Light-sensitive membrane potentials in onion guard cells

Zeiger, Eduardo; Moody, William J.; Hepler, Peter K.; Varela, Filipa

doi:10.1038/270270a0

Cited by 44 publications

(9 citation statements)

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“…Another inhibitor, vanadate, blocked the acidification of the medium by epidermal tissue (6). Light-induced hyperpolarization of guard cell membrane potential was observed (14,30). These results support the operation of light-driven H4 pump in guard cells (29).…”

Section: Discussionsupporting

confidence: 70%

Light-Induced Alkalinization of the Suspending Medium of Guard Cell Protoplasts from Vicia faba L.

Gotow¹,

Sakaki²,

Kondo³

et al. 1985

Plant Physiol.

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The light-dependent pH changes in the suspending medium of guard cell protoplasts (GCP) from Viciafaba were studied. Upon illumination, the medium was initially slightly alklinied and then acidified. The extent of alkalinization was lower in COrfree air than in normal air. This initial alkalinization was inhibited by DCMU. Acidification in COr free air became observable in shorter duration of light exposure than that in normal air. The rate of acidification was higher in COrfree air than in normal air. The CO2 level of the medium decreased in the light, and increased in the dark. 'I4C()2 uptake was enhanced 2-to 3-fold by light, but not in the presence of DCMU. These results indicate that photosynthetic CO2 fixation does take place in GCP and that the initial alkalinization is due to this photosynthetic CO2 uptake. Diethylstilbestrol, a nonmitochondrial membrane-bound ATPase inhibitor, inhibited the acidification, suggesting that the acidification resulted from H' extrusion by GCP (12).We studied the light-induced changes in pH of the suspending medium ofGCP. GCP with pure and homogeneous populations are experimental materials suitable for the study of ion transport through the plasma membrane, because the lack of cell walls facilitate ion movements. We carried out the experiments in normal air as well as in C02-free air. We now report initial alkalinization of the medium of Vicia GCP on illumination and light-enhanced CO2 fixation by GCP, and discuss the effect of CO2 on H+ extrusion. MATERIALS AND METHODSIsolation of Guard Cell Protoplasts. Guard cell protoplasts were isolated from the lower epidermis of Vicia faba leaves as reported previously (9). Purified protoplasts were kept in the dark on ice until use.Measurement of pH Changes in the Suspending Medium. The pH ofthe suspending medium was continuously monitored using a small flat surface pH electrode (Fuji Kagaku Keisoku Co., Japan) at 25C. The measurements were conducted in a closed vessel (22). A l-ml sample of protoplast suspension was continuously stirred and illuminated with a white light from 300-W slide projector (Elmo Co., Japan) through a 50-mm water layer at 300 gE m-2 s-'. The suspension medium contained 0.4 M mannitol, 10 mm KCI, and 1 mM CaCl2, and was weakly buffered by 0.1 mM Mes-Tris (pH 6.5).Experiments in COrFree Air. Water-saturated CO2-free air, obtained by passing commercial C02-free air through water, was bubbled into the medium through a hypodermic needle until the pH rise in the medium stopped. Then GCP were transferred into the medium to a final volume of 1 ml, and incubated with continuous flow of C02-free air over the surface of the medium in the vessel (capacity 8 ml) covered with Parafilm (American Can Co.). The gas flow rate was 300 ml min-'.CO2 Concentrations. The CO2 concentration in the suspending medium was determined from the increase in pH that could be induced by bubbling N2 through a supernatant (13). GCP suspension (1.2 ml) was transferred into centrifuge tube (capacity 1.5 ml) and centrifuged at 10,000g for 10 s (M...

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Section: Discussionsupporting

confidence: 70%

Light-Induced Alkalinization of the Suspending Medium of Guard Cell Protoplasts from Vicia faba L.

Gotow¹,

Sakaki²,

Kondo³

et al. 1985

Plant Physiol.

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“…The voltage sensitivity of K+ channels as described here implies that K+ fluxes into and out of guard cells may be regulated by mechanisms that produce changes in the membrane potential. K+ channels may be activated by displacements of the membrane potential from VK+, induced by physiological effectors of stomatal aperture such as light, C02, and abscisic acid (28)(29)(30).…”

Section: Resultsmentioning

confidence: 99%

Voltage dependence of K ⁺ channels in guard-cell protoplasts

Schroeder

Raschke

Neher

1987

Proc. Natl. Acad. Sci. U.S.A.

405

363

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Stomatal pores in leaves enable plants to regulate the exchange of gases with their environment. Variations of the pore aperture are mediated by controlled changes of potassium salt concentrations in the surrounding guard cells. The voltage-dependent gating of K+-selective channels in the plasma membrane (plasmalemma) of cell-wall-free guard cells (protoplasts) was studied at the molecular level in order to investigate the regulation of K+ fluxes during stomatal movements. Inward and outward K+ currents across the plasmalemma of guard cells were identified by using the whole-cell configuration of the patch-clamp technique. Depolarizations of the membrane potential from a holding potential of -60 mV to values more positive than -40 mV produced outward currents that were shown to be carried by KV. Hyperpolarizations elicited inward K+ currents. Inward and outward currents were selective for K+ over Na' and could be partially blocked by exposure to extracellular Ba2 . In cell-attached and excised membrane patches, previously identified KV-selective single channels in guard cells were studied. Averaging of singlechannel currents during voltage pulses resulted in activation and deactivation kinetics that were similar to corresponding kinetics of inward and outward currents in whole cells, showing that K+-selective channels were the molecular pathways for the K+ currents recorded across the plasmalemma of single guardcell protoplasts. Estimates demonstrate that K+ currents through the voltage-gated K+ channels recorded in whole guard cells can account for physiological K+ fluxes reported to occur during stomatal movements in leaves.Elucidation of the molecular mechanisms involved in the transport of K+ across membranes of higher plant cells is fundamental to the understanding of osmoregulation and cell-volume control. Our studies have focused on the specialized guard cells surrounding stomatal pores in the lower epidermis of leaves of Viciafaba. Two guard cells modulate the aperture ofthe pore they surround, allowing the exchange of CO2 and 02 for photosynthesis, while minimizing water loss to the atmosphere. Light, among other physiological factors, causes guard cells to accumulate K+, leading to the opening of the pore (for reviews, see refs. 1-3). Darkening, high CO2 concentrations, and the phytohormone abscisic acid (under a condition of drought) mediate pore closure by inducing the release of K+ and counter ions (4, 5). Recently, ion channels have been identified in the plasmalemma of guard cells that are selectively permeable to K+ and through which K+ can be accumulated and released (6). In the present study, we have investigated the voltage-dependent activation of K+-selective channels in guard-cell protoplasts to explore possible mechanisms involved in the regulation of stomatal apertures. METHODSGuard-Cell Protoplasts. These were isolated from the lower epidermis of leaves from 2-to 3-week-old Vicia faba plants by 30-to 40-min incubation in 0.3 M D-mannitol/1 mM CaCl2/2% cellulase Onozuka RS (Yakult ...

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“…A characterization of the conditions under which the coupling between photosynthesis and stomatal conductance is altered should enhance our understanding of the physiology of the intact leaf and provide insight into the mechanisms controlling epidermis-mesophyll interactions. Experiments with isolated epidermal peels and guard cell protoplasts have shown that guard cells can respond to light in the absence of mesophyll (7,11,27,28). The wavelength dependence of these responses points to the activity of two separate photosystems, one sensitive to PAR and the other responsive specifically to blue light (5,9,15).…”

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confidence: 99%

Photocontrol of the Functional Coupling between Photosynthesis and Stomatal Conductance in the Intact Leaf

Zeiger¹,

Field²

1982

Plant Physiol.

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The photocontrol of the functional coupUng between photosynthesis and stomatal conductance in the leaf was investigated in gas exchange experiments using monochromatic light provided by lasers. Net photosynthesis and stomatal conductance were measured in attached leaves of Malva parviflora L. as a function of photon irradiance at 457.9 and 640.0 nanometers.Photosynthetic rates and quantum yields of photosynthesis were higher under red light than under blue, on an absorbed or incident basis.Stomatal conductance was higher under blue than under red light at all intensities. Based on a calculated apparent photon efficiency of conductance, blue and red light had similar effects on conductance at intensities higher than 0.02 milimoles per square meter per second, but blue light was several-fold more efficient at very low photon irradiances. Red Ught had no effect on conductance at photon irradiances below 0.02 mililmoles per square meter per second. These observations support the hypothesis that stomatal conductance is modulated by two photosystems: a blue lightdependent one, driving stomatal opening at low light intensities and a photosynthetically active radiation (PAR)-dependent one operating at higher irradiances.When low intensity blue light was used to illuminate a leaf already irradiated with high intensity, 640 nanometers light, the leaf exhibited substantial increases in stomatal conductance. Net photosynthesis changed only slightly. Additional far-red light increased net photosynthesis without affecting stomatal conductance. These observations indicate that under conditions where the PAR-dependent system is driven by high intensity red light, the blue light-dependent system has an additive effect on stomatal conductance.The wavelength dependence of photosynthesis and stomatal conductance demonstrates that these processes are not obligatorily coupled and can be controlled by light, independent of prevailing levels of intercellular CO2.The blue light-dependent system in the guard cells may function as a specific light sensor while the PAR-dependent system supplies a C02-modulated energy source providing functional coupling between the guard cells and the photosynthesizing mesophyll.Stomatal conductance in the intact leaf is tightly coupled to photosynthetic rates in the mesophyll.

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Light-sensitive membrane potentials in onion guard cells

Cited by 44 publications

References 16 publications

Light-Induced Alkalinization of the Suspending Medium of Guard Cell Protoplasts from Vicia faba L.

Light-Induced Alkalinization of the Suspending Medium of Guard Cell Protoplasts from Vicia faba L.

Voltage dependence of K ⁺ channels in guard-cell protoplasts

Photocontrol of the Functional Coupling between Photosynthesis and Stomatal Conductance in the Intact Leaf

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Light-sensitive membrane potentials in onion guard cells

Cited by 44 publications

References 16 publications

Light-Induced Alkalinization of the Suspending Medium of Guard Cell Protoplasts from Vicia faba L.

Light-Induced Alkalinization of the Suspending Medium of Guard Cell Protoplasts from Vicia faba L.

Voltage dependence of K + channels in guard-cell protoplasts

Photocontrol of the Functional Coupling between Photosynthesis and Stomatal Conductance in the Intact Leaf

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Voltage dependence of K ⁺ channels in guard-cell protoplasts