Field production, purification and analysis of high-oleic acetyl-triacylglycerols from transgenic Camelina sativa

Industrial Crops and Products 65 (2015) 259–268

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Field production, purification and analysis of high-oleic acetyl-triacylglycerols from transgenic Camelina sativa Jinjie Liu a,b,1 , Henrik Tjellström a,b,1 , Kathleen McGlew a , Vincent Shaw a , Adam Rice a , Jeffrey Simpson a,b , Dylan Kosma a , Wei Ma a,b , Weili Yang a , Merissa Strawsine a , Edgar Cahoon c , Timothy P. Durrett d , John Ohlrogge a,b,∗ a

Department of Plant Biology, Michigan State University, East Lansing, MI, USA Great Lakes Bioenergy Research Center, East Lansing, MI, USA c Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE 68583, USA d Department of Biochemistry and Molecular Biophysics, Kansas State University, KS, USA b

a r t i c l e

i n f o

Article history: Received 28 August 2014 Received in revised form 27 October 2014 Accepted 10 November 2014 Keywords: Camelina Metabolic engineering Emulsifier Acetylglyceride ACETEM

a b s t r a c t A diacylglycerol acetyltransferase, EaDAcT, from Euonymus alatus, synthesizes sn-3 acetyl triacylglycerols (acetyl-TAG) when expressed in Arabidopsis, Camelina and soybean. Compared to most vegetable oils, acetyl-TAGs have reduced viscosity and improved cold temperature properties that confer advantages in applications as biodegradable lubricants, food emulsifiers, plasticizers, and ‘drop-in’ fuels for some diesel engines. A high-oleic Camelina line was engineered to express the EaDAcT gene in order to produce acetyl-TAG oils with fatty acid compositions and physiochemical properties complementary to wild-type acetyl-TAG. The accumulation of acetyl-TAGs at 70 mol% of seed TAG in field-grown high-oleic Camelina had minor or no effect on seed weight, oil content, harvest index and seed yield. The total moles of TAG increased up to 27% reflecting the ability to synthesize more acetyl-TAG from the same supply of long-chain fatty acid. Acetyl-TAG could be separated from long-chain TAG by silica column or by reverse phase chromatography. The predominant acetyl-TAG molecular species produced in high-oleic Camelina was acetyl-dioleoyl-glycerol. The crystallization temperature of high-oleic acetyl-TAG (by differential scanning calorimetry at 1.0 ◦ C/min) was reduced by 30 ◦ C compared to control TAG. The viscosity of higholeic acetyl-TAG was 27% lower than TAG from the high-oleic control and the caloric content was reduced by 5%. Field production of T4 and T5 transgenic plants yielded over 250 kg seeds for oil extraction and analysis. © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction Seed oils from nearly all plant species contain three acyl chains esterified to the three hydroxyl groups of glycerol. In major oilseed crops, five fatty acids (16:0, 18:0, 18:1, 18:2, and 18:3) predominate, but within the plant kingdom, over 200 ‘unusual’ oils occur in seeds, of which most are characterized by the presence of modified or uncommon fatty acids (Jaworski and Cahoon, 2003; Singh

Abbreviations: DAG, diacylglycerol; DGAT1, diacylglycerol acyltransferase1; DW, dry weight; EaDAcT, Euonymus alatus diacylglycerol acetyl transferase; LcTAG, long chain triacylglycerol; PDAT1, phosphatidylcholine:diacylglycerol acyltransferase1. ∗ Corresponding author at: Department of Plant Biology, Michigan State University, East Lansing, MI, USA. Tel.: +1 517 353 0611. E-mail address: [email protected] (J. Ohlrogge). 1 These authors contributed equally to this work.

et al., 2005). These modifications include atypical chain length (C8–C22), unusual double bond positions, and addition of functional groups (e.g. hydroxyl, epoxy, etc.). Some of these modified structures have added-value properties that are currently used in a range of industrial applications (Gunstone, 1998). 3-acetyl1,2-diacyl-sn-glycerols (acetyl-TAG) possess a particularly striking variation where the long-chain fatty acid at the sn-3 position of TAG is replaced by a two-carbon acetyl group (Fig. 1). Acetyl-TAG was discovered as a major component of oil from the seeds of Euonymus alatus (burning bush) and other species of Celastraceae and Lardizabalaceae (Kleiman et al., 1966, 1967; Miller et al., 1974). In addition, up to 36% of neutral lipids of the insect Eurosta solidaginis occur in the form of acetyl-TAG (Marshall et al., 2014). The enzyme that synthesizes acetyl-TAG, E. alatus diacylglycerol acetyl transferase (EaDAcT), was identified by analysis of RNASeq data from developing seeds of E. alatus (Durrett et al., 2010). Its activity was demonstrated by the synthesis of acetyl-TAG in

http://dx.doi.org/10.1016/j.indcrop.2014.11.019 0926-6690/© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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of isolation of acetyl-TAG are described and the viscosity, thermotropic phase transitions, molecular species, caloric content and oxidative stability of acetyl-TAG are reported. 2. Materials and methods 2.1. Plant transformation vectors and generation of transgenic plants

Fig. 1. Comparison of typical plant triacylglycerol structure with acetyltriacylglycerol. Camelina sativa plants engineered in this study produce 3-acetyl1,2-diacyl-sn-glycerol in which an acetyl group replaces a long chain fatty acid at the sn-3 position.

both yeast and in Arabidopsis seeds engineered to express the EaDAcT gene. We recently reported that over-expression of EaDAcT in Camelina resulted in up to 85 mol% acetyl-TAG in the seed oil (Liu et al., 2014), representing the highest accumulation of an unusual oil achieved so far in transgenic plants. This contrasts with many previous attempts to engineer unusual plant oils, which have achieved comparatively low levels of accumulation of the desired end product (Singh et al., 2005; Vanhercke et al., 2013; Dyer et al., 2008). The substitution of an acetyl group for a long acyl chain at the sn-3 position of TAG confers changes in the physical and chemical properties of the oil, and these in turn open up a range of possible applications. For example, acetyl-TAGs have lower viscosity and improved low temperature properties compared to oils from the major commodity oilseeds. These properties may enable direct ‘drop-in’ use of acetyl-TAG as a biofuel in some diesel engines. Vegetable oils also have a number of advantages over petrochemical-based lubricants including biodegradability, low toxicity, and excellent lubricity (Cermak et al., 2013). Their high flash and fire points provide added safety where fire or explosions are a possibility. The lower viscosity and cold-temperature properties of acetyl-TAG may provide benefits over other vegetable oils, particularly in low-temperature applications. In addition, structures similar to acetyl-TAG (e.g. acetylated mono- and diglycerides, ACETEM) are currently used in the food industry as emulsifiers (Gaupp and Adams, 2004) and as plasticizers for food packaging materials (Lundsgaard, 2010). As an initial step toward development of a novel oilseed crop that produces acetyl-TAG oil, Camelina sativa was selected as a transgenic host plant. Camelina is grown in much of the world and has drawn increasing interest because of its minimal requirements for irrigation and fertilizer and its short life cycle (Putnam et al., 1993; Zubr, 1997). In addition, Camelina can be readily transformed and several modifications in oil compositions have already been achieved in transgenic lines (Snapp et al., 2014; Nguyen et al., 2013; Iskandarov et al., 2014). In this study we report the engineering of acetyl-TAG in a high-oleic line of Camelina. A goal of this research was to evaluate and compare acetyl-TAG production and field performance of high-oleic and wild-type Camelina lines expressing EaDAcT. Engineering acetyl-TAG in high-oleic Camelina provided an opportunity to produce oil with greater oxidative stability, and other physical properties that can complement acetyl-TAG oils produced in the wild-type background. The field production presented here provided multi-liter quantities of oils suitable for testing in a range of applications and with different fatty acid compositions. Methods

Constructs for the seed-specific expression of EaDAcT and for EaDAcT together with RNAi of DGAT1 and/or RNAi of PDAT1 were assembled in a T-DNA binary vector (pBinGlyRed3: http://www.camelinagene.org). The orientation of transgenes and promoters used for EaDAcT expression and for RNAi constructs are summarized schematically in Fig. S1. The sequences for RNAi suppression contained the two arms of a reverse complement sequence from the Camelina DGAT1 and PDAT1 genes interrupted by the pdk intron from pHANNIBAL (Wesley et al., 2001). In order to select transformants from T1 seeds and to easily monitor segregation, the vector included dsRed (35S promoter) as selection marker (Jach et al., 2001). High-oleic C. sativa (Celine background) (Nguyen et al., 2013) was transformed via floral dip vacuum infiltration using Agrobacterium tumefaciens (Lu and Kang, 2008). Positive transformants were selected based on expression of dsRED fluorescence in T1 seeds. Seeds were examined for red fluorescence under a green light (Night Sea, Dual FP DFP-1) and red filter (Vivitar RED 52 mm, Japan). Camelina plants were cultivated in growth chambers, with 16/8 h, light (200–250 ␮E m−2 s−1 )/dark and at 25/18 ◦ C. To assess acetyl-TAG production in the different transgenic events, T2 seeds from each transformation event were collected from individual plants cultivated in growth chambers. Based on dsRED-fluorescence screening, null seeds were removed and oil was extracted (Section 2.4) from 20 transgenic seeds from each T2 plant. The approximate proportions of acetyl-TAG and TAG in the oil were analyzed by thin layer chromatography (TLC). Visual examination of primuline-stained TLC plates allowed the identification of transgenic plants producing high levels of acetyl-TAG. The acetyl-TAG and lcTAG bands from 2 to 4 T2 plants (per construct) with high acetyl-TAG/lcTAG ratios were recovered by scraping and transmethylated directly using a modified acid-catalyzed method (Li et al., 2006). Care was taken to minimize oxidation of polyunsaturated fatty acids by adding 0.2% butylated hydroxytoluene for the transmethylation reaction. Fatty acid methyl esters were analyzed by GC-FID on a DB23 column to obtain quantitative measures of acetyl-TAG and lcTAG and their fatty acid compositions (Li et al., 2006). 2.2. Camelina seed amplification Seeds of transgenic Camelina lines harvested from growth chambers were planted in the greenhouse for two successive generations to provide enough seeds for field tests and oil production. Seeds harvested from the first greenhouse cycle were examined for dsRED fluorescence to distinguish between homozygous, heterozygous and null lines and to determine the segregation ratio of the heterozygous lines. Seeds from at least 50 randomly selected plants from each line were harvested and 50 seeds per plant were examined for red fluorescence. Based on the absence of null phenotypes, seeds from homozygous (or near homozygous) plants were selected for growth in a second greenhouse generation that provided 2.5 kg seeds for field planting. 2.3. Field planting, cultivation and harvesting Field plots were established on Michigan State University Farms (East Lansing, Michigan, USA) in a 0.9 ha field. Four replicate 24 m2

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plots for each line were arranged in a random block design in a 60 m × 25 m section of the field. Both non-transformed and vectoronly lines were planted as controls. Seeds harvested from the greenhouse were planted in plots in early May and harvested by early August 2013. Larger individual plots (0.06–0.19 ha) of some lines were planted to achieve greater oil production. During field growth, the average high temperature was 24.7 ◦ C, the average low temperature was 12.9 ◦ C, and total rainfall was 312 mm. No irrigation was performed. The high-oleic lines expressed the bar (bialaphos resistance) gene (Nguyen et al., 2013) and therefore gluphosinate herbicide (Liberty, BayerCropSciences) was applied 3 weeks after seedling establishment to control weeds. To avoid losses of the small Camelina seeds through shattering or during machine harvest, the mature Camelina plants were harvested using a modified hedge trimmer and the seeds were recovered with a plot thresher (Almaco LPR with grass concave) operated without air flow and the seeds were cleaned with a custom-built seed cleaner (http://plantbiology.msu.edu/ 2013CamelinaFieldTrial.html). After harvest, seed water content was monitored by weighing before and after drying in an oven at 90 ◦ C to constant weight. Seeds with moisture content above 7% were dried in the greenhouse at 30–35 ◦ C with constant air circulation. Seed water content of both transgenic and control lines ranged from 5.7 to 7.6 at harvest and remained at 6.1–7.3% during storage in an air-conditioned room at 20–22 ◦ C. Harvest index was calculated from five randomly selected plants from each replicate 24 m2 plot, for a total of 20 plants per line. The dry plants were individually weighed, and then seeds harvested and weighed. The average ratio of seed weight per dry weight of each plant provided the harvest index value per line. 2.4. Seed lipid analysis: field-grown plants Fifty seeds were counted and weighed from each harvested replicate block. Tripentadecanoin and di17:0 sn-3-acetyl-TAG were added as internal standards. Lipids were extracted with 1 mL of hexane:isopropanol (7:2) by bead beating for 5 min at 30 Hz (Retsch) in 2 mL screw cap micro tubes. The steel bead was removed and the extractions were centrifuged with a tabletop centrifuge for 3 min at 13,000 rpm. 50 ␮L of each extract was loaded onto K6 TLC plates, and developed in hexane: ethyl ether:acetic acid (70:30:1). Lipids were visualized under UV light after spraying with 0.01% Primuline. Tri 21:0 TAG was added as an external standard to acetyl-TAG and TAG bands, which were then scraped into screw-cap test tubes and transmethylated. The resulting FAMEs were analyzed by GCFID. The accuracy of the quantification method for acetyl-TAG and TAG was verified by analysis of six known ratios of synthetic di18:1 sn-3-acetyl-TAG, tri 15:0 and tri 21:0 TAG. Oil content by dry weight was determined gravimetrically in triplicate. 100 seeds were counted and weighed, and were then extracted in hexane: isopropanol (7:2) with a Polytron (Kinamatica). Pooled extracts were combined in pre-weighed tubes and dried under nitrogen until the oil reached a constant weight. The oil contents were corrected for 6.5% seed moisture content. 2.5. Larger-scale extraction and purification of acetyl-TAG and lcTAG by chromatography Seeds (400–500 g) were extracted by grinding for 10 min in two volumes hexane with a Polytron (35 mm probe). Particulates were removed by filtration and the residual seed cake was re-extracted twice with hexane. The volume of the combined hexane extracts was reduced by rotary evaporation and the oil mixture was further purified by silica gel column chromatography. The silica gel (230–40 mesh, 60 A˚ pore, Silicycle) was activated at 100 ◦ C and

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725 g was added to a 90 mm diameter glass column. After conditioning the column with hexane, the oil was applied to the column at a loading of 0.20 g oil g−1 silica. The components of the oil (predominantly lcTAG, acetyl-TAG, DAG, FFA and polar lipids) were separated using a hexane/diethyl ether step gradient from 100:0, 85:15, 80:20, 75:25, to 70:30 (v/v) as eluent. The column was pressurized with 4 psi of argon gas providing a flow rate of 200 mL/min. Fractions of 1/2 column volume were collected and analyzed by TLC. In the example shown in Fig. 5 for high-oleic acetyl-TAG purification, fractions 3 and 4 are heavily enriched in lcTAG and essentially void of acetyl-TAG. Fraction 5 contains both TAG and acetyl-TAG. Fractions 6 and 7 were essentially pure acetyl-TAG with only trace amount of TAG, DAG, FFA and pigments. For physical characterization, fractions 3 and 4 and fractions 6 and 7 were pooled and represent lcTAG (3 + 4) and acetyl-TAG (6 + 7), respectively. Residual solvent in these fractions was removed by rotary evaporation under a high vacuum pump of >760 mmHg at 50 ◦ C for 1.5 h. The resulting neat lcTAG and acetyl-TAG oils were used for measurements of viscosity, thermal properties and energy content. 2.6. Reverse phase chromatography Lipids were loaded on KC18 reverse phase TLC plates (Whatman) and triple developed (first 50%, then 75% and finally 100%) using acetone: acetonitrile (70:30) for all three development steps. Lipids were visualized by iodine staining. Tri-heptadecanoic TAG was spotted to adjacent non-iodine stained bands and lipids were extracted from the KC18-TLC plate using chloroform:methanol:water (5:5:1). Chloroform and 0.88% (aq) KCl were added to induce phase separation. The organic phase was dried under nitrogen and transmethylated and quantified as above. Synthetic di18:1 sn-3-acetyl-TAG was synthesized by Bridge Organics, Kalamazoo, MI, USA and triolein was from Sigma–Aldrich. 2.7. Physical property measurements Viscosity was determined by Iowa Central Fuel Testing Laboratory (Fort Dodge, IA, USA) using ASTM method D96 at 40 ◦ C. Thermograms were determined in triplicate by differential scanning calorimetry (TA Instruments Q2000) with a thermal cycle starting at 40 ◦ C, and data collected as the sample was cooled and then re-heated across the range of −80 to 40 ◦ C at 1 ◦ C/min. For each DSC peak, an onset, peak and completion temperature was determined directly from the scan by use of the system software. The crystallization point was defined by the temperature at the peak of the major exotherm in the DSC cooling curves. Heat of combustion was determined with a Parr 1341 Oxygen Bomb Calorimeter. 3. Results and discussion 3.1. Camelina transformation A commercial variety of Camelina can be engineered to express the EaDAcT gene under the control of the soybean glycinin promoter resulting in accumulation of high levels of acetyl-TAG (Liu et al., 2014). In this study, a high-oleic Camelina line derived from the commercial variety “Celine” was engineered to produce acetylTAG. The high-oleic fatty acid composition was achieved by RNAi suppression of the FATTY ACID DESATURASE2 (FAD2) and FATTY ACID ELONGASE1 (FAE1) (Nguyen et al., 2013). High-oleic Camelina plants were transformed with empty vectors and with constructs designed to express EaDAcT under control of the strong, seed-specific, soybean glycinin promoter. Diacylglycerol (DAG) is the immediate substrate for EaDAcT. Because diacylglycerol acyltransferase 1 (DGAT1) and phosphatidylcholine:diacylglycerol acyltransferase1 (PDAT1) acyltransferases

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likely compete with EaDAcT for its DAG substrate, we assembled constructs that also included hairpin RNAi sequences that were designed to suppress DGAT1 and PDAT1 expression (Fig. S1). All constructs included the dsRED fluorescent protein as a visible reporter that allowed transformed seeds to be readily identified at the T1 and later generations. In high-oleic Camelina transformed with EaDAcT plus RNAi of DGAT and PDAT, seven transgenes were introduced from two T-DNA constructs: EaDAcT, RNAi-DGAT1, RNAi-PDAT1 and dsRED (Fig. S1), and RNAi-FAD2, RNAi-FAE1, and bar resistance from the high-oleic background. After an initial screen for T1-transgenic seeds, the number of dsRED positive, T1 seeds carried forward for further investigation ranged from 9 to 31 from the different transformed plants (‘events’) depending on the number of transgenic seeds obtained from each transformed plant. Initial analysis of segregation of the dsRED marker in T2 seeds revealed that seeds from approximately half of the plants fit the expected Chi-square test with a 3:1 segregation ratio of the dsRED marker. Higher transgene segregation ratios were apparent in a number of transformation events, suggesting that there were multiple independent copies of inserts in the genome. Although some T1 seeds did not germinate, no reduction in germination efficiency was observed in T2 seeds of plants generated from the constructs in Fig. S1. 3.2. Identification and analysis of lines producing high acetyl-TAG To assess acetyl-TAG production in the different transgenic events, T2 seeds were collected from individual plants cultivated in growth chambers. Based on dsRED-fluorescence screening, null seeds were removed and oil was extracted from transgenic seeds from each individual plant. Thin layer chromatography of oil extracts of over 100 T2 plants from EaDAcT and control constructs was used as an initial screen to determine the approximate proportions of acetyl-TAG and TAG in the oil. The acetyl-TAG and lcTAG bands from two to four T2 plants (per construct) that displayed high acetyl-TAG/lcTAG ratios were recovered. Fatty acid methyl esters were analyzed by GC-FID to obtain quantitative measures of acetyl-TAG and lcTAG and their fatty acid compositions. Expression of EaDAcT alone in the high-oleic background resulted in an average of 53 ± 4 mol% acetyl-TAG in T2 plant seeds (Fig. 2A). The level of acetyl-TAG increased to 78 ± 2 mol% in higholeic lines expressing both EaDAcT and DGAT1 RNAi. These results are similar to the expression of EaDAcT alone and with RNAi in the Camelina wild-type background (Liu et al., 2014). Thus, in both the high-oleic and wild-type backgrounds, the RNAi suppression of DGAT1 was successful in increasing acetyl-TAG production. The inclusion of PDAT1 RNAi either alone or together with DGAT1 RNA did not increase acetyl-TAG accumulation in the high-oleic background (Fig. 2A). However, it is not known if the PDAT1 RNAi construct was successful in suppressing endogenous PDAT1 mRNA. The fatty acid composition of the acetyl-TAG fraction isolated from the oil was reduced in saturates and C20 and C22 fatty acids, and was enriched in 18:1 compared to the lcTAG fraction (Fig. S2). These results are consistent with displacement by acetate of acyl chains from the sn-3 position where saturated and C20 and C22 fatty acids are enriched in Brassicacea species. 3.3. Field production of acetyl-TAG A field trial was designed to evaluate growth, yield, and acetylTAG production of different transgenic lines under field conditions and to produce sufficient oil for a range of application tests. To obtain seeds for field production, two cycles of amplification of seeds of T2 plants with high mol% acetyl-TAG producing were conducted in a greenhouse. The second greenhouse cycle yielded

approximately 2.5 kg seeds from 50 to 60 m2 with an average seed weight of 1.18 ± 0.02 mg. At the T4 generation, all lines were judged to be homozygous or near-homozygous based on the absence of null dsRED phenotypes in the seed population. For each selected transgenic and control line, four replicate 24 m2 (8 m × 3 m) blocks were arranged randomly in the field. Larger individual plots were also planted to provide greater quantities of seed for oil production (total planted area of 0.7 ha). Seeds were planted early May 2013 and harvested by early August, 2013. Harvest equipment and procedures were designed or modified to maximize recovery of the small Camelina seeds. The high-oleic line expressing only EaDAcT accumulated 70 ± 0.9 mol% acetyl-TAG in the field. The increase over the T2 acetyl-TAG level may reflect increased gene dosage as T2 plants producing heterozygous seeds were eliminated at the T3 and T4 generations. The line transformed with both EaDAcT and DGAT1 RNAi accumulated 69 ± 3.0 mol% acetyl-TAG (Fig. 2B). Under the same field conditions, the wild-type line transformed with only the EaDAcT gene accumulated 65 mol% acetyl-TAG whereas expression of EaDAcT plus DGAT1 RNAi produced seeds with up to 85 mol% acetyl-TAG (Liu et al., 2014. For the wild-type background, the acetyl-TAG phenotype observed in T2 plants was maintained in the field through the T4 and T5 generations (Liu et al., 2014). However, decreases were observed in acetyl-TAG levels for some lines in the high-oleic background. For example, the EaDAcT+ DGAT1 RNAi line in the high-oleic background that accumulated 78% acetyl-TAG at T2, produced 69% in the field. Another line, which had been transformed with the same construct, was reduced in acetyl-TAG content in the field (to <20%). These decreases were accompanied by reduced dsRED florescence of seeds. Reductions in acetyl-TAG accumulation were not observed with the same constructs in the wild-type background. It is possible that trait instability in the high-oleic lines is related to use of the same promoters for multiple genes. Constructs transformed into the wild-type background contain at most one glycinin promoter. In the high-oleic background, in addition to GlyEaDAcT the glycinin promoter is also used for RNAi suppression of FAE1 (Nguyen et al., 2013). Although other factors cannot be ruled out, we speculate that this instability in acetyl-TAG production in some lines might result from gene silencing that is related to the multiple use of the same promoter in transgenic lines (Peremarti et al., 2010) or to multiple inserts of the same T-DNA (Matzke and Matzke, 1995).

3.4. Production of acetyl-TAG has minor or no impact on agronomic performance The production of acetyl-TAG resulted in less than a 4% difference in seed weight in either the high-oleic or wild-type backgrounds (Fig. 3A). Seed weight of the high-oleic control lines averaged 1.36 ± 0.01 mg, compared to 1.31 ± 0.01 mg for the higholeic EaDAcT and EaDAcT plus DGAT1 RNAi lines. We also observed that the average weight per seed from plants grown in the field was 20% higher than that of the T2 seeds harvested from growth chambers (1.14 mg). Seed weights are comparable to previous agronomic studies of non-transgenic Camelina (Berti et al., 2011; Johnson et al., 2009). Germination of seeds harvested from field-grown lines was above 80% and seedlings metabolized acetyl-TAG at a rate similar to or higher than lcTAG (Liu et al., 2014). Harvest index was determined from the ratio of the weight of seeds produced by each plant to the total above ground plant weight. The average harvest index of the Camelina lines in field plots ranged from 33 to 37%, and was not statistically different between the transgenic lines and corresponding control lines (Fig. 3B). Harvest index data are within the range previously

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Fig. 2. Accumulation of acetyl-TAG in Camelina sativa high-oleic transgenic lines that express EaDAcT only or EaDAcT with RNAi of DGAT1 (dgat1) or PDAT1 (pdat1) or RNAi of both (dgat1/pdat1). (A) Seeds of T2 plants grown in growth chambers; (B) seeds harvested from field-grown T4 and T5 generation plants. Data represent mean ± SE.

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Fig. 3. Field production of acetyl-TAG has little or no effect on weight per seed, harvest index or yield. Values presented are the average of data pooled from high-oleic transgenic lines and from high-oleic control lines. (A) Seed weight; (B) harvest index (seed weight/plant weight); (C) yield of seeds per 24 m2 test plots. Data represent mean ± SE.

high-oleic and WT) were reduced by 5–10% in oil per dry weight. These results suggest that the RNAi of DGAT1 has a negative impact that is not compensated by the EaDAcT activity. One possible reason is the timing of the different promoters. RNAi expression was driven by an oleosin promoter whereas EaDAcT was controlled by the

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reported for Camelina (Berti et al., 2011) and similar to Canola harvest index (Hocking et al., 1997). The accumulation of acetyl-TAG did not significantly alter the seed yield: 2.39 ± 0.19 and 2.25 ± 0.11 kg of seeds were harvested per 24 m2 area for control and acetyl-TAG-accumulating high-oleic plants, respectively (Fig. 3C). These values were not statistically different by T-test at P = 0.05. The high-oleic lines carried the bialaphos resistance (bar) gene providing resistance to glufosinate herbicides (Nguyen et al., 2013). This allowed for partial control of weed pressure that was not available for wild-type lines, and resulted in yields that were on average 42% higher. A greater-than-expected yield variation was observed between replicate 24 m2 blocks. This variation most likely resulted from variability in seedling establishment and weed pressure. We observed that seedling establishment was strongly impacted by planting depth. Areas of uniform growth had 13 plants per m2 and produced 570 kg seed/ha, which can be compared to previously reported values of 650 kg/ha for this seedling density (Johnson et al., 2009). Seed yield per ha data presented here should be considered preliminary and additional evaluation of agronomic performance of acetyl-TAG-producing plants will require multiple locations and growing seasons. The oil content as a percentage of dry weight (DW) in the high-oleic control lines was 36.8 ± 0.2%. Expressing EaDAcT without RNAi slightly increased oil yield per DW to 37.6 ± 0.5% in the high-oleic background (Fig. 4). However, all RNAi lines (both

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Fig. 4. Total moles of TAG accumulated (acetyl-TAG plus lcTAG) and oil content as percentage of dry weight for field grown plants. (A) Total moles of TAG per dry weight are increased in lines producing acetyl-TAG; (B) oil content as % of dry weight in lines expressing EaDAcT alone, or EaDAcT plus DGAT1-RNAi. Data represent mean ± SE.

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glycinin seed storage promoter. Oleosin transcripts are expressed later in seed development than storage proteins such as glycinin (Troncoso-Ponce et al., 2011). Therefore, we speculate that RNAi of DGAT1 may have reduced overall TAG synthesis at the later developmental stage when EaDAcT was less active, leading to a reduction in final oil content. This can be tested in the future using different promoters.

3.5. Total moles of TAG and oil content increased in lines producing acetyl-TAG The accumulation of acetyl-TAG in Camelina was accompanied by an increase in the total moles of TAG (acetyl-TAG plus lcTAG) in seeds. Control high-oleic lines accumulated 416 ± 3 ␮mol TAG g−1 seeds. This increased to 527 ± 6 ␮mol in the high-oleic line expressing only EaDAcT (Fig. 4A). An increase in moles of TAG is expected if the amount of fatty acid produced by non-transgenic seeds is maintained in lines producing acetyl-TAG. This is because 6 moles of fatty acid will produce only 2 moles of lcTAG, compared to 3 moles of acetyl-TAG. An increase in the mass of total oil per seed in acetyl-TAG over lcTAG would also be predicted because synthesis of acetylTAG from the same moles of fatty acid requires added glycerol and acetate. Indeed, as noted above (Section 3.4) in the higholeic line expressing only EaDAcT, a small increase in oil mass per DW over the control was observed (Fig. 4B). However, the increases in moles of TAG and oil mass were lower than is predicted if the same amount of fatty acid had been produced in the transgenics and controls. In lines that produced 70 mol% acetylTAG a 31% increase in moles of TAG and a 7% increase in oil mass would be expected if no reduction in fatty acid accumulation occurred. The lower-than-expected increases may reflect an insufficient supply of acetyl-CoA, glycerol-3-phosphate or other precursors for acetyl-TAG production (discussed below, Section 3.9). In summary, field production of up to 70 mol% acetyl-TAG in oil of high-oleic Camelina had minor or no impact on the seed weight, oil content, seed yield and harvest index.

3.6. An increase in DAG is associated with acetyl-TAG accumulation In all acetyl-TAG-accumulating plants (both high-oleic and wild-type backgrounds) an increase in DAG levels in the oil was observed (Fig. S3A). Whereas the control high-oleic lines accumulated 2.5 ␮mol DAG/g seed (0.6 mol% of total fatty acid), the DAG levels increased to 9.5 ␮mol/g (1.9 mol%) in lines expressing only EaDAcT. The amount of DAG further increased in DGAT1 RNAi lines to 15–19 ␮mol/g, representing 4 mol% of all lipids. The increased DAG pool is likely biosynthetic, rather than from lipolysis because increased DAG was not only observed in mature seeds, but also in developing seeds. Furthermore, the fatty acid composition of DAG (Fig. S3B) was similar to acetyl-TAG (Fig. S2), with lower proportions of C20 and C22 chain lengths than TAG and was not significantly different from the DAG fatty acid composition of nontransgenic control seeds. The 4% DAG levels were only observed in seeds expressing DGAT1 RNAi, and not in EaDAcT only seeds that had almost the same mol% acetyl-TAG. Therefore, 4% DAG accumulation is likely a response to RNAi of DGAT1 rather than to acetyl-TAG accumulation. It is notable that a similar increase in DAG was reported for the Arabidopsis dgat1 mutants, which accumulate up to 19 ␮mol DAG/g (5% of total lipids) (Katavic et al., 1995).

Fig. 5. Distinct separation of acetyl-TAG from lcTAG by silica gel column chromatography. (A) TLC of oil extract from high-oleic line expressing EaDAcT plus DGAT1-RNAi; (B) fractions from silica gel column eluted with hexane and ether step gradient.

3.7. Acetyl-TAG produced in Camelina has lower viscosity, crystallization temperature, and caloric content The analysis of Camelina oil presented above was based on thin layer chromatography plus GC analysis of oil from 100 mg or less of seeds. In order to prepare sufficient oil for physical testing, largerscale extraction and purification procedures were developed. Oil was extracted with hexane from 0.5 kg seeds and purified on a 1.2 L silica gel column. As shown in Fig. 5, acetyl-TAG could be separated from lcTAG and from DAG and FFA using a hexane/diethyl ether step gradient from 100% hexane to 30% ether/hexane. This provided a method for preparation of purified acetyl-TAG for the tests described below. Viscosity is a key physical parameter of vegetable oils that influences their use as biodegradable lubricants (Cermak et al., 2013), hydraulic fluids (Cermak et al., 2013), transformer oils (Bertrand and Hoang, 2003) and fuels (Knothe, 2008). The viscosity of major commercially produced vegetable oils is near or above 30 mm2 s−1 at 40 ◦ C (Blin et al., 2013; Franco and Nguyen, 2011). Because their viscosity prevents the direct use of vegetable oils in most diesel engines, the oil is either heated before entering the engine and fuel filters, or is transesterified to methyl or ethyl esters to produce biodiesel. To determine the kinematic viscosity of high-oleic and of TAG of control lines, the oils were tested at 40 ◦ C using the ASTM D96 method. Acetyl-TAG fractions from high-oleic lines had 27% lower viscosity than the high-oleic control background (Fig. 6A). Diesel #4 fuel is used in locomotive, ship, generator and other engines run at lower rpm. The viscosity of high-oleic-acetylTAG (26.3 mm2 s−1 at 40 ◦ C) was slightly above the specifications of diesel #4 fuel (5–24 mm2 s−1 at 40 ◦ C). Consistent with its higher polyunsaturated fatty acid content, acetyl-TAG from the wild-type background (20.3 mm2 s−1 at 40 ◦ C) falls within diesel #4 specifications. Introduction of EaDAcT into lines producing shorter chain fatty acids should provide further reduction of viscosity. Another key parameter that influences the use of vegetable oils for many applications is their response to low temperature. Vegetable oil crystallization can plug filters, prevent use of vegetable oils as low temperature lubricants, and can create a cloudy appearance in food oils. Crystallization of vegetable oils is complex with several polymorphic forms and with interconversions of these forms that depend both on the temperature and rate of temperature change. We examined the thermal properties of acetyl-TAG and control oils by differential scanning calorimetry. During cooling at 1.0 ◦ C/min, acetyl-TAGs had markedly lower crystallization

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Fig. 6. Physical properties of acetyl-TAG. (A) Kinematic viscosity determined at 40 ◦ C; (B) crystallization temperature determined by differential scanning calorimetry of acetyl-TAG and lcTAG isolated from high-oleic Camelina seeds expressing EaDAcT and empty vector, respectively. Cooling curves at 1.0 ◦ C/min presented with crystallization temperature defined as the peak of the major exotherm upon cooling. Vertical black bar represents y-axis scale of 0.25 W/g; (C) caloric content determined by bomb calorimetry; (D) calculated oxidation stability index. Data represent mean ± SE. Data for wild-type lines (WT) are included for comparison and are adapted from Liu et al. (2014).

temperatures than the corresponding conventional TAG. High-oleic acetyl-TAG underwent a major crystallization exotherm at minus 64 ◦ C compared to minus 34 ◦ C for lcTAG (Fig. 6B). Similar reduced temperatures compared to controls were obtained with synthetic standards of di18:1 acetyl-TAG, as well as with wild-type acetylTAG (Liu et al., 2014). The much lower crystallization temperature of acetyl-TAG will likely be reflected in improved pour point and cold filter plug points. Thus, these structures may provide improved low temperature performance for acetyl-TAG oils as lubricants, and fuels and also as alpha-tending emulsifiers in food applications. The low level of erucic acid in Camelina oil allows it to meet standards for food use in Europe and North America. In addition, synthetic acetylated mono- and diglycerides are approved for food use. Because acetyl-TAG contains fewer carbon-hydrogen bonds than lcTAG it is expected that it would have a lower caloric content. Indeed, based on bomb calorimetry, high-oleic acetylTAG (9.05 ± 0.07) contains 4.5% lower kcal/g compared to lcTAG (9.50 ± 0.14) (Fig. 6C). This corresponds to a reduction of 29% on a molar basis. The lower caloric content of acetyl-TAG may prove useful in the formulation of reduced-calorie foods. Acetyl-TAG may also have the ability to reduce accumulation of body fat by other mechanisms as observed for diacylglycerol (Lo et al., 2008). It is also noteworthy that the structure of fully hydrogenated acetyl-TAG will be rich in stearic acid and very similar to Benefat/SalatrimTM , a fat substitute. A lower intestinal absorption of these saturated oils leads to a reduction in available calories to 4.5–6.0 kcal/g (Finley et al., 1994). Future feeding studies will evaluate if acetyl-TAG

produced in Camelina is also suitable in some foods as a strategy to reduce caloric content and fat absorption. The oxidative stability of vegetable oils strongly influences their suitability for applications including lubrication, frying oils, fuel and for longer-term storage. The unsaturated fatty acid content is the main determinant of the oxidative stability of plant oils. In particular, polyunsaturated fatty acids are at least 10-fold more susceptible to oxidation than monounsaturates (Knothe and Dunn, 2003). The oxidative stability index (OSI) can be determined by measuring the increase in conductivity of water in contact with oil during oxidation for up to 90 h. Alternatively, it has been shown that OSI values calculated based on “bis-allylic position equivalences (BAPE)” correlate very well (R2 = 0.983) with experimental values (Knothe and Dunn, 2003). For this study, OSI was calculated based on BAPE for the acetyl-TAG produced by high-oleic lines and by wild-type lines. A higher OSI indicates more stable oils. The OSI index for high-oleic acetyl-TAG is more than 7-fold higher compared to non-transgenic Camelina oil and 24% higher than the high-oleic background line (Fig. 6D). This increase in oxidative stability of high-oleic acetyl-TAG is expected to be an important characteristic for potential uses at high temperatures and in other oxidative environments. Further improvements in the oxidative stability of acetyl-TAG are expected based on current efforts toward increasing oleic acid levels in Camelina by transgenic and other approaches (Iskandarov et al., 2014).

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Fig. 7. 3-acetyl-1,2-dioloyl-sn-glycerol is the most abundant acetyl-TAG molecular species in high-oleic Camelina expressing EaDAcT. (A) RPTLC of standards and of acetylTAG isolated from high-oleic and wild-type Camelina expressing EaDAcT; (B) separation of lcTAG and acetyl-TAG from high-oleic Camelina; (C)–(E) fatty acid composition of indicated bands. n.d. = not detected.

3.8. di18:1 acetyl-TAG is the most abundant acetyl-TAG molecular species Reverse phase thin layer chromatography (RPTLC) separates lipids based on hydrophobicity and can partially resolve TAG molecular species with different unsaturation and chain lengths (Christie and Han, 2010). As shown in Fig. 7A, synthetic di18:1 acetyl-TAG migrates above and is well resolved from triolein (Rf of 0.65 and 0.41, respectively) reflecting the difference in number of acyl carbons. In addition, there was little or no overlap of bands of acetyl-TAG and lcTAG produced in high-oleic Camelina (Fig. 7B). RPTLC also provided a method to examine the distribution of acyl chains in different acetyl-TAG structures. The fatty acid composition in high-oleic acetyl-TAG (68 mol% 18:1) is less complex compared to wild-type acetyl-TAG and, as expected, fewer acetylTAG bands were observed. Acetyl-TAG from high-oleic Camelina was resolved into three major bands compared to five acetyl-TAG bands from wild-type (Fig. 7A). Recovery of these three major bands allowed us to identify the most abundant acetyl-TAG molecular species (Fig. 7C–E). GC-FID analysis of the lower, most-abundant band indicated that it contains 54% of total fatty acids in the acetylTAG fraction (Fig. 7E). 18:1 constitutes 85% of the fatty acids in this band. Based on these values it can be calculated that di18:1-acetylTAG represents approximately 47% of all acetyl-TAG molecular species in the oil and is the most abundant species. The second most prevalent molecular species is 18:1/18:3-acetyl-TAG (from band C) which constitutes approximately 17% of all acetyl-TAG molecular species. Taken together, these results indicate that reverse phase chromatography can be a complementary method to silica gel to purify acetyl-TAG with the added advantage of providing fractions

enriched in specific molecular species. These purified fractions will allow testing of acetyl-TAG with a range of fatty acid compositions.

3.9. Can acetyl-TAG accumulate to higher levels? The acetyl-TAG level in E. alatus endosperm is over 95 mol% (Durrett et al., 2010). Can these levels be achieved in Camelina or other crop seeds? The supply of cytosolic acetyl-CoA substrate for EaDAcT might be one limit to the level of acetyl-TAG accumulation that can be achieved. Expression of EaDAcT in the high-oleic line provided a partial test of this possibility. AcetylCoA provides the carbon for cytosolic elongation of 18 carbon fatty acids to C20 and C22. In wild-type control Camelina, C20 and C22 fatty acids are present at 17.3 mol% and 2.7 mol% whereas in the high-oleic control line this is reduced to 5.1% and 0.5%. This difference represents a reduction in the consumption of acetyl-CoA by 243 nmol per seed in the high-oleic lines. The synthesis of 70 mol% acetyl-TAG will require 439 nmol acetyl-CoA/seed. Because higholeic lines did not accumulate higher levels of acetyl-TAG than wild-type lines transformed with the same constructs, this study provides preliminary evidence that acetyl-CoA supply is not limiting, at least for production of 70% acetyl-TAG. However, it remains to be determined if acetyl-CoA may limit acetyl-TAG accumulation to higher levels similar to those of Euonymus endosperm. Other potential metabolic limitations to acetyl-TAG accumulation are the supply of glycerol-3-phosphate or/and in the activity of enzymes responsible for the synthesis of other intermediates in TAG biosynthesis. Use of promoters that express EaDAcT over a wider range of seed development than the storage protein

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promoter used here may also help to increase acetyl-TAG accumulation.

3.10. Summary Development of new or improved crops that produce ‘addedvalue’ oils has been an objective of plant breeders, agronomists, and molecular biologists for many decades (Zanetti et al., 2013). In addition to developing or adapting Cuphea, Lunaria, Meadowfoam, and other plants for agricultural production, there have been many efforts to isolate genes from these and other species that produce added-value oils and to transfer these genes into conventional oilseed crop. Important achievements have been made, for example, in production of 60% lauric acid in Canola (Knutzon et al., 1999) and 65% omega-7 monoenes in Camelina (Nguyen et al., 2014) and in increasing erucic acid content to more than 75% in Crambe (Li et al., 2012). Engineering crops to produce high levels of hydroxy, epoxy, acetylenic, conjugated and a number of other modified fatty acids has been less successful and in some cases has resulted in reduced oil content and germination (e.g. Cahoon et al., 2006, 2007; Bates et al., 2014). In this study and in Liu et al. (2014) the engineering and field production of some of the highest levels of an unusual oil so far achieved in a transgenic oilseed crops is presented. Attaining these high levels of acetyl-TAG was likely facilitated because it was necessary to modify only the final step in TAG synthesis (including down regulation of the endogenous reactions), and because acetyl-TAG biosynthesis does not require the modification of intermediates in membrane glycerolipid or de novo fatty acid synthesis. Importantly, these high levels were obtained with no or only minor impacts on oil content, seed yield or germination. The establishment of crop production of acetyl-TAG in both high-oleic- and wild-type Camelina provides for the first time an abundant source of two novel seed oils that can be tested in a range of applications. Oil compositions with 70–80 mol% acetyl-TAG may be suitable for several applications. Alternatively, more highly enriched acetyl-TAG can be obtained by molecular distillation, a common industrial process for large-scale vegetable oil separations (Compton et al., 2008). The availability of acetyl-TAG with a high-oleic fatty acid composition will be advantageous for applications where oxidative stability is important. The acetyl-TAG from the wild-type background may have wider use in the food industry. For example, related structures (e.g. GRINSTEADTM ACETEM) are produced semi-synthetically from soybean oil (as well as palm and coconut oils) and are now used for water retention on food surfaces, as emulsifiers, as a chewing gum base, as foam stabilizers, and as plasticizers for food packaging (Gaupp and Adams, 2004; www.danisco.com/productrange/emulsifiers/grindstead-acetem). Semi-synthetic acetylated mono- and di-glycerides currently used in the food industry have a market price substantially above that of vegetable oils. Direct production of acetyl-TAG in crops might therefore offer an alternative, lower-cost and abundant supply of acetyl-TAG for these markets. In addition, the lower viscosity and cold-temperature properties of acetyl-TAG will likely confer advantages for its use as biodegradable lubricants, hydraulic fluids, transformer oils and other products. Production of acetyl-TAG in this study provides a first step toward testing high-oleic acetyl-TAG in a range of non-food and food applications. Results from these studies will also provide guidance toward possible future commercial development of acetyl-TAG producing Camelina as a novel oilseed crop.

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Acknowledgements We are grateful to Mike Pollard (Michigan State University) for initiating this project and for extensive advice and discussions. We thank Brian Graff, Todd Martin, James Kelly and Kurt Thelen of the Crop and Soil Sciences Department for assistance with field production, Rachael Sak, Biosystems Engineering Department, for guidance in bomb calorimetry and Michael Rich, Composite Materials & Structures Center, for assistance with differential scanning calorimetry (all of Michigan State University) and Xiaowen Kong and Carol Ohlrogge also for assistance with field production. We are grateful to Tom Clemente (University of Nebraska–Lincoln) for advice on obtaining permits for transgenic field work and on growing Camelina. This work was supported in part by Department of Energy-Great Lakes Bioenergy Research Center Cooperative Agreement DE-FC02-07ER64494.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.indcrop. 2014.11.019.

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