Very long chain polyunsaturated fatty acids (VLCPUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are valuable commodities that provide important human health benefits. We report the transgenic production of significant amounts of AA and EPA in Brassica juncea seeds via a stepwise metabolic engineering strategy. Using a series of transformations with increasing numbers of transgenes, we demonstrate the incremental production of VLCPUFAs, achieving AA levels of up to 25% and EPA levels of up to 15% of total seed fatty acids. Both fatty acids were almost exclusively found in triacylglycerols, with AA located preferentially at sn-2 and sn-3 positions and EPA distributed almost equally at all three positions. Moreover, we reconstituted the DHA biosynthetic pathway in plant seeds, demonstrating the practical feasibility of large-scale production of this important omega-3 fatty acid in oilseed crops.

Industrial oil flax (Linum usitatissimum) and edible oil or solin flax differ markedly in seed linolenic acid levels. Despite the economic importance of low-linolenic-acid or solin flax, the molecular mechanism underlying this trait has not been established. Two independently inherited genes control the low-linolenic-acid trait in flax. Here, we identified two genes, LuFAD3A and LuFAD3B that encode microsomal desaturases capable of desaturating linoleic acid. The deduced proteins encoded by these genes shared 95.4% identity. In the low-linolenic-acid line solin 593-708, both LuFAD3A and LuFAD3B carry point mutations that produce premature stop codons. Expression of these genes in yeast (Saccharomyces cerevisiae) demonstrated that, while the wild-type proteins were capable of desaturating linoleic acid, the truncated proteins were inactive. Furthermore, the low-linolenic-acid phenotype in flax was complemented by transformation with a wild-type gene. Codominant DNA markers were developed to distinguish between null and wild-type alleles of both genes, and linolenic acid levels cosegregated with genotypes, providing further proof that LuFAD3A and LuFAD3B are the major genes controlling linolenic acid levels in flax. The level of LuFAD3 transcripts in seeds peaked at about 20 d after flowering, and transcripts were not detectable in leaf, root, or stem tissue. A dramatic reduction in transcript levels of both genes occurred in the low-linolenicacid solin line, which was likely due to nonsense-mediated decay.The seed oil of flax (Linum usitatissimum) is notable for its high level of linolenic acid, generally around 45% to 65%, which gives it a high drying quality, making it useful for industrial purposes. The development of low-linolenic-acid flax lines (Green, 1986;Rowland, 1991), known as solin or linola types, expanded the potential markets for flaxseed oil. Compared to oil from traditional flax cultivars, low-linolenic-acid oil is less subject to rapid oxidation and is thus more competitive as a cooking or salad oil (Bhatty, 1995). Two recessive genes at independent loci control the low-linolenic-acid trait in flax (Rowland, 1991). Following ethyl methanesulfonate (EMS) mutagenesis, Rowland (1991) identified an M2 seed carrying mutations in both these genes. The resulting line had a linolenic acid content of less than 2% compared to levels of approximately 49% in the wild-type parent McGregor. Similarly, Green and Marshall (1984) isolated two EMS-derived mutants with linolenic acid contents of approximately 30% and by recombining these lines were able to obtain plants with linolenic acid contents below 2% (Green, 1986). In contrast to the dramatic reduction of linolenic acid in seeds, the levels in leaf tissue remained unchanged (Tonnet and Green, 1987).Linolenic acid is produced through the desaturation of linoleic acid by omega-3/delta-15 desaturases. In plants, this reaction occurs both in the plastids and in the endoplasmic reticulum (ER). Arabidopsis (Arabidopsis thaliana) carries two plastidial desaturases...

Studies of waxy mutations in wheat and other cereals have shown that null mutations in genes encoding granule-bound starch synthase I (GBSSI) result in amylose-free starch in endosperm and pollen grains, whereas starch in other tissues may contain amylose. We have isolated a cDNA from waxy wheat that encodes GBSSII, which is thought to be responsible for the elongation of amylose chains in non-storage tissues. The deduced amino acid sequences of wheat GBSSI and GBSSII were almost 66% identical, while those of wheat GBSSII and potato GBSSI were 72% identical. GBSSII was expressed in leaf, culm, and pericarp tissue, but transcripts were not detected in endosperm tissue. In contrast, GBSSI expression was high in endosperm tissue. The expression of GBSSII mRNA in pericarp tissue was similar at the midpoints of the day and night periods. The GBSSII genes were mapped to chromosomes 2AL, 2B, and 2D, whereas GBSSI genes are located on group 7 chromosomes. Gelblot analysis indicated that genes related to GBSSII also occur in barley, rice, and maize. The possible role of GBSSII in starch synthesis is discussed.Starch is composed of two distinct polymers; amylopectin, which consists of long chains of (1-4)-linked ␣-Dglucopyranosyl units with extensive branching resulting from (1-6) linkages, and amylose, which is a relatively linear molecule of (1-4)-linked ␣-d-glucopyranosyl units (Whistler and Daniel, 1984). Both types of chains are elongated by starch synthases that transfer ␣-d-Glc from ADPGlc to the growing chain, and specific starch synthases are active in the synthesis of each type of polymer. Whereas a number of starch synthases are thought to catalyze amylopectin synthesis (Dry et al

Mutations in the three homeologous waxy loci Wx-A1, Wx-B1, and Wx-D1 of a waxy wheat line have previously been characterized at the molecular level. Using combinations of these mutations, six types of partial waxy wheat plus wild type and waxy wheat (types 1-8) can be produced. Here, we describe primer sets for all three loci that can be used under a single set of conditions, allowing 32 lines to be characterized as types 1-8 in a single PCR run using a 96-well plate. Using multiplex PCR, mutations at the Wx-B1 and Wx-D1 loci can be identified in a single PCR, reducing the number of reactions necessary to identify and select the desired partial waxy wheat line. A single multiplex PCR can be used to detect all three mutations when products are analyzed using capillary electrophoresis on a microchip device. The PCR conditions and primers are effective with a number of cultivars from other countries, indicating that the mutations found at the Wx-A1 and Wx-B1 loci of these cultivars likely have the same origins as the mutations in the corresponding loci of the waxy wheat line used in this study. The PCR selection method described here is an easy and effective alternative to the commonly used SDS-PAGE methods for identification of null alleles.

Waxy wheat (Triticum aestivum L.) lacks the waxy protein, which is also known as granule-bound starch synthase I (GBSSI). The starch granules of waxy wheat endosperm and pollen do not contain amylose and therefore stain red-brown with iodine. However, we observed that starch from pericarp tissue of waxy wheat stained blue-black and contained amylose. Significantly higher starch synthase activity was detected in pericarp starch granules than in endosperm starch granules. A granule-bound protein that differed from GBSSI in molecular mass and isoelectric point was detected in the pericarp starch granules but not in granules from endosperm. This protein was designated GBSSII. The N-terminal amino acid sequence of GBSSII, although not identical to wheat GBSSI, showed strong homology to waxy proteins or GBSSIs of cereals and potato, and contained the motif KTGGL, which is the putative substratebinding site of GBSSI of plants and of glycogen synthase of Escherichia coli. GBSSII cross-reacted specifically with antisera raised against potato and maize GBSSI. This study indicates that GBSSI and GBSSII are expressed in a tissue-specific manner in different organs, with GBSSII having an important function in amylose synthesis in the pericarp.