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Soybean somatic embryos were developed as a model for investigating the developmental relationships of lipid biosynthesis and accumulation in this important crop. Batch cultures of embryos grown for 8 wk in liquid culture medium exhibited typical sigmoidal growth kinetics as they passed through characteristic globular, heart, torpedo, and cotyledon stages. Exponential growth occurred for the first 4 wk in culture with net growth terminating when total embryo fresh weight per culture flask reached a maximum of 4–4.5 g at 6 wk. This was followed by a slight decrease in embryo fresh weight (FW) and the onset of apparent tissue senescence as judged by yellowing and browning of embryos. On a FW basis, embryos accumulated up to 4% protein, 2.5% soluble sugars, 1.9% starch, and 1.5% lipid relatively early in development. Levels decreased to 0.8% protein, 0.5% soluble sugars, 0.03% starch, and 0.09% lipid at the end of the culture period. On a mass percent basis, lipid extracts were comprised of approximately 80–90% polar lipid early in embryo development. This shifted to 56% storage lipid (triacylglycerol) and 44% polar lipid after 4–5 wk in culture and then reverted back to 91% and 9% polar vs. storage lipid, respectively, by the end of the 8-wk culture period. On the average, polar and storage lipid fractions were comprised of 24% palmitic acid, 7% stearic acid, 8% oleic acid, 36% linoleic acid, and 26% linolenic acid. However, the amounts of linoleic and linolenic acids declined sharply during embryo senescence at the end of the culture period, with corresponding increases in the combined amounts of palmitic and stearic acids. This is the first report that documents the progress of storage reserve accumulation in soybean somatic embryos in relation to their continuous growth in liquid batch cultures.

Glucosinolates are defensive compounds found in several plant families. We recently described five distinct isoforms of a novel plant enzyme, thiol methyltransferase (TMT), which methylate the hydrolysis products of glucosinolates to volatile sulfur compounds that have putative anti-insect and anti-pathogen roles. In the work presented here, two cDNAs encoding these enzymes (cTMT1 and cTMT2) were isolated by screening a cabbage cDNA library with an ArabidopsisEST showing high sequence homology to one TMT isoform. The genomic clone of cTMT1 was subsequently amplified by PCR. Both cDNAs encoded polypeptides of identical lengths (227 amino acids) and similar predicted masses (ca. 25 kDa), but differing in 13 residues. The cDNAs contained the typical methyltransferase signatures, but were otherwise distinct from conventionally known N-, O-or S-methyltransferases. A chloride methyl transferase was the only gene with an assigned function that shared significant similarity with the TMT cDNAs. Southern analysis indicated single copy for each TMT gene. The two cDNAs were expressed in Escherichia coli. The substrate range, kinetic properties and molecular sizes of the purified recombinant proteins were comparable to those of the native enzyme. These data, together with the detection of the sequenced amino acid motif of one native TMT peptide in the cDNAs, confirmed that the latter were authentic TMTs. The expression pattern of the TMTs in various cabbage tissues was consistent with their association with glucosinolates. The cloning of this new class of plant genes furnishes crucial molecular tools to understand the role of this metabolic sector in plant defenses against biotic stress.

Cottonseed typically contains about 15% oleic acid. Here we report the development of transgenic cotton plants with higher seed oleic acid levels. Plants were generated by Agrobacterium-mediated transformation. A binary vector was designed to suppress expression of the endogenous cottonseed †-12 desaturase (fad2) by subcloning a mutant allele of a rapeseed fad2 gene downstream from a heterologous, seedspecific promoter (phaseolin). Fatty acid profiles of total seed lipids from 43 independent transgenic lines were analyzed by gas chromatography. Increased seed oleic acid content ranged from 21 to 30% (by weight) of total fatty acid content in 22 of the primary transformants. The increase in oleic acid content was at the expense of linoleic acid, consistent with reduced activity of cottonseed FAD2. Progeny of some lines yielded oleic acid content as high as 47% (three times that of standard cottonseed oil). Molecular analyses of nuclear DNA from transgenics confirmed the integration of the canola transgene into the cotton genome. Collectively, our results extend the metabolic engineering of vegetable oils to cottonseed and should provide the basis for the development of a family of novel cottonseed oils.

Five functional isoforms of a novel plant thiol methyltransferase from the leaves of cabbage (Brassica oleracea L.) were purified to electrophoretic homogeneity. Pooled, partly purified preparations of the enzyme were previously shown to methylate thiol compounds released upon the hydrolysis of glucosinolates. The enzyme could also accept halide ions as substrates. The estimated molecular masses of the purified isoforms ranged between 26 and 31 kDa. The three most abundant isoforms of the enzyme could all catalyze the S-adenosyl-l-methionine-dependent methylation of thiocyanate, a number of organic thiols and iodide. However, the kinetic properties of these forms toward various substrates differed widely. None of the isoforms examined methylated the O- and N-equivalents of the thiol substrates. The three isoforms also had distinct pH optima, covering the range from 5 to 9. Their kinetic analysis indicated that they shared a sequential substrate binding mechanism and an Ordered Bi Bi mechanism for substrate binding and product release. Partial internal amino acid sequence from one isoform showed high similarity to an Arabidopsis EST of unknown function, and to a recently cloned methyl chloride transferase from Batis maritima. The differences in the pH optima and kinetic properties of the isoforms suggest that each may methylate a specific substrate or a narrow group of substrates under cellular conditions.

The physiological relevance of a novel thiol methyltransferase from cabbage, and its possible role in sulphur metabolism have been investigated. The enzyme was absent from the chloroplast, the site of sulphate reduction, and was localized in the cytosol. Potential substrates were initially screened on the basis of their ability to inhibit the methylation of iodide, a previously known substrate for the enzyme. Thiocyanate, 4,4′-thiobisbenzenethiol, thiophenol, and thiosalicylic acid were identified as possible substrates. Methylation of these thiols by the purified enzyme using [Methyl-3H]S-adenosyl- L-methionine confirmed their nature as substrates. The purified enzyme strongly preferred thiocyanate as a methyl acceptor. The enzyme had Km values of 11, 51, 250 and 746 mmol m−3 for thiocyanate, 4,4′-thiobisbenzenethiol, thiophenol and thiosalicylic acid, respectively. The identity of methylthiocyanate as the product of thiocyanate methylation by the purified enzyme was confirmed by mass spectrometry. The enzyme was strictly associated with glucosinolate-containing plants. Thiol substrates of the enzyme are known products of glucosinolate hydrolysis. Our observations indicate that this enzyme could be involved in the detoxification of reactive thiols produced upon glucosinolate degradation in these plants.

The first two oxygenation steps post-trichodiene in the biosyntheses of the trichothecenes 3-acetyldeoxynivalenol and sambucinol were investigated. The plausible intermediates 2-hydroxytrichodiene (2alpha- and 2beta-) and 12,13-epoxytrichodiene and the dioxygenated compounds 12,13-epoxy-9,10-trichoene-2-ol (2alpha- and 2beta-) were prepared specifically labeled with stable isotopes. They were then fed separately and/or together to Fusarium culmorum cultures, and the derived trichothecenes were isolated, purified, and analyzed. The stable isotopes enable easy localization of the labels in the products by 2H NMR, 13C NMR, and mass spectrometry. We found that 2alpha-hydroxytrichodiene is the first oxygenated step in the biosynthesis of both 3-acetyldeoxynivalenol and sambucinol. The stereoisomer 2beta-hydroxytrichodiene and 12,13-epoxytrichodiene are not biosynthetic intermediates and have not been isolated as metabolites. We also demonstrated that the dioxygenated 12, 13-epoxy-9,10-trichoene-2alpha-ol is a biosynthetic precursor to trichothecenes as had been suggested in a preliminary work. Its stereoisomer was not found in the pathway. A further confirmation of our results was the isolation of both oxygenated trichodiene derivatives 2alpha-hydroxytrichodiene and 12,13-epoxy-9, 10-trichoene-2alpha-ol as natural metabolites in F. culmorum cultures.

The pyruvate dehydrogenase complex (PDC) and acetyl-CoA carboxylase (ACC, EC 6.4.1.2) have been characterized in pea root plastids. PDC activity was optimum in the presence of 1.0 mM pyruvate, 1.5 mM NAD + , 0.1 mM CoA, 0.1 mM TPP, 5 mM MgCI2, 3.0 mM cysteine-HCI, and 0.1 M Tricine (pH 8.0) and represents approximately 47% of the total cellular activity. ACC activity was greatest in the presence of 1.0 mM acetyl-CoA, 4 mM NaHC03, 3 mM ATP, 10 mM MgCI2, 2.5 mM dithiothreito l, and 100 mM Tricine (pH 8.0). Both enzymes were stimulated by reduced sutphydryl reagents and inhibited by sulphydryl inhibitors. ACC was also inhibited by malonyl-CoA while PDC was inhibited by both malonyl-CoA and NADH. Both enzymes were stimulated by DHAP and UDP-galactose while ACC was also stimulated by PEP and F1,6P. Palmitic acid and oleic acid both inhibited ACC, but had essentially no effect on PDC. Palmitoyl-CoA inhibited both enzymes while PA and Lyso-PA inhibited PDC, but stimulated ACC. The results presented support the hypothesis that PDC and ACC function in a co-ordinated fashion to promote glycolytic carbon flow to fatty acid biosynthesis in pea root plastids.

Fatty acid and glycerolipid biosynthesis from [ 14 C]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

Glucosinolates are defensive compounds found in several plant families. We recently described five distinct isoforms of a novel plant enzyme, thiol methyltransferase (TMT), which methylate the hydrolysis products of glucosinolates to volatile sulfur compounds that have putative anti-insect and anti-pathogen roles. In the work presented here, two cDNAs encoding these enzymes (cTMT1 and cTMT2) were isolated by screening a cabbage cDNA library with an Arabidopsis EST showing high sequence homology to one TMT isoform. The genomic clone of cTMT1 was subsequently amplified by PCR. Both cDNAs encoded polypeptides of identical lengths (227 amino acids) and similar predicted masses (ca. 25 kDa), but differing in 13 residues. The cDNAs contained the typical methyltransferase signatures, but were otherwise distinct from conventionally known N-, O- or S-methyltransferases. A chloride methyl transferase was the only gene with an assigned function that shared significant similarity with the TMT cDNAs. Southern analysis indicated single copy for each TMT gene. The two cDNAs were expressed in Escherichia coli. The substrate range, kinetic properties and molecular sizes of the purified recombinant proteins were comparable to those of the native enzyme. These data, together with the detection of the sequenced amino acid motif of one native TMT peptide in the cDNAs, confirmed that the latter were authentic TMTs. The expression pattern of the TMTs in various cabbage tissues was consistent with their association with glucosinolates. The cloning of this new class of plant genes furnishes crucial molecular tools to understand the role of this metabolic sector in plant defenses against biotic stress.

The glycerolipid composition of pea (Pisum sativum L.) root plastids and their capacity to synthesize glycerolipids from [UL-14C]glycerol-3-phosphate were determined. Pea root plastids primarily consist of monogalactosyldiacylglycerol, triacylglycerol, phosphatidylcholine, digalactosyldiacylglycerol, and diacylglycerol. Maximum rates of total glycerolipid biosynthesis were obtained in the presence of 2.4 mM glycerol-3-phosphate, 15 mM KHCO3, 0.2 mM sodium-acetate, 0.5 mM each of NADH and NADPH, 0.05 mM coenzyme A, 2 mM MgCl2, 1 mM ATP, 0.1 M Bis-Tris propane (pH 7.5), and 0.31 M sorbitol. Glycerolipid biosynthesis was completely dependent on exogenously supplied ATP, coenzyme A, and a divalent cation, whereas the remaining cofactors improved their activity from 1.3- to 2.4-fold. Radioactivity from glycerol-3-phosphate was recovered predominantly in phosphatidic acid, phosphatidylglycerol, diacylglycerol, and triacylglycerol with lesser amounts in phosphatidylcholine and monoacylglycerol. The proportions of the various radiolabeled lipids that accumulated were dependent on the pH and the concentration of ATP and glycerol-3-phosphate. The data presented indicate that pea root plastids can synthesize almost all of their component glycerolipids and that glycerolipid biosynthesis is tightly coupled to de novo fatty acid biosynthesis. pH and the availability of ATP may have important roles in the regulation of lipid biosynthesis at the levels of phosphatidic acid phosphatase and in the reactions that are involved in phosphatidylglycerol and triacylglycerol biosynthesis.

Radiolabeled pyruvate, glucose, glucose-6-phosphate, acetate, and malate are all variously utilized for fatty acid and glycerolipid biosynthesis by isolated pea (Pisum sativum L.) root plastids. At the highest concentrations tested (3-5mM), the rates of incorporation of these precursors into fatty acids were 183, 154, 125, 99 and 57 nmol h-1 mg-1 protein, respectively. In all cases, cold pyruvate consistently caused the greatest reduction, whereas cold acetate consistently caused the least reduction, in 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 10 and 20% dependent on added NAD(P)H. In contrast, the utilization of all precursors was greatly dependent (85-95%) 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 in 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

Fatty acid biosynthesis from Na[1-(14)C]acetate was characterized in plastids isolated from primary roots of 7-day-old germinating pea (Pisum sativum L.) seeds. Fatty acid synthesis was maximum at 82 nanomoles per hour per milligram protein in the presence of 200 micromolar acetate, 0.5 millimolar each of NADH, NADPH, and coenzyme A, 6 millimolar each of ATP and MgCl(2), 1 millimolar each of MnCl(2) and glycerol-3-phosphate, 15 millimolar KHCO(3), 0.31 molar sucrose, and 0.1 molar Bis-Tris-propane, pH 8.0, incubated at 35 degrees C. At the standard incubation temperature of 25 degrees C, fatty acid synthesis was essentially linear for up to 6 hours with 80 to 120 micrograms per milliliter plastid protein. ATP and coenzyme A were absolute requirements, whereas divalent cations, potassium bicarbonate, and reduced nucleotides all variously improved activity two- to 10-fold. Mg(2+) and NADH were the preferred cation and nucleotide, respectively. Glycerol-3-phosphate had little effect, whereas dithiothreitol and detergents generally inhibited the incorporation of [(14)C]acetate into fatty acids. 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.

Intact mitochondria from the endosperm of castor bean were isolated on linear sucrose gradients. These mitochondria were ruptured and the membranes separated on discontinuous sucrose gradients into outer membrane, intact inner membrane, and ruptured inner membrane fractions. Each membrane fraction was examined for its capacity to synthesize phosphatidylglycerol, CDP-diglyceride, phosphatidylcholine via methylation, and phosphatidic acid. The syntheses of phosphatidylglycerol, CDP-diglyceride, and phosphatidylcholine were localized exclusively in the inner mitochondrial membrane fractions while phosphatidic acid synthesis occurred in both the inner and outer mitochondrial membranes.

Pyrenochaeta terrestris (Hansen) Gorenz, Walker, and Larson, causal agent of pink root of onion ( Allium cepa L) produces three pyrones (pyrenocines A, B and C) showing varying degrees of phytotoxicity. These metabolites accumulate only in the growth medium. No pyrenocines are produced during the first four days of growth. Subsequently, pyrenocine levels sharply increase and reach maxima after 10–12 days and then steadily decline and are barely detectable beyond 22 days of growth. Initially, pyrenocine A predominates and ultimately pyrenocine B predominates. The proportion of pyrenocine C remains essentially constant from day 6 to 18 and subsequently increases to 30% of the total pyrenocines. Pyrenocines A and B spontaneously interconvert in control growth medium and reach equilibria consisting of 40–45% A and 55–60% B. The accumulation of the pyrenocines varies considerably from one isolate to another, Pyrenocine A is the most inhibitory to onion and radish seedling elongation with an effective concentration for 50% inhibition of 5 and 40 μg ml −1 respectively, Pyrenocine A is toxic to onion protoplasts with a concentration of 0·4 mg ml −1 , causing 50% reduction in protoplast viability. Similar concentrations of pyrenocines B and C are without effect.

Phosphatidylethanolamine synthesis by CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) from the endoplasmic reticulum of castor bean (Ricinus communis L. var. Hale) endosperm was characterized. The Michaelis-Menten constant of the enzyme for CDP-ethanolamine was approximately 8.0 micromolar. The pH optimum was 6.5 and a divalent cation was an absolute requirement for activity, with Mg(2+) giving the greatest stimulation at 3 millimolar. Sulfhydryl reagents variously affected enzyme activity. No discernible differences were detected between the responses of the ethanolaminephosphotransferase and CDP-choline:1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) to a variety of treatments. CDP-choline and CDP-ethanolamine were competitive inhibitors of the ethanolaminephosphotransferase and cholinephosphotransferase reactions, respectively.

Subcellular fractions from pea (Pisum sativum L.) roots have been prepared by differential centrifugation techniques. Greater than 50% of the recovered plastids can be isolated by centrifugation at 500g for 5 minutes. Plastids of this fraction are largely free from mitochondrial and microsomal contamination as judged by marker enzyme analysis. De novo fatty acid biosynthesis in pea roots occurs in the plastids. Isolated pea root plastids are capable of fatty acid synthesis from acetate at rates up to 4.3 nanomoles per hour per milligram protein. ATP, bicarbonate, and either Mg(2+) or Mn(2+) are all absolutely required for activity. Coenzyme A at 0.5 millimolar improved activity by 60%. Reduced nucleotides were not essential but activity was greatest in the presence of 0.5 millimolar of both NADH and NADPH. The addition of 0.5 millimolar glycerol-3-phosphate increased activity by 25%. The in vitro and in vivo products of fatty acid synthesis from acetate were primarily palmitate, stearate, and oleate, the proportions of which were dependent on experimental treatments. Fatty acids synthesized by pea root plastids were recovered in primarily phosphatidic acid and diacylglycerol or as water soluble derivatives and the free acids. Lesser amounts were found in phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and monogalactosyldiacylglycerol.

[(14)C]Acetate, [(14)C]formate, and methyl[(14)C]methionine all serve as precursors of pyrenocines A, B, and C when added to cultures of Pyrenochaeta terrestris (Hansen) Gorenz, Walker, and Larson, the pathogen responsible for disease known as pink root of onion (Allium cepa L.). This information supports the hypothesis that these metabolites are methyl-substituted polyketides in origin. Pyrenocine A arises from acetate via uncharacterized intermediates and is subsequently converted to pyrenocine B. The biosynthetic role of pyrenocine C remains uncertain.

Relatively little information is available concerning patterns of lipid and fatty acid metabolism in roots in comparison to leaves. We report here our findings on the in vivo lipid metabolism from 14C- acetate and 14C-glycerol in pea roots as part of a comprehensive approach towards under-standing root lipid metabolism.

Pyrenocine A, a phytotoxin produced by Pyrenochaeta terrestris (Hansen) Gorenz, Walker and Larson, possesses general antibiotic activity against plants, fungi, and bacteria. Effective doses for 50% inhibition (ED50s) are 4 μg/mL for onion seedling elongation; 14, 20, 20, and 25 μg/mL for the germination of asexual spores of Fusarium oxysporum f. sp. cepae, Fusarium solani f. sp. pisi, Mucor hiemalis, and Rhizopus stolonifer, respectively. Pyrenocine A also inhibits the linear mycelial growth of both P. terrestris and F. oxysporum with ED50s calculated as 77 and 54 μg/mL, respectively. Gram-positive bacteria are more susceptible to pyrenocine A than Gram-negative bacteria. ED50s are estimated as 30, 45, and 200 μg/mL for the inhibition of growth of Bacillus subtilis, Staphylococcus aureus, and Escherichia coli, respectively, with Pseudomonas aeruginosa resistant to those concentrations tested. Pyrenocine A acts primarily as a biostatic rather than a biocidal agent with all organisms tested showing some degree of recovery when released from pyrenocine A. Pyrenocines B and C show little antibiotic activity in the bioassays performed.

Publisher Summary This chapter describes methods for the interpretation of phosphatidylglycerol (PG) synthesis in the chloroplast. The PG from plant material has a unique characteristic in the presence of trans-3-hexadecenoic acid at the sn-2 position of the molecule. Although PG is found in the mitochondria and the endoplasmic reticulum of higher plants, the PG of the plastids contains the trans-3-hexadecenoic acid. The synthesis of PG from acetate or G-3-P by isolated chloroplasts is stimulated by illumination of the chloroplasts. Breakage of the chloroplasts by osmotic shock eliminated the synthesis of PG. G-3-P is acylated using acyl-ACP in the stroma. This reaction is specific for the sn -1 position, and in the cases so far described is very specific for oleyl-ACP. The second acylation takes place in the inner envelope membrane and is specific for palmitoyl-ACP. The product, 1-oleoyl-2-palmitoyl-sn-G-3-P, is the precursor of PG. Fatty acid analysis of PG from isolated chloroplasts reflects this pathway of synthesis.

CDPcholine:1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and CDPethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) were detected in mitochondrial fractions from castor bean (Ricinus communis) endosperm. These activities were not due to contamination of the fractions with endoplasmic reticulum. The enzymes were localized on both the inner and outer mitochondrial membranes.Only minor kinetic differences between the phosphatidylcholine-synthesizing activities of intact mitochondria and of the endoplasmic reticulum were found. The K(m) of the mitochondrial enzyme for CDP-choline was about 2 units less than that for the enzyme of the endoplasmic reticulum (8.0 and 10.0 micromolar, respectively). The mitochondrial enzyme activity was maximal above 10 millimolar Mg(2+), whereas maximum endoplasmic reticulum activity was achieved by 4 millimolar. The endoplasmic reticulum enzyme was more stable at 37 C than was that of the mitochondria. The mitochondrial cholinephosphotransferase represented about 1 to 2% of the total activity isolated from castor bean endosperm.

Intact chloroplasts from spinach (Spinacia oleracea L., hybrid 424) readily incorporate [(14)C]glycerol-3-phosphate and [(14)C]acetate 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. Glycerol-3-phosphate-labeled phosphatidylglycerol is equally labeled in the two glycerol moieties of the molecule. Acetate-labeled phosphatidylglycerol is equally labeled in both acyl groups. Position one contains primarily oleate, linoleate and small amounts of palmitate. Position two contains primarily palmitate. No radioactive trans-Delta(3)-hexadecenoate was detected. The labeling patterns indicate that the radioactive phosphatidylglycerol is the product of de novo chloroplast lipid biosynthesis and furthermore, phosphatidylglycerol may be a substrate for fatty acid desaturation.

The structure of pyrenocine C, a new metabolite isolated from onion pink root fungus, Pyrenochaeta terrestris (Hansen) has been elucidated as (±)-(2′E)-5-(1′-hydroxybut-2′-enyl)-4-methoxy-6-methyl-2-pyrone by spectroscopic methods and chemical correlation with pyrenocine A.