Effect of K + , its Counter Anion, and pH on Sodium Efflux from Barley Root Tips

322

172

The pH-dependent fluorescence quenching of acridine orange was used to study the Na'-and KI-dependent H' fluxes in tonoplast vesicles isolated from storage tissue of red beet and sugar beet (Beta vulgaris L.). The Na'-dependent H' flux across the tonoplast membrane could be resolved into two components: (a) a membrane potential-mediated flux through conductive pathways; and (b) an electroneutral flux which showed Michaelis-Menten kinetics relationship to Na' concentration and was competitively inhibited by amiloride (K1 = 0.1 millimolar). The potentialdependent component of H' flux showed an approximately linear dependence on Na' concentration. In contrast, the K-dependent H' flux apparently consisted of a single component which showed an approximately linear dependence on K concentration, and was insensitive to amiloride. Based on the Na'-and Ktdependent H' fluxes, the passive permeability of the vesicle preparation to Na was about half of that to K+.The apparent K,for Na of the electroneutral Nat/H+ exchange varied by more than 3-fold (7.5-26.5 millimolar) when the internal and external pH values were changed in parallel. The results suggest a simple kinetic model for the operation of the Na+/H+ antiport which can account for the estimated in vivo accumulation ratio for Na into the vacuole.Plant cells typically maintain a high K+/Na+ ratio in the cytoplasm, and there is evidence that Na+ is actively transported out ofthe cytoplasm at both the plasmalemma and the tonoplast membranes (10,14,15,17,24,25). While some moderately sodium-tolerant species depend mainly on exclusion of sodium at the plasma membrane, halophytes such as Beta vulgaris may accumulate large amounts of sodium which are sequestered into the vacuole to serve as osmoticum. Thus, transport of sodium at the tonoplast may be particularly important in these species (9).The mechanism of sodium transport at the tonoplast has not yet been elucidated. There is evidence supporting an H+/Na+ antiport mechanism at the plasma membrane of barley roots (19). The existence of a proton transporting ATPase at the tonoplast of various species (5, 7) including B. vulgaris (2,18,27) suggests the possibility of a similar mechanism for sodium transport into the vacuole. In the present work, we present evidence for an Na+/H+ antiport in tonoplast vesicles isolated from B. vulgaris. Membrane Preparations. Tonoplast vesicles were isolated as described elsewhere (20). For the identification of the membrane preparation as tonoplast, see "Discussion." The vesicles were preloaded with a buffer of desired ionic composition by suspension and sedimentation at 100,OOOg for 30 min. Final membrane pellets were resuspended in the same media to 6 mg protein/ml and incubated at 4°C for 3 h.Fluorescence Assays. The fluorescence quenching of acridine orange was used to monitor the formation and dissipation of inside-acid pH gradients (3, 18) across the membranes of the tonoplast vesicles. In all experiments, tonoplast vesicles (60 yg protein) were added to 2 ml of buffer con...

mentioning

confidence: 99%

Na⁺/H⁺ Antiport in Isolated Tonoplast Vesicles from Storage Tissue of Beta vulgaris

Blumwald¹,

Poole²

1985

322

172

“…3,10,11,16). A general model suggested by Hanson (6) includes three types of transport: (a) active proton efflux which is electrogenic, probably associated directly with activity of an ATPase and possibly coupled closely to active K uptake (see also refs.…”

mentioning

confidence: 99%

Mathematical Analysis of the Dependence of Cell Potential on External Potassium in Corn Roots

Cheeseman¹,

Hanson²

1979

The K+ dependence of normal (4) and diffusion (4,) Following the great rise in popularity of the chemiosmotic hypothesis and noting the great complexity of the transport data across higher plant cell membrane systems, Mitchellian transport systems have been suggested by several investigators (e.g. 3, 10, 11, 16). A general model suggested by Hanson (6) includes three types of transport: (a) active proton efflux which is electrogenic, probably associated directly with activity of an ATPase and possibly coupled closely to active K uptake (see also refs. I and 11); (b) electrophoretic movement (uniport); and (c) neutral exchange systems making use of the chemical gradient for protons. To date, however, no single element of the system has been characterized in such a way as to be useful to the model. The object of the present study was to characterize changes in 4 which occur over a range of external K activities. This is a first step in understanding and separating the electrically non-neutral elements of the models.We have found it useful to analyze (-exp(Ff/RT). As a result we have been able to describe mathematically the behavior of 4 as a function of K°; to show that in the presence of 10 tLM FCCP,2 4 behaves as expected of a diffusion potential; and to characterize 4,p, the deviation of 4, from 4r) due to an electrogenic system. MATERIALS AND METHODS TISSUE PREPARATIONMethods for raising corn seedling [Zea mays L., hybrid (A619 x Oh43) x A632] were previously described (8).

“…Although use has been made of the relatively cytoplasmic cells in root tips (e.g. 23) and in nematode-infected plants (10), these materials are difficult to prepare in quantity and lack homogeneity. On the other hand, cytoplasmic algal cells such as Chlorella (2) After centrifugation, 0.5 ml of the supernatant was diluted to 10 ml, and 1.0 ml of this was assayed for protein by the method of Lowry et al (17).…”

mentioning

confidence: 99%

Regulation of Cytoplasmic and Vacuolar Volumes by Plant Cells in Suspension Culture

Owens¹,

Poole²

1979

This study has three objectives: (a) to investigate a plant cell suspension culture as a course of cytoplasmic cells for transport studies; (b) to assess alternative methods of estimating cytoplasmic and vacuolar volumes; and (c) to make a start toward understanding the process of vacuolation in plant cells, and the control of cytoplasmic volume.Studies of the solute composition of the cytoplasm of plant cells, and of transport across the plasmalemma, have been greatly limited by the fact that the cytoplasm typically occupies such a small proportion of the cell volume. Although use has been made of the relatively cytoplasmic cells in root tips (e.g. 23) and in nematode-infected plants (10), these materials are difficult to prepare in quantity and lack homogeneity. On the other hand, cytoplasmic algal cells such as Chlorella (2)