The Influence of Internal Ion Concentration on Potassium Accumulation and Salt Respiration of Red Beet Root Tissue

It is shown that during a period of washing in serated distilledwater disks of Red Beet root tissue acquire the capacity toabsorb potassium rapidly. Subsequently the rate of accumulationof this ion is closely related to the internal salt-contentof the material. On the other hand, whilst the level of saltrespiration increases during the preliminary washing, it isnot influenced significantly by the internal potassium concentration.The implications of these observations are discussed in relationto a possible mechanism of mineral salt absorption.     

Carrier-mediated Potassium Efflux Across the Cell Membrane of Red Beet

The flux ratio (influx/efflux) of K⁺ across the plasmalemma of beet cells at an external potassium concentration of 0.6 mm does not respond to changes of membrane potential in the manner expected for the free diffusion of ions. The K⁺ efflux is affected by the presence of adsorbed Ca²⁺, but is apparently unrelated to the electrical potential or to the net uptake of potassium. The K⁺ efflux is greater than the efflux of the sulfate and organic anions which are accumulated with potassium, and is partially dependent on the presence of external potassium. Thus the loss of ⁴²K from the cell does not appear to be a leakage of freely diffusing K⁺ ions, nor a leakage of ion pairs, but a carrier-mediated transport or exchange of potassium across the cell membrane.     

Potassium Fluxes during Potassium Absorption by Intact Barley Plants of Increasing Potassium Content

The presence of previously absorbed K in plants caused a marked reduction in the short term influx of ⁸⁶Rb-labeled K into roots of barley seedlings. The influx values agreed with net K absorption rates into intact plants, thus suggesting that K efflux was negligible in comparison with influx.     

The Selective Uptake of Alkali Cations by Red Beet Root Tissue

The selective uptake of alkali cations by red beet root tissuefrom solutions of chlorides has been investigated. It is shownthat when disks are transferred from distilled water to a solutionof salts, there is a rapid initial uptake of cations which isneither particularly selective, nor directly related to metabolism.On the other hand, the prolonged active accumulation of cationsexhibits strong selectivity, Na being preferred to other ions. Evidence is presented to show that the alkali cations competewith one another for the same absorption mechanism. In thisrespect the material apparently differs from that investigatedby Epstein and Hagen, in which the operation of distinct mechanismsfor some of these ions was visualized. The validity of Epsteinand Hagen's conclusion is discussed in relation to the resultspresented here.     

ATP Levels and their Effects on Plasmalemma Influxes of Potassium Chloride in Red Beet

Tissue ATP concentrations in slices of red beet increase progressively with time for up to 7 days after cutting the root. ATP levels are higher in slices taken from stored roots than in slices from fresh roots. ATP is reduced during incubation in salt solutions.     

Syringomycin-Stimulated Phosphorylation of the Plasma Membrane H-ATPase from Red Beet Storage Tissue

The syringomycin-stimulated in vitro protein phosphorylation of the plasma membrane H⁺-ATPase of red beet (Beta vulgaris L.) storage tissue was investigated. Peptides representing the H⁺-ATPase N and C termini and nucleotide binding site (P-2, P-3, and P-1, respectively) were synthesized, and rabbit antisera against each were produced. In western immunoblots of purified plasma membranes, these antisera immunoreacted with the 100-kilodalton polypeptide of the H⁺-ATPase and with other smaller polypeptides. The smaller polypeptides appeared to be degraded forms of the intact 100-kilodalton polypeptide. Immunoprecipitation experiments showed that plasma membranes treated with syringomycin had increased protein phosphorylation rates of the 100-kilodalton polypeptide. Optimal phosphorylation levels were achieved with 25 micromolar free Ca²⁺. Phosphoserine and phosphothreonine were detected in the immunoprecipitates. Washed immunoprecipitates generated with anti-P-1 possessed protein phosphorylation activity. This immunoprecipitate activity was not stimulated by syringomycin, but it was inhibited when plasma membranes were treated with sodium deoxycholate before immunoprecipitation. The findings show that syringomycin stimulates the phosphorylation of the plasma membrane H⁺-ATPase and that specific protein kinase(s) are probably associated with the enzyme.     

The Effects of Ouabain on Sodium and Potassium Fluxes in Excised Root Tissue 2

Washed carrot slices take up more K than Na during accumulationof C1 salts from a mixed salt solution. Ouabain does not affectthe overall influx of labelled K or Na to the vacuole, but inhibitsthe efflux of labelled Na from the vacuole. The simplest hypothesisis the ouabain inhibits a Na efflux pump at the plasmalemma.Some suggestions to explain the transient changes in Na effluxafter the addition of ouabain are put forward.     

Potassium Fluxes in Excised Barley Roots

The method of the modified compartmental analysis for excisedroots has been adopted for measuring K⁺-fluxes and compartmentationin barley (Hordeum distichon) roots. Efflux of ⁴²K and ⁸⁶Rbindicated that more than two intracellular compartments wereinvolved in the tracer exchange; the ⁴²K data clearly showedthe components. On the basis of the efflux behaviour of theapical and more basal tissues of the roots, the three componentsof efflux were attributed to the cytoplasm of differentiated(fast) and meristematic tissues (intermediate) and to the vacuoles(slow exchange) of the roots. A model is proposed on the basisof which, the fluxes corresponding to the meristematic and differentiatedtissues of the root can be estimated. Additionally, fluxes ofthe differentiated root tissues were determined by using effluxdata obtained with root segments without apical tissues. Thedata obtained in both ways compare reasonably well and agreeto independent chemical measurements. Comparison of the ⁴²K and ⁸⁶Rb efflux data show strong discriminationof K^– in favour of Rb⁺ and indicate that ⁸⁶Rb is not suitableas a tracer for K⁺ in efflux measurements, at least with barleyroots.     

Intensive DNA Replication and Metabolism during the Lag Phase in Cyanobacteria

Unlike bacteria such as Escherichia coli and Bacillus subtilis, several species of freshwater cyanobacteria are known to contain multiple chromosomal copies per cell, at all stages of their cell cycle. We have characterized the replication of multi-copy chromosomes in the cyanobacterium Synechococcus elongatus PCC 7942 (hereafter Synechococcus 7942). In Synechococcus 7942, the replication of multi-copy chromosome is asynchronous, not only among cells but also among multi-copy chromosomes. This suggests that DNA replication is not tightly coupled to cell division in Synechococcus 7942. To address this hypothesis, we analysed the relationship between DNA replication and cell doubling at various growth phases of Synechococcus 7942 cell culture. Three distinct growth phases were characterised in Synechococcus 7942 batch culture: lag phase, exponential phase, and arithmetic (linear) phase. The chromosomal copy number was significantly higher during the lag phase than during the exponential and linear phases. Likewise, DNA replication activity was higher in the lag phase cells than in the exponential and linear phase cells, and the lag phase cells were more sensitive to nalidixic acid, a DNA gyrase inhibitor, than cells in other growth phases. To elucidate physiological differences in Synechococcus 7942 during the lag phase, we analysed the metabolome at each growth phase. In addition, we assessed the accumulation of central carbon metabolites, amino acids, and DNA precursors at each phase. The results of these analyses suggest that Synechococcus 7942 cells prepare for cell division during the lag phase by initiating intensive chromosomal DNA replication and accumulating metabolites necessary for the subsequent cell division and elongation steps that occur during the exponential growth and linear phases.     

Potassium Fluxes in Desheathed Frog Sciatic Nerve

Desheathed frog (R. pipiens) sciatic nerves were soaked in Na-deficient solutions, and measurements were made of their Na and K contents and of the movements of K⁴². When a nerve is in Ringer's solution, the Na fluxes are equal to the K fluxes, and about 75 per cent of the K influx is due to active transport. The Na content and the Na efflux are linearly related to the Na concentration of the bathing solution, while the K content and the K fluxes are not so related. When a nerve is in a solution in which 75 per cent of the NaCl has been replaced by choline chloride or sucrose, the active K influx exceeds the active Na efflux, and the K content is maintained. When a nerve is soaked in a solution that contains Li, the K⁴² uptake is inhibited, and the nerve loses K and gains Li. When a Li-loaded nerve recovers in a Li-free solution, K is taken up in exchange for Li. This uptake of K requires Na in the external solution. It is concluded that the active transports of K and of Na may be due to different processes, that an accumulation of K occurs only in exchange for an intracellular cation, which need not be Na, and that Na plays a specific, but unknown, role in K transport.     

The Exchangeability of Potassium and Bromide Ions in Cells of Red Beetroot Tissue

The exchangeability of potassium and bromide ions accumulatedby cells of red beetroot tissue has been examined under variousexperimental conditions by means of radioactive tracers. It is established that the cells contain a certain amount ofeasily exchanged ions which is largely independent of the saltcontent of the tissue. The remainder of the salt does not exchangeappreciably within 24 hours either during absorption or whenaccumulation is stopped by low temperature, potassium cyanide,high internal salt content, or by an insufficient preliminarywashing of the material. An hypothesis is proposed that the easily exchanged ions aredistributed throughout the intercellular spaces, cell walls,and in parts of the protoplasm, whilst those which do not readilyexchange may be situated in the cell vacuoles, or else stronglyassociated with protoplasmic constituents. The results suggestthat a considerable barrier to the free diffusion and exchangeof ions is located in the region of the tonoplast of a plantcell, and the metabolic transport of ions across this membraneis discussed. An examination of the changes which occur in the amounts ofreadily exchanged potassium during the washing of freshly cutbeet disks in aerated distilled water indicates that the protoplasmprobably acquires a capacity to fix ions more strongly as aresult of this treatment. This supports the view that an effectof washing on the capacity of cells to absorb salts metabolicallyis to increase the number of ‘absorption centres’involved.     

Fluxes of Sodium and Potassium in Acetabularia mediterranea

Sodium efflux in Acetabularia mediterranea occurs against agradient of electrochemical potential and is a light-stimulated,temperature-sensitive process; it is not sensitive to the uncouplerCCCP. Sodium influx is stimulated in CCCP and at low temperature.Potassium influx is temperature- and uncoupler-sensitive, butis not light-stimulated. Tracer K efflux shows complex kinetics,which cannot be explained by any arrangement of intracellularcompartments; it appears to be stimulated at low temperatureand is insensitive to light and uncouplers. There is no evidencefor any chemical linkage between fluxes of Na⁺, K⁺, or Cl^–.It is concluded that Na^– efflux at the plasmalemma isan active process, but no consistent explanation can be advancedto account for the results of K⁺ flux measurements.     

Occurrence of Localized Intense Cation Uptake Sites in the Vascular Rings of Red Beet Storage Tissue 2

Development and decline of cation uptake capacity in discs takenfrom the vascular and parenchyma rings of storage tissue ofred table beet (Beta vulgaris L.) were observed during 12 dof ageing. Uptake capacity for Na⁺ and Rb⁺ showed a steady risereaching maximums by the fourth to fifth days of ageing. Thereafter,there was a steady decline in the uptake rates. Vascular ringtissues were able to develop a greater uptake capacity for bothNa⁺ and Rb⁺ than the tissues of parenchyma rings. This difference,which was more pronounced for Rb⁺ than for Na⁺ uptake, is attributedto a combination of variations in cell density and differencesin the acquisition and retention of the cation uptake capacity.Respiration of tissue discs showed no significant rise duringageing, nor were there significant differences in the respirationof vascular and parenchyma tissues. Vascular tissues containedsignificantly more betacyanin than parenchyma tissues; and theyretained their pigment, as well as their acquired cation uptakecapacity, for a longer period during the ageing process. Key words: Cation uptake, Red beet, Vascular rings, Ageing     

Effects of Prolonged Washing on Primary and Secondary Transport Processes at the Plasma Membrane in Red Beet Storage Tissue

Changing patterns of enzyme activity and solute transport in response to washing were investigated in red beet (Beta vulgaris L.) storage tissue. Washing had a pronounced effect on the plasma membrane (PM) H+-ATPase with an increase in both hydrolytic and proton-pumping activities. Immunoblotting indicated that this may be due, in part, to a higher amount of this enzyme in the PM of washed tissue. Activities of the tonoplast (V)H+-ATPase and pyrophosphatase fluctuated during a 4-d washing period, but overall showed no marked change in activity. In tissue discs sucrose (Suc), glucose (Glc), and fructose uptakes increased significantly in response to washing. Cycloheximide, cordycepin, and tunicamycin inhibited both Glc- and Suc-inducible uptake. Monensin also strongly inhibited inducible Glc uptake, but the effect on Suc was less marked. N-Ethylmaleimide inhibited both Suc and Glc uptake, with its effects being more pronounced in fresh tissue. Other protein-modifying reagents showed no significant difference in their level of inhibition between fresh and washed tissue. Transport studies, carried out using energized PM vesicles from fresh and washed tissue, indicated that there was no rise in Suc and Glc uptake rates in response to washing. Results with a range of inhibitors indicated that there was no marked change in transporter sensitivity in vesicles isolated from fresh and washed tissue. The results indicate that the well-described enhancement of solute transport in washed storage tissue may be due to an increased PM H+-ATPase activity rather than to changes in PM carrier activity or to changes in metabolism such as invertase activity.     

A Comparison of Methods for Measuring Turgor Pressures and Osmotic Pressures of Cells of Red Beet Storage Tissue

Two methods for measuring the turgor pressures of cells in discsof storage tissue of red beet (Beta vulgaris L.) were compared,and a centrifugation method for extracting sap from frozen andthawed tissue was evaluated. Turgor pressures were measureddirectly using a pressure probe, or indirectly using a vapourpressure osmometer. With the latter, discs were placed directlyin the osmometer chamber and turgor was calculated as the differencein osmotic pressure before and after freezing and thawing. Turgorin freshly cut discs, measured with the pressure probe, wasbetween 0-012 MPa and 0.118 MPa with a mean ±s.d. of0.092±;0.032 MPa (n = 24). That measured with the osmometervaried between 0.08 MPa and 0.12 MPa with a mean ±s.d.of 0.09±0.10 MPa (n = 54). After vacuum infiltrationof discs with distilled water, the turgor measured with thepressure probe increased to 1.05–1.12 MPa. Turgor measuredwith the osmometer also increased after vacuum infiltrationbut was, on average, 12% lower than that measured with the pressureprobe. Overall, the results suggest that for routine measurements,the osmometer can provide reasonable estimates of the turgorof cells in beet discs. This is because a number of factorsthat, potentially, could interfere with this method have onlya small effect in this tissue. None of the measured turgorsis indicative of that occurring in intact storage roots becauseboth excision and vacuum infiltration of discs alter the concentrationsof solutes in the extracellular space. The osmotic pressureof sap extracted by centrifugation from frozen and thawed discswas not significantly different from that measured by placingfrozen and thawed discs directly in the osmometer. Solute concentrationsin the sap were not significantly different from those measuredby chemical extraction of discs. Key words: Beta vulgaris, Osmotic pressure, Turgor pressure     

Development and Characteristics of Sodium-selective Transport in Red Beet

Slices of storage tissue of red beet (Beta vulgaris L.) washed for only 1 day in distilled water readily absorb K⁺ but lack a mechanism for rapid Na⁺ uptake. A Na⁺ transport mechanism develops if the tissue is washed for several days, and the tissue then excludes K⁺ during Na⁺ uptake.     

Change in Target Molecular Size of the Red Beet Plasma Membrane ATPase during Solubilization and Reconstitution

The plasma membrane ATPase from red beet (Beta vulgaris L.) storage tissue associated with either native plasma membrane vesicles, a detergent-solubilized enzyme preparation or reconstituted liposomes was subjected to radiation inactivation analysis to determine if changes in target molecular size occurred with modification of its amphipathic environment. For each preparation of the enzyme, the decline in ATP hydrolytic activity with increasing dose of γ-ray radiation demonstrated a simple exponential profile indicating the presence of a single target size. Analysis of the radiation inactivation profiles for the plasma membrane associated, solubilized, and reconstituted enzyme revealed target molecular sizes of 225 kilodaltons (kD), 129 kD, and 218 kD, respectively. These results suggest that the plasma membrane associated and reconstituted ATPase preparations consist of enzyme present as a dimer of 100 kD subunits while the solubilized enzyme is present in the monomeric form. These results also indicate that the 100 kD catalytic subunit most likely represents the minimal unit of ATP hydrolytic activity.     

Potassium Fluxes in Chlamydomonas reinhardtii (II. Compartmental Analysis)

42K+ and 86Rb+ were used to determine the subcellular distribution of potassium in Chlamydomonas reinhardtii by compartmental analysis. In both wild type and a mutant strain, three distinct compartments (referred to as I, II, and III) were apparent. Using 42K+, we found that these had half-lives for K+ exchange of 1.07 min, 12.8 min, and 2.9 h, respectively, in wild-type cells and 0.93 min, 14.7 min, and 9.8 h, respectively, for the mutants. Half-lives were not significantly different when 86Rb+ was used to trace K+. Compartments I and II probably correspond to the cell wall and cytoplasm, respectively. Based on the lack of a large central vacuole in Chlamydomonas, the effect of a dark pretreatment on the kinetic properties of compartment III and the similarity between the [K+] of compartment III and that of isolated chloroplasts, this slowly exchanging compartment was identified as the chloroplast. Growth of wild-type cells at 100 [mu]M (instead of 10 mM K+) caused no change of cytoplasmic [K+] but reduced chloroplast [K+] very substantially. The mutants failed to grow at 100 [mu]M K+.