Critical Metabolic Pathways And Genes Cooperate For Epoxy Fatty Acid-Enriched Oil Production In Developing Seeds of Vernonia Galamensis, An Industrial Oleaginous Plant

Background: Vernonia galamensis, originated from Africa, is an annual oleaginous plant of Asteraceae family. As a newly-establishing industrial oil crop, this plant produces high level (> 70%) of vernolic acid (cis-12-epoxyoctadeca-cis-9-enoic acid), an unusual epoxy fatty acid (EFA) with multiple industrial applications. Here, transcriptome analysis and fatty acid profiling from developing V. galamensis seeds were integrated to uncover the critical metabolic pathways responsible for high EFA accumulation and identify the targets that could be used in assembly of EFA biosynthesis pathway in existing oilseed crops. Results: Based on oil accumulation dynamics of V. galamensis seeds, we harvested seed samples from three stages (17, 38, and 45 days after pollination, DAP) representing the initial, fast and final EFA accumulation phases, and one mixed sample from different tissues for RNA-sequencing, with three biological replicates at each sample. Using Illumina platform, we have generated a total of 265 million raw cDNA reads. After filtering process, de novo assembly of clean reads yielded 67,114 unigenes with an N50 length of 1,316 nt. Functional annotation resulted in the identification of almost all genes involved in diverse lipid-metabolic pathways, including the novel fatty acid desaturase/epoxygenase, diacylglycerol acyltransferase 1 and 2, and phospholipid:diacylglycerol acyltransferases. By comparison, we found that various genes associated with acyl editing, fatty acid β-oxidation, triacylglycerol assembly and oil-body formation had greater expression levels at middle developmental stage (38 DAP), which are consistent with the fast accumulation of EFA in V. galamensis developing seed, implying their fundamental roles in EFA production. Additionally, we isolated some transcription factors (such as WRI1, FUS3 and ABI4), which putatively regulated the production of V. galamensis seed oils. The transient assay of selected genes resulted in a synergistic increase of EFA-enriched TAG accumulation in tobacco leaves. Transcriptome data were further confirmed by quantitative real-time PCR for twelve key genes in EFA biosynthesis. Finally, a comprehensive network for high EFA accumulation in V. galamensis seed was established.Conclusions: Our results are of significance with regard to understanding the molecular mechanisms underlying the natural epoxy oil production in this plant and provide valuable reference for developing other oilseeds with high level of valued epoxy oil.

Optimization of Partial Epoxidation on Jatropha curcas Oil Based Methyl Linoleate Using Urea-hydrogen Peroxide and Methyltrioxorhenium Catalyst

Natural epoxy fatty acids such as Coronaric acid (9,10-epoxy-12Z-octadecenoic acid) and vernolic acid (12,13-epoxy-9Z-octadecenoic acid) are rich in of Vernolia galamensis, Vernolia anthelmintica and Chrysanthemums coronanium. The two fatty acids each contains an oxirana ring and a double bond C = C. The oil or its derivatives are suitable for industrial usage as reactive diffluent of alkyd resins, plasticizers and stabilizers, surface coatings, surfactants and lubricants, as intermediates in chemical reactions for making linear epoxides of composite materials and polymers. However, the use of such oils on an industrial scale is impossible due to limited resources. Therefore, epoxidation reactions need to be carried out to overcome the demand for partial epoxide fatty acids. Partially epoxidation of methyl linoleate at room temperature (30°C) in the presence of pyridine, methyltrioxorhenium (MTO) as catalyst and urea-hydrogen peroxide (UHP) as oxidant was studied by using response surface methodology (RSM). A five-level-four-factors central composite rotatable design (CCRD) was used to optimize the partially epoxidation conditions and study the effect of MTO, UHP, pyridine and reaction time on relative conversion to oxirane (RCO). Quadratic polynomial model was employed to generate response surface plots for RCO. At optimal condition, 79.05% monoepoxide was formed at the RCO of 58.15% under condition of 0.75 mol% mole ratio of MTO, 300 mol% mole ratio of UHP and 9 mol% of pyridine at 120 min reaction time. It can be concluded that the effect of UHP mole ratios was the dominant factor to control the degree of partial epoxidation of methyl linoleate followed by mole ratio of MTO, reaction time and mole ratio of pyridine to formed methyl 12,13-epoxy-9Z-octadecenoate or/and methyl 9,10-epoxy-12Z-octadecenoate.

Combined transgenic expression of Δ12-desaturase and Δ12-epoxygenase in high linoleic acid seeds leads to increased accumulation of vernolic acid

The transgenic production of unusual fatty acids in oil seed crops offers an alternative, renewable resource for industry. However, transgenic expression of genes catalysing the synthesis of unusual fatty acids has generally resulted in these fatty acids accumulating at levels significantly below the levels in the wild species from which the genes were sourced. This study reports expression of additional copies of any of three Δ12-desaturase genes (FAD2) from Crepis palaestina Bornm., cotton (Gossypium hirsutum L.) or Arabidopsis thaliana (L.) Heynh. with C. palaestina Δ12-epoxygenase gene (Cpal2), in an Arabidopsis mutant having a significantly higher level of linoleic acid substrate. This resulted in the highest levels of vernolic acid accumulation, 21% of total fatty acids, reported so far in any transgenic plant expressing the Δ12-epoxygenase. Similarly, the co-expression of C. palaestina Cpal2 and a transgenic copy of FAD2 in cotton seed that contains large amounts of linoleic acid substrate also resulted in greater accumulation of vernolic acid in seed than did expression of C. palaestina Cpal2 alone.

Genotypic and phenotypic variability in Vernonia galamensis germplasm collected from eastern Ethiopia

Ethiopia is the centre of origin and diversity of Vernonia galamensis, which is a new potential industrial oilseed crop. Seeds of Vernonia are rich in vernolic acid, a naturally epoxidized fatty acid with unique chemical (epoxy) and physical (low viscosity) properties. Vernolic acid is a useful raw material for manufacturing paints and coatings. No effort has been made so far in Ethiopia or elsewhere to improve this plant. Therefore, an experiment was conducted in three contrasting agroclimatic zones (Alemaya, Harar and Babile) using eight accessions to assess the genotypic (GCV) and phenotypic (PCV) coefficients of variation, to estimate broad sense heritability (h2B) and genetic advance (GA) as a per cent of the mean for 17 pheno-morphic and agronomic traits.Considering GCV, h2B and GA (as per cent of the mean) simultaneously as the best estimators of the amount of advance expected from selection, secondary head number (28·90, 0·90 and 56·60), secondary branch number (13·90, 0·67 and 51·97), and seed number per head (15·97, 0·72 and 27·92) gave the highest values at Alemaya, Harar and Babile, respectively. This shows that a satisfactory selection program for improvement of these characters is possible in V. galamensis var. ethiopica at each specific location.