Mixed-Linkage Glucan Is the Main Carbohydrate Source and Starch Is an Alternative Source during Brachypodium Grain Germination.

Seeds of the model grass Brachypodium distachyon are unusual because they contain very little starch and high levels of mixed-linkage glucan (MLG) accumulated in thick cell walls. It was suggested that MLG might supplement starch as a storage carbohydrate and may be mobilised during germination. In this work, we observed massive degradation of MLG during germination in both endosperm and nucellar epidermis. The enzymes responsible for the MLG degradation were identified in germinated grains and characterized using heterologous expression. By using mutants targeting MLG biosynthesis genes, we showed that the expression level of genes coding for MLG and starch-degrading enzymes was modified in the germinated grains of knocked-out cslf6 mutants depleted in MLG but with higher starch content. Our results suggest a substrate-dependent regulation of the storage sugars during germination. These overall results demonstrated the function of MLG as the main carbohydrate source during germination of Brachypodium grain. More astonishingly, cslf6 Brachypodium mutants are able to adapt their metabolism to the lack of MLG by modifying the energy source for germination and the expression of genes dedicated for its use.

Assembly of tomato fruit cuticles: a cross-talk between the cutin polyester and cell wall polysaccharides.

The cuticle is an essential and ubiquitous biological polymer composite covering aerial plant organs, whose structural component is the cutin polyester entangled with cell wall polysaccharides. The nature of the cutin-embedded polysaccharides (CEPs) and their association with cutin polyester are still unresolved Using tomato fruit as a model, chemical and enzymatic pretreatments combined with biochemical and biophysical methods were developed to compare the fine structure of CEPs with that of the noncutinized polysaccharides (NCPs). In addition, we used tomato fruits from cutin-deficient transgenic lines cus1 (cutin synthase 1) to study the impact of cutin polymerization on the fine structure of CEPs. Cutin-embedded polysaccharides exhibit specific structural features including a high degree of esterification (i.e. methylation and acetylation), a low ramification of rhamnogalacturonan (RGI), and a high crystallinity of cellulose. In addition to decreasing cutin deposition and polymerization, cus1 silencing induced a specific modification of CEPs, especially on pectin content, while NCPs were not affected. This new evidence of the structural specificities of CEPs and of the cross-talk between cutin polymerization and polysaccharides provides new hypotheses concerning the formation of these complex lipopolysaccharide edifices.

Correction: Cherkaoui, M., Lollier, V., Geairon, A., Bouder, A., Larré, C., Rogniaux, H., Jamet, E., Guillon, F. and Francin-Allami, M. Cell Wall Proteome of Wheat Grain Endosperm and Outer Layers at Two Key Stages of Early Development. Int. J. Mol. Sci. 2020, 21, 239.

The authors wish to make the following corrections to this paper [...].

Enhancement of corn stover conversion to carboxylates by extrusion and biotic triggers in solid-state fermentation.

Solid-state fermentation is a potential technology for developing lignocellulosic biomass-based biorefineries. This work dealt with solid-state fermentation for carboxylates production from corn stover, as building blocks for a lignocellulosic feedstock-based biorefinery. The effect of extrusion pretreatment, together with the action of a microbial consortia and hydrolytic enzymes as biotic triggers, was investigated on corn stover conversion, microbial metabolic pathways, and populations. The extrusion caused changes in the physical and morphological characteristics, without altering the biochemical composition of the corn stover. Extrusion also led to remarkable differences in the composition of the indigenous microbial population of the substrate. Consequently, it affected the structure of community developed after fermentation and the substrate conversion yield, which increased by 118% (from 23 ± 4 gCOD/kgVSi obtained with raw substrate to 51 ± 1 gCOD/kgVSi with extruded corn stover) with regard to self-fermentation experiments. The use of activated sludge as inoculum further increased the total substrate conversion into carboxylates, up to 60 ± 2 gCOD/kgVSi, and shaped the microbial communities (mainly composed of bacteria from the Clostridia and Bacteroidia classes) with subsequent homogenization of the fermentation pathways. The addition of hydrolytic enzymes into the reactors further increased the corn stover conversion, leading to a maximum yield of 142 ± 1 gCOD/kgVSi. Thus, extrusion pretreatment combined with the use of an inoculum and enzyme addition increased by 506% corn stover conversion into carboxylates. Beside biomass pretreatment, the results of this study indicated that biotic factor greatly impacted solid-state fermentation by shaping the microbial communities and related metabolic pathways.

Cell Wall Proteome of Wheat Grain Endosperm and Outer Layers at Two Key Stages of Early Development.

The cell wall is an important compartment in grain cells that fulfills both structural and functional roles. It has a dynamic structure that is constantly modified during development and in response to biotic and abiotic stresses. Non-structural cell wall proteins (CWPs) are key players in the remodeling of the cell wall during events that punctuate the plant life. Here, a subcellular and quantitative proteomic approach was carried out to identify CWPs possibly involved in changes in cell wall metabolism at two key stages of wheat grain development: the end of the cellularization step and the beginning of storage accumulation. Endosperm and outer layers of wheat grain were analyzed separately as they have different origins (maternal and seed) and functions in grains. Altogether, 734 proteins with predicted signal peptides were identified (CWPs). Functional annotation of CWPs pointed out a large number of proteins potentially involved in cell wall polysaccharide remodeling. In the grain outer layers, numerous proteins involved in cutin formation or lignin polymerization were found, while an unexpected abundance of proteins annotated as plant invertase/pectin methyl esterase inhibitors were identified in the endosperm. In addition, numerous CWPs were accumulating in the endosperm at the grain filling stage, thus revealing strong metabolic activities in the cell wall during endosperm cell differentiation, while protein accumulation was more intense at the earlier stage of development in outer layers. Altogether, our work gives important information on cell wall metabolism during early grain development in both parts of the grain, namely the endosperm and outer layers. The wheat cell wall proteome is the largest cell wall proteome of a monocot species found so far.

Cell Wall Proteome Investigation of Bread Wheat (Triticum Aestivum) Developing Grain in Endosperm and Outer Layers.

The remodeling of cell wall polysaccharides is controlled by cell wall proteins (CWPs) during the development of wheat grain. This work describes for the first time the cell wall proteomes of the endosperm and outer layers of the wheat developing grain, which have distinct physiological functions and technological uses. Altogether 636 nonredundant predicted CWPs are identified with 337 proteins in the endosperm and 594 proteins in the outer layers, among which 295 proteins are present in both tissues, suggesting both common and tissue specific remodeling activities. These proteins are distributed into eight functional classes. Approximatively a quarter of them were predicted to act on cell wall polysaccharides, with many glycosylhydrolases and also expansin, lyases, and carbohydrate esterases. Therefore, these results provide crucial data to go further in the understanding of relationship between tissue-specific morphogenesis and cell wall remodeling in cereals. Data are available via ProteomeXchange with identifier PXD010367.

Distribution of cell wall hemicelluloses in the wheat grain endosperm: a 3D perspective.

MAIN CONCLUSION: Uneven distribution of AX and BG in lateral and longitudinal dimensions of a wheat grain was observed by three-dimensional MS imaging, presumably related to specific physicochemical properties of cell walls. Arabinoxylans (AX) and ß-glucans (BG) are the main hemicelluloses that comprise the primary walls of starchy endosperm. These components are not evenly distributed in the endosperm, and the impact of their distribution on cell wall properties is not yet fully understood. Combined with on-tissue enzymatic degradation of the cell walls, mass spectrometry imaging (MSI) was used to monitor the molecular structure of AX and BG in thirty consecutive cross-sections of a mature wheat grain. A 3D image was built from the planar images, showing the distribution of these polymers at the full-grain level, both in lateral and longitudinal dimensions. BGs were more abundant at the vicinity of the germ and in the central cells of the endosperm, while AX, and especially highly substituted AX, were more abundant close to the brush and in the cells surrounding the crease (i.e., the transfer cells). Compared with the previously reported protocol, significant improvements were made in the tissue preparation to better preserve the shape of the fragile sections. This allowed to us achieve a good-quality 3D reconstruction from the consecutive 2D images. By providing a continuous view of the molecular distribution of the cell wall components across and along the grain, the three-dimensional images obtained by MSI may help understand the structure-function relationships of cell walls. The method should be readily extendable to other parietal polymers by selecting the appropriate enzymes.

Mutation in Brachypodium caffeic acid O-methyltransferase 6 alters stem and grain lignins and improves straw saccharification without deteriorating grain quality.

Cereal crop by-products are a promising source of renewable raw material for the production of biofuel from lignocellulose. However, their enzymatic conversion to fermentable sugars is detrimentally affected by lignins. Here the characterization of the Brachypodium Bd5139 mutant provided with a single nucleotide mutation in the caffeic acid O-methyltransferase BdCOMT6 gene is reported. This BdCOMT6-deficient mutant displayed a moderately altered lignification in mature stems. The lignin-related BdCOMT6 gene was also found to be expressed in grains, and the alterations of Bd5139 grain lignins were found to mirror nicely those evidenced in stem lignins. The Bd5139 grains displayed similar size and composition to the control. Complementation experiments carried out by introducing the mutated gene into the AtCOMT1-deficient Arabidopsis mutant demonstrated that the mutated BdCOMT6 protein was still functional. Such a moderate down-regulation of lignin-related COMT enzyme reduced the straw recalcitrance to saccharification, without compromising the vegetative or reproductive development of the plant.

Cell wall proteomic of Brachypodium distachyon grains: A focus on cell wall remodeling proteins.

Cell walls play key roles during plant development. Following their deposition into the cell wall, polysaccharides are continually remodeled according to the growth stage and stress environment to accommodate cell growth and differentiation. To date, little is known concerning the enzymes involved in cell wall remodeling, especially in gramineous and particularly in the grain during development. Here, we investigated the cell wall proteome of the grain of Brachypodium distachyon. This plant is a suitable model for temperate cereal crops. Among the 601 proteins identified, 299 were predicted to be secreted. These proteins were distributed into eight functional classes; the class of proteins that act on carbohydrates was the most highly represented. Among these proteins, numerous glycoside hydrolases were found. Expansins and peroxidases, which are assumed to be involved in cell wall polysaccharide remodeling, were also identified. Approximately half of the proteins identified in this study were newly discovered in grain and were not identified in the previous proteome analysis conducted using the culms and leaves of B. distachyon. Therefore, the data obtained from all organs of B. distachyon infer a global cell wall proteome consisting of 460 proteins. At present, this is the most extensive cell wall proteome of a monocot species.

The Deconstruction of Pectic Rhamnogalacturonan I Unmasks the Occurrence of a Novel Arabinogalactan Oligosaccharide Epitope.

Rhamnogalacturonan I (RGI) is a pectic polysaccharide composed of a backbone of alternating rhamnose and galacturonic acid residues with side chains containing galactose and/or arabinose residues. The structure of these side chains and the degree of substitution of rhamnose residues are extremely variable and depend on species, organs, cell types and developmental stages. Deciphering RGI function requires extending the current set of monoclonal antibodies (mAbs) directed to this polymer. Here, we describe the generation of a new mAb that recognizes a heterogeneous subdomain of RGI. The mAb, INRA-AGI-1, was produced by immunization of mice with RGI oligosaccharides isolated from potato tubers. These oligomers consisted of highly branched RGI backbones substituted with short side chains. INRA-AGI-1 bound specifically to RGI isolated from galactan-rich cell walls and displayed no binding to other pectic domains. In order to identify its RGI-related epitope, potato RGI oligosaccharides were fractionated by anion-exchange chromatography. Antibody recognition was assessed for each chromatographic fraction. INRA-AGI-1 recognizes a linear chain of (1→4)-linked galactose and (1→5)-linked arabinose residues. By combining the use of INRA-AGI-1 with LM5, LM6 and INRA-RU1 mAbs and enzymatic pre-treatments, evidence is presented of spatial differences in RGI motif distribution within individual cell walls of potato tubers and carrot roots. These observations raise questions about the biosynthesis and assembly of pectin structural domains and their integration and remodeling in cell walls.

Endomembrane proteomics reveals putative enzymes involved in cell wall metabolism in wheat grain outer layers.

Cereal grain outer layers fulfil essential functions for the developing seed such as supplying energy and providing protection. In the food industry, the grain outer layers called 'the bran' is valuable since it is rich in dietary fibre and other beneficial nutriments. The outer layers comprise several tissues with a high content in cell wall material. The cell wall composition of the grain peripheral tissues was investigated with specific probes at a stage of active cell wall synthesis. Considerable wall diversity between cell types was revealed. To identify the cellular machinery involved in cell wall synthesis, a subcellular proteomic approach was used targeting the Golgi apparatus where most cell wall polysaccharides are synthesized. The tissues were dissected into outer pericarp and intermediate layers where 822 and 1304 proteins were identified respectively. Many carbohydrate-active enzymes were revealed: some in the two peripheral grain fractions, others only in one tissue. Several protein families specific to one fraction and with characterized homologs in other species might be related to the specific detection of a polysaccharide in a particular cell layer. This report provides new information on grain cell walls and its biosynthesis in the valuable outer tissues, which are poorly studied so far. A better understanding of the mechanisms controlling cell wall composition could help to improve several quality traits of cereal products (e.g. dietary fibre content, biomass conversion to biofuel).

Immunolabelling of intervessel pits for polysaccharides and lignin helps in understanding their hydraulic properties in Populus tremula × alba.

BACKGROUND AND AIMS: The efficiency and safety functions of xylem hydraulics are strongly dependent on the pits that connect the xylem vessels. However, little is known about their biochemical composition and thus about their hydraulic properties. In this study, the distribution of the epitopes of different wall components (cellulose, hemicelluloses, pectins and lignins) was analysed in intervessel pits of hybrid poplar (Populus tremula × alba). METHODS: Immunogold labelling with transmission electron microscopy was carried out with a set of antibodies raised against different epitopes for each wall polysaccharide type and for lignins. Analyses were performed on both immature and mature vessels. The effect of sap ionic strength on xylem conductance was also tested. KEY RESULTS: In mature vessels, the pit membrane (PM) was composed of crystalline cellulose and lignins. None of the hemicellulose epitopes were found in the PM. Pectin epitopes in mature vessels were highly concentrated in the annulus, a restricted area of the PM, whereas they were initially found in the whole PM in immature vessels. The pit border also showed a specific labelling pattern, with higher cellulose labelling compared with the secondary wall of the vessel. Ion-mediated variation of 24 % was found for hydraulic conductance. CONCLUSIONS: Cellulose microfibrils, lignins and annulus-restricted pectins have different physicochemical properties (rigidity, hydrophobicity, porosity) that have different effects on the hydraulic functions of the PM, and these influence both the hydraulic efficiency and vulnerability to cavitation of the pits, including ion-mediated control of hydraulic conductance. Impregnation of the cellulose microfibrils of the PM with lignins, which have low wettability, may result in lower cavitation pressure for a given pore size and thus help to explain the vulnerability of this species to cavitation.

New insights into the structural and spatial variability of cell-wall polysaccharides during wheat grain development, as revealed through MALDI mass spectrometry imaging.

Arabinoxylans (AX) and (1→3),(1→4)-ß-glucans (BG) are the major components of wheat grain cell walls. Although incompletely described at the molecular level, it is known that the chemical and distributional heterogeneity of these compounds impacts the quality and use of wheat. In this work, an emerging technique based on MALDI mass spectrometry imaging (MSI) was employed to map variations in the quantity, localization, and structure of these polysaccharides in the endosperm during wheat maturation. MALDI MSI couples detailed structural information with the spatial localization observed at the micrometer scale. The enzymic hydrolysis of AX and BG was performed directly on the grain sections, resulting in the efficient formation of smaller oligosaccharides that are easily measurable through MS, with no relocation across the grain. The relative quantification of the generated oligosaccharides was achieved. The method was validated by confirming data previously obtained using other analytical techniques. Furthermore, in situ analysis of grain cell walls through MSI revealed previously undetectable intense acetylation of AX in young compared to mature grains, together with findings concerning the feruloylation of AX and different structural features of BG. These results provide new insights into the physiological roles of these polysaccharides in cell walls and the specificity of the hydrolytic enzymes involved.

Maize Internode Autofluorescence at the Macroscopic Scale: Image Representation and Principal Component Analysis of a Series of Large Multispectral Images.

A quantitative histology of maize stems is needed to study the role of tissue and of their chemical composition in plant development and in their end-use quality. In the present work, a new methodology is proposed to show and quantify the spatial variability of tissue composition in plant organs and to statistically compare different samples accounting for biological variability. Multispectral UV/visible autofluorescence imaging was used to acquire a macroscale image series based on the fluorescence of phenolic compounds in the cell wall. A series of 40 multispectral large images of a whole internode section taken from four maize inbred lines were compared. The series consisted of more than 1 billion pixels and 11 autofluorescence channels. Principal Component Analysis was adapted and named large PCA and score image montages at different scales were built. Large PCA score distributions were proposed as quantitative features to compare the inbred lines. Variations in the tissue fluorescence were clearly displayed in the score images. General intensity variations were identified. Rind vascular bundles were differentiated from other tissues due to their lignin fluorescence after visible excitation, while variations within the pith parenchyma were shown via UV fluorescence. They depended on the inbred line, as revealed by the first four large PCA score distributions. Autofluorescence macroscopy combined with an adapted analysis of a series of large images is promising for the investigation of the spatial heterogeneity of tissue composition between and within organ sections. The method is easy to implement and can be easily extended to other multi-hyperspectral imaging techniques. The score distributions enable a global comparison of the images and an analysis of the inbred lines' effect. The interpretation of the tissue autofluorescence needs to be further investigated by using complementary spatially resolved techniques.

Substituent-specific antibody against glucuronoxylan reveals close association of glucuronic acid and acetyl substituents and distinct labeling patterns in tree species.

Immunolabeling can be used to locate plant cell wall carbohydrates or other components to specific cell types or to specific regions of the wall. Some antibodies against xylans exist; however, many partly react with the xylan backbone and thus provide limited information on the type of substituents present in various xylans. We have produced a monoclonal antibody which specifically recognizes glucopyranosyl uronic acid (GlcA), or its 4-O-methyl ether (meGlcA), substituents in xylan and has no cross-reactivity with linear or arabinofuranosyl-substituted xylans. The UX1 antibody binds most strongly to (me)GlcA substitutions at the non-reducing ends of xylan chains, but has a low cross-reactivity with internal substitutions as well, at least on oligosaccharides. The antibody labeled plant cell walls from both mono- and dicotyledons, but in most tissues an alkaline pretreatment was needed for antibody binding. The treatment removed acetyl groups from xylan, indicating that the vicinity of glucuronic acid substituents is also acetylated. The novel labeling patterns observed in the xylem of tree species suggested that differences within the cell wall exist both in acetylation degree and in glucuronic acid content.

Real-time imaging of enzymatic degradation of pretreated maize internodes reveals different cell types have different profiles.

This work presents a dynamic view of the enzymatic degradation of maize cell walls, and sheds new light on the recalcitrance of hot water pretreated maize stem internodes. Infra-red microspectrometry, mass spectrometry, fluorescence recovery after photobleaching and fluorescence imaging were combined to investigate enzymatic hydrolysis at the cell scale. Depending on their polymer composition and organisation, cell types exhibits different extent and rate of enzymatic degradation. Enzymes act sequentially from the cell walls rich in accessible cellulose to the most recalcitrant cells. This phenomenon can be linked to the heterogeneous distribution of enzymes in the liquid medium and the adsorption/desorption mechanisms that differ with the type of cell.

Impact of cell wall non-cellulosic and cellulosic polymers on the mechanical properties of flax fibre bundles.

Fibre bundles are groups of elementary fibres glued together thanks to the middle lamella, and are the main fraction in plant fibre composites. In this study, relationship between the mechanical properties of flax fibre bundles, chemical composition and cellulose structure were investigated. To do so, a sequential biopolymer extraction was implemented. Fibre bundles were first depectinated by oxalate extraction, and then the hemicelluloses were extracted by LiCl/dimethyl sulfoxide (DMSO) and KOH. The oxalate extract consisted of homogalacturonans and type I rhamnogalacturonans, while the LiCl extract was composed mainly of glucomannans and the KOH extract of xyloglucans. The KOH stage resulted in the appearance of cellulose II in flax bundles. The extraction of pectin and hemicelluloses led to the disappearance of the middle lamella concomitant with a decrease in the tensile Young's modulus and maximum strength. Finally, the fibre bundle composition, ultrastructure and mechanical properties are discussed together in view of the thin middle lamella.

Combined meta-genomics analyses unravel candidate genes for the grain dietary fiber content in bread wheat (Triticum aestivum L.).

Grain dietary fiber content in wheat not only affects its end use and technological properties including milling, baking and animal feed but is also of great importance for health benefits. In this study, integration of association genetics (seven detected loci on chromosomes 1B, 3A, 3D, 5B, 6B, 7A, 7B) and meta-QTL (three consensus QTL on chromosomes 1B, 3D and 6B) analyses allowed the identification of seven chromosomal regions underlying grain dietary fiber content in bread wheat. Based either on a diversity panel or on bi-parental populations, we clearly demonstrate that this trait is mainly driven by a major locus located on chromosome 1B associated with a log of p value >13 and a LOD score >8, respectively. In parallel, we identified 73 genes differentially expressed during the grain development and between genotypes with contrasting grain fiber contents. Integration of quantitative genetics and transcriptomic data allowed us to propose a short list of candidate genes that are conserved in the rice, sorghum and Brachypodium chromosome regions orthologous to the seven wheat grain fiber content QTL and that can be considered as major candidate genes for future improvement of the grain dietary fiber content in bread wheat breeding programs.

Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat.

(1,3;1,4)-beta-d-Glucan (beta-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative beta-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total beta-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable beta-glucan was also reduced by about 50% in the transgenic lines, and the M(r) distribution of the fraction was decreased from an average of 79 to 85 x 10(4) g/mol in the controls and 36 to 57 x 10(4) g/mol in the transgenics. Immunolocalization of beta-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a beta-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.