Expression of a Lychee PHOSPHATIDYLCHOLINE:DIACYLGLYCEROL CHOLINEPHOSPHOTRANSFERASE with an Escherichia coli CYCLOPROPANE SYNTHASE Enhances Cyclopropane Fatty Acid Accumulation in Camelina Seeds

Abstract

Cyclopropane fatty acids (CPAs) are useful feedstocks for biofuels and bioproducts such as lubricants and biodiesel. Our goal is to identify factors that can facilitate the accumulation of CPA in seed triacylglycerol (TAG) storage oil. We hypothesized that the poor metabolism of CPA through the TAG biosynthetic network could be overcome by the addition of enzymes from species that naturally accumulate CPA in their seed oil, such as lychee (Litchi chinensis), which contains approximately 40% CPA in TAG. Our previous work on engineering CPA accumulation in crop and model plants identified a metabolic bottleneck between phosphatidylcholine (PC), the site of CPA biosynthesis, diacylglycerol (DAG), and TAG. Here, we report the cloning and heterologous expression in camelina (Camelina sativa) of a lychee PHOSPHATIDYLCHOLINE:DIACYLGLYCEROL CHOLINEPHOSPHOTRANSFERASE (PDCT), which encodes the enzyme that catalyzes the transfer of the phosphocholine headgroup from PC to DAG. Camelina lines coexpressing LcPDCT and Escherichia coli CYCLOPROPANE SYNTHASE (EcCPS) showed up to a 50% increase of CPA in mature seed, relative to the EcCPS background. Stereospecific lipid compositional analysis showed that the expression of LcPDCT strongly reduced the level of C18:1 substrate at PC-sn-1 and PC-sn-2 (i.e. the sites of CPA synthesis), while the levels of CPA increased in PC-sn-2, DAG-sn-1 and DAG-sn-2, and both sn-1/3 and sn-2 positions in TAG. Taken together, these data suggest that the addition of PDCT facilitates more efficient movement of CPA from PC to DAG and establishes LcPDCT as a useful factor to combine with others to enhance CPA accumulation in plant seed oil.

Figure 1.

Plant CPA biosynthesis network. Acyl editing can provide PC-mFAs for DAG/TAG synthesis. Substrate abbreviations not defined in the text are as follows: CPT, CDP-choline:DAG choline phosphotransferase; DGAT, acyl-CoA:DAG acyltransferase; FAS, fatty acid synthase; G3P, glycerol-3-phosphate; GPAT, acyl-CoA:G3P acyltransferase; LPA, lyso-phosphatidic acid; LPC, lyso-phosphatidylcholine; LPCAT, acyl-CoA:LPC acyltransferase; PA, phosphatidic acid; PAP, PA phosphatase; PDAT, phospholipid:DAG acyltransferase; PLA, phospholipase A; PLC, phospholipase C; PLD, phospholipase D.

Figure 2.

Comparison of PDCT homologs from land plants. A, Alignment of protein sequences of LcPDCT, RcROD1, AtROD1, LuPDCT1, and LuPDCT2. Sequences were aligned using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). Identical amino acids are marked with asterisks, and conserved substitutions are marked with colons. The five highly conserved residues in the C2 and C3 domains of the lipid phosphatase/phosphotransferase family are highlighted in yellow and blue, respectively. B, Phylogenetic tree showing the evolutionary relationship between PDCT proteins from different plant species, as follows: Arabidopsis (AtRod1; At3g15820), Glycine max (XP_003531718.1), lychee (LcPDCT; KU926346.1 and AXM43881.1), Ricinus communis (RcRod1; XP_002517643.1), Momordica charantia (XP_022153394.1), Brassica napus (CDY30114.1), Linum usitatissimum (LuPDCT1 [AHE80679.1] and LuPDCT2 [AHE80680.1]), Vitis vinifera (XP_002266079.2), Populus trichocarpa (XP_006385307.1 and XP_002299689.2), Gossypium hirsutum (isoform 2 [XP_016725742.1] and isoform 1 [XP_016725741.1], camelina (XP_010465574.1), Medicago truncatula (XP_003604371.1), Theobroma cacao (EOY25833.1), Cucumis sativus (XP_004152194.1), Oryza sativa (XP_015644243.1), Sorghum bicolor (XP_021306179.1), Zea mays (AQK82308.1), and Brachypodium distachyon (XP_003563650.1).

Figure 3.

CPA accumulation in T2 and T3 progeny upon the expression of EcCPS in fad2/fae1 plants. CPA accumulation is shown in T2 (A) and T3 (B) progeny. Fatty acid methyl esters (FAMEs) were analyzed by gas chromatography-mass spectrometry; CPA (CFA) is presented as a percentage of the total fatty acids. Values represent means ± sd (n = 3).

Figure 4.

CPA accumulation and transgene expression in transgenic plants. A, CPA in mature seeds. CPA is expressed as a weight percentage of the total seed fatty acids. Values represent means ± sd (n = 3 pooled sets of 100 seeds). B and C, EcCPS and LcPDCT expression in seeds of transgenic plants. RT-qPCR analysis is shown for EcCPS (B) and LcPDCT (C) expression levels in seeds of camelina fad2/fae1, EcCPS transgenic seeds, and three transgenic lines harboring EcCPS and LcPDCT, as indicated. The relative expression levels are reported relative to the expression of the Actin transcript. RT-qPCR values are presented as percentages of untransformed fad2/fae1 as described in “Materials and Methods.” All data are means ± sd (n = 3). Asterisks represent values found to differ significantly (*, P < 0.05; **, P < 0.01) from controls using mean crossing point deviation analysis computed by the relative expression software tool (REST) algorithm (Pfaffl et al., 2002).

Figure 5.

Seed weight and fatty acid content in transgenic camelina seeds. A, Mean weight of transgenic seeds determined by three pooled sets of 100 seeds each. B, Oil content in transgenic camelina seeds as a proportion of dry seed weight. Seed oil content, represented by total acyl lipids, was quantified by gas chromatography of FAMEs. Values represent means ± sd (n = 3). **, P < 0.01 by Student’s t test.

Figure 6.

CPA distribution in transgenic seeds. CPA in TAG (A) and polar lipids (B) was expressed as a weight percentage of the total fatty acids. Values represent means ± sd of three biological replicates. **, P < 0.01 and *, P < 0.05 by Student’s t test.