Cellulose-rich Cell Walls Research Articles

Mesostructural changes in cellulose within wood cell wall upon hydrothermal treatment at 200 °C

Hydrothermal treatment between 150 °C and 230 °C is widely used in wood processing, from the steam treatment of timber for better dimensional stability and durability to the pretreatment for enzymatic saccharification and chemical pulping. Understanding the ultrastructural changes of wood cell walls through hydrothermal treatments is crucial for controlling and optimizing these hydrothermal treatment-based processes. Here, we studied the ultrastructure of wood cell walls of 24 hardwood species using simultaneous small- and wide-angle X-ray scattering measurements before and after the hydrothermal treatment at 200 °C. Most hardwoods show similar equatorial scattering features, representing the structure in the cross-section of the cell walls. In a water-saturated native state, there is a prominent correlation peak between 0.1 and 0.2 Å−1 and a second peak between 0.2 and 0.4 Å−1. The hydrothermal treatment above 160 ˚C drastically altered the structure at this nanometric scale: the two native correlation peaks disappeared, coincident with a buildup of a correlation peak in the 0.03–0.04 Å−1 range. The hydrothermal treatment likely removed the cell wall matrix component between the microfibrils through autohydrolysis and phase separation, leading to the collapse of microfibrils with each other in the normal wood. In cellulose-rich cell walls, such as the G-layer in tension wood, cellulose microfibrils are already collated in the native state.

X-ray microtomography reveals the 3D enzymatic deconstruction pathway of raw lignocellulosic biomass

In the context of the development of a circular bioeconomy, plant biomass constitutes a vast potential source of renewable molecules of interest. The industrial process essentially consists in using enzymes for its deconstruction. However, we are still lacking a precise understanding of the enzymes' action on such a complex, multiscale porous material. Here, we demonstrate that X-ray microtomography tackles this question in a unique way, making it possible to follow precisely the enzymatic degradation of a model biomass, wheat straw. We perform a thorough analysis of the imaging results over the full length scale of the sample and as a function of degradation time. In particular, we observe the progressive disappearance of specific cellulose-rich cell walls, with distinct 3D heterogeneities in the enzymatic action at different times and length scales. Those 3D pieces of information about the degradation process contrast clearly with those obtained from classical 2D imaging techniques.

Laser Capture Microdissection-Based RNA-Seq of Barley Grain Tissues.

Spatiotemporal patterning throughout the plant body depends to a large degree on cell- and tissue-specific expression of genes. Subsequently, for a better understanding of cell and tissue differentiation processes during plant development it is important to conduct transcript analyses in individual cells or tissue types rather than in bulk tissues. Laser capture microdissection (LCM) provides a useful method for isolating specific cell types from complex tissue structures for downstream applications. Contrasting to mammalian cells, the texture of plant cells is more critical due to hard, cellulose-rich cell walls, large vacuoles, and air spaces which complicates tissue preparation and extraction of macromolecules, like DNA and RNA. In particular, developing barley seeds (i.e. grains) depict cell types with differences in osmomolarity (meristematic, differentiating and degenerating tissues) and contain high amounts of the main storage product starch. In this study, we report about methods allowing tissue-specific transcriptome profiling by RNA-seq of developing barley grain tissues from low-input RNA amounts. Details on tissue preparation, laser capture microdissection, RNA isolation, and linear mRNA amplification to produce high-quality samples for Illumina sequencing are provided. Particular emphasis was placed on the influence of the mRNA amplification step on the transcriptome data and the fidelity of deduced expression levels obtained by the developed methods. Analysis of RNA-seq data confirmed sample processing as a highly reliable and reproducible procedure that was also used for transcriptome analyses of different tissue types from barley plants.

Transcriptional Regulation of Cellulose Biosynthesis during the Early Phase of Nitrogen Deprivation in Nannochloropsis salina

Microalgal photosynthesis provides energy and carbon-containing precursors for the biosynthesis of storage carbohydrates such as starch, chrysolaminarin, lipids, and cell wall components. Under mild nitrogen deficiency (N−), some Nannochloropsis species accumulate lipid by augmenting cytosolic fatty acid biosynthesis with a temporary increase in laminarin. Accordingly, biosynthesis of the cellulose-rich cell wall should change in response to N− stress because this biosynthetic pathway begins with utilisation of the hexose phosphate pool supplied from photosynthesis. However, few studies have characterised microalgal cell wall metabolism, including oleaginous Nannochloropsis sp. microalgae subjected to nitrogen deficiency. Here, we investigated N-induced changes in cellulose biosynthesis in N. salina. We observed that N− induced cell wall thickening, concurrently increased the transcript levels of genes coding for UDPG pyrophosphorylase and cellulose synthases, and increased cellulose content. Nannochloropsis salina cells with thickened cell wall were more susceptible to mechanical stress such as bead-beating and sonication, implicating cellulose metabolism as a potential target for cost-effective microalgal cell disruption.

Sugarcane cell wall structure and lignin distribution investigated by confocal and electron microscopy

Lignocellulosic plant cell wall is considered a potential source for second generation biofuels. The plant cell wall is a highly complex structure mainly composed of cellulose, hemicelluloses, and lignin that form a network of crosslinked fibers. The structural organization of the sugarcane cell wall has not been previously analyzed in detail, and this analysis is a prerequisite for further studies on the recalcitrance and deconstruction of its biomass. In this work, cellulose and lignin localization were investigated by confocal laser scanning microscopy. In addition, the internode sugarcane cell wall structural organization was analyzed by electron microscopy. Internode stem anatomy showed a typical monocot structure consisting of epidermis, hypoderm, and vascular bundles scattered throughout ground parenchyma tissue and surrounded by sclerenchyma fibers. Confocal images of safranin labeled sugarcane showed that lignin distribution was predominant in the vessel elements, cell wall corners (CC), and middle lamella (ML), while cellulose-rich cell walls were randomly distributed in the ML and organized in the other cell wall layers. KMnO4 cytochemistry revealed that lignin was predominantly distributed in secondary cell walls, ML and CC. Cell wall sublayers (S1, S2, and S3) were identified and measured by transmission electron microscopy. Our results provide insights that may help further understanding of sugarcane cell wall organization, which is crucial for the research and technology of plant-based biofuel production.

Genetic and hormonal regulation of cambial development

The stems and roots of most dicot plants increase in diameter by radial growth, due to the activity of secondary meristems. Two types of meristems function in secondary plant body formation: the vascular cambium, which gives rise to secondary xylem and phloem, and the cork cambium, which produces a bark layer that replaces the epidermis and protects the plant stem from mechanical damage and pathogens. Cambial development, the initiation and activity of the vascular cambium, leads to an accumulation of wood, the secondary xylem tissue. The thick, cellulose-rich cell walls of wood provide a source of cellulose and have the potential to be used as a raw material for sustainable and renewable energy production. In this review, we will discuss what is known about the mechanisms regulating the cambium and secondary tissue development.

Safranine fluorescent staining of wood cell walls

Safranine is an azo dye commonly used for plant microscopy, especially as a stain for lignified tissues such as xylem. Safranine fluorescently labels the wood cell wall, producing green/yellow fluorescence in the secondary cell wall and red/orange fluorescence in the middle lamella (ML) region. We examined the fluorescence behavior of safranine under blue light excitation using a variety of wood- and fiber-based samples of known composition to interpret the observed color differentiation of different cell wall types. We also examined the basis for the differences in fluorescence emission using spectral confocal microscopy to examine lignin-rich and cellulose-rich cell walls including reaction wood and decayed wood compared to normal wood. Our results indicate that lignin-rich cell walls, such as the ML of tracheids, the secondary wall of compression wood tracheids, and wood decayed by brown rot, tend to fluoresce red or orange, while cellulose-rich cell walls such as resin canals, wood decayed by white rot, cotton fibers and the G-layer of tension wood fibers, tend to fluoresce green/yellow. This variation in fluorescence emission seems to be due to factors including an emission shift toward red wavelengths combined with dye quenching at shorter wavelengths in regions with high lignin content. Safranine fluorescence provides a useful way to differentiate lignin-rich and cellulose-rich cell walls without counterstaining as required for bright field microscopy.

Conformation and mobility of the arabinan and galactan side-chains of pectin

The function of the arabinan and galactan side-chains of pectin remains unknown. We describe 13C NMR experiments designed to yield spectra from the most mobile polymer components of hydrated cell walls isolated from a range of plant species. In pectin-rich cell walls, these corresponded to the pectic side-chains. The arabinan side-chains were in general more mobile than the galactans, but the long galactan side-chains of potato pectin showed high mobility. Due to motional line-narrowing effects these arabinan and galactan chains gave 13C NMR spectra of higher resolution than has previously been observed from ‘solid’ biopolymers. These spectra were similar to those reported for the arabinan and galactan polymers in the solution state, implying time-averaged conformations resembling those found in solution. The mobility of the highly esterified galacturonan in citrus cell walls overlapped with the lower end of the mobility range characteristic of the pectic side-chains. The cellulose-rich cell walls of flax phloem fibres gave spectra of low intensity corresponding to mobile type II arabinogalactans. Cell walls from oat coleoptiles appeared to contain no polymers as mobile as the pectic arabinans and galactans in primary cell walls of the other species examined. These properties of the pectic side-chains suggest a role in interacting with water.

Boron in Plant Biology

Abstract: The interest of biologists in boron (B) has largely been focused on its role in plants for which B was established as essential in 1923 (Warington, 1923[296]). Evidence that B has a biological role in other organisms was first indicated by the establishment of essentiality of B for diatoms (Smyth and Dugger, 1981[296]) and cyanobacteria (Bonilla et al., 1990[296]; Garcia‐Gonzalez et al., 1991[296]; Bonilla et al., 1997[296]). Recently, B was shown to stimulate growth in yeast (Bennett et al., 1999[296]) and to be essential for zebrafish (Danio rerio) (Eckhert and Rowe, 1999[296]; Rowe and Eckhert, 1999[296]) and possibly for trout (Oncorhynchus mykiss) (Eckhert, 1998[296]; Rowe et al., 1998[296]), frogs (Xenopus laevis) (Fort et al., 1998[296]) and mouse (Lanoue et al., 2000[296]). There is also preliminary evidence to suggest that B has at least a beneficial role in humans (Nielsen, 2000[296]).While research into the role of B in plants has been ongoing for 80 years it has only been in the past 5 years that the first function of B in plants has been defined. Boron is now known to be essential for cell wall structure and function, likely through its role as a stabilizer of the cell wall pectic network and subsequent regulation of cell wall pore size. A role for B in plant cell walls, however, is inadequate to explain all of the effects of B deficiency seen in plants. The suggestion that B plays a broader role in biology is supported by the discovery that B is essential for animals where a cellulose‐rich cell wall is not present. Careful consideration of the physical and chemical properties of B in biological systems, and of the experimental data from both plants and animals suggests that B plays a critical role in membrane structure and hence function. Verification of B association with membranes would represent an important advance in modern biology.For several decades there has been uncertainty as to the mechanisms of B uptake and transport within plants. This uncertainty has been driven by a lack of adequate methodology to measure membrane fluxes of B at physiologically relevant concentrations. Recent experimentation provides the first direct measurement of membrane permeability of B and illustrates that passive B permeation contributes sufficient B at adequate levels of B supply, but would be inadequate at conditions of marginal B supply. The hypothesis that an active, carrier mediated process is involved in B uptake at low B supply is supported by research demonstrating that B uptake can be stimulated by B deprivation, that uptake rates follow a Michaelis‐Menton kinetics, and can be inhibited by application of metabolic inhibitors. Since the mechanisms of element uptake are generally conserved between species, an understanding of the processes of B uptake is relevant to studies in both plants and animals.The study of B in plant biology has progressed markedly in the last decade and we are clearly on the cusp of additional, significant discoveries. Research in this field will be greatly stimulated by the discovery that B is essential for animals, a discovery that will not only encourage the participation of a wider cadre of scientists but will refocus the efforts of plant biologists toward a determination of roles for B outside the plant cell wall. Determination of the function of B in biology and of the mechanisms of B uptake in biological systems, is essential to our understanding and management of B deficiency and toxicity in plants and animals in both agricultural and natural environments. Through an analysis of existing data and the development of new hypotheses, this review aims to provide a vision of the future of research into the biology of boron.

Thermal transitions in freeze-dried carrot and its cell wall components

Differential scanning calorimetry (DSC) was used to measure the glass transition temperature, T g and a sub- T g event in freeze-dried carrot material and different carrot cell wall components, at moistures ranging from 0% to 20% (w.w.b.). Two glass transition temperatures were detected in the freeze-dried carrot material associated with two phases: a sugar-rich phase and a cell wall-rich phase. The water distribution in the different phases is also important and sorption isotherms of freeze-dried carrot and different cell wall components were determined and compared with published results produced on sugar, pectins and cellulose. For the insoluble carrot cell wall materials, a single T g was detected which increased in the order: a Ca 2+ bound pectin-free residue, an esterified pectin-free residue and a cellulose-rich residue. In all cases T g increased with decreasing moisture. Additionally, a sub- T g endothermic event was observed in all the materials which disappeared on rescanning, consistent with results published on ageing of other biopolymers. The T g of the different cell wall residues was modelled, using the Gordon–Taylor and Kwei equations. The latter showed that water–biopolymers interactions are less prominent in the cellulose-rich cell wall residue, shown by a negative value of q.