Endogenous Ammonium Generation in Maize Roots and its Relationship to other Ammonium Fluxes

An investigation to determine the magnitude of the back reactionswhich occur during net ammonium uptake by roots and during netammonium assimilation within roots was undertaken with maize(Zea mays L.). Ten-day-old seedlings, which had been grown on250 mmol m^–3 ammonium at pH 4 or 6, were pretreated for3 h in the absence or presence of 500 mmol m ^–3 MSX (methionine-DL-sulphoximine),an inhibitor of the glutamine synthetase-catalysed pathway ofammonium assimilation. They were then exposed for 2 h to 99A% ¹⁵N-ammonium ± MSX. Substantial ammonium cycling occurredduring net ammonium uptake. Efflux was enhanced by MSX treatment,reflecting a 2- to 3-fold accumulation of ammonium in the roottissue. Influx of ammonium was also increased by treatment withMSX, indicating that influx was enhanced when products of ammoniumassimilation were dissipated. The decline in root ¹⁴N-ammoniumaccounted for only a small fraction of the ¹⁴N-ammonium recoveredin the ambient ¹⁵N-ammonium solution, revealing a substantialgeneration of endogenous ¹⁴N-ammonium during the 2 h exposure.The net quantity of ammonium generated was increased appreciablywhen assimilation of ammonium was restricted by MSX and it wasestimated to occur at least 50% faster than net ammonium uptake.Presence of MSX severely decreased translocation of ¹⁵N to shootsbut had a smaller influence on incorporation of ¹⁵N into macromoleculesof the root tissue. The various ammonium flux rates were notgreatly affected by growth at pH 4.0, implying a considerableresistance of ammonium assimilation processes in these maizeroots to the high ambient acidity commonly induced by exposureto ammonium Key words: Ammonium generation, uptake, assimilation     

Inward and Outward Movement of Ammonium in Root Systems: Transient Responses during Recovery from Nitrogen Deprivation in Presence of Ammonium

Morgan, M. A. and Jackson, W. A. 1988. Inward and outward movementof ammonium in root systems: transient responses during recoveryfrom nitrogen deprivation in presence of ammonium.— J.exp. Bot. 39: 179-191. Net ammonium uptake by 20-d-old wheat (Triticum aestivum cv.Kleiber) and oat (Avena sativa cv. Tarok) seedlings was increased5- to 10-fold when the seedlings were deprived of nitrate duringthe 14-20 d period. The effect of nitrogen deprivation was toincrease net ¹⁵N-ammonium influx and decrease net ¹⁴N-ammoniumefflux during a 1 h assay period. The sizeable rate of net ¹⁵N-ammoniuminflux resulting from nitrogen deprivation was stimulated furtherby prior exposure of the seedlings to ¹⁴N-ammonium for 5 h.Additional exposure to ¹⁴N-ammonium caused the stimulated rateof ¹⁵N-ammonium influx to decline. During the 1 h assays in¹⁵N-ammonium, net ¹⁴N-ammonium efflux increased after 2 h exposureto ¹⁴N-ammonium, peaked at 5–10 h, and then declined.The consequence of the differential response of the influx andefflux processes in wheat was a marked decrease in net ammoniumuptake in the initial 2–5 h, followed by a recovery which,in turn, was followed by a slow decline. In oat, there was norecovery in net ammonium uptake after 2–5 h. Interference in ammonium assimilation by presence of methioninesulphoximine after 5 h did not inhibit expression of the ammonium-stimulatednet ¹⁵N-ammonium influx at 10 h but did substantially increasenet ¹⁴N-ammonium efflux. In nitrogen depleted seedlings, andin those exposed to ¹⁴N-ammonium for 2 h, subsequent net ¹⁴N-ammoniumefflux during 1 h in ¹⁵N-ammonium exceeded the quantity of ¹⁴N-ammoniuminitially in the roots. The increase in ¹⁵N-ammonium influx upon nitrogen deprivation,its further stimulation with 5-10 h exposure to ammonium andits subsequent decline, are discussed as possibly resultingfrom (a) the operation of two ammonium influx systems (b) theinterplay of tissue ammonium and a product of its assimilationrespectively acting as positive and negative effectors of asingle influx system and (c) variations in energy supply fromthe shoots. Key words: Net ammonium uptake, stimulated ammonium influx, ammonium efflux, tissue ammonium     

Reciprocal Ammonium Transport Into and Out of Plant Roots: Modifications by Plant Nitrogen Status and Elevated Root Ammonium Concentration

Wheat and oat were grown for 20 d on a nitrate-containing solution(nitrogen-replete plants) or for the last 6 d of this periodon a nitrate-free solution (nitrogen-depleted plants). Exposureof the nitrogen-depleted plants on day 20 to nitrate-free solutionscontaining 500 mmol m^–3 ammonium (96 A% ¹⁵N) resultedin a cumulative net influx of ¹⁵N-ammonium over an 8 h periodthat was appreciably greater than that of the nitrogen-repleteplants. Both the initial rate and the more restricted rate afterthe first hour were enhanced by nitrogen deprivation. In thenitrogen-replete plants, cumulative net efflux of endogenous¹⁴N-ammonium was approximately equivalent to net ammonium uptakeduring the first hour, and was essentially complete after 1–2h. Pretreating nitrogen-depleted plants for 5 h in 500 mmolm^{–3 15}N-ammonium (99 A% ¹⁵N) resulted in root ammoniumconcentrations of 12.7?1.1 and 16.0?0.4 µmol for wheat and oat, respectively. Subsequent net efflux of ¹⁵N-ammoniumto 500 mmol m^–3 exogenous ¹⁴N-ammonium exceeded theseinitial amounts within 2 h. Increasing ambient ¹⁴N-ammoniumto 5000 mmol m^–3 increased net ¹⁵N-ammonium efflux suchthat net loss of the maximal original amount in the root tissuewas exceeded within 0.75 h. The data for both species indicatesubstantial reciprocal transfers of ammonium into and out ofroots of ammonium-treated plants and a significant degradationof recently synthesized products of ammonium assimilation concurrentwith ammonium assimilation. Key words: Accumulation, ammonium, efflux, oat, root, uptake, wheat     

Synthesis and 15N-labelling of glutamine and glutamate in maize roots during early stages of 15N-ammonium assimilation

The concentrations of glutamine and glutamate, and the abundanceof ¹⁵N in these compounds, were measured in roots of intact,nitrate-grown maize plants fed with ¹⁵N-nitrate or ¹⁵N-ammoniumfor periods of 3–80 min. On supplying ¹⁵N-ammonium therewas a rapid and almost linear accumulation of glutamine, itsconcentration in the roots rising 3-fold over 1 h. Supplying¹⁵N-nitrate instead of ¹⁵N-ammonium did not increase root glutamine,and the concentration of glutamate was not affected by eithertreatment. The time-course of amide ¹⁵N-labelling seen in glutamine extractedfrom roots which had been supplied with ¹⁵N-ammonium could bestbe explained by a model in which (a) the ‘additional’glutamine which accumulates rapidly during ¹⁵N-ammonium feedingis heavily amide-labelled from the outset, and (b) of the glutaminealready present in the roots, only a small proportion (c. 10–15%)incorporates ¹⁵N during the initial 60–80 min of ¹⁵N-ammoniumfeeding, the remainder (c. 85–90%) remaining essentiallyunlabelled over this period. The latter is assumed to be locatedin the vacuoles. Even though prior N-starvation stimulated ammonium net uptakemarkedly, the data were not of sufficient quality to show whetherthe relative sizes of the extra-vacuolar and vacuolar glutaminefractions were altered by this treatment. For that reason itwas not possible to determine whether cytosolic glutamine hasa role in regulating N-absorption. Key words: Subcellular compartmentation, regulation, N-absorption     

Nitrogen-stimulated potassium influx into maize roots: differential response of components resistant and sensitive to ambient ammonium1

Abstract Potassium (⁸⁶Rb) influx from 200 mmol m ^?3 KCl into dark grown, decapitated maize seedlings 6 d old) was stimulated by nitrate pretreatment. The stimulus was clearly evident by 6h exposure to nitrate and required 12–24 h for maximal expression. Decay of the nitrate-stimulated potassium influx was more than 50% complete within 3 h after transfer to nitrogen-free solutions. The stimulation of potassium influx was entirely accounted for by an increase in the influx component that was resistant to inhibition by presence of 200 mmol m^?3 ambient ammonium. In contrast, the component of potassium influx that was sensitive to inhibition by ambient ammonium was unaffected by nitrate pretreatment. Exposure to the glutamine synthetase inhibitor L-methionine-dl-sulphoximine (MSX) during nitrate pretreatment stimulated the resistant component but the sensitive component was nearly eliminated. Pretreatment with ammonium increased the resistant component of potassium influx within 3 h, i.e. before it was increased by nitrate pretreatment, but the sensitive component was concomitantly restricted. The latter recovered partially during extended pretreatment with ammonium. The data indicate that the resistant component responded positively to increases in tissue ammonium concentrations whereas the sensitive component was unaffected by tissue ammonium except at concentrations in excess of 10μmol g^?1. Ammonium influx was also stimulated by nitrate pretreatment and to a greater extent than potassium influx. Presence of MSX with nitrate during pretreatment resulted in a further stimulation in ammonium influx. The parallel increases in root ammonium concentrations with the two pretreatments imply that part of the increase in ammonium influx was a consequnce of increased counter-transport with endogenous ammonium.     

Synthetic and Maintenance Respiratory Losses of 14CO2 in Uniculm Barley and Maize

The respiratory losses of ¹⁴CO₂ from whole plants of uniculmbarley and maize were measured following exposure of the wholeplant or a single leaf to air containing a uniform specificactivity of ¹⁴CO₂ for 30–60 min during normal photosynthesis.The total respiratory efflux of ¹⁴CO₂ could be described interms of two main components: an intense efflux characterizedby a half-life of 4–8 h, which was identified with thebiosynthesis of new tissue in meristems; and a much less intenseefflux characterized by a half-life of 26–120 h, whichwas primarily identified with the maintenance of metabolic activity.The (bio)synthetic efflux of ¹⁴CO₂ totalled between 25 and 35per cent of the labelled assimilate (¹⁴C: ¹⁴C), and was generallyinsensitive to temperature and light intensity. The maintenanceefflux totalled between 12 and 27 per cent of the labelled assimilateor its derivatives: the total generally increased with hightemperature and low light intensity. The rate of the maintenanceefflux showed a normal temperature response (Q102). It is concludedthat the efficiency of conversion of assimilate into new growthis unlikely to exceed 65 per cent in the long term, and willgenerally be less.     

Depression of Nitrate and Ammonium Transport in Barley Plants with Diminished Sulphate Status. Evidence of Co-regulation of Nitrogen and Sulphate Intake

When barley plants were grown in a solution with nitrate asthe sole N-source but deprived of sulphate (–Splants)for 1 to 5 d, the capacity for sulphate transport by the rootsincreased very markedly; subsequent measurement of influx using³⁵S-labelled showed increases of > 10-fold compared to plants continuously supplied with sulphate (+S plants).There were only small effects on plant growth over a 5 d periodand yet the influx of , labelled with the short-lived tracer ¹³N, was diminished by approximately 30%.By contrast, the influx of phosphate was little affected bysulphate-deprivation. When a sulphate supply was restored to– S plants, the sulphate influx was quickly repressedover the subsequent 24 h and the nitrate influx was restoredto >90% of the value in +S plants. When plants were grown in a solution with a mixed nitrate andammonium supply and deprived of sulphate for 1 d or 5 d thedepression of nitrate influx was more strongly marked (up to55% depression). The influx of ammonium was also depressed after5 d of sulphate-deprivation, but not at 1 d, nor where the concentrationof ammonium in the uptake solution was lowered to 20 mmol m^–3or less. Additional measurements with ¹⁵N-labelled nitrate and ammoniumover longer periods were used to determine net uptake. Net uptakeof nitrate was depressed to a similar extent to efflux, butnet ammonium uptake was depressed only in unbuffered uptakesolution where the pH decreased to pH 4.9 during the uptakeperiod. The ¹⁵N-tracer experiments showed that the translocationof label to the shoot, from both nitrate and ammonium, was depressedto a greater extent than net uptake in –S plants. Thedepression of nitrate influx, caused by 5 d of sulphate deprivation,could be relieved almost completely by providing plants with1.0 mol m^–3 L-methionine during the day prior to influxmeasurement. This treatment substantially decreased sulphateand potassium (⁸⁶Rb-labelled) influx in both +S and –Splants, but greatly increased total S-status of the plants.This methionine treatment had no effect on ammonium influx ornet uptake in – S plants but increased influx significantlyin +S ones. When plants were grown with sulphate but deprived of nitratefor 4 d there was a marked depression of the sulphate influx(by 48–65%) but a smaller effect on phosphate influx (21–37%of +N). The results are discussed in relation to the effects of sulphate-deprivationon growth rate and the root: shoot weight ratio. It is concludedthat the effects on influx and net uptake of nitrogen are moresevere than could be accounted for by these factors. The decreasedtranslocation of either nitrate, or the products of nitrateand ammonium assimilation from the roots, is suggested as areason for the depression of influx. The restoration of nitrateinflux and net uptake by methionine suggests that, for thision at least, a shortage of S-amino acids within the plant maylead to the accumulation of inhibitory concentrations of non-Samino acids in the transport pool. Key words: ¹³N, sulphate, nitrate, ammonium, ion-uptake, barley     

A field study on the fate of 15N-ammonium to demonstrate nitrification of atmospheric ammonium in an acid forest soil

To demonstrate the contribution of atmospheric ammonium to soil acidification in acid forest soils, a field study with¹³N-ammonium as tracer was performed in an oak-birch forest soil. Monitoring and analysis of soil solutions from various depths on the¹³N-ammonium and¹⁵N-nitrate contents, showed that about 54% of the applied¹⁵N-ammonium was oxidized to nitrate in the forest floor. Over a period of one year about 20% of the¹⁵N remained as organic nitrogen in this layer. The percentage¹⁵N enrichment in ammonium and nitrate were in the same range in all the forest floor percolates, indicating that even in extremely acid forest soils (pH < 4) nitrate formation from ammonium can occur. Clearly, atmospheric ammonium can contribute to soil acidification even at low soil pH.     

Rapid, Reversible Inhibition of Nitrate Influx in Barley by Ammonium

The rate of influx of nitrate into the roots of intact barleyplants was measured over a period of 3–5 min from externalnitrate concentrations of 1–150 mmol m^–3, using¹³N-labelled nitrate as tracer. Ammonium at external concentrationsof 0.005–50 mol m^–3 inhibited nitrate influx ina manner which did not conform to a simple kinetic model butincreased approximately as the logarithm of the ammonium concentration.At any particular ammonium concentration, inhibition of nitrateinflux reached its full extent within 3 min of the ammoniumbeing supplied and was not made more severe by up to 17 minpre-treatment with ammonium. On removing the external ammonium,nitrate influx returned to its original rate within about 3min. Potassium at 0.005–50 mol m^–3 did not reproducethe rapid effect of ammonium on nitrate influx. Net uptake of nitrate also decreased when ammonium was supplied,over a similar timescale and to a similar extent as nitrateinflux. The decrease in nitrate influx caused by ammonium wassufficient to account for the observed reduction in net uptake,without necessitating any acceleration of nitrate efflux. Key words: Hordeum vulgare, roots, ion transport, short-lived isotopes, ¹³N     

Na+ fluxes in Chara under salt stress

The influx and efflux of Na⁺ across the plasma membrane of Characorallina and Chara longifolia were examined under mild saltstress conditions. Na⁺ influx was found to be rapid in bothspecies with the freely exchangeable cytoplasmic Na⁺ cominginto isotopic equilibrium with external ²²Na⁺ within 1 h ofexposure to isotope. Cytoplasmlc Na⁺ concentration and Na⁺ influxwere greater in C. corallina than in C. longifolla under thesame conditions. Na⁺ influx across the tonoplast was much lowerthan the flux across the plasma membrane. Na⁺ efflux was stimulatedat pH 5 relative to pH 7 by 218% in C. coralllna and 320% inC. longifolia. In both species externally applied Li⁺ inhibitedNa⁺ efflux at pH 5 but not at pH 7. Na⁺ etflux was not significantlyinhibited by amiloride. Key words: Na⁺ influx, Na⁺ efflux, Na⁺/H⁺ antiport, Chara     

31P NMR Measurements of Cytoplasmic pH Changes in Maize Root Tips

³¹P nuclear magnetic resonance (NMR) spectroscopy has been usedto measure the changes caused by sodium azide and anaerobiosisin the cytoplasmic pH of maize (Zea mays L.) root tips. Measurementsof H⁺ influx, lactate accumulation and ATP hydrolysis, togetherwith estimates of the cytoplasmic volume and the cytoplasmicbuffering capacity, were used to calculate the expected pH changesunder the same conditions. In the case of azide inhibition,where there was a large H⁺ influx, there was good agreementbetween the calculated and measured pH changes. In contrastduring anoxia, where the accumulation of lactate was the mostimportant source of protons, the predicted cytoplasmic pH changewas greater than the observed change. It was concluded thatin the absence of a compensating H⁺ efflux, the excess H⁺ productionwas offset by the production of H⁺-consuming metabolites suchas -aminobutyrate. Key words: Cytoplasmic pH, ³¹P NMR, Membrane H⁺ fluxes.     

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.Paper No. 6722 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh     

Nitrogen-13 Studies of Nitrate Fluxes in Barley Roots: I. COMPARTMENTAL ANALYSIS FROM MEASUREMENTS OF 13N EFFLUX

The short-lived radio-isotope nitrogen-13 (half-life 10 min)was used as a tracer in studying fluxes of N in the roots ofintact barley plants. After supplying the plants with ¹³N-nitratefor 30 min, efflux of ¹³N into an unlabelled (wash) solutionwas followed under steady-state conditions for a further 10min. Tests with ion exchange resins suggested that all of the¹³N released during this period was in the form of nitrate. In addition to nitrate from a surface film of solution and fromthe free space of the roots, efflux from another compartmentwas detected, tentatively identified as the cytoplasmic nitratepool. In plants grown with nitrate as the only external N-source,efflux from this compartment decreased with a rate constantabout 0·17 min^–1 (half-time 4 min). Adding ammoniumsulphate to the wash solution alone did not significantly affecteither the initial rate, or the rate constant, of efflux of¹³N from these roots. However, ¹³N efflux decreased more rapidly(rate constant about 0·32 min^–1, half-time 2·2min) in roots grown in, and subsequently washed with, solutioncontaining ammonium nitrate. In barley plants grown with 1·5 mol m^–3 nitrate,the cytoplasmic nitrate pool was estimated to contain about2% of the total nitrate in the roots, corresponding to a cytoplasmicnitrate concentration 26 mol m^–3. Nitrate efflux was equivalentto almost 40% of nitrate influx in the roots of these plants. Key words: Ion transport, nitrate, ammonium, efflux analysis, compartmentation     

Evidence of ammonium assimilation via the glutamine synthetase-glutamate synthase system in rice seedling roots

When rice seedling roots were fed ¹⁵N-ammonium for 1 hr, theamide nitrogen of glutamine showed the highest ¹⁵N abundance.Moreover, glutamine amino, glutamic acid, aspartic acid andalanine showed higher ¹⁵N abundance than ammonium did. In roots whose GS activity was inhibited with MS, both the amountof ammonium and its ¹⁵N abundance were increased. In contrast,both the amount of all examined amino acids containing glutamicacid and their ¹⁵N abundance decreased in roots whose GS activitywas inhibited. From these results, it could be concluded thatthe first step of ammonium assimilation in rice seedling rootswas mainly glutamine synthesis by GS and the second was glutamicacid formation by the GOGAT system. The results of an experiment using ¹⁵N glutamine also supportedthis conclusion. (Received February 23, 1977; )     

The regulation of ammonia uptake in Chara australis

The regulation of ammonia uptake was investigated in internodalcells of the freshwater alga Chara australis. Ammonia uptakewas estimated by monitoring (i) its depletion from the bathingsolution, (ii) the uptake of radiolabelled methylamine, an analogueof ammonia, and (iii) depletion of ammonia in the unstirredlayer with the microelectrode ion-flux estimation technique(MIFE). Distribution of methylamine (¹⁴CH₃NH₃⁺) between thevacuole and cytoplasm was estimated with efflux analysis. Whencells were bathed continuously in solutions containing ammoniaor methylamine, the uptake rates of both amines decreased over12 to 48 h despite the continuing existence of a large electrochemicalgradient favouring influx of the NH⁺₄ and CH₃NH⁺₄ cations. Treatmentwith 1.0 to 10.0 mM MSX, an inhibitor of glutamine synthetase,caused the internal ammonia concentration to rise and reducedthe subsequent uptake of ammonia and methylamine by up to 70%within 2 h. These results suggest that the permease facilitatingNH⁺₄/CH₃NH⁺₄ influx is under feedback or kinetic regulationfrom either internal ammonia or an intermediate of nitrogenassimilation. Treatment with metabolic inhibitors (CCCP, azide and DCMU) andsome weak acids (DMO and butyric acid) for 30 to 60 min inhibitedmethylamine uptake, but the changes in the electrical potentialdifference across the plasma membrane could not account forthe magnitude of inhibition. The rate of cytopiasmic streaming,which is an indicator of the cellular ATP concentration in Chara,was inhibited by many of these treatments. However, under certainconditions of external pH and concentration, butyric acid couldreversibly inhibit ammonia and methylamine uptake without affectingcytoplasmic streaming, demonstrating that a decrease in cytoplasmicATP concentration was not responsible for the inhibition. Theeffect of butyric acid was rapid, causing a 60% inhibition ofuptake in 15 min. We conclude that weak acids can inhibit theNH⁺₄/CH₃NH⁺₄ permease by acidifying the cytoplasm and suggestthat this may also explain the effects of the metabolic inhibitorson ammonia and methylamine uptake. Key words: Ammonia, methylamine, uptake, regulation, Chara     

Respiration-Dependent H+ Efflux from Intact Cells of Cyanidium caldarium

An active H⁺ efflux depending on respiration was found in anacidophilic unicellular alga, Cyanidium caldarium. Alkalizationof the medium due to passive H⁺ transport into the cells wasobserved when the respiratory activity was inhibited by addingrespiratory poisons, such as rotenone or antimycin A, or byintroducing pure nitrogen into the cell suspension. The extentof the H⁺ influx increased as the pH of the medium was loweredto 2.9, indicating that H⁺ leaks into the cells according tothe pH gradient across the plasma membrane. The medium pH whichhad increased under anaerobic condition returned to the originallevel with aeration of the cell suspension. This suggests thatan active H⁺ transport, related to respiration, pumps out theexcess H⁺ accumulated in the cells during anaerobic preincubation.The pH changes in the cell suspension were related to the intracellularATP level. From these results it was concluded that active H⁺efflux dependent upon oxidative phosphorylation functions inthe dark to maintain a constant intracellular pH against passiveH⁺ leakage through the plasma membrane. The light-induced H⁺ efflux and the respiration-dependent H⁺efflux were also compared in relation to the physiological roleof the active H⁺ efflux, especially with respect to the intracellularpH regulation in this alga. ¹The data in this paper are included in the Ph. D. dissertationsubmitted by M. Kura-Hotta to Tokyo Metropolitan University. (Received February 3, 1984; Accepted June 14, 1984)     

Characterization of Photosynthetic 14Carbon Assimilation by Potamogeton lucens L.

Photosynthetic assimilation of exogenous ¹⁴CO₂ and H¹⁴CO₃^–by the aquatic angiosperm Potamogeton lucens L. is reported.Equivalent maximum rates of assimilation (1.5 µmol s^–1m^–2) were obtained in the presence of saturating levelsof ¹⁴CO₂ (1.0 mol m^–3, pH 5.3) or H¹⁴CO₃^– (1.5 molm^–3, pH, 9.2). Under subsaturating ¹⁴CO₂ levels, bothgaseous diffusion and H¹⁴CO₃^– transport were shown tooperate simultaneously, such that maximal photosynthetic rateswere established. An induction lag of approximately 3 min was observed when exogenous¹⁴CO₂ was assimilated. A longer lag of approximately 12 minwas required, however, before linear assimilation rates wereestablished when H¹⁴CO₃ acted as the carbon source. The light-activatedH¹⁴CO₃^– transport system was found to be quite labile.A brief (5 min) dark treatment returned the system to the inactivestate. Bicarbonate transport was shown to be competitively inhibitedby CO₃^2–ions. The possibility is discussed that this formof inhibition may be common to many HCO₃^– assimilators. Preliminary polar cation transport studies (from lower to upperleaf surface) indicated an almost exact one to one relationshipbetween the rates of Na⁺ influx and efflux and H¹⁴CO₃^–assimilation. The possible relationship(s) between these transportprocesses and the requirement for electrical neutrality is brieflydiscussed.     

Amino acid metabolism in plant leaf IV. The effect of light on ammonium assimilation and glutamine metabolism in the cells isolated from spinach leaves

Comparison of incorporation of ¹⁵N-labeled ammonium into aminoacids in cells isolated from spinach leaves showed that ammoniumwas most actively incorporated into the amido-group of glutamine.The ¹⁵N contents of other amino acids were less than one-tenththat of the amido-group of glutamine. L-Methionine-DL-sulfoximine(MS) suppressed the incorporation of ammonium not only intothe amido-group of glutamine, but also into glutamic acid. Turningoff the light after 1 min illumination increased die 15N contentof glutamine while it decreased that of the glutamic acid, asparticacid and alanine. Illumination of the cells after die applicationof ammonium had a more significant effect on ammonium assimilationthan illumination before the application of ammonium. When ¹⁴C-U-¹⁵N(amido labeled)-glutamine was added to the cell suspension,the transfer of amido-group of glutamine was completely inhibitedin the dark, but no difference in the flow of ¹⁴C was observed. These results suggest that glutamine synthetase (GS) and glutamatesynthase (GOGAT) pathways operate in ammonium assimilation inthe cells isolated from spinach leaves, and that the formeris light-independent but die latter is light-dependent. (Received December 23, 1977; )     

Restricted nitrate influx and reduction in corn seedlings exposed to ammonium

The effect of ambient ammonium (0.5 millimolar [¹⁴NH₄]₂SO₄) added to a nutrient solution containing 1.0 millimolar K¹⁵NO₃, 99 atom per cent ¹⁵N, upon [¹⁵N]nitrate assimilation and utilization of previously accumulated [¹⁴N]nitrate was investigated. Corn seedlings, 5-day-old dark-grown decapitated (experiment I) and 10-day-old light-grown intact (experiment II), which had previously been grown on K¹⁴NO₃ nutrient solution, were used. In both experiments, the presence of ambient ammonium decreased [¹⁵N]nitrate influx (20% after 6 hours) without significantly affecting the efflux of previously accumulated [¹⁴N]nitrate. In experiment I, relative reduction of [¹⁵N]nitrate (reduction as a percentage of influx) was inhibited more than was [¹⁵N]nitrate influx. Nevertheless, in experiment I, where all reduction could be assigned to the root system, the absolute inhibition of reduction during the 12 hours (13 micromoles/root) was less than the absolute inhibition in influx (24 micromoles/root). The data suggest that the influence of ammonium on [¹⁵N]nitrate influx could not be totally accounted for by the decrease in the potential driving force which resulted from restricted reduction; an additional impact on the influx process is indicated. Reduction of [¹⁵N]nitrate in experiment II after 6 hours accounted for 30 and 18% of the tissue excess ¹⁵N in the control and ammonium treatments, respectively. Relative distribution of ¹⁵N between roots and exudate (experiment I), or between roots and shoots (experiment II) was not affected by ammonium. On the other hand, the accumulation of [¹⁵N]nitrate in roots, shoots, and xylem exudate was enhanced by ammonium treatment compared to the control, whereas the accumulation of reduced ¹⁵N was inhibited.     

Biophysical and Biochemical Regulation of Cytoplasmic pH in Chara corallina during Acid Loads

The contribution of membrane transport to regulation of cytoplasmicpH in Chara corallina has been measured during proton-loadingby uptake of butyric acid. In the short-term (i.e. up to 20min) uptake of butyric acid is not affected by removal of externalK⁺, Na⁺ or Cl^– but over longer periods uptake is decreased(by 20–50% in different experiments) in the absence ofexternal Na⁺ or, sometimes, K⁺. Influxes of both Na⁺ and K⁺increase temporarily after addition of butyrate, Na⁺ immediatelyand K⁺ after a lag. Effects on Cl^– influx are small butCl^– efflux increases enormously after a short lag. Anapproximate comparison of internal butyrate with changes inthe concentration of K⁺, Na⁺, and Cl^– suggests that initially(i.e. for a few min) cytoplasmic pH is determined by bufferingand possibly by some decarboxylation of organic acids (biochemicalpH regulation), and that biophysical pH regulation involvingefflux of H⁺ balanced by influxes of K⁺, Na⁺ and especiallyefflux of Cl^– progressively becomes dominant. When butyric acid is washed out of the cells, cytoplasmic pHis restored completely or partially (depending on the butyrateconcentration used) and this is independent of the presenceor absence of external Cl^–. Where Cl^– is present,its influx is relatively small. It is suggested that cytoplasmicpH is then controlled biochemically, involving the synthesisof an (unidentified) organic acid and the accumulation of acidicanions in place of butyurate lost from the cell. During thesecond application of butyrate, net Cl^– efflux is small:it is suggested that control of cytoplasmic pH then involvesdecarboxylation of the organic acid anions. The questions of the source of Cl^– lost from the cell(cytoplasm or vacuole) and of possible cytoplasmic swellingassociated with the accumulation of butyrate are discussed. Key words: Chara corallina, butyric acid, cytoplasmic pH, membrane transport