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 co‐expressing 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. Plants co‐expressing 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.     

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.     

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.     

Winter oilseed production for biofuel in the US Corn Belt: opportunities and limitations

Interest from the US commercial aviation industry and commitments established by the US Navy and Air Force to use renewable fuels has spurred interest in identifying and developing crops for renewable aviation fuel. Concern regarding greenhouse gas emissions associated with land‐use change and shifting land grown for food to feedstock production for fuel has encouraged the concept of intensifying current prominent cropping systems through various double cropping strategies. Camelina (Camelina sativa L.) and field pennycress (Thlaspi arvense L.) are two winter oilseed crops that could potentially be integrated into the corn (Zea mays L.)–soybean [(Glycine max (L.) Merr.] cropping system, which is the prominent cropping system in the US Corn Belt. In addition to providing a feedstock for renewable aviation fuel production, integrating these crops into corn–soybean cropping systems could also potentially provide a range of ecosystem services. Some of these include soil protection from wind and water erosion, soil organic C (SOC) sequestration, water quality improvement through nitrate reduction, and a food source for pollinators. However, integration of these crops into corn–soybean cropping systems also carries possible limitations, such as potential yield reductions of the subsequent soybean crop. This review identifies and discusses some of the key benefits and constraints of integrating camelina or field pennycress into corn–soybean cropping systems and identifies generalized areas for potential adoption in the US Corn Belt.     

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.     

Insight into the evolution and functional characteristics of the pan‐genome assembly from sesame landraces and modern cultivars

Sesame (Sesamum indicum L.) is an important oil crop renowned for its high oil content and quality. Recently, genome assemblies for five sesame varieties including two landraces (S. indicum cv. Baizhima and Mishuozhima) and three modern cultivars (S. indicum var. Zhongzhi13, Yuzhi11 and Swetha), have become available providing a rich resource for comparative genomic analyses and gene discovery. Here, we employed a reference‐assisted assembly approach to improve the draft assemblies of four of the sesame varieties. We then constructed a sesame pan‐genome of 554.05 Mb. The pan‐genome contained 26 472 orthologous gene clusters; 15 409 (58.21%) of them were core (present across all five sesame genomes), whereas the remaining 41.79% (11 063) clusters and the 15 890 variety‐specific genes were dispensable. Comparisons between varieties suggest that modern cultivars from China and India display significant genomic variation. The gene families unique to the sesame modern cultivars contain genes mainly related to yield and quality, while those unique to the landraces contain genes involved in environmental adaptation. Comparative evolutionary analysis indicates that several genes involved in plant‐pathogen interaction and lipid metabolism are under positive selection, which may be associated with sesame environmental adaption and selection for high seed oil content. This study of the sesame pan‐genome provides insights into the evolution and genomic characteristics of this important oilseed and constitutes a resource for further sesame 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.     

Using lipidomics to reveal details of lipid accumulation in developing Siberian apricot (Prunus sibirica L.) seed kernels

Siberian apricot seed kernel (SASK) contains a high of 50% oil with suitable fuel properties conformed to biodiesel standard. To date, Prunus sibirica is a novel non‐crop feedstock for biodiesel production in China. Here, oil contents and fatty acid (FA) compositions were identified in developing SASK from AS‐80 and AS‐84, at intervals of 1 week from 3 weeks after anthesis (WAA) to 9 weeks. The major differences in oil content between C18:1 and C18:2 levels were greater among the AS‐80 (32.69/15.48 g/100 g) than among the AS‐84 (25.78/13.15 g/100 g). Subsequently, the SASKs from 4, 6, and 8 WAA, respectively, representing early, middle, and late phases of oil accumulation, were selected as optimal samples for lipidomics analysis. It was notable that 18:1/18:1/18:2, 18:1/18:1/18:3, and 18:2/18:2/18:2 were the prominent compositions in triacylglycerol (TAG), and their higher content found among the AS‐80 was consistent with FA results. Although phosphatidic acid (PA) is directly connected with diacylglycerol (DAG) in Kennedy pathway, we found significant difference between PA and DAG compositions. The resulting molecular species differ in acyl composition depending on whether they were generated via phosphatidylcholine (PC) or Kennedy pathway. By qRT‐PCR analysis, the expression levels of FAD3, PDCT, and DAG‐CPT related to the biosynthesis of polyunsaturated fatty acids (PUFAs) showed a gradual decrease with SASK mature, explaining the drastic change of DAG‐18:3/18:3 content. Additionally, the lipidomics data coupled with qRT‐PCR analysis suggested that phospholipid:DAG acyltransferase may play a critical role in incorporation of PUFAs into sn‐3 of TAG. Our data contribute significantly to understand the underlying mechanisms of lipid accumulation in P. sibirica, and may also present strategies for engineering oil accumulation in oilseed plants.     

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.     

Extending the rapeseed gene pool with resynthesized <Emphasis Type="Italic">Brassica napus</Emphasis> II: Heterosis

Hybrid breeding relies on the combination of parents from two differing heterotic groups. However, the genetic diversity in adapted oilseed rape breeding material is rather limited. Therefore, the use of resynthesized Brassica napus as a distant gene pool was investigated. Hybrids were derived from crosses between 44 resynthesized lines with a diverse genetic background and two male sterile winter oilseed rape tester lines. The hybrids were evaluated together with their parents and check cultivars in 2 years and five locations in Germany. Yield, plant height, seed oil, and protein content were monitored, and genetic distances were estimated with molecular markers (127 polymorphic RFLP fragments). Resynthesized lines varied in yield between 40.9 dt/ha and 21.5 dt/ha, or between 85.1 and 44.6% of check cultivar yields. Relative to check cultivars, hybrids varied from 91.6 to 116.6% in yield and from 94.5 to 103.3% in seed oil content. Mid-parent heterosis varied from −3.5 to 47.2% for yield. The genetic distance of parental lines was not significantly correlated with heterosis or hybrid yield. Although resynthesized lines do not meet the elite rapeseed standards, they are a valuable source for hybrid breeding due to their large distance from present breeding material and their high heterosis when combined with European winter oilseed rape.     

Genetic transformation of the oilseed crop <Emphasis Type="Italic">Crambe abyssinica</Emphasis>

Engineering oilseed crops for industrial purposes requires a suitable crop that does not outcross to any food oilseed crop, thus eliminating problems of gene flow. Crambe abyssinica is such a dedicated crop as it does not hybridize with any existing food oilseed crops. However, lack of regeneration and transformation protocols has limited the use of C. abyssinica in genetic manipulation studies. In this study, efficient regeneration and transformation protocols for Crambe have been developed. Hypocotyls of C. abyssinica cv. Galactica were incubated on a Murashige and Skoog medium supplemented with various plant growth regulators (PGRs). Among the different PGR combinations tested, 10 μM thidiazuron and 2.7 μM α-naphthaleneacetic acid promoted highest frequency of regeneration, up to 60%. Among six Agrobacterium stains evaluated, each harbouring the cloning vector containing the neomycin phosphotransferase (nptII) and β-glucuronidase (gus) genes. EHA101 and AGL-1 yielded the highest transformation frequencies of 1.3 and 2.1%, respectively. Putative transgenic lines were recovered, and confirmed as transgenic by Southern blot analysis. Subsequently, Agrobacterium-mediated transformation of hypocotyls of cv. Galactica with constructs harbouring the wax synthase and fatty acid reductase genes have also successfully recovered confirmed transgenic plants carrying these transgenes.     

The coexpression of two desaturases provides an optimized reduction of saturates in camelina oil

Reducing the saturate content of vegetable oils is key to increasing their utility and adoption as a feedstock for the production of biofuels. Expression of either the FAT5 16 : 0-CoA desaturase from Caenorhabditis elegans, or an engineered cyanobacterial 16 : 0/18 : 0-glycerolipid desaturase, DES9*, in seeds of Arabidopsis (Arabidopsis thaliana) substantially lowered oil saturates. However, because pathway fluxes and regulation of oil synthesis are known to differ across species, translating this transgene technology from the model plant to crop species requires additional investigation. In the work reported here, we found that high expression of FAT5 in seeds of camelina (Camelina sativa) provided only a moderate decrease in saturates, from 12.9% of total oil fatty acids in untransformed controls to 8.6%. Expression of DES9* reduced saturates to 4.6%, but compromised seed physiology and oil content. However, the coexpression of the two desaturases together cooperatively reduced saturates to only 4.0%, less than one-third of the level in the parental line, without compromising oil yield or seedling germination and establishment. Our successful lowering of oil saturates in camelina identifies strategies that can now be integrated with genetic engineering approaches that reduce polyunsaturates to provide optimized oil composition for biofuels in camelina and other oil seed crops.     

Constitutive or seed‐specific overexpression of Arabidopsis G‐protein γ subunit 3 (AGG3) results in increased seed and oil production and improved stress tolerance in Camelina sativa

Heterotrimeric G‐proteins consisting of Gα, Gβ and Gγ subunits play an integral role in mediating multiple signalling pathways in plants. A novel, recently identified plant‐specific Gγ protein, AGG3, has been proposed to be an important regulator of organ size and mediator of stress responses in Arabidopsis, whereas its potential homologs in rice are major quantitative trait loci for seed size and panicle branching. To evaluate the role of AGG3 towards seed and oil yield improvement, the gene was overexpressed in Camelina sativa, an oilseed crop of the Brassicaceae family. Analysis of multiple homozygous T4 transgenic Camelina lines showed that constitutive overexpression of AGG3 resulted in faster vegetative as well as reproductive growth accompanied by an increase in photosynthetic efficiency. Moreover, when expressed constitutively or specifically in seed tissue, AGG3 was found to increase seed size, seed mass and seed number per plant by 15%–40%, effectively resulting in significantly higher oil yield per plant. AGG3 overexpressing Camelina plants also exhibited improved stress tolerance. These observations draw a strong link between the roles of AGG3 in regulating two critical yield parameters, seed traits and plant stress responses, and reveal an effective biotechnological tool to dramatically increase yield in agricultural crops.     

Pollinators and pollination of oilseed rape crops (Brassica napus L.) in Ireland: ecological and economic incentives for pollinator conservation

Pollinators are beneficial for many wild and crop plants. As a mass-flowering crop, oilseed rape has received much focus in terms of its pollination requirements but despite a threefold increase in area of cultivation of this crop in Ireland over the past 5 years, little is known about its pollination here. We surveyed the flower visiting insects found in commercial winter oilseed rape fields and evaluated the importance of different pollinator groups, investigated the contribution of insect pollination to oilseed rape seed production, and estimated the economic value of insect pollination to the crop at a national level. Our data showed that winter oilseed rape is visited by a wide variety of insect species, including the honeybee, bumblebees, solitary bees, and hoverflies. The honeybee, Eristalis hoverflies and bumblebees (especially Bombus sensu stricto and B. lapidarius) were the best pollinators of winter oilseed rape based on the number of pollen grains they carry, visitation rates per flower and their relative abundance per field. Exclusion of pollinators resulted in a 27 % decrease in the number of seeds produced, and a 30 % decrease in seed weight per pod in winter crops, with comparable values from a spring oilseed rape field also. The economic value of insect pollination to winter oilseed rape was estimated as €2.6 million per annum, while the contribution to spring oilseed rape was €1.3 million, resulting in an overall value of €3.9 million per annum. We can suggest the appropriate conservation and management of both honeybees and wild pollinators in agricultural areas to ensure continued provision of pollination services to oilseed rape, as a decrease in insect numbers has the potential to negatively influence crop yields.