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John Ralph - One of the best experts on this subject based on the ideXlab platform.
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Lignin monomers from beyond the canonical Monolignol biosynthetic pathway: Another brick in the wall
ACS Sustainable Chemistry & Engineering, 2020Co-Authors: José C. Del Río, Hoon Kim, Thomas Elder, Jorge Rencoret, Ana Gutiérrez, John RalphAbstract:Lignin is conventionally defined as being formed by the oxidative polymerization of three main Monolignols, p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, that are derived from the general phenylpropanoid biosynthetic pathway. Many other phenolic compounds that are also derived from the phenylpropanoid pathway are also known to perform as genuine lignin monomers in many plants, as is the case of the Monolignol ester conjugates, phenolic compounds arising from the truncated biosynthesis of Monolignols, or ferulate esters. Recent investigations, however, have indicated that phenolic compounds arising from beyond the canonical phenylpropanoid pathway, namely flavonoids, hydroxystilbenes, and hydroxycinnamic amides, may also behave as authentic lignin monomers and are incorporated into the lignin in some plants, further challenging the traditional definition of lignin. This is the case of the flavone tricin that is incorporated into the lignin of grasses and other monocots, the hydroxystilbene piceatannol (together with resveratrol and isorhapontigenin, at lower levels) that has been found in the lignins of palm fruit shells, their respective O-glucosides (astringin, piceid, and isorhapontin) that are present in the lignin of Norway spruce bark, or the ferulic amides feruloyltyramine, incorporated into the lignin of tobacco and potato tubers, and diferuloylputrescine, which appears to be incorporated into maize kernel lignin. These valuable compounds are potentially available in high amounts and at low cost and may be obtained from the waste products from the processing of agricultural or forest biomass.
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Radical coupling reactions of piceatannol and Monolignols: A density functional theory study
Phytochemistry, 2019Co-Authors: Thomas Elder, Hoon Kim, John Ralph, José C. Del Río, Jorge Rencoret, Gregg T. BeckhamAbstract:Recent experimental work has revealed that the hydroxystilbene piceatannol can function as a monomeric unit in the lignification of palm fruit endocarp tissues. Results indicated that piceatannol homo-couples and cross-couples with Monolignols through radical reactions and is integrally incorporated into the lignin polymer. The current work reports on the thermodynamics of the proposed reactions using density functional theory calculations. The results indicated that, in general, the energetics of both homo-coupling and cross-coupling are not dissimilar from those of the Monolignol coupling, demonstrating the compatibility of piceatannol with the lignification process. Moreover, the DFT methods appear to predict the correct courses of post-coupling rearomatization reactions.
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Reductive Cleavage Method for Quantitation of Monolignols and Low-Abundance Monolignol Conjugates.
ChemSusChem, 2018Co-Authors: Matt Regner, John Ralph, Dharshana Padmakshan, Allison Bartuce, Steven D. KarlenAbstract:As interest in biomass utilization has grown, the manipulation of lignin biosynthesis has received significant attention, such that recent work has demanded more robust lignin analytical methods. As the derivatization followed by reductive cleavage (DFRC) method is particularly effective for structurally characterizing natively acylated lignins, we used an array of synthetic β-ether γ-acylated model compounds to determine theoretical yields for all Monolignol conjugates currently known to exist in lignin, and we synthesized a new set of deuterated analogs as internal standards for quantification using GC-MS/MS. Yields of the saturated ester conjugates ranged from 40 to 90 %, and NMR analysis revealed the presence of residual unsaturated conjugates in yields of 20 to 35 %. In contrast to traditional selected-ion-monitoring, we demonstrated the superior sensitivity and accuracy of multiple-reaction-monitoring detection methods, and further highlighted the inadequacy of traditional standards relative to isotopically labeled analogs.
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Defining the Diverse Cell Populations Contributing to Lignification in Arabidopsis Stems
Plant physiology, 2017Co-Authors: Rebecca A. Smith, John Ralph, Steven D. Karlen, Shawn D. Mansfield, Mathias Schuetz, Naohito Tokunaga, David Bird, Yashushi Sato, A. Lacey SamuelsAbstract:Many land plants evolved tall and sturdy growth habits due to specialized cells with thick lignified cell walls: tracheary elements that function in water transport and fibers that function in structural support. The objective of this study was to define how and when diverse cell populations contribute lignin precursors, Monolignols, to secondary cell walls during lignification of the Arabidopsis (Arabidopsis thaliana) inflorescence stem. Previous work demonstrated that, when lignin biosynthesis is suppressed in fiber and tracheary element cells with thickened walls, fibers become lignin-depleted while vascular bundles still lignify, suggesting that nonlignifying neighboring xylem cells are contributing to lignification. In this work, we dissect the contributions of different cell types, specifically xylary parenchyma and fiber cells, to lignification of the stem using cell-type-specific promoters to either knock down an essential Monolignol biosynthetic gene or to introduce novel Monolignol conjugates. Analysis of either reductions in lignin in knockdown lines, or the addition of novel Monolignol conjugates, directly identifies the xylary parenchyma and fiber cell populations that contribute to the stem lignification and the developmental timing at which each contribution is most important.
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Engineering Monolignol p-Coumarate Conjugates into Poplar and Arabidopsis Lignins.
Plant Physiology, 2015Co-Authors: Rebecca A. Smith, John Ralph, Curtis G. Wilkerson, Ji Young Park, Eliana Gonzales-vigil, Steven D. Karlen, Lacey Samuels, Shawn D. MansfieldAbstract: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 by 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 plant growth and development has not yet been examined in plants that do not inherently possess p-coumarates on their lignins. The rice (Oryza sativa) p-COUMAROYL-Coenzyme A Monolignol TRANSFERASE gene was introduced into two eudicots, Arabidopsis (Arabidopsis thaliana) and poplar (Populus alba × grandidentata), and a series of analytical methods was used to show 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 Monolignol p-hydroxybenzoates. Plants expressing the p-COUMAROYL-Coenzyme A Monolignol TRANSFERASE transgene can therefore produce Monolignol p-coumarate conjugates essentially without competing with the formation of other acylated Monolignols and without drastically impacting normal Monolignol production.
Hoon Kim - One of the best experts on this subject based on the ideXlab platform.
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Lignin monomers from beyond the canonical Monolignol biosynthetic pathway: Another brick in the wall
ACS Sustainable Chemistry & Engineering, 2020Co-Authors: José C. Del Río, Hoon Kim, Thomas Elder, Jorge Rencoret, Ana Gutiérrez, John RalphAbstract:Lignin is conventionally defined as being formed by the oxidative polymerization of three main Monolignols, p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, that are derived from the general phenylpropanoid biosynthetic pathway. Many other phenolic compounds that are also derived from the phenylpropanoid pathway are also known to perform as genuine lignin monomers in many plants, as is the case of the Monolignol ester conjugates, phenolic compounds arising from the truncated biosynthesis of Monolignols, or ferulate esters. Recent investigations, however, have indicated that phenolic compounds arising from beyond the canonical phenylpropanoid pathway, namely flavonoids, hydroxystilbenes, and hydroxycinnamic amides, may also behave as authentic lignin monomers and are incorporated into the lignin in some plants, further challenging the traditional definition of lignin. This is the case of the flavone tricin that is incorporated into the lignin of grasses and other monocots, the hydroxystilbene piceatannol (together with resveratrol and isorhapontigenin, at lower levels) that has been found in the lignins of palm fruit shells, their respective O-glucosides (astringin, piceid, and isorhapontin) that are present in the lignin of Norway spruce bark, or the ferulic amides feruloyltyramine, incorporated into the lignin of tobacco and potato tubers, and diferuloylputrescine, which appears to be incorporated into maize kernel lignin. These valuable compounds are potentially available in high amounts and at low cost and may be obtained from the waste products from the processing of agricultural or forest biomass.
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Radical coupling reactions of piceatannol and Monolignols: A density functional theory study
Phytochemistry, 2019Co-Authors: Thomas Elder, Hoon Kim, John Ralph, José C. Del Río, Jorge Rencoret, Gregg T. BeckhamAbstract:Recent experimental work has revealed that the hydroxystilbene piceatannol can function as a monomeric unit in the lignification of palm fruit endocarp tissues. Results indicated that piceatannol homo-couples and cross-couples with Monolignols through radical reactions and is integrally incorporated into the lignin polymer. The current work reports on the thermodynamics of the proposed reactions using density functional theory calculations. The results indicated that, in general, the energetics of both homo-coupling and cross-coupling are not dissimilar from those of the Monolignol coupling, demonstrating the compatibility of piceatannol with the lignification process. Moreover, the DFT methods appear to predict the correct courses of post-coupling rearomatization reactions.
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CAD1 and CCR2 protein complex formation in Monolignol biosynthesis in Populus trichocarpa.
New Phytologist, 2018Co-Authors: Xiaojing Yan, Hoon Kim, Jie Liu, Baoguang Liu, Xiong Huang, Zhichang Yang, Ying-chung Jimmy Lin, Hao Chen, Chenmin Yang, Jack P. WangAbstract:Lignin is the major phenolic polymer in plant secondary cell walls and is polymerized from monomeric subunits, the Monolignols. Eleven enzyme families are implicated in Monolignol biosynthesis. Here, we studied the functions of members of the cinnamyl alcohol dehydrogenase (CAD) and cinnamoyl-CoA reductase (CCR) families in wood formation in Populus trichocarpa, including the regulatory effects of their transcripts and protein activities on Monolignol biosynthesis. Enzyme activity assays from stem-differentiating xylem (SDX) proteins showed that RNAi suppression of PtrCAD1 in P. trichocarpa transgenics caused a reduction in SDX CCR activity. RNAi suppression of PtrCCR2, the only CCR member highly expressed in SDX, caused a reciprocal reduction in SDX protein CAD activities. The enzyme assays of mixed and coexpressed recombinant proteins supported physical interactions between PtrCAD1 and PtrCCR2. Biomolecular fluorescence complementation and pull-down/co-immunoprecipitation experiments supported a hypothesis of PtrCAD1/PtrCCR2 heterodimer formation. These results provide evidence for the formation of PtrCAD1/PtrCCR2 protein complexes in Monolignol biosynthesis in planta.
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An engineered Monolignol 4-o-methyltransferase depresses lignin biosynthesis and confers novel metabolic capability in Arabidopsis.
The Plant Cell, 2012Co-Authors: Kewei Zhang, Mohammad Wadud Bhuiya, Hoon Kim, John Ralph, Jorge Rencoret Pazo, Yu-chen Miao, Chang-jun LiuAbstract:Although the practice of protein engineering is industrially fruitful in creating biocatalysts and therapeutic proteins, applications of analogous techniques in the field of plant metabolic engineering are still in their infancy. Lignins are aromatic natural polymers derived from the oxidative polymerization of primarily three different hydroxycinnamyl alcohols, the Monolignols. Polymerization of lignin starts with the oxidation of Monolignols, followed by endwise cross-coupling of (radicals of) a Monolignol and the growing oligomer/polymer. The para-hydroxyl of each Monolignol is crucial for radical generation and subsequent coupling. Here, we describe the structure-function analysis and catalytic improvement of an artificial Monolignol 4-O-methyltransferase created by iterative saturation mutagenesis and its use in modulating lignin and phenylpropanoid biosynthesis. We show that expressing the created enzyme in planta, thus etherifying the para-hydroxyls of lignin monomeric precursors, denies the derived Monolignols any participation in the subsequent coupling process, substantially reducing lignification and, ultimately, lignin content. Concomitantly, the transgenic plants accumulated de novo synthesized 4-O-methylated soluble phenolics and wall-bound esters. The lower lignin levels of transgenic plants resulted in higher saccharification yields. Our study, through a structure-based protein engineering approach, offers a novel strategy for modulating phenylpropanoid/lignin biosynthesis to improve cell wall digestibility and diversify the repertories of biologically active compounds.
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Identification of Grass-specific Enzyme That Acylates Monolignols with p-Coumarate
Journal of Biological Chemistry, 2012Co-Authors: Saunia Withers, Hoon Kim, John Ralph, Yimin Zhu, Curtis G. WilkersonAbstract:Lignin is a major component of plant cell walls that is essential to their function. However, the strong bonds that bind the various subunits of lignin, and its cross-linking with other plant cell wall polymers, make it one of the most important factors in the recalcitrance of plant cell walls against polysaccharide utilization. Plants make lignin from a variety of Monolignols including p-coumaryl, coniferyl, and sinapyl alcohols to produce the three primary lignin units: p-hydroxyphenyl, guaiacyl, and syringyl, respectively, when incorporated into the lignin polymer. In grasses, these Monolignols can be enzymatically preacylated by p-coumarates prior to their incorporation into lignin, and these Monolignol conjugates can also be “monomer” precursors of lignin. Although Monolignol p-coumarate-derived units may comprise up to 40% of the lignin in some grass tissues, the p-coumarate moiety from such conjugates does not enter into the radical coupling (polymerization) reactions of lignification. With a greater understanding of Monolignol p-coumarate conjugates, grass lignins could be engineered to contain fewer pendent p-coumarate groups and more Monolignol conjugates that improve lignin cleavage. We have cloned and expressed an enzyme from rice that has p-coumarate Monolignol transferase activity and determined its kinetic parameters.
John H Grabber - One of the best experts on this subject based on the ideXlab platform.
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Hydroxycinnamate Conjugates as Potential Monolignol Replacements: In vitro Lignification and Cell Wall Studies with Rosmarinic Acid
ChemSusChem, 2012Co-Authors: Yuki Tobimatsu, John H Grabber, Sasikumar Elumalai, Christy L. Davidson, Xuejun Pan, John RalphAbstract:The plasticity of lignin biosynthesis should permit the inclusion of new compatible phenolic monomers, such as rosmarinic acid (RA) and analogous catechol derivatives, into cell-wall lignins that are consequently less recalcitrant to biomass processing. In vitro lignin polymerization experiments revealed that RA readily underwent peroxidase-catalyzed copolymerization with Monolignols and lignin oligomers to form polymers with new benzodioxane inter-unit linkages. Incorporation of RA permitted extensive depolymerization of synthetic lignins by mild alkaline hydrolysis, presumably by cleavage of ester intra-unit linkages within RA. Copolymerization of RA with Monolignols into maize cell walls by in situ peroxidases significantly enhanced alkaline lignin extractability and promoted subsequent cell wall saccharification by fungal enzymes. Incorporating RA also improved cell wall saccharification by fungal enzymes and by rumen microflora even without alkaline pretreatments, possibly by modulating lignin hydrophobicity and/or limiting cell wall cross-linking. Consequently, we anticipate that bioengineering approaches for partial Monolignol substitution with RA and analogous plant hydroxycinnamates would permit more efficient utilization of plant fiber for biofuels or livestock production.
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Identifying new lignin bioengineering targets: 1. Monolignol-substitute impacts on lignin formation and cell wall fermentability
BMC Plant Biology, 2010Co-Authors: John H Grabber, Paul F. Schatz, Hoon Kim, Fachuang Lu, John RalphAbstract:BACKGROUND: Recent discoveries highlighting the metabolic malleability of plant lignification indicate that lignin can be engineered to dramatically alter its composition and properties. Current plant biotechnology efforts are primarily aimed at manipulating the biosynthesis of normal Monolignols, but in the future apoplastic targeting of phenolics from other metabolic pathways may provide new approaches for designing lignins that are less inhibitory toward the enzymatic hydrolysis of structural polysaccharides, both with and without biomass pretreatment. To identify promising new avenues for lignin bioengineering, we artificially lignified cell walls from maize cell suspensions with various combinations of normal Monolignols (coniferyl and sinapyl alcohols) plus a variety of phenolic Monolignol substitutes. Cell walls were then incubated in vitro with anaerobic rumen microflora to assess the potential impact of lignin modifications on the enzymatic degradability of fibrous crops used for ruminant livestock or biofuel production.\n\nRESULTS: In the absence of anatomical constraints to digestion, lignification with normal Monolignols hindered both the rate and extent of cell wall hydrolysis by rumen microflora. Inclusion of methyl caffeate, caffeoylquinic acid, or feruloylquinic acid with Monolignols considerably depressed lignin formation and strikingly improved the degradability of cell walls. In contrast, dihydroconiferyl alcohol, guaiacyl glycerol, epicatechin, epigallocatechin, and epigallocatechin gallate readily formed copolymer-lignins with normal Monolignols; cell wall degradability was moderately enhanced by greater hydroxylation or 1,2,3-triol functionality. Mono- or diferuloyl esters with various aliphatic or polyol groups readily copolymerized with Monolignols, but in some cases they accelerated inactivation of wall-bound peroxidase and reduced lignification; cell wall degradability was influenced by lignin content and the degree of ester group hydroxylation.\n\nCONCLUSION: Overall, Monolignol substitutes improved the inherent degradability of non-pretreated cell walls by restricting lignification or possibly by reducing lignin hydrophobicity or cross-linking to structural polysaccharides. Furthermore some Monolignol substitutes, chiefly readily cleaved bi-phenolic conjugates like epigallocatechin gallate or diferuloyl polyol esters, are expected to greatly boost the enzymatic degradability of cell walls following chemical pretreatment. In ongoing work, we are characterizing the enzymatic saccharification of intact and chemically pretreated cell walls lignified by these and other Monolignol substitutes to identify promising genetic engineering targets for improving plant fiber utilization.
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Identifying new lignin bioengineering targets: 1. Monolignol-substitute impacts on lignin formation and cell wall fermentability
BMC Plant Biology, 2010Co-Authors: John H Grabber, Paul F. Schatz, Hoon Kim, John RalphAbstract:Recent discoveries highlighting the metabolic malleability of plant lignification indicate that lignin can be engineered to dramatically alter its composition and properties. Current plant biotechnology efforts are primarily aimed at manipulating the biosynthesis of normal Monolignols, but in the future apoplastic targeting of phenolics from other metabolic pathways may provide new approaches for designing lignins that are less inhibitory toward the enzymatic hydrolysis of structural polysaccharides, both with and without biomass pretreatment. To identify promising new avenues for lignin bioengineering, we artificially lignified cell walls from maize cell suspensions with various combinations of normal Monolignols (coniferyl and sinapyl alcohols) plus a variety of phenolic Monolignol substitutes. Cell walls were then incubated in vitro with anaerobic rumen microflora to assess the potential impact of lignin modifications on the enzymatic degradability of fibrous crops used for ruminant livestock or biofuel production. In the absence of anatomical constraints to digestion, lignification with normal Monolignols hindered both the rate and extent of cell wall hydrolysis by rumen microflora. Inclusion of methyl caffeate, caffeoylquinic acid, or feruloylquinic acid with Monolignols considerably depressed lignin formation and strikingly improved the degradability of cell walls. In contrast, dihydroconiferyl alcohol, guaiacyl glycerol, epicatechin, epigallocatechin, and epigallocatechin gallate readily formed copolymer-lignins with normal Monolignols; cell wall degradability was moderately enhanced by greater hydroxylation or 1,2,3-triol functionality. Mono- or diferuloyl esters with various aliphatic or polyol groups readily copolymerized with Monolignols, but in some cases they accelerated inactivation of wall-bound peroxidase and reduced lignification; cell wall degradability was influenced by lignin content and the degree of ester group hydroxylation. Overall, Monolignol substitutes improved the inherent degradability of non-pretreated cell walls by restricting lignification or possibly by reducing lignin hydrophobicity or cross-linking to structural polysaccharides. Furthermore some Monolignol substitutes, chiefly readily cleaved bi-phenolic conjugates like epigallocatechin gallate or diferuloyl polyol esters, are expected to greatly boost the enzymatic degradability of cell walls following chemical pretreatment. In ongoing work, we are characterizing the enzymatic saccharification of intact and chemically pretreated cell walls lignified by these and other Monolignol substitutes to identify promising genetic engineering targets for improving plant fiber utilization.
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coniferyl ferulate incorporation into lignin enhances the alkaline delignification and enzymatic degradation of cell walls
Biomacromolecules, 2008Co-Authors: John H Grabber, Ronald D HatfieldAbstract: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.
Norman G Lewis - One of the best experts on this subject based on the ideXlab platform.
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allyl propenyl phenol synthases from the creosote bush and engineering production of specialty commodity chemicals eugenol isoeugenol in escherichia coli
Archives of Biochemistry and Biophysics, 2014Co-Authors: Sung-jin Kim, Laurence B. Davin, Daniel Giddings Vassão, Syed G. A. Moinuddin, Diana L. Bedgar, Norman G LewisAbstract:Abstract The creosote bush (Larrea tridentata) harbors members of the Monolignol acyltransferase, allylphenol synthase, and propenylphenol synthase gene families, whose products together are able to catalyze distinct regiospecific conversions of various Monolignols into their corresponding allyl- and propenyl-phenols, respectively. In this study, co-expression of a Monolignol acyltransferase with either substrate versatile allylphenol or propenylphenol synthases in Escherichia coli established that various Monolignol substrates were efficiently converted into their corresponding allyl/propenyl phenols, as well as providing proof of concept for efficacious conversion in a bacterial platform. This capability thus potentially provides an alternate source to these important plant phytochemicals, whether for flavor/fragrance and fine chemicals, or ultimately as commodities, e.g., for renewable energy or other intermediate chemical purposes. Previous reports had indicated that specific and highly conserved amino acid residues 84 (Phe or Val) and 87 (Ile or Tyr) of two highly homologous allyl/propenyl phenol synthases (circa 96% identity) from a Clarkia species mainly dictate their distinct regiospecific catalyzed conversions to afford either allyl- or propenyl-phenols, respectively. However, several other allyl/propenyl phenol synthase homologs isolated by us have established that the two corresponding amino acid 84 and 87 residues are not, in fact, conserved.
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Allyl/propenyl phenol synthases from the creosote bush and engineering production of specialty/commodity chemicals, eugenol/isoeugenol, in Escherichia coli.
Archives of biochemistry and biophysics, 2013Co-Authors: Sung-jin Kim, Laurence B. Davin, Daniel Giddings Vassão, Syed G. A. Moinuddin, Diana L. Bedgar, Norman G LewisAbstract:Abstract The creosote bush (Larrea tridentata) harbors members of the Monolignol acyltransferase, allylphenol synthase, and propenylphenol synthase gene families, whose products together are able to catalyze distinct regiospecific conversions of various Monolignols into their corresponding allyl- and propenyl-phenols, respectively. In this study, co-expression of a Monolignol acyltransferase with either substrate versatile allylphenol or propenylphenol synthases in Escherichia coli established that various Monolignol substrates were efficiently converted into their corresponding allyl/propenyl phenols, as well as providing proof of concept for efficacious conversion in a bacterial platform. This capability thus potentially provides an alternate source to these important plant phytochemicals, whether for flavor/fragrance and fine chemicals, or ultimately as commodities, e.g., for renewable energy or other intermediate chemical purposes. Previous reports had indicated that specific and highly conserved amino acid residues 84 (Phe or Val) and 87 (Ile or Tyr) of two highly homologous allyl/propenyl phenol synthases (circa 96% identity) from a Clarkia species mainly dictate their distinct regiospecific catalyzed conversions to afford either allyl- or propenyl-phenols, respectively. However, several other allyl/propenyl phenol synthase homologs isolated by us have established that the two corresponding amino acid 84 and 87 residues are not, in fact, conserved.
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a lignin specific peroxidase in tobacco whose antisense suppression leads to vascular tissue modification
Phytochemistry, 2003Co-Authors: Kristopher A Blee, Joon W Choi, Ann P Oconnell, Norman G Lewis, Wolfgang Schuch, Paul G BolwellAbstract:Abstract A tobacco peroxidase isoenzyme ( TP60 ) was down-regulated in tobacco using an antisense strategy, this affording transformants with lignin reductions of up to 40–50% of wild type (control) plants. Significantly, both guaiacyl and syringyl levels decreased in essentially a linear manner with the reductions in lignin amounts, as determined by both thioacidolysis and nitrobenzene oxidative analyses. These data provisionally suggest that a feedback mechanism is operative in lignifying cells, which prevents build-up of Monolignols should oxidative capacity for their subsequent metabolism be reduced. Prior to this study, the only known rate-limiting processes in the Monolignol/lignin pathways involved that of Phe supply and the relative activities of cinnamate-4-hydroxylase/ p -coumarate-3-hydroxylase, respectively. These transformants thus provide an additional experimental means in which to further dissect and delineate the factors involved in Monolignol targeting to precise regions in the cell wall, and of subsequent lignin assembly. Interestingly, the lignin down-regulated tobacco phenotypes displayed no readily observable differences in overall growth and development profiles, although the vascular apparatus was modified.
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trends in lignin modification a comprehensive analysis of the effects of genetic manipulations mutations on lignification and vascular integrity
Phytochemistry, 2002Co-Authors: Aldwin M. Anterola, Norman G LewisAbstract:A comprehensive assessment of lignin configuration in transgenic and mutant plants is long overdue. This review thus undertook the systematic analysis of trends manifested through genetic and mutational manipulations of the various steps associated with Monolignol biosynthesis; this included consideration of the downstream effects on organized lignin assembly in the various cell types, on vascular function/integrity, and on plant growth and development. As previously noted for dirigent protein (homologs), distinct and sophisticated Monolignol forming metabolic networks were operative in various cell types, tissues and organs, and form the cell-specific guaiacyl (G) and guaiacyl-syringyl (G-S) enriched lignin biopolymers, respectively. Regardless of cell type undergoing lignification, carbon allocation to the different Monolignol pools is apparently determined by a combination of phenylalanine availability and cinnamate-4-hydroxylase/"p-coumarate-3-hydroxylase" (C4H/C3H) activities, as revealed by transcriptional and metabolic profiling. Downregulation of either phenylalanine ammonia lyase or cinnamate-4-hydroxylase thus predictably results in reduced lignin levels and impaired vascular integrity, as well as affecting related (phenylpropanoid-dependent) metabolism. Depletion of C3H activity also results in reduced lignin deposition, albeit with the latter being derived only from hydroxyphenyl (H) units, due to both the guaiacyl (G) and syringyl (S) pathways being blocked. Apparently the cells affected are unable to compensate for reduced G/S levels by increasing the amounts of H-components. The downstream metabolic networks for G-lignin enriched formation in both angiosperms and gymnosperms utilize specific cinnamoyl CoA O-methyltransferase (CCOMT), 4-coumarate:CoA ligase (4CL), cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) isoforms: however, these steps neither affect carbon allocation nor H/G designations, this being determined by C4H/C3H activities. Such enzymes thus fulfill subsidiary processing roles, with all (except CCOMT) apparently being bifunctional for both H and G substrates. Their severe downregulation does, however, predictably result in impaired Monolignol biosynthesis, reduced lignin deposition/vascular integrity, (upstream) metabolite build-up and/or shunt pathway metabolism. There was no evidence for an alternative acid/ester O-methyltransferase (AEOMT) being involved in lignin biosynthesis. The G/S lignin pathway networks are operative in specific cell types in angiosperms and employ two additional biosynthetic steps to afford the corresponding S components, i.e. through introduction of an hydroxyl group at C-5 and its subsequent O-methylation. [These enzymes were originally classified as ferulate-5-hydroxylase (F5H) and caffeate O-methyltransferase (COMT), respectively.] As before, neither step has apparently any role in carbon allocation to the pathway; hence their individual downregulation/manipulation, respectively, gives either a G enriched lignin or formation of the well-known S-deficient bm3 "lignin" mutant, with cell walls of impaired vascular integrity. In the latter case, COMT downregulation/mutation apparently results in utilization of the isoelectronic 5-hydroxyconiferyl alcohol species albeit in an unsuccessful attempt to form G-S lignin proper. However, there is apparently no effect on overall G content, thereby indicating that deposition of both G and S moieties in the G/S lignin forming cells are kept spatially, and presumably temporally, fully separate. Downregulation/mutation of further downstream steps in the G/S network [i.e. utilizing 4CL, CCR and CAD isoforms] gives predictable effects in terms of their subsidiary processing roles: while severe downregulation of 4CL gave phenotypes with impaired vascular integrity due to reduced Monolignol supply, there was no evidence in support of increased growth and/or enhanced cellulose biosynthesis. CCR and CAD downregulation/mutations also established that a depletion in Monolignol supply reduced both lignin contents supply reduced both lignin contents and vascular integrity, with a concomitant shift towards (upstream) metabolite build-up and/or shunting. The extraordinary claims of involvement of surrogate monomers (2-methoxybenzaldehyde, feruloyl tyramine, vanillic acid, etc.) in lignification were fully disproven and put to rest, with the investigators themselves having largely retracted former claims. Furthermore analysis of the well-known bm1 mutation, a presumed CAD disrupted system, apparently revealed that both G and S lignin components were reduced. This seems to imply that there is no Monolignol specific dehydrogenase, such as the recently described sinapyl alcohol dehydrogenase (SAD) for sinapyl alcohol formation. Nevertheless, different CAD isoforms of differing homology seem to be operative in different lignifying cell types, thereby giving the G-enriched and G/S-enriched lignin biopolymers, respectively. For the G-lignin forming network, however, the CAD isoform is apparently catalytically less efficient with all three Monolignols than that additionally associated with the corresponding G/S lignin forming network(s), which can more efficiently use all three Monolignols. However, since CAD does not determine either H, G, or S designation, it again serves in a subsidiary role-albeit using different isoforms for different cell wall developmental and cell wall type responses. The results from this analysis contrasts further with speculations of some early investigators, who had viewed lignin assembly as resulting from non-specific oxidative coupling of Monolignols and subsequent random polymerization. At that time, though, the study of the complex biological (biochemical) process of lignin assembly had begun without any of the (bio)chemical tools to either address or answer the questions posed as to how its formation might actually occur. Today, by contrast, there is growing recognition of both sophisticated and differential control of Monolignol biosynthetic networks in different cell types, which serve to underscore the fact that complexity of assembly need not be confused any further with random formation. Moreover, this analysis revealed another factor which continues to cloud interpretations of lignin downregulation/mutational analyses, namely the serious technical problems associated with all aspects of lignin characterization, whether for lignin quantification, isolation of lignin-enriched preparations and/or in determining monomeric compositions. For example, in the latter analyses, some 50-90% of the lignin components still cannot be detected using current methodologies, e.g. by thioacidolysis cleavage and nitrobenzene oxidative cleavage. This deficiency in lignin characterization thus represents one of the major hurdles remaining in delineating how lignin assembly (in distinct cell types) and their configuration actually occurs.
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Transcriptional control of Monolignol biosynthesis in Pinus taeda: factors affecting Monolignol ratios and carbon allocation in phenylpropanoid metabolism.
Journal of Biological Chemistry, 2002Co-Authors: Aldwin M. Anterola, Jae-heung Jeon, Laurence B. Davin, Norman G LewisAbstract:Transcriptional profiling of the phenylpropanoid pathway in Pinus taeda cell suspension cultures was carried out using quantitative real time PCR analyses of all known genes involved in the biosynthesis of the two Monolignols, p-coumaryl and coniferyl alcohols (lignin/lignan precursors). When the cells were transferred to a medium containing 8% sucrose and 20 mm potassium iodide, the Monolignol/phenylpropanoid pathway was induced, and transcript levels for phenylalanine ammonia lyase, cinnamate 4-hydroxylase, p-coumarate 3-hydroxylase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase, and cinnamyl alcohol dehydrogenase were coordinately up-regulated. Provision of increasing levels of exogenously supplied Phe to saturating levels (40 mm) to the induction medium resulted in further up-regulation of their transcript levels in the P. taeda cell cultures; this in turn was accompanied by considerable increases in both p-coumaryl and coniferyl alcohol formation and excretion. By contrast, transcript levels for both cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase were only slightly up-regulated. These data, when considered together with metabolic profiling results and genetic manipulation of various plant species, reveal that carbon allocation to the pathway and its differential distribution into the two Monolignols is controlled by Phe supply and differential modulation of cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase activities, respectively. The coordinated up-regulation of phenylalanine ammonia lyase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase in the presence of increasing concentrations of Phe also indicates that these steps are not truly rate-limiting, because they are modulated according to metabolic demand. Finally, the transcript profile of a putative acid/ester O-methyltransferase, proposed as an alternative catalyst for O-methylation leading to coniferyl alcohol, was not up-regulated under any of the conditions employed, suggesting that it is not, in fact, involved in Monolignol biosynthesis.
Simon Hawkins - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the UDP-glycosyltransferase UGT72 Family in Poplar and Identification of Genes Involved in the Glycosylation of Monolignols
International Journal of Molecular Sciences, 2020Co-Authors: Nathanaël Speeckaert, Simon Hawkins, Nassirou Mahamadou Adamou, Hadjara Amadou Hassane, Fabien Baldacci-cresp, Adeline Mol, Geert Goeminne, Wout Boerjan, Pierre Duez, Godfrey NeutelingsAbstract:Monolignols are the building blocks for lignin polymerization in the apoplastic domain. Monolignol biosynthesis, transport, storage, glycosylation, and deglycosylation are the main biological processes partaking in their homeostasis. In Arabidopsis thaliana, members of the uridine diphosphate-dependent glucosyltransferases UGT72E and UGT72B subfamilies have been demonstrated to glycosylate Monolignols. Here, the poplar UGT72 family, which is clustered into four groups, was characterized: Group 1 UGT72AZ1 and UGT72AZ2, homologs of Arabidopsis UGT72E1-3, as well as group 4 UGT72B37 and UGT72B39, homologs of Arabidopsis UGT72B1-3, glycosylate Monolignols. In addition, promoter-GUS analyses indicated that poplar UGT72 members are expressed within vascular tissues. At the subcellular level, poplar UGT72s belonging to group 1 and group 4 were found to be associated with the nucleus and the endoplasmic reticulum. However, UGT72A2, belonging to group 2, was localized in bodies associated with chloroplasts, as well as possibly in chloroplasts. These results show a partial conservation of substrate recognition between Arabidopsis and poplar homologs, as well as divergent functions between different groups of the UGT72 family, for which the substrates remain unknown.
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BLISS: Shining a light on lignification in plants
Plant Signaling and Behavior, 2017Co-Authors: Clémence Simon, Cedric Lion, Brigitte Huss, Anne-sophie Blervacq, Corentin Spriet, Yann Guerardel, Christophe Biot, Simon HawkinsAbstract:Lignin is a polyphenolic polymer of the plant cell wall formed by the oxidative polymerization of 3 main monomers called Monolignols that give rise to the lignin H-, G- and S-units. Together with cellulose and hemicelluloses, lignin is a major component of plant biomass that is widely exploited by humans in numerous industrial processes. Despite recent advances in our understanding of Monolignol biosynthesis, our current understanding of the spatio-temporal regulation of their transport and polymerization is more limited. In a recent publication, we have reported the development of an original Bioorthogonal Labeling Imaging Sequential Strategy (BLISS) that allows us to visualize the simultaneous incorporation dynamics of H and G Monolignol reporters into lignifying cell walls of the flax stem. (11) Here, we extend the application of this strategy to other plant organs such as roots and rapidly discuss some of the contributions and perspectives of this new technique for improving our understanding of the lignification process in plants.
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Plant cell wall lignification and Monolignol metabolism
Frontiers in Plant Science, 2013Co-Authors: Yin Wang, Maxime Chantreau, Richard Sibout, Simon HawkinsAbstract:Plants are built of various specialized cell types that differ in their cell wall composition and structure. The cell walls of certain tissues (xylem, sclerenchyma) are characterized by the presence of the heterogeneous lignin polymer that plays an essential role in their physiology. This phenolic polymer is composed of different monomeric units - the Monolignols - that are linked together by several covalent bonds. Numerous studies have shown that Monolignol biosynthesis and polymerization to form lignin are tightly controlled in different cell types and tissues. However, our understanding of the genetic control of Monolignol transport and polymerization remains incomplete, despite some recent promising results. This situation is made more complex since we know that Monolignols or related compounds are sometimes produced in non-lignified tissues. In this review, we focus on some key steps of Monolignol metabolism including polymerization, transport, and compartmentation. As well as being of fundamental interest, the quantity of lignin and its nature are also known to have a negative effect on the industrial processing of plant lignocellulose biomass. A more complete view of Monolignol metabolism and the relationship that exists between lignin and other Monolignol-derived compounds thereby appears essential if we wish to improve biomass quality.
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Natural hypolignification is associated with extensive oligolignol accumulation in flax stems
Plant Physiology, 2012Co-Authors: Rudy Huis, Francois Mesnard, Brigitte Chabbert, Kris Morreel, Godfrey Neutelings, Ophelie Fliniaux, Anca Lucau, Stephane Fenart, Sébastien Grec, Simon HawkinsAbstract:Flax (Linum usitatissimum) stems contain cells showing contrasting cell wall structure: lignified in inner stem xylem tissue and hypolignified in outer stem bast fibers. We hypothesized that stem hypolignification should be associated with extensive phenolic accumulation and used metabolomics and transcriptomics to characterize these two tissues. 1H nuclear magnetic resonance clearly distinguished inner and outer stem tissues and identified different primary and secondary metabolites, including coniferin and p-coumaryl alcohol glucoside. Ultrahigh-performance liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry aromatic profiling (lignomics) identified 81 phenolic compounds, of which 65 were identified, to our knowledge, for the first time in flax and 11 for the first time in higher plants. Both aglycone forms and glycosides of Monolignols, lignin oligomers, and (neo)lignans were identified in both inner and outer stem tissues, with a preponderance of glycosides in the hypolignified outer stem, indicating the existence of a complex Monolignol metabolism. The presence of coniferin-containing secondary metabolites suggested that coniferyl alcohol, in addition to being used in lignin and (neo)lignan formation, was also utilized in a third, partially uncharacterized metabolic pathway. Hypolignification of bast fibers in outer stem tissues was correlated with the low transcript abundance of Monolignol biosynthetic genes, laccase genes, and certain peroxidase genes, suggesting that flax hypolignification is transcriptionally regulated. Transcripts of the key lignan genes Pinoresinol-Lariciresinol Reductase and Phenylcoumaran Benzylic Ether Reductase were also highly abundant in flax inner stem tissues. Expression profiling allowed the identification of NAC (NAM, ATAF1/2, CUC2) and MYB transcription factors that are likely involved in regulating both Monolignol production and polymerization as well as (neo)lignan production.