Abscisic acid biosynthesis during corn embryo development

In this study we examined the biosynthesis of abscisic acid (ABA) by developing corn (Zea mays L.) embryos. Three comparisons were made: ABA biosynthesis in embryos isolated from kernels grown in vitro with those grown in the field; the developmental profile of ABA content with that of biosynthesis; and ABA biosynthesis in corn embryos lacking carotenoid precursors with ABA biosynthesis in normal embryos. Embryos were harvested at various times during seed development and divided into two groups. Endogenous levels of ABA were measured in one group of embryos and ABA biosynthetic capacity was measured in the other group. The ABA biosynthetic capacity was measured with and without tetcyclacis (an inhibitor of ABA degradation) in embryos from both field-grown and in-vitro-grown corn kernels. Reduced-carotenoid (either fluridone-treated or genetically viviparous) embryos were also included in the study. Corn kernels developing under field and in-vitro conditions differed from each other in their responses to tetcyclacis and in their profiles of ABA biosynthesis during development. Therefore, in-vitro kernel culture may not be an appropriate substitute for field conditions for studies of embryo development. The developmental profiles of endogenous ABA content differed from those of ABA biosynthesis in isolated embryos of both in-vitro-and field-grown kernels. This indicated that ABA levels in the developing embryos were determined by import from the maternal tissues available to the embryos rather than by in-situ biosynthesis. In embryos with reduced levels of carotenoids, either fluridone-treated or genetically viviparous embryos, ABA biosynthesis was low or nonexistent. This result is expected for the presence of an indirect pathway of ABA biosynthesis and in the absence of ABA precursors.Abbreviations ABA abscisic acid - DAP days after pollination     

Recent progress in cellulose biosynthesis

Cellulose comprises the major polymer of the plant cell wall. It consists of a set of parallel chains composed of glucans and these chains are highly oriented to form a structure known as a microfibril. The orientation of the microfibrils controls the extension of the direction of the plant cell. Extensive studies on the cellulose biosynthesis have been carried out for over three decades, and recently (1996) genes for cellulose biosynthesis in plants (CesA) were isolated. In the year 2002, a specific primer for cellulose biosynthesis reaction has been discovered and cellulose synthetic activity has been also confirmed by recombinant protein derived from the plant CesA gene. Furthermore, other proteins involved in cellulose biosynthesis besides CesA proteins were also proposed at the same time. One of these proteins, Korrigan cellulase, was suggested to act by removing sitosterol from the primer for biosynthesis reaction of cellulose. A membrane-bound sucrose synthase was also suggested to provide UDP-glucose as a substrate for cellulose biosynthesis. On the basis of these results, a new pathway for cellulose biosynthesis was proposed. Now, the research field of cellulose biosynthesis is facing a major turning point. Electronic Publication     

Biosynthesis of Biotin from Dethiobiotin by Intact Cells of Biotin-producing Bacterium

Intact cells of a biotin-producing bacterium, KY–21–1–25, were found to synthesize biotin from dethiobiotin. Optimal conditions for the biosynthesis of biotin from dethiobiotin by intact cells were investigated. Intact cells harvested from adenine-supplemented medium showed intensive biosynthesis. However, the biosynthesis of biotin by intact cells was strongly inhibited by the addition of adenine or adenosine. The inhibitory activity of adenine was about 10-fold greater than that of adenosine. Formation of several unidentified biotin-vitamers was observed in both reaction mixtures incubated with and/or without addition of adenine.     

In vivo and in vitro effects of thiolactomycin on fatty acid biosynthesis in Streptomyces collinus.

A stable-isotope assay was used to analyze the effectiveness of various perdeuterated short-chain acyl coenzyme A (acyl-CoA) compounds as starter units for straight- and branched-chain fatty acid biosynthesis in cell extracts of Streptomyces collinus. In these extracts perdeuterated isobutyryl-CoA was converted to isopalmitate (a branched-chain fatty acid), while butyryl-CoA was converted to palmitate (a straight-chain fatty acid). These observations are consistent with previous in vivo analyses of fatty acid biosynthesis in S. collinus, which suggested that butyryl-CoA and isobutyryl-CoA function as starter units for palmitate and isopalmitate biosynthesis, respectively. Additionally, in vitro analysis demonstrated that acetyl-CoA can function as a starter unit for palmitate biosynthesis. Palmitate biosynthesis and isopalmitate biosynthesis in these cell extracts were both effectively inhibited by thiolactomycin, a known type II fatty acid synthase inhibitor. In vivo experiments demonstrated that concentrations of thiolactomycin ranging from 0.1 to 0.2 mg/ml produced both a dramatic decrease in the cellular levels of branched-chain fatty acids and a surprising three- to fivefold increase in the cellular levels of the straight-chain fatty acids palmitate and myristate. Additional in vivo incorporation studies with perdeuterated butyrate suggested that, in accord with the in vitro studies, the biosynthesis of the palmitate from butyryl-CoA decreases in the presence of thiolactomycin. In contrast, in vivo incorporation studies with perdeuterated acetate demonstrated that the biosynthesis of palmitate from acetyl-CoA increases in the presence of thiolactomycin. These observations clearly demonstrate that isobutyryl-CoA is a starter unit for isopalmitate biosynthesis and that either acetyl-CoA or butyryl-CoA can be a starter unit for palmitate biosynthesis in S. collinus. However, the pathway for palmitate biosynthesis from acetyl-CoA is less sensitive to thiolactomycin, and it is suggested that the basis for this difference is in the initiation step.     

Role of fatty acid de novo biosynthesis in polyhydroxyalkanoic acid (PHA) and rhamnolipid synthesis by pseudomonads: establishment of the transacylase (PhaG)-mediated pathway for PHA biosynthesis in Escherichia coli.

Since Pseudomonas aeruginosa is capable of biosynthesis of polyhydroxyalkanoic acid (PHA) and rhamnolipids, which contain lipid moieties that are derived from fatty acid biosynthesis, we investigated various fab mutants from P. aeruginosa with respect to biosynthesis of PHAs and rhamnolipids. All isogenic fabA, fabB, fabI, rhlG, and phaG mutants from P. aeruginosa showed decreased PHA accumulation and rhamnolipid production. In the phaG (encoding transacylase) mutant rhamnolipid production was only slightly decreased. Expression of phaG from Pseudomonas putida and expression of the beta-ketoacyl reductase gene rhlG from P. aeruginosa in these mutants indicated that PhaG catalyzes diversion of intermediates of fatty acid de novo biosynthesis towards PHA biosynthesis, whereas RhlG catalyzes diversion towards rhamnolipid biosynthesis. These data suggested that both biosynthesis pathways are competitive. In order to investigate whether PhaG is the only linking enzyme between fatty acid de novo biosynthesis and PHA biosynthesis, we generated five Tn5 mutants of P. putida strongly impaired in PHA production from gluconate. All mutants were complemented by the phaG gene from P. putida, indicating that the transacylase-mediated PHA biosynthesis route represents the only metabolic link between fatty acid de novo biosynthesis and PHA biosynthesis in this bacterium. The transacylase-mediated PHA biosynthesis route from gluconate was established in recombinant E. coli, coexpressing the class II PHA synthase gene phaC1 together with the phaG gene from P. putida, only when fatty acid de novo biosynthesis was partially inhibited by triclosan. The accumulated PHA contributed to 2 to 3% of cellular dry weight.     

Activities of 3-hydroxyl-3-methylglutaryl-CoA reductase and acetyl-CoA carboxylase and the rate of mevalonic acid, squalene, sterol and fatty acid biosynthesis from [1-14C]acetyl-CoA and [2-14C]malonyl-CoA in rat liver: effects of Triton WR 1339, starvation and cholesterol diet

The effects of Triton WR 1339, starvation and cholesterol diet on the activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and acetyl-CoA carboxylase and on the rates of mevalonic acid (MVA) biosynthesis from acetyl-CoA and malonyl-CoA in the soluble (140 000 g) and microsomal fractions of rat liver, on the rate of incorporation of these substrates into squalene, cholesterol and lanosterol in the rat liver postmitochondrial fraction and on the rate of fatty acid biosynthesis was studied. The administration of Triton WR 1339 (200 mg per 100 g of body weight twice) stimulated the activity of HMG-CoA reductase and MVA biosynthesis from acetyl-CoA and malonyl-CoA in the intact and solubilized microsomal fractions and had no effect on these parameters in the soluble fraction. Starvation for 36 hrs did not cause inhibition of the reductase activity or MVA biosynthesis from both substrates in the soluble fraction. Alimentary cholesterol significantly increased the activity of HMG-CoA reductase, had no effect on the rate of MVA biosynthesis from acetyl-CoA and stimulated the malonyl-CoA incorporation in to MVA in the soluble fraction. Starvation an alimentary cholesterol inhibited the HMG-CoA reductase activity and MVA biosynthesis from both substrates in the solubilized microsomal fraction. Triton WR 1339 stimulated 4--19-fold the lipid formation in the total unsaponified fraction and its components i.e. squalene, lanosterol, cholesterol, from acetyl-CoA and only insignificantly (1,2--1,7-fold) increased malonyl-CoA incorporation into these compounds. Starvation and alimentary cholesterol repressed lanosterol and cholesterol biosynthesis from acetyl-CoA, decreased malonyl-CoA incorporation into these sterols and had no influence on squalene biosynthesis from the two substrates. Triton WR 1339 and starvation inhibited the acetyl-CoA carboxylase activity, unaffected by alimentary cholesterol. No significant changes in the rate of fatty acid biosynthesis from the substrates were observed. The data obtained provide evidence for the existence of autonomic pathways of MVA biosynthesis localized in the soluble and microsomal fractions of rat liver. The pathway of MVA biosynthesis in the soluble fraction is less sensitive to regulatory factors. Sterol biosynthesis from malonyl-CoA is also more resistant to regulatory effects than sterol biosynthesis from acetyl-CoA. This suggests that HMG-CoA reductase localized in the soluble fraction takes part in MVA and sterol biosynthesis from malonyl-CoA.     

Elucidation of the dual role of Mycobacterial MoeZR in molybdenum cofactor biosynthesis and cysteine biosynthesis

The pathway of molybdenum cofactor biosynthesis has been studied in detail by using proteins from Mycobacterium species, which contain several homologs associated with the first steps of Moco biosynthesis. While all Mycobacteria species contain a MoeZR, only some strains have acquired an additional homolog, MoeBR, by horizontal gene transfer. The role of MoeBR and MoeZR was studied in detail for the interaction with the two MoaD-homologs involved in Moco biosynthesis, MoaD1 and MoaD2, in addition to the CysO protein involved in cysteine biosynthesis. We show that both proteins have a role in Moco biosynthesis, while only MoeZR, but not MoeBR, has an additional role in cysteine biosynthesis. MoeZR and MoeBR were able to complement an E. coli moeB mutant strain, but only in conjunction with the Mycobacterial MoaD1 or MoaD2 proteins. Both proteins were able to sulfurate MoaD1 and MoaD2 in vivo, while only MoeZR additionally transferred the sulfur to CysO. Our in vivo studies show that Mycobacteria have acquired several homologs to maintain Moco biosynthesis. MoeZR has a dual role in Moco- and cysteine biosynthesis and is involved in the sulfuration of MoaD and CysO, whereas MoeBR only has a role in Moco biosynthesis, which is not an essential function for Mycobacteria.     

Evidence that cyclic AMP-induced inhibition of phosphatidylcholine biosynthesis is caused by a decrease in cellular diacylglycerol levels in cultured rat hepatocytes.

The mechanism by which glucagon and cAMP analogues inhibit phosphatidylcholine biosynthesis was investigated in rat hepatocytes. The studies were facilitated by preparation of an antibody to a synthetic peptide (D-F-V-A-H-D-D-I-P-Y-S-S-A) corresponding to residues 164-176 of CTP:phosphocholine cytidylyl-transferase. The antibody, which was purified by affinity chromatography, quantitatively immunoprecipitated cytidylyltransferase from rat liver cytosol. Various analogues of cAMP had no effect on the labeling of cytidylyltransferase with 32Pi in rat hepatocytes. Nor did the cAMP analogues have any effect on the distribution of cytidylyltransferase between cytosol and membranes. These results indicate that the supply of CDP-choline does not limit phosphatidylcholine biosynthesis in hepatocytes treated with cAMP analogues. A decreased supply of diacylglycerol was considered as an alternative mechanism for inhibition of phosphatidylcholine biosynthesis. An approximately 30% decrease in diacylglycerol concentration was observed in hepatocytes treated with the cAMP analogues or glucagon, compared with controls. A similar decrease of phosphatidylcholine biosynthesis was observed. The cAMP-mediated decrease in diacylglycerol levels and inhibition of phosphatidylcholine biosynthesis were reversed by addition of 0.5-1.5 mM oleic acid to the treated hepatocytes. A correlation coefficient of 0.93 was calculated between the levels of diacylglycerol and the rate of phosphatidylcholine biosynthesis. In another approach, the diacylglycerol levels were increased by an inhibitor of diacylglycerol lipase (U-57908) which also reversed the cAMP effects on diacylglycerol levels and phosphatidylcholine biosynthesis. We conclude that the cAMP-mediated inhibition of phosphatidylcholine biosynthesis was not due to an effect on the phosphorylation of cytidylyltransferase. Instead, phosphatidylcholine biosynthesis appears to be inhibited due to a decreased level of diacylglycerol, a substrate for CDP-choline: 1,2-diacylglycerol cholinephosphotransferase.     

Ascorbic acid within chromaffin granules. In situ kinetics of norepinephrine biosynthesis

Ascorbic acid requirements for norepinephrine biosynthesis were investigated in intact bovine chromaffin granules using the physiologic substrate dopamine and a novel coulometric electrochemical detection high pressure liquid chromatography system for ascorbic acid. 10 mM external dopamine, 1 mM Mg-ATP, and 1 mM ascorbic acid produced maximal norepinephrine biosynthesis without granule lysis. When external ascorbic acid was omitted, intragranular ascorbic acid was consumed in a 1:1 ratio with respect to norepinephrine biosynthesis. The initial concentration of intragranular ascorbic acid was 10.5 mM, which was depleted in stepwise fashion to 15 lower concentrations over the range of 9.2-0.2 mM. Chromaffin granules containing these varying concentrations of intragranular ascorbic acid were then incubated with 1 mM exogenous ascorbic acid, and norepinephrine biosynthesis from dopamine was determined. The apparent Km of norepinephrine biosynthesis for intragranular ascorbic acid was 0.57 mM by Eadie-Hofstee analysis and 0.68 mM by Lineweaver-Burk analysis. These data indicate that intragranular ascorbic acid is available and required for norepinephrine biosynthesis, that ascorbic acid is a true co-substrate for dopamine beta-monooxygenase, and that intragranular ascorbic acid is maintained by extragranular ascorbic acid. Continued norepinephrine biosynthesis in granules is dependent on both intragranular and extragranular concentrations of the vitamin. Furthermore, in situ kinetics of dopamine beta-monooxygenase for ascorbic acid may be most accurately determined using intact granules and the true physiologic substrate.     

Differential inhibitory effects of antibiotics on the biosynthesis of envelope proteins of Escherichia coli

Inhibitory effects of six antibiotics (kasugamycin, tetracycline, chloramphenicol, sparsomycin, puromycin and rifampicin) on the biosynthesis of envelope proteins of Escherichia coli were examined and compared with those on the biosynthesis of cytoplasmic proteins. Kasugamycin, puromycin and rifampicin were much more inhibitory to the over-all biosynthesis of cytoplasmic proteins than to that of envelope proteins. On the contrary, tetracycline and sparsomycin showed much stronger inhibitory effects on the biosynthesis of envelope proteins than on that of cytoplasmic proteins. Chloramphenicol showed little difference in its inhibitory effect on the biosynthesis of envelope proteins and cytoplasmic proteins.The envelope proteins were labeled with [³H]arginine in the presence of the antibiotics and separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The inhibitory effects of the antibiotics on the biosynthesis of individual envelope proteins were then examined. Inhibition patterns were found to be widely different from one envelope protein to the other. For example, the biosynthesis of one major envelope protein of molecular weight 38,000 was more resistant to kasugamycin, chloramphenicol and sparsomycin than that of the other envelope proteins. On the other hand, the biosynthesis of another major envelope protein (lipoprotein) of about 7500 molecular weight was much more resistant to puromycin and rifampicin than that of the other envelope proteins. In the case of tetracycline, little differential inhibitory effect on the biosynthesis of individual envelope proteins was observed.Stability of messenger RNAs for individual envelope proteins was also determined from the inhibitory effect of rifampicin on their biosynthesis. It was found that the average of half lives of mRNAs for major envelope proteins examined (5.5 minutes) is twice as long as the average of those of mRNAs for cytoplasmic proteins (2 minutes), except for the lipoprotein of about 7500 molecular weight which has extremely stable mRNA with a half life of 11.5 minutes. From these results the envelope proteins of E. coli appear to be biosynthesized in a somewhat different manner from that of the cytoplasmic proteins. Furthermore, at least some envelope proteins may have their own specific biosynthetic systems.     

Candicidin biosynthesis in <Emphasis Type="Italic">Streptomyces griseus</Emphasis>

The biosynthesis of the aromatic polyene macrolide antibiotic candicidin, produced by Streptomyces griseus IMRU 3570, begins with a p-aminobenzoic acid (PABA) molecule which is activated to PABA-CoA and used as starter for the head-to-tail condensation of four propionate and 14 acetate units to produce a polyketide molecule to which the deoxysugar mycosamine is attached. Using the gene coding for the PABA synthase ( pabAB) from S. griseusIMRU 3570 as the probe, a 205-kb region of continuous DNA from the S. griseus chromosome was isolated and partially sequenced. Some of the genes possibly involved in the biosynthesis of candicidin were identified including part of the modular polyketide synthase (PKS), genes for thioesterase, deoxysugar biosynthesis, modification, transport, and regulatory proteins. The regulatory mechanisms involved in the production of candicidin, such as phosphate regulation, were studied using internal probes for some of the genes involved in the biosynthesis of the three moieties of candicidin (PKS, aromatic moiety and amino sugar). mRNAs specific for these genes were detected only in the production medium (SPG) but not in the SPG medium supplemented with phosphate or in the inoculum medium, indicating that phosphate represses the expression of genes involved in candicidin biosynthesis. The modular architecture of the candicidin PKS and the availability of the PKSs involved in the biosynthesis of three polyene antibiotics (pimaricin, nystatin, and amphotericin B) shall make possible the creation of new, less toxic and more active polyene antibiotics through combinatorial biosynthesis and targeted mutagenesis.     

Induction of holomycin production and complex metabolic changes by the argR mutation in Streptomyces clavuligerus NP1

In bacteria, arginine biosynthesis is tightly regulated by a universally conserved regulator, ArgR, which regulates the expression of arginine biosynthetic genes, as well as other important genes. Disruption of argR in Streptomyces clavuligerus NP1 resulted in complex phenotypic changes in growth and antibiotic production levels. To understand the metabolic changes underlying the phenotypes, comparative proteomic studies were carried out between NP1 and its argR disruption mutant (designated CZR). In CZR, enzymes involved in holomycin biosynthesis were overexpressed; this is consistent with its holomycin overproduction phenotype. The effects on clavulanic acid (CA) biosynthesis are more complex. Several proteins from the CA cluster were moderately overexpressed, whereas several proteins from the 5S clavam biosynthetic cluster and from the paralog cluster of CA and 5S clavam biosynthesis were severely downregulated. Obvious changes were also detected in primary metabolism, which are mainly reflected in the altered expression levels of proteins involved in acetyl-coenzyme A (CoA) and cysteine biosynthesis. Since acetyl-CoA and cysteine are precursors for holomycin synthesis, overexpression of these proteins is consistent with the holomycin overproduction phenotype. The complex interplay between primary and secondary metabolism and between secondary metabolic pathways were revealed by these analyses, and the insights will guide further efforts to improve production levels of CA and holomycin in S. clavuligerus.     

Tn10 insertional mutations of Bacillus subtilis that block the biosynthesis of bacilysin

Transposon mutagenesis was employed to isolate the gene(s) related with the biosynthesis of dipeptide antibiotic in Bacillus subtilis PY79 (a prototrophic derivative of the standard 168 strain). The blocked mutants were phenotypically selected from the transposon library by bioassay and the complete loss of biosynthetic ability was verified through ESI-mass spectrometry analysis. Four different bacilysin nonproducer mutants (Bac(-)::Tn10(ori-spc)) were isolated from the transposon library. The genes involved in bacilysin biosynthesis were identified as thyA (thymidilate synthetase), ybgG (unknown; similar to homocysteine methyl transferase) and oppA (oligopeptide permease), respectively. The other blocked gene was yvgW (unknown; similar to heavy metal-transporting ATPase); however, backcross studies did not verify its involvement in bacilysin biosynthesis. This gene, on the other hand, appeared to be necessary for efficient sporulation and transformation. Opp involvement was significant as it suggested that bacilysin biosynthesis is under or a component of the quorum sensing pathway which has been shown to be responsible for the establishment of sporulation, competence development and onset of surfactin biosynthesis. For verification, it was necessary to check the involvement of peptide pheromones (PhrA or PhrC) internalized by the Opp system and response regulator ComA as the essential components of this global control. phrA, phrC and comA deleted mutants of PY79 were thus constructed and the latter two genes were shown to be essential for bacilysin biosynthesis.     

Cloning and characterization of the polyether salinomycin biosynthesis gene cluster of Streptomyces albus XM211

Salinomycin is widely used in animal husbandry as a food additive due to its antibacterial and anticoccidial activities. However, its biosynthesis had only been studied by feeding experiments with isotope-labeled precursors. A strategy with degenerate primers based on the polyether-specific epoxidase sequences was successfully developed to clone the salinomycin gene cluster. Using this strategy, a putative epoxidase gene, slnC, was cloned from the salinomycin producer Streptomyces albus XM211. The targeted replacement of slnC and subsequent trans-complementation proved its involvement in salinomycin biosynthesis. A 127-kb DNA region containing slnC was sequenced, including genes for polyketide assembly and release, oxidative cyclization, modification, export, and regulation. In order to gain insight into the salinomycin biosynthesis mechanism, 13 gene replacements and deletions were conducted. Including slnC, 7 genes were identified as essential for salinomycin biosynthesis and putatively responsible for polyketide chain release, oxidative cyclization, modification, and regulation. Moreover, 6 genes were found to be relevant to salinomycin biosynthesis and possibly involved in precursor supply, removal of aberrant extender units, and regulation. Sequence analysis and a series of gene replacements suggest a proposed pathway for the biosynthesis of salinomycin. The information presented here expands the understanding of polyether biosynthesis mechanisms and paves the way for targeted engineering of salinomycin activity and productivity.     

Juvenile Hormone biosynthesis and utilization of farnesoic acid during the final larval stadium of the ring-legged earwig

Abstract. The regulation of Juvenile Hormone (JH) HI biosynthesis and release by the corpora allata (CA) was studied in final instar male and female larvae of the earwig, Euborellia annulipes , using a radiochemical assay in vitro. In males, maximal biosyntiiesis of JH IH occurred on day 1, then declined to virtually undetectable levels for the following 12 days of the stadium, and finally increased on days 14–16. In females, peaks of biosynthesis were detected on days 0–1 and on day 12. A further investigation of the 12-day-old larvae demonstrated mat in nonmoulting males and females, JH UJ biosynthesis was undetectable. However, for males and females undergoing ecdysis, the biosynthesis of JH III was detected and quantified.
The addition of 60 μM farnesoic acid to the incubation medium significantly increased the production of JH III by CA taken from females from day 8 until the end of the stadium. Glands from 12-day old females that had initiated ecdysis were stimulated by farnesoic acid. By contrast, we could detect no stimulation of production of JH III by farnesoic acid in CA taken from males, even very late in the stadium. CA from newly emerged adult males and females were more active than those of larvae, and were greatly stimulated by farnesoic acid. CA from females immediately after emergence were stimulated significantly more by farnesoic acid man were glands from newly emerged males. These results suggest fundamental differences in the synmetic activity of CA for males and females in this insect.     

The effect of l-methionine concentration on juvenile hormone biosynthesis by corpora allata of the cockroach Diploptera punctata

The effect of varying l-methionine (l-met) concentration on rates of juvenile hormone (JH) biosynthesis/release by corpora allata of females of the viviparous cockroach Diploptera punctata has been studied using a radiochemical assay. Both high activity glands (corpora allata from day 5 females) and low activity glands (corpora allata from day 11 females) were used to study the dose dependence of JH biosynthesis on l-met concentrations, under both de novo (spontaneous) conditions of JH biosynthesis and stimulated conditions (in the presence of the exogenous JH III precursor farnesoic acid). Maximal rates of JH biosynthesis/release were observed at l-met concentration of 20 μM (spontaneous) and 40 μM (stimulated). Below these concentrations, rates of JH biosynthesis declined linearly with decreasing l-met concentration. Optimal concentration of l-met appeared to be similar for both high and low activity corpora allata, under spontaneous and stimulated conditions of biosynthesis. Above 40 μM l-met, no increase in rates of JH biosynthesis was observed. It appears that the corpora allata of D. punctata are efficient scavengers of l-met and are able to utilize even low concentrations of the substrate for JH biosynthesis. The corpora allata of D. punctata may prove useful for the biosynthesis of authentic JH III, radiolabelled in the methyl position using as methyl donor, l[methyl-³H]met of high specific activity.     

Enhancement by farnesol and farnesoic acid of juvenile-hormone biosynthesis in induced low-activity locust corpora allata

Exogenous farnesol or farnesoic acid (FA) stimulates juvenile hormone III (JH III) biosynthesis by isolated corpora allata from Locusta migratoria in a dose-dependent manner. Farnesol and FA also stimulate a dose-dependent accumulation of substantial amounts of methyl farnesoate (MF), identified by gas chromatography-mass spectroscopy (GCMS) analysis, in the corpora allata. Lower quantities of MF were found in the incubation medium. Corpora allata, denervated 2 days prior to assay, showed low spontaneous rates of JH biosynthesis which were stimulated by farnesol and FA. The dose-response curves for control and denervated corpora allata were similar. During oocyte maturation the rate of farnesol and FA stimulation of JH biosynthesis increased gradually. However, after transection of nervus corporis allati 1 (NCA-1), the rate of stimulated JH synthesis was maintained at preoperative levels. Although the spontaneous rate of JH biosynthesis decreased rapidly after NCA-1 transection, denervated glands could still be stimulated by farnesol or FA to produce large amounts of JH. These results suggest that the low spontaneous rate of JH biosynthesis in denervated corpora allata is not caused by inhibition of the final steps of JH biosynthesis.     

Molecular cloning of N-methylputrescine oxidase from tobacco

Nicotine biosynthesis in Nicotiana species requires an oxidative deamination of N-methylputrescine, catalyzed by N-methylputrescine oxidase (MPO). In a screen for tobacco genes that were down-regulated in a tobacco mutant with altered regulation of nicotine biosynthesis, we identified two homologous MPO cDNAs which encode diamine oxidases of a particular subclass. Tobacco MPO genes were expressed specifically in the root, and up-regulated by jasmonate treatment. Recombinant MPO protein expressed in Escherichia coli formed a homodimer and deaminated N-methylputrescine more efficiently than symmetrical diamines. These results indicate that MPO evolved from general diamine oxidases to function effectively in nicotine biosynthesis.