Developing Sunflower Seeds Research Articles

Identification and integrated analysis of mRNAs, lncRNAs, and microRNAs of developing seeds in high oleic acid sunflower (Helianthus annuus L.)

High unsaturated fatty acid content of sunflower seed is desirable for human-healthy high oleic acid oil production. Knowledge of lipid metabolic regulatory mechanisms is key to improving oil content and quality. This study is expected to explore regulatory mechanisms of lipid metabolism in high oleic acid seeds. First, we analyzed the oil contents and detected the fatty acid compositions in developing sunflower seeds with high oleic acid content. Next, high-throughput sequencing (HTS) of seed RNA samples from three key seed developmental stages relevant to oil content and quality was performed. Pairwise comparisons of differentially expressed mRNAs, long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) yielded respective differentially expressed transcripts as follows: 52293, 120625, 95 (L7d vs L22d); 57398, 123485, 127 (L7 vs L37d); 15417, 9554, 77 (L22d vs L37d). 15652 novel mRNAs, 123888 novel lncRNAs, and 98 novel miRNAs were identified, which enriched the RNA library of sunflower. Furthermore, gene functions were predicted using GO and KEGG analyses. These prediction analysis revealed that differentially expressed ncRNAs and mRNAs were mainly included in fatty acid metabolism and oil accumulation. Moreover, 9072 pairs of competing endogenous RNA (ceRNA) relationships comprised of interacting lncRNAs, miRNAs, and mRNAs. Pathway network analysis of differentially expressed lipid metabolism-associated ceRNAs revealed several important enzymes including glycerol-3-phosphate O-acyltransferase, linoleate 9S-lipoxygenase, acyl-CoA oxidase, and others that were enriched in fatty acid synthesis and oil accumulation, which highlighted competitive interactions in lipid metabolism. This study will elucidate potential regulatory mechanisms of lipid metabolism in sunflower seeds.

Genome-Wide Mapping of Histone H3 Lysine 4 Trimethylation (H3K4me3) and Its Involvement in Fatty Acid Biosynthesis in Sunflower Developing Seeds.

Histone modifications are of paramount importance during plant development. Investigating chromatin remodeling in developing oilseeds sheds light on the molecular mechanisms controlling fatty acid metabolism and facilitates the identification of new functional regions in oil crop genomes. The present study characterizes the epigenetic modifications H3K4me3 in relationship with the expression of fatty acid-related genes and transcription factors in developing sunflower seeds. Two master transcriptional regulators identified in this analysis, VIV1 (homologous to Arabidopsis ABI3) and FUS3, cooperate in the regulation of WRINKLED 1, a transcriptional factor regulating glycolysis, and fatty acid synthesis in developing oilseeds.

Molecular and biochemical characterization of the sunflower (Helianthus annuus L.) cytosolic and plastidial enolases in relation to seed development

In the present study, we describe the molecular and biochemical characterization of sunflower (Helianthus annuus L.) enolase (ENO, EC 4.2.1.11) proteins, which catalyze the formation of phosphoenolpyruvate, the penultimate intermediate in the glycolytic pathway. We cloned and characterized three cDNAs encoding different ENO isoforms from developing sunflower seeds. Studies using fluorescently tagged ENOs confirmed the predicted subcellular localization of ENO isoforms: HaENO1 in the plastid while HaENO2 and HaENO3 were found in the cytosol. The cDNAs were used to express the corresponding 6(His)-tagged proteins in Escherichia coli. The proteins were purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Recombinant HaENO1 and HaENO2, but not HaENO3 were shown to have enolase activity, in agreement with data obtained with the Arabidopsis homolog proteins. Site directed mutagenesis of several critical amino acids was used to attempt to recover enolase activity in recombinant HaENO3, resulting in very small increases that were not additive. A kinetic characterization of the two active isoforms showed that pH had similar effect on their velocity, that they had similar affinity for 2-phosphoglycerate, but that the kcat/Km of the plastidial enzyme was higher than that of the cytosolic isoform. Even though HaENO2 was always the most highly expressed transcript, the levels of expression of the three ENO genes were remarkably distinct in all the vegetative and reproductive tissues studied. This indicates that in seeds the conversion of 2-phosphoglycerate to phosphoenolpyruvate takes place through the cytosolic and the plastidial pathways therefore both routes could contribute to the supply of carbon for lipid synthesis. The identity of the main source of carbon during the period of stored products synthesis is discussed.

Acyl carrier proteins from sunflower (Helianthus annuus L.) seeds and their influence on FatA and FatB acyl-ACP thioesterase activities.

The kinetics of acyl-ACP thioesterases from sunflower importantly changed when endogenous ACPs were used. Sunflower FatB was much more specific towards saturated acyl-ACPs when assayed with them. Acyl carrier proteins (ACPs) are small (~9kDa), soluble, acidic proteins involved in fatty acid synthesis in plants and bacteria. ACPs bind to fatty acids through a thioester bond, generating the acyl-ACP lipoproteins that are substrates for fatty acid synthase (FAS) complexes, and that are required for fatty acid chain elongation, acting as important intermediates in de novo fatty acid synthesis in plants. Plants, usually express several ACP isoforms with distinct functionalities. We report here the cloning of three ACPs from developing sunflower seeds: HaACP1, HaACP2, and HaACP3. These proteins were plastidial ACPs expressed strongly in seeds, and as such they are probably involved in the synthesis of sunflower oil. The recombinant sunflower ACPs were expressed in bacteria but they were lethal to the prokaryote host. Thus, they were finally produced using the GST gene fusion system, which allowed the apo-enzyme to be produced and later activated to the holo form. Radiolabelled acyl-ACPs from the newly cloned holo-ACP forms were also synthesized and used to characterize the activity of recombinant sunflower FatA and FatB thioesterases, important enzymes in plant fatty acids synthesis. The activity of these enzymes changed significantly when the endogenous ACPs were used. Thus, FatA importantly increased its activity levels, whereas FatB displayed a different specificity profile, with much high activity levels towards saturated acyl-CoA derivatives. All these data pointed to an important influence of the ACP moieties on the activity of enzymes involved in lipid synthesis.

Glycolytic enzymatic activities in developing seeds involved in the differences between standard and low oil content sunflowers (Helianthus annuus L.)

As opposed to other oilseeds, developing sunflower seeds do not accumulate starch initially. They rely on the sucrose that comes from the mother plant to synthesise lipid precursors. Glycolysis is the principal source of carbon skeletons and reducing power for lipid biosynthesis. In this work, glycolytic initial metabolites and enzyme activities from developing seed of two different sunflower lines, of high and low oil content, were compared during storage lipid synthesis. These two lines showed different kinetic lipid accumulation in the developing embryos. Fatty acids levels during the initial and final stage of lipid synthesis were higher in CAS-6 than in ZEN-8. The analysis of the photosynthate and sugars content suggests that, although the hexoses levels were quite similar in both lines, the amount of sucrose produced by the mother plant and available for lipid synthesis was higher in CAS-6. Although, a smaller amount of sucrose is available in the ZEN-8 line, its seeds maintain the levels of intermediate sugars in the initial steps of glycolysis due to an increase in the levels of the invertase, hexokinase and phosphoglucose isomerase activities in ZEN-8, with respect to CAS-6. Also, a readjustment in the final part of this metabolic route took place, with the activities of phosphoglycerate kinase and enolase in CAS-6 being higher, allowing increased synthesis of phosphoenolpiruvate, the intermediate carbon donor for fatty acid synthesis. In addition, recently, it has been shown that Arabidopsis mutants with a lower fat content in their seeds have a higher amount of sucrose. These data together point to these last two enzymatic activities, phosphoglycerate kinase and enolase, as being responsible for the lower fat content in the ZEN-8 line.

Cloning, biochemical characterisation, tissue localisation and possible post-translational regulatory mechanism of the cytosolic phosphoglucose isomerase from developing sunflower seeds

Lipid biosynthesis in developing sunflower (Helianthus annuus L.) seeds requires reducing power. One of the main sources of cellular NADPH is the oxidative pentose phosphate pathway (OPPP), generated from the oxidation of glucose-6-phosphate. This glycolytic intermediate, which can be imported to the plastid and enter in the OPPP, is the substrate and product of cytosolic phosphoglucose isomerase (cPGI, EC 5.3.1.9). In this report, we describe the cloning of a full-length cDNA encoding cPGI from developing sunflower seeds. The sequence was predicted to code for a protein of 566 residues characterised by the presence of two sugar isomerase domains. This cDNA was heterologously expressed in Escherichia coli as a His-tagged protein. The recombinant protein was purified using immobilised metal ion affinity chromatography and biochemically characterised. The enzyme had a specific activity of 1,436 micromol min(-1) mg(-1) and 1,011 micromol min(-1) mg(-1) protein when the reaction was initiated with glucose-6-phosphate and fructose-6-phosphate, respectively. Activity was not affected by erythrose-4-phosphate, but was inhibited by 6-P gluconate and glyceraldehyde-3-phosphate. A polyclonal immune serum was raised against the purified enzyme, allowing the study of protein levels during the period of active lipid synthesis in seeds. These results were compared with PGI activity profiles and mRNA expression levels obtained from Q-PCR studies. Our results point to the existence of a possible post-translational regulatory mechanism during seed development. Immunolocalisation of the protein in seed tissues further indicated that cPGI is highly expressed in the procambial ring.

Characterization of glycolytic initial metabolites and enzyme activities in developing sunflower (Helianthus annuus L.) seeds

Unlike other oilseeds (e.g. Arabidopsis), developing sunflower seeds do not accumulate a lot of starch and they rely on the sucrose that comes from the mother plant to synthesise lipid precursors. Between 10 and 25days after flowering (DAF), when sunflower seeds form and complete the main period of storage lipid synthesis, the sucrose content of seeds is relatively constant. By contrast, the glucose and fructose content falls from day 20 after flowering and it is always lower than that of sucrose, with glucose being the minor sugar at the end of the seed formation. By studying the apparent kinetic parameters and the activity of glycolytic enzymes in vitro, it is evident that all the components of the glycolytic pathway are present in the crude seed extract. However, in isolated plastids important enzymatic activities are missing, such as the glyceraldehyde-3-phosphate dehydrogenase, involved in the conversion of glyceraldehyde 3-phosphate into 1,3-biphospho-glycerate, or the enolase that converts 2-phosphoglycerate into phosphoenolpyruvate. Hence, phosphoenolpyruvate or one of its derivatives, like pyruvate and malate from the cytosol, may be the primary carbon sources for lipid biosynthesis. Accordingly, the glucose-6-P imported into the plastid is likely to be used in the pentose phosphate pathway to produce the reducing power for lipid biosynthesis in the form of NADPH. Data from crude seed extracts indicate that enolase activity increased during seed formation, from 16days after flowering, and that this activity was well correlated with the period of storage lipid synthesis. In addition, while the presence of some glycolytic enzymes increased during lipid synthesis, others decreased, remained constant, or displayed irregular temporal behaviour.

Temperature-dependent endogenous oxygen concentration regulates microsomal oleate desaturase in developing sunflower seeds

Oleoyl-phosphatidylcholine desaturase (FAD2) is a key enzyme involved in fatty acid desaturation in oilseeds, which is affected by environmental temperature. The results of this study show that FAD2 is regulated in vivo via temperature-dependent endogenous oxygen concentrations in developing sunflower (Helianthus annuus L.) seeds. By combining in vivo oxygen profiling, in situ hybridization of FAD2 genes, an assay of energy status, fatty acid analysis, and an in vitro FAD2 enzyme activity assay, it is shown that: (i) the oil-storing embryo is characterized by a very low oxygen level that is developmentally regulated. Oxygen supply is mainly limited by the thin seed coat. (ii) Elevations of external oxygen supply raised the energy status of seed and produced a dramatic increase of the FAD2 enzyme activity as well as the linoleic acid content. (iii) A clear negative correlation exists between temperature and internal oxygen concentration. The changes occurred almost instantly and the effect was fully reversible. The results indicate that the internal oxygen level acts as a key regulator for the activity of the FAD2 enzyme. It is concluded that a major mechanism by which temperature modifies the unsaturation degree of the sunflower oil is through its effect on dissolved oxygen levels in the developing seed.

Regulation of polyunsaturated fatty acids accumulation and characterization of linolenic acid after germination of sunflower seed

The metabolism of triacylglycerols (TAG) and their conversion into new triglycerides and polar lipids such as linolenic acid take place during the first phase of development of Helianthus annuus L. seed after seeding. Oleate desaturase and linoleate desaturase are two major enzymes responsible for the synthesis of these compounds. To determine whether enzymatic mechanisms function the same in catabolizing seed store lipids of normal and mutant sunflower lines, modifications of fatty acids composition of seeds and the initial stages of growth (seeding-VE) cultivated under conditions of controlled temperature were assessed. The increase of linoleic acid content during all stages of development in a mutant line revealed a higher activity of oleic acid desaturase (Δ12-desaturase) in transforming oleic acid to linoleic acid probably due to higher availability of the substrate of this enzyme in this line; however, its amount increased moderately in a normal line. The activity of FAD2-3 in developing sunflower seeds is recognized even two days after seeding (DAS) in both lines; however, linolenic acid accumulation starts seven DAS.

Increase of the Stearic Acid Content in High-Oleic Sunflower (Helianthus annuus) Seeds

We have performed an "in vivo" study of storage lipid synthesis in developing sunflower seeds, from several high-oleic genetic backgrounds, using radioactive acetate in conjunction with methyl viologen as an inhibitor of the stearoyl-ACP desaturase. As such, some backgrounds showed stronger acyl-ACP thioesterase activity on stearoyl-ACP. We have developed a saturation coefficient that quantifies stearoyl-ACP thioesterase activity among sunflower lines based on their ability to synthesize saturated fatty acids under conditions when the competing stearoyl-ACP desaturase is inhibited by methyl viologen. The saturation coefficient is defined as the ratio of sum of the stearic, araquidic, and behenic saturated fatty acid contents to the unsaturated fatty acid content. On the basis of this coefficient, we were able to select high-oleic lines that, when crossed with the high-stearic CAS-3 line, developed progeny with high-stearic content on a high-oleic background. This approach has enabled us to identify lines with a combination of alleles that synthesized oils with more stearic acid in a high-oleic background, 21% stearic and 62% oleic contents. In contrast, lines with a lower index produced progeny that contained less stearic acid, similar to those obtained previously, that were 13% stearic acid content in high-oleic background. This method could also be used for other metabolic pathways where the blockage of a principal pathway may activate a secondary pathway. However, it should be emphasized that although the stearic acid content could be augmented it was not possible to break the association or the epistatic relationship that exists between the genes that permit a high-stearic phenotype and those that determine a high-oleic background.

Changes in Lipid Status and Glass Properties in Cotyledons of Developing Sunflower Seeds

Biochemical events involved in the acquisition of germinability and storability during orthodox seed development are well documented; however, the roles played by the physical organization of lipids and water are poorly characterized. The aim of this work was to determine, using a thermodynamic approach, whether changes in thermal properties of lipid reserves, and intracellular glasses might play a role in sunflower (Helianthus annuus L.) seed development. Triacyglycerols (TAGs) accumulated in cotyledons until the end of seed filling, which occurred 42 days after anthesis (DAA). Further seed development, leading to mature seed at 58 DAA, was mainly associated with an enlargement of lipid bodies without significant changes either in the lipid content or in their composition. When cooled to -100 degrees C, lipid reserves from cotyledons of mature seeds displayed alpha and beta' polymorphic crystalline structures; however, the ability to form alpha crystals, which was an indicator of lipid purity, progressively appeared during seed development. Characteristics of lipid melting confirmed that seed maturation drying was associated with changes in TAG physical organization. Cotyledon development was associated with an increase in the temperature of glass to rubber transition (Tg), thus suggesting a decrease in molecular mobility during maturation drying. This phenomenon was concomitant with an increase in raffinose content. Our results demonstrate that physical characteristics of lipid reserves and glasses of sunflower cotyledons are developmentally regulated and might play a role in acquisition of seed germinability and storability.

Inhibitors of fatty acid biosynthesis in sunflower seeds

During de novo fatty acid synthesis in sunflower seeds, saturated fatty acid production is influenced by the competition between the enzymes of the principal pathways and the saturated acyl-ACP thioesterases. Genetic backgrounds with more efficient saturated acyl-ACP thioesterase alleles only express their phenotypic effects when the alleles for the enzymes in the main pathway are less efficient. For this reason, we studied the incorporation of [2-(14)C]acetate into the lipids of developing sunflower seeds (Helianthus annuus L.) from several mutant lines in vivo. The labelling of different triacylglycerol fatty acids in different oilseed mutants reflects the fatty acid composition of the seed and supports the channelling theory of fatty acid biosynthesis. Incubation with methyl viologen diminished the conversion of stearoyl-ACP to oleoyl-ACP in vivo through a decrease in the available reductant power. In turn, this led to the accumulation of stearoyl-ACP to the levels detected in seeds from high stearic acid mutants. The concomitant reduction of oleoyl-ACP content inside the plastid allowed us to study the activity of acyl-ACP thioesterases on saturated fatty acids. In these mutants, we verified that the accumulation of saturated fatty acids requires efficient thioesterase activity on saturated-ACPs. By studying the effects of cerulenin on the in vivo incorporation of [2-(14)C]acetate into lipids and on the in vitro activity of beta-ketoacyl-ACP synthase II, we found that elongation to very long chain fatty acids can occur both inside and outside of the plastid in sunflower seeds.

Cloning, characterization and structural model of a FatA-type thioesterase from sunflower seeds (Helianthus annuus L.)

The substrate specificity of acyl-acyl carrier protein (ACP) thioesterases (EC 3.1.2.14) determines the fatty acids available for the biosynthesis of storage and membrane lipids in seeds. In order to determine the mechanisms involved in the biosynthesis of fatty acids in sunflower seeds (Helianthus annuus L.), we isolated, cloned and sequenced a cDNA clone of acyl-ACP thioesterase from developing sunflower seeds, HaFatA1. Through the heterologous expression of HaFatA1 in Escherichia coli we have purified and characterized this enzyme, showing that sunflower HaFatA1 cDNA encodes a functional thioesterase with preference for monounsaturated acyl-ACPs. The HaFatA1 thioesterase was most efficient (kcat/K(m)) in catalyzing oleoyl-ACP, both in vivo and in vitro. By comparing this sequence with those obtained from public databases, we constructed a phylogenetic tree that included FatA and FatB thioesterases, as well as related prokaryotic proteins. The phylogenetic relationships support the endosymbiotic theory of the origin of eukaryotic cells and the suggestion that eubacteria from the delta-subdivision were the guest cells in the symbiosis with archaea. These prokaryotic proteins are more homologous to plant FatB, suggesting that the ancient thioesterases were more similar to FatB. Finally, using the available structure prediction methods, a 3D model of plant acyl-ACP thioesterases is proposed that reflects the combined data from direct mutagenesis and chimera studies. In addition, the model was tested by mutating the residues proposed to interact with the ACP protein in the FatA thioesterase by site-directed mutagenesis. The results indicate that this region is involved in the stabilization of the substrate at the active site.

Catalase activity and expression in developing sunflower seeds as related to drying.

Changes in catalase (CAT) activity and in CAT isoform pattern and expression were investigated in developing sunflower (Helianthus annuus L.) seeds during desiccation on the mother plant and after artificial drying on the flowerheads. Seeds regularly desiccated during their development on the mother plant and reached mass maturity at c. 42 d after flowering (DAF). Freshly harvested seeds did not germinate at any stage of development because they were dormant, but their dormancy was broken after 5-6 months of dry storage. Immature seeds were desiccation-tolerant at 24 DAF since they were able to germinate fully after artificial drying on the flowerheads followed by dry storage. CAT activity increased in non-dehydrated seeds during their development, reaching a maximum a little after seed mass maturity and after artificial drying in immature seeds. This stimulation of CAT activity by natural and artificial drying was related to changes in CAT isoform pattern. Of the four constitutive CAT subunits, that of 59 kDa was always present, but dehydration induced the synthesis of a 55 kDa subunit. This synthesis of the CAT 55 kDa subunit resulted from an activation of the CATA1 gene, suggesting that the regulation of catalase activity and synthesis by drying occurred at the transcriptional level. The increase in CAT activity induced by seed drying was associated with a decrease in hydrogen peroxide level and in lipid peroxidation. These results suggest that CAT plays a role during seed desiccation by preventing dehydration-related oxidative damage and that H(2)O(2) may play a role in the regulation of CAT gene expression and the transduction pathway of the dehydration signal.

Half-seed analysis for comparing linolenic acid synthesis between high and low oleic acid sunflower inbred lines

The modification of fatty acid composition of two sunflowers inbred lines HA89 and R978, low oleic acid (normal) and high oleic acid (mutant) respectively, in seeds and during the first stages of growth (A1-B2) was studied under controlled conditions. Enzymatic mechanisms have great effect on the catabolism of seed stored lipids. Temperature and oxygen regulation influence developing sunflower seeds. For simultaneous study of seed and developing seed, half-seed analysis technique was used. The behavior of the fatty acids during the germination in cotyledon of seed showed the increase of linolenic acid in both lines, demonstrating the activity of linoleic acid desaturase (Δ6-desaturase). But linoleic acid as a substrate for linoleate desaturase increased during all stages of developing only in mutant line that revealed higher activity of oleic acid desaturase (Δ12-desaturase) in transforming oleic acid to linoleic acid in this line, and lower activity of this enzyme in low oleic acid line, the reasons probably being the low availability of substrate of this enzyme in low oleic acid line and the complexity of enzymatic mechanisms. The modification of fatty acids in developing sunflower, depends not only on ambient conditions such as temperature and oxygen regulation as described by many authors, but also on the genotype.

Respiration of Mitochondria in Developing Sunflower Seeds

We studied the oxidative and phosphorylating activity of mitochondria in the seeds of three sunflower cultivars (Polevik, Peredovik, and Yubileinyi) during development of the seed embryo within 1 to 54 days after flowering. The rates of succinate oxidation by the mitochondria were 1.5–2 times those of malate or α-ketoglutarate oxidation. The ratio of substrate oxidation rates underwent changes during the seed growth. The differences were recorded between cultivars as concerns the times when the maximum oxidation rates were reached. Oxidation was coupled with phosphorylation during the entire period of seed development: the value of respiratory control after Chance changed from 1.4 to 7. By the time of transition to maturation, the rates of oxidation of both substrates and the values of respiratory control and ADP/O decreased. The results we obtained suggest that by days 13–15 of seed embryo growth, the rate of ATP production decreases upon oxidation of Krebs cycle products.

Diacylglycerol acyltransferase in maturing sunflower seeds

Developing sunflower seeds exhibit a high diacylglycerol acyltransferase (DAGAT, EC 2.3.1.20) activity. The distribution of the enzyme has been studied in subcellular fractions prepared by differential centrifugation of seed homogenate. Its activity was characterized using [1-(14)C]oleoyl-CoA and diolein dispersed in Tween 20. Some properties of the microsomal fraction of DAGAT were investigated. Hyperbolic kinetics were observed, the apparent K(m) was 60 microM and the specific activity of the reaction 15 pmol/min/mg of protein. Addition of BSA (0.1%) stimulated oleate incorporation, which was not dependent on the presence of exogenous diacylglycerol. Detergents which might solubilize DAGAT, Triton X-100 and CHAPS, were tested for enzyme inhibition, and CHAPS was found to be the least denaturing.

Diacylglycerol acyltransferase in maturing sunflower seeds

Developing sunflower seeds exhibit a high diacylglycerol acyltransferase (DAGAT, EC 2.3.1.20) activity. The distribution of the enzyme has been studied in subcellular fractions prepared by differential centrifugation of seed homogenate. Its activity was characterized using [1-14C]oleoyl-CoA and diolein dispersed in Tween 20. Some properties of the microsomal fraction of DAGAT were investigated. Hyperbolic kinetics were observed, the apparent Km was 60 μM and the specific activity of the reaction 15 pmol/min/mg of protein. Addition of BSA (0.1%) stimulated oleate incorporation, which was not dependent on the presence of exogenous diacylglycerol. Detergents which might solubilize DAGAT, Triton X-100 and CHAPS, were tested for enzyme inhibition, and CHAPS was found to be the least denaturing.

Temperature and oxygen regulation of microsomal oleate desaturase (FAD2) from sunflower

The effect of temperature and oxygen on the in vivo oleate desaturation and microsomal oleate desaturase (FAD2) activity was studied in peeled developing sunflower seeds. Using an oxygen concentration that was saturating for FAD2 enzyme, the amount of linoleic acid increased for all studied temperatures, being maximal at 20 degrees C. Under these conditions, FAD2 activity increased at the beginning of the incubation, remaining constant for the rest of the time, but reaching a lower level at 30 degrees C. Anoxia brought about a decrease in the FAD2 activity for all studied temperatures, becoming faster as the temperature increased. All these data suggest that temperature and oxygen control the level of FAD2 activity by separate mechanisms.

Oleate desaturase activity in sunflower ( Helianthus annuus) seeds and its relation to associated constitutents during seed development

The locus and specific acitivity of oleate desaturase (ODS) activity of developing sunflower seeds has been studied using differential centrifugation. The ratio of activity recovered in the crude pellet, oil body and microsomal fractions was consistently about 3:1:2 throughout seed development. Microsomes always contained the highest specific activity of the enzyme, reaching a maximum at 20–25 days after flowering and declining rapidly thereafter. The change in desaturase specific activity (in vitro) with seed development slightly preceded linoleate accumulation (in vivo) but was synchronous with the changing levels of cytochrome b. Although the associated metabolic enzymes CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CPT) and acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) reached maximum activities at the same time as the desaturase enzyme, these activities decreased less markedly during subsequent seed maturation. An unrelated microsomal enzyme, NADPH-cytochrome c-reductase (NCR), did not reach maximal activities until 40 days after flowering.