Characterization of the fiber-like cortical cells in moss gametophytes.

MAIN CONCLUSION: Fiber-like cells with thickened cell walls of specific structure and polymer composition that includes (1 → 4)-ß-galactans develop in the outer stem cortex of several moss species gametophytes. The early land plants evolved several specialized cell types and tissues that did not exist in their aquatic ancestors. Of these, water-conducting elements and reproductive organs have received most of the research attention. The evolution of tissues specialized to fulfill a mechanical function is by far less studied despite their wide distribution in land plants. For vascular plants following a homoiohydric trajectory, the evolutionary emergence of mechanical tissues is mainly discussed starting with the fern-like plants with their hypodermal sterome or sclerified fibers that have xylan and lignin-based cell walls. However, mechanical challenges were also faced by bryophytes, which lack lignified cell-walls. To characterize mechanical tissues in the bryophyte lineage, following a poikilohydric trajectory, we used six wild moss species (Polytrichum juniperinum, Dicranum sp., Rhodobryum roseum, Eurhynchiadelphus sp., Climacium dendroides, and Hylocomium splendens) and analyzed the structure and composition of their cell walls. In all of them, the outer stem cortex of the leafy gametophytic generation had fiber-like cells with a thickened but non-lignified cell wall. Such cells have a spindle-like shape with pointed tips. The additional thick cell wall layer in those fiber-like cells is composed of sublayers with structural evidence for different cellulose microfibril orientation, and with specific polymer composition that includes (1 → 4)-ß-galactans. Thus, the basic cellular characters of the cells that provide mechanical support in vascular plant taxa (elongated cell shape, location at the periphery of a primary organ, the thickened cell wall and its peculiar composition and structure) also exist in mosses.

Rhamnogalacturonan I with ß-(1,4)-Galactan Side Chains as an Ever-Present Component of Tertiary Cell Wall of Plant Fibers.

The cellulose-enriched tertiary cell walls present in many plant fibers have specific composition, architecture, machinery of formation, and function. To better understand the mechanisms underlying their mode of action and to reveal the peculiarities of fibers from different plant species, it is necessary to more deeply characterize the major components. Next to overwhelming cellulose, rhamnogalacturonan I (RG-I) is considered to be the key polymer of the tertiary cell wall; however, it has been isolated and biochemically characterized in very few plant species. Here, we add RG-I to the list from the phloem fibers of the Phaseolus vulgaris stem that was isolated and analyzed by nuclear magnetic resonance (NMR), dynamic light scattering, and immunolabeling, both within tissue and as an isolated polymer. Additionally, fibers with tertiary cell walls from nine species of dicotyledonous plants from the orders Malphigiales, Fabales, and Rosales were labeled with RG-I-related antibodies to check the presence of the polymer and compare the in situ presentation of its backbone and side chains. The obtained results confirm that RG-I is an obligatory polymer of the tertiary cell wall. However, there are differences in the structure of this polymer from various plant sources, and these peculiarities may be taxonomically related.

Plant Polysaccharide Array for Studying Carbohydrate-Binding Proteins.

The specificity of the most plant carbohydrate-binding proteins (CBP), many of which are known only through bioinformatic analysis of the genome, has either not been studied at all or characterized to a limited extent. The task of deciphering the carbohydrate specificity of the proteins can be solved using glycoarrays composed of many tens or even hundreds of glycans immobilized on a glass surface. Plant carbohydrates are the most significant natural ligands for plant proteins; this work shows that plant polysaccharides without additional modification can be immobilized on the surface, bearing N-hydroxysuccinimide activated carboxyl groups. As a result, an array of 113 well-characterized polysaccharides isolated from various plant cell walls, 23 mono- and oligosaccharides - components of polysaccharides, and glycans - ligands for widely known plant lectins was designed. Upon chemical immobilization of polysaccharides, their functional activity was preserved, which was confirmed by the results of interaction with antibodies and the plant lectin ricin. Using the constructed array, a previously unknown ability of ricin to bind polysaccharides was found, which significantly expands the knowledge of its specificity, and it was also found that a large variety of antibodies to plant polysaccharides are present in human peripheral blood.

Growing Maize Root: Lectins Involved in Consecutive Stages of Cell Development.

Proteins that carry specific carbohydrate-binding lectin domains have a great variety and are ubiquitous across the plant kingdom. In turn, the plant cell wall has a complex carbohydrate composition, which is subjected to constant changes in the course of plant development. In this regard, proteins with lectin domains are of great interest in the context of studying their contribution to the tuning and monitoring of the cell wall during its modifications in the course of plant organ development. We performed a genome-wide screening of lectin motifs in the Zea mays genome and analyzed the transcriptomic data from five zones of primary maize root with cells at different development stages. This allowed us to obtain 306 gene sequences encoding putative lectins and to relate their expressions to the stages of root cell development and peculiarities of cell wall metabolism. Among the lectins whose expression was high and differentially regulated in growing maize root were the members of the EUL, dirigent-jacalin, malectin, malectin-like, GNA and Nictaba families, many of which are predicted as cell wall proteins or lectin receptor-like kinases that have direct access to the cell wall. Thus, a set of molecular players was identified with high potential to play important roles in the early stages of root morphogenesis.

A flax mutant with impaired intrusive growth of phloem and xylem fibres demonstrates constitutive gravitropic response.

Intrusive growth is a type of growth in which a cell exceeds the growth rate of its neighbours and intrudes between them, reaching a much greater length. This process provides plant fibres with their exceptional length. Fibres are the most abundant cell type in the mechanical tissues of plants. At the same time, the plant fibres are of fundamental importance for the production of textiles, paper, biocomposites, etc. Here we describe a mutant of flax (reduced fibre 1, rdf) in which intrusive growth of fibres is impaired in both phloem and xylem. In addition to the intrinsic differences in fibre length, the mutant is characterized by a constitutive gravitropic response, mechanical aberrations at the macro- and nanolevels, disruption of the cambium and uneven transition of xylem cells to secondary cell wall formation. Gelatinous cell walls in both phloem and xylem of mutant plants have disturbed structure and reduced elasticity. The existence of this mutant-control pair offers both prospects for finding the molecular players involved in triggering intrusive growth, cell wall thickening and for understanding the principles of plant mechanical tissue functioning.

Dynamics of cell wall polysaccharides during the elongation growth of rye primary roots.

MAIN CONCLUSION: In cells of growing rye roots, xyloglucans and homogalacturonans demonstrate developmental stage specificity, while different xylans have tissue specificity. Mannans, arabinans and galactans are also detected within the protoplast. Mannans form films on sections of fresh material. The primary cell walls of plants represent supramolecular exocellular structures that are mainly composed of polysaccharides. Cell wall properties and architecture differ between species and across tissues within a species. We revised the distribution of cell wall polysaccharides and their dynamics during elongation growth and histogenesis in rye roots using nonfixed material and the spectrum of antibodies. Rye is a member of the Poaceae family and thus has so-called type II primary cell walls, which are supposed to be low in pectins and xyloglucans and instead have arabinoxylans and mixed-linkage glucans. However, rye cell walls at the earliest stages of cell development were enriched with the epitopes of xyloglucans and homogalacturonans. Mixed-linkage glucan, which is often considered an elongation growth-specific polysaccharide in plants with type II cell walls, did not display such dynamics in rye roots. The cessation of elongation growth and even the emergence of root hairs were not accompanied by the disappearance of mixed-linkage glucans from cell walls. The diversity of xylan motifs recognized by different antibodies was minimal in the meristem zone of rye roots, but this diversity increased and showed tissue specificity during root growth. Antibodies specific for xyloglucans, galactans, arabinans and mannans bound the cell content. When rye root cells were cut, the epitopes of xyloglucans, galactans and arabinans remained within the cell content, while mannans developed net-like or film-like structures on the surface of sections.

The Modification of Plant Cell Wall Polysaccharides in Potato Plants during Pectobacterium atrosepticum-Caused Infection.

Our study is the first to consider the changes in the entire set of matrix plant cell wall (PCW) polysaccharides in the course of a plant infectious disease. We compared the molecular weight distribution, monosaccharide content, and the epitope distribution of pectic compounds and cross-linking glycans in non-infected potato plants and plants infected with Pectobacterium atrosepticum at the initial and advanced stages of plant colonization by the pathogen. To predict the gene products involved in the modification of the PCW polysaccharide skeleton during the infection, the expression profiles of potato and P. atrosepticum PCW-related genes were analyzed by RNA-Seq along with phylogenetic analysis. The assemblage of P. atrosepticum biofilm-like structures-the bacterial emboli-and the accumulation of specific fragments of pectic compounds that prime the formation of these structures were demonstrated within potato plants (a natural host of P. atrosepticum). Collenchyma was shown to be the most "vulnerable" tissue to P. atrosepticum among the potato stem tissues. The infection caused by the representative of the Soft Rot Pectobacteriaceae was shown to affect not only pectic compounds but also cross-linking glycans; the content of the latter was increased in the infected plants compared to the non-infected ones.

Cell Wall Layer Induced in Xylem Fibers of Flax Upon Gravistimulation Is Similar to Constitutively Formed Cell Walls of Bast Fibers.

In the fibers of many plant species after the formation of secondary cell walls, cellulose-enriched cell wall layers (often named G-layers or tertiary cell walls) are deposited which are important in many physiological situations. Flax (Linum usitatissimum L.) phloem fibers constitutively develop tertiary cell walls during normal plant growth. During the gravitropic response after plant inclination, the deposition of a cellulose-enriched cell wall layer is induced in xylem fibers on one side of the stem, providing a system similar to that of tension wood in angiosperm trees. Atomic force microscopy (AFM), immunochemistry, and transcriptomic analyses demonstrated that the G-layer induced in flax xylem fibers was similar to the constitutively formed tertiary cell wall of bast (phloem) fibers but different from the secondary cell wall. The tertiary cell walls, independent of tissue of origin and inducibility, were twice as stiff as the secondary cell walls. In the gravitropic response, the tertiary cell wall deposition rate in xylem was higher than that of the secondary cell wall. Rhamnogalacturonan I (RG-I) with galactan side chains was a prominent component in cellulose-rich layers of both phloem and xylem flax fibers. Transcriptomic events underlying G-layer deposition in phloem and xylem fibers had much in common. At the induction of tertiary cell wall deposition, several genes for rhamnosyltransferases of the GT106 family were activated in xylem samples. The same genes were expressed in the isolated phloem fibers depositing the tertiary cell wall. The comparison of transcriptomes in fibers with both inducible and constitutive tertiary cell wall deposition and xylem tissues that formed the secondary cell walls is an effective system that revealed important molecular players involved in the formation of cellulose-enriched cell walls.

Elongation of wood fibers combines features of diffuse and tip growth.

Xylem fibers are highly elongated cells that are key constituents of wood, play major physiological roles in plants, comprise an important terrestrial carbon reservoir, and thus have enormous ecological and economic importance. As they develop, from fusiform initials, their bodies remain the same length while their tips elongate and intrude into intercellular spaces. To elucidate mechanisms of tip elongation, we studied the cell wall along the length of isolated, elongating aspen xylem fibers and used computer simulations to predict the forces driving the intercellular space formation required for their growth. We found pectin matrix epitopes (JIM5, LM7) concentrated at the tips where cellulose microfibrils have transverse orientation, and xyloglucan epitopes (CCRC-M89, CCRC-M58) in fiber bodies where microfibrils are disordered. These features are accompanied by changes in cell wall thickness, indicating that while the cell wall elongates strictly at the tips, it is deposited all over fibers. Computer modeling revealed that the intercellular space formation needed for intrusive growth may only require targeted release of cell adhesion, which allows turgor pressure in neighboring fiber cells to 'round' the cells creating spaces. These characteristics show that xylem fibers' elongation involves a distinct mechanism that combines features of both diffuse and tip growth.

Gene Expression Patterns for Proteins With Lectin Domains in Flax Stem Tissues Are Related to Deposition of Distinct Cell Wall Types.

The genomes of higher plants encode a variety of proteins with lectin domains that are able to specifically recognize certain carbohydrates. Plants are enriched in a variety of potentially complementary glycans, many of which are located in the cell wall. We performed a genome-wide search for flax proteins with lectin domains and compared the expression of the encoding genes in different stem tissues that have distinct cell wall types with different sets of major polysaccharides. Over 400 genes encoding proteins with lectin domains that belong to different families were revealed in the flax genome; three quarters of these genes were expressed in stem tissues. Hierarchical clustering of the data for all expressed lectins grouped the analyzed samples according to their characteristic cell wall type. Most lectins differentially expressed in tissues with primary, secondary, and tertiary cell walls were predicted to localize at the plasma membrane or cell wall. These lectins were from different families and had various architectural types. Three out of four flax genes for proteins with jacalin-like domains were highly upregulated in bast fibers at the stage of tertiary cell wall deposition. The dynamic changes in transcript level of many genes for lectins from various families were detected in stem tissue over the course of gravitropic response induced by plant gravistimulation. The data obtained in this study indicate a large number of lectin-mediated events in plants and provide insight into the proteins that take part in tissue specialization and reaction to abiotic stress.

Gradients of cell wall nano-mechanical properties along and across elongating primary roots of maize.

To test the hypothesis that particular tissues can control root growth, we analysed the mechanical properties of cell walls belonging to different tissues of the apical part of the maize root using atomic force microscopy. The dynamics of properties during elongation growth were characterized in four consecutive zones of the root. Extensive immunochemical characterization and quantification were used to establish the polysaccharide motif(s) related to changes in cell wall mechanics. Cell transition from division to elongation was coupled to the decrease in the elastic modulus in all root tissues. Low values of moduli were retained in the elongation zone and increased in the late elongation zone. No relationship between the immunolabelling pattern and mechanical properties of the cell walls was revealed. When measured values of elastic moduli and turgor pressure were used in the computational simulation, this resulted in an elastic response of the modelled root and the distribution of stress and strain similar to those observed in vivo. In all analysed root zones, cell walls of the inner cortex displayed moduli of elasticity that were maximal or comparable with the maximal values among all tissues. Thus, we propose that the inner cortex serves as a growth-limiting tissue in maize roots.

Forgotten Actors: Glycoside Hydrolases During Elongation Growth of Maize Primary Root.

Plant cell enlargement is coupled to dynamic changes in cell wall composition and properties. Such rearrangements are provided, besides the differential synthesis of individual cell wall components, by enzymes that modify polysaccharides in muro. To reveal enzymes that may contribute to these modifications and relate them to stages of elongation growth in grasses, we carried out a transcriptomic study of five zones of the primary maize root. In the initiation of elongation, significant changes occur with xyloglucan: once synthesized in the meristem, it can be linked to other polysaccharides through the action of hetero-specific xyloglucan endotransglycosidases, whose expression boosts at this stage. Later, genes for xyloglucan hydrolases are upregulated. Two different sets of enzymes capable of modifying glucuronoarabinoxylans, mainly bifunctional α-arabinofuranosidases/ß-xylosidases and ß-xylanases, are expressed in the maize root to treat the xylans of primary and secondary cell walls, respectively. The first set is highly pronounced in the stage of active elongation, while the second is at elongation termination. Genes encoding several glycoside hydrolases that are able to degrade mixed-linkage glucan are downregulated specifically at the active elongation. It indicates the significance of mixed-linkage glucans for the cell elongation process. The possibility that many glycoside hydrolases act as transglycosylases in muro is discussed.

The Toolbox for Fiber Flax Breeding: A Pipeline From Gene Expression to Fiber Quality.

The goal of any plant breeding program is to improve quality of a target crop. Crop quality is a comprehensive feature largely determined by biological background. To improve the quality parameters of crops grown for the production of fiber, a functional approach was used to search for genes suitable for the effective manipulation of technical fiber quality. A key step was to identify genes with tissue and stage-specific pattern of expression in the developing fibers. In the current study, we investigated the relationship between gene expression evaluated in bast fibers of developing flax plants and the quality parameters of technical fibers measured after plant harvesting. Based on previously published transcriptomic data, two sets of genes that are upregulated in fibers during intrusive growth and tertiary cell wall deposition were selected. The expression level of the selected genes and fiber quality parameters were measured in fiber flax, linseed (oil flax) cultivars, and wild species that differ in type of yield and fiber quality parameters. Based on gene expression data, linear regression models for technical stem length, fiber tensile strength, and fiber flexibility were constructed, resulting in the identification of genes that have high potential for manipulating fiber quality. Chromosomal localization and single nucleotide polymorphism distribution in the selected genes were characterized for the efficacy of their use in conventional breeding and genome editing programs. Transcriptome-based selection is a highly targeted functional approach that could be used during the development of new cultivars of various crops.

Elongating maize root: zone-specific combinations of polysaccharides from type I and type II primary cell walls.

The dynamics of cell wall polysaccharides may modulate the cell wall mechanics and thus control the expansion growth of plant cells. The unique composition of type II primary cell wall characteristic of grasses suggests that they employ specific mechanisms for cell enlargement. We characterized the transcriptomes in five zones along maize root, clustered the expression of genes for numerous glycosyltransferases and performed extensive immunohistochemical analysis to relate the changes in cell wall polysaccharides to critical stages of cell development in Poaceae. Specific patterns of cell wall formation differentiate the initiation, realization and cessation of elongation growth. Cell walls of meristem and early elongation zone represent a mixture of type I and type II specific polysaccharides. Xyloglucans and homogalacturonans are synthesized there actively together with mixed-linkage glucans and glucuronoarabinoxylans. Rhamnogalacturonans-I with the side-chains of branched 1,4-galactan and arabinan persisted in cell walls throughout the development. Thus, the machinery to generate the type I primary cell wall constituents is completely established and operates. The expression of glycosyltransferases responsible for mixed-linkage glucan and glucuronoarabinoxylan synthesis peaks at active or late elongation. These findings widen the number of jigsaw pieces which should be put together to solve the puzzle of grass cell growth.

The Living Fossil Psilotum nudum Has Cortical Fibers With Mannan-Based Cell Wall Matrix.

Cell wall thickening and development of secondary cell walls was a major step in plant terrestrialization that provided the mechanical support, effective functioning of water-conducting elements and fortification of the surface tissues. Despite its importance, the diversity, emergence and evolution of secondary cell walls in early land plants have been characterized quite poorly. Secondary cell walls can be present in different cell types with fibers being among the major ones. The necessity for mechanical support upon increasing plant height is widely recognized; however, identification of fibers in land plants of early taxa is quite limited. In an effort to partially fill this gap, we studied the fibers and the composition of cell walls in stems of the sporophyte of the living fossil Psilotum nudum. Various types of light microscopy, combined with partial tissue maceration demonstrated that this perennial, rootless, fern-like vascular plant, has abundant fibers located in the middle cortex. Extensive immunodetection of cell wall polymers together with various staining and monosaccharide analysis of cell wall constituents revealed that in P. nudum, the secondary cell wall of its cortical fibers is distinct from that of its tracheids. Primary cell walls of all tissues in P. nudum shoots are based on mannan, which is also common in other extant early land plants. Besides, the primary cell wall contains epitope for LM15 specific for xyloglucan and JIM7 that binds methylesterified homogalacturonans, two polymers common in the primary cell walls of higher plants. Xylan and lignin were detected as the major polymers in the secondary cell walls of P. nudum tracheids. However, the secondary cell wall in its cortical fibers is quite similar to their primary cell walls, i.e., enriched in mannan. The innermost secondary cell wall layer of its fibers but not its tracheids has epitope to bind the LM15, LM6, and LM5 antibodies recognizing, respectively, xyloglucan, arabinan and galactan. Together, our data provide the first description of a mannan-based cell wall in sclerenchyma fibers, and demonstrate in detail that the composition and structure of secondary cell wall in early land plants are not uniform in different tissues.

Stimulation of adventitious root formation by the oligosaccharin OSRG at the transcriptome level.

Oligosaccharins, which are biologically active oligosaccharide fragments of cell wall polysaccharides, may regulate the processes of growth and development as well as the response to stress factors. We characterized the effect of the oligosaccharin that stimulates rhizogenesis (OSRG) on the gene expression profile in the course of IAA-induced formation of adventitious roots in hypocotyl explants of buckwheat (Fagopyrum esculentum Moench.). The transcriptomes at two stages of IAA-induced root primordium formation (6 h and 24 h after induction) were compared after either treatment with auxin alone or joint treatment with auxin and OSRG. The set of differentially expressed genes indicated the special importance of oligosaccharin at the early stage of auxin-induced adventitious root formation. The list of genes with altered mRNA abundance in the presence of oligosaccharin included those, which Arabidopsis homologs encode proteins directly involved in the response to auxin as well as proteins that contribute to redox regulation, detoxification of various compounds, vesicle trafficking, and cell wall modification. The obtained results contribute to understanding the mechanism of adventitious root formation and demonstrate that OSRG is involved in fine-tuning of ROS and auxin regulatory modes involved in root development.

Assessment of Primary Cell Wall Nanomechanical Properties in Internal Cells of Non-Fixed Maize Roots.

The mechanical properties of cell walls play a vital role in plant development. Atomic-force microscopy (AFM) is widely used for characterization of these properties. However, only surface or isolated plant cells have been used for such investigations, at least as non-embedded samples. Theories that claim a restrictive role of a particular tissue in plant growth cannot be confirmed without direct measurement of the mechanical properties of internal tissue cell walls. Here we report an approach of assessing the nanomechanical properties of primary cell walls in the inner tissues of growing plant organs. The procedure does not include fixation, resin-embedding or drying of plant material. Vibratome-derived longitudinal and transverse sections of maize root were investigated by AFM in a liquid cell to track the changes of cell wall stiffness and elasticity accompanying elongation growth. Apparent Young's modulus values and stiffness of stele periclinal cell walls in the elongation zone of maize root were lower than in the meristem, i.e., cell walls became more elastic and less resistant to an applied force during their elongation. The trend was confirmed using either a sharp or spherical probe. The availability of such a method may promote our understanding of individual tissue roles in the plant growth processes.

AFM analysis reveals polymorphism of purified flax rhamnogalacturonans I of distinct functional types.

Functionally distinct polymers organized on the basis of rhamnogalacturonan I (RG-I) backbone with more than a half of rhamnose residues substituted by the side chains containing mostly galactose were purified from flaxseed mucilage, the primary cell wall of young hypocotyls and tertiary cell walls of bast fibers and characterized by atomic force microscopy. Seed mucilage RG-I with short side chains and unusual O3 substitution showed loose coils or star-like conformations. Primary cell wall RG-I, which included polygalacturonan (PGA) fragments, represented micellar objects and rare long chains. Pure RG-I with long galactan side chains, which was isolated as nascent polysaccharide before its incorporation into the tertiary cell wall of bast fibers was observed as long unbranched objects. RG-I entrapped by cellulose microfibrils in tertiary cell wall was visualized as compact micellar complexes. All types of flax RGs-I tended to aggregate. Relationships between RG-I structure and morphology are discussed.

Intrusive Growth of Phloem Fibers in Flax Stem: Integrated Analysis of miRNA and mRNA Expression Profiles.

Phloem fibers are important elements of plant architecture and the target product of many fiber crops. A key stage in fiber development is intrusive elongation, the mechanisms of which are largely unknown. Integrated analysis of miRNA and mRNA expression profiles in intrusivelygrowing fibers obtained by laser microdissection from flax (Linum usitatissimum L.) stem revealed all 124 known flax miRNA from 23 gene families and the potential targets of differentially expressed miRNAs. A comparison of the expression between phloem fibers at different developmental stages, and parenchyma and xylem tissues demonstrated that members of miR159, miR166, miR167, miR319, miR396 families were down-regulated in intrusively growing fibers. Some putative target genes of these miRNA families, such as those putatively encoding growth-regulating factors, an argonaute family protein, and a homeobox-leucine zipper family protein were up-regulated in elongating fibers. miR160, miR169, miR390, and miR394 showed increased expression. Changes in the expression levels of miRNAs and their target genes did not match expectations for the majority of predicted target genes. Taken together, poorly understood intrusive fiber elongation, the key process of phloem fiber development, was characterized from a miRNA-target point of view, giving new insights into its regulation.