Thioesterase overexpression in Nicotiana benthamiana leaf increases the fatty acid flux into triacylgycerol

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1 Thioesterase overexpression in Nicotiana benthamiana leaf increases the fatty acid flux into triacylgycerol Anna El Tahchy, Kyle B. Reynolds, James R. Petrie, Surinder P. Singh and Thomas Vanhercke Agriculture and Food, CSIRO, Canberra, Australian Capital Territory, Australia Correspondence A. El Tahchy, Agriculture and Food, CSIRO, GPO Box 1700, Australian Capital Territory 2601, Australia Fax: Tel: anna.eltahchy@csiro.au (Received 1 December 2016, revised 15 December 2016, accepted 20 December 2016, available online 12 January 2017) doi: / Edited by Ulf-Ingo Fl ugge Increasing the oil content of leafy biomass is emerging as a sustainable source of vegetable oil to meet global demand. Transient gene expression in leaf provides a reproducible platform to study the effect of transgenes on lipid biosynthesis. We first generated a transgenic Nicotiana benthamiana line containing high levels of triacylglycerol in the leaf tissue (31.4% by dry weight) by stably expressing WRI1, DGAT1 and OLEOSIN. We then used this line as a platform to test the effect of three Arabidopsis thaliana thioesterases (FATA1, FATA2 and FATB). Further increases in leaf oil content were observed with biochemical and lipid assays revealing an increase in the export of fatty acids from the chloroplast and a modification in the oil profile. Keywords: diacylglycerol; leaf oil; lipid biosynthesis; Nicotiana benthamiana; thioesterase; triacylglycerol Genetically engineered oil crops offer the potential to deliver new and improved crop varieties with increased oil contents and modified oil profiles, not only for food use but also as oleochemicals and biofuel feedstocks [1]. One novel strategy for the production of oils through agricultural production is to elevate oil content in tissues where oil does not normally accumulate, for example, leaves and stems [2]. This is particularly attractive because of the large amounts of biomass that these tissues typically represent. Over the last 10 years, a range of transgenic strategies that increase leaf oil contents have been reported, including single gene approaches as well as synergistic, multigene pathways [3]. These approaches include the upregulation of lipid biosynthesis by overexpressing seed transcription factors (LEC2, WRI1), upregulation of oil accumulation pathways (DGAT) and reducing catabolism of oil [2]. Acyl-ACP thioesterases (FATs) mediate the release of fatty acids (FAs) for transport from their site of synthesis within the chloroplast to the cytoplasm and endoplasmic reticulum (ER) [4]. Acyl-ACP thioesterases are classified into two general families termed FATA and FATB [5,6]. FATAs generally act on acyl chains of 18 carbons in length, saturated or unsaturated, while FATBs release acyl chains of 16 carbons in lengths [7]. FATA orthologues possess similar substrate specificities among different species, with high activity upon 18 : 1 D9 -ACP substrate [8,9]. FATB enzymes are further classified into two subclasses: FATB1, with preference towards saturated acyl-acps, especially 16 : 0-ACP, and FATB2 with preference towards short/medium-chain saturated acyl-acps [10]. Therefore, acyl-acp thioesterases are key enzymes in determining which FAs are exported to the cytosol and subsequently incorporated into glycerolipids Abbreviations DAG, diacylglycerol; DAS, days after sowing; DW, dry weight; ER, endoplasmic reticulum; FAME, fatty acid methyl esters; FAT, fatty acid thioesterase; PC, phosphatidylcholine; TAG, triacylglycerol; TLC, thin-layer chromatography; WE, wax esters; WT, wild-type. 448 FEBS Letters 591 (2017) ª 2016 Federation of European Biochemical Societies

2 A. El Tahchy et al. Thioesterases push the fatty acid flux to yield more oil synthesized in the ER, including triacylglycerol (TAG) [11]. The overexpression of specific acyl-acp thioesterases has previously been demonstrated to effectively modify oil profile in transgenic plants [6]. As one example, the overexpression of FATB1 from the California Bay laurel tree in oilseed rape increased the laurate levels in the seed oil of this crop from negligible levels to over 50% of the total FAs [11]. Interest in the use of acyl-acp thioesterases in lipid biotechnology has led to a very active research on their different forms coming from a variety of sources, especially in oil seeds [12 15]. Transient expression in Nicotiana benthamiana infiltrated leaf has been widely used as a reliable platform to test various gene candidates for plant oil production and other plant products [16]. Recently, transient expression of fatty acyl-reductases and wax esters (WE) synthases in N. benthamiana was effective in producing significant amount of WE [16]. This platform was also used to alter the leaf oil profile to produce industrially relevant FAs in leaves, such as medium-chain FAs [17]. We have previously reported a synergistic effect upon the transient coexpression of WRI1 and DGAT1 in N. benthamiana leaves which resulted in 22-fold increase in TAG [18]. This synergy was later followed by the addition of OLEOSIN in stably transformed Nicotiana tabacum which provided an improvement in oil content of transgenic leaves. This strategy has been summarized as push, pull and protect [5], and resulted in a maximum accumulation of 15% oil on a dry weight (DW) basis in mature leaves. One possible way to further increase the lipid yield in such transgenic plants could be increasing FA export from the plastids by the coexpression of a specific acyl-acp thioesterase [6]. However, increasing the acyl flux in such high oil transgenic plants could interfere with membrane lipids leading to severe physiological phenotypes. Therefore, leaf transient assays represent a very useful alternative to the stable transformation by facilitating the rapid analysis of the resulting phenotype in a high oil background. In this study, we tested the additive effect of transiently coexpressing thioesterases with the WRI/DGAT synergy in wild-type (WT) N. benthamiana. We then generated a stable transgenic event expressing WRI1, DGAT1 and OLEOSIN to use as high oil platform for leaf-based transient assays. We subsequently used this new platform to test the transient overexpression of three Arabidopsis thaliana thioesterases (FATA1, FATA2 and FATB). This resulted in an increase in TAG levels in the plant leaf, further corroborated by biochemical studies including radiolabelled pulse chase experiment. Materials and methods Nicotiana benthamiana stable transformation Agrobacterium tumefaciens-mediated transformation of N. benthamiana with the binary vector pjp3502 was essentially carried out as described by [19,20]. Shoots were developed on selective ½ MS agar supplemented with 1% sucrose to reduce any abnormal phenotypic effects from the expression of the WRI1 gene [21]. Plants were grown in a plant growth cabinet with 1000 lmol photons m 2 s 1 of light intensity for 16 h light and 8 h dark periods. Following segregation, T 2 seeds of the selected line (AT001) were identified as homozygous according to increased oil content in leaf tissue and to digital PCR analyses as described by [22]. T 3 (AT001) homozygous plants were then used for transient experiments. Genes and expression vectors The A. thaliana FATA1 and FATA2 genes were amplified from silique cdna using primers containing EcoRI and PstI sites and subsequently cloned into pjp3343 downstream of the 35S promoter using the same restriction sites [18]. The resulting expression vectors were designated poil079 and poil080 respectively. The gene coding for the A. thaliana FATB thioesterase was amplified using primers containing NotI and SacI flanking sites and cloned into the corresponding restriction sites of pjp3343, resulting in poil081. Transient expression in N. benthamiana Transient expression in N. benthamiana leaves was performed as described [23] with some minor modifications. A. tumefaciens cultures containing the gene coding for the p19 viral suppressor protein and the chimeric gene(s) of interest were mixed such that the final OD 600 of each culture was equal to prior to infiltration [18]. For lipid analyses, a total of nine leaves from three plants were infiltrated with the different gene combinations. Samples being compared were randomly located on the same leaf. After infiltrations, N. benthamiana plants were grown for a further 5 days before leaf discs were harvested, pooled across the three leaves from the same plant, freeze-dried, weighed and stored at 80 C. Lipid analysis Total lipids were extracted from leaf tissues using chloroform : methanol : 0.1 M KCl (2 : 1 : 1 v : v : v). Freeze- FEBS Letters 591 (2017) ª 2016 Federation of European Biochemical Societies 449

3 Thioesterases push the fatty acid flux to yield more oil A. El Tahchy et al. dried leaf tissues were first homogenized in chloroform : methanol in a microcentrifuge tube containing a metallic ball using a Reicht tissue lyser (Qiagen, Hilden, Germany) for 3 min at 20 frequencys 1. After mixing homogenate at g (Vibramax 10; Heidolph, Schwabach, Germany) for 10 min, KCl was added and mixed for a further 5 min. Finally, the mixture was centrifuged for 5 min at g and the lower lipid phase collected. The remaining phase was washed once with CHCl 3 and lipid phase pooled with the earlier extract. Solvent of lipid phase was evaporated completely using N 2 flow and a known volume of CHCl 3 was added for per mg leaf DW. TAG were fractionated by TLC (silica gel 60; MERCK, Darmstadt, Germany) in hexane : diethylether : acetic acid (70 : 30 : 1 v : v : v) and visualized by spraying primuline [Sigma, Taufkirchen, Germany 5 mg/100 ml acetone : water (80 : 20 v : v)] and exposing plate under UV. Fatty acid methyl esters (FAME) of TAG were produced by incubating corresponding bands in 1 N methanolic HCl (Supelco, Bellefonte, PA, USA) at 80 C for 2 h together with known amount of heptadecanoin (Nu-Chek PREP, Inc., Waterville, MN, USA) as an internal standard to quantify TAG. To quantify total FAs in total lipids, known amount of Triheptadecanoin (Nu-Chek PREP, Inc.) was used as an internal standard. FAME were analysed by GC-FID (7890A GC; Agilent Technologies, Palo Alto, CA, USA) equipped with a 30 m BPX70 column (0.25 mm inner diameter, 0.25 mm film thickness, SGE, Austin, Tx, USA) as described previously [18]. Peaks were integrated with Agilent Technologies CHEMSTATION software (Rev B.04.03, Agilent Technologies, Inc., Wilmington, DE, USA). In vitro [ 14 C] acetate feeding and pulse chase assays Following infiltration, leaf discs were floated in potassium phosphate buffer (0.1 M ph 7.2) containing [ 14 C] acetate (56 mcimmol 1 from American Radiolabeled Chemicals, St. Louis, MO, USA) as a substrate at a concentration of 0.4 mm. The assays were carried out for 15 min at room temperature with gentle mixing. Another batch was pulsed with [ 14 C] acetate for 10 min and chased for 20 and 30 min after washing three times with buffer. At each time point, three leaf discs were pooled randomly across the two leaves. After incubation, the reaction was stopped by adding 300 ll chloroform : methanol (2 : 1 v : v). Total lipids were then extracted as described by [24]. Lipid samples were loaded on a TLC plate ( cm, silica gel 60; Merck) and developed in hexane : diethyl ether : acetic acid (90 : 7.5 : 1 v : v : v). The TLC plate was exposed to phosphor imaging screens overnight and analysed by a Fujifilm FLA-5000 phosphorimager. Radiolabelled lipid spots were measured using a Beckman-Coulter Ready Safe liquid scintillation cocktail and Beckman-Coulter LS 6500 Multipurpose Scintillation Counter. Results Characterization of thioesterase activity in transient expression assays We first characterized the biochemical activity of the three A. thaliana thioesterases by coinfiltration of N. benthamiana WT leaves with 35S::WRI1 and 35S:: DGAT1 expression vectors (Fig. 1). The oil content in transient leaf assays was measured, confirming that the WRI1 and DGAT1 synergy was increasing the oil content. The transient overexpression of FATA2 in combination with WRI1 and DGAT1 led to a further 2.5-fold increase in TAG relative to p19 + WRI1 + DGAT1 control, or a 50-fold increase relative to p19 alone (Fig. 1A). FATA1 transient expression increased TAG by twofold compared to p19 + WRI1 + DGAT1, and 40-fold increase compared to p19 alone. FATB transient expression improved TAG accumulation by 1.6- fold relative to p19 + WRI1 + DGAT1, and 32-fold increase relative to p19 control. Thioesterase coexpression was also found to result in modified leaf FA profile (Fig. 1B). FATA1 was found to increase the C16:0 and C18:0 percentage at the expense of saturated FAs. FATA2 also increased the proportion of C18:0 but did not have as great an effect on C16:0. In contrast, FATB was more specific for channelling C16:0 into leaf oils, with little activity observed for C18:0. FATA1, FATA2 and FATB all reduced C18:1 levels. Overall, C16:0 percentage increased from 28.4% in p19 + WRI1 + DGAT1 to 43.8% with the addition of FATA1, to 34.4% with the addition of FATA2 and to 46.3% with the addition of FATB. Effect of transient thioesterase expression in a high oil background Thioesterase genes were also tested in a homozygous high oil N. benthamiana (AT001) plant stably expressing WRI1, DGAT1 and OLEOSIN genes (Fig. 2). Thirty plants from T2 transgenic AT001 seeds were grown in a random design alongside WT controls. At young vegetative stage, 53 days after sowing (DAS), we observed 8.7% TAG DW in transgenic leaves compared to 0.03% TAG DW in WT (Fig. 2). In WT, TAG levels did not exceed 0.03% DW. In the transgenic line, TAG levels increased from 11.2 to 21.3% DW during flowering stages. It then increased progressively reaching a maximum level of 31.4% TAG DW at late seed development stage. At senescence, TAG percentage seemed to decrease to 19.6% DW. The analysis of the TAG FA profile showed an overall decrease in C18:3n3 and an increase in C18:1 450 FEBS Letters 591 (2017) ª 2016 Federation of European Biochemical Societies

4 A. El Tahchy et al. Thioesterases push the fatty acid flux to yield more oil A TAG (% DW) p19 p19+wri+dgat p19+wri+dgat+fata1 p19+wri+dgat+fata2 p19+wri+dgat+fatb B TAG FA profile (%) WT - p19 WT - p19+wri+dgat WT - p19+wri+dgat+fata1 WT - p19+wri+dgat+fata2 WT - p19+wri+dgat+fatb Fig. 1. Thioesterase transient expression in WT Nicotiana benthamiana leaves. (A) TAG accumulation as a DW percentage. (B) TAG FA profile. Error bars denote standard deviation with n = 9. 0 C14:0 C16:0 C16:1 C16:3 C18:0 C18:1 C18:1d 11 C18:2 C18:3n 3 C20:0 C20:1 C22:0 C24:0 WT - p WT - p19+wri+dgat WT - p19+wri+dgat+fata WT - p19+wri+dgat+fata WT - p19+wri+dgat+fatb Fig. 2. TAG accumulation and FA profile in transgenic Nicotiana benthamiana overexpressing WRI1, DGAT1 and OLEOSIN transgenes and in WT control leaves at different plant development stages. Error bars represent standard deviation (n = 11). when compared to WT (Fig. 2). The FA profile shift from polyunsaturated FA to monounsaturated FA upon WRI1 and DGAT1 coexpression has already been reported in N. benthamiana [22]. These results are in coherence with the earlier findings with WRI1 and DGAT1 coexpression. In order to test the effect of thioesterases overexpression in high oil background, leaves of young homozygous plants were infiltrated with p19, FATA1, FATA2 and FATB expression vectors at 49 DAS. Leaves at this stage typically had an oil level of 3.1% TAG DW (Fig. 3). Five days after thioesterase infiltration, leaves were harvested and analysed for TAG content and also used in radiolabel feeding assays. Similar to the WT results mentioned above, FATA2 overexpression in AT001 N. benthamiana showed a FEBS Letters 591 (2017) ª 2016 Federation of European Biochemical Societies 451

5 Thioesterases push the fatty acid flux to yield more oil A. El Tahchy et al. significant TAG increase up to 4.4% TAG DW when compared to the p19 control (3.1% TAG DW; Fig. 3). FATA1 further increased TAG content up to 3.9% TAG DW. However, FATB transient expression showed no significant effect on TAG accumulation (3.1% TAG DW) in AT001 infiltrated leaves (Fig. 3). The FA profile shift was not as pronounced in the high oil background as compared to the WT transient assay (data not shown). The additional effect of thioesterase expression on the FA profile may not have been observed due to the short expression window of the transient assay hence needs further investigation in a stable context. [ 14 C] Acetate pulse chase experiment [ 14 C] Acetate was added in a 10 min pulse to floating leaf discs of AT001 leaves, infiltrated prior with p19 plus FATA1, FATA2 or FATB. This pulse was followed by a 20 min chase (Fig. 4). Lipid extracts were prepared at each time point followed by separation of labelled lipid classes on TLC (Fig. 4A). Quantification of the labelled reaction products showed no additional transient effect of FATA1, FATA2 and FATB overexpression on diacylglycerol (DAG) levels in AT001 transgenic leaves. In line with the earlier GC results, increases in radiolabelled TAG were observed in AT001 leaves transiently expressing FATA1 (602 DPM), FATA2 (762 DPM) and FATB (559 DPM) compared to p19 control (283 DPM; Fig. 4B). Discussion Reprogramming vegetative tissue to produce increased amounts of TAG represents a potential way of substantially increasing the global supply of renewable TAG (% DW) p19 p19 + FATA1 Fig. 3. Thioesterase overexpression in AT001 Nicotiana benthamiana leaves, expressing WRI1, DGAT1 and OLEOSIN transgenes. Error bars denote standard deviation with n = 9. *Significant at P < (Student s T-test). * p19 + FATA2 p19 + FATB liquid fuel energy, especially if this could be achieved in high-biomass crops [2,25]. Vanhercke et al. (2013) demonstrated that coexpression of A. thaliana WRI1 and DGAT1 using N. benthamiana transient leaf expression system, increased TAG levels by 22-fold, and the FA profile of leaf tissue shifted from polyunsaturated FAs to monounsaturated FAs. More recently, Vanhercke et al. (2014) [20] combined the expression of WRI1, DGAT1 and OLEOSIN to engineer N. tabacum leaves capable of producing more than 15% TAG DW content without severe effects on plant development. In addition, the combined expression of WRI1, DGAT1 and OLEOSIN in N. tabacum resulted in the production of leaf TAG more enriched in C18:1 at the expense of C18:3n3 [20]. In our study, TAG levels reached 31.4% DW during seed setting in N. benthamiana. Similar to that reported in N. tabacum [20], high TAG levels (19.6% DW) were detected in dry brown leaves at senescence. The higher levels in TAG in N. benthamiana species compared to the previously reported N. tabacum levels could be the result of higher temperature and light conditions. This present study was performed in confined growth cabinets to replicate summer-like conditions with 1000 lmol photons m 2 s 1 of light intensity for 16 h light and 8 h dark periods while the earlier tobacco study was performed under glasshouse conditions [20]. Recent studies have shown that heat stress can lead to increased accumulation of TAG in A. thaliana vegetative tissue [26,27]. Higashi et al. (2015) [26] reported complementary transcriptomic data which indicated that the expression of genes encoding most of the Kennedy pathway enzymes increased during heat stress. TAG biosynthesis can be divided into two parts in which the first part can be categorized as FA production inside plastids ( push ) and the second part as TAG assembly in the ER ( pull ) [28]. The hydrolysis of FAs exiting the chloroplast represents one of the limiting factors for TAG synthesis. As a result, improvements in FA flux at this step represent one possible method by which TAG accumulation could be further increased [29]. Thioesterase expression could also potentially have an additive effect when combined with WRI1 and DGAT1 expression to increase TAG in vegetative tissues. First, we demonstrated this concept with a series of transient thioesterase coexpression experiments with WRI1 and DGAT1 in WT N. benthamiana leaves. An additive effect on TAG levels was observed in transient assays with up to 2.5-fold increase relative to p19 + WRI1 + DGAT1 control. Second, we used a combination of WRI1, DGAT1 and OLEOSIN to stably transform N. benthamiana, 452 FEBS Letters 591 (2017) ª 2016 Federation of European Biochemical Societies

6 A. El Tahchy et al. Thioesterases push the fatty acid flux to yield more oil A TAG DAG Origin Fig. 4. [ 14 C] acetate pulse chase experiment on AT001 Nicotiana benthamiana leaves overexpressing WRI1, DGAT1 and OLEOSIN transgenes and infiltrated with p19, FATA1, FATA2, orfatb. (A) Radiolabelled lipid classes separated on TLC and exposed to phosphor image screen. (B) Radiolabel accumulation in TAG and DAG neutral lipids. Infiltrated leaves were pulsed for 10 min with [ 14 C] acetate and chased for 20 min after washing and replacement of buffer. Error bars denote standard deviation with n = 9. B Counts (DPM) TAG DAG p19 p19+fata1 p19+fata2 p19+fatb providing a high oil matrix in a plant that is well suited for rapid assemblies of metabolic pathways [30]. In this study, the overexpression of thioesterase increased the flux of FAs exiting the chloroplast, visible as higher incorporation of radiolabel in TAG in the 14 C- acetate pulse chase experiment. The overexpression of thioesterases is therefore likely improving the metabolic link with the WRI1-mediated push and DGAT1-driven pull strategies [18]. Overexpression of specific thioesterases has been demonstrated to be effective for modifying the profile of bioengineered oil [6]. The specificity of the acyl- ACP thioesterases is important in defining the FA profile, and thus, these enzymes are relevant targets to manipulate the FA composition of seed oils [10]. The composition of FAs exported from the plastid in different plants, or different tissues within a plant, is dependent on the relative activities of the acyl-acp thioesterases and acyl-acp desaturases [31,32] to produce various saturated and monounsaturated FAs [33]. Arabidopsis FATB1 overexpression has been reported to increase the accumulation of palmitate in the Arabidopsis seeds [34]. These and other results indicate that acyl-acp thioesterases can be key enzymes to produce specialty bioengineered oils not only in seed but also in leaf tissue as well, provided acyl-acp thioesterases displaying high activities towards the target FAs are available [6]. Here, the overexpression of thioesterases resulted in a modification of the FA profile and improved the fluxes of FAs into oils. Once in the extra-plastidic acyl-coa pool, FAs are utilized for glycerolipid assembly via FEBS Letters 591 (2017) ª 2016 Federation of European Biochemical Societies 453

7 Thioesterases push the fatty acid flux to yield more oil A. El Tahchy et al. acyl-editing or the Kennedy pathway [35,36]. In addition, it is known that the de novo synthesis of FAs, through the Kennedy pathway can assemble oil rich in saturated and monounsaturated FAs [37]. It has also been suggested that the shift towards C18:1 and the decrease in C18:3n3 in total FAs could be the cause of the limited availability of the ER desaturases responsible for polyunsaturated FA formation [38]. The modification of the FA profile with thioesterase overexpression resulted in predominantly saturated FAs that are readily accessible to the Kennedy pathway and do not require passage through membrane lipid such as phosphatidylcholine (PC). However, further quantitative analysis of PC production need to be investigated. We also observed no increase in radiolabel accumulation in DAG after overexpression of thioesterases. This indicates that the increased flow FAs are able to pass from DAG to TAG very efficiently in N. benthamiana. This is probably due to additional pull capacity created by the overexpression of DGAT1 [18]. Finally, the overexpression of thioesterases in N. benthamiana WT and AT001 leaves did not affect the leaf morphology (Fig. S1). Thioesterases could then be used in combination with WRI1, DGAT1 and OLEOSIN to stably transform a highbiomass crop to produce high oil yields. In conclusion, we demonstrated high levels of TAG during the seed setting stage of a bioengineered leafy biomass. We were able to generate a very useful, reproducible and prominent leaf system producing a homogenous oil content that can be used for further lipid biosynthesis studies. Furthermore, by overexpressing FATA2, FATA1 or FATB genes, we were able to further enhance the oil content. Our results also highlight a modification of the FA profile and an increase in the acyl flux towards TAG assembly. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Authors contributions AET conceptualized and carried out the experiments, and drafted the manuscript. KR conducted initial transformation of N. benthamiana and performed lipid analyses for the selection of homozygous line. JP and SS provided guidance and participated in critical review of the manuscript. TV conceptualized and contributed to critical review of the manuscript. Acknowledgements This work was supported by post-doctoral fellowship of the CSIRO Agriculture Business Unit. We thank Xue-Rong Zhou and Yoko Kennedy who kindly selected the homozygous line. References 1 Priti M and Kovalchuk I (2014) Genetic engineering of oilseed crops. Biocatal Agric Biotechnol 3, Ohlrogge J and Chapman K (2011) Expanding the contribution of plant oils as biofuels. The seeds of green energy. Biochemist 33, Weselake RJ (2016) Chapter 15 - Engineering oil accumulation in vegetative tissue. In Industrial Oil Crops, pp st edn. Elsevier Inc. ISBN: Voelker TA, Jones A, Cranmer AM, Davies HM and Knutzon DS (1997) Broad-range and binary-range acylacyl-carrier-protein thioesterases suggest an alternative mechanism for medium-chain production in seeds. Plant Physiol 114, Jones A, Davies HM and Voelker TA (1995) Palmitoylacyl carrier protein (acp) thioesterase and the evolutionary origin of plant acyl-acp thioesterases. Plant Cell 7, Salas JJ and Ohlrogge JB (2002) Characterization of substrate specificity of plant FatA and FatB acyl-acp thioesterases. Arch Biochem Biophys 403, Sanchez-Garcia A, Moreno-Perez AJ, Muro-Pastor AM, Salas JJ, Garces R and Martinez-Force E (2010) Acyl- ACP thioesterases from castor (Ricinus communis L.): an enzymatic system appropriate for high rates of oil synthesis and accumulation. Phytochemistry 71, Hawkins DJ and Kridl JC (1998) Characterization of acyl-acp thioesterases of mangosteen (Garcinia mangostana) seed and high levels of stearate production in transgenic canola. Plant J 13, Knutzon DS, Bleibaum JL, Nelsen J, Kridl JC and Thompson GA (1992) Isolation and characterization of 2 safflower oleoyl-acyl carrier protein thioesterase cdna clones. Plant Physiol 100, Rodrıguez-Rodrıguez MF, Salas JJ, Garces R and Martınez-Force E (2014) Acyl-ACP thioesterases from Camelina sativa: cloning, enzymatic characterization and implication in seed oil fatty acid composition. Phytochemistry 107, Voelker TA, Hayes TR, Cranmer AM, Turner JC and Davies HM (1996) Genetic engineering of a quantitative trait: metabolic and genetic parameters influencing the accumulation of laurate in rapeseed. Plant J 9, Mandal MNA, Santha IM, Lodha ML and Mehta SL (2000) Cloning of acyl-acyl carrier protein (ACP) 454 FEBS Letters 591 (2017) ª 2016 Federation of European Biochemical Societies

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9 Thioesterases push the fatty acid flux to yield more oil A. El Tahchy et al. 38 Andrianov V, Borisjuk N, Pogrebnyak N, Brinker A, Dixon J, Spitsin S, Flynn J, Matyszczuk P, Andryszak K, Laurelli M et al. (2010) Tobacco as a production platform for biofuel: overexpression of Arabidopsis DGAT and LEC2 genes increases accumulation and shifts the composition of lipids in green biomass. Plant Biotechnol J 8, Supporting information Additional Supporting Information may be found online in the supporting information tab for this article: Fig. S1. Wild-type and AT001 transgenic Nicotiana benthamiana plants at 28 days after sowing. 456 FEBS Letters 591 (2017) ª 2016 Federation of European Biochemical Societies