The capacity of pea root plastids for galactolipid synthesis was investigated utilizing radiolabelled acetate and UDP-galactose. Galactolipid biosynthesis was completely dependent on an exogenous supply of UDP-galactose. UDP-galactose stimulated both total lipid biosynthesis from acetate and the proportion of radioactivity accumulated in monogalactosyldiacylglycerol (MGDG). The proportion of MGDG synthesized was saturated at 30$ mu$M UDP-galactose and represented approximately 30% of the total lipid radioactivity after a one hour incubation. However, total lipid biosynthesis continued to increase with concentrations of UDP-galactose up to 75$ mu$M while the proportion of radioactivity in MGDG remained at 30%. MGDG biosynthesis was always accompanied by a corresponding decrease in the amount of diacylglycerol (DAG) accumulated. Digalactosyldiacylglycerol (DGDG) synthesis was not routinely observed in these experiments. These results suggest that the in vitro pathway for MGDG synthesis in the root plastids of pea (an 18:3 plant) is similar to 16:3 plants (FFA's$ to$PA$ to$DAG$ to$MGDG). The endogenous lipids, consistent with the thought of pea as an 18:3 plant, contained 80% C$ sb{18}$ in the fatty acids of MGDG, DGDG, TG and PC. However, in labelled acetate experiments palmitate was the predominately labelled fatty acid in all lipids except PC (where 80% was 18:1). The precursors PA and DAG had ratios of 16:0, 18:0, and 18:1 similar to that of MGDG. 70-80% of the label was associated with the sn-2 position of glycerolipids. The cofactors required for fatty acid synthesis were generally not as required for galactolipid synthesis. The results suggest that galactolipid synthesis relies primarily on endogenous DAG and only partly involves de novo fatty acid synthesis. (Abstract shortened by UMI.)

Pea root plastids were isolated by differential centrifugation and resulting crude plastid fraction was purified by centrifugation through 10%(v/v) Percoll. Marker enzymes indicated that greater than 50% of the plastids were recovered essentially free from mitochondrial and endoplasmic reticulum contamination. The optimum in vitro conditions for glycerolipid biosynthesis from (U-$ sp{14}$C) glycerol-3-phosphate have been determined. Total glycerolipid biosynthesis was approximately 15 nmole/hr/mg protein in the presence of 200 $ mu$M glycerol-3-phosphate, 0.5 mM each of NADH and NADPH, 15 mM KH$ sb2$CO$ sb3$, 0.05 mM CoA, and 2 mM each of ATP and MgCl$ sb2$, 100 mM Bis Tris Propane (pH 7.5) and incubated at the standard temperature of 25$ sp circ$C. ATP, Coenzyme A and a divalent cation are absolutely required for glycerolipid biosynthesis, whereas reduced nucleotides and bicarbonate improve the synthesis to varying degrees. Dihydroxyacetone phosphate had little effect, while dithiothreitol, detergent and Mn$ sp{2+}$ inhibited activity. Under the optimum conditions, isolated pea root plastids mainly synthesized approximately 15% phosphatidic acid, 16% phosphatidylcholine, 13% phosphatidylglycerol, 32% triacylglycerol. Galactolipid synthesis occurred only when UDP-galactose was supplied. Different concentrations of some cofactors resulted in alterations of glycerolipid distribution. Phospholipase A$ sb2$ and Rhizopus lipase digestions of phospholipids and neutral lipids revealed that radioactive fatty acids were preferentially esterified to position sn 2 of each glycerolipid with generally 2-4 times as much radioactivity as position sn 1. Pea root plastids are composed of approximately 62% phospholipid, 24% neutral lipid and 14% glycolipid. Within these classes PG, TAG, and the galactolipids are the major components representing 24, 12, and 12% of the total plastid lipids.

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Germinating pea (Pisum sativum L.) roots were divided into five sequential 0.5 cm segments from the root tip. Pooled segments were analyzed for their protein, starch and lipid content as an indirect indication of plastid function. Fresh weights of root segments were lowest in the tips (4.45mug per segment) and progressively higher up to the fifth segment (11.09mug per segment). Total protein, starch and lipid content, on a per segment basis, were all highest in zone 1 (tip segment) and progressively lower up to zone 5. Plastids were isolated from each of the five root segments and analyzed for their capacity for lipid biosynthesis under several different in vitro conditions. Collectively, the observations presented here suggest that the relative contributions of plastids to the overall physiology of germinating pea roots gradually diminishes as root development proceeds, and that plastids isolated from progressively older root zones have increased capacity for glycolytic and/or pentose phosphate metabolism. (Abstract shortened by UMI.)

The interaction between the glycolytic metabolism and fatty acid and glycerolipid biosynthesis in pea root (Pisum sativum L.) plastids was assessed in this study. When various glycolytic intermediates were used to substitute for the APT requirement for fatty acid synthesis from acetate, phosphoenolpyruvate, 2-phosphoglycerate, fructose-6-phosphate and glucose-6-phosphate each gave 48, 17, 23 and 17%, respectively, of the ATP-control activity. Similarly, in the absence of exogenously supplied ATP, the optimized triose-phosphate shuttle, which consists of 2 mM dihydroxyacetone phosphate, 2 mM oxaloacetic acid and 4 mM inorganic phosphate, gave up to 44% the ATP-control activity in promoting fatty acid synthesis from acetate. These results suggest that 3-phosphoglycerate kinase and pyruvate kinase in these plastids can function in intraplastidic ATP production through substrate level phosphorylation. However, in all cases, exogenously supplied ATP gave the greatest rates of fatty acid and glycerolipid synthesis. Radiolabeled pyruvate, glucose, glucose-6-phosphate, and malate in comparison to acetate were all variously utilized for fatty acid and glycerolipid biosynthesis by the root plastid. At the highest concentrations tested (3-5 mM), the rates of incorporation of pyruvate, glucose-6-phosphate and acetate into fatty acids were 183, 154, 125 nd 99 nmol $ rm cdot h sp{-1} cdot mg sp{-1}$, respectively. Malate was the least effective precursor, giving less than 55 nmol $ rm cdot h sp{-1} cdot mg sp{-1}$. Acetate incorporation was approximately 55% dependent on exogenously supplied reduced nuclotides (NADPH and NADH), whereas the utilization of the remaining precursors was only approximately 10-20% dependent on NAD(P)H. These results indicate that the entire pathway of carbon flow from glycolysis, including pyruvate dehydrogenase (PDHase), to fatty acids is operating in pea root plastids. Further, the intraplastidic glycolytic pathway plays an important role in provi

Fatty acid biosynthesis from (1-$ sp{14}$C) acetate was optimized in plastids isolated from primary root tips of 7-day-old germinating pea seeds. Fatty acid synthesis was maximum at 82.3 nmol/hr/mg protein in the presence of 200$ mu$M acetate, 0.5mM each of NADH, NADPH and CoA, 6mM each of ATP and MgCl$ sb2$, 1mM each of MnCl$ sb2$ and glycerol-3-phosphate (G3P), 15mM KHCO$ sb3$, and 0.1M Bis tris propane, pH 8.0 incubated at 35C. At the standard incubation temperature of 25C, fatty acid synthesis was linear for up to 6 hours with 80 to 120 $ mu$g/ml plastid protein. ATP and CoA were absolute requirements, whereas divalent cations, potassium bicarbonate and reduced nucelotides all improved activity by 2 to 10 fold. Mg$ sp{2+}$ and NADH were the preferred cation and nucleotide, respectively. G3P and dihydroxyacetone phosphate had little effect, and dithiothreitol and detergents generally inhibited incorporation of $ sp{14}$C-acetate into fatty acid. / Glycerolipid synthesis was obtained from $ sp{14}$C-acetate, (U-$ sp{14}$C) G3P and (U-$ sp{14}$C) glycerol at relative rates of 3.7:1.0:0.1, respectively. (Abstract shortened by UMI.)

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