©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Discovery of an Epidermal Stearoyl-Acyl Carrier Protein Thioesterase
ITS POTENTIAL ROLE IN WAX BIOSYNTHESIS (*)

Dehua Liu , Dusty Post-Beittenmiller (§)

From the (1)Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73402

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 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.

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.


EXPERIMENTAL PROCEDURES

Plant Materials

Fresh leeks (Allium porrum) were obtained at a local grocery store and the leaves were removed 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.

Total leaf samples from Brassica campestris and pea (Pisum sativum, A783-161, arg, 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

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-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) .

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.

Enzyme Assays

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-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.

After some assays the hexane-extracted radioactive products were dried under a gentle stream of N, 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

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.

Determination of Native Molecular Mass

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: -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

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 and Wax Analyses

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, 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.

Wax was removed from leaf sections by dipping leaf tissue in CHCl 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.


RESULTS

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.

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.


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.

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.

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.


DISCUSSION

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.


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.

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.

  
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



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This research was supported by the Samuel Roberts Noble Foundation.

§
To whom correspondence should be addressed. Tel.: 405-223-5810 (ext. 377); Fax: 405-221-7380; E-mail: DPOST@NOBLE.ORG.

The abbreviations used are: ACP, acyl carrier protein; DTT, dithiothreitol; MCTE, medium chain acyl-ACP thioesterase; OTE, oleoyl-ACP thioesterase; STE, stearoyl-ACP thioesterase; FPLC, fast performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; MOPS, 2-(N-morpholino)ethanesulfonic acid; bis-Tris, bis[2-hydroxyethyl]imino-tris[hydroxymethyl] methane; dpm, disintegrations/min; gfw, grams fresh weight. The designation of Cx:y for fatty acids and their derivatives indicate chain lengths of x carbons long and with y number of double bonds.


ACKNOWLEDGEMENTS

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.


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