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 seg- ments. 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 exam- ined 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 study- ing 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 vac- uum 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 des- motubule 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 1021-4437/05/5203-0326 © 2005 MAIK “Nauka /Interperiodica” 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 Ӷ 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 coeffi- cient (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.