Download Very-long-chain fatty acid biosynthesis is controlled through the

The Plant Journal (1997) 12(1), 121-131
Very-long-chain fatty acid biosynthesis is controlled
through the expression and specificity of the condensing
enzyme
Anthony A. Millar and Ljerka Kunst*
Department of Botany, University of British Columbia,
Vancouver, BC V6T IZ4, Canada
Summary
The Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene
encodes a putative seed-specific condensing enzyme. It
is the first of four enzyme activities that comprise the
microsomal fatty acid elongase (FAE) involved in the
biosynthesis of very-long-chain fatty acids (VLCFAs). FAEI
has been expressed in yeast and in tissues of Arabidopsis
and tobacco, where significant quantities of VLCFAs are
not found. The introduction of FAE1 alone in these systems
is sufficient for the production of VLCFAs, for wherever
FAE1 was expressed, VLCFAs accumulated. These results
indicate that FAE1 is the rate-limiting enzyme for VLCFA
biosynthesis in Arabidopsis seed, because introduction of
extra copies of FAE1 resulted in higher levels of the
VLCFAs. Furthermore, the condensing enzyme is the activity of the elongase that determines the acyl chain length
of the VLCFAs produced. In contrast, it appears that the
other three enzyme activities of the elongase are found
ubiquitously throughout the plant, are not rate-limiting
and play no role in the control of VLCFA synthesis. The
ability of yeast containing FAE1 to synthesize VLCFAs
suggests that the expression and the acyl chain length
specificity of the condensing enzyme, along with the
apparent broad specificities of the other three FAE activities, may be a universal eukaryotic mechanism for regulating the amounts and acyl chain length of VLCFAs
synthesized.
Introduction
Very-long-chain fatty acids (VLCFAs) have chain lengths
from 20 to 30 carbons, or greater. The major site of VLCFA
synthesis is the epidermal cells where they are utilized for
the production of waxes embedded in the suberin and
cutin and the epicuticular waxes which cover the aerial
surfaces of the plant. The VLCFAs are either directly incorporated into waxes, or act as precursors for other aliphatic
hydrocarbons found in waxes, including alkanes, second-
Received7 November1996;revised 13 February1997;accepted3 March
1997.
*For correspondence(fax + 1 604 822 6089;e-mail [email protected]).
ary and primary alcohols, ketones, aldehydes and acylesters (for review see Post-Beittenmiller, 1996). VLCFAs
also accumulate in the seed oil of some plant species,
where they are incorporated into triacylglyerols (TAGs),
as in the Brassicaceae, or into wax esters, as in jojoba
(Simmondsia chinensis). These seed VLCFAs include the
agronomically important erucic acid (C22:1), with oils containing this fatty acid used in the manufacture of lubricants,
nylon, cosmetics, pharmaceuticals and plasticisers (Battey
et aL, 1989; Johnson and Fritz, 1989). Conversely, VLCFAs
have detrimental nutritional effects and are therefore
undesirable in edible oils. This has led to the breeding of
canola rapeseed varieties that are almost devoid of VLCFAs
(Stefansson et al., 1961).
VLCFAs are synthesized by a microsomal fatty acid
elongation (FAE) system by sequential additions of C2
moieties from malonyl coenzyme A (CoA) to pre-existing
C18 fatty acids derived from the de novofatty acid synthesis
(FAS) pathway of the plastid. Analogous to de novo FAS,
it is thought that each cycle of FAE involves four enzymatic
reactions: (i) condensation of malonyl CoA with a long
chain acyl CoA; (ii) reduction to 13-hydroxyacyl CoA; (iii)
dehydration to an enoyl CoA; and (iv) reduction of the
enoyl CoA, resulting in an elongated acyl CoA (Fehling and
Mukherjee, 1991). Together, these four activities are termed
the elongase (von Wettstein-Knowles, 1982).
The seeds of Arabidopsis contain approximately 28%
(w/w of total fatty acids (FA)) of VLCFAs, esicosenoic acid
(20:1) being the predominant VLCFA (21% w/w of total FA).
To identify the gene products that are involved in the
synthesis of seed VLCFAs and establish the VLCFA biosynthetic pathway, several groups performed mutational analysis and screened for seed that had reduced VLCFA
content. Each group independently identified the FATTY
ACID ELONGATION1 gene (FAEl;James and Dooner, 1990;
Kunst et aL, 1992; Lemieux et aL, 1990). A mutation at this
locus resulted in reduced VLCFA levels (<1% w/w of total
FA) in the seed. Several other mutations that were nonallelic to FAElwere also isolated. However, these mutations
had a less pronounced effect, and VLCFAs still constituted
6-7% (w/w of total FA) of the seed fatty acid (Katavic et aL,
1995; Kunst et aL, 1992). Thus, despite the fact that four
enzymatic activities are required for each elongation step,
the FAE1 gene was the only one found by mutant analysis
that resulted in almost complete abolishment of VLCFA
synthesis in the seed.
The Arabidopsis FAE1 gene was subsequently cloned
121
122
A n t h o n y A. Millar and Ljerka Kunst
(James et al., 1995), and showed homology to three condensing enzymes: chalcone synthase, stilbene synthase
and 13-ketoacyl-[acyl carrier protein] synthase III (17 amino
acids were identical to a 50 amino acid region of a consensus sequence for condensing enzymes). Based on this
homology, it was proposed that FAE1 encodes a J3-ketoacylCoA synthase (KCS), the condensing enzyme which catalyses the first reaction of the microsomal fatty acid elongation system (James etaL, 1995). As determined by Northern
analysis, the FAE1 gene is expressed in seeds of Arabidopsis, but is absent from leaves (James et al., 1995). This
result is consistent with the fact that the fael mutation
affects only the fatty acid composition of the developing
seed, having no pleiotropic effects on fatty acid composition of the vegetative or floral parts of the plant. Thus,
FAE1 is regarded as a seed-specific condensing enzyme.
However, to date, the function of the FAE1 gene product
has not been confirmed, nor has FAE1 been used in
transgenic experiments to determine its activity in vivo.
Recently, a cDNA from jojoba seeds involved in the
synthesis of VLCFAs has been isolated (Lassner et al.,
1996). The protein encoded by this cDNA showed high
homology to FAE1 (52% amino acid identity), and biochemical analysis demonstrated that it has a KCS activity. Using
the jojoba KCS cDNA, Lassner et aL (1996) were able to
complement the mutation in a canola variety of Brassica
napus, restoring a low erucic acid rapeseed to a line that
contained higher levels of VLCFAs. This suggests that, in
canola, the mutation is in the structural gene encoding
KCS, or a gene affecting KCS activity. Thus, both in Arabidopsis and Brassica napus, the mutations that result in
the abolishment of VLCFA synthesis seem to affect the
condensing enzyme. If four enzyme activities are necessary
for an elongation step, and FAEI and jojoba KCS 0nly
encode the KCS activity, one might expect to find other
complementation groups that result in very low levels of
VLCFA synthesis. There are several possibilities as to why
mutations in gene(s) encoding the reductases and the
dehydrase have not been found. For example, these
enzymes may be encoded by members of gene families.
Alternatively, they may not be seed-specific, but ubiquitously present throughout the plant and shared with other
FAE systems involved in VLCFA formation. If any of these
VLCFA products were essential for plant viability, a mutation in one of the reductase or dehydrase genes would be
lethal, and go undetected.
If the dehydrase and the reductase activities are ubiquitously present, it would be the condensing enzyme that is
unique to each FAE system, and the expression of this
enzyme would determine whether VLCFAs are synthesized
or not. To examine this possibility, we have expressed the
FAE1 gene ectopically in vegetative and floral tissues of
Arabidopsis, seeds of tobacco and yeast. We demonstrate
that FAE1 activity is sufficient to result in the synthesis of
VLCFAs in tissues where they are not usually found.
Results
A p35S-FAE1 transgene directs the synthesis of VLCFAs
in vegetative and floral tissues in Arabidopsis
The fatty acyl composition was determined in leaves, roots,
flowers and stems of wild-type Arabidopsis to ascertain
the levels of VLCFAs present (Figure 1). In whole flowers,
leaves and stems, only very low levels of saturated VLCFAs
were present. These VLCFAs are believed to originate from
the cuticular waxes whose biosynthesis occurs almost
exclusively in epidermal cells (Lessire et al., 1982). However, in roots, high levels of saturated VLCFAs were found,
with 22:0 being the major fatty acid. It is likely that VLCFAs
in roots are components of the waxes associated with the
suberin, and are presumably made by a FAE system similar
to the one found in seeds. The mutation in the fael gene
does not affect VLCFA levels in roots (Millar and Kunst,
unpublished results), indicating that a condensing enzyme
other than FAE1 is involved in VLCFA biosynthesis in
the roots.
To determine whether the expression of FAE1 in vegetative and floral tissues could result in the synthesis of
VLCFAs, we used the binary vector construct p35S-FAE1
to constitutively express FAE1 in Arabidopsis. Using a
combination of in planta and vacuum-infiltration methods,
more than 100 kanamycin-resistant Arabidopsis plants
were obtained. The fatty acyl composition of leaves from
all transformants was determined. Compared with wildtype leaves, the leaves from many of the transgenic plants
had a dramatically altered fatty acid profile, with eight
additional peaks being present in the GC chromatograms.
The retention times of these eight peaks coincided with
those corresponding to VLCFAs that are found in the seed
of Arabidopsis. GC-MS of the pentafluorobenzyl ester
derivatives confirmed these peaks to be 20:0, 20:1, 20:2,
20:3, 22:0, 22:1, 22:2 and 22:3 fatty acids. Thus, the expression of FAE1 alone is able to direct the synthesis of VLCFAs
in tissues which do not normally produce these fatty acids,
which is consistent with the hypothesis that FAE1 is a
condensing enzyme involved in VLCFA synthesis.
One transgenic line was examined in detail. T2 seeds
were sown and the fatty acyl composition was determined
in leaves, flowers, roots and stems of 7-week-old plants
(Figure 1). The increases in proportion of VLCFAs in all
four parts of the plant were similar, with the highest
increase occurring in the flowers. Both saturated and
unsaturated VLCFAs were synthesized, demonstrating that
18:0 and 18:1 fatty acids could serve as primers for elongation by FAE1 in all parts of the plant. The fact that the
synthesis of the saturated VLCFAs occurs at the expense
Condensing enzyme controls VLCFA biosynthesis 123
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Figure 1. The accumulation of VLCFAs in vegetative and floral tissues of transgenic p35S-FAEIArabidopsis plants.
Fatty acid compositions were determined in leaves, roots, flowers and stems of wild-type and one transgenic line. Fatty acid levels are shown as the weight
percentage of total fatty acids. Each bar represents the average of five samples, with each sample composed of tissues pooled from at least five plants.
of 16:0 suggests that FAE1 is able to elongate C16 fatty
acids. The synthesis of the unsaturated VLCFAs seemed to
occur at the expense of 18:3 in leaves, stems and flowers.
In roots, the proportion of 18:2 also decreased in the
transgenic plants when compared to wild-type levels.
These results indicated that in all parts of the plant the
dehydrase and two reductases necessary for elongation
are constitutively present (or are induced by FAE1). In
addition, fatty acids, malonyl CoA and the co-factors that
are required for elongation by FAE1 were all available.
Independent transgenic lines accumulated different
quantities of VLCFAs. In the leaves of 7-week-old transgenic
124
Anthony A. Millar and Ljerka Kunst
found between transgenic lines are due to varying levels
of transgene expression.
Finally, we found that independently isolated lines with
the highest levels of VLCFAs had a very unusual morphological phenotype. These plants had reduced apical dominance, stunted growth, waxless inflorescences, and altered
floral morphology (Millar and Kunst, unpublished results),
and are presently being characterized in detail.
A pNAPIN-FAE1 transgene is able to direct the synthesis
of VLCFAs in the seeds of tobacco
Figure2. Presenceof the FAEItranscriptin leavesof transgenicplants.
(a) RNA gel blot analysisof total RNA isolatedfrom leavesof wild-type
plants (wt) and three transgeniclines (#9. 36 and 60) hybridizedto the
FAEI gene.
(b) Ethidiumbromidestainingof RNA usedfor the RNAgel btot in (a).
plants, we observed from 4-5% (w/w of total FA) of VLCFAs
in some lines to approximately 30% (w/w of total FA) in
others. Northern analyses were performed on leaves of
several transgenic lines to confirm the presence of the FAE1
transcript in transgenic tissue, and to examine whether the
levels of expression of the 35S-FAE1 transgenes correlated
with the level of accumulation of VLCFAs. RNA was isolated
from leaves of 7-week-old wild-type plants and three independent transgenic lines (#9, 36 and 60) that were accumulating 13.8%, 11.7% and 19.4% (w/w of total FA) of VLCFAs
respectively. The leaves of all three transgenic lines contained FAE1 transcript, whereas wild-type leaves did not
(Figure 2). In addition, the line accumulating the highest
level of VLCFAs also had the highest level of FAEltranscript
(Figure 2, #60), implying that the different levels of VLCFAs
Unlike Arabidopsis, VLCFAs are not present at significant
levels in seeds of tobacco. GC analysis of wild-type tobacco
seeds showed that the peaks whose retention times coincide with those of methyl-20:0, -20:1, -20:2, -22:0 and 22:1 total less than 0.5% of the fatty acyl content (Figure
3a). To determine whether the addition of FAE1 activity to
the seeds of tobacco is sufficient for the production of
VLCFAs, we generated transgenic plants expressing FAE1
in their seeds.
For targeting of FAE1 gene product to the seed, we
cloned the FAE1 coding region behind the Napin seed
storage protein promoter of Brassica napus in the plant
expression vector pNGKM (Kohno-Murase et al., 1994).
Tobacco was transformed using Agrobacterium, and 23
different transgenic plants were regenerated. The fatty acid
compositions of pooled mature seeds from all primary
transformants were determined. The majority of transformants had a seed oil that contained three extra fatty
acids whose retention times coincided with those of
methyl-20:0, -20:1 and -20:2 (Figure 3b). These fatty acids
accounted for 1-2.5% (w/w) of the total fatty acids present.
Although VLCFAs were being synthesized, their accumulation was low when compared to wild-type Arabidopsis.
Low VLCFA levels may be due to weak expression of
FAE1, as the strength of the Brassica napus Napin promoter
in tobacco is unknown. Because fatty acid compositional
analysis was performed on T2 seed segregating for the
presence of the trangene(s), we anticipated that T3 seeds
would contain higher levels of VLCFA. Therefore, seeds
from the T2 line with the highest levels of VLCFAs (2.5%
w/w of total FA) were sown, and pooled seed fatty acid
compositions of the progeny were determined. Several T3
lines had a VLCFA content of over 4% (w/w of total FA).
We then analysed single T4 seeds from one of these lines,
and found seeds that were accumulating over 4.8% VLCFAs
(w/w of total FA).
The Arabidopsis FAE1 gene is able to direct the synthesis
of VLCFAs in yeast
For expression in yeast, the coding region of FAE1 was
linked to the galactose-inducible GALl promoter in the
Condensing enzyme controls VLCFA biosynthesis
(a)
125
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10
Retention time (min)
Retention time (min)
Figure 3. FAE1 directs VLCFA synthesis in tobacco seeds.
Gas chromatograms of fatty acid methyl esters prepared from (a) wild-type tobacco seed and (b) transgenic pNapin-FAElseed.
Saccharomyces cerevisiae expression vector, pYES2. When
S. cerevisiae cells harbouring this pYES-FAE1 plasmid
were grown in the presence of galactose, fatty acids with
retention times that coincided with VLCFAs were synthesized. Yeast showed accumulation of 20:1, 22:1 and 20:0
VLCFAs that were not normally present and were not
detected in cultures of yeast transformed with the pYES2
ptasmid alone (Table 1). Therefore, in yeast, the dehydrase
and two reductases necessary for elongation are also
constitutively present, or are induced by FAE1.
Introduction of a pNapin-FAE1 transgene results in
increased synthesis of VLCFAs in Arabidopsis seed
The semi-dominant nature of the FAEI mutation in Arabidopsis (Kunst et al., 1992) points to the condensing enzyme
activity as the limiting factor for VLCFA biosynthesis. This
suggested that over, expression of FAE1 should result in
an increase in the VLCFA content in the seed oil. A detailed
analysis of independently isolated p35S-FAE1 transgenic
lines showed that the best VLCFA accumulators in leaves
exhibited only modest increases (5-6% w/w of total FA) in
VLCFA content of their seeds. Since p35S-FAE1 plants have
an unusual morphology, and high levels of FAE1 expression
in vegetative parts of the plant may even be lethal (Millar
and Kunst, unpublished results), it is possible that higher
VLCFA accumulation in seeds could not be achieved using
the 35S promoter. Therefore, we decided to use the pNapin-FAE1 construct to specifically target FAE1 expression
to the seeds. From e vacuum-infiltration transformation
experiment, 35 kanamycin-resistant T2 plants were reco-
126
Anthony A. Millar and Ljerka Kunst
Table 1. VLCFAcomposition of transgenic (pYES-FAE1)yeast
pYES (no insert)
pYES-FAEI
16:0
16:1
18:0
18:1
20:1
22:1
10.12
8.52
54.97
60.14
3.13
2.13
28.15
19.52
0
4.38
0
0.45
Values are the weight percentage of total fatty acids.
vered. The T3 seed was collected individually from each
transformant and the fatty acyl composition determined.
The majority of transgenic lines either contained wild-type
VLCFA levels, or exhibited a decrease in their VLCFA
content, the latter presumably due to the mechanism
termed co-suppression. However, four independent lines
(#35, 12, 13, 8) were found to have a significantly higher
content of VLCFAs. Instead of 27-28% (w/w) of the total
fatty acids found in wild-type Arabidopsis (Columbia-4)
seeds, the proportion of VLCFA in these lines increased to
38-40% (w/w) of the total fatty acids. Because T2 plants
are hemizygous for every T-DNA insertion, which will
segregate in T3 seed, we expected that the lines homozygous for T-DNA inserts would have a higher FAE1 activity
and thus a higher proportion of VLCFA. To establish this,
T4 seeds from the "1"3 plants were analysed for all four
lines, #35, 12, 13 and 8. In line 13, the VLCFA content of
the T4 progeny fell into one of three distinct groups. Eight
plants had wild-type levels of VLCFAs (approximately 27%),
23 plants had VLCFAs levels similar to the T3 parent plant
(approximately 38%) and seven plants had a new higher
VLCFA content (approximately 42.5% w/w of total FA)
(Figure 4). The ratios of these three groups suggest that
the original T2 line 13 had one functional T-DNA locus and
that the T3 progeny with the higher level of VLCFAs in
their seed were homozygous at this T-DNA locus. However,
the increase in the proportion of VLCFA content from the
hemizygote to the homozygote (4.5%) was significantly
less than the corresponding increase from the wild-type to
the hemizygote (11%). Since the increase in the proportion
of VLCFAs did not follow the increase in pNapin-FAE1 gene
dosage, it may be that some other factor became limiting
in the accumulation of VLCFAs. In addition, none of the
other three T2 lines examined, (#35, 12 and 8) had progeny
that accumulated VLCFA levels higher than 43% (w/w of
total FA). Collectively, these data suggest that 42-43%
represents the limit of VLCFA accumulation that can be
obtained in seeds by over-expressing FAE1 alone.
Discussion
This paper examines the control and organization of VLCFA
biosynthesis. Four enzymatic activities are thought to be
required for each fatty acid elongation step. However, we
found that, in plants, it is the expression of the condensing
enzyme (or ~-ketoacyl CoA synthase) that determines
whether VLCFAs are synthesized in a particular cell. Furthermore, the condensing enzyme is the activity of the elongase
that determines the amounts and chain lengths of VLCFAs
produced.
Expression of FAE1 protein results in the synthesis of
VLCFAs
Both in transgenic Arabidopsis and tobacco, ecotopic
expression of FAE1, a putative Arabidopsis seed-specific
condensing enzyme (James et al., 1995), results in the
accumulation of VLCFAs in parts of the plant where they
are not usually present. These data support the hypothesis
that FAE1 is indeed a condensing enzyme involved in
VLCFA synthesis. Further, the data demonstrate that the
introduction of a condensing enzyme alone is sufficient
for the synthesis of VLCFAs. The high levels of VLCFA
accumulation in the p35S-FAEltransgenic plants suggests
that VLCFA biosynthesis is not confined to the epidermal
cells as in the wild-type, but rather takes place in the
majority of the plant's cells. This is surprising, because it
implies that the other three activities of the elongase, the
two reductases and the dehydrase, must be expressed
throughout the plant. Consistent with this finding are the
results obtained by mutational analyses of the VLCFA
biosynthetic pathway. Since no mutations were found in
the genes encoding the reductases or the dehydrase, the
possibility was raised that mutations in these genes result
in a lethal phenotype, because these enzymes were shared
with other, presently undefined, essential FAE pathways.
In principle, the three activities could be common to all
microsomal FAE systems, including those that make
VLCFAs for waxes and sphingolipids. Sphinigolipids are
found in membranes of eukaryotic cells (Lynch, 1993), thus
providing a rationale for the ubiquitous presence of these
three activities. Therefore, the organization of the microsomal FAE systems would be similar to that of plastidic
FAS, with the condensing enzyme being the only unique
component of each elongase.
Condensing enzyme controls VLCFA biosynthesis
50
127
...........................................................................................................................................................................................................................
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18:1
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18:3
20:0
20:1
20:2
20:3
22:0
1
22:1
Total
VLCFAs
Fatty Acid
Figure 4. Over-expression of FAE1 in Arabidopsi$seeds results in a higher proportion of VLCFA accumulation.
.Fatty acid composition of total seed lipids from wild-type Arabidopsis and transganic pNapin-FAE1 plants. Each bar represents the average of five
measurements using seed pooled from five different plants, Napin-FAE1-(13-T3) are plants that are homozygous for the Napin-FAE1 transgene, whereas in
Napin-FAE1-(13-T2) plants, the transgene is hemizygous.
FAEI protein controls the amounts and the acyl chain
length of VLCFA products
Incomplete dominance of the wild-type FAEI gene over
mutant fael alleles implies that FAE1 activity is the ratelimiting factor of VLCFA synthesis in Arabidopsis seed.
Transgenic plants were obtained with a significantly higher
proportion of seed VLCFAs (Figure 4), confirming that FAE1
activity is the rate-limiting factor of VLCFA synthesis in
wild-type Arabidopsis. In addition, independently derived
p35S-FAE1 transgenic lines contain very different amounts
of VLCFAs in their leaves, presumably due to different
levels of FAE1 expression. These results support the notion
that the proportion of fatty acids channelled into VLCFA
synthesis is dependent on the levels of FAE1 activity.
This is not unexpected, because regulation of metabolic
pathways often occurs at the first committed step of a
pathway, and FAE1 catalyses the first, irreversible reaction
of VLCFA biosynthesis.
In addition to controlling the amount of VLCFAs, the
condensing enzyme also appears to control the acyl
chain length of VLCFAs made. To date, two different
condensing enzymes have been expressed in Arabidopsis
seed under a Napin promoter - FAE1 from this study,
and the jojoba KCS from the study by Lassner et aL
(1996). A comparison of the seed fatty acid profiles of
these two populations of transgenic Arabidopsis plants
reveals that, on average, VLCFAs synthesized by plants
with the jojoba KCS enzyme have a greater chain length
than those made by plants over-expressing FAE1. For
example, seed of transgenic pNapin-jojoba-KCS plants
produces 62.7% C20, 27.6% C22 and 9.6% C24 of total
VLCFA (Lassner et al., 1996), whereas seed of pNapinFAE1 transformants contain approximately 81.6% of C20,
and 17.7% of C22 of total VLCFA. Very low levels of C24
acyl groups (0.3%) are also present in pNapin-FAE1
transgenic plants. However, similar levels are found in
wild-type Arabidopsis, and even in fael mutants. Thus,
since the levels of FAE1 expression do not seem to
affect C24 content, we suspect that C24 VLCFAs are
made by a condensing enzyme other than FAEI. In
addition, in contrast to the jojoba KCS, FAE1 is unable
to synthesize C24 fatty acids in p35S-FAE1 Arabidopsis,
in transgenic pNapin-FAE1 tobacco, or yeast. In all cases,
the expression of FAE1 results only in C20 and C22
VLCFA production. These data strongly suggest that
FAE1 and jojoba KCS have different acyl chain length
specificities in vivo, and that the specificity of the
condensing enzyme is the major factor determining the
lengths of VLCFAs made by each FAE system. This
situation is analogous to the FAS in the plastid, where
there are three condensing enzymes with strict acyl chain
length specificities, KASIII (C2 to C4), KASII (C16 to C18)
and KASI (C4 to C16) (Ohlrogge et al., 1993). In contrast,
the reductases and the dehydrase are shared between
all three elongating systems, apparently having broad
acyl chain length specificities (Shimakata and Stumpf,
1982; Stumpf, 1984).
128
Anthony A. Millar and Ljerka Kunst
A model for the organization and control of the VLCFA
biosynthetic pathway
Based on our findings described above, we propose a
model for the organization and regulation of VLCFA synthesis in plants. Of the four enzymes involved in VLCFA
biosynthesis, the two reductases and the dehydrase are
expressed throughout the plant and are common to all
microsomal FAE systems. They have broad substrate specificities and are expressed at levels that will not be ratelimiting for VLCFA biosynthesis. In contrast, the condensing
enzymes seem to be differentially expressed, are probably
unique to each FAE system, and have limited substrate
specificities. Their level of expression determines the
amount of fatty acids channelled into the VLCFA pathway.
If this model is correct, the plant would only have to
regulate the activity of one enzyme to adjust the rate
of VLCFA biosynthesis and specify the types of VLCFAs
produced by each elongase. In support of this hypothesis,
we investigated the expression patterns of two cDNAs
from the Arabidopsis expressed sequenced tag (EST)
sequencing project, which had high sequence similarity
to FAEI. Northern analyses showed that they are both
differentially expressed, one being specific to flowers
whereas the other exhibits its highest levels of expression
in stems (Millar and Kunst, data not shown). So, together
with FAE1, all three condensing enzymes analysed are
differentially expressed.
The control of VLCFA biosynthesis by the condensing
enzyme may not just be limited to plants. Our results show
that FAE1 expression in yeast results in the formation of
the same types of VLCFAs characteristic of the Arabidopsis
seed. Therefore, the levels of expression and acyl chain
length specificity of the condensing enzymes may be
a universal mechanism for determining the amounts of
VLCFAs and the acyl chain length of the VLCFAs synthesized
in a particular cell.
Additional factors influence the types and amounts of
VLCFAs produced in a cell
In addition to condensing enzymes, the source and sink
will also affect the spectrum of VLCFAs and VLCFA levels
synthesized by each individual cell. For instance, FAE1 is
involved in the synthesis of both saturated and unsaturated
VLCFAs in wild-type seeds (Kunst et al., 1992), as well
as in all the examined tissues of the p35S-FAE1 plants.
However, in the leaves of p35S-FAE1 plants, saturated
VLCFAs constitute a higher proportion of the final VLCFA
content compared with seeds. This change in the proportion of saturated versus unsaturated VLCFAs synthesized
by FAE1 probably reflects the composition of the fatty acyl
CoA pool available for elongation.
In all three transgenic systems, tobacco, Arabidopsis and
yeast, VLCFAs were able to accumulate to significant levels,
indicating that there was a sink available for them. In this
paper, we have not addressed how the accessibility and
size of the sink will affect the amount of VLCFAs produced.
However, a recent study of an Arabidopsis mutant with
low diacylglycerol acyltransferase activity demonstrated
that a sink is an important factor, because reduced incorporation of VLCFAs into TAGs results in a severe repression
of VLCFA biosynthesis (Katavic et aL, 1995). Conversely,
expression of a yeast sn-2 acyltransferase (SLCl-1) in
Arabidopsis resulted in a significantly higher total oil content, as well as an increase in proportions and absolute
amounts of VLCFAs (Zou et aL, 1996), suggesting that
the size of the sink has a profound effect on VLCFA
accumulation.
Functional expression of FAE1 protein in yeast
The expression of FAE1 in yeast represents the first
example of a plant membrane-bound condensing enzyme
active in a microbial system. It indicates that the yeast
host not only provides a suitable membrane and redox
environment for the enzyme, but also that FAE1 is able to
interact with the endogenous yeast reductase and dehydrase enzymes to carry out VLCFA synthesis. This is surprising given that the elongase enzymes are thought to be
functionally organized in a complex (Bessoule et aL, 1989;
Fehling et a/.,1992). In yeast, there are no known proteins
with amino acid similarity to FAEI. Thus, the FAE complex
is either extremely flexible, or FAE1 is able to functionally
interact with the other elongase enzyme(s) without being
in a complex. The effective expression of FAE1 in yeast,
together with site-directed mutagenesis, should provide
a powerful system for the study of structure-function
relationships of plant condensing enzymes, and also of the
subcellular organization of fatty acid elongation.
The utility of condensing enzymes for metabolic
engineering
Because condensing enzymes are pivotal enzymes in the
synthesis of VLCFAs, they maybe useful for biotechnology.
For instance, the accumulation of VLCFAs in p35S-FAE1
tobacco raises the possibility of producing VLCFAs in
plant species that currently do not synthesize VLCFAs. The
availability of the ESTs may allow identification of enzyme
activities that synthesize VLCFAs up to C34 in length.
Targeting of these VLCFA condensing enzymes to seeds
may eventually lead to the production of crop plants
capable of synthesizing new, agronomically important
VLCFAs.
Development of transgenic rapeseed varieties showing
dramatically higher levels of VLCFAs, especially 22:1 for
industrial applications, is one of the major goals of oilseed
Condensing enzyme controls VLCFA biosynthesis
metabolic engineering. Currently, the best Brassica napus
cultivars are approaching 67% (w/w of the total FA) of
VLCFAs in their oil. One of the factors that precludes
further increase of VLCFAs in rapeseed oil is thought to
be lysophosphatidic acid acyltransferase (LPAAT), which
exhibits high specificity for C18 fatty acids and does not
use VLCFAs for incorporation into the sn-2 position of TAG.
Recently, Lassner et aL (1995) reported that the expression
of meadowfoam LPAAT in rapeseed resulted in incorporation of 22:1 into the sn-2 position of TAGs in transgenic
seed and very limited production of trierucin. However,
the introduction of the meadowfoam LPAAT did not cause
an increase in seed oil 22:1 content. One possible reason
for this could be that the levels of 22:1 in the seed acyl
CoA pool may be too I o w t o support high levels of trierucin
biosynthesis. If so, over-expression of VLCFA condensing
enzymes will be required to boost VLCFA production. On
the other hand, unlike meadowfoam LPAAT, expression
of a yeast sn-2 acyltransferase (SLCl-1) in Arabidopsis
resulted in an increase in proportions and absolute
amounts of VLCFAs, as well as an increase in total oil
content (Zou et aL, 1996). Thus, the co-expression of the
yeast acyltransferase and FAE1 condensing enzyme may
allow a unique opportunity to achieve high levels of VLCFA
accumulation.
In addition to directing VLCFA production for seed oils,
one would anticipate that condensing enzymes will also
play key roles in the synthesis of VLCFAs for incorporation
into waxes. Waxes play important roles in many physiological processes in plants, including water balance, plantinsect interactions, protection from UV light, and defence
against bacterial and fungal pathogens (Post-Beittenmiller,
1996). The availability of condensing enzymes with different acyl chain length specificities, together with the ability
to manipulate their levels of expression in epidermal cells,
may allow us to alter wax composition and/or accumulation. This may in turn result in the production of crops
with increased tolerance to environmental stresses and/or
resistance to pathogens.
Experimental procedures
Construction of transformation vectors
The coding region of the FAE1 gene was amplified by PCR using
10 ng of Arabidopsis thaliana (L.) Heynh. (ecotype Columbia) DNA
and 50 pmol of oligonucleotide primers 5'-CGTCGACCAAAGAGGATACATAC-3' and 5'-GGCGGCCGGAACGTTGGCACTCAGATACATC-3', designed according to the sequence of the FAEI gene as
determined by James et aL (1995). Conditions used were 95°C for
5 min, 60°C for 4 min, then 30 cycles of denaturation (94°C for 45
s), annealing (50°C for 1 min), and extension (72°C for 45 s),
followed by a final 72°C incubation for 6 min. DNA was amplified
with the high fidelity Pfu polymerase derived from Pyrococcus
furiosus (Stratagene) in 50 mM Tris-HCI pH 8.3, 2 mM MgCI2, 50
mM KCI, 1 mM DTT and 0.2 mM of each dNTP. The resulting 1.6
129
kb PCR fragment product was blunt-end cloned into the Hincll
site of pT7T3 18U (Pharmacia) in both orientations, resulting in
the plasmids pT3FAE1 (5' end of FAEI adjacent to the Xbal site
of the polylinker) and pT7FAE1 (5' end of FAE1 adjacent to the
Psi1 site of the polylinker). Sequencing confirmed that the PCR
product was indeed the FAEI gene.
For constitutive expression in Arabidopsis, we used the vector
pJD330 described by Shaul and Galili (1992), which contained the
35S promoter of CaMV and the Q translational enhancer (Gallie
et al., 1987). Using the Sail and Sad restriction sites, FAE1
was cleaved from pT7FAE1 and inserted into the Sail and Sad
restriction sites of pJD330, replacing the ~-glucuronidase (GUS)
gene in the process. This plasmid was subcloned into the EcoRI/
Hindlll sites of pBIN19, resulting in the p35S-FAE1 binary vector.
For seed-specific expression, we used the binary vector pNGKM,
which contains the promoter of the gene encoding the Napin seed
storage protein from Brassica napus (Kohno-Murase et aL, 1994).
To generate the pNapin-FAE1 construct, FAE1 was first released
from pT7FAE1following digestion with Sail and Sacl, and inserted
into the Xhol and Sacl sites of pGEM-7Zf(+) (Promega). This
placed an Xbal site Upstream of FAE1 which was needed for
subcloning into pNGKM. FAE1was then cleaved from this recombinant plasmid with Xbal and Sacl and inserted into the Xbal and
Sact sites of pNGKM, replacing the GUS gene.
The FAE1 gene was also subcloned into the Saccharomyces
cerevisiae expression vector pYES2 (Invitrogen) which contained
the GALl inducible promoter. FAE1 was cleaved from pT3FAEI
with Sphl and Kpnl and inserted into the Sphl and Kpnl sites of
the polylinker of pYES2, resulting in the plasmid pYES-FAEI. The
construct was transformed into the S. cerevisiae LRB520 strain by
standard methods (Schiestl and Gietz, 1989) and transformants
selected on minimal medium agar plates lacking uracil (Ausubel
et aL, 1995).
Plant transformation
Both binary vectors were electroporated into Agrobacterium tumefaciens strain GV3101 (Koncz and Schell, 1986). The p35S-FAE1
Agrobacterium transformants were selected on LB medium containing 25 p,g/mL-1 gentamycin and 50 I~g m1-1 kanamycin. The
pNapin-FAE1 Agrobacterium transformants were selected on 50
p.g m1-1 spectinomycin and 25 I~g m1-1gentamycin.
For transformation of tobacco, Agrobacterium harbouring the
pNapin-FAE1 construct was co-cultivated with leaf pieces of
Nicotiana tabacum SR1 and transformants were selected with
kanamycin (100I~g m I-1) on solid medium (Lee and Douglas, 1996).
Arabidopsis thaliana (L.) Heynh. ecotype Columbia was transformed with both the p35S-FAEI and pNapin-FAE1 binary vectors
using a combination of in planta (Chang et aL, 1994, Katavic et aL,
1994) and vacuum-inflitration methods (Bechtold et aL, 1993).
Plants were grown until the primary inflorescence shoots reached
1-5 cm in height, when these bolts were cut off. The wound site
was inoculated with 50 ml of an overnight Agrobacterium culture.
After 4-6 days, a number of secondary inflorescences that
appeared were cut off, and vacuum inflitration was performed on
these plants using the conditions described by Bechtold et aL
(1993). Screening for transformed seed was done as described
previously (Katavic et aL, 1994). Briefly, seeds from infiltrated
plants were plated out (approximately 1500 seeds/plate) on solid
minimal salts nutrient medium supplemented with 50 ~g ml-~
kanamycin. Seedlings that showed resistance were visible after
approximately 8 days, because they turned green and were
elongated. Plants that were derived from seed harvested from
different pots were considered as independent lines. We follow
130
A n t h o n y A. Mfllar a n d Ljerka Kunst
the designations of the infiltrated plant being T1, primary transformants as T2, etc., as outlined in Katavic et a/. (1994). Plants
were grown at 20°C under continuous fluorescent illumination
(100 t~ E m-2 s-l).
Fatty acid analysis
For the determination of the cell fatty acid composition, fatty acid
methyl esters were prepared according to Browse et aL (1986) by
transmethylation in methanolic-HCI (Supelco), extracted in hexane
and analysed by gas-liquid chromatography (Kunst et al., 1992).
The conditions used allowed the detection of VLCFAs of up to C24
in length. For GC-MS analysis, leaf lipid extracts were saponified
in 10% methanolic KOH at 80°C and extracted with ether to remove
any non-fatty acyl contaminants. After acidification with 6 N HCI,
the free fatty acids were derivatized with pentafluorobenzyl (PFB)
bromide, according to the Pierce Product protocol, to yield the
corresponding fatty acyI-PBF esters. These were then subjected
to GC-MS on a Trio 2000 GC/MS (Micromass Canada Inc.) in the
negative CI mode (Carrier et aL, 1995).
Northern blot analysis
Total RNA from leaves of 6-week-old Arabidopsis plants was isolated using the RNeasy plant mini kit (Qiagen) and the concentration
was determined spectrophotometricallyat 260 nm. RNA was separated in agarose gels containing 15% formaldehyde and blotted onto
HybondTM-N÷ filters (Amersham) using the alkali blotting procedure as specified by the manufacturer. The membrane was hybridized to the FAEI gene which was labelled with 32P-dATPaccording
to Feinberg and Vogelstein (1983). Hybridization was performed at
65°C following the protocol of the manufacturer (Amersham). The
blot was washed at high stringency (0.1 ×SSC and 0.1% SDS at
65°C) and then exposed to X-ray film (DuPont).
Acknowledgements
We thank Drs G.W. Haughn and D.C. Taylor for critical reading of
the manuscript and helpful discussions, and D.W. Reed, Dr D.C.
Taylor, L. Hogge and D. Olson for performing GC-MS on the fatty
acid extracts. We also thank Dr J. Kohno-Murase for providing
the pNGKM vector. Valuable comments by two anonymous
reviewers are gratefully acknowledged. This work was supported
by Natural Sciences and Engineering Research Council of Canada
through a research grant to L.K.
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