Loading of quin2 into the oat protoplast and measurement of cytosolic calcium ion concentration changes by phytochrome action.
The loading of quin2 into oat protoplasts was carried out in an incubation medium (0.6 M sorbitol, 1 mM CaCl2, 5 mM Mes, 5 mM Tris, 0.05% BSA, 1 mM KCl, 1 mM MgSO4 (pH 6.8)), in which we found the best viability of the protoplast and the highest membrane permeability of quin2/AM, compared with the results obtained from any other incubation medium we had tried to use. 50 microns of quin2/AM was added in the suspension medium containing 5 x 10(5)/ml of oat protoplasts, and incubation at 4 degrees C was performed for 24 h. From atomic absorption data, we confirmed that quin2 loading was 1.78 mmol per liter of cells. Red-light (660 nm) irradiation for 5 min caused an increase of the cytosolic Ca2+ concentration from 30 to 193 nM. On the other hand, a subsequent irradiation with far-red light (730 nm) for 5 min decreased it by about 48 nM. Even when the extracellular Ca2+ was completely chelated with 1 mM EDTA, red light increased the cytosolic Ca2+ concentration by about 51 nM and far-red light decreased it to 3 nM. These results imply that the Pfr form of phytochrome functions not only in the process of influx of Ca2+, but also in the mobilization process of Ca2+ from the intracellular Ca2+ pools. The fact that the Pr form of phytochrome lowers the cytosolic Ca2+ concentration is also presented in this report.
Chae
Q
Q
Department of Biochemistry, Chungbuk National University, Cheongju, Korea.
Park
H J
HJ
Hong
S D
SD
D000634:Aminoquinolines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D002523:Edible Grain; D005456:Fluorescent Dyes; D010834:Phytochrome / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D011523:Protoplasts / Q000187:drug effects / Q000378:metabolism*
D000634:Aminoquinolines; D005456:Fluorescent Dyes; D010940:Plant Proteins; D010834:Phytochrome; C035738:Quin2-acetoxymethyl ester; D002118:Calcium