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PubMed1999

Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis.

Vitamin C (L-ascorbic acid; AsA) acts as a potent antioxidant and cellular reductant in plants and animals. AsA has long been known to have many critical physiological roles in plants, yet its biosynthesis is only currently being defined. A pathway for AsA biosynthesis that features GDP-mannose and L-galactose has recently been proposed for plants. We have isolated a collection of AsA-deficient mutants of Arabidopsis thaliana that are valuable tools for testing of an AsA biosynthetic pathway. The best-characterized of these mutants (vtc1) contains approximately 25% of wild-type AsA and is defective in AsA biosynthesis. By using a combination of biochemical, molecular, and genetic techniques, we have demonstrated that the VTC1 locus encodes a GDP-mannose pyrophosphorylase (mannose-1-P guanyltransferase). This enzyme provides GDP-mannose, which is used for cell wall carbohydrate biosynthesis and protein glycosylation as well as for AsA biosynthesis. In addition to genetically defining the first locus involved in AsA biosynthesis, this work highlights the power of using traditional mutagenesis techniques coupled with the Arabidopsis Genome Initiative to rapidly clone physiologically important genes.

Amino Acid Sequence↗
PubMed1997

An animal cell mutant with a deficiency in acyl/alkyl-dihydroxyacetone-phosphate reductase activity. Effects on the biosynthesis of ether-linked and diacyl glycerolipids.

In the accompanying paper (James, P. F., and Zoeller, R. A. (1997) J. Biol. Chem. 272, 23532-23539), we reported the isolation of a series of mutants from the fibroblast-like cell line, CHO-K1, that are deficient in the incorporation of the long chain fatty alcohol, hexadecanol, into complex lipids. All but one of these mutants, FAA. K1B, were deficient in long-chain-fatty alcohol oxidase (FAO) activity. We have further characterized this FAO+ isolate. FAA.K1B cells displayed a 40% decrease in [9,10-3H]hexadecanol uptake when compared with the parent strain. Although incorporation of hexadecanol into the phospholipid fraction was decreased by 52%, the cells accumulated label in alkylglycerol (20-fold over wild type). The increase in 1-alkylglycerol labeling corresponded to a 4-fold increase in alkylglycerol mass. Short term labeling with 32Pi showed a 45-50% decrease in overall phospholipid biosynthesis in FAA.K1B. Both diacyl- and ether-linked species were affected, suggesting a general defect in phospholipid biosynthesis. Mutant cells were able to partially compensate for the decreased biosynthesis by decreasing the turnover of the phospholipid pools. The primary lesion in FAA. K1B was identified as a 95% reduction in acyl/alkyl-dihydroxyacetone-phosphate reductase activity. Whole cell homogenates from FAA.K1B were unable to reduce either acyl-dihydroxyacetone phosphate (DHAP) or alkyl-DHAP, supporting the notion that the reduction of these two compounds is catalyzed by a single enzyme. These data suggest that the biosynthesis of diacyl phospholipids, in Chinese hamster ovary cells, begins with the acylation of dihydroxyacetone phosphate as well as glycero-3-phosphate and that the "DHAP pathway" contributes significantly to diacyl glycerolipid biosynthesis. Also, the severe reduction in acyl/alkyl-DHAP reductase activity in FAA.K1B resulted in only a moderate decrease in ether lipid biosynthesis. These latter data together with the observed increase in alkylglycerol levels support the existence of a shunt pathway that is able to partially bypass the enzymatic lesion.

Animals↗
PubMed2000

5-hydroxyconiferyl aldehyde modulates enzymatic methylation for syringyl monolignol formation, a new view of monolignol biosynthesis in angiosperms.

S-Adenosyl-L-methionine-dependent caffeate O-methyltransferase (COMT, EC 2.1.1.6) has traditionally been thought to catalyze the methylation of caffeate and 5- hydroxyferulate for the biosynthesis of syringyl monolignol, a lignin constituent of angiosperm wood that enables efficient lignin degradation for cellulose production. However, recent recognition that coniferyl aldehyde prevents 5-hydroxyferulate biosynthesis in lignifying tissue, and that the hydroxylated form of coniferyl aldehyde, 5-hydroxyconiferyl aldehyde, is an alternative COMT substrate, demands a re-evaluation of the role of COMT during monolignol biosynthesis. Based on recombinant aspen (Populus tremuloides) COMT enzyme kinetics coupled with mass spectrometry analysis, this study establishes for the first time that COMT is in fact a 5-hydroxyconiferyl aldehyde O-methyltransferase (AldOMT), and that 5-hydroxyconiferyl aldehyde is both the preferred AldOMT substrate and an inhibitor of caffeate and 5-hydroxyferulate methylation, as measured by K(m) and K(i) values. 5-Hydroxyconiferyl aldehyde also inhibited the caffeate and 5-hydroxyferulate methylation activities of xylem proteins from various angiosperm tree species. The evidence that syringyl monolignol biosynthesis is independent of caffeate and 5-hydroxyferulate methylation supports our previous discovery that coniferyl aldehyde prevents ferulate 5-hydroxylation and at the same time ensures a coniferyl aldehyde 5-hydroxylase (CAld5H)-mediated biosynthesis of 5-hydroxyconiferyl aldehyde. Together, our results provide conclusive evidence for the presence of a CAld5H/AldOMT-catalyzed coniferyl aldehyde 5-hydroxylation/methylation pathway that directs syringyl monolignol biosynthesis in angiosperms.

Acrolein↗
PubMed2003

Regulation of anthraquinone biosynthesis in cell cultures of Morinda citrifolia.

Cell cultures of Morinda citrifolia L. are capable of accumulating substantial amounts of anthraquinones. Chorismate formed by the shikimate pathway is an important precursor of these secondary metabolites. Isochorismate synthase (EC 5.4.99.6), the enzyme that channels chorismate into the direction of the anthraquinones, is involved in the regulation of anthraquinone biosynthesis. Other enzymes of the shikimate pathway such as deoxy-D-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) and chorismate mutase (EC 5.4.99.5) do not play a regulatory role in the process. The accumulation of anthraquinones is correlated with isochorismate synthase activity under a variety of conditions, which indicates that under most circumstances the concentration of the branchpoint metabolite chorismate is not a rate-limiting factor. Anthraquinone biosynthesis in Morinda is strongly inhibited by 2,4-D, but much less by NAA. Both auxins inhibit the activity of isochorismate synthase proportionally to the concomitant reduction in the amount of anthraquinone accumulated. However, the correlation between enzyme activity and rate of biosynthesis is less clear when the activity of the enzyme is very high. In this case, a limiting concentration of precursor may determine the extent of anthraquinone accumulation. Partial inhibition of chorismate biosynthesis by glyphosate leads to less anthraquinone accumulation, but also to a reduction in ICS activity. The complexity of the interference of glyphosate with anthraquinone biosynthesis is illustrated by the effect of the inhibitor in cell cultures of the related species Rubia tinctorum L. in these cells, glyphosate leads to an increase in anthraquinone content and a concomitant rise in ICS activity. All data indicate that the main point of regulation in anthraquinone biosynthesis is located at the entrance of the specific secondary route.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1989

Inhibitory effect of a new mycotic agent, piritetrate on ergosterol biosynthesis in pathogenic fungi.

A new antimycotic agent piritetrate, a thiocarbamate derivative, was found to interfere with fungal sterol biosynthesis. Sterol biosynthesis was measured by means of incorporation of radioactivity from [14C]acetate into individual sterol fractions and their precursor compounds extracted from cells of Candida albicans, Cryptococcus neoformans, and Trichophyton mentagrophytes. Piritetrate was a much more potent inhibitor of fungal squalene epoxidation than a related drug, naphthiomate. The greater antifungal efficacy of the former was reflected in its greater inhibitory action on sterol biosynthesis than the latter. Such inhibitory effects were also demonstrated in in vitro labelling experiments with [14C]mevalonate on the S10 fraction (10,000 g supernatant of homogenate) of C. albicans cells. At 5 x 10(-7) M concentration, piritetrate gave almost 90% inhibition of ergosterol biosynthesis in the S10 fraction of C. albicans cells. Furthermore, this agent was highly specific for fungal enzymes involved in ergosterol biosynthesis, with no detectable effects on mammalian cholesterol biosynthesis in rat liver at the therapeutic concentration.

Animals↗
PubMed1997

Suppression of leukotriene B4 biosynthesis by endogenous adenosine in ligand-activated human neutrophils.

Adenosine (Ado) has been shown to suppress several functional responses of human polymorphonuclear leukocytes (PMNs). The current study investigated whether endogenous Ado regulates the biosynthesis of leukotriene (LT)B4 in ligand-stimulated PMNs. Measurements of Ado in PMN resuspended in Hanks' buffered salt solution (HBSS) or plasma showed a cell concentration- and time-dependent accumulation of the nucleoside. The removal of endogenous Ado with either Ado deaminase or the blockade of its action by the Ado A2a receptor antagonist, 8-(3-chlorostyryl) caffeine, markedly increased LTB4 biosynthesis upon ligand stimulation in HBSS. Similarly, LTB4 synthesis by ligand-stimulated PMNs in plasma (containing recombinant LTA4 hydrolase to allow the conversion of protein-bound LTA4) was strongly enhanced by addition of Ado deaminase. Addition of red blood cells to suspensions of PMNs in plasma mimicked the effect of adding Ado deaminase and LTA4 hydrolase in enhancing LTB4 biosynthesis upon ligand stimulation. This effect of red blood cells on LTB4 biosynthesis was blocked by dipyridamole, an inhibitor of Ado transport, or captopril, an inhibitor of LTA4 hydrolase. These results demonstrate that endogenous Ado efficiently downregulates ligand-stimulated LTB4 biosynthesis in PMN suspensions, pointing out a potentially important regulatory function of Ado in inflammatory exudates. These results also unveil a dual role for red blood cells in upregulating LTB4 biosynthesis, namely, the removal of endogenous Ado and the conversion of LTA4 released by activated PMNs.

Adenosine↗
PubMed2004

Jasmonate and ethylene signalling and their interaction are integral parts of the elicitor signalling pathway leading to beta-thujaplicin biosynthesis in Cupressus lusitanica cell cultures.

Roles of jasmonate and ethylene signalling and their interaction in yeast elicitor-induced biosynthesis of a phytoalexin, beta-thujaplicin, were investigated in Cupressus lusitanica cell cultures. Yeast elicitor, methyl jasmonate, and ethylene all induce the production of beta-thujaplicin. Elicitor also stimulates the biosynthesis of jasmonate and ethylene before the induction of beta-thujaplicin accumulation. The elicitor-induced beta-thujaplicin accumulation can be partly blocked by inhibitors of jasmonate and ethylene biosynthesis or signal transduction. These results indicate that the jasmonate and ethylene signalling pathways are integral parts of the elicitor signal transduction leading to beta-thujaplicin accumulation. Methyl jasmonate treatment can induce ethylene production, whereas ethylene does not induce jasmonate biosynthesis; methyl jasmonate-induced beta-thujaplicin accumulation can be partly blocked by inhibitors of ethylene biosynthesis and signalling, while blocking jasmonate biosynthesis inhibits almost all ethylene-induced beta-thujaplicin accumulation. These results indicate that the ethylene and jasmonate pathways interact in mediating beta-thujaplicin production, with the jasmonate pathway working as a main control and the ethylene pathway as a fine modulator for beta-thujaplicin accumulation. Both the ethylene and jasmonate signalling pathways can be regulated upstream by Ca(2+). Ca(2+) influx negatively regulates ethylene production, and differentially regulates elicitor- or methyl jasmonate-stimulated ethylene production.

Cells, Cultured↗
PubMed1996

The excessive production of indole-3-acetic acid and its significance in studies of the biosynthesis of this regulator of plant growth and development.

Because of the importance of indole-3-acetic acid (IAA) in the growth and development of plants, extensive studies of the biosynthesis of IAA have been performed during the four decades since the discovery of IAA as a plant hormone. The pathway for the biosynthesis of IAA in plants remains, however, to be unelucidated, even though studies within the past decade have revealed unexpected aspects of such biosynthesis. By contrast, two pathways to IAA have been characterized in bacteria at the molecular level: the indole-3-acetamide (IAM) pathway (L-tryptophan-->IAM-->IAA); the indole-3-pyruvic acid pathway (L-tryptophan-->indole-3-pyruvic acid-->indole-3-acetaldehyde-->IAA) (Fig. 1). In both pathways, the details of the biosynthesis of IAA were clarified using IAA-overproducing bacteria. After a description of recent advances of the studies of the biosynthesis of IAA in plants, this review focuses on the excessive production of IAA in several organisms and its significance in the studies of the biosynthesis of IAA.

Forecasting↗
PubMed2006

Co-regulation of brassinosteroid biosynthesis-related genes during xylem cell differentiation.

To understand the regulatory mechanisms of brassinosteroid (BR) biosynthesis in specific plant developmental processes, we first investigated the accumulation profiles of BRs and sterols in xylem differentiation in a Zinnia culture. The amounts of many substances in the late C28 sterol biosynthetic pathway to campesterol (CR), such as episterol and 24-methylenecholesterol, as well as those in the BR-specific biosynthetic pathway from CR to brassinolide (BL), were elevated in close association with tracheary element differentiation. Among them, 6-deoxotyphasterol (6-deoxoTY) accumulated to unusually high levels within cells cultured in tracheary element-inductive medium, while castasterone (CS) was not elevated either within or outside cells. To identify the molecular basis of this co-up-regulation of BRs and C28 sterols, we isolated Zinnia genes for the key enzymes of BR biosynthesis, ZeSTE1, ZeDIM, ZeDWF4, ZeCPD1 and ZeCPD2. RNA gel blot analysis of these genes indicated a coordinated increase in transcripts for ZeSTE1, ZeDIM, ZeDWF4 and ZeCPD1, and a tracheary element differentiation-specific increase in transcripts for ZeDWF4 and ZeCPD1. In situ hybridization experiments of ZeDWF4 and ZeCPD1 mRNAs revealed their preferential accumulation in procambium cells, immature xylem cells and xylem parenchyma cells. These results suggest that BR biosynthesis during tracheary element differentiation may be regulated by the coordinated regulation of broad sterol biosynthesis and specific regulation of BR biosynthesis, which occurs in part by elevated transcript levels of genes encoding BR biosynthetic enzymes, specifically ZeDWF4 and ZeCPD1. These data provide new insights into the regulation of BR biosynthesis and BR signaling during plant development.

Amino Acid Sequence↗
PubMed2000

Specific suppression of interleukin 2 biosynthesis by synthetic antisense oligodeoxynucleotides does not influence allograft rejection.

BACKGROUND: Interleukin (IL)-2 supplementation can reverse both blood transfusion-induced tolerance to kidney allografts and spontaneous tolerance to liver allografts in rats. Moreover, IL-2 expression is frequently suppressed in models of allograft tolerance. The failure of IL-2 biosynthesis might therefore play a critical role in tolerance induction. METHODS: Three antisense oligodeoxynucleotides (AS-1, AS-2, AS-3) to rat IL-2, and a control oligo (C-1) consisting of a scrambled version of AS-1, were evaluated for gene-specific suppression of IL-2 biosynthesis in vitro and in vivo, and for their effects on kidney allograft survival. Reverse transcriptase-polymerase chain reaction and IL-2 protein assays were used to assay concanavalin A-driven IL-2 biosynthesis by lymph node lymphocytes in vitro. PVG recipients of Dark Agouti kidney allografts were treated with the oligos. Graft survival and IL-2 biosynthesis by reverse transcriptase-polymerase chain reaction in spleen and graft biopsy specimens were assessed. RESULTS: The AS-1 oligo, but not the AS-2, AS-3 or C-1 oligos, suppressed concanavalin A-driven IL-2 biosynthesis for the 4 days of culture. This effect was dependent on delivery of the AS-1 oligo with lipofectamine. Supplementation with exogenous IL-2 reversed the suppression of lymphocyte proliferation in AS-1-treated cultures. Administration of AS-1 intravenously at 10 mg/kg/day to PVG recipients of Dark Agouti kidney allografts suppressed IL-2 (but not IL-6, interferon-gamma, or tumor necrosis factor-alpha) synthesis in the grafts of seven of nine rats, as measured in biopsy specimens taken at days 2-7. By contrast, all nine control grafts strongly expressed IL-2. However, neither graft histopathology nor graft survival was affected. CONCLUSIONS: Antisense oligonucleotides can powerfully suppress IL-2 biosynthesis in vitro and in allograft recipients in vivo, but this does not affect kidney allograft rejection.

Animals↗
PubMed1994

Studies of the biosynthesis of tentoxin by Alternaria alternata.

Biosynthesis of the phytotoxin, tentoxin, its regulation and the enzymic synthesis steps were studied in vivo and in vitro. The physiology of biosynthesis of tentoxin in vivo was investigated by using sections of mycelial mats incubated in buffer. Differentiated mycelia could be studied under defined conditions. The de novo synthesis of tentoxin was measured by incorporation of [U-14C]leucine into tentoxin. The investigation system was stable for 10 h. Biosynthesis and the growth of biomass started before day 5 of culture, with the maximum between days 9 and 12. After this, biosynthesis quickly declined. pH values about 7 were optimal, and pH values above and below this led to an increased release of tentoxin stored in the cells. The formation of tentoxin by older mycelia was not regulated by acetate, phosphate or glucose, which was not utilized. Precursor amino acids, applied at the start of the culture, slightly activated the synthesis of tentoxin. Older mycelia were inhibited. Substances from the host plant (Brassica chinensis) reduced the de novo synthesis of tentoxin. Enzyme separation studies suggested that biosynthesis of tentoxin involves a multienzyme (> or = 400 kDa), which is a polyfunctional protein without subunits. Experiments suggested that the synthetase contains active SH-groups and an integrated activity of methyltransferase. The precursor amino acids are activated by ATP and bound at the enzyme. N-Methylation occurs with the enzyme-bound amino acids or during the elongation of the growing peptide chain. Methionine is the primary donor of the methyl groups, but the immediate methylation reaction needs 5-adenosyl methionine (SAM). The methylation is essential for the continuation of biosynthesis. The elongation proceeds either stepwise from glycine by binding alanine/methylalanine, phenylalanine/methylphenylalanine and leucine or by formation and linkage of two dipeptides glycine-alanine/methylalanine and phenylalanine/methylphenylalanine-leucine. At the end of this process dihydrotentoxin, the direct precursor of tentoxin, is released from the synthetase probably by cyclization. Independent of this first enzyme, dihydrotentoxin is transformed into tentoxin. This last reaction step is reversible. The rate of transformation of dihydrotentoxin to tentoxin is higher, but in this direction the native turnover is relatively low.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed2004

Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation.

Five ORFs were detected in a fragment from the Streptomyces venezuelae ISP5230 genomic DNA library by hybridization with a PCR product amplified from primers representing a consensus of known halogenase sequences. Sequencing and functional analyses demonstrated that ORFs 11 and 12 (but not ORFs 13-15) extended the partially characterized gene cluster for chloramphenicol (Cm) biosynthesis in the chromosome. Disruption of ORF11 (cmlK) or ORF12 (cmlS) and conjugal transfer of the insertionally inactivated genes to S. venezuelae gave mutant strains VS1111 and VS1112, each producing a similar series of Cm analogues in which unhalogenated acyl groups replaced the dichloroacetyl substituent of Cm. 1H-NMR established that the principal metabolite in the disrupted strains was the alpha-N-propionyl analogue. The sequence of CmlK implicated the protein in adenylation, and involvement in halogenation was inferred from biosynthesis of analogues by the cmlK-disrupted mutant. A role in generating the dichloroacetyl substituent was supported by partial restoration of Cm biosynthesis when a cloned copy of cmlK was introduced in trans into VS1111. Complementation of the mutant also indicated that inactivation of cmlK rather than a polar effect of the disruption on cmlS expression had interfered with dichloroacetyl biosynthesis. The deduced CmlS sequence resembled sequences of FADH2-dependent halogenases. Conjugal transfer of cmlK or cmlS into S. venezuelae cml-2, a chlorination-deficient strain with a mutation mapped genetically to the Cm biosynthesis gene cluster, did not complement the cml-2 lesion, suggesting that one or more genes in addition to cmlK and cmlS is needed to assemble the dichloroacetyl substituent. Insertional inactivation of ORF13 did not affect Cm production, and the products of ORF14 and ORF15 matched Streptomyces coelicolor A3(2) proteins lacking plausible functions in Cm biosynthesis. Thus cmlS appears to mark the downstream end of the gene cluster.

Amino Acid Sequence↗
PubMed1992

Regulation of plant Fatty Acid biosynthesis : analysis of acyl-coenzyme a and acyl-acyl carrier protein substrate pools in spinach and pea chloroplasts.

In previous work (D. Post-Beittenmiller, J.G. Jaworski, J.B. Ohlrogge [1991] J Biol Chem 266: 1858-1865), the in vivo acyl-acyl carrier protein (ACP) pools were measured in spinach (Spinacia oleracea) leaves and changes in their levels were compared to changes in the rates of fatty acid biosynthesis. To further examine the pools of substrates and cofactors for fatty acid biosynthesis and to evaluate metabolic regulation of this pathway, we have now examined the coenzyme A (CoA) and short chain acyl-CoA pools, including acetyl- and malonyl-CoA, in isolated spinach and pea (Pisum sativum) chloroplasts. In addition, the relationships of the acetyl- and malonyl-CoA pools to the acetyl- and malonyl-ACP pools have been evaluated. These studies have led to the following conclusions: (a) Essentially all of the CoA (31-54 mum) in chloroplasts freshly isolated from light-grown spinach leaves or pea seedling was in the form of acetyl-CoA. (b) Chloroplasts contain at least 77% of the total leaf acetyl-CoA, based on comparison of acetyl-CoA levels in chloroplasts and total leaf. (c) CoA-SH was not detected either in freshly isolated chloroplasts or in incubated chloroplasts and is, therefore, less than 2 mum in the stroma. (d) The malonyl-CoA:ACP transacylase reaction is near equilibrium in both light- and dark-incubated chloroplasts, whereas the acetyl-CoA:ACP transacylase reaction is far from equilibrium in light-incubated chloroplasts. However, the acetyl-CoA:ACP transacylase reaction comes nearer to equilibrium when chloroplasts are incubated in the dark. (e) Malonyl-CoA and -ACP could be detected in isolated chloroplasts only during light incubations, and increased with increased rates of fatty acid biosynthesis. In contrast, both acetyl-CoA and acetyl-ACP were detectable in the absence of fatty acid biosynthesis, and acetyl-ACP decreased with increased rates of fatty acid biosynthesis. Together these data have provided direct in situ evidence that acetyl-CoA carboxylase plays a regulatory role in chloroplast fatty acid biosynthesis.

Journal Article↗
PubMed1989

Ethylene biosynthesis-inducing protein from cellulysin is an endoxylanase.

The proteinaceous ethylene biosynthesis-inducing factor (EIF) that was purified from Cellulysin was also shown to contain a xylanase activity. In all nondenaturing protein separation methods employed (Sephacryl S-200 chromatography, and preparative isoelectric focusing and agarose electrophoresis), xylanase activity copurified with the ethylene biosynthesis-inducing activity. Treatment with heat (60 degrees C) or proteases in 8 molar urea inhibited both ethylene-inducing and xylanase activities. Antibodies raised against purified EIF, which contains three polypeptides of 18, 14, and 10 kilodaltons, immunoprecipitated both ethylene biosynthesis-inducing and xylanase activities. The purified EIF contained no detectable cellulase, polygalacturonase, or protease activity. Other hydrolytic activities as estimated by using p-nitrophenyl derivatives of several sugars as substrates also were not detected. Different commercially available hydrolytic enzyme preparations were tested for both ethylene biosynthesis-inducing and xylanase activities. All enzymes tested contained xylanase activity, but only a few induced ethylene biosynthesis. Western blots of proteins separated by SDS-PAGE, using antibodies prepared against the non-denatured purified EIF, revealed two major bands of about 18 and 14 kilodaltons in EIF. These antibodies seem to be specific for these proteins from Trichoderma viride, because there was little cross-reactivity with the other proteins in Cellulysin and other commercial enzyme preparations. Based on these data, we suggest that EIF contains a specific xylanase activity which is involved in inducing ethylene biosynthesis.

Journal Article↗
PubMed1991

ent-Kaurene Biosynthesis in Germinating Barley (Hordeum vulgare L., cv Himalaya) Caryopses and Its Relation to alpha-Amylase Production.

ent-Kaurene biosynthesis as a prerequisite for gibberellin (GA) biosynthesis was studied in germinating Hordeum vulgare L., cv Himalaya caryopses and correlated, in time, with the appearance of alpha-amylase activity. The rate of ent-kaurene biosynthesis was estimated by inhibiting its further metabolism with plant growth retardants (triapenthenol or tetcyclacis) and measuring its accumulation by isotope dilution using combined gas chromatographymass spectrometry. In the inhibitor-treated caryopses, ent-kaurene accumulation began approximately 24 hours after imbibition and proceeded at a rate of about 1 to 2 picomoles per hour per caryopsis, depending on the batch of seeds. In the absence of inhibitor, ent-kaurene did not accumulate, indicating that it is normally turned over rapidly, presumably to further intermediates of the GA biosynthesis pathway and eventually to GAs. ent-Kaurene accumulation occurred almost exclusively in the shoot, which is, therefore, probably the site of biosynthesis. alpha-Amylase production began between 30 and 36 hours after imbibition and, thus, correlated well with de novo GA biosynthesis, as estimated from ent-kaurene accumulation. However, inhibition of ent-kaurene oxidation by plant growth retardants did not reduce the alpha-amylase production significantly, although it did reduce shoot elongation. We conclude that ent-kaurene is produced in the shoot and is continuously converted to GA, which is essential for normal shoot elongation, but not for the production of alpha-amylase in the aleurone layer.

Journal Article↗
PubMed1992

Energy requirements for Fatty Acid and glycerolipid biosynthesis from acetate by isolated 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 <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.

Journal Article↗
PubMed2004

Regulation of phospholipid biosynthesis by phosphatidylinositol transfer protein Sec14p and its homologues. A critical role for phosphatidic acid.

Transcription of yeast phospholipid biosynthesis structural genes, which contain an inositol-sensitive upstream activating sequence in their promoters, responds to the availability of the soluble precursors inositol and choline and to changes in phospholipid metabolism. The INO1 gene is deregulated (derepressed when inositol is present) under the conditions of increased phosphatidylcholine (PtdCho) turnover, as occurs in the sec14Delta cki1Delta strain (SEC14 encodes the major yeast phosphatidylinositol transfer protein; CKI1 encodes choline kinase of the cytidine diphosphate choline pathway of PtdCho biosynthesis). Five proteins (Sfhp) share sequence homology with phosphatidylinositol transfer protein Sec14p. Two (Sfh2p and Sfh4p), when overexpressed largely complement the otherwise essential Sec14p requirement concerning growth and secretion. In this study, we analysed the ability of Sec14 homologues to correct the defect in regulation of phospholipid biosynthesis resulting from defective or missing Sec14p. We also analysed how PtdCho turnover relates to the transcriptional regulation of phospholipid biosynthesis. The results show that (a) none of the Sec14 homologues was able to substitute for Sec14p in its regulatory aspects of phospholipid biosynthesis, (b) removal of phospholipase D activity corrected the aberrant INO1 gene regulation in yeast strains with otherwise high PtdCho turnover, and (c) increased steady-state phosphatidic acid levels correlated with derepressed levels of the INO1 gene. Overall, the results support the model in which high phosphatidic acid levels lead to derepression of the genes of phospholipid biosynthesis [Henry, S.A. & Patton-Vogt, J.L. (1998) Prog. Nucleic Acid Res. Mol. Biol.61, 133-179].

Gene Expression Regulation, Enzymologic↗
PubMed1984

Insensitivity of ubiquinone biosynthesis in glioblastoma cells to an epileptogenic drug, U18666A.

To investigate the perturbation of ubiquinone biosynthesis by a hypocholesterolemic drug, 3 beta-(2-diethylaminoethoxy)androst-5-en-17-one hydrochloride (U18666A), we measured the incorporation of radioactive mevalonate, methionine, tyrosine, and 4-hydroxybenzoic acid into ubiquinone in glioblastoma cells. These four precursors unanimously showed that ubiquinone biosynthesis was not significantly altered by U18666A, which blocked cholesterol biosynthesis at steps beyond mevalonate formation. The fluctuation of the endogenous mevalonate level had little effect on ubiquinone biosynthesis, implying the relative stability of cellular ubiquinone biosynthesis. Furthermore, exogenously added mevalonate did not have an appreciable effect on ubiquinone biosynthesis. The major ubiquinone produced in rat glioblastoma cells was identified as ubiquinone-9. The mevalonate-derived products accumulated in the U18666A-treated cells differed significantly from those reported in a broken cell study, suggesting the existence of delicate mechanisms regulating the formation of cholesterol intermediates.

Androstenes↗