Production of arachidonic and eicosapentaenoic acids in plants using bryophyte fatty acid Delta6-desaturase, Delta6-elongase, and Delta5-desaturase genes.

The liverwort Marchantia polymorpha L. synthesizes arachidonic (ARA) and eicosapentaenoic acids (EPA) from linoleic and alpha-linolenic acids respectively by a series of reactions catalyzed by Delta6-desaturase, Delta6-elongase, and Delta5-desaturase. Overexpression of the M. polymorpha genes encoding these enzymes in transgenic M. polymorpha plants resulted in 3- and 2-fold accumulation of ARA and EPA respectively, as compared to those in the wild type. When these three genes were introduced and co-expressed in tobacco plants, in which long-chain polyunsaturated fatty acids (LCPUFAs) are not native cellular components, ARA and EPA represented up to 15.5% and 4.9% respectively of the total fatty acid in the leaves. Similarly in soybean, C20-LCPUFAs represented up to 19.5% of the total fatty acids in the seeds. These results suggest that M. polymorpha can provide genes crucial to the production of C20-LCPUFAs in transgenic plants.

Gene organization of the liverwort Y chromosome reveals distinct sex chromosome evolution in a haploid system.

Y chromosomes are different from other chromosomes because of a lack of recombination. Until now, complete sequence information of Y chromosomes has been available only for some primates, although considerable information is available for other organisms, e.g., several species of Drosophila. Here, we report the gene organization of the Y chromosome in the dioecious liverwort Marchantia polymorpha and provide a detailed view of a Y chromosome in a haploid organism. On the 10-Mb Y chromosome, 64 genes are identified, 14 of which are detected only in the male genome and are expressed in reproductive organs but not in vegetative thalli, suggesting their participation in male reproductive functions. Another 40 genes on the Y chromosome are expressed in thalli and male sexual organs. At least six of these genes have diverged X-linked counterparts that are in turn expressed in thalli and sexual organs in female plants, suggesting that these X- and Y-linked genes have essential cellular functions. These findings indicate that the Y and X chromosomes share the same ancestral autosome and support the prediction that in a haploid organism essential genes on sex chromosomes are more likely to persist than in a diploid organism.

A front-end desaturase from Chlamydomonas reinhardtii produces pinolenic and coniferonic acids by omega13 desaturation in methylotrophic yeast and tobacco.

Pinolenic acid (PA; 18:3Delta(5,9,12)) and coniferonic acid (CA; 18:4Delta(5,9,12,15)) are Delta(5)-unsaturated bis-methylene-interrupted fatty acids (Delta(5)-UBIFAs) commonly found in pine seed oil. They are assumed to be synthesized from linoleic acid (LA; 18:2Delta(9,12)) and alpha-linolenic acid (ALA; 18:3Delta(9,12,15)), respectively, by Delta(5)-desaturation. A unicellular green microalga Chlamydomonas reinhardtii also accumulates PA and CA in a betain lipid. The expressed sequence tag (EST) resource of C. reinhardtii led to the isolation of a cDNA clone that encoded a putative fatty acid desaturase named as CrDES containing a cytochrome b5 domain at the N-terminus. When the coding sequence was expressed heterologously in the methylotrophic yeast Pichia pastoris, PA and CA were newly detected and comparable amounts of LA and ALA were reduced, demonstrating that CrDES has Delta(5)-desaturase activity for both LA and ALA. CrDES expressed in the yeast showed Delta(5)-desaturase activity on 18:1Delta(9) but not 18:1Delta(11). Unexpectedly, CrDES also showed Delta(7)-desaturase activity on 20:2Delta(11,14) and 20:3Delta(11,14,17) to produce 20:3Delta(7,11,14) and 20:4Delta(7,11,14,17), respectively. Since both the Delta(5) bond in C18 and the Delta(7) bond in C20 fatty acids are 'omega13' double bonds, these results indicate that CrDES has omega13 desaturase activity for omega9 unsaturated C18/C20 fatty acids, in contrast to the previously reported front-end desaturases. In order to evaluate the activity of CrDES in higher plants, transgenic tobacco plants expressing CrDES were created. PA and CA accumulated in the leaves of transgenic plants. The highest combined yield of PA and CA was 44.7% of total fatty acids, suggesting that PA and CA can be produced in higher plants on a large scale.

Isolation and characterization of delta(6)-desaturase, an ELO-like enzyme and delta(5)-desaturase from the liverwort Marchantia polymorpha and production of arachidonic and eicosapentaenoic acids in the methylotrophic yeast Pichia pastoris.

The liverwort Marchantia polymorpha contains high proportions of arachidonic and eicosapentaenoic acids. In general, these C20 polyunsaturated fatty acids (PUFA) are synthesized from linoleic and alpha -linolenic acids, respectively, by a series of reactions catalyzed by Delta(6)-desaturase, an ELO-like enzyme involved in Delta(6) elongation and Delta(5)-desaturase. Here we report the isolation and characterization of the cDNAs, MpDES6, MpELO1 and MpDES5, coding for the respective enzymes from M. polymorpha. Co-expression of the MpDES6, MpELO1 and MpDES5 cDNAs resulted in the accumulation of arachidonic and eicosapentaenoic acids in the methylotrophic yeast Pichia pastoris. Interestingly, Delta(6) desaturation by the expression of the MpDES6 cDNA appears to occur both in glycerolipids and the acyl-CoA pool, although other lower-plant Delta(6)-desaturases are known to have a strong preference for glycerolipids.