Production of g–Linolenic Acid (GLA) in Novel or Conventional Crops
- Current Sources of g–Linolenic Acid
- Options for Increasing g–Linolenic Acid Production
Production of Dyes from Traditional Crops
- Actual and Potential Markets
- The Crops and Dyes
- Biochemistry of Dye Biosynthesis
- Improving the Production of Dye Plants in the UK
- Application of New Technology
Surpluses in the production of conventional cereals and oilseeds in the EU have stimulated interest in the development and production of crops for specific non-food end uses. Much of the discussion has centred on the modification of conventional crops, using the new technology of genetic engineering. This has the advantage that the crops are already adapted to EU conditions with high yields and well established systems for production, harvest, storage and fractionation. Thus a relatively simple modification, such as the insertion of a single gene, could lead to the synthesis of a novel product. A large number of laboratories worldwide have adopted this approach, with targets ranging from high value proteins for biomedical and pharmaceutical use through industrial oils to novel compounds for the production of plastics.
A major disadvantage of this approach is that many compounds are the products of metabolic pathways which are still poorly understood at the biochemical level, or require the activities of many enzymes, all of which would need to be expressed in the modified plant. in addition, the products themselves may be toxic to cells unless the plant has developed specific preventative mechanisms (e.g. modified enzymes or compartmentation). These mechanisms are currently poorly understood and it is therefore difficult to predict the consequences of expressing many novel compounds in non-adapted plants.
Because of the above considerations it is important to consider an alternative approach : the improvement and adaptation to EU conditions of obsolete crops or wild species. This is of particular interest for the production of specialist oils for industrial, pharmaceutical and food use. For example, there is a large demand for medium chain (C8–C12) fatty acids, for the production of detergents. These are currently obtained mainly from palm oil, imported to the EU from the tropics. Such fatty acids are also present in seeds of European plants such as Cuphea (Röbbelen, 1988), and the domestication and improvement of such species is consequently being studied.
in the present paper we discuss the advantages and drawbacks of these two approaches, using as examples two contrasting systems that are currently being studied in our laboratories: the production of dyes from traditional plants to replace synthetic products and improving the production of g–linolenic acid (GLA) either from current sources (evening primrose and borage) or by engineering conventional oilseeds (such as oilseed rape and sunflower).
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The triacylglycerols (TAGS) stored in oilseeds are esters of glycerol and fatty acids. Although there is considerable variation in the types and proportions of fatty acids present in different species, the major fatty acids present in the triacylglycerols of temperate oilseeds consist of eighteen carbon atoms (C18) with either one double bond at position 9 (counting from the carboxyl end) (C18:1 D9 oleic acid) or two double bonds at positions 9 and 12 (C18:2 D9, 12 linoleic acid). In fact, oleic and linoleic acids together account for over 60% of the total fatty acids in most oilseeds. Linoleic acid may be further desaturated in some oilseeds to give a–linolenic acid (C18:3 D9,12,15), although this is not usually a major component.
Whereas all plants are able to synthesise linoleic acid (Fig. 1), this cannot be synthesised by animals and is therefore an essential fatty acid. The importance of linoleic acid for animals stems from the fact that it is the precursor of the n–6 family. The first step in this pathway is the desaturation of linoleic acid at the D6 position to give g–linolenic acid (C18:3 D6,9,12) (GLA), catalysed by a D6 fatty acid desaturase. This is followed by chain elongation and further desaturation to give dihomo–g–linolenic acid (C20:3 D6,9,12) and arachidonic acid (C22:4 D6,9,12,15)
The n–6 essential fatty acids are active in animal cells in at least four areas : in membrane structure and function, the regulation of cholesterol synthesis and transport. prevention of water loss from the skin and in the synthesis of eicosanoids. The eicosanoids are short-lived molecules that regulate aspects of cellular function and include the prostaglandins and leucotrienes.
Most humans are able to synthesise adequate amounts of n–6 fatty acids for all their requirements, provided they have adequate dietary levels of linoleic acid. However, in some situations the amounts may be inadequate, due either to reduced synthesis or increased consumption. The activity of the D6 desaturase may be reduced due to ageing, diabetes, stress, viral infections, eczema or other conditions, while increased consumption may result from oxidation or high rates of cell division (e.g. in inflammation or cancer). In such situations dietary supplementation with GLA may be beneficial. In fact, clinical trials have shown that GLA may have therapeutic effects for treating atopic eczema, mastalgia, diabetic neuropathy, inflammation, viral infections and forms of cancer (Lapinskas, 1993).
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The major plant source of GLA is currently evening primrose (Oenothera spp. Onagraceae) in which GLA accounts for 8–10% of the total seed oil. Evening primrose oil is widely used as a pharmaceutical and dietary supplement, and has been used extensively in clinical trials. Although higher levels of GLA are present in seeds of blackcurrant (Ribes nigrum, Rosaceae) and borage (Borago officinalis, Boraginaceae, also called starflower) and in oil produced by species of the fungus Mucor, these appear to be less effective in stimulating prostaglandin E1 production (Lapinskas, 1993). The reason for this is not clear. It may relate, however, to the intramolecular position of the GLA on the triacylglycerol and to the presence of other fatty acids in the oil. The most attractive species for future exploitation is borage. Borage oil, containing some 20–25% GLA, constitutes 25–40% of the seed weight and is marketed as an alternative to evening primrose oil.
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Although both evening primrose and borage have been improved by traditional plant breeding methods (Lapinskas, 1993; Fieldsend, 1995), the current yields (700–1300 kg/ha for evening primrose, 300–600 kg/ha for borage) are still greatly below those of conventional oilseed crops such as oilseed rape (c. 3 tonnes/ha in the UK). The production of GLA in a conventional oilseed is therefore a very attractive proposition. The feasibility of this approach is supported by the presence of the substrate, linoleic acid in all temperate oil crops. In particular, sunflower oil contains about 65% linoleic acid and no a–linolenic acid, and would therefore be an ideal background for the introduction of a D6 desaturase gene. Furthermore, Reddy and Thomas (1996) have recently demonstrated that low levels of GLA and of octadecatetraenoic acid (C18:4 D6,9,12,15) accumulated when a cyanobacterial gene encoding a D6 desaturase was expressed in leaves of tobacco plants. However, the combined levels of these two fatty acids varied from about 2–4% of the leaf C:18 fatty acids, with minor differences depending on whether the protein was targeted to the plastid, cytoplasm or lumen of the endoplasmic reticulum (ER). Also, no accumulation of GLA occurred in the seed oil of the transgenic plants.
The failure by Reddy and Thomas (1996) to achieve high levels of GLA accumulation using a cyanobacterial gene may not be surprising because of the differences between the cyanobacterial and higher plant D6 desaturases. The borage D6 desaturase probably uses cytochrome b5 as an electron donor (Stobart et al., (1992) and is located in the cytoplasmic face of the ER (Stobart and Stymne, 1986; Smith et al., 1990; Kearns et al., 1991). In contrast, the cyanobacterial enzyme uses ferredoxin as a cofactor (Wada et al., 1993), similar to the plastidic desaturases of higher plants (Schmidt and Heinz 1990). In addition the enzymes utilise different substrates, the sn–1 position of monogalactosyl diglyceride for the cyanobacterial enzyme and the sn–2 position of phosphatidyl choline for the borage enzyme. It is difficult to envisage how the cyanobacterial enzyme would function efficiently in the higher plant ER (the site of oleic and linoleic acid desaturation), even if correct targeting could be achieved. Nevertheless the results obtained by Reddy and Thomas (1996) are very encouraging.
A more promising approach would be to isolate a cDNA or gene for the D6 desaturase from developing seeds of borage or evening primrose and transfer this to sunflower or another conventional oilseed. A number of ER–located fatty acid desaturases have been cloned from seeds, including the D12 desaturase from Arabidopsis (Okuley et al., 1994) and D15 desaturases from Arabidopsis, oilseed rape, soybean and mung bean (Arondel et al., 1992; Yadav et al., 1992; Iba et al., 1993; Yamamoto et al., 1992). The availability of these clones should assist in the isolation of clones for the D6 desaturase, which is expected to be related to the other ER–located higher plant desaturases. Similarly, the use of specific gene promoters should give the correct pattern and level of expression if the cDNAs or genes are transferred to conventional oil crops. However, it is not sufficient to synthesise a high level of free GLA, as this must be incorporated into TAGS. We do not know whether the TAG assembly processes in conventional oilseeds would be capable of synthesising GLA-rich TAGs.
Despite the uncertainties discussed above it is no doubt worthwhile attempting to produce GLA in conventional oilseeds. However, an alternative approach should not be dismissed : this is to increase the yield and GLA content of evening primrose or borage using modern biotechnological approaches.
Improvement of evening primrose by plant breeding is limited by the presence of multiple reciprocal translocations in the terminal regions of the chromosomes which result in the 14 chromosomes forming a ring at meiosis. This results in a whole set of maternal or paternal chromosomes migrating to each pole, thus eliminating chromosome reassortment. Heterozygosity is maintained by genes which are lethal in homozygotes. This leads to serious problems for conventional plant breeders. Nevertheless, some progress has been made, utilising lines in which the series of translocations is incomplete, leading to the formation of smaller rings and one or more chromosome pairs, which permit limited chromosomal reassortment and hence genetic segregation (see Lapinskas, 1993).
Targets for improvement include seed dormancy, bolting (i.e. transition from vegetative to flowering state) and seed loss due to pod splitting (Lapinskas, 1993). Seed shedding is also a major problem in borage, with up to 50% of the yield lost during harvest (Fieldsend, 1995). Although borage exhibits classical Mendelian genetics and is therefore amenable to plant breeding, the ability to improve this and other characters depends on the availability of variation in the germplasm.
In fact, molecular genetic approaches to all these problems may soon be available based on studies of other crops (bolting in sugar beet, pod-shatter in oilseed rape) and model systems such as Arabidopsis and tobacco. These studies are leading to the identification of genes involved in the control of processes such as the vegetative to flowering transition, seed dormancy and abscission. The agronomic performance of both evening primrose and borage may also be improved by introducing genes for herbicide resistance, as discussed below for dye plants.
In addition to increasing the yield of borage and evening primrose, it would also be of value to increase their GLA content. This may be achieved by increasing the level of expression of the D6 desaturase gene, perhaps by the introduction of additional gene copies. This would require the isolation of a cDNA or gene as discussed above. In addition, it would be necessary to develop transformation systems for these species. Transformation of evening primrose callus with Agrobacterium-based vectors has recently been reported (Pavingerová et al., 1996), although regeneration was not achieved. Similarly, borage has been successfully micropropagated by tissue culture, but not yet used for transformation (Janick et al., 1987). Further work is required to evaluate the prospects for improving these species by gene transfer. The advantage of using borage or evening primrose is, of course, that the seeds of both species already have efficient systems for the assembly of GLA-rich TAGs, and are already used for dietary or healthcare purposes.
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It is perhaps not accurate to describe dye plants as new crops because they have been grown throughout the world for thousands of years. However, for all practical purposes, dye plants have not been grown in Europe, as commercial agricultural crops, since the middle of the nineteenth-century, although some dye crops are still grown in other parts of the world. As a consequence of the lack of commercial interest there has been no systematic scientific research on plants as an economic alternative source of dyes for industrial purposes. Thus, they should be considered as novel crops, from the agronomic and scientific points of view.
Recent changes in legislation on the use of some toxic synthetic dyestuffs and an increased preference by consumers for natural products have combined to reawaken an interest in dyes derived from crops, with the limited demand, by craft workers, being met by small-scale market gardening, resulting in high prices. In order for natural dyes to move beyond such niche applications optimum conditions for the growth, harvesting and processing of dye plants must be determined and modern agricultural methods adapted to their requirements.
It is a common misconception that natural dyes are inferior in quality to synthetic dyes, giving only pale, earthy colours, rather than strong, bright colours. In fact, when nylon was first developed, it was necessary to use natural dyes to obtain the best shades demanded by the consumer, as no synthetic alternatives were available. The earliest dyed textiles, preserved to the present day, used natural dyes from plants such as woad, madder and weld. Their colours are still recognisable and some vibrant, after many years.
There is also a growing consumer-led demand for naturally dyed textiles and producers estimate that they will account for up to 10% of the total within the next 10 years, (Das spezifische EG programm für Forschung, 1994). Similarly, there is an industry-led demand for a wide range of other products, such as non-toxic dyes for foodstuffs. The objectives of the work being done at IACR-Long Ashton are to optimise methods for cultivating dye crops and to refine and scale-up the extraction of the colouring matters, to underpin natural dye production as an economic alternative to synthetic dye manufacture.
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At present, the total world market for dyestuffs is 800,000 tonnes, of which, indigo represents 80,000 tonnes (Prof. D.M. Lewis, University of Leeds, Pers. comm.). Unpublished market research showed that approximately 5% of consumers expressed a preference for products coloured with natural dyes. Based on this level of consumption and an estimated cost of US $50 kg-1 for natural indigo, the world market would be US $200 m. Assuming an equivalent price for other natural dyes, the total world market for natural dyes would be US $2 bn. Similarly, the European market, estimated at about 10% of the world market, would be US $200 m. In the next 10 years this market is expected to grow to a possible total of US $600 m. However, it is not known whether the consumer would be prepared to pay more for naturally dyed products.
Although the increase in cost of dyeing textiles with natural dyes over synthetic dyes is considerable (although less so when colouring other products) this will represent a much smaller proportional increase when passed on to the consumer. At present, the use of natural dyes is likely to increase the price of textiles to the consumer by less than 10%. However, increases in the yields of dye plants and in their contents of dyes should result in decreases in the price of dyes and hence the added cost to the consumer.
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Four species of dye plants are currently being studied at IACR-Long Ashton: woad (Isatis tinctoria), Chinese woad (Isatis indigotica), madder (Rubia tinctorum) and weld (Reseda luteola). These species produce the three primary colours, blue, red and yellow.
Isatis tinctoria (woad, Cruciferae) is indigenous to most of northern Europe and grows well in a temperate climate. It produces two compounds which are precursors of indigo, isatan B and indican. Isatan B is oxidised in the presence of air to give indigo, under alkaline conditions. It is therefore extracted from leaves by steeping in warm acidified water and indigo is then formed by making the solution alkaline and aeration, a process used by home dyers today. Indican can also be extracted by steeping, but requires enzymic hydrolysis to break a glucosic bond. The product, indoxyl, then also requires alkaline conditions for oxidation to indigo. Originally the only source of blue pigment, woad was grown in Britain as a source of indigo until the middle of the 17th Century. In fact, references by Pliny, the Roman historian, to the `fearsome' ancient Britons with blue faces indicates that woad has been used for over two thousand years. In mediaeval Europe indigo derived from woad was a valuable commodity, traded through the many routes, earning it the title `the king of colours' (Sandberg, 1989). In warmer climates other plants which yielded higher amounts of indigo were used, notably Indigofera, but their use was initially prohibited in Europe to protect the local woad industry.
Isatis indigotica (Chinese woad, Cruciferae) is a close relative of woad and, in the 19th century, was grown in China, near Shanghai. The leaves are glaucous and broader than its European counterpart and it grows well in the temperate climate of Britain.
Rubia tinctorum (dyers madder, Rubiaceae) a perennial plant native to Asia Minor. Its roots produce a complex red dye of nineteen separate components. Unlike indigo, madder requires a mordant for fixation to the textile, which makes the dyeing process more complicated. In northern climates, madder sets only a small amount of seed which makes propagation difficult. At present, the most effective method of propagation is to plant cuttings at the beginning of the season, which is time-consuming and laborious. The dye is obtained from two to three-year-old roots, which are dried and powdered. Large areas of madder have recently been grown in the Netherlands where the sandy soil makes the roots easy to remove. Methods to remove roots from heavier soils must be developed if commercial cultivation of madder is to be achieved in the UK.
Reseda luteola (weld, Resedaceae) is a perennial plant which produces a yellow dye in its stems, foliage and flowers. It has been used as a source of dye for a thousand years or more and remains have been found in archaeological sites around Britain. In the past it was harvested in the middle of its flowering season, dried and tied into bundles, which were sold to textile dyers who extracted and used the dye. The yellow dye from weld has at least five components and is very intense. Although it requires a mordant, it is one of the better natural sources of yellow colour and is valuable because it can be used with woad to produce green, green dyes being uncommon.
In addition to woad, weld and madder, a number of other plants are potentially valuable sources of dyes.
Polygonum tinctorium (ai, Polygonaceae) is an annual semi-tropical plant, grown in Japan as a source of indigo. It produces indican, as in woad, but not isatan B. Ai will grow in Britain if it is irrigated regularly to give high humidity, but is frost sensitive. The yield of indigo is about three times that of woad (Hill, 1992) and, therefore, careful consideration must be given to its economic viability.
Solidago canadensis (golden rod, Compositae), a common garden plant, is a tall perennial shrub which will grow throughout the British Isles. It produces a different yellow dye to that of weld which also requires a mordant for fixation. The flowers are yellow brush-like structures on long thick stems and are produced annually. The dye can be extracted from the foliage and flowers using hot water. Golden rod thrives under poor nutritional conditions making it suitable for growing in low-input farming systems.
Anthemis tinctoria (yellow chamomile, Compositae) is a hardy perennial which produces a mass of foliage from which many yellow flowers emerge. It produces a deep yellow dye, which is extracted from the blossoms and requires mordanting for fixation. It also grows well on poor soils and provides an alternative yellow dye to those extracted from weld or golden rod.
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Although the structures of the major plant dyes are known, their biosynthetic pathways are still incompletely understood.
Isatis species contain two compounds, indoxyl–3–b–D–glucoside (indican) and indoxyl–5–ketogluconate (isatan B), which are thought to be synthesised either via tryptophan (Maier et al., 1990) or indole (Xia and Zenk, 1992). These are hydrolysed to give indoxyl, two molecules of which combine in the presence of oxygen to form indigo (Strobel and Gröger, 1989). A small quantity of a red compound, indirubin, is also formed, possibly via the isomerisation of isatan B to isatan.
Reseda species contain yellow flavonoids, mainly luteolin and apigenin but also at least three other components including quercetin (Leistner, 1980). Variation in the proportions of these components may influence the final colour of the dye.
Rubia species contain some 19 anthraquinones, including alizarin, pseudopurpurin and lucidin (Thomson, 1987), which may be synthesised via the decarboxylation of prenylated 1, 4–dihydroxy–2–naphthonic acid (Leistner, 1980).
In all cases further research is required on the basic biosynthetic pathways and on how they are regulated during plant growth and development and by environmental factors. The complexity of the pathways, and the presence in some dye plants of multiple components, means that it is unlikely that dye production could be transferred to conventional crops in the foreseeable future. Consequently research should focus on improving production from traditional dye plants. This will require optimisation of dye content and composition as well as improvement of yield and agronomic performance.
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Adaptation of traditional dye plants to modern agricultural systems poses important problems, some of which are being studied at IACR-Long Ashton.
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The dye plants discussed here have been essentially unimproved since historical times. Consequently there is great potential for improvement by classical breeding, selecting the best strains and progeny of crosses. Biotechnology can assist this improvement in various ways. The use of molecular markers may allow traits of interest, such as colour, yield and composition to be followed and selected, without the need for expensive and time consuming biochemical screening. Genetic engineering can also be used to improve the agronomic performance of the plants. In particular, the transfer of genes for herbicide resistance would greatly facilitate weed control using conventional herbicides such as basta.
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The choice of whether to adapt and improve obsolete crops or wild species or to introduce novel compounds into conventional crops will depend on several considerations.
Production of novel compounds in conventional crops is currently feasible if the compound of interest can be derived from a substrate already present in the recipient species by the introduction of one, or a small number, of genes encoding key biosynthetic enzymes. This is clearly an option with GLA, which could be
synthesised from a substrate (linoleic acid) present in oilseed rape and sunflower by the addition of a single enzyme, the D6 desaturase. However, an additional problem may arise with novel chemicals: toxicity or interference with growth or metabolism. In this case care must be taken to ensure that the novel product is directed to a "safe" subcellular location such as the vacuole.
In contrast to GLA, natural dyes are the products of complex biosynthetic pathways not specifically present in most plants, and may also be mixtures of structurally related components whose precise balance is important. In this case improvement and adaptation of traditional wild species is the only option, although genetic engineering may be used to assist conventional breeding efforts. A similar approach can also be used to improve the yield and agronomic performance of plants currently used to produce GLA, borage and evening primrose, but in all cases the application of genetic engineering to novel crops will require the development of efficient transformation procedures.
In view of the uncertainties discussed above it is essential to explore various options for the production of new crops for EU agriculture rather than rely solely on the application of genetic engineering to modify conventional crops.
Work on dye plants at IACR-Long Ashton is funded by an EU grant (AIR2-CT940981) to Dr. D.J. Hill. IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.
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(Note: I allowed my name to be added to the list of authors of this chapter under protest, as my contribution was minor. I have however reproduced it here as I believe it makes some important points and deserves wider publicity. PL )
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