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Biochemical characterization of a high-palmitoleic acid Helianthus annuus mutant
Plant Physiology and Biochemistry 42 (2004) 373–381 www.elsevier.com/locate/plaphy
Original article
Biochemical characterization of a high-palmitoleic acid Helianthus annuus mutant Joaquín J. Salas, Enrique Martínez-Force, Rafael Garcés * Dep. Fisiología y Tecnología de Productos Vegetales, Instituto de la Grasa, Av. Padre García Tejero, 4, 41012 Sevilla, Spain Received 5 January 2004; accepted 1 March 2004 Available online 09 April 2004
Abstract In the present work we carried out analytical and biochemical studies on a new high-n-7 monounsaturated fatty acid sunflower (Helianthus annuus L.) mutant. This new line, which has been selected by classical methods of breeding and mutagenesis, shows contents of unusual acyl chains up to 20% (12% of 16:1D9, 5% of 16:2D9,12 and 6% of 18:1D11), whereas those fatty acids are found in negligible amounts in common sunflower cultivars. This characterization involved in vivo incubations with radiolabeled acetate and measurement of the last enzymes involved in the intraplastidial de novo fatty acid synthesis: b-ketoacyl-ACP synthase II, stearoyl-ACP desaturase (EC 1.14.19.2) and acyl-ACP thioesterases (EC 3.1.2.14). Results indicated that the high-palmitoleic acid phenotype was associated with a concerted reduction in the fatty acid synthase II activity with respect to the control lines and an increase of stearoyl-ACP desaturase activity with respect to the high-palmitate mutant line. © 2004 Elsevier SAS. All rights reserved. Keywords: Acyl-ACP thioesterases; cis-vaccenate; b-ketoacyl synthase II; Helianthus annuus; Palmitoleate; Stearoyl-ACP desaturase
1. Introduction Seed oils provide a great variety of fatty acids (up to 200, including oxygenated, monounsaturated, polyunsaturated, conjugated and acetylenic derivatives) that could have applications in the food and chemical industry as renewable, clean and safer alternatives to carbon chains of mineral origin [1]. Among these compounds, unusual monoenoic fatty acids are of a special interest due to the variety of their applications. Thus, short chained monounsaturated fatty acids like myristoleate or palmitoleate combine low melting points with a high stability towards oxidation, which makes them a good base for the preparation of biodegradable lubricants. Other industrial stocks that can be produced from these fatty acids are lauric acid, margarines and plastic monomers [21]. Plant fatty acid synthesis takes place in the plastids through a process involving the sequential condensation of
Abbreviations: ACP, acyl carrier protein; DAF, days after flowering; FW, fresh weight; KAS II, b-ketoacyl synthase II; SAD, stearoyl-ACP desaturase; TLC, thin layer chromatography. * Corresponding author. E-mail address: [email protected] (R. Garcés). © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2004.03.001
malonyl-ACP on acyl-ACP derivatives in a reaction catalyzed by different dissociated forms of fatty acid synthase. The final products of these elongation cycles are 16:0-ACP and 18:0-ACP [16], which can be desaturated by the action of D9 stearoyl-ACP desaturases (SAD: EC 1.14.19.2) to produce the corresponding acyl-ACP derivatives of palmitoleate and oleate [29]. Oleate is the main product of plastidial fatty acid synthesis whereas palmitoleate is found only in trace amounts in most plant tissues; being abundant in the seed endosperm of some tropical species like Macadamia integrifolia and Doxantha unguis-cati or cat’s claw [7]. Intraplastidial reactions of elongation and desaturation are terminated by the acyl-ACP thioesterases (EC 3.1.2.14), enzymes that hydrolyze the acyl-ACP intermediates in their fatty acid and ACP moieties. The released free acyl residues are then exported out of the plastid to be incorporated into glycerolipids [17]. Although this pathway is made of discrete elements, previous results indicate that there are interactions between such components expediting the dynamic channeling of the metabolic flux through both the elongation and the desaturation steps [19,25]. It is well known that most unusual monoenoic fatty acids are produced by the action of diverged acyl-ACP desaturases with different substrate specificity. This divergence affects
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both the chain length and the position of the double bond of the preferred substrate of the enzyme. Diverged acyl-ACP desaturases have been cloned from different sources, like endosperm of Coriandrum sativum (D4 16:0-ACP desaturase [6]), Thumbergia alata (D6 16:0-ACP desaturase [5]) or cat’s claw (D9 16:0-ACP desaturase [7]) as well as trichomes of Pelargonium xhortorum (D9 14:0-ACP desaturase [27]). Since plants producing these fatty acids are not adapted for agricultural production, a first attempt to provide a large scale supply of these compounds would be the transformation of more productive oil crops with these desaturases. However, even when levels of expression of the putative enzymes were assessed to be high, the yield of the desired products was often very poor [28]. The possible reasons for these disappointing results have been reviewed recently, concluding that monoene production in oil crops is not a single step dependent process and that many other facts including substrate availability, cofactor specificity, b-oxidation, protein folding factors or metabolic discrimination have to be considered [32]. In the present work, we have characterized biochemically a sunflower mutant that displays a high content in palmitoleate and its derivative by elongation cis-vaccenate, both of them n-7 fatty acids. This mutant (called CAS-37) was selected by classical techniques of breeding and chemical mutagenesis from common sunflower lines. Several sunflower mutants with modified fatty acid composition have been described including high-oleic [13,31], high-stearic [22] and high-palmitic traits [12]. The total content of n-7 fatty acids of the new line ranged from 20% to 25%, which is similar to, or even an improvement over the yield obtained in transgenic plants expressing diverged desaturases [8,28]. The characterization of this mutant involved labeling experiments with radioactive acetate as well as the determination of the enzyme activities involved in the last steps of the plastidial de novo fatty acid synthesis pathway: 16:0-ACP elongation, SAD and acyl-ACP thioesterases. Enzyme activities in the high-n-7 line were compared with those in lines with different fatty acid composition, including common (CAS-6), high-palmitic (CAS-12) and high-oleic (CAS-9) sunflower plants. Results indicated that the increase of such fatty acids was due to a concerted change in several activities and could contribute to a better understanding of the interactions between substrates and enzymes of fatty acid biosynthesis.
2. Results 2.1. Fatty acid composition of different sunflower lines
Table 1 Oil fatty acid composition of different sunflower lines Line
Fatty acid (mol%) 16:0 16:1 16:2 18:0 CAS-6 4.9 0.1 – 6.5 CAS-9 3.9 0.1 – 5.6 CAS-12 31.0 6.1 – 1.7 CAS-37 29.5 12.3 4.6 1.4
18:1D11 18:2 0.3 50.9 0.6 1.7 6.0 12.5 5.8 38.7
18:3 0.1 0.3 0.6 0.7
20:0 0.3 0.4 1.0 0.3
22:0 Rn-7 0.6 0.4 0.9 0.7 1.1 12.1 1.2 22.7
of oleate of about 85% of the total fatty acids, with a saturated fatty acid composition similar to that found in the CAS-6 line (Table 1). Both the common sunflower and high-oleic lines presented only traces (0.4–0.7%) of n-7 fatty acids. In this regard, we paid special attention to the high-palmitate higholeic sunflower line, CAS-12, which produces oils with 30% palmitate, 2% stearate and 40% oleate (Table 1), showing contents of n-7 fatty acids of about 12% of the total fatty acid fraction. Closely related to this mutant was the more recently developed high-n-7 line, CAS-37. The palmitate content of this line was also 30%, but showed a content of n-7 and n-7 derived fatty acids that doubled that in the CAS-12 line: 20–24% of the total oil fatty acids (Table 1). These n-7 fatty acids included hexadecadienoic acid (16:2D9, D12), resulting from the desaturation of palmitoleate by the action of microsomal oleate desaturase [20]. Furthermore, the C16 total percentage significantly increased in the CAS-37 line when compared with the high-palmitate mutant (Table 1). 2.2. Incubations with [2-14C]acetate As the first approach to establish the biochemical differences that determined the oil composition of the CAS-37 sunflower mutant, in vivo radiolabeling experiments were carried out. Different incorporation patterns were observed when endosperm of developing seeds from different mutants were incubated with the labeled precursor. CAS-37 and CAS-12 lines displayed lower incorporation rates into triacylglycerols than control and high-oleic sunflower lines (Table 2). Out of the total incorporation rates we paid special attention to the distribution of the label into the acyl fragments esterified to triacylglycerols. The distribution of radioactivity among acyl lipids was significantly different in CAS-12 and CAS-37 lines as compared with control and high-oleic lines. Oleate and stearate were the main products resulting from CAS-6 and CAS-9, which did not display detectable incorporations into labeled n-7 fatty acids Table 2 Incubation of sunflower seed endosperm with [2-14C]acetate. Total incorporation and label distribution among different lipid species. Results corresponded to developing endosperm dissected 15 DAF Line
Common sunflower (CAS-6) endosperm shows a typical fatty acid composition with high contents of linoleate and oleate, which approximately account for 40% and 50% of the total acyl composition, respectively, (Table 1). The very high-oleic line used in this work (CAS-9) displayed contents
18:1 36.3 86.4 40.0 5.4
CAS-6 CAS-9 CAS-12 CAS-37
Incorporation (pmol acetate s–1 g–1 FW) Total Hydrocarbons TAG 11.9 ± 0.5 2.4 ± 0.1 4.6 ± 0.3 (38.6%) 9.5 ± 1.5 1.9 ± 0.3 3.3 ± 0.2 (34.3%) 6.3 ± 0.3 1.4 ± 0.2 2.1 ± 0.2 (33.3%) 9.0 ± 0.6 1.7 ± 0.3 2.2 ± 0.1 (24.4%)
Polar lipids 4.9 ± 0.5 (41.2%) 2.7 ± 0.1 (28.4%) 2.7 ± 0.1 (42.9%) 5.1 ± 0.4 (56.6%)
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Fig. 2. Palmitoyl-ACP elongation activity in the endosperm of different sunflower mutant lines along the oil cumulating period. Activities were expressed on a fresh endosperm mass basis. (-♦-), CAS-9; (-X-), CAS-6; (-m-), CAS-37; (- -), CAS-12. Data corresponded to the average of three determinations. Error bars represent standard deviation.
Fig. 1. Radioactivity distribution among different fatty acids in the triacylglycerol fraction of the endosperm of different sunflower lines incubated with [2-14C]acetate. (A) Control lines CAS-6 ( ) and CAS-9 ( ); (B) CAS-37 ( ) and CAS-12 ( ). Data corresponded to the average of three determinations. Error bars represent standard deviation. Results corresponded to developing endosperm dissected 15 DAF.
(Fig. 1A). CAS-12 and CAS-37 mutants accumulated palmitate as the main acyl product in the triacylglycerol fraction, followed by oleate or stearate (Fig. 1B). In this case palmitoleate and cis-vaccenate were also present among the radiolabeled fatty acid products. The most remarkable quantitative difference between these two lines was observed in the incorporation into monoenoic fatty acids. Thus, the incorporation into oleate and palmitoleate was about twofold higher in the high-n-7 CAS-37 line. This increase took place at the expense of palmitate and stearate that were lower in CAS-37, as was the total incorporation into C16 fatty acids when compared with the high-palmitate CAS-12 control (Fig. 1B). 2.3. Palmitoyl-ACP elongation activity Palmitate elongation was assayed in extracts from developing endosperm of the above mentioned sunflower lines during a period ranging from 15 to 35 days after flowering (DAF) (Fig. 2). All lines studied displayed different developmental profiles attending both to the activity levels and the shape of the curves. The highest activity of the complex was found in the high-oleic mutant, in which the activity peaked about 21–23 DAF and was fivefold and fourfold higher than
those activities displayed by CAS-12 and CAS-37 lines, respectively, along the studied period. Moreover, the common sunflower line used as a control displayed high activity at 15–18 DAF, when it was even higher than the activity found in the high-oleic line, and abruptly dropped at more than 20 DAF to a low level of activity at longer maturation times. CAS-12 and CAS-37 lines presented profiles with lower activities, which was about twofold lower in the former mutant. In both cases palmitoyl-ACP elongation activity did not display remarkable changes during development but remained at an approximately constant activity level during the oil accumulation period. 2.4. Stearoyl-ACP desaturase activity As with other enzymes mentioned in the present work, SAD was assayed in extracts prepared from the oil accumulating endosperm of different mutants of sunflower. The first step for the characterization of this activity involved substrate specificity studies. SAD was examined with the substrates 18:0-ACP and 16:0-ACP, and showed similar relative desaturation rates in all sunflower lines analyzed (Table 3). Thus, they displayed desaturation rates towards 18:0-ACP that were about 100-fold higher than those found for 16:0ACP. Furthermore, SAD activity was measured during the period of oil accumulation using 16:0-ACP as the substrate in order to evaluate the capacity of these lines for the production of n-7 fatty acids. These determinations showed differences among the studied mutant lines involving a lower SAD activity in the high-palmitate CAS-12 mutant, which desaturated the exogenous substrate at half the rate of the CAS-37 line (Fig. 3B). The SAD activity displayed by the CAS-37 line was similar to the control and the high-oleic lines. All these activities peaked between 20 and 24 DAF but the developmental curves presented different shapes. High-C16 lines
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Table 3 Stearoyl-ACP desaturase substrate specificity in extracts from endosperm of different sunflower lines. Extracts were prepared from seeds harvested 24 DAF Line CAS-6 CAS-9 CAS-12 CAS-37
Stearoyl-ACP desaturase activity (pkat mg prot–1) 18:0-ACP % 16:0-ACP % 16.7 ± 0.9 100 0.17 ± 0.01 1.0 17.8 ± 0.8 100 0.19 ± 0.02 1.1 8.7 ± 0.6 100 0.11 ± 0.01 1.3 20.3 ± 0.1 100 0.27 ± 0.01 1.3
displayed a maximum at 24 DAF, whereas the curve corresponding to the common sunflower line peaked 2 days earlier and decreased quickly, in a similar way to the rapid decrease in the palmitoyl-ACP elongation activity. On the contrary the high-oleic sunflower mutant again maintained a higher level of activity during a longer period of time (Fig. 3A). 2.5. Acyl-ACP thioesterase activity Acyl-ACP thioesterase was assayed in extracts prepared from developing endosperm of sunflower seeds. The first stage of this study involved the characterization of the substrate specificity of this enzyme. Therefore, thioesterase activity was assayed with a variety of acyl-ACP substrates that included 16:0-ACP and 18:0-ACP and their D9 monounsaturated homologous 16:1-ACP and 18:1-ACP, resulting in the specificity profiles shown in Fig. 4. The substrate hydrolyzed at the highest rate was 18:1D9-ACP followed by 16:1D9-ACP, with lower activities towards saturated acyl-ACPs. The substrate specificity did not significantly change among these lines, showing hydrolysis rates towards 16:1D9-ACP that were around 13% of that measured with 18:1D9-ACP. These relative activities decreased for saturated acyl-ACPs to 4% and 2% of the maximum specificity for 18:0-ACP and 16:0ACP, respectively. Furthermore, acyl-ACP thioesterase was measured during the period of oil accumulation (15– 30 DAF) in the sunflower lines under study using 18:1D9ACP as the substrate (Fig. 5). The resulting developmental curves were similar among different lines, with the highest activities at approximately 20 DAF. None of the lines acylACP thioesterases activities were significantly different than the others during the whole period of oil accumulation,
Fig. 4. Substrate specificity of the acyl-ACP thioesterase activity in extracts from endosperm of different sunflower lines. Extracts were prepared from seeds harvested 20 DAF. ( ), CAS-6; ( ), CAS-9; ( ), CAS-37; ( ), CAS-12. Data corresponded to the average of three determinations. Error bars represent standard deviation.
Fig. 5. Acyl-ACP thioesterase activity in the endosperm of different sunflower mutant lines during the oil accumulating period. Activities were expressed on a fresh endosperm mass basis, 18:1D9-ACP was used as the substrate. (-♦-), CAS-9; (-X-), CAS-6; (-m-), CAS-37; (- -), CAS-12. Data corresponded to the average of three determinations. Error bars represent standard deviation.
except for the CAS-9 line, which stayed higher at more advanced developmental stages (Fig. 5).
3. Discussion
Fig. 3. Desaturation rates of palmitoyl-ACP in the endosperm of different sunflower lines during the oil accumulating period. Activities were expressed on a fresh endosperm mass basis. (A) (-♦-), CAS-9; (-X-), CAS-6; (B) (-m-), CAS-37; (- -), CAS-12. Data corresponded to the average of three determinations. Error bars represent standard deviation.
One of the most remarkable features of the high-palmitate sunflower mutant is its high content of palmitoleic and cisvaccenic acids, both present in negligible amounts in the common or high-oleic sunflower lines (Table 1). Further rounds of breeding and mutagenesis produced new lines that doubled the n-7 monoene level, yielding contents of these fatty acids in the range of 20–25% of the total acyl fraction.
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These results, obtained by classical methods in nontransgenic lines, contrasted with earlier attempts aimed at the production of different monoenes in oil crops by genetic engineering, in which lower amounts of unusual fatty acids were obtained [28]. In this regard, the only work in which higher amounts of unusual monoenes were reported in a transgenic crop corresponded to the expression of an engineered desaturase from castor bean in Arabidopsis thaliana seeds in which n-7 fatty acids and its derivatives accounted for 25–30% of the total oil fatty acids [8]. Nevertheless, comparative biochemical characterization of different sunflower lines with distinct monoene content could provide interesting information about the way these compounds are synthesized and accumulated, being a good basis for future projects aimed at the production of newer specialty oils useful for industry. The first approach for the biochemical characterization of n-7 fatty acid accumulation consisted of studies about the incorporation of [2-14C]acetate into lipids by the developing endosperm of the different sunflower lines. Although recent works indicating that acetate is not the substrate used in vivo for fatty acid synthesis [2,10] this precursor has been extensively used because it is readily incorporated into the glycerolipids fraction. Nevertheless, our hypothesis only involved the last steps of the fatty acid synthesis pathway and so the source of carbon used for them should not significantly affect the final results obtained in these experiments. Differences in the total incorporation rates into triacylglycerols were found between the lines under study. Thus, those lines richer in C18 fatty acids (common and high-oleic lines) incorporated acetate into triacylglycerols at higher rates than high-C16 lines (Table 2). This means that the latter cultivars might be inhibited in some step in the de novo fatty acid synthesis pathway. As described previously, this step could correspond to the one catalyzed by the KAS II, which has been previously reported to be reduced in the CAS-12 line [18], similarly to the fab 1 Arabidopsis mutant [33]. More important for the object of the present work was the distribution of label among the different fatty acids present in triacylglycerols. The common and high-oleic sunflower lines presented very similar incorporation patterns (Fig. 1A) in which, as expected, oleate was the most abundant product. The low incorporation rates into linoleate indicates that studies of [2-14C]acetate metabolism reflects mainly the intraplastidial de novo synthesis and that the extraplastidial modifications of the acyl chains are underrepresented, in this regard the differences in 18:2 content between CAS-12 and CAS-37 (Table 1) should not be an impediment in order to compare the n-7 fatty acid metabolism of these lines. Furthermore, the profiles corresponding to the mentioned lines were free of n-7 fatty acids, which means that the intraplastidial fatty acid synthesis was efficiently channeled to oleate without any side reaction. The incorporation patterns corresponding to CAS-12 and CAS-37 lines were, on the other hand, very different. Radiolabeled palmitate was the main
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accumulated product in these mutants followed by oleate, which pointed again towards an inhibition of the 16:0-ACP elongation step. Moreover, the incorporation of acetate into n-7 fatty acids took place at high rates in these lines, accounting for the 3% and 8% of the label in CAS-12 and CAS-37 lines, respectively. In this regard, the main difference between these two mutants was that in the incorporation into monounsaturated fatty acids was higher in the CAS-37 line (Fig. 1B). These increments occurred at the expense of the saturated homologs, palmitate and stearate, which suggests an increase of the SAD activity in that line. Taken together, these results outline a global picture of the metabolic changes involving KAS II and SAD. However, these in vivo experiments might be distorted by artifacts like the lack of reducing equivalents at long incubation times, producing high 18:0/18:1 incorporation ratios, or the increment of very long-chain fatty acid labeling produced by the accumulation of the radioactive precursor in the cells cytosol. These limitations of in vivo studies made necessary a contrast with in vitro determinations, in which enzyme activities are assayed in cell-free extracts and are supplied with the appropriate cofactors. The first step potentially involved in the phenotypes described here was the one catalyzed by b-ketoacyl-ACP synthase (KAS II). Previous studies indicated that a decrease of the KAS II activity was involved in the high-palmitic phenotype [18]. Since our metabolism studies pointed that the CAS-37 line is also KAS II-deficient, the elongation of 16:0-ACP, associated to this condensing enzyme, was measured in the endosperm of the four lines at different stages of development. Results in Fig. 2 confirmed this hypothesis, indicating that the control lines CAS-6 and CAS-9 displayed activities that were four- to fivefold higher than those found in CAS-37 or CAS-12, respectively, during most of the period analyzed. In addition, the CAS-37 mutant displayed palmitate elongation activities that were double than those found in the CAS-12 line. This suggested that the decrease of palmitoyl elongation came probably from a different mutation and agreed with the labeling experiments, in which incorporation into C16 fatty acids was higher in the CAS-12 mutant. Furthermore, the fact that no accumulation of any intermediate of the elongation cycle was found when palmitoyl-ACP elongation was assayed in both CAS-12 and CAS-37 lines indicated that the decrease of activity is associated with the condensing enzyme, ketoacyl-ACP synthase II. On the other hand, palmitoyl-ACP elongation profiles displayed by high-oleic and common sunflower differed in some aspects: the activity in CAS-6 peaked 2–3 days earlier than in the other plants and abruptly decreased in the period beyond 20 DAF. That was probably due to the fact CAS-6 has a shorter life cycle, in which the seeds dry earlier than in other lines. The differences in the incorporation into n-7 or n-9 monoenes that appeared in the labeling experiments between the high-palmitoleic and high-palmitic lines indicated that the
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SAD activity was probably involved in the former phenotype. It has been reported that point mutations can significantly alter the substrate specificity of this enzyme [7]. Since the CAS-37 line came from several rounds of mutagenesis there was the possibility of a mutation that altered the substrate specificity of this enzyme in sunflower. In order to investigate this point the specificity for 18:0-ACP and 16:0ACP substrates was examined for the different lines studied. Results in Table 3 showed that for crude extracts prepared from all lines, the substrate specificity was similar, with 18:0-ACP as the best substrate. The shorter chained 16:0ACP substrate was desaturated at a rate that was 1–1.3% of that found for 18:0-ACP. Because SAD specificity was not significantly different among the four lines, the activity level of this enzyme was investigated in the developing endosperm of the different mutants during the oil accumulation period. The rate of desaturation of 16:0-ACP was found to be about twofold higher in the CAS-37 line than in the CAS-12 line on a fresh weight basis during most of the developmental period (Fig. 3B), a similar increase of the n-7 fatty acid content in the seed oil, which indicates that, at this stage of development, the yield of those fatty acids was very sensitive to changes in the intraplastidial desaturation rates and there is still room for it to be improved. Activity of SAD in the CAS-37 line was, on the other hand, similar to that shown by control, common and high-oleic sunflower lines (Fig. 3B). The decrease of activity in the CAS-12 line was probably selected on the basis of high-saturated fatty acid content criteria. This decrease of SAD activity in CAS-12 was not a constraint for this line to display high contents of 18:1, which meant that the synthesis of that fatty acid is limited by the 16:0-ACP elongation instead of stearate desaturation. Once palmitoleic acid is synthesized in the plastids it has to be exported to the cytosol for its incorporation into triacylglycerols. The key enzymes for plastid fatty acid export are acyl-ACP thioesterases, which hydrolyze the thioester bond between the acyl moiety and the ACP. Acyl-ACP thioesterases are soluble plastid-targeted enzymes that have been classified into two families called FatA and FatB [17]. FatB, which displays specificity towards shorter chained saturated acyl-ACPs, has been recently demonstrated to play an important role in plant growth and development [3]. On the other hand, FatA-type thioesterases present high specificity towards 18:1-ACP and are responsible for most of the plastidial fatty acid export [26]. A good flow of palmitoleate from the plastid is essential to obtain optimum yields of this fatty acid in sunflower oil since it would prevent feedback inhibition of the SAD as well as further intraplastidial modifications of the product (i.e.: elongation to cis-vaccenic acid) [9,21]. Acyl-ACP thioesterase was assayed in the above mentioned sunflower lines in order to evaluate their potential for the export of palmitoleate. As the first approach substrate specificity studies on the different lines were carried out. Resulting specificity profiles were homogeneous among the different lines (Fig. 4), with the highest activities observed
towards 18:1D9-ACP and 16:1D9-ACP, which fits with data previously reported for FatA type thioesterases [26]. Saturated acyl-ACPs were found to be poorly processed, with rates of about 4% and 2% of that measured with the monounsaturated derivative for 18:0-ACP and 16:0-ACP, respectively. These profiles were similar to those previously described for Coriandrum sativum FatA [26], presenting 16:1ACP/16:0-ACP activity ratios with values of 6–8, which are very favorable to 16:1-ACP hydrolysis. This probably means that export was not a limiting factor in the overproduction of palmitoleate in sunflower. This point seemed to be confirmed by the fact that the content of 18:1D11 remained constant between CAS-12 and CAS-37, indicating that there was an increase of the 16:1 export rate parallel to the increase of 16:0-ACP desaturation in the CAS-37, which was probably associated with intraplastidial concentrations of 16:1-ACP much lower than the Km value of FatA for that substrate. Acyl-ACP thioesterase studies were completed with the activity levels of this enzyme at different stages of seed development for the distinct mutants. Thioesterase activity levels were similar in all cases (Fig. 5), indicating that the selection of the different traits did not affect FatA thioesterases. Additional factors inducing an increase of n-7 monoenes in CAS-37 could involve changes in the channeling of the fatty acid synthesis, as it has been also proposed for CAS-12 [19]. Thus, a decrease of fourfold in the palmitoyl-ACP elongation activity paralleled with a 50-fold increase in the n-7 fatty acid content of CAS-37 with respect to control lines, which suggests that there are additional factors inducing an increase of the internal pool of 16:0-ACP beyond what is expected attending the decrease of the enzymatic activities. Furthermore, the fact that palmitoleate was desaturated by the oleate desaturases, enzymes that act on phosphatidylcholine-esterified fatty acids, to produce hexadecadienoic acid (Table 1) seemed to indicate that this fatty acid finds a good accommodation in the membranes and is not identified as an alien element as has been described for other unusual fatty acids [11]. Summarizing, in vivo and in vitro determinations indicated that the high palmitoleic trait was caused to a decrease of KAS II together with an increase of SAD activity in the CAS-37 sunflower line. On this basis, the future prospect for the production of sunflower lines with higher contents of palmitoleate involves finding lines with a higher SAD/KAS II activity ratio, although the low specificity of endogenous sunflower desaturases towards 16:0-ACP could limit palmitoleate synthesis and accumulation. In this regard, the biochemical studies presented here indicate that the highpalmitoleate line, presenting a large pool of substrate for the synthesis of this product, could be an excellent host for engineered or exogenous acyl-ACP desaturases displaying a higher specificity towards 16:0-ACP in order to produce sunflower lines with an improved content of palmitoleic acid.
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4. Methods 4.1. Plant material and growth conditions Sunflower (Helianthus annuus L.) plants from standard sunflower line CAS-6, high-oleic CAS-9 [13], high-palmitic CAS-12 [12] and the one with high-n-7 fatty acid content (CAS-37) were cultivated in growth chambers at 25/15 °C (day/night), with a 16-h photoperiod, photon flux density of 300 µmole m–2 s–1 and endowed with fertirrigation lines. 4.2. Analysis of fatty acid methyl esters Fatty acid methyl esters were prepared by treating the sunflower endosperm or lipid sample with 3 ml of methanol/toluene/sulfuric acid (88/10/2 v/v/v) for 1 h at 80 °C [14]. After cooling, fatty acid methyl esters were extracted twice with 2 ml of heptane and analyzed by gas– liquid chromatography using a Supelco SP-2380 capillary column (30 m length; 0.25 mm i.d.; 0.20 µm film thickness) of fused silica (Bellefonte, PA). Hydrogen was used as the carrier gas at 28 cm s–1 Detector and injector temperatures were 200 °C whereas the oven temperature was 170 °C. Different methyl esters were identified by comparison with known standards.
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in the presence of 660 MBeq of [2-14C]acetate (2.15 Gbeq mmol–1) in a final volume of 0.5 ml, at 20 °C for 3 h. Then the endosperm was inactivated by heating at 80 °C for 10 min [23]. Labeled lipids were extracted from the endosperm using a modification of the method described by Hara and Radin [15]. The supernatant containing the remaining radioactive precursor was removed and the tissue was homogenized in 3 ml hexane/2-propanol (3/2, v/v). Then of 2 ml of 6.7% sodium sulfate was added and lipids extracted. The aqueous phase was re-extracted with 2.25 ml of hexane/2propanol (7/2, v/v) and organic phases combined. Lipids were then fractionated by TLC on silica gel plates using a double development. Neutral lipids were developed with hexane/ethyl-ether (80/20, v/v) until the mobile phase front reached the 80% of the plate length, then another run for polar lipids was applied with chloroform/methanol/acetic acid/water (85/15/5/3.5 v/v/v/v) covering 25% of the plate length. Radioactive bands were detected and quantified using a digital autoradiography scanner (Instant Imager, Packard, Canberra). The band corresponding to triacylglycerols was scraped from the plate and methylated as indicated above. Methyl esters were fractionated by argentation TLC. Radioactive bands corresponding to unsaturated fatty acids were located and quantified as above. The band corresponding to saturated fatty acids was scrapped from the plate, further fractionated by reversed-phase TLC and quantified in the same instrument.
4.3. Thin-layer chromatography fractionation 4.6. Preparation of acyl-ACP substrates Different TLC fractionation techniques were used in this study. Unsaturated fatty acid methyl esters were separated by argentation TLC on silica gel plates coated by immersion in 15% AgNO3 in acetonitrile. A triple development at –20 °C using toluene as the mobile phase allowed a complete separation of the different monounsaturated and polyunsaturated species of fatty acid methyl esters. Saturated fatty acids methyl esters were separated on the basis of their chain length by reversed phase TLC on silica gel plates previously coated with 2.5% vaseline oil in hexane, using acetonitrile/ hexane (90/10, v/v) as the solvent. 4.4. Preparation of enzyme extracts Typically 100 mg of developing sunflower endosperm was ground in 1 ml of 50 mM Tris (pH 8.0), 5 mM DTT using an ice cooled glass homogenator. The resulting homogenate was centrifuged for 10 min at 10,000 × g, the clear supernatant was used for enzyme assays. Protein was quantified by the colorimetric method of Bradford [4] using bovine gamma globulin as the standard. 4.5. Studies of in vivo metabolism using [2-14C]acetate A mass of 100 mg of fresh sunflower endosperm harvested 16 DAF, was incubated in triplicate in 50 mM MES (pH 6.0)
Labeled acyl-ACP substrates were prepared using a recombinant acyl-ACP synthetase from Escherichia coli kindly provided by John Shanklin (Brookhaven National Laboratory). Acylation reactions contained 50 µg of recombinant ACP-I from spinach (Sigma), 660 MBeq (approx. 0.1 µmol) of [1-14C] fatty acid ammonium salt (2.07 Gbeq mmol–1, [3H]fatty acid in the case of 16:1D9), 5 mM ATP, 2 mM DTT, 4 mM LiCl2, 10 mM MgCl2, 100 mM Tris (pH 8.0) and 10 µg acyl-ACP synthetase in a final volume of 0.5 ml. Reactions were carried out at room temperature for 3–4 h and acyl-ACPs were purified and concentrated by ion exchange chromatography on DEAE-sepharose as described by Rock and Garwin [24]. 4.7. Acyl-ACP desaturase assay Acyl-ACP desaturase activity was assayed in the soluble fraction of sunflower endosperm following a variation of the method described by Cahoon et al. [5]. The assay medium contained 33 mM pipes pH 6.0, 3.3 mM ascorbate, 0.7 mM DTT, 8000 U catalase, 5 µg BSA, 20 µg spinach ferredoxin (Sigma), 80 mU spinach ferredoxin-NADP reductase (Sigma), 1.25 mM NADPH, 0.72 MBeq of [1-14C]16:0-ACP or 18:0-ACP and 1–30 µg of soluble protein in a final volume of 0.15 ml. Assays were run for a time long enough to convert
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about the 10% of the initial substrate (10–30 min) and then terminated and saponified with 0.85 ml of 2.35 M NaOH at 80 °C for 30 min. After saponification, the assay medium was acidified with 0.35 ml sulfuric acid and free fatty acids were extracted with 2 ml of hexane three times. Organic phases were combined and evaporated under nitrogen. Free fatty acids were then methylated for 1 h at 80 °C in 1 ml 2.5% methanolic sulfuric acid. Then, 3 ml of 5% NaCl were added and methyl esters were extracted three times with 2 ml hexane. The solvent was again evaporated under nitrogen and the resulting lipid extract fractionated by argentation TLC. The conversion to palmitoleate or oleate was quantified in an Instant Imager scanner (Packard, Canberra). 4.8. Palmitoyl-ACP elongation assay The fatty acid synthase II activity was determined using a modification of the method described by Slabaugh et al. [30] by monitoring the conversion of the acyl moiety of radiolabeled 16:0-ACP to 18:0 by the action of the soluble protein supernatant isolated from sunflower seed endosperm. The assay media consisted of 50 mM Tris (pH 8.0), 1 mM DTT, 1 mM NADH, 1 mM NADPH, 50 µM malonyl-CoA, 50 µM E. coli acyl carrier protein, 1.2 MBeq [1-14C]16:0-ACP, 0.1 mM cerulenin and 1–30 µg of soluble protein in a final volume of 0.1 ml. Assays were run at 25 °C for 15 min and stopped by adding 0.85 ml of 2.35 M NaOH. Acyl moieties were then saponified and methylated as described for the acyl-ACP desaturase assay. Finally, the resulting radioactive fatty acid methyl esters were fractionated on reversed-phase TLC plates. Radioactive bands corresponding to fatty acid methyl esters were found and quantified in a Packard Instant Imager. 4.9. Acyl-ACP thioesterase assay Acyl-ACP thioesterase (E.C. 3.1.2.14) was assayed by incubating 0.6 MBeq of [1-14C]acyl-ACP in 50 mM Tris pH 8.0, 5 mM DTT and 0.1–10 µg of soluble protein in a final volume of 0.1 ml. Assays were run at 25 °C for 5 min. Reactions were stopped by adding 0.2 ml 1 M acetic acid in 2-propanol and free fatty acids extracted twice with 0.35 ml of hexane. Radioactivity in the organic phase was quantified in a calibrated scintillation counter (Rackbeta II; LKB, Finland), after adding 3 ml scintillant solvent.
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[10] P.J. Eastmond, S. Rawsthorne, Coordinate changes in carbon partitioning and plastidial metabolism during the development of oilseed rape embryos, Plant Physiol. 122 (2000) 767–774. [11] V.S. Eccleston, J.B. Ohlrogge, Expression of lauryl-acyl carrier protein thioesterases in Brassica napus seeds induces pathways for both fatty acid oxidation and biosynthesis and implies a set point for triacylglycerol accumulation, Plant Cell 10 (1998) 613–622. [12] J. Fernández-Martínez, M. Mancha, J. Osorio, R. Garcés, Sunflower mutant containing high levels of palmitic acid in high oleic background, Euphytica 97 (1997) 113–116. [13] R. Garcés, J.M. García, M. Mancha, Lipid characterization in seeds of a high oleic acid sunflower mutant, Phytochemistry 28 (1989) 2597– 2600. [14] R. Garcés, M. Mancha, One-step lipid extraction and fatty acid methyl esters preparation from fresh plant tissues, Anal. Biochem. 211 (1993) 139–143. [15] A. Hara, N.S. Radin, Lipid extraction of tissues with a low-toxicity solvent, Anal. Biochem. 90 (1978) 420–424. [16] J.L. Harwood, Recent advances in the biosynthesis of plant fatty acids, Biochim. Biophys. Acta 1301 (1996) 7–56. [17] A. Jones, H.M. Davies, T.A. Voelker, Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases, Plant Cell 7 (1995) 359–371. [18] E. Martínez-Force, R. Álvarez-Ortega, R. Garcés, Enzymatic characterisation of high-palmitic acid sunflower (Helianthus annuus L.) mutants, Planta 207 (1999) 533–538. [19] E. Martínez-Force, R. Garcés, Dynamic channelling during de novo fatty acid biosynthesis in Helianthus annuus seeds, Plant Physiol. Biochem. 40 (2000) 383–391. [20] M. Miquel, J. Browse, Arabidopsis mutant deficient in polyunsaturated fatty acid synthesis. Biochemical and genetic characterization of a plant oleoyl-phosphatidylcholine desaturase, J. Biol. Chem. 267 (1992) 1502–1509. [21] J.B. Ohlrogge, Design of new plant products: engineering of fatty acid metabolism, Plant Physiol. 104 (1994) 821–826. [22] M. Osorio, J. Fernández-Martínez, M. Mancha, R. Garcés, Mutant sunflower with high concentration of saturated fatty acids in the oil, Crop Sci (1995) 739–742.
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Original article
Biochemical characterization of a high-palmitoleic acid Helianthus annuus mutant Joaquín J. Salas, Enrique Martínez-Force, Rafael Garcés * Dep. Fisiología y Tecnología de Productos Vegetales, Instituto de la Grasa, Av. Padre García Tejero, 4, 41012 Sevilla, Spain Received 5 January 2004; accepted 1 March 2004 Available online 09 April 2004
Abstract In the present work we carried out analytical and biochemical studies on a new high-n-7 monounsaturated fatty acid sunflower (Helianthus annuus L.) mutant. This new line, which has been selected by classical methods of breeding and mutagenesis, shows contents of unusual acyl chains up to 20% (12% of 16:1D9, 5% of 16:2D9,12 and 6% of 18:1D11), whereas those fatty acids are found in negligible amounts in common sunflower cultivars. This characterization involved in vivo incubations with radiolabeled acetate and measurement of the last enzymes involved in the intraplastidial de novo fatty acid synthesis: b-ketoacyl-ACP synthase II, stearoyl-ACP desaturase (EC 1.14.19.2) and acyl-ACP thioesterases (EC 3.1.2.14). Results indicated that the high-palmitoleic acid phenotype was associated with a concerted reduction in the fatty acid synthase II activity with respect to the control lines and an increase of stearoyl-ACP desaturase activity with respect to the high-palmitate mutant line. © 2004 Elsevier SAS. All rights reserved. Keywords: Acyl-ACP thioesterases; cis-vaccenate; b-ketoacyl synthase II; Helianthus annuus; Palmitoleate; Stearoyl-ACP desaturase
1. Introduction Seed oils provide a great variety of fatty acids (up to 200, including oxygenated, monounsaturated, polyunsaturated, conjugated and acetylenic derivatives) that could have applications in the food and chemical industry as renewable, clean and safer alternatives to carbon chains of mineral origin [1]. Among these compounds, unusual monoenoic fatty acids are of a special interest due to the variety of their applications. Thus, short chained monounsaturated fatty acids like myristoleate or palmitoleate combine low melting points with a high stability towards oxidation, which makes them a good base for the preparation of biodegradable lubricants. Other industrial stocks that can be produced from these fatty acids are lauric acid, margarines and plastic monomers [21]. Plant fatty acid synthesis takes place in the plastids through a process involving the sequential condensation of
Abbreviations: ACP, acyl carrier protein; DAF, days after flowering; FW, fresh weight; KAS II, b-ketoacyl synthase II; SAD, stearoyl-ACP desaturase; TLC, thin layer chromatography. * Corresponding author. E-mail address: [email protected] (R. Garcés). © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2004.03.001
malonyl-ACP on acyl-ACP derivatives in a reaction catalyzed by different dissociated forms of fatty acid synthase. The final products of these elongation cycles are 16:0-ACP and 18:0-ACP [16], which can be desaturated by the action of D9 stearoyl-ACP desaturases (SAD: EC 1.14.19.2) to produce the corresponding acyl-ACP derivatives of palmitoleate and oleate [29]. Oleate is the main product of plastidial fatty acid synthesis whereas palmitoleate is found only in trace amounts in most plant tissues; being abundant in the seed endosperm of some tropical species like Macadamia integrifolia and Doxantha unguis-cati or cat’s claw [7]. Intraplastidial reactions of elongation and desaturation are terminated by the acyl-ACP thioesterases (EC 3.1.2.14), enzymes that hydrolyze the acyl-ACP intermediates in their fatty acid and ACP moieties. The released free acyl residues are then exported out of the plastid to be incorporated into glycerolipids [17]. Although this pathway is made of discrete elements, previous results indicate that there are interactions between such components expediting the dynamic channeling of the metabolic flux through both the elongation and the desaturation steps [19,25]. It is well known that most unusual monoenoic fatty acids are produced by the action of diverged acyl-ACP desaturases with different substrate specificity. This divergence affects
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both the chain length and the position of the double bond of the preferred substrate of the enzyme. Diverged acyl-ACP desaturases have been cloned from different sources, like endosperm of Coriandrum sativum (D4 16:0-ACP desaturase [6]), Thumbergia alata (D6 16:0-ACP desaturase [5]) or cat’s claw (D9 16:0-ACP desaturase [7]) as well as trichomes of Pelargonium xhortorum (D9 14:0-ACP desaturase [27]). Since plants producing these fatty acids are not adapted for agricultural production, a first attempt to provide a large scale supply of these compounds would be the transformation of more productive oil crops with these desaturases. However, even when levels of expression of the putative enzymes were assessed to be high, the yield of the desired products was often very poor [28]. The possible reasons for these disappointing results have been reviewed recently, concluding that monoene production in oil crops is not a single step dependent process and that many other facts including substrate availability, cofactor specificity, b-oxidation, protein folding factors or metabolic discrimination have to be considered [32]. In the present work, we have characterized biochemically a sunflower mutant that displays a high content in palmitoleate and its derivative by elongation cis-vaccenate, both of them n-7 fatty acids. This mutant (called CAS-37) was selected by classical techniques of breeding and chemical mutagenesis from common sunflower lines. Several sunflower mutants with modified fatty acid composition have been described including high-oleic [13,31], high-stearic [22] and high-palmitic traits [12]. The total content of n-7 fatty acids of the new line ranged from 20% to 25%, which is similar to, or even an improvement over the yield obtained in transgenic plants expressing diverged desaturases [8,28]. The characterization of this mutant involved labeling experiments with radioactive acetate as well as the determination of the enzyme activities involved in the last steps of the plastidial de novo fatty acid synthesis pathway: 16:0-ACP elongation, SAD and acyl-ACP thioesterases. Enzyme activities in the high-n-7 line were compared with those in lines with different fatty acid composition, including common (CAS-6), high-palmitic (CAS-12) and high-oleic (CAS-9) sunflower plants. Results indicated that the increase of such fatty acids was due to a concerted change in several activities and could contribute to a better understanding of the interactions between substrates and enzymes of fatty acid biosynthesis.
2. Results 2.1. Fatty acid composition of different sunflower lines
Table 1 Oil fatty acid composition of different sunflower lines Line
Fatty acid (mol%) 16:0 16:1 16:2 18:0 CAS-6 4.9 0.1 – 6.5 CAS-9 3.9 0.1 – 5.6 CAS-12 31.0 6.1 – 1.7 CAS-37 29.5 12.3 4.6 1.4
18:1D11 18:2 0.3 50.9 0.6 1.7 6.0 12.5 5.8 38.7
18:3 0.1 0.3 0.6 0.7
20:0 0.3 0.4 1.0 0.3
22:0 Rn-7 0.6 0.4 0.9 0.7 1.1 12.1 1.2 22.7
of oleate of about 85% of the total fatty acids, with a saturated fatty acid composition similar to that found in the CAS-6 line (Table 1). Both the common sunflower and high-oleic lines presented only traces (0.4–0.7%) of n-7 fatty acids. In this regard, we paid special attention to the high-palmitate higholeic sunflower line, CAS-12, which produces oils with 30% palmitate, 2% stearate and 40% oleate (Table 1), showing contents of n-7 fatty acids of about 12% of the total fatty acid fraction. Closely related to this mutant was the more recently developed high-n-7 line, CAS-37. The palmitate content of this line was also 30%, but showed a content of n-7 and n-7 derived fatty acids that doubled that in the CAS-12 line: 20–24% of the total oil fatty acids (Table 1). These n-7 fatty acids included hexadecadienoic acid (16:2D9, D12), resulting from the desaturation of palmitoleate by the action of microsomal oleate desaturase [20]. Furthermore, the C16 total percentage significantly increased in the CAS-37 line when compared with the high-palmitate mutant (Table 1). 2.2. Incubations with [2-14C]acetate As the first approach to establish the biochemical differences that determined the oil composition of the CAS-37 sunflower mutant, in vivo radiolabeling experiments were carried out. Different incorporation patterns were observed when endosperm of developing seeds from different mutants were incubated with the labeled precursor. CAS-37 and CAS-12 lines displayed lower incorporation rates into triacylglycerols than control and high-oleic sunflower lines (Table 2). Out of the total incorporation rates we paid special attention to the distribution of the label into the acyl fragments esterified to triacylglycerols. The distribution of radioactivity among acyl lipids was significantly different in CAS-12 and CAS-37 lines as compared with control and high-oleic lines. Oleate and stearate were the main products resulting from CAS-6 and CAS-9, which did not display detectable incorporations into labeled n-7 fatty acids Table 2 Incubation of sunflower seed endosperm with [2-14C]acetate. Total incorporation and label distribution among different lipid species. Results corresponded to developing endosperm dissected 15 DAF Line
Common sunflower (CAS-6) endosperm shows a typical fatty acid composition with high contents of linoleate and oleate, which approximately account for 40% and 50% of the total acyl composition, respectively, (Table 1). The very high-oleic line used in this work (CAS-9) displayed contents
18:1 36.3 86.4 40.0 5.4
CAS-6 CAS-9 CAS-12 CAS-37
Incorporation (pmol acetate s–1 g–1 FW) Total Hydrocarbons TAG 11.9 ± 0.5 2.4 ± 0.1 4.6 ± 0.3 (38.6%) 9.5 ± 1.5 1.9 ± 0.3 3.3 ± 0.2 (34.3%) 6.3 ± 0.3 1.4 ± 0.2 2.1 ± 0.2 (33.3%) 9.0 ± 0.6 1.7 ± 0.3 2.2 ± 0.1 (24.4%)
Polar lipids 4.9 ± 0.5 (41.2%) 2.7 ± 0.1 (28.4%) 2.7 ± 0.1 (42.9%) 5.1 ± 0.4 (56.6%)
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Fig. 2. Palmitoyl-ACP elongation activity in the endosperm of different sunflower mutant lines along the oil cumulating period. Activities were expressed on a fresh endosperm mass basis. (-♦-), CAS-9; (-X-), CAS-6; (-m-), CAS-37; (- -), CAS-12. Data corresponded to the average of three determinations. Error bars represent standard deviation.
Fig. 1. Radioactivity distribution among different fatty acids in the triacylglycerol fraction of the endosperm of different sunflower lines incubated with [2-14C]acetate. (A) Control lines CAS-6 ( ) and CAS-9 ( ); (B) CAS-37 ( ) and CAS-12 ( ). Data corresponded to the average of three determinations. Error bars represent standard deviation. Results corresponded to developing endosperm dissected 15 DAF.
(Fig. 1A). CAS-12 and CAS-37 mutants accumulated palmitate as the main acyl product in the triacylglycerol fraction, followed by oleate or stearate (Fig. 1B). In this case palmitoleate and cis-vaccenate were also present among the radiolabeled fatty acid products. The most remarkable quantitative difference between these two lines was observed in the incorporation into monoenoic fatty acids. Thus, the incorporation into oleate and palmitoleate was about twofold higher in the high-n-7 CAS-37 line. This increase took place at the expense of palmitate and stearate that were lower in CAS-37, as was the total incorporation into C16 fatty acids when compared with the high-palmitate CAS-12 control (Fig. 1B). 2.3. Palmitoyl-ACP elongation activity Palmitate elongation was assayed in extracts from developing endosperm of the above mentioned sunflower lines during a period ranging from 15 to 35 days after flowering (DAF) (Fig. 2). All lines studied displayed different developmental profiles attending both to the activity levels and the shape of the curves. The highest activity of the complex was found in the high-oleic mutant, in which the activity peaked about 21–23 DAF and was fivefold and fourfold higher than
those activities displayed by CAS-12 and CAS-37 lines, respectively, along the studied period. Moreover, the common sunflower line used as a control displayed high activity at 15–18 DAF, when it was even higher than the activity found in the high-oleic line, and abruptly dropped at more than 20 DAF to a low level of activity at longer maturation times. CAS-12 and CAS-37 lines presented profiles with lower activities, which was about twofold lower in the former mutant. In both cases palmitoyl-ACP elongation activity did not display remarkable changes during development but remained at an approximately constant activity level during the oil accumulation period. 2.4. Stearoyl-ACP desaturase activity As with other enzymes mentioned in the present work, SAD was assayed in extracts prepared from the oil accumulating endosperm of different mutants of sunflower. The first step for the characterization of this activity involved substrate specificity studies. SAD was examined with the substrates 18:0-ACP and 16:0-ACP, and showed similar relative desaturation rates in all sunflower lines analyzed (Table 3). Thus, they displayed desaturation rates towards 18:0-ACP that were about 100-fold higher than those found for 16:0ACP. Furthermore, SAD activity was measured during the period of oil accumulation using 16:0-ACP as the substrate in order to evaluate the capacity of these lines for the production of n-7 fatty acids. These determinations showed differences among the studied mutant lines involving a lower SAD activity in the high-palmitate CAS-12 mutant, which desaturated the exogenous substrate at half the rate of the CAS-37 line (Fig. 3B). The SAD activity displayed by the CAS-37 line was similar to the control and the high-oleic lines. All these activities peaked between 20 and 24 DAF but the developmental curves presented different shapes. High-C16 lines
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Table 3 Stearoyl-ACP desaturase substrate specificity in extracts from endosperm of different sunflower lines. Extracts were prepared from seeds harvested 24 DAF Line CAS-6 CAS-9 CAS-12 CAS-37
Stearoyl-ACP desaturase activity (pkat mg prot–1) 18:0-ACP % 16:0-ACP % 16.7 ± 0.9 100 0.17 ± 0.01 1.0 17.8 ± 0.8 100 0.19 ± 0.02 1.1 8.7 ± 0.6 100 0.11 ± 0.01 1.3 20.3 ± 0.1 100 0.27 ± 0.01 1.3
displayed a maximum at 24 DAF, whereas the curve corresponding to the common sunflower line peaked 2 days earlier and decreased quickly, in a similar way to the rapid decrease in the palmitoyl-ACP elongation activity. On the contrary the high-oleic sunflower mutant again maintained a higher level of activity during a longer period of time (Fig. 3A). 2.5. Acyl-ACP thioesterase activity Acyl-ACP thioesterase was assayed in extracts prepared from developing endosperm of sunflower seeds. The first stage of this study involved the characterization of the substrate specificity of this enzyme. Therefore, thioesterase activity was assayed with a variety of acyl-ACP substrates that included 16:0-ACP and 18:0-ACP and their D9 monounsaturated homologous 16:1-ACP and 18:1-ACP, resulting in the specificity profiles shown in Fig. 4. The substrate hydrolyzed at the highest rate was 18:1D9-ACP followed by 16:1D9-ACP, with lower activities towards saturated acyl-ACPs. The substrate specificity did not significantly change among these lines, showing hydrolysis rates towards 16:1D9-ACP that were around 13% of that measured with 18:1D9-ACP. These relative activities decreased for saturated acyl-ACPs to 4% and 2% of the maximum specificity for 18:0-ACP and 16:0ACP, respectively. Furthermore, acyl-ACP thioesterase was measured during the period of oil accumulation (15– 30 DAF) in the sunflower lines under study using 18:1D9ACP as the substrate (Fig. 5). The resulting developmental curves were similar among different lines, with the highest activities at approximately 20 DAF. None of the lines acylACP thioesterases activities were significantly different than the others during the whole period of oil accumulation,
Fig. 4. Substrate specificity of the acyl-ACP thioesterase activity in extracts from endosperm of different sunflower lines. Extracts were prepared from seeds harvested 20 DAF. ( ), CAS-6; ( ), CAS-9; ( ), CAS-37; ( ), CAS-12. Data corresponded to the average of three determinations. Error bars represent standard deviation.
Fig. 5. Acyl-ACP thioesterase activity in the endosperm of different sunflower mutant lines during the oil accumulating period. Activities were expressed on a fresh endosperm mass basis, 18:1D9-ACP was used as the substrate. (-♦-), CAS-9; (-X-), CAS-6; (-m-), CAS-37; (- -), CAS-12. Data corresponded to the average of three determinations. Error bars represent standard deviation.
except for the CAS-9 line, which stayed higher at more advanced developmental stages (Fig. 5).
3. Discussion
Fig. 3. Desaturation rates of palmitoyl-ACP in the endosperm of different sunflower lines during the oil accumulating period. Activities were expressed on a fresh endosperm mass basis. (A) (-♦-), CAS-9; (-X-), CAS-6; (B) (-m-), CAS-37; (- -), CAS-12. Data corresponded to the average of three determinations. Error bars represent standard deviation.
One of the most remarkable features of the high-palmitate sunflower mutant is its high content of palmitoleic and cisvaccenic acids, both present in negligible amounts in the common or high-oleic sunflower lines (Table 1). Further rounds of breeding and mutagenesis produced new lines that doubled the n-7 monoene level, yielding contents of these fatty acids in the range of 20–25% of the total acyl fraction.
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These results, obtained by classical methods in nontransgenic lines, contrasted with earlier attempts aimed at the production of different monoenes in oil crops by genetic engineering, in which lower amounts of unusual fatty acids were obtained [28]. In this regard, the only work in which higher amounts of unusual monoenes were reported in a transgenic crop corresponded to the expression of an engineered desaturase from castor bean in Arabidopsis thaliana seeds in which n-7 fatty acids and its derivatives accounted for 25–30% of the total oil fatty acids [8]. Nevertheless, comparative biochemical characterization of different sunflower lines with distinct monoene content could provide interesting information about the way these compounds are synthesized and accumulated, being a good basis for future projects aimed at the production of newer specialty oils useful for industry. The first approach for the biochemical characterization of n-7 fatty acid accumulation consisted of studies about the incorporation of [2-14C]acetate into lipids by the developing endosperm of the different sunflower lines. Although recent works indicating that acetate is not the substrate used in vivo for fatty acid synthesis [2,10] this precursor has been extensively used because it is readily incorporated into the glycerolipids fraction. Nevertheless, our hypothesis only involved the last steps of the fatty acid synthesis pathway and so the source of carbon used for them should not significantly affect the final results obtained in these experiments. Differences in the total incorporation rates into triacylglycerols were found between the lines under study. Thus, those lines richer in C18 fatty acids (common and high-oleic lines) incorporated acetate into triacylglycerols at higher rates than high-C16 lines (Table 2). This means that the latter cultivars might be inhibited in some step in the de novo fatty acid synthesis pathway. As described previously, this step could correspond to the one catalyzed by the KAS II, which has been previously reported to be reduced in the CAS-12 line [18], similarly to the fab 1 Arabidopsis mutant [33]. More important for the object of the present work was the distribution of label among the different fatty acids present in triacylglycerols. The common and high-oleic sunflower lines presented very similar incorporation patterns (Fig. 1A) in which, as expected, oleate was the most abundant product. The low incorporation rates into linoleate indicates that studies of [2-14C]acetate metabolism reflects mainly the intraplastidial de novo synthesis and that the extraplastidial modifications of the acyl chains are underrepresented, in this regard the differences in 18:2 content between CAS-12 and CAS-37 (Table 1) should not be an impediment in order to compare the n-7 fatty acid metabolism of these lines. Furthermore, the profiles corresponding to the mentioned lines were free of n-7 fatty acids, which means that the intraplastidial fatty acid synthesis was efficiently channeled to oleate without any side reaction. The incorporation patterns corresponding to CAS-12 and CAS-37 lines were, on the other hand, very different. Radiolabeled palmitate was the main
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accumulated product in these mutants followed by oleate, which pointed again towards an inhibition of the 16:0-ACP elongation step. Moreover, the incorporation of acetate into n-7 fatty acids took place at high rates in these lines, accounting for the 3% and 8% of the label in CAS-12 and CAS-37 lines, respectively. In this regard, the main difference between these two mutants was that in the incorporation into monounsaturated fatty acids was higher in the CAS-37 line (Fig. 1B). These increments occurred at the expense of the saturated homologs, palmitate and stearate, which suggests an increase of the SAD activity in that line. Taken together, these results outline a global picture of the metabolic changes involving KAS II and SAD. However, these in vivo experiments might be distorted by artifacts like the lack of reducing equivalents at long incubation times, producing high 18:0/18:1 incorporation ratios, or the increment of very long-chain fatty acid labeling produced by the accumulation of the radioactive precursor in the cells cytosol. These limitations of in vivo studies made necessary a contrast with in vitro determinations, in which enzyme activities are assayed in cell-free extracts and are supplied with the appropriate cofactors. The first step potentially involved in the phenotypes described here was the one catalyzed by b-ketoacyl-ACP synthase (KAS II). Previous studies indicated that a decrease of the KAS II activity was involved in the high-palmitic phenotype [18]. Since our metabolism studies pointed that the CAS-37 line is also KAS II-deficient, the elongation of 16:0-ACP, associated to this condensing enzyme, was measured in the endosperm of the four lines at different stages of development. Results in Fig. 2 confirmed this hypothesis, indicating that the control lines CAS-6 and CAS-9 displayed activities that were four- to fivefold higher than those found in CAS-37 or CAS-12, respectively, during most of the period analyzed. In addition, the CAS-37 mutant displayed palmitate elongation activities that were double than those found in the CAS-12 line. This suggested that the decrease of palmitoyl elongation came probably from a different mutation and agreed with the labeling experiments, in which incorporation into C16 fatty acids was higher in the CAS-12 mutant. Furthermore, the fact that no accumulation of any intermediate of the elongation cycle was found when palmitoyl-ACP elongation was assayed in both CAS-12 and CAS-37 lines indicated that the decrease of activity is associated with the condensing enzyme, ketoacyl-ACP synthase II. On the other hand, palmitoyl-ACP elongation profiles displayed by high-oleic and common sunflower differed in some aspects: the activity in CAS-6 peaked 2–3 days earlier than in the other plants and abruptly decreased in the period beyond 20 DAF. That was probably due to the fact CAS-6 has a shorter life cycle, in which the seeds dry earlier than in other lines. The differences in the incorporation into n-7 or n-9 monoenes that appeared in the labeling experiments between the high-palmitoleic and high-palmitic lines indicated that the
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SAD activity was probably involved in the former phenotype. It has been reported that point mutations can significantly alter the substrate specificity of this enzyme [7]. Since the CAS-37 line came from several rounds of mutagenesis there was the possibility of a mutation that altered the substrate specificity of this enzyme in sunflower. In order to investigate this point the specificity for 18:0-ACP and 16:0ACP substrates was examined for the different lines studied. Results in Table 3 showed that for crude extracts prepared from all lines, the substrate specificity was similar, with 18:0-ACP as the best substrate. The shorter chained 16:0ACP substrate was desaturated at a rate that was 1–1.3% of that found for 18:0-ACP. Because SAD specificity was not significantly different among the four lines, the activity level of this enzyme was investigated in the developing endosperm of the different mutants during the oil accumulation period. The rate of desaturation of 16:0-ACP was found to be about twofold higher in the CAS-37 line than in the CAS-12 line on a fresh weight basis during most of the developmental period (Fig. 3B), a similar increase of the n-7 fatty acid content in the seed oil, which indicates that, at this stage of development, the yield of those fatty acids was very sensitive to changes in the intraplastidial desaturation rates and there is still room for it to be improved. Activity of SAD in the CAS-37 line was, on the other hand, similar to that shown by control, common and high-oleic sunflower lines (Fig. 3B). The decrease of activity in the CAS-12 line was probably selected on the basis of high-saturated fatty acid content criteria. This decrease of SAD activity in CAS-12 was not a constraint for this line to display high contents of 18:1, which meant that the synthesis of that fatty acid is limited by the 16:0-ACP elongation instead of stearate desaturation. Once palmitoleic acid is synthesized in the plastids it has to be exported to the cytosol for its incorporation into triacylglycerols. The key enzymes for plastid fatty acid export are acyl-ACP thioesterases, which hydrolyze the thioester bond between the acyl moiety and the ACP. Acyl-ACP thioesterases are soluble plastid-targeted enzymes that have been classified into two families called FatA and FatB [17]. FatB, which displays specificity towards shorter chained saturated acyl-ACPs, has been recently demonstrated to play an important role in plant growth and development [3]. On the other hand, FatA-type thioesterases present high specificity towards 18:1-ACP and are responsible for most of the plastidial fatty acid export [26]. A good flow of palmitoleate from the plastid is essential to obtain optimum yields of this fatty acid in sunflower oil since it would prevent feedback inhibition of the SAD as well as further intraplastidial modifications of the product (i.e.: elongation to cis-vaccenic acid) [9,21]. Acyl-ACP thioesterase was assayed in the above mentioned sunflower lines in order to evaluate their potential for the export of palmitoleate. As the first approach substrate specificity studies on the different lines were carried out. Resulting specificity profiles were homogeneous among the different lines (Fig. 4), with the highest activities observed
towards 18:1D9-ACP and 16:1D9-ACP, which fits with data previously reported for FatA type thioesterases [26]. Saturated acyl-ACPs were found to be poorly processed, with rates of about 4% and 2% of that measured with the monounsaturated derivative for 18:0-ACP and 16:0-ACP, respectively. These profiles were similar to those previously described for Coriandrum sativum FatA [26], presenting 16:1ACP/16:0-ACP activity ratios with values of 6–8, which are very favorable to 16:1-ACP hydrolysis. This probably means that export was not a limiting factor in the overproduction of palmitoleate in sunflower. This point seemed to be confirmed by the fact that the content of 18:1D11 remained constant between CAS-12 and CAS-37, indicating that there was an increase of the 16:1 export rate parallel to the increase of 16:0-ACP desaturation in the CAS-37, which was probably associated with intraplastidial concentrations of 16:1-ACP much lower than the Km value of FatA for that substrate. Acyl-ACP thioesterase studies were completed with the activity levels of this enzyme at different stages of seed development for the distinct mutants. Thioesterase activity levels were similar in all cases (Fig. 5), indicating that the selection of the different traits did not affect FatA thioesterases. Additional factors inducing an increase of n-7 monoenes in CAS-37 could involve changes in the channeling of the fatty acid synthesis, as it has been also proposed for CAS-12 [19]. Thus, a decrease of fourfold in the palmitoyl-ACP elongation activity paralleled with a 50-fold increase in the n-7 fatty acid content of CAS-37 with respect to control lines, which suggests that there are additional factors inducing an increase of the internal pool of 16:0-ACP beyond what is expected attending the decrease of the enzymatic activities. Furthermore, the fact that palmitoleate was desaturated by the oleate desaturases, enzymes that act on phosphatidylcholine-esterified fatty acids, to produce hexadecadienoic acid (Table 1) seemed to indicate that this fatty acid finds a good accommodation in the membranes and is not identified as an alien element as has been described for other unusual fatty acids [11]. Summarizing, in vivo and in vitro determinations indicated that the high palmitoleic trait was caused to a decrease of KAS II together with an increase of SAD activity in the CAS-37 sunflower line. On this basis, the future prospect for the production of sunflower lines with higher contents of palmitoleate involves finding lines with a higher SAD/KAS II activity ratio, although the low specificity of endogenous sunflower desaturases towards 16:0-ACP could limit palmitoleate synthesis and accumulation. In this regard, the biochemical studies presented here indicate that the highpalmitoleate line, presenting a large pool of substrate for the synthesis of this product, could be an excellent host for engineered or exogenous acyl-ACP desaturases displaying a higher specificity towards 16:0-ACP in order to produce sunflower lines with an improved content of palmitoleic acid.
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4. Methods 4.1. Plant material and growth conditions Sunflower (Helianthus annuus L.) plants from standard sunflower line CAS-6, high-oleic CAS-9 [13], high-palmitic CAS-12 [12] and the one with high-n-7 fatty acid content (CAS-37) were cultivated in growth chambers at 25/15 °C (day/night), with a 16-h photoperiod, photon flux density of 300 µmole m–2 s–1 and endowed with fertirrigation lines. 4.2. Analysis of fatty acid methyl esters Fatty acid methyl esters were prepared by treating the sunflower endosperm or lipid sample with 3 ml of methanol/toluene/sulfuric acid (88/10/2 v/v/v) for 1 h at 80 °C [14]. After cooling, fatty acid methyl esters were extracted twice with 2 ml of heptane and analyzed by gas– liquid chromatography using a Supelco SP-2380 capillary column (30 m length; 0.25 mm i.d.; 0.20 µm film thickness) of fused silica (Bellefonte, PA). Hydrogen was used as the carrier gas at 28 cm s–1 Detector and injector temperatures were 200 °C whereas the oven temperature was 170 °C. Different methyl esters were identified by comparison with known standards.
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in the presence of 660 MBeq of [2-14C]acetate (2.15 Gbeq mmol–1) in a final volume of 0.5 ml, at 20 °C for 3 h. Then the endosperm was inactivated by heating at 80 °C for 10 min [23]. Labeled lipids were extracted from the endosperm using a modification of the method described by Hara and Radin [15]. The supernatant containing the remaining radioactive precursor was removed and the tissue was homogenized in 3 ml hexane/2-propanol (3/2, v/v). Then of 2 ml of 6.7% sodium sulfate was added and lipids extracted. The aqueous phase was re-extracted with 2.25 ml of hexane/2propanol (7/2, v/v) and organic phases combined. Lipids were then fractionated by TLC on silica gel plates using a double development. Neutral lipids were developed with hexane/ethyl-ether (80/20, v/v) until the mobile phase front reached the 80% of the plate length, then another run for polar lipids was applied with chloroform/methanol/acetic acid/water (85/15/5/3.5 v/v/v/v) covering 25% of the plate length. Radioactive bands were detected and quantified using a digital autoradiography scanner (Instant Imager, Packard, Canberra). The band corresponding to triacylglycerols was scraped from the plate and methylated as indicated above. Methyl esters were fractionated by argentation TLC. Radioactive bands corresponding to unsaturated fatty acids were located and quantified as above. The band corresponding to saturated fatty acids was scrapped from the plate, further fractionated by reversed-phase TLC and quantified in the same instrument.
4.3. Thin-layer chromatography fractionation 4.6. Preparation of acyl-ACP substrates Different TLC fractionation techniques were used in this study. Unsaturated fatty acid methyl esters were separated by argentation TLC on silica gel plates coated by immersion in 15% AgNO3 in acetonitrile. A triple development at –20 °C using toluene as the mobile phase allowed a complete separation of the different monounsaturated and polyunsaturated species of fatty acid methyl esters. Saturated fatty acids methyl esters were separated on the basis of their chain length by reversed phase TLC on silica gel plates previously coated with 2.5% vaseline oil in hexane, using acetonitrile/ hexane (90/10, v/v) as the solvent. 4.4. Preparation of enzyme extracts Typically 100 mg of developing sunflower endosperm was ground in 1 ml of 50 mM Tris (pH 8.0), 5 mM DTT using an ice cooled glass homogenator. The resulting homogenate was centrifuged for 10 min at 10,000 × g, the clear supernatant was used for enzyme assays. Protein was quantified by the colorimetric method of Bradford [4] using bovine gamma globulin as the standard. 4.5. Studies of in vivo metabolism using [2-14C]acetate A mass of 100 mg of fresh sunflower endosperm harvested 16 DAF, was incubated in triplicate in 50 mM MES (pH 6.0)
Labeled acyl-ACP substrates were prepared using a recombinant acyl-ACP synthetase from Escherichia coli kindly provided by John Shanklin (Brookhaven National Laboratory). Acylation reactions contained 50 µg of recombinant ACP-I from spinach (Sigma), 660 MBeq (approx. 0.1 µmol) of [1-14C] fatty acid ammonium salt (2.07 Gbeq mmol–1, [3H]fatty acid in the case of 16:1D9), 5 mM ATP, 2 mM DTT, 4 mM LiCl2, 10 mM MgCl2, 100 mM Tris (pH 8.0) and 10 µg acyl-ACP synthetase in a final volume of 0.5 ml. Reactions were carried out at room temperature for 3–4 h and acyl-ACPs were purified and concentrated by ion exchange chromatography on DEAE-sepharose as described by Rock and Garwin [24]. 4.7. Acyl-ACP desaturase assay Acyl-ACP desaturase activity was assayed in the soluble fraction of sunflower endosperm following a variation of the method described by Cahoon et al. [5]. The assay medium contained 33 mM pipes pH 6.0, 3.3 mM ascorbate, 0.7 mM DTT, 8000 U catalase, 5 µg BSA, 20 µg spinach ferredoxin (Sigma), 80 mU spinach ferredoxin-NADP reductase (Sigma), 1.25 mM NADPH, 0.72 MBeq of [1-14C]16:0-ACP or 18:0-ACP and 1–30 µg of soluble protein in a final volume of 0.15 ml. Assays were run for a time long enough to convert
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about the 10% of the initial substrate (10–30 min) and then terminated and saponified with 0.85 ml of 2.35 M NaOH at 80 °C for 30 min. After saponification, the assay medium was acidified with 0.35 ml sulfuric acid and free fatty acids were extracted with 2 ml of hexane three times. Organic phases were combined and evaporated under nitrogen. Free fatty acids were then methylated for 1 h at 80 °C in 1 ml 2.5% methanolic sulfuric acid. Then, 3 ml of 5% NaCl were added and methyl esters were extracted three times with 2 ml hexane. The solvent was again evaporated under nitrogen and the resulting lipid extract fractionated by argentation TLC. The conversion to palmitoleate or oleate was quantified in an Instant Imager scanner (Packard, Canberra). 4.8. Palmitoyl-ACP elongation assay The fatty acid synthase II activity was determined using a modification of the method described by Slabaugh et al. [30] by monitoring the conversion of the acyl moiety of radiolabeled 16:0-ACP to 18:0 by the action of the soluble protein supernatant isolated from sunflower seed endosperm. The assay media consisted of 50 mM Tris (pH 8.0), 1 mM DTT, 1 mM NADH, 1 mM NADPH, 50 µM malonyl-CoA, 50 µM E. coli acyl carrier protein, 1.2 MBeq [1-14C]16:0-ACP, 0.1 mM cerulenin and 1–30 µg of soluble protein in a final volume of 0.1 ml. Assays were run at 25 °C for 15 min and stopped by adding 0.85 ml of 2.35 M NaOH. Acyl moieties were then saponified and methylated as described for the acyl-ACP desaturase assay. Finally, the resulting radioactive fatty acid methyl esters were fractionated on reversed-phase TLC plates. Radioactive bands corresponding to fatty acid methyl esters were found and quantified in a Packard Instant Imager. 4.9. Acyl-ACP thioesterase assay Acyl-ACP thioesterase (E.C. 3.1.2.14) was assayed by incubating 0.6 MBeq of [1-14C]acyl-ACP in 50 mM Tris pH 8.0, 5 mM DTT and 0.1–10 µg of soluble protein in a final volume of 0.1 ml. Assays were run at 25 °C for 5 min. Reactions were stopped by adding 0.2 ml 1 M acetic acid in 2-propanol and free fatty acids extracted twice with 0.35 ml of hexane. Radioactivity in the organic phase was quantified in a calibrated scintillation counter (Rackbeta II; LKB, Finland), after adding 3 ml scintillant solvent.
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