Apoplastic pH of intact leaves of Vicia faba as influenced by light

The fluorochrome FITC-dextran was used to measure the effectof light on the apoplastic pH of intact Vicia faba leaves withthe ratio imaging technique. In darkadapted leaves the apoplasticpH varied depending on the leaf between 5.2 and 5.9. Red light(660 nm, 4–12 W m^–2) leads to multiphasic responses:in the first seconds an alkalinization ({small tilde}0.3 pHunits), and thereafter an acidification of the leaf apoplast({small tilde}0.4 pH units) were observed. Both effects couldbe inhibited by DCMU. While variation of CO₂ concentration revealedno effect on light-induced apoplastic pH changes, a decreasein O₂ concentration decreased the effect. On the basis of ourdata it is suggested that the influence of photosynthesis onplasmalemma H⁺ ATPase is responsible for the observed effects,rather than altered CO₂ uptake. Key words: Leaf apoplast, apoplastic pH, light, ratio imaging, pH-sensitive fluorescent dye, Vicia fab     

The membrane potential of Arabidopsis thaliana guard cells; depolarizations induced by apoplastic acidification

The apoplastic pH of guard cells probably acidifies in response to light, since light induces proton extrusion by both guard cells and epidermal leaf cells. From the data presented here, it is concluded that these apoplastic pH changes will affect K⁺ fluxes in guard cells of Arabidopsis thaliana (L.) Heynh. Guard cells of this species were impaled with double-barrelled microelectrodes, to measure the membrane potential (E_m) and the plasma-membrane conductance. Guard cells were found to exhibit two states with respect to their E_m, a depolarized and a hyperpolarized state. Apoplastic acidification depolarized E_m in both states, though the origin of the depolarization differed for each state. In the depolarized state, the change in E_m was the result of a combined pH effect on instantaneously activating conductances and on the slow outward rectifying K⁺ channel (s-ORC). At a more acidic apoplastic pH, the current through instantaneously activated conductances became more inwardly directed, while the maximum conductance of s-ORC decreased. The effect on s-ORC was accompanied by an acceleration of activation and deactivation of the channel. Experiments with acid loading of guard cells indicated that the effect on s-ORC was due to a lowered intracellular pH, caused by apoplastic acidification. In the hyperpolarized state, the pH-induced depolarization was due to a direct effect of the apoplastic pH on the inward rectifying K⁺ channel. Acidification shifted the threshold potential of the channel to more positive values. This effect was accompanied by a decrease in activation times and an increase of deactivation times, of the channel. From the changes in E_m and membrane conductance, the expected effect of acidification on K⁺ fluxes was calculated. It was concluded that apoplastic acidification will increase the K⁺-efflux in the depolarized state and reduce the K⁺-influx in the hyperpolarized state. Received: 28 April 1997 / Accepted: 10 November 1997     

Iron availability in plant tissues-iron chlorosis on calcareous soils

The article describes factors and processes which lead to Fe chlorosis (lime chlorosis) in plants grown on calcareous soils. Such soils may contain high HCO₃ ^- concentrations in their soil solution, they are characterized by a high pH, and they rather tend to accumulate nitrate than ammonium because due to the high pH level ammonium nitrogen is rapidly nitrified and/or even may escape in form of volatile NH₃. Hence in these soils plant roots may be exposed to high nitrate and high bicarbonate concentrations. Both anion species are involved in the induction of Fe chlorosis.Physiological processes involved in Fe chlorosis occur in the roots and in the leaves. Even on calcareous soils and even in plants with chlorosis the Fe concentration in the roots is several times higher than the Fe concentration in the leaves. This shows that the Fe availability in the soil is not the critical process leading to chlorosis but rather the Fe uptake from the root apoplast into the cytosol of root cells. This situation applies to dicots as well as to monocots. Iron transport across the plasmamembrane is initiated by Fe^III reduction brought about by a plasmalemma located Fe^III reductase. Its activity is pH dependent and at alkaline pH supposed to be much depressed. Bicarbonate present in the root apoplast will neutralize the protons pumped out of the cytosol and together with nitrate which is taken up by a H⁺/nitrate cotransport high pH levels are provided which hamper or even block the Fe^III reduction.Frequently chlorotic leaves have higher Fe concentrations than green ones which phenomenon shows that chlorosis on calcareous soils is not only related to Fe uptake by roots and Fe translocation from the roots to the upper plant parts but also dependent on the efficiency of Fe in the leaves. It is hypothesized that also in the leaves Fe^III reduction and Fe uptake from the apoplast into the cytosol is affected by nitrate and bicarbonate in an analogous way as this is the case in the roots. This assumption was confirmed by the highly significant negative correlation between the leaf apoplast pH and the degree of iron chlorosis measured as leaf chlorophyll concentration. Depressing leaf apoplast pH by simply spraying chlorotic leaves with an acid led to a regreening of the leaves.     

Effects of white,blue, red light and darkness on pH of the apoplast in the Samanea pulvinus

Leaflet movements in Samanea saman (Jacq.) Merrill are driven by fluxes of K⁺, anions, and water through membranes of motor cells in the pulvinus (R.L. Satter et al., 1974, J. Gen. Physiol. 64, 413–430). Extensor cells take up K⁺ and swell in white light (WL) while flexor cells take up K⁺ and swell in darkness (D). Excised strips of extensor and flexor motor tissue acidify their bathing medium under conditions that normally promote increase in K⁺ in the intact tissue, and alkalize the medium under conditions that normally induce decrease in K⁺ (A. Iglesias and R.L. Satter, 1983, Plant Physiol. 72, 564). To obtain information on pH changes in the whole pulvinus, we measured effects of light on pH of the apoplast, using liquid membrane microelectrodes sensitive to H⁺. We report the following: (1) The pH of the extensor apoplast was higher than that of the flexor apoplast in WL and in D (pH gradient of 1.0 units in WL and 2.0 units in D). Apoplastic pH might affect K⁺ transport through the plasma membranes of Samanea motor cells, since the conductance, gating, and selectivity of ionic channels in other systems depend upon external pH. (2) Extensor cells acidified and flexor cells alkalized their environment in response to irradiation with WL, while the reverse changes occurred in response to D. These results are consistent with the results of Iglesias and Satter (1983), and support the physiological relevance of data obtained with excised tissue. (3) The pH changes in response to irradiation with red light were similar to those obtained with D; also, the pH changes in response to blue light were similar to those obtained with WL. The pulvinus closed in red light as in darkness and opened in WL, but failed to open in blue light. The advantages and limitations of apoplastic pH measurements for assaying H⁺ transport are discussed.Abbreviations BL blue light - D darkness - RL red light - WL white light     

Cell walls as reservoirs of potassium ions for reversible volume changes of pulvinar motor cells during rhythmic leaf movements

The laminar pulvinus of primary leaves of Phaseolus coccineus L. was investigated with respect to the total K⁺ content, the apoplastic K⁺ content, and the water potential of extensor and flexor sections in relation to the leaf positions in a circadian leaf-movement cycle, as well as the cation-exchange properties of isolated extensor- and flexor-cell walls. Turgid tissue showed a high total but low apoplastic K⁺ content, shrunken tissue a low total but high apoplastic K⁺ content. Thus, part of the K⁺ transported into and out of the swelling or shrinking protoplasts is shuttled between the protoplasts and the surrounding walls, another part between different regions of the pulvinus. The K⁺ fraction shuttled between protoplasts and walls was found to be 30–40% of the total transported K⁺ fraction. Furthermore, 15–20% of the total K⁺ content of the tissue is located in the apoplast when the apoplastic reservoir is filled, 5–10% when the apoplastic reservoir is depleted. The ion-exchange properties of walls of extensor and flexor cells appear identical in situ and in isolated preparations. The walls behave as cation exchangers of hhe weak-acid type with a strong dependence of the activity of fixed negative charges as well as of the K⁺-storing capacity on pH and [K⁺] of the equilibration solution. The high apoplastic K⁺ contents of freshly cut tissues reflect the cation-storing capacity of the isolated walls. We suggest that K⁺ ions of the Donnan free space are used for the reversible volume changes (mediating the leaf movement) mainly by an electrogenic proton pump which changes the pH and-or the [K⁺] in the water free space of the apoplast.Abbreviations and symbols DFS Donnan free space - DW dry weight - pK negative logarithm of the equilibrium constant K of the acidic group - WFS water free space - water potential; Indices - cw cell wall - t tissue     

Abscisic Acid Movement into the Apoplastic solution of Water-Stressed Cotton Leaves: Role of Apoplastic pH

Leaves of cotton (Gossypium hirsutum L.) were subjected to overpressures in a pressure chamber, and the exuded sap was collected and analyzed. The exudate contained low concentrations of solutes that were abundant in total leaf extracts, and photosynthetic rates and stomatal conductance were completely unaffected by a cycle of pressurization and rehydration. These criteria and others indicate that the experimental techniques inflicted no damage upon the leaf cells. The pH and abscisic acid (ABA) content of the apoplastic fluid both increased greatly with pressure-induced dehydration. Although ABA concentrations did not reach a steady state, the peak levels were above 1 micromolar, an order of magnitude greater than bulk ABA concentrations of the leaf blades. Treatment of leaves with fusicoccin decreased the K⁺ concentration, greatly reduced the pH rise, and completely eliminated the increase in ABA in the apoplast upon dehydration. When water-stressed leaves were pressurized, the pH of the exuded sap was increased by 0.2 units per 1 megapascal decrease in initial leaf water potential. Buffer capacity of the sap was least in the pH range of interest (6.5-7.5), allowing extremely small changes in H⁺ fluxes to create large changes in apoplastic pH. The data indicate that dehydration causes large changes in apoplastic pH, perhaps by effects on ATPases; the altered pH then enhances the release of ABA from mesophyll cells into the apoplastic fluid.     

Potassium Transport and Regulation of Intracellular pH in Elodea densa Leaves

The effects of extracellular K⁺ concentration ([K⁺]_o) on the pH of cell sap, “bulk cytoplasm” and vacuole have been investigated in Elodea densa leaves under conditions of either low or high activity of the plasmalemma electrogenic H⁺ pump. Cell sap pH was evaluated directly in the cell sap expressed after freezing and thawing. Cytoplasmic and vacuolar pH were calculated by the weak base and weak acid distribution method, DMO and benzylamine appearing to be a suitable acid and base, respectively, for this purpose in this material. When added to the basal medium (no rapidly permeating ions present), 5 mM K⁺ induced an increase in intracellular pH, larger for the cell sap and the vacuole (about 0.2 units), and smaller but still significant for the cytoplasm (0.07 units). This alkalinizing effect of K⁺ was thus associated with a significant decrease in the pH difference across the tonoplast. The alkalinizing effect of K⁺ was markedly and synergistically enhanced by the presence of fusicoccin, a condition inducing a marked activation of H⁺ extrusion and of K⁺ uptake. The correlation between these effects of [K⁺]_o on intracellular pH and those on H⁺ extrusion indicates that changes in extracellular K⁺ concentration, and thus in K⁺ influx, can influence cytoplasmic and vacuolar pH by modulating the rate of H⁺ extrusion by the plasmalemma H⁺ pump.     

PHOTOCONTROL OF STOMATAL MOVEMENTS

1. Opening in light is a feature common to the majority of functional stomata, but the current argument is against the traditional view that light is the principal environmental promoter of opening, because stomata can open in the dark in response to CO₂ removal and/or temperature increase. In this review, evidence is provided that light is more efficient and effective than other physical factors in both producing and maintaining wide opening. However, light acts on stomata both directly and indirectly, in conjunction with changes in, for example, CO₂ balance, water regime and temperature of the leaf tissue. 2. Three general categories of light effects on stomata are recognized: (a) photosynthetic effects driven by metabolic processes, induced or enhanced by light, (b) hydrophotic effects mediating through light-induced changes in epidermal turgor, and (c) photothermal effects arising from light-dependent changes in leaf temperature. 3. Photosynthetic effects involve both CO₂ depletion, and starch mobilization, malate synthesis, H⁺ extrusion, and accumulation of K⁺ and C1^- in guard cells; these processes are triggered by light of different qualities: (a) Both blue and red light are involved in photosynthetic CO₂ fixation, utilizing energy from photosynthetic light reaction(s), which provides C precursors for synthesis of stornatal starch. (b) Blue light, but not red, enhances starch mobilization, PEP carboxylase activity and respiration. Accordingly, blue light is postulated to enhance hydrolysis of stornatal starch providing C₃ precursors for malate synthesis via PEP-fixation of endogenous CO₂; the active extrusion of H⁺, derived from malate, is coupled with K⁺ influx to guard cells. Malate and C1^- are competitive anions, for K⁺, and one begins to play a progressively more important role as the other becomes limiting; in intact leaves, however, malate plays a more decisive role. These processes are driven by the energy from blue-light-enhanced respiration. (c) Both photosynthetic fixation and PEP carboxylation act as CO₂ sensors, but the exact role of CO₂ in the stornatal mechanism has yet to be determined. 4. Hydrophotic and photothermal effects facilitate guard cell expansion by releasing epidermal pressure through enhanced evaporative water loss, and are, therefore, indirect effects of light; photothermal effects may also contribute to metabolic processes outlined in paragraph 3. 5. Stomatal closure in the dark accompanies starch synthesis, malate reduction, efflux of K⁺ and C1^- from guard cells, and accumulation of CO₂ in substomatal cavities. Malate may be converted to starch via C₂ compounds. Guard cells release K⁺ and C1- into apoplastic space, from which they are removed by neighbouring cells. The entry of K⁺ into neighbouring cells is supposed to be coupled with H⁺ extrusion. These processes are dependent on respiratory energy. 6. The differential abaxial and adaxial stomatal light responses are related to inherent metabolic differences between the two epidermes, but the biochemical basis is not known.     

Changes of the Apoplastic pH and K+ Concentration in the Phaseolus Pulvinus in situ in Relation to Rhythmic Leaf Movements

The apoplastic pH and K⁺ concentration of the extensor of thePhaseolus primary-leaf pulvinus in relation to rhythmic leafmovements have been investigated with double-barrelled ion-sensitivemicro-electrodes. Simultaneous measurements of leaf movementand ion activities in a fine hole of the extensor in situ showedco-existence of ultradian and circadian leaf movements as wellas of ultradian and circadian pH changes in the Water Free Space(WFS) of the extensor apoplast in situ. During circadian leafmovement the H⁺ and K⁺ activities in the WFS of the extensorchange in an antagonistic manner. When extensor cells swell(upward movement of the lamina) the H⁺ activity increases fromapproximately pH 6.7 to 5.9 and the K⁺ concentration decreasesfrom approximately 50 to 10 mol m^–3 and vice versa whenextensor cells shrink. These changes in the ionic activitiesin the WFS must be correlated with large changes in the ioncontent of the DFS and thus support the hypothesis that thecell walls of pulvinar cells serve as reservoirs for K⁺ andH⁺. Key words: Phaseolus pulvinus, apoplastic ionic activities, rhythmic leaf movements, ion-sensitive micro-electrodes (double-barrelled)     

Role of apoplast acidification by the H+ pump

We have studied the mechanism of the response to iron deficiency in rape (Brassica napus L.), taking into account our previous results: net H⁺ extrusion maintains a pH shift between the root apoplast and the solution, and the magnitude of the pH shift decreases as the buffering power in the solution increases. The ferric stress increased the ability of roots to reduce Fe[III]EDTA. Buffering the bulk solution (without change in pH) inhibited Fe[III]EDTA reduction. At constant bulk pH, the inhibition (ratio of the Fe[III]EDTA-reduction rates measured in the presence and in the absence of buffer) increased with the rate of H⁺ extrusion (modulated by the length of a pretreatment in 0.2 mM CaSO₄). These results support the hypothesis that the apoplastic pH shift caused by H⁺ excretion stimulated Fe[III] reduction. The shape of the curves describing the pH-dependency of Fe[III]EDTA reduction in the presence and in the absence of a buffer fitted this hypothesis. When compared to the titration curves of Fe[III]citrate and of Fe[III]EDTA, the curves describing the dependency of the reduction rate of these chelates on pH indicated that the stimulation of Fe[III] reduction by the apoplastic pH shift due to H⁺ excretion could result from changes in electrostatic interactions between the chelates and the fixed chargers of the cell wall and-or plasmalemma. Blocking H⁺ excretion by vanadate resulted in complete inhibiton of Fe[III] reduction, even in an acidic medium in which there was neither a pH shift nor an inhibitory effect of a buffer. This indicates that the apoplastic pH shift resulting from H⁺ pumping is not the only mechanism which is involved in the coupling of Fe[III] reduction to H⁺ transport. Our results shed light on the way by which the strong buffering effect of HCO ₃ ^- in some soils may be involved in iron deficiency encountered by some of the plants which grow in them.     

Determination of apoplastic K+ in intact leaves by ratio imaging of PBFI fluorescence

The tetraammonium salt of the K⁺ binding fluorescent dye benzofuranisophthalate (PBFI) was used to investigate the influence ofpotassium nutrition (0.1–2.1 mol m^–3) on apoplasticK⁺ inVicia faba leaves by means of ratio imaging. As a referencethe infiltration-centrifugation method was used. Both methodsreflected the influence of K⁺ supply on apoplastic K⁺ concentration.The abaxial leaf side revealed significantly higher K⁺ concentrations(20-25 mol m^–3) than the adaxial side (5–8 mol m^–3).Application of CCCP led to an immediate increase in apoplasticK⁺ demonstrating the reliability of the PBFI method. Key words: Vicia faba, leaf, apoplast, K⁺, PBFI, ratio imaging, ratiometric fluorescence microscopy     

Physiological regulation of plant-atmosphere ammonia exchange

Plants have a compensation point for NH₃ which ranges from 0.1 to 20 nmol mol^-1, and may be several-fold higher or lower than naturally occurring atmospheric NH₃ concentrations. This implies that NH₃ fluxes over vegetated surfaces are bi-directional and that ammonia exchange with the atmosphere in many cases contributes significantly to the nitrogen economy of vegetation. Physiological regulation of plant–atmosphere NH₃ fluxes is mediated via processes involved in nitrogen uptake, transport and metabolism. A rapid turnover of NH₃ ⁺ in plant leaves leads to the establishment of a finite NH₃ ⁺ concentration in the leaf apoplastic solution. This concentration determines, together with that of H⁺, the size of the NH₃ compensation point. Barley and oilseed rape plants with access to NH₃ ⁺ in the root medium have higher apoplastic NH₃ ⁺ concentrations than plants absorbing NO₃ ^-. Furthermore, the apoplastic NH₃ ⁺ concentration increases with the external NH₃ ⁺ concentration. Inhibition of GS leads to a rapid and substantial increase in apoplastic NH₃ ⁺ and barley mutants with reduced GS activity have higher apoplastic NH₃ ⁺ than wild-type plants. Increasing rates of photorespiration do not affect the steady-state NH₃ ⁺ or H⁺ concentration in tissue or apoplast of oilseed rape, indicating that the NH₃ ⁺ produced is assimilated efficiently. Nevertheless, NH₃ emission increases due to a temperature-mediated displacement of the chemical equilibrium between gaseous and aqueous NH₃ in the apoplast. Sugarbeet plants grown with NO₃ ^- seem to be temporarily C-limited in the light due to a repression of respiration. As a consequence, the activity of chloroplastic GS declines during the day causing a major part of NH₃ ⁺ liberated in photorespiration to be assimilated during darkness when 2-oxoglutarate is supplied in high rates by respiration. This revised version was published online in June 2006 with corrections to the Cover Date.     

Rapid apoplastic pH measurement in Arabidopsis leaves using a fluorescent dye

In plants, the extracellular space (apoplast) is one of the main places where exchange of molecules occurs between cells. Not only is this compartment involved in the storage of multiple metabolites and ions, including calcium and protons, but it also plays a role in the transmission of signaling molecules for cell-to-cell communication. It has recently been shown multiple times that these two aspects are linked and can influence each other. In particular, apoplast pH was shown as a primary regulator of auxin (IAA) transport in Arabidopsis thaliana. To prove the role of apoplastic pH, we have developed a protocol for apoplastic fluid extraction from Arabidopsis leaves, followed by pH determination using the 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) fluorescent dye. This technique successfully allows one to monitor apoplastic pH variations among different plant lines and to link changes in apoplastic pH to cellular responses in the plant.     

The effect of light on membrane potential, apoplastic pH and cell expansion in leaves of Pisum sativum L. var. Argenteum.

The connection between three light responses of green leaf cells-membrane potential (V_m), H⁺ net efflux and growth, was analyzed. Illumination of mesophyll cells in leaves from Argenteum peas caused two rapid responses: (i) a de- and repolarization of V_m and (ii) an alkalinization of the apoplast. The rapid responses were completely eliminated by the photosynthetic inhibitor 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) but not affected by ortho-vanadate, an inhibitor of the plasma membrane (PM) H⁺-ATPase. The rapid changes were followed by a set of delayed responses: (i) a slow, gradual hyperpolarization of V_m, (ii) a gradual acidification of the mesophyll apoplast and (iii) an increased rate of elongation. These three light responses persisted under DCMU but were completely eliminated by vanadate. The data show that the delayed (in contrast to the rapid) responses were due to a stimulation of PM H⁺ pumps which occurred independently of non-cyclic photosynthetic electron transport and the “dark” processes depending on it. When the rapid responses were blocked by DCMU, light-induced acidification, hyperpolarization of the membrane potential and growth proceeded simultaneously. A shared (4-min) lag phase indicated slower signal processing in mesophyll than in epidermal cells where light stimulation of PM H⁺ pumps was rapid. Received: 3 September 1998 / Accepted: 15 October 1998     

Light-Induced Polar pH Changes in Leaves of Elodea canadensis: II. Effects of Ferricyanide: Evidence for Modulation by the Redox State of the Cytoplasm

The effect of an extracellular electron acceptor, ferricyanide, on the light-induced polar leaf pH changes of the submerged angiosperm Elodea canadensis in light and in darkness was determined. The rate of transmembrane ferricyanide reduction was stimulated by increased light intensity and was inhibited by inorganic carbon, indicating that changes in the redox state of the chloroplast were reflected at the plasma membrane. The addition of ferricyanide inhibited the light-induced polar leaf pH reaction. This effect could be balanced by increasing the light intensity. In the dark, the acidification induced by ferricyanide was not influenced by diethylstilbestrol at concentrations that completely inhibited the polar leaf pH changes. This indicates that the ferricyanide-induced H⁺ extrusion and the H⁺ transport during the polar reaction were mediated by different mechanisms.     

Apoplast Acidification in Growing Barley (Hordeum vulgare L.) Leaves

Apoplast acidification associated with growth is well documented in roots, coleoptiles, and internodes but not in leaves. In the present study, advantage was taken of the high cuticle permeability in the elongation zone of barley leaves to measure apoplast pH and growth in response to application of test reagents. The role of the plasma membrane H⁺-ATPase (PM-H⁺-ATPase) and K⁺ in this process was of particular interest. pH microelectrodes and an in vitro gel system with bromocresol purple as pH indicator were used to monitor apoplast pH. Growth was measured in parallel or in separate experiments using a linear variable differential transformer. Test reagents that blocked (vanadate) or stimulated (fusicoccin) PM-H⁺-ATPase or that reduced (Cs⁺, tetraethylammonium) K⁺ uptake were applied. Apoplast pH was lower in growing than in nongrowing leaf tissue and increased in the elongation zone with increasing apoplast K⁺. Vanadate increased apoplast pH and reduced growth, whereas fusicoccin caused the opposite effects. It is concluded that barley leaves exhibit acid-growth-type mechanisms in that apoplast pH is lower in elongating leaf tissue. Both growth and apoplast pH depend on the activity of the PM-H⁺-ATPase and K⁺ transport processes. However, not all of the growth displayed by leaves is dependent on a lower apoplast pH in the elongation zone; up to 50 % of growth is retained when apoplast pH in the elongation zone increases to a value observed in mature tissue.     

Close coupling between extrusion of H+ and uptake of K+ by barley roots

Extrusion of H⁺ by intact barley (Hordeum vulgare L.) roots was automatically titrated. Simultaneously, uptake of K⁺ into the roots, transport of K⁺ through the roots, and (as a residual term) accumulation of K⁺ within the root tissue were determined. When no monovalent cation was present in the medium the steady rate of H⁺ release was close to zero. Addition of K⁺ stimulated H⁺ extrusion within less than 1 min. The stimulation of H⁺ release was apparently limited only by the movement of K⁺ through the apoplast of the roots. The steady rate of H⁺ extrusion depended on the availability of external K⁺ and saturated at a K⁺ concentration of about 100 mol· dm^-3. Half-maximum rates of net K⁺ uptake and H⁺ extrusion were reached at a K⁺ concentration of about 10 mol·dm^-3. With (slowly absorbable) sulfate as the only anion present, the stoichoimetry between H⁺ release and net K⁺ uptake was one. In conclusion, the uptake of K⁺ across the plasmalemma of the cells of the root cortex is electrically coupled to H⁺ extrusion.     

Energized Uptake of Sugars from the Apoplast of Leaves: A Study of Some Plants Possessing Different Minor Vein Anatomy

Solutions of sucrose, glucose, raffinose, and stachyose were fed via the petiole to detached leaves of plant species known to transfer sugars during photosynthesis into the phloem using either the apoplastic or the symplastic pathway of phloem loading. Symplastic phloem loaders, which translocate raffinose-type oligosaccharides and sucrose in the phloem, and apoplastic plants, translocating exclusively sucrose, were selected for this study. As the sugars arrived with the transpiration stream in the leaf blade within little more than a minute, dark respiration increased. Almost simultaneously, fluorescence of a potential-indicating dye, which had been infiltrated into the leaves, indicated membrane depolarization. Another fluorescent dye used to record the apoplastic pH revealed apoplastic alkalinization that occurred with a slight lag phase after respiration and membrane depolarization responses. Occasionally, alkalinization was preceded by transient apoplastic acidification. Whereas membrane depolarization and apoplastic acidification are interpreted as initial responses of the proton motive force across the plasma membrane to the advent of sugars in the leaf apoplast, the following apoplastic alkalinization showed that sugars were taken up from the apoplast into the symplast in cotransport with protons. This was true not only for glucose and sucrose, but also for raffinose and stachyose. Similar observations were made for sugar uptake not only in leaves of plants known to export sugars by symplastic phloem loading but also of plants using the apoplastic pathway. Increased respiration during sugar uptake revealed tight coupling between respiratory ATP production and ATP consumption by proton-translocating ATPase of the plasma membrane, which exports protons into the apoplast, thereby compensating for the proton loss in the apoplast when protons are transported together with sugars into the symplast. The extent of stimulation of respiration by sugars indicated that sugar uptake was not limited to phloem tissue. Ratios of the extra CO₂ released during sugar uptake to the amounts of sugars taken up were variable, but lowest values were lower than 0.2. When a ratio of 0.2 is taken as a basis to calculate rates of sugar uptake from observed maxima of sugar-dependent increases in respiration, rates of sugar uptake approached 350 nmol/(m² leaf surface s). Sugar uptake rates were half-saturated at sugar concentrations in the feeding solutions of about 10–25 mM indicating a low in vivo affinity of sugar uptake systems for sugars.     

Simulation of the light-induced oscillations of the membrane potential in Potamogeton leaf cells

Summary An attempt has been made to simulate the light-induced oscillations of the membrane potential of Potamogeton lucens leaf cells in relation to the apoplastic pH changes. Previously it was demonstrated that the membrane potential of these cells can be described in terms of proton movements only. It is hypothesized that the membrane potential is determined by an electrogenic H⁺-ATPase with a variable H⁺/ATP stoichiometry. The stoichiometry shifts from a value of two in the dark to a value of one in the light. Moreover, this H⁺ pump shows the characteristics of either a pump or a passive H⁺ conductance: the mode of operation of the H⁺ translocator is considered to be regulated by the external pH. The pump conductance is assumed to be dominant at low or neutral pH, while the passive H⁺ conductance becomes more significant at alkaline pH. The pH dependence of the transport characteristic is expressed by protonation reactions in the plasma membrane. The proposed model can account for most features of the light-induced oscillations but not for the absolute level of the membrane potential.This research was supported by the Foundation of Biophysics, part of the Dutch Organization for Scientific Research (NWO) ECOTRANS publication No. 34.     

Role of the Apoplast in the Control of Assimilate Transport,Photosynthesis, and Plant Productivity

A concept is suggested, which supposes that assimilates are transferred within the plant downward through phloem sieve tubes and, after entering the stem apoplast, are carried up with the ascending flow of transpiration water. After entering the apoplast of fully expanded leaves, these solutes are reexported through the phloem. Thus, a common pool of assimilates with uniform concentration is formed in the plant apoplast. According to this concept, the mechanism of assimilate demand represents a response of photosynthetic apparatus to changes in the apoplastic level of metabolites consumed by sink organs. The ratios of labeled photoassimilates differ between the apoplast and mesophyll cells. Most of the apoplastic labeled carbon is contained in sucrose, less in amino acids, and even less in hexoses. The ¹⁴C-labeling of amino acids increases and the sucrose/hexose labeling ratio decreased under conditions of enhanced nitrate supply. The well-known effect of relative inhibition of assimilate export from leaves under conditions of enhanced nitrogen supply is explained by an enhanced hydrolysis of apoplast-derived sucrose due to the increase in invertase activity, rather than by diversion of primary photosynthetic products from sucrose synthesis to other pathways required for activated growth processes in leaves. This notion is based on observations that the sucrose/hexose ratio is reduced to a greater extent in the apoplast than in the symplast. The last assumption was supported by data obtained after artificial changes in the apoplastic pH. In these experiments intact plants were placed in the atmosphere of NH₃ or HCl vapors, which induced opposite changes in relative content of labeled assimilates in the apoplast and in the photosynthetic rate.