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S M Assmann

Publications and source records attributed to S M Assmann.

At least 37 records · Page 2Linked to original sources
PubMed1997

Laser-assisted patch clamping: a methodology.

Laser microsurgery can be used to perform both cell biological manipulations, such as targeted cell ablation, and molecular genetic manipulations, such as genetic transformation and chromosome dissection. In this report, we describe a laser microsurgical method that can be used either to ablate single cells or to ablate a small area (1-3 microns diameter) of the extracellular matrix. In plants and microorganisms, the extracellular matrix consists of the cell wall. While conventional patch clamping of these cells, as well as of many animal cells, requires enzymatic digestion of the extracellular matrix, we illustrate that laser microsurgery of a portion of the wall enables patch clamp access to the plasma membrane of higher plant cells remaining situated in their tissue environment. What follows is a detailed description of the construction and use of an economical laser microsurgery system, including procedures for single cell and targeted cell wall ablation. This methodology will be of interest to scientists wishing to perform cellular or subcellular ablation with a high degree of accuracy, or wishing to study how the extracellular matrix affects ion channel function.

Cell Fusion↗
PubMed1997

Seal-promoting solutions and pipette perfusion for patch clamping plant cells.

Patch-clamp technology has greatly increased our knowledge of plant membrane transport. However, the success of patch clamping crucially relies on establishing a high resistance (G omega) seal between the membrane and the patch-clamp pipette. This can prove problematic in many plant-cell preparations. It is therefore of great importance to develop protocols for protoplast isolation, maintenance and seal formation that improve seal rate. This study investigated whether the pH and the K+ and the Cl(-)concentration of the pipette solution had an effect on the seal formation. High pH and absence of K+ significantly promoted membrane sealing, whereas the concentration of Cl- had no effect. To reap the benefit of seal-promoting pipette solutions and yet retain the option to adjust this solution to experimental requirements, a pipette perfusion apparatus was implemented. The perfusion system was successfully applied in cell-attached patch, excised-patch and whole-cell configurations, using plasma membrane and tonoplast of three different species. The system enables complete solution exchange within minutes and is potentially of great benefit in the study of channel selectivity, the application of (cytoplasmic) channel blockers and the study of primary and secondary transport.

Arabidopsis↗
PubMed1996

A membrane-delimited effect of internal pH on the K+ outward rectifier of Vicia faba guard cells.

ABA stimulation of outward K+ current (IK,out) in Vicia faba guard cells has been correlated with a rise in cytosolic pH (pHi). However, the underlying mechanism by which IK,out is affected by pHi has remained unknown. Here, we demonstrate that pHi regulates outward K+ current in isolated membrane patches from Vicia faba guard cells. The stimulatory effect of alkalinizing pHi was voltage insensitive and independent of the two free calcium levels tested, 50 nM and 1 microM. The single-channel conductance was only slightly affected by pHi. Based on single-channel measurements, the kinetics of time-activated whole-cell current, and the analysis of current noise in whole-cell recordings, we conclude that alkaline pHi enhances the magnitude of IK,out by increasing the number of channels available for activation. The fact that the pHi effect is seen in excised patches indicates that signal transduction pathways involved in the regulation of IK,out by pHi, and by implication, components of hormonal signal transduction pathways that are downstream of pHi, are membrane-delimited.

Calcium↗
PubMed1996

Transport proteins of the plant plasma membrane.

Recently developed molecular and genetic approaches have enabled the identification and functional characterization of novel genes encoding ion channels, ion carriers, and water channels of the plant plasma membrane.

Biological Transport↗
PubMed1996

Laser microsurgery of higher plant cell walls permits patch-clamp access.

Plasma membranes of guard cells in epidermal peels of Vicia faba and Commelina communis can be made accessible to a patch-clamp pipet by removing a small portion (1-3 micrometers in diameter) of the guard cell wall using a microbeam of ultraviolet light generated by a nitrogen laser. Using this laser microsurgical technique, we have measured channel activity across plasma membranes of V. faba guard cells in both cell-attached and isolated patch configurations. Measurements made in the inside-out patch configuration revealed two distinct K(+)-selective channels. Major advantages of the laser microsurgical technique include the avoidance of enzymatic protoplast isolation, the ability to study cell types that have been difficult to isolate as protoplasts or for which enzymatic isolation protocols result in protoplasts not amenable to patch-clamp studies, the maintenance of positional information in single-channel measurements, reduced disruption of cell-wall-mediated signaling pathways, and the ability to investigate intercellular signaling through studies of cells remaining situated within tissue.

Cell Membrane↗
PubMed1994

A membrane-delimited pathway of G-protein regulation of the guard-cell inward K+ channel.

GTP-binding protein (G-protein) regulation of inward rectifying K+ channels in the plasma membrane of Vicia (Vicia faba L.) guard cells has previously been demonstrated at the whole-cell level. However, whether a cytosolic signal transduction chain is required for G-protein regulation of K+ channels in Vicia guard cells, or in any plant cell type, remains unknown. In the present study, we assayed effects of several G-protein regulators on inward K+ channels in isolated inside-out membrane patches from Vicia guard cell protoplasts. Guanosine 5'-[gamma-thio]triphosphate, a nonhydrolyzable GTP analog that locks G proteins into their activated state, decreased the open state probability (Po) of single inward K+ channels. This decrease in Po was accompanied by an increase in one of the closed time constants of the K+ channel. Guanosine 5'-[beta-thio]diphosphate, a GDP analog that locks G proteins into their inactivated state, slightly increased the Po of the inward K+ channel and shortened the closed time constants. Pertussis toxin and cholera toxin, which ADP-ribosylate G proteins at different sites, decreased the Po of the inward K+ channel. Our data indicate that G proteins can act via a membrane-delimited pathway to regulate inward K+ channels in the guard-cell plasma membrane.

Adenosine Triphosphate↗
PubMed1994

Inhibition of inward K+ channels and stomatal response by abscisic acid: an intracellular locus of phytohormone action.

Abscisic acid (ABA), a plant hormone whose production is stimulated by water stress, reduces the apertures of stomatal pores in the leaf surface, thereby lessening transpirational water loss. It has been thought that inhibition of stomatal opening and promotion of stomatal closure by ABA are initiated by the binding of extracellular ABA to a receptor located in the guard-cell plasma membrane. However, in the present research, we employ three distinct experimental approaches to demonstrate that ABA can act from within guard cells to regulate stomatal apertures. (i) The extent to which ABA inhibits stomatal opening and promotes stomatal closure in Commelina communis L. is proportional to the extent of ABA uptake, as assayed with [3H]ABA. (ii) Direct microinjection of ABA into the cytoplasm of Commelina guard cells precipitates stomatal closure. (iii) Application of ABA to the cytosol of Vicia faba L. guard-cell protoplasts via patch-clamp techniques inhibits inward K+ currents, an effect sufficient to inhibit stomatal opening. These results demonstrate an intracellular locus of phytohormone action and imply that the search for hormone receptor proteins should be extended to include intracellular compartments.

Abscisic Acid↗
PubMed1994

Cyclic AMP stimulates K+ channel activity in mesophyll cells of Vicia faba L.

Whole-cell patch-clamp recordings from Vicia faba mesophyll protoplasts reveal that outward K+ current is increased in a dose-dependent fashion by intracellular application of cAMP. The enhancement of the outward current by cAMP is specific and it cannot be mimicked by a series of nucleotides that includes AMP, cGMP, and GMP. The enhancement is evoked by micromolar concentrations of cAMP in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine. PKI or Walsh inhibitor, a specific peptide inhibitor of cAMP-dependent protein kinase (PKA), inhibits the outward K+ current. Adenosine 3',5'-phosphothioate, a competitive inhibitor of PKA, has a similar effect. Conversely, the catalytic subunit of PKA (cAMP independent) from bovine brain enhances the magnitude of the outward K+ current in the absence of added cAMP. Our results indicate that cAMP modulates K+ channel activity in mesophyll cells and suggest that this modulation occurs through a cAMP-regulated protein kinase.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Evidence for protein phosphatase 1 and 2A regulation of K+ channels in two types of leaf cells.

Ion channels control ion fluxes across membranes, membrane potential, and signal transduction between and within cells. Protein kinases and phosphatases are important regulators involved in stimulus-response coupling in eukaryotic organisms. We have identified in extracts of Vicia faba leaf cells protein phosphatase activities inhibited by okadaic acid (OA) and calyculin A (CA), two inhibitors of protein phosphatases 1 and 2A. Using whole-cell patch-clamp techniques, we have demonstrated that inward K+ currents in guard cells are inhibited by nanomolar concentrations of OA or CA, whereas outward K+ currents are not affected. However, the same inhibitors enhance the magnitude of outward K+ currents in mesophyll cells. A phosphatase antagonist, adenosine-5'-O-(3-thiotriphosphate), has an effect similar to OA and CA on outward K+ currents in mesophyll cells. Our findings suggest that protein phosphatases 1 and/or 2A play different physiological roles in modulating the activity of K+ channels in mesophyll cells and guard cells.

Adenosine Triphosphate↗
PubMed1994

Analysis of a soluble calmodulin binding protein from fava bean roots: identification of glutamate decarboxylase as a calmodulin-activated enzyme.

The identity of a soluble 62-kD Ca(2+)-dependent calmodulin binding protein (CaM-BP) from fava bean seedlings was determined. Using 125I-CaM overlay assays, a class of soluble CaM-BPs was detected in extracts of tissues comprising the axis of 1.5-week-old seedlings, excluding the root tip and emergent leaves. The size of these CaM-BPs was not uniform within all parts of the plant; the apparent molecular masses were 62 kD in roots, 60 kD in stems, and 64 kD in nodules. The root 62-kD CaM-BP was purified, and internal microsequence analysis was performed on the protein. A tryptic peptide derived from the CaM-BP consisted of a 13-residue sequence corresponding to a highly conserved region of glutamate decarboxylase (GAD), an enzyme that catalyzes the alpha-decarboxylation of glutamate to form the stress-related metabolite gamma-aminobutyrate. Activity assays of partially purified, desalted, root GAD revealed a 50% stimulation by the addition of 100 microM Ca2+, a 100% stimulation by the addition of 100 microM Ca2+ plus 100 nM CaM, and no appreciable stimulation by CaM in the absence of added Ca2+. The demonstration that plant GAD is a Ca(2+)-CaM-stimulated enzyme provides a model in which stress-linked metabolism is modulated by a Ca(2+)-mediated signal transduction pathway.

Amino Acid Sequence↗
PubMed1994

Inhibition of guard-cell inward K+ channels by abscisic acid: links and gaps in the signal transduction chain.

Abscisic acid (ABA) affects a number of ion transport mechanisms in guard cells. One effect of ABA is to inhibit inward K+ channels, an effect that can contribute to the inhibition of stomatal opening. One model of the signal transduction cascade mediating this response involves ABA-activation of a G-protein, which in turn activates phospholipase C, resulting in production of inositol 1,4,5-trisphosphate (IP3), elevation of cytosolic free Ca2+ levels (Cai), and activation of a Ca(2+)-dependent phosphatase (protein phosphatase 2B; PP2B) which mediates channel inhibition. A review of the literature reveals that several of the links in this putative signal transduction chain have been established. The G-protein activator, GTP gamma S, inhibits inward K+ currents. Exogenous IP3 inhibits inward K+ currents. Exogenous IP3 elevates Cai. Elevated Cai inhibits inward K+ currents, and the inhibition by ABA of inward K+ currents does require Ca2+. Exogenous PP2B inhibits inward K+ currents, and an endogenous Ca(2+)-dependent phosphatase activity is present in guard cells. However, significant gaps in the chain remain. There is no direct evidence that ABA activates a G-protein. The presence or absence of a G-protein-activated phospholipase C in guard cells has not been investigated. Elevation of Cai by ABA is variable, and the reasons for this variability remain to be established. It is not known whether or not activation of a guard-cell PP2B homolog is the exclusive mechanism by which Ca2+ inhibits the channels.

Abscisic Acid↗
PubMed1993

Immunosuppressants implicate protein phosphatase regulation of K+ channels in guard cells.

The elevation of Ca2+ levels in the cytoplasm inactivates inward-rectifying K+ channels that play a central role in regulating the apertures of stomatal pores in higher plants. However, the mechanism for the Ca(2+)-mediated inhibition of K(+)-channel function is unknown. Using patch-clamp techniques, we show that cyclophilin-cyclosporin A and FK506-binding protein-FK506 complexes, which are highly specific inhibitors of protein phosphatase 2B (calcineurin), block Ca(2+)-induced inactivation of K+ channels in Vicia faba guard cells. A constitutively active calcineurin fragment that is Ca(2+)-independent inhibits K(+)-channel activity in the absence of Ca2+. We have also identified an endogenous Ca(2+)-dependent phosphatase activity from V. faba that is inhibited by the cyclophilin-cyclosporin A and FK506-binding protein-FK506 complexes. Our findings implicate a Ca(2+)-dependent, calcineurin-like protein phosphatase in a Ca2+ signal-transduction pathway of higher plants.

Amino Acid Isomerases↗
PubMed1993

Characterization of a G-protein-regulated outward K+ current in mesophyll cells of vicia faba L.

Whole-cell voltage-dependent currents in isolated mesophyll protoplasts of Vicia faba were investigated by patch-clamp techniques. With 104 mM K+ in the cytosol and 13 mM K+ in the external solution, depolarization of the plasma membrane from -47 mV to potentials between -15 and +85 mV activated a voltage- and time-dependent outward current (Iout). The average magnitude of Iout at +85 mV was 28.5 +/- 3.3 pA.pF-1. No inward voltage-dependent current was observed upon hyperpolarization of the plasma membrane from -55 mV to potentials as negative as -175 mV. Time-activated outward current was blocked by Ba2+ (1 mM BaCl2) and was not observed when K+ was eliminated from the external and internal solutions, indicating that this outward current was carried primarily by K+ ions. The voltage dependency of outward K+ current revealed a possible mechanism for K+ efflux from mesophyll cells. A GDP analogue guanosine 5'-[beta-thio]diphosphate (500 microM) significantly enhanced outward K+ current. The outward K+ current was inhibited by the GTP analogue guanosine 5'-[gamma-thio]triphosphate (500 microM) and by an increase in cytoplasmic free Ca2+ concentrations. Cholera toxin, which ADP-ribosylates guanine nucleotide-binding regulatory proteins, also inhibited outward K+ current. These findings illustrate the presence in mesophyll cells of outward-rectifying K+ channels that are regulated by GTP-binding proteins and calcium.

Adenosine Diphosphate↗
PubMed1992

Permeation of Ca2+ through K+ channels in the plasma membrane of Vicia faba guard cells.

The whole-cell patch-clamp method has been used to measure Ca2+ influx through otherwise K(+)-selective channels in the plasma membrane surrounding protoplasts from guard cells of Vicia faba. These channels are activated by membrane hyperpolarization. The resulting K+ influx contributes to the increase in guard cell turgor which causes stomatal opening during the regulation of leaf-air gas exchange. We find that after opening the K+ channels by hyperpolarization, depolarization of the membrane results in tail current at voltages where there is no electrochemical force to drive K+ inward through the channels. Tail current remains when the reversal potential for permeant ions other than Ca2+ is more negative than or equal to the K+ equilibrium potential (-47 mV), indicating that the current is due to Ca2+ influx through the K+ channels prior to their closure. Decreasing internal [Ca2+] (Cai) from 200 to 2 nM or increasing the external [Ca2+] (Cao) from 1 to 10 mM increases the amplitude of tail current and shifts the observed reversal potential to more positive values. Such increases in the electrochemical force driving Ca2+ influx also decrease the amplitude of time-activated current, indicating that Ca2+ permeation is slower than K+ permeation, and so causes a partial block. Increasing Cao also (i) causes a positive shift in the voltage dependence of current, presumably by decreasing the membrane surface potential, and (ii) results in a U-shaped current-voltage relationship with peak inward current ca. -160 mV, indicating that the Ca2+ block is voltage dependent and suggesting that the cation binding site is within the electric field of the membrane. K+ channels in Zea mays guard cells also appear to have a Cai- and Cao-dependent ability to mediate Ca2+ influx. We suggest that the inwardly rectifying K+ channels are part of a regulatory mechanism for Cai. Changes in Cao and (associated) changes in Cai regulate a variety of intracellular processes and ion fluxes, including the K+ and anion fluxes associated with stomatal aperture change.

Calcium↗
PubMed1992

Cellular distribution of calmodulin and calmodulin-binding proteins in Vicia faba L.

The distribution of calmodulin (CaM) and CaM-binding proteins within Vicia faba was investigated. Both CaM and CaM-binding proteins were found to be differentially distributed among organs, tissues, and protoplast types. CaM levels, on a per protein basis, were found to be the highest in leaf epidermis, containing 3-fold higher levels of CaM than in total leaf. Similarly, guard cell and epidermal cell protoplasts were also found to have higher levels of CaM than mesophyll cell protoplasts. 125I-CaM blot overlay assays were performed to qualitatively examine CaM-binding proteins in these protoplast types as well as in whole tissues and organs. CaM-binding proteins with Mr 52,000, 78,000, and 115,000 were common in all metabolically active plant parts. Unique CaM-binding protein bands were detected in guard cell protoplasts (Mr 39,000, 88,000), stems (Mr 45,000, 60,000, 64,000), and roots (Mr 62,000), suggesting the presence of specialized CaM-dependent processes in these cells and organs.

Calmodulin↗
PubMed1992

Synergistic effect of light and fusicoccin on stomatal opening : epidermal peel and patch clamp experiments.

Upon incubation of epidermal peels of Commelina communis in 1 millimolar KCl, a synergistic effect of light and low fusicoccin (FC) concentrations on stomatal opening is observed. In 1 millimolar KCl, stomata remain closed even in the light. However, addition of 0.1 micromolar FC results in opening up to 12 micrometers. The same FC concentration stimulates less than 5 micrometers of opening in darkness. The synergistic effect (a) decreases with increasing FC or KCl concentrations; (b) is dark-reversible; (c) like stomatal opening in high KCl concentrations (120 millimolar) is partially inhibited by the K(+) channel blocker, tetraethyl-ammonium(+) (20 millimolar). In whole-cell patch-clamp experiments with guard cell protoplasts of Vicia faba, FC (1 or 10 micromolar) stimulates an increase in outward current that is essentially voltage independent between - 100 and +60 millivolts, and occurs even when the membrane potential is held at a voltage (-60 millivolts) at which K(+) channels are inactivated. These results are indicative of FC activation of a H(+) pump. FC effects on the magnitude of inward and outward K(+) currents are not observed. Epidermal peel and patch clamp data are both consistent with the hypothesis that the plasma membrane H(+) ATPase of guard cells is a primary locus for the FC effect on stomatal apertures.

Journal Article↗