Plant Cell Rep (2016) 35:213–226 DOI 10.1007/s00299-015-1880-z

ORIGINAL ARTICLE

Enhancement of a-linolenic acid content in transgenic tobacco seeds by targeting a plastidial x-3 fatty acid desaturase (fad7) gene of Sesamum indicum to ER Rupam Kumar Bhunia1,2,4 • Anirban Chakraborty1 • Ranjeet Kaur1,2 Mrinal K. Maiti1,2,3 • Soumitra Kumar Sen1,2

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Received: 24 June 2015 / Revised: 25 September 2015 / Accepted: 7 October 2015 / Published online: 31 October 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract Key message Expression of sesame plastidial FAD7 desaturase modified with the endoplasmic reticulum targeting and retention signals, enhances the a-linolenic acid accumulation in seeds of Nicotiana tabacum. Abstract In plants, plastidial x-3 fatty acid desaturase-7 (FAD7) catalyzes the formation of C16 and C18 trienoic fatty acids using organellar glycerolipids and participate in the membrane lipid formation. The plastidial x-3 desaturases (FAD7) share high sequence homology with the microsomal x-3 desaturases (FAD3) at the amino acid level except the N-terminal organelle transit peptide. In the present study, the predicted N-terminal plastidial signal peptide of fad7 gene was replaced by the endoplasmic reticulum signal peptide and an endoplasmic reticulum retention signal was placed at the C-terminal. The Communicated by M. Petersen. A. Chakraborty and R. Kaur contributed equally.

Electronic supplementary material The online version of this article (doi:10.1007/s00299-015-1880-z) contains supplementary material, which is available to authorized users. & Soumitra Kumar Sen [email protected] 1

Advanced Laboratory for Plant Genetic Engineering, Indian Institute of Technology, Kharagpur 721302, India

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Advanced Technology Development Centre, Indian Institute of Technology, Kharagpur 721302, India

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Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302, India

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Present Address: Department of Biochemistry, Biophysics and Molecular Biology (BBMB), Iowa State University, Ames, IA 50011, USA

expression of the modified sesame x-3 desaturase increases the a-linolenic acid content in the range of 4.78–6.77 % in the seeds of transgenic tobacco plants with concomitant decrease in linoleic acid content. The results suggested the potential of the engineered plastidial x-3 desaturase from sesame to influence the profile of a-linolenic acid in tobacco plant by shifting the carbon flux from linoleic acid, and thus it can be used in suitable genetic engineering strategy to increase the a-linolenic acid content in sesame and other vegetable oils. Keywords Endoplasmic reticulum  Plastid  Sesamum indicum  Nicotiana tabacum  x-3 Fatty acid desaturase Abbreviations CaMV Cauliflower mosaic virus DHA Docosahexaenoic acid DPA Docosapentaenoic acid EPA Eicosapentaenoic acid FAME Fatty acid methyl ester ER Endoplasmic reticulum FA Fatty acids FAD3 Fatty acid desaturase-3 FAD7 Fatty acid desaturase-7 FAD8 Fatty acid desaturase-8 hptII Hygromycin phosphotransferase system II MCS Multiple cloning site MGDG Monogalactosyldiacylglycerol PC Phosphatidyl choline PUFA Polyunsaturated fatty acids Se Sesame TAG Triacylglycerols TFA Total fatty acid TA Trienoic fatty acid C18:1 Oleic acid

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C18:2 C18:3

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Linoleic acid a-Linolenic acid (ALA)

Introduction Human beings evolved on a diet that was balanced in x-6 and x-3 essential fatty acids intake, whereas western diets have a ratio of x-6/x-3 of 16.74 (Simopoulos 2001). The high percentage of x-6 and trans-FA combined with a lower percentage of x-3 FA in the human diet has been implicated in chronic diseases such as obesity, diabetes, cardiovascular and inflammatory diseases, whereas increased levels of x-3 fatty acid (a low x-6:x-3 ratio) exert suppressive effects (Simopoulos 2002; Harbige 2003). In plants, proportion of linoleic acid (C18:2, x-6 fatty acid) and a-linolenic acid (C18:3, x-3 fatty acid) varies significantly within different oil seed crops (Gunstone et al. 1994). The C18:3 is the precursor of some of the most important long-chain (LC) x-3 polyunsaturated FA (PUFA), viz., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are not synthesized in human (Sinclair et al. 2002; Liu et al. 2012). Humans cannot convert C18:2 into C18:3 because of the absence of x-3 fatty acid desaturase (x-3 FAD), which catalyzes the conversion of C18:2 into C18:3 in plants (Yadav et al. 1993); therefore, humans must obtain C18:3 from their diet. Long-chain polyunsaturated fatty acids (LC-PUFAs) are health beneficial for maintaining the cellular membrane by regulating cholesterol synthesis (Simopoulos 1991) and eicosanoid synthesis (Kankaanpaa et al. 1999; Damude et al. 2007). Research has shown that C18:2 or x-6 FAderived eicosanoids have general pro-inflammatory effects, whereas C18:3 or x-3 FA-derived eicosanoids have antiinflammatory effects (Tapiero et al. 2002). As a consequence, dietary recommendations now include a balanced proportion of x-6 and x-3 FA. Considering the increasing population of the world, consumption of edible plant oils is expected to double over the next 30 years (Chapman and Ohlrogge 2012). Hence, crop yield and nutritional quality need to be improved. Thus, development of oil seed crop plants with health-beneficial proportion of x-6 and x-3 FA is the dire need of the hour. Sesame (Sesamum indicum L.) of Pedaliaceae family is regarded as one of the most ancient oil seed crop and is cultivated worldwide. India stands as one of the major producers of this oil seed crop (3419.35 hg/ha) (FAOSTAT 2013). The oil content ranges from 35 to 55 % in different cultivars, with an average of 50 % of the seed weight (Ashri 1989). Furthermore, the sesame seed oil contains a group of compounds called lignans (dimers of phenyl

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propane units), which possess important health-beneficial effects like anti-oxidant property (Nakai et al. 2003), cholesterol lowering activity (Hirata et al. 1996), anti-inflammatory (Shimizu et al. 1991), anti-carcinogenic (Miyahara et al. 2000) and many more related activities. In one of our recent studies, analysis of FA compositions of Indian germplasms of sesame revealed presence of high level of both oleic acid (C18:1) and C18:2, but substantially low level (\1 %) of C18:3 (Bhunia et al. 2015a). The corresponding ratio of C18:2 to C18:3 is also very high (*32.18). Therefore, sesame cultivars with a balanced proportion of x-6 and x-3 FA could definitely add more value to the nutritional merit of this cheap and healthbeneficial source of vegetable oil, and thus could contribute significantly in ameliorating the world dietary x-3 fatty acid deficiency. The mechanism of FA biosynthesis and oil formation in plant cells have been extensively studied (Browse et al. 1986; Bates et al. 2013). Our group has recently reviewed such metabolic pathways and discussed possibilities of fatty acid metabolic engineering in sesame to generate nutritionally superior oil (Bhunia et al. 2015b). Membrane bound desaturases are of two categories: carboxyl-directed desaturases and methyl-directed desaturases. The carboxyldirected desaturases (front end desaturases) introduce a new double bond between the existing double bond and carboxyl-terminus of the fatty acyl chain, e.g., D6 desaturases. Methyl-end-directed desaturases introduce a new double bond between the existing double bond and methylterminus of the fatty acyl chain, e.g., D9, D12 and D15 (x-3) desaturases (Sayanova et al. 1997; Qui et al. 2001). These are the key enzymes in the production of PUFAs and therefore, hold significant biotechnological implication in plants. The nuclear-encoded x-3 fatty acid desaturases are localized in two separate cell organelles: FA desaturase-3 (FAD3) is specific to the endoplasmic reticulum (ER), while fatty acid desaturase-7 (FAD7) is plastid specific and localized in thylakoid membrane (Andreu et al. 2007). Microsomal x-3 desaturase and specific transcription factors (Nookaraju et al. 2014) catalyzes desaturation of C18:2 to C18:3 at position x-3 (Wakita et al. 2001; Anai et al. 2003) and control the level of C18:3 in TAG. Plastidial x-3 desaturase converts dienoic FA (C16:2 and C18:2) to trienoic FA (C16:3 and C18:3) (Browse et al. 1986). However, this C18:3 is not accumulated in the seeds, rather it participates in membrane lipid formation. In Arabidopsis as well as in other plant species, three x-3 FA desaturase (FAD) enzymes have been characterized. Two are plastidial enzymes (FAD7 and FAD8), located in the inner membrane of the chloroplast envelope (McConn et al. 1994; Chi et al. 2011) and one is microsomal (FAD3), located in the ER, facing the cytosol (Dyer and Mullen 2001; McCartney et al. 2004). The FAD3 uses cytochrome

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b5 as an electron donor to act upon FA esterified to phosphatidylcholine (PC). On the other hand, FAD7 utilizes ferredoxin as the intermediate electron donor to act upon fatty acids esterified to galactolipids, sulfolipid, and phosphatidylglycerol. It was speculated that despite the differences in electron donor, both the desaturases use C18:2 as a substrate with minor changes in the primary structure. It may be possible to functionally alternate one desaturase enzyme for the other by expressing genes with modified transit peptide sequences in transgenic plants (Iba et al. 1993). Presently, the major sources of x-3 FA comprise some deep sea fishes (e.g., salmon and tuna) and some edible oilseed plants (e.g., soybean, rape and flax). Among them, flax seed oil contains as high as 45–65 % of C18:3, making it useful for industrial purposes. However, this is not a popular edible source of x-3 FA, and thus there is urgent need to develop and expand plant sources with the recommended levels of C18:3 for human diet. In one of our recent studies, the seed-specific expression of heterologous soybean fad3 gene in sesame increased the C18:3 content from 1.30 to 6.21 % (Bhunia et al. 2014). Unfortunately, despite recent advances in sesame genome information (Zhang et al. 2013; Wang et al. 2014), the current understanding of the identity of the gene(s) in sesame that can facilitate conversion of C18:2 into C18:3 is still far from comprehensive. It has been reported that nuclear-encoded FAD7 protein contains N-terminal transit peptide sequence with all the genetic information required for their import into plastids (Iba et al. 1993). In a similar way, ER resident proteins also contain upstream signal sequences through which they are targeted to ER and additionally, they contain retention signals that prevent mislocalization to postER compartments such as Golgi, vacuolar and plasma membranes (Gomord et al. 1999; Pelham 2000). In the present study, the predicted chloroplast N-terminal transit peptide of sesame fad7 gene was replaced by the ER N-terminal transit peptide and KDEL ER-retention signal was placed at the C-terminal end to direct and subsequently localize the product in ER. Similar strategy was adopted earlier, where removal of the chloroplast targeting sequence and the addition of ER-retention signal to the C-terminus of sunflower FAD7 increased the activity by tenfold with concomitant enhancement of C18:3 FA profile as compared to over-expression of the native protein in yeast (Venegas-Calero´n et al. 2010). The modified gene thus could be used for the purpose of altering the C18:3 content in sesame seeds, given the present knowledge gap about the identity of fad3 gene in sesame. The potential of this molecular strategy was put to test in transgenic tobacco plants, most often used as a model for gene expression studies. The C18:3 content in the seeds of transgenic tobacco lines were indeed found to increase in the range of

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4.78–6.77 % of total seed lipid after expression of the modified fad7 gene. Thus, this approach will offer an opportunity to test an endogenous genetic element in enhancing the C18:3 content of seed storage lipid in sesame and also in related oil crop plants. Given the preferred choice of endogenous genetic element(s) in transgenic approach for better genetic regulation under the similar genetic background, the outcome of this experiment holds significance.

Materials and methods Plant and bacterial materials Sesame (Sesamum indicum, Var-9) and tobacco (Nicotiana tabacum, Jayanti) constituted the experimental plant materials. The super virulent Agrobacterium tumefaciens strain EHA105 and binary vector pCAMBIA1300 were used in the tobacco transformation experiment. Escherichia coli strain DH10B, and cloning bacterial vector pUC18 were applied in gene cloning. Isolation of coding DNA sequence (CDS) of sesame fad7 gene without chloroplast targeting signal Young tender leaf tissues from S. indicum were used for total RNA isolation using RNeasy Mini Kit (Qiagen) according to manufacturer’s protocol. 2 lg of total RNA was used for cDNA synthesis with gene-specific reverse primer using Transcriptor 1st strand cDNA synthesis kit version 6.0 (Roche Molecular biochemicals) in a total volume of 20 ll using manufacturer’s protocol. Isolation of 1125 bp engineered fad7 gene CDS (without predicted chloroplast transit signal) from sesame was carried out by RT-PCR using 2 ll of the 1st strand cDNA with primer sets fad7 forward primer and fad7 reverse primer by the following thermal profile: initial denaturation at 98 °C for 2 min, followed by 30 cycles of 98 °C/15 s, 65 °C/1 min 40 s, 72 °C/1 min 30 s (1 min/kb) and a final extension at 72 °C for 7 min in an Applied Biosystems Veriti gradient thermocycler. Modification of Sefad7 CDS with ER-targeting/ retention signals, construct preparation and tobacco transformation The Sefad7 gene without the chloroplast transit peptide was placed in the multiple cloning site (MCS) of Impact Vector 1.3 (Wageningen UR, Netherlands) in frame with N-terminal ER-signal peptide sequence within NotI and SacI restriction sites. However, during the cloning of Sefad7 gene in frame with N-terminal signal peptide,

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KDEL ER-retention signal along with stop codon got deleted. So, a separate set of specific primers (IV1.3fad7 FP and IV1.3-fad7 RP) were designed where the KDEL sequence (AAG-TTC-GTC-CTT) was placed in the reverse primer along with a stop codon (TAA). This reverse primer along with the forward primer led to amplification of the full-length engineered Sefad7 gene with N-terminal ER-signal peptide sequence and C-terminal KDEL ER-retention signal followed by stop codon. Engineered full-length fad7 gene was placed under the control of 29 CaMV35S promoter at 50 end and nos terminator at 30 end within the PstI/SalI sites in the pCAMBIA1300 vector. The expression vectors were transferred to Agrobacterium tumefaciens strain EHA105 by freeze–thaw method (Burow et al. 1990) and subsequently transformed into tobacco leaf discs (Horsch et al. 1985). Selected explants (50 mg/l hygromycin) with green elongated shoots were transferred to root inducing medium (without plant growth regulators) for rooting. The plants were grown in greenhouse after proper acclimatization for molecular and phenotypic evaluation. Progeny and statistical analysis Segregation analysis for integrated transgene in T1 progeny plants was carried out using the v2 (Chi-square) test in which observed values were compared to the corresponding theoretical values. The goodness of fit of the observed segregation ratio of the transgene was tested against the Mendelian segregation ratio (3:1) using the v2 test using the formula: X v2 ¼ fðobserved frequency . o expected frequencyÞ2 expected frequency : To compare the different levels of C18:3 content a onetailed Student’s t test was performed and significance (P value) was evaluated at 5 % level. Southern hybridization Southern hybridization (Sambrook et al. 1989) was performed at 65 °C in CHURCH buffer using truncated Sefad7 gene probe for representative T0 and T1 tobacco transformant lines. 12 lg of genomic DNA was digested with SacI and run on 0.8 % gel overnight. The radiolabeling of the probe DNA was carried out with P32dCTP (3500 ci/mmol) by random priming using rediprime II DNA labeling system (GE Healthcare, USA) following manufacturer’s instructions.

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Transcript analysis by qRT-PCR Real-time PCR was carried out in an Eppendorf Realplex2 Master Cycler using SYBR green-based relative quantification method using 5 prime kit (Eppendorf) in a 20-ll reaction volume. Total RNA was isolated from developing seed and young leaf tissues of different transgenic plants using RNeasy Mini Kit (Qiagen) following manufacturer’s protocol. 1st strand cDNA was synthesized by using primer fad7qRT RP using transcriptor 1st strand cDNA synthesis kit (Roche). PCR was carried out using primer sets, IV-ERT.P-fad7qRT FP and fad7 qRT RP for monitoring fad7 gene expression. Thermal cycling conditions were 2 min at 94 °C followed by 40 cycles of 94 °C for 30 s, 50 °C for 15 s, and 68 °C for 30 s. For each reading, duplicate CT values were averaged. Melting curve analysis in each case confirmed the amplification of specific product. The 2-DDCT method for relative quantification (Livak and Schmittgen 2001) was adopted to estimate the relative expression levels of fad7 gene in different transgenic tobacco lines. b-actin (accession no.: AB158612) gene was used as the internal control for normalization in each case. The average CT was calculated for both reference and target genes and the DCt (Ct, target gene - Ct, b-actin) was determined. Oligonucleotides used in the study The oligonucleotides used in the present study are listed in Supplementary Table 1. Western blot analysis The total protein was isolated from developing seeds and young leaf tissues of transgenic tobacco lines. Protein was quantitated by the Coomassie Blue method (Bradford 1976). An aliquot of 50 lg was loaded onto 15 % SDSPAGE, electrophoresed and blotted onto Hybond C nylon membrane (Amersham Pharmacia Biotech) using the wet transfer method. Affinity-purified polyclonal antibody raised against Sefad7 peptide (GHEDKLPWYRGKEWSYLRG) in rabbit (Imgenex India Pvt. Ltd) was used as the primary antibody (1:1000 dilution) and anti-rabbit IgGPOD (horseradish peroxidase) was used as the secondary antibody. Mouse monoclonal plant actin antibody (Sigma, mabGPa) was used as loading control (1:500 dilution) with corresponding anti-mouse IgG-POD. Immunodetection was carried out with the Lumi-LightPLUS western blotting kit (Roche Molecular Biochemicals), according to the manufacturer’s instructions. The band intensities were quantified using ImageJ software and normalized with that of the loading control to get an estimate of relative level of

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expression in independent transgenic lines (in case of seed samples). Analysis of fatty acid methyl esters by GC Mature control and transgenic tobacco seeds (in three independent seed batches, each comprising 1 g seeds, for each sample) and young, developing leaves were used to prepare fatty acid methyl esters (FAME) according to reported protocol (Abbadi et al. 2004) with slight modifications. About 5–10 ll methyl esterified sample was manually injected into a GC-Clarus 500 instrument (PerkinElmer, Waltham, MA), fitted with a manual injector and fused silica capillary column (30 m 9 0.25 mm 0.25 lm) (Omegawax TM 250, Supelco). Eluants were detected on a flame ionization detector (FID). Conditions set for analysis include split mode of injection (split ratio20). High-grade hydrogen was used as carrier gas at a pressure of 114.9 kPa with a column flow rate of 1.29 ml/ min. The initial FID temperature of the column was set to 140 °C and then was increased at a rate of 10 °C/min to a terminal temperature of 260 °C and the operating temperature was maintained at 220 °C. Data processing was performed by the software Total-Chrom Navigator from Perkin Elmer (Waltham, MA).

Results Bioinformatic analysis of the sesame x-3 fatty acid desaturase-7 (FAD7) protein and prediction-based deletion to obtain the engineered Sefad7 gene FAD7 sequence of S. indicum (accession no.: AAA70334) was subjected to ClustalW alignment (Larkin et al. 2007) with the FAD7 and FAD3 protein sequences of Nicotiana tabacum (FAD7 accession no.: AIA22325 and FAD3 accession no.: BAA05515), Arabidopsis thaliana (FAD7 accession no.: NP_187727 and FAD3 accession no.: NP_180559), Brassica napus (FAD7 accession no.: ACS26170 and FAD3 accession no.: AAT09135) and Glycine max (FAD7 accession no.: AEE25911 and FAD3 accession no.: ABV00681). The derived amino acid sequence of SeFAD7 revealed 85–90 % sequence homology with other known FAD7 and FAD3 amino acid sequences (Fig. 1) and also close similarity in terms of various transmembrane (TM) domains essential for their structure and function. This constituted a case for Sefad7 gene as a potential candidate for further genetic modification in terms of its organellar targeting and localization. ChloroP 1.1 Prediction Server (Emanuelsson et al. 1999) revealed a putative 72-amino acid-long N-terminal chloroplast transit peptide in the sesame FAD7 sequence instrumental for its

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import into the chloroplast. This predicted chloroplast targeting signal sequence was deleted from Sefad7 CDS by PCR (using Sefad7 FP and Sefad7 RP). The truncated CDS were engineered further by incorporation of the N-terminal ER-targeting signal and C-terminal ER-retention signal along with stop codon to generate the engineered Sefad7 gene. This modification is expected to target the gene product to ER and accumulate there. The engineered Sefad7 was cloned in binary vector pCAMBIA1300 under the control of 29 CaMV35S promoter element (Fig. 2) and transformed into tobacco via Agrobacterium-mediated plant transformation method. It is noteworthy to mention here that we tried another shorter deletion of 25 amino acids from the N-terminal of the Sefad7 gene. However, subsequent analysis (data not shown) revealed this to be ineffective as there was no significant alteration in seed fatty acid profile (data not shown). Southern blot analysis in T0 and T1 progeny plants A number of putative transgenic tobacco lines were generated following stringent selection by hygromycin (50 mg/l) and they were screened through specific primers (hptII-fad7 FP and hptII-fad7 RP) encompassing a region of fad7 sequence and hptII marker gene with an expected product length of about 1400 bp. Four putative transgenic tobacco lines, harboring engineered Sefad7 plant expression cassette, were subjected to Southern hybridization (Fig. 3). The lower uniform bands signify the endogenous tobacco fad7 gene present in control and transgenic lines. All the four lines were found to represent independent transgenic events with single integration site of Sefad7 transgene. Seeds obtained from four independent T0 tobacco lines were germinated under selection of hygromycin (50 mg/l) and surviving plantlets were further screened through transgene-specific PCR as described above (Supplementary Fig. 1). This PCR strategy helped to verify proper segregation of the full-length transgene cassette. Representative surviving seedlings were grown to maturity (n = 4–5) in greenhouse after proper acclimatization. The segregation pattern of the transgene was analyzed by v2 test to determine the number of functional transgene loci on tobacco genome. Chi-square ( v2) analysis indicated a 3:1 segregation for the hygromycin resistance trait, a ratio that suggested Mendelian mode of inheritance for a single dominant gene (Supplementary Table 2). Randomly chosen T1 progenies (n = 2) from each independent transgenic event were then subjected to Southern hybridization. The autoradiogram showed similar pattern of Sefad7 transgene integration (Fig. 3) in all T1 transgenic plants as was found in T0 lines. This provided evidence for stable integration of the engineered Sefad7 transgene as well as its transfer to next generation through seeds. Although representative independent transgenic lines

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Fig. 1 a Multiple sequence alignment of deduced amino acids of SeFAD7 with other plant plastidial FAD7 (tobacco NtFAD7, soybean GmFAD7, Brassica BnFAD7 and Arabidopsis AtFAD7). Sequences were aligned using ClustalW program and white boxes show the putative membrane-spanning domains while grey boxes indicate histidine boxes. b Multiple sequence alignment of deduced amino

acids of SeFAD7 with other plant FAD3 (tobacco NtFAD3, soybean GmFAD3, Brassica BnFAD3 and Arabidopsis AtFAD3). Sequences were aligned using ClustalW program and white boxes show the putative membrane-spanning domains while grey boxes indicate histidine boxes. In each case, putative chloroplast targeting sequences of SeFAD7 are indicted by arrows at the N-terminal

having single site of transgene integration has been documented, plants with multiple sites of transgene integration were also obtained in the present study (data not shown). However, plants with single site of integration were chosen for further analysis to compare between various transgenic events.

designed from CDS of the gene having lowest homology to endogenous tobacco fad7 gene sequence. The expected product length was about 200 bp. The fold expression for Sefad7 transgene, after normalization with b-actin gene (using b-actin FP and b-actin RP), was found to be in the range of 6.57–9.62 (taking normalized expression in untransformed control line as unity). The highest expression of Sefad7 gene was observed in fad7# T1-4#1 (Fig. 4). To monitor the expression profile in protein level, Western blot analysis was carried out with seed proteins from above mentioned T1 transgenic tobacco plants. A band was detected in the untransformed control plant in the similar size range indicating expression of the endogenous NtFAD7 protein of tobacco (Fig. 5). This was expected as the peptide sequence used to raise the polyclonal antibody against SeFAD7 shared sequence homology with the tobacco endogenous FAD7 protein sequence. The

Analysis of Sefad7 gene expression in tobacco seed and leaf tissues by qRT-PCR and Western blot analysis The developing seeds from one randomly chosen T1 transgenic plant (confirmed for transgene inheritance by Southern blot) from each category were used for qRT-PCR (quantitative real-time PCR). The 50 forward primer was designed from N-terminal ER-signal peptide region (IV-ER-T.Pfad7qRT FP) and 30 reverse primer (fad7qRT RP) was

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Fig. 2 Modification of sesame fad7 gene. a Deletion of chloroplast transit peptide from sesame fad7 gene; b cloning of the fad7 gene fragment in Impact Vector 1.3 in frame with N-terminal ER-targeting signal sequence; c insertion of C-terminal KDEL ER-retention signal

in sesame fad7 gene by PCR; d fad7 gene fragment with N-terminal ER-targeting signal and C-terminal ER-retention signal (KDEL) was cloned in pCAM1300 under 50 29 CaMV35S promoter and 30 nos terminator (asterisk indicates stop codon)

endogenous FAD7 protein of tobacco contained 442 amino acids, while the engineered SeFAD7 protein contained 421 amino acids. The size difference between the endogenous and the transgenic proteins is expected to be approx. 2.3 kDa, and thus they remained inseparable in our replicate Western blot experiments. However, bands with higher intensity were detected in all four transgenic lines, indicating expression of the transgene in the tobacco seeds. b-actin was used as an internal control and it showed similar band intensity in the transgenic and control plants, further pointing to expression of the modified SeFAD7 in transgenic tobacco plants. The normalized fold expression level followed a similar pattern as revealed by qRT-PCR analysis (Fig. 4). As constitutive 29 CaMV35S promoter was used for the study, the expression of engineered Sefad7 gene was scored from young leaf tissues (early maturation stage) of transgenic plants in RNA and protein level. Significant expression was observed in leaf also. The fold expression for Sefad7 transgene, after normalization with b-actin gene as before, was found to be in the range of 9.2–13.31 (taking normalized expression in untransformed control line as unity) (Supplementary Fig. 2a). Western blot analysis revealed expression of the SeFAD7 protein in all the transgenic lines (Supplementary Fig. 2b). A band in

the untransformed control plant in the similar size range most probably indicated expression of endogenous NtFAD7 protein. Although sharp increase was observed in SeFAD7 expression in transgenic lines as compared to control plant, variation of expression (as indicated by qRTPCR data) was not very clearly evident, probably due to saturation of the detected bands. As the RNA was sampled from seed and leaf tissues in different developmental stages as mentioned, hence, caution should be maintained in extrapolating the data for comparative expression profiling between seed and leaf tissues. This is more important keeping in mind developmental regulation of CaMV35S promoter activity as reported earlier (Hraska et al. 2008). However, the above results indicate constitutive transgenic expression of the engineered FAD7 desaturase in various tissues, including seed tissues. Alteration in the C18:3 content in seeds vs leaves of control and transgenic tobacco plants GC-FID analyses were performed using FAME prepared from the mature seeds and young, developing leaves of T1 transgenic plants of each category and untransformed control. Analysis of GC chromatograms revealed that C18:3

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Fig. 3 Representative Southern blot showing the integration pattern of Sefad7 transgene in T0 and T1 transgenic tobacco lines. Plant genomic DNA was digested with SacI and electrophoresed in 0.8 % agarose gel and hybridized with *1260 bp fad7 gene probe. Control untransformed tobacco plants, lane M EcoRI ? HindIII digested k-DNA molecular weight marker

Fig. 4 Representative histogram for qRT-PCR analysis of sesame FA desaturase-7 (Sefad7) expression in transgenic tobacco seeds obtained from T1 transgenic plants. Total RNA was isolated from 10- to 14-dayold seeds. The b-actin gene was used as the internal control for normalization. UC untransformed control. Error bars represent the standard deviation of the mean for triplicate readings in each case

content in storage lipid (Supplementary Fig. 3) was increased from 0.8 to 4.78–6.77 % in the transgenic tobacco seeds compared to the untransformed tobacco plants (Fig. 6;

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Table 1). The analysis also showed decrease in C18:2 and C18:1 profile in the seed lipids thereby indicating fatty acid pool shift in favor of C18:3 due to the expression of Sefad7.

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Fig. 5 Analysis of SeFAD7 expression by Western blot. a Upper panel UC untransformed control and rest of the lanes represent independent T1 transgenic lines; lower panel equal loading of proteins shown with anti-b-actin antibody. Approx. sizes of the proteins are indicated in right. b Bar diagram representing fold changes of expression level of SeFAD7 (normalized with loading control) quantified by ImageJ

However, GC-FID analyses showed no such significant alteration in the C18:3 content in leaf tissues (Supplementary Fig. 4) of transgenic tobacco plants compared to the untransformed control (Supplementary Table 3).

Discussion Trienoic (TA) and dienoic FA constitute 70–80 % of the total FA from leaves or root lipids (Douce et al. 1990). They participate in the formation of membrane lipid and maintain membrane fluidity. TAs also serve as precursor molecules for plant hormones like jasmonic acid (JAs) involved in defense signaling against several stress responses (Jung et al. 2007). TAs are synthesized from dienoics FA by the activity of two x-3 desaturases encoded by fad3 and fad7 genes in different organelles (Browse et al. 1986). The synthesis of C18:3 and C16:3 FA in plant cells occurs by the sequential desaturation of the saturated FA, viz., palmitic acid (C16:0) and stearic acid (C18:0), respectively. The N-terminal sequence of the FAD7 contained a consensus chloroplastidial transit peptide. Except for the N-terminal domain, the deduced amino acid sequence of the FAD7 desaturase had high sequence homology to the microsomal FAD3 desaturase, indicating their evolution from a common ancestral origin (Iba et al. 1993; Venegas-Caleron et al. 2010). In the present study,

homology-based sequence alignment was performed to identify the sequence similarity of FAD7 protein of sesame with other available FAD7 and FAD3 (Fig. 1) deduced amino acid sequences. The SeFAD7 polypeptide sequence was found to contain four transmembrane domains and three histidine-rich motifs characteristic of membrane desaturases like other related desaturase gene products and it further suggests that the conserved region may be essential for the catalytic activity of this family of desaturase gene. The soybean fad7 gene is a nuclear-encoded gene, and in situ molecular evidence proved that the gene product gets localized in plastid (Andreu et al. 2007). Therefore, same is expected to be true for the Sefad7 gene product, as N-terminal domains of chloroplastidial FAD7 desaturases are reported to contain such cleavable targeting peptides (Pagny et al. 1999). Analysis of a large number of chloroplast signal sequences revealed the presence of serine and threonine (with hydroxyl groups in their side chain), leucine and alanine residues (Hitz et al. 1994) and they also feature in the N-terminal of SeFAD7. ChloroP 1.1 server-based prediction could detect one 72-amino acidlong chloroplastidial transit peptide sequence at the N-terminus of SeFAD7. Therefore, the replacement of the chloroplast transit peptide from the upstream of fad7 gene by the ER transit peptide along with addition of KDEL ERretention signal at the C-terminus is expected to target this gene product to accumulate in ER and execute similar

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Fig. 6 Representative bar diagram showing the trends in the alteration of FA composition of tobacco T2 seeds from control and transgenic plants as determined by GC-FID analysis of FAME. Each data point is an average of three replicates and error bar represents standard deviation. Asterisks denote significant differences of C18:3 with P \ 0.05

Table 1 FA composition of seeds obtained from T1 tobacco transgenic lines of each category, showing significant alterations in FA composition of T1 transgenic lines compared to the untransformed control (UC) tobacco plants Fatty acids

UC

fad7# T1-1#1

fad7# T1-2#1

fad7# T1-3#1

fad7# T1-4#1

C16:0

7.1 ± 0.2

8.8 ± 0.1

7.5 ± 0.8

8.7 ± 0.6

8 ± 0.3

C16:1

0.3 ± 0.1

0.8 ± 0.1

0.7 ± 0.1

0.9 ± 0.2

0.6 ± 0.1

C18:0

2.7 ± 0.5

2 ± 0.5

2.5 ± 0.6

1.9 ± 0.2

2.8 ± 0.3

C18:1 C18:2

12.1 ± 0.7 76.9 ± 1.1

10.1 ± 0.6 73 ± 1.6

10.9 ± 1.3 72.3 ± 0.8

9.8 ± 0.5 72.8 ± 1

10.6 ± 0.5 71.3 ± 1.2

C18:3

0.8 ± 0.2

5 ± 0.7

5.8 ± 0.3

4.8 ± 0.5

6.7 ± 0.5

Values are mol% ± SD (n = 3, indicating three seed batches of 1 g seeds each)

activities as a regular fad3 gene would do, provided their otherwise structural and functional similarity. The precise modification of FAD7 was crucial for subsequent trafficking and activity of the protein. One of our shorter deletions (25 amino acid sequence from N-terminal) proved to be unsuccessful in localizing the end product in ER, reflected in unaltered seed fatty acid profile (data not shown). This is also in accordance with some earlier observations where deletion up to 16 amino acids from N-terminus of chloroplast precursor proteins led to neither import into chloroplasts nor mis-sorting to any other organelles, thereby emphasizing the importance of entire N-terminal transit peptide for proper localization as well as mis-sorting (Jarvis and Robinson 2004; Bhushan et al. 2006). The effect of predicted N-terminal 72 amino acid

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deletion was monitored in the transgenic tobacco lines. Southern hybridization revealed integration of Sefad7 gene at single integration sites and their stable inheritance through seeds. This provided us with a common genetic premise to compare their expression level and phenotypic attributes. The expression of the modified Sefad7 gene was checked in the transcript and protein level by qRT-PCR and Western blot, respectively. Expression of the modified Sefad7 transgene in seeds was evident in the transcript level as the relative level of the Sefad7 transcript increased in the range of 6.57–9.62 fold in different transgenic lines compared to untransformed control line (normalized expression was taken as unity). This enhanced level of transcript ultimately reflected in the level of FAD7 protein. As the various x-3 desaturases from several plant sources

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share very high sequence homology in the amino acid level, it was difficult to choose an epitope for specific antibody generation against SeFAD7. Although in the present study, the sequence of microsomal FAD3 and plastidial FAD7 proteins were analyzed, the sequence of another plastidial x-3 desaturase, viz., FAD8 is also expected to contain significant sequence homology with FAD3 and FAD7, thereby causing further difficulty in specific detection of expressed FAD7 in the background of endogenous proteins. Thus, the transgenic protein could not be separated from the endogenous FAD7 protein of tobacco due to very small size difference. However, the increased band intensity of FAD7 protein in the backdrop of equal amount of protein in each lane clearly indicated transgene expression in the protein level. Quantitative estimation of normalized protein band intensity also followed similar expression pattern in different transgenic lines as revealed by qRT-PCR. This variation of fad7 gene expression could be well attributed to differential integration sites (position effect) (Chen et al. 2015). Most importantly, GC analyses revealed significant increase (from 0.8 to 4.78–6.77 %) in the content of C18:3 in seed storage lipid in the transgenic tobacco lines compared to the control plants. This is indicative of engineered transgene expression driven by 29 CaMV35S promoter in tobacco immature and nearly mature seeds and thereby, microsomal localization of the translated product. A segregation ratio of 3:1 was observed for the hptII gene among the T1 progenies, indicating a single transgene locus and heterozygosity in the T0 parents. However, we could not confirm the zygosity status (homozygous vs heterozygous) of the individual T1 plants as no subsequent progeny analysis (T1 to T2) was performed. After the complete transgene segregation in subsequent generations, the homozygous transgenic lines are expected to perform much better in terms of enhancement of C18:3 seed storage lipid content. Among other FA, C18:1 content markedly decreased along with the slight decrease of the C18:2. The moderate increase of C18:3 in seed tissues could be related to efficiency of this gene modification in terms of percent of transgenic protein actually transported and localized in ER and activity of the engineered protein in ER. The use of constitutive 29 CaMV35S promoter offered an opportunity for comparison of the FA profile in seed and leaf tissues in terms of C18:3, and thus would help in determining the efficiency of the gene modification. The 29 CaMV35S is known to be efficient in constitutive transgene expression in all tissue parts of both monocot and in dicot plants (Odell et al. 1985; Jefferson et al. 1987; Hraska et al. 2008). In the present study, the constitutive expression was evident, as significant expression (confirmed in RNA and in protein level) was observed in leaf tissue also, beside seeds. However, some earlier reports

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suggest developmental regulation of transgene expression under CaMV35S promoter, as transgene activity was found to increase from younger to mature leaves (Hraska et al. 2008). In higher plants, triacylglycerol (TAG, storage lipid) biosynthesis occurs primarily in the seeds, anthers and pollen, providing energy and carbon skeletons to support germination and early growth (Bao and Ohlrogge 1999; Piffanelli and Murphy 1999; Footitt et al. 2007). In tobacco leaf cells, spherical lipid-based particles were identified (Wahlroos et al. 2003). These bonafide oil bodies are not TAG-based particles and resulted due to cell membrane catabolism (Yao et al. 1991). In case of Arabidopsis, TAG is found in the leaves, however, it accounts for less than 1 % of leaf glycerolipids (Yang and Ohlrogge 2009; Tjellstrom et al. 2015). The plastidial fad7 gene participates in the leaf membrane lipid formation and transgenic tobacco plants with elevated levels of C18:3 in chloroplast lipid were observed due to the expression of the Atfad7 cDNA (Kodama et al. 1994). In the present study, nearly mature seeds and young leaves of tobacco plants were used as experimental materials to isolate the seed neutral lipids (TAG) and leaf total lipids, to investigate the increased content of ALA (C18:3) in different tissue parts. Significant increase in the total seed storage lipid was observed. As the native fad7 gene participates in the membrane lipid formation, leaf total lipid was analyzed to estimate the C18:3 content. As there was no significant change in the leaf lipid profile it indicates proper gene modification, as otherwise C18:3 would have been directed to plastid and incorporated within membrane lipid, like monogalactosyl diacylglycerol (MGDG), which would reflect in the total lipid composition. Thus, keeping in mind the developmental regulation of CaMV35S activity along with the presence of an efficient TAG accumulation system in the developing seeds, it becomes imperative that C18:3 synthesized in transgenic tobacco plants are incorporated in glycerol backbone to form TAG and predominantly be accumulated in seeds instead of leaves. Although TAG is the major lipid component of seeds, extraplastidic membrane lipids like phosphatidyl choline (PC) can also be present. It will be intriguing to explore the lipid compartmentalization between TAG and PC in seeds by analyses of various lipid classes individually, which remained beyond the scope of the present study. Thus, the present study demonstrated the efficiency of this engineered Sefad7 gene to enhance ALA (x-3; C18:3) content in seeds of tobacco plants. Although a number of different strategies have been employed in the past to engineer fatty acid biosynthetic pathways (reviewed in Bhunia et al. 2015b), mostly involving expression of endogenous/heterologous fad3 gene(s) from various plant sources, to enhance x-3 PUFA, till date there is no report of genetic engineering-based organelle retargeting of an endogenous protein to achieve

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the same in the plant system. With the proof of principle now validated in tobacco, the implementation of the similar strategy in sesame, using engineered Sefad7 gene, is expected to advance the present state of the art of the field in a significant manner. Although the recommended dietary x-6:x-3 fatty acid ratio is 5:1–10:1, the ratio in the modern diet has increased up to 15:1–20:1 (Simopoulos 2006). Our present study qualified, as a proof of principle, the genetic engineering strategy to employ modified Sefad7 to increase the C18:3 content of seed vegetable oils. The x-6:x-3 fatty acid ratio achieved in the present study was 10.64:1 (for the transgenic line with highest expression of Sefad7 gene), which is still higher as would be desirable from a health point of view. However, with the use of seed-specific endogenous 2Salbumin promoter from sesame (Bhunia et al. 2014) and endogenous expression of modified Sefad7 gene in sesame, we expect to reproduce the results in sesame with higher efficiency, and thus achieve the desired fatty acid ratio beneficial for human health. Author contribution statement SKS, RKB and AC conceived and designed the research. RKB, RK and AC performed the experiments. SKS, RKB, AC and RK analyzed the data. MKM provided valuable scientific inputs during the course of the study. RKB and AC wrote the manuscript in consultation with SKS and MKM. Acknowledgments The authors are thankful to Professor Debabrata Basu and Professor Sampa Das, Bose Institute, Kolkata, for critical reading of the manuscript and valuable comments, Sona Dogra and Gayatri Aditya for technical assistance in plant tissue culture and molecular biology experiments, Manoj Aditya, Sudarshan Maity and Subhas Ghosh for greenhouse logistic support. Financial assistance from National Agricultural Innovation Project, Indian Council of Agricultural Research (NAIP/ICAR), in terms of grant support (Project component code 4C1090) to laboratory and fellowship to RKB is thankfully acknowledged. Finally, the authors express their sincere gratitude to the two anonymous reviewers, whose critical comments helped to improve the clarity of the manuscript. Compliance with ethical standards Conflict of interest interests.

Authors declare no financial or competing

References Abbadi A, Domergue F, Bauer J, Napier JA, Welti R, Zahringer U, Cirpus P, Heinz E (2004) Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: constraints on their accumulation. Plant Cell 16:2734–2748 Anai T, Koga M, Tanaka H, Kinoshita T, Rahman SM, Takagi Y (2003) Improvement of rice (Oryza sativa L.) seed oil quality through introduction of a soybean microsomal omega-3 fatty acid desaturase gene. Plant Cell Rep 21:988–992 Andreu V, Collados R, Testillano SP, Risueno CM, Picorel R, Alfonso M (2007) In situ molecular identification of the plastid

123

x-3 fatty acid desaturase FAD7 from soybean: evidence of thylakoid membrane localization. Plant Physiol 145:1336–1344 Ashri A (1989) Sesame. In: Robbelen G, Downey RK, Ashri A (eds) Oil crops of the world: their breeding and utilization. McGraw Hill, New York Bao X, Ohlrogge J (1999) Supply of fatty acid is one limiting factor in the accumulation of triacylglycerol in developing embryos. Plant Physiol 120:1057–1062 Bates DP, Stymne S, Ohlrogge J (2013) Biochemical pathways in seed oil synthesis. Curr Opin Plant Biol 16:358–364 Bhunia RK, Chakraborty A, Kaur R, Gayatri T, Bhattacharyya J, Basu A, Maiti MK, Sen SK (2014) Seed-specific increased expression of 2S albumin promoter of sesame qualifies it as a useful genetic tool for fatty acid metabolic engineering and related transgenic intervention in sesame and other oil seed crops. Plant Mol Biol 86:351–365 Bhunia RK, Chakraborty A, Kaur R, Gayatri T, Basu A, Maiti MK, Sen SK (2015a) Analysis of fatty acid and lignan composition of Indian germplasm of sesame in terms of their nutritional merits. J Am Oil Chem Soc 92:65–76 Bhunia RK, Kaur R, Maiti MK (2015b) Metabolic engineering of fatty acid biosynthetic pathway in sesame (Sesamum indicum L.): assembling tools to develop nutritionally desirable sesame seed oil. Phytochem. doi:10.1007/s11101-015-9424-2 Bhushan S, Kuhn C, Berglund AK, Roth C, Glaser E (2006) The role of the N-terminal domain of chloroplast targeting peptides in organellar protein import and miss-sorting. FEBS Lett 580:3966–3972 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254 Browse J, McCourt P, Somerville C (1986) A mutant of Arabidopsis deficient in C16:3 and C18:3, leaf lipids. Plant Physiol 81:859–864 Burow MD, Chlan CA, Sen P, Lisca A, Murai N (1990) High frequency generation of transgenic tobacco plants after modified leaf disk co-cultivation with Agrobacterium tumefaciens. Plant Mol Biol Rep 8:124–139 Chapman KD, Ohlrogge JB (2012) Compartmentation of triacylglycerol accumulation in plants. J Biol Chem 287:2288–2294 Chen Y, Zhou XR, Zang Z-J, Dribnenki P, Singh S, Green A (2015) Development of high oleic oil crop platform in flax through RNAi-mediated multiple FAD2 gene silencing. Plant Cell Rep 34:643–653 Chi X, Yang Q, Pan L, Chen M, He Y, Yang Z, Yu S (2011) Isolation and characterization of fatty acid desaturase genes from peanut (Arachis hypogaea L.). Plant Cell Rep 30:1393–1404 Damude HG, Kinney AJ (2007) Engineering oilseed plants for a sustainable, land-based source of long Chain polyunsaturated fatty acids. Lipids 42:179–185 Douce R, Joyard J, Block MA, Dorne AJ, Harwood JL, Bowyer JR (1990) Glycolipid analyses and synthesis in plastids. In: Harwood JL, Bowyer JR (eds) Methods in plant biochemistry. Academic Press, London, pp 71–103 Dyer JM, Mullen RT (2001) Immunocytological localization of two plant fatty acid desaturases in the endoplasmic reticulum. FEBS Lett 494:44–47 Emanuelsson O, Nielsen H, Von Heijne G (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8:978–984 FAOSTAT Data (2013) Food and Agriculture Organization of the United Nations. Statistical database. http://faostat3.fao.org/home/E Footitt S, Dietrich D, Fait A, Fernie AR, Holdsworth MJ, Baker A, Theodoulou FL (2007) The COMATOSE ATP-binding cassette transporter is required for full fertility in Arabidopsis. Plant Physiol 144:1467–1480

Plant Cell Rep (2016) 35:213–226 Gomord V, Wee E, Faye L (1999) Protein retention and localization in the endoplasmic reticulum and the Golgi apparatus. Biochimie 81:607–618 Gunstone F, Harwood JL, Padley FB (1994) The lipid handbook, 2nd edn. Chapman and Hall, London Harbige LS (2003) Fatty acids, the immune response, and autoimmunity: a question of n-6 essentiality and the balance between n-6 and n-3. Lipids 38:323–341 Hirata F, Fujita K, Ishikura Y, Hosoda K, Ishikawa T, Nakamura H (1996) Hypocholesterolemic effect of sesame lignan in humans. Atherosclerosis 122:135–136 Hitz WD, Carlson TJ, Booth JR, Kinney AJ, Stecca KL, Yadav NS (1994) Cloning of a higher-plant plastid x-6 fatty acid desaturase cDNA and its expression in a cyanobacterium. Plant Physiol 105:635–641 Horsch RB, Fry JE, Hoffman NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231 Hraska E, Rakousky S, Curn V (2008) Tracking of the CaMV-35S promoter performance in GFP transgenic tobacco, with a special emphasis on flowers and reproductive organs, confirmed its predominant activity in vascular tissues. Plant Cell Tiss Organ Cult 94:239–251 Iba K, Gibson S, Nishiuchi T, Fuse T, Nishimura M, Aronde V, Hugly S, Somerville C (1993) A gene encoding a chloroplast x-3 fatty acid desaturase complements alterations in fatty acid desaturation and chloroplast copy number of the fad7 mutants of Arabidopsis thaliana. J Biol Chem 268:24099–24105 Jarvis P, Robinson C (2004) Mechanisms of protein import and routing in chloroplasts. CurrBiol 14:R1064–R1077 Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: bglucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907 Jung C, Lyou SH, Yeu SY, Kim MA, Rhee S, Kim M, Lee JS, Choi YD, Cheong J-J (2007) Microarray-based screening of jasmonate-responsive genes in Arabidopsis thaliana. Plant Cell Rep 26:1053–1063 Kankaanpaa P, Sutas Y, Salminen S, Isolauri E (1999) Dietary fatty acids and allergy. Ann Med 31:282–287 Kodama H, Hamada T, Horiguchi G, Nishimura M, Iba K (1994) Genetic enhancement of cold tolerance by expression of a gene for chloroplast x-3 fatty acid desaturase in transgenic tobacco. Plant Physiol 105:601–605 Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and clustal X version 2.0. Bioinformatics 23:2947–2948 Liu HL, Yin ZJ, Xiao L, Xu YN, Qu LQ (2012) Identification and evaluation of x-3 fatty acid desaturase genes for hyperfortifying a-linolenic acid in transgenic rice seed. J Exp Bot 63:3279–3287 Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-DDCT method. Methods 25:402–408 McCartney AW, Dyer JM, Dhanoa PK, Kim PK, Andrews DW, McNew JA, Mullen RT (2004) Membrane-bound fatty acid desaturases are inserted cotranslationally into the ER and contain different ER retrieval motifs at their carboxy termini. Plant J 37:156–173 McConn M, Hugly S, Browse J, Somerville C (1994) A mutation at the fad8 locus of Arabidopsis identifies a second chloroplast x-3 desaturase. Plant Physiol 106:1609–1614 Miyahara Y, Komiya T, Katsuzaki H, Imai K, Nakagawa M, Ishii Y, Hibasami H (2000) Sesamin and episesamin induce apoptosis in human lymphoid leukemia. Int J Mol Med 6:43–46 Nakai M, Harada M, Nakahara K, Akimoto K, Shibata H, Miki W, Kiso Y (2003) Novel antioxidative metabolites in rat liver with ingested sesamin. J Agric Food Chem 51:1666–1670

225 Nookaraju A, Pandey SK, Fujino T, Kim JY, Suh MV, Joshi CP (2014) Enhanced accumulation of fatty acids and triacylglycerols in transgenic tobacco stems for enhanced bioenergy production. Plant Cell Rep 33:1041–1052 Odell JT, Nagy F, Chua NH (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810–812 Pagny S, Lerouge P, Faye L, Gomord V (1999) Signals and mechanisms for protein retention in the endoplasmic reticulum. J Exp Bot 50:157–164 Pelham HRB (2000) Using sorting signals to retain proteins in the endoplasmic reticulum. Meth Enzymol 327:279–283 Piffanelli P, Murphy DJ (1999) Lipid accumulation and related gene expression in gametophytic and sporophytic anther tissues. In: Cresti M, Cai G, Moscatelli A (eds) Fertilization in higher plants: molecular and cytological aspects. Springer, Heidelberg Qui X, Hong H, MacKenzie SL (2001) Identification of a D4 fatty acid desaturase from Thraustochytrium sp. involved in the synthesis of docosahexaenoic acid by heterologous expression in Saccharomyces cerevisiae and Brassica juncea. J Biol Chem 276:31561–31566 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor Sayanova O, Smith MA, Lapinskas P, Stobart AK, Dobson G, Christie WW, Shewry PR, Napier JA (1997) Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain results in the accumulation of high levels of delta 6-desaturated fatty acids in transgenic tobacco. Proc Natl Acad Sci 94:4211–4216 Shimizu S, Akimoto K, Shinmen Y, Kawashima H, Sugano M, Yamada H (1991) Sesamin is a potent and specific inhibitor of delta 5 desaturase in polyunsaturated fatty acid biosynthesis. Lipids 26:512–516 Simopoulos AP (1991) Omega-3 fatty acids in health and disease and in growth and development. Am J Clin Nutr 54:438–463 Simopoulos AP (2001) n-3 fatty acids and human health: defining strategies for public policy. Lipids 36:S83–S89 Simopoulos AP (2002) The importance of the ratio of omega-6/ omega-3 essential fatty acids. Biomed Pharmacother 56:365–379 Simopoulos AP (2006) Evolutionary aspects of diet, the omega-6/ omega-3 ratio and genetic variation: nutritional implications for chronic diseases. Biomed Pharmacother 60:502–507 Sinclair AJ, Attar-Bashi NM, Li D (2002) what is the role of alinolenic acid for mammals? Lipids 37:1113–1123 Tapiero A, Ba NG, Couvreur P, Tew KD (2002) Polyunsaturated fatty acids (PUFA) and eicosanoids in human health and pathologies. Biomed Pharmacother 56:215–222 Tjellstrom H, Strawsine M, Ohlrogge JB (2015) Tracking synthesis and turnover of triacylglycerol in leaves. J Exp Bot. doi:10.1093/ jxb/eru500 Venegas-Caleron M, Beaudoin F, Garces R, Napier JA, MartinezForce E (2010) The sunflower plastidial omega-3 fatty acid desaturase (HaFAD7) contains the signalling determinants required for targeting to, and retention in, the endoplasmic reticulum membrane in yeast but requires co-expressed ferredoxin for activity. Phytochemistry 71:1050–1058 Wahlroos T, Soukka J, Denesyuk A, Wahlroos R, Korpela T, Kilby NJ (2003) Oleosin expression and trafficking during oil body biogenesis in tobacco leaf cells. Genesis 35:125–132 Wakita Y, Otani M, Hamada T, Mori M, Iba K, Shimada T (2001) A tobacco microsomal x-3 fatty acid desaturase gene increases the linolenic acid content in transgenic sweet potato (Ipomoea batatas). Plant Cell Rep 20:244–249 Wang L, Yu S, Tong C, Zhao Y, Liu Y, Song C, Zhang Y, Zhang X, Wang Y, Hua W, Li D, Li D, Li F, Yu J, Xu C, Han X, Huang S,

123

226 Tai S, Wang J, Xu X, Li Y, Liu S, Varshney RK, Wang J, Zhang X (2014) Genome sequencing of the high oil crop sesame provides insight into oil biosynthesis. Genome Biol 15:R39 Yadav NS, Wierzbicki A, Aegerter M, Caster CS, Perez-Grau L, Kinney AJ, Hitz WD, Jr-Booth JR, Schweiger B, Stecca KL, Allen SM, Blackwell M, Reiter RS, Carlson TJ, Russell SH, Feldmann KA, Pierce J, Browse J (1993) Cloning of higher plant x-3 fatty acid desaturases. Plant Physiol 103:467–476 Yang Z, Ohlrogge JB (2009) Turnover of fatty acids during natural senescence of Arabidopsis, Brachypodium, and switchgrass and in Arabidopsis beta-oxidation mutants. Plant Physiol 150:1981–1989

123

Plant Cell Rep (2016) 35:213–226 Yao KG, Paliyath G, Humphrey RW, Hallett FR, Thompson JE (1991) Identification and characterization of non sedimentable lipidprotein microvesicles. Proc Natl Acad Sci 88:2269–2273 Zhang H, Miao H, Wang L, Qu L, Liu H, Wang Q, Yue M (2013) Genome sequencing of the important oilseed crop Sesamum indicum L. Genome Biol 14:401