The biochemistry of headgroup exchange during triacylglycerol synthesis in canola

Many pathways of primary metabolism are substantially conserved within and across plant families. However, significant differences in organization and fluxes through a reaction network may occur, even between plants in closely related genera. Assessing and understanding these differences is key to appreciating metabolic diversity, and to attempts to engineer plant metabolism for higher crop yields and desired product profiles. To better understand lipid metabolism and seed oil synthesis in canola (Brassica napus), we have characterized four canola homologues of the Arabidopsis (Arabidopsis thaliana) ROD1 gene. AtROD1 encodes phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), the enzyme that catalyzes a major flux of polyunsaturated fatty acids (PUFAs) in oil synthesis. Assays in yeast indicated that only two of the canola genes, BnROD1.A3 and BnROD1.C3, encode active isozymes of PDCT, and these genes are strongly expressed during the period of seed oil synthesis. Loss of expression of BnROD1.A3 and BnROD1.C3 in a double mutant, or by RNA interference, reduced the PUFA content of the oil to 26.6% compared with 32.5% in the wild type. These results indicate that ROD1 isozymes in canola are responsible for less than 20% of the PUFAs that accumulate in the seed oil compared with 40% in Arabidopsis. Our results demonstrate the care needed when translating results from a model species to crop plants.     

The control of seed oil polyunsaturate content in the polyploid crop species Brassica napus

Many important plant species have polyploidy in their recent ancestry, complicating inferences about the genetic basis of trait variation. Although the principal locus controlling the proportion of polyunsaturated fatty acids (PUFAs) in seeds of Arabidopsis thaliana is known (fatty acid desaturase 2; FAD2), commercial cultivars of a related crop, oilseed rape (Brassica napus), with very low PUFA content have yet to be developed. We showed that a cultivar of oilseed rape with lower than usual PUFA content has non-functional alleles at three of the four orthologous FAD2 loci. To explore the genetic basis further, we developed an ethyl methanesulphonate mutagenised population, JBnaCAB_E, and used it to identify lines that also carried mutations in the remaining functional copy. This confirmed the hypothesised basis of variation, resulting in an allelic series of mutant lines showing a spectrum of PUFA contents of seed oil. Several lines had PUFA content of ~6 % and oleic acid content of ~84 %, achieving a long-standing industry objective: very high oleic, very low PUFA rapeseed without the use of genetic modification technology. The population contains a high rate of mutations and represents an important resource for research in B. napus.     

Down‐regulation of crambe fatty acid desaturase and elongase in Arabidopsis and crambe resulted in significantly increased oleic acid content in seed oil

High oleic oil is an important industrial feedstock that has been one of the main targets for oil improvement in a number of oil crops. Crambe (Crambe abyssinica) is a dedicated oilseed crop, suitable for industrial oil production. In this study, we down‐regulated the crambe fatty acid desaturase (FAD) and fatty acid elongase (FAE) genes for creating high oleic seed oil. We first cloned the crambe CaFAD2, CaFAD3 and CaFAE1 genes. Multiple copies of each of these genes were isolated, and the highly homologous sequences were used to make RNAi constructs. These constructs were first tested in Arabidopsis, which led to the elevated oleic or linoleic levels depending on the genes targeted, indicating that the RNAi constructs were effective in regulating the expression of the target genes in nonidentical but closely related species. Furthermore, down‐regulation of CaFAD2 and CaFAE1 in crambe with the FAD2–FAE1 RNAi vector resulted in even more significant increase in oleic acid level in the seed oil with up to 80% compared to 13% for wild type. The high oleic trait has been stable in subsequent five generations and the GM line grew normally in greenhouse. This work has demonstrated the great potential of producing high oleic oil in crambe, thus contributing to its development into an oil crop platform for industrial oil production.     

High-Oleic Peanut Oils Produced by HpRNA-Mediated Gene Silencing of Oleate Desaturase

The quality of peanut oil largely depends on the quantity of oleic (18:1) and linoleic acids (18:2). These two acids comprise more than 80% of the total fatty acids in peanuts. The oleate desaturase (FAD2) gene is important for maintaining high oleic acid content. A partial conservative sequence of the FAD2 gene from peanut was selected. The sense and antisense 260-bp fragments were amplified and subcloned into pFGC1008 binary expression vectors. A total of 21 transgenic plants were obtained via Agrobacterium-mediated transformation. The resulting down-regulation of the FAD2 gene resulted in a 70% increase in oleic acid content in the seeds of transformed plants compared with a 37.93% increase in untransformed plants. The results demonstrated that the target genes were likely suppressed by hpRNA interference, a pathway capable of achieving phenotypic changes. The silencing of FAD2 enabled the development of peanut oils having novel combinations of oleic acid content that can be used in high-value applications, making this approach a reliable technique for the genetic modification of seed quality and the potential for enhancement of other traits as well.     

Natural variation of GmFATA1B regulates seed oil content and composition in soybean

Soybean (Glycine max) produces seeds that are rich in unsaturated fatty acids and is an important oilseed crop worldwide. Seed oil content and composition largely determine the economic value of soybean. Due to natural genetic variation, seed oil content varies substantially across soybean cultivars. Although much progress has been made in elucidating the genetic trajectory underlying fatty acid metabolism and oil biosynthesis in plants, the causal genes for many quantitative trait loci (QTLs) regulating seed oil content in soybean remain to be revealed. In this study, we identified GmFATA1B as the gene underlying a QTL that regulates seed oil content and composition, as well as seed size in soybean. Nine extra amino acids in the conserved region of GmFATA1B impair its function as a fatty acyl–acyl carrier protein thioesterase, thereby affecting seed oil content and composition. Heterogeneously overexpressing the functional GmFATA1B allele in Arabidopsis thaliana increased both the total oil content and the oleic acid and linoleic acid contents of seeds. Our findings uncover a previously unknown locus underlying variation in seed oil content in soybean and lay the foundation for improving seed oil content and composition in soybean.     

Overexpression of <Emphasis Type="Italic">FAD2</Emphasis> promotes seed germination and hypocotyl elongation in <Emphasis Type="Italic">Brassica napus</Emphasis>

The enzyme fatty acid desaturase 2 (FAD2) transforms oleic acid (C18:1) to linoleic acid (C18:2) in plants and as such is involved in fatty acid synthesis. It is also involved in plant development and self-defense, such as seed germination, leaf expansion and cold resistance. We have cloned the full coding region of the Brassica napus FAD2 gene and ectopically expressed it in B. napus expressing low levels of FAD2. Overexpression of FAD2 under the control of the CaMV 35S promoter resulted in an up-regulated FAD2 mRNA level in B. napus as expected. Further analysis revealed that the FAD2 transgenic lines varied greatly in terms of their physiological characteristics, such as enhanced seed germination and increased hypocotyl length, compared to non-transgenic plants, suggesting that up-regulated FAD2 can promote seed germination and hypocotyl elongation in B. napus. Our results demonstrate the possible roles of FAD2 in plant development and also provide a platform for further analysis of fatty acid synthesis in plants.     

Influence of salt stress on seed yield and oil quality of two sunflower hybrids

Sunflower is a major oil seed crop worldwide, and it is also an important crop in Mediterranean areas where salinity is an increasing problem. In this paper, the effect of saline irrigation water on seed yield and quality of sunflower was evaluated. A pot experiment was carried out over two crop seasons on two hybrids – a standard one (Carlos) and a high oleic one (Tenor) – submitted to five salinity levels of irrigation water (0.6, 3, 6, 9 and 12 dS m^?1). Soil salinity was monitored over the entire crop cycle, and leaf ion content was determined at maturity. Tenor showed higher Na⁺ and Mg²⁺ content but lower K⁺ values. No difference between the two hybrids was observed for Cl^? content. A progressive increase in leaf Na⁺, K⁺ and Cl^? contents and Na⁺/K⁺ ratio with increasing salinity level was observed. Seed weight per head, 1000 achene weight, number of seeds per plant and oil yield significantly decreased under salt stress in both hybrids. The percent seed yield decrease was higher per unit increase in electrical conductivity of irrigation water, ECw (8%), than per unit increase in electrical conductivity of saturated‐soil extracts, ECe (5%). Concerning oil fatty acid composition, the main significant difference as result of salt stress was a progressive increase in oleic acid content, from 82.2% to 86.7% for Tenor and from 21.8% to 27.3% for Carlos, which was consistent with a decrease in linoleic acid content, from 5.9% to 3% for Tenor and from 66% to 61.3% for Carlos. These results confirm the possible inhibition of oleate desaturase under salt stress.     

Quantification of seed oil from species with varying oil content using supercritical fluid extraction

Introduction: The quantity and composition of seed oil affects seed viability and storability and hence the value of a species as a resource for nutrition and plant conservation. Supercritical fluid extraction with carbon dioxide (SFE‐CO₂) offers a rapid, environmentally friendly alternative to traditional solvent extraction. Objective: To develop a method using SFE‐CO₂ to quantify the seed oil content in a broad range of species with high to low oil contents. Methodology: Seed oil was extracted using SFE‐CO₂ from four crop species representing high, medium and low oil content: Helianthus annuus, Asteraceae, with ca. 55% oil; Brassica napus, Brassicaceae, with ca. 50% oil; Glycine max, Fabaceae, with ca. 20% oil; and Pisum sativum, Fabaceae, with ca. 2% oil. Extraction pressures of 5000, 6000 and 7500 psi and temperatures of 40, 60 and 80°C were examined and a second step using 15% ethanol as a modifier included. Oil yields were compared with that achieved from Smalley Butt extraction. The optimised SFE‐CO₂ method was validated on six species from taxonomically distant families and with varying oil contents: Swietenia humilis (Meliaceae), Stenocereus thurberi (Cactaceae), Sinapis alba (Brassicaceae), Robinia pseudoacacia (Fabaceae), Poa pratensis (Poaceae) and Trachycarpus fortunei (Arecaceae). Results: The two‐step extraction at 6000 psi and 80°C produced oil yields equivalent to or higher than Smalley Butt extraction for all species, including challenging species from the Brassicaceae family. Conclusion: SFE‐CO₂ enables the rapid analysis of seed oils across a broad range of seed oil contents. Copyright © 2008 John Wiley & Sons, Ltd.     

A novel <Emphasis Type="Italic">FAD2</Emphasis>-<Emphasis Type="Italic">1 A</Emphasis> allele in a soybean plant introduction offers an alternate means to produce soybean seed oil with 85% oleic acid content

The alteration of fatty acid profiles in soybean to improve soybean oil quality has been a long-time goal of soybean researchers. Soybean oil with elevated oleic acid is desirable because this monounsaturated fatty acid improves the nutrition and oxidative stability of soybean oil compared to other oils. In the lipid biosynthetic pathway, the enzyme fatty acid desaturase 2 (FAD2) is responsible for the conversion of oleic acid precursors to linoleic acid precursors in developing soybean seeds. Two genes encoding FAD2-1A and FAD2-1B were identified to be expressed specifically in seeds during embryogenesis and have been considered to hold an important role in controlling the seed oleic acid content. A total of 22 soybean plant introduction (PI) lines identified to have an elevated oleic acid content were characterized for sequence mutations in the FAD 2-1A and FAD2-1B genes. PI 603452 was found to contain a deletion of a nucleotide in the second exon of FAD2-1A. These important SNPs were used in developing molecular marker genotyping assays. The assays appear to be a reliable and accurate tool to identify the FAD 2-1A and FAD2-1B genotype of wild-type and mutant plants. PI 603452 was subsequently crossed with PI 283327, a soybean line that has a mutation in FAD2-1B. Interestingly, soybean lines carrying both homozygous insertion/deletion mutation (indel) FAD2-1A alleles and mutant FAD2-1B alleles have an average of 82–86% oleic acid content, compared to 20% in conventional soybean, and low levels of linoleic and linolenic acids. The newly identified indel mutation in the FAD2-1A gene offers a simple method for the development of high oleic acid commercial soybean varieties.     

Modification of starch metabolism in transgenic Arabidopsis thaliana increases plant biomass and triples oilseed production

We have identified a novel means to achieve substantially increased vegetative biomass and oilseed production in the model plant Arabidopsis thaliana. Endogenous isoforms of starch branching enzyme (SBE) were substituted by either one of the endosperm‐expressed maize (Zea mays L.) branching isozymes, ZmSBEI or ZmSBEIIb. Transformants were compared with the starch‐free background and with the wild‐type plants. Each of the maize‐derived SBEs restored starch biosynthesis but both morphology and structure of starch particles were altered. Altered starch metabolism in the transformants is associated with enhanced biomass formation and more‐than‐trebled oilseed production while maintaining seed oil quality. Enhanced oilseed production is primarily due to an increased number of siliques per plant whereas oil content and seed number per silique are essentially unchanged or even modestly decreased. Introduction of cereal starch branching isozymes into oilseed plants represents a potentially useful strategy to increase biomass and oilseed production in related crops and manipulate the structure and properties of leaf starch.     

Two high linolenic mutants of Arabidopsis thaliana contain megabase-scale genome duplications encompassing the FAD3 locus

Understanding the quantitative control of fatty acid desaturation during the biosynthesis of seed storage oil has become a priority area for research, as a consequence of its importance for both human health and the substitution of mineral oil for industrial applications. We have analysed the genome structure of two mutants in Arabidopsis thaliana that show substantially elevated content of the omega‐3 polyunsaturated fatty acid linolenic acid in their seed oil. In one, rfc4, sequences totalling approximately 2 Mb from chromosome 2 have been duplicated and inserted into chromosome 3. In the other mutant, ife, chromosome 2 sequences totalling approximately 1.4 Mb have been duplicated and inserted into a linked position. In both cases, the duplications encompass the FAD3 locus, which encodes the linoleate desaturase responsible for the biosynthesis of linolenic acid for accumulation in seed storage oil. The results show that mutagens such as fast neutrons (used for the induction of rfc4) and T‐DNA (used for the induction of ife, which is not linked to the T‐DNA present in the line) can result in the duplication of very large genome segments. They also show that increasing the dosage of the FAD3‐containing genomic region results in an increase in the linolenic acid content of seed oil. Consequently, screening methods for duplication of FAD3 orthologues in oil crops may be an appropriate approach for the identification of germplasm for breeding varieties with increased proportions of linolenic acid in the oil that they produce.     

The impact of reducing fatty acid desaturation on the composition and thermal stability of rapeseed oil

Oilseed rape (Brassica napus) is the third largest source of vegetable oil globally. In addition to food uses, there are industrial applications that exploit the ability of the species to accumulate the very‐long‐chain fatty acid (VLCFA) erucic acid in its seed oil, controlled by orthologues of FATTY ACID ELONGASE 1 (Bna.FAE1.A8 and Bna.FAE1.C3). The proportion of polyunsaturated fatty acids (PUFAs) in rapeseed oil is predicted to affect its thermal stability and is controlled by orthologues of FATTY ACID DESATURASE 2, particularly Bna.FAD2.C5. Our aim was to develop rapeseed lines combining high erucic and low PUFA characters and to assess the impact on thermal stability of the oil they produce. The new type of rapeseed oil (high erucic low polyunsaturate; HELP) contained a substantially greater proportion of erucic acid (54%) compared with high erucic rapeseed oil (46%). Although the total VLCFA content was greater in oil from HELP lines (64%) than from high erucic rapeseed (57%), analysis of triacylglycerol composition showed negligible incorporation of VLCFAs into the sn‐2 position. Rancimat analysis showed that the thermal stability of rapeseed oil was improved greatly as a consequence of reduction of PUFA content, from 3.8 and 4.2 h in conventional low erucic and high erucic rapeseed oils, respectively, to 11.3 and 16.4 h in high oleic low PUFA (HOLP) and HELP oils, respectively. Our results demonstrate that engineering of the lipid biosynthetic pathway of rapeseed, using traditional approaches, enables the production of renewable industrial oils with novel composition and properties.     

Synthesis of oleyl oleate wax esters in Arabidopsis thaliana and Camelina sativa seed oil

Seed oil composed of wax esters with long‐chain monoenoic acyl moieties represents a high‐value commodity for industry. Such plant‐derived sperm oil‐like liquid wax esters are biodegradable and can have excellent properties for lubrication. In addition, wax ester oil may represent a superior substrate for biodiesel production. In this study, we demonstrate that the low‐input oil seed crop Camelina sativa can serve as a biotechnological platform for environmentally benign wax ester production. Two biosynthetic steps catalysed by a fatty alcohol‐forming acyl‐CoA reductase (FAR) and a wax ester synthase (WS) are sufficient to achieve wax ester accumulation from acyl‐CoA substrates. To produce plant‐derived sperm oil‐like liquid wax esters, the WS from Mus musculus (MmWS) or Simmondsia chinensis (ScWS) were expressed in combination with the FAR from Mus musculus (MmFAR1) or Marinobacter aquaeolei (MaFAR) in seeds of Arabidopsis thaliana and Camelina sativa. The three analysed enzyme combinations Oleo3:mCherry:MmFAR1?c/Oleo3:EYFP:MmWS, Oleo3:mCherry:MmFAR1?c/ScWS and MaFAR/ScWS showed differences in the wax ester molecular species profiles and overall biosynthetic performance. By expressing MaFAR/ScWS in Arabidopsis or Camelina up to 59% or 21% of the seed oil TAGs were replaced by wax esters, respectively. This combination also yielded wax ester molecular species with highest content of monounsaturated acyl moieties. Expression of the enzyme combinations in the Arabidopsis fae1 fad2 mutant background high in oleic acid resulted in wax ester accumulation enriched in oleyl oleate (18:1/18:1 > 60%), suggesting that similar values may be obtained with a Camelina high oleic acid line.     

Phospholipase Dε enhances Braasca napus growth and seed production in response to nitrogen availability

Phospholipase D (PLD), which hydrolyses phospholipids to produce phosphatidic acid, has been implicated in plant response to macronutrient availability in Arabidopsis. This study investigated the effect of increased PLDε expression on nitrogen utilization in Brassica napus to explore the application of PLDε manipulation to crop improvement. In addition, changes in membrane lipid species in response to nitrogen availability were determined in the oil seed crop. Multiple PLDε over expression (PLDε‐OE) lines displayed enhanced biomass accumulation under nitrogen‐deficient and nitrogen‐replete conditions. PLDε‐OE plants in the field produced more seeds than wild‐type plants but have no impact on seed oil content. Compared with wild‐type plants, PLDε‐OE plants were enhanced in nitrate transporter expression, uptake and reduction, whereas the activity of nitrite reductase was higher under nitrogen‐depleted, but not at nitrogen‐replete conditions. The level of nitrogen altered membrane glycerolipid metabolism, with greater impacts on young than mature leaves. The data indicate increased expression of PLDε has the potential to improve crop plant growth and production under nitrogen‐depleted and nitrogen‐replete conditions.     

Determination of fatty acid composition in seed oil of rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) by mutated alleles of the FAD3 desaturase genes

One of the goals in oilseed rape programs is to develop genotypes producing oil with low linolenic acid content (C18:3, ≤3%). Low linolenic mutant lines of canola rapeseed were obtained via chemical mutagenesis at the Plant Breeding and Acclimatization Institute – NRI, in Poznan, Poland, and allele-specific SNP markers were designed for monitoring of two statistically important single nucleotide polymorphisms detected by SNaPshot analysis in two FAD3 desaturase genes, BnaA.FAD3 and BnaC.FAD3, respectively. Strong negative correlation between the presence of mutant alleles of the genes and linolenic acid content was revealed by analysis of variance. In this paper we present detailed characteristics of the markers by estimation of the additive and dominance effects of the FAD3 genes with respect to particular fatty acid content in seed oil, as well as by calculation of the phenotypic variation of seed oil fatty acid composition accounted by particular allele-specific marker. The obtained percentage of variation in fatty acid composition was considerable only for linolenic acid content and equaled 35.6% for BnaA.FAD3 and 39.3% for BnaC.FAD3, whereas the total percentage of variation in linolenic acid content was 53.2% when accounted for mutations in both genes simultaneously. Our results revealed high specificity of the markers for effective monitoring of the wild-type and mutated alleles of the Brassica napus FAD3 desaturase genes in the low linolenic mutant recombinants in breeding programs.     

Punicic acid production in Brassica napus

Punicic acid (PuA; 18:3Δ^{9cis,11trans,13cis}), a conjugated linolenic acid isomer bearing three conjugated double bonds, is associated with various health benefits and has potential for industrial use. The major nature source of this unusual fatty acid is pomegranate (Punica granatum) seed oil, which contains up to 80% (w/w) of its fatty acids as PuA. Pomegranate seed oil, however, is low yielding with unstable production and thus limits the supply of PuA. Metabolic engineering of established temperate oil crops for PuA production, therefore, has the potential to be a feasible strategy to overcome the limitations associated with sourcing PuA from pomegranate. In this study, the cDNAs encoding a pomegranate fatty acid conjugase and a pomegranate oleate desaturase were co-expressed in canola-type Brassica napus. Transgenic B. napus lines accumulated up to 11% (w/w) of the total fatty acids as PuA in the seed oil, which is the highest level of PuA reported in metabolically engineered oilseed crops so far. Levels of seed oil PuA were stable over two generations and had no negative effects on seed germination. The transgenic B. napus lines with the highest PuA levels contained multiple transgene insertions and the PuA content of B. napus seed oil was correlated with efficiency of oleic acid desaturation and linoleic acid conjugation. In addition, PuA accumulated at lower levels in polar lipids (5.0–6.9%) than triacylglycerol (7.5–10.6%), and more than 60% of triacylglycerol-associated PuA was present at the sn-2 position. This study provides the basis for the commercial production of PuA in transgenic oilseed crops and thus would open new prospects for the application of this unusual fatty acid in health and industry.