G. A. Velikanov

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.

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 α-, δ-, and γ-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 (D2 and D3) 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.

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.

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 Study of the Hydraulic Conductivity of the Plasmodesmal Transport Channels by the Pulse NMR Method

Radial self-diffusion of water in the absorbing zone of the roots of winter wheat (Triticum aestivumL.) seedlings was studied by the pulse-gradient-spin-echo NMR method. At the fixed time of diffusion observation, the diffusion decay of proton spin-echo was nonexponential; however, it could be reliably separated into three exponential components differing in the self-diffusion coefficients (SDC) of water molecules. Our experimental data corroborate the modern concept of two transport channels in plant plasmodesmata, which connect cytoplasmic and vacuolar (endoplasmic) compartments of adjacent cells into the unified supracellular continuums. Two SDC obtained by the kinetic analysis of diffusion decay were shown to depend on the expected changes in the hydraulic conductivity of the two above-mentioned plasmodesmal channels. To elucidate the role of ATP-dependent actomyosin proteins in the regulation of the hydraulic conductivity of plasmodesmata, we followed the changes in the water SDC induced by treating the roots with cytochalasin B (5 μM, 30 min), the inhibitor of actin polymerization; 2,3-butanedione monoxime (10 mM, 1 h), the inhibitor of myosin ATPase activity; and antimycin A (5 μM, 1 h) and sodium azide (10 mM, 30 min), the inhibitors of energy generation. The data thus obtained provided the basis for elaborating a new methodological approach to simultaneously monitoring the functional state of both plasmodesmal channels without any wound effect impairing their functions.

Stromule-like protrusions of plastid membrane envelope in root cells

The work presents the results of the electron-microscopy visualization of stromule-like protrusions of plastid membrane envelope in root cells. Cases of the appearance of a long, narrow protrusion of the outer membrane, in which the shorter protrusion of the plastid envelope inner membrane was located, are discussed. The possible role of cytoskeleton and plastoskeleton in formation of outer and inner protrusions, respectively, is considered. It is concluded that items of the structure and functions of stromules in plant cells are to be considered to be the same as the structure and functions of the intracavity space of endoplasmic reticulum.

Vacuolar symplast formation is due to highly permeable gap junctions between the tonoplast and endoplasmic reticulum membrane

An NMR method with a pulsed magnetic field gradient was applied to study changes in water permeability of the vacuolar symplast in maize (Zea mays L.) seedling roots treated with various inhibitors of cell metabolism. The results were qualitatively analogous to literature data on conductivity changes of intercellular gap junctions in animal cells exposed to similar treatments. Electron microscopy examination of root cells provided evidence for the existence of membrane contacts between the endoplasmic reticulum and the tonoplast. It is supposed that vacuoles of neighboring plant cells are interconnected through highly dynamical gap junctions between the tonoplast and the endoplasmic reticulum membrane.

Membrane contacts of the endoplasmic reticulum and their possible functions in the plant cell

Close contacts of the endoplasmic reticulum membrane and plasmalemma have been visualized inside plant cells by means of electron microscopy. The qualitative similarity of these contacts to high-permeable intercellular contacts in animals has been shown. New data confirming the hypothesis of the identity of stromules, i.e., dynamic tubular protuberances of the plastid membrane of the plant cell, and tubular elements of the endoplasmic reticulum have been presented. New possible functions of the contacts of the endoplasmic reticulum membrane with other membranes inside the cell have been discussed on the basis of this hypothesis.

Adjustable channel for diffusion between vacuoles of next cells: Vacuolar symplast

The problem of the existence of a continuous transcellular association of internal spaces of endoplasmic reticulum (ER) and central vacuoles of next cells into a single system (vacuolar symplast) containing within the plastids and mitochondria in plant tissues is discussed. Based on the original experimental data obtained by NMR spectroscopy and electron microscopy, a device model of a vacuolar symplast is proposed. The model is based on the transport distributive function of the internal space of the ER and the ability of the ER membrane to form close contacts with the other membranes in cell. For these contacts, a qualitative analogy with highly permeable intercellular contacts in animals was preliminarily established.

Contacts of the endoplasmic reticulum membrane with plasmalemma in plant cells.

It is shown by electron microscopy that, in maize root cells, there is close contact between the membrane of the endoplasmic reticulum and the plasmalemma. A qualitative preliminary comparison is conducted between these contacts and high-permeability intercellular contacts in animals.