fluxes across the plasmalemma and the tonoplast. In the present study we report estimates of compartmental concentrations and fluxes in oat coleoptile tissue and present evidence that ion selectivity resides primarily in the plasmalemma. MATERIALS AND METHODSOat (Avena sativa L. cv. Victory) coleoptile segments were chosen for this work because they are cylinders averaging about six cells in thickness; thus the problems associated with diffusion from more deeply seated cells are minimized. Seedlings were grown in darkness at 25 C with a 20-min exposure to weak red light at 72 to 76 hr and coleoptiles were harvested at 96 hr. The substrate was vermiculite watered generously with a nutrient solution having the following composition in mM: KCI, 10; NaH2PO4, 9.04; Na2HPO4, 0.48; Ca(NO3)2, 10; MgSO4, 2.5.The pH was 5.3 to 5.5. The solution used for tracer loading and for efflux had the same composition.The upper 0.5 cm of the coleoptile was discarded, and the subjacent 2-cm portion-after removal of the enclosed leaf-was cut into 2.5-mm segments. These segments were pretreated in unlabeled nutrient solution for 4 to 8 hr except in experiment II where isotope loading began immediately upon completion of cutting. Each tissue sample consisted of about 35 segments weighing approximately 0.8 g. In two experiments, one done in duplicate, tracer loading was with 42K and 22Na; in another experiment done in duplicate, loading was with 42K and 36Cl. The specific radioactivity of the labeling solution at the start of washout was about 20,000 cpm,',ueq for 42K and 22Na; and for 36C1 about 5,000 cpm, 'geq. The loading period was 12 or 16 hr with each sample in a 125-ml flask containing 50 ml of solution agitated on a reciprocating shaker at 20 C. At the end of this period the segments were screened, gently blotted, then placed without rinsing into a cylindrical filter tube having a chamber of 29 mm diameter by 70 mm high, fitted with a drainage tap 8 mm in diameter; these also were mounted on a shaker. Aliquots of unlabeled nutrient solution (about 10 ml) were added and drained off periodically to provide measurements of tracer loss with time. Initially the washout periods were about 30 sec each but were gradually increased to 2 hr ( Figs. 1 and 2). The sum of the radioactivity values of the washout samples plus that remaining in the tissue at the end provides the measure of activity at to (i.e., at start of efflux).For assay of isotopes each drainage sample was evaporated to dryness in stainless steel planchets and counted with a NuclearChicago automatic planchet changer and D-47 gas flow detector. When "fCl was present, tris was added to prevent volatilization of Cl-. Initial counts included both 42K and 22Na or 42K and 36C1. Seven to 10 days later, after complete decay of 42K, another count was made of the 22Na or 36C1; thus the difference represented 42K activity only. All 42K counts were corrected for decay.Chemical assays of parallel tissue samples were made at the start of tracer loading, at the start of efflux, and at the ...

Summary. The relationships of concentration gradients to electropotential gradients resulting from passive diffusion processes, after equilibration, are described by the Nernst equation. The primary criterion for the hypothesis that any given ion is actively transported is to establish that it is not diffusing passively. A test was made of how closely the Nernst equation describes the electrochemical equilibrium in seedling tissues. Segments of roots and epicotyl internodes of pea (Pisumn sativum var. Alaska) and of roots and coleoptiles of oat (Avena sativa var. Victory) seedlings were immersed and shaken in defined nutrient solutions containing eight major nutrients (K+, Na+, Ca2+, Mg2+, Cl-, NO3-, H2PO4-and SO42-) at 1-fold and 10-fold concentrations. The tissue content of each ion was assayed at 0, 8, 24, and 48 hours. A near-equilibrium condition was approached by roots for most ions; however, the segments of shoot tissue generally continued to show a net accumulation of some ions, mainly K+ and NO3-. Only K+ approached a reasonable fit to the Nernst equation and this was true for the 1-fold concentration but not the 10-fold. The data suggest that for Na+, Mg2+, and Ca2+ the electrochemical gradient is from the external solution to the cell interior; thus passive diffusion should be in an inward direction. Consequently, some mechanism must exist in plant tissue either to exclude these cations or to extrude them (e.g., by an active efflux pump). For each of the anions the electrochemical gradient is from the tissue to the solution; thus an active influx pump for anions seems required. Root segments approach ionic equilibrium with the solution concentration in which the seedlings were grown. Segments of shoot tissue, however, are far removed from such equilibration. Thus in the intact seedling the extracellular (wall space) flttid must be very different from that of the nutrient solution bathing the segments; it would appear that the root is the site of regulation of ion uptake in the intact plant although other correlative mechanisms may be involved.The significance of cell electropotentials. PDs, as an important force having definite relationships to ionic gradients in plant cells has been discussed recently by Dainty (4) and by Briggs, Hope, and Robertson (3). The existence of cell transmembrane PDs in higher plants has been established (6,7,10,11,12,20)

No abstract

Cyanide (CN) and dinitrophenol (DNP) rapidly depolarize the cells of oat coleoptiles (Avena sativa L., cultivar Victory) and of pea epicotyls (Pisum sativum L., cultivar Alaska); the effect is reversible. This indicates that electrogenesis is metabolic in origin, and, since active transport is blocked in the presence of CN and DNP, perhaps caused by interference with ATP synthesis, that development of cell potential may be associated with active ion transport. Additional evidence for an electrogenic pump is as follows. (1) Cell electropotentials are higher than can be accounted for by ionic diffusion. (2) Inhibition of potential, respiration, andactive ion transport is nearly maximal, but a potential of -40 to -80 mV remains. This is probably a passive diffusion potential since, under these conditions, a fairly close fit to the Goldman constant-field equation is found in oat coleoptile cells.

Measurements of the difference in electropotential between the interior of the cell and the external solution have been made for the first time in cells of several crop plants (1). The interiors of cells of Avena, Pisum, and Zea seedling tissues all have potentials of about -80 to -115 mv relative to that of an external solution of 0.1 mmole of KC1 per liter, bathing the tissue. The potential difference of Avena coleoptiles varies with the concentration of external KC1 and is depressed by 2,4-dinitrophenol. The potential difference occurs between the cytoplasmic layer and the exterior; the potential of the vacuole does not appear to be significantly different from that of the cytoplasm. Obviously a relatively large cation accumulation ratio could be accounted for in plant cells by this large potential without invoking a chemical cation transport scheme.

Transmembrane electropotential difference (PD) was measured in whole roots of barley (Hordeum vulgare L. cvs. Compana and Himalaya). Seedlings were grown 4 to 5 days in aerated 0.5 mM CaSO4 or a nutrient solution. Measurements of PD were made with roots bathed in CaSO4, KCI + CaSO4, or the nutrient solution. The following results were found. (a) There was a radial PD gradient with epidermal cells being 10 to 58 millivolts less negative than ceUls in the third layer of the cortex (outside to inside). There was no longitudinal PD gradient in the region 0.5 to 4 cm from the root tip, nor was there any difference between the PD of young root hairs and other epidermal cells. (b) Cell PD in excised whole roots was not detectably different from that found in roots attached to the shoot, and was unchanged for 2 hours from excision. (c) In 1-centimeter sections of root, cel PD at the freshly cut surface was depolarized by 90 millivolts from that in the intact root; cells farther than 1 millimeter from the cut surface were not depolarized. The PD of ceUls at the cut surface became more negative upon aging the segment in 0.5 mM CaSO4, eventually becoming greater by -25 milivolts than that in cells of intact roots. Cells in segments to which the root tips were attached had less negative PDs after aging than those in subapical segments, indicating a possible hormonal effect. PDs in aged, excised segments are not equivalent to those in intact roots. (d) Creeping of cytoplasm over electrode tips inserted into the vacuole gave measurements of vacuole-tocytoplasm PD of +9 millivolts in 0.5 mM CaSO4 and +35 millivolts in 1 mM KCI + 0.5 mM CaSO4. Most of the cel PD was across the plasmalemma. (e) The reducing supr content of roots in CaSO4 solution was greater than that of roots in the nutrient solution in which ion uptake, parficularly K+ occurred. Many electrophysiologists measure the transmembrane electropotential difference in slices or segments of plant organs such as roots, stems, and leaves. The segments are often aged for periods varying from none to several hours after excision in order to minimize possible side effects due to injury to the tissue during excision. This procedure has led to other problems. For example, in a study using 1-centimeter segments of low-salt barley roots (24), the PD4 of cortex cells along the cut surface became significantly more negative with time after exci- (24). Seeds were surface-sterilized for 20 min in 1% NaOCI, rinsed in deionized H20, and germinated for 24 hr at 20 C in the dark in aerated deionized H20. Germinated seedlings were grown hydroponically at 20 C in the dark for an additional 3 to 6 days. For most studies, the growing solution was 0.5 mm CaSO4 (pH 5.5). Deionized H20 and a nutrient solution referred to as IX (13) were used in a few experiments. The IX solution contained in mM: I KCI, I Ca(NO3)2, 0.25 MgSO4, 0.904 NaH2PO4, and 0.048 Na2HPO, (pH 5.5).The hydroponic growth chamber, illustrated elsewhere (18), was constructed as follows. The base was a 35 x 30 x 14 ...

A number of studies have shown that an electropotential difference, PD, of about 100 mv (interior negative), is present across plant cell membranes (2,3,7,8,10,11,13,21), similar to that across animal celtl membranes. Thus, if minerals move through cell membranes as ions, they are subject not only to concentration gradients but also to electrical fields.

A study has been made of the effects of the inhibitors carbonylcyanide n-chlorophenylhydrazone (CCCP), 3-(3,4-dichkophenyl)-1,14dnethyl urea (DCMU), and of anoxia on the lgt-sensitive membrane potential of VaNsneria leaf cells. The present results are compared with the known effects of these inbibitors on ion transport and photosynthesis (Prins 1974 Ph.D thesis). The membrane potential is composed of a diffusion potential plus an electrogenic component. The electrogenic potential is about -13 millivolts in the dark and -80 millivolts in the light. The inhibitory effect of DCMU and CCCP on the electrogenic mechanisms strongly depends on the light intensity used, the inhibition being less at a higher Light intensity. This is of significance in view of the often conflicting results obtained with these inhibitors. With The present results show that the sensitivity of the membrane potential to inhibitors depends very much on the light intensity used. The meaning of this will be discussed. For Nitella it has been shown that the more negative potential in the light is caused by an electrogenic pump (19,20). Generally, it is assumed that this electrogenic pump is a proton extrusion pump; this may be true for Vallisneria also. MATERIALS AND METHODSPlants of V. spiralis were grown aquatically in a plastic tank on a slightly alkaline soil at room temperature (about 20 C).Leaf strips of 20 x 4 mm were mounted in a modified Mertz chamber (12), except for experiments on the effect of 02, in which small (2-mm) leaf strips were used from the margins of the leaves which are devoid of gas-filled intercellar spaces. By doing this a more rapid exchange of 02 between the cells and the medium was obtained. Cells of the epidermis or outer layer of mesophyll were used. Membrane potentials were measured using 3 M KCl-filled glass microelectrodes. The cells were too small to use the classic two-electrode method for membrane resistance measurements, therefore electrical resistances were measured with the single electrode method of Anderson et al. (1), using 10-kHz current pulses between +5 and -5 namp. Over this range the resistance was ohmic and no rectification was observed. Resistances lower than 1.5 Mgl could not be measured accurately with the set-up used.Despite the recent criticism on this method (6)