Identification of hydroxy fatty acid and triacylglycerol metabolism-related genes in lesquerella through seed transcriptome analysis
© Kim and Chen; licensee BioMed Central. 2015
Received: 5 October 2014
Accepted: 27 February 2015
Published: 24 March 2015
Castor oil is the only commercial source of hydroxy fatty acid that has industrial value. The production of castor oil is hampered by the presence of the toxin ricin in its seed. Lesquerella seed also accumulates hydroxy fatty acid and is free of ricin, and thus it is being developed as a new crop for hydroxy fatty acid production. A high-throughput, large-scale sequencing of transcripts from developing lesquerella seeds was carried out by 454 pyrosequencing to generate a database for quality improvement of seed oil and other agronomic traits. Deep mining and characterization of acyl-lipid genes were conducted to uncover candidate genes for further studies of mechanisms underlying hydroxy fatty acid and seed oil synthesis.
A total of 651 megabases of raw sequences from an mRNA sample of developing seeds was acquired. Bioinformatic analysis of these sequences revealed 59,914 transcripts representing 26,995 unique genes that include nearly all known seed expressed genes. Based on sequence similarity with known plant proteins, about 74% (19,861) genes matched with annotated coding genes. Among them, 95% (18,868) showed highest sequence homology with Arabidopsis genes, which will allow translation of genomics and genetics findings from Arabidopsis to lesquerella. Using Arabidopsis acyl-lipid genes as queries, we searched the transcriptome assembly and identified 615 lesquerella genes involved in all known pathways of acyl-lipid metabolism. Further deep mining the transcriptome assembly led to identification of almost all lesquerella genes involved in fatty acid and triacylglycerol synthesis. Moreover, we characterized the spatial and temporal expression profiles of 15 key genes using the quantitative PCR assay.
We have built a lesquerella seed transcriptome that provides a valuable reference in addition to the castor database for discovering genes involved in the synthesis of triacylglycerols enriched with hydroxy fatty acids. The information obtained from data mining and gene expression profiling will provide a resource not only for the study of hydroxy fatty acid metabolism, but also for the biotechnological production of hydroxy fatty acids in existing oilseed crops.
KeywordsHydroxy fatty acid Lesquerella Physaria fendleri Seed Transcriptome Triacylglycerol Gene expression Quantitative polymerase chain reaction
Lesquerella [Physaria fendleri, formerly Lesquerella fendleri (Gray) Wats.] , is a potential Brassicaceae oilseed crop for the southwest region of the United States. The seed oil of lesquerella is rich in lesquerolic acid (14-hydroxy-eicos-cis-11-enoic acid: 20:1-OH), a hydroxy fatty acid (HFA) comprising 55-60% of total seed fatty acids [2-6]. The conventional source of HFA is castor (Ricinus communis) seeds; 90% of castor oil is ricinoleic acid (12-hydroxy-octadec-cis-9-enoic acid: 18:1-OH). Ricinoleic acid and its derivatives are used as raw materials for numerous industrial products, such as lubricants, plastics and surfactants . The production of castor oil, however, is hampered by the presence of the toxin ricin and hyper-allergic 2S albumins in its seed. Lesquerella on the other hand, does not have such biologically toxic compounds, and thus its oil represents a safe source of HFA. With the development of clean and renewable energy, hydroxy fatty acid methyl esters of lesquerella oil were found to be excellent lubricity enhancers in diesel fuels [8,9] that replace sulfur-based petroleum lubricity additives, and thus reduce environmental pollution. Besides the HFA, several co-products can be obtained from lesquerella. Seed meal after oil extraction is high in protein and the amino acid lysine and could be used as livestock feed [10,11]. Gums from the seed coat and seed meal could be used as thickening or gelling agents in food and pharmaceutical products [12-15].
Considerable efforts have been made to improve the agronomics of lesquerella through plant breeding [2,16-19]. Furthermore, stable genetic transformation has been established in lesquerella , which provides means to quickly improve this crop through genetic engineering. Currently, the United States Department of Agriculture (USDA) National Plant Germplasm System (NPGS) has a Phyasaria germplasm collection of over 212 accessions representing 32 species. Variation in fatty acids among species was reported. In species P. lindheimeri and P. pallida, 20:1-OH was the most abundant, comprising over 80% in seed oil . Some species have seeds with oil rich in other HFAs, such as densipolic acid (12-hydroxy-octadec-cis-9,15-enoic acid: 18:2OH) in P. perforata, P. stonensis, P. densipila, P. lyrata, and P. lescurii (average over 40%) [21-23]. In species P. auriculata and P. densiflora, auricolic acid (14-hydroxyeicos-cis-11,17-enoic acid: 20:2-OH) was the prevalent HFA, at 34-40% levels [24,25]. These species with different HFA profiles are valuable genetic resource and may contribute to the improvement of lesquerella cultivars.
Seed oil is stored as triacylglycerol (TAG). Biosynthesis of TAG in lesquerella follows the pathways for fatty acid (FA) in the plastid and TAG in the endoplasmic reticulum (ER) [26,27]. After the FAs are synthesized in the plastid (mostly oleic acid 18:1 with small amounts of palmitic acid 16:0 and stearic acid 18:0), they are released and then converted to acyl-Co-enzyme A (CoA). The newly synthesized acyl-CoAs can be incorporated into TAG through the glycerol-3-phosphate (G3P) pathway also known as the Kennedy pathway [28,29]. Briefly, G3P is first acylated by glycerol-3-phosphate acyltransferase (GPAT), followed by a second acylation by the acyl-CoA:acylglycerol-3-phosphate acyltransferase (LPAT), yielding phosphatidic acid (PA). PA is then hydrolyzed to form diacylglycerol (DAG), which is finally used as a substrate for the diacylglycerol acyltranstransferase (DGAT) to produce TAG. The newly synthesized acyl-CoAs can also be incorporated directly into membrane lipid phospatidylcholine (PC) by the acyl editing reactions or Lands cycle [30-32]. These acyl editing reactions can be catalyzed either by forward and reverse reactions of lyso-PC acyltransferase (LPCAT) to yield acyl-CoA, or by a phospholipase A–type activity to yield a free FA that then is activated to acyl-CoA. Since PC is the substrate for many FA-modifying enzymes (desaturase, hydroxylase, etc.), rapid de-acylation and re-acylation of PC results in an acyl-CoA pool enriched with modified FAs, which are then utilized for TAG synthesis [33,34]. Additionally, accumulating evidence indicates that many plants utilize PC-derived DAG to synthesize TAG. The main PC to DAG conversion is catalyzed by phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) through the phosphocholine headgroup exchange between PC and DAG [35,36]. Thus acyl editing and PC-DAG interconversion through LPCAT and PDCT, respectively, may co-contribute to the formation of TAGs with enriched modified FAs. Besides, TAG synthesis is not as simple as the sequential acylation of glycerol with GPAT, LPAT, and DGAT by the Kennedy pathway. The enzyme Phospholipid:DAG acyltransferase (PDAT) also syntheses TAG by transacylation of the sn-2 FA from PC onto sn-3 position of DAG, with lyso-PC as a co-product .
The molecular and biochemical bases of HFA synthesis have been investigated mostly in castor, lesquerella, and Arabidopsis (review) . Based on studies in castor, 18:1-OH is formed by the hydroxylation of 18:1 esterified to the sn-2 position of PC [39,40]. Then, the 18:1-OH is released from 18:1-OH-PC and activated to 18:1-OH-CoA. In lesquerella, due to an efficient microsomal elongation system, newly formed 18:1-OH-CoA is elongated to 20:1-OH-CoA [24,25,41]. Genes encoding the oleate 12-hydroxylase (FAH) have been isolated from castor (RcFAH12)  and lesquerella (PfFAH12) . (LFAH12 or LfFAH12 were used in publications before this report). Arabidopsis is a model oilseed that usually does not produce HFA. Expression of the RcFAH12 in Arabidopsis leading to HFA accumulation thus demonstrated that this enzyme is directly responsible for synthesis of 18:1-OH [42,44]. Expression of PfFAH12 in Arabidopsis [43,44] and yeast  has revealed that the lesquerella enzyme is bifunctional and can catalyze ∆12 hydroxylation to produce 18:1-OH and ∆12 desaturation to produce 18:2. In lesquerella, a gene encoding a condensing enzyme, PfKCS18 (LfKCS3 was used in publications before this report) has been isolated, and its activity has been shown to specifically catalyze elongation of 18:1-OH-CoA . Besides 18:1-OH and 20:1-OH, lesquerella seed accumulates a low level of 20:2-OH, which is formed by a microsomal ∆15 desaturase [24,25,44].
Although enzymatic reactions and key genes involved in the HFA synthesis have been elucidated, mechanisms contributing to the accumulation of HFA in TAG are largely unknown. Transgenic experiments have consistently failed to achieve high yields of desired HFAs. Seed-specific expression of RcFAH12 in Arabidopsis resulted in HFA accumulation at 17% of total seed lipids [44,46-48], which is much lower than 90% level of 18:1-OH in castor seeds. Efforts have been made to search for additional genes, especially those involved in a final step of TAG synthesis. It was shown that co-expression of a second gene, RcDGAT2  or a RcPDAT [50,51] boosted HFA content from 17% to nearly 30% or 25-27%, respectively. When triple transgenic Arabidopsis (carrying RcFAH, RcDGAT2 and RcPDAT1A) is compared with a double transgenic line (carrying RcFAH and RcPDAT1A), HFA increased slightly from 25.4% to 26.7% [50,51]. With the discovery of PDCT, a castor gene RcPDCT was co-expressed in the transgenic Arabidopsis line carrying RcFAH. It indeed increased HFA from 17% to 23% in Arabidopsis . Additional expression of RcDGAT2 further enhanced the HFA content to 28% .
Broadening our knowledge on HFA-containing TAG biosynthesis undoubtedly requires the identification of more genes involved in HFA and TAG metabolism. The high-throughput 454 GX FLX pyrosequencing is a superior technology for transriptome analysis. It revolutionizes science by enabling users to acquire massive genome-wide data rapidly with low cost and labor. Because the method increases sequencing depth and coverage, it allows assembly of overlapping reads without a references sequence. It is particularly suitable for use in organisms whose genomic sequences are unknown. Prior to our work, there are only 71 lesquerella microsatellite sequences and ESTs in GenBank (http://www.ncbi.nlm.nih.gov/nucest/). In this study, we adopted 454 GX FLX pyrosequencing to analyze the seed transcriptome of lesquerella. We describe here identification of 26,995 unique transcripts from a total of 651 mega-base raw sequences, including transcripts for the majority of enzymes involved in lipid biosynthesis and metabolism. We further characterize the expression profiles of 15 key lipid genes in various tissues of lesquerella, including developing seeds, leaf, stem, root, and flower buds using quantitative PCR (qPCR) assays. Our results provide information on key target genes that can be useful in the design of future studies involving manipulation of HFA production in plants.
Results and discussion
The transcriptome represents a major source for lesquerella seed genes
Summary of sequencing reads
Number of reads
Number of bases
Partially assembled reads
Reads too short to assemble
Summary of de novo assembly
Average contig count
Largest contig count
Number with one contig
Average isotig count
Largest isotig count
Number with one isotig
Number of bases (nt)
Average isotig size (bp)
N50# isotig size (bp)
Largest isotig size (bp)
List of gene products for the 50 most abundant isotigs
# of istotigs
HSD5 (hydroxysteroid dehydrogenase 5)
FDH (formate dehydrogenase)
Physaria fendleri 3-ketoacyl-CoA synthase
CESA3 (cellulose synthase 3)
Class-II DAHP synthetase family
UAP56B (homolog of human UAP56 B)
UBQ1 (ubiquitin extension protein 1)
splicing factor PWI domain-containing protein
12S seed storage protein CRU4
FDM1 (factor of DNA methylation 1)
XYP1 (xylogen protein 1)
HAI1 (highly ABA-induced PP2C gene 1)
RING-type Zinc finger protein
ALPHA-TIP (alpha-tonoplast intrinsic protein)
ACO2 (ACC oxidase 2)
CESA1 (cellulose synthase 1)
BBD2 (bifuctional nuclease in basal defense response 2)
Heat shock protein 81-2,
BGLU37 (beta glucosidase 37)
NLP4 (nin-like protein 4)
TUA6 (tubulin alpha-6)
Pyruvate kinase family
UBQ1 (ubiquitin extension protein 1)
PEPR1 (PEP1 receptor 1)
Methionine synthesis 1
GTP binding Elongation factor Tu family
TCTP (translationally controlled tumor protein)
DRP2B (dynamin related protein 2B)
AOAT2 (alanine-2-oxoglutarate aminotransferase 2)
2S seed storage protein 3
Papain family cysteine protease
Phosphoinositide phosphatase family
ALAAT1(alanine aminotransferase 1)
GTP binding Elongation factor Tu family protein
Leucine-rich repeat (LRR) family protein
HSP70 (heat shock protein 70)
BGLU37 ( beta glucosidase 37) ,
GTP binding Elongation factor Tu family
F-box/RNI-like superfamily protein
Copper amine oxidase family
AAE17(acyl-activating enzyme 17)
The transcriptome covers a broad spectrum of genes involved in acyl-lipid metabolism
Number of genes and transcripts involved in acyl-lipid metabolism
#of At genes*
#of expressed Pf genes in seed
#of detected Pf transcripts (isotigs and singletons)
#of transcripts per Pf gene
Fatty acid synthesis
Fatty acid elongation, Desaturation & export from plastid
Prokaryotic galactolipid, Sulfolipid, & Phospholipid synthesis
Eukaryotic galactolipid & Sulfolipid synthesis
Eukaryotic phospholipid synthesis & editing
Triacylglycerol & fatty acid degradation
Fatty acid elongation & wax biosynthesis
Mitochondria fatty acid & lipoic acid synthesis
Mitochondria phospholipid synthesis
Cutin synthesis & transport
Suberin synthesis & transport
Genes involved in fatty acid and TAG biosynthesis are well-represented in the transcriptome
List of expressed genes involved in fatty acid and TAG biosynthesis in lesquerella seed
%of nucleotide identity
Partial (P) or Full length (F)
De novo fatty acid biosynthesis and export from plastid
Endoplasmic reticulum-hydroxylase, desaturase, and elongase
Acyl-CoA- dependent TAG synthesis in Kennedy pathway
PC-mediated TAG synthesis
Fatty acid biosynthesis in plastids
We examined our collection of lesquerella transcripts for representatives of key genes encoding the known steps of fatty acid biosynthesis in plastids (Figure 3). Fatty acid biosynthesis begins with the rate-limiting conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase, a heteromeric complex enzyme composed of 4 subunits: 1 beta-carboxyltransferase (β-CT) encoded by the plastid genome and biotin carboxyl-carrier protein (BCCP), biotic carboxylase (BC), and alpha-carboxyltransferase (α-CT), each encoded by the nuclear genome. In the lesquerella transcriptome, transcripts encoding BCCP and α-CT subunits were identified with 10 isotigs and 17 isotigs, respectively (Table 5). In Arabidopsis, two paralogous of BCCP genes, BCCP1 (At5g16390) and BCCP2 (At5g15530) were characterized with BCCP1 being the more highly expressed during embryo development . In lesquerella seed, 7 isotigs of PfBCCP1 and 3 isotigs of PfBCCP2 were identified, and the longest, isotig19563 and isotig07553, shared 88% and 89% nucleotide sequence identity, respectively, to Arabidopsis homologous. We did not detect BC and β-CT in the lesquerella seed transcriptome, which could be due to a low level of their transcripts in 30 DAP seeds. We detected lesquerella homologs of all five isoforms of plastid acyl-carrier proteins (ACP) reported in Arabidopsis (At3g05020, At1g54580, At1g54630, At4g25050, At5g27200) . Among them, PfACP5 (6 isotigs), corresponding to At5g27200 isoform, was the mostly expressed (Table 5). A gene encoding malonyl-CoA ACP transferase (MCMT) was also identified in lesquerella with 5 isotigs, and the longest isotig17972 showed 93% nucleotide sequence identify with its Arabidopsis MCMT homolog (At2g30200).
Fatty acid synthesis is continued by an acyl-chain specific condensing enzyme subunit (KASIII, I, and II), and the common component of 3-ketoacyl-ACP reductase (KAR), 3-hydroxyacyl-ACP dehydratase (HAD), and 2-enoyl-ACP reductase (ER) (Figure 3). We tried to identify transcripts for three key fatty acid synthases, 3-ketoacyl-ACP synthase (KAS) III, I, II using Arabidopsis KASIII (At1g62640), KASI (At5g46290) and KASII (At1g74960) genes as queries, but only PfKASII was detected. It had 6 isotigs, and the longest, isotig15566, showed 91% identity with Arabidopsis seed homolog KASII . The other two KAS transcripts are apparently missing or rare in the 30-day seed transcriptome. Based on Arabidopsis KAR (At1g24360), HAD (At5g10160), and ER (At2g05990), we detected PfKAR (5 isotigs) , PfHAD (9 isotigs) and PfER (4 isotigs) in lesquerella seed. Stearoyl-ACP desaturase (SAD) catalyzes the conversion of 18:0-ACP to 18:1-ACP in plastids (Figure 3). Arabidopsis has seven SAD family genes including FAB2 (At2g43710) and DES5 (At1g02630); FAB2 is the most highly expressed . Indeed the FAB2 plays a major role in the reaction . In the seed transcriptome, lesquerella FAB2 homologues were detected with 6 isotigs; No homologous isotig was detected for DES5 (Table 5, Figure 3). Two fatty acid thioesteases, FatA (homologue of At3g25110) and FatB (homologue of At1g08510), were detected with 2 and 4 isotigs, respectively in lesquerella seed (Table 5, Figure 3). Long chain acyl-CoA synthase (LACS) is located the membrane of plastid outer envelopes and catalyzes addition of CoA to free fatty acids to produce the fatty acyl-CoA’s utilized in the endoplasmic reticulum. Two Arabidopsis plastid-localized LACS9 (At1g77590) and ER-localized LACS8 (At2g04350) have been reported [65,66]. In the lesquerella seed transcriptome, 1 isotig of PfLACS8 and 2 isotigs of PfLACS9 were identified (Table 5, Figure 3).
Endoplasmic reticulum-associated fatty acid hydroxylase, desaturases and elongase
Seed oil of lesquerella contains 55-60% 20:1-OH, and two key genes, PfFHA12 and PfKSC18, directly responsible for synthesis of this unusual fatty acid have been previously identified [41,43]. In our seed transcriptome, we found 7 and 28 isotigs representing PfFAH12 and PfKCS18, respectively (Table 5, Figure 3). The detailed temporal expression patterns of PfFAH12 and PfKCS18 during lesquerella seed development were reported . Both of the genes showed a bell-shaped expression pattern with a peak at 35 DAP. The increased expression of PfFAH12 and PfKCS18 coincided with the increased synthesis and accumulation of HFA-containing TAG during lesquerella seed development .
Conventional Kennedy pathway for TAG synthesis in ER
The conventional Kennedy pathway for TAG synthesis utilizes three acyl-CoA-dependent acyltransferases, GPAT, LPAT and DGAT, that sequentially acylate the sn-1- and sn-2- and then sn-3-position of G3P with acyl-CoA (Figure 3, red arrows). Since the synthesis of membrane glycerolipids also begins with sequential acylation of the sn-1- and sn-2- positions of G3P, GPAT and LPAT are common to synthesis of TAG and membrane glycerolipids. Using a bioinformatics approach, a new GPAT (At5g60620) was identified in Arabidopsis that exhibited extensive homology with a GPAT from mammalian cells involved in storage oil formation; that GPAT was postulated to be a putative AtGPAT9 for ER associated membrane and storage lipid biosynthesis in plants . For the second acyl-CoA transferase, Arabidopsis LPAT2 (At3g57650) was found to be an ER-localized and involved in TAG and membrane lipid biosynthesis . Using Arabidopsis genes (At5g60620 and At3g57650), we identified lesquerella orthologs of PfGPAT9 and PfLPAT2, each represented by 2 isotigs (Table 5). Result of gene expression analysis indicated that both genes were expressed at a similar level in most samples examined, including leaf, stem, flower bud, and developing seeds from 14 DAP to 42 DAP, with the exception of PfLPAT2 levels in stem tissue, where expression was only about 50% that detected in leaf (Figure 4B, 4C). Low levels of expression were detected in root and developing seeds at 49 DAP (Figure 4B, 4C). Our spatial and temporal expression profiles of PfGPAT9 and PfLPAT2 were similar to those from Arabidopsis [59,73-75]. Based on the overall spatial and temporal expression profiles of PfGPAT9 and PfLPAT2, we suggest both genes playing essential housekeeping roles in membrane and storage lipid biosynthesis throughout plant life.
It is general accepted that depending on the plant species, DGAT1 or DGAT2 is a major enzyme responsible for the accumulation of seed TAG . DGAT3 was recently demonstrated to be active in recycling of 18:2 and 18:3 FAs into TAG through a cytosolic pathway in peanut . Our results of gene expression analysis showed that PfDGAT1-1 and PfDGAT1-2 had distinct expression patterns. PfDGAT1-1 was expressed in all stages during seed development and in leaf, stem, and flower bud, but it was expressed more in leaf, stem, and in immature seeds prior to active oil biosynthesis and became a predominant DGAT mRNA at late-maturation/desiccation stages (42-49 DAP) (Figure 6B). In contrast, PfDGAT1-2 had expression levels higher in developing seeds than in other tissues such as leaf, stem, root and flower buds (Figure 6C). PfDGAT1-2 may specifically contribute to TAG synthesis in seed. Indeed, our PfDGAT1-2 is the same gene as PfDGAT1a identified in a lesquerella seed cDNA library  and found to complement the Arabidopsis AS11 mutant . AS11 had reduced DGAT activity and seed oil content due to a deletion in AtDGAT1 gene [79-81]. Seed-specific over-expression of an Arabidopsis cDNA encoding AtDGAT1 not only restored the oil content in AS11 but also enhanced seed oil content and seed weight in wild-type plants . The expression profile of PfDGAT2 was overall similar to that of PfDGAT1-1, except in the late-maturation/desiccation stages where PfDGAT2 expression dropped to undetectable levels or trace amounts (42 and 49 DAP, respectively) (Figure 6D). The results indicate that both PfDGAT1-1 and PfDGAT2 may function in other physiological processes besides seed oil synthesis, and that they clearly contribute differently in lipid metabolism during late-maturation/desiccation stages of seed development. PfDGAT3 was ubiquitously expressed in all samples and showed a moderate dynamic pattern compared with the other PfDGATs. In leaf, stem, flower bud, and developing seeds at early stages (14-21 DAP), PfDGAT3 transcripts were detected at levels similar to that of PfDGAT1-1 (Figure 6B, 6E). In developing seeds at 35 DAP, their levels rose 2- to 4-fold before declining steadily at late stages 42-49 DAP (Figure 6B, 6E). The boosted expression of PfDGAT3 may be associated with increasing demands of membrane and storage lipids synthesis at 35 DAP, when seeds had attained their maximum size and storage compounds have accumulated to a high plateau . The temporal and spatial expression pattern of PfDGAT3 suggests its role of house-keeping in most organs of lesquerella. Similar expression profile was reported for DGAT3 in peanut , Arbidopsis  and tung tree . None of these DGAT3s were hypothesized to play a significant role in seed oil synthesis; rather it was proposed that they are involved in general TAG metabolism. Among all samples, root tissue had the lowest number of transcripts of all PfDGATs. While it is clear that PfDGAT1-2 plays a role in seed TAG assembly, it remains an open question as to whether or not PfDGAT1-1, PfDGAT2 or PfDGAT3 also contribute. Measurements of enzyme activity and substrate specificity in various tissues are needed to better elucidate the functions of the different PfDGATs. The results of such studies combined with our sequence characterization and expression profiling will provide the molecular basis for future identification of PfDGAT candidates for genetic engineering oilseeds for hydroxy fatty acid production.
PC-mediated TAG synthesis
As described, PC is the substrate for many FA-modifying enzymes (desaturase, hydroxylase, etc.). The FA fluxes into and out of PC are crucial for the production of TAG esterified with modified FAs, such as HFA. Based on current knowledge, there are three routes allowing PC-derived FA to be incorporated into TAG. First, The FA esterified to PC undergoes constant deacylation and reacylation by LPCAT in so called acyl editing . Thus modified FA released by LPCAT can enter Kennedy pathway for TAG assembly (Figure 3, orange arrows). Second, direct transfer by PDAT of a FA from the sn-2 position of PC to the sn-3 position of DAG produces TAG (Figure 3, green arrows). Third, PDCT catalyzes the inter-conversion between DAG and PC by phosphocholine head group exchange (Figure 3, blue arrows). Thus FA on PC can be incorporated into the sn-1 and sn-2 positions of TAG by the PC derived DAG.
Fatty acids at the sn-1 and sn-2 positions in PC can be directly transferred to TAG through DAG converted by PDCT. The PDCT enzyme, encoded in Arabidopsis by the Reduced Oleate Desaturation1 (ROD1) gene, catalyzes the inter-conversion between DAG and PC by phosphocholine head group exchange . In castor, the 18:1-OH is produced by the hydroxylation of 18:1 that is esterified to the sn-2 position of PC . Since PDCT catalyzes the shuffling of acyl groups between PC and DAG, it provides a mechanism of making HFA-DAG from HFA-PC, thus the HFA-DAG can be subsequently converted to HFA-TAG. A castor PDCT enzyme gene was isolated and co-expressed in a transgenic Arabidopsis line carrying RcFAH12. The doubly transformed line had increases of 17-23% in seed HFA content . The authors noted that co-expression of AtPDCT did not increase HFA in transgenic Arabidopsis, indicating that RcPDCT had evolved to effectively convert HFA-PC to HFA-DAG . In the lesquerella seed transcriptome, we have identified one isotig25038 showing high homology with Arabidopsis PDCT and have designated it PfPDCT. The PfPDCT sequence shares 89% and 73% identify with AtPDCT and RcPDCT, respectively (Additional file 1: Figure S5). Gene expression analysis indicated that PfPDCT is expressed ubiquitously in developing seeds and other organs examined (Figure 4D). Among most samples, the expression levels ranged from 100-243 relative copy number, with exception of the seed sample at 42 DAP and the root sample, which had levels of about 365 and 11, respectively. A similar expression profile for AtPDCT was reported . It is known that the sn-2 position of TAG in lesquerella consists almost all of C18 unsaturated acyl groups including 18:1, 18:2 and 18:3 . Thus PfPDCT would not be a major enzyme involved in channeling HFA into lesquerella TAGs. It is possible that PfPDCT contributes FA flux through PC-derived DAG in TAG assembly in lesquerella. However, based on the expression profile of PfPDCT, it is likely that PfPDCT plays a general house-keeping function in lesquerella acyl-lipid metabolism.
Lesquerella is valued for its unusual HFA in seeds. Deep sequencing of cDNAs from developing lesquerella seeds was carried out to identify candidate genes that are associated with the synthesis of seed TAG enriched with HFA. A total of 26,995 unique genes from 651 mega-base raw sequences were assembled and 74% of them (19,861) had homology with known genes. The vast majority (95%, 18,868) of the matched genes showed highest homology to Arabidopsis genes, confirming the close relationship between the two species. The results provide a molecular basis for translating findings from the model plant Arabidopsis to facilitate lesquerella crop improvement. Genes involved in the synthesis of FA and TAG were identified and annotated by detailed sequence alignments. We have identified nearly all of the known genes for de novo FA biosynthesis and export from the plastid, and all of the known genes for TAG assembly in ER. In addition, we characterized the temporal and spatial expression profiles of 15 key genes in TAG metabolism using quantitative RT-PCR. The sequence and gene expression data presented in this study will serve as a useful resource for future research on lesquerella and other oilseed crops and promote their development into safe sources of HFA.
Plant material and general growth conditions
The P. fendleri seeds, WCL-LY2 , were kindly provided by Dr. David Dierig (USDA-ARS, National Center for Genetic Resources Preservation, Fort Collins, Colorado 80521, USA). Plants were germinated and grown in a greenhouse at temperatures between 28°C (day) and 18°C (night), with supplemental metal halide lighting to provide a 15-h-day length (1000 to 1250 μmol m-2 s-1). Mature flowers were individually hand-pollinated and tagged, and the tagging dates were recorded as 0 day after pollination (0 DAP). Developing seeds at 7, 14, 21, 28, 30, 35, 42 and 49 DAP were frozen immediately in liquid nitrogen after harvest and stored at -80°C. Leaf and stem tissues were obtained from mature plants, and root tissue was obtained from 2 month old seedlings cultured in half-strength MS liquid medium . Our flower sample consists of mature flower buds. Once the tissues were harvested, they were frozen immediately in liquid nitrogen and stored at -80°C.
RNA preparation, cDNA library construction and sequencing
Total RNA was extracted from developing seeds using TRIzol Reagent (Invitrogen, Carlsbad, CA). RNA pellets were dissolved in RNAse-free water, quantified by NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE). RNA quality was checked by 2% agarose gel electrophoresis. Total RNA from the 30 DAP sample was used for preparing an mRNA sample and subsequently constructing of a cDNA library using Illumina ® TruSeq™ RNA Sample Preparation Kit (Illumina Inc., San Diego, CA ). In brief, the mRNA was purified using poly-T oligo attached to magnetic beads. Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then went through an end repair process, the addition of a single ‘A’ base, and then ligation of the adapters. The products were then purified and enriched with PCR to create the final cDNA library. The cDNA library was sequenced on a GS FLX Titanium sequencing platform (Roche, Branford, CT).
Assembly and gene annotation
High quality sequence reads from seed libraries were assembled into isotigs and singletons using GS De Novo Assembler (v 2.6) software with the option for de novo transcriptome assembly. Clean singletons were processed to obtain high quality clean sequences, SeqClean was used to trim adapter sequences and Lucy (version 1.20p) was used to remove low quality sequences and those < 100 bp. As a result, total 21,912 singletons were generated.
To annotate the detected genes, a BLASTx search against the NCBI non-redundant protein (NR) database (http://www.ncbi.nlm.nih.gov/refseq/) was performed with an E-value threshold of less than 10-3. NR annotation was used to obtain GO annotation of genes according to molecular function, biological process and cellular component ontologies (http://www.geneontology.org/).
Total RNA was reverse transcribed using the QuantiTect Reverse Transcription Kit (QIAGEN, Valencia, CA) according to manufacturer’s directions. The resulted cDNA samples were used in PCR reactions. Standard PCR amplification reactions were carried out in a volume of 25 μL containing 20 ng of cDNA, 0.5 μM each of forward and reverse primers and 1× SYBR Select Master Mix, CFX (Applied Biosystems) using a 7500 Fast Real-Time PCR system (Applied Biosystems) and standard default thermal cycling conditions [initial step, 95°C for 10 min for polymerase activation; 40 cylces of PCR, 95°C, 15 s for melting, 60°C, 1 min for annealing and extending; and dissociation step set by the system software]. Putative oligonucleotide primers were designed using Primer Express, version 3 software (Applied Biosystems). To ensure maximum specificity and efficiency during quantitative PCR, putative primer pairs were further tested for linearity of response by constructing standard curves on five or six serial 10-fold dilutions. The templates used for the standard curve analysis were mixed cDNAs from developing seeds, leaf and flower samples with a starting concentration of 20 ng/μL. For each primer set, standard curves were analyzed independently at least three times, and standard curves repeatedly showing correlation coefficients of 0.99 or higher and PCR efficiencies between 83 and 107% were accepted. PCR product specificity was confirmed by melting-curve analysis and by electrophoresis on 4% agarose gel to ensure that PCR reactions were free of primer dimers and non-specific amplicons. Information on primer pairs and their PCR efficiencies is listed in Additional file 2: Table S1. The method of Pfaffl  was applied to calculate comparative expression levels between samples. The P. fendleri 18S gene was used as internal reference to normalize the relative amount of RNAs for all samples. For each selected gene, triplicate sets of PCR reaction samples including the 18S controls, and duplicate negative controls (reaction samples without cDNA templates), were prepared and run in a 96-well plate. The average CT from 28 DAP measurements were calibrated as 100 or 1000 copy numbers, and the relative copy numbers of a gene were averaged over triplicates. The PCR experiments were repeated three times for each plate to ensure that similar results could be obtained.
Availability of supporting data
The sequence raw data from this study have been submitted to the NCBI Sequence Read Archive (SRA) http://www.ncbi.nlm.nih.gov/bioproject/260225) under the BioProject ID PRJNA260225.
This study was conducted with support from the National Academy of Agricultural Science (project no. PJ01007505) and International Cooperative Research Project Program (Project No. PJ00855602) of the National Academy of Agricultural Science and “Next-Generation BioGreen 21 Program (SSAC, Project No. PJ01108101)” of the Rural development Administration (RDA), Republic of Korea, from the US Department of Agriculture-Agricultural Research Service-Current Research Information System Project 5325-21410-020-00D, and from the USDA Trust Fund Cooperative Agreement with RDA (Agreement number: 58 0212 9 036 F). The authors wish to thank Ann Blechl and Mark Smith for critical reading of the manuscript, and Kumiko Johnson for assisting qPCR analysis. Thanks also go to Dr. David Dierig and Dr. Mark Cruz for providing the cover photo of leaquerella grown in a research field of US Department of Agriculture. USDA is an equal opportunity provider and employer. Mention of a specific product name by the United States Department of Agriculture does not constitute an endorsement and does not imply a recommendation over other suitable products.
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