Phosphatidylglycerol Synthesis in Pea Chloroplasts

Abstract

Isolated intact pea chloroplasts synthesized phosphatidyiglycerol from either I'4Clacetate or j'Clglycerol 3-phosphate. Both time-course and pulse-chase labeling studies demonstrated a precursor-product relationship between newly synthesized phosphatidic acid and newly synthesized phosphatidylglycerol.The synthesis both of CDP-diacylglycerol from exogenous phosphatidic acid and CTIP, and of phosphatidylglycerol from exogenous CDPdiacyiglycerol and glycerol 3-phosphate, could be assayed in fractions obtained from disrupted chloroplasts. Moreover, the enzymes catalyzing these reactions were localized in the inner envelope membrane. Exoge- Purification and Fractionation of Chloroplasts. Intact chloroplasts were obtained from homogenates of 14-to 17-d-old pea seedlings (Pisum sativum var Laxton's Progress No. 9) by differential centrifugation followed by Percoll density gradient centrifugation as described (8), with the following exceptions: 1. the pH of the buffer was adjusted with KOH instead of NaOH; 2. the centrifugation speeds were increased and the times decreased, as modified from the protocol described by Heinz and Roughan (17) to: (a) first sedimentation, 3300g, 90 s; (b) Percoll gradient sedimentation, 13,000g, 5 min; (c) dilution and recovery, and wash sedimentations, 3300g, 90 and 75 s, respectively. Those chloroplasts used directly for lipid synthesizing assays were resuspended in 0.3 M sorbitol in 33.4 mM Tricine-NaOH (pH 7.9).Those chloroplasts which were fractionated were suspended in 0.6 M sucrose (2 mg Chl/ml), and broken by slow freezing (-20°C, about 45 min) and thawing (22-24C, about 30 min) (8). The suspension was diluted with buffer to 0.36 M sucrose, and 5 ml were pipetted over the following sucrose step-density gradients: 1.3 M, 2 ml; 1.2 M, 1 ml; 1.1 M, 4 ml; 0.5 M, 5 ml (as modified from Joyard and Douce [ 18]). The gradients were then centrifuged at 1 16,000gma for 90 min. The soluble stromal components remained within the 0.36 M layer, the envelopes sedimented to the 0.5/1.1 M interface, while the thylakoids sedimented to the 1.3 M layer. A small green pellet, which accounted for 12 to 40% of the total Chl, was assumed to be unbroken chloroplasts. The envelope membranes were collected with a Pasteur pipette, diluted 4-to 5-fold with buffer, and collected by centrifugation (90,000g,,,. 60 min); they were then resuspended in either buffer only or 0.2 M sucrose (usually to about 0.5-1 mg protein/ml). All

Low Temperature Development Induces a Specific Decrease in trans-Δ3-Hexadecenoic Acid Content which Influences LHCII Organization

“…Phosphatidylglycerol is the major phospholipid present in thylakoid membranes ( 14) and its biosynthesis has recently been shown to take place within isolated chloroplasts (2). This lipid is characterized by the presence of the unusual fatty acid, trans-16:1, which is present only in plastids (14).…”

Metabolic Remodeling of Membrane Glycerolipids in the Microalga Nannochloropsis oceanica under Nitrogen Deprivation

“…Three enzymes-CDP-DAG synthase (CDS), PG-phosphate synthase (PGPS), and PG-phosphate phosphatase (PGPPS)-were proposed to be involved in PG synthesis (Andrews and Mudd, 1985). In contrast to the absence of the candidate PGPPS in plants Arabidopsis and C. reinhardtii (Riekhof et al, 2005b), all the genes required for PG biosynthesis, including two copies of putative PGPPS, were identified in IMET1.…”

Molecular aspects of plastid envelope biochemistry

“…2), i.e. with C I 8 fatty acid at the sn-I and C16 fatty acid at the sn-2 position of the glycerol, and the enzymes involved in its synthesis are localized in the inner envelope membrane [66]. Therefore, in addition to glycerol-3-phosphate acyltransferase and 1-acylglycerol-3-phosphate acyltransferase, which catalyze the formation of phosphatidic acid, phosphatidylglycerol synthesis requires the coordinated functioning of the following three enzymes ( Fig.…”