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... 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...
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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.... 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...
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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... 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.
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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.... 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...
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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... 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 climate change have challenged scientists to develop new crops, employing revolutionary technologies in combination with traditional methods. In... 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.
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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... 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.
Research Interests: Science and Ferulic Acid
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Research Interests: Genetics, Plant Biology, Biology, Membrane Proteins, Cell Biology, and 14 moreMedicine, Cytoskeleton, Signal Transduction, Mutation, Gravitropism, Arabidopsis, Auxin, Plant cell, Membrane Protein, Hydrogen-Ion Concentration, Cytoplasm, Polar Auxin Transport, Protein Transport, and Biochemistry and cell biology
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... 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...
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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... 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...
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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,... 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 in the off-season throughout temperate regions of the world. With its diploid genome and ease of directed mutagenesis using molecular... 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 nucleation has been the major focus of study for new microtubule generation, severing has been proposed as an alternative mechanism to create... 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 provide both important ecosystem services and intensify farmland output. Through the use of high throughput sequencing and phenotyping, along... 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. Although nucleation has been the major focus of study for microtubule generation, severing has been proposed as an alternative mechanism to create... 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...
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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... 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...
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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... 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...
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Brachypodium distachyon (Brachypodium) has emerged as a useful model system for studying traits unique to graminaceous species including bioenergy crop grasses owing to its amenability to laboratory experimentation and the availability of... more
Brachypodium distachyon (Brachypodium) has emerged as a useful model system for studying traits unique to graminaceous species including bioenergy crop grasses owing to its amenability to laboratory experimentation and the availability of extensive genetic and germplasm resources. Considerable natural variation has been uncovered for a variety of traits including flowering time, vernalization responsiveness, and above-ground growth characteristics. However, cell wall composition differences remain underexplored. Therefore, we assessed cell wall-related traits relevant to biomass conversion to biofuels in seven Brachypodium inbred lines that were chosen based on their high level of genotypic diversity as well as available genome sequences and recombinant inbred line (RIL) populations. Senesced stems plus leaf sheaths from these lines exhibited significant differences in acetyl bromide soluble lignin (ABSL), cell wall polysaccharide-derived sugars, hydroxycinnamates content, and syrin...
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Triacylglycerol (TAG) is a storage lipid used for food purposes and as renewable feedstock for biodiesel production. WRINKLED1 (WRI1) is a transcription factor, which governs fatty acid (FA) synthesis and indirectly TAG accumulation in... more
Triacylglycerol (TAG) is a storage lipid used for food purposes and as renewable feedstock for biodiesel production. WRINKLED1 (WRI1) is a transcription factor, which governs fatty acid (FA) synthesis and indirectly TAG accumulation in oil storing plant tissues and its ectopic expression has led to TAG accumulation in vegetative tissues of different dicotyledonous plants. The ectopic expression of BdWRI1 in the grass Brachypodium (Brachypodium distachyon) induced the transcription of predicted genes involved in glycolysis and FA biosynthesis, and TAG content was increased up to 32.5-fold in 8-week-old leaf blades. However, the ectopic expression of BdWRI1 also caused cell death in leaves, which has not been previously observed in dicotyledonous plants such as Arabidopsis (Arabidopsis thaliana). Lipid analysis indicated that the free FA content was 2.0-fold elevated in BdWRI1-expressing leaf blades of Brachypodium. The transcription of predicted genes involved in β-oxidation was indu...
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Background/Question/Methods Nutrient recycling is an important mechanism for nitrogen (N) retention in plants and to reduce N losses and fertilizer inputs in perennial bioenergy cropping systems. N resorption in perennial plants varies... more
Background/Question/Methods Nutrient recycling is an important mechanism for nitrogen (N) retention in plants and to reduce N losses and fertilizer inputs in perennial bioenergy cropping systems. N resorption in perennial plants varies greatly and is attributed to both genetic and environmental factors. Soil N availability is of particular interest, as crops will likely be fertilized, and the effects of N availability on resorption remain unclear. The timing of N resorption also has significant agronomic implications, since delaying harvest reduces biomass yields. Therefore, we set out to answer 1) how variable is the timing and magnitude of N resorption and 2) do genetic and environmental factors affect N resorption? We collected tissue samples of switchgrass (Panicum virgatum) from naturally-occurring populations across Wisconsin, with sites ranging in soil type and productivity. We also collected samples from cultivated plants, where known genotypes of both upland and lowland eco...
We have analyzed annual Brachypodium for flowering time and nitrogen content when grown under a nitrogen gradient. Flowering time is correlated to whole plant N content, likely due to greater leaf mass in later flowering accessions. We... more
We have analyzed annual Brachypodium for flowering time and nitrogen content when grown under a nitrogen gradient. Flowering time is correlated to whole plant N content, likely due to greater leaf mass in later flowering accessions. We hope the easily transformable annuals can assist in gene characterizations of loci identified in perennial species. We have analyzed nitrogen content in perennial Brachypodium species to identify accessions that differ in nitrogen content following senescence. We are focusing on perennial Brachypodium species where simple genetic analysis is possible and the accessions are candidates for sequencing by JGI. To identify loci involved in flowering/senescence/nutrient recycling in dedicated bioenergy crops, we have analyzed nitrogen content in switchgrass and Miscanthus from locations that vary in soil type. Our goal is to understand how the timing of developmental processes influence nutrient recycling in bioenergy crops. We have extended our switchgrass...
Plants use the energy from sunlight to fix carbon dioxide and generate a number of energy rich molecules that are incorporated into cell walls (polysaccharides and lignin) as well as used for energy storage (eg. triacylglycerides).... more
Plants use the energy from sunlight to fix carbon dioxide and generate a number of energy rich molecules that are incorporated into cell walls (polysaccharides and lignin) as well as used for energy storage (eg. triacylglycerides). Researchers at the Great Lakes Bioenergy Research Center (GLBRC) are pursuing fundamental research that will allow for the engineering and breeding of bioenergy crop plants with increased amounts of easily accessible and convertible polysaccharides, and that accumulate energy-dense oils in vegetative tissues. This presentation will highlight Center efforts to 1) engineer lignin to contain zips that can readily be chemically cleaved thereby releasing cell wall polysaccharides for saccharification and fermentation; 2) identify genes and gene variants affecting polysaccharide composition, accumulation, and enzymatic digestibility; and 3) engineer plants to synthesize and retain up to 20% oil in vegetative tissues such as leaves, stems, and tubers.
The phenylpropanoid pathway in plants synthesizes a variety of structural and defence compounds, and is an important target in efforts to reduce cell wall lignin for improved biomass conversion to biofuels. Little is known concerning the... more
The phenylpropanoid pathway in plants synthesizes a variety of structural and defence compounds, and is an important target in efforts to reduce cell wall lignin for improved biomass conversion to biofuels. Little is known concerning the trade-offs in grasses when perturbing the function of the first gene family in the pathway, PHENYLALANINE AMMONIA LYASE (PAL). Therefore, PAL isoforms in the model grass Brachypodium distachyon were targeted, by RNA interference (RNAi), and large reductions (up to 85%) in stem tissue transcript abundance for two of the eight putative BdPAL genes were identified. The cell walls of stems of BdPAL-knockdown plants had reductions of 43% in lignin and 57% in cell wall-bound ferulate, and a nearly 2-fold increase in the amounts of polysaccharide-derived carbohydrates released by thermochemical and hydrolytic enzymic partial digestion. PAL-knockdown plants exhibited delayed development and reduced root growth, along with increased susceptibilities to the f...
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Oilseed crops are sources of oils and seed meal having a multitude of uses. While the domestication of soybean and rapeseed took extended periods of time, new genome-based techniques have ushered in an era where crop domestication can... more
Oilseed crops are sources of oils and seed meal having a multitude of uses. While the domestication of soybean and rapeseed took extended periods of time, new genome-based techniques have ushered in an era where crop domestication can occur rapidly. One attractive target for rapid domestication is the winter annual plant Field Pennycress (Thlaspi arvense L.; pennycress; Brassicaceae). Pennycress grows widespread throughout temperate regions of the world and could serve as a winter oilseed-producing cover crop. If grown throughout the USA Midwest Corn Belt, for example, pennycress could produce as much as 840L/ha oils and 1470kg/ha press-cake annually on 16 million hectares of farmland currently left fallow during the fall through spring months. However, wild pennycress strains have inconsistent germination and stand establishment, un-optimized maturity for a given growth zone, suboptimal oils and meal quality for biofuels and food production, and significant harvest loss due to pod ...
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Mutations at five loci named PEANUT1-5 (PNT) were identified in a genetic screen for radially swollen embryo mutants. pnt1 cell walls showed decreased crystalline cellulose, increased pectins, and irregular and ectopic deposition of... more
Mutations at five loci named PEANUT1-5 (PNT) were identified in a genetic screen for radially swollen embryo mutants. pnt1 cell walls showed decreased crystalline cellulose, increased pectins, and irregular and ectopic deposition of pectins, xyloglucans, and callose. Furthermore, pnt1 pollen is less viable than the wild type, and pnt1 embryos were delayed in morphogenesis and showed defects in shoot and root meristems. The PNT1 gene encodes the Arabidopsis thaliana homolog of mammalian PIG-M, an endoplasmic reticulum-localized mannosyltransferase that is required for synthesis of the glycosylphosphatidylinositol (GPI) anchor. All five pnt mutants showed strongly reduced accumulation of GPI-anchored proteins, suggesting that they all have defects in GPI anchor synthesis. Although the mutants are seedling lethal, pnt1 cells are able to proliferate for a limited time as undifferentiated callus and do not show the massive deposition of ectopic cell wall material seen in pnt1 embryos. Th...
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When Arabidopsis thaliana seedlings grow embedded in an agar-based medium, their roots grow vertically downward. This reflects their ability to sense the gravity vector and to position their tip parallel to it (gravitropism). We have... more
When Arabidopsis thaliana seedlings grow embedded in an agar-based medium, their roots grow vertically downward. This reflects their ability to sense the gravity vector and to position their tip parallel to it (gravitropism). We have isolated a number of mutations affecting root gravitropism in Arabidopsis thaliana. One of these mutations, named arg1, affects root and hypocotyl gravitropism without promoting defects in starch content or in the ability of seedlings' organs to respond to plant hormones. The ARG1 gene was cloned and shown to code for a protein with a J domain at its amino terminus and a second sequence motif found in several cytoskeleton binding proteins. Mutations in the AGR1 locus promote a strong defect in root gravitropism. Some alleles also confer an increased root resistance to exogenous ethylene and an increased sensitivity to auxin. AGR1 was cloned and found to encode a putative transmembrane protein which might be involved in polar auxin transport, or in r...
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Several lines of evidence indicate that the adaxial leaf domain possesses a unique competence to form shoot apical meristems. Factors required for this competence are expected to cause a defect in shoot apical meristem formation when... more
Several lines of evidence indicate that the adaxial leaf domain possesses a unique competence to form shoot apical meristems. Factors required for this competence are expected to cause a defect in shoot apical meristem formation when inactivated and to be expressed or active preferentially in the adaxial leaf domain. PINHEAD, a member of a family of proteins that includes the translation factor eIF2C, is required for reliable formation of primary and axillary shoot apical meristems. In addition to high-level expression in the vasculature, we find that low-level PINHEAD expression defines a novel domain of positional identity in the plant. This domain consists of adaxial leaf primordia and the meristem. These findings suggest that the PINHEAD gene product may be a component of a hypothetical meristem forming competence factor. We also describe defects in floral organ number and shape, as well as aberrant embryo and ovule development associated with pinhead mutants, thus elaborating o...
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ABSTRACT Gravity is an important cue that plant organs use to guide their growth. Each organ is characterized by a defined gravity set point angle that dictates its optimal orientation within the gravity field. Specialized cells, named... more
ABSTRACT Gravity is an important cue that plant organs use to guide their growth. Each organ is characterized by a defined gravity set point angle that dictates its optimal orientation within the gravity field. Specialized cells, named statocytes, enable this directional growth response by perceiving gravity via the sedimentation of, and/or tension/pressure exerted by, starch-filled plastids within their cytoplasm. Located in the columella region of the cap in roots and in the endodermis of hypocotyls and stems, these cells modulate the lateral transport of auxin across the corresponding organ in a gravistimulus-dependent manner. Upon plant reorientation within the gravity field, a gravity signal transduction pathway is activated within those cells, which in roots leads to a relocalization of the PIN3 auxin efflux carrier toward the lower membrane and an alkalinization of the cytoplasm. In turn, these events appear to promote a lateral transport of auxin toward the bottom side of the stimulated organ, which promotes a curvature. We previously uncovered ARG1 and ARL2 as essential contributors to these cellular processes. Mutations in these genes result in altered root and hypocotyl gravitropism. In roots, this abnormal growth behavior is associated with a lack of PIN3 relocalization within the statocytes and an absence of preferential downward auxin transport upon gravistimulation. These two genes encode paralogous J-domain proteins that are associated with the plasma membrane and other membranes of the vesicular trafficking pathway, and appear to modulate protein trafficking within the statocytes. An analysis of the root gravitropic phenotypes associated with different double mutant configurations affecting ARG1, ARL2 and PIN3 suggest that all three proteins function in a common gravity-signaling pathway. Surprisingly, when a mutation that affects starch biosynthesis (pgm) is introgressed into an arg1-2 mutant, the gravitropic defects are dramatically enhanced relative to those of the single mutants. We used this observation to design a genetic screen for the identification of new loci that contribute to the pgm gravity-signaling pathway. Two genetic enhancers of arg1-2 were identified this way, called mar1-1 and mar2-1. These mutations were shown to affect components of the protein-import complex found in the outer membrane of plastids. Interestingly, the columellar amyloplasts of arg1-2 mar2-1 mutant roots display wild-type ultra-structure, accumulate starch and sediment at wild-type rates upon gravistimulation. We conclude that the plastid outer envelope may contribute directly to gravity signal transduction within the statocytes.
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The microtubule plus-end tracking proteins (+TIPs) END BINDING1b (EB1b) and SPIRAL1 (SPR1) are required for normal cell expansion and organ growth. EB proteins are viewed as central regulators of +TIPs and cell polarity in animals; SPR1... more
The microtubule plus-end tracking proteins (+TIPs) END BINDING1b (EB1b) and SPIRAL1 (SPR1) are required for normal cell expansion and organ growth. EB proteins are viewed as central regulators of +TIPs and cell polarity in animals; SPR1 homologs are specific to plants. To explore if EB1b and SPR1 fundamentally function together, we combined genetic, biochemical, and cell imaging approaches in Arabidopsis thaliana. We found that eb1b-2 spr1-6 double mutant roots exhibit substantially more severe polar expansion defects than either single mutant, undergoing right-looping growth and severe axial twisting instead of waving on tilted hard-agar surfaces. Protein interaction assays revealed that EB1b and SPR1 bind each other and tubulin heterodimers, which is suggestive of a microtubule loading mechanism. EB1b and SPR1 show antagonistic association with microtubules in vitro. Surprisingly, our combined analyses revealed that SPR1 can load onto microtubules and function independently of EB1...
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ARG1 ( ALTERED RESPONSE TO GRAVITY ) is required for normal root and hypocotyl gravitropism. Here, we show that tar- geting ARG1 to the gravity-perceiving cells of roots or hypocotyls is sufficient to rescue the gravitropic defects in the... more
ARG1 ( ALTERED RESPONSE TO GRAVITY ) is required for normal root and hypocotyl gravitropism. Here, we show that tar- geting ARG1 to the gravity-perceiving cells of roots or hypocotyls is sufficient to rescue the gravitropic defects in the corre- sponding organs of arg1-2 null mutants. The cytosolic alkalinization of root cap columella cells that normally occurs very rapidly upon
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The sku6-1 mutant of Arabidopsis thaliana exhibits altered patterns of root and organ growth. sku6 roots, etiolated hypocotyls, and leaf petioles exhibit right-handed axial twisting, and root growth on inclined agar media is strongly... more
The sku6-1 mutant of Arabidopsis thaliana exhibits altered patterns of root and organ growth. sku6 roots, etiolated hypocotyls, and leaf petioles exhibit right-handed axial twisting, and root growth on inclined agar media is strongly right skewed. The touch-dependent sku6 root skewing phenotype is suppressed by the antimicrotubule drugs propyzamide and oryzalin, and right skewing is exacerbated by cold treatment. Cloning revealed that sku6-1 is allelic to spiral1-1 (spr1-1). However, modifiers in the Columbia (Col) and Landsberg erecta (Ler) ecotype backgrounds mask noncomplementation in sku6-1 (Col)/spr1-1 (Ler) F1 plants. The SPR1 gene encodes a plant-specific 12-kD protein that is ubiquitously expressed and belongs to a six-member gene family in Arabidopsis. An SPR1:green fluorescent protein (GFP) fusion expressed in transgenic seedlings localized to microtubules within the cortical array, preprophase band, phragmoplast, and mitotic spindle. SPR1:GFP was concentrated at the growi...
Research Interests: Genetics, Plant Biology, Arabidopsis thaliana, Mutation, Microtubules, and 15 morePlant Roots, Green Fluorescent Protein, Arabidopsis, Phenotype, Fluorescent Protein, Plant cell, Amino Acid Sequence, Gene Family, Benzamides, Cell Expansion, Biochemistry and cell biology, COL, Cell Size, Molecular Sequence Data, and Mitotic Spindle
To investigate how roots respond to directional cues, we characterized a T-DNA–tagged Arabidopsis mutant named sku5 in which the roots skewed and looped away from the normal downward direction of growth on inclined agar surfaces. sku5... more
To investigate how roots respond to directional cues, we characterized a T-DNA–tagged Arabidopsis mutant named sku5 in which the roots skewed and looped away from the normal downward direction of growth on inclined agar surfaces. sku5 roots and etiolated hypocotyls were slightly shorter than normal and exhibited a counterclockwise (left-handed) axial rotation bias. The surface-dependent skewing phenotype disappeared when the roots penetrated the agar surface, but the axial rotation defect persisted, revealing that these two directional growth processes are separable. The SKU5 gene belongs to a 19-member gene family designated SKS (SKU5 Similar) that is related structurally to the multiple-copper oxidases ascorbate oxidase and laccase. However, the SKS proteins lack several of the conserved copper binding motifs characteristic of copper oxidases, and no enzymatic function could be assigned to the SKU5 protein. Analysis of plants expressing SKU5 reporter constructs and protein gel blo...
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Tobacco and Arabidopsis plants, expressing a transgene for the calcium-sensitive luminescent protein apoaequorin, revealed circadian oscillations in free cytosolic calcium that can be phase-shifted by light-dark signals. When apoaequorin... more
Tobacco and Arabidopsis plants, expressing a transgene for the calcium-sensitive luminescent protein apoaequorin, revealed circadian oscillations in free cytosolic calcium that can be phase-shifted by light-dark signals. When apoaequorin was targeted to the chloroplast, circadian chloroplast calcium rhythms were likewise observed after transfer of the seedlings to constant darkness. Circadian oscillations in free calcium concentrations can be expected to control many calcium-dependent enzymes and processes accounting for circadian outputs. Regulation of calcium flux is therefore fundamental to the organization of circadian systems.
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Research Interests: Arabidopsis thaliana, Biological Sciences, Plant Physiology, Plant, Polymerase Chain Reaction, and 11 moreArabidopsis, Molecular cloning, Genomic DNA, Potassium Channels, Base Sequence, Recombinant Proteins, PLANT PROTEINS, Transgenic plant, Molecular Sequence Data, Restriction Mapping, and Reporter gene
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Research Interests:
Research Interests: Arabidopsis thaliana, Biological Sciences, Plant Physiology, Low Dose, Mutation, and 15 moreEndoplasmic Reticulum, Microtubules, Plant Roots, Gravitropism, Arabidopsis, Phenotype, Base Sequence, Epidermal Stem Cells, Plasma Membrane, Gtp Binding Proteins, Cell Expansion, Plant Epidermis, Molecular Sequence Data, Root growth, and root hair
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Research Interests: Renewable Energy, Genomics, Molecular Evolution, Life Sciences, Multidisciplinary, and 15 moreNature, Human Nutrition, renewable Energy sources, Plant Genome Project, Genome evolution, Sorghum, Oryza Sativa, Life Cycle, Poaceae, Model System, Gene fusion, Genome sequence, Synteny, Molecular Sequence Data, and Sequence Comparison
Previously, we showed that the yeast Saccharomyces cerevisiae cold-sensitive mutation tcp1-1 confers growth arrest concomitant with cytoskeletal disorganization and disruption of microtubule-mediated processes. We have identified two new... more
Previously, we showed that the yeast Saccharomyces cerevisiae cold-sensitive mutation tcp1-1 confers growth arrest concomitant with cytoskeletal disorganization and disruption of microtubule-mediated processes. We have identified two new recessive mutations, tcp1-2 and tcp1-3, that confer heat- and cold-sensitive growth. Cells carrying tcp1 alleles were analyzed after exposure to the appropriate restrictive temperatures by cell viability tests, differential contrast microscopy, fluorescent, and immunofluorescent microscopy of DNA, tubulin, and actin and by determining the DNA content per cell. All three mutations conferred unique phenotypes indicative of cytoskeletal dysfunction. A causal relationship between loss of Tcp1p function and the development of cytoskeletal abnormalities was established by double mutant analyses. Novel phenotypes indicative of allele-specific genetic interactions were observed when tcp1-1 was combined in the same strain with tub1-1, tub2-402, act1-1, and a...
Research Interests: Molecular Biology, Cell Cycle, Biological Sciences, Molecular chaperones, Saccharomyces cerevisiae, and 14 moreMutation, Mice, Animals, Temperature, Microtubules, Phenotype, Cell nucleus, Tubulin, Amino Acid Sequence, Cytoplasm, Chaperonins, Molecular Sequence Data, alleles, and Medical and Health Sciences
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Research Interests:
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... 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.