Developmental changes in lignin composition are driven by both monolignol supply and laccase specificity.
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ABSTRACT:
SUBMITTER: Zhuo C
PROVIDER: S-EPMC8906750 | biostudies-literature | 2022 Mar
REPOSITORIES: biostudies-literature
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Developmental changes in lignin composition are driven by both monolignol supply and laccase specificity.
Zhuo Chunliu C Wang Xin X Docampo-Palacios Maite M Sanders Brian C BC Engle Nancy L NL Tschaplinski Timothy J TJ Hendry John I JI Maranas Costas D CD Chen Fang F Dixon Richard A RA
Science advances 20220309 10
The factors controlling lignin composition remain unclear. Catechyl (C)-lignin is a homopolymer of caffeyl alcohol with unique properties as a biomaterial and precursor of industrial chemicals. The lignin synthesized in the seed coat of <i>Cleome hassleriana</i> switches from guaiacyl (G)- to C-lignin at around 12 to 14 days after pollination (DAP), associated with a rerouting of the monolignol pathway. Lack of synthesis of caffeyl alcohol limits C-lignin formation before around 12 DAP, but coni ...[more]
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Project description:BackgroundBast fibres are characterized by very thick secondary cell walls containing high amounts of cellulose and low lignin contents in contrast to the heavily lignified cell walls typically found in the xylem tissues. To improve the quality of the fiber-based products in the future, a thorough understanding of the main cell wall polymer biosynthetic pathways is required. In this study we have carried out a characterization of the genes involved in lignin biosynthesis in flax along with some of their regulation mechanisms.ResultsWe have first identified the members of the phenylpropanoid gene families through a combination of in silico approaches. The more specific lignin genes were further characterized by high throughput transcriptomic approaches in different organs and physiological conditions and their cell/tissue expression was localized in the stems, roots and leaves. Laccases play an important role in the polymerization of monolignols. This multigenic family was determined and a miRNA was identified to play a role in the posttranscriptional regulation by cleaving the transcripts of some specific genes shown to be expressed in lignified tissues. In situ hybridization also showed that the miRNA precursor was expressed in the young xylem cells located near the vascular cambium. The results obtained in this work also allowed us to determine that most of the genes involved in lignin biosynthesis are included in a unique co-expression cluster and that MYB transcription factors are potentially good candidates for regulating these genes.ConclusionsTarget engineering of cell walls to improve plant product quality requires good knowledge of the genes responsible for the production of the main polymers. For bast fiber plants such as flax, it is important to target the correct genes from the beginning since the difficulty to produce transgenic material does not make possible to test a large number of genes. Our work determined which of these genes could be potentially modified and showed that it was possible to target different regulatory pathways to modify lignification.
| S-EPMC5513022 | biostudies-literature
Project description: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.
| S-EPMC9245121 | biostudies-literature
Project description:Lignin is a complex polymer derived from the oxidative coupling of three classical monolignols. Lignin precursors are methylated exclusively at the meta-positions (i.e. 3/5-OH) of their phenyl rings by native O-methyltransferases, and are precluded from substitution of the para-hydroxyl (4-OH) position. Ostensibly, the para-hydroxyls of phenolics are critically important for oxidative coupling of phenoxy radicals to form polymers. Therefore, creating a 4-O-methyltransferase to substitute the para-hydroxyl of monolignols might well interfere with the synthesis of lignin. The phylogeny of plant phenolic O-methyltransferases points to the existence of a batch of evolutionarily "plastic" amino acid residues. Following one amino acid at a time path of directed evolution, and using the strategy of structure-based iterative site-saturation mutagenesis, we created a novel monolignol 4-O-methyltransferase from the enzyme responsible for methylating phenylpropenes. We show that two plastic residues in the active site of the parental enzyme are vital in dominating substrate discrimination. Mutations at either one of these separate the evolutionarily tightly linked properties of substrate specificity and regioselective methylation of native O-methyltransferase, thereby conferring the ability for para-methylation of the lignin monomeric precursors, primarily monolignols. Beneficial mutations at both sites have an additive effect. By further optimizing enzyme activity, we generated a triple mutant variant that may structurally constitute a novel phenolic substrate binding pocket, leading to its high binding affinity and catalytic efficiency on monolignols. The 4-O-methoxylation of monolignol efficiently impairs oxidative radical coupling in vitro, highlighting the potential for applying this novel enzyme in managing lignin polymerization in planta.
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Project description:Eucalyptus species constitutes the most widely planted hardwood trees in temperate and subtropical regions. In this study, we compared the transcript levels of genes involved in lignocellulose formation such as cellulose, hemicellulose and lignin biosynthesis in two selected 3-year old hybrid Eucalyptus (Eucalyptus urophylla × Eucalyptus grandis) genotypes (AM063 and AM380) that have different lignin content. AM063 and AM380 had 20.2 and 35.5% of Klason lignin content and 59.0 and 48.2%, α-cellulose contents, respectively. We investigated the correlation between wood properties and transcript levels of wood formation-related genes using RNA-seq with total RNAs extracted from developing xylem tissues at a breast height. Transcript levels of cell wall construction genes such as cellulose synthase (CesA) and sucrose synthase (SUSY) were almost the same in both genotypes. However, AM063 exhibited higher transcript levels of UDP-glucose pyrophosphorylase and xyloglucan endotransglucoxylase than those in AM380. Most monolignol biosynthesis-related isozyme genes showed higher transcript levels in AM380. These results indicate monolignol biosynthesis-related genes may regulate wood composition in Eucalyptus. Flavonoids contents were also observed at much higher levels in AM380 as a result of the elevated transcript levels of common phenylpropanoid pathway genes, phenylalanine ammonium lyase, cinnamate-4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL). Secondary plant cell wall formation is regulated by many transcription factors. We analyzed genes encoding NAC, WRKY, AP2/ERF, and KNOX transcription factors and found higher transcript levels of these genes in AM380. We also observed increased transcription of some MYB and LIM domain transcription factors in AM380 compared to AM063. All these results show that genes related to monolignol biosynthesis may regulate the wood composition and help maintain the ratio of cellulose and lignin contents in Eucalyptus plants.
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Project description:BackgroundThe phenolic polymer lignin is one of the primary chemical constituents of the plant secondary cell wall. Due to the inherent plasticity of lignin biosynthesis, several phenolic monomers have been shown to be incorporated into the polymer, as long as the monomer can undergo radicalization so it can participate in coupling reactions. In this study, we significantly enhance the level of incorporation of monolignol ferulate conjugates into the lignin polymer to improve the digestibility of lignocellulosic biomass.ResultsOverexpression of a rice Feruloyl-CoA Monolignol Transferase (FMT), OsFMT1, in hybrid poplar (Populus alba x grandidentata) produced transgenic trees clearly displaying increased cell wall-bound ester-linked ferulate, p-hydroxybenzoate, and p-coumarate, all of which are in the lignin cell wall fraction, as shown by NMR and DFRC. We also demonstrate the use of a novel UV-Vis spectroscopic technique to rapidly screen plants for the presence of both ferulate and p-hydroxybenzoate esters. Lastly we show, via saccharification assays, that the OsFMT1 transgenic p oplars have significantly improved processing efficiency compared to wild-type and Angelica sinensis-FMT-expressing poplars.ConclusionsThe findings demonstrate that OsFMT1 has a broad substrate specificity and a higher catalytic efficiency compared to the previously published FMT from Angelica sinensis (AsFMT). Importantly, enhanced wood processability makes OsFMT1 a promising gene to optimize the composition of lignocellulosic biomass.
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Project description:Lignin is a key secondary cell wall chemical constituent, and is both a barrier to biomass utilization and a potential source of bioproducts. The Arabidopsis transcription factors MYB58 and MYB63 have been shown to upregulate gene expression of the general phenylpropanoid and monolignol biosynthetic pathways. The overexpression of these genes also results in dwarfism. The vascular integrity, soluble phenolic profiles, cell wall lignin, and transcriptomes associated with these MYB-overexpressing lines were characterized. Plants with high expression of MYB58 and MYB63 had increased ectopic lignin and the xylem vessels were regular and open, suggesting that the stunted growth is not associated with loss of vascular conductivity. MYB58 and MYB63 overexpression lines had characteristic soluble phenolic profiles with large amounts of monolignol glucosides and sinapoyl esters, but decreased flavonoids. Because loss of function lac4 lac17 mutants also accumulate monolignol glucosides, we hypothesized that LACCASE overexpression might decrease monolignol glucoside levels in the MYB-overexpressing plant lines. When laccases related to lignification (LAC4 or LAC17) were co-overexpressed with MYB63 or MYB58, the dwarf phenotype was rescued. Moreover, the overexpression of either LAC4 or LAC17 led to wild-type monolignol glucoside levels, as well as wild-type lignin levels in the rescued plants. Transcriptomes of the rescued double MYB63-OX/LAC17-OX overexpression lines showed elevated, but attenuated, expression of the MYB63 gene itself and the direct transcriptional targets of MYB63. Contrasting the dwarfism from overabundant monolignol production with dwarfism from lignin mutants provides insight into some of the proposed mechanisms of lignin modification-induced dwarfism.
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Project description:Poplar (Populus) lignin is naturally acylated with p-hydroxybenzoate ester moieties. However, the enzyme(s) involved in the biosynthesis of the monolignol-p-hydroxybenzoates have remained largely unknown. Here, we performed an in vitro screen of the Populus trichocarpa BAHD acyltransferase superfamily (116 genes) using a wheatgerm cell-free translation system and found five enzymes capable of producing monolignol-p-hydroxybenzoates. We then compared the transcript abundance of the five corresponding genes with p-hydroxybenzoate concentrations using naturally occurring unrelated genotypes of P. trichocarpa and revealed a positive correlation between the expression of p-hydroxybenzoyl-CoA monolig-nol transferase (pHBMT1, Potri.001G448000) and p-hydroxybenzoate levels. To test whether pHBMT1 is responsible for the biosynthesis of monolignol-p-hydroxybenzoates, we overexpressed pHBMT1 in hybrid poplar (Populus alba × P. grandidentata) (35S::pHBMT1 and C4H::pHBMT1). Using three complementary analytical methods, we showed that there was an increase in soluble monolignol-p-hydroxybenzoates and cell-wall-bound monolignol-p-hydroxybenzoates in the poplar transgenics. As these pendent groups are ester-linked, saponification releases p-hydroxybenzoate, a precursor to parabens that are used in pharmaceuticals and cosmetics. This identified gene could therefore be used to engineer lignocellulosic biomass with increased value for emerging biorefinery strategies.
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Project description:The evolutionary plant-pathogen arms race has equipped plants with the immune system that can defend against pathogens. Pattern-triggered immunity and effector-triggered immunity are two major branches of innate immunity that share immune responses, including oxidative bursts, transcriptional reprogramming, and cell wall modifications such as lignin deposition. In a previous study, we reported that lignin rapidly accumulates in pathogen-infected Arabidopsis leaves and acts as a mechanical barrier, spatially restricting pathogens and cell death. Lignin deposition into the cell wall is a three-step process: monolignol biosynthesis, transport, and polymerization. While monolignol biosynthesis and polymerization are relatively well understood, the mechanism of monolignol transport remains unclear. In this study, we show that macroautophagy/autophagy modulates pathogen-induced lignin formation. Lignification and other immune responses were impaired in autophagy-defective atg (autophagy-related) mutants. In microscopy analyses, monolignols formed punctate structures in response to pathogen infection and colocalized with autophagic vesicles. Furthermore, autophagic activity and lignin accumulation were both enhanced in dnd1 (defense, no death 1) mutant with elevated disease resistance but no cell death and crossing dnd1-1 with atg mutants resulted in a lignin deficit, further supporting that lignin formation requires autophagy. Collectively, these findings demonstrate that lignification, particularly monolignol transport, is achieved through autophagic membrane trafficking in plant immunity.Abbreviations: ABC transporter: ATP-binding cassette transporter; ACD2/AT4G37000: accelerated cell death 2; ATG: autophagy-related; C3'H/AT2G40890: p-coumaroyl shikimate 3-hydroxylase; C4H/AT2G30490: cinnamate 4-hydroxylase; CA: coniferyl alcohol; CaMV: cauliflower mosaic virus; CASP: Casparian strip membrane domain protein; CASPL: CASP-like protein; CBB: Coomassie Brilliant Blue; CCoAOMT1/AT4G34050: caffeoyl-CoA O-methyltransferase 1; CCR1/AT1G15950: cinnamoyl-CoA reductase 1; CFU: colony-forming unit; COMT1/AT5G54160: caffeic acid O-methyltransferase 1; Con A: concanamycin A; DMAC: dimethylaminocoumarin; DND1/AT5G15410: defense, no death 1; CNGC2: cyclic nucleotide-gated channel 2; ER: endoplasmic reticulum; ESB1/AT2G28670/DIR10: enhanced suberin 1; ETI: effector-triggered immunity; EV: extracellular vesicle; F5H/AT4G36220: ferulate-5-hydroxylase; Fluo-3 AM: Fluo-3 acetoxymethyl ester; GFP: green fluorescent protein; HCT/AT5G48930: p-hydroxycinnamoyl-CoA:quinate/shikimate p-hydroxycinnamoyltransferase; HR: hypersensitive response; LAC: laccase; LTG: LysoTracker Green; LSD1/AT4G200380: lesion stimulating disease 1; PAL1/AT2G37040: phenylalanine ammonia-lyase 1; PAMP: pathogen-associated molecular patterns; PCD: programmed cell death; PE: phosphatidylethanolamine; PRX: peroxidase; Pst DC3000: Pseudomonas syringe pv. tomato DC3000; PTI: pattern-triggered immunity; SA: salicylic acid; SD: standard deviation; SID2/AT1G7410: SA induction-deficient 2; UGT: UDP-glucosyltransferase; UPLC: ultraperformance liquid chromatography; UPS: unconventional protein secretion; V-ATPase: vacuolar-type H+-translocating ATPase.
| S-EPMC9851231 | biostudies-literature
Project description:Laccase-mediator systems (LMS) have been widely studied for their capacity to oxidize the nonphenolic subunits of lignin (70-90% of the polymer). The phenolic subunits (10-30% of the polymer), which can also be oxidized without mediators, have received considerably less attention. Consequently, it remains unclear to what extent the presence of a mediator influences the reactions of the phenolic subunits of lignin. To get more insight in this, UHPLC-MS was used to study the reactions of a phenolic lignin dimer (GBG), initiated by a laccase from Trametes versicolor, alone or in combination with the mediators HBT and ABTS. The role of HBT was negligible, as its oxidation by laccase occurred slowly in comparison to that of GBG. Laccase and laccase/HBT oxidized GBG at a comparable rate, resulting in extensive polymerization of GBG. In contrast, laccase/ABTS converted GBG at a higher rate, as GBG was oxidized both directly by laccase but also by ABTS radical cations, which were rapidly formed by laccase. The laccase/ABTS system resulted in Cα oxidation of GBG and coupling of ABTS to GBG, rather than polymerization of GBG. Based on these results, we propose reaction pathways of phenolic lignin model compounds with laccase/HBT and laccase/ABTS.
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Project description:Lignin plays important structural and functional roles in plants by forming a hydrophobic matrix in secondary cell walls that enhances mechanical strength and resists microbial decay. While the importance of the lignin matrix is well documented and the biosynthetic pathways for monolignols are known, the process by which lignin precursors or monolignols are transported and polymerized to form this matrix remains a subject of considerable debate. In this study, we have synthesized and tested an analog of coniferyl alcohol that has been modified to contain an ethynyl group at the C-3 position. This modification enables fluorescent tagging and imaging of this molecule after its incorporation into plant tissue by click chemistry-assisted covalent labeling with a fluorescent azide dye, and confers a distinct Raman signature that could be used for Raman imaging. We found that this monolignol analog is incorporated into in vitro-polymerized dehydrogenation polymer (DHP) lignin and into root epidermal cell walls of 4-day-old Arabidopsis seedlings. Incorporation of the analog in stem sections of 6-week-old Arabidopsis thaliana plants and labeling with an Alexa-594 azide dye revealed the precise locations of new lignin polymerization. Results from this study indicate that this molecule can provide high-resolution localization of lignification during plant cell wall maturation and lignin matrix assembly.
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