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1.
Plant Biotechnol J ; 19(11): 2221-2234, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34160888

RESUMO

Lignins are cell wall-located aromatic polymers that provide strength and hydrophobicity to woody tissues. Lignin monomers are synthesized via the phenylpropanoid pathway, wherein CAFFEOYL SHIKIMATE ESTERASE (CSE) converts caffeoyl shikimate into caffeic acid. Here, we explored the role of the two CSE homologs in poplar (Populus tremula × P. alba). Reporter lines showed that the expression conferred by both CSE1 and CSE2 promoters is similar. CRISPR-Cas9-generated cse1 and cse2 single mutants had a wild-type lignin level. Nevertheless, CSE1 and CSE2 are not completely redundant, as both single mutants accumulated caffeoyl shikimate. In contrast, the cse1 cse2 double mutants had a 35% reduction in lignin and associated growth penalty. The reduced-lignin content translated into a fourfold increase in cellulose-to-glucose conversion upon limited saccharification. Phenolic profiling of the double mutants revealed large metabolic shifts, including an accumulation of p-coumaroyl, 5-hydroxyferuloyl, feruloyl and sinapoyl shikimate, in addition to caffeoyl shikimate. This indicates that the CSEs have a broad substrate specificity, which was confirmed by in vitro enzyme kinetics. Taken together, our results suggest an alternative path within the phenylpropanoid pathway at the level of the hydroxycinnamoyl-shikimates, and show that CSE is a promising target to improve plants for the biorefinery.


Assuntos
Populus , Sistemas CRISPR-Cas/genética , Carboxilesterase , Regulação da Expressão Gênica de Plantas , Lignina/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Populus/genética , Populus/metabolismo
2.
PLoS Comput Biol ; 16(4): e1007197, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32275650

RESUMO

Accurate manipulation of metabolites in monolignol biosynthesis is a key step for controlling lignin content, structure, and other wood properties important to the bioenergy and biomaterial industries. A crucial component of this strategy is predicting how single and combinatorial knockdowns of monolignol specific gene transcripts influence the abundance of monolignol proteins, which are the driving mechanisms of monolignol biosynthesis. Computational models have been developed to estimate protein abundances from transcript perturbations of monolignol specific genes. The accuracy of these models, however, is hindered by their inability to capture indirect regulatory influences on other pathway genes. Here, we examine the manifestation of these indirect influences on transgenic transcript and protein abundances, identifying putative indirect regulatory influences that occur when one or more specific monolignol pathway genes are perturbed. We created a computational model using sparse maximum likelihood to estimate the resulting monolignol transcript and protein abundances in transgenic Populus trichocarpa based on targeted knockdowns of specific monolignol genes. Using in-silico simulations of this model and root mean square error, we showed that our model more accurately estimated transcript and protein abundances, in comparison to previous models, when individual and families of monolignol genes were perturbed. We leveraged insight from the inferred network structure obtained from our model to identify potential genes, including PtrHCT, PtrCAD, and Ptr4CL, involved in post-transcriptional and/or post-translational regulation. Our model provides a useful computational tool for exploring the cascaded impact of single and combinatorial modifications of monolignol specific genes on lignin and other wood properties.


Assuntos
Biologia Computacional/métodos , Lignina/genética , Lignina/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Técnicas de Silenciamento de Genes/métodos , Lignina/biossíntese , Modelos Teóricos , Populus/genética , Madeira/genética
3.
New Phytol ; 222(1): 244-260, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30276825

RESUMO

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.


Assuntos
Lignina/metabolismo , Proteínas de Plantas/metabolismo , Populus/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Regulação para Baixo/genética , Regulação da Expressão Gênica de Plantas , Espectroscopia de Ressonância Magnética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Populus/genética , Interferência de RNA , Proteínas Recombinantes/metabolismo , Xilema/metabolismo
4.
Proc Natl Acad Sci U S A ; 112(27): 8481-6, 2015 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26109572

RESUMO

Although phosphorylation has long been known to be an important regulatory modification of proteins, no unequivocal evidence has been presented to show functional control by phosphorylation for the plant monolignol biosynthetic pathway. Here, we present the discovery of phosphorylation-mediated on/off regulation of enzyme activity for 5-hydroxyconiferaldehyde O-methyltransferase 2 (PtrAldOMT2), an enzyme central to monolignol biosynthesis for lignification in stem-differentiating xylem (SDX) of Populus trichocarpa. Phosphorylation turned off the PtrAldOMT2 activity, as demonstrated in vitro by using purified phosphorylated and unphosphorylated recombinant PtrAldOMT2. Protein extracts of P. trichocarpa SDX, which contains endogenous kinases, also phosphorylated recombinant PtrAldOMT2 and turned off the recombinant protein activity. Similarly, ATP/Mn(2+)-activated phosphorylation of SDX protein extracts reduced the endogenous SDX PtrAldOMT2 activity by ∼ 60%, and dephosphorylation fully restored the activity. Global shotgun proteomic analysis of phosphopeptide-enriched P. trichocarpa SDX protein fractions identified PtrAldOMT2 monophosphorylation at Ser(123) or Ser(125) in vivo. Phosphorylation-site mutagenesis verified the PtrAldOMT2 phosphorylation at Ser(123) or Ser(125) and confirmed the functional importance of these phosphorylation sites for O-methyltransferase activity. The PtrAldOMT2 Ser(123) phosphorylation site is conserved across 93% of AldOMTs from 46 diverse plant species, and 98% of the AldOMTs have either Ser(123) or Ser(125). PtrAldOMT2 is a homodimeric cytosolic enzyme expressed more abundantly in syringyl lignin-rich fiber cells than in guaiacyl lignin-rich vessel cells. The reversible phosphorylation of PtrAldOMT2 is likely to have an important role in regulating syringyl monolignol biosynthesis of P. trichocarpa.


Assuntos
Acroleína/análogos & derivados , Catecóis/metabolismo , Lignina/biossíntese , Metiltransferases/metabolismo , Proteínas de Plantas/metabolismo , Populus/metabolismo , Acroleína/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Biocatálise , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Metiltransferases/genética , Microscopia Confocal , Dados de Sequência Molecular , Mutação , Fosfoproteínas/metabolismo , Fosforilação , Proteínas de Plantas/genética , Populus/enzimologia , Populus/genética , Proteômica/métodos , Protoplastos/enzimologia , Protoplastos/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Massas em Tandem
5.
Planta ; 245(5): 927-938, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28083709

RESUMO

MAIN CONCLUSION: Co-expression networks based on transcriptomes of Populus trichocarpa major tissues and specific cell types suggest redundant control of cell wall component biosynthetic genes by transcription factors in wood formation. We analyzed the transcriptomes of five tissues (xylem, phloem, shoot, leaf, and root) and two wood forming cell types (fiber and vessel) of Populus trichocarpa to assemble gene co-expression subnetworks associated with wood formation. We identified 165 transcription factors (TFs) that showed xylem-, fiber-, and vessel-specific expression. Of these 165 TFs, 101 co-expressed (correlation coefficient, r > 0.7) with the 45 secondary cell wall cellulose, hemicellulose, and lignin biosynthetic genes. Each cell wall component gene co-expressed on average with 34 TFs, suggesting redundant control of the cell wall component gene expression. Co-expression analysis showed that the 101 TFs and the 45 cell wall component genes each has two distinct groups (groups 1 and 2), based on their co-expression patterns. The group 1 TFs (44 members) are predominantly xylem and fiber specific, and are all highly positively co-expressed with the group 1 cell wall component genes (30 members), suggesting their roles as major wood formation regulators. Group 1 TFs include a lateral organ boundary domain gene (LBD) that has the highest number of positively correlated cell wall component genes (36) and TFs (47). The group 2 TFs have 57 members, including 14 vessel-specific TFs, and are generally less correlated with the cell wall component genes. An exception is a vessel-specific basic helix-loop-helix (bHLH) gene that negatively correlates with 20 cell wall component genes, and may function as a key transcriptional suppressor. The co-expression networks revealed here suggest a well-structured transcriptional homeostasis for cell wall component biosynthesis during wood formation.


Assuntos
Proteínas de Plantas/genética , Populus/genética , Transcriptoma , Madeira/genética , Parede Celular/metabolismo , Celulose/metabolismo , Análise por Conglomerados , Regulação da Expressão Gênica de Plantas , Lignina/metabolismo , Anotação de Sequência Molecular , Especificidade de Órgãos , Floema/genética , Floema/crescimento & desenvolvimento , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Populus/crescimento & desenvolvimento , Análise de Sequência de RNA , Fatores de Transcrição/genética , Madeira/crescimento & desenvolvimento , Xilema/genética , Xilema/crescimento & desenvolvimento
6.
Plant Cell ; 26(3): 894-914, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24619611

RESUMO

We established a predictive kinetic metabolic-flux model for the 21 enzymes and 24 metabolites of the monolignol biosynthetic pathway using Populus trichocarpa secondary differentiating xylem. To establish this model, a comprehensive study was performed to obtain the reaction and inhibition kinetic parameters of all 21 enzymes based on functional recombinant proteins. A total of 104 Michaelis-Menten kinetic parameters and 85 inhibition kinetic parameters were derived from these enzymes. Through mass spectrometry, we obtained the absolute quantities of all 21 pathway enzymes in the secondary differentiating xylem. This extensive experimental data set, generated from a single tissue specialized in wood formation, was used to construct the predictive kinetic metabolic-flux model to provide a comprehensive mathematical description of the monolignol biosynthetic pathway. The model was validated using experimental data from transgenic P. trichocarpa plants. The model predicts how pathway enzymes affect lignin content and composition, explains a long-standing paradox regarding the regulation of monolignol subunit ratios in lignin, and reveals novel mechanisms involved in the regulation of lignin biosynthesis. This model provides an explanation of the effects of genetic and transgenic perturbations of the monolignol biosynthetic pathway in flowering plants.


Assuntos
Lignina/metabolismo , Proteínas de Plantas/metabolismo , Populus/metabolismo , Proteoma , Cinética , Espectrometria de Massas , Polimorfismo de Nucleotídeo Único
7.
Plant Cell ; 26(3): 876-93, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24619612

RESUMO

As a step toward predictive modeling of flux through the pathway of monolignol biosynthesis in stem differentiating xylem of Populus trichocarpa, we discovered that the two 4-coumaric acid:CoA ligase (4CL) isoforms, 4CL3 and 4CL5, interact in vivo and in vitro to form a heterotetrameric protein complex. This conclusion is based on laser microdissection, coimmunoprecipitation, chemical cross-linking, bimolecular fluorescence complementation, and mass spectrometry. The tetramer is composed of three subunits of 4CL3 and one of 4CL5. 4CL5 appears to have a regulatory role. This protein-protein interaction affects the direction and rate of metabolic flux for monolignol biosynthesis in P. trichocarpa. A mathematical model was developed for the behavior of 4CL3 and 4CL5 individually and in mixtures that form the enzyme complex. The model incorporates effects of mixtures of multiple hydroxycinnamic acid substrates, competitive inhibition, uncompetitive inhibition, and self-inhibition, along with characteristic of the substrates, the enzyme isoforms, and the tetrameric complex. Kinetic analysis of different ratios of the enzyme isoforms shows both inhibition and activation components, which are explained by the mathematical model and provide insight into the regulation of metabolic flux for monolignol biosynthesis by protein complex formation.


Assuntos
Coenzima A Ligases/metabolismo , Ácidos Cumáricos/metabolismo , Lignina/biossíntese , Populus/metabolismo , Biologia de Sistemas , Coenzima A Ligases/genética , Imunoprecipitação , Espectrometria de Massas , Modelos Biológicos , Propionatos , RNA Mensageiro/genética , Especificidade por Substrato
8.
Proc Natl Acad Sci U S A ; 110(26): 10848-53, 2013 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-23754401

RESUMO

Laccases, as early as 1959, were proposed to catalyze the oxidative polymerization of monolignols. Genetic evidence in support of this hypothesis has been elusive due to functional redundancy of laccase genes. An Arabidopsis double mutant demonstrated the involvement of laccases in lignin biosynthesis. We previously identified a subset of laccase genes to be targets of a microRNA (miRNA) ptr-miR397a in Populus trichocarpa. To elucidate the roles of ptr-miR397a and its targets, we characterized the laccase gene family and identified 49 laccase gene models, of which 29 were predicted to be targets of ptr-miR397a. We overexpressed Ptr-MIR397a in transgenic P. trichocarpa. In each of all nine transgenic lines tested, 17 PtrLACs were down-regulated as analyzed by RNA-seq. Transgenic lines with severe reduction in the expression of these laccase genes resulted in an ∼40% decrease in the total laccase activity. Overexpression of Ptr-MIR397a in these transgenic lines also reduced lignin content, whereas levels of all monolignol biosynthetic gene transcripts remained unchanged. A hierarchical genetic regulatory network (GRN) built by a bottom-up graphic Gaussian model algorithm provides additional support for a role of ptr-miR397a as a negative regulator of laccases for lignin biosynthesis. Full transcriptome-based differential gene expression in the overexpressed transgenics and protein domain analyses implicate previously unidentified transcription factors and their targets in an extended hierarchical GRN including ptr-miR397a and laccases that coregulate lignin biosynthesis in wood formation. Ptr-miR397a, laccases, and other regulatory components of this network may provide additional strategies for genetic manipulation of lignin content.


Assuntos
Regulação para Baixo/genética , Lacase/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Populus/enzimologia , Populus/genética , RNA de Plantas/genética , RNA de Plantas/metabolismo , Sequência de Bases , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Genes de Plantas , Lacase/antagonistas & inibidores , Lignina/antagonistas & inibidores , Lignina/química , Lignina/metabolismo , Filogenia , Proteínas de Plantas/genética
9.
J Proteome Res ; 14(10): 4158-68, 2015 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-26325666

RESUMO

Cellulose, the main chemical polymer of wood, is the most abundant polysaccharide in nature.1 The ability to perturb the abundance and structure of cellulose microfibrils is of critical importance to the pulp and paper industry as well as for the textile, wood products, and liquid biofuels industries. Although much has been learned at the transcript level about the biosynthesis of cellulose, a quantitative understanding at the proteome level has yet to be established. The study described herein sought to identify the proteins directly involved in cellulose biosynthesis during wood formation in Populus trichocarpa along with known xylem-specific transcription factors involved in regulating these key proteins. Development of an effective discovery proteomic strategy through a combination of subcellular fractionation of stem differentiating xylem tissue (SDX) with recently optimized FASP digestion protocols, StageTip fractionation, as well as optimized instrument parameters for global proteomic analysis using the quadrupole-orbitrap mass spectrometer resulted in the deepest proteomic coverage of SDX protein from P. trichocarpa with 9,146 protein groups being identified (1% FDR). Of these, 20 cellulosic/hemicellulosic enzymes and 43 xylem-specific transcription factor groups were identified. Finally, selection of surrogate peptides led to an assay for absolute quantification of 14 cellulosic proteins in SDX of P. trichocarpa.


Assuntos
Celulose/biossíntese , Proteínas de Plantas/isolamento & purificação , Populus/genética , Proteoma/isolamento & purificação , Fatores de Transcrição/isolamento & purificação , Madeira/metabolismo , Metabolismo dos Carboidratos , Celulose/genética , Cromatografia Líquida , Regulação da Expressão Gênica de Plantas , Anotação de Sequência Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Populus/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteômica , Espectrometria de Massas em Tandem , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Madeira/química , Xilema/genética , Xilema/metabolismo
10.
Plant Biotechnol J ; 12(9): 1174-92, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25330253

RESUMO

Lignocelluloses from plant cell walls are attractive resources for sustainable biofuel production. However, conversion of lignocellulose to biofuel is more expensive than other current technologies, due to the costs of chemical pretreatment and enzyme hydrolysis for cell wall deconstruction. Recalcitrance of cell walls to deconstruction has been reduced in many plant species by modifying plant cell walls through biotechnology. These results have been achieved by reducing lignin content and altering its composition and structure. Reduction of recalcitrance has also been achieved by manipulating hemicellulose biosynthesis and by overexpression of bacterial enzymes in plants to disrupt linkages in the lignin-carbohydrate complexes. These modified plants often have improved saccharification yield and higher ethanol production. Cell wall-degrading (CWD) enzymes from bacteria and fungi have been expressed at high levels in plants to increase the efficiency of saccharification compared with exogenous addition of cellulolytic enzymes. In planta expression of heat-stable CWD enzymes from bacterial thermophiles has made autohydrolysis possible. Transgenic plants can be engineered to reduce recalcitrance without any yield penalty, indicating that successful cell wall modification can be achieved without impacting cell wall integrity or plant development. A more complete understanding of cell wall formation and structure should greatly improve lignocellulosic feedstocks and reduce the cost of biofuel production.


Assuntos
Biocombustíveis , Biotecnologia/métodos , Lignina/metabolismo , Plantas/metabolismo , Parede Celular/metabolismo , Plantas/enzimologia , Engenharia de Proteínas
11.
Plant Physiol ; 161(3): 1501-16, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23344904

RESUMO

4-Coumaric acid:coenzyme A ligase (4CL) is involved in monolignol biosynthesis for lignification in plant cell walls. It ligates coenzyme A (CoA) with hydroxycinnamic acids, such as 4-coumaric and caffeic acids, into hydroxycinnamoyl-CoA thioesters. The ligation ensures the activated state of the acid for reduction into monolignols. In Populus spp., it has long been thought that one monolignol-specific 4CL is involved. Here, we present evidence of two monolignol 4CLs, Ptr4CL3 and Ptr4CL5, in Populus trichocarpa. Ptr4CL3 is the ortholog of the monolignol 4CL reported for many other species. Ptr4CL5 is novel. The two Ptr4CLs exhibited distinct Michaelis-Menten kinetic properties. Inhibition kinetics demonstrated that hydroxycinnamic acid substrates are also inhibitors of 4CL and suggested that Ptr4CL5 is an allosteric enzyme. Experimentally validated flux simulation, incorporating reaction/inhibition kinetics, suggested two CoA ligation paths in vivo: one through 4-coumaric acid and the other through caffeic acid. We previously showed that a membrane protein complex mediated the 3-hydroxylation of 4-coumaric acid to caffeic acid. The demonstration here of two ligation paths requiring these acids supports this 3-hydroxylation function. Ptr4CL3 regulates both CoA ligation paths with similar efficiencies, whereas Ptr4CL5 regulates primarily the caffeic acid path. Both paths can be inhibited by caffeic acid. The Ptr4CL5-catalyzed caffeic acid metabolism, therefore, may also act to mitigate the inhibition by caffeic acid to maintain a proper ligation flux. A high level of caffeic acid was detected in stem-differentiating xylem of P. trichocarpa. Our results suggest that Ptr4CL5 and caffeic acid coordinately modulate the CoA ligation flux for monolignol biosynthesis.


Assuntos
Vias Biossintéticas , Coenzima A Ligases/metabolismo , Coenzima A/metabolismo , Simulação por Computador , Ácidos Cumáricos/metabolismo , Lignina/biossíntese , Populus/enzimologia , Regulação Alostérica/efeitos dos fármacos , Sítios de Ligação , Vias Biossintéticas/efeitos dos fármacos , Western Blotting , Ácidos Cafeicos/farmacologia , Coenzima A Ligases/antagonistas & inibidores , Ácidos Cumáricos/química , Ácidos Cumáricos/farmacologia , Cinética , Lignina/química , Fenilpropionatos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Extratos Vegetais , Populus/efeitos dos fármacos , Propionatos , Proteômica , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato/efeitos dos fármacos , Xilema/efeitos dos fármacos , Xilema/metabolismo
12.
Proc Natl Acad Sci U S A ; 108(52): 21253-8, 2011 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-22160716

RESUMO

The hydroxylation of 4- and 3-ring carbons of cinnamic acid derivatives during monolignol biosynthesis are key steps that determine the structure and properties of lignin. Individual enzymes have been thought to catalyze these reactions. In stem differentiating xylem (SDX) of Populus trichocarpa, two cinnamic acid 4-hydroxylases (PtrC4H1 and PtrC4H2) and a p-coumaroyl ester 3-hydroxylase (PtrC3H3) are the enzymes involved in these reactions. Here we present evidence that these hydroxylases interact, forming heterodimeric (PtrC4H1/C4H2, PtrC4H1/C3H3, and PtrC4H2/C3H3) and heterotrimeric (PtrC4H1/C4H2/C3H3) membrane protein complexes. Enzyme kinetics using yeast recombinant proteins demonstrated that the enzymatic efficiency (V(max)/k(m)) for any of the complexes is 70-6,500 times greater than that of the individual proteins. The highest increase in efficiency was found for the PtrC4H1/C4H2/C3H3-mediated p-coumaroyl ester 3-hydroxylation. Affinity purification-quantitative mass spectrometry, bimolecular fluorescence complementation, chemical cross-linking, and reciprocal coimmunoprecipitation provide further evidence for these multiprotein complexes. The activities of the recombinant and SDX plant proteins demonstrate two protein-complex-mediated 3-hydroxylation paths in monolignol biosynthesis in P. trichocarpa SDX; one converts p-coumaric acid to caffeic acid and the other converts p-coumaroyl shikimic acid to caffeoyl shikimic acid. Cinnamic acid 4-hydroxylation is also mediated by the same protein complexes. These results provide direct evidence for functional involvement of membrane protein complexes in monolignol biosynthesis.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Lignina/biossíntese , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Populus/metabolismo , Transcinamato 4-Mono-Oxigenase/metabolismo , Xilema/metabolismo , Hidrolases de Éster Carboxílico/química , Cromatografia Líquida , Ácidos Cumáricos , Primers do DNA/genética , Hidroxilação , Imunoprecipitação , Cinética , Espectrometria de Massas , Proteínas de Membrana/química , Microscopia Confocal , Estrutura Molecular , Complexos Multiproteicos/química , Fenóis , Fenilpropionatos , Plasmídeos/genética , Propionatos , Transcinamato 4-Mono-Oxigenase/química , Leveduras
13.
Mol Plant ; 17(1): 112-140, 2024 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-38102833

RESUMO

Cell walls in plants, particularly forest trees, are the major carbon sink of the terrestrial ecosystem. Chemical and biosynthetic features of plant cell walls were revealed early on, focusing mostly on herbaceous model species. Recent developments in genomics, transcriptomics, epigenomics, transgenesis, and associated analytical techniques are enabling novel insights into formation of woody cell walls. Here, we review multilevel regulation of cell wall biosynthesis in forest tree species. We highlight current approaches to engineering cell walls as potential feedstock for materials and energy and survey reported field tests of such engineered transgenic trees. We outline opportunities and challenges in future research to better understand cell type biogenesis for more efficient wood cell wall modification and utilization for biomaterials or for enhanced carbon capture and storage.


Assuntos
Lignina , Madeira , Madeira/genética , Madeira/metabolismo , Lignina/metabolismo , Ecossistema , Plantas/metabolismo , Parede Celular/metabolismo , Árvores/genética
14.
Science ; 381(6654): 216-221, 2023 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-37440632

RESUMO

The domestication of forest trees for a more sustainable fiber bioeconomy has long been hindered by the complexity and plasticity of lignin, a biopolymer in wood that is recalcitrant to chemical and enzymatic degradation. Here, we show that multiplex CRISPR editing enables precise woody feedstock design for combinatorial improvement of lignin composition and wood properties. By assessing every possible combination of 69,123 multigenic editing strategies for 21 lignin biosynthesis genes, we deduced seven different genome editing strategies targeting the concurrent alteration of up to six genes and produced 174 edited poplar variants. CRISPR editing increased the wood carbohydrate-to-lignin ratio up to 228% that of wild type, leading to more-efficient fiber pulping. The edited wood alleviates a major fiber-production bottleneck regardless of changes in tree growth rate and could bring unprecedented operational efficiencies, bioeconomic opportunities, and environmental benefits.


Assuntos
Edição de Genes , Lignina , Populus , Madeira , Carboidratos/análise , Lignina/genética , Madeira/genética , Sistemas CRISPR-Cas , Populus/genética , Papel , Crescimento Sustentável
15.
Planta ; 236(3): 879-85, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22729823

RESUMO

Lignin content and composition are largely determined by the composition and quantity of the monolignol precursors. Individual enzymes of the monolignol biosynthetic pathway determine the composition and quantity of monolignols. Monolignol biosynthesis in angiosperms is mediated by ten enzyme families. We developed a method using a total protein extract (soluble and microsomal) for the comprehensive and simultaneous analysis of these ten enzyme activities in a single target tissue, stem differentiating xylem (SDX) of Populus trichocarpa. As little as 300 mg fresh weight of SDX is sufficient for triplicate assays of all ten enzyme activities. To expand the effectiveness of the analysis, we quantified the reaction products directly by HPLC and developed a universal method that can separate the substrates and products of all enzymes. The specific activities measured with this simple approach are similar to those obtained with the optimum conditions previously established for each individual enzyme. This approach is applicable to the enzyme activity analysis for both P. trichocarpa (angiosperm) and Pinus taeda (gymnosperm) and is particularly useful when a large number of samples need to be analyzed for all monolignol biosynthetic enzymes.


Assuntos
Lignina/biossíntese , Pinus taeda/enzimologia , Proteínas de Plantas/análise , Caules de Planta/enzimologia , Populus/enzimologia , Xilema/enzimologia , Vias Biossintéticas , Cromatografia Líquida de Alta Pressão , Ensaios Enzimáticos , Metiltransferases/metabolismo , Oxigenases de Função Mista/metabolismo , Oxirredutases/metabolismo , Especificidade por Substrato
16.
Planta ; 236(3): 795-808, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22628084

RESUMO

Flowering plants have syringyl and guaiacyl subunits in lignin in contrast to the guaiacyl lignin in gymnosperms. The biosynthesis of syringyl subunits is initiated by coniferaldehyde 5-hydroxylase (CAld5H). In Populus trichocarpa there are two closely related CAld5H enzymes (PtrCAld5H1 and PtrCAld5H2) associated with lignin biosynthesis during wood formation. We used yeast recombinant PtrCAld5H1 and PtrCAld5H2 proteins to carry out Michaelis-Menten and inhibition kinetics with LC-MS/MS based absolute protein quantification. CAld5H, a monooxygenase, requires a cytochrome P450 reductase (CPR) as an electron donor. We cloned and expressed three P. trichocarpa CPRs in yeast and show that all are active with both CAld5Hs. The kinetic analysis shows both CAld5Hs have essentially the same biochemical functions. When both CAld5Hs are coexpressed in the same yeast membranes, the resulting enzyme activities are additive, suggesting functional redundancy and independence of these two enzymes. Simulated reaction flux based on Michaelis-Menten kinetics and inhibition kinetics confirmed the redundancy and independence. Subcellular localization of both CAld5Hs as sGFP fusion proteins expressed in P. trichocarpa differentiating xylem protoplasts indicate that they are endoplasmic reticulum resident proteins. These results imply that during wood formation, 5-hydroxylation in monolignol biosynthesis of P. trichocarpa requires the combined metabolic flux of these two CAld5Hs to maintain adequate biosynthesis of syringyl lignin. The combination of genetic analysis, absolute protein quantitation-based enzyme kinetics, homologous CPR specificity, SNP characterization, and ER localization provides a more rigorous basis for a comprehensive systems understanding of 5-hydroxylation in lignin biosynthesis.


Assuntos
Lignina/biossíntese , Oxigenases de Função Mista/metabolismo , Populus/metabolismo , Xilema/enzimologia , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Hidroxilação , Cinética , Lignina/análise , Plantas Geneticamente Modificadas , Leveduras/metabolismo
17.
Anal Bioanal Chem ; 402(2): 983-7, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22071606

RESUMO

A new robust, noninvasive, Raman microspectroscopic method is introduced to analyze the structure of native lignin. Lignin spectra of poplar, Arabidopsis, and Miscanthus were recovered and structural differences were unambiguously detected. Compositional analysis of 4-coumarate-CoA ligase suppressed transgenic poplar showed that the syringyl-to-guaiacyl ratio decreased by 35% upon the mutation. A cell-specific compositional analysis of basal stems of Arabidopsis showed similar distributions of S and G monolignols in xylary fiber cells and interfascicular cells.


Assuntos
Lignina/análise , Lignina/química , Análise Espectral Raman/métodos , Arabidopsis/química , Estrutura Molecular
18.
Anal Chem ; 83(18): 7020-6, 2011 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-21851065

RESUMO

As a major component in plant cell walls, lignin is an important factor in numerous industrial processes, especially in wood saccharification and fermentation to biofuels. The ability to chemically differentiate and spatially locate lignins in wood cell structures provides an important contribution to the effort to improve these processes. The spatial distribution of the syringyl (S) and guaiacyl (G) lignins, both over larger regions and within a single cell wall, on poplar ( Populus trichocarpa ) wood cross-sections was determined via time-of-flight secondary ion mass spectrometry (ToF-SIMS). This is the first time that direct chemically specific mass spectrometric mapping has been employed to elucidate the spatial distribution of S and G lignins. In agreement with results obtained by UV microscopy, ToF-SIMS images clearly show that the guaiacyl lignin is predominantly located in the vessel cell walls of poplar wood while syringyl lignin is mainly located in the fiber cell walls. The G/S ratio in vessel cell walls was determined to be approximately twice that found in fiber cell walls. A combination of Bi ToF-SIMS spectral image acquisition and C(60) sputtering provided the ability to attain the combination of spatial resolution and signal-to-noise necessary to determine the distribution of S and G lignins in a single cell wall. By this technique, it was possible to demonstrate that more guaiacyl lignin is located in the middle lamella layer and more syringyl lignin is located in the inner cell wall area.


Assuntos
Lignina/análise , Populus/química , Espectrometria de Massa de Íon Secundário/métodos , Parede Celular/química , Fulerenos/química , Caules de Planta/química
19.
Plant Cell Physiol ; 51(1): 144-63, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19996151

RESUMO

As a step toward a comprehensive description of lignin biosynthesis in Populus trichocarpa, we identified from the genome sequence 95 phenylpropanoid gene models in 10 protein families encoding enzymes for monolignol biosynthesis. Transcript abundance was determined for all 95 genes in xylem, leaf, shoot and phloem using quantitative real-time PCR (qRT-PCR). We identified 23 genes that most probably encode monolignol biosynthesis enzymes during wood formation. Transcripts for 18 of the 23 are abundant and specific to differentiating xylem. We found evidence suggesting functional redundancy at the transcript level for phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL), p-hydroxycinnamoyl-CoA:quinate shikimate p-hydroxycinnamoyltransferase (HCT), caffeoyl-CoA O-methyltransferase (CCoAOMT) and coniferyl aldehyde 5-hydroxylase (CAld5H). We carried out an enumeration-based motif identification and discriminant analysis on the promoters of all 95 genes. Five core motifs correctly discriminate the 18 xylem-specific genes from the 77 non-xylem genes. These motifs are similar to promoter elements known to regulate phenylpropanoid gene expression. This work suggests that genes in monolignol biosynthesis are regulated by multiple motifs, often related in sequence.


Assuntos
Vias Biossintéticas/genética , Lignina/biossíntese , Lignina/genética , Populus/genética , Populus/metabolismo , RNA de Plantas/genética , Motivos de Aminoácidos/genética , Enzimas/biossíntese , Enzimas/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Genoma de Planta/genética , Floema/enzimologia , Floema/genética , Brotos de Planta/enzimologia , Brotos de Planta/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/análise , RNA de Plantas/metabolismo , Transcrição Gênica/fisiologia , Xilema/enzimologia , Xilema/genética
20.
BMC Plant Biol ; 9: 124, 2009 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-19788757

RESUMO

BACKGROUND: The monolignol biosynthetic pathway interconnects with the biosynthesis of other secondary phenolic metabolites, such as cinnamic acid derivatives, flavonoids and condensed tannins. The objective of this study is to evaluate whether genetic modification of the monolignol pathway in silver birch (Betula pendula Roth.) would alter the metabolism of these phenolic compounds and how such alterations, if exist, would affect the ectomycorrhizal symbiosis. RESULTS: Silver birch lines expressing quaking aspen (Populus tremuloides L.) caffeate/5-hydroxyferulate O-methyltransferase (PtCOMT) under the 35S cauliflower mosaic virus (CaMV) promoter showed a reduction in the relative expression of a putative silver birch COMT (BpCOMT) gene and, consequently, a decrease in the lignin syringyl/guaiacyl composition ratio. Alterations were also detected in concentrations of certain phenolic compounds. All PtCOMT silver birch lines produced normal ectomycorrhizas with the ectomycorrhizal fungus Paxillus involutus (Batsch: Fr.), and the formation of symbiosis enhanced the growth of the transgenic plants. CONCLUSION: The down-regulation of BpCOMT in the 35S-PtCOMT lines caused a reduction in the syringyl/guaiacyl ratio of lignin, but no significant effect was seen in the composition or quantity of phenolic compounds that would have been caused by the expression of PtCOMT under the 35S or UbB1 promoter. Moreover, the detected alterations in the composition of lignin and secondary phenolic compounds had no effect on the interaction between silver birch and P. involutus.


Assuntos
Betula/metabolismo , Lignina/biossíntese , Micorrizas/fisiologia , Fenóis/metabolismo , Betula/genética , Betula/microbiologia , Regulação da Expressão Gênica de Plantas , Metiltransferases/genética , Metiltransferases/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Populus/genética , RNA de Plantas/genética , Análise de Sequência de DNA , Simbiose , Taninos/biossíntese
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