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Pennycress ( Thlaspi arvense L.) is being domesticated as an oilseed cash cover crop to be grown in the off-season throughout temperate regions of the world. With its diploid genome and ease of directed mutagenesis using molecular approaches, pennycress seed oil composition can be rapidly tailored for a plethora of food, feed, oleochemical and fuel uses. Here, we utilized Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology to produce knockout mutations in the FATTY ACID DESATURASE2 ( FAD2 ) and REDUCED OLEATE DESATURATION1 ( ROD1 ) genes to increase oleic acid content. High oleic acid (18:1) oil is valued for its oxidative stability that is superior to the polyunsaturated fatty acids (PUFAs) linoleic (18:2) and linolenic (18:3), and better cold flow properties than the very long chain fatty acid (VLCFA) erucic (22:1). When combined with a FATTY ACID ELONGATION1 ( fae1 ) knockout mutation, fad2 fae1 and rod1 fae1 double mutants produced ∼90% and ∼60% oleic acid in seed oil, respectively, with PUFAs in fad2 fae1 as well as fad2 single mutants reduced to less than 5%. MALDI-MS spatial imaging analyses of phosphatidylcholine (PC) and triacylglycerol (TAG) molecular species in wild-type pennycress embryo sections from mature seeds revealed that erucic acid is highly enriched in cotyledons which serve as storage organs, suggestive of a role in providing energy for the germinating seedling. In contrast, PUFA-containing TAGs are enriched in the embryonic axis, which may be utilized for cellular membrane expansion during seed germination and seedling emergence. Under standard growth chamber conditions, rod1 fae1 plants grew like wild type whereas fad2 single and fad2 fae1 double mutant plants exhibited delayed growth and overall reduced heights and seed yields, suggesting that reducing PUFAs below a threshold in pennycress had negative physiological effects. Taken together, our results suggest that combinatorial knockout of ROD1 and FAE1 may be a viable route to commercially increase oleic acid content in pennycress seed oil whereas mutations in FAD2 will likely require at least partial function to avoid fitness trade-offs.

Summary Thlapsi arvense L. (pennycress) is being developed as a profitable oilseed cover crop for the winter fallow period throughout the temperate regions of the world, controlling soil erosion and nutrients run‐off on otherwise barren farmland. We demonstrate that pennycress can serve as a user‐friendly model system akin to Arabidopsis that is well‐suited for both laboratory and field experimentation. We sequenced the diploid genome of the spring‐type Spring 32‐10 inbred line (1C DNA content of 539 Mb; 2n = 14), identifying variation that may explain phenotypic differences with winter‐type pennycress, as well as predominantly a one‐to‐one correspondence with Arabidopsis genes, which makes translational research straightforward. We developed an Agrobacterium‐mediated floral dip transformation method (0.5% transformation efficiency) and introduced CRISPR‐Cas9 constructs to produce indel mutations in the putative FATTY ACID ELONGATION1 (FAE1) gene, thereby abolishing erucic acid production and creating an edible seed oil comparable to that of canola. We also stably transformed pennycress with the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene, producing low‐viscosity acetyl‐triacylglycerol‐containing seed oil suitable as a diesel‐engine drop‐in fuel. Adoption of pennycress as a model system will accelerate oilseed‐crop translational research and facilitate pennycress’ rapid domestication to meet the growing sustainable food and fuel demands.

Background False flax, or gold-of-pleasure ( Camelina sativa ) is an oilseed that has received renewed research interest as a promising vegetable oil feedstock for liquid biofuel production and other non-food uses. This species has also emerged as a model for oilseed biotechnology research that aims to enhance seed oil content and fatty acid quality. To date, a number of genetic engineering and gene editing studies on C. sativa have been reported. Among the most common targets for this research are genes, encoding fatty acid desaturases, elongases, and diacylglycerol acyltransferases. However, the majority of these genes in C. sativa are present in multiple copies due to the allohexaploid nature of the species. Therefore, genetic manipulations require a comprehensive understanding of the diversity of such gene targets. Results Here we report the detailed analysis of FAD2 , FAD3 and FAE1 gene diversity in five Camelina species, including hexaploid C. sativa and four diploids, namely C. neglecta , C. laxa , C. hispida var. hispida and var. grandiflora . It was established that FAD2 , FAD3 and FAE1 homeologs in C. sativa retain very high conservancy, despite their allohexaploid inheritance. High sequence conservancy of the identified genes along with their different expression patterns in C. sativa suggest that subfunctionalization of these homeologs is mainly grounded on the transcriptional balancing between subgenomes. Finally, fatty acid composition of seed lipids in different Camelina species was characterized, suggesting potential variability in the activity of fatty acid elongation/desaturation pathways may vary among these taxa. Conclusion It was shown that the FAD2 , FAD3 and FAE1 genes retain high conservation, even in allohexaploid C. sativa after polyploidzation, in which the subfunctionalization of the described homeologs is mainly grounded on the expressional differences. The major differences in FA accumulation patterns within the seeds of different species were identified as well. These results provide a foundation for future precise gene editing, which would be based on targeting of particular FAD2 , FAD3 and FAE1 gene copies in C. sativa that allow regulating the dosage of the mentioned genes, thus shaping the desired FA composition in cultivated false flax.

Feedstocks for industrial applications ranging from polymers to lubricants are largely derived from petroleum, a non-renewable resource. Vegetable oils with fatty acid structures and storage forms tailored for specific industrial uses offer renewable and potentially sustainable sources of petrochemical-type functionalities. A wide array of industrial vegetable oils can be generated through biotechnology, but will likely require non-commodity oilseed platforms dedicated to specialty oil production for commercial acceptance. Here we show the feasibility of three Brassicaceae oilseeds crambe, camelina, and carinata, none of which are widely cultivated for food use, as hosts for complex metabolic engineering of wax esters for lubricant applications. Lines producing wax esters >20% of total seed oil were generated for each crop and further improved for high temperature oxidative stability by down-regulation of fatty acid polyunsaturation. Field cultivation of optimized wax ester-producing crambe demonstrated commercial utility of these engineered crops and a path for sustainable production of other industrial oils in dedicated specialty oilseeds.

Camelina neglecta is a new diploid Brassicaceae species, which has great research value because of its close relationship with the hexaploid oilseed crop Camelina sativa . Here, we report a chromosome-level assembly of C. neglecta with a total length of 210 Mb. By adopting PacBio sequencing and Hi-C technology, the C. neglecta genome was assembled into 6 chromosomes with scaffold N50 of 29.62 Mb. C. neglecta has undergone the whole-genome triplication (γ) shared among eudicots and two whole-genome duplications (α and β) shared by crucifers, but it has not undergone a specific whole-genome duplication event. By synteny analysis between C. neglecta and C. sativa , we successfully used the method of calculating Ks to distinguish the three subgenomes of C. sativa and determined that C. neglecta was closest to the first subgenome (SG1) of C. sativa . Further, transcriptomic analysis revealed the key genes associated with seed oil biosynthesis and its transcriptional regulation, including SAD , FAD2 , FAD3 , FAE1 , ABI3 , WRI1 and FUS3 displaying high expression levels in C. neglecta seeds . The high representability of C. neglecta as a model species for Camelina- based biotechnology research has been demonstrated for the first time. In particular, floral Agrobacterium tumefaciens infiltration-based transformation of C. neglecta , leading to overexpression of CvLPAT2 , CpDGAT1 and CvFatB1 transgenes, was demonstrated for medium-chain fatty acid accumulation in C. neglecta seed oil. This study provides an important genomic resource and establishes C. neglecta as a new model for oilseed biotechnology research.

Protein superfamilies can exhibit considerable diversification of function among their members in various organisms. The DJ-1 superfamily is composed of proteins that are principally involved in stress response and are widely distributed in all kingdoms of life. The model flowering plant Arabidopsis thaliana contains three close homologs of animal DJ-1, all of which are tandem duplications of the DJ-1 domain. Consequently, the plant DJ-1 homologs are likely pseudo-dimeric proteins composed of a single polypeptide chain. We report that one A. thaliana DJ-1 homolog (AtDJ1C) is the first DJ-1 homolog in any organism that is required for viability. Homozygous disruption of the AtDJ1C gene results in non-viable, albino seedlings that can be complemented by expression of wild-type or epitope-tagged AtDJ1C. The plastids from these dj1c plants lack thylakoid membranes and granal stacks, indicating that AtDJ1C is required for proper chloroplast development. AtDJ1C is expressed early in leaf development when chloroplasts mature, but is downregulated in older tissue, consistent with a proposed role in plastid development. In addition to its plant-specific function, AtDJ1C is an atypical member of the DJ-1 superfamily that lacks a conserved cysteine residue that is required for the functions of most other superfamily members. The essential role for AtDJ1C in chloroplast maturation expands the known functional diversity of the DJ-1 superfamily and provides the first evidence of a role for specialized DJ-1-like proteins in eukaryotic development.

Seeds of castor (Ricinus communis) are enriched in oil with high levels of the industrially valuable fatty acid ricinoleic acid (18:1OH), but production of this plant is limited because of the cooccurrence of the ricin toxin in its seeds. Lesquerella (Physaria fendleri) is being developed as an alternative industrial oilseed because its seeds accumulate lesquerolic acid (20:1OH), an elongated form of 18:1OH in seed oil which lacks toxins. Synthesis of 20:1OH is through elongation of 18:1OH by a lesquerella elongase, PfKCS18. Oleic acid (18:1) is the substrate for 18:1OH synthesis, but it is also used by fatty acid desaturase 2 (FAD2) and FAD3 to sequentially produce linoleic and linolenic acids. To develop lesquerella that produces 18:1OH-rich seed oils such as castor, RNA interference sequences targeting KCS18, FAD2 and FAD3 were introduced to lesquerella to suppress the elongation and desaturation steps. Seeds from transgenic lines had increased 18:1OH to 1.1–26.6% compared with that of 0.4–0.6% in wild-type (WT) seeds. Multiple lines had reduced 18:1OH levels in the T2 generation, including a top line with 18:1OH reduced from 26.7% to 19%. Transgenic lines also accumulated more 18:1 than that of WT, indicating that 18:1 is not efficiently used for 18:1OH synthesis and accumulation. Factors limiting 18:1OH accumulation and new targets for further increasing 18:1OH production are discussed. Our results provide insights into complex mechanisms of oil biosynthesis in lesquerella and show the biotechnological potential to tailor lesquerella seeds to produce castor-like industrial oil functionality.

Summary Biomass crops engineered to accumulate energy‐dense triacylglycerols (TAG or ‘vegetable oils’) in their vegetative tissues have emerged as potential feedstocks to meet the growing demand for renewable diesel and sustainable aviation fuel (SAF). Unlike oil palm and oilseed crops, the current commercial sources of TAG, vegetative tissues, such as leaves and stems, only transiently accumulate TAG. In this report, we used grain (Texas430 or TX430) and sugar‐accumulating ‘sweet’ (Ramada) genotypes of sorghum, a high‐yielding, environmentally resilient biomass crop, to accumulate TAG in leaves and stems. We initially tested several gene combinations for a ‘push‐pull‐protect’ strategy. The top TAG‐yielding constructs contained five oil transgenes for a sorghum WRINKLED1 transcription factor (‘push’), a Cuphea viscosissima diacylglycerol acyltransferase (DGAT; ‘pull’), a modified sesame oleosin (‘protect’) and two combinations of specialized Cuphea lysophosphatidic acid acyltransferases and medium‐chain acyl‐acyl carrier protein thioesterases. Though intended to generate oils with medium‐chain fatty acids, engineered lines accumulated oleic acid‐rich oil to amounts of up to 2.5% DW in leaves and 2.0% DW in stems in the greenhouse, 36‐fold and 49‐fold increases relative to wild‐type (WT) plants, respectively. Under field conditions, the top‐performing event accumulated TAG to amount to 5.5% DW in leaves and 3.5% DW in stems, 78‐fold and 58‐fold increases, respectively, relative to WT TX430. Transcriptomic and fluxomic analyses revealed potential bottlenecks for increased TAG accumulation. Overall, our studies highlight the utility of a lab‐to‐field pipeline coupled with systems biology studies to deliver high vegetative oil sorghum for SAF and renewable diesel production.

Summary Ketocarotenoids, including astaxanthin, are red lipophilic pigments derived from the oxygenation of β‐carotene ionone rings. These carotenoids have exceptional antioxidant capacity and high commercial value as natural pigments, especially for aquaculture feedstocks to confer red flesh colour to salmon and shrimp. Ketocarotenoid biosynthetic pathways occur only in selected bacterial, algal, fungal and plant species, which provide genetic resources for biotechnological ketocarotenoid production. Toward pathway optimization, we developed a transient platform for ketocarotenoid production using Agrobacterium infiltration of Nicotiana benthamiana leaves with plant (Adonis aestivalis) genes, carotenoid β‐ring 4‐dehydrogenase 2 (CBFD2) and carotenoid 4‐hydroxy‐β‐ring 4‐dehydrogenase (HBFD1), or bacterial (Brevundimonas) genes, β‐carotene ketolase (crtW) and β‐carotene hydroxylase (crtZ). In this test system, heterologous expression of the plant‐derived astaxanthin pathway conferred higher astaxanthin production with fewer ketocarotenoid intermediates than the bacterial pathway. We evaluated the plant‐derived pathway for ketocarotenoid production using the oilseed camelina (Camelina sativa) as a production platform. Genes for CBFD2 and HBFD1 and maize phytoene synthase were introduced under the control of seed‐specific promoters. In contrast to prior research with bacterial pathways, our strategy resulted in nearly complete conversion of β‐carotene to ketocarotenoids, including primarily astaxanthin. Tentative identities of other ketocarotenoids were established by chemical evaluation. Seeds from multi‐season US and UK field sites maximally accumulated ~135 μg/g seed weight of ketocarotenoids, including astaxanthin (~47 μg/g seed weight). Although plants had no observable growth reduction, seed size and oil content were reduced in astaxanthin‐producing lines. Oil extracted from ketocarotenoid‐accumulating seeds showed significantly enhanced oxidative stability and was useful for food oleogel applications.

Diacylglycerol acyltransferase (DGAT) catalyses the last step in acyl-CoA-dependent triacylglycerol (TAG) biosynthesis and is an important determinant of cellular oil content and quality. In this study, a gene, designated TaDGAT2, encoding a type 2 DGAT (DGAT2)-related enzyme was identified from the oleaginous marine protist Thraustochytrium aureum. The deduced TaDGAT2 sequence contains a ~460 amino acid domain most closely related to DGAT2s from Dictyostelium sp. (45–50% identity). Recombinant TaDGAT2 restored TAG biosynthesis to the Saccharomyces cerevisiae H1246 TAG-deficient mutant, and microsomes from the complemented mutant displayed DGAT activity with C16 and C18 saturated and unsaturated fatty acyl-CoA and diacylglycerol substrates. To examine its biotechnological potential, TaDGAT2 was expressed under control of a strong seed-specific promoter in wild-type Arabidopsis thaliana and the high linoleic acid fad3fae1 mutant. In both backgrounds, little change was detected in seed oil content, but a striking increase in oleic acid content of seeds was observed. This increase was greatest in fad3fae1 seeds, where relative amounts of oleic acid increased nearly 2-fold to >50% of total fatty acids. In addition, >2-fold increase in oleic acid levels was detected in the triacylglycerol sn-2 position and in the major seed phospholipid phosphatidylcholine. These results suggest that increased seed oleic acid content mediated by TaDGAT2 is influenced in part by the fatty acid composition of host cells and occurs not by enhancing oleic acid content at the TAG sn-3 position directly but by increasing total oleic acid levels in seeds, presumably by limiting flux through phosphatidylcholine-based desaturation reactions.