The Effects of Fusicoccin on Anion Fluxes in Isolated Guard Cells of Commelina communis L.

Clint, G. M. 1987. The effects of fusicoccin on anion fluxesin isolated guard cells of Commelina communis L.—J. exp.BoL 38: 863–876. The effects of 3?10^–2 mol m^–3 fusicoccin (FC) onbromide fluxes and contents in isolated guard cells of Commelinacommunis L. have been studied using K⁸²Br at pH 3?9 and pH 6?7.At pH 3?9 FC caused a reduction in both the influx and the effluxof ⁸²Br, whereas at pH 6?7 FC had no effect on the influx butcaused a transient increase in the efflux of ⁸²Br. There wasno obvious change in bromide content with FC treatment at eitherpH. The behaviour of the anion fluxes in response to FC suggeststhat FC does not act solely via a hyperpolarization at the plasmalemma.A redistribution of bromide between the intracellular compartmentssuggests that anion flux from the cytoplasm to the vacuole maybe stimulated by FC at pH 3?9. The failure of guard cells toincrease their anion content on treatment with FC despite anincrease in stomatal aperture and in cation content suggeststhat in FC-induced stomatal opening excess cation is balancedby organic acid synthesis within the guard cell. Key words: Fusicoccin, guard cells, ion fluxes, Commelina communis     

Effects of Light/Dark on Anion Fluxes in Isolated Guard Cells of Commelina communis L.

The effects of light/dark on anion fluxes in isolated guardcells of Commelina communis L. have been studied, using ⁸²Brand ³⁶Cl. Transfer of open guard cells from light to dark hasno effect on the ⁸²Br influx, but produces a marked transientstimulation of ⁸²Br or ³⁶Cl efflux, similar to the effect ofsuch transfer on the ⁸⁶Rb fluxes, and to the effects on both⁸⁶Rb and ⁸²Br fluxes of adding ABA. On return of guard cellsto light, after the transient, there is a further reductionin Cl/Br efflux. It is argued that control of a specific processof ion extrusion is important in regulating the ability of guardcells to stay open. In three out of four batches of steady-statetissue labelled with ⁸²Br, the plasmalemma fluxes were highenough, relative to the tonoplast fluxes, for the efflux kineticsto be separable into two exponential components, allowing estimationof bromide contents in cytoplasm and vacuole (Q_c and Q_v), andfluxes at plasmalemma and tonoplast. With opening in light,Q_c increased by 3.9 ± 0.4 pmol mm^–2 µm^–1and Qy by 5.2 ± 0.6 pmol mm^–2 µm^–1(change in content per mm² of epidermis perµm change inaperture). Using rough estimates for the volumes of cytoplasmand vacuole these figures suggest that at 6.1 µm in thedark the concentrations were about 63 mol m^–3 in the cytoplasmand 35 mol m^–3 in the vacuole, rising to about 185 molm^–3 in the cytoplasm and 125 mol m^–3 in the vacuole,at 16.7 µm aperture in light. Neither increase can providean adequate increase in salt concentration to account for theosmotic change required, and some solute other than potassiumsalt must also be involved. In one experiment with 82Br andin the only experiment with 36Cl the plasmalemma flux was lower,and not high enough relative to the tonoplast flux to allowseparation of two phases in the efflux curves, and calculationof cytoplasmic and vacuolar contents and fluxes. The effectsof transfer from light to dark were, nevertheless, similar inboth types of tissue. Key words: Commelina communis L., Light/dark effects, Anion fluxes, Guard cells     

Effects of Light/Dark on Cation Fluxes in Guard Cells of Commelina communis L.

The effects of light/dark on cation fluxes in isolated guardcells of Commelina communis L. have been studied, using ⁸⁶RbCland ²²NaCl. Transfer to the dark has no effect on ⁸⁶Rb influx,but produces a marked transient stimulation of ⁸⁶Rb efflux,similar to that seen previously on adding ABA. The ⁸⁶Rb effluxfalls on return to light only during the period of stimulatedflux; after the transient, return to light has no effect onefflux. The ability to produce this transient stimulation ontransfer to the dark is recovered in a subsequent light period.In general, in Na-loaded cells, the stimulated efflux is notseen. and the cells do not close in the dark. The results arenot consistent with a simple permeability or potential change,but suggest a specific ion excretion activated by the transferto the dark. Key words: Commelina communis L., Light/dark effects, Cation flux, Guard cells     

Ion Fluxes in 'Isolated' Guard Cells of Commelina communis L.

Ion fluxes have been measured in ‘isolated’ guardcells of Commelina communis L. using ⁸⁶RbCl and K⁸²Br, in epidermalstrips in which all cells other than guard cells have been killedby treatment at low pH. To avoid problems of slow free spaceexchange most fluxes have been measured at pH 3.9, at whichstomata open well in K(Rb) Cl(Br) and are stable for many hours.At pH 3.9 the intracellular ⁸⁶Rb exchanged as a single compartmentwith a half-time of 2–3 h, independent of external concentration(C_o). The influx of ⁸⁶Rb rose with concentration, to a V_maxof about 23 pmol mm^–2 h^–1. The efflux curve of ⁸²Brcould be well fitted by two exponential terms, with half-timesof 38 min (independent of C_o), and 5–35 h (falling withincreasing C_o). Bromide contents of cytoplasm and vacuole (Q_cand Q_v), and fluxes at plasmalemma and tonoplast, were calculatedfrom the efflux kinetics. Over C_o 20–60 mM, as the apertureincreased from 7 µm to 17 µm, the tonoplast flux(0.5–11.5 pmol mm^–2h^–1) was always much lessthan the plasmalemma flux (7–77 pmol mm^–2 h^–1).Q_c and Q_v both increased with aperture. The increase in Q_c of10.3 pmol mm^–2 µm^–1 is adequate to accountfor the osmotic changes required to change the aperture, aspreviously estimated. However, the change in vacuolar contentof only 5.9 pmol mm^–2 µm^–1 is much too smallto account for the osmotic changes required, or to balance thecytoplasmic changes. It appears therefore that increasing KBroutside not only increases the cytoplasmic salt content, andthe Br flux at the tonoplast, but also stimulates the vacuolaraccumulation of some other solute.     

Effects of ABA in 'Isolated' Guard Cells of Commelina communis L.

The effects of 2 x 10^–3 M ABA on ion fluxes in isolatedguard cells of Commelina communis L. have been studied, using⁸⁶RbCl and K⁸²Br, in epidermal strips in which all cells otherthan guard cells have been killed by treatment at low pH. Theeffect of ABA on influx is small, if present, and the majoreffect is a marked transient stimulation of the efflux of both⁸⁶Rb and ⁸²Br at the plasmalemma; there is also an increasein the flux of ⁸²Br from vacuole to cytoplasm. The stimulationis transient, and the cells do not simply become more leaky.The results are not consistent with previous speculations onthe mechanism by which ABA reduces aperture.     

Change in Levels of Starch and Sugars in Epidermis of Commelina benghalensis during Fusicoccin Stimulated Stomatal Opening

The concentrations of malate, starch and sugars were determinedin presonicated epidermal strips of Commelina benghalensis.During opening, the starch content of epidermis decreased whilethe level of sugars or malate increased. Fusicoccin (FC) stimulatedstomatal opening and elevated the levels of malate and sugars.However, the contribution from sugars was nearly 50% of theosmotic effect of malate and it increased to more than 60% inthe presence of FC. We conclude that FC stimulates stomatalopening by enhancing not only potassium influx into guard cellsbut also hydrolysis of starch into sugars (and malate). Significantcorrelations were noticed between the width of stomatal apertureand epidermal starch (negative), malate and sugars (both positive).The negative relationship between starch and malate or sugarswithin epidermis indicated that starch hydrolysis lead to formationof sugars as well as malate. Starch—sugar interconversioncan therefore play a significant role in modulating the solutepotential of guard cells. Key words: Commelina benghalensis, Stomatal opening, Fusicoccin, Epidermal starch and sugars     

A Critical Assessment of the Role of Potassium and Osmolarity in Stomatal Opening

An attempt has been made to separate the osmotic effect fromthe ionic effect of KCI in stomatal responses. For this purposeisolated illuminated epidermis from species with and withoutsubsidiary cells were treated with KCI (0-250 mOs kg^–1)and with mannitol (0-250 mOs kg^–1). Since osmolarity wasmade the basis of comparison, the effect of mannitol had tobe observed immediately, before guard cell contents could haveleached into the incubation medium. When plotting aperturesagainst osmolarity sigmoid curves were obtained with KCI, butwith mannitol straight lines resulted provided that prior tostripping and incubation leaves were briefly illuminated. Whilst in lower concentrations (60 mOs kg^–1 for Viciafaba; 90 mOs kg^–1 for Zantedeschia aethiopica; 190 mOskg^–1 for Commelina communis) pores were wider in mannitolthan in KCI, in concentrations above these values the situationwas reversed. It appeared therefore that KCI had either an inhibitoryor a promoting effect. Inhibition was most pronounced when atthe beginning of incubation stomata were closed; the inhibitoryeffect on stomata without subsidiary cells occurred at low concentrations(0-60 mOs kg^–1) whereas when subsidiary cells were presentinhibition occurred at up to 190mOs kg^–1. Other experiments started with KCI solutions of 50 mOs kg^–1for Vicia faba, 85 mOs kg^–1 for Zantedeschia aethiopicaand 115 mOs kg^–1 for Commelina communism; mannitol wasadditionally used to give the progressive increases in osmolarity.Degrees of opening were then reached which with KCI alone couldonly be attained at the very highest concentrations. Starch disappearance was followed using the periodic-acid-silvertest; by using either ⁸⁶Rb or ⁴³K it was shown that ion uptakewas restricted to guard cells alone only at osmolarities exceeding200 mOs kg^–1. On the basis of these observations it was concluded that K transportdoes not represent the major mechanism of stomatal regulation. Key words: Stomata, Potassium, Osmolarity     

Effects of Fusicoccin and Abscisic Acid on Sugar and lon Transport from Plant Glands

Fusicoccin (FC) inhibited net excretion of Cl^– by theglands of the pitchers of the carnivorous plant Nepenthes hookeriana;of Na⁺ and Cl^– by the salt glands of the halophytes Limoniumvulgare and L. pectinatum and of K⁺ in the nectar of Acer platanoidesflowers. It had no effect on K⁺ elimination with nectar of Impatienswalleriana (extrafloral nectaries) and Abutilon striatum. Abscisicacid (ABA) stimulated net excretion of K⁺ and Cl^– in Nepenthesand of Na⁺ and Cl^– in Limonium but had no effects on K⁺in nectar. Thus, FC and ABA had opposing effects on ion excretionby the salt eliminating glands of Limonium and Nepenthes. Bothcompounds, however, had similar effects on sugar secretion ofnectary glands which was either inhibited or unaffected by FCand ABA. It is suggested that the effects of FC and ABA on ion excretionby gland cells could be reconciled with literature showing FC-stimulationand possible ABA-inhibition of proton pumps at the plasmalemmaof plant cells. Nepenthes hookeriana, Limonium vulgare, Limonium pectinatum, Acer platanoides, salt-glands, nectaries, excretion, fusicoccin, abscisic acid, proton pump     

The Effects of Temperature on 86Rb Uptake by Two Species of Chlamydomonas (Chlorophyta, Chlorophyceae)

⁸⁶Rb uptake was examined in two species of unicellular greenalgae, Chlamydomonas nivalis isolated from snow, and a cellwall-less mutant of the temperate freshwater Chlamydomonas reinhardii.In C. reinhardii cells grown at 20°C and cooled rapidlyto 0°C, ⁸⁶Rb uptake was abolished. Cells cooled rapidlyto –5°C in the absence of ice accumulated ⁸⁶Rb veryrapidly but the time course of this uptake suggested non-selectiveaccumulation through a damaged plasmalemma. Cells grown at 8°Cwere viable, able to divide and motile; they showed no signsof cold-shock and ⁸⁶Rb uptake, albeit slow, was measurable at–5°C in the absence of extracellular ice. Cells ofC. nivalis grown at 20°C were damaged at sub-zero temperaturesalthough they did show an enhanced ⁸⁶Rb uptake at 0°C. Cellsgrown at 5°C were able to accumulate ⁸⁶Rb from media undercooledto -5°C in the absence of extracellular ice, and again showedenhanced uptake at 0°C. The process of acclimation to lowtemperature appears to differ in the two species. Key words: Chlamydomonas, temperature, ⁸⁶Rb uptake, membrane     

Functional characterization of the neuronal-specific K-Cl cotransporter: implications for [K+]o regulation

The neuronal K-Cl cotransporter isoform (KCC2) was functionallyexpressed in human embryonic kidney (HEK-293) cell lines. Two stablytransfected HEK-293 cell lines were prepared: one expressing anepitope-tagged KCC2 (KCC2-22T) and another expressing theunaltered KCC2 (KCC2-9). The KCC2-22T cells produced aglycoprotein of ~150 kDa that was absent from HEK-293 control cells.The ⁸⁶Rb influx in both cell lineswas significantly greater than untransfected control HEK-293 cells. TheKCC2-9 cells displayed a constitutively active⁸⁶Rb influx that could beincreased further by 1 mMN-ethylmaleimide (NEM) but not by cellswelling. Both furosemide [inhibition constant (K_i) ~25µM] and bumetanide (K_i~55 µM) inhibited the NEM-stimulated ⁸⁶Rb influx in the KCC2-9cells. This diuretic-sensitive⁸⁶Rb influx in theKCC2-9 cells, operationally defined as KCC2 mediated, required external Clbut not external Na⁺ and exhibiteda high apparent affinity for externalRb⁺(K⁺)[Michaelis constant(K_m) = 5.2 ± 0.9 (SE) mM; n = 5] but alow apparent affinity for externalCl(K_m >50 mM). Onthe basis of thermodynamic considerations as well as the unique kineticproperties of the KCC2 isoform, it is hypothesized that KCC2 may servea dual function in neurons: 1) themaintenance of low intracellularCl concentration so as toallow Cl influx vialigand-gated Cl channelsand 2) the buffering of externalK⁺ concentration([K⁺]_o) in the brain.

     

Measurement of the Apoplastic Activity of K+ and Cl- in the Leaf Epidermis of Commelina communis in Relation to Stomatal Activity

Bowling, D. J. F. 1987. Measurement of the apoplastic activityof K⁺ and Cl^– in the leaf epidermis of Commelina communisin relation to stomatal activity.–J. exp. Bot. 38: 1351–1355. Ionic activity of K⁺ and Cl^– in the apoplast of the lowerepidermis of the leaf of Commelina communis was measured usingion selective micro-electrodes. Large gradients across the stomatalcomplex were observed which were related to stomatal aperture.On stomatal closure the activity of K⁺ and Cl^– in theapoplast of the guard cell rose from 3·0 mol m^–3to 100 mol m^–3 and 33 mol m^–3 respectively. It wasconcluded that the apoplast is an important pathway for iontransport between the cells. Key words: Stomata, ionic activity, leaves, apoplast     

An Examination, by Compartmental Flux Analysis, of the Development of Sodium and Chloride Absorption Capacities in Beetroot Disks

Using beetroot, Beta vulgaris L. var. Avon Early, grown in radioactivelylabelled nutrient solutions, concentrations and fluxes of Na⁺and Cl^– were estimated for cells of freshly cut storageroot disks, by compartmental analysis of radioisotope elutionmeasurements. These values were compared with results obtainedin a similar manner from beetroot grown in non-labelled nutrientsolution and loaded instead during an aging period in ²²Na-or ³⁶Cl- labelled 1 mM NaCl solution. In accord with the generallyaccepted, but never properly tested, view, it was found thatnet Na⁺ influx followed from a reduction in efflux with agingand net Cl^– uptake depended on a marked increase in influx.However, both these important changes took place at the tonoplast,and, although aging led to a reduction of plasmalemma fluxes,at no time was entry of Cl^– into the cytoplasm, or lossof Na⁺ from the cytoplasm, significantly restricted.     

Orthophosphate Relations of Root: NO-3 Effects on Orthophosphate Influx, Accumulation and Secretion into the Xylem

Lamaze, T., Sentenac, H. and Grignon, C. 1987. Orthophosphaterelations of root: NO^–₃effects on orthophosphate influx,accumulation and secretion into the xylem.—J. exp. Bot.38: 923–934. Orthophosphate (Pi) accumulation by barley (Hordeum vulgareL.) roots was specifically inhibited by NO^–₃ as comparedto Cl^– and SO₄^{2 –}, and Pi secretion into the xylemwas stimulated. The inhibition of Pi accumulation by NO^–₃was also observed in roots of intact photosynthesizing horsebean(Vicia faba L.), rice (Oryza sativa L.) and soybean (Glycinemax L.) plants. NO^–₃ effects on Pi transport by rootswere more thoroughly investigated with corn (Zea mays L.). Theywere due to intracellular NO^–₃. Pi secretion was stillstimulated by NO^–₃ after Pi withdrawal from the absorptionsolution. ³²Pi influx decreased during Pi accumulation, supportingthe hypothesis that this ion allosterically regulated its owntransport system by feedback control. This control was modulatedby other anions: the decrease was more pronounced in the presenceof nitrate. Chronologically, the depressive effect of NO^–₃on ³²Pi influx appeared after the inhibition of Pi accumulation.Furthermore, under conditions where Pi accumulation was notaffected by NO^–₃, ³²Pi influx and Pi secretion into thexylem became insensitive to the presence of nitrate. Our hypothesisis that the stimulative effect of NO^–₃ on Pi secretionand the depressive one on ³²Pi influx are the repercussionsof an increase in the Pi cytosolic concentration due to an NO^–₃-induced decrease in Pi uptake by the vacuoles. Key words: Root, orthophosphate fluxes, orthophosphate accumulation, nitrate, ionic interaction     

Calcium Inhibits Ion-Stimulated Stomatal Opening in Epidermal Strips of Commelina communis L.

Inoue, H. and Katoh, Y. 1987. Calcium inhibitsion-stimulatedstomatal opening in epidermal strips of Commelina communis L.—J.exp. Bot. 38: 142–149. Ca²⁺ suppressed both the ion-stimulated stomatal opening andH⁺ extrusion of pre-illuminated epidermal strips isolated fromCommelina communis L. In the absence of Ca²⁺, the rate of H⁺release was 18 nmol H⁺ cm^–2 h^–1 per epidermal stripunit area in 150 mol m^–3 KCL at pH 7?4. Half-maximum inhibitionof stomatal opening was observed with 220 mmol m^–3 ofCa²⁺. The hexavalent dye, ruthenium red, showed concentration-dependentprevention of the inhibition by Ca²⁺ of the ion-stimulated stomatalopening. The effect of ruthenium red was non-competitive, andthe K₁ for the calcium inhibition was found to be 3?6 mmol m^–3.The calcium inhibition of H⁺ extrusion was also prevented byruthenium red. These results suggest that Ca²⁺ inhibits theactivity of electrogenic H⁺ translocating ATPase of the guardcell plasma membrane and leads to the suppression of stomatalopening. Key words: Calcium, Commelina communis, ruthenium red, stomata     

Tentoxin Suppresses Stomatal Opening by Inhibiting Photophosphorylation

Tentoxin and, to a lesser extent, dihydrotentoxin (both at 10mmol m^–3) reduce stomatal opening in epidermal stripsof Commelina communis in the light but not in darkness. Thiseffect was significantly greater in normal air than in CO₂-freeair. Fusicoccin overcame the tentoxin effect. However, tentoxindid not inhibit stomatal opening in the light in epidermal stripsof Paphiopedilum harrisianum, a species which lacks guard cellchloroplasts. It is concluded that tentoxin exerts its actionon stomata not by an ionophorous effect in the plasmalemma ofguard cells but by the inhibition of photophosphorylation intheir chloroplasts. The effects of DCMU and tentoxin on guardcells are discussed in terms of their effects on chloroplastsand the extent to which energy is supplied from this organelleduring stomatal opening in the light. The results indicate thatneither photophosphorylation nor non-cyclic electron transportin guard cell chloroplasts are essential for stomatal opening. Key words: Commelina, epidermal strips, Paphiopedilum, photophosphorylation, stomata, tentoxin     

Inhibitory effect of fusicoccin on cell division

Rapid interactions in cell division and cytodifferentiationare induced by hormone treatments in dark-cultured explantsof Jerusalem artichoke. Fusicoccin, at concentrations between10^–6 and 10^–5 M, markedly inhibited the division-promotingactivity induced by plant hormones. Further, fusicoccin-treatedmeristematic root tips of Vicia faba and Allium cepa showeda rapid decrease in the mitotic index. Fusicoccin seems to inhibitsome hormone-sensitive processes required during the inductionand regulation of cell division. (Received March 28, 1979; )     

Responses of Commelina communis Stomata In Vitro

Weyers, J. D. B. and Paterson, N. W. 1987. Responses of Commelinacommunis stomata in vitro.— J. exp. Bot. 38: 631–641. Analysis of the kinetics of movements of Commelina communisL. stomata in vitro revealed a sequence of opening and closingphases dependent on the incubation medium used and the physiologicalstate of the plant material. In buffer containing 50 mol m ^–3KC1 the sequence of aperture changes appeared to be influencedby equilibration of cell water potentials with that of the mediumand by solute fluxes (dependent and independent on metabolicactivity). The results indicate that the stomatal aperture afterseveral hours of incubation may not always provide a reliablequantitative estimate of the ability of the stomata to operate.As a consequence, modifications are suggested to the ways inwhich experiments using epidermal strips are carried out andreported. Key words: Epidermal strips, guard cells, hydroactive, hydropassive, kinetics, potassium chloride, mannitol, osmotic effects, solute fluxes.     

Coupling of Proton Fluxes in the Polar Leaves of Potamogeton lucens L

An attempt has been made to quantify the light-induced H⁺ effluxand influx observed in polar leaves of Potamogeton lucens.Theseproton fluxes are spatially separated. The H⁺ efflux, mediatedby a plasmalemma bound H⁺ –ATPase, occurs across theplasmamembrane at the morphological lower epidermis and is accompaniedby an H⁺ influx (or OH^– efflux) at the upper side oftheleaf. As a result, these leaves exhibit a remarkable pH–polarityin the light. The pH near the lower epidermis may drop to avalueas low as 3.5, while a pH of about 10.5 can be observed at theupper epidermis. Obviously this phenomenon requires theco–ordinationof transport processes in the different cell layers of the leaftissue. These observations led to quantitative studies oftherelation between the H⁺ fluxes at either plasmalemma. Thesefluxes were calculated from the pH values recorded at twodistancesfrom the leaf surface. Although the H⁺ influx always exceededthe efflux, a coupling between the transport processesacrosseither plasma membrane became evident from the time–coursesof the two fluxes. Key words: Potamogeton lucens, proton flux, flux coupling, pH–;polarity     

Stomatal Movement and Sucrose Uptake by Guard Cell Protoplasts of Commelina benghalensis L.

Sucrose concentration in guard cells of epidermal strips ofCommelina benghalensis increased with stomatal opening. Sucroseuptake patterns were investigated using guard cell protoplastsof C. benghalensis. Sucrose (0.5 mM) uptake into these protoplastswas sensitive to pH, with an optimum at pH 6. Uptake of sucroseinto guard cell protoplasts was inhibited by 2,4-dinitrophenol(DNP), diethylstilbestrol (DES) and (ptrifluoromethoxy)carbonylcyanide phenylhydrozone (FCCP), while DCMU and o-phenanthrolinehad no effect on the uptake of sucrose. Fusicoccin (FC) stimulatedsucrose influx. The influence of pH and the effect of the metabolicinhibitors on the sucrose uptake into the guard cell protoplastsare consistent with an energy dependent membrane-function. (Received July 7, 1986; Accepted September 26, 1986)     

Utilization of the Vibrating Probe and Ion-Selective Microelectrode Techniques to Investigate Electrophysiological Responses to Wounding in Pea Roots

This paper examines the ionic composition of wound-induced electricalcurrents in higher plant tissue, using two non-injurious electrophysiologicaltechniques. By simultaneous recording of K⁺, H⁺ , and Ca²⁺ ionfluxes with extracellular ion-selective microelectrodes, wehave determined that a Ca²⁺ influx (2.4 µA cm^–2),a small H⁺ influx (0.17 µA cm^–2) and a large K⁺efflux (16 µA cm^–2) occur immediately after woundingin roots of Pisum sativum L. var. Greenfeast. Using an extracellularvibrating probe at the wound site, net ion currents of 26 µAcm^–2 were measured 5 min after wounding. In a more concentratedbathing medium (1/4 rather than 1/16 strength Hoagland's solution),net ion currents of 59 µA cm^–2 were measured, andthese would appear to be the largest extracellular currentsthat have been measured in plants. We made a quantitative comparisonof the summed ion fluxes with the net ion currents and thisrevealed that ion fluxes, in addition to those measured here,occur after wounding. Key words: Wounding, ion flux, electric current, calcium, potassium