RESUMO
Lignin is an abundant polymer in plant secondary cell walls. Prototypical lignins derive from the polymerization of monolignols (hydroxycinnamyl alcohols), mainly coniferyl and sinapyl alcohol, via combinatorial radical coupling reactions and primarily via the endwise coupling of a monomer with the phenolic end of the growing polymer. Hydroxycinnamaldehyde units have long been recognized as minor components of lignins. In plants deficient in cinnamyl alcohol dehydrogenase, the last enzyme in the monolignol biosynthesis pathway that reduces hydroxycinnamaldehydes to monolignols, chain-incorporated aldehyde unit levels are elevated. The nature and relative levels of aldehyde components in lignins can be determined from their distinct and dispersed correlations in 2D 1H-13C-correlated nuclear magnetic resonance (NMR) spectra. We recently became aware of aldehyde NMR peaks, well resolved from others, that had been overlooked. NMR of isolated low-molecular-weight oligomers from biomimetic radical coupling reactions involving coniferaldehyde revealed that the correlation peaks belonged to hydroxycinnamaldehyde-derived benzofuran moieties. Coniferaldehyde 8-5-coupling initially produces the expected phenylcoumaran structures, but the derived phenolic radicals undergo preferential disproportionation rather than radical coupling to extend the growing polymer. As a result, the hydroxycinnamaldehyde-derived phenylcoumaran units are difficult to detect in lignins, but the benzofurans are now readily observed by their distinct and dispersed correlations in the aldehyde region of NMR spectra from any lignin or monolignol dehydrogenation polymer. Hydroxycinnamaldehydes that are coupled to coniferaldehyde can be distinguished from those coupled with a generic guaiacyl end-unit. These benzofuran peaks may now be annotated and reported and their structural ramifications further studied.
Assuntos
Acroleína/análogos & derivados , Benzofuranos , Cinamatos , Lignina , Lignina/metabolismo , Aldeídos , PolímerosRESUMO
The biosynthesis of cellulose, lignin, and hemicelluloses in plant secondary cell walls (SCWs) is regulated by a hierarchical transcriptional regulatory network. This network features orthologous transcription factors shared between poplar and Arabidopsis, highlighting a foundational similarity in their genetic regulation. However, knowledge on the discrepant behavior of the transcriptional-level molecular regulatory mechanisms between poplar and Arabidopsis remains limited. In this study, we investigated the function of PagMYB128 during wood formation and found it had broader impacts on SCW formation compared to its Arabidopsis ortholog, AtMYB103. Transgenic poplar trees overexpressing PagMYB128 exhibited significantly enhanced xylem development, with fiber cells and vessels displaying thicker walls, and an increase in the levels of cellulose, lignin, and hemicelluloses in the wood. In contrast, plants with dominant repression of PagMYB128 demonstrated the opposite phenotypes. RNA sequencing and reverse transcription - quantitative polymerase chain reaction showed that PagMYB128 could activate SCW biosynthetic gene expression, and chromatin immunoprecipitation along with yeast one-hybrid, and effector-reporter assays showed this regulation was direct. Further analysis revealed that PagSND1 (SECONDARY WALL-ASSOCIATED NAC-DOMAIN PROTEIN1) directly regulates PagMYB128 but not cell wall metabolic genes, highlighting the pivotal role of PagMYB128 in the SND1-driven regulatory network for wood development, thereby creating a feedforward loop in SCW biosynthesis.
Assuntos
Parede Celular , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas , Populus , Madeira , Populus/genética , Populus/metabolismo , Populus/crescimento & desenvolvimento , Parede Celular/metabolismo , Madeira/crescimento & desenvolvimento , Madeira/genética , Madeira/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Xilema/metabolismo , Xilema/genética , Lignina/biossíntese , Lignina/metabolismo , Plantas Geneticamente Modificadas , Genes de Plantas , Celulose/biossíntese , Celulose/metabolismoRESUMO
The complexity of lignin structure impedes efficient cell wall digestibility. Native lignin is composed of a mixture of three dominant monomers, coupled together through a variety of linkages. Work over the past few decades has demonstrated that lignin composition can be altered through a variety of mutational and transgenic approaches such that the polymer is derived almost entirely from a single monomer. In this study, we investigated changes to lignin structure and digestibility in Arabidopsis thaliana in near-single-monolignol transgenics and mutants and determined whether novel monolignol conjugates, produced by a FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) or a p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (PMT), could be integrated into these novel polymers to further improve saccharification efficiency. Monolignol conjugates, including a new conjugate of interest, p-coumaryl p-coumarate, were successfully integrated into high-H, high-G and high-S lignins in A. thaliana. Regardless of lignin composition, FMT- and PMT-expressing plants produced monolignol ferulates and monolignol p-coumarates, respectively, and incorporated them into their lignin. Through the production and incorporation of monolignol conjugates into near-single-monolignol lignins, we demonstrated that substrate availability, rather than monolignol transferase substrate preference, is the most important determining factor in the production of monolignol conjugates, and lignin composition helps dictate cell wall digestibility.
Assuntos
Arabidopsis , Lignina , Arabidopsis/metabolismo , Parede Celular/metabolismo , Lignina/metabolismo , Transferases/análise , Transferases/metabolismoRESUMO
Lignocellulosic biomass is mainly composed of polysaccharides and lignin. The complexity and diversity of the plant cell wall polymers makes it difficult to isolate the components in pure form for characterization. Many current approaches to analyzing the structure of lignocellulose, which involve sequential extraction and characterization of the resulting fractions, are time-consuming and labor-intensive. The present study describes a new and facile system for rationally derivatizing and dissolving coarsely ground plant cell wall materials. Using ionic liquids (EmimAc) and dichloroacetyl chloride as a solvent/reagent produced mildly acetylated whole cell walls without significant degradation. The acetylated products were soluble in DMSO-d6 from which they can be characterized by solution-state two-dimensional nuclear magnetic resonance (2D NMR) spectrometry. A distinct advantage of the procedure is that it realizes the dissolution of whole lignocellulosic materials without requiring harsh ball milling, thereby allowing the acquisition of high-resolution 2D NMR spectra to revealing structural details of the main components (lignin and polysaccharides). The method is therefore beneficial to understanding the composition and structure of biomass aimed at its improved utilization.
Assuntos
Parede Celular/química , Dimetil Sulfóxido/química , Líquidos Iônicos/química , Lignina/análise , Polissacarídeos/análise , Populus/química , Acetatos/química , Acetilação , Espectroscopia de Ressonância Magnética , Populus/citologia , Solubilidade , SoluçõesRESUMO
The function of the transcription factor KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) is still unclear since it appears to be either a negative or a positive regulator for secondary cell wall deposition with its loss-of-function mutant displaying thicker interfascicular and xylary fiber cell walls but thinner vessel cell walls in inflorescence stems. To explore the exact function of KNAT7, class II KNOTTED1-LIKE HOMEOBOX (KNOX II) genes in Arabidopsis including KNAT3, KNAT4, and KNAT5 were studied together. By chimeric repressor technology, we found that both KNAT3 and KNAT7 repressors exhibited a similar dwarf phenotype. Both KNAT3 and KNAT7 genes were expressed in the inflorescence stems and the knat3 knat7 double mutant exhibited a dwarf phenotype similar to the repressor lines. A stem cross-section of knat3 knat7 displayed an enhanced irregular xylem phenotype as compared with the single mutants, and its cell wall thickness in xylem vessels and interfascicular fibers was significantly reduced. Analysis of cell wall chemical composition revealed that syringyl lignin was significantly decreased while guaiacyl lignin was increased in the knat3 knat7 double mutant. Coincidently, the knat3 knat7 transcriptome showed that most lignin pathway genes were activated, whereas the syringyl lignin-related gene Ferulate 5-Hydroxylase (F5H) was down-regulated. Protein interaction analysis revealed that KNAT3 and KNAT7 can form a heterodimer, and KNAT3, but not KNAT7, can interact with the key secondary cell wall formation transcription factors NST1/2, which suggests that the KNAT3-NST1/2 heterodimer complex regulates F5H to promote syringyl lignin synthesis. These results indicate that KNAT3 and KNAT7 synergistically work together to promote secondary cell wall biosynthesis.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Lignina , Proteínas Nucleares , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genéticaRESUMO
Stretchable and compressible hydrogels based on natural polymers have received immense considerations for electronics. The feasibility of using pure natural polymer-based hydrogels could be improved if their mechanical behaviors satisfy the requirements of practical applications. Herein, we report highly stretchable (tensile strain â¼126%) and compressible (compression strain â¼80%) cellulose ionic hydrogels (CIHs) among pure natural polymer-based hydrogels including cellulose, chitin, and chitosan via chemical cross-linking based on free radical polymerization of allyl cellulose in NaOH/urea aqueous solution. In addition, the hydrogels have good transparency (transmittance of â¼89% at 550 nm) and ionic conductivity (â¼0.16 mS cm-1) and can be worked at -20 °C without freezing and visual loss of transparency. Moreover, the CIHs can serve as reliable and stable strain sensors and have been successfully used to monitor human activities. Significantly, the various properties of hydrogel can be controlled through rationally adjusting the chemically cross-linked density. Our methodology will prove useful in developing the satisfied mechanical and transparent CIHs for a myriad of applications in flexible electronics.
Assuntos
Celulose/análogos & derivados , Força Compressiva , Hidrogéis/química , Resistência à Tração , Quitosana/análogos & derivados , Elastômeros/química , Condutividade ElétricaRESUMO
Conifers (softwoods) naturally lack syringyl units in their lignins, rendering lignocellulosic materials from such species more difficult to process than syringyl-rich hardwood species. Using a transformable Pinus radiata tracheary element (TE) system as an experimental platform, we investigated whether metabolic engineering can be used to create syringyl lignin in conifers. Pyrolysis-GC/MS and 2D-NMR analysis of P. radiata TE cultures transformed to express ferulate 5-hydroxylase (F5H) and caffeic acid O-methyltransferase (COMT) from Liquidambar styraciflua confirmed the production and incorporation of sinapyl alcohol into the lignin polymer. Transformation with F5H was sufficient for the production of syringyl lignin in TEs, but cotransformation with COMT improved its formation. In addition, lower levels of the pathway intermediate 5-hydroxyconiferyl alcohol were evidenced in cotransformation experiments, indicating that the introduction of the COMT overcame the inefficiency of the native pine methyltransferases for supporting sinapyl alcohol production.Our results provide the proof of concept that it is possible to generate a lignin polymer that contains syringyl units in softwood species such as P. radiata, suggesting that it might be possible to retain the outstanding fiber properties of softwoods while imbuing them with the lignin characteristics of hardwoods that are more favorable for industrial processing.
Assuntos
Álcoois/química , Lignina/biossíntese , Engenharia Metabólica , Biocombustíveis , Biomassa , Parede Celular/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Regulação da Expressão Gênica de Plantas , Espectroscopia de Ressonância Magnética , Pinus , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Polímeros/química , Traqueófitas , TransgenesRESUMO
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 ß-1,3-glucan synthesis in vivo and in vitro, apparently by directly binding ß-1,3-glucan. Through its activity on the glucan layer, poacic acid inhibits growth of the fungi Sclerotinia sclerotiorum and Alternaria solani as well as the oomycete Phytophthora sojae. A single application of poacic acid to leaves infected with the broad-range fungal pathogen S. sclerotiorum substantially reduced lesion development. The discovery of poacic acid as a natural antifungal agent targeting ß-1,3-glucan highlights the potential side use of products generated in the processing of renewable biomass toward biofuels as a source of valuable bioactive compounds and further clarifies the nature and mechanism of fermentation inhibitors found in lignocellulosic hydrolysates.
Assuntos
Ácidos Cumáricos/química , Fungicidas Industriais/química , Poaceae/química , Saccharomyces cerevisiae/efeitos dos fármacos , Estilbenos/química , beta-Glucanas/química , Caspofungina , Membrana Celular/metabolismo , Parede Celular/metabolismo , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Equinocandinas/química , Genômica , Hidrólise , Concentração Inibidora 50 , Lignina/química , Lipopeptídeos , Extratos Vegetais/química , Saccharomyces cerevisiae/metabolismoRESUMO
Chinese Angelica is a significant medical plant due to the various therapeutic constituents in its root; whereas the aerial part is considered worthless and often discarded as agricultural waste. In this work, phytochemicals from the stem were first systematically analyzed by means of GCâ»MS after derivatization and HPLCâ»MS/MS in multiple reaction monitoring (MRM) mode. Phthalides, ferulic acid, and coniferyl ferulate were detected in the stem; although their content is relatively low in comparison with the root. Some specific compounds, such as p-hydroxybenzoic acid, vanillic acid, protocatechuic acid, caffeic acid, 4-hydroxyphenyl-1, 2-ethanediol, thymol-ß-d-glucopyranoside, etc. and a significant amount of phytosterols (1.36 mg/g stem, mainly ß-sitosterol) were detected in the stem. The extracted oil from the stem contained a considerable amount of phthalides (48.5 mg/g), ß-sitosterol (56.21 mg/g), and stigmasterol (14.03 mg/g); no other bioactive compounds were found that could be potentially used as pharmaceuticals or additives to healthcare food.
Assuntos
Angelica/química , Benzofuranos/química , Fitosteróis/química , Extratos Vegetais/química , Caules de Planta/química , Benzofuranos/isolamento & purificação , Benzofuranos/farmacologia , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Estrutura Molecular , Fitosteróis/isolamento & purificação , Fitosteróis/farmacologia , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Raízes de Plantas/químicaRESUMO
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'-O-ß)-ether bonds and the degradation of tricin under the corresponding reaction conditions were evaluated. A hexadeuterated 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 Orchidaceae (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.
Assuntos
Flavonoides/metabolismo , Lignina/metabolismo , Filogenia , Cromatografia Líquida , Espectrometria de Massas , Poaceae/classificação , Poaceae/metabolismoRESUMO
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 ß-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 ß-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.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Lignina/metabolismo , Sphingomonas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação da Expressão Gênica de Plantas , Engenharia Genética/métodos , Glucose/metabolismo , Lignina/química , Espectroscopia de Ressonância Magnética , Redes e Vias Metabólicas/genética , Plantas Geneticamente Modificadas/genéticaRESUMO
Lignin is an abundant aromatic plant cell wall polymer consisting of phenylpropanoid units in which the aromatic rings display various degrees of methoxylation. Tricin [5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4H-chromen-4-one], a flavone, was recently established as a true monomer in grass lignins. To elucidate the incorporation pathways of tricin into grass lignin, the metabolites of maize (Zea mays) were extracted from lignifying tissues and profiled using the recently developed 'candidate substrate product pair' algorithm applied to ultra-high-performance liquid chromatography and Fourier transform-ion cyclotron resonance-mass spectrometry. Twelve tricin-containing products (each with up to eight isomers), including those derived from the various monolignol acetate and p-coumarate conjugates, were observed and authenticated by comparisons with a set of synthetic tricin-oligolignol dimeric and trimeric compounds. The identification of such compounds helps establish that tricin is an important monomer in the lignification of monocots, acting as a nucleation site for starting lignin chains. The array of tricin-containing products provides further evidence for the combinatorial coupling model of general lignification and supports evolving paradigms for the unique nature of lignification in monocots.
Assuntos
Flavonas/metabolismo , Flavonoides/metabolismo , Lignina/metabolismo , Zea mays/metabolismo , Acilação , Vias Biossintéticas , Parede Celular/química , Parede Celular/metabolismo , Flavonas/química , Flavonoides/química , Lignina/química , Polímeros/química , Polímeros/metabolismo , Zea mays/químicaRESUMO
Plant metabolomics is increasingly used for pathway discovery and to elucidate gene function. However, the main bottleneck is the identification of the detected compounds. This is more pronounced for secondary metabolites as many of their pathways are still underexplored. Here, an algorithm is presented in which liquid chromatography-mass spectrometry profiles are searched for pairs of peaks that have mass and retention time differences corresponding with those of substrates and products from well-known enzymatic reactions. Concatenating the latter peak pairs, called candidate substrate-product pairs (CSPP), into a network displays tentative (bio)synthetic routes. Starting from known peaks, propagating the network along these routes allows the characterization of adjacent peaks leading to their structure prediction. As a proof-of-principle, this high-throughput cheminformatics procedure was applied to the Arabidopsis thaliana leaf metabolome where it allowed the characterization of the structures of 60% of the profiled compounds. Moreover, based on searches in the Chemical Abstract Service database, the algorithm led to the characterization of 61 compounds that had never been described in plants before. The CSPP-based annotation was confirmed by independent MS(n) experiments. In addition to being high throughput, this method allows the annotation of low-abundance compounds that are otherwise not amenable to isolation and purification. This method will greatly advance the value of metabolomics in systems biology.
Assuntos
Arabidopsis/metabolismo , Cromatografia de Fase Reversa , Metabolômica , Espectrometria de Massas por Ionização por Electrospray , Especificidade por SubstratoRESUMO
Phenylcoumaran benzylic ether reductase (PCBER) is one of the most abundant proteins in poplar (Populus spp) xylem, but its biological role has remained obscure. In this work, metabolite profiling of transgenic poplar trees downregulated in PCBER revealed both the in vivo substrate and product of PCBER. Based on mass spectrometry and NMR data, the substrate was identified as a hexosylated 8-5-coupling product between sinapyl alcohol and guaiacylglycerol, and the product was identified as its benzyl-reduced form. This activity was confirmed in vitro using a purified recombinant PCBER expressed in Escherichia coli. Assays performed on 20 synthetic substrate analogs revealed the enzyme specificity. In addition, the xylem of PCBER-downregulated trees accumulated over 2000-fold higher levels of cysteine adducts of monolignol dimers. These compounds could be generated in vitro by simple oxidative coupling assays involving monolignols and cysteine. Altogether, our data suggest that the function of PCBER is to reduce phenylpropanoid dimers in planta to form antioxidants that protect the plant against oxidative damage. In addition to describing the catalytic activity of one of the most abundant enzymes in wood, we provide experimental evidence for the antioxidant role of a phenylpropanoid coupling product in planta.
Assuntos
Oxirredutases/metabolismo , Populus/enzimologia , Xilema/enzimologia , Aminoácidos/metabolismo , Parede Celular/metabolismo , Cisteína/metabolismo , Regulação para Baixo , Ensaios Enzimáticos , Immunoblotting , Lignanas/biossíntese , Lignanas/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Oxirredução , Estresse Oxidativo , Oxirredutases/química , Fenótipo , Plantas Geneticamente Modificadas , Reprodutibilidade dos Testes , Especificidade por SubstratoRESUMO
Tricin was recently discovered in lignin preparations from wheat (Triticum aestivum) 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-ß-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-ß-coupling with sinapyl alcohol and then coniferyl alcohol. Tricin was also found to cross couple with monolignols to form tricin-(4'-O-ß)-linked dimers in biomimetic oxidations using peroxidase/hydrogen peroxide or silver (I) oxide. Nuclear magnetic resonance characterization of gel permeation chromatography-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 with lignification reactions, is an authentic lignin monomer, and, because it can only start a lignin chain, functions as a nucleation site for lignification in monocots. This initiation role helps resolve a long-standing dilemma that monocot lignin chains do not appear to be initiated by monolignol homodehydrodimerization as they are in dicots that have similar syringyl-guaiacyl compositions. The term flavonolignin is recommended for the racemic oligomers and polymers of monolignols that start from tricin (or incorporate other flavonoids) in the cell wall, in analogy with the existing term flavonolignan that is used for the low-molecular mass compounds composed of flavonoid and lignan moieties.
Assuntos
Flavonoides/metabolismo , Lignina/metabolismo , Triticum/química , Zea mays/química , Acetilação , Vias Biossintéticas , Parede Celular/metabolismo , Flavonoides/síntese química , Flavonoides/química , Lignina/química , Espectroscopia de Ressonância Magnética , Peso Molecular , Fenóis/química , Fenóis/metabolismo , Polímeros/metabolismo , Triticum/metabolismo , Zea mays/metabolismoRESUMO
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.
Assuntos
Arabidopsis/metabolismo , Ácidos Cumáricos/metabolismo , Lignina/metabolismo , Populus/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Arabidopsis/genética , Parede Celular/genética , Parede Celular/metabolismo , Cromatografia Gasosa , Ácidos Cumáricos/química , Lignina/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Engenharia Metabólica/métodos , Oryza/enzimologia , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Populus/genética , Propionatos , Reprodutibilidade dos TestesRESUMO
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 ß-O-4-, ß-ß-, and ß-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.
Assuntos
Reagentes de Ligações Cruzadas/química , Lignina , Modelos Químicos , Picea/química , Pinus taeda/química , Madeira/química , Lignina/síntese química , Lignina/química , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Solventes/químicaRESUMO
Wood shortages, environmental pollution and high energy consumption remain major obstacles hindering the development of today's pulp and paper industry. Energy-saving and environmental friendly pulping processes are still needed, especially for non-woody materials. In this study, soda-oxygen pulping of bagasse was investigated and a successful soda-oxygen pulping process for bagasse at 100 °C was established. The pulping parameters of choice were under active alkali charge of 23%, maximum cooking temperature 100 °C, time hold at maximum temperature 180 min, initial pressure of oxygen 0.6 MPa, MgSO4 charge 0.5%, and de-pithed bagasse consistency 12%. Properties of the resultant pulp were screened yield 60.9%, Kappa number 14, viscosity 766 dm³/kg, and brightness 63.7% ISO. Similar pulps were also obtained at 110 °C or 105 °C with a cooking time of 90 min. Compared with pulps obtained at higher temperatures (115-125 °C), this pulp had higher screened yield, brightness, and acceptable viscosity, while the delignification degree was moderate. These results indicated that soda-oxygen pulping at 100 °C, the lowest cooking temperature reported so far for soda-oxygen pulping, is a suitable process for making chemical pulp from bagasse. Pulping at lower temperature and using oxygen make it an environmental friendly and energy-saving pulping process.
Assuntos
Celulose/química , Química Verde/métodos , Indústria Manufatureira/métodos , Papel , Álcalis/química , Temperatura Alta , Humanos , Oxigênio/química , Pressão , ViscosidadeRESUMO
Glutathione-dependent enzymes play important protective, repair, or metabolic roles in cells. In particular, enzymes in the glutathione S-transferase (GST) superfamily function in stress responses, defense systems, or xenobiotic detoxification. Here, we identify novel features of bacterial GSTs that cleave ß-aryl ether bonds typically found in plant lignin. Our data reveal several original features of the reaction cycle of these GSTs, including stereospecific substrate recognition and stereoselective formation of ß-S-thioether linkages. Products of recombinant GSTs (LigE, LigP, and LigF) are ß-S-glutathionyl-α-keto-thioethers that are degraded by a ß-S-thioetherase (LigG). All three Lig GSTs produced the ketone product (ß-S-glutathionyl-α-veratrylethanone) from an achiral side chain-truncated model substrate (ß-guaiacyl-α-veratrylethanone). However, when ß-etherase assays were conducted with a racemic model substrate, ß-guaiacyl-α-veratrylglycerone, LigE- or LigP-catalyzed reactions yielded only one of two potential product (ß-S-glutathionyl-α-veratrylglycerone) epimers, whereas the other diastereomer (differing in configuration at the ß-position (i.e. its ß-epimer)) was produced only in the LigF-catalyzed reaction. Thus, ß-etherase catalysis causes stereochemical inversion of the chiral center, converting a ß(R)-substrate to a ß(S)-product (LigE and LigP), and a ß(S)-substrate to a ß(R)-product (LigF). Further, LigG catalyzed glutathione-dependent ß-S-thioether cleavage with ß-S-glutathionyl-α-veratrylethanone and with ß(R)-configured ß-S-glutathionyl-α-veratrylglycerone but exhibited no or significantly reduced ß-S-thioether-cleaving activity with the ß(S)-epimer, demonstrating that LigG is a stereospecific ß-thioetherase. We therefore propose that multiple Lig enzymes are needed in this ß-aryl etherase pathway in order to cleave the racemic ß-ether linkages that are present in the backbone of the lignin polymer.
Assuntos
Proteínas de Bactérias/metabolismo , Glutationa/metabolismo , Oxirredutases/metabolismo , Transdução de Sinais , Sphingomonadaceae/enzimologia , Lignina/química , Lignina/metabolismo , Proteínas Recombinantes/metabolismo , Sphingomonadaceae/química , Sphingomonadaceae/metabolismo , Estereoisomerismo , Especificidade por Substrato , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismoRESUMO
Grass lignins contain substantial amounts of p-coumarate (pCA) that acylate the side-chains of the phenylpropanoid polymer backbone. An acyltransferase, named p-coumaroyl-CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol-pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages; however p-coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was involved specifically in monolignol acylation. A sodium azide-generated Bdpmt-1 missense mutant had no (<0.5%) residual pCA on lignin, and BdPMT RNAi plants had levels as low as 10% of wild-type, whereas the amounts of pCA acylating arabinosyl units on arabinoxylans in these PMT mutant plants remained unchanged. pCA acylation of lignin from BdPMT-overexpressing plants was found to be more than three-fold higher than that of wild-type, but again the level on arabinosyl units remained unchanged. Taken together, these data are consistent with a defined role for grass PMT genes in encoding BAHD (BEAT, AHCT, HCBT, and DAT) acyltransferases that specifically acylate monolignols with pCA and produce monolignol p-coumarate conjugates that are used for lignification in planta.