Accumulation of D6-unsaturated fatty acids in transgenic tobacco plants expressing a D6-desaturase from Borago officinalis

Transcription

1 Journal of Experimental Botany, Vol. 0, No. 340, pp , 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 CV3 9EF, UK Received 19 April 1999; Accepted 12 July 1999 Abstract Transgenic tobacco lines expressing the borage D6- fatty acid desaturase were characterized for the accu- mulation 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 D6- desaturated 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 differ- ent 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. Key words: Borage, fatty acid desaturase, c-linolenic acid, transgenic tobacco. Introduction 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 bio- technology is the modification of the lipid profile of oilseeds ( Topfer et al., 199). 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; 183D6,9,12) 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; 204D,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 4 To whom correspondence should be addressed. Fax: jon.napier@bbsrc.ac.uk 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

2 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 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; 184D6,9,12,1). 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; 182D9,12) and a-linolenic acid ( ALA; 183D9,12,1) (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., 199). Total fatty acids were analysed as fatty acid methyl esters (FAMEs) using a modified extraction method (Bligh and Dyer, 199). 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 g of leaf tissue was used for extraction (using the method of Bligh and Dyer, 199), and TLC of the chloroform phase was used to purify either monogalactosyldiacylglycerol (MGDG) or phosphatidylcholine (PC). MGDG was eluted from the silica in chloroformmeth- anol (12, vv), 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 2 C. Results Transgenic tobacco plants expressing the borage D6-fatty acid desaturase under the control of the cauliflower mosaic virus constitutive (3S) 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% GLAcontaining. 3 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.% and 14.9% in young and mature leaves, respectively. GLA and OTA

3 D6-unsaturated fatty acids 1649 Table 1. Total fatty acid compositions of leaves and mature seeds usually mirrored by a decrease in the levels of LA of either control transformed (WT) or a homozygous line and/or ALA. expressing the borage D6-desaturase (c2) In view of the low levels of GLA and OTA observed Fatty acid methyl esters were analysed by GC and quantified against in the mature seeds of the c2 tobacco line, the fatty acid 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 composition of developing transgenic tobacco seeds was 3% of that of the mean. determined, at three defined stages of seed development Fatty acid Young leaves Old leaves Mature seeds (Bearson and Lamppa, 1993; Napier et al., 199). These were categorized as: S1 (early seed development; white WT c2 WT c2 WT c2 seeds), S2 (mid-stage seed development; opaque seeds) and S3 ( late stage development; brown seeds). The distri bution of fatty acids shows (Table 3) that there are significant amounts of GLA (16.6%) and OTA (2.%) at the early stages (S1) of seed development, but that the proportions of both decline rapidly over a short develop mental period. Although this decline coincides with a GLA major increase in the level of LA, this LA clearly does OTA 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 were not observed in the leaves of the control tobacco development when expressed as nmol fatty acid mg 1 plants. There were also decreases in the levels of LA and fresh weight ( Table 4), although the increase was small ALA in the c2 lines, compared with the control. The in comparison with the substantial increase in the total occurrence of GLA and OTA in mature (i.e. fully amounts of fatty acids synthesized over this period. developed and desiccated) seeds was also determined. Analysis of the lipid classes of the S1 seeds showed that However, only a low level of accumulation of GLA the major class was PC, and that this glycerolipid con- (2.6%) in these mature seeds with insignificant amounts tained most of the GLA. In contrast, the GLA in the of OTA ( Table 1) was observed. Moreover, there were mature tobacco seeds was present predominantly in the no other changes in the fatty acid profile of the tobacco TAG, which is the major lipid class in the seed at this seeds, the major component being LA. (ultimate) stage of development (data not shown). Other tissues of the transgenic tobacco plants were also To determine the distribution of GLA and OTA within examined for the presence of D6-unsaturated fatty acids, and accumulation of these two fatty acids was observed Table 3. Total fatty acid composition of developing seeds of in stems, roots and floral tissues ( Table 2). The highest homozygous line c2 expressing the borage D6-desaturase amount of GLA was observed in stem tissue (27.2% of FAMEs were analysed and quantified as described in Table 1. Seeds total fatty acids), with roots also showing a high level were taken at three stages of development (S1 being the youngest, S3 (21.9%), whilst OTA in these tissues was 8.7% and.9%, the oldest), and compared with mature seeds. respectively. This indicated that the presence of these novel fatty acids was widely tolerated throughout the Fatty acid S1 S2 S3 Mature 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 GLA expressing the borage D6-desaturase (c2) OTA FAMEs were analysed and quantified as described in Table 1. Fatty acid Stems Roots Flowers Table 4. Accumulation of GLA and OTA during seed development WT c2 WT c2 WT c2 of transgenic line c The amounts of GLA, OTA and total fatty acids are expressed as nmol fatty acid mg 1 fresh weight Fatty acid Stage of seed development S1 S2 S3 Mature GLA GLA OTA OTA Total FAs

4 160 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 composi- tions determined ( Table ). 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 propor- tions 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 phospho- lipase 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 8.3%, respectively). Table. 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 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 3S 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 D6- unsaturated 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 con- trasts with the constitutive expression of a castor D12- hydroxylase 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., 199). 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. Lipid class Unlike the housekeeping fatty acids, however, ricinoleic PG MGDG PC PE TAG acid is rapidly removed from membrane PC and only accumulates in TAG (Bafor et al., 1991). Thus, mechan isms 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 GLA and OTA accumulate in membrane lipids of non- OTA seed tissues, whereas the distribution of D6-unsaturated fatty acids is restricted almost exclusively to the seeds of Table 6. Positional analysis of MGDG and PC from the lipids of Oenothera biennis (evening primrose). leaves of homozygous line c2 The low accumulation of GLA in mature seeds is The amounts of constituent fatty acids at the sn-1 and sn-2 positions perhaps surprising, given the levels of this fatty acid in 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 non-seed tissues and in seeds at early stages of develop- sn-2 position (%sn-2) is also given. ment. It is possible that this is due to the use of the viral CaMV 3S promoter, which is known to show a plateau Fatty acid MGDG PC of activity in the developing seeds of transgenic tobacco sn-1 sn-2 %sn-2 sn-1 sn-2 %sn-2 (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 desatur % % % ases. It has recently been observed that seed expression % % of a D12 hydroxylase (under the control of the CaMV-3S GLA % % promoter) resulted in higher levels of hydroxylated fatty OTA % % acids when carried out in the fad2 mutant compared with

5 D6-unsaturated fatty acids 161 the wild type ( Broun et al., 1998). The authors concluded In conclusion, this study indicates that the expression that this was due to competition between the oleate of the borage D6-desaturase results in the accumulation desaturase (i.e. FAD2) and the oleate hydroxylase. It of D6-unsaturated fatty acids in a range of different lipid may also be that tobacco seed acyltransferases for TAG classes. It is clear from expression of the D6-desaturase in assembly are less selective for the GLA or OTA than for yeast (Napier et al., 1998; O Sayanova et al., unpublished their own endogenous fatty acids, or that potential substrates data) that this enzyme is localized to the ER, it also for the D6-desaturase (such as LA) are rapidly implies that fatty acids such as GLA and OTA are transferred into TAG and rendered unavailable for further synthesized by the eukaryotic pathway and are then desaturation. subsequently transferred to the plastid for MGDG synthesis. The presence of GLA and OTA in MGDG and PC Since the observed accumulation of these novel indicates that these fatty acids are present in both plastidic fatty acids in the lipids of transgenic tobacco or borage (MGDG) and extraplastidic ( PC) membranes, demonstrating is likely to reflect the steady-state of the process, it is exchange of fatty acids between these two com- also possible that more dynamic (short-term) fluxes of partments. No GLA or OTA was detected in the PG fatty acids take place within the cell. 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 Acknowledgements desaturase. Positional analysis of plastidic galactolipids IACR-Long Ashton Research Station and Horticulture (MGDG) revealed the predominant occurrence of GLA Research International receive grant-aided support from the and OTA on the sn-2 position, which is consistent with Biotechnology and Biological Sciences Research Council the observed positions of these fatty acids in borage leaf (BBSRC) of the UK. This work was partially supported by a grant from the BBSRC under the Collaboration with Industry lipids (Griffiths et al., 1996). Although it has been sug- Scheme and Scotia Pharmaceuticals Ltd. gested 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 References form results in the incorporation of D6-desaturated fatty acids into both ER and plastidic lipids, confirming the Bafor M, Smith MA, Jonsson L, Stobart K, Stymne S Ricinoleic acid biosynthesis and triacylglycerol assembly in existence of a flux between the two compartments (Miquel microsomal preparations from developing castor-bean endoand Browse, 1998). This is in contrast to the situation sperms. Biochemical Journal 280, observed for the unicellular alga Isochrysis galbana, in Bearson SR, Lamppa GK Developmental regulation of which the complete series of desaturation reactions from an acyl carrier protein gene promoter in vegetative and 181to184 appear to take place on MGDG, implying reproductive tissue. Plant Molecular Biology 22, Bligh EG, Dyer WJ A rapid method of total lipid a plastidial D6-desaturase for that organism (Stern and extraction and purification. Canadian Journal of Biochemistry Tietz, 1993). It is also interesting to note that, whilst it and Physiology 37, is possible to envisage the desaturation of 163 fatty acids Broun P, Boddupalli S, Somerville C A bifunctional oleate (which are usually confined to the sn-2 position of 12-hydroxylase: desaturase from Lesquerella fendleri. The MGDG), if a D6-desaturase had a plastidial location, the Plant Journal 13, Christie WW Structural analysis of lipids by means of formation of any 164 fatty acids was not observed. Since enzymatic hydrolysis. In: Lipid analysis. Oxford: Pergamon no transfer of 163 fatty acids to the ER occurs, this also Press, indicates an endomembrane location for the borage D6- Gill I, Valivety R Polyunsaturated fatty acids: occurrence, desaturase. biological activities and applications. Trends in Biotechnology The D6-desaturase has also been proposed to use two 1, Griffiths G, Brechany EY, Jackson FM, Christie WW, Stymne fatty acids as substrates, LA for the production of GLA, S, Stobart AK Distribution and biosynthesis of and ALA for the production of OTA (Griffiths et al., stearidonic acid. Phytochemistry 43, ), based on data from in vitro experiments which Horrobin DF Gamma linolenic acid: an intermediate in indicated that GLA could not be further desaturated by essential fatty acid metabolism with potential as an ethical a D1-desaturase to yield OTA. These data show no pharmaceutical and as a food. Reviews in Contemporary Pharmacotherapy 1, 1 4. obvious correlation between the levels of GLA and OTA Miquel M, Browse J Arabidopsis lipids: a fat chance. and that of the substrates ( linoleic acid and ALA), Plant Physiology and Biochemistry 36, indicating that the flux through the pathway is not Napier JA, Hey SJ, Lacey DJ, Shewry PR Identification determined solely by substrate levels. Similarly, it is not of a Caenhorhabditis elegans D6 fatty acid desaturase by known whether the presence of GLA or OTA convey any heterologous expression in Saccharomyces cerevisiae. Bio- chemical Journal 330, selective advantage, but their distribution in species of Napier JA, Sayanova O, Stobart AK, Shewry PR A new botanically-unrelated families suggests they may be select- class of cytochrome b fusion proteins. Biochemical Journal ively neutral. 328,

6 162 Sayanova et al. Napier JA, Smith MA, Stobart AK, Shewry PR Isolation unicellular alga Isochrysis galbana: studies with intact and of a cdna encoding a cytochrome b specifically expressed broken chloroplasts. Biochimica et Biophysica Acta 1167, in developing tobacco seeds. Planta 197, Sayanova O, Smith MA, Lapinskas P, Stobart AK, Dobson G, Stymne S, Stobart AK Triacylglycerol biosynthesis. In: Christie WW, Shewry PR, Napier JA Expression of Shewry PR, Stobart AK, eds. Seed storage compounds. a borage desaturase cdna containing an N-terminal Oxford University Press, cytochrome b domain results in the accumulation of high Topfer R, Martini N, Schell J Modification of plant lipid levels of D6-desaturated fatty acids in transgenic tobacco. synthesis. Science 268, Proceedings of the National Academy of Sciences, USA 94, van de Loo FJ, Broun P, Turner S, Somerville C An oleate 12-hydroxylase from Ricinus communis L. is a fatty Shanklin J, Cahoon EB Desaturation and related acid desaturase homolog. Proceedings of the National modifications of fatty acids. Annual Reviews of Plant Academy of Sciences, USA 92, Physiology and Plant Molecular Biology 49, van de Loo FJ, Fox BG, Somerville C Unusual fatty acids Slocombe SP, Piffanelli P, Fairburn D, Bowra S, Hatzopoulos in plants. In: Moore TS, ed. Lipid metabolism in plants. Boca P, Tsiantis M, Murphy DJ Temporal and tissue-specific Raton: CRC Press, regulation of a Brassica napus stearoyl-acyl carrier protein Vogel G, Browse J Cholinephosphotransferase and desaturase gene. Plant Physiology 104, diacylglycerol acyltransferase. Plant Physiology 110, Stern N, Tietz A Octadecatetraenoic synthesis in the