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Summary The pulvinus of bean ( Phaseolus vulgaris L) responds to unilateral photo-excitation by phototropic curvature. Osmotically active solutes and water are transported from its exposed to the opposite sector of its motor tissue, resulting in differential changes in turgor pressure in these sectors and generation of a trans-pulvinar torque. A null-point approach was used to non-invasively study these dynamic changes in the terminal leaflet of bean. A variable torque was applied perpendicular to the midrib, to restrain laminar movement by precisely and continuously counteracting the generated torque. This equilibrium prevented curvature of the pulvinus and the associated opposite axial changes in volume in the opposite sectors of its motor tissue. The laminar torques measured were used to estimate stresses (changes in turgor pressure) generated within the motor tissue. These stress values were used to derive the corresponding changes in osmotic pressures and in solute concentration. Skotonastically downfolded leaflets were excited with white light to study their combined dynamic response to photonastic and phototropic excitation. Photonastically unfolded (horizontal) leaflets were excited with blue and red light, alone and in combination, to determine the spectral dependence of the dynamic pulvinar responses tophototropic excitation by itself.

Leaf movements in Mimosa pudica, are in response to thermal stress, touch, and light or darkness, appear to be regulated by electrical, hydrodynamical, and chemical signal transduction. The pulvinus of the M. pudica shows elastic properties. We have found that the movements of the petiole, or pinnules, are accompanied by a change of the pulvinus morphing structures. After brief flaming of a pinna, the volume of the lower part of the pulvinus decreases and the volume of the upper part increases due to the redistribution of electrolytes between these parts of the pulvinus; as a result of these changes the petiole falls. During the relaxation of the petiole, the process goes in the opposite direction. Ion and water channel blockers, uncouplers as well as anesthetic agents diethyl ether or chloroform decrease the speed of alert wave propagation along the plant. Brief flaming of a pinna induces bidirectional propagation of electrical signal in pulvini. Transduction of electrical signals along a pulvinus induces generation of an action potential in perpendicular direction between extensor and flexor sides of a pulvinus. Inhibition of signal transduction and mechanical responses in M. pudica by volatile anesthetic agents chloroform or by blockers of voltage gated ion channels shows that the generation and propagation of electrical signals is a primary effect responsible for turgor change and propagation of an excitation. There is an electrical coupling in a pulvinus similar to the electrical synapse in the animal nerves.

Summary The light-driven responses of the terminal leaflet of bean were analyzed spectrally and functionally. Laminar elevation increases rapidly in response to continuous overhead exposure of its pulvinus to blue light. This response is enhanced in its early stages by simultaneous exposure to red light. The pulvinus responds similarly to continuous overhead unmixed red, or far-red light, albeit at much lower rates. The response to overhead red, alone, or during enhancement of the response to blue, was not affected by simultaneous far-red. However, the response to blue alone, or enhanced by mixture with red, was partially inhibited by simultaneous exposure to far-red. The results suggest that the response to blue resulted mostly from a blue-absorbing pigment system, but may involve some absorption by phytochrome, while responses to red or far-red, with and without blue, may be mediated by high-irradiance responses of phytochrome. Functional differences between the responses to red and blue become apparent when the abaxial (lower), or lateral sectors of the pulvinus are exposed to them, separately and in combination. These differences suggest that red controls the photonastic unfolding of the pulvinus, whereas blue controls its phototropic responses. These responses co-exist in the same tissue, but are separate and additive.

Gravitropic curvature of pulvini of wheat and oat stem segments gradually declined with decreasing atmospheric O₂ concentration and was almost completely blocked under anoxia, whereas that of rice stem segments was enhanced under hypoxia and anoxia. Anoxia substantially increased the ethanol content in pulvini of gravistimulated stem segments in rice, wheat and oat, but the ethanol content showed no marked difference between rice pulvini and wheat and oat pulvini. The concentrations of exogenous ethanol and acetaldehyde required to inhibit the gravitropic curvature of pulvini were significantly higher in rice segments than in wheat and oat segments. However, in all three species, the concentrations of ethanol and acetaldehyde required to completely inhibit curvature were several-fold higher than the endogenous levels that accumulated in pulvini gravistimulated in N₂. The pulvini of rice segments gravistimulated in N₂ did not contain much more ATP than those of wheat or oat segments gravistimulated in N₂. When applied unilaterally to the pulvini of vertically oriented stem segments incubated in N₂, indole-3-acetic acid induced bending in rice stem segments but not in wheat and oat stem segments. Transference of graviresponsive pulvini of rice, as well as those of wheat and oat, from aerobic conditions to anaerobic conditions led to cessation of gravitropic curvature within several minutes, but subsequently only gravitropic curvature of anoxic rice pulvini was completely recovered within 2 h. A large portion of this recovery was blocked by cordycepin, a transcription inhibitor. These results suggested that anoxia-induced expression of any gene or genes enables rice pulvini to respond to gravistimulation under anaerobic conditions, and that such a gene or genes might be unrelated to ethanol fermentation and ATP production in anaerobic conditions.

Summary Movements of the terminal and lateral leaflets in the trifoliate leaf of pinto bean (Phaseolus vulgaris L.) cv. Bulgarian, in response to directional light, were studied by kinetic analysis. The pulvinule responds to unilateral light by contraction of the sector that intercepts the light and concomitant expansion of the opposite sector. The rate of the resulting laminar reorientation depends on the excitation of the photo-receptors in the pulvinule, which changes with angle of the incident light and as pulvinar topology changes in response to light. This dependence provides feed-back control of the eventual steady-state of the response. The pulvinule perceives directional light from all sides, but only as unilateral signals, and integrates these signals in its response. Phototropic responses of the pulvinule are modified by its asymmetry, which differs in the terminal and lateral leaflets. The asymmetry is expressed by axial torsion, superimposed on the positive phototropic curvature. The resulting laminar reorientations reflect this asymmetry. Laminar reorientation in directional light is a passive result of the pulvinar response and may combine changes in azimuth and axial rotation with changes in elevation.

Summary Methylxanthines (theophylline, aminophylline, caffeine) inhibit the seismonastic reaction of primary pulvini of Mimosa pudica leaves, showing that these compounds act on rapid turgor variations and the concomitant ion migrations. Compared with theophylline, aminophylline is more effective and caffeine is less effective in triggering the inhibitory effect. These compounds alter H + -ATPase activity as a rapid dose-dependent proton influx was noted about 4 min after application, indicating that an early impact may be at the plasmalemma. However, they have almost no effect on the motor cell membrane potential. Moreover, the compounds hinder the calcium transport. Not all of these inhibitory effects are mediated through an action on general cell metabolism since respiration was not affected even by rather long treatment (about 2 h).

Summary Proteins have been extracted from the distal pulvinus: petiole abscission zone and adjacent pulvinus and petiole tissues of primary leaves of Phaseolus vulgaris L. These have been compared by gel electrophoresis and immuno-blotting to determine if the polypeptide profile of the abscission zone differs, either qualitatively or quantitatively, from those of the other two tissues. Enrichment of the abscission zone extract by immuno-absorption with antibodies to petiole and pulvinus extracts has revealed a polypeptide of c. 68 kDa which is prominent only in the abscission zone. By Western blotting and probing with a monoclonal antibody of predetermined specificity, two further polypeptides, of c. 45 kDa and c. 36 kDa, have been shown also to be expressed preferentially in the abscission zone. The c. 36 kDa polypeptide appears to be membrane-bound. The identification of such proteins is discussed with respect to evidence that specific target cells for the plant hormone ethylene are differentiated at the abscission zone.

An asymmetric (top vs. bottom) increase in invertase activity is elicited by gravistimulation in oat-shoot pulvini starting within 3 h after treatment. In order to analyze the regulation of invertase gene expression in this system, we examined the effect of gravistimulation on invertase mRNA induction. Total RNA and poly (A)+RNA, isolated from oat pulvini, and two oligonucleotide primers, corresponding to two conserved amino-acid sequences (NDPNG and WECPD) found in invertase from other species, were used for the polymerase chain reaction (PCR). A partial-length cDNA (550 base pairs) was obtained and characterized. There was a 52% deduced amino-acid sequence homology to that of carrot beta-fructosidase and a 48% homology to that of tomato invertase. Northern blot analysis showed that there was an obvious transient accumulation of invertase mRNA elicited by gravistimulation of oat pulvini. The mRNA was rapidly induced to a maximum level at 1 h following gravistimulation treatment and gradually decreased afterwards. The mRNA level in the bottom half of the oat pulvinus was significantly higher (five-fold) than that in the top half of the pulvinus tissue. The induction of invertase mRNA was consistent with the transient enhancement of invertase activity during the graviresponse of the pulvinus. These data indicate that the expression of the invertase gene(s) could be regulated by gravistimulation at the transcriptional and/or translational levels. Southern blot analysis showed that there were four genomic DNA fragments hybridized to the invertase cDNA. This suggests that an invertase gene family may exist in oat plants.

The role of auxin redistribution in the graviresponse of the leaf-sheath pulvinus of oat (Avena sativa L.) was assessed using 3H-indole-3-acetic acid (3H-IAA) preloaded into isolated pulvini. When pulvini were totally isolated from subtending nodal tissue as well as leaf-sheath and internode, gravistimulation failed to induce an asymmetric growth response. Presence of either the nodal tissue or the internode/leaf-sheath tissue was sufficient to restore a normal graviresponse. Gravistimulation of totally isolated pulvini inhibited basipetal export of label (i.e., 3H-IAA) without generating any asymmetry of label within the pulvinus. In contrast, gravistimulation of pulvini with nodes intact generated an asymmetric distribution of label (initiation by 1 h; final ratio, lower/upper = 1.5) as well as the upward bending response. The kinetics of formation of the asymmetry of label paralleled the kinetics of initiation of the asymmetric growth response. The addition of 0.1 M sucrose to all agar blocks shortened both the time to initiation of label redistribution and the time to initial upward bending. However, sucrose did not change the final magnitude of label asymmetry although it increased the final steady state bending rate four fold. The inhibitors of polar auxin transport N-1-naphthylphthalamaic acid (NPA), 2,3,5-triiodobenzoic acid (TIBA), morphactin, naringenin, kaempferol and myricetin all significantly decreased the bending response of oat pulvini, but this inhibition was less than 50%. In contrast, TIBA and naringenin (each at 100 micromoles), effectively eliminated the redistribution of label, but did not eliminate the bending response. These results indicate that the active basipetal export of auxin is inhibited by gravistimulation of the oat pulvinus, while active lateral transport is induced. It is concluded that, while lateral transport of auxin occurs following gravistimulation, it is not necessary for a graviresponse. Other processes, such as localized changes in tissue responsiveness or the conversion of conjugated hormone to free (active) hormone, may suffice to drive the graviresponse.

Summary The activity of fixed charges of isolated, morphologically intact extensor cell walls of the laminar pulvinus of the primary leaves of Phaseolus coccineus L. was determined by measuring the electric potential generated by the fixed negative charges. The activity of fixed charges depends strongly on the pH and KCl concentration of the bathing solution. The maximum activity measured was approx. 570 meq 1 -1 at pH 5 in 100 mM KCl and 400 meq 1 -1 at pH 6 in 10 mM KCl, respectively. The apparent maximum activity of fixed charges in the walls of living extensor cells (250 meq 1 -1 in 10 mM KCl) is lower than in isolated cell walls. The apparent pKa value of 3.1 calculated from the titration curves of isolated extensor cell walls in both 100 mM and 10 mM KCl indicates that the carboxyl-groups of pectins are the source of the fixed charges. In isolated cell walls the mean concentration of fixed anions as calculated from the K + concentration in the walls is lower than that calculated from the wall potential. This indicates that a large fraction of the cell wall solution is unaffected by the fixed anions, i.e. may be divided into a water free space and Donnan free space. The volume of the Donnan free space is estimated to be 55 % of the total wall water or 2.9 ml g -1 dry weight. We suggest that, due to the high activity of fixed negative charges, the Donnan free space of the extensor cell wall serves as a K + reservoir which is used to change the cell volume via the pH of the apoplastic solution.

Summary Vanden Driessche (1978) proposed that EDTA prevents the seismonastic leaf movement (the closing movement) of the pinnules (tertiary pulvini) of Mimosa via inhibition of the contraction of Ca 2 ?,?-regulated contractile proteins. However, the effects of EGTA and EDTA on the leaf movement of the laminar pulvinus of Phaseolus coccineus L. and the primary pulvinus of Mimosa pudica L. are contrary to the effects measured at the tertiary pulvini (pinnules) of Mimosa or Al- bizzia. EGTA and EDTA (5 to 25 m M) inhibit the upward (opening) movement and induce a downward (closing) movement of the laminar pulvinus of Phaseolus and the primary pulvinus of Mimosa. High EGTA and EDTA concentrations «freeze» these pulvini in the night (closed) position of the leaves, whereas the tertiary pulvini of Mimosa and Albizzia in the day (open) position. However, there is evidence that these contrary leaf movement reactions of the different pulvini are due to structural differences and not to different actions of these substances. In excised flexor and extensor tissues of the laminar pulvinus of Phaseolus EDTA and EGTA induce a netto efflux of osmotica in hypotonic solutions. DMSO (5 to 10%, v/v), which change also the permeability of membranes, induces, similar to EDTA, an opening of the pinnules (tertiary pulvini) of Mimosa in light and dark and inhibit the seismonastic or dark-induced closing movement. However, in the laminar pulvinus of Phaseolus , DMSO (3 to 5 %) induces a closing movement and «freezes» the lamina in the physiological night position. We conclude therefore, that leakage of osmotica induces night (closed) position in such pulvini, which are lowered during the night and elevated during the day, and day (open) position in such pulvini, which are elevated during the night and lowered during the day. Thus experiments with EDTA give no evidence that contractile proteins play an essential role in the mechanism of circadian, nyctin- astic, or seismonastic leaf movements.

Summary Auxins were the only growth regulators capable of inducing root formation in leaf cuttings with a basal pulvinus. These roots were usually formed in the petiole above the pulvinus. Although NAA and IAA stimulated root initiation IBA showed the most marked promotive effect. Application of ABA did not affect rooting and was found in higher levels in the lower part of the petiole than in the pulvinus. The transport of labelled material (IAA and ABA) did not help the understanding of the inhibitory effect of the pulvinus. The presence of substances with similar chromatographic pattern of standard oestrogens in the basic fraction of extracts of the pulvinus suggests the possibility of its involvement in the inhibitory effect of the pulvinus on the rooting process.

Summary Leaves of Lavatera cretica L. were exposed to different constant levels of TO (at Al = +15° +30° or + 60°C), or BO excitation (at AI = −15°, −30°, or −60°), along their midvein. After 90, or 150min, they were transferred to light regimes that were not vectorially effective (white, red or darkness) and the time-course of the subsequent changes in LE was recorded. These changes are the expression of solute (and water) fluxes in the sector of pulvinar motor tissue subtending the midvein, whose associated photoreceptors had been vectorially excited. The diaphototropic response was non-inductive and required continuous VE. When such VE was terminated by darkness, the immediate reorientation was inertial, i.e. its direction was dictated by that of the preceding VE. This short-lived phase was replaced by the primary post-inertial phase, during which LE fell, irrespective of whether the preceding VE had been TO, or BO. However, the subsequent reorientation differed qualitatively when preceded by TO, or BO. Kinetic analysis showed that the post-inertial phases in laminar reorientation were quantitatively modified by the level (AI) and duration of the preceding VE. Thus, the laminar reorientation in darkness was not nyctinastic, but induced by the preceding VE. Non vectorial white or red light, had marked and different effects on the kinetics of both post-inertial phases, suggesting the involvement of different photoreceptors.

Summary Microelectrode technique was used to determine the activity of fixed charges of cell walls of various tissues of primary leaves of Phaseolus coccineus L. by measuring the electric potential differences generated by the fixed negative charges of the isolated but morphologically intact walls. An unequal distribution of the activity of fixed charges was found in the walls of parenchyma and collenchyma cells of the petiole: at the layer facing the cell lumen the activity was low (approx. 220 meq 1 −1 in 100 mM KCl at pH 7) compared to that of the wall layer facing the intercellularies in parenchyma cells (approx. 550 meq 1 −1 in 100 mM KCI, pH 7) or of the thickened corners of the collenchyma cell walls (approx. 610 meq 1 -1 in 100 mM KCI, pH 7). In contrast, the activity of fixed charges determined in pulvinar motor cells at the wall layers facing the cell lumen was high. In both the primary and secondary pulvinus the walls of motor cells located above the stele exhibited a higher activity of fixed charges (approx. 640–680 meq 1 −1 in 100 mM KCI, pH 7) than the motor cells located below the stele (approx. 550–600 meq 1 −1 in 100 mM KCI, pH 7). The results indicate that wall layers rich in pectic substances exhibit high fixed charge activities. Consequently, the walls of most tissues should be characterized by a layering of fixed charge activities corresponding to the layering in the biochemical composition. The functional importance of the negative fixed charges of the cell wall and of their heterogenous distribution is discussed with respect to the activity of wall-bound enzymes, membrane transport processes and the role of walls as cation reservoirs.

Summary Earlier work suggested that glycine uptake by the pulvinus motor cell of Mimosa pudica is a H + -cotransport mechanism. This hypothesis is sustained by the present investigations showing that glycine is transported electrogenically across the motor cell membrane as evidenced by concentration — dependent depolarizations observed upon addition of glycine in the range 1–10 mM. On the contrary, fusicoccin (0.1–10μM)elicited fast and great hyperpolarization. This antagonistic action of glycine and fusicoccin on membrane potential is in agreement with the antagonistic effects of these substances on the pulvinus movements previously observed. The data corroborate the assumption that glycine is taken up by pulvinus tissue by a H + -cotransport mechanism driven by a proton-electrochemical gradient at the plasmalemma and provide support for the model proposed previously on pulvinus functioning involving proton fluxes.

Summary Microautoradiographic methods applied to the pulvinar region of Phaseolus vulgaris L. revealed specific patterns of ion distribution according to the phases of upward or downward movements of the leaves: When feeding was taking place during the dark phase of the diurnal cycle, the main source of radioactivity was the dorsal groove of the vascular core on the flexor side. During the phase of leaf opening, 86 Rb and 43 K were mainly found within the parenchymatous bundle sheath. As an additional similarity to the guard cell physiology, toxic metal ions were accumulated in the cells of the bundle sheath when introduced into the plant during the light phase. Additional experiments on the influence of osmotic gradients supported the view that the characteristic distribution of ions during the two phases of the diurnal cycle is the result of regulatory processes controlling water and solute flux from the sites of storage to the evaporating surfaces. The conclusion was that in light the cells of the bundle sheath removed the ions from the flexor sites and that this process is substantially controlled by transpiration rates. Findings of other authors were discussed in view of the hypothesis that K + movements in the pulvinar region depend on regulatory systems other than active ion pumps. Among the processes probably involved in the initiation of primary water flux changes are the import of carbohydrates into the cells of the bundle sheath and starch hydrolysis.

Summary Change of extracellular ion concentration in the main pulvinus of Mimosa pudica L. during a slow downward movement induced by white light (0.24 mW cm-2) was investigated using K+- and Cl--selective electrodes and a pH electrode. The photostimulation induced a transient and concurrent efflux of K+ and Cl- from the motor cells and acidification of extracellular pH in the main pulvinus.