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Compartments and Fluxes of K+, NA+, and CL- in Avena Coleoptile Cells
Abstract
By the compartmental analysis method of MacRobbie and Dainty, and Pitman, estimates of K(+), Na(+), and Cl(-) concentrations and fluxes were obtained for the cytoplasm and vacuole of coleoptile cells of oat, Avena sativa L. cv. Victory. Double labeling was used in experiments with (42)K plus (22)Na and with (42)K plus (36)Cl in a complete nutrient solution. At the plasmalemma, according to the Ussing-Teorell flux ratio equation, Na(+) is pumped out and Cl(-) is actively transported inward. The results with K(+) are less conclusive, but it is probably pumped in. At the tonoplast there is an active inward transport of Na(+) and probably of K(+), but the status of Cl(-) is uncertain, depending upon whether there is an electrical potential difference between the cytoplasm and vacuole. The results suggest that ion selectivity resides mostly in the plasmalemma. Possible errors in the estimates and interpretations are discussed.
... CATE simultaneously provides information not only about influx, but also about multiple other fluxes (efflux, net flux, and flux to the xylem), as well as kinetic constants of exchange, and subcellular compartmentation, of the traced ion. CATE has been used extensively to investigate a wide range of plant processes, including: ion fluxes and compartmentation across membranes for K + , NH 4 + , Na + , NO 3 -, and Cl - (Cram, 1968;Pallaghy and Scott, 1969;Macklon and Higinbotham, 1970;Pierce and Higinbotham, 1970;Pitman, 1971;Macklon, 1975;Behl and Jeschke, 1982;Jeschke, 1982;Mills et al., 1985;Lee and Clarkson, 1986;Siddiqi et al., 1991;Kronzucker et al., 1995a, b, c, d;Min et al., 1999;Britto et al., 2001Britto et al., , 2002Britto et al., , 2004Kronzucker et al., 2003b;Ritchie, 2006); salinity stress (Hajibagheri et al., 1988); stomatal function (MacRobbie, 1981(MacRobbie, , 1995; forest succession (Kronzucker et al., 1997(Kronzucker et al., , 2003a; metal tolerance (Lasat et al., 1998(Lasat et al., , 2000Zhu et al., 2000;Pedas et al., 2005); synergistic effects of mineral nutrition (Kronzucker et al., 1999); action of hormones (Hellwege and Hartung, 1997;Jovanovic et al., 2000); and compartmentation of herbicides (DiTomaso et al., 1993;Lasat et al., 1997). ...
... Prior to engaging in CATE analysis, rigorous phase testing must take place, in order to appropriately assign compartment identity (Pitman and Saddler, 1967;Pallaghy and Scott, 1969;Pierce and Higinbotham, 1970;Macklon et al., 1975;Memon et al., 1985a;Mills et al., 1985;Kronzucker et al., 1995a, b, c, d;Britto et al., 2001;Kronzucker et al., 2003b). While each compartment has a unique half-time of exchange, various experimental conditions can adjust these values, reducing the difference between compartments. ...
... Chloride is an important anion in opening and closing of stomatal guard cells (Roelfsema and Hedrich, 2005 ). Opening and closure of stomata is mediated by fl uxes of potassium and accompanying anions such as malate and chloride, and it has long been hypothesized that tonoplast Cl − /H + antiporters mediate stomatal opening (Pierce and Higinbotham, 1970 ). Recently, the chloride transporters AtCLC -c and SLAC1 have been localized to the guard cell vacuole, and endomembrane compartments though their function in chloride transport and stomatal opening has not been resolved. ...
... Field evidence suggests that chlorine status has a signifi cant impact on disease resistance in wheat (Fixen, 1993 ;Heckman, 2007 ). An interaction between disease and chlorine have been observed for 15 foliar diseases in 11 crops, and the presence of disease pressure can result in an apparent early physiological observations that predicted the presence of guard cell tonoplast Cl − /H − antiporters (Pierce and Higinbotham, 1970 ). Colmenero -Flores et al. (2007) cloned an Arabidopsis cDNA encoding a member of the CCC family. ...
... There is currently no definitive technique that provides a precise determination of the intracellular compartmentation of compounds such as paraquat. Efflux analysis, however, can yield results that may be interpreted as movement of compounds out of three cellular compartments in series: the cell wall, cytoplasm, and vacuole (30)(31)(32)(33). While recognizing the limitations of applying a technique based on radiotracer flux analysis in single cells to a complex, multicellular organ such as a root, we used this technique to approximate intracellular fluxes and compartmentation of paraquat in maize roots. ...
... We recognize that this approach can be subject to criticism because flux equilibrium was not maintained. However, in earlier studies on compartmental analysis in oat coleoptiles and carrot root tissue in which net ionic fluxes were occurring, the authors provided good arguments for the applicability of the technique, if there was a clear separation of the slopes and rate constants for the putative cytoplasmic and vacuolar compartments (32,33). Despite the relatively brief loading period, the results yielded [14C]paraquat efflux curves that fit the three-compartment model (Fig. 6), yielding compartments with widely different kinetic parameters ( Table 1). ...
Uptake and compartmentation of paraquat was investigated in intact roots of hydroponically grown maize (Zea mays L.) seedlings. Because this investigation focused on the transport of a potentially phytotoxic species, electrophysiological studies were conducted to determine the effect of paraquat exposure on root-cell membrane integrity. Exposure of roots to 1 mM paraquat for up to 40 min or 0.1 mM paraquat for up to 140 min had little effect on the root-cell membrane potential, which indicates that the relatively brief paraquat exposures used for this study (up to 2 hr) had little effect on membrane integrity. The time course for [14C]paraquat accumulation in roots was linear over a 75-min period. Concentration-dependent kinetics for paraquat influx were nonsaturating up to 1 mM and could be resolved into a linear and a saturable component. The linear component was determined to be radiolabeled paraquat remaining in the apoplasm of root epidermal and cortical cells following a 15-min desorption period that was employed to remove cell wall [14C]paraquat. The saturable component displayed Michaelis-Menten kinetics with Km = 90 μM and Vmax = 458 nmol g fresh wt−1 hr−1. Compartmental analysis of [14C]paraquat efflux from intact roots revealed the following estimated distribution of [14C]paraquat at the end of a 2-hr loading period: 76% in the cell wall free space, 16% in the cytoplasm, and about 8% in the vacuole. Estimated efflux half-times were 7.6 min, 29 min, and 4.7 hr from the cell wall, cytoplasm, and vacuole, respectively. These results suggest that paraquat enters the symplasm of plant roots via a carrier-mediated system similar to that proposed for animal tissues. In addition, efflux data indicate that while paraquat slowly accumulates in the vacuole, it can also move back out across the tonoplast and plasmalemma.
... The membrane potential ('PM) can be generated from three sources (Nicholls, 1982). One is due to diffusion potentials which may contribute 30 to 40% of measured membrane potential (Pierce and Higinbotham, 1970;Higinbotham et aL, 1970). Salts (e.g. ...
... Plant salt tolerance is determined not only by the ability to reduce cytosolic Na + accumulation, but also by maintaining a high K + concentration (Pierce and Higinbotham 1970). Because K + plays a pivotal role in growth and enhancing the salt tolerance of plants, the The phenotype of Arabidopsis seedlings grown vertically on MS medium supplemented with 0 mmol/L NaCl, 75 mmol/L NaCl and 100 mmol/L NaCl for 10 d. (E and F) Root length and fresh weight analysis of Arabidopsis seedlings. ...
U‐box E3 ubiquitin ligases play important roles in the ubiquitin/26S proteasome machinery and in abiotic stress responses. TaPUB1‐overexpressing wheat (Triticum aestivum L.) were generated to evaluate its function in salt tolerance. These plants were more salt stress tolerance during seedling and flowering stages, whereas the TaPUB1‐RNAi‐mediated knock‐down transgenic wheat showed more salt stress sensitivity than the wild type (WT). TaPUB1 overexpression up‐regulated the expression of genes related to ion channels and increased the net root Na⁺ efflux, but decreased the net K⁺ efflux and H⁺ influx, thereby maintaining a low cytosolic Na⁺/K⁺ ratio, compared with the WT. However, RNAi‐mediated knock‐down plants showed the opposite response to salt stress. TaPUB1 could induce the expression of some genes that improved the antioxidant capacity of plants under salt stress. TaPUB1 also interacted with TaMP (Triticum aestivum.α‐mannosidase protein), a regulator playing an important role in salt response in yeast and in plants. Thus, low cytosolic Na⁺/K⁺ ratios and better antioxidant enzyme activities could be maintained in wheat with overexpression of TaPUB1 under salt stress. Therefore, we conclude that the U‐box E3 ubiquitin ligase TaPUB1 positively regulates salt stress tolerance in wheat.
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... Compartmental analysis of efflux has been used to study ion compartmentation for several decades (MacRobbie & Dainty, 1958a;Pitman, 1963;Pierce & Higinbotham, 1970;Cheeseman, 1982;Schubert & Läuchli, 1988). With this method, measurement of fluxes across membranes and of the contents of compartments of cells relies to a great extent on tracer techniques (Walker & Pitman, 1976). ...
... Vacuole is commonly known as a storage organ, occupying up to 99% of the cell volume and can be used as a store for inorganic ions which are accumulated in the mature plant cell under saline conditions (Flowers et al., 1977;Karley et al., 2000b;James et al., 2006b). The theory of intercellular compartmentation of inorganic solutes in the vacuole was first postulated by researchers in early 1970's (Jennings, 1968;Pierce and Higinbotham, 1970;Flowers, 1972;Greenway and Osmond, 1972;Shepherd and Bowling, 1973), however, direct evidence for K + /Na + exchange across the tonoplast and compartmentation in the vacuole was first reported by Jeschke and Stelter (1976) in barley and Atriplex root cells. Subsequently, many other studies have studied intercellular compartmentation of Na + and other ions in other cereal crops (Huang and Van Steveninck, 1989;Leigh and Storey, 1993;Fricke et al., 1996;Colmer et al., 2005;James et al., 2006b). ...
Soil salinity causes osmotic and ion specific stresses and significantly affects growth, yield and productivity of wheat. The visual symptoms of salinity stressed wheat include stunted shoot growth, dark green leaves with thicker laminar surfaces, wilting and premature leaf senescence. There are three major components of salinity tolerance that contribute to plant adaptation to saline soils: osmotic tolerance, Na⁺ exclusion and tissue tolerance. However, to date, research into improving the salinity tolerance of wheat cultivars has focused primarily on Na⁺ exclusion and little work has been carried out on osmotic or tissue tolerance. This was partly due to the subjective nature of scoring for plant health using the human eye. In this project, commercially available imaging equipment has been used to monitor and record the growth and health of salt stressed plants in a quantitative, non-biased and non-destructive way in order to dissect out the components of salinity tolerance. Using imaging technology, a high throughput salt screening protocol was developed to screen osmotic tolerance, Na⁺ exclusion and tissue tolerance of 12 different accessions of einkorn wheat (T. monococcum), including parents of the existing mapping populations. Three indices were used to measure the tolerance level of each of the three major components of salinity tolerance. It was identified that different lines used different combinations of the three major salinity tolerance components as a means of increasing their overall salinity tolerance. A positive correlation was observed between a plant’s overall salinity tolerance and its proficiency in Na⁺ exclusion, osmotic tolerance and tissue tolerance. It was also revealed that MDR 043 as the best osmotic and tissue tolerant parent and MDR 002 as a salt sensitive parent for further mapping work. Accordingly, the F₂ population of MDR 002 × MDR 043 was screened to understand the genetic basis of osmotic tolerance and tissue tolerance in T. monococcum. Wide variation in osmotic tolerance and tissue tolerance was observed amongst the progenies. The broad sense heritability for osmotic tolerance was identified as 0.82. Similar, salinity tolerance screening assays were used to quantify and identify QTL for major components of salinity tolerance in Berkut × Krichauff DH mapping population of bread wheat (T. aestivum). Phenotyping and QTL mapping for Na⁺ exclusion and osmotic tolerance has been successfully done in this mapping population. There existed a potential genetic variability for osmotic tolerance and Na⁺ exclusion in this mapping population. The broad sense heritability of osmotic tolerance was 0.70; whereas, it was 0.67 for Na⁺ exclusion. The composite interval mapping (CIM) identified a total of four QTL for osmotic tolerance on 1D, 2D and 5B chromosomes. For Na⁺ exclusion, CIM identified a total of eight QTL with additive effects for Na+ exclusion on chromosomes 1B, 2A, 2D, 5A, 5B, 6B and 7A. However, there were QTL inconsistencies observed for both osmotic tolerance and Na⁺ exclusion across the three different experimental time of the year. It necessitates re-estimating the QTL effect and validating the QTL positions either in the same or different mapping population.
... and this may be further reduced by the substitution of other monovalent cations for K such as Na and Rb (Evans and Sorger, 1966i Wyn Jones and Pollard, 1983b). It is reported that protein synthesis i,l yi/ro in plant systems is maximized at 100 mM of K or higher (Pierce and Higinbotham, 1970;Gibson et at., 1984;Wyn Jones, 1999). Potassium exists as a monovalent cation in biological systems and does not participate in covalent bonding. ...
Plant scientists usually classify plant mineral nutrients based on the concept of "essentiality" defined by Arnon and Stout as those elements necessary to complete the life cycle of a plant. Certain other elements such as Na have a ubiquitous presence in soils and waters and are widely taken up and utilized by plants, but are not considered as plant nutrients because they do not meet the strict definition of " essentiality." Sodium has a very specific function in the concentration of carbon dioxide in a limited number of C4 plants and thus is essential to these plants, but this in itself is insufficient to generalize that Na is essential for higher plants. The unique set of roles that Na can play in plant metabolism suggests that the basic concept of what comprises a plant nutrient should be reexamined. We contend that the class of plant mineral nutrients should be comprised not only of those elements necessary for completing the life cycle, but also those elements which promote maximal biomass yield and/or which reduce the requirement (critical level) of an essential element. We suggest that nutrients functioning in this latter manner should be termed "functional nutrients." Thus plant mineral nutrients would be comprised of two major groups, "essential nutrients" and "functional nutrients." We present an array of evidence and arguments to support the classification of Na as a "functional nutrient," including its requirement for maximal biomass growth for many plants and its demonstrated ability to replace K in a number of ways, such as being an osmoticium for cell enlargement and as an accompanying cation for long-distance transport. Although in this paper we have only attempted to make the case for Na being a "functional nutrient," other elements such as Si and Se may also confirm to the proposed category of "functional nutrients".
... In Hippeastrum petal and Tulipa petal and leaves, the majority of the glucose and fructose accumulate in the vacuoles [9]. In oat coleoptiles, where vacuoles occupy around 90% of the cell volume, large amounts of Na + , K + , and Cl − cross the tonoplast and accumulate in the vacuole [10]. In the storage root of sugar beets (Beta vulgaris), most of the Na + , K + , sucrose and acid invertase for sucrose metabolism accumulate in the vacuoles [11,12]. ...
The vacuole is an essential organelle for plant growth and development. It is the location for the storage of nutrients; such as sugars and proteins; and other metabolic products. Understanding the mechanisms of vacuolar trafficking and molecule transport across the vacuolar membrane is of great importance in understanding basic plant development and cell biology and for crop quality improvement. Proteins play important roles in vacuolar trafficking; such proteins include Rab GTPase signaling proteins; cargo recognition receptors; and SNAREs (Soluble NSF Attachment Protein Receptors) that are involved in membrane fusion. Some vacuole membrane proteins also serve as the transporters or channels for transport across the tonoplast. Less understood but critical are the roles of lipids in vacuolar trafficking. In this review, we will first summarize molecular composition of plant vacuoles and we will then discuss our latest understanding on the role of lipids in plant vacuolar trafficking and a surprising connection to ribosome function through the study of ribosomal mutants.
From compartmental analysis of radioisotope elution measurements, concentrations and fluxes of K(+), Na(+) and Cl(-) were estimated for cortical cells in root segments of onion, Allium cepa L., relative to a complete nutrient solution. The transported fraction of the total efflux was estimated separately. With the Ussing-Teorell flux ratio equation as the criterion, it was concluded that all three ions were actively accumulated from the outside medium into the cytoplasm and that only Na(+) was actively accumulated into the vacuole. K(+) and Cl(-) moved passively, in both directions across the tonoplast. Failure to account for leakage from the stele via the segment cut ends resulted in an over-estimate of exchange across the tonoplast but did not alter the conclusions qualitatively. The consequences of changing the assumed value of the tonoplast electrical potential (from 0 to+10- mV), and the effects of different experimental procedures, were also assessed, and found not to affect the main conclusions significantly. Separate measurement of ions leaking from the segment ends revealed that Na(+) was transported almost exclusively in an acropetal direction in the stele. Cl(-) appeared at both ends of the segments in similar amounts and K(+) was transported mainly in the basipetal direction. The implications of these findings for the mechanism and site of ion selectivity are discussed.
A method is described by which the Na(+) and K(+) content in 0.5 mm sections of single roots of Hordeum distichon L. and Atriplex hortensis L. can be determined by use of flameless atomic absorption spectroscopy. By this method the longitudinal profiles of K(+) and Na(+) along low salt roots and roots which had been equilibrated with or grown in K(+)-free 1 mM Na(+)-solution were determined. The profiles reveal that high K(+)/Na(+) ratios in the cytoplasm are maintained also in K(+)-free solutions. In solutions containing 1 mM Na(+) a high K(+)/Na(+) selectivity was found to be dependent on sufficient aeration. From the ion profiles the cytoplasmic (110 mM) and vacuolar (20 mM) K(+) concentration in low salt barley roots-values which are unobtainable by compartmental analysis-could be estimated.
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.
An excess of NaCl in the growth medium of melon (Cucumis melo L.) seedlings affected growth rate, morphology, and contents of dry weight, chlorophyll, potassium, sodium and chloride in the root and leaf tissues. In roots of seedlings grown in NaCl the electrolyte leakage to iso-osmotic medium was more rapid. Fluorescence polarization measurements revealed that root membranes isolated from these seedlings were significantly less fluid. Chemical analysis disclosed a lower lipid to protein weight ratio in these membranes. The relative contents of phospholipids and free sterols were only slightly affected by excess NaCl. Accordingly, the fluidity of liposomes prepared from lipid extracts of membranes isolated from control and from salt-grown seedlings was similar. The extent of partitioning of the root microsomal membranes between the two phases of aqueous polymer mixtures revealed differences in the surface properties of the root plasma membrane of control and salt-grown seedlings. Possible relationships between the changes in membrane properties and the response to excess salt are discussed.
The effect of Li+, K+, Rb+ and Cs+ on the plasmalemma Na+ efflux Øco in cortical cells of Na+-loaded barley roots has been studied. K+ and Rb+ effectively stimulated Øco and subsequently inhibited the trans-root Na+ transport. Cs+ had a similar but smaller, Li+ almost no effect on Øco. As a function of [K+]0, [Rb+]0 and [Cs+]0 the cation-dependent part of Øco followed Michaelis-Menten kinetics within the experimental error. The apparent Km′s corresponded to the Km′s of the influx (initial net uptake) of the stimulating ions indicating a linkage between part of cation influx and the cation-dependent Na+ efflux or a K+-Na+ exchange system with a certain affinity to alkali cations other than K+. From the Km′s the selectivity sequence K+ > Rb+ > Cs+ ≫ Li+ of the external site of the exchange system was derived. This sequence corresponds qualitatively to that of isolated plant (and animal) membrane ATPases. The significance of the analogy is discussed. It does not preclude although not yet definitely prove the involvement of an ATPase similar to the isolated plant enzymes in K+-Na+ exchange and in K+-Na+-selectivity of barley roots.
Measurements of the effect of [K+]0 on the steady state Na+ fluxes and contents showed the plasmalemma efflux Øco and the vacuolar content of Na+ to be less decreased in the continuous presence of external K+ than other fluxes and the cytoplasmic content. This is taken to indicate a) a continuous operation of K+-Na+ exchange in the presence of external K+and Na+ and b) the involvement of other mechanism(s) besides the plasmalemmal K+-Na+ exchange in selective accumulation of K+ and Na+ by barley roots.
After removal of the embryo from developing seeds of Vicia faba L. and Pisum sativum L., the ‘empty’ ovules were filled with a substitute medium (pH 5.5) and the effect of the osmolality of the medium on K+ and Mg2+ release from the seed coat was examined. In long-term experiments (12 h or longer), with both attached and detached seed
coats, the rate of K+ and Mg2+ release from seed coats filled with a solution without osmoticum was enhanced, in comparison with release from seed coats
filled with a solution containing 400 mol m⊟3 mannitol. In relatively short experiments (2.5 h) with excised seed-coat halves and cotyledons of pea, the stimulatory effect
of a low osmolality of the medium on K+ and Mg2+ efflux into the bathing medium could also be observed in cotyledons. Quantitatively, efflux stimulation was most important
in experiments with seed coats and cotyledons from ovules in an early stage of development. In experiments of 7 h, compartmental
analysis was used for a more detailed analysis of efflux stimulation. Half-times for exchange (t½) of K+ and Mg2+ from the slowest exchanging compartment (the vacuole) were much lower in tissues incubated in a low-osmolality medium than
in tissues incubated in a high-osmolality medium. There was no evidence for an effect on the permeability of the plasmalemma.
An increase in the tonoplast permeability at high cell turgor, for all solutes measured (K+ , Mg2+, sucrose, amino acids), will help to maintain high solute concentrations in the seed apoplast.
Salinity is a major problem confronting agriculture in arid environments. Sensitivity to high levels of salt in plants is associated with an inability to effectively remove Na+ ions from the cell cytoplasm. The ability to compartmentalize Na+ may result, in part, from stimulation of the H+-ATPases on the plasma membrane (PM-ATPase) and vacuolar membrane (V- ATPase). These H+-pumping ATPases may provide the driving force for Na+ transport via Na+-H+ exchangers. In a salt-sensitive line of cotton (Gossypium hirsutum L.), greater relative reductions in root length and root fresh weight than in hypocotyl length of seedlings grown in 75 mM NaCl indicated that the root was most affected by salt stress. To determine if the H+-ATPases are involved in the response to salt, we compared activities of the PM- and V-ATPases from roots in salt-sensitive cotton seedlings grown with or without 75 mM NaCl. Higher PM-ATPase activity (42%) was observed in seedlings grown in 75 mM NaCl. This stimulation was specific for Na+, was not observed when Na+ was added to membrane fractions, and was not due to an increase in PM-ATPase protein levels. V-ATPase protein accumulation was unaffected by growth in the presence of Na+, and activity was unaffected by Na+ in the growth medium or by Na+ added to membrane fractions. These studies suggest that although the PM-ATPase responds to increased Na+, activity of the transport proteins on the plasma membrane alone may be insufficient to regulate intracellular Na+ levels. In addition, the inability of the V-ATPase to respond to increased levels of Na+ indicates that salt sensitivity in cotton seedlings may result, in port, from a lack of effective driving force for compartmentalization of Na+.
Some ionic relations of the filamentous green alga Mougeotia sp. have been analyzed under different light conditions. Data from influx and efflux measurements using 86Rb+ and 36Cl- fit the model of three cellular compartments (cell wall, cytoplasm, vacuole) in series. This result is remarkable, since in a Mougeotia cell at least two thirds of the cytoplasmic compartment are occupied by the cell-filling, flat and nearly rectangular chloroplast which is axially oriented. The chloroplast is concluded to be part of the cytoplasmic flux compartment.
Photosynthetically saturating irradiances of continuous white light enhance the active and passive fluxes of K+ and Cl- at the plasmalemma by a factor of 3. Photosystem II is responsible for the light-dependent increase of the uptake of Cl- (36Cl-) whereas the uptake of K+ (86Rb+) depends additionally on energy from photosystem I.
Ion flux measurements performed after irradiations with red and far-red, respectively, show that the fluxes of K+ and Cl- across the plasmalemma are not affected by the state of phytochrome.
The transmembrane potential of mesophyll cell protoplasts from Avena sativa was altered in two different ways: first by raising the external KCI concentration with and without the addition of valinomycin
and, secondly, by using the proton carrier CCCP and varying the external pH. The percentage of unlysed protoplasts was determined
after decreasing the osmotic pressure from 1.2 to 0.7 MPa in order to characterize their ability to enlarge their surface
area. Lysis strongly increased when the KCI concentration was raised from 0 to 90 mol m−3. This effect did not depend on the presence of valinomycin. Using CCCP, lysis increased similarly if the external pH was
lowered from 7.2 to 5.5. In the presence of CCCP the influence of the external KCI concentration disappeared completely. This
led to the conclusion that depolarization inhibited surface area expansion. Since the expansion is thought to be based on
exocytotic-like membrane incorporation, these results suggest that exocytosis is controlled, at least in part, by the transmembrane
potential.
The time-course of exchange of sodium and potassium ions from root and leaf material of the halophyte Suaeda maritima has been followed and the data analysed according to the phenomenology of efflux, or compartmental, analysis. Sodium ions
were exchanged much more slowly (c. 4 times) from the vacuoles of leaf cells of plants grown in sodium chloride than were
potassium ions from the vacuoles of leaf cells of plants grown either in similar concentrations of potassium chloride or in
low concentrations of potassium. In plants grown in sodium chloride, sodium ions were exchanged 9 times more slowly from the
vacuoles of leaf cells than from the vacuoles of root cells.
The concentration of sodium ions in the cytoplasm of leaf cells of plants growing in 340 mol m−3 sodium chloride was estimated to be 165 mol m−3 when the average concentration in the leaf tissue was about 600 mol m−3.
As measured by movement from mature to developing leaves in intact plants; there was less in vivo retranslocation of 22Na and 36CI in plants growing in sodium chloride than there was of 86Rb in plants growing either in potassium chloride or in non-saline conditions.
The results are discussed in terms of the concept and energetics of compartmentation of ions in the cells of halophytes.
The relationship of the electrogenic pump to the ATP concentration in barley roots was examined. Excision, salt accumulation
and changes in temperature produced changes in the ATP concentration which did not correlate with changes in membrane potential
(ψm). Illumination of seedlings prior to excision of the root elevated the ATP level and caused ψm to hyperpolarize. Metabolic inhibition by sodium azide resulted in a fall in ATP concentration and membrane depolarization.
With treatment in azide for longer than 10 min there was a linear relationship between ATP concentration and ψm. Time-courses of the effects of azide and carbon monoxide showed that this relationship did not hold at short treatment times
because the ATP concentration fell more rapidly than the decay of ψm. Application of butyrate or fusicoccin produced little or no change in the ATP concentration but both caused significant
changes in ψm.
The effects on ψm of butyrate, fusicoccin, external pH and metabolic inhibitors were considered to be consistent with regulation of electrogenic
pumping by cytoplasmic pH.
Pea plants (Pisum sativum L.) were supplied with external phosphate for differing periods of time, so that their phosphorus status varied, and the
intracellular distribution of inorganic phosphate (P1) in the roots was examined by 31P nuclear magnetic resonance. Over the range of phosphorus nutrition investigated, the quantity of vacuolar P1 per unit fresh weight of root tip changed considerably, whereas the quantity of cytoplasmic P1 per unit fresh weight of root tip did not alter. The relative volumes of the cytoplasm and the vacuole in pea root tips seemed
to be little affected by differences in phosphorus nutrition, and this implied that the concentration of P1 in the cytoplasm was kept almost constant, at a level estimated to be ∼ 18 mM.
The rate of absorption of 32P-labelled phosphate was negatively correlated with the vacuolar P1 concentration, but there was no clear correlation with the concentration of P1 in the cytoplasm.
ABSTRACT Hajibagheri, M. A., Flowers, T. J., Collins, J. C. and Yeo, A. R. 1988. A comparison of the methods of X-ray microanalysis,
compartmental analysis and longitudinal ion profiles to estimate cytoplasnuc ion concentrations in two maize varieties.—J.
exp. Bot. 39: 279-290.
The ion content of compartments within plant root cells has been studied by three different methods; flux analysis using radioactive
isotopes, longitudinal ion profiles and X-ray microanalysis. The data provide estimates of the concentrations of K+ and CI− in the cytoplasm of roots of culture solution and salt grown maize by three independent methods.
In the cultivar LG11 grown in 50 mol m−3 NaCl X-ray microanalysis, compartmental analysis and longitudinal profiles yielded approximately agreeing values for cytoplasmic
K+ of 90, 70 and 62 mol m−33 of tissue volume respectively. However, the methods disagreed on cytoplasmic Cl− where the value obtained by compartmental analysis was about four times that from X-ray microanalysis and longitudinal profiles.
Possible reasons for this are discussed.
Using the compartmental analysis the unidirectional Na+ fluxes in cortical cells of barley roots, the cytoplasmic and vacuolar Na+ contents Qc and Qv, and the trans-root Na+ transport R′ have been studied as a function of the external Na+ concentration. Using the re-elution technique the effect of low K+ concentrations on the plasmalemma efflux πco of Na+ (K+-Na+ exchange) and on R' was investigated at different Na+ concentrations and correspondingly different values of the
cytoplasmic sodium content Qc. The relation of the K+-dependent Na+ efflux coK+-dep to Qc or to the cytoplasmic Na+ concentration
obeyed Michaelis-Menten kinetics. This is consistent with a linkage of πco, K+-dep to K+ influx by a K+-Na+ exchange system. The apparent Km corresponded to a cytoplasmic Na+ concentration of 28 mM at 0·2 mM K+ and about 0·2 mM Na+ in the external solution. 0·2 mM K+ stimulated the plasma-lemma efflux of Na+ and inhibited Na+ transport selectively even in the presence of 10 mM Na+ in the external medium showing the high efficiency of the K+-Na+ exchange system. However, πco, K+-dep was inhibited at 10 mM Na1 compared to lower Na1 concentrations suggesting some competition of Na1 with K1 at the external site of the exchange system. The effect of the Na+ concentration on Na1 influx πoc is discussed with respect to kinetic models of uuptake.
Techniques have been developed for continuous recording of electrical potential difference in maize root preparations. In this way the continuous p.d. profile along the lateral axis of the root could be obtained. The features of the profile are resolved in terms of the root morphology.Analysis of the p.d. profile indicates (a) that the phases within the root are electrically negative with respect to the bathing medium, (b) that the vacuoles are positive relative to the cytoplasm, i.e. a p.d. exists across the tonoplast, (c) that all cytoplasmic phases are equipotential, which indicates a continuity within and between the cortical and stelar symplasms, (d) that the extracellular space of the root is presumably only slightly influenced by the concentration of the external medium since the p.d. profile within the root does not react to changes of concentration in the external medium, (e) that the potential of the stelar vacuoles is negative relative to the cortical one and this is consistent with the accumulation of surplus ions in the stelar vacuoles.The implications of the present findings to the elucidation of ion transport mechanism across the root are briefly discussed.
The intracellular distribution of K+ and Na+ ions has been determined by compartmental analysis of isotope exchange. The simultaneous measurement of electrical potentials allowed us to show that the distribution of K+ was close to thermodynamic equilibrium while the internal concentration of Na+ was well below the value predicted for the equilibrium. The efflux of Na+ was more sensitive to temperature than its influx. Both ouabain and variations in the external levels of KCl produced weak and inconsistent effects, observations which would emphasize the difference between the Na+ extrusion mechanism of plants and animals. The Na+ extrusion system of Acer cells ceased to be functional in Na+-depleted cells but recovered its function if the cells were placed in 10 mM NaCl, which suggests that the extrusion system was induced by the level of internal Na+ ions.
Treatment with sodium chloride solutions led to loss of intracellular potassium and uptake of sodium in both the halophytic moss Grimmia maritima and the glycophyte G. pulvinata. In the presence of calcium, potassium loss and sodium uptake were considerably reduced in G. maritima, but in G. pulvinata, while potassium loss was reduced, net uptake of sodium was stimulated at high external sodium concentrations. In G. pulvinata sodium uptake from artificial seawater solutions was considerably reduced and potassium loss stimulated by increasing the external magnesium concentration. Magnesium had little effect on the retention or uptake of cations by G. maritima. Prewetting had no effect on G. maritima but it reduced subsequent seawater-induced sodium uptake and potassium loss in G. pulvinata and stimulated sodium uptake from calcium-free sodium chloride solutions.Experiments with the metabolic inhibitors, 2:4 dinitrophenol, malonate and arsenate demonstrated that sodium uptake by G. pulvinata and potassium retention by both species may be under metabolic control. An experiment in which the intracellular sodium contents of G. maritima and G. pulvinata were artificially increased and then sodium loss measured indicated that sodium efflux is inhibitor-sensitive in G. maritima but not in G. pulvinata. The results suggest that G. maritima maintains normal cation levels under saline conditions by a combination of a low permeability to cations and the possession of a sodium efflux pump.
From compartmental analysis of radioisotope elution measurements, concentrations and fluxes of Ca(2+) were estimated for cortical cells in root segments of onion, Allium cepa L., relative to a complete nutrient solution containing 1 mM Ca(2+). Five compartments for Ca(2+) in the cortex were revealed. These were identified, in order of increasing rates of exchange, with the vacuole and cytoplasm of the cortical parenchyma, the Donnan free space in the cell walls, the water free space in the tissue and the superficial film of solution on the segments. With the Ussing-Teorell flux ratio equation as the criterion, it was concluded that Ca(2+) entered the cytoplasm passively and was actively pumped back to the external solution. Ca(2+) concentration in the vacuole could only be estimated as lying between wide limits (1.0 to 7.5 μeq. ml(-1)), but even at the maximum concentration, it was concluded that entry was passive and content limited by an efflux pump across the tonoplast. Net flux was zero and the vacuolar concentration of Ca(2+) compatible with this was found to be 2.6 μeq. ml(-1). The transported fraction of the total efflux, appearing at the segment cut ends, was estimated separately. Calcium was found to be transported almost exclusively in the basipetal direction.
The aim of the work described in this thesis was to characterise the mechanism of Na+ transport at the vacuole membrane (tonoplast) of red beet using isolated tonoplast vesicles. It was confirmed that pure tonoplast vesicles could be prepared from red beet storage root. However, it was found that the KI treatment used in published tonoplast vesicle preparation protocols was deleterious to tonoplast, so this was subsequently modified. Attempts to characterise a Na+/H+ antiport reported by Blumwald and Poole (1985a; Plant Physiol. 78, 163-167) by studying the effect of Na+ on pH gradients imposed by a pH jump were not successful. Under the conditions used by these workers the fluorescent pH gradient probe acridine orange misreported the pH gradient and apparently good evidence for a Na+/H+ antiport was shown to be artefactual. With modifications to the media used for these experiments, acridine orange could be used to monitor the pH gradient and these results were confirmed using another pH gradient probe, [14C] methyl amine. However, no evidence was found for a Na+/H+ antiport as it was not possible to distinguish between changes in the pH gradient due to an Na+/H+ antiport and changes caused by electrically-coupled Na+ and H+ fluxes. In an attempt to avoid these problems, +Na uptake by tonoplast vesicles was studied in response to pH gradients generated by the H+-ATPase. No pH gradient-dependent Na+ uptake was found using this approach. It is concluded from these studies that previous evidence for a tonoplast Na+/H+ antiport in red beet is artef actual. It is suggested that Na+ is passively distributed across the tonoplast in red beet.
The enhancement by indoleacetic acid (IAA) of (36)Cl(-) uptake into Avena coleoptile sections was used to study the effects of a hormone on a membrane-controlled phenomenon. Compared to sections in phosphate buffer only, Cl(-) content of the cells increases 15 min after addition of IAA; the promotion is seen only with growth-active auxins and is saturated at 3 μM IAA. The percent enhancement by IAA is the same over a wide range of Cl(-) concentrations. The hormone effect is not observed at ice-bath temperature and is not correlated with growth or water movement into the cells. IAA does not influence the movement of Cl(-) in the section. While auxin must be present within the tissue in order to maintain the enhancement, there is no relationship between the total amount of auxin and the accelerated Cl(-) uptake that results. A polarity in the auxin effect is implied since only apical applications of IAA promote Cl(-) uptake.
The plasma membrane ATPase of oat roots requires Mg2+ and is further stimulated by salts of monovalent ions such as KCL (LEONARD and HODGES, 1973). The monovalent ion stimulation appeared to be due to the cation, however, this result was equivocal since substances such as Tris chloride and choline chloride also stimulated the ATPase. Other studies have shown that a semi-purified membrane fraction of turnip roots exhibited ATPase activity that was more sensitive to anions than to cations (RUNGIE and WISKICH, 1973), and it has also been claimed that organic cations can stimulate the ATPase activity of crude membrane preparations of cereal roots, including oats (RATNER and JACOBY, 1973). Thus, further clarification of the ion sensitivity of the plasma membrane ATPase of oat roots was sought.
The solution conditions such as the ion concentration and pH have a profound effect on the behaviour of the silica/water interface, which dictates many of the surface properties of mesoporous silica nanoparticles (MSNs) and their utility in a range of nanotechnology applications. The interaction of water molecules with a model silica surface, α-quartz (101), at different surface charge densities, is investigated to evaluate the influence of pH on structure (density profile, radial distribution function) and dynamics (diffusion coefficient D) of the interfacial water in the presence of biologically relevant ions, Na+, K+ and Rb+ ions. Classical molecular dynamics were performed using a recently developed force field (Kroutil et al, J. Phys. Chem C, 2015, 119, 9274-9286). Our results show the interfacial water is more structured and the diffusion of interfacial water molecules becomes slower as the surface charge becomes more negatively charged, as we increase the pH from acidic to neutral to basic pH. The self-coefficient diffusion (D) of water molecules and ions decrease with an increase in pH and is affected by concentration and type of ions in the system. The diffusion of water molecules around deprotonated oxygen atoms is slower than the diffusion of water molecules around oxygen protonated. The presence of the ions near to deprotonated oxygen atoms further decreases the diffusion of water molecules.
Im zweiten Kapitel haben wir vorwiegend ein einfaches, zweikompartimentelles Außen-Innen-Modell betrachtet und gesehen, wie nützlich es für die Untersuchung grundlegender Probleme des Membrantransportes ist. Lebende Zellen sind aber durch Membranen in zahlreiche Kompartimente gegliedert. Die Mannigfaltigkeit der Transportprozesse an den Grenzen dieser Kompartimente ist noch um ein Vielfaches größer, weil an jeder Membranbarriere die verschiedensten Stoffe transportiert werden müssen. Man kann versuchen, möglichst viele dieser Membrantransporte zu analysieren und sich überlegen, wie sie zusammen oder gegeneinander wirken.
The majority of investigations of anion transport at the tonoplast have involved examination of the effects of anions on ATP-dependent H+-transport in sealed tonoplast vesicles (e.g. Bennett and Spanswick, 1983; Churchill and Sze, 1984; Blumwald and Poole, 1985; Lew and Spanswick, 1985). These studies have shown that anions stimulate ΔpH formation and dissipate membrane potential (ΔΨ) and that these effects are sensitive to anion channel blockers such as DIDS and SITS. This is thought to indicate that anions move trough selective channels in the tonoplast in response to the Air generated by ATPase, causing dissipation of the ΔΨ and thus an increase in ΔpH, as expected from the chemiosmotic hypothesis (Mitchell, 1966).
In relation to ions, plant roots have two main functions. At the surface of the root ions are absorbed from the soil or solution; from within the root, ions are transported to other parts of the plant. Active processes maintain concentrations of ions in the vacuole and cytoplasm of cortical cells as well as participating in the general movement across the root to the stele.
Transport is an important feature of the contact between plants and their environment. In many lower plants, the plant body, or the population of cells is predominantly of a single type and this cell type mediates all the exchanges between the plant and its environment. This is partly the reason why certain algal cells (Part A, Chap. 6) and fungal hyphae (Part A, Chap. 7) have become standard systems for study of transport processes (e.g. Chara, Chlorella, Neurospora).
Data on transtonoplastic potential are rather rare for vacuoles in situ, and can be contradictory for protoplasts and isolated vacuoles.
Compartimentai analysis of ion fluxes has been successful in many studies on plant cells (HIGINBOTHAM, 1973; LÜTTGE, 1973); yet it is uncertain whether large cell organelles, such as chloroplasts or cytoplasmic vesicles, constitute autonomous ion flux compartments and hence are not represented by the current model of three compartments in series (free space, cytoplasm, vacuole). Such additional flux compartments could lead to wrong estimates of the intracellular ionic relations (MacROBBIE, 1971).
In den Jahren 1968–70, der Zeit nach dem Bericht von SCHILDE (Fortschr. Botan. 30, 44), erlebte die Membranbiologie eine Buchflut, an der die Elektrophysiologie naturgemäß teilhatte. COLE schrieb eine “Spezielle Elektrophysiologie”; PLONSEY betont die physikalischen, LAKSHMINARAYANAIAH die physiologischen Aspekte. PASSOW u. STÄMPFLI sowie LAVALLEE et al. demonstrieren ausgewählte Techniken. Neu aufgelegt wurden die einführenden Werke von BURES et al. und KATZ (in dt. Übers.). Das nach Ablieferung dieses Beitrags erschienene Büchlein von HOPE ist eine vorbildliche Einführung in die elektrischen Aspekte des Ionentransportes der Pflanzenzelle.
Barley roots grown on a nutrient solution containing 1 mM Na(+) but no K(+) are capable of a considerable Na(+) transport via the symplasm of the root and the xylem vessels. K(+) added to the medium surrounding the root cortex severely inhibits this transport after a lag period at a high rate constant (Fig. 3).It is likely that the fluxes of Na(+) are changed drastically during this transition from low to high K(+) status. Although originally limited to steady state fluxes, the extended method of efflux analysis for excised roots (Pitman, 1971) has been applied to the non-steady fluxes which occur upon the addition of K(+) to the roots. It is shown that besides other changes the efflux of (22)Na(+) through the cortex of barley roots is stimulated instantaneously (Fig. 5) by the addition of K(+) and presumably by an influx of K(+) ions. From this a transient, K(+)-stimulated Na(+) efflux at the plasmalemma of the cortical cells can be estimated. It amounts to 10.9 μ moles/g fw · h compared to the control efflux of 3.3 μ moles/g fw · h without K(+).The stimulated efflux is attributed to a Na(+) efflux pump at the plasmalemma and is thus related to the K-Na-selectivity of barley plants. The inhibition of the Na(+) transport by K(+) is probably a consequence of this increased efflux of Na(+) from the symplasm through the root cortex.
This chapter focuses on ion absorption by cells and other tissues—such as storage roots, leaves, algae—and in organelles such as mitochondria and chloroplasts. Ion absorption by cells, including the flux of ions across both the plasma membrane and tonoplast is presented. The chapter provides an overview of nutrient absorption by excised roots. Excised roots usually function for several hours, at least with regard to ion absorption, as if they had never been removed from the shoot. The value of using excised roots that are low in salt content for studying nutrient absorption is determined. These roots accumulate salts in a short time and the increase in ion content can be measured easily either chemically or by using the radioisotope of a specific element. The chapter discusses the kinetics and selectivity of ion absorption and proposes a model for ion absorption by roots. This model consists of two main features: a cation-activated ATPase in the plasma membrane and an anion carrier that brings about the exchange of anions across the plasma membrane.
Leaves, roots and seeds of soybean and cotton were subjected to a series of temperature stress of 30, 34, 38, 42, 46 and 50°C to measure the ion efflux from the cell. Soybean leaves leaked more electrolytes at 313 μS/cm than cotton leaves which recorded the electrical conductivity of 143 μS/cm at 50°C. Below the stress at 42°C relatively less ions were effluxed as compared to 42°C or above where severe damage to cell membrane occurred and more than 50% electrolytes leaked from leaves, seeds and roots of both crop plants.
It seems a truism to say that every organism must have its biochemical and structural properties adapted so that it is able to function in its habitat. Nevertheless, when investigating the causal relationships between ecological conditions and biochemical responses, one ends up with information relevant to our understanding of the regulation of basic cell functions, such as membranes and metabolism. Apart from the immediate importance of studying major ecological problems such as salinity from the biochemical point of view, it should be possible to utilize the data to shed light on fundamental theoretical aspects of life.
Respected and known worldwide in the field for his research in plant nutrition, Dr. Horst Marschner authored two editions of Mineral Nutrition of Higher Plants. His research greatly advanced the understanding of rhizosphere processes and trace element uptake by plants and he published extensively in a variety of plant nutrition areas. While doing agricultural research in West Africa in 1996, Dr. Marschner contracted malaria and passed away, and until now this legacy title went unrevised. Despite the passage of time, it remains the definitive reference on plant mineral nutrition. Great progress has been made in the understanding of various aspects of plant nutrition and in recent years the view on the mode of action of mineral nutrients in plant metabolism and yield formation has shifted. Nutrients are not only viewed as constituents of plant compounds (constructing material), enzymes and electron transport chains but also as signals regulating plant metabolism via complex signal transduction networks. In these networks, phytohormones also play an important role. Principles of the mode of action of phytohormones and examples of the interaction of hormones and mineral nutrients on source and sink strength and yield formation are discussed in this edition. Phytohormones have a role as chemical messengers (internal signals) to coordinate development and responses to environmental stimuli at the whole plant level. These and many other molecular developments are covered in the long-awaited new edition. Esteemed plant nutrition expert and Horst Marschner's daughter, Dr. Petra Marschner, together with a team of key co-authors who worked with Horst Marschner on his research, now present a thoroughly updated and revised third edition of Marschner's Mineral Nutrition of Higher Plants, maintaining its value for plant nutritionists worldwide. A long-awaited revision of the standard reference on plant mineral nutrition Features full coverage and new discussions of the latest molecular advances Contains additional focus on agro-ecosystems as well as nutrition and quality.
Recretohalophytes with specialized salt-secreting structures (salt glands) can secrete excess salts from plant, while discriminating between Na(+) and K(+). K(+)/Na(+) ratio plays an important role in plant salt tolerance, but the distribution and role of K(+) in the salt gland cells is poorly understood. In this article, the in situ subcellular localization of K and Na in the salt gland of the recretohalophyte Limonium bicolor Kuntze is described. Samples were prepared by high-pressure freezing (HPF), freeze substitution (FS) and analyzed using NanoSIMS. The salt gland of L. bicolor consists of sixteen cells. Higher signal strength of Na(+) was located in the apoplast of salt gland cells. Compared with control, 200mM NaCl treatment led to higher signal strength of K(+) and Na(+) in both cytoplasm and nucleus of salt gland cells although K(+)/Na(+) ratio in both cytoplasm and nucleus were slightly reduced by NaCl. Moreover, the rate of Na(+) secretion per salt gland of L. bicolor treated with 200mM NaCl was five times that of controls. These results suggest that K(+) accumulation both in the cytoplasm and nucleus of salt gland cells under salinity may play an important role in salt secretion, although the exact mechanism is unknown.
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By washing out potassium and magnesium from the free space of stem segments of Vicia faba, parasitised stem parts were shown to have an efflux pattern different from that of normal ones.Using the graphical method of compartmental analysis it could be shown that the K+ and Mg++ efflux from parasitised stem parts does not occur in accordance with the regularities displayed by non-parasitised stem parts and known for other higher plant tissues.The process causing the different efflux pattern is apparently under metabolic control since it appeared to be inhibited at 0°C and after addition of 2,4-dinitrophenol or sodium azide. The possibility is discussed that the increased efflux is at least partly caused by an enhanced unloading rate for K+ and Mg++ by the host phloem as was shown in a previous study for 14C-solutes.
The vacuole is the largest compartment of a mature plant cell and serves as an internal reservoir of metabolites and nutrients. In the last years transport of solutes across the tonoplast has been intensively investigated. It was shown that two different proton pumps reside in the tonoplast. These pumps generate an electrochemical gradient which can be used as an energy-source to accumulate solutes. Cation uptake is driven by an H+ antiport mechanism. Anions are accumulated in response to the inside positive membrane potential. In addition, the existence of ion channels was shown using the patch clamp technique. The aim of this review is to compare and to discuss the present state of our knowledge of solute transport across the tonoplast.
Spring wheat (Triticum aestivum L. cv. Svenno), oat (Avena sativa L. cv. Brighton) and glasshouse cucumber (Cucumis sativus L. cv. Bestseller F1) were cultured for a week after germination on complete nutrient solutions of three different dilutions (1, 25 and 50% of the full strength medium). K+(86Rb) and 45Ca were present during the whole culture period. Relative humidity (RH) was 50% except during the last day, when half the material was transferred to 90% RH. Efflux of labelled ions was then followed during eight hours on unlabelled solutions of the same composition as before, and at both 50% and 90% RH in the atmosphere. – Uptake of K+(86Rb) during growth tended to be saturated in the 25% medium. Contrariwise, the level of Ca2+ in the roots increased continuously with strength of the medium. At low concentrations cucumber roots were higher in Ca2+ than roots of oat or wheat, whereas all three species showed similar levels of Ca2+ in 50% medium. – At the lowest ionic strength, smooth efflux curves were obtained that could be resolved according to the three-compartment theory. At higher ionic strength, irregularities were observed, and more for Ca2+ than for K+; but for practical purposes compartment analysis with the same time constants could be applied as for the lowest concentration. – Discrimination between K+ and Rb+ differed between the roots, but not much between the shoots of different species. The roots of oat and wheat took up Rb+ preferentially over K+ in the 25% and 50% media; whereas K+ was preferred over Rb+ or little discrimination made in 1% medium and for cucumber. The shoots generally showed less discrimination than the roots. The main variability in discrimination between K+ and Rb+ thus appears to be localized in the tonoplasts of the roots cells. – Low RH around the shoots increased efflux of K+(86Rb) from the cytoplasm and vacuoles of the root cells as compared to the efflux at high RH. DNP (2,4-dinitrophenol) in the medium had the same effect as high RH around the shoots. The signal system that must exist between shoots and roots is discussed as a response to “drought” conditions. In relation to investigations of others, it is assumed that the effect of DNP may indicate that part of the chain between roots and shoots consists of metabolically influenced sites, whose output is influenced by the rate of water transport.
The accumulation of several major nutrient ions by the roots of some aquatic plants was investigated. Five species growing in freshwater lochs on the north coast of Scotland were studied. Electrical potential differences between root epidermal cells and the surrounding loch water were measured using micro-electrodes. From these measurements, electrochemical potential gradients between the roots and the loch water were calculated, and also the driving forces on each ion. For each species investigated, the results indicate that sodium is actively accumulated by the roots. This contrasts with results of studies which provide evidence for a sodium efflux pump in plant cells. It is suggested that active accumulation of sodium is evident in plant roots when the external sodium concentration is low. The results showed that K+, Cl-, and NO3- are actively accumulated; Ca++ and Mg++ appear to be in electrochemical equilibrium with the external medium.
Intracellular potentials were measured in beetroot tissue during the steady-state uptake of K(+) from various solutions. In solutions containing bicarbonate, the membrane potential becomes up to 70 mv more negative than the estimated equilibrium potential for K(+). The uptake of K(+) from such solutions is correlated with variations in the potential, both when the bicarbonate concentration is changed and also when the metabolic activity of the tissue is changed by washing in water for various periods. However, the estimated permeability to K(+) varies from 0.4 x 10(-7) to 1.5 x 10(-7) cm.sec(-1). It is postulated that the change of potential arises from the metabolic transport of HCO(3) (-) into the cell or H(+) outwards, and that the associated uptake of K(+) is partly or entirely by passive diffusion across the cell membrane. In contrast, K(+) uptake from KCl solutions is not accompanied by any significant change in the membrane potential, which remains relatively close to the K(+) equilibrium potential. In solutions containing both KHCO(3) and KCl, it appears that an amount of K(+) equal to the influx of Cl(-) is taken up independently of the potential, while the component of K(+) uptake which is not balanced by Cl(-) uptake is related to the potential in the manner described. These results suggest that K(+) uptake is linked to Cl(-) uptake in an electrically neutral active transport process.
An attempt is made to apply the Ussing-Teorell criterion for passive ion movement to root cells of young broad bean seedlings. This requires the estimation of ion concentrations in the cells, ion fluxes between cellular compartments and the external medium, and membrane potential differences.
An analysis of the rate at which isotope diffuses out of disks of beetroot tissue shows that there are at least two components of the non-free space. As tlwse components are not due to differences in cell type within the tissue, it is suggested they are due to a cytoplasmic phase in the parenchymatou's cells, and to the vacuoles.
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.
SUMMARY The potential differences across the tonoplast and plasmalemma membranes have been measured in the single cells of Nitella translucens, the cells being immersed in an artificial pond water (composition: NaCl 1.0 mM., KC1 0.1 mM., CaCl2, 0.1 mM.). The potential of the cytoplasm is –138 m V with respect to the bathing medium and –18 mV with respect to the vacuole.
The concentrations of Na, K, and Cl have been measured in the two cell fractions. The concentrations in the flowing cytoplasm
are: Na 14 mM., K 119 mM., and Cl 65 mM.; the vacuolar concentrations are: Na 65 mM., K 75 mM.,and Cl 160 mM.
The observed potential differences across the two membranes are compared with the Nernst potentials for all three ions. This
analysis shows that all three ions are actively transported at the plasmalemma: Na is pumped outwards while K and Cl are pumped
inwards. At the tonoplast Na is pumped into the vacuole while K and Cl are close to electrochemical equilibrium.
The inhibitor, ouabain, has no effect on the cell resting potential.
IN electrophysiology the term `polarization' often has an ambiguous meaning. Here it will be used in the physical sense as an overall definition to include changes in the `resting potential' of the object (membrane, nerve, skin, etc.), as well as the appearance of rectification, capacity and inductivity effects under the influence of an externally applied current. The potential changes and the rectification effects have mostly been interpreted in a qualitative manner in terms of selective ion permeability, and the capacity as `double layers', `dielectrics', etc.1,2,3. Cole4 has recently made the unexpected observation of an inductance component in the squid axon, which he seems inclined to explain as analogous to piezoelectric behaviour. Lorente de Nó5 prefers more unspecific concepts implying also chemical reactions.
A more complete study of ionic concentrations and fluxes in the giant internodal cells of Nitella translucens has been made. The vacuolar concentrations were 76 mM K and 170 mM Cl. The content of the chloroplast layer was 135 mmicromoles K/cm(2) and 215 mmicromoles Cl/cm(2); in a layer 9 micro thick these correspond to concentrations of 150 mM K and 240 mM Cl. Such a high level of chloride requires active transport of chloride into the cytoplasm, either at the plasmalemma or at the membranes bounding the cytoplasmic particles; it cannot be achieved by active transport of chloride only at the tonoplast. With concentrations of 0.1 mM K and 1.3 mM Cl outside, the fluxes into the cytoplasm had mean values of 1.0 to 1.4 micromicromoles K/cm(2)sec. and 2.1 to 2.8 micromicromoles Cl/cm(2)sec.; the corresponding fluxes from the cytoplasm to the vacuole were about 110 micromicromoles K/cm(2)sec. and 175 micromicromoles Clcm(2)sec. The transfer of both potassium and chloride to the vacuole under different conditions appeared to be correlated with the uptake of chloride into the cytoplasm. It is suggested that two separate processes are involved in the active accumulation of salts in the vacuole-an active uptake of chloride in the cytoplasm and a subsequent transfer of salt to the vacuole. It may be that the second process involves the formation of small vesicles in the cytoplasm and their subsequent discharge into the central vacuole.
Impedance and potential measurements have been made on a number of artificial membranes. Impedance changes were determined as functions of current and of the composition of the environmental solutions. It was shown that rectification is present in asymmetrical systems and that it increases with the membrane potential. The behavior in pairs of solutions of the same salt at different concentrations has formed the basis for the studies although a few experiments with different salts at the same concentrations gave results consistent with the conclusions drawn. A theoretical picture has been presented based on the use of the general kinetic equations for ion motion under the influence of diffusion and electrical forces and on a consideration of possible membrane structures. The equations have been solved for two very simple cases; one based on the assumption of microscopic electroneutrality, and the other on the assumption of a constant electric field. The latter was found to give better results than the former in interpreting the data on potentials and rectification, showing agreement, however, of the right order of magnitude only. Although the indications are that a careful treatment of boundary conditions may result in better agreement with experiment, no attempt has been made to carry this through since the data now available are not sufficiently complete or reproducible. Applications of the second theoretical case to the squid giant axon have been made showing qualitative agreement with the rectification properties and very good agreement with the membrane potential data.
Flux and flux-ratio equations are derived on the basis of the phenomenological equations of irreversible thermodynamics. Deviations of flux-ratios from that given by the often quoted Ussing (1949) relation are predicted, even in the absence of active transport, by considering the dependence of coupled fluxes on the membrane potential. The treatment is extended to include the interpretation of fluxes measured with tracers. Estimation of the numerical values of the resistance coefficients show that the voltage dependence of the entrainment terms can adequately account for the departures from the Ussing relation and the discrepancies between isotopically and electrically measured membrane conductances.
1.1. An attemp has been made to provide an experimental basis for interpreting the kinetics of the exchange of Cl− in excised carrot tissue.2.2. The fastest component of the efflux of tracer Cl− from carrot tissue is shown to be extracellular in origin. The two slower components are shown to be subcellular in origin, and are equated with the cytoplasm and the vacuole.3.3. When tissue is transferred from salt solution to water, the efflux of tracer Cl− is much reduced. On transfer back to salt, there is an initial burst before the Cl− efflux returns to the original level in salt. The initial burst is shown to represent loss of Cl− from the cytoplasm. This indicates that Cl− can move directly between the cytoplasm and the vacuole, and that the inhibition of Cl− efflux in water is an effect mainly at the plasmalemma. Further quantitative consideration ssuggest that there is no direct connection between the vacuole and the external solution.4.4. With the use of a model in which the cytoplasm and vacuole are arranged exclusively in series, the one-way fluxes at the plasmalemma and tonoplast and the cytoplasmic and vacuolar contents are estimated over a range of external Cl− concentrations. In conjuction with electrical measurements, these results show that Cl− is probably actively transported inwards across both plasmalemma and tonoplast, and that there is a 1-for-1 exchange diffusion component of the Cl− fluxes at the plasmalemma. Anions, other than halide are much less effective than Cl− in exchanging for internal 36Cl−.
Three intracellular compartments for potassium exchange have been observed in intact cells of the giant-celled alga, Nitella axillaris. These compartments have been compared with the exchange properties of isolated subcellular structures. The smallest and fastest compartment (apparent half-time, 23 seconds) appears to involve passive absorption on the cell wall. The next largest (apparent half-time, 5 hours) may represent exchange with the cytoplasmic layer through the plasma membrane, the chloroplasts being in rapid equilibrium with the surrounding cytoplasm. The largest and slowest compartment (apparent half-time, 40 days) has been identified with the central vacuole. The vacuolar membrane and the plasma membrane have similar properties with respect to K permeability. Thus, the experimental data from the whole cell can be accounted for by a structural model of the compartments. Cyanide in concentrations up to 10(-3)M causes no net loss of K. The fastest compartment in Nitella and in higher plants is compared, and the ecological significance of the slow rate of potassium transport in Nitella is discussed.
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.
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 (Pisum 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(+), Ca(2+), Mg(2+), Cl(-), NO(3) (-), H(2)PO(4) (-) and SO(4) (2-)) 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 NO(3) (-). 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(+), Mg(2+), and Ca(2+) 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) fluid 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 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. Measurements of the rates at which (42)K 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 MacRobbie 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.
Glass capillary microelectrodes were used to study the electrical potential difference (PD) between the xylem exudate of excised corn roots, Zea mays L. Golden Bantam hybrid, and the external solution. A survey of the effects of various ions on the PD was made. With 1 mm single salt solutions, the PD was between 25 and 50 mv, exudate negative. The PD responded to concentration differences in single salt solutions of K(+), Na(+), and Ca(2+) in a manner suggestive of cation selectivity and cation diffusion potentials. With Ca(2+) present, the PD was insensitive to concentration changes of other cations. Substitution of NO(3) (-) for Cl(-) in K(+) solutions increased the PD by 2 to 5 mv, although in general the PD showed little response to anion concentration changes. The PD was partially abolished by cyanide. The remaining fraction of the PD was sensitive to concentration changes in external K(+), and we postulate that the PD is the result of both a diffusion potential and an electrogenic pump.
In contrast to intact etiolated pea seedling tissue (Pisum sativum L.), excised segments immersed in a complete nutrient solution show marked increases in ion content, largely of K(+) and NO(3) (-), over a 72-hour period. During this time there is increase in cell electropotential difference, PD. During the initial 6 to 8 hours there is a lag in ion uptake; cell PD, however, increases rapidly from approximately -50 to -100 mv then increases more slowly. The increase in PD precedes and thus may be a prerequisite for the rapid ion accumulation phase. Cell PD increases in either water or nutrient solution but eventually reaches higher levels in the latter. Following water pretreatment of sufficient duration K(+) accumulation shows no lag period. The lag phase noted here appears dissimilar to that of storage tissues.
Steady state effluxes of potassium and sodium ions were measured on Pisum sativum var. Alaska root segments excised from seedlings which had grown in a nutrient solution containing the major inorganic ions and either (86)Rb as a tracer for K or (22)Na as a tracer for Na. Fluxes appeared to be from 2 cellular compartments, a small compartment with a high flux rate and a larger compartment with a slow flux rate. Cell wall exchange fluxes are believed to have been negligible. Efflux rates for 11.3% and 88.7% of cellular potassium ions were 6 x 10(-7) and 1.32 x 10(-7) respectively; rates for 33.7% and 66.3% of cellular sodium ions were 1.48 x 10(-7) and 3.83 x 10(-8) respectively, (equivalents per gram fr wt per hr). The sodium flux measurements, with previous measurements of ionic concentrations and transmembrane potentials, support the theory that sodium is transported actively from Pisum roots.
Mineral ion contents and cell transmembrane electropotentials of pea and oat seedling tissue Evidence for an electro-genic pump in cells of higher plants Ion transport Potassium and nitrate uptake and cell transmembrane electropotential in excised pea epicotyls
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The electrochemical rstate of cells of broad bean roots. I. Investigations of elongating roots of young rseedlings Electrical potentials and Na, K, and Cl concentrations in the vacuole and cytoplasm of Nitella traslucenls Membrane electrophoresis in relation to bio-electrical polariza-tion effects
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Scorr, B. I. H., H. GULLINE, AND C. K. PALLAGHY. 1968. The electrochemical rstate of cells of broad bean roots. I. Investigations of elongating roots of young rseedlings. Aust. J. Biol. Sci. 21: 185-200. 20. SPANSWICK, R. M. AND E. J. WILLIAMS. 1964. Electrical potentials and Na, K, and Cl concentrations in the vacuole and cytoplasm of Nitella traslucenls. J. Exp. Bot. 15: 193-200. 21. TEORELL, T. 1949. Membrane electrophoresis in relation to bio-electrical polariza-tion effects. Arch. Sci. Physiol. 3: 205-219.
The electrochemical rstate of cells of broad bean roots. I. Investigations of elongating roots ofyoung rseedlings
- B I H Scorr
- H Gulline
Scorr, B. I. H., H. GULLINE, AND C. K. PALLAGHY. 1968. The electrochemical rstate of cells of broad bean roots. I. Investigations of elongating roots ofyoung rseedlings. Aust. J. Biol. Sci. 21: 185-200
Potassium and nitrate uptake and cell transmembrane electropotential in excised pea epicotyls
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MACKLON, A. E. S. AND N. HIBINBOTHAM. 1968. Potassium and nitrate uptake and
cell transmembrane electropotential in excised pea epicotyls. Plant Physiol.
43: 888-892.
Active sodium and potassium transport in cells of barley roots
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PITMAN, M. G. AND H. D. W. SADDLER. 1967. Active sodium and potassium transport in cells of barley roots. Proc. Nat. Acad. Sci. U. S. A. 57: 44-49.