Planta

Planta (1984)162:487-494

9 Springer-Verlag 1984

Synthesis of wax esters by a cell-free system from barley (Hordeum vulgateL.) Pinarosa Avato * Department of Physiology, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark

Abstract. Experimental evidence for a membranebound microsomal ester synthetase from Bonus barley primary leaves is reported. The results are consistent with at least two mechanisms for the synthesis of barley wax esters: an acyl-CoA-fattyalcohol-transacylase-type reaction and an apparent direct esterification of alcohols with fatty acids. Biosynthesis of wax esters was not specific with regard to the chain length of the tested alcohols. The microsomal preparation readily catalyzed the esterification of C16-, C18- , C22- or Cz4-1abelled alcohols with fatty acids of endogenous origin. Exogenous long-chain alcohols were exclusively incorporated into the alkyl moieties of the esters. Addition of ATP, CoA and-or free fatty acids was not effective in stimulating or depressing the esterifying activity of the microsomal fraction. Partial solubilization of the ester synthetase was obtained using phosphate-buffered saline. Key words: Alcohol (long chain) - Ester synthetase - H o r d e u m (wax esters) - Wax ester biosynthesis.

Introduction

Wax esters are widely distributed in nature and are common constituents of plant (Tulloch 1976) and animal (Downing 1976; Jackson and Blomquist 1976; Jacob 1976) surface lipids and, in a few instances, of the reserve lipids of seeds (Yermanos 1975; Richter et al. 1981). Esters have been also isolated from algae (Weete 1976) and a few species of bacteria (Wang et al. 1972; Albro 1976). They are usually made up of long-chain fatty alco* Present address. Dipartimento di Chimica Organica, UniversitA di Pavia, Viale Taramelli 10, 1-27100 Pavia, Italy Abbreviations: P = pellet; PBS = phosphate-buffered saline; S = supernatant; SDS = sodium dodecyl sulphate

hols and fatty acids having 10-32 carbons. Two types of long-chain esters have been characterized as components of barley epicuticular waxes : those containing alkan-l-ols (mainly C3s-C4s) and those containing alkan-2-ols (mainly C33-C35 ) (yon Wettstein-Knowles and Netting 1976). The latter have been identified in the wax from all barley organs except the awns and leaf blades. Determination of the chemical composition of wax from various eceriferum mutants of barley and the use of radioisotopes in combination with chemical inhibitots of wax fbrmation indicate that two different enzymatic systems are involved in the biosynthesis of the two series of esters found in barley wax (von Wettstein-Knowles 1971, 1979, 1982a, b). Little is known about the esterification process of fatty acids with fatty alcohols giving rise to plant wax esters. Nevertheless, three mechanisms have been shown to be active in the synthesis of wax esters in Brassica oleracea leaves (Kolattukudy 1967, 1968a). These are i) a direct esterification of free fatty acids with fatty alcohols by a reversal of an esterase-type reaction, ii) an acyl-CoA-fattyalcohol transacylase and iii) a fatty-alcohol-acyltransferase esterification reaction (Kolattukudy 1967). In the present study, a method for preparing a cell-free ester-synthetase system from barley leaves is described and some of its properties delineated. Material and methods Plant material and growth conditions. Seeds of barley (Hordeum vulgate L.) cv. Bonus were germinated and grown on moist vermiculite at 23 ~ C in complete darkness. Six clays later, seedlings grown in the dark were transferred to the light and illuminated for 12 h with Osram (Miinchen, FRG) L-Fluora model 120-W77 lamps (2400 lux). Primary leaves, extending 7-10 cm above the coleoptile were harvested. Chemicals. [1-14C]Palmitic acid (2.18 x 1 0 9 Bq mmo1-1) and [1-1~C]stearic acid (2.06x 109 Bq mmo1-1) were purchased from the Radiochemical Centre, Amersham, UK. [1-14C]Doco -

488

P. Avato: Synthesis of wax esters

sanoic acid (2.15 x 10 9 Bq mmol -~) and [1-14C]tetracosanoic acid (1.85 • 109 Bq mmol 2) were supplied by Rosechem Products, Los Angeles, Calif., USA and by Service des Molecules Marquees, Gif-Sur Yvette, France, respectively. Biochemicals were obtained from Sigma Chemical Co., St. Louis, Mo., USA. Unlabelled acids and alcohols were purchased from Merck, Darmstadt, FRG.

Primary-leaf homogenate

I

20 min I 4080 g r

I S

P

20 min

Synthesis ofsubstrates. [1-'4C]Hexadecanol, -octadecanoi, -docosanol and -tetracosanol were prepared by reduction of the corresponding labelled fatty acids with lithium aluminium hydride according to the following procedure. Free fatty acid (10 lamol) was dissolved in 2 ml of anhydrous, freshly distilled tetrahydrofuran and an excess of lithium aluminium hydride was added. The reaction was carried out at room temperature for 1 h. Saturated sodium chloride was used to end the reaction after which the primary alcohols were extracted with n-hexane (3 x 3 ml). The n-hexane extracts were combined and the solvent evaporated under a stream of nitrogen. Purity of the labelled alcohols was checked by silica-gel-H thin-layer chromatography (TLC) in amylene-stabilized chloroform (Merck). The longchain alcohols were dispersed by sonication in the grinding buffer (see below) containing Triton X-100 (250 pg ml-1) using a PG 100 MSE Model 150 W ultrasonic disintegrator, Sussex, UK, at maximum power 3 x 30 s. The acyl-CoA thioesters of [1-14C]stearic acid and unlabelled palmitic acid were synthesized from the corresponding free acids, following the procedure described by Sanchez etal. (i973). Their purity was checked by TLC on cellulose plates (Merck), developed in butanol : acetic acid : water (5 : 2 : 3, by vol.).

Preparation of subcellular fractions. Primary leaves from wildtype barley were homogenized in a blender with replaceable razor blades (Kannangara et al. 1977) using a buffer containing 10 mM 2-(N-morpholino)ethane sulfonic acid (Mes), 0.32 M sucrose, I mM MgCI2, 2 mM dithiothreitol (DTT) at pH 6.0. After filtration through a double layer of nylon gauze (20 pm mesh), the homogenate was centrifuged in a Sorval (Bad Nauhelm, FRG) RC 213 centrifuge for 20 rain at 4080 g. The supernatant was then centrifuged at 14727 g for 20 rain. The crude microsomai pellet was obtained from the 14727-g supernatant by recentrifugation at 130000 g for 60 rain in a Beckman Model L5/75 ultracentrifuge using a Ti 70 rotor (Fig. 1). The microsomal pellet (P13o000) was, unless specified, resuspended in the grinding buffer. To solubilize the ester synthetase, the 130000-g pellet was resuspended in phosphate-buffered saline (PBS buffer), containing 0.1 M NaCI at pH 7.2 and recentrifuged at 130000 g for 1 h (Fig. 1). The resulting pellet and supernatant are referred to as PpBs and SpBs, respectively. All operations were carried out at 4-5 ~ C.

Enzyme assay. Ester-synthetase activity was assayed by following the transfer of label from 1-14C-labelled C~6, ClS , C22 or C24 alcohol (1 gmol) into esters. The labelled alcohol was mixed with the selected subcellular fraction (4-6 mg of protein in a final volume of 2 mt) with or without the additional cofactors and was incubated with shaking under aerobic condition at 30 ~ C. Each experiment included controls in which the selected subcellular fraction was boiled for 5 rain at 100 ~ C. At the end of the desired experimental time, the enzymatic reaction was terminated by extracting the lipids following the procedure of Bligh and Dyer (1959). To facilitate the recovery of lipids, an unlabelled wax mixture was added to the sample before the extraction. In some experiments unlabelled free acids (C16 or Cls), or stearoyl-CoA or dipalmitoyl-~-lecitihin were added to the reaction mixture. Alternatively, ester-synthetase activity was measured using 2 gmol of [1-~4C]stearic acid or [l-l~C]stear-

I

I

P

S 60 min

130000 g

S (soluble fraction)

P13oooo

(crude microsomal pellet)

I Resuspended in PBS i

60 rain I 130000 g

SpBs

PPBs

Fig. 1[. Fractionation procedure and designation of subcellular fractions. P = pellet, S = supernatant oyl-CoA plus / p.mol unlabelled docosanol. Finally, the effect of ATP (5 gmol) and CoA (1 gmol) addition on the ester synthesis was also assayed.

Radioactivity determination. Total recovery of the ~4C-labelled lipids was determined by liquid scintillation counting. Aliquots from the isolated lipid solutions (10 gl m l - 1) were transferred to counting vials. The solvent was removed under nitrogen and I ml Insta Fluor (United Technologies Packard, Ill., USA) added before quantitation of the total radioactivity as previously described (Avato et al. 1980, 1982). Radioactive lipid classes after separation on TLC plates were located and quantitated following described procedures (Mikkelsen and von WettsteinKnowles 1978; Avato et al. 1982). Radio-gas liquid chomatography separation and identification with standards of alcohols, free acids and esters, plus their quantification were also carried out following published methods (Avato et al. 1982).

Sodium dodecyl sulfate (SDSj-polyacrylamide gel etectrophoresis. The degree of solubilization of the various enzymes in Spzs and PPBS was investigated by SDS-polyacrylamide gel electrophoresis using the technique of Chua and Bennoun (1975).

Protein and chlorophyll determination. Protein content was assayed by the procedure of Lowry et al. (1951) using bovine serum albumin (Merck) as a reference standard. Interference by DTT was calculated according to Spector (1978) and Bradford (1976). Chlorophyll was measured by the method of Bruinsma (1961).

Electron microscopy. The 130000-g pellet was fixed for 1 h in 2% buffered glutaraldehyde (0.06 M phosphate buffer, pH 7.4), washed and post-fixed in the same buffer by 2% osmium tetroxide for 2 h. The sample was then dehydrated in a graded series of ethanol and propylene oxide. Staining with uranyl acetate was carried out while dehydrating with 95 % ethanol. The speci-

P. Avato: Synthesis of wax esters

489

men was then embedded in Spurr's resin (Spurr 1969) and sections were cut with a Reichert (Wien, Austria) Model OM U3 ultramicrotome. The thin sections were re-stained with uranyl acetate/lead citrate in an LKB 2168 Ultrastainer, System Carlsberg (LKB, Bromma, Sweden) and examined in an Elmiskop 102 electron microscope (Siemens, Berlin, West Germany) at 80 kV.

Table 1. Esterification of [1-14C]octadecanol by subcellular fractions from barley leaves. The reaction mixture was incubated for 20 h at 30 ~ C

Results and discussion

Soluble Microsomal

Subcellular distribution of enzyme activity. Substantial evidence exists indicating that the epicuticular wax lipid chains are synthesized in the epidermal cells of the leaf (Kolattukudy 1968b; Cassagne 1972). Additional observations lead to the conclusion that a de-novo fatty-acid-synthesizing system plus the different elongases contributing to the fatty acyl chains found in wax tipids, as well as the enzymatic machinery for the synthesis of given wax classes therefrom, are microsomal (von WettsteinKnowles 1979, 1982a, b). To localize the subcellular site(s) of the ester synthetase activity in barley leaves, the soluble and the crude microsomal fractions (Fig. 1) were firstly incubated with [1-14C]hexadecanol or -octadecanol. Although both preparations showed esterilying activity, the recovery of esters per unit of protein was three times larger when the microsomes were used (Table 1). Therefore, all further studies were performed using the crude microsomal preparation ( P 1 3 0 0 o o ) a s the enzyme source. Boiled subcellular preparations showed no activity (Fig. 2). This result parallels that of others using different systems. For example, synthesis of internally stored esters by dark-grown Euglena gracilis was also shown to take place in the microsomes. The latter, moreover, catalyze de-novo synthesis of fatty acids and the reduction of the resulting acylchains (Khan and Kolattukudy 1975; Kolattukudy 1970). In developing jojoba cotyledons (Pollard et al. 1979; Wu et al. 1981), the membranous material composing the floating wax pad from the 12000-g spin of the cell homogenate has been reported as the major site of ester synthesis. Furthermore, microsomes have been shown to be the site of wax-ester-synthetase activity in systems other than plants (Friedberg and Greene 1967; Sawaya and Kolattukudy 1973; Grigor and Harris 1977; Blomquist and Ries 1979). In contrast, the presently observed esterification activity of the microsomes was much higher than that reported for Brassica oleracea leaves in which 90% was in the soluble fraction (Kolattukudy 1967). Characterization of the microsomalfraction by electron microscopy. Ultrastructural examination of

Esterification (% total cpm recovered)

cpm m g - 1 protein ( x 10 3)

21 55

35 106

0 tb) E

I

POH18

y Fig. 2a, b. Separation by radio-thin-layer chromatography of lipids isolated after incubation of [1-1r with barley-leaf microsomes (P13o0oo)" The reaction mixture contained 5 mg of protein and 1 gmol of labelled alcohols in a final volume of 2 ml. The incubation was performed for 20 h at 30 ~ C using boiled (a) and non-boiled (b) microsomal preparations. O = origin; POHj8 = [1-14C]octadecanol; E = esters

the P13oo0o microsomal preparation from barley leaves showed primarily membrane fragments. Most of the membranes appeared as smooth and rough vescicles (Fig. 3). Structures clearly attributable to chloroplasts were very rare. Since the preparation had a pale green colour, however, some chloroplast structures were assumed to be

490

P. Avato: Synthesis of wax esters

|

i= .E

4O"

30

>

-

20

t~

.s x~ e~

4

5

6

7

8

pH Fig. 4. Effect o f p H on [1-14C]octadecanol esterification by barley-leaf microsomes. 9 0.32 M Citrate-phosphate; 9 0.32 M 2-(N-morpholino)ethane sutfonic acid (Mes); | 0.32 M 4-(2hydroxyethyl)-l-piperazineethane sulfonic acid (Hepes); zx 0.32 M 2-(N-cyclohexylamino)-ethane sulfonic acid. Each reaction mixture contained 5 mg of protein and 1 gmol of [114C]octadecanol in a total volume of 2 ml. Incubation was carried out for 6 h at 30 ~ C

Fig. 3. Electron micrograph of microsomal pellet from barley primary leaves. B a r = 0.2 gm, x 85000

present. The contamination must have been very small as only 0.02 ~tg chlorophyll per mg of protein could be measured. The membranes were, therefore, deduced to be of endoplasmic reticulum andor plasmalemma origin. The electron-microscopic analysis indicates that the enzyme-esterifying alkan-l-ol is membrane bound. No attempt was made, however, to assay the ester-synthetase activity of microsomal fractions from barley primary leaves enriched in either plasmalemma or endoplasmic reticulum.

Effect of temperature, pH and time. After 3 h of incubation at 30 ~ C, about 30% of [ 1 - 1 4 C ] d o c o sanol was esterified, whereas, in parallel experiments, only 5% of the total radioactivity was recovered in the esters when the incubation was carried out at room temperature. No esters were synthesized at 0 ~ C, but more t h a n / 0 % of the alcohol was converted into esters at 50 ~ C. This is in marked contrast to the ester synthetase in cell-free homogenates of developingjojoba seeds. The latter was fairly stable at 0 ~ C, less stable at room temperature and labile above 30 ~ C (Wu et al. /981).

The pH optimum for barley microsomal estersynthetase activity was determined to be 6.0 (Fig. 4). Activity declined sharply as the pH was raised or lowered, none taking place at pH 4 or less, or at pH 8 or greater. The effect of pH on the enzymatic activity of this system appears similar to that reported for wax-ester synthesis in broccoli (Kolattukudy 1967) and in honeybee (Blomquist and Ries 1979) microsomal preparations. Ester synthetases from several other sources however retained high activity up to pH 9.0 (Sawaya and Kolattukudy 1973). The effect of time on the incorporation of [/14C]hexadecanol into barley-leaf wax esters was also studied. After / h of incubation, over 10% of the labelled alcohol was already converted into esters. The reaction rate was approximately linear for the next 4 h after which it decreased, presumably because of a lower substrate concentration or an inhibition by the accumulation of the reaction product. Long incubation times ( > 10 h) were necessary to get 50% alcohol esterification.

Requirements for ester synthesis. To determinate the mechanism of esterification of alkan-l-ols in barley leaves, the microsomal fraction plus [l14C]docosanol was incubated with different combinations of free palmitic acid, CoA and ATP (Table 2). No appreciable stimulation or depression of ester synthesis catalyzed by the microsomes of

P. Avato: Synthesis of wax esters

49t

Table 2. Effect of palmitic acid, ATP and-or CoA addition on the incorporation of label by barley-leaf microsomes from the [1-14C]docosanol (POH) substrate into the esters (E). Incubation was carried out for 6 h. See Material and methods for the other experimental conditions. ATP, 5 gmol; CoA, I gmol; Palmitic acid, 3 p.mol

Additions %

None

Palmitic acid

Palmitic Palmitic acid, acid, ATP, CoA ATP

ATP, CoA

POH E

64 36

66 33

63 37

60 40

66 34

barley primary leaves was observed under any of the conditions tested. These results imply a direct esterification of endogenous acid moieties with exogenous fatty alcohols. To define the origin of the ester acid moieties, [1-14C]stearic acid plus unlabelled docosanol were used together as substrates. The enzymatic conversion into ester by the microsomal ester synthetase was however unsatisfactory (2%). This result indicates that the physiological substrate for the acid moiety which is to be esterified is other than a free fatty acid: that is, an activated acid is required. To obtain further evidence indicating that waxester synthesis in barley primary leaves may occur in vivo by the transfer of acyl groups from acylCoA to primary alcohols, the microsomal preparation (P13o00o) was then incubated with [1-14C]do cosanol and palmitoyl-CoA. In this case, 5-10% of the total label was incorporated in the esters, while the remaining radioactivity was associated with the alcohol substrate. Similarly, when [1~4C]stearoyl-CoA and cold docosanol were used as the substrates, the amount of label associated with the esters was very low. Previous tissue-slice experiments with Bonus barley primary leaves also gave extremely variable total incorporation of palmitoyl-CoA and stearoyl-CoA into the wax and 37-98% of the label was recovered in the free fatty acids (Avato et al. 1982). The low activity might arise because the exogenous acyl-CoAs are inaccesine to the wax-synthesizing enzymes, or because the microsomal preparation has a high thioesterase activity so that the acyl-CoAs are rapidly hydrolyzed to give free fatty acids. These could negatively affect the enzyme activity, as was proposed to explain the inhibition of ester formation by microsomes from the pheasant uropygial gland (Sawaya and Kolattukudy 1973). However, such an explanation does not seem consistant with the results we obtained (Table 2). More likely, the observed

depression of ester synthesis by barley-leaf microsomes in the presence of exogenous acyl-CoAs might be the result of their detergent action on the cellular enzyme system (Paude and Mead 1968; Dorsey and Porter 1968). No consistent evidence of an acyl-transferasecatalyzed esterification could be observed with Bonus barley primary-leaf microsomes in the tested experimental conditions when lecithin was incubated with [1-~4C]octadecanol as the labelled substrate.

Chain-length specificity and characterization of the reaction product. Since high amounts of esters could be obtained by incubating the microsomal fraction with a labelled alcohol but without addition of any cofactors, these conditions were used to characterize the chain-length specificity of the ester synthetase. Labelled alcohols of 16, 18, 22 and 24 carbon atoms were used as substrates. No absolute specificity in their esterification was observed; that is, the microsomal preparation readily catalyzed the esterification of all of them with fatty acids of endogenous origin. The naturally occurring alkan-l-ol-containing esters in barley primary-leaf wax are characterized by hexacosanol (75%) as the predominant alcohol moiety and C16, Cls, C20 and Cz2 as the major esterified acids (Giese 1975). The easy incorporation into the esters of alcohols which are not usual components of these lipids, indicates a lack of specificity for the alcohol moiety of the studied ester synthetase. More likely, the chain-length distribution of esterified primary alcohols is controlled by the substrate specificity of the preceding enzyme, the acylCoA reductase. Hexacosanol is in fact the predominant homologue (88%) of the free primary alcohols in barley wax (Giese 1975). A broccoli acetone-powder preparation was able to esterify indistinctly both C16 and C18 primary alcohols (Kolattukudy 1967) in a similar manner to barley microsomes. A jojoba seed homogenate was shown to incorporate into esters cis-ll-eicosenol, which is the endogenous alcohol of jojoba wax, as well as the non-endogenous tetradecanol, cis-9-octadecanol, dodecanol and cis-13-eicosenol (Wu et al. 1981). Furthermore, studies with grasshopper (Jackson and Blomquist 1976) and honeybee (Blomquist and Ries 1979; Lambremont and Wykle 1979) revealed an ester synthetase having only a moderate chain-length specificity. Table 3 shows the composition of esters synthesized by the microsomal preparation when [114C]octadecanol or -docosanol were used as substrates. The C34 (60%) and C38 (68%) esters were

492

P. Avato: Synthesis of wax esters

Table 3. Percent composition of esters synthesized from [l14C]octadecanol and [1-14C]docosanol by barley primary-leaf microsomes. ( - = not detected) Ester chain length

34 36 38 40

Substrate [1-14C]Octadecanol

[1-14C]Docosanol

60 40 ---

1 2 68 29

Table 4. Esterification of [l-14C]docosanol by SpBS and PPBS fractions from barley-leaf microsomes. The reaction mixture contained 4 mg of protein and 1 gmoi [1-14C]docosanol in a final volume of 2 ml. Stearic acid and stearoyl-CoA (2 ~tmol) were added to the reaction mixture as specified. The incubation was performed for 6 h at 30 ~ C

Additions None Stearic acid Stearoyl-CoA

Estcrification (% of total cpm recovered)

cpm rag- i protein ( x 10 -3)

PPBS

SpBs

29 39 < 1

11 35

Synthesis of wax esters by a cell-free system from barley (Hordeum vulgare L.).

Experimental evidence for a membranebound microsomal ester synthetase from Bonus barley primary leaves is reported. The results are consistent with at...
1MB Sizes 0 Downloads 0 Views