Summary: High biomass crops have recently attracted significant attention as an alternative platform for the renewable production of high energy storage lipids such as triacylglycerol (TAG). While TAG typically accumulates in seeds as storage compounds fuelling subsequent germination, levels in vegetative tissues are generally low. Here, we report the accumulation of more than 15% TAG (17.7% total lipids) by dry weight in Nicotiana tabacum (tobacco) leaves by the co‐expression of three genes involved in different aspects of TAG production without severely impacting plant development. These yields far exceed the levels found in wild‐type leaf tissue as well as previously reported engineered TAG yields in vegetative tissues of Arabidopsis thaliana and N. tabacum. When translated to a high biomass crop, the current levels would translate to an oil yield per hectare that exceeds those of most cultivated oilseed crops. Confocal fluorescence microscopy and mass spectrometry imaging confirmed the accumulation of TAG within leaf mesophyll cells. In addition, we explored the applicability of several existing oil‐processing methods using fresh leaf tissue. Our results demonstrate the technical feasibility of a vegetative plant oil production platform and provide for a step change in the bioenergy landscape, opening new prospects for sustainable food, high energy forage, biofuel and biomaterial applications.

We have genetically modified the fatty acid composition of cottonseed oil using the recently developed technique of hairpin RNA-mediated gene silencing to down-regulate the seed expression of two key fatty acid desaturase genes, ghSAD-1-encoding stearoyl-acyl-carrier protein Δ9-desaturase andghFAD2-1-encoding oleoyl-phosphatidylcholine ω6-desaturase. Hairpin RNA-encoding gene constructs (HP) targeted against either ghSAD-1 or ghFAD2-1 were transformed into cotton (Gossypium hirsutum cv Coker 315). The resulting down-regulation of the ghSAD-1 gene substantially increased stearic acid from the normal levels of 2% to 3% up to as high as 40%, and silencing of the ghFAD2-1gene resulted in greatly elevated oleic acid content, up to 77% compared with about 15% in seeds of untransformed plants. In addition, palmitic acid was significantly lowered in both high-stearic and high-oleic lines. Similar fatty acid composition phenotypes were also achieved by transformation with conventional antisense constructs targeted against the same genes, but at much lower frequencies than were achieved with the HP constructs. By intercrossing the high-stearic and high-oleic genotypes, it was possible to simultaneously down-regulate both ghSAD-1 and ghFAD2-1to the same degree as observed in the individually silenced parental lines, demonstrating for the first time, to our knowledge, that duplex RNA-induced posttranslational gene silencing in independent genes can be stacked without any diminution in the degree of silencing. The silencing of ghSAD-1 and/or ghFAD2-1 to various degrees enables the development of cottonseed oils having novel combinations of palmitic, stearic, oleic, and linoleic contents that can be used in margarines and deep frying without hydrogenation and also potentially in high-value confectionery applications.

As a resource allocation strategy, plant growth and defense responses are generally mutually antagonistic. Brassinosteroid (BR) regulates many aspects of plant development and stress responses, however, genetic evidence of its integrated effects on plant growth and stress tolerance is lacking. We overexpressed the Arabidopsis BR biosynthetic gene AtDWF4 in the oilseed plant Brassica napus and scored growth and stress response phenotypes. The transgenic B. napus plants, in comparison to wild type, displayed increased seed yield leading to increased overall oil content per plant, higher root biomass and root length, significantly better tolerance to dehydration and heat stress and enhanced resistance to necrotrophic fungal pathogens Leptosphaeria maculans and Sclerotinia sclerotiorum. Transcriptome analysis supported the integrated effects of BR on growth and stress responses; in addition to BR responses associated with growth, a predominant plant defense signature, likely mediated by BES1/BZR1, was evident in the transgenic plants. These results establish that BR can interactively and simultaneously enhance abiotic and biotic stress tolerance and plant productivity. The ability to confer pleiotropic beneficial effects that are associated with different agronomic traits suggests that BR–related genes may be important targets for simultaneously increasing plant productivity and performance under stress conditions.

Understanding the fundamental nature of a molecular process or a biological pathway is often a catalyst for the development of new technologies in biology. Indeed, studies from late 1990s to early 2000s have uncovered multiple overlapping but functionally distinct RNA silencing pathways in plants, including the posttranscriptional microRNA and small interfering RNA pathways and the transcriptional RNA-directed DNA methylation pathway. These findings have in turn been exploited for developing artificial RNA silencing technologies such as hairpin RNA, artificial microRNA, intrinsic direct repeat, 3’ UTR inverted repeat, artificial trans-acting siRNA, and virus-induced gene silencing technologies. Some of theseRNA silencing technologies, such as the hairpin RNA technology, have already been widely used for genetic improvement of crop plants in agriculture. For horticultural plants, RNA silencing technologies have been used to increase disease and pest resistance, alter plant architecture and flowering time, improve commercial traits of fruits and flowers, enhance nutritional values, remove toxic compounds and allergens, and develop high-value industrial products. In this article we aim to provide an overview of the RNA silencing pathways in plants, summarize the existing RNA silencing technologies, and review the current progress in applying these technologies for the improvement of agricultural crops particularly horticultural crops.

Omega-3 long-chain (≥C20) polyunsaturated fatty acids (ω3 LC-PUFA) such as eicosapentaenoic acid (EPA) and docosapentaenoic acid (DHA) are critical for human health and development. Numerous studies have indicated that deficiencies in these fatty acids can increase the risk or severity of cardiovascular, inflammatory and other diseases or disorders. EPA and DHA are predominantly sourced from marine fish although the primary producers are microalgae. Much work has been done to engineer a sustainable land-based source of EPA and DHA to reduce pressure on fish stocks in meeting future demand, with previous studies describing the production of fish oil-like levels of DHA in the model plant species, Arabidopsis thaliana. In this study we describe the production of fish oil-like levels (>12%) of DHA in the oilseed crop species Camelina sativa achieving a high ω3/ω6 ratio. The construct previously transformed in Arabidopsis as well as two modified construct versions designed to increase DHA production were used. DHA was found to be stable to at least the T₅ generation and the EPA and DHA were found to be predominantly at the sn-1,3 positions of triacylglycerols. Transgenic and parental lines did not have different germination or seedling establishment rates. DHA can be produced at fish oil-like levels in industrially-relevant oilseed crop species using multi-gene construct designs which are stable over multiple generations. This study has implications for the future of sustainable EPA and DHA production from land-based sources.