Vacuolar Symplast As a Regulated Pathway for Water Flows in Plants G

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Russian Journal of Plant Physiology, Vol. 52, No. 3, 2005, pp. 326–331. Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 372–377. Original Russian Text Copyright © 2005 by Velikanov, Volobueva, Belova, Gaponenko.

Vacuolar Symplast as a Regulated Pathway for Water Flows in Plants G. A. Velikanov, O. V. Volobueva, L. P. Belova, and E. M. Gaponenko Kazan Institute of Biochemistry and Biophysics, Kazan Research Center, Russian Academy of Sciences, a/ya 30, ul. Lobachevskogo 2/31, Kazan, Tatarstan, 420111 Russia; fax: 7 (8432) 38-7577; e-mail: [email protected] Received August 6, 2004

Abstract—Indirect immunofluorescent microscopy and a tonoplast-specific marker enzyme were used to dem- onstrate 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 con- trolled 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.

Key words: Zea mays - roots - plasmodesmata - desmotubules - vacuolar symplast - NMR

INTRODUCTION spaces into a supracellular continuum. This means that vacuoles of neighboring cells are also interconnected. Until recently, many problems of plant physiology The diffusive contact of cytoplasmic compartments is were considered from the principle that the central vac- confined to the ring-shaped periphery of the plas- uole is an enclosed intracellular compartment. The modesma section. The Gamalei’s studies raised the plasmodesmata, responsible for the symplastic contin- hypothesis on the occurrence in plant tissues of the vacuum in tissues, were regarded to ensure diffusive com- uolar symplast in addition to the cytoplasmic symplast. munications only between the cytoplasmic compart- If this hypothesis turns true, the classic views should be ments of cells. The root structure models, envisaging revised in many aspects regarding mineral nutrition, vacuoles as enclosed intracellular compartments, pro- water balance, and intercellular transport in plants. vide the basis for theoretical concepts on the transport None of these problems was ever considered from the of water and mineral nutrients into the aboveground standpoint of existence of two regulated transport organs [1]. On the other hand, electron microscopy routes within the plasmodesma. The NMR data on studies of plant tissues revealed an additional transport intercellular water transport in wheat roots can be route within the plasmodesma. The structure located in readily interpreted within the framework of this con- the center of the plasmodesma cross-section was shown cept [7]. to be a tubule, not a rod as was previously thought [2, 3]. A long-lasting discussion over reliability of elec- The idea of vacuolar symplast and the view of the tron-microscopic evidence for the existence of a central plasmodesma desmotubule as an important transport desmotubule was finished after confirmative data were pathway remain unadopted by many researchers. Even obtained with the confocal microscopy [4]. However, the most reputed experts in transport properties of plas- the question of what compartments of neighboring cells modesmata consider these structures as pathways inter- are interconnected by the central desmotubule is still a connecting the cytoplasmic compartments only [8]. matter of discussion. Gamalei [5, 6] presented the most The aim of this study is to present experimental evi- convincing arguments on this matter. Ample data of his dence in support of the idea of vacuolar symplast as a own and other researchers' studies provide evidence regulated pathway for water transport in plants. that the vacuolar membrane (tonoplast) is an element of the endoplasmic membrane network and that the desmotubule is a pathway interconnecting endoplasmic MATERIALS AND METHODS We used the roots of 4-day-old etiolated maize (Zea Abbreviations: DD—diffusive spin-echo decay; PBS—phos- mays L.) seedlings. The seeds were soaked for 8 h in phate-buffered saline; PFG NMR—pulsed field gradient NMR running tap water, placed in rolls of moistened filter method; SDC—self-diffusion coefficient; td—observation time of diffusion. paper, and germinated in a thermostat at 25°ë. The

VACUOLAR SYMPLAST AS A REGULATED PATHWAY 327 roots were slightly blotted with a filter paper, and the time at a constant temperature. According to the afore- elongation zone (beyond 8 mm from the root tip) was mentioned Einstein equation, the distance L equals to cut into 7-mm-long segments. the square root of the product 2D0td, where D0 is SDC The self-diffusion of water molecules was assessed of the molecules in the infinite volume. with a spin echo proton NMR using a pulsed magnetic In the regime of short-term diffusion, the displace- field gradient (PFG–NMR method). The root segments ments of molecules are much smaller than the linear were placed in the sensor unit of NMR diffusometer in dimension a of a constricted compartment; i.e., L a. such a way that the magnetic field gradient was directed In this regime, the effective SDC “does not see” the parallel to the root radius; this orientation allowed us to obstacles, and its value is similar to SDC of pure liquid monitor self-diffusion of water in the radial direction. (water in our case) in a large volume. Self-diffusion of molecules was assessed by analyzing The regime of intermediate times of diffusion is the diffusive decay of spin echo signal (R/R0, see equa- realized when the diffusive length L is approximately tion below) as a function of parameters of the pulsed ≈ ≈ 2 δ equal to the compartment size a: L a or td a /D. In field gradient (its amplitude g and the pulse width ) at this case, unlike the short-term regime, many molecules fixed observation times of diffusion td. The quantity R0 have a chance to hit the wall of the compartment. The designates the initial amplitude of spin echo signal in effective (apparent) SDC in this case is sensitive to the the absence of magnetic field gradient. The method is presence of obstacles and becomes a descending func- described in detail in [9]. tion of the observation time of diffusion. When self-diffusion of molecules occurs in a large In the regime of long-term diffusion, the diffusive volume, and there is no obstacle to impede their move- length is larger than the linear dimensions of the com- ment, the mean square dispacement of the molecules 2 〈∆X2〉 along one coordinate obeys the Einstein equa- partment; and td > a /D. If the compartment is com- 〈∆ 2〉 pletely insulated (its walls are impermeable), the appar- tion: X = 2Dtd, where D is the self-diffusion coefficient (SDC). In this case, the diffusive spin-echo decay ent mean square dispacement should be restricted by 〈∆ 2〉 (DD) is represented by the Gaussian function: the linear dimension of the compartment: X = 2Dtd = const = a. Therefore, a characteristic indicator of the γ2δ2 2 R = R0exp(– g tdD), long-term regime for isolated compartments is the where γ is a gyromagnetic ratio for proton. When any inverse relationship between the apparent SDC and the δ –1 parameter of the pulse sequence—g, , or td—is subject period of observing diffusion: D ~ td . If there is a dif- to variation at other parameters kept constant, the loga- fusive pathway that interconnects the compartments (as rithm of DD becomes a straight line. The self-diffusion it occurs in cells interconnected with plasmodesmata), coefficient D can be obtained from the tangent (slope) the mean square dispacement of molecules can increase of this line. indefinitely with prolongation of the observation period If self-diffusion of molecules occurs in a system (diffusion throughout the symplast). Thus, one can with spatial restrictions (e.g., self-diffusion of water in write the Einstein equation with some effective SDC a plant tissue), the logarithm of DD does not follow a value. This procedure is equivalent to measurements in straight line. In the case of plant tissues, the most sim- the situation when the actual porous medium, having a ple and interpretatively rational procedure is to record connectivity between the compartments (cells of the DD upon variation of the amplitude of magnetic field plant tissue), is replaced with a homogenous medium δ gradient at fixed values of and td parameters [10]. It featuring some effective SDC. This SDC represents a was also shown for such experimental protocol that the macroscopic characteristic of the real porous medium; initial portion of DD curve (at g 0) contains infor- it is independent of the diffusion time, and its value is mation on the mean square dispacement of molecules, substantially lower than SDC of the bulk phase. In this irrespective of particular structural features of the case, the effective SDC is called the coefficient of con- porous medium (the medium with spatial restrictions) nectivity (permeability) of the porous medium [12]. We [11]. Determinations of effective SDC in porous media adopted this notion of a long-term regime of restricted from the tangent of the initial portion of DD curve and diffusion and used it here for characterizing the water analysis of SDC as a function of diffusion time repre- permeability of symplastic systems in plant tissues. sent a simple yet important stage of the investigation. In order to visualize the tonoplast, we employed

When the effective SDC is determined from the ini- indirect immunoﬂuorescent microscopy and used the tial slope of DD curve, three regimes of apparent SDC tonoplast marker,^ pyrophosphatase, according to the as a function of the observation time can be distin- method of Balus ka et al. [13]. The root segments were guished: the regimes of short-term diffusion, intermedi- vacuum-inﬁltrated for 2 min with 4% formaldehyde ate times of diffusion, and long-term diffusion. Before dissolved in a stabilizing buffer of the following com- considering these regimes, a term of “diffusive length” position: 50 mM Pipes, pH 6.9, 50 mM MgSO4, 50 mM should be introduced. The diffusive length L is a dis- EGTA, and 12% dimethyl sulfoxide. Following a tance covered by a diffusing molecule (water in our 60-min incubation in this solution, root samples were case) in the unlimited volume over given observation immersed into a pure stabilizing buffer for 30 min, and

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wax–ethanol mixture was infiltrated for 12 h, and then a pure wax was applied for 1–2 h. The root segments were placed into special molds and allowed wax to polymerize for 8 h. The root sections (10 µm thick) cut with a microtome were placed into a drop of dis- tilled water, adhered to Super-Frost*/plus glasses (no. 041300, Serva, Germany) covered with glycerol albumin, and wiped to remove water. After the removal of wax and rehydration of sections in a series of ethanol–PBS solutions (97, 90, and 50% ethanol), with 10-min exposure at each rehydration step, the glass plates with samples were washed in PBS for 10 min, and then the targeted proteins were labeled. The antibodies against pyrophosphatase, used as primary antibodies, were the gift from Ratajczak [15]. The primary antibodies were dissolved in PBS at a ratio of 1 : 100 or 1 : 200 and incubated for 1 h. After 10-min washing of sections in PBS, they were treated with second antibod- Fig. 1. Visualization of pyrophosphatase in cells of maize ies—anti-rabbit immunoglobulins conjugated with flu- root segments excised from the elongation zone under nor- orescein (Sigma, United States) and dissolved in PBS at mal cell turgor (control conditions). Magnification ×100. a ratio of 1 : 100. The samples were examined under an Axiovert 405 M microscope (Zeiss, Germany) equipped with epifluo- rescent illuminator and standard filters (a band pass filter 450–490 nm and a long-wavelength filter passing above 520 nm). Photographs were made with a digital camera AxioCam (Zeiss). The immunofluorescent microscopy experiments were performed at the Botan- ical Institute of Bonn University (Germany).

RESULTS In a search for new evidence that the plasmodesma comprises the vacuolar membrane (tonoplast), we made use of antibodies against pyrophosphatase as a specific marker of this membrane. We applied indirect immunofluorescent microscopy and attempted to visualize the distribution of pyrophosphatase on root sections under conditions of normal cell turgor. Figure 1 shows the typical result. Under normal turgor condi- Fig. 2. Visualization of pyrophosphatase in cells of maize tions, the detection of pyrophosphatase inside the plas- root segments excised from the elongation zone after cell plasmolysis in the presence of mannitol (700 mM, 2.5 h). modesmal pore turned out problematic. Experiments Arrows indicate the location of the proteins under study with plasmolyzed cells were more successful in this within the cell walls, namely, near the plasmodesmal pores. respect. As Fig. 2 shows, after plasmolyzing cells with Magnification ×100. mannitol solution, the occurrence of pyrophosphatase within the cell walls was clearly visualized in the region of plasmodesmata location. then kept for 15 min in a phosphate buffer saline (PBS) In the next series of experiments, we studied the dif- containing 0.14 M NaCl, 2.7 mM KCl, and 6.5 mM fusive decay of the proton echo in maize root segments K2HPO4/KH2PO4 (pH 7.3). Next, the segments were under conditions of long-term diffusion; i.e., at rather dehydrated in series of ethanol–PBS solutions (30, 50, long observation times td that would be sufficient for 70, 90, and 97% ethanol); the treatment duration at each water molecules in a large volume to diffuse over dis- stage was 30 min. An ethanol-soluble Stedman’s wax tances exceeding cell dimensions. Figure 3 (curve 1) featuring a low melting point was used as a fixative displays the diffusive decay of echo signal for the dif- [14]. The Stedman’s wax was prepared by mixing fusion time td equal to 700 ms. The decay is not expo- PEG-400 distearate (melted at 65°ë prior to use) and nential, which is a typical manifestation of restricted hexadecanol at a ratio of 9 : 1. The impregnation of wax diffusion [9]. The final portion of the DD curve is an into tissues was performed at 37°ë; at the first stage the exponent (straight line in a logarithmic scale). Figure 4

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1 R/R0 1

0.1

0 2 0.1 R / R 0.01 0.01 900 ms 1 500 ms 700 ms 0 200 400 600 800 1000 1200 × –8 γδ 2 2 8.3 10 ( g) td, s/m 0 200 400 600 800 1000 1200 × –8 γδ 2 2 8.3 10 ( g) td, s/m Fig. 3. Diffusive decay of the proton echo in root segments excised from the elongation zone of 4-day-old etiolated Fig. 4. Diffusive decay of the proton echo in root segments maize seedlings: (1) untreated samples and (2) segments of 4-day-old etiolated maize seedlings at different times of treated with sodium azide (10 mM, 30 min). observing diffusion (shown with arrows). The effective SDC (Deff) was determined from the slope of the initial portion of DD curve. The values of Deff were 6.2 × 10–10 m2/s and 1.1 × 10–10 m2/s for untreated and tion of this enzyme in the plasmodesmal pores provides azide-treated roots, respectively. The diffusion under study additional evidence that the desmotubule, an element of occurred in the direction orthogonal to the root longitudinal endoplasmic membrane network, is also an element of axis. The observation time of diffusion was 700 ms. the vacuolar membrane. The presence of pyrophosphatase in plasmodesmata is an essential condition and strong evi- shows similar DD curves that were measured upon dence for the existence of the vacuolar symplast. variation of the diffusion time in the range from 500 to We obtained even stronger evidence by studying 900 ms. Obviously, the initial slope of DD curve (at self-diffusion of water molecules in root segments with g 0), i.e., the effective SDC (see Materials and PFG NMR technique. The period of diffusion (td) in our Methods section) was independent of the diffusion NMR experiment is limited by the relaxation time of duration. The calculation yielded the effective SDC longitudinal magnetization. The times of longitudinal value of (6.2 ± 0.2) × 10–10 m2/s, which is substantially relaxation differ for various compartments of maize smaller than the SDC of pure water in a large volume root cells. The longest relaxation times (up to 1.5 s) (2.6 × 10–9 m2/s at 25°ë). were attributed to vacuoles; intermediate values (300−400 ms), to the cytoplasm; and the shorter ones The kinetics of DD in root segments was strongly (50 ms) were assigned to the apoplast [17]. The coeffi- affected by sodium azide (10 mM, 30 min); this treat- cient of self-diffusion is determined in the PFG NMR ment is known to reduce the endogenous ATP level in method by plotting the relative amplitude of spin-echo root cells by 80% or even more [16]. The effective SDC signal as a function of the pulse gradient parameters— value, as calculated from the slope of the initial portion g, δ, and t (see Materials and Methods section). The of DD curve, was significantly reduced compared to the d × –10 2 SDC assayed with this technique characterizes those control value and equaled to 1.1 10 m /s. The slope molecules whose magnetic moments participated in the of the DD final portion remained the same as in formation of the echo signal. Hence, in our experiments untreated (control) samples. on root segments at td = 700 ms (Fig. 3), the signal can Figure 5 shows the DD curves that were measured at be assigned to the vacuolar water. The cytoplasmic and variable observation periods of diffusion td on root seg- apoplastic water pools have their relaxation completed ments treated with sodium azide. In this case, the initial by the end of this period and, therefore, do not contrib- slope of DD curve decreased sharply as the period of ute appreciably to the echo signal. diffusion td was increased, which is in a marked con- According to the Einstein equation, the time of trast to the observations with untreated samples 700 ms is sufficient for water molecules to diffuse in a (Fig. 4). At the same time, the slope of the final portion large volume to a distance of 60 µm. The diameter of of DD curve remained constant, irrespective of the td the largest cells in the root cortex did not exceed 35 µm, period. and the parenchymal cells of the central cylinder (stele) measured 15–20 µm [18]. Thus, a formal reasoning DISCUSSION based on cell dimensions led us to conclude that the regime of restricted diffusion at td = 700 ms can be con- It is commonly accepted that pyrophosphatase is a sidered as a long-term diffusion (see Materials and specific marker of the tonoplast. Therefore, the detec- Methods section). This conclusion provides an expla-

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R/R0 of desmotubule and, possibly, the transition of the open 1 desmotubule to the closed state [5]. Thus, the experiments performed with PFG NMR method indicate that vacuoles of untreated roots are interconnected by highly permeable diffusive links. 0.1 These links are effective within one NMR-phase (i.e., within the continuum characterized by a single relax- 900 ms 700 ms ation time) and disappear after the azide treatment. 500 ms 0.01 Analysis of the data presented above allows us to 200 ms draw the following conclusions. (1) The maize roots comprise a vacuolar symplast that represents an inter- 100 ms cellular continuum and a pathway for unrestricted dif- 0 200 400 600 800 1000 1200 fusion of water within the plant tissue; this symplast is 8.3 × 10–8(γδg)2t , s/m2 formed by interconnected vacuoles of neighboring d cells, with plasmodesmata as connectivity elements. (2) The permeability of desmotubule of the vacuolar Fig. 5. Diffusive decay of the proton echo at different times of observing diffusion in root segments treated with sodium symplast to water is under the control of an ATP-depen- azide (10 mM, 30 min). dent process. (3) The experimental data obtained with PFG NMR method provide the methodological basis for studying the permeability of the vacuolar symplast nation to experimental observations that the effective to water. SDC is independent on the diffusion time td (Fig. 4) and is much smaller than SDC of bulk water diffusion (cf. 6.2 × 10–10 m2/s and 2.6 × 10–9 m2/s; see Results ACKNOWLEDGMENTS section). Obviously, these observations are related to This work was supported by the Russian Foundation the existence of a high diffusive connectivity between for Basic Research, project no. 03-04-48174. the diffusion-limiting compartments (vacuoles) (see Materials and Methods section and [19]). REFERENCES Interestingly, time-dependent changes of effective 1. Vakhmistrov, D.B., Prostranstvennaya organizatsiya SDC, as calculated from the initial slope of DD curves, ionnogo transporta v korne. 49-e Timiryazevskoe chtenie were revealed in the presence of sodium azide, an (Spatial Organization of Ion Transport in the Root, the inhibitor of energy metabolism (Fig. 5). In root samples 49th Timiryazev Lecture), Moscow: Nauka, 1988. treated with azide, in contrast to untreated samples 2. Robards, A.W., A New Interpretation of Plasmodesmatal (Fig. 4), the effective SDC was clearly dependent on the Ultrastructure, Planta, 1968, vol. 82, pp. 200–210. diffusion period t : the initial slope of DD curves d 3. Robards, A.W., Plasmodesmata in Higher Plants, Inter- decreased as the diffusion time was raised. Obviously, cellular Communication in Plants: Studies on Plas- the decrease of the initial slope should be limited in our modesmata, Guning, B.E.S. and Robards, A.W., Eds., case. This slope cannot be smaller than the slope of the Berlin: Springer-Verlag, 1976, pp. 15–58. terminal, td-independent exponential portion of DD 4. Lassaro, M.D. and Thomson, W.W., The Vacuolar–Tubu- curve (see Fig. 5). Indeed, the effective SDC in azide- lar Continuum in Living Trichomes of Chickpea (Cicer treated roots decreased with the prolongation of the dif- arietinum) Provides a Rapid Means of Solute Delivery from fusion period td and approached a limiting (asymptotic) Base to Tip, Protoplasma, 1996, vol. 193, pp. 181–190. value deﬁned by the slope of the terminal, exponential 5. Gamalei, Yu.V., Supercellular Plant Organization, Fiziol. portion of DD. This dependence corresponded to the Rast. (Moscow), 1997, vol. 44, pp. 819–846 (Russ. J. Plant Physiol., Engl. Transl., pp. 706–730). asymptotic behavior D ~ t–1 predicted for the regime of d 6. Gamalei, Yu.V., Photosynthesis and Export of Photoas- long-term diffusion in the medium with isolated com- similates: Development of the Transport System and partments (vacuoles); i.e., for the regime of completely Source–Sink Relations, Fiziol. Rast. (Moscow), 1998, restricted diffusion (see Materials and Methods sec- vol. 45, pp. 614–631 (Russ. J. Plant Physiol., Engl. tion). Apparently, the azide treatment disrupts the inter- Transl., pp. 525–541). vacuolar diffusive connectivity, and the vacuolar sym- 7. Velikanov, G.A., Volobueva, O.V., and Khokhlova, L.P., plast disintegrates into separate compartments. The Study of the Hydraulic Conductivity of the Plas- modesmal Transport Channels by the Pulse NMR Method, Such an effect of azide treatment is not entirely Fiziol. Rast. (Moscow), 2001, vol. 48, pp. 375–383 (Russ. unexpected. The existence of actomyosin sphincter in J. Plant Physiol., Engl. Transl., pp. 318–325). the cytoplasmic annulus within the plasmodesmata is 8. Holdaway-Clarke, T.L., Walker, N.A., Hepler, P.K., and not doubted nowadays [5, 20]. The ATP-dependent Overall, R.L., Physiological Elevations in Cytoplasmic actomyosin sphincter is thought to control the diameter Free Calcium by Cold or Ion Injection Result in Tran-

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