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John Browse - One of the best experts on this subject based on the ideXlab platform.
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castor lpcat and pdat1a act in concert to promote transacylation of Hydroxy Fatty Acid onto triacylglycerol
Plant Physiology, 2020Co-Authors: Daniel Lunn, James G Wallis, John BrowseAbstract:Oilseeds produce abundant triacylglycerol (TAG) during seed maturation to fuel the establishment of photoautotrophism in the subsequent generation. Commonly, TAG contains 18-carbon polyunsaturated Fatty Acids (FA), but plants also produce oils with unique chemical properties highly desirable for industrial processes. Unfortunately, plants that produce such oils are poorly suited to agronomic exploitation, leading to a desire to reconstitute novel oil biosynthesis in crop plants. Here, we studied the production and incorporation of Hydroxy-Fatty Acids (HFA) onto TAG in Arabidopsis (Arabidopsis thaliana) plants expressing the castor (Ricinus communis) FAH12 Hydroxylase. One factor limiting HFA accumulation in these plants is the inefficient removal of HFA from the site of synthesis on phosphatidylcholine (PC). In Arabidopsis, lysophosphatidic Acid acyltransferase (LPCAT) cycles FA to and from PC for modification. We reasoned that the castor LPCAT (RcLPCAT) would preferentially remove HFA from PC, resulting in greater incorporation onto TAG. However, expressing RcLPCAT in Arabidopsis expressing FAH12 alone (line CL37) or together with castor acyl:coenzyme A:diacylglycerol acyltransferase2 reduced HFA and total oil yield. Detailed analysis indicated that RcLPCAT reduced the removal of HFA from PC, possibly by competing with the endogenous LPCAT isozymes. Significantly, coexpressing RcLPCAT with castor phospholipid:diacylglycerol acyltransferase increased novel FA and total oil contents by transferring HFA from PC to diacylglycerol. Our results demonstrate that a detailed understanding is required to engineer modified FA production in oilseeds and suggest that phospholipase A2 enzymes rather than LPCAT mediate the highly efficient removal of HFA from PC in castor seeds.
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development defects of Hydroxy Fatty Acid accumulating seeds are reduced by castor acyltransferases
Plant Physiology, 2018Co-Authors: Daniel Lunn, James G Wallis, Gracen A Smith, John BrowseAbstract:Researchers have long endeavored to produce modified Fatty Acids in easily managed crop plants where they are not natively found. An important step toward this goal has been the biosynthesis of these valuable products in model oilseeds. The successful production of such Fatty Acids has revealed barriers to the broad application of this technology, including low seed oil and low proportion of the introduced Fatty Acid and reduced seed vigor. Here, we analyze the impact of producing Hydroxy-Fatty Acids on seedling development. We show that germinating seeds of a Hydroxy-Fatty Acid-accumulating Arabidopsis (Arabidopsis thaliana) line produce chlorotic cotyledons and suffer reduced photosynthetic capacity. These seedlings retain Hydroxy-Fatty Acids in polar lipids, including chloroplast lipids, and exhibit decreased Fatty Acid synthesis. Triacylglycerol mobilization in seedling development also is reduced, especially for lipids that include Hydroxy-Fatty Acid moieties. These developmental defects are ameliorated by increased flux of Hydroxy-Fatty Acids into seed triacylglycerol created through the expression of either castor (Ricinus communis) acyltransferase enzyme ACYL-COA:DIACYLGLYCEROL ACYLTRANSFERASE2 or PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1A. Such expression increases both the level of total stored triacylglycerol and the rate at which it is mobilized, fueling Fatty Acid synthesis and restoring photosynthetic capacity. Our results suggest that further improvements in seedling development may require the specific mobilization of triacylglycerol-containing Hydroxy-Fatty Acids. Understanding the defects in early development caused by the accumulation of modified Fatty Acids and providing mechanisms to circumvent these defects are vital steps in the development of tailored oil crops.
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overexpression of seipin1 increases oil in Hydroxy Fatty Acid accumulating seeds
Plant and Cell Physiology, 2018Co-Authors: Daniel Lunn, James G Wallis, John BrowseAbstract:While plant oils are an important source of food, plants also produce oils containing specialized Fatty Acids with chemical and physical properties valued in industry. Ricinoleic Acid, a Hydroxy Fatty Acid (HFA) produced in the seed of castor (Ricinus communis), is of particular value, with a wide range of applications. Since castor cultivation is currently successful only in tropical climates, and because castor seed contain the toxin ricin, there are ongoing efforts to develop a temperate crop capable of HFA biosynthesis. In castor, ricinoleic Acid is incorporated into triacylglycerol (TAG) which accumulates in the seed lipid droplets. Research in the model plant Arabidopsis (Arabidopsis thaliana) has successfully produced HFA constituting 30% of the total seed oil, but this is far short of the level required to engineer commercially viable crops. Strategies to increase HFA have centered on co-expression of castor TAG biosynthesis enzymes. However, since lipid droplets are the location of neutral lipid storage, manipulating droplets offers an alternative method to increase oil that contains specialized Fatty Acids. The Arabidopsis Seipin1 protein modulates TAG accumulation by affecting lipid droplet size. Here, we overexpress Seipin1 in a Hydroxylase-expressing Arabidopsis line, increasing seed HFA by 62% and proportionally increasing total oil. Increased seed oil was concomitant with a 22% increase in single seed weight and a 69% increase in harvest weight, while seed germination rose by 45%. Because Seipin1 function is unaffected by the structure of the HFA, these results demonstrate a novel strategy that may increase accumulation of many specialized seed oils.
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metabolic engineering of Hydroxy Fatty Acid production in plants rcdgat2 drives dramatic increases in ricinoleate levels in seed oil
Plant Biotechnology Journal, 2008Co-Authors: Julie Jeannine Burgal, Jay M Shockey, John M Dyer, Tony R Larson, Ian A Graham, John BrowseAbstract:A central goal of green chemistry is to produce industrially useful Fatty Acids in oilseed crops. Although genes encoding suitable Fatty Acid-modifying enzymes are available from many wild species, progress has been limited because the expression of these genes in transgenic plants produces low yields of the desired products. For example, Ricinus communis Fatty Acid Hydroxylase 12 (FAH12) produces a maximum of only 17% Hydroxy Fatty Acids (HFAs) when expressed in Arabidopsis. cDNA clones encoding R. communis enzymes for additional steps in the seed oil biosynthetic pathway were identified. Expression of these cDNAs in FAH12 transgenic plants revealed that the R. communis type-2 acyl-coenzyme A:diacylglycerol acyltransferase (RcDGAT2) could increase HFAs from 17% to nearly 30%. Detailed comparisons of seed neutral lipids from the single- and double-transgenic lines indicated that RcDGAT2 substantially modified the triacylglycerol (TAG) pool, with significant increases in most of the major TAG species observed in native castor bean oil. These data suggest that RcDGAT2 prefers acyl-coenzyme A and diacylglycerol substrates containing HFAs, and biochemical analyses of RcDGAT2 expressed in yeast cells confirmed a strong preference for HFA-containing diacylglycerol substrates. Our results demonstrate that pathway engineering approaches can be used successfully to increase the yields of industrial feedstocks in plants, and that members of the DGAT2 gene family probably play a key role in this process.
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a high throughput screen for genes from castor that boost Hydroxy Fatty Acid accumulation in seed oils of transgenic arabidopsis
Plant Journal, 2006Co-Authors: Chaofu Lu, Martin Fulda, James G Wallis, John BrowseAbstract:Summary It is desirable to produce high homogeneity of novel Fatty Acids in oilseeds through genetic engineering to meet the increasing demands of the oleo-chemical industry. However, expression of key enzymes for biosynthesis of industrial Fatty Acids usually results in low levels of desired Fatty Acids in transgenic oilseeds. The abundance of derivatized Fatty Acids in their natural species suggests that additional genes are needed for high production in transgenic plants. We used the model oilseed plant Arabidopsis thaliana expressing a castor Fatty Acid Hydroxylase (FAH12) to identify genes that can boost Hydroxy Fatty Acid accumulation in transgenic seeds. Here we describe a high-throughput approach that, in principle, can allow testing of the entire transcriptome of developing castor seed endosperm by shotgun transforming a full-length cDNA library into an FAH12-expressing Arabidopsis line. The resulting transgenic seeds were screened by high-throughput gas chromatography. We obtained several lines transformed with castor cDNAs that contained increased amounts of Hydroxy Fatty Acids in transgenic Arabidopsis. These cDNAs were then isolated by PCR and retransformed into the FAH12-expressing line, thus confirming their beneficial contributions to Hydroxy Fatty Acid accumulation in transgenic Arabidopsis seeds. Although we describe an approach that is targeted to oilseed engineering, the methods we developed can be applied in many areas of plant biotechnology and functional genomic research.
Grace Q. Chen - One of the best experts on this subject based on the ideXlab platform.
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variant castor lysophosphatidic Acid acyltransferases acylate ricinoleic Acid in seed oil
Industrial Crops and Products, 2020Co-Authors: Hyun Uk Kim, Mideum Park, Kyeongryeol Lee, Mi Chung Suh, Grace Q. ChenAbstract:Abstract Determining the role of castor lysophosphatidic Acid acyltransferases (RcLPATs) provides information that aid in understanding the biosynthesis mechanism of castor oil (triacylglycerols, TAG), which contains 90 % ricinoleic Acid (18:1OH), a Hydroxy Fatty Acid (HFA) with numerous industrial applications. The entire family of seven RcLPATs was shown to encode functional enzymes using in vitro assays. Gene expression analysis suggested that RcLPATs play roles in various vegetative and reproductive organs by associating with membrane lipid and TAG biosynthesis. To identify isoforms of RcLPATs capable of acylating 18:1OH, individual RcLPATs were expressed in CL37, an Arabidopsis line containing approximately 17 % HFA in seed TAG. Transgenics expressing RcLPAT2, RcLPAT3B, or RcLPATB increased total HFA to 18.2 %–20.3 %. Furthermore, different accumulation levels of 18:1OH and densipolic Acid (18:2OH) were detected among these three transgenic backgrounds. The mechanisms of substrate selectivity among RcLPAT2, RcLPAT3B, and RcLPATB are discussed.
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transcriptome analysis of lesquerella a potential industrial oilseed crop for Hydroxy Fatty Acid production
Advances in Crop Science and Technology, 2016Co-Authors: Grace Q. ChenAbstract:S oil of Lesquerella (Physaria fendelri) contains 55-60% Hydroxy Fatty Acid (HFA) that has important industrial application. A high throughput, large scale sequencing of transcripts from developing Lesquerella seeds was carried out by 454 pyrosequencing to generate a database for quality improvement of seed oil and other agronomic traits. Deep mining and characterization of acyl lipid genes were conducted to uncover candidate genes for further studies of mechanisms underlying HFA and seed oil synthesis. A total of 651 mega bases of raw sequences from an mRNA sample of developing seeds were acquired. Bioinformatic analysis of these sequences revealed 59,914 transcripts representing 26,995 unique genes that include nearly all known seed expressed genes. Based on sequence similarity with known plant proteins, about 74% (19,861) genes matched with annotated coding genes. Among them, 95% (18,868) showed highest sequence homology with Arabidopsis genes, which will allow translation of genomics and genetics findings from Arabidopsis to Lesquerella. Using Arabidopsis acyl lipid genes as queries, we searched the transcriptome assembly and identified 615 Lesquerella genes involved in all known pathways of acyl lipid metabolism. Further deep mining the transcriptome assembly led to identification of almost all Lesquerella genes involved in Fatty Acid and triacylglycerol synthesis. Moreover, we characterized the spatial and temporal expression profiles of 15 key genes using the quantitative PCR assay. The information obtained from data mining and gene expression profiling will provide a resource not only for the study of HFA metabolism but also for the biotechnological improvement of HFA production in Lesquerella.
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identification of Hydroxy Fatty Acid and triacylglycerol metabolism related genes in lesquerella through seed transcriptome analysis
BMC Genomics, 2015Co-Authors: Hyun Uk Kim, Grace Q. ChenAbstract:Castor oil is the only commercial source of Hydroxy Fatty Acid that has industrial value. The production of castor oil is hampered by the presence of the toxin ricin in its seed. Lesquerella seed also accumulates Hydroxy Fatty Acid and is free of ricin, and thus it is being developed as a new crop for Hydroxy Fatty Acid production. A high-throughput, large-scale sequencing of transcripts from developing lesquerella seeds was carried out by 454 pyrosequencing to generate a database for quality improvement of seed oil and other agronomic traits. Deep mining and characterization of acyl-lipid genes were conducted to uncover candidate genes for further studies of mechanisms underlying Hydroxy Fatty Acid and seed oil synthesis. A total of 651 megabases of raw sequences from an mRNA sample of developing seeds was acquired. Bioinformatic analysis of these sequences revealed 59,914 transcripts representing 26,995 unique genes that include nearly all known seed expressed genes. Based on sequence similarity with known plant proteins, about 74% (19,861) genes matched with annotated coding genes. Among them, 95% (18,868) showed highest sequence homology with Arabidopsis genes, which will allow translation of genomics and genetics findings from Arabidopsis to lesquerella. Using Arabidopsis acyl-lipid genes as queries, we searched the transcriptome assembly and identified 615 lesquerella genes involved in all known pathways of acyl-lipid metabolism. Further deep mining the transcriptome assembly led to identification of almost all lesquerella genes involved in Fatty Acid and triacylglycerol synthesis. Moreover, we characterized the spatial and temporal expression profiles of 15 key genes using the quantitative PCR assay. We have built a lesquerella seed transcriptome that provides a valuable reference in addition to the castor database for discovering genes involved in the synthesis of triacylglycerols enriched with Hydroxy Fatty Acids. The information obtained from data mining and gene expression profiling will provide a resource not only for the study of Hydroxy Fatty Acid metabolism, but also for the biotechnological production of Hydroxy Fatty Acids in existing oilseed crops.
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Hydroxy Fatty Acid synthesis and lipid gene expression during seed development in lesquerella fendleri
Industrial Crops and Products, 2011Co-Authors: Grace Q. Chen, Jianntsyh LinAbstract:Abstract Lesquerella fendleri is a developing oilseed crop in the U.S. The seed oil of L. fendleri is rich in lesquerolic Acid (14-Hydroxy-eicos-cis-11-enoic Acid: 20:1OH), a Hydroxy Fatty Acid (HFA) with potential uses in industrial materials. Although the synthesis pathway of HFA is extensively studied, the regulatory mechanism underlying synthesis and accumulation of 20:1OH is largely unknown. In this study, we investigated the Fatty Acid composition and lipid gene expression pattern in developing seeds of L. fendleri, from 7 days after pollination (DAP) to desiccation (49 DAP). The results showed that accumulation of 20:1OH started at 21 DAP, increased quickly between 21 and 35 DAP and reached a plateau, about 50–55% of total lipids, at 35–49 DAP. In addition, two other HFAs, ricinoleic (12-Hydroxy-octadec-cis-9-enoic Acid: 18:1OH) and auricolic (14-Hydroxy-eicos-cis-11,17-enoic Acid: 20:2OH) Acids were present at low levels during various stages of seed development. Ricinoleic Acid could be detected as early as 14 DAP whereas 20:2OH began to accumulate at 21 DAP or later. Using real-time polymerase chain reaction, we quantified the transcript level of three lipid genes, LFAH12 (bifunctional oleate 12-Hydroxylase:desaturase), LfKCS3 (3-ketoacyl-CoA synthase) and LfFen1 (oleate 12-desaturase). While all of these genes displayed a bell-shaped expression pattern with a peak at 35 DAP and a sharp decline at 42–49 DAP, they had different expression levels during early seed development and maximum inductions. Based on the defined time course of seed development, the relationship between gene expression and HFA accumulation is discussed.
Daniel Lunn - One of the best experts on this subject based on the ideXlab platform.
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castor lpcat and pdat1a act in concert to promote transacylation of Hydroxy Fatty Acid onto triacylglycerol
Plant Physiology, 2020Co-Authors: Daniel Lunn, James G Wallis, John BrowseAbstract:Oilseeds produce abundant triacylglycerol (TAG) during seed maturation to fuel the establishment of photoautotrophism in the subsequent generation. Commonly, TAG contains 18-carbon polyunsaturated Fatty Acids (FA), but plants also produce oils with unique chemical properties highly desirable for industrial processes. Unfortunately, plants that produce such oils are poorly suited to agronomic exploitation, leading to a desire to reconstitute novel oil biosynthesis in crop plants. Here, we studied the production and incorporation of Hydroxy-Fatty Acids (HFA) onto TAG in Arabidopsis (Arabidopsis thaliana) plants expressing the castor (Ricinus communis) FAH12 Hydroxylase. One factor limiting HFA accumulation in these plants is the inefficient removal of HFA from the site of synthesis on phosphatidylcholine (PC). In Arabidopsis, lysophosphatidic Acid acyltransferase (LPCAT) cycles FA to and from PC for modification. We reasoned that the castor LPCAT (RcLPCAT) would preferentially remove HFA from PC, resulting in greater incorporation onto TAG. However, expressing RcLPCAT in Arabidopsis expressing FAH12 alone (line CL37) or together with castor acyl:coenzyme A:diacylglycerol acyltransferase2 reduced HFA and total oil yield. Detailed analysis indicated that RcLPCAT reduced the removal of HFA from PC, possibly by competing with the endogenous LPCAT isozymes. Significantly, coexpressing RcLPCAT with castor phospholipid:diacylglycerol acyltransferase increased novel FA and total oil contents by transferring HFA from PC to diacylglycerol. Our results demonstrate that a detailed understanding is required to engineer modified FA production in oilseeds and suggest that phospholipase A2 enzymes rather than LPCAT mediate the highly efficient removal of HFA from PC in castor seeds.
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development defects of Hydroxy Fatty Acid accumulating seeds are reduced by castor acyltransferases
Plant Physiology, 2018Co-Authors: Daniel Lunn, James G Wallis, Gracen A Smith, John BrowseAbstract:Researchers have long endeavored to produce modified Fatty Acids in easily managed crop plants where they are not natively found. An important step toward this goal has been the biosynthesis of these valuable products in model oilseeds. The successful production of such Fatty Acids has revealed barriers to the broad application of this technology, including low seed oil and low proportion of the introduced Fatty Acid and reduced seed vigor. Here, we analyze the impact of producing Hydroxy-Fatty Acids on seedling development. We show that germinating seeds of a Hydroxy-Fatty Acid-accumulating Arabidopsis (Arabidopsis thaliana) line produce chlorotic cotyledons and suffer reduced photosynthetic capacity. These seedlings retain Hydroxy-Fatty Acids in polar lipids, including chloroplast lipids, and exhibit decreased Fatty Acid synthesis. Triacylglycerol mobilization in seedling development also is reduced, especially for lipids that include Hydroxy-Fatty Acid moieties. These developmental defects are ameliorated by increased flux of Hydroxy-Fatty Acids into seed triacylglycerol created through the expression of either castor (Ricinus communis) acyltransferase enzyme ACYL-COA:DIACYLGLYCEROL ACYLTRANSFERASE2 or PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1A. Such expression increases both the level of total stored triacylglycerol and the rate at which it is mobilized, fueling Fatty Acid synthesis and restoring photosynthetic capacity. Our results suggest that further improvements in seedling development may require the specific mobilization of triacylglycerol-containing Hydroxy-Fatty Acids. Understanding the defects in early development caused by the accumulation of modified Fatty Acids and providing mechanisms to circumvent these defects are vital steps in the development of tailored oil crops.
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overexpression of seipin1 increases oil in Hydroxy Fatty Acid accumulating seeds
Plant and Cell Physiology, 2018Co-Authors: Daniel Lunn, James G Wallis, John BrowseAbstract:While plant oils are an important source of food, plants also produce oils containing specialized Fatty Acids with chemical and physical properties valued in industry. Ricinoleic Acid, a Hydroxy Fatty Acid (HFA) produced in the seed of castor (Ricinus communis), is of particular value, with a wide range of applications. Since castor cultivation is currently successful only in tropical climates, and because castor seed contain the toxin ricin, there are ongoing efforts to develop a temperate crop capable of HFA biosynthesis. In castor, ricinoleic Acid is incorporated into triacylglycerol (TAG) which accumulates in the seed lipid droplets. Research in the model plant Arabidopsis (Arabidopsis thaliana) has successfully produced HFA constituting 30% of the total seed oil, but this is far short of the level required to engineer commercially viable crops. Strategies to increase HFA have centered on co-expression of castor TAG biosynthesis enzymes. However, since lipid droplets are the location of neutral lipid storage, manipulating droplets offers an alternative method to increase oil that contains specialized Fatty Acids. The Arabidopsis Seipin1 protein modulates TAG accumulation by affecting lipid droplet size. Here, we overexpress Seipin1 in a Hydroxylase-expressing Arabidopsis line, increasing seed HFA by 62% and proportionally increasing total oil. Increased seed oil was concomitant with a 22% increase in single seed weight and a 69% increase in harvest weight, while seed germination rose by 45%. Because Seipin1 function is unaffected by the structure of the HFA, these results demonstrate a novel strategy that may increase accumulation of many specialized seed oils.
James G Wallis - One of the best experts on this subject based on the ideXlab platform.
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castor lpcat and pdat1a act in concert to promote transacylation of Hydroxy Fatty Acid onto triacylglycerol
Plant Physiology, 2020Co-Authors: Daniel Lunn, James G Wallis, John BrowseAbstract:Oilseeds produce abundant triacylglycerol (TAG) during seed maturation to fuel the establishment of photoautotrophism in the subsequent generation. Commonly, TAG contains 18-carbon polyunsaturated Fatty Acids (FA), but plants also produce oils with unique chemical properties highly desirable for industrial processes. Unfortunately, plants that produce such oils are poorly suited to agronomic exploitation, leading to a desire to reconstitute novel oil biosynthesis in crop plants. Here, we studied the production and incorporation of Hydroxy-Fatty Acids (HFA) onto TAG in Arabidopsis (Arabidopsis thaliana) plants expressing the castor (Ricinus communis) FAH12 Hydroxylase. One factor limiting HFA accumulation in these plants is the inefficient removal of HFA from the site of synthesis on phosphatidylcholine (PC). In Arabidopsis, lysophosphatidic Acid acyltransferase (LPCAT) cycles FA to and from PC for modification. We reasoned that the castor LPCAT (RcLPCAT) would preferentially remove HFA from PC, resulting in greater incorporation onto TAG. However, expressing RcLPCAT in Arabidopsis expressing FAH12 alone (line CL37) or together with castor acyl:coenzyme A:diacylglycerol acyltransferase2 reduced HFA and total oil yield. Detailed analysis indicated that RcLPCAT reduced the removal of HFA from PC, possibly by competing with the endogenous LPCAT isozymes. Significantly, coexpressing RcLPCAT with castor phospholipid:diacylglycerol acyltransferase increased novel FA and total oil contents by transferring HFA from PC to diacylglycerol. Our results demonstrate that a detailed understanding is required to engineer modified FA production in oilseeds and suggest that phospholipase A2 enzymes rather than LPCAT mediate the highly efficient removal of HFA from PC in castor seeds.
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development defects of Hydroxy Fatty Acid accumulating seeds are reduced by castor acyltransferases
Plant Physiology, 2018Co-Authors: Daniel Lunn, James G Wallis, Gracen A Smith, John BrowseAbstract:Researchers have long endeavored to produce modified Fatty Acids in easily managed crop plants where they are not natively found. An important step toward this goal has been the biosynthesis of these valuable products in model oilseeds. The successful production of such Fatty Acids has revealed barriers to the broad application of this technology, including low seed oil and low proportion of the introduced Fatty Acid and reduced seed vigor. Here, we analyze the impact of producing Hydroxy-Fatty Acids on seedling development. We show that germinating seeds of a Hydroxy-Fatty Acid-accumulating Arabidopsis (Arabidopsis thaliana) line produce chlorotic cotyledons and suffer reduced photosynthetic capacity. These seedlings retain Hydroxy-Fatty Acids in polar lipids, including chloroplast lipids, and exhibit decreased Fatty Acid synthesis. Triacylglycerol mobilization in seedling development also is reduced, especially for lipids that include Hydroxy-Fatty Acid moieties. These developmental defects are ameliorated by increased flux of Hydroxy-Fatty Acids into seed triacylglycerol created through the expression of either castor (Ricinus communis) acyltransferase enzyme ACYL-COA:DIACYLGLYCEROL ACYLTRANSFERASE2 or PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1A. Such expression increases both the level of total stored triacylglycerol and the rate at which it is mobilized, fueling Fatty Acid synthesis and restoring photosynthetic capacity. Our results suggest that further improvements in seedling development may require the specific mobilization of triacylglycerol-containing Hydroxy-Fatty Acids. Understanding the defects in early development caused by the accumulation of modified Fatty Acids and providing mechanisms to circumvent these defects are vital steps in the development of tailored oil crops.
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overexpression of seipin1 increases oil in Hydroxy Fatty Acid accumulating seeds
Plant and Cell Physiology, 2018Co-Authors: Daniel Lunn, James G Wallis, John BrowseAbstract:While plant oils are an important source of food, plants also produce oils containing specialized Fatty Acids with chemical and physical properties valued in industry. Ricinoleic Acid, a Hydroxy Fatty Acid (HFA) produced in the seed of castor (Ricinus communis), is of particular value, with a wide range of applications. Since castor cultivation is currently successful only in tropical climates, and because castor seed contain the toxin ricin, there are ongoing efforts to develop a temperate crop capable of HFA biosynthesis. In castor, ricinoleic Acid is incorporated into triacylglycerol (TAG) which accumulates in the seed lipid droplets. Research in the model plant Arabidopsis (Arabidopsis thaliana) has successfully produced HFA constituting 30% of the total seed oil, but this is far short of the level required to engineer commercially viable crops. Strategies to increase HFA have centered on co-expression of castor TAG biosynthesis enzymes. However, since lipid droplets are the location of neutral lipid storage, manipulating droplets offers an alternative method to increase oil that contains specialized Fatty Acids. The Arabidopsis Seipin1 protein modulates TAG accumulation by affecting lipid droplet size. Here, we overexpress Seipin1 in a Hydroxylase-expressing Arabidopsis line, increasing seed HFA by 62% and proportionally increasing total oil. Increased seed oil was concomitant with a 22% increase in single seed weight and a 69% increase in harvest weight, while seed germination rose by 45%. Because Seipin1 function is unaffected by the structure of the HFA, these results demonstrate a novel strategy that may increase accumulation of many specialized seed oils.
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a high throughput screen for genes from castor that boost Hydroxy Fatty Acid accumulation in seed oils of transgenic arabidopsis
Plant Journal, 2006Co-Authors: Chaofu Lu, Martin Fulda, James G Wallis, John BrowseAbstract:Summary It is desirable to produce high homogeneity of novel Fatty Acids in oilseeds through genetic engineering to meet the increasing demands of the oleo-chemical industry. However, expression of key enzymes for biosynthesis of industrial Fatty Acids usually results in low levels of desired Fatty Acids in transgenic oilseeds. The abundance of derivatized Fatty Acids in their natural species suggests that additional genes are needed for high production in transgenic plants. We used the model oilseed plant Arabidopsis thaliana expressing a castor Fatty Acid Hydroxylase (FAH12) to identify genes that can boost Hydroxy Fatty Acid accumulation in transgenic seeds. Here we describe a high-throughput approach that, in principle, can allow testing of the entire transcriptome of developing castor seed endosperm by shotgun transforming a full-length cDNA library into an FAH12-expressing Arabidopsis line. The resulting transgenic seeds were screened by high-throughput gas chromatography. We obtained several lines transformed with castor cDNAs that contained increased amounts of Hydroxy Fatty Acids in transgenic Arabidopsis. These cDNAs were then isolated by PCR and retransformed into the FAH12-expressing line, thus confirming their beneficial contributions to Hydroxy Fatty Acid accumulation in transgenic Arabidopsis seeds. Although we describe an approach that is targeted to oilseed engineering, the methods we developed can be applied in many areas of plant biotechnology and functional genomic research.
Chaofu Lu - One of the best experts on this subject based on the ideXlab platform.
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The phosphatidylcholine diacylglycerol cholinephosphotransferase is required for efficient Hydroxy Fatty Acid accumulation in transgenic Arabidopsis.
Plant Physiology, 2012Co-Authors: Zhaohui Hu, Chaofu LuAbstract:We previously identified an enzyme, phosphatidylcholine diacylglycerol cholinephosphotransferase (PDCT), that plays an important role in directing Fatty acyl fluxes during triacylglycerol (TAG) biosynthesis. The PDCT mediates a symmetrical interconversion between phosphatidylcholine (PC) and diacylglycerol (DAG), thus enriching PC-modified Fatty Acids in the DAG pool prior to forming TAG. We show here that PDCT is required for the efficient metabolism of engineered Hydroxy Fatty Acids in Arabidopsis (Arabidopsis thaliana) seeds. When a Fatty Acid Hydroxylase (FAH12) from castor (Ricinus communis) was expressed in Arabidopsis seeds, the PDCT-deficient mutant accumulated only about half the amount of Hydroxy Fatty Acids compared with that in the wild-type seeds. We also isolated a PDCT from castor encoded by the RcROD1 (Reduced Oleate Desaturation1) gene. Seed-specific coexpression of this enzyme significantly increased Hydroxy Fatty Acid accumulation in wild type-FAH12 and in a previously produced transgenic Arabidopsis line coexpressing a castor diacylglycerol acyltransferase 2. Analyzing the TAG molecular species and regiochemistry, along with analysis of Fatty Acid composition in TAG and PC during seed development, indicate that PDCT acts in planta to enhance the fluxes of Fatty Acids through PC and enrich the Hydroxy Fatty Acids in DAG, and thus in TAG. In addition, PDCT partially restores the oil content that is decreased in FAH12-expressing seeds. Our results add a new gene in the genetic toolbox for efficiently engineering unusual Fatty Acids in transgenic oilseeds.
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a high throughput screen for genes from castor that boost Hydroxy Fatty Acid accumulation in seed oils of transgenic arabidopsis
Plant Journal, 2006Co-Authors: Chaofu Lu, Martin Fulda, James G Wallis, John BrowseAbstract:Summary It is desirable to produce high homogeneity of novel Fatty Acids in oilseeds through genetic engineering to meet the increasing demands of the oleo-chemical industry. However, expression of key enzymes for biosynthesis of industrial Fatty Acids usually results in low levels of desired Fatty Acids in transgenic oilseeds. The abundance of derivatized Fatty Acids in their natural species suggests that additional genes are needed for high production in transgenic plants. We used the model oilseed plant Arabidopsis thaliana expressing a castor Fatty Acid Hydroxylase (FAH12) to identify genes that can boost Hydroxy Fatty Acid accumulation in transgenic seeds. Here we describe a high-throughput approach that, in principle, can allow testing of the entire transcriptome of developing castor seed endosperm by shotgun transforming a full-length cDNA library into an FAH12-expressing Arabidopsis line. The resulting transgenic seeds were screened by high-throughput gas chromatography. We obtained several lines transformed with castor cDNAs that contained increased amounts of Hydroxy Fatty Acids in transgenic Arabidopsis. These cDNAs were then isolated by PCR and retransformed into the FAH12-expressing line, thus confirming their beneficial contributions to Hydroxy Fatty Acid accumulation in transgenic Arabidopsis seeds. Although we describe an approach that is targeted to oilseed engineering, the methods we developed can be applied in many areas of plant biotechnology and functional genomic research.
