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S M Assmann

Publications and source records attributed to S M Assmann.

At least 19 recordsLinked to original sources
PubMed2001

G protein regulation of ion channels and abscisic acid signaling in Arabidopsis guard cells.

The phytohormone abscisic acid (ABA) promotes plant water conservation by decreasing the apertures of stomatal pores in the epidermis through which water loss occurs. We found that Arabidopsis thaliana plants harboring transferred DNA insertional mutations in the sole prototypical heterotrimeric GTP-binding (G) protein alpha subunit gene, GPA1, lack both ABA inhibition of guard cell inward K(+) channels and pH-independent ABA activation of anion channels. Stomatal opening in gpa1 plants is insensitive to inhibition by ABA, and the rate of water loss from gpa1 mutants is greater than that from wild-type plants. Manipulation of G protein status in guard cells may provide a mechanism for controlling plant water balance.

Abscisic Acid↗
PubMed2001

From milliseconds to millions of years: guard cells and environmental responses.

During the past year, significant advances have been made in our understanding of stomatal development and its response to climate change, and in our knowledge of how guard cell Ca(2+) oscillations encode environmental signals. Recent studies on (de)phosphorylation mechanisms have provided new information on how guard cells respond to abscisic acid and blue light.

Abscisic Acid↗
PubMed2001

Apparent absence of a redox requirement for blue light activation of pump current in broad bean guard cells.

In guard cells, membrane hyperpolarization in response to a blue light (BL) stimulus is achieved by the activation of a plasma membrane H(+)-ATPase. Using the patch clamp technique on broad bean (Vicia faba) guard cells we demonstrate that both steady-state- and BL-induced pump currents require ATP and are blocked by vanadate perfused into the guard cell during patch clamp recording. Background-pump current and BL-activated currents are voltage independent over a wide range of membrane potentials. During BL-activated responses significant hyperpolarization is achieved that is sufficient to promote K(+) uptake. BL activation of pump current becomes desensitized by three or four pulses of 30 s x 100 micromol m(-2) s(-1) BL. This desensitization is not a result of pump inhibition as maximal responses to fusicoccin are observed after full BL desensitization. BL treatments prior to whole cell recording show that BL desensitization is not due to washout of a secondary messenger by whole cell perfusion, but appears to be an important feature of the BL-stimulated pump response. We found no evidence for an electrogenic BL-stimulated redox chain in the plasma membrane of guard cells as no steady-state- or BL-activated currents are detected with NADH or NADPH added to the cytosol in the absence of ATP. Steady-state- nor BL-activated currents are affected by the inclusion along with ATP of 1 mM NADH in the pipette under saturating red light or by including NADPH in the pipette under darkness or saturating red light. These data suggest that reduced products of photosynthesis do not significantly modulate plasma membrane pump currents and are unlikely to be critical regulators in BL-stimulation of the plasma membrane H(+)-ATPase in guard cells.

Cell Membrane↗
PubMed2000

Regulation of abscisic acid-induced stomatal closure and anion channels by guard cell AAPK kinase.

Abscisic acid (ABA) stimulates stomatal closure and thus supports water conservation by plants during drought. Mass spectrometry-generated peptide sequence information was used to clone a Vicia faba complementary DNA, AAPK, encoding a guard cell-specific ABA-activated serine-threonine protein kinase (AAPK). Expression in transformed guard cells of AAPK altered by one amino acid (lysine 43 to alanine 43) renders stomata insensitive to ABA-induced closure by eliminating ABA activation of plasma membrane anion channels. This information should allow cell-specific, targeted biotechnological manipulation of crop water status.

Abscisic Acid↗
PubMed2000

Increases in cytosolic Ca2+ are not required for abscisic acid-inhibition of inward K+ currents in guard cells of Vicia faba L.

The inward K+ channels (IKin) of guard cells are inhibited upon application of abscisic acid (ABA). It has been postulated that I(Kin) inhibition requires an elevation in cytosolic free Ca2+ levels ([Ca2+]c) because: (i) experimental increases in [Ca2+]c can mimic the ABA effect, and; (ii) ABA can trigger an elevation of [Ca2+]c in guard cells. However, not all guard cells respond to ABA with a [Ca2+]c increase, and the magnitude of the increases that do occur is variable. Therefore, an obligate role for Ca2+ in the regulation of downstream effectors of ABA response, such as the I(Kin) channels, remains in question. In this study, we developed a methodology for simultaneous patch clamping and confocal ratiometric Ca2+ imaging of Vicia faba L. guard-cell protoplasts. This allowed us to directly assess the relationship between ABA-induced changes in [Ca2+]c and I(Kin) inhibition. In the presence of extracellular Ca2+, the extent of [Ca2+]c elevation correlated with the extent of I(Kin) inhibition. However, upon chelation of either extracellular Ca2+, [Ca2+]c or both, extracellular Ca2+ and [Ca2+]c, [Ca2+]c elevation did not occur in response to ABA yet I(Kin) currents were still strongly inhibited. These data illustrate that Ca2+-independent regulation is involved in ABA-inhibition of stomatal opening processes.

Abscisic Acid↗
PubMed2000

Kinetic analysis of the K(+)-selective outward rectifier in Arabidopsis mesophyll cells: a comparison with other plant species.

This paper gives a kinetic analysis of the K(+)-selective outward-rectifier (IK,out) in the plasma membrane of Arabidopsis thaliana mesophyll cells in terms of the Hodgkin-Huxley formalism. We compared the kinetic characteristics of IK,out in Arabidopsis with IK,out channels in three other plant species that were subjected to a similar analysis: tobacco suspension cells, Vicia faba guard cells and Plantago media root cells. Because the activation kinetics of IK,out shows a clear voltage dependence, the time constant of half-activation (tau 1/2) and the elementary rate constant of channel opening (a) were calculated at the potential of half-activation (V1/2). The Arabidopsis IK,out activates relatively slowly and this is reflected in a tau 1/2 of approximately 1 s. The reason for this slow activation is twofold. Firstly, the value of a is 1.5 s-1 falls at the lower end of the range of values obtained for tobacco, Vicia and Plantago: 1.1 to 3.0 s-1. Secondly, IK,out in Arabidopsis has four closed states, while tobacco and Vicia have only two. As observed in other plant species, the activation kinetics of IK,out in Arabidopsis are sensitive to external K+: V1/2 shifts with EK but remains approximately 50 mV more positive than EK.

Arabidopsis↗
PubMed2000

Distinct abscisic acid signaling pathways for modulation of guard cell versus mesophyll cell potassium channels revealed by expression studies in Xenopus laevis oocytes.

Regulation of guard cell ion transport by abscisic acid (ABA) and in particular ABA inhibition of a guard cell inward K(+) current (I(Kin)) is well documented. However, little is known concerning ABA effects on ion transport in other plant cell types. Here we applied patch clamp techniques to mesophyll cell protoplasts of fava bean (Vicia faba cv Long Pod) plants and demonstrated ABA inhibition of an outward K(+) current (I(Kout)). When mesophyll cell protoplast mRNA (mesophyll mRNA) was expressed in Xenopus laevis oocytes, I(Kout) was generated that displayed similar properties to I(Kout) observed from direct analysis of mesophyll cell protoplasts. I(Kout) expressed by mesophyll mRNA-injected oocytes was inhibited by ABA, indicating that the ABA signal transduction pathway observed in mesophyll cells was preserved in the frog oocytes. Co-injection of oocytes with guard cell protoplast mRNA and cRNA for KAT1, an inward K(+) channel expressed in guard cells, resulted in I(Kin) that was similarly inhibited by ABA. However, oocytes co-injected with mesophyll mRNA and KAT1 cRNA produced I(Kin) that was not inhibited by ABA. These results demonstrate that the mesophyll-encoded signaling mechanism could not substitute for the guard cell pathway. These findings indicate that mesophyll cells and guard cells use distinct and different receptor types and/or signal transduction pathways in ABA regulation of K(+) channels.

Abscisic Acid↗
PubMed1999

Ozone inhibits guard cell K+ channels implicated in stomatal opening.

Ozone (O3) deleteriously affects organisms ranging from humans to crop plants, yet little is understood regarding the underlying mechanisms. In plants, O3 decreases CO2 assimilation, but whether this could result from direct O3 action on guard cells remained unknown. Potassium flux causes osmotically driven changes in guard cell volume that regulate apertures of associated microscopic pores through which CO2 is supplied to the photosynthetic mesophyll tissue. We show in Vicia faba that O3 inhibits (i) guard cell K+ channels that mediate K+ uptake that drives stomatal opening; (ii) stomatal opening in isolated epidermes; and (iii) stomatal opening in leaves, such that CO2 assimilation is reduced without direct effects of O3 on photosynthetic capacity. Direct O3 effects on guard cells may have ecological and agronomic implications for plant productivity and for response to other environmental stressors including drought.

Journal Article↗
PubMed1999

Abscisic acid signal transduction in guard cells is mediated by phospholipase D activity.

In guard cells, the plant hormone abscisic acid (ABA) inhibits stomatal opening and induces stomatal closure through the coordinated regulation of ion transport. Despite this central role of ABA in regulating stomatal function, the signal transduction events leading to altered ion fluxes remain incompletely understood. We report that the activity of the enzyme phospholipase D (PLD) transiently increased in guard cell protoplasts at 2.5 and 25 min after ABA application. Treatment of guard cell protoplasts with phosphatidic acid (PtdOH), one of the products of PLD activity, led to an inhibition of the activity of the inward K+ channel. PtdOH also induced stomatal closure and inhibited stomatal opening when added to epidermal peels. Application of 1-butanol (1-buOH), a selective inhibitor of PtdOH production by PLD, inhibited the increase in PtdOH production elicited by ABA. 1-BuOH treatment also partially prevented ABA-induced stomatal closure and ABA-induced inhibition of stomatal opening. This inhibitory effect of buOH was enhanced by simultaneous application of nicotinamide, an inhibitor of cADP ribose action. These results suggest that in the guard cell, ABA activates the enzyme PLD, which leads to the production of PtdOH. This PtdOH is then involved in triggering subsequent ABA responses of the cell via a pathway operating in parallel to cADP ribose-mediated events.

Journal Article↗
PubMed1999

A laser microsurgical method of cell wall removal allows detection of large-conductance ion channels in the guard cell plasma membrane.

Application of patch clamp techniques to higher-plant cells has been subject to the limitation that the requisite contact of the patch electrode with the cell membrane necessitates prior enzymatic removal of the plant cell wall. Because the wall is an integral component of plant cells, and because cell-wall-degrading enzymes can disrupt membrane properties, such enzymatic treatments may alter ion channel behavior. We compared ion channel activity in enzymatically isolated protoplasts of Vicia faba guard cells with that found in membranes exposed by a laser microsurgical technique in which only a tiny portion of the cell wall is removed while the rest of the cell remains intact within its tissue environment. "Laser-assisted" patch clamping reveals a new category of high-conductance (130 to 361 pS) ion channels not previously reported in patch clamp studies on plant plasma membranes. These data indicate that ion channels are present in plant membranes that are not detected by conventional patch clamp techniques involving the production of individual plant protoplasts isolated from their tissue environment by enzymatic digestion of the cell wall. Given the large conductances of the channels revealed by laser-assisted patch clamping, we hypothesize that these channels play a significant role in the regulation of ion content and electrical signalling in guard cells.

Biodegradation, Environmental↗
PubMed1999

Arabidopsis thaliana 'extra-large GTP-binding protein' (AtXLG1): a new class of G-protein.

Heterotrimeric GTP-binding proteins, composed of alpha, beta, and gamma subunits, are involved in signal transduction pathways in animal and plant systems. In plants, physiological analyses implicate heterotrimeric G-proteins in ion channel regulation, light signaling, and hormone and pathogen responses. However, only one class of plant G alpha genes has been identified to date. We have cloned a novel gene, 'Arabidopsis thaliana extra-large GTP-binding protein' (AtXLG1). AtXLG1 appears to be a member of a small gene family and is transcribed in all tissues assayed: roots, leaves, stems, flowers, and fruits. The conceptually translated protein from AtXLG1 is 99 kDa, twice as large as typical G alpha proteins. The carboxy-terminal half of the AtXLG1 protein has significant homology to animal and plant G alpha proteins. This region includes a GTP-binding domain, a predicted helical domain, and an aspartate/glutamate-rich loop, which are characteristics of G alpha's. Despite the absence of some of the amino acids implicated in GTP binding and hydrolysis by crystallographic and mutational analyses of mammalian G alpha's, recombinant AtXLG1 binds GTP with specificity. The amino-terminal region of AtXLG1 contains domains homologous to the bacterial TonB-box, which is involved in energy transduction between the inner and outer bacterial membranes, and to zinc-finger proteins. Given the unique structure of AtXLG1, it will be of interest to uncover its physiological functions.

Amino Acid Sequence↗
PubMed1998

The calculation of intracellular ion concentrations and membrane potential from cell-attached and excised patch measurements. Cytosolic K+ concentration and membrane potential in Vicia faba guard cells.

Ion channel activity in cell-attached patch recordings shows channel behavior under more physiological conditions than whole-cell and excised patch measurements. Yet the analysis of cell-attached patch measurements is complicated by the fact that the system is ill defined with respect to the intracellular ion activities and the electrical potential actually experienced by the membrane patch. Therefore, of the several patch-clamp configurations, the information that is obtained from cell-attached patch measurements is the most ambiguous. The present study aims to achieve a better understanding of cell-attached patch measurements. Here we describe a method to calculate the intracellular ion concentration and membrane potential prevailing during cell-attached patch recording. The first step is an analysis of the importance of the input resistance of the intact cell on the cell-attached patch measurement. The second step, and actual calculation, is based on comparison of the single channel conductance and reversal potential in the cell-attached patch and excised patch configurations. The method is demonstrated with measurements of membrane potential and cytosolic K+ concentrations in Vicia faba guard cells. The approach described here provides an attractive alternative to the measurement of cytosolic ion concentrations with fluorescent probes or microelectrodes.

Cell Adhesion↗
PubMed1998

Ca(2+)-permeable, outwardly-rectifying K+ channels in mesophyll cells of Arabidopsis thaliana.

Arabidopsis thaliana has become a powerful tool in genetics and molecular biology. In order to use Arabidopsis as a model system for electrophysiological studies on plant cells, a detailed characterization of the transporters present in the plasma and vacuolar membranes of this species is required. We used the patch-clamp technique to study ion channels in the plasma membrane of Arabidopsis mesophyll cells. The most prominent conductance in these cells was a K(+)-selective, voltage-dependent, outwardly-rectifying channel (IK,out). In the whole-cell configuration, IK,out was observed in 100% of the cells assayed. In contrast, inward current was observed in less than 50% of the cells which were bathed in 100 mM K+, and was totally absent from cells bathed in 10 mM K+. The activation kinetics of IK,out were modulated by the external K+ concentration with a faster activation at low external K+. Tail-current analysis revealed that in addition to K+, IK,out is also permeable to Ca2+ and Ba2+. Externally applied Ba2+ also caused a voltage-dependent decrease in current magnitude, indicating that IK,out is also partially blocked by this classic K+ channel blocker. Single channels studied in outside-out patches showed Ca2+ and Ba2+ sensitivity, voltage dependence and time activation similar to that of IK,out in the whole-cell configuration. Given their permeability to Ca2+, these channels may function as an avenue for Ca2+ influx as well as K+ efflux, both of which may affect photosynthesis.

Arabidopsis↗
PubMed1998

Guard cells possess a calcium-dependent protein kinase that phosphorylates the KAT1 potassium channel.

Increasing evidence suggests that changes in cytosolic Ca2+ levels and phosphorylation play important roles in the regulation of stomatal aperture and as ion transporters of guard cells. However, protein kinases responsible for Ca2+ signaling in guard cells remain to be identified. Using biochemical approaches, we have identified a Ca(2+)-dependent protein kinase with a calmodulin-like domain (CDPK) in guard cell protoplasts of Vicia faba. Both autophosphorylation and catalytic activity of CDPK are Ca2+ dependent. CDPK exhibits a Ca(2+)-induced electrophoretic mobility shift and its Ca(2+)-dependent catalytic activity can be inhibited by the calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide. Antibodies to soybean CDPK alpha cross-react with CDPK. Micromolar Ca2+ concentrations stimulate phosphorylation of several proteins from guard cells; cyclosporin A, a specific inhibitor of the Ca(2+)-dependent protein phosphatase calcineurin enhances the Ca(2+)-dependent phosphorylation of several soluble proteins. CDPK from guard cells phosphorylates the K+ channel KAT1 protein in a Ca(2+)-dependent manner. These results suggest that CDPK may be an important component of Ca2+ signaling in guard cells.

Calcium↗
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↗