ABSTRACT
It is now generally accepted that a phosphoinositide cycle is involved in the transduction of a variety of signals in plant cells. In animal cells, the binding of D-myo-inositol 1,4,5-trisphosphate (InsP(3)) to a receptor located on the endoplasmic reticulum (ER) triggers an efflux of calcium release from the ER. Sites that bind InsP(3) with high affinity and specificity have also been described in plant cells, but their precise intracellular locations have not been conclusively identified. In contrast to animal cells, it has been suggested that in plants the vacuole is the major intracellular store of calcium involved in signal induced calcium release. The aim of this work was to determine the intracellular localization of InsP(3)-binding sites obtained from 3-week-old Chenopodium rubrum leaves. Microsomal membranes were fractionated by sucrose density gradient centrifugation in the presence and absence of Mg(2+) and alternatively by free-flow electrophoresis. An ER-enriched fraction was also prepared. The following enzymes were employed as specific membrane markers: antimycin A-insensitive NADH-cytochrome c reductase for ER, cytochrome c oxidase for mitochondrial membrane, pyrophosphatase for tonoplast, and 1,3-beta-D-glucansynthase for plasma membrane. In all membrane separations, InsP(3)-binding sites were concentrated in the fractions that were enriched with ER membranes. These data clearly demonstrate that the previously characterized InsP(3)-binding site from C. rubrum is localized on the ER. This finding supports previous suggestions of an alternative non-vacuolar InsP(3)-sensitive calcium store in plant cells.
Subject(s)
Chenopodiaceae/metabolism , Inositol 1,4,5-Trisphosphate/metabolism , Binding Sites , Biomarkers , Cell Fractionation , Cell Membrane/metabolism , Centrifugation, Density Gradient , Endoplasmic Reticulum/metabolism , Enzymes/metabolism , Intracellular Membranes/metabolism , Microsomes/metabolism , Plant Leaves/metabolism , Signal TransductionABSTRACT
Different forms of phospholipase D (dependent on and independent of the presence of phosphatidylinositol 4,5-bisphosphate, PIP(2)) were identified in maturing and germinating seeds of Brassica napus. Both forms were present in cytosolic and membrane fractions of maturing seeds. PIP(2)-dependent activity increased continuously during seed germination, while PIP(2)-independent activity appeared mostly at the very beginning of seed maturation. PIP(2)-dependent activity was detected mainly in the plasma-membrane fraction. Phosphatidylinositol-specific phospholipase C (PI-PLC) was found only in membrane fractions of both types of developing rape seed tissues. The increasing activities of PLC and PIP(2)-dependent PLD were mainly detected in hypocotyls of seedlings. Some biochemical characteristics of both described enzymes are also presented.
