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. cervisiaeale1 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.

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.

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.

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.

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.

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.