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The mhqPOD gene cluster in lignin-degrading Paenibacillus sp. B2 encodes a pathway for the degradation of lignin-derived 5,5'-di(dehydrovanillic acid) (DDVA).

Lignin-degrading bacteria Paenibacillus sp. B2, Agrobacterium sp. B1, and Ochrobactrum sp. each contain mhqO genes encoding ring cleavage dioxygenase enzymes whose biochemical function is unknown. Each of these strains was found to degrade the biphenyl-containing lignin fragment 5,5'-di(dehydrovanillic acid) (DDVA) on solid media. An operon of five mhq genes in Paenibacillus sp. B2 was analysed via gene expression using quantitative PCR, and all five genes were highly induced (400-1000-fold overexpression) by the presence of DDVA. Recombinant azoreductase MhqP was found to demethylate DDVA to its monodemethylated derivative. Hence, these genes are proposed to be responsible for DDVA degradation, via a pathway involving the same biochemical steps as that studied in Sphingobium lignivorans SYK-6, but using several unrelated genes. Decarboxylation of later pathway intermediate 5-carboxyvanillic acid in Paenibacillus sp. B2 is proposed to be catalysed by decarboxylase UbiD, whose gene is also upregulated in the presence of DDVA. Degradation of the other fragment 4-carboxy-2-hydroxypentadienoic acid is proposed to occur via hydratase UxuA, whose gene is also upregulated by DDVA, and 4-hydroxy-4-methyl-2-oxoglutarate aldolase.

Paenibacillus

Bacterial decomposition of synthetic 14C-labeled lignin and lignin monomer derivatives.

Nocardia sp. which was isolated from soil is capable of degrading synthetic lignin and utilizing its monomer derivatives. Decomposition was monitored by measuring the 14CO2 evolved and O2 consumed, when the bacterium was grown on a medium containing specifically 14C-labeled ligning or monomer phenolic compounds as major carbon source. The time course of the 14CO2 release and O2 uptake indicates a significant depolymerization and utilization of lignin by the Nocardia sp.

Anisoles

Addressing lignin composition and content via Arabidopsis arogenate dehydratase knockout and over-expression genotypes.

Following the down-selection of 14 Arabidopsis thaliana arogenate dehydratase (ADT) knockout and over-expression (OE) genotypes, the most highly contrasting quadruple knockout adt3/4/5/6 and ADT OE genotypes were subjected to proteomics, metabolomics, and scanning electron microscopy (SEM) analyses as needed, with results compared to Columbia wild-type (WT). The basal adt3/4/5/6 stem cross-sections, ∼70% lignin content reduced, exhibited buckled vessel cell walls and partially detached xylary fibers, in contrast to WT and ADT4m/5 m OE genotypes that did not. Anatomical defects primarily resulted from guaiacyl lignin level reductions in vessels with concomitant increased stem syringyl:guaiacyl (S/G) ratios. Phenylpropanoid and various upstream shikimate-chorismate pathway enzyme abundances, as well as specific monolignol oxidases (laccases/peroxidases), generally increased in adt3/4/5/6 at different stem and rosette leaf growth/development stages, relative to WT. Opposite effects were largely observed with the ADT5m OE genotype. By contrast, flavonoid and glucosinolate pathway enzyme amounts varied. Such enzyme abundance increases were overall unproductive as adt3/4/5/6 was unable to restore WT, ADT4 OE, ADT5 OE, ADT5m OE, and ADT4m/5 m OE secondary metabolite (lignin, phenylpropanoid, lignan, flavonoid, phenolic acid, and glucosinolate) levels. Conversely, ADT OE genotypes did not significantly increase programmed lignin levels or alter S/G compositions. In sum, proteomics analyses of adt3/4/5/6 and adt5 'perceived' that lignin and low molecular weight secondary metabolite amounts were not at 'programmed' levels as for WT and ADT OE genotypes but observed increases in relevant pathway protein abundances were futile. Notably though, proteomics analyses did not lead to predicting that lignin and associated biochemical pathways would have reduced metabolite levels, relative to WT and ADT OE genotypes. Genotype adt3/4/5/6, possibly the highest lignin level reduced genotype reported, did not utilize other phenolics to compensate. By contrast, the differential temporal and spatial deposition of cell wall oxidases again indicate the exquisite control over lignin deposition, and our lack of knowledge of precise lignin structure and assembly in subcellular regions of the lignified cell walls.

Lignin

GA4+7 alleviates pear fruit semi-russeting partly by suppressing PRX-mediated lignin deposition.

Pear fruit semi-russeting is a surface disorder that frequently occurs during fruit development and significantly diminishes fruit appearance quality and commercial value. Although Gibberellin 4 + 7 (GA4+7) has been used to reduce fruit surface defects in horticultural crops, the physiological and molecular mechanisms underlying its inhibitory effect on pear fruit semi-russeting remain poorly understood. In this study, preharvest GA4+7 treatment of 'Cuiguan' pear significantly reduced russet coverage and lignin accumulation in mature fruit skin without adversely affecting fruit size, fruit shape index, or total soluble solids content. Integrated metabolomic and transcriptomic analyses revealed that GA4+7 treatment was associated with the repression of phenylpropanoid and lignin biosynthesis at both metabolic and transcriptional levels. Among the lignin-related differentially expressed genes, two class III peroxidase genes, PpyPRX22 and PpyPRX65, were strongly downregulated by both GA4+7 and bagging treatments. Both proteins localized to the cell wall, and transient expression assays in pear fruit skin supported positive roles for PpyPRX22 and PpyPRX65 in lignin deposition. Furthermore, dual-luciferase reporter assays combined with transient overexpression experiments suggested that several PpyMYB transcription factors may regulate PpyPRX expression and lignin accumulation, with PpyMYB138 and PpyMYB139 significantly activating PpyPRX22 and/or PpyPRX65 promoter activity. Taken together, these results suggest that GA4+7 alleviates pear fruit semi-russeting at least partly by reducing lignin deposition in the fruit skin, with PpyPRX22 and PpyPRX65 potentially contributing to this process.

Class III peroxidase

Factors underlying a latitudinal gradient in the S/G lignin monomer ratio in natural poplar variants.

The chemical composition of wood plays a pivotal role in the adaptability and structural integrity of trees. However, few studies have investigated the environmental factors that determine lignin composition and its biological significance in plants. Here, we examined the lignin syringyl-to-guaiacyl (S/G) ratio in members of a Populus trichocarpa population sourced from their native habitat and conducted a genome wide association study to identify genes linked to lignin formation. Our results revealed many significant associations, suggesting that lignin biosynthesis is a complex polygenic trait. Additionally, we found an increase in the S/G ratio from northern to southern geographic origin of the trees sampled, along with a corresponding metabolic and transcriptional reprogramming of xylem cell wall biosynthesis. Further molecular analysis identified a mutation in a cell wall laccase genetically associated with higher S/G ratios that predominate in trees from warmer lower latitudes. Collectively, our findings suggest that lignin heterogeneity arises from an evolutionary process enabling poplar adaptation to different climatic challenges.

Populus

Genetic modification of the shikimate pathway to reduce lignin content in switchgrass (Panicum virgatum L.) significantly impacts plant microbiomes.

UNLABELLED: Switchgrass (Panicum virgatum L.) is considered a sustainable biofuel feedstock, given its fast-impact growth, low input requirements, and high biomass yields. Improvements in bioenergy conversion efficiency of switchgrass could be made by reducing its lignin content. Engineered switchgrass that expresses a bacterial 3-dehydroshikimate dehydratase (QsuB) has reduced lignin content and improved biomass saccharification due to the rerouting of the shikimate pathway towards the simple aromatic protocatechuate at the expense of lignin biosynthesis. However, the impacts of this QsuB trait on switchgrass microbiome structure and function remain unclear. To address this, wild-type and QsuB-engineered switchgrass were grown in switchgrass field soils, and samples were collected from inflorescences, leaves, roots, rhizospheres, and bulk soils for microbiome analysis. We investigated how QsuB expression influenced switchgrass-associated fungal and bacterial communities using high-throughput Illumina MiSeq amplicon sequencing of ITS and 16S rDNA. Compared to wild-type, QsuB-engineered switchgrass hosted different microbial communities in roots, rhizosphere, and leaves. Specifically, QsuB-engineered plants had a lower relative abundance of arbuscular mycorrhizal fungi (AMF). Additionally, QsuB-engineered plants had fewer Actinobacteriota in root and rhizosphere samples. These findings may indicate that changes in the plant metabolism impact both AMF and Actinobacteriota similarly or potential interactions between AMF and the bacterial community. This study enhances understanding of plant-microbiome interactions by providing baseline microbial data for developing beneficial bioengineering strategies and by assessing nontarget impacts of engineered plant traits on the plant microbiome. IMPORTANCE: Bioenergy crops provide an important strategy for mitigating climate change. Reducing the lignin in bioenergy crops could improve fermentable sugar yields for more efficient conversion into bioenergy and bioproducts. In this study, we assessed how switchgrass engineered for low lignin impacted aboveground and belowground switchgrass microbiome. Our results show unexpected reductions in mycorrhizas and actinobacteria in belowground tissues, raising questions on the resilience and function of genetically engineered plants in agricultural systems.

Panicum

DfCAD16 controls guaiacyl lignin biosynthesis during shoot development in Dendrocalamus farinosus.

Although bamboo can be an ideal raw material for pulp and paper industry, the depolymerization of its complex polymers needs to be facilitated. The deposition of lignin is influenced by cinnamyl alcohol dehydrogenase (CAD), an enzyme that catalyzes the formation of monolignol precursors. Here, we identified 18 DfCAD genes in Dendrocalamus farinosus and revealed using bioinformatics methods, DfCAD16 functions as the primary enzyme in the synthesis pathway of guaiacyl (G)-lignin. Phenotypic analysis of plants overexpression DfCAD16 exhibited remarkable increasing in G-lignin. Furthermore, we demonstrated that an R2R3-type MYB transcription factor DfMYB12 could directly bind to the promoter region of DfCAD16 and activate its expression both in vitro and in vivo. Our findings revealed that DfMYB12-DfCAD16 is a key regulatory factor governing G-lignin biosynthesis in D. farinosus. These insights can be used for improving bamboo varieties for pulp production.

Lignin

Abscisic acid promotes RBOH-dependent reactive oxygen species production and lignin biosynthesis in pears via the PuABI5-PuMYB169 module.

Pear stone cell lignification, a critical determinant of fruit texture and quality, is regulated by developmental and environmental cues, with abscisic acid (ABA) playing a central role. However, the molecular mechanisms underlying its role in reactive oxygen species (ROS)-mediated lignification remain unclear. Here, we show that PuABI5, a key component in ABA signaling, directly combines with PuMYB169, the master regulator of stone cell lignification, to modulate ROS production and lignin biosynthesis in pear fruit. Exogenous application of ABA enhances H2O2 and lignin accumulation in both pear fruits and calli, and ABA-activated PuABI5 positively regulates stone cell lignification. We demonstrate that ABA-induced PuABI5 binds directly to the PuMYB169 promoter and activates its expression to promote the transcription of PuRBOHF and lignin-related genes, thereby enhancing ROS production and lignin accumulation. Notably, PuABI5 interacted with PuMYB169 to enhance the induction of PuRBOHF expression, leading to elevated levels of H2O2, which feedback to strengthen the interaction between PuABI5 and PuMYB169. Collectively, our findings elucidate that ABA induces ROS-mediated lignification of stone cells in pears by activating the PuABI5-PuMYB169 transcriptional module.

Lignin

Antibiotic properties of lignin components.

Inhibitory effects of compounds with guaiacyl and syringyl structure, representing the structure of native lignin, were studied on model cultures of bacteria, yeasts, yeast-like microorganisms and moulds. Isoeugenol exhibited the most pronounced inhibitory effect on growth of the studied microorganisms.

Anti-Bacterial Agents

Influence of molasses lignin-hemicellulose fractions in rat nutrition.

Previous reports have shown that fractionation of the non-sugar, non-dialyzable components of cane molasses yielded a fraction designated as a black phenolic-carbohydrate complex. Incorporation of 0.03% of this complex into diets fed weanling male rats significantly increased the growth rate above that of rats fed the basal diet alone. This study was conducted to determine the chemical nature and growth stimulating action of the black phenol-carbohydrate complex. Alkaline cleavage under nitrogen yielded a mixture of phenols and a carbohydrate fraction which was recovered by precipitation at pH 6 in four volumes of ethanol. The alkaline cleaved, free hemicellulose was non-diayzable and stimulated growth when incorporated into rat diets at the 0.03% levels. Acid hydrolysis of the complex yielded an insoluble product identified as lignin and found to represent 18% to 20% of the entire complex. The chemical nature of this ligninhemicellulose fraction and the previously reported growth-enhancing acid resistant hemicellulose fraction isolated from various plant sources were found to be similar.

Animal Nutritional Physiological Phenomena

Enzymic synthesis of lignin precursors. Purification and properties of a cinnamoyl-CoA: NADPH reductase from cell suspension cultures of soybean (Glycinemax).

A cinnamoyl-coenzyme A reductase catalyzing the NADPH-dependent reduction of substituted cinnamoyl-CoA thiol esters to the corresponding cinnamaldehydes was isolated from cell suspension cultures of soybean (Glycine max L. var. Mandarin). A 1660-fold purification of the enzyme was achieved by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, hydroxyapatite and Sephadex G-100 and affinity chromatography on 5'-AMP-Sepharose. The apparent molecular weight of the reductase was found to be about 38 000 on the basis of the elution volume from a Sephadex G-100 column. Maximum rate of reaction was observed between pH 6.0 and 6.2 in 0.1-0.2 M citrate buffer at 30 degrees C. The enzyme was markedly inhibited by thiol reagents. The reductase showed a high degree of specificity for cinnamoyl-CoA esters. Feruloyl-CoA was the substrate with the lowest Km value (73 muM) and highest V (230 nkat/mg) followed by 5-hydroxy-feruloyl-CoA, sinapoyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA and cinnamoyl-CoA. No reaction took place with acetyl-CoA. The Km value for NADPH varied with the type of substrate. Km values of 28, 120, and 290 muM were found with feruloyl-CoA, sinapoyl-CoA, and p-coumaroyl-CoA, respectively. The rate of reaction observed with NADH was only about 5% of that found with NADPH. The reaction products CoASH and NADP+ inhibited the reaction. The Ki values were in the range of 0.5-1 mM and the inhibition was of a noncompetitive (mixed) type. The role of the reductase in the biosynthesis of lignin precursors is discussed.

Aldehyde Oxidoreductases

Resistance of Populus davidiana × P. bolleana overexpressing cinnamoyl-CoA reductase gene to Lymantria dispar larvae.

Lignin is a crucial defense phytochemical against phytophagous insects. Cinnamoyl-CoA reductase (CCR) is a key enzyme in lignin biosynthesis. In this study, transgenic Populus davidiana × P. bolleana overexpressing the PdbCCR gene were generated via Agrobacterium-mediated transformation. Successful integration of PdbCCR into the poplar genome was confirmed by PCR amplification and quantitative reverse transcription PCR (qRT-PCR). The lignin content in the transgenic poplar leaves was significantly higher than that in the wild poplar, and after L. dispar larvae fed on the transgenic poplar, the CCR activity was clearly induced. The L. dispar larvae grew slowly after feeding on transgenic poplar and the laccase, cellulase and three detoxifying enzymes were induced compared with larvae after feeding on wild-type poplar. The bioassay further revealed that transgenic poplar plants overexpressing PdbCCR showed a high level of resistance to L. dispar larvae. These results confirmed that PdbCCR is a candidate gene for breeding insect resistant poplar.

Populus

Integrated multi-omics analyses provide new insights into genomic variation landscape and regulatory network candidate genes associated with walnut endocarp.

Persian walnut (Juglans regia) is an economically important nut oil tree; the fruit has a hard endocarp/shell to protect seeds, thus playing a key role in its evolution, and the shell thickness is an important trait for walnut breeding. However, the genomic landscape and the gene regulatory networks associated with walnut shell development remain to be systematically elucidated. Here, we report a high-quality genome assembly of the walnut cultivar 'Xiangling' and construct a graphic structure pan-genome of eight Juglans species to reveal the genetic variations at the genome level. We re-sequence 285 accessions to characterize the genomic variation landscape. Through genome-wide association studies (GWAS), we identified 19 loci associated with more than 268 loci that underwent selection during walnut domestication and improvement. Multi-omics analyses, including transcriptomics, metabolomics, DNA methylation, and spatial transcriptomics across eleven developmental stages, revealed several candidate genes related to secondary cell biosynthesis and lignin accumulation. This integrated multi-omics approach revealed several candidate genes associated with secondary cell biosynthesis and lignin accumulation, such as UGP, MYB308, MYB83, NAC043, NAC073, CCoAOMT1, CCoAOMT7, CHS2, CESA7, LAC7, COBL4, and IRX12. Overexpression of JrUGP and JrMYB308 in Arabidopsis thaliana confirmed their roles in lignin biosynthesis and cell wall thickening. Consequently, our comprehensive multi-omics findings offer novel insights into walnut genetic variation and network regulation of endocarp development and shell thickness, which enable further genome-informed breeding strategies for walnut cultivar improvement.

Juglans