Side streams from wheat processing, such as the bran and gluten fractions, show great potential as a feedstock for the production of novel food ingredients and materials. In this study, we prepared hybrid polysaccharide-protein hydrogels via enzymatic crosslinking of wheat bran arabinoxylan and gluten fractions. Arabinoxylan was first isolated from wheat bran via subcritical water extraction, which preserved the covalently bound ferulic acid moieties to the arabinoxylan core amenable for laccase crosslinking. Gluten was fractionated into its main protein components (glutenin and gliadin) via treatment with aqueous ethanol. Hydrogels with different contents of arabinoxylan and gluten were prepared, demonstrating the integration of the protein fractions within the polysaccharide gel network. Increased addition of gluten led to gradually softer hydrogels, suggesting that the gluten fractions were not involved in the covalent crosslinking with the ferulic acid moieties to any noticeable level. Freeze-drying and regeneration of the hydrogels led to a 3-fold–10-fold increase in the storage and loss moduli, depending on the sample. Analysis of the structure of the hydrogels revealed that the addition of gluten upon enzymatic crosslinking impacted the physical interactions and crystallinity of the arabinoxylan populations, resulting in phase separation of the protein and polysaccharide components. This study demonstrates that tunable hydrogels can be prepared from cereal side streams, with potential as functional plant-based food hydrocolloids with improved nutritional properties, combining dietary fibre and protein components.

Alterations in the structure and chemistry of cell wall polymers during wood decay by white-rot fungi could be one of the best experimental systems to studying the association between different cell wall polymers and the biology of plant-microbe interactions. We investigated the spatial and temporal changes in the distribution patterns of matrix polysaccharides and lignin in the fibre cell walls of D. sissoo wood subjected to preferential delignification and simultaneous decay by two species of white rot fungi. Transmission electron microscopy analysis of fibre walls affected with L. betulina showed removal of lignin from the S1 layer of the secondary walls (SW), resulting in cell separation. Subsequently, preferential removal of lignin from the S2 and S3 layers was observed. The structural changes in the SW of fibres inoculated with D. flavida directly correlated with the simultaneous degradation of all wall polymers. Immunogold labelling-TEM analysis revealed degradation of xyloglucan in the compound middle lamellae (CML) region, undergoing preferential delignification. Weak labelling for less substituted heteroxylans was evident in S2 and S3 layers of preferentially delignified fibre walls. Highly substituted heteroxylans showed a higher distribution in the outer layers of SW even at late stages of degradation. The degradation pattern of cell wall polymers suggests a close association between lignin-heteroxylans in the SW as they were removed simultaneously during preferential delignification in the xylem fibres.

Cuticle-a hydrophobic barrier of cutin and waxes covering the outer cell wall surface of plants-enables survival in terrestrial habitats. However, it is not understood how the hydrophobic cuticle precursors travel through the homogalacturonan-rich hydrophilic cell wall. To elucidate the role of homogalacturonan in cuticle development, we disrupted its integrity by overexpressing a pectate lyase, PtxtPL1-27, in aspen. PtxtPL1-27 had pleiotropic effects on shoot development, including the reduction of cuticle thickness and changes in cutin and wax composition, but the expression of cutin biosynthetic genes was little affected. Despite a reduction in homogalacturonan content in the leaves, labeling with the homogalacturonan-specific antibody JIM5 in the outer epidermal cell wall layer increased and displayed an altered pattern. Moreover, the ultra-structure of cell walls was changed concomitant with lipid accumulation. We propose that the disruption of homogalacturonan integrity affected the cutinsome-dependent transport and polymerization of cutin monomers in the cell wall.

Wood is the most abundant renewable natural resource composed of different polysaccharides and lignin, but its utilisation is hampered by intermolecular linkages between these components forming lignin‐carbohydrate complexes (LCCs) causing recalcitrance. The links between glucuronoxylan and the γ‐C of lignin (γ‐ester linkages) are thought to contribute to one‐third of LCCs, but direct evidence for their natural occurrence and their role in recalcitrance has been scarce so far. To address these issues, Phanerochaete carnosa glucuronoyl esterase ( Pc GCE), hydrolysing γ‐ester linkages, was expressed in cell walls of developing wood in hybrid aspen ( Populus tremula L. × tremuloides Michx.). The enzyme reduced HSQC 2D NMR signals corresponding to the γ‐esters and xylan in dioxane‐extracted LCCs without altering glucuronoxylan content or structure. This increased acid solubility of lignin and lignin content. Reduced wood recalcitrance was shown by increased sugar yields and glucose production rates (by approx. 20%) in saccharification without pretreatment and increased xylan extractability by subcritical water (by approx. 70%). Moreover, trees expressing Pc GCE exhibited greater primary and secondary growth. Transcriptomics and metabolomics analyses in developing wood suggested that growth could have been induced by a higher transcription of SMR2 and RPOTmp, which was likely triggered by the secondary cell wall integrity signalling. The results provide evidence for the natural existence of LCC γ‐esters and their significant contribution to lignocellulose recalcitrance. Furthermore, they show that reducing γ‐ester linkages could increase plant productivity.

Spatial information on wood structure and chemistry is crucial for understanding wood functionality. We present a high-throughput and high-resolution near-infrared (NIR) method for combined imaging of the physical and chemical properties of stem sections from Populus trees. Pyrolysis-GC/MS data was used for sensitive and spatially resolved calibration of wood chemistry while SilviScan™ analyses provided reference data for wood physical properties with 25 μm resolution for wood density and 0.2–2.0 mm for microfibril angle (MFA). NIR prediction models were trained and calibrated on material from both field- and greenhouse-grown trees. Thus, the method was developed for NIR imaging of stem samples as small as 4 mm in diameter with an image resolution of 0.03 mm for small-diameter samples and 0.5 mm for samples with multiple annual rings. The NIR model performance, tested against data not used in the training set, reached the coefficient of determination (   R   pred   2  ) values for wood density and MFA of 0.60 and 0.72, respectively. The NIR models for wood chemistry showed    R   pred   2   values of 0.78 and 0.77 for carbohydrates and lignin, respectively. Models for the G-, S- and H-type lignin had    R   pred   2   values between 0.58 and 0.86. In addition, we developed a prediction model for the determination of tension wood distribution. According to this model, tension wood was frequently observed in young greenhouse samples, which might explain the higher variation found in the chemical and physical properties of wood in greenhouse-grown compared to field-grown trees. The study also demonstrated that NIR-model estimations in image format can capture spatial variations that are not detectable in bulk analyses of wood properties. Examples of the method applied to greenhouse-grown trees highlight the efforts to develop NIR models with good prediction accuracies based on high-resolution data.

Wood of broad-leaf tree species is a valued source of renewable biomass for biorefinery and a target for genetic improvement efforts to reduce its recalcitrance. Glucuronoxylan (GX) plays a key role in recalcitrance through its interactions with cellulose and lignin. To reduce recalcitrance, we modified wood GX by expressing GH10 and GH11 endoxylanases from Aspergillus nidulans in hybrid aspen (Populus tremula L. × tremuloides Michx.) and targeting the enzymes to cell wall. The xylanases reduced tree height, modified cambial activity by increasing phloem and reducing xylem production, and reduced secondary wall deposition. Xylan molecular weight was decreased, and the spacing between acetyl and MeGlcA side chains was reduced in transgenic lines. The transgenic trees produced hypolignified xylem having thin secondary walls and deformed vessels. Glucose yields of enzymatic saccharification without pretreatment almost doubled indicating decreased recalcitrance. The transcriptomics, hormonomics and metabolomics data provided evidence for activation of cytokinin and ethylene signalling pathways, decrease in ABA levels, transcriptional suppression of lignification and a subset of secondary wall biosynthetic program, including xylan glucuronidation and acetylation machinery. Several candidate genes for perception of impairment in xylan integrity were detected. These candidates could provide a new target for uncoupling negative growth effects from reduced recalcitrance. In conclusion, our study supports the hypothesis that xylan modification generates intrinsic signals and evokes novel pathways regulating tree growth and secondary wall biosynthesis.

Glucuronoxylan is the main hemicellulose in the secondary cell wall of angiosperms. Elucidating its molecular structure provides a basis for more accurate plant cell wall models and the utilization of xylan in biorefinery processes. Here, we investigated the spacing of acetyl, glucuronopyranosyl and galactopyranosyl substitutions on Eucalyptus glucuronoxylan using sequential extraction combined with enzymatic hydrolysis and mass spectrometry. We found that the acetyl groups are preferentially spaced with an even pattern and that consecutive acetylation is present as a minor motif. Distinct odd and even patterns of glucuronidation with tight and sparse spacing were observed. Furthermore, the occurrence of consecutive glucuronidation is reported, which adds to the growing body of evidence that this motif is not only present in gymnosperms but also in angiosperms. In addition, the presence of terminal galactopyranosyl units, which can be released by β-galactosidase, altered the digestibility of the glucuronoxylan by GH30 and GH10 xylanase and appeared to be clustered within the polymeric backbone. These findings increase our understanding of the complex structure of glucuronoxylans and its effect on the extractability and biological degradation of Eucalyptus wood.

Background: Microbial expansin-related proteins include fungal loosenins, which have been previously shown to disrupt cellulose networks and enhance the enzymatic conversion of cellulosic substrates. Despite showing beneficial impacts to cellulose processing, detailed characterization of cellulosic materials after loosenin treatment is lacking. In this study, small-angle neutron scattering (SANS) was used to investigate the effects of three recombinantly produced loosenins that originate from Phanerochaete carnosa, PcaLOOL7, PcaLOOL9, and PcaLOOL12, on the organization of holocellulose preparations from Eucalyptus and Spruce wood samples. Results: Whereas the SANS analysis of Spruce holocellulose revealed an increase in inter-microfibril spacing of neighboring cellulose microfibrils following treatment with PcaLOOL12 and to a lesser extent PcaLOOL7, the analysis of Eucalyptus holocellulose revealed a reduction in the ordered arrangement of microfibrils following treatment with PcaLOOL12 and to a lesser extent PcaLOOL9. Parallel SEC-SAXS characterization of PcaLOOL7, PcaLOOL9, and PcaLOOL12 indicated the proteins likely function as monomers; moreover, all appear to retain a flexible disordered N-terminus and folded C-terminal region. The comparatively high impact of PcaLOOL12 motivated its NMR structural characterization, revealing a double-psi β-barrel (DPBB) domain surrounded by three α-helices—the largest nestled against the DPBB core and the other two part of loops extending from the core. Conclusions: The SANS analysis of PcaLOOL action on holocellulose samples confirms their ability to disrupt cellulose fiber networks and suggests a progression from reducing regular order in the microfibril arrangement to increasing inter-microfibril spacing. The most impactful PcaLOOL, PcaLOOL12, was previously observed to be the most highly expressed loosenin in P. carnosa. Its structural characterization herein reveals its stabilization through two disulfide linkages, and an extended N-terminal region distal to a negatively charged and surface accessible polysaccharide binding groove.