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  • 1
    Electronic Resource
    Electronic Resource
    THE PRESSURE PROBE: A Versatile Tool in Plant Cell Physiology (1999)
    Palo Alto, Calif. : Annual Reviews
    Annual Review of Plant Physiology and Plant Molecular Biology 50 (1999), S. 447-472 
    Details
    ISSN: 1040-2519
    Source: Annual Reviews Electronic Back Volume Collection 1932-2001ff
    Topics: Biology
    Notes: Abstract This review discusses how the pressure probe has evolved from an instrument for measuring cell turgor and other water relations parameters into a device for sampling the contents of individual higher plant cells in situ in the living plant. Together with a suite of microanalytical techniques it has permitted the mapping of water and solute relations at the resolution of single cells and has the potential to link quantitatively the traditionally separate areas of water relations and metabolism. The development of the probe is outlined and its modification to measure root pressure and xylem tension described. The deployment of the pressure probe to determine and map turgor, hydraulic conductivity, reflection coefficient, cell rheological properties, solute concentrations and enzyme activities at the resolution of single cells is discussed. The controversy surrounding the interpretation of results obtained with the xylem-pressure probe is included. Possible further developments of the probe and applications of single cell sampling are suggested.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1146/annurev.arplant.50.1.447
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  • 2
    Electronic Resource
    Electronic Resource
    Characterisation of a Ca2+-dependent ATP hydrolysis associated with the vacuolar membrane of wheat roots (1995)
    Oxford, UK : Blackwell Publishing Ltd
    Physiologia plantarum 94 (1995), S. 0 
    Details
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Microsomal fractions from wheat tissues exhibit a higher level of ATP hydrolytic activity in the presence of Ca2+ than Mg2+. Here we characterise the Ca2+-dependent activity from roots of Triticum aestivum lev. Troy) and investigate its possible function. Ca2+-dependent ATP hydrolysis in the microsomal fraction occurs over a wide pH range with two slight optima at pH 5.5 and 7.5. At these pHs the activity co-migrates with the major peak of nitrate-inhibited Mg2+. Cl-ATPase on continuous sucrose gradients indicating that it is associated with the vacuolar membrane. Ca2+-dependent ATP hydrolysis can be distinguished from an inhibitory effect of Ca2+ on the plasma membrane K+, Mg2+-ATPase following microsomal membrane separation using aqueous polymer two phase partitioning. The Ca2+-dependent activity is stimulated by free Ca2+ with a Km of 8.1 μM in the absence of Mg2+ ([CaATP] = 0.8 mM). Vacuoiar membrane vacuolar preparations contain a higher Ca2+-dependent than Mg2+-dependent ATP hydrolysis, although the two activities are not directly additive. The nucleotide specificity of the divalent ion-dependent activities in vacuolar membrane-enriched fractions was low. hydrolysis of CTP and UTP being greater than ATP hydrolysis with both Ca2+ and Mg2+ The Ca2+-dependent activity did discriminate against dinucleotides, and mononucleotides. and failed to hydrolyse phosphatase substrates. Despite low nucleotide specificity the Mg2+-dependent activity functioned as a bafilomycin sensitive H+-pump in vacuolar membrane vesicles.Ca2+-dependent ATP hydrolysis was not inhibited by the V-, P-, or F-type ATPase inhibitors bafilomycin. vanadate and azide, respectively. nor by the phosphatase inhibitor molybdate, but was inhibited 20% at pH 7.5 by K+. Possible functions of Ca2+-dependent hydrolysis as a H+-pump or a Ca2+-pump was investigated using vacuolar membrane vesicles. No H+ or Ca2+ translocating activity was observed under conditions when the Ca2+-dependent ATP hydrolysis was active.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1399-3054.1995.tb00961.x
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  • 3
    Electronic Resource
    Electronic Resource
    Methods, progress and potential for the use of isolated vacuoles in studies of solute transport in higher plant cells (1983)
    Oxford, UK : Blackwell Publishing Ltd
    Physiologia plantarum 57 (1983), S. 0 
    Details
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1399-3054.1983.tb02306.x
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  • 4
    Electronic Resource
    Electronic Resource
    Characterisation of a salt-stimulated ATPase activity associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.) (1981)
    Springer
    Planta 153 (1981), S. 140-149 
    Details
    ISSN: 1432-2048
    Keywords: ATPase ; Beta ; Vacuole
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Ion stimulation and some other properties of an ATPase activity associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.) have been determined. The ATPase had a specific requirement for Mg2+ and in the presence of Mg2+ it was stimulated by salts of monovalent cations. The degree of stimulation by monovalent salts was influenced mainly by the anion and the order of effectiveness of the anions tested was Cl-〉HCO 3 - 〉Br-〉malate〉acetate〉SO 4 2- . For any given series of anions the magnitude of the stimulation obtained was influenced by the accompanying cation (NH 4 + ≫ Na+〉K+). This cation effect was abolished by 0.01% (v/v) Triton X-100 and it is suggested that it is the result of different permeabilities of membrane vesicles to the cations. There was no evidence of synergistic stimulation of the ATPase by mixtures of Na+ and K+. KCl- and NaCl-stimulation was maximal with salt concentrations in the range 60–150 mM. The true substrate of the enzyme was shown to be MgATP. It was shown that KCl stimulation was the result of an increase in Vmax rather than a change in the affinity of the enzyme for MgATP. The ATPase was inhibited by N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, mersalyl and KNO3 but other inhibitors tested (azide, oligomycin, orthovanadate, K3[Cr(oxalate)6] and ethyl-3-[3-dimethylaminopropyl]carbodiimide) were without effect or caused only partial inhibition at the highest concentration tested. The ATPase activity was equally distributed between pellet and supernatant fractions obtained after the subfractionation of vacuoles but the properties of the ATPase in each fraction were the same. It is suggested that beet vacuoles possess only one ATPase. The properties of the ATPase are compared with those of ATPases associated with other plant membranes and organelles and its possible role in transport at the tonoplast is discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00384095
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  • 5
    Electronic Resource
    Electronic Resource
    Concentrations of inorganic and organic solutes in extracts from individual epidermal, mesophyll and bundle-sheath cells of barley leaves (1994)
    Springer
    Planta 192 (1994), S. 310-316 
    Details
    ISSN: 1432-2048
    Keywords: Epidermis (leaf) ; Hordeum ; Mesophyll ; Osmolality ; Single-cell sampling ; Solute compartmentation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The distribution of solutes between epidermal, mesophyll and bundle-sheath cells in barley (Hordeum vulgare L. cv. Klaxon) leaves was studied by analysing extracts obtained from single cells with a modified pressure probe. Activity of the cytoplasmic marker enzyme, malate dehydrogenase, revealed that epidermal cell extracts were completely vacuolar in origin, but extracts from mesophyll cells also contained cytoplasmic constituents. The extracts were analysed for osmolality and the concentrations of K, Na, Ca, Cl, P, S, NO 3 − , sugars and total amino acids. Epidermal and mesophyll cell extracts had similar osmolalities but these varied between 420 and 565 mosmol, kg 1 depending on the leaf developmental stage; the osmolality of bundle-sheath extracts was approximately 100 mosmol, kg−1 lower. Under the growth conditions used, K and NO 3 − were found in all three cell types and their concentrations generally ranged between 180 and 230 mM. In contrast, Ca was almost restricted to epidermal cells, where it increased to 70 mM during leaf ageing. Phosphorus was only detectable (≥ 5 mM) in extracts from mesophyll and bundle-sheath cells, while Cl concentrations were highest in epidermal and lowest in mesophyll cell extracts. The concentrations of sugars and amino acids were close to the detection limit (approx. 2 mM) in epidermal cells but mesophyll cells contained total sugar (glucose, fructose and sucrose) of up to 78 mM and total amino-acid concentrations of up to 13.5 mM. Concentrations in bundle-sheath cells were intermediate between those in the epidermis and mesophyll.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00198565
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  • 6
    Electronic Resource
    Electronic Resource
    Mg2+-Dependent, cation-stimulated inorganic pyrophosphatase associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.) (1981)
    Springer
    Planta 153 (1981), S. 150-155 
    Details
    ISSN: 1432-2048
    Keywords: Beta ; Pyrophosphatase ; Vacuole
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Vacuoles isolated from storage roots of red beet (Beta vulgaris L.) posess a Mg2+-dependent, alkaline pyrophosphatase (PPase) activity which is further stimulated by salts of monovalent cations. The requirement for Mg2+ is specific. Mn2+ and Zn2+ permitted only 20% and 12%, respectively, of the PPase activity obtained in the presence of Mg2+ while Ca2+, Co2+ and Cu2+ were ineffective. Stimulation of Mg2+-PPase activity by salts of certain monovalent cations was due to the cation and the order of effectiveness of the cations tested was K+=Rb+=NH 4 + 〉Cs+. Salts of Li+ and Na+ inhibited Mg2+-PPase activity by 44% and 24%, respectively. KCl-stimulation of Mg2+-PPase activity was maximal with 60–100 mM KCl. There was a sigmoidal relationship between PPase activity and Mg2+ concentrations which resulted in markedly non-linear Lineweaver-Burk plots. At pH 8.0, the optimal [Mg2+]:[PPi] ratio for both Mg2+-PPase and (Mg2++KCl)-PPase activities was approximately 1:1, which probably indicates MgP2O7 2- is the true substrate.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00384096
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  • 7
    Electronic Resource
    Electronic Resource
    An attempt to use isolated vacuoles to determine the distribution of sodium and potassium in cells of storage roots of red beet (Beta vulgaris L.) (1983)
    Springer
    Planta 159 (1983), S. 469-475 
    Details
    ISSN: 1432-2048
    Keywords: Beta (ion compartmentation) ; Extracellular space ; Ion compartmentation ; Turgor ; Vacuole
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Vacuoles isolated from red beet (Beta vulgaris L.) storage roots contain Na+ and K+ but their analysis does not give reliable information about the size of vacuolar pools of these ions in vivo. Analyses of isolated vacuoles indicated that between 53% and 90% of the Na+ was located in the vacuole and that the vacuolar concentrations of Na+ ranged between 4 and 45 mol m-3. Calculated concentrations of K+ in the vacuoles varied between 32 and 72 mol m-3 but, in contrast to Na+, only about 50% of the K+ was located in the vacuole. Considerations of the likely cytoplasmic concentrations of Na+ and K+ suggest that if these results indicate conditions in vivo a large proportion of these ions must be located in the extracellular space, where they would exert considerable osmotic pressure. To test this, the effect of washing on cell turgor (measured directly with a pressure probe) and on loss of Na+ and K+ was determined. Washing caused an increase in turgor of 5 bar but losses of Na+ and K+ were less than predicted by the experiments with isolated vacuoles. It is concluded that beet vacuoles leak Na+ and K+ when isolated resulting in an underestimation of the size of vacuolar pools of these cations in vivo. Nonetheless, the turgor measurements provide evidence for the presence of osmotically active solute in the extracellular space. The possible contribution of extracellular Na+ and K+ to the observed turgor reduction is calculated and the physiological importance of the accumulation of extracellular solutes is discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00392083
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  • 8
    Electronic Resource
    Electronic Resource
    The regulation of turgor pressure during sucrose mobilisation and salt accumulation by excised storage-root tissue of red beet (1987)
    Springer
    Planta 170 (1987), S. 353-361 
    Details
    ISSN: 1432-2048
    Keywords: Beta (storage root) ; Osmotic pressure ; Salt uptake ; Sucrose mobilisation ; Turgor pressure ; Vacuole (solute content)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The changes in turgor pressure that accompany the mobilisation of sucrose and accumulation of salts by excised disks of storage-root tissue of red beet (Beta vulgaris L.) have been investigated. Disks were washed in solutions containing mannitol until all of their sucrose had disappeared and then were transferred to solutions containing 5 mol·m-3 KCl+5 mol·m-3 NaCl in addition to the mannitol. Changes in solute contents, osmotic pressure and turgor pressure (measured with a pressure probe) were followed. As sucrose disappeared from the tissue, reducing sugars were accumulated. For disks in 200 mol·m-3 mannitol, the final reducing-sugar concentration equalled the initial sucrose concentration so there was no change in osmotic pressure or turgor pressure. At lower mannitol concentrations, there was a decrease in tissue osmotic pressure which was caused by a turgor-driven leakage of solutes. At concentrations of mannitol greater than 200 mol·m-3, osmotic pressure and turgor pressure increased because reducing-sugar accumulation exceeded the initial sucrose concentration. When salts were provided they were absorbed by the tissue and reducing-sugar concentrations fell. This indicated that salts were replacing sugars in the vacuole and releasing them for metabolism. The changes in salf and sugar concentrations were not equal because there was an increase in osmotic pressure and turgor pressure. The amount of salt absorbed was not affected by the external mannitol concentration, indicating that turgor pressure did not affect this process. The implications of the results for the control of turgor pressure during the mobilisation of vacuolar sucrose are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00395027
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  • 9
    Electronic Resource
    Electronic Resource
    The use of a chloride-sensitive fluorescent probe to measure chloride transport in isolated tonoplast vesicles (1988)
    Springer
    Planta 176 (1988), S. 451-460 
    Details
    ISSN: 1432-2048
    Keywords: Anion channels ; Chloride transport ; Beta (Cl- transport) ; Hordeum (Cl- transport) ; 6-Methoxy-1-(3-sulfonatopropyl)quinolinium ; Tonoplast ; Vesicle (isolated)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A fluorescence method for the direct measurement of Cl- transport in isolated tonoplast vesicles is described. This technique utilises the Cl--sensitive fluorescent compound, 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ). This is a water-soluble compound with excitation and emission wavelengths of 350 and 440 nm, respectively. Its fluorescence is quenched by Cl-, Br-, I-, SCN-, NO 2 - and tetraphenylborate but not by NO 3 - , SO 4 2- , iminodiacetate or malate. These effects are independent of pH. This compound was loaded into tonoplast vesicles from red beet (Beta vulgaris L.) storage roots or from barley (Hordeum vulgare L.) roots by incubation at 37° C and the external probe was then removed by repeated centrifugation of the vesicles in SPQ-free medium. In this way a large proportion of the observed fluorescence signal was from the interior of the vesicles, and its quenching could be used to monitor, quantitatively, and in real time, the intravesicular Cl- concentration. In this paper we describe some of the problems encountered in using this probe to measure Cl- transport in tonoplast vesicles, how these were overcome and some characteristics of Cl- transport at the tonoplast as measured by the probe.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00397651
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  • 10
    Electronic Resource
    Electronic Resource
    Compartmental nitrate concentrations in barley root cells measured with nitrate-selective microelectrodes and by single-cell sap sampling (1991)
    Springer
    Planta 185 (1991), S. 356-361 
    Details
    ISSN: 1432-2048
    Keywords: Hordeum (intracellular nitrate) ; Nitrate compartmentation (cytosol, vacuole) ; Root (intracellular nitrate)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Nitrate-selective microelectrodes were used to measure intracellular nitrate concentrations (as activities) in epidermal and cortical cells of roots of 5-d-old barley (Hordeum vulgare L.) seedlings grown in nutrient solution containing 10 mol · m−3 nitrate. Measurements in each cell type grouped into two populations with mean (±SE) values of 5.4 ± 0.5 mol · m−3 (n=19) and 41.8 ± 2.6 mol · m−3 (n = 35) in epidermal cells, and 3.2 ± 1.2 mol · m−3 (n = 4) and 72.8 ± 8.4 mol · m−3 (n = 13) in cortical cells. These could represent the cytoplasmic and vacuolar nitrate concentrations, respectively, in each cell type. To test this hypothesis, a single-cell sampling procedure was used to withdraw a vacuolar sap sample from individual epidermal and cortical cells. Measurement of the nitrate concentration in these samples by a fluorometric nitrate-reductase assay confirmed a mean vacuolar nitrate concentration of 52.6 ± 5.3 mol · m−3 (n = 10) in epidermal cells and 101.2 ± 4.8 mol · m−3 (n = 44) in cortical cells. The nitrate-reductase assay gave only a single population of measurements in each cell type, supporting the hypothesis that the higher of the two populations of electrode measurements in each cell type are vacuolar in origin. Differences in the absolute values obtained by these methods are probably related to the fact that the nitrate electrodes were calibrated against nitrate activity but the enzymic assay against concentration. Furthermore, a 28-h time course for the accumulation of nitrate measured with electrodes in epidermal cells showed the apparent cytoplasmic measurements remained constant at 5.0 ± 0.7 mol · m−3, while the vacuole accumulated nitrate to 30–50 mol · m−3. The implications of the data for mechanisms of nitrate transport at the plasma membrane and tonoplast are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00201056
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