Transcriptome Analysis and Identification of Lipid Genes in Physaria lindheimeri, a Genetic Resource for Hydroxy Fatty Acids in Seed Oil.

Hydroxy fatty acids (HFAs) have numerous industrial applications but are absent in most vegetable oils. Physaria lindheimeri accumulating 85% HFA in its seed oil makes it a valuable resource for engineering oilseed crops for HFA production. To discover lipid genes involved in HFA synthesis in P. lindheimeri, transcripts from developing seeds at various stages, as well as leaf and flower buds, were sequenced. Ninety-seven percent clean reads from 552,614,582 raw reads were assembled to 129,633 contigs (or transcripts) which represented 85,948 unique genes. Gene Ontology analysis indicated that 60% of the contigs matched proteins involved in biological process, cellular component or molecular function, while the remaining matched unknown proteins. We identified 42 P. lindheimeri genes involved in fatty acid and seed oil biosynthesis, and 39 of them shared 78-100% nucleotide identity with Arabidopsis orthologs. We manually annotated 16 key genes and 14 of them contained full-length protein sequences, indicating high coverage of clean reads to the assembled contigs. A detailed profiling of the 16 genes revealed various spatial and temporal expression patterns. The further comparison of their protein sequences uncovered amino acids conserved among HFA-producing species, but these varied among non-HFA-producing species. Our findings provide essential information for basic and applied research on HFA biosynthesis.

Specialized Functions of Olive FAD2 Gene Family Members Related to Fruit Development and the Abiotic Stress Response.

Three different cDNA sequences, designated OepFAD2-3, OepFAD2-4 and OepFAD2-5, encoding three microsomal oleate desaturases (FAD2) have been isolated from olive (Olea europaea cv. Picual). Sequence analysis and functional expression in yeast of the corresponding cDNAs confirm that they encode microsomal oleate desaturases. Gene expression and lipid analysis indicate that these three genes are not involved in the linoleic acid present in seed lipids, while OeFAD2-5, together with OeFAD2-2, contributes mostly to the linoleic acid present in the mesocarp and, therefore, in the olive oil. Our results have also shown that olive FAD2-3, FAD2-4 and FAD2-5 gene expression is not only spatially and temporally regulated in olive fruit, but also is cultivar-dependent, as well as regulated by water regime, temperature, light and wounding. All these data suggest specialized physiological roles for the olive FAD2 gene family members with respect to both aspects of the biosynthesis of the linoleic acid, either present in storage lipids that constitute the olive oil or being part of membrane lipids, which are involved in the response to abiotic stresses, and highlight the differences on FAD2 gene regulation between oilseeds and oil fruits.

Devolopmental and growth temperature regulation of omega-3 fatty acid desaturase genes in safflower (Carthamus tinctorius L.).

Three ω-3 fatty acid desaturase genes (CtFAD3, CtFAD7, and CtFAD8) were isolated from safflower (Carthamus tinctorius L.). Transcript analysis showed that the highest transcript levels were detected for CtFAD3 and the low transcript levels were detected for CtFAD7 and CtFAD8 in flowers. This result indicates that CtFAD3 enzyme activity is important for fatty acid desaturation in flowers. The low transcript level of CtFAD3 in developing seeds was consistent with the recorded high level of linoleic acid (18:2) and lack of linolenic acid (18:3) in safflower seed oil. At low temperatures, the induced transcription levels of ω-3 fatty acid desaturase genes in the stems and petioles were consistent with increased polyunsaturated fatty acids (PUFAs). In the roots, ω-3 fatty acid desaturase noticeably increased at low temperatures, whereas PUFA levels decreased. Interestingly, C18:3(Δ9,12,15) alcohol was specifically found in safflower roots, and showed a significant increase, indicating a flux in the acid to alcohol ratio of this compound in safflower roots.

Long chain polyunsaturated fatty acid synthesis in a marine vertebrate: ontogenetic and nutritional regulation of a fatty acyl desaturase with Δ4 activity.

Solea senegalensis is an unusual marine teleost as it has very low dietary requirement for long-chain polyunsaturated fatty acids (LC-PUFA) during early development. Aquaculture is rapidly becoming the main source of health-beneficial fish products for human consumption. This, associated with limited supply of LC-PUFA-rich ingredients for fish feeds, render S. senegalensis a highly interesting species in which to study the LC-PUFA biosynthesis pathway. We have cloned and functionally characterized fatty acyl desaturase and elongase cDNAs corresponding to Δ4fad (with some Δ5 activity for the n-3 series) and elovl5 with the potential to catalyze docosahexaenoic acid (DHA) biosynthesis from eicosapentaenoic acid (EPA). Changes in expression of both transcripts were determined during embryonic and early larval development, and transcriptional regulation in response to higher or lower dietary n-3 LC-PUFA was assessed during larval and post-larval stages. There was a marked pattern of regulation during early ontogenesis, with both transcripts showing peak expression coinciding with the start of exogenous feeding. Although elovl5 transcripts were present in fertilized eggs, Δ4fad only appeared at hatching. However, eggs have high proportions of DHA (~20%) and high DHA/EPA ratio (~11) to meet the high demands for early embryonic development. The fatty acid profile of larvae after the start of exogenous feeding closely reflected dietary composition. Nonetheless, Δ4fad was significantly up-regulated in response to LC-PUFA-poor diets, which may suggest biological relevance of this pathway in reducing LC-PUFA dietary requirements in this species, compared to other marine teleosts. These results indicate that sole is capable of synthesizing DHA from EPA through a Sprecher-independent pathway.

Vertebrate fatty acyl desaturase with Δ4 activity.

Biosynthesis of the highly biologically active long-chain polyunsaturated fatty acids, arachidonic (ARA), eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids, in vertebrates requires the introduction of up to three double bonds catalyzed by fatty acyl desaturases (Fad). Synthesis of ARA is achieved by Δ6 desaturation of 182n - 6 to produce 183n - 6 that is elongated to 203n - 6 followed by Δ5 desaturation. Synthesis of EPA from 183n - 3 requires the same enzymes and pathway as for ARA, but DHA synthesis reportedly requires two further elongations, a second Δ6 desaturation and a peroxisomal chain shortening step. This paper describes cDNAs, fad1 and fad2, isolated from the herbivorous, marine teleost fish (Siganus canaliculatus) with high similarity to mammalian Fad proteins. Functional characterization of the cDNAs by heterologous expression in the yeast Saccharomyces cerevisiae showed that Fad1 was a bifunctional Δ6/Δ5 Fad. Previously, functional dual specificity in vertebrates had been demonstrated for a zebrafish Danio rerio Fad and baboon Fad, so the present report suggests bifunctionality may be more widespread in vertebrates. However, Fad2 conferred on the yeast the ability to convert 225n - 3 to DHA indicating that this S. canaliculatus gene encoded an enzyme having Δ4 Fad activity. This is a unique report of a Fad with Δ4 activity in any vertebrate species and indicates that there are two possible mechanisms for DHA biosynthesis, a direct route involving elongation of EPA to 225n - 3 followed by Δ4 desaturation, as well as the more complicated pathway as described above.