Proceedings of 2022 AOCS Annual Meeting & Expo
Acetyl-triacylglycerols (acetyl-TAG) produced in the seeds of different Euonymus species are tria... more Acetyl-triacylglycerols (acetyl-TAG) produced in the seeds of different Euonymus species are triacylglycerols (TAG) that possess an sn-3 acetate group instead of a long chain fatty acid. This unusual structure confers useful properties to acetyl-TAG, including reduced kinematic viscosity and improved cold temperature performance. Acetyl-TAG are synthesized by unique diacylglycerol acetyltransferases (DAcTs), expressed in the endosperm of Euonymus seeds, that use acetyl-CoA to acetylate the sn-3 position of diacylglycerol (DAG) molecules. Isolation and expression of DAcT enzymes from different Euonymus species has resulted in the successful accumulation of high levels of acetyl-TAG in different oil seed crops. For example, expression of EfDAcT isolated from E. fortunei caused acetyl-TAG levels of 81 mol% in camelina seeds and 51 mol% in pennycress. To increase acetyl-CoA supply for EfDAcT, CRISPR-based genome editing was used to generate mutations in FATTY ACID ELONGASE1 (FAE1) genes...
Thlapsi arvense L. (pennycress) is being developed as a profitable oilseed cover crop for the win... more Thlapsi arvense L. (pennycress) is being developed as a profitable oilseed cover crop for the winter fallow period throughout the temperate regions of the world, controlling soil erosion and nutrients run-off on otherwise barren farmland. We demonstrate that pennycress can serve as a user-friendly model system akin to Arabidopsis that is well-suited for both laboratory and field experimentation. We sequenced the diploid genome of the spring-type Spring 32-10 inbred line (1C DNA content of 539 Mb; 2n = 14), identifying variation that may explain phenotypic differences with winter-type pennycress, as well as predominantly a one-to-one correspondence with Arabidopsis genes, which makes translational research straightforward. We developed an Agrobacterium-mediated floral dip transformation method (0.5% transformation efficiency) and introduced CRISPR-Cas9 constructs to produce indel mutations in the putative FATTY ACID ELONGATION1 (FAE1) gene, thereby abolishing erucic acid production a...
Thlaspi arvense (field pennycress) is being domesticated as a winter annual oilseed crop capable ... more Thlaspi arvense (field pennycress) is being domesticated as a winter annual oilseed crop capable of improving ecosystems and intensifying agricultural productivity without increasing land use. It is a selfing diploid with a short life cycle and is amenable to genetic manipulations, making it an accessible field-based model species for genetics and epigenetics. The availability of a high quality reference genome is vital for understanding pennycress physiology and for clarifying its evolutionary history within the Brassicaceae. Here, we present a chromosome-level genome assembly of var. MN106-Ref with improved gene annotation, and use it to investigate gene structure differences between two accessions (MN108 and Spring32-10) that are highly amenable to genetic transformation. We describe small RNAs, pseudogenes, and transposable elements, and highlight tissue specific expression and methylation patterns. Resequencing of forty wild accessions provides insights into genome-wide genetic variation as well as QTL regions for flowering time and a seedling color phenotype. Altogether, these data will serve as a tool for pennycress improvement in general and for translational research across the Brassicaceae.
Thlaspi arvense L. (pennycress) is a cold-tolerant Brassicaceae that produces large amounts of se... more Thlaspi arvense L. (pennycress) is a cold-tolerant Brassicaceae that produces large amounts of seeds rich in triacylglycerols and protein, making it an attractive target for domestication into an offseason oilseed cash cover crop. Pennycress is easily genetically transformed, enabling synthetic biology approaches to tailor oil properties for specific biofuel and industrial applications. To test the feasibility in pennycress of producing TAGs and acetyl-TAGs rich in medium-chain fatty acids (MCFAs; C6–C14) for industrial, biojet fuel and improved biodiesel applications, we generated transgenic lines with seed-specific expression of unique acyltransferase (LPAT and diacylglycerol acyltransferase) genes and thioesterase (FatB) genes isolated from Cuphea viscosissima, Cuphea avigera var. pulcherrima, Cuphea hookeriana, Coco nucifera, and Umbellularia californica. Wild-type pennycress seed TAGs accumulate no fatty acids shorter than 16C and less than 5 mol percent C16 as palmitic acid (1...
Project Goals: Our goal is to identify and characterize lines having traits that will improve pen... more Project Goals: Our goal is to identify and characterize lines having traits that will improve pennycress efficiency and utility as a biofuel feedstock species and make the seed easier for producers to handle, namely: 1) Increased seed size and 2) Increased seed oil content. To advance towards this goal, we are: 1) Investigating the genetic control of these traits in wildgermplasm collections using quantitative trait loci (QTL) and association mapping; 2) Identifying and characterizing EMS-induced lines for these traits using high-throughput NIRS and seed analyzer screening in combination with genomics tools; 3) Generate CRISPR-Cas9 knockouts in the genes known and found to regulate these traits in Brassica species.
Growing concerns over food insecurity and ecosystems health related to population growth and clim... more Growing concerns over food insecurity and ecosystems health related to population growth and climate change have challenged scientists to develop new crops, employing revolutionary technologies in combination with traditional methods. In this review, we discuss the domestication of the oilseed-producing cover crop pennycress, which along with the development of other new crops and improvements to farming practices can provide sustainable solutions to address malnutrition and environmental impacts of production agriculture. We highlight some of the new technologies such as bioinformatics-enabled next-generation sequencing and CRISPR genome editing in combination with traditional mutation breeding that has accelerated pennycress development as a new crop and a potential model system. Furthermore, we provide a brief overview of the technologies that can be integrated for improving pennycress and other crops and the status of pennycress development using these technologies.
Angiosperms represent most of the terrestrial plants and are the primary research focus for the c... more Angiosperms represent most of the terrestrial plants and are the primary research focus for the conversion of biomass to liquid fuels and coproducts. Lignin limits our access to fibers and represents a large fraction of the chemical energy stored in plant cell walls. Recently, the incorporation of monolignol ferulates into lignin polymers was accomplished via the engineering of an exotic transferase into commercially relevant poplar. We report that various angiosperm species might have convergently evolved to natively produce lignins that incorporate monolignol ferulate conjugates. We show that this activity may be accomplished by a BAHD feruloyl-coenzyme A monolignol transferase, OsFMT1 (AT5), in rice and its orthologs in other monocots.
Proceedings of the National Academy of Sciences, 1999
Gravitropism allows plant organs to direct their growth at a specific angle from the gravity vect... more Gravitropism allows plant organs to direct their growth at a specific angle from the gravity vector, promoting upward growth for shoots and downward growth for roots. Little is known about the mechanisms underlying gravitropic signal transduction. We found that mutations in the ARG1 locus of Arabidopsis thaliana alter root and hypocotyl gravitropism without affecting phototropism, root growth responses to phytohormones or inhibitors of auxin transport, or starch accumulation. The positional cloning of ARG1 revealed a DnaJ-like protein containing a coiled-coil region homologous to coiled coils found in cytoskeleton-interacting proteins. These data suggest that ARG1 participates in a gravity-signaling process involving the cytoskeleton. A combination of Northern blot studies and analysis of ARG1-GUS fusion-reporter expression in transgenic plants demonstrated that ARG1 is expressed in all organs. Ubiquitous ARG1 expression in Arabidopsis and the identification of an ortholog in Caenor...
Proceedings of the National Academy of Sciences, 1998
Auxins are plant hormones that mediate many aspects of plant growth and development. In higher pl... more Auxins are plant hormones that mediate many aspects of plant growth and development. In higher plants, auxins are polarly transported from sites of synthesis in the shoot apex to their sites of action in the basal regions of shoots and in roots. Polar auxin transport is an important aspect of auxin functions and is mediated by cellular influx and efflux carriers. Little is known about the molecular identity of its regulatory component, the efflux carrier [Estelle, M. (1996) Current Biol. 6, 1589–1591]. Here we show that mutations in the Arabidopsis thaliana AGRAVITROPIC 1 ( AGR1 ) gene involved in root gravitropism confer increased root-growth sensitivity to auxin and decreased sensitivity to ethylene and an auxin transport inhibitor, and cause retention of exogenously added auxin in root tip cells. We used positional cloning to show that AGR1 encodes a putative transmembrane protein whose amino acid sequence shares homologies with bacterial transporters. When expressed in Saccharom...
Additional file 1. Figure 1. Molecular characterization of the mu1013391 insertion in Zmccr1. A) ... more Additional file 1. Figure 1. Molecular characterization of the mu1013391 insertion in Zmccr1. A) Scale diagram of the ZmCCR1 locus GRMZM2G131205, Chr1: 211567137..211573211. Black and white boxes represent exons and untranslated regions, respectively, while lines indicate introns. Shown is the location of the Uniform Mu insertion mu1013391 (triangle) in seed lots UFMu00732 (Zmccr 1a) and UFMu01379 (Zmccr 1b) along with relative primer locations (arrows). B) Shown are agarose gel-electrophoresed PCR products indicating either the presence (primers T1 + R1, 375 bp) or absence (primers F1 + R1, 537 bp) of the mu1013391 insertion in either plants homozygous for the mu1013391 insertion (Zmccr 1a and 1b) or wild-type W22. (C) Agarose gel-electrophoresed PCR products semi-quantitatively amplified from reverse-transcribed first-strand cDNA from plants either homozygous for the mu1013391 insertion (Zmccr 1a and 1b) or WT B73 (primers F2 + R2, ~408 bp). Note that the Zmccr 1a and 1b PCR produ...
Pennycress (Thlaspi arvense L.) is being domesticated as an oilseed cash cover crop to be grown i... more Pennycress (Thlaspi arvense L.) is being domesticated as an oilseed cash cover crop to be grown in the off-season throughout temperate regions of the world. With its diploid genome and ease of directed mutagenesis using molecular approaches, pennycress seed oil composition can be rapidly tailored for a plethora of food, feed, oleochemical and fuel uses. Here, we utilized Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology to produce knockout mutations in the FATTY ACID DESATURASE2 (FAD2) and REDUCED OLEATE DESATURATION1 (ROD1) genes to increase oleic acid content. High oleic acid (18:1) oil is valued for its oxidative stability that is superior to the polyunsaturated fatty acids (PUFAs) linoleic (18:2) and linolenic (18:3), and better cold flow properties than the very long chain fatty acid (VLCFA) erucic (22:1). When combined with a FATTY ACID ELONGATION1 (fae1) knockout mutation, fad2 fae1 and rod1 fae1 double mutants produced ∼90% and ∼60% oleic aci...
Central to building and reorganizing cytoskeletal arrays is the creation of new polymers. While n... more Central to building and reorganizing cytoskeletal arrays is the creation of new polymers. While nucleation has been the major focus of study for new microtubule generation, severing has been proposed as an alternative mechanism to create new polymers, a mechanism recently shown to drive the reorientation of cortical arrays of higher plants in response to blue light perception. As severing produces new plus ends behind the stabilizing GTP-cap, an important and unanswered question is how these are stabilized in vivo to promote net microtubule generation. Here we identify the conserved protein CLASP as a potent stabilizer of new plus ends created by katanin severing and find that CLASP is required for rapid cortical array reorientation. In clasp mutants both rescue of shrinking plus ends and the regrowth of plus ends immediately after severing are reduced, computational modeling reveals that it is the specific stabilization of severed ends that explains CLASP’s function in promoting mi...
The oilseed species Thlaspi arvense (pennycress) is being domesticated as a new crop that can pro... more The oilseed species Thlaspi arvense (pennycress) is being domesticated as a new crop that can provide both important ecosystem services and intensify farmland output. Through the use of high throughput sequencing and phenotyping, along with classical mutagenesis key traits needed for pennycress domestication have been identified. Domestication traits identified herein include reduced pod shatter, early maturity, reduced seed glucosinolate levels, and improved oil fatty acid content. By taking advantage of pennycress close genetic relationship with Arabidopsis thaliana, the causative mutations responsible for each of these traits have been identified. These mutations have been used to develop molecular markers to begin to stack the traits into individual lines.
Central to the building and reorganizing cytoskeletal arrays is creation of new polymers. Althoug... more Central to the building and reorganizing cytoskeletal arrays is creation of new polymers. Although nucleation has been the major focus of study for microtubule generation, severing has been proposed as an alternative mechanism to create new polymers, a mechanism recently shown to drive the reorientation of cortical arrays of higher plants in response to blue light perception. Severing produces new plus ends behind the stabilizing GTP-cap. An important and unanswered question is how these ends are stabilized in vivo to promote net microtubule generation. Here we identify the conserved protein CLASP as a potent stabilizer of new plus ends created by katanin severing in plant cells. Clasp mutants are defective in cortical array reorientation. In these mutants, both rescue of shrinking plus ends and the stabilization of plus ends immediately after severing are reduced. Computational modeling reveals that it is the specific stabilization of severed ends that best explains CLASP’s functio...
Thlapsi arvense L. (pennycress) is being developed as a profitable oilseed cover crop for the win... more Thlapsi arvense L. (pennycress) is being developed as a profitable oilseed cover crop for the winter fallow period throughout temperate regions of the world, controlling soil erosion and nutrients runoff on otherwise barren farmland. We demonstrate that pennycress can serve as a user-friendly model system akin to Arabidopsis that is well-suited for both laboratory and field experimentation. We sequenced the diploid genome of the spring-type Spring 32-10 inbred line (1C DNA content of 539 Mb; 2n=14), identifying variation that may explain phenotypic differences with winter-type pennycress, as well as predominantly a one-to-one correspondence with Arabidopsis genes, which makes translational research straightforward. We developed an Agrobacterium-mediated floral dip transformation method (0.5% transformation efficiency) and introduced CRISPR-Cas9 constructs to produce indel mutations in the putative FATTY ACID ELONGATION1 (FAE1) gene, thereby abolishing erucic acid production and crea...
The cell wall polymer lignin provides structural support and rigidity to plant cell walls, and th... more The cell wall polymer lignin provides structural support and rigidity to plant cell walls, and therefore to the plant body. However, the recalcitrance associated with lignin impedes the extraction of polysaccharides from the cell wall to make plant-based biofuels and biomaterials. The cell wall digestibility can be improved by introducing labile ester bonds into the lignin backbone that can be easily broken under mild base treatment at room temperature. The FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) enzyme, which may be naturally found in many plants, uses feruloyl-CoA and monolignols to synthesize the ester-linked monolignol ferulate conjugates. A mutation in the first lignin-specific biosynthetic enzyme, CINNAMOYL-CoA REDUCTASE (CCR), results in an increase in the intracellular pool of feruloyl-CoA. Maize (Zea mays) has a native putative FMT enzyme, and its ccr mutants produce an increased pool of feruloyl-CoA that can be used for conversion to monolignol ferulate conjugates. The d...
Proceedings of 2022 AOCS Annual Meeting & Expo
Acetyl-triacylglycerols (acetyl-TAG) produced in the seeds of different Euonymus species are tria... more Acetyl-triacylglycerols (acetyl-TAG) produced in the seeds of different Euonymus species are triacylglycerols (TAG) that possess an sn-3 acetate group instead of a long chain fatty acid. This unusual structure confers useful properties to acetyl-TAG, including reduced kinematic viscosity and improved cold temperature performance. Acetyl-TAG are synthesized by unique diacylglycerol acetyltransferases (DAcTs), expressed in the endosperm of Euonymus seeds, that use acetyl-CoA to acetylate the sn-3 position of diacylglycerol (DAG) molecules. Isolation and expression of DAcT enzymes from different Euonymus species has resulted in the successful accumulation of high levels of acetyl-TAG in different oil seed crops. For example, expression of EfDAcT isolated from E. fortunei caused acetyl-TAG levels of 81 mol% in camelina seeds and 51 mol% in pennycress. To increase acetyl-CoA supply for EfDAcT, CRISPR-based genome editing was used to generate mutations in FATTY ACID ELONGASE1 (FAE1) genes...
Thlapsi arvense L. (pennycress) is being developed as a profitable oilseed cover crop for the win... more Thlapsi arvense L. (pennycress) is being developed as a profitable oilseed cover crop for the winter fallow period throughout the temperate regions of the world, controlling soil erosion and nutrients run-off on otherwise barren farmland. We demonstrate that pennycress can serve as a user-friendly model system akin to Arabidopsis that is well-suited for both laboratory and field experimentation. We sequenced the diploid genome of the spring-type Spring 32-10 inbred line (1C DNA content of 539 Mb; 2n = 14), identifying variation that may explain phenotypic differences with winter-type pennycress, as well as predominantly a one-to-one correspondence with Arabidopsis genes, which makes translational research straightforward. We developed an Agrobacterium-mediated floral dip transformation method (0.5% transformation efficiency) and introduced CRISPR-Cas9 constructs to produce indel mutations in the putative FATTY ACID ELONGATION1 (FAE1) gene, thereby abolishing erucic acid production a...
Thlaspi arvense (field pennycress) is being domesticated as a winter annual oilseed crop capable ... more Thlaspi arvense (field pennycress) is being domesticated as a winter annual oilseed crop capable of improving ecosystems and intensifying agricultural productivity without increasing land use. It is a selfing diploid with a short life cycle and is amenable to genetic manipulations, making it an accessible field-based model species for genetics and epigenetics. The availability of a high quality reference genome is vital for understanding pennycress physiology and for clarifying its evolutionary history within the Brassicaceae. Here, we present a chromosome-level genome assembly of var. MN106-Ref with improved gene annotation, and use it to investigate gene structure differences between two accessions (MN108 and Spring32-10) that are highly amenable to genetic transformation. We describe small RNAs, pseudogenes, and transposable elements, and highlight tissue specific expression and methylation patterns. Resequencing of forty wild accessions provides insights into genome-wide genetic variation as well as QTL regions for flowering time and a seedling color phenotype. Altogether, these data will serve as a tool for pennycress improvement in general and for translational research across the Brassicaceae.
Thlaspi arvense L. (pennycress) is a cold-tolerant Brassicaceae that produces large amounts of se... more Thlaspi arvense L. (pennycress) is a cold-tolerant Brassicaceae that produces large amounts of seeds rich in triacylglycerols and protein, making it an attractive target for domestication into an offseason oilseed cash cover crop. Pennycress is easily genetically transformed, enabling synthetic biology approaches to tailor oil properties for specific biofuel and industrial applications. To test the feasibility in pennycress of producing TAGs and acetyl-TAGs rich in medium-chain fatty acids (MCFAs; C6–C14) for industrial, biojet fuel and improved biodiesel applications, we generated transgenic lines with seed-specific expression of unique acyltransferase (LPAT and diacylglycerol acyltransferase) genes and thioesterase (FatB) genes isolated from Cuphea viscosissima, Cuphea avigera var. pulcherrima, Cuphea hookeriana, Coco nucifera, and Umbellularia californica. Wild-type pennycress seed TAGs accumulate no fatty acids shorter than 16C and less than 5 mol percent C16 as palmitic acid (1...
Project Goals: Our goal is to identify and characterize lines having traits that will improve pen... more Project Goals: Our goal is to identify and characterize lines having traits that will improve pennycress efficiency and utility as a biofuel feedstock species and make the seed easier for producers to handle, namely: 1) Increased seed size and 2) Increased seed oil content. To advance towards this goal, we are: 1) Investigating the genetic control of these traits in wildgermplasm collections using quantitative trait loci (QTL) and association mapping; 2) Identifying and characterizing EMS-induced lines for these traits using high-throughput NIRS and seed analyzer screening in combination with genomics tools; 3) Generate CRISPR-Cas9 knockouts in the genes known and found to regulate these traits in Brassica species.
Growing concerns over food insecurity and ecosystems health related to population growth and clim... more Growing concerns over food insecurity and ecosystems health related to population growth and climate change have challenged scientists to develop new crops, employing revolutionary technologies in combination with traditional methods. In this review, we discuss the domestication of the oilseed-producing cover crop pennycress, which along with the development of other new crops and improvements to farming practices can provide sustainable solutions to address malnutrition and environmental impacts of production agriculture. We highlight some of the new technologies such as bioinformatics-enabled next-generation sequencing and CRISPR genome editing in combination with traditional mutation breeding that has accelerated pennycress development as a new crop and a potential model system. Furthermore, we provide a brief overview of the technologies that can be integrated for improving pennycress and other crops and the status of pennycress development using these technologies.
Angiosperms represent most of the terrestrial plants and are the primary research focus for the c... more Angiosperms represent most of the terrestrial plants and are the primary research focus for the conversion of biomass to liquid fuels and coproducts. Lignin limits our access to fibers and represents a large fraction of the chemical energy stored in plant cell walls. Recently, the incorporation of monolignol ferulates into lignin polymers was accomplished via the engineering of an exotic transferase into commercially relevant poplar. We report that various angiosperm species might have convergently evolved to natively produce lignins that incorporate monolignol ferulate conjugates. We show that this activity may be accomplished by a BAHD feruloyl-coenzyme A monolignol transferase, OsFMT1 (AT5), in rice and its orthologs in other monocots.
Proceedings of the National Academy of Sciences, 1999
Gravitropism allows plant organs to direct their growth at a specific angle from the gravity vect... more Gravitropism allows plant organs to direct their growth at a specific angle from the gravity vector, promoting upward growth for shoots and downward growth for roots. Little is known about the mechanisms underlying gravitropic signal transduction. We found that mutations in the ARG1 locus of Arabidopsis thaliana alter root and hypocotyl gravitropism without affecting phototropism, root growth responses to phytohormones or inhibitors of auxin transport, or starch accumulation. The positional cloning of ARG1 revealed a DnaJ-like protein containing a coiled-coil region homologous to coiled coils found in cytoskeleton-interacting proteins. These data suggest that ARG1 participates in a gravity-signaling process involving the cytoskeleton. A combination of Northern blot studies and analysis of ARG1-GUS fusion-reporter expression in transgenic plants demonstrated that ARG1 is expressed in all organs. Ubiquitous ARG1 expression in Arabidopsis and the identification of an ortholog in Caenor...
Proceedings of the National Academy of Sciences, 1998
Auxins are plant hormones that mediate many aspects of plant growth and development. In higher pl... more Auxins are plant hormones that mediate many aspects of plant growth and development. In higher plants, auxins are polarly transported from sites of synthesis in the shoot apex to their sites of action in the basal regions of shoots and in roots. Polar auxin transport is an important aspect of auxin functions and is mediated by cellular influx and efflux carriers. Little is known about the molecular identity of its regulatory component, the efflux carrier [Estelle, M. (1996) Current Biol. 6, 1589–1591]. Here we show that mutations in the Arabidopsis thaliana AGRAVITROPIC 1 ( AGR1 ) gene involved in root gravitropism confer increased root-growth sensitivity to auxin and decreased sensitivity to ethylene and an auxin transport inhibitor, and cause retention of exogenously added auxin in root tip cells. We used positional cloning to show that AGR1 encodes a putative transmembrane protein whose amino acid sequence shares homologies with bacterial transporters. When expressed in Saccharom...
Additional file 1. Figure 1. Molecular characterization of the mu1013391 insertion in Zmccr1. A) ... more Additional file 1. Figure 1. Molecular characterization of the mu1013391 insertion in Zmccr1. A) Scale diagram of the ZmCCR1 locus GRMZM2G131205, Chr1: 211567137..211573211. Black and white boxes represent exons and untranslated regions, respectively, while lines indicate introns. Shown is the location of the Uniform Mu insertion mu1013391 (triangle) in seed lots UFMu00732 (Zmccr 1a) and UFMu01379 (Zmccr 1b) along with relative primer locations (arrows). B) Shown are agarose gel-electrophoresed PCR products indicating either the presence (primers T1 + R1, 375 bp) or absence (primers F1 + R1, 537 bp) of the mu1013391 insertion in either plants homozygous for the mu1013391 insertion (Zmccr 1a and 1b) or wild-type W22. (C) Agarose gel-electrophoresed PCR products semi-quantitatively amplified from reverse-transcribed first-strand cDNA from plants either homozygous for the mu1013391 insertion (Zmccr 1a and 1b) or WT B73 (primers F2 + R2, ~408 bp). Note that the Zmccr 1a and 1b PCR produ...
Pennycress (Thlaspi arvense L.) is being domesticated as an oilseed cash cover crop to be grown i... more Pennycress (Thlaspi arvense L.) is being domesticated as an oilseed cash cover crop to be grown in the off-season throughout temperate regions of the world. With its diploid genome and ease of directed mutagenesis using molecular approaches, pennycress seed oil composition can be rapidly tailored for a plethora of food, feed, oleochemical and fuel uses. Here, we utilized Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology to produce knockout mutations in the FATTY ACID DESATURASE2 (FAD2) and REDUCED OLEATE DESATURATION1 (ROD1) genes to increase oleic acid content. High oleic acid (18:1) oil is valued for its oxidative stability that is superior to the polyunsaturated fatty acids (PUFAs) linoleic (18:2) and linolenic (18:3), and better cold flow properties than the very long chain fatty acid (VLCFA) erucic (22:1). When combined with a FATTY ACID ELONGATION1 (fae1) knockout mutation, fad2 fae1 and rod1 fae1 double mutants produced ∼90% and ∼60% oleic aci...
Central to building and reorganizing cytoskeletal arrays is the creation of new polymers. While n... more Central to building and reorganizing cytoskeletal arrays is the creation of new polymers. While nucleation has been the major focus of study for new microtubule generation, severing has been proposed as an alternative mechanism to create new polymers, a mechanism recently shown to drive the reorientation of cortical arrays of higher plants in response to blue light perception. As severing produces new plus ends behind the stabilizing GTP-cap, an important and unanswered question is how these are stabilized in vivo to promote net microtubule generation. Here we identify the conserved protein CLASP as a potent stabilizer of new plus ends created by katanin severing and find that CLASP is required for rapid cortical array reorientation. In clasp mutants both rescue of shrinking plus ends and the regrowth of plus ends immediately after severing are reduced, computational modeling reveals that it is the specific stabilization of severed ends that explains CLASP’s function in promoting mi...
The oilseed species Thlaspi arvense (pennycress) is being domesticated as a new crop that can pro... more The oilseed species Thlaspi arvense (pennycress) is being domesticated as a new crop that can provide both important ecosystem services and intensify farmland output. Through the use of high throughput sequencing and phenotyping, along with classical mutagenesis key traits needed for pennycress domestication have been identified. Domestication traits identified herein include reduced pod shatter, early maturity, reduced seed glucosinolate levels, and improved oil fatty acid content. By taking advantage of pennycress close genetic relationship with Arabidopsis thaliana, the causative mutations responsible for each of these traits have been identified. These mutations have been used to develop molecular markers to begin to stack the traits into individual lines.
Central to the building and reorganizing cytoskeletal arrays is creation of new polymers. Althoug... more Central to the building and reorganizing cytoskeletal arrays is creation of new polymers. Although nucleation has been the major focus of study for microtubule generation, severing has been proposed as an alternative mechanism to create new polymers, a mechanism recently shown to drive the reorientation of cortical arrays of higher plants in response to blue light perception. Severing produces new plus ends behind the stabilizing GTP-cap. An important and unanswered question is how these ends are stabilized in vivo to promote net microtubule generation. Here we identify the conserved protein CLASP as a potent stabilizer of new plus ends created by katanin severing in plant cells. Clasp mutants are defective in cortical array reorientation. In these mutants, both rescue of shrinking plus ends and the stabilization of plus ends immediately after severing are reduced. Computational modeling reveals that it is the specific stabilization of severed ends that best explains CLASP’s functio...
Thlapsi arvense L. (pennycress) is being developed as a profitable oilseed cover crop for the win... more Thlapsi arvense L. (pennycress) is being developed as a profitable oilseed cover crop for the winter fallow period throughout temperate regions of the world, controlling soil erosion and nutrients runoff on otherwise barren farmland. We demonstrate that pennycress can serve as a user-friendly model system akin to Arabidopsis that is well-suited for both laboratory and field experimentation. We sequenced the diploid genome of the spring-type Spring 32-10 inbred line (1C DNA content of 539 Mb; 2n=14), identifying variation that may explain phenotypic differences with winter-type pennycress, as well as predominantly a one-to-one correspondence with Arabidopsis genes, which makes translational research straightforward. We developed an Agrobacterium-mediated floral dip transformation method (0.5% transformation efficiency) and introduced CRISPR-Cas9 constructs to produce indel mutations in the putative FATTY ACID ELONGATION1 (FAE1) gene, thereby abolishing erucic acid production and crea...
The cell wall polymer lignin provides structural support and rigidity to plant cell walls, and th... more The cell wall polymer lignin provides structural support and rigidity to plant cell walls, and therefore to the plant body. However, the recalcitrance associated with lignin impedes the extraction of polysaccharides from the cell wall to make plant-based biofuels and biomaterials. The cell wall digestibility can be improved by introducing labile ester bonds into the lignin backbone that can be easily broken under mild base treatment at room temperature. The FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) enzyme, which may be naturally found in many plants, uses feruloyl-CoA and monolignols to synthesize the ester-linked monolignol ferulate conjugates. A mutation in the first lignin-specific biosynthetic enzyme, CINNAMOYL-CoA REDUCTASE (CCR), results in an increase in the intracellular pool of feruloyl-CoA. Maize (Zea mays) has a native putative FMT enzyme, and its ccr mutants produce an increased pool of feruloyl-CoA that can be used for conversion to monolignol ferulate conjugates. The d...
Angiosperms represent most of the terrestrial plants and are the primary research focus for the c... more Angiosperms represent most of the terrestrial plants and are the primary research focus for the conversion of biomass to liquid fuels and coproducts. Lignin limits our access to fibers and represents a large fraction of the chemical energy stored in plant cell walls. Recently, the incorporation of monolignol ferulates into lignin polymers was accomplished via the engineering of an exotic transferase into commercially relevant poplar. We report that various angiosperm species might have convergently evolved to natively produce lignins that incorporate monolignol ferulate conjugates. We show that this activity may be accomplished by a BAHD feruloyl–coenzyme A monolignol transferase, OsFMT1 (AT5), in rice and its orthologs in other monocots.
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Papers by John Sedbrook