lysophosphatidate acyltransferaseactivities microsomes from … · lysophosphatidate...

Plant Physiol. (1989) 91, 1288-12950032-0889/89/91/1 288/08/$01 .00/0

Received for publication June 12, 1989and in revised form August 8, 1989

Lysophosphatidate Acyltransferase Activities in theMicrosomes from Palm Endosperm, Maize Scutellum, and

Rapeseed Cotyledon of Maturing Seeds1

Khaik-Cheang 002 and Anthony H. C. Huang*Department of Botany and Plant Sciences, University of California, Riverside, California 92521

ABSTRACT

Lysophosphatidate (LPA) acyltransferase (EC 2.3.1.51) in themicrosomes from palm endosperm (Syagrus cocoides Martius),maize scutellum (Zea mays L.), and rapeseed cotyledon (Brassicanapus L.) of maturing seeds were studied for their specificitiestoward the acyl moiety of the substrates lysophosphatidate andacyl coenzyme A (CoA). The LPA acceptor greatly influenced theacyl CoA specificity of the enzyme and vice versa. With 1-oleoyl-lysophosphatidate (LPA-18:1), the palm enzyme was equally ac-tive on oleoyl CoA and lauroyl CoA, whereas the maize andrapeseed enzymes were more active on oleoyl CoA than onlauroyl CoA. With 1-lauroyl-lysophosphatidate (LPA-12), whichgenerated less activity than LPA-18:1, the palm enzyme wasthree times more active on lauroyl CoA than on oleoyl CoA. LPA-12 was an inactive substrate for the maize and rapeseed en-zymes. The selectivity of the enzymes was also studied using amixture of LPA-18:1 and LPA-12, as well as lauroyl CoA andoleoyl CoA. Under this selectivity condition and compared to thespecificity condition, the enzymes from all the three seeds ex-erted stronger preference for oleoyl moiety in either the LPA oracyl CoA, and again, only the palm enzyme could act on LPA-12.Similar studies, although in lesser detail, showed that the en-zymes from soybean and castor bean were similar to the maizeand rapeseed enzymes in having little activity on substratescontaining lauroyl moiety. The results demonstrate the impor-tance of the acyl group in the sn-I position of LPA in determiningthe acyl preference in the sn-2 position in phosphatidate synthe-sis. The palm enzyme appears to be the only one capable ofsynthesizing phosphatidates containing high amounts of lauricmoieties.

In oil seeds, TAG3 synthesis proceeds via the Kennedypathway (20). Glycerol-3-P is first acylated by glycerol-3-PAT at the sn-l position to form lysophosphatidate, whichundergoes a second acylation by LPA-AT to form phospha-tidate. Dephosphorylation of phosphatidate by PA phospha-tase gives DAG, which undergoes a third acylation by DAG-

' Supported by National Science Foundation grant DMB 88-05618.2Cuffent address: Department of Biochemistry, University of Ma-

laya, Kuala Lumpur, Malaysia.3 Abbreviations: TAG, triacylglycerol; LPA-AT, lysophosphatidate

acyltransferase; LPA- 12, 1-lauroyl-lysophosphatidate; LPA-18: 1, 1-

oleoyl-lysophosphatidate; PA- 18:1/12, 1 -oleoyl-2-lauroylphosphati-date; PA- 18:1/18: 1, dioleoyl-phosphatidate; PA- 12/18: 1, I -lauroyl-2-oleoyl-phosphatidate; DAG, diacylglycerol; PL, phospholipids.

AT to produce TAG. All four enzymes in the pathway arefound in microsomal preparations of maturing seeds (20, 22).

In oil seeds, the first and third acyltransferase enzymes,glycerol-3-P AT and DAG-AT, were studied by various work-ers (3, 4, 7, 10, 12, 15). These studies show that, in general,glycerol-3-P AT preferentially uses palmitoyl CoA over C- 18CoAs (7, 8, 10). On the other hand, DAG-AT shows a broadspecificity toward acyl CoA donors, and the acyl donor usedin this acylation step is thought to depend largely upon theacyl CoA pool (12). In contrast, only one direct study on thesecond acyltransferase (LPA-AT) was reported (1 1). This en-zyme from maturing safflower seeds has some substrate spec-ificity toward both acyl CoA and LPA acceptor of C16 andC18 chain length (11).The fatty acyl moieties in the storage TAG in oil seeds are

species specific and are distributed in a nonrandom mannerin the three sn positions (20). The nonrandom distribution ofthe fatty acyl moieties in TAG is controlled by a number offactors, including the in vivo pool size of available acyl CoAs,the specificity of individual acyltransferases and, for polyun-saturated C-18 fatty acids, the acyl exchange reaction betweenoleoyl CoA and PC (8, 20, 21). While most seed oils containfatty acids ofC-16 and C-18 chain length, in some species theprincipal fatty acids may be shorter (C-8 to C-14) or longer(C-20 to C-24) (13). Our recent studies suggest that theseunusual fatty acids may not be readily utilized for TAGsynthesis in seeds where they are not normally found. In ourstudies, we tested the ability of the enzymes from threeselected oil seeds, each containing a different abundant fattyacid in their storage TAG (palm or Cuphea, Cl 2; maize,Cl 8: 1, rapeseed C22: 1), to utilize those fatty acids of unusualchain length for TAG synthesis in vitro (3, 22). The resultsshow that while DAG-AT from Cuphea, maize, and rapeseedcan utilize lauroyl CoA and oleoyl CoA for TAG synthesis,only the rapeseed enzyme has activity with erucoyl CoA (3).Also, when radioactive glycerol-3-P was incubated with palm,maize, or rapeseed microsomes in the presence ofnonradioac-tive acyl CoA, all three preparations synthesized radioactiveLPA, PA, DAG, and TAG from oleoyl CoA (22). However,with lauroyl CoA instead ofoleoyl CoA, the three microsomalpreparations synthesized LPA but only the palm microsomescould carry the synthesis further to form PA, DAG, and TAG.The observation suggests that acylation of lauroyl CoA at thesn-2 position ofLPA could only be carried out by palm LPA-AT. An alternate explanation for the failure of the maize andrapeseed enzymes to synthesize TAG from glycerol-3-P and

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LYSOPHOSPHATIDATE ACYLTRANSFERASE OF OIL SEEDS

lauroyl CoA is that the LPA- 12 rather than lauroyl CoA wasthe inactive substrate for LPA-AT.

In view of the importance of LPA-AT in TAG synthesisdue to its high substrate specificity and of the very limitedstudy of LPA-AT in oil seed tissues, we have made a directcomparative study of this enzyme in three plant species con-taining diverse fatty acyl moieties in seed TAG. We comparedthe enzyme specificity and selectivity toward acyl CoA andLPA which contain C- 12 and C- 18: 1 acyl moieties. We foundthat the acyl moiety of the LPA greatly influences the acylCoA specificity and selectivity of the enzyme and vice versa;this feature has not been demonstrated previously in thestudies of acyl preference of glyceride acyltransferases. Theresults are discussed in terms of the species-dependent acylpreference of LPA-AT and the use of genetic engineering toproduce temperate crop synthesizing a high lauric oil.

MATERIALS AND METHODS

Plant Materials

The scutella of maize (Zea mays L. inbreed Mo-17), thecotyledons of rapeseed (Brassica napus L. var Dwarf Essex)and soybean (Glycine max L. merr cv Coker 237), and theendosperm of castor bean (Ricinus communis L. var Hale)were obtained from plants grown in the greenhouse. Theendosperm of palm (Syagrus cocoides Martius) seeds wascollected from a local plant. The seeds were harvested whenthe oil content in the storage tissues ofthe maturing seeds wasapproximately half the values in the mature seeds.

Preparation of Microsomes

The tissues were chopped with a razor blade in a Petri dishcontaining 0.15 M Tricine-NaOH (pH 7.5) and 0.16 M sucrose(2-4 mL per g tissue), and then were homogenized gentlywith a mortar and pestle. The homogenate was filteredthrough a Nitex cloth (20 x 20 ,um). The filtrate was centri-fuged at 10,000g for 15 min, and the supernatant was recen-trifuged at 100,000g for 90 min. The pellet was resuspendedin a small volume of grinding medium. All operations wereperformed at 0 to 4°C. The microsomal preparations wereused immediately or kept frozen at -80°C until required.

Substrates

Nonradioactive lauroyl CoA, oleoyl CoA, erucoyl CoA,CoA, 1 -oleoyl-LPA, and dilauroyl-PA were obtained fromSigma Chemical Corp. Radioactive [1-'4C]oleoyl CoA (59.9Ci/mol) was purchased from New England Nuclear Corp. [ 1-'4C]Lauric acid (56 Ci/mol) was purchased from ResearchProducts International Corp. (Mount Prospect, IL) and con-verted to [1-'4C]lauroyl CoA by the acyl chloride method (1).

Preparation of 1-Lauroyl-LPA

LPA-12 was prepared by digestion of dilauroyl-PA withphospholipase A2 from Naja naja venom (Sigma) (1 1). Dilau-royl-PA (50 mg) was dissolved in 0.2 mL methanol and 3.6mL diethyl ether. Phospholipase A2 (1000 units), dissolved in50,uL 0.5 M Tricine-NaOH (pH 7.5) containing 0.02 M CaCl2,

was added, and the mixture was stirred at 30°C for 6 h. TheLPA-12 product was precipitated with 0.04 mL 1 M CaC12,converted to the sodium salt, and extracted with chloroform/methanol (17/3, v/v) as described (1 1). The extract was addedto 2 g activated silicic acid packed in a small column (0.8 cmdiameter, 4 mL bed volume). Elution was carried out stepwisewith 40 mL chloroform, 60 mL chloroform/methanol (95/5,v/v) and 60 mL chloroform/methanol (1/3, v/v). TLCshowed only one spot (LPA-12) in the eluate from the thirdsolvent; free fatty acids and unreacted dilauroyl-PA werefound in the first and second eluates, respectively. The con-centration of the synthesized LPA-12 preparation and com-mercial LPA- 18:1 (from Sigma) were estimated by measuringthe acyl ester bond as ferric hydroxamate derivatives (5).To see if the acyl group of the synthesized LPA- 12 was

indeed at the sn- 1 position, the LPA- 12 preparation wasincubated with ['4C]oleoyl CoA and palm microsomes, andthe ['4C]-PA produced was separated by TLC (see "Assay ofLPA-AT Activity") This PA was eluted from the TLC silicagel with chloroform/methanol (2/1, v/v) and then digestedwith phospholipase A2 (Sigma). The digestion mixture wasrechromatographed by TLC and the plate radioautographed.About 88% of the original ['4C]-PA was digested. Of thedigested PA, 99% of the radioactivity was found as free fattyacid, the remaining 1% as LPA. Thus the LPA- 12 preparationhad the lauroyl group in the sn- 1 position, and isomerizationof l-lauroyl-LPA to 2-lauroyl-LPA did not occur.

Assay of LPA-AT Activity

The standard reaction mixture (1 mL) contained 0.15 Mbuffer (Mops-NaOH [pH 6.5] for palm and maize micro-somes; Tricine-NaOH [pH 7.5] for rapeseed microsomes), 1mM MgCl2, 20 jAM LPA, 10 uM ['4C]acyl CoA (approximately100,000 dpm), and microsomes (approximately 40, 40, and 9,ug proteins from palm, maize, and rapeseed, respectively).The reaction was started by the addition of microsomes. Afterincubation at 30C for 10 min (palm and maize microsomes)or 4 min (rapeseed microsomes), the reaction was stopped.Modifications of the reaction mixture and incubation time inindividual experiments are described in the text. The reactionwas stopped by adding 2.5 mL of chloroform/methanol (1/1,v/v). The chloroform layer was partitioned by vigorouslyshaking the whole mixture with 1.1 mL of 1 M KCI-0.2 MH3PO4 (6) and centrifuging for 5 min at top speed in an IECHN-SII centrifuge. The lower phase was removed and theaqueous layer washed with 1 mL of chloroform. The com-bined chloroform layers were dried under nitrogen gas andquantitatively transferred onto precoated silica gel plates(Whatman silica gel 60A). The plates were developed inchloroform/methanol/acetic acid/water (85/15/10/3.5, v/v/v/v). In the experiments where PA containing mixed acylgroups (PA-18:1/18:1, PA-18:1/12 and PA-12/18:1, and PA-12/12) were formed, the PA were separated into differentspots in the TLC plate by this solvent system. Radioactivelipid spots were located by radioautography. Gel containingthe PA spots were scraped into scintillation vials and countedin 7 mL ofAquasol cocktail (NEN Research Products, Boston,MA).

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Plant Physiol. Vol. 91,1989

RESULTS

LPA-AT Activities on LPA-18:1 and Oleoyl CoA

As earlier reported (22), microsomal preparations frommaturing seeds of palm, maize, and rapeseed are capable ofcarrying out all the enzymic reactions ofthe Kennedy pathwayfor TAG synthesis in the presence of glycerol-3-P and anappropriate acyl CoA donor. In the chain reaction, LPA isbarely detectable, and PA accumulates before its conversionto DAG and TAG. The kinetics suggest that LPA-AT activityis high relative to the activities of the other enzymes. Similarkinetics have also been observed in in vivo labeling experi-ments (9, 19).

In the current study, we characterized the LPA-AT activityin the microsomes from palm, maize, and rapeseed usingLPA- 18:1 and oleoyl CoA; these are the two substrates knownto be reactive in the enzymatic reaction (22). In our assay ofLPA-AT activity, microsomes were incubated with radioac-tive oleoyl CoA and nonradioactive LPA- 18: 1, and the radio-active PA formed was detected by radioautography after TLC.The assay was performed at pH 7.5 for the rapeseed enzymeand pH 6.5 for the palm and maize enzymes. The amount ofPA formed in the absence of exogenous LPA was in generalless than 4% of that formed with exogenous LPA. Since weroutinely used radioautography to locate the radioactive PAspots before we scraped off the silica gel for radioactivitycounting, we had the opportunity to observe visually anyappreciable increases in the radioactivity in other lipids dueto a change in assay conditions. In all assays, we did notobserve appreciable increases in the radioactivities in DAGand TAG (from PA by further enzymatic reactions), FFA (byacyl CoA hydrolase), and PC (by exchange reactions).With the palm and maize enzymes, formation of PA was

linear for at least 15 min (Fig. 1), and using a reaction timeof 10 min, the formation was linear from 5 to 30 ,ug micro-somal proteins in the 1 mL reaction mixture (Fig. 2). Withthe rapeseed enzyme, formation ofPA was linear from 0 to 5

a-° 0.4

min, and from 5 min onward this formation leveled offrapidly(Fig. 1). After a 5 min incubation, about 42% of radioactivitywas recovered in PA. Using a reaction time of 4 min, forma-tion of PA was linear from 2 to 8 g.g of microsomal proteinsin the 1 mL reaction mixture (Fig. 2). There was a slow, butsteady, formation ofDAG and TAG, as well as PC, in all the3 preparations (Fig. 1). The formation ofPC was also observedby others using similar assay conditions (16, 18, 22), and thereaction was attributed to the acylation of endogenous accep-tors or by an acyl exchange reaction of endogenous PC (21).The pH for optimal LPA-AT activity was about 6 to 6.5

for the palm and maize enzymes (Fig. 3). In both preparations,there was some activity at pH 10-11. This activity was genu-ine, since the product PA was identified on a radioautogramand was eliminated if boiled microsomes were used. The pHactivity curve for rapeseed microsomes (Fig. 3) shows that theenzyme was active over a wide pH range, even above pH 10.There is only one report on the pH for optimal activity ofseed LPA-AT activity; in safflower, the microsomal enzymehas optimal activities at pH 8-10 (1 1).The effects of various compounds on LPA-AT activities are

shown in Table I. EDTA inhibited the palm enzyme activity,but slightly stimulated the maize and rapeseed enzyme activi-ties. The activities in all three preparations were inhibited bythe divalent cations Ca2 , Mn2 , and Zn2+. It was reportedearlier that safflower LPA-AT activity was inhibited by Mg2'(1 1). In our study, Mg2' stimulated or maintained the palmand maize enzyme activities, but inhibited the rapeseed en-zyme activity. The sulfhydryl compounds, DTT and 2-mer-captoethanol, had little effect on the palm enzyme activity,slightly inhibited the maize enzyme activity, but stimulatedthe rapeseed enzyme activity about twofold. On the otherhand, p-chloromercuribenzoate severely inhibited both thepalm and maize enzyme activities but had little effect on therapeseed enzyme activity. Spermidine, a polyamine, is knownto stimulate LPA-AT activity in safflower microsomes (1 1).In our study, spermine, a related polyamine, did not substan-

5 10 15 5 10 15Time (min)

5 10 15

Figure 1. Kinetics of formation of PA, PC, and neutral glycerides (DAG + TAG) from LPA-1 8:1 and [14C]oleoyl CoA. Microsomes from palm (40,Mg protein), maize (40 jug), and rapeseed (9 Mug) were used. Data are expressed in nmol compounds produced in a 1 -mL reaction mixture.

1290 OO AND HUANG




E

E._

0

E0la

0.4

0.2

Protein (jg)Figure 2. PA formation at increasing amounts of microsomal proteinsfrom palm, maize, and rapeseed. Reaction was carried out for 10 min(palm and maize) or 4 min (rapeseed).

tially affect the enzyme activities in the three microsomalpreparations. Fatty acid-free bovine serum albumin has oftenbeen added to reaction mixtures containing acyl CoA sub-strates in order to modulate the hydrophobicity ofthe reactionmixture. In our LPA-AT assay, however, this protein at 0.1 %almost completely inhibited the activities in all the threeenzyme preparations. The detergents, deoxycholate, and Tr-ton X-100, at concentrations above the CMC, were alsopowerful inhibitors of LPA-AT activities.

Specificity of LPA-AT Toward Lauroyl CoA/Oleoyl CoAand LPA-12/LPA-18:1An earlier report on LPA-AT from safflower microsomes

shows that the enzyme prefers LPA-18:1 and LPA-18:2 to

LPA-16:0 as the acyl acceptor, and oleoyl CoA and linoleoylCoA to palmitoyl CoA as the acyl donor (1 1). In the currentstudy, we used enzymes from three diverse seeds and extendedthe test on substrate specificity of LPA-AT to acyl donor andacceptor of shorter chain length (C- 12). The results are shownin Figure 4.When LPA- 18:1 was used as an acceptor, all the three

enzyme preparations were active with either oleoyl CoA orlauroyl CoA as the acyl donor (Fig. 4). With the maize andrapeseed enzymes, maximal activity was obtained at an acyfCoA concentration of about 10 Mm. At all the concentrationstested (10-40 jAM), the activity was about three times higherwith oleoyl CoA than with lauroyl CoA. With the palmenzyme, maximal activity was also obtained at about 10 Mmacyl CoA, and the activity was slightly higher with oleoyl CoAthan with lauroyl CoA. However, when the acyl CoA concen-tration was increased from 10 to 40 ,M, the activity on lauroylCoA remained about the same whereas the activity on oleoylCoA was inhibited. This latter inhibition did not appear to bedue to substrate insolubility, since the inhibition was notobserved in the maize and rapeseed assays.When LPA-12 was used as an acceptor, only the palm

enzyme showed detectable activity, and the activity was aboutthree times higher with lauroyl CoA than with oleoyl CoA(Fig. 4). Again, maximal activity was obtained at about 10 AMacyl CoA.The activity ofpalm LPA-AT toward increasing concentra-

tions of LPA-12 or LPA-18:1 in the presence of 10 Mm ofeither oleoyl CoA or lauroyl CoA as the acyl donor is shownin Figure 5. In all cases the palm enzyme activities reachedmaxima around 20 to 40 Mm of LPA. The order of productformation was 18:1/18:1 > 18:1/12 > 12/12 > 12/18:1 at allconcentrations of LPA used. The results are quite similar tothose in Figure 4, in which the palm enzyme activity wasstudied using a fixed concentration of LPA and increasingconcentrations of acyl CoA.

0._E 0.030E

0.020)E04-0.01

04

6 8 10

0.15

0.10

0.05

6 8 10

0.6

0.4

0.2

6 8 10

pHFigure 3. LPA-AT activities at various pHs. The following buffers were used: acetate-Na (0), Mops-NaOH (O), Tricine-NaOH (O), and Caps-NaOH (A). Reaction time was 10 min (palm, maize) or 4 min (rapeseed). Microsomes from palm (40 /g proteins), maize (40 jg), and rapeseed (9jig protein) were used.

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Plant Physiol. Vol. 91,1989

Table I. Effects of Various Compounds on LPA-AT ActivityThe reaction mixture (1 mL) contained 20 Mm LPA-18:1, 10 AM

(14C]oleoyl CoA, microsomes (palm, 45 Mg protein; maize, 49 ,ugprotein; rapeseed, 8 Mug protein), and 0.15 M Mops-NaOH (pH 6.5)(palm and maize microsomes) or 0.15 M Tricine-NaOH (pH 7.5)(rapeseed microsomes). Additions were made as indicated at the finalconcentrations. For palm, maize, and rapeseed enzymes, 100%activity was 0.08, 0.22, and 0.44 nmol, respectively.

Enzyme ActivityAddition

Palm Maize Rapeseed

None 100 100 100EDTA, 1.2 mM 54 118 129CaCl2, 1 mM 19 51 41MnC12, 1 mM 21 50 40ZnCI2, 1 mM 2 12 55MgC92, 1 mM 151 205 72MgCI2, 5 mm 96 144 44DTT, 5 mM 116 78 2072-Mercaptoethanol, 5 mm 109 76 174p-Chloromercuribenzoate, 1 mm 15 13 98Spermine, 2 mm 143 84 95BSA (fatty acid free), 0.1% 6 9 7Deoxycholate, 0.1% 1 1 84Triton X-100, 0.1O% 6 1 10

Very similar to the maize and rapeseed enzymes, the en-

zymes from soybean and castor bean were incapable of utiliz-ing LPA- 12 as an acceptor, and with LPA- 18: 1 as an acceptor,the activity on oleoyl CoA was several times higher than thaton lauroyl CoA (Table II).

Selectivity of LPA-AT Toward Oleoyl CoA and LauroylCoA

The selectivity of LPA-AT toward acyl CoAs was studiedusing a mixture of oleoyl CoA and lauroyl CoA at a fixed

combined concentration of 10 Mm but at varying proportions(Table III). With LPA- 18:1 as the acceptor, the enzymes fromall the three species behaved quite similarly. The maize andrapeseed enzymes showed especially strong selectivity towardoleoyl CoA. At an equal molar ratio of the two acyl CoAs, 91to 96% of the maize and rapeseed enzyme activities weretoward oleoyl CoA. Even at a concentration ratio of 4 to 1 oflauroyl CoA/oleoyl CoA, more than 50% of the maize andrapeseed enzyme activities were toward oleoyl CoA. The palmenzyme also showed strong selectivity toward oleoyl CoA,although not as strong as the maize and rapeseed enzymes.Overall, all the three enzymes had selectivity toward oleoylCoA stronger than their respective specificity. In addition, thetotal enzyme activity decreased as the concentration ratio oflauroyl CoA increased; this suggests that lauroyl CoA was a

less effective substrate for the enzyme and was actually inhib-iting the enzyme activity on oleoyl CoA.LPA- 12 was also used as an acceptor to study the acyl CoA

selectivity of palm LPA-AT (Table III). The study was notdone on the maize and rapeseed enzymes since they had littleactivity on LPA-12 (Fig. 4; Table II). At a fixed combinedoleoyl CoA and lauroyl CoA concentration of 10 uM but atvarying proportions, the palm enzyme had only a slight or noselectivity toward oleoyl CoA (Table III). However, the totalenzyme activity increased as the concentration ratio oflauroylCoA increased. The absence of selectivity toward lauroyl CoAin the presence of LPA-12 is different from the enzyme

specificity of being in favor of lauroyl CoA (Fig. 4; Table II).

Selectivity of LPA-AT Toward LPA-12 and LPA-18:1

The selectivity of palm LPA-AT toward LPA acceptors wasstudied using a mixture of LPA-12 and LPA-18:1 at varyingproportions but at a fixed total concentration of40 Mm. Eitheroleoyl CoA or lauroyl CoA at 10 gM was used as the acyldonor. The enzyme showed a strong selectivity toward LPA-18:1 acceptor, irrespective or whether oleoyl CoA or lauroyl

10 20 30 40

0.3

0.2

0.1

0.3

0.2

0.1

10 20 30 40

I I I I

1 0 20 30 40

Acyl CoA conc (pM)Figure 4. Effects of increasing acyl CoA concentrations on LPA-AT activities. The reaction mixture contained 20 gM LPA-1 8:1 or LPA-1 2.Microsomes from palm (40 Mig proteins), maize (40 Mg protein), and rapeseed (9 Mg protein) were used. PA-18:1/18:1 (0), PA-18:1/12 (A), PA-12/18:1 (0), PA-12/12 (A).

0.09

0.06

0.03

0

E

E*-

0

Rapeseed

* 1 8:1/18:1

0~0

18:1/12

12/18 1 12/12

0

1 292 OO AND HUANG




.C 0.10

E0

Ec

laE

0.05 A

Figure ir

activity.

[14C]lauroyl F

18:1/12 (A), PA-12/18

Table II. Activities of ISpecies on LPA-18:1 cCoA (10 AM)

The different PAs f18:1/18:1 formed. Fcsoybean enzymes, 1 0(nmol/min, respectively

Species PalriPA-18:1 /18:1 1 00PA-18:1/12 100PA-12/18:1 10PA-12/12 30

CoA was used (Tabland LPA-18:1, 86 t418:1. Even at a coi

LPA-18:1, 61-85% (

18:1. The total prodtion ratio of LPA-1

LPA- 12 was a poor,,

inhibiting the enzyn

iments were repeateitially all the PA pro

at a concentration r;not shown).

We have studiedoil seeds for using:shorter (C-12) chaiisubstrate specificityacceptor greatly infliversa. Overall, the er

palm exhibited strong preference for LPA and acyl CoA ofacyl chain length C-18:1 over C-12. The palm enzyme also

18:1/18:1 showed preference for 18:1, although not as strong as that ofthe other enzymes. In all cases, the preference of the enzymesfor C- 18:1 over C- 12 is stronger when assaying with a mixtureof substrates (substrate selectivity) than with individual sub-strates (substrate specificity). It is clear that LPA-AT from oil

* seeds which do not contain shorter fatty acid (C- 12) in TAG18:1/12:0 are incapable of using effectively LPA or acyl CoA of C- 12

A for the synthesis of PA. The ability ofpalm LPA-AT to utilizeA LPA and acyl CoA of C- 12 is genuine, although the enzyme

still prefers utilizing C- 18:1 substrates.1 2:0/12:0 Our work characterizes the substrate preference ofLPA-AT

in an in vitro system. Whether or not the findings are totally12:0/18:1 applicable to the in vivo conditions remains to be seen. So

o 0 far, all the results of our in vitro experiments in the currentII I and earlier studies are consistent with the in vivo observations:

2

I3 4

(a) There are close similarities between the in vitro (22) and1 0 20 30 40 in vivo (9, 19) labeling kinetics of LPA, PA, DAG, and TAG

LPA conc (pM)from glycerol-3-P. (b) The ability of palm LPA-AT to uselauroyl CoA (Figs. 4 and 5) is consistent with the presence of

icreasing LPA concentrations on palm LPA-AT lauroyl moiety in the sn-2 position of palm TAG (2). (c) Themixture contained 10 MM [14C]oleoyl CoA or inability of rapeseed LPA-AT to use erucoyl CoA (22) agrees)alm microsomes (9,Mg). PA-18:1/18:1 (0), PA- with the complete absence of erucoyl moiety in the sn-2:1 (0), PA-12/12 (A). position of rapeseed TAG (14). (d) The ability of rapeseed

DAG-AT to use C-22: 1 CoA (3) conforms to the presence ofLPA-AT from Maturing Seeds of Various erucoyl moiety in the sn-3 position of rapeseed TAG (14). (e)or LPA-12 (20 AM) and Oleoyl CoA or Lauroyl The low acyl CoA preference of the first (22) and third (3)

acyltransferases concurs with the low acyl specificity in theFormed are expressed as percentages of PA- sn- 1 and sn-3 positions of seed TAG (20).)r palm, maize, rapeseed, castor bean, and The product of LPA-AT reaction, PA, is a common inter-0% activity was 0.08, 0.23, 0.23, 0.59 and 0.42 mediate for the synthesis of storage TAG and membrane PLf. (21). Apparently, the seeds have a selective mechanism forn Maize Rapeseed Castor Bean Soybean channeling PA to the synthesis of either TAG or membrane

100 100 100 100 PL, since those seeds that contain uncommon acyl moieties24 36 18 18 in storage TAG do not have these acyl moieties in the mem-

1 3 1 1 brane PL (21). It is unknown if the selective mechanism is0 1 1 0 purely physical (e.g. instability of PL containing the uncom-

mon acyl moieties in membranes) or biological (e.g. differentle IV). At an equal molar ratio of LPA- 12 isozymes for the synthesis ofPL and TAG). Ifthe latter aspecto 94% of the activities were toward LPA- is valid, our microsomal preparations would contain twoncentration ratio of 19 to 1 of LPA- 12/ different LPA-AT for PL and TAG synthesis. The TAG-of the enzyme activity were toward LPA- specific LPA-AT would be much more active than the PL-luction of PA decreased as the concentra- specific LPA-AT, since the microsomes were prepared from02/LPA-18:1 increased; this suggests that seeds at a stage when they were actively synthesizing storagesubstrate for the enzyme and was actually TAG. If so, the palm TAG-specific LPA-AT would be morene activity on LPA- 18:1. When the exper- active on lauroyl CoA and LPA- 12 than what we have found,d with maize or rapeseed enzymes, essen- but the maize and rapeseed enzyme would still be very inac-ducts were derived from LPA-e18:1, even tive on lauroyl CoA and LPA- 12.atio of 19 to 1 of LPA- 1 2/LPA- 18:1 (data A major objective in seed oil biotechnology is to use genetic

engineering to transform an existing temperate-zone crop tosynthesize a high-lauric oil (17). In this endeavor, the focus

DISCUSSION has been on genetic engineering of the fatty acid synthetasecomplex which catalyses the elongation of fatty acid. Assum-

the preference of LPA-AT from several ing that this is successful such that the target seeds couldLPA and acyl CoA of usual (C- 18) and produce medium chain fatty acids, it is still necessary for then length acyl groups. In studies of both cell machinery to be capable of incorporating the modifiedand selectivity of the enzyme, the LPA fatty acids into TAG. Microsomes from maturing seeds ofuenced the acyl CoA preference, and vice various species except palm cannot utilize lauroyl CoA forizyme from all the oil seeds studied except TAG formation from glycerol-3-P, although the same prepa-

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Plant Physiol. Vol. 91, 1989

Table Ill. Acyl CoA Selectivity of LPA-AT in the Microsomes from the Maturing Seeds of VariousSpecies

Varying proportions of lauroyl CoA and oleoyl CoA at a combined concentration of 10 Mm were usedwith 20 AM LPA acceptor.

Acyl CoA mixture LPA-1 8:1 as Acceptor LPA-1 2 as Acceptor

12 18:1 Total PA PA Total PA PA*formed 18:1/12 18:1/18:1 formed 12/12 12/18:1

% nmol/min % nmol/min %

Maize20 80 0.33 2 9850 50 0.24 9 9180 20 0.18 47 53

Rapeseed20 80 0.44 1 9950 50 0.40 4 9680 20 0.34 37 63

Palm20 80 0.27 4 96 0.01 20 8050 50 0.23 19 81 0.02 49 5180 20 0.20 56 44 0.04 73 27

Table IV. LPA Selectivity of LPA-AT in Palm MicrosomesVarying proportions of LPA-12 and LPA-18:1 at a total concentration of 40 Mm were used with 10

AM acyl CoA donor as indicated.LPA mixture Oleoyl CoA as Donor Lauroyl CoA as Donor

LPA 12 LPA-1 8:1 Total PA PA Total PA PAformed 12/18:1 18:1/18:1 formed 12/12 18:1/12

% nmol/min % nmol/min %

20 80 0.51 5 95 0.15 10 9050 50 0.14 6 94 0.10 14 8690 10 0.07 11 89 0.06 24 7695 5 0.05 15 85 0.06 39 61

rations are capable of acylating glycerol-3-P (22) and DAG(3) with lauroyl CoA. The blocking step appears to be theLPA-AT reaction. We have now shown that LPA-AT is highlyspecific toward the two substrates, LPA acceptor and the acylCoA donor. LPA- 12 is not utilized as an acceptor by LPA-AT from various seeds examined except palm. With LPA-18:1, only palm LPA-AT showed activity towards lauroylCoA comparable to, or slightly less than, that toward oleoylCoA. If seeds like rapeseed and soybean had been engineeredto produce a high amount of lauric acid, they would still haveto produce a sufficient amount of oleic acid for the synthesisof membrane phospholipids. Under this condition and evenin the presence of a high molar ratio of lauroyl CoA to oleoylCoA, glycerol-3-P AT would produce LPA- 12 and LPA- 18:1(22), and LPA-AT would produce no PA-12/12, little or noPA-12/18:1, a small amount of PA-18:1/12, and a largeamount of PA- 18:1/18:1. Since DAG-AT has very low acylCoA preference between C- 12 and C- 18:1 (3), the TAG pro-duced in the presence of lauroyl CoA and oleoyl CoA wouldbe roughly proportional to the available PA and acyl CoA.This reasoning suggests that the TAG synthesized in thegenetically engineered seeds would contain a low amount oflauroyl moiety. Our findings pose new problems for a suc-

cessful genetic engineering of a new high lauric oil seed. Primeconsideration must also be given to the second acylation stepin TAG synthesis.

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