Vacuolar symplast and methodological approach to monitoring water self-diffusion between vacuoles of contacting root cells

New concepts of structural-functional organization of the transport system in higher plants were evolved at the current stage of investigations. In addition to the classical (cytoplasmic) symplast, another supra-cellular continuum was supposed to exist in the plant tissue, which interconnects vacuoles of neighboring cells through desmotubules and represents the second transport pathway within the plasmodesmata. This study describes and experimentally validates the method for monitoring the self-diffusion of water molecules between vacuoles of contacting cells in the maize (Zea mays L.) root by means of NMR method with a pulsed magnetic field gradient. The method is based on the fact that, at long period of self-diffusion observation, when water molecules in the apoplast and cytoplasm had already completed their relaxation and did not contribute significantly to the proton echo signal, the slope of the initial portion of the diffusional decay is independent of water permeability of the vacuolar membrane and is determined exclusively by water permeability of intervacuolar pathway through the desmotubules.     

PFG-NMR measurements of the self-diffusion coefficients of water in equilibrium poly(HEMA-co-THFMA) hydrogels

The self-diffusion coefficients for water in a series of copolymers of 2-hydroxyethyl methacrylate, HEMA, and tetrahydrofurfuryl methacrylate, THFMA, swollen with water to their equilibrium states have been studied at 310 K using PFG-NMR. The self-diffusion coefficients calculated from the Stejskal-Tanner equation, D(obs), for all of the hydrated polymers were found to be dependent on the NMR storage time, as a result of spin exchange between the proton reservoirs of the water and the polymers, reaching an equilibrium plateau value at long storage times. The true values of the diffusion coefficients were calculated from the values of D(obs) in the plateau regions by applying a correction for the fraction of water protons present, obtained from the equilibrium water contents of the gels. The true self-diffusion coefficient for water in polyHEMA obtained at 310 K by this method was 5.5 x 10(-10) m(2)s-1. For the copolymers containing 20% HEMA or more a single value of the self-diffusion coefficient was found, which was somewhat larger than the corresponding values obtained for the macroscopic diffusion coefficient from sorption measurements. For polyTHFMA and copolymers containing less than 20% HEMA, the PFG-NMR stimulated echo attenuation decay curves and the log-attenuation plots were characteristic of the presence of two diffusing water species. The self-diffusion coefficients of water in the equilibrium-hydrated copolymers were found to be dependent on the copolymer composition, decreasing with increasing THFMA content.     

State of water and its diffusion across lipid bilayers: effect of hydration degree

The state of adsorbed water (estimated from the dependence of the shape of the 1H NMR spectrum on the angle between the normal to the bilayers and the direction of the magnetic field) and the diffusion of water molecules in the direction of the normal to the bilayers (estimated by 1H NMR spectroscopy with the impulse gradient of magnetic field) in microscopically oriented dioleoylphosphatidylcholine bilayers have been studied depending on hydration. The dependences of the shape of the NMR spectrum on angle differ qualitatively only at concentrations of water greater and less than the concentration that is achieved upon hydration from saturated vapors chi(eq) (about 23 weight %). At concentrations below chi(eq), all water present in samples enters the hydrate shells of polar "heads" of lipids or is in the state of "rapid exchange" with the water of hydrate shells, with the result that the signal of spin echo for water is observed only in a narrow range of angles close to the "magic angle", 54 degrees C. At concentrations above xhi(eq), the signal of spin echo for water is retained at all orientations, indicating probably that part of water between the bilayers ("quasi-free water") is in the state of a "slow exchange" with water "bound" to polar "heads". It was found that the coefficient of self-diffusion of water across the system of bilayers inversely depends on the degree of hydration, which is described in the Tanner model with consideration of the self-diffusion of water molecules in the hydrophobic moiety of the bilayer. The permeability of the bilayer, the coefficient of distribution of molecules between the water and lipid phases, and the coefficient of self-diffusion of water in the hydrophobic moiety of the bilayer were estimated.     

Regulation of Intercellular Water Exchange in Various Zones of Maize Root under Stresses

Apical root meristems and segments of root elongation zone were sampled from 4- to 5-day-old Zea mays L. seedlings. The vacuolar ATPase and pyrophosphatase, the tonoplast marker enzymes, and the tonoplast -, -, and -aquaporins were visualized by means of indirect immunofluorescent microscopy with the use of the respective antibodies. Following cell plasmolysis (700 mM mannitol, 2.5 h), the vacuolar ATPase and pyrophosphatase were detected in cell wall pores where plasmodesmata remained detached from the plasmolyzed protoplasts. This finding provides further evidence for existence of the vacuolar symplast in the elongation zone of maize root, which may ensure intercellular continuity of plant tissues. The pulsed NMR method was used to study the self-diffusion of water molecules. The diffusive decay in the root elongation zone was nonexponential, and it was transformed to three exponential terms with characteristic coefficients of self-diffusion; two of these coefficients (D ₂ and D ₃) characterize the water self-diffusion in the cytoplasmic and vacuolar symplasts of root, respectively. The root apical meristem was also investigated with NMR technique by virtue of paramagnetic doping of the apoplast. This approach allowed selective studying of water diffusion within the symplast compartments. Partial dehydration with PEG-6000, 12 and 20%, for 2.5 h and chemical stressors (ABA and salicylic acid, 0.1 mM, 24 h) were applied to modify water permeability of plasmodesmata and tonoplast aquaporins. The transcellular water permeability increased in the root meristem under the action of all stress factors. In the root elongation zone exposed to partial dehydration, the water exchange in the apoplast became the dominant component. Other stress factors affected water relations in different manners. ABA elevated the water permeability of the vacuolar symplast, in contrast to salicylic acid that decreased water conductance of both the cytoplasmic and vacuolar symplasts.     

Some biophysical applications of motional contrast in n.m.r. microscopy

The principal advantage of the n.m.r. imaging method lies in the specific contrasts which are available. In this work we describe the use of velocity and diffusion contrast methods in biophysical applications and at microscopic spatial resolution. In the first example, involving water-protein interactions, the relationship between water self-diffusion and water concentration, as measured using pulsed gradient spin echo n.m.r., is shown. It is demonstrated that this relationship can be used to provide a water concentration image. The result is compared with the conventional proton density and transverse relaxation maps. The next example concerns the use of dynamic n.m.r. microscopy to obtain water diffusion and velocity maps for wheat grain in vivo. Finally we suggest how the method may be used in the study of polymer-water interactions in an unusual adjunct to conventional polymer self-diffusion studies.     

Translation mobility of water isolated in the cells of lyophilized Cryptococcus albidus var. diffluens yeasts by the NMR impulse method

The method of NMR spin echo in a combination with the impulse gradient of the magnetic field was used to study the self-diffusion of water isolated in the cells of lyophylized Criptococcus. A relative fall of the amplitude of spin echo (factor R) in relation to the value of magnetic field impulse gradient (g), their duration (delta) and temporary distance between them (delta). The studies were carried out in the temperature range of 20 degrees divided by 80 degrees C. It has been shown that the fraction of isolated mobile water is located in intracellular permeable compartments with an average size of 0.6.10--4 cm at 20 degrees C. The coefficient of selfdiffusion of isolated water of Criptococcus (0.25.10--5 cm2S--1 at 20 degrees C) and the activation energy of selfdiffusion (4.4 ccal/mole at 20 degrees divided by 60 degrees C) were determined, permeability of walls of water-containing compartments taken into account.     

Measurement of the Self-Diffusion Coefficient of Water as a Function of Position in Wheat Grain Using Nuclear Magnetic Resonance Imaging

A pulsed field gradient spin echo sequence has been incorporated in a nuclear magnetic resonance (NMR) imaging experiment to provide an image contrast dependent on local molecular self-diffusion. The consequent image attenuation is shown to exhibit a dependence on applied magnetic field gradient consistent with the Stejskal-Tanner relationship. The method used represents a novel extension of microscopic imaging and demonstrates the possibility of measuring localized motion.

Water self-diffusion rates normal to the transverse 1.3-mm section of a wheat grain have been measured in structural features at 150-μm resolution. The results are consistent with averaged measurements in the bulk grain obtained by other methods while local differences in water mobility correlate with differences in physiological function.

     

NMR imaging of roots: methods for reducing the soil signal and for obtaining a 3‐dimensional description of the roots

Studies of root morphology or root architecture are limited by the problems of observing roots in soil. NMR imaging can preferentially detect the water in roots rather than soil and can therefore produce a map of the distribution of the water in roots, even though there is much more water in the soil than in the roots. The gradient echo imaging technique was shown to detect less signal from water in soil than the more usual spin‐echo technique, probably because the signal loss due to magnetic field inhomogeneities was not refocused in the gradient echo experiments. The gradient echo sequence produced images of a water‐filled capillary tube in soil and of the main roots of a 31‐day‐old oat plant, images which had better contrast than those from the spin‐echo sequence. The improvement was particularly noticeable in the wetter soils. The finer roots could only be observed in the driest of soil and with the spin‐echo sequence. Magnetic field inhomogeneities have a greater effect on thinner roots than on thicker ones, and because the initial signal from thin roots was so small, the additional signal loss due to magnetic field inhomogeneities made the fine roots undetectable with the gradient echo sequence. The problem of loss of perspective associated with projection images was overcome by taking a number of images with different projection planes. By comparing images, a set of (x, y, z) coordinates was obtained for the 4 main roots of a 31‐day‐old oat plant. Computer graphics methods made it possible to compare the digitised root coordinate data set with the original images, thereby confirming the accuracy of the digitised data.     

Self-diffusion of water into silk fibers from magnetic field pulse gradient data

Self-diffusion of water was studied in fibers of natural silk (Bombyx mori) with a water content of 0.18 g H2O/g dried material. Self-diffusion measurements were conducted by pulsed gradient of magnetic field (stimulated echo) at diffusion times from 10 to 200 mc. The dependence of experimental diffusion coefficients Dexp = f(delta) (observed decrease when delta increased) was determined to be responsible for the restricted diffusion. A model of planar and regularly spaced permeable barriers to diffusion of water molecules was applied to estimate the barrier spacing a and the permeability constant p. The maximal value of Dexp (at short diffusion time) in B. mori silk fibres was about 0.06 of the value of Dexp in bulk free water. The results obtained are compared to literature data on self-diffusion of water in hydrated biopolymer fibers and are discussed in connection with molecular mobility in natural macromolecular systems with low water content.     

Calculation of self-diffusion coefficients of the [BMIM][TFSA]/water system by molecular dynamics simulation

We performed a molecular dynamics simulation to calculate the self-diffusion coefficients of 1-Butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide and water in a water–ionic liquid mixture. We then compared the simulated self-diffusion coefficients of cation, anion and water molecules with experimental data and with simulated data from the literature. Although the simulation overestimated the self-diffusion coefficients of ions, the simulated results qualitatively reproduced the enhancement of the self-diffusion coefficients of water as the water molar fraction increased. We also calculated the radial distribution functions to investigate the solution structure, i.e. the clustering of water molecules. The clustering of water in ionic liquid was found to play an important role in the enhancement of the diffusion of water molecules in the ionic liquid.     

Diffusion properties of water in the human crystalline lens during cataract development

Normal and cataractal decapsulated lenses of man were studied by NMR method-spin echo to obtain information concerning efficient coefficients of self diffusion, times of spin-spin relaxation of water protons (T2) and water content in the lens (c) at 25 degrees C in the course of cataractogenesis. It has been found that the values T2 and c at 25 degrees C are much higher in the nuclei of completely turbid lenses than in the transparent ones; the self-diffusion coefficients in the turbid lenses were also higher. At -9 degrees C a significant decrease of the content of undestroyed by frost (bound) water was observed at the stage of mature cataract as compared to transparent lenses. It is suggested that the most specific differences between the nuclei of transparent and completely turbid lenses are related to increased diffusion mobility of water molecules, apparently, at the expense of damaged plasmic membranes of the lens fibres noted during cataract development.     

Diffusional attenuation of proton echo and control of changes in the cytoskeleton hydration level

Using the pulsed NMR-method with pulse gradient of the magnetic field, a study was made of changes in diffusional attenuation (DA) of proton echo in germs of triticum grains under the influence of agents depolymerizing cytoskeleton elements or inhibiting their ATP-activity. At a short diffusion time, we observed respective alterations of NMR-population in a fraction of cell water molecules contributing to DA at large values of pulse gradient. It is shown that the presence in DA of a site with essentially smaller coefficient of self-diffusion, as compared with basic cell water fraction, is not a consequence of restricted diffusion phenomenon. The observed changes in NMR-population of the appropriate fraction of cell water molecules are interpreted as changes in the level of cytoskeleton hydratation occurring under the influence of the used agents. A hypothesis is proposed for the functional role of similar changes in the level of hydratation of cytoskeleton elements located inside plamodesmata in the mechanism of plasmodesmatal water permeability regulation.     

Vacuolar Symplast as a Regulated Pathway for Water Flows in Plants

Indirect immunofluorescent microscopy and a tonoplast-specific marker enzyme were used to demonstrate the occurrence of pyrophosphatase within the plasmodesmata in the elongation zone of maize root segments. The pulsed field gradient NMR method (PFG NMR) was applied to study restricted self-diffusion of water molecules in the root segments under normal conditions and after the inhibition of respiration with sodium azide (10 mM NaN₃, 30 min). The results led to the conclusion that vacuoles in the root segments examined are interconnected into a unified intercellular continuum and that intervacuolar connections are formed by desmotubules within the plasmodesmata. The water permeability of the vacuolar symplast appears to be controlled by an ATP-dependent process. The experimental data can provide a methodological approach to studying water permeability of the vacuolar symplast with the PFG NMR technique.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 372–377.Original Russian Text Copyright © 2005 by Velikanov, Volobueva, Belova, Gaponenko.     

NMR investigations of the 4-ethyl guaicol self-diffusion in iota (ι)-carrageenan gels

Self-diffusion coefficient of an aroma molecule (4-ethyl guaicol) was measured using the pulsed field gradient spin echo NMR (PGSE-NMR) method in order to investigate the influence of a macromolecular matrix on its diffusion and release processes. Iota (ι)-carrageenan was used for its ability to form thermoreversible gels in aqueous salt solutions. Variations of the ι-carrageenan and the salt concentrations permitted various gels with different thermal and rheological properties to be obtained. These latter were modified by an isotope effect obtained by preparing gels in D₂O. The NMR self-diffusion measurements realised for water and the aroma molecules indicated neither chemical interactions with ι-carrageenan, nor obstruction effects from the polysaccharide chains. In ι-carrageenan gels, the diffusional phenomenon was highly dependent on the heterogeneous gel structure and controlled by hydrodynamic interactions due to frictional drag between each molecule of the system and water microviscosity changes.     

Radial diffusion transport of water in various zones of maize root and its sensitivity to mercury chloride

NMR-spin echo method was used for comparative study of radial water diffusion in various zones of maize (Zea mays L., cv. Donskaya 1) primary root. Coefficients of water diffusion varied strongly along the root length; the pattern of variations depended on the period during which the diffusion of water molecules was traced. Water diffusion transport in various root zones was unevently sensitive to mercury chloride, an aquaporin inhibitor. The discovered variations in the mobility of water molecules were assigned to morphological and functional features of cells and tissues in the root zones examined; they were interpreted in terms of variable contribution and redistribution of water flows along several transport pathways. The decrease in diffusional water flows could be caused by cell wall modifications (deposition of suberin) that emerge in the endoderm regions distant from the root apex and diminish the contribution of apoplastic transport.     

Self-diffusion of polymers in cartilage as studied by pulsed field gradient NMR

Pulsed field gradient (PFG) nuclear magnetic resonance (NMR) was used to investigate the self-diffusion behaviour of polymers in cartilage. Polyethylene glycol and dextran with different molecular weights and in different concentrations were used as model compounds to mimic the diffusion behaviour of metabolites of cartilage. The polymer self-diffusion depends extremely on the observation time: The short-time self-diffusion coefficients (diffusion time Delta approximately 15 ms) are subjected to a rather non-specific obstruction effect that depends mainly on the molecular weights of the applied polymers as well as on the water content of the cartilage. The observed self-diffusion coefficients decrease with increasing molecular weights of the polymers and with a decreasing water content of the cartilage. In contrast, the long-time self-diffusion coefficients of the polymers in cartilage (diffusion time Delta approximately 600 ms) reflect the structural properties of the tissue. Measurements at different water contents, different molecular weights of the polymers and varying observation times suggest that primarily the collagenous network of cartilage but also the entanglements of the polymer chains themselves are responsible for the observed restricted diffusion. Additionally, anomalous restricted diffusion was shown to occur already in concentrated polymer solutions.     

Low- and high-resolution nuclear magnetic resonance (NMR) characterisation of hyaluronan-based native and sulfated hydrogels

Hyaluronan-based hydrogels were synthesised using different crosslinking agents, such as 1,3-diaminopropane (1,3-DAP) and 1,6-diaminohexane (1,6-DAE). The hydrogels were sulfated to provide materials (Hyal-1,3-DAP, Hyal-1,6-DAE, HyalS-1,3-DAP and HyalS-1,6-DAE) that were characterised by both high- and low-resolution nuclear magnetic resonance (NMR) spectroscopy. The (13)C NMR spectra of the materials were analysed to identify, characterise and study the crosslinking degree of the hydrogels. The crosslinking degree was also determined by potentiometric titration and the effectiveness of the two techniques was compared. Measurements of longitudinal relaxation times (spin-lattice) and of NOE enhancement were used to study the mobility of the hydrogels. Low-resolution NMR studies allowed the determination of the water transport properties in the hydrogels. In addition, the swelling degree for the various hydrogels was calculated as a function of the longitudinal and transversal relaxation times of the water molecules. Lastly, the self-diffusion coefficients of the water in interaction with the four polysaccharides were measured by the pulsed field gradient spin echo (PFGSE) sequence.     

Water self-diffusion behavior in yeast cells studied by pulsed field gradient NMR

The water self-diffusion behavior in yeast cell water suspension was investigated by pulsed field gradient NMR techniques. Three types of water were detected, which differ according to the self-diffusion coefficients: bulk water, extracellular and intracellular water. Intracellular and extracellular water self-diffusion was restricted; the sizes of restriction regions were approximately 3 and 15-20 microm, respectively. The smallest restriction size was determined as inner cell size. This size and also cell permeability varied with the growth phase of yeast cell. Cell size increased, but permeability decreased with increasing growth time. The values of cell permeabilities P(1)(d) obtained from time dependence of water self-diffusion coefficient were in good agreement with the permeabilities obtained from the exchange rate constants P(1)(eff). The values of P(1)(eff) were 7 x 10(-6), 1.2 x 10(-6) and 1.6 x 10(-6) m/s, and P(1)(d) were 6.3 x 10(-6), 8.4 x 10(-7), 1.5 x 10(-6) m/s for yeast cells incubated for 9 h (exponential growth phase), 24 h (end of exponential growth phase), and 48 h (stationary growth phase), respectively.     

Molecular dynamics simulation of liquid water under the influence of an external electric field

Molecular dynamics simulations of liquid water were performed at 258K and a density of 1.0?g/cm³ under various applied external electric field, ranging 0～10¹⁰?V/m. The influence of external field on structural and dynamical properties of water was investigated. The simple point charge (SPC) model is used for water molecules. An enhancement of the water hydrogen bond structure with increasing strength of the electric field has been deduced from the radial distribution functions and the analysis of hydrogen bonds structure. With increasing field strength, water system has a more perfect structure, which is similar to ice structure. However, the electrofreezing phenomenon of liquid water has not been detected since the self-diffusion coefficient was very large. The self-diffusion coefficient decreases remarkably with increasing strength of electric field and the self-diffusion coefficient is anisotropic.     

Study of water and oil diffusion in rape seeds with sorption-desorption and NMR techniques.

The sorption and desorption of water in rape seeds was measured. From the sorption isotherm it follows that for water content greater than about 6% the water molecules tend to form clusters. The mutual diffusion coefficient of water into and out of the seeds was determined from the time dependence of sorption and desorption. There is a pronounced hysteresis in the sorption-desorption process, desorption proceeds faster than sorption. The self-diffusion of water (at maximum humidity of the seeds) and oil within the seeds was investigated by the pulsed field gradient NMR. The measurement of oil self-diffusion shows restricted diffusion of the oil within droplets and allows the determination of the droplet radii and their distribution width.