Uptake and metabolism of clomazone in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures

Studies were conducted to determine the uptake and metabolism of the pigment synthesis inhibiting herbicide clomazone in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) photomixotrophic cell suspensions. Soybean and cotton on a whole plant level are tolerant and susceptible to clomazone, respectively. Preliminary studies indicated that I₅₀ values for growth, chlorophyll (Chl), β-carotene, and lutein were, respectively, >22, 14, 19, and 23 times greater for the soybean cell line (SB-M) 8 days after treatment (DAT) compared to the cotton cell line (COT-M) 16 DAT. Differences in [¹⁴C]clomazone uptake cannot account for selectivity since there were significantly greater levels of clomazone absorbed by the SB-M cells compared to the COT-M cells for each treatment. The percentage of absorbed clomazone converted to more polar metabolite(s) was significantly greater by the SB-M cells relative to COT-M cells at 6 and 24 hours after treatment, however, only small differences existed between the cell lines by 48 hours after treatment. Nearly identical levels of parental clomazone was recovered from both cell lines for all treatments. A pooled metabolite fraction isolated from SB-M cells had no effect on the leaf pigment content of susceptible velvetleaf (Abutilon theophrasti Medic.) or soybean seedlings. Conversely, a pooled metabolite fraction from COT-M cells reduced the leaf Chl content of velvetleaf. Soybean tolerance to clomazone appears to be due to differential metabolism (bioactivation) and/or differences at the site of action.     

Studies on isoprenoid biosynthesis with bacterial intact cells

For the study on the regulation of isoprenoid biosynthesis with intact cells, some strains of bacteria capable of growing on mevalonate as a sole carbon source were isolated from soil. Many of them incorporated [¹⁴C]-mevalonate, [¹⁴C]isopentenyl- and [¹⁴C]farnesyl pyrophosphates into the cells. However, radioactivity was found in their degradation products but not in isoprenoids. Addition of [¹⁴C]isopentenyl pyrophosphate, farnesyl pyrophosphate and Mg²⁺ ions in combination to the culture of a strain of Arthrobacter gave rise to ¹⁴C-incorporation into isoprenoids. Radioactivity was found in polyprenol, its pyrophosphate, monophosphate and fatty acid esters. The reactions of isopentenyl- and farnesyl pyrophosphates syntheses seemed to be rate-limiting steps.     

Diterpene biosynthesis in maize seedlings in response to fungal infection

A cell-free system which catalyzes the biosynthesis of terpene hydrocarbons when supplemented with mevalonate, Mn²⁺, and ATP was prepared from the scutellum-embryonic axis region of maize seedlings. The capacity of this system for the production of terpene hydrocarbons was enhanced 50- to 100-fold when the seedlings were exposed for 48 hours to the fungus Rhizopus stolonifer prior to tissue homogenization. The fungi Aspergillus niger, Fusarium moniliforme, and Verticillium albo-atrum also elicited this biosynthetic enhancement. The terpene hydrocarbon products were separable into six fractions by argentation thin layer chromatography. Radioactivity was contributed to five of these fractions when either geranylgeranyl pyrophosphate or copalyl pyrophosphate was supplied as substrate, suggesting that polycyclic diterpenoid hydrocarbons were the main products. Large scale biosynthetic reactions led to the acquisition of about 1 milligram of terpene hydrocarbon products plus some more polar terpenoid products. Analysis of the hydrocarbon products by gas chromatography and mass spectrometry led to the separation of six distinct diterpene hydrocarbons plus a fraction with a molecular weight of about 550. Three of the diterpene hydrocarbons were identified as kaur-16-ene, kaur-15-ene (isokaurene), and pimara-8(14),15-diene. None of the terpene hydrocarbon fractions tested displayed antifungal activity in the Cladosporium cucumerinum thin layer plate assay.     

Machine-learning guided elucidation of contribution of individual steps in the mevalonate pathway and construction of a yeast platform strain for terpenoid production

The production of terpenoids from engineered microbes contributes markedly to the bioeconomy by providing essential medicines, sustainable materials, and renewable fuels. The mevalonate pathway leading to the synthesis of terpenoid precursors has been extensively targeted for engineering. Nevertheless, the importance of individual pathway enzymes to the overall pathway flux and final terpenoid yield is less known, especially enzymes that are thought to be non-rate-limiting. To investigate the individual contribution of the five non-rate-limiting enzymes in the mevalonate pathway, we created a combinatorial library of 243 Saccharomyces cerevisiae strains, each having an extra copy of the mevalonate pathway integrated into the genome and expressing the non-rate-limiting enzymes from a unique combination of promoters. High-throughput screening combined with machine learning algorithms revealed that the mevalonate kinase, Erg12p, stands out as the critical enzyme that influences product titer. ERG12 is ideally expressed from a medium-strength promoter which is the ‘sweet spot’ resulting in high product yield. Additionally, a platform strain was created by targeting the mevalonate pathway to both the cytosol and peroxisomes. The dual localization synergistically increased terpenoid production and implied that some mevalonate pathway intermediates, such as mevalonate, isopentyl pyrophosphate (IPP), and dimethylallyl pyrophosphate (DMAPP), are diffusible across peroxisome membranes. The platform strain resulted in 94-fold, 60-fold, and 35-fold improved titer of monoterpene geraniol, sesquiterpene α-humulene, and triterpene squalene, respectively. The terpenoid platform strain will serve as a chassis for producing any terpenoids and terpene derivatives.     

The role of kidneys in mevalonate metabolism: fact and artifact

This laboratory has previously demonstrated that circulating mevalonate is metabolized primarily by the kidneys by both the sterol and nonsterol pathways of mevalonate metabolism. Bardenheier and Popják recently challenged this conclusion, claiming that a contaminant in commercially obtained [5-¹⁴C]mevalonate can artifactually account for much of the ¹⁴C reported as ¹⁴CO₂ in such experiments.In the present study, this contaminant was shown to occur at levels averaging only 0.08% and even if not corrected for, would cause less than a 4% error in calculating renal mevalonate oxidation. Further, regardless of the minor degree of contamination of commercial ¹⁴C-mevalonate, the kidney oxidized mevalonate to CO₂ at rates averaging 24 times that of the liver. Finally, purified (R)-[5-¹⁴C]mevalonate was shown to yield results virtually identical, both in vitro and in vivo, to those obtained with commercial [5-¹⁴C]mevalonate. These data, therefore, fully confirm our previous conclusion that the kidney represents the primary organ site of mevalonate metabolism by the nonsterol, or shunt, pathway.     

Quantitative assessment of crosstalk between the two isoprenoid biosynthesis pathways in plants by NMR spectroscopy

Plants have been shown to use the mevalonate pathway for the biosynthesis of sterols and triterpenes in the cytoplasm and the recently discovered deoxyxylulose phosphate pathway for the biosynthesis of a variety of hemiterpenes, monoterpenes, diterpenes, as well as for the biosynthesis of carotenoids and the phytol side chain of chlorophyll in plastids. Despite the compartmental separation, at least one terpene precursor can be exchanged between the two pathways. In order to assess quantitatively the crosstalk between the two isoprenoid pathways, [2-¹³C₁]mevalonolactone or [U-¹³C₆]glucose were supplied to cell cultures of Catharanthus roseus grown under illumination or in darkness. Sitosterol, lutein and phytol were isolated and analysed by NMR spectroscopy. The incorporations of exogenous [2-¹³C₁]mevalonolactone were 48% and 7% into the DMAPP and IPP precursors of sitosterol and lutein, respectively. With [U-¹³C₆]glucose as precursor, at least 95% of sitosterol precursors were obtained via the mevalonate pathway, whereas phytol appeared to be biosynthesised via the deoxyxylulose phosphate pathway (approximately 60%) as well via the mevalonate pathway (approximately 40%). The apparent ratios for the contribution of the two pathways depend on the nature of the precursor supplied as well as the nature of the target compound. Thus, crosstalk between the two terpenoid pathways cannot be explained in detail by a simple two compartment model and requires an additional in depth study of complex regulatory mechanisms.     

Synthesis of monoterpene hydrocarbons from [1-3H]linalyl pyrophosphate by carbocyclase from Citrus limonum

A partially purified enzyme preparation from the flavedo of Citrus limonum utilized [1-³H]linalyl pyrophosphate as a substrate for cyclic terpene hydrocarbon formation more efficiently than the pyrophosphates of nerol and geraniol. The products formed from all three substrates are α-pinene, β-pinene, limonene, and γ-terpinene. Neryl and geranyl pyrophosphate inhibit the formation of these products from linalyl pyrophosphate. No free linalyl pyrophosphate could be detected during the enzymatic formation of cyclic terpene hydrocarbons from geranyl pyrophosphate. Mn²⁺ catalyzes the nonenzymatic solvolysis of linalyl pyrophosphate, forming myrcene and ocymenes and no bicyclic hydrocarbons. Linalyl pyrophosphate is a sterically plausible precursor of cyclic hydrocarbons, but the present data support only its role as an alternative substrate and not as an obligatory free intermediate in terpene biosynthesis.     

Metabolism of Linoleic Acid or Mevalonate and 6-Pentyl-α-Pyrone Biosynthesis by Trichoderma Species

The understanding of the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species was achieved by using labelled linoleic acid or mevalonate as a tracer. Incubation of growing cultures of Trichoderma harzianum and T. viride with [U-¹⁴C]linoleic acid or [5-¹⁴C]sodium mevalonate revealed that both fungal strains were able to incorporate these labelled compounds (50 and 15%, respectively). Most intracellular radioactivity was found in the neutral lipid fraction. At the initial time of incubation, the radioactivity from [¹⁴C]linoleic acid was incorporated into 6-pentyl-α-pyrone more rapidly than that from [¹⁴C]mevalonate. No radioactivity incorporation was detected in 6-pentyl-α-pyrone when fungal cultures were incubated with [1-¹⁴C]linoleic acid. These results suggested that β-oxidation of linoleic acid was a probable main step in the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species.     

Phytoene synthase and phytoene dehydrogenase associated with envelope membranes from spinach chloroplasts

Envelope membranes of spinach chloroplasts contain appreciable activities of the carotenogenic enzymes phytoene synthase (formation of phytoene by condensation of two molecules geranylgeranyl pyrophosphate) and phytoene dehydrogenase (formation of lycopene from phytoene), plus a phosphatase activity. These results were obtained by coincubation experiments using isolated envelope membranes and either a phytoene-forming in vitro system (from [1-¹⁴C]isopentenyl pyrophosphate) or [¹⁴C]geranylgeranyl pyrophosphate or a geranylgeranyl-pyrophosphate-forming in vitro system (from [1-¹⁴C]isopentenyl pyrophosphate). Within thylakoids carotenogenic enzymes could not be detected. It is concluded that the chloroplast envelope is at least a principal site of the membrane-bound steps of carotenoid biosynthesis in chloroplasts.Abbreviastions Chlorophyll a_GC Chlorophyll a, esterified with geranylgeraniol - GGPP geranylgeranyl pyrophosphate - HPLC high pressure liquid chromatography - IPP isopentenyl pyrophosphate     

Relationships among the biosyntheses of ubiquinone, carotene, sterols, and triacylglycerols in Zygomycetes

The Zygomycetes Phycomyces blakesleeanus and Blakeslea trispora are actual or potential sources of β-carotene, ergosterol, ubiquinone, edible oil, and other compounds. By feeding [¹⁴C]acetyl-CoA, L-[¹⁴C]leucine, or R-[¹⁴C]mevalonate in the presence of excess unlabeled glucose, we found that ubiquinone (the terpenoid moiety), β-carotene, and triacylglycerols were made from separate pools of all their common intermediates; the pools for ubiquinone and ergosterol were indistinguishable. Fatty acids were not labeled from mevalonate, showing the absence in these fungi of a shunt pathway that would recycle carbon from mevalonate and its products back to central metabolism. The overproduction of carotene in a Phycomyces mutant and in sexually mated cultures of Blakeslea modified the relative use of labeled and unlabeled carbon sources in the production of carotene, but not of the other compounds. We concluded that carotene, ubiquinone, and triacylglycerols are synthesized in separate subcellular compartments, while sterols and ubiquinone are synthesized in the same compartments or in compartments that exchange precursors. Carotene biosynthesis was regulated specifically and not by flow diversion in a branched pathway.     

Biosynthesis of geraniol and nerol and their β-d-glucosides in Perlargonium graveolens and Rosa dilecta

1. 3R-[2-(14)C]Mevalonate was incorporated into geranyl and neryl beta-d-glucosides in petals of Rosa dilecta in up to 10.6% yield, and the terpenoid part was specifically and equivalently labelled in the moieties derived from isopentenyl pyrophosphate and 3,3-dimethylallyl pyrophosphate. A similar labelling pattern, with incorporations of 0.06-0.1% was found for geraniol or nerol formed in leaves of Pelargonium graveolens The former results provide the best available evidence for the mevalonoid route to regular monoterpenes in higher plants. 2. Incorporation studies with 3RS-[2-(14)C,(4R)-4-(3)H(1)]-mevalonate and its (4S)-isomer showed that the pro-4R hydrogen atom of the precursor was retained and the pro-4S hydrogen atom was eliminated in both alcohols and both glucosides. These results suggest that the correlation of retention of the pro-4S hydrogen atom of mevalonate with formation of a cis-substituted double bond, such as has been found in certain higher terpenoids, does not apply to the biosynthesis of monoterpenes. It is proposed that either nerol is derived from isomerization of geraniol or the two alcohols are directly formed by different prenyltransferases. Possible mechanisms for these processes are discussed. 3. The experiments with [(14)C,(3)H]mevalonate also show that in these higher plants, as has been previously found in animal tissue and yeast, the pro-4S hydrogen atom of mevalonate was lost in the conversion of isopentenyl pyrophosphate into 3,3-dimethylallyl pyrophosphate.     

Conversion of geranylgeranyl pyrophosphate to ent-kaurene in enzyme extracts of sonicated chloroplasts

Farnesyl pyrophosphate-[¹⁴C] and geranylgeranyl pyrophosphate-[¹⁴C] were biosynthesized from mevalonic acid-[2-¹⁴C] by cell-free enzyme extracts of pea (Pisum sativum) cotyledons containing MgCl₂, MnCl₂, ATP and AMO-1618. Maximum yields of farnesyl pyrophosphate were obtained after 30 min incubation while geranylgeranyl pyrophosphate was the primary product after 180 min. Biosynthesized geranylgeranyl pyrophosphate-[¹⁴C] served as an efficient substrate for ent-kaurene biosynthesis in reaction mixtures containing cotyledon enzymes when AMO-1618 was omitted. Enzyme extracts from green pea shoot tips and chloroplasts also converted geranylgeranyl pyrophosphate to ent-kaurene in very low yields. Ent-kaurene production from mevalonic acid-[2-¹⁴C] in extracts of pea shoot tips was also enhanced by addition of chloroplast enzymes. This evidence indicates that kaurene synthetase is present in pea chloroplasts and adds to the possibility that some gibberellin biosynthesis may be compartmentalized in those organelles.     

Herbicide clomazone does not inhibit in vitro geranylgeranyl synthesis from mevalonate

Clomazone reduced the chlorophyll and carotenoid contents of spinach (Spinacia oleracea L.), barley (Hordeum vulgare L.), velvetleaf (Abutilon theophrasti Medik.), and soybean (Glycine max L. Merr.) seedlings. The order of species sensitivity was velvetleaf > spinach > barley > soybean. Clomazone (100 micromolar) did not affect the in vitro activities of spinach isopentenyl pyrophosphate isomerase or prenyl transferase. Clomazone also did not affect the synthesis of isopentenyl pyrophosphate from mevalonic acid. Thus, clomazone had no direct in vitro effect on the synthesis of geranylgeranyl pyrophosphate from mevalonic acid. Greening seedlings of both soybean and velvetleaf metabolized clomazone. No qualitative differences in the metabolites were detected between soybean and velvetleaf. Thus, differential metabolism of clomazone to a toxic chemical that inhibits terpenoid synthesis is unlikely. Clomazone has either a mode of action not yet identified or a metabolite that is selective in that it is much more active in sensitive than tolerant species.     

Trichodiene biosynthesis and the stereochemistry of the enzymatic cyclization of farnesyl pyrophosphate

Cyclization of trans,trans-[1-³H₂,12,13-¹⁴C]farnesyl pyrophosphate (2a) by a preparation of trichodiene synthetase isolated from the fungus, Trichothecium roseum, gave trichodiene (5a), which was shown by chemical degradation to retain both tritium atoms of the precursor at C-11. Incubation of 1S-[1-³H,12,13-¹⁴C]farnesyl pyrophosphate (2b) and 1R-[1-³H,12,13-¹⁴C]farnesyl pyrophosphate (2c) with trichodiene synthetase and degradation of the resulting labeled trichodienes, 5b and 5c, established that the displacement of the pyrophosphate moiety from C-1 of the precursor and formation of the new C-C bond in the formation of trichodiene takes place with net retention of configuration. These results are accounted for by an isomerization-cyclization mechanism involving the intermediacy of nerolidyl pyrophosphate (4).     

Biosynthesis of precocenes-I and -II,anti-juvenile hormones

Administration of [2-¹⁴C]-sodium acetate and [2-¹⁴C]-mevalonic acid to Ageratum conyzoides plants has shown that the aromatic moiety of the precocenes is derived from acetate and the other five carbon atoms are of terpenoid origin.     

Sesquiterpene biosynthesis in maritime pine needles

The biosynthesis of sesquiterpene hydrocarbons was studied in maritime pine (Pinus pinaster) needles by incorporation of ¹⁴CO₂, [1-¹⁴C] acetate and [2-¹⁴C] mevalonate. It was shown that the mechanisms of sesquiterpene biosynthesis are different according to the applied tracer. The important role of the acyclic compound, trans-β-farnesene, before cyclisation processes is discussed.     

Metabolism of Mevalonic Acid in Vegetative and Induced Plants of Xanthium strumarium

The metabolism of mevalonic acid in Xanthium strumarium L. Chicago plants was studied to determine how mevalonate was metabolized and whether metabolism was related to induction of flowering. Leaves of vegetative, photoperiodically induced, and chemically inhibited cocklebur plants were supplied with [¹⁴C]mevalonic acid prior to or during a 16-hour inductive dark period. Vegetative, induced, and Tris(2-diethylaminoethyl)phosphate trihydrochloride-treated plants did not differ significantly in the amount of [¹⁴C]mevalonic acid they absorbed, nor in the distribution of radioactivity among the leaf blade (97%), petiole (2.3%), or shoot tip (0.7%). [¹⁴C]Mevalonic acid was rapidly metabolized and transported out of the leaves. Possible metabolites of mevalonate were mevalonic acid phosphates and sterols. No detectable ¹⁴C was found in gibberellins, carotenoids, or the phytol alcohol of chlorophyll. Chemically inhibited plants accumulated ¹⁴C compounds not found in vegetative or induced plants. When ethanol extracts of leaves, petioles, and buds were chromatographed, comparisons of chromatographic patterns did not show significant differences between vegetative and induced treatments.     

Evidence for negative feedback control of cholesterogenesis from mevalonate in liver: Absence in the intestine of guinea pigs fed A 0,5 % cholesterol diet

The in vitro rate of incorporation of [2-¹⁴C]-acetate and [2-¹⁴C]-mevalonate into cholesterol of liver, ileum and caecum was determined in guinea pigs. In control animals, contrary to the situation observed when acetate was used as precursor, the rate of conversion of mevalonate to cholesterol was higher in liver than in intestine. In this latter tissue, the cholesterogenesis varied depending on the portion tested. The distribution of radiolabel derived from mevalonate between esterified and unesterified cholesterol differed among the various tissues. In cholesterol-fed guinea pigs, the plasma, liver, intestine and aorta cholesterol contents increased significantly. In addition, a negative feedback control existed for hepatic cholesterol synthesis for mevalonate and acetate. This control was absent in intestinal tissues.     

Evidence that Isolated Developing Chloroplasts Are Capable of Synthesizing Chlorophyll b from 5-Aminolevulinic Acid

Developing chloroplasts isolated from cucumber (Cucumis sativus L. var Beit Alpha) cotyledons are capable of incorporating [¹⁴C]5-aminolevulinic acid into chlorophyll (Chl) b and Chl a when incubated under photosynthetic illumination. Thin layer chromatography and high pressure liquid chromatography were employed to analyze the pigments. The specific radioactivity in Chl a was over three times higher than that found in Chl b. Both Chl a and b synthesizing activities in organello decayed rapidly at approximately the same rate. We conclude that concomitant synthesis of Chl a/b-binding apoprotein is not required for Chl b synthesis.     

Cholesterol biosynthesis in human lymphocytes,monocytes, and granulocytes

Lymphocytes, monocytes and granulocytes were separated by counter-flow centrifugation from the blood of normal individuals and were incubated in full serum medium or lipid-depleted medium. The monocytes incorporated about five times more [2-¹⁴C]acetate into sterols than did the lymphocytes in full serum medium and approximately twenty times more than the lymphocytes in lipid-depleted medium. The granulocytes were unable to synthesize sterols from either [2-¹⁴C]acetate or [2-¹⁴C]mevalonate, but they were able to use these substrates for the synthesis of squalene and demonstrated approximately a two fold increase in the incorporation of [2-¹⁴C]acetate (but not [2-¹⁴C]mevalonate) into squalene when incubated in the lipid-depleted medium as compared to the full serum medium.