Molecular Characterization of Two Lysophospholipid:acyl-CoA Acyltransferases Belonging to the MBOAT Family in Nicotiana benthamiana

Abstract

In the remodeling pathway for the synthesis of phosphatidylcholine (PC), acyl-CoA-dependent lysophosphatidylcholine (lysoPC) acyltransferase (LPCAT) catalyzes the reacylation of lysoPC. A number of genes encoding LPCATs have been cloned and characterized from several plants in recent years. Using Arabidopsis and other plant LPCAT sequences to screen the genome database of Nicotiana benthamiana, we identified two cDNAs encoding the putative tobacco LPCATs (NbLPCAT1 and NbLPCAT2). Both of them were predicted to encode a protein of 463 amino acids with high similarity to LPCATs from other plants. Protein sequence features such as the presence of at least eight putative transmembrane regions, four highly conserved signature motifs and several invariant residues indicate that NbLPCATs belong to the membrane bound O-acyltransferase family. Lysophospholipid acyltransferase activity of NbLPCATs was confirmed by testing lyso-platelet-activating factor (lysoPAF) sensitivity through heterologous expression of each full-length cDNA in a yeast mutant Y02431 (lca1△) disrupted in endogenous LPCAT enzyme activity. Analysis of fatty acid profiles of phospholipids from the NbLPCAT-expressing yeast mutant Y02431 cultures supplemented with polyunsaturated fatty acids suggested more incorporation of linoleic acid (18:2n6, LA) and α-linolenic acid (18:3n3, ALA) into PC compared to yeast mutant harbouring empty vector. In vitro enzymatic assay demonstrated that NbLPCAT1had high lysoPC acyltransferase activity with a clear preference for α-linolenoyl-CoA (18:3), while NbLPCAT2 showed a high lysophosphatidic acid (lysoPA) acyltransferase activity towards α-linolenoyl-CoA and a weak lysoPC acyltransferase activity. Tissue-specific expression analysis showed a ubiquitous expression of NbLPCAT1 and NbLPCAT2 in roots, stems, leaves, flowers and seeds, and a strong expression in developing flowers. This is the first report on the cloning and characterization of lysophospholipid acyltransferases from N. benthamiana.

Fig 1. Sequence alignment of NbLPCATs with the related LPCATs from higher plants.

The amino acid sequences of NbLPCATs were aligned, using the software Clustal X v1.83 with those of characterized LPCATs from B. napus and A. thaliana. The Jalview v2.8.2 program was used to highlight the homology between LPCAT protein sequences. Conserved motifs and the putative ER signal are boxed. Invariant residues are marked with black triangle stars.

Fig 2. Predicted transmembrane domains for N. benthamiana LPCAT sequences.

The TMHMM web tools of the Center for Biological Sequence Analysis, Technical University of Denmark TMHMM Server plot the probability of the ALDH sequence forming a membrane-spanning helix (0–1.0 on the y-axis). The transmembrane regions are shown in red, whereas regions of those sequences predicted to be located inside or outside the membrane are shown in blue and pink, respectively.

Fig 3. Phylogenetic relationship among deduced amino acid sequences of NbLPCAT1, NbLPCAT2 and LPCATs from other organisms.

The tree was constructed according to the Neighbor-Joining algorithm. The percentages of bootstrap support, derived from 1000 replicates, are shown at branch points. AtLPCAT1 (Arabidopsis thaliana, GenBank accession number F4IDU4), AtLPCAT2 (Arabidopsis thaliana, GenBank accession number Q9CAN8), HsLPCAT1 (Homo sapiens, GenBank accession number BAE94688), HsLPCAT2 (Homo sapiens, GenBank accession number BAF47696), HsLPCAT3 (Homo sapiens, GenBank accession number NP_005759), HsLPCAT4 (Homo sapiens, GenBank accession number NP_620154), MmLPCAT1 (Mus musculus, GenBank accession number BAE94687), MmLPCAT2 (Mus musculus, GenBank accession number BAF47695), MmLPCAT3 (Mus musculus, GenBank accession number BAG12120), MmLPCAT4 (Mus musculus, GenBank accession number BAG12122), OsLPCAT (Oryza sativa, GenBank accession number Q6EP89), PtLPCAT1 (Populus trichocarpa, GenBank accession number B9GW66), PtLPCAT2 (Populus trichocarpa, GenBank accession number B9GKN7), RcLPCAT (Ricinus communis, GenBank accession number B9RC25), ScALE1 (Saccharomyces cerevisiae, GenBank accession number NP_014818).

Fig 4. LysoPAF sensitivity test for yeast mutant Y02431 (lca1△) expressing NbLPCAT or harbouring the pESC-Ura empty vector.

Yeast cells grown overnight and induced for the expression of NbLPCAT for 24 h were suspended in sterile distilled water and adjusted to an OD₆₀₀ of 2, 1, 0.5, and 0.1. The resulting 2 μL yeast solution was spotted on a SC-Ura agar plate containing 5, 10, 25, and 30 μg/mL lysoPAF. The growth of yeast cells was evaluated after 72 h at 28°C.

Fig 5. Distribution of exogenously supplemented ALA (A) or LA (B) in phospholipids from cultures of yeast mutant Y02431 (lca1△) expressing NbLPCAT or harbouring the pESC-Ura empty vector.

Cultures were supplemented with 500 μM PUFA in the presence of 1% (w/v) Tween 40. The value was expressed as the amount of PUFA (mg, isolated from PC, PE, or PI) per g dry cell weight (mg/g). The data represent the mean±S.E. of three measurements. ALA, α-linolenic acid (18:3n3); LA, linoleic acid (18:2n6); PUFA, polyunsaturated fatty acid.

Fig 6. LPAAT activity (sn-1-18:1-LPA and¹⁴C-labeled 18:1-CoA as substrates) of microsomal preparation from yeast overexpressed with empty plasmid (control), NbLPCAT2 and NbLPCAT1.

Fig 7. LPCAT activity (sn-1-18:1-LPC and¹⁴C-labeled 18:1-CoA as substrates) of microsomal preparation from yeast overexpressed with NbLPCAT1 and NbLPCAT2.

Fig 8. LPCAT activity of microsomal preparation from yeast overexpressed with NbLPCAT1 and NbLPCAT2 towards different acyl-CoAs.

A, expressed as pmol of de novo synthesised [¹⁴C] PC/mg microsomal protein/min. B, expressed as % of activity towards 18:1-CoA.

Fig 9. LPAAT activity of microsomal preparation from yeast overexpressed with empty plasmid (control) and with NbLPCAT2 towards different acyl-CoAs.

A, expressed as pmol of de novo synthesised [¹⁴C] PA/mg microsomal protein/min. B, expressed as % of activity towards 18:1-CoA.

Fig 10. Tissue-specific expression patterns of NbLPCAT genes in different tissues of 13-week-old (A, B), 16-week-old (C, D), and 19-week-old (E, F), tissue-cultivated N. benthamiana.

The expression levels of NbLPCATs were analyzed by the real-time quantitative RT-PCR method. X-axis indicates different tissues and y-axis indicates relative expression levels. Gene encoding elongation factor 1α signal was used as the reference gene. Error bars represent standard deviations of mean value from three technical replicates. W, week.

Fig 11. Expression patterns of NbLPCAT genes in roots (A,B), stems (C,D), flowers and seeds (E,F) during different developing periods of tissue-cultivated N. benthamiana.

The expression levels of NbLPCATs were analyzed by the real-time quantitative RT-PCR method. X-axis indicates different developing periods and y-axis indicates relative expression levels. Gene encoding elongation factor 1α signal was used as the reference gene. Error bars represent standard deviations of mean value from three technical replicates. W, week.