Search

Early prediction of compound absorption by cells is of considerable importance in the building of an integrated scheme describing the impact of a compound on intracellular biological processes. In this scope, we study the structure-activity relationships of several benzoic acid-related phenolics which are involved in many plant biological phenomena (growth, flowering, allelopathy, defense processes). Using the partial least squares (PLS) regression method, the impact of molecular descriptors that have been shown to play an important role concerning the uptake of pharmacologically active compounds by animal cells was analyzed in terms of the modification of membrane potential, variations in proton flux, and inhibition of the osmocontractile reaction of pulvinar cells of Mimosa pudica leaves. The hydrogen bond donors (HBD) and hydrogen bond acceptors (HBA), polar surface area (PSA), halogen ratio (Hal ratio), number of rotatable bonds (FRB), molar volume (MV), molecular weight (MW), and molar refractivity (MR) were considered in addition to two physicochemical properties (logD and the amount of non-dissociated form in relation to pKa). HBD + HBA and PSA predominantly impacted the three biological processes compared to the other descriptors. The coefficient of determination in the quantitative structure-activity relationship (QSAR) models indicated that a major part of the observed seismonasty inhibition and proton flux modification can be explained by the impact of these descriptors, whereas this was not the case for membrane potential variations. These results indicate that the transmembrane transport of the compounds is a predominant component. An increasing number of implicated descriptors as the biological processes become more complex may reflect their impacts on an increasing number of sites in the cell. The determination of the most efficient effectors may lead to a practical use to improve drugs in the control of microbial attacks on plants.

The fourth basic circuit element, a memristor, is a resistor with memory that was postulated by Chua in 1971. Here we found that memristors exist in vivo. The electrostimulation of the Mimosa pudica by bipolar sinusoidal or triangle periodic waves induce electrical responses with fingerprints of memristors. Uncouplers carbonylcyanide-3-chlorophenylhydrazone and carbonylcyanide-4-trifluoromethoxy-phenyl hydrazone decrease the amplitude of electrical responses at low and high frequencies of bipolar sinusoidal or triangle periodic electrostimulating waves. Memristive behavior of an electrical network in the Mimosa pudica is linked to the properties of voltage gated ion channels: the channel blocker TEACl reduces the electric response to a conventional resistor. Our results demonstrate that a voltage gated K(+) channel in the excitable tissue of plants has properties of a memristor. The discovery of memristors in plants creates a new direction in the modeling and understanding of electrical phenomena in plants.

Salicylic acid (o-hydroxy benzoic acid) (SA) induced a rapid dose-dependent membrane hyperpolarization (within seconds) and a modification of the proton secretion (within minutes) of Mimosa pudica pulvinar cells at concentrations higher than 0.1mM. Observations on plasma membrane vesicles isolated from pulvinar tissues showed that SA acted directly at the membrane level through a protonophore action as suggested by the inhibition of the proton gradient and the lack of effect on H(+)-ATPase catalytic activity. Comparative data obtained with protonophores (carbonylcyanide-m-chlorophenylhydrazone and 2,4-dinitrophenol) and inhibitors of ATPases (vanadate, N,N'-dicyclohexylcarbodiimide, and diethylstilbestrol) corroborated this conclusion. Consequently, the collapse of the proton motive force led to an impairment in membrane functioning. This impairment is illustrated by the inhibition of the ion-driven turgor-mediated seismonastic reaction of the pulvinus following SA treatment. SA acted in a specific manner as its biosynthetic precursor benzoic acid induced much milder effects and the m- and p-OH benzoic acid derivatives did not trigger similar characteristic effects. Therefore, SA may be considered both a membrane signal molecule and a metabolic effector following its uptake in the cells.

Background and Aims: Two aspects of the competence of abscission zone cells as a specific class of hormone target cell are examined. The first is the competence of these target cells to respond to a remote stele-generated signal, and whether ethylene acts in concert with this signal to initiate abscission of the primary leaf in Phaseolus vulgaris. The second is to extend the concept of dual control of abscission cell competence. Can the concept of developmental memory that is retained by abscission cell of Phaseolus vulgaris post-separation in terms of the inductive/repressive control of beta-1,4-glucan endohydrolase (cellulase) activity exerted by ethylene/auxin be extended to the rachis abscission zone cells of Sambucus nigra?, Methods: Abscission assays were performed using the leaf petiole-pulvinus explants of P. vulgaris with the distal pulvinus stele removed. These (-stele) explants do not separate when treated with ethylene and require a stele-generated signal from the distal pulvinus for separation at the leaf petiole-pulvinis abscission zone. Using these explants, the role of ethylene was examined, using the ethylene action blocker, 1-methyl cyclopropene, as well as the significance of the tissue from which the stele signal originates. Further, leaf rachis abscission explants were excised from the compound leaves of S. nigra, and changes in the activity of cellulase in response to added ethylene and auxin post-separation was examined., Key Results: The use of (-stele) explants has confirmed that ethylene, with the stele-generated signal, is essential for abscission. Neither ethylene alone nor the stelar signal alone is sufficient. Further, in addition to the leaf pulvinus distal to the abscission zone, mid-rib tissue that is excised from senescent or green mid-rib tissue can also generate a competent stelar signal. Experiments with rachis abscission explants of S. nigra have shown that auxin, when added to cells post-separation can retard cellulase activity, with activity re-established with subsequent ethylene treatment., Conclusions: The triggers that initiate and regulate the separation process are complex with, in bean leaves at least, the generation of a signal (or signals) from remote tissues, in concert with ethylene, a requisite part of the process. Once evoked, abscission cells maintain a developmental memory such that the induction/repression mediated by ethylene/auxin that is observed prior to separation is also retained by the cells post-separation.

The seismonastic movement of Mimosa pudica is triggered by a sudden loss of turgor pressure. In the present study, we compared the cell cytoskeleton by immunofluorescence analysis before and after movement, and the effects of actin- and microtubule-targeted drugs were examined by injecting them into the cut pulvinus. We found that fragmentation of actin filaments and microtubules occurs during bending, although the actin cytoskeleton, but not the microtubules, was involved in regulation of the movement. Transmission electron microscopy revealed that actin cables became loose after the bending. We injected phosphatase inhibitors into the severed pulvinus to examine the effects of such inhibitors on the actin cytoskeleton. We found that changes in actin isoforms, fragmentation of actin filaments and the bending movement were all inhibited after injection of a tyrosine phosphatase inhibitor. We thus propose that the phosphorylation status of actin at tyrosine residues affects the dynamic reorganization of actin filaments and causes seismonastic movement.

In Zea mays L., changes in orientation of stems are perceived by the pulvinal tissue, which responds to the stimulus by differential growth resulting in upward bending of the stem. Gravity is perceived in the bundle sheath cells, which contain amyloplasts that sediment to the new cell base when a change in the gravity vector occurs. The mechanism by which the mechanical signal is transduced into a physiological response is so far unknown for any gravity perceiving tissue. It is hypothesized that this involves interactions of amyloplasts with the plasma membrane and/or ER via cytoskeletal elements. To gain further insights into this process we monitored amyloplast movements in response to gravistimulation. In a pharmacological approach we investigated how the dynamics of plastid sedimentation are affected by actin and microtubule (MT) disrupting drugs. Dark grown caulonemal filaments of the moss Physcomitrella patens respond to gravity vector changes with a reorientation of tip growth away from the gravity vector. MT distributions in tip cells were monitored over time and MTs were seen to accumulate preferentially on the lower flank of the tip 30 min after a 90 degree turn. Using a self-referencing Ca2+ selective ion probe, we found that growing caulonemal filaments exhibit a Ca2+ influx at the apical dome, similar to that reported previously for other tip growing cells. However, in gravistimulated Physcomitrella filaments the region of Ca2+ influx is not confined to the apex, but extends about 60 micrometers along the upper side of the filament. Our results indicate an asymmetry in the Ca2+ flux pattern between the upper and side of the filament suggesting differential activation of Ca2+ permeable channels at the plasma membrane., (c2003 COSPAR. Published by Elsevier Ltd. All rights reserved.)

Maize (Zea mays) stem gravitropism involves differential elongation of cells within a highly specialized region, the stem internodal pulvinus. In the present study, we investigated factors that control gravitropic responses in this system. In the graviresponding pulvinus, hexose sugars (D-Glc and D-Fru) accumulated asymmetrically across the pulvinus. This correlated well with an asymmetric increase in acid invertase activity across the pulvinus. Northern analyses revealed asymmetric induction of one maize acid invertase gene, Ivr2, consistent with transcriptional regulation by gravistimulation. Several lines of evidence indicated that auxin redistribution, as a result of polar auxin transport, is necessary for gravity-stimulated Ivr2 transcript accumulation and differential cell elongation across the maize pulvinus. First, the auxin transport inhibitor, N-1-naphthylphthalamic acid, inhibited gravistimulated curvature and Ivr2 transcript accumulation. Second, a transient gradient of free indole-3-acetic acid (IAA) across the pulvinus was apparent shortly after initiation of gravistimulation. This temporarily free IAA gradient appears to be important for differential cell elongation and Ivr2 transcript accumulation. This is based on the observation that N-1-naphthylphthalamic acid will not inhibit gravitropic responses when applied to pulvinus tissue after the free IAA gradient peak has occurred. Third, IAA alone can stimulate Ivr2 transcript accumulation in non-gravistimulated pulvini. The gravity- and IAA-stimulated increase in Ivr2 transcripts was sensitive to the protein synthesis inhibitor, cycloheximide. Based on these results, a two-phase model describing possible relationships between gravitropic curvature, IAA redistribution, and Ivr2 expression is presented.

This study focuses on the characterization of protein phosphorylation in the gravitropic response in oat shoot pulvini through the use of inhibitors of this process, namely staurosporine, okadaic acid and sodium fluoride. These three inhibitors reduce gravitropic curvature and cause changes in the phosphorylation of 38- and 50-kDa soluble proteins which show different levels of phosphorylation between lower and upper halves of gravistimulated pulvini. A kinetic analysis of phosphorylation shows that the 38- and 50-kDa soluble proteins exhibit different levels of phosphorylation between lower and upper halves of graviresponsive pulvini at 5 min after initiation of gravistimulation of stems. In addition, the phosphorylation of 63- and 70-kDa proteins from a total membrane preparation increases in lower halves of the pulvini following gravistimulation. These phosphoproteins are not found in the plasma membrane fraction. Taken together, at least four kinds of phosphoproteins are gravi-related. Of these, the 38- and 50-kDa soluble phosphoproteins may be involved in the regulation of early stages of the gravitropic response.

Segments can be cut from the peduncular-1 internode of oat (Avena sativa L.) shoots so as to contain the graviresponsive leaf-sheath pulvinus and gibberellin-sensitive internodal tissue. Incorporation of [14C]glucose was used to monitor cell wall synthesis in these two tissues as affected by gravistimulus, indoleacetic acid (IAA), gibberellic acid (GA3), and fusicoccin (FC). Pulvinar cell wall synthesis was promoted by IAA and FC (both within about 1 h), as well as by gravistimulus (starting between 3 and 6 h), whereas GA3 had no effect on nongravistimulated pulvini. In contrast, GA3 and FC promoted internodal cell wall synthesis (initiated between 1 and 2 h), whereas IAA and gravistimulus caused a decrease in internodal uptake. FC preferentially promoted incorporation into the matrix component of the wall in both tissues. Gravistimulus failed to increase responsiveness of pulvinar tissue to IAA, whereas GA3 partially overcame gravistimulus-promoted incorporation into pulvinar cell wall, probably because of preferential movement of label into the rapidly elongating internode. The results demonstrate that these eight stimulus/tissue combinations can be examined easily in an isolated 10-mm stem segment, providing new opportunities for the comparative study of tissue- and stimulus-specific events in gene regulation and signal transduction in agronomically important cereals.

During the gravitropic response, auxin-sensitivity of the lower flanks of leaf-sheath pulvini of Avena sativa (oat) is at least 1000-fold higher than those of the upper flanks and non-gravistimulated pulvini. When the pulvini are treated with 1 mM Ca2+, a 10-fold increase in auxin-sensitivity of the pulvini is observed. Related to this difference in auxin-sensitivity, in vitro activation of the vanadate-sensitive H(-)-ATPase by IAA was observed. Results show that the activation of the H(+)-ATPase by IAA is probably mediated by soluble protein factors and that the H(+)-ATPase prepared from the lower flanks is activated by IAA with a 1000-fold higher auxin-sensitivity as compared with that from the upper flanks of the graviresponding pulvini. Ammonium sulfate fractionation experiments show that these soluble protein factors are in the 30 to 60% fraction. Auxin-binding assays reveal that lower flanks contain more high-affinity soluble auxin-binding sites (kD; on the order of 10(-9) M) and less low-affinity soluble auxin-binding sites (kD; on the order of 10(-6) M) than upper flanks. It is concluded that differential auxin-sensitivity of graviresponding oat-shoot pulvini is achieved by the modulation of affinities of auxin-binding sites in upper and lower flanks of the pulvini, that Ca2+ is involved in such modulation, and that one of the probable cellular functions of these auxin binding sites is the activation of the proton pump on the plasma membranes.

Leaf-sheath pulvini of excised segments from oat (Avena sativa L.) were induced to grow by treatment with 10 micromoles indole-3-acetic acid (IAA), gravistimulation, or both, and the effects of calcium, EGTA, and calcium channel blockers on growth were evaluated. Unilaterally applied calcium (10 mM CaCl2) significantly inhibited IAA-induced growth in upright pulvini but had no effect on growth induced by either gravity or gravity plus IAA. Calcium alone had no effect on upright pulvini. The calcium chelator EGTA alone (10 mM) stimulated growth in upright pulvini. However, EGTA had no effect on either IAA- or gravity-induced growth but slightly diminished growth in IAA-treated gravistimulated pulvini. The calcium channel blockers lanthanum chloride (25 mM), verapamil (2.5 mM), and nifedipine (2.5 mM) greatly inhibited growth as induced by IAA (> or = 50% inhibition) or IAA plus gravity (20% inhibition) but had no effect on gravistimulated pulvini. Combinations of channel blockers were similar in effect on IAA action as individual blockers. Since neither calcium ions nor EGTA significantly affected the graviresponse of pulvini, we conclude that apoplastic calcium is unimportant in leaf-sheath pulvinus gravitropism. The observation that calcium ions and calcium channel blockers inhibit IAA-induced growth, but have no effect on gravistimulated pulvini, further supports previous observations that gravistimulation alters the responsiveness of pulvini to IAA.

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.

Using Avena sativa L. cv. Victory oat seedlings and excised p-1 stem segments (including the p-1 and p-2 internodes) the effect of exogenously supplied ethylene and the removal of ethylene on internodal extension and gravitropic bending was assessed. Similarly, the ability of the excised system to respond to gravistimulation was assessed in the presence of inhibitors of ethylene action (AgNO3) and ethylene synthesis (3,5-diiodo-4-hydroxybenzoic acid and benzyl isothiocyanate; BITC). The production of ethylene from both intact and excised systems was also measured from 0 to 48 h after gravistimulation, relative to vertical controls. Although gravitropic curvature is initiated, and indeed enters the most rapid phase of upward bending during the first 6 h, there is no difference in ethylene production between vertical and geostimulated plants during this period. The ethylene production of gravistimulated plants rises sharply to a maximum at 24 h, then decreases steeply to almost the control level by 48 h, at which time the rate of upward curvature is diminishing. Neither the addition nor removal of ethylene, nor the addition of inhibitors affecting ethylene-action (AgNO3) or synthesis (DIHB) influence gravitropic bending or internodal extension in excised segments. Although the ethylene synthesis inhibitor BITC slowed down the rate of upward bending, this effect could not be reversed by addition of ethylene. We conclude that the burst in ethylene production that develops in leaf-sheath bases (pulvini) after they have started to curve upwards is not primary to the induction of curvature. We further suggest that ethylene has no major effect or role in the induction of upward bending after gravistimulation. The metabolism of high specific activity gibberellin A1 ([3H]-GA1) in the excised system was assessed during 1, 2 and 4 h of gravistimulation. Changes in endogenous GAs and GA metabolism have been shown previously to be correlated (at the later stages) with gravistimulated bending in intact Avena shoots. The excised segments "leaked" free [3H]-GAs and [3H]-GA glucosyl conjugate-like substances into the bathing medium, and this was a confounding factor. Nevertheless, gravistimulated stem segments, and especially the bottom half of the segment, were significantly less leaky then vertical segments. Thus, just 1 h after gravistimulation, bottom segment halves retained 22% more precursor [3H]-GA1, 36% more free [3H]-GA-like metabolites, and 48% more [3H]-GA glucosyl conjugate-like metabolites than vertical segments. In contrast, the 1 h gravistimulated top halves retained slightly less (1-4%) precursor [3H]-GA1 and free [3H]-GA metabolites, but 21% more [3H]-GA glucosyl conjugate-like radioactivity than vertical segments.

Starch in pulvinus amyloplasts of barley (Hordeum vulgare cv Larker) disappears when 45-day-old, light-grown plants are given 5 days of continuous darkness. The effect of this loss on the pulvinus graviresponse was evaluated by following changes in the kinetics of response during the 5-day dark period. Over 5 days of dark pretreatment, the lag to initial graviresponse and the subsequent half-time to maximum steady state bending rate increased significantly while the maximum bending rate did not change. The change in response to applied indoleacetic acid (100 micromolar) plus gibberellic acid (10 micromolar) without gravistimulation, under identical dark pretreatments, was used as a model system for the response component of gravitropism. Dark pretreatment did not change the lag to initial response following hormone application to vertical pulvini, but both the maximum bending rate and the half-time to the maximum rate were significantly reduced. Also, after dark pretreatment, significant bending responses following hormone application were observed in vertical segments with or without added sucrose, while gravistimulation produced a response only if segments were given sucrose. These results indicate that starch-filled amyloplasts are required for the graviresponse of barley pulvini and suggest that they function in the stimulus perception and signal transduction components of gravitropism.

Pulvini of excised segments from oats (Avena sativa L. cv Victory) were treated unilaterally with indoleacetic acid (IAA) or gibberellic acid (GA3) with or without gravistimulation to assess the effect of gravistimulation on hormone action. Optimum pulvinus elongation growth (millimeters) and segment curvature (degrees) over 24 hours were produced by 100 micromolar IAA in vertical segments. The curvature response to IAA at levels greater than 100 micromolar, applied to the lower sides of gravistimulated (90 degrees) pulvini, was significantly less than the response to identical levels in vertical segments. Furthermore, the bending response of pulvini to 100 micromolar IAA did not vary significantly over a range of presentation angles between 0 and 90 degrees. In contrast, the response to IAA at levels less than 10 micromolar, with gravistimulation, was approximately the sum of the responses to gravistimulation alone and to IAA without gravistimulation. This was observed over a range of presentation angles. Also, GA3 (0.3-30 micromolar) applied to the lower sides of horizontal segments significantly enhanced pulvinus growth and segment curvature, although exogenous GA3 over a range of concentrations had no effect on pulvinus elongation growth or segment curvature in vertical segments. The response to GA3 (10 micromolar) plus IAA (1.0 or 100 micromolar) was additive for either vertical or horizontal segments. These results indicate that gravistimulation produces changes in pulvinus responsiveness to both IAA and GA3 and that the changes are unique for each growth regulator. It is suggested that the changes in responsiveness may result from processes at the cellular level other than changes in hormonal sensitivity.

Pulvini of excised stem segments from barley (Hordeum vulgare cv Larker') were pretreated with 1 millimolar coumarin before gravistimulation to reduce longitudinal cell expansion and exaggerate radial cell enlargement. The cellular localization and pattern of graviresponse across individual pulvini were then evaluated by cutting the organ in cross-section, photographing the cross-section, and then measuring pulvinus thickness and the radial width of cortical and epidermal cells in enlargements of the photomicrographs. With respect to orientation during gravistimulation, we designated the uppermost point of the cross-section 0 degrees and the lowermost point 180 degrees. A gravity-induced increase in pulvinus thickness was observable within 40 degrees of the vertical in coumarin-treated pulvini. In upper halves of coumarin-treated gravistimulated pulvini, cells in the inner cortex and inner epidermis had increased radial widths, relative to untreated gravistimulated pulvini. In lower halves of coumarin-treated pulvini, cells in the central and outer cortex and in the outer epidermis showed the greatest increase in radial width. Cells comprising the vascular bundles also increased in radial width, with this pattern following that of the central cortex. These results indicate (a) that all cell types are capable of showing a graviresponse, (b) that the graviresponse occurs in both the top and the bottom of the responding organ, and (c) that the magnitude of the response increases approximately linearly from the uppermost point to the lowermost. These results are also consistent with models of gravitropism that link the pattern and magnitude of the graviresponse to graviperception via statolith sedimentation.

The negative gravitropic response in the grass leaf-sheath pulvinus is a consequence of cell elongation involving all cells except those of the uppermost region of the upper flank of an horizontally oriented pulvinus. The lowermost layer of cells elongate maximally, and the regions in between elongate to intermediate extents. The resulting curvatures of a responding pulvinus can be expressed mathematically by relating the angle of curvature (theta) to the original length (L0) and the maximal length of the lower surface (L1) and the diameter of the organ (D), using the equation, theta = (L1-L0)/D, where theta is in radians. The elongation response (S) of any individual cells within the pulvinus can be expressed by the equation, S = 0.5 - r cos theta, where r is the radius of the pulvinus and theta is in degrees. Microscopic measurement of cell lengths in different regions of the pulvinus supports the mathematical predictions. Indirect support is also obtained from the use of colchicine, coumarin, dichlorobenzonitrile (DCBN) and isopropyl N-chlorophenyl carbamate which exaggerate the inherent asymmetry during gravitropic response. Coumarin and DCBN also induce thickenings in the radial walls which appear first in the statenchyma, and later, in cells located towards the outer periphery of the pulvinus. The distribution patterns of these thickenings suggest that the asymmetric growth response of the pulvinus may be due to a differential and radial, centrifugal transport of growth promotors from the central statenchyma region.