The Rhythmic Leaf Movements after Regeneration of Partially Excised Pulvinus in Phaseolus vulgaris L.

Unlike the petiole or stem, the laminar pulvinus of the primaryleaves of Phaseolus vulgaris L. regenerated after a partialexcision. The histological and physiological aspects of theseregeneration processes have been studied. On the third day afterthe excision of the flexor (or extensor) region, the pulvinuswas regenerated. When the major part of the extensor was cutaway, the period and phase of the circadian leaf movements wereunchanged whereas the amplitude was greatly reduced. When theflexor region was excised, period, phase and amplitude weremaintained. Some changes could be seen in the ultradian movementsafter excision of flexor as well extensor regions. (Received August 31, 1988; Accepted March 30, 1989)     

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_½ = 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.     

Protoplasts from Phaseolus coccineus L. Pulvinar Motor Cells Show Circadian Volume Oscillations

The circadian movement of the lamina of the primary leaf of Phaseolus coccineus is mediated by circadian volume changes of the extensor and flexor cells in the upper and lower half of the laminar pulvinus. Isolated protoplasts from the extensor, flexor, and flank cells of the pulvinus showed a circadian volume rhythm with a period longer than 24 h. In the case of the flexor protoplasts, we found a period length of 28 h, which is similar to that of the pulvinar cells in situ. In the extensor protoplasts, the volume rhythm was synchronized with 14-h light/10-h dark cycles. The larger volume was correlated with the early hours in the light period and the smaller volume with the dark period, as would be expected from the behavior of the extensor cells in situ.     

Water Relations in Pulvini from Samanea saman: I. Intact Pulvini

The movement of Samanea leaflets depends upon changes in the curvature of the pulvinus at the base of each leaflet. Pulvinar bending and straightening, in turn, are driven by the movement of water between opposing (extensor and flexor) sides of the pulvinus. Although water movement depends on water potential (Ψ) and thus on osmotic potential (π) and hydrostatic pressure (P), none of these parameters have been measured in Samanea. In this investigation, Ψ and π were measured and P was calculated for extensor and flexor tissues of excised, whole pulvini that were open in the light and closed in the dark. In fully open pulvini, π in the extensor was generally between 800 and 1000 milliosmol per kilogram and exceeded π in the flexor by 300 to 450 milliosmol per kilogram. In fully closed pulvini the reverse was true, with π in the flexor between 800 and 1000 milliosmol per kilogram, exceeding π in the extensor by 300 to 450 milliosmol per kilogram. To obtain approximate values of Ψ of pulvinar tissues, shallow cuts in extensor and flexor sides of oil-covered pulvini were filled with droplets of polyethylene glycol solutions of known Ψ. Droplets maintaining constant size were assumed to have the same Ψ as the tissue. Extensor and flexor halves of open pulvini had very different Ψ (extensor, about −1.4 MPa; flexor, about −0.3 MPa), but both sides of closed pulvini had similar Ψ (about −0.3 MPa). Measurements of Ψ and π and calculations of P indicate: (a) In open pulvini, P is about the same in extensor and flexor. The large Ψ gradient is caused by a large osmotic gradient. (b) In closed pulvini, P is approximately 50% higher in the flexor than in the extensor. This difference in P compensates for differences in π such that the Ψ gradient is small. (c) Pulvini close as P increases in the flexor and reopen as flexor P decreases; extensor P values are similar in open and closed pulvini.     

Effects of Light on the Membrane Potential of Protoplasts from Samanea saman Pulvini : Involvement of K Channels and the H -ATPase

Rhythmic light-sensitive movements of the leaflets of Samanea saman depend upon ion fluxes across the plasma membrane of extensor and flexor cells in opposing regions of the leaf-movement organ (pulvinus). We have isolated protoplasts from the extensor and flexor regions of S. saman pulvini and have examined the effects of brief 30-second exposures to white, blue, or red light on the relative membrane potential using the fluorescent dye, 3,3′-dipropylthiadicarbocyanine iodide. White and blue light induced transient membrane hyperpolarization of both extensor and flexor protoplasts; red light had no effect. Following white or blue light-induced hyperpolarization, the addition of 200 millimolar K⁺ resulted in a rapid depolarization of extensor, but not of flexor protoplasts. In contrast, addition of K⁺ following red light or in darkness resulted in a rapid depolarization of flexor, but not of extensor protoplasts. In both flexor and extensor protoplasts, depolarization was completely inhibited by tetraethylammonium, implicating channel-mediated movement of K⁺ ions. These results suggest that K⁺ channels are closed in extensor plasma membranes and open in flexor plasma membranes in darkness and that white and blue light, but not red light, close the channels in flexor plasma membranes and open them in extensor plasma membranes. Vanadate treatment inhibited hyperpolarization in response to blue or white light, but did not affect K⁺ -induced depolarization. This suggests that white or blue light-induced hyperpolarization results from activation of the H⁺ -ATPase, but this hyperpolarization is not the sole factor controlling the opening of K⁺ channels.     

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.     

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     

Potassium Flux and Leaf Movement in Samanea saman : I. Rhythmic Movement

Samanea leaflets usually open in white light and fold together when darkened, but also open and dose with a circadian rhythm during prolonged darkness. Leaflet movement results from differential changes in the turgor and shape of motor cells on opposite sides of the pulvinus; extensor cells expand during opening and shrink during closure, while flexor cells shrink during opening and expand during closure but change shape more than size. Potassium in both open and closed pulvini is about 0.4 N. Flame photometric and electron microprobe analyses reveal that rhythmic and light-regulated postassium flux is the basis for pulvinar turgor movements. Rhythmic potassium flux during darkness in motor cells in the extensor region involves alternating predominance of inwardly directed ion pumps and leakage outward through diffusion channels, each lasting ca 12 h. White light affects the system by activating outwardly directed K⁺ pumps in motor cells in the flexor region.     

K+ Gradients in the Pulvinus of Phaseolus coccineus during Leaf Movement*

We used quantitative histochemistry to investigate the tissue-specific compartmentation of potassium in the laminar pulvinus of Phaseolus coccineus L. at day and night positions of diurnal leaf movement. The assay was based on the potassium-dependent activation of pyruvate kinase. Total potassium levels of pulvini were higher in the light than in the dark [0.88 and 0.57mol (kg dry weight)^?1, respectively]. Transverse compartmentation of potassium was studied on three tissue slices, representing the middle part, petiolar and laminar sides of individual pulvini. These were dissected further into 10 distinct subsamples (bundle; motor tissues: extensor, flexor; flanks). In the day position the amount of potassium in the extensor was higher than in the night position [1.92 and 1.50 mol (kg dry weight)^?1, respectively]. Flexor changes were opposite [1.13 and 1.65 mol (kg dry weight)^?1, respectively]. In the day position there was a steep and consistent increase in potassium content from the innermost to the outermost zones of the extensor. In the night position this was much more variable. Comparable gradients were not detected in flexor samples. Here highest amounts of potassium were recovered from the middle of the motor tissue. The data specify distinct tissue regions involved in osmotic adjustment during leaf movement in Phaseolus coccineus.     

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.     

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)     

Protoplasts from Phaseolus coccineus L. Pulvinar Motor Cells Show Circadian Volume Oscillations

The circadian movement of the lamina of the primary leaf of Phaseolus coccineus is mediated by circadian volume changes of the extensor and flexor cells in the upper and lower half of the laminar pulvinus. Isolated protoplasts from the extensor, flexor, and flank cells of the pulvinus showed a circadian volume rhythm with a period longer than 24 h. In the case of the flexor protoplasts, we found a period length of 28 h, which is similar to that of the pulvinar cells in situ. In the extensor protoplasts, the volume rhythm was synchronized with 14-h light/10-h dark cycles. The larger volume was correlated with the early hours in the light period and the smaller volume with the dark period, as would be expected from the behavior of the extensor cells in situ.     

Potassium-linked Chloride Fluxes during Rhythmic Leaf Movement of Albizzia julibrissin

Transverse sections of Albizzia pulvinules were examined with an electron microprobe to determine ion fluxes associated with turgorcontrolled leaflet movements. K⁺ and Cl⁻ concentrations are high in the flexor and low in the extensor region of closed pulvini. Both ions migrate out of the flexor and into the extensor during opening as previously described for K⁺. The distribution of these elements is significantly correlated in each phase of the rhythmic cycle examined, but only 50 to 60% of the ionic charge of potassium is balanced by chloride. This value increases to 65 to 85% if one considers only the mobile fraction of the potassium.     

SECONDARY PULVINUS OF ROBINIA PSEUDOACACIA (LEGUMINOSAE): STRUCTURAL AND ULTRASTRUCTURAL FEATURES

The structure of the secondary pulvinus of Robinia pseudoacacia has been examined together with ultrastructural features of motor cells both in open and closed pulvini, to identify ultrastructural changes associated with leaflet movement. Pulvini have a central vascular core bordered by thick-walled collenchyma cells, which in turn are surrounded by several layers of cortical parenchyma cells. Cortical motor cells exhibit ultrastructural features similar to those reported in homologous cells of other pulvini. The vacuolar compartment contains two kinds of vacuoles: nontannin vacuoles, which change both in number and size during leaflet movement, and tannin vacuoles, which may act as an ion reservoir. No differences in wall thickness were found between flexor and extensor motor cells. Thick walls of collenchyma cells show numerous pits with plasmodesmata through which the phloem parenchyma cells and the inner cortical motor cells are connected. Tannin vacuoles and calcium oxalate crystals are common inclusions of phloem parenchyma cells. The tissue arrangement and the occurrence of pits with plasmodesmata in the central cylinder cells provide evidence of symplastic continuity through the central cylinder between the extensor and flexor regions of the motor organs. The greater amplitude of Robinia leaflet movements may be related to the extension of motor regions, the scarcity of lignification in the central vascular core, and the thin flexor walls.     

Isolation of functional extensor and flexor protoplasts fromPhaseolus coccineus L. pulvini: potassium induced swelling

Methods are described for the isolation of functional protoplasts from secondary pulvinus tissue (flexor and extensor) and from leaf mesophyll tissue of primary leaves ofPhaseolus coccineus L. Integrity of the protoplasts was shown by vital staining and their ability to evolve oxygen in the light. Extensor-cell protoplasts increased their volume for up to 60% upon addition of 10 mM KCl. This K⁺-induced swelling was accompanied by increased rates of proton extrusion.     

Pulvinus activity,leaf movement and leaf water-use efficiency of bush bean (<Emphasis Type="Italic">Phaseplus vulgaris</Emphasis> L.) in a hot environment

Pulvinus activity of Phaseolus species in response to environmental stimuli plays an essential role in heliotropic leaf movement. The aims of this study were to monitor the continuous daily pulvinus movement and pulvinus temperature, and to evaluate the effects of leaf movements, on a hot day, on instantaneous leaf water-use efficiency (WUE_i), leaf gas exchange, and leaf temperature. Potted plants of Phaseolus vulgaris L. var. Provider were grown in Chicot sandy loam soil under well-watered conditions in a greenhouse. When the second trifoliate leaf was completely extended, one plant was selected to measure pulvinus movement using a beta-ray gauging (BRG) meter with a point source of thallium-204 (²⁰⁴Tl). Leaf gas exchange measurements took place on similar leaflets of three plants at an air temperature interval of 33–42°C by a steady-state LI-6200 photosynthesis system. A copper-constantan thermocouple was used to monitor pulvinus temperature. Pulvinus bending followed the daily diurnal rhythm. Significant correlations were found between the leaf-incident angle and the stomatal conductance (R ² = 0.54; P < 0.01), and photosynthesis rate (R ² = 0.84; P < 0.01). With a reduction in leaf-incidence angle and increase in air temperature, WUE_i was reduced. During the measurements, leaf temperature remained below air temperature and was a significant function of air temperature (r = 0.92; P < 0.01). In conclusion, pulvinus bending followed both light intensity and air temperature and influenced leaf gas exchange.     

Changes in Morphometry and Elemental Composition of Robinia pseudoacacia Pulvinar Motor Cells During Leaflet Movements

Changes in shape and size of Robinia pulvinar cortical cellsin relation to leaflet movements have been investigated usingan image processing system applied to drawings of transverseand longitudinal pulvinar sections. Both the size and shapeof cell sections underwent change during movement. The dorsal-leftside region of the cortex has been characterized as the extensorregion which increases turgor during opening. Morphometric changesoccur throughout the cortical motor cells except in the threeor four inner layers. K, Cl, S, and Ca distribution in cellwalls and protoplasts of inner and outer motor cells have beenmeasured with X-ray microanalysis. The distribution patternof K and Cl shows that these ions are mainly responsible forturgor changes. K and Cl were simultaneously depleted in apoplastand protoplast, which suggests that cell walls do not possessa high enough ionic reservoir during Robinia leaflet movements.Ca was always higher in flexor cell walls than in extensor regionsof closed pulvini. This fact could be related to a lower abilityto extend of flexor cells which underwent fewer morphomerticchangrs during movement.     

Diurnal Changes in the Malic Acid Concentration in Phaseolus coccineus L. Pulvini

Concentration of malic acid was determined in pulvini and petiolesand in isolated parts of the pulvinus, i.e. extensor and flexorregions, in Phaseolus coccineus. In the light period of thecircadian cycle, the concentration of malic acid in whole pulvinireached the highest value of 35.1 mmole CW while in the darkphase the respective value was 21.0 mmole CW. In the petiole,the highest concentration of malic acid was only 15.3% of themaximum concentration in the whole pulvinus. In isolated regions of motor cells, a cyclic alternation inthe concentration of malic acid was observed. In the light phase,the maximum acid concentration of 43.7 mmole CW in the extensorzone corresponds with the lowest concentration of 15.5 mmoleCW in the flexor region. The lowest value of acid concentrationof 30.8 mmole CW in the extensor part corresponds with the highestacid concentration of 31.1 mmole CW in the flexor part in thedark phase. About 22% of the total concentration of malic acid was transportedbetween the two opposite parts of the pulvinus as dependingupon the phases of leaf movement. (Received February 28, 1986; Accepted May 23, 1986)     

A mechanism for the leaching of calcium from foliage

Young bean plants (Phaseolus vulgaris) containing root-absorbed ⁴⁵Ca and ⁸⁶Rb were leached to determine the pathway and mechanism of cation loss by leaching. Calcium is leached from the exchangeable calcium fraction within the plant by a process of ion exchange and diffusion involving exchange sites both within the leaf and on the leaf surface. Leaching of cations is primarily a passive process, although some metabolites may be deposited upon leaf surfaces by active processes. The exchange and diffusion explanation is compatible with current theories of ion uptake and translocation and explains the results of numerous experiments on leaching reported in the literature.     

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.