Interaction of the Depolarization-Activated K Channel of Samanea saman with Inorganic Ions: A Patch-Clamp Study.

A depolarization-activated K(+) channel capable of carrying the large K(+) currents that flow from shrinking cells during movements of Samanea saman leaflets has been described in the plasmalemma of Samanea motor cell protoplasts (N Moran et al [1988] Plant Physiol 88:643-648). We now characterize this channel in greater detail. It is selective for K(+) over other monovalent ions, with the following order of relative permeability: K(+) > Rb(+) > Na(+) approximately Cs(+) approximately Li(+). It is blocked by Cs(+) and by Ba(2+) in a voltage dependent manner, exhibiting a ;long-pore' behavior, similarly to various types of K(+) channels in animal systems. Cadmium, known for its blockage of Ca(2+) channels in animal systems, and Gd(3+), closely related to La(3+), which also blocks Ca(2+) channels in animal cells, both block K(+) currents in Samanea in a voltage-independent manner, and without interfering with the kinetics of the currents. The suggested mechanism of block is either (a) by a direct interaction with the K(+) channel, but external to its lumen, or, alternatively, (b) by blocking putative Ca(2+) channels, and preventing the influx of Ca(2+), on which the activation of the K(+) channels may be dependent.

Separation and Characterization of Inositol Phospholipids from the Pulvini of Samanea saman.

To supplement current thin-layer chromatographic methods for separation and quantitation of plant phospholipids, an alternative method, high-performance liquid chromatography was developed. The major inositol-containing lipids from the pulvini of Samanea saman Merr. were identified as phosphatidylinositol, phosphatidylinositol phosphate, and phosphatidylinositol bisphosphate based on comigration with authentic standards on high-performance liquid chromatography and on thin-layer chromatography. The patterns of incorporation of radioactivity into the putative phosphatidylinositol and phosphatidylinositol phosphate were consistent with these identifications when pulvini were labeled with [(3)H]glycerol, [(3)H]inositol, or [(32)P]orthophosphate. Analysis of the products of enzymic hydrolysis, of chemical deacylation, and of ;fingerprint' methanolysis of these phospholipids confirmed the identifications.

Light-Stimulated Inositol Phospholipid Turnover in Samanea saman Pulvini : Increased Levels of Diacylglycerol.

Leaflet movement in Samanea saman is driven by an endogenous circadian clock and by light. We are investigating whether the effects of light on leaflet movement are mediated by increased inositol phospholipid turnover. We demonstrated previously that irradiation of excised pulvini with 15 to 30 seconds of white light decreases the levels of phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate and increases the levels of inositol phosphates. We now report that the diacylglycerol level increases after 30 seconds of white light but returns to below the control level after 10 minutes of white light.

Light-promoted changes in apoplastic K(+) activity in the Samanea saman pulvinus, monitored with liquid membrane microelectrodes.

The movement of Samanea saman (Jacq.) Merrill leaflets is a consequence of the re-distribution of K(+) and anions between motor cells on opposite sides of the pulvinus. We used a K(+)-sensitive microelectrode to study dynamic changes in K(+) transport through motor-cell membranes during and immediately after change in illumination. Potassium-ion-sensitive and reference microelectrodes were inserted into extensor or flexor tissue of a whole pulvinus in white light (WL). A brief pulse of red light (RL) followed by darkness (D) (a) increased K(+) activity in the extensor apoplast, indicating K(+) release by the protoplast; and (b) decreased K(+) activity in the flexor apoplast, indicating K(+) uptake by the protoplast. White light after 35-40 min D reversed K(+) activity in the extensor apoplast to approximately its original value. Blue light substituted partially for WL in this regard. Potassium-ion activity in the flexor apoplast reverted to approximately its original value after 2 h, with or without white illumination. Our data support the hypothesis that K(+) efflux from extensor cells and K(+) uptake by flexor cells following a WL→RL→D transition occurs by way of K(+) channels.

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.

Potassium Channels in Motor Cells of Samanea saman: A Patch-Clamp Study.

Leaflet movements in Samanea saman are driven by the shrinking and swelling of cells in opposing (extensor and flexor) regions of the motor organ (pulvinus). Changes in cell volume, in turn, depend upon large changes in motor cell content of K(+), Cl(-) and other ions. We performed patch-clamp experiments on extensor and flexor protoplasts, to determine whether their plasma membranes contain channels capable of carrying the large K(+) currents that flow during leaflet movement. Recordings in the "whole-cell" mode reveal depolarization-activated K(+) currents in extensor and flexor cells that increase slowly (t((1/2)) = ca. 2 seconds) and remain active for minutes. Recordings from excised patches reveal a single channel conductance of ca. 20 picosiemens in both cell types. The magnitude of the K(+) currents is adequate to account quantitatively for K(+) loss, previously measured in vivo during cell shrinkage. The K(+) channel blockers tetraethylammonium (5 millimolar) or quinine (1 millimolar) blocked channel opening and decreased light- and dark-promoted movements of excised leaflets. These results provide evidence for the role of potassium channels in leaflet movement.

Effects of temperature on h uptake and release during circadian rhythmic movements of excised samanea motor organs.

A previous study revealed that Samanea saman leaflets open more completely and close less completely as temperature is increased. We now demonstrate that, as temperature is increased, extensor cells release more H(+) during their swelling phase (opening), but flexor motor cells release less H(+) during their swelling phase (closure).

Effects of Temperature on H Secretion and Uptake by Excised Flexor Cells during Dark-Induced Closure of Samanea Leaflets.

Previous studies reveal that dark-induced closure of Samanea leaflets is accompanied by H(+) secretion from flexor motor cells. We now report that flexor tissue excised in the light, incubated in a weakly buffered bathing solution, and then darkened at different temperatures (18 degrees C-30 degrees C) acidified the medium (indicating net H(+) efflux) at all temperatures tested, but most rapidly at the highest temperature. However, pH changes reversed direction after 20 to 70 minutes; the lower the temperature, the later pH reversal occurred, and the lower the pH at reversal and after 45 minutes. These data provide a basis for the previously reported promotive effect of low temperature on dark-induced leaflet closure, assuming net H(+) and K(+) fluxes are opposite in direction. Net H(+) efflux at all temperatures tested was greater when the impermeant molecule iminodiacetate replaced small permeant anions in the bathing solution, suggesting that H(+) uptake is coupled to anion uptake, probably via a H(+)/anion symport system. When permeant anions were deficient, the amount of malate in the tissue increased, presumably by new synthesis. Malate synthesis would substitute for H(+)/anion uptake in charge balance and in providing H(+) for cytoplasmic pH regulation.

Light-stimulated inositolphospholipid turnover in Samanea saman leaf pulvini.

Leaflets of Samanea saman open and close rhythmically, driven by an endogenous circadian clock. Light has a rapid, direct effect on the movements and also rephases the rhythm. We investigated whether light signals might be mediated by increased inositolphospholipid turnover, a mechanism for signal transduction that is widely utilized in animal systems. Samanea motor organs (pulvini) labeled with [(3)H]inositol were irradiated briefly (5-30 sec) with white light, and membrane-localized phosphatidylinositol phosphates and their aqueous breakdown products, the inositol phosphates, were examined. After a 15-sec or longer light pulse, labeled phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate decreased and their labeled metabolic products inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate increased, changes characteristic of inositolphospholipid turnover. We conclude that inositolphospholipid turnover may act as a phototransduction mechanism in Samanea pulvini in a manner that is similar to that reported in animal systems.

H Uptake and Release during Circadian Rhythmic Movements of Excised Samanea Motor Organs : Effects of Mannitol, Sorbitol, and External pH.

We investigated H(+) fluxes during circadian rhythmic movements of Samanea saman leaflets by monitoring the pH of a weakly buffered medium bathing extensor or flexor motor tissue excised at different times during 51 hours of darkness. Experiments were made in media of two different osmotic potentials: -0.3 megapascal (control medium) and -1.2 megapascals (control medium supplemented with 0.4 molar mannitol or sorbitol). Both extensor and flexor tissue took up H(+) from the control medium at all times when the initial pH was 5.5. Rates of uptake by the extensor varied rhythmically in phase with the leaflet movement rhythm, whereas rates for the flexor were similar at all times. Addition of 0.4;molar mannitol (or sorbitol) to the medium magnified the amplitude of the rhythm in H(+) uptake and release by extensor tissue and revealed a rhythm with flexor tissue. In the flexor, mannitol promoted H(+) release (or reduced H(+) uptake) at all times. We propose that mannitol reduces flexor cell turgor, and that low turgor activates the H(+) pump. The magnitude and/or direction of pH changes varied with the initial pH of the medium. The pH values after 60 minutes converged to a narrow range, suggesting that cell wall pH might be regulated.

Phosphatidylinositol Cycle Metabolites in Samanea saman Pulvini.

The major metabolites of the phosphatidylinositol cycle from extracts of [(32)PO(4)]- and [(3)H]-inositol-labeled Samanea saman pulvini were separated. The membrane localized phosphoinositides were separated by thin layer chromatography, identified by comparison with purified lipid standards, and quantitated based on incorporation of radiolabel. The ratio of radioactivity in phosphatidylinositol:phosphatidylinositol 4-phosphate:phosphatidylinositol 4,5-bisphosphate is about 32:8:1. The aqueous inositol phosphates were separated by anion exchange chromatography using conventional liquid chromatography and by high performance liquid chromatography (HPLC) and were identified by comparison with standards. Analysis by HPLC reveals that (32)P-labeled pulvini have inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate that co-migrate with red blood cell inositol phosphates, but (3)H-inositol-labeled pulvini appear to have a variant profile.

Isolation of soluble metabolites of the phosphatidylinositol cycle from Samanea saman.

An improved protocol for the separation of inositol phosphates by high performance liquid chromatography was used to resolve inositol phosphates from pulvini (motor organs) of the legume, Samanea saman. The pulvini contained inositol phosphate, inositol bisphosphate, and inositol trisphosphate isomers which co-migrated with those of mammalian red blood cells, and one or more other inositol metabolites which, to our knowledge, have not been previously noted in preparations of inositol phosphates. The finding of inositol phosphates in Samanea which comigrate with mammalian inositol phosphates supports the possibility that the phosphatidylinositol cycle may function in signal transduction in plants as well as in animals.

Chronobiology of Aging in Albizzia julibrissin: I. An Automated, Computerized System for Monitoring Leaflet Movement; The Rhythm in Constant Darkness.

We are using leaflet movements in Albizzia julibrissin as a model system for investigating the chronobiology of aging. To monitor leaflet movements during long dark periods with adequate temporal and spatial resolution, we designed an automated, computerized photoelectric monitoring system. Each of 12 leaflet pairs was positioned in an individual light-proof container, with one leaflet immobilized. The angle of the mobile leaflet was monitored by a photosensor array using a low intensity infrared beam. Leaflet position was determined by custom-developed software, using information on the shading patterns of the sensors. Data on leaflet angle as a function of time were collected and stored on a floppy disc and then printed in numerical and graphical form.Oscillations of young, middle-aged, and old leaflets persist during 7 d of darkness with a periodicity close to 24 h. Period length appears to be age-independent, but rhythmic wave form is age-dependent. The older the leaflet, the earlier and more completely it opens and the less completely it closes.

Extensor and flexor protoplasts from samanea pulvini : I. Isolation and initial characterization.

Protoplasts were isolated from extensor and flexor regions of open pulvini of the nyctinastic tree Samanea saman. Both types of protoplasts undergo many changes during isolation. Extensor protoplasts are univacuolate in vivo, but some become multivacuolate. All flexor protoplasts are univacuolate. In an open pulvinus, extensor cells have a higher osmotic pressure than flexor cells. However, both types of protoplasts can be isolated with optimal yield using the same osmoticum (0.5 molar sorbitol) in the digestion medium. This suggests that some leakage of osmoticum occurs during harvest or digestion, especially from extensor tissue. Despite these changes, both types of protoplasts extrude protons in response to 10 micromolar fusicoccin (1.6-1.8 nanoequivalent/10(6) protoplasts/minute), demonstrating that the protoplasts are metabolically active and that proton transport mechanisms must be at least partially functional. The changes in vacuolar structure and osmotic pressure are what one might expect if the protoplasts, which are isolated from open pulvini, take on characteristics of cells in a closed pulvinus.

Extensor and Flexor Protoplasts from Samanea Pulvini : II. X-Ray Analysis of Potassium, Chlorine, Sulfur, Phosphorus, and Calcium.

Concentrations of K, Cl, P, S, and Ca in extensor and flexor protoplasts from open pulvini of the nyctinastic tree Samanea saman were estimated using x-ray microanalysis. This technique is particularly suitable when absolute numbers of protoplasts are low, because less than 100 protoplasts are required to obtain statistically significant data. Flexor protoplasts contain similar concentrations of P and S but almost twice as much K and Cl as extensor protoplasts. Low levels of total measurable osmoticum suggest that extensive leakage has occurred during protoplast isolation. Both extensor and flexor protoplasts appear to contain some unidentified osmoticum not detectable by x-ray analysis. Extensor protoplasts must have more unidentified osmoticum to compensate for their lower levels of K and Cl.

H fluxes in excised samanea motor tissue : I. Promotion by light.

Previous investigators revealed that white light-promoted leaflet opening in Samanea saman (Jacq) Merrill depends upon K(+) uptake by extensor cells and efflux from flexor cells of the pulvinus, while dark-promoted closure depends upon K(+) fluxes in the opposite directions. We now monitored H(+) fluxes during pulvinar movement to test a model proposing coupled H(+)/K(+) fluxes. H(+) fluxes were monitored by measuring changes in the pH of a weakly buffered solution (initial pH = 5.5) bathing excised strips of extensor or flexor tissue. White light at hour 3 of the usual dark period promoted pulvinar opening, H(+) efflux from extensor cells and uptake by flexor cells, while darkness at hours 2 to 4 of the usual light period promoted pulvinar closure, H(+) uptake by extensor cells and efflux from flexor cells. The following conditions altered H(+) fluxes during dark-promoted closure. (a) Light reversed the directions of the fluxes in both extensor and flexor cells. (b) Anoxia increased the rate of H(+) uptake by extensor cells and promoted H(+) uptake (rather than efflux) by flexor cells, consistent with an outwardly directed H(+) pump. KCN showed similar effects initially, but they were transient. (c) Increase in external pH from 5.5 to 6.7 promoted H(+) efflux (rather than uptake) by extensor cells and increased the rate of H(+) efflux from flexor cells, presumably by decreasing the rate of inward diffusion. (d) Change in external K(+) did not alter H(+) fluxes by extensor cells, but removal of external K(+) decreased the rate of H(+) efflux from flexor cells by 70%. These observations support a model for coupled H(+)/K(+) fluxes in pulvinar cells during light-and dark-promoted leaflet movements.

H Fluxes in Excised Samanea Motor Tissue : II. Rhythmic Properties.

Homogeneous groups of cells were excised at regular intervals from opposing (extensor and flexor) motor tissue of Samanea saman (Jacq) Merrill maintained in white light for 34 hours. H(+) fluxes between the tissue and bathing solution were then monitored during 30 minutes of darkness. Flux rates in both cell types vary with circadian rhythms. Flexor cells secrete H(+) to the medium during two-thirds of the circadian cycle and take up H(+) during the remainder of the cycle, while extensor cells take up H(+) from the medium during the entire cycle.

Effects of Age, Water Stress, and 1-Aminocyclopropane-1-carboxylic Acid on Leaflet Movement in Albizzia julibrissin.

The movement patterns of Albizzia julibrissin leaflets transferred from light to darkness differ for leaflets of different age: the older the leaflet, the slower and less completely it closes. Water stress, which enhances ethylene synthesis in other plants, and 1-aminocyclopropane-1-carboxylic acid (ACC), precursor of ethylene, both mimic the effect of aging by reducing the rate and extent of dark-induced closure. Brief far-red compared to red irradiation before darkness does not appear to alter the closure of young leaflets, but far-red preirradiation retards the closure of middle-aged and old leaflets floating on water, and middle-aged leaflets treated with ACC. A change in some membrane property and/or cell wall lignification are suggested as possible explanations for the alteration of leaflet movement.

Elemental analysis of freeze-dried thin sections of Samanea motor organs: barriers to ion diffusion through the apoplast.

Leaflet movements in the legume Samanea saman are dependent upon massive redistribution of potassium (K), chloride (Cl), and other solutes between opposing (extensor and flexor) halves of the motor organ (pulvinus). Solutes are known to diffuse through the apoplast during redistribution. To test the possibility that solute diffusion might be restricted by apoplastic barriers, we analyzed elements in the apoplast in freeze-dried cryosections of pulvini using scanning electron microscopy/x-ray microanalysis. Large discontinuities in apoplastic K and Cl at the extensor-flexor interface provide evidence for a barrier to solute diffusion. The barrier extends from the epidermis on upper and lower sides of the pulvinus to cambial cells in the central vascular core. It is completed by hydrophobic regions between phloem and cambium, and between xylem rays and surrounding vascular tissue, as deduced by discontinuities in apoplastic solutes and by staining of fresh sections with lipid-soluble Sudan dyes. Thus, symplastic pathways are necessary for ion redistribution in the Samanea pulvinus during leaflet movement. In pulvini from leaflets in the closed state, all cells on the flexor side of the barrier have high internal as well as external K and Cl, whereas cells on the extensor side have barely detectable internal or external K or Cl. Approximately 60% of these ions are known to migrate to the extensor during opening; all return to the flexor during subsequent closure. We propose that solutes lost from shrinking cells in the outer cortex diffuse through the apoplast to plasmodesmata-rich cells of the inner cortex, collenchyma, and phloem; and that solutes cross the barrier by moving through plasmodesmata.

Apoplastic transport of ions in the motor organ of Samanea.

Turgor-mediated leaf movements of the legume Samanea saman are associated with the migration of K(+) and Cl(-) between opposing sides of the motor organs (pulvini). We have investigated the pathway of this ion migration by localizing K(+) and Cl(-) within the secondary pulvinus at various times during leaf movements. Ion distributions in freeze-dried cryosections of pulvini were determined by scanning electron microscopy/x-ray microanalysis. The results indicate that the apoplast is a major pathway for the migration of K(+) and Cl(-) within the pulvinus.