Background Lignification of secondary cell walls is a major factor conferring recalcitrance of li... more Background Lignification of secondary cell walls is a major factor conferring recalcitrance of lignocellulosic biomass to deconstruction for fuels and chemicals. Genetic modification can reduce lignin content and enhance saccharification efficiency, but usually at the cost of moderate-to-severe growth penalties. We have developed a method, using a single DNA construct that uses CRISPR–Cas9 gene editing to knock-out expression of an endogenous gene of lignin monomer biosynthesis while at the same time expressing a modified version of the gene’s open reading frame that escapes cutting by the Cas9 system and complements the introduced mutation in a tissue-specific manner. Results Expressing the complementing open reading frame in vessels allows for the regeneration of Arabidopsis plants with reduced lignin, wild-type biomass yield, and up to fourfold enhancement of cell wall sugar yield per plant. The above phenotypes are seen in both homozygous and bi-allelic heterozygous T1 lines, an...
Grape seed extract (GSE) is rich in flavonoids and has been recognized to possess human health be... more Grape seed extract (GSE) is rich in flavonoids and has been recognized to possess human health benefits. Our group and others have demonstrated that GSE is able to attenuate the development of Alzheimer's disease (AD). Moreover, our results have disclosed that the anti-Alzheimer's benefits are not directly/solely related to the dietary flavonoids themselves, but rather to their metabolites, particularly to the glucuronidated ones. To facilitate the understanding of regioisomer/stereoisomer-specific biological effects of (epi)catechin glucuronides, we here describe a concise chemical synthesis of authentic standards of catechin and epicatechin metabolites 3−12. The synthesis of glucuronides 9 and 12 is described here for the first time. The key reactions employed in the synthesis of the novel glucuronides 9 and 12 include the regioselective methylation of the 4′-hydroxyl group of (epi)catechin (≤1.0/99.0%; 3′-OMe/4′-OMe) and the regioselective deprotection of the tert-butyldimethylsilyl (TBS) group at position 5 (yielding up to 79%) over the others (3, 7 and 3′ or 4′).
Lignin is a major component of secondarily thickened plant cell walls and is considered to be the... more Lignin is a major component of secondarily thickened plant cell walls and is considered to be the second most abundant biopolymer on the planet. At one point believed to be the product of a highly controlled polymerization procedure involving just three potential monomeric components (monolignols), it is becoming increasingly clear that the composition of lignin is quite flexible. Furthermore, the biosynthetic pathways to the major monolignols also appear to exhibit flexibility, particularly as regards the early reactions leading to the formation of caffeic acid from coumaric acid. The operation of parallel pathways to caffeic acid occurring at the level of shikimate esters or free acids may help provide robustness to the pathway under different physiological conditions. Several features of the pathway also appear to link monolignol biosynthesis to both generation and detoxification of hydrogen peroxide, one of the oxidants responsible for creating monolignol radicals for polymeriza...
Bioactive dietary polyphenols have health benefits against a variety of disorders, but some benef... more Bioactive dietary polyphenols have health benefits against a variety of disorders, but some benefits of polyphenols may be not directly related to them but rather to their metabolites. Recently, we have identified the brain-available phenol glucuronide metabolite deoxyrhapontigenin-3-O-β-D-glucuronide (5) in perfused rat brains following subacute treatment with the stilbene resveratrol (1). However, the role of such a metabolite in the neuroprotective activity of resveratrol (1) is not understood, in part due to the noncommercial availability of 5 for performing biological evaluation in animal models of Alzheimer's disease or other neurological disorders. Here, we describe a concise chemical synthesis of deoxyrhapontigenin-3-O-β-Dglucuronide (5) and its precursor 4-O-Me-resveratrol (2), accomplished in four and six steps with 74 and 21% overall yields, respectively, starting from commercially available 3,5-dihydroxybenzaldehyde. Pivotal reactions employed in the synthesis include the palladium-catalyzed C−C coupling between 3,5-di-tert-butyldiphenylsilyloxystyrene and p-iodoanisole in the presence of tributylamine and the acid-catalyzed glucuronidation between the trichloroacetimidate-activated glucuronic acid and 4-O-Me-resveratrol.
Proanthocyanidins (PAs) are oligomeric plant natural products mostly derived from epicatechin and... more Proanthocyanidins (PAs) are oligomeric plant natural products mostly derived from epicatechin and/or catechin monomers. In studies aimed at engineering PAs into plant tissues that do not naturally make these compounds, we have expressed PA biosynthetic and regulatory genes in tobacco, alfalfa ( Medicago sativa) and the model legume Medicago truncatula. Because engineered tannins may be produced in small quantities and it is often necessary to screen many independent plant lines, we have developed an improved, highly sensitive method to quantify and determine the composition of oligomeric PAs in plant extracts. The method involves normal-phase HPLC separation of semi-purified PAs followed by post-column reaction with the PA-specific reagent DMACA (dimethylaminocinnamaldehyde). This procedure allows for accurate and sensitive quantification of individual oligomeric PAs and, unlike currently used methods, does not require exhaustive sample preparation and clean-up. Compositional data a...
This paper originates from an address at the 8th International Symposium on Nitrogen Fixation wit... more This paper originates from an address at the 8th International Symposium on Nitrogen Fixation with Non-Legumes, Sydney, NSW, December 2000 Arabidopsis thaliana L. has many features favoring its use as a model in studies of plant-growth-promoting rhizobacteria (PGPR), such as diazotrophs. Several niches are colonized in the root system of Arabidopsis, including xylem, and intact colonized roots can be observed microscopically without sectioning of tissues. Studies of plant genes involved in colonization are facilitated by the ease with which plants are transformed and by the availability of mutant lines and other accessions obtainable from stock centers. Lines of Arabidopsis carrying reporter gene fusions are helping to reveal the pattern of expression of previously cloned plant genes induced by rhizobacteria. Studies utilizing indole-3-acetic acid (IAA)-producing PGPR and Arabidopsis that contain an auxin-responsive GUS fusion suggest that plants perceive IAA released by bacteria in...
Lignin, a major component of lignocellulosic biomass, is crucial to plant growth and development ... more Lignin, a major component of lignocellulosic biomass, is crucial to plant growth and development but is a major impediment to efficient biomass utilization in various processes. Valorizing lignin is increasingly realized as being essential. However, rapid condensation of lignin during acidic extraction leads to the formation of recalcitrant condensed units that, along with similar units and structural heterogeneity in native lignin, drastically limits product yield and selectivity. Catechyl lignin (C-lignin), which is essentially a benzodioxane homopolymer without condensed units, might represent an ideal lignin for valorization, as it circumvents these issues. We discovered that C-lignin is highly acid-resistant. Hydrogenolysis of C-lignin resulted in the cleavage of all benzodioxane structures to produce catechyl-type monomers in near-quantitative yield with a selectivity of 90% to a single monomer.
Downregulation of lignin in alfalfa (Medicago sativa L.) is associated with increased availabilit... more Downregulation of lignin in alfalfa (Medicago sativa L.) is associated with increased availability of cell wall polysaccharides in plant cells. We tested transgenic alfalfa plants downregulated for Caffeoyl-CoA O-methyltransferase (CCoAOMT) against an economically important fungal disease of alfalfa, Fusarium wilt caused by Fusarium oxysporum f. sp. medicaginis, and found it more resistant to this disease. Transcriptomic and metabolomic analyses indicated that the improved disease resistance against Fusarium wilt is due to increased accumulation and/or spillover of flux towards the (iso)flavonoid pathway. Some (iso)flavonoids and their pathway intermediate compounds showed strong accumulation in CCoAOMT downregulated plants after F. oxysporum f. sp. medicaginis inoculation. The identified (iso)flavonoids, including medicarpin and 7,4'-dihydroxyflavone, inhibited the in vitro growth of F. oxysporum f. sp. medicaginis. These results suggested that the increased accumulation and/or...
Cell wall recalcitrance is the major challenge to improving saccharification efficiency in conver... more Cell wall recalcitrance is the major challenge to improving saccharification efficiency in converting lignocellulose into biofuels. However, information regarding the transcriptional regulation of secondary cell wall biogenesis remains poor in switchgrass (Panicum virgatum), which has been selected as a biofuel crop in the United States. In this study, we present a combination of computational and experimental approaches to develop gene regulatory networks for lignin formation in switchgrass. To screen transcription factors (TFs) involved in lignin biosynthesis, we developed a modified method to perform co-expression network analysis using 14 lignin biosynthesis genes as bait (target) genes. The switchgrass lignin co-expression network was further extended by adding 14 TFs identified in this study, and seven TFs identified in previous studies, as bait genes. Six TFs (PvMYB58/63, PvMYB42/85, PvMYB4, PvWRKY12, PvSND2 and PvSWN2) were targeted to generate overexpressing and/or down-reg...
A reduction in the lignin content in transgenic plants induces the ectopic expression of defense ... more A reduction in the lignin content in transgenic plants induces the ectopic expression of defense genes, but the importance of altered lignin composition in such phenomena remains unclear. Two Arabidopsis lines with similar lignin contents, but strikingly different lignin compositions, exhibited different quantitative and qualitative transcriptional responses. Plants with lignin composed primarily of guaiacyl units overexpressed genes responsive to oomycete and bacterial pathogen attack, whereas plants with lignin composed primarily of syringyl units expressed a far greater number of defense genes, including some associated with cis-jasmone-mediated responses to aphids; these plants exhibited altered responsiveness to bacterial and aphid inoculation. Several of the defense genes were differentially induced by water-soluble extracts from cell walls of plants of the two lines. Glycome profiling, fractionation and enzymatic digestion studies indicated that the different lignin compositi...
Genetic engineering of switchgrass ( L.) for reduced cell wall recalcitrance and improved biofuel... more Genetic engineering of switchgrass ( L.) for reduced cell wall recalcitrance and improved biofuel production has been a long pursued goal. Up to now, constitutive promoters have been used to direct the expression of cell wall biosynthesis genes toward attaining that goal. While generally sufficient to gauge a transgene's effects in the heterologous host, constitutive overexpression often leads to undesirable plant phenotypic effects. Green tissue-specific promoters from switchgrass are potentially valuable to directly alter cell wall traits exclusively in harvestable aboveground biomass while not changing root phenotypes. We identified and functionally characterized three switchgrass green tissue-specific promoters and assessed marker gene expression patterns and intensity in stably transformed rice ( L.), and then used them to direct the expression of the switchgrass () transcription factor gene in transgenic switchgrass to endow reduced recalcitrance in aboveground biomass. Th...
Lignin is a natural polymer that is interwoven with cellulose and hemicellulose within plant cell... more Lignin is a natural polymer that is interwoven with cellulose and hemicellulose within plant cell walls. Due to this molecular arrangement, lignin is a major contributor to the recalcitrance of plant materials with respect to the extraction of sugars and their fermentation into ethanol, butanol, and other potential bioenergy crops. The lignin biosynthetic pathway is similar, but not identical in different plant species. It is in each case comprised of a moderate number of enzymatic steps, but its responses to manipulations, such as gene knock-downs, are complicated by the fact that several of the key enzymes are involved in several reaction steps. This feature poses a challenge to bioenergy production, as it renders it difficult to select the most promising combinations of genetic manipulations for the optimization of lignin composition and amount. Here, we present several computational models than can aid in the analysis of data characterizing lignin biosynthesis. While minimizing ...
The mission of the BioEnergy Science Center (BESC) was to enable efficient lignocellulosic-based ... more The mission of the BioEnergy Science Center (BESC) was to enable efficient lignocellulosic-based biofuel production. One BESC goal was to decrease poplar and switchgrass biomass recalcitrance to biofuel conversion while not affecting plant growth. A transformation pipeline (TP), to express transgenes or transgene fragments (constructs) in these feedstocks with the goal of understanding and decreasing recalcitrance, was considered essential for this goal. Centralized data storage for access by BESC members and later the public also was essential. A BESC committee was established to codify procedures to evaluate and accept genes into the TP. A laboratory information management system (LIMS) was organized to catalog constructs, plant lines and results from their analyses. One hundred twenty-eight constructs were accepted into the TP for expression in switchgrass in the first 5 years of BESC. Here we provide information on 53 of these constructs and the BESC TP process. Eleven of the co...
Plant cell walls contribute the majority of plant biomass that can be used to produce transportat... more Plant cell walls contribute the majority of plant biomass that can be used to produce transportation fuels. However, the complexity and variability in composition and structure of cell walls, particularly the presence of lignin, negatively impacts their deconstruction for bioenergy. Metabolic and genetic changes associated with secondary wall development in the biofuel crop switchgrass (Panicum virgatum) have yet to be reported. Our previous studies have established a cell suspension system for switchgrass, in which cell wall lignification can be induced by application of brassinolide (BL). We have now collected cell wall composition and microarray-based transcriptome profiles for BL-induced and non-induced suspension cultures to provide an overview of the dynamic changes in transcriptional reprogramming during BL-induced cell wall modification. From this analysis, we have identified changes in candidate genes involved in cell wall precursor synthesis, cellulose, hemicellulose, and ...
Elicitor-active fractions from a culture filtrate of Colletotrichum lindemuthianum were separated... more Elicitor-active fractions from a culture filtrate of Colletotrichum lindemuthianum were separated on the basis of their affinity for Concanavalin A Sepharose, and were compared to unfractionated culture filtrate and elicitor from cell walls of the fungus in terms of their effects on induced protein synthesis in cultured bean cells. In vitro translation of polysomal mRNA followed by one-and twodimensional electrophoretic analysis revealed both similarities and differences in the patterns of mRNA induction in response to different elicitor preparations. All elicitors induced mRNA activities encoding the phytoalexin biosynthetic enzymes phenylalanine ammonia-lyase, chalcone synthase and chalcone isomerase. Chalcone synthase subunits, synthesized in vivo and in vitro from mRNA were resolved into a number of charge isoforms whose relative proportions, although not constant, did not appear to reflect substantial differential induction by specific elicitor fractions. Chromatofocussing analysis revealed that culture filtrate and cell wall elicitors induced similar patterns of phenylalanine ammonia-lyase and chalcone synthase intact enzyme charge iso-forms which were, however, different from those observed in unelicited cells. The results indicate that the elicitor preparations contain components, separable on the basis ofstructural criteria, which exhibit both common and differential effects on host protein synthesis .
Background Lignification of secondary cell walls is a major factor conferring recalcitrance of li... more Background Lignification of secondary cell walls is a major factor conferring recalcitrance of lignocellulosic biomass to deconstruction for fuels and chemicals. Genetic modification can reduce lignin content and enhance saccharification efficiency, but usually at the cost of moderate-to-severe growth penalties. We have developed a method, using a single DNA construct that uses CRISPR–Cas9 gene editing to knock-out expression of an endogenous gene of lignin monomer biosynthesis while at the same time expressing a modified version of the gene’s open reading frame that escapes cutting by the Cas9 system and complements the introduced mutation in a tissue-specific manner. Results Expressing the complementing open reading frame in vessels allows for the regeneration of Arabidopsis plants with reduced lignin, wild-type biomass yield, and up to fourfold enhancement of cell wall sugar yield per plant. The above phenotypes are seen in both homozygous and bi-allelic heterozygous T1 lines, an...
Grape seed extract (GSE) is rich in flavonoids and has been recognized to possess human health be... more Grape seed extract (GSE) is rich in flavonoids and has been recognized to possess human health benefits. Our group and others have demonstrated that GSE is able to attenuate the development of Alzheimer's disease (AD). Moreover, our results have disclosed that the anti-Alzheimer's benefits are not directly/solely related to the dietary flavonoids themselves, but rather to their metabolites, particularly to the glucuronidated ones. To facilitate the understanding of regioisomer/stereoisomer-specific biological effects of (epi)catechin glucuronides, we here describe a concise chemical synthesis of authentic standards of catechin and epicatechin metabolites 3−12. The synthesis of glucuronides 9 and 12 is described here for the first time. The key reactions employed in the synthesis of the novel glucuronides 9 and 12 include the regioselective methylation of the 4′-hydroxyl group of (epi)catechin (≤1.0/99.0%; 3′-OMe/4′-OMe) and the regioselective deprotection of the tert-butyldimethylsilyl (TBS) group at position 5 (yielding up to 79%) over the others (3, 7 and 3′ or 4′).
Lignin is a major component of secondarily thickened plant cell walls and is considered to be the... more Lignin is a major component of secondarily thickened plant cell walls and is considered to be the second most abundant biopolymer on the planet. At one point believed to be the product of a highly controlled polymerization procedure involving just three potential monomeric components (monolignols), it is becoming increasingly clear that the composition of lignin is quite flexible. Furthermore, the biosynthetic pathways to the major monolignols also appear to exhibit flexibility, particularly as regards the early reactions leading to the formation of caffeic acid from coumaric acid. The operation of parallel pathways to caffeic acid occurring at the level of shikimate esters or free acids may help provide robustness to the pathway under different physiological conditions. Several features of the pathway also appear to link monolignol biosynthesis to both generation and detoxification of hydrogen peroxide, one of the oxidants responsible for creating monolignol radicals for polymeriza...
Bioactive dietary polyphenols have health benefits against a variety of disorders, but some benef... more Bioactive dietary polyphenols have health benefits against a variety of disorders, but some benefits of polyphenols may be not directly related to them but rather to their metabolites. Recently, we have identified the brain-available phenol glucuronide metabolite deoxyrhapontigenin-3-O-β-D-glucuronide (5) in perfused rat brains following subacute treatment with the stilbene resveratrol (1). However, the role of such a metabolite in the neuroprotective activity of resveratrol (1) is not understood, in part due to the noncommercial availability of 5 for performing biological evaluation in animal models of Alzheimer's disease or other neurological disorders. Here, we describe a concise chemical synthesis of deoxyrhapontigenin-3-O-β-Dglucuronide (5) and its precursor 4-O-Me-resveratrol (2), accomplished in four and six steps with 74 and 21% overall yields, respectively, starting from commercially available 3,5-dihydroxybenzaldehyde. Pivotal reactions employed in the synthesis include the palladium-catalyzed C−C coupling between 3,5-di-tert-butyldiphenylsilyloxystyrene and p-iodoanisole in the presence of tributylamine and the acid-catalyzed glucuronidation between the trichloroacetimidate-activated glucuronic acid and 4-O-Me-resveratrol.
Proanthocyanidins (PAs) are oligomeric plant natural products mostly derived from epicatechin and... more Proanthocyanidins (PAs) are oligomeric plant natural products mostly derived from epicatechin and/or catechin monomers. In studies aimed at engineering PAs into plant tissues that do not naturally make these compounds, we have expressed PA biosynthetic and regulatory genes in tobacco, alfalfa ( Medicago sativa) and the model legume Medicago truncatula. Because engineered tannins may be produced in small quantities and it is often necessary to screen many independent plant lines, we have developed an improved, highly sensitive method to quantify and determine the composition of oligomeric PAs in plant extracts. The method involves normal-phase HPLC separation of semi-purified PAs followed by post-column reaction with the PA-specific reagent DMACA (dimethylaminocinnamaldehyde). This procedure allows for accurate and sensitive quantification of individual oligomeric PAs and, unlike currently used methods, does not require exhaustive sample preparation and clean-up. Compositional data a...
This paper originates from an address at the 8th International Symposium on Nitrogen Fixation wit... more This paper originates from an address at the 8th International Symposium on Nitrogen Fixation with Non-Legumes, Sydney, NSW, December 2000 Arabidopsis thaliana L. has many features favoring its use as a model in studies of plant-growth-promoting rhizobacteria (PGPR), such as diazotrophs. Several niches are colonized in the root system of Arabidopsis, including xylem, and intact colonized roots can be observed microscopically without sectioning of tissues. Studies of plant genes involved in colonization are facilitated by the ease with which plants are transformed and by the availability of mutant lines and other accessions obtainable from stock centers. Lines of Arabidopsis carrying reporter gene fusions are helping to reveal the pattern of expression of previously cloned plant genes induced by rhizobacteria. Studies utilizing indole-3-acetic acid (IAA)-producing PGPR and Arabidopsis that contain an auxin-responsive GUS fusion suggest that plants perceive IAA released by bacteria in...
Lignin, a major component of lignocellulosic biomass, is crucial to plant growth and development ... more Lignin, a major component of lignocellulosic biomass, is crucial to plant growth and development but is a major impediment to efficient biomass utilization in various processes. Valorizing lignin is increasingly realized as being essential. However, rapid condensation of lignin during acidic extraction leads to the formation of recalcitrant condensed units that, along with similar units and structural heterogeneity in native lignin, drastically limits product yield and selectivity. Catechyl lignin (C-lignin), which is essentially a benzodioxane homopolymer without condensed units, might represent an ideal lignin for valorization, as it circumvents these issues. We discovered that C-lignin is highly acid-resistant. Hydrogenolysis of C-lignin resulted in the cleavage of all benzodioxane structures to produce catechyl-type monomers in near-quantitative yield with a selectivity of 90% to a single monomer.
Downregulation of lignin in alfalfa (Medicago sativa L.) is associated with increased availabilit... more Downregulation of lignin in alfalfa (Medicago sativa L.) is associated with increased availability of cell wall polysaccharides in plant cells. We tested transgenic alfalfa plants downregulated for Caffeoyl-CoA O-methyltransferase (CCoAOMT) against an economically important fungal disease of alfalfa, Fusarium wilt caused by Fusarium oxysporum f. sp. medicaginis, and found it more resistant to this disease. Transcriptomic and metabolomic analyses indicated that the improved disease resistance against Fusarium wilt is due to increased accumulation and/or spillover of flux towards the (iso)flavonoid pathway. Some (iso)flavonoids and their pathway intermediate compounds showed strong accumulation in CCoAOMT downregulated plants after F. oxysporum f. sp. medicaginis inoculation. The identified (iso)flavonoids, including medicarpin and 7,4'-dihydroxyflavone, inhibited the in vitro growth of F. oxysporum f. sp. medicaginis. These results suggested that the increased accumulation and/or...
Cell wall recalcitrance is the major challenge to improving saccharification efficiency in conver... more Cell wall recalcitrance is the major challenge to improving saccharification efficiency in converting lignocellulose into biofuels. However, information regarding the transcriptional regulation of secondary cell wall biogenesis remains poor in switchgrass (Panicum virgatum), which has been selected as a biofuel crop in the United States. In this study, we present a combination of computational and experimental approaches to develop gene regulatory networks for lignin formation in switchgrass. To screen transcription factors (TFs) involved in lignin biosynthesis, we developed a modified method to perform co-expression network analysis using 14 lignin biosynthesis genes as bait (target) genes. The switchgrass lignin co-expression network was further extended by adding 14 TFs identified in this study, and seven TFs identified in previous studies, as bait genes. Six TFs (PvMYB58/63, PvMYB42/85, PvMYB4, PvWRKY12, PvSND2 and PvSWN2) were targeted to generate overexpressing and/or down-reg...
A reduction in the lignin content in transgenic plants induces the ectopic expression of defense ... more A reduction in the lignin content in transgenic plants induces the ectopic expression of defense genes, but the importance of altered lignin composition in such phenomena remains unclear. Two Arabidopsis lines with similar lignin contents, but strikingly different lignin compositions, exhibited different quantitative and qualitative transcriptional responses. Plants with lignin composed primarily of guaiacyl units overexpressed genes responsive to oomycete and bacterial pathogen attack, whereas plants with lignin composed primarily of syringyl units expressed a far greater number of defense genes, including some associated with cis-jasmone-mediated responses to aphids; these plants exhibited altered responsiveness to bacterial and aphid inoculation. Several of the defense genes were differentially induced by water-soluble extracts from cell walls of plants of the two lines. Glycome profiling, fractionation and enzymatic digestion studies indicated that the different lignin compositi...
Genetic engineering of switchgrass ( L.) for reduced cell wall recalcitrance and improved biofuel... more Genetic engineering of switchgrass ( L.) for reduced cell wall recalcitrance and improved biofuel production has been a long pursued goal. Up to now, constitutive promoters have been used to direct the expression of cell wall biosynthesis genes toward attaining that goal. While generally sufficient to gauge a transgene's effects in the heterologous host, constitutive overexpression often leads to undesirable plant phenotypic effects. Green tissue-specific promoters from switchgrass are potentially valuable to directly alter cell wall traits exclusively in harvestable aboveground biomass while not changing root phenotypes. We identified and functionally characterized three switchgrass green tissue-specific promoters and assessed marker gene expression patterns and intensity in stably transformed rice ( L.), and then used them to direct the expression of the switchgrass () transcription factor gene in transgenic switchgrass to endow reduced recalcitrance in aboveground biomass. Th...
Lignin is a natural polymer that is interwoven with cellulose and hemicellulose within plant cell... more Lignin is a natural polymer that is interwoven with cellulose and hemicellulose within plant cell walls. Due to this molecular arrangement, lignin is a major contributor to the recalcitrance of plant materials with respect to the extraction of sugars and their fermentation into ethanol, butanol, and other potential bioenergy crops. The lignin biosynthetic pathway is similar, but not identical in different plant species. It is in each case comprised of a moderate number of enzymatic steps, but its responses to manipulations, such as gene knock-downs, are complicated by the fact that several of the key enzymes are involved in several reaction steps. This feature poses a challenge to bioenergy production, as it renders it difficult to select the most promising combinations of genetic manipulations for the optimization of lignin composition and amount. Here, we present several computational models than can aid in the analysis of data characterizing lignin biosynthesis. While minimizing ...
The mission of the BioEnergy Science Center (BESC) was to enable efficient lignocellulosic-based ... more The mission of the BioEnergy Science Center (BESC) was to enable efficient lignocellulosic-based biofuel production. One BESC goal was to decrease poplar and switchgrass biomass recalcitrance to biofuel conversion while not affecting plant growth. A transformation pipeline (TP), to express transgenes or transgene fragments (constructs) in these feedstocks with the goal of understanding and decreasing recalcitrance, was considered essential for this goal. Centralized data storage for access by BESC members and later the public also was essential. A BESC committee was established to codify procedures to evaluate and accept genes into the TP. A laboratory information management system (LIMS) was organized to catalog constructs, plant lines and results from their analyses. One hundred twenty-eight constructs were accepted into the TP for expression in switchgrass in the first 5 years of BESC. Here we provide information on 53 of these constructs and the BESC TP process. Eleven of the co...
Plant cell walls contribute the majority of plant biomass that can be used to produce transportat... more Plant cell walls contribute the majority of plant biomass that can be used to produce transportation fuels. However, the complexity and variability in composition and structure of cell walls, particularly the presence of lignin, negatively impacts their deconstruction for bioenergy. Metabolic and genetic changes associated with secondary wall development in the biofuel crop switchgrass (Panicum virgatum) have yet to be reported. Our previous studies have established a cell suspension system for switchgrass, in which cell wall lignification can be induced by application of brassinolide (BL). We have now collected cell wall composition and microarray-based transcriptome profiles for BL-induced and non-induced suspension cultures to provide an overview of the dynamic changes in transcriptional reprogramming during BL-induced cell wall modification. From this analysis, we have identified changes in candidate genes involved in cell wall precursor synthesis, cellulose, hemicellulose, and ...
Elicitor-active fractions from a culture filtrate of Colletotrichum lindemuthianum were separated... more Elicitor-active fractions from a culture filtrate of Colletotrichum lindemuthianum were separated on the basis of their affinity for Concanavalin A Sepharose, and were compared to unfractionated culture filtrate and elicitor from cell walls of the fungus in terms of their effects on induced protein synthesis in cultured bean cells. In vitro translation of polysomal mRNA followed by one-and twodimensional electrophoretic analysis revealed both similarities and differences in the patterns of mRNA induction in response to different elicitor preparations. All elicitors induced mRNA activities encoding the phytoalexin biosynthetic enzymes phenylalanine ammonia-lyase, chalcone synthase and chalcone isomerase. Chalcone synthase subunits, synthesized in vivo and in vitro from mRNA were resolved into a number of charge isoforms whose relative proportions, although not constant, did not appear to reflect substantial differential induction by specific elicitor fractions. Chromatofocussing analysis revealed that culture filtrate and cell wall elicitors induced similar patterns of phenylalanine ammonia-lyase and chalcone synthase intact enzyme charge iso-forms which were, however, different from those observed in unelicited cells. The results indicate that the elicitor preparations contain components, separable on the basis ofstructural criteria, which exhibit both common and differential effects on host protein synthesis .
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Papers by Richard Dixon