Microsomal f Brassica oleracea Cultivar lncorporates Erucic Acid

Plant Physiol. (1 995) 109: 409-420

Microsomal f yso-Phosphatidic Acid Acyltransferase from a Brassica oleracea Cultivar lncorporates Erucic Acid into the

sn-2 Position of Seed Triacylglycerols'

David C. Taylor*, Dennis L. Barton, E. Michael Giblin, Samuel L. MacKenzie, Cornelius G. J. van den Berg, and Peter B. E. McVetty

National Research Council of Canada, Plant Biotechnology Institute, 11 O Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N OW9 (D.C.T., E.M.C., S.L.M.); CanAmera Foods, Inc., Oakville, Ontario,

Canada L6L 5N1 (D.L.B.); and the University of Manitoba, Department of Plant Science, Winnipeg, Manitoba, Canada R3T 2N2 (C.G.J.v.d.B., P.B.E.M.)

~~ ~ ~ ~~ ~

Developing seeds from Brassica oleracea (1.) var botrytis cv Sesam were examined for the ability to biosynthesize and incorporate erucic acid into triacylglycerols (TAGs). Seed embryos at mid- development contained a high concentration of erucic acid in dia- cylglycerols and TACs, and substantial levels were also detected in free fatty acids, acyl-coenzyme A (COA), phosphatidic acid, and phosphatidylcholine. Homogenates and microsomal fractions from seeds at mid-development produced ['4Cleicosenoyl- and [14C]erucoyl-COAS from ['4C]oleoyl-CoA in the presence of malonyl-COA and reducing equivalents in vitro. These fatty acids were incorporated into TAGs via the Kennedy pathway. However, unlike most Brassicaceae, the 6. oleracea was able to insert significant erucic acid into the sn-2 position of TACs. It was shown that the lyso- phosphatidic acid acyltransferase (LPAT) incorporated erucic acid into the sn-2 position of lyso-phosphatidic acid. The erucoyl-COA: LPAT activity during seed development and the sn-2 erucic acid content of the TAC fraction in mature seed were compared to those in 6. napus, Tropaeolum majus, and Limnanthes douglasii. There was a correlation between the in vitro erucoyl-CoA:LPAT activity and the sn-2 erucic acid content in seed TACs. To our knowledge, this i s the first member of the Brassicaceae reported to have an LPAT able to use erucoyl-COA. This observation has important implications for efforts being made to increase the erucic acid content in B. napus, to supply strategic industrial feedstocks.

~~

A major objective of our seed oil modification and breeding programs is to increase production of 221 in Brassica napus to provide strategic industrial feedstocks (Scarth et al., 1992; Taylor et al., 1992b; Sonntag, 1991). The biosynthesis of 22:l in B. napus and other members of the Brassi- caceae has been shown to occur via successive condensa- tions of malonyl-COA with oleoyl-COA in the presence of a reductant (Agrawal and Stumpf, 1985; Mukherjee, 1986; Murphy and Mukherjee, 1988; Kunst et al., 1992; Taylor et

This work was partially supported by National Research Council Biotechnology Contributions Agreement No. GC103-3- 2024 awarded to CanAmera Foods, Inc. (Oakville, Ontario). This is National Research Council of Canada No. 38923 and Department of Plant Science contribution No. 974.

* Corresponding author; e-mail dtaylor8pbi.nrc.ca; fax 1-306- 975-4839.

al., 1992a). Then the very long chain acyl-CoAs are incorporated into TAGs via the Kennedy pathway (Fehling et al., 1990; Taylor et al., 1992a; Taylor and Weber, 1994).

In general, stereospecific analyses have shown that among most members of the Brassicaceae, 22:l is virtually excluded from the sn-2 position of TAGs (Taylor et al., 1994). Severa1 studies have shown that in B. napus this is due to the inability of the LPAT to utilize erucoyl-COA (00 and Huang, 1989; Bernerth and Frentzen, 1990; Taylor et al., 1990a, 1992a). Accordingly, various groups worldwide are attempting to transform rapeseed with an LPAT gene from meadowfoam (Limnanthes douglasii), which has the desired capacity to utilize erucoyl-COA during TAG bioassembly (Cao et al., 1990; Lohden et al., 1990; Taylor et al., 1990a, 199213; Peterek et al., 1992; Murphy et al., 1994).

Recently, we identified Brassica oleracea accession lines which, for the first time among Brassicaceae, contained seed oil TAGs with 30 to 35% of the total 22:l in the sn-2 position (Taylor et al., 1994). This prompted a further study, using developing seed from one of these lines, of the biosynthesis of TAGs containing 22:l and a comparison of the erucoyl-CoA:LPAT activity with that of other species found to have significant sn-2 22:l in seed TAGs. Here we report that the erucoyl-CoA:LPAT activity of the B. oleracea cultivar can be correlated with the unusually high sn-2 221

Abbreviations: DAG, diacylglycerol; DCI, direct chemical ionization; DGAT, diacylglycerol acyltransferase (EC 2.3.1.20); d.p.a., days postanthesis; FAME, fatty acid methyl ester; FFA, free fatty acid; G-3-P, glycerol-3-phosphate; GPAT, glycerol-3-phosphate acyltransferase (EC 2.3.1.15); LPA, lyso-phosphatidic acid; LPAT, lyso-phosphatidic acid acyltransferase (EC 2.3.1.51); MAG, mono- acylglycerol; NH,+-CI-MS, ammonia-chemical ionization-MS; PA, phosphatidic acid; PC, phosphatidylcholine; PUFA, polyunsaturated fatty acid; TAG, triacylglycerol; TLE, total acyl lipid extract; VLCMFA, very long-chain monounsaturated (>18:1) fatty acid; 18:lc 9, oleic acid, cis A 9-octadecenoic acid; 18:lc 11, vaccenic acid, cis A 11-octadecenoic acid; 20:1, cis A 11-eicosenoic acid; 22:1, erucic acid, cis A 13-docosenoic acid; 24:1, nervonic acid, cis A 15-tetracosenoic acid; 18:2, linoleic acid, cis A 9, A 12-octadecadi- enoic acid; 18:3, a-linolenic acid, cis A 9, A 12, A 15octadecatrienoic acid; a11 other fatty acyl groups are designated by the number of carbons:number of double bonds.

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41 O Taylor et al. Plant Physiol. Vol. 109, 1995

content. This germplasm provides an alternative to L. douglasii for the isolation of a gene encoding an LPAT with suitable flexibility in its specificity to lend itself to transformation of high erucic cultivars of B. napus.

MATERIALS A N D METHODS

Substrates and Biochemicals

[l-’4C]Erucic acid (52 mCi/mmol) was purchased from New England Nuclear, and [l-’4C]oleic acid (58 mCi/ mmol) and most other 1-I4C-fatty acids (50-60 mC/mmol) were purchased from Amersham. [ l-’4ClEicosenoic acid was synthesized as described previously (Taylor et al., 1992a). All l-I4C-fatty acids were converted to [l-’4C]acyl- COAS by an enzymatic method described previously (Taylor et al., 1990b). 22:l-LPA and 22:1/22:1-PA were synthesized as described previously (Taylor et al., 1991). Unlabeled acyl-CoAs, polyvinylpolypyrrolidone, ATP, COA, NADH, NADPH, and most other biochemicals were purchased from Sigma. Neutra1 lipid standards were obtained from NuChek Prep, Inc. (Elysian, MN), and polar lipids were purchased from Sigma. sn-1,2-Dierucin and -diolein were purified as described previously (Taylor et al., 1991). HPLC-grade solvents (Omni-Solv; BDH Chemi- cals) were used throughout these studies.

Plant Material

Seed of Brassica oleracea (L.) var botrytis (accession No. PI 372890) was obtained courtesy of Dr. J.R. McFerson (U.S. Department of Agriculture-Agricultura1 Research Service Plant Genetic Resources Unit, Cornell University, Geneva, NY). (PI 372890 is a cauliflower cultivar named ”Sesam,” originally developed by Broersen Brothers [The Nether- lands] and acquired by the Plant Genetic Resources Unit in 1972.) Seed of Brassica napus L. cv Reston, a high 22:l cultivar accumulating both C,, and C,, fatty acids in developing seeds, was obtained from the University of Mani- toba (Winnipeg, MB, Canada). Seed of Tropaeolum majus L. (cv Dwarf Double Golden Jewel) and Limnanthes douglasii R. Br. were purchased from Early’s Farm and Garden Cen- tre, Inc. (Saskatoon, Saskatchewan, Canada) and Floragran B.V. (Ma Lochem, The Netherlands), respectively.

B. napus, T . majus, and L. douglasii seeds were germinated and plants were grown in controlled growth chambers with a 16-h photoperiod (photon flux density of 400 pE

[General Electric CW 1500 Cool-Whitel and incandescent [Sylvania 60 W] lights) and day/night temperatures of 22/18”C.

S, seeds of the B. oleracea accession line (originally described by Taylor et al., 1994) were initially germinated, transplanted, and allowed to grow for about 4 weeks in a growth chamber with day/night temperatures of 20/15”C and a 16-h photoperiod (photon flux density of 500 pE m-’ s-I , supplied by a 1:l mixture of fluorescent [Sylvania Gro-Lite F96T12/GL/ WS/VHO] and incandescent [Sylva- nia 60-W lights]). At the “2 true leaf” stage, the plants were vernalized for 14 weeks in a low-temperature growth chamber at 2 to 4°C under low-light conditions (9-h pho-

m--2 s -1 [400-700 nm], supplied by a mixture of fluorescent

toperiod with a photon flux density of only 20 p E m-2 s-’, supplied by fluorescent [Sylvania Gro-Lite F96T12/(3L/ WS/VHOl light). After vernalization, plants were trans- ferred to a growth cabinet with day/night temperatures of 22/15”C and a 16-h photoperiod (photon flux density of 400 p E m-‘ s-’, supplied by a 1:l mixture of Sylvania Gro-Lite F96T12/GL/WS/VHO and General Electric CW 1500 fluorescent lamps), and plants flowered 6 to 8 weeks thereafter. Flowering plants were bud pollinated and mature seed was harvested 10 to 12 weeks after flowering. Thus, the total time taken to go from seed to seed in B. oleracea was approximately 35 to 38 weeks.

Developing seeds for metabolic experiments were se- lected at stages of development at which 22:l was synthesized at high rates and incorporated efficiently into TAGs. Under the growth conditions used in this study, this stage was 18 to 22 d.p.a. for B. napus, 16 to 20 d.p.a. for L. douglasii, 14 to 17 d.p.a. for T. majus, and 35 to 38 d.p.a. for B. oleracea. Seeds were harvested and developing embryos were excised and frozen at -80°C. Frozen seed was used for lipid analyses or for the preparation of homogenates or subcellular fractions for in vitro studies.

Preparation of Homogenates and Subcellular Fractionation

Harvested developing embryos were rinsed with dis- tilled water and suction filtered to remove excess moisture. After weighing, embryos (1-3 g fresh weight) were hcimog- enized at 4°C in a mortar and pestle in the presence of a small amount of acid-washed silica and grinding medium (80 mM Hepes-NaOH, pH 7.4, containing 0.32 M SUC, 1 mM DTT, 1 mM EDTA) a t a ratio of 5 mL g-’ fresh weight tissue and polyvinylpolypyrrolidone (100 mg mL-’ grinding medium). After the sample was homogenized, an equal volume of grinding medium was added to the paste, and the slurry was filtered through two layers of Miracloth (Cal- biochem). The filtered homogenates (cell-free extracts) were used directly for in vitro biosynthesis studies or were subjected to subcellular fractionation by differential centrifugation, or were extracted immediately for analysis of lipid components as described below.

For acyltransferase studies, homogenates were further fractionated by differential centrifugation in an SS-34 rotor on a Sorvall RC5C refrigerated centrifuge (Sorvall, Du- Pont). The homogenate was first centrifuged at 3,OOOg for 20 min and the pellet discarded. The supernatant was centrifuged at 30,OOOg for 30 min, and the pellet was resus- pended, using a small paint brush, in a volume of grinding medium equivalent to one-fifth of the original homogenate volume. This 3,000 to 30,OOOg pellet fraction was found to be enriched in microsomal LPAT activity. Proteins in homogenates or subcellular fractions were estimated by the method of Bradford (1976) using BSA as a standard.

Extraction and Analysis of Fatty Acyl Composition of Embryo Lipids

Total acyl lipids were extracted immediately from fresh homogenates of developing seed embryos (1-mL aliquots equivalent to 100-125 mg fresh weight). To the homoge-

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Brassica oleracea 22:l -CoA:lyso-Phosphatidate Acyltransferase 41 1

nate, 2.5 mL iso-propanol was added, the tube capped, and the mixture placed in a boiling water bath for 5 min. The solution was cooled briefly, 1.25 mL of CH,Cl, was added, and the mixture was allowed to sit for 30 min at room temperature, with occasional vortexing. The organic and aqueous phases were then separated by the sequential addition of 2 mL of CH,C1, and 2 mL of 1 M KC1 in 0.2 M

HJO,. After the sample was centrifuged (5008 for 5 min), the lower organic phase was saved and the aqueous phase was washed twice with 2 mL of CH,Cl,. The original organic phase was combined with the washes and dried under nitrogen to yield the TLE.

An aliquot of the TLE was transmethylated directly to determine the overall acyl composition. An interna1 standard of 17:O FFA was added to the TLE to permit quanti- tative fatty acid analysis. The sample was transmethylated directly in the presence of 2 mL of 3 N methanolic HC1 (Supelco Canada Ltd., Mississauga, Ontario) at 80°C for 1 h. Reaction mixtures were cooled on ice, 2 mL of 0.9% (w/v) NaCl was added, the mixture was extracted three times with 2 mL of hexane, and the hexane extracts were combined and dried on a cooling block at -10°C under nitrogen. The acyl composition was determined by GC of the FAMEs on a Hewlett-Packard model 5880 gas chro- matograph fitted with a DB-23 column (30 m X 0.25 mm; film thickness 0.25 pm; J & W Scientific, Folsom, CA). The GC conditions were: injector temperature and flame ionization detector temperature, 250°C; running temperature program, 180°C for 1 min, then increasing at 4"C/min to 240°C and holding at this temperature for 10 min.

The remaining portion of the TLE was further separated into its polar and neutral lipid components by TLC on Silica Gel H as previously described (Taylor et al., 1991). Individual lipid classes were further analyzed by treatment with pancreatic lipase or phospholipase A, (positional analyses) as described by Christie (1982) or transmethylated and the acyl composition analyzed by GC as described above.

Assay for Biosynthesis of 22:l and lncorporation into Glycerolipids

In the standard reaction mixture, homogenate (0.5-2.0 mg) or microsomal (100-200 pg) protein was incubated in open tubes in a shaking water bath (100 rpm) at 30°C for 1 to 2 h at pH 7.4 with a solution containing 90 mM Hepes- NaOH, 0.5 mM ATP, 0.5 mM COA, 0.5 mM NADH, 0.5 mM NADPH, 1 mM MgCl,, 200 p~ G-3-P, 1 mM malonyl-COA, and 18 p~ [1-'4C]18:1-CoA (10-50 nCi/nmol) in a final volume of 0.5 mL. Reactions were stopped by adding 2 mL of CH,Cl,:iso-propanol (1:2). The organic and aqueous phases were separated, the ',C-TLE was isolated and fractionated into its polar and neutral lipid components by TLC, and then portions were either transmethylated or subjected to positional analyses with pancreatic lipase or phospholipase A, (Christie, 1982). The aqueous phase, containing [14Clacyl-CoAs (Taylor et al., 1991, 1992a), was dried on a Speed-Vac overnight, and the residue was treated with 2 mL of 5% (w/v) KOH in methanol and heated at 80°C for 2 h to saponify the acyl-COA thioesters.

Tubes were cooled on ice, and the contents were acidified with 1 mL of 6 N HC1 and extracted three times with 2 mL of hexane. The hexane extract containing the ',C-fatty acids was dried under nitrogen, and the fatty acids were transmethylated in the presence of 3 N methanolic HCl as described above. A11 ',C-FAMEs were then separated and quantified by radio-HPLC as described previously (Taylor et al., 1992a).

Acyltransferase Assays

LPAT activity was measured by incubating homogenate or 30,OOOg pellet protein fractions in a shaking water bath (100 rpm.) at 30°C for 30 min at pH 7.4 with 90 mM Hepes-NaOH, 0.5 mM ATP, 0.5 mM COA, 45 p~ 18:l-LPA or 45 p~ 22:1-LPA, and 18 p~ [1-'4C]18:1-CoA and/or 18 p~ [1-'4C]22:1-CoA (each at a specific activity of 10-50 nCi/nmol) in a final volume of 0.5 mL. Reactions were stopped by adding 2 mL of CH,Cl,:iso-propanol (1:2). Phases were separated and the TLE was isolated as described above. Following TLC isolation of individual polar and neutral lipid species (Taylor et al., 19911, the I4C- labeled PA was further purified by TLC on Silica H in ethyl acetate:iso-0ctane:acetic acid (45:15:10) (Bocckino et al., 1989), scraped, and counted in 4 mL of Aquasol 2 (New England Nuclear) on an LKB 1219 Rack-peta instrument (LKB-Wallac Oy, Turku, Finland). In replicate treatments, after TLC, the ['4C]PA was eluted from the silica using an acidic (modified) Bligh and Dyer procedure (Taylor et al., 1991) for positional analysis with phospholipase A,. The resulting sn-2 FFA and sn-1-LPA fractions were transmethylated and the FAMEs analyzed by radio-HPLC as described previously (Taylor et al., 1992a). The relative LPAT activities in the developing seed homogenates or subcellular fractions are expressed as the rate of incorporation of the radiolabeled fatty acid into the sn-2 position of sn-1 acyl-LPA. In some cases, individual species of ['4C]PA ( e g dierucoyl-PA versus dioleoyl-PA) were identified by their migration distances on C,, reverse-phase TLC plates relative to authentic standards, as described by Cao et al. (1990).

[l4C1DAG and [14ClTAG fractions synthesized by further metabolism of the LPAT reaction product ([14C]PA) were analyzed by radio-HPLC (Taylor et al., 1991) or subjected to positional analyses with pancreatic lipase (Christie, 1982).

DGAT assays were conducted using 200 FM sn-1,2-diolein or -dierucin as the acyl acceptor and ['4C]18:1-CoA or ['4C122:1-CoA as the acyl donor, and radiolabeled TAG fractions were analyzed by reverse-phase C,, HPLC with a Radiomatic Flo-One p detector as described previously (Weselake et al., 1991).

Analyses of Developing and Mature Seed DAGs and TAGs

Intact DAGs and TAGs were isolated and purified by TLC as described by Taylor et al. (1991,1994). DAG species were analyzed by high-temperature GC as described previously (Katavic et al., 1995). Samples of TAGs were subjected to pancreatic lipase (Christie, 1982) and/or



41 2 Taylor et al. Plant Physiol. Vol. 109, 11395

Grignard-based stereospecific analyses (Taylor et al., 1994) or analyzed intact by high-temperature GC (Katavic et al., 1995) or reverse-phase HPLC (Taylor et al., 1991).

TAGs were also analyzed by direct probe NH,+-CI-MS as described previously (Taylor et al., 1995) but using a DCI probe. The analyses were performed on a Fisons 70-250 SEG hybrid mass spectrometer (Fisons, Manchester, UK) in the chemical ionization mode (NH,+ reagent gas). The source temperature was 200°C and the source pressure was 1 x l O P 4 Torr. Each sample, dissolved in hexane, was deposited on the heating coil of the DCI probe (Fisons standard DCI probe) and allowed to air dry. After the probe was inserted into the source, the heating coil was heated rapidly by applying a current rate of 32 mA/s. Heating was continued until the total ion current returned to baseline. Repetitive scans were acquired from mass 1200 to 700 at a rate of 20 s/decade.

RESULTS AND DISCUSSION

Endogenous Lipids Accumulating in Developing B. oleracea Seed

Developing seed of the B. oleracea cultivar had a relatively high VLCMFA content in a11 neutra1 lipid species (MAGs, DAGs, TAGs) (Table I). It should be noted that the MAG pool was very small (<1% of the TLE), indicating that there was negligible phospholipase activity (or PA phosphatase [EC 3.1.3.41 activity acting on LPAs) during isolation of the TLE. It was also possible to detect 22:l in the polar lipids LPA/PA and PC (Table I). This finding is unusual in that for most of the Brassicaceae studied thus far, 22:l is not detected in the polar lipid fraction in developing seeds (Taylor et al., 1991). However, 20:l was report- edly detected in the PC fraction of Brassica rapa (Griffiths et al., 1988). The very small pool of phosphatidylethano- lamine present in developing B. oleracea seed contained only saturated fatty acids: 16:O (80.0%), 18:O (15.3%), and 22:o (4.7%).

The finding of 22:l in a11 of the endogenous pools of LPA/PA, DAG, PC, and TAG in developing B. oleracea steed is, to our knowledge, the first clear evidence beyoncl that from in vitro radiolabeling experiments (Murphy and Mukherjee, 1988; Fehling et al., 1990; Taylor et al., 1992a) to support the involvement of Kennedy pathway intermediates, as well as PC, in the biosynthesis of TAGs containing 22:l in the Brassicaceae. Normally, 22:l is detected primarily in the TAGs and, to a much lesser extent, DAGs of developing seed in the Brassicaceae.

The 22:l content in TAGs of developing seed at 35 d.p.a. was 51 pg/mg fresh weight and about 170 pg/mg dry weight. This is about 55% of the 221 leve1 present in mature seed of this cultivar (approximately 310 pg/mg dry weight) and indicates that the seed was at mid-development, both temporally (maturity reached at approximaí ely 10-12 weeks postanthesis) and in terms of accumulation of 22:l. Positional analyses of the TAGs and DAGs from developing seed at 35 d.p.a. indicated that 221 was present at the sn-[l+31 and sn-2 positions of TAGs and at both positions in DAGs (Table 11). However, at about mid- development, the proportions of VLCMFAs at the sn-2 position of TAGs were not yet as high as found in the mature seed (Table 11).

The major species of DAGs and TAGs, as analyzed intact by GC, are shown in Table 111. In particular, there was an enrichment in dierucoyl TAGs (C6,) at this stage of development. An analysis of the TAG content in developing B. oleracea seed at both 35 d.p.a (Fig. 1A) and 45 d.p.a. (data not shown) by NH,+-CI-MS indicated ion clusters for various monoerucoyl TAG species (centered at [M+18]' = 931,959, 987) and a prominent cluster for dierucoyl-TAGs (centered at [M+18]+ = 1013). There was also a small cluster for C,oC,,C,, TAGs (centered at [M+181+ = 1041) and a trace of trierucin ([M+18]+ = 1071) among other CG6 TAGs. At maturity, (Fig. lB), the B. oleracea TAG profile had shifted to one in which the major prominent ion cluster was for C,,C,,C,, dierucoyl-TAGs, whereas those for mo-

Table 1. Acvl composition of lipid species in developing B. oleracea seed (Cv PI No. 372890) at 35 d.p.a.

FAME

2O:l 22:l 21:l 20:o (20:2P 22:o f22:21b (21:O)

18:2 18:3 Lipid Species 18:l c9

fc l l ) 16:O 18:0

Wt % TLE' 6.1 1.2 13.6 14.4 8.2 0.6

6.0 LPA plus PA 51.8 27.8 4.0 0.9 -

PC 45.8 27.2 6.5 1.4 1.9 5.5

FFA plus acyl-COA 6.0 1.2 12.7 13.9 9.0 0.7

MAG 10.8 2.5 22.4 26.7 13.1 0.6

DAG 7.6 1.6 21.9 21 .o 11.2 -

TAG 4.4 0.8 12.8 13.7 8.1 0.6

d (1.4)

(4

(4

(1.4)

(3.5)

(2.2)

7.4 0.5 (0.6) - 2.4 (4 - 2.9 (4 7.3 0.5

(0.7) 4.9 0.4 (0.5) 6.6 -

(4 7.1 0.5

44.0 1.2 (0.7) (-) 5.0 i!.2 (4 6.8 1.9 (-)

44.5 1.2 (0.8) (0.3)

13.3 0.5 (0.4) (0.3)

27.9 - (-) ( -)

47.9 1.5 (1 .O) (0.6) (0.8) (0.3)

a cis A 1 1 , A 14. cis A 13, A 16. TLE, 156 ? 7.8 udmF: fresh weight. -, Not detected.



Brassica oleracea 22:l -CoA:Lyso-Phosphatidate Acyltransferase 41 3

Table II. Stereospecific analyses of 20:l and 22:l distribution in endogenous DAGs and TAGs of developing seed of i?. oleracea at 35 d.p.a. and in TAGs of mature seed and lJ4C/20:l and lJ4C/Z2:l distribution in [J4C]DAGs and IJ4C]TAGs produced in metabolism experiments (cf. Fig. 2)

14C-VLCMFA distributions are shown in bold italics. DACs TACs

Seed Age/ Seed Age/ Acyl Moiety Percentage Percentage Acyl Moiety Percentage Percentage

sn-1 sn-2 sn-I1 + 31 sn-2

35 d.p.a. 20:l 86 14 P4CI 20: 48 52

22:l 89 11 1'4C122: 25 75

1

1

noerucoyl TAG species were present at lower intensities. Again, a small trierucin peak was detectable among other C,, TAGs.

In a previous report utilizing electron impact-MS (Taylor et al., 1994), we were unable to detect trierucin in mature seed of this B. oleruceu line. In the present study, trierucin was not readily detectable by high-temperature GC in TAGs of developing or mature seed unless the column was severely overloaded; only in such cases were c66 TAGs detected and represented <0.3% of the total TAG in developing seed and only about 2% in mature seed (Table 111). Trierucin was only detected utilizing NH,+-CI-MS, which has been shown to give a stronger molecular (adduct) ion than electron impact-MS (Taylor et al., 1995). Detection was also enhanced by the use of the DCI probe in performing the NH,+-CI-MS, which aids in volatilizing the high molecular weight TAGs directly at the MS source. Based on the overall proportion of C,, TAGs by GC and the relative CI-MS peak intensities for trierucin ([M+1811 = 1071) versus other C,, TAGs (e.g. 22:2/22:1/22:1 at [M+18]+ = 1069; 22:0/22:1/22:1 at [M+18]+ = 1073), trierucin was calculated to be present in only trace amounts in developing or mature seed TAGs (about 0.08 and 0.6% of total TAGs, respectively) in this B. oleruceu cultivar. Given that the amounts of C,, TAGs were too low to allow MS/MS to be performed, the calculated proportions for trierucin content are probably overestimates.

Biosynthesis of TACs Containing 22:l in B. oleracea

Initially, homogenate and 30,OOOg pellet protein fractions were tested for the capacity to biosynthesize VLCMFAs by chain extension of ['4C]18:1-CoA with malonyl-COA and, in the presence of G-3-P, to incorporate these newly synthesized VLCMFAs into glycerolipids. After a 2-h incubation with homogenate protein, about 30% of the ['4C]18:1-CoA had been elongated to ['4C120:1-CoA and ['4C]22:1-CoA (acyl-COA plus FFA fractions), and similar proportions of l4C-VLCMFAs were detected, primarily in TAGs. Incubations with the 30,OOOg microsomal fraction showed similar proportions of newly synthesized

35 d.p.a. 20:l 91 9 I'4CI2O:l 91 9

['4C122:1 89 11 22:l 92 8

Mature seed 20:l 69 31 22:l 71 29

['4C]20:1 and ['*C]22:1 in the acyl-CoA/FFA and TAG pools after 1 h (Fig. 2). In addition, there was also evi- dente of significant desaturation of ['4C]18:1 to [14C]18:2 and ['4C]18:3. The ',C-VLCMFAs were also found in LPA/PA, DAG, and PC fractions. Although the proportions of l4C-VLCMFAs were highest in TAGs, the polyunsaturates, ['4C]18:2 and ['4C]18:3 were more prominent in PC, LPA/PA, and DAG and not yet prevalent in TAG. An analysis of the intact [',C]TAG species by radio- HPLC showed that, whereas various mono- and dierucoyl TAGs were biosynthesized, no radiolabeled trierucin could be detected (data not shown). However, unlike previous studies with B. nupus (Taylor et al., 1992a), significant [I4C122:1 was found in the sn-2 position of 14C- labeled DAGs and TAGs synthesized in vitro by the B. olerucea microsomal protein fraction (Table 11). The presence of I4C-VLCMFAs at both the sn-1 and sn-2 positions of DAGs is consistent with the operation of the Kennedy pathway (Kennedy, 1961; Stymne and Stobart, 1987), suggesting that, in the presence of G-3-P, both the GPAT and LPAT enzymes of this pathway are capable of utilizing both 20:l-COA and 22:l-COA. Furthermore, the proportions of newly synthesized ['4C]20:1 and [l4C]22:1 in the sn-2 position of DAGs showed an enrichment over the endogenous pool, suggesting that enhanced incorpo-

Table 111. DAG and TAG species present in developing seed of i?. oleracea at 35 d.p.a. and TAG species present in mature seed

percentage TAG Percentage of Total Of 'IJecies 35 d o a . Mature

DAG Species

cl gC1 6 2.5 c,, C18C16 21 .o c52

c18c18 Plus c16c20 33.0 c54

C18C22 P l U S C20C2, 19.1 C58

c22c22 6.0 C62

C,,

CI6C22 P l U S Cl8C20 16.4 '56

c20c22 2.0 c60

'64

0.8 0.4 1.9 0.9 3.8 2.0

13.5 9.7 17.6 9.4 18.5 11.2 38.3 46.8

3.2 12.0 0.3 2.4



414 T a y l o r et al. Plant Physiol. Vol. 109, 1!395

1013 1 O0 A

80 n E .- o

60 a, S * -

40 m - 2

20

O 900 920 940 960 980 1000 1020 1040 1060 1080 1100

m /z

J 1013

I B

II 1071 (trace) I...

900 920 940 960 980 1000 1020 1040 1060 1080 1100

m /z Figure 1. Spectra of molecular ion region of TAGs from developing (35 d.p.a., A) and mature (6) 6. oleracea seed of line PI No. 372890, analyzed by NH,+-CI-MS.

ration of VLCMFAs into the sn-2 position was beginning at this stage of development.

LPAT Studies

synthesized ''C-erucoyl moieties into DAGs and TAGs in B. oleruceu homogenate and microsomal fractions prepared from developing seed (Fig. 2; Table 11) were consistent with the operation of the Kennedy pathway. More importantly, the presence of significant 22:l in the sn-2 position of IIAG and TAG fractions suggested that this member of the Dras- sicaceae possesses an LPAT capable of utilizing 22:l-CoA. This was confirmed by direct in vitro assays of LPAT

The presence of 22:l in the endogenous LPA/PA, DAG, PC, and TAG pools of developing seed of the B. oleracea line (Tables I and 11) and the pattern of incorporation of newly




rzzzza 18:3 18:2

msi 18:l 0 20:l - 22:l

Acyl-CoA I FFA LPA I PA DAG PC TAG

Figure 2. Proportions of 14C-fatty acyl moieties accumulating in fatty acyl-CoA/FFA pools and in glycerolipid species of the Kennedy pathway, as well as PC, during incubation of a 30,OOOg microsomal protein fraction prepared from developing B. oleracea (35 d.p.a.) seeds with ['4C118:1-CoA in the presence of reducing equivalents, malonyl-COA and G-3-P as described in "Materials and Methods."

activity (Table IV). In reaction mixtures in which homogenates were supplied with 18:l-LPA and [14C]22:1-CoA, more than 95% of the [l4C]22:1 present in the radiolabeled PA product of the LPAT reaction was found in the sn-2 position, as shown by stereospecific analysis using phospholipase A,. Similarly, the major proportion of radiolabel was found at the sn-2 position of PA produced in reaction mixtures in which [14C]18:1-CoA was the acyl donor. This indicated that, although there was some capacity for acylation of endogenous G-3-P at the sn-1 position, the exogenously supplied 18:l-LPA was the primary acyl acceptor and that there was minimal acyl migration during PA isolation and analysis. [I4C1DAG and [l4C1TAG fractions from these reaction mixtures were further analyzed using pancreatic lipase (Table IV). The DAGs showed a consis- tently high proportion of [14C]22:1 or ['4C]18:1 at the sn-2 position of DAG, essentially identical with that proportion found in PA. Thus, there was no significant acyl migration from the sn-2 position during DAG isolation and handling. In TAG, [14C]22:1 or ['4C]18:1 were found at both the sn-[l+3] and sn-2 positions. These findings are again consistent with the involvement of the Kennedy pathway enzymes in the conversion of PA to TAG. More importantly, they indicate that in the B. oleracea cultivar, 22:1 can be incorporated into the sn-2 position of LPA and that the PA product is subsequently converted to DAGs and TAGs containing 22:l in this position. The proportion of [14C]22:1 at the sn-2 position of TAGs arising from further metabolism of the [14C]PA (Table IV) is quite comparable to that

observed during de novo synthesis of [14C]22:1 from [14C]18:1-CoA followed by incorporation onto the glycerol backbone (Table 11). In both cases, the relatively high proportion of [I4C122:1 found in the sn-[1+31 position of TAGs compared to that in the sn-2 position is likely due to a superimposed sn-3 acylation of endogenous DAGs present in the homogenate/microsomal fractions, a reaction catalyzed by DGAT using [14C]22:1-CoA. Such high specificity/selectivity of DGAT for erucoyl-COA has been docu- mented previously in B. napus cv Reston (Weselake et al., 1991), and the DGAT reaction was also shown to be enhanced by the addition of exogenous acyl acceptors such as G-3-P, 18:1-LPA, or 22:l-LPA (Taylor et al., 1991).

The low but measurable amount of [14C]22:1-labeled PC produced in the reaction in which [14C]22:1-CoA was the acyl donor (Table IV) is consistent with the detection of significant 22:l in the endogenous PC pool (cf. Table I) and also agrees with the findings in a previous metabolism study conducted in B. napus, which showed that a small pool of radiolabeled PC containing this nonmembrane fatty acid was synthesized (Taylor et al., 1992a). However, in the latter study, the radiolabeled 22:l was found only in the sn-1 position of PC, consistent with the absence of significant 22:l-CoA:LPAT activity in B. napus (Taylor et al., 1990a). Thus, whereas erucoyl moieties normally accumulate primarily in neutra1 lipids (cf. Fig. Z), it may be that membrane or other polar lipids (e.g. PC or PA) containing this fatty acid are marked for rapid mobilization into DAGs via CDP-choline sn-1,2-diacylglycerol cholinephospho-



41 6 Taylor et al. Plant Physiol. Vol. 109, 1'395

Table IV. lncorporation of ['4CIZ2:l-CoA or ['4C118:l-CoA into glycerolipids PA, DAG, PC, and TAG b y B. oleracea developing seed homogenates supplied with 18: 1 -L PA

Reactions were performed as described for LPAT assays in "Materials and Methods." Values are expressed as pmol of 14C-fatty acid incorporated into each glycerolipid fraction min-' mg-' protein ? SE. The proportion of "'C-fatty acid at each sn position was determiried by analysis of phospholipids with phospholipase A,, and neutra1 lipids with pancreatic lipase.

Reaction Mixture PA DAC PC TAC

['4C]22:1-CoA plus 18:l-LPA 7.2 t 0.3 39.5 2 1.5 1.9 ? 0.1 72.0 ? 2.1 sn-1 : 5% sn-1 : 4% sn-1 : N.D." sn-[l + 31: 88Yo

sn-2: 95% sn-2: 96% sn-2: N.D. sn-2: 12Y0

['4C118:1-C~A plus 18:l-LPA 86.6 L 2.7 51.7 2 1.1 19.2 ? 0.6 23.2 ? 0.6 sn-1 : 18% sn-1 : 20% sn-1 : 40% sn-[l + 31: 52%

sn-2: 48Y0 sn-2: 82% sn-2: 80% sn-2: 60% __ a N.D., Not determined: insufficient labeled PC (<2500 d.p.m.) to perform accurate phospholipase analysis.

transferase (EC 2.7.8.2) (Taylor et al., 1992b) or for degra- dation by phospholipases (Banas et al., 1992), either or both of which possess some specificity for these "unusual" polar lipids. Such mechanisms have been shown to operate in sequestering ricinoleic acid into TAGs in castor bean (Bafor et al., 1991; Baras et al., 1992), and similar mechanisms may operate to channel 22:l away from membranes and into TAGs in the Brassicaceae.

In the LPAT reaction with ['4CJ18:1-CoA as the acyl donor, there was significant redistribution of total radiolabel at the sn-l/sn-2 positions of PC (Table IV), and overall, a large proportion of the ['4C]18:1 was further desaturated to 18:2 and 18:3. In PC, of the 60% sn-2 labeled fatty acids, PUFAs were enriched 159% (['4C118:2 + 18:3); 41% ['4C]18:1] compared to PA, where, of the 82% sn-2 labeled fatty acids, more of this was still present as 18:l [62% ['4C]18:1; 38% ([I4C]18:2 + 18:3). In DAG, of the 80% sn-2 labeled fatty acids, about 50% was (['4C118:2 + 18:3) and 50% was ['4C]18:1. In TAG, the overall leve1 of labeled polyunsaturates was too low to allow an accurate determi- nation of their positional distribution.

These findings are consistent with the well-known sequential synthesis of 18:2 and 18:3 from 18:1, catalyzed by microsomal A12 and A15 desaturases, respectively, acting primarily at the sn-2 position of PC. Then, via a combination of the reversible interconversion of DAG to PC as catalyzed by CDP-choline sn-1,2-diacylglycero1 choline- phosphotransferase, and the enrichment of the acyl-COA pool with polyunsaturated acyl-CoAs (released from PC via exchange with 18:l-CoA catalyzed by the reversible acyl-CoA:lyso-PC acyltransferase [EC 2.3.1.231), Kennedy intermediates enriched in PUFAs are synthesized (Stymne and Stobart, 1987). These pathways have been elegantly characterized in oilseeds that accumulate PUFAs, such as safflower and flax (Stymne and Stobart, 1987; Stymne et al., 1992), and have also been shown to be present in developing embryos of other Brassicaceae (Lemieux et al., 1990; Browse and Somerville, 1991; Taylor et al., 1991, 1993). In the current study, during the in vitro experiments in which ['4C]18:1-CoA was both desaturated and elongated (cf. Fig. 2), only low levels of ''C-PUFAs, but relatively larger proportions of I4C-VLCMFAs, accumulated in TAGs during the short incubation time. This contrasts strongly with the enrichment of endogenous DAG and TAG pools with

22:1, 18:2 (and to a lesser extent, 20:l and 18:3), and the relatively low proportions of PUFAs present in endogenous PC (cf. Table I). Given that the in vitro studies are, of necessity, a static view of ongoing metabolism, taken to- gether, the data would suggest that VLCMFAs are more efficiently channeled into TAGs than are PUFAs but that ultimately both VLCMFAs and PUFAs accumulate in TAGs in the B. oleracea line.

A comparison of the LPAT activities in homogenates of the B. oleracea line with those of rapeseed (B . napus), nas- turtium (T. majus), and meadowfoam ( L . douglasii) ('Table V) indicated that, although the relative 18:l-COA activities were of the same order of magnitude among the four species tested, there were dramatic differences in the ability to utilize erucoyl-COA. The B. oleracea cultivar had an 22:l-CoA:LPAT activity that was about 10-fold higher than the low, barely detectable activity found in B. napus and 2-fold higher than that found in T. majus. The highest 22:l-CoA:LPAT activity was found in L. douglasii, about 7-fold higher than that observed in B. oleracea.

When subcellular fractions were prepared, it was found that the total oleoyl- and erucoyl-CoA:LPAT activities re- covered in a microsomal 3,000 to 30,OOOg pellet fraction were 3- to 4-fold higher than those found in a 30,000 to 100,OOOg pellet fraction (data not shown). These findings are consistent with previous studies in a number of oilseeds, in which l0,OOOg (Taylor et al., 199213) and 20,OOOg (Ichihara et al., 1987; Bernerth and Frentzen, 1990; Lohden and Frentzen, 1992) differential centrifugation fractions had higher total LPAT activities than those membrane fractions sedimenting at 100,OOOg. In the present study, the 30,OOOg microsomal fraction from B. oleruceu displayed a 22:l-CoA:LPAT activity about 70-fold higher than that observed in B. napus and 7-fold higher than that in T. rnajus (Table V). The 22:l-CoA:LPAT activity in L. douglasii ini- crosomes was about 2-fold higher than in B. oleracea. The observation that the specific activity of the B. napus erucoyl-CoA:LPAT was not significantly enriched in a micro- soma1 preparation confirms the very low specificity of this enzyme for erucoyl-COA.

The finding of a significant 22:l-CoA:LPAT activity in B. oleracea is, to our knowledge, a first among members of the Brassicaceae. Previously, B. napus (00 and Huang, 1989; Bernerth and Frentzen, 1990; Taylor et al., 1990a,




Table V. Relative LPAT activities in developing oilseed homogenate and microsomal protein fractions supplied with 45 p~ 18:l-LPA and either ['4C118:1-CoA or [14C]22:1-CoA, and sn-2 22:l content in mature seed TAGs

Activity is expressed as pmol 14C-fatty acid incorporated into the sn-2 position of PA. SE 5 5% in all cases (n = 2 or 3). Homogenate Studies: LPAT Stereospecific

Activity Microsome Studies: 22:l-COA Analyses: Sn-2 22:l Content in Mature LPAT Activity

pino/ min- mg- ' protein pmol min- mg- protein mo/ % of total 22:7

Oilseed Species

22:l -COA 18: l -COA Seed TAGs

B. oleracea PI No. 372890 6.9 107 53.2 29.4b

B. napus cv Reston 0.65 156 0.8 3.0' (1 5.5)a

(240.0)a

(32.5)"

(2.6)"

T. majus cv Dwarf Double Colden Jewel 3.7 120 8.3 35.4c

L. douglasii 48.5 127 102.3 67.0d

a Ratio of 18:l-CoA:LPAT activity/22:l-CoA:LPAT activity. bTaylor et al. (1994). 'Taylor et al. (1992b). Phillips et al. (1971).

1992a, 1992b), Sinapis alba (Fehling et al., 1990), and Lunaria annua (Fehling and Mukherjee, 1990; Fehling et al., 1990) were shown to have LPATs incapable of inserting erucoyl moieties into the sn-2 position on the glycerol backbone during TAG biosynthesis. Furthermore, the widely dispar- ate 22:l-CoA:LPAT activities in B. napus (low) and L. douglasii (high) are collectively consistent with findings from previous studies (00 and Huang, 1989; Bernerth and Frent- zen, 1990; Cao et al., 1990; Lohden et al., 1990; Taylor et al., 1990a, 1992b). However, the current (Table V) and previous (Cao et al., 1990; Taylor et al., 1990a, 1992b) findings with T. majus conflict with a study by Lohden and Frentzen (1992), who failed to detect significant 22:l-CoA:LPAT activity in microsomal fractions, regardless of the LPA donor supplied. Although we are unable to explain the lack of activity observed in the latter study (Lohden and Frentzen, 19921, our findings would suggest that T. majus LPAT does indeed possess a moderate capacity to utilize 22:l-COA. Combined with other studies of the biosynthesis of TAGs containing sn-2 22:l in T. majus (Pollard and Stumpf, 1980; Murphy and Mukherjee, 1988; Whitfield and Murphy, 1990), it appears that such TAGs are probably synthesized via the Kennedy pathway and not via acyl exchange at the sn-2 position of DAGs and TAGs as suggested previously (Lohden and Frentzen, 1992). Thus, in a11 oilseeds studied thus far that accumulate TAGs containing VLCMFAs, the data are consistent with VLCMFA biosynthesis by elongation of oleoyl moieties to give VLCMFA-COAS, which are then used in the synthesis of TAGs via the Kennedy pathway.

In general, there was a correlation between the proportion of 22:l found at the sn-2 position of seed TAGs by stereospecific analysis and the 22:l-CoA:LPAT activity observed in vitro and also a negative correlation between the proportion of sn-2 22:l and the ratio of 18:1-CoA:22:1-CoA LPAT activities (Table V). Thus, as one might expect that the relative LPAT activities can serve as a reasonable gauge of the ability to biosynthesize TAGs containing 22:l at the sn-2 position.

The LPAT from the B. oleracea line was capable of utilizing a range of [14Clacyl-CoA donors in vitro, including 16:O-, 18:O-, 18:1-, 18:2-, 18:3-, 2O:l-, and 22:1-CoAs, to vary-

ing degrees (data not shown). With 22:l-LPA as the acyl acceptor, for example, the relative rates of incorporation into PA were: 16:O-CoA, 16%; 18:O-CoA, 23%; 18:1-CoA, 100%; 18:2-CoA, 69%; 20:1-CoA, 13%; 22:1-CoA, 7%. These findings confirm that the B. oleracea LPAT has a broad acyl-COA specificity, as was suggested previously based on the sn-2 composition of TAGs by stereospecific analysis (Taylor et al., 1994).

To test the acyl selectivity of the B. oleracea LPAT, the microsomal fraction was assayed in the presence of 45 FM 18:l-LPA and equimolar quantities (18 KM) of ['4C]18:1- COA and ['4C]22:1-CoA. Under these conditions, the enzyme incorporated 90 to 95% ['4C]18:1 and 5 to 10% [l4C122:1 into the sn-2 position of PA. Thus, although the B. oleracea LPAT could utilize 22:1-CoA, 18:l-CoA was still the preferred substrate in vitro. Similar findings were reported in selectivity studies with the meadowfoam (Lim- nanthes) LPAT, in which, under similar conditions, 18:l- COA was preferred 9:l over erucoyl-COA (Cao et al., 1990).

The B. oleracea microsomal LPAT was able to insert 22:l into the sn-2 position of 22:1-LPA, producing dierucoyl-PA (Table VI). The activity using 45 WM sn-1 22:l-LPA as an acyl acceptor was about 50% of that observed when 18:l-

Table VI. lncorporation o f [14Clerucoyl moieties%to lr4F glycerolipids b y microsomal fractions from developing embryos o f B. oleracea and 8. napus supplied with 18 p~ [14C]22:l-CoA as acyl donor and either 45 p~ 22:l-LPA or 200 p~ 1,2-dierucin as acyl acceptor

determined.

~

Results in parentheses are percentages of ['4C]trierucin. n.d., Not

B. oleracea 6. napus cv Reston Reaction Tested PI No. 372890

PA DAG TAG PA DAC TAG

pmol min- mg- pmol min- mg- protein profein

22:l -LPA PIUS 26.8 143 196 O O 300 [14C122:1 -COA (0) (0)

1,2-Dierucin plus n.d. n.d. 95 n.d. n.d. 100 [14C]22:1-CoA (1.4) (35.0)



41 8 Taylor et al. Plant Physiol. Vol. 109, 1!195

LPA was supplied at the equivalent concentration (cf. Ta- ble V). In contrast, the B. napus microsomal preparation was unable to insert 22:l into the sn-2 position of sn-1-22:1-LPA, confirming the inability of the LPAT in this species to use erucoyl-COA, regardless of the acyl acceptor supplied (Table VI). In meadowfoam, similar specificity with respect to LPA acyl acceptors was found, in that the 22:l-CoA:LPAT activity in the presence of 40 p~ 22:l-LPA was reported to be about 50% of that observed in the presence of 40 p~ 18:l-LPA (Cao et al., 1990). Thus, in B. oleracea as in meadowfoam, 18:l-LPA is the preferred acyl acceptor in vitro. However, the ability of the B. oleracea LPAT to produce dierucoyl-PA from 22:l-LPA is important for any scheme in which transgenic expression in high 22:l rapeseed is planned. In this regard, over the range of oilseed species examined thus far, to our knowledge only the LPAT preparations from meadowfoam (Cao et al., 1990) and now B. oleracea, have been shown to synthesize significant levels of dierucoyl-PA from 22:l-LPA and erucoyl-COA in vitro. The ['4C]PA produced by the LPAT reaction was catalyzed further in the B. oleracea microsomal protein preparation to [l4C1DAGs (143 pmol min-' mg-' protein) and [I4C]TAGs (196 pmol min-' mg-' protein). However, among the radiolabeled TAGs synthesized by this protein preparation in vitro, although there were mono- and dierucoyl TAGs present, surprisingly, there was no detectable ['4C]trierucin (Table VI).

DCAT Studies

The microsomal €3. oleracea DGAT, when supplied with ['4C]22:1-CoA and dierucin, synthesized significant I4C- labeled TAG (95 pmol min-' mg-' protein), but only only a fraction of this (1.4%) was ['4C]trierucin (Table VI). The ['*C]TAC profile on radio-HPLC indicated about 94% monoerucoyl TAGs and 3% dierucoyl TAGs and <2% trierucin, which indicated that the DGAT preferred to use endogenous DAG substrates over the exogenously supplied dierucin. The calculated rate of synthesis of ['4C]trierucin in vitro was only 1.3 pmol min-' mg-' protein in the B. oleracea microsomes. When diolein was supplied as the acyl acceptor in the presence of ['4C]22:1-CoA (data not shown), although the rate of synthesis of [I4C]TAG was similar (105 pmol min-' mg-' protein), much higher proportions of the expected 1,2 dioleoyl, [-'4C]erucoyl-TAG were produced (19% of total [I4C]TAG or about 20 pmol min-' mg-' protein). Collectively, the preliminary comparative data (Table VI) would suggest that it is, perhaps, the relative selectivity of the B. oleracea DGAT against the combination of dierucin as an acyl acceptor and 22:l-COA as the acyl donor that precludes synthesis of significant trierucin in this species. It may be that when dierucin is present the DGAT prefers to utilize acyl-CoAs other than 22:l-COA. This contrasts with B. napus cv Reston, in which the DGAT was previously shown to catalyze the synthesis of 25 to 35 pmol trierucin min-' mg-' protein, when dierucin was supplied as an acyl acceptor in vitro in the presence of ['4C]22:1-CoA (Taylor et al., 1992~). Thus, although B. napus cannot make 1,2-dierucin because of its LPAT specific-

ity (which precludes effective incorporation of 22:l inio the sn-2 position of the glycerol backbone), the DGAT in this species can utilize 1,2 dierucin effectively (Table VI). Fbr- ther studies of the DGAT specificity/selectivity in the B. oleracea cultivar are warranted. Clearly, in any str,ategy to develop a super high erucic oil, the specificity/seLzctiv- ity of the DGAT must also be considered.

CONCLUSIONS

Many groups worldwide are attempting to insert genes encoding erucoyl-CoA:LPAT activity into B. napus in an effort to produce the industrial feedstock trierucin (Sonntag, 1991). L. douglasii (Cao et al., 1990; Lohden et al., 1990; Taylor et al., 1990a, 1992b; Peterek et al., 1992; Murphy et al., 1994) and T. majus (Kridl et al., 1993) have previously been cited as potential gene donors. We pro- pose that the B. oleracea cultivar characterized in the present study is an attractive alternative source for a gene encoding a microsomal LPAT capable of utilizing 221- COA. Given that such specificity is now known to be present in a related member of the Brassicaceae, it may, in fact, represent the preferred source of a gene for compatible transformation of B. napus. Combined with the knowledge that the B. napus GPAT will place erucoyl moieties in the sn-1 position (Taylor et al., 1992a) and that the B. rzapus DGAT can incorporate erucoyl-COA to produce trierucin from dierucin (Taylor et al., 1992~) and recent evidence that the B. napus microsomal PA phosphatase can convert dierucoyl-PA to dierucin (M.G. Kocsis, R.J. Weselake, J.A. Eng, T.L. Furukawa-Stoffer, and M.K. Pomeroy, unpub- lished data), this makes B. napus an excellent "host crop" for transformation with a gene encoding an LPAT with the capacity to utilize erucoyl-COA.

In addition to being exploited for cDNA library construc- tion and gene isolation, the B. oleracea germplasm identified in the present study is also being used in our laboratories to perform interspecific crosses in attempts to introduce the sn-2 erucic trait into artificial B. napus. These artificial B. napus plants will provide interesting germplasm for the study of nonallelic interaction. For example, in artificial B. napus, the activity of LPAT and DGAT could be influenced by nonallelic interaction between the genes derived from the parental genomes of B. rapa (n = 10) and B. oleracea (n = 9), the progenitors of B. napus (n = 19). Since the TAGs in a11 of the lines of high erucic B. rapa studied thus far are also devoid of significant sn-2 22:1, it is possible that the capacity for the LPAT to utilize erucoyl-COA in B. napus has been lost through nonallelic interaction. Similarly, the relative inactivity of the B. oleracea DGAT with dierucin may affect the capacity to synthesize significant trierucin in B. napus seed produced via interspecific crosses. With this information, appropriate strategies could be chosen to overcome any nonallelic interaction in transformed plants. Studies on the interaction of the parental genomes and their effects on 22:l content in resynthesized B. napus con- tinue in our laboratories and those of others (Liihs and Friedt, 1994).



B r a s s i c a o l e r a c e a 22:l -CoA:Lyso-Phosphatidate Acyltransferase 41 9

ACKNOWLEDGMENTS

The authors thank L. Hogge and D. Olson for performing the direct probe MS, Dr. J.R. McFerson (U.S. Department of Agricul- ture-Agricultura1 Research Service) for additional ”passport” information on B. oleracea cultivar PI 372890, and Drs. W. A. Keller and P. Covello for critica1 reviews of the manuscript.

Received March 31, 1995; accepted June 23, 1995. Copyright Clearance Center: 0032-0889/95/109/0409/12.

LITERATURE ClTED

Agrawal VP, Stumpf PK (1985) Elongation systems involved in the biosynthesis of erucic acid from oleic acid in developing Brassica juncea seeds. Lipids 20: 361-366

Bafor M, Smith MA, Jonssen L, Stobart K, Stymne S (1991) Ricinoleic acid biosynthesis and triacylglycerol assembly in microsomal preparations from developing castor bean (Ric inus communis ) endosperm. Biochem J 280: 507-514

Banas A, Johansson I, Stymne S (1992) Plant microsomal phospholipases exhibit preference for phosphatidylcholine with oxy- genated acyl groups. Plant Sci 8 4 137-144

Bernerth R, Frentzen M (1990) Utilization of erucoyl-COA by acyltransferases from developing seeds of Brassica nnpus (L.) involved in triacylglycerol biosynthesis. Plant Sci 67: 21-28

Bocckino S, Wilson P, Exton JH (1989) An enzymatic assay for picomole leveIs of phosphatidate. Ana1 Biochem 180: 24-27

Bradford MM (1976) A rapid and sensitive method for the quan- titation of microgram quantities of protein utilizing the principle of protein-dye binding. Ana1 Biochem 72: 248-254

Browse J, Somerville C (1991) Glycerolipid synthesis: biochemistry and regulation. Annu Rev Plant Physiol Plant Mo1 Biol 4 2

Cao Y-2, 00 K-C, Huang AHC (1990) Lysophosphatidate acyltransferase in the microsomes from maturing seeds of meadowfoam (Limnanthes alba). Plant Physiol 9 4 1199-1206

Christie WW (1982) Lipid Analysis, Ed 2. Pergamon Press, Toronto, Ontario, Canada

Fehling E, Mukherjee KD (1990) Biosynthesis of triacylglycerols containing very long chain mono-unsaturated fatty acids in seeds of Lunaria annua. Phytochemistry 2 9 1525-1527

Fehling E, Murphy DJ, Mukherjee KD (1990) Biosynthesis of triacylglycerols containing very long chain monounsaturated acyl moieties in developing seeds. Plant Physiol94 492498

Griffiths G, Stymne S , Stobart K (1988) Biosynthesis of triglycer- ides in plant storage tissue. In H Applewhite, ed, Proceedings of the World Conference on Biotechnology for the Fats and Oil Industry. American Oil Chemists’ Society, Champaign, IL, pp

Ichihara K, Asahi T, Fujii S (1987) 1-Acyl-sn-glycerol-3-phosphate acyltransferase in maturing safflower seeds and its contribution to the non-random fatty acid distribution of triacylglycerol. Eur J Biochem 167: 339-347

Katavic V, Reed DW, Taylor DC, Giblin EM, Barton DL, Zou J-T, MacKenzie SL, Covello PS, Kunst L (1995) Alteration of seed fatty acid composition by an ethyl methane-sulfonate-induced mutation in Arabidopsis thaliana affecting diacylglycerol acyltransferase activity. Plant Physiol 108: 399-409

Kennedy EP (1961) Biosynthesis of complex lipids. Fed Proc 20:

Kridl JC, Knauf VC, Thompson GA (1993) Progress in expression of genes controlling fatty acid biosynthesis to alter oil composition and content in transgenic rapeseed. In DPS Verma, ed, Control of Plant Gene Expression. CRC Press, Boca Raton, FL, pp 481-498

Kunst L, Taylor DC, Underhill EW (1992) Fatty acid elongation in developing seeds of Arabidopsis thaliana. Plant Physiol Biochem 3 0 425-434

Lemieux B, Miquel M, Somerville C, Browse J (1990) Mutants of Arabidopsis with alterations in seed lipid fatty acid composition. Theor Appl Genet 80: 234-240

467-506

23-29

934-940

Lohden 1, Bernerth R, Frentzen M (1990) Acyl-CoA:l-acylglycerol-3-phosphate acyltransferase from developing seeds of Lim- nanthes douglasii (R.Br.) and Brassica napus (L.) In PJ Quinn, JL Harwood, eds, Plant Lipid Biochemistry, Structure and Utiliza- tion. Portland Press, London, pp 175-177

Lohden I, Frentzen M (1992) Triacylglycerol biosynthesis in developing seeds of Tropaeolum majus L. and Limnanthes douglasii R. Br. Planta 188: 215-224

Lühs W, Friedt W (1994) The use of resynthesized Brassica napus in breeding of high-erucic acid rapeseed. Proceedings of the 2nd Eurolipid and 50th Deutschen Gesellschaft fiir Fettwissenschaft, e. v. Conference, Miinster, Germany, Sept 26-28, 1994, p 11

Mukherjee KD (1986) Glycerolipid synthesis by homogenate and oil bodies from developing mustard (Sinapis alba L.) seed. Planta

Murphy DJ, Mukherjee KD (1988) Biosynthesis of very long chain monounsaturated fatty acids by subcellular fractions of developing seeds. FEBS Lett 230: 101-104

Murphy DJ, Richards D, Taylor R, Capdevielle J, Guillmont J-C, Grison R, Fairbairn D, Bowra S (1994) Manipulation of seed oil content to produce industrial crops. Ind Crops Products 3:

00 K-C, Huang AHC (1989) Lysophosphatidate acyltransferase activities in the microsomes from palm endosperm, maize scute- Ilum, and rapeseed cotyledons of maturing seeds. Plant Physiol 91: 1288-1295

Peterek G, Schmidt V, Wolter FP, Frentzen M (1992) Approaches for cloning 1-acylglycerol acyltransferase from oilseeds. In A Cherif, ed, Metabolism, Structure and Utilization of Plant Lipids. CNP Press, Tunis, Tunisia, pp 401404

Phillips BE, Smith CR Jr, Tallent WH (1971) Glycerides of Lim- nathes douglasii seed oil. Lipids 6 93-99

Pollard MR, Stumpf PK (1980) Long chain (Czo and Cz2) fatty acid biosynthesis in developing seeds of Tropaeolum majus, an in vivo study. Plant Physiol 6 6 641448

Scarth R, McVetty PBE, Rimmer SR, Daun J (1992) Breeding for special oil quality in Canola/rapeseed: the University of Mani- toba program. In SL MacKenzie, DC Taylor, eds, Seed Oils for the Future. American Oil Chemists’ Society Press, Champaign,

Sonntag NOV (1991) Erucic, behenic: Feedstocks of the 21st cen- tury. Inform 2: 449-463

Stymne S, Stobart AK (1987) Triacylglycerol biosynthesis, In PK Stumpf, EE Conn, eds, The Biochemistry of Plants, Vol 9. Aca- demic Press, New York, pp 175-214

Stymne S , Tonnet ML, Green AG (1992) Biosynthesis of linolenate in developing embryos and cell-free preparations of high-linolenate linseed ( L i n u m us i ta t i ss imum) and low-linolenate mutants. Arch Biochem Biophys 294 557-563

Taylor DC, Barton DL, Rioux KP, MacKenzie SL, Reed DW, Underhill EW, Pomeroy MK, Weber N (1992a) Biosynthesis of acyl lipids containing very long chain fatty acids in microspore- derived and zygotic embryos of Brassicn napus L. cv Reston. Plant Physiol 99: 1609-1618

Taylor DC, Ferrie AMR, Keller WA, Giblin EM, Pass EW, Mac- Kenzie SL (1993) Bioassembly of acyl lipids in microspore- derived embryos of Brassica campestris L. Plant Cell Rep 1 2

Taylor DC, Giblin EM, Reed DW, Hogge LR, Olson DJ, Mac- Kenzie SL (1995) Stereospecific analysis and mass spectrometry of triacylglycerols from Arabidopsis thaliana (L.) Heynh. Colum- bia seed. J Am Oil Chem SOC 72: 305-308

Taylor DC, MacKenzie SL, McCurdy AR, McVetty PBE, Giblin EM, Pass EW, Stone SJ, Scarth R, Rimmer SR, Pickard MD (1994) Stereospecific analyses of triacylglycerols from high erucic Brassicaceae: detection of erucic acid at the sn-2 position in B. oleracea L. genotypes. J Am Oil Chem Soc 71: 163-167

Taylor DC, Magus JR, Bhella R, Zou J-T, MacKenzie SL, Giblin EM, Pass EW, Crosby WL (1992b) Biosynthesis of triacylglycer- 01s in Brassica napus L. cv Reston; Target: trierucin. In SL Mac- Kenzie, DC Taylor, eds, Seed Oils for the Future. American Oil Chemists’ Society Press, Champaign, IL, pp 77-102

167: 279-283

17-27

IL, pp 171-176

375-384



420 Taylor et al. Plant Physiol. Vol. 109, 1995

Taylor DC, Thomson LW, MacKenzie SL, Pomeroy MK, Weselake RJ (1990a) Target enzymes for modification of seed storage lipids. In JR McFerson, S Kresovich, SG Dwyer, eds, Sixth Crucifer Genetics Workshop Proceedings. U.S. Depart- ment of Agriculture-Agricultura1 Research Service Plant Genetic Resources Unit, Cornell University, Geneva, NY, pp 38-39

Taylor DC, Weber N (1994) Microspore-derived embryos of the Brassicaceae-model systems for studies of storage lipid bioassembly and its regulation. Fat Sci Technol 9 6 228-235

Taylor DC, Weber N, Barton DL, Underhill EW, Hogge LR, Weselake RJ, Pomeroy MK (1991) Triacylglycerol bioassembly in microspore-derived embryos of Brassica napus L-. cv Reston. Plant Physiol 97: 65-79

Taylor DC, Weber N, Hogge LR, Underhill EW (1990b) A sim- ple enzymatic method for the preparation of radiolabeled

erucoyl-COA and other long-chain fatty acyl-CoAs and iheir characterization by mass spectrometry. Ana1 Biochem 184:

Taylor DC, Weber N, Hogge LR, Underhill EW, Pomeroy MK (1992~) Formation of trierucoylglycerol (trierucin) frorn 1,Z- dierucoylglycerol by a homogenate of microspore-derived embryos of Brussica napus L. J Am Oil Chem SOC 69: 355-358

Weselake RJ, Taylor DC, Pomeroy MK, Lawson SL, Underhill EW (1991) Properties of diacylglycerol acyltransferase from microspore-derived embryos of Brussica napus L. Phytochemistry

Whitfield HV, Murphy DJ (1990) Storage lipid synthesis i n nas- turtium (Tropaeolum majus). In PJ Quinn, JL Harwood, eds, Plant Lipid Biochemistry, Structure and Utilization. Portland Press, London, pp 225-227

311-316

30: 3533-3538



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