From the
Plant epicuticular, or surface, waxes are synthesized primarily,
if not exclusively, by epidermal cells. The epicuticular wax
constitutes almost 20% of the chloroform-extractable lipids in
developing leek leaf and is derived predominantly from saturated fatty
acids. The significant requirement for saturated fatty acids in
epidermal tissues led us to investigate whether or not epidermal
extracts have thioesterase activities that prefer saturated acyl-acyl
carrier protein (ACP) substrates, rather than the 18:1-ACP more
commonly hydrolyzed by total leaf extracts. Epidermal extracts from Brassica, pea, and leek exhibited higher activities toward
saturated acyl-ACPs relative to 18:1-ACP when compared to total leaf or
leaf parenchymal extracts. We identified and purified a stearoyl-ACP
(18:0-ACP)-specific thioesterase from leek epidermal extracts which
could be separated from 18:1-ACP thioesterase using hydroxyapatite
chromatography. The stearoyl-ACP thioesterase exhibited a high
preference for 18:0-ACP, having less than 10% of the 18:0-ACP
hydrolyzing activity when presented with 18:1-ACP, 16:0-ACP, or
18:0-CoA substrates. The stearoyl-ACP thioesterase was predominantly,
if not exclusively, expressed in epidermis and may play a role in
generating the saturated fatty acid pool required for wax production.
In most plant tissues, the primary products of de novo fatty acid synthesis are the mono-unsaturated fatty acid oleate
(C18:1) and, to a much lesser extent, the saturated fatty acids,
palmitate (C16:0), and stearate (C18:0). The acyl chains are
synthesized in plastids esterified to the protein cofactor, acyl
carrier protein (ACP).
The control mechanisms directing termination of de novo fatty acid synthesis, and therefore defining the pools of various
acyl-CoA precursors, are not well understood. The acyl-CoA pools may be
controlled, in part, by the substrate specificities of acyl-ACP
hydrolases (thioesterases). For example, in a variety of oilseeds, such
as California Bay (Umbellularia california) and Cuphea, medium chain fatty acids (the major acyl components of
the storage triacylglycerols), are the products of medium chain
acyl-ACP thioesterases(1, 2) . In addition,
transformation of Arabidopsis with a medium chain thioesterase
(under the direction of a seed-specific napin promoter) produces high
quantities of medium chain fatty acids in seed
triacylglycerols(3) . Medium chain fatty acids in lactating
mammary glands (4, 5) and in the uropygial gland of
mallard ducks (6, 7) are also the products of medium
chain thioesterases.
In membrane lipids of leaves, and most
vegetative tissues, the major fatty acids are C18 polyunsaturates,
derived from de novo synthesis of oleoyl-ACP. Oleate is then
released by a specific oleoyl-ACP hydrolase (or thioesterase, OTE) (EC
3.1.2.14). Although this enzyme prefers oleoyl-ACP, it also has
activity (10-20%) with palmitoyl-ACP and stearoyl-ACP
substrates(8, 9, 10, 11) . There has
been speculation that the minor pools of palmitic and stearic acids in
whole leaf tissue are generated by OTE and its reduced specificity for
palmitoyl- and stearoyl-ACPs. However, leek epidermal tissue which
constitutes only 4% of the leaf fresh weight is solely responsible for
wax production which is more than 15% of the total leaf lipid.
Furthermore, the wax is derived almost exclusively from saturated fatty
acids. Therefore, while the minor OTE activity may be sufficient for
generating saturated fatty acid pools in whole leaves, it is unlikely
that it is sufficient to supply the saturated fatty acids required for
wax biosynthesis by epidermal cells.
In order to determine if
epidermal tissue has thioesterases which are specific for, or have a
greater preference for, saturated acyl-ACPs, we compared thioesterase
activities in total leaf and in epidermal tissue extracts prepared from Brassica, pea, and leek. From these initial studies we have
discovered an epidermal stearoyl-ACP thioesterase. This enzyme has been
purified to homogeneity and shown to be highly specific for 18:0-ACP.
It is expressed primarily, if not exclusively in epidermal tissue, and
may be responsible, in part, for generating the saturated fatty acid
precursor pools for epicuticular wax production.
Fresh leeks (Allium porrum) were obtained at a local
grocery store and the leaves were removed
Total leaf samples from Brassica campestris and pea (Pisum sativum, A783-161, arg
Recombinant spinach ACP-I and Escherichia coli ACP
were isolated from E. coli E103S, a gift from J. B. Ohlrogge,
and purified using DEAE-cellulose chromatography according to Clough et al.(12) . The preparation of ACP was essentially
pure as determined by SDS-PAGE and visualization by Coomassie staining
(data not shown). [1-
ACP (10 mg) was bound to 1 ml of
Affi-Gel 15 (Bio-Rad) according to Clough et al.(12) .
Before use, the ACP affinity column was incubated for 15 min with 20
mM Tris, pH 7.2, 5 mM DTT to ensure the ACP
phosphopanthetheine prosthetic group was fully reduced, and then
equilibrated with 10 ml of 20 mM Tris, pH 7.2, 0.5 mM
DTT, 0.1% Triton X-100.
Acyl-ACP thioesterases activities were assayed as
described(8) . Assays were performed under initial velocity
conditions, with less than 10% of substrate typically hydrolyzed. The
assay for acyl-ACP hydrolysis contained 1.0 µM
[1-
After some assays the
hexane-extracted radioactive products were dried under a gentle stream
of N
The active fractions from the Mono Q column were pooled and
desalted with PD10 columns in Buffer D (25 mM bis-Tris,
adjusted to pH 7.1 by iminodiacetic acid). The sample was applied to a
Mono P column (HR 5/20) equilibrated with Buffer D, at a flow rate of 1
ml/min at 4 °C. The column was washed with Buffer D, and the
activity was eluted with 60 ml of 10% (v/v) polybuffer 74 (Pharmacia)
adjusted to pH 4.0 by iminodiacetic acid, at a flow rate of 1 ml/min.
One-ml fractions were collected into tubes containing 50 µl of 1 M bis-Tris, pH 9.9, to immediately neutralize the eluant.
Active fractions were concentrated and stored at -20 °C.
The native molecular mass of the saturated acyl-ACP
thioesterase was determined using a Superdex 200 FPLC gel filtration
column (HR 10/30, Pharmacia). The desalted active fraction from the
Mono Q column was loaded onto the column which had been equilibrated
with 50 mM Tris-HCl, pH 7.0, 0.10 M NaCl buffer and
calibrated with the following standards:
Proteins from each purification step were separated by
SDS-PAGE (15) and stained using a Silver Stain Plus kit (Bio-Rad)
according to manufacturer's recommendations. Immunoblot analyses
were performed as described by Post-Beittenmiller et
al.(16) . The polyclonal antibodies used as the primary
antibodies were raised against one of three thioesterases: 1) an Arabidopsis thioesterase with a broad substrate range from
14:0-ACP to 18:1-ACP(17) , 2) coriander OTE, and 3) California
bay medium chain acyl-ACP thioesterase (MCTE). Both Arabidopsis and coriander thioesterase antibodies were kind gifts from Peter
Dörmann and John Ohlrogge (Michigan State University), and the
antibody to California bay MCTE was a kind gift from Toni Voelker
(Calgene, Davis, CA).
Fatty acid methyl esters were prepared from frozen tissue
using methanolic boron trichloride (10%, w/v, Alltech). Frozen tissue
(100 mg) was ground in liquid nitrogen to a fine powder with a mortar
and pestle and added to 1 ml of methanolic boron trichloride.
Heptadecanoic acid (Sigma) was added as an internal standard at a
concentration of 300 ng/mgfw. The suspension was heated to 60-80
°C for 1 h. After extraction into hexane and drying under a gentle
stream of N
Wax was removed from leaf sections by
dipping leaf tissue in CHCl
We initially compared
PEG-8000 precipitation and ammonium sulfate precipitation.
Approximately 50% of the total thioesterase activity was precipitated
by 25% PEG, while over 90% of the total thioesterase activity was
precipitated by 75% ammonium sulfate. Therefore, the latter was used as
the initial purification step giving a 1.8-fold enrichment of total
thioesterase specific activity, and an enrichment of 1.3% for the
saturated acyl-ACP thioesterase activity.
We demonstrated that the
hydroxyapatite-purified saturated acyl-ACP thioesterase activity was
due to a separate enzyme, rather than an OTE with broad substrate
specificity. This step reliably separated the saturated acyl-ACP
thioesterase and OTE thioesterase activities. When a linear NaCl
gradient was used to separate the two thioesterase activities,
resolution was poor, while a step-wise elution was found to be more
efficient. After hydroxyapatite chromatography, the partially purified
saturated acyl-ACP thioesterase had very low activity with 18:1-ACP
(<10%), while the OTE fraction retained significant activity with
both 18:0- and 16:0-ACPs (30-50% of the activity with 18:1-ACP).
The saturated acyl-ACP thioesterase was further purified by FPLC
Mono S and Mono Q chromatography and finally by ACP affinity
chromatography. In some experiments the order of columns after
hydroxyapatite chromatography was changed without significantly
altering the final purification result. Fig. 2shows a
silver-stained SDS-polyacrylamide gel comparing enrichment of the
saturated acyl-ACP thioesterase activity during several steps of
purification. The arrow indicates the band which corresponds
to the single 40 kDa band after ACP affinity column chromatography.
This 40 kDa band is consistently found in all active fractions when
alternative chromatographic separations are used. Fig. 3shows
the final ACP affinity purification of the saturated acyl-ACP
thioesterase. After hydroxyapatite chromatography the saturated
acyl-ACP thioesterase activity was enriched only 20-fold ( and Fig. 3A, lane 1). A further
500-fold enrichment was achieved with two ion-exchange columns. After
Mono Q chromatography, many protein bands could still be detected;
however, most of the proteins from Mono Q pooled fractions did not bind
the ACP affinity column (Fig. 3A, lanes 2 and 3). After two passes through the ACP affinity column, the
saturated acyl-ACP thioesterase subunit was purified to homogeneity, as
judged by SDS-PAGE and silver staining (Fig. 3B), giving
rise to a single protein band at 40 kDa. The binding of the saturated
acyl-ACP thioesterase to the ACP affinity matrix was relatively weak as
the 0.1 M NaCl eluted 40-50% of the activity (data not
shown). Typically, about 50% of the applied thioesterase activity was
released with 1.0 M NaCl.
The optimal pH for STE activity was 8.5. This is lower than
the pH preference of 9.0-9.6 reported for avocado OTE(8) ,
California bay MCTE(18) , rapeseed OTE(10) , and squash
OTE(19) . This also contrasts with Cuphea MCTEs which
have a broader pH range(2) . The optimal temperature for STE was
37 °C.
Leek epicuticular, or surface, waxes are comprised of a
mixture of very long chain hydrocarbons (fatty acids, alkanes, and
ketones), all of which are derived from saturated fatty acids. The
major component of leek epicuticular wax is a saturated C31 ketone,
which comprises more than 50% of the total chloroform-extractable wax
and as much as 16% of total leaf lipids. Epidermis is the primary, if
not exclusive site, of wax biosynthesis, and as such, requires
significantly more saturated fatty acids precursors for wax production
than the total leaf requires for glycerolipid biosynthesis. De novo fatty acid synthesis has been studied most extensively in whole
leaf where OTE is the major acyl-ACP thioesterase and unsaturated fatty
acids are the major products. In the epidermis, however, a mechanism
must exist whereby increased levels of saturated fatty acids are made
available for wax production.
There are at least three simple
mechanisms which might increase the levels of saturated fatty acids: 1)
an increase in 3-ketoacyl-ACP synthase II (KAS II) activity; 2) a
decrease in stearoyl-ACP desaturase activity; or 3) an increase in the
hydrolysis of saturated acyl-ACPs (Fig. 9). Any one of these
steps, or combination of steps, could lead to increased levels of
saturated fatty acids. KAS II catalyzes the extension of 16:0-ACP to
18:0-ACP. An increase in KAS II activity could lead to increased
synthesis of 18:0-ACP; its subsequent utilization, however, would
depend on the relative activities of stearoyl-ACP desaturase and a
saturated acyl-ACP thioesterase. Although an increase in OTE activity
may increase 18:0 production because of its minor activity with
18:0-ACP, an increase in OTE activity would presumably also lead to an
increase in 18:1 production. Therefore, we postulated that a saturated
acyl-ACP thioesterase may exist in the epidermis such that C18
saturated and mono-unsaturated fatty acid pools could be controlled
separately.
The leek stearoyl-ACP thioesterase is unusual in
its narrow substrate range, preferring a single acyl-ACP substrate. Two
other Class B thioesterases, California bay (1) and Arabidopsis broad range thioesterase(2) , hydrolyze
significant levels of several acyl-ACP substrates. The greater
specificity of leek STE has implications in the regulation of
individual fatty acid pools for wax and glycerolipid biosynthesis. For
example, differential control of STE activity (expression) could
dramatically influence stearic acid production without significantly
altering the fatty acid pools required for glycerolipid biosynthesis.
Furthermore, the significantly greater preference of STE for a single
substrate may offer unique opportunities for engineering seed oils and
influencing wax production. The broad range of substrate specificity of
other thioesterases may pose problems in genetically engineered plants
when overall rates of fatty acid synthesis are increased since pools of
several acyl-ACPs may be increased simultaneously.
We thank Richard Dixon, Maria Harrison, Lining Guo,
and Kimberly Evenson for critical reading of the manuscript, Maelor
Davies, Jean Kridl, Janet Nelsen, and Toni Voelker for helpful
discussions, and Jan Jaworksi and John Ohlrogge for in depth and
thoughtful discussions and for critical reading of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)The plastidial extension
of an acyl chain is terminated by hydrolysis or transfer of the acyl
chain from ACP, either before or after desaturation, and therefore, the
terminal steps of plastidial de novo fatty acid synthesis
determine the proportion of saturated and unsaturated fatty acids
produced. The free fatty acids are exported out of the plastid and
subsequently esterified to coenzyme A. These acyl-CoAs are the
substrates for membrane and storage lipid synthesis, and in the case of
epidermal cells, they may be further elongated to very long chain fatty
acids (C20 to C30 and greater) for production of epicuticular wax.
Plant Materials
5-10 cm above the
root stock with a razor blade. The leek bulbs were then planted in
Metro Mix (Grace/Sierra) potting soil such that the roots were buried
to a depth of approximately 2.5-3.5 cm. The epidermis of the cut
leaves was peeled in wide strips with a pair of fine-tipped forceps and
rapidly frozen in liquid nitrogen. The remaining parenchyma was frozen
separately. These tissues were defined as ``mature.'' The
planted leeks were placed in a growth chamber (50% humidity and 16 h
photoperiod) and later used as a source of rapidly expanding
``regenerated'' leaf. Epidermis and parenchyma of the
regenerated leaf were harvested after 10-14 days, as described
for mature tissue. All samples were stored at -70 °C until
used.
,
Norman Weeden, Cornell University) were harvested from growth
chamber-grown plants (50% humidity, 16 h photoperiod) and rapidly
frozen in liquid nitrogen. Epidermal tissues were peeled from stems of
6-10-week-old Brassica plants or from leaves of
3-6-week-old pea plants and frozen as described above.
Chemicals
C]Acyl-ACPs were
synthesized from [1-
C]fatty acids (palmitic,
stearic, and oleic acid, SA 2.1 GBq/mmol, Dupont-NEN) and plant or E. coli ACP with E. coli acyl-ACP synthetase 13. Over
90% of the ACP was routinely acylated by this procedure. The
[1-
C]acyl-ACPs were purified by DE52-cellulose
(Whatman) chromatography and desalted with a Microdialyzer System 500
(Pierce) against 20 mM MOPS buffer, pH 6.1.
[1-
C]Stearoyl-CoA was prepared according to
Taylor et al.(14) .
Enzyme Assays
C]acyl-ACP, and the incubation was for 15 min
at 37 °C. One unit of thioesterase activity was defined as 1 nmol
of fatty acid hydrolyzed/min/mg protein under the assay conditions.
Stearoyl-CoA thioesterase activity was assayed under identical
conditions except 1 µM
[1-
C]stearoyl-CoA was used in place of
[1-
C]acyl-ACP. Protein concentration was
measured using a Protein Assay kit (Bio-Rad) according to
manufacturer's recommendations.
, dissolved in a small volume (
50 µl) of
chloroform and separated by thin layer chromatography on a silica gel
plate (J. T. Baker Si250 (19C)) using hexane/diethyl ether/glacial
acetic acid (70:30:1 (v/v/v)) as the developing solvent. The
radiolabeled product was visualized after exposure to x-ray film (Kodak
X-OMAT AR) and was found to comigrate with the free fatty acid standard
([1-
C]18:0). The radiolabeled band was scraped,
eluted from the silica gel with chloroform, and applied to a reversed
phase (Whatman KC18) plate to determine the chain length of the
product. Radiolabeled standards ([1-
C]12:0,
[1-
C]14:0, [1-
C]16:0,
[1-
C]18:0) were used to identify the chain
length of the product band by comigration. The products of all
thioesterase assays were free fatty acids whose chain lengths were
identical to the chain lengths of the added acyl-ACP substrates.
Purification of a Saturated Acyl-ACP Thioesterase
Step 1
All purification steps were carried out
at 4 °C. Frozen leek epidermis (70-200 g) was ground to a
powder in liquid nitrogen using a prechilled mortar and pestle. The
powder was added to 10 volumes of ice-cold 50 mM sodium
phosphate, pH 7.5, 2 mM DTT, plus protease inhibitors (0.2
mM phenylmethylsulfonyl fluoride, 5 mM EDTA, 1 mM benzamidine, 1 µM pepstatin, 100 µM
phosphoramidon), and 5% (w/v) water-insoluble polyvinylpolypyrolidone,
and the sample was homogenized with a polytron. After homogenization
Triton X-100 (Pierce Surfact-Amps X-100) was added to a final
concentration of 0.01% (v/v). The homogenate was centrifuged for 30 min
at 20,000 g. After centrifugation, the supernatant was
filtered through miracloth and centrifuged again for 30 min at 20,000
g. The supernatant was collected, and the proteins
were precipitated with 75% ammonium sulfate, collected by
centrifugation, and redissolved in 30 ml of Buffer A (20 mM KPO
, pH 6.8, 2 mM DTT, containing the
protease inhibitors as indicated above without the
polyvinylpolypyrolidone). The resuspended extract was desalted by PD10
columns (Pharmacia) using Buffer A.
Step 2
The sample was loaded onto a Bio-Gel HT
hydroxyapatite (Bio-Rad, 5 100 cm) column which had been
equilibrated with 20 mM KPO
, pH 6.8. The column
was washed with 1 bed volume of Buffer A to remove unbound proteins.
The saturated acyl-ACP thioesterase was then eluted with 2 bed volumes
0.1 M NaCl in Buffer A. OTE was eluted with 2 bed volumes (400
ml) 0.3 M KPO
. The active OTE fraction was not
purified further. The active saturated acyl-ACP thioesterase fractions
were pooled and concentrated using an Amicon PM-30 (Danvers, MA)
ultrafiltration membrane. The sample was desalted with PD10 columns
into 20 mM succinate buffer, pH 5.0, 2 mM DTT
containing the protease inhibitors and 5% glycerol (Buffer B).
Step 3
Fifteen ml of concentrated protein were
loaded onto a Mono S HR 5/5 column (Pharmacia) which had been
equilibrated with Buffer B. The column was washed with Buffer B to
remove unbound protein. The enzyme was eluted with a linear NaCl
gradient from 0 to 400 mM] in 20 mM Tris-HCl, pH
8.0, 2 mM DTT, containing the protease inhibitors and 5%
glycerol. The active fractions were pooled, desalted in 20 mM Tris-HCI, pH 8.5, and loaded onto a Mono Q HR 5/5 column
(Pharmacia). The bound proteins were eluted with a linear NaCl gradient
from 0 to 400 mM in 20 mM Tris-HCl, pH 8.5,
containing 2 mM DTT, 5 mM EDTA, 1 mM benzamidine, and 5% glycerol. The active fractions were pooled and
concentrated in 20 mM Tris, pH 7.2, 0.5 mM DTT, 20%
glycerol, 0.01% Triton X-100 buffer for ACP affinity chromatography.
Step 4
One bed volume (1 ml) of enzyme was loaded
onto the ACP affinity column and allowed to incubate for 10-20
min at 4 °C. The column was sequentially washed stepwise with
Buffer C (20 mM Tris-HCl, pH 7.2, 5% glycerol, 0.5 mM DTT, 0.01% Triton X-100) and then with 3 ml of 0.1 and 0.2 M NaCl in Buffer C to remove nonspecifically bound proteins. The
saturated acyl-ACP thioesterase was eluted with 1.0 M NaCl in
Buffer C. After dialysis against Buffer C, the sample was reapplied to
the affinity column, bound, and eluted by the same procedure. Saturated
acyl-ACP thioesterase was purified to homogeneity (as determined by
silver-staining of SDS-polyacrylamide gels) after the second elution
from the column.
FPLC Mono P Chromatography
Determination of Native Molecular Mass
-amylase (200 kDa),
alcohol dehydrogenase (150 kDa), albumin (66 kDa), carbonic anhydrase
(29 kDa), and cytochrome c (12.4 kDa). Fractions (0.5 ml) were
collected, and aliquots were assayed for thioesterase activity. Only
one peak of activity was detected.
SDS-PAGE and Immunoblot Analyses
Fatty Acid and Wax Analyses
, samples were resuspended in 200 µl of
hexane, and 1-µl aliquots were analyzed using a Hewlett Packard
5890 gas chromatograph and a 5971 mass spectrometer. Samples were
separated using a DB23 (J& Scientific) 20 m
180-µm
capillary column. The injector temperature was 275 °C, and the oven
temperature was ramped at 10 °C/min from 125 to 250 °C. Total
ion monitoring and comparison of retention times with standards were
used to identify components.
for 20-30 s.
Heptadecanoic acid was added as an internal standard. The solvent was
evaporated under a gentle stream of N
. Samples were
dissolved in hexane and analyzed directly, or an aliquot was treated
with methanolic boron trichloride for analysis of fatty acid methyl
esters. Waxes were analyzed using an HP5 30 m
250 µm
diameter capillary column. Injector was at 250 °C and the oven
ramped from 150 to 300 °C at 5 °C/min.
Comparison of Acyl-ACP Thioesterase Activities from
Different Plant Species
The primary acyl-ACP thioesterase
activity in total leaf is oleoyl-ACP
thioesterase(8, 9, 10, 11) . However,
because of the increased requirement for very long chain saturated
fatty acid precursors for epicuticular wax production, we postulated
that epidermal tissues may have thioesterase activities which prefer
saturated acyl-ACP substrates. Such activities could be due to either
an OTE with a broad substrate range that includes hydrolysis of
saturated fatty acids or a thioesterase specific for saturated fatty
acids. We measured thioesterase activities in Brassica, pea,
and leek tissues using 16:0-ACP, 18:0-ACP, and 18:1-ACP substrates to
determine whether or not epidermal tissue was enriched for saturated
acyl-ACP thioesterase activity when compared to total leaf. As shown in Fig. 1, the hydrolysis of 18:1-ACP was 3-5-fold higher than
the hydrolysis of the saturated acyl-ACPs in total leaf extracts of Brassica, pea, or leek parenchymal tissue. Compared to total
leaf, the epidermal extracts had higher thioesterase activity with
saturated acyl-ACPs relative to 18:1-ACP. Leek epidermal extracts had
the highest relative activities for the saturated substrates.
Furthermore, the ratios of saturated acyl-ACP thioesterase activities
to 18:1-ACP activity were greater in leek epidermis than in leek
parenchyma. Consequently, because of the relatively higher level of
saturated acyl-ACP thioesterase activities, the relative ease of
peeling leek epidermis, and the higher level of wax production compared
to either Brassica or pea, leeks were used for the further
characterization of this activity.
Figure 1:
Comparison of thioesterase activities
in Brassica, pea, and leek tissues. Extracts were prepared
from Brassica leaves and epidermis, pea leaves and epidermis,
leek parenchyma, and epidermis as described under ``Experimental
Procedures.'' Hydrolysis of [1-C]16:0-ACP
(
), [1-
C]18:0-ACP (
), and
[1-
C]18:1-ACP (
) were determined and
are reported here as percent of activity with 18:1-ACP. For Brassica leaf and epidermis, 100% values were 10.4 and 17.4
nmol/mg/h, respectively. For pea leaf and epidermis, 100% values were
12.6 and 16.2 nmol/mg/h, respectively. For leek parenchyma and
epidermis, 100% values were 16.0 and 11.6 nmol/mg/h, respectively.
Results shown are the average of two or three
experiments.
Purification of the Saturated Acyl-ACP
Thioesterase
An acyl-ACP thioesterase specific for saturated
acyl-ACPs was purified to homogeneity from leek epidermis and shown to
be distinct from 18:1-ACP thioesterase. A summary of the chromatography
steps and the fold-purification are presented in . The
purification procedure was repeated five times with different amounts
of starting material (70-200 g). Starting with 200 g, this
procedure yielded approximately 3600-fold purification with a recovery
of 1-2%. The saturated acyl-ACP thioesterase and OTE activities
were followed by hydrolyses of 18:0-ACP and 18:1-ACP, respectively. The
crude extract contains several thioesterases, each with some 18:0-ACP
activity; therefore, the saturated acyl-ACP thioesterase activity at
all purification steps (beyond hydroxyapatite chromatography) was
calculated based on the ratio of saturated acyl-ACP thioesterase
activity to OTE activity with 18:0-ACP as the substrate. We estimated
that 80% of the total 18:0-ACP hydrolysis can be attributed to OTE
and/or other thioesterases. Because there are losses before the
hydroxyapatite column, these are only estimates of the actual
activities. Differential inactivation of the two thioesterases during
purification may also affect these estimates.
Figure 2:
SDS-PAGE of samples from crude extract and
from different column chromatography steps. Samples were loaded
according to the enzyme activity, and the gel was silver-stained.
Molecular weights of protein standards are indicated at the left. Lane 1, crude extract of leek epidermis (14 µg of
protein). Lane 2, fraction from Mono S (60,000 dpm). Lane
3, fraction from Mono Q (80,000 dpm). Lane 4, fraction
from Mono P (160,000 dpm).
Figure 3:
SDS-PAGE of fractions from ACP affinity
purification. Fractions were separated on 10% (A) or 12% (B) acrylamide gel followed by silver staining. A, lane 1. Fraction from hydroxyapatite chromatography (activity
loaded: 50,000 dpm). Lane 2, unbound proteins from first pass
through ACP Affinity column (fraction from Mono Q loaded on to the ACP
affinity column (activity loaded 100,000 dpm). Lane 3, 1 M NaCl elution from ACP affinity column (activity loaded 180,000
dpm). B, lane 1, second pass through ACP affinity
column. The active fraction from the first pass was desalted,
concentrated, and applied to the ACP affinity column. The column was
washed, and the saturated acyl-ACP thioesterase was eluted as described
for the first pass.
Determination of Molecular Mass
Gel filtration was
used to determine the native molecular mass of the saturated acyl-ACP
thioesterase. Comparison with elution times of standard proteins
indicated that the saturated acyl-ACP thioesterase had a native
molecular mass of 84 kDa (Fig. 4). Comparison of the sizes of the
saturated acyl-ACP thioesterase by SDS-PAGE and by gel filtration
suggests that it may function as a dimer. This is similar to safflower
OTE which has a subunit mass of 41 kDa as determined by SDS-PAGE and a
molecular mass of 74 kDa as determined by gel filtration (9).
Thioesterases from California bay (MCTE) and Cuphea (OTE) have
similar molecular masses (42 and 53 kDa, respectively), while the Cuphea MCTE is slightly smaller, 28 kDa(1, 2) .
Avocado OTE has a molecular mass of 70-80 kDa(8) .
Figure 4:
Standard curve of Superdex 200 gel
filtration. The native molecular weight of STE was determined using
calibrated Superdex 200 FPLC gel filtration column (HR 10/30,
Pharmacia) with the following standards: -amylase (200 kDa),
alcohol dehydrogenase (150 kDa), albumin (66 kDa), carbonic anhydrase
(29 kDa), cytochrome c (12.4 kDa). Elution of STE is indicated
by the arrow.
Substrate Specificity
The substrate specificity of
the purified saturated acyl-ACP thioesterase was determined under
initial velocity conditions using 1 µM
[1-C]acyl-ACPs. The purified saturated acyl-ACP
thioesterase showed a strong preference for stearoyl-ACP (Fig. 5A). The very narrow specificity exhibited by the
leek epidermal saturated acyl-ACP thioesterase is in sharp contrast to
the broader specificity reported for a variety of OTEs and has not been
reported previously. The partially purified OTE (Fig. 5B) from leek used 14:0- to 18:1-ACPs as
substrates with the highest activity against oleoyl-ACP. Due to its
significant preference for stearoyl-ACP, we now refer to the saturated
acyl-ACP thioesterase as stearoyl-ACP thioesterase (STE).
Figure 5:
Substrate specificity of the purified STE (A) and partial purified OTE (B). Substrates at a
concentration of 1 µM were assayed with the purified STE
and the partially purified OTE. Activities are reported as percent of
maximum activity for the preferred substrate. For STE (A) the
100% value for [1-C]18:0-ACP was 8640 nmol/mg/h.
For OTE (B) the 100% value for
[1-
C]18:1-ACP was 1740 nmol/mg/h. Results are
from three or four experiments.
To further
characterize STE, we assessed its activity with 18:0-CoA. Activity was
assayed with [1-C]stearoyl-CoA and
[1-
C]stearoyl-ACP after various purification
steps. Hydrolysis of 18:0-CoA was greater than hydrolysis of 18:0-ACP
during the initial purification steps (). After the Mono Q
step, however, the 18:0-CoA hydrolyzing activity decreased
significantly compared to the 18:0-ACP hydrolyzing activity. The
activity with 18:0-CoA was 30,000-fold lower than 18:0-ACP hydrolysis
after the enzyme was purified by ACP affinity chromatography. These
data indicate that the purified STE strongly prefers 18:0-ACP and that
the 18:0-CoA hydrolase activity was most likely due to a separate
enzyme.
Immunological Cross-reactivity
To assess the
relatedness of various cloned plant thioesterases with the leek STE,
immunoblots were probed with antibodies raised to three different
thioesterases. Hydroxyapatite-purified STE was used, which allowed STE
to be analyzed without interference from OTE. In Fig. 6the
cross-reactivity of the samples probed with three polyclonal antisera
is shown. The three different antisera were raised against 1) MCTE
(California bay medium chain acyl-ACP thioesterase), 2) OTE (coriander
oleoyl-ACP thioesterase), and 3) an Arabidopsis thioesterase
which has a broad range of acyl-ACP substrates. These three enzymes
represent two classes of thioesterases based on their amino acid
sequences. Thioesterases specific for oleoyl-ACP are designated Class
A, while the medium chain and broad range acyl-ACP thioesterases are
Class B. The lanes loaded with the hydroxyapatite fractions had 50
µg of protein of which we estimate 1-10% (50-500 ng) of
the protein was STE based on total activities recovered after ACP
affinity chromatography. As positive controls on the blots, 10 ng of
purified MCTE, 10 ng of OTE, and 3 ng of the broad range thioesterase
were loaded. A band at 40 kDa (indicated by solid arrowhead)
in the partially purified leek STE preparation cross-reacted with
antibody directed against the broad range Arabidopsis thioesterase (Fig. 6B, lane 1), but showed
no cross-reactivity with either the MCTE antibody, or the OTE antibody (Fig. 6A, lane 1; C, lane 1).
An open arrowhead indicates where a 40 kDa band would be if it
cross-reacted with either MCTE or OTE antibody. These results suggest
that leek STE may be more similar to the broad range Arabidopsis thioesterase than to either the California bay MCTE or the
coriander OTE. Furthermore, they suggest that the STE may fall into the
Class B category of thioesterases. Since this is the first report of an
acyl-ACP thioesterase purified from a monocot, the lack of
cross-reactivity with several antibodies raised to dicot thioesterases
may reflect dicot/monocot differences or structural differences related
to the narrow substrate range of STE. Protein sequencing and/or cloning
will be necessary to answer some of these questions.
Figure 6:
Immunoblot analyses using three
antibodies. Protein samples were separated by SDS-PAGE, blotted to
nitrocellulose, and developed as described under ``Experimental
Procedures'' using primary antibodies directed against the
following: A, California bay MCTE; B, Arabidopsis broad range TE; or C, coriander OTE. Lane 1,
hydroxyapatite fraction enriched for STE activity (50 µg). Lane
2A, purified California bay MCTE (10 ng). Lane 2B, Arabidopsis broad range thioesterase (3 ng). Lane 2C,
purified coriander OTE (10 ng).
Comparison of Thioesterase Activities in Leek Epidermis
and Parenchyma
The hydrolysis of 18:0-ACP relative to 18:1-ACP
was significantly higher in crude epidermal extracts compared to that
in total leaf or leaf parenchymal extracts (Fig. 1). It was
difficult to assess the relative contributions of OTE and STE
activities, however, since OTE has some activity against 18:0-ACP.
Therefore, hydroxyapatite chromatography was used to separate these two
enzymes in extracts of leek epidermis and leek parenchyma. Relative
activity levels were compared in the hydroxyapatite-purified fractions
with identical [1-C]acyl-ACP substrate
preparations. When STE and OTE peaks in epidermal or parenchymal
preparations were separated, only the epidermal preparation (Fig. 7A) had significant STE activity in both STE and
OTE peaks, while the parenchymal preparations (Fig. 7B)
had little activity in the STE peak.
Figure 7:
Comparison of thioesterase activities with
stearoyl-ACP and oleoyl-ACP in leek epidermis and parenchyma. STE and
OTE activities were separated and partially purified from epidermal (A) and parenchymal (B) extracts by hydroxyapatite
chromatography. Enzyme assays were under initial velocity conditions
using 1 µM [1-C]acyl-ACPs as
described under ``Experimental Procedures.'' The values given
are nanomole/milligram/hour as percent of OTE activity in the crude
extract.
, 18:0-ACP;
, 18:1-ACP. Results are from two
experiments.
One of the primary functions of
the epidermis is to synthesize wax, therefore, STE may be instrumental
in providing an adequate pool of saturated fatty acids for wax
biosynthesis. Waxes are synthesized primarily during rapid expansion of
young leaves, so 18:0- and 18:1-ACP hydrolyzing activities in epidermis
from mature (store-purchased) leaf and in epidermis from actively
expanding leaf (regenerated 2, 5, 7, or 10 days) were compared. In
general, the enzyme activities measured in purchased leek were more
variable than in regenerated leek. In crude epidermal extracts from 2-
and 5-day regenerated leaf, stearoyl-ACP hydrolysis was similar to
oleoyl-ACP hydrolysis (Fig. 8, A and B). By 7
days, however, 18:0-ACP hydrolysis declines and 18:1-ACP hydrolysis
increases (Fig. 8A). By 10 days, 18:0-ACP hydrolysis was
75% of 18:1-ACP hydrolysis. Furthermore, the ratio of 18:0- to 18:1-ACP
hydrolysis in epidermis from 7- to 10-day regenerated tissue decreased (Fig. 8B). This change in the ratio of thioesterase
activities correlated with the decrease in rate of leaf elongation. It
should be noted that a decrease in the ratio of 18:0-ACP/18:1-ACP
hydrolysis was due to increases in 18:1-ACP hydrolysis rather than to
decreases in 18:0-ACP hydrolysis. These data suggested that epidermal
OTE levels increased as tissue expansion declined, whereas STE activity
remained constant.
Figure 8:
Comparison of epidermal thioesterase
activities with various acyl-ACP substrates during regeneration of leek
plants. A, thioesterase activities during leaf elongation
().
, 16:0-ACP;
, 18:0-ACP;
, 18:1-ACP). B, ratio of 18:0-ACP hydrolysis to 18:1-ACP hydrolysis during
leaf elongation (
). Values reported are averages of three
plants.
Fatty Acid Analyses of Leek Epidermis and
Parenchyma
Total lipids from leek epidermis, parenchyma, and
total leaf were extracted, transmethylated, and analyzed by gas
chromatography-mass spectrometry as described under ``Experimental
Procedures'' (I). Results are reported as mol %. Only
wax components which could be identified unequivocally were included;
consequently, the values reported may underestimate the total wax load.
Total surface wax in epidermis constitutes approximately 120
µmol/gfw compared to epidermal C16 and C18 fatty acids which
constitute 27 µmol/gfw, and parenchymal C16 and C18 fatty acids
which constitute 13 µmol/gfw. When leek leaf was dipped in
chloroform and the extracted lipids were analyzed, the major component
was a saturated C31 ketone, comprising 45-68% of the wax. The
remainder of the wax components consisted primarily of fatty acids and
alkanes greater than 18 carbons in length. Similarly, in epidermis,
most lipid (>50%) was identified as a saturated C31 ketone. The C31
ketone was not detected in leek parenchymal extracts but constituted
16% of total leaf lipid. Calculations based on moles of C31 ketone/gram
fresh weight of epidermis and per gram fresh weight of total leaf,
indicate that epidermis is 4% of the total tissue weight in leek leaf.
Despite its relatively small proportion of the leaf, epidermis produces
essentially all of the epicuticular wax (i.e. components
>C20), accounting for more than 19% of the total lipid. Since
virtually all of the wax components identified are derived from
saturated fatty acids (presumably 18:0), it is reasonable to expect
that a saturated acyl-ACP thioesterase such as STE is present in the
epidermis as part of the wax biosynthetic machinery.
Figure 9:
Terminal steps of fatty acids biosynthesis
and initiation of wax biosynthesis. Three terminal steps of plastidial de novo fatty acid biosynthesis and two possible pathways for
utilization of C18 fatty acids in epidermis are indicated. The
mono-unsaturated fatty acids are primarily used in phospholipid
biosynthesis while the saturated fatty acids are in general destined
for wax production. KAS II, 3-ketoacyl-ACP synthase II; STE, stearoyl-ACP thioesterase; and desaturase,
stearoyl-ACP desaturase.
This study has clearly demonstrated the presence of a
leek stearoyl-ACP thioesterase which preferred 18:0-ACP as a substrate
and was predominantly, if not exclusively, expressed in epidermis. In
addition, the ratio of 18:0-ACP to 18:1-ACP hydrolyzing activity in
regenerating leek was significantly higher than in mature
(non-expanding) leek. Indeed, the epidermal thioesterase activities
toward saturated fatty acids were equivalent to the 18:1-ACP
hydrolyzing activity in 2-5-day regenerating leek leaf. The ratio
of activities decreased as leaf expansion and the need for wax
production declined. The higher activity ratio was consistent with our
proposed role for STE in wax production. Additional in vivo analyses of epidermal acyl-ACP and acyl-CoA pools will be needed
to further elucidate the role(s) of STE, KAS II, and stearoyl-ACP
desaturase in generating the precursors for wax biosynthesis. If,
however, STE plays a significant role in generating saturated fatty
acids in the epidermis, similar activities are likely to exist in other
plant species, and our preliminary evidence (Fig. 1) suggests a
similar STE activity may be present in Brassica and pea
epidermal tissues.
Table: Purification of saturated acyl-ACP thioesterase
from leek epidermis
Table: Comparison of thioesterase activities with
stearoyl-CoA and stearoyl-ACP substrates during purification
Table: Total lipid analyses of leek epidermis,
parenchyma, and total leaf
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.