Characterization of Fatty Acid Biosynthesis in Isolated Pea Root Plastids

Stahl, Richard; Sparace, Salvatore A.

doi:10.1104/pp.96.2.602

Cited by 18 publications

(20 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although in vitro fatty acid biosynthesis in these plastids is dependent on exogenously supplied ATP (17,18), the results clearly demonstrate that these plastids can independently generate sufficient ATP to support high rates of fatty acid biosynthesis. At least two mechanisms appear to be involved in this ATP synthesis.…”

Section: Resultsmentioning

confidence: 78%

“…When measurable, varying the nucleotide energy sources had essentially no effect on the proportions of radioactive fatty acids synthesized except that ADP + PPi caused a marked increase in the proportion ofoleic acid accumulated with a corresponding decrease in palmitic acid, suggesting that fatty acid synthetase II was somehow stimulated. Alternatively, by inhibiting the triose phosphate translocator (7), PPi might reduce the availability of glycerol-3-phosphate in the standard incubation mixture, which could also cause an increase in the proportion of oleic acid accumulated (18).…”

Section: Resultsmentioning

confidence: 99%

“…Roots were homogenized and plastids isolated by a method modified from Stahl and Sparace (18) and Emes and England (6) as follows. Roots (6-10 g per experiment) were homogenized in 2 mL of homogenization medium (6) Fatty acid biosynthesis was measured under reaction conditions described earlier ( 18) except that the incubation mixture contained 0.33 M sorbitol instead ofsucrose. The reaction was terminated by the addition of 3 mL of chloroform/ methanol/acetic acid (1:2:0.1, v/v).…”

Section: Plant Materials and Plastid Isolationmentioning

confidence: 99%

See 2 more Smart Citations

Energy Requirements for Fatty Acid and Glycerolipid Biosynthesis from Acetate by Isolated Pea Root Plastids

Kleppinger-Sparace¹,

Stahl²,

Sparace³

1992

Plant Physiol.

Self Cite

View full text Add to dashboard Cite

Fatty acid and glycerolipid biosynthesis from [14C]acetate by isolated pea root plastids is completely dependent on exogenously supplied ATP. CTP, GTP, and UTP are ineffective in supporting fatty acid biosynthesis, all resulting in <3% of the activity obtained with ATP. However, ADP alone or in combination with inorganic phosphate (Pi) or pyrophosphate (PPi) gave up to 28% of the ATP control activity, whereas AMP + PPi, PPi alone, or Pi alone were ineffective in promoting fatty acid biosynthesis. The components of the dihydroxyacetonephosphate (DHAP) shuttle (DHAP, oxaloacetate, and Pi), which promote intraplastidic ATP synthesis, restored 41% of the control ATP activity, whereas the omission of any of the shuttle components abolished this activity.When the DHAP shuttle components were supplemented with ADP, the rate of fatty acid biosynthesis was completely restored to that observed in the presence of ATP. Under the conditions of ADP + DHAP shuttle-driven fatty acid biosynthesis, exogenously supplied ATP gave only a 6% additional stimulation of activity. In general, variations in the energy source had only small effects on the proportions of radioactive fatty acids and glycerolipids synthesized. Most notably, higher amounts of radioactive oleic acid, free fatty acids, and diacylglycerol and lower amounts of phosphatidic acid were observed when ADP and/or the DHAP shuttle were substituted for ATP. The results presented here indicate that, although isolated pea root plastids readily utilize exogenously supplied ATP for fatty acid biosynthesis, these plastids can also synthesize sufficient ATP when provided with the appropriate cofactors.Fatty acid and glycerolipid biosynthesis from acetate are strictly energy dependent in all plastids. ATP is required for the synthesis of both acetyl-CoA and malonyl-CoA by acetylCoA synthetase and acetyl-CoA carboxylase, respectively (20). Similarly, the reduced nucleotides NADPH and NADH are required in the 13-ketoacyl-ACP2 reductase and 2-enoyl-ACP reductase steps ofde novo fatty acid biosynthesis, respectively, as well as the desaturation of stearoyl-ACP (20 tors are only indirectly required for plastidic glycerolipid biosynthesis in so far as this process is dependent on fatty acid synthesis. In chloroplasts, ATP and reduced nucleotides are supplied during photosynthesis and glycolytic metabolism, whereas glycolytic and oxidative pentose phosphate metabolism provides these cofactors in developing oilseed plastids (3,4). Similar metabolism may be involved in other nonphotosynthetic plastids (1,8,9, 1 1); however, the extent to which these metabolic pathways are involved remains to be fully defined.Fatty acid and glycerolipid biosynthesis from acetate in isolated pea root plastids is completely dependent on exogenously supplied ATP

show abstract

Section: Resultsmentioning

confidence: 78%

Section: Resultsmentioning

confidence: 99%

Section: Plant Materials and Plastid Isolationmentioning

confidence: 99%

See 1 more Smart Citation

Energy Requirements for Fatty Acid and Glycerolipid Biosynthesis from Acetate by Isolated Pea Root Plastids

Kleppinger-Sparace¹,

Stahl²,

Sparace³

1992

Plant Physiol.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Under optimum conditions for fatty acid synthesis from acetate (Stahl and Sparace, 1991), pea root plastids readily incorporate radiolabeled pyruvate, Glc, and Glc-6-P into fatty acids ( Table 1). Pyruvate generally gave the greatest rates of fatty acid biosynthesis at a11 concentrations tested, whereas malate was relatively ineffective as a substrate for fatty acid synthesis.…”

Section: Results and Discussion Lncorporation Of Clycolytic Lntermedimentioning

confidence: 99%

“…Radiolabeled precursors were diluted with unlabeled substrates to a specific activity of 1 pCi/mmol. The in vitro incubation conditions for the assay and analysis of the incorporation of the labeled precursors into fatty acids and glycerolipids were as described elsewhere (Stahl and Sparace, 1991;Kleppinger-Sparace et al, 1992 …”

Section: Plant Materials and Lsolation Of Plastidsmentioning

confidence: 99%

The Utilization of Glycolytic Intermediates as Precursors for Fatty Acid Biosynthesis by Pea Root Plastids

Kleppinger-Sparace

Sparace

1995

Plant Physiol.

View full text Add to dashboard Cite

Radiolabeled pyruvate, glucose, glucose-6-phosphate, acetate, and malate are all variously utilized for fatty acid and glycerolipid biosynthesis by isolated pea (Pisum safivum 1.) root plastids. At the highest concentrations tested (3-5 mM), the rates of incorporation of these precursors into fatty acids were 183, 154, 125, 99, and 57 nmol h-' mg-' protein, respectively. In all cases, cold pyruvate consistently caused the greatest reduction, whereas cold acetate consistently caused the least reduction, i n the amounts of each of the other radioactive precursors utilized for fatty acid biosynthesis.Acetate incorporation into fatty acids was approximately 55% dependent on exogenously supplied reduced nucleotides (NADH and NADPH), whereas the utilization of the remaining precursors was only approximately 1 O and 20% dependent on added NAD(P)H. In contrast, the utilization of all precursors was greatly dependent (85-95O/0) on exogenously supplied ATP. Palmitate, stearate, and oleate were the only fatty acids synthesized from radioactive precursors. Higher concentrations of each precursor caused increased proportions of oleate and decreased proportions of palmitate synthesized. Radioactive fatty acids from all precursors were incorporated into glycerolipids. The data presented indicate that the entire pathway from glucose, including glycolysis, to fatty acids and glycerolipids is operating i n pea root plastids. This pathway can supply both carbon and reduced nucleotides required for fatty acid biosynthesis but only a small portion of the ATP required.Plastids, including chloroplasts and chromoplasts, oilseed plastids, and root plastids, are the site of de novo fatty acid biosynthesis in plants. This subject has been thoroughly reviewed in a number of works (Stumpf, 1984;Harwood, 1988;Dennis, 1989; Sparace and KleppingerSparace, 1993). Traditionally, most in vitro studies of fatty acid biosynthesis utilize acetate as a radioactive tracer largely because it is efficiently incorporated into fatty acids. This has been justified by the belief that extraplastidic acetate originates through the action of mitochondrial pyruvate dehydrogenase and acetyl-COA hydrolase (Murphy and Stumpf, 1981), as well as by the occurrence of envelope-bound acetyl-COA synthetase (Kuhn et al., 1981). However, more recent studies indicate that most plastids contain their own pyruvate dehydrogenase (Reid et al

show abstract

Uncommon properties of lipid biosynthesis of isolated plastids in the unicellular red alga Cyanidioschyzon merolae

2018

View full text Add to dashboard Cite

Red algae are a large group of photosynthetic eukaryotes that diverged from green algae over one billion years ago, and have various traits distinct from those of both green algae and land plants. Although most red algae are marine species (both unicellular and macrophytic), the Cyanidiales class of red algae includes unicellular species which live in hot springs, such as Cyanidioschyzon merolae , which is a model species for biochemical and molecular biological studies. Lipid metabolism in red algae has previously been studied in intact cells. Here, we present the results of radiolabeling and stable isotope labeling experiments in intact plastids isolated from the unicellular red alga C. merolae . We focused on two uncommon features: First, the galactose moiety of monogalactosyldiacylglycerol was efficiently labeled with bicarbonate, indicating that an unknown pathway for providing UDP‐galactose exists within the plastid. Second, saturated fatty acids, namely, palmitic and stearic acids, were the sole products of fatty acid synthesis in the plastid, and they were efficiently exported. This finding suggests that the endoplasmic reticulum is the sole site of desaturation. We present a general principle of red algal lipid biosynthesis, namely, ‘indigenous C18 fatty acids are neither desaturated nor directly utilized within the plastid'. We believe that this is valid in both C. merolae lacking polyunsaturated fatty acids and marine red algae with a high content of arachidonic and eicosapentaenoic acids.

show abstract

Characterization of Fatty Acid Biosynthesis in Isolated Pea Root Plastids

Abstract: On the average, the principal radioactive products of fatty acid biosynthesis were approximately 39% palmitic, 9% stearic, and 52% oleic acid. The proportions of these fatty acids synthesized depended on the experimental conditions.

Cited by 18 publications

References 13 publications

Energy Requirements for Fatty Acid and Glycerolipid Biosynthesis from Acetate by Isolated Pea Root Plastids

Energy Requirements for Fatty Acid and Glycerolipid Biosynthesis from Acetate by Isolated Pea Root Plastids

The Utilization of Glycolytic Intermediates as Precursors for Fatty Acid Biosynthesis by Pea Root Plastids

Uncommon properties of lipid biosynthesis of isolated plastids in the unicellular red alga Cyanidioschyzon merolae

Contact Info

Product

Resources

About