Multiphasic uptake of potassium by barley roots of low and high potassium content: Separate sites for uptake and transitions

In the range 10^?6M - 5 × 10^?2M uptake of K⁺ in excised roots of barley (Hordeum vulgare L. cv. Herta) with low and high K content could in both cases be represented by an isotherm with four phases. Uptake, especially in the range of the lower phases, was reduced in high K roots through decreases in V_max and increases in K_m. Similar data for other plants are also shown to be consistent with multiphasic kinetics. The concentrations at which transitions occurred were not affected by the K status, indicating the existence of separate uptake and transition sites. Uptake was markedly reduced in the presence of 10^?5M 2,4-dinitrophenol, especially at low K⁺ concentrations, but the isotherms remained multiphasic. This contraindicates major contributions from a non-carrier-mediated, passive flux. A tentative hypothesis for multiphasic ion uptake envisions a structure which changes conformation as a result of all-or-none changes in a separate transition site. The structure is “tight” at low external ion concentrations (low V_max. low K_m. active uptake, allosteric regulation) and “loose” at high concentrations (high V_max- high K_m- facilitated diffusion, no regulation).     

Membrane ATPase Activity of Barley Roots and Potassium Uptake by Isolated Stele and Cortex

Uptake of potassium ions by isolated stelar tissues of barley from 0.5 and 10 mM K⁺ was respectively 13 and 3.6% of that of the cortical tissues. 0.1 mM H₂PO₄, LO mM ATP and 10 mM Ca(NO₃)₂ did not increase the potassium uptake of either stele or cortex during 5 h of uptake period. A time-course incubation for histological demonstration of the ATPase activity of the plasmalemma and tonoplast of the matured sections of the roots demonstrated a greater activity for the cortical than the stelar tissue. In the stelar parenchyma cells, the plasma lemma showed a higher activity than the tonoplast. These results, which support the “leakiness hypothesis” of the stele, are discussed in relation to the proposed mechanisms of radial ion transport in roots.     

Characteristics of Zinc Uptake by Barley Roots

The linear uptake of zinc by excised barley roots (Hordeum distichon L.) in the time range from 20–120 min is not continued over periods of 20–28 h. In concentration dependent uptake experiments with intact barley roots three phases in the range up to 1.38 mM zinc could be detected independent of the tested uptake period. The kinetic constants increased with higher phases and the transition points were lowered with increasing time. The presence of copper did not inhibit the uptake of zinc competitively. On the contrary a slight stimulation in the uptake rates was observed indicating an interaction with the transition site. It is concluded that zinc and copper are taken up by separate mechanisms.     

Invalidity of the multiphasic concept of ion absorption in plants

Abstract. According to the multiphasic concept uptake isotherms consist of a number of successive Michaelis-Menten phases separated by discontinuous transitions. In this paper evidence is provided that the concept is most probably due to an erroneous interpretation of continuous uptake isotherms that yield non-linear, concave downward Lineweaver-Burk plots. With reference to the isotherm of sulphate uptake in barley roots (Nissen, 1971) uptake isotherms were simulated in which the uptake rates, computed from the sum of two Michaelis-Menten terms and a linear term, contained a 4% (or 5%) random error. The same arguments that have been adduced in support of the multiphasic concept would also lead to a multiphasic interpretation of the simulated isotherms. The conclusions concerning a multiplicity of transition points and the multiphasic concept follow from the particular form of statistical test that had been applied rather than from the set of data itself. Characteristics that have been noted in multiphasic uptake appear to follow simply from the multiphasic interpretation of uptake isotherms that yield non-linear, concave downward Lineweaver–Burk plots.     

Mechanisms of Sodium and Rubidium Uptake by Excised Barley Roots

Accumulation of sodium and rubidium by excised barley roots was investigated. The concentration isotherm yielded one absorption shoulder. Nevertheless, it is suggested that two mechanisms take part in the uptake of sodium and rubidium: One non-metabolic mechanism with an apparent participation at low external salt concentrations (< 1 mM) and at high concentrations (> 20 mM). Such a mechanism is almost unaffected by low temperature conditions and by metabolic inhibitors. Rubidium possesses a high affinity toward this non-metabolic system. The second mechanism is sensitive to metabolic inhibitors and to low temperature conditions. It dominates at intermediate external concentrations (1–20 mM). Sodium possesses high affinity towards this mechanism. The two mechanisms operate in a parallel manner beyond a diffusion barrier (= plasmamembrane) surrounding the cells. It is assumed that both the metabolic and the non-metabolic mechanisms operate in the entire concentration spectrum, but their relative contribution to the total uptake varies at different ranges.     

Multiphasic Uptake of Amino Acids by Barley Roots

Concentration-dependence and other characteristics of uptake of ³H-labeled l -lysine, l -methionine and l -proline by excised roots of barley (Hordeum vulgare L.) were studied. Use of relatively short uptake and wash periods and low solute concentrations ensured good estimates of influx across the plasmalemma. Uptake in the range of 10^?7M– 6.3 × 10^?3M can be precisely represented by four or five phases of single, multiphasic mechanisms. The mechanisms appear to be relatively specific as judged from the competition by unlabeled analogues. Structural requirements for interaction of a compound with the uptake site for methionine are given, as are the effects of analogues on the phase pattern for this amino acid. There is no indication of separate uptake and transition sites for methionine or lysine. i.e. phase transitions seem in this case to be caused by binding of molecule(s) to the uptake site. Uptake, but not phase patterns, was highly pH-dependent. The optima were pH 5 for lysine, pH 3–5 (a broad peak) for methionine and about pH 5.5 for proline. Uptake of the three amino acids was strongly inhibited by 2,4-dinitrophenol. sulfhydryl reagents and deoxycholate.     

Ion Uptake Efficiency of Sunflower Roots

The term ion uptake efficiency is used for the rate of uptake of a particular ion from nutrient solutions holding a standard concentration of that ion (0.5 mM sulphate, 1.5 mM phosphate or 2.0 mM rubidium). The uptake efficiency for rubidium and phosphate in roots of intact sunflower plants depended on the salt status of the plants and on the concentration of the ion under investigation in pretreatment solutions. The effect of pretreatment was a rapid process causing differences of more than 300% in ion uptake efficiency within 1 h, depending on the composition of the pretreatment solution. At concentrations above 0.1 mM the rate of uptake of rubidium in the root was higher than the net potassium uptake necessary for adequate growth. The rate of sulphate uptake was related to potassium uptake but not to phosphate uptake. It is suggested that ion uptake of the roots is regulated by internal factors as well as by direct interactions between the medium and the absorbing surfaces.     

Multiphasic Uptake of Sulfate by Barley Roots

Uptake of sulfate by excised barley roots increases upon their washing in aerated water or dilute CaCl₂ solutions. Washing increases the values for V_max and the sulfate concentrations required for transition between the lower phases, but the K_M-values remain essentially constant. At low sulfate concentrations, phase transitions do not occur in the absence of calcium or other divalent cations. These ions are about equally effective in enhancing short-term sulfate uptake. Phase transitions were not principally altered by sulfhydryl or protein reagents. These concentration-dependent transitions appear unrelated to temperature-dependent phase transitions as evidenced by similar multiphasic patterns at low and high temperature.     

Uptake of Sulfate by Roots and Leaf Slices of Barley: Mediated by Single, Multiphasic Mechanisms

The uptake of 10^?1M–2.5 × 10^?1M sulfate by roots and leaf slices of barley can be described by a single isotherm having, respectively, 8 and 5 phases with regularly increasing kinetic constants. Each phase covers a limited concentration range and obeys Michaelis-Menten kinetics. The uptake of sulfate is proposed to be rate-limited by a single mechanism or structure which is located in the plasmalemma (or cytoplasm) and which changes characteristics at certain discrete external concentrations (inflection points). Examination of published data indicates that the uptake of other inorganic ions by higher plant cells is also mediated by single, multi-phasic mechanisms.     

PHOSPHATE UPTAKE BY THE OCEANIC DINOFLAGELLATE PYROCYSTIS NOCTILUCA1

Phosphate uptake by P-replete and P-depleted Pyrocystis noctiluca (Murray) Schütt grown at different ambient N:P ratios was multiphasic between 0.1 and 100 μM PO₄^3-. Within each of the kinetic phases, the saturated uptake rate (V_max), but not the half saturation constant (K_m) was affected by the cellular-P status and light. Uptake rates in the dark were ca. 50% of that in the light and respiratory activity accounted for the observed basal uptake. The combination of multiphasic uptake, and the uncoupling of short term transient uptake from growth resulting in maximum specific uptake rates of 50 h^?1 may help explain the abundance of P. noctiluca in oligotrophic regions.     

Multiple or multiphasic uptake mechanisms in plants?

Abstract Borstlap (1983) has alleged that (a) there are no abrupt changes in curves for the concentration dependence of solute uptake in plants, and (b) many such uptake isotherms may be described by the sum of two Michaelis-Menten terms and a linear term. These claims are considered in detail in connection with the recent finding (Nandi, Pant & Nissen, 1987) that phosphate uptake by corn roots increased more rapidly within the higher phases, i.e. at high external phosphate concentrations, but also levelled off faster than predicted from Michaelis-Menten kinetics. Similar deviations are, in retrospect, also found for uptake of other solutes and result in fewer phases at high external solute concentrations. The simplified and strikingly similar multiphasic patterns in the present paper show that (a) the abrupt changes in published isotherms are not due to error in the data, and (b) uptake isotherms cannot be adequately represented by the sum of two Michaelis-Menten terms and a linear term, or by similar continuous functions, if sufficiently detailed and precise data are used. These findings are not consistent with the existence of multiple uptake mechanisms, including free diffusion, in the plasmalemma. Uptake occurs instead by a single, multiphasic mechanism for each solute or group of related solutes. The similarities in the multiphasic patterns indicate, furthermore, that influx of the various solutes may be coupled.     

Effect of calcium on sulfate uptake by barley roots

Abstract Sulfate uptake by excised roots of barley (Hordeum vulgare L.) was maximal in the presence of about 3x10^-3M CaCl₂. Kinetic studies contraindicate a stoichiometric binding of calcium to the carrier for sulfate, in contrast to findings of Cuppoletti and Segel (Biochemistry 14: 471–4718, 1975) for the filamentous fungus Penicillium notatum. In barley, calcium affects the K_m but not the V_max for sulfate uptake, presumably by altering the conformation and, thereby, the affinity of the carrier. Calcium also affects the transition site for sulfate uptake.     

Distribution of 45Ca in the superior cervical ganglion of the cat

Superior cervical ganglia isolated from immature cats accumulated 0.9 ng atoms of ⁴⁵Ca per mg wet weight during 10-min incubations at 37°C; when expressed as an equivalent volume of medium the accumulation was four times the uptake of ³H-inulin. Orthodromic stimulation of the ganglia doubled ⁴⁵Ca accumulation, whereas excitation with 50 mM KCl, 5 mM glutamate, or antidromic stimulation increased accumulation by one-half. Hexamethonium reduced the increment in ⁴⁵Ca accumulation due to orthodromic stimulation only, but another ganglionic blocking agent, tetraethylammonium, did not reduce accumulation in any case. Both agents blocked ganglionic transmission monitored electrophysiologically. To resolve this discrepancy, and to approach the localization of ⁴⁵Ca within the ganglia, the efflux of previously accumulated ⁴⁵Ca was examined. The data could be fitted by an equation incorporating the sum of three exponentials, representing a rapidly exchanging compartment plus two more slowly exchanging ones. The latter two appeared to reflect the pre- and postganglionic elements in the ganglia: ⁴⁵Ca content of the “preganglionic” compartment was increased by orthodromic but not by antidromic stimulation, and was not decreased by either blocking agent; conversely, ⁴⁵Ca content of the “postganglionic” compartment was increased by both orthodromic and antidromic stimulation, and was decreased by both blocking agents after orthodromic stimulation. The lack of effect of tetraethylammonium on the whole ganglion resulted from an increase in “preganglionic” accumulation that offset the “postganglionic” decrease. After preganglionic denervation, the ⁴⁵Ca content of the “preganglionic” compartment was reduced by two-thirds, while the ⁴⁵Ca content of the “postganglionic” compartment was unchanged. Chemical stimulation increased ⁴⁵Ca accumulation in both compartments. Diphenylhydantoin, 0.1 mM, decreased the increment in ⁴⁵Ca accumulation due to electrical stimulation and to 50 m^M KCl; this inhibition occurred in the “preganglionic” compartment (and perhaps also in the “postganglionic”), and was accompanied by an increased efflux of ⁴⁵Ca.     

Nitrite Uptake by Nitrogen-Depleted Wheat Seedlings

Intact, 14-day-old nitrogen-depleted wheat (Triticum vulgare cv. Blueboy) seedlings were exposed to solutions of 0.5 mM KNO₂, 0.05 mM CaSO₄ and 1 mM sodium 2-[N-morpholino]-ethanesulfonate, pH 6.1. Nitrite uptake was determined from depletion of the ambient solution or from incorporation of ¹⁵N in the tissue. An initial nitrite uptake shoulder was followed by a relatively slow uptake rate which subsequently increased to a substantially greater rate. This accelerated phase was maintained through 24 h. Nitrite accumulated to a slight extent in the root tissues during the first few hours but declined to low values when the accelerated rate was fully developed, indicating an increase in nitrite reductase activity paralleling the increase in nitrite uptake capacity. About 50% of the nitrogen absorbed as nitrite was translocated to the shoots by 9–12 h. Development of the accelerated nitrite uptake rate was restricted in excised roots, in intact plants kept in darkness, by 400 μg puromycin ml^?1 and by 1 mM L-ethionine. When puromycin and L-ethionine were added after the accelerated phase had been initiated, their effects were not as detrimental as when they were added at first exposure to KNO₂. The two inhibitors restricted translocation more than uptake. The data indicate an involvement of protein synthesis and a requirement for movement of a substance from shoots to roots for maximal development of the accelerated nitrite uptake phase. A requirement for protein synthesis in the transport of soluble organic nitrogen from roots to shoots is also suggested.     

Kinetics of Potassium and Magnesium Uptake by Intact Soybean Roots

The kinetics of uptake of K⁺ and Mg²⁺ were studied by using intact soybean [Glycine max (L.) Merr. cv. Amsoy] roots. Uptake of K⁺ in the concentration range 1.29 × 10^?5 to 1.82 × 10^?3 M can be represented by two phases of a single, multiphasic mechanism. Similarly, uptake of Mg²⁺ in the concentration range 4.10 × 10^?6 to 2.49 × 10^?4M was biphasic.     

Multiphasic Uptake of Sulfate by Barley Roots I. Effects of Analogues, Phosphate, and pH

For sulfate uptake by barley roots, competition studies reveal that uptake and phase transitions are caused by interaction of ions with separate sites on or in the plasmalemma. Uptake is competitively, and unequally, inhibited by sulfate analogues but not by other divalent anions. In contrast, divalent phosphate and di- and trivalent pyrophosphate are equally effective in causing transitions. Phosphate is taken up mainly or entirely as H₂PO₄^? by a similar but separate multiphasic mechanism. At pH 8, sulfate uptake is mediated by fewer phases than at low and intermediate pH.     

Uptake of Proteins by Plant Roots

The patterns of uptake of fluorescein-labelled lysozyme (Fl-lysozyme) by barley, maize, onion, tomato and vetch are similar as revealed by fluorescence microscopy. Penetration of the root cap and through the epidermis into the cortex increases with time of exposure and decreases with higher salt concentrations. In fact, one molar ethylammonium chloride can remove most of the absorbed protein from treated roots and the space observed to be stained by Fl-lysozyme in this manner can be visualized as “free space”. Results with sterile and non-sterile barley roots were indistinguishable. At low ionic strength, Fl-lysozyme can penetrate cells and complex with nucleoli. Such cell protoplasts appear “coagulated”. Uptake results with fluorescein per se were unlike those with protein. The uptake of a much larger molecule, ferritin, is confined to the epidermis and root cell walls. Localized, absorbed protein and root growth inhibition by basic proteins have yet to be related.     

Quasielastic light scattering by biopolymers. V. Interparticle interactions between polynucleosomes

Quasielastic light scattering and electrophoretic light scattering experiments were performed on chicken erythrocyte polynucleosome solutions at various temperatures and ionic strengths. The apparent diffusion coefficient, D_app, was found to depend on the scattering vector K. In general, D_app can be described as a damped oscillatory function of K in the ionic strength range of 10 to 60 mM and over the temperature range of 10 to 40°C. Electrophoretic light scattering studies on total digest chromatin samples indicate the apparent charge on the polynucleosomes increases as the ionic strength is lowered from 10 to 1 mM. These data are interpreted in terms of fluctuations in the surface charge distribution of the polyion and subsequent inducement of an asymmetric distribution of small ions about the polyion. These fluctuation components lead to the formation of “clusters” of polyions.     

Evidence for channel mediated transport of boric acid in squash (Cucurbita pepo)

Boron (B) is taken up by plant roots as undissociated boric acid which is a non-electrolyte of similar size to urea and other non-electrolytes. In animal systems, non-electrolytes are transported across biological membranes through aquaporins or through non-aquaporin channels. In artificial lipids boric acid is known to diffuse directly through the lipid bilayer at a rate that is determined by lipid composition. A possible role for channel proteins in in-vitro B uptake is suggested by recent work in which B uptake into isolated membrane vesicles was inhibited by channel blockers and by demonstration that the expression of the plant channel protein PIP1 in Xenopus oocytes increases boric acid uptake by 30%. This study examines whether B transport is a channel-mediated process in intact plants. In the presence of the channel inhibitors HgCl₂, phloretin, and DIDS, B uptake by squash plants was reduced by 40–90% by HgCl₂ (as HgCl₂ varied from 50 M to 1 mM), 44% by phloretin (250 M) and 58% by DIDS (250 M). The effect of Hg ions on B uptake was reversed by 2-mercaptoethanol. The addition of other non-electrolytes in size ranges similar to boric acid inhibited B uptake to various degrees. Addition of urea resulted in 54% decrease in B uptake, while, acetamide, formamide, thiourea and glycerol inhibited uptake by 50, 35, 53 and 44%, respectively. The effect of HgCl₂ on B uptake was greater at high B concentrations than at low B concentrations. These data and information from in-vivo studies suggest two possible mechanisms of B uptake: passive diffusion through lipid bilayers and channel-mediated transport.