Plant protoplasts as a model system to study phytochrome-regulated changes in the plasma membrane

Protoplasts, isolated from the primary leaves of dark-grown wheat ( Triticum aestivum L.), have been used as a model system to study phytochrome-regulated changes of the plasma membrane. Such protoplasts only swelled after red light (R)-irradiation, when Ca2+was present in the medium. Far-red light (FR), after R, prevented swelling, indicating phytochrome involvement. Swelling was inhibited when La3+or the Ca 2+-channelblocker Verapamil were added. Swelling was induced in darkness by the Ca 2+-ionophore A23187 and the calmodulin antagonist W 7 . It is proposed that Rirradiation leads to opening of Ca 2+-channels, resulting in an increase of the cytoplasmic [Ca 2+] and protoplast swelling.The effect of modulators of G-proteins and the phosphatidylinositol cycle, as known in animal cells, on the swelling response was examined. The R-induced swelling was inhibited by GDP-β-S and by neomycin, Li +and H 7 . In darkness, swelling was found when GTP-γ-S or PMA were added to the protoplasts. All agonists and antagonists used, influenced the swelling response, as predicted by transposition of the animal model to plants. This suggests that R-irradiation, leads to activation of a G- protein, which results in the opening of Ca 2+-channels.Plant hormones also induced protoplast swelling in the presence of Ca 2+, while swelling was inhibited by GDP-β-S Acetylcholine induced, contrary to R-irradiation, swelling in the absence of Ca 2+, when K +or Na +were present in the medium. This swelling was not inhibited by GDP-β-S The Ca 2+-sensitive dye murexide, has been used to monitor phytochrome-regulated changes in the [Ca 2+] of the medium. Red light induced a Ca 2+-efflux, while FR reversed this effect. The R-induced efflux was inhibited by Verapamil and W 7 by approx. 75%. Therefore, the efflux, via a Ca 2+-ATPase, appears to be dependent on the activation of Ca 2+-channels and a Ca 2+-influx.The fluidity of the protoplast plasma membrane was studied, using the fluorescent membrane probe DPH. After R the anisotropy of DPH (r f ) was higher, indicating a decrease in membrane fluidity. In darkness, r f also increased upon osmotically induced protoplast swelling. It is not clear, whether R causes changes in membrane fluidity, independent of changes in volume.The observed changes in plasma membrane properties after R-irradiation, show that protoplasts are an useful tool for studying phytochrome action in higher plants.