The face value of ion fluxes: the challenge of determining influx in the low-affinity transport range

The existence of distinct high- and low-affinity transport systems (HATS and LATS) is well established for major nutrient ions. However, influx mediated by these systems is usually estimated using uniformly simple tracer protocols. Two (42)K radiotracer methods to measure potassium influxes in the HATS and LATS ranges in intact barley (Hordeum vulgare L.) roots are compared here: a direct influx (DI) method, and an integrated flux analysis (IFA), which is designed to account for tracer efflux from labelled roots and differential tracer accumulation along the plant axis. Methods showed only minor discrepancies for influx values in the HATS range, but large discrepancies in the LATS range, revealing striking distinctions in the cellular exchange properties dominated by the operation of the two transport systems. It is shown that accepted DI protocols are associated with very large errors in the high-conductance LATS range, underestimating influx at least 6-fold due to four characteristics of this transport mode: (i) accelerated cellular (42)K exchange; (ii) a greatly increased ratio of efflux to influx; (iii) increased (42)K loss during the removal of water from roots in preweighing centrifugation or blotting protocols; and (iv) increased (42)K retention at the root-shoot interface, a region of the plant frequently disregarded in DI determinations. The findings warrant a re-evaluation of a large body of literature reporting influx in the LATS range, and are of fundamental importance to ion flux experimentation in plant physiology.     

Ammonium Uptake by Rice Roots (II. Kinetics of 13NH4+ Influx across the Plasmalemma)

Short-term influxes of 13NH4+ were measured in intact roots of 3-week-old rice (Oryza sativa L. cv M202) seedlings that were hydroponically grown at 2, 100, or 1000 [mu]M NH4+. Below 1 mM external concentration ([NH4+]0), influx was saturable and due to a high-affinity transport system (HATS). For the HATS, Vmax values were negatively correlated and Km values were positively correlated with NH4+ provision during growth and root [NH4+]. Between 1 and 40 mM [NH4+]0, 13NH4+ influx showed a linear response due to a low-affinity transport system (LATS). The 13NH4+ influxes by the HATS, and to a lesser extent the LATS, are energy-dependent processes. Selected metabolic inhibitors reduced influx of the HATS by 50 to 80%, but of the LATS by only 31 to 51%. Estimated values for Q10 (the ratio of rates at temperatures differing by 10[deg]C) for HATS were greater than 2.4 at root temperatures from 5 to 10[deg]C and were constant at approximately 1.5 between 5 and 30[deg]C for the LATS. Influx of 13NH4+ by the HATS was insensitive to external pH in the range from 4.5 to 9.0, but influx by the LATS declined significantly beyond pH 6.0. The data presented are discussed in the context of the kinetics, energy dependence, and the regulation of ammonium influx.     

Studies of the Uptake of Nitrate in Barley : IV. Electrophysiology

Transmembrane electrical potential differences (Δψ) of epidermal and cortical cells were measured in intact roots of barley (Hordeum vulgare L. cv Klondike). The effects of exogenous NO₃⁻ on Δψ (in the concentration range from 100 micromolar to 20 millimolar) were investigated to probe the mechanisms of nitrate uptake by the high-affinity (HATS) and low-affinity (LATS) transport systems for NO₃⁻ uptake. Both transport systems caused depolarization of Δψ, demonstrating that the LATS (like the HATS) for NO₃⁻ uptake is probably mediated by an electrogenic cation (H⁺?) cotransport system. Membrane depolarization by the HATS was “inducible” by NO₃⁻, and saturable with respect to exogenous [NO₃⁻]. By contrast, depolarization by the LATS was constitutive, and first-order in response to external [NO₃⁻]. H⁺ fluxes, measured in 200 micromolar and in 5 millimolar Ca(NO₃)₂ solutions, failed to alkalinize external media as anticipated for a 2 H⁺:1 NO₃⁻ symport. However, switching from K₂SO₄ solutions (which were strongly acidifying) to KNO₃ solutions at the same K⁺ concentration caused marked reductions in H⁺ efflux. These observations are consistent with NO₃⁻ uptake by the HATS and the LATS via 2 H⁺:1 NO₃⁻ symports. These observations establish that the HATS for nitrate uptake by barley roots is essentially similar to those reported for Lemna and Zea mays by earlier workers. There are, nevertheless, distinct differences between barley and corn in their quantitative responses to external NO₃⁻.     

Characterization and regulation of ammonium transport systems in Citrus plants

We have investigated both the kinetics and regulation of 15NH4+ influx in roots of 3-month-old hydroponically grown Citrus (Citrus sinensis L. Osbeck x Poncirus trifoliata Blanco) seedlings. The 15NH4+ influx is saturable below an external ammonium concentration of 1 mM, indicating the action of a high-affinity transport system (HATS). The HATS is under feedback repression by the N status of the plant, being down-regulated in plants adequately supplied with N during growth, and up-regulated by N-starvation. When assayed between 1 and 50 mM [15NH4+]0, the 15NH4+ influx showed a linear response typical of a low-affinity transport system (LATS). The activity of the LATS increased in plants supplied with NH4+ as compared with plants grown on an N-free medium. Transfer of the plants to N-free solution resulted in a marked decrease in the LATS-mediated 15NH4+ influx. Accordingly, resupply of NH4+ after N-starvation triggered a dramatic stimulation of the activity of the LATS. These data provide evidence that in Citrus plants, the LATS or at least one of its components is inducible by NH4+. Even when up-regulated, both the HATS and the LATS displayed a limited capacity, as compared with that usually found in herbaceous species. The use of various metabolic uncouplers or inhibitors indicated that 15NH4+ influx mediated by the HATS is strongly dependent on energy metabolism and H+ transmembrane electrochemical gradient. By contrast, the LATS is not affected by protonophores or inhibitors of the H(+)-ATPase, suggesting that its activity is mostly driven by the NH4+/NH3 transmembrane gradient. In agreement with these hypotheses, the HATS-mediated 15NH4+ influx was strongly inhibited when the solution pH was raised from 4 to 7, whereas influx mediated by the LATS was slightly stimulated.     

Determination of Unidirectional Sodium Fluxes in Roots of Intact Sunflower Seedlings

The method of compartmental analysis was applied to study sodiumfluxes in roots of intact seedlings of Helianthus annuus L.By measuring sodium uptake and transport to the shoots of theseedlings in parallel experiments, transport of tracer sodiumto shoots and net accumulation of Na⁺ in the roots during theflux measurements was accounted for. The steady-state sodiumfluxes in the intact sunflower roots were similar in size tothose in excised roots but in general they were somewhat higher.This indicates more metabolic activity in the intact tissues.Using whole plants it is possible to study the response of ionfluxes in roots to ecophysiological stimuli received by theshoots, and in the present experiments the effect of continuouslight versus long-day growth conditions was investigated. Potassium,when continually present, depressed all fluxes and the cytoplasmiccontent of sodium but tended to increase the vacuolar sodiumcontent, in particular when this was related to the cytoplasmiccontent. When added to sodium-loaded roots, potassium stimulatedthe plasmalemma sodium efflux but slightly, suggesting a lowefficiency of K⁺-Na⁺ exchange across the plasmalemma in intactas well as excised sunflower roots. Subsequently, however, potassiuminduced a transient decrease in the ²²Na efflux that was followedby oscillations in tracer efflux. These changes were attributedto potassium-induced transfer of sodium to vacuoles. Moreover,the oscillations seem to indicate the operation of negativefeedback control of sodium fluxes.     

Cercospora beticola Toxins (X. Inhibition of Plasma Membrane H+-ATPase by Beticolin-1)

Influxes of 13NH4+ across the root plasmalemma were measured in intact seedlings of Picea glauca (Moench) Voss. Two kinetically distinct uptake systems for NH4+ were identified. In N-deprived plants, a Michaelis-Menten-type high-affinity transport system (HATS) operated in a 2.5 to 350 [mu]M range of external NH4+ concentration ([NH4 +]o). The Vmax of this HATS was 1.9 to 2.4 [mu]mol g-1 h-1, and the Km was 20 to40 [mu]M. At [NH4+]o from 500 [mu]M to 50 mM, a linear low-affinity system (LATS) was apparent. Both HATS and LATS were constitutive. A time-dependence study of NH4+ influx in previously N-deprived seedlings revealed a small transient increase of NH4+ influx after 24 h of exposure to 100 [mu]M [NH4+]o. This was followed by a decline of influx to a steady-state value after 4 d. In seedlings exposed to 100 [mu]M external NO3- concentration for 3 d, the Vmax for NH4+ uptake by HATS was increased approximately 30% compared to that found in N-deprived seedlings, whereas LATS was down-regulated. The present study defines the much higher uptake capacity for NH4+ than for N03- in seedlings of this species.     

Kinetics of NO3(-) net fluxes in Pinus pinaster, Rhizopogon roseolus and their ectomycorrhizal association, as affected by the presence of NO3(-) and NH4+

The impact of mineral N supply, N-free or NO3(-) with or without NH4+, on the subsequent uptake of NO3(-) by maritime pine seedlings associated with the ectomycorrhizal fungus Rhizopogon roseolus was studied using ion-selective microelectrodes. NO3(-) net fluxes into N-starved non-mycorrhizal short roots (NMSRs) were low and measurable only over the NO3(-) concentration range of 0-70 microM. The simple kinetics observed in those roots may reflect the dominant operation of a high-affinity NO3(-) transport system (HATS) which is constitutive. NO3(-) pretreatment increased the NO3(-) net fluxes and led to a complex kinetics that may reflect the operation of other HATS. A simple kinetics was observed in plants pre-incubated at high NH4+ concentration. In contrast, NO3(-) uptake kinetics presented only one saturation phase in the fungus, whether associated with the plant or not. NO3(-) uptake was greater after a pretreatment in N-free or NO3 (-) solution, but NH4+ pretreatment led to a threefold reduction in NO3 (-) uptake. These results suggest that the regulation of NO3(-) transport systems varies between the host and the fungal partner. This variation is likely to contribute to the positive effect of mycorrhizal association on N uptake in plants when the N supply is low and fluctuating.     

Salt-induced NO<Subscript>3</Subscript><Superscript>-</Superscript> uptake inhibition in cowpea roots is dependent on the ionic composition of the salt and its osmotic effect

Salinity remarkably inhibits NO₃ ^- uptake but the mechanisms are not well understood. This study was addressed to elucidate the role of ionic and osmotic components of salinity on NO₃ ^- influx and efflux employing classic kinetics involving a low affinity transport system (LATS) and a high affinity transport system (HATS). In the presence of KCl, NaCl, and Na₂SO₄ at 100 mM concentrations, in both LATS and HATS, Michaelis constant (K_m) was similar for the three salts and maximum rate (V_max) decreased as follows: KCl > NaCl > Na₂SO₄, compared to control indicating a non-competitive interaction with NO₃ ^-. Unexpectedly, iso-osmotic solutions (osmotic potential Ψ_π = -0.450) of polyethylene glycol (PEG, 17.84 %, v/v) and mannitol (100 mM) remarkably increased K_m in both the LATS and the HATS, but V_max did not change indicating a competitive inhibition. Under the PEG and mannitol treatments, K_m and V_max were higher than under the salt treatments. The salts increased slightly NO₃ ^- efflux in the following order KCl > NaCl > Na₂SO₄. In contrast, mannitol strongly stimulated and the PEG inhibited NO₃ ^- efflux. The obtained data reveal that salinity effects were not dependent on the anion type (Cl^- versus SO₄ ^2-) indicating a non-competitive inhibition mechanism between Cl^- and NO₃ ^-. In contrast, the cation types (K⁺ versus Na⁺) had a pronounced effect. The osmotic component is important to net NO₃ ^- uptake affecting remarkably the influx in both LATS and HATS components of cowpea roots.     

Regulation of Nitrate Transport in Citrus Rootstocks Depending on Nitrogen Availability

Previously, we reported that in Citrus plants, nitrate influx through the plasmalemma of roots cells follows a biphasic pattern, suggesting the existence of at least two different uptake systems, a high and low affinity transport system (HATS and LATS, respectively). Here, we describe a novel inducible high affinity transport system (iHATS). This new nitrate transport system has a high capacity to uptake nitrate in two different Citrus rootstocks (Cleopatra mandarin and Troyer citrange). The iHATS was saturable, showing higher affinity than constitutive high affinity transport system (cHATS) to the substrate NO₃⁻. The V_max for this saturable component iHATS was higher than cHATS, reaching similar values in both rootstocks.Additionally, we studied the regulation of root NO₃⁻ uptake mediated by both HATS (iHATS and cHATS) and LATS. In both rootstocks, cHATS is constitutive and independent of N-status. Concerning the regulation of iHATS, this system is upregulated by NO₃⁻ and down-regulated by the N status and by NO₃⁻ itself when plants are exposed to it for a longer period of time. LATS in Cleopatra mandarin and Troyer citrange rootstocks is repressed by the N-status.The use of various metabolic uncouplers or inhibitors indicated that NO₃⁻ net uptake mediated by iHATS and LATS was an active transport system in both rootstocks.Key Words: Citrus, inducible high affinity transport system (iHATS), constitutive high affinity transport system (cHATS), nitrate uptake, regulation     

Futile Na+ cycling at the root plasma membrane in rice (Oryza sativa L.): kinetics, energetics, and relationship to salinity tolerance

Globally, over one-third of irrigated land is affected by salinity, including much of the land under lowland rice cultivation in the tropics, seriously compromising yields of this most important of crop species. However, there remains an insufficient understanding of the cellular basis of salt tolerance in rice. Here, three methods of 24Na+ tracer analysis were used to investigate primary Na+ transport at the root plasma membrane in a salt-tolerant rice cultivar (Pokkali) and a salt-sensitive cultivar (IR29). Futile cycling of Na+ at the plasma membrane of intact roots occurred at both low and elevated levels of steady-state Na+ supply ([Na+]ext=1 mM and 25 mM) in both cultivars. At 25 mM [Na+]ext, a toxic condition for IR29, unidirectional influx and efflux of Na+ in this cultivar, but not in Pokkali, became very high [>100 micromol g (root FW)(-1) h(-1)], demonstrating an inability to restrict sodium fluxes. Current models of sodium transport energetics across the plasma membrane in root cells predict that, if the sodium efflux were mediated by Na+/H+ antiport, this toxic scenario would impose a substantial respiratory cost in IR29. This cost is calculated here, and compared with root respiration, which, however, comprised only approximately 50% of what would be required to sustain efflux by the antiporter. This suggests that either the conventional 'leak-pump' model of Na+ transport or the energetic model of proton-linked Na+ transport may require some revision. In addition, the lack of suppression of Na+ influx by both K+ and Ca2+, and by the application of the channel inhibitors Cs+, TEA+, and Ba2+, questions the participation of potassium channels and non-selective cation channels in the observed Na+ fluxes.     

Compartmental analysis of efflux of K+(86Rb+) and Rb+(86Rb+) from roots of intact plants of barley (Hordeum vulgare) of high and low K+ status, and after excision of the shoot

The classic compartment analysis of ion efflux from roots is often applied with the assumption that there is a system of 3 compartments in series. However, complex ion transport across the root tissues, as well as influences from the shoot, may complicate the picture. The present experiments were performed to study the immediate effects that excision of the shoot before the experiment exerts on the efflux of Rb⁺(⁸⁶Rb⁺) and of K⁺(⁸⁶Rb⁺) from 9-day-old roots of plants of barley (Hordeum vulgare L. cv. Salve). The efflux from high K⁺ and low K⁺ roots of intact and detopped plants were compared. After excision of the shoot of high K⁺ plants, a marked increase in efflux was observed after 2.5 h with a maximum at about 7 h. The increase in efflux was seen as a peak in plots of efflux versus time. Excision of the shoot from low K⁺ roots did not give rise to a consistent increase in efflux. Regular K⁺ ion efflux curves were observed from roots of intact plants of high or low K⁺ status. Furthermore, after a pulse treatment of 9-day-old roots of intact plants of high or low K⁺ status with a solution containing Rb⁺(⁸⁶Rb⁺), the Rb⁺(⁸⁶Rb⁺) transport to the shoots was not reduced during the following 3 h in unlabelled solution. It is suggested that both the peak appearing in the efflux plots and the maintained tracer transport to the shoots after transfer of the roots to an unlabelled solution indicate the existence of a K⁺/Rb⁺ transport system in the symplasm of the roots that has only a slow exchange with the bulk cytoplasm and vacuoles.     

Effect of azetidine 2-carboxylic Acid on ion uptake and ion release to the xylem of excised barley roots

Azetidine 2-carboxylic acid (AZ) was used as an analog of proline to investigate further the relationship between protein synthesis and ion transport. AZ does not inhibit protein assembly, but the proteins formed are ineffective as enzymes. At relatively low concentrations (50 μM) AZ was a potent inhibitor of release of ions to the xylem of excised roots of barley (Hordeum vulgare L.) and intact plants. Uptake to the root was also inhibited but to a lesser degree. A procedure was introduced for estimating unidirectional fluxes from measurements of net tracer uptake, net transport to the xylem, and net efflux from the roots. It was shown that inhibition of release to the xylem was not caused by reduction in influx at the plasmalemma or to stimulation of influx to the vacuoles. It was suggested that AZ was acting on the process of release from symplast to the xylem. The action of AZ is compared with similar effects on ion transport produced by p-fluorophenylalanine, cycloheximide, and abscisic acid.     

The Kinetics of Chlorate Uptake by XD Tobacco Cells

The uptake of [³⁶Cl]chlorate by the 14U variant of the XD cell line of Nicotiana tobaccum L. cv Xanthi was investigated to examine the use of chlorate as a nitrate analog in transport studies. The kinetics of chlorate uptake against concentration was complex. Evidence was obtained, e.g., by means of nitrate competition, that these kinetics could be resolved into two components indicating the existence of two influx mechanisms: a saturable high affinity transport system (HATS) and a low affinity transport system (LATS) that showed first order kinetics. HATS has an apparent K_m for chlorate of 0.3 millimolar, and a marked pH dependence. The V_max dropped about fivefold as the pH was changed from the optimum pH (5.5-6.5), while the K_m remained virtually unchanged. The activity of HATS was completely inhibited by 15 millimolar nitrate and was less sensitive to chloride. LATS was inhibited by chloride and showed some inhibition by nitrate. It was concluded that [³⁶Cl]chlorate can be used as an analog for nitrate uptake studies only in a limited low concentration range where HATS is the main route for chlorate influx.     

The characterization of inhibition of net nitrate uptake by salt in salt-tolerant barley (Hordeum vulgare L. cv. California Mariout)

Barley seedlings (Hordeum vulgare L. cv. California Mariout) grown hydroponically for 14-19 d without addition of NaCl were used for describing the effects of salt application on net nitrate uptake and for the calculation of kinetic parameters. The addition of NaCl, KCl, CaCl2, and Na2SO4 to the uptake solution in the experiments led to similar inhibition of nitrate uptake, only at low and very high salt concentrations were ion-specific effects found. The same decrease in nitrate uptake can also be achieved by sorbitol or betaine at corresponding osmolalities. Thus it was concluded that the inhibition of uptake was caused mainly by the osmotic effects of salts. Differences in the mechanisms of inhibition were detected between the two systems of nitrate uptake (high affinity system: HATS, and low affinity system: LATS). The HATS was inhibited non-competitively by NaCl, an apparent Ki of 60 mol m^-3 was calculated using a Dixon-plot. Fitting an equation assuming a non-competitively inhibited HATS by computer program to the raw data resulted in an apparent Ki of about 37 mol m^-3. In contrast, the LATS was affected in a complex way: up to 60 mol m^-3 NaCl the affinity was increased, which led to a stimulation of nitrate uptake at low nitrate concentrations (<2 mol m^-3). An inhibition of the LATS became obvious at concentrations above 3 mol m^-3 nitrate (for all applied salt concentrations) or with 100 mol m^-3 NaCl (throughout the whole nitrate range). Related plots of the data pointed to a competitive effect.Key words: Hordeum vulgare L., net nitrate uptake, high affinity transport system (HATS), low affinity transport system (LATS), salt, inhibition, apparent kinetic parameters.      

Identification and expression analyses of two genes encoding putative low-affinity nitrate transporters from Nicotiana plumbaginifolia

Higher plants have both high- and low-affinity nitrate uptake systems (HATS and LATS respectively). Here we report the isolation and characterization of two genes, NpNRT1.1 and NpNRT1.2, from Nicotiana plumbaginifolia whose structural features suggest that they both belong to the NRT1 gene family, which is involved in the LATS. Amino acid sequence alignment showed that the N. plumbaginifolia proteins have greater similarity to their corresponding tomato homologues than to each other. Genomic Southern blot analysis indicates that there are probably more than two members of this family in N. plumbaginifolia. Northern blot analysis shows that NpNRT1.2 expression is restricted strictly to roots, whereas NpNRT1.1, in addition to roots, is expressed at a basal level in all other plant organs. Likewise, differential expression in response to external treatments with various N sources was observed for these two genes: NpNRT1.1 can be considered as a constitutively expressed gene whereas NpNRT1.2 expression is dependent strictly on high nitrate concentrations. Finally, over-expression of a gene involved in the HATS does not lead to any modification of LATS gene expression.     

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K⁺) as a nutrient, and ammonia/ammonium (NH₃/NH₄⁺) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.     

Kinetics of NH 4 + uptake by the arbuscular mycorrhizal fungus Rhizophagus irregularis

The kinetics and energetics of (15)NH (4) (+) uptake by the extraradical mycelium of the arbuscular mycorrhizal fungus Rhizophagus irregularis were investigated. (15)NH (4) (+) uptake increased with increasing substrate concentration over the concentration range of 0.002 to 25?mM. Eadie-Hofstee plots showed that ammonium (NH (4) (+) ) uptake over this range was biphasic. At concentrations below 100?μM, NH (4) (+) uptake fits a Michaelis-Menten curve, typical of the activity of a saturable high-affinity transport system (HATS). At concentrations above 1?mM, NH (4) (+) influx showed a linear response typical of a nonsaturable low-affinity transport system (LATS). Both transport systems were dependent on external pH. The HATS and, to a lesser extent, the LATS were inhibited by the ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP) and the ATP-synthesis inhibitor 2,4-dinitrophenol. These data indicate that the two NH (4) (+) transport systems of R. irregularis are dependent on metabolic energy and on the electrochemical H(+) gradient. The HATS- and the LATS-mediated (15)NH (4) (+) influxes were also regulated by acetate. This first report of the existence of active high- and low-affinity NH4(+) transport systems in the extraradical mycelium of an arbuscular mycorrhizal fungus and provides novel information on the mechanisms underlying mycosymbiont uptake of nitrogen from the soil environment.     

NO3- and ClO3- fluxes in the chl1-5 mutant of Arabidopsis thaliana. Does the CHL1-5 gene encode a low-affinity NO3- transporter?

The CHL1 gene is considered to encode a low-affinity transport system (LATS) for NO3- in Arabidopsis thaliana (Y.-F. Tsay, J.I. Schroeder, K.A. Feldmann, N.M. Crawford [1993] Cell 72: 705-713). However, the anticipated reduced NO3- uptake by the LATS associated with loss of CHL1 gene activity in chl1-5 deletion mutants was evident only when plants were grown on NH4NO3. When KNO3 was the sole N source, NO3- accumulation and short-term tracer influx (using 13NO3- and 15NO3-) in leaves and roots of wild-type and mutant plants were essentially identical. Nevertheless, root uptake of 36CIO3- by the LATS and CIO3- accumulation in roots and shoots of mutant plants were significantly lower than in wild-type plants when grown on KNO3. One explanation for these results is that a second LATS is able to compensate for the chl1-5 deficiency in KNO3-grown plants. Growth on NH4NO3 may down-regulate the second LATS enough that the anticipated difference in NO3- uptake becomes apparent.