Planta (2014) 240:599–610 DOI 10.1007/s00425-014-2122-2

ORIGINAL ARTICLE

A fatty acid condensing enzyme from Physaria fendleri increases hydroxy fatty acid accumulation in transgenic oilseeds of Camelina sativa Anna R. Snapp • Jinling Kang • Xiaoli Qi Chaofu Lu

•

Received: 19 May 2014 / Accepted: 4 July 2014 / Published online: 15 July 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Main conclusion Co-expression of a lesquerella fatty acid elongase and the castor fatty acid hydroxylase in camelina results in higher hydroxy fatty acid containing seeds with normal oil content and viability.

HFA-TAG molecules. These effects of LfKCS3 thus may effectively relieve the bottleneck for HFA utilization in TAG biosynthesis and the feedback inhibition to fatty acid synthesis, result in higher HFA accumulation and restore oil content and seed viability.

Producing hydroxy fatty acids (HFA) in oilseed crops has been a long-standing goal to replace castor oil as a renewable source for numerous industrial applications. A fatty acid hydroxylase, RcFAH, from Ricinus communis, was introduced into Camelina sativa, but yielded only 15 % of HFA in its seed oil, much lower than the 90 % found in castor bean. Furthermore, the transgenic seeds contained decreased oil content and the germination ability was severely affected. Interestingly, HFA accumulation was significantly increased in camelina seed when coexpressing RcFAH with a fatty acid condensing enzyme, LfKCS3, from Physaria fendleri, a native HFA accumulator relative to camelina. The oil content and seed germination of the transgenic seeds also appeared normal compared to non-transgenics. LfKCS3 has been previously characterized to specifically elongate the hydroxylated ricinoleic acid to lesquerolic acid, the 20-carbon HFA found in lesquerella oil. The elongation reaction may facilitate the HFA flux from phosphatidylcholine (PC), the site of HFA formation, into the acyl-CoA pool for more efficient utilization in triacylglycerol (TAG) biosynthesis. This was demonstrated by increased HFA accumulation in TAG concurrent with reduced HFA content in PC during camelina seed development, and increased C20-HFA in

Keywords Camelina sativa  Hydroxy fatty acid  Phosphatidylcholine  Physaria fendleri  Transgenic plant  Triacylglycerol

A. R. Snapp  J. Kang  X. Qi  C. Lu (&) Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT 59717-3150, USA e-mail: [email protected]

Abbreviations DAG Diacylglycerol ER Endoplasmic reticulum FAE1 Fatty acid elongation 1 FAMEs Fatty acid methyl esters GC–MS Gas chromatography–mass spectrometry HFA Hydroxy fatty acid LfKCS3 Lesquerella (Physaria) fendleri fatty acid condensing enzyme MAG Monoacylglycerol ODP Oleate derivative proportion PC Phosphatidylcholine RcFAH Ricinus communis fatty acid hydroxylase TAG Triacylglycerol VLCFA Very-long-chain fatty acid (C20 and longer) X:Y A fatty acid containing X carbons with Y double bonds

Introduction Plant oils containing hydroxy fatty acids (HFA) are desirable for a wide variety of renewable industrial applications such as lubricants and chemical derivatives such as nylon-11 (Ogunniyi 2006). The main commercial

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source of HFA is from the castor plant, Ricinus communis, which accumulates nearly 90 % ricinoleic acid, 12-hydroxyoctadec-cis-9-enoic acid (18:1OH), in its seed triacylglycerols (TAG) (Severino et al. 2012). Castor is considered to be unsuitable for large scale agricultural production due to the presence of the toxin ricin and highly allergenic 2S albumins in its seed (Chan et al. 2010). Another potential source of HFA may be from lesquerella (Physaria fendleri), whose seed oil contains up to 60 % lesquerolic acid, 14-hydroxyeicos-cis-11-enoic acid (20:1OH) (Dierig et al. 2011). Lesquerella oil is desirable for some of the same reasons as castor oil, the presence of the hydroxy group and monounsaturation of the oil, but additionally, the 2 carbons longer fatty acid chains of lesquerolic acid can impart better qualities to the oil such as increased resistance to friction and wear (Cermak et al. 2006; Dierig et al. 2011). However, lesquerella is not yet a commercial crop and requires more breeding efforts and research to improve its agronomic properties; additionally, the production will be limited to hot regions (Dierig et al. 2011). The desire to synthesize HFA and other industrial fatty acids in a more agronomically favorable oilseed crop has been the focus of research in the past decades (Snapp and Lu 2013). The results of these experiments have typically been disappointing, producing in most cases, plant lines with very low yields of the desired fatty acids (Broun and Somerville 1997; Cahoon et al. 1999; Lee et al. 1998; Suh et al. 2002). The biosynthesis of ricinoleic acid in castor is catalyzed by the oleate D12-hydroxylase (RcFAH) (van de Loo et al. 1995). Compared to *90 % HFA in castor seeds, heterologous expression of RcFAH in Arabidopsis seeds yielded disappointing levels of 17 % or less (Lu et al. 2006; Smith et al. 2003). These results underlie the need to understand the fundamental aspects of how plant fatty acids are synthesized and accumulated in seed oils. In oilseeds, fatty acid synthesis occurs exclusively in plastids and produces mostly oleic acid (18:1) and a small amount of palmitic acid (16:0) and stearic acid (18:0) (Ohlrogge and Browse 1995). These fatty acids are exported into the cytosol and may be subject to further modification before being incorporated into triacylglycerol: (1) 18:1 may be elongated into 20:1–22:1 by fatty acid elongase FAE1 (Kunst et al. 1992); (2) the majority of nascent fatty acids enter the membrane lipid phosphatidylcholine (PC) (Roughan and Slack 1982), where they can be modified by the ER-localized fatty acid desaturases to produce the polyunsaturated fatty acids linoleic acid (18:2) and a-linolenic acid (18:3) (Browse et al. 1993; Okuley et al. 1994; Sperling et al. 1993). As shown in Fig. 1, in many plant species, FAs esterified on PC are modified with

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Fig. 1 The proposed roles of LfKCS3 in channeling HFA (thick arrows) into triacylglycerol. The hydroxylated 18:1OH is formed from 18:1 on PC by RcFAH, then exits PC to enter the acyl-CoA pool and is elongated into 20:1OH by the LfKCS3. HFA may enter DAG through the Kennedy pathway by sequential acylation of glycerol-3phosphate (G3P) to produce lysophosphatidic acid (LPA) and phosphatidic acid (PA). The majority of 20:1OH is used to acylate the sn-3 position on DAG by DGAT. The efficient utilization of HFA may relieve the feedback inhibition (I) to fatty acid synthesis in the plastid

unusual chemical structures besides desaturation, such as hydroxylation in castor and lesquerella (Bafor et al. 1991; Jaworski and Cahoon 2003). The modified FAs then exit the PC to be used by the TAG biosynthetic acyltransferases (Weselake 2005), or may be transferred directly onto the sn-3 position of diacylglycerol (DAG) by the acyl-CoAindependent phospholipid:diacylglycerolacyltransferase (PDAT) (Dahlqvist et al. 2000; Zhang et al. 2009). It is therefore not surprising that the flux of FAs through PC and subsequent removal of modified FAs from PC have been identified as a major bottleneck for HFA accumulation in TAG (Bates and Browse 2011; Cahoon et al. 2006). Breaking this bottleneck would presumably need the enzymes from the native species that may have been coevolved with the unusual fatty acid modification and TAG assembly pathways (Lu et al. 2006). This hypothesis has been supported by experiments that significantly increased HFA accumulation in transgenic Arabidopsis seeds by coexpressing RcFAH with castor diacylglycerol acyltransferase (RcDGAT2), phospholipid:diacylglycerol acyltransferase (RcPDAT1A), or phosphatidylcholine: diacylglycerol cholinephosphotransferase (RcPDCT) (Burgal et al. 2008; Hu et al. 2012; van Erp et al. 2011). These enzymes either prefer HFA substrates, or may effectively remove HFA from PC, the site of its formation, to be accessible by acyltransferases during TAG biosynthesis.

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Another major hurdle that impedes oilseed biotechnology for the production of industrial FAs is the decreased oil content in transgenic seeds (Dauk et al. 2007; van Erp et al. 2011). The RcFAH-expressing Arabidopsis line CL37 exhibits roughly 30 % less oil than its parent fae1 lines (van Erp et al. 2011). This reduced oil accumulation was previously hypothesized as a result from b-oxidation of unusual FAs in transgenic plants (Moire et al. 2004). Recent experiments demonstrated that it was primarily due to inefficient utilization of unusual FAs during TAG bio assembly on the ER, which may consequently trigger an unknown mechanism that activates an endogenous pathway for post-translational reduction of FA synthesis within the plastid (Bates et al. 2014). Intriguingly, the castor enzymes RcGDAT2, RcPDAT1A and RcPDCT not only increased HFA content but also relieved the feedback inhibition of FA synthesis and at least partially restored TAG accumulation (Hu et al. 2012; van Erp et al. 2011). Here, we describe the transgenic production of HFA in an industrial oilseed crop camelina (Camelina sativa). For its favorable agronomic attributes such as low input and drought and cold tolerance, camelina has recently been the focus of research and development for potential bioenergy production (Lu et al. 2011; Moser 2010; Shonnard et al. 2010). Camelina is also an ideal platform for novel industrial products including unusual FAs due to its amenability to genetic transformation and a short life cycle (Collins-Silva et al. 2011; Lu and Kang 2008). Camelina is related to lesquerella, both are in the Brassica family, but camelina does not accumulate HFA-containing oils in their embryos. In lesquerella, a bifunctional fatty acid hydroxylase/desaturase is responsible for the HFA formation on 18:1-PC (Broun et al. 1998). In contrast to the highly homogeneous ricinoleic acid (18:1OH) in the endosperm of castor seeds, oils accumulated in lesquerella embryos contain predominantly 20 carbons (C20-HFA) (Hayes et al. 1995). The elongation in lesquerella is conducted by a fatty acid condensation enzyme, LfKCS3, which specifically acts on C18-HFAs (Moon et al. 2001). In this report, we generated transgenic camelina seeds producing hydroxy fatty acids. When LfKCS3 was coexpressed with RcFAH, the double-gene transgenic seeds accumulated nearly 150 % HFA level of the RcFAH only expressing lines, and showed normal germination ability. LfKCS3 also successfully restored the decreased oil content in the RcFAH lines, which was presumably caused by inefficient utilization of HFA and its subsequent inhibition to FA synthesis (Fig. 1). Our results opened a gateway to understand the biochemical mechanisms of HFA biosynthesis in lesquerella, which will help engineer these valuable fatty acids in an agronomically favorable crop.

601

Materials and methods Plasmid constructs A binary plasmid vector, pGP-RcFAH-LfKCS3, was constructed based on the pGATE-PHAS-RcFAH (Lu et al. 2006), and contained the R. communis fatty acid hydroxylase gene, RcFAH, and the Physaria (formerly Lesquerella) fendleri condensing enzyme gene, LfKCS3. Spectinomycin resistance was used for bacterial selection, and glufosinate resistance through the bar gene for plant selection. The LfKCS3 gene was expressed under the control of its native promoter and terminator, while the RcFAH gene was inserted under the control of the seed-specific phaseolin promoter and terminator. Lesquerella genomic DNA for the KCS3 gene was kindly provided by Dr. Ljerka Kunst (University of British Columbia), as part of construct MHS15: pGEM7-Lf in XL1-Blue E. coli cells (Moon et al. 2001). The LfKCS3 gene was excised by the restriction enzyme EcoRI, and inserted into the pGate-Phas-RcFAH (Lu et al. 2006) at the EcoRI site. The gene-specific primers, LfKCS3-1 (50 -TTACGTCCCGGATCTTAAGC-30 ) and LfKCS3-2 (50 -ATGAATAAACGGCCTGCCTG-30 ) were used to ascertain transformation events, and to check LfKCS3 gene expression by RT-PCR. Plant materials A C. sativa variety, Suneson, released by Montana State University, was used as the wild-type plant line. Plants were germinated as 5 seeds to a 600 or 800 pot and grown in a 1:1 mix of MSU soil (equal parts by volume of loam soil:washed concrete sand:Canadian sphagnum peat moss with AquaGro 2000 G wetting agent blended in at 1 lb/ cubic yard of soil. Aerated steam pasteurized at 70 °C for 1 h) and Sunshine Mix #1 (Bellevue, WA, USA). Greenhouse conditions were 228/18 ± 1 °C for day/night temperatures, a relative humidity of 30 %, and a 16 h photoperiod of natural lighting supplemented when necessary by season. Transgenic lines were created by transforming camelina plants following the established protocol in our lab (Lu and Kang 2008). Seed from mature Agrobacterium treated plants was harvested, and then planted out directly in flats to screen transgenics by spraying with glufosinate herbicide at a concentration of 23.4 ml/l (5.78 % glufosinate ammonium, Power Force Grass and Weed Killer, Bayer, Birmingham, AL, USA). Surviving plants were also tested for the presence of the RcFAH gene by PCR using primers 50 -ATGGGAGGTGGTGGTCGCAT-30 and 50 -TTAA TACTTGTTCCGGTACC-30 . The individual plant showing the highest hydroxy fatty acid amount was advanced to the T3 generation to obtain homozygous lines.

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Lipid analysis Total lipids were extracted from developing and mature seeds using a modified Blight and Dyer (1959) method as previously described (Hu et al. 2012). Two different methods were used to create fatty acid methyl esters (FAMEs) from total lipid samples to separate and identify fatty acids by gas chromatography (GC) and gas chromatography mass spectrometry (GC–MS). (1) TMSH method: trimethylsulfonium hydroxide (TMSH) preparation was used for the 96-well plate screenings as described previously (Lu et al. 2006). (2) Acid derivation method: FAMEs were derived from seeds in 1 ml of 2.5 % sulfuric acid in methanol at 80 °C for 90 min (Browse et al. 1986). FAMEs were injected into a Shimadzu 2010 GC fitted with a flame ion detector and a narrow-bore column (HP-Innowax 19091N-133; 30 m 9 0.25 mm i.d. 9 0.25 lm, 260 °C max temperature; Agilent Technologies). The oven temperature was programmed at 190 °C initially followed by an increase of 20 °C/min to 250 °C and maintained for 9 min. FAMEs were also run on GC–MS comprising an Agilent 7890A GC fitted with the same HP-Innowax column and a 5975C inert MSP with triple ONS detector. GC was programmed for a 12 min starting oven temperature of 190 °C and hold for 0.6 min then increase to 250 °C with a ramp time of 25 °C/min and hold at 250 °C for 7 min. A split– splitless inlet and helium carrier gas was used to run samples. Phosphatidylcholine (PC) was resolved by thin layer chromatography (TLC) on silica gel plates (silica gel 60, 20 9 20 cm, EMD Chemicals, Darmstadt, Germany) with a solvent system consisting of CHCl3/MeOH/H2O/30 % ammonium hydroxide, 65:35:3:2.5 by volume (Cahoon et al. 2006). The solvents used for separation of TAG from HFA-TAG were Hexane/anhydrous ethyl ether/formic acid, 70:30:1 by volume. Lipid bands were visualized under UV light after spraying with 0.005 % primuline in 80 % acetone, and TAG and PC were collected for GC analysis as below. Stereochemical analysis of TAG The HFA-TAG band fractions were obtained by TLC separation of lipids extracted from dry seed samples using castor oil as the standard, followed by the Blight and Dyer extraction from the silica gel into chloroform. All HFATAG samples were completely dried down under nitrogen gas to remove chloroform, then resuspended in 300 ll anhydrous ethyl ether. One ml of Tris buffer pH 7.8 with 500 mM CaCl2 was added to all samples. Quickly, 200 ll of Rhizomucor miehei lipase (Sigma, St. Louis, MO, USA) was added to the tubes and they were placed on a vortex

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shaker (Vortex Genie 2, Scientific Industries, Bohemia, NY, USA) at speed of 3 for 20 min. The digestion was stopped by the addition of 3 ml of chloroform/methanol, 2:1 by volume, and lipids were extracted by Blight and Dyer methods. Samples were run on TLC plates using the solvents of hexane/anhydrous ethyl ether/formic acid, 35:70:1 by volume. Non-digested castor and lesquerella oil as well as non-digested RcFAH-LfKCSoil samples were run as standards to aid in band identification. GC analysis was run on the GC–MS using the Agilent HP-Innowax 1909 1N-133 column. Oil content measurement and germination test Seed oil content was determined by a bench-top NMR seed analyzer (MQC23, Oxford Instruments, Concord, MA, USA) and by GC analysis using heptadecanoic acid (17:0) as internal standard (10 mg/ml), which was added to test tubes prior to FAMEs derivatization. For the germination test, 100 seeds of each of the three RcFAH-LfKCS lines were used and compared to three control RcFAH lines and non-transgenic seeds. Seeds were placed on filter paper dampened with sterile water in covered petri dishes. Water was replenished as needed when filter paper dried and seeds were left to germinate for 7 days at room temperature. Germination was determined by radicle emergence. All seeds used for comparison of oil content and germination rates were harvested from plants grown at the same time in the greenhouse.

Results and discussion LfKCS3 causes an increase in very-long-chain HFA content The Physaria (formerly Lesquerella) fendleri LfKCS3 has been shown previously functioning as a condensing enzyme which has little effect on 18:1 elongation but specifically elongates the hydroxy fatty acid 18:1OH into the very-long-chain hydroxy fatty acid, 20:1OH (Moon et al. 2001). Camelina has been successfully transformed with a castor fatty acid hydroxylase RcFAH and resulted in accumulation of mainly C18 hydroxy fatty acids (C18HFA) in its seed (Lu and Kang 2008). To determine the effect of 18:1OH elongation on HFA accumulation in transgenic camelina seed oil, we transformed LfKCS3 into camelina variety Suneson along with RcFAH. The construct pGP-RcFAH-LfKCS3 includes the RcFAH gene coding sequence behind a strong seed-specific phaseolin promoter (Sengupta-Gopalan et al. 1985), and the LfKCS3 genomic sequence including its native promoter, introns

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and the terminator. LfKCS3 expression is also seed-specific (Moon et al. 2001). For comparison, transgenic plants containing the RcFAH gene only were also created using the construct pGate-Phas-DsRed-RcFAH (Lu et al. 2006). A total of 25 RcFAH-LfKCS transgenic lines were obtained via the vacuum floral infiltration method and glufosinate herbicide (basta) selection. Also, 22 RcFAH transgenic lines were obtained by DsRed selection as described previously (Lu and Kang 2008). The fatty acid profiles in the seed oils of the T1 transgenic lines were determined in single seeds by gas chromatography–mass spectrometry (GC–MS). Both groups of transgenic seeds showed distinct peaks on the chromatographs compared to wild-type seeds, which were identified as hydroxy fatty acids, ricinoleic acid (18:1OH), densipolic acid (18:2OH), lesquerolic acid (20:1OH), and auricolic acid (20:2OH). The transgenic RcFAH-LfKCS lines contained a higher percentage of 20-carbon (C20) hydroxy fatty acids 20:1OH and 20:2OH than the control RcFAH only lines, with the majority of lines containing greater than twice the C20-HFA levels seen in RcFAH only plants (Fig. 2a). The highest C20-HFA level from the RcFAH lines was measured at 1.4 %, whereas the highest RcFAHLfKCS line was almost six times greater at 8.1 % verylong-chain hydroxy FAs, 20:1OH and 20:2OH (Fig. 2a). To determine whether the difference in C20-HFA between the RcFAH and the RcFAH-LfKCS lines was attributed to the presence of the LfKCS3 gene, we did the RT-PCR analysis of the RcFAH-LfKCS transgenic lines using RNA extracted from developing seeds of 16 days after flowering (DAF). The primers are specific for the Physaria LfKCS3 gene and do not recognize the native camelina FAE1 gene. Positive expression of the LfKCS3 gene was confirmed in RcFAH-LfKCS transgenics but not

Fig. 2 The percentage of HFA amounts in RcFAH and RcFAH-LfKCS T1 transgenic seeds. a C20-HFA content. b C18-HFA content. c Total HFA content. Each transgenic line is represented by the average value of HFA percentage in total oils of six single HFA-producing seeds. Average values of each group (RcFAH, squares; RcFAHLfKCS, round dots) are presented above straight lines

in the RcFAH only control lines (data not shown). These results indicated that the LfKCS3 gene was expressed in transgenic camelina at the early stages of oil filling, and resulted in a much higher accumulation of 20:1OH in the oil than is seen in lines lacking a LfKCS3 gene. The unsaturated 20:2OH may result from elongation of 18:2OH, a desaturated product of 18:1OH by the camelina FAD3 desaturase, as speculated in Arabidopsis (Smith et al. 2003). The native camelina elongase FAE1 does not have significant effects on HFA accumulation as very low amounts of C20-HFA were detected in RcFAH only lines (Table 1). Previous results also suggest that the Arabidopsis FAE1, which is almost identical to camelina FAE1 at sequence levels (Hutcheon et al. 2010), is not efficient in catalyzing the 18:1OH elongation (Broun and Somerville 1997). Therefore, we would not expect high levels of C20HFA even if the camelina FAE1 were overexpressed. We concluded that the lesquerella LfKCS3 gene, when transformed in its native genomic form, was functionally expressed in camelina. The LfKCS3 effectively elongated C18-HFA produced by the castor RcFAH and resulted in high levels of C20-HFA in transgenic camelina seeds. Co-expression of LfKCS3 with RcFAH causes an increase in total HFA As seen in our previous results (Lu and Kang 2008), expression of RcFAH alone in camelina resulted in the accumulation of *15 % HFA, which consisted almost entirely of 18:1OH and 18:2OH (Table 1). Co-expression of the LfKCS3 gene with RcFAH resulted in total HFA measures of up to *23 % in the highest HFA-accumulating RcFAH-LfKCS lines, an over 50 % increase (Fig. 2c). While total HFA levels differed, the amount of

a

b

c

9.0

16.0

25.0

8.0

14.0

13.6 12.7

7.0

19.4

20.0

12.0

6.0

5.7

10.0

15.0

13.8

5.0 8.0 4.0

10.0

6.0

3.0

4.0

2.0

5.0 1.1

1.0

2.0 0.0

0.0

C20-HFA

0.0

C18-HFA

Total HFA

123

604 Table 1 Fatty acid composition of HFA-accumulating homozygous T4 lines of RcFAH-LfKCS

Previously made T5 RcFAH line #7-1 (Lu and Kang 2008) is shown as a control. Data are mean values of six individual seeds from each plant. Minor FAs and SDs are not shown

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Line

Fatty acid composition (mol %) 16:0

18:0

18:1

18:2

18:3

20:1

Suneson

7.5

4.3

14.1

24.7

30.1

12.5

RcFAH#7-1

9.8

7.9

20.4

13.0

9.9

18:2OH

20:1OH

18.8

8.3

5.1

1.5

20:2OH

Total HFA

14.9

RcFAH-LfKCS #14-1

7.5

7.3

22.1

11.7

10.8

15.6

7.8

7.6

3.6

1.8

#17-6

7.1

6.7

22.8

11.8

10.9

16.2

7.1

6.9

4.4

2.1

20.5

#19-3

5.9

6.5

25.6

9.4

9.8

18.7

6.8

6.6

4.1

1.7

19.2

C18-HFA was relatively similar between the highest RcFAH-LfKCS lines and the control RcFAH only lines at 13–15 % (Fig. 2b). The transgenic lines did, however, differ greatly in the amount of C20-HFA they accumulated (Fig. 2a), and the distribution of hydroxy fatty acids contributing to the total HFA measurement (Fig. 2c). RcFAHLfKCS lines with a similar amount of C18-HFA to the RcFAH lines contained higher total HFA levels due to the addition of the extra C20-HFA to the hydroxy fatty acid profile. Homozygous RcFAH-LfKCS lines were obtained by pedigree analysis using herbicide resistance and the HFA contents in 12 single seeds per plant. Three homozygous lines were advanced to T4 generation, and their FA composition is shown in Table 1. All the three lines contained 5.4–6.5 % C20-HFA and about 20 % total HFA, compared with 1.5 and 14.9 %, respectively, in the control RcFAH line. These results indicate that the increase of HFA accumulation mediated by LfKCS3 is stably inherited through multiple generations. To examine the relationship of oleic acid (18:1) and its derivatives, desaturated and hydroxylated fatty acids, we calculated the oleate derivative proportion (ODP) as the proportion of the derivatives of oleate (18:2 ? 18:3 ? 20:1 ? 18:1OH ? 18:2OH ? 20:1OH ? 20:2OH) in the sum of oleate and all its derivatives. The ODP of the RcFAH-LfKCS lines was similar to that of the RcFAH lines at about 0.7, but both were lower than that of the nontransgenic line at *0.8. This was reflected in the higher accumulation of 18:1 and consequently 20:1 in all HFAcontaining seeds, as observed previously in transgenic Arabidopsis (Lu et al. 2006). The amount of saturated FAs was also increased in HFA-accumulating seeds as noted before (Table 1). These results suggest that HFA production in transgenic seeds affects metabolic flux towards desaturation, which mechanisms are unclear but may partly due to competition of the 18:1 substrate by the fatty acid desaturase and hydroxylase (Broun and Somerville 1997). However, the similar ODP between RcFAH and RcFAHLfKCS indicated that the increased HFA accumulation in the latter was not due to an increase in the amount of oleate

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18:1OH

20.9

used to make HFA, but rather a more efficient incorporation of HFA in the RcFAH-LfKCS lines. Taken together, it is clear that the addition of the C20HFA by co-expressing LfKCS3 helps achieve higher total content of hydroxy fatty acids in the oil profile of transgenic RcFAH-LfKCS lines. Stereochemical distribution of HFA in HFA-containing TAG To understand the mechanisms by which LfKCS3 increased HFA accumulation in transgenic camelina, we used thin layer chromatography (TLC) to separate total oils of homozygous RcFAH-LfKCS and RcFAH seeds. Visualization of lipids on TLC plates sprayed with primuline showed two major HFA-containing triacylglycerols, 1-hydroxy TAG (1-OH TAG) and a lower amount of 2-OH TAG, that co-migrated with the castor oil control (Fig. 3a). The bands were scraped from TLC plates and analyzed by GC. These two HFA-TAG fractions accounted for 37 and 34 % of total lipids in seeds of RcFAH-KCS (line 17-6) and RcFAH (line 7-1), respectively (Table 2). In both lines, the 1-OH TAG can be clearly separated from other lipids, and the HFA levels were close to the expected amounts of 33.3 %. However, the HFA levels were lower than the expected 66.7 % in 2-OH TAG at 55–56 %, probably because the 2-OH TAG fractions were found near the origin on the TLC plate and may contain other lipid species especially DAG (Fig. 3a). In fact, some HFAs were found in other lipids such as DAG and unesterified fatty acids (Table 2). The lower than expected HFA percentage for RcFAH lines was also observed in RcFAH-expressing Arabidopsis lines (Smith et al. 2003). GC analysis of the 1- and 2-OH TAG, and non-HFAcontaining TAG bands from the TLC plates revealed a differing composition of HFA between oil fractions (Table 2). The 1-OH TAG and 2-OH TAG contained all four types of HFA, 18:1OH, 18:2OH, 20:1OH, and 20:2OH, but the ratios of each HFA differed between the 1-OH to 2-OH TAG fractions. In the RcFAH-LfKCS oils, the 1-OH TAG derived most of its HFA content from

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605

a

b

c

TAG

18:1OH

FA

OH-FA

18:2OH

3OH-TAG 1OH-TAG

1OH-DAG

DAG

2OH-DAG

2OH-TAG

MAG OH-MAG

3OH-TAG

Polar lipids RcFAH -LfKCS

Suneson

Castor oil

Castor oil

Fig. 3 Seed lipids and their molecular species in transgenic camelina. a TLC separation of neutral lipids in RcFAH-LfKCS camelina line #17-6. Suneson camelina (untransformed control) and castor oils

RcFAH -LfKCS

are shown as controls. b TLC separation of TAG species in castor and RcFAH-LfKCS oils after partial digestion with lipase. c Gas chromatography of OH-MAG separated from RcFAH-LfKCS oil

Table 2 Fatty acid composition of different TLC oil fractions of neutral and polar lipids Line lipid classa

% of total lipids

Fatty acid composition (mol %)b 16:0

18:0

18:1

18:2

18:3

20:1

18:1OH

18:2OH

20:1OH

20:2OH

Total HFA

2.4

0.8

32.9 54.8

RcFAH TAG

56.9

9.7

11.1

26.5

12.7

12.9

18.2

1-OH TAG

31.1

8.4

9.9

16.4

4.8

5.4

15.5

17.8

11.9

2-OH TAG

2.8

4.8

5.7

15.5

4.5

5.6

7.7

28.4

13.7

9.8

3.0

Polar lipids

4.3

12.7

7.8

29.0

22.0

12.7

6.4

5.5

2.0

0

0

7.5

Other lipids

4.9

8.4

10.4

32.3

10.5

9.0

13.6

8.0

4.3

0.4

0

12.7

TAG

55.4

9.4

10.6

25.9

14.0

12.7

18.1

1-OH TAG

31.5

7.0

8.6

16.4

6.1

5.9

15.3

14.0

9.8

6.2

3.1

33.1

2-OH TAG Polar lipids

5.3 4.0

4.7 12.3

5.8 5.7

16.2 34.7

4.1 25.1

4.0 12.8

8.2 6.1

16.4 3.1

12.1 0

18.7 0

8.9 0

56.0 3.1

Other lipids

3.7

9.3

9.9

24.3

10.1

9.1

15.0

7.5

4.5

3.5

1.5

17.0

RcFAH-LfKCS

a

Other lipids include DAG and unesterified fatty acids

b

Minor fatty acids, e.g., 20:0, 20:2, 20:3, 22:0 and 22:1 are not listed

18:1OH to 18:2OH at 14.0 and 9.8 %, respectively. The 20:1OH and 20:2OH fatty acids only made up a combined 9.3 % of the HFA possible in the 1-OH TAG fraction. While a rise in HFA is to be expected in 2-OH TAG compared to 1-OH TAG, the amount of increase was not equal between the C18- and C20-HFAs. Incorporation of 18:1OH and 18:2OH into 2-OH TAG was measured at less than 15 % higher, but the incorporation of 20:1OH and 20:2OH almost tripled to a combined total of 27.6 %

(Table 2). A similar trend was observed in the RcFAH line where the C18-HFA level in 2-OH TAG was 1.4 times higher than in 1-OH TAG, and the C20-HFA level was four times higher despite only accumulating to 12.8 % of the total HFA level present in 2-OH TAG (Table 2). The 1-OH and 2-OH TAG bands were recovered from the TLC plate and digested with lipase to further elucidate the position of the HFA in TAG. Digestion with a sn-1 and sn-3 position-specific lipase revealed free HFA and the

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presence of sn-2 OH-MAG in the RcFAH-LfKCS samples (Fig. 3b). This result confirms that the HFA must be accumulating at both the sn-2 position, as evidenced by the presence of OH-MAG, as well as the sn-1 or sn-3 position to yield free HFA. The band corresponding to 1-OH MAG was analyzed via GC–MS and found to contain mainly C18-HFA with little to no C20-HFA (Fig. 3c). This result is consistent with similar findings in Arabidopsis and the knowledge that the RcFAH hydroxylates sn-2 position fatty acids on the PC (Bafor et al. 1991; Smith et al. 2003), and that the very-long-chain FAs including VLCHFAs are generally prevented from being incorporated onto the sn-2 position due to discrimination against such substrates by lysophosphatidic acid acyltransferases (Weselake 2005). Compared to the 1-OH TAG fraction, the 2-OH TAG fraction showed about 50 % decreased levels of the usual very-long-chain fatty acid 20:1 in both RcFAH and RcFAH-LfKCS oils. The decrease in 20:1 could be due to competition between the RcFAH and the native elongase (FAE1) for the same 18:1 substrate, or modified acyl pool competition between the 20:1 and 20:1OH fatty acids for incorporation into 2-OH TAG. These results also point to a preference for utilization of the longer chain HFA to make 2-OH TAG as compared to 1-OH TAG, which would cause increased C20-HFA thus a higher amount of total HFA for the RcFAH-LfKCS lines (Table 2). Based on recent research suggesting that most of the DAG used for TAG synthesis is derived from PC but not from de novo synthesis (Bates et al. 2009; Bates and Browse 2011), our results suggest, as shown in Fig. 1, that most of the 2-OH TAG may have resulted from a PC derived DAG containing a sn-2 C18-HFA that has a HFA added onto the sn-3 position by a DGAT. For 1-OH TAG, the sn-3 position of HFA-DAG was filled with anon-HFA. Alternatively, 1-OH TAG can be formed from 0HFA-DAG by DGAT using a HFA from the acyl-CoA pool, or by a PDAT using a C18-HFA from sn-2 position of HFA-PC. A smaller amount of sn-1-HFA-DAG should also be available for forming HFA-TAG. An increase of total HFA in TAG of the RcFAH-LfKCS lines may be mainly caused by efficient utilization of C20-HFA in the Kennedy pathway. Besides being used by the DGAT enzymes, C20-HFA may also be incorporated through de novo synthesis onto the sn1 position on DAG, which are inefficiently used for PC synthesis but are used directly for TAG formation. In lesquerella oils that contain *60 % of C20-HFA, these HFAs are found mostly on the sn-1/3 positions of TAG (Hayes et al. 1995), suggesting the major role of the Kennedy pathway in HFA accumulation in lesquerella. However, it is not clear how 18:1OH is almost completely removed from PC to enter the acyl-CoA pools in this native HFA accumulator.

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HFA accumulation in TAG and PC during seed development The increased HFA accumulation in the RcFAH-LfKCS lines raised the possibility that the LfKCS may enhance the C18-HFA flux from PC into the acyl-CoA pools for HFA elongation. Results shown in Table 2 indicate that the RcFAH-LfKCS polar lipids (mainly PC) contain only 40 % of HFA found in those of the RcFAH line. We thus investigated changes in the HFA content of both total oil and the polar PC fraction of transgenic seed during seed development. Seeds were collected at 12 days after flowering (DAF) when seed reached maximum size and started rapid accumulation of oil until maturation at around 35 DAF (Rodriguez–Rodriguez et al. 2013). The HFA does not start to accumulate at measureable levels until between 12 and 16 DAF, after which it increases rapidly (Fig. 4). The total oil samples showed a peak HFA content at 30 DAF, which may be followed by a slight decrease in HFA content in the mature seed oil. The RcFAH-LfKCS lines displayed a higher level of HFA in total oil throughout all developmental stages when compared to the RcFAH line (Fig. 4a). An increase in HFA content was also observed in the PC fraction during early stages of seed development (Fig. 4b). The highest HFA content was detected at 20 DAF for the RcFAH-LfKCS lines at nearly 7 %, but rapidly decreased afterwards and accumulated at *1.5 % in mature seeds. In contrast, in the RcFAH seeds, the HFA content continued to rise rapidly after 20 DAF and reached apeak amount of over 10 % at 24 DAF before it started to decrease. The mature RcFAH seeds still contained a considerably higher amount of HFA at nearly 5 %. These results clearly indicate that the lesquerella LfKCS3 causes an effect on a more efficient removal of HFA from PC, the site of its formation, in the RcFAH-LfKCS lines than in the RcFAH lines, and consequently more HFA is accumulated in the TAG of the LfKCS-expressing camelina lines. LfKCS3 restored oil accumulation and germination abilities To determine if HFA production in transgenic camelina affects seed quality, we quantified oil content and tested germination ability of the RcFAH and RcFAH-LfKCS lines. Three homozygous lines from each group were used in the experiments, which were grown side by side in the greenhouse. Seeds harvested from greenhouses weighed about 0.5–0.6 mg/seed, which was lower than those of camelina seeds harvested from field grown plants for Suneson (about 1 mg/seed) and those reported in the literature (Vollmann et al. 2007), probably due to suboptimal growth conditions in greenhouses. The seed weight differences between transgenic and wild-type seeds harvested at the same time

Planta (2014) 240:599–610

a 25

b 12 RcFAH

RcFAH RcFAH-LfKCS

RcFAH-LfKCS

10

HFA% in PC fraction

20

HFA% in Total oil

Fig. 4 Changes in the measured hydroxy fatty acid content of total oil and PC fraction in developing seeds in RcFAH#7-1 and RcFAHLfKCS #17-6 lines. a Percentage of HFA in total oil; b Percentage of HFA present in PC. Data represent mean ± SD of three replicates

607

15

10

5

8

6

4

2

0

0 12

16

20

24

30

Days after flowering

were not significant. We therefore compared the amounts of HFA and total FA in single seeds of different lines. As shown in Fig. 5b, RcFAH-LfKCS seeds accumulated about 150 % of HFA (*36 lg/seed) of the RcFAH seeds (*24 lg/seed). This increase of HFA content in the RcFAH-LfKCS seeds concurred with the increase of their total FA content to *175 lg/seed from the much reduced *145 lg/seed in RcFAH seeds, a level near the *185 lg/ seed of oil content found in non-transgenic seeds (Fig. 5c). The *20 % reduction of oil accumulation in the RcFAH camelina seeds in comparison with the wild type was also observed in RcFAH-expressing Arabidopsis (van Erp et al. 2011). It has been recently recognized that the inefficient utilization of HFA for TAG synthesis may be detected by an unknown mechanism that activates an endogenous pathway for post-translational reduction of fatty acid synthesis within the plastid (Bates et al. 2014). Another study in Brassica napus also proposed a mechanism for feedback inhibition of fatty acid synthesis by the accumulation of 18:1-acyl carrier proteins in the plastid that directly inhibits acetyl-CoA carboxylase (Andre et al. 2012). Our results of restored TAG accumulation in RcFAH-LfKCS suggested that LfKCS3 may effectively relieve the feedback inhibition in HFA-producing camelina seeds. As shown in Fig. 1, the LfKCS3 utilizes C18-HFA to synthesize C20-HFA that may be readily available to acyltransferases for TAG assembly. This increased efficiency of HFA utilization may reduce the rate of possible HFA degradation by b-oxidation (Moire et al. 2004), and also relieve the bottleneck proposed by Bates et al. (2014) that the futile cycle of C18-HFA in the acyl-CoA pool, in which HFA can be used for de novo DAG, but resulting HFA-DAG cannot be efficiently used for PC and TAG synthesis (Bates and Browse 2011).

35

12

16

20

24

30

35

Days after flowering

The oil content is a very important trait for oilseed crops. Some studies have identified quantitative trait loci (QTLs) for oil content that are associated with the FAE1 genes because of their correlation with the very-long-chain fatty acid contents of 20:1 in camelina (Gehringer et al. 2006) and erucic acid (22:1) in B. napus (Cao et al. 2010). Although we cannot rule out the contribution of LfKCS3, an FAE1-type condensing enzyme in lesquerella, to restored oil content in the RcFAH-LfKCS lines, our results strongly suggest its roles in relieving the feedback inhibition by ineffective utilization of HFA in TAG synthesis. LfKCS3 specifically elongates 18:1OH but has little effect on 18:1 (Moon et al. 2001). Also, the 20:1 content in the RcFAH line increased in comparison with non-transgenic seeds despite reduced oil content. We also tested germination of homozygous T4 or T5 transgenic camelina seeds that accumulate HFAs (Fig. 6). When seeds were plated on damp filter paper, nearly all non-transgenic seeds germinated the next day showing radicle emergence and followed by seedling growth (data not shown). RcFAH seeds showed much delayed germination, with a few seeds that showed radicle emergence on the second to third days, and reached a maximum germination rate ranging from 6 to 65 % by 7 days after imbibition. In contrast, all three of the homozygous RcFAHLfKCS lines tested displayed a rapid germination at ratios of 91–98 %, similar to non-transgenic seeds. Once germinated, no obvious difference in seedling growth was observed in all lines tested. It is obvious that LfKCS3 had rescued germination tardiness observed in RcFAH lines, but the cause is difficult to determine. Seed oil mobilization is not essential for germination in Arabidopsis (Kelly et al. 2011). Successful germination of the RcFAH-LfKCS lines indicates that HFA-TAG can be used to supply energy and

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20

80

Germination rate (%)

100

% total HFA

a 25

15

10

5

60

40

20

0 b 40 0 1

µg HFA/seed

30

2

3

4

5

6

7

Days after imbibition Fig. 6 Comparison of germination rates of transgenic camelina RcFAH (square) and RcFAH-LfKCS (diamond) lines

20

10

HFA in PC may disrupt membrane integrity and thus affect seed viability of the RcFAH lines.

0

c 200

Conclusion

µg total FA/seed

150

100

50

0 Suneson

RcFAH

RcFAHLfKCS

Fig. 5 Comparison of fatty acid accumulation in homozygous transgenic RcFAH #7-1, RcFAH-LfKCS #17-6 and non-transgenic camelina seeds. a Percentage of total HFA (see Table 1 for FA composition). b Amount of HFA per seed. c Amount of total FA per seed. Data represent mean ± SD of single seed analyses of 10 seeds from three independent plants for each genotype

carbon resource for seedling development. One plausible explanation is the difference in HFA remaining in PC between the two lines in developing seeds. It has been hypothesized that seed viability may be determined during seed maturation (Rajjou et al. 2012). The greater amount of

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It has been made clear that engineering high amounts of unusual fatty acids requires additional genes besides the FA modifier genes from their native accumulators (Lu et al. 2006). Here, we investigated the effect on hydroxy fatty acid accumulation in transgenic camelina, a non-HFA accumulator, by incorporating a fatty acid condensing enzyme from its relative species, P. fendleri, a native high HFA accumulator. The LfKCS3 enzyme, which specifically elongates the C18-HFA into very-long-chain C20HFA, effectively increased total HFA levels compared to RcFAH only transgenic camelina seed by adding C20-HFA content and creating more 2-OH TAG molecules. The increase of HFA in TAG was concurrent with decrease of HFA in PC at all stages of oil accumulation during seed development and in the mature seed. Our results suggest that LfKCS3 may play an important role in accumulating high amounts of HFA in its native host lesquerella, and coexpressing LfKCS3 with RcFAH may relieve the previously hypothesized bottleneck of the HFA flux through PC into TAG in transgenic seed (Bates and Browse 2011). Upon exiting PC, 18:1OH (and its desaturated product 18:2OH) enter the acyl-CoA pool, where they are elongated to 20:1OH (and 20:2OH)-CoA by the LfKCS3 enzyme. This may effectively prevent the HFA from re-entering PC through the acyl-editing cycle (Bates et al. 2012) and place

Planta (2014) 240:599–610

them in a different modified acyl-CoA pool that could be more readily utilized by TAG-synthesizing enzymes in the Kennedy pathway. Removal of these very-long-chain HFA-CoAs from the modified acyl-CoA pool may cause a net movement of HFA out of PC and into TAG due to the dynamic flux between the PC and acyl-CoA pools (Bates et al. 2012). The enhanced efficiency of HFA utilization in transgenic camelina also relieved the detrimental effect of HFA-induced feedback inhibition to fatty acid synthesis and decreased seed viability, as shown by restored seed oil content and germination ability. Our study opens an opportunity to understand the HFA-accumulating mechanisms in a native plant species, lesquerella, and demonstrates the possibility of creating higher HFA-containing oils in an agriculturally amenable relative crop plant, camelina. Acknowledgments The authors thank Dr. Ljerka Kunst (University of British Columbia) for kindly providing the lesquerella LfKCS3 gene construct MHS15. This work was supported by grants from U.S. Department of Agriculture and the Plant Genome Research Program of the U.S. National Science Foundation.

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