Redistribution of potassium,chloride and calcium during the gravitropically induced movement of Mimosa pudica pulvinus

When the leaves of Mimosa pudica are changed from their normal position in the gravitational field, they perform reversible compensatory movements by means of pulvini. These movements are not the result of growth processes but involve reversible turgor variations. These variation are concomitant with ion migrations within pulvini: during the gravitropic movement, K⁺ and Cl^- shift towards the adaxial half of the motor organ whereas Ca²⁺ shifts towards the abaxial half. Compounds known to affect K⁺ transport, tetraethylammonium chloride and valinomycin, do not hinder the gravitropic movement but inhibit strongly the seismonastic reaction. The same general result is obtained with compounds affecting anion transport, disulfonic stilbenes and 9-anthracene carboxylic acid. Calcium chelators inhibit the gravitropic movement more efficiently than the seismonastic reaction and the calcium ionophore A 23 187 increases both movements. The data obtained with these various compounds indicate that ions do not have the same functional importance in the regulation of the two different pulvinar movements.Abbreviations abx abaxial half of the pulvinus - adx adaxial half of the pulvinus - 9-AC 9-anthracene carboxylic acid - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - SITS 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid - TEA tetraethylammonium chloride     

Uptake of glycine by excised pulvini of Mimosa pudica in relation to proton pump activity

Uptake of [³H]-glycine by sections of Mimosa pudica L. pulvini is pH dependent (maximum at pH 5.5) and exhibits biphasic saturation kinetics in the range of concentrations tested (1–75 m M ). Effects of compounds which increase [fusicoccin (FC)] or decrease (uncouplers, ATPase inhibitors) the proton-motive force were tested both on the pH variations induced in the incubation medium and on glycine uptake by the pulvinar tissues: there is a close relationship between the time required for the effect of these compounds on the acidification (for FC) and the pH rise (for the inhibitors) of the medium and that needed respectively for promotion and inhibition of glycine uptake. Experiments with sulfhydryl-reacting compounds show that N-ethylmaleimide induces a large rise in pH in the incubation medium and strongly inhibits glycine uptake, whereas p -chloromercuribenzenesulfonic acid has less effect on these processes. These results argue for a proton-glycine symport mechanism in the pulvinar tissue and thus support the previously postulated involvement of a proton pump in the regulation of pulvinar movement.     

Ultrastructural features of the starch sheath cells of the primary pulvinus after gravistimulation of the sensitive plant (Mimosa pudica L.)

Summary InMimosa pudica the primary and secondary motor organs (pulvini) of fully grown leaves are capable of graviresponse. These organs possess sedimentable amyloplasts in their starch sheath cells.In the primary pulvinus these cells are characterized by a structural polarity induced by the localization of nucleus at their (morphologically) apical part and the localization of amyloplasts at their (physically) basal part. These cells also display structural peculiarities including plasmodesmatal disposition, little development of the endoplasmic reticulum and an absence of vacuolar tannins; moreover, the sedimentation of the amyloplasts, induced by gravistimulation, is accompanied by the variation of localization of the cytoplasm, vacuole and mitochondria and by structural modifications of the nucleus and endoplasmic reticulum.     

Effects of blue light on electrical potential and turgor in pulvinar motor cells ofPhaseolus

Pulvinar motor cells ofPhaseolus vulgaris L display transient depolarization of the membrane potential and a turgor pressure decrease when exposed to a pulse of blue light. To analyze the mechanism of the transient depolarization, the effects of some factors such as anoxia, metabolic inhibitors and specific inhibitors of H⁺-ATPase have been examined. The findings have led to the conclusion that blue light inactivates the electrogenic H⁺-pumping ATPase in the plasma membrane of the motor cells. This inactivation seems to suppress ion uptake and decrease the turgor pressure of the motor cells.     

Calcium-sensitivity of the plasmalemmal delayed rectifier potassium current suggests that calcium influx in pulvinar protoplasts from Mimosa pudica L. can be revealed by hyperpolarization

Isolated protoplasts from pulvinar motor cells of Mimosa pudica were studied using conventional whole-cell patch clamp techniques. With internal solutions weakly buffered for Ca²⁺ (0.2 mm EGTA), a run-down of the outward delayed rectifier K⁺ current was induced by hyperpolarizing the holding potential, and this effect was strongly promoted by high external Ca²⁺ concentrations. This rundown could be reversed by coming back to less hyperpolarized holding potentials or by lowering the external [Ca²⁺]. Such rundown was absent when pipette internal solutions strongly buffered (10 mm EGTA) for Ca²⁺ were used. Ionomycin induced run-down of the K⁺ current with internal solutions containing 0.2 mm but not 10 mm EGTA. The hyperpolarization-associated rundown was reversibly blocked by Gd³⁺ and La³⁺.We thank Christophe Untereiner and Denis Wagner for expert technical assistance in facilitating the experiments and data acquisition and analysis.     

Early time course and specificity of auxin effects on turgor movement of the bean pulvinus

Auxin application to the upper side of the pulvinus of primary leaves of Phaseolus vulgaris L. promoted bending away from the place of application. The effect had a latency of less than 20 min and was specifically induced by substances known as active auxins in growth tests (indoleacetic and 1-naphthaleneacetic acid) but not by inactive auxin analogs (2-naphthaleneacetic, 3-indolepropionic and benzoic acid); 2,4-dichlorophenoxyacetic acid, and L-(-)-2,4-dichlorophenoxyisopropionic acid were of intermediate activity. Auxin-promoted bending was reversible and presumably caused by turgor increase in the treated cells.Abbreviations IAA 3-indolacetic acid - NAA naphthylacetic acid     

Absence of plasma membrane H+-ATPase in plasmodesmata located in pit-fields of the young reactive pulvinus ofMimosa pudica L.

Summary Immunocytochemical techniques were employed to study the spatial distribution of the plasma membrane H⁺-ATPase within various cell types of the young reactive primary pulvinus ofMimosa pudica L. These cells were interconnected by large numbers of plasmodesmata, being concentrated within pit-fields. Although we could routinely detect evidence of the H⁺-ATPase along the plasma membrane, immunolabelling was rarely, if ever, observed along the plasma membranes of the plasmodesmata. This finding is discussed with respect to the likely specialized supramolecular structure of the plasmodesma.Abbreviations SEL size exclusion limit of plasmodesmata     

Chloride ion efflux during an action potential in the main pulvinus ofMimosa pudica

The shortening, action potential and Cl⁻-efflux of the excised lower half cortex in the main pulvinus ofMimosa pudica were simultaneously recorded. The mean values±(S.E.) for Cl⁻-efflux and shortening were 183±18 picomoles/mg fresh weight/impulse and 87.0±2.2 μm, respectively.     

Specialized formations of endoplasmic reticulum in parenchyma cells ofMimosa pudica L.

Summary Parenchyma cells ofMimosa pudica display close associations between two or more cisternae of the endoplasmic reticulum. These associations form simplified types of lamellar bodies in which inner paired lamellae have lost their ribonucleoprotein granules and are separated by a dense layer.     

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     

Microautoradiographic localisation of [3H]sucrose and [3H]mannitol in Robinia pseudoacacia pulvinar tissues during phytochrome-mediated nyctinastic closure

Summary. We have analysed the incorporation of [³H]sucrose and [³H]mannitol in pulvinar motor cells of Robinia pseudoacacia L. during phytochrome-mediated nyctinastic closure. Pairs of leaflets, excised 2 h after the beginning of the photoperiod, were fed with 50 mM [³H]sucrose or [³H]mannitol, irradiated with red (15 min) or far-red (5 min) light and placed in the dark for 2–3 h. Label uptake was measured in whole pulvini by liquid scintillation counting. The distribution of labelling in pulvinar sections was assessed by both light and electron microautoradiography. [³H]Sucrose uptake was twice that of [³H]mannitol incorporation in both red- and far-red-irradiated pulvini. In the autoradiographs, [³H]sucrose and [³H]mannitol labelling was localised in the area from the vascular bundle to the epidermis, mainly in vacuoles, cytoplasm, and cell walls. Extensor and flexor protoplasts displayed a different distribution of [³H]sucrose after red and far-red irradiation. Far-red light drastically reduced the [³H]sucrose incorporation in extensor protoplasts and caused a slight increase in internal flexor protoplasts. After red light treatment, no differences in [³H]sucrose labelling were found between extensor and flexor protoplasts. Our results indicate a phytochrome control of sucrose distribution in cortical motor cells and seem to rule out the possibility of sucrose acting as an osmoticum. Correspondence and reprints: Unidad de Fisiología Vegetal, Facultad de Biología, Universidad de Barcelona, Avenida Diagonal 645, 08028 Barcelona, Spain.     

Effects of osmotic concentrations and IAA on the rapid shortening of the cortex in the main pulvinus ofMinosa pudica

In 0.8 M mannitol solution, no rapid shortening was recorded, while normal action potential was recorded. This result suggests that motive force of rapid shortening is an elastic stretch of the cell wall caused by the turgor pressure, and that osmotic concentration of motor cell is 0.6–0.7 M. IAA increased the rapid shortening, recovery rate and Cl⁻-efflux. The magnitudes of rapid shortening in IAA were 3–10 times as large as those in APW. The mean and maximum values of the rapid shortening in IAA were 87.0±2.2μm and 207 μm, respectively, or 1.6 and 3.9% of the whole length of motor tissue. When the large rapid shortening occurred, the large recovery rate was observed. These results suggest that both mechanisms, expulsion and re-entry of cell sap, are enhanced by IAA treatment. IAA-induced hyperpolarization was observed with a short time lag, which suggests that IAA enhances the electrogenic ion pump.     

Evidence for amino-acid: proton cotransport in Ricinus cotyledons

During germination and early growth of the castor-bean (Ricinus communis L.), protein in the endosperm is hydrolyzed and the amino acids are transferred into the cotyledons and then via the translocation stream to the axis of the growing seedling. The cotyledons retain the ability to absorb amino acids after removal of the endosperm and hypocotyl, exhibiting rates of transport up to 70 mol g^-1 h^-1. The transport of L-glutamine was not altered by KCl or NaCl in low concentrations (0–20 mM). High concentrations of KCl (100 mM) inhibited transport, presumably by decreasing the membrane potential. An increase in the pH of the medium bathing the cotyledons was observed for 10 min following addition of L-glutamine but not with D-glutamine, which is not transported. The rate of proton uptake was dependent on the concentration of L-glutamine in the external solution. Inhibitors and uncouplers of respiration (azide, 2, 4-dinitrophenol, carbonyl cyanide phenylhydrazone and N-ethylmaleimide) inhibited both L-glutamine uptake and L-glutamine-induced proton uptake. Amino acids other than L-glutamine also caused a transient pH rise and the rate of proton uptake was proportional to the rate of amino-acid uptake. The stoichiometry was 0.3 protons per amino acid transported. Addition of sucrose also caused proton uptake but the alkalisation by sucrose and by amino acids were not additive. Nevertheless, when sucrose was added 60 min after providing L-glutamine at levels saturating its uptake system, a rise in pH was again observed. The results were consistent with amino-acid transport and sucrose transport in castor-bean cotyledons both occurring by a proton cotransport in the same membrane system but involving separate carriers.     

The shortening and action potential of the cortex in the main pulvinus ofMimosa pudica

The shortening and action potential of the upper and lower cortices of the main pulvinus ofMimosa pudica were recorded simultaneously. The shortening and action potential were observed only in the lower cortex. The extensibility and the excitability of the cortex are discussed.     

Changes in the extracellular ion concentration in the main pulvinus of Mimosa pudica during rapid movement and recovery

A decrease in electric resistance and an increase in the extracellularCl^– concentration ([Cl^–]) in the main pulvinus ofMimosa occurred immediately after the action potential of themotor cells. The beginnings of both changes were almost coincidentalwith the beginning of the rapid movement. A remarkable increasein [Cl^–] was seen in the lower half of the pulvinus, butonly a slight increase in the upper half. Release of Cl^–,probably with K⁺; and other ions, from the motor cells impliesan ejection of liquid from the vacuole. When recovery of thepulvinus following rapid movement was fast in light, [Cl^–]decreased to its initial level within 20 min. When the recoverywas slow in darkness, [Cl^–] decreased at a slow rate andmaintained a higher level than its initial one for a long time.Photosynthetic inhibitors delayed recovery and the decreasein [Cl^–] even in light. These facts suggest that the re-entryof ions into motor cells during recovery partially requiresa photosynthetic energy supply. (Received February 12, 1980; )     

Effects of prostaglandins E,precursors and some inhibitors of prostaglandin biosynthesis on dark- and light-induced leaflet movements in Cassia fasciculata Michx.

Prostaglandin E₁ and prostaglandin E₂ speed up the dark-induced (scotonastic) and light-induced (photonastic) leaflet movements of Cassia fasciculata. The precursors of prostaglandin biosynthesis, homo -linolenic and arachidonic acids, and an intermediary product, prostaglandin-interm-5, act in the same manner on these movements. Inhibitors of prostaglandin biosynthesis, indomethacin and phenylbutazone, inhibited the scotonastic but promoted the photonastic movements in an unexpected way. Since the pulvinar movements are mediated by water and ion migrations, the observed modifications of these movements indicate that prostaglandins and their precursors may affect, as in animal cells, processes linked to a variation of membrane permeability.Abbreviations PGE₁ prostaglandin E₁ - PGE₂ prostaglandin E₂     

Transport processes in stimulated and non-stimulated leaves of Mimosa pudica

Summary Using energy-dispersive X-ray microanalysis, the concentrations of ions, especially potassium and chlorine, were determined in different tissues of primary and tertiary pulvini of Mimosa pudica. It was shown that stimulating the leaf was followed by ion displacements which were most striking in the outer extensor cells, resulting in turgor loss. Since Ca concentration remains relatively constant in cell walls of collapsed cells, the changes of K concentration are best described by the K:Ca ratio. After stimulation the K:Ca ratio dropped in the outer extensor of the primary pulvinus from 775.3 to 2.37 in the cytoplasm, and from 542.2 to 9.25 in the cell wall. Changes in chlorine content were less striking in the primary pulvinus. The KCl ratios in some cases were lower than 1.0, which indicates that Cl content can increase, while K content is diminished. In the non-stimulated tertiary pulvini the outer extensor cells show high concentrations of Cl, but much lower Cl concentrations were found after stimulation. In contrast to the primary pulvinus the K content of the tertiary pulvini is very low. In the vascular tissues of both primary and tertiary pulvini stimulation is followed by a release of K and Cl out of the sieve element cytoplasm into the apoplast. K then appears accumulated in the cell walls of the collenchymatous tissue. These displacements lead to the assumption that the collenchymatous apoplast temporarily functions as a reservoir for K and to a lesser extent for Cl. With regard to the mechanism of leaf movement after stimulation, the accumulation of ions in the apoplast seems to be initiated by the decrease of water potential triggered by an apoplastic accumulation of unloaded sucrose (Fromm and Eschrich 1988a). The resulting turgor release in the outer extensor is accompanied by an efflux of ions.Supported by the Deutsche Forschungsgemeinschaft     

Effect of 2,4-dichlorophenoxyacetic acid on the dark- and light-induced pulvinar movements in Cassia fasciculata Michx

2,4-dichlorophenoxyacetic (2,4-D) applied to excised leaves of Cassia fasciculata modified the dark-induced (scotonasty) and light-induced (photonasty) leaflet movements, showing that this compound acts on rapid turgor variation and the concomitant ion migrations, in particular K⁺. 2,4-D inhibited the scotonastic closure in a dose-dependent manner from 10^–8 M to 10^–5 M and promoted the photonastic opening in the same range of concentrations. The compound acted rapidly since a treatment as short as 5 min gave an obvious effect on the motile reaction; however, a lag period of 45–60 min was needed to observe its effect. Although 2,4-D is a weak acid, its greatest physiological efficiency was obtained with pH values close to neutrality. The physiological results are discussed in relation to the chemical properties and the characteristics of transport of the molecule.Abbreviations ABA abscisic acid - 6-BAP 6-benzylaminopurine - 2,4-D 2,4 dichlorophenoxyacetic acid - GA₃ gibberellic acid - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethanesulphonic acid] - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - MES 2-(N-morpholino)-ethanesulphonic acid     

Sodium-alanine cotransport in oocytes ofXenopus laevis: Correlation of alanine and sodium fluxes with potential and current changes

Summary The sodium-dependentl-alanine transport across the plasma membrane of oocytes ofXenopus laevis was studied by means of [¹⁴C]-l-alanine,²²Na⁺ and electrophysiological measurements. At fixed sodium concentrations, the dependence of alanine transport on alanine concentration follows Michaelis-Menten kinetics; at fixed alanine concentrations, the transport varies with sodium concentration with a Hill coefficient of 2. In the presence of sodium the uptake of alanine is accompanied by a depolarization of the membrane. Under voltage-clamp conditions this depolarization can be compensated by an inward-directed current. Assuming that this current is carried by sodium we arrive at a 21 stoichiometry for the sodium-alanine cotransport. The assumption was confirmed by direct measurements of both sodium and alanine fluxes at saturating concentrations of the two substrates, which also yielded a stoichiometry close to 21. The sodium-l-alanine cotransport is neither inhibited by furosemide (0.5 mmol/liter) nor by N-methyl amino isobutyric acid (5 mmol/liter). A 20-fold excess ofd-alanine overl-alanine caused about 60% inhibition.     

Transport processes in stimulated and non-stimulated leaves ofMimosa pudica

Summary Mature leaves ofMimosa pudica L. or parts of them were exposed to¹⁴CO₂, and translocation was recorded by macroautoradiography. It was observed that considerable amounts of labelled photoassimilates were accumulated in pulvini when the leaf was stimulated. In non-stimulated leaves, no such accumulation of label was observed.Microautoradiographs of pulvinar regions of the non-stimulated leaf showed¹⁴C- label restricted to the phloem. When stimulated, the¹⁴C- label was unloaded from the phloem of the pulvini. Labelled photoassimilates appeared most concentrated in the walls of the collenchymatous cells and beyond in the extensor region of the motor cortex. There, label was accumulated in the apoplastic compartments. Stimulation causes a sudden phloem unloading of sucrose, and its accumulation in the apoplast lowers the water potential which eventually exceeds the osmotic potential of the extensor cells of the motor cortex. By removal of cytoplasmic water the motor cells lose turgidity which results in the closing movement of the leaflets, and — some seconds later — in the bending down of the petiole. In late afternoon night-stimulation triggers sucrose unloading in secondary pulvini. During phases of relaxation, labelled material is taken up by motor cells of the extensor, which concomitantly gain turgor.Part of the doctoral dissertation of Jörg Fromm supported by the Deutsche Forschungsgemeinschaft