Anne M. Marpeau

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.

General characteristics of Pinus spp. seed fatty acid compositions, and importance of Δ5-olefinic acids in the taxonomy and phylogeny of the genus.

The Δ5-unsaturated polymethylene-interrupted fatty acid (Δ5-UPIFA) contents and profiles of gymnosperm seeds are useful chemometric data for the taxonomy and phylogeny of that division, and these acids may also have some biomedical or nutritional applications. We recapitulate here all data available on pine (Pinus; the largest genus in the family Pinaceae) seed fatty acid (SFA) compositions, including 28 unpublished compositions. This overview encompasses 76 species, subspecies, and varieties, which is approximately one-half of all extant pines officially recognized at these taxon levels. Qualitatively, the SFA from all pine species analyzed so far are identical. The genus Pinus is coherently united—but this qualitative feature can be extended to the whole family Pinaceae—by the presence of Δ5-UPIFA with C18 [taxoleic (5,9–18∶2) and pinolenic (5,9,12–18∶3) acids] and C20 chains [5,11–20∶2, and sciadonic (5,11,14–20∶3) acids]. Not a single pine species was found so far with any of these acids missing. Linoleic acid is almost always, except in a few cases, the prominent SFA, in the range 40–60% of total fatty acids. The second habitual SFA is oleic acid, from 12 to 30%. Exceptions, however, occur, particularly in the Cembroides subsection, where oleic acid reaches ca. 45%, a value higher than that of linoleic acid. α-Linolenic acid, on the other hand, is a minor constituent of pine SFA, almost always less than 1%, but that would reach 2.7% in one species (P. merkusii). The sum of saturated acids [16∶0 (major) and 18∶0 (minor) acids principally] is most often less than 10% of total SFA, and anteiso-17∶0 acid is present in all species in amounts up to 0.3%. Regarding C18 Δ5-UPIFA, taxoleic acid reaches a maximum of 4.5% of total SFA, whereas pinolenic acid varies from 0.1 to 25.3%. The very minor coniferonic (5,9,12,15–18∶4) acid is less than 0.2% in all species. The C20 elongation product of pinolenic acid, bishomo-pinolenic (7,11,14–20∶3) acid, is a frequent though minor SFA constituent (maximum, 0.7%). When considering C20 Δ5-UPIFA, a difference is noted between the subgenera Strobus and Pinus. In the former subgenus, 5,11–20∶2 and sciadonic acids are ≤0.3 and ≤1.9%, respectively, whereas in the latter subgenus, they are most often ≥0.3 and ≥2.0%, respectively. The highest values for 5,11–20∶2 and sciadonic acids are 0.5% (many species) and 7.0% (P. pinaster). The 5,11,14,17–20∶4 (juniperonic) acid is present occasionally in trace amounts. The highest level of total Δ5-UPIFA is 30–31% (P. sylvestris), and the lowest level is 0.6% (P. monophylla). Uniting as well as discriminating features that may complement the knowledge about the taxonomy and phylogeny of pines are emphasized.

Fatty acid composition of pinaceae as taxonomic markers

Following our previous review on Pinus spp. seed fatty acid (FA) compositions, we recapitulate here the seed FA compositions of Larix (larch), Picea (spruce), and Pseudotsuga (Douglas fir) spp. Numerous seed FA compositions not described earlier are included. Approximately 40% of all Picea taxa and one-third of Larix taxa have been analyzed so far for their seed FA compositions. Qualitatively, the seed FA compositions in the three genera studied here are the same as in Pinus spp., including in particular the same Δ5-olefinic acids. However, they display a considerably lower variability in Larix and Picea spp. than in Pinus spp. An assessment of geographical variations in the seed FA composition of P. abies was made, and intraspecific dissimilarities in this species were found to be of considerably smaller amplitude than interspecific dissimilarities among other Picea species. This observation supports the use of seed FA compositions as chemotaxonomic markers, as they practically do not depend on edaphic or climatic conditions. This also shows that Picea spp. are coherently united as a group by their seed FA compositions. This also holds for Larix spp. Despite a close resemblance between Picea and Larix spp. seed FA compositions, principal component analysis indicates that the minor differences in seed FA compositions between the two genera are sufficient to allow a clear-cut individualization of the two genera. In both cases, the main FA is linoleic acid (slightly less than one-half of total FA), followed by pinolenic (5,9,12-18:3) and oleic acids. A maximum of 34% of total Δ5-olefinic acids is reached in L. sibirica seeds, which appears to be the highest value found in Pinaceae seed FA. This apparent limit is discussed in terms of regio- and stereospecific distribution of Δ5-olefinic acids in seed triacylglycerols. Regarding the single species of Pseudotsuga analyzed so far (P. menziesii), its seed FA composition is quite distinct from that of the other two genera, and in particular, it contains 1.2% of 14-methylhexadecanoic (anteiso-17:0) acid. In the three genera studied here, as well as in most Pinus spp., the C18Δ5-olefinic acids (5,9-18:2 and 5,9,12-18:3 acids) are present in considerably higher amounts than the C20Δ5-olefinic acids (5,11-20:2 and 5,11,14-20:3 acids).

Conifer seeds : Oil content and fatty acid composition

The seed oils from twenty-five Conifer species (from four families—Pinaceae, Cupressaceae, Taxodiaceae, and Taxaceae) have been analyzed, and their fatty acid compositions were established by capillary gas-liquid chromatography on two columns with different polarities. The oil content of the seeds varied from less than 1% up to 50%. Conifer seed oils were characterized by the presence of several Δ5-unsaturated polymethylene-interrupted polyunsaturated fatty acids (Δ5-acids) with either 18 (cis-5,cis-9, 18∶2,cis-5,cis-9,cis-12 18∶3, andcis-5,cis-9,cis-12,cis-15 18∶4 acids) or 20 carbon atoms (cis-5,cis-11 20∶2,cis-5,cis-11,cis-14, 20∶3, andcis-5,cis-11,cis-14,cis-17 20∶4 acids). Pinaceae seed oils contained 17–31% of Δ5-acids, mainly with 18 carbon atoms. The 20-carbon acids present were structurally derived from 20∶1n-9 and 20∶2n-6 acids. Pinaceae seed oils were practically devoid of 18∶3n-3 acid and did not contain either Δ5-18∶4 or Δ5-20∶4 acids. Several Pinaceae seeds had a Δ5-acid content higher than 50 mg/g of seed. The only Taxaceae seed oil studied (Taxus baccata) had a fatty acid composition related to those of Pinaceae seed oils. Cupressaceae seed oils differed from Pinaceae seed oils by the absence of Δ5-acids with 18 carbon atoms and high concentrations in 18∶3n-3 acid and in Δ5-acids with 20 carbon atoms (Δ5-20∶3 and Δ5-20∶4 acids). Δ5-18∶4 Acid was present in minute amounts. The highest level of Δ5-20∶4 acid was found inJuniperus communis seed oil, but the best source of Δ5-acids among Cupressaceae wasThuja occidentalis. Taxodiaceae seed oils had more heterogeneous fatty acid compositions, but the distribution of Δ5-acids resembled that found in Cupressaceae seed oils. Except forSciadopytis verticillata, other Taxodiaceae species are not interesting sources of Δ5-acids. The distribution profile of Δ5-acids among different Conifer families appeared to be linked to the occurrence of 18∶3n-3 acid in the seed oils.

Arachidonic, Eicosapentaenoic, and Biosynthetically Related Fatty Acids in the Seed Lipids from a Primitive Gymnosperm, Agathis robusta

The fatty acid composition of the seeds from Agathis robusta, an Australian gymnosperm (Araucariaceae), was determined by a combination of chromatographic and spectrometric techniques. These enabled the identification of small amounts of arachidonic (5,8,11,14–20∶4) and eicosapentaenoic (5,8,11,14,17–20∶5) acid for the first time in the seed oil of a higher plant. They were apparently derived from γ-linolenic (6,9,12–18∶3) and stearidonic (6,9,12,15–18∶4) acids, which were also present, via chain elongation and desaturation, together with other expected biosynthetic intermediates [bis-homo-γ-linolenic (8,11,14–20∶3) and bishomo-stearidonic (8,11,14,17–20∶4) acids]. Also present were a number of C20 fatty acids, known to occur in most gymnosperm families, i.e., 5,11–20∶2, 11,14–20∶2 (bishomo-linoleic), 5,11,14–20∶3 (sciadonic), 11,14,17–20∶3 (bishomo-α-linolenic), and 5,11,14,17–20∶4 (juniperonic) acids. In contrast to most other gymnosperm seed lipids analyzed so far, A. robusta seed lipids did not contain C18 Δ5-desaturated acids [i.e., 5,9–18∶2 (taxoleic), 5,9,12–18∶3 (pinolenic), or 5,9,12,15–18∶4 (coniferonic)]. These structures support the simultaneous existence of Δ6- and Δ5-desaturase activities in A. robusta seeds. The Δ6-ethylenic bond is apparently introduced into C18 polyunsaturated acids, whereas the Δ5-ethylenic bond is introduced into C20 polyunsaturated acids. A general metabolic pathway for the biosynthesis of unsaturated fatty acids in gymnosperm seeds is proposed. When compared to Bryophytes, Pteridophytes (known to contain arachidonic and eicosapentaenoic acids), and species from other gymnosperm families (without such acids), A. robusta appears as an “intermediate,” with the C18 Δ6-desaturase/C18→C20 elongase/C20 Δ5-desaturase system in common with the former subphyla, and the unsaturated C18→C20 elongase/C20 Δ5-desaturase system specific to gymnosperms. The following hypothetical evolutionary sequence for the C18 Δ6/Δ5-desaturase class in gymnosperm seeds is suggested: Δ6 (initial)→Δ6/Δ5 (intermediate)→Δ5 (final).

Chemotaxonomic differentiation of conifer families and genera based on the seed oil fatty acid compositions: multivariate analyses

Abstract The fatty acid compositions of seed oils from 34 conifer species, mainly Pinaceae and secondarily Cupressaceae, have been determined by gas-liquid chromatography of the methyl esters. As noted in earlier studies, these oils were characterized by the presence of several Δ5-olefinic acids, i.e., 5,9-18:2, 5,9,12-18:3, 5,9,12,15-18:4, 5,11-20:2, 5,11,14-20:3, and 5,11,14,17-20:4 acids, in addition to the more common saturated, oleic, linoleic and α-linolenic acids. Based on these fatty acid compositions, and on those established in earlier systematic studies (totalling 82 species), we established a chemotaxonomic grouping of the main conifer families, i.e., of the Pinaceae, Taxodiaceae, Cupressaceae, and Taxaceae. This was achieved using multivariate analyses (principal component analysis and discriminant analysis). The fatty acids that discriminate best in this classification are the 5,11,14,17-20:4, 9,12,15-18:3 and 5,9,12-18:3 acids. Moreover, it was possible to differentiate between several genera of the Pinaceae: Pinus (including Tsuga and Pseudotsuga), Abies, Cedrus, and Picea plus Larix, represented quite distinct groups. Other fatty acids such as oleic, linoleic, and 5,9-18:2 acids were also important for this purpose. The fatty acid compositions, and particularly the Δ5-olefinic acid contents of conifer seed oils, may thus be applied to the chemosystematic distinction among conifer families as well as genera of the Pinaceae.

Taxonomy of Pinus species based on the seed oil fatty acid compositions

Abstract The fatty acid compositions of the seed oils from ten pine species have been established by capillary gas-liquid chromatography of the methyl esters. With regard to either normal fatty acids or Δ5-olefinic acids, the general pattern of fatty acids did not differ from that of other pine seed oils reported previously. The main fatty acid was linoleic (9,12–18:2) acid (44.4–57.1%), followed by either oleic (9–18:1) acid (13.4–24.5%) or pinolenic (5,9,12–18:3) acid (1.5–25.2%). When applying multivariate analyses to the chemometric data (13 variables) of 49 pine species (ca. 40% of the living pine species), it was possible to distinguish between several sections: Pinea, Longifolia, Halepensis, Ponderosa-Banksiana, Sylvestris, and Cembra. The latter section was clearly divided into two sub-groups. A few species that presented a low overall content of Δ5-olefinic acids, and that grow in warm-temperate regions, were isolated from the bulk of other pine species. It is hypothesized that Δ5-olefinic acids might be related to cold-acclimation.

Geographic variability of terpene composition in Abies cephalonica Loudon and Abies species around the Aegean: hypotheses for their possible phylogeny from the Miocene

SummaryCortical oleoresin composition of lateral shoots (mono- and sesquiterpenes) was analysed by gas chromatography on 18 Mediterranean fir populations grown in experimental fields in southern France: 13 Abies cephalonica Loudon provenances and one A. borisii regis Mattfeld provenance from Greece, 2 A. alba Miller provenances from Romania and Bulgaria, 1 Abies bornmuelleriana Mattfeld provenance and 1 A. equi trojani Ascherson and Sintenis provenances from Turkey. Terpenes with the highest between-provenance variability were limonene, β-pinene, longifolene, δ-cadinene and α-humulene. Both similarity in terpene composition of all the firs studied and paleogeographical data demonstrated the possible existence of a unique Eastern Mediterranean tertiary ancestor for present-day Aegean species. The Pliocene marine transgression of the Aegean basin could have caused this geographical disjunction. According to this hypothesis, A. alba would have appeared in the Northern part of the Aegean and then colonized all of Europe while A. bornmuelleriana would have appeared in its Eastern part. Frequent Pleistocene contacts between the two species could have contributed to the individualization of A. equi trojani and given rise to the particular morphology of the south-eastern ecotypes of A. alba. In the western part of the basin, A. cephalonica populations would have appeared. Southern migrations of A. alba during the Ice Age and contact with A. cephalonica in refugia probably led to the development of A. borisii regis hybrid populations. Evidence for an A. alba gene flow can be found in the southernmost A. cephalonica populations.

Δ5-Olefinic acids in the seed lipids from four Ephedra species and their distribution between the α and β positions of triacylglycerols. Characteristics common to coniferophytes and cycadophytes

The fatty acid compositions of the seed lipids from four Ephedra species, E. nevadensis, E. viridis, E. przewalskii, and E. gerardiana (four gymnosperm species belonging to the Cycadophytes), have been established with an emphasis on Δ5-unsaturated polymethylene-interrupted fatty acids (Δ5-UPIFA). Mass spectrometry of the picolinyl ester derivatives allowed characterization of 5,9- and 5,11–18∶2; 5,9,12–18∶3; 5,9,12,15–18∶4; 5,11–20∶2; 5,11,14–20∶3; and 5,11,14,17–20∶4 acids. Δ5-UPIFA with a Δ11-ethylenic bond (mostly C20 acids) were in higher proportions than δ5-UPIFA with a δ9 double bond (exclusively C18 acids) in all species. The total δ5-UPIFA content was 17–31% of the total fatty acids, with 5, 11, 14–20∶3 and 5, 11, 14, 17–20∶4 acids being the principal δ5-UPFIA isomers. The relatively high level of cis-vaccenic (11–18∶1) acid found in Ephedra spp. seeds, the presence of its δ5-desaturation product, 5, 11–18∶2 acid (proposed trivial name: ephedrenic acid), and of its elongation product, 13–20∶1 acid, were previously shown to occur in a single other species, Ginkgo biloba, among the approximately 170 gymnosperm species analyzed so far. Consequently, Ephedraceae and Coniferophytes (including Ginkgoatae), which have evolved separately since the Devonian period (≈300 million yr ago), have kept in common the ability to synthesize C18 and C20 δ5-UPIFA. We postulate the existence of two δ5-desaturases in gymnosperm seeds, one possibly specific for unsaturated acids with a δ9-ethylenic bond, and the other possibly specific for unsaturated acids with a δ11-ethylenic bond. Alternatively, the δ5-desaturases might be specific for the chain length with C18 unsaturated acids on the one hand and C20 unsaturated acids on the other hand. The resulting hypothetical pathways for the biosynthesis of δ5-UPIFA in gymnosperm seeds are only distinguished by the position of 11–18∶1 acid. Moreover, 13C nuclear magnetic resonance spectroscopy of the seed oil from two Ephedra species has shown that δ5-UPIFA are essentially excluded from the internal position of triacylglycerols, a characteristic common to all of the Coniferophytes analyzed so far (more than 30 species), with the possibility of an exclusive esterification at the sn-3 position. This structural feature would also date back to the Devonian period, but might have been lost in those rare angiosperm species containing δ5-UPIFA.

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.