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Pod dehiscence facilitates seed dispersal in wild legumes while indehiscence is a key domestication trait in cultivated ones. However, the evolutionary genetic mechanisms underlying its diversity are largely unclear. In this study, we compared transcriptomes of two warm-season (Glycine spp. and Phaseolus spp.) and two cool-season (Pisum spp. and Medicago ruthenica) legumes in analysis of dehiscent and indehiscent pod genotypes. Differentially expressed genes in AP2/ERF-like transcription factors and seven structural gene families, including lactoperoxidase, laccase, and cellulose synthase-interactive proteins, which are involved in secondary cell wall component accumulation, were identified to exert key roles in pod dehiscence variation. In accordance with this, higher lignin and cellulose contents were observed in pod secondary cell wall of dehiscent accessions of soybean and pea; however, the variation patterns of lignin polymers in soybean (accumulation) and pea (proportion) differed between dehiscent and indehiscent pods. Moreover, genome-wide comparative analysis revealed that orthogroups represented <1% of all identified differentially expressed genes could be traced among the four genera of legumes, while recruiting paralogous members may constitute the genetic robustness of legume pod dehiscence. This study compared the genetic mechanism among several legumes in pod dehiscence formation and revealed a compensating role of paralogous redundancy of involved gene families in seed dispersal, which can guide crop breeding., Competing Interests: Conflict of Interest The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Understanding the responses of olive trees to drought stress is crucial for improving cultivation and developing drought-tolerant varieties. Water transport and storage within the plant is a key factor in drought-tolerance strategies. Water management can be based on a variety of factors such as stomatal control, osmoprotectant molecules, proteins and wood properties. The aim of the study was to evaluate the water management strategy under drought stress from an anatomical and biochemical point of view in three young Italian olive cultivars (Giarraffa, Leccino and Maurino) previously distinguished for their physiological and metabolomic responses. For each cultivar, 15 individuals in pots were exposed or not to 28 days of water withholding. Every 7 days, the content of sugars (including mannitol), proline, aquaporins, osmotins, and dehydrins, in leaves and stems, as well as the chemical and anatomical characteristics of the wood of the three cultivars, were analyzed. 'Giarraffa' reduced glucose levels and increased mannitol production, while 'Leccino' accumulated more proline. Both 'Leccino' and 'Maurino' increased sucrose and aquaporin levels, possibly due to their ability to remove embolisms. 'Maurino' and 'Leccino' accumulated more dehydrins and osmotins. While neither genotype nor stress affected wood chemistry, 'Maurino' had a higher vessel-to-xylem area ratio and a larger hydraulic diameter, which allows it to maintain a high transpiration rate but may make it more susceptible to cavitation. The results emphasized the need for an integrated approach, highlighting the importance of the relative timing and sequence of each parameter analyzed, allowing, overall, to define a "strategy" rather than a "response" to drought of each cultivar.

Oat straw, a residue of Avena sativa L., is recognized for its abundance in cellulose, hemicelluloses, and lignin. However, its potential as a source of lipophilic compounds within the framework of a biorefinery concept still remains unexplored. In this study, we conducted an extensive investigation into the content and chemical composition of the lipophilic compounds present in acetone extracts from oat straws of two distinct oat varieties, namely, Karen and Isaura. Furthermore, we examined their seasonal variability in content and composition in straw samples from oats planted in both spring and winter seasons. The extracted lipophilic compounds were predominantly composed of high molecular weight esters (26.0-38.1%), steroids (16.6-24.0%), n -fatty alcohols (10.9-20.7%), n -fatty acids (10.9-16.0%), and n -aldehydes (10.7-15.8%), with lower amounts of n -alkanes (1.1-3.0%), acylglycerides (2.3-3.8%), phytol and phytyl esters (0.6-2.9%), β-diketones (0.1-2.5%), triterpenoids (0.9-1.2%), tocopherols and tocopheryl esters (0.2-0.7%), 2-hydroxy fatty acids (0.1-0.2%), and n -alkylresorcinols (0.1%). Notably, these different classes of compounds exhibited variations in their contents depending on the oat variety and the specific planting season. Of particular interest was the Karen variety, which presented significant amounts of high molecular weight esters, free fatty acids, and acylglycerols, especially when it was cultivated during the winter season. These findings underline the potential of oat straw as a valuable resource for lipid extraction within a biorefinery context and emphasize the importance of selecting the appropriate variety and season for optimal lipid yield.

The lignin from tritordeum straw, a hybrid cereal from crossbreeding of durum wheat and wild barley, was isolated and chemically characterized. Its composition and structure were studied by analytical pyrolysis (Py-GC/MS), nuclear magnetic resonance spectroscopy (NMR), Derivatization Followed by Reductive Cleavage (DFRC) method, and gel permeation chromatography (GPC). The data revealed an enrichment of guaiacyl (G) units (H:G:S of 3:61:36), which had a significant impact on the distribution of inter-unit linkages. The predominant linkages were the β-O-4' alkyl-aryl ethers (78 % of all linkages), with substantial proportions of condensed linkages such as phenylcoumarans (11 %), resinols (4 %), spirodienones (4 %), and dibenzodioxocins (2 %). Moreover, DFRC revealed that tridordeum straw lignin was partly acylated at the γ-OH with both acetates and p-coumarates. Acetates were principally attached to G-units, whereas p-coumarates were predominantly attached to S-units. Furthermore, and more importantly, tritordeum lignin incorporates remarkable amounts of a valuable flavone, tricin, exceeding 30 g per kilogram of straw. Given the diverse industrial applications associated with this high-value molecule, tritordeum straw emerges as a promising and sustainable resource for its extraction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Plant vascular pathogens use different ways to reach the xylem vessels and cause devastating diseases in plants. Resistant and tolerant plants have evolved various defense mechanisms against vascular pathogens. Inducible physico-chemical structures, such as the formation of tyloses and wall reinforcements with phenolic polymers, are very effective barriers that confine the pathogen and prevent colonization. Here, we use a combination of classical histochemistry along with bright-field and fluorescence microscopy and two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy to visualize and characterize wall reinforcements containing phenolic wall polymers, namely, lignin, ferulates, and suberin, which occur in different xylem vasculature in response to pathogen attack., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Lignins released in the black liquors of kraft pulp mills are an underutilised source of aromatics. Due to their phenol oxidase activity, laccases from ligninolytic fungi are suitable biocatalysts to depolymerise kraft lignins, which are characterised by their elevated phenolic content. However, the alkaline conditions necessary to solubilise kraft lignins make it difficult to use fungal laccases whose activity is inherently acidic. We recently developed through enzyme-directed evolution high-redox potential laccases active and stable at pH 10. Here, the ability of these tailor-made alkaliphilic fungal laccases to oxidise, demethylate, and depolymerise eucalyptus kraft lignin at pH 10 is evidenced by the increment in the content of phenolic hydroxyl and carbonyl groups, the methanol released, and the appearance of lower molecular weight moieties after laccase treatment. Nonetheless, in a second assay carried out with higher enzyme and lignin concentrations, these changes were accompanied by a strong increase in the molecular weight and content of β-O-4 and β-5 linkages of the main lignin fraction, indicating that repolymerisation of the oxidised products prevails in one-pot reactions. To prevent it, we finally conducted the enzymatic reaction in a bench-scale reactor coupled to a membrane separation system and were able to prove the depolymerisation of kraft lignin by high-redox alkaliphilic laccase.

The differences in the composition and structure of the lignins from straws of different oat (Avena sativa L.) varieties, planted in two seasons (winter and spring), were studied in detail by different analytical techniques such as pyrolysis coupled to gas chromatography-mass spectrometry (Py-GC/MS), two-dimensional nuclear magnetic resonance (2D-NMR), derivatization followed by reductive cleavage (DFRC), and gel permeation chromatography (GPC). Overall, the analyses revealed that oat straw lignins were enriched in guaiacyl (G; 50-56 %) and syringyl (S; 39-44 %) units, with relatively lower amounts of p-hydroxyphenyl (H; 4-6 %) units. The lignins also incorporated significant quantities of p-coumarates (8-14 % of total lignin units), which are acylating the γ-OH of the lignin side chains, and predominantly over the S units. Furthermore, oat straw lignins also incorporated considerable amounts of the flavone tricin (5-12 % of total lignin units). Interestingly, this study revealed that the lignin content and composition of the oat straws vary with genotype and planting season. Since p-coumarates and tricin are high-value aromatic compounds especially attractive from a biorefinery point of view, the information disclosed here is highly relevant to plant breeding programs aimed at developing functional foods and lignin modifications for improved biorefinery applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

The pruning of sweet orange trees ( Citrus sinensis ) generates large amounts of lignocellulosic residue. Orange tree pruning (OTP) residue presents a significant lignin content (21.2%). However, there are no previous studies describing the structure of the native lignin in OTPs. In the present work, the "milled-wood lignin" (MWL) was extracted from OTPs and examined in detail via gel permeation chromatography (GPC), pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), and two-dimensional nuclear magnetic resonance (2D-NMR). The results indicated that the OTP-MWL was mainly composed of guaiacyl (G) units, followed by syringyl (S) units and minor amounts of p -hydroxyphenyl (H) units (H:G:S composition of 1:62:37). The predominance of G-units had a strong influence on the abundance of the different linkages; therefore, although the most abundant linkages were β- O -4' alkyl-aryl ethers (70% of total lignin linkages), the lignin also contained significant amounts of phenylcoumarans (15%) and resinols (9%), as well as other condensed linkages such as dibenzodioxocins (3%) and spirodienones (3%). The significant content of condensed linkages will make this lignocellulosic residue more recalcitrant to delignification than other hardwoods with lower content of these linkages.

Hydroxystilbenes are a class of polyphenolic compounds that behave as lignin monomers participating in radical coupling reactions during the lignification. Here, we report the synthesis and characterization of various artificial copolymers of monolignols and hydroxystilbenes, as well as low-molecular-mass compounds, to obtain the mechanistic insights into their incorporation into the lignin polymer. Integrating the hydroxystilbenes, resveratrol and piceatannol, into monolignol polymerization in vitro, using horseradish peroxidase to generate phenolic radicals, produced synthetic lignins [dehydrogenation polymers (DHPs)]. Copolymerization of hydroxystilbenes with monolignols, especially sinapyl alcohol, by in vitro peroxidases notably improved the reactivity of monolignols and resulted in substantial yields of synthetic lignin polymers. The resulting DHPs were analyzed using two-dimensional NMR and 19 synthesized model compounds to confirm the presence of hydroxystilbene structures in the lignin polymer. The cross-coupled DHPs confirmed both resveratrol and piceatannol as authentic monomers participating in the oxidative radical coupling reactions during polymerization.

Papyrus ( Cyperus papyrus L.) is a sedge plant with a high rate of biomass productivity that represents an interesting raw material to produce chemicals, materials and fuels, which are currently still obtained from fossil resources, in the context of a lignocellulosic biorefinery. In this work, the content and chemical composition of the lipids present in papyrus stems were thoroughly studied. For this, the papyrus stems were separated into the rind and the pith. The lipid content accounted for 4.1% in the rind and 4.9% in the pith (based on dry matter). The main compounds identified in both parts of the papyrus stem were hydrocarbons, n -fatty acids, 2-hydroxyfatty acids, alcohols, alkylamides, mono- and diglycerides, steroids (sterols, ketones, hydrocarbons, esters and glycosides), tocopherols, tocopherol esters, phytol, phytol esters, alkyl ferulates, ω-carboxyalkyl ferulates (and their monoglycerides), and acylglycerol glycosides. The rind presented a predominance of n -fatty acids (6790 mg/kg; that represented 28.6% of all identified compounds), steroid compounds (6255 mg/kg; 26.3%), phytol and phytol esters (4985 mg/kg; 21.0%), and isoprenoid hydrocarbons, namely phytadiene and squalene (2660 mg/kg; 11.2%), while the most abundant lipids in the pith were steroids (8600 mg/kg; 44.4% of all identified compounds) and fatty acids (6245 mg/kg; 32.2%). Due to the great diversity and significant abundance of the compounds identified in papyrus, it can be considered as a potential raw material for biorefineries to obtain valuable phytochemicals of interest to various industrial sectors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rosado, Marques, Rencoret, Gutiérrez, Bausch, Rosenau, Potthast and del Río.)

The molecular mechanisms associated with secondary cell wall (SCW) deposition in sorghum remain largely uncharacterized. Here, we employed untargeted metabolomics and large-scale transcriptomics to correlate changes in SCW deposition with variation in global gene expression profiles and metabolite abundance along an elongating internode of sorghum, with a major focus on lignin and phenolic metabolism. To gain deeper insight into the metabolic and transcriptional changes associated with pathway perturbations, a bmr6 mutant [with reduced cinnamyl alcohol dehydrogenase (CAD) activity] was analyzed. In the wild type, internode development was accompanied by an increase in the content of oligolignols, p-hydroxybenzaldehyde, hydroxycinnamate esters, and flavonoid glucosides, including tricin derivatives. We further identified modules of genes whose expression pattern correlated with SCW deposition and the accumulation of these target metabolites. Reduced CAD activity resulted in the accumulation of hexosylated forms of hydroxycinnamates (and their derivatives), hydroxycinnamaldehydes, and benzenoids. The expression of genes belonging to one specific module in our co-expression analysis correlated with the differential accumulation of these compounds and contributed to explaining this metabolic phenotype. Metabolomics and transcriptomics data further suggested that CAD perturbation activates distinct detoxification routes in sorghum internodes. Our systems biology approach provides a landscape of the metabolic and transcriptional changes associated with internode development and with reduced CAD activity in sorghum., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Tomato varieties resistant to the bacterial wilt pathogen Ralstonia solanacearum have the ability to restrict bacterial movement in the plant. Inducible vascular cell wall reinforcements seem to play a key role in confining R. solanacearum into the xylem vasculature of resistant tomato. However, the type of compounds involved in such vascular physico-chemical barriers remain understudied, while being a key component of resistance. Here we use a combination of histological and live-imaging techniques, together with spectroscopy and gene expression analysis to understand the nature of R. solanacearum-induced formation of vascular coatings in resistant tomato. We describe that resistant tomato specifically responds to infection by assembling a vascular structural barrier formed by a ligno-suberin coating and tyramine-derived hydroxycinnamic acid amides. Further, we show that overexpressing genes of the ligno-suberin pathway in a commercial susceptible variety of tomato restricts R. solanacearum movement inside the plant and slows disease progression, enhancing resistance to the pathogen. We propose that the induced barrier in resistant plants does not only restrict the movement of the pathogen, but may also prevent cell wall degradation by the pathogen and confer anti-microbial properties, effectively contributing to resistance., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Diferuloylputrescine has been found in a variety of plant species, and recent work has provided evidence of its covalent bonding into lignin. Results from nuclear magnetic resonance spectroscopy revealed the presence of bonding patterns consistent with homo-coupling of diferuloylputrescine and the possibility of cross-coupling with lignin. In the present work, density functional theory calculations have been applied to assess the energetics associated with radical coupling, rearomatization, and dehydrogenation for possible homo-coupled dimers of diferuloylputrescine and cross-coupled dimers of diferuloylputrescine and coniferyl alcohol. The values obtained for these reaction energetics are consistent with those reported for monolignols and other novel lignin monomers. As such, this study shows that there would be no thermodynamic impediment to the incorporation of diferuloylputrescine into the lignin polymer and its addition to the growing list of non-canonical lignin monomers., (Published by Elsevier Ltd.)

Rice ( Oryza sativa L.) straw is a highly abundant, widely available, and low cost agricultural waste that can be used as a source to extract valuable phytochemicals of industrial interest. Hence, in the present work, the chemical composition of the lipophilic compounds present in rice straw was thoroughly characterized by gas chromatography and mass spectrometry using medium-length high-temperature capillary columns, which allowed the identification of a wide range of lipophilic compounds, from low molecular weight fatty acids to high molecular weight sterols esters, sterol glucosides, or triglycerides in the same chromatogram. The most abundant lipophilic compounds in rice straw were fatty acids, which accounted for up to 6,400 mg/kg (41.0% of all identified compounds), followed by free sterols (1,600 mg/kg; 10.2%), sterol glucosides (1,380 mg/kg; 8.8%), fatty alcohols (1,150 mg/kg; 7.4%), and triglycerides (1,140 mg/kg; 7.3%), along with lower amounts of high molecular weight wax esters (900 mg/kg; 5.8%), steroid ketones (900 mg/kg; 5.8%), monoglycerides (600 mg/kg; 3.8%), alkanes (400 mg/kg; 2.6%), diglycerides (380 mg/kg; 2.4%), sterol esters (380 mg/kg; 2.4%), tocopherols (340 mg/kg; 2.2%), and steroid hydrocarbons (60 mg/kg; 0.4%). This information is of great use for the valorization of rice straw to obtain valuable lipophilic compounds of interest for the nutraceutical, pharmaceutical, cosmetic, and chemical industries. Moreover, this knowledge is also useful for other industrial uses of rice straw, as in pulp and papermaking, since some lipophilic compounds are at the origin of the so-called pitch deposits during pulping., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rosado, Marques, Rencoret, Gutiérrez and del Río.)

Recent studies demonstrate that several polyphenolic compounds produced from beyond the canonical monolignol biosynthetic pathways can behave as lignin monomers, participating in radical coupling reactions and being incorporated into lignin polymers. Here, we show various classes of flavonoids, the chalconoid naringenin chalcone, the flavanones naringenin and dihydrotricin, and the flavone tricin, incorporated into the lignin polymer of papyrus (Cyperus papyrus L.) rind. These flavonoids were released from the rind lignin by Derivatization Followed by Reductive Cleavage (DFRC), a chemical degradative method that cleaves the β-ether linkages, indicating that at least a fraction of each was integrated into the lignin as β-ether-linked structures. Due to the particular structure of tricin and dihydrotricin, whose C-3' and C-5' positions at their B-rings are occupied by methoxy groups, these compounds can only be incorporated into the lignin through 4'-O-β bonds. However, naringenin chalcone and naringenin have no substituents at these positions and can therefore form additional carbon-carbon linkages, including 3'- or 5'-β linkages that form phenylcoumaran structures not susceptible to cleavage by DFRC. Furthermore, Nuclear Magnetic Resonance analysis indicated that naringenin chalcone can also form additional linkages through its conjugated double bond. The discovery expands the range of flavonoids incorporated into natural lignins, further broadens the traditional definition of lignin, and enhances the premise that any phenolic compound present at the cell wall during lignification could be oxidized and potentially integrated into the lignin structure, depending only on its chemical compatibility. This study indicates that papyrus lignin has a unique structure, as it is the only lignin known to date that integrates such a diversity of phenolic compounds from different classes of flavonoids. This discovery will open up new ways to engineer and design lignins with specific properties and for enhanced value., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

In the present work, lignin-like fractions were isolated from several ancestral plants -including moss ( Hypnum cupressiforme and Polytrichum commune ), lycophyte ( Selaginella kraussiana ), horsetail ( Equisetum palustre ), fern ( Nephrolepis cordifolia and Pteridium aquilinum ), cycad ( Cycas revoluta ), and gnetophyte ( Ephedra fragilis ) species- and structurally characterized by pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS) and two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy. Py-GC/MS yielded marker compounds characteristic of lignin units, except in the H. cupressiforme , P. commune and E. palustre "lignins," where they were practically absent. Additional structural information on the other five samples was obtained from 2D-NMR experiments displaying intense correlations signals of guaiacyl (G) units in the fern and cycad lignins, along with smaller amounts of p -hydroxyphenyl (H) units. Interestingly, the lignins from the lycophyte S. kraussiana and the gnetophyte E. fragilis were not only composed of G- and H-lignin units but they also incorporated significant amounts of the syringyl (S) units characteristic of angiosperms, which appeared much later in plant evolution, most probably due to convergent evolution. The latter finding is also supported by the abundance of syringol derivatives after the Py-GC/MS analyses of these two samples. Regarding lignin structure, β- O -4' alkyl-aryl ethers were the most abundant substructures, followed by condensed β-5' phenylcoumarans and β-β' resinols (and dibenzodioxocins in the fern and cycad lignins). The highest percentages of alkyl-aryl ether structures correlated with the higher S/G ratio in the S. Kraussiana and E. fragilis lignin-like fractions. More interestingly, apart from the typical monolignol-derived lignin units (H, G and S), other structures, assigned to flavonoid compounds never reported before in natural lignins (such as amentoflavone, apigenin, hypnogenol B, kaempferol, and naringenin), could also be identified in the HSQC spectra of all the lignin-like fractions analyzed. With this purpose, in vitro synthesized coniferyl-naringenin and coniferyl-apigenin dehydrogenation polymers were used as standards. These flavonoids were abundant in H. cupressiforme appearing as the only constituents of the moss lignin-like fraction (including 84% of dimeric hypnogenol B) and their abundance decreased in those of S. Kraussiana (with amentoflavone and naringenin representing 14% of the total aromatic units), and the two ancient gymnosperms (0.4-1.2%) and ferns (0-0.7%)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rencoret, Gutiérrez, Marques, del Río, Tobimatsu, Lam, Pérez-Boada, Ruiz-Dueñas, Barrasa and Martínez.)

Lignin biodegradation has been extensively studied in white-rot fungi, which largely belong to order Polyporales. Among the enzymes that wood-rotting polypores secrete, lignin peroxidases (LiPs) have been labeled as the most efficient. Here, we characterize a similar enzyme (ApeLiP) from a fungus of the order Agaricales (with ~13,000 described species), the soil-inhabiting mushroom Agrocybe pediades . X-ray crystallography revealed that ApeLiP is structurally related to Polyporales LiPs, with a conserved heme-pocket and a solvent-exposed tryptophan. Its biochemical characterization shows that ApeLiP can oxidize both phenolic and non-phenolic lignin model-compounds, as well as different dyes. Moreover, using stopped-flow rapid spectrophotometry and 2D-NMR, we demonstrate that ApeLiP can also act on real lignin. Characterization of a variant lacking the above tryptophan residue shows that this is the oxidation site for lignin and other high redox-potential substrates, and also plays a role in phenolic substrate oxidation. The reduction potentials of the catalytic-cycle intermediates were estimated by stopped-flow in equilibrium reactions, showing similar activation by H 2 O 2 , but a lower potential for the rate-limiting step (compound-II reduction) compared to other LiPs. Unexpectedly, ApeLiP was stable from acidic to basic pH, a relevant feature for application considering its different optima for oxidation of phenolic and nonphenolic compounds.

Papyri belong to the oldest writing grounds in history. Their conservation is of the highest importance in preserving our cultural heritage, which is best achieved based on an extensive knowledge of the materials' constituents to choose a tailored conservation approach. Thermogravimetric Analysis (TGA) has been widely employed to quantify cellulose and lignin in papyrus sheets, yielding reported lignin contents of 25% to 40%. In this work, the TGA method conventionally used for papyrus samples was repeated and compared to other lignin determination approaches (Klason-lignin and acetyl bromide-soluble lignin). TGA can lead to a large overestimation of the lignin content of commercial papyrus sheets (~27%) compared to the other methods (~5%). A similar overestimation of the lignin content was found for the pith and rind of the native papyrus plant. We concluded that the TGA method should, therefore, not be used for lignin quantification.

Pleurotus eryngii is a grassland-inhabiting fungus of biotechnological interest due to its ability to colonize non-woody lignocellulosic material. Genomic, transcriptomic, exoproteomic, and metabolomic analyses were combined to explain the enzymatic aspects underlaying wheat-straw transformation. Up-regulated and constitutive glycoside-hydrolases, polysaccharide-lyases, and carbohydrate-esterases active on polysaccharides, laccases active on lignin, and a surprisingly high amount of constitutive/inducible aryl-alcohol oxidases (AAOs) constituted the suite of extracellular enzymes at early fungal growth. Higher enzyme diversity and abundance characterized the longer-term growth, with an array of oxidoreductases involved in depolymerization of both cellulose and lignin, which were often up-regulated since initial growth. These oxidative enzymes included lytic polysaccharide monooxygenases (LPMOs) acting on crystalline polysaccharides, cellobiose dehydrogenase involved in LPMO activation, and ligninolytic peroxidases (mainly manganese-oxidizing peroxidases), together with highly abundant H 2 O 2 -producing AAOs. Interestingly, some of the most relevant enzymes acting on polysaccharides were appended to a cellulose-binding module. This is potentially related to the non-woody habitat of P. eryngii (in contrast to the wood habitat of many basidiomycetes). Additionally, insights into the intracellular catabolism of aromatic compounds, which is a neglected area of study in lignin degradation by basidiomycetes, were also provided. The multiomic approach reveals that although non-woody decay does not result in dramatic modifications, as revealed by detailed 2D-NMR and other analyses, it implies activation of the complete set of hydrolytic and oxidative enzymes characterizing lignocellulose-decaying basidiomycetes.

The monolignols, p -coumaryl, coniferyl, and sinapyl alcohol, arise from the general phenylpropanoid biosynthetic pathway. Increasingly, however, authentic lignin monomers derived from outside this process are being identified and found to be fully incorporated into the lignin polymer. Among them, hydroxystilbene glucosides, which are produced through a hybrid process that combines the phenylpropanoid and acetate/malonate pathways, have been experimentally detected in the bark lignin of Norway spruce ( Picea abies ). Several interunit linkages have been identified and proposed to occur through homo-coupling of the hydroxystilbene glucosides and their cross-coupling with coniferyl alcohol. In the current work, the thermodynamics of these coupling modes and subsequent rearomatization reactions have been evaluated by the application of density functional theory (DFT) calculations. The objective of this paper is to determine favorable coupling and cross-coupling modes to help explain the experimental observations and attempt to predict other favorable pathways that might be further elucidated via in vitro polymerization aided by synthetic models and detailed structural studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Elder, Rencoret, del Río, Kim and Ralph.)

Rice ( Oryza sativa L.) is a major cereal crop used for human nutrition worldwide. Harvesting and processing of rice generates huge amounts of lignocellulosic by-products such as rice husks and straw, which present important lignin contents that can be used to produce chemicals and materials. In this work, the structural characteristics of the lignins from rice husks and straw have been studied in detail. For this, whole cell walls of rice husks and straw and their isolated lignin preparations were thoroughly analyzed by an array of analytical techniques, including pyrolysis coupled to gas chromatography-mass spectrometry (Py-GC/MS), nuclear magnetic resonance (NMR), and derivatization followed by reductive cleavage (DFRC). The analyses revealed that both lignins, particularly the lignin from rice husks, were highly enriched in guaiacyl (G) units, and depleted in p -hydroxyphenyl (H) and syringyl (S) units, with H:G:S compositions of 7:81:12 (for rice husks) and 5:71:24 (for rice straw). These compositions were reflected in the relative abundances of the different interunit linkages. Hence, the lignin from rice husks were depleted in β- O -4' alkyl-aryl ether units (representing 65% of all inter-unit linkages), but presented important amounts of β-5' (phenylcoumarans, 23%) and other condensed units. On the other hand, the lignin from rice straw presented higher levels of β- O -4' alkyl-aryl ethers (78%) but lower levels of phenylcoumarans (β-5', 12%) and other condensed linkages, consistent with a lignin with a slightly higher S/G ratio. In addition, both lignins were partially acylated at the γ-OH of the side-chain (ca. 10-12% acylation degree) with p -coumarates, which overwhelmingly occurred over S-units. Finally, important amounts of the flavone tricin were also found incorporated into these lignins, being particularly abundant in the lignin of rice straw., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rosado, Rencoret, Marques, Gutiérrez and del Río.)

Plant lignocellulosic biomass, mostly composed of polysaccharide-rich secondary cell walls (SCWs), provides fermentable sugars that may be used to produce biofuels and biomaterials. However, the complex chemical composition and physical structure of SCWs hinder efficient processing of plant biomass. Understanding the molecular mechanisms underlying SCW deposition is, thus, essential to optimize bioenergy feedstocks. Here, we establish a xylogenic culture as a model system to study SCW deposition in sugarcane; the first of its kind in a C4 grass species. We used auxin and brassinolide to differentiate sugarcane suspension cells into tracheary elements, which showed metaxylem-like reticulate or pitted SCW patterning. The differentiation led to increased lignin levels, mainly caused by S-lignin units, and a rise in p -coumarate, leading to increased p -coumarate:ferulate ratios. RNAseq analysis revealed massive transcriptional reprogramming during differentiation, with upregulation of genes associated with cell wall biogenesis and phenylpropanoid metabolism and downregulation of genes related to cell division and primary metabolism. To better understand the differentiation process, we constructed regulatory networks of transcription factors and SCW-related genes based on co-expression analyses. Accordingly, we found multiple regulatory modules that may underpin SCW deposition in sugarcane. Our results provide important insights and resources to identify biotechnological strategies for sugarcane biomass optimization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Simões, Ferreira, Grandis, Rencoret, Persson, Floh, Ferraz, del Río, Buckeridge and Cesarino.)

Lignins from different tree barks, including Norway spruce (Picea abies), eucalyptus (Eucalyptus globulus), mimosa (Acacia dealbata) and blackwood acacia (A. melanoxylon), are thoroughly characterized. The lignin from E. globulus bark is found to be enriched in syringyl (S) units, with lower amounts of guaiacyl (G) and p-hydroxyphenyl (H) units (H/G/S ratio of 1:26:73), which produces a lignin that is highly enriched in β-ether linkages (83 %), whereas those from the two Acacia barks have similar compositions (H/G/S ratio of ≈5:50:45), with a predominance of β-ethers (73-75 %) and lower amounts of condensed carbon-carbon linkages; the lignin from A. dealbata bark also includes some resorcinol-related compounds, that appear to be incorporated or intimately associated to the polymer. The lignin from P. abies bark is enriched in G units, with lower amounts of H units (H/G ratio of 14:86); this lignin is thus depleted in β-O-4' alkyl-aryl ether linkages (44 %) and enriched in condensed linkages. Interestingly, this lignin contains large amounts of hydroxystilbene glucosides that seem to be integrally incorporated into the lignin structure. This study indicates that lignins from tree barks can be seen as an interesting source of valuable phenolic compounds. Moreover, this study is useful for tailoring conversion technologies for bark deconstruction and valorization., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Although cell wall polymers play important roles in the tolerance of plants to abiotic stress, the effects of salinity on cell wall composition and metabolism in grasses remain largely unexplored. Here, we conducted an in-depth study of changes in cell wall composition and phenolic metabolism induced upon salinity in maize seedlings and plants. Cell wall characterization revealed that salt stress modulated the deposition of cellulose, matrix polysaccharides and lignin in seedling roots, plant roots and stems. The extraction and analysis of arabinoxylans by size-exclusion chromatography, 2D-NMR spectroscopy and carbohydrate gel electrophoresis showed a reduction of arabinoxylan content in salt-stressed roots. Saponification and mild acid hydrolysis revealed that salinity also reduced the feruloylation of arabinoxylans in roots of seedlings and plants. Determination of lignin content and composition by nitrobenzene oxidation and 2D-NMR confirmed the increased incorporation of syringyl units in lignin of maize roots. Salt stress also induced the expression of genes and the activity of enzymes enrolled in phenylpropanoid biosynthesis. The UHPLC-MS-based metabolite profiling confirmed the modulation of phenolic profiling by salinity and the accumulation of ferulate and its derivatives 3- and 4-O-feruloyl quinate. In conclusion, we present a model for explaining cell wall remodeling in response to salinity., (© 2020 John Wiley & Sons Ltd.)

A comparison of sequenced Agaricomycotina genomes suggests that efficient degradation of wood lignin was associated with the appearance of secreted peroxidases with a solvent-exposed catalytic tryptophan. This hypothesis is experimentally demonstrated here by resurrecting ancestral fungal peroxidases, after sequence reconstruction from genomes of extant white-rot Polyporales, and evaluating their oxidative attack on the lignin polymer by state-of-the-art analytical techniques. Rapid stopped-flow estimation of the transient-state constants for the 2 successive one-electron transfers from lignin to the peroxide-activated enzyme ( k 2app and k 3app ) showed a progressive increase during peroxidase evolution (up to 50-fold higher values for the rate-limiting k 3app ). The above agreed with 2-dimensional NMR analyses during steady-state treatments of hardwood lignin, showing that its degradation (estimated from the normalized aromatic signals of lignin units compared with a control) and syringyl-to-guaiacyl ratio increased with the enzyme evolutionary distance from the first peroxidase ancestor. More interestingly, the stopped-flow estimations of electron transfer rates also showed how the most recent peroxidase ancestors that already incorporated the exposed tryptophan into their molecular structure (as well as the extant lignin peroxidase) were comparatively more efficient at oxidizing hardwood (angiosperm) lignin, while the most ancestral "tryptophanless" enzymes were more efficient at abstracting electrons from softwood (conifer) lignin. A time calibration of the ancestry of Polyporales peroxidases localized the appearance of the first peroxidase with a solvent-exposed catalytic tryptophan to 194 ± 70 Mya, coincident with the diversification of angiosperm plants characterized by the appearance of dimethoxylated syringyl lignin units., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Recent experimental work has revealed that the hydroxystilbene piceatannol can function as a monomeric unit in the lignification of palm fruit endocarp tissues. Results indicated that piceatannol homo-couples and cross-couples with monolignols through radical reactions and is integrally incorporated into the lignin polymer. The current work reports on the thermodynamics of the proposed reactions using density functional theory calculations. The results indicated that, in general, the energetics of both homo-coupling and cross-coupling are not dissimilar from those of the monolignol coupling, demonstrating the compatibility of piceatannol with the lignification process. Moreover, the DFT methods appear to predict the correct courses of post-coupling rearomatization reactions., (Published by Elsevier Ltd.)

Recent investigations have revealed that, in addition to monolignols, some phenolic compounds derived from the flavonoid and hydroxystilbene biosynthetic pathways can also function as true lignin monomers in some plants. In this study, we found that the hydroxystilbene glucosides isorhapontin (isorhapontigenin- O -glucoside) and, at lower levels, astringin (piceatannol- O -glucoside) and piceid (resveratrol- O -glucoside) are incorporated into the lignin polymer in Norway spruce ( Picea abies ) bark. The corresponding aglycones isorhapontigenin, piceatannol, and resveratrol, along with glucose, were released by derivatization followed by reductive cleavage, a chemical degradative method that cleaves β-ether bonds in lignin, indicating that the hydroxystilbene glucosides are (partially) incorporated into the lignin structure through β-ether bonds. Two-dimensional NMR analysis confirmed the occurrence of hydroxystilbene glucosides in this lignin, and provided additional information regarding their modes of incorporation into the polymer. The hydroxystilbene glucosides, particularly isorhapontin and astringin, can therefore be considered genuine lignin monomers that participate in coupling and cross-coupling reactions during lignification in Norway spruce bark., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Tricin [5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)-4H-chromen-4-one] is a flavone that has been found to be incorporated in grass lignin polymers via 4'⁻O⁻β coupling. Herein, we investigated the tricin-lignin structure using nuclear magnetic resonance (NMR) methods by comparing the 1H⁻13C heteronuclear correlation (HSQC) NMR spectra of the isolated lignin with a series of dimeric and trimeric tricin-4'⁻O⁻β-ether model compounds. Results showed that the tricin moiety significantly affects the chemical shift of the Cβ/Hβ of 4'⁻O⁻β unit, producing peaks at around δC/δH 82.5⁻83.5/4.15⁻4.45, that differ from the Cβ/Hβ correlations from normal 4⁻O⁻β units formed solely by monolignols, and that have to date been unassigned.

Background: Preparing multiple products from lignocellulosic biomass feedstock enhances the profit and sustainability of future biorefineries. Grasses are suitable feedstocks for biorefineries as they permit a variety of possible by-products due to their particular chemical characteristics and morphology. Elucidating the fate of p -hydroxycinnamates (ferulates-FAs and p -coumarates- p CAs) and major structural components during bioprocessing helps to discriminate the sources of recalcitrance in grasses and paves the way for the recovery of p -hydroxycinnamates, which have multiple applications. To address these subjects, we assessed sugarcane bagasse biorefining under alkaline-sulfite chemithermomechanical (AS-CTM) pretreatment and enzymatic saccharification., Results: The mass balances of the major bagasse components were combined with 2D-NMR structural evaluation of process solids to advance our understanding of sugarcane bagasse changes during biorefining. AS-CTM pretreatment provided a high yield and thoroughly digestible substrates. The pretreated material was depleted in acetyl groups, but retained 62 and 79% of the original lignin and xylan, respectively. Forty percent of the total FAs and p CAs were also retained in pretreated material. After pretreatment and enzymatic hydrolysis, the residual solids contained mostly lignin and ester-linked p CAs, with minor amounts of FAs and non-digested polysaccharides. Saponification of the residual solids, at a higher alkali load, cleaved all the ester linkages in the p CAs; nevertheless, a significant fraction of the p CAs remained attached to the saponified solids, probably to lignin, through 4- O ether-linkages., Conclusion: AS-CTM pretreatment provided soundly digestible substrates, which retain substantial amounts of xylans and lignin. Acetyl groups were depleted, but 40% of the total FAs and p CAs remained in pretreated material. Ester-linked p CAs detected in pretreated material also resisted to the enzymatic hydrolysis step. Only a more severe saponification reaction cleaved ester linkages of p CAs from residual solids; nevertheless, p CAs remained attached to the core lignin through 4- O ether-linkages, suggesting the occurrence of an alkali-stable fraction of p CAs in sugarcane bagasse.

The structure of the phenolic polymer in maize grain fibers, with 5.5% Klason lignin content, has been studied. For this, the milled wood lignin (MWL) and dioxane lignin (DL) preparations were isolated and analyzed. The data indicated that the lignin in maize fibers was syringyl rich, mostly involved in β-aryl ether, resinol, and phenylcoumaran substructures. 2D NMR and derivatization followed by reductive cleavage (DFRC) also revealed the occurrence of associated ferulates together with trace amounts of p-coumarates acylating the γ-OH of lignin side chains, predominantly on S-lignin units. More interesting was the occurrence of diferuloylputrescine, a ferulic acid amide, which was identified by 2D NMR and comparison with a synthesized standard, that was apparently incorporated into this lignin. A phenylcoumaran structure involving a diferuloylputrescine coupled through 8-5' linkages to another diferuloylputrescine (or to a ferulate or a guaiacyl lignin unit) was found, providing compelling evidence for its participation in radical coupling reactions. The occurrence of diferuloylputrescine in cell walls of maize kernels and other cereal grains appears to have been missed in previous works, perhaps due to the alkaline hydrolysis commonly used for composition studies.

The lignins from different anatomical parts of macaúba (Acrocomia aculeata) palm fruit, namely stalks, epicarp, and endocarp, were studied. The lignin from stalks was enriched in S-lignin units (S/G 1.2) and β-ether linkages (84% of the total) and was partially acylated at the γ-OH of the lignin side-chains (26% lignin acylation), predominantly with p-hydroxybenzoates and acetates. The epicarp lignin was highly enriched in G-lignin units (S/G 0.2) and consequently depleted in β-ethers (65%) and enriched in condensed structures such as phenylcoumarans (24%) and dibenzodioxocins (3%). The endocarp lignin was strikingly different from the rest and presented large amounts of piceatannol units incorporated into the polymer. This resulted in a lignin polymer depleted in β-ethers but enriched in condensed structures and linked piceatannol moieties. The incorporation of piceatannol into the lignin polymer seems to have a role in seed protection.

Protein polymers exist in every plant cell wall preparation, and they interfere with lignin characterization and quantification. Here, we report the structural characterization of the residual protein peaks in 2D NMR spectra in corn cob and kenaf samples and note that aromatic amino acids are ubiquitous and evident in spectra from various other plants and tissues. The aromatic correlations from amino acid residues were identified and assigned as phenylalanine and tyrosine. Phenylalanine's 3/5 correlation peak is superimposed on the peak from typical lignin p-hydroxyphenyl (H-unit) structures, causing an overestimation of the H units. Protein contamination also occurs when using cellulases to prepare enzyme lignins from virtually protein-free wood samples. We used a protease to remove the protein residues from the ball-milled cell walls, and we were able to reveal H-unit structures in lignins more clearly in the 2D NMR spectra, providing a better basis for their estimation.

Background: New biorefinery concepts are necessary to drive industrial use of lignocellulose biomass components. Xylan recovery before enzymatic hydrolysis of the glucan component is a way to add value to the hemicellulose fraction, which can be used in papermaking, pharmaceutical, and food industries. Hemicellulose removal can also facilitate subsequent cellulolytic glucan hydrolysis., Results: Sugarcane bagasse was pretreated with an alkaline-sulfite chemithermomechanical process to facilitate subsequent extraction of xylan by enzymatic or alkaline procedures. Alkaline extraction methods yielded 53% (w/w) xylan recovery. The enzymatic approach provided a limited yield of 22% (w/w) but produced the xylan with the lowest contamination with lignin and glucan components. All extracted xylans presented arabinosyl side groups and absence of acetylation. 2D-NMR data suggested the presence of O -methyl-glucuronic acid and p -coumarates only in enzymatically extracted xylan. Xylans isolated using the enzymatic approach resulted in products with molecular weights (Mw) lower than 6 kDa. Higher Mw values were detected in the alkali-isolated xylans. Alkaline extraction of xylan provided a glucan-enriched solid readily hydrolysable with low cellulase loads, generating hydrolysates with a high glucose/xylose ratio., Conclusions: Hemicellulose removal before enzymatic hydrolysis of the cellulosic fraction proved to be an efficient manner to add value to sugarcane bagasse biorefining. Xylans with varied yield, purity, and structure can be obtained according to the extraction method. Enzymatic extraction procedures produce high-purity xylans at low yield, whereas alkaline extraction methods provided higher xylan yields with more lignin and glucan contamination. When xylan extraction is performed with alkaline methods, the residual glucan-enriched solid seems suitable for glucose production employing low cellulase loadings.

Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases and the new dye-decolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate proteins. Nevertheless, basidiomycete unspecific peroxygenases have the highest biotechnological interest due to their ability to catalyze a variety of regio- and stereo-selective monooxygenation reactions with H 2 O 2 as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating H 2 O 2 that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other H 2 O 2 generating enzymes - copper-radical oxidases - together with classical laccases that are the oxidoreductases with the largest number of reported applications to date. However, the recently described lytic polysaccharide monooxygenases have attracted the highest attention among copper oxidoreductases, since they are capable of oxidatively breaking down crystalline cellulose, the disintegration of which is still a major bottleneck in lignocellulose biorefineries, along with lignin degradation. Interestingly, some flavin-containing dehydrogenases also play a key role in cellulose breakdown by directly/indirectly "fueling" electrons for polysaccharide monooxygenase activation. Many of the above oxidoreductases have been engineered, combining rational and computational design with directed evolution, to attain the selectivity, catalytic efficiency and stability properties required for their industrial utilization. Indeed, using ad hoc software and current computational capabilities, it is now possible to predict substrate access to the active site in biophysical simulations, and electron transfer efficiency in biochemical simulations, reducing in orders of magnitude the time of experimental work in oxidoreductase screening and engineering. What has been set out above is illustrated by a series of remarkable oxyfunctionalization and oxidation reactions developed in the frame of an intersectorial and multidisciplinary European RTD project. The optimized reactions include enzymatic synthesis of 1-naphthol, 25-hydroxyvitamin D 3 , drug metabolites, furandicarboxylic acid, indigo and other dyes, and conductive polyaniline, terminal oxygenation of alkanes, biomass delignification and lignin oxidation, among others. These successful case stories demonstrate the unexploited potential of oxidoreductases in medium and large-scale biotransformations., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Lignin, the plant cell wall polymer that binds fibers together but makes processing difficult, is traditionally formed from three monomers, the so-called monolignols ( p -coumaryl, coniferyl, and sinapyl alcohols). Recently, we discovered, in grass lignins, a phenolic monomer that falls outside the canonical lignin biosynthetic pathway, the flavone tricin. As we show here, palm fruit (macaúba [ Acrocomia aculeata ], carnauba [ Copernicia prunifera ], and coconut [ Cocos nucifera ]) endocarps contain lignin polymers derived in part from a previously unconsidered class of lignin monomers, the hydroxystilbenes, including the valuable compounds piceatannol and resveratrol. Piceatannol could be released from these lignins upon derivatization followed by reductive cleavage, a degradative method that cleaves β-ether bonds, indicating that at least a fraction is incorporated through labile ether bonds. Nuclear magnetic resonance spectroscopy of products from the copolymerization of piceatannol and monolignols confirms the structures in the natural polymer and demonstrates that piceatannol acts as an authentic monomer participating in coupling and cross-coupling reactions during lignification. Therefore, palm fruit endocarps contain a new class of stilbenolignin polymers, further expanding the definition of lignin and implying that compounds such as piceatannol and resveratrol are potentially available in what is now essentially a waste product., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Lignins were isolated from spruce, wheat straw, and eucalyptus by using the milled wood lignin (MWL) method. Functional groups and compositional analyses were assessed via 2D NMR and 31 P NMR to realize their effect on enzyme binding. Films of the lignins were fabricated and ellipsometry, atomic force microscopy, and water contact angle measurements were used for their characterization and to reveal the changes upon enzyme adsorption. Moreover, lignin thin films were deposited on quartz crystal microgravimetry (QCM) and surface plasmon (SPR) resonance sensors and used to gain further insights into the lignin-cellulase interactions. For this purpose, a commercial multicomponent enzyme system and a monocomponent Trichoderma reesei exoglucanase (CBH-I) were considered. Strong enzyme adsorption was observed on the various lignins but compared to the multicomponent cellulases, CBH-I displayed lower surface affinity and higher binding reversibility. This resolved prevalent questions related to the affinity of this enzyme with lignin. Remarkably, a strong correlation between enzyme binding and the syringyl/guaiacyl (S/G) ratio was found for the lignins, which presented a similar hydroxyl group content ( 31 P NMR): higher protein affinity was determined on isolated spruce lignin (99% G units), while the lowest adsorption occurred on isolated eucalyptus lignin (70% S units). The effect of electrostatic interactions in enzyme adsorption was investigated by SPR, which clearly indicated that the screening of charges allowed more extensive protein adsorption. Overall, this work furthers our understanding of lignin-cellulase interactions relevant to biomass that has been subjected to no or little pretreatment and highlights the widely contrasting effects of the nature of lignin, which gives guidance to improve lignocellulosic saccharification and related processes.

Caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) and caffeic acid-O-methyltransferase (COMT) are key enzymes in the biosynthesis of coniferyl and sinapyl alcohols, the precursors of guaiacyl (G) and syringyl (S) lignin subunits. The function of these enzymes was characterized in single and double mutant maize plants. In this work, we determined that the comt (brown-midrib 3) mutant plants display a reduction of the flavonolignin unit derived from tricin (a dimethylated flavone), demonstrating that COMT is a key enzyme involved in the synthesis of this compound. In contrast, the ccoaomt1 mutants display a wild-type amount of tricin, suggesting that CCoAOMT1 is not essential for the synthesis of this compound. Based on our data, we suggest that CCoAOMT1 is involved in lignin biosynthesis at least in midribs. The phenotype of ccoaomt1 mutant plants displays no alterations, and their lignin content and composition remain unchanged. On the other hand, the ccoaomt1 comt mutant displays phenotypic and lignin alterations similar to those already described for the comt mutant. Although stems from the three mutants display a similar increase of hemicelluloses, the effect on cell wall degradability varies, the cell walls of ccoaomt1 being the most degradable. This suggests that the positive effect of lignin reduction on cell wall degradability of comt and ccoaomt1 comt mutants is counteracted by changes occurring in lignin composition, such as the decreased S/G ratio. In addition, the role of the flavonolignin unit derived from tricin in cell wall degradability is also discussed., (© The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Lignin, a cross-linked macromolecule of hydrophobic aromatic structure, provides additional rigidity to a plant cell wall. Although it is an integral part of the plant cell, presence of lignin considerably reduces the quality of the fiber of fiber-yielding plants. Decreasing lignin in such plants holds significant commercial and environmental potential. This study aimed at reducing the lignin content in jute-a fiber crop, by introducing hpRNA-based vectors for downregulation of two monolignoid biosynthetic genes- cinnamate 4-hydroxylase (C4H) and caffeic acid O-methyltransferase (COMT). Transgenic generations, analyzed through Southern, RT-PCR and northern assays showed downregulation of the selected genes. Transgenic lines exhibited reduced level of gene expression with ~ 16-25% reduction in acid insoluble lignin for the whole stem and ~13-14% reduction in fiber lignin content compared to the control lines. Among the two transgenic plant types one exhibited an increase in cellulose content and concomitant improvement of glucose release. Composition of the lignin building blocks was found to alter and this alteration resulted in a pattern, different from other plants where the same genes were manipulated. It is expected that successful COMT-hpRNA and C4H-hpRNA transgenesis in jute will have far-reaching commercial implications leading to product diversification and value addition.

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-β)-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., (© 2016 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The composition and structure of lignin in different tissues-phellem (cork), phloem and xylem (wood)-of Quercus suber was studied. Whole cell walls and their respective isolated milled lignins were analyzed by pyrolysis coupled with gas chromatography/mass spectrometry (Py-GC/MS), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) and derivatization followed by reductive cleavage (DFRC). Different tissues presented varied p -hydroxyphenyl:guaiacyl:syringyl (H:G:S) lignin compositions. Whereas lignin from cork has a G-rich lignin (H:G:S molar ratio 2:85:13), lignin from phloem presents more S-units (H:G:S molar ratio of 1:58:41) and lignin from xylem is slightly enriched in S-lignin (H:G:S molar ratio 1:45:55). These differences were reflected in the relative abundances of the different interunit linkages. Alkyl-aryl ethers (β- O -4') were predominant, increasing from 68% in cork, to 71% in phloem and 77% in xylem, as consequence of the enrichment in S-lignin units. Cork lignin was enriched in condensed structures such as phenylcoumarans (β-5', 20%), dibenzodioxocins (5-5', 5%), as corresponds to a lignin enriched in G-units. In comparison, lignin from phloem and xylem presented lower levels of condensed linkages. The lignin from cork was highly acetylated at the γ-OH of the side-chain (48% lignin acetylation), predominantly over G-units; while the lignins from phloem and xylem were barely acetylated and this occurred mainly over S-units. These results are a first time overview of the lignin structure in xylem, phloem (generated by cambium), and in cork (generated by phellogen), in agreement with literature that reports that lignin biosynthesis is flexible and cell specific.