Proton excretion and cell expansion in bean leaves

Light-induced expansion of Phaseolus vulgaris L. leaf cells is accompanied by increased cell-wall plasticity. The possibility that leaf-cell walls are loosened by excreted protons has been investigated. First, light causes acidification, detected at the leaf surface, within 5–15 min. Growth starts 10–20 min after exposure to light. Second, exogenous acid induces loosening of isolated leaf cell walls. Third, infiltration of the tissue with a neutral buffer inhibits light-induced growth. Fourth, fusicoccin stimulates growth of as well as H⁺ excretion by bean leaf cells, without light. These findings show that the acid-growth theory is applicable to light-induced growth of leaf cells, and indicate that light-induced proton excretion initiates cell enlargement in leaves.Abbreviations FC fusicoccin - RL red light - WEx wall extensibility - WL white light     

Light-Regulated Gravitropism in Seedling Roots of Maize

Red light-induced changes in the gravitropism of roots of Zea mays variety Merit is a very low fluence response with a threshold of 10⁻⁹ moles per square meter and is not reversible by far red light. Blue light also affects root gravitropism but the sensitivity of roots to blue is 50 to 100 times less than to an equal fluence of red. In Z. mays Merit we conclude that phytochrome is the sole pigment associated with light-induced changes in root gravitropism.     

Bacterial rhodopsins monitored with fluorescent dyes in vesicles andin Vivo

Summary Three retinal-containing pigments have been detected inHalobacterium halobium membranes: bacteriorhodopsin (bR), halorhodopsin (hR), and slow-cycling rhodopsin (sR). The first two hyperpolarize the cell membrane by electrogenic transport of H⁺ and Cl^–, respectively. The third pigment, sR, may be a photosensory receptor since mutants lacking bR and hR retain their retinal-dependent phototaxis responses. We monitored light-induced changes in fluorescence of several voltage-sensitive dyes in cells and membrane vesicles. Red light-induced potential changes generated by bR and hR were similar to signals described previously. Signals generated by hR could be identified using four criteria: wavelength dependence, Cl^– dependence, shunting by valinomycin and K⁺, and the absence of these signals in hR-deficient mutants. The absence (detection limit 0.5 mV) of hyperpolarization signals in bR^–hR^–sR⁺ vesicles and cells shows that sR photochemical reactions are nonelectrogenic. Two signals independent of bR and hR were measured: blue light caused a decrease and red light an increase in dye fluorescence. Both signals appear to derive from sR on the basis of their retinal-dependence and action spectra. In a retinal-deficient mutant strain (Flx3R), both sR signals appeared after addition of all-trans retinal. In this strain retinal also restores phototaxis sensitivity within the same time scale. The retinal concentration dependence for all four parameters monitored—the attractant (red) and repellent (blue) phototaxis, and the red light and blue light-induced fluorescence signals—is the same. This correlation is consistent with the hypothesis that both attractant and repellent responses are mediated by sR, as suggested by Bogomolni and Spudich (Proc. Natl. Acad. Sci. USA.79:6250–6254 (1982)).     

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.     

ADVENTITIOUS ROOT DEVELOPMENT FROM THE COLEOPTILAR NODE IN ZEA MAYS L.

Department of Botany and Bacteriology, University of Arkansas, Fayetteville, Arkansas 72701 Zea mays L. root development from the coleoptilar node was observed by light and electron microscopy. Roots developed opposite collateral vascular bundles in the coleoptilar nodal region. Three distinct histogens (stelar, cortical-protoderm, and root cap) became evident in early development. In median sections of the young roots, root cap and cortical regions formed a “hat” configuration over the stelar region. As the root matured, this “hat” developed centripetally to encapsulate the stelar region. Central core cells of the root cap were characterized by having numerous dictyosomes, amyloplasts, vacuoles, and thin cell walls. As these cells matured into outer or peripheral cap cells, the Golgi vesicles became hypertrophied. These hypertrophied vesicles contained a granular PAS-positive material which accumulated between the plasma membrane and the cell wall and formed a thick layer. As the PAS-positive material passed through the cell wall, it changed to a fibrillar texture. A PAS-positive material similar to that in the outer root cap cells was found adjacent to the outer walls of the protodermal cells. In median sections, PAS-positive material was not present in the promeristem region. Root cap cells as well as parent cortical cells were crushed as the young root forced its way through the parent tissue.     

Transient light-induced changes in ion channel and proton pump activities in the plasma membrane of tobacco mesophyll protoplasts

Rapid, transient changes of the membrane potential upon lighttransitions are generally observed in microelectrode studies.In a patch-clamp study similar responses to light transitionswere found in current clamp. Corresponding with the changesof membrane potential, light-induced current changes in voltageclamp were observed. This paper evaluates the involvement ofoutward rectifying conductances and plasma membrane bound H⁺-ATpases(proton pump) to these light responses in mesophyll protoplastsof Nicotiana tabacum L. The contribution of K⁺-channels to theseresponses, could be minimized by variation of the holding potentialor addition of the K⁺-channel blocker verapamil. It was concludedthat light transitions modulate both proton pump and K⁺-channelactivity. Effects of light on membrane current were not observedin root cells and chlorophyll-deficient cells, suggesting thatthe response requires photosynthetic activity. However, blockersof photosystems I and II did not affect current changes. Key words: Light, patch-clamp, plasma membrane, tobacco, whole cell     

Interactions between a membrane sialoglycoprotein and planar lipid bilayers

Summary Bilayer membranes formed from lipids dissolved in decane were exposed to glycophorin, a sialoglycoprotein which had been extracted from human red cell membranes. The interaction with the bilayer produced an increase in the steady state electrical conductance of the membrane proportional to the amount added. Fluctuations in membrane current when the electrical potential difference was constant were observed concommitantly with this increase in membrane conductance. The minimum size of the fluctuations corresponds to a conductance of 10^–10 mho. The increase in conductance as well as the current fluctuations persisted after extensive washout of the chamber containing the protein (cisside). Subsequent addition of lectins (wheat germ agglutinin and phytohemoagglutinin) to the cis-side produced rupture of the membranes, whilst these hemoagglutinins added to the trans-side failed to produce an effect. Measurements of changes in surface potential using K⁺ nonactin as a probe indicated that glycophorin induces a negative surface charge. At high protein concentrations, the magnitude of the induced surface potential became independent of glycophorin concentration. The maximum number of charges introduced onto the membrane under these conditions was 1.4×10⁵/m². Cis (but not trans)-side addition of neuraminidase abolished these charges, indicating that they can be ascribed to the sialic acid residues that the protein bears. These results suggest that glycophorin incorporates into bilayer membranes with its N-terminal end (where the sialic acid and carbohydrates are located) facing the cis-side. Spectrin reversibly lowered the glycophorin-induced membrane conductance when added to the trans-side. Cis-side additions failed to produce an effect. Trypsin present on the trans-side irreversibly lowered the membrane conductance. These results indicate that parts of the glycophorin molecule, probably the C-terminal end, are accessible to reagents in the solution bathing the trans-side of the membrane. Thus glycophorin spans the planar bilayer in much the same way as it spans the red cell membrane.     

Comparative structure of vesicular-arbuscular mycorrhizas and ectomycorrhizas

During the establishment of vesicular-arbuscular mycorrhizas, fungal hyphae contact the root surface, form appressoria and initiate the internal colonization phase. Structural changes occur in the cell wall, the cytoplasm and the nucleus as the fungus progresses from a presymbiotic to a symbiotic phase. Nuclei in spores are in G₁ whereas in intraradical hyphae they are in G₁ and G₂. Changes in nuclear organization are evident in various stages in the colonization process. Dramatic changes in both symbionts occur as the nutrient exchange interface is established between arbuscules and root cortical cells. An interfacial matrix, consisting of molecules common to the primary wall of the cortical cell, separates the cortical cell plasma membrane from the fungal cell wall. Ectomycorrhizas are characterized structurally by the presence of a mantle of fungal hyphae enclosing the root and usually an Hartig net of intercellular hyphae characterized by labyrinthine branching. As hyphae contact the root surface, they may respond by increasing their diameter and switching from apical growth to precocious branching. The site of initial contact of hyphae may be either the root cap or the ‘mycorrhiza infection zone’. The mantle varies considerably in structure depending on both the plant and fungus genome. In some ectomycorrhizas, the mantle may be a barrier to apoplastic transport, and in most it may store polyphosphate, glycogen, lipids and perhaps protein.     

Red light enhancement of the phototropic response of etiolated pea stems

In the subapical third internode of 7-day-old etiolated pea seedlings, the magnitude of phototropic curvature in response to continuous unilateral blue illumination is increased when seedlings are pre-exposed to brief red light. The effect of red light on blue light-induced phototropism becomes manifest maximally 4 or more hours after red illumination, and closely parallels the promotive action of red light on the elongation of the subapical cells. Ethylene inhibits phototropic curvature by an inhibitory action on cell elongation without affecting the lateral transport of auxin. Pretreatment of seedlings with gibberellic acid causes increased phototropic curvature, but experiments using ¹⁴C-gibberellic acid indicate that gibberellic acid itself is not laterally transported under phototropic stimuli. Neither red light nor gibberellic acid treatment has any promotive effect on blue light-induced lateral transport of ³H-indoleacetic acid. Under conditions where phototropic curvature is increased by red light treatment, low concentrations of indoleacetic acid applied in lanolin paste to the apical cut end of the seedling cause an increased elongation response in subapical tissue. This could explain increased phototropic curvature caused by red light treatment.     

Root growth inhibition and ultrastructural changes in radish root tips after treatment with aqueous extracts of Fallopia japonica and F. ×bohemica rhizomes

Allelopathic compounds released by invasive alien plants can suppress the growth of plants in their vicinity. The aim of this study was to investigate changes in tissue and cell structure in roots of radish seedlings treated with 10% aqueous extracts of rhizomes from the invasive knotweeds Fallopia japonica and F. ×bohemica. After 7 days of growth without and with aqueous extracts from these rhizomes, the anatomical and ultrastructural changes in the radish seedling roots were analyzed with light and transmission electron microscopy, and hydrogen peroxide was localized with diaminobenzidine, to define oxidative stress. The roots of radish seedlings treated with the knotweed extracts were shorter and thicker, due to the shorter and wider shapes of their cortex cells, which were organized in more columns than the control roots. There were signs of cell damage and oxidative stress in the root cap cells, and to a lesser extent in the meristematic zone. As well as the irregularly shaped nuclei and plasma membrane detached from the cell wall, the most prominent ultrastructural effects in the root cap cells of these aqueous rhizome extracts were the ring-shaped form of the mitochondria and large endoplasmic reticulum bodies. Excessive vacuolization was seen for the cells of the root apical meristem.

     

Localization of intracellular calcium in root meristematic cells and root cap cells of maize

The localization of Ca²⁺ in cells of the periblem and dermatogen in the root meristem and the columella and peripheral cells of the root cap of maize was examined by the precipitation method of potassium pyroantimonate and EGTA-treatment. In periblem and dermatogen cells, Ca²⁺ was found to be localized in the nucleoplasm and granular zone of the nucleolus. Ca²⁺ was also found in most cell organelles: in the matrix in mitochondria, on the thylakoid membrane in proplastids, in the vacuoles and on the plasma membranes. Ca²⁺ was also distributed throughout the cytoplasmic ground matrix. Much Ca²⁺ was present in the cell wall soon after its formation during the cell division. Ca²⁺ was also conspicuous in the vesicles of Golgi in the dermatogen cells. In columella and peripheral cells, there was less Ca²⁺ in the organelles and cytoplasmic ground matrix, but Ca²⁺ was present in Golgi vesicles in the peripheral cells. Electron microscopic and X-ray microanalysis showed that Ca²⁺ was also present in the mucilaginous layer, the outermost cell wall of the peripheral cells.     

Extracellular charge adsorption influences intracellular electrochemical homeostasis in amphibian skeletal muscle

The membrane potential measured by intracellular electrodes, E_m, is the sum of the transmembrane potential difference (E₁) between inner and outer cell membrane surfaces and a smaller potential difference (E₂) between a volume containing fixed charges on or near the outer membrane surface and the bulk extracellular space. This study investigates the influence of E₂ upon transmembrane ion fluxes, and hence cellular electrochemical homeostasis, using an integrative approach that combines computational and experimental methods. First, analytic equations were developed to calculate the influence of charges constrained within a three-dimensional glycocalyceal matrix enveloping the cell membrane outer surface upon local electrical potentials and ion concentrations. Electron microscopy confirmed predictions of these equations that extracellular charge adsorption influences glycocalyceal volume. Second, the novel analytic glycocalyx formulation was incorporated into the charge-difference cellular model of Fraser and Huang to simulate the influence of extracellular fixed charges upon intracellular ionic homeostasis. Experimental measurements of E_m supported the resulting predictions that an increased magnitude of extracellular fixed charge increases net transmembrane ionic leak currents, resulting in either a compensatory increase in Na⁺/K⁺-ATPase activity, or, in cells with reduced Na⁺/K⁺-ATPase activity, a partial dissipation of transmembrane ionic gradients and depolarization of E_m.     

Inhibition by white light of rb absorption in the root apex of corn

Measurements of cell lengths made at 0.5 millimeter intervals in median longitudinal sections of the primary roots of corn (Zea mays) were used to construct a growth curve. The region 1.5 to 4.0 millimeters from the apex contained the largest number of elongating cells. Absorption of ⁸⁶Rb⁺ was measured using intact, dark-grown corn seedlings. Following uptake and exchange, the terminal 8.0 millimeters of each root was cut into four 2.0 millimeter segments. Maximum ⁸⁶Rb⁺ uptake occurred in the region from 0.0 to 4.0 millimeter from the root tip. Washing the intact primary root in fresh 2.0 millimolar CaSO₄ for 2 hours prior to uptake augmented the rate of ⁸⁶Rb⁺ uptake in all regions. Illumination with white light during washing caused a reduction of ⁸⁶Rb⁺ uptake as compared with controls washed in darkness, and the region of greatest light response was the region of elongation. Removal of the coleoptile prior to washing did not prevent the light inhibition of subsequent ⁸⁶Rb⁺ uptake. Removal of the root cap prior to washing in light partially reversed the light-induced inhibition of the washing response.     

Role of acid efflux during growth promotion of primary leaves of Phaseolus vulgaris L. by hormones and light

The white-light-(WL) induced enlargement of dicotyledonous leaf cells is known to occur via an acid-growth mechanism; i.e., WL causes leaf cells to excrete protons which lead to an increase in wall extensibility and thus cell enlargement. Gibberellic acid (GA₃) and N⁶-benzyladenine (BA) also induce leaf cell enlargement. To see if they also act via acid-induced cell wall loosening, a comparison has been made of WL-, GA₃-and BA-induced growth of strips, taken from primary leaves of bean (Phaseolus vulgaris L.) plants raised in continuous red light for 10 d. White light, GA₃ and BA all increased wall extensibility as measured by the Instron technique, and this change preceded the increase in growth rate. However, whereas WL induced significant proton excretion, neither GA₃ nor BA caused any acidification of the apoplast. Furthermore, neutral buffers, which effectively inhibited the growth induced by WL, were without effect on growth promoted by either GA₃ or BA. These results indicate that while WL, GA₃ and BA all initiate growth in bean leaves by altering cell-wall properties, GA₃ and BA do so through some wall loosening mechanism other than wall acidification. Neither gibberellin nor cytokinin is likely to play a major role in light-induced cell enlargement of dicotyledonous leaves.Abbreviations BA N^o-benzyladenine - FC fusicoccin - GA₃ gibberellic acid - RL red light - SK medium 10 mM sucrose+10mM KCl - WL white light     

Negative phototropism of rice root and its influencing factors

Some characteristics of the rice (Oryza sativa L.) root were found in the experiment of unilaterally irradiating the roots which were planted in water: (i) All the seminal roots, adventitious roots and their branched roots bent away from light, and their curvatures ranged from 25° to 60°. The curvature of adventitious root of the higher node was often larger than that of the lower node, and even larger than that of the seminal root, (ii) The negative phototropic bending of the rice root was mainly due to the larger growth increment of root-tip cells of the irradiated side compared with that of the shaded side, (iii) Root cap was the site of light perception. If root cap was shaded while the root was irradiated the root showed no negative phototropism, and the root lost the characteristic of negative phototropism when root cap was divested. Rice root could resume the characteristic of negative phototropism when the new root cap grew up, if the original cells of root cap were well protected while root cap was divested, (iv) The growth increment and curvature of rice root were both influenced by light intensity. Within the range of 0–100 μmol · m² -s⁻¹, the increasing of light intensity resulted in the decreasing of the growth increment and the increasing of the curvature of rice root, (v) The growth increment and the curvature reached the maximum at 30°C with the temperature treatment of 10–40°C. (vi) Blue-violet light could prominently induce the negative phototropism of rice root, while red light had no such effect. (vii) The auxin (IAA) in the solution, as a very prominent influencing factor, inhibited the growth, the negative phototropism and the gravitropism of rice root when the concentration of IAA increased. The response of negative phototropism of rice root disappeared when the concentration of IAA was above 10 mg · L⁻¹     

Surface charges on chloroplast membranes as studied by particle electrophoresis

1. Particle microelectrophoresis mobility studies have been conducted with chloroplast thylakoid membranes and with isolated intact chloroplasts.2. The pH dependence of the electrophoretic mobility indicated that at pH values above 4.3 both membrane systems carry a net negative charge.3. Chemical treatment of thylakoids has shown that neither the sugar residues of the galactolipids in the membrane nor the basic groups of the membrane proteins having pK values between 6 and 10 are exposed at the surface.4. However, treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide, together with glycine methyl ester, neutralized the negative charges on the thylakoid membrane surface indicating the involvement of carboxyl groups which, because of their pH sensitivity, are likely to be the carboxyl groups of aspartic and glutamic acid residues.5. The nature of the protein giving rise to the negative surface charges on the thylakoids is not known but is shown not to involve the coupling factor or the light harvesting $\text{chlorophyl}\text{a}\text{chlorophyll}\text{b}\text{pigment · protein complex}$.6. No significant effect of light was observed on the electrophoretic mobility of either thylakoids or intact chloroplasts.7. The striking difference in the ability of divalent and monovalent cations to screen the surface charges was demonstrated and explained in terms of the Gouy-Chapman theory.8. Calculations of the ζ-potentials for thylakoid membranes gave values for the charge density at the plane of shear to be in the region of one electronic charge per 1500–2000 Ų.9. The significance of the results is discussed in terms of cation distribution in chloroplasts and the effect of cations on photosynthetic phenomena.     

Red and blue light-stimulated proton efflux by epidermal leaf cells of the Argenteum mutant of Pisum sativum

Light stimulates leaf expansion in dicotyledons by increasingapoplastic acidification, cell wall loosening and solute accumulationfor turgor maintenance. Red and blue light enhance growth viadifferent photo-systems, but the cellular location and modesof action of these systems is not known. Here, the effect of red and blue light was studied on transportprocesses in epidermal cells of expanding leaves of the Argenteummutant of Pisum satlvum. Both red and blue light caused extraceiiuiaracidification by isolated epidermal tissue, which was stimulatedby extracellular K⁺ and inhibited by DCCD at 0.1 mol m^–3.Acidification induced by red compared with blue light showeddifferent saturating kinetics in fluence rate-response curves.Under near saturating light conditions the effects of red andblue light were additive. The red light-induced acidificationwas inhibited by far-red light while the blue light-inducedacidification was not. Light caused a hyperpoianzation of themembrane potential in epidermal strips, and stimulated ⁸⁶Rb⁺uptake by epidermal protoplasts. These results show that phytochromeand an additional blue light-photoreceptor function in isolatedepidermal cells to promote proton efflux, hyperpolarization,and cation uptake. Key words: Pisum sativum, light-induced acidification, ion transport, epidermis, photoreceptor     

Effects of lysolecithin on the surface properties of human erythrocytes

Human red blood cells (RBCs), transformed by incubation with the amphiphatic compound lysolecithin from their normal discocyte shape into echinocytes, have increased rates of agglutination in the presence of either poly- -lysine (PLL) or soybean agglutinin (SBA). Removal of lysolecithin by washing caused a reversal of shape back to the discocyte configuration and a lowering of agglutination rates. Methochlorpromazine, another amphiphatic echinocytogenic substance produced a similar increase in agglutination rates, suggesting that increased agglutinability may be a general property of echinocytes. Lysolecithin treatment of RBCs caused a decrease in the binding of cationized ferritin (CF) particles/μm² of RBC surface. The decrease in CF binding is due to a rearrangement of negative charge bearing molecules on the RBC surface rather than shedding of charged groups. These observations support the hypothesis that integral membrane proteins which bear negative charges and receptors are associated with a cytoskeleton within the red cell. Alterations in cell shape which result in distortion of the cytoskeleton may cause a redistribution of integral membrane proteins which bear charged groups at the RBC surface.     

Structural and quantitative changes of carbohydrate chain of erythrocyte membrane glycoproteins in experimental diabetes mellitus after treatment with agmatine

It was shown that the development of experimental diabetes mellitus accompanied by increase of desialylation of carbohydrate determinants of erythrocytes membrane glycoproteins, removal of both O-linked and N-linked oligosaccharides from the glycoproteins and decrease of erythrocyte membrane negative charges. Treatment of streptozotocin-induced diabetic rats with agmatine led to enhance the content of N-and O-glycans in the erythrocyte glycoproteins, increase in sialic acid content and restore the negative charge of the cell membrane. Detected changes in configuration of membrane components of red blood cells in diabetic animals after treatment with agmatine indicate circulating in the bloodstream cells with a repertoire of adhesion molecules and glycoprotein receptors, which are inherent to the population of young erythrocyte.     

A theory of charge separation,ion, electron and proton transport in photosynthetic membranes based on asymmetry of surface charges

We present a model for the light-induced charge separation, proton and ion transport across photosynthetic membranes based on an assumption of the transmembrane surface charge asymmetry. In dark equilibrium, this asymmetry gives rise to an internal membrane electric field whose direction is perpendicular to the membrane surfaces. The role of the field in the light-induced charge separation is similar to the function of the built-in electric field across a solid-state p-n junction. Light-generated free charge carriers in the membrane flow according to its direction and upon recombination on the surface give rise to an electrochemical potential difference for electrons across the membrane. The associated coupled electron-proton transport, and ion diffusion can be viewed as a response of the system to the light-induced redox and electric potential changes.