Manipulation of seed oil content to produce industrial crops

INDUSTRIALCROPS ANDPRODUCTS

ANINTERNATIONALJOURNAL

ELSEVIER Industrial Crops and Products 3 (1994) 17-27

Manipulation of seed oil content to produce industrial crops

Denis J. Murphy ay*, Donald Richards a, Rebecca Taylor a, Jo61 Capdevielle b, Jean-Claude Guillemot b, RenC Grison ‘, David Fairbairn a, Steve Bowra a

a Department of Bmssica & Oilseeds Research, John Innes Centre, Nonvich NR4 7UH, UK b Laboratoire de Biochimie des ProtCines, Sanoji Elf Bio Recherches, Labege Innopole, BP 137, 31676 Lubege Cedex, France

c Laboratoim de Phytotechnologie, Sanofi Elf Bio Recherches, Labege Innopole, BP 137, 31676 Labege Ceder, Fmnce

Received 20 December 1993; accepted 8 February 1994

Abstract

Three examples are presented of the modification of seed oil quality by molecular genetics, in order to create potentially valuable industrial feedstocks. (a) Cloning of the desaturase gene responsible for the formation of petroselinic acid from the spice plant Coriander has allowed for the insertion of this gene into rapeseed, in an attempt to obtain transgenic rapeseed plants with a high seed petroselinic acid phenotype. The possibility that additional genes other than this desaturase may be required for petroselinic acid formation is discussed. (b) Attempts to achieve very high erucic acid phenotypes in rapeseed are concentrated around cloning an acyltransferase gene from meadowfoam. The transfer of this gene into rapeseed may result in the accumulation of a very high erucic acid seed oil, in contrast to current high erucic varieties which only contain between 45% and 55% of this useful industrial feedstock. (c) Attempts to clone the hydroxylase gene responsible for the accumulation of ricinoleic acid in castorbean are described. A biochemical strategy based upon solubilisation and purification of the hydroxylase protein was unsuccessful due to the loss of hydroxylase activity in the presence of detergents. A differential screening approach was therefore adopted. Antibodies were raised against microsomal membrane fractions from castorbean seeds at a late stage of development corresponding to their maximum oleate hydroxylase activity. The purified IgG fraction was then incubated in the presence of microsomal membrane fractions from either very young or mature castorbean seeds which had no oleate hydroxylase activity. This resulted in the isolation of a population of castorbean IgGs specific for microsomal membrane proteins which were only present at the stage of maximum oleate hydroxylase activity. The IgG fraction was then used to screen a A-ZAP cDNA expression library prepared from poly A+-enriched RNA, extracted from seeds at the late developmental stage. From these antibody screens, 43 positive cDNA clones were isolated and sequenced from the 5’ end to an average of 250 bases each. All but two of the cDNA clones were eliminated due to (a) sequence similarity with previously known proteins or (b) inappropriate expression patterns of the corresponding mRNAs following Northern blot analysis. Potential sequence motifs in the oleate hydroxylase, which may be useful for the isolation or identification of candidate hydroxylase cDNA clones, such as the two clones isolated in this study, are discussed.

Keywords: Coriander; Rapeseed; Castorbean; Petroselinic acid; Erucic acid; Ricinoleic acid; Differential screening; Industrial oilseeds

* Corresponding author. Fax: 44 603 259 882. E-mail: [email protected].

0926-6690/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved. SSDI 0926-6690(94)00017-S

18 D.J. Murphy et al. I Industrial Crops and Products 3 (1994) 17-27

1. Introduction

There is considerable interest in the manipulation of the oil content of certain crop species in order to produce potentially valuable industrial raw materials. This interest has been heightened in recent years due to the convergence of a number of different factors all of which favour the development of non-edible oil crops (Murphy, 1993a, 1994). The most decisive factor in the long term will be the inevitable depletion and eventual ex- haustion of the global fossil hydrocarbon reserves that are currently the basis of the overwhelming majority of oil-based industrial commodities. Such commodities include building materials, plastics, paints, lubricants, cosmetics and even pharmaceu- ticals and are regarded as vital components of any advanced industrial society (Liihs and Fried& 1994). Industrial oil crops are potential sources of renewable oleochemical feedstocks that will be available into the indefinite future.

A second important factor favouring industrial oil crops is the growing consumer awareness of environmental issues which is leading to demand for industrial products that are biodegradable, partially or totally recyclable, and are produced by environmentally friendly processes. In some cases, consumers are prepared to pay a significant price premium for such products.

Thirdly, industrial oil crops have the potential to produce partially refined oleochemicals which may reduce downstream refining processes and hence their cost. This may also lead to reduced environmental consequences, for example lower levels of pollution from oil refineries.

An important way to obtain novel industrial oils is to exploit the enormous level of natural diversity found in the seed oil contents of many different plant species (Kleiman, 1988). Unfortu- nately, very few such species have yet to be domes- ticated and, in general, their agronomic perfor- mance is markedly inferior to that of established crop plants. The domestication of these novel oil crops is an important objective, but one which may take several decades.

The advent of modern biotechnological methods including marker-assisted selection and gene transfer between organisms allows for the modi-

fication of an existing high-yielding oilseed crop species, so that it may produce a wide range of potentially valuable industrial oils. This new strategy may allow plant breeders to manipulate a limited number of major oilseed crop species, so that each species is able to produce a very wide range of industrial products (Murphy, 1994). This concept of designer oil crops must be regarded as com- plementary to the domestication of novel oilseed crops since the latter will probably take place over a much longer time scale, but will be important in maintaining agricultural diversity during the Zlst century.

The purpose of this paper is to describe some of the progress that has recently been made, both in our own laboratories and elsewhere, in the manipulation of seed oil quality. We include a detailed case study of a strategy to clone a Al2 oleate hydroxylase gene responsible for the production of ricinoleic acid in castorbean. This study illustrates some of the complexities which arise, even in biotechnological projects that on the surface appear to be relatively straightforward.

2. Manipulation of seed oil quality

The manipulation of the quality of a seed oil involves the alteration of its fatty acid profile. It is the fatty acids which determine the chemical properties responsible for the particular end use of the seed oil. The scope for the alteration of seed fatty acid content in a given crop species is enormous. Even with the use of conventional plant breeding, it has been possible to effect dramatic changes in fatty acid content in a number of important oil crops. Examples include the high erucic and high oleic varieties of rapeseed, the high linoleic and high oleic varieties of sunflower and the high linolenic and high linoleic varieties of linseed. Nevertheless, there are limits to the repertoire of the conventional plant breeder, which are deter- mined by the genetic make up of the individual crop species. For example, the enzyme(s) responsible for the formation of high petroselinic oils are virtually confined to the Umbelliferae family, while long-chain storage wax esters are only found in the desert shrub, jojoba (Simmondsia chinen- sis). The advent of recombinant DNA technology

D.J. Murphy et al. IIndustrial Crops and Products 3 (1994) 17-27 19

now makes it possible to clone the genes encoding these enzymes from their species of origin and to insert them into a suitable established crop plant species such as rapeseed.

There are many hundreds of actual or potential fatty acid raw materials produced by oilseeds (Ltihs and Fried& 1994). In attempting to demon- strate the effectiveness of biotechnology for producing new industrial oil crops, it is therefore important to decide initially upon a relatively limited number of target fatty acids to be produced. It is equally important to select an appropriate host species which is both an established high- yielding oil crop and is amenable to genetic transformation and regeneration. At present, the only major oil crop which can be transformed rou- tinely at relatively high efficiencies, i.e. lo-40%, is rapeseed (Murphy, 1994). Soybean transformation has made rapid progress over the past two years and some transformants with altered seed lipid contents have been reported (A. Kinney, pers. commun., 1993), although progress still lags be- hind rapeseed. The transformation of other major annual oil crops such as sunflower, maize and linseed, has also been reported, but is as yet relatively inefficient and far from being a routine procedure. In the meantime, prospects for the transformation of the important high-yielding perennial oil crops, such as palm, coconut and olive suggest that this is unlikely to become routine for many years (Mur- phy, 1994). For the immediate future, therefore, rapeseed will continue to be the most suitable major oil crop for molecular manipulation to produce industrial oils.

2.1. Petroselinic acid

Amongst the target fatty acids selected for new designer rapeseed varieties in our laboratories are petroselinic, erucic and ricinoleic acids. Petroselinic acid is an isomer of oleic acid, the major fatty acid in conventional edible varieties of rapeseed. The advantage of petroselinic acid as a target for fatty acid modification is that the desaturase gene responsible for its formation has proved relatively straightforward to clone and insert into rapeseed plants (Fairbairn et al., 1994). The disadvantages are the following.

(a) It may be necessary to insert additional genes other than the desaturase, in order to achieve the very high levels of petroselinic acid formation in rapeseed that are desirable if it is to serve as an industrial feedstock (Dormann and Ohlrogge, 1993). The insertion of several new genes into rapeseed is not particularly problemati- cal. However, the initial cloning of these genes and the sexual crosses necessary to get all the genes into the desired rapeseed background could add 3-5 years onto the timescale of this project.

(b) Petroselinic-rich seed oils have not been available hitherto in large quantities or at the appropriate price for the chemical industry. This means that there is no established market for petroselinic acid, although it has the potential to serve as a feedstock for both polymer and detergent production and possibly for additional industrial processes. During the coming months, it is likely that we will know whether it is possible to produce a high petroselinic rapeseed variety using only the inserted coriander desaturase gene. If, however, it is necessary to clone and insert three or four additional genes to create a high petroselinic rapeseed, it is unlikely that petroselinic acid would be available as a bulk industrial feedstock for many more years.

2.2. Erucic acid

In contrast to petroselinic acid, erucic acid has an established worldwide market currently esti- mated at over 100,000 tonnes of high erucic rapeseed oil per annum. This market is growing at a rate of 5% per annum, due to the increased use of erucic acid for the production of erucamides, lubricants, surfactants and cosmetics (Leonard, 1993). The typical erucic acid content of most high erucic acid rapeseed (HEAR) varieties is between 45% and 55%. It may be possible for plant breeders to increase this proportion to as high as 60%. Nevertheless, it is unlikely that higher levels of erucic acid can be achieved using traditional sources of genetic variation. The reason is that rapeseed appears to lack an acyltransferase enzyme capable of inserting erucic acid onto the second carbon of the triacylglycerol molecule. This means that the theoretical maximum amount of

20 D.J. Murphy et al. /Industrial Crops and Products 3 (1994) 17-27

erucic acid in rapeseed is 66%. An acyltransferase which is capable of this reaction has been found in the garden ornamental plant Limnanthes alba (poached-egg plant or meadowfoam). Efforts are now underway in our laboratories (M.J. Hills, R. Chamberlain and R. Dan, pet-s. commun., 1994) and elsewhere (Taylor et al., 1992; Peterek et al., 1992; Morand and German, 1993) to clone this gene from Limnanthes or other suitable donors, for insertion into rapeseed. It is hoped that this will result in a new rapeseed variety containing very high levels, i.e. far greater than 60%, of eru- tic acid in its seed oil. The availability of very high erucic oil will substantially increase the yield per hectare of erucic acid and should simplify (and hence cheapen) its downstream processing costs. In turn, such improvements may lead to an expan- sion in the market for erucic-derived products and provide opportunities for more farmers to grow HEAR varieties on set-aside land in Europe.

hydroxylated oils from a more reliable and potentially higher yielding annual oilseed crop such as rapeseed. One approach to achieving this objective has been the use of chemical hydroxylation of high oleic rapeseed oil from conventional varieties (Keene and Hignott, 1993). Although this involves extra processing costs, sufficient added value may be obtained from the hydroxylated oil to make this a commercially viable operation. A longer-term alternative to the chemical hydroxylation of rapeseed oil is to isolate the hydroxylase gene from castorbean and to insert this into rapeseed, so that a new transgenic rapeseed variety producing a high-ricinoleic acid seed oil is obtained.

3.2. Materials and methods

3.2.1. Plant material and enzyme assays

3. Ricinoleic acid - a case study

Castorbean, Ricinus communis, plants were grown either in glasshouses or in the field. Seeds were collected and processed for enzyme assays as described previously (Richards et al., 1993).

3.1. Background 3.2.2. Preparation of antibodies

Like erucic acid, ricinoleic acid has an established industrial market as a feedstock for the manufacture of polymers, cosmetics, pharmaceu- ticals, lubricants, plasticisers, coatings and surfactants. Ricinoleic acid is currently derived from the castorbean plant, where it forms up to 90% of the seed oil fraction (Achaya et al., 1964). Castorbean is grown as a crop in countries such as Brazil and India, and its cultivation is now being carried out on a limited scale in Mediter- ranean countries such as Italy, Spain and France. Current annual global production of castor oil is about 460,000 tonnes (Anonymous, 1993). Unfor- tunately, the castorbean crop is very susceptible to climatic variations. This has led to an erratic supply and wide fluctuations in commodity prices which have ranged from $450 to $1200/tonne of seed over the past decade (L. Ramos, pers. commun., 1993). This market instability has led to a reluctance of companies to invest in research and development to expand further the uses of castor oil (Vignolo and Naughton, 1991). For this reason, the chemical industry would prefer to obtain

An aliquot of the microsomal proteins (about 1 mg total protein) from the optimum stage of hydroxylase activity was mixed with Freund’s coadju- vant, and injected into rabbits. The injections were repeated twice at intervals of two weeks. After this period, the antiserum was tested in West- ern blots of microsomal proteins from castorbean. From this antiserum, an IgG-enriched fraction was prepared by precipitation in 16% (w/v) sodium sulphate. The pelleted fraction was dissolved in a minimum volume of sodium phosphate 10 mM, NaCl 150 mM, pH 7.2 at RT (PBS), and dialyzed for 48 h against 500 volumes and two changes of PBS. The IgG fraction was then adsorbed against E. coli XL-Blue (Stratagene) proteins, according to the Stratagene protocol, and kept frozen at -70°C. Control experiments showed that in West- ern blots of microsomal proteins of castorbeans, the IgG fraction behaved as the original antiserum.

3.2.3. Preparation of the castorbean library The castorbean library was prepared in the h-

ZAP cloning vector (Stratagene) according to the

D.J. Murphy et al. /Industrial Crops and Products 3 (1994) 17-27 21

manufacturer’s instructions, using poly A+ RNA from castorbean seeds at the stage of maximal hydroxylase activity.

3.2.4. Adsorption of ZgGs Adsorption of IgGs with proteins from the dif-

ferent stages of castorbean development was done essentially as described by Choi et al. (1987). Ei- ther mature, dry, or very immature castorbean seeds (two stages where the hydroxylase activity is not detectable) were collected and microso- ma1 proteins prepared as above. The pellets were washed twice with PBS, and resuspended at about 20 mg protein/ml in PBS. These proteins were then diluted in TBS (where Tris replaces sodium phosphate of PBS) containing 0.1% (v/v) SDS, and incubated at about 1 mg protein/ml, with strips (10 x 5 cm) of nitrocellulose paper for 30 min. After washing, and blocking in PBS containing 1% (w/v) bovine serum albumin, these filters were incubated with the IgG fraction obtained above. After four l-h incubations with two filters each time, the IgG fraction was collected. By this method, three IgG fractions were prepared: one adsorbed against proteins from very early seeds, another adsorbed against dry seeds, and a third one adsorbed against proteins from both early and dry seeds.

3.2.5. Library screening Library screening with the pre-adsorbed IgG

preparations was done according to Strata- gene, using the phosphatase colour development method. Positive cDNAs were excised from the lambda vector as p-Bluescript (SK-) plasmid (Stratagene). Sequencing of positive clones was done with Sequenase II according to the manufacturer’s instructions (BRL). All other molecular biology techniques were performed as described in Sambrock et al. (1989).

3.2.6. Analysis of sequences The sequences obtained were compared to se-

quences in existing databases using the DAPJOB facility at the University of Kent. The sequences considered homologous to sequences in the databases had values greater than 15 for the standard deviation difference between the score for

the alignment and that expected from the distribu- tion of fortuitous alignments.

3.2.7. Two-dimensional gels Two-dimensional gel electrophoresis of micro-

somal proteins was done according to the manufacturer’s instructions (BioRad). Analysis of the gels, and proteolysis, extraction and sequencing of peptides was done as described by Rosenfeld et al. (1992). Protein contents were measured by the bicinchoninic acid method (Smith et al., 1985) using bovine serum albumin as standard.

3.3. Results

3.3.1. Biochemical approach The initial objective of this project was to char-

acterise the enzyme responsible for ricinoleic acid formation in castorbean, in order to allow for the eventual cloning of the corresponding gene. When the project was first started, relatively lit- tle was known about the properties and even the subcellular localisation of the Ai2 oleate hydroxylase responsible for ricinoleic acid biosynthesis. It was even necessary to develop a new assay procedure in order to obtain suitable reaction rates in vitro. Using this new assay system (Richards et al., 1993) the subcellular localisation of the hydroxylase enzyme was studied using differential cen- trifugation on sucrose density gradients. The results of these studies showed that the enzyme was present in two discrete subcellular fractions of the endoplasmic reticulum. Interestingly, a similar dis- tribution was found for another membrane-bound fatty acid modification enzyme system, i.e. oleoyl- CoA elongase, in several species including rapeseed and nasturtium (Whitfield et al., 1993). The significance of this finding is unclear at present, but it may reflect a heterogeneity in the organisa- tion of endoplasmic reticulum membranes under conditions of active storage oil deposition.

Extensive attempts to solubilise the hydroxylase activity from these membrane fractions proved fruitless. This failure was probably due to the detergent solubilisation causing the disso- ciation of the hydroxylase from other accessory enzymes required for the conversion of oleoyl- CoA to ricinoleate. Further studies in our own

22 D.J. Mtqohy et al. lhadustrial Crops and Products 3 (1994) 17-27

laboratories and elsewhere (Bafor et al., 1991; Richards et al., 1992) have shown that the substrate for the hydroxylase reaction is oleoyl- phosphatidylcholine. Therefore, the participation of a lyso-phosphatidylcholine acyltransferase is required. In addition, the hydroxylation reaction re- quires reducing equivalents which are probably supplied from a cytochrome bs/cytochrome b5 re- ductase couple (Smith et al., 1992). It appears that these four enzymes are not tightly bound together and become dissociated following detergent solubilisation, even in mild non-ionic detergents such as octyl glucoside. In principle, it would be possible to assay the hydroxylase by reconstituting it in the presence of extracts containing the other three accessory enzymes. However, this would require at least the partial purification of these enzymes, which was ruled out from the present project on the basis of time and cost.

3.3.2. Differential screening approach For the above reasons, an alternative strategy

was developed based on differential screening of endoplasmic reticulum (microsomal) membrane fractions known to be enriched in the oleate hydroxylase. This approach was feasible following the demonstration that oleate hydroxylase activity could only be detected in two out of the six developmental stages analysed in castorbean seeds (see Fig. 1). This meant that endoplasmic reticulum membrane fractions from the mid-late stages of seed development could be screened against similar fractions from dry seeds and young seeds. The two screening strategies used were (a) two-dimensional electrophoresis using isoelectric focusing (IEF) plus SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by the identification and partial sequencing of proteins unique to the mid-late stages of castorbean seed development, and (b) the production of a differential antibody library, enriched in antibodies raised against proteins abundant during the mid-late stages of castorbean seed development. This library was then used to screen a cDNA library made from mRNA isolated from mid-late developing castorbean seeds.

A typical two-dimensional IEF/SDS-PAGE chromatogram of endoplasmic reticulum mem-

--- <lcm lcm 2 cm g/b brown dry lsaf

seed

Developmental stage

Fig. 1. Oleate hydroxylase activity of castorbean seeds at different stages of development. The measurements indicate length of seed. g/b: seed length about 2 cm with green/brown testa; brown: dark-brown test. A measurement in leaf is also included.

brane proteins from mid-late developing castorbean seeds is shown in Fig. 2. The most abundant species are relatively neutral proteins of molecular weight range 30-40 kDa. These probably corre- spond to subunits of the major seed storage proteins, possibly undergoing processing in the endoplasmic reticulum. These abundant proteins were present at most developmental stages. However, a number of other proteins, some of which are shown arrowed, were found only in membranes from mid-late developing seeds. Initially, three separate proteins were identified for microse- quencing. This was performed following the in-gel partial digestion of the candidate proteins with endoproteases in order to yield internal amino acid sequence data, which is often more informa- tive than N-terminal sequence analysis (Rosenfeld et al., 1992). The sequence data obtained showed that these proteins had homology respectively with the 2s albumin precursor from castorbean (Irwin et al., 1990), the oat globulin precursor (Shotwell et al., 1988) and a disulphide isomerase from alfalfa. Therefore, the first three microsomal proteins sequenced by this method could be ruled out

D.J. Murphy et al. IIndustrial Crops and Products 3 (1994) 17-27 23

tiw ( KD) zoo

116 97

66

6.5

PH 3 b pHl0

Fig. 2. Two-dimensional IEF/SDS-PAGE gel of endoplasmic reticulum from castorbean seeds, at the stage of maximum oleate hydroxylase activity. Several protein bands appeared to be unique to this developmental stage when compared with similar protein fractions from very young and dry castorbean seeds. Three of these unique proteins, which were sequenced, were found to have homology with: A, the 2s albumin precursor from castorbean; B, the oat globulin precusror; C, a disulphide isomerase from alfalfa.

as potential hydroxylases, since their sequences corresponded to previously known proteins.

The second screening approach to isolating a hydroxylase gene involved the use of differential immuno-adsorption. This involved the injection of rabbits with an endoplasmic reticulum protein preparation from castorbean seeds at the mid- late developmental stage, at which they had been shown to have maximum hydroxylase activity. The IgG fraction from these animals was then adsorbed against endoplasmic reticulum membrane proteins from (a) dry seeds, (b) young seeds, and (c) a mixture of (a) and (b). It was expected that the immuno-adsorbed IgG would become enriched in antibodies against proteins which were uniquely

present in the endoplasmic reticulum protein fraction exhibiting the highest levels of hydroxylase activity. As shown by the Western blot in Fig. 3, the number of bands detected by the IgG fraction was substantially reduced following immuno- adsorption, confirming that the procedure had increased the specificity of the antibody probe.

The resultant subtractively screened IgG was used to screen a castorbean cDNA expression library produced in the vector A-ZAP This library had been produced from poly A”-enriched RNA extracted from seeds at the stage of maximal hydroxylase activity. From these antibody screens, 43 positive cDNA clones were isolated. All of these clones were then sequenced from the 5’ end to

24 D.J. Murphy et aL IIndustrial Crops and Products 3 (1994) 17-27

1 2 3 45 6

Fig. 3. Western blot of castorbean microsomal proteins using purified IgGs (left-hand picture) and using purified IgGs pre-adsorbed with dry seed microsomal proteins (righ-hand picture): 1 = microsomal proteins from very early seeds, less than 1 cm length; 2 = microsomal proteins from seeds at the stage of maximum hydroxylase activity; 3 = microsomal proteins from dry seeds. For further deatils see Sect. 3.2.

an average of about 250 bases and the sequences analysed with reference to both the amino acid and nucleotide international sequence databases.

The majority of the clones isolated by this method proved to have a greater or lesser degree of sequence homology with previously isolated genes. For example, clones were isolated corresponding to a 2S albumin (Irwin et al., 1990), ricin (Lamb et al., 198S), and a metallothionien (Weig and Komor, 1992), all from castorbean. Other clones were homologous with genes isolated from other plant species. For example, a vicilin precursor from cacao (McHenry and Fritz, 1992) an intrinsic tonoplast protein from common bean (Johnson et al., 1990), a glucanase from tobacco (De Loose et al., 1988), a late embryogenesis abundant protein (LEA) from radish (Raynal et al., 1989), and an ATP-dependent protease from tomato (Gottesman et al., 1990). The presence amongst these clones of genes encoding proteins which are normally soluble may reflect the fact that some of these proteins are post-translationally modified during their passage through the endoplasmic reticulum. The fact that several of the

clones isolated here have homology with the precursor forms of seed storage proteins is consistent with this hypothesis. However, it is also possible that the endoplasmic reticulum membrane preparations used to raise the antibodies became partially contaminated with soluble proteins during their isolation from the seeds.

In addition to the 30 cDNA clones having homology to previously identified genes, we were able to identify 13 cDNA clones, whose sequences had no homology with any known genes in the databases. A secondary screen of these clones was used in order to eliminate classes of gene which were not subject to the same expression patterns as the hydroxylase gene. This was done using a Northern blot method, since it was known that the hydroxylase gene was (a) only expressed in developing seeds and (b) was expressed specifically towards the end of seed development. Total RNA extracts were therefore prepared from a number of tissues of castorbean, including leaves, roots, seeds and germinated cotyledons, plus the six developmental stages of the seeds. Using these RNA samples, Northern blots were performed for all of the unknown clones, one example of which is shown in Fig. 4. Only three of the thirteen clones had the required temporal, spatial and species specificity to qualify as candidate clones for the castorbean hydroxylase. Of these three clones, one of them, E35, the longest of the three, showed no homology to any known protein for the first 800 base pairs that were sequenced. However, subsequent screening of a further 600 base pairs showed that it had a high homology to ricin, a soluble seed storage protein in castorbean. This clone was therefore discarded from the screen. The remaining two clones are currently undergoing further sequencing in order to attempt to find motifs which would be expected in the castorbean oleate hydroxylase.

3.4. Discussion

The search for expected structural motifs in the castorbean A12 oleate hydroxylase may be assisted by recent reports that this enzyme shares many common features (including subcellular localisation, electron donor and substrate) with the A12

D.J. Murphy et aL IIndustrial Crops and Products 3 (1994) 17-27 25

1 2 3 4 5 6 76

-7OObp

Fig. 4. Northern blot analysis of a castorbean clone with homology to an LEA protein: 1 = seeds of less than 1 cm length; 2 = seeds of about 2 cm length, with green testa; 3 = stage of maximum hydroxylase activity; 4 = dry seeds; 5 = castorbean root; 6 = castorbean cotyledons; 7 = castorbean leaf; 8 = rapeseed at stage of maximal oil production. Each lane of the gel was loaded with 20 pg of total RNA. For further details see Sect. 3.2. Note that the 700 bp mRNA was only present in dry seeds (lane 4). In contrast, oleate hydroxylase mRNA would be expected to be maximally abundant in late-stage seeds (lane 3) and absent from non-seed tissues (lanes 5-7).

oleate desaturase found ubiquitously in plants. Even more interesting is the proposal that all plant desaturases may share a common reaction mechanism similar to that of methane monooxygenase hydroxylase (Fox, 1990; Fox et al., 1993) and involving a hydroxylated intermediate. This has led to the suggestion that the castorbean oleate hydroxylase is a modified or mutated desaturase which is only able to carry out part of the reaction sequence, resulting in the accumulation of what would normally be a short-lived intermediate, i.e. ricinoleic acid (Murphy, 1993b). The significance of this for the cloning of the castorbean hydroxylase gene is that the latter may be expected to contain conserved sequence motifs similar to those already described in the many plant desaturase genes isolated in the past few years (Murphy, 1993b).

Should such motifs be found, the putative hydroxylase clone would be transformed into a suitable heterologous expression system which does not normally contain a Ai2 oleate hydroxylase ac-

tivity. The accumulation of even small amounts of ricinoleic acid in such cells would confirm that the clone did indeed encode a Al2 oleate hydroxylase. It is only when the identity of the clone is con- firmed as the castorbean hydroxylase that we will be in a position to insert it into rapeseed explants in order to produce transgenic rapeseed plants containing ricinoleic acid in their seed oil.

The above case study illustrates the complexities involved in the initial cloning of a single gene in order to modify seed oil composition. In many cases, several genes will need to be cloned for this purpose. There is then the often formidable task of inserting these genes under the control of suitable promotors to achieve the appropriate level of expression and the correct spatial and temporal regulation in the transgenic crop plant. The problems to be overcome are by no means trivial and progress is often hampered by a lack of information about the basic mechanisms of both the biochemical regulation of the metabolic path- ways responsible for seed oil accumulation and the developmental regulation of gene expression in seeds. Nevertheless, the results of initial attempts elsewhere to engineer new high-stearic and high-lauric varieties of rapeseed are encouraging (Knutzon et al., 1992; Voelker et al., 1992). It is probably only a matter of time before these techniques are refined to allow for the engineering of virtually any desired oil composition in the annual oilseed crop of choice.

Acknowledgements

The authors are grateful to other members of the Department of Brassica and Oilseeds Re- search for their assistance. Funding was provided by Plant Science Research Limited, Agricultural and Food Research Council, Ministry of Agricul- ture, Fisheries and Food, SociCtC Nationale Elf Aquitaine and other private contractors whose sponsorship is gratefully acknowledged.

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Manipulation of seed oil content to produce industrial crops