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.

The Effect of Leaf Plasticity on the Isolation of Apoplastic Fluid from Leaves of Tartary Buckwheat Plants Grown In Vivo and In Vitro.

Vacuum infiltration-centrifugation (VIC) is the most reproducible technique for the isolation of apoplast washing fluid (AWF) from leaves, but its effectiveness depends on the infiltration-centrifugation conditions and the anatomical and physiological peculiarities of leaves. This study aimed to elaborate an optimal procedure for AWF isolation from the leaves of Tartary buckwheat grown in in vivo and in vitro conditions and reveal the leaf anatomical and physiological traits that could contribute to the effectiveness of AWF isolation. Here, it was demonstrated that leaves of buckwheat plants grown in vitro could be easier infiltrated, were less sensitive to higher forces of centrifugation (900× g and 1500× g), and produced more AWF yield and apoplastic protein content than in vivo leaves at the same forces of centrifugation (600× g and 900× g). The extensive study of the morphological, anatomical, and ultrastructural characteristics of buckwheat leaves grown in different conditions revealed that in vitro leaves exhibited significant plasticity in a number of interconnected morphological, anatomical, and physiological features, generally driven by high RH and low lighting; some of them, such as the reduced thickness and increased permeability of the cuticle of the epidermal cells, large intercellular spaces, increase in the size of stomata and in the area of stomatal pores, higher stomata index, drop in density, and area of calcium oxalate druses, are beneficial to the effectiveness of VIC. The size of stomata pores, which were almost twice as large in in vitro leaves as those in in vivo ones, was the main factor contributing to the isolation of AWF free of chlorophyll contamination. The opening of stomata pores by artificially created humid conditions reduced damage to the in vivo leaves and improved the VIC of them. For Fagopyrum species, this is the first study to develop a VIC technique for AWF isolation from leaves.

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.

Root growth of monocotyledons and dicotyledons is limited by different tissues.

Plant growth and morphogenesis are determined by the mechanical properties of its cell walls. Using atomic force microscopy, we have characterized the dynamics of cell wall elasticity in different tissues in developing roots of several plant species. The elongation growth zone of roots of all species studied was distinguished by a reduced modulus of elasticity of most cell walls compared to the meristem or late elongation zone. Within the individual developmental zones of roots, there were also significant differences in the elasticity of the cell walls of the different tissues, thus identifying the tissues that limit root growth in the different species. In cereals, this is mainly the inner cortex, whereas in dicotyledons this function is performed by the outer tissues-rhizodermis and cortex. These differences result in a different behaviour of the roots of these species during longitudinal dissection. Modelling of longitudinal root dissection using measured properties confirmed the difference shown. Thus, the morphogenesis of monocotyledonous and dicotyledonous roots relies on different tissues as growth limiting, which should be taken into account when analyzing the localization of associated molecular events. At the same time, no matrix polysaccharide was found whose immunolabelling in type I or type II cell walls would predict their mechanical properties. However, assessment of the degree of anisotropy of cortical microtubules showed a striking correlation with the elasticity of the corresponding cell walls in all species studied.

Technogenic Fiber Wastes for Optimizing Concrete.

A promising method of obtaining mineral fiber fillers for dry building mixtures is the processing of waste that comes from the production of technogenic fibrous materials (TFM). The novelty of the work lies in the fact that, for the first time, basalt production wastes were studied not only as reinforcing components, but also as binder ones involved in concrete structure formation. The purpose of the article is to study the physical and mechanical properties of waste technogenic fibrous materials as additives for optimizing the composition of raw concrete mixes. To assess the possibility of using wastes from the complex processing of TFM that were ground for 5 and 10 min as an active mineral additive to concrete, their chemical, mineralogical, and granulometric compositions, as well as the microstructure and physical and mechanical characteristics of the created concretes, were studied. It is established that the grinding of TFM for 10 min leads to the grinding of not only fibers, but also pellets, the fragments of which are noticeable in the total mass of the substance. The presence of quartz in the amorphous phase of TFM makes it possible to synthesize low-basic calcium silicate hydrates in a targeted manner. At 90 days age, at 10-20% of the content of TFM, the strength indicators increase (above 40 MPa), and at 30% of the additive content, they approach the values of the control composition without additives (above 35 MPa). For all ages, the ratio of flexural and compressive strengths is at the level of 0.2, which characterizes a high reinforcing effect. Analysis of the results suggests the possibility of using waste milled for 10 min as an active mineral additive, as well as to give better formability to the mixture and its micro-reinforcement to obtain fiber-reinforced concrete.

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.

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.

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.

Rearrangement of the Cellulose-Enriched Cell Wall in Flax Phloem Fibers over the Course of the Gravitropic Reaction.

The plant cell wall is a complex structure consisting of a polysaccharide network. The rearrangements of the cell wall during the various physiological reactions of plants, however, are still not fully characterized. Profound changes in cell wall organization are detected by microscopy in the phloem fibers of flax (Linum usitatissimum) during the restoration of the vertical position of the inclined stems. To characterize the underlying biochemical and structural changes in the major cell wall polysaccharides, we compared the fiber cell walls of non-inclined and gravistimulated plants by focusing mainly on differences in non-cellulosic polysaccharides and the fine cellulose structure. Biochemical analysis revealed a slight increase in the content of pectins in the fiber cell walls of gravistimulated plants as well as an increase in accessibility for labeling non-cellulosic polysaccharides. The presence of galactosylated xyloglucan in the gelatinous cell wall layer of flax fibers was demonstrated, and its labeling was more pronounced in the gravistimulated plants. Using solid state NMR, an increase in the crystallinity of the cellulose in gravistimulated plants, along with a decrease in cellulose mobility, was demonstrated. Thus, gravistimulation may affect the rearrangement of the cell wall, which can enable restoration in a vertical position of the plant stem.

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.

Plant 'muscles': fibers with a tertiary cell wall.

Plants, although sessile organisms, are nonetheless able to move their body parts; for example, during root contraction of geophytes or in the gravitropic reaction by woody stems. One of the major mechanisms enabling these movements is the development of specialized structures that possess contractile properties. Quite unlike animal muscles, for which the action is driven by protein-protein interactions in the protoplasma, the action of plant 'muscles' is polysaccharide-based and located in the uniquely designed, highly cellulosic cell wall that is deposited specifically in fibers. This review describes the development of such cell walls as a widespread phenomenon in the plant kingdom, gives reasons why it should be considered as a tertiary cell wall, and discusses the mechanism of action of the 'muscles'. The origin of the contractile properties lies in the tension of the axially oriented cellulose microfibrils due to entrapment of rhamnogalacturonan-I aggregates that limits the lateral interaction of microfibrils. Long side chains of the nascent rhamnogalacturonan-I are trimmed off during cell wall maturation leading to tension development. Similarities in the tertiary cell wall design in fibers of different plant origin indicate that the basic principles of tension creation may be universal in various ecophysiological situations.

Phloem fibres as motors of gravitropic behaviour of flax plants: level of transcriptome.

Restoration of stem vertical position after plant inclination is a widely spread version of plant orientation in accordance with gravity vector direction. Gravitropic behaviour of flax plants involves the formation of curvature in stem region that has ceased elongation long in advance of stem inclination. The important participants of such behaviour are phloem fibres with constitutively formed tertiary cell wall (G-layer). We performed the large-scale transcriptome profiling of phloem fibres isolated from pulling and opposite sides of gravitropic curvature and compared with control plant fibres. Significant changes in transcript abundance take place for genes encoding proteins of several ion channels, transcription factors and other regulating elements. The largest number of upregulated genes belonged to the cell wall category; many of those were specifically upregulated in fibres of pulling stem side. The obtained data permit to suggest the mechanism of fibre participation in gravitropic reaction that involves the increase of turgor pressure and the rearrangements of cell wall structure in order to improve contractile properties, and to identify the regulatory elements that operate specifically in the fibres of the pulling stem side making gelatinous phloem fibres an important element of gravitropic response in herbaceous plants.

Brown Mycelial Mat as an Essential Morphological Structure of the Shiitake Medicinal Mushroom Lentinus edodes (Agaricomycetes).

We show here, to our knowledge for the first time, that the brown mycelial mat of the xylotrophic shiitake medicinal mushroom, Lentinus edodes, not only performs a protective function owing to significant changes in the ultrastructure (thickening of the cell wall, increased density, and pigmentation of the fungal hyphae) but also is a metabolically active stage in the development of the mushroom. The cells of this morphological structure exhibit repeated activation of expression of the genes lcc4, tir, exp1, chi, and exg1, coding for laccase, tyrosinase, a specific transcription factor, chitinase, and glucanase, which are required for fungal growth and morphogenesis. This study revealed the maximum activity of functionally important proteins with phenol oxidase and lectin activities, and the emergence of additional laccases, tyrosinases, and lectins, which are typical of only this stage of morphogenesis and have a regulatory function in the development and formation of fruiting bodies.

Pectobacterium atrosepticum exopolysaccharides: identification, molecular structure, formation under stress and in planta conditions.

In the present study, we identified exopolysaccharides of the harmful phytopathogenic bacterium Pectobacterium atrosepticum SCRI1043 and characterized the molecular structure of these polymers. The synthesis of the target polysaccharides was shown to be induced under starvation conditions. Moreover, intensive accumulation of exopolysaccharides occurred during the colonization by bacteria of the xylem vessels of infected plants, where microorganisms formed specific 3D "multicellular" structures-bacterial emboli. Thus, the identified polymers are likely to be involved in the adaptation and virulence of bacteria of Pectobacterium genus.

Alteration in the ultrastructural morphology of mycelial hyphae and the dynamics of transcriptional activity of lytic enzyme genes during basidiomycete morphogenesis.

The morphogenesis of macromycetes is a complex multilevel process resulting in a set of molecular-genetic, physiological-biochemical, and morphological-ultrastructural changes in the cells. When the xylotrophic basidiomycetes Lentinus edodes, Grifola frondosa, and Ganoderma lucidum were grown on wood waste as the substrate, the ultrastructural morphology of the mycelial hyphal cell walls differed considerably between mycelium and morphostructures. As the macromycetes passed from vegetative to generative development, the expression of the tyr1, tyr2, chi1, chi2, exg1, exg2, and exg3 genes was activated. These genes encode enzymes such as tyrosinase, chitinase, and glucanase, which play essential roles in cell wall growth and morphogenesis.

Development of gravitropic response: unusual behavior of flax phloem G-fibers.

The major mechanism of gravitropism that is discussed for herbal plants is based on the nonuniform elongation of cells located on the opposite stem sides, occurring in the growing zone of an organ. However, gravitropic response of flax (Linum usitatissimum L.) is well-pronounced in the lower half of developing stem, which has ceased elongation long in advance of plant inclination. We have analyzed the stem curvature region by various approaches of microscopy and found the undescribed earlier significant modifications in primary phloem fibers that have constitutively developed G-layer. In fibers on the pulling stem side, cell portions were widened with formation of "bottlenecks" between them, leading to the "sausage-like" shape of a cell. Lumen diameter in fiber widening increased, while cell wall thickness decreased. Callose was deposited in proximity to bottlenecks and sometimes totally occluded their lumen. Structure of fiber cell wall changed considerably, with formation of breaks between G- and S-layers. Thick fibrillar structures that were revealed in fiber cell wall by light microscopy got oblique orientation instead of parallel to the fiber axis one in control plants. The described changes occurred at various combinations of gravitational and mechanical stimuli. Thus, phloem fibers with constitutively formed gelatinous cell wall, located in nonelongating parts of herbal plant, are involved in gravitropism and may become an important element in general understanding of the gravity effects on plants. We suggest flax phloem fibers as the model system to study the mechanism of plant position correction, including signal perception and transduction.

Alternative scenarios of starvation-induced adaptation in Pectobacterium atrosepticum.

Bacteria have high adaptive potential that ensures their survival during various environmental challenges. To adapt, bacteria activate a physiological program of stress response that makes them able to persist under adverse conditions. The present study sought to examine the ability of a particular bacterial species to induce a stress response in alternative scenarios. Cells of the phytopathogenic microorganism Pectobacterium atrosepticum were taken as a model. The cells were exposed to starvation in different physiological states (actively growing exponential phase and stationary phase cells), and the resulting starving cultures were monitored using CFU counting, quantitative PCR and electron microscopy. When exponential phase cells were subjected to starvation, the nucleoids of the cells became condensed and their DNA was detected by qPCR less effectively than that of cells growing in nutrient-rich medium, or stationary phase cells after starvation. Exponential phase cells subjected to starvation showed increased expression of genes encoding DNA binding histone-like proteins, whereas, in cultures inoculated by stationary phase cells, cell-wall-deficient forms that were inefficient at colony forming and that had a non-culturable phenotype were formed. The cell-wall-deficient forms displayed reduced expression of genes encoding synthases of cell wall components.

Intrusive growth of primary and secondary phloem fibres in hemp stem determines fibre-bundle formation and structure.

Plant fibres-cells with important mechanical functions and a widely used raw material-are usually identified in microscopic sections only after reaching a significant length or after developing a thickened cell wall. We characterized the early developmental stages of hemp (Cannabis sativa) stem phloem fibres, both primary (originating from the procambium) and secondary (originating in the cambium), when they still had only a primary cell wall. We gave a major emphasis to the role of intrusive elongation, the specific type of plant cell growth by which fibres commonly attain large cell length. We could identify primary phloem fibres at a distance of only 1.2-1.5 mm from the shoot apical meristem when they grew symplastically with the surrounding tissues. Half a millimeter further downwards along the stem, fibres began their intrusive elongation, which led to a sharp increase in fibre numbers visible within the stem cross-sections. The intrusive elongation of primary phloem fibres was completed within the several distal centimetres of the growing stem, before the onset of their secondary cell wall formation. The formation of secondary phloem fibres started long after the beginning of secondary xylem formation. Our data indicate that only a small portion of the fusiform cambial initials (<10 %) give rise directly or via their derivatives to secondary phloem fibres. The key determinant of final bundle structure, both for primary and secondary phloem fibres, is intrusive growth. Through bi-directional elongation, fibres join other fibres initiated individually in other stem levels, thus forming the bundles. Our results provide the specific developmental basis for further biochemical and molecular-genetic studies of phloem fibre development in hemp, but may be applied to many other species.

Dissociation of a population of Pectobacterium atrosepticum SCRI1043 in tobacco plants: formation of bacterial emboli and dormant cells.

The population dynamics of Pectobacterium atrosepticum SCRI1043 (Pba) within tobacco plants was monitored from the time of inoculation until after long-term preservation of microorganisms in the remnants of dead plants. We found and characterised peculiar structures that totally occlude xylem vessels, which we have named bacterial emboli. Viable but non-culturable (VBN) Pba cells were identified in the remnants of dead plants, and the conditions for resuscitation of these VBN cells were established. Our investigation shows that dissociation of the integrated bacterial population during plant colonisation forms distinct subpopulations and cell morphotypes, which are likely to perform specific functions that ensure successful completion of the life cycle within the plant.