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A Specialized Diacylglycerol AcyltransferaseContributes to the Extreme Medium-Chain FattyAcid Content ofCuphea Seed OilUmidjon IskandarovUniversity of Nebraska-Lincoln, uiskandarov2@unl.edu

Jillian E. SilvaUniversity of Nebraska - Lincoln

Hae Jin KimUniversity of Nebraska-Lincoln, hkim6@unl.edu

Mariette AnderssonUniversity of Nebraska - Lincoln

Rebecca E. CahoonUniversity of Nebraska-Lincoln, rcahoon2@unl.edu

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Iskandarov, Umidjon; Silva, Jillian E.; Kim, Hae Jin; Andersson, Mariette; Cahoon, Rebecca E.; Mockaitis, Keithanne; and Cahoon,Edgar B., "A Specialized Diacylglycerol Acyltransferase Contributes to the Extreme Medium-Chain Fatty Acid Content of Cuphea SeedOil" (2017). Faculty Publications from the Center for Plant Science Innovation. 173.http://digitalcommons.unl.edu/plantscifacpub/173

AuthorsUmidjon Iskandarov, Jillian E. Silva, Hae Jin Kim, Mariette Andersson, Rebecca E. Cahoon, KeithanneMockaitis, and Edgar B. Cahoon

This article is available at DigitalCommons@University of Nebraska - Lincoln: http://digitalcommons.unl.edu/plantscifacpub/173

A Specialized Diacylglycerol AcyltransferaseContributes to the Extreme Medium-Chain Fatty AcidContent of Cuphea Seed Oil1[OPEN]

Umidjon Iskandarov, Jillian E. Silva, Hae Jin Kim, Mariette Andersson, Rebecca E. Cahoon,Keithanne Mockaitis, and Edgar B. Cahoon*

Center for Plant Science Innovation and Department of Biochemistry, University of Nebraska-Lincoln, Lincoln,Nebraska 68588 (U.I., J.E.S., H.J.K., R.E.C., E.B.C.); Department of Plant Breeding, Swedish University ofAgricultural Sciences, Alnarp, Sweden (M.A.); and Pervasive Technology Institute and Department of Biology,Indiana University, Bloomington, Indiana 47405 (K.M.)

ORCID IDs: 0000-0001-7456-2127 (H.J.K.); 0000-0003-3392-5766 (R.E.C.); 0000-0001-6123-8424 (K.M.); 0000-0002-7277-1176 (E.B.C.).

Seed oils of many Cuphea sp. contain .90% of medium-chain fatty acids, such as decanoic acid (10:0). These seed oils,which are among the most compositionally variant in the plant kingdom, arise from specialized fatty acid biosyntheticenzymes and specialized acyltransferases. These include lysophosphatidic acid acyltransferases (LPAT) and diacylglycerolacyltransferases (DGAT) that are required for successive acylation of medium-chain fatty acids in the sn-2 and sn-3positions of seed triacylglycerols (TAGs). Here we report the identification of a cDNA for a DGAT1-type enzyme,designated CpuDGAT1, from the transcriptome of C. avigera var pulcherrima developing seeds. Microsomes of camelina(Camelina sativa) seeds engineered for CpuDGAT1 expression displayed DGAT activity with 10:0-CoA and thediacylglycerol didecanoyl, that was approximately 4-fold higher than that in camelina seed microsomes lackingCpuDGAT1. In addition, coexpression in camelina seeds of CpuDGAT1 with a C. viscosissima FatB thioesterase(CvFatB1) that generates 10:0 resulted in TAGs with nearly 15 mol % of 10:0. More strikingly, expression ofCpuDGAT1 and CvFatB1 with the previously described CvLPAT2, a 10:0-CoA-specific Cuphea LPAT, increased 10:0amounts to 25 mol % in camelina seed TAG. These TAGs contained up to 40 mol % 10:0 in the sn-2 position, nearlydouble the amounts obtained from coexpression of CvFatB1 and CvLPAT2 alone. Although enriched in diacylglycerol,10:0 was not detected in phosphatidylcholine in these seeds. These findings are consistent with channeling of 10:0 intoTAG through the combined activities of specialized LPAT and DGAT activities and demonstrate the biotechnologicaluse of these enzymes to generate 10:0-rich seed oils.

Fatty acid and triacylglycerol (TAG) biosyntheticpathways in species of the Cuphea genus are amongthe most highly divergent in the plant kingdom(Graham, 1989). Seed oils of Cuphea species such asC. avigera var pulcherrima and C. viscosissima accu-mulate the medium-chain length fatty acids octanoic(8:0) and decanoic (10:0) acids to .90 mol % of TAG

fatty acids (Graham, 1989; Kim et al., 2015b). Bycontrast, seed oils of most plants, including those ofthe major commercial oilseed crops, are enriched infatty acids with 16- and 18-carbon atoms. The Cupheamedium-chain fatty acids are produced by variationsof the plastid-localized fatty acid synthase that re-sults in the release of medium-chain fatty acids fromacyl carrier protein by specialized FatB-type thioes-terases before their extension to C16 and C18 (Deheshet al., 1996a, 1996b; Leonard et al., 1997; Slabaughet al., 1998). In addition to their synthesis, medium-chain fatty acids must accumulate to high levels at thethree stereospecific positions of the glycerol back-bone to generate TAGs with .90 mol % of medium-chain fatty acids found in C. avigera var pulcherrimaand C. viscosissima. These reactions are catalyzed bythe ER-localized glycerol 3-P acyltransferase (GPAT;EC 2.3.1.15), lysophosphatidic acid acyltransferase(LPAT; EC 2.3.1.51), and diacylglycerol acyltransfer-ase (DGAT; EC 2.3.1.20) that use acyl-CoA substratesfor successive acylation of the three stereospecificpositions of glycerol intermediates in the Kennedypathway of TAG biosynthesis (Kennedy, 1961). Theproduction of TAGs with .90 mol % medium-chain

1 The research was supported by the Center for Advanced BiofuelSystems, an Energy Frontier Research Center funded by the U.S.Department of Energy, Office of Basic Energy Sciences under Awardno. DE-SC0001295, and the National Science Foundation (PlantGenome no. IOS-13-39385 to E.B.C.).

* Address correspondence to ecahoon2@unl.edu.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Edgar B. Cahoon (ecahoon2@unl.edu).

U.I., J.E.S., H.J.K., and E.B.C. contributed to the conception of theresearch; U.I., J.E.S., H.J.K., M.A., R.E.C., and K.M. conducted theresearch; U.I. and E.B.C. analyzed the data andwrote the manuscript;E.B.C. supervised the research.

[OPEN] Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.16.01894

Plant Physiology�, May 2017, Vol. 174, pp. 97–109, www.plantphysiol.org � 2017 American Society of Plant Biologists. All Rights Reserved. 97

fatty acids requires specialized acyltransferases withhigh specificities for medium-chain acyl-CoAs andmedium-chain fatty acid-containing glycerol inter-mediates, as previously shown by enzymatic assaysand radiolabeling using C. lanceolata seed microsomes(Bafor et al., 1990; Vogel and Browse, 1996). Thesestudies are consistent with direct channeling of 10:0in C. lanceolata seeds through the Kennedy pathwayacyltransferases for TAG synthesis (Bafor et al., 1990),rather than through phosphatidylcholine (PC) as occursin most seeds for polyunsaturated fatty acid synthesis(Slack et al., 1978; Stymne and Appelqvist, 1978; Bateset al., 2009).

We have previously identified a LPAT from C. vis-cosissima (CvLPAT2) that has high activity with 10:0-CoA (Kim et al., 2015a). The CvLPAT2 activity differsfrom that of typical ER-localized LPATs that in-stead primarily use C18 substrates with D9 unsatu-ration, and have only low activity with saturatedfatty acyl-CoA substrates (Ichihara et al., 1987; Sunet al., 1988; Oo and Huang, 1989). Transgenic ex-pression of CvLPAT2 along with a 10:0 producingC. viscosissima FatB (CvFatB1) in camelina (Camelinasativa) seeds resulted in the production of TAGs with10:0 in the sn-2 position, which were absent fromthis position in seeds expressing only CvFatB1 (Kimet al., 2015a).

Previous analyses of DGAT activities in Cuphea sp.seed microsomes have also indicated substrate prefer-ence for medium-chain acyl-CoAs and medium-chainfatty acid-containing diacylglycerols (DAGs; Cao andHuang, 1986; Bafor et al., 1990). These findings point toa role for DGAT as amajor contributor to the high levelsof medium-chain fatty acids accumulated in TAGs ofmany Cuphea sp. Two major forms of DGAT are as-sociated with TAG biosynthesis in seeds DGAT1 andDGAT2, and a third DGAT, DGAT3, has also beenascribed a role in TAG biosynthesis (Saha et al., 2006;Cao, 2011; Liu et al., 2012). DGAT1 and DGAT2, incontrast to the soluble DGAT3, are ER-localized butare structurally distinct (Shockey et al., 2006). TypicalDAGAT1 polypeptides are .150 amino acids largerthan DGAT2 and have eight to ten predicted trans-membrane domains versus three to six in DGAT2(Cao, 2011; Liu et al., 2012). Both DGAT1 and DGAT2enzymes from a variety of species have been linked tothe preferential accumulation of unusual fatty acids inseed TAGs (Burgal et al., 2008; Li et al., 2010; Ayméet al., 2015). Recently, a palm (Elaeis guineensis) kernelDGAT1 was shown to promote increased accumula-tion of the medium-chain fatty acid lauric acid (12:0)relative to the Arabidopsis (Arabidopsis thaliana)DGAT1 when expressed in the oleaginous yeast Yar-rowia lipolytica. This preferential activity for lauric acid(12:0) was proposed to be related to variations in theN-terminal region observed in diverse DGAT1 poly-peptides (Aymé et al., 2015). In addition, a DGAT2from castor bean (Ricinus communis) was shown toincrease accumulation of the hydroxy fatty acid rici-noleic acid in TAG when expressed in transgenic

Arabidopsis seeds (Burgal et al., 2008). Similarly, anironweed (Vernonia galamensis) DGAT2 was shown toincrease accumulation of the epoxy fatty acid vernolicacid in TAG when expressed in transgenic soybeanseeds (Li et al., 2010).

Here we report the identification of a cDNA for aseed-specific DGAT1 enzyme designated CpuDGAT1from the transcriptome of developing C. avigera varpulcherrima seeds that is active with 10:0-CoA anddidecanoin (10:0-DAG). In addition, we show thatrecombinant expression of this enzyme enhances10:0 accumulation in camelina seeds engineered forCvFatB1 expression. Consistent with channeling of10:0 through the Kennedy pathway, coexpression ofthe CpuDGAT1 and CvLPAT2 enzymes not onlyincreased total 10:0 accumulation in camelina seedTAG, but also doubled the total content of 10:0 inthe sn-2 position in TAG relative to expression ofCvLPAT2 alone. This was achieved with no detect-able accumulation of 10:0 in PC of engineered cam-elina seeds. Results of analyses of fatty acid content,seed weight, and germination assays of engineeredcamelina seeds are also reported. Overall, thesefindings expand the functional diversity of DGAT1enzymes and demonstrate the concerted capacity ofspecialized DGAT and LPAT activities for conferringenhanced medium-chain fatty acid accumulation inan engineered oilseed crop.

RESULTS

Identification of a Seed-specific DGAT1 in C. avigeravar pulcherrima

The previously described transcriptome of develop-ing C. avigera var pulcherrima seeds (Kim et al., 2015b)was mined for candidate genes encoding DGATs thatare specialized for medium-chain fatty acid metabo-lism. Sequences for one DGAT1-like gene designatedCpuDGAT1 and three DGAT2-like genes designated

Figure 1. Expression ofCpuDGAT1,CpuDGAT2_A,CpuDGAT2_B, andCpuDGAT2_C transcripts in different organs of C. avigera var pulcher-rima. RT-PCR analysis of DGAT1 and DGAT2 transcripts in different or-gans of C. avigera var pulcherrima. Eukaryotic initiation factor 4A (eIF4)and actin genes were used as internal controls. PCR products wereobtained with gene-specific primers for CpuDGAT1, CpuDGAT2_A,CpuDGAT2_B, or CpuDGAT2_C (Supplemental Table S1).

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Figure 2. Alignment of deduced amino acid sequence of CpuDGAT1with selected plant andmammalian homologs. Amino acidalignment of CpuDGAT1 with plant and mammalian DGAT1 homologs. Amino acid sequences were obtained from the NCBI.DGAT sequences are shown and NCBI accession numbers are as follows: CpuDGAT1, C. avigera var pulcherrima, CpuDGAT1(KU055625); AtDGAT1, Arabidopsis, AtDGAT1 (CAB44774); BnDGAT1, B. napus, BnDGAT1 (AF164434); RcDGAT1,R. communis RcDGAT1 (XP002514132); VfDGAT1, Vernicia fordii, VfDGAT1 (ABC94471); OeDGAT1, Olea europaea,OeDGAT1(AAS01606); EgDGAT1, E. guineensisDGAT1 (XP010924968); RnDGAT1, Rattus norvegicus, RnDGAT1 (NP445889);HsDGAT1,Homo sapiens, HsDGAT1 (NP036211). The residues blocked on red background are 100% conserved bases in sevenknown motifs (in dotted boxes) of DGAT1s. Black arrows show the CpuDGAT1 residues differing from those of other plantDGAT1s within the seven motifs. White triangles show the catalytic active site residues (Asn-381) and (His-417) found in allMBOAT family enzymes (Chang et al., 2011). The ER retrieval motif is indicated with an orange box (within motif VII).

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CpuDGAT2_A, CpDGAT2_B, and CpuDGAT2_C, wereidentified from these studies. Based on RT-PCR analy-sis, CpuDGAT1 was determined to be seed specific,whereasCpuDGAT2_A,CpuDGAT2_B, andCpuDGAT2_Cwere found to be constitutively expressed inC. avigera varpulcherrima (Fig. 1). Based on its expression patternthat reflects the seed-specific occurrence of medium-chain fatty acids inC. avigera var pulcherrima,CpuDGAT1was chosen for functional characterization. CpuDGAT1encodes a 493 amino acid polypeptide with 59%, 54%,and 50% amino acid sequence identity with DGAT1polypeptides from Arabidopsis, Brassica napus, andE. guineensis and # 39% identity with mammalianDGAT1s (Supplemental Fig. S1). As shown in Figure 2,CpuDGAT1 has seven motifs that are conserved amongdicot and monocot DGAT1 polypeptides, includingcatalytic active site residues (Asn-381) and (His-417)in Motif VII that are characteristic of members ofthe membrane-bound O-acyltransferase (MBOAT)enzyme family. The 78-amino acid terminus ofCpuDGAT1 upstream of Motif I shares little or noidentity with other plant DGAT1s and containsapproximately 30 fewer amino acids than found inthe N-terminal region of the Arabidopsis DGAT1(AtDGAT1).

Functional Characterization of CpuDGAT1

CpuDGAT1 was initially expressed in the TAG-deficient Saccharomyces cerevisiae H1246 mutant(Sandager et al., 2002) to determine if it encodes anactive DGAT1. No TAG production was detected byTLC analysis of S. cerevisiae H1246 cells expressingCpuDGAT1 (Fig. 3A). Given the variant nature of theCpuDGAT1 N-terminus, a truncated form lacking thefirst 70 amino acids was tested and found to restoreTAG biosynthesis to S. cerevisiae H1246 cells (Fig. 3A).Although no TAG production was detected by TLCanalysis, expression of CpuDGAT1 was able to rescuelipotoxicity of S. cerevisiae H1246 cells grown in ex-ogenous 250 mM 8:0 or 10:0, but not 18:1. By contrast,expression of AtDGAT1 rescued lipotoxicity associatedwith supplementation of 18:1 in media, but not with 8:0or 10:0 supplementation (Supplemental Fig. S2). Amongthe three DGAT2-like genes, only CpuDGAT2_C func-tionally complemented TAG biosynthesis in S. cerevisiaeH1246 cells (Fig. 3, B and C).

As an alternative approach for characterizingthe function of CpuDGAT1, extracts of developingwild-type camelina seeds or transgenic camelinaseeds expressing CvFatB1, CvFatB1+CpuDGAT1, orCvFatB1+CvLPAT2+CpuDGAT1 (described below) wereassayed for DGAT activity using oleoyl (18:1)-CoA anddiolein or decanoyl (10:0)-CoA and didecanoin as sub-strates. Extracts of seeds expressing CpuDGAT1 hadapproximately 4-fold higher activity with the 10:0substrates than those lacking CpuDGAT1, but nosignificant difference in activity was detected with18:1 substrates among seed extracts of the different

lines (Fig. 4). As additional evidence that CpuDGAT1is associated with medium-chain fatty acid metabolism,Agrobacterium infiltration of Nicotiana benthamianaleaves with cDNAs for CpuDGAT1, the 8:0- and10:0-producing FatB from C. hookeriana, ChFatB2,and the Arabidopsis transcription factor Wrinkled1(AtWRI1), all under control of CamV35S promoters,resulted in the accumulation of TAGs containing8:0 and 10:0, which were not detected in leavesinfiltrated with expression cassettes containingcDNAs for AtWRI1 only or AtWRI1 and ChFatB2(Supplemental Fig. S3).

Seed-specific CpuDGAT1 Overexpression Enhances 10:0Content in Camelina Seeds

CpuDGAT1 was further examined for its ability toenhance 8:0 and 10:0 accumulation in camelina seedsresulting from expression of CvFatB1 encoding a spe-cialized Cuphea thioesterase (Kim et al., 2015b). We alsoexamined the ability of CpuDGAT1 to increase the ac-cumulation of these fatty acids when coexpressed withthe previously described CvLPAT2 that encodes anLPAT specialized for in planta accumulation of 8:0and 10:0 (Kim et al., 2015a). These transgenes wereexpressed in camelina under control of strong seed-specific promoters. Seeds obtained from independenttransgenic lines at the T2 generation were screened forexpression of the transgenes (Supplemental Fig. S4) and

Figure 3. Analysis of neutral lipids from S. cerevisiae H1246 strainexpressing Cuphea and Arabidopsis DGATs. Thin layer chromato-graphic analysis of neutral lipids of S. cerevisiae H1246 strainexpressing Cuphea DGAT2 cDNAs (CpuDGAT1_A, CpuDGAT2_B,and CpuDGAT2_C ), full-length CpuDGAT1, CpuDGAT1 lackingthe coding sequence for the first 70 amino acids (CpuDGAT1trunc),and the Arabidopsis DGAT1 (AtDGAT1) in the vector pYes2.CpuDGAT1trunc, CpuDGAT2_C, and AtDGAT1 expression func-tionally complement TAG biosynthesis in S. cerevisiae H1246mutant. A, Neutral lipids from yeast cells expressing CpuDGAT1 andCpuDGAT1trunc. B, Neutral lipids from control yeast cells (pYes2)and yeast cells expressing CpuDGAT2_A and CpuDGAT2_B. C,Neutral lipids from yeast cells expressing CpuDGAT2_C andAtDGAT1.

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for fatty acid composition (Supplemental Fig. S5). In-creases in amounts of 10:0 were observed in seeds ofthese lines (Supplemental Fig. S5). Amounts of 10:0ranged from 4 mol % to 13.5 mol % in T2 single seeds oflines from CvFatB1+CpuDGAT1 expression, which wasnearly 2-fold higher than from expression of CvFatB1alone (Supplemental Fig. S5 A; Kim et al., 2015b).Amounts of 10:0 ranged from 12mol % to 21.5 mol % inT2 single seedsof lines fromCvFatB1+CvLPAT2+CpuDGAT1expression, which was up to 3-fold higher than fromexpression of CvFatB1 alone (Supplemental Fig, S5;Kim et al., 2015b).Subsequent analyses of fatty acid compositions were

conducted on seeds from homozygous T4 lines. Thelines chosen were those that were found to accumulatethe highest amounts of 10:0 from screening of seedsat the T2 generation. The amount of 10:0 in seedTAGs from the CvFatB1+CpuDGAT1 line was 14.5mol % versus 8.0 mol % of 10:0 in seeds from CvFatB1expression alone. In addition, seed TAGs of theCvFatB1+CvLPAT2+CpuDGAT1 line contained 23.7mol%of 10:0 mol % versus 11.1 mol % of this fatty acid in seedTAGsof theCvFatB1+CvLPAT2 line (Fig. 5A).Alsonotablewas the detection of 8:0 in seed TAGs at amounts of2.8 mol % in the CvFatB1+CvLPAT2+CpuDGAT1 line,which were not detected in seed TAGs from CvFatB1 ex-pression alone. In addition, seed TAGs from all lines

expressing CvFatB1 had increased amounts of 12:0, 14:0,and 16:0with partially compensating decreases in amountsof linoleic (18:2), a-linolenic (18:3), and gondoic (20:1) acids(Fig. 5A).

Coexpression of CpuDGAT1 and CvLPAT2 PromotesAccumulation of 10:0 at the TAG sn-2 Position

Stereospecific analyses of fatty acid species at thesn-2 position of seed TAGs of engineered lines wasconducted using a standard TAG lipase digestion pro-tocol to assess the effects of CpuDGAT1 expression on

Figure 4. DGATactivity in extracts of developing seeds from transgeniccamelina plants expressingCpuDGAT1. Measurement of DGATactivityin crude extracts from developing wild-type camelina seeds (22 DAF)and developing camelina seeds expressing CvFatB1 alone, coexpress-ing CvFatB1 and CpuDGAT1 (CvFatB1+CpuDGAT1), or coexpressingCvFatB1, CvLPAT2, and CpuDGAT1 (CvFatB1+CvLPAT2+CpuDGAT1).Results are for assays using [1-14C] 10:0-CoA andDAG species: 10:0/10:0and 18:1/18:1, respectively. Values are mean 6 SD (n = 3). Asterisks de-note statistically significant differences (*P, 0.0001) as compared towildtype, as determined by two-tailed Student’s t test. Wt, wild type.

Figure 5. Fatty acid composition of TAG and sn-2 position of TAG inmature seeds of wild-type camelina and camelina lines engineered forexpression of CvFatB1 alone or with combinations of CpuDGAT1 andCvLPAT2. A, Fatty acid composition of TAG inwild-type camelina seedsand camelina seeds engineered for expression of CvFatB1 alone, withCvLPAT2 (CvFatB1+CvLPAT2) or CpuDGAT1 (CvFatB1+CpuDGAT1),and with the combination of CvLPAT2 and CpuDGAT1 (CvFatB1+CvLPAT2+CpuDGAT1). Values shown are the means of mol % of each fatty acidand 6 SD of the mean for three biological replicates. Asterisks denotestatistically significant difference fromwild type (or CvFatB1 in the caseof 10:0) values at *P, 0.001, **P, 0.03 based on two-tailed Student’st test. B, Fatty acid composition of TAG sn-2 position of wild-typecamelina seeds and camelina seeds engineered for expression ofCvFatB1 alone, with CvLPAT2 (CvFatB1+CvLPAT2) or CpuDGAT1(CvFatB1+CpuDGAT1), and with the combination of CvLPAT2 andCpuDGAT1 (CvFatB1+CvLPAT2+CpuDGAT1). Values shown are themeans of mol % of each fatty acid and 6 SD of the mean for three bi-ological replicates. Asterisks denote statistically significant differencefrom wild type (or CvFatB1 in the case of 10:0) values at *P , 0.002,**P , 0.02 based on two-tailed Student’s t test. Wt, wild type.

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the TAG stereospecific accumulation of 10:0. Very lowamounts (#2 mol %) of 10:0 were detected in the TAGsn-2 position from seeds expressing CvFaB1 CvFatB1alone or with CpuDGAT1. More strikingly, the amountof 10:0 detected in the TAG sn-2 position was 38.4 mol% in seeds of the CvFatB1+CvLPAT2+CpuDGAT1 line,which was nearly 2-fold the amount found in the TAGsn-2 position of the CvFatB1+CvLPAT2 (Fig. 5B). Seedsfrom all of the transgenic lines had large reductions in18:3 amounts in the TAG sn-2 position relative to TAGsfrom wild-type seeds, but oleic acid (18:1) amountswere approximately 2-fold higher in the TAG sn-2 positionin CvFatB1, CvFatB1+CvLPAT2, and CvFatB1+CpuDGAT1seeds, relative to wild-type seeds (Fig. 5B).

To confirm the positional distribution of 10:0 in TAG,liquid chromatography (LC) electrospray ionization-massspectrometry/mass spectrometry (ESI-MS/MS)was usedto identify the TAG molecular species in the engineeredcamelina seeds. TAG containing 10:0 in all three stereo-specific positions (tridecanoin) was detectable in seeds ofthe CvFatB1+CvLPAT2+CpuDGAT1 line, but not detect-able in seeds of theCvFatB1+CpuDGAT1 line (Fig. 6). TAGsfrom the CvFatB1+CvLPAT2+CpuDGAT1 seeds were alsomore enriched in molecular species containing 10:0/10:0in combination with the saturated fatty acids 12:0,myristic acid (14:0), palmitic acid (16:0), and stearic acid(18:0) as well as with C20 and C22 very long-chain fattyacids (Fig. 6). These results support the channeling ofDAG containing 10:0 in the sn-2 position for TAG bio-synthesis due to the apparent selectivity of CpuDGAT1for these molecular species.

CpuDGAT1 and CvLPAT2 Coexpression Increases 10:0Accumulation in DAG, but Little 10:0 Accumulation IsDetected in PC

The fatty acid composition of DAG, a substrate forPC and TAG synthesis, was examined in camelina

seeds expressing CvFatB1 in combination withCpuDGAT1 or CpuDGAT1 and CvLPAT2 to gain in-sights into howCpuDGAT1mediates fatty acid flux inseeds. In seeds from all of the transgenic lines, 10:0,12:0, and 14:0 were detected in DAG, but absent fromDAG of wild-type seeds (Fig. 7A). The largest differ-ence observed in DAG from seeds of the transgeniclines was the 2-fold higher amounts of 10:0 in DAG inCvFatB1+CvLPAT2+CpuDGAT1 seeds compared toDAG of seeds from other transgenic lines includingCvFATB1+CpuDGAT1 (Fig. 7A). These results sug-gest that, although CpuDGAT1 does not directlyparticipate in DAG synthesis, the coordinate activitiesof CvLPAT2 and CpuDGAT1 pull increased flux of10:0 through DAG. Despite the presence of 20 mol %of 10:0 in DAG of CvFatB1+CvLPAT2+CpuDGAT1seeds, no 10:0 was detected in PC of mature seeds(Fig. 7B), and in CvFatB1+CpuDGAT1 developingseeds at 22 d after flowering (DAF; SupplementalFig. S6). This observation indicates that 10:0-DAG iseither excluded from PC or is efficiently removed byacyl-editing.

10:0-CoA Is a Minor Component of acyl-CoA Pools inDeveloping Seeds Coexpressing CpuDGAT1 andCvLPAT2

Acyl-CoA pools were monitored in seeds at fourdevelopmental stages (10, 17, 22, and 30 DAF) in wild-type camelina plants and plants engineered for ex-pression of CvFatB1 alone or with CpuDGAT1 orCpuDGAT1 and CvLPAT2 (Fig. 8, A to D). In all of theselines, seed oil content was highest at 22 DAF, and17 DAF corresponded to approximately the midstageof seed oil accumulation. In addition, seeds of theCvFatB1+CpuDGAT1+CvLPAT2 lines had 10:0 amountsof 24 mol % at 17 DAF and 30 mol % at 22 DAF(Supplemental Fig. S7). Despite these levels of 10:0 in

Figure 6. ESI-MS/MS profiling of 10:0/10:0DAG-containing TAG molecular species inseeds of camelina lines engineered for ex-pression of CvFatB1 alone or with combi-nations of CpuDGAT1 and CvLPAT2. TAGspecies from camelina seeds engineeredfor expression of CvFatB1 alone (A), withCpuDGAT1 (B), or with CvLPAT2 andCpuDGAT1 (C) were analyzed by ESI-MS/MS. Shown are precursor 383.3 m/z pre-cursor scans of TAG species containing10:0/10:0 DAGs. The labeled TAG speciesindicate fatty acid composition, but thestereospecific arrangement cannot be de-termined from the analyses. Asterisks in-dicate unknown compounds.

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the seed oil, amounts of 10:0-CoA were typically,5% of the total acyl-CoA pools in the seeds ofthe transgenic lines at all stages of seed develop-ment. In addition, relative amounts of 10:0-CoAwere generally lower in developing seeds ofCvFatB1+CpuDGAT1+CvLPAT2 lines compared toseeds expressing only CvFatB1. This was most pro-nounced at 17 DAF and 30 DAF. At these devel-opmental stages, relative amounts of 10:0-CoA inCvFatB1+CpuDGAT1+CvLPAT2 seeds were 50% (17 DAF)

and 80% (30 DAF) lower than those in CvFatB1 seeds(Fig. 8, B and D). These results suggest that CpuDGAT1and CvLPAT2 efficiently metabolizes 10:0-CoA indeveloping seeds and that accumulation of 10:0 inthe engineered seeds is limited by the supply of10:0-CoA.

CpuDGAT1 and CvLPAT2 Coexpression Results in 10:0Production with Modest Effects on Seed Properties

Seed weight, fatty acid content, and germinationwere examined for wild-type camelina plants andcamelina lines engineered for expression of CvFatB1alone or in combination with CpuDGAT1, CvLPAT2,or CpuDGAT1+CvLPAT2 (Fig. 9). For these studies,plants were grown under the same greenhouseconditions. The average weight of 100 seeds fromwild-type plants and CvFatB1+CvLPAT2+CpuDGAT1differed by ,3% (79.0 mg wild-type versus 76.8 mgCvFatB1+CvLPAT2+CpuDGAT1; Fig. 9A). The largestdifference in seed weight between the transgenic linesand wild-type plants was observed in the CvFatB1+CvLPAT2 line, which had approximately 17% lower100-seed weight than wild-type plants. Weight for100 seeds from CvFatB1 was 10% lower, and forthat from CvFatB1+CpuDGAT1, it was 6% lower thanthose from wild-type plants (Fig. 9A). Germination ofseeds from wild-type plants and CvFatB1 and CvFatB1+CvLPAT2+CpuDGAT1 lines were not significantlydifferent, with germination efficiency of seeds rang-ing from 93% to 97% in these lines. Seed germina-tion, however, was reduced by approximately 16% inCvFatB1+CvLPAT2 and CvFatB1+CpuDGAT1 linesrelative to wild-type plants (Fig. 9B; Supplemental Fig.S8). Seed fatty acid content was calculated on a weightand molar basis (Fig. 9C), as well as moles and mg perseed (Supplemental Fig. S9). The latter calculationwas done because of the lower Mr of 10:0 in seeds oftransgenic plants relative to the C16-C20 fatty acidsfound in seeds of wild-type plants. Using both cal-culations, fatty acid content was significantly re-duced in seeds of lines expressing CvFatB1 alone aswell as lines expressing CvFatB1 in combination withCvLPAT2 or with CpuDGAT1. The largest differencesin fatty acid content relative to that in wild-typeseeds was detected in seeds coexpressing CvFatB1+CvLPAT2 (Fig. 9C). Seed fatty acid content was re-duced in these lines by 30% on a molar basis and 34%on a weight basis compared to wild-type plants. Thismay reflect a limited ability of the native camelinaDGAT to use DAG species containing 10:0 in thesn-2 position. By contrast, the fatty acid content ofseeds coexpressing CvFatB1+CvLPAT2+CpuDGAT1was not significantly different from that of wild-type seeds on a molar basis, but was 16% lower ona weight basis relative to the fatty acid content ofwild-type seeds. Overall, these findings suggestthat apparent CvLPAT2- and CpuDGAT1-mediatedchanneling of 10:0 through the Kennedy pathway for

Figure 7. Fatty acid composition of DAG and PC in mature seeds ofwild-type and engineered camelina lines expressing CvFatB1 aloneor in combinations with CvLPAT2 and CpuDGAT1. A, Fatty acidcomposition of DAG in wild-type camelina seeds and camelinaseeds engineered for expression of CvFatB1 alone, with CvLPAT2(CvFatB1+CvLPAT2) or CpuDGAT1 (CvFatB1+CpuDGAT1), and withthe combination of CvLPAT2 and CpuDGAT1 (CvFatB1+CvLPAT2+CpuDGAT1). B, Fatty acid composition of PC in wild-type camelinaseeds and camelina seeds engineered for expression of CvFatB1alone, with CvLPAT2 (CvFatB1+CvLPAT2) or CpuDGAT1 (CvFatB1+CpuDGAT1), and with the combination of CvLPAT2 and CpuDGAT1(CvFatB1+CvLPAT2+CpuDGAT1). Values shown are the means ofmol % of each fatty acid and 6 SD of the mean for three biologicalreplicates. Asterisks denote statistically significant difference fromwild-type values or CvFatB1 for 10:0 values at *P, 0.01, **P, 0.05based on two-tailed Student’s t test. Wt, wild type.

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TAG synthesis results in no or modest effects on seedcomposition and performance.

DISCUSSION

We identified a cDNA for C. avigera var pulcherrimaDGAT1 (CpuDGAT1) from transcriptomic data that isactive in vitro and in planta with 10:0-containingDAG and 10:0-CoA. We also showed that the coex-pression of CpuDGAT1 with the CvLPAT2 in a 10:0-producing camelina background not only enhancesthe total accumulation of 10:0 in TAG but also doublesthe content of 10:0 in the TAG sn-2 position relative toCvLPAT2 expression alone. These results provide di-rect genetic evidence for metabolic cooperation ofLPAT and DGAT for channeling of 10:0 through theKennedy pathway for TAG synthesis (Bafor et al.,1990; Fig. 10). This metabolic route is coupled with anapparent inability of the PC biosynthetic enzymesto use 10:0-containing DAGs. In addition to 10:0,CpuDGAT1 was also found to enhance accumulationof 8:0 in TAG in N. benthamiana agrobacterium-infiltration studies (Supplemental Fig. S3), indicating

that this enzyme is readily able to use 8:0-containingsubstrates, if sufficient pools of these substrates areavailable. These findings contribute to an increasedunderstanding of the extreme metabolic specializa-tion that enables many Cuphea species to accumulatemedium-chain fatty acids, including 8:0 and 10:0, toamounts .90 mol % in TAG.

The primary sequences of enzymes that are asso-ciated with the biosynthesis and metabolism ofmedium-chain fatty acids in seeds of Cuphea speciesprovide tools for dissecting the structural basis forthe substrate specificities of enzyme classes such asFatB thioesterases, LPATs, and DGATs. In the case ofthe DGAT1 enzyme, structural characterization ofthe substrate properties of plant and mammalianmembers of this enzyme class in plants and mam-mals has been conducted, despite the challenges as-sociated with their occurrence as integral membraneproteins. Of most significance to the findings repor-ted here, a cytosolic N-terminal region encompassinga predicted acyl-CoA binding domain has beenlinked to substrate specificities of mouse and B. napusDGAT1 (Weselake et al., 2006; Siloto et al., 2008;McFie et al., 2010). The predicted acyl-CoA binding

Figure 8. Acyl-CoA species in camelina developing seeds engineered for expression of CvFatB1 alone and with CpuDGAT1 andCvLPAT2+CpuDGAT1. Acyl-CoA species of camelina developing seeds at 10, 17, 22, and 30DAF fromwild-type, and transgeniclines expressing CvFatB1 alone, with CpuDGAT1, and with CvLPAT2+CpuDGAT1 were analyzed by LC-MS/MS. The data aremeans of mol % of each acyl-CoA species6 SD of three biological replicates. Asterisks denote statistically significant differencefrom wild type (or CvFatB1 in the case of 10:0) values at *P, 0.02, **P, 0.05 based on two-tailed Student’s t test. A, Acyl-CoAspecies of camelina seeds at 10 DAF. B, Acyl-CoA species of camelina seeds at 17 DAF. C, Acyl-CoA species of camelina seeds at22 DAF. D, Acyl-CoA species of camelina seeds at 30 DAF. Wt, wild type.

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domain of CpuDGAT1 has at least two residuesthat differ from conserved residues in other knownplant DGAT1s, including Asn-97 (Ser in other plantDGAT1s) and Glu-99 (Ala in other plant DGAT1s) aswell as one residue Arg-91 that differs from the Aspconserved in other plant DGAT1 and the human andmouse DGAT1s. The importance of these and otherlikely amino acid differences between CpuDGAT1and other plant and mammalian DGAT1s for deter-mination of substrate specificity remains to be de-termined. In addition, although the mechanism wasnot studied, the ability of CpuDGAT1 (SupplementalFig. S2), in contrast to AtDGAT1, to rescue the lipo-toxicity of exogenous 8:0 and 10:0 to S. cerevisiaestrain H1246 may provide the basis for screening forengineered DGAT1s with activity for 8:0- and 10:0-CoA, using procedures similar to those previouslydescribed (Siloto et al., 2009).The use of camelina in these studies allowed for ex-

amination of acyltransferase properties in a 10:0-nullbackground. Just as importantly, expression of genesin camelina allowed for agronomic and metabolic

assessment of 10:0 production in an oilseed crop. Tothis end, we showed that the coexpression of a 10:0-producing FatB thioesterase along with CpuDGAT1 andCvLPAT2 results in the accumulation of 10:0 to 25 mol %of TAG with no measurable effect on seed germinationand weight and modest effect on seed oil content forplants grown under greenhouse conditions. It is possiblethat the accumulation of higher concentrations of 10:0in TAG can be achieved by inclusion of a specializedGPAT with increased activity for 10:0-CoA to acylate thesn-1 position of the glycerol backbone in the Kennedypathway. Although such activity has been previouslydetected in seeds of one Cuphea species (Bafor andStymne, 1992), we did not detect a GPAT9 gene with seed-specific expression in seed transcriptomes of C. viscosissimaand C. avigera var pulcherrima. Down-regulation ofthe native LPAT and DGAT activities in the ER ofcamelina seeds may also enhance amounts of 10:0 ac-cumulation. These enzymes maintain an apparentmetabolically separate pathway for TAG synthesisinvolving typical C16 and C18 fatty acid-containingsubstrates. In addition, suppression of native

Figure 9. Seed weight, germinationefficiency, and total fatty acid contentof wild-type camelina seeds and seedsof engineered camelina lines express-ing CvFatB1 alone or in combinationswith CvLPAT2 and CpuDGAT1.Shown are 100-seed weight (A), ger-mination efficiency (B), and total fattyacid content (C) of mature seeds fromwild-type camelina and camelinaseeds engineered for expressionof CvFatB1 alone, with CvLPAT2(CvFatB1+CvLPAT2) or CpuDGAT1(CvFatB1+CpuDGAT1), and withthe combination of CvLPAT2 andCpuDGAT1 (CvFatB1+CvLPAT2+CpuDGAT1). Values shown for100-seed weight are the meansand 6 SD of the mean for four bi-ological replicates. Values for seedfatty acid content are the meansand 6 SD of the mean for three bio-logical replicates. Asterisks indicatestatistically significant difference(*P , 0.005, **P , 0.025) as com-pared to the wild type based ontwo-tailed Student’s t test. Valuesfor germination efficiency are themeans and 6 SD of the mean forthree biological replicates. Asterisksdenote statistically significant differ-ence (*P , 0.005, ***P , 0.05) ingermination efficiency of transgenicseeds as compared to the CvFatB1+CvLPAT2+CpuDGAT1 seeds basedon two-tailed Student’s t test. Wt,wild type.

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camelina acyl-ACP synthetase activity may reduce thepossible recycling of 10:0 to fatty acid synthase, asindicated by recent studies in Arabidopsis (Tjellströmet al., 2013). Furthermore, the relatively low content of10:0 in acyl-CoA pools in the developing engineeredcamelina seeds (Fig. 8) suggests that plastid production

of 10:0 is the major limiting factor for accumulation ofthis fatty acid in the transgenic seeds. Although di-vergent FatBs, such as CvFatB1, are primary deter-minants of 10:0 content in Cuphea seeds, it is likely thatadditional specialization is present inCuphea fatty acidsynthase to generate elevated amounts of the FatB10:0-ACP substrate in C. viscosissima seeds or FatB8:0-ACP substrate in C. avigera var pulcherrima seeds.For example, the Cuphea b-ketoacyl-ACP synthaseIV has been previously implicated in contributing toproduction of high levels of medium-chain fatty acidsin seeds of different Cuphea species (Dehesh et al.,1998; Leonard et al., 1998; Slabaugh et al., 1998). Inaddition, fatty acyl-CoA synthetases with specific-ities for medium-chain fatty acyl-CoAs cannot beexcluded as a factor limiting 10:0 accumulation incamelina seeds, given the reported importance offatty acyl-CoA synthetases with specificity for CoAesters of 16:0, 18:0 and longer chain fatty acids forseed oil accumulation (Schnurr et al., 2002; Jessenet al., 2015).

A notable observation from the studies with cam-elina is the absence of detectable amounts of 10:0 inPC in mature seeds of 10:0-producing lines express-ing CvLPAT2 and CpuDGAT1 alone or together.These findings suggest that 10:0-containing DAGs areprecluded from PC synthesis by CDP-choline phos-photransferase (CPT) or phospholipid:DAG choline-phosphotransferase (PDCT) activities or that 10:0 isefficiently edited or removed from PC by lipolyticactivities. Even in developing seeds at 20 DAF,amounts of 10:0 detected in PC in seeds expressingCvFatB1 alone or in combination with CpuDGAT1were approximately 1 mol % of the total PC fattyacids (Supplemental Fig. S6). This finding differsfrom results in engineered B. napus seeds that ac-cumulated 10:0 in PC to approximately 12 mol %during seed development, but at seed maturity, 10:0accounted for 1.3 mol % of PC fatty acids (Wiberget al., 2000). Our findings can be interpreted that 10:0flux largely occurs through the Kennedy pathway forTAG synthesis rather than being directed firstthrough PC, as is the typical route for fatty acids suchas 18:1 (Bates, 2016; Fig. 10). However, confirmationof this requires more detailed flux analyses. Otherpossibilities to explain the lack or low amounts of 10:0in PC include rapid editing of PC for selective re-moval of 10:0 or direct transacylation of 10:0-DAG toTAG, precluding flux through PC.

Overall, our findings point to a viable path formetabolic engineering of 10:0-rich oils in existing oil-seed crops for biofuel and industrial applications thatappears to only be limited currently by the ability offatty acid synthase to generate 10:0 substrate. Ourstudies also provide information to guide under-standing of the structural basis for extreme functionaldivergence in the DGAT1 family. Finally, our findingshighlight the synergy of LPAT and DGAT substratespecificities for generating preferential intermediatesto drive production of novel oil compositions.

Figure 10. A proposed pathway for 10:0-rich TAG assemblyin transgenic camelina seeds coexpressing CvLPAT2 andCpuDGAT1. Acyl-CoA species of 10:0 and 18:1 are sequentiallyesterified to glycerol-3-P (G3P) at sn-1 and sn-2 positions by GPATand LPAT, forming LPA and PA. The release of P from PA by phos-phatidic acid phosphatase forms DAG. The subsequent acylationof DAG at sn-3 catalyzed by DGAT forms TAG. DAG can be con-verted to PC via the action of DAG/CPT and/or PDCT. DAG mole-cules containing 10:0 at the sn-2 position arise from thespecialized activity of the Cuphea LPAT CvLPAT2. DAGs contain-ing 10:0 at the sn-2 position are selectively used for acylation with10:0-CoA at the sn-3 position by activity of the specialized Cupheadiacylglycerol acyltransferase CpuDGAT1 to form TAG speciesenriched in 10:0. DAG molecules containing 10:0 appear to bepoor substrates for PC synthesis, or 10:0 is rapidly removed fromPC formed from 10:0-DAG species. As such, 10:0 accumulation inTAG appears to proceed primarily by the Kennedy pathway ratherthan through PC via activity of enzymes including PDCT andphospholipid/diacylglycerol acyltransferase, which catalyzes thetransfer of fatty acids at the sn-2 position of PC to the sn-3 positionof DAG producing TAG and lysophosphatidylcholine. The PC routeof fatty acid flux for TAG synthesis involves primarily flux of 18:1for desaturation to 18:2 and 18:3. LPC, lysophosphatidylcholine;PAP, phosphatidic acid phosphatase; PDAT, phospholipid/diacylglycerolacyltransferase.

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MATERIALS AND METHODS

Plant Material and Growth Conditions

Studies were conducted with camelina (Camelina sativa var Suneson) thatwas grown under greenhouse conditions with supplemental lighting as de-scribed in Kim et al. (2015b). Except where indicated, seeds analyzed were fromhomozygous transgenic lines in the T4 generation.

RNA Isolation

Total RNA was isolated from developing seeds, leaves, stems, flowers, androots of Cuphea avigera var pulcherrima and cDNA for semiquantitative PCRamplification of CpuDGAT1, CpuDGAT2_A, CpuDGAT2_B, and CpuDGAT2_Cwas prepared from DNAase-digested total RNA as described in Kim et al.(2015a). Total RNA from developing seeds of transgenic camelina lines wasisolated using the method described in Bekesiova et al. (1999).

Cloning of DGATs from C. avigera var pulcherrima

The CpuDGAT1 (1482 bp), CpuDGAT2_A (1005 bp), CpuDGAT2_B (948 bp),and CpuDGAT2_C (1005 bp) gene sequences were identified among the lipidbiosynthesis genes in the C. avigera var pulcherrima 454 transcriptome sequencedata (Kim et al., 2015b). The open reading frames corresponding to the iden-tified DGATs were PCR amplified from C. avigera var pulcherrima cDNA usingthe gene-specific primers listed in Supplemental Table S1.

Preparation of CpuDGAT1 Yeast and PlantExpression Constructs

For yeast expression studies, the open-reading frames of the full-lengthCpuDGAT1, CpuDGAT1 truncated for the 70-amino acid N-terminal codingsequence (CpuDGAT1trunc), CpuDGAT2_A, CpuDGAT2_B, CpuDGAT2_C,and the ArabidopsisDGAT1were amplified using oligonucleotides provided inSupplemental Table S1 and cloned into the corresponding restriction sites of theyeast expression vector pYES2 under control of the GAL1 promoter.

For preparation of plant expression vectors, the open-reading frame forCpuDGAT1 was amplified using oligonucleotides in Supplemental Table S1and subcloned into the NotI site of pKMS3 (Nguyen et al., 2013) downstreamof the seed-specific soybean GLYCININ-1 promoter and upstream of theGLYCININ-1 39 UTR. This cassette was subsequently digested from pKMS3with AscI and cloned into the MluI site of the previously describedpBinGlyRed3_CvFatB1 (Kim et al., 2015b) yielding the plasmid pBinGlyRed3_CvFatB1+CpuDGAT1. The CvFATB1 coding sequence in this vector is undercontrol of the GLYCININ-1 promoter. An AscI cassette containing the CvLPAT2ORF under control of the seed-specific promoter for the soybean oleosin gene,generated as described in Kim et al. (2015a), was ligated into the AscI siteof pBinGlyRed3_CvFatB1+CpuDGAT1 to form pBinGlyRed3_CvFatB1+CpuDGAT1+CvLPAT2. The backbone of the vector is derived from pCAM-BIA0380 and was engineered with the DsRed marker gene under the controlof the constitutively expressed cassava mosaic virus promoter (Nguyen et al.,2013) for selection of transgenic seeds by fluorescence (Lu and Kang, 2008).

Camelina Transformation and Selection

The binary vector containing a cassette for seed-specific expression ofCvFatB1, CvLPAT2, and CpuDGAT1 was introduced into Agrobacterium tume-faciens by electroporation. Transformation of camelina plants was performedusing the floral vacuum infiltration method and transgenic mature seeds wereselected using DsRed protein fluorescence, the selection marker, as described inLu and Kang (2008). Expression of transgenes in developing seeds was con-firmed by RT-PCR (Supplemental Fig. S4) using gene-specific primers(Supplemental Table S1).

Phylogenetic Analysis

An unrooted phylogenetic tree of CpuDGAT1, CpuDGAT2_A, CpuDG-AT2_B, and CpuDGAT2_C deduced amino acid sequences, along with otheramino acid sequences homologous to DGAT1 or DGAT2, was generated usingthe neighbor-joining program in MEGA4 (http://www.megasoftware.net/mega4/; Tamura et al., 2007) with 1000 bootstrap replicates (Supplemental Fig. S1).

Yeast Transformation and Selection

Yeast expression constructs were introduced into Saccharomyces cerevisiaestrain H1246 (W303; MATa are1-D::HIS3 are2-D::LEU2 dga1::KanMX4 lro1-D::TRP1 ADE2 met ura3; Sandager et al., 2002) using the PEG/lithium acetatemethod (Gietz et al., 1995). Transformed cells were selected on minimal me-dium (YSM) containing 2% (w/v) dextrose and lacking uracil (Invitrogen). YSMcontaining 2% (w/v) raffinose was inoculated with transformed yeast cells andgrown at 28°C for 24 h with shaking (350 rpm). For induction, YSM containing2% (w/v) Gal was seeded atOD600 � 0.2 with the cells grown in raffinose. Cellswere then grown at 28°C for 40 h. For fatty acid feeding experiments, cultureswere grown for 2.5 h in YSM containing 2% Gal before adding 1% (w/v)Tergitol-40 and 250 mM of 8:0, 10:0, or 18:1. Cells were harvested by centrifu-gation, washed twice with 0.1% NaHCO3, freeze-dried, and used for fatty acidand TAG analysis. Lipids were extracted from cell pellets as described in Blighand Dyer (1959) and Zhang et al. (2013). Neutral lipids including TAG wereresolved on Silica Gel 60 (Merck) TLC plates using a solvent consisting of70:30:1 v/v) heptane:ethyl ether:acetic acid and detected with iodine vapor.

Agrobacterium Infiltration of Nicotiana benthamiana

TheChFatB2 andCpuDGAT1 open-reading frameswere cloned in the binaryvector pXZP393 and Arabidopsis thaliana WRINKLED1 (AtWri1; At3G54320synthetically produced by Eurofins MW Operon/Eurofins Genomics) wascloned in the binary vector pART27 downstream of a 35S promoter using theGATEWAY recombination system (Invitrogen). p19 (Qu and Morris, 2002), V2(Naim et al., 2012), and GFP (Wood et al., 2009) are described elsewhere.A. tumefaciens GV3101:pMP90 (Koncz and Schell, 1986), harboring the individualvectors, were grown in LB broth supplemented with appropriate antibiotics at28°C overnight. Coexpression was made with AtWRI1, GFP, and p19 or V2with Cuphea candidate genes, at a concentration of OD600 � 0.2 for each culture.Before infiltration, 100 mM acetosyringone was added to the mixture of cells.

A. tumefaciens infiltration was performed as described in Wood et al. (2009).Wild-type N. benthamiana plants were grown at 26°C, 60% relative humidity,and with a 12 h photoperiod (250 mmol/m2/s) for 6 weeks. Infiltration wasmade on young developing leaves on freshly watered plants until an area ofat least 7 cm2was infiltrated. The plants were returned to the growth cabinet foran additional 5 d, after which the infiltrated leaf areas were excised. When theinfiltrated area was not visible, a fluorescent protein flashlight (DFP-1; Night-Sea) was used to track the GFP expression. The harvested leaf tissues were flashfrozen and freeze dried.

Lipids were extracted (Bligh and Dyer, 1959), separated with TLC, andanalyzed using gas chromatography (model no. GC-17A; Shimadzu) as de-scribed in Doan et al. (2012) and Grimberg et al. (2015). Fatty acid profiles andquantities of TAG were calculated using reference mixture Me 63 (LGC Stan-dards) and internal standard triheptadecanoin (50 to 100 nmol).

Collection of Developing Seeds from Transgenic CamelinaLines for Fatty Acid Profiling

Seeds from transgenic camelina lines CvFatB1, CvFatB1+CpuDGAT1, andCvFatB1+CvLPAT2+CpuDGAT1 were grown in a greenhouse at the same time.Fully opened flowers were tagged for developing seeds to be collected at timepoints of 10, 17, 22, and 30 DAF.

Acyl-CoA Analyses

Acyl-CoAs were extracted and analyzed bymass spectrometry as describedin Larson et al. (2002) and Kim et al. (2013) from camelina seeds at 10 and17 DAF that were flash frozen in liquid N2 and stored at 280°C until used.

DGAT Assay

DGAT activity assays were conducted according to a method described inZhang et al. (2013). [1-14C] Acyl-CoA substrates (50 to 60 mCi/mmol) wereprepared as described in Zhang et al. (2013) or purchased (American Radiola-beled Chemicals). Assays were done using extracts from developing seeds(22 DAF). Seed extracts were prepared by grinding seeds in 50 mM HEPESpH 7.5, 50 mM NaCl, 20% glycerol containing 10 mL/mL protease inhibitor (forplants; Sigma-Aldrich). The homogenates were filtered through Miracloth(Calbiochem). Protein concentration in seed extracts was measured using a

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bicinchoninic acid assay (Smith et al., 1985) with purchased reagents (Sigma-Aldrich).

Total Fatty Acid Quantification and Lipid Analyses

Total oil was quantified by gas chromatographic analysis of fatty acid methylesters generated from camelina seeds and a triheptadecanoin internal standardusing the methodology described in Kim et al. (2015b). Total lipids were extractedfor analysis of TAG, DAG, and PC fatty acid composition (Bligh and Dyer, 1959;Zhang et al., 2013). Camelina seeds (30 mg) in 13 3 100-mm glass tubes wereground using a TH Homogenizer (Omni) in 3 mL methanol:chloroform (2:1 v/v).After 1 h incubation at 25°C, 1 mL of chloroform and 1.9 mL of water were added.The solution was mixed thoroughly and spun at 4000 rpm in a clinical centrifugefor 10min. The organic phase containing total lipids was transferred to a new glasstube, and used for separation of TAG, DAG, and PC using solid phase extractioncolumns (Supelco Supel Clean LC-Si SPE columns; Sigma-Aldrich). Lipid extractsdried under N2 were redissolved in 1 mL of heptane and loaded onto solid phaseextraction columns preequilibrated with heptane. The TAG fraction was elutedwith 5 mL of heptane:ethyl ether 80:20 (v/v). DAGwas subsequently eluted with3 mL chloroform:acetone 80:20 (v/v). Columns were washed with 6 mL of ace-tone followed by elution of phospholipids with 5 mLmethanol:chloroform:water100:50:40 (v/v/v). To the phospholipid fraction, 1.3 mL chloroform and 1.3 mLwater were added. After mixing and centrifugation, the phospholipids recoveredin the organic layer were dried under N2. The phospholipids were redissolved in100 mL chloroform and separated by TLC on Silica 60 plates (Merck) in a solventsystem consisting of chloroform:methanol:water:30% ammonium hydroxide(65:35:3:2.5 v/v/v/v). Bands from the TLC plates corresponding to PC werescraped and transferred to 13 3 100-mm glass tubes for fatty acid com-positional analyses. Transesterification of total lipids, TAG, DAG, and PCfractions was done in 1 mL of 2.5% sulfuric acid in methanol by heating at90°C for 30 min. Upon cooling, 1 mL H2O and 0.5 mL heptane were addedfollowed by vortexing and centrifuging at 4000 rpm for 5 min. Heptanephase-containing FAMEs was transferred to autosampler vials. FAMEswere analyzed on a model no. 7890A gas chromatograph (Agilent Tech-nologies) fitted with a 30-m length 3 0.25 mm i.d. HP-INNOWax Column(Agilent Technologies) using instrument conditions as described in Kimet al. (2015b).

The purified TAG described above was also used for stereospecific analysesas described in Cahoon et al. (2006) and Kim et al. (2015b).

Neutral Loss ESI-MS/MS Analysis of TAG Species

MSanalyseswere conducted using amodel no. 4000QTRAPLinear Ion TrapQuadrupole Mass Spectrometer (Applied Biosystems) to characterize TAGmolecular species according to the method described in Kim et al. (2015a).

Accession Numbers

GenBank Accession Numbers are as follows: CpuDGAT1, KU055625;CpuDGAT2_A, KU055626; CpDGAT2_B, KU055627; CpuDGAT2 C, KU055628.

Supplemental Data

The following supplemental materials are available.

Supplemental Table S1. Oligonucleotides used in these studies.

Supplemental Figure S1. Unrooted phylogram of C. avigera var pulcherrimaDGAT1 and DGAT2s with other hypothetical and functionally charac-terized DGATs.

Supplemental Figure S2. Lipotoxicity resistance of yeast cells expressingCpuDGAT1 to medium-chain fatty acids.

Supplemental Figure S3. Fatty acid composition of TAG from N. benthami-ana leaves infiltrated with AtWRI1 alone, with ChFatB2, or CpuDGAT1.

Supplemental Figure S4. Transcript analysis of Cuphea genes expressed intransgenic camelina lines.

Supplemental Figure S5. Medium-chain fatty acid profile in seeds of T2camelina lines expressing CpuDGAT1.

Supplemental Figure S6. Fatty acid composition of phosphatidylcholine indeveloping seeds (22 d after flowering) of wild-type camelina seeds and

camelina seeds engineered for expression of CvFatB1 alone and withCpuDGAT1 (CvFatB1+CpuDGAT1). Wt, wild type.

Supplemental Figure S7. Decanoic acid accumulation during seed devel-opment in wild-type and transgenic camelina seeds expressing CvFatB1alone and with CpuDGAT1 or CvLPAT2+CpuDGAT1.

Supplemental Figure S8. Growth of camelina transgenic lines expressingCvFatB1 alone and with CpuDGAT1 or CvLPAT2+CpuDGAT1.

Supplemental Figure S9. Total fatty acid content of wild-type camelinaseeds and camelina seeds engineered for expression of CvFatB1 alone orin combination with CvLPAT2 and CpuDGAT1.

ACKNOWLEDGMENTS

We thank Tara Nazarenus for technical assistance and for maintenance ofcamelina.

Received December 14, 2016; accepted March 20, 2017; published March 21,2017.

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