pH Gradients in the Stomatal Complex of Tradescantia virginiana

pH of the vacuolar sap, cytoplasm, and apoplast of the cellsof the stomatal complex of Tradescantia virginiana was measuredusing micro-electrodes. Marked differences in vacuolar and apoplasticpH were observed between leaves with open and closed stomata.Cytoplasmic pH appeared to be uniform in all the cells and didnot change with stomatal aperture. The information obtainedenabled proton motive force across the plasmalemma and tonoplastof the guard cells to be calculated. The results are discussedin relation to the accumulation of potassium by the guard cellon stomatal opening. Key words: Stomata, pH gradients, Proton pumps     

An Electrophysiological Study of the Stomatal Complex of Tradescantia virginiana

Measurements of electrical potential difference (PD) and potassiumactivity were carried out on the intact leaf of Tradescantiavirginiana. PD gradients across the stomatal complex were observedwith both open and closed stomata. The guard cell PD appearedto be linearly related to stomatal aperture. With the stomataopen a gradient of potassium activity across the stomatal complexwas observed which became reversed on stomatal closure. Calculationof the driving forces on potassium suggested that it was distributedpassively between the vacuoles of the cells of the stomatalcomplex. The electrophysiological data obtained from this investigationenabled potassium activity in the apoplast to be calculated.The results showed that on stomatal closure there was a massiveincrease in the potassium activity in the guard cell wall. Key words: Stomata, Ionic gradients, Electrical potentials     

Evidence for a CO2 Inhibited Proton Extrusion Pump in the Stomatal Cells of Tradescantia virginiana

The effect of CO₂ on the electrical potential difference (p.d.)of the cells of the stomatal complex of Tradescantia virginianahas been investigated. Enhanced levels of CO₂ affected all thecells equally by depolarizing the p.d. On removal of the CO₂there was an immediate hyperpolarization, the degree of whichwas linearly related to the length of time the CO₂ was given.Changes in the pH of the cytoplasm and the vacuolar sap wereobserved during and after treatment with CO₂. The metabolicinhibitor 2,4-DNP eliminated the effect of CO₂ on the p.d. Theseresults wuld be explained by postulating an electrogenic protonpump in the plasmalemma which is inhibited by CO₂. Key words: CO₂, Proton pump, Stomata     

Correlations Between the Unbound Water Content of Guard Cells and Stomatal Aperture in Tradescantia virginiana L.

When the apertures of stomata in an epidermal strip changedthere was found to be a simultaneous change in the unbound watercontent of the tissue. This occurred even when only the guardcells were living. It was concluded that the change in unboundwater occurred in the guard cells, and possibly the guard cellwalls.     

Cell Potentials and Turgor Pressures in Epidermal Cells of Tradescantia and Commelina

Cell membrane potentials have been measured both in epidermalstrips and intact leaf sections of Tradescantia virginiana andCommelina communis, and in epidermal cells over green and overalbino mesophyll cells of T. albiflora var. albovittata. Membranepotentials (_cell) in strips were considerably lower than thosein intact sections and were insensitive to light and to theabsence or presence of calcium. Their response to external cationlevels was indifferent to ionic species. However, in intactleaf sections incubated with calcium present, membrane potentialsresponded to K⁺ levels but not to Na⁺. were more negative thancells in epidermal strips, and responded to changes in illumination. Long-term recordings of _cell and vacuolar K⁺ levels in T. virginianaduring stomatal closure suggest that the fluctuations of _cellwere unrelated to K⁺ movement (which we could not detect) andthus probably to stomatal movement as well. Turgor pressures measured in epidermal cells of intact leafsections of T. virginiana were found to be of the same magnitudeas those previously reported for epidermal strips. It is concludedthat epidermal cells maintain their solute contents during strippingwithout the involvement of an electrophysiological transportsystem. With the possible exception of lateral subsidiary cells,there was no evidence suggesting that ordinary epidermal cellsare capable of osmotic adjustment even when additional KCI wassupplied in the osmoticum. Absolute turgor levels in intactleaf sections kept at constant external KCI were unrelated tosteady state _cell.     

Potassium Loss from Stomatal Guard Cells at Low Water Potentials

The potassium content of guard cells and the resistance to viscousflow of air through the leaf were determined in sunflower (Helianthusannuus) subjected to low leaf water potentials under illuminatedconditions. In intact plants desiccated slowly by withholdingwater from the soil, large losses in guard cell K occurred asleaf water potentials decreased. Leaf viscous resistance increased,indicating stomatal closure. Similar results were obtained whendetached leaf segments were desiccated rapidly. Upon rehydrationof leaves, no stomatal opening was observed initially, despiteleaf water potentials at predesiccated levels. After severalhours, however, re-entry of K occurred and stomata became fullyopen. Turgid leaf segments floated on an ABA solution showedlosses of guard cell K and closure of stomata as rapidly andcompletely as those brought about by desiccation. It is concludedthat stomatal closure at low water potentials under illuminatedconditions is not controlled solely by water loss from the tissuebut involves the loss of osmoticum from the guard cells as well.This in turn decreases the turgor difference between the guardcells and the surrounding cells, and closing occurs.     

Hydrogen Peroxide-Dependent Oxidation of Flavonoids and Hydroxycinnamic Acid Derivatives in Epidermal and Guard Cells of Tradescantia virginiana L.

Luteolin, kaempferol, quercetin, caffeic acid and ferulic acidwere identified in acid-hydrolyzed epidermal strips of Tradescantiavirginiana using HPLC and spectrophotometry. The amount of flavonoidswas much smaller than that of cinnamic acid derivatives. Morethan 80% of the flavonoids were found in methanol extracts ofepidermal strips. Caffeic acid was found in both methanol extractsand the residues in nearly equal amounts, while more than 80%of the ferulic acid was found in the residues after methanolextraction. These data suggest that most of the ferulic acidand part of the caffeic acid bind to macromolecules as estersin the cell wall and that flavonoids are localized mainly inthe cytoplasm. The localization of esters of hydroxycinnamicacids in cell walls was ascertained by fluorometric analysis.These phenolic compounds were oxidized by H₂O₂ (0.025–1mM) in epidermal and guard cells and the oxidation was inhibitedby KCN and NaN₃: luteolin glycosides were less sensitive toH₂O₂ than quercetin and kaempferol glycosides in flavonoids.Ferulic acid esters were more sensitive to H₂O₂ than caffeicacid esters in hydroxycinnamic acid derivatives. On the basisof these data, the physiological significance of the oxidationof phenolic compounds by H₂O₂ is discussed. (Received October 9, 1987; Accepted February 3, 1988)     

The Hydraulic Pathways in Nicotiana glauca (Grah.) and Tradescantia virginiana (L.) Leaves, and Water Potentials in Leaf Epidermes

The hydraulic conductances of leaves of a species which exhibitsstomatal responses to humidity (Nicotiana glauca) are significantlylower than the conductances in a species which does not exhibitsuch responses (Tradescantia virginiana). This difference couldat least partly account for their difference in stomatal responseto humidity. In both species, the hydraulic conductance betweenthe leaf bulk and its epidermis is much lower than the conductancein any other part of the pathway. The apparently conflictingresults, reported in recent literature, on the hydraulic conductancesand water pathways in leaves are reinterpreted, and shown tobe due to misinterpretation of results. The recently publishedcriticisms of a technique used to measure hydraulic conductivityare commented on and refuted. An examination of the factors that influence the water potentialat the sites of evaporation from the inner walls of the epidermisnear stomatal pores showed that the water potential at thesesites is lower than the bulk epidermal water potential. Thewater potential at these sites changes in a complex way as stomatalaperture changes. As it is reduced the ratio of: ‘waterpotential at sites of evaporation on the inner walls of theepidermis near stomatal pores/bulk leaf water potential‘increases. The positive feedback effect of this phenomenon,which tends to keep stomatal water potential constant as thestomata close and therefore enhances closure, and two other‘passive’ positive feedback effects on the waterpotential at sites of evaporation near stomata that have beenreported in the literature are briefly discussed. Nicotiana glauca (Grah.), Tradescantia virginiana (L.), sub-stomatal cavities, peristomatal evaporation, stomata, humidity response, leaf hydraulic conductance, water potential     

Direct turgor pressure measurements in individual leaf cells of Tradescantia virginiana

The water relations of leaves of Tradescantia virginiana were studied using the miniaturized pressure probe (Hüsken, E. Steudle, Zimmermann, 1978 Plant Physiol. 61, 158–163). Under well-watered conditions cell turgor pressures, P _o, ranged from 2 to 8 bar in epidermal cells. In subsidiary cells P _o was about 1.5 to 4.5 bar and in mesophyll cells about 2 to 3.5 bar. From the turgor pressure, relaxation induced in individual cells by changing the turgor pressure directly by means of the pressure probe, the half-time of water exchange was measured to be between 3 and 100 s for the epidermal, subsidiary, and mesophyll cells. The volumetric elastic modulus, , of individual cells was determined by changing the cell volume by a defined amount and simultaneously measuring the corresponding change in cell turgor pressure. The values for the elastic modulus for epidermal, subsidiary, and mesophyll cells are in the range of 40 to 240 bar, 30 to 200 bar, and 6 to 14 bar, respectively. Using these values, the hydraulic conductivity, L _p, for the epidermal, subsidiary, and mesophyll cells is calculated from the turgor pressure relaxation process (on the basis of the thermodynamics of irreversible processes) to be between 1 and 55·10^-7 cm s^-1 bar^-1. The data for the volumetric elastic modulus of epidermal and subsidiary cells indicate that the corresponding elastic modulus for the guard cells should be considerably lower due to the large volume changes of these cells during opening or closing. Recalculation of experimental data obtained by K. Raschke (1979, Encycl. Plant Physiol. N.S., vol. 7, pp 383–441) on epidermal strips of Vicia faba indicates that the elastic modulus of guard cells of V. faba is in the order of 40–80 bar for closed stomata. However, with increasing stomatal opening, i.e., increasing guard cell volume, decreases. Therefore, in our opinion Raschke's results would indicate a relationship between guard cell volume and which would be inverse to that for plant cells known in the literature. assumes values between 20–40 bar when the guard cell colume is soubled.     

Water Pathways in Leaves of Hedera helix L. and Tradescantia virginiana L.

Hydraulic conductances of leaf tissues of Hedera helix and Tradescantiavirginiana leaves were measured. It was found that water couldflow most easily through the veins, but that the cell wallsof at least the ventral epidermis were more efficient at resupplyingwater lost from the epidermal tissue than was the mesophyllat rehydrating itself. Vein and bundle-sheath extensions, whichare characteristic of mesomorphic leaves (e.g. T. virginiana),seem to be important in maintaining a close hydraulic connectionbetween the epidermis and the vascular tissue. In leaves notcontaining vein and bundle-sheath extensions, typically xeromorphicleaves (e.g. H. helix), there is not such a close connectionbetween the epidermis and vascular tissue. This was shown inexperiments involving the sudden application of a reduced pressurepotential to either the epidermis or the other tissues of leaves,and the measurement of transient stomatal opening.     

Nuclear localization of profilin during the cell cycle in Tradescantia virginiana stamen hair cells

Summary. The localization of the actin-monomer-binding protein profilin during the cell cycle of living Tradescantia virginiana stamen hair cells has been studied by microinjection of a fluorescently labeled analog of the protein. In contrast to previously published studies performed on chemically fixed animal cells, we do not find a specific colocalization of profilin with actin filament arrays. Our results show that, besides a general cytoplasmic distribution, profilin specifically accumulates in the nucleus in interphase and prophase cells. This nuclear localization was confirmed by means of electron microscopic immunolocalization of endogenous profilin (in Gibasis scheldiana stamen hair cells). During mitosis, as the nuclear envelope and nuclear matrix break down at the onset of prometaphase, the nuclear profilin redistributes equally into the accessible volume (cytosol) of the cell. During metaphase and anaphase no specific localization of profilin can be observed associated with the mitotic apparatus. However, during telophase, as nuclear envelopes and nuclear matrices re-form and the sister chromatids start to decondense, a subset of the microinjected profilin again localizes to the nucleus. No accumulation of profilin could be observed in the phragmoplast, where a distinct array of actin filaments exists. The function of profilin in the nucleus remains unclear.Correspondence and reprints: Department of Biology, 221 Morrill Science Center II, University of Massachusetts, Amherst, MA 01003, U.S.A.Received September 30, 2002; accepted February 12, 2003 Published online September 23, 2003     

Stomatal Responses of Tradescantia albiflora to Changing Air Humidity in Light and in Darkness

Tradescantia albiflora has green variegated and white leaves.Its stomatal apparatus consists of the guard cells and two pairsof subsidiary cells. Investigations were carried out by observingthe stomata microscopically by means of a video system in situin a CO₂ exchange chamber and by simultaneously measuring thegas exchange of the leaves. In response to air humidity changes,stomatal movements in T. albiflora begin, owing to turgor changes,in the polar and lateral subsidiary cells. The stomatal responseof green leaves to changes of air humidity showed typical transientand oscillatory phases prior to steady-state reactions. In darkness,stomata closed when air humidity decreased; however, they didnot reopen when air humidity was raised again. Stomata of illuminatedwhite leaves responded like those of green leaves in darkness.With increasing soil water stress stomata responded to changingair humidity with reductions of the transient phases and a decreasingtendency to reopen when air humidity became high again. CO₂deficiency of the air caused the stomata to open in the dark,and interacted with the air humidity effect in such a way thatstomata of green leaves responded to air humidity changes indarkness in a similar way as they did in light. Key words: Stomata, humidity response, green and white leaf areas, CO₂ deficient air     

Isolation of H+ -translocating ATPase in tonoplast of Tradescantia virginiana L. leaf cells

The tonoplast of Tradescantia virginiana L. was prepared from leaf cells and then solubilized with deoxycholate (DOC) and n-octyl-beta-D-glucoside (n-OG). Three major polypeptides (68, 60, 16 kDa) and several other minor components were isolated. These polypeptides were reconstituted in soybean phospholipids (asolectin). The H(+) pump activity was investigated with the reconstituted system as well as with the tonoplast. In both cases, the quinacrine-fluorescence quenching was observed in the presence of ATP-Mg(2+), indicating the H(+) pumping. The H(+) pump activity was inhibited by gramicidin D, a channel-forming ionophore, and by KNO(3), an inhibitor specific to tonoplast-type (V-type) H(+)-ATPase.     

Focus on Water: Polarity of Water Transport across Epidermal Cell Membranes in Tradescantia virginiana

Using the automated cell pressure probe, small and highly reproducible hydrostatic pressure clamp (PC) and pressure relaxation (PR) tests (typically, applied step change in pressure = 0.02 MPa and overall change in volume = 30 pL, respectively) were applied to individual Tradescantia virginiana epidermal cells to determine both exosmotic and endosmotic hydraulic conductivity (L_pOUT and L_pIN, respectively). Within-cell reproducibility of measured hydraulic parameters depended on the method used, with the PR method giving a lower average coefficient of variation (15.2%, 5.8%, and 19.0% for half-time, cell volume [V_o], and hydraulic conductivity [L_p], respectively) than the PC method (25.4%, 22.0%, and 24.2%, respectively). V_o as determined from PC and PR tests was 1.1 to 2.7 nL and in the range of optically estimated V_o values of 1.5 to 4.9 nL. For the same cell, V_o and L_p estimates were significantly lower (about 15% and 30%, respectively) when determined by PC compared with PR. Both methods, however, showed significantly higher L_pOUT than L_pIN (L_pOUT/L_pIN ≅ 1.20). Because these results were obtained using small and reversible hydrostatically driven flows in the same cell, the 20% outward biased polarity of water transport is most likely not due to artifacts associated with unstirred layers or to direct effects of externally applied osmotica on the membrane, as has been suggested in previous studies. The rapid reversibility of applied flow direction, particularly for the PR method, and the lack of a clear increase in L_pOUT/L_pIN over a wide range of L_p values suggest that the observed polarity is an intrinsic biophysical property of the intact membrane/protein complex.The conductivity of membranes to water (hydraulic conductivity [L_p]) is an important property of the cells of all organisms, and whether plant cell membranes exhibit a polarity in this property has been debated for a number of decades (Dainty and Hope, 1959; Steudle, 1993). Most early evidence for polarity was based on transcellular osmotic experiments using giant algal cells in the Characeae, in which the relative areas of cell membrane exposed to conditions of osmotic inflow (endosmosis) or outflow (exosmosis) could be varied and, hence, L_p for both directions determined (Tazawa and Shimmen, 2001). Interpretation of these experiments is complicated by unstirred layer (USL) effects (Dainty, 1963), but even after accounting for these, it was concluded that inflow L_p (L_pIN) was higher than outflow L_p (L_pOUT) in these cells, with L_pOUT/L_pIN of about 0.65 (Dainty, 1963). When using osmotic driving forces in algal cells, L_pOUT/L_pIN values of between 0.5 and 0.91 have been reported in many studies (Steudle and Zimmermann, 1974; Steudle and Tyerman, 1983; Tazawa et al., 1996), and the same direction of polarity was also reported using osmotic driving forces in whole roots of maize (Zea mays; Steudle et al., 1987). When applying hydrostatic driving forces in algal cells using the pressure probe (Steudle, 1993), which is less influenced by USL effects (Steudle et al., 1980), L_pOUT/L_pIN has been closer to 1 (0.83–1; Steudle and Zimmermann, 1974; Steudle and Tyerman, 1983). However, in higher plant cells, an analysis of the data presented by Steudle et al. (1980, 1982) and Tomos et al. (1981) indicates the opposite polarity, with L_pOUT/L_pIN averaging from 1.2 to 1.4. Moore and Cosgrove (1991) used two contrasting hydrostatic methods to measure L_p in sugarcane (Saccharum spp.) stem cells: (1) the most commonly used pressure relaxation (PR) method, in which cell turgor pressure (P_cell) changes during the measurement, and (2) the more technically demanding pressure clamp (PC) method, in which P_cell is maintained constant. Consistent with other studies in higher plant cells, Moore and Cosgrove (1991) reported average L_pOUT/L_pIN from 1.15 (PC) to 1.65 (PR). Using the PR method in epidermal cells of barley (Hordeum vulgare), Fricke (2000) reported only a modest L_pOUT/L_pIN (based on reported half-time [T_1/2]) of 1.08. In view of the contribution of proteins (e.g. aquaporins) to overall membrane L_p, Tyerman et al. (2002) suggested that polarity may result either from asymmetry in the pores themselves or from an active regulation of the conductive state of the pores in response to the experimental conditions that cause inflow or outflow. Either of these mechanisms may explain the wide range of values reported in the literature for L_pOUT/L_pIN. Cosgrove and Steudle (1981) reported that a substantial (6-fold) and rapid (within 20 s) reduction in L_p could occur in the same cell, and in hindsight, this presumably reflected the influence of aquaporins. Cosgrove and Steudle (1981) did not consider the lower L_p as indicative of the L_p in situ, and Wan et al. (2004) reported that a reduction in L_p was associated with perturbations to P_cell on the order of 0.1 MPa. Hence, if measured membrane L_p itself can exhibit substantial changes over relatively short periods of time in the same cell, then further study of systematic differences between L_pOUT and L_pIN will require a robust hydrostatic methodology (PC or PR) that can reversibly and reproducibly apply small perturbations in pressure (P) to individual cells over short periods of time.For the PR method, a T_1/2 of water exchange is measured by fitting an exponential curve to the observed decay in P_cell over time following a step change in volume, and membrane L_p can be calculated if cell surface area (A), cell volume (V_o), and volumetric elastic modulus (ε) are known (Steudle, 1993). In practice, A and V_o are typically calculated from optical measurements of individual cell dimensions or estimates using average values, and ε is calculated based on V_o and an empirical change in pressure (dP) to change in volume (dV) relation for each cell (Steudle, 1993; Tomos and Leigh, 1999). In the PC method, first developed by Wendler and Zimmermann (1982), V_o (and, given reasonable assumptions about cell geometry, A) is estimated without the need for optical measurements, and L_p can be measured without the need to determine dP/dV or ε. However, this method is technically more demanding because it requires precise P control as well as a continuous record of the volume flow of water across the cell membrane (as measured by changes in the position of the cell solution/oil meniscus within the glass capillary over time) and has rarely been used (Wendler and Zimmermann, 1982, 1985; Cosgrove et al., 1987; Moore and Cosgrove, 1991; Zhang and Tyerman, 1991; Murphy and Smith, 1998). Since volume (V) is continuously changing over time, this approach may also be influenced by the hydraulic conductance of the capillary tip (K_h) used to make the measurements as well as surface tension effects due to the progressive changes in capillary diameter with meniscus position, and these influences have not been quantitatively addressed.Automation of the pressure probe operation, particularly automatic tracking of the meniscus location in the glass microcapillary tip, would address many of the above-mentioned issues, and to date, several attempts have been made to monitor the meniscus location using electrical resistance (Hüsken et al., 1978) or hardware-based image analysis (Cosgrove and Durachko, 1986; Murphy and Smith, 1998). Recently, Wong et al. (2009) redesigned the automated cell pressure probe (ACPP), originally proposed by Cosgrove and Durachko (1986), using a software-based meniscus detection system and a precise pressure control system. In the new ACPP system, both the position of the meniscus and oil pressure (P_oil) are recorded frequently (typically at 10 Hz), and P_oil is controlled with a resolution of ±0.002 MPa. We have combined the ACPP with a new technique to reproducibly fabricate microcapillary tips of known hydraulic properties (Wada et al., 2011) in order to correct for K_h and surface tension effects in both PC and PR estimates of the water relations parameters of Tradescantia virginiana epidermal cells and have determined the relation of L_pOUT to L_pIN in these cells.     

Dynamics of adaptation of stomatal behaviour to moderate or high relative air humidity in Tradescantia virginiana

Chlorophyll fluorescence imaging was used to measure stomatalclosure in response to desiccation of Tradescantia virginianaleaves grown under high (90%) and moderate (55%) relative humidities(RHs), or transferred between these humidities. Stomata in leavesgrown at high RH were less responsive to desiccation than thoseof leaves grown at moderate RH. Stomata of plants transferredfrom moderate RH conditions to high RH showed the same diminishedclosure in response to desiccation as did stomata that developedat high RH. This response was found both when the leaves werefully expanded and when still actively expanding during themoderate RH pre-treatment. Four days of exposure to high RHwas the minimal exposure time to induce the diminished closureresponse. When leaves were grown in high RH prior to a 10 dmoderate RH treatment, the reduced stomatal closure responseto desiccation was only reversed in leaves (regions) which wereactively expanding during moderate RH treatment. This indicatesthat with respect to stomatal responses to desiccation, highRH leaf regions have a limited capacity to adapt to moderateRH conditions. The decrease in responsiveness to desiccationof the stomata, induced by long-term exposure to high RH, wasnot due to osmotic adjustment in the leaves. Within 1 d aftertransferring moderate RH-grown plants to a high RH, the abscisicacid (ABA) concentration of their leaves decreased to the lowlevel of ABA found in high RH-grown leaves. The closure responsein leaves exposed to high RH for 5 d, however, could not befully restored by the application of ABA. Transferring plantsfrom high to moderate RH resulted in increased ABA levels within2 d without a recovery of the stomatal closing response. Itis discussed that the diminished stomatal closure in plantsexposed to high RH could be due to changes in the signallingpathway for ABA-related closure of stomata or to an increasedsequestration of ABA by mesophyll tissue or the symplast inthe epidermis, induced by a longer period (several days) ofa low ABA level. Key words: Abscisic acid, desiccation, PSII efficiency, relative water content, stomatal closure, vapour pressure deficit, water potential Received 8 October 2007; Revised 5 November 2007 Accepted 9 November 2007     

The Structure and Orientation of Guard Cells in Plants Showing Stomatal Responses to Changing Vapour Pressure Difference

Transmission electron micrographs revealed that a substantialpart of the guard cell wall of both Quercus robur L. and Populusnigra ‘italica’ L. was either free of cuticle orcovered with a greatly reduced cuticular layer. In Quercus thestructure of the guard cell was such that the area of limitedcuticular development would be exposed to the evaporating powerof the atmosphere even when the stomata were closed. Lanthanumstaining confirmed that this area might be an important siteof evaporation. A similar evaporation site was identified inthe guard cell wall of Pinus sylvestris L. Light micrographsrevealed that this area could also be exposed on the outsideof the leaf when the stomata were closed. It appears that guardcell orientation with respect to the epidermal plane dependsupon epidermal turgor. Changes in orientation of the guard cellcoupled with the exact location of the cuticle-free area inthe guard cell wall may explain the nature of the stomatal responseof individual species to changing VPD and the effect of othervariables, e.g. water deficit, on this response. Quercus robur L, oak, Populus nigra L, poplar, stomata, guard cells, cuticle, evaporation, vapour pressure difference     

Solute Transport Process in Intestinal Epithelial Cells

In rat small intestine, the active transport of organic solutes results in significant depolarization of the membrane potential measured in an epithelial cell with respect to a grounded mucosal solution and in an increase in the transepithelial potential difference. According to the analysis with an equivalent circuit model for the epithelium, the changes in emf's of mucosal and serosal membranes induced by active solute transport were calculated using the measured conductive parameters. The result indicates that the mucosal cell membrane depolarizes while the serosal cell membrane remarkably hyperpolarizes on the active solute transport. Corresponding results are derived from the calculations of emf's in a variety of intestines, using the data that have hitherto been reported. The hyperpolarization of serosal membrane induced by the active solute transport might be ascribed to activation of the serosal electrogenic sodium pump. In an attempt to determine the causative factors in mucosal membrane depolarization during active solute transport, cell water contents and ion concentrations were measured. The cell water content remarkably increased and, at the same time, intracellular monovalent ion concentrations significantly decreased with glucose transport. Net gain of glucose within the cell was estimated from the restraint of osmotic balance between intracellular and extracellular fluids. In contrast to the apparent decreases in intracellular Na⁺ and K⁺ concentrations, significant gains of Na⁺ and K⁺ occurred with glucose transport. The quantitative relationships among net gains of Na⁺, K⁺ and glucose during active glucose transport suggest that the coupling ratio between glucose and Na⁺ entry by the carrier mechanism on the mucosal membrane is approximately 1:1 and the coupling ratio between Na⁺-efflux and K⁺-influx of the serosal electrogenic sodium pump is approximately 4:3 in rat small intestine. In addition to the electrogenic ternary complex inflow across the mucosal cell membrane, the decreases in intracellular monovalent ion concentrations, the temporary formation of an osmotic pressure gradient across the cell membrane and the streaming potential induced by water inflow through negatively charged pores of the cell membrane in the course of an active solute transport in intestinal epithelial cells are apparently all possible causes of mucosal membrane depolarization.     

Control of Volume and Turgor in Stomatal Guard Cells

Water loss from plants is determined by the aperture of stomatal pores in the leaf epidermis, set by the level of vacuolar accumulation of potassium salt, and hence volume and turgor, of a pair of guard cells. Regulation of ion fluxes across the tonoplast, the key to regulation of stomatal aperture, can only be studied by tracer flux measurements. There are two transport systems in the tonoplast. The first is a Ca²⁺-activated channel, inhibited by phenylarsine oxide (PAO), responsible for the release of vacuolar K⁺(Rb⁺) in response to the “drought” hormone, abscisic acid (ABA). This channel is sensitive to pressure, down-regulated at low turgor and up-regulated at high turgor, providing a system for turgor regulation. ABA induces a transient stimulation of vacuolar ion efflux, during which the flux tracks the ion content (volume, turgor), suggesting ABA reduces the set-point of a control system. The second system, which is PAO-insensitive, is responsible for an ion flux from vacuole to cytoplasm associated with inward water flow following a hypo-osmotic transfer. It is suggested that this involves an aquaporin as sensor, and perhaps also as responder; deformation of the aquaporin may render it ion-permeable, or, alternatively, the deformed aquaporin may signal to an associated ion channel, activating it. Treatment with inhibitors of aquaporins, HgCl₂ or silver sulfadiazine, produces a large transient increase in ion release from the vacuole, also PAO-insensitive. It is suggested that this involves the same aquaporin, either rendered directly ion-permeable, or signalling to activate an associated ion channel.     

Control of Ion Fluxes in Stomatal Guard Cells

Opening and closing of the stomatal pore is associated with very large changes in K-salt accumulation in stomatal guard cells. This review discusses the ionic relations of guard cells in relation to the general pattern of transport processes in plant cells, in plasmalemma and tonoplast, involving primary active transport of protons, proton-linked secondary active transport, and a number of gated ion channels. The evidence available suggests that the initiation of stomatal opening is regulated through the uptake mechanisms, whereas initiation of stomatal closing is regulated by control of ion efflux at the plasmalemma, and of fluxes to and from the vacuole. In response to a closing signal there are large transient increases in efflux of both Cl^? (or Br^?) and Rb⁺ (K⁺) at the plasmalemma, with also a probable increase in anion flux from vacuole to cytoplasm and decrease in anion flux from cytoplasm to vacuole. A speculative hypothetical sequence of events is discussed, by which the primary response to a closing signal is an increase in Ca²⁺ influx at the plasmalemma, producing depolarisation and increase in cytoplasmic Ca²⁺. The consequent opening of Ca²⁺-sensitive Cl^? channels, and voltage-sensitive K⁺ channels (also Ca²⁺-sensitive?) in the plasmalemma, and of a Ca²⁺-sensitive nonspecific channel in the tonoplast, could produce the flux effects identified by the tracer work; this speculation is also consistent with the Ca²⁺-sensitivity of the response to closing signals and with evidence from patch clamping that such channels exist in at least some plant cells, though not yet all shown in guard cells.     

Stomatal Responses to Humidity and the Water Potentials of Epidermal and Mesophyll Tissue

Effects of ambient humidity on relative water contents (RWC)and water potentials were measured separately for epidermaland mesophyll tissues in leaves of two species. Water potentialsdid not always change in the same direction as RWC within thesame tissue and rarely were the changes in water potential andRWC of one tissue correlated with those of the other. The natureof the humidity responses of stomata in certain species is discussedin relation to these results and to the anatomical propertiesof epidermal tissues.