Partial purification and properties of a microsomal

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Partial purification and properties of a microsomal lysophosphatidic acid acyltransferase from oilseed rape EIRA WYN EDWARDS and DENIS J. MURPHY Oilseeds Research Group, Department of Biological Sciences, University of Durham DHI 3LE, U.K. Storage oils in seeds are synthesized via the Kennedy pathway localized in the endoplasmic reticulum (Gurr, 1980). Triacylglycerol is formed by the sequential addition of three acyl groups to a glycerol 3-phosphate backbone. The esterifications are catalysed by three separate acyltransferase enzymes. It is believed that the enzyme catalysing esterification of acyl groups to the sn-2 position of glycerol, acyl-CoA: lysophosphatidic acid acyltransferase (LPA.AT ), plays an important role in regulating the acyl composition of seed oils. LPA.AT has the greatest degree of substrate specificity for acyl-CoAs of the three acyltransferases. Selectivity is a measure of substrate preference shown when the enzyme is incubated with a mixture of fatty acyl-CoAs. When incubated with a mixture of saturated and unsaturated fatty acylCoAs (Stobart & Stymne, 1985), safflower LPA.AT shows the following preference: linoleate > linolenate > oleate, with no incorporation of saturated fatty acids at position sn-2. However, if the levels of unsaturated fatty acids are limiting, the LPA.AT can use any available saturated fatty acids. It does not show absolute specificity for unsaturated fatty acids. Therefore the fatty acyl composition of lipids depends on a complex balance of selectivities and specificities. Complete exclusion of some fatty acyl-CoAs from position sn-2 has been demonstrated (Sun et al., 1988).Maturing seeds of palm, maize and rapeseed were incubated with lauroyl- ( 12:0),oleoyl- ( 18:1) and erucoyl- (22:1 j CoAs. No 22:l could be incorporated at position sn-2 in any of the plants tested, even in rapeseed, which can contain up to 65% erucic acid in triacylglycerol. Also, only the palm was capable of esterifying 1 2 9 CoA to sn-2. The other two acyltransferases showed much lower substrate preferences. It is important to know the substrate preference of these acyltransferase enzymes, before strategies for modifying oil composition of seed storage oils by genetic manipulation can be worked out. If the composition of the cellular fatty acylCoA pool is modified, it is necessary to ensure that the existing acyltransferases can utilize the available fatty acids. Introducing acyltransferases with different substrate preferences from other plant species is a possible solution to this problem. Preliminary results on substrate specificity showed that the LPA.AT from crude rapeseed microsomal preparations had a four-fold preference for oleoyl-CoA over palmitoylCoA. This indicates that the acyltransferase activity in the membrane preparation was localized mainly in the endoplasmic reticulum. A second Kennedy pathway exists in the plastid, having a different set of enzymes from that of the endoplasmic reticulum. The LPA.AT activity associated with the inner membrane of the plastid has a preference for unsaturated over saturated acyl groups (Cronan & Roughan, 1987). This difference in the substrate specificities provides a means of testing that the purified protein does in fact originate in the endoplasmic reticulum, and that the plastid enzyme has not been inadvertently purified. After sucrose density gradient centrifugation, most of the LPA.AT activity was found in fractions with the same buoyant density as that of the endoplasmic reticulum. This supports the results on

Abbreviations used: LPA. AT. acyl-CoA: lysophosphatidic acid acyltransferase; PEG, polyethylene glycol.

Table 1. Partial purification ofLI'A.AT LPA.AT Bctivity was assayed by measuring [ ''C]oleoyl-CoA incorporation into phosphatidic acid (PA) (Moreau & Stumpf, 1982) Activity in PA (% (a)

PA formed (nmol)

Elution of LPA.AT from DEAE-Trisacryl

Control

16.0

300 600

33.2 0

Control

20.6 21.5 10.1

0.596 0.368 0.259

0

0

4.2 6.4 13.7 1 7 .o

0.073 0.164 0.339 0.3X1

200 300 400

( b ) Effect of PEG precipitation Control 5"/0 (w/v) PEG 7.5% (w/v) PEG 10% ( W / V ) PEG

0.579 0.444

0

substrate utilization, which indicated most of the activity was associated with the endoplasmic reticulum. The LPA.AT from the endoplasmic reticulum is an integ: ral membrane protein, and so far little progress has been made in its purification. Before purification can be achieved, the enzyme must first be isolated in a soluble form, which retains its activity. Moreau & Stumpf (1982) succeeded in solubilizing and characterizing the microsomal LPA.AT from developing safflower seeds. In the present study, the zwitterionic detergent, CHAPS, was found to solubilize the rapeseed microsomal enzyme in an active form. Investigations into CHAPS solubulization of chloroplast membranes (Coves et al., 1988)show that it is capable of selective solubilization, removing some proteins in preference to others. Incubation of a microsomal preparation from rapeseed with 0.6% (w/v) CHAPS also produced selective solubilization and so provided an initial purification step. It was found that CHAPS caused significant inhibition of the enzyme activity. This could be reversed by reducing the CHAPS concentration of the solubilized fraction. Reduction of the CHAPS concentration from 0.6% to 0.04% resulted in a three-fold increase in the enzyme activity. Having solubilized the LPA.AT, several methods were developed in an attempt to purify it:

Ion-exchange chromatography (Table l a ) LPA.AT bound to the anion exchanger DEA E-Trisacryl. Elution was found only to be possible using batch elution techniques, very low recoveries of activity being obtained with gradient elution. This could be due to the CHAPS affecting the charge distribution on the protein, resulting in elution over a broad range of salt concentrations. Alternatively, denaturation could be occurring, due to increased lability of the protein whilst bound to the column. Polyethylene glycol (PEG) precipitation (Table 16) PEG precipitation was carried out, followed by one-step batch elution from an ion-exchange column on the nonprecipitated fraction, containing the LPA.AT activity. The 1989

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ion-exchange step was necessary, to remove the PEG, which interfered with the thin-layer chromatography step involved in the enzyme assay. The large pore size of the Trisacryl resin allows the PEG (6000) to pass freely through, the LPA.AT activity being retained on the column. It is interesting to note the stimulation (three- to five-fold) of LPA.AT activity by PEG. This may be due to some stabilization affect, or rearrangement of the detergent molecules surrounding the protein on contacting the PEG. Further studies aimed at a more complete purification of the LPA.AT enzyme are now underway in our laboratory.

Coves, J., Pineau, B., Joyard, J. & Douce, R. (1988) Plant Physiol. Biochem. 26,151-163 Cronan, J. E., Jr & Roughan, P. G. (1987) Plant l’hysiol. 83, 676-680 Gurr, M. I. ( 1980) The Biochemistryof Plants (Stumpf,P. J. & Conn, E. E., eds.),vol. 4, pp. 205-248, Academic Press, New York Moreau, R. A. & Stumpf, P. K. (1982) Plant Physiol. 69, 1293- 1297 Stobart, A. K. & Stymne, S. ( 1985) Riochern. J. 232,217-221 Sun, C., Cao, Y. & Huang, H. C. ( 1988) Plant Physiol. 88,56-60 Received 24 November I988

Are the promoter regions of seed storage protein genes suitable for the expression of genes involved in storage lipid synthesis? DENIS J. MURPHY 0il.seeds Research Group, Department of Biological Sciences, Univtwity c f Durham, Durham D / l l 3LE, U.K. Rapeseed can be transformed by a variety of methods including ( 1j use of the vector Agrobacterium tumefaciens (Pua et al., 1987; Radke et ul., 1988), ( 2 ) electroporation (Guerche et al., 1987)and (3)micro-injection into embryoids (Neuhaus ef al., 1987). Although it is now possible to produce transgenic rapeseed plants, to date only ‘marker genes‘ have been inserted. Before it will be possible to make a considered attempt at the production of a commercially useful transgenic variety of rapeseed, it will be necessary to elucidate further the mechanisms involved in the timing, control and regulation of storage oil and protein synthesis during seed development. We have attempted to elucidate these mechanisms by employing a combined biochemical, cell biological and molecular biological approach.

Biochemical studies The major storage constituents of mature rapeseeds are triacyglycerols (45%, w/w) and proteins (25%, w/w). Most of the seed protein is made up of the two polar storage proteins, cruciferin (40%, w/w, total protein) and napin (24% w/w), and the hydrophobic protein, olein (20%, w/w). The timing of the synthesis of each of these storage products during the course of seed development was followed as shown in Fig. 1. These data show that there were three distinct phases in the synthesis of storage products in developing rapeseed embryos. The onset of the respective synthesis of ( 1 ) triacyl-

-4

~

Olein

Cruciferin and napin

Triacylglycerols

Maturitv

Anthesis

2

4

6 8 Time after anthesis (weeks)

lb

i2

Fig. 1. Timing of storage product formution in developing rapeseed Triacylglycerols were assayed by quantitative g.1.c. and proteins by e.1.i.s.a. and SDS/polyacrylamide-gel densitometry. Arrows indicate the start of storage product synthesis and hatched areas indicate the period of maximum biosynthetic activity. Vol. 17

glycerols, ( 2 ) cruciferin and napin and (3) olein were each displaced by about 2 weeks. By the time that cruciferin and napin synthesis started, at 5 weeks after anthesis, about half of the total storage triacylglycerol had already been deposited. Olein synthesis started at about 7 weeks after arithesis, when over three-quarters of the total cruciferin and napin had already been synthesized.

Cell biological studies The synthesis of rapeseed storage products in developing embryos was followed visually by means of conventional electron microscopy and immunocytochemistry. The results confirmed the biochemical data in showing that storage oil bodies were deposited very early in embryo development, whereas protein deposition was not observable until week 5. Antibodies raised against cruciferin and napin were used in immunogold-labelling studies which demonstrated that both proteins were synthesized at the same time and that both were deposited immediately into the central vacuole of embryo cells, where they eventually formed large protein bodies. Antibodies raised against olein specifically labelled the proteinaceous membranes of oil-storage bodies. These membranes were only formed after week 7, i.e. at the same time as olein was synthesized. Therefore, whereas cruciferin and napin are ‘classical’seed storage proteins deposited into large protein bodies, olein is a member of a novel class of seed protein, associated exclusively with oil-storage bodies (Murphy et al., 1989).

Molecular biological studies Genornic clones for the major rapeseed storage proteins, napin and cruciferin, were isolated and sequenced. The nucleotide sequence derived from napin was almost identical to that reported previously by other groups (Ericson et al., 1986; Schofield & Crouch, 1987).The role of the napin gene promotor in the tissue-specific and temporal regulation of napin gene expression was studied following the insertion of the glucuronidase (GUS) gene, under control of the napin promotor, into the transgenic tobacco plants. It was found that the napin promotor did indeed confer the appropriate tissue and temporal specificities of gene expression in this heterologous system. Further studies involving re-insertion of marker genes or ‘tagged’ napin genes, under the control of the napin promotor, into transgenic rapeseed plants are now underway. The conclusions from these and other (Higgins, 1985; Hamapel& Ohlrogge, 1988)studies are as follows: ( 1 ) The onset of storage protein and oil formation in developing seeds is due to increased levels of gene expression, resulting in increased levels of the appropriate mRNA species.