Tammy Francis

Cloning and characterization of an acyl‐CoA‐dependent diacylglycerol acyltransferase 1 (DGAT1) gene from Tropaeolum majus, and a study of the functional motifs of the DGAT protein using site‐directed mutagenesis to modify enzyme activity and oil content

SUMMARY A full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) was obtained from Tropaeolum majus (garden nasturtium). The 1557-bp open reading frame of this cDNA, designated TmDGAT1, encodes a protein of 518 amino acids showing high homology to other plant DGAT1s. The TmDGAT1 gene was expressed exclusively in developing seeds. Expression of recombinant TmDGAT1 in the yeast H1246MATalpha quadruple mutant (DGA1, LRO1, ARE1, ARE2) restored the capability of the mutant host to produce triacylglycerols (TAGs). The recombinant TmDGAT1 protein was capable of utilizing a range of (14)C-labelled fatty acyl-CoA donors and diacylglycerol acceptors, and could synthesize (14)C-trierucin. Collectively, these findings confirm that the TmDGAT1 gene encodes an acyl-CoA-dependent DGAT1. In plant transformation studies, seed-specific expression of TmDGAT1 was able to complement the low TAG/unusual fatty acid phenotype of the Arabidopsis AS11 (DGAT1) mutant. Over-expression of TmDGAT1 in wild-type Arabidopsis and high-erucic-acid rapeseed (HEAR) and canola Brassica napus resulted in an increase in oil content (3.5%-10% on a dry weight basis, or a net increase of 11%-30%). Site-directed mutagenesis was conducted on six putative functional regions/motifs of the TmDGAT1 enzyme. Mutagenesis of a serine residue in a putative SnRK1 target site resulted in a 38%-80% increase in DGAT1 activity, and over-expression of the mutated TmDGAT1 in Arabidopsis resulted in a 20%-50% increase in oil content on a per seed basis. Thus, alteration of this putative serine/threonine protein kinase site can be exploited to enhance DGAT1 activity, and expression of mutated DGAT1 can be used to enhance oil content.

Triacylglycerol synthesis by PDAT1 in the absence of DGAT1 activity is dependent on re-acylation of LPC by LPCAT2

BackgroundThe 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.MethodsBiochemical, 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.ResultsThrough 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.ConclusionsCollectively, 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.

Molecular cloning and characterization of a KCS gene from Cardamine graeca and its heterologous expression in Brassica oilseeds to engineer high nervonic acid oils for potential medical and industrial use.

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.

Hairpin-RNA mediated silencing of endogenous FAD2 gene combined with heterologous expression of Crambe abyssinica FAE gene causes an increase in the level of erucic acid in transgenic Brassica carinata seeds

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.

Cloning and Characterization of a Constitutive Lysophosphatidic Acid Acyltransferase 2 (LPAT2) Gene from Tropaeolum majus L

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 polypep- tide; 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.

Genetic characterization of type II Fusarium head blight resistance derived from transgressive segregation in a cross between Eastern and Western Canadian spring wheat

Fusarium head blight (FHB) caused by Fusarium graminearum is a devastating disease affecting global wheat production, causing significant losses to yield and grain quality. The Eastern Canadian line FL62R1 was developed using a systemic breeding approach and boasts high levels of FHB resistance with good yield, desirable agronomics, and end-use quality traits. The objective of this study was to identify genetic determinants of type II resistance in a cross between FL62R1 and cv. Stettler, a Canada Western Red Spring variety rated moderately susceptible to FHB. Although neither parent displayed strong resistance to FHB spread within spikes following point inoculation (type II resistance) in greenhouses, strong type II resistance was observed in a large number of progeny, including 6% with resistance comparable to the best check line, Sumai 3. Quantitative trait locus (QTL) mapping identified a locus from chromosome 2BL of Stettler which provides Sumai 3 level type II resistance when combined with favorable FHB resistance QTLs with minor effects from FL62R1. This study provides insight into transgressive segregation for FHB, which despite its importance is poorly understood, rare, and difficult to predict. It also begins to dissect the genetic architecture of FHB resistance in wheat derived from the systemic breeding approach.