Core Ideas Carinata growth and yield was influenced more by row spacing than seeding rate. Rows spaced at 36 cm maximized carinata yield. Carinata branching was favored by wider row spacing. Carinata (Brassica carinata A. Braun) is an oilseed crop with potential as a winter crop in the southeastern United States to diversify crop rotations and provide biofuel production and livestock feed. The objective was to evaluate the effects of row spacing and seeding rate on carinata yield and oil composition. Field experiments were conducted in Jay and Quincy, FL, from 2013 to 2016 evaluating carinata growth, seed and oil yield, and oil composition grown in a factorial arrangement of four seeding rates (3, 6, 9, and 12 kg ha−1) and four row spacings (18, 36, 53, and 89 cm). No interactions between seeding rate and row spacing were detected. Seeding rate did not influence any of the variables studied. In contrast, row spacing affected seed and oil yield, branch production, and pods per plant. Seed yield (ranked from highest to lowest) was 2761, 2286, 1851, and 1572 kg ha−1 for rows spaced at 36, 18, 53, and 89 cm, respectively. Branching and pods per plant increased with row spacing. Neither seeding rate nor row spacing affected oil concentration and quality. Oil concentration averaged 40%, of which more than a third was erucic acid (C22:1). Protein concentration was 31%, and glucosinolate concentration was 93 μmol g−1. The results of the present study demonstrated that carinata can be successfully grown in the southeastern United States, reaching yields and oil quality similar to those reported at other latitudes, and can be a source of biofuel, protein for animal feed, and cropping system diversification for growers.

Background The Arabidopsis thaliana dgat1 mutant, AS11 , has an oil content which is decreased by 30%, and a strongly increased ratio of 18:3/20:1, compared to wild type. Despite lacking a functional DGAT1, AS11 still manages to make 70% of WT seed oil levels. Recently, it was demonstrated that in the absence of DGAT1 , PDAT1 was essential for normal seed development, and is a dominant determinant in Arabidopsis TAG biosynthesis. Methods Biochemical, metabolic and gene expression studies combined with genetic crossing of selected Arabidopsis mutants have been carried out to demonstrate the contribution of Arabidopsis PDAT1 and LPCAT2 in the absence of DGAT1 activity. Results Through microarray and RT-PCR gene expression analyses of AS11 vs. WT mid-developing siliques, we observed consistent trends between the two methods. FAD2 and FAD3 were up-regulated and FAE1 down-regulated, consistent with the AS11 acyl phenotype. PDAT1 expression was up-regulated by ca 65% while PDAT2 expression was up-regulated only 15%, reinforcing the dominant role of PDAT1 in AS11 TAG biosynthesis. The expression of LPCAT2 was up-regulated by 50-75%, while LPCAT1 expression was not significantly affected. In vitro LPCAT activity was enhanced by 75-125% in microsomal protein preparations from mid-developing AS11 seed vs WT. Co-incident homozygous knockout lines of dgat1 / lpcat2 exhibited severe penalties on TAG biosynthesis, delayed plant development and seed set, even with a functional PDAT1; the double mutant dgat1/lpcat1 showed only marginally lower oil content than AS11 . Conclusions Collectively, the data strongly support that in AS11 it is LPCAT2 up-regulation which is primarily responsible for assisting in PDAT1-catalyzed TAG biosynthesis, maintaining a supply of PC as co-substrate to transfer sn -2 moieties to the sn -3 position of the enlarged AS11 DAG pool.

The cloning and characterization of a lyso-phosphatidic acid acyltransferase (LPAT2; EC 2.3.1.51)from Tropaeolum majus from a 20,000 EST collection is described.The 1358 bp TmLPAT2 gene encodes a 42.6 kD polypeptide; the primary sequence has a membrane bound O-acyltransferase, MBOAT, Box I motif, and Boxes II, III and IV typical of LPATs from various species.Unlike many LPAT2s, the gene was constitutively expressed in all tissues.The TmLPAT2 functionality was confirmed by expression in a yeast LPAT deletion (SLC1 -) mutant.The TmLPAT2 could use a range of acyl-CoAs as acyl donor, including 22:1-CoA and 20:1-CoA and either 18:1-LPA or 22:1-LPA as acyl acceptor.This new LPAT2 could enable the production of Brassica seed oils with enhanced levels of very long-chain fatty acids.

Crop development and species diversity are important aspects of the emerging global bioeconomy, as is maximizing crop value through total crop utilization. We advocate development of Brassica carinata as a biorefinery and bioindustrial oils platform using traditional and molecular breeding techniques and tools. We review genetic studies and breeding efforts to develop elite B. carinata germplasm, work involving development of transformation and regeneration protocols, target gene isolation, and transgene expression. Genetic modification strategies using a B. carinata breeding line as a delivery platform for very long‐chain fatty acid‐enhanced/modified oils are presented as case studies. The target oil products are erucic acid (22:1 Δ13), docosadienoic acid (22:2 Δ5, Δ13) and nervonic acid (24:1 Δ15); in addition transgenic efforts to enhance B. carinata seed oil content are discussed. The overall advantages and current limitations to utilizing this crop are delineated. Other anticipated biobased products from a B. carinata platform may include, but are not limited to, the production of biolubricants, biofuels and biopolymers from the oil, biopesticides, antioxidants, as well as plant gums, and vegetable protein‐based bioplastics and novel food and feed products. In summation, this collaborative B. carinata breeding/germplasm development/value‐added molecular modification effort will not only contribute to the development of renewable feedstocks for the emerging Canadian bioeconomy (biorefinery/bioproducts), but also promises to generate positive economic and environmental benefits. Published in 2010 by John Wiley & Sons, Ltd.

Nervonic acid 24:1 Delta15 (cis-tetracos-15-enoic acid) is a very long-chain monounsaturated fatty acid and exists in nature as an elongation product of oleic acid. There is an increasing interest in production of high nervonic acid oils for pharmaceutical, nutraceutical and industrial applications. Using a polymerase chain reaction approach, we have isolated a gene from Cardamine graeca L., which encodes a 3-ketoacyl-CoA synthase (KCS), the first component of the elongation complex involved in synthesis of nervonic acid. Expression of the Cardamine KCS in yeast resulted in biosynthesis of nervonic acid, which is not normally present in yeast cells. We transformed Arabidopsis and Brassica carinata with the Cardamine KCS under the control of the seed-specific promoter, napin. The T(3) generations of transgenic Arabidopsis and B. carinata plants expressing the Cardamine KCS showed that seed-specific expression resulted in relatively large comparative increases in nervonic acid proportions in Arabidopsis seed oil, and 15-fold increase in nervonic acid proportions in B. carinata seed oil. The highest nervonic acid level in transgenic B. carinata lines reached 44%, with only 6% of residual erucic acid. In contrast, similar transgenic expression of the Cardamine KCS in high erucic B. napus resulted in 30% nervonic acid but with 20% residual erucic acid. Experiments using the Lunaria KCS gene gave results similar to the latter. In both cases, the erucic acid content is too high for human or animal consumption. Thus, the Cardamine KCS: B. carinata high nervonic/highly reduced erucic transgenic seed oils will be the most suitable for testing in pharmaceutical/nutraceutical applications to improve human and animal health.

The 3′-UTR of the FAD2 gene from Brassica carinata was cloned by PCR and used to prepare an intron-spliced hairpin RNA (ihpRNA) construct. Compared to that of the wild type (WT) background, this construct, when expressed in B. carinata, resulted in a high degree of FAD2 gene silencing accompanied by strong increases of up to 16 and 10% in oleic acid and erucic acid proportions, respectively. The increase in 18:1 was accompanied by a concomitant proportional reduction in 18:2. A second construct containing ihpRNA targeted to the endogenous FAD2 gene in addition to the heterologous Crambe abyssinica FAE gene under the control of seed specific napin promoter, was used to transform B. carinata. This approach resulted in an even greater increase in erucic acid proportions, by up to 16% in T1 segregating seeds as compared to that of the WT control. To our knowledge, this is currently the highest accumulation of erucic acid achieved in B. carinata seeds using transgenic approaches, making it an increasingly-attractive alternative to high erucic B. napus cultivars as an industrial oil crop.

Among the GFPs used for imaging green fluorescence, the Emerald version has been considered the best GFP to use but there is no formal report on its construction or the relevance of the amino acid (aa) substitutions in it relative to the commonly used GFPs. Here, we have shown that a version of Emerald makes Escherichia coli host cells visibly green even under dim room light conditions. Exploiting this feature, we have determined for the first time whether the changes in the structure of Emerald protein brought about by the aa substitutions are all indeed essential for brightness. F64L and S72A accompanying the classical S65T substitution on the chromophore-bearing helix are essential. Two amino acid changes, one on the surface (N149K) of the beta barrel that encases the helix and the other (I167T) near the chromophore enhance the visible green colour individually and additively when present together. The other two substitutions, M153T (on the surface) and H231L (on the surface), do not contribute to the visible green phenotype, even though in earlier studies M153T has been reported to enhance GFP fluorescence. The GFP version with F64L-S65T-S72A-N149K-I167T is referred to as VisGreen. We found VisGreen and Emerald to be indistinguishable in their quantum yield, molar extinction coefficient, folding efficiency, or photosensitivity. VisGreen rendered bacterial, plant, and animal cells highly fluorescent. Interestingly, N149K in the above combination was not essential to render bacterial cells highly fluorescent.