Melanie Dauk, Patricia Lam, and Mark A. Smith. Introduction

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1 552 The role of diacylglycerol acyltransferase-1 and phospholipid:diacylglycerol acyltransferase-1 and -2 in the incorporation of hydroxy fatty acids into triacylglycerol in Arabidopsis thaliana expressing a castor bean oleate 12-hydroxylase gene 1 Melanie Dauk, Patricia Lam, and Mark A. Smith Abstract: Expression of oleate 12-hydroxylase genes in Arabidopsis results in the accumulation of hydroxy fatty acids in seed triacylglycerol (TAG). The pathways by which these unusual fatty acids become incorporated into TAG are not well understood. We expressed a fatty acid hydroxylase cdna in Arabidopsis mutant lines to assess the role of three enzymes implicated in TAG assembly in this species. Plants deficient in the expression of phospholipid:diacylglycerol acyltransferase-1 or -2 accumulated hydroxy fatty acids and showed no differences to equivalent transformed wild-type plants. Plants lacking diacylglylcerol acyltransferase activity were also able to accumulate hydroxy fatty acids in seed neutral lipids. Triacylglycerol species containing one and two hydroxy fatty acids were abundant, and small amounts of trihydroxy-tag were detected. These results indicate that individually, the three enzymes do not play a major role in the incorporation of hydroxy fatty acids into TAG. Key words: Arabidopsis thaliana, diacylglycerol acyltransferase, hydroxy fatty acids, phospholipid:diacylglycerol acyltransferase, triacylglycerol. Résumé : L expression des gènes de l oléate 12-hydroxylase dans l Arabidopsis conduit à l accumulation d acides gras hydroxylés dans le triacylglycérol de la graine (TAG). On connaît mal les sentiers par lesquels ces acides gras inhabituels s incorporent dans le TAG. Les auteurs ont exprimé un cadn de l hydroxylase d acides gras chez une lignée mutante de l Arabidopsis afin d évaluer le rôle de trois enzymes impliquées dans l assemblage du TGA chez cette espèce. Les plants déficients pour l expression du phospholipide:diacylglycérol acyltransférase-1 ou -2 accumulent des acides gras hydroxylés et ne montrent aucune différence avec des plants sauvages transforméséquivalents. Les plantes sans activité diacylglycérol acyltransférase sont également capables d accumuler des acides gras hydroxylés dans les lipides neutres de la graine. On observe une abondance en espèces de triacylglycérol contenant des acides gras hydroxylés 1 et 2, ainsi que de petites quantités de trihydroxy-tag. Ces résultats indiquent qu individuellement les trois enzymes ne jouent pas de rôle majeur dans l incorporation des acides gras hydroxylés dans le TAG. Mots-clés : Arabidopsis thaliana, diacylglycérol acyltransférase, acides gras hdroxylés, phospholipide:diacylglycérol acyltransférase, triacylglycérol. [Traduit par la Rédaction] Introduction Ectopic expression in Arabidopsis thaliana of oleate 12- hydroxylase genes isolated from plants and fungi leads to the accumulation of hydroxy fatty acids in the seed triacylglycerol (TAG), (van de Loo et al. 1995; Broun and Somerville 1997; Broun et al. 1998; Smith et al. 2003; Dauk et al. 2007; Meesapyodsuk and Qiu 2008). Despite many attempts, levels above 25% of total seed fatty acids have not been achieved by expression of a hydroxylase gene alone. The factors limiting the accumulation of higher levels of hydroxy fatty acids are not understood. Castor bean (Ricinus communis L.) seed oil contains nearly 90% ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid:18:1-oh) and this spe- Received 31 July Published on the NRC Research Press Web site at botany.nrc.ca on 9 June M. Dauk and M.A. Smith. 2 National Research Council Canada, Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada. P. Lam. Department of Botany, University of British Columbia, 6270 University Boulevard, Vancouver, BC V6T 1Z4, Canada. 1 This paper is one of a selection of papers published in a Special Issue from the National Research Council of Canada Plant Biotechnology Institute. 2 Corresponding author ( mark.smith@nrc-cnrc.gc.ca). Botany 87: (2009) doi: /b09-011

2 Dauk et al. 553 cies has been used as a model to study the pathways of hydroxy fatty acid biosynthesis and TAG assembly. Ricinoleic acid synthesis is catalysed by an oleate 12-hydroxylase enzyme (CFAH12) that utilizes oleic acid esterified to the membrane lipid phosphatidylcholine (PC) as substrate (Bafor et al. 1991). The hydroxy fatty acid is efficiently removed from PC and incorporated into TAG. In vitro assays have suggested that hydroxy fatty acids are removed from PC via a microsomal phospholipase A 2 activity, with subsequent activation of the free hydroxy fatty acid to ricinoleoyl-coa (Bafor et al. 1991). TAG assembly is currently considered to occur largely via the pathway proposed by Kennedy (Kennedy 1961) based on the sequential acylation of glycerol-3-phosphate catalysed by acyl-coa dependant acyltransferases (Bafor et al. 1991; McKeon et al. 1999). In Arabidopsis plants engineered to produce hydroxy fatty acids, endogenous enzyme activities must be responsible for the relocalization of hydroxy fatty acids from their site of synthesis on PC to TAG. The pathways by which hydroxy fatty acid containing TAG (OH-TAG) molecules are synthesized in Arabidopsis have not been characterized and the relative roles of the endogenous enzymes of TAG assembly are not known. To attempt to address the role of enzymes known or suggested to be involved in TAG biosynthesis in Arabidopsis, we expressed the CFAH12 gene in EMS mutant or T-DNA insertion lines deficient in the production of selected enzymes. Characterization of hydroxy fatty acid production in three such transformed lines is described. Target enzymes are diacylglycerol acyltransferase-1 (DGAT1), phospholipid: diacylglycerol acyltransferase-1 (PDAT1) and a close homologue of PDAT1 referred to as phospholipid:diacylglycerol acyltransferase-2 (PDAT2). The DGAT1 enzyme catalyses the final step of TAG assembly, the acylation of the sn-3 position of diacylglycerol (DAG) using acyl-coa (Stymne and Stobart 1987; Coleman and Lee 2004). In Arabidopsis this enzyme is encoded by a single gene at the TAG1 locus (At2g19450). Loss of DGAT1 expression results in a 30% 40% decrease in seed oil content and a dramatic change in seed fatty acid composition characterized by reduced oleic (18:1 D9 ) and eicosenoic acid (20:1 D11 ) levels and increased percentage of linolenic acid (18:3 D9,D12,D15 ) (Katavic et al. 1995; Hobbs et al. 1999; Routaboul et al. 1999; Zou et al. 1999). PDAT activity was first characterized in the yeast Saccharomyces cerevisiae, where the enzyme plays a major role in TAG assembly, catalyzing the transfer of acyl groups directly from phospholipids to DAG to produce TAG and lyso-phospholipids (Dahlqvist et al. 2000). In yeast, this represents an alternative, non-acyl-coa dependant, step in the TAG biosynthetic pathway. Two close homologues in Arabidopsis of the yeast PDAT were identified and are referred to as AtPDAT1 (At5g13640) and AtPDAT2 (At3g44830) (Stahl et al. 2004). AtPDAT1 has been characterized in vitro and has been demonstrated to catalyze the transfer of fatty acids from the sn-1 and sn-2 position of phospholipids to DAG, having highest activity with PC containing acyl groups with several double bonds, epoxy groups, or hydroxy groups (Stahl et al. 2004). Arabidopsis lines containing T- DNA insertions in the At5g13640 locus and deficient in PDAT1 expression have barely detectable microsomal Table 1. Oligonucleotide primer sequences. Name UR TC1NCO1 RCOH1 AtPDAT1F AtPDAT1R PDAT2FP PDAT2RP ADS2F ADS2R Sequence 5 -GGAAACAGCTATGACCATG-3 5 -GTGCCGACCATGGCCTCTGATGTTTTAT- CCT-3 5 -TGGTCCCATGGCTACTGTCATAACCAG- CAA-3 5 -CGCGAATTCATGCCCCTTATTCATCGGA-3 5 -GCGTCTAGACAGCTTCAGGTCAATACGC-3 5 -CCGGTTTAGAAAACTATCGTCCTTC-3 5 -CTCTCCGACATCCTCATCACATC-3 5 -GACATCAACGGTGGAGGAGAAC-3 5 -GTTGGCACTTTCACATCGGTC-3 PDAT activity, but no obvious seed oil phenotype (Stahl et al. 2004; Mhaske et al. 2005). These results suggest that while PDAT1 is not a major contributing enzyme in TAG biosynthesis in Arabidopsis, it may still play a role in controlling the fatty acid composition of membrane phospholipids. Although the activity of AtPDAT2 has not been reported, expression analysis indicates that the gene is highly expressed in developing seeds and a possible role in TAG biosynthesis has been suggested (Stahl et al. 2004). Materials and methods Vectors for plant transformation The constructs used for plant transformation (pms589 and pms755) encode a synthetic fusion of the soluble domain of tobacco cytochrome b 5 connected to the amino terminus of the castor bean oleate 12-hydroxylase. To create this fusion protein, a DNA fragment encoding the first 109 amino acids of Nicotiana tabacum (L.) cytochrome b 5 (accession X71441) was synthesized by PCR using primers UR and TC1NCO (Table 1) with the vector pntcyb5-tc1 (Smith et al. 1994) as a template. A fragment encoding the complete castor oleate 12-hydroxylase open reading frame was synthesized by PCR using primers UR and RCOH1 (Table 1) with the castor oleate hydroxylase cdna (accession U22378), cloned in vector pgem11zf ( ) (Promega Ltd., Southampton, UK), as a template. After restriction digestion, the PCR products were ligated together to generate the sequence encoding the b5::cfah12 fusion protein. The fusion protein was shown to be a functional oleate 12-hydroxylase by expression in the yeast Saccharomyces cereviseae (data not shown). Expression cassettes comprising the Lesquerella hydroxylase promoter (Broun et al. 1998), b5::cfah12 and an Arabidopsis oleosin terminator (kindly provided by M. Moloney, SemBioSys Inc., Calgary, Alta.) were cloned into the binary vector prd400 (Datla et al. 1992) to create pms589 (kanamycin selection), or into a binary vector derived from pbib-hyg (Becker 1990) to create pms755 (hygromycin selection). These vectors were used to transform Agrobacterium tumefaciens strain GV3101 (pmp90) (Koncz and Schell 1986) by heat shock. Arabidopsis lines and plant transformation The Arabidopsis thaliana (L.) Heynh mutant line AS11, containing an EMS-induced insertion in the TAG1 locus encoding DGAT1 (Katavic et al. 1995; Zou et al. 1999) was

3 554 Botany Vol. 87, 2009 Table 2. Total seed fatty acid composition of Arabidopsis lines expressing the b5::cfah12 transgene, and untransformed control lines. Fatty acid (mol %) Line n 16:0 16:1 18:0 18:1 D9 18:1 D11 18:2 18:3 20:0 20:1 D11 Col WT ± ± ± ± ± ± ± ± ±0.03 WT+b5::CFAH12 3* 8.83± ± ± ± ± ± ± ± ±0.02 WS PDAT1-KO ± ± ± ± ± ± ± ± ±0.12 PDAT1-KO+b5:: 3* 8.43± ± ± ± ± ± ± ± ±0.09 CFAH12 Col PDAT2-KO ± ± ± V ± ± ± ± ±1.75 PDAT2-KO+b5:: 3* 9.56± ± ± ± ± ± ± ± ±0.31 CFAH12 AS ± ± ± ± ± ± ± ± ±0.52 AS11+b5::CFAH12 3* 8.01± ± ± ± ± ± ± ± ±0.02 AS11+b5::CFAH12(T4) ± ± ± ± ± ± ± ± ±0.12 Note: Col, Columbia ecotype (accession); WS, Wassilewska ecotype (accession); n = number of samples analyzed from individual plants; asterisk (*), indicated. obtained from L. Kunst (University of British Columbia, B.C.). The Arabidopsis PDAT1-knockout (KO) line, a homozygous line containing a T-DNA insertion in the PDAT1 locus At5g13640 was provided by Prof. M. Pollard (Michigan State University, Mich.). An Arabidopsis PDAT2-KO line, homozygous for a T-DNA insertion in the PDAT2 locus At3g44830 (SALK_010854) was provided by D. Taylor and J. Xu (National Research Council of Canada Plant Biotechnology Institute). The ecotype of the Arabidopsis lines is given in Table 2. Wild-type (WT), AS11, and KO plants were transformed using the Agrobacterium-mediated floral dip method (Clough and Bent 1998). Transformed plants were identified by sprinkling seed from the dipped plants onto agar plates containing 50 mgml 1 kanamycin or 30 mgml 1 hygromycin as appropriate. Following vernalization at 4 8C for 64 h, the plates were transferred to a growth chamber with a controlled temperature of 24 8C and a 16 h photoperiod. After approximately 10 d, the surviving seedlings were transplanted to pots containing Sunshine Mix 3 soil (Sun Gro Horticulture; Vancouver, B.C.) and grown to maturity in a controlled growth chamber at 22 8C under constant light. Reverse-transcriptase polymerase chain reaction (RT- PCR) To obtain material for RT-PCR, plants were grown in soil, as described above. Total RNA was isolated from 2- week-old seedlings, or from developing siliques at the midstage of oil deposition, using the RNeasy Plant Mini Kit (Qiagen Inc., Mississauga, Ont.). One microgram of total RNA was used as a template for cdna synthesis using oligo dt and SuperScript II as described in the manufacturer s protocol (Invitrogen Inc., Burlington, Ont.). RT-PCR was carried out using 2 ml of cdna and Pfu Turbo (Stratagene, La Jolla, Calif.). Primers used to amplify the AtPDAT1 transcript were PDAT1F and PDAT1R (giving a product of 2031 bp), and to amplify the AtPDAT2 transcript were PDAT2FP and PDAT2RP (giving a product of 1966 bp). Primers used to amplify a control transcript (At2g31360) were ADS2F and ADS2R. All primer sequences are given in Table 1. Seed lipid analysis To determine total seed fatty acid composition, intact Arabidopsis seeds were transmethylated using methanolic HCl (1 moll 1 ), and fatty acid methyl esters (FAMEs) were separated by gas chromatography (GC) as described previously (Dauk et al. 2007). For analysis of glycerolipids by thin-layer chromatography (TLC), seeds were crushed and the oil extracted using chloroform methanol or hexane (Smith et al. 2003). Neutral lipids were separated using TLC with aluminium backed silica gel G60 plates (Whatman, Kent, UK) and hexane diethyl ether acetic acid (70:140:3 v/v) as developing solvent. Lipid spots were visualized by exposure to iodine. To determine the fatty acid composition of individual lipids, samples were applied to the TLC plates in bands. After development, slices were cut from the edges and centre of the plate and lipids in these slices were visualized with iodine. Lipids were then scraped from corresponding unstained areas of the plate and FAME prepared and analysed by GC as described above. Single seed TLC was conducted as described by Dauk et al. (2007). Data analysis Statistical analysis was conducted using Microsoft Excel. Calculation of the oleate derivative proportion (ODP) values was conducted using a slight modification of that described by Singh et al. (2001). The value is the sum of all fatty acids derived from oleic acid divided by the sum of oleic plus its derivatives (18:2% + 18:3% + 20:1 D11 %+ 20:2% + 20:3% + 22:1% + 18:1-OH% + 18:2-OH% + 20:1- OH% + 20:2-OH%) / (18:1% + 18:2% + 18:3% + 20:1 D11 %+ 20:2% + 20:3% + 22:1% + 18:1-OH% + 18:2-OH% + 20:1- OH% + 20:2-OH%). Results Verification of homozygous knockout lines The DGAT1 mutant line AS11 has been extensively characterized (Katavic et al. 1995; Zou et al. 1999) and has a characteristic seed fatty acid phenotype (Table 2) and reduced oil content. The fatty acid profile of elevated 18:3

4 Dauk et al :1 D13 20:2 20:3 22:0 22:1 D13 18:1-OH 18:2-OH 20:1-OH 20:2-OH Total OH ODP 1.94± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.02 T ± ± ± ± ± ± ± ± ± ± ±0.01 samples from independent samplings of seeds from the same plant; T, trace amount. Seed analysis was conducted using T 2 seed, except where and reduced 18:1 and 20:1, compared with WT, was used to confirm the identity of this line prior to transformation with the hydroxylase gene. Homozygous KO lines for PDAT1 and PDAT2 did not show an obvious seed fatty acid composition phenotype (Table 2; Mhaske et al. 2005) or change in oil content (Mhaske et al. 2005; D.C. Taylor, personal communication 2006). To confirm the identity of these lines we conducted RT-PCR using cdna from seedling (PDAT1) or developing silique (PDAT2). Material was chosen based on predicted expression patterns (Stahl et al. 2004). Results (Fig. 1) indicated that transcript was not being produced and verified that these were homozygous KO lines. Fig. 1. Verification of Arabidopsis knockout lines. RT-PCR was conducted using cdna from wild-type (WT) and two individual plants of the PDAT1-KO population (lanes 1 and 2, panel A) and PDAT2-KO population (lanes 3 and 4, panel B). Control primers were used to demonstrate the integrity of the cdna. Lane M contains molecular weight markers. Seed lipid profiles of primary transformants Primary transformants for the four lines (WT, AS11, PDAT1-KO, and PDAT2-KO) were grown to maturity and seeds were harvested. FAMEs were prepared from batches of around 100 seeds from each plant by direct transmethylation and fatty acid composition of the seed lipids was determined. In all lines, plants producing hydroxy fatty acids were observed (Fig. 2). The hydroxy fatty acids formed were ricinoleic acid, densipolic acid (12-hydroxyoctadec-cis-9,15-dienoic acid:18:2-oh), lesquerolic acid (14-hydroxyeicos-cis-11- enoic acid:20:1-oh), and auricolic acid (14-hydroxyeicoscis-11, 17-dienoic acid: 20:2-OH), as described previously (Broun and Somerville 1997; Smith et al. 2003). Transformed AS11 showed the largest range with samples containing up to 22.5% hydroxy fatty acids. Highest producing plants in the PDAT1-KO population were slightly higher than the highest plants in the WT population and the range of hydroxy fatty acid content in the PDAT2-KO population was slightly less than in the WT population. The seed fatty acid profiles of representative plants from each population, and from untransformed plants, is given in Table 2. All lines showed an increase in the percentage of 18:1 in the seed lipids and a decrease in polyunsaturated fatty acids 18:2 and 18:3 corresponding to the production of hydroxy fatty acids. Decrease in 18:3 levels was most dramatic in the AS11 line. A slight decrease in 20:1 was observed in all transformed lines except

5 556 Botany Vol. 87, 2009 Fig. 2. Distribution of hydroxy fatty acid content in populations of transformed Arabidopsis expressing the castor hydroxylase. Each point represents an individual plant. Fig. 3. TLC separation of seed neutral lipids. Lane 1, castor bean; lane 2, Transformed Arabidopsis AS11 plant expressing the castor hydroxylase (21.3% hydroxy fatty acid); lane 3, TAG standard. AS11 where levels increased. Long chain saturated fatty acid content (16:0 and 18:0) was slightly increased in all lines. Calculation of oleate derivative proportion (ODP) values showed a reduction in ODP for all transformed lines compared to their untransformed controls, indicating a reduced conversion of oleic acid to polyunsaturated or longer chain fatty acids. Transformed AS11 lines showed relatively lower accumulation of 20-carbon hydroxy fatty acids compared to the other lines. Glycerolipid analysis of AS11-hydroxylase lines To characterize the seed lipids of transformed AS11 plants, a line producing over 20% hydroxy fatty acids, and estimated by single seed TLC of segregation ratios to contain the transgene at two loci, was selected and taken to the T 4 generation by repeated self pollination. Pooled T 5 seed contained 21.3% hydroxy fatty acid (Table 2). As shown in Fig. 3, the predominant lipids in these seeds were TAGs containing zero, one, or two hydroxy fatty acids, accounting for approximately 38%, 35%, and 15% of total seed fatty acids, respectively. TLC separation of the seed lipids also revealed additional spots which were tentatively identified as 3-hydroxy-TAG (3-OH-TAG) and DAG containing a single hydroxy fatty acid (1-OH-DAG). Lipids separated by TLC were recovered and their fatty acid composition was determined by GC of FAME (Table 3). The hydroxy fatty acid content of the putative 1-OH-TAG, 2-OH-TAG, and 1- OH-DAG were close to the expected values of 33%, 66%, and 50%, respectively, whereas the spot corresponding to 3- OH-TAG contained only 63% hydroxy fatty acid. This lipid accounted for less than 1% of the total seed fatty acids. Discussion The pathways by which hydroxy fatty acids are incorporated into TAG in Arabidopsis plants engineered to produce these unusual fatty acids are not clear. The characterization and cloning of genes encoding enzymes involved in Arabidopsis seed lipid metabolism has enabled a reverse genetics approach to be taken to further understand this process. Ectopic expression of a hydroxylase cdna in plants with lesions in known genes is an approach that can be used to assess the relative roles of specific enzymes of seed lipid metabolism. In this report we examined the role of three enzymes that have been implicated in the last step of TAG assembly, the acylation of the sn-3 position of DAG to form TAG. As reviewed by Lung and Weselake (2006) there appear to be at least two possible pathways that can result in the transfer of a fatty acid to the sn-3 position of DAG. These are referred to as the acyl-coa dependant pathway, in which the acyl donor is acyl-coa, and the acyl-coa independent pathway, in which an acyl group is transferred directly from a phospholipid such as PC or neutral lipid such as DAG. In plants, multiple enzymes appear to be involved in both pathways.

6 Dauk et al. 557 Table 3. Fatty acid composition of neutral lipids extracted from seeds of a transformed AS11 line expressing the b5::cfah12 gene. Fatty acid (mol %) Fraction of seed Lipid neutral lipids (%) 16:0 18:0 18:1 18:2 18:3 20:0 20:1 18:1-OH 18:2-OH 20:1-OH Total OH- TAG OH-TAG OH-TAG OH-TAG OH-DAG Other* Note: Other*, includes DAG. The enzyme DGAT1 is the most extensively characterized component of the acyl-coa mediated pathway in plants and has been shown to play a major role in TAG assembly in Arabidopsis. Loss of DGAT1 activity reduces, but does not abolish, TAG biosynthesis (Katavic et al. 1995) suggesting that other TAG forming activities play a significant role in TAG assembly in this species. A second enzyme from plants that has been clearly demonstrated to have acyl-coa dependant DAG acyltransferase activity is DGAT2, identified by homology to fungal and mammalian DGATs (Lardizabal et al. 2001). Based on evidence from tung tree (Shockey et al. 2006) and from castor bean (Kroon et al. 2006; Burgal et al. 2008) it has been suggested that DGAT2 isoforms play an important role in the incorporation of unusual fatty acids into TAG. Co-expression in Arabidopsis, for example, of cdnas encoding DGAT2 and the oleate hydroxylase from castor bean enhances the accumulation of hydroxy fatty acids in seed oil (Burgal et al. 2008). The role of DGAT2 in plants that do not make unusual fatty acids has not been determined. In vitro assays in which the activity of Arabidopsis DGAT2 was compared with that of castor DGAT2 indicated that the Arabidopsis enzyme is essentially inactive towards hydroxy fatty acid containing substrates (Burgal et al. 2008). Activity of this enzyme towards other substrates is unknown. Our study also includes two enzymes, PDAT1 and PDAT2, which may represent an alternative, non-acyl-coa dependant pathway for TAG assembly in which the acyl group is transferred to DAG directly from a phospholipid (Stahl et al. 2004). PDAT2 has not been characterized in detail; however, in vitro assays with PDAT1 suggest a role for this enzyme in the removal of hydroxy fatty acids from membrane phospholipids (Stahl et al. 2004). Enzymes catalyzing transacylation reactions between DAG molecules (Stobart et al. 1997) in which an acyl group from DAG is transferred to the sn-3 position of a second DAG molecule to produce TAG and a monoacylglycerol have not yet been identified in plants. Expression of the castor hydroxylase in the seeds of PDAT1 and PDAT2 KO lines, resulted in plants containing hydroxy fatty acids at levels similar to transformed WT plants. Owing to the relatively small population sizes, it is not possible to say whether the difference in the range of hydroxy fatty acid production in the populations is significant. Analysis of fatty acid profiles from T 2 seeds suggests that there is no obvious difference in the accumulation of hydroxy fatty acids compared to the transformed WT lines. All showed the increased 18:1 content and lowering of ODP characteristic of the expression of oleate 12-hydroxylase genes in Arabidopsis (Broun and Somerville 1997; Smith et al. 2003). Relative levels of ricinoleic acid, densipolic acid, and lesquerolic acid were also similar in the three lines. Plants lacking PDAT1 or PDAT2 expression did not appear to show any changes in oil content compared with WT, and at the hydroxy fatty acid levels reported in Table 2 (around 6.5%) we did not see significant differences in oil content correlating to hydroxy fatty acid production (results not shown). The oil content of lines containing higher percentages of hydroxy fatty acids was not determined. We have previously reported a reduction in seed oil content in

7 558 Botany Vol. 87, 2009 lines of Arabidopsis expressing an oleate hydroxylase and accumulating hydroxy fatty acids in excess of 15% of total seed fatty acids (Dauk et al. 2007). The suggested role of PDAT in plants is the removal of fatty acids from PC (Stahl et al. 2004). We did not examine in detail the level of hydroxy fatty acid in membrane phospholipids during seed development. However, any changes in the ability of the plant to remove ricinoleic acid from PC is likely to be reflected in changes in the ratios of hydroxy fatty acids in the seed oil and in ODP values, and we saw no evidence of this. For example, 18:1-OH remaining on PC for an increased length of time is more likely to be converted to 18:2-OH by the action of the endogenous FAD3 desaturase (linoleate D15 desaturase). Based on these results, PDAT1 and PDAT2 appear unlikely to play a significant role in hydroxy fatty acid metabolism in developing Arabidopsis seeds. Transformed Arabidopsis plants deficient in DGAT1 activity were able to accumulate hydroxy fatty acids in their seed TAG and, like WT plants expressing the hydroxylase, exhibited a decrease in ODP correlating to the amount of hydroxy fatty acid present. The range of hydroxy fatty acid content in seed from the transformed DGAT1-deficient line appears to be greater than we have observed in transformed WT and in our previous work with various fatty acid desaturase mutant lines (Smith et al. 2003). These observations merit further investigation. It seems unlikely that this is simply related to availability of 18:1 substrate, as the lines accumulated hydroxy fatty acids at levels equal to or higher than our previously transformed lines that lacked FAD2 desaturase and FAE1 elongase activity, and that accumulated over 80% oleic acid. It is possible that the higher percentage of hydroxy fatty acids reflects the relative activity of the hydroxylation reaction compared to the reduced rate of TAG assembly in this mutant line. As DGAT1-deficient lines accumulate less oil, the proportion of hydroxy fatty acids in the oil would appear higher. Further analysis of the transformed plants indicated that relative levels of the hydroxy fatty acids were not the same as those in transformed WT plants containing equivalent levels of hydroxy fatty acids. Considerably lower levels of 20:1-OH were found in the seed TAG. This fatty acid is synthesized by the elongation of ricinoleic acid catalysed in transformed Arabidopsis by the activity of the endogenous FAE1 condensing enzyme (Smith et al. 2003). Elongation is considered to occur while the acyl group is esterified to Coenzyme A. Reduced levels of 20:1-OH may reflect a reduction in the level of ricinoleoyl-coa or alternatively, could be related to the acyl selectivity of DGAT1. In Arabidopsis, studies of fatty acid distribution in storage lipids have shown that the major endogenous C20 fatty acid (20:1 D11 ) is preferentially incorporated into position sn-3 of TAG (Taylor et al. 1995). DGAT1 silencing leads to a decrease in overall 20:1 content corresponding to a reduction in the amount of 20:1 in the sn-3 position of TAG (Katavic et al. 1995). DGAT1 may therefore play a role in the incorporation of 20:1-OH into TAG in the transformed lines. As the enzymes responsible for sn-3 acylation of DAG in the AS11 mutant line have not been identified, reduced incorporation of the C20 fatty acids into TAG is also likely to be related to the acyl selectivity of the unknown enzymes responsible for this activity. The high 18:3 phenotype of the DGAT1-deficient lines has been suggested to be a result of the increased DAG levels allowing a greater flux between PC and DAG pools (via the activity of CDP-choline; sn-1,2 diacylglycerol choline phosphotransferase). This would result in enrichment of C18 polyunsaturated fatty acids (C18-PUFAs: 18:2 D9,D12 and 18:3 D9,D12,D15 ) owing to the activity of the FAD2 (oleate D12 desaturase) and FAD3 desaturases (Katavic et al. 1995). This mechanism would also be expected to result in an increase in relative levels of 18:2-OH in AS11 lines expressing the hydroxylase, as 18:1-OH can act as a substrate for FAD3-catalysed desaturation. A major increase in 18:2- OH content was not obvious in the transformed lines, except in lines with relatively low total hydroxy fatty acid content. In Table 2, for example, 18:2-OH was the predominant hydroxy fatty acid in the AS11 transformants in lines containing around 6.5% total hydroxy fatty acid, whereas in equivalent WT and PDAT-KO lines, ricinoleic acid predominated. In AS11 plants with higher total hydroxy fatty acid content, ricinoleic acid predominated. A more direct method to assess this activity is required. Transformed plants accumulated TAG with one and two hydroxy fatty acids with the relative amount of 2-OH-TAG appearing to be higher than observed in previous work where the hydroxylase was expressed in fad2/fae1 double mutant and fad3 mutant lines of Arabidopsis (Smith et al. 2003). The significance of this observation is unclear. TLC separation of lipids also suggested the presence of 3- OH-TAG in the transformed AS11 lines. GC analysis of FAME prepared from the spot isolated from the TLC plate was inconclusive. Using MALDI-TOF MS we detected an ion with a molecular mass corresponding to 3-OH-TAG (results not shown), but other unidentified components were also present. This lipid fraction represented less than 1% of the total seed neutral lipids. The 1-OH-DAG fraction from the transformed line is enriched in 18:1-OH, with relatively low levels of 18:2-OH and 20:1-OH. As this is a minor component of the oil we were unable to recover enough material to determine the stereospecific location of the hydroxy fatty acids in the 1-OH-DAG. The Arabidopsis DGAT1-mutant line is still able to accumulate TAG in its seed oil. It has not yet been reported whether there is up-regulation of alternative enzymes of TAG assembly to compensate for the loss of DGAT1 activity. A relative increase in 2-OH-TAG for example could be a result of altered DAG:DAG transacylase activity resulting from a larger pool of DAG, or up-regulation of transacylase activity. A more detailed study of this mutant line may help to clarify the role of the enzymes of the TAG biosynthetic pathways in TAG and OH-TAG assembly. The results of this study indicate that none of the three enzymes investigated appear to play a major role in the incorporation of hydroxy fatty acids into TAG. Hydroxy fatty acids accumulate in their absence, and the transformants show the characteristic decrease in ODP associated with hydroxy fatty acid production and suggested to be a result of their inefficient removal from PC (Thomaeus et al. 2001; Drexler et al. 2003; Cahoon et al. 2007). Similar investigations of other components of the TAG biosynthetic pathway are in progress.

8 Dauk et al. 559 Acknowledgments The authors acknowledge the financial support of National Research Council of Canada and Linnaeus Plant Sciences Inc.; and thank Miss Jelena Pistolic for assistance with plant transformation; Dr. J. Xu and Dr. M. Pollard for providing Arabidopsis KO-lines; D. Schwab for primer synthesis; Prof L. Kunst for helpful discussions; and Dr. David Taylor and Dr. Jitao Zou for critical review of the manuscript. Plant Biotechnology Institute publication number References Bafor, M., Smith, M.A., Jonsson, L., Stobart, K., and Stymne, S Ricinoleic acid biosynthesis and triacylglycerol assembly in microsomal preparations from developing castor bean (Ricinus communis) endosperm. Biochem. J. 280: PMID: Becker, D Binary vectors which allow the exchange of plant selectable markers and reporter genes. 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