Cuticle architecture and mechanical properties: a functional relationship delineated through correlated multimodal imaging.

Cuticles are multifunctional hydrophobic biocomposites that protect the aerial organs of plants. During plant development, plant cuticles must accommodate different mechanical constraints combining extensibility and stiffness, and the corresponding relationships with their architecture are unknown. Recent data showed a fine-tuning of cuticle architecture during fruit development, with several chemical clusters which raise the question of how they impact the mechanical properties of cuticles. We investigated the in-depth nanomechanical properties of tomato (Solanum lycopersicum) fruit cuticle from early development to ripening, in relation to chemical and structural heterogeneities by developing a correlative multimodal imaging approach. Unprecedented sharps heterogeneities were evidenced including an in-depth mechanical gradient and a 'soft' central furrow that were maintained throughout the plant development despite the overall increase in elastic modulus. In addition, we demonstrated that these local mechanical areas are correlated to chemical and structural gradients. This study shed light on fine-tuning of mechanical properties of cuticles through the modulation of their architecture, providing new insight for our understanding of structure-function relationships of plant cuticles and for the design of bioinspired material.

The conformation of glutenin polymers in wheat grain: some genetic and environmental factors associated with this important characteristic.

In a previous study we used asymmetric-flow field-flow fractionation to determine the polymer mass (Mw), gyration radius (Rw) and the polydispersity index of glutenin polymers (GPs) in wheat (Triticum aestivum). Here, using the same multi-location trials (4 years, 11 locations, and 192 cultivars), we report the factors that are associated with the conformation (Conf) of the polymers, which is the slope of Log(Rw) versus a function of Log(Mw). We found that Conf varied between 0.285 and 0.740, it had low broad-sense heritability (H2=16.8), and it was significantly influenced by the temperature occurring over the last month of grain filling. Higher temperatures were found to increase Rw and the compactness and sphericity of GPs. Alleles for both high- and low-molecular-weight glutenin subunits had a significant influence on the Conf value. Assuming a Gaussian distribution for Mw, the number of polymers present in wheat grains was computed for different kernel weights and protein concentrations, and it was found to exceed 1012 GPs per grain. Using atomic force microscopy and cryo-TEM, images of GPs were obtained for the first time. Under higher average temperature, GPs became larger and more spherical and consequently less prone to rapid hydrolysis. We propose some orientations that could be aimed at potentially reducing the impact of numerous GPs on people suffering from non-celiac gluten sensitivity.

The cutin polymer matrix undergoes a fine architectural tuning from early tomato fruit development to ripening.

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.

Dextran-based polyelectrolyte multilayers: Effect of charge density on film build-up and morphology.

We have studied the growth process of thin polyelectrolyte (PE) films fabricated by the layer-by-layer assembly (LbL) and composed of Dextran sulfate with high (DexS H) and low (DexS L) sulfation rate and poly(allylamine hydrochloride) (PAH). Film growths were monitored by combining Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), Surface Plasmon Resonance (SPR) and Atomic Force Microscopy (AFM). Even though, the two films growth up to 10 bilayers, QCM-D showed that polyelectrolyte pairs do not display similar behaviours. (PAH/DexS H) systems lead to linear growth, i.e. amounts deposited increase both for PAH and DexS H, while the PAH/DexS L pair generated zig-zag shaped asymmetric growth. Film water contents were determined by QCM-D solvent exchange and SPR experiments. DexS L contains less water than DexS H and in agreement with the QCM-D dissipation values that suggest the formation of more rigid films in the case of DexS L than DexS H. Surface morphology investigated by AFM display distinct surface patterns since DexS H form thin films with fibril-like morphology covering all the surface while heterogeneous films with "puddle-like" aggregates were imaged in the case of DexS L. Difference of charge compensation and charge neutralisation between both systems likely lead to dissimilar growth mechanisms that are tentatively proposed in this paper.

BdERECTA controls vasculature patterning and phloem-xylem organization in Brachypodium distachyon.

BACKGROUND: The vascular system of plants consists of two main tissue types, xylem and phloem. These tissues are organized into vascular bundles that are arranged into a complex network running through the plant that is essential for the viability of land plants. Despite their obvious importance, the genes involved in the organization of vascular tissues remain poorly understood in grasses. RESULTS: We studied in detail the vascular network in stems from the model grass Brachypodium distachyon (Brachypodium) and identified a large set of genes differentially expressed in vascular bundles versus parenchyma tissues. To decipher the underlying molecular mechanisms of vascularization in grasses, we conducted a forward genetic screen for abnormal vasculature. We identified a mutation that severely affected the organization of vascular tissues. This mutant displayed defects in anastomosis of the vascular network and uncommon amphivasal vascular bundles. The causal mutation is a premature stop codon in ERECTA, a LRR receptor-like serine/threonine-protein kinase. Mutations in this gene are pleiotropic indicating that it serves multiple roles during plant development. This mutant also displayed changes in cell wall composition, gene expression and hormone homeostasis. CONCLUSION: In summary, ERECTA has a pleiotropic role in Brachypodium. We propose a major role of ERECTA in vasculature anastomosis and vascular tissue organization in Brachypodium.

Influence of the carbohydrate-binding module on the activity of a fungal AA9 lytic polysaccharide monooxygenase on cellulosic substrates.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Cellulose nanocrystals-starch nanocomposites produced by extrusion: Structure and behavior in physiological conditions.

Different amounts of cellulose nanocrystals (CNCs) were added to glycerol-plasticized thermoplastic starch (TPS) to obtain bio-based nanocomposites. First, nanocomposites are prepared by extrusion and their structure is studied at different scales using WAXS (Wide Angle X-ray Scattering) and solid-state NMR (Nuclear Magnetic Resonance) for local/crystalline organization, AF4 (Asymmetrical Flow Field-Flow Fractionation) for molecular weight and chain length, and SEM (Scanning Electron Microscopy) for the morphology at a larger scale. Then, relevant mechanical properties and behavior in physiological conditions (swelling, enzymatic degradation) are characterized. The results show that the incorporation of cellulose nanocrystals up to 2.5 wt% causes a mechanical reinforcement as determined by DMTA (Dynamic Mechanical Thermal Analysis) and reduces the swelling and the enzymatic degradation of the materials compared to reference TPS. This could be linked to the formation of starch-cellulose hydrogen and hydroxyl bonds. Conversely, above 5 wt% CNC content nanocrystals seem to aggregate which in turn worsens the behavior in physiological conditions.

Inactivation of LACCASE8 and LACCASE5 genes in Brachypodium distachyon leads to severe decrease in lignin content and high increase in saccharification yield without impacting plant integrity.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

AFM-Nano Manipulation of Plasmonic Molecules Used as "Nano-Lens" to Enhance Raman of Individual Nano-Objects.

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.