Intercellular communication in Chara: factors affecting transnodal electrical resistance and solute fluxes

The permeability of plasmodesmata in the nodal complex of branch cells of Chara corallina was examined by measuring both the transnodal electrical resistance and transnodal fluxes of ³⁶CI and ¹⁴C-buty-rate. Under normal circumstances, the resistance across the node was low, but increased rapidly in response to metabolic inhibition, pressure gradients across the node or excision of one of the cells. For each of these treatments, there was a substantial reduction in solute transport between the cells. Acidification of the cytoplasm by weak acids or alkalinization by amines did not affect either the electrical resistance or the flux of solutes through the node between whorl cells. The transnodal resistance was significantly higher in older cell pairs, but was unaffected by large transnodal voltage differences or by the passage of action potentials. There was no evidence that short-term increases in cytoplasmic calcium have any effect on plasmodesmatal permeability.     

Physiological elevations in cytoplasmic free calcium by cold or ion injection result in transient closure of higher plant plasmodesmata

The concentration of cytoplasmic free calcium ([Ca²⁺]_cyt) required to close higher plant plasmodesmata was investigated using corn (Zea mays L. cv. Black Mexican Sweet) suspension-culture cells. Physiological elevations of [Ca²⁺]_cyt were applied by cold treatment, and ion injection was also used to increase [Ca²⁺]_cyt, by diffusion (for small increases) or by iontophoresis (for larger increases). The impact of such treatments on [Ca²⁺]_cyt was measured by ratiometric ion imaging. Intercellular communication during treatments was monitored using our recently developed electrophysiological technique that allows the electrical resistance of plasmodesmata and the plasma membranes of a sister-cell pair to be measured. A 4-fold increase in the calculated resistance of single plasmodesmata was observed in response to cold treatment that caused a 2-fold increase in average [Ca²⁺]_cyt (from 107 to 210 nM). In response to iontophoresis of Ca²⁺, plasmodesmata were observed to go from “open” (low resistance) to “shut” (high resistance) and then back “open” within 10 s. Our results thus indicate that higher plant plasmodesmata respond quickly to physiological changes in [Ca²⁺]_cyt. Received: 2 June 1999 / Accepted: 16 July 1999     

Effect of low oxygen concentration on the electrical properties of cortical cells of wheat roots

Short-term effect of oxygen-deficiency on the membrane potential difference (PD), membrane resistance of cortical cells and electrical coupling between cortical cells was investigated using excised wheat roots. Hypoxia rapidly depolarised the membrane potential of the cortical cells by about 60 mV, while hypoxia had little effect on the membrane resistance of the cells. No significant change in membrane resistance by potassium channel blockers, TEA+ and verapamil, under hypoxia was observed. The electrical coupling ratio, which is a measure of plasmodesmatal resistance, between cortical cells of wheat roots was 5.9 % in aerated solution and was not affected by the low oxygen treatment, suggesting that solute transport through cytoplasmic annulus of plasmodesmata could not be affected. The possible involvement of the endoplasmic reticulum in intercellular transport of solute and water is discussed.     

Fine structure of plasmodesmata in mature leaves of sugarcane

The fine structure of plasmodesmata in vascular bundles and contiguous tissues of mature leaf blades of sugarcane (Saccharum interspecific hybrid L62–96) was studied with the transmission electron microscope. Tissues were fixed in glutaraldehyde, with and without the addition of tannic acid, and postfixed in OsO₄. The results indicate that the fine structure of plasmodesmata in sugarcane differs among various cell combinations in a cell-specific manner, but that three basic structural variations can be recognized among plasmodesmata in the mature leaf: 1) Plasmodesmata between mesophyll cells. These plasmodesmata possess amorphous, electron-opaque structures, termed sphincters, that extend from plasma membrane to desmotubule near the orifices of the plasmodesmata. The cytoplasmic sleeve is filled by the sphincters where they occur; elsewhere it is open and entirely free of particulate or spokelike components. The desmotubule is tightly constricted and has no lumen within the sphincters, but between the sphincters it is a convoluted tubule with an open lumen. 2) Plasmodesmata that traverse the walls of chlorenchymatous bundle-sheath cells and mestome-sheath cells. In addition to the presence of sphincters, these plasmodesmata are modified by the presence of suberin lamellae in the walls. Although the plasmodesmata are quite narrow and the lumens of the desmotubules are constricted where they traverse the suberin lamellae, the cytoplasmic sleeves are still discernible and appear to contain substructural components there. 3) Plasmodesmata between parenchymatous cells of the vascular bundles. These plasmodesmata strongly resemble those found in the roots of Azolla, in that their desmotubules are closed for their entire length and their cytoplasmic sleeves appear to contain substructural components for their entire length. The structural variations exhibited by the plasmodesmata of the sugarcane leaf are compared with those proposed for a widely-adopted model of plasmodesmatal structure.Abbreviation ER endoplasmic reticulum This study was supported by National Science Foundation grants DCB 87-01116 and DCB 90-01759 to R.F.E. and a University of Wisconsin-Madison Dean's Fellowship to K. R.-B. We also thank Claudia Lipke and Kandis Elliot for photographic and artistic assistance, respectively.     

Artificial control of cytoplasmic pH and its bearing on cytoplasmic streaming,electrogenesis and excitability of characeae cells

Effects of changing the cytoplasmic pH on the cytoplasmic streaming, membrane potential and membrane excitability were studied in tonoplast-free cells ofChara australis andNitellopsis obtusa. The cytoplasmic pH was varied by internal perfusion of pH-buffered media.Nitellopsis cells were perfused only once, whileChara cells were perfused twice to control the pH more accurately. In both materials the rate of cytoplasmic streaming was maximum at about pH 7, low at pH 8.5–9 and almost zero at pH 5–5.5. The membrane potential was most negative at about pH 7. InChara the membrane potential supported by Mg·ATP was strongly inhibited at pH 5.5, and almost zero at pH 9, supporting the results obtained by Fujiiet al. (1979) on cells ofChara australis which were perfused once. The action potential could be induced by electrical stimulation inChara at pH 6.0–9.0 and inNitellopsis at pH 6.6–7.9. The membrane resistance ofNitellopsis was high at acidic and neutral pH values and low at alkaline pH, while that ofChara was low at both acidic and alkaline pH values.     

Intercellular communication inAzolla roots: II. Electrical coupling

Summary There is a predictable and well defined variation in numbers of plasmodesmata in roots ofAzolla. As the apical cell of the root ages, it lays down walls with progressively fewer plasmodesmata, thereby gradually cutting itself off from the rest of the root (Gunning 1978). Electrical coupling was examined between the apical cell and an adjacent merophyte in roots of various lengths. The apical cell becomes increasingly electrically isolated from the rest of the root as it ages. Electrical coupling is strongly correlated with the number of the plasmodesmata between the coupled cells. The resistance of a plasmodesma, as estimated from equivalent electrical circuits, was 150–600 times more resistive than a value based on theoretical considerations. No evidence was found for a change in the physiology of plasmodesmata as the root ages. Coupling experiments, both on root hairs and at the apex, gave some suggestion that plasmodesmata may be less resistive towards the apical cell than away from it.     

Hydrogen peroxide－induced changes in intracellular pH of guard cells precede stomatal closure

INTRODUCTIONEven under optimal conditions, many metabolicprocesses, including chloroplastic, mitochondrial,and plasma membrane-linked electron transportsystems, produce reactive oxygen species (ROS)such as the superoxide radical (OZ--), hydrogenperoxide (HZOZ), and the hydroxyl free radical(OH--)[1, 2]. Furthermore, the imposition of bioticand abiotic stress conditions can give rise to ex-cess concentrations of ROS, resulting in oxidativedamage at the cellular level. Interestingly, R…     

The formation of symplasmic domains by plugging of plasmodesmata: a general event in plant morphogenesis?

Summary The plasmodesmal network was examined in multicellular protoplast-derived calluses of the dicotyledonSolanum nigrum which had not yet formed any visible adventitious organs and in globular proembryogenic structures developed from scutellar calluses of the monocotyledonMolinia caerulea. Electron microscopical analyses revealed that both calluses and proembryos consisted of small, undifferentiated cells. The interconnecting plasmodesmata at many cell interfaces were structurally inconspicuous in both systems; in particular cell walls, however, all plasmodesmata were occluded with an osmiophilic, dense material. As the blocking material was obviously located in the microchannels of the plasmodesmal cytoplasmic sleeves, the plugged plasmodesmata can be assumed to be nonfunctional. Thus, selective occlusion of all the plasmodesmata in specific cell walls resulted in the symplasmic disconnection of particular adjacent cells. Complex patterns of symplasmic continuity and discontinuity were established within the developing tissues. Some cells or groups of cells were entirely symplasmically disconnected from the surrounding cells by plugged plasmodesmata and might function as independent domains. However, blockage of plasmodesmata was achieved by the surrounding cells rather than by those cells belonging to the isolated domains. The demarcation of symplasmic domains might be a general prerequisite for differential morphogenesis, since they were found to be established very early in the course of morphogenetic processes.     

Butyric acid mediated induction of enhanced transendothelial resistance in an in vitro model blood-brain barrier system.

Previously we reported that the co-culture of non-brain vascular endothelial cells with glioma cells leads to the induction of a more differentiated endothelial cell phenotype which exhibits important properties of the blood-brain barrier (BBB). Recognising the potential for improving the model barrier system with agents known to modify the growth and differentiation of cells in culture we examined the effects of four differentiating agents (butyric acid, dexamethasone, retinoic acid, and dimethyl sulfoxide) on barrier function. Of these agents only butyric acid and dexamethasone resulted in an enhancement (depending on the dose used) of transendothelial electrical resistance (barrier function). The greatest effect was observed with butyric acid in a dose-dependent manner and was slow in onset and only occurred in the endothelial/glial cell co-cultures. These data indicate that butyric acid may be a beneficial agent in optimising conditions necessary for induction of BBB properties in in vitro barrier systems.     

Susceptibility of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> to organic acids and monoacylglycerols

Organic acids can be used as feed supplements or for treatment of poultry carcasses in processing plants. The antimicrobial activity of nineteen organic acids and two monoacylglycerols in cultures of Campylobacter jejuni CCM 6214^T (ATCC 33560) was determined using a SYBR Green-based real-time PCR assay. The IC₅₀ was a concentration at which only 50 % of a bacteria specific DNA sequence was amplified. Caprylic, capric and lauric acids were the most efficient antimicrobials among the compounds tested (IC₅₀ ≤ 0.1 mg/mL). In a weakly acidic environment (pH 5.5), the antimicrobial activity was more pronounced than at pH 6.5. At pH 5.5, oleic and fumaric acid also had clear antimicrobial activity, as did monocaprylin. The antimicrobial activity of acetic, butyric, stearic and succinic acid was low. In cells treated with fumaric acid, the potential of potassium and tetraphenylphosphonium ion-selective electrodes changed, indicating an increase in cytoplasmic and outer membrane permeability, respectively. No changes in membrane permeability were observed in cells treated with capric acid or monocaprin. Transmission electron microscopy revealed separation of the inner and outer membrane in cells treated with capric and fumaric acid, as well as cytoplasmic disorganization in cells exposed to capric acid.     

Studies on graft unions

Summary De novo formation of cytoplasmic cell connections are studied at the graft interface of 5 day old in vitro heterografts ofVicia faba onHelianthus annuus. Continuous and half plasmodesmata, both branched and unbranched, are described at various stages of development in non-division walls between unlike and like dedifferentiated callus cells. In apical portions of protruding callus cells and in the contact zone between opposing cells extremely thin wall parts with a striking ER/plasmalemma contact are observed. During subsequent thickening of the modified wall parts cytoplasmic strands enclosing constricted ER cisternae are entrapped within the newly deposited wall material. These cytoplasmic strands represent half plasmodesmata which—in case of fusion with corresponding structures of adjoining cells across the loosened wall matrix — form continuous cell connections. Golgi vesicles secreting wall material are involved in the process of forming half and continuous plasmodesmata, thus following the same mechanism of plasmodesmata development as described for isolated protoplasts in cell cultures. The findings suggest the existence of a unifying mechanism of secondary formation of plasmodesmata showing far-reaching similarities with the establishment of primary cell connections.     

The neck constriction in plasmodesmata

Simple plasmodesmata between mesophyll and bundle sheath cells in actively expanding leaves of Salsola kali L. and roots of Epilobium hirsutum L. are shown to possess specialized structures, called sphincters, around their neck regions. The sphineters are made visible by the combined effects of tannic acid and heavy metal staining; they are localized just outside that area of the plasmalemma, which forms the collar around the entrance to each plasmodesmos. This localization corresponds to a very active area of the plasmodesmos/olasmalemma complex (i.e. enzyme activity and/or presence of strongly reducing substances).Evidence is presented that these ring structures are structural equivalents to hypothetical sphincters performing some valve function; i.e. participating in the control of rates and directions of symplastic transport of solutes through plasmodesmata. The middle layer of the plasmalemma in the neck region is composed of closely-packed, globular subunits appearing in negative contrast. Apparently, these subunits correspond to particle clusters observed at the plasmodesmatal entrance in freeze-fracture preparations. They appear similar to particle clusters in animal tight junctions, and their possible function in providing electrical coupling via low resistance junctions between plant cells is discussed.     

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.     

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.     

Influence of minerals on cytoplasmic streaming in root hairs of intact wheat seedlings (Triticum aestivum L.)

The calcium dependency of the cytoplasmic streaming of wheat root hairs was demonstrated by adding the Ca-Ionophore A 23187. Within three minutes the streaming velocity was decreased dramactically. The influence of ammonium on the cytoplasmic streaming is highly pH-dependent. While at a pH of 9.0 an inhibitory effect was observed even at low ammonium concentrations (0.5 mM) no effect could be measured at a pH of 6.5. Nitrate, independently of medium pH had no effect on the cytoplasmic streaming. The same is true for aluminium. It is suggested that at pH 9 ammonium permiates the plasmalemma as NH₃. Due to higher cytoplasmic pH ( 7.5), NH₃ is protonated leading to an increase in cytoplasmic pH. Ammonium may displace sorbed calcium leading to an increase in the free cytoplasmic calcium responsible for the cessation of the streaming. Alternative explanations are discussed.Abbreviations HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid     

Integument cell differentiation in dandelions (<Emphasis Type="Italic">Taraxacum</Emphasis>, Asteraceae,Lactuceae) with special attention paid to plasmodesmata

The aim of the paper is to determine what happens with plasmodesmata when mucilage is secreted into the periplasmic space in plant cells. Ultrastructural analysis of the periendothelial zone mucilage cells was performed on examples of the ovule tissues of several sexual and apomictic Taraxacum species. The cytoplasm of the periendothelial zone cells was dense, filled by numerous organelles and profiles of rough endoplasmic reticulum and active Golgi dictyosomes with vesicles that contained fibrillar material. At the beginning of the differentiation process of the periendothelial zone, the cells were connected by primary plasmodesmata. However, during the differentiation and the thickening of the cell walls (mucilage deposition), the plasmodesmata become elongated and associated with cytoplasmic bridges. The cytoplasmic bridges may connect the protoplast to the plasmodesmata through the mucilage layers in order to maintain cell-to-cell communication during the differentiation of the periendothelial zone cells.     

Germination and pH of intracellular compartments in seeds of Phacelia tanacetifolia

³¹ P nuclear magnetic resonance spectroscope (NMR) was used to study the response of Phacelia tanacetifolia seeds to dark and light conditions during the first 72 h of incubation. Changes in the chemical shifts (δ) of the pH-dependent ³¹P-NMR signals from the vacuolar and the cytoplasmic orthophosphate pools were correlated with the different incubation conditions. In the dark (favorable to germination), the cytoplasmic pH remained nearly constant over the whole period considered, while the vacuolar pH shitted to more acidic values after the 24th h of incubation. In the light (inhibiting germination), the values of cytoplasmic pH tended to become more acidic than in the dark after the 24th h of incubation, while the vacuolar pH remained practically constant. When seed germination was inhibited in the dark by butyric acid (BA). a permeant weak acid, the values of cytoplasmic and vacuolar pH were similar to those of the ungerminated seeds incubated in the light. When, vice versa, seed germination was promoted in the light by fusicoccin (FC), the values of cytoplasmic and vacuolar pH were similar to those of the dark-germinated seeds. A progressive augmentation of P, metabolism occurred both in the dark and in the light up to the 24th h of incubation. Subsequently, light blocked any further evolution of this parameter. Treatment with butyric acid in the dark again mimicked the effect of light, while FC reversed the negative effect of light. The data show that in Phacelia tanacetifolia seeds germination is linked to a more alkaline cytoplasmic pH. The finding that the light-dependent metabolic inhibition occurs after an early activation of metabolism, i.e. after the first 24 h. suggests that the effects of light on the cytoplasmic and vacuolar pH depend on the early metabolic processes involved in the control of the homeostasis of cell pH and/or on the inhibition of the reactivation of the transport mechanisms.     

洋葱花粉母细胞中胞间连丝和胞质通道内的胞质骨架

用DGD包埋去包埋方法,观察了洋葱花粉母细胞中胞间连丝和胞质通道内的胞质骨架分布。结果发现,在花粉母细胞的胞间连丝内有胞质骨加分布,这些骨架纤维集结成束,穿过胞间连丝。在胞质通道内也有胞质骨架分布,但与胸间连丝内的骨困分布有所不同,主要表现为两种形式;骨架纤维致密或稀少。研究讨论了胞质骨架在胞间连丝和胞质通道内的作用。     

Production of butyric acid by batch fermentation of cheese whey withClostridium beijerinckii

Summary The effect of pH on the fermentation of butyric acid byClostridium beijerinckii using cheese whey as a substrate was studied. Maximum concentrations of the acid were produced when the pH was controlled at 5.5. Raising or lowering of pH was found to reduce the total acid formation. This particular strain ofC. beijerinckii produced insignificant amounts of butanol in all the pure culture cases investigated. A comparative study of the fermentation in a synthetic glucose medium and in cheese whey showed the whey to produce more butyric acid.     

H+-ATPase-Mediated Cytoplasmic pH-Responses Associated with Elevation of Cytoplasmic Calcium in Cultured Rabbit Nonpigmented Ciliary Epithelium

Studies were conducted to test whether an increase of cytoplasmic calcium concentration influences H⁺-ATPase activity in cultured rabbit nonpigmented ciliary epithelium (NPE). Cytoplasmic calcium concentration or cytoplasmic pH was measured by a fluorescence ratio technique in cells loaded with either Fura-2 or BCECF. Cytoplasmic calcium was increased in three ways; by exposure to BAY K 8644 (1 μm), by exposure to a mixture of epinephrine (1 μm) + acetylcholine (10 μm) or by depolarization with potassium-rich solution. In each case cytoplasmic pH increased significantly. In all three cases 100 nm bafilomycin A₁, a specific H⁺-ATPase inhibitor, significantly inhibited the pH increase. These results suggest an increase of cytoplasmic calcium might initiate events that lead to activation of proton export from the cytoplasm by a mechanism involving H⁺-ATPase. This notion is supported by the observation that the pH increase was suppressed when either verapamil or nifedipine was used to prevent the cytoplasmic calcium increase in cells exposed to potassium-rich solution. Protein kinase C activation might also be involved in the mechanism of H⁺-ATPase stimulation since staurosporine suppressed the pH response to potassium-rich solution. A transient rise of cytoplasmic calcium concentration was observed when cytoplasmic acidification was induced by exposure to high pCO₂. This suggests a rise of cytoplasmic calcium might represent part of a physiological mechanism to stimulate H⁺-ATPase-mediated protein export under acid conditions. Received: 11 August 2000/Revised: 29 March 2001