Gene Isolation And Enzymatic Study Of Lysophosphatidylcholine Acyltransferase In Brassica Napus

Phosphatidylcholine (PC) performs not only as a structural and functional component of cellular membranes, but also as a significant fatty acyl donor for neutral lipid biosynthesis and an essential substrate for modification of fatty acids (e.g. desaturation and hydroxylation) in plant cells. The sn-2acyl moiety of PC undergoes continuous deacylation and reacylation cyclic processes which causes the rapid turnover and remodeling of PC and is known as Lands cycle. Lysophosphatidylcholine acyltransferase (LPCAT) catalyzing the acylation of lysophosphatidylcholine (LPC) to produce PC is a key enzyme in the Lands cycle. Through modulating the composition of acyl moieties at the sn-2position of PC, LPCAT can affect the fatty acid profile and properties of PC as well as the acyl exchange between PC and acyl-CoA pool. It has been shown that acyl exchange involving LPCAT dominates acyl flux during triacylglycerol (TAG) synthesis in seeds. Understanding the role of LPCAT in acyl exchange is important for engineering the accumulation of fatty acids, especially the unusual fatty acids, in transgenic plants. In this study, we devised a screening approach to identify LPCAT genes through functionally complementing the yeast mutant deficient in LPCAT activity, and successfully isolated the LPCAT genes from Arabidopsis and Brassica napus. To further study the B. napus LPCATs (BnLPCATs) isolated, the expression profile of BnLPCATs and the enzymatic activity of BnLPCAT proteins were investigated. The main results are summarised as follows:1. Based on the previous report that the yeast lca1Δ mutant is hypersensitive to lyso-PAF, an ester-linked analogue of LPC, we complemented the lea1Δ mutant with an Arabidopsis seedling cDNA library and obtained two previously reported lysophospholipid acyltransferase (LPLAT) genes, AtLPLATl (Atlg12640) and AtLPLAT2(Atlg63050). Although many other LPLAT genes have been confirmed in Arabidopsis, our results suggested that AtLPLATl and AtLPLAT2are the only two Arabidopsis genes that rescue the deficiency of lea1Δ in the presence of lyso-PAF, and the complementation selection method was stringent and efficient.2. To further identify the LPCAT genes in B. napus, a cDNA library was generated with RNA isolated from B. napus cv Hero seeds at different developmental stages, then was used to complement the lea1Δ mutant. Three BnLPCAT genes, named BnLPCATl-1, BnLPCAT1-2and BnLPCAT1respectively, were obtained after the screening. Sequence alignment revealed that BnLPCATl-1and BnLPCATl-2are orthologs of AtLPLATl while BnLPCAT2is orthologous to AtLPLAT2. In the complementation assay, we found cDNAs derived from BnLPCATl-2showed several single nucleotide differences. However, sequence differences were not observed in cDNAs of BnLPCAT1-1or BnLPCAT2.3. The deduced amino acid sequences of BnLPCATs showed that BnLPCATs belong to membrane-bound O-acyltransferase (MBOAT) family and possess three conserved MBOAT motifs (motifs A-C) that are essential for LPLAT activities. In motif B, we found a highly conserved histine residue, which is suggested to be a putative active-site residue. A hylogenetic analysis of BnLPCATs and their homologous proteins from other plant species revealed that the homologs of BnLPCATs are widely distributed among dicots, monocots, gymnosperm, bryophyte and algae.4. Enzymatic assays were conducted to measure the activity of protein BnLPCAT1-1and BnLPCAT2in the presence of different lysophospholipids or acyl-CoAs. Of lysophospholipids tested, both BnLPCAT1-1and BnLPCAT2showed higher activities with LPC as acyl acceptor which confirmed that BnLPCAT1-1and BnLPCAT2are lysophosphatidylcholine acyltransferases. Moreover, both had an approximately equal preference for16:0-LPC,18:0-LPC and18:1-LPC, suggesting that BnLPCAT1-1and BnLPCAT2do not discriminate sn-1acyl species of those LPCs. In the present of all tested acyl-CoAs, both BnLPCAT1-1and BnLPCAT2preferred unsaturated fatty acyl-CoAs to saturated fatty acyl-CoAs. Both enzymes showed limited activities with22:1-CoA, which suggested that this very-long-chain unsaturated fatty acyl-CoA is not an efficient substrate for the two BnLPCATs. In all enzymatic assays, BnLPCAT2constantly exhibited a higher activity when compared with BnLPCAT1-1.5. Experiments of quantitative real-time RT-PCR were performed to investigate the expression patterns of BnLPCAT1-1and BnLPCAT2in different tissues. The transcripts of BnLPCAT1-1and BnLPCAT2were widely distributed in all tissues tested, and BnLPCAT1-1was expressed higher in tissues examined than BnLPCAT2. BnLPCAT1-1 showed its highest expression level in1-week-old seedlings, followed by leaves, buds and flowers, while the highest transcript of BnLPCAT2was found in buds.