Plant Material
Vicia faba ‘Witkiem’, Cucurbita pepo ‘GelberZentner’, and Nicotiana tabacum plants were grown in potsin a greenhouse at temperatures varying between 20°C and30°C at 60% to 70% humidity and a 14-h/10-h light/dark periodwith supplementary lamp light (model SONT Agro 400 W; Phillips).The irradiance level was 200 to 250 µmol m–2 s–1at the plant apex. Plants were all taken in the vegetative periodjust before flowering.
Protoplast Isolation
Internodes were excised from 3- to 4-week-old V. faba plants.Then, tangential cuts were made to split the internodes. Forcoarse mechanical isolation of stem phloem strands, tangentialtissue sheets with a thickness of approximately 300 µmwere sliced with a razor blade from the fracture face of thesplit internode. After preincubation of the slices for 15 minin a standard medium (WM) containing 600 mol m–3 mannitol,1 mol m–3DL-dithiotreitol (DTT), and 25 mol m–3MES/NaOH, pH 5.7, the tissue was transferred into an enzymemixture containing 400 mol m–3 mannitol, 100 mol m–3KCl, 5 mol m–3 MgCl2, 1 mol m–3 DTT, 0.2% (w/v)polyvinylpyrrolidone-25, 0.5% (w/v) bovine serum albumin, 0.5%(w/v) cellulase ‘Onuzuka’ RS (Yakult Honsha), 0.03%(w/v) pectolyase Y-23 (Seishin), and 25 mol m–3 MES/NaOH,pH 5.7 (compare with Hafke et al., 2003
).
After incubation for 10 h at 28°C, disintegrating phloemstrands were filtered through a 80-µm nylon mesh and washedtwo times with the appropriate experimentation solution. Forpatch-clamp experiments, protoplasts were washed with standardbath solution and collected by centrifugation (Pico Fuge; Stratagene)twice.
The mechanism of protoplast formation and detachment were observedunder microscopic surveillance (Leica DM-LB, fluorescence microscope,equipped with a special water immersion objective, HCX APO L40x/0.80W U-V-I objective; Leica). The protoplasts were transferredinto a small volume of WM in a bathing chamber equipped witha microperfusion system. Here, SE protoplasts were treated withvarious solutions and permanent microscopic surveillance.
Light micrographs were taken with a digital camera (Canon PowerShot S40) connected to a computer (Canon Digital Camera SolutionDisk v8.0 software package).
SE protoplasts of N. tabacum and C. pepo were isolated as describedfor V. faba with slight modifications of the enzyme mixture,incubation time, and isolation temperature. SE protoplasts ofN. tabacum were isolated over a period of 4 h and a temperatureof 31°C in the above-mentioned enzyme mixture containing0.55% cellulase and 0.035% pectolyase. SE precursors of Cucurbitawere isolated over a period of 14 h and a temperature of 4°Cin the above-mentioned enzyme mixture containing 0.6% cellulaseand 0.04% pectolyase.
Staining of Protoplasts with CFDA, RH-414, and CW
CFDA
To test their membrane integrity, SE protoplasts were loadedwith CFDA-AM ester (Molecular Probes) as described for intactphloem tissue (Knoblauch and van Bel, 1998). The solution wasprepared from a CFDA-AM ester stock solution dissolved in WMto give a final concentration of 2.1 µM CFDA. After applicationof the CFDA-AM, protoplasts were incubated for 45 min at roomtemperature. During this period, the ester was cleaved by endogenousSE esterases into the membrane-impermeant and fluorescent formCF (Oparka and Read, 1994). Following thorough washing withWM, protoplast fluorescence was examined using CLSM (Leica TCS4D) with a Krypton-Argon laser (Omnichrome) at 488 nm as describedbefore (Knoblauch and van Bel, 1998).
RH-414
The protoplast plasma membrane was stained using the membrane-solublefluorochrome RH-414 (Molecular Probes). RH-414 was diluted froma stock solution in WM to give a final concentration of 4.3µM in a manner described before for the membrane-solublefluorochrome RH-160 (Knoblauch and van Bel, 1998). Protoplastswere incubated for 5 min in RH-414 before washing with WM andscanning with CLSM (excitation 564 nm).
CW Staining for Detection of Cellulose and Callose
Isolated protoplasts or intact tissues were stained with 0.1%(w/v) CW (Choi and O'Day, 1984) dissolved in 400 mM mannitolfor 15 min. After thorough washing with WM, cells were observedunder an epifluorescence microscope (Leica DMLB) using a BP340-380 excitation filter and an LP 425 barrier filter combination(Leica Microsystems).
Osmotic Experiments
SE protoplasts were bathed in a hyperosmotic solution containing600 mol m–3 mannitol, 1 mol m–3 DTT, 1 mol m–3CaCl2, and 25 mol m–3 MES/NaOH, pH 5.7. An abrupt bathchange to a hypo-osmotic medium containing 50 mol m–3mannitol, 1 mol m–3 DTT, 1 mol m–3 CaCl2, and 25mol m–3 MES/NaOH, pH 5.7, by a homemade microperfusionsystem imposed an osmotic shock. As a control experiment forSE protoplast swelling in a calcium-free solution, protoplastswere preincubated in the hyperosmotic solution and osmoticallyshocked with a solution containing 50 mol m–3 mannitol,1 mol m–3 DTT, 4 mol m–3 EGTA, and 25 mol m–3MES/NaOH, pH 5.7.
Gd3+ Experiments
SE protoplasts were bathed in the hyperosmotic standard solution(see above) containing 600 mol m–3 mannitol, 1 mol m–3DTT, 1 mol m–3 CaCl2, and 25 mol m–3 MES/NaOH, pH5.7. Mechanical stress (suction) was exerted on SE protoplastsvia patch-clamp microcapillaries connected to a pressure device(Cell Tram Oil microinjector; Eppendorf).
A microcapillary filled with the respective bathing medium wasmaneuvered to the protoplast by means of an LN SM-1 micromanipulator(Luigs & Neumann). Contact between protoplast and microcapillarywas made by suction with the aid of a Cell Tram Oil microinjector(Eppendorf). As a control, protoplasts were incubated in theabove-mentioned hyperosmotic solution supplied with 2 mol m–3of the calcium channel blocker Gd3+ (as GdCl3). Protoplastswere observed using an epifluorescence microscope (Leica DM-LB,fluorescence microscope, equipped with a special water immersionobjective, HCX APO L40x/0.80 W U-V-I objective; Leica). Micrographswere taken with a digital camera (Canon Power Shot S40).
Patch-Clamp Experiments
Membrane currents were recorded using standard patch-clamp techniquesaccording to Hamill et al. (1981). Micropipettes were pulledfrom borosilicate glass microcapillaries (GC150F-10; Clark ElectromedicalInstruments) using an L/M-3P-A puller (List-Medical). The standardexperimental solutions contained 60 mol m–3 potassium-gluconate,2 MgCl2 mol m–3, 300 mol m–3 mannitol, 10 mol m–3Bis-Tris propane, titrated with MES to pH 7.5 in the pipette,and 6 mol m–3 potassium-gluconate, 1 mol m–3 MgCl2,1 mol m–3 CaCl2, 400 mol m–3 mannitol, 5 mol m–3MES, titrated with Bis-Tris propane to pH 5.5 in the bath. TheAg/AgCl electrode was connected to the bath by a 3% (w/v) agarbridge filled with 100 mol m–3 KCl. While applying a positivepressure to the pipette, the pipette tip was dipped into thebath and brought into contact with a SE protoplast with theaid of an LN-SM-1 micromanipulator. After compensation of theoffset potential of the pipette, contact was made between thepipette tip and the protoplast and gentle suction was appliedto obtain a gigaseal. The membrane under the patch was brokenby a short bipolar voltage pulse (±720 mV, 2.5 ms each)and simultaneous suction to obtain the whole-cell configuration.Whole-cell currents were recorded with an EPC-9 patch-clampamplifier (HEKA Elektronik), filtered with an eight-pole Besselfilter with a cutoff frequency of 200 Hz, and sampled five timesthe filter frequency at 1 kHz on a personal computer. Data weredigitized (ITC-16; Instrutech) and analyzed using PULSE andPULSFIT software (HEKA Elektronik). Voltages and currents weregiven with reference to extracellular side of the membrane asground (Bertl et al., 1992). All membrane voltages were correctedoff-line for liquid junction potential (Neher, 1992) using anLJP-calculator (Ng and Barry, 1995). All measurements were carriedout at ambient temperatures between 20°C and 22°C.
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