Identification of bottlenecks in the accumulation of cyclic fatty acids in camelina seed oil

Abstract

Modified fatty acids (mFA) have diverse uses; for example, cyclopropane fatty acids (CPA) are feedstocks for producing coatings, lubricants, plastics and cosmetics. The expression of mFA-producing enzymes in crop and model plants generally results in lower levels of mFA accumulation than in their natural-occurring source plants. Thus, to further our understanding of metabolic bottlenecks that limit mFA accumulation, we generated transgenic Camelina sativa lines co-expressing Escherichia coli cyclopropane synthase (EcCPS) and Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT). In contrast to transgenic CPA-accumulating Arabidopsis, CPA accumulation in camelina caused only minor changes in seed weight, germination rate, oil accumulation and seedling development. CPA accumulated to much higher levels in membrane than storage lipids, comprising more than 60% of total fatty acid in both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) versus 26% in diacylglycerol (DAG) and 12% in triacylglycerol (TAG) indicating bottlenecks in the transfer of CPA from PC to DAG and from DAG to TAG. Upon co-expression of SfLPAT with EcCPS, di-CPA-PC increased by ~50% relative to lines expressing EcCPS alone with the di-CPA-PC primarily observed in the embryonic axis and mono-CPA-PC primarily in cotyledon tissue. EcCPS-SfLPAT lines revealed a redistribution of CPA from the sn-1 to sn-2 positions within PC and PE that was associated with a doubling of CPA accumulation in both DAG and TAG. The identification of metabolic bottlenecks in acyl transfer between site of synthesis (phospholipids) and deposition in storage oils (TAGs) lays the foundation for the optimizing CPA accumulation through directed engineering of oil synthesis in target crops.

Keywords: Camelina sativa; cyclopropane fatty acid; lipid metabolism; lipid synthesis; triacylglycerol; unusual fatty acid.

Figure 1

Cyclopropane fatty acid accumulation and transgene expression in T3 and T4 progeny upon the expression of indicated gene(s) in fad2/fae1 plants. (a) Cyclopropane fatty acid accumulation in mature seeds of T3 and T4 progeny compared to camelina fad2/fae1 (ctrl). Cyclopropane fatty acid (CPA) is expressed as a mol percentage of the total seed FA. Values represent the mean ± SD (n = 3 pooled sets of 100 seeds). (b) EcCPS and SfLPAT expression in seeds of transgenic plants. qRT‐PCR analysis of EcCPS (top) and SfLPAT (bottom) expression levels in seeds of camelina fad2/fae1 (ctrl) and three transgenic lines harbouring EcCPS or EcCPS and SfLPAT as indicated. The relative expression levels are reported relative to the expression of the Actin transcript. The values represent the mean ± SD of at least three biological replicates.

Figure 2

Seed weight and FA content in T4 camelina seeds. (a) Mean weight of transgenic seeds determined by five pooled sets of 100 seeds each. (b) Total fatty acid content in transgenic camelina seeds as a proportion of seed weight (panel A). Seed fatty acid content was quantified by GC of fatty acid methyl esters. Values represent means ± SD (n = 3). **Student's t‐test P < 0.01; *Student's t‐test, P < 0.05.

Figure 3

Cyclopropane fatty acid distribution in transgenic seeds. Cyclopropane fatty acid in polar lipids and TAG was expressed as a percentage of the total FA in (a). The percentage of CPA deposited in TAG was of total cyclopropane fatty acid (b). The values represent the mean and standard deviation of three replicates. **Student's t‐test P < 0.01; *Student's t‐test, P < 0.05.

Figure 4

MSI imaging of selected phosphatidylcholine (PC) molecular species in camelina seeds. (a), (f) and (k). Bright‐field cross‐sectional image. Abbreviations: co, cotyledons; ea, embryonic axis. (b–e), (g–j) and (l–o). Relative distribution profiles of selected PC molecular species. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5

MS analysis of CPA‐containing DAG. CPA‐containing DAGs were detected by neutral loss of CPA (m/z 313.2, [M+NH ₃]) with electrospray ionization source under positive ion mode by direct infusion. The values represent the mean and standard deviation of three replicates.

Figure 6

TAG profiles: Q1 scans showing M+NH4 intact TAG species. Abundant M+NH4 ions in the fad2/fae1 sample are 927.0 (18 : 3/18 : 1/20 : 1 or 56 : 5), 903.1 (tri‐18 : 1), 899.0 (18 : 3/18 : 1/18 : 1 or 54 : 5), 877.0 (16 : 0/18 : 1/18 : 1) and 873.0 (16 : 0/18 : 1/18 : 3 or 52 : 4). *TAG species with odd‐numbered fatty acid species (c19 : 0 primarily but may also contain c21 : 0 and c23 : 0 (elongation products of c19 : 0 DHSA).

Figure 7

Plant triacylglycerol biosynthesis network Acyl editing can provide PC‐modified FAs for de novo DAG/TAG synthesis. Substrate abbreviations: DAG, diacylglycerol; G3P, glycerol‐3‐phosphate; LPA, lyso‐phosphatidic acid; LPC, lyso‐phosphatidylcholine; mFA, modified FA; PA, phosphatidic acid; PC, phosphatidylcholine; TAG, triacylglycerol; FAS, Fatty acid synthase; CPT, CDP‐choline: DAG choline phosphotransferase; DGAT, acyl‐CoA:DAG acyltransferase; GPAT, acyl‐CoA:G3P acyltransferase; PLA, phospholipase A; LPC, phospholipase C; LPCAT, acyl‐CoA:LPC acyltransferase; PAP, PA phosphatase; PDAT, phospholipid:DAG acyltransferase.