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Two mechanisms for etherification of hydroxycinnamates (illustrated with ferulate) during lignification. a) The ‘passive’ mechanism in which ferulate sits around during the radical coupling and opportunistically adds to quinone methide intermediates produced during lignification, leads to α -ferulate ethers 7A . b) ‘Active’ radical coupling mechanisms directly couple ferulate with, e.g., a monolignol, resulting in β -ferulate ethers 7B .
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Peroxidases are heavily implicated in plant cell wall cross-linking reactions, altering the properties of the wall and impacting its utilization. Polysaccharide-polysaccharide cross-linking in grasses is achieved by dehydrodimerization of hydroxycinnamate-polysaccharide esters; a complex array of hydroxycinnamic acid dehydrodimers are released by s...
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... More extensive cross-linking involving higher oligomers may also be possible. Another area of cell wall cross-linking via similar peroxidase-mediated cross-linking mechanisms occurs during lignification, particularly in grasses. Lignification is a radical-coupling process mediated by peroxidases; being a solely chemical process, the polymerization is not controlled by enzymes or proteins and no exact primary structure is therefore stip- ulated – it is essentially a combinatorial process like the coupling of the ferulates themselves (to give the various DFAs) (Ralph et al., 2004). As a result, any phenolic present in the lignifying zone has the potential to incorporate into the lignification process, subject to its ability to form a radical and its com- patibility with the other phenolics undergoing radical (cross-)coupling reactions. Ferulates are compatible with the mono- and oligolignols, enter the lignin by the same types of radical coupling reactions that typify lignification, and become integral to the structure, only being partially releasable by known cleavage methods. Since ferulates are tethered to polysaccharides, the result is the cross-linking of the two diverse polymers, polysaccharides and lignin. Ferulates are not unique; their dehydrodimers, the DFAs, are also compatible phenolics (Quideau and Ralph, 1997). Incorporation of DFAs into lignins can result in more extensive cell wall cross-linking, between lignins and multiple polysaccharide chains. It is now well established that cross-coupling of the ferulates and dehydrodiferulates with lignin monomers (and perhaps oligomers) is a mechanism for cross-linking lignins and polysaccharides in grasses, as reviewed (Ralph et al., 1998b; Hatfield et al., 1999). It is too early yet to tell whether dehydrodisinapates can also enter into lignification, but it seems likely. Lignin-polysaccharide cross-linking has already been reviewed several times, so only a few topics will be added here. Cross-linking of lignins and polysaccharides via ferulates has been well studied. However, the DFAs also enter into radical cross-coupling reactions during lignification, becoming intimately incorporated into the polymer (Quideau and Ralph, 1997; Grabber et al., 2000). During lignification, ferulate and 5–5-coupled DFA copolymerized more rapidly and formed fewer ether-linked structures with coniferyl alcohol than 8–5-, 8–O–4- and 8–8-DFAs. The potential incorporation of most ferulates and diferulates into lignin exceeded 90%. As a result, xylans in grasses become extensively cross-linked by ferulate dehydrodimerization and incorporation into lignins, but only a small and variable proportion of these cross-links is measurable, since solvolytic cleavage does not release analyzable products. There are two divergent mechanisms, Figure 6, by which polysaccharides can become cross-linked to phenolics (notably lignin) during plant cell wall growth and development (Ralph and Helm, 1993). The only mechanism considered early on was the nucleophilic addition of the ferulate phenols to quinone methides (Yamamoto et al., 1989; Iiyama et al., ...
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... In addition, hydroxycinnamates are found in grass cell walls at the C5 position of the arabinofuranosyl moiety of the xylan backbone. These esters are mainly ferulates, with a smaller proportion of p-coumarates (Buanafina 2009;Chandrakanth et al. 2023;Ralph et al. 2004). In relation to the formation of acylated lignin, the enzymes that acylate monolignols to produce γ-hydroxycinnamoylmonolignols, p-coumaroyl- (Smith et al. 2022) and SbFMT (Smith et al. 2022); and switchgrass (P. ...
The sustainable production and utilization of lignocellulose biomass are indispensable for establishing sustainable societies. Trees and large-sized grasses are the major sources of lignocellulose biomass, while large-sized grasses greatly surpass trees in terms of lignocellulose biomass productivity. With an overall aim to improve lignocellulose usability, it is important to increase the lignin content and simplify lignin structures in biomass plants via lignin metabolic engineering. Rice (Oryza sativa) is not only a representative and important grass crop, but also is a model for large-sized grasses in biotechnology. This review outlines progress in lignin metabolic engineering in grasses, mainly rice, including characterization of the lignocellulose properties, the augmentation of lignin content and the simplification of lignin structures. These findings have broad applicability for the metabolic engineering of lignin in large-sized grass biomass plants.
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... Dehydrodimerization involves one electron oxidation of the monomeric precursors to give dehydrogenated radicals (Bunzel, 2010;Ralph et al., 2004). Therefore, the FT-ICR-MS data were further scru- Next, we assessed whether the rise of dehydrodimers could explain the observed BG fluorescence emission during HCD. ...
... It was shown before that dehydrodimers are bound to the cell wall by esterification (Buanafina, 2009;Ralph et al., 2004). To test whether this also occurs in Arabidopsis, we saponified cell wall fractions and Previous research focussed mainly on dehydrodimers of ferulates, whereas much less is known about dimers of sinapates (Buanafina, 2009;Bunzel et al., 2003;Santiago & Malvar, 2010). ...
... Dehydrodiferulates facilitate cell wall cross-linking during growth and development (Buanafina, 2009;Ralph et al., 2004). Available evidence additionally argues for a role of these molecules in plant defense responses. ...
Infection of Arabidopsis with avirulent Pseudomonas syringae and exposure to nitrogen dioxide (NO 2 ) both trigger hypersensitive cell death (HCD) that is characterized by the emission of bright blue‐green (BG) autofluorescence under UV illumination. The aim of our current work was to identify the BG fluorescent molecules and scrutinize their biosynthesis, localization, and functions during the HCD. Compared with wild‐type (WT) plants, the phenylpropanoid‐deficient mutant fah1 developed normal HCD except for the absence of BG fluorescence. Ultrahigh resolution metabolomics combined with mass difference network analysis revealed that WT but not fah1 plants rapidly accumulate dehydrodimers of sinapic acid, sinapoylmalate, 5‐hydroxyferulic acid, and 5‐hydroxyferuloylmalate during the HCD. FAH1‐dependent BG fluorescence appeared exclusively within dying cells of the upper epidermis as detected by microscopy. Saponification released dehydrodimers from cell wall polymers of WT but not fah1 plants. Collectively, our data suggest that HCD induction leads to the formation of free BG fluorescent dehydrodimers from monomeric sinapates and 5‐hydroxyferulates. The formed dehydrodimers move from upper epidermis cells into the apoplast where they esterify cell wall polymers. Possible functions of phenylpropanoid dehydrodimers are discussed.
... Recently, natural enzymes involved in incorporation of monolignols esterified with ferulate groups into the lignin backbone biosynthesis were found in wild type plants, even though the incorporation of the monolignol ferulate ( Fig. 1a) into the lignin had previously only been achieved by biosynthesis pathway perturbations and engineering (Karlen et al., 2016;Wilkerson et al., 2014). More evidence shows that the role of some pedant units of lignin such as pcoumarate, acetates and p-hydroxybenoylates serve as radical transfer intermediates to other monolignols for lignin polymer growth (Hatfield et al., 2008;Ralph et al., 2004a). Additionally, they have relatively lower activity for radical formation, making them normally present but less abundance in lignin. ...
Searching for renewable alternatives for fossil carbon resources to produce chemicals, fuels and materials is essential for the development of a sustainable society. Lignin, a major component of lignocellulosic biomass, is an abundant renewable source of aromatics and is currently underutilized as it is often burned as an undesired side stream in the production of paper and bioethanol. This lignin harbors great potential as source of high value aromatic chemicals and materials. Biorefinery schemes focused on lignin are currently under development with aim of acquiring added value from lignin. However, the performance of these novel lignin-focused biorefineries is closely linked with the quality of extracted lignin in terms of the level of degradation and modification. Thus, the reactivity including the degradation pathways of the native lignin contained in the plant material need to be understood in detail to potentially achieve higher value from lignin. Undegraded native-like lignin with an as close as possible structure to native lignin contained in the lignocellulosic plant material serves as a promising model lignin to support detailed studies on the structure and reactivity of native lignin, yielding key understanding for the development of lignin-focused biorefineries. The aim of this review is to highlight the different methods to attain "native-like" lignins that can be valuable for such studies. This is done by giving a basic introduction on what is known about the native lignin structure and the analytical used to analyze it followed by an overview of the fractionation and isolation methods to isolate native-like lignin. Finally, a perspective on the isolation and use of native-like lignin is provided, showing the great potential that this type of lignin brings for understanding the effect of different biomass treatments on the native lignin structure.
... Based on other fungal delignification studies [6,40], it is further hypothesized that A. bisporus lignin degradation mechanisms can be understood based on structural characteristics of (remaining) modified lignin. Wheat straw lignin consists of p-hydroxyphenyl (H), syringyl (S) and guaiacyl (G) subunits that maybe linked to p-coumarates, (di)ferulates, and tricin [12,15,16]. Typically, the β-O-4 aryl ether is the most abundant interunit linkage amongst phenylcoumaran (β-5′) and resinol (β-β′) linkages, with the exact relative abundance largely depending on subunit composition and degree of Cγ-acylation (by acetate or p-coumarate) [12,15,17,18]. ...
Fungi are main lignin degraders and the edible white button mushroom, Agaricus bisporus, inhabits lignocellulose-rich environments. Previous research hinted at delignification when A. bisporus colonized pre-composted wheat straw-based substrate in an industrial setting, assumed to aid subsequent release of monosaccharides from (hemi-)cellulose to form fruiting bodies. Yet, structural changes and specific quantification of lignin throughout A. bisporus mycelial growth remain largely unresolved. To elucidate A. bisporus routes of delignification, at six timepoints throughout mycelial growth (15 days), substrate was collected, fractionated, and analyzed by quantitative pyrolysis-GC-MS, 2D-HSQC NMR, and SEC. Lignin decrease was highest between day 6 and day 10 and reached in total 42 % (w/w). The substantial delignification was accompanied by extensive structural changes of residual lignin, including increased syringyl to guaiacyl (S/G) ratios, accumulated oxidized moieties, and depleted intact interunit linkages. Hydroxypropiovanillone and hydroxypropiosyringone (HPV/S) subunits accumulated, which are indicative for β-|O-4' ether cleavage and imply a laccase-driven ligninolysis. We provide compelling evidence that A. bisporus is capable of extensive lignin removal, have obtained insights into mechanisms at play and susceptibilities of various substructures, thus we were contributing to understanding fungal lignin conversion.
... It comprises one unit of cinnamyl alcohol group, phenylcoumaran (β-5), resinol, and biphenyl ether e (4-O-5). It also contains sixteen β-ether units a (β-O-4), but it does not contain any spirodienones and dibenzodioxocins units [39]; since their radicals primarily perform transfer processes instead of radical couplings, and their phenolic units remain mostly free [40]. ...
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... Although p-coumarate and p-hydroxybenzoate esters, being phenolic, can form radicals and undergo radical coupling under in vitro polymerization conditions, under radicallimited conditions their primary fate is radical transfer (to other more stable phenolics) rather than radical coupling Ralph 2010;Ralph et al. 2004a). They are therefore present entirely or largely as free-phenolic appendages acylating lignin sidechains (Ralph and Landucci 2010;Ralph et al. 1994). ...
... Lignification is the process by which phenolic monomers polymerize, in planta, to form lignin polymers (Boerjan et al. 2003;Freudenberg and Neish 1968;Ralph et al. 2004b;Sarkanen and Ludwig 1971). Dehydrogenation (1-electron oxidation) using H 2 O 2 -requiring peroxidases or O 2 -requiring laccases provides the required phenolic radicals from monolignols, oligolignols, and/or polymers, as shown in Figure 1.8a for guaiacyl lignins from coniferyl alcohol 1. Direct contact of the enzymes with the oligomeric/polymeric phenolic substrate may not be required as radical-transfer may occur via radical shuttles such as Mn (III) oxalate (Önnerud et al. 2002), via intermediaries that are more easily oxidized such as p-coumarate Ralph 2010;Ralph et al. 2004a;Takahama et al. 1996), or via monomer radicals themselves (Boerjan et al. 2003;Ralph 2010;Ralph et al. 2004bRalph et al. , 2019Sasaki et al. 2004;Takahama 1995;Vanholme et al. 2012). ...
As ever more component monomers are discovered, lignin can no longer be regarded as deriving from just the three canonical monolignols. Pathway intermediates such as the hydroxycinnamaldehydes and their derived hydroxybenzaldehydes, and additional products of truncated biosynthesis such as caffeyl and 5‐hydroxyconiferyl alcohols, are now established lignin monomers. The array of acylated monolignols continues to expand with evidence for monolignol benzoates, vanillates, and ferulates that complement the accepted monolignol acetates, p ‐hydroxybenzoates, and p ‐coumarates. Game‐changing findings have demonstrated that phenolics from alternative pathways, including flavonoids and hydroxystilbenes, are also involved in lignification, expanding the traditional concept. Beyond the basic science intrigue, these findings propound exciting new avenues for valorizing lignins, or for producing more readily extractable/depolymerizable lignins, in crop and bioenergy plants. Delineating the components that plants are already conscripting for lignification and detailing new components they may be induced to utilize are stimulating current research in this area.
... This variation confirms that these components are sources of lignin and shows the propensity of ferulic and p-coumaric acids to convert to a bound form usually. Some authors have also reported on the absence of ferulic acid in Fragaria leaves even in the cell-wall-bound fraction; they explained this phenomenon by extremely tight bonds via ether linkages in lignin and by consequent difficulties with extraction [46,47]. It is noteworthy that p-coumaric acid, along with some other compounds (ellagic acid, gallic acid, and kaempferol hexoside), has been also identified in the cell-wall-bound fraction [47]. ...
The positive effect of silicon on plants is thought to be mediated by a modification of phenolic metabolism. The purpose of the study was to evaluate the effect of a silicon-based mechanocomposite (MC) on alterations of the phenolic profile of strawberry plants in the course of development under in vitro, ex vitro, and in vivo conditions. Aqueous ethanol extracts of aboveground parts of in vitro–derived plants (Fragaria × ananassa cv. ‘Solnechnaya polyanka’) were subjected to HPLC. Nineteen individual phenolic compounds (hydroxybenzoic and hydroxycinnamic acids, catechins, ellagic acid derivatives, and flavonol glycosides) were quantified. The results revealed phenolic profiles specific to each studied stage and significant transformations of the profiles by the MC. It induced strong upregulation of hydroxycinnamic acid during in vitro rooting and of catechins and hydroxybenzoic acids during ex vitro acclimation. At ex vitro and in vivo stages, the emergence of quercetin glycosides and ellagitannins was registered, and the MC elevated their levels during ex vitro acclimation and field growth. Principal component analysis confirmed the significant effect of the MC on the phenolic profile at all stages, and this effect was the strongest during ex vitro acclimation. The results are consistent with previous reports on the modification of phenolic profiles of plants by silicon-derived biostimulants.
... Results revealed that the products observed in UV led to m/z = 387 for P1, P2 and P3, corresponding to FA-dehydrodimers [M-H] + . The ion having m/z = 404 for P5 corresponds to the FA-dimer having m/z = 387, which is related with the water molecule (H 2 O) as explained by another study [4,22]. These authors showed the possibility of nucleophilic attachment of a water molecule on the FA-tetrahydrofuran depending on the reaction pH. ...
... Moreover, this FA-dimer was identified during FA oxidation in aqueous and organic media [3,8,17]. The P5 dimer has been isolated from plant cell walls and identified under the name 8-8 -tetrahydrofuran [22]. As two (P1, P5) dimers of the five dehydrodimers identified result from C 8 -C 8 radical coupling, this mechanism of radical coupling is favored by FA chemistry according to a previous study [24]. ...
The ferulic acid (FA)-oxidation by Myceliophthora thermophila laccase was performed in phosphate buffer at 30 °C and pH 7.5 as an eco-friendly procedure. LC-MS analysis showed that oxidation products were four dehydrodimers (P1, P2, P3, P5) at MM = 386 g/mol, two dehydrotetramers (P6, P7) at MM = 770 g/mol and one decarboxylated dehydrodimer (P4) at MM = 340 g/mol. Structural characterization showed that FA-dehydrodimers were symmetric for P1 and P5 while asymmetric for P2, P3 and P4. Physicochemical characterization showed that oxidation products presented a higher lipophilicity than that of FA. Moreover, symmetric dimers and tetra dimers had a higher melting point compared to FA and its asymmetric dimers. Antioxidant and anti-proliferative assessments indicated that enzymatic oligomerization increased antioxidant and anti-proliferative properties of oxidation products for P2, P3 and P6 compared to FA. Finally, this enzymatic process in water could produce new molecules, having good antiradical and anti-proliferative activities.
... Grass and cereal bran LCCs are considered to exist via ferulate and diferulate 'bridges' between GAX and lignin (Ralph et al., 2004;Underlin et al., 2020). The (di) ferulates (i.e., esterified to the O-5 position in arabinosyl units) can directly participate in the combinatorial coupling reactions during lignin biosynthesis as described for the monolignols and their conjugates (Ralph, 2010). ...
The development and sustainability of second-generation biorefineries are essential for the production of high added value compounds and biofuels and their application at the industrial level. Pretreatment is one of the most critical stages in biomass processing. In this specific case, hydrothermal pretreatments (liquid hot water [LHW] and steam explosion [SE]) are considered the most promising process for the fractionation, hydrolysis and structural modifications of biomass. This review focuses on architecture of the plant cell wall and composition, fundamentals of hydrothermal pretreatment, process design integration, the techno-economic parameters of the solubilization of lignocellulosic biomass (LCB) focused on the operational costs for large-scale process implementation and the global manufacturing cost. In addition, profitability indicators are evaluated between the value-added products generated during hydrothermal pretreatment, advocating a biorefinery implementation in a circular economy framework. In addition, this review includes an analysis of environmental aspects of sustainability involved in hydrothermal pretreatments.
... Ferulic acid (FA) is found at a relatively high concentration in the cell walls of Graminaceae, Solanaceae and Chenopodiaceae (Mathew and Abraham 2004). These FAs are commonly linked to the branch or side chains of l-arabinofuranose-containing polysaccharides, such as l-arabinod-xylans and l-arabinans, through ester or ether bonds, but can also be attached to lignin and protein (Bento-Silva et al. 2018;Ralph et al. 2004;Topakas et al. 2007). This increases the branching and cross-linking degree of lignocellulose, and makes the hydrolytic sites less accessible to hydrolytic enzymes, resulting in decreased digestibility of plant tissues (Wong et al. 2011). ...
Feruloyl esterase (FAE; EC 3.1.1.73) cleaves the ester bond between ferulic acid (FA) and sugar, to assist the release of FAs and degradation of plant cell walls. In this study, two FAEs (Fae13961 and Fae16537) from the anaerobic fungus Pecoramyces sp. F1 were heterologously expressed in Pichia pastoris (P. pastoris). Compared with Fae16537, Fae13961 had higher catalytic efficiency. The optimum temperature and pH of both the FAEs were 45 ℃ and 7.0, respectively. They showed good stability—Fae16537 retained up to 80% activity after incubation at 37 ℃ for 24 h. The FAEs activity was enhanced by Ca²⁺ and reduced by Zn²⁺, Mn²⁺, Fe²⁺ and Fe³⁺. Additionally, the effect of FAEs on the hydrolytic efficiency of xylanase and cellulase was also determined. The FAE Fae13961 had synergistic effect with xylanase and it promoted the degradation of xylan substrates by xylanase, but it did not affect the degradation of cellulose substrates by cellulase. When Fae13961 was added in a mixture of xylanase and cellulase to degrade complex agricultural biomass, it significantly enhanced the mixture's ability to disintegrate complex substrates. These FAEs could serve as superior auxiliary enzymes for other lignocellulosic enzymes in the process of degradation of agricultural residues for industrial applications.
