INTRACELLULAR-LOCALIZATION OF PHOSPHOLIPASE-D IN LEAVES AND SEEDLING TISSUES OF CASTOR BEAN

The intracellular distribution of phospholipase D (PLD; EC 3.1.4.4) in castor bean (Ricinus communis L.) tissues was investigated by subcell ular fractionation and by immune-electron microscopy. Centrifugal frac tionation revealed that most PLD in young leaves was soluble, whereas in mature leaves a majority of PLD was associated with microsomal memb ranes. Further separation of microsomal membranes by a two-phase parti tioning system indicated that PLD was associated with both plasma and intracellular membranes. Sucrose gradient separation of intracellular membranes showed PLD present in the endoplasmic reticulum, a submicros omal band, and in soluble fractions but not in mitochondria and glyoxy somes of postgermination endosperm. Immunocytochemical studies found h igh gold labeling in vacuoles in young leaves, suggesting that the hig h level of soluble PLD in young leaves is due to release of PLD from v acuoles during tissue disruption. In addition to the labeling in vacuo les, gold particles were also found in the cytoplasmic matrices and pl asma membrane in leaves and in 2-d postgermination seedlings. Collecti vely, these results show that PLD in castor bean leaf and seedling tis sues is localized in the vacuole and is associated with the endoplasmi c reticulum and plasma membrane and that the relative distribution bet ween the soluble and membrane compartments changes during castor bean leaf development.