Experiments were conducted to characterize the distribution of N compounds in the xylem sap of nodulated and nonnodulated soybean plants through development and to determine the effects of exogenous N on the distribution of N compounds in the xylem. Xylem sap was collected from nodulated and nonnodulated greenhouse-grown soybean plants (Glycine allantoin and allantoic acid have suggested that the greater portion of ureide synthesis occurs in the nodules or that nodules stimulate the production of ureides in the root tissue (7,15). Evidence that ureide synthesis occurs in the nodules was provided by the observation that the incorporation of 15N from '5N2 into allantoin and allantoic acid in nodules was higher than that in the basal portion of roots (14). Further, nodules of 4-week-old soybean plants have been reported to have significant activities of xanthine oxidase and uricase, which catalyze the oxidative decomposition of xanthine into allantoin, whereas roots and stems have only low activity levels of these enzymes (25).It is apparent that allantoin and allantoic acid play a central role in the N metabolism of nodulated soybean plants, and thus possibly are important in the translocation of N from nodulated roots to the shoot. Matsumoto et al. (12) and Ishisuka (10) have reported that soybean xylem sap contained considerable amounts of allantoin and allantoic acid. Streeter (23) reported that asparagine was the principal N compound in the xylem sap of fieldgrown nodulated soybean plants. However, no total N analysis was performed. Recent reanalysis (24) of the sap from this study (which had been stored frozen) showed that ureide-N concentrations during reproductive development were two to six times greater than amino acid plus nitrate-N concentrations. The present studies were designed to: (a) characterize the distribution of all nitrogenous compounds in the xylem sap of nodulated and nonnodulated soybean plants throughout development; and (b) determine the effect of exogenous N upon the distribution of nitrogenous compounds in the xylem.The form in which N is transported from the assimilatory root to the shoot has been found to vary widely among higher plants.Nitrate, amino acids, amides, and ureides all have been implicated as principal forms of N in the xylem sap of various plants (2,(19)(20)(21). Work from a number of laboratories in Japan has led to the hypothesis that the ureides, allantoin and allantoic acid, are important in the translocation of N in nodulated soybean plants. Large quantities of allantoin and allantoic acid were accumulated in the soluble N fraction of nodulated soybean plants, whereas very little ureide-N was accumulated in a nonnodulating soybean variety (13). It was also demonstrated (15) MATERIALS AND METHODSPLANT CULTURE Experiment 1. Soybean seeds (Glycine max [L.] Merr., "Ransom") were germinated in paper towels saturated with 0.5 mM CaS04 in a chamber maintained at 30 C and 90%Yo RH. Three days after imbibition (May 20, 1977) seedlings (three per pot) were transpla...

The use of the relative ureide content of xylem sap l(ureide-N/total N) x 1001 as an indicator of N2 fixation in soybeans (Merr.) was examined under greenhouse conditions. Acetylene treatments to inhibit N2 fixation were imposed upon the root systems of plants totally dependent upon N2 fixation as their source of N and of plants dependent upon both N2 fixation and uptake of exogenous nitrate. Significant decreases in the total N concentration of xylem sap from plants of the former type were observed, but no significant decrease was observed in the total N concentration of sap from the latter type of plants. In both types of plants, acetylene treatment caused significant decreases in the relative ureide content of xylem sap. The results provided further support for a link between the presence of ureides in the xylem and the occurrence of N2 fixation in soybeans. The relative ureide content of xylem sap from plants totally dependent upon N2 fixation was shown to be insensitive to changes in the exudation rate and total N concentration of xylem sap brought about by diurnal changes in environmental factors. There was little evidence of soybean cultivars or nodulating strains affecting the relative ureide content of xylem sap. 'Ransom' soybeans nodulated with Rhizobium japonicum strain USDA 110 were grown under conditions to obtain plants exhibiting a wide range of dependency upon N2 fixation. The relative ureide content of xylem sap was shown to indicate reliably the N2 fixation of these plants during vegetative growth using a 1"N method to measure N2 fixation activity. The use of the relative ureide content of xylem sap for quantification of N2 fixation in soybeans should be evaluated further.Soybean plants acquire N from their environment by the uptake of nitrogenous compounds from the soil solution and by the symbiotic fixation of atmospheric N2 within their root nodules.Although several methods have been developed to estimate the contribution made by N2 fixation to the total N accumulation of soybean and other leguminous plants, a need still exists for a rapid, inexpensive, and quantitative method that will separate the contributions made by the uptake of soil and fertilizer N and the fixation of N2 in field-grown soybeans. The C2H2 reduction assay (6, 10, 22) provides a sensitive, relatively inexpensive, and simple method for measuring instantaneous nitrogenase activity. However, application of this method to the quantitative measurement of seasonal profiles of N2 fixation in field situations has a number of shortcomings. The procedure underestimates N2 fixation due to the negative effect of root excision on C2H2 reduction (7, 18) and the difficulty in extracting all nodules from the soil, especially at later developmental stages when nodules may exist on lateral roots at some distance from the root crown. Overestimations arise when roots are extracted from the soil due to decreased resistance to gaseous diffusion (7) and when legume root nodules which evolve H2 under ambient conditions are assayed (23)...

The electrical response of nitrate-grown maize (Zea mays L.) roots to 0.1 millimolar nitrate was comprised of two sequential parts: a rapid and transient depolarization of the membrane potential, followed by a slower, net hyperpolarization to a value more negative than the original resting potential. The magnitude of the response was smaller in roots of seedlings grown in the absence of nitrate, but, within 3 hours of initial exposure to 0.1 millimolar nitrate, increased to that of nitrate-grown roots. Chloride elicited a separate electrical response with a pattern similar to that of the nitrate response. However, the results presented in this study strongly indicate that the electrical response to nitrate reflects the activity of a nitrate-inducible membrane transport system for nitrate which is distinct from that for chloride. Inhibitors of the plasmalemma H+-ATPase (vanadate, diethylstilbestrol) completely inhibited both parts of the electrical response to nitrate, as did alkaline extemal pH. The magnitude of the initial nitrate-dependent, membrane potential depolarization was independent of nitrate concentration, but the subsequent nitratedependent hyperpolarization showed saturable dependence with an apparent Km of 0.05 millimolar. These results support a model for nitrate uptake in maize roots which includes a depolarizing NO3-/H symport. The model proposes that the nitrate-dependent membrane potential hyperpolarization is due to the plasma membrane proton pump, which is secondarily stimulated by the operation of the NO3-/H+ symport.The absorption of nitrate by roots of higher plants is generally thought to be thermodynamically active and to require a significant input of energy (2, .11). The mechanism of absorption, however, is a matter of controversy.Because root nitrate absorption often leads to an alkalinization of the external solution, a popular early hypothesis was that an OH-/NO3-or HCO3-/NO3-exchange mechanism mediated the process (12). More recently, however, Ullrich, Novacky and coworkers (20,25,26) became less negative). The transient depolarization was followed by a gradual repolarization of the membrane potential. In nitrogen-starved plants, the degree of depolarization was enhanced by nitrate pretreatment, in correlation with observations of nitrate-induced acceleration of nitrate uptake. These authors explained these results by the operation of a nitrate-inducible, NO3-/H' symport mechanism (H+:NO3-stoichiometry > 1), in which active nitrate influx was coupled to passive influx of protons across the plasma membrane. The subsequent repolarization was proposed to be due to a stimulation of the H+-translocating, plasma membrane ATPase caused by changes in either cytoplasmic pH or the membrane potential itself.Results from other studies of nitrate uptake by plants, however, have led to alternative proposals. In studies of nitrate-starved and nitrate-induced excised maize roots, Thibaud and Grignon (24) reported that nitrate-starved (noninduced) roots excreted protons in the presence of...

We report here on an investigation of net nitrate and proton fluxes in root cells of maize (Zea mays L.) seedlings grown without (noninduced) and with (induced) 0.1 millimolar nitrate. A microelectrode system described previously (IA Newman, LV Kochian, MA Grusak, WJ Lucas [1987] by a slower, net hyperpolarization ofthe membrane potential. The electrical response was nitrate-inducible and influenced by ambient pH and nitrate concentration.Previous observations of nitrate-dependent membranepotential depolarizations in Lemna fronds (17, 27) and tissues of other higher plant species (26) have been offered to support the hypothesis that nitrate transport across the plasma membrane proceeds via a NO3-/H' symport mechanism. Our observations of nitrate-dependent depolarizations of the membrane potential in maize roots suggest that such a mechanism also operates in this species.The current research utilized a microelectrode method described previously (7, 15) to measure electrochemical potential gradients for NO3-and H+ within the unstirred layer at the root surface with ion-selective microelectrodes. Net NO3-and H+ fluxes were then calculated based on the measurements of ionic gradients. The objectives of the work described herein were: (a) to characterize the nitrate-inducibility, pH dependence and concentration dependence of net nitrate uptake in maize roots using nitrate-selective microelectrodes; (b) to determine if changes in the electrical response to nitrate are correlated with changes in net nitrate uptake; and (c) to examine the influence of external nitrate on the net flux of protons in maize roots. MATERIALS AND METHODS Plant MaterialsZea mays L. seeds (3377 Pioneer) were germinated and the seedlings were grown as described in the accompanying paper (14). Measurement of Net Nitrate and Proton FluxesIn a companion study (14), we have characterized the electrical response of maize root cells to nitrate. Nitrate elicited a rapid, transient depolarization of the membrane potential in nitrate-induced roots.

Eighty-five maize lines were evaluated in a primary screen for differences in uptake rate at 1 mM KNO 3 ; 15 were statistically separable into either "high" or "low" populations. Kinetic parameters were determined by iterative, least-squares regression using a model combining both a saturable and a linear component. Using the inbred B73 as an arbitrary standard, it was determined that the saturable component had a Km of 224 μM and a Vmax of 0.107 μmolNO 3 /mg root dry weight/hr. The ,linear component had a first order rate constant of 6.1 × 10 -2 μmol NO 3 /mg root dry weight-hr/mM. These values compared favorably with other published determinations of nitrate uptake kinetic parameters for saturable systems. No other constants have been reported for a linear component of nitrate uptake in higher plants.