Michel Gleizes

Monoterpene hydrocarbon biosynthesis by isolated leucoplasts of Citrofortunella mitis.

A plastid vesicle preparation isolated from exocarpium of young Citrofortunella mitis (calamondin) fruits was able to synthesise monoterpene hydrocarbons when incubated with isopentenyl pyrophosphate. The electron-microscope comparison between this organelle fraction and the various plastid classes present in the peel tissues has shown the structural identity between these plastid vesicles and the leucoplasts of the epithelial cells lining the secretory pockets. The monoterpene biosynthesis required the presence of dimethylallyl pyrophosphate, Mn2+ or Mg2+ and was increased by addition of 2-mercaptoethanol. Evidence is provided that the leucoplast vesicles act as a complete system in which occur all the successive steps involved in monoterpene hydrocarbon elaboration from isopentenyl pyrophosphate.

Identification des constituants de l'essence des aiguilles de Pinus pinaster

Abstract The chromatographic analysis of the volatile leaf oil of Pinus pinaster Ait. showed 42% of monoterpene hydrocarbons (α-pinene, camphene, β-pinene, myrcene, 3-carene, limonene, cis -ocimene, terpinolene, para -cymene, 35% of sesquiterpene hydrocarbons (cubebene, copaene, caryophyllene, humulene, germacrene D, α- and γ-muurolenes, δ- and γ-cadinenes) and 23% of oxygenated compounds including esters (linalyl, bornyl, geranyl, neryl and farnesyl acetates), alcohols ( cis -hexenol, linalool, α-fenchol, trans -pinocarveol, terpinen-4-ol, α-terpineol, dihydrocarveol, guaiol, junenol and α-cadinol), one aldehyde (hexenal) and one ketone (piperitone). Three non terpenoid phenylethyl esters were also identified: phenylethyl isovalerate, methyl-2 burtyate and 3-3 dimethylacrylate. Some alcohols and mainly α-terpineol and linalool seemed to be formed during the steam distillation process, they were absent when the leaf oil was obtained by maceration of small portions of leaves in the usual solvents of terpenes.

Evidence for a geranyl-diphosphate synthase located within the plastids of Vitis vinifera L. cultivated in vitro

Intact plastids from cell suspensions of Vitis vinifera L. cv. Muscat de Frontignan, free of detectable contamination by other particles as judged by the distribution of organelle-specific marker enzymes and by electron microscopy, exhibit geranyl-diphosphate synthase activity (EC 2.5.1.1). This synthase activity remains stable after tryptic digestion of unlysed organelles and is enhanced by plastid disruption. We conclude that the enzyme is located within the organelle. The possibility of an isopentenyl diphosphate/dimethylallyl diphosphate translocating system which would play a major role in the regulation of monoterpene metabolism is discussed.

In vivo formation of sesquiterpene hydrocarbons in the endoplasmic reticulum of pine

Abstract Memebrane fractions were isolated from an homogenate of primary leaves of maritime pine (Pinus pinaster Ait.) seedlings, labelled in vivo by [1-14C]acetate. The various membranes from the 38 000 × g pellet were isolated by discontinuous density gradient, characterized with enzymic markers and identified by electron microscopy. It is shown that two fractions containing labelled sesquiterpene hydrocarbons were enriched in endoplasmic reticulum. A fraction devoid of radioactivity located between these fractions is very probably enriched in tonoplast.

Isolation by an Aqueous Two-Polymer Phase System and Identification of Endomembranes from Citrofortunella mitis Fruits for Sesquiterpene Hydrocarbon Synthesis

Summary A dextran-polyethylene glycol two-phase system was used to separate an endomembrane fraction involved in sesquiterpene hydrocarbon biosynthesis, from organelles elaborating monoterpene hydrocarbons in the exocarp of CitrofortunelLa mitis (calamondin) fruits. Endomembranes showed a high affinity for the polyethylene glycol-rich upper phase whereas organelles partitioned in the dextran-rich lower phase. The membranes obtained in the polyethylene glycol phase were purified on a sucrose density gradient. A fraction enriched in endoplasmic reticulum, characterized by enzyme markers, was isolated. In the presence of [1- 14 C] isopentenyl pyrophosphate, this fraction was able to synthesize ,β-selinene and germacrene D, the main CIS hydrocarbons present in the oil of calamondin fruits.

Purification and Characterization of Trans-β-farnesene Synthase from Maritime Pine (Pinus pinaster Ait.) Needles

Summary The trans-β-farnesene synthase has been purified from basal and middle parts of growing needles of maritime pine ( Pinus pinaster Ait.). This enzyme catalyses the conversion of farnesyl diphosphate to trans-β-farnesene, an acyclic sesquiterpene hydrocarbon. Its acidic property facilited its isolation by ionexchange chromatography. Its purification was continued on hydroxylapatite column and gel permeation and was greatly improved by dye binding chromatography. SDS-gel electrophoresis revealed a single band in the range of 45 kD for this enzyme which has been 1,030-fold purified. Trans-β-farnesene synthase requires for divalent cations with Mg 2+ preferred to Mn 2+ and for thiol reagents. Effects of inhibitors were examined and the enzyme activity was strongly affected by p-hydroxymercuribenzoic acid. The K m for farnesyl diphosphate was in the range of 5 μM and the optimum pH of activity was between 7-7.3.

Monoterpene formation by leucoplasts of Citrofortunella mitis and Citrus unshiu : Steps and conditions of biosynthesis.

Leucoplasts of immature calamondin and satsuma fruits were incubated with [1-14C] isopentenyl pyrophosphate under various conditions. Optimal incorporation of the tracer into geranyl pyrophosphate and monoterpene hydrocarbons occurred in the presence of exogenous dimethylallyl pyrophosphate and Mn2+ which was more effective than Mg2+. The dependence of dimethylallyl pyrophosphate showed that about 10 moles were required for 1 mole of isopentenyl pyrophosphate for the best recovery in monoterpene hydrocarbon biosynthesis. A time-course incorporation of isopentenyl pyrophosphate revealed that the C10 hydrocarbon elaboration was dependent on the geranyl pyrophosphate production and at no time neryl pyrophosphate was synthesized by leucoplasts. The amount of labelled farnesyl pyrophosphate was rather low whatever the conditions used in the experiments and sesquiterpene hydrocarbon biosynthesis was never observed.

Partial purification and properties of the sesquiterpene β-selinene cyclase from Citrofortunella mitis fruits

Abstract From a supernatant fraction of the peripheral flavedo of immature calamondin ( Citrofortunella mitis ) fruits were extracted by sesquiterpene cyclase catalyzing the conversion of farnesyl diphosphate to β-selinene. This enzyme was partially purified by ion exchange chromatography and its properties were investigated. The molecular weight was estimated to be in the range of 67 000 by gel filtration. The K m for the substrate farnesyl diphosphate was found to be 45 μM. This protein exhibited an isoelectric point of 6.0. It required the presence of divalent metal cations and Mg 2+ was preferred to Mn 2+ .

Role of acyclic compounds in monoterpene biosynthesis in Pinus pinaster

Abstract The biosynthesis of monoterpene hydrocarbons was studied in maritime pine needles by incorporation of 14CO2. It was shown that the acyclic terpenes β-myrcene and trans-β-ocimene, act as transitory compounds before the biosynthesis of cyclic monoterpenes such as α- and β-pinene. The mechanisms of biosynthesis are genetically controlled.

Effects of wounding on the terpene content of twigs of maritime pine (Pinus pinaster Ait.)

SummaryAfter wounding the cortical tissues of twigs of Pinus pinaster, we investigated changes in diterpene resin acid content and the ultrastructural modification of secretory structures. There was an increase in total resin acid content in the cortical and woody tissues located near the wound. Not all resin acids responded in the same way, but in wounded tissues the amounts of isopimaric acid and of the group of dehydroabietic, levopimaric and palustric acids significantly increased. The composition of cortical tissues becomes closely related to that of woody tissues. The resin acid enrichment of cortical tissues is correlated with the reactivation of the epithelial cells of the resin ducts and the de novo synthesis of resin acids demonstrated by labelling with 14CO2.