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John Ralph

University of Wisconsin-Madison, Biochemistry, Faculty Member

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Educational Background
1976 B.Sc. (Hons). Chemistry, Canterbury U., New Zealand (Thesis: Lithium Aluminium Hydride Reduction of Propargyl Alcohols. Supervisor: Michael P. Hartshorn)
1982 Ph.D. Chemistry/Forestry, U. Wisconsin-Madison (Thesis: Reactions of Lignin Model Quinone Methides and NMR Studies of Lignins. Supervisor: Raymond A. Young)

Professional Experience
1974-1987 Research Scientist, Forest Research Institute (F.R.I.), Rotorua, New Zealand.
1987-1988 Scientific Head of Research Laboratory for Nuclear Magnetic Resonance. Spectroscopy, Chemistry Department, U. California-Berkeley, Berkeley, CA.
1988-2008 Research Chemist, USDA-ARS, U.S. Dairy Forage Research Center.
1988-1995 Assistant Professor, Department of Forestry, U. Wisconsin-Madison.
1995-1999 Associate Professor, Dept Forest Ecology and Management, U. Wisconsin-Madison.
1999-2006 Full Professor, Dept Forest Ecology and Management, U. Wisconsin-Madison.
2006-present Full Professor, Dept. Biological Systems Engineering, U. Wisconsin-Madison.
2008-present Full Professor, Dept. of Biochemistry, U. Wisconsin-Madison
2008-present ‘Improved Plant Biomass’ Area Leader, Great Lakes Bioenergy Research Center.

Specialization and Areas of Professional Experience
• General plant cell wall (CW) chemistry/biochemistry.
• Lignin Biosynthesis (including pathway delineation), Lignin Structure, Lignin Chemistry, Lignin Reactions.
• Delineation of effects of perturbing lignin biosynthesis, and extensions aimed at redesigning lignins to be more readily degraded to improve lignocellulosics bioprocessing.
• Development of synthetic methods for biosynthetic products, precursors, intermediates, molecular markers, cell wall model compounds, etc.
• Solution-state NMR (particularly of CW components, especially lignins); methods development; NMR methods applied to unfractionated cell walls.
• Plant cell wall cross-linking mechanisms.
• Methods for wall structural analysis (chemical/degradative, NMR, GC-MS, etc.).
• Processes such as biomass to bioenergy, pulping, and valorization of cell wall components.
Supervisors: Raymond A. Young (U. Wisconsin-Madison, Ph.D.) and Michael P. Hartshorn (U. Canterbury, New Zealand, B.Sc.(Hons))

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2017 Papers by John Ralph

Supporting Information Selective Oxidation of Lignin Model Compounds

Characteristics of Hot Water Extracts from the Bark of Cultivated Willow (Salix sp

Karlen PlantPhys 177 513 (Commelinid Monocotyledon Lignins are Acylated by p Coumarate)

Gao ChemSusChem 11 2045 (Selective oxidation of lignin model compounds)

Structural Characterization of Lignins from Willow Bark and Wood

Gold-catalyzed conversion of lignin to low molecular weight aromatics

An "ideal lignin" facilitates full biomass utilization

Complimentary and personal copy

An "ideal lignin" facilitates full biomass utilization

Letter Synthesis of Nepetoidin B Vitaliy Timokhin a Matthew Regner a 0 0 0 0 - 0 0 0 1 - 7 7 9 9 - 2 7 0 4

Tissue and cell-specific transcriptomes in cotton reveal the subtleties of gene regulation underlying the diversity of plant secondary cell walls

by John Ralph and Colleen MacMillan

Background: Knowledge of plant secondary cell wall (SCW) regulation and deposition is mainly base... more Background: Knowledge of plant secondary cell wall (SCW) regulation and deposition is mainly based on the Arabidopsis model of a 'typical' lignocellulosic SCW. However, SCWs in other plants can vary from this. The SCW of mature cotton seed fibres is highly cellulosic and lacks lignification whereas xylem SCWs are lignocellulosic. We used cotton as a model to study different SCWs and the expression of the genes involved in their formation via RNA deep sequencing and chemical analysis of stem and seed fibre. Results: Transcriptome comparisons from cotton xylem and pith as well as from a developmental series of seed fibres revealed tissue-specific and developmentally regulated expression of several NAC transcription factors some of which are likely to be important as top tier regulators of SCW formation in xylem and/or seed fibre. A so far undescribed hierarchy was identified between the top tier NAC transcription factors SND1-like and NST1/2 in cotton. Key SCW MYB transcription factors, homologs of Arabidopsis MYB46/83, were practically absent in cotton stem xylem. Lack of expression of other lignin-specific MYBs in seed fibre relative to xylem could account for the lack of lignin deposition in seed fibre. Expression of a MYB103 homolog correlated with temporal expression of SCW CesAs and cellulose synthesis in seed fibres. FLAs were highly expressed and may be important structural components of seed fibre SCWs. Finally, we made the unexpected observation that cell walls in the pith of cotton stems contained lignin and had a higher S:G ratio than in xylem, despite that tissue's lacking many of the gene transcripts normally associated with lignin biosynthesis. Conclusions: Our study in cotton confirmed some features of the currently accepted gene regulatory cascade for 'typical' plant SCWs, but also revealed substantial differences, especially with key downstream NACs and MYBs. The lignocellulosic SCW of cotton xylem appears to be achieved differently from that in Arabidopsis. Pith cell walls in cotton stems are compositionally very different from that reported for other plant species, including Arabidopsis. The current definition of a 'typical' primary or secondary cell wall might not be applicable to all cell types in all plant species.

Degradation of lignin b-aryl ether units in Arabidopsis thaliana expressing LigD, LigF and LigG from Sphingomonas paucimobilis SYK-6

by John Ralph, Ruben Vanholme, and Wout Boerjan

Lignin is a major polymer in the secondary plant cell wall and composed of hydrophobic interlinke... more Lignin is a major polymer in the secondary plant cell wall and composed of hydrophobic interlinked hydroxyphenylpropanoid units. The presence of lignin hampers conversion of plant biomass into biofuels; plants with modified lignin are therefore being investigated for increased digestibility. The bacterium Sphingomonas paucimobilis produces lignin-degrading enzymes including LigD, LigF and LigG involved in cleaving the most abundant lignin interunit linkage, the b-aryl ether bond. In this study, we expressed the LigD, LigF and LigG (LigDFG) genes in Arabidopsis thaliana to introduce postlignification modifications into the lignin structure. The three enzymes were targeted to the secretory pathway. Phenolic metabolite profiling and 2D HSQC NMR of the transgenic lines showed an increase in oxidized guaiacyl and syringyl units without concomitant increase in oxidized b-aryl ether units, showing lignin bond cleavage. Saccharification yield increased significantly in transgenic lines expressing LigDFG, showing the applicability of our approach. Additional new information on substrate specificity of the LigDFG enzymes is also provided.

Suppression of CINNAMOYL-CoA REDUCTASE increases the level of monolignol ferulates incorporated into maize lignins

Background: The cell wall polymer lignin provides structural support and rigidity to plant cell w... more Background: 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. Results: 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 decreased lignin content and monomers did not, however, impact the plant growth or biomass. The increase in monolignol conjugates correlated with an improvement in the digestibility of maize stem rind tissue. Conclusions: Together, increased monolignol ferulates and improved digestibility in ccr1 mutant plants suggests that they may be superior biofuel crops.

Lignin-Derived Thioacidolysis Dimers: Reevaluation, New Products, Authentication, and Quantification

Different Routes for Conifer-and Sinapaldehyde and Higher Saccharification upon Deficiency in the Dehydrogenase CAD1 1[OPEN

by John Ralph and Wout Boerjan

In the search for renewable energy sources, genetic engineering is a promising strategy to improv... more In the search for renewable energy sources, genetic engineering is a promising strategy to improve plant cell wall composition for biofuel and bioproducts generation. Lignin is a major factor determining saccharification efficiency and, therefore, is a prime target to engineer. Here, lignin content and composition were modified in poplar (Populus tremula 3 Populus alba) by specifically down-regulating CINNAMYL ALCOHOL DEHYDROGENASE1 (CAD1) by a hairpin-RNA-mediated silencing approach, which resulted in only 5% residual CAD1 transcript abundance. These transgenic lines showed no biomass penalty despite a 10% reduction in Klason lignin content and severe shifts in lignin composition. Nuclear magnetic resonance spectroscopy and thioacidolysis revealed a strong increase (up to 20-fold) in sinapaldehyde incorporation into lignin, whereas coniferaldehyde was not increased markedly. Accordingly, ultra-high-performance liquid chromatography-mass spectrometry-based phenolic profiling revealed a more than 24,000-fold accumulation of a newly identified compound made from 8-8 coupling of two sinapaldehyde radicals. However, no additional cinnamaldehyde coupling products could be detected in the CAD1-deficient poplars. Instead, the transgenic lines accumulated a range of hydroxycinnamate-derived metabolites, of which the most prominent accumulation (over 8,500-fold) was observed for a compound that was identified by purification and nuclear magnetic resonance as syringyl lactic acid hexoside. Our data suggest that, upon down-regulation of CAD1, coniferaldehyde is converted into ferulic acid and derivatives, whereas sinapaldehyde is either oxidatively coupled into S9(8-8)S9 and lignin or converted to sinapic acid and derivatives. The most prominent sink of the increased flux to hydroxycinnamates is syringyl lactic acid hexoside. Furthermore, low-extent saccharification assays, under different pretreatment conditions, showed strongly increased glucose (up to +81%) and xylose (up to +153%) release, suggesting that down-regulating CAD1 is a promising strategy for improving lignocellulosic biomass for the sugar platform industry.

Altering carbon allocation in hybrid poplar (Populus alba 3 grandidentata) impacts cell wall growth and development

Galactinol synthase is a pivotal enzyme involved in the synthesis of the raffinose family of olig... more Galactinol synthase is a pivotal enzyme involved in the synthesis of the raffinose family of oligosaccharides (RFOs) that function as transport carbohydrates in the phloem, as storage compounds in sink tissues and as soluble metabolites that combat both abiotic and biotic stress in several plant species. Hybrid poplar (Populus alba 9 grandidentata) overexpressing the Arabidopsis thaliana GolS3 (AtGolS3) gene showed clear effects on development; the extreme overexpressing lines were stunted and had cell wall traits characteristic of tension wood, whereas lines with only moderate up-regulation grew normally and had moderately altered secondary cell wall composition and ultrastructure. Stem cross-sections of the developing xylem revealed a significant increase in the number of vessels, as well as the clear presence of a G-layer in the fibres. Furthermore, AtGolS3-OE lines possessed higher cellulose and lower lignin contents, an increase in cellulose crystallinity, and significantly altered hemicellulose-derived carbohydrates, notably manifested by their mannose and xylose contents. In addition, the transgenic plants displayed elevated xylem starch content. Transcriptome interrogation of the transgenic plants showed a significant up-regulation of genes involved in the synthesis of myo-inositol, along with genes involved in sucrose degradation. The results suggest that the overexpression of GolS and its product galactinol may serve as a molecular signal that initiates metabolic changes, culminating in a change in cell wall development and potentially the formation of tension wood.

Suppression of CINNAMOYL-CoA REDUCTASE increases the level of monolignol ferulates incorporated into maize lignins

Background: The cell wall polymer lignin provides structural support and rigidity to plant cell w... more Background: 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. Results: 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 decreased lignin content and monomers did not, however, impact the plant growth or biomass. The increase in monolignol conjugates correlated with an improvement in the digestibility of maize stem rind tissue. Conclusions: Together, increased monolignol ferulates and improved digestibility in ccr1 mutant plants suggests that they may be superior biofuel crops.

Silencing CHALCONE SYNTHASE in Maize Impedes the Incorporation of Tricin into Lignin and Increases Lignin Content 1[OPEN

by John Ralph and Wout Boerjan

Lignin is a phenolic heteropolymer that is deposited in secondary-thickened cell walls, where it ... more Lignin is a phenolic heteropolymer that is deposited in secondary-thickened cell walls, where it provides mechanical strength. A recent structural characterization of cell walls from monocot species showed that the flavone tricin is part of the native lignin polymer, where it is hypothesized to initiate lignin chains. In this study, we investigated the consequences of altered tricin levels on lignin structure and cell wall recalcitrance by phenolic profiling, nuclear magnetic resonance, and saccharification assays of the naturally silenced maize (Zea mays) C2-Idf (inhibitor diffuse) mutant, defective in the CHALCONE SYNTHASE Colorless2 (C2) gene. We show that the C2-Idf mutant produces highly reduced levels of apigenin-and tricin-related flavonoids, resulting in a strongly reduced incorporation of tricin into the lignin polymer. Moreover, the lignin was enriched in b-b and b-5 units, lending support to the contention that tricin acts to initiate lignin chains and that, in the absence of tricin, more monolignol dimerization reactions occur. In addition, the C2-Idf mutation resulted in strikingly higher Klason lignin levels in the leaves. As a consequence, the leaves of C2-Idf mutants had significantly reduced saccharification efficiencies compared with those of control plants. These findings are instructive for lignin engineering strategies to improve biomass processing and biochemical production.

Silencing CAFFEOYL SHIKIMATE ESTERASE Affects Lignification and Improves Saccharification in Poplar 1[OPEN

by John Ralph, Ruben Vanholme, and Wout Boerjan

Caffeoyl shikimate esterase (CSE) was recently shown to play an essential role in lignin biosynth... more Caffeoyl shikimate esterase (CSE) was recently shown to play an essential role in lignin biosynthesis in Arabidopsis (Arabidopsis thaliana) and later in Medicago truncatula. However, the general function of this enzyme was recently questioned by the apparent lack of CSE activity in lignifying tissues of different plant species. Here, we show that down-regulation of CSE in hybrid poplar (Populus tremula 3 Populus alba) resulted in up to 25% reduced lignin deposition, increased levels of p-hydroxyphenyl units in the lignin polymer, and a relatively higher cellulose content. The transgenic trees were morphologically indistinguishable from the wild type. Ultra-high-performance liquid chromatography-mass spectrometry-based phenolic profiling revealed a reduced abundance of several oligolignols containing guaiacyl and syringyl units and their corresponding hydroxycinnamaldehyde units, in agreement with the reduced flux toward coniferyl and sinapyl alcohol. These trees accumulated the CSE substrate caffeoyl shikimate along with other compounds belonging to the metabolic classes of benzenoids and hydroxycinnamates. Furthermore, the reduced lignin amount combined with the relative increase in cellulose content in the CSE down-regulated lines resulted in up to 62% more glucose released per plant upon limited saccharification when no pretreatment was applied and by up to 86% and 91% when acid and alkaline pretreatments were used. Our results show that CSE is not only important for the lignification process in poplar but is also a promising target for the development of improved lignocellulosic biomass crops for sugar platform biorefineries.

Short-term facilitation of microbial litter decomposition by ultraviolet radiation

Supporting Information Selective Oxidation of Lignin Model Compounds

Characteristics of Hot Water Extracts from the Bark of Cultivated Willow (Salix sp

Karlen PlantPhys 177 513 (Commelinid Monocotyledon Lignins are Acylated by p Coumarate)

Gao ChemSusChem 11 2045 (Selective oxidation of lignin model compounds)

Structural Characterization of Lignins from Willow Bark and Wood

Gold-catalyzed conversion of lignin to low molecular weight aromatics

An "ideal lignin" facilitates full biomass utilization

Complimentary and personal copy

An "ideal lignin" facilitates full biomass utilization

Letter Synthesis of Nepetoidin B Vitaliy Timokhin a Matthew Regner a 0 0 0 0 - 0 0 0 1 - 7 7 9 9 - 2 7 0 4

Tissue and cell-specific transcriptomes in cotton reveal the subtleties of gene regulation underlying the diversity of plant secondary cell walls

by John Ralph and Colleen MacMillan

Background: Knowledge of plant secondary cell wall (SCW) regulation and deposition is mainly base... more Background: Knowledge of plant secondary cell wall (SCW) regulation and deposition is mainly based on the Arabidopsis model of a 'typical' lignocellulosic SCW. However, SCWs in other plants can vary from this. The SCW of mature cotton seed fibres is highly cellulosic and lacks lignification whereas xylem SCWs are lignocellulosic. We used cotton as a model to study different SCWs and the expression of the genes involved in their formation via RNA deep sequencing and chemical analysis of stem and seed fibre. Results: Transcriptome comparisons from cotton xylem and pith as well as from a developmental series of seed fibres revealed tissue-specific and developmentally regulated expression of several NAC transcription factors some of which are likely to be important as top tier regulators of SCW formation in xylem and/or seed fibre. A so far undescribed hierarchy was identified between the top tier NAC transcription factors SND1-like and NST1/2 in cotton. Key SCW MYB transcription factors, homologs of Arabidopsis MYB46/83, were practically absent in cotton stem xylem. Lack of expression of other lignin-specific MYBs in seed fibre relative to xylem could account for the lack of lignin deposition in seed fibre. Expression of a MYB103 homolog correlated with temporal expression of SCW CesAs and cellulose synthesis in seed fibres. FLAs were highly expressed and may be important structural components of seed fibre SCWs. Finally, we made the unexpected observation that cell walls in the pith of cotton stems contained lignin and had a higher S:G ratio than in xylem, despite that tissue's lacking many of the gene transcripts normally associated with lignin biosynthesis. Conclusions: Our study in cotton confirmed some features of the currently accepted gene regulatory cascade for 'typical' plant SCWs, but also revealed substantial differences, especially with key downstream NACs and MYBs. The lignocellulosic SCW of cotton xylem appears to be achieved differently from that in Arabidopsis. Pith cell walls in cotton stems are compositionally very different from that reported for other plant species, including Arabidopsis. The current definition of a 'typical' primary or secondary cell wall might not be applicable to all cell types in all plant species.

Degradation of lignin b-aryl ether units in Arabidopsis thaliana expressing LigD, LigF and LigG from Sphingomonas paucimobilis SYK-6

by John Ralph, Ruben Vanholme, and Wout Boerjan

Lignin is a major polymer in the secondary plant cell wall and composed of hydrophobic interlinke... more Lignin is a major polymer in the secondary plant cell wall and composed of hydrophobic interlinked hydroxyphenylpropanoid units. The presence of lignin hampers conversion of plant biomass into biofuels; plants with modified lignin are therefore being investigated for increased digestibility. The bacterium Sphingomonas paucimobilis produces lignin-degrading enzymes including LigD, LigF and LigG involved in cleaving the most abundant lignin interunit linkage, the b-aryl ether bond. In this study, we expressed the LigD, LigF and LigG (LigDFG) genes in Arabidopsis thaliana to introduce postlignification modifications into the lignin structure. The three enzymes were targeted to the secretory pathway. Phenolic metabolite profiling and 2D HSQC NMR of the transgenic lines showed an increase in oxidized guaiacyl and syringyl units without concomitant increase in oxidized b-aryl ether units, showing lignin bond cleavage. Saccharification yield increased significantly in transgenic lines expressing LigDFG, showing the applicability of our approach. Additional new information on substrate specificity of the LigDFG enzymes is also provided.

Suppression of CINNAMOYL-CoA REDUCTASE increases the level of monolignol ferulates incorporated into maize lignins

Background: The cell wall polymer lignin provides structural support and rigidity to plant cell w... more Background: 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. Results: 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 decreased lignin content and monomers did not, however, impact the plant growth or biomass. The increase in monolignol conjugates correlated with an improvement in the digestibility of maize stem rind tissue. Conclusions: Together, increased monolignol ferulates and improved digestibility in ccr1 mutant plants suggests that they may be superior biofuel crops.

Lignin-Derived Thioacidolysis Dimers: Reevaluation, New Products, Authentication, and Quantification

Different Routes for Conifer-and Sinapaldehyde and Higher Saccharification upon Deficiency in the Dehydrogenase CAD1 1[OPEN

by John Ralph and Wout Boerjan

In the search for renewable energy sources, genetic engineering is a promising strategy to improv... more In the search for renewable energy sources, genetic engineering is a promising strategy to improve plant cell wall composition for biofuel and bioproducts generation. Lignin is a major factor determining saccharification efficiency and, therefore, is a prime target to engineer. Here, lignin content and composition were modified in poplar (Populus tremula 3 Populus alba) by specifically down-regulating CINNAMYL ALCOHOL DEHYDROGENASE1 (CAD1) by a hairpin-RNA-mediated silencing approach, which resulted in only 5% residual CAD1 transcript abundance. These transgenic lines showed no biomass penalty despite a 10% reduction in Klason lignin content and severe shifts in lignin composition. Nuclear magnetic resonance spectroscopy and thioacidolysis revealed a strong increase (up to 20-fold) in sinapaldehyde incorporation into lignin, whereas coniferaldehyde was not increased markedly. Accordingly, ultra-high-performance liquid chromatography-mass spectrometry-based phenolic profiling revealed a more than 24,000-fold accumulation of a newly identified compound made from 8-8 coupling of two sinapaldehyde radicals. However, no additional cinnamaldehyde coupling products could be detected in the CAD1-deficient poplars. Instead, the transgenic lines accumulated a range of hydroxycinnamate-derived metabolites, of which the most prominent accumulation (over 8,500-fold) was observed for a compound that was identified by purification and nuclear magnetic resonance as syringyl lactic acid hexoside. Our data suggest that, upon down-regulation of CAD1, coniferaldehyde is converted into ferulic acid and derivatives, whereas sinapaldehyde is either oxidatively coupled into S9(8-8)S9 and lignin or converted to sinapic acid and derivatives. The most prominent sink of the increased flux to hydroxycinnamates is syringyl lactic acid hexoside. Furthermore, low-extent saccharification assays, under different pretreatment conditions, showed strongly increased glucose (up to +81%) and xylose (up to +153%) release, suggesting that down-regulating CAD1 is a promising strategy for improving lignocellulosic biomass for the sugar platform industry.

Altering carbon allocation in hybrid poplar (Populus alba 3 grandidentata) impacts cell wall growth and development

Galactinol synthase is a pivotal enzyme involved in the synthesis of the raffinose family of olig... more Galactinol synthase is a pivotal enzyme involved in the synthesis of the raffinose family of oligosaccharides (RFOs) that function as transport carbohydrates in the phloem, as storage compounds in sink tissues and as soluble metabolites that combat both abiotic and biotic stress in several plant species. Hybrid poplar (Populus alba 9 grandidentata) overexpressing the Arabidopsis thaliana GolS3 (AtGolS3) gene showed clear effects on development; the extreme overexpressing lines were stunted and had cell wall traits characteristic of tension wood, whereas lines with only moderate up-regulation grew normally and had moderately altered secondary cell wall composition and ultrastructure. Stem cross-sections of the developing xylem revealed a significant increase in the number of vessels, as well as the clear presence of a G-layer in the fibres. Furthermore, AtGolS3-OE lines possessed higher cellulose and lower lignin contents, an increase in cellulose crystallinity, and significantly altered hemicellulose-derived carbohydrates, notably manifested by their mannose and xylose contents. In addition, the transgenic plants displayed elevated xylem starch content. Transcriptome interrogation of the transgenic plants showed a significant up-regulation of genes involved in the synthesis of myo-inositol, along with genes involved in sucrose degradation. The results suggest that the overexpression of GolS and its product galactinol may serve as a molecular signal that initiates metabolic changes, culminating in a change in cell wall development and potentially the formation of tension wood.

Suppression of CINNAMOYL-CoA REDUCTASE increases the level of monolignol ferulates incorporated into maize lignins

Background: The cell wall polymer lignin provides structural support and rigidity to plant cell w... more Background: 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. Results: 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 decreased lignin content and monomers did not, however, impact the plant growth or biomass. The increase in monolignol conjugates correlated with an improvement in the digestibility of maize stem rind tissue. Conclusions: Together, increased monolignol ferulates and improved digestibility in ccr1 mutant plants suggests that they may be superior biofuel crops.

Silencing CHALCONE SYNTHASE in Maize Impedes the Incorporation of Tricin into Lignin and Increases Lignin Content 1[OPEN

by John Ralph and Wout Boerjan

Lignin is a phenolic heteropolymer that is deposited in secondary-thickened cell walls, where it ... more Lignin is a phenolic heteropolymer that is deposited in secondary-thickened cell walls, where it provides mechanical strength. A recent structural characterization of cell walls from monocot species showed that the flavone tricin is part of the native lignin polymer, where it is hypothesized to initiate lignin chains. In this study, we investigated the consequences of altered tricin levels on lignin structure and cell wall recalcitrance by phenolic profiling, nuclear magnetic resonance, and saccharification assays of the naturally silenced maize (Zea mays) C2-Idf (inhibitor diffuse) mutant, defective in the CHALCONE SYNTHASE Colorless2 (C2) gene. We show that the C2-Idf mutant produces highly reduced levels of apigenin-and tricin-related flavonoids, resulting in a strongly reduced incorporation of tricin into the lignin polymer. Moreover, the lignin was enriched in b-b and b-5 units, lending support to the contention that tricin acts to initiate lignin chains and that, in the absence of tricin, more monolignol dimerization reactions occur. In addition, the C2-Idf mutation resulted in strikingly higher Klason lignin levels in the leaves. As a consequence, the leaves of C2-Idf mutants had significantly reduced saccharification efficiencies compared with those of control plants. These findings are instructive for lignin engineering strategies to improve biomass processing and biochemical production.

Silencing CAFFEOYL SHIKIMATE ESTERASE Affects Lignification and Improves Saccharification in Poplar 1[OPEN

by John Ralph, Ruben Vanholme, and Wout Boerjan

Caffeoyl shikimate esterase (CSE) was recently shown to play an essential role in lignin biosynth... more Caffeoyl shikimate esterase (CSE) was recently shown to play an essential role in lignin biosynthesis in Arabidopsis (Arabidopsis thaliana) and later in Medicago truncatula. However, the general function of this enzyme was recently questioned by the apparent lack of CSE activity in lignifying tissues of different plant species. Here, we show that down-regulation of CSE in hybrid poplar (Populus tremula 3 Populus alba) resulted in up to 25% reduced lignin deposition, increased levels of p-hydroxyphenyl units in the lignin polymer, and a relatively higher cellulose content. The transgenic trees were morphologically indistinguishable from the wild type. Ultra-high-performance liquid chromatography-mass spectrometry-based phenolic profiling revealed a reduced abundance of several oligolignols containing guaiacyl and syringyl units and their corresponding hydroxycinnamaldehyde units, in agreement with the reduced flux toward coniferyl and sinapyl alcohol. These trees accumulated the CSE substrate caffeoyl shikimate along with other compounds belonging to the metabolic classes of benzenoids and hydroxycinnamates. Furthermore, the reduced lignin amount combined with the relative increase in cellulose content in the CSE down-regulated lines resulted in up to 62% more glucose released per plant upon limited saccharification when no pretreatment was applied and by up to 86% and 91% when acid and alkaline pretreatments were used. Our results show that CSE is not only important for the lignification process in poplar but is also a promising target for the development of improved lignocellulosic biomass crops for sugar platform biorefineries.

Short-term facilitation of microbial litter decomposition by ultraviolet radiation

Lignocellulose pretreatment in a fungus-cultivating termite

Depolymerizing lignin, the complex phenolic polymer fortifying plant cell walls, is an essential ... more Depolymerizing lignin, the complex phenolic polymer fortifying plant cell walls, is an essential but challenging starting point for the lignocellulosics industries. The variety of ether– and carbon–carbon interunit linkages produced via radical coupling during lignification limit chemical and biological depolymerization efficiency. In an ancient fungus-cultivating termite system, we reveal unprecedentedly rapid lignin depolymerization and degradation by combining laboratory feeding experiments, lignocellulosic compositional measurements , electron microscopy, 2D-NMR, and thermochemolysis. In a gut transit time of under 3.5 h, in young worker termites, poplar lignin sidechains are extensively cleaved and the polymer is significantly depleted, leaving a residue almost completely devoid of various condensed units that are traditionally recognized to be the most recalcitrant. Subsequently, the fungus-comb microbiome preferentially uses xylose and cleaves polysaccharides, thus facilitating final utilization of easily digestible oligosaccharides by old worker termites. This complementary symbiotic pretreatment process in the fungus-growing termite symbiosis reveals a previously unappreciated natural system for efficient lignocellulose degradation. lignin | carbohydrate | NMR | symbiosis | age polyethism

Isolation and characterization of new lignin streams derived from extractive-ammonia (EA) pretreatment

One of the key challenges facing lignin conversion to fuels and chemicals is related to the level... more One of the key challenges facing lignin conversion to fuels and chemicals is related to the level of carbohydrate and ash impurities found in extracted lignin. Structural modifications of lignin may also occur as a result of biomass pretreatment and harsh lignin extraction protocols. Extractive-Ammonia (EA) is a new pretreatment technology that uses liquid ammonia to cleave lignin–carbohydrate complexes, decrystallize cellulose, solubilize lignin, and selectively extract lignin from lignocellulosic biomass, enabling better utilization of both lignin and carbohydrate components in a biorefinery. The EA-based biorefinery produces two different lignin-rich streams, with different properties, that could potentially be upgraded to fuels and chemicals using green processes. In this work, a water/ethanol-based fractionation method was developed to enrich the ammonia-soluble extractives, resulting in a major product stream containing 92% lignin. Detailed characterization of the various streams resulting from EA treatment, including compositional analysis, structural characterization by nuclear magnetic resonance (NMR) spectrometry, elemental analysis , molecular weight analysis, and thermo-gravimetric analysis provides a broad evaluation of the EA-derived lignin product stream structures and properties, assessing their potential for commercial applications. In summary, EA-derived lignins preserve much of lignin's functionality, including the sensitive β-aryl ether units. Nitrogen incorporation was observed in the lignin-rich streams, notably due to the presence of hydroxycinnamoyl amides formed during ammonia pretreatment.

Showcasing research led by L. da Costa Sousa, V. Balan and B. E. Dale group in collaboration with various groups from the As featured in

Next-generation ammonia pretreatment enhances cellulosic biofuel production Enzyme loading reduct... more Next-generation ammonia pretreatment enhances cellulosic biofuel production Enzyme loading reduction is an important requirement for improving the economic viability of lignocellulosic biofuels. One approach to achieve this goal is to reduce biomass recalcitrance via development of more effective pretreatment methods. This communication article presents a novel Extractive Ammonia (EA) pretreatment method and includes a comprehensive overview of the physicochemical events contributing for the effectiveness of EA pretreatment on achieving high biofuel yields at reduced enzyme loadings.

Identification of 4−O−5-Units in Softwood Lignins via Definitive Lignin Models and NMR

Lignins are complex and heterogeneous natural polymers in which the major units are characterized... more Lignins are complex and heterogeneous natural polymers in which the major units are characterized by certain prominent interunit linkages. Previous attempts to identify and quantify 4−O−5-linked units in softwood lignins by NMR were not successful. In this work, various lignin model compounds, including the tetramers formed by the 4−O−5-coupling of β−O−4-, β−β-, and β−5-model dimers, were synthesized. Such compounds are better able to model the corresponding structures in lignins than those used previously. 4−O−5-Linked structures could be clearly observed and identified in real softwood lignin samples by comparison of their 2D HSQC NMR spectra with those from the model compounds. When comparing NMR data of phenol-acetylated versus phenol-etherified model compounds with those of acetylated lignins, it was apparent that most of the 4−O−5-linked structures in softwood lignins are present as free-phenolic end units.

2016-Shuai-Science-354-329 w-SI.pdf

Ligninverwertung Wege zur Verwertung von Lignin: Fortschritte in der Biotechnik, der Bioraffination und der Katalyse

Lignin Valorisation Paving the Way for Lignin Valorisation: Recent Advances in Bioengineering, Biorefining and Catalysis

Angewandte Chemie Keywords: bioengineering · biorefining · catalysis · lignin · lignocellulose An... more

Sugarcane transgenics expressing MYB transcription factors show improved glucose release

Background: Sugarcane, a tropical C4 perennial crop, is capable of producing 30–100 tons or more ... more Background: Sugarcane, a tropical C4 perennial crop, is capable of producing 30–100 tons or more of biomass per hectare annually. The lignocellulosic residue remaining after sugar extraction is currently underutilized and can provide a significant source of biomass for the production of second-generation bioethanol. Results: MYB31 and MYB42 were cloned from maize and expressed in sugarcane with and without the UTR sequences. The cloned sequences were 98 and 99 % identical to the published nucleotide sequences. The inclusion of the UTR sequences did not affect any of the parameters tested. There was little difference in plant height and the number of internodes of the MYB-overexpressing sugarcane plants when compared with controls. MYB transgene expression determined by qPCR exhibited continued expression in young and maturing internodes. MYB31 down-regulated more genes within the lignin biosynthetic pathway than MYB42. MYB31 and MYB42 expression resulted in decreased lignin content in some lines. All MYB42 plants further analyzed showed significant increases in glucose release by enzymatic hydrolysis in 72 h, whereas only two MYB31 plants released more glucose than control plants. This correlated directly with a significant decrease in acid-insoluble lignin. Soluble sucrose content of the MYB42 transgenic plants did not vary compared to control plants. Conclusions: This study demonstrates the use of MYB transcription factors to improve the production of bioethanol from sugarcane bagasse remaining after sugar extraction.

Rapid Py-GC/MS assessment of the structural alterations of lignins in genetically modified plants

Genetic modifications for perturbing the lignin pathway in three different species of angiosperm ... more Genetic modifications for perturbing the lignin pathway in three different species of angiosperm plants, including non-woody (Arabidopsis and alfalfa) and woody (poplar) plants, were readily evaluated by analytical pyrolysis coupled to gas chromatography-mass spectrometry (Py-GC/MS). Pyrolysis showed that the composition of Arabidopsis plants was severely altered when the expression of the gene encoding the enzyme caffeic acid O-methyltransferase (COMT) was downregulated, resulting in a lignin largely enriched in guaiacyl (G) units (88%). Alfalfa plants in which lignin biosynthesis was modified by down-regulation of the p-coumarate 3-hydroxylase (C3H) gene, showed extremely high proportions of p-hydroxyphenyl (H) units (71%) relative to the naturally prevailing guaiacyl (G) and syringyl (S) units. Finally, Py-GC/MS analyses indicated that overexpression in poplar of the gene that encodes the enzyme ferulate 5-hydroxylase (F5H) resulted in a lignin with a higher content of syringyl lignin units (88%) compared to the wild-type control (71%). In conclusion, Py-GC/MS is a useful and convenient tool for the rapid evaluation of compositional changes in lignin from genetically modified plants.

BdCESA7, BdCESA8, and BdPMT Utility Promoter Constructs for Targeted Expression to Secondary Cell-Wall-Forming Cells of Grasses

Utility vectors with promoters that confer desired spatial and temporal expression patterns are u... more Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the β-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels in stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8 Open Reading Frames into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. The identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels confirms that the BdCESA8 gene is a secondary-cell-wall-forming cellulose synthase.

Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin -Aryl Ether Cleavage Pathway from Sphingobium sp. SYK-6 * □ S

There has been great progress in the development of technology for the conversion of lignocellulo... more There has been great progress in the development of technology for the conversion of lignocellulosic biomass to sugars and subsequent fermentation to fuels. However, plant lignin remains an untapped source of materials for production of fuels or high value chemicals. Biological cleavage of lignin has been well characterized in fungi, in which enzymes that create free radical intermediates are used to degrade this material. In contrast, a catabolic pathway for the stereospecific cleavage of-aryl ether units that are found in lignin has been identified in Sphingobium sp. SYK-6 bacteria.-Aryl ether units are typically abundant in lignin, corresponding to 50 –70% of all of the intermonomer linkages. Consequently, a comprehensive understanding of enzymatic-aryl ether (ether) cleavage is important for future efforts to biologically process lignin and its breakdown products. The crystal structures and biochemical characterization of the NAD-dependent dehydrogenases (LigD, LigO, and LigL) and the glutathione-dependent lyase LigG provide new insights into the early and late enzymes in the-ether degradation pathway. We present detailed information on the cofactor and substrate binding sites and on the catalytic mechanisms of these enzymes, comparing them with other known members of their respective families. Information on the Lig enzymes provides new insight into their catalysis mechanisms and can inform future strategies for using aromatic oligomers derived from plant lignin as a source of valuable aromatic compounds for biofuels and other bioproducts.

Tricin-lignins: occurrence and quantitation of tricin in relation to phylogeny

SUMMARY Tricin [5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4H-chromen-4-one], a flavone, was... more SUMMARY Tricin [5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4H-chromen-4-one], a flavone, was recently established as an authentic monomer in grass lignification that likely functions as a nucleation site. It is linked onto lignin as an aryl alkyl ether by radical coupling with monolignols or their acylated analogs. However, the level of tricin that incorporates into lignin remains unclear. Herein, three lignin characterization methods: acidolysis; thioacidolysis; and derivatization followed by reductive cleavage; were applied to quantitatively assess the amount of lignin-integrated tricin. Their efficiencies at cleaving the tricin-(4 0 –O–b)-ether bonds and the degradation of tricin under the corresponding reaction conditions were evaluated. A hexa-deuterated tricin analog was synthesized as an internal standard for accurate quantitation purposes. Thioacidolysis proved to be the most efficient method, liberating more than 91% of the tricin with little degradation. A survey of different seed-plant species for the occurrence and content of tricin showed that it is widely distributed in the lignin from species in the family Poaceae (order Poales). Tricin occurs at low levels in some commelinid monocotyledon families outside the Poaceae, such as the Arecaceae (the palms, order Arecales) and Bromeliaceae (Poales), and the non-commelinid monocotyledon family Orchi-daceae (Orchidales). One eudicotyledon was found to have tricin (Medicago sativa, Fabaceae). The content of lignin-integrated tricin is much higher than the extractable tricin level in all cases. Lignins, including waste lignin streams from biomass processing, could therefore provide a large and alternative source of this valuable flavone, reducing the costs, and encouraging studies into its application beyond its current roles.

Structural Basis of Stereospecificity in the Bacterial Enzymatic Cleavage of -Aryl Ether Bonds in Lignin * □ S

by John Ralph, Daniel Noguera, and Blake Simmons

Lignin is a combinatorial polymer comprising monoaromatic units that are linked via covalent bond... more Lignin is a combinatorial polymer comprising monoaromatic units that are linked via covalent bonds. Although lignin is a potential source of valuable aromatic chemicals, its recalcitrance to chemical or biological digestion presents major obstacles to both the production of second-generation biofuels and the generation of valuable coproducts from lignin's monoaromatic units. Degradation of lignin has been relatively well characterized in fungi, but it is less well understood in bacteria. A catabolic pathway for the enzy-matic breakdown of aromatic oligomers linked via-aryl ether bonds typically found in lignin has been reported in the bacterium Sphingobium sp. SYK-6. Here, we present x-ray crystal structures and biochemical characterization of the glutathione-dependent-etherases, LigE and LigF, from this pathway. The crystal structures show that both enzymes belong to the canonical two-domain fold and glutathione binding site architecture of the glutathione S-transferase family. Mutagenesis of the conserved active site ser-ine in both LigE and LigF shows that, whereas the enzymatic activity is reduced, this amino acid side chain is not absolutely essential for catalysis. The results include descriptions of cofactor binding sites, substrate binding sites, and catalytic mechanisms. Because-aryl ether bonds account for 50 –70% of all interunit linkages in lignin, understanding the mechanism of enzymatic-aryl ether cleavage has significant potential for informing ongoing studies on the valorization of lignin.

2016-Harman-Ware-BiotechJ-11.pdf

An essential role of caffeoyl shikimate esterase in monolignol biosynthesis in Medicago truncatula

SUMMARY Biochemical and genetic analyses have previously identified caffeoyl shikimate esterase (... more SUMMARY Biochemical and genetic analyses have previously identified caffeoyl shikimate esterase (CSE) as an enzyme in the monolignol biosynthesis pathway in Arabidopsis thaliana, although the generality of this finding has been questioned. Here we show the presence of CSE genes and associated enzyme activity in barrel medic (Medicago truncatula, dicot, Leguminosae), poplar (Populus deltoides, dicot, Salicaceae), and switchgrass (Panicum virgatum, monocot, Poaceae). Loss of function of CSE in transposon insertion lines of M. truncat-ula results in severe dwarfing, altered development, reduction in lignin content, and preferential accumulation of hydroxyphenyl units in lignin, indicating that the CSE enzyme is critical for normal lignification in this species. However, the model grass Brachypodium distachyon and corn (Zea mays) do not possess orthologs of the currently characterized CSE genes, and crude protein extracts from stems of these species exhibit only a weak esterase activity with caffeoyl shikimate. Our results suggest that the reaction catalyzed by CSE may not be essential for lignification in all plant species.

Low temperature hydrogenation of pyrolytic lignin over Ru/TiO 2 : 2D HSQC and 13 C NMR study of reactants and products

Pyrolytic lignin and hydrogenated pyrolytic lignin were characterized by 2D 1 H– 13 C HSQC and qu... more Pyrolytic lignin and hydrogenated pyrolytic lignin were characterized by 2D 1 H– 13 C HSQC and quantitative 13 C NMR techniques. The pyrolytic lignin was produced from a mixed maple wood feedstock and separated from the bio-oil by water extraction. p-Hydroxyphenyl (H), guaiacyl (G), and syringyl (S) aromatics were the basic units of pyrolytic lignin. The native lignin β-aryl ether, phenylcoumaran and resinol structures were not present in the pyrolytic lignin. The hydrogenation was conducted with a Ru/TiO 2 catalyst at temperatures ranging from 25–150 °C with higher temperatures exhibiting higher levels of hydrogenation. Solid coke formed on the catalyst surface (1% coke yield) even for hydrogenation at 25 °C. The carbon yield of pyrolytic lignin to coke increased from 1% to 5% as the hydrogenation temperature increased from 25 to 150 °C. A single-step hydrogenation at 150 °C resulted in a reduction from 65% to 39% aromatic carbons. A three-step hydrogenation scheme at this same temperature resulted in a reduction of aromatic carbons from 65% to 17%. The decrease in the aromatic carbon corresponded with an increase in the aliphatic carbon. Coke formation reduced from a 5% carbon yield of pyrolytic lignin in the first hydrogenation step to a 1% carbon yield in each of the second and third hydrogenation steps. The pyrolytic lignin could be separated into a high and low molecular weight fraction. The coke yield from the high molecular weight fraction was twice as much as that from the low molecular weight fraction.

Cell Wall Composition and Biomass Recalcitrance Differences Within a Genotypically Diverse Set of Brachypodium distachyon Inbred Lines

Brachypodium distachyon (Brachypodium) has emerged as a useful model system for studying traits u... 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 syringyl:guaiacyl:p-hydroxyphenyl (S:G:H) lignin ratios. Free glucose, sucrose, and starch content also differed significantly in senesced stems, as did the amounts of sugars released from cell wall polysaccharides (digestibility) upon exposure to a panel of thermochemical pretreatments followed by hydrolytic enzymatic digestion. Correlations were identified between inbred line lignin compositions and plant growth characteristics such as biomass accumulation and heading date (HD), and between amounts of cell wall polysaccharides and biomass digestibility. Finally, stem cell wall p-coumarate and ferulate contents and free-sugars content changed significantly with increased duration of vernalization for some inbred lines. Taken together, these results show that Brachypodium displays substantial phenotypic variation with respect to cell wall composition and biomass digestibility, with some compositional differences correlating with growth characteristics. Moreover, besides influencing HD

Enhancing digestibility and ethanol yield of Populus wood via expression of an engineered monolignol 4-O-methyltransferase

Producing cellulosic biofuels and bio-based chemicals from woody biomass is impeded by the presen... more Producing cellulosic biofuels and bio-based chemicals from woody biomass is impeded by the presence of lignin polymer in the plant cell wall. Manipulating the monolignol biosynthetic pathway offers a promising approach to improved processability, but often impairs plant growth and development. Here, we show that expressing an engineered 4-O-methyl-transferase that chemically modifies the phenolic moiety of lignin monomeric precursors, thus preventing their incorporation into the lignin polymer, substantially alters hybrid aspens' lignin content and structure. Woody biomass derived from the transgenic aspens shows a 62% increase in the release of simple sugars and up to a 49% increase in the yield of ethanol when the woody biomass is subjected to enzymatic digestion and yeast-mediated fermentation. Moreover, the cell wall structural changes do not affect growth and biomass production of the trees. Our study provides a useful strategy for tailoring woody biomass for bio-based applications.

RNAi downregulation of three key lignin genes in sugarcane improves glucose release without reduction in sugar production

Background: Sugarcane is a subtropical crop that produces large amounts of biomass annually. It i... more Background: Sugarcane is a subtropical crop that produces large amounts of biomass annually. It is a key agricultural crop in many countries for the production of sugar and other products. Residual bagasse following sucrose extraction is currently underutilized and it has potential as a carbohydrate source for the production of biofuels. As with all ligno-cellulosic crops, lignin acts as a barrier to accessing the polysaccharides, and as such, is the focus of transgenic efforts. In this study, we used RNAi to individually reduce the expression of three key genes in the lignin biosynthetic pathway in sugarcane. These genes, caffeoyl-CoA O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H) and caffeic acid O-methyltransferase (COMT), impact lignin content and/or composition. Results: For each RNAi construct, we selected three events for further analysis based on qRT-PCR results. For the CCoAOMT lines, there were no lines with a reduction in lignin content and only one line showed improved glucose release. For F5H, no lines had reduced lignin, but one line had a significant increase in glucose release. For COMT, one line had reduced lignin content, and this line and another released higher levels of glucose during enzymatic hydroly-sis. Two of the lines with improved glucose release (F5H-2 and COMT-2) also had reduced S:G ratios. Conclusions: Along with improvements in bagasse quality for the production of lignocellulosic-based fuels, there was only one line with reduction in juice sucrose extraction, and three lines with significantly improved sucrose production , providing evidence that the alteration of sugarcane for improved lignocellulosic ethanol production can be achieved without negatively impacting sugar production and perhaps even enhancing it.

The Structure and Catalytic Mechanism of Sorghum bicolor Caffeoyl-CoA O-Methyltransferase 1

Caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT) is an S-adenosyl methionine (SAM)-dependent O... more Caffeoyl-coenzyme A 3-O-methyltransferase (CCoAOMT) is an S-adenosyl methionine (SAM)-dependent O-methyltransferase responsible for methylation of the meta-hydroxyl group of caffeoyl-coenzyme A (CoA) on the pathway to monolignols, with their ring methoxylation status characteristic of guaiacyl or syringyl units in lignin. In order to better understand the unique class of type 2 O-methyltransferases from monocots, we have characterized CCoAOMT from sorghum (Sorghum bicolor; SbCCoAOMT), including the SAM binary complex crystal structure and steady-state enzyme kinetics. Key amino acid residues were validated with site-directed mutagenesis. Isothermal titration calorimetry data indicated a sequential binding mechanism for SbCCoAOMT, wherein SAM binds prior to caffeoyl-CoA, and the enzyme showed allosteric behavior with respect to it. 5-Hydroxyferuloyl-CoA was not a substrate for SbCCoAOMT. We propose a catalytic mechanism in which lysine-180 acts as a catalytic base and deprotonates the reactive hydroxyl group of caffeoyl-CoA. This deprotonation is facilitated by the coordination of the reactive hydroxyl group by Ca 2+ in the active site, lowering the pK a of the 39-OH group. Collectively, these data give a new perspective on the catalytic mechanism of CCoAOMTs and provide a basis for the functional diversity exhibited by type 2 plant OMTs that contain a unique insertion loop (residues 208–231) conferring affinity for phenylpropanoid-CoA thioesters. The structural model of SbCCoAOMT can serve as the basis for protein engineering approaches to enhance the nutritional, agronomic, and industrially relevant properties of sorghum.

A highly diastereoselective oxidant contributes to Ligninolysis by the white rot basidiomycete Ceriporiopsis subvermispora

by John Ralph and Carl Houtman

Applied and environmental microbiology, 2014

The white rot basidiomycete Ceriporiopsis subvermispora delignifies wood selectively and has pote... more The white rot basidiomycete Ceriporiopsis subvermispora delignifies wood selectively and has potential biotechnological applications. Its ability to remove lignin before the substrate porosity has increased enough to admit enzymes suggests that small diffusible oxidants contribute to delignification. A key question is whether these unidentified oxidants attack lignin via single-electron transfer (SET), in which case they are expected to cleave its propyl side chains between Cα and Cβ and to oxidize the threo-diastereomer of its predominating β-O-4-linked structures more extensively than the corresponding erythro-diastereomer. We used two-dimensional solution-state nuclear magnetic resonance (NMR) techniques to look for changes in partially biodegraded lignin extracted from spruce wood after white rot caused by C. subvermispora. The results showed that (i) benzoic acid residues indicative of Cα-Cβ cleavage were the major identifiable truncated structures in lignin after decay and (ii...

Introduction of chemically labile substructures into Arabidopsis lignin through the use of LigD, the Cα-dehydrogenase from Sphingobium sp. strain SYK-6

Plant biotechnology journal, Jan 8, 2015

Bacteria-derived enzymes that can modify specific lignin substructures are potential targets to e... more Bacteria-derived enzymes that can modify specific lignin substructures are potential targets to engineer plants for better biomass processability. The Gram-negative bacterium Sphingobium sp. SYK-6 possesses a Cα-dehydrogenase (LigD) enzyme that has been shown to oxidize the α-hydroxy functionalities in β-O-4-linked dimers into α-keto analogues that are more chemically labile. Here, we show that recombinant LigD can oxidize an even wider range of β-O-4-linked dimers and oligomers, including the genuine dilignols, guaiacylglycerol-β-coniferyl alcohol ether and syringylglycerol-β-sinapyl alcohol ether. We explored the possibility of using LigD for biosynthetically engineering lignin by expressing the codon-optimized ligD gene in Arabidopsis thaliana. The ligD cDNA, with or without a signal peptide for apoplast targeting, has been successfully expressed, and LigD activity could be detected in the extracts of the transgenic plants. UPLC-MS/MS-based metabolite profiling indicated that lev...

Engineering monolignol p-coumarate conjugates into Poplar and Arabidopsis lignins

Plant physiology, Jan 28, 2015

Lignin acylation, the decoration of hydroxyls on lignin structural units with acyl groups, is com... more Lignin acylation, the decoration of hydroxyls on lignin structural units with acyl groups, is common in many plant species. Monocot lignins are decorated with p-coumarates via the polymerization of monolignol p-coumarate conjugates. The acyltransferase involved in the formation of these conjugates has been identified in a number of model monocot species, but the effect of monolignol p-coumarate conjugates on lignification, and on plant growth and development, has not yet been examined in plants that do not inherently possess p-coumarates on their lignins. The Oryza sativa p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE gene (OsPMT) was introduced into two eudicots, Arabidopsis thaliana and Populus alba × grandidentata, and a series of analytical methods was used to demonstrate the incorporation of the ensuing monolignol p-coumarate conjugates into the lignin of these plants. In poplar, specifically, the addition of these conjugates did not occur at the expense of the naturally incorporated m...

Plant-derived antifungal agent poacic acid targets β-1,3-glucan

Proceedings of the National Academy of Sciences of the United States of America, Jan 9, 2015

A rise in resistance to current antifungals necessitates strategies to identify alternative sourc... more A rise in resistance to current antifungals necessitates strategies to identify alternative sources of effective fungicides. We report the discovery of poacic acid, a potent antifungal compound found in lignocellulosic hydrolysates of grasses. Chemical genomics using Saccharomyces cerevisiae showed that loss of cell wall synthesis and maintenance genes conferred increased sensitivity to poacic acid. Morphological analysis revealed that cells treated with poacic acid behaved similarly to cells treated with other cell wall-targeting drugs and mutants with deletions in genes involved in processes related to cell wall biogenesis. Poacic acid causes rapid cell lysis and is synergistic with caspofungin and fluconazole. The cellular target was identified; poacic acid localized to the cell wall and inhibited β-1,3-glucan synthesis in vivo and in vitro, apparently by directly binding β-1,3-glucan. Through its activity on the glucan layer, poacic acid inhibits growth of the fungi Sclerotinia ...

Analysis of a Modern Hybrid and an Ancient Sugarcane Implicates a Complex Interplay of Factors in Affecting Recalcitrance to Cellulosic Ethanol Production

PLOS ONE, 2015

Abundant evidence exists to support a role for lignin as an important element in biomass recalcit... more Abundant evidence exists to support a role for lignin as an important element in biomass recalcitrance. However, several independent studies have also shown that factors apart from lignin are also relevant and overall, the relative importance of different recalcitrance traits remains in dispute. In this study we used two genetically distant sugarcane genotypes, and performed a correlational study with the variation in anatomical parameters, cell wall composition, and recalcitrance factors between these genotypes. In addition we also tracked alterations in these characteristics in internodes at different stages of development. Significant differences in the development of the culm between the genotypes were associated with clear differential distributions of lignin content and composition that were not correlated with saccharification and fermentation yield. Given the strong influence of the environment on lignin content and composition, we hypothesized that sampling within a single plant could allow us to more easily interpret recalcitrance and changes in lignin biosynthesis than analysing variations between different genotypes with extensive changes in plant morphology and culm anatomy. The syringyl/guaiacyl (S/G) ratio was higher in the oldest internode of the modern genotype, but S/G ratio was not correlated with enzymatic hydrolysis yield nor fermentation efficiency. Curiously we observed a strong positive correlation between ferulate ester level and cellulose conversion efficiency. Together, these data support the hypothesis that biomass enzymatic hydrolysis recalcitrance is governed by a quantitative heritage rather than a single trait.

Tricin, A Flavonoid Monomer in Monocot Lignification

Plant physiology, Jan 9, 2015

Tricin was recently discovered in lignin preparations from wheat straw, and subsequently in all m... more Tricin was recently discovered in lignin preparations from wheat straw, and subsequently in all monocot samples examined. To provide proof that tricin is involved in lignification and establish the mechanism by which it incorporates into the lignin polymer, the 4'-O-β-coupling products of tricin with the monolignols (p-coumaryl, coniferyl, and sinapyl alcohols) were synthesized along with the trimer that would result from its 4'-O-β-coupling with sinapyl alcohol and then coniferyl alcohol. Tricin was also found to cross-couple with monolignols to form tricin-(4'-O-β)-linked dimers in biomimetic oxidations using peroxidase/H2O2 or silver (I) oxide. NMR characterization of GPC-fractionated acetylated maize (Zea mays) lignin revealed that the tricin moieties are found in even the highest molecular weight fractions, ether-linked to lignin units, demonstrating that tricin is indeed incorporated into the lignin polymer. These findings suggest that tricin is fully compatible wi...

Small Glycosylated Lignin Oligomers Are Stored in Arabidopsis Leaf Vacuoles

The Plant cell, Jan 19, 2015

Lignin is an aromatic polymer derived from the combinatorial coupling of monolignol radicals in t... more Lignin is an aromatic polymer derived from the combinatorial coupling of monolignol radicals in the cell wall. Recently, various glycosylated lignin oligomers have been revealed in Arabidopsis thaliana. Given that monolignol oxidation and monolignol radical coupling are known to occur in the apoplast, and glycosylation in the cytoplasm, it raises questions about the subcellular localization of glycosylated lignin oligomer biosynthesis and their storage. By metabolite profiling of Arabidopsis leaf vacuoles, we show that the leaf vacuole stores a large number of these small glycosylated lignin oligomers. Their structural variety and the incorporation of alternative monomers, as observed in Arabidopsis mutants with altered monolignol biosynthesis, indicate that they are all formed by combinatorial radical coupling. In contrast to the common believe that combinatorial coupling is restricted to the apoplast, we hypothesized that the aglycones of these compounds are made within the cell. ...

Effects of PHENYLALANINE AMMONIA LYASE (PAL) knockdown on cell wall composition, biomass digestibility, and biotic and abiotic stress responses in Brachypodium

Journal of experimental botany, Jan 19, 2015

The phenylpropanoid pathway in plants synthesizes a variety of structural and defence compounds, ... 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...

Manipulation of Guaiacyl and Syringyl Monomer Biosynthesis in an Arabidopsis Cinnamyl Alcohol Dehydrogenase Mutant Results in Atypical Lignin Biosynthesis and Modified Cell Wall Structure

by John Ralph and Bryon Donohoe

The Plant Cell, 2015

Modifying lignin composition and structure is a key strategy to increase plant cell wall digestib... more Modifying lignin composition and structure is a key strategy to increase plant cell wall digestibility for biofuel production. Disruption of the genes encoding both cinnamyl alcohol dehydrogenases (CADs), including CADC and CADD, in Arabidopsis thaliana results in the atypical incorporation of hydroxycinnamaldehydes into lignin. Another strategy to change lignin composition is downregulation or overexpression of ferulate 5-hydroxylase (F5H), which results in lignins enriched in guaiacyl or syringyl units, respectively. Here, we combined these approaches to generate plants enriched in coniferaldehyde-derived lignin units or lignins derived primarily from sinapaldehyde. The cadc cadd and ferulic acid hydroxylase1 (fah1) cadc cadd plants are similar in growth to wild-type plants even though their lignin compositions are drastically altered. In contrast, disruption of CAD in the F5H-overexpressing background results in dwarfism. The dwarfed phenotype observed in these plants does not appear to be related to collapsed xylem, a hallmark of many other lignin-deficient dwarf mutants. cadc cadd, fah1 cadc cadd, and cadd F5H-overexpressing plants have increased enzyme-catalyzed cell wall digestibility. Given that these CAD-deficient plants have similar total lignin contents and only differ in the amounts of hydroxycinnamaldehyde monomer incorporation, these results suggest that hydroxycinnamaldehyde content is a more important determinant of digestibility than lignin content.

Chemical and structural changes associated with Cu-catalyzed alkaline-oxidative delignification of hybrid poplar

Biotechnology for biofuels, 2015

Alkaline hydrogen peroxide pretreatment catalyzed by Cu(II) 2,2'-bipyridine complexes has pre... more Alkaline hydrogen peroxide pretreatment catalyzed by Cu(II) 2,2'-bipyridine complexes has previously been determined to substantially improve the enzymatic hydrolysis of woody plants including hybrid poplar as a consequence of moderate delignification. In the present work, cell wall morphological and lignin structural changes were characterized for this pretreatment approach to gain insights into pretreatment outcomes and, specifically, to identify the extent and nature of lignin modification. Through TEM imaging, this catalytic oxidation process was shown to disrupt cell wall layers in hybrid poplar. Cu-containing nanoparticles, primarily in the Cu(I) oxidation state, co-localized with the disrupted regions, providing indirect evidence of catalytic activity whereby soluble Cu(II) complexes are reduced and precipitated during pretreatment. The concentration of alkali-soluble polymeric and oligomeric lignin was substantially higher for the Cu-catalyzed oxidative pretreatment. Thi...

A group of sequence-related sphingomonad enzymes catalyzes cleavage of β-aryl ether linkages in lignin β-guaiacyl and β-syringyl ether dimers

Systematic Structural Characterization of Metabolites in Arabidopsis via Candidate Substrate-Product Pair Networks

The Plant Cell, 2014

Catalytic Alkaline Oxidation of Lignin and its Model Compounds: a Pathway to Aromatic Biochemicals

BioEnergy Research, 2013

Structure-guided analysis of catalytic specificity of the abundantly secreted chitosanase SACTE_5457 from Streptomyces sp. SirexAA-E

by John Ralph, Lai Bergeman, and B. Fox

Proteins: Structure, Function, and Bioinformatics, 2013

Improved saccharification and ethanol yield from field-grown transgenic poplar deficient in cinnamoyl-CoA reductase

Proceedings of the National Academy of Sciences, 2014

Towards lignin-protein crosslinking: amino acid adducts of a lignin model quinone methide

Cellulose, 2014

Disruption of Mediator rescues the stunted growth of a lignin-deficient Arabidopsis mutant

by John Ralph and Bryon Donohoe

Nature, 2014

Stereochemical Features of Glutathione-dependent Enzymes in the Sphingobium sp. Strain SYK-6 β-Aryl Etherase Pathway

Journal of Biological Chemistry, 2014

Influence of Populus genotype on gene expression by the wood decay fungus Phanerochaete chrysosporium

Applied and environmental microbiology, 2014

We examined gene expression patterns in the lignin-degrading fungus Phanerochaete chrysosporium w... more We examined gene expression patterns in the lignin-degrading fungus Phanerochaete chrysosporium when it colonizes hybrid poplar (Populus alba × tremula) and syringyl (S)-rich transgenic derivatives. A combination of microarrays and liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowed detection of a total of 9,959 transcripts and 793 proteins. Comparisons of P. chrysosporium transcript abundance in medium containing poplar or glucose as a sole carbon source showed 113 regulated genes, 11 of which were significantly higher (>2-fold, P < 0.05) in transgenic line 64 relative to the parental line. Possibly related to the very large amounts of syringyl (S) units in this transgenic tree (94 mol% S), several oxidoreductases were among the upregulated genes. Peptides corresponding to a total of 18 oxidoreductases were identified in medium consisting of biomass from line 64 or 82 (85 mol% S) but not in the parental clone (65 mol% S). These results demonstrate that P. chryso...

Determination of the Structure and Catalytic Mechanism of Sorghum bicolor Caffeic Acid O-Methyltransferase and the Structural Impact of Three brown midrib12 Mutations

Plant physiology, Jan 19, 2014

Using S-adenosyl-methionine as the methyl donor, caffeic acid O-methyltransferase from sorghum (S... more Using S-adenosyl-methionine as the methyl donor, caffeic acid O-methyltransferase from sorghum (Sorghum bicolor; SbCOMT) methylates the 5-hydroxyl group of its preferred substrate, 5-hydroxyconiferaldehyde. In order to determine the mechanism of SbCOMT and understand the observed reduction in the lignin syringyl-to-guaiacyl ratio of three brown midrib12 mutants that carry COMT gene missense mutations, we determined the apo-form and S-adenosyl-methionine binary complex SbCOMT crystal structures and established the ternary complex structure with 5-hydroxyconiferaldehyde by molecular modeling. These structures revealed many features shared with monocot ryegrass (Lolium perenne) and dicot alfalfa (Medicago sativa) COMTs. SbCOMT steady-state kinetic and calorimetric data suggest a random bi-bi mechanism. Based on our structural, kinetic, and thermodynamic results, we propose that the observed reactivity hierarchy among 4,5-dihydroxy-3-methoxycinnamyl (and 3,4-dihydroxycinnamyl) aldehyde,...

A gel-state 2D-NMR method for plant cell wall profiling and analysis: a model study with the amorphous cellulose and xylan from ball-milled cotton linters

RSC Advances, 2014

Phenylcoumaran Benzylic Ether Reductase Prevents Accumulation of Compounds Formed under Oxidative Conditions in Poplar Xylem

p-Coumaroyl-CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon

by John Ralph and John Sedbrook

The Plant Journal, 2014

Laccases direct lignification in the discrete secondary cell wall domains of protoxylem

by John Ralph and Taku Demura

Plant physiology, 2014

Plants precisely control lignin deposition in spiral or annular secondary cell wall domains durin... more Plants precisely control lignin deposition in spiral or annular secondary cell wall domains during protoxylem tracheary element (TE) development. Because protoxylem TEs function to transport water within rapidly elongating tissues, it is important that lignin deposition is restricted to the secondary cell walls in order to preserve the plasticity of adjacent primary wall domains. The Arabidopsis (Arabidopsis thaliana) inducible VASCULAR NAC DOMAIN7 (VND7) protoxylem TE differentiation system permits the use of mutant backgrounds, fluorescent protein tagging, and high-resolution live-cell imaging of xylem cells during secondary cell wall development. Enzymes synthesizing monolignols, as well as putative monolignol transporters, showed a uniform distribution during protoxylem TE differentiation. By contrast, the oxidative enzymes LACCASE4 (LAC4) and LAC17 were spatially localized to secondary cell walls throughout protoxylem TE differentiation. These data support the hypothesis that p...

Mutation of the Inducible ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE2 Alters Lignin Composition and Improves Saccharification

PLANT PHYSIOLOGY, 2014

A click chemistry strategy for visualization of plant cell wall lignification

Chem. Commun., 2014

Accumulation of N-acetylglucosamine oligomers in the plant cell wall affects plant architecture in a dose-dependent and conditional manner

Plant physiology, 2014

To study the effect of short N-acetylglucosamine (GlcNAc) oligosaccharides on the physiology of p... more To study the effect of short N-acetylglucosamine (GlcNAc) oligosaccharides on the physiology of plants, N-ACETYLGLUCOSAMINYLTRANSFERASE (NodC) of Azorhizobium caulinodans was expressed in Arabidopsis (Arabidopsis thaliana). The corresponding enzyme catalyzes the polymerization of GlcNAc and, accordingly, β-1,4-GlcNAc oligomers accumulated in the plant. A phenotype characterized by difficulties in developing an inflorescence stem was visible when plants were grown for several weeks under short-day conditions before transfer to long-day conditions. In addition, a positive correlation between the oligomer concentration and the penetrance of the phenotype was demonstrated. Although NodC overexpression lines produced less cell wall compared with wild-type plants under nonpermissive conditions, no indications were found for changes in the amount of the major cell wall polymers. The effect on the cell wall was reflected at the transcriptome level. In addition to genes encoding cell wall-mo...

Monolignol Ferulate Transferase Introduces Chemically Labile Linkages into the Lignin Backbone

Science, 2014

Benzoyl Coenzyme A Pathway-Mediated Metabolism of meta-Hydroxy-Aromatic Acids in Rhodopseudomonas palustris

Journal of Bacteriology, 2013

Simulated Moving Bed Chromatography: Separation and Recovery of Sugars and Ionic Liquid from Biomass Hydrolysates

ChemSusChem, 2013

Preparation of monolignol γ-acetate, γ-p-hydroxycinnamate, and γ-p-hydroxybenzoate conjugates: selective deacylation of phenolic acetates with hydrazine acetate

RSC Advances, 2013

Two-Dimensional NMR Evidence for Cleavage of Lignin and Xylan Substituents in Wheat Straw Through Hydrothermal Pretreatment and Enzymatic Hydrolysis

by John Ralph, Christopher Hunt, and Nuria Cortés-francisco

BioEnergy Research, 2012

Breeding with rare defective alleles (BRDA): a natural Populus nigra HCT mutant with modified lignin as a case study

New Phytologist, 2013

Visualization of plant cell wall lignification using fluorescence-tagged monolignols

The Plant Journal, 2013

Novel seed coat lignins in the Cactaceae: structure, distribution and implications for the evolution of lignin diversity

The Plant Journal, 2013

Coexistence but Independent Biosynthesis of Catechyl and Guaiacyl/Syringyl Lignin Polymers in Seed Coats

The Plant Cell, 2013

Lignins are phenylpropanoid polymers, derived from monolignols, commonly found in terrestrial pla... more Lignins are phenylpropanoid polymers, derived from monolignols, commonly found in terrestrial plant secondary cell walls. We recently reported evidence of an unanticipated catechyl lignin homopolymer (C lignin) derived solely from caffeyl alcohol in the seed coats of several monocot and dicot plants. We previously identified plant seeds that possessed either C lignin or traditional guaiacyl/syringyl (G/S) lignins, but not both. Here, we identified several dicot plants (Euphorbiaceae and Cleomaceae) that produce C lignin together with traditional G/S lignins in their seed coats. Solution-state NMR analyses, along with an in vitro lignin polymerization study, determined that there is, however, no copolymerization detectable (i.e., that the synthesis and polymerization of caffeyl alcohol and conventional monolignols in vivo is spatially and/or temporally separated). In particular, the deposition of G and C lignins in Cleome hassleriana seed coats is developmentally regulated during seed maturation; C lignin appears successively after G lignin within the same testa layers, concurrently with apparent loss of the functionality of O-methyltransferases, which are key enzymes for the conversion of C to G lignin precursors. This study exemplifies the flexible biosynthesis of different types of lignin polymers in plants dictated by substantial, but poorly understood, control of monomer supply by the cells.

Profiling of diferulates (plant cell wall cross-linkers) using ultrahigh-performance liquid chromatography-tandem mass spectrometry

The Analyst, 2013

Caffeoyl Shikimate Esterase (CSE) is an Enzyme in the Lignin Biosynthetic Pathway in Arabidopsis

Science, 2013

Ptr-miR397a is a negative regulator of laccase genes affecting lignin content in Populus trichocarpa

by John Ralph and S. Tunlaya-anukit

Proceedings of the National Academy of Sciences, 2013

Loss of function of cinnamyl alcohol dehydrogenase 1 leads to unconventional lignin and a temperature-sensitive growth defect in Medicago truncatula

Proceedings of the National Academy of Sciences, 2013

There is considerable debate over the capacity of the cell wall polymer lignin to incorporate unn... more There is considerable debate over the capacity of the cell wall polymer lignin to incorporate unnatural monomer units. We have identified Tnt1 retrotransposon insertion mutants of barrel medic (Medicago truncatula) that show reduced lignin autofluorescence under UV microscopy and red coloration in interfascicular fibers. The phenotype is caused by insertion of retrotransposons into a gene annotated as encoding cinnamyl alcohol dehydrogenase, here designated M. truncatula CAD1. NMR analysis indicated that the lignin is derived almost exclusively from coniferaldehyde and sinapaldehyde and is therefore strikingly different from classical lignins, which are derived mainly from coniferyl and sinapyl alcohols. Despite such a major alteration in lignin structure, the plants appear normal under standard conditions in the greenhouse or growth chamber. However, the plants are dwarfed when grown at 30 °C. Glycome profiling revealed an increased extractability of some xylan and pectin epitopes from the cell walls of the cad1-1 mutant but decreased extractability of others, suggesting that aldehyde-dominant lignin significantly alters cell wall structure.

Suppression of CCR impacts metabolite profile and cell wall composition in Pinus radiata tracheary elements

Plant Molecular Biology, 2013

Chemoselective Metal-Free Aerobic Alcohol Oxidation in Lignin

Journal of the American Chemical Society, 2013

Structural Characterization of Lignin Isolated from Coconut (Cocos nucifera) Coir Fibers

Journal of Agricultural and Food Chemistry, 2013

The structure of the isolated milled &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp... more The structure of the isolated milled &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;wood&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; lignin from coconut coir has been characterized using different analytical methods, including Py-GC/MS, 2D NMR, DFRC, and thioacidolysis. The analyses demonstrated that it is a p-hydroxyphenyl-guaiacyl-syringyl (H-G-S) lignin, with a predominance of G units (S/G ratio 0.23) and considerable amounts of associated p-hydroxybenzoates. Two-dimensional NMR indicated that the main substructures present in this lignin include β-O-4&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; alkyl aryl ethers followed by phenylcoumarans and resinols. Two-dimensional NMR spectra also indicated that coir lignin is partially acylated at the γ-carbon of the side chain with p-hydroxybenzoates and acetates. DFRC analysis showed that acetates preferentially acylate the γ-OH in S rather than in G units. Despite coir lignin&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s being highly enriched in G-units, thioacidolysis indicated that β-β&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; resinol structures are mostly derived from sinapyl alcohol. Finally, we find evidence that the flavone tricin is incorporated into the coconut coir lignin, as has been recently noted for various grasses.

Plant cell wall profiling by fast maximum likelihood reconstruction (FMLR) and region-of-interest (ROI) segmentation of solution-state 2D 1H–13C NMR spectra

Biotechnology for Biofuels, 2013

Facile Synthesis of 4-Hydroxycinnamaldehydes

BioEnergy Research, 2011

Environmental Stresses of Field Growth Allow Cinnamyl Alcohol Dehydrogenase-Deficient Nicotiana attenuata Plants to Compensate for their Structural Deficiencies

PLANT PHYSIOLOGY, 2012

Identification of Grass-specific Enzyme That Acylates Monolignols with p-Coumarate

Journal of Biological Chemistry, 2012

Rapid Syntheses of Dehydrodiferulates via Biomimetic Radical Coupling Reactions of Ethyl Ferulate

Journal of Agricultural and Food Chemistry, 2012

Whole plant cell wall characterization using solution-state 2D NMR

Nature protocols, 2012

Recent advances in nuclear magnetic resonance (NMR) technology have made it possible to rapidly s... more Recent advances in nuclear magnetic resonance (NMR) technology have made it possible to rapidly screen plant material and discern whole cell wall information without the need to deconstruct and fractionate the plant cell wall. This approach can be used to improve our understanding of the biology of cell wall structure and biosynthesis, and as a tool to select plant material for the most appropriate industrial applications. This is particularly true in an era when renewable materials are vital to the emerging bio-based economies. This protocol describes procedures for (i) the preparation and extraction of a biological plant tissue, (ii) solubilization strategies for plant material of varying composition and (iii) 2D NMR acquisition (for typically 15 min-5 h) and integration methods used to elucidate lignin subunit composition and lignin interunit linkage distribution, as well as cell wall polysaccharide profiling. Furthermore, we present data that demonstrate the utility of this new ...

Lignification and Lignin Manipulations in Conifers

Advances in Botanical Research, 2012

ABSTRACT Lignification is integral to wood formation and has been studied in great detail in coni... more ABSTRACT Lignification is integral to wood formation and has been studied in great detail in conifers for decades. This effort has resulted in detailed knowledge about the chemical composition, structure and content of lignin in different cell and wood types. Lignin distribution and biochemical composition has been resolved at an ultra-structural level, and structural models for conifer lignin have been established. Recent years have seen significant advances in our molecular-level understanding of lignification, and in conifer monolignol biosynthesis. The majority of the genes involved have been identified and the molecular functions of several have been experimentally verified. Suppression of lignin-related genes confirmed that lignin is vital for plant fitness and vascular integrity in conifers and established that conifers do not tolerate substantial reductions in lignin content. Significant gaps in our understanding of conifer lignification nevertheless remain. Aspects of lignification about which we still know relatively little include: the regulatory cascades that trigger lignification, metabolic connections between monolignol biosynthesis and other metabolic processes, the cellular biology of monolignol biosynthesis, the transport of monolignols to the apoplast, the role of monolignol glucosides in lignification, the process of lignin initiation, and the interaction of lignin with other cell wall polymers such as non-cellulosic polysaccharides. These significant gaps in our understanding provide ample opportunity for new and exciting discoveries on lignification in conifers.

Biosynthesis and incorporation of side-chain-truncated lignin monomers to reduce lignin polymerization and enhance saccharification

by John Ralph and Özgül Çetinkol

Plant Biotechnology Journal, 2012

An Engineered Monolignol 4-O-Methyltransferase Depresses Lignin Biosynthesis and Confers Novel Metabolic Capability in Arabidopsis

The Plant Cell, 2012

A polymer of caffeyl alcohol in plant seeds

Proceedings of the National Academy of Sciences, 2012

Metabolic engineering of novel lignin in biomass crops

New Phytologist, 2012

Syntheses of Lignin-Derived Thioacidolysis Monomers and Their Uses as Quantitation Standards

Journal of Agricultural and Food Chemistry, 2012

Structural Characterization of Wheat Straw Lignin as Revealed by Analytical Pyrolysis, 2D-NMR, and Reductive Cleavage Methods

Journal of Agricultural and Food Chemistry, 2012

The structure of the lignin in wheat straw has been investigated by a combination of analytical p... more The structure of the lignin in wheat straw has been investigated by a combination of analytical pyrolysis, 2D-NMR, and derivatization followed by reductive cleavage (DFRC). It is a p-hydroxyphenyl-guaiacyl-syringyl lignin (with an H:G:S ratio of 6:64:30) associated with p-coumarates and ferulates. 2D-NMR indicated that the main substructures present are β-O-4&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-ethers (∼~75%), followed by phenylcoumarans (~11%), with lower amounts of other typical units. A major new finding is that the flavone tricin is apparently incorporated into the lignins. NMR and DFRC indicated that the lignin is partially acylated (~10%) at the γ-carbon, predominantly with acetates that preferentially acylate guaiacyl (12%) rather than syringyl (1%) units; in dicots, acetylation is predominantly on syringyl units. p-Coumarate esters were barely detectable (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;1%) on monomer conjugates released by selectively cleaving β-ethers in DFRC, indicating that they might be preferentially involved in condensed or terminal structures.

Structural Characterization of the Lignin in the Cortex and Pith of Elephant Grass (Pennisetum purpureum) Stems

Journal of Agricultural and Food Chemistry, 2012

Hydroxycinnamate Conjugates as Potential Monolignol Replacements: In vitro Lignification and Cell Wall Studies with Rosmarinic Acid

ChemSusChem, 2012

Synthesis and Characterization of New 5-Linked Pinoresinol Lignin Models

Chemistry - A European Journal, 2012

Cellulose microfibril angles and cell-wall polymers in different wood types of Pinus radiata

Cellulose, 2012

Epigallocatechin gallate incorporation into lignin enhances the alkaline delignification and enzymatic saccharification of cell walls

Biotechnology for Biofuels, 2012

Science, society and biosafety of a field trial with transgenic biofuel poplars

BMC Proceedings, 2011

Independent Recruitment of an O-Methyltransferase for Syringyl Lignin Biosynthesis in Selaginella moellendorffii

The Plant Cell, 2011

Reactions of dehydrodiferulates with ammonia

Organic & Biomolecular Chemistry, 2011

Delineating pMDI model reactions with loblolly pine via solution-state NMR spectroscopy. Part 1. Catalyzed reactions with wood models and wood polymers

Holzforschung, 2000

Stereoselective synthesis of 1-O-β-feruloyl and 1-O-β-sinapoyl glucopyranoses

The charophycean green algae provide insights into the early origins of plant cell walls

The DUF579 domain containing proteins IRX15 and IRX15-L affect xylan synthesis in Arabidopsis

The Plant Journal, 2011

CCoAOMT suppression modifies lignin composition in Pinus radiata

The Plant Journal, 2011

Molecular and Biochemical Basis for Stress-Induced Accumulation of Free and Bound p-Coumaraldehyde in Cucumber

PLANT PHYSIOLOGY, 2011

Lignin Composition and Structure in Young versus Adult Eucalyptus globulus Plants

PLANT PHYSIOLOGY, 2011

Multidimensional NMR analysis reveals truncated lignin structures in wood decayed by the brown rot basidiomycete Postia placenta

Environmental Microbiology, 2011

Multi-scale visualization and characterization of lignocellulosic plant cell wall deconstruction during thermochemical pretreatment

by John Ralph and Bryon Donohoe

Energy & Environmental Science, 2011

Fluorescence-Tagged Monolignols: Synthesis, and Application to Studying In Vitro Lignification

Biomacromolecules, 2011

Significant Alteration of Gene Expression in Wood Decay Fungi Postia placenta and Phanerochaete chrysosporium by Plant Species

by John Ralph and Jill Gaskell

Applied and Environmental Microbiology, 2011

Mass spectrometry-based sequencing of lignin oligomers

Plant physiology, 2010

Although the primary structure of proteins, nucleic acids, and carbohydrates can be readily deter... more Although the primary structure of proteins, nucleic acids, and carbohydrates can be readily determined, no sequencing method has been described yet for the second most abundant biopolymer on earth (i.e. lignin). Within secondary-thickened plant cell walls, lignin forms an aromatic mesh arising from the combinatorial radical-radical coupling of monolignols and many other less abundant monomers. This polymerization process leads to a plethora of units and linkage types that affect the physicochemical characteristics of the cell wall. Current methods to analyze the lignin structure focus only on the frequency of the major monomeric units and interunit linkage types but do not provide information on the presence of less abundant unknown units and linkage types, nor on how linkages affect the formation of neighboring linkages. Such information can only be obtained using a sequencing approach. Here, we describe, to our knowledge for the first time, a sequencing strategy for lignin oligome...

Modeling lignin polymerization. I. Simulation model of dehydrogenation polymers

Plant physiology, 2010

Lignin is a heteropolymer that is thought to form in the cell wall by combinatorial radical coupl... more Lignin is a heteropolymer that is thought to form in the cell wall by combinatorial radical coupling of monolignols. Here, we present a simulation model of in vitro lignin polymerization, based on the combinatorial coupling theory, which allows us to predict the reaction conditions controlling the primary structure of lignin polymers. Our model predicts two controlling factors for the beta-O-4 content of syringyl-guaiacyl lignins: the supply rate of monolignols and the relative amount of supplied sinapyl alcohol monomers. We have analyzed the in silico degradability of the resulting lignin polymers by cutting the resulting lignin polymers at beta-O-4 bonds. These are cleaved in analytical methods used to study lignin composition, namely thioacidolysis and derivatization followed by reductive cleavage, under pulping conditions, and in some lignocellulosic biomass pretreatments.

Lignin Biosynthesis and Structure

PLANT PHYSIOLOGY, 2010

Engineering traditional monolignols out of lignin by concomitant up-regulation of F5H1 and down-regulation of COMT in Arabidopsis

by John Ralph and Antje Rohde

Biochemistry of Hydroxycinnamates and Their Role in Plant, Food, Nutritional and Agricultural Sciences

Convergent Evolution of Syringyl Lignin Biosynthesis via Distinct Pathways in the Lycophyte Selaginella and Flowering Plants

The Plant Cell, 2010

Sequencing around 5-Hydroxyconiferyl Alcohol-Derived Units in Caffeic Acid O-Methyltransferase-Deficient Poplar Lignins

PLANT PHYSIOLOGY, 2010

Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d6/pyridine-d5

Organic & Biomolecular Chemistry, 2010

NMR fingerprinting of the components of finely divided plant cell walls swelled in DMSO has been ... more NMR fingerprinting of the components of finely divided plant cell walls swelled in DMSO has been recently described. Cell wall gels, produced directly in the NMR tube with perdeutero-dimethylsulfoxide, allowed the acquisition of well resolved/dispersed 2D (13)C-(1)H correlated solution-state NMR spectra of the entire array of wall polymers, without the need for component fractionation. That is, without actual solubilization, and without apparent structural modification beyond that inflicted by the ball milling and ultrasonication steps, satisfactorily interpretable spectra can be acquired that reveal compositional and structural details regarding the polysaccharide and lignin components in the wall. Here, the profiling method has been improved by using a mixture of perdeuterated DMSO and pyridine (4 : 1, v/v). Adding pyridine provided not only easier sample handling because of the better mobility compared to the DMSO-d(6)-only system but also considerably elevated intensities and improved resolution of the NMR spectra due to the enhanced swelling of the cell walls. This modification therefore provides a more rapid method for comparative structural evaluation of plant cell walls than is currently available. We examined loblolly pine (Pinus taeda, a gymnosperm), aspen (Populus tremuloides, an angiosperm), kenaf (Hibiscus cannabinus, an herbaceous plant), and corn (Zea mays L., a grass, i.e., from the Poaceae family). In principle, lignin composition (notably, the syringyl : guaiacyl : p-hydroxyphenyl ratio) can be quantified without the need for lignin isolation. Correlations for p-coumarate units in the corn sample are readily seen, and a variety of the ferulate correlations are also well resolved; ferulates are important components responsible for cell wall cross-linking in grasses. Polysaccharide anomeric correlations were tentatively assigned for each plant sample based on standard samples and various literature data. With the new potential for chemometric analysis using the 2D NMR fingerprint, this gel-state method may provide the basis for an attractive approach to providing a secondary screen for selecting biomass lines and for optimizing biomass processing and conversion efficiencies.

Isolation of Cellulolytic Enzyme Lignin from Wood Preswollen/Dissolved in Dimethyl Sulfoxide/N-Methylimidazole

Journal of Agricultural and Food Chemistry, 2010

Advances in modifying lignin for enhanced biofuel production

Current Opinion in Plant Biology, 2010

Identifying new lignin bioengineering targets: 1. Monolignol-substitute impacts on lignin formation and cell wall fermentability

BMC Plant Biology, 2010

Comparative study of SPORL and dilute-acid pretreatments of spruce for cellulosic ethanol production

Bioresource Technology, 2010

Understanding the impact of ionic liquid pretreatment on cellulose and lignin via thermochemical analysis

by John Ralph and Özgül Çetinkol

Biomass and Bioenergy, 2013

Comparative Transcriptome and Secretome Analysis of Wood Decay Fungi Postia placenta and Phanerochaete chrysosporium

Applied and Environmental Microbiology, 2010

Mass Spectrometry-Based Fragmentation as an Identification Tool in Lignomics

by John Ralph and Ruben Vanholme

Analytical Chemistry, 2010

Sequencing around 5-Hydroxyconiferyl Alcohol-Derived Units in Caffeic Acid O-Methyltransferase-Deficient Poplar Lignins

NMR database of lignin and cell wall model compounds

Genetic and molecular basis of grass cell-wall degradability. I. Lignin-cell wall matrix interactions

Lignification limits grass cell-wall digestion by herbivores. Lignification is spatially and temp... more Lignification limits grass cell-wall digestion by herbivores. Lignification is spatially and temporally regulated, and lignin characteristics differ between cell walls, plant tissues, and plant parts. Grass lignins are anchored within walls by ferulate and diferulate cross-links, p-coumarate cyclodimers, and possibly benzyl ester and ether cross-links. Cell-wall degradability is regulated by lignin concentration, cross-linking, and hydrophobicity but not directly by most

Effects of Coumarate 3-Hydroxylase Down-regulation on Lignin Structure

Journal of Biological Chemistry, 2006

Phenolic Compounds as Cross-Links of Plant Derived Polysaccharides

A potential role of sinapyl p-coumarate in grass lignin formation

Structural Characterization of Lignin during Pinus taeda Wood Treatment with Ceriporiopsis subvermispora

by John Ralph and Andre Ferraz

Applied and Environmental Microbiology, 2004

Coniferyl Ferulate Incorporation into Lignin Enhances the Alkaline Delignification and Enzymatic Degradation of Cell Walls

Biomacromolecules, 2008

Incorporating ester interunit linkages into lignin could facilitate fiber delignification and uti... more Incorporating ester interunit linkages into lignin could facilitate fiber delignification and utilization. In model studies with maize cell walls, we examined how partial substitution of coniferyl alcohol (a normal monolignol) with coniferyl ferulate (an ester conjugate from lignan biosynthesis) alters the formation and alkaline extractability of lignin and the enzymatic hydrolysis of structural polysaccharides. Coniferyl ferulate moderately reduced lignification and cell-wall ferulate copolymerization with monolignols. Incorporation of coniferyl ferulate increased lignin extractability by up to 2-fold in aqueous NaOH, providing an avenue for producing fiber with less noncellulosic and lignin contamination or of delignifying at lower temperatures. Cell walls lignified with coniferyl ferulate were more readily hydrolyzed with fibrolytic enzymes, both with and without alkaline pretreatment. Based on our results, bioengineering of plants to incorporate coniferyl ferulate into lignin should enhance lignocellulosic biomass saccharification and particularly pulping for paper production.

Genetic and molecular basis of grass cell wall biosynthesis and degradability. II. Lessons from brown-midrib mutants

by John Ralph and Valérie Méchin

Comptes Rendus Biologies, 2004

Genetic and molecular basis of grass cell-wall degradability. I. Lignin–cell wall matrix interactions

Comptes Rendus Biologies, 2004

Lignification limits grass cell-wall digestion by herbivores. Lignification is spatially and temp... more Lignification limits grass cell-wall digestion by herbivores. Lignification is spatially and temporally regulated, and lignin characteristics differ between cell walls, plant tissues, and plant parts. Grass lignins are anchored within walls by ferulate and diferulate cross-links, p-coumarate cyclodimers, and possibly benzyl ester and ether cross-links. Cell-wall degradability is regulated by lignin concentration, cross-linking, and hydrophobicity but not directly by most variations in lignin composition or structure. Genetic manipulation of lignification can improve grass cell-wall degradability, but the degree of success will depend on genetic background, plant modification techniques employed, and analytical methods used to characterize cell walls.

Discovery of Lignin in Seaweed Reveals Convergent Evolution of Cell-Wall Architecture

Current Biology, 2009

Evidence for cleavage of lignin by a brown rot basidiomycete

Environmental Microbiology, 2008

Isolation and identification of a ferulic acid dehydrotrimer from saponified maize bran insoluble fiber

European Food Research and Technology, 2003

Cell wall hydroxycinnamates in wild rice ( Zizania aquatica L.) insoluble dietary fibre

European Food Research and Technology, 2002

2D-NMR (HSQC) difference spectra between specifically 13C-enriched and unenriched protolignin of Ginkgo biloba obtained in the solution state of whole cell wall material

by John Ralph, Ulla Westermark, and Magnus Paulsson

Holzforschung, 2000

Simplified Preparation of Coniferyl and Sinapyl Alcohols

Journal of Agricultural and Food Chemistry, 2005

Coniferyl and sinapyl alcohols were prepared from commercially available coniferaldehyde and sina... more Coniferyl and sinapyl alcohols were prepared from commercially available coniferaldehyde and sinapaldehyde using borohydride exchange resin in methanol. This reduction is highly regioselective and exceptionally simple, making these valuable monolignols readily available to researchers lacking synthetic chemistry expertise.

Structural Identification of Dehydrotriferulic and Dehydrotetraferulic Acids Isolated from Insoluble Maize Bran Fiber

Journal of Agricultural and Food Chemistry, 2006

Two new dehydrotriferulic acids and two dehydrotetraferulic acids were isolated from saponified m... more Two new dehydrotriferulic acids and two dehydrotetraferulic acids were isolated from saponified maize bran insoluble fiber using size exclusion chromatography on Bio-Beads S-X3 followed by Sephadex LH-20 chromatography and semipreparative phenyl-hexyl reversed phase high-performance liquid chromatography. On the basis of UV spectroscopy, mass spectrometry, and one- and two-dimensional NMR experiments, the structures were identified as 8-5(noncyclic)/5-5-dehydrotriferulic acid, 8-8(tetrahydrofuran)/5-5-dehydrotriferulic acid, and 4-O-8/5-5/8-O-4-dehydrotetraferulic acid. The second tetramer was tentatively identified as 4-O-8/5-5/8-5(noncyclic)-dehydrotetraferulic acid. Compounds containing an 8-5(noncyclic)-coupled dimeric unit probably do not exist in planta but are formed from their phenylcoumaran precursors containing an 8-5(cyclic)-coupled dimeric unit during saponification. The presented dehydrotrimers are the first dehydrotriferulates that do not contain an 8-O-4-coupled dimeric unit. The ferulate dehydrotetramers that are reported for the first time are presumed, like the dimers and trimers, to cross-link polysaccharides in the plant. Because both tetramers contain a 5-5/8-O-4-dehydrotriferulate moiety, the predominant dehydrotrimer in maize bran, it is not possible to deduce whether tetramers are formed by coupling of a fourth unit to a preformed dehydrotriferulate or by 5-5-coupling of preformed 8-O-4- and 8-5-dehydrodiferulates. Nevertheless, such compounds document expanded roles for ferulates in cross-linking polysaccharides in plant cell walls.

Model Studies of Ferulate−Coniferyl Alcohol Cross-Product Formation in Primary Maize Walls: Implications for Lignification in Grasses

Journal of Agricultural and Food Chemistry, 2002

Ferulate and diferulates mediate cell wall cross-linking in grasses, but little is known about th... more Ferulate and diferulates mediate cell wall cross-linking in grasses, but little is known about their cross-coupling reactions with monolignols and their role in lignin formation in primary cell walls. Feruloylated primary walls of maize were artificially lignified and then saponified to release ferulate and diferulates and their cross-products with coniferyl alcohol for analysis by GC-FID, GC-MS, and NMR spectroscopy. Ferulate and 5-5-coupled diferulate had a greater propensity than 8-coupled diferulates to copolymerize with coniferyl alcohol, forming mostly 4-O-beta&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; and 8-beta&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; and some 8-O-4&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; and 8-5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; cross-coupled structures. Some 8-beta&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; structures de-esterified from xylans, but these cross-links were subsequently replaced as 8-coupled diferulates formed stable cross-coupled structures with lignin. Based on the incorporation kinetics of ferulate and diferulates and the predicted growth of lignin, cross-products formed at the onset of lignification acted as nucleation sites for lignin polymerization.

NMR Characterization of Lignins Isolated from Fruit and Vegetable Insoluble Dietary Fiber

Journal of Agricultural and Food Chemistry, 2006

Compositional information for lignins in food is rare and concentrated on cereal grains and brans... more Compositional information for lignins in food is rare and concentrated on cereal grains and brans. As lignins are suspected to have important health roles in the dietary fiber complex, the confusing current information derived from nonspecific lignin determination methods needs to be augmented by diagnostic structural studies. For this study, lignin fractions were isolated from kiwi, pear, rhubarb, and, for comparison, wheat bran insoluble dietary fiber. Clean pear and kiwi lignin isolates allowed for substantive structural profiling, but it is suggested that the significance of lignin in wheat has been overestimated by reliance on nonspecific analytical methods. Volume integration of NMR contours in two-dimensional (13)C-(1)H correlation spectra shows that pear and wheat lignins have comparable guaiacyl and syringyl contributions and that kiwi lignins are particularly guaiacyl-rich (approximately 94% guaiacyl) and suggest that rhubarb lignins, which could not be isolated from contaminating materials, are as syringyl-rich (approximately 96% syringyl) as lignins from any known natural or transgenic fiber source. Typical lignin structures, including those newly NMR-validated (glycerols, spirodienones, and dibenzodioxocins), and resinols implicated as possible mammalian lignan precursors in the gut are demonstrated via their NMR correlation spectra in the fruit and vegetable samples. A novel putative benzodioxane structure appears to be associated with the kiwi lignin. It is concluded that the fruits and vegetables examined contain authentic lignins and that the detailed structural analysis exposes limitations of currently accepted analytical methods.

NMR Evidence for Benzodioxane Structures Resulting from Incorporation of 5-Hydroxyconiferyl Alcohol into Lignins of O -Methyltransferase-Deficient Poplars

Journal of Agricultural and Food Chemistry, 2001

Identification of 4-O-5‘-Coupled Diferulic Acid from Insoluble Cereal Fiber

Journal of Agricultural and Food Chemistry, 2000

The extracts of saponified cereal fibers of whole grains of corn (Zea mays cv. microsperma KOERN.... more The extracts of saponified cereal fibers of whole grains of corn (Zea mays cv. microsperma KOERN.), wheat (Triticum aestivum L.), spelt (Triticum spelta L.), and rice (Oryza sativa L.) were investigated for dehydrodimers of ferulic acid using gas-liquid chromatography (GLC) with mass spectrometric detection (GLC-MS) and flame ionization detection (GLC-FID). In addition to the 8,5&#39;-, 8, 8&#39;-, 5,5&#39;-, and 8-O-4&#39;-coupled diferulic acids previously identified from other plant materials the 4-O-5&#39;-coupled diferulic acid (E)-3-[4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy]-4-hydroxy-5-methoxyci nnamic acid (4-O-5&#39;-DFA) was identified in all fibers investigated. This new diferulate was authenticated by comparison of its mass spectrum and its relative GLC retention time with those of the synthesized compound. Semiquantitative determination of 4-O-5&#39;-DFA showed that it is present at 8-30 microg/g, approximately 70-100 times lower concentrations than the sum of 8,5&#39;-coupled diferulic acids, the major diferulic acids in the investigated fibers.

Ferulate cross-linking in cell walls isolated from maize cell suspensions

by Jerzy Zoń, Ronald Hatfield, and John Ralph

Phytochemistry, 1995

< i> p</i>-coumaroylated syringyl units in maize lignin: Implications for β-ether cleavage by thioacidolysis

Synthesis of methyl 5-< i> O-trans</i>-feruloyl-α-l-arabinofuranoside and its use as a substrate to assess feruloyl esterase activity

Improving forage fiber digestibility

Stereoselectivity in benzyl 1, 2-diaryl ether cleavage by bromotrimethylsilane

Lignin-ferulate cross-links in grasses: active incorporation of ferulate polysaccharide esters into ryegrass lignins

Carbohydrate Research, 1995

Active incorporation of ferulate polysaccharide esters into ryegrass lignins has been demonstrate... more

Synthesis of feruloylated and p-coumaroylated methyl glycosides

Carbohydrate Research, 1992

Formation of ferulic acid dehydrodimers through oxidative cross-linking of sugar beet pectin

Carbohydrate Research, 1997

Cell Wall Structural Foundations: Molecular Basis for Improving Forage Digestibilities

Crop Science, 1999

Unexpected variation in lignin

Current Opinion in Plant Biology, 1999

Recent studies on mutant and transgenic plants indicate that lignification may be far more flexib... more Recent studies on mutant and transgenic plants indicate that lignification may be far more flexible than previously realized. Pines with a mutation affecting the biosynthesis of the major lignin precursor, coniferyl alcohol, show a high level of an unusual subunit, dihydroconiferyl alcohol. These results argue in favor of an increased potential for genetic modification of lignin and indicate that our knowledge of the biosynthesis of lignin is far from complete.

Dimeric ß-Ether Thioacidolysis Products Resulting from Incomplete Ether Cleavage

Holzforschung, 1996

A Biomimetic Route to Lignin Model Compounds via Silver (I) Oxide Oxidation. 2. NMR Characterization of Non-Cyclic Benzyl Aryl Ether Trimers and Tetramers

Holzforschung, 1994

Accuracy of Klason Lignin and Acid Detergent Lignin Methods As Assessed by Bomb Calorimetry †

Journal of Agricultural and Food Chemistry, 1999

An accurate method for estimation of lignin concentration is important for prediction of the dige... more An accurate method for estimation of lignin concentration is important for prediction of the digestible energy content of livestock feeds. The accuracy of lignin concentration estimates based on the Klason lignin and acid detergent lignin methods was compared. Ten diverse forage samples were analyzed for protein, carbohydrates, lipids, organic acids, ash, lignin (by both methods), and gross energy. The accuracy of the two lignin concentration estimates was examined by comparing the measured forage gross energy to a gross energy value calculated from the compositional analysis. Use of the acid detergent lignin estimate in this gross energy calculation accounted for 68-84% of the forage gross energy compared to 85-97% of the gross energy using Klason lignin. These results indicate that while Klason lignin estimates are substantially higher than acid detergent lignin estimates, Klason lignin is the more accurate lignin method and does not overestimate lignin because gross energy recoveries were less than 100%.

Facile large-scale synthesis of coniferyl, sinapyl, and p-coumaryl alcohol

Journal of Agricultural and Food Chemistry, 1992

Ferulate Cross-Links Limit the Enzymatic Degradation of Synthetically Lignified Primary Walls of Maize

Journal of Agricultural and Food Chemistry, 1998

Ferulate cross-linking of arabinoxylans to lignin may restrict the enzymatic degradation of struc... more Ferulate cross-linking of arabinoxylans to lignin may restrict the enzymatic degradation of structural polysaccharides, limiting the utilization of graminaceous crops and crop residues as feedstuffs and as feedstocks for fuel and chemical production. Maize walls from nonlignified cell suspensions with ca. 5.1 or 17.6 mg g-1 of ferulates were synthetically lignified with coniferyl alcohol and H2O2 to form dehydrogenation polymer-cell wall complexes with ca. 4.8 or 15.8 mg g-1 of ferulates incorporated into lignin. Ferulate concentrations in cell walls were reduced from normal levels by growing cell suspensions with 2-aminoindan-2-phosphonic acid or by methylating wall ferulates with diazomethane prior to complex formation. A 70% reduction in ferulate-lignin cross-linking increased carbohydrate solubilization by 24-46% after 6 h and by 0-25% after 72 h of hydrolysis with two fungal enzyme mixtures. Reduced cross-linking enhanced the hydrolysis of xylans and, to a lesser degree, cellulose from walls. The results presented indicate that reduced feruloylation of arabinoxylans will significantly improve the hydrolysis of lignified grass walls.

The DFRC Method for Lignin Analysis. 4. Lignin Dimers Isolated from DFRC-Degraded Loblolly Pine Wood

Journal of Agricultural and Food Chemistry, 1998

Dehydrogenation Polymer−Cell Wall Complexes as a Model for Lignified Grass Walls

Journal of Agricultural and Food Chemistry, 1996

... Pollet, B.; Monties, B.; Rolando, C. Thioacidolysis of spruce lignin: GC-MS analysis of the m... more

Rapid proton NMR method for determination of threo:erythro ratios in lignin model compounds and examination of reduction stereochemistry

Journal of Agricultural and Food Chemistry, 1991

... Also, we continue to be grateful to John C. Vederas, Chemistry Department, University of Albe... more

p -Hydroxyphenyl, Guaiacyl, and Syringyl Lignins Have Similar Inhibitory Effects on Wall Degradability

Journal of Agricultural and Food Chemistry, 1997

... J. Chem. Soc., Perkin Trans. 1, 1994, 3485−3498. [CrossRef]. Sewalt, VJH; Ni, W.; Jung, HG; D... more

Using the Acetyl Bromide Assay To Determine Lignin Concentrations in Herbaceous Plants: Some Cautionary Notes

Journal of Agricultural and Food Chemistry, 1999

The acetyl bromide assay was developed to provide a rapid and sensitive method for quantifying li... more The acetyl bromide assay was developed to provide a rapid and sensitive method for quantifying lignin in woody plant species. The original procedure cautioned against prolonged reaction times and advised keeping the reaction temperature at 70 degrees C to prevent excessive carbohydrate degradation that would skew the absorption spectra. Characterization of the reaction conditions revealed that the acetyl bromide reagent readily degrades xylans, a prominent polysaccharide group within all lignified plants. This degradation results in increased absorbance in the 270-280 nm region that is used to quantify lignin. The degradation of xylans is temperature dependent and is exacerbated by the addition of perchloric acid. Lowering the reaction temperature to 50 degrees C and increasing the reaction time from 2 to 4 h allows complete lignin solubilization but minimizes degradation of the xylans.

Synthesis of the Lignin Model Compounds threo-Guaiacylglycerol-β-Guaiacyl Ether and threo-Veratrylglycerol-β-Guaiacyl Ether

A One- and Two-Dimensional 13 C and 1 H N.M.R. Study of Some Triterpenes of the Hopane, Stictane and Flavicene Groups

Australian Journal of Chemistry, 1989

Stereochemical Aspects Op Addition Reactions Involving Lignin Model Quinone Methides

by R. Young and John Ralph

Journal of Wood Chemistry and Technology, 1983

... R R = CH ,OH ... The predominant isomer uas expected to be the threo-adduct based on results ... more

Reactions of lignin model quinone methides and NMR studies of lignins

NMR of lignin model compounds: application of long-range C—H correlations through oxygen

ChemInform Abstract: Preparation of Synthetic Lignins with Superior NMR Characteristics via Isotopically Labeled Monolignols

Cheminform, 2010

Characterization of Hardwood Lignin: Investigation of Syringyl/Guaiacyl Composition by 13 C Nuclear Magnetic Resonance Spectroscopy

Holzforschung, 1983

The Stereochemistry of β—5 Lignin Model Compounds

Holzforschung, 1987

Synthesis of Trimeric Lignin Model compounds Composed of β-Aryl Ether and Phenylcoumaran Structures

Holzforschung, 1986

Rotenone NMR assignments

Journal of Heterocyclic Chemistry, 1988

Adducts of anthrahydroquinone and anthranol with lignin model quinone methides. 2. Dehydration derivatives. Proof of threo configuration

The Journal of Organic Chemistry, 1983

... Adducts of Anthrahydroquinone and Anthranol with Lignin Model Quinone Methides. 1. Synthesis ... more

Adducts of Anthrahydroquinone and Anthranol with Lignin Model Quinone Methides. 9,10-13C Labelled Anthranol-Lignin Adducts; Examination of Adduct Formation and Stereochemistry in the Polymer

Journal of Wood Chemistry and Technology, 1986

... Alkylation of TMS Anthranol with Bromide 9 1-br om-1-( 4 -hyd roxy-3-me t hoxyp heny 1) - 2-(... more

Reactions of β-ARYL Lignin Model Quinone Methides with Anthrahydroquinone and Anthranol

Journal of Wood Chemistry and Technology, 1987

Quinone methides prepared in situ from phenylcoumaran and ß-C-l lignin models which did not conta... more Quinone methides prepared in situ from phenylcoumaran and ß-C-l lignin models which did not contain a ß-hydroxymethyl group, readily formed addition products with anthranol but not with anthrahydroquinone. For ß-aryl lignin models containing the hydroxymethyl group, the retro-aldol reaction (liberating formaldehyde) was so facile under the conditions used that stilbene formation from the quinone methide took precedence over adduct formation.

Anthraquinone Losses During Alkaline Pulping

Journal of Wood Chemistry and Technology, 1984

Determination of the Conformation and Isomeric Composition of Lignin Model Quinone Methides By NMR

Journal of Wood Chemistry and Technology, 1983

Proton and C NMR of representative guaiacyl and syringyl β-aryl ether quinone methides have shown... more Proton and C NMR of representative guaiacyl and syringyl β-aryl ether quinone methides have shown that guaiacyl quinone methides, generated from the corresponding benzyl bromides, exist as isomeric pairs in approximately a 70:30 ratio, the major isomer having the 3-methoxyl group syn with respect to the side chain. The ring protons at the 2- and 6-positions in the syn-and the

Isochroman lignin trimers from DFRC-degraded< i> Pinus taeda</i>

Modifying lignin in conifers: the role of HCT during tracheary element formation in Pinus radiata

Modeling cross-links within wall matrices: impact upon polysaccharide degradation

A novel approach for analysis of poplar wood using twodimensional NMR spectroscopy on dissolved whole cell walls in combination with chemometrics

5-Hydroxyconiferyl alcohol as a monolignol in COMT-deficient angiosperms

Should lignin be redefined?

Ferulates and diferulates as nucleation sites for lignification in grasses

Ferulate cross-linking limits degradation of lignified grass walls by fungal hydrolases

An improved 13C-tracer method for the study of lignin structure and reactions: differential 13C-NMR

by John Ralph and Magnus Paulsson

Incorporation of hydroxy-cinnamaldehydes into lignins

Variations in lignin: what do recent studies on lignin-biosynthetic pathway mutans and transgenics revbeal about lignification

Effect of diferulate cross-linking on the hydrolysis of xylans and nonlignified walls by a fungal enzyme mixture containing feruloyl esterase and potent xylanase activities

Altering Lignin Composition, Structure and Cross-Linking: Potential Impact on Cell Wall Degradation

Kenaf’s amazing lignin

NMR methods in food chemistry

Tools for lignin structural analysis; applications to C3H down regulation

Incorporation of 5-hydroxyconiferyl alcohol into lignins of COMT-deficient poplar

Severe Inhibition of Fiber Degradation by Cinnamyl Aldehyde-Containing Lignins

Order and Randomness in Lignin and Lignification: Is a New Paradigm for Lignification Required?

by John Ralph and Ronald Sederoff

$Research paper thumbnail of High-Resolution Solution-State Nmr of Unfractionated Plant Cell Walls: Potential for Biomass Selection and Process Optimization$

High-Resolution Solution-State Nmr of Unfractionated Plant Cell Walls: Potential for Biomass Selection and Process Optimization

by John Ralph and Dino Ress

Description: Detailed structural studies on the plant cell wall have traditionally been difficult... more

Incorporation of Monolignol Conjugates into Lignin for Improved Processing

METHOD FOR MODIFYING LIGNIN STRUCTURE USING MONOLIGNOL FERULATE CONJUGATES

FERULOYL-CoA: MONOLIGNOL TRANSFERASE

NMR DATABASE OF PLANT-CELL WALL MODEL COMPOUNDS

Potential Monolignol Replacements: Synthesis and Lignification of Monolignol γ-Glucosides and β-Hydroxycinnamoyl Glucosides

ABSTRACT Bioengineering plants to have more readily hydrolysable ligno-cellulosics is needed to r... more ABSTRACT Bioengineering plants to have more readily hydrolysable ligno-cellulosics is needed to reap the potential of biomass for liquid biofuels generation. Lignin is a hydrophobic biopolymer and contributes much to the recalcitrance to lignocellulosics toward hydrolysis. We proposed that increasing the hydrophilicity of lignin would increase the ener-gy efficiency of lignocellulose processing. To test this hypothesis, we have synthesized monolignol γ-glucosides and hydroxycinnamoyl glu-cosides. We have also demonstrated that these compounds are readily incorporated into synthetic lignin, i.e., during in vitro lignification. Our studies have successfully paved the road toward studying the in vitro incorporation of these hydrophilic monolignol surrogates to plant cell walls to explore their influence on lignocellulose hydrolysis.

Passive membrane transport of lignin-related compounds

Proceedings of the National Academy of Sciences

Lignin is an abundant aromatic polymer found in plant secondary cell walls. In recent years, lign... more Lignin is an abundant aromatic polymer found in plant secondary cell walls. In recent years, lignin has attracted renewed interest as a feedstock for bio-based chemicals via catalytic and biological approaches and has emerged as a target for genetic engineering to improve lignocellulose digestibility by altering its composition. In lignin biosynthesis and microbial conversion, small phenolic lignin precursors or degradation products cross membrane bilayers through an unidentified translocation mechanism prior to incorporation into lignin polymers (synthesis) or catabolism (bioconversion), with both passive and transporter-assisted mechanisms postulated. To test the passive permeation potential of these phenolics, we performed molecular dynamics simulations for 69 monomeric and dimeric lignin-related phenolics with 3 model membranes to determine the membrane partitioning and permeability coefficients for each compound. The results support an accessible passive permeation mechanism fo...

Lignin‐based barrier restricts pathogens to the infection site and confers resistance in plants

The EMBO Journal

Pathogenic bacteria invade plant tissues and proliferate in the extracellular space. Plants have ... more Pathogenic bacteria invade plant tissues and proliferate in the extracellular space. Plants have evolved the immune system to recognize and limit the growth of pathogens. Despite substantial progress in the study of plant immunity, the mechanism by which plants limit pathogen growth remains unclear. Here, we show that lignin accumulates in Arabidopsis leaves in response to incompatible interactions with bacterial pathogens in a manner dependent on Casparian strip membrane domain protein (CASP)‐like proteins (CASPLs). CASPs are known to be the organizers of the lignin‐based Casparian strip, which functions as a diffusion barrier in roots. The spread of invading avirulent pathogens is prevented by spatial restriction, which is disturbed by defects in lignin deposition. Moreover, the motility of pathogenic bacteria is negatively affected by lignin accumulation. These results suggest that the lignin‐deposited structure functions as a physical barrier similar to the Casparian strip, trapping pathogens and thereby terminating their growth.

Poacic Acid Suppresses Dollar Spot and Snow Mould in Amenity Turfgrass

Plant Pathology

Plant‐based antifungal agents offer an alternative to synthetic fungicides in amenity turfgrass d... more Plant‐based antifungal agents offer an alternative to synthetic fungicides in amenity turfgrass disease management. Poacic acid is a by‐product of the biofuel production process that has exhibited antifungal activity, and the objective of this research was to determine its ability to serve as an effective management tool for economically important turfgrass diseases such as dollar spot and snow moulds. In vitro and field tests were conducted in Wisconsin and Michigan, USA from 2015 to 2017 to determine the efficacy of poacic acid in suppressing the economically important turfgrass pathogens Clarireedia jacksonii and Microdochium nivale. Poacic acid demonstrated strong antifungal activity against both pathogens in vitro, inhibiting growth of C. jacksonii and M. nivale by 93% and 74% relative to nonamended media, respectively. Poacic acid reduced dollar spot in the field in one of two years, but failed to suppress snow mould when applied alone. Poacic acid was an effective mix partner for snow mould control when combined with a synthetic fungicide, an important attribute because no single fungicide currently on the market provides acceptable snow mould control under heavy disease pressure. Future research should focus on improving poacic acid field efficacy so that it can be incorporated into plant‐based disease management strategies for amenity turfgrass.

A comparative study of secondary depolymerization methods on oxidized lignins

Green Chemistry

DDQ oxidized lignins were used as substrates to assess the effectiveness of secondary depolymeriz... more

Hydroxystilbene Glucosides Are Incorporated into Norway Spruce Bark Lignin

Plant Physiology

Editorial overview: Plant biotechnology – lignin engineering

Current Opinion in Biotechnology

Lignin biosynthesis and its integration into metabolism

Current Opinion in Biotechnology

Lignin is a principal structural component of cell walls in higher terrestrial plants. It reinfor... more Lignin is a principal structural component of cell walls in higher terrestrial plants. It reinforces the cell walls, facilitates water transport, and acts as a physical barrier to pathogens. Lignin is typically described as being composed of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units that derive from the polymerization of the hydroxycinnamyl alcohols, p-coumaryl, coniferyl, and sinapyl alcohol, respectively. However, lignin also derives from various other aromatic monomers. Here, we review the biosynthetic pathway to the lignin monomers, and how flux through the pathway is regulated. Upon perturbation of the phenylpropanoid pathway, pathway intermediates may successfully incorporate into the lignin polymer, thereby affecting its physicochemical properties, or may remain soluble as such or as derivatized molecules that might interfere with physiological processes.

Lignin structure and its engineering

Current Opinion in Biotechnology

Studies on lignin structure and its engineering are inextricably and bidirectionally linked. Pert... more Studies on lignin structure and its engineering are inextricably and bidirectionally linked. Perturbations of genes on the lignin biosynthetic pathway may result in striking compositional and structural changes that in turn suggest novel approaches for altering lignin and even 'designing' the polymer to enhance its value or with a view toward its simpler removal from the cell wall polysaccharides. Basic structural studies on various native lignins increasingly refine our knowledge of lignin structure, and examining lignins in different species reveals the extent to which evolution and natural variation have resulted in the incorporation of 'non-traditional' phenolic monomers, including phenolics from beyond the monolignol biosynthetic pathway. As a result, the very definition of lignin continues to be expanded and refined.

Characteristics of Hot Water Extracts from the Bark of Cultivated Willow (Salix sp.)

ACS Sustainable Chemistry & Engineering

Willow bark is a rich source of heterogeneous polyphenolic compounds and a potential feedstock fo... more Willow bark is a rich source of heterogeneous polyphenolic compounds and a potential feedstock for biorefinery processes aiming at chemicals and fiber production. Here, mild hot water treatment of willow hybrid Karin was studied to find a practical means of isolating its non-cell-wall components for their utilization in a willow biorefinery proposed to aid valorization of the willow biomass. A short aqueous treatment of the bark at 80 °C liberated the extract in >20% yield under unpressurized conditions. The extract was characterized using mainly gas chromatography-mass spectrometry and one- and two-dimensional NMR techniques. Authentic analytes were applied to confirm the identification and quantification of the main components that were picein, (+)-catechin, triandrin, glucose, and fructose. Fructose was converted into 5-hydroxymethylfurfural in an acidic treatment which led to its condensation with the phenolic components and formation of a recalcitrant precipitate that should be avoided.

Structural Characterization of Lignins from Willow Bark and Wood

Journal of Agricultural and Food Chemistry

Understanding the chemical structure of lignin in willow bark is an indispensable step to design ... more Understanding the chemical structure of lignin in willow bark is an indispensable step to design how to separate its fiber bundles. The whole cell wall and enzyme lignin preparations sequentially isolated from ball-milled bark, inner bark, and wood were comparatively investigated by nuclear magnetic resonance (NMR) spectroscopy and three classical degradative methods, i.e., alkaline nitrobenzene oxidation, derivatization followed by reductive cleavage, and analytical thioacidolysis. All results demonstrated that the guaiacyl (G) units were predominant in the willow bark lignin over syringyl (S) and minor p-hydroxyphenyl (H) units. Moreover, the monomer yields and S/G ratio rose progressively from bark to inner bark and wood, indicating that lignin may be more condensed in bark than in other tissues. Additionally, major interunit linkage substructures (β-aryl ethers, phenylcoumarans, and resinols) together with cinnamyl alcohol end groups were relatively quantitated by two-dimensional NMR spectroscopy. Bark and inner bark were rich in pectins and proteins, which were present in large quantities and also in the enzyme lignin preparations.

An “ideal lignin” facilitates full biomass utilization

Science Advances

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.

Gold-catalyzed conversion of lignin to low molecular weight aromatics

Chemical Science

Gold nanoparticles supported on lithium–aluminum layered double hydroxide function as a heterogen... more

Characterizing phenol–formaldehyde adhesive cure chemistry within the wood cell wall

International Journal of Adhesion and Adhesives

Short-term facilitation of microbial litter decomposition by ultraviolet radiation

The Science of the total environment, Jan 6, 2017

Solar radiation plays an important role in carbon cycling by increasing the decomposition rates o... more Solar radiation plays an important role in carbon cycling by increasing the decomposition rates of plant litter and soil organic matter (i.e. photodegradation). Previous work suggests that exposure to radiation can facilitate microbial decomposition of litter by altering litter chemistry and consequently litter degradability (i.e. photopriming). However, it remains unclear to what extent photopriming contributes to litter decomposition processes and on what timescale photopriming operates. We conducted laboratory experiments to compare the effects of UV photopriming at two temporal scales (months versus days). In one experiment, we found that four months of UV exposure induced a significant but small (3-4%) mass loss in two of three litter species commonly found in California oak savanna; however, UV exposure did not alter litter degradability as measured by microbial respiration in an incubation experiment. We also found that UV exposure had limited effects on lignin and other cell...

Characterization and Elimination of Undesirable Protein Residues in Plant Cell Wall Materials for Enhancing Lignin Analysis by Solution-State Nuclear Magnetic Resonance Spectroscopy

Biomacromolecules, Jan 8, 2017

Protein polymers exist in every plant cell wall preparation, and they interfere with lignin chara... more Protein polymers exist in every plant cell wall preparation, and they interfere with lignin characterization and quantification. Here, we report the structural characterization of the residual protein peaks in 2D NMR spectra in corn cob and kenaf samples and note that aromatic amino acids are ubiquitous and evident in spectra from various other plants and tissues. The aromatic correlations from amino acid residues were identified and assigned as phenylalanine and tyrosine. Phenylalanine's 3/5 correlation peak is superimposed on the peak from typical lignin p-hydroxyphenyl (H-unit) structures, causing an overestimation of the H units. Protein contamination also occurs when using cellulases to prepare enzyme lignins from virtually protein-free wood samples. We used a protease to remove the protein residues from the ball-milled cell walls, and we were able to reveal H-unit structures in lignins more clearly in the 2D NMR spectra, providing a better basis for their estimation.

2D NMR characterization of wheat straw residual lignin after dilute acid pretreatment with different severities

Holzforschung

The chemical characteristics of wheat straw lignin pretreated under dilute acid conditions were c... more

Chemical Pulping Advantages of Zip-lignin Hybrid Poplar

ChemSusChem

Silencing CAFFEOYL SHIKIMATE ESTERASE affects lignification and improves saccharification

Plant Physiology

Different metabolic routes for coniferaldehyde and sinapaldehyde with CINNAMYL ALCOHOL DEHYDROGENASE1 deficiency

Plant Physiology

Estimation of Syringyl Units in Wood Lignins by FT-Raman Spectroscopy

Syringyl (S) lignin content and the syringyl-to-guaiacyl (S/G) lignin ratio are important charact... more Syringyl (S) lignin content and the syringyl-to-guaiacyl (S/G) lignin ratio are important characteristics of wood and lignocellulosic biomass. Although numerous methods are available for estimating S lignin units and the S/G ratio, in this work, a new method based on Raman spectroscopy that uses the 370 cm −1 Raman band-area intensity (370-area) was developed. The reliability of the Raman approach for determining S content was first tested by the quantitative analysis of three syringyl lignin models by sampling them, separately, in dioxane and in Avicel. Good linear correlations between the 370 cm −1 intensity and model concentrations were obtained. Next, the percent syringyl (%S) lignin units in various woods were measured by correlating the 370 cm −1 Raman intensity data with values of S units in lignin determined by three regularly used methods, namely, thioacidolysis, DFRC, and 2D-HSQC NMR. The former two methods take into account only the monomers cleaved from β−O−4-linked lignin units, whereas the NMR method reports S content on the whole cell wall lignin. When the 370-area intensities and %S values from the regularly used methods were correlated, good linear correlations were obtained (R 2 = 0.767, 0.731, and 0.804, respectively, for the three methods). The correlation with the highest R 2 , i.e., with the 2D NMR method, is proposed for estimating S units in wood lignins by Raman spectroscopy as, in principle, both represent the whole cell wall lignin and not just the portion of lignin that gets cleaved to release monomers. The Raman analysis method is quick, uses minimal harmful chemicals, is carried out nondestructively, and is insensitive to the wet or dry state of the sample. The only limitations are that the sample of wood contains at least 30% S and not be significantly fluorescent, although the latter can be mitigated in some cases.

Editorial overview: Plant biotechnology -lignin engineering ScienceDirect

by John Ralph and Wout Boerjan

It is particularly timely to publish a special issue on lignin biosynthesis and its engineering g... more It is particularly timely to publish a special issue on lignin biosynthesis and its engineering giving the depth and breadth of amazing breakthroughs in fundamental understanding and applications that have occurred in recent years. Lignins are polymers that are mainly deposited in secondary-thickened plant cell walls. There, they are produced in a polymerization step (lignifica-tion) from their component monomers by a process of simple combinatorial radical coupling that was once considered an enigma. The monomers that have been known for decades are the p-coumaryl, coniferyl, and sinapyl alcohols. However, the development of new analytical tools along with analyses of lignin structures from a broader range of plant species has led to the discovery of many additional monomers, giving rise to a rich diversity of polymers with different physicochemical properties. As a consequence, the very definition of lignin continues to evolve as new species of plants are characterized in greater detail. The fact that lignin polymerization is a combinatorial radical coupling process, and that so many monomers naturally couple into the lignin polymer, has given rise to the concept that lignin structure is malleable and that plants can be engineered to make lignin structures that are tailored for various end-use applications. Indeed, in industrial operations producing pulp or fermentable sugars from wood, lignin needs to be extracted by expensive chemical treatments, and the efficiency of this process heavily depends on lignin's amount and composition. On the other hand, lignin is increasingly considered as a valuable source of aromatic building blocks for the chemical industry. As is now becoming evident, particular lignin structures can be designed and engineered into plants to maximize lignin-derived aromatic monomers streams. The acceleration in the interest in lignin and its engineering, caused by the urgent need for a transition from a fossil-based to a biobased economy, is illustrated by the steadily increasing number of papers covering 'lignin' with 'gene' terms, from 725 in 2010 to 1516 in 2017, with citations escalating at a far greater rate. When we conceived this issue, our objective was to cover lignin research extending from biosynthetic pathway revelations up to its valorization, but always with a focus on biotechnology, and with minimal overlap among the individual reviews. Given that several research groups work on the very same topic, it was, unfortunately, not possible to invite all of the main players in the lignin field and we apologize for this. The following are just a few of the highlights covered in the various contributions to this special issue as a testament to the state-of-the-art advances and practical implications of lignin bioengineering. Vanholme et al. [1] review the biosynthetic pathways towards the 35 metabolites from over 10 metabolic classes that have now been identified as lignin building blocks. They examine (Belgium). He specializes in lignin biosynthesis (gene and pathway discovery) and in lignin engineering in model plants (Arabidopsis), and crops (maize and poplar) to speed up the transition from a fossil-based to a bio-based economy.

Poacic acid suppresses dollar spot and snow mould in amenity turfgrass

by John Ralph and Paul Koch

Plant-based antifungal agents offer an alternative to synthetic fungicides in amenity turfgrass d... more Plant-based antifungal agents offer an alternative to synthetic fungicides in amenity turfgrass disease management. Poacic acid is a by-product of the biofuel production process that has exhibited antifungal activity, and the objective of this research was to determine its ability to serve as an effective management tool for economically important turfgrass diseases such as dollar spot and snow moulds. In vitro and field tests were conducted in Wisconsin and Michigan, USA from 2015 to 2017 to determine the efficacy of poacic acid in suppressing the economically important turfgrass pathogens Clarireedia jacksonii and Microdochium nivale. Poacic acid demonstrated strong antifungal activity against both pathogens in vitro, inhibiting growth of C. jacksonii and M. nivale by 93% and 74% relative to nonamended media, respectively. Poacic acid reduced dollar spot in the field in one of two years, but failed to suppress snow mould when applied alone. Poacic acid was an effective mix partner for snow mould control when combined with a synthetic fungicide , an important attribute because no single fungicide currently on the market provides acceptable snow mould control under heavy disease pressure. Future research should focus on improving poacic acid field efficacy so that it can be incorporated into plant-based disease management strategies for amenity turfgrass.

Lignin-based barrier restricts pathogens to the infection site and confers resistance in plants

Pathogenic bacteria invade plant tissues and proliferate in the extracellular space. Plants have ... more Pathogenic bacteria invade plant tissues and proliferate in the extracellular space. Plants have evolved the immune system to recognize and limit the growth of pathogens. Despite substantial progress in the study of plant immunity, the mechanism by which plants limit pathogen growth remains unclear. Here, we show that lignin accumulates in Arabidopsis leaves in response to incompatible interactions with bacterial pathogens in a manner dependent on Casparian strip membrane domain protein (CASP)-like proteins (CASPLs). CASPs are known to be the organizers of the lignin-based Casparian strip, which functions as a diffusion barrier in roots. The spread of invading avirulent pathogens is prevented by spatial restriction, which is disturbed by defects in lignin deposition. Moreover , the motility of pathogenic bacteria is negatively affected by lignin accumulation. These results suggest that the lignin-deposited structure functions as a physical barrier similar to the Casparian strip, trapping pathogens and thereby terminating their growth.

Lignin structure and its engineering

by John Ralph and Wout Boerjan

Hydroxystilbene Glucosides Are Incorporated into Norway Spruce Bark Lignin 1[OPEN

Recent investigations have revealed that, in addition to monolignols, some phenolic compounds der... more Recent investigations have revealed that, in addition to monolignols, some phenolic compounds derived from the flavonoid and hydroxystilbene biosynthetic pathways can also function as true lignin monomers in some plants. In this study, we found that the hydroxystilbene glucosides isorhapontin (isorhapontigenin-O-glucoside) and, at lower levels, astringin (piceatannol-O-glucoside) and piceid (resveratrol-O-glucoside) are incorporated into the lignin polymer in Norway spruce (Picea abies) bark. The corresponding aglycones isorhapontigenin, piceatannol, and resveratrol, along with glucose, were released by derivatization followed by reductive cleavage, a chemical degradative method that cleaves b-ether bonds in lignin, indicating that the hydroxystilbene glucosides are (partially) incorporated into the lignin structure through b-ether bonds. Two-dimensional NMR analysis confirmed the occurrence of hydroxystilbene glucosides in this lignin, and provided additional information regarding their modes of incorporation into the polymer. The hydroxystilbene glucosides, particularly isorhapontin and astringin, can therefore be considered genuine lignin monomers that participate in coupling and cross-coupling reactions during lignification in Norway spruce bark. Lignin is a complex aromatic polymer derived essentially from the oxidative coupling of three mono-lignols, p-coumaryl, coniferyl, and sinapyl alcohols (Boerjan et al., 2003; Ralph et al., 2004). Other phenolic compounds, including monolignol ester conjugates (with acetates, p-hydroxybenzoates, p-coumarates, or ferulates) or compounds derived from the truncated biosynthesis of monolignols (such as caffeyl alcohol or 5-hydroxyconiferyl alcohol and the hydrox-ycinnamaldehydes), have also been widely found to act as true lignin monomers in the lignins of many plants (Ralph et al.

A comparative study of secondary depolymerization methods on oxidized lignins

Selective oxidation of lignin's β-aryl ether units combined with secondary chemical treatment for... more Selective oxidation of lignin's β-aryl ether units combined with secondary chemical treatment for depoly-merization can generate valuable oxygen-rich aromatics. Although there have been many reports of the successful oxidative depolymerization of lignin, an accurate assessment of the merits of each method is hampered by the wide array of lignins used. Here, we test a selection of literature methods for secondary lignin depolymerization using a common set of lignin substrates. In an initial step, the lignins were oxidized using 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ)/tert-butyl nitrite (t BuONO)/O 2. The oxidized lignins were then subjected to a variety of depolymerization methods, the yield of aromatic monomers being quantified and compared to lignin depolymerized using an Au/Li-Al LDH catalyst followed by hydrolysis without prior C α-OH oxidation. The Au/Li-Al LDH system gave the highest monomer yield for the untreated lignins, moreover, for DDQ-oxidized lignins, the Au/Li-Al LDH method produced similar monomer yields with high selectivity towards aromatic acids and aldehydes.

SOLUTION-STATE MULTIDIMENSIONAL NMR OF LIGNINS: APPROACHES AND APPLICATIONS

by John Ralph and Yuki Tobimatsu

Among numerous analytical techniques developed for lignins, solution-state multidimensional NMR p... more Among numerous analytical techniques developed for lignins, solution-state multidimensional NMR provides unparalleled details of the polymers' structural features to be elucidated. With an ability to diagnostically identify and approximate the diverse array of the structural elements in lignin polymers, the NMR techniques are becoming essential in numerous lignin research settings including those aiming to investigate the biosynthesis, bioengineering and biodegradation lignins, as well as the chemistry of lignins in various chemical and biochemical contexts in the light of the biorefinery concept. This chapter review will consider some basic and practical aspects of such NMR implementations in current lignin research. Specifically, a handful of key 2D and 3D NMR experiments for characterizing lignins and basic strategies for preparing suitable cell wall lignin samples to apply these NMR techniques are discussed.

Lignin biosynthesis and its integration into metabolism

by John Ralph and Wout Boerjan

Systematic parameterization of lignin for the CHARMM force field

Lignin is an abundant aromatic biopolymer within plant cell walls formed through radical coupling... more Lignin is an abundant aromatic biopolymer within plant cell walls formed through radical coupling chemistry , whose composition and topology can vary greatly depending on the biomass source. Computational modeling provides a complementary approach to traditional experimental techniques to probe lignin interactions, lignin structure, and lignin material properties. However, current modeling approaches are limited based on the subset of lignin chemistries covered by existing lignin force fields. To fill the gap, we developed a comprehensive lignin force field that accounts for more lignin-lignin and lignin-carbohydrate interlinkages than existing lignin force fields, and also greatly expands the lignin monomer chemistries that can be modeled beyond simple alcohols and into the rich mixture of natural lignin varieties. The development of this force field utilizes recent developments in parameterization methodology, and synthesizes them into a workflow that combines target data from multiple molecules simultaneously into a single consistent and comprehensive parameter set. The parameter set represents a significant improvement to alternatives for atomic modeling of diverse lignin topologies, more accurately reproducing experimental observables while also significantly reducing the error relative to quantum calculations. The improved energetics, as well as the rigid adherence to CHARMM parameterization philosophy , enables simulation of lignin within its biological context with greater accuracy than was previously possible. The lignin force field presented here is therefore a crucial first step towards modeling lignin structure across a broad range of environments, including within plant cell walls where lignin is com-plexed with carbohydrates and deconstructed by bacterial or fungal enzymes, or as it exists within industrial solvent mixtures. Future simulations enabled by this updated lignin force field will thus lead to better chemical and structural understanding of lignin, providing new insight into its role in biomass recalcitrance or probing the potential for lignin to be used within industrial processes.

Passive membrane transport of lignin- related compounds

by John Ralph and Wout Boerjan

Lignin is an abundant aromatic polymer found in plant secondary cell walls. In recent years, lign... more Lignin is an abundant aromatic polymer found in plant secondary cell walls. In recent years, lignin has attracted renewed interest as a feedstock for bio-based chemicals via catalytic and biological approaches and has emerged as a target for genetic engineering to improve lignocellulose digestibility by altering its composition. In lignin biosynthesis and microbial conversion, small phenolic lignin precursors or degradation products cross membrane bilayers through an unidentified translocation mechanism prior to incorporation into lignin polymers (synthesis) or catabolism (bioconversion), with both passive and transporter-assisted mechanisms postulated. To test the passive permeation potential of these phenolics, we performed molecular dynamics simulations for 69 monomeric and dimeric lignin-related phe-nolics with 3 model membranes to determine the membrane partitioning and permeability coefficients for each compound. The results support an accessible passive permeation mechanism for most compounds, including monolignols, dimeric phenolics, and the flavonoid, tricin. Computed lignin partition coefficients are consistent with concentration enrichment near lipid car-bonyl groups, and permeability coefficients are sufficient to keep pace with cellular metabolism. Interactions between methoxy and hydroxy groups are found to reduce membrane partitioning and improve permeability. Only carboxylate-modified or glyco-sylated lignin phenolics are predicted to require transporters for membrane translocation. Overall, the results suggest that most lignin-related compounds can passively traverse plant and microbial membranes on timescales commensurate with required biological activities, with any potential transport regulation mechanism in lignin synthesis, catabolism, or bioconversion requiring compound functionalization. molecular dynamics | lignin permeability | lignin biosynthesis | biological funneling | free energy calculation