Synthesis and release of hydrocarbons by the oenocytes of the desert locust, Schistocerca gregaria

When incubated in vitro, the oenocytes in the peripheral fat body of the desert locust incorporate Na-¹⁴C-acetate into hydrocarbons (paraffins). The presence of haemolymph in the incubation medium greatly stimulates the release of the ¹⁴C-hydrocarbons into the medium. The labelled hydrocarbons appear to be rapidly released by the cells into the incubation medium as a function of time provided that haemolymph is present. The fact that the oenocytes not only synthesize ¹⁴C-hydrocarbons but also release them into the medium supports the hypothesis that the oenocytes of the desert locust synthesize cuticle lipids.     

INORGANIC CARBON AND ACETATE ASSIMILATION IN BOTRYOCOCCUS BRAUNII(CHLOROPHYTA)1

Assimilation of ¹⁴CO₂ or ¹⁴C-acetate by the hydrocarbon producing alga Botryococcus braunii Kützing was investigated to determine the allocation of incorporated ¹⁴C among early metabolites of photosynthesis and secondary metabolites. When the cells were exposed to NaH¹⁴CO₃ for 10 sec, over 90% of incorporated ¹⁴C was detected in phosphoglycerate, suggesting that this alga assimilates inorganic carbon by the C-3 pathway. The distribution pattern of ¹⁴C in the number of metabolites revealed that organic acids, neutral sugars and amino acids were first labelled with ¹⁴C, and, after lag periods of a few minutes, lipids including hydrocarbon were increasingly labelled. Addition of 5 mM acetate to the culture medium did not affect the growth of this alga but enhanced cellular respiration. The incorporation of ¹⁴CO₂ into the lipid fraction was stimulated, but net photosynthesis was inhibited by the addition of acetate. ¹⁴C-acetate was incorporated into lipids at a very low rate in comparison with the rate of ¹⁴CO₂ incorporation.     

RADIOLABELING STUDIES OF LIPIDS AND FATTY ACIDS IN NANNOCHLOROPSIS (EUSTIGMATOPHYCEAE), AN OLEAGINOUS MARINE ALGA1

The synthesis of fatty acids and lipids in Nannochloropsis sp. was investigated by labeling cells in vivo with [¹⁴C]-bicarbonate or [¹⁴C]-acetate. [¹⁴C]-bicarbonate was incorporated to the greatest extent into 16:0, 16:1, and 14:0 fatty acids, which are the predominant fatty acids of triacylglycerols. However, more than half of the [¹⁴C]-acetate was incorporated into longer and more desaturated fatty acids, which are constituents of membrane lipids. [¹⁴C]-acetate was incorporated most strongly into phosphatidylcholine, which rapidly lost label during a 5-h chase period. The label associated with phosphatidylethanolamine also decreased during the chase period, whereas label in other membrane lipids and triacylglycerol increased. The dynamics of labeling, along with information regarding the acyl compositions of various lipids, suggests that 1) the primary products of chloroplast fatty acid synthesis are 14:0, 16:0, and 16:1; 2) C₂₀ fatty acids are formed by an elongation reaction that can utilize externally supplied acetate; 3) phosphatidylcholine is a site for desaturation of C₁₈ fatty acids; and 4) phosphatidylethanolamine may be a site for desaturation of C₂₀ fatty acids.     

Incorporation of 2-14C-acetate into isolated nuclei and cytosolic lipids of rat thymus cells

It is generally recognized nowadays that active lipid metabolism takes place in the nucleus of a mammalian cell. Experimental data testify to the biosynthesis of polyphosphoinositides and phosphatidylcholine and reveal corresponding enzymes within nuclei of mammalian cells. These findings suggest that lipidmediated signaling pathways in nuclei operate independently of lipid-mediated regulatory mechanisms functioning in membranes and cytosol. To explore the pathways of intranuclear lipid biosynthesis, we studied incorporation of 2-¹⁴C-acetate into lipids of cytosol and isolated nuclei of rat thymus cells after separate and combined incubation with the labeled precursor. The most efficient incorporation of 2-¹⁴C-acetate into lipids (cholesterol, free fatty acids, and phospholipids) was observed in a reaction mixture containing cytosol. When the reaction mixture contained only nuclei, incorporation of the radioactive precursor into lipids also took place, but specific radioactivity of the lipids was essentially lower than in the cytosol. In both cases, 2-¹⁴C-acetate incorporated into phosphatidylethanolamine, sphingomyelin, phosphatidylserine, phosphatidylinositol, and cardiolipin. Phosphatidylcholine, the most abundant membrane phospholipid, demonstrated the lowest radioactivity, which was significantly lower than that of phosphatidylethanolamine. Incorporation of newly synthesized free fatty acids in nuclear phospholipids was inhibited, if nuclei were incubated with cytosol. As a result, radioactive free fatty acids were accumulated in nuclei, while in cytosol they were efficiently incorporated into phospholipids. The levels of phospholipids and cholesterol remained constant regardless of incubation protocol, while the overall yield of free fatty acids decreased after combined incubation of nuclear and cytosolic fractions or after incubation of cytosol without nuclei. Putative mechanisms underlying the appearance of radioactive lipids in isolated nuclei of thymus cells are discussed.     

Further evidence for an elongation-decarboxylation mechanism in the biosynthesis of paraffins in leaves

In isolated tobacco leaves l-valine-U-¹⁴C gave rise to labeled even-numbered isobranched fatty acids containing 16 to 26 carbon atoms and iso C₂₉, iso C₃₁, and iso C₃₃ paraffins. l-Isoleucine-U-¹⁴C on the other hand produced labeled odd-numbered anteiso C₁₇ to C₂₇ fatty acids and anteiso C₃₀ and C₃₂ paraffins. Trichloroacetic acid inhibited the incorporation of isobutyrate into C₂₀ and higher fatty acids and paraffins without affecting the synthesis of the C₁₆ and C₁₈ fatty acids. Thus the very long branched fatty acids are biosynthetically related to the paraffins. In Senecio odoris leaves acetate-1-¹⁴C was incorporated into the paraffins (mainly n-C₃₁) only in the epidermis although acetate was readily incorporated into fatty acids in the mesophyll tissue. Similarly only the epidermal tissue incorporated acetate into fatty acids longer than C₁₈ suggesting that the epidermis is the site of synthesis of both paraffins and the very long fatty acids. In broccoli leaves n-C₁₂ acid labeled with ¹⁴C in the carboxyl carbon and ³H in the methylene carbons was incorporated into C₂₉ paraffin without the loss of ¹⁴C relative to ³H. Since n-C₁₈ acid is known to be incorporated into the paraffin without loss of carboxyl carbon these results suggest that the condensation of C₁₂ acid with C₁₈ acid is not responsible for n-C₂₉ paraffin synthesis in this tissue. Thus all the experimental evidence thus far obtained strongly suggests that elongation of fatty acids followed by decarboxylation is the most likely pathway for paraffin biosynthesis in leaves.     

Alternate pathways of linolenic acid biosynthesis in growing cultures of Penicillium chrysogenum

Evidence was obtained that Penicillium chrysogenum can produce linolenate by two biosynthetic pathways, i.e., by elongation of a shorter trienoic acid as well as direct desaturation of 18-C acids. In oxygen deficient cultures, exogenous hexadecatrienoate stimulated [1-¹⁴C]acetate incorporation into labeled octadecatrienoate and [U-¹⁴C]hexadecatrienoate with nonlabeled acetate yielded linolenate that had relatively little label in the 1-C position. With [1-¹⁴C]acetate as the only added substrate, oxygen deficiency inhibited incorporation of label into monoenoic and dienoic acids but not into trienoic acids. Incorporation of the [U-¹⁴C]linoleate into linolenate also was inhibited.In aerated cultures, 1-¹⁴C-label from laurate, palmitate, stearate, oleate, linoleate, and hexadecatrienoate was readily incorporated into linolenate. Decarboxylation and oxidation studies indicated that the longer acids were incorporated largely intact. [U-¹⁴C]Linoleate was incorporated into linolenate in which the fraction of label in 1-C was similar to that of the substrate. These data suggest that this mold has broader synthetic capabilities than do some chloroplast systems for the biosynthesis of linolenate.     

Diel Variations in Carbon Metabolism by Green Nonsulfur-Like Bacteria in Alkaline Siliceous Hot Spring Microbial Mats from Yellowstone National Park

Green nonsulfur-like bacteria (GNSLB) in hot spring microbial mats are thought to be mainly photoheterotrophic, using cyanobacterial metabolites as carbon sources. However, the stable carbon isotopic composition of typical Chloroflexus and Roseiflexus lipids suggests photoautotrophic metabolism of GNSLB. One possible explanation for this apparent discrepancy might be that GNSLB fix inorganic carbon only during certain times of the day. In order to study temporal variability in carbon metabolism by GNSLB, labeling experiments with [¹³C]bicarbonate, [¹⁴C]bicarbonate, and [¹³C]acetate were performed during different times of the day. [¹⁴C]bicarbonate labeling indicated that during the morning, incorporation of label was light dependent and that both cyanobacteria and GNSLB were involved in bicarbonate uptake. ¹³C-labeling experiments indicated that during the morning, GNSLB incorporated labeled bicarbonate at least to the same degree as cyanobacteria. The incorporation of [¹³C]bicarbonate into specific lipids could be stimulated by the addition of sulfide or hydrogen, which both were present in the morning photic zone. The results suggest that GNSLB have the potential for photoautotrophic metabolism during low-light periods. In high-light periods, inorganic carbon was incorporated primarily into Cyanobacteria-specific lipids. The results of a pulse-labeling experiment were consistent with overnight transfer of label to GNSLB, which could be interrupted by the addition of unlabeled acetate and glycolate. In addition, we observed direct incorporation of [¹³C]acetate into GNSLB lipids in the morning. This suggests that GNSLB also have a potential for photoheterotrophy in situ.     

Incorporation of Label from Acetate and Laurate into the Mannan of Leishmania donovani via the Glyoxylate Cycle

Leishmania donovani promastigotes in late-stationary phase incorporated label from [2-¹⁴C]acetate and [1-¹⁴C]laurate into the mannose residues of mannan, thus confirming the presence of a functional glyoxylate bypass in these parasitic protozoa. Isolated, washed calls also incorporated label from [2-¹⁴C]acetate and [1-¹⁴C]laurate into mannan during a 1-hr incubation in buffer. Glucose had no effect on label incorporation into mannan, but glutamate caused over a four-fold increase in incorporation from [2-¹⁴C]acetate and a 2.4-fold increase from [1-¹⁴C]laurate. Staurosporine, a protein kinase inhibitor that inhibits glutamate and alanine oxidation, did not inhibit label incorporation from [2-¹⁴C]acetate into mannan. Hyperosmolality caused about a 33% inhibition of label incorporation into mannan. These results show the glyoxylate cycle and/or the subsequent biosynthetic pathway from fructose-6-phosphate to mannan are subject to regulation.     

Synthesis of lipids in isolated nuclei from rat thymus and liver cells

Synthesis of lipids was studied in isolated nuclei from rat thymus and liver cells. On incubation of the isolated nuclei with [2-¹⁴C]acetate and [1-¹⁴C]glycerol, the label was intensively incorporated into phospholipids and with a significantly lower intensity into fatty acids and cholesterol. Only trace amounts of radioactivity were detected in the lipids of chromatin prepared from isolated thymus nuclei after their incubation, and this suggested that lipids were mainly synthesized on the nuclear membrane. On the preincubation of thymus tissue homogenate with [2-¹⁴C]acetate and the subsequent isolation of the nuclei and chromatin, the radioactivity of chromatin lipids was comparable to the radioactivity of nuclear lipids. The findings suggested that in the isolated nuclei the newly synthesized lipids were not transported into chromatin from the nuclear membrane. The specific radioactivities of individual phospholipids and fatty acids were different in the isolated nuclei and in nuclei obtained from preincubated homogenate. Mechanisms of lipid synthesis in isolated nuclei and causes of the different radioactivities of lipids in the isolated nuclei and in the nuclei obtained from the preincubated homogenate are discussed.     

Migration of the monarch butterfly, Danaus plexippus: energy sources,

The origin and nature of the metabolic reserves of the monarch butterfly during its autumn migration through Missouri were examined. Information was obtained about the butterfly's protein, glycogen, lipid, and lipid-soluble pigment composition. The principal results showed that lipids, which made up ca. 45 per cent of the insect's dry weight, were the major reserve. Analyses of the lipid classes showed that triglycerides were the dominant class in each sex. Separate analyses of the thorax and abdomen showed that >90 per cent of the lipids were present in the massive fat body which is restricted to the abdomen. The adult female rapidly incorporated U-¹⁴C-d-glucose into abdominal glycerides. The rôle of larval reserves and nectar consumption in migration and ovarial maturation was discussed.     

[14C]Acetate incorporation by cultured normal,familial hypercholesterolemia and Down's syndrome fibroblasts

Fibroblasts from patients with homozygous familial hypercholesterolemia (FH), a disease characterized by accelerated atherogenesis, are known to lack functional low-density-lipoprotein receptors, which ultimately results in increased cholesterol biosynthesis in the cultured cells. [¹⁴C]Acetate incorporation in these cells was compared to that of normal fibroblasts and to fibroblasts from patients with Down's syndrome, a disease in which atherosclerosis is rare. Total [¹⁴C]acetate incorporation did not differ significantly between normal and Down's fibroblasts, nor did its partitioning into the hexane-extractable and aqueous fractions of the cell hydrolysates. [¹⁴C]Acetate incorporation was much greater in FH cells in both the aqueous and hexane-extractable fractions. Preincubation in fetal bovine serum increased acetate incorporation only by FH cells, while 50 μg low-density lipoprotein/ml medium depressed acetate incorporation in all three groups. A C₂₇ sterol, identified by gas chromatography-mass spectrometry as a probable isomer of cholesterol, was present in small amounts in FH fibroblasts, but was not detectable in the normal or Down's cells. The absolute amounts of [¹⁴C]acetate incorporated into the non-sterol lipids were greater in the FH fibroblasts, indicating that these cells may have to synthesize, in addition to cholesterol, other required cellular lipids which are delivered to the normal cells by low-density lipoproteins.     

Specific inhibition of alkane synthesis with accumulation of very long chain compounds by dithioerythritol, dithiothreitol, and mercaptoethanol in Pisum sativum

Sodium [1-¹⁴C]acetate and [1-¹⁴C]stearic acid were readily incorporated into hydrocarbons, secondary alcohols, wax esters, aldehydes, primary alcohols, and fatty acids in young pea leaves (Pisum sativum). Dithioerythritol, dithiothreitol, and mercaptoethanol (but not glutathione and cysteine) severely inhibited the incorporation of labeled acetate into alkanes and secondary alcohols with accumulation of label in wax ester and aldehyde fractions. Detailed radio gas-chromatographic analyses of the fatty acids of both the surface lipid components and internal lipids showed that dithioerythritol and mercaptoethanol specifically inhibited n-hentriacontane (C₃₁) synthesis and caused accumulation of C₃₂ aldehyde, suggesting that the inhibition was at or near the terminal step in alkane biosynthesis, presumably decarboxylation. Trichloroacetate, at a concentration that inhibited C₃₁ alkane synthesis but not the synthesis of alcohols (C₂₆ and C₂₈) specifically inhibited the formation of C₃₂ aldehyde but not that of the C₂₆ or C₂₈ aldehyde. From these results, it is concluded that the C₃₂ aldehyde is derived from the C₃₂ acyl derivative which is the precursor of C₃₁ alkane.     

Site of Monoterpene Biosynthesis in Majorana hortensis Leaves

Excised epidermis of Majorana hortensis Moench (sweet marjoram) leaves incorporates label from [U-¹⁴C]sucrose into monoterpenes as efficiently as do leaf discs, while mesophyll tissue has only a very limited capacity to synthesize monoterpenes from exogenous sucrose. These results strongly suggest that epidermal cells, presumably the epidermal oil glands, are the primary site of monoterpene biosynthesis in marjoram. Using a leaf disc assay, it was demonstrated that label from [U-¹⁴C]sucrose is incorporated into monoterpenes most efficiently in very young leaves.     

The Incorporation of Glycolytic Intermediates into Lipids by Plastids Isolated from the Developing Endosperm of Castor Oil Seeds (Ricinus communis L.)

Plastids were isolated from the developing endosperm of Ricinuscommunis L. and purified by rate-zonal centrifugation on discontinuoussucrose gradients. Assay conditions were optimized for the uptakeand incorporation of ¹⁴C-acetate into lipids by intact plastids.Using the optimized conditions, the uptake and incorporationof several ¹⁴C-glycolytic intermediates into lipids were examined.Neither sucrose nor glucose-6-phosphate was incorporated intolipids. In order of increasing magnitude of incorporation, glucose,fructose, 3-phosphoglycerate, acetate, and pyruvate were metabolizedto chloroform-methanol (2: 1 v/v) soluble products. Pyridoxal-5'-phosphateinhibited the incorporation of 3-phosphoglycerate into lipidswhereas -cyano-4 hydroxycinnamate was without effect on pyruvateincorporation. Avidin and cerulenin did not inhibit the incorporationof acetate into lipids by intact plastids. Key words: Ricinus communis, Plastids, Lipid synthesis, Glycolytic intermediates     

In vivo incorporation of acetate into the acyl moieties of polar lipids from etiolated leek seedlings

Seven-day-old leek seedlings actively synthesize lipids in vivo from [1-¹⁴C]acetate, both in the light and in the dark. In the dark, phospholipid synthesis is more effective than galactolipid synthesis. Whatever the time of acetate incorporation by the etiolated seedlings, very long chain fatty acids having from 20 to 26 carbon atoms are found in all the polar lipids, including the acyl-CoAs. All of the labelled very long chain fatty acids incorporated into the polar lipids are saturated. On the other hand, the labelled C₁₈-fatty acids are unsaturated in phospholipids and galactolipids and almost no label is found in the saturated or unsaturated C₁₈-fatty acids of the acyl-CoAs.     

INCORPORATION OF LIPIDS INTO SUBCELLULAR FRACTIONS OF BRAIN OF QUAKING MUTANT

The synthesis of lipids and their assembly into subcellular membrane fractions of the myelin deficient Quaking mutant and control brains was studied in 18-, 24- and 41-day-old animals using a double label methodology with¹⁴C and ³H acetate as precursors. As a general procedure, Quaking mutants were injected intracranially with 50 μCi [¹⁴C]acetate and their littermate controls with 300 μCi [³H]acetate. The animals were killed 3 h post-injection, their brains were pooled and subcellular fractions prepared from the common homogenate. An 80-90% decrease in the incorporation of acetate into eleven lipids of myelin in the Quaking mutant was found. This occurred in the face of apparent normal incorporation (relative to microsomes) into lipids of the other main subcellular fractions (nuclear. mitochondrial and synaptosomal) with the exception of decreased incorporation into the myelin-like fraction at 18 and 24 days. Cholesterol and cerebroside were less readily incorporated into Quaking myelin than the other lipids. Although the microsomal synthesis of cholesterol and cerebroside was depressed by about 30% in the Quaking mutant, the incorporation of cholesterol into nuclear, synaptosomal and mitochondrial fractions was unaffected in the mutant. This indicates that sufficient cholesterol is synthesized for the normal assembly of these organelles. In contrast the incorporation of acetate into cholesterol and cerebroside of Quaking myelin was decreased much more than microsomal synthesis. This latter result is consistent with a defect in the process of myclin membrane assembly     

Effect of BASF 13-338, a Substituted Pyridazinone, on Lipid Metabolism in Leaf Tissue of Spinach, Pea, Linseed, and Wheat

A substituted pyridazinone (BASF 13-338) inhibited photosynthesis in spinach (Spinacia oleracea, Hybrid 102 Arthur Yates Ltd.) leaf discs and reduced the incorporation of [1-¹⁴C]acetate into trienoic acids of diacylgalactosylglycerol while causing radioactivity to accumulate in diacylgalac-tosylglycerol dienoic acids. Although BASF 13-338 inhibited photosynthesis in isolated spinach chloroplasts, it did not prevent dienoate desaturation. In discs, the labeling of fatty acids was affected by the inhibitor only in diacylgalactosylglycerol. Very little radioactivity was incorporated into trienes of phosphatidylcholine and the proportion of the label recovered in the fatty acids of phosphatidylcholine was not changed by BASF 13-338. The herbicides caused an increase in the proportion of the lipid ¹⁴C incorporated into diacylgalactosylglycerol and a decrease in labeling of phosphatidylcholine, whereas the proportion of ¹⁴C recovered in other lipids remained unchanged. Similar results were obtained with pea (Pisum sativum cv. Victory Freeze), linseed (Linum usitatissimum cv. Punjab), and wheat (Triticum aestivum cv. Karamu). With these species, a greater proportion of the label was incorporated into phosphatidylcholine and less into diacylgalactosylglycerol than with spinach. The data indicate that trienoate synthesis uses diacylgalactosylglycerol as substrate. BASF 13-338 appears to act at that step, and seems to cause in spinach a shift in polyenoate synthesis from the pathway involving microsomal phosphatidylcholine to the pathway operating inside the chloroplast.     

Lipid synthesis by oligodendrocytes from adult pig brain maintained in long-term culture

Oligodendrocytes were isolated from adult pig brain and cultivated for 18–24 days. [¹⁴C]acetate, [³H]galactose or [³⁵S]sulfate were added to the medium for an additional 24 h. Lipids were extracted and separated by high-performance thin-layer chromatography. The labeled lipids were studied by fluorography and scintillation counting. [¹⁴C]acetate was incorporated in decreasing order into neutral lipids, phosphatidylcholine, ethanolamine phosphatides, galactocerebrosides, phosphatidylinositol, phosphatidylserine, sulfatides and sphingomyelin. From the [¹⁴C]acetate incorporated into ethanolamine and choline phosphatides, 71.6 and 14.8%, respectively, were found in plasmalogens. Among neutral lipids, [¹⁴C]acetate labeled not only cholesterol but also large amounts of triglycerides. No cholesterol esters were synthesized. [³H]galactose primarily labeled galactocerebrosides, sulfatides, and monogalactosyl diglyceride. [³⁵S]sulfate incorporation was restricted to sulfatides. Together with our previous results concerning proteins, these data show that: (1) oligodendrocytes remain highly differentiated in long-term cultures; (2) they are able to synthesize the major components of myelin; (3) they synthesize surprisingly high amounts of triglycerides and of monogalactosyl diglyceride, a marker for myelination.     

Biochemistry of Suberization: Incorporation of [1-C]Oleic Acid and [1-C]Acetate into the Aliphatic Components of Suberin in Potato Tuber Disks (Solanum tuberosum)

Biosynthesis of the aliphatic components of suberin was studied in suberizing potato (Solanum tuberosum) slices with [1-¹⁴C]oleic acid and [1-¹⁴C]acetate as precursors. In 4-day aged tissue, [1-¹⁴C]oleic acid was incorporated into an insoluble residue, which, upon hydrogenolysis (LiA1H₄), released the label into chloroform-soluble products. Radio thin layer and gas chromatographic analyses of these products showed that ¹⁴C was contained exclusively in octadecenol and octadecene-1, 18-diol. OsO₄ treatment and periodate cleavage of the resulting tetraol showed that the labeled diol was octadec-9-ene-1, 18-diol, the product expected from the two major components of suberin, namely 18-hydroxyoleic acid and the corresponding dicarboxylic acid. Aged potato slices also incorporated [1-¹⁴C]acetate into an insoluble material. Hydrogenolysis followed by radio chromatographic analyses of the products showed that ¹⁴C was contained in alkanols and alkane-α,ω-diols. In the former fraction, a substantial proportion of the label was contained in aliphatic chains longer than C₂₀, which are known to be common constituents of suberin. In the labeled diol fraction, the major component was octadec-9-ene-1,18-diol, with smaller quantities of saturated C₁₆, C₁₈, C₂₀, C₂₂, and C₂₄-α,ω-diols. Soluble lipids derived from [1-¹⁴C]acetate in the aged tissue also contained labeled very long acids from C₂₀ to C₂₈, as well as C₂₂ and C₂₄ alcohols, but no labeled ω-hydroxy acids or dicarboxylic acids were detected. Label was also found in n-alkanes isolated from the soluble lipids, and the distribution of label among them was consistent with the composition of n-alkanes found in the wound periderm of this tissue; C₂₁ and C₂₃ were the major components with lesser amounts of C₁₉ and C₂₅. The amount of ¹⁴C incorporated into these bifunctional monomers in 0-, 2-, 4-, 6-, and 8-day aged tissue were 0, 1.5, 2.5, 0.8, and 0.3% of the applied [1-¹⁴C]oleic acid, respectively. Incorporation of [1-¹⁴C]acetate into the insoluble residue was low up to the 3rd day of aging, rapid during the next 4 days of aging, and subsequently the rate decreased. These changes in the rates of incorporation of exogenous oleic acid and acetate reflected the development of diffusion resistance of the tissue surface to water vapor. As the tissue aged, increasing amounts of the [1-¹⁴C]acetate were incorporated into longer aliphatic chains of the residue and the soluble lipids, but no changes in the distribution of radioactivity among the α-ω-diols were obvious. The above results demonstrated that aging potato slices constitute a convenient system with which to study the biochemistry of suberization.     

Manipulating the incorporation of [1-C]acetate into different leaf glycerolipids in several plant species

During short term labeling of expanding leaves of seven plant species with [1-¹⁴C]acetate, 35 to 64% of the label incorporated into lipids was found in phosphatidylcholine and 5 to 24% in phosphatidylglycerol. In pumpkin, sunflower, broad bean, and maize, only 4 to 12% of the label was found in diacylgalactosylglycerol, but in tomato, parsley, and spinach, the proportion was 17 to 31%. The latter group was further distinguished by having diacylgalactosylglycerol containing C16:3.

The proportions of total incorporated [1-¹⁴C]acetate entering the lipids could be manipulated in a predictable manner. Phosphatidylcholine labeling was depressed by treating intact leaves with glycerol or ethylene glycol monomethyl ether or by incubating leaf discs in vitro. An associated increase in phosphatidylglycerol labeling occurred within the first group of plants, whereas an increase in labeling of either diacylgalactosylglycerol, phosphatidylglycerol, or sulfolipid occurred within the second group. Treating intact leaves with glycerol or incubating leaf discs in vitro was shown to elevate cellular concentrations of sn-glycerol 3-phosphate.

These results have been interpreted in terms of the two-pathway hypothesis for glycerolipid biosynthesis, in which it is proposed that phosphatidylcholine is synthesized via a different pathway (eukaryotic) to that for synthesis of phosphatidylglycerol (prokaryotic). Both pathways may contribute toward the synthesis of diacylgalactosylglycerol, with the contribution of each being assessed from the proportion of hexadecatrienoic acid found in the particular plant.