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.

Arabidopsis (Arabidopsis thaliana) contains two enzymes (encoded by the At1g80950 and At2g45670 genes) preferentially acylating lysophosphatidylethanolamine (LPE) with acyl-coenzyme A (CoA), designated LYSOPHOSPHATIDYLETHANOLAMINE ACYLTRANSFERASE1 (LPEAT1) and LPEAT2. The transfer DNA insertion mutant lpeat2 and the double mutant lpeat1 lpeat2 showed impaired growth, smaller leaves, shorter roots, less seed setting, and reduced lipid content per fresh weight in roots and seeds and large increases in LPE and lysophosphatidylcholine (LPC) contents in leaves. Microsomal preparations from leaves of these mutants showed around 70% decrease in acylation activity of LPE with 16:0-CoA compared with wild-type membranes, whereas the acylation with 18:1-CoA was much less affected, demonstrating that other lysophospholipid acyltransferases than the two LPEATs could acylate LPE. The above-mentioned effects were less pronounced in the single lpeat1 mutant. Overexpression of either LPEAT1 or LPEAT2 under the control of the 35S promotor led to morphological changes opposite to what was seen in the transfer DNA mutants. Acyl specificity studies showed that LPEAT1 utilized 16:0-CoA at the highest rate of 11 tested acyl-CoAs, whereas LPEAT2 utilized 20:0-CoA as the best acyl donor. Both LPEATs could acylate either sn position of ether analogs of LPC. The data show that the activities of LPEAT1 and LPEAT2 are, in a complementary way, involved in growth regulation in Arabidopsis. It is shown that LPEAT activity (especially LPEAT2) is essential for maintaining adequate levels of phosphatidylethanolamine, LPE, and LPC in the cells.

Main conclusion The transfer of polyunsaturated fatty acids from phosphatidylcholine to other lipids involves several enzymes. In Camelina sativa seeds, acyl-CoA:lysophosphatidylcholine acyltransferases could be one of the most important players in this process. Abstract The transfer of polyunsaturated fatty acids from the location of their synthesis (phosphatidylcholine) to other lipids, e.g., triacylglycerol, remains insufficiently understood. Several enzymes could be involved in this process. One of these enzymes is acyl-CoA:lysophosphatidylcholine acyltransferases (LPCATs). In Camelina sativa seeds, LPCATs could be one of the most important players in this process. Our data clearly indicate that the CsLPCATs present in developing seeds have the potential to transfer almost all polyunsaturated fatty acids synthesised on phosphatidylcholine to the acyl-CoA pool. CsLPCAT activity is the highest at 30 °C, and the enzymes operate well at a pH of 7.0–11.0, with the best activity at a pH of 9.0. The activity of CsLPCATs was inhibited by calcium and magnesium ions at a concentration of 0.05–2 mM. In the forward reaction, CsLPCATs preferentially utilise 18:2-CoA; however, other C18 unsaturated fatty acids are also well accepted. In the backward reactions, there is no clear discrimination between the C18 unsaturated fatty acids utilised by the enzymes for phosphatidylcholine remodelling. The activity of CsLPCATs does not differ much between the stages of seed development.

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.

Yellow lupine is a great model for abscission-related research given that excessive flower abortion reduces its yield. It has been previously shown that the EPIP peptide, a fragment of LlIDL (INFLORESCENCE DEFICIENT IN ABSCISSION) amino-acid sequence, is a sufficient molecule to induce flower abortion, however, the question remains: What are the exact changes evoked by this peptide locally in abscission zone (AZ) cells? Therefore, we used EPIP peptide to monitor specific modifications accompanied by early steps of flower abscission directly in the AZ. EPIP stimulates the downstream elements of the pathway—HAESA and MITOGEN-ACTIVATED PROTEIN KINASE6 and induces cellular symptoms indicating AZ activation. The EPIP treatment disrupts redox homeostasis, involving the accumulation of H₂O₂ and upregulation of the enzymatic antioxidant system including superoxide dismutase, catalase, and ascorbate peroxidase. A weakening of the cell wall structure in response to EPIP is reflected by pectin demethylation, while a changing pattern of fatty acids and acyl lipids composition suggests a modification of lipid metabolism. Notably, the formation of a signaling molecule—phosphatidic acid is induced locally in EPIP-treated AZ. Collectively, all these changes indicate the switching of several metabolic and signaling pathways directly in the AZ in response to EPIP, which inevitably leads to flower abscission.

The search of the Phaeodactylum tricornutum genome database revealed the existence of six genes potentially encoding lysophospholipid acyltransferases. One of these genes, Phatr3_J20460, after introduction to yeast ale1 mutant disrupted in the LPCAT gene, produced a very active acyl-CoA:lysophosphatidylcholine (LPCAT) enzyme. Using in vitro assays applying different radioactive and non-radioactive substrates and microsomal fractions from such yeast, we have characterized the biochemical properties and substrate specificities of this PtLPCAT1. We have found that the substrate specificity of this enzyme indicates that it can completely supply phosphatidylcholine (PC) with all fatty acids connected with a biosynthetic pathway of very long-chain polyunsaturated fatty acids (VLC-PUFAs) used further for the desaturation process. Additionally, we have shown that biochemical properties of the PtLPCAT1 in comparison to plant LPCATs are in some cases similar (such as the dependency of its activity on pH value), differ moderately (such as in response to temperature changes), or express completely different properties (such as in reaction to calcium and magnesium ions or toward some acyl-CoA with 20C polyunsaturated fatty acids). Moreover, the obtained results suggest that cloned “Phatr3_J20460” gene can be useful in oilseeds plant engineering toward efficient production of VLC-PUFA as LPCAT it encodes can (contrary to plant LPCATs) introduce 20:4-CoA (n-3) to PC for further desaturation to 20:5 (EPA, eicosapentaenoic acid).