Phosphatidylglycerol Synthesis in Spinach Chloroplasts: Characterization of the Newly Synthesized Molecule

Abstract

Intact chloroplasts from spinach (Spuiacia oleracea L., hybrid 424) readily incorporate I'4Ciglycerol-3-phosphate and 114CIacetate into diacylglycerol, monoacylglycerol, diacylglycrol, free fatty acids (only when acetate is the precursor), phosphatidic acid, phosphatidylcholine, and most notably phosphatidylglycerol. The fraction of phosphatidylglycerol synthesized is greatly increased by the presence of manganese chloride in the reaction mixture. PG2 is an important lipid component ofchloroplasts. Depending on the tissue, it can comprise 60 to 70% of the total chloroplast phospholipid (10). Characterized by the presence of the unusual trans-A3-hexadecenoic acid (11) and its increase during chloroplast development, this unique species of PG has been suggested to have a role in granal stacking (9).While chloroplasts from Euglena can synthesize PG (4-6), conflicting reports (7,16,25) left some question as to whether or not chloroplasts from higher plants can synthesize their own PG. The earlier works of Douce and Guillot-Salomon (7) and Sastry and Kates (25) suggested that PG was synthesized by chloroplasts. These results were cast in doubt when Marshall and Kates (16) determined that PG was not synthesized by chloroplasts, and since then, chloroplasts from higher plants generally have been considered incapable of PG synthesis. Recently, Mudd and DeZacks (20) reexamined chloroplasts from spinach for their capacity to synthesize PG and presented evidence that these higher plant chloroplasts could in fact synthesize their own PG.We have continued these investigations using both radiolabeled G3P and acetate to further establish optimal conditions for PG ' synthesis. We have also subjected the biosynthesized PG to a number of degradative procedures in order to understand the pathway to the newly synthesized PG molecule.MATERIALS AND METHODS Spinach Culture. Spinach seeds (Spinacia oleracea L., hybrid 424; Ferry Morse Seed Company, Mountain View, CA) were germinated and maintained for 10 to 12 d in vermiculite moistened with half-strength Hoagland nutrient solution. The seedlings were transferred to hydroponic culture dishes containing nutrient solution and supported by plastic sheets with 1-cm holes and cotton plugs. Seedlings and plants were grown in a growth chamber with a 12-h daylength and maintained at 23°C and 18°C during the daytime and nighttime, respectively.Chloroplast Isolation. Spinach leaves were harvested and homogenized and chloroplasts isolated and purified as described previously (20). Chl determinations were as described by Holden (12).Chloroplast Incubation Conditions and Lipid Extraction. The composition of the standard reaction mixture conditions of incubation and lipid extraction were all as described earlier (20). Variations of these procedures were the use of 2 mm MnCl2 in the standard reaction mixture and substitution of 1 M KCI for the water of the extraction mixtures when phospholipase A2 and Rhizopus arrhizus digestions were performed. When necessary, approximately 500,000 to 60...

Involvement of Ferredoxin in Desaturation of Lipid-Bound Oleate in Chloroplasts

“…This resembles fatty acid labeling of procaryotic MGD in spinach leaves, which contained 18:1, 18:2, and 18:3 at the sn-l position, whereas 16:0 at the sn-2 position was not desaturated ( 15). In previous studies with intact organelles, desaturation of newly formed acyl groups was not only observed in MGD but also in SQD (1,14), PG (1,21,26), and PC (13). With ruptured chloroplasts we also detected desaturation in lipids other than MGD.…”

The phosphatidic acid phosphatase of the chloroplast envelope is located on the inner envelope membrane

“…These observations are strengthened by the fact that phosphatidic acid and diacylglycerol synthesized ['4C]acetate [8] or a mixture of different acyl-CoA and/or acyl-ACP species [3] have a specific fatty acid pattern (i.e., with Cl8 fatty acids and Cl6 fatty acids respectively, on the sn-I and sn-2 position of the glycerol backbone). This pattern is identical to that found in MGDG from 16: 3-containing plants and in sulfolipids and phosphatidylglycerol synthesized either in vivo [9-121 or in vitro [6,7,13,14]. Therefore, it is important to localize precisely the enzymes involved in diacylglycerol biosynthesis in order to understand their role in plastid glycerolipid metabolism.…”

Phosphatidylglycerol Synthesis in Pea Chloroplasts

“…The addition of MnCl2 was reported to stimulate PG synthesis in spinach chloroplasts (28). However, the addition of MnC12 ( 1-4 mM) to pea chloroplasts had no significant effect on the proportion of label incorporated into PG, although the proportion of label present in PA and LPA appeared to increase slightly (data not shown).…”

“…It has been clearly demonstrated recently that isolated intact spinach chloroplasts can synthesize PG de novo from either acetate or glycerol 3-P under conditions of fatty acid synthesis (25,28). We have now utilized pea chloroplasts to investigate the pathway and localization of PG synthesis.…”