of nitrogen, contributing 55% of the embryo nitrogen requirement. Asparagine was the second most important, contributing 20%. The ureides allantoin and allantoic acid directly contributed only insignificantly to the total nitrogen requirement of the embryo. These conclusions were based upon analyses of tissue extracts, translocation studies of radiolabeled solutes, analysis of in vivo seed coat exudate collected from the freespace of attached, surgically altered seeds, and the in vitro culture of isolated immature soybean embryos.

A technique has been developed which permits mechanistic studies of phloem unloading in developing seeds of soybean (Glyrune mar cv Clark) and other legumes. An opening is cut in the pod wall and the embryo surgically removed from the seedcoat without diminishing the capacity of that tissue for assimilate import, phloem unloading, or efflux. The sites of phloem unloading were accessible via the seedcoat apoplast and were challenged with inhibitors, solutes, buffers, etc., to characterize the unloading process.Unloading is stimulated by divalent metal chelators and diethylsdibestrol, and inhibited by metabolic uncouplers and sulfhydryl group modifiers. Solutes released from the seed coat had a carbon/nitrogen ratio of 31 milligrams carbon per milligram nitrogen; sucrose represented 90% of the carbon present and various nitrogenous solutes contributed the remaining 10%. Unloading could be maintained for up to 8 hours at rates of 0.5 to 1.0 micromoles per hour, providing a valid, convenient in viva technique for studies of phloem unloading and seed growth mechanisms.The study of solute exit from the phloem ('unloading') in sink tissue has been severely hampered by the lack of convenient, valid techniques. Access to the phloem for experimental manipulation of the unloading process has been gained in but few systems, most notably sugar cane stalks (6) and Cuscuta-parasitized stems of Viciafaba (16). While much has been learned of the kinetics of photosynthate import, anatomy of the tissues involved, and uptake of assimilates by sink consumer cells (4,5,15), little is known of the mechanisms controlling the exit of solutes from the phloem sieve tubes.The phloem retains its solutes with minimal radial exchange;sites of unloading are notable exceptions. A popular hypothesis is that the sieve tube/companion cell/phloem parenchyma complex counters leakage by continuously reloading assimilates along the entire vascular length, and that sinks permit unloading by locally inhibiting the reloading mechanism (5, 10). This model is consistent with the data suggesting passive efflux from the phloem of developmentally immature sinks incapable of reloading, and for storage sinks where rapid hydrolysis or compartmentation prevents reloading of sucrose (4, 5, 15). However, other studies have provided evidence that, under some conditions, sinks directly effect solute exit from the phloem complex in a controlled, energydependent manner (13, 16). Given the diversity of sink tissues ' Visiting Scientist.which exist, it is probable that more than one unloading mechanism operates. This paper characterizes a novel technique for studying phloem unloading and maternal/embryo transfer of photosynthate in legume fruit. MATERIALS AND METHODSSoybean (Glycine max cv Clark) were grown as previously described (14) except that some plants were grown symbiotically by inoculating the seed at sowing with an appropriate strain of Rhizobium Japonicum and deleting nitrogen from the nutrient solution. Plants were utilized for experiments at various...

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).15N2-and 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 "GO2-feeding 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 10-or 15-min period. Roots were then excavated and the nodules harvested.

Xylem sap composition was examined in nodulated and nonnodulated cowpea ( Vigna unguicd L.I Walp.) plants receiving a range of levels of NO3 and in eight other ureide-forming legumes udlizing NO3 or N2 as sole source of nitrogen. A 15N dilution technique determined the proportions of plant nitrogen derived from N2 in the nodulated cowpeas fed NO. Xylem sap composition of N03-fed, nodulated cowpea varied predictably with the relative extents to which N2 and NO3 were being utilized. The ratios of asparagine to glutamine (N/N) and of NO3 to ureide (N/N) in xylem sap increased with increasing dependence on NO3 whereas per cent of xylem nitrogen as ureide and the ratio of ureide plus glutamine to asparagine plus NO3 (N/N) in xylem sap increased with increasing dependence on N2 fixation. The amounts of NO3 and ureides stored in leaflets, stems plus petioles, and roots of cowpea varied in a complex manner with level of NO3 and the presence or absence of N2 fixation. All species showed higher proportions of organic nitrogen as ureide and several-fold lower ratios of asparagine to glutamine in their xylem sap when relying on N2 than when utilizing NO3. In nodulated (minus nitrate) cowpea and mung bean (Vigna radiata [L.1 Wilczek) the percentage of xylem nitrogen as ureide remained constant during growth but the ratio of asparagine to glutamine varied considerably. The biochemical significance of the above differences in xylem sap compositdon was discussed.A number of legumes, especially tropical species, synthesize allantoin and allantoic acid from currently fixed N2 in nodules and use these compounds for transport and storage of N (1, 3, 6, 10). In soybean (Glycine max [L.] Merr.) the ureides are more prominent in nodulated plants than in plants grown with NO3 or NH4 salts (8, 11), suggesting a special association between N2 fixation and ureide formation (9), and the possibility of using ureide levels in plant tissues or transport fluids to determine the extent to which a field-grown crop is relying on symbiosis as opposed to combined forms of N from soil or fertilizer. This paper describes xylem sap composition of N2 and N03-fed cowpea (Vigna unguiculata [L.] Walp.) and of other grain legumes which form ureides, and suggests how compositional features of the sap might be used to evaluate the N2-fixing status of nodulated plants grown in the presence of NO3. MATERIALS AND METHODSThe species studied were cowpea (V. unguiculata [L.] Plants were grown in pots of heat sterilized sand during summer in a naturally lit glasshouse maintained within the temperature range 20-35 C by supplementary heating (night) and evaporative cooling (day). One set of plants of each species was inoculated with an effective Rhizobium (strain CB437, for soybean, strain NGR234 for horse gram, and strain CB756 for all other species) and grown with a N-free culture solution. A matching set of uninoculated plants received culture solution containing 10 mM NO3. By using this level of NO3, sterile sand and effective surface sterilization of s...