Physaria fendleri and Ricinus communis lecithin:cholesterol acyltransferase-like phospholipases selectively cleave hydroxy acyl chains from phosphatidylcholine.

Production of hydroxy fatty acids (HFAs) in transgenic crops represents a promising strategy to meet our demands for specialized plant oils with industrial applications. The expression of Ricinus communis (castor) OLEATE 12-HYDROXYLASE (RcFAH12) in Arabidopsis has resulted in only limited accumulation of HFAs in seeds, which probably results from inefficient transfer of HFAs from their site of synthesis (phosphatidylcholine; PC) to triacylglycerol (TAG), especially at the sn-1/3 positions of TAG. Phospholipase As (PLAs) may be directly involved in the liberation of HFAs from PC, but the functions of their over-expression in HFA accumulation and distribution at TAG in transgenic plants have not been well studied. In this work, the functions of lecithin:cholesterol acyltransferase-like PLAs (LCAT-PLAs) in HFA biosynthesis were characterized. The LCAT-PLAs were shown to exhibit homology to LCAT and mammalian lysosomal PLA2 , and to contain a conserved and functional Ser/His/Asp catalytic triad. In vitro assays revealed that LCAT-PLAs from the HFA-accumulating plant species Physaria fendleri (PfLCAT-PLA) and castor (RcLCAT-PLA) could cleave acyl chains at both the sn-1 and sn-2 positions of PC, and displayed substrate selectivity towards sn-2-ricinoleoyl-PC over sn-2-oleoyl-PC. Furthermore, co-expression of RcFAH12 with PfLCAT-PLA or RcLCAT-PLA, but not Arabidopsis AtLCAT-PLA, resulted in increased occupation of HFA at the sn-1/3 positions of TAG as well as small but insignificant increases in HFA levels in Arabidopsis seeds compared with RcFAH12 expression alone. Therefore, PfLCAT-PLA and RcLCAT-PLA may contribute to HFA turnover on PC, and represent potential candidates for engineering the production of unusual fatty acids in crops.

An integrated omics analysis reveals the gene expression profiles of maize, castor bean, and rapeseed for seed oil biosynthesis.

BACKGROUND: Seed storage lipids are valuable for human diet and for the sustainable development of mankind. In recent decades, many lipid metabolism genes and pathways have been identified, but the molecular mechanisms that underlie differences in seed oil biosynthesis in species with developed embryo and endosperm are not fully understood. RESULTS: We performed comparative genome and transcriptome analyses of castor bean and rapeseed, which have high seed oil contents, and maize, which has a low seed oil content. These results revealed the molecular underpinnings of the low seed oil content in maize. First of all, transcriptome analyses showed that more than 61% of the lipid- and carbohydrate-related genes were regulated in castor bean and rapeseed, but only 20.1% of the lipid-related genes and 22.5% of the carbohydrate-related genes were regulated in maize. Then, compared to castor bean and rapeseed, fewer lipid biosynthesis genes but more lipid metabolism genes were regulated in the maize embryo. More importantly, most maize genes encoding lipid-related transcription factors, triacylglycerol (TAG) biosynthetic enzymes, pentose phosphate pathway (PPP) and Calvin Cycle proteins were not regulated during seed oil synthesis, despite the presence of many homologs in the maize genome. Additionally, we observed differential regulation of vital oil biosynthetic enzymes and extremely high expression levels of oil biosynthetic genes in castor bean, which were consistent with the rapid accumulation of oil in castor bean developing seeds. CONCLUSIONS: Compared to high-oil seeds (castor bean and rapeseed), less oil biosynthetic genes were regulated during the seed development in low-oil seed (maize). These results shed light on molecular mechanisms of lipid biosynthesis in maize, castor bean, and rapeseed. They can provide information on key target genes that may be useful for future experimental manipulation of oil production in oil plants.

Genome-Wide Identification of Eucalyptus Heat Shock Transcription Factor Family and Their Transcriptional Analysis under Salt and Temperature Stresses.

Heat shock transcription factors (HSFs) activate heat shock protein gene expression by binding their promoters in response to heat stress and are considered to be pivotal transcription factors in plants. Eucalyptus is a superior source of fuel and commercial wood. During its growth, high temperature or other abiotic stresses could impact its defense capability and growth. Hsf genes have been cloned and sequenced in many plants, but rarely in Eucalyptus. In this study, we used bioinformatics methods to analyze and identify Eucalyptus Hsf genes, their chromosomal localization and structure. The phylogenetic relationship and conserved domains of their encoded proteins were further analyzed. A total of 36 Hsf genes were identified and authenticated from Eucalyptus, which were scattered across 11 chromosomes. They could be classified into three classes (A, B and C). Additionally, a large number of stress-related cis-regulatory elements were identified in the upstream promoter sequence of HSF, and cis-acting element analysis indicated that the expression of EgHsf may be regulated by plant growth and development, metabolism, hormones and stress responses. The expression profiles of five representative Hsf genes, EgHsf4, EgHsf9, EgHsf13, EgHsf24 and EgHsf32, under salt and temperature stresses were examined by qRT-PCR. The results show that the expression pattern of class B genes (EgHsf4, EgHsf24 and EgHsf32) was more tolerant to abiotic stresses than that of class A genes (EgHsf9 and EgHsf13). However, the expressions of all tested Hsf genes in six tissues were at different levels. Finally, we investigated the network of interplay between genes, and the results suggest that there may be synergistic effects between different Hsf genes in response to abiotic stresses. We conclude that the Hsf gene family played an important role in the growth and developmental processes of Eucalyptus and could be vital for maintaining cell homeostasis against external stresses. This study provides basic information on the members of the Hsf gene family in Eucalyptus and lays the foundation for the functional identification of related genes and the further investigation of their biological functions in plant stress regulation.

Reorganization of Acyl Flux through the Lipid Metabolic Network in Oil-Accumulating Tobacco Leaves.

The triacylglycerols (TAGs; i.e. oils) that accumulate in plants represent the most energy-dense form of biological carbon storage, and are used for food, fuels, and chemicals. The increasing human population and decreasing amount of arable land have amplified the need to produce plant oil more efficiently. Engineering plants to accumulate oils in vegetative tissues is a novel strategy, because most plants only accumulate large amounts of lipids in the seeds. Recently, tobacco (Nicotiana tabacum) leaves were engineered to accumulate oil at 15% of dry weight due to a push (increased fatty acid synthesis)-and-pull (increased final step of TAG biosynthesis) engineering strategy. However, to accumulate both TAG and essential membrane lipids, fatty acid flux through nonengineered reactions of the endogenous metabolic network must also adapt, which is not evident from total oil analysis. To increase our understanding of endogenous leaf lipid metabolism and its ability to adapt to metabolic engineering, we utilized a series of in vitro and in vivo experiments to characterize the path of acyl flux in wild-type and transgenic oil-accumulating tobacco leaves. Acyl flux around the phosphatidylcholine acyl editing cycle was the largest acyl flux reaction in wild-type and engineered tobacco leaves. In oil-accumulating leaves, acyl flux into the eukaryotic pathway of glycerolipid assembly was enhanced at the expense of the prokaryotic pathway. However, a direct Kennedy pathway of TAG biosynthesis was not detected, as acyl flux through phosphatidylcholine preceded the incorporation into TAG. These results provide insight into the plasticity and control of acyl lipid metabolism in leaves.

Nitric oxide affects seed oil accumulation and fatty acid composition through protein S-nitrosation.

Nitric oxide (NO) is a key signaling molecule regulating several plant developmental and stress responses. Here, we report that NO plays an important role in seed oil content and fatty acid composition. RNAi silencing of Arabidopsis S-nitrosoglutathione reductase 1 (GSNOR1) led to reduced seed oil content. In contrast, nitrate reductase double mutant nia1nia2 had increased seed oil content, compared with wild-type plants. Moreover, the concentrations of palmitic acid (C16:0), linoleic acid (C18:2), and linolenic acid (C18:3) were higher, whereas those of stearic acid (C18:0), oleic acid (C18:1), and arachidonic acid (C20:1) were lower, in seeds of GSNOR1 RNAi lines. Similar results were obtained with rapeseed embryos cultured in vitro with the NO donor sodium nitroprusside (SNP), and the NO inhibitor NG-Nitro-L-arginine Methyl Ester (L-NAME). Compared with non-treated embryos, the oil content decreased in SNP-treated embryos, and increased in L-NAME-treated embryos. Relative concentrations of C16:0, C18:2 and C18:3 were higher, whereas C18:1 concentration decreased in rapeseed embryos treated with SNP. Proteomics and transcriptome analysis revealed that three S-nitrosated proteins and some key genes involved in oil synthesis, were differentially regulated in SNP-treated embryos. Therefore, regulating NO content could be a novel approach to increasing seed oil content in cultivated oil crops.

Molecular Network for Regulation of Ovule Number in Plants.

In seed-bearing plants, the ovule ("small egg") is the organ within the gynoecium that develops into a seed after fertilization. The gynoecium located in the inner compartment of the flower turns into a fruit. The number of ovules in the ovary determines the upper limit or the potential of seed number per fruit in plants, greatly affecting the final seed yield. Ovule number is an important adaptive characteristic for plant evolution and an agronomic trait for crop improvement. Therefore, understanding the mechanism and pathways of ovule number regulation becomes a significant research aspect in plant science. This review summarizes the ovule number regulators and their regulatory mechanisms and pathways. Specially, an integrated molecular network for ovule number regulation is constructed, in which phytohormones played a central role, followed by transcription factors, enzymes, other protein and micro-RNA. Of them, AUX, BR and CK are positive regulator of ovule number, whereas GA acts negatively on it. Interestingly, many ovule number regulators have conserved functions across several plant taxa, which should be the targets of genetic improvement via breeding or gene editing. Many ovule number regulators identified to date are involved in the diverse biological process, such as ovule primordia formation, ovule initiation, patterning, and morphogenesis. The relations between ovule number and related characteristics/traits especially of gynoecium/fruit size, ovule fertility, and final seed number, as well as upcoming research questions, are also discussed. In summary, this review provides a general overview of the present finding in ovule number regulation, which represents a more comprehensive and in-depth cognition on it.

Engineering Trienoic Fatty Acids into Cottonseed Oil Improves Low-Temperature Seed Germination, Plant Photosynthesis and Cotton Fiber Quality.

Alpha-linolenic acid (ALA, 18:3Δ9,12,15) and γ-linolenic acid \ (GLA, 18:3Δ6,9,12) are important trienoic fatty acids, which are beneficial for human health in their own right, or as precursors for the biosynthesis of long-chain polyunsaturated fatty acids. ALA and GLA in seed oil are synthesized from linoleic acid (LA, 18:2Δ9,12) by the microsomal ω-3 fatty acid desaturase (FAD3) and Δ6 desaturase (D6D), respectively. Cotton (Gossypium hirsutum L.) seed oil composition was modified by transforming with an FAD3 gene from Brassica napus and a D6D gene from Echium plantagineum, resulting in approximately 30% ALA and 20% GLA, respectively. The total oil content in transgenic seeds remained unaltered relative to parental seeds. Despite the use of a seed-specific promoter for transgene expression, low levels of GLA and increased levels of ALA were found in non-seed cotton tissues. At low temperature, the germinating cottonseeds containing the linolenic acid isomers elongated faster than the untransformed controls. ALA-producing lines also showed higher photosynthetic rates at cooler temperature and better fiber quality compared to both untransformed controls and GLA-producing lines. The oxidative stability of the novel cottonseed oils was assessed, providing guidance for potential food, pharmaceutical and industrial applications of these oils.

The Phospholipid:Diacylglycerol Acyltransferase-Mediated Acyl-Coenzyme A-Independent Pathway Efficiently Diverts Fatty Acid Flux from Phospholipid into Triacylglycerol in Escherichia coli.

Researchers have long endeavored to accumulate triacylglycerols (TAGs) or their derivatives in easily managed microbes. The attempted production of TAGs in Escherichia coli has revealed barriers to the broad applications of this technology, including low TAG productivity and slow cell growth. We have demonstrated that an acyl-CoA-independent pathway can divert phospholipid flux into TAG formation in E. coli mediated by Chlamydomonas reinhardtii phospholipid:diacylglycerol acyltransferase (CrPDAT) without interfering with membrane functions. We then showed the synergistic effect on TAG accumulation via the acyl-CoA-independent pathway mediated by PDAT and the acyl-CoA-dependent pathway mediated by wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT). Furthermore, CrPDAT led to synchronous TAG accumulation during cell growth, and this could be enhanced by supplementation of arbutin. We also showed that rationally mutated CrPDAT was capable of decreasing TAG lipase activity without impairing PDAT activity. Finally, ScPDAT from Saccharomyces cerevisiae exhibited similar activities as CrPDAT in E. coli Our results suggest that the improvement in accumulation of TAGs and their derivatives can be achieved by fine-tuning of phospholipid metabolism in E. coli Understanding the roles of PDAT in the conversion of phospholipids into TAGs during the logarithmic growth phase may enable a novel strategy for the production of microbial oils.IMPORTANCE Although phospholipid:diacylglycerol acyltransferase (PDAT) activity is presumed to exist in prokaryotic oleaginous bacteria, the corresponding gene has not been identified yet. In this article, we have demonstrated that an acyl-CoA-independent pathway can divert phospholipid flux into TAG formation in Escherichia coli mediated by exogenous CrPDAT from Chlamydomonas reinhardtii without interfering with membrane functions. In addition, the acyl-CoA-independent pathway and the acyl-CoA-dependent pathway had the synergistic effect on TAG accumulation. Overexpression of CrPDAT led to synchronous TAG accumulation during cell growth. In particular, CrPDAT possessed multiple catalytic activities, and the rational mutation of CrPDAT led to the decrease of TAG lipase activity without impairing acyltransferase activity. The present findings suggested that applying PDAT in E. coli or other prokaryotic microbes may be a promising strategy for accumulation of TAGs and their derivatives.

Harnessing Biotechnology for the Development of New Seed Lipid Traits in Brassica.

The seed oil quality of Brassica oilseed species has been improved in the last few decades, using conventional breeding approaches. Modern biotechnology has enabled the significant development of new seed lipid traits in many oil crops. Alternation of seed lipid component with gene knockout by RNAi gene silencing, artificial microRNA or gene editing within the crop is relative straightforward. Introducing a new pathway from an exogenous source via biotechnology enables the creation of a new trait, where the biosynthetic pathway for such a new trait is not available in the host crop. This review updates the recent development of new seed lipid traits in six major Brassica species and highlights the capability of biotechnology to improve the composition of important fatty acids for both industrial and nutritional purposes.

Up-regulation of lipid biosynthesis increases the oil content in leaves of Sorghum bicolor.

Synthesis and accumulation of the storage lipid triacylglycerol in vegetative plant tissues has emerged as a promising strategy to meet the world's future need for vegetable oil. Sorghum (Sorghum bicolor) is a particularly attractive target crop given its high biomass, drought resistance and C4 photosynthesis. While oilseed-like triacylglycerol levels have been engineered in the C3 model plant tobacco, progress in C4 monocot crops has been lagging behind. In this study, we report the accumulation of triacylglycerol in sorghum leaf tissues to levels between 3 and 8.4% on a dry weight basis depending on leaf and plant developmental stage. This was achieved by the combined overexpression of genes encoding the Zea mays WRI1 transcription factor, Umbelopsis ramanniana UrDGAT2a acyltransferase and Sesamum indicum Oleosin-L oil body protein. Increased oil content was visible as lipid droplets, primarily in the leaf mesophyll cells. A comparison between a constitutive and mesophyll-specific promoter driving WRI1 expression revealed distinct changes in the overall leaf lipidome as well as transitory starch and soluble sugar levels. Metabolome profiling uncovered changes in the abundance of various amino acids and dicarboxylic acids. The results presented here are a first step forward towards the development of sorghum as a dedicated biomass oil crop and provide a basis for further combinatorial metabolic engineering.

Comparison of the Substrate Preferences of ω3 Fatty Acid Desaturases for Long Chain Polyunsaturated Fatty Acids.

Omega-3 long chain polyunsaturated fatty acids (ω3 LC-PUFAs) such as eicosapentaenoic acid (EPA; 20:5ω3) and docosahexaenoic acid (DHA; 22:6ω3) are important fatty acids for human health. These ω3 LC-PUFAs are produced from their ω3 precursors by a set of desaturases and elongases involved in the biosynthesis pathway and are also converted from ω6 LC-PUFA by omega-3 desaturases (ω3Ds). Here, we have investigated eight ω3-desaturases obtained from a cyanobacterium, plants, fungi and a lower animal species for their activities and compared their specificities for various C18, C20 and C22 ω6 PUFA substrates by transiently expressing them in Nicotiana benthamiana leaves. Our results showed hitherto unreported activity of many of the ω3Ds on ω6 LC-PUFA substrates leading to their conversion to ω3 LC-PUFAs. This discovery could be important in the engineering of EPA and DHA in heterologous hosts.

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.

Cellular and Molecular Mechanisms of Calcium/Calmodulin-Dependent Protein Kinase II in Chronic Pain.

Chronic pain, often defined as any pain lasting more than 3 months, is poorly managed because of its multifaceted and complex mechanisms. Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine kinase that plays a fundamental role in synaptic plasticity, learning, and memory. Recent emerging evidence demonstrates increased expression and activity of CaMKII in the spinal cord and dorsal root ganglia of various chronic pain models. Moreover, our previous studies also find that inhibiting CaMKII could attenuate inflammatory pain and neuropathic pain. In this review, we provide evidence for the involvement of CaMKII in the initiation and development of chronic pain, including neuropathic pain, bone cancer pain, and inflammatory pain. Novel CaMKII inhibitors with potent inhibitory effect and high specificity may be alternative therapeutic strategies for the management of chronic pain in the future.

Step changes in leaf oil accumulation via iterative metabolic engineering.

Synthesis and accumulation of plant oils in the entire vegetative biomass offers the potential to deliver yields surpassing those of oilseed crops. However, current levels still fall well short of those typically found in oilseeds. Here we show how transcriptome and biochemical analyses pointed to a futile cycle in a previously established Nicotiana tabacum line, accumulating up to 15% (dry weight) of the storage lipid triacylglycerol in leaf tissue. To overcome this metabolic bottleneck, we either silenced the SDP1 lipase or overexpressed the Arabidopsis thaliana LEC2 transcription factor in this transgenic background. Both strategies independently resulted in the accumulation of 30-33% triacylglycerol in leaf tissues. Our results demonstrate that the combined optimization of de novo fatty acid biosynthesis, storage lipid assembly and lipid turnover in leaf tissue results in a major overhaul of the plant central carbon allocation and lipid metabolism. The resulting further step changes in oil accumulation in the entire plant biomass offers the possibility of delivering yields that outperform current oilseed crops.

Deep Sequencing of the Fruit Transcriptome and Lipid Accumulation in a Non-Seed Tissue of Chinese Tallow, a Potential Biofuel Crop.

Chinese tallow (Triadica sebifera) is a valuable oilseed-producing tree that can grow in a variety of conditions without competing for food production, and is a promising biofuel feedstock candidate. The fruits are unique in that they contain both saturated and unsaturated fat present in the tallow and seed layer, respectively. The tallow layer is poorly studied and is considered only as an external fatty deposition secreted from the seed. In this study we show that tallow is in fact a non-seed cellular tissue capable of triglyceride synthesis. Knowledge of lipid synthesis and storage mechanisms in tissues other than seed is limited but essential to generate oil-rich biomass crops. Here, we describe the annotated transcriptome assembly generated from the fruit coat, tallow and seed tissues of Chinese tallow. The final assembly was functionally annotated, allowing for the identification of candidate genes and reconstruction of lipid pathways. A tallow tissue-specific paralog for the transcription factor gene WRINKLED1 (WRI1) and lipid droplet-associated protein genes, distinct from those expressed in seed tissue, were found to be active in tallow, underpinning the mode of oil synthesis and packaging in this tissue. Our data have established an excellent knowledge base that can provide genetic and biochemical insights for engineering non-seed tissues to accumulate large amounts of oil. In addition to the large data set of annotated transcripts, the study also provides gene-based simple sequence repeat and single nucleotide polymorphism markers.

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.

Effect of cerulenin on fatty acid composition and gene expression pattern of DHA-producing strain Colwellia psychrerythraea strain 34H.

BACKGROUND: Colwellia psychrerythraea 34H is a psychrophilic bacterium able to produce docosahexaenoic acid (DHA). Polyketide synthase pathway is assumed to be responsible for DHA production in marine bacteria. RESULTS: Five pfa genes from strain 34H were confirmed to be responsible for DHA formation by heterogeneous expression in Escherichia coli. The complexity of fatty acid profile of this strain was revealed by GC and GC-MS. Treatment of cells with cerulenin resulted in significantly reduced level of C16 monounsaturated fatty acid (C16:1(Δ9t), C16:1(Δ7)). In contrast, the amount of saturated fatty acids (C10:0, C12:0, C14:0), hydroxyl fatty acids (3-OH C10:0 and 3-OH C12:0), as well as C20:4ω3, C20:5ω3 and C22:6ω3 were increased. RNA sequencing (RNA-Seq) revealed the altered gene expression pattern when C. psychrerythraea cells were treated with cerulenin. Genes involved in polyketide synthase pathway and fatty acid biosynthesis pathway were not obviously affected by cerulenin treatment. In contrast, several genes involved in fatty acid degradation or ß-oxidation pathway were dramatically reduced at the transcriptional level. CONCLUSIONS: Genes responsible for DHA formation in C. psychrerythraea was first cloned and characterized. We revealed the complexity of fatty acid profile in this DHA-producing strain. Cerulenin could substantially change the fatty acid composition by affecting the fatty acid degradation at transcriptional level. Acyl-CoA dehydrogenase gene family involved in the first step of ß-oxidation pathway may be important to the selectivity of degraded fatty acids. In addition, inhibition of FabB protein by cerulenin may lead to the accumulation of malonyl-CoA, which is the substrate for DHA formation.

Development of high oleic oil crop platform in flax through RNAi-mediated multiple FAD2 gene silencing.

KEY MESSAGE: Simultaneous gene silencing of both FAD2 genes in high linoleic acid flax leads to high level of oleic acid, which is stable across multiple generations. High oleic oil is one of the preferred traits in oil crop engineering due to its stability and multiple applications as an industrial feedstock. Flax possesses two isoforms of FAD2 enzymes that desaturate monounsaturated oleic acid to polyunsaturated linoleic acid. These two enzymes are encoded by two FAD2 genes. By simultaneous gene silencing both FAD2 genes in high linoleic acid flax, Linola, high level of oleic acid up to 80% was achieved in 69 silencing lines. The high oleic trait was stable across multiple generations with oleic acid reaching up to 77% in homozygote T3 progeny. The RNAi-mediated gene-silencing approach generated high oleic linseed oil, as well as a high oleic platform that can be exploited for further fatty acid engineering.

Diversity of Δ12 fatty acid desaturases in santalaceae and their role in production of seed oil acetylenic fatty acids.

Plants in the Santalaceae family, including the native cherry Exocarpos cupressiformis and sweet quandong Santalum acuminatum, accumulate ximenynic acid (trans-11-octadecen-9-ynoic acid) in their seed oil and conjugated polyacetylenic fatty acids in root tissue. Twelve full-length genes coding for microsomal Δ12 fatty acid desaturases (FADs) from the two Santalaceae species were identified by degenerate PCR. Phylogenetic analysis of the predicted amino acid sequences placed five Santalaceae FADs with Δ12 FADs, which include Arabidopsis thaliana FAD2. When expressed in yeast, the major activity of these genes was Δ12 desaturation of oleic acid, but unusual activities were also observed: i.e. Δ15 desaturation of linoleic acid as well as trans-Δ12 and trans-Δ11 desaturations of stearolic acid (9-octadecynoic acid). The trans-12-octadecen-9-ynoic acid product was also detected in quandong seed oil. The two other FAD groups (FADX and FADY) were present in both species; in a phylogenetic tree of microsomal FAD enzymes, FADX and FADY formed a unique clade, suggesting that are highly divergent. The FADX group enzymes had no detectable Δ12 FAD activity but instead catalyzed cis-Δ13 desaturation of stearolic acid when expressed in yeast. No products were detected for the FADY group when expressed recombinantly. Quantitative PCR analysis showed that the FADY genes were expressed in leaf rather than developing seed of the native cherry. FADs with promiscuous and unique activities have been identified in Santalaceae and explain the origin of some of the unusual lipids found in this plant family.