Uptake Mechanisms: Inorganic and Organic

The effect of the exogenous and endogenous N03-concentration on net uptake, influx, and efflux of N03-and on nitrate reductase activity (NRA) in roots was studied in Phaseolus vulgars L. cv. Witte Krombek.After exposure to an Beyond 100 micromoles per cubic decimeter, root NRA was not affected by exogenous NO3-indicating that N03-uptake was not coupled to root NRA, at least not at high concentrations.Nitrate utilization in higher plants is essentially the integration of three processes: acquisition, conversion, and translocation of nitrate-nitrogen. The amount of N03-taken up by the roots represents the upper limit of a plant's N03-utilization capacity. Despite its great biological and agricultural significance, the uptake of NO3 has been studied much less extensively than the uptake ofother important ions. It is noteworthy that recent reviews on the concentration kinetics of ion transport do not give data on NO3 (14,26). Although kinetic parameters of NO3 uptake have been reviewed (7,18) sufficiently rigorous analytical procedures.In this study, several techniques were combined, e.g. measurement of net uptake, influx, and efflux of N03-and the assay of nitrate reductase activity, to construct a kinetic picture of the uptake and reduction of N03 by bean roots. Additionally, a comparison of characteristics of net N03 uptake with those of unidirectional fluxes was made and the kinetic behavior of plants during environmental depletion of NO3 was studied. Short reports of our findings have appeared (4, 5). MATERIALS AND METHODSPlant Cultivation. Phaseolus vulgaris L. cv. Witte Krombek was germinated in Perlite and transferred to N-free basal medium (3), the pH of which was kept between 4.2 and 6.5 with dilute KOH or at 5.0 ± 0.1 with a pH stat. Plants were grown for 10 d in the former and 7 d in the latter treatment. After these periods, the roots were unnodulated, the primary leaves had expanded, and the contribution of trifoliate leaves to plant weight or plant NRA2 was negligible (3). No major differences in plant weight, development, rate of N03 uptake, and NRA occurred between plants grown with manual or automatic pH control. Barley (Hordeum vulgare L. cv. Herta) was grown for 10 d with manual pH control.Nitrate utilization was initiated by adding Ca(NO3)2 to plants grown in basal medium. Germination, cultivation, and experiments were performed at 20 ± 1VC, 65 ± 10%1o RH, and a 16-h photoperiod at 30 w m-2. Dwarf bean was grown for 10 d on CaCl2 solutions (0.5 mmol dm-3) to produce low-salt roots. We refer to previous papers for detailed conditions of cultivation and experimentation (2, 3).Experiments. Net uptake of N03 and H2PO4 was assayed by following nutrient depletion in the ambient solution for several hours. In experiments with various starting concentrations, N03 was regularly replenished to the initial levels in long-term experiments, while in short-term experiments the depletion in a certain interval was plotted against the average concentration in that interval. In experiments on the time ...

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confidence: 99%

Effect of Exogenous and Endogenous Nitrate Concentration on Nitrate Utilization by Dwarf Bean

Breteler¹,

Nissen²

1982

“…In fact, the kinetics of K+ uptake and K+ stimulation of ATPase activity in corn roots (28) have been shown to be similarly, if not identically, multiphasic (39). It should perhaps be noted that multiphasic kinetics are not due to artifacts of any kind (38,44). It has not been possible to eliminate the discontinuities by obtaining additional points in the range of the jumps (6).…”

Section: Resultsmentioning

confidence: 94%

“…17, 31), it seems that the basis for multiphasic kinetics must differ markedly from that for cooperative kinetics. Multiphasic kinetics have been taken (33,38) to arise from abrupt changes in a single structure (carrier or site). These changes are brought about by all-or-none transitions in a separate site.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Stimulation of Plasmalemma Adenosine Triphosphatase from Oat Roots by Inorganic and Organic Cations: Concentration-Dependence, Selectivity, and Sites

Håvarstein¹,

Nissen²

1981

K-stimulated ATPase activity of a plasmalemma-enriched fraction from excised roots of oat was triphasic in the range 5 to 80 m;limolar KCI. The phases obeyed MlchaeLs-Menten kinetics and were separated from each other by jumps or sharp breaks at about 10 and 20 nillilar. Stimulation by alkali cations was in the order K+ > Rb+ > Na+ > Cs' > Li' or in a closely related sequence. The specificity reflected differences in ,,.,, not in affinity (K.'). Stimulation by the organic cations ethanolamine and choline in the interval 11 to 80 mllmolar appeared monophasic rather than biphasic. Substitution on the quatemary nitrogen of the amino alcohols decreased their effectiveness, as did extension and brhing of the chain. Stimulation was maximal at about pH 7 both for K' and choline.The kinetics of K' stimulation are multiphasic, not cooperative, as was also found for uptake. The ATPase is also stimulated by organic cations, but the difference in kinetics indicates the existence of separate sites for stimulation and transition.Membrane-bound ATPases have been implicated in the transport of ions in plants (17,18,30). Particularly close correlations exist between a K+-stimulated ATPase and uptake of K+ across the plasmalemma (13,27,28). The role of this enzyme in ion transport remains unclear, however (25, 43).The present paper examines some unresolved questions concerning this relationship. The complex kinetics of ion uptake and ATPase stimulation have been described variously as cooperative (17,27,28) MATERIALS AND METHODS Seedlings of oat (Avena sativa L. cv. Titus) were grown in 0.5 mM CaC12 for 6 to 8 days at 16 to 22 C in the dark (40).A plasmalemma-enriched membrane fraction was isolated as described by Hodges and Leonard (19) with some minor modifications (32). The excised roots were ground with a mortar and pestle in a solution of 250 mm sucrose, 3 mm EDTA, 2.5 mm DTT, and 25 mm Tris-Mes buffer (pH 7.2). The filtered homogenate was centrifuged successively at 13,000g for 15 min and 40,000g for 30 min. The 40,000g pellet was suspended in 34% sucrose (w/w) and layered onto a discontinuous gradient consisting of 20 ml of 45% sucrose (w/w), 8 ml of 40%o sucrose, and 8 ml of 34% sucrose. All sucrose solutions contained 1 mm DTT and 1 mM Tris-Mes (pH 7.2). The gradient was centrifuged for 2 h at 80,000g in a Spinco SW 27 rotor, and the membrane fraction was collected at the 34/ 40% sucrose interface. For most experiments the membrane fraction was frozen at -18 C and used the following day (cf. Figs. 1 and 2).ATPase activity was measured in a l-ml volume containing 3 mM ATP (Tris salt, pH 6.5), 30 mm Tris-Mes (pH 6.5), 3 mM MgSO4, varying concentrations of monovalent inorganic or organic cations (as chlorides), and 10 to 25 ,tg of membrane protein.Reactions, initiated by the addition of ATP, were carried out at 38 C in a shaking water bath for 30 to 45 min, depending on the amount of protein. (ATPase activity was linear for at least 1 h, and the ATP concentrations remained essentially constant.) Pi released was de...

“…Since this pioneering work, the kinetic parameters for the transport of a variety of ions in many different species have been published (16).…”

mentioning

confidence: 99%

Rubidium (Potassium) Uptake by Arabidopsis

Polley

Hopkins²

1979

Expriments are reported in which the uptake of 'Rb', used as an analog of K , into cultured cels of Arabidopsis thaiana is investigated. A single transport system is found with KM = 0.34 mliolar and V.. -14 nmoles per miigram of protein per hour. This system is blocked by the metabolic inhibitor carbonyl cyanide n-chlorophenyl hydrazone (CCCP) and by cold. At high concentrations of external K' (above 1 milllar), a significant fraction of total uptake is energy-independent. No evidence is found for more than one energy-dependent uptake system or for concentration-dependent modifications of a carrier as postulated in multiphasic transport models.Rb' uptake was also examined in cultured cells derived from an "osmotic mutant" of Arabidpsis The with NaOH, and 20 g/l glucose. Suspension cultures were grown in Bellco spinner flasks in the dark at 25 C. Large aggregates of cells were removed from suspension by gravity and discarded; the cells remaining in suspension were pelleted by low speed centrifugation and washed three times with HP-7 medium lacking K+ (NH4' substituted for K+). The final suspension used for uptake studies consisted mostly of single cells with some two-and fourcell aggregates. Cells were harvested in log phase at densities between 1 x 105 and 5 x 10' cells/ml. Cell viability was estimated by microscopic inspection and a dye exclusion test using erythrocin B. Viability by these criteria was consistently about 80%Yo.Culture 105 cells/ml. One-ml aliquots of the suspension were withdrawn at appropriate intervals and the cells were collected on Millipore filters and washed three times with 3.0 ml unlabeled HP-7. The amount of isotope taken up was determined by either liquid scintillation or gas flow counting. For experiments in the presence of CCCP2 or at 4 C, there was a 30-min preincubation period before isotope was added. Seedlings were incubated completely immersed with shaking (approximately 100 oscillations/min) at 25 C in medium containing MRbCl (10 mCi/mg) at a concentration of 3 isCi/ml. They were removed from the labeling medium and placed in desorption medium (K+-free growth medium) for 10 min at 4 C. After desorption, seedlings were blotted dry, placed in scintillation vials with scintillation fluid, and the radioactivity measured. Figure 1. Cells were incubated with shaking at 25 C in HP-7 medium containing 10 ,UM KCI and 3 ,uCi/ml 'RbCl. Samples (1.0 ml) were taken at the indicated times and the radioactivity determined. Two observations are apparent: (a) both cold and the metabolic inhibitor, CCCP, at 0.01 mm, strikingly inhibit uptake; this supports the notion that transport is an active process; and (b) there is a lag of 40 to 60 min after which transport is linear. We do not understand the reason for the lag. However, we find that uptake of Rb+ into roots of intact seedlings has a similar lag, which disappears when cells are incubated in the assay medium for 2 h prior to the addition of the radioisotope (Fig. IA)