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Instituto de Agricultura Sostenible, Apartado 4084, E-14080 Córdoba, Spain
* Corresponding author (ia2veval{at}uco.es).
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: FAD2, microsomal oleate desaturase • FAD3, microsomal linoleate desaturase • FAD6, chloroplastic oleate desaturase • FAD7 and FAD8, chloroplastic linoleate desaturases • wt, wild type
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genetic improvement of oilseed crops for producing seed oils with a high concentration of oleic acid and low concentrations of polyunsaturated fatty acids has been a major breeding goal in recent years (Velasco et al., 1999). Improved germplasm with elevated levels of oleic acid and concomitantly reduced levels of linoleic acid has been developed for a wide range of oilseed crops such as safflower (Carthamus tinctorius L.; Knowles and Mutwakil, 1963), sunflower (Helianthus annuus L.; Soldatov, 1976), soybean [Glycine max (L.) Merr.; Rahman et al., 1994; Mazur et al., 1999], canola (Brassica napus L.; Wong and Swansson, 1991; Auld et al., 1992; Tanhuanpää et al., 1996; Rücker and Röbbelen, 1997; Stoutjesdijk et al., 1999), Ethiopian mustard; Velasco et al., 1997a, 2003), maize (Zea mays L.; Wright, 1995), and peanut (Arachis hypogea L.; Gorbet and Knauft, 1997). Similarly, a considerable reduction of linolenic acid (18:3) levels has been achieved in linseed (Linum usitatissimum L.; Green, 1986; Rowland, 1991), soybean (Wilcox et al., 1984; Fehr et al., 1992; Rahman and Takagi, 1997; Ross et al., 2000), canola (Rakow, 1973; Röbbelen and Nitsch, 1975; Wong and Swanson, 1991; Auld et al., 1992; Rücker and Röbbelen, 1997), and Ethiopian mustard (Velasco et al., 1997a, 1997b, 2003).
Increased levels of oleic acid in the seed oil have been obtained by alteration of the desaturation step from oleic acid to linoleic acid, which is mediated by specific oleate desaturase enzymes. Plant cells typically possess two different types of membrane-bound oleate desaturases: the microsomal oleate desaturase (FAD2), located in the endoplasmic reticulum, and the chloroplastic oleate desaturase (FAD6), located in the plastid (Somerville et al., 2000). FAD2 operates in all plant cells, including green tissues, whereas FAD6 only is present in the latter (Miquel and Browse, 1992). Studies with Arabidopsis mutants have concluded that genetic modifications affecting FAD2 activity result in a rise of oleic acid concentration and a concomitant reduction of linoleic acid and linolenic acid concentrations that are mainly expressed in nonphotosynthetic tissues (seeds and roots), and to a lesser extent in leaves (Lemieux et al., 1990; Okuley et al., 1994; Horiguchi et al., 2001). Contrarily, genetic alterations affecting FAD6 activity are exclusively expressed in photosynthetic tissues, which exhibit increased concentrations of oleic acid and palmitoleic acid (16:1) and a drastic reduction of hexadecatrienoic acid (16:3) concentration (Browse et al., 1989; Falcone et al., 1994).
Reduction of linolenic acid concentration in seed oils has been produced by genetic modifications in the desaturation step from linoleic acid to linolenic acid, controlled by linoleate or 
-3 fatty acid desaturases. Like for oleate desaturases, there are two different types of linoleate desaturases in plant cells: FAD3, located at the endoplasmic reticulum, and FAD7 and FAD8, located in the plastid (Somerville et al., 2000). FAD8 has a substrate specificity similar to FAD7 but has more activity at low temperature (McConn et al., 1994). Studies on Arabidopsis revealed that mutations affecting FAD3 activity resulted in increased linoleic acid and reduced linolenic acid concentration in roots, whereas the expression of the mutations in leaves is weak (Lemieux et al., 1990; Browse et al., 1993). Conversely, mutations affecting the chloroplast membrane-bound desaturases FAD7 and FAD8 are only expressed in leaves, resulting in a considerable reduction of the concentration of both linolenic acid and hexadecatrienoic acid (Browse et al., 1986; McConn et al., 1994).
Ethiopian mustard is an oilseed crop that naturally contains high levels of erucic acid (22:1) in seed triacylglycerols (Fernández-Martínez et al., 2001). Zero-erucic acid forms of this crop, developed by different breeding approaches, are characterized by a seed oil fatty acid profile mainly made up of oleic acid (330 g kg–1), linoleic acid (370 g kg–1), and linolenic acid (210 g kg–1) (Alonso et al., 1991; Getinet et al., 1994; Fernández-Martínez et al., 2001). Additionally, Ethiopian mustard mutants with very high concentration of oleic acid (839 g kg–1; Velasco et al., 2003) or with very low concentration of linolenic acid in seed triacylglycerols (15 g kg–1; Velasco et al., 2004) have been developed. No studies have been done to determine if the mutations underlying such contrasting fatty acid profiles in Ethiopian mustard are seed specific or if they are expressed in other plant tissues. Similarly, no studies have been performed that monitor the level of expression of the altered fatty acid traits at different stages of seed development or after seed germination. Such information will be of great value for the management of the novel fatty acid traits in breeding programs. Additionally, it could provide a valuable insight into the enzymatic alterations underlying the modified fatty acid profiles. Accordingly, the objective of the present research was to monitor the relative expression of altered fatty acid profiles in different plant tissues and stages of seed development and germination in a high oleic acid mutant and a low linolenic acid mutant of Ethiopian mustard.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Analysis of Roots, Leaves, and Pollen
Six seeds per line were germinated on moist filter paper and their fatty acid composition was determined by gas-liquid chromatography (GLC) using the half-seed technique (Downey and Harvey, 1963). Two-day-old seedlings were planted in small pots (7 x 7 x 8 cm) containing a mixture of sand and peat (1:1, v/v). After 15 d in a growth chamber with a 16-h photoperiod, photon flux density of 300 µmol m–2 s–1 (fluorescent lamps Sylvania F36W/GRO, SLI Lichtsysteme GmbH, Erlangen, Germany) at 25/20°C (day/night), the plants were transplanted to larger pots containing 3 L of fertilized sand-silt-peat (2:1:1, v/v/v) soil mixture. Root samples were collected at the time of the transplant and thoroughly rinsed with distillated water. Leaf samples were collected from two fully expanded leaves. Pollen samples were collected from about 20 flowers per plant. In all cases, the samples were oven dried at 90°C for 18 h. Such a temperature does not alter the fatty acid profile of the samples. Dried tissue samples were finely crushed with a stainless-steel rod and analyzed for fatty acid composition by GLC as described below.
Analysis of Cotyledons after Seed Germination
Sixty seeds per line were placed on moist filter paper in Petri dishes. The seeds were maintained in the dark for 2 d at 25°C. After this time, the seedlings were planted in small pots (7 x 7 x 8 cm) containing vermiculite as inert medium. The plants were periodically watered with deionized water in a growth chamber at 25°C/18°C (day/night) and a 14-h photoperiod. Both cotyledons were removed from four seeds/seedlings per line every 24 h from 0 to 10 d after germination (DAG). The fatty acid composition of the oven-dried cotyledons was determined by GLC as described below.
Analysis of Developing Seeds
Three plants per line were grown in pots in a growth chamber at 25°C/18°C (day/night) and a 14-h photoperiod. Flowers in the main stem were tagged with the date of flowering. Two pods per plant were randomly collected at 10, 15, 20, 25, 30, 35, 40, and 45 d after flowering (DAF). The seeds were separated from the pods and the number of seeds was registered. Dry weight was determined after drying the seeds in an oven at 60°C for 24 h. Dried seeds were finely crushed with a stainless-steel rod and analyzed for fatty acid composition by GLC.
Gas-Liquid Chromatography of Fatty Acid Methyl Esters
The fatty acid composition of the seed oil was determined by GLC of fatty acid methyl esters, prepared by simultaneous extraction and methylation following the procedure of Garcés and Mancha (1993). Fatty acid methyl esters were analyzed on a PerkinElmer Autosystem gas-liquid chromatograph (PerkinElmer Corporation, Norwalk, CT, USA) with a 2-m-long column packed with 3% SP-2310/2% SP-2300 on Chromosorb WAW (Supelco, Bellefonte, PA, USA). A temperature program starting at 190°C for 10 min, increasing by 2°C min–1 up to 210°C, and maintained at this temperature for 10 min was used. The injector and flame ionization detector were held at 275 and 250°C, respectively. Fatty acids were identified by comparison of retention times with standards.
Statistical Analyses
Statistical significance of differences among concentrations of fatty acids in different plant tissues or at different stages was computed by analysis of variance through the general linear model (GLM) and Duncan's multiple range test. The analyses were conducted by SAS statistical package (SAS Institute, 2001).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The fatty acid profile of root tissues followed a similar trend to that of pollen grains (Table 1). Whereas roots of the standard line 25X-1 showed predominance of linolenic acid (389 g kg–1) and palmitic acid (276 g kg–1), those of the high oleic acid line AB-1 exhibited a dramatic increase of oleic acid concentration (782 g kg–1 compared with 108 g kg–1 in 25X-1), with simultaneous reduction of both linoleic acid (22 g kg–1 compared with 108 g kg–1 in 25X-1) and linolenic acid concentration (64 g kg–1 compared with 389 g kg–1 in 25X-1). Similarly, roots of the low linolenic acid line AB-4 were characterized by an increased linoleic acid (563 g kg–1 compared with 209 g kg–1 in 25X-1) and a reduced linolenic acid concentration (66 g kg–1 compared with 389 g kg–1 in 25X-1). It is noteworthy that the mutant lines AB-1 and AB-4 showed similarly reduced levels of linolenic acid in their root tissues (Table 1).
Leaves of the standard line 25X-1 were characterized by high concentrations of linolenic acid (558 g kg–1), palmitic acid (155 g kg–1), and hexadecatrienoic acid (130 g kg–1), the latter not detected in roots and seeds and only in small amounts in pollen grains (Table 1). Leaves of the high oleic acid line AB-1 showed increased levels of oleic acid (97 g kg–1 compared with 12 g kg–1 in 25X-1) and reduced levels of linoleic acid (69 g kg–1 compared with 126 g kg–1 in 25X-1). Linolenic acid concentration in AB-1 leaves was not different to that in 25X-1 (Table 1). Conversely, linolenic acid concentration was reduced in the leaves of the low linolenic acid mutant AB-4 (495 g kg–1 compared with 558 g kg–1 in 25X-1), which was accompanied by an increased linoleic acid concentration (198 g kg–1 compared with 126 g kg–1 in 25X-1).
Fatty Acid Composition after Seed Germination
Seed germination was accompanied by a rapid change of the fatty acid composition of seed cotyledons (Fig. 1)
. At 2 d after germination (DAG), the concentration of linolenic acid in cotyledon lipids of the standard line 25X-1 started increasing, accounting for 540 g kg–1 at 10 DAG compared with the initial value of 230 g kg–1 in cotyledons from mature seeds. The rise of linolenic acid concentration was paralleled by a reduction of oleic acid and linoleic acid concentration. The high oleic acid mutant AB-1 and the low linolenic acid mutant AB-4 also exhibited dramatic increases of linolenic acid concentration in the cotyledons after germination, which showed both lines had a similar linolenic acid concentration to the standard line 25X-1 at 10 DAG. In the AB-4 mutant, the reduced linolenic acid phenotype was maintained in comparison to 25X-1 until 8 DAG, when it was still possible to differentiate both lines according to their fatty acid profile. More pronounced was the expression of the high oleic/low linoleic acid phenotype in the AB-1 mutant, which was expressed even at 10 DAG. At this time, cotyledons of the AB-1 mutant contained 220 g kg–1 oleic acid and 80 g kg–1 linoleic acid, compared with 60 g kg–1 oleic acid and 220 g kg–1 linoleic acid in the cotyledons of the standard line 25X-1 (Fig. 1).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In B. napus, Schierholt et al. (2001) identified two different types of mutants with similarly increased levels of oleic acid in the seeds, but differing for the oleic acid concentration at the root and leaf level. The first type showed a slight increase of oleic acid concentration in roots (230 g kg–1 compared with 180 g kg–1 in the wild type) and leaves (60 g kg–1 compared with 30 g kg–1 in the wild type), whereas the second type exhibited a more pronounced expression of the high oleic acid phenotype both in roots (up to 500 g kg–1) and leaves (up to 180 g kg–1), as well as a reduced concentration of linolenic acid in root tissues (146 g kg–1 compared with 178 g kg–1 in the wild type). A comparison of the expression of the altered fatty acid profile in these B. napus mutants with that in the B. carinata AB-1 mutant indicates a closer similarity between AB-1 and the second mutant type of B. napus. Schierholt et al. (2001) hypothesized the alteration of a single gene affecting FAD2 in the first mutant type, and two genes in the second mutant type, one of them affecting FAD2 and the other one probably affecting another fatty acid biosynthesis enzyme.
Jourdren et al. (1996a) examined the fatty acid composition of pollen lipids in the low linolenic acid B. napus mutant Stellar, which is characterized by altered desaturase activity at the FAD3 level (Jourdren et al., 1996b). The authors found a linolenic acid concentration in pollen lipids of 390 g kg–1 compared with 510 g kg–1 in the wild type. Such a reduced concentration of linolenic acid is equivalent to the reduction observed in AB-1 and AB-4 pollen lipids. These data support the hypothesis that FAD3 activity might be altered in both AB-1 and AB-4 mutants.
An Arabidopsis mutant JB9, with altered FAD2 activity, was developed and characterized by Lemieux et al. (1990). This mutant showed increased oleic acid levels in seeds (535 g kg–1 compared with 154 g kg–1 in the wild type), roots (559 g kg–1 compared with 68 g kg–1 in the wild type), and leaves (209 g kg–1 compared with 23 g kg–1 in the wild type). The level of expression of increased oleic acid in different plant tissues of the Arabidopsis mutant JB9 is similar to that found in the present research for the Ethiopian mustard mutant AB-1, which suggests that the latter mutant may also have altered FAD2 activity.
Arabidopsis mutants with altered activity of the chloroplastic FAD6 desaturase have been developed (Browse et al., 1989; Falcone et al., 1994). These mutants showed increased levels of palmitoleic acid (16:1) and absence of hexadecatrienoic acid (16:3) in leaf lipids. Such fatty acid modifications were not observed in AB-1 leaf lipids, which suggests that FAD6 activity is not altered in this mutant.
Lemieux et al. (1990) identified and characterized the Arabidopsis mutant BL1, with deficient microsomal FAD3 activity. In that mutant, reduced linolenic acid concentration was mainly expressed in seeds (23 g kg–1 compared with 203 g kg–1 in the wild type) and roots (105 g kg–1 compared with 291 g kg–1 in the wild type), whereas linolenic acid reduction in leaf lipids was much less marked (472 g kg–1 compared with 508 g kg–1 in the wild type). These data are similar to those observed in Ethiopian mustard mutants AB-1 and AB-4, which showed reduced linolenic acid concentration in seeds, roots, and pollen. Comparison of Arabidopsis FAD3 mutant BL1 with B. carinata AB-1 and AB-4 mutants suggests that the microsomal FAD3 activity might be also altered in the latter mutants.
High oleic acid concentration in AB-1 seeds was expressed during the whole seed filling period, being therefore feasible to identify developing embryos carrying the high oleic acid trait at early stages of seed development. Similarly, both AB-1 and AB-4 mutants expressed the low linolenic acid trait at early stages of seed development, from 15 DAF onwards. These results are similar to those reported by Rakow and McGregor (1975), who found that B. napus lines with contrasting seed linolenic acid concentration expressed the trait as early as 20 DAF.
The expression of the high oleic acid and the low linolenic acid traits in both photosynthetic and nonphotosynthetic tissues opens up new ways for early selection of target phenotypes in segregating populations and identification of out-of-type plants in seed multiplication programs. Jourdren et al. (1996a) concluded clear advantages in early selection for low linolenic acid concentration in pollen instead of seeds in materials derived from the rapeseed cultivar Stellar. Because of the gametophytic control of seed fatty acid concentration, the possibility of selecting for fatty acid profile in the first stages of seed development will enable the speed-up of breeding programs through early fatty acid analysis coupled with embryo rescue.
The expression of the mutations for high oleic and low linolenic acid in plant organs other than seeds may have disadvantages as well. Mutants with altered FAD6 activity have been reported to have a simultaneous reduction in chlorophyll content (Hugly et al., 1989). However, the results of the present research suggest that FAD6 is not altered in AB-1 nor AB-4 mutants. Plants of AB-4 are phenotypically indistinguishable from the wild type with standard seed oil fatty acid profile. Conversely, plants of AB-1 were initially characterized by deleterious chlorosis, apparently produced by iron deficiency (Velasco et al., 2003). Such a deficiency, however, could be removed through selection, which suggests that it was produced as a side effect of the mutagenic treatment rather than as a direct effect of the mutations altering desaturases activity.
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ACKNOWLEDGMENTS |
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Received for publication April 30, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6 desaturase activity. Plant Physiol. 106:1453–1459.[Abstract]-3 desaturase. Plant Physiol. 106:1609–1614.[Abstract]Related articles in Crop Science:
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