Isolation of Guard Cell Protoplasts from Mechanically Prepared Epidermis of <i>Vicia faba</i> Leaves

Kruse, Tamara N.; Tallman, Gary; Zeiger, Eduardo

doi:10.1104/pp.90.4.1382

Cited by 74 publications

(62 citation statements)

References 21 publications

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“…The youngest, fully expanded leaves of 3-4 wk-old plants were harvested. Protoplasts were isolated according to procedure of Kruse, Tallman, and Zeiger (1989). We measured the diameters of the spherical protoplasts using a microscope micrometer with a resolution of 0.22 p,m (at a magnification of 400).…”

Section: Experimental Methodsmentioning

confidence: 99%

External pH effects on the depolarization-activated K channels in guard cell protoplasts of Vicia faba.

Ilan

Schwartz

Moran

1994

The Journal of General Physiology

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Previous studies reveal that the pH of the apoplastic solution in the guard cell walls may vary between 7.2 and 5.1 in closed and open stomata, respectively. During these aperture and pH changes, massive K § fluxes cross the cellular plasma membrane driving the osmotic turgor and volume changes of guard cells. Therefore, we examined the effect of extracellular pH on the depolarizationactivated K channels (KD channels), which constitute the K § efflux pathway, in the plasma membrane of Viciafaba guard cell protuplasts. We used patch clamp, both in whole cells as well as in excised outside-out membrane patches.Approximately 500 KD channels, at least, could be activated by depolarization in one protoplast (density: ~0.6 ~m-2). Acidification from pH 8.1 to 4.4 decreased markedly the whole-cell conductance, GK, of the KD channels, shifted its voltage dependence, GK-EM, to the right on the voltage axis, slowed the rate of activation and increased the ,rate of deactivation, whereas the single channel conductance was not affected significantly. Based on the GK-EM shifts, the estimated average negative surface charge spacing near the KD channel is 39 /~. To quantify the effects of protons on the rates of transitions between the hypothesized conformational states of the channels, we fitted the experimental macroscopic steady state conductancevoltage relationship and the voltage dependence of time constants of activation and deactivation, simultaneously, with a sequential three-state model CCO. In terms of this model, protonation affects the voltage-dependent properties via a decrease in localized, rather than homogeneous, surface charge sensed by the gating moieties. In terms of either the CO or CCO model, the protonation of a site with a pKa of 4.8 decreases the voltage-independent number of channels, N, that are available for activation by depolarization.

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Section: Experimental Methodsmentioning

confidence: 99%

External pH effects on the depolarization-activated K channels in guard cell protoplasts of Vicia faba.

Ilan

Schwartz

Moran

1994

The Journal of General Physiology

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“…Guard cell protoplasts were isolated following a procedure modified from that of Kruse et al (1989). The youngest fully expanded leaflets of 2-to 3-week-old plants were used.…”

Section: Lsolation Of Guard Cell Protoplasts From V Fabamentioning

confidence: 99%

Actin Filaments Modulate Both Stomatal Opening and Inward K+-Channel Activities in Guard Cells of Vicia faba L

et al. 1997

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Actin antagonists have previously been shown to alter responses of Commelina communis stomata to physiological stimuli, implicating actin filaments i n the control of guard cell volume changes (M. Kim, P.K. Hepler, 5-0. Eun, K.S. Ha, Y. Lee [1995] Plant Physiol 109: 1077-1084). Since K+ channels i n the guard cell play an important role i n stomatal movements, we examined the possible regulation of K+-channel activities by the state of actin polymerization. Agents affecting actin polymerization altered light-induced stomatal opening and inward K+-channel activities measured by patch clamping in Vicia faba. Cytochalasin D, which induces depolymerization of actin filaments, promoted light-induced stomatal opening and potentiated the inward K+ current in guard cell protoplasts. Phalloidin, a stabilizer of filamentous actin, inhibited both light-induced stomatal opening and inward K+ current. lnward K+-channel activities in outside-out membrane patches showed responses to these agents that support results at the whole-cell current levei, suggesting that cytochalasin D facilitates and phalloidin inhibits K+ influx in intact guard cells, thus resulting in enhancement and inhibition of stomatal opening, respectively. To our knowledge, this is the first report that provides evidence that actin filaments may regulate an important physiological process by modulating the activities of ion channels i n plant cells.The regulation of stomatal aperture is critica1 to a plant's ability to balance the need for a C source while avoiding the deleterious effects of water loss. The size of the stomatal opening is established through volume changes of stomatal guard cells under the concerted influence of light, temperature, CO,, and phytohormones (Assmann, 1993). Recently, Kim et al. (1995) showed that cortical actin filaments are distributed radially, fanning out from the stomatal pore site in mature guard cells of Commelinu communis. Moreover, fungal toxins that interfere with the polymerization or depolymerization of actin filaments brought about altered stomatal responses to physiological stimuli. Thus, dynamic changes in the actin cytoskeleton

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“…Light intensity was 200 pmol m-'s-l for a 10-h daily light period and day and night temperatures were 20 (9) and 16°C (+2"C), respectively. Guard-cell protoplasts were isolated from the epidermes of young expanded leaves from 3-to 4-week-old plants as described by Kruse et al (1989). Before use in patch-clamp experiments, isolated guard-cell protoplasts were kept in the dark at O to 2°C in a solution containing 5 mM Mes (KOH, pH 5.5), 0.45 M sorbitol, 1 mM MgCl,, and 1 mM CaC1,.…”

Section: Materials a N D M E T H O D S Preparation Of Cuard-cell Protmentioning

confidence: 99%

Is ATP Required for K+ Channel Activation in Vicia Guard Cells?

Assmann

1995

Plant Physiol.

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In vivo, K+ entry into guard cells via inward-rectifying K+ channels is indirectly driven by ATP via an H+-ATPase that hyperpolarizes the membrane potential. However, whether activation of the K+ channels of guard cells requires ATP remains unknown. In the present study, both whole-cell and single-channel patch-clamp techniques were used to address this question. Exogenous ATP, ADP, and adenosine-5'-0(3-thiotriphosphate) applied to the cytoplasm had no effect on whole-cell K+ currents of Vicia faba 1. guard cells. Azide, an inhibitor of oxidative phosphorylation, also had no effect. However, an ATP-scavenging system, glucose plus hexokinase, inhibited whole-cell inward K+ currents by 30 to 40%. Singlechannel results acquired from cytoplasm-free inside-out membrane patches showed definite activation of inward K+ channels by ATP. Other nucleotides, such as ADP, adenosine-5'-0(3-thiotriphosphate), and CTP, did not increase channel activity in the membrane patches. lnward K+ channel activity in membrane patches preactivated by exogenous ATP was inhibited by glucose plus hexokinase.These results suggest that a low concentration of ATP is requjred for activation of the inward K+ channels of the guard-cell plasma membrane. The issue of how ATP as a signal regulates these K+ channels is discussed.Stomatal movement is an energy-dependent process (Weyers et al., 1982;Assmann and Zeiger, 1987;Karlsson and Schwartz, 1988). Under conditions that favor stomatal opening, such as illumination, H'-ATPases in the plasma membrane of guard cells are activated (Assmann et al., 1985;Shimazaki et al., 1986;Serrano et al., 1988;Schwartz et al., 1991;Lohse and Hedrich, 1992), which utilize ATP to pump H+ out of the cytoplasm and build up an H+ gradient across the guard-cell plasma membrane. The resultant electrical gradient drives Kf entry, which is accompanied by C1-influx and malate*-synthesis from starch breakdown. As the concentrations of intracellular osmotica increase, guard cells take up water and bend, opening the stomatal pore (Outlaw, 1983). Therefore, ATP-driven H+ extrusion is a vital process for stomatal regulation in vivo. entry into guard cells other than to fuel the H+-ATPase. Whether ATP also plays a role as a signal chemical that regulates guard-cell K+ channels has not been investigated.In mesophyll protoplasts of Arabidopsis thaliana, ATP activation of the K+ channel PKC1, which can mediate both K+ influx and K+ efflux, has been reported (Spalding and Goldsmith, 1993), and an association between K+ channel activity and photosynthetic activity was inferred. In the same cell type, ATP activation of an outward-rectifying Kf channel, designated PKCZ, was also reported (Spalding and Goldsmith, 1993). Luan et al. (1993) reported evidence for inhibition of the inward-rectifying K+ channels of guard cells by a Ca2+-dependent phosphatase, which suggests that ATP might be involved in Kf channel regulation by phosphorylation/dephosphorylation pathways. Hedrich et al. (1990) reported that anion channels of Vicia fabu guard cell...

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Isolation of Guard Cell Protoplasts from Mechanically Prepared Epidermis of Vicia faba Leaves

Cited by 74 publications

References 21 publications

External pH effects on the depolarization-activated K channels in guard cell protoplasts of Vicia faba.

External pH effects on the depolarization-activated K channels in guard cell protoplasts of Vicia faba.

Actin Filaments Modulate Both Stomatal Opening and Inward K+-Channel Activities in Guard Cells of Vicia faba L

Is ATP Required for K+ Channel Activation in Vicia Guard Cells?

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