Modification of a Marine Pine Kraft Lignin Sample by Enzymatic Treatment with a Pycnoporus cinnabarinus Laccase.

Here, we report work on developing an enzymatic process to improve the functionalities of industrial lignin. A kraft lignin sample prepared from marine pine was treated with the high-redox-potential laccase from the basidiomycete fungus Pycnoporus cinnabarinus at three different concentrations and pH conditions, and with and without the chemical mediator 1-hydroxybenzotriazole (HBT). Laccase activity was tested in the presence and absence of kraft lignin. The optimum pH of PciLac was initially 4.0 in the presence and absence of lignin, but at incubation times over 6 h, higher activities were found at pH 4.5 in the presence of lignin. Structural changes in lignin were investigated by Fourier-transform infrared spectroscopy (FTIR) with differential scanning calorimetry (DSC), and solvent-extractable fractions were analyzed using high-performance size-exclusion chromatography (HPSEC) and gas chromatography-mass spectrometry (GC-MS). The FTIR spectral data were analyzed with two successive multivariate series using principal component analysis (PCA) and ANOVA statistical analysis to identify the best conditions for the largest range of chemical modifications. DSC combined with modulated DSC (MDSC) revealed that the greatest effect on glass transition temperature (Tg) was obtained at 130 U g cm-1 and pH 4.5, with the laccase alone or combined with HBT. HPSEC data suggested that the laccase treatments led to concomitant phenomena of oligomerization and depolymerization, and GC-MS revealed that the reactivity of the extractable phenolic monomers depended on the conditions tested. This study demonstrates that P. cinnabarinus laccase can be used to modify marine pine kraft lignin, and that the set of analytical methods implemented here provides a valuable tool for screening enzymatic treatment conditions.

Dual Antioxidant Properties and Organic Radical Stabilization in Cellulose Nanocomposite Films Functionalized by In Situ Polymerization of Coniferyl Alcohol.

A new biobased material based on an original strategy using lignin model compounds as natural grafting additive on a nanocellulose surface through in situ polymerization of coniferyl alcohol by the Fenton reaction at two pH values was investigated. The structural and morphological properties of the materials at the nanoscale were characterized by a combination of analytical methods, including Fourier transform infrared spectroscopy, liquid chromatography combined with mass spectrometry, nuclear molecular resonance spectroscopy, electron paramagnetic resonance spectroscopy, water sorption capacity by dynamic vapor sorption, and atomic force microscopy (topography and indentation modulus measurements). Finally, the usage properties, such as antioxidant properties, were evaluated in solution and the nanostructured casted films by radical 2,2'-diphenyl-1-picrylhydrazyl (DPPH•) scavenging tests. We demonstrate the structure-function relationships of these advanced CNC-lignin films and describe their dual functionalities and characteristics, namely, their antioxidant properties and the presence of persistent phenoxy radicals within the material.

Real Time and Quantitative Imaging of Lignocellulosic Films Hydrolysis by Atomic Force Microscopy Reveals Lignin Recalcitrance at Nanoscale.

Lignocellulosic biomass is considered as a sustainable source of energy and chemicals, but its recalcitrance to bioconversion still limits its use. In this paper, a strategy based on two aspects was developed to improve our knowledge on the lignin recalcitrance to enzymatic hydrolysis. First, lignocellulosic films of cellulose nanofibrils (CNFs) with increasing content of lignin (up to 40%) were prepared. Thanks to in situ real time Atomic Force Microscopy (AFM) measurements during the hydrolysis and by comparison with biochemical assays, the use of such films allows to fully assess the importance of the lignin content and of the arrangement between CNFs and lignin on the hydrolysis efficiency. In a second time, contrary to other studies by AFM which only followed a specific structure during enzymatic processes mostly on simple systems (CNFs or cellulose nanocrystals), a quantitative analysis of in-situ time-lapse measurements was developed. It enables to accurately address lignocellulosic biomass recalcitrance mechanisms mediated by lignin at nanoscale. Such analysis could pave the way for the use of a quantitative criteria to visualize in situ deconstruction of complex lignocellulosic substrates. Coupling the use of lignocellulosic films and dynamical AFM quantitative analysis to follow the evolution of the structure at nanoscale might lead to an effective targeting of new promising bioconversion strategies.

Langmuir-Blodgett Procedure to Precisely Control the Coverage of Functionalized AFM Cantilevers for SMFS Measurements: Application with Cellulose Nanocrystals.

Atomic force microscopy (AFM) experiments with functionalized tips are currently one of the most powerful tools to locally measure adhesion forces via single-molecule force spectroscopy (SMFS) measurements. The main difficulty is to precisely control the attachment of biomolecules to the cantilever. Different chemistry procedures have been developed including the use of spacer molecules. Even if a process works well for small biomolecules such as antibodies, issues remain regarding nanoparticles or larger objects such as cellulose nanocrystals because it is difficult to precisely control their coverage and homogeneity. In this work, an original procedure based on the Langmuir-Blodgett (LB) technique was implemented for lever functionalization with cellulose nanocrystals and compared with classical chemical strategies. LB shows to be almost 6.0-fold more efficient than chemical procedure in terms of cellulose nanocrystals coverage attachment. Moreover, the LB technology provides advantage of not requiring linker molecules, which could have detrimental effects such as overestimation of the interaction force. The structural characterization and SMFS measurements of lignocellulosic polymers show that this strategy enables the precise control of the lever coverage, which improves the accuracy of the adhesion measurements. Such methodology is expected to strongly impact the AFM tip/tipless functionalization and SMFS measurements in different fields.

Distribution of Lignin, Hemicellulose, and Arabinogalactan Protein in Hemp Phloem Fibers.

The distribution of lignin, 8-5' and 8-8' linked lignin substructure, and noncellulosic polysaccharides in hemp (Cannabis sativa L.) phloem fibers were explored based on histochemical and immunological methods. Ultraviolet absorption and potassium permanganate staining were observed mainly in the compound middle lamella (CML) and S1 layers, and rarely in the G-layer of phloem fibers, suggesting that lignin concentration is high at the CML and S1 layers, and very low at the G-layer of hemp fibers. Acriflavine staining, uniform KM1 labeling (8-5' linked lignin substructure), and no KM2 labeling (8-8' linked structure) were observed in the G-layer, suggesting that there is a small amount of lignin-like compound with 8-5' linked structure in the G-layer. In addition, some fiber cells showed a multilayered structure. Uniform arabinogalactan protein (AGP) labeling was observed on the S1 layers and G-layers using JIM14, but little appeared in the CML of hemp fibers, indicating that these layers of the phloem fibers contain AGP. Immunogold labeling of xylan (LM11) and glucomannan (LM21) showed that xylan and glucomannan were mainly present in the S1 layers and the G-layers, respectively. In some phloem fibers, LM21 immunofluorescence labeling showed multilayered structure, suggesting the heterogeneous distribution of glucomannan.

Ectopic lignification in the flax lignified bast fiber1 mutant stem is associated with tissue-specific modifications in gene expression and cell wall composition.

Histochemical screening of a flax ethyl methanesulfonate population led to the identification of 93 independent M2 mutant families showing ectopic lignification in the secondary cell wall of stem bast fibers. We named this core collection the Linum usitatissimum (flax) lbf mutants for lignified bast fibers and believe that this population represents a novel biological resource for investigating how bast fiber plants regulate lignin biosynthesis. As a proof of concept, we characterized the lbf1 mutant and showed that the lignin content increased by 350% in outer stem tissues containing bast fibers but was unchanged in inner stem tissues containing xylem. Chemical and NMR analyses indicated that bast fiber ectopic lignin was highly condensed and rich in G-units. Liquid chromatography-mass spectrometry profiling showed large modifications in the oligolignol pool of lbf1 inner- and outer-stem tissues that could be related to ectopic lignification. Immunological and chemical analyses revealed that lbf1 mutants also showed changes to other cell wall polymers. Whole-genome transcriptomics suggested that ectopic lignification of flax bast fibers could be caused by increased transcript accumulation of (1) the cinnamoyl-CoA reductase, cinnamyl alcohol dehydrogenase, and caffeic acid O-methyltransferase monolignol biosynthesis genes, (2) several lignin-associated peroxidase genes, and (3) genes coding for respiratory burst oxidase homolog NADPH-oxidases necessary to increase H2O2 supply.

Functional analyses of cellulose synthase genes in flax (Linum usitatissimum) by virus-induced gene silencing.

Flax (Linum usitatissimum) bast fibres are located in the stem cortex where they play an important role in mechanical support. They contain high amounts of cellulose and so are used for linen textiles and in the composite industry. In this study, we screened the annotated flax genome and identified 14 distinct cellulose synthase (CESA) genes using orthologous sequences previously identified. Transcriptomics of 'primary cell wall' and 'secondary cell wall' flax CESA genes showed that some were preferentially expressed in different organs and stem tissues providing clues as to their biological role(s) in planta. The development for the first time in flax of a virus-induced gene silencing (VIGS) approach was used to functionally evaluate the biological role of different CESA genes in stem tissues. Quantification of transcript accumulation showed that in many cases, silencing not only affected targeted CESA clades, but also had an impact on other CESA genes. Whatever the targeted clade, inactivation by VIGS affected plant growth. In contrast, only clade 1- and clade 6-targeted plants showed modifications in outer-stem tissue organization and secondary cell wall formation. In these plants, bast fibre number and structure were severely impacted, suggesting that the targeted genes may play an important role in the establishment of the fibre cell wall. Our results provide new fundamental information about cellulose biosynthesis in flax that should facilitate future plant improvement/engineering.

Combined hemicellulolytic and phenoloxidase activities of Thermobacillus xylanilyticus enable growth on lignin-rich substrates and the release of phenolic molecules.

Major challenge in biorefineries is the use of all lignocellulosic components, particularly lignins. In this study, Thermobacillus xylanilyliticus grew on kraft lignin, steam-exploded and native wheat straws produced different sets of phenoloxidases and xylanases, according to the substrate. After growth, limited lignin structural modifications, mainly accompanied by a decrease in phenolic acids was observed by Nuclear Magnetic Resonance spectroscopy. The depletion of p-coumaric acid, vanillin and p-hydroxybenzaldehyde combined to vanillin production in the culture media indicated that the bacterium can transform some phenolic compounds. Proteomic approaches allowed the identification of 29 to 33 different hemicellulases according to the substrates. Twenty oxidoreductases were differentially expressed between kraft lignin and steam-exploded wheat straw. These oxidoreductases may be involved in lignin and aromatic compound utilization and detoxification. This study highlights the potential value of Thermobacillus xylanilyticus and its enzymes in the simultaneous valorization of hemicellulose and phenolic compounds from lignocelluloses.

PT-Flax (phenotyping and TILLinG of flax): development of a flax (Linum usitatissimum L.) mutant population and TILLinG platform for forward and reverse genetics.

BACKGROUND: Flax (Linum usitatissimum L.) is an economically important fiber and oil crop that has been grown for thousands of years. The genome has been recently sequenced and transcriptomics are providing information on candidate genes potentially related to agronomically-important traits. In order to accelerate functional characterization of these genes we have generated a flax EMS mutant population that can be used as a TILLinG (Targeting Induced Local Lesions in Genomes) platform for forward and reverse genetics. RESULTS: A population of 4,894 M2 mutant seed families was generated using 3 different EMS concentrations (0.3%, 0.6% and 0.75%) and used to produce M2 plants for subsequent phenotyping and DNA extraction. 10,839 viable M2 plants (4,033 families) were obtained and 1,552 families (38.5%) showed a visual developmental phenotype (stem size and diameter, plant architecture, flower-related). The majority of these families showed more than one phenotype. Mutant phenotype data are organised in a database and can be accessed and searched at UTILLdb (http://urgv.evry.inra.fr/UTILLdb). Preliminary screens were also performed for atypical fiber and seed phenotypes. Genomic DNA was extracted from 3,515 M2 families and eight-fold pooled for subsequent mutant detection by ENDO1 nuclease mis-match cleavage. In order to validate the collection for reverse genetics, DNA pools were screened for two genes coding enzymes of the lignin biosynthesis pathway: Coumarate-3-Hydroxylase (C3H) and Cinnamyl Alcohol Dehydrogenase (CAD). We identified 79 and 76 mutations in the C3H and CAD genes, respectively. The average mutation rate was calculated as 1/41 Kb giving rise to approximately 9,000 mutations per genome. Thirty-five out of the 52 flax cad mutant families containing missense or codon stop mutations showed the typical orange-brown xylem phenotype observed in CAD down-regulated/mutant plants in other species. CONCLUSIONS: We have developed a flax mutant population that can be used as an efficient forward and reverse genetics tool. The collection has an extremely high mutation rate that enables the detection of large numbers of independant mutant families by screening a comparatively low number of M2 families. The population will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in flax.

Natural hypolignification is associated with extensive oligolignol accumulation in flax stems.

Flax (Linum usitatissimum) stems contain cells showing contrasting cell wall structure: lignified in inner stem xylem tissue and hypolignified in outer stem bast fibers. We hypothesized that stem hypolignification should be associated with extensive phenolic accumulation and used metabolomics and transcriptomics to characterize these two tissues. (1)H nuclear magnetic resonance clearly distinguished inner and outer stem tissues and identified different primary and secondary metabolites, including coniferin and p-coumaryl alcohol glucoside. Ultrahigh-performance liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry aromatic profiling (lignomics) identified 81 phenolic compounds, of which 65 were identified, to our knowledge, for the first time in flax and 11 for the first time in higher plants. Both aglycone forms and glycosides of monolignols, lignin oligomers, and (neo)lignans were identified in both inner and outer stem tissues, with a preponderance of glycosides in the hypolignified outer stem, indicating the existence of a complex monolignol metabolism. The presence of coniferin-containing secondary metabolites suggested that coniferyl alcohol, in addition to being used in lignin and (neo)lignan formation, was also utilized in a third, partially uncharacterized metabolic pathway. Hypolignification of bast fibers in outer stem tissues was correlated with the low transcript abundance of monolignol biosynthetic genes, laccase genes, and certain peroxidase genes, suggesting that flax hypolignification is transcriptionally regulated. Transcripts of the key lignan genes Pinoresinol-Lariciresinol Reductase and Phenylcoumaran Benzylic Ether Reductase were also highly abundant in flax inner stem tissues. Expression profiling allowed the identification of NAC (NAM, ATAF1/2, CUC2) and MYB transcription factors that are likely involved in regulating both monolignol production and polymerization as well as (neo)lignan production.

Modeling progression of fluorescent probes in bioinspired lignocellulosic assemblies.

Progression of enzymes in lignocellulosic biomass is a crucial parameter in biorefinery processes, and it appears to be one of the limiting factors in optimizing lignocellulose degradation. In order to assay the importance of the chemical and structural features of the substrate matrix on enzyme mobility, we have designed bioinspired model assemblies of secondary plant cell walls, which have been used to measure the mobility of fluorescent probes while modifying different parameters (probe size, water content, polysaccharide concentration). The results were used to construct a model of probe mobility and to rank the parameters in order of importance. Water content and probe size were shown to have the greatest effect. Although these assemblies are simplified templates of the plant cell walls, our strategy paves the way for proposing new approaches for optimizing biomass saccharification, such as selecting enzymes with suitable properties.

Automated quantification of fluorescence and morphological changes in pretreated wood cells by fluorescence macroscopy.

BACKGROUND: Lignocellulosic biomass is a complex network of polysaccharides and lignin that requires a pretreatment step to overcome recalcitrance and optimize valorisation into biobased products. Pretreatment of biomass induces chemical and morphological changes. Quantification of these changes is critical to understand biomass recalcitrance and to predict lignocellulose reactivity. In this study, we propose an automated method for the quantification of chemical and morphological parameters through fluorescence macroscopy, which was applied on wood samples (spruce, beechwood) pretreated with steam explosion. RESULTS: Results in fluorescence macroscopy highlighted the impact of steam explosion on spruce and beechwood: fluorescence intensity of samples was highly altered, especially for the most severe conditions. Morphological changes were also revealed: shrinkage of cells and deformation of cell walls manifested as the loss of rectangularity or circular shape, for tracheids in spruce and vessels in beechwood respectively. Quantification of fluorescence intensity of cell walls and quantification of morphological parameters related to cell lumens were carried out accurately by applying the automated method onto the macroscopic images. The results showed that lumens area and circularity could be considered as complementary markers of cell deformation, and that fluorescence intensity of the cell walls could be related to morphological changes and to the conditions of pretreatment. CONCLUSIONS: The developed procedure allows simultaneous and effective quantification of morphological parameters and fluorescence intensity of the cell walls. This approach can be applied to fluorescence macroscopy as well as other imaging techniques and provides encouraging results towards the understanding of biomass architecture.

Influence of Surface Chemistry of Fiber and Lignocellulosic Materials on Adhesion Properties with Polybutylene Succinate at Nanoscale.

The production of bio-based composites with enhanced characteristics constitutes a strategic action to minimize the use of fossil fuel resources. The mechanical performances of these materials are related to the specific properties of their components, as well as to the quality of the interface between the matrix and the fibers. In a previous research study, it was shown that the polarity of the matrix played a key role in the mechanisms of fiber breakage during processing, as well as on the final properties of the composite. However, some key questions remained unanswered, and new investigations were necessary to improve the knowledge of the interactions between a lignocellulosic material and a polar matrix. In this work, for the first time, atomic force microscopy based on force spectroscopy measurements was carried out using functionalized tips to characterize the intermolecular interactions at the single molecule level, taking place between poly(butylene succinate) and four different plant fibers. The efficiency of the tip functionalization was checked out by scanning electron microscopy and energy-dispersive X-ray spectroscopy, whereas the fibers chemistry was characterized by Fourier-transform infrared spectroscopy. Larger interactions at the nanoscale level were found between the matrix and hypolignified fibers compared to lignified ones, as in control experiments on single lignocellulosic polymer films. These results could significantly aid in the design of the most appropriate composite composition depending on its final use.

Identification of new potential molecular actors related to fiber quality in flax through Omics.

One of the biggest challenges for a more widespread utilization of plant fibers is to better understand the different molecular factors underlying the variability in fineness and mechanical properties of both elementary and scutched fibers. Accordingly, we analyzed genome-wide transcription profiling from bast fiber bearing tissues of seven different flax varieties (4 spring, 2 winter fiber varieties and 1 winter linseed) and identified 1041 differentially expressed genes between varieties, of which 97 were related to cell wall metabolism. KEGG analysis highlighted a number of different enriched pathways. Subsequent statistical analysis using Partial Least-Squares Discriminant Analysis showed that 73% of the total variance was explained by the first 3 X-variates corresponding to 56 differentially expressed genes. Calculation of Pearson correlations identified 5 genes showing a strong correlation between expression and morphometric data. Two-dimensional gel proteomic analysis on the two varieties showing the most discriminant and significant differences in morphometrics revealed 1490 protein spots of which 108 showed significant differential abundance. Mass spectrometry analysis successfully identified 46 proteins representing 32 non-redundant proteins. Statistical clusterization based on the expression level of genes corresponding to the 32 proteins showed clear discrimination into three separate clusters, reflecting the variety type (spring-/winter-fiber/oil). Four of the 32 proteins were also highly correlated with morphometric features. Examination of predicted functions for the 9 (5 + 4) identified genes highlighted lipid metabolism and senescence process. Calculation of Pearson correlation coefficients between expression data and retted fiber mechanical measurements (strength and maximum force) identified 3 significantly correlated genes. The genes were predicted to be connected to cell wall dynamics, either directly (Expansin-like protein), or indirectly (NAD(P)-binding Rossmann-fold superfamily protein). Taken together, our results have allowed the identification of molecular actors potentially associated with the determination of both in-planta fiber morphometrics, as well as ex-planta fiber mechanical properties, both of which are key parameters for elementary fiber and scutched fiber quality in flax.

Characterization of arabinoxylan/cellulose nanocrystals gels to investigate fluorescent probes mobility in bioinspired models of plant secondary cell wall.

Biomass from lignocellulose (LC) is a highly complex network of cellulose, hemicellulose, and lignin, which is considered to be a sustainable source of fuels, chemicals and materials. To achieve an environmental friendly and efficient LC upgrading, a better understanding of the LC architecture is necessary. We have devised some LC bioinspired model systems, based on arabinoxylan gels, in which mobility of dextrans and BSA grafted with FITC has been studied by FRAP. Our results indicate that the probes diffusion is more influenced by their hydrodynamic radius than by the gel mesh size. The addition of some cellulose nanocrystals (CNCs) decreases polymer chain mobility and has low effect on the probes diffusion, suggesting that the gels are better organized in the presence of CNCs, as shown by rheological measurements and scanning electronic microscopy observations. This demonstrates that the FRAP analysis can be a powerful tool to screen the architecture of LC model systems.

Natural organic UV-absorbent coatings based on cellulose and lignin: designed effects on spectroscopic properties.

Novel nanocomposite coatings composed of cellulose nanocrystals (CNCs) and lignin (either synthetic or fractionated from spruce and corn stalks) were prepared without chemical modification or functionalization (via covalent attachment) of one of the two biopolymers. The spectroscopic properties of these coatings were investigated by UV-visible spectrophotometry and spectroscopic ellipsometry. When using the appropriate weight ratio of CNC/lignin (R), these nanocomposite systems exhibited high-performance optical properties, high transmittance in the visible spectrum, and high blocking in the UV spectrum. Atomic force microscopy analysis demonstrated that these coatings were smooth and homogeneous, with visible dispersed lignin nodules in a cellulosic matrix. It was also demonstrated that the introduction of nanoparticles into the medium increases the weight ratio and the CNC-specific surface area, which allows better dispersion of the lignin molecules throughout the solid film. Consequently, the larger molecular expansion of these aromatic polymers on the surface of the cellulosic nanoparticles dislocates the π-π aromatic aggregates, which increases the extinction coefficient and decreases the transmittance in the UV region. These nanocomposite coatings were optically transparent at visible wavelengths.

A thermostable feruloyl-esterase from the hemicellulolytic bacterium Thermobacillus xylanilyticus releases phenolic acids from non-pretreated plant cell walls.

A gene (Tx-est1) encoding a thermostable feruloyl-esterase was isolated from the genome of the gram-positive hemicellulolytic thermophilic bacterium Thermobacillus xylanilyticus. This gene contains an open reading frame of 1,020 bp encoding a protein with molecular mass of 37.4 kDa, similar to feruloyl-esterases from cellulolytic bacteria and fungi. The recombinant enzyme Tx-Est1 was expressed and produced in Escherichia coli. Tx-Est1 contains the conserved putative lipase residues Ser 202, Asp 287, and His 322 which act as catalytic triad in its C-terminus part. Purified Tx-Est1 was active against phenolic acid derivatives and stable at high temperatures. Optimal activity was observed at 65 °C and the optimal pH was around 8.5. The kinetic parameters of the esterase were determined on various substrates. The enzyme displayed activity against methyl esters of hydrocinnamic acids and feruloylated arabino-xylotetraose, exhibiting high specificity and affinity for the latter. Our results showed that Tx-Est1 is a thermostable feruloyl-esterase which could be useful to hydrolyze arabinoxylans from graminaceous plant cell walls as the enzyme is able to release phenolic acids from a lignocellulose biomass.

Development and validation of a flax (Linum usitatissimum L.) gene expression oligo microarray.

BACKGROUND: Flax (Linum usitatissimum L.) has been cultivated for around 9,000 years and is therefore one of the oldest cultivated species. Today, flax is still grown for its oil (oil-flax or linseed cultivars) and its cellulose-rich fibres (fibre-flax cultivars) used for high-value linen garments and composite materials. Despite the wide industrial use of flax-derived products, and our actual understanding of the regulation of both wood fibre production and oil biosynthesis more information must be acquired in both domains. Recent advances in genomics are now providing opportunities to improve our fundamental knowledge of these complex processes. In this paper we report the development and validation of a high-density oligo microarray platform dedicated to gene expression analyses in flax. RESULTS: Nine different RNA samples obtained from flax inner- and outer-stems, seeds, leaves and roots were used to generate a collection of 1,066,481 ESTs by massive parallel pyrosequencing. Sequences were assembled into 59,626 unigenes and 48,021 sequences were selected for oligo design and high-density microarray (Nimblegen 385K) fabrication with eight, non-overlapping 25-mers oligos per unigene. 18 independent experiments were used to evaluate the hybridization quality, precision, specificity and accuracy and all results confirmed the high technical quality of our microarray platform. Cross-validation of microarray data was carried out using quantitative qRT-PCR. Nine target genes were selected on the basis of microarray results and reflected the whole range of fold change (both up-regulated and down-regulated genes in different samples). A statistically significant positive correlation was obtained comparing expression levels for each target gene across all biological replicates both in qRT-PCR and microarray results. Further experiments illustrated the capacity of our arrays to detect differential gene expression in a variety of flax tissues as well as between two contrasted flax varieties. CONCLUSION: All results suggest that our high-density flax oligo-microarray platform can be used as a very sensitive tool for analyzing gene expression in a large variety of tissues as well as in different cultivars. Moreover, this highly reliable platform can also be used for the quantification of mRNA transcriptional profiling in different flax tissues.

Targeted Metagenomics of Retting in Flax: The Beginning of the Quest to Harness the Secret Powers of the Microbiota.

The mechanical and chemical properties of natural plant fibers are determined by many different factors, both intrinsic and extrinsic to the plant, during growth but also after harvest. A better understanding of how all these factors exert their effect and how they interact is necessary to be able to optimize fiber quality for use in different industries. One important factor is the post-harvest process known as retting, representing the first step in the extraction of bast fibers from the stem of species such as flax and hemp. During this process microorganisms colonize the stem and produce hydrolytic enzymes that target cell wall polymers thereby facilitating the progressive destruction of the stem and fiber bundles. Recent advances in sequencing technology have allowed researchers to implement targeted metagenomics leading to a much better characterization of the microbial communities involved in retting, as well as an improved understanding of microbial dynamics. In this paper we review how our current knowledge of the microbiology of retting has been improved by targeted metagenomics and discuss how related '-omics' approaches might be used to fully characterize the functional capability of the retting microbiome.

Multiscale modeling of microbial degradation of outer tissues of fiber-crop stems during the dew retting process.

Dew retting of fiber crops, such as hemp or flax, in the field after harvest promotes the microbial biodegradation of the tissues surrounding cellulosic fibers, which helps preserve the quality of fibers during their extraction and valorization for industry. This bioprocess is currently the bottleneck for plant fiber valorization because it is empirically managed and its controlling factors have not been properly quantified. A novel multiscale model representing tissue and polymer biodegradation was developed to simulate microbial growth on the stem during retting. The model was evaluated against experimental hemp retting data. It consistently simulated the mass loss of eight plant polymers belonging to two tissues of the stem outer layer, i.e., parenchyma and fiber bundles. Microbial growth was modeled by Monod equations and modulated by the functions of temperature and moisture. This work provides a tool for gaining more insights into microorganism behavior during retting under local climate conditions.