A rapid method to screen for cell-wall mutants using discriminant analysis of Fourier transform infrared spectra

We have developed a rapid method to screen large numbers of mutant plants for a broad range of cell wall phenotypes using Fourier transform infrared (FTIR) microspectroscopy of leaves. We established and validated a model that can discriminate between the leaves of wild-type and a previously defined set of cell-wall mutants of Arabidopsis . Exploratory principal component analysis indicated that mutants deficient in different cell-wall sugars can be distinguished from each other. Discrimination of cell-wall mutants from wild-type was independent of variability in starch content or additional unrelated mutations that might be present in a heavily mutagenised population. We then developed an analysis of FTIR spectra of leaves obtained from over 1000 mutagenised flax plants, and selected 59 plants whose spectral variation from wild-type was significantly out of the range of a wild-type population, determined by Mahalanobis distance. Cell wall sugars from the leaves of selected putative mutants were assayed by gas chromatography-mass spectrometry and 42 showed significant differences in neutral sugar composition. The FTIR spectra indicated that six of the remaining 17 plants have altered ester or protein content. We conclude that linear discriminant analysis of FTIR spectra is a robust method to identify a broad range of structural and architectural alterations in cell walls, appearing as a consequence of developmental regulation, environmental adaptation or genetic modification.     

3,4-Dehydroproline inhibits cell wall assembly and cell division in tobacco protoplasts.

We investigated the function of cell wall hydroxyproline-rich glycoproteins by observing the effects of a selective inhibitor of prolyl hydroxylase, 3,4-dehydro-L-proline (Dhp), on wall regeneration by Nicotiana tabacum mesophyll cell protoplasts. Protoplasts treated with micromolar concentrations of Dhp do not develop osmotic stability and do not initiate mitosis. The architecture of regenerated cell walls was examined using deep-etch, freeze-fracture electron microscopy of rapidly frozen tobacco cells. Untreated protoplasts assemble a dense fibrillar cell wall consisting of laterally associating subelementary fibrils. In contrast, treatment of protoplasts with Dhp alters the structure of the regenerated wall fibrils in several ways: first, the microfibrils are coated with globular knobs; second, some larger fiber bundles have an open ribbon-like appearance; and third, the smallest subelementary fibrils were not visible. Tobacco cells develop an abnormal morphology as a consequence of this abnormal cell wall structure. Thus, inhibition of prolyl hydroxylase results in the regeneration of a cell wall with abnormal structural and functional properties. These data provide experimental evidence that hydroxyproline-rich glycoproteins are important for the structural integrity of primary cell walls and for the correct assembly of other wall polymers, and that wall structure is an important regulator of cell division and cell morphology.     

Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth

Advances in determination of polymer structure and in preservation of structure for electron microscopy provide the best view to date of how polysaccharides and structural proteins are organized into plant cell walls. The walls that form and partition dividing cells are modified chemically and structurally from the walls expanding to provide a cell with its functional form. In grasses, the chemical structure of the wall differs from that of all other flowering plant species that have been examined. Nevertheless, both types of wall must conform to the same physical laws. Cell expansion occurs via strictly regulated reorientation of each of the wall's components that first permits the wall to stretch in specific directions and then lock into final shape. This review integrates information on the chemical structure of individual polymers with data obtained from new techniques used to probe the arrangement of the polymers within the walls of individual cells. We provide structural models of two distinct types of walls in flowering plants consistent with the physical properties of the wall and its components.     

On the Cytochemistry of Cell Wall Formation in Poplar Trees

Abstract: The ultrastructure of cell walls and the mechanisms of cell wall formation are still not fully understood. The objective of our study was therefore to obtain additional fine structural details on the deposition of cell wall components during the differentiation of xylem cells in hybrid aspen ( Populus tremula L. × P. tremuloides Michx.) we used as a model tree. At the electron microscope level, PATAg staining revealed a successive deposition of polysaccharides with increasing distance from the cambium. Staining with potassium permanganate and UV microspectrophotometry showed that the cell walls were lignified, with some delay to the deposition of polysaccharides. Immunogold labelling of three lignin types in developing cell walls varied with progressive deposition of cell wall layers. Condensed lignin subunits were localized in corners of cells adjacent to the cambium prior to S1 formation, whereas non-condensed lignin subunits became labelled only in later stages - in secondary walls near cell corners and simultaneously with the completion of S1 formation. As S2 polysaccharide deposition progressed, the labelling extended towards the lumen. Labelling of peroxidases revealed their presence in cell corner regions of young xylem cells, still lacking a secondary wall, implying that peroxidases are incorporated into the developing cell wall at early developmental stages. A weak labelling of middle lamella regions and secondary walls could also be seen at later stages. The results are discussed in relation to current knowledge on the succession of polysaccharide and lignin deposition in woody cell walls.     

Enzymatic processes involved in the incorporation of hydroxycinnamates into grass cell walls

Many plant species have one or more types of acylation of cell wall polymers. Grasses (Poaceae family) are unique with abundant acylation of specific cell wall polymers by hydroxycinnamates. The most common hydroxycinnamates found in a wide range of grasses are ferulates (trans-4-hydroxy-3-methoxycinnamate) and p-coumarates (trans-4-hydroxycinnamate). These two hydroxycinnamates are synthesized by the phenylpropanoid pathway. Though structurally related, they seem to have different functional roles within the cell wall. Ferulates have been shown to have a critical role in cross-linking cell wall components; forming links between structural polysaccharides and links between structural polysaccharides and lignin. They are incorporated into the cell wall by distinctly different mechanisms. Ferulic acid is incorporated into cell walls as ester linked substituents on arabinoxylans. The exact role p-coumarates play in plant cell walls is unknown, but it has been shown that p-coumaric acid is ester-linked to monolignols and shuttled out to the wall to become incorporated into newly forming lignin polymers. Both processes require the activity of specific hydroxycinnamoyl transferases utilizing CoA derivatives to drive the transferase reactions.     

Structural differentiation of the Bacillus subtilis 168 cell wall.

Exponential-growth-phase cultures of Bacillus subtilis 168 were probed with polycationized ferritin (PCF) or concanavalin A (localized by the addition of horseradish peroxidase conjugated to colloidal gold) to distinguish surface anionic sites and teichoic acid polymers, respectively. Isolated cell walls, lysozyme-digested cell walls, and cell walls treated with mild alkali to remove teichoic acid were also treated with PCF. After labelling, whole cells and walls were processed for electron microscopy by freeze-substitution. Thin sections of untreated cells showed a triphasic, fibrous wall extending more than 30 nm beyond the cytoplasmic membrane. Measurements of wall thickness indicated that the wall was thicker at locations adjacent to septa and at pole-cylinder junctions (P < 0.001). Labelling studies showed that at saturating concentrations the PCF probe labelled the outermost limit of the cell wall, completely surrounding individual cells. However, at limiting PCF concentrations, labelling was observed at only discrete cell surface locations adjacent to or overlying septa and at the junction between pole and cylinder. Labelling was rarely observed along the cell cylinder or directly over the poles. Cells did not label along the cylindrical wall until there was visible evidence of a developing septum. Identical labelling patterns were observed by using concanavalin A-horseradish peroxidase-colloidal gold. Neither probe appeared to penetrate between the fibers of the wall. We suggest that the fibrous appearance of the wall seen in freeze-substituted cells reflects turnover of the wall matrix, that the specificity of labelling to discrete sites on the cell surface is indicative of regions of extreme hydrolytic activity in which alpha-glucose residues of the wall teichoic acids and electronegative sites (contributed by phosphate and carboxyl groups of the teichoic acids and carboxyl groups of the peptidoglycan polymers) are more readily accessible to our probes, and that the wall of exponentially growing B. subtilis cells contains regions of structural differentiation.     

Evolutionary divergence of β–expansin structure and function in grasses parallels emergence of distinctive primary cell wall traits

Expansins are wall‐loosening proteins that promote the extension of primary cell walls without the hydrolysis of major structural components. Previously, proteins from the EXPA (α–expansin) family were found to loosen eudicot cell walls but to be less effective on grass cell walls, whereas the reverse pattern was found for EXPB (β–expansin) proteins obtained from grass pollen. To understand the evolutionary and structural bases for the selectivity of EXPB action, we assessed the extension (creep) response of cell walls from diverse monocot families to EXPA and EXPB treatments. Cell walls from Cyperaceae and Juncaceae (families closely related to grasses) displayed a typical grass response (‘β–response’). Walls from more distant monocots, including some species that share with grasses high levels of arabinoxylan, responded preferentially to α–expansins (‘α–response’), behaving in this regard like eudicots. An expansin with selective activity for grass cell walls was detected in Cyperaceae pollen, coinciding with the expression of genes from the divergent EXPB–I branch that includes grass pollen β–expansins. The evolutionary origin of this branch was located within Poales on the basis of phylogenetic analyses and its association with the ‘sigma’ whole‐genome duplication. Accelerated evolution in this branch has remodeled the protein surface in contact with the substrate, potentially for binding highly substituted arabinoxylan. We propose that the evolution of the divergent EXPB–I group made a fundamental change in the target and mechanism of wall loosening in the grass lineage possible, involving a new structural role for xylans and the expansins that target them.     

Molecular domains of the cellulose/xyloglucan network in the cell walls of higher plants

Cellulose and xyloglucan (XG) assemble to form the cellulose/XG network, which is considered to be the dominant load-bearing structure in the growing cell walls of non-graminaceous land plants. We have extended the most commonly accepted model for the macromolecular organization of XG in this network, based on the structural and quantitative analysis of three distinct XG fractions that can be differentially extracted from the cell walls isolated from etiolated pea stems. Approximately 8% of the dry weight of these cell walls consists of XG that can be solubilized by treatment of the walls with a XG-specific endoglucanase (XEG). This material corresponds to an enzyme-susceptible XG domain, proposed to form the cross-links between cellulose microfibrils. Another 10% of the cell wall consists of XG that can be solubilized by concentrated KOH after XEG treatment. This material constitutes another XG domain, proposed to be closely associated with the surface of the cellulose microfibrils. An additional 3% of the cell wall consists of XG that can be solubilized only when the XEG- and KOH-treated cell walls are treated with cellulase. This material constitutes a third XG domain, proposed to be entrapped within or between cellulose microfibrils. Analysis of the three fractions indicates that metabolism is essentially limited to the enzyme-susceptible domain. These results support the hypothesis that enzyme-catalyzed modification of XG cross-links in the cellulose/XG network is required for the growth and development of the primary plant cell wall, and demonstrate that the structural consequences of these metabolic events can be analyzed in detail.     

Characterization of the Sclerotinia sclerotiorum cell wall proteome

We used a proteomic analysis to identify cell wall proteins released from Sclerotinia sclerotiorum hyphal and sclerotial cell walls via a trifluoromethanesulfonic acid (TFMS) digestion. Cell walls from hyphae grown in Vogel's glucose medium (a synthetic medium lacking plant materials), from hyphae grown in potato dextrose broth and from sclerotia produced on potato dextrose agar were used in the analysis. Under the conditions used, TFMS digests the glycosidic linkages in the cell walls to release intact cell wall proteins. The analysis identified 24 glycosylphosphatidylinositol (GPI)‐anchored cell wall proteins and 30 non‐GPI‐anchored cell wall proteins. We found that the cell walls contained an array of cell wall biosynthetic enzymes similar to those found in the cell walls of other fungi. When comparing the proteins in hyphal cell walls grown in potato dextrose broth with those in hyphal cell walls grown in the absence of plant material, it was found that a core group of cell wall biosynthetic proteins and some proteins associated with pathogenicity (secreted cellulases, pectin lyases, glucosidases and proteases) were expressed in both types of hyphae. The hyphae grown in potato dextrose broth contained a number of additional proteins (laccases, oxalate decarboxylase, peroxidase, polysaccharide deacetylase and several proteins unique to Sclerotinia and Botrytis) that might facilitate growth on a plant host. A comparison of the proteins in the sclerotial cell wall with the proteins in the hyphal cell wall demonstrated that sclerotia formation is not marked by a major shift in the composition of cell wall protein. We found that the S. sclerotiorum cell walls contained 11 cell wall proteins that were encoded only in Sclerotinia and Botrytis genomes.     

Evolution of xyloglucan-related genes in green plants

Background  

The cell shape and morphology of plant tissues are intimately related to structural modifications in the primary cell wall that are associated with key processes in the regulation of cell growth and differentiation. The primary cell wall is composed mainly of cellulose immersed in a matrix of hemicellulose, pectin, lignin and some structural proteins. Xyloglucan is a hemicellulose polysaccharide present in the cell walls of all land plants (Embryophyta) and is the main hemicellulose in non-graminaceous angiosperms.     

A Raman-scattering study on the net orientation of biomacromolecules in the outer epidermal walls of mature wheat stems (Triticum aestivum)

BACKGROUND AND AIMS: Raman spectroscopy can be used to examine the orientation of biomacromolecules using relatively thick samples of material, whereas more traditional means of analysing molecular structure require prior isolation of the components, which often destroys morphological features. In this study, Raman spectroscopy was used to examine the outer epidermal cell walls of wheat stems. METHODS: Polarized Raman spectra from the epidermal cell walls of wheat stem were obtained using near-infrared-Fourier transform Raman scattering. By comparing spectra taken with Raman light polarized perpendicular or parallel to the longitudinal axis of the cell, the orientation of macromolecules in the cell wall was investigated. KEY RESULTS: The net orientation of macromolecules varies in the epidermal cell walls of the different components of wheat stem. The net orientation of cellulose is parallel to the longitudinal axis of the cells, whereas the xylan and the phenylpropane units of lignin tend to lie perpendicular to the longitudinal axis of the cells, i.e. perpendicular to the net orientation of cellulose in the epidermal cell walls. CONCLUSIONS: The results imply that cellulose, lignin and xylan form a relatively ordered network that defines the mechanical and structural properties of the cell wall. Such results are likely to have a significant impact on the formulation of definitive models for the static and growing cell wall.     

Nonaqueous titration of amino groups in polymeric matrix of plant cell walls

Nonaqueous titration was used for detection of free amino groups in the polymeric matrix of plant cell walls. The content of amino groups varied in the range 0.54–0.91 and total nitrogen in the range 1.0–4.2 mmol per gram dry mass of cell walls depending on the plant species. However, these data on the high content of free amino groups do not correlate with the present day concept that the nitrogen fraction in charged amino groups in plant cell wall proteins, which are assumed to be mainly amino groups of lysine and arginine residues, is about 10%. It is supposed that most detected free amino groups belong to the hydroxy-amino acids hydroxyproline and tyrosine that can be bound at the hydroxyl group with the carbohydrate part of glycoprotein or another structural cell wall polymer.     

Differential expression of cell-wall-related genes during the formation of tracheary elements in the Zinnia mesophyll cell system

Plants, animals and some fungi undergo processes of cell specialization such that specific groups of cells are adapted to carry out particular functions. One of the more remarkable examples of cellular development in higher plants is the formation of water-conducting cells that are capable of supporting a column of water from the roots to tens of metres in the air for some trees. The Zinnia mesophyll cell system is a remarkable tool with which to study this entire developmental pathway in vitro. We have recently applied an RNA fingerprinting technology, to allow the detection of DNA fragments derived from RNA using cDNA synthesis and subsequent PCR-amplified fragment length polymorphisms (cDNA-AFLP), to systematically characterize hundreds of the genes involved in the process of tracheary element formation. Building hoops of secondary wall material is the key structural event in forming functional tracheary elements and we have identified over 50 partial sequences related to cell walls out of 600 differentially expressed cDNA fragments. The Zinnia system is an engine of gene discovery which is allowing us to identify and characterize candidate genes involved in cell wall biosynthesis and assembly.     

Observations of the structural changes that occur during charcoalification: implications for identifying charcoal in the fossil record

All of our current understanding of fossil charcoal structure comes from observations of modern wood charcoal produced in furnaces. These charcoals consistently show cell wall homogenization after prolonged heating (>325°C) and this is therefore considered to be a key identifying feature of fossil charcoal. Yet furnaces are unable to replicate the full combustion processes that occur during a wildfire. Here, for the first time, we have studied the microscopic structural evolution of charcoal produced using calorimetry, wherein the wood is ignited under controlled conditions and the heat release rate and other parameters measured, and the resulting charcoal studied using reflected light microscopy. We show that homogenization of cell walls is actually only a short‐lived phase of charcoal formation that occurs during the early heating stages as the pyrolysis front traverses through the wood. Cell wall homogenization is then rapidly overprinted by the thinning, distortion and breakdown of cell walls, and a notable visual increase in reflectance. Our preliminary study therefore suggests that we need to first improve our understanding of charcoal formation in order to better understand the fossil record of wildfires.     

Visualizing lignin coalescence and migration through maize cell walls following thermochemical pretreatment

Plant cell walls are composed primarily of cellulose, hemicelluloses, lignins, and pectins. Of these components, lignins exhibit unique chemistry and physiological functions. Although lignins can be used as a product feedstock or as a fuel, lignins are also generally seen as a barrier to efficient enzymatic breakdown of biomass to sugars. Indeed, many pretreatment strategies focus on removing a significant fraction of lignin from biomass to better enable saccharification. In order to better understand the fate of biomass lignins that remain with the solids following dilute acid pretreatment, we undertook a structural investigation to track lignins on and in biomass cell walls. SEM and TEM imaging revealed a range of droplet morphologies that appear on and within cell walls of pretreated biomass; as well as the specific ultrastructural regions that accumulate the droplets. These droplets were shown to contain lignin by FTIR, NMR, antibody labeling, and cytochemical staining. We provide evidence supporting the idea that thermochemical pretreatments reaching temperatures above the range for lignin phase transition cause lignins to coalesce into larger molten bodies that migrate within and out of the cell wall, and can redeposit on the surface of plant cell walls. This decompartmentalization and relocalization of lignins is likely to be at least as important as lignin removal in the quest to improve the digestibility of biomass for sugars and fuels production.     

Distribution of pectic epitopes in cell walls of the sugar beet root

Immunolabelling techniques with antibodies specific to partially methyl-esterified homogalacturonan (JIM5: unesterified residues flanked by methylesterified residues. JIM7: methyl-esterified residues flanked by unesterified residues), a blockwise de-esterified homogalacturonan (2F4), 1,4-galactan (LM5) and 1,5-arabinan (LM6) were used to map the distribution of pectin motifs in cell walls of sugar beet root (Beta vulgaris). PME and alkali treatments of sections were used in conjunction with JIM5-7 and 2F4. The JIM7 epitope was abundant and equally distributed in all cells. In storage parenchyma, the JIM5 epitope was restricted to some cell junctions and the lining of intercellular spaces while in vascular tissues it occurred at cell junctions in some phloem walls and in xylem derivatives. After secondary wall formation, the JIM5 epitope was restricted to inner cell wall regions between secondary thickenings. The 2F4 epitope was not detected without de-esterification treatment. PME treatments prior to the use of 2F4 indicated that HG at cell corners was not acetylated. The LM5 epitope was mainly present in the cambial zone and when present in storage parenchyma, it was restricted to the wall region closest to the plasma membrane. The LM6 epitope was widely distributed throughout primary walls but was more abundant in bundles than in medullar ray tissue and storage parenchyma. These data show that the occurrence of oligosaccharide motifs of pectic polysaccharides are spatially regulated in sugar beet root cell walls and that the spatial patterns vary between cell types suggesting that structural variants of pectic polymers are involved in the modulation of cell wall properties.     

The functions of cell wall polysaccharides in composition and architecture revealed through mutations

The plant cell wall is a highly organized composite of many different polysaccharides, proteins and aromatic substances. These complex matrices define the shape of each individual cell, and ultimately, they are the determinants of plant morphology. The fine structures of the major angiosperm cell wall polysaccharides have been characterized, but it is not well understood how these polysaccharides are assembled into a metabolically active architecture. Cell wall biogenesis and remodeling may be partitioned into six major stages of development (precursor synthesis, polymerization, secretion, assembly, rearrangement and disassembly), and to date, a handful of mutations have been identified that affect the composition and structure in each of these stages. To greatly augment this collection, we have initiated a program to use Fourier transform infrared spectroscopy as a high through-put screen to identify a broad range of cell-wall mutants of Arabidopsis and maize. We anticipate that such mutants will be useful to probe the impact of the individual components and their metabolism on basic processes of plant growth and development. The structures of dicot and grass walls, the identification of representative cell wall mutants, and the use of a novel spectroscopic screen to identify many more cell wall mutants, are briefly reviewed.     

Dynamics of intracellular mannan and cell wall folding in the drought responses of succulent Aloe species

Plants have evolved a multitude of adaptations to survive extreme conditions. Succulent plants have the capacity to tolerate periodically dry environments, due to their ability to retain water in a specialized tissue, termed hydrenchyma. Cell wall polysaccharides are important components of water storage in hydrenchyma cells. However, the role of the cell wall and its polysaccharide composition in relation to drought resistance of succulent plants are unknown. We investigate the drought response of leaf‐succulent Aloe (Asphodelaceae) species using a combination of histological microscopy, quantification of water content, and comprehensive microarray polymer profiling. We observed a previously unreported mode of polysaccharide and cell wall structural dynamics triggered by water shortage. Microscopical analysis of the hydrenchyma cell walls revealed highly regular folding patterns indicative of predetermined cell wall mechanics in the remobilization of stored water and the possible role of homogalacturonan in this process. The in situ distribution of mannans in distinct intracellular compartments during drought, for storage, and apparent upregulation of pectins, imparting flexibility to the cell wall, facilitate elaborate cell wall folding during drought stress. We conclude that cell wall polysaccharide composition plays an important role in water storage and drought response in Aloe.     

Analysis of the proteins involved in the structure and synthesis of the cell wall of Ustilago maydis

A study of the proteins involved in the synthesis and structure of the cell wall of Ustilago maydis was made by in silico analysis of the fungal genome, with reference to supporting experimental evidence. The composition of the cell wall of U. maydis shows similarities with the structural composition of the walls of Ascomycetes, but also shows important differential features. Accordingly, the enzymes involved in the synthesis of the U. maydis wall polysaccharides chitin and beta-1,6 glucans displayed some differential characteristics. The most salient difference in protein composition was the predicted absence of Pir proteins, an important class of proteins present in the Ascomycetes. Other classes of proteins that are covalently-linked to the wall in Ascomycetes, including those bound through disulfide linkages, joined by alkali-labile bonds, and GPI proteins, were predicted to be present in the U. maydis walls. The main characteristic of the exo-cellular, non-covalently-bound proteins was their relative low number, especially for hydrolytic enzymes.     

Cell wall proteins: a new insight through proteomics

Cell wall proteins are essential constituents of plant cell walls; they are involved in modifications of cell wall components, wall structure, signaling and interactions with plasma membrane proteins at the cell surface. The application of proteomic approaches to the cell wall compartment raises important questions: are there technical problems specific to cell wall proteomics? What kinds of proteins can be found in Arabidopsis walls? Are some of them unexpected? What sort of post-translational modifications have been characterized in cell wall proteins to date? The purpose of this review is to discuss the experimental results obtained to date using proteomics, as well as some of the new questions challenging future research.