W. A. Jackson

A mechanism for nitrate transport and reduction

Abstract It is proposed that a tetrahedron-shaped, transmembrane nitrate reductase tetramer functions as a carrier for nitrate transport. Reduction and transport are thereby brought about by the same enzyme complex. An ATPase is visualized to be closely associated with the nitrate reductase tetramer. The tetramer is apparently oriented such that one monomer is exposed to the outside of the plasmalemma while the other three are exposed to the cytoplasmic side. This orientation yields a reaction mechanism where the transport and reduction of one nitrate ion is accompanied by the transport of two additional nitrate ions (i.e. a 3 : 1 transport-reduction ratio). The proportion of transported nitrate that is reduced is apparently modulated by thiol reversible ADP inhibition of reduction. This inhibition, however, is probably the result of adenylate binding at sites on the proposed nitrate-activated ATPase to which nitrate reductase is tightly coupled. An analogous system consisting of a nitrate reductase dimer that spans a unit membrane plus an ATPase is proposed to be responsible for nitrate transport and reduction in some algae and chloroplasts.

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.

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.

The influence of ammonium on nitrate reduction in wheat seedlings

SummaryAmmonium markedly inhibited nitrate absorption by nitrogenstarved wheat seedlings but did not decrease the proportion of absorbed nitrate that was reduced. Seedlings high in nitrate (absorbed prior to the experimental periods) reduced similar amounts of this nitrate regardless of whether or not ammonium was present and being absorbed during the period of measurement. Ammonium or products of ammonium assimilation did not interfere with the induction, stability, or activity of nitrate reductase. Consequently, the hypothesis that ammonium depresses nitrate uptake indirectly by inhibiting nitrate reduction is rejected, and it is suggested that the ammonium effect is directly on the nitrateuptake process.

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 reduction in the roots and shoots of wheat seedlings.

SummaryIntact wheat seedlings cultured in high nitrate solutions (high-NO3-cultures) reduced NO3-when placed for 24 hr in dilute CaSO4 solutions although they leaked NO3-back to solution during this period. Most of the reduction seemed to occur in shoots since in parallel experiments detached shoots reduced nearly as much of the previously absorbed NO3-as intact cultures. Detached roots leaked greater quantities of NO3-back to solution than did intact cultures, and failed to reduce any of their previously absorbed NO3-during the experimental period. Seedlings of identical age cultured without a nitrogen source and rich in carbohydrate reserves (low-N cultures) rapidly absorbed NO3-from dilute Ca(NO3)2 solutions and reduced 80% of that absorbed. Detached low-N roots also absorbed NO3-and reduced 40% of that absorbed. Total NO3-reduction by intact low-N cultures over the 24-hr period was comparable to that of high-NO3-cultures in spite of the fact that in vitro nitrate reductase activity of the former did not reach the levels found initially or at the end of the period in the latter, and that the total NO3-absorbed by low-N cultures was less than that initially present in high-NO3-cultures.

Inhibition of nitrate uptake by aluminium in maize

Experiments with two maize (Zea mays L.) hybrids were conducted to determine (a) if the inhibition of nitrate uptake by aluminium involved a restriction in the induction (synthesis/assemblage) of nitrate transporters, and (b) if the magnitude of the inhibition was affected by the concurrent presence of ambient ammonium. At pH 4.5, the rate of nitrate uptake from 240 μM NH4NO3 was maximally inhibited by 100 μM aluminium, but there was little measurable effect on the rate of ammonium uptake. Presence of ambient aluminium did not eliminate the characteristic induction pattern of nitrate uptake upon first exposure of nitrogen-depleted seedlings to that ion. Removal of ambient aluminium after six hours of induction resulted in recovery within 30 minutes to rates of nitrate uptake that were similar to those of plants induced in absence of aluminium. Addition of aluminium to plants that had been induced in absence of aluminium rapidly restricted the rate of nitrate uptake to the level of plants that had been induced in the presence of aluminium. The data are interpreted as indicating that aluminium inhibited the activity of nitrate transporters to a greater extent than the induction of those transporters. When aluminium was added at initiation of induction, the effect of ambient ammonium on development of the inhibition by aluminium differed between the two hybrids. The responses indicate a complex interaction between the aluminium and ammonium components of high acidity soils in their influence on nitrate uptake. ei]{gnA C}{fnBorstlap}

Aluminum inhibition of shoot lateral branches ofGlycine max and reversal by exogenous cytokinin

Aluminum effects on the morphological development of soybean (Glycine max (L.) Merr.) were characterized in greenhouse and growth chamber experiments. An Al-sensitive cultivar, ‘Ransom’, was grown in an acid soil (Aeric Paleudult) adjusted to 3 levels of exchangeable Al. Lateral shoot development at the nodes of the main stem was extensive in the limed soil containing 0.06 cmol(+) Alkg−1. However, lateral shoot length and weight were severely inhibited in the unlimed soil containing 2.19 cmol(+) Alkg−1, and in the unlimed soil amended to 2.63 cmol(+) Alkg−1 with AlCl3. This inhibition by the high Al/low pH condition was reversed by the exogenous application of a synthetic cytokinin 6-benzylaminopurine (BA). The daily application of 20 μg mL−1 BA applied locally to the lateral meristems of plants grown in the unlimed soil stimulated lateral shoot growth substantially, such that it was either comparable to or greater than that observed in the limed treatment without BA. Accumulation of K, Ca, and Mg in lateral shoot branches was also stimulated by the local application of BA. The inhibitory effects of Al on lateral shoot development were confirmed in solution culture. In addition, differential sensitivity to Al was evident among the primary root, first order lateral roots, and second order lateral roots. The length of the primary root was only slightly decreased by increasing concentrations of Al up to 30 μM. In contrast, the length of basipetally located first order lateral roots was restricted to greater extent; up to 50% by 30 μM Al. Second order lateral lengths were inhibited even more severely; up to 86% by 30 μM Al. Substantial evidence in the literature indicates that the root apex is a major site for the biosynthesis of cytokinin that is supplied to shoots, and cellular function and development in this region of the root are impaired during Al toxic conditions. This suggests that one mode of action by which Al may affect shoot growth is by inhibiting the synthesis and subsequent translocation of cytokinin to the meristematic regions of the shoot. The present observation of a reversal of Al-inhibited lateral shoot development by exogenously applied cytokinin supports this hypothesis. However, the inability of applied cytokinin to counter the restriction imposed by Al on total shoot dry matter production implies the impairment by Al toxicity of other root functions, such as ion and water transport, also played an important role in altering shoot morphology.

Growth and Nitrogen Assimilation of Soybeans in Response to Ammonium and Nitrate Nutrition

Plants supplied with moderate concentrations of NH4 + in solution generally grow poorly compared with plants supplied with other sources of nitrogen. Experiments were conducted with a flowing solution culture system to determine whether growth restrictions could be avoided over an extended period in the presence of NH4 + if root-zone pH were strictly controlled and if plants were exposed to NH4 + during exponential growth when carbohydrate fluxes to the root are coordinated with the rate of nitrogen acquisition. Vegetative soybeans (Glycine max [L.] Merrill) initially were exposed to complete nutrient solutions containing NO3 - until the exponential growth stage and then were exposed for 4 wk to solutions in which nitrogen was supplied as either 1.0 mM NH4 +, 1 0 mM NO3 -, or 0.5 mM NH4 + plus 0.5 mM NO3 -. Acidity of the solutions was constantly maintained at pH 5 8 ± 0 1 by automated control In separate experiments, irradiance (photosynthetic photon flux density [PPFD] of 700 and 325 μE m-2 s-1) levels were controlled to produce distinct steady-state rates of leaf, root, and whole-plant growth The source of nitrogen did not alter growth or nitrogen accumulation within either environment Growth of whole plants and plant parts and accumulation of nitrogen remained exponential The results support the conclusion that plants can effectively utilize NH4 + as a nitrogen source as long as root-zone pH is strictly controlled and a balance is maintained between carbohydrate availability and acquisition of NH4 +

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.