The Effect of Lignin Composition on Ruminal Fiber Fractions Degradation from Different Roughage Sources in Water Buffalo (Bubalus bubalis)

The water buffalo (Bubalus bubalis) is known for its unique utilization of low-quality fibrous feeds and outstanding digestion performance, highlighting its role as an animal model in studying fiber fractions degradation. Among roughage, lignin attracted wide attention in ruminant nutrition studies, which affects animal digestibility. Therefore, the present study aims to investigate the functional relation between three lignin monomeric compositions of coniferyl alcohol (G), ρ-coumaryl alcohol (H) and sinapyl alcohol (S) and ruminal fiber degradation in water buffalo. Hence, three female water buffaloes (Nili-Ravi × Mediterranean, five years old, 480 ± 20 kg) were assigned for an in vivo study by utilizing the nylon-bag method, examining eight kinds of roughage. All the experimental roughage types were analyzed for the effective degradability (ED) of neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), cellulose (CEL) and hemicellulose (HC) fractions. Then, prediction models for the roughage fiber degradation were established based on the characteristics of lignin monomer content. The results showed that S, S/G and S/(G+S+H) were positively correlated with the ED of NDF, ADF, CEL and HC; H/S was negatively correlated. For the effective degradability of ADL (ADLD), S and S/(G+S+H) were positively correlated with it; H, H/G, H/S and H/(G+S+H) were negatively correlated. The model with the highest fitting degree was ADLD = 0.161 − 1.918 × H + 3.152 × S (R2 = 0.758, p < 0.01). These results indicated that the lignin monomer composition is closely related to the utilization rate of roughage fiber. S-type lignin monomer plays a vital role in the fiber degradation of roughage. The experiment found the effect of lignin monomer composition on the degradation of fiber fractions using buffalo as the experimental animal and constructed prediction models, providing a scientific basis for building a new technological method using lignin composition to evaluate buffalo roughage. Furthermore, the capacity of ADL degradation of buffalo was proved in this experiment. In order to further explore the ability of lignin degradation by the buffalo, the DNA of rumen microorganisms was extracted for sequencing. The top three composition of rumen microorganisms at the genus level were Prevotella_1, 226, Rikenellaceae_RC9_gut_group and Ruminococcaceae_UCG-011. Six strains with lignin degradation ability were screened from buffalo rumen contents. This experiment also revealed that the buffalos possess rumen microorganisms with lignin degradation potential.

Biochemical and Histological Insights into the Interaction Between the Canker Pathogen Neofusicoccum parvum and Prunus dulcis L.

The number of reports associated with wood dieback caused by fungi in the Botryosphaeriaceae in numerous perennial crops worldwide has significantly increased in the past years. In this study, we investigated the interactions between the canker pathogen Neofusicoccum parvum and the almond tree host (Prunus dulcis L.), with an emphasis on varietal resistance and host response at the cell wall biochemical and histological levels. Plant bioassays in a shaded house showed that among the four commonly planted commercial almond cultivars (cvs. ‘Butte’, ‘Carmel’, ‘Monterey’ and ‘Nonpareil’), there was no significant varietal difference with respect to resistance to the pathogen. Gummosis was only triggered by fungal infection, and not by wounding. A two-dimensional NMR and liquid chromatography determination of cell wall polymers showed that infected almond trees differed significantly in their glycosyl and lignin composition compared to healthy, non-infected trees. Response to fungal infection involved a significant increase in lignin, a decrease in glucans, and an overall enrichment in other carbohydrates with a profile similar to those observed in gums. Histological observations revealed the presence of guaiacyl-rich cell wall reinforcements. Confocal microscopy suggested that N. parvum mainly colonized the lumina of xylem vessels and parenchyma cells, and to a lesser extent the gum ducts. We discuss the relevance of these findings in the context of the CODIT model in almond and its potential involvement in the vulnerability of the host toward fungal wood canker diseases.

Fiber Cell-Specific Expression of the VP16-Fused Ethylene Response Factor 41 Protein Increases Biomass Yield and Alters Lignin Composition

Arabidopsis thaliana transcription factors belonging to the ERFIIId and ERFIIIe subclade (ERFIIId/e) of the APETALA 2/ethylene response factor (AP2/ERF) family enhance primary cell wall (PCW) formation. These transcription factors activate expression of genes encoding PCW-type cellulose synthase (CESA) subunits and other genes for PCW biosynthesis. In this study, we show that fiber-specific expression of ERF035-VP16 and ERF041-VP16, which are VP16-fused proteins of ERFIIId/e members, promote cell wall thickening in a wild-type background with a concomitant increase of alcohol insoluble residues (cell wall content) per fresh weight (FW) and monosaccharides related to the PCW without affecting plant growth. Furthermore, in the ERF041-VP16 lines, the total amount of lignin and the syringyl (S)/guaiacyl (G) ratio decreased, and the enzymatic saccharification yield of glucose from cellulose per fresh weight improved. In these lines, PCW-type CESA genes were upregulated and ferulate 5-hydropxylase1 (F5H1), which is necessary for production of the S unit lignin, was downregulated. In addition, various changes in the expression levels of transcription factors regulating secondary cell wall (SCW) formation were observed. In conclusion, fiber cell-specific ERF041-VP16 improves biomass yield, increases PCW components, and alters lignin composition and deposition and may be suitable for use in future molecular breeding programs of biomass crops.

Knockout of the lignin pathway gene BnF5H decreases the S/G lignin composition ratio and improves S. sclerotiorum resistance in B. napus

Ferulate-5-hydroxylase (F5H) is a key enzyme involved in the conversion of the guaiacyl monolignol (G-monolignol) to the syringyl monolignol (S-monolignol) in angiosperms. The monolignol ratio has been proposed to affect biomass recalcitrance and the resistance to plant disease. Sclerotinia sclerotiorum (S. sclerotiorum) stem rot of Brassica napus (B. napus) causes heavy damage in oilseed rape production. To date, there is no information about the effect of the lignin monomer ratio on the resistance to S. sclerotiorum in B. napus. Four dominantly expressed BnF5H genes were knocked out by CRISPR/Cas9 simultaneously in B. napus, and the f5h mutant KO-7 was generated. The S/G lignin composition ratio was decreased compared to that of the wild type (WT) based on the results of Mӓule staining and 2D-NMR profiling. The resistance to S. sclerotiorum in stems and leaves increased in KO-7. Furthermore, we found that the stem strength of KO-7 was significantly increased compared to that of the WT. Collectively, for the first time, we demonstrate that knockout of the lignin pathway gene F5H decreases the S/G ratio, improves S. sclerotiorum resistance in B. napus, and increases stem strength.

Comparison of methodologies used to determine aromatic lignin unit ratios in lignocellulosic biomass

Background Multiple analytical methods have been developed to determine the ratios of aromatic lignin units, particularly the syringyl/guaiacyl (S/G) ratio, of lignin biopolymers in plant cell walls. Chemical degradation methods such as thioacidolysis produce aromatic lignin units that are released from certain linkages and may induce chemical changes rendering it difficult to distinguish and determine the source of specific aromatic lignin units released, as is the case with nitrobenzene oxidation methodology. NMR methods provide powerful tools used to analyze cell walls for lignin composition and linkage information. Pyrolysis-mass spectrometry methods are also widely used, particularly as high-throughput methodologies. However, the different techniques used to analyze aromatic lignin unit ratios frequently yield different results within and across particular studies, making it difficult to interpret and compare results. This also makes it difficult to obtain meaningful insights relating these measurements to other characteristics of plant cell walls that may impact biomass sustainability and conversion metrics for the production of bio-derived fuels and chemicals. Results The authors compared the S/G lignin unit ratios obtained from thioacidolysis, pyrolysis-molecular beam mass spectrometry (py-MBMS), HSQC liquid-state NMR and solid-state (ss) NMR methodologies of pine, several genotypes of poplar, and corn stover biomass. An underutilized approach to deconvolute ssNMR spectra was implemented to derive S/G ratios. The S/G ratios obtained for the samples did not agree across the different methods, but trends were similar with the most agreement among the py-MBMS, HSQC NMR and deconvoluted ssNMR methods. The relationship between S/G, thioacidolysis yields, and linkage analysis determined by HSQC is also addressed. Conclusions This work demonstrates that different methods using chemical, thermal, and non-destructive NMR techniques to determine native lignin S/G ratios in plant cell walls may yield different results depending on species and linkage abundances. Spectral deconvolution can be applied to many hardwoods with lignin dominated by S and G units, but the results may not be reliable for some woody and grassy species of more diverse lignin composition. HSQC may be a better method for analyzing lignin in those species given the wealth of information provided on additional aromatic moieties and bond linkages. Additionally, trends or correlations in lignin characteristics such as S/G ratios and lignin linkages within the same species such as poplar may not necessarily exhibit the same trends or correlations made across different biomass types. Careful consideration is required when choosing a method to measure S/G ratios and the benefits and shortcomings of each method discussed here are summarized.

Accurate determination of genotypic variance of cell wall characteristics of a Populus trichocarpa pedigree using high-throughput pyrolysis-molecular beam mass spectrometry

Background Pyrolysis-molecular beam mass spectrometry (py-MBMS) analysis of a pedigree of Populus trichocarpa was performed to study the phenotypic plasticity and heritability of lignin content and lignin monomer composition. Instrumental and microspatial environmental variability were observed in the spectral features and corrected to reveal underlying genetic variance of biomass composition. Results Lignin-derived ions (including m/z 124, 154, 168, 194, 210 and others) were highly impacted by microspatial environmental variation which demonstrates phenotypic plasticity of lignin composition in Populus trichocarpa biomass. Broad-sense heritability of lignin composition after correcting for microspatial and instrumental variation was determined to be H2 = 0.56 based on py-MBMS ions known to derive from lignin. Heritability of lignin monomeric syringyl/guaiacyl ratio (S/G) was H2 = 0.81. Broad-sense heritability was also high (up to H2 = 0.79) for ions derived from other components of the biomass including phenolics (e.g., salicylates) and C5 sugars (e.g., xylose). Lignin and phenolic ion abundances were primarily driven by maternal effects, and paternal effects were either similar or stronger for the most heritable carbohydrate-derived ions. Conclusions We have shown that many biopolymer-derived ions from py-MBMS show substantial phenotypic plasticity in response to microenvironmental variation in plantations. Nevertheless, broad-sense heritability for biomass composition can be quite high after correcting for spatial environmental variation. This work outlines the importance in accounting for instrumental and microspatial environmental variation in biomass composition data for applications in heritability measurements and genomic selection for breeding poplar for renewable fuels and materials.

Comparison of Methodologies used to Determine Aromatic Lignin Unit Ratios in Lignocellulosic Biomass

Background Multiple analytical methods have been developed to determine the ratios of aromatic lignin units, particularly the syringyl/guaiacyl (S/G) ratio, of lignin biopolymers in plant cell walls. Chemical degradation methods such as thioacidolysis produce aromatic lignin units that are released from certain linkages and may induce chemical changes rendering it difficult to distinguish and determine the source of specific aromatic lignin units released, as is the case with nitrobenzene oxidation methodology. NMR methods provide powerful tools used to analyze cell walls for lignin composition and linkage information. Pyrolysis-mass spectrometry methods are also widely used, particularly as high-throughput methodologies. However, the different techniques used to analyze aromatic lignin unit ratios frequently yield different results within and across particular studies, making it difficult to interpret and compare results. This also makes it difficult to obtain meaningful insights relating these measurements to other characteristics of plant cell walls that may impact biomass sustainability and conversion metrics for the production of bio-derived fuels and chemicals. Results The authors compared the S/G lignin unit ratios obtained from thioacidolysis, pyrolysis-molecular beam mass spectrometry (py-MBMS), HSQC liquid-state NMR and solid-state (ss) NMR methodologies of pine, several genotypes of poplar, and corn stover biomass. An underutilized approach to deconvolute ssNMR spectra was implemented to derive S/G ratios. The S/G ratios obtained for the samples did not agree across the different methods, but trends were similar with the most agreement among the py-MBMS, HSQC NMR and deconvoluted ssNMR methods. The relationship between S/G, thioacidolysis yields, and linkage analysis determined by HSQC is also addressed. Conclusions This work demonstrates that different methods using chemical, thermal, and nondestructive NMR techniques to determine native lignin S/G ratios in plant cell walls may yield different results depending on species and linkage abundances. Spectral deconvolution can be applied to many hardwoods with lignin dominated by S and G units, but the results may not be reliable for some woody and grassy species of more diverse lignin composition. HSQC may be a better method for analyzing lignin in those species given the wealth of information provided on additional aromatic moieties and bond linkages. Additionally, trends or correlations in lignin characteristics such as S/G ratios and lignin linkages within the same species such as poplar may not necessarily exhibit the same trends or correlations made across different biomass types. Careful consideration is required when choosing a method to measure S/G ratios and the benefits and shortcomings of each method discussed here are summarized.

Accurate Determination of Genotypic Variance of Cell Wall Characteristics of a Populus trichocarpa Pedigree Using High-Throughput Pyrolysis-Molecular Beam Mass Spectrometry

Background Pyrolysis-molecular beam mass spectrometry (py-MBMS) analysis of a pedigree of Populus trichocarpa was performed to study the phenotypic plasticity and heritability of lignin content and lignin monomer composition. Instrumental and microspatial environmental variability were observed in the spectral features and corrected to reveal underlying genetic variance of biomass composition.Results Lignin-derived ions (including m/z 124, 154, 168, 194, 210 and others) were highly impacted by microspatial environmental variation which demonstrates phenotypic plasticity of lignin composition in Populus trichocarpa biomass. Broad-sense heritability of lignin composition after correcting for microspatial and instrumental variation was determined to be H2 = 0.56 based on py-MBMS based on ions known to derive from lignin. Heritability of lignin monomeric syringyl/guaiacyl ratio (S/G) was H2 = 0.81. Broad-sense heritability was also high (up to H2 = 0.79) for ions derived from other components of the biomass including phenolics (e.g., salicylates) and C5 sugars (e.g., xylose). Lignin and phenolic ion abundances were primarily driven by maternal effects, and paternal effects were either similar or stronger for the most heritable carbohydrate-derived ions.Conclusions We have shown that many biopolymer-derived ions from py-MBMS show substantial phenotypic plasticity in response to microenvironmental variation in plantations. Nevertheless, broad-sense heritability for biomass composition can be quite high after correcting for spatial environmental variation. This work outlines the importance in accounting for instrumental and microspatial environmental variation in biomass composition data for applications in heritability measurements and genomic selection for breeding poplar for renewable fuels and materials.

A mechanistic study of cellulase adsorption onto lignin

To explore the effect of lignin composition on cellulase adsorption, in this study, dehydrogenation polymers (DHPs) were first prepared from p-glucocoumaryl alcohol/coniferin/syringin, giving rise to H-DHP, G-DHP, and S-DHP, respectively.