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Journal of Experimental Botany, Vol. 50, No. 340, pp. 1647–1652, November 1999 Accumulation of D6-unsaturated fatty acids in transgenic tobacco plants expressing a D6-desaturase from Borago officinalis Olga Sayanova1, Gayle M. Davies1,2, Mark A. Smith1,2, Gareth Griffiths3, A. Keith Stobart2, Peter R. Shewry1 and Johnathan A. Napier1,4 1 IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS41 9AF, UK 2 School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK 3 Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK Received 19 April 1999; Accepted 12 July 1999 Abstract Introduction Transgenic tobacco lines expressing the borage D6fatty acid desaturase were characterized for the accumulation of novel D6-unsaturated fatty acids. Two such fatty acids, c-linolenic acid and octadecatetraenoic acid, were found to be present in a range of seed and non-seed tissues, though accumulation in seed tissues was low. The distribution of these novel fatty acids amongst the various lipid classes of the tobacco leaf was examined. D6-Unsaturated fatty acids were found in both plastidic and microsomal lipids and positional analysis revealed that the novel fatty acids accumulated predominantly at the sn-2 position of the glycerolipid backbone. These data indicate that D6desaturated fatty acids are readily incorporated into a range of membrane lipids, unlike other unusual plant fatty acids. However, it may be that a number of different factors regulate the accumulation of D6-desaturated fatty acids in the storage triacylglycerols of seeds. As the borage D6-desaturase is most probably located in the endoplasmic reticulum, the present data suggest that D6-unsaturated fatty acids present in the plastid lipids are likely to have arisen as a result of import into this organelle after desaturation in the ER. Advances in recombinant DNA technology and plant transformation over the past few years have allowed the introduction of ‘novel’ traits into plant species. One area of interest in this rapidly expanding field of plant biotechnology is the modification of the lipid profile of oilseeds (Topfer et al., 1995). This is particularly attractive as a target for manipulation because the end-products have significant commercial value (as either foods, pharmaceuticals or industrial raw material ) and because the lipids of oilseeds are synthesized by a well-defined pathway (Shanklin and Cahoon, 1998; Miquel and Browse, 1998). Plants, unlike animals, produce a large array of different fatty acids and these are usually found in the storage lipid triacylglycerol ( TAG) (Stymne and Stobart, 1993). To date, over 300 different types of fatty acid have been reported in plants, a number of which are of interest as targets for exploitation (van de Loo et al., 1993). One such fatty acid is c-linolenic acid (GLA; 1853D6,9,12) which is used as a general health supplement and is also a registered pharmaceutical used for the treatment of conditions such as eczema and mastalgia (Horrobin, 1990). One reason for the biologically active role of GLA in animals is that it is required for the synthesis of arachidonic acid (AA; 2054D5,8,11,14), which in turn is the precursor of a class of compounds called the eicosanoids, which include the prostaglandins and thromboxanes and are involved in cellular ‘moment-tomoment’ regulation (Gill and Valivety, 1997). No such Key words: Borage, fatty acid desaturase, c-linolenic acid, transgenic tobacco. 4 To whom correspondence should be addressed. Fax: +44 1275 394281. E-mail: [email protected] Abbreviations: FAME, fatty acid methyl ester; GLA, c-linolenic acid; MGDG, monoacyldigalactosyldiacylglycerol; OTA, octadecatetraenoic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidyl-glycerol; TAG, triacylglycerol. © Oxford University Press 1999 1648 Sayanova et al. function has been to ascribed to GLA in plants, where it is considered to be mainly a storage compound present in the seed lipids of a limited number of species from a botanically-unrelated range of plant families (including the Onagraceae, Boraginaceae and Primulaceae), although GLA has been reported to occur in the membrane lipids of leaves of common borage (Griffiths et al., 1996). Recently, a cDNA clone was identified from borage (Borago officinalis) which encoded a member of a new class of higher plant fatty acid desaturases (Sayanova et al., 1997). The predicted amino acid sequence of this desaturase differed from all previously described plant desaturases with the presence of an additional N-terminal domain of ~100 residues which showed homology to the electron transport protein, cytochrome b (Sayanova 5 et al., 1997; Napier et al., 1997). When this borage cDNA was expressed in transgenic tobacco plants, analysis of total fatty acids revealed the presence of novel desaturation products, which were identified by GCMS as GLA and the related compound octadecatetraenoic acid (OTA; 1854D6,9,12,15). This identified the borage desaturase as being a D6-fatty acid desaturase, responsible for the introduction of a double bond at the D6-position of linoleic acid (LA; 1852D9,12) and a-linolenic acid (ALA; 1853D9,12,15) (Sayanova et al., 1997). It also confirmed that the synthesis of GLA and OTA could be attributed to the activity of one gene product, the D6-fatty acid desaturase. Although a previous study demonstrated the accumulation of GLA and OTA in tobacco plants expressing the borage D6-fatty acid desaturase, the distribution of these fatty acids in different lipid classes was not determined and, in particular, whether there were any constraints on the accumulation of GLA in specific lipid classes (such as TAG or phosphatidylcholine; PC ) and on the positions that these fatty acids were able to occupy on the glycerol backbone. This is particularly important since a number of studies have indicated that unusual fatty acids may be actively excluded either from some classes of lipids (usually membrane lipids like phosphatidylcholine) or from specific positions within the glycerolipids themselves ( Vogel and Browse, 1996). A detailed analysis of the lipid classes present in the seeds and vegetative tissues of homozygous lines of transgenic tobacco plants expressing the borage D6-fatty acid desaturase is reported here. Materials and methods Transgenic tobacco plants (Nicotiana tabacum cv. NVS ) were produced as described previously (Sayanova et al., 1997). Homozygous lines used in this study were produced by selffertilizing selected lines, selection on kanamycin-containing media and Northern blotting. Leaf material was taken from young (<3 cm long) or mature leaves. Homozygous lines were grown in a containment glasshouse and seeds harvested at different stages of seed development (according to Napier et al., 1995). Total fatty acids were analysed as fatty acid methyl esters (FAMEs) using a modified extraction method (Bligh and Dyer, 1959). The fatty acid content was determined by gas chromatography of fatty acid methyl esters with flame ionization detection using previously described methods (Sayanova et al., 1997). In all cases, a minimum number of three runs were carried out and quantified against known standards. All the results are expressed as mol% of total fatty acids, with a maximum SE of 3% of the mean value. Glycerolipids were separated according to the method of Griffiths et al. (Griffiths et al., 1996); lipids were extracted and separated by thin layer chromatography (TLC ). Individual lipid classes were identified by iodine staining and mobility when compared with authentic standards (Sigma). The lipids were then scraped from the plate, trans-methylated and analysed as FAMEs as described above. Positional analysis of the fatty acid composition of lipids was carried out as follows. Approximately 5 g of leaf tissue was used for extraction (using the method of Bligh and Dyer, 1959), and TLC of the chloroform phase was used to purify either monogalactosyldiacylglycerol (MGDG) or phosphatidylcholine (PC ). MGDG was eluted from the silica in chloroform5methanol (152, v5v), dried under nitrogen and then resuspended in digestion buffer (40 mM TRIS/HCl, pH 7.2 containing 3 mg Triton X-100). Rhizopus arrhizus lipase (3.4 mg) was then added to the sample and incubated with mixing at RT for 30 min (according to the method of Christie, 1982). R. arrhizus lipase displays an extremely high specificity for the sn-1 position of glycerolipids, regardless of the actual fatty acids present at this position. The digestion products were extracted in acidified butanol (Griffiths et al., 1996) then separated by TLC and quantified by methylation and GC analysis; for PC analysis, lipase digestion was carried out using phospholipase A2 (Naja naja), incubating for 80 min at 25 °C. Results Transgenic tobacco plants expressing the borage D6-fatty acid desaturase under the control of the cauliflower mosaic virus constitutive (35S ) promoter were selfed to produce homozygous T lines. The D6-desaturase 2 co-segregated with the kanamycin-resistance gene, neomycin phosphotransferase II, as well as the expected presence of GLA. Confirmation of homozygosity was obtained by further selfing of selected lines, resulting in T populations which were 100% KanR and 100% GLA3 containing. Total fatty acids were extracted from either leaves or mature seeds of glasshouse-grown T transgenic tobacco 2 plants expressing the borage transgene ( line c2) or from control transformed material. The fatty acids were analysed by GC, identified against known standards and expressed as mole percentages of total fatty acids ( Table 1). GLA accumulated in the leaves of the transgenic tobacco plants expressing the borage D6 desaturase ( line c2), at between 12.9% of total fatty acids in young leaves and 20.1% in mature leaves. Similar results were observed for OTA which accumulated to 8.5% and 14.9% in young and mature leaves, respectively. GLA and OTA D6-unsaturated fatty acids Table 1. Total fatty acid compositions of leaves and mature seeds of either control transformed (WT) or a homozygous line expressing the borage D6-desaturase (c2) Fatty acid methyl esters were analysed by GC and quantified against known standards; amounts are expressed as mol% and are the average of a minimum of three runs. In all tabulated data, the SE is less than 3% of that of the mean. Fatty acid 1650 1651 1653 1850 1851 1852 1853 GLA OTA Young leaves Old leaves Mature seeds WT c2 WT c2 WT c2 17.2 3.1 9.2 1.2 0.9 12.5 55.8 – 8.5 24.6 2.2 6.7 1.9 0.5 6.4 36.3 12.9 – 29.9 3.8 2.8 2.0 1.1 17.4 42.8 – 14.9 31.9 2.0 2.0 3.6 1.5 8.6 15.5 20.1 – 13.5 – – 2.3 10.1 72.7 1.2 – – 12.4 – – 2.4 8.5 72.9 1.3 2.6 were not observed in the leaves of the control tobacco plants. There were also decreases in the levels of LA and ALA in the c2 lines, compared with the control. The occurrence of GLA and OTA in mature (i.e. fully developed and desiccated ) seeds was also determined. However, only a low level of accumulation of GLA (2.6%) in these mature seeds with insignificant amounts of OTA ( Table 1) was observed. Moreover, there were no other changes in the fatty acid profile of the tobacco seeds, the major component being LA. Other tissues of the transgenic tobacco plants were also examined for the presence of D6-unsaturated fatty acids, and accumulation of these two fatty acids was observed in stems, roots and floral tissues ( Table 2). The highest amount of GLA was observed in stem tissue (27.2% of total fatty acids), with roots also showing a high level (21.9%), whilst OTA in these tissues was 8.7% and 5.9%, respectively. This indicated that the presence of these novel fatty acids was widely tolerated throughout the plant, and that accumulation of GLA and/or OTA was Table 2. Total fatty acid compositions of stems, roots and flowers of either control transformed (WT) or a homozygous line expressing the borage D6-desaturase (c2) 1649 usually mirrored by a decrease in the levels of LA and/or ALA. In view of the low levels of GLA and OTA observed in the mature seeds of the c2 tobacco line, the fatty acid composition of developing transgenic tobacco seeds was determined, at three defined stages of seed development (Bearson and Lamppa, 1993; Napier et al., 1995). These were categorized as: S1 (early seed development; white seeds), S2 (mid-stage seed development; opaque seeds) and S3 ( late stage development; brown seeds). The distribution of fatty acids shows ( Table 3) that there are significant amounts of GLA (16.6%) and OTA (2.5%) at the early stages (S1) of seed development, but that the proportions of both decline rapidly over a short developmental period. Although this decline coincides with a major increase in the level of LA, this LA clearly does not serve as a substrate for the D6-desaturase. It did not appear to be due to the reduced activity of the transgene promoter, as the levels of GLA actually increased during development when expressed as nmol fatty acid mg−1 fresh weight ( Table 4), although the increase was small in comparison with the substantial increase in the total amounts of fatty acids synthesized over this period. Analysis of the lipid classes of the S1 seeds showed that the major class was PC, and that this glycerolipid contained most of the GLA. In contrast, the GLA in the mature tobacco seeds was present predominantly in the TAG, which is the major lipid class in the seed at this (ultimate) stage of development (data not shown). To determine the distribution of GLA and OTA within Table 3. Total fatty acid composition of developing seeds of homozygous line c2 expressing the borage D6-desaturase FAMEs were analysed and quantified as described in Table 1. Seeds were taken at three stages of development (S1 being the youngest, S3 the oldest), and compared with mature seeds. Fatty acid S1 S2 S3 Mature 1650 1850 1851 1852 1853 GLA OTA 28.0 6.3 4.2 38.7 3.7 16.6 2.5 12.9 2.8 8.2 72.0 1.8 1.9 0.3 13.5 4.8 10.0 68.7 1.0 2.0 – 13.2 3.0 10.2 70.4 1.0 2.1 – FAMEs were analysed and quantified as described in Table 1. Fatty acid 1650 1651 1653 1850 1851 1852 1853 GLA OTA Stems Roots Flowers WT c2 WT c2 WT c2 29.9 – – 3.9 2.1 25.5 38.6 – – 34.0 – – 3.5 0.8 13.8 11.9 27.2 8.7 37.1 – – 3.5 0.9 39.1 19.4 – – 39.8 – – 3.7 1.2 21.0 6.6 21.9 5.9 21.0 7.5 – 5.5 4.1 45.8 16.9 – – 23.6 1.8 1.2 3.6 5.7 40.5 13.4 8.3 1.8 Table 4. Accumulation of GLA and OTA during seed development of transgenic line c2 The amounts of GLA, OTA and total fatty acids are expressed as nmol fatty acid mg−1 fresh weight. Fatty acid GLA OTA Total FAs Stage of seed development S1 S2 S3 Mature 4.1 0.7 15.4 4.8 1.3 218 8.4 0.3 282 14.0 – 556 1650 Sayanova et al. the different lipid species of the leaves, phosphatidylcholine (PC ), phosphatidyl-glycerol (PG), phosphatidylethanolamine (PE), monoacyldigalactosyldiacylglycerol (MDGD), and triacylglycerol ( TAG) were isolated from transgenic tobacco line c2 and their fatty acid compositions determined ( Table 5). This showed that both GLA and OTA accumulated in MGDG, PC, TAG, and PE (in decreasing order of mol%), but not in PG. However, there was no obvious relationship between the proportions of these fatty acids and those of their precursors (LA and ALA). Similarly, the ratios of GLA and OTA in the different lipid species varied. In order to determine the positions of GLA and OTA on the glycerol moiety of PC and MGDG, these lipid fractions from c2 leaf material was subjected to phospholipase or lipase digestion, respectively, followed by GC analysis of the reaction products. This revealed ( Table 6) that GLA and OTA were present predominantly at the sn-2 position (respectively, 79.7% and 86.4% at sn-2 as a percentage of total ) of MGDG. A similar situation was observed for PC, in which the majority of GLA and OTA was at postion sn-2 (83.3% and 85.3%, respectively). Table 5. Distribution of fatty acids amongst the different classes of lipids present in the leaves of homozygous line c2 Lipid classes were identified and separated by TLC and FAMEs of each lipid type analysed by GC as before; results are expressed as mol%. Fatty acid 1650 1651 1653 1850 1851 1852 1853 GLA OTA Lipid class PG MGDG PC PE TAG 46.5 15.8 0.8 8.5 12.7 6.4 9.4 – – 8.6 1.6 17.9 2.6 1.1 2.7 38.2 14.3 13.0 37.3 – 1.4 3.8 2.2 7.0 29.5 10.7 8.1 41.3 1.2 – 8.0 8.9 23.4 10.5 5.1 1.6 40.2 15.3 – 21.3 6.7 5.6 2.6 5.6 2.6 Table 6. Positional analysis of MGDG and PC from the lipids of leaves of homozygous line c2 The amounts of constituent fatty acids at the sn-1 and sn-2 positions were determined as before. Figures for fatty acids at the sn-1 and sn-2 positions are given as mol%, and the percentage of the fatty acid at the sn-2 position (%sn-2) is also given. Fatty acid 1650 1651 1653 1850 1851 1852 1853 GLA OTA MGDG PC sn-1 sn-2 %sn-2 sn-1 sn-2 %sn-2 6.0 2.8 – – – 4.0 82.5 3.0 2.0 1.6 1.0 26.7 – – 2.0 43.5 11.8 12.8 21.0% 26.3% 100% 73.1 – – 4.9 1.3 7.5 8.1 1.3 1.0 2.4 – – 0.8 2.8 24.9 55.5 6.5 5.8 3.2% 33.3% 34.5% 79.7% 86.4% 14.0% 68.3% 76.9% 87.3% 83.3% 85.3% Discussion The results presented here demonstrate that the expression of a B. officinalis D6-fatty acid desaturase in transgenic tobacco plants results in the accumulation of D6-unsaturated fatty acids in all the tissues examined, consistent with the use of the ‘constitutive’ viral 35S promoter. Accumulation patterns in the c2 lines are similar to those in borage, both fatty acids being present in many nonseed tissues (Griffiths et al., 1996; O Sayanova et al., unpublished data). Thus, the borage D6-fatty acid desaturase is not expressed in an exclusively seed-specific manner, unlike some other ‘unusual’ fatty acid desaturases (van de Loo et al., 1993). This ability of GLA and OTA to accumulate in non-seed tissues demonstrates that D6unsaturated fatty acids do not compromise the fitness of either the borage plant (Griffiths et al., 1996) or the transgenic tobacco plants used in this study. This contrasts with the constitutive expression of a castor D12hydroxylase in transgenic tobacco, which resulted in no detectable accumulation in tissues other than seeds, even though the transgene was expressed at high levels (van de Loo et al., 1995). This is consistent with the observation that ricinoleic acid is only accumulated in the endosperm of castor bean and that the hydroxylase gene is seed-specific. It is also of interest that in castor bean the hydroxylase is closely related to the D12-( oleate) desaturase and utilizes fatty acid substrates in microsomal PC. Unlike the ‘housekeeping’ fatty acids, however, ricinoleic acid is rapidly removed from membrane PC and only accumulates in TAG (Bafor et al., 1991). Thus, mechanisms appear to exist for removing uncommon and/or deleterious fatty acids from membrane lipids such as PC and channelling them towards storage TAG (Stymne and Stobart, 1993). In that respect it is intriguing that in borage (and the transgenic tobacco used in this study) GLA and OTA accumulate in membrane lipids of nonseed tissues, whereas the distribution of D6-unsaturated fatty acids is restricted almost exclusively to the seeds of Oenothera biennis (evening primrose). The low accumulation of GLA in mature seeds is perhaps surprising, given the levels of this fatty acid in non-seed tissues and in seeds at early stages of development. It is possible that this is due to the use of the viral CaMV 35S promoter, which is known to show a plateau of activity in the developing seeds of transgenic tobacco (Slocombe et al., 1994). However, this plateau occurs at a very high level of activity and does not parallel the temporal accumulation and decline of GLA observed in the tobacco seeds. Other possible explanations are the competition for substrate from other fatty acid desaturases. It has recently been observed that seed expression of a D12 hydroxylase (under the control of the CaMV-35S promoter) resulted in higher levels of hydroxylated fatty acids when carried out in the fad2 mutant compared with D6-unsaturated fatty acids the wild type (Broun et al., 1998). The authors concluded that this was due to competition between the oleate desaturase (i.e. FAD2) and the oleate hydroxylase. It may also be that tobacco seed acyltransferases for TAG assembly are less selective for the GLA or OTA than for their own endogenous fatty acids, or that potential substrates for the D6-desaturase (such as LA) are rapidly transferred into TAG and rendered unavailable for further desaturation. The presence of GLA and OTA in MGDG and PC indicates that these fatty acids are present in both plastidic (MGDG) and extraplastidic (PC ) membranes, demonstrating exchange of fatty acids between these two compartments. No GLA or OTA was detected in the PG fraction, which although a marker for the ‘prokaryotic’ (i.e. plastid) synthesized lipids, does not contain any LA at the sn-2 position to act as a substrate for the borage desaturase. Positional analysis of plastidic galactolipids (MGDG) revealed the predominant occurrence of GLA and OTA on the sn-2 position, which is consistent with the observed positions of these fatty acids in borage leaf lipids (Griffiths et al., 1996). Although it has been suggested that there may be two forms ( ER and plastidial ) of the D6-desaturase in borage leaves (Griffiths et al., 1996), these results indicate that expression of a single form results in the incorporation of D6-desaturated fatty acids into both ER and plastidic lipids, confirming the existence of a flux between the two compartments (Miquel and Browse, 1998). This is in contrast to the situation observed for the unicellular alga Isochrysis galbana, in which the complete series of desaturation reactions from 1851 to 1854 appear to take place on MGDG, implying a plastidial D6-desaturase for that organism (Stern and Tietz, 1993). It is also interesting to note that, whilst it is possible to envisage the desaturation of 1653 fatty acids (which are usually confined to the sn-2 position of MGDG), if a D6-desaturase had a plastidial location, the formation of any 1654 fatty acids was not observed. Since no transfer of 1653 fatty acids to the ER occurs, this also indicates an endomembrane location for the borage D6desaturase. The D6-desaturase has also been proposed to use two fatty acids as substrates, LA for the production of GLA, and ALA for the production of OTA (Griffiths et al., 1996), based on data from in vitro experiments which indicated that GLA could not be further desaturated by a D15-desaturase to yield OTA. These data show no obvious correlation between the levels of GLA and OTA and that of the substrates ( linoleic acid and ALA), indicating that the flux through the pathway is not determined solely by substrate levels. Similarly, it is not known whether the presence of GLA or OTA convey any selective advantage, but their distribution in species of botanically-unrelated families suggests they may be selectively neutral. 1651 In conclusion, this study indicates that the expression of the borage D6-desaturase results in the accumulation of D6-unsaturated fatty acids in a range of different lipid classes. It is clear from expression of the D6-desaturase in yeast (Napier et al., 1998; O Sayanova et al., unpublished data) that this enzyme is localized to the ER, it also implies that fatty acids such as GLA and OTA are synthesized by the ‘eukaryotic’ pathway and are then subsequently transferred to the plastid for MGDG synthesis. Since the observed accumulation of these novel fatty acids in the lipids of transgenic tobacco or borage is likely to reflect the ‘steady-state’ of the process, it is also possible that more dynamic (short-term) fluxes of fatty acids take place within the cell. 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