The Measurement of Permeability to water in Disks of Storage Tissues4

1. Experiments have been performed to examine methods to determinehydraulic conductivity and diffusional permeability in disksof red beet (Beta vulgaris) and Jerusalem artichoke (Helianthustuberosus). 2. The half-time for the weight change caused by transferringdisks between pure water and a sucrose solution, or vice-versa,has been shown to be linearly dependent on the square of thedisk thickness. 3. The efflux kinetics of sucrose from these disks are alsocharacterized by a half-time linearly dependent on the squareof the disk thickness. 4. It is concluded that extracellular solute diffusion, andnot hydraulic conductivity, is the principal rate-controllingfactor in experiments of this type. 5. Efflux studies with tritiated water also show a characteristichalf-time which is linearly dependent on the square of the diskthickness. The cell membrane does not constitute a sufficientbarrier to diffusion fot its diffusional permeability to bedetermined.     

Regulation of Water Permeability of Vacuolar Symplast

Effect of exogenous ABA and an inhibitor of energy metabolism NaN₃ on water permeability of the desmotubules and tonoplast as the structural elements of vacuolar symplast ensuring water permeability of this transport system was investigated. The methodological approach based on the use of NMR with magnetic field pulse gradient is described in detail. It was shown that ABA affects water permeability of the vacuolar symplast in the root cells of maize (Zea mays L.) seedlings by temporary increase in water permeability of its membrane (tonoplast) and does not modify water permeability of desmotubules. At the same time, the effect of sodium azide is related to the disturbance of water permeability in the latter, and this evidence is corroborated by the additivity in the effects of the two above-mentioned agents on diffusion decay of spin echo produced by vacuolar symplast water molecules. ABA effect was detected only at high exogenous concentrations (10^?4 M). The effect of ABA on water permeability of the tonoplast did not depend on or was weakly related to intracellular concentration of ATP, whereas the open state of desmotubules was ATP-dependent. Observations were made on the role of aquaporins in the ABA influence on tonoplast water permeability and the physiological role of high ABA concentrations.     

The Relationship between Water Binding and Desiccation Tolerance in Tissues

In an effort to define the nature of desiccation tolerance, a comparison of the water sorption characteristics was made between tissues that were resistant and tissues that were sensitive to desiccation. Water sorption isotherms were constructed for germinated and ungerminated soybean axes and also for fronds of several species of Polypodium with varying tolerance to dehydration. The strength of water binding was determined by van't Hoff as well as D'Arcy/Watt analyses of the isotherms at 5, 15, and/or 25°C. Tissues which were sensitive to desiccation had a poor capacity to bind water tightly. Tightly bound water can be removed from soybean and pea seeds by equilibration at 35°C over very low relative humidities; this results in a reduction in the viability of the seed. We suggest that region 1 water (i.e. water bound with very negative enthalpy values) is an important component of desiccation tolerance.     

Effect of {alpha}-Tomatine on the Permeability of Plant Storage Tissues

The effect of the steroidal glycoalkaloid -tomatine on effluxof cell material from disks of beetroot, potato, apple, andcarrot has been investigated. A concentration of 1 mM tomatinecaused large increases in efflux from all tissues but 10 µMhad no effect. Leakage from potato and apple, but not from beetrootand carrot, was stimulated by 100 µM. tomatine, althoughlosses were smaller. Alkaloid-induced efflux of cell materialwas reduced considerably by addition of 1 mM CaCl₂ and to alesser extent by 1 mM MgCl₂. Cholesterol and its acetate didnot reduce leakage from beetroot but higher concentrations ofthe former caused small reductions in loss of material frompotato. The significance of these findings in relation to theaction of other membrane-active compounds is discussed.     

Response of Tissues2

Efflux of K⁺ from tomato fruit pericarp disks was not affectedby 100 µM tomatine, and only slightly by 1 mM. No differentialresponses to the alkaloid were observed in disks obtained fromfruits at different stages of development, and there was noevidence of any correlation between the level of endogenoustomatine and the effect of exogenous tomatine. Growth of tomatoseedlings in liquid nutrient medium was unaffected by 1 mM tomatinewhereas the growth oflettuce seedlings was stunted in the presenceof 100 µM alkaloid. The polyene antibiotic nystatin gavesimilar resultsto tomatine, having little effect on tomato tissuesbut causing marked inhibition of lettuce seedling development.     

Determinants of Water Permeability through Nanoscopic Hydrophilic Channels

Naturally occurring pores show a variety of polarities and sizes that are presumably directly linked to their biological function. Many biological channels are selective toward permeants similar or smaller in size than water molecules, and therefore their pores operate in the regime of single-file water pores. Intrinsic factors affecting water permeability through such pores include the channel-membrane match, the structural stability of the channel, the channel geometry and channel-water affinity. We present an extensive molecular dynamics study on the role of the channel geometry and polarity on the water osmotic and diffusive permeability coefficients. We show that the polarity of the naturally occurring peptidic channels is close to optimal for water permeation, and that the water mobility for a wide range of channel polarities is essentially length independent. By systematically varying the geometry and polarity of model hydrophilic pores, based on the fold of gramicidin A, the water density, occupancy, and permeability are studied. Our focus is on the characterization of the transition between different permeation regimes in terms of the structure of water in the pores, the average pore occupancy and the dynamics of the permeating water molecules. We show that a general relationship between osmotic and diffusive water permeability coefficients in the single-file regime accounts for the time averaged pore occupancy, and that the dynamics of the permeating water molecules through narrow non single file channels effectively behaves like independent single-file columns.     

Rehydration versus Growth-induced Water Uptake in Plant Tissues

Experiments show that the rate of water uptake by living tissues external to mature xylem of cotton stems (Gossypium hirsutum L. Auburn 7-683) is very similar to the corresponding curves for leaf tissue. In both cases one obtains a two-phase curve with phase I corresponding to passive rehydration and phase II pertaining to active growth.     

Water Permeability in Resting and Stimulated Crayfish Nerve

The hydraulic conductivity, L_p, was determined in single axons of the crayfish, Procambarus clarkii, by injecting a hypertonic sample between two drops of silicone oil and photographing the volume increase of the sample. The method has the advantage of minimizing errors due to hydrostatic pressure differences across the membrane. In resting axons an L_p of 0.236 x 10^-8 cm/ sec per cm H₂O was found and similar values were obtained with low external calcium concentration and when the nerve was continuously stimulated at 20–30 impulses/sec. Thus the experiments have failed to demonstrate any change of water permeability in cases in which the ionic conductance is known to change. Some possible implications of this are discussed.     

Permeability of the Ehrlich Ascites Tumor Cell to Water

The osmotic permeability coefficient for water has been measured for the Ehrlich mouse ascites tumor cell. Measurements were made of the rate of cell shrinkage in hyperosmotic solutions of NaCI, a functionally impermeable solute. During the first 9 months of weekly serial transplantation the mean was 6.4 µ³/µ³/atm. ± 0.8 (S.E.). By the end of the 2nd year the permeability coefficient was much lower and averaged 1.6 ± 0.09. There were no significant differences in the volume of the tumor cells which could explain the discrepancy on the basis of a change in the volume to surface area ratio. Studies of the effect of temperature were done and Eyring's theory of absolute reaction rates was applied to the data. The apparent energy of activation was 9.6 kcal./mol and ΔS‡ was 39.1 entropy units. The thermodynamic data are twice as high as data reported by Wang for self-diffusion and viscous properties of water. Two alternate explanations have been advanced based on the pore hypothesis of membrane permeability. One explains the thermodynamic data from a change in the A'/Δx available for water movement; the other assumes A'/Δx constant and bases the results on the interaction of water dipoles with each other and the membrane.     

Growth-induced Water Potentials in Plant Cells and Tissues

A physical analysis of water movement through elongating soybean (Glycine max L. Merr.) hypocotyls was made to determine why significant water potentials persist in growing tissues even though the external water potentials were zero and transpiration is virtually zero. The analysis was based on a water transport theory modified for growth and assumed that water for growing cells would move through and along the cells in proportion to the conductivity of the various pathways.

Water potentials calculated for individual cells were nearly in local equilibrium with the water potentials of the immediate cell surroundings during growth. However, water potentials calculated for growing tissue were 1.2 to 3.3 bars below the water potential of the vascular supply in those cells farthest from the xylem. Only cells closest to the xylem had water potentials close to that of the vascular supply. Gradients in water potential were steepest close to the xylem because all of the growth-sustaining water had to move through this part of the tissue. Average water potentials calculated for the entire growing region were −0.9 to −2.2 bars depending on the tissue diffusivity.

For comparison with the calculations, average water potentials were measured in elongating soybean hypocotyls using isopiestic thermocouple psychrometers for intact and excised tissue. In plants having virtually no transpiration and growing in Vermiculite with a water potential of −0.1 bar, rapidly growing hypocotyl tissue had water potentials of −1.7 to −2.1 bars when intact and −2.5 bars when excised. In mature, nongrowing hypocotyl tissue, average water potentials were −0.4 bar regardless of whether the tissue was intact or excised.

The close correspondence between predicted and measured water potentials in growing tissue indicates that significant gradients in water potential are required to move growth-associated water through and around cells over macroscopic distances. The presence of such gradients during growth indicates that cells must have different cell wall and/or osmotic properties at different positions in the tissue in order for organized growth to occur. The mathematical development used in this study represents the philosophy that would have to be followed for the application of contemporary growth theory when significant tissue water potential gradients are present.

     

Measurement of the Water Potential of Intact Plant Tissues1

A modified design of a thermocouple psychrometer is describedwhich utilizes the Peltier effect for producing an exceedinglysmall wet-bulb thermometer. The thermocouple assembly has beenreduced to miniature dimensions and the thermocouple chamberconsists of a silver cylinder (volume 0.06 cm³) whose base isformed by the material under observation. This makes it possibleto measure water potentials over limited surfaces of intactplant tissues, e.g. germinating pea seeds (Pisum sativum L.).The apparatus is capable of measuring water potentials downto a magnitude of about –6,000 joules/kg.³     

Effect of Water Deficit and Membrane Destruction on Water Diffusion in the Tissues of Maize Seedlings

We investigated diffusion of water in maize seedlings (Zea mays L. cv. Dnepropetrovskaya) following addition of polyethylene glycol (PEG) 6000 (osmotic potential –0.1 and –0.3 MPa) to the root medium by NMR method with pulsed gradient of magnetic field. Diffusion coefficients of different water phases in plant tissues (water of apoplast and vacuoles, water transported through the membranes) have been estimated from multicomponent decays of echo amplitude. Different signs of changes of water diffusion coefficients of fast and slow components of diffusional echo decay in roots and leaves under the influence of PEG-induced water deficits were shown. It has been supposed that under water deficit a sharing of water flows takes places through the different pathways (apoplastic, symplastic and transmembrane). In roots, 1-h water deficit increased the rate of fast diffusing water (water of apoplasm, vacuoles and, perhaps, water contained in intercellular endoplasm system), and decreased the rate of slowly diffusing water (water passing across the membranes). A long-term water deficit increased to a small extent the rate of water transmembrane transfer in root tissue. Leaf response to water stress was in the intensification of rate of transmembrane water transport that could be connected with the expression of water channels, and in the decrease of apoplastic water flow and flow along endoplasm. The possibility of estimation of plant tissue (membrane) integrity on the basis of diffusional data has been demonstrated.     

Water Permeability of Brush Border Membrane Vesicles from Kidney Proximal Tubule

Brush border membrane vesicles (BBMV) maintain an initial hydrostatic pressure difference between the intra- and extravesicular medium, which causes membrane strain and surface area expansion (Soveral, Macey & Moura, 1997). This has not been taken into account in prior osmotic water permeability P _f evaluations. In this paper, we find further evidence for the pressure in the variation of stopped-flow light scattering traces with different vesicle preparations. Response to osmotic shock is used to estimate water permeability in BBMV prepared with buffers of different osmolarities (18 and 85 mosM). Data analysis includes the dissipation of both osmotic and hydrostatic pressure gradients. P _f values were of the order of 4 × 10⁻³ cm sec⁻¹ independent of the osmolarity of the preparation buffer. Arrhenius plots of P _f vs. 1/T were linear, showing a single activation energy of 4.6 kcal mol⁻¹. The initial osmotic response which is significantly retarded is correlated with the period of elevated hydrostatic pressure. We interpret this as an inhibition of P _f caused by membrane strain and suggest how this inhibition may play a role in cell volume regulation in the proximal tubule. Received: 8 August 1996/Revised: 4 March 1997     

Visualization of AqpZ-Mediated Water Permeability in Escherichia coli by Cryoelectron Microscopy

Transport of water across the plasma membrane is a fundamental process occurring in all living organisms. In bacteria, osmotic movement of water across the cytoplasmic membrane is needed to maintain cellular turgor; however, the molecular mechanisms of this process are poorly defined. Involvement of aquaporin water channels in bacterial water permeability was suggested by the recent discovery of the aquaporin gene, aqpZ, in Escherichia coli. By employing cryoelectron microscopy to compare E. coli cells containing (AqpZ+) and lacking (AqpZ-) aquaporin, we show that the AqpZ water channel rapidly mediates large water fluxes in response to sudden changes in extracellular osmolarity. These findings (i) demonstrate for the first time functional expression of a prokaryotic water channel, (ii) evidence the bidirectional water channel feature of AqpZ, (iii) document a role for AqpZ in bacterial osmoregulation, and (iv) define a suitable model for studying the physiology of prokaryotic water transport.     

Permeability to Water of the Fibre Cell Wall Material of Two Hardwoods

The permeability to water of the fibre cell wall of birch (Betulapubescens Ehrh.) and lime (Tilia x vulgaris Hayne) wood, wasmeasured in wood slices in which most of the void space wasfilled with paraffin wax or a polymerizing silicone elastomer.An osmotic technique was used with solutes of molecular weightgreat enough to prevent their molecules from penetrating thepores in the water-swollen cell wall. The solutes used weredextran and polyethylene glycol with molecular weights of 40000 and 6000 respectively. Values for permeability k x 10²¹, as defined by the Darcy equation,ranged between 3.6 m² for the tangential direction in birch,to 27 m² for the longitudinal direction in birch. These resultsare in good agreement with previously measured values but areat least ten times less than theoretical values. It is calculatedthat total emptying or filling of a wood cell through the wallmight be possible in as little as 5 min under a pressure differenceof 0.1 MPa if other flow pathways were blocked. Key words: Betula pubescens, Tilia x vulgaris, Water permeability, Fibre cell wall