Cellulose-active lytic polysaccharide monooxygenases (LPMOs) secreted by filamentous fungi play a key role in the degradation of recalcitrant lignocellulosic biomass. They can occur as multidomain proteins fused to a carbohydrate-binding module (CBM). From a biotech perspective, LPMOs are promising innovative tools for producing nanocelluloses and biofuels, but their direct action on cellulosic substrates is not fully understood. In this study, we probed the role of the CBM from family 1 (CBM1) appended to the LPMO9H from Podospora anserina (PaLPMO9H) using model cellulosic substrates. Deletion of the CBM1 weakened the binding to cellulose nanofibrils, amorphous and crystalline cellulose. Although the release of soluble sugars from cellulose was drastically reduced under standard conditions, the truncated LPMO retained some activity on soluble oligosaccharides. The cellulolytic action of the truncated LPMO was demonstrated using synergy experiments with a cellobiohydrolase (CBH). The truncated LPMO was still able to improve the efficiency of the CBH on cellulose nanofibrils in the same range as the full-length LPMO. Increasing the substrate concentration enhanced the performance of PaLPMO9H without CBM in terms of product release. Interestingly, removing the CBM also altered the regioselectivity of PaLPMO9H, significantly increasing cleavage at the C1 position. Analysis of the insoluble fraction of cellulosic substrates evaluated by optical and atomic force microscopy confirmed that the CBM1 module was not strictly required to promote disruption of the cellulose network. Absence of the CBM1 does not preclude the activity of the LPMO on cellulose but its presence has an important role in driving the enzyme to the substrate and releasing more soluble sugars (both oxidized and non-oxidized), thus facilitating the detection of LPMO activity at low substrate concentration. These results provide insights into the mechanism of action of fungal LPMOs on cellulose to produce nanocelluloses and biofuels.

The cuticle is a complex polymer matrix that protects all aerial organs of plants, fulfills multiple roles in plant–environment interactions, and is critical for plant development. These functions are associated with the structural features of cuticles, and the architectural modeling of cuticles during plant development is crucial for understanding their physical properties and biological functions. In this work, the in-depth architecture of the cutin polymer matrix during fruit development was investigated. Using cherry tomato fruit (Solanum lycopersicum) as a model from the beginning of the cell expansion phase to the red ripe stage, we designed an experimental scheme combining sample pretreatment, Raman mapping, multivariate data analyses, and biochemical analyses. These approaches revealed clear chemical areas with different contributions of cutin, polysaccharides, and phenolics within the cutin polymer matrix. Besides, we demonstrated that these areas are finely tuned during fruit development, including compositional and macromolecular rearrangements. The specific spatiotemporal accumulation of phenolic compounds (p-coumaric acid and flavonoids) suggests that they fulfill distinct functions during fruit development. In addition, we highlighted an unexpected dynamic remodeling of the cutin-embedded polysaccharides pectin, cellulose, and hemicellulose. Such structural tuning enables consistent adaption of the cutin-polysaccharide continuum and the functional performance of the fruit cuticle at the different developmental stages. This study provides insights into the plant cuticle architecture and in particular into the organization of the epidermal cell wall-cuticle.

Dedicated lignocellulosic feedstock from grass crops for biofuel production is extensively increasing. However, the access to fermentable cell wall sugars by carbohydrate degrading enzymes is impeded by lignins. These complex polymers are made from reactive oxidized monolignols in the cell wall. Little is known about the laccase-mediated oxidation of monolignols in grasses, and inactivation of the monolignol polymerization mechanism might be a strategy to increase the yield of fermentable sugars. LACCASE5 and LACCASE8 are inactivated in a Brachypodium double mutant. Relative to the wild type, the lignin content of extract-free mature culms is decreased by 20–30% and the saccharification yield is increased by 140%. Release of ferulic acid by mild alkaline hydrolysis is also 2.5-fold higher. Interfascicular fibers are mainly affected while integrity of vascular bundles is not impaired. Interestingly, there is no drastic impact of the double mutation on plant growth. This work shows that two Brachypodium laccases with clearly identified orthologs in crops are involved in lignification of this model plant. Lignification in interfascicular fibers and metaxylem cells is partly uncoupled in Brachypodium. Orthologs of these laccases are promising targets for improving grass feedstock for cellulosic biofuel production.

Adsorption of hyperbranched arabinogalactan-proteins (AGPs) from two plant exudates, A. senegal and A. seyal, was thoroughly studied at the solid–liquid interface using quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR), and atomic force microscopy (AFM). Isotherms of the adsorption reveal that 3.3 fold more AGPs from A. seyal (500 ppm) are needed to cover the gold surface compared to A. senegal (150 ppm). The pH and salt concentration of the environment greatly affected the adsorption behavior of both gums, with the surface density ranging from 0.92 to 3.83 mg m⁻² using SPR (i.e., “dry” mass) and from 1.16 to 19.07 mg m⁻² using QCM-D (wet mass). Surprisingly, the mass adsorbed was the highest in conditions of strong electrostatic repulsions between the gold substrate and AGPs, i.e., pH 7.0, highlighting the contribution of other interactions involved in the adsorption process. Structural changes of AGPs induced by pH would result in swelling of the polysaccharide blocks and conformational changes of the polypeptide backbone, therefore increasing the protein accessibility and hydrophobic interactions and/or hydrogen bonds with the gold substrate.

This paper explores the enhancement of Raman signals using individual nano-plasmonic structures and demonstrates the possibility to obtain controlled gold plasmonic nanostructures by atomic force microscopy (AFM) manipulation under a confocal Raman device. By manipulating the gold nanoparticles (Nps) while monitoring them using a confocal microscope, it is possible to generate individual nano- structures, plasmonic molecules not accessible currently by lithography at these nanometer scales. This flexible approach allows us to tune plasmonic resonance of the nanostructures, to generate localized hot spots and to circumvent the effects of strong electric near field gradients intrinsic to Tip Enhanced Raman Spectroscopy (TERS) or Surface Enhanced Raman Spectroscopy (SERS) experiments. The inter Np distances and symmetry of the plasmonic molecules in interaction with other individual nano-objects control the resonance conditions of the assemblies and the enhancement of their Raman responses. This paper shows also how some plasmonic structures generate localized nanometric areas with high electric field magnitude without strong gradient. These last plasmonic molecules may be used as "nano-lenses" tunable in wavelength and able to enhance Raman signals of neighbored nano-object. The positioning of one individual probed nano-object in the spatial area defined by the nano-lens becomes then very non-restrictive, contrary to TERS experiments where the spacing distance between tip and sample is crucial. The experimental flexibility obtained in these approaches is illustrated here by the enhanced Raman scatterings of carbon nanotube.