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Today, plant fibers are considered as an important new renewable resource that can compete with some synthetic fibers, such as glass, in fiber-reinforced composites. In previous works, it was noted that the pectin-enriched middle lamella (ML) is a weak point in the fiber bundles for plant fiber-reinforced composites. ML is strongly bonded to the primary walls of the cells to form a complex layer called the compound middle lamella (CML). In a composite, cracks preferentially propagate along and through this layer when a mechanical loading is applied. In this work, middle lamellae of several plant fibers of different origin (flax, hemp, jute, kenaf, nettle, and date palm leaf sheath), among the most used for composite reinforcement, are investigated by atomic force microscopy (AFM). The peak-force quantitative nanomechanical property mapping (PF-QNM) mode is used in order to estimate the indentation modulus of this layer. AFM PF-QNM confirmed its potential and suitability to mechanically characterize and compare the stiffness of small areas at the micro and nanoscale level, such as plant cell walls and middle lamellae. Our results suggest that the mean indentation modulus of ML is in the range from 6 GPa (date palm leaf sheath) to 16 GPa (hemp), depending on the plant considered. Moreover, local cell-wall layer architectures were finely evidenced and described.

Previous studies have shown the interest in using Atomic Force Microscopy (AFM) to investigate the parietal mechanicalproperties of hemp or flax fibres, in correlation with their structure or maturity (1–3), especially in PeakForce QuantitativeNanomechanical Property Mapping (PF-QNM) mode. However, this technique is relatively new, and its application is oftenlimited to the engineering and biological fields, but the small quantity of the sample required make it a useful tool also to studymaterials from the cultural heritage. In this research, two yarns of few centimetres were taken from the back of four Italianpaintings of the art gallery of Ascoli Piceno, dated around 1600-1700, and one yarn of each pair was selected for the AFMmechanical characterization. PF-QNM analysis was performed with a Multimode AFM (Bruker Corporation, USA) equipped witha RTESPA-525 probe (Bruker Corporation, USA) and a maximum load of 200 nN; the cantilever was carefully calibrated using theSader method and the indentation modulus of Highly Oriented Pyrolytic Graphite (HOPG) was taken as reference.

Tension wood (TW) is produced by temperate hardwood trees in order to support theirincreasing weight, orient their axes and cope with environmental cues such as wind. Poplar TWfibres harbour a supplemental layer, the G-layer, rich in crystalline cellulose, containing matrixpolysaccharides but no lignin. The tensile force responsible for the specific mechanicalproperties of TW originates from the G-layer and is transmitted to cellulose microfibrils soonafter their deposition, during G-fibre maturation (Clair et al, 2011). This force is likely tooriginate from physical changes in the high porosity hydrogel recently identified in the G-layer.RG-I type pectins appear as good candidate molecules responsible for the formation of this gel.Indeed, during G-layer maturation, LM5 labelling (specific to RG-I side chains) decreased,while RU1 labelling (specific to RG-I backbone) increased (Guedes et al, 2017). This suggesteda hydrolysis of the RG-I side chains during G-fibre maturation possibly by a β-galactosidase asdemonstrated in flax phloem fibers (Roach et al, 2011). Flax phloem fibres and TW G-fibresexhibit many similar features and, in flax, it has been shown that the hydrolysis of the sidechains of RG-I type pectins was associated to the very peculiar mechanical properties of bastfibres.In order to determine if RG-I pectins were effectively involved in the building of the G-layertensile force, we carried out different measurements on a common sampling during G-fibredevelopment: i) β-galactosidase activities using a histochemical test, ii) the evolution of RG-Iimmunolabelling profiles using LM5 and RU1 as probes and iii) the stiffening of the differentcell wall layers using Atomic Force Microscopy (AFM). We found a good correlation betweenβ-galactosidase activities and RG-I immunological labelling but we failed to establish a directassociation between RG-I hydrolysis and cell wall stiffening.

The aim of the present study is to improve the understanding of the evolution of flax fibre(Linum usitatissimum L.) ultrastructure and properties caused by the retting process at the cellwall scale. The dew retting is a natural bioprocess, where the various microorganisms presentin the soil colonize the flax stems. The enzymes they secrete make it easier to extract the bastfibre bundles during the scutching stage.In this study, six different stages of retting were investigated. Differences between rettingstage 1 (R1, earlier) and the retting stage 6 (R6, over retted) are +17% and +23% with AFMPF-QNM and nanoindentation measurements, respectively.An over retting results in the loss of the cellulose that makes up the fibres (Placet et al. 2017),arguably due to secretion of cellulase. To obtain information on the supramolecular structureof cellulose fibrils in the plant cell walls, the model of Larsson and Wickholm (Larsson et al.,1997) could be used for deconvoluate the C4 region between 77 and 92 ppm. In addition, themechanical properties of flax cell walls during could be investigated by AFM Peak ForceQuantitative Nano-Mechanical property mapping (AFM PF-QNM) (Goudenhooft et al. 2018).Two complementary structural investigating technic will be presented, namely using X-RayDiffraction (XRD) and solid state Nuclear Magnetic Resonance (NMR). An estimation of thecellulose crystallinity index by XRD measurements, confirmed by NMR, shows an increaseof 8% in crystallinity with retting, mainly due to the disappearance of amorphous polymer. Inaddition, NMR investigations show a compaction of inaccessible cell wall polymers,combined with an increase in the relaxation times of the C4 carbon.One highlight of the work is that NMR results show a significant evolution in the cell wallultrastructure of flax during the retting stages. The improvement observed in the mechanicalperformance of flax cell walls can be explained by the compaction of inaccessible zones.Based on the NMR results, a schematic representation of the flax cellulose elementarymicrofibrils structure of R1 and R6 was hypothesised. In this flax cellulose evolution modelduring retting, we have considered in particular the lateral fibril dimension (LFD) and the thelateral fibril aggregate dimension (LFAD). As illustration, the LFD of samples R1 and R6 are4.1 and 4.5 nm, respectively, while the LFAD slightly decreases during retting from 17.1 nmfor R1 to 16.3 nm for R6.

ABSTRACTThe aim of the present study is to improve the understanding of the evolution of fibreproperties and ultrastructure caused by the retting process at the plant cell wall scale. Twocomplementary structural investigating technic are presented here, namely using X-RayDiffraction (XRD) and solid state Nuclear Magnetic Resonance (NMR). An estimation of thecellulose crystallinity index by XRD measurements, confirmed by NMR, shows an increaseof 8% in crystallinity with retting mainly due to the disappearance of amorphous polymer. Inaddition, NMR investigations show a compaction of inaccessible cell wall polymers,combined with an increase in the relaxation times of the C4 carbon. This densificationprovides a structural explanation for the observed improvement in mechanical performance ofthe secondary wall of flax fibres during the field retting process.INTRODUCTIONDuring retting, the various microorganisms present in the soil colonize the flax stems. Theenzymes they secrete, and particularly polygalacturonases, gradually degrade the pecticcompounds located mainly in the middle lamellae of the fibre bundles. This damage to theplant tissues structure makes it easier to extract the fibre bundles during the scutching stage.An over retting results in the loss of the cellulose that makes up the fibres (Placet et al. 2017),arguably due to secretion of cellulase. To obtain information on the supramolecular structureof cellulose fibrils in the plant cell walls, the model of Larsson and Wickholm (Larsson et al.,1997) could be used for deconvoluate the C4 region between 77 and 92 ppm. In addition, themechanical properties of flax cell walls during could be investigated by AFM Peak ForceQuantitative Nano-Mechanical property mapping (AFM PF-QNM) (Goudenhooft et al. 2018).Here we explore the evolution of the flax cell wall behaviour during a controlled dew rettingphase. Structural and mechanical investigations were conducted at the cell wall scale using XRayDiffraction (XRD) and solid-state Nuclear Magnetic Resonance (NMR), whilenanomechanical measurements were performed by nanoindentation and AFM PF-QNM).RESULTS AND CONCLUSIONSThe macroscopic illustration of the flax lots is displayed in Fig 1. NMR results show asignificant evolution in the cell wall ultrastructure of flax during the retting stages. Theimprovement in the mechanical performance of plant cell walls can be explained by thecompaction of inaccessible zones (Fig 2).