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Summary: The Brassicaceae Camelina sativa (gold of pleasure) is now an established niche crop and being used as a transgenic host for a range of novel seed traits. Most notable of these is the accumulation of omega‐3 long‐chain polyunsaturates such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), fatty acids normally only found in marine organisms. As part of continued efforts to optimize the accumulation of these non‐native fatty acids via seed‐specific expression of algal genes, a new series of iterative constructs was built and introduced into Camelina. Seed fatty acid composition was determined, and the presence of EPA and DHA was confirmed. To provide an additional level of evaluation, full environmental release was carried out on selected events, providing a real‐world gauntlet against which to assess the performance of these novel lines. Composition of the seed oil triacylglycerol was determined by mass spectrometry, allowing for conclusions as to the contribution of different activities to the final accumulation of EPA and DHA. Since these data were derived from field‐grown material, they also represent a robust demonstration of the stability of the omega‐3 LC‐PUFA trait in Camelina. We propose that field trialling should be routinely incorporated in the plant synthetic biology 'design–build–test–learn' cycle. [ABSTRACT FROM AUTHOR]

Yellow seed is a desirable trait with great potential for improving seed quality in Brassica crops. Unfortunately, no natural or induced yellow seed germplasms have been found in Brassica napus, an important oil crop, which likely reflects its genome complexity and the difficulty of the simultaneous random mutagenesis of multiple gene copies with functional redundancy. Here, we demonstrate the first application of CRISPR/Cas9 for creating yellow‐seeded mutants in rapeseed. The targeted mutations of the BnTT8 gene were stably transmitted to successive generations, and a range of homozygous mutants with loss‐of‐function alleles of the target genes were obtained for phenotyping. The yellow‐seeded phenotype could be recovered only in targeted mutants of both BnTT8 functional copies, indicating that the redundant roles of BnA09.TT8 and BnC09.TT8b are vital for seed colour. The BnTT8 double mutants produced seeds with elevated seed oil and protein content and altered fatty acid (FA) composition without any serious defects in the yield‐related traits, making it a valuable resource for rapeseed breeding programmes. Chemical staining and histological analysis showed that the targeted mutations of BnTT8 completely blocked the proanthocyanidin (PA)‐specific deposition in the seed coat. Further, transcriptomic profiling revealed that the targeted mutations of BnTT8 resulted in the broad suppression of phenylpropanoid/flavonoid biosynthesis genes, which indicated a much more complex molecular mechanism underlying seed colour formation in rapeseed than in Arabidopsis and other Brassica species. In addition, gene expression analysis revealed the possible mechanism through which BnTT8 altered the oil content and fatty acid composition in seeds. [ABSTRACT FROM AUTHOR]

Summary: Modified fatty acids (mFA) have diverse uses; for example, cyclopropane fatty acids (CPA) are feedstocks for producing coatings, lubricants, plastics and cosmetics. The expression of mFA‐producing enzymes in crop and model plants generally results in lower levels of mFA accumulation than in their natural‐occurring source plants. Thus, to further our understanding of metabolic bottlenecks that limit mFA accumulation, we generated transgenic Camelina sativa lines co‐expressing Escherichia coli cyclopropane synthase (EcCPS) and Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT). In contrast to transgenic CPA‐accumulating Arabidopsis, CPA accumulation in camelina caused only minor changes in seed weight, germination rate, oil accumulation and seedling development. CPA accumulated to much higher levels in membrane than storage lipids, comprising more than 60% of total fatty acid in both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) versus 26% in diacylglycerol (DAG) and 12% in triacylglycerol (TAG) indicating bottlenecks in the transfer of CPA from PC to DAG and from DAG to TAG. Upon co‐expression of SfLPAT with EcCPS, di‐CPA‐PC increased by ~50% relative to lines expressing EcCPS alone with the di‐CPA‐PC primarily observed in the embryonic axis and mono‐CPA‐PC primarily in cotyledon tissue. EcCPS‐SfLPAT lines revealed a redistribution of CPA from the sn‐1 to sn‐2 positions within PC and PE that was associated with a doubling of CPA accumulation in both DAG and TAG. The identification of metabolic bottlenecks in acyl transfer between site of synthesis (phospholipids) and deposition in storage oils (TAGs) lays the foundation for the optimizing CPA accumulation through directed engineering of oil synthesis in target crops. [ABSTRACT FROM AUTHOR]