Vacuolar and Cytoplasmic Potassium Concentrations in Pea Roots in Relation to Cell-to-Medium Electrical Potentials.

The cause of the cell-to-medium electrical potentials of plant and animal cells is incompletely understood. In some cases, the potentials appear to be diffusion potentials in which potassium ions play a major role (3, 7.) This is indicated A) by potentials which are close to the Nernst potentials for potassium and B) by cell-to-medium potentials which become markedly less negative when the external potassium ion concentration is increased (2, 3, 5, 6, 8, 13). Some evidence indicates that the cell-to-medium potentials are not potassium diffusion potentials. In the squid axon, for example, a large potential remains after the axoplasm has been replaced with a potassium-free solution (14) [There is conflicting evidence here however (1)]. Similar results have been found in the case of vacuolar perfusion of Halicystis and Nitella cells (2,15). The cause of the cell-to-medium electrical potentials of higher plants has not been extensively investigated. Evidence for Avena roots indicates that most of the potential difference occurs between the cytoplasm and the medium, that a negligible potential change occurs between the cytoplasm and the vacuole, and that the cell-to-medium potentials of Avena coleoptiles become less negative when the potassium concentration in the medium is increased (5). Also, flame photometric analysis of extracts from roots and tops of pea and oat seedlings show that the potassium concentrations in the cells are much higher than the concentrations of other inorganic ions (7). The above factors indicate that the cell-to-mediuiii potentials could be diffusion potentials caused principally by the cell-to-medium potassium concentration ratios. In pea roots and stems, however, the cellular potassium concentrations under some conditions did not appear to be high enough to be a major factor in a diffusion potential svstem i(4). This finding has to be qualified, however, because only the average cellular potassium concentration was measured and not the specific cytoplasmic and vacuolar concentrations. Because the vacuole is large relative to the cytoplasm, the cellular potassium concentration probably approximates the concentration in the vacuole. If the major potential difference is between the cytoplasm and the medium, as the data for Avena roots suggest, the critical potassium concentration would be in the cytoplasm. In the experiments reported here, an attempt was made to measure this value. Root tissues were obtained from dark-grown 7-day-old Pisurn sativum cv. Alaska seedlings. They had grown for 4 days with their roots in an aerated nutrient solution containing (in mmoles per liter) K0CI, 10; Ca(N03 )2, 10; NaH,PO4, 9.05; Na2HPO4, 0.48; MgSO4, 2.5; (pH 5.3). Previous treatment included washing the seeds and soaking them for 4 hours in distilled water, followed by a 4-day germination period on moistened filter paper. The temperature was 250 for seedling growth and subsequent experimental operations. The potassium concentrations in the cytoplasm and vacuoles of the root cells were derived from measurements of the potential difference between potassium-sensitive and reference microelectrodes which were inserted with micromanipulators into the cytoplasm or vacuole of cortical parenchyma cells. Electrode insertion was approximately perpendicular to the transverse cut surface (5 mm behind the tip) of a root which was in the above nutrient solution in a lucite chamber. Electrode placement was observed with a microscope having a 40X long-working-distance objective (Unitron Company). The potassium-sensitive microelectrodes were prepared from 'Corning NAS 27-4 glass by a method similar to that used by Khuri and co-workers (9). The 1 mm glass tubing was made into microcapillaries with a microelectrode puller like the model PE-2 of Narishige Scientific 'Company. Open tips of the capillaries (diameter less than 1 u) were sealed with a microforge and the tip was assumed to be sealed if water would not move into it by capillarity.