Metabolic engineering of omega-3 long chain polyunsaturated fatty acids in plants using different ∆6- and ∆5-desaturases co-expressed with LPCAT from the marine diatom Phaeodactylum tricornutum.

Continuous research on obtaining an even more efficient production of very long-chain polyunsaturated fatty acids (VLC-PUFAs) in plants remains one of the main challenges of scientists working on plant lipids. Since crops are not able to produce these fatty acids due to the lack of necessary enzymes, genes encoding them must be introduced exogenously from native organisms producing VLC-PUFAs. In this study we reported, in tobacco leaves, the characterization of three distinct ∆6-desaturases from diatom Phaeodactylum tricornutum, fungi Rhizopus stolonifer and microalge Osterococcus tauri and two different ∆5-desaturases from P. tricornutum and single-celled saprotrophic eukaryotes Thraustochytrium sp. The in planta agroinfiltration of essential ∆6-desaturases, ∆6-elongases and ∆5-desaturases allowed for successful introduction of eicosapentaenoic acid (20:5∆5,8,11,14,17) biosynthesis pathway. However, despite the desired, targeted production of ω3-fatty acids we detected the presence of ω6-fatty acids, indicating and confirming previous results that all tested desaturases are not specifically restricted to neither ω3- nor ω6-pathway. Nevertheless, the additional co-expression of acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) from Phaeodactylum tricornutum boosted the proportion of ω3-fatty acids in newly synthesized fatty acid pools. For the most promising genes combinations the EPA content reached at maximum 1.4% of total lipid content and 4.5% of all fatty acids accumulated in the TAG pool. Our results for the first time describe the role of LPCAT enzyme and its effectiveness in alleviating a bottleneck called 'substrate dichotomy' for improving the transgenic production of VLC-PUFAs in plants.

Biochemical characterization of acyl-CoA:diacylglycerol acyltransferase2 from the diatom Phaeodactylum tricornutum and its potential effect on LC-PUFAs biosynthesis in planta.

BACKGROUND: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), belonging to ω-3 long-chain polyunsaturated fatty acids (ω3-LC-PUFAs), are essential components of human diet. They are mainly supplemented by marine fish consumption, although their native producers are oleaginous microalgae. Currently, increasing demand for fish oils is insufficient to meet the entire global needs, which puts pressure on searching for the alternative solutions. One possibility may be metabolic engineering of plants with an introduced enzymatic pathway producing ω3-LC-PUFAs. RESULT: In this study we focused on the acyl-CoA:diacylglycerol acyltransferase2b (PtDGAT2b) from the diatom Phaeodactylum tricornutum, an enzyme responsible for triacylglycerol (TAG) biosynthesis via acyl-CoA-dependent pathway. Gene encoding PtDGAT2b, incorporated into TAG-deficient yeast strain H1246, was used to confirm its activity and conduct biochemical characterization. PtDGAT2b exhibited a broad acyl-CoA preference with both di-16:0-DAG and di-18:1-DAG, whereas di-18:1-DAG was favored. The highest preference for acyl donors was observed for 16:1-, 10:0- and 12:0-CoA. PtDGAT2b also very efficiently utilized CoA-conjugated ω-3 LC-PUFAs (stearidonic acid, eicosatetraenoic acid and EPA). Additionally, verification of the potential role of PtDGAT2b in planta, through its transient expression in tobacco leaves, indicated increased TAG production with its relative amount increasing to 8%. Its co-expression with the gene combinations aimed at EPA biosynthesis led to, beside elevated TAG accumulation, efficient accumulation of EPA which constituted even 25.1% of synthesized non-native fatty acids (9.2% of all fatty acids in TAG pool). CONCLUSIONS: This set of experiments provides a comprehensive biochemical characterization of DGAT enzyme from marine microalgae. Additionally, this study elucidates that PtDGAT2b can be used successfully in metabolic engineering of plants designed to obtain a boosted TAG level, enriched not only in ω-3 LC-PUFAs but also in medium-chain and ω-7 fatty acids.

Phospholipid:diacylglycerol acyltransferase1-overexpression stimulates lipid turnover, oil production and fitness in cold-grown plants.

BACKGROUND: Extensive population growth and climate change accelerate the search for alternative ways of plant-based biomass, biofuel and feed production. Here, we focus on hitherto unknow, new promising cold-stimulated function of phospholipid:diacylglycerol acyltransferase1 (PDAT1) - an enzyme catalyzing the last step of triacylglycerol (TAG) biosynthesis. RESULT: Overexpression of AtPDAT1 boosted seed yield by 160% in Arabidopsis plants exposed to long-term cold compared to standard conditions. Such seeds increased both their weight and acyl-lipids content. This work also elucidates PDAT1's role in leaves, which was previously unclear. Aerial parts of AtPDAT1-overexpressing plants were characterized by accelerated growth at early and vegetative stages of development and by biomass weighing three times more than control. Overexpression of PDAT1 increased the expression of SUGAR-DEPENDENT1 (SDP1) TAG lipase and enhanced lipid remodeling, driving lipid turnover and influencing biomass increment. This effect was especially pronounced in cold conditions, where the elevated synergistic expression of PDAT1 and SDP1 resulted in double biomass increase compared to standard conditions. Elevated phospholipid remodeling also enhanced autophagy flux in AtPDAT1-overexpresing lines subjected to cold, despite the overall diminished autophagy intensity in cold conditions. CONCLUSIONS: Our data suggest that PDAT1 promotes greater vitality in cold-exposed plants, stimulates their longevity and boosts oilseed oil production at low temperature.

Phosphatidylcholine:diacylglycerol cholinephosphotransferase's unique regulation of castor bean oil quality.

Castor bean (Ricinus communis) seed oil (triacylglycerol [TAG]) is composed of ∼90% of the industrially important ricinoleoyl (12-hydroxy-9-octadecenoyl) groups. Here, phosphatidylcholine (PC):diacylglycerol (DAG) cholinephosphotransferase (PDCT) from castor bean was biochemically characterized and compared with camelina (Camelina sativa) PDCT. DAGs with ricinoleoyl groups were poorly used by Camelina PDCT, and their presence inhibited the utilization of DAG with "common" acyl groups. In contrast, castor PDCT utilized DAG with ricinoleoyl groups similarly to DAG with common acyl groups and showed a 10-fold selectivity for DAG with one ricinoleoyl group over DAG with two ricinoleoyl groups. Castor DAG acyltransferase2 specificities and selectivities toward different DAG and acyl-CoA species were assessed and shown to not acylate DAG without ricinoleoyl groups in the presence of ricinoleoyl-containing DAG. Eighty-five percent of the DAG species in microsomal membranes prepared from developing castor endosperm lacked ricinoleoyl groups. Most of these species were predicted to be derived from PC, which had been formed by PDCT in exchange with DAG with one ricinoleoyl group. A scheme of the function of PDCT in castor endosperm is proposed where one ricinoleoyl group from de novo-synthesized DAG is selectivity transferred to PC. Nonricinoleate DAG is formed and ricinoleoyl groups entering PC are re-used either in de novo synthesis of DAG with two ricinoleoyl groups or in direct synthesis of triricinoleoyl TAG by PDAT. The PC-derived DAG is not used in TAG synthesis but is proposed to serve as a substrate in membrane lipid biosynthesis during oil deposition.

Manufacturing specialized wax esters in plants.

Biologically produced wax esters can fulfil different industrial purposes. These functionalities almost drove the sperm whale to extinction from hunting. After the ban on hunting, there is a niche in the global market for biolubricants with properties similar to spermaceti. Wax esters can also serve as a mechanism for producing insect sex pheromone fatty alcohols. Pheromone-based mating disruption strategies are in high demand to replace the toxic pesticides in agriculture and manage insect plagues threatening our food and fiber reserves. In this study we set out to investigate the possibilities of in planta assembly of wax esters, for specific applications, through transient expression of various mix-and-match combinations of genes in Nicotiana benthamiana leaves. Our synthetic biology designs were outlined in order to pivot plant lipid metabolism into producing wax esters with targeted fatty acyl and fatty alcohols moieties. Through this approach we managed to obtain industrially important spermaceti-like wax esters enriched in medium-chain fatty acyl and/or fatty alcohol moieties of wax esters. Via employment of plant codon-optimized moth acyl-CoA desaturases we also managed to capture unusual, unsaturated fatty alcohol and fatty acyl moieties, structurally similar to moth pheromone compounds, in plant-accumulated wax esters. Comparison between outcomes of different experimental designs identified targets for stable transformation to accumulate specialized wax esters and helped us to recognize possible bottlenecks of such accumulation.

LPEATs Tailor Plant Phospholipid Composition through Adjusting Substrate Preferences to Temperature.

Acyl-CoA:lysophosphatidylethanolamine acyltransferases (LPEATs) are known as enzymes utilizing acyl-CoAs and lysophospholipids to produce phosphatidylethanolamine. Recently, it has been discovered that they are also involved in the growth regulation of Arabidopsis thaliana. In our study we investigated expression of each Camelina sativa LPEAT isoform and their behavior in response to temperature changes. In order to conduct a more extensive biochemical evaluation we focused both on LPEAT enzymes present in microsomal fractions from C. sativa plant tissues, and on cloned CsLPEAT isoforms expressed in yeast system. Phylogenetic analyses revealed that CsLPEAT1c and CsLPEAT2c originated from Camelina hispida, whereas other isoforms originated from Camelina neglecta. The expression ratio of all CsLPEAT1 isoforms to all CsLPEAT2 isoforms was higher in seeds than in other tissues. The isoforms also displayed divergent substrate specificities in utilization of LPE; CsLPEAT1 preferred 18:1-LPE, whereas CsLPEAT2 preferred 18:2-LPE. Unlike CsLPEAT1, CsLPEAT2 isoforms were specific towards very-long-chain fatty acids. Above all, we discovered that temperature strongly regulates LPEATs activity and substrate specificity towards different acyl donors, making LPEATs sort of a sensor of external thermal changes. We observed the presented findings not only for LPEAT activity in plant-derived microsomal fractions, but also for yeast-expressed individual CsLPEAT isoforms.

Camelina sativa phosphatidylcholine:diacylglycerol cholinephosphotransferase-catalyzed interconversion does not discriminate between substrates.

Phosphatidylcholine:diacylglycerol cholinephosphotransferases (PDCT) regulate the fatty acid composition of seed oil (triacylglycerol, TAG) by interconversion of diacylglycerols (DAG) and phosphatidylcholine (PtdCho). PtdCho is the substrate for polyunsaturated fatty acid biosynthesis, as well as for a number of unusual fatty acids. By the action of PDCT, these fatty acids can be transferred into the DAG pool to be utilized in TAG biosynthesis by the action of acyl-CoA:DAG and phospholipid:diacylglycerol acyltransferases. Despite its importance in regulating seed oil composition, biochemical characterization of PDCT enzymes has been lacking. We characterized Camelina sativa PDCT in microsomal preparations of a yeast strain expressing Camelina PDCT and lacking the capacity of producing TAG. Camelina PDCT was specific for PtdCho and the sn-1,2 enantiomer of DAG and could not utilize ceramide. The interconversion reaches equilibrium within 15 min of incubation, indicating that only distinct pools of DAG and PtdCho were available for exchange. However, the pool sizes of DAG and PtdCho involved in the exchange were not fixed but increased with the amount of exogenous DAG or PtdCho added. Camelina PDCT showed about the same selectivity for di-oleoyl, di-linoleoyl, and di-linolenoyl species in both PtdCho and DAG substrates, suggesting that no unidirectional transfer of particular unsaturated substrates occurred. Camelina PDCT had a good activity with erucoyl-DAG as a substrate despite low erucic acid levels in PtdCho in plant species accumulating a high amount of this fatty acid in the seed oil.

Subcellular Localization of Acyl-CoA: Lysophosphatidylethanolamine Acyltransferases (LPEATs) and the Effects of Knocking-Out and Overexpression of Their Genes on Autophagy Markers Level and Life Span of A. thaliana.

Arabidopsis thaliana possesses two acyl-CoA:lysophosphatidylethanolamine acyltransferases, LPEAT1 and LPEAT2, which are encoded by At1g80950 and At2g45670 genes, respectively. Both single lpeat2 mutant and double lpeat1 lpeat2 mutant plants exhibit a variety of conspicuous phenotypes, including dwarfed growth. Confocal microscopic analysis of tobacco suspension-cultured cells transiently transformed with green fluorescent protein-tagged versions of LPEAT1 or LPEAT2 revealed that LPEAT1 is localized to the endoplasmic reticulum (ER), whereas LPEAT2 is localized to both Golgi and late endosomes. Considering that the primary product of the reaction catalyzed by LPEATs is phosphatidylethanolamine, which is known to be covalently conjugated with autophagy-related protein ATG8 during a key step of the formation of autophagosomes, we investigated the requirements for LPEATs to engage in autophagic activity in Arabidopsis. Knocking out of either or both LPEAT genes led to enhanced accumulation of the autophagic adaptor protein NBR1 and decreased levels of both ATG8a mRNA and total ATG8 protein. Moreover, we detected significantly fewer membrane objects in the vacuoles of lpeat1 lpeat2 double mutant mesophyll cells than in vacuoles of control plants. However, contrary to what has been reported on autophagy deficient plants, the lpeat mutants displayed a prolonged life span compared to wild type, including delayed senescence.

Phospholipid:Diacylglycerol Acyltransferase1 Overexpression Delays Senescence and Enhances Post-heat and Cold Exposure Fitness.

In an alternative pathway to acyl-CoA: diacylglycerol acyltransferase (DGAT)-mediated triacylglycerol (TAG) synthesis from diacylglycerol, phospholipid:diacylglycerol acyltransferase (PDAT) utilizes not acyl-CoA but an acyl group from sn-2 position of a phospholipid, to form TAG. The enzyme's activity in vitro matches DGAT's in a number of plant species, however its main function in plants (especially in vegetative tissue) is debatable. In the presented study, we cultivated PDAT1-overexpressing, pdat1 knockout and wild-type lines of Arabidopsis thaliana through their whole lifecycle. PDAT1 overexpression prolonged Arabidopsis lifespan in comparison to wild-type plants, whereas knocking out pdat1 accelerated the plant's senescence. After subjecting the 3-week old seedlings of the studied lines (grown in vitro) to 2-h heat stress (40°C) and then growing them for one more week in standard conditions, the difference in weight between wild-type and PDAT1-overexpressing lines increased in comparison to the difference between plants grown only in optimal conditions. In another experiment all lines exposed to 2-week cold stress experienced loss of pigment, except for PDAT1-overexpressing lines, which green rosettes additionally weighed 4 times more than wild-type. Our results indicate that plants depleted of PDAT1 are more susceptible to cold exposure, while PDAT1 overexpression grants plants a certain heat and cold resilience. Since it was shown, that lysophospholipids may be intertwined with stress response, we decided to also conduct in vitro assays of acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) and acylCoA:lysophosphatidylethanolamine acyltransferase (LPEAT) activity in microsomal fractions from the PDAT1-overexpressing Arabidopsis lines in standard conditions. The results show significant increase in LPEAT and LPCAT activity in comparison to wild-type plants. PDAT1-overexpressing lines' rosettes also present twice as high expression of LPCAT2 in comparison to control. The presented study shows how much heightened expression of PDAT1 augments plant condition after stress and extends its lifespan.

A predicted transmembrane region in plant diacylglycerol acyltransferase 2 regulates specificity toward very-long-chain acyl-CoAs.

Triacylglycerols are the main constituent of seed oil. The specific fatty acid composition of this oil is strongly impacted by the substrate specificities of acyltransferases involved in lipid synthesis, such as the integral membrane enzyme diacylglycerol acyltransferase (DGAT). Two forms of DGAT, DGAT1 and DGAT2, are thought to contribute to the formation of seed oil, and previous characterizations of various DGAT2 enzymes indicate that these often are associated with the incorporation of unusual fatty acids. However, the basis of DGAT2's acyl-donor specificity is not known because of the inherent challenges of predicting structural features of integral membrane enzymes. The recent characterization of DGAT2 enzymes from Brassica napus reveals that DGAT2 enzymes with similar amino acid sequences exhibit starkly contrasting acyl-donor specificities. Here we have designed and biochemically tested a range of chimeric enzymes, substituting parts of these B. napus DGAT2 enzymes with each other, allowing us to pinpoint a region that dramatically affects the specificity toward 22:1-CoA. It may thus be possible to redesign the acyl-donor specificity of DGAT2 enzymes, potentially altering the fatty acid composition of seed oil. Further, the characterization of a DGAT2 chimera between Arabidopsis and B. napus demonstrates that the specificity regulated by this region is transferrable across species. The identified region contains two predicted transmembrane helices that appear to reoccur in a wide range of plant DGAT2 orthologues, suggesting that it is a general feature of plant DGAT2 enzymes.

Editing of phosphatidic acid and phosphatidylethanolamine by acyl-CoA: lysophospholipid acyltransferases in developing Camelina sativa seeds.

MAIN CONCLUSIONS: The main source of polyunsaturated acyl-CoA in cytoplasmic acyl-CoA pool of Camelina sativa seeds are fatty acids derived from phosphatidylcholine followed by phosphatidic acid. Contribution of phosphatidylethanolamine is negligible. While phosphatidylethanolamine (PE) is the second most abundant phospholipid, phosphatidic acid (PA) only constitutes a small fraction of C. sativa seeds' polar lipids. In spite of this, the relative contribution of PA in providing fatty acids for the synthesis of acyl-CoA, supplying cytosolic acyl-CoA pool seems to be much higher than the contribution of PE. Our data indicate that up to 5% of fatty acids present in mature C. sativa seeds are first esterified with PA, in comparison to 2% first esterified with PE, before being transferred into acyl-CoA pool via backward reactions of either acyl-CoA:lysophosphatidic acid acyltransferases (CsLPAATs) or acyl-CoA:lysophoshatidylethanolamine acyltransferases (CsLPEATs). Those acyl-CoAs are later reused for lipid biosynthesis or remodelling. In the forward reactions both aforementioned acyltransferases display the highest activity at 30 °C. The spectrum of optimal pH differs for both enzymes with CsLPAATs most active between pH 7.5-9.0 and CsLPEATs between pH 9.0 to 10.0. Whereas addition of magnesium ions stimulates CsLPAATs, calcium and potassium ions inhibit them in concentrations of 0.05-2.0 mM. All three types of ions inhibit CsLPEATs activity. Both tested acyltransferases present the highest preferences towards 16:0-CoA and unsaturated 18-carbon acyl-CoAs in forward reactions. However, CsLPAATs preferentially utilise 18:1-CoA and CsLPEATs preferentially utilise 18:2-CoA while catalysing fatty acid remodelling of PA and PE, respectively.

Crambe hispanica Subsp. abyssinica Diacylglycerol Acyltransferase Specificities Towards Diacylglycerols and Acyl-CoA Reveal Combinatorial Effects That Greatly Affect Enzymatic Activity and Specificity.

Crambe is an oil crop suitable for industrial purposes due to the high content of erucic acid (22:1) in the seed oil. The final acylation of diacylglycerols (DAG) with acyl-CoA in the production of triacylglycerols (oil) is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. We identified eight forms of DGATs in crambe and characterized them in microsomal preparations of yeast expressing the enzymes using various acyl-CoAs and both di-6:0-DAG and long-chain DAG species as acyl acceptors. All DGATs accepted 22:1-CoA when using di-6:0-DAG as acyl acceptor. When di-22:1-DAG was the acyl acceptor, the DGAT1 type of enzyme utilized 22:1-CoA at a much-reduced rate compared to assays with sn-1-22:1-sn-2-18:1(oleoyl)-DAG, the most frequently available DAG precursor in crambe seeds. None of the DGAT2 enzymes was able to acylate di-22:1-DAG. Our results indicate that formation of trierucin by crambe DGATs is a limiting step for further increasing the levels of 22:1 in the previously developed transgenic crambe lines due to their poor abilities to acylate di-22:1-DAG. We also show that the acyl-CoA specificities and the enzymatic activities are highly influenced by the fatty acid composition of the DAG acyl acceptor. This finding implies that the use of artificial acyl acceptors (e.g. di-6:0-DAG) may not always reflect the actual acyl-CoA specificities of DGATs in planta. The relevance of the here reported pronounced specificities for specific DAG species exerted by DGAT enzymes is discussed in the context of the findings of DAG pools of distinct catalytic origin in triacylglycerol biosynthesis in the seed oil.

Isoforms of Acyl-CoA:Diacylglycerol Acyltransferase2 Differ Substantially in Their Specificities toward Erucic Acid.

In most oilseeds, two evolutionarily unrelated acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, are the main contributors to the acylation of diacylglycerols in the synthesis of triacylglycerol. DGAT1 and DGAT2 are both present in the important crop oilseed rape (Brassica napus), with each type having four isoforms. We studied the activities of DGAT isoforms during seed development in microsomal fractions from two oilseed rape cultivars: edible, low-erucic acid (22:1) MONOLIT and nonedible high-erucic acid MAPLUS. Whereas the specific activities of DGATs were similar with most of the tested acyl-CoA substrates in both cultivars, MAPLUS had 6- to 14-fold higher activity with 22:1-CoA than did MONOLIT. Thus, DGAT isoforms with different acyl-CoA specificities are differentially active in the two cultivars. We characterized the acyl-CoA specificities of all DGAT isoforms in oilseed rape in the microsomal fractions of yeast cells heterologously expressing these enzymes. All four DGAT1 isoforms showed similar and broad acyl-CoA specificities. However, DGAT2 isoforms had much narrower acyl-CoA specificities: two DGAT2 isoforms were highly active with 22:1-CoA, while the ability of the other two isoforms to use this substrate was impaired. These findings elucidate the importance, which a DGAT isoform with suitable acyl-CoA specificity may have, when aiming for high content of a particular fatty acid in plant triacylglycerol reservoirs.

Possible Role of Different Yeast and Plant Lysophospholipid:Acyl-CoA Acyltransferases (LPLATs) in Acyl Remodelling of Phospholipids.

Recent results have suggested that plant lysophosphatidylcholine:acyl-coenzyme A acyltransferases (LPCATs) can operate in reverse in vivo and thereby catalyse an acyl exchange between the acyl-coenzyme A (CoA) pool and the phosphatidylcholine. We have investigated the abilities of Arabidopsis AtLPCAT2, Arabidopsis lysophosphatidylethanolamine acyltransferase (LPEAT2), S. cerevisiae lysophospholipid acyltransferase (Ale1) and S. cerevisiae lysophosphatidic acid acyltransferase (SLC1) to acylate lysoPtdCho, lysoPtdEtn and lysoPtdOH and act reversibly on the products of the acylation; the PtdCho, PtdEtn and PtdOH. The tested LPLATs were expressed in an S. cervisiae ale1 strain and enzyme activities were assessed in assays using microsomal preparations of the different transformants. The results show that, despite high activity towards lysoPtdCho, lysoPtdEtn and lysoPtdOH by the ALE1, its capacities to operate reversibly on the products of the acylation were very low. Slc1 readily acylated lysoPtdOH, lysoPtdCho and lysoPtdEtn but showed no reversibility towards PtdCho, very little reversibility towards PtdEtn and very high reversibility towards PtdOH. LPEAT2 showed the highest levels of reversibility towards PtdCho and PtdEtn of all LPLATs tested but low ability to operate reversibly on PtdOH. AtLPCAT2 showed good reversible activity towards PtdCho and PtdEtn and very low reversibility towards PtdOH. Thus, it appears that some of the LPLATs have developed properties that, to a much higher degree than other LPLATs, promote the reverse reaction during the same assay conditions and with the same phospholipid. The results also show that the capacity of reversibility can be specific for a particular phospholipid, albeit the lysophospholipid derivatives of other phospholipids serve as good acyl acceptors for the forward reaction of the enzyme.

The utilization of the acyl-CoA and the involvement PDAT and DGAT in the biosynthesis of erucic acid-rich triacylglycerols in Crambe seed oil.

The triacylglycerol of Crambe abyssinica seeds consist of 95% very long chain (>18 carbon) fatty acids (86% erucic acid; 22:1∆13) in the sn-1 and sn-3 positions. This would suggest that C. abyssinica triacylglycerols are not formed by the action of the phospholipid:diacylglycerol acyltransferase (PDAT), but are rather the results of acyl-CoA:diacylglycerol acyltransferase (DGAT) activity. However, measurements of PDAT and DGAT activities in microsomal membranes showed that C. abyssinica has significant PDAT activity, corresponding to about 10% of the DGAT activity during periods of rapid seed oil accumulation. The specific activity of DGAT for erucoyl-CoA had doubled at 19 days after flowering compared to earlier developmental stages, and was, at that stage, the preferred acyl donor, whereas the activities for 16:0-CoA and 18:1-CoA remained constant. This indicates that an expression of an isoform of DGAT with high specificity for erucoyl-CoA is induced at the onset of rapid erucic acid and oil accumulation in the C. abyssinica seeds. Analysis of the composition of the acyl-CoA pool during different stages of seed development showed that the percentage of erucoyl groups in acyl-CoA was much higher than in complex lipids at all stages of seed development except in the desiccation phase. These results are in accordance with published results showing that the rate limiting step in erucic acid accumulation in C. abyssinica oil is the utilization of erucoyl-CoA by the acyltransferases in the glycerol-3-phosphate pathway.