Phosphatidate phosphatase activity was first solubilized from spinach chloroplast envelope membranes using a zwitterionic detergent, CHAPS. We have set up a method to assay the solubilized enzyme, using [P-32]phosphatidic acid as a substrate. Addition of phosphatidylglycerol to the incubation medium was essential for optimal enzyme activity, probably because it was responsible for a better solubilization of the substrate by CHAPS. However, the possibility of a specific role for this phospholipid as a physiological activator cannot be ruled out since the inner envelope membrane from spinach chloroplasts contains significant amounts of phosphatidylglycerol. The biochemical properties of the solubilized phosphatidate phosphatase from chloroplast envelope membranes were investigated. As in the native membranes, the solubilized phosphatidate phosphatase was inhibited by Mg2+ and also by a wide range of metal ions such as Mn2+ and Zn2+. A partial purification of the enzyme was obtained, using hydroxyapatite chromatography. Our results demonstrate that the biochemical properties of the envelope phosphatidate phosphatase are rather different from those of phosphatidate phosphatase described in other systems, and especially in extraplastidial compartments from plant tissues. Finally, the question of whether the two envelope enzymes which catalyze the conversion of phosphatidic acid into monogalactosyldiacylglycerol, i.e. phosphatidate phosphatase and 1,2-diacylglycerol galactosyltransferase (or MGDG synthase), could be associated together within the inner envelope membrane is discussed.

Photosynthetic membranes, or thylakoids, are the most extensive membrane system found in the biosphere. They form flattened membrane cisternae in the cytosol of cyanobacteria and in the stroma of chloroplasts. The efficiency of light energy capture and conversion, critical for primary production in ecosystems, relies on the rapid expansion of thylakoids and their versatile reorganization in response to light changes. Thylakoid biogenesis results from the assembly of a lipid matrix combined with the incorporation of protein components. Four lipid classes are conserved from cyanobacteria to chloroplasts: mono- and digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol, and phosphatidyldiacylglycerol. This review focuses on the production and biophysical properties of galactolipids, making them determinant factors for the nonvesicular/nonlamellar biogenesis and for the three-dimensional architecture of nascent thylakoids. The regulation of MGD1, the committing enzyme of galactolipid biosynthesis in Arabidopsis, via feedback regulatory loops and control of protein binding to membranes, is also detailed.

Because the envelope phosphatidate phosphatase plays a pivotal role in chloroplast glycerolipid metabolism, we have analyzed whether diacylglycerol could be a regulatory factor of the enzyme. Using isolated envelope membranes in which the level of diacylglycerol was modified by thermolysin treatment of intact chloroplasts to destroy the galactolipid:galactolipid galactosyltransferase, we have demonstrated that phosphatidate phosphatase activity was reduced when the membrane was enriched in diacylglycerol. All 1,2-diacylglycerol molecular species assayed were demonstrated to inhibit the enzyme to about the same extent. Kinetic studies with envelope from thermolysin-treated chloroplasts were performed in the absence and presence of diacylglycerol, and diacylglycerol was shown to be a powerful competitive inhibitor of the reaction. Finally, using isolated intact spinach chloroplasts, we have demonstrated that in situ phosphatidate phosphatase activity can be modulated by the level of diacylglycerol present in the membrane. The relevance of phosphatidate phosphatase inhibition by diacylglycerol in the regulation of chloroplast glycerolipid biosynthesis is discussed.