Identification of Brassica napus lysophosphatidylcholine acyltransferase genes through yeast functional screening.
Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), which acylates lysophosphatidylcholine (LPC) to produce phosphatidylcholine (PC), is a key enzyme in the Lands cycle. There is evidence that acyl exchange involving LPCAT is a prevailing metabolic process during triacylglycerol (TAG) synthesis in seeds. In this study, by complementing the yeast lca1Δ mutant deficient in LPCAT activity with an Arabidopsis seedling cDNA library, it was found that the previously reported lysophospholipid acyltransferases (LPLATs), At1g12640 and At1g63050, were the only two acyltransferase genes that restored hyposensitivity of the lca1Δ mutant to lyso-platelet-activating factor (lyso-PAF). A developing seed cDNA library from Brassica napus L. cv Hero was constructed to further explore the heterologous yeast complementation approach. Three B. napusLPCAT homologs were identified, of which BnLPCAT1-1 and BnLPCAT1-2 are orthologous to ArabidopsisAtLPLAT1 (At1g12640) while BnLPCAT2 is an ortholog of AtLPLAT2 (At1g63050). The proteins encoded by BnLPCAT1-1 and BnLPCAT2 were chosen for further study. Enzymatic assays demonstrated that both proteins exhibited a substrate preference for LPCs and unsaturated fatty acyl-CoAs. In addition to the enzymatic properties of plant lysophosphatidylcholine acyltransferases uncovered in this study, this report describes a useful technique that facilitates subsequent analyses into the role of LPCATs in PC turnover and seed oil biosynthesis.
National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, No. 1, Shizi Shan Street, Wuhan, Hubei 430070, China.
Qian
Q
Zheng
John Qiang
JQ
Li
Michael
M
Kazachkov
Kede
K
Liu
Jitao
J
Zou
D008243:1-Acylglycerophosphocholine O-Acyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D055162:Biocatalysis; D001681:Biological Assay / Q000379:methods*; D029688:Brassica napus / Q000201:enzymology*; D015723:Gene Library; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
D010713:Phosphatidylcholines; D008243:1-Acylglycerophosphocholine O-Acyltransferase