Polar lipid and fatty acid distribution in carotenolipoprotein complexes extracted from sea buckthorn fruits.

Sea buckthorn (Hippophae rhamnoides L., fam. Elaeagnaceae) fruits are rich in pigments and lipoproteins located in membranes and the fleshy mesocarp. In spite of many reports concerning the neutral lipids in the mesocarp, no data about the polar lipids and their fatty acid composition are available even though they play important structural and physiological roles in cell membranes and may offer interesting applications as emulsifiers and nutrients in cosmetic preparations. Carotenolipoprotein complexes are located particularly in fruit membranes where polar lipids may function as bridge compounds between the polar (protein) and non-polar (carotenoid) moieties. The fatty acid compositions of total and individual polar lipids separated from carotenolipoprotein complexes were determined by HPTLC and GC. The polar lipids included 61% phospholipids and 39% galactolipids, which contained mainly 16:0, 16:1 (9c), 18:1 (9c), 18:1 (11c) and 18:2 (9c, 12c) fatty acids. Almost all polar lipids showed high ratios of 16:0/16:1 (11c) and 18:1 (9c)/18:1 (11c), and higher quantities of 18 carbon unsaturated fatty acids than of the saturated analogue. Galactolipids proved to be richest in 18:1 (9c) and 18:3 (9c, 12c, 15c) fatty acids, while phospholipids contained higher concentrations of 16:0 and 18:1 (9c).

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 delta5-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 delta5-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 delta5-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 C18 delta5-olefinic acids (5,9-18:2 and 5,9,12-18:3 acids) are present in considerably higher amounts than the C20 delta5-olefinic acids (5,11-20:2 and 5,11,14-20:3 acids).

General characteristics of Pinus spp. seed fatty acid compositions, and importance of delta5-olefinic acids in the taxonomy and phylogeny of the genus.

The delta5-unsaturated polymethylene-interrupted fatty acid (delta5-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 delta5-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. Alpha-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 delta5-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 delta5-UPIFA, a difference is noted between the subgenera Strobus and Pinus. In the former subgenus, 5,11-20:2 and sciadonic acids are < or =0.3 and < or =1.9%, respectively, whereas in the latter subgenus, they are most often > or =0.3 and > or =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 delta5-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.

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) acids for the first time in the seed oil of a higher plant. They were apparently derived from gamma-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-gamma-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-alpha-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 delta5-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 delta6- and delta5-desaturase activities in A. robusta seeds. The delta6-ethylenic bond is apparently introduced into C18 polyunsaturated acids, whereas the delta5-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 delta6-desaturase/C18-->C20 elongase/C20 delta5-desaturase system in common with the former subphyla, and the unsaturated C18-->C20 elongase/C20 delta5-desaturase system specific to gymnosperms. The following hypothetical evolutionary sequence for the C18 delta6/delta5-desaturase class in gymnosperm seeds is suggested: delta6 (initial)-->delta6/delta5 (intermediate)-->delta5 (final).

Delta5-olefinic acids in the seed lipids from four Ephedra species and their distribution between the alpha and beta 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 delta5-unsaturated polymethylene-interrupted fatty acids (delta5-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. Delta5-UPIFA with a delta11-ethylenic bond (mostly C20 acids) were in higher proportions than delta5-UPIFA with a delta9 double bond (exclusively C18 acids) in all species. The total delta5-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 delta5-UPIFA isomers. The relatively high level of cis-vaccenic (11-18:1) acid found in Ephedra spp. seeds, the presence of its delta5-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 (approximately 300 million yr ago), have kept in common the ability to synthesize C18 and C20 delta5-UPIFA. We postulate the existence of two delta5-desaturases in gymnosperm seeds, one possibly specific for unsaturated acids with a delta9-ethylenic bond, and the other possibly specific for unsaturated acids with a delta11-ethylenic bond. Alternatively, the delta5-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 delta5-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 delta5-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 delta5-UPIFA.

Genetic determinism of δ3-carene in maritime pine using RAPD markers.

An F2 progeny of maritime pine (Pinus pinaster Aïton) was used to investigate the mode of inheritance of δ3-carene using a quantitative and a qualitative approach. A previously reported genetic map constructed with random amplified polymorphic DNA (RAPD) markers made it possible to locate one major quantitative trait locus (QTL) accounting for most of the phenotypic variation of this trait on linkage group 5. In the qualitative approach, the "C" locus that controls the relative quantity of δ3-carene (C+ for the richness allele and C− for the poorness allele) was found to be strongly associated with RAPD markers in the same genomic region of linkage group 5. The colocation between the QTL and the "C" locus suggests that a major gene or closely linked loci affect the variation in δ3-carene expression. Key words : Pinus pinaster, terpenes, QTL, RAPD, linkage analysis.

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-(14)C] 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 Mn(2+) which was more effective than Mg(2+). 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.

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, Mn(2+) or Mg(2+) 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.