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Backgrounds and aims Because stomata in bryophytes occur on sporangia, they are subject to different developmental and evolutionary constraints from those on leaves of tracheophytes. No conclusive experimental evidence exists on the responses of hornwort stomata to exogenous stimulation. Methods Responses of hornwort stomata to abscisic acid (ABA), desiccation, darkness and plasmolysis were compared with those in tracheophyte leaves. Potassium ion concentrations in the guard cells and adjacent cells were analysed by X-ray microanalysis, and the ontogeny of the sporophytic intercellular spaces was compared with those of tracheophytes by cryo-scanning electron microscopy. Key results The apertures in hornwort stomata open early in development and thereafter remain open. In hornworts, the experimental treatments, based on measurements of >9000 stomata, produced only a slight reduction in aperture dimensions after desiccation and plasmolysis, and no changes following ABA treatments and darkness. In tracheophytes, all these treatments resulted in complete stomatal closure. Potassium concentrations are similar in hornwort guard cells and epidermal cells under all treatments at all times. The small changes in hornwort stomatal dimensions in response to desiccation and plasmolysis are probably mechanical and/or stress responses of all the epidermal and spongy chlorophyllose cells, affecting the guard cells. In contrast to their nascent gas-filled counterparts across tracheophytes, sporophytic intercellular spaces in hornworts are initially liquid filled. Conclusions Our experiments demonstrate a lack of physiological regulation of opening and closing of stomata in hornworts compared with tracheophytes, and support accumulating developmental and structural evidence that stomata in hornworts are primarily involved in sporophyte desiccation and spore discharge rather than the regulation of photosynthesis-related gaseous exchange. Our results run counter to the notion of the early acquisition of active control of stomatal movements in bryophytes as proposed from previous experiments on mosses.

In plant cells, molecular connections link the cell wall-plasma membrane-actin cytoskeleton to form a continuum. It is hypothesized that the cell wall provides stable anchor points around which the actin cytoskeleton remodels. Here we use live cell imaging of fluorescently labelled marker proteins to quantify the organization and dynamics of the actin cytoskeleton and to determine the impact of disrupting connections within the continuum. Labelling of the actin cytoskeleton with green fluorescent protein (GFP)-fimbrin actin-binding domain 2 (FABD2) resulted in a network composed of fine filaments and thicker bundles that appeared as a highly dynamic remodelling meshwork. This differed substantially from the GFP-Lifeact-labelled network that appeared much more sparse with thick bundles that underwent 'simple movement', in which the bundles slightly change position, but in such a manner that the structure of the network was not substantially altered during the time of observation. Label-dependent differences in actin network morphology and remodelling necessitated development of two new image analysis techniques. The first of these, 'pairwise image subtraction', was applied to measurement of the more rapidly remodelling actin network labelled with GFP-FABD2, while the second, 'cumulative fluorescence intensity', was used to measure bulk remodelling of the actin cytoskeleton when labelled with GFP-Lifeact. In each case, these analysis techniques show that the actin cytoskeleton has a decreased rate of bulk remodelling when the cell wall-plasma membrane-actin continuum is disrupted either by plasmolysis or with isoxaben, a drug that specifically inhibits cellulose deposition. Changes in the rate of actin remodelling also affect its functionality, as observed by alteration in Golgi body motility.

The growing pollen tube is central to plant reproduction and is a long-standing model for cellular tip growth in biology. Rapid osmotically driven growth is maintained under variable conditions, which requires osmosensing and regulation. This study explores the mechanism of water entry and the potential role of osmosensory regulation in maintaining pollen growth. The osmotic permeability of the plasmalemma of Lilium pollen tubes was measured from plasmolysis rates to be 1.32±0.31×10(-3) cm s(-1). Mercuric ions reduce this permeability by 65%. Simulations using an osmotic model of pollen tube growth predict that an osmosensor at the cell membrane controls pectin deposition at the cell tip; inhibiting the sensor is predicted to cause tip bursting due to cell wall thinning. It was found that adding mercury to growing pollen tubes caused such a bursting of the tips. The model indicates that lowering the osmotic permeability per se does not lead to bursting but rather to thickening of the tip. The time course of induced bursting showed no time lag and was independent of mercury concentration, compatible with a surface site of action. The submaximal bursting response to intermediate mercuric ion concentration was independent of the concentration of calcium ions, showing that bursting is not due to a competitive inhibition of calcium binding or entry. Bursting with the same time course was also shown by cells growing on potassium-free media, indicating that potassium channels (implicated in mechanosensing) are not involved in the bursting response. The possible involvement of mercury-sensitive water channels as osmosensors and current knowledge of these in pollen cells are discussed.

In mesophyll cells of the aquatic angiosperm Vallisneria gigantea Graebner, a rapid and transient inhibition of cytoplasmic streaming was induced by hypertonic treatment with sorbitol. Higher concentrations of sorbitol induced the response more rapidly and in more cells. The response to hypertonic treatment was strictly dependent on the presence of extracellular Ca 2 + and was sensitive to Ca 2 + -channel blockers, including the stretch-activated Ca 2 + -channel blocker Gd 3 + . Deplasmolyzed cells never responded to a second hypertonic treatment administered immediately after plasmolysis and subsequent deplasmolysis. Responsiveness was gradually recovered during 24 h of incubation; however, cycloheximide, cordycepin, and trypsin completely suppressed the recovery. Although an Arg-Gly-Asp (RGD) hexapeptide markedly disturbed the pattern of cytoplasmic streaming, it exhibited no specific effects on the response to hypertonic treatment or on the recovery of responsiveness. Taken together, our results demonstrate that leaf mesophyll cells in a multicellular plant can respond to mechanical stimuli and that a Ca 2 + influx through stretch-activated Ca 2 + channels plays an indispensable role in the response. Furthermore, the possible involvement of RGD-insensitive but trypsin-sensitive protein factor(s), whose function is impaired by detachment of the plasma membrane from the cell wall, is suggested.

Microtubules (MTs) of cells of Spirogyra sp. were depolymerized by treatment with amiprophos-methyl (APM) for 1 h and then reorganized in 0.30 M mannitol solution. The reorganized MTs after 1.5 h incubation showed an oblique/longitudinal orientation and then became transversely oriented as the incubation was prolonged. During this incubation, the osmotic pressure of cells was measured by the plasmolysis method. The cell osmotic pressure increased with time. The calculated turgor pressure at 1.5 h was 0.11 M (mannitol equivalent) and, at 13.5 h, 0.25 M. Similar changes in MT orientation and recovery of the turgor pressure were also observed in 0.30 M sorbitol solution. These results suggest that the MT orientation may be correlated with the turgor pressure. Among fresh water algae sensitive to a saline environment, this Spirogyra was the first species shown to have a turgor regulating mechanism, although the recovery of turgor pressure was incomplete. The recovery of turgor pressure in mannitol solutions was also observed without APM treatment.

Minor vein ultrastructure and phloem loading were studied in leaves of the tulip tree (Liriodendron tulipifera; Magnoliaceae). Plasmodesmatal frequencies leading into minor vein companion cells are higher than in species known to load via the apoplast. However, these companion cells are not specialized as "intermediary cells" as they are in species in which the best evidence for symplastic phloem loading has been documented. Mesophyll cells plasmolyzed in 600 mM sorbitol, whereas sieve elements and companion cells did not plasmolyze even in 1.2 M sorbitol, indicating that solute accumulates in the phloem against a steep concentration gradient. Both [(14)C]sucrose and (14)C-labeled photo-assimilate accumulated in the minor vein network, as demonstrated by autoradiography. [(14)C]sucrose accumulation was prevented by p-chloromercuribenzenesulfonic acid, an inhibitor of sucrose-proton cotransport from the apoplast. p-Chloromercuribenzenesulfonic acid largely, but not entirely, inhibited exudation of radiolabeled photoassimilate. The evidence is most consistent with the presence of an apoplastic component to phloem loading in this species, contrary to speculation that the more basal members of the angiosperms load by an entirely symplastic mechanism.

Optical microsurgical techniques were employed to investigate the mechanical properties of Hechtian strands in tobacco (Nicotiana tabacum) and Ginkgo biloba callus cells. Using optical tweezers, a 1.5-μm diameter microsphere coated with concanavalin A was inserted though an ablated hole in the cell wall of a plasmolyzed cell and attached to a Hechtian strand. By displacing the adhered microsphere from equilibrium using the optical trapping force, the tensions of individual strands were determined. Measurements were made using both normal and cold-hardened cells, and in both cases, tensions were on the order of 10−12 N. Significant differences were found in the binding strengths of cold-hardened and normal cultured cells. An increased number density of strands in cold-hardened G. biloba compared with normal cultured cells was also observed. Although no Hechtian strands were detected in any Arabidopsis callus cells, strands were present in leaf epidermal cells. Finally, the movement of attached microspheres was monitored along the outside of a strand while cycling the osmotic pressure.

‘Peel pitting’ in the flavedo (the coloured epicarp) of ‘Encore’ mandarin arises as chlorotic spots. These spots correspond to parenchymal flattening and collapse of a variable number of sub-epidermal cell layers, which increase in parallel with the epidermis, extending between apparently healthy zones. The first signs of cellular damage are associated with internal membrane disorganization of the plastids. Great vesiculation of the cytoplasm occurs, followed by degradation of cytoplasmic membranes and accumulation of abundant osmiophilic material, yielding amorphous and dense masses. The disorder started by September. Spotting was only observed on the outer side of the fruit and on portions of the rind most exposed to external factors. Radiation and high temperature of the rind over long periods may induce localized dehydration, plasmolysis and cell collapse.

Salinity and high temperature stresses adversely affect growth and development of rice plants. To investigate the response of rice cells to these stresses, we have analysed short-term stress-induced subcellular alterations in undifferentiated leaf cells of rice seedlings by transmission electron microscopy. Perturbations noted particularly with respect to plasma membrane, mitochondrial membranes, endoplasmic reticulum, polyribosomes and dictyosomes are highlighted. The subcellular changes evoked by both stresses after 4 h were lysis of the cytoplasm, accumulation of electron-dense granules in the cytoplasm, distension in the ER membranes, enhanced association of ribosomes with the endoplasmic reticulum, reduction in the number of mitochondrial cristae, as well as disorganization of cell wall fibrillar material. Certain changes were found to be unique to either the salinity or high temperature stress. Plasmolysis and increased cytoplasmic vesiculation were seen only in response to salinity stress, while discontinuity in the plasma membrane with close association of the osmiophilic granules were observed only in response to high temperature.

The electrophysiology of non-turgid cells of Chara australis was investigated. The data fell into two categories. (i) 20% of cells; these were relatively unaffected by plasmolysis (with the exception of a small depolarization) and retained many aspects of normal electrophysiological behaviour, such as excitation and K + -state I/V characteristics when exposed to 5 mol m -3 K + . (ii) 80% of cells; these showed electrophysiology significantly altered by plasmolysis. These cells exhibited a large depolarization and became highly conductive after plasmolysis. The classic current relaxation kinetics upon voltage step changes diminished. The channels constituting this high conductance state appeared to be weakly anion selective. Application of Ca 2+ channel blocker LaCl 3 did not prevent development of the high conductance. Upon exposure to 5 mol m -3 K + , the negative conductance regions were much less pronounced than those in turgid cells. Excitation was abolished. Cells in both categories showed a steady increase in conductance with time. The effects of plasmolysis on cell membrane conductance were irreversible. The information gathered from turgorless Chara cells can be extrapolated to explain depolarized or positive resting potentials and predominance of weakly anion selective channels in some higher plant cell protoplasts.

The extent of post-phloem solute transport through the coat symplasts of developing seeds of Vicia faba L. and Phaseolus vulgaris L. was evaluated. For Vicia seed coats, the membrane-impermeant fluorochrome, CF, moved radially from the chalazal vein to reach the chlorenchyma and thin-walled parenchyma transfer cell layers. Thereafter, the fluorochrome moved laterally in these two cell layers around the entire circumference of the seed coat. Transfer of CF from the chalazal vein was inhibited by plasmolysis of attached «empty» seed coats. In contrast, the spread of phloemimported CF was restricted to the ground parenchyma of Phaseolus seed coats. Fluorochrome loaded into the outermost ground parenchyma cell layer was rendered immobile following plasmolysis of excised seed-coat halves. Phloem-imported [ 14 C]sucrose and the slowly membrane permeable sugar, L-[ 14 C]glucose, were partitioned identically between the vascular and non-vascular regions of intact Vicia seed coats. For 14 C-photosynthates, these partitioning patterns in attached «empty» Vicia seed coats were unaffected by PCMBS, but inhibited by plasmolysis. Tissue autoradiographs of intact Phaseolus seed coats demonstrated that a pulse of 14 C-photosynthate moved from the veins to the ground tissues. In excised Vicia seed coats, preloaded with 14 C-photosynthates, the cellular distribution of residual 14 C-label was unaffected by PCMBS. In contrast, PCMBS caused the 14 C-photosynthate levels to be elevated in the veins and ground parenchyma relative to the branch parenchyma of Phaseolus seed coat halves. Based on the above findings, it is concluded that the phloem of Vicia seed coats is interconnected to two major symplastic domains; one comprises the chlorenchyma, the other the thin-walled parenchyma plus thin-walled parenchyma transfer cells. For Phaseolus seed coats, the phloem forms a major symplastic domain with the ground parenchyma

Biolistic transfer and subsequent reporter gene expression was enhanced 3-20-fold in embryogenic suspension cultures of Picea abies by treatment with osmotica before and after bombardment. Myoinositol was more effective than mannitol + sorbitol and about as effective as sucrose in enhancing expression 1 d after bombardment. For both myoinositol and sucrose the optimal concentration was 0,25 M. Abscisic acid interacted with myoinositol to give a further enhancement. Maintaining the cells on 0.25 M myoinositol after bombardment enhanced the expression measured 10 d later. These results provide evidence for a metabolic component of the mechanism of osmotic enhancement in addition to an effect depending on partial plasmolysis at the time of bombardment. Expression driven by the polyubiquitin promoter 10 d after bombardment was more than twice that driven by an enhanced 35S, and 20 times that driven by a simple 35S promoter. Experiments using plasmids with and without scaffold attachment regions indicated that a greater proportion of the cells that expressed the reporter gene at 6 d compared with 1 d after bombardment had been integratively transformed

The respiration of Escherichia coli is severely inhibited, during hyperosmotic stress period, as a consequence of plasmolysis; deplasmolysis allows the cell to recover respiration. A mutant lacking all K+ transport systems can neither deplasmolyze nor recover respiration unless betaine is present in the medium. Betaine, in these conditions, increases both cytoplasmic volume and respiration; this suggests a control of respiration by cytoplasmic volume.

Our previous work (E. Shedletzky, M. Shmuel, D.P. Delmer, D.T.A. Lamport [1990] Plant Physiol 94:980-987) showed that suspension-cultured tomato cells adapted to growth on the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) have a markedly altered cell wall composition, most notably a markedly reduced level of the cellulose-xyloglucan network. This study compares the adaptation to DCB of two cell lines from dicots (tomato [Lycopersicon esculentum] and tobacco [Nicotiana tabacum]) and a Graminaceous monocot (barley [Hordeum bulbosum] endosperm). The difference in wall structures between the dicots and the monocot is reflected in the very different types of wall modifications induced by growth on DCB. The dicots, having reduced levels of cellulose and xyloglucan, possess walls the major integrity of which is provided by Ca(2+)-bridged pectates because protoplasts can be prepared from these cells simply by treatment with divalent cation chelator and a purified endopolygalacturonase. The tensile strength of these walls is considerably less than walls from nonadapted cells, but wall porosity is not altered. In contrast, walls from adapted barley cells contain very little pectic material and normal to elevated levels of noncellulosic polysaccharides compared with walls from nonadapted cells. Surprisingly, they have tensile strengths higher than their nonadapted counterpart, although cellulose levels are reduced by 70%. Evidence is presented that these walls obtain their additional strength by an altered pattern of cross-linking of polymers involving phenolic components. Such cross-linking may also explain the observation that the porosity of these walls is also considerably reduced. Cells of adapted lines of both the dicots and barley are resistant to plasmolysis, suggesting that they possess very strong connections between the wall and the plasma membrane.

High-resolution nuclear magnetic resonance images (using very short spin-echo times of 3.8 milliseconds) of cross-sections of excised roots of the halophyte Aster tripolium showed radial cell strands separated by air-filled spaces. Radial insertion of the pressure probe (along the cell strands) into roots of intact plants revealed a marked increase of the turgor pressure from the outermost to the sixth cortical layer (from about 0.1-0.6 megapascals). Corresponding measurements of intracellular osmotic pressure in individual cortical cells (by means of a nanoliter osmometer) showed an osmotic pressure gradient of equal magnitude to the turgor pressure. Neither gradient changed significantly when the plants were grown in, or exposed for 1 hour to, media of high salinity. Differences were recorded in the ability of salts and nonelectrolytes to penetrate the apoplast in the root. The reflection coefficients of the cortical cells were approximately 1 for all the solutes tested. Excision of the root from the stem resulted in a collapse of the turgor and osmotic pressure gradients. After about 15 to 30 minutes, the turgor pressure throughout the cortex attained an intermediate (quasistationary) level of about 0.3 megapascals. This value agreed well with the osmotic value deduced from plasmolysis experiments on excised root segments. These and other data provided conclusions about the driving forces for water and solute transport in the roots and about the function of the air-filled radial spaces in water transport. They also showed that excised roots may be artifactual systems.

The unusual nature of protoplasm to generate subprotoplasts spontaneously from disintegrated Bryopsis cells was examined. Protoplasm extruded from algal cells aggregated rapidly in cell sap which was derived mainly from huge central vacuoles of the cells. Electron microscopic observations revealed extensive agglutination of algal cellular membranes in the protoplasmic masses, suggesting that this is of primary importance for the wound-healing ability of the alga. Seawater caused spheration of the resultant protoplasmic aggregates. Gelatinous sheaths were formed temporarily surrounding the spherical protoplasmic masses before reformation of cell membrane. Staining with phosphotungstic and chromic acids suggested that new cell membrane was formed by fusion of the disintegrated original cell membrane with cytoplasmic vesicles on the surfaces of the protoplasmic masses. Both pH and salts were found to be essentially important at the two steps of subprotoplast generation. The newly formed cell membranes were responsible for subsequent notable plasmolysis of the wounded cells in seawater. Thus, it is suggested that unicellular marine algae Bryopsis spp. naturally contain effective materials for agglutinating and fusing particular cellular membranes through the sequential aid of acidic cell sap and alkaline seawater after disintegration of the giant cells.

Phloem loading in Coleus blumei Benth. leaves cannot be explained by carrier-mediated transport of export sugar from the apoplast into the sieve element-companion cell complex, the mechanism by which sucrose is thought to load in other species that have been studied in detail. Uptake profiles of the export sugars sucrose, raffinose, and stachyose into leaf discs were composed of two components, one saturable and the other not. Saturable (carrier-mediated) uptake of all three sugars was almost completely eliminated by the inhibitor p-chloromercuribenzenesulfonic acid (PCMBS). However, when PCMBS was introduced by transpiration into mature leaves it did not prevent accumulation of (14)C-photosynthate in minor veins or translocation of labeled photosynthate from green to nonchlorophyllous regions of the leaf following exposure to (14)CO(2). The efficacy of introducing inhibitor solutions in the transpiration stream was proven by observing saffranin O and calcofluor white movement in the minor veins and leaf apoplast. PCMBS introduced by transpiration completely inhibited phloem loading in tobacco leaves. Phloem loading in C. blumei was also studied in plasmolysis experiments. The carbohydrate content of leaves was lowered by keeping plants in the dark and then increased by exposing them to light. The solute level of intermediary cells increased in the light (phloem loading) in both PCMBS-treated and control tissues. A mechanism of symplastic phloem loading is proposed for species that translocate the raffinose series of oligosaccharides.

The time course of turgor regulation of the euryhaline giant-celled alga, Chara buckellii, is presented. Isolated intermodal cells were challenged by increasing or decreasing the external osmotic pressure by 150 milliosmoles per kilogram with all ions in the media or by dilution, respectively. Regulation following hypotonic stress was complete within 48 hours whereas regulation following hypertonic stress required between 96 and 144 hours. The change in internal osmotic pressure could be entirely accounted for by changes in vacuolar KCl in response to hypotonic stress, but this ion pair only accounted for 45% of the change in response to hypertonic stress. The membrane potential of C. buckellii is normally hyperpolarized with respect to the equilibrium potential for K+ (EK). The membrane depolarized to a level close to EK in response to hypotonic treatment and this was accompanied by a transient increase in membrane conductance. In response to hypertonic stress, the membrane hyperpolarized transiently, then repolarized to a level close to the control. This was accompanied by a temporary decrease in membrane conductance. The data are discussed with respect to the ecological significance of the time course and ion transport mechanisms during turgor regulation.

After plasmolysis of Escherichia coli cells, the adhesion zones were characterized using the cytochemical PTA and SP procedures which stain peptidoglycan and lipopolysaccharides (LPS) respectively. A PTA-stained layer was detected at the adhesion sites. This layer was visualized irrespective of the electron microscopy procedure used. Also, using SP staining an outer membrane in which LPS molecules were asymmetrically distributed, was observed.

The effect of plasma membrane contraction and re-expansion on redox activity was investigated. This was done by determination of ferricyanide reduction in non-plasmolysed epidermal strips, after plasmolysis and after plasmolysis followed by a subsequent re-hydratation

Autoradiographic, plasmolysis, and 14C-metabolite distribution studies indicate that the majority of exogenously supplied 14C-sucrose enters the phloem directly from the apoplast in source leaf discs of Beta vulgaris. Phloem loading of sucrose is pH-dependent, being markedly inhibited at an apoplast pH of 8 compared to pH 5. Kinetic analyses indicate that the apparent Km of the loading process increases at the alkaline pH while the maximum velocity, Vmax, is pH-independent. The pH dependence of sucrose loading into source leaf discs translates to phloem loading in and translocation of sucrose from intact source leaves. Studies using asymmetrically labeled sucrose 14C-fructosyl-sucrose, show that sucrose is accumulated intact from the apoplast and not hydrolyzed to its hexose moieties by invertase prior to uptake. The results are discussed in terms of sucrose loading being coupled to the co-transport of protons (and membrane potential) in a manner consistent with the chemiosmotic hypothesis of nonelectrolyte transport.

Pseudomonas aeruginosa NCTC6750 and Escherichia coli K12 were used to study permeability of whole, intact cells to a series of labelled oligosaccharides. Stationary phase, oxygen depleted simple salts batch cultures were used. An efflux method was used to compare diffusion from cells of various 3H-labelled sugars (an homologous series based on isomaltitol) with diffusion of [14C]sucrose. Both plasmolysed and unplasmolysed cell suspensions were used. The data are consistent with an E. coli pore exclusion limit of approx. 833 Da for unplasmolysed cells and of about 670 Da for plasmolysed cells. For P. aeruginosa the data indicated a relatively small pore exclusion limit about the same size as sucrose with plasmolysis having little effect. These findings were confirmed with P. aeruginosa PAO1 grown in nutrient broth.

Ultrastructural studies on oat coleoptile parenchyma cells (Avena sativa L. cv. Victory) reveal that severe plasmolysis either breaks plasmodesmatal connections or leaves the protoplasts still connected via strands of cytoplasm (Hechtian strands). Plasmolysis also induces the formation of callose around the plasmodesmata. The callose remains for several hours after recovery of the cells to full turgor. Immediately following recovery of turgor, intercellular electrical coupling cannot be detected. However, during the next 6 h, some degree of coupling is restored. These results indicate that, while plasmolysis does not necessarily break all plasmodesmatal connections, the treatment probably does disrupt them sufficiently to interfere, at least temporarily, with symplastic transport.

Rates of solute leakage from excised discs of cucumber (Cucumis sativus L. cv Straight Eight) cotyledons were altered by temperature during plasmolysis in the manner of a simple diffusion phenomenon; the log of the leakage rate increased in proportion to the temperature. During deplasmolysis, however, leakage rates responded to temperature with a very different pattern: chilling conditions (below about 20 degrees C) caused large increases in leakage rates, indicating disruption of membrane integrity in the tissues. The time course of restoration of normal leakage rates after deplasmolysis/chilling damage indicated a rapid repair of the lesions. A similar sensitivity to low temperatures was found during rehydration after leaf desiccation, with low temperatures again causing high leakage rates. It is suggested that low temperatures interfere with membrane expansion, possibly by lowering elasticity and hindering the incorporation of lipid material into the expanding membrane. The expansion of tissues at low temperatures may cause lesions in cellular membranes, contributing to chilling injury.

The wilty tomato mutant flacca and the normal variety Rheinlands Ruhm were compared in terms of: (1) potassium transport into and out of the guard cells, (2) cell wall properties which include protein, hydroxyproline and peroxidase activity, and (3) activity of indol 3yl-acetic acid oxidase. Also studied were the effects of auxin on stomatal behaviour and peroxidase activity when applied to normal plants during development, and the short-term effect of abscisic acid on the resistance of flacca stomata to closure under plasmolysis. Potassium transport, wall protein and hydroxyproline all seemed to be equal in mutant and normal plants. Peroxidase activity was higher in the soluble and wall fractions of the mutant, and decreased toward normal in the mutant treated with abscisic acid. More stomata were open and peroxidase activity was higher in normal plants treated with auxin during development. The percentage of open stomata under plasmolysis was lower and their aperture size was smaller in the epidermal strips taken from abscisic-acid-treated mutant plants than from control mutant plants.

Osmotic shock, a 15-minute plasmolysis followed by a 15-minute rehydration in the cold, is a nondestructive technique which inhibits fusicoccin-stimulated H + excretion from oat mesophyll cells ( Avena sativa L.). Osmotic shock also causes a loss of intracellular solutes and stimulates H + uptake, but osmoregulation can still occur, and enhanced H + uptake is observed only at low external pH. It is concluded that osmotic shock interferes directly with the excretion of H + rather than affecting only H + or counter ion uptake. Plasmolysis alone does not inhibit fusicoccin-enhanced H + excretion, and the rehydration step must be rapid and in the cold for maximum inhibition. This suggests that the plasma membrane is perturbed, possibly due to release or rearrangement of membrane protein. Compared to corresponding osmolarities of sorbitol or NaCl, polyethylene glycol 4000 is much less effective during the plasmolysis step; the possibility is discussed that the ineffectiveness of polyethylene glycol 4000 is due to its preservation of plasmodesmata.

The present studies provide the first measurements of the resistance to diffusive flux of metabolites between mesophyll and bundle sheath cells of C(4) plants. Species examined were Panicum miliaceum, Urochloa panicoides, Atriplex spongiosa, and Zea mays. Diffusive flux of metabolites into isolated bundle sheath cells was monitored by following their metabolic transformation. Evidence was obtained that the observed rapid fluxes occurred via functional plasmodesmata. Diffusion constants were determined from the rate of transformation of limiting concentrations of metabolites via cytosolic enzymes with high potential velocities and favorable equilibrium constants. Values on a leaf chlorophyll basis ranged between 1 and 5 micromoles per minute per milligram of chlorophyll per millimolar gradient depending on the molecular weight of the metabolite and the source of bundle sheath cells. Diffusion of metabolites into these cells was unaffected by a wide variety of compounds including respiratory inhibitors, monovalent and divalent cations, and plant hormones, but it was interrupted by treatments inducing cell plasmolysis. The molecular weight exclusion limit for permeation of compounds into bundle sheath cells was in the range of 850 to 900. These cells provide an ideal system for the quantitative study of plasmodesmatal function.

Abscisic acid (ABA) accumulated in detached, wilted leaves of spinach ( Spinacia oleracea L. cv Savoy Hybrid 612) and reached a maximum level within 3 to 4 hours. The increase in ABA over that found in detached turgid leaves was approximately 10-fold. The effects of water stress could be mimicked by the use of thin slices of spinach leaves incubated in the presence of 0.6 molar mannitol, a compound which causes plasmolysis (loss of turgor). About equal amounts of ABA were found both in the leaf slices and in detached leaves, whereas 2 to 4 times more ABA accumulated in the medium than in the slices. When spinach leaf slices were incubated with ethylene glycol, a compound which rapidly penetrates the cell membrane causing a decrease in the osmotic potential of the tissue and only transient loss of turgor, no ABA accumulated. Ethylene glycol was not inhibitory with respect to ABA accumulation. Spinach leaf slices incubated in both ethylene glycol and mannitol had ABA levels similar to those found when slices were incubated with mannitol alone. Increases similar to those found with mannitol also occurred when Aquacide III, a highly purified form of polyethylene glycol, was used. Aquacide III causes cytorrhysis, a situation similar to that found in wilted leaves. Thus, it appears that loss of turgor is essential for ABA accumulation. When spinach leaf slices were incubated with solutes which are supposed to disturb membrane integrity (KHSO 3 , 2-propanol, or KCl) no increase in ABA was observed. These data indicate that, with respect to the accumulation of ABA, mannitol caused a physical stress (loss of turgor) rather than a chemical stress (membrane damage).

Attempts were made to disrupt the plasmodesmata between oat coleoptile cells (Avena sativa L. cv. Victory) by severe plasmolysis. Coleoptiles, allowed to regain turgor after plasmolysis, were able to execute geotropic and phototropic curvatures and segments would grow in response to applied auxin. In coleoptiles similarly treated, studies with [14C]IAA have shown that longitudinal, basipetal transport of auxin still takes place and, as in controls, IAA is preferentially redistributed laterally within coleoptiles orientated horizontally. Physical continuity of the symplast of oat coleoptile cells may not always be disrupted by severe plasmolysis. Nevertheless, functional continuity appears to be interrupted. Despite this, all the processes involved in the execution of tropistic curvatures remain intact, including transport of hormones. Plasmodesmatal continuity between oat coleoptile cells appears not to be a necessary requirement for auxin transport.

Electron microscopy of negatively stained Bacteroides fragilis cells treated with hypertonic solutions of di- or trisaccharides showed the shrinkage of whole cells, whereas the pentose- or hexose-treated cells shrank less significantly. Electron microscopy of thin sections of the monosaccharide-treated cells showed plasmolysis. Determination of the diffusion rate by the liposome-swelling method suggested that the apparent exclusion limit of the outer membrane pore is close to the size of uncharged saccharides of approx. Mr 250. These results suggested that the outer membrane of B. fragilis contains small diffusion pores.

A bioassay to measure the incorporation of [14C]leucine into acid-precipitable polymers of suspension-cultured sycamore (Acer pseudoplatanus L.) cells is described. Using this assay, cell wall fragments solubilized from sycamore cell walls by partial acid hydrolysis are shown to contain components that inhibit the incorporation of [14C]leucine into the acid-precipitable polymers. This inhibition was not attributable to a suppression of [14C]leucine uptake. The effectiveness of the wall fragments in inhibiting [14C]leucine incorporation was substantially relieved by plasmolysis of the cells. Fragments released from starch and citrus pectin are shown not to possess such inhibitory activities.

Time-course experimenls demonstrate that sporangial emptying in Achlya inlricata is inhibited by experimental depression of the external osmotic potential. The sporangial wall is a macromolecular sieve, impermeable to polyethylene glycols (PEGs) with molecular weight 3350. The process of emptying can be precisely manipulated using PEG-8000 which suggests that spore discharge in an aqueous environment is effected by a reduction in the osmotic potential of the sporangial lumen relative to the external solution.

Morris, G. J., Coulson, G. E., and Engels, M. 1986. A cryomicroscopic study of Cylindrocystis brebissonii De Bary and two species of Micrasterias Ralfs (Conjugatophyceae, Chlorophyta) during freezing and thawing.—J. exp. Bot. 37: 842-856. The changes in morphology of the unicellular algae Cylindrocystis brebissonii and two species of Micrasterias during freezing and thawing were observed on a light microscope fitted with a temperature controlled stage. At slow rates of cooling extensive shrinkage of the protoplast was observed. The response of the cell wall varied with cell-type. In C. brebissonii plasmolysis was not observed and the cell wall and protoplast shrank together. In Micrasterias the cell wall did not contract and a distinct plasmolysis was observed. Following freezing to and thawing from —25 °C cells of C. brebissonii were non-viable but remained osmotically responsive. Cooling at faster rates induced intracellular ice formation in all cell-types. The critical rate of cooling varied with cell-type and was determined by cell volume and suface area. Intracellular gas bubbles were observed during thawing following both rapid and slow cooling. Following cooling in dimethylsulphoxide cells of C. brebissonii were protected against freezing injury. The recovery on thawing from —196 °C being determined by the rate of cooling, an optimum rate of 1°C min-1 was observed. During slow rates of cooling ( 20°C min"1).

The walls of normal root hairs of white mustard, corn and tomato seedlings developed in moist air following seed treatment with 45CaCl2 were tested by radioautography for incorporated calcium. Radioactivity was absent or faint over the growing tips but became concentrated immediately behind this point. It extended back along the walls of the hairs, increasing in concentration towards the base. Since the hairs were subjected to plasmolysis and prolonged extraction with hot water prior to making the radioautographs, the distribution of radioactivity indicated the presence of bound 45Ca. These findings are compatible with the view that gradual calcification is essential for the normal growth and form of the hair.

Incubation of Zea coleoptiles in 0-5 M mannitol totally inhibits growth and geotropic curva ture, but does not affect the development of the geoelectric effect. This pre-treatment also inhibits the curvature induced by the asymmetrical application of IAA to the apical end of decapitated vertical coleoptiles, but it does not prevent the IAA from giving rise to an electropotential difference between the two sides of the coleoptile. Neither the normal geoelectric effect, nor the auxin-induced potential difference in vertical coleoptiles, can therefore arise as the result of the different rates of cell extension in the two halves of the organ. They must be the result of the change of IAA concentration affecting some other aspect of the cell's physiology or metabolism. The abolition of the electrical responses in coleoptiles which have been plasmolysed in TO M mannitol strongly suggests that both longitudinal and lateral transport of IAA are severely depressed by this degree of plasmolysis. Asymmetrical application of 10~5 M mersalyl and several other substances to the apical end of a decapitated vertical coleoptile gave rise to a marked electropotential difference between the two sides of the coleoptile, the side beneath the donor being positively charged with respect to the other side. Mersalyl does not promote the growth of Zea coleoptiles. These results provide additional evidence that the electropotentials do not arise from differential growth, and suggest that such substances, especially the diuretics used in clinical medicine, may provide useful tools in the further study of the induction of surface electro potentials in plant tissues at the cellular level.

Vacuolated and nonvacuolated root tissues of Zea mays were exposed to low water potentials by addition of mannitol or glycerol. Temporary increases were observed for O(2) uptake, but CO(2) evolution remained steady. This increase in O(2) uptake ceased after 15 minutes. Further treatment induced decreases in respiration, with similar reductions in O(2) uptake and CO(2) evolution.Removal of osmotica from nonvacuolated tissues restored high rates of respiration, uptake of glucose, and synthesis of methanol-insoluble compounds. In contrast, rates of all these processes decreased to very low values when vacuolated tissues were returned to high water potentials. Deplasmolysis also induced rapid leakage of metabolic intermediates from vacuolated tissues, but leakage from nonvacuolated tissues was only slightly increased. It is suggested that these contrasting responses of vacuolated and nonvacuolated tissues are related to differences in structural changes, during either plasmolysis or deplasmolysis.

The distribution of solutes in the various cells of sugar beet (Beta vulgaris L.) source leaves, petioles, and sink leaves was studied in tissue prepared by freeze-substitution. The differences in degree of cryoprotection indicated that sieve elements and companion cells of the source leaf, petiole, and sink leaf contain a high concentration of solute. The osmotic pressure of various types of cells was measured by observing incipient plasmolysis in freeze-substituted tissues equilibrated with a series of mannitol solutions prior to rapid freezing. Analysis of source leaf tissue revealed osmotic pressure values of 13 bars for the mesophyll and 30 bars for the sieve elements and companion cells. The osmotic pressure of the mesophyll of sink leaves was somewhat higher.The sharp concentration increase at the membrane of the sieve element-companion cell complex of the source leaf indicates active phloem loading from the free space at this site. Active loading of the phloem is presumably needed to move the sugar from the chloroplasts of the mesophyll to the sieve tubes against the concentration gradient. The osmotic pressure of the mature sieve element-companion cell complex appears to be approximately the same in source leaf, path, and sink leaf tissue. There is a distinct difference in concentration between the mature sieve element-companion cell complex in the sink and the surrounding mesophyll. The solute distribution suggests that sugar is actively accumulated from the free space by the developing sink leaf tissue.The osmotic values observed in the various cells are consistent with the operation of a mass flow mechanism of translocation driven by active phloem loading and by active accumulation of sugar by sink tissues.

Inhibition of hyphal growth in Aspergillus fumigatus due to the presence of living cells of Pseudomonas aeruginosa correlates with ultrastructural abnormalities which include plasmolysis, vacuolation and degeneration of organelles. Hyphal cells adjacent to and distant from bacteria are equally affected, indicating translocation of the inhibitor or its diffusion through agar.

SUMMARY The absorption of potassium ions by cells of red beetroot tissue plasmolysed in various media has been examined and compared with that of unplasmolysed cells under similar experimental conditions. It is established that although the plasmolysing agents in themselves tend to promote the absorption of ions at the concentrations employed, the effect of plasmolysis is to inhibit the rate of potassium uptake. Evidence is provided that this is due to an increase in the rate of leakage of ions from plasmolysed cells, and to a reduction of gross uptake. These results are discussed in terms of the structural and physiological changes which are associated with plasmolysis. It is concluded that alterations in the surface area, and thickness or density of the protoplasts, modifications of the vacuolar concentration of ions, and respiratory influences are all involved. Another factor, the nature of which has not been elucidated, also appears to be involved.

Cellular tests which are used in place of actual freezing tests to estimate the resistance to injury by freezing that a plant tissue will show are based on the observations of Levitt and Scarth (2, 5) and Levitt, Scarth and SiMiNoviTCH (3, 6, 7, 10) that the most characteristic physical property of frost hardy cells is their capacity to withstand injury by dehydration. The frost hardiness test described in the first papers of the present series (1, 11) was based on a determination of the percentage survival of cells after dehydration by plasmolysis, as effected by subjecting the tissues to a graded series of strong salt solutions. In subsequent studies hardiness has been determined also by estimating the survival of the cells after desiccation in atmospheres of varied relative humidities produced over a graded series of sulphuric acid solutions. The degree of hardiness has been assumed to be measurable by the extent to which the cells possess the property of surviving dehydration. Experimental verification of the validity of this assumption by comparison of dehydration tests with actual freezing tests has been obtained previously (?, 9) with other plant tissues but not with the bark cells of the black locust, the experimental tissue employed in these studies.

In 1884 de Vries (10) introduced a new method, that of determining the osmotic value of plant cells by immersion in solutions of known concentrations of cane sugar or potassium nitrate. He determined the concentration of each solute which would bring about plasmolysis or the withdrawal of the protoplasm from the walls of the cells. He reasoned that all solutions inducing the same minimum degree of visible plasmolysis of cells must be of equal strength. Various aspects of the phenomenon of plasmolysis have been elaborately studied since then by de Vries (11), Fitting (4), Hannig (5), Iljin (8), Hofler (6, 7), Ursprung and Blum (9), Beck (1, 2), Weber (12, 13), and many others. These workers have interpreted the results obtained by this method as furnishing adequate data for the estimation of the osmotic value of the cell sap. Objection may be raised to this interpretation on two grounds. In the first place, the material used by de Vries, and by most of his followers, cannot be considered to have been in normal, uninjured condition. Thin sections were prepared for microscopic examination by sectioning, thus subjecting the cells to the deleterious osmotic and other actions of sap exuded from adjacent ruptured cells. That this influence may be important has been pointed out elsewhere (3). Moreover, the phenomenon commonly designated as plasmolysis, although usually regarded as purely osmotic in nature, may be induced by several other causes. Mechanical injury induced by pressure brings about a contraction of the protoplasm which in its early stages resembles plasmolysis and which is often reversible if the injury is slight. It passes through a plasmolysis-like stage to an irreversible granulation if the pressure is considerable or of long duration. Strong light (13) and high temperature likewise cause retraction of the protoplasm from the cell walls, and light and temperature conditions which are not markedly injurious may influence the time required for plasmolysis. In the second place, the assumption made by Ursprung and Blum (9), and with few exceptions accepted without experimental proof by subsequent workers, that the cell contents and applied solution come into equilibrium within 20-30 minutes, can be shown to be untenable both on theoretical i This work was done at the Imperial College of Science and Technology, London. The writer wishes to express her thanks to Professor V. H. Blackman for his. interest and advice.

An investigation was made into the relationship between extracellular solute concentration and transfer frequency in F-type mating in Escherichia coli . Solute concentrations above a certain level correlated with a sharp decrease in transfer frequency which was not due to a loss of cell viability; this effect was obtained both with electrolyte (sodium chloride) and with osmotically equivalent non-electrolyte ( myo -inositol), despite the contrasting effects of these solutes at lower concentrations. It is hypothesized that the inhibition of conjugal transfer at high solute concentrations is due to plasmolytic rupture of adhesion sites (“Bayer's junctions”).