The Initial Uptake of Ions by Barley Roots: III. THE UPTAKE OF CATIONS

Experiments have been carried out to determine the extent towhich rubidium and strontium, recently absorbed by excised barleyroots, can be divided into discrete fractions, namely, ionswhich are extractable with water, those which are exchangeable,and those remaining in the tissue at the end of the experiment.The object of this study was to determine whether the behaviourof cations could be validly interpreted in terms of the electricaldouble-layer theory as had been previously demonstrated foranions. To facilitate the interpretation of the results obtained, comparableexperiments were carried out on a cation-exchange resin andon gelatin. The behaviour of strontium accorded qualitatively with the theoryproviding allowance was made for effects due to the hydrolysisof negative sites and for the presence of positive charges inthe roots. For rubidium, on the other hand, the situation was much morecomplex. Not only was there evidence even at 0.2°C for siteswhich retained a high specificity for this ion, but also movementof the ion due to physical diffusion was masked by concurrentmetabolic activity. It was concluded that a meaningful separation of the over-alluptake of rubidium into successive steps of exchange absorptionand metabolic accumulation cannot be made.     

The Initial Uptake of Ions by Barley Roots: II. APPLICATION OF MEASUREMENTS ON SORPTION OF ANIONS TO ELUCIDATE THE STRUCTURE OF THE FREE SPACE

In previous experiments on excised barley roots, results werepresented showing the effect of varying the concentration ofthe uptake solution on the amount of anion which could subsequentlybe extracted in water. It was concluded that the system couldbe more satisfactorily analysed by means of the Goüy–Chapmanelectrical double-layer theory than by the Donnan theory. Onthe basis that ions diffuse into a system of negatively chargedpores within the root, the relationship between the quantityof water-extractable anion and its concentration in the uptakesolution has been examined in the light of the electrical double-layertheory. For potassium iodide and sulphate the experimental resultsconformed reasonably well with a structural model for the FreeSpace of parallel, charged surfaces separated by a mean distanceof about 200 A. The internal surface area of the free spacewas estimated as 2x10⁶ cm²/g dry weight. The uptake of iodidefrom calcium iodide was somewhat greater than that predictedby the theory; this may be ascribed to interaction between theanions and divalent cations within the double layer.     

Boron Uptake by Excised Barley Roots

Active uptake of boron (B) by excised barley roots is linear with time for at least 1.5 h. Although no evidence was found for accumulation of B against a concentration gradient. this component of B uptake does satisfy other criteria for an active transport process. Transport is inhibited by 0.05 mM 2,4-dinitrophenol, 0.05 mM azide, 5 mM arsenate and 5 mM dicoumarol. Also, uptake is temperature-sensitive, being nil at 2°C and maximal at 34 to 38°C. Boron uptake by barley roots increases with time when they are washed in aerated 0.5 mM CaSO₄ solution. A double reciprocal plot of the B uptake data manifests a series of phases separated by sharp transitions or “jumps”, and is compatible with the concept of multiphasic uptake mechanisms. Kinetic constants and transition points for the various phases were calculated accordingly. The fit of these data was compared statistically to three other relevant models, viz, the dual model, the “single + diffusion” model (a Michaelis–Menten term and a diffusion term), and the negative cooperativity model. In each case, the data were better represented by the multiphasic model.     

Extrusion of Sodium Ions by Barley Roots I. Characteristics of the Extrusion Mechanism

Evidence is presented for an outwardly directed sodium ion pumpin excised barley roots. The efflux has a Q₁₀ of about 2 andis inhibited by ouabain at concentrations of the inhibitor downto 10^–5 M. A threefold stimulation of the sodium losswas observed both with ATP and inorganic phosphate at concentrationsof 10^–3 M. A stimulatory effect of different concentrationsof DNP on the extrusion of sodium has been observed and is attributedto permeability changes resulting from the use of this inhibitor.It is suggested that the sodium extrusion mechanism reportedhere for excised barley roots is similar to the sodium pumpsfound in animal tissues and certain algal cells.     

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.     

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.     

The effect of Aging on Ion Uptake by Excised Barley Roots

When excised barley (Hodeum Vulgare L.) roots were aged, the rate of uptake of K and C1 increased raching a maximum in about 14 to 18 h. At its maximum the uptake rate was approxiamately twice that of the freshly excised root materila. Respiratory activity declined markedly during the aging period. Although excision was an essential requirement for uptake enhancement, washing was not. Both the uptake and aging processes were shown ot be unresponsive to the presence or absence of Ca in the tratment solutions. Because of cation exchange properties, the change in the total cation content of the root material was a more meaningful measure of the metabolically mediated cation uptake than was the change in content of the test cation itself. The pluggin of xylem vessels with protein and pectin-like substances was observed to increase with aging. It is proposed that the occlusion of the vessels may account for an apparent increaase in uptake since the obstruction could reduce a concurrent loss of ions through the cut ends of the root segments.     

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.     

The Interaction of pH and Aeration in CI Uptake by Barley Roots

The uptake of Cl by excised roots of barley (Hordeum vulgare L.) from KC1 solution maintained at high pH was markedly reduced by high rates of aeration, whereas K uptake was scarcely affected. Aeration rate had relatively minor effects at low pH. The effect of high aeration rate at pH 9 could be overcome by the use of buffered solutions. In unbuffered solutions the H resulting from the excess cation uptake together with that produced from respiratory CO₂ was sufficient to materially reduce the pH of the solution. The reduction in pH favored the uptake of Cl which is adversely affected by high pH. The effect of aeration rate could be explained in terms of root induced pH changes and film diffusion involving the solution film adjacent to the root surface.     

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.     

The Effects of pH and Carbon Dioxide on Ion Uptake by Excised Barley Roots

The effects of carbon dioxide concentrations up to 8 per centin air on uptake of potassium and chloride at two pH levels,nominally pH 6 and 8, werestudied. In all experiments, enhanced uptake of potassium occurred atthe higher pH level with carbon dioxidefree air, but chlorideuptake was generally unaffected. At nominal pH 6, 1 per cent carbon dioxide reduced and 6 percent increased potassium uptake. There was no effect on chlorideuptake except with 1 per cent carbon dioxide where a markedenhancement was recorded. At nominal pH 8, l and 2 per cent carbon dioxide increased potassiumuptake whereas 6 and 8 per cent were inhibitory. Chloride uptakewas favoured by 1 and 8 per cent concentrations of the gas.     

The Uptake of Growth Substances: I. FACTORS CONTROLLING THE UPTAKE OF PHENOXYACETIC ACIEDS BY LEMNA MINOR

An analysis has been made of the factors controlling the uptakeof 2:4-dichloro-phenoxyacetic acid (2:4-D) and related compoundsby Lemna minor, grown under controlled conditions. All compoundswere labelled with ¹⁴C in the carboxyl group and the ¹⁴C inthe plants, after liquid combustion, assayed as barium carbonate. With 2:4-D the rate of entry from concentrations of 8 to 32mg./l. is maximal in the first 20 minutes, then falls progressivelyuntil the rate is zero between 1 and 2 hours and in the thirdphase (up to 24 hours) there is a net loss from the plants dueto an outward movement of the compound back into the externalsolution. When the chlorine substitutions are in the 2:6-positionthe course of uptake is similar, save that loss to the solutiontakes place later. For the 2-chloro-substitution, after theinitial phase of uptake there is some loss but this is followedby a period of recovery and uptake again proceeds, but slowly.In contrast, the course of uptake over 24 hours of phenoxyaceticacid is normal, i.e. there is a steady accumulation. When plants are pretreated with labelled 2:4-D for 30–60minutes and transferred to water or culture solution then over90 per cent. of the ¹⁴C is found in the external medium after4:5 hours and this release cannot be accounted for in termsof exchange processes since the addition of unlabelled compoundto the medium retards the rate of loss. This loss is slowedbut not stopped at 1.25°C. and up to 22.5°C. the rangeof the Q₁₀ is 1.6–1.9. Uptake in the first 30 minutes is temperature sensitive between7.5 and 30°C. (Q₁₀ 2.3–2.6) and in general is positivelyand curvilinearly related to the external concentration. Pretreatmentwith unlabelled compound up to 2 hours progressively depressesthe subsequent initial uptake of labeled growth regulator. Itis concluded that initially the rate of entry greatly exceedsthe rate of loss but that with time the ratio steadily diminishesto less than unity. Initial uptake of 2:4-D is markedly dependent on the pH of thesolution and closely but not completely correlated with theexternal concentration of undissociated molecules. On the otherhand, the outward movement is relatively unaffected by the externalpH. Combinations of concentration and time of exposure which bringabout loss to the solution need not cause any permanent retardationof growth. In fact, exposure for 1 hour to 8 mg./l. significantlyaccelerated growth in the next 8 days. These results are discussed in relation to previous findingsand it is concluded that the pattern of uptake is determinedon the one hand by the chemical structure of the growth substanceand on the other by specific physiological differences at celllevel.     

Constitutive Uptake of Choline Sulfate by Roots and Leaf Slices of Barley

Barley roots take up choline sulfate constitutively via 3 separate, homogeneous mechanisms which obey Michaelis-Menten kinetics and have, respectively, low, high, and very high affinity for the sulfate ester. Leaf slices possess only the low-and the high-affinity mechanism. These are both inhibited by dini-trophenol and NaF but are differently affected by solute analogues. Uptake via the high-affinity mechanism is active; the low-affinity mechanism has also characteristics of facilitated diffusion.     

Extrusion of Sodium Ions by Barley Roots: III. The Effect of High Salinity on Long-distance Sodium Ion Transport

The quantity of sodium transported to the shoots of intact barleyplants was stimulated by 0·5 mM ouabain when the sodiumchloride level of the bathing medium was below 100 mM. At sodiumchloride concentrations of 100 mM or more this ouabain-stimulatedsodium transport was not observed. Equiosmotic mannitol, equimolarpotassium chloride or equivalent calcium chloride solutionsdid-not affect the ouabain-stimulated sodium transport froma basic medium containing 10 mM sodium chloride. It is suggestedthat under the present experimental conditions the increasedsodium uptake by the root cells at sodium chloride concentrationsof 100 mM or more masks the extrusion process.     

Simulation of Cl Uptake by Low-salt Barley Roots as a Test of Models of Salt Uptake

Computing techniques are used to simulate the course of uptake of K⁺, Na⁺, and Cl⁻ by low-salt roots. Measurements of the fluxes of these ions in high-salt roots are used to calculate membrane permeabilities, which are then used to calculate cell uptake. In this way it is possible to test the predictive value of different models for the location of sites of salt uptake in the cell.     

The Nature of the pH EfFect on the Uptake of Rubidium by Excised Barley Roots

The specific effects of pH on the accumulation mechanism of Rb+ by excised barley roots were investigated. Exposure of roots to solutions of a wide range of pH values did not result in any noticeable damage. It is therefore suggested that even pH 3.5 is within the physiological range of pH for barley roots. It is assumed that the inhibitory effect of pH on Rb+ accumulation is due to competition of H ions on binding sites of the temperature dependent metabolic accumulation mechanism. When this mechanism is saturated with Rb ions, no inhibition of H ions occurs, even at pH 3.5. Temperature coefficients provide hints that the inhibitory effects of Na and H ions on Rb+ accumulation are similar.     

Variations in Uptake of Sodium and Rubidium Along Barley Roots

Variations in uptake of sodium and rubidium were ob) served along the apical 8 cm segments of barley roots. Dif) ferent patterns of uptake were obtained for low- and for high-salt roots. The validity of using batches of entire barley roots for kinetic analysis of ion uptake is questioned.     

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.     

The Effect of Cyclopropane on Ion Uptake and Protoplasmic Streaming in Barley Roots

Increasing concentrations of cyclopropane up to about 95 % C₃H₆—5 % O₂ caused an increasing inhibition of uptake by excised barley roots from KC1 solution, much more so for Cl than K. No inhibition of K uptake from K₂SO₄ occurred under similar conditions. It is suggested that the small decrease in K uptake from KC1 is related to the large decrease in Cl uptake and that C₃H₆ primarily inhibits Cl uptake with little or no effect on K uptake. Time curves of uptake in 80 % C₃H₆ 20 % O₂ revealed no indication of cumulative injury in either KC1 or K₂SO₄ for periods up to 3 hours. The amount of organic acid production associated with K uptake from K₂SO₄ was about the same in 80 % C₃H_g— 20 % O₂ as in air. Oxygen consumption in K₂SO₄ was unaffected by the gas mixture and only slightly affected in KC1. Protoplasmic streaming in epidermal cells was rapidly stopped at C₃H₆ concentrations of 20 % or higher. In 80 % C₃H₆–20 % O₂ the effect on streaming was confined mostly to the outer two or three layers of root cells. A connection between the cessation of streaming and the inhibition of Cl uptake appeared to exist but very little if any relationship between streaming and K uptake was observed.