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Antoni R. Slabas - One of the best experts on this subject based on the ideXlab platform.
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Limnanthes douglasii lysophosphatidic acid acyltransferases: immunological quantification, acyl selectivity and functional replacement of the Escherichia coli plsC gene.
Biochemical Journal, 2002Co-Authors: Adrian P. Brown, Simon Carnaby, Clare L. Brough, Melissa Brazier, Antoni R. SlabasAbstract:Antibodies were raised against the two membrane-bound lysophosphatidic acid acyltransferase (LPAAT) enzymes from Limnanthes douglasii (meadowfoam), LAT1 and LAT2, using the predicted soluble portion of each protein as recombinant protein antigens. The antibodies can distinguish between the two acyltransferase proteins and demonstrate that both migrate in an anomalous fashion on SDS/PAGE gels. The antibodies were used to determine that LAT1 is present in both leaf and developing seeds, whereas LAT2 is only detectable in developing seeds later than 22 daf (days after flowering). Both proteins were found exclusively in microsomal fractions and their amount was determined using the recombinant antigens as quantification standards. LAT1 is present at a level of 27 pg/microg of membrane protein in leaf tissue and
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Limnanthes douglasii erucic acid specific lysophospatidic acid acyltransferase activity in oilseed rape an analysis of biochemical effects
Biochemical Society Transactions, 2000Co-Authors: J A Wilmer, Adrian P. Brown, Simon Carnaby, Tina L. Barsby, K Forsyth, Antoni R. SlabasAbstract:It has been shown previously that erucic acid accumulates in the sn -2 position of triglycerides after introduction of a Limnanthes lysophosphatidic acid acyltransferase. However, in none of the studies to date has a proper evaluation been made of the relationship between transgene expression and the erucic acid accumulation at sn -2. One of the necessary tools to study the variability is the presence of a specific antibody raised against the introduced acyltransferase. In the present study, we have looked at the correlation between erucic acid accumulation at sn -2, production of trierucin and the expression of the transgene, as detected by Western blotting. The data presented include comparisons of progeny of individual lines grown under various conditions and at various times, and an analysis of a number of lines during development. We also present calculations that could indicate from which position the erucic acid found at position sn -2 is derived.
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Engineering Trierucin into Oilseed Rape by the Introduction of a 1-Acyl-sn-Glycerol-3-Phosphate Acyltransferase from Limnanthes Douglasii
Physiology Biochemistry and Molecular Biology of Plant Lipids, 1997Co-Authors: Clare L. Brough, Johan T.m. Kroon, Jane M. Coventry, William W. Christie, Tina L. Barsby, Antoni R. SlabasAbstract:Currently available cultivars of high erucic acid rape (HEAR) have a theoretical maximum of 66% erucic acid (22:1, Δ13) in their seed oil due to the specificity of the membrane-bound 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPA-AT) enzyme. In HEAR the LPA-AT does not incorporate erucic acid at the s n-2 position of triacylglycerols (TAG) but preferentially incorporates oleic acid (18:1, Δ9), even if this is only a minor component of the total fatty acid pool (1). However, some plant species, e.g Limnanthes, can utilise erucoyl-CoA as a substrate and effectively incorporate erucic acid at the sn-2 position (2, 3).
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towards the genetic engineering of triacylglycerols of defined fatty acid composition major changes in erucic acid content at the sn 2 position affected by the introduction of a 1 acyl sn glycerol 3 phosphate acyltransferase from Limnanthes douglasii
Molecular Breeding, 1996Co-Authors: Clare L. Brough, Adrian P. Brown, Johan T.m. Kroon, Jane M. Coventry, William W. Christie, Tina L. Barsby, Antoni R. SlabasAbstract:A cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii was introduced into oil seed rape (Brassica napus) under the control of a napin promoter. Seed triacylglycerols from transgenic plants were analysed by reversed-phase HPLC and trierucin was detected at a level of 0.4% and 2.8% in two transgenic plants but was not found in untransformed rape seed. Total fatty acid composition analysis of seeds from these selected plants revealed that the erucic acid content was no higher than the maximum found in the starting population. Analysis of fatty acids at the sn-2 position showed no erucic acid in untransformed rape but in the selected transgenic plants 9% (mol/mol) and 28.3% (mol/mol) erucic acid was present. These results conclusively demonstrate that the gene from L. douglasii encodes a 1-acyl-sn-glycerol-3-phosphate acyltransferase which can function in rape and incorporate erucic acid at the sn-2 position of triacylglycerols in seed. Additional modifications may further increase levels of trierucin.
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identification of a cdna that encodes a 1 acyl sn glycerol 3 phosphate acyltransferase from Limnanthes douglasii
Plant Molecular Biology, 1995Co-Authors: Adrian P. Brown, Clare L. Brough, Johan T.m. Kroon, Antoni R. SlabasAbstract:Two different techniques were used to isolate potential cDNAs for acyl-CoA: 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPA-AT) enzymes from Limnanthes douglasii. Both heterologous screening with the maize pMAT1 clone and in vivo complementation of the Escherchia coli mutant JC201 which is deficient in LPA-AT activity, were carried out. Clones identified by these procedures were different. Homology searches demonstrated that the clone isolated by heterologous probing, pLAT1, encodes a protein which is most similar to the maize (open reading frame in pMAT1) and yeast SLC1 proteins, which are putative LPA-AT sequences. This L. douglasii sequence shows much lower homology to the E. coli LPA-AT protein PlsC, which is the only LPA-AT sequence confirmed by over-expression studies. The clone isolated by complementation, pLAT2, encodes a protein with homology to both SLC1 and PlsC. It was not possible to over-express the complementing protein encoded by pLAT2 but further experimentation on membranes from complemented JC201 demonstrated that they possess a substrate specificity distinctly different from PlsC and similar to Limnanthes sp. microsome specificity. This data strongly supports the contention that pLAT2 is an LPA-AT clone. Northern blot analysis revealed different expression patterns for the two genes in pLAT1 and pLAT2. Transcription of the gene encoding the insert of pLAT2 occurred almost exclusively in developing seed tissue, whilst the cDNA of pLAT1 hybridised to poly(A)+ mRNA from seed, stem and leaf, demonstrating more widespread expression throughout the plant. Southern blot analysis indicated that the cDNA of pLAT2 was transcribed from a single-copy gene while that for pLAT1 was a member of a small gene family.
Jan F Stevens - One of the best experts on this subject based on the ideXlab platform.
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identification and phytotoxicity of a new glucosinolate breakdown product from meadowfoam Limnanthes alba seed meal
Journal of Agricultural and Food Chemistry, 2014Co-Authors: Suphannika Intanon, Ralph L Reed, Jan F Stevens, Andrew G Hulting, Carol A MallorysmithAbstract:Meadowfoam (Limnanthes alba Hartw. ex Benth.) is an oilseed crop grown in the Willamette Valley of Oregon. Meadowfoam seed meal (MSM), a byproduct after oil extraction, contains 2-4% glucosinolate (glucolimnanthin). Activated MSM, produced by adding freshly ground myrosinase-active meadowfoam seeds to MSM, facilitates myrosinase-mediated formation of glucosinolate-derived degradation products with herbicidal activity. In the activated MSM, glucolimnanthin was converted into 3-methoxybenzyl isothiocyanate ("isothiocyanate") within 24 h and was degraded by day three. 3-Methoxyphenylacetonitrile ("nitrile") persisted for at least 6 days. Methoxyphenylacetic acid (MPAA), a previously unknown metabolite of glucolimnanthin, appeared at day three. Its identity was confirmed by LC-UV and high resolution LC-MS/MS comparisons with a standard of MPAA. Isothiocyanate inhibited lettuce germination 8.5- and 14.4-fold more effectively than MPAA and nitrile, respectively. Activated MSM inhibited lettuce germination by 58% and growth by 72% compared with the control. Results of the study suggest that MSM has potential uses as a pre-emergence bioherbicide.
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Identification and Phytotoxicity of a New Glucosinolate Breakdown Product from Meadowfoam (Limnanthes alba) Seed Meal
2014Co-Authors: Suphannika Intanon, Ralph L Reed, Jan F Stevens, Andrew G. Hulting, Carol A. Mallory-smithAbstract:Meadowfoam (Limnanthes alba Hartw. ex Benth.) is an oilseed crop grown in the Willamette Valley of Oregon. Meadowfoam seed meal (MSM), a byproduct after oil extraction, contains 2–4% glucosinolate (glucolimnanthin). Activated MSM, produced by adding freshly ground myrosinase-active meadowfoam seeds to MSM, facilitates myrosinase-mediated formation of glucosinolate-derived degradation products with herbicidal activity. In the activated MSM, glucolimnanthin was converted into 3-methoxybenzyl isothiocyanate (“isothiocyanate”) within 24 h and was degraded by day three. 3-Methoxyphenylacetonitrile (“nitrile”) persisted for at least 6 days. Methoxyphenylacetic acid (MPAA), a previously unknown metabolite of glucolimnanthin, appeared at day three. Its identity was confirmed by LC-UV and high resolution LC-MS/MS comparisons with a standard of MPAA. Isothiocyanate inhibited lettuce germination 8.5- and 14.4-fold more effectively than MPAA and nitrile, respectively. Activated MSM inhibited lettuce germination by 58% and growth by 72% compared with the control. Results of the study suggest that MSM has potential uses as a pre-emergence bioherbicide
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activity of meadowfoam Limnanthes alba seed meal glucolimnanthin degradation products against soilborne pathogens
Journal of Agricultural and Food Chemistry, 2012Co-Authors: Inga A Zasada, Ralph L Reed, Jerry E Weiland, Jan F StevensAbstract:Meadowfoam (Limnanthes alba L.) is a herbaceous winter−spring annual grown as a commercial oilseed crop. The meal remaining after oil extraction from the seed contains up to 4% of the glucosinolate glucolimnanthin. Degradation of glucolimnanthin yields toxic breakdown products, and therefore the meal may have potential in the management of soilborne pathogens. To maximize the pest-suppressive potential of meadowfoam seed meal, it would be beneficial to know the toxicity of individual glucolimnanthin degradation products against specific soilborne pathogens. Meloidogyne hapla second-stage juveniles (J2) and Pythium irregulare and Verticillium dahliae mycelial cultures were exposed to glucolimnanthin as well as its degradation products. Glucolimnanthin and its degradation product, 2-(3-methoxyphenyl)acetamide, were not toxic to any of the soilborne pathogens at concentrations up to 1.0 mg/mL. Two other degradation products, 2-(3-methoxymethyl)ethanethioamide and 3- methoxyphenylacetonitrile, were toxic to M. hapla and P. irregulare but not V. dahliae. The predominant enzyme degradation product, 3-methoxybenzyl isothiocyanate, was the most toxic compound against all of the soilborne pathogens, with M. hapla being the most sensitive with EC50 values (0.0025 ± 0.0001 to 0.0027 ± 0.0001 mg/mL) 20−40 times lower than estimated EC50 mortality values generated for P. irregulare and V. dahliae (0.05 and 0.1 mg/mL, respectively). The potential exists to manipulate meadowfoam seed meal to promote the production of specific degradation products. The conversion of glucolimnanthin into its corresponding isothiocyanate should optimize the biopesticidal properties of meadowfoam seed meal against M. hapla, P. irregulare, and V. dahliae.
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glucosinolates in the new oilseed crop meadowfoam natural variation in section inflexae of Limnanthes a new glucosinolate in l floccosa and qtl analysis in l alba
Plant Breeding, 2011Co-Authors: Pablo Velasco, Mary B Slabaugh, Ralph L Reed, J G Kling, Venkata K Kishore, Jan F Stevens, Steven J KnappAbstract:Meadowfoam, an oilseed crop grown in the Willamette Valley of Oregon, was developed from Limnanthes alba (Benth.), an herbaceous winter annual native to the west coast of North America. Meadowfoam oil is valued for unique properties attributed to the predominance of very long-chain fatty acids and desaturation at the D5 position. Seed meal remaining after commercial oil extraction contains intact glucosinolates and has potential pesticidal properties owing to isothiocyanate and nitrile glucosinolate derivatives. We identified and quantitated seed glucosinolates in four species of Section Inflexae of Limnanthes to assess variation for this trait in primary and secondary gene pools of cultivated meadowfoam. Glucosinolate content in seeds ranged from 30 to 204 lmol/g dry weight. Only glucolimnanthin (3-methoxybenzyl glucosinolate) was detected in L. alba, L. montana and L. gracilis, but L. floccosa contained glucolepigramin (3-hydroxybenzyl glucosinolate) as well as glucolimnanthin. Two QTL affecting seed glucosinolate content were identified in an inter-subspecific BC1 population derived from a cross between L. alba subsp. alba and L. alba subsp. versicolor.
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herbicidal activity of glucosinolate degradation products in fermented meadowfoam Limnanthes alba seed meal
Journal of Agricultural and Food Chemistry, 2009Co-Authors: Jan F Stevens, Ralph L Reed, Susan Alber, Larry Pritchett, Stephen MachadoAbstract:Meadowfoam (Limnanthes alba) is an oilseed crop grown in western Oregon. After extraction of the oil from the seeds, the remaining seed meal contains 2−4% of the glucosinolate glucolimnanthin. This study investigated the effect of fermentation of seed meal on its chemical composition and the effect of the altered composition on downy brome (Bromus tectorum) coleoptile emergence. Incubation of enzyme-inactive seed meal with enzyme-active seeds (1% by weight) resulted in complete degradation of glucolimnanthin and formation of 3-methoxybenzyl isothiocyanate in 28% yield. Fermentation in the presence of an aqueous solution of FeSO4 (10 mM) resulted in the formation of 3-methoxyphenylacetonitrile and 2-(3-methoxyphenyl)ethanethioamide, a novel natural product. The formation of the isothiocyanate, the nitrile, and the thioamide, as a total, correlated with an increase of herbicidal potency of the seed meal (r2 = 0.96). The results of this study open new possibilities for the refinement of glucosinolate-contain...
Ralph L Reed - One of the best experts on this subject based on the ideXlab platform.
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identifying rates of meadowfoam Limnanthes alba seed meal needed for suppression of meloidogyne hapla and pythium irregulare in soil
Plant Disease, 2014Co-Authors: Yurdagul şimsek Ersahin, Ralph L Reed, Jerry E Weiland, Inga A Zasada, Fred J StevensAbstract:Şimsek Ersahin, Y., Weiland, J. E., Zasada, I. A., Reed, R. L., and Stevens, J. F. 2014. Identifying rates of meadowfoam (Limnanthes alba) seed meal needed for suppression of Meloidogyne hapla and Pythium irregulare in soil. Plant Dis. 98:1253-1260. Meadowfoam (Limnanthes alba) is a commercial oilseed annual crop grown in Oregon. After extracting oil from seed, the remaining seed meal is rich in the secondary plant metabolite glucolimnanthin, which can be converted into pesticidal compounds such as 3-methoxybenzyl isothiocyanate (ITC) and 3-methoxyphenylacetonitrile (nitrile) in the presence of the enzyme myrosinase. In previous studies, we demonstrated that ITC and nitrile, produced by mixing freshly ground meadowfoam seed with meadowfoam seed meal, are toxic to the plantparasitic nematode Meloidogyne hapla and the plant pathogen Pythium irregulare. In this study, we evaluated factors that might influence the implementation of meadowfoam seed meal into agricultural production systems for soilborne pathogen and nematode control. Rate-finding experiments demonstrated that a minimum 1.0% seed/seed meal formulation (wt/wt) was necessary to achieve nematode and pathogen suppression; seed meal alone was insufficient for complete control of M. hapla and stimulated the growth of P. irregulare. When this 1.0% seed/seed meal formulation was used, a greater soil amendment rate was required to cause 100% mortality of P. irregulare (1.0% wt/wt) than for M. hapla (0.5% wt/wt). In phytotoxicity experiments, soil amended with the 1.0% seed/seed meal formulation was consistently phytotoxic to wheat, cucumber, and tomato. However, phytotoxic effects were mitigated by a delayed planting into the amended soil. A final assay to monitor concentrations of ITC and nitrile in conjunction with assessing M. hapla and P. irregulare mortality was conducted over a 6-day period in soils amended at 0.5 and 1.0% (wt/wt) with the 1.0% seed/seed meal formulation. The response was rapid, with 100% mortality of both organisms within 2 h after exposure to amended soil. Concentrations of nitrile remained relatively constant over the 6-day period (approximately 0.017 and 0.032 mg/ml at 0.5 and 1.0% amendment rates, respectively), whereas ITC production increased rapidly and peaked 12 to 24 h after amendment (0.083 and 0.171 mg/ml at 0.5 and 1.0% amendment rates, respectively) before returning to near undetectable levels.
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identification and phytotoxicity of a new glucosinolate breakdown product from meadowfoam Limnanthes alba seed meal
Journal of Agricultural and Food Chemistry, 2014Co-Authors: Suphannika Intanon, Ralph L Reed, Jan F Stevens, Andrew G Hulting, Carol A MallorysmithAbstract:Meadowfoam (Limnanthes alba Hartw. ex Benth.) is an oilseed crop grown in the Willamette Valley of Oregon. Meadowfoam seed meal (MSM), a byproduct after oil extraction, contains 2-4% glucosinolate (glucolimnanthin). Activated MSM, produced by adding freshly ground myrosinase-active meadowfoam seeds to MSM, facilitates myrosinase-mediated formation of glucosinolate-derived degradation products with herbicidal activity. In the activated MSM, glucolimnanthin was converted into 3-methoxybenzyl isothiocyanate ("isothiocyanate") within 24 h and was degraded by day three. 3-Methoxyphenylacetonitrile ("nitrile") persisted for at least 6 days. Methoxyphenylacetic acid (MPAA), a previously unknown metabolite of glucolimnanthin, appeared at day three. Its identity was confirmed by LC-UV and high resolution LC-MS/MS comparisons with a standard of MPAA. Isothiocyanate inhibited lettuce germination 8.5- and 14.4-fold more effectively than MPAA and nitrile, respectively. Activated MSM inhibited lettuce germination by 58% and growth by 72% compared with the control. Results of the study suggest that MSM has potential uses as a pre-emergence bioherbicide.
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application of meadowfoam Limnanthes alba seed meal as a soil amendment for management of pythium irregulare
Türk Tarım ve Doğa Bilimleri Dergisi, 2014Co-Authors: Yurdagul şimsek Ersahin, Ralph L Reed, Jerry E Weiland, And Fred J StevensAbstract:Meadowfoam (Limnanthes alba Hartw. ex Benth.) is a member of the order Brassicales and has been grown as a commercial oil seed annual crop in the Willamette Valley of Oregon, USA since the 1980’s. After harvest, the seed is pressed to yield oil containing unique long chain fatty acids (20:1 and 22:1) of high quality and commercial value in cosmetics and lubricants, making meadowfoam a high-value oilseed crop. After the oil has been extracted from seed, the remaining seed meal contains the glucosinolate glucolimnanthin. When plant cells containing glucolimnanthin are physically damaged and exposed to moisture and the enzyme myrosinase, this secondary plant metobolite degrades into toxic breakdown products. In a previous study, we demonstrated the toxicity of the glucolimnanthin degradation products nitrile, thioamide, and isothiocyanage (ITC) to the plant pathogen Pythium irregulare. The ITC was the most toxic to this pathogen while glucolimnanthin and its degradation product acetamide were not toxic to the pathogen. This research demonstrated the potential to utilize meadowfoam seed meal (MSM) as a soil amendment to manage this soilborne pathogen
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Identification and Phytotoxicity of a New Glucosinolate Breakdown Product from Meadowfoam (Limnanthes alba) Seed Meal
2014Co-Authors: Suphannika Intanon, Ralph L Reed, Jan F Stevens, Andrew G. Hulting, Carol A. Mallory-smithAbstract:Meadowfoam (Limnanthes alba Hartw. ex Benth.) is an oilseed crop grown in the Willamette Valley of Oregon. Meadowfoam seed meal (MSM), a byproduct after oil extraction, contains 2–4% glucosinolate (glucolimnanthin). Activated MSM, produced by adding freshly ground myrosinase-active meadowfoam seeds to MSM, facilitates myrosinase-mediated formation of glucosinolate-derived degradation products with herbicidal activity. In the activated MSM, glucolimnanthin was converted into 3-methoxybenzyl isothiocyanate (“isothiocyanate”) within 24 h and was degraded by day three. 3-Methoxyphenylacetonitrile (“nitrile”) persisted for at least 6 days. Methoxyphenylacetic acid (MPAA), a previously unknown metabolite of glucolimnanthin, appeared at day three. Its identity was confirmed by LC-UV and high resolution LC-MS/MS comparisons with a standard of MPAA. Isothiocyanate inhibited lettuce germination 8.5- and 14.4-fold more effectively than MPAA and nitrile, respectively. Activated MSM inhibited lettuce germination by 58% and growth by 72% compared with the control. Results of the study suggest that MSM has potential uses as a pre-emergence bioherbicide
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activity of meadowfoam Limnanthes alba seed meal glucolimnanthin degradation products against soilborne pathogens
Journal of Agricultural and Food Chemistry, 2012Co-Authors: Inga A Zasada, Ralph L Reed, Jerry E Weiland, Jan F StevensAbstract:Meadowfoam (Limnanthes alba L.) is a herbaceous winter−spring annual grown as a commercial oilseed crop. The meal remaining after oil extraction from the seed contains up to 4% of the glucosinolate glucolimnanthin. Degradation of glucolimnanthin yields toxic breakdown products, and therefore the meal may have potential in the management of soilborne pathogens. To maximize the pest-suppressive potential of meadowfoam seed meal, it would be beneficial to know the toxicity of individual glucolimnanthin degradation products against specific soilborne pathogens. Meloidogyne hapla second-stage juveniles (J2) and Pythium irregulare and Verticillium dahliae mycelial cultures were exposed to glucolimnanthin as well as its degradation products. Glucolimnanthin and its degradation product, 2-(3-methoxyphenyl)acetamide, were not toxic to any of the soilborne pathogens at concentrations up to 1.0 mg/mL. Two other degradation products, 2-(3-methoxymethyl)ethanethioamide and 3- methoxyphenylacetonitrile, were toxic to M. hapla and P. irregulare but not V. dahliae. The predominant enzyme degradation product, 3-methoxybenzyl isothiocyanate, was the most toxic compound against all of the soilborne pathogens, with M. hapla being the most sensitive with EC50 values (0.0025 ± 0.0001 to 0.0027 ± 0.0001 mg/mL) 20−40 times lower than estimated EC50 mortality values generated for P. irregulare and V. dahliae (0.05 and 0.1 mg/mL, respectively). The potential exists to manipulate meadowfoam seed meal to promote the production of specific degradation products. The conversion of glucolimnanthin into its corresponding isothiocyanate should optimize the biopesticidal properties of meadowfoam seed meal against M. hapla, P. irregulare, and V. dahliae.
Adrian P. Brown - One of the best experts on this subject based on the ideXlab platform.
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Limnanthes douglasii lysophosphatidic acid acyltransferases: immunological quantification, acyl selectivity and functional replacement of the Escherichia coli plsC gene.
Biochemical Journal, 2002Co-Authors: Adrian P. Brown, Simon Carnaby, Clare L. Brough, Melissa Brazier, Antoni R. SlabasAbstract:Antibodies were raised against the two membrane-bound lysophosphatidic acid acyltransferase (LPAAT) enzymes from Limnanthes douglasii (meadowfoam), LAT1 and LAT2, using the predicted soluble portion of each protein as recombinant protein antigens. The antibodies can distinguish between the two acyltransferase proteins and demonstrate that both migrate in an anomalous fashion on SDS/PAGE gels. The antibodies were used to determine that LAT1 is present in both leaf and developing seeds, whereas LAT2 is only detectable in developing seeds later than 22 daf (days after flowering). Both proteins were found exclusively in microsomal fractions and their amount was determined using the recombinant antigens as quantification standards. LAT1 is present at a level of 27 pg/microg of membrane protein in leaf tissue and
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Limnanthes douglasii erucic acid specific lysophospatidic acid acyltransferase activity in oilseed rape an analysis of biochemical effects
Biochemical Society Transactions, 2000Co-Authors: J A Wilmer, Adrian P. Brown, Simon Carnaby, Tina L. Barsby, K Forsyth, Antoni R. SlabasAbstract:It has been shown previously that erucic acid accumulates in the sn -2 position of triglycerides after introduction of a Limnanthes lysophosphatidic acid acyltransferase. However, in none of the studies to date has a proper evaluation been made of the relationship between transgene expression and the erucic acid accumulation at sn -2. One of the necessary tools to study the variability is the presence of a specific antibody raised against the introduced acyltransferase. In the present study, we have looked at the correlation between erucic acid accumulation at sn -2, production of trierucin and the expression of the transgene, as detected by Western blotting. The data presented include comparisons of progeny of individual lines grown under various conditions and at various times, and an analysis of a number of lines during development. We also present calculations that could indicate from which position the erucic acid found at position sn -2 is derived.
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towards the genetic engineering of triacylglycerols of defined fatty acid composition major changes in erucic acid content at the sn 2 position affected by the introduction of a 1 acyl sn glycerol 3 phosphate acyltransferase from Limnanthes douglasii
Molecular Breeding, 1996Co-Authors: Clare L. Brough, Adrian P. Brown, Johan T.m. Kroon, Jane M. Coventry, William W. Christie, Tina L. Barsby, Antoni R. SlabasAbstract:A cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii was introduced into oil seed rape (Brassica napus) under the control of a napin promoter. Seed triacylglycerols from transgenic plants were analysed by reversed-phase HPLC and trierucin was detected at a level of 0.4% and 2.8% in two transgenic plants but was not found in untransformed rape seed. Total fatty acid composition analysis of seeds from these selected plants revealed that the erucic acid content was no higher than the maximum found in the starting population. Analysis of fatty acids at the sn-2 position showed no erucic acid in untransformed rape but in the selected transgenic plants 9% (mol/mol) and 28.3% (mol/mol) erucic acid was present. These results conclusively demonstrate that the gene from L. douglasii encodes a 1-acyl-sn-glycerol-3-phosphate acyltransferase which can function in rape and incorporate erucic acid at the sn-2 position of triacylglycerols in seed. Additional modifications may further increase levels of trierucin.
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identification of a cdna that encodes a 1 acyl sn glycerol 3 phosphate acyltransferase from Limnanthes douglasii
Plant Molecular Biology, 1995Co-Authors: Adrian P. Brown, Clare L. Brough, Johan T.m. Kroon, Antoni R. SlabasAbstract:Two different techniques were used to isolate potential cDNAs for acyl-CoA: 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPA-AT) enzymes from Limnanthes douglasii. Both heterologous screening with the maize pMAT1 clone and in vivo complementation of the Escherchia coli mutant JC201 which is deficient in LPA-AT activity, were carried out. Clones identified by these procedures were different. Homology searches demonstrated that the clone isolated by heterologous probing, pLAT1, encodes a protein which is most similar to the maize (open reading frame in pMAT1) and yeast SLC1 proteins, which are putative LPA-AT sequences. This L. douglasii sequence shows much lower homology to the E. coli LPA-AT protein PlsC, which is the only LPA-AT sequence confirmed by over-expression studies. The clone isolated by complementation, pLAT2, encodes a protein with homology to both SLC1 and PlsC. It was not possible to over-express the complementing protein encoded by pLAT2 but further experimentation on membranes from complemented JC201 demonstrated that they possess a substrate specificity distinctly different from PlsC and similar to Limnanthes sp. microsome specificity. This data strongly supports the contention that pLAT2 is an LPA-AT clone. Northern blot analysis revealed different expression patterns for the two genes in pLAT1 and pLAT2. Transcription of the gene encoding the insert of pLAT2 occurred almost exclusively in developing seed tissue, whilst the cDNA of pLAT1 hybridised to poly(A)+ mRNA from seed, stem and leaf, demonstrating more widespread expression throughout the plant. Southern blot analysis indicated that the cDNA of pLAT2 was transcribed from a single-copy gene while that for pLAT1 was a member of a small gene family.
Clare L. Brough - One of the best experts on this subject based on the ideXlab platform.
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Limnanthes douglasii lysophosphatidic acid acyltransferases: immunological quantification, acyl selectivity and functional replacement of the Escherichia coli plsC gene.
Biochemical Journal, 2002Co-Authors: Adrian P. Brown, Simon Carnaby, Clare L. Brough, Melissa Brazier, Antoni R. SlabasAbstract:Antibodies were raised against the two membrane-bound lysophosphatidic acid acyltransferase (LPAAT) enzymes from Limnanthes douglasii (meadowfoam), LAT1 and LAT2, using the predicted soluble portion of each protein as recombinant protein antigens. The antibodies can distinguish between the two acyltransferase proteins and demonstrate that both migrate in an anomalous fashion on SDS/PAGE gels. The antibodies were used to determine that LAT1 is present in both leaf and developing seeds, whereas LAT2 is only detectable in developing seeds later than 22 daf (days after flowering). Both proteins were found exclusively in microsomal fractions and their amount was determined using the recombinant antigens as quantification standards. LAT1 is present at a level of 27 pg/microg of membrane protein in leaf tissue and
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Engineering Trierucin into Oilseed Rape by the Introduction of a 1-Acyl-sn-Glycerol-3-Phosphate Acyltransferase from Limnanthes Douglasii
Physiology Biochemistry and Molecular Biology of Plant Lipids, 1997Co-Authors: Clare L. Brough, Johan T.m. Kroon, Jane M. Coventry, William W. Christie, Tina L. Barsby, Antoni R. SlabasAbstract:Currently available cultivars of high erucic acid rape (HEAR) have a theoretical maximum of 66% erucic acid (22:1, Δ13) in their seed oil due to the specificity of the membrane-bound 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPA-AT) enzyme. In HEAR the LPA-AT does not incorporate erucic acid at the s n-2 position of triacylglycerols (TAG) but preferentially incorporates oleic acid (18:1, Δ9), even if this is only a minor component of the total fatty acid pool (1). However, some plant species, e.g Limnanthes, can utilise erucoyl-CoA as a substrate and effectively incorporate erucic acid at the sn-2 position (2, 3).
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towards the genetic engineering of triacylglycerols of defined fatty acid composition major changes in erucic acid content at the sn 2 position affected by the introduction of a 1 acyl sn glycerol 3 phosphate acyltransferase from Limnanthes douglasii
Molecular Breeding, 1996Co-Authors: Clare L. Brough, Adrian P. Brown, Johan T.m. Kroon, Jane M. Coventry, William W. Christie, Tina L. Barsby, Antoni R. SlabasAbstract:A cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii was introduced into oil seed rape (Brassica napus) under the control of a napin promoter. Seed triacylglycerols from transgenic plants were analysed by reversed-phase HPLC and trierucin was detected at a level of 0.4% and 2.8% in two transgenic plants but was not found in untransformed rape seed. Total fatty acid composition analysis of seeds from these selected plants revealed that the erucic acid content was no higher than the maximum found in the starting population. Analysis of fatty acids at the sn-2 position showed no erucic acid in untransformed rape but in the selected transgenic plants 9% (mol/mol) and 28.3% (mol/mol) erucic acid was present. These results conclusively demonstrate that the gene from L. douglasii encodes a 1-acyl-sn-glycerol-3-phosphate acyltransferase which can function in rape and incorporate erucic acid at the sn-2 position of triacylglycerols in seed. Additional modifications may further increase levels of trierucin.
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identification of a cdna that encodes a 1 acyl sn glycerol 3 phosphate acyltransferase from Limnanthes douglasii
Plant Molecular Biology, 1995Co-Authors: Adrian P. Brown, Clare L. Brough, Johan T.m. Kroon, Antoni R. SlabasAbstract:Two different techniques were used to isolate potential cDNAs for acyl-CoA: 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPA-AT) enzymes from Limnanthes douglasii. Both heterologous screening with the maize pMAT1 clone and in vivo complementation of the Escherchia coli mutant JC201 which is deficient in LPA-AT activity, were carried out. Clones identified by these procedures were different. Homology searches demonstrated that the clone isolated by heterologous probing, pLAT1, encodes a protein which is most similar to the maize (open reading frame in pMAT1) and yeast SLC1 proteins, which are putative LPA-AT sequences. This L. douglasii sequence shows much lower homology to the E. coli LPA-AT protein PlsC, which is the only LPA-AT sequence confirmed by over-expression studies. The clone isolated by complementation, pLAT2, encodes a protein with homology to both SLC1 and PlsC. It was not possible to over-express the complementing protein encoded by pLAT2 but further experimentation on membranes from complemented JC201 demonstrated that they possess a substrate specificity distinctly different from PlsC and similar to Limnanthes sp. microsome specificity. This data strongly supports the contention that pLAT2 is an LPA-AT clone. Northern blot analysis revealed different expression patterns for the two genes in pLAT1 and pLAT2. Transcription of the gene encoding the insert of pLAT2 occurred almost exclusively in developing seed tissue, whilst the cDNA of pLAT1 hybridised to poly(A)+ mRNA from seed, stem and leaf, demonstrating more widespread expression throughout the plant. Southern blot analysis indicated that the cDNA of pLAT2 was transcribed from a single-copy gene while that for pLAT1 was a member of a small gene family.