Significant enhancement of fatty acid composition in seeds of the allohexaploid,Camelina sativa,using CRISPR/Cas9 gene editing

The CRISPR/Cas9 nuclease system is a powerful and flexible tool for genome editing, and novel applications of this system are being developed rapidly. Here, we used CRISPR/Cas9 to target the FAD2 gene in Arabidopsis thaliana and in the closely related emerging oil seed plant, Camelina sativa, with the goal of improving seed oil composition. We successfully obtained Camelina seeds in which oleic acid content was increased from 16% to over 50% of the fatty acid composition. These increases were associated with significant decreases in the less desirable polyunsaturated fatty acids, linoleic acid (i.e. a decrease from ~16% to <4%) and linolenic acid (a decrease from ~35% to <10%). These changes result in oils that are superior on multiple levels: they are healthier, more oxidatively stable and better suited for production of certain commercial chemicals, including biofuels. As expected, A. thaliana T₂ and T₃ generation seeds exhibiting these types of altered fatty acid profiles were homozygous for disrupted FAD2 alleles. In the allohexaploid, Camelina, guide RNAs were designed that simultaneously targeted all three homoeologous FAD2 genes. This strategy that significantly enhanced oil composition in T₃ and T₄ generation Camelina seeds was associated with a combination of germ‐line mutations and somatic cell mutations in FAD2 genes in each of the three Camelina subgenomes.     

Identification,characterization and field testing of Brassica napus mutants producing high‐oleic oils

Producing healthy, high‐oleic oils and eliminating trans‐fatty acids from foods are two goals that can be addressed by reducing activity of the oleate desaturase, FAD2, in oilseeds. However, it is essential to understand the consequences of reducing FAD2 activity on the metabolism, cell biology and physiology of oilseed crop plants. Here, we translate knowledge from studies of fad2 mutants in Arabidopsis (Arabidopsis thaliana) to investigate the limits of non‐GMO approaches to maximize oleic acid in the seed oil of canola (Brassica napus), a species that expresses three active FAD2 isozymes. A series of hypomorphic and null mutations in the FAD2.A5 isoform were characterized in yeast (Saccharomyes cerevisiae). Then, four of these were combined with null mutations in the other two isozymes, FAD2.C5 and FAD2.C1. The resulting mutant lines contained 71–87% oleic acid in their seed oil, compared with 62% in wild‐type controls. All the mutant lines grew well in a greenhouse, but in field experiments we observed a clear demarcation in plant performance. Mutant lines containing less than 80% oleate in the seed oil were indistinguishable from wild‐type controls in growth parameters and seed oil content. By contrast, lines with more than 80% oleate in the seed oil had significantly lower seedling establishment and vigor, delayed flowering and reduced plant height at maturity. These lines also had 7–11% reductions in seed oil content. Our results extend understanding of the B. napusFAD2 isozymes and define the practical limit to increasing oil oleate content in this crop species.     

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.     

Mutant alleles of <Emphasis Type="Italic">FAD2-1A</Emphasis> and <Emphasis Type="Italic">FAD2-1B</Emphasis>combine to produce soybeans with the high oleic acid seed oil trait

Background  

The alteration of fatty acid profiles in soybean [Glycine max (L.) Merr.] to improve soybean oil quality is an important and evolving theme in soybean research to meet nutritional needs and industrial criteria in the modern market. Soybean oil with elevated oleic acid is desirable because this monounsaturated fatty acid improves the nutrition and oxidative stability of the oil. Commodity soybean oil typically contains 20% oleic acid and the target for high oleic acid soybean oil is approximately 80% of the oil; previous conventional plant breeding research to raise the oleic acid level to just 50-60% of the oil was hindered by the genetic complexity and environmental instability of the trait. The objective of this work was to create the high oleic acid trait in soybeans by identifying and combining mutations in two delta-twelve fatty acid desaturase genes, FAD2-1A and FAD2-1B.     

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.     

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.     

Detection of induced mutations in CaFAD2 genes by next‐generation sequencing leading to the production of improved oil composition in Crambe abyssinica

Crambe abyssinica is a hexaploid oil crop for industrial applications. An increase of erucic acid (C22:1) and reduction of polyunsaturated fatty acid (PUFA) contents in crambe oil is a valuable improvement. An increase in oleic acid (C18:1), a reduction in PUFA and possibly an increase in C22:1 can be obtained by down‐regulating the expression of fatty acid desaturase2 genes (CaFAD2), which code for the enzyme that converts C18:1 into C18:2. We conducted EMS‐mutagenesis in crambe, followed by Illumina sequencing, to screen mutations in three expressed CaFAD2 genes. Two novel analysis strategies were used to detect mutation sites. In the first strategy, mutation detection targeted specific sequence motifs. In the second strategy, every nucleotide position in a CaFAD2 fragment was tested for the presence of mutations. Seventeen novel mutations were detected in 1100 one‐dimensional pools (11 000 individuals) in three expressed CaFAD2 genes, including non‐sense mutations and mis‐sense mutations in CaFAD2‐C1, ‐C2 and ‐C3. The homozygous non‐sense mutants for CaFAD2‐C3 resulted in a 25% higher content of C18:1 and 25% lower content of PUFA compared to the wild type. The mis‐sense mutations only led to small changes in oil composition. Concluding, targeted mutation detection using NGS in a polyploid was successfully applied and it was found that a non‐sense mutation in even a single CaFAD2 gene can lead to changes in crambe oil composition. Stacking the mutations in different CaFAD2 may gain additional changes in C18:1 and PUFA contents.     

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.     

Seed‐specific RNAi in safflower generates a superhigh oleic oil with extended oxidative stability

Vegetable oils extracted from oilseeds are an important component of foods, but are also used in a range of high value oleochemical applications. Despite being biodegradable, nontoxic and renewable current plant oils suffer from the presence of residual polyunsaturated fatty acids that are prone to free radical formation that limit their oxidative stability, and consequently shelf life and functionality. Many decades of plant breeding have been successful in raising the oleic content to ~90%, but have come at the expense of overall field performance, including poor yields. Here, we engineer superhigh oleic (SHO) safflower producing a seed oil with 93% oleic generated from seed produced in multisite field trials spanning five generations. SHO safflower oil is the result of seed‐specific hairpin‐based RNA interference of two safflower lipid biosynthetic genes, FAD2.2 and FATB, producing seed oil containing less than 1.5% polyunsaturates and only 4% saturates but with no impact on lipid profiles of leaves and roots. Transgenic SHO events were compared to non‐GM safflower in multisite trial plots with a wide range of growing season conditions, which showed no evidence of impact on seed yield. The oxidative stability of the field‐grown SHO oil produced from various sites was 50 h at 110°C compared to 13 h for conventional ~80% oleic safflower oils. SHO safflower produces a uniquely stable vegetable oil across different field conditions that can provide the scale of production that is required for meeting the global demands for high stability oils in food and the oleochemical industry.     

Membrane lipid polyunsaturation mediated by FATTY ACID DESATURASE 2 (FAD2) is involved in endoplasmic reticulum stress tolerance in Arabidopsis thaliana

Unsaturation of membrane glycerolipid classes at their hydrophobic fatty acid tails critically affects the physical nature of the lipid molecule. In Arabidopsis thaliana, 7 fatty acid desaturases (FADs) differently desaturate each glycerolipid class in plastids and the endoplasmic reticulum (ER). Here, we showed that polyunsaturation of ER glycerolipids is required for the ER stress response. Through systematic screening of FAD mutants, we found that a mutant of FAD2 resulted in a hypersensitive response to tunicamycin, a chemical inducer of ER stress. FAD2 converts oleic acid to linoleic acid of the fatty acyl groups of ER‐synthesized phospholipids. Our functional in vivo reporter assay revealed the ER localization and distinct tissue‐specific expression patterns of FAD2. Moreover, glycerolipid profiling of both mutants and overexpressors of FAD2 under tunicamycin‐induced ER stress conditions, along with phenotypic screening of the mutants of the FAD family, suggested that the ratio of monounsaturated fatty acids to polyunsaturated fatty acids, particularly 18:1 to 18:2 species, may be an important factor in allowing the ER membrane to cope with ER stress. Therefore, our results suggest that membrane lipid polyunsaturation mediated by FAD2 is involved in ER stress tolerance in Arabidopsis.     

Metabolic engineering of oilseed crops to produce high levels of novel acetyl glyceride oils with reduced viscosity,freezing point and calorific value

Seed oils have proved recalcitrant to modification for the production of industrially useful lipids. Here, we demonstrate the successful metabolic engineering and subsequent field production of an oilseed crop with the highest accumulation of unusual oil achieved so far in transgenic plants. Previously, expression of the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene in wild‐type Arabidopsis seeds resulted in the accumulation of 45 mol% of unusual 3‐acetyl‐1,2‐diacyl‐sn‐glycerols (acetyl‐TAGs) in the seed oil (Durrett et al., 2010 PNAS 107:9464). Expression of EaDAcT in dgat1 mutants compromised in their ability to synthesize regular triacylglycerols increased acetyl‐TAGs to 65 mol%. Camelina and soybean transformed with the EaDAcT gene accumulate acetyl‐triacylglycerols (acetyl‐TAGs) at up to 70 mol% of seed oil. A similar strategy of coexpression of EaDAcT together with RNAi suppression of DGAT1 increased acetyl‐TAG levels to up to 85 mol% in field‐grown transgenic Camelina. Additionally, total moles of triacylglycerol (TAG) per seed increased 20%. Analysis of the acetyl‐TAG fraction revealed a twofold reduction in very long chain fatty acids (VLCFA), consistent with their displacement from the sn‐3 position by acetate. Seed germination remained high, and seedlings were able to metabolize the stored acetyl‐TAGs as rapidly as regular triacylglycerols. Viscosity, freezing point and caloric content of the Camelina acetyl‐TAG oils were reduced, enabling use of this oil in several nonfood and food applications.     

Functional Characterization of Flax Fatty Acid Desaturase FAD2 and FAD3 Isoforms Expressed in Yeast Reveals a Broad Diversity in Activity

With 45 % or more oil content that contains more than 55 % alpha linolenic (LIN) acid, linseed (Linum usitatissimum L.) is one of the richest plant sources of this essential fatty acid. Fatty acid desaturases 2 (FAD2) and 3 (FAD3) are the main enzymes responsible for the Δ12 and Δ15 desaturation in planta. In linseed, the oilseed morphotype of flax, two paralogous copies, and several alleles exist for each gene. Here, we cloned three alleles of FAD2A, four of FAD2B, six of FAD3A, and seven of FAD3B into a pYES vector and transformed all 20 constructs and an empty construct in yeast. The transformants were induced in the presence of oleic (OLE) acid substrate for FAD2 constructs and linoleic (LIO) acid for FAD3. Conversion rates of OLE acid into LIO acid and LIO acid into LIN acid were measured by gas chromatography. Conversion rate of FAD2 exceeded that of FAD3 enzymes with FAD2B having a conversion rate approximately 10 % higher than FAD2A. All FAD2 isoforms were active, but significant differences existed between isoforms of both FAD2 enzymes. Two FAD3A and three FAD3B isoforms were not functional. Some nonfunctional enzymes resulted from the presence of nonsense mutations causing premature stop codons, but FAD3B-C and FAD3B-F seem to be associated with single amino acid changes. The activity of FAD3A-C was more than fivefold greater than the most common isoform FAD3A-A, while FAD3A-F was fourfold greater. Such isoforms could be incorporated into breeding lines to possibly further increase the proportion of LIN acid in linseed.     

Gene editing of the wheat homologs of TONNEAU1‐recruiting motif encoding gene affects grain shape and weight in wheat

Grain size and weight are important components of a suite of yield‐related traits in crops. Here, we showed that the CRISPR‐Cas9 gene editing of TaGW7, a homolog of rice OsGW7 encoding a TONNEAU1‐recruiting motif (TRM) protein, affects grain shape and weight in allohexaploid wheat. By editing the TaGW7 homoeologs in the B and D genomes, we showed that mutations in either of the two or both genomes increased the grain width and weight but reduced the grain length. The effect sizes of mutations in the TaGW7 gene homoeologs coincided with the relative levels of their expression in the B and D genomes. The effects of gene editing on grain morphology and weight traits were dosage dependent with the double‐copy mutant showing larger effect than the respective single copy mutants. The TaGW7‐centered gene co‐expression network indicated that this gene is involved in the pathways regulating cell division and organ growth, also confirmed by the cellular co‐localization of TaGW7 with α‐ and β‐tubulin proteins, the building blocks of microtubule arrays. The analyses of exome capture data in tetraploid domesticated and wild emmer, and hexaploid wheat revealed the loss of diversity around TaGW7‐associated with domestication selection, suggesting that TaGW7 is likely to play an important role in the evolution of yield component traits in wheat. Our study showed how integrating CRISPR‐Cas9 system with cross‐species comparison can help to uncover the function of a gene fixed in wheat for allelic variants targeted by domestication selection and select targets for engineering new gene variants for crop improvement.     

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.     

Construction and Functional Analysis of CRISPR/Cas9 Vector of <i>FAD2</i> Gene Family in Soybean

Soybean oleic acid content is one of the important indexes to evaluate the quality of soybean oil. In the synthesis pathway of soybean fatty acids, the FAD2 gene family is the key gene that regulates the production of linoleic acid from soybean oleic acid. In this study, CRISPR/Cas9 gene editing technology was used to regulate FAD2 gene expression. Firstly, the CRISPR/Cas9 single knockout vectors GmFAD2-1B and GmFAD2-2C and double knockout vectors GmFAD2-2A-3 were constructed. Then, the three vectors were transferred into the recipient soybean variety Jinong 38 by Agrobacterium-mediated cotyledon node transformation, and the mutant plants were obtained. Functional analysis and comparison of the mutant plants of the T2 and T3 generations were carried out. The results showed that there was no significant difference in agronomic traits between the CRISPR/Cas9 single and double knockout vectors and the untransformed CRISPR/Cas9 receptor varieties. The oleic acid content of the plants that knocked out the CRISPR/Cas9 double gene vector was significantly higher than that of the single gene vector.