Temperature effect on a high stearic acid sunflower mutant.

Vegetable oil with elevated saturated fatty acid content may be useful for producing solid fat without hydrogenation or transesterification. Under the nutritional point of view stearic acid is preferred to other saturated fatty acids because of its neutral effect on serum cholesterol lipoproteins. Selection of a very high stearic acid sunflower (Helianthus annuus L.) line (CAS-14), with up to a 37.3% of stearic acid in the seed oil, and the relationship between the expression of this character and the growth temperature are presented. The mutant was selected from the M(2) progeny of 3000 mutagenized seeds (4 mM sodium azide mutagenesis treatment) by analysing the fatty acid composition of half-seed by gas liquid chromatography. In order to genetically fix the mutant character, plants were grown at high day/night temperatures during seed formation. We found that temperatures higher than 30/20 degrees C are required for good expression of the phenotype, the maximum stearic acid content being obtained at 39/24 degrees C. This behaviour is totally opposed to that observed in normal and previously isolated high-stearic acid sunflower lines that contain more stearic acid at low temperature. Thus, a new type of temperature regulation on the stearate desaturation must occur. This line is the sunflower mutant with the highest stearic acid content reported so far.     

Epistatic interaction among loci controlling the palmitic and the stearic acid levels in the seed oil of sunflower

Two sunflower (Helianthus annuus L.) mutants with high concentrations of saturated fatty acids in their seed oil have been identified and studied extensively. The mutant line CAS-5 has high concentrations of palmitic acid (C16:0) (>25% compared with 7% in standard sunflower seed oil) and low-C18:0 values (3%). CAS-3 is characterized by its high levels of stearic acid (C18:0) (>22% compared with 4% in standard sunflower seed oil) and a low-C16:0 content (5%). CAS-5 also possesses elevated levels of palmitoleic acid (C16:1) (>5%), which is absent in standard sunflower seed oil. The objective of this study was to determine the relationships between the loci controlling the high-C16:0 and the high-C18:0 traits in these mutants. Plants of both mutants were reciprocally crossed. Gas chromatographic analyses of fatty acids from the seed oil of F₁, F₂, F₃ and the BC₁F₁ to CAS-5 generations indicated that the loci controlling the high-C16:0 trait exerted an epistatic effect over the loci responsible for the high-C18:0 character. As a result, the phenotypic combination containing both the high-C16:0 levels of CAS-5 and the high-C18:0 levels of CAS-3 was not possible. However, phenotypes with a saturated fatty acid content of 44% (34.5% C16:0+9.5% C18:0) were identified in the F₃ generation. These are the highest saturated (C16:0 and C18:0) levels reported so far in sunflower seed oil. When F₃ C16:0 segregating generations in both a high- and a low-C18:0 background were compared, the high-C16:1 levels were not expressed as expected in the high-C18:0 background (CAS-3 background). In this case, the C16:1 content decreased to values below 1.5%, compared with >5% in a low-C18:0 background. As the stearoyl-ACP desaturase has been reported to catalyze the desaturation from C16:0-ACP to C16:1-ACP, these results suggested that a decrease in its activity was involved in the accumulation of C18:0 in the high-C18:0 mutant CAS-3. Received: 10 March 1999 / Accepted: 16 June 1999     

Enzymatic studies of high stearic acid sunflower seed mutants

Three high stearic acid sunflower (Helianthus annuus L.) mutants, CAS-3, CAS-4 and CAS-8, accumulating 28, 15 and 14 % of stearic acid in the seed lipids have been biochemically characterised. In vivo conversion rate of palmitic acid into stearic acid is not altered in the mutants but the conversion rate of stearic acid into oleic acid shows a reduction that correlated with the total stearic acid content of seed lipid mutants. Two enzymatic activities are found to be involved in the mutant phenotype, the acyl-ACP thioesterase (EC 3.1.2.14) and the stearoyl-ACP desaturase (EC 1.12.99.6). Our data suggest that the high stearic phenotype is due to the combined effect of a reduced stearoyl-ACP desaturase activity and an acyl-ACP thioesterase with higher activity on stearoyl-ACP. The same thioesterase activity increment, found on stearoyl-ACP, was also found on palmitoyl-ACP, suggesting that the affected thioesterase activity could be a FatB type.     

Molecular analysis of the high stearic acid content in sunflower mutant CAS-14

Increasing the stearic acid content to improve sunflower (Helianthus annuus L.) oil quality is a desirable breeding objective for food-processing applications. CAS-14 is a sunflower mutant line with a high stearic acid content in its seed oil (>35% vs. <6% in currently grown sunflower hybrids), which is controlled by the Es3 gene. However, the expression of the high stearic acid character in CAS-14 is strongly influenced by temperature during seed maturation and it is not uniform along the seed. The objectives of this study were (1) to identify PCR-based molecular markers linked to the Es3 gene from CAS-14, (2) to map this gene on the sunflower genetic map, and (3) to characterize the interaction between CAS-14 and CAS-3, a sunflower high stearic acid (about 26%) mutant line with the Es1 and Es2 genes determining this trait. Two F₂ mapping populations were developed from crosses between CAS-14 and P21, a nuclear male sterile line with the Ms₁₁ gene controlling this character, and between CAS-14 and CAS-3. One hundred and thirty-three individuals from P21×CAS-14, and 164 individuals from CAS-3×CAS-14 were phenotyped in F₂ and F₃ seed generations for fatty acid composition using gas–liquid chromatography, and they were then genotyped with microsatellite [simple sequence repeat (SSR)] and insertion–deletion (INDEL) markers. Bulk segregant analysis in the P21×CAS-14 population identified two markers on LG 8 putatively linked to Es3. A large linkage group was identified using additional markers mapping to LG 8. Es3 mapped to the distal half of LG 8 and was flanked by the SSR markers ORS243 and ORS1161 at genetic distances of 0.5, and 3.9 cM, respectively. The Ms₁₁ gene was also mapped to LG 8 and genetic distance between this gene and Es3 was found to be 7.4 cM. In the CAS-3×CAS-14 population, two QTLs were identified on LG 1 and LG 8, which underlie the Es1 gene from CAS-3 and the Es3 gene from CAS-14, respectively. A significant epistatic interaction between these two QTLs was found. Results from this study provided a basis for determining CAS-14 efficient breeding strategies.     

Molecular basis of the high-palmitic acid trait in sunflower seed oil

Sunflower (Helianthus annuus L.) seed oil with high palmitic acid content has enhanced thermo-oxidative stability, which makes it well suited to high-temperature uses. CAS-5 is a sunflower mutant line that accumulates over 25 % palmitic acid in its seed oil, compared to 5–8 % in conventional cultivars. The objective of this study was to investigate the molecular basis of the high-palmitic acid trait in CAS-5 through both candidate gene and QTL mapping approaches. An F₂ population derived from the cross between CAS-5 and the conventional line HA-89 was developed. A 3-ketoacyl-ACP synthase II (KASII) locus on a telomeric region of linkage group (LG) 9 of the sunflower genetic map was found to co-segregate with palmitic acid content in this population. The KASII locus explained the vast majority of the phenotypic variation (98 %) of the trait. Two minor QTL affecting palmitic acid content were also found on the lower half of LG 9 and on LG 17. Additionally, QTL associated with other major fatty acids (stearic, oleic, and linoleic acid) were identified on LG 1, 6, and 10. This result may reflect untapped genetic variation that could exist among sunflower cultivars for genes determining fatty acid composition. In addition to demonstrating the major role of a KASII locus in the accumulation of high levels of palmitic acid in CAS-5 seeds, this study stressed the importance of characterizing genes with minor effects on fatty acid profile in order to establish optimal breeding strategies for modifying fatty acid composition in sunflower seed oil.     

Lipid characterization of a wrinkled sunflower mutant

As part of a sunflower mutagenesis program carried out to obtain lines with fatty acid profiles in their oils, the half-palmitic CAS-7 line, with ca. 14% palmitic acid content, was isolated. Attempts to obtain a homozygotic line proved to be futile due to the lack of growth of the seedlings 10-12 days after germination. At this age, the seedlings stop growing, displayed a lack of chlorophyll and poor linolenic acid content, a fatty acid intimately linked to photosynthetic membranes. Accordingly, this line has only been maintained through heterozygotic seeds. Likewise, the cotyledons of seeds from this line with medium levels of palmitic acid present a characteristic wrinkled phenotype. In the oil of these seeds, the triacylglycerol content displayed a reduction of approximately 57% with respect to the control line, although a similar reduction was not observed in the polar lipids. Furthermore this mutant has 40.0% of trilinolein, the higher content found until today in sunflower seeds. These data indicate that the CAS-7 mutant possesses a multiple phenotype having a reduced triacylglycerol seed content, a modified intraplastidial fatty acid synthesis, together with a seedling blocked growth and poor green colour and reduced chloroplast development.     

Stearoyl-ACP and oleoyl-PC desaturase genes cosegregate with quantitative trait loci underlying high stearic and high oleic acid mutant phenotypes in sunflower

The genetic control of the synthesis of stearic acid (C18:0) and oleic acid (C18:1) in the seed oil of sunflower was studied through candidate-gene and QTL analysis. Two F₂ mapping populations were developed using the high C18:0 mutant CAS-3 crossed to either HA-89 (standard, high linoleic fatty acid profile), or HAOL-9 (high C18:1 version of HA-89). A stearoyl-ACP desaturase locus (SAD17A), and an oleoyl-PC de-saturase locus (OLD7) were found to cosegregate with the previously described Es1 and Ol genes controlling the high C18:0 and the high C18:1 traits, respectively. Using linkage maps constructed from AFLP and RFLP markers, these loci mapped to LG1 (SAD17A) and to LG14 (OLD7) and were found to underlie the major QTLs affecting the concentrations of C18:0 and C18:1, explaining around 80% and 56% of the phenotypic variance of these fatty acids, respectively. These QTLs pleiotropically affected the levels of other primary fatty acids in the seed storage lipids. A minor QTL affecting both C18:0 and C18:1 levels was identified on LG8 in the HAOL-9×CAS-3 F₂. This QTL showed a significant epistatic interaction for C18:1 with the QTL at the OLD7 locus, and was hypothesized to be a modifier of Ol. Two additional minor C18:0 QTLs were also detected on LG7 and LG3 in the HA-89×CAS-3 and the HAOL-9×CAS-3 F₂ populations, respectively. No association between a mapped FatB thioesterase locus and fatty acid concentration was found. These results provide strong support about the role of fatty acid desaturase genes in determining fatty acid composition in the seed oil of sunflower. Received: 7 December 2000 / Accepted: 21 May 2001     

Mapping minor QTL for increased stearic acid content in sunflower seed oil

Increased stearic acid (C18:0) content in the seed oil of sunflower would improve the oil quality for some edible uses. The sunflower line CAS-20 (C18:0 genotype Es1Es1es2es2), developed from the high C18:0 mutant line CAS-3 (C18:0 genotype es1es1es2es2; 25% C18:0), shows increased C18:0 levels in its seed oil (8.6%). The objective of this research was to map quantitative trait loci (QTL) conferring increased C18:0 content in CAS-20 in an F₂ mapping population developed from crosses between HA-89 (wild type Es1Es1Es2Es2; low C18:0) and CAS-20, which segregates independently of the macromutation Es1 controlling high C18:0 content in CAS-3. Seed oil fatty acid composition was measured in the F₂ population by gas-liquid chromatography. A genetic linkage map of 17 linkage groups (LGs) comprising 80 RFLP and 19 SSR marker loci from this population was used to identify QTL controlling fatty acid composition. Three QTL affecting C18:0 content were identified on LG3, LG11, and LG13, with all alleles for increased C18:0 content inherited from CAS-20. In total, these QTL explained 43.6% of the C18:0 phenotypic variation. Additionally, four candidate genes (two stearate desaturase genes, SAD6 and SAD17, and a FatA and a FatB thioesterase gene) were placed on the QTL map. On the basis of positional information, QTL on LG11 was suggested to be a SAD6 locus. The results presented show that increased C18:0 content in sunflower seed oil is not a simple trait, and the markers flanking these QTL constitute a powerful tool for plant breeding programs.     

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.     

Genetic possibilities for altering sunflower oil quality to obtain novel oils

The sunflower is one of the four most important oilseed crops in the world, and the nutritional quality of its edible oil ranks among the best vegetable oils in cultivation. Typically up to 90% of the fatty acids in conventional sunflower oil are unsaturated, namely oleic (C 18:1, 16%-19%) and linoleic (C 18:2, 68%-72%) fatty acids. Palmitic (C 16:0, 6%), stearic (C 18:0, 5%), and minor amounts of myristic (C 14:0), myristoleic (C 14:1), palmitoleic (C 16:1), arachidic (C 20:0), behenic (C 22:0), and other fatty acids account for the remaining 10%. Advances in modern genetics, most importantly induced mutations, have altered the fatty acid composition of sunflower oil to a significant extent. Treating sunflower seeds with gamma- and X-rays has produced mutants with 25%-30% palmitic acid. Sunflower seed treatment with X-rays has also resulted in mutants having 30% palmitoleic acid, while treatments with mutagenic sodium azide have produced seeds containing 35% stearic acid. The most important mutations have been obtained by treatment with dimethyl sulfate, which produced genotypes with more than 90% oleic acid. Mutants have also been obtained that have a high linoleic acid content (>80%) by treating seeds with X-rays and ethyl methanesulfonate. Of the vitamin E family of compounds, sunflower oil is known to predominantly contain alpha-tocopherol (>90%). Spontaneous mutations controlled by recessive genes have been discovered that significantly alter tocopherol forms and levels. The genes in question are tph(1) (50% alpha- and 50% beta-tocopherol), tph(2) (0%-5% alpha- and 95%-100% gamma-tocopherol), and tph(1)tph(2) (8%-40% alpha-, 0%-25% beta-, 25%-84% gamma-, and 8%-50% delta-tocopherol). The existence of (mutant) genes for increased levels of individual fatty acids and for different forms and levels of tocopherol enables the development of sunflower hybrids with different oil quality. The greatest progress has been made in developing high-oleic hybrids (>90% oleic acid). There has been considerable work done recently on the development of high-oleic hybrids with altered tocopherol levels, the oil of which will have 10-20 times greater oxidative stability than that of conventional sunflower oil. While sunflower breeders work on developing hybrids with altered oil quality, medical scientists in general and nutritionists in particular will determine the parameters for the use of these novel types of oil that can improve human nutrition and be used in the prevention of cardiovascular diseases.     

Oleate desaturation in seeds of two genotypes of sunflower

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.     

Unsaturated Fatty Acid Synthesis During the Development of Isolated Sunflower (Helianthus annuus L.) Seeds

The effect of temperature on unsaturated fatty acid synthesisin developing sunflower seed embryos (Helianthus annuus L.)has been studied using isolated seeds grown in culture. Variabilitybetween individual embryos in the response to temperature wasalso investigated. Oil and dry matter accumulation in cultured embryos were similarto those of embryos allowed to develop in intact plants, andthe effect of increasing temperature in lowering the amountof linoleic acid in seed oil was reproduced in cultured embryos.The isolated seed culture system, therefore, constitutes a suitablemodel system for studies of oil synthesis in developing sunflowerembryos. The decrease in linoleic acid synthesis in response to highertemperature was detectable after only 18 day-degrees incubation,and the incorporation of labelled substrates suggests that alterationsin the fatty acid composition of seed oil in response to temperatureare produced by an effect on the desaturation of newly synthesizedoleate rather than through turnover of existing lipid. Variation in fatty acid composition between individual embryosgrown at constant temperature was considerable. The detectionof embryos with high linoleic acid levels following growth athigh temperature indicates that potential may exist for theselection of cultivars for temperature-stable fatty acid compositionin sunflower oil. Key words: Fatty acid synthesis, Helianthus annuus, Sunflower seeds     

Metabolic control analysis of de novo sunflower fatty acid biosynthesis

We have obtained a simulation of the final steps of de novo fatty acid biosynthesis in sunflower control line RHA-274. For this simulation, we have used data from the evolution of fatty acids during seed formation and from the biochemical characterization of beta-keto-acyl-ACP synthetase II (FASII), stearoyl-ACP desaturase (SAD) and acyl-ACP thioesterase activities and the program GEPASI (based on the metabolic control-analysis theory). When physiological data from high- and medium-stearic acid mutants seed development were used with this model the predicted changes in SAD and TE were very similar to those actually found in the biochemical characterization of these mutants. However, the model had to be modified when results from high-palmitic mutants, accumulating unusual fatty acids like palmitoleic, asclepic and palmitolinoleic acids, were used. The emerging model, that fits all of our results, predicts the existence of a dynamic channelling between the FASII complex and SAD, that channelling being responsible for the alternative pathway starting with the desaturation of palmitic acid by the stearoyl-ACP desaturase. This channelling is consistent with our previous results. For instance, the determination of SAD activity on sunflower seed crude extracts only rendered oleic acid when the stearic acid used as a substrate was obtained from a KASII assay, but not when the stearic acid came from in vitro synthesis using acyl-ACP synthetase from Escherichia coli. This theoretical approximation will be very useful in predicting the evolution of the system when introducing new or modified activities; similar approximations in other oil-seed crops could be of great interest.     

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.     

Sunflower (Helianthus annuus) long‐chain acyl‐coenzyme A synthetases expressed at high levels in developing seeds

Long chain fatty acid synthetases (LACSs) activate the fatty acid chains produced by plastidial de novo biosynthesis to generate acyl‐CoA derivatives, important intermediates in lipid metabolism. Oilseeds, like sunflower, accumulate high levels of triacylglycerols (TAGs) in their seeds to nourish the embryo during germination. This requires that sunflower seed endosperm supports very active glycerolipid synthesis during development. Sunflower seed plastids produce large amounts of fatty acids, which must be activated through the action of LACSs, in order to be incorporated into TAGs. We cloned two different LACS genes from developing sunflower endosperm, HaLACS1 and HaLACS2, which displayed sequence homology with Arabidopsis LACS9 and LACS8 genes, respectively. These genes were expressed at high levels in developing seeds and exhibited distinct subcellular distributions. We generated constructs in which these proteins were fused to green fluorescent protein and performed transient expression experiments in tobacco cells. The HaLACS1 protein associated with the external envelope of tobacco chloroplasts, whereas HaLACS2 was strongly bound to the endoplasmic reticulum. Finally, both proteins were overexpressed in Escherichia coli and recovered as active enzymes in the bacterial membranes. Both enzymes displayed similar substrate specificities, with a very high preference for oleic acid and weaker activity toward stearic acid. On the basis of our findings, we discuss the role of these enzymes in sunflower oil synthesis.     

Dynamic channelling during de novo fatty acid biosynthesis in Helianthus annuus seeds

We have obtained a simulation of the final intraplastidial steps of de novo fatty acid biosynthesis in sunflower (Helianthus annuus L.) seeds. For this simulation, we have used data from the fatty acid content of normal and high-saturated seed formation and from the enzymatic characterization of the stearoyl-ACP desaturase (SAD; EC 1.12.99.6), acyl-ACP thioesterase (TE; EC 3.1.2.14) and fatty acid synthase II complex (FAS II), and the program GEPASI (based on the metabolic control analysis theory). When developmental data from high-stearic acid mutant seeds were analysed and compared to those predicted with this model, the changes in SAD and TE actually found in the biochemical characterization of these mutants were very similar to the predictions. However, the model had to be modified when results from high-palmitic mutants, accumulating unusual fatty acids like palmitoleic, asclepic and palmitolinoleic acids, were used. The emerging model, consistent with all of our results, predicts the existence of a dynamic channelling between the FAS II complex and SAD, that channelling being responsible for an alternative pathway starting with the desaturation of palmitoyl-ACP by the SAD. For instance, the determination of SAD activity on crude extracts from sunflower seeds only rendered oleoyl-ACP when stearoyl-ACP used as a substrate was obtained from an FAS II assay but not when in vitro synthesized stearoyl-ACP was provided as a substrate. This theoretical approximation will be very useful in order to predict the evolution of the system when introducing new or modified activities; similar approximations in other oilseed crops could be of great interest.     

Enzymatic characterisation of high-palmitic acid sunflower (Helianthus annuus L.) mutants

Two high-palmitic acid sunflower (Helianthus annuus L.) mutants, CAS-5 and CAS-12, have been biochemically characterised. The enzymatic activities found to be responsible for the mutant characteristics are β-keto-acyl-acyl carrier protein synthetase II (KASII; EC 2.3.1.41) and acyl-acyl carrier protein thioesterase (EC 3.1.2.14). Our data suggest that the high-palmitic acid phenotype observed in both mutant lines is due to the combined effect of a lower KASII activity and a higher thioesterase activity with respect to palmitoyl-acyl carrier protein (16:0-ACP). The level of the latter enzyme appeared to be insufficient to hydrolyse the produced 16:0-ACP completely. As a consequence of this, three new fatty acids appear: palmitoleic acid (16:1 Δ9), asclepic acid (18:1 Δ11), and palmitolinoleic acid (16:2 Δ9 Δ12). These fatty acids should be synthesised from palmitoyl-ACP or a derivative by the action of the stearoyl-ACP desaturase, fatty acid synthetase II and oleoyl-phosphatidylcholine desaturase, respectively. Received: 11 July 1998 / Accepted: 10 October 1998     

Acyl-acyl carrier protein thioesterase activity from sunflower (Helianthus annuus L.) seeds

During sunflower (Helianthus annuus L.) seed formation there was an active period of lipid biosynthesis between 12 and 28 days after flowering (DAF). The maximum in-vitro acyl-acyl carrier protein (ACP) thioesterase activities (EC 3.1.2.14) were found at 15 DAF, preceding the largest accumulation of lipid in the seed. Data from the apparent kinetic parameters, V _max and K _m, from seeds of 15 and 30 DAF, showed that changes in acyl-ACP thioesterase activity are not only quantitative, but also qualitative, since, although the preferred substrate was always oleoyl-ACP, the affinity for palmitoyl-ACP decreased, whereas that for stearoyl-ACP increased with seed maturation. Bisubstrate assays carried out at 30 DAF seemed to indicate that the total activity found in mature seeds is due to a single enzyme with 100/75/15 affinity for oleoyl-ACP/stearoyl-ACP/palmitoyl-ACP. In contrast, at 15 DAF, enzymatic data together with partial sequences from cDNAs indicated the presence of at least two enzymes with different properties, a FatA-like thioesterase, with a high affinity for oleoyl-ACP, plus a FatB-like enzyme, with preference for long-chain saturated fatty acids, both being expressed during the active lipid biosynthesis period. Competition assays carried out with CAS-5, a mutant with a higher content of palmitic acid in the seed oil, indicated that a modified FatA-type thioesterase is involved in the mutant phenotype. Received: 17 December 1999 / Accepted: 25 February 2000