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1.
Catherine P. Darley Lorna A. Skiera Frederick D. Northrop Dale Sanders Julia M. Davies 《Planta》1998,206(2):272-277
Lysed guard-cell protoplasts of Vicia faba L. exhibited hydrolytic activity characteristic of tonoplast inorganic pyrophosphatase (V-PPase; EC 3.6.1.1). Activity was
inhibited by the specific V-PPase inhibitor aminomethylenediphosphonate, stimulated by K+ (K
m
= 51 mM) and inhibited by Ca2+ (80 nM free Ca2+ was required for 50% inhibition at 0.27 mM free Mg2+). Patch-clamp measurements of electrogenic activity confirmed enzyme localisation at the tonoplast. This is the first report
of V-PPase activity in guard cells; its possible involvement in stomatal opening is discussed.
Received: 12 February 1998 / Accepted: 24 April 1998 相似文献
2.
The dynamic changes of tonoplasts in guard cells are important for stomatal movement in Vicia faba
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Stomatal movement is important for plants to exchange gas with environment. The regulation of stomatal movement allows optimizing photosynthesis and transpiration. Changes in vacuolar volume in guard cells are known to participate in this regulation. However, little has been known about the mechanism underlying the regulation of rapid changes in guard cell vacuolar volume. Here, we report that dynamic changes in the complex vacuolar membrane system play a role in the rapid changes of vacuolar volume in Vicia faba guard cells. The guard cells contained a great number of small vacuoles and various vacuolar membrane structures when stomata closed. The small vacuoles and complex membrane systems fused with each other or with the bigger vacuoles to generate large vacuoles during stomatal opening. Conversely, the large vacuoles split into smaller vacuoles and generated many complex membrane structures in the closing stomata. Vacuole fusion inhibitor, (2s,3s)-trans-epoxy-succinyl-l-leucylamido-3-methylbutane ethyl ester, inhibited stomatal opening significantly. Furthermore, an Arabidopsis (Arabidopsis thaliana) mutation of the SGR3 gene, which has a defect in vacuolar fusion, also led to retardation of stomatal opening. All these results suggest that the dynamic changes of the tonoplast are essential for enhancing stomatal movement. 相似文献
3.
K+ transport properties of K+ channels in the plasma membrane of Vicia faba guard cells 总被引:23,自引:3,他引:20
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J I Schroeder 《The Journal of general physiology》1988,92(5):667-683
Electrical properties of the plasma membrane of guard cell protoplasts isolated from stomates of Vicia faba leaves were studied by application of the whole-cell configuration of the patch-clamp technique. The two types of K+ currents that have recently been identified in guard cells may allow efflux of K+ during stomatal closing, and uptake of K+ during stomatal opening (Schroeder et al., 1987). A detailed characterization of ion transport properties of the inward-rectifying (IK+,in) and the outward-rectifying (IK+,out) K+ conductance is presented here. The permeability ratios of IK+,in and IK+,out currents for K+ over monovalent alkali metal ions were determined. The resulting permeability sequences (PK+ greater than PRb+ greater than PNa+ greater than PLi+ much greater than PCs+) corresponded closely to the ion specificity of guard cell movements in V. faba. Neither K+ currents exhibited significant inactivation when K+ channels were activated for prolonged periods (greater than 10 min). The absence of inactivation may permit long durations of K+ fluxes, which occur during guard cell movements. Activation potentials of inward K+ currents were not shifted when external K+ concentrations were changed. This differs strongly from the behavior of inward-rectifying K+ channels in animal tissue. Blue light and fusicoccin induce hyperpolarization by stimulation of an electrogenic pump. From slow-whole-cell recordings it was concluded that electrogenic pumps require cytoplasmic substrates for full activation and that the magnitude of the pump current is sufficient to drive K+ uptake through IK+,in channels. First, direct evidence was gained for the hypothesis that IK+,in channels are a molecular pathway for K+ accumulation by the finding that IK+,in was blocked by Al3+ ions, which are known to inhibit stomatal opening but not closing. The results presented in this study strongly support a prominent role for IK+,in and IK+,out channels in K+ transport across the plasma membrane of guard cells. 相似文献
4.
Potassium channels were resolved in Vicia faba guard cell protoplasts by patch voltage-clamp. Whole-cell currents and single K+ channels had linear instantaneous current-voltage relations, reversing at the calculated Nernst potential for K+. Whole cell K+ currents activated exponentially during step depolarizations, with half-activation times of 400-450 msec at +80 mV and 90-110 msec at +150 mV. Single K+ channel conductance was 65 +/- 5 pS with a mean open time of 1.25 +/- 0.30 msec at 150 mV. Potassium channels were blocked by internal Cs+ and by external TEA+, but they were insensitive to external 4-aminopyridine. Application of 10 microM abscisic acid increased mean open time and caused long-lasting bursts of channel openings. Since internal and external composition can be controlled, patch-clamped protoplasts are ideal systems for studying the role of ion channels in plant physiology. 相似文献
5.
Summary 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+] (Ca
i
) from 200 to 2 nm or increasing the external [Ca2+] (Ca
o
) 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 Ca
o
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 Ca
i
-, and Ca
o
-dependent ability to mediate Ca2+ influx. We suggest that the inwardly rectiying K+ channels are part of a regulatory mechanism for Ca
i
. Changes in Ca
o
and (associated) changes in Ca
i
regulate a variety of intracellular processes and ion fluxes, including the K+ and anion fluxes associated with stomatal aperture change.This work was supported by grants to S.M.A. from NSF (DCB-8904041) and from the McKnight Foundation. K.F.-G. is a Charles Gilbert Heydon Travelling Fellow. The authors thank Dr. R. MacKinnon (Harvard Medical School) and two anonymous reviewers for helpful comments. 相似文献
6.
7.
W. G. Allaway 《Planta》1973,110(1):63-70
Summary The level of malate in the epidermis from illuminated leaves of Vicia faba was greater than in that from dark-treated leaves. A difference in the malate level was still detected after the epidermis had been treated by rolling so that only the guard cells remained alive. The results suggest that malate may accumulate in guard cells on illumination. In subsequent experiments, stomatal apertures were measured, and potassium as well as malate was analysed in extracts of epidermis. In illuminated leaves, the potassium content of rolled epidermis increased from about 90 to about 335 picoequivalents mm-2 of epidermis whele malate increased from about zero to about 71 pmoles mm-2 and the stomata opened; in dark-treated leaves, the potassium content of rolled epidermis decreased slightly, the malate level remained about zero, and the stomata showed very slight further closure. The measured increase in potassium is likely to represent an increase in potassium concentration in the guard cells of about 0.4 Eq l-1 with stomatal opening; the increase in malate could correspond to 0.23 Eq l-1 (with respect to potassium) in the guard cells. Thus, malate accumulating in guard cells could balance about half of the potassium taken up by guard cells when stomata open in the light. 相似文献
8.
External pH effects on the depolarization-activated K channels in guard cell protoplasts of Vicia faba 总被引:2,自引:0,他引:2
《The Journal of general physiology》1994,103(5):807-831
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 depolarization-activated K channels (KD channels), which constitute the K+ efflux pathway, in the plasma membrane of Vicia faba guard cell protoplasts. 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: approximately 0.6 micron-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 A. 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 conductance-voltage 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. 相似文献
9.
Furuichi T Mori IC Muto S 《Zeitschrift für Naturforschung. C, Journal of biosciences》2005,60(9-10):769-773
Protein kinases are involved in signal transduction for environmental stress responses. In response to drought and salinity, a 48-kDa protein kinase (AAPK; abscisic acid-activated protein kinase (AAPK) in guard cells is activated by abscisic acid (ABA) and phosphorylates several targets such as the carboxy-terminus of inward-rectifying K+ channel and heterogeneous mRNA binding protein to adopt to the changing environment. The AAPK expressed specifically in guard cells, and recombinant AAPK was phosphorylated only with the extract from ABA-treated guard cells but not from untreated cells. This indicates the presence of an AAPK kinase (AAPKK), which is activated by ABA and phosphorylates AAPK preceding the activation of AAPK. Both AAPK and AAPKK are involved in the protein kinase cascade for the rapid ABA-signaling. 相似文献
10.
Stomatal movement is controlled by external and internal signals such as light, phytohormones or cytoplasmic Ca2+. Using Vicia faba L., we have studied the dose-dependent effect of auxins on the modulation of stomatal opening, mediated through the activity of the plasma-membrane H+-ATPase. The patch-clamp technique was used to elucidate the electrical properties of the H+-ATPase as effected by growth regulators and seasonal changes. The solute composition of cytoplasmic and extracellular media was selected to record pump currents directly with high resolution. Proton currents through the ATPase were characterized by a voltage-dependent increase in amplitude, positive to the resting potential, reaching a plateau at more depolarized values. Upon changes in extracellular pH, the resting potential of the cell shifted with a non-Nernst potential response (±21 mV), indicating the contribution of a depolarizing ionic conductance other than protons to the permeability of the plasma membrane. The use of selective inhibitors enabled us to identify the currents superimposing the H+-pump as carried by Ca2+. Auxinstimulation of this electroenzyme resulted in a rise in the outwardly directed H+ current and membrane hyperpolarization, indicating that modulation of the ATPase by the hormone may precede salt accumulation as well as volume and turgor increase. Annual cycles in pump activity (1.5–3.8 μA · cm-2) were expressed by a minimum in pump current during January and February. Resting potentials of up to -260 mV and plasmamembrane surface area, on the other hand, did not exhibit seasonal changes. The pump activity per unit surface area was approximately 2- to 3-fold higher in guard cells than in mesophyll cells and thus correlates with their physiological demands. 相似文献
11.
Stretch-activated chloride,potassium, and calcium channels coexisting in plasma membranes of guard cells of Vicia faba L. 总被引:27,自引:0,他引:27
Mechanosensitive ion channels in the plasma membrane of Vicia faba guard cell protoplasts were studied by use of the patch clamp technique. Stretch-activated (SA) channels in outside-out patches were analyzed for channel conductance, kinetics and ion selectivity. We found three distinct SA channels, permeable to Cl–, K+ and Ca2+ and distinguishable from spontaneous (non-SA) channels for these ions on the basis of conductance, kinetics, and voltage-dependence, as well as sensitivity to membrane stretch. In the attached patch configuration, light suction (2 to 10 kPa) reversibly induced channel opening with multiple amplitudes and complex kinetics. The open probability for SA channels increased nonlinearly with pipette suction. In guard cells in situ, these SA channels may mediate ion transport across the plasma membrane directly, as well as influence the activity of non-SA channels via effects on membrane voltage and cytoplasmic calcium. Through such effects, SA channels likely influence volume and turgor regulation of guard cells, and thereby control of leaf gas exchange.Abbreviations EK
equilibrium potential for potassium transport
- ECl
equilibrium potential for chloride transport
- SA
stretchactivated
Dedicated to the 80. birthday of Franz HedrichSupported by a grant from the Deutsche Forschungsgemeinschaft to R.H. and a Department of Energy grant to D.J.C. gratefully acknowledges a John S. Guggenheim Fellowship and Fulbright Kommission Senior Professor Award. We thank Ingrid Baumann and Angela Schön for technical assistance, and Klaus Raschke and Heiner Busch for spirited discussions and support. 相似文献
12.
The effects of elevated CO2 concentrations on stomatal movement, anion- and K+-channel activities were examined in guard cells from epidermal strips of Vicia faba. Membrane voltage was measured using intracellular, double-barrelled microelectrodes and ion-channel currents were recorded
under voltage clamp during exposure to media equilibrated with ambient (350 μl · l−1), 1000 μl · l−1 and 10 000 μl · l−1 CO2 in 20% O2 and 80% N2. The addition of 1000 μl · l−1 CO2 to the bathing solution caused stomata to close with a halftime of approx. 40 min, and with 10 000 μl · l−1 CO2 closure occurred with a similar time course. Under voltage clamp, exposure to 1000 μl · l−1 and 10 000 μl · l−1 CO2 resulted in a rapid increase (mean, 1.5 ± 0.2-fold, n = 8; range 1.3- to 2.5-fold) in the magnitude of current carried by outward-rectifying K+ channels (IK,out). The effect of CO2 on IK,out was essentially complete within 30 s and was independent of clamp voltage, but was associated with 25–40% (mean, 30 ± 4%)
decrease in the halftime for current activation. Exposure to CO2 also resulted in a four-fold increase in background current near the free-running membrane voltage, recorded as the instantaneous
current at the start of depolarising and hyperpolarising voltage steps, and a decrease in the magnitude of current carried
by inward-rectifying K+ channels (IK,in). The effect of CO2 on IK,in was generally slower than on IK,out; it was allied with a transient acceleration of its activation kinetics during the first 60–120 s of treatment; and it was
associated with a negative shift in the voltage-sensitivity of gating over a period of 3–5 min. Measurements carried out to
isolate the background currents attributable to anion channels (ICl), using tetraethylammonium chloride and CsCl, showed that CO2 also stimulated ICl and dramatically altered its relaxation kinetics. Within the timeframe of CO2 action at the membrane, no significant effect was observed on cytosolic pH, measured using the fluorescent dye 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyflourescein
(BCECF) and ratio fluorescence microphotometry. These results are broadly consistent with the pattern of guard-cell response
to abscisic acid, and indicate that guard cells control both anion and K+ channels to achieve net solute loss in CO2. By contrast with the effects of abscisic acid, however, the data indicate that CO2 action is not mediated through changes in cytosolic pH and thereby implicate new and, as yet, unidentified pathway(s) for
channel regulation in the guard cells.
Received: 8 January 1997 / Accepted: 28 February 1997 相似文献
13.
The distribution of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in different leaf‐cell‐types and tissues of Vicia faba L. cv. 3‐fach Weiße was studied. The highest specific PEPCase activity was found in guard cell protoplasts (16.3 µmol mg−1 protein h−1 ) whereas for epidermal and mesophyll protoplasts remarkably lower specific activities were found (1.6 and 1.0 µmol mg−1 protein h−1 , respectively). On chlorophyll and protoplast basis, a similar distribution of enzyme activity was observed. Compared with epidermal extracts, the specific PEPCase activity of mesophyll tissue was 17‐fold lower. Immunological studies with polyclonal antibodies to PEPCase indicated 3 immunoreactive proteins in epidermal tissue and guard cell protoplasts with molecular masses of 107 000, 110 000, and 112 000. Only the Mr 107 000 protein was found in extracts of mesophyll and epidermis protoplasts. Western immunoblots after native electrophoresis of epidermal and mesophyll proteins showed a significant difference in PEPCase mobility. It is assumed, that the immunostained proteins of Mr 110 000 and 112 000 represent isoforms or subunits of the PEPCase and that they are involved in stomatal movements. 相似文献
14.
15.
16.
The use of a discontinuous single electrode voltage-clamp (dSEVC) offers an attractive alternative to the patch-clamp technique, since whole-cell measurements can be performed with a single sharp electrode. Comparison of current-voltage relations, however, revealed a weaker voltage dependence of channels measured with the dSEVC compared to patch clamp. The accuracy of the dSEVC was tested on Vicia faba guard cells impaled with double-barrelled electrodes. The actual clamp potential was measured independently of the dSEVC, at the second barrel. The weaker voltage dependence of ion channels appeared to be due to an overestimation of the clamp potential by the dSEVC. The deviation between the intended and actual clamp potential showed a linear relationship with the injected current; on average a 126 mV deviation was found for a clamp current of 1 nA. The deviation was probably caused by a slow settling capacity at the electrode, not compensated by the dSEVC amplifier. It is concluded that the dSEVC method in its current state is only suited for the study of small ion conductances in plant cells. 相似文献
17.
18.
19.
The inward rectified potassium current of Vicia faba guard cell protoplasts treated with acetylcholine (ACh) or the antagonists of its receptors were recorded by employing the patch clamp technique. The results show that ACh at lower concentrations increases the inward K current, in contrast, ACh at higher concentrations inhibits it. Treated with d-Tubocurarine (d-Tub), an antagonist of the nicotine ACh receptor (nAChR) inhibits the inward K current by 30%. Treated with atropine (Atr), an antagonist of the muscarine (Mus) ACh receptor (mAChR) also inhibits it by 36%. However, if guard cell protoplasts are treated with d-Tub and Atr together, the inward K current is inhibited by 60%-75%. Tetraethylammonium chloride (TEA), a strong inhibitor of K channels has no effect on the inward K current regulated by ACh, suggesting that there are inward K channels modulated by AChRs on the membrane of the guard cell protoplasts. These data demonstrate an ACh-regulated mechanism for stomatal movement. 相似文献
20.
Furuichi T Tatsumi H Sokabe M 《Biochemical and biophysical research communications》2008,366(3):758-762
In the bright fields, stomata of the plants are fully opened to raise the transpiration rate and CO2 uptake required for photosynthesis. Stomatal opening is driven by the activation of plasma membrane H+-ATPase and K+in channels, and the Ca2+-dependent inactivation and blockage of both components were supposed to be inevitable function to regulate the stomatal aperture. Although, it is still obscure how these activities are regulated at the open state. Application of an amphipathic membrane creator, trinitrophenol (TNP), instantly generates the convex curvature in the plasma membrane, which occurs in the phases of stomatal opening and closure. TNP surely activates mechanosensitive Ca2+-permeable channels and attenuates the promotion of stomatal opening, but does not inhibit and promote stomatal closure. These results suggest that activation of mechanosensitive Ca2+-permeable channels regulates the opening phase of stomata in plants. 相似文献