Figure 4 - uploaded by Harrie Van Erp
Content may be subject to copyright.
Simplified figure showing the effect of RcLPAT2 on the TAG biosynthetic pathway in lesquerella focusing on the Kennedy pathway. In the Kennedy pathway sequential acylation of a glycerol backbone takes place, by the GPAT, LPAT and DGAT enzymes in order to generate TAG. (A) In lesquerella the GPAT and DGAT enzymes have high selectivity for 20:1-OH leading to acylation of the sn-1 and sn-3 positions of the glycerol backbone with 20:1-OH. LPAT has specificity for non-hydroxylated FAs, preventing the incorporation of significant amounts of HFA (~2%18:1-OH and ~1% 20:1-OH) at the sn-2 position of the glycerol backbone (Figure 3); (B) In transgenic lesquerella seeds expressing RcLPAT2, e.g., line 3-1, approximately 19% of FAs at the sn-2 position of TAG consist of HFAs (~17% 18:1-OH and ~2% 20:1-OH) (Figure 3). However, the total amount of HFAs in the seed oil is not increased, which leads to a decrease in acylation of 20:1-OH the sn-1 and sn-3 positions (Tables 1 and 2). This indicates that RcLPAT2 has a high specificity for acylating 18:1-OH to the sn-2 position of LPA in lesquerella, which results in an increased amount of tri-HFA-TAGs in transgenic seed oil (Figure 2). 18:n means 18:1, 18:2, or 18:3, described as in Table 2. Other abbreviations are as in Figure 1. Red words show the effect of RcLPAT2.
Source publication
Abstract: Lesquerella is a potential industrial oilseed crop that makes hydroxy fatty acid (HFA).
Unlike castor its seeds are not poisonous but accumulate lesquerolic acid mostly at the sn-1 and
sn-3 positions of triacylglycerol (TAG), whereas castor contains ricinoleic acid (18:1OH) at all
three positions. To investigate whether lesquerella can be...
Similar publications
Abstract:
Carissa carandas (L.) belonging to the Apocynaceae family and it is represented about 89 species in
India. It is distributed throughout India in dry, sandy and rocky grounds. It naturally grows in the Himalayas
at a height of 300 to 1800 meters in the Siwalik Hills from sea level and require fully exposure to sun and
unfavorable to humidi...
The Panel on Food Additives and Nutrient Sources added to Food (ANS) provides a scientific opinion re-evaluating the safety of polyglycerol polyricinoleate (PGPR, E 476) used as a food additive. In 1978, the Scientific Committee for Food (SCF) established an acceptable daily intake (ADI) of 7.5 mg/kg body weight (bw) per day for PGPR. PGPR is hydro...
Ricinoleic acid (RA), a hydroxyl fatty acid, is suitable for medical and industrial uses and is produced in high-oil-accumulating organisms such as castor bean and the ergot fungus Claviceps. We report here the efficient production of RA in a transgenic diatom Chaetoceros gracilis expressing the fatty acid hydroxylase gene (CpFAH) from Claviceps pu...
In the perspective to valorize Cameroonian castor bean (Ricinus communis L), physico-chemical properties of oil and cake from seeds of three accessions of this plant originated from the Sudano-sahelian and Sudano-guinean zones of Cameroon were assessed. Seeds oil extracted by soxhlet method using hexane exhibited contents varying significantly (p<0...
(R)-Ricinoleic acid (RA) [(12R,9Z)-hydroxyoctadecenoic acid], the main compound of castor seed oil, due to its unusual structure readily undergoes multi-directional chemical and biochemical transformations to produce derivatives with the retained carbon skeleton or with its degradation. Many of these are of high biological activity, as documented b...
Citations
... We have produced 17 transgenic lesquerella lines expressing RcLPAT2 under the control of a seed specific promoter [21]. Our results indicate that RcLPAT2 enables the incorporation of 18:1OH at sn-2 position of LPA which increases the accumulation of 18:1OH and also tri-HFA-TAGs in lesquerella (Figures 2 and 3). ...
... Besides, less dynamic changes between average increase of 18:1OH and average decrease of total HFA were observed in the 3-dsRNA group, showing averages of 4.7% and 48.9%, respectively, ( Table 2), compared with that of 7.7% and 53% in lines expressing 2-dsRNA, respectively ( Table 1). FA composition in WT seeds (Tables 1 and 2) were similar to described [21,36]. There was no change of growth phenotype for transgenic lesquerella expressing CsFAD2 RNAi, CsFAD3 RNAi and AtFEA1 RNAi. ...
... On the other hand, the accumulation of 18:1 could also be due to a lesquerella LPAT that has substrate preference for 18:1-CoA, resulting in efficient incorporation of 18:1-CoA into TAG through Kennedy pathway. We already showed that castor RcLPAT2 increased 18:1OH in lesquerella [20,21]. Besides, castor RcLPAT3B and RcLPATB also showed substrate preference to 18:1OH in Arabidopsis [40]. ...
Hydroxy fatty acid (HFA) is a vital raw material for numerous industrial products, such as lubricants, plasticizers and surfactants. Castor oil is the current commercial source of HFA which contains 90% ricinoleic acid (18,1OH). Castor seeds contain the toxin ricin and hyperallergic 2S albumins; it is detrimental to castor oil production. Lesquerella is a potential industrial oilseed crop for a safe source of HFA, because lesquerella seeds contain a valuable HFA, lesquerolic acid (20,1OH), at 55–60% in seed oil. This chapter describes current progress on improving HFA production in lesquerella through metabolic engineering.
... The Nicaud group engineered Yarrowia lipolytica by co-expression of CpFAH12 and PDAT to accumulate RA to 43% of total lipids, and over 60 mg/g DCW [102]. RA accumulation attempts were also tested in microalgae and plants by expressing castor LPAT or CpFAH [103,104]. ...
Microbial lipids have been a hot topic in the field of metabolic engineering and synthetic biology due to their increased market and important applications in biofuels, oleochemicals, cosmetics, etc. This review first compares the popular hosts for lipid production and explains the four modules for lipid synthesis in yeast, including the fatty acid biosynthesis module, lipid accumulation module, lipid sequestration module, and fatty acid modification module. This is followed by a summary of metabolic engineering strategies that could be used for enhancing each module for lipid production. In addition, the efforts being invested in improving the production of value-added fatty acids in engineered yeast, such as cyclopropane fatty acid, ricinoleic acid, gamma linoleic acid, EPA, and DHA, are included. A discussion is further made on the potential relationships between lipid pathway engineering and consequential changes in cellular physiological properties, such as cell membrane integrity, intracellular reactive oxygen species level, and mitochondrial membrane potential. Finally, with the rapid development of synthetic biology tools, such as CRISPR genome editing tools and machine learning models, this review proposes some future trends that could be employed to engineer yeast with enhanced intracellular lipid production while not compromising much of its cellular health.
... As such, efforts have been made through plant breeding to develop lesquerella as a new oilseed crop that is a safe source of HFA [12,13]. With the success of lesquerella biotechnology [14,15] and the deep knowledge of genes for fatty acid and seed oil biosynthesis [16][17][18], lesquerella oil can be improved through metabolic engineering [15]. ...
... As such, efforts have been made through plant breeding to develop lesquerella as a new oilseed crop that is a safe source of HFA [12,13]. With the success of lesquerella biotechnology [14,15] and the deep knowledge of genes for fatty acid and seed oil biosynthesis [16][17][18], lesquerella oil can be improved through metabolic engineering [15]. ...
... Lesquerella TAGs contain~60% 20:1OH, and almost all of 20:1OH are acylated to the sn-1 and sn-3 positions, and the sn-2 positions of lesquerella TAGs are exclusively occupied by unsaturated FAs, i.e., 18:1, 18:2 and 18:3 [34][35][36][37]. The reason for lack of HFA at the sn-2 position of TAG has been suggested, in part, by the selectivity of lesquerella LPAT (PfLPAT) for unsaturated FA [15], which is a common feature for most plant microsomal LPAT [38]. PC can be converted to DAG (PC-derived DAG) through the removal of the head group from the PC by PC:DAG cholinephosphotransferase (PDCT) [39][40][41] (Figure 1); therefore, acyl-CoAs on the PC are directed to DAG for TAG synthesis. ...
Seeds of castor (Ricinus communis) are enriched in oil with high levels of the industrially valuable fatty acid ricinoleic acid (18:1OH), but production of this plant is limited because of the cooccurrence of the ricin toxin in its seeds. Lesquerella (Physaria fendleri) is being developed as an alternative industrial oilseed because its seeds accumulate lesquerolic acid (20:1OH), an elongated form of 18:1OH in seed oil which lacks toxins. Synthesis of 20:1OH is through elongation of 18:1OH by a lesquerella elongase, PfKCS18. Oleic acid (18:1) is the substrate for 18:1OH synthesis, but it is also used by fatty acid desaturase 2 (FAD2) and FAD3 to sequentially produce linoleic and linolenic acids. To develop lesquerella that produces 18:1OH-rich seed oils such as castor, RNA interference sequences targeting KCS18, FAD2 and FAD3 were introduced to lesquerella to suppress the elongation and desaturation steps. Seeds from transgenic lines had increased 18:1OH to 1.1–26.6% compared with that of 0.4–0.6% in wild-type (WT) seeds. Multiple lines had reduced 18:1OH levels in the T2 generation, including a top line with 18:1OH reduced from 26.7% to 19%. Transgenic lines also accumulated more 18:1 than that of WT, indicating that 18:1 is not efficiently used for 18:1OH synthesis and accumulation. Factors limiting 18:1OH accumulation and new targets for further increasing 18:1OH production are discussed. Our results provide insights into complex mechanisms of oil biosynthesis in lesquerella and show the biotechnological potential to tailor lesquerella seeds to produce castor-like industrial oil functionality.
... Plant cytoplasmic LPAAT-As have been cloned from Limnanthes douglasii, Arabidopsis thaliana, Brassica napus, Echium pitardii, and Ricinus communis. They are normally expressed ubiquitously in plants with broad substrates (Brown et al., 2002a,b;Kim et al., 2005;Maisonneuve et al., 2010;Arroyo-Caro et al., 2013;Mañas-Fern andez et al., 2013;Chen et al., 2016) with two exceptions that cytoplasmic LPAAT-As from R. communis and Vernicia fordii have specific activity towards ricinoleic acid and α-eleostearic acid, respectively (Shockey et al., 2019). Plant cytoplasmic LPAAT-Bs have been identified in L. douglasii (Brown et al., 1995;Hanke et al., 1995) and Cocos nucifera where they are usually involved in the incorporation of unusual FAs into storage TAGs in seeds with an exception that a cytoplasmic LPAAT-B from R. communis is constitutively expressed and exhibits a broad range of substrates . ...
... Therefore, substrate specificity of this enzyme has been recognized to be a limiting factor in the incorporation of specialty fatty acids into the position of glycerolipids, thereby constraining the production of TAGs with unusual fatty acids. For instance, in some plant species, specific LPAATs are required to incorporate erucic acid (Brown et al., 1995), lauric acid , ricinoleic acid (Chen et al., 2016) and sterculic acid (Yu et al., 2014) into triacylglycerols. In this study, co-expression of EhELO1 and EhLPAAT2 could effectively synthesize and incorporate two unusual VLCPUFAs, DDA and DTA into the sn-2 position of TAGs, thereby improving the production of the two fatty acids in B. carinata seeds. ...
Docosadienoic acid (DDA, 22:2n-6) and docosatrienoic acid (DTA, 22:3n-3) are two very long chain polyunsaturated fatty acids (VLCPUFAs) that are recently shown to possess strong anti-inflammatory and antitumor properties. An ELO type elongase (EhELO1) from wild plant Eranthis hyemalis can synthesize the two fatty acids by sequential elongation of linoleic acid and alpha-linolenic acid, respectively. Seed-specific expression of this gene in oilseed crop Brassica carinata produced a considerable amount of DDA and DTA in transgenic seeds. However, these fatty acids were excluded from the sn-2 position of triacylglycerols (TAGs). To improve the production level and nutrition value of the VLCPUFAs in the transgenic oilseed crop, a cytoplasmic lysophosphatidic acid acyltransferase (EhLPAAT2) for the incorporation of the two fatty acids into the sn-2 position of triacylglycerols was identified from E. hyemalis. RT-PCR analysis showed that it was preferentially expressed in developing seeds where EhELO1 was exclusively expressed in E. hyemalis. Seed specific expression of EhLPAAT2 along with EhELO1 in B. carinata resulted in not only the effective incorporation of DDA and DTA at the sn-2 position of TAGs, and the increased amount of DDA and DTA reaching 35% of the total fatty acids in transgenic seeds, but also increased seed oil and seed weight relative to the control with EhELO1 alone. Improved production of DDA and DTA in the oilseed crop using EhLPAAT2 and EhELO1 provides a real commercial opportunity for high value agriculture products for nutraceutical uses.
... P. lindheimeri is also crossed with lesquerella, but there is no significant increase in HFA level in the hybrid off-springs of lesquerella [16]. With the success of lesquerella biotechnology [17,18], genes and regulatory elements from P. lindheimeri can be used as excellent targets for improving lesquerella through Agrobacterium-mediated genetic transformation. Besides the HFAs found in plant seeds, families of saturated hydroxy fatty acids (SHFAs) have been recently discovered in cow, goat, and human milks [19,20], and the SHFAs from human milk inhibit the growth of human cancer cells and suppress beta-cell apoptosis [19], indicating that SHFAs may play a role in the promotion and protection of human health. ...
... Lesquerella TAGs contain~60% 20:1OH and almost all of it is esterified to the sn-1 and sn-3 positions [54] (Figure 1). The absence of HFAs at the sn-2 position of TAG might be caused by the preference of endogenous lesquerella LPATs (PfLPATs) for common FAs [18]. To increase the HFA content at the sn-2 position of lesquerella TAGs, we have overexpressed RcLPAT2 in lesquerella seeds, and the resulting transgenic seed oil increases 3-HFA-TAGs (TAGs with all three sn positions acylated with HFAs) from 5% to 13-14% [18]. ...
... The absence of HFAs at the sn-2 position of TAG might be caused by the preference of endogenous lesquerella LPATs (PfLPATs) for common FAs [18]. To increase the HFA content at the sn-2 position of lesquerella TAGs, we have overexpressed RcLPAT2 in lesquerella seeds, and the resulting transgenic seed oil increases 3-HFA-TAGs (TAGs with all three sn positions acylated with HFAs) from 5% to 13-14% [18]. Regiochemical analysis reveals that RcLPAT2 increases the 3-HFA-TAGs content by enhancing the acylation of 18:1OH at the sn-2 position of 20:1OH-LPA for the subsequent generation of sn-1/3-20:1OH-sn-2-18:1OH-TAG [55]. ...
Hydroxy fatty acids (HFAs) have numerous industrial applications but are absent in most vegetable oils. Physaria lindheimeri accumulating 85% HFA in its seed oil makes it a valuable resource for engineering oilseed crops for HFA production. To discover lipid genes involved in HFA synthesis in P. lindheimeri, transcripts from developing seeds at various stages, as well as leaf and flower buds, were sequenced. Ninety-seven percent clean reads from 552,614,582 raw reads were assembled to 129,633 contigs (or transcripts) which represented 85,948 unique genes. Gene Ontology analysis indicated that 60% of the contigs matched proteins involved in biological process, cellular component or molecular function, while the remaining matched unknown proteins. We identified 42 P. lindheimeri genes involved in fatty acid and seed oil biosynthesis, and 39 of them shared 78–100% nucleotide identity with Arabidopsis orthologs. We manually annotated 16 key genes and 14 of them contained full-length protein sequences, indicating high coverage of clean reads to the assembled contigs. A detailed profiling of the 16 genes revealed various spatial and temporal expression patterns. The further comparison of their protein sequences uncovered amino acids conserved among HFA-producing species, but these varied among non-HFA-producing species. Our findings provide essential information for basic and applied research on HFA biosynthesis.
... The Siberian apricot seed oil FAMEs was determined using gas chromatography-mass spectrometry (GC-MS). The determination and analysis of FAMEs are performed in Agilent 7890A gas chromatograph-Flame Ionization Detector (Chen et al., 2016). One microliter of the hexane extract was transferred to a vial and injected into a highly polar HP Innowax capillary column (30 m × 0.32 mm ID × 0.25 mm film thickness) with a split ratio of 1:20. ...
Siberian apricot (Prunus sibirica L.), an excellent woody oil plant unique to Asia, is well known for its ability to produce high‐oil seeds for use as a promising feedstock of biodiesel. Based on the investigation of natural Siberian apricot resources in China in the early stage, seeds of Siberian apricot from 74 geographic provenances which can fully reflect the overall information were collected. In this research, seeds oil content, fatty acid composition and biodiesel properties were evaluated, and the key environmental factors that caused the variation of these in different geographic provenance were analyzed. The oil content of Siberian apricot seeds is 45.48%–61.07%, and the average was 50.95% for all provenances. The characteristics of oil can identify and quantify eight fatty acids. The most abundant fatty acids were oleic acid (C18:1; 54.02%–76.54%), followed by linoleic acid (C18:2; 16.78%–38.49%) and erucic acid (C16:0; from 3.27% to 6.12%). Monounsaturated fatty acids are the most abundant in 54.75%–77.03% compared with saturated fatty acids and polyunsaturated fatty acids. The biodiesel properties of most provenance seeds meet the standards of the ASTM D6751 and GB/T 20828, and a few meet the standards of the EN14214. Through the clustering of oil content and fatty acid composition and the analysis of biodiesel properties indexes, it is concluded that KSK provenance is the most suitable for biodiesel production. The XBZ, HHE, AES, ZLQ and LD provenances may be preserved as potential biodiesel. RDA and VPA showed that the effects of environmental factors on the oil properties of Siberian apricot were ranked as terrain factor > climate factor > soil factor, among which longitude, latitude and altitude are the main terrain indicators. These evaluations can provide reference for the effective utilization and further development of Siberian apricot as a bioenergy feedstock.
... However, major drawbacks of castor oil production lie in the lethal toxin ricin and allergenic proteins in seeds (Chen et al., 2004(Chen et al., , 2005Lord et al., 2003;Machado and Silva, 1992). To develop safe crops for castor oil production, metabolic engineering studies regarding HFA production have been attempted by introducing key genes from castor to the non-HFA-producing Arabidopsis model and that of other oilseeds (Aryal and Lu, 2018;Bayon et al., 2015;Chen et al., 2016;Lee et al., 2015;Shockey et al., 2019). ...
Determining the role of castor lysophosphatidic acid acyltransferases (RcLPATs) provides information that aid in understanding the biosynthesis mechanism of castor oil (triacylglycerols, TAG), which contains 90 % ricinoleic acid (18:1OH), a hydroxy fatty acid (HFA) with numerous industrial applications. The entire family of seven RcLPATs was shown to encode functional enzymes using in vitro assays. Gene expression analysis suggested that RcLPATs play roles in various vegetative and reproductive organs by associating with membrane lipid and TAG biosynthesis. To identify isoforms of RcLPATs capable of acylating 18:1OH, individual RcLPATs were expressed in CL37, an Arabidopsis line containing approximately 17 % HFA in seed TAG. Transgenics expressing RcLPAT2, RcLPAT3B, or RcLPATB increased total HFA to 18.2 %–20.3 %. Furthermore, different accumulation levels of 18:1OH and densipolic acid (18:2OH) were detected among these three transgenic backgrounds. The mechanisms of substrate selectivity among RcLPAT2, RcLPAT3B, and RcLPATB are discussed.
... Up [498] The table includes the fatty acid or lipid product engineered in particular plants and respective tissues. It also includes the maximum relative amount of FA or lipid product obtained, along with the reactions/genes, their sources, and the respective manipulations employed (i.e. ...
Plant lipids have versatile applications and provide essential fatty acids in human diet. Therefore, there has been a growing interest to better characterize the genetic basis, regulatory networks, and metabolic pathways that shape lipid quantity and composition. Addressing these issues is challenging due to context-specificity of lipid metabolism integrating environmental, developmental, and tissue-specific cues. Here we systematically review the known metabolic pathways and regulatory interactions that modulate the levels of storage lipids in oilseeds. We argue that the current understanding of lipid metabolism provides the basis for its study in the context of genome-wide plant metabolic networks with the help of approaches from constraint-based modeling and metabolic flux analysis. The focus is on providing a comprehensive summary of the state-of-the-art of modeling plant lipid metabolic pathways, which we then contrast with the existing modeling efforts in yeast and microalgae. We then point out the gaps in knowledge of lipid metabolism, and enumerate the recent advances of using genome-wide association and quantitative trait loci mapping studies to unravel the genetic regulations of lipid metabolism. Finally, we offer a perspective on how advances in the constraint-based modeling framework can propel further characterization of plant lipid metabolism and its rational manipulation.
... Kim et al. studied the four cytoplasmic AtLPAT (LPAT2-5) genes of Arabidopsis thaliana, and found that heterozygous mutants of LPAT2 would produce shorter siliques as well as cause abortion in the female gametophyte [9]. Chen et al. found that overexpression of RcLPAT2 would increase the accumulation of ricinoleic acid (18:1OH) at the sn-2 position of LPA in the transgenic Lesquerella seeds [10]. Overexpression of AtLPAT1-5 under phosphate starvation revealed that only AtLPAT2 could significantly contribute to root development, and a significantly increased level of PC and PE in rosette leaves in Arabidopsis [11]. ...
... However, overexpressing two rapeseed LPAAT isoforms (BAT1.13 and BAT1.5) in Arabidopsis seeds, a 16% significant increase was detected in mean total fatty acid content compared with the mean of the combined nontransformed plants [53]. Moreover, in the unicellular green microalga Chlamydomonas reinhardtii, overexpressing CrLPAAT1 in plastids resulted to a > 20% increase in oil content under nitrogen-deficient conditions [10]. It seems that any increase in oil content is hard-won in model or simple organisms, let alone in allotetraploid rapeseed. ...
... They could possibly prevent the coalescence of normal oil bodies to form large oil bodies that increase the ratio of total oil bodies to cell area, which in turn might ultimately increase the total oil content. Additionally, a significant increase was detected in C18:0 and C20:0, while obvious decreases were detected in C18:1, C18:2 and C18:3 in the Bnlpat2/Bnlpat5 lines in the present study, which was similar to the previous result that an increase in 18:1OH but a decrease in 20:1OH was observed when RcLPAT2 was over expressed in Lesquerella [10]. This shows that the decrease in the final oil content was mostly derived from C18 and C20:0 FAs, which probably resulted in an increased conversion of FAs to other compounds, such as sucrose, in mature seeds. ...
Background:
Brassica napus is one of the most important oilseed crops, and can supply considerable amounts of edible oil as well as provide raw materials for the production of biodiesel in the biotechnology industry. Lysophosphatidic acid acyltransferase (LPAT), a key enzyme in the Kennedy pathway, catalyses fatty acid chains into 3-phosphoglycerate and promotes further production of oil in the form of triacylglycerol. However, because B. napus is an allotetraploid with two subgenomes, the precise genes which involved in oil production remain unclear due to the intractability of efficiently knocking out all copies with high genetic redundancy. Therefore, a robust gene editing technology is necessary for gene function analysis.
Results:
An efficient gene editing technology was developed for the allotetraploid plant B. napus using the CRISPR-Cas9 system. Previous studies showed poor results in either on-target or off-target activity in B. napus. In the present study, four single-gRNAs and two multi-gRNAs were deliberately designed from the conserved coding regions of BnLPAT2 which has seven homologous genes, and BnLPAT5, which has four homologous genes. The mutation frequency was found to range from 17 to 68%, while no mutation was observed in the putative off-target sites. The seeds of the Bnlpat2/Bnlpat5 mutant were wizened and showed enlarged oil bodies, disrupted distribution of protein bodies and increased accumulation of starch in mature seeds. The oil content decreased, with an average decrease of 32% for Bnlpat2 lines and 29% for Bnlpat5 lines in single-gRNA knockout lines, and a decline of 24% for Bnlpat2 mutant lines (i.e., g123) and 39% for Bnlpat2/Bnlpat5 double mutant lines (i.e., g134) in multi-gRNA knockout lines.
Conclusions:
Seven BnLPAT2 homologous genes and four BnLPAT5 homologous genes were cleaved completely using the CRISPR-Cas9 system, which indicated that it is effective for editing all homologous genes in allotetraploid rapeseed, despite the relatively low sequence identities of both gene families. The size of the oil bodies increased significantly while the oil content decreased, confirming that BnLPAT2 and BnLPAT5 play a role in oil biosynthesis. The present study lays a foundation for further oil production improvement in oilseed crop species.
... As lesquerella oil contains 55-60% C20:1-OH, over 50% of C18:1 on PC are hydroxylated by the fatty acid hydroxylase, FAH12 (Broun and Somerville, 1997;Kim and Chen, 2015), to form ricinoleic acid (12-hydroxy-9-cis-octadecenoic acid, C18:1-OH) which then be released and activated to C18:1-OH -CoA into the cytosol. Due to an efficient lesquerella condensing enzyme 3-ketoacyl-CoA synthase 3 (KCS3) elongase (Moon et al., 2001;Reed et al., 1997), C18:1-OH -CoA is rapidly elongated to 20:1-OH -CoA for TAG assembly (Chen et al., 2016;Kim and Chen, 2015). In addition to 20:1-OH, lesquerella accumulates two minor HFAs, densipolic acid (12-hydroxy-octadec-cis-9,15enoic acid, C18:2-OH) around˜1% and auricolic acid (14-hydroxyeicos-cis-11,17-enoic acid, C20:2-OH) around 3% (Chen et al., 2011). ...
Lesquerella (Physaria fendleri) contains a major unusual hydroxy fatty acid, lesquerolic acid (14-hydroxy-eicos-cis-11-enoic acid, C20:1-OH), at 55–60% of seed oil which has industrial value. The remaining seed oil comprises mainly common fatty acids including α-linolenic acid (octadec-cis-9,12,15-enoic acid, C18:3) at 10.7–15.8%. C18:3 is produced from linoleic acid (octadec-cis-9,12-enoic acid, C18:2) by FATTY ACID DESATURASE3. Previous seed transcriptome analysis uncovers two fatty acid desaturase 3 (FAD3) transcripts, PfFAD3-1 and PfFAD3-2. To determine the activity of PfFAD3-1 and PfFAD3-2, PfFAD3-1 and PfFAD3-2 were introduced into an Arabidopsis FAD3-deficient mutant (fad3-2) which has reduced C18:3 from 20.0% in wild-type to 1.6% in fad3-2. Among 20 T2 transgenic lines expression PfFAD3-1, C18:3 increased variably from 2.5 to 29.9% demonstrating that PfFAD3-1 acted as a functional FAD3. Among 32 T2 transgenic lines expressing PfFAD3-2, C18:3 content ranged from 1.0 to 3.6%, showing that PfFAD3-2 failed to recover the loss of C18:3 in fad3-2. Sequence comparison among known FAD3s revealed putative variation in PfFAD3-2 which might cause the absence of PfFAD3-2. In addition, lesquerella accumulates a minor hydroxy fatty acid, densipolic acid (12-hydroxy-octadec-cis-9,15-enoic acid, C18:2OH) at about 1%. C18:2OH has been shown to be produced by a FAD3 in Arabidopsis (AtFAD3) using ricinoleic acid (12-hydroxy-9-cis-octadecenoic acid, 18:1-OH) as substrate. To test if either of PfFAD3s is able to convert C18:1-OH to C18:2-OH, PfFAD3-1 or PfFAD3-2 was transferred into a CL37 Arabidopsis which already expresses a castor (Ricinus communis) fatty acid hrdroxylase FAH12 gene (RcFAH12) and consequently accumulates C18:1-OH and C18:2-OH at 13.7% and 3.4%, respectively. Among 43 transgenic CL37 lines expressing PfFAD3-1, C18:2-OH level varied from 0.2 to 7.2%, and four of these lines exceeded to the background level of 3.4% in CL37. Whereas among 23 transgenic CL37 lines expressing PfFAD3-2, C18:2-OH level ranged from 0.4 to 3.4%, none exceeding 3.4%. The results consist with our notion that PfFAD3-1, not PfFAD3-2, exerts FAD3 function which includes converting C18:1-OH to C18:2-OH. Factors limiting PfFAD3s function in CL37 are discussed.
