Engineering Camelina sativa (L.) Crantz for enhanced oil and seed yields by combining diacylglycerol acyltransferase1 and glycerol‐3‐phosphate dehydrogenase expression

Plant seed oil‐based liquid transportation fuels (i.e., biodiesel and green diesel) have tremendous potential as environmentally, economically and technologically feasible alternatives to petroleum‐derived fuels. Due to their nutritional and industrial importance, one of the major objectives is to increase the seed yield and oil production of oilseed crops via biotechnological approaches. Camelina sativa, an emerging oilseed crop, has been proposed as an ideal crop for biodiesel and bioproduct applications. Further increase in seed oil yield by increasing the flux of carbon from increased photosynthesis into triacylglycerol (TAG) synthesis will make this crop more profitable. To increase the oil yield, we engineered Camelina by  the Arabidopsis thaliana (L.) Heynh. diacylglycerol acyltransferase1 (DGAT1) and a yeast cytosolic glycerol‐3‐phosphate dehydrogenase (GPD1) genes under the control of seed‐specific promoters.  DGAT1 and GPD1 exhibited up to 13% higher seed oil content and up to 52% increase in seed mass compared to wild‐type plants. Further, DGAT1‐ and GDP1‐co‐expressing lines showed significantly higher seed and oil yields on a dry weight basis than the wild‐type controls or plants expressing DGAT1 and GPD1 alone. The oil harvest index (g oil per g total dry matter) for DGTA1‐ and GPD1‐co‐expressing lines was almost twofold higher as compared to wild type and the lines expressing DGAT1 and GPD1 alone. Therefore, combining the overexpression of TAG biosynthetic genes, DGAT1 and GPD1, appears to be a positive strategy to achieve a synergistic effect on the flux through the TAG synthesis pathway, and thereby further increase the oil yield.

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title: Multigene Engineering of Triacylglycerol Metabolism Boosts Seed Oil Content in Arabidopsis1[W][OPEN]
abstract: Transgenes specifically targeting fatty acid synthesis, triacylglycerol synthesis, and triacylglycerol breakdown lead to an additive effect on seed oil content in Arabidopsis. Increasing the yield of oilseed crops is an important objective for biotechnologists. A number of individual genes involved in triacylglycerol metabolism have previously been reported to enhance the oil content of seeds when their expression is altered. However, it has yet to be established whether specific combinations of these genes can be used to achieve an additive effect and whether this leads to enhanced yield. Using Arabidopsis (Arabidopsis thaliana) as an experimental system, we show that seed-specific overexpression of WRINKLED1 (a transcriptional regulator of glycolysis and fatty acid synthesis) and DIACYLGLYCEROL ACYLTRANSFERASE1 (a triacylglycerol biosynthetic enzyme) combined with suppression of the triacylglycerol lipase SUGAR-DEPENDENT1 results in a higher percentage seed oil content and greater seed mass than manipulation of each gene individually. Analysis of total seed yield per plant suggests that, despite a reduction in seed number, the total yield of oil is also increased.

title: Increasing seed oil content in oil-seed rape (Brassica napus L.) by over-expression of a yeast glycerol-3-phosphate dehydrogenase under the control of a seed-specific promoter.
abstract: Previous attempts to manipulate oil synthesis in plants have mainly concentrated on the genes involved in the biosynthesis and use of fatty acids, neglecting the possible role of glycerol-3-phosphate supply on the rate of triacylglycerol synthesis. In this study, a yeast gene coding for cytosolic glycerol-3-phosphate dehydrogenase (gpd1) was expressed in transgenic oil-seed rape under the control of the seed-specific napin promoter. It was found that a twofold increase in glycerol-3-phosphate dehydrogenase activity led to a three- to fourfold increase in the level of glycerol-3-phosphate in developing seeds, resulting in a 40% increase in the final lipid content of the seed, with the protein content remaining substantially unchanged. This was accompanied by a decrease in the glycolytic intermediate dihydroxyacetone phosphate, the direct precursor of glycerol-3-phosphate dehydrogenase. The levels of sucrose and various metabolites in the pathway from sucrose to fatty acids remained unaltered. The results show that glycerol-3-phosphate supply co-limits oil accumulation in developing seeds. This has important implications for strategies that aim to increase the overall level of oil in commercial oil-seed crops for use as a renewable alternative to petrol.

title: Molecular modification of triacylglycerol accumulation by over-expression of DGAT1 to produce canola with increased seed oil content under field conditions
abstract: The final step in the Kennedy pathway for seed oil synthesis is catalyzed by an acyl-CoA-dependent diacylglycerol acyltransferase, DGAT1 (EC. 2.3.1.20). We have cloned DGAT1 genes from both Arabido...

title: Plant triacylglycerols as feedstocks for the production of biofuels.
abstract: Triacylglycerols produced by plants are one of the most energy-rich and abundant forms of reduced carbon available from nature. Given their chemical similarities, plant oils represent a logical substitute for conventional diesel, a non-renewable energy source. However, as plant oils are too viscous for use in modern diesel engines, they are converted to fatty acid esters. The resulting fuel is commonly referred to as biodiesel, and offers many advantages over conventional diesel. Chief among these is that biodiesel is derived from renewable sources. In addition, the production and subsequent consumption of biodiesel results in less greenhouse gas emission compared to conventional diesel. However, the widespread adoption of biodiesel faces a number of challenges. The biggest of these is a limited supply of biodiesel feedstocks. Thus, plant oil production needs to be greatly increased for biodiesel to replace a major proportion of the current and future fuel needs of the world. An increased understanding of how plants synthesize fatty acids and triacylglycerols will ultimately allow the development of novel energy crops. For example, knowledge of the regulation of oil synthesis has suggested ways to produce triacylglycerols in abundant non-seed tissues. Additionally, biodiesel has poor cold-temperature performance and low oxidative stability. Improving the fuel characteristics of biodiesel can be achieved by altering the fatty acid composition. In this regard, the generation of transgenic soybean lines with high oleic acid content represents one way in which plant biotechnology has already contributed to the improvement of biodiesel.

title: Rubisco without the Calvin cycle improves the carbon efficiency of developing green seeds
abstract: Efficient storage of carbon in seeds is crucial to plant fitness and to agricultural productivity. Oil is a major reserve material in most seeds, and these oils provide the largest source of renewable reduced carbon chains available from nature. However, the conversion of carbohydrate to oil through glycolysis results in the loss of one-third of the carbon as CO2. Here we show that, in developing embryos of Brassica napus L. (oilseed rape), Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) acts without the Calvin cycle and in a previously undescribed metabolic context to increase the efficiency of carbon use during the formation of oil. In comparison with glycolysis, the metabolic conversion we describe provides 20% more acetyl-CoA for fatty-acid synthesis and results in 40% less loss of carbon as CO2. Our conclusions are based on measurements of mass balance, enzyme activity and stable isotope labelling, as well as an analysis of elementary flux modes.

title: Normal growth of Arabidopsis requires cytosolic invertase but not sucrose synthase
abstract: The entry of carbon from sucrose into cellular metabolism in plants can potentially be catalyzed by either sucrose synthase (SUS) or invertase (INV). These 2 routes have different implications for cellular metabolism in general and for the production of key metabolites, including the cell-wall precursor UDPglucose. To examine the importance of these 2 routes of sucrose catabolism in Arabidopsis thaliana (L.), we generated mutant plants that lack 4 of the 6 isoforms of SUS. These mutants (sus1/sus2/sus3/sus4 mutants) lack SUS activity in all cell types except the phloem. Surprisingly, the mutant plants are normal with respect to starch and sugar content, seed weight and lipid content, cellulose content, and cell-wall structure. Plants lacking the remaining 2 isoforms of SUS (sus5/sus6 mutants), which are expressed specifically in the phloem, have reduced amounts of callose in the sieve plates of the sieve elements. To discover whether sucrose catabolism in Arabidopsis requires INVs rather than SUSs, we further generated plants deficient in 2 closely related isoforms of neutral INV predicted to be the main cytosolic forms in the root (cinv1/cinv2 mutants). The mutant plants have severely reduced growth rates. We discuss the implications of these findings for our understanding of carbon supply to the nonphotosynthetic cells of plants.

title: Control of storage-product synthesis in seeds.
abstract: Seeds provide humans with much of their diet and have been targets for improvement for millennia. The recent development of a range of methodologies for investigating the control of seed metabolism will allow rapid progress towards understanding this process in the future. In situ measurements of metabolite concentrations, in combination with the localisation of gene expression, in developing legume seeds have led to the description of detailed models of the control of starch and protein synthesis. In oilseeds, the application of recently developed 13C-labelling methods allows the quantification of carbon fluxes through individual pathways in the cytosol and plastid. Molecular and genetic approaches are being used in combination to probe both the importance of individual steps in the pathways of storage-product synthesis and potential regulators of the entire process.

title: Secondary structure of oleosins in oil bodies isolated from seeds of safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.).
abstract: Oil bodies were isolated from mature seeds of sunflower (Helianthus annuus L.) and safflower (Carthamus tinctorius L.). Oil body preparations containing only oleosin proteins could be obtained from safflower seeds by salt-washing followed by centrifugation on discontinuous sucrose density gradients. However, it was necessary to treat sunflower oil bodies with urea to obtain preparations of similar purity. Incubation of the oil bodies with proteinases gave two fragments with molecular masses of 6 and 8 kDa which were protected from digestion. These fragments represented the hydrophobic domain of the oleosins, as determined by N-terminal sequencing. Intact and proteinase-treated oil bodies of both species were analysed by Fourier-transform infrared spectroscopy, as dry films and in aqueous medium, the spectra being compared with those obtained for pure oil samples in order to identify the bands resulting from the oleosin proteins and protected peptides. This investigation showed that the hydrophobic domain of the oleosins in intact oil bodies is predominantly alpha-helical in structure and that the conformation was not greatly affected by washing the oil bodies with urea during preparation.

title: Metabolic control analysis is helpful for informed genetic manipulation of oilseed rape (Brassica napus) to increase seed oil content
abstract: Top–down control analysis (TDCA) is a useful tool for quantifying constraints on metabolic pathways that might be overcome by biotechnological approaches. Previous studies on lipid accumulation in oilseed rape have suggested that diacylglycerol acyltransferase (DGAT), which catalyses the final step in seed oil biosynthesis, might be an effective target for enhancing seed oil content. Here, increased seed oil content, increased DGAT activity, and reduced substrate:product ratio are demonstrated, as well as reduced flux control by complex lipid assembly, as determined by TDCA in Brassica napus (canola) lines which overexpress the gene encoding type-1 DGAT. Lines overexpressing DGAT1 also exhibited considerably enhanced seed oil content under drought conditions. These results support the use of TDCA in guiding the rational selection of molecular targets for oilseed modification. The most effective lines had a seed oil increase of 14%. Moreover, overexpression of DGAT1 under drought conditions reduced this environmental penalty on seed oil content.

title: Changes in Kennedy pathway intermediates associated with increased triacylglycerol synthesis in oil-seed rape
abstract: The rates of triacylglycerol synthesis in maturing oil-seed rape (Brassica napus cv Shiralee) were manipulated by light/dark treatments. Under conditions of high lipid accumulation the amounts of the Kennedy pathway intermediates, phosphatidate and particularly, diacylglycerol were increased significantly. At the same time there were no significant changes in the activities of the four pathway enzymes, of which diacylglycerol acyltransferase had the lowest detectable activity. The alteration in carbon flux was accompanied by some changes in the acyl quantity of the diacylglycerol pool but not in that of other intermediates. The results provide additional evidence for our proposal that diacylglycerol acyltransferase can exert significant flux control at times of high lipid accumulation in oil-seed rape.

title: Type 1 diacylglycerol acyltransferases of Brassica napus preferentially incorporate oleic acid into triacylglycerol
abstract: Highlight Fatty acid composition determines oil qualities. Not only the selectivity of BnDGAT1 enzymes, but also the concentration of the fatty acid substrates, determines the oil composition in Brassica napus seeds.

title: Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight.
abstract: We recently reported the cloning and characterization of an Arabidopsis (ecotype Columbia) diacylglycerol acyltransferase cDNA (Zou et al., 1999) and found that in Arabidopsis mutant line AS11, an ethyl methanesulfonate-induced mutation at a locus on chromosome II designated as Tag1 consists of a 147-bp insertion in the DNA, which results in a repeat of the 81-bp exon 2 in the Tag1 cDNA. This insertion mutation is correlated with an altered seed fatty acid composition, reduced diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) activity, reduced seed triacylglycerol content, and delayed seed development in the AS11 mutant. The effect of the insertion mutation on microsomal acyl-coenzyme A-dependent DGAT is examined with respect to DGAT activity and its substrate specificity in the AS11 mutant relative to wild type. We demonstrate that transformation of mutant AS11 with a single copy of the wild-type Tag1 DGAT cDNA can complement the fatty acid and reduced oil phenotype of mutant AS11. More importantly, we show for the first time that seed-specific over-expression of the DGAT cDNA in wild-type Arabidopsis enhances oil deposition and average seed weight, which are correlated with DGAT transcript levels. The DGAT activity in developing seed of transgenic lines was enhanced by 10% to 70%. Thus, the current study confirms the important role of DGAT in regulating the quantity of seed triacylglycerols and the sink size in developing seeds.

title: Cloning and characterization of an acyl-CoA-dependent diacylglycerol acyltransferase 1 (DGAT1) gene from Tropaeolum majus, and a study of the functional motifs of the DGAT protein using site-directed mutagenesis to modify enzyme activity and oil content.
abstract: A full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) was obtained from Tropaeolum majus (garden nasturtium). The 1557-bp open reading frame of this cDNA, designated TmDGAT1, encodes a protein of 518 amino acids showing high homology to other plant DGAT1s. The TmDGAT1 gene was expressed exclusively in developing seeds. Expression of recombinant TmDGAT1 in the yeast H1246MATalpha quadruple mutant (DGA1, LRO1, ARE1, ARE2) restored the capability of the mutant host to produce triacylglycerols (TAGs). The recombinant TmDGAT1 protein was capable of utilizing a range of (14)C-labelled fatty acyl-CoA donors and diacylglycerol acceptors, and could synthesize (14)C-trierucin. Collectively, these findings confirm that the TmDGAT1 gene encodes an acyl-CoA-dependent DGAT1. In plant transformation studies, seed-specific expression of TmDGAT1 was able to complement the low TAG/unusual fatty acid phenotype of the Arabidopsis AS11 (DGAT1) mutant. Over-expression of TmDGAT1 in wild-type Arabidopsis and high-erucic-acid rapeseed (HEAR) and canola Brassica napus resulted in an increase in oil content (3.5%-10% on a dry weight basis, or a net increase of 11%-30%). Site-directed mutagenesis was conducted on six putative functional regions/motifs of the TmDGAT1 enzyme. Mutagenesis of a serine residue in a putative SnRK1 target site resulted in a 38%-80% increase in DGAT1 activity, and over-expression of the mutated TmDGAT1 in Arabidopsis resulted in a 20%-50% increase in oil content on a per seed basis. Thus, alteration of this putative serine/threonine protein kinase site can be exploited to enhance DGAT1 activity, and expression of mutated DGAT1 can be used to enhance oil content.

title: The Arabidopsis thaliana TAG1 mutant has a mutation in a diacylglycerol acyltransferase gene.
abstract: In Arabidopsis thaliana (ecotype Columbia) mutant line AS11, an EMS-induced mutation at a locus on chromosome II results in a reduced diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) activity, reduced seed triacylglycerol, an altered seed fatty acid composition, and delayed seed development. A mutation has been identified in AS11 in a gene, which we designated as TAG1, that encodes a protein with an amino acid sequence which is similar to a recently reported mammalian DGAT, and, to a lesser extent, to acyl CoA:cholesterol acyltransferases. Molecular analysis revealed that the mutant allele in AS11 has a 147 bp insertion located at the central region of intron 2. At the RNA level, an 81 bp insertion composed entirely of an exon 2 repeat was found in the transcript. While the seed triacylglycerol content is reduced by the lesion in AS11, there is no apparent effect on sterol ester content in the mutant seed. The TAG1 cDNA was over-expressed in yeast, and its activity as a microsomal DGAT confirmed. Therefore, the TAG1 locus encodes a diacylglycerol acyltransferase, and the insertion mutation in the TAG1 gene in mutant AS11 results in its altered lipid phenotype.

title: Overexpression of patatin-related phospholipase AIIIδ altered plant growth and increased seed oil content in camelina.
abstract: Camelina sativa is a Brassicaceae oilseed species being explored as a biofuel and industrial oil crop. A growing number of studies have indicated that the turnover of phosphatidylcholine plays an important role in the synthesis and modification of triacylglycerols. This study manipulated the expression of a patatin-related phospholipase AIIIδ (pPLAIIIδ) in camelina to determine its effect on seed oil content and plant growth. Constitutive overexpression of pPLAIIIδ under the control of the constitutive cauliflower mosaic 35S promoter resulted in a significant increase in seed oil content and a decrease in cellulose content. In addition, the content of major membrane phospholipids, phosphatidylcholine and phosphatidylethanolamine, in 35S::pPLAIIIδ plants was increased. However, these changes in 35S::pPLAIIIδ camelina were associated with shorter cell length, leaves, stems, and seed pods and a decrease in overall seed production. When pPLAIIIδ was expressed under the control of the seed specific, β-conglycinin promoter, the seed oil content was increased without compromising plant growth. The results suggest that pPLAIIIδ alters the carbon partitioning by decreasing cellulose content and increasing oil content in camelina.

title: Metabolic control analysis of developing oilseed rape (Brassica napus cv Westar) embryos shows that lipid assembly exerts significant control over oil accumulation.
abstract: Metabolic control analysis allows the study of metabolic regulation. We applied both single- and double-manipulation top-down control analysis to examine the control of lipid accumulation in developing oilseed rape (Brassica napus) embryos. The biosynthetic pathway was conceptually divided into two blocks of reactions (fatty acid biosynthesis (Block A), lipid assembly (Block B)) connected by a single system intermediate, the acyl-coenzyme A (acyl-CoA) pool. Single manipulation used exogenous oleate. Triclosan was used to inhibit specifically Block A, whereas diazepam selectively manipulated flux through Block B. Exogenous oleate inhibited the radiolabelling of fatty acids from [1-(14)C]acetate, but stimulated that from [U-14C]glycerol into acyl lipids. The calculation of group flux control coefficients showed that c. 70% of the metabolic control was in the lipid assembly block of reactions. Monte Carlo simulations gave an estimation of the error of the resulting group flux control coefficients as 0.27±0.06 for Block A and 0.73±0.06 for Block B. The two methods of control analysis gave very similar results and showed that Block B reactions were more important under our conditions. This contrasts notably with data from oil palm or olive fruit cultures and is important for efforts to increase oilseed rape lipid yields.

title: Targeting of the Arabidopsis Homomeric Acetyl-Coenzyme A Carboxylase to Plastids of Rapeseeds
abstract: Acetyl-coenzyme A carboxylase (ACCase) occurs in at least two forms in rapeseed (Brassica napus): a homomeric (HO) and presumably cytosolic isozyme and a heteromeric, plastidial isozyme. We investigated whether the HO-ACCase of Arabidopsis can be targeted to plastids of B. napus seeds. A chloroplast transit peptide and the napin promoter were fused to the Arabidopsis ACC1 gene and transformed into B. napus, with the following results. (a) The small subunit transit peptide was sufficient to provide import of this very large protein into developing seed plastids. (b) HO-ACCase in isolated plastids was found to be biotinylated at a level comparable to extraplastidial HO-ACCase. (c) In vitro assays of HO-ACCase in isolated plastids from developing seeds indicate that it occurs as an enzymatically active form in the plastidial compartment. (d) ACCase activity in mature B. napus seeds is normally very low; however, plants expressing the SSU/ACC1 gene had 10- to 20-fold higher ACCase activity in mature seeds, suggesting that plastid localization prevents the turnover of HO-ACCase. (e) ACCase over-expression altered seed fatty acid composition, with the largest effect being an increase in oleic acid. (f) The total oil content of seeds was increased approximately 5% by the expression of HO-ACCase in plastids.

title: Field testing of transgenic rapeseed cv. Hero transformed with a yeast sn-2 acyltransferase results in increased oil content, erucic acid content and seed yield
abstract: A major goal of our research is to produce, by genetic manipulation, Brassica napus L. cultivars with higher levels of 22:1 in their seed oil than in present Canadian HEA cultivars developed through traditional breeding. Previously, we reported that transgenic expression of a mutated yeast sn-2 acyltransferase (SLC1-1) in industrial rapeseed cv. Hero resulted in increased seed oil content, increased proportions of erucic acid and increased average seed weight (Zou et al. 1997). Those results were reported only for plants grown in a controlled greenhouse setting. Here we report a summary of the results from two successive years of field trials with T4 and T5 generations of B. napus cv. Hero transformed with the SLC1-1 gene. These trials, conducted at Rosthern, Saskatchewan, in two very different growing seasons, show that the SLC1-1 transgenics clearly and consistently out-performed controls, with much increased oil and 22:1 contents, as well as yield, under varying field conditions.

title: Changes in Oil Content of Transgenic Soybeans Expressing the Yeast SLC1 Gene
abstract: The wild type (Wt) and mutant form of yeast (sphingolipid compensation) genes, SLC1 and SLC1-1, have been shown to have lysophosphatidic acid acyltransferase (LPAT) activities (Nageic et al.  1993). Expression of these LPAT genes was reported to increase oil content in transgenic Arabidopsis and Brassica napus. It is of interest to determine if the TAG content increase would also be seen in soybeans. Therefore, the wild type SLC1 was expressed in soybean somatic embryos under the control of seed specific phaseolin promoter. Some transgenic somatic embryos and in both T2 and T3 transgenic seeds showed higher oil contents. Compared to controls, the average increase in triglyceride values went up by 1.5% in transgenic somatic embryos. A maximum of 3.2% increase in seed oil content was observed in a T3 line. Expression of the yeast Wt LPAT gene did not alter the fatty acid composition of the seed oil.

title: Diseases of Camelina sativa (false flax)
abstract: There is renewed interest in the crucifer Camelina sativa (false flax, camelina, gold of pleasure) as an alternative oilseed crop because of its potential value for food, feed, and industrial applications. This species is adapted to canola-growing areas in many regions of the world and is generally considered to be resistant to many diseases. A review of the literature indicates that C. sativa is highly resistant to alternaria black spot and blackleg of crucifers. Genotypes resistant to sclerotinia stem rot, brown girdling root rot, and downy mildew can be found among C. sativa accessions, raising the possibility of developing cultivars resistant to these diseases. However, C. sativa is susceptible to clubroot, white rust, and aster yellows disease. Until resistant cultivars or effective management practices have been developed, the susceptibility of C. sativa to these diseases will limit the cultivation of the crop in areas where these diseases are prevalent.  false flax, alternaria black spot, sclerotinia stem rot, root rot, white rust, blackleg, downy mildew, clubroot, aster yellows, Brassicaceae. La cameline (Camelina sativa) est une espèce oléagineuse de plus en plus recherchée pour l’alimentation humaine et animale et pour des fins industrielles. L’espèce est adaptée aux zones de culture du Colza et de la Navette dans plusieurs régions du monde et est généralement considérée comme étant résistante aux maladies. Une recherche bibliographique a révélé que C. sativa est fortement résistante à l’alternariose et à la nécrose du collet. Certains génotypes de C. sativa sont résistants à la sclérotiniose, au rhizoctone commun ou au mildiou, ce qui permet d’entrevoir le développement de cultivars résistants à ces maladies. Camelina sativa est cependant sensible à la hernie des crucifères, l’albugine et la jaunisse de l’aster. La culture de C. sativa sera limitée dans les régions où ces maladies sont importantes. Mots-clés : cameline, alternariose, sclérotioniose, rhizoctone commun, albugine, nécrose du collet, mildiou, hernie, jaunisse de l’aster, Brassicaceae.

title: Transcriptome profiling of Camelina sativa to identify genes involved in triacylglycerol biosynthesis and accumulation in the developing seeds
abstract: BackgroundCamelina sativa is an emerging dedicated oilseed crop designed for biofuel and biodiesel applications as well as a source for edible and general-purpose oils. Such valuable oilseed crop is subjected to plant breeding programs and is suggested for large-scale production of better seed and oil quality. To accomplish this objective and to further enhance its oil content, a better understanding of lipid metabolism at the molecular level in this plant is critical. Here, we applied tissue transcriptomics and lipid composition analysis to identify and profile the genes and gene networks associated with triacylglycerol (TAG) biosynthesis, and to investigate how those genes are interacting to determine the quantity and quality of Camelina oil during seed development.ResultsOur Camelina transcriptome data analysis revealed an approximate of 57,854 and 57,973 genes actively expressing in developing seeds (RPKM ≥ 0.1) at 10–15 (Cs-14) and 16–21 (Cs-21) days after flowering (DAF), respectively. Of these, 7932 genes showed temporal and differential gene expression during the seed development (log2 fold change ≥1.5 or ≤−1.5; P ≤ 0.05). The differentially expressed genes (DEGs) were annotated and were found to be involved in distinct functional categories and metabolic pathways. Furthermore, performing quantitative real-time PCR for selected candidate genes associated with TAG biosynthesis validated RNA-seq data. Our results showed strong positive correlations between the expression abundance measured using both qPCR and RNA-Seq technologies. Furthermore, the analysis of fatty-acid content and composition revealed major changes throughout seed development, with the amount of oil accumulate rapidly at early mid seed development stages (from 16–28 DAF onwards), while no important changes were observed in the fatty-acid profile between seeds at 28 DAF and mature seeds.ConclusionsThis study is highly useful for understanding the regulation of TAG biosynthesis and identifying the rate-limiting steps in TAG pathways at seed development stages, providing a precise selection of candidate genes for developing Camelina varieties with improved seed and oil yields.

title: Identification of three genes encoding microsomal oleate desaturases (FAD2) from the oilseed crop Camelina sativa.
abstract: Camelina sativa is a re-emerging low-input oilseed crop that may provide economical vegetable oils for industrial applications. It is desirable to increase the monounsaturated oleic acid (cis-9-octadecenoic acid, 18:1), and to decrease polyunsaturated fatty acids (PUFA), linoleic (cis, cis-9,12-octadecadienoic acid, 18:2) and α-linolenic (all-cis-9,12,15-octadecatrienoic acid, 18:3) acids, in camelina oils to improve oxidative stability. 18:1 desaturation is mainly controlled by the microsomal oleate desaturase (FAD2; EC 1.3.1.35) encoded by the FAD2 gene. Three FAD2 genes, designated CsFAD2-1 to 3, were identified in camelina. Functional expression of these genes in yeast confirmed that they all encode microsomal oleate desaturases. Although the three CsFAD2 genes share very high sequence similarity, they showed different expression patterns. Expression of CsFAD2-1 was detected in all tissues examined, including developing seed, flower, as well as in vegetable tissues such as leaf, root, and stem. Transcripts of CsFAD2-2 and CsFAD2-3 were mainly detected in developing seeds, suggesting their major roles in storage oil desaturation in seed. The introns of the three CsFAD2 genes, which showed greater sequence variations, may provide additional resources for designing molecular markers in breeding. Furthermore, the roles of CsFAD2 in PUFA synthesis were demonstrated by mutant analysis and by antisense gene expression in camelina seed.

title: Generation of transgenic plants of a potential oilseed crop Camelina sativa by Agrobacterium-mediated transformation
abstract: Camelina sativa is an alternative oilseed crop that can be used as a potential low-cost biofuel crop or a source of health promoting omega-3 fatty acids. Currently, the fatty acid composition of camelina does not uniquely fit any particular uses, thus limit its commercial value and large-scale production. In order to improve oil quality and other agronomic characters, we have developed an efficient and simple in planta method to generate transgenic camelina plants. The method included Agrobacterium-mediated inoculation of plants at early flowering stage along with a vacuum infiltration procedure. We used a fluorescent protein (DsRed) as a visual selection marker, which allowed us to conveniently screen mature transgenic seeds from a large number of untransformed seeds. Using this method, over 1% of transgenic seeds can be obtained. Genetic analysis revealed that most of transgenic plants contain a single copy of transgene. In addition, we also demonstrated that transgenic camelina seeds produced novel hydroxy fatty acids by transforming a castor fatty acid hydroxylase. In conclusion, our results provide a rapid means to genetically improve agronomic characters of camelina, including fatty acid profiles of its seed oils. Camelina may serve as a potential industrial crop to produce novel biotechnology products.

title: The emerging biofuel crop Camelina sativa retains a highly undifferentiated hexaploid genome structure
abstract: Camelina sativa is an oilseed with desirable agronomic and oil-quality attributes for a viable industrial oil platform crop. Here we generate the first chromosome-scale high-quality reference genome sequence for C. sativa and annotated 89,418 protein-coding genes, representing a whole-genome triplication event relative to the crucifer model Arabidopsis thaliana. C. sativa represents the first crop species to be sequenced from lineage I of the Brassicaceae. The well-preserved hexaploid genome structure of C. sativa surprisingly mirrors those of economically important amphidiploid Brassica crop species from lineage II as well as wheat and cotton. The three genomes of C. sativa show no evidence of fractionation bias and limited expression-level bias, both characteristics commonly associated with polyploid evolution. The highly undifferentiated polyploid genome of C. sativa presents significant consequences for breeding and genetic manipulation of this industrial oil crop.

title: Camelina seed transcriptome: a tool for meal and oil improvement and translational research.
abstract: Camelina (Camelina sativa), a Brassicaceae oilseed, has received recent interest as a biofuel crop and production platform for industrial oils. Limiting wider production of camelina for these uses is the need to improve the quality and content of the seed protein-rich meal and oil, which is enriched in oxidatively unstable polyunsaturated fatty acids that are deleterious for biodiesel. To identify candidate genes for meal and oil quality improvement, a transcriptome reference was built from 2047 Sanger ESTs and more than 2 million 454-derived sequence reads, representing genes expressed in developing camelina seeds. The transcriptome of approximately 60K transcripts from 22 597 putative genes includes camelina homologues of nearly all known seed-expressed genes, suggesting a high level of completeness and usefulness of the reference. These sequences included candidates for 12S (cruciferins) and 2S (napins) seed storage proteins (SSPs) and nearly all known lipid genes, which have been compiled into an accessible database. To demonstrate the utility of the transcriptome for seed quality modification, seed-specific RNAi lines deficient in napins were generated by targeting 2S SSP genes, and high oleic acid oil lines were obtained by targeting FATTY ACID DESATURASE 2 (FAD2) and FATTY ACID ELONGASE 1 (FAE1). The high sequence identity between Arabidopsis thaliana and camelina genes was also exploited to engineer high oleic lines by RNAi with Arabidopsis FAD2 and FAE1 sequences. It is expected that these transcriptomic data will be useful for breeding and engineering of additional camelina seed traits and for translating findings from the model Arabidopsis to an oilseed crop.

title: Over-expression of AtPAP2 in Camelina sativa leads to faster plant growth and higher seed yield
abstract: BackgroundLipids extracted from seeds of Camelina sativa have been successfully used as a reliable source of aviation biofuels. This biofuel is environmentally friendly because the drought resistance, frost tolerance and low fertilizer requirement of Camelina sativa allow it to grow on marginal lands. Improving the species growth and seed yield by genetic engineering is therefore a target for the biofuels industry. In Arabidopsis, overexpression of purple acid phosphatase 2 encoded by Arabidopsis (AtPAP2) promotes plant growth by modulating carbon metabolism. Overexpression lines bolt earlier and produce 50% more seeds per plant than wild type. In this study, we explored the effects of overexpressing AtPAP2 in Camelina sativa.ResultsUnder controlled environmental conditions, overexpression of AtPAP2 in Camelina sativa resulted in longer hypocotyls, earlier flowering, faster growth rate, higher photosynthetic rate and stomatal conductance, increased seed yield and seed size in comparison with the wild-type line and null-lines. Similar to transgenic Arabidopsis, activity of sucrose phosphate synthase in leaves of transgenic Camelina was also significantly up-regulated. Sucrose produced in photosynthetic tissues supplies the building blocks for cellulose, starch and lipids for growth and fuel for anabolic metabolism. Changes in carbon flow and sink/source activities in transgenic lines may affect floral, architectural, and reproductive traits of plants.ConclusionsLipids extracted from the seeds of Camelina sativa have been used as a major constituent of aviation biofuels. The improved growth rate and seed yield of transgenic Camelina under controlled environmental conditions have the potential to boost oil yield on an area basis in field conditions and thus make Camelina-based biofuels more environmentally friendly and economically attractive.

title: Sensitive and high throughput metabolite assays for inorganic pyrophosphate, ADPGlc, nucleotide phosphates, and glycolytic intermediates based on a novel enzymic cycling system.
abstract: Metabolite assays are required to characterise how metabolism changes between genotypes during development and in response to environmental perturbations. They provide a springboard to identify important regulatory sites and investigate the underlying mechanisms. Due to their small size, Arabidopsis seeds pose a technical challenge for such measurements. A set of assays based on a novel enzymic cycling system between glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate oxidase have been developed and optimised for use with growing Arabidopsis seeds. In combination with existing assays they provide a suite of high throughput, sensitive assays for the immediate precursors for starch (adenine diphosphate glucose) and lipid (acetyl coenzyme A, glycerol-3-phosphate) synthesis, as well as pyrophosphate, ATP, ADP and most of the glycolytic intermediates. A method is also presented to rapidly quench intact siliques, lyophilise them and then manually separate seeds for metabolite analysis. These techniques are used to investigate changes in overall seed metabolite levels during development and maturation, and in response to a stepwise decrease of the external oxygen concentration.

title: Increased levels of glycerol-3-phosphate lead to a stimulation of flux into triacylglycerol synthesis after supplying glycerol to developing seeds of Brassica napus L. in planta
abstract: Glycerol-3-phosphate (glycerol-3P) is a primary substrate for triacylglycerol synthesis. In the present study, changes in the levels of glycerol-3P during rape (Brassica napus L.) seed development and the influence of manipulating glycerol-3P levels on triacylglycerol synthesis were investigated. (i) Glycerol-3P levels were high in young seeds and decreased during seed development at 30 and 40 days after flowering (DAF), when lipid accumulation was maximal. (ii) To manipulate glycerol-3P levels in planta, various concentrations of glycerol were injected directly into 30-DAF seeds, which remained otherwise intact within their siliques and attached to the plant. Injection of 0–10 nmol glycerol led to a progressive increase in seed glycerol-3P levels within 28 h. (iii). Increased levels of glycerol-3P were accompanied by an increase in the flux of injected [14C]sucrose into total lipids and triacylglycerol, whereas fluxes to organic acids, amino acids, starch, protein and cell walls were not affected. (iv) When [14C]acetate was injected into seeds, label incorporation into total lipids and triacylglycerol increased progressively with increasing glycerol-3P levels. (v) There was a strong correlation between the level of glycerol-3P and the incorporation of injected [14C]acetate and [14C]sucrose into triacylglycerol. (v) The results provide evidence that the prevailing levels of glycerol-3P co-limit triacylglycerol synthesis in developing rape seeds.

title: A phenylalanine in DGAT is a key determinant of oil content and composition in maize
abstract: Plant oil is an important renewable resource for biodiesel production and for dietary consumption by humans and livestock. Through genetic mapping of the oil trait in plants, studies have reported multiple quantitative trait loci (QTLs) with small effects, but the molecular basis of oil QTLs remains largely unknown. Here we show that a high-oil QTL (qHO6) affecting maize seed oil and oleic-acid contents encodes an acyl-CoA:diacylglycerol acyltransferase (DGAT1-2), which catalyzes the final step of oil synthesis. We further show that a phenylalanine insertion in DGAT1-2 at position 469 (F469) is responsible for the increased oil and oleic-acid contents. The DGAT1-2 allele with F469 is ancestral, whereas the allele without F469 is a more recent mutant selected by domestication or breeding. Ectopic expression of the high-oil DGAT1-2 allele increases oil and oleic-acid contents by up to 41% and 107%, respectively. This work provides insights into the molecular basis of natural variation of oil and oleic-acid contents in plants and highlights DGAT as a promising target for increasing oil and oleic-acid contents in other crops.

title: Transgenic increases in seed oil content are associated with the differential expression of novel Brassica-specific transcripts
abstract: BackgroundSeed oil accumulates primarily as triacylglycerol (TAG). While the biochemical pathway for TAG biosynthesis is known, its regulation remains unclear. Previous research identified microsomal diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) as controlling a rate-limiting step in the TAG biosynthesis pathway. Of note, overexpression of DGAT1 results in substantial increases in oil content and seed size. To further analyze the global consequences of manipulating DGAT1 levels during seed development, a concerted transcriptome and metabolome analysis of transgenic B. napus prototypes was performed.ResultsUsing a targeted Brassica cDNA microarray, about 200 genes were differentially expressed in two independent transgenic lines analyzed. Interestingly, 24–33% of the targets showing significant changes have no matching gene in Arabidopsis although these represent only 5% of the targets on the microarray. Further analysis of some of these novel transcripts indicated that several are inducible by ABA in microspore-derived embryos. Of the 200 Arabidopsis genes implicated in lipid biology present on the microarray, 36 were found to be differentially regulated in DGAT transgenic lines. Furthermore, kinetic reverse transcriptase Polymerase Chain Reaction (k-PCR) analysis revealed up-regulation of genes encoding enzymes of the Kennedy pathway involved in assembly of TAGs. Hormone profiling indicated that levels of auxins and cytokinins varied between transgenic lines and untransformed controls, while differences in the pool sizes of ABA and catabolites were only observed at later stages of development.ConclusionOur results indicate that the increased TAG accumulation observed in transgenic DGAT1 plants is associated with modest transcriptional and hormonal changes during seed development that are not limited to the TAG biosynthesis pathway. These might be associated with feedback or feed-forward effects due to altered levels of DGAT1 activity. The fact that a large fraction of significant amplicons have no matching genes in Arabidopsis compromised our ability to draw concrete inferences from the data at this stage, but has led to the identification of novel genes of potential interest.

title: Alteration of Seed Fatty Acid Composition by an Ethyl Methanesulfonate-Induced Mutation in Arabidopsis thaliana Affecting Diacylglycerol Acyltransferase Activity
abstract: In characterizing the enzymes involved in the formation of very long-chain fatty acids (VLCFAs) in the Brassicaceae, we have generated a series of mutants of Arabidopsis thaliana that have reduced VLCFA content. Here we report the characterization of a seed lipid mutant, AS11, which, in comparison to wild type (WT), has reduced levels of 20:1 and 18:1 and accumulates 18:3 as the major fatty acid in triacylglycerols. Proportions of 18:2 remain similar to WT. Genetic analyses indicate that the fatty acid phenotype is caused by a semidominant mutation in a single nuclear gene, designated TAG1, located on chromosome 2. Biochemical analyses have shown that the AS11 phenotype is not due to a deficiency in the capacity to elongate 18:1 or to an increase in the relative [delta]15 or [delta]12 desaturase activities. Indeed, the ratio of desaturase/elongase activities measured in vitro is virtually identical in developing WT and AS11 seed homogenates. Rather, the fatty acid phenotype of AS11 is the result of reduced diacylglycerol acyltransferase activity throughout development, such that triacylglycerol biosynthesis is reduced. This leads to a reduction in 20:1 biosynthesis during seed development, leaving more 18:1 available for desaturation. Thus, we have demonstrated that changes to triacylglycerol biosynthesis can result in dramatic changes in fatty acid composition and, in particular, in the accumulation of VLCFAs in seed storage lipids.

title: Identification of potential substrate-binding sites in yeast and human acyl-CoA sterol acyltransferases by mutagenesis of conserved sequences.
abstract: In mammals, the esterification of sterols by ACAT plays a critical role in eukaryotic lipid homeostasis. Using the predominant isoform of the yeast ACAT-related enzyme family, Are2p, as a model, we targeted phylogenetically conserved sequences for mutagenesis in order to identify functionally important motifs. Deletion, truncation, and missense mutations implicate a regulatory role for the amino-terminal domain of Are2p and identified two carboxyl-terminal motifs as required for catalytic activity. A serine-to-leucine mutation in the (H/Y)SF motif (residues 338-340), unique to sterol esterification enzymes, nullified the activity and stability of yeast Are2p. Similarly, a tyrosine-to-alanine change in the FYxDWWN motif of Are2p (residues 523-529) produced an enzyme with decreased activity and apparent affinity for oleoyl-CoA. Mutagenesis of the tryptophan residues in this motif completely abolished activity. In human ACAT1, mutagenesis of the corresponding motifs (residues 268-270, and 403-409, respectively) also nullified enzymatic activity. On the basis of their critical roles in enzymatic activity and their sequence conservation, we propose that these motifs mediate sterol and acyl-CoA binding by this class of enzymes.

title: Oleate desaturase enzymes of soybean: evidence of regulation through differential stability and phosphorylation.
abstract: The endoplasmic reticulum-associated oleate desaturase FAD2 (1-acyl-2-oleoyl-sn-glycero-3-phosphocholine Delta12-desaturase) is the key enzyme responsible for the production of linoleic acid in non-photosynthetic tissues of plants. Little is known, however, concerning the post-transcriptional mechanisms that regulate the activity of this important enzyme. The soybean genome possesses two seed-specific isoforms of FAD2, designated FAD2-1A and FAD2-1B, which differ at only 24 amino acid residues. Expression studies in yeast revealed that the FAD2-1A isoform is more unstable than FAD2-1B, particularly when cultures were maintained at elevated growth temperatures. Analysis of chimeric FAD2-1 constructs led to the identification of two domains that appear to be important in mediating the temperature-dependent instability of the FAD2-1A isoform. The enhanced degradation of FAD2-1A at high growth temperatures was partially abrogated by treating the cultures with the 26S proteasome-specific inhibitor MG132, and by expressing the FAD2-1A cDNA in yeast strains devoid of certain ubiquitin-conjugating activities, suggesting a role for ubiquitination and the 26S proteasome in protein turnover. In addition, phosphorylation state-specific antipeptide antibodies demonstrated that the Serine-185 of FAD2-1 sequences is phosphorylated during soybean seed development. Expression studies of phosphopeptide mimic mutations in yeast suggest that phosphorylation may downregulate enzyme activity. Collectively, the results show that post-translational regulatory mechanisms are likely to play an important role in modulating FAD2-1 enzyme activities.

title: Physiological and developmental regulation of seed oil production.
abstract: Triacylglycerols (TAGs) constitute a highly efficient form of energy storage. In seeds of angiosperms, they can act as a reserve of carbon and energy allowing to fuel post-germinative seedling growth until photosynthesis becomes effective. They also constitute the economic value of seeds in many crops. In the past years, extensive tools allowing the molecular dissection of plant metabolism have been developed together with analytical and cytological procedures adapted for seed material. These tools have allowed gaining a comprehensive overview of the metabolic pathways leading to TAG synthesis. They have also unravelled factors limiting oil production such as metabolic bottlenecks and light or oxygen availability in seed tissues. Beyond these physiological aspects, accumulation of TAGs is developmentally regulated in seeds. The oil biosynthetic process is initiated at the onset of the maturation phase, once embryo morphogenesis is achieved. A wealth of recent studies has shed new lights on the intricate regulatory network controlling the seed maturation phase, including reserve deposition. This network involves a set of regulated transcription factors that crosstalk with physiological signaling. The knowledge thus acquired paves the way for the genetic engineering of oilseed crops dedicated to food applications or green chemistry.

title: Alterations in Seed Development Gene Expression Affect Size and Oil Content of Arabidopsis Seeds1[C][W][OPEN]
abstract: Analysis of mutant and overexpressing lines of Arabidopsis seed development genes uncovers the impact of a leucine-rich repeat kinase on seed size and oil content. Seed endosperm development in Arabidopsis (Arabidopsis thaliana) is under control of the polycomb group complex, which includes Fertilization Independent Endosperm (FIE). The polycomb group complex regulates downstream factors, e.g. Pheres1 (PHE1), by genomic imprinting. In heterozygous fie mutants, an endosperm develops in ovules carrying a maternal fie allele without fertilization, finally leading to abortion. Another endosperm development pathway depends on MINISEED3 (a WRKY10 transcription factor) and HAIKU2 (a leucine-rich repeat kinase). While the role of seed development genes in the embryo and endosperm establishment has been studied in detail, their impact on metabolism and oil accumulation remained unclear. Analysis of oil, protein, and sucrose accumulation in mutants and overexpression plants of the four seed development genes revealed that (1) seeds carrying a maternal fie allele accumulate low oil with an altered composition of triacylglycerol molecular species; (2) homozygous mutant seeds of phe1, mini3, and iku2, which are smaller, accumulate less oil and slightly less protein, and starch, which accumulates early during seed development, remains elevated in mutant seeds; (3) embryo-specific overexpression of FIE, PHE1, and MINI3 has no influence on seed size and weight, nor on oil, protein, or sucrose content; and (4) overexpression of IKU2 results in seeds with increased size and weight, and oil content of overexpressed IKU2 seeds is increased by 35%. Thus, IKU2 overexpression represents a novel strategy for the genetic manipulation of the oil content in seeds.

title: Analyzing real-time PCR data by the comparative CT method
abstract: Two different methods of presenting quantitative gene expression exist: absolute and relative quantification. Absolute quantification calculates the copy number of the gene usually by relating the PCR signal to a standard curve. Relative gene expression presents the data of the gene of interest relative to some calibrator or internal control gene. A widely used method to present relative gene expression is the comparative CT method also referred to as the 2−ΔΔCT method. This protocol provides an overview of the comparative CT method for quantitative gene expression studies. Also presented here are various examples to present quantitative gene expression data using this method.

title: Wax ester profiling of seed oil by nano-electrospray ionization tandem mass spectrometry
abstract: BackgroundWax esters are highly hydrophobic neutral lipids that are major constituents of the cutin and suberin layer. Moreover they have favorable properties as a commodity for industrial applications. Through transgenic expression of wax ester biosynthetic genes in oilseed crops, it is possible to achieve high level accumulation of defined wax ester compositions within the seed oil to provide a sustainable source for such high value lipids. The fatty alcohol moiety of the wax esters is formed from plant-endogenous acyl-CoAs by the action of fatty acyl reductases (FAR). In a second step the fatty alcohol is condensed with acyl-CoA by a wax synthase (WS) to form a wax ester. In order to evaluate the specificity of wax ester biosynthesis, analytical methods are needed that provide detailed wax ester profiles from complex lipid extracts.ResultsWe present a direct infusion ESI-tandem MS method that allows the semi-quantitative determination of wax ester compositions from complex lipid mixtures covering 784 even chain molecular species. The definition of calibration prototype groups that combine wax esters according to their fragmentation behavior enables fast quantitative analysis by applying multiple reaction monitoring. This provides a tool to analyze wax layer composition or determine whether seeds accumulate a desired wax ester profile. Besides the profiling method, we provide general information on wax ester analysis by the systematic definition of wax ester prototypes according to their collision-induced dissociation spectra. We applied the developed method for wax ester profiling of the well characterized jojoba seed oil and compared the profile with wax ester-accumulating Arabidopsis thaliana expressing the wax ester biosynthetic genes MaFAR and ScWS.ConclusionsWe developed a fast profiling method for wax ester analysis on the molecular species level. This method is suitable to screen large numbers of transgenic plants as well as other wax ester samples like cuticular lipid extracts to gain an overview on the molecular species composition. We confirm previous results from APCI-MS and GC-MS analysis, which showed that fragmentation patterns are highly dependent on the double bond distribution between the fatty alcohol and the fatty acid part of the wax ester.

title: Study of phenology, harvest index, yield, yield components and oil content of different cultivars of rain-fed safflower.
abstract: 3  To study phenology, harvest index, yield, yield components and oil content of different safflower rain-fed cultivars, an experiment was carried out in Zanjan Rain-fed Farming Research Station during 2006-7. Seventeen safflower cultivars supplied by Maraqeh Rain-fed Farming Research Station were evaluated in a completely Randomized Block Design with three replications. Variance analysis of phenological and morphological traits, yield and yield components showed that safflower cultivars had significant differences on the probability level of 1% regarding days to branching, days to flowering, branch no per plant, grain no per boll and thousand-seed weight and they had significant differences on the probability level of 5% regarding days to boll-bearing, harvest index and oil yield. But no significant differences were observed regarding days to germination, days to Rosset termination, boll no per plant and oil content. The cultivar PI-537598 had the highest grain and oil yield (800 and 236.8 kg ha in average, respectively) and Isfahan variety had the lowest 1