In algae, the biosynthesis of docosahexaenoic acid (22:6 3; DHA) proceeds via the elongation of eicosapentaenoic acid (20:5 3; EPA) to 22:5 3, which is required as a substrate for the final ⌬ 4 desaturation. To isolate the elongase specific for this step, we searched expressed sequence tag and genomic databases from the algae Ostreococcus tauri and Thalassiosira pseudonana , from the fish Oncorhynchus mykiss , from the frog Xenopus laevis , and from the sea squirt Ciona intestinalis using as a query the elongase sequence PpPSE1 from the moss Physcomitrella patens . The open reading frames of the identified elongase candidates were expressed in yeast for functional characterization. By this, we identified two types of elongases from O. tauri and T. pseudonana : one specific for the elongation of ( ⌬ 6-)C18-PUFAs and one specific for ( ⌬ 5-)C20-PUFAs, showing highest activity with EPA. The clones isolated from O. mykiss , X. laevis , and C. intestinalis accepted both C18-and C20-PUFAs. By coexpression of the ⌬ 6-and ⌬ 5-elongases from T. pseudonana and O. tauri , respectively, with the ⌬ 5-and ⌬ 4-desaturases from two other algae we successfully implemented DHA synthesis in stearidonic acid-fed yeast. This may be considered an encouraging first step in future efforts to implement this biosynthetic sequence into transgenic oilseed crops. -Meyer, A., H. Kirsch, F. Domergue, A. Abbadi, P. Sperling, J. Bauer, P. Cirpus, T. K. Zank, H. Moreau, T. J. Roscoe, U. Zähringer, and E. Heinz. Novel fatty acid elongases and their use for the reconstitution of docosahexaenoic acid biosynthesis.

The LAFL (i.e. LEC1, ABI3, FUS3, and LEC2) master transcriptional regulators interact to form different complexes that induce embryo development and maturation, and inhibit seed germination and vegetative growth in Arabidopsis. Orthologous genes involved in similar regulatory processes have been described in various angiosperms including important crop species. Consistent with a prominent role of the LAFL regulators in triggering and maintaining embryonic cell fate, their expression appears finely tuned in different tissues during seed development and tightly repressed in vegetative tissues by a surprisingly high number of genetic and epigenetic factors. Partial functional redundancies and intricate feedback regulations of the LAFL have hampered the elucidation of the underpinning molecular mechanisms. Nevertheless, genetic, genomic, cellular, molecular, and biochemical analyses implemented during the last years have greatly improved our knowledge of the LALF network. Here we summarize and discuss recent progress, together with current issues required to gain a comprehensive insight into the network, including the emerging function of LEC1 and possibly LEC2 as pioneer transcription factors.

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Addendum to: Maisonneuve S, Bessoule J-J, Lessire R, Delseny M, Roscoe TJ. Expression of rapeseed microsomal lysophosphatidic acid acyltransferase isozymes enhances seed oil content in Arabidopsis.

The biosynthesis of phosphatidic acid, a key intermediate in the biosynthesis of lipids, is controlled by lysophosphatidic acid (LPA, or 1-acyl-glycerol-3-P) acyltransferase (LPAAT, EC 2.3.1.51). We have isolated a cDNA encoding a novel LPAAT by functional complementation of the Escherichia coli mutant plsC with an immature embryo cDNA library of oilseed rape (Brassica napus). Transformation of the acyltransferase-deficient E. coli strain JC201 with the cDNA sequence BAT2 alleviated the temperature-sensitive phenotype of the plsC mutant and conferred a palmitoyl-coenzyme A-preferring acyltransferase activity to membrane fractions. The BAT2 cDNA encoded a protein of 351 amino acids with a predicted molecular mass of 38 kD and an isoelectric point of 9.7. Chloroplast-import experiments showed processing of a BAT2 precursor protein to a mature protein of approximately 32 kD, which was localized in the membrane fraction. BAT2 is encoded by a minimum of two genes that may be expressed ubiquitously. These data are consistent with the identity of BAT2 as the plastidial enzyme of the prokaryotic glycerol-3-P pathway that uses a palmitoyl-ACP to produce phosphatidic acid with a prokaryotictype acyl composition. The homologies between the deduced protein sequence of BAT2 with prokaryotic and eukaryotic microsomal LAP acytransferases suggest that seed microsomal forms may have evolved from the plastidial enzyme.Phosphatidic acid is a key intermediate in the biosynthesis of phospho-and glycerolipids, essential components of all cellular membranes and of triacylglycerols. In most plant species, palmitoyl-ACP and oleoyl-ACP are the predominant products of de novo fatty acid biosynthesis in the chloroplast (Ohlrogge et al., 1993). These fatty acids may enter the prokaryotic pathway of lipid biosynthesis by transfer of the acyl group from ACP to glycerol-3-P, which is mediated by a stromal glycerol-3-P acyltransferase to form LPA or to position sn-2 of glycerol-3-P mediated by LPAAT (EC 2.3.1.51) to form phosphatidic acid. The phosphatidic acid produced by the prokaryotic pathway of the chloroplast is then used to produce phosphatidylglycerol (for review, see Ohlrogge and Browse, 1995) or is dephosphorylated to diacylglycerol, from which the glycodiacylglycerols characteristic of the thylakoid membrane are derived (for review, see Douce and Joyard, 1990).Alternatively, the elongation of fatty acids may be terminated by the action of an acyl-ACP thioesterase, which hydrolyzes the acyl-ACP to release a free fatty acid, which then leaves the plastid. The fatty acid in the form of an acyl-CoA thioester may participate in the synthesis of glycerolipids via the eukaryotic pathway located at the ER. The phosphatidic acid of the cytoplasmic pathway is used to produce the phospholipids characteristic of extrachloroplastic lipids. The phosphatidylcholine produced by the eukaryotic pathway is a substrate for desaturation, after which the diacylglycerol moiety may be returned to the chloroplast. The contribution of each pathway to the syn...

Low erucic acid rapeseed (LEAR) is characterised by a near absence of very long chain fatty acids in the seed oil which has been correlated with a lack of acyl-CoA elongation activity. Here we show that the absence of acyl-CoA and ATP-dependent elongation activities in microsomes isolated from LEAR embryos is associated with an absence of L L-ketoacyl-CoA synthase activity encoded by the Bn-fatty acid elongation 1 (FAE1) genes. Size exclusion chromatography of solubilised microsomes revealed the presence of a high molecular mass acyl-CoA elongase complex in high erucic acid rapeseed which was absent in microsomes isolated from LEAR seeds. Although transcripts for the Bn-FAE1 genes were detected in LEAR embryos, immunoblots using antisera raised against the L L-ketoacyl-CoA synthase indicated an absence of this protein. Comparison of the deduced amino acid sequences of immature embryo cDNAs reveals that LEAR alleles of Bn-FAE1 encode variant L Lketoacyl-CoA synthase proteins. ß

Networks controlling developmental or metabolic processes in plants are often complex as a consequence of the duplication and specialisation of the regulatory genes as well as the numerous levels of transcriptional and post-transcriptional controls added during evolution. Networks serve to accommodate multicellular complexity and increase robustness to environmental changes. Mathematical simplification by regrouping genes or pathways in a limited number of hubs has facilitated the construction of models for complex traits. In a complementary approach, a biological simplification can be achieved by using genetic modification to understand the core and singular ancestral function of the network, which is likely to be more prevalent within the plant kingdom rather than specific to a species. With this viewpoint, we review examples of simplification successfully undertaken in yeast and other organisms. A strategy of progressive complementation of single, double and triple mutants of seed maturation confirmed the fundamental role of the AFL sub-family of B3 transcription factors as master regulators of seed maturation, illustrating that biological simplification of complex networks could be more widely applied in plants. Defining minimal control networks will facilitate evolutionary comparisons of regulatory processes and the identification of an essential gene set for synthetic biology.

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