Active and Passive Transport of Potassium in Cells of Excised Pea Epicotyls 1

The Ussing-Theorell equation, which provides a fundamental test for the independent passive movement of ions under conditions of nonequilibrium, has been used to assess the active and passive components of K+ uptake by segments of pea epicotyl (Pisum sativum L. cultivar Alaska), incubated for 24 hours in both 1-fold and 10-fold concentrations of a complete nutrient solution. Mleasurements of the rates at which 41K diffused out of the segments provided data from which were estimated the K+ content of, and the fluxes to and from, the nonfree space compartments, interpreted as being cytoplasm and vacuole. For this analysis the serial model of IacRobbie and Dainty and Pitman for the spatial arrangement of cell compartments was used. On the basis of these values, and measurements of electrical potential across the cell membranes, the vacuolar K+ concentration was found to be fairly close to that expected as a result of passive diffusion between the cytoplasm and vacuole provided that no potential exists across the tonoplast. Cytoplasmic K+ concentration, however, was much too high in both treatments to be accounted for in passive terms. It was concluded, therefore, that, on the basis of the model, the high ratio of influx to efflux was maintained in the cells by an active K+ pump located at the plasmalemma. There is some reason to question the applicability of this model for flux analysis to the conditions of high net influx as encountered here; nonetheless, it provides a first approach to an over-all flux analysis in pea stem tissue. The most straightforward situation in which to assess the significance of cell electropotential, E (=PD), in determining the transmembrane concentration gradient of an ion is one in which the cells under study are at ionic equilibrium. In these circumstances the relationship between E and the concentration gradient of an ion resulting from passive diffusion is described by the Nernst equation: E =-ln ] zjF Cj' (1) where E is the electrical potential difference between the inside 1 This work was supported by National Science Foundation Grant GB 5117X and by funds provided for biological and medical research by the State of Washington Initiative Measure 171. 2 On leave of absence (September, 1966, to September, 1967) from the Macaulay Institute for Soil Research, Aberdeen, Scotland. and outside of the cell; zj is the valance of ion j; R is the gas constant; T, the absolute temperature; and F, the faraday. Cjo and Cji are the external and internal concentrations of the ionj. In a study of the mineral ion contents and cell transmembrane electrical potentials of pea and oat seedlings, Higinbotham et al. (6) found little net change in the content of most ions during the incubation of excised root segments in the same nutrient solution as that in which the plants had been grown. Thus, for these tissues, a comparison between observed ionic concentration gradients and the gradients predicted by the Nernst equation, on the basis of observed PD, gave a good indication of how far passive diffusion accounted for the ionic distribution observed. In contrast to root segments, segments of shoot tissue from these seedlings were found to be far removed from ionic equilibrium when immersed in the nutrient solution, there being considerable uptake of several ions. This is most notable for K+ content in pea epicotyl segments, the time course of which has been further examined by Macklon and Higinbotham (8). In an accumulating system such as this, the Nernst equation alone is not a sufficient criterion for evaluating the active or passive nature of ion transport. Where there is a net flux across a membrane, a better test for the independent passive movement of an ion is given by the following expression, deduced by both Ussing (16) and Teorell (15): Ji CjO A)jo Jo C.z exp (zjFEIRT) H/i (2) where Ji is the flux of the ion j from outside to inside the membrane and Jo is the flux from inside to outside the membrane; Aji is the electrochemical activity of ion j outside (where E, by convention, is zero) and uji (= Cji exp [zj FE/RT]) is the electrochemical activity inside. In order to test whether or not an ion moves in accord with the Ussing flux ratio equation in plant cells, it is necessary to have information on the fluxes, concentration gradients, and electropotential gradients across both the plasmalemma and tonoplast. In this paper we present data from such an analysis ofK+ in pea epicotyls in complete nutrient solutions. The compartmental analysis scheme of MacRobbie and Dainty (10), Pitman (11), MacRobbie (9), and Cram (1) was utilized. The results suggest that another model may be required for a system such as that encountered here in which high net uptake occurs. MATERIALS AND METHODS Pea seedlings (Pisum sativum L. cultivar Alaska) were grown, and samples of etiolated epicotyl segments 1 cm long (except as noted) were taken in the manner described in an earlier paper (8). The nutrient solution used for growing the seedlings and incubating the segments, and for the efflux experiments, was of the following composition in mM: KCI, 1.0; Ca(NO3)2, 1.0; MgSO4, 133 www.plantphysiol. org on June 27, 2017 Published by Downloaded from Copyright © 1970 American Society of Plant Biologists. All rights reserved. MACKLON AND HIGINBOTHAM 0.25; NaH2PO4, 0.904; Na2HPO4, 0.048; pH between 5.5 and 5.8. Used at this concentration, the solution is referred to as 1 X. Experiments were also performed with nutrient solutions of 10 times this strength (10X). Samples of segments, amounting to about 0.6 g, which were to be used for efflux measurements, were pretreated in nutrient solution for 12 hr and then transferred to a similar solution containing the radioisotope 4K for a 12-hr loading period. Parallel samples were used to follow influx of labeled K+ at intervals up to 24 hr, and these samples were also used for chemical assay of K+. Details of the assay procedures are given in a previous paper (8). All experiments were carried out in a constant temperature room at 200. At the end of the 12-hr tracer-loading period, the samples to be used for efflux measurements were drained and placed in a cylindrical filter tube having a chamber 29 mm in diameter by 70 mm high, fitted with a drainage tap 8 mm in diameter. The tubes were