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

M.E. Bossen

    Research output: Thesisinternal PhD, WU

    Abstract

    <p><TT>Protoplasts, isolated from the primary leaves of dark-grown wheat ( <em>Triticum aestivum</em> 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 Ca</TT><sup>2+</SUP><TT>was present in the medium. Far-red light (FR), after R, prevented swelling, indicating phytochrome involvement. Swelling was inhibited when La</TT><sup>3+</SUP><TT>or the Ca <sup>2+</SUP>-channelblocker Verapamil were added. Swelling was induced in darkness by the Ca <sup>2+</SUP>-ionophore A23187 and the calmodulin antagonist W <sub>7</sub> . It is proposed that Rirradiation leads to opening of Ca <sup>2+</SUP>-channels, resulting in an increase of the cytoplasmic [Ca <sup>2+</SUP>] and protoplast swelling.</TT><p><TT>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 <sup>+</SUP>and H <sub>7</sub> . 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 <sup>2+</SUP>-channels.</TT><p><TT>Plant hormones also induced protoplast swelling in the presence of Ca <sup>2+</SUP>, while swelling was inhibited by GDP-β-S Acetylcholine induced, contrary to R-irradiation, swelling in the absence of Ca <sup>2+</SUP>, when K <sup>+</SUP>or Na <sup>+</SUP>were present in the medium. This swelling was not inhibited by GDP-β-S</TT><br/> <p><TT>The Ca <sup>2+</SUP>-sensitive dye murexide, has been used to monitor phytochrome-regulated changes in the [Ca <sup>2+</SUP>] of the medium. Red light induced a Ca <sup>2+</SUP>-efflux, while FR reversed this effect. The R-induced efflux was inhibited by Verapamil and W <sub>7</sub> by approx. 75%. Therefore, the efflux, via a Ca <sup>2+</SUP>-ATPase, appears to be dependent on the activation of Ca <sup>2+</SUP>-channels and a Ca <sup>2+</SUP>-influx.</TT><p><TT>The fluidity of the protoplast plasma membrane was studied, using the fluorescent membrane probe DPH. After R the anisotropy of DPH (r <sub>f</sub> ) was higher, indicating a decrease in membrane fluidity. In darkness, r <sub>f</sub> also increased upon osmotically induced protoplast swelling. It is not clear, whether R causes changes in membrane fluidity, independent of changes in volume.</TT><p><TT>The observed changes in plasma membrane properties after R-irradiation, show that protoplasts are an useful tool for studying phytochrome action in higher plants.</TT>
    Original languageEnglish
    QualificationDoctor of Philosophy
    Awarding Institution
    Supervisors/Advisors
    • Vredenberg, W.J., Promotor
    • Kendrick, R.E., Promotor
    Award date28 Mar 1990
    Place of PublicationS.l.
    Publisher
    Publication statusPublished - 1990

    Keywords

    • plant physiology
    • photosynthesis
    • protoplasts
    • cells
    • cell membranes

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