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.

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.

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.

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.

Darkfield and Fluorescence Macrovision of a Series of Large Images to Assess Anatomical and Chemical Tissue Variability in Whole Cross-Sections of Maize Stems.

The proportion and composition of plant tissues in maize stems vary with genotype and agroclimatic factors and may impact the final biomass use. In this manuscript, we propose a quantitative histology approach without any section labelling to estimate the proportion of different tissues in maize stem sections as well as their chemical characteristics. Macroscopic imaging was chosen to observe the entire section of a stem. Darkfield illumination was retained to visualise the whole stem cellular structure. Multispectral autofluorescence images were acquired to detect cell wall phenolic compounds after UV and visible excitations. Image analysis was implemented to extract morphological features and autofluorescence pseudospectra. By assimilating the internode to a cylinder, the relative proportions of tissues in the internode were estimated from their relative areas in the sections. The approach was applied to study a series of 14 maize inbred lines. Considerable variability was revealed among the 14 inbred lines for both anatomical and chemical traits. The most discriminant morphological descriptors were the relative amount of rind and parenchyma tissues together with the density and size of the individual bundles, the area of stem and the parenchyma cell diameter. The rind, as the most lignified tissue, showed strong visible-induced fluorescence which was line-dependant. The relative amount of para-coumaric acid was associated with the UV-induced fluorescence intensity in the rind and in the parenchyma near the rind, while ferulic acid amount was significantly correlated mainly with the parenchyma near the rind. The correlation between lignin and the tissue pseudospectra showed that a global higher amount of lignin resulted in a higher level of lignin fluorescence whatever the tissues. We demonstrated here the potential of darkfield and autofluorescence imaging coupled with image analysis to quantify histology of maize stem and highlight variability between different lines.

Evaluating polymer interplay after hot water pretreatment to investigate maize stem internode recalcitrance.

BACKGROUND: Biomass recalcitrance is governed by various molecular and structural factors but the interplay between these multiscale factors remains unclear. In this study, hot water pretreatment (HWP) was applied to maize stem internodes to highlight the impact of the ultrastructure of the polymers and their interactions on the accessibility and recalcitrance of the lignocellulosic biomass. The impact of HWP was analysed at different scales, from the polymer ultrastructure or water mobility to the cell wall organisation by combining complementary compositional, spectral and NMR analyses. RESULTS: HWP increased the kinetics and yield of saccharification. Chemical characterisation showed that HWP altered cell wall composition with a loss of hemicelluloses (up to 45% in the 40-min HWP) and of ferulic acid cross-linking associated with lignin enrichment. The lignin structure was also altered (up to 35% reduction in ß-O-4 bonds), associated with slight depolymerisation/repolymerisation depending on the length of treatment. The increase in [Formula: see text], [Formula: see text] and specific surface area (SSA) showed that the cellulose environment was looser after pretreatment. These changes were linked to the increased accessibility of more constrained water to the cellulose in the 5-15 nm pore size range. CONCLUSION: The loss of hemicelluloses and changes in polymer structural features caused by HWP led to reorganisation of the lignocellulose matrix. These modifications increased the SSA and redistributed the water thereby increasing the accessibility of cellulases and enhancing hydrolysis. Interestingly, lignin content did not have a negative impact on enzymatic hydrolysis but a higher lignin condensed state appeared to promote saccharification. The environment and organisation of lignin is thus more important than its concentration in explaining cellulose accessibility. Elucidating the interactions between polymers is the key to understanding LB recalcitrance and to identifying the best severity conditions to optimise HWP in sustainable biorefineries.

The endosperm cavity of wheat grains contains a highly hydrated gel of arabinoxylan.

Cereal grains provide a substantial part of the calories for humans and animals. The main quality determinants of grains are polysaccharides (mainly starch but also dietary fibers such as arabinoxylans, mixed-linkage glucans) and proteins synthesized and accumulated during grain development in a specialized storage tissue: the endosperm. In this study, the composition of a structure localized at the interface of the vascular tissues of the maternal plant and the seed endosperm was investigated. This structure is contained in the endosperm cavity where water and nutrients are transferred to support grain filling. While studying the wheat grain development, the cavity content was found to autofluoresce under UV light excitation. Combining multispectral analysis, Fourier-Transform infrared spectroscopy, immunolabeling and laser-dissection coupled with wet chemistry, we identified in the cavity arabinoxylans and hydroxycinnamic acids. The cavity content forms a "gel" in the developing grain, which persists in dry mature grain and during subsequent imbibition. Microscopic magnetic resonance imaging revealed that the gel is highly hydrated. Our results suggest that arabinoxylans are synthesized by the nucellar epidermis, released in the cavity where they form a highly hydrated gel which might contribute to regulate grain hydration.

Enzymatic degradation of maize shoots: monitoring of chemical and physical changes reveals different saccharification behaviors.

BACKGROUND: The recalcitrance of lignocellulosics to enzymatic saccharification has been related to many factors, including the tissue and molecular heterogeneity of the plant particles. The role of tissue heterogeneity generally assessed from plant sections is not easy to study on a large scale. In the present work, dry fractionation of ground maize shoot was performed to obtain particle fractions enriched in a specific tissue. The degradation profiles of the fractions were compared considering physical changes in addition to chemical conversion. RESULTS: Coarse, medium and fine fractions were produced using a dry process followed by an electrostatic separation. The physical and chemical characteristics of the fractions varied, suggesting enrichment in tissue from leaves, pith or rind. The fractions were subjected to enzymatic hydrolysis in a torus reactor designed for real-time monitoring of the number and size of the particles. Saccharification efficiency was monitored by analyzing the sugar release at different times. The lowest and highest saccharification yields were measured in the coarse and fine fractions, respectively, and these yields paralleled the reduction in the size and number of particles. The behavior of the positively- and negatively-charged particles of medium-size fractions was contrasted. Although the amount of sugar release was similar, the changes in particle size and number differed during enzymatic degradation. The reduction in the number of particles proceeded faster than that of particle size, suggesting that degradable particles were degraded to the point of disappearance with no significant erosion or fragmentation. Considering all fractions, the saccharification yield was positively correlated with the amount of water associated with [5-15 nm] pore size range at 67% moisture content while the reduction in the number of particles was inversely correlated with the amount of lignin. CONCLUSION: Real-time monitoring of sugar release and changes in the number and size of the particles clearly evidenced different degradation patterns for fractions of maize shoot that could be related to tissue heterogeneity in the plant. The biorefinery process could benefit from the addition of a sorting stage to optimise the flow of biomass materials and take better advantage of the heterogeneity of the biomass.

The TaCslA12 gene expressed in the wheat grain endosperm synthesizes wheat-like mannan when expressed in yeast and Arabidopsis.

Mannan is a class of cell wall polysaccharides widespread in the plant kingdom. Mannan structure and properties vary according to species and organ. The cell walls of cereal grains have been extensively studied due to their role in cereal processing and to their beneficial effect on human health as dietary fiber. Recently, we showed that mannan in wheat (Triticum aestivum) grain endosperm has a linear structure of ß-1,4-linked mannose residues. The aim of this work was to study the biosynthesis and function of wheat grain mannan. We showed that mannan is deposited in the endosperm early during grain development, and we identified candidate mannan biosynthetic genes expressed in the endosperm. The functional study in wheat was unsuccessful therefore our best candidate genes were expressed in heterologous systems. The endosperm-specificTaCslA12 gene expressed in Pichia pastoris and in an Arabidopsis thaliana mutant depleted in glucomannan led to the production of wheat-like linear mannan lacking glucose residues and with moderate acetylation. Therefore, this gene encodes a mannan synthase and is likely responsible for the synthesis of wheat endosperm mannan.

Parametric mapping of cellular morphology in plant tissue sections by gray level granulometry.

BACKGROUND: The cellular morphology of plant organs is strongly related to other physical properties such as shape, size, growth, mechanical properties or chemical composition. Cell morphology often vary depending on the type of tissue, or on the distance to a specific tissue. A common challenge in quantitative plant histology is to quantify not only the cellular morphology, but also its variations within the image or the organ. Image texture analysis is a fundamental tool in many areas of image analysis, that was proven efficient for plant histology, but at the scale of the whole image. RESULTS: This work presents a method that generates a parametric mapping of cellular morphology within images of plant tissues. It is based on gray level granulometry from mathematical morphology for extracting image texture features, and on Centroidal Voronoi Diagram for generating a partition of the image. Resulting granulometric curves can be interpreted either through multivariate data analysis or by using summary features corresponding to the local average cell size. The resulting parametric maps describe the variations of cellular morphology within the organ. CONCLUSIONS: We propose a methodology for the quantification of cellular morphology and of its variations within images of tissue sections. The results should help understanding how the cellular morphology is related to genotypic and / or environmental variations, and clarify the relationships between cellular morphology and chemical composition of cell walls.

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 [...].

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.

Challenging the putative structure of mannan in wheat (Triticum aestivum) endosperm.

In wheat endosperm, mannan, is poorly documented. Nevertheless, this hemicellulosic polysaccharide might have a determinant role in wheat grain development since, in Arabidopsis thaliana, mutants with a reduced amount of mannan show an altered seed development. In order to gain knowledge about mannan in wheat, we have determined its biochemical structure in wheat endosperm where mannose content is about 0.2% (dry weight basis). We developed a method of enzymatic fingerprinting and isolated mannan-enriched fractions to decipher its fine structure. Although it is widely accepted that the class of mannan present in grass cell walls is glucomannan, our data indicate that, in wheat endosperm, this hemicellulose is only represented by short unsubstituted chains of 1,4 linked D-mannose residues and is slightly acetylated. Our study provides information regarding the interactions of mannan with other cell wall components and help to progress towards the understanding of monocot cell wall architecture and the mannan synthesis in wheat endosperm.

Changes in cell walls lignification, feruloylation and p-coumaroylation throughout maize internode development.

Plant cell walls development is an integrated process during which several components are deposited successively. In the cell walls in grass, the accessibility of structural polysaccharides is limited by the cell walls structure and composition mainly as a result of phenolic compounds. Here, we studied the patterns of cell walls establishment in the internode supporting the ear in three distinct maize genotypes. The developmental patterns observed in the internode cell walls in terms of its composition are reported with an emphasis on lignification, p-coumaroylation and feruloylation. We combined biochemical and histological approaches and revealed that internode cell walls development in maize before flowering is characterized by the rapid deposition of secondary cell walls components and robust lignification in both the pith and the rind. After flowering and until silage maturity, the slow deposition of secondary walls components occurs in the cortical region, and the deposited lignins are rich in ß-O-4 bonds and are highly p-coumaroylated. We conclude the paper by proposing a revised spatiotemporal model based on that proposed by Terashima et al. (1993) for cell walls development in grass.

Multi-excitation hyperspectral autofluorescence imaging for the exploration of biological samples.

Many plant tissues can be observed thanks to autofluorescence of their cell wall components. Hyperspectral autofluorescence imaging using confocal microscopy is a fast and efficient way of mapping fluorescent compounds in samples with a high spatial resolution. However a huge spectral overlap is observed between molecular species. As a consequence, a new data analysis approach is needed in order to fully exploit the potential of this spectroscopic technique and extract unbiased chemical information about complex biological samples. The objective of this work is to evaluate multi-excitation hyperspectral autofluorescence imaging to identify biological components in wheat grains during their development through their spectral profiles and corresponding contribution maps using Multivariate Curve Resolution - Alternating Least-Squares (MCR-ALS), a signal unmixing algorithm under proper constraints. For this purpose two different scenarios are used: 1) analyzing the total spectral domain of data sets using MCR-ALS under non negativity constraint in both spectral and spatial modes; 2) analyzing a reduced spectral domain of data sets using MCR-ALS under non negativity in both modes and trilinearity constraint in spectral mode. Considering the original instrumental setup and our data analysis approach, we will demonstrate that extracted contribution maps and spectral profiles of constituents can provide complementary information used to identify molecules in complex biological samples.

Spatial and temporal distribution of cell wall polysaccharides during grain development of Brachypodium distachyon.

Brachypodium distachyon (Brachypodium) is now well considered as being a suitable plant model for studying temperate cereal crops. Its cell walls are phylogenetically intermediate between rice and poaceae, with a greater proximity to these latter. By microscopic and biochemical approaches, this work gives an overview of the temporal and spatial distribution of cell wall polysaccharides in the grain of Brachypodium from the end of the cellularization step to the maturation of grain. Variation in arabinoxylan chemical structure and distribution were demonstrated according to development and different grain tissues. In particular, the kinetic of arabinoxylan feruloylation was shown occuring later in the aleurone layers compared to storage endosperm. Mixed linked ß-glucan was detected in whole the tissues of Brachypodium grain even at late stage of development. Cellulose was found in both the storage endosperm and the outer layers. Homogalacturonan and rhamnogalacturonan I epitopes were differentially distributed within the grain tissues. LM5 galactan epitope was restricted to the aleurone layers contrary to LM6 arabinan epitope which was detected in the whole endosperm. A massive deposition of highly methylated homogalacturonans in vesicular bodies was observed underneath the cell wall of the testa t2 layer at early stage of development. At maturity, low-methylated homogalacturonans totally fulfilled the lumen of the t2 cell layer, suggesting pectin remodeling during grain development. Xyloglucans were only detected in the cuticle above the testa early in the development of the grain while feruloylated arabinoxylans were preferentially deposited into the cell wall of t1 layer. Indeed, the circumscribed distribution of some of the cell wall polysaccharides raises questions about their role in grain development and physiology.

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.

Ferulate and lignin cross-links increase in cell walls of wheat grain outer layers during late development.

Important biological, nutritional and technological roles are attributed to cell wall polymers from cereal grains. The composition of cell walls in dry wheat grain has been well studied, however less is known about cell wall deposition and modification in the grain outer layers during grain development. In this study, the composition of cell walls in the outer layers of the wheat grain (Triticum aestivum Recital cultivar) was investigated during grain development, with a focus on cell wall phenolics. We discovered that lignification of outer layers begins earlier than previously reported and long before the grain reaches its final size. Cell wall feruloylation increased in development. However, in the late stages, the amount of ferulate releasable by mild alkaline hydrolysis was reduced as well as the yield of lignin-derived thioacidolysis monomers. These reductions indicate that new ferulate-mediated cross-linkages of cell wall polymers appeared as well as new resistant interunit bonds in lignins. The formation of these additional linkages more specifically occurred in the outer pericarp. Our results raised the possibility that stiffening of cell walls occur at late development stages in the outer pericarp and might contribute to the restriction of the grain radial growth.