Richard J. Volk

Nitrate influx and efflux by intact wheat seedlings: Effects of prior nitrate nutrition.

SummaryWheat (Triticum vulgare L., cv. Blueboy) seedlings, grown with 0.25, 1.0 and 15 mM nitrate in complete nutrient solutions, were transferred 10 days after germination to 1.0 mM K15NO3 (∼99 A% 15N) plus 0.1 mM CaSO4 at pH 6.0. The solutions were replaced periodically over a 6-h period (5 mW cm-2; 23°). Changes in the [15N]- and [14N]nitrate in the solution were determined by nitrate reductase and mass-spectrometric procedures and potassium by flame photometry. Influx of [15N]nitrate was depressed in plants grown at 1.0 mM nitrate relative to those grown at 0.25 mM, but there was no appreciably difference in [14N]nitrate efflux. Prior growth at 15 mM further restricted [15N]nitrate influx which, together with a substantial increase in [14N]nitrate efflux, resulted in no net nitrate uptake during the course of the experiment. Efflux of [14N]nitrate occurred to solutions containing no nitrate but it was significantly enhanced upon exposure to [15N]nitrate in the external solution. Influx of [15N]nitrate was more restricted at 5°, relative to 23°, than was [14N]nitrate efflux. The nitrate concentrations of the root tissue immediately before exposure to the K15NO3 solutions did not give a precise indication of the subsequent [15N]nitrate influx rates nor of the [14N]nitrate efflux rates. Net K+ uptake was related to the magnitude of the net nitrate uptake, not to the initial K+ concentration in the roots. The data are interpreted as indicating that [15N]nitrate influx and [14N]nitrate efflux are largely independent processes, subject to different controls, and that net nitrate uptake provides the driving force for net potassium uptake.

Nitrogen-15 Dioxide Uptake and Incorporation by Phaseolus vulgaris (L.)

The sorption rate and metabolic fate of nitrogen dioxide, a major air pollutant, have been determined for Phaseolus vulgaris (L.). Sorption was determined kinetically by chemiluminescent monitoring of 15NO2 removal from the test atmosphere and directly by mass spectrometric analysis of nitrogen derived from the plant tissue. Sorptive processes were first order with respect to 15NO2 concentration. Virtually all of the 15NO2 taken up was metabolized.

Daily changes in nitrate influx, efflux and metabolism in maize and pearl millet

Maize (Zea mays L.) and pearl millet (Pennisetum americanum (L.) Leeke) seedlings were exposed to [15N]nitrate for 1-h periods at eight times during a 24-h period (16–8 h light-dark for maize; 14–10 h for millet). Influx of [15N]nitrate as well as its reduction and translocation were determined during each period. The efflux of previously absorbed [14N]nitrate to the uptake solution was also estimated. No marked diurnal changes in [14N]nitrate efflux or [15N]nitrate influx were evident in maize. In contrast, [14N]nitrate efflux from millet increased and eventually exceeded [15N]nitrate influx during the late dark and early light periods, resulting in net nitrate efflux from the roots. The dissimilarity of their diurnal patterns indicates that influx and efflux are independently regulated. In both species, [15N]nitrate reduction and 15N translocation to shoots were curtailed more by darkness than was [15N]nitrate influx. In the light, maize reduced 15% and millet 24% of the incoming [15N]nitrate. In darkness, reduction dropped to 11 and 17%, respectively. Since the accumulation of reduced-15N in shoots declined abruptly in darkness, whereas that in roots was little affected, it is suggested that in darkness [15N]nitrate reduction occurred primarily in roots. The decrease in nitrate uptake and reduction in darkness was not related to efflux, which remained constant in maize and did not respond immediately to darkness in pearl millet.

Simultaneous influx of ammonium and potassium into maize roots: kinetics and interactions

The interaction between ammonium and potassium during influx was examined in roots of dark-grown decapitated corn seedlings (Zea mays L., cv. Pioneer 3369A). Influx was measured during a 10-min exposure to either (15NH4)2SO4 ranging from 10 to 200 μM NH4+with and without 200 μM K(86Rb)Cl or to K(86Rb)Cl ranging from 10 to 200 μM K+ with and without 200 μM NH4+as (15NH4)2SO4. The simple Michaelis-Menten model described the data well only for potassium influx in the presence of ambient ammonium. For the other three instances, the data were improved by assuming that a second influx mechanism became operative as the low-concentration phase approached saturation. Two distinct mechanisms are thus indicated for both ammonium and potassium influx within the range of 10 to 200 μM.The influx mechanism operating at low concentrations showed greater affinity for potassium than for ammonium, even though the capacity for ammonium transport was twice as large as that for potassium. It is suggested that this phase involved a common transport system for the two ions and that localized low acidity next to the internal surface, following H+ extrusion, favored ammonium deprotonation and dissociation from the transport system-ammonium complex. Parallel decreases in Vmax and increases in Km of the low-concentration saturable phase occurred for ammonium influx when ambient potassium was present and for potassium influx when ambient ammonium was present. The data support a mixed-type inhibition in each case. Simultaneous measurement of potassium and ammonium influx showed that they were highly negatively correlated at the lower concentrations, indicating that the extent to which influx of the inhibited ion was restricted was associated with influx of the inhibitor ion. Presence of ambient ammonium eliminated the second phase of potassium influx. In contrast, the presence of ambient potassium decreased the concentration at which the second phase of ammonium influx was initiated but did not restrict the rate.

Nitrate assimilation by decapitated corn root systems: effects of ammonium during induction☆

Abstract Development of the accelerated phase of nitrate uptake by darkgrown decapitated corn seedlings upon first exposure to nitrate (1.0 mM, pH 6, 30°C) was restricted by the presence of ambient ammonium (0.5 mM (NH 4 ) 2 SO 4 ). Upon transfer to ammonium-free solutions, the increase in the nitrate uptake rate paralleled the increase in seedlings not exposed to ammonium, but did not, within 6 h, completely recover to yield the rate occuring in the absence of ammonium throughout. In contrast, the restriction in potassium uptake was completely eliminated within 2 h following transfer. Presence of ammonium decreased reduction of [ 15 N]nitrate and in vitro nitrate reductase activity (NRA) of the root tissue, but the decrease in [ 15 N]nitrate reduction was not sufficient to account for the decrease in uptake. The data are interpreted as indicating that ammonium exerted a detrimental effect on the formation of the nitrate uptake system.

Partitioning of previously-accumulated nitrate to translocation, reduction, and efflux in corn roots.

The effect of nitrate uptake, or its absence, on the utilization of nitrate previously accumulated by dark-grown, decpitated maize (Zea mays L., cv. DeKalb XL-45) seedlings was examined. Five-d-old plants that had been pretreated with 50 mM 14NO3−for 20 h were exposed for 8 h to nutrient solutions containing either no nitrate or 50 mM 15NO3−, 98.7 atom % 15N. The ambient solution, xylem exudate, and plant tissue were analyzed to determine the quantities of previously-accumulated (endogenous) 14NO3−that were translocated to the xylem, lost to the solution, or reduced within the tissue during the 8-h period. Energy was continuously available to the roots from the attached endosperm. In the absence of incoming nitrate, appreciable reduction and translocation of the endogenous 14NO3−occurred, but efflux of 14NO3−to the external solution was minimal. In contrast, during 15NO3−uptake, there was considerable efflux of 14NO3−as well as translocation of 14NO3−to the xylem, but little 14NO3−was reduced. Thus there appeared to be an inverse relationship between 14NO3−efflux and reduction. The data are tentatively interpreted on the basis of a model which envisages (a) two storage locations within roots, one of which primarily supplies nitrate for translocation and the other of which primarily supplies nitrate for outward passage through plasmalemma, and (b) the majority of nitrate reduction as occurring during or immediately following influx across the plasmalemma, with endogenous 14NO3−initially moving outward being recycled inward and thereby being reduced.

Uptake of nitrogen by flue-cured tobacco during maturation and senescence

A field experiment with flue-cured tobacco,Nicotiana tabacum L., was conducted to estimate the uptake and partitioning of nitrogen during maturation and senescence. On the 83rd day after transplanting (crop day 83), nitrate which had been leached from the plow layer was replaced with an equivalent amount of15N-labeled nitrate. Plants were harvested at crop day 83, 90, 96, 106, 113, and 127, and each of 11 plant parts was analyzed for nitrogen derived from the soil (NDS) and from the applied15N-labeled fertilizer (NDF). Equivalent quantities of NDF and NDS were taken up during the initial week after15N-fertilizer application; in the subsequent 5 weeks, ten times more NDS than NDF were taken up. It appears likely that the leached nitrate (NDS) accumulated below the hard pan where it became available to plants as their roots penetrated this layer via fractures originating from prior deep chiseling. Of the NDF taken up during the initial week, 20% was partitioned to the root and 42%, 24%, 14% respectively, to the upper, middle and bottom node positions (leaves plus stems). The partitioning reflected the respective growth rates of the tissues. Little change in partitioning was evident during the subsequent 5-week period, indicating that little remobilization of NDF from older to younger tissues occurred. In contrast, some remobilization of NDS was apparent between crop day 96 and 106 when the uptake of both NDF and NDS was negligible. During this period root growth was sustained by the apparent transfer of NDS from the root stump and from the adjacent lower leaf and stem tissues. These responses occurred in tobacco grown under higher nitrogen fertility levels than those usually considered optimal for the growth of flue-cured tobacco, but under conditions which are sometimes encountered.

Unidirectional fluxes of nitrate into and out of maize roots: measurement and regulation by prior nitrate nutrition

Abstract A method for rapid assay of concurrent fluxes of nitrate into and out of intact plant roots has been developed. Maize ( Zea mays L.) seedlings were grown on ‘normal’ 14 N-nitrate prior to exposure for 5 min to 99 at.% 15 N-nitrate. Influx and efflux rates were calculated from the depletion of nitrate and its 15 N enrichment in the external solution. Once developed, the assay was used to examine the regulation of nitrate fluxes by prior nitrate nutrition. Maize seedlings were pretreated for 24 h with 0, 20 or 200 μM 14 N-nitrate, providing root nitrate concentrations of 5.0, 8.2 and 30.7 μmol/g fw, respectively. Nitrate influx rates increased 55% when the root nitrate concentration rose from 5.0 to 8.2 μmol/g fw, but remained constant when root nitrate increased to 30.7 μmol/g fw. By contrast, efflux of nitrate continued to accelerate as root nitrate concentrations increased. As a consequence of these differential responses, net nitrate uptake was 26–30% higher at a root nitrate concentration of 8.2 μmol/g fw than at the lower and higher concentrations.

Concentration-dependence of the nitrate assimilation pathway in maize roots

Abstract The effect of nitrate concentration on uptake and assimilation of nitrate by dark-grown, decapitated maize ( Zec mays L.) seedlings was examined. During the 12-h exposure period, twice as much nitrate was taken up from 20 mM as from 0.05 mM K 15 NO 3 (98.7 atom% 15 N). The higher concentration had little effect on nitrate reduction, but nitrate translocation was more than doubled. Thus, partitioning to translocation accounted for 80% of the increased uptake which occured from 20 mM nitrate. At both concentrations a relatively constant percentage, 22–27%, of the reduced- 15 N was incorporated into protein and other insoluble nitrogenous compounds. Concurrently, translocation of soluble reduced- 15 N increased from less than 10% of the total reduced- 15 N at 4 h to more than 50% at 12 h. Neither the translocation of reduced- 15 N nor its incorporation into protein was affected appreciably by the 400-fold difference in exogenous nitrate concentration. The results may be interpreted in one of two ways. At 0.05 mM, uptake occurred mainly via ‘mechanism 1’ of ion uptake, whereas at 20 mM, ‘mechanism 2’ also contributed: nitrate reduction was predominantly associated with uptake via the former mechanism and nitrate translocation with the latter. Alternatively, it is possible that at the low nitrate concentration uptake occurred primarily via root cells which contained nitrate reductase (NR), whereas at the higher concentration, uptake occurred in addition via cells which were dificient in NR and from which translocation of unreduced nitrate was thus more likely.

Alterations in enrichment of NO3− and reduced-N in xylem exudate during and after extended plant exposure to 15NO3−

Distinctly different patterns of 15N enrichment were observed in the nitrate and reduced-N fractions of xylem exudate from soybean plants during and after 5 to 6 days of exposure to 15NO 3 − . Within 1 d after changes in solution NO 3 − label, more than 90% of the exudate NO 3 − originated from the exogenous supply. Alterations in the enrichment of exudate reduced-N were much slower, however, and the enrichment reached only 40% even after 5 d of continuous exposure to 15NO 3 − . Taking into account possible reduction of endogenous NO 3 − and delayed translocation of NO 3 − reduction products, it was concluded that root reduction could have contributed only 30 to 42% of the reduced-N found in the exudate.