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.     

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 M, 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 M, 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.     

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.     

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.     

Influence of inhibitors of cytoskeleton proteins on water exchange of wheat roots under the after-action of water stress

The dynamics of water molecular state and transport in winter wheat (Triticum aestivum L.) of roots different resistance cultivars was studied by a biophysical method, Nuclear Magnetic Resonance (NMR), and a physiological method, Water-Holding Capacity (WHC). The effective coefficient of water self-diffusion (D(eff)), spin-spin relaxation times (T(2)) and WHC were measured after structural modification of cytoskeleton by colchicine and cytochalasin B after the action of water stress. New information about molecular mechanisms of water state and water transport regulation determined by the influence of dynamic cytoskeleton structure has been obtained. This is very important for the development of a fundamental theory of water exchange in plants, and for the ways of its optimization under conditions of environmental stress.     

Signalling via plasmodesmata—the neglected pathway

This review considers recent studies on the role of plasmodesmata in the conduction of small solutes and signalling molecules between plant cells. The substructure of plasmodesmata is described in relation to the potential pathways available for symplastic signalling between cells. At least two discrete pathways are available for transport through plasmodesmata, the cytoplasmic sleeve between the desmotubule and the plasmalemma, and the endoplasmic reticulum which connects contiguous cells via the central desmotubule. This latter pathway has been shown recently to function as a dynamic continuum for the movement of lipids and lipid-signalling molecules between plant cells. The role of plasmodesmata in the conduction of hormones and electrical signals is also considered, as is the potential for movement of macromolecular signalling molecules via the symplast. The factors which regulate plasmodesmatal conductance and the significance of symplast 'domains' are discussed in relation to the control of movement of signalling molecules in the symplast.     

Ethylene enhances water transport in hypoxic aspen

Water transport was examined in solution culture grown seedlings of aspen (Populus tremuloides) after short-term exposures of roots to exogenous ethylene. Ethylene significantly increased stomatal conductance, root hydraulic conductivity (L(p)), and root oxygen uptake in hypoxic seedlings. Aerated roots that were exposed to ethylene also showed enhanced L(p). An ethylene action inhibitor, silver thiosulphate, significantly reversed the enhancement of L(p) by ethylene. A short-term exposure of excised roots to ethylene significantly enhanced the root water flow (Q(v)), measured by pressurizing the roots at 0.3 MPa. The Q(v) values in ethylene-treated roots declined significantly when 50 microM HgCl(2) was added to the root medium and this decline was reversed by the addition of 20 mM 2-mercaptoethanol. The results suggest that the response of Q(v) to ethylene involves mercury-sensitive water channels and that root-absorbed ethylene enhanced water permeation through roots, resulting in an increase in root water transport and stomatal opening in hypoxic seedlings.     

Water permeability and revolving movement in Phaseolus vulgaris L. twining shoots

Osmotic water permeability (Pos) was measured in protoplasts isolated from different tissues of Phaseolus vulgaris twining shoot. Parenchyma protoplasts exhibited more frequently high Pos values than epidermis protoplasts did. Water channels could facilitate water movement between parenchyma cells whereas cell-to-cell water transport mostly occurs through plasmodesmata in epidermis.     

胞间连丝：共质体运输的动态通道

胞间连丝作为一种细胞质结构将相邻的细胞连系起来而形成植物的共质体。胞间连丝通过调控许多离子和分子的共质体运输而广泛地参与植物的生命活动。胞间连丝的主要构成部分是细胞质膜、连丝小管、以及位于二之间的环层细胞质。这三都很容易在电子显微镜下观察到。细胞骨架的成分（肌动蛋白和肌球蛋白）起到稳定胞间连丝的作用。同时,钙结合蛋白可能具有调节间连丝功能的作用。在胞间连丝里,环层细胞质为大多数溶质提供共质体运输的通道,而有些 共质体运输则可能是通过连丝小管的内腔、连丝小管的壳层、甚或是细胞质膜来实现的。共质体可以细分为数个区块,它们各自允许不同大小的分子（从低于1000到高于10000道尔顿）通过。从发生上看,胞间连丝可以是初生的,也可以是次生的。前是伴随着新细胞壁的形成则产生的,而后则是在已有的细胞壁上产生的。胞间连丝的动态性质还表现在它们的频率是处于变化之中,这是由于组织或植物整体的发育和生理状态决定的。虽然共质体运输的基本形式是扩散,但胞间连丝对于某些离子和分子却是选择性的。在病毒感染细胞时,病毒的移动蛋白作用于胞间连丝的受体蛋白,结果,胞间连丝被显地扩张（其机理尚不清楚）。于是,病毒的移动蛋白连同与之结合在一起的病毒基因组进入毗邻的健康细胞。一些植物源性的蛋白质也能够通过胞间连丝来运输;推测其方式类似于病毒的移动蛋白。有些植物蛋白质本身就是信号分子,它们调节分化和其他活动。与此相反,还有一些植物蛋白质的共质体运输并不是通过特异的方式来实现的。     

The NMR spin-echo method is used for measurements of the translational water diffusion selectively along the apoplast and the vacuolar and cytoplasmic symplasts of plant tissue

Simple methods for the registration of translational diffusion of water in apoplast and vacuolar and cytoplasmic symplasts were developed. The methods are based on spin-echo NMR with a pulsed magnetic field gradient and are realized by preliminary inversion of magnetization and the use of paramagnetic doping. It was shown that the diffusion of a part of root water in segments of mais roots is more enhanced than that for the bulk water. The results are explained by the appearance of the rotational movement of the protoplasm and in terms of the hypothesis of water transfer along the apoplast, which balances the cytoplasmic symplast.     

Electrical Conductance of Cell-to-Cell Junctions and the Cytoskeleton of Plant Cells

In the submerged trichomes of floating-moss (Salvinia auriculataAubl.) and the roots of the higher water plant Trianea bogotensisKarst., the dependence of the electrical resistance of intercellular junctions on the presence of the agents that destroy microfilaments (cytochalasin B) and microtubules (colchicine) was investigated using the microelectrode technique. The resistance of the junctions (R _c) was estimated taking into account the input resistance and the coefficient of intercellular electrical communication. Should the cells be connected via symplast, R _cwill describe the resistance of plasmodesmata. Cytochalasin B (3–30 g/ml) reversibly changed R _cduring the first minutes after application. The extent of the change depended on the concentration of the inhibitor; its character of action depended on the initial strength of intercellular communication. When the initial conductance of the contact was high, cytochalasin B elevated the resistance; when it was low, the inhibitor decreased it. In all the experiments, cytochalasin B reduced the input resistance (R _i) that suggests the dependence of plasma membrane resistance on actin cytoskeleton. The effect of colchicine (0.1–1.0 mM) on R _iand R _cwas observed only when the cellular membrane was hyperpolarized or after a prolonged action of the inhibitor (for about 0.5 h). It was concluded that the electrical conductance of plasmodesmata and plasma membrane depended on the state of actin cytoskeleton. A complex and probably mediated interaction of microtubules with the processes affecting these characteristics of the cells was suggested.     

Current perspectives on plasmodesmata: structure and function

Recent studies on plasmodesmata have shown that these important intercellular passages for communication and transport are much more sophisticated in both structure and regulatory abilities than previously imagined. A complex, but not well understood, substructure has been revealed by a variety of increasingly reliable ultrastructural techniques. Proteinaceous particles are seen within the cytoplasmic sleeve surrounding the desmotubule. Dye-coupling studies have provided experimental evidence for the physical pathway of solute movement, supporting conclusions about substructural dimensions within plasmodesmata drawn from the ultrastructural studies. Calcium has been identified as a major factor in the regulation of intercellular communication via plasmodesmata. Evidence from studies on virus movement through plasmodesmata suggests a direct interaction between virallycoded movement proteins and plasmodesmata in the systemic spread of many viruses. There is increasing evidence, albeit indirect, that in some plant species phloem loading may involve transport of photoassimilate entirely within the symplast from mesophyll cells to the sieve element-companion cell complexes of minor veins.     

The Cellular Pathway of Short-distance Transfer of Photosynthates and Potassium in the Elongating Stem of Phaseolus vulgaris L. A Structural Assessment

The potential cellular pathway of radial transfer of photosynthateand potassium delivered in the phloem to the elongation zone(apical 0.5–2.5 cm) of internode 2 ofPhaseolus vulgarisL. seedlings was elucidated. This was achieved using ultrastructuralobservations of the cell types that constitute the radial pathwayand estimates of potential sucrose and potassium fluxes throughthe cross-sectional area of interconnecting plasmodesmata andacross the plasma membrane surface areas of selected cell types.The investigation relied on predicting the relative roles ofthe mature and developing sieve elements as conduits for theaxial delivery of solutes to the elongation zone. In turn, thesepredictions led to formulation of two transport models whichwere subsequently evaluated. It was found that unloading ofsucrose and potassium from the protophloem sieve elements cannotbe through the symplast due to the absence of plasmodesmata.On the other hand, mature metaphloem sieve element-companioncell complexes have the potential capacity to unload eitherthrough the stem symplast or apoplast. The potential symplasticroute is proposed to be via the companion cells to the adjacentlarge phloem parenchyma cells. Continued radial transfer couldoccur either by exchange to the stem apoplast from the largephloem parenchyma cells or continue in the symplast to the groundtissues. It was further predicted that sucrose utilized forthe development of the procambial/small phloem parenchyma cellscould be delivered axially by them and not by the mature sieveelements. Phaseolus vulgaris ; apoplast; elongating stem; photosynthates; potassium; transport; symplast     

Cytoskeleton-induced alterations of the lectin activity in winter wheat under cold hardening and abscisic acid (ABA)

The roots and leaves of 7-day seedlings of three winter wheat cultivars differing in frost resistant were used to study changes in lectin activity under cytoskeleton modifiers (DMSO-7%; colchicine-1 m m; oryzalin-15 microm; cytochalasin B-15 microm) of non-hardened (23 degrees C) and hardened (2-3 degrees C, 3-7 day) plants. Plants were grown with ABA (30 microm) or without ABA. Pretreatment with colchicine, oryzalin [inhibitors of microtubules (MT) polymerization], cytochalasin B [inhibitor of microfilament (MF) polymerization] increased the activity of cell wall lectins, although pretreatment with DMSO (stabilizer of microtubules) decreased the activity. Both hardening and ABA decreased the effect of the cytoskeletal modifiers. These results could be explained by the appearance of tolerant MTs with less affinity. It is probable that increase in the activity of cell wall lectins may be the compensatory mechanism which stabilizes the cytoskeleton structure in conditions tending to disrupt it. The genotype with low resistance had higher sensitivity of lectin activity to cytoskeleton modifiers than the frost resistant genotype. The results suggest that leaves have more stable MTs and MFs and stronger MT-MF binding than roots.     

Abscisic acid in the xylem: where does it come from, where does it go to?

Abscisic acid is a hormonal stress signal that moves in the xylem from the root to the different parts of the shoot where it regulates transpirational water loss and leaf growth. The factors that modify the intensity of the ABA signal in the xylem are of particular interest because target cells recognize concentrations. ABA(xyl), will be decreased as radial water flow through the roots is increased, assuming that radial ABA transport occurs in the symplast only. Such dilutions of the plant hormone concentration can be compensated in different ways, which help to keep the ABA-concentrations in the xylem constant: (i) apoplastic bypass flows of ABA, (ii) ABA flows between the stem parenchyma and the xylem during transport and (iii) the action of beta-D-glucosidases that release free ABA from its conjugates to the root cortex and the leaf apoplast. The significance of reflection coefficients (sigma(ABA)), permeability coefficients of membranes (P(S)(ABA)) and apoplastic barriers for ABA is discussed.     

Plasmodesmal widening accompanies the short-term increase in symplasmic phloem unloading in pea root tips under osmotic stress

Summary Root tips ofPisum sativum seedlings were exposed to 350 mM mannitol, which was shown to effect a transient but dramatic increase in phloem unloading, and investigated by electron microscopy. After chemical fixation and embedding, extremely thin sections of the root extension zone were examined. Outer, inner, and desmotubule diameters of 830 primary plasmodesmata in transverse walls of cortical cells were measured. Statistical analysis indicated that the majority of plasmodesmata had no neck constriction during osmoregulation. Compared to controls, a highly significant increase in mean plasmodesmata diameter was found, but the desmotubule diameter remained unchanged. Both loss of neck constriction and widening of the cytoplasmic sleeve indicate an increase in effective passage area of plasmodesmata. Spokes between plasma membrane and desmotubule were preserved. Continued exposure of the root tips to mannitol led to a return to control values for plasmodesmal diameters. In contrast to these responses, plasmolysis of cortical cells by 1,000 mM sucrose, diminishing phloem unloading, was accompanied by a reduction in those plasmodesmata classified as open. This is the first report showing a correlation between the ultrastructure of plasmodesmata and the rate of symplasmic transport. The role of the different plasmodesmal components in controlling the passage area of symplasmic transport is discussed.     

Microfilament network is needed for the endocytosis of water channels and not for apical membrane insertion upon vasopressin action

In the current study, a novel role for the microfilaments in vasopressin-induced water transport in toad urinary bladders, a popular model for the mammalian collecting duct, was established. Vasopressin-induced water transport was not affected by cytochalasin D (CD, 20 microM) or latrunculin B (Lat B, 0.5-2 microM), microfilament-disrupting reagents, suggesting that the initial trafficking of vesicles containing water channels and insertion of membranes into the apical membrane are microfilament-independent. After the removal of vasopressin, bladders treated with CD or Lat B continued to transport water at least 2-3-fold greater than those that received the vehicle. Furthermore, the enhanced water transport was inhibited by HgCl2 (1 mM), a potent inhibitor of water channel-mediated water flow, suggesting that the enhanced water flow was through water channels. In addition, Lat B and CD inhibited vasopressin-induced endocytosis of horseradish peroxidase (HRP), a fluid endocytotic marker. These results suggested that although microfilaments are not needed for the initial trafficking of water channels to the apical side, the microfilament network is essential for the retrieval of water channels following their insertion into apical membranes.     

How do plant virus nucleic acids move through intercellular connections?

In addition to their function in transport of water, ions, small metabolites, and growth factors in normal plant tissue, the plasmodesmata presumably serve as routes for cell-to-cell movement of plant viruses in infected tissue. Virus cell-to-cell spread through plasmodesmata is an active process mediated by specialized virus encoded movement proteins; however, the mechanism by which these proteins operate is not clear. We incorporate recent information on the biochemical properties of plant virus movement proteins and their interaction with plasmodesmata in a model for transport of nucleic acids through plasmodesmatal channels. We propose that only single stranded (ss) nucleic acids can be transported efficiently through plasmodesmata, and that movement proteins function as molecular chaperones for ss nucleic acids to form unfolded movement protein-ss nucleic acid complexes. These complexes are targeted to plasmodesmata. Plasmodesmatal permeability is then increased following interaction with movement protein and the entire movement complex or its nucleic acid component is translocated across the plasmodesmatal channel.