Manuel Mancha

In vitro oleate desaturase in developing sunflower seeds

Abstract The in vitro oleoyl phosphatidylcholine desaturase (ODS) activity in developing seeds of normal and high oleic sunflower lines has been studied. Lysophosphatidylcholine and sorbitol stimulated the enzyme activity, while mg2+ was an absolute requirement. The microsomal fraction was the main site of ODS localization. The oil bodies fraction also retained some activity which could be removed after sonication. In the seeds of the normal genotype the microsomal ODS showed good activity throughout the period of active lipid synthesis. On the contrary, in the high oleic seeds this activity was lost at the beginning of this period. The microsomal ODS was strongly affected by incubation temperature and showed maximal activity at 20°, suggesting that sunflower seeds could be a good model system to study temperature control of ODS enzyme activity.

Sunflower mutant containing high levels of palmitic acid in high oleic background

A new sunflower mutant, CAS-12, was obtained, which has both high palmitic (≈30%) and high oleic acid contents, and also a substantial amount of palmitoleic acid (≈7%). The mutant was selected after X-ray irradiation of dry seeds of the inbred line BSD-2-423, which had normal palmitic (≈3%) and high oleic (≈88%) acid levels. The increase of palmitic and palmitoleic acids occurred at the expense of the oleic acid content, which decreased to around 55% in respect to the original line. Linoleic acid content is always under 5%. Palmitic and palmitoleic acid levels were similar to those of the high palmitic mutant CAS-5 obtained in a previous programme from a low oleic line isogenic to BSD-2-423 using a similar mutagenic treatment. In that previous programme we also selected three high stearic acid mutants using chemical mutagenic treatment on the same sunflower line (RDF-1-532). We attempted to obtain mutants in other lines but were unsuccessful. The isolation of similar mutants in isogenic parental lines illustrates the importance of the genetic background in the development of specific mutants with an altered seed oil fatty acid composition. The oil of this mutant will increase the range of potential uses of sunflower oil.

Temperature regulation of oleate desaturase in sunflower (Helianthus annuus L.) seeds

The effect of temperature on oleate desaturation in developing sunflower (Helianthus annuus L.) seeds has been examined. When seeds from plants grown at low (20/10° C, day/night) temperature were transferred for 24 h to 10° C, an increase in the linoleate/oleate ratio in phosphatidylcholine and triacylglycerol was observed, but not when transfer was to 20 or 30° C. The same effect was observed in triacylglycerol, phosphatidylcholine and phosphatidylethanolamine in the newly synthesized lipids after in-vivo incubation with [1-14C]oleate at 10° C. The microsomal oleoyl phosphatidylcholine desaturase (ODS) activity of the seeds maintained at 10† C was also enhanced. The stimulation was observed after only 3 h in plants grown at high temperature (30/20° C). This effect was inhibited by cycloheximide, implying that the low-temperature stimulation of the ODS activity was caused by the synthesis of new enzyme. As a consequence, seeds from plants grown at low temperature had higher ODS activities and linoleate contents than those grown at high temperature. The microsomal ODS activity of seeds from plants grown at low temperature was dependent on incubation temperature and showed a maximum at 20° C. By contrast, this activity was almost temperature-insensitive in seeds from plants grown at high temperature. These results could explain how temperature regulates the fatty-acid composition in sunflower-seed lipids.

Genetic analysis of the high oleic acid content in cultivated sunflower (Helianthus annuus L.)

SummarySunflower lines breeding true for very high oleic acid content in their oil (average levels higher than 85%) were crossed with standard sunflower lines with mean oleic acid levels of 30%. Analysis of the oil of F1 seeds indicated dominance for high oleic levels and control of the genotype of the embryo. Segregating generations were obtained selfing heterozygous high oleic BCnF1 plants from several generations of a backcrossing program to incorporate the high oleic character to standard inbred lines and testcrossing these plants to low oleic material. Analysis of F2 and testcrossed seeds showed three kind of segregations, in both F2 and testcrossed populations, with different proportions of low, intermediate and high oleic types. Genetic analysis of these data supported the hypothesis, that the high oleic character is controlled by three dominant complementary genes OL1, OL2 and OL3. Additional data showing F1 seeds with intermediate oleic content and segregations for high oleic in progenies of intermediate types, suggest the presence of major factors modifying high oleic acid content.

Lipid characterization in seeds of a high oleic acid sunflower mutant

Abstract The expression of the ‘high oleic’ character in a sunflower mutant takes place exclusively in the developing seeds, during the synthesis of reserve lipids. The fatty acid composition of different lipid classes was similar in each genotype and depended on growth temperature. In the mutant seeds triacylglycerols contained up to 90% of oleic acid and less than 0.5% of linoleic acid. The content of oleic acid was 2.5 ± 0.7 mg/g of fresh seed all over the developing period.

Effect of different environmental stresses on the expression of oleate desaturase genes and fatty acid composition in olive fruit.

The regulation of microsomal and plastidial oleate desaturases by low and high temperature, darkness, and wounding was investigated. To this end, their gene expression levels and the fatty acid composition was determined in the mesocarp tissue of olive fruit from the Picual and Arbequina varieties subjected to the corresponding stress treatments. Firstly, a plastidial oleate desaturase from olive was cloned and its functional identity was confirmed by overexpression in Escherichia coli. The results showed that temperature and light regulate olive oleate desaturase genes at transcriptional level. However, no correlation between their expression levels and the linoleic acid content in microsomal and plastidial lipids was found. In addition, the involvement of microsomal but not plastidial oleate desaturases in the wounding response of olive fruit mesocarp is demonstrated. The fatty acid analysis revealed the appearance of palmitolinoleic acid only in microsomal lipids, reaching a maximum 3h after wounding.

Oleate desaturation in seeds of two genotypes of sunflower

Abstract In vivo oleate incorporation and desaturation in developing seeds of normal and the high oleic acid mutant of sunflower have been studied. In seeds less than 15 days after flowering (DAF) of both genotypes, incorporation and desaturation was similar and took place mainly in polar lipids. Seeds 15–35 DAF incorporated fatty acids preferently into triacylglycerols. During this period mutant seeds lacked oleate desaturation capacity but it was recovered after the cotyledon started a special process of differentiation.

Oleate desaturation and acyl turnover in sunflower (Helianthus annuus L.) seed lipids during rapid temperature adaptation

Abstract. In-vivo experiments with developing sunflower (Helianthus annuus L.) seeds demonstrated that oleate desaturase activity was stimulated by low temperature (10 °C), repressed by high temperature (30 °C) and rapidly restored by returning the seeds to low temperature. Within time periods of 2–4 h, in which the de-novo fatty acid synthesis was negligible, the percentages of oleate (18:1) and linoleate (18:2) were modified in the seed lipids as a consequence of temperature adaptation. When the seeds were transferred to low temperature, the 18:2 content increased in all lipids from both microsomal membranes and oil bodies. After shifting to high temperature, the overall 18:2 content remained constant, but the 18:2 content decreased in diacylglycerols, phosphatidylcholine (PC) and other polar lipids of the two fractions and also in triacylglycerols (TAGs) of the microsomes but increased in TAGs of the oil bodies. The results indicate that the mechanism for the rapid adaptation of sunflower seeds to temperature changes involves (i) the synthesis or activation of oleate desaturase at low temperature and the reversible inhibition of this enzyme at high temperature and (ii) the exchange of 18:1 and 18:2 between TAGs and PC. Under both low and high temperature, 18:1 is transferred from reserve TAGs to PC and 18:2 is transferred from PC to reserve TAGs. At low temperature, 18:1 is desaturated to 18:2 thus allowing the enrichment of membrane lipids with 18:2, the excess being stored in reserve TAGs. At high temperature, however, and provided that oleate desaturase is repressed, the membrane lipids become enriched in 18:1 and the oil-body TAGs become enriched in 18:2.

The utilization and desaturation of oleate and linoleate during glycerolipid biosynthesis in olive (Olea europaea L.) callus cultures

Callus cultures from olive (Olea europaea L.) were used to study characteristics of desaturation in this oil-rich tissue. The incorporation of [1-14C]oleate and [1-14C]linoleate into complex lipids and their further desaturation was followed in incubations of up to 48 h. Both radiolabelled fatty acids were rapidly incorporated into lipids, especially phosphatidylcholine and triacylglycerol. Radiolabelling of these two lipids peaked after 1–4 h, after which it fell. In contrast, other phosphoglycerides and the galactosylglycerides were labelled in a more sustained manner. [1-14C]Linoleate was almost exclusively found in the galactolipids. With [1-14C]linoleate as a precursor, the only significant desaturation to linolenate was in the galactolipids. Monogalactosyldiacylglycerol was the first lipid in which [1-14C]linoleate and [1-14C]linolenate appeared after incubation of the calli with [1-14C]oleate and [1-14C]linoleate, respectively. The presence of radioactivity in the plastidial lipids shows that both [1-14C]oleate and [1-14C]linoleate can freely enter the chloroplast. Two important environmental effects were also examined. Raised incubation temperatures (30–35 °C) reduced oleate desaturation and this was also reflected in the endogenous fatty acid composition. Low light also caused less oleate desaturation. The data indicate that lysophosphatidylcholine acyltransferase is important for the entry of oleate and linoleate into olive callus lipid metabolism and phospholipid:diacylglycerol acyltransferase may be involved in triacylglycerol biosynthesis. In addition, it is shown that plastid desaturases are mainly responsible for the production of polyunsaturated fatty acids. Individual fatty acid desaturases were differently susceptible to environmental stresses with FAD2 being reduced by both high temperature and low light, whereas FAD7 was only affected by high temperature.

Oleate from triacylglycerols is desaturated in cold-induced developing sunflower (Helianthus annuus L.) seeds

For the first time, an active fatty-acid metabolism is indicated for triacylglycerols (TAG) of developing sunflower (Helianthus annuus L.) seeds. When the developing seeds were transferred to low temperature, the total amount of oleate found in TAG decreased as that of linoleate increased, while the contents of total lipids and TAG remained unchanged. These results suggest that oleate from TAG was used for desaturation. This occurred first in microsomal TAG, but after a long cold period it was observed mainly in the oil-body fraction. Thesn-2 position of TAG was preferentially enriched in linoleate. Apparently, more linoleate than necesary for the maintenance of membrane fluidity was synthesized at the expense of TAG oleate.