Download Accumulation of D6-unsaturated fatty acids in transgenic tobacco

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.
Acknowledgements
IACR-Long Ashton Research Station and Horticulture
Research International receive grant-aided support from the
Biotechnology and Biological Sciences Research Council
(BBSRC ) of the UK. This work was partially supported by a
grant from the BBSRC under the Collaboration with Industry
Scheme and Scotia Pharmaceuticals Ltd.
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