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.

Transcriptome profiling of celery petiole tissues reveals peculiarities of the collenchyma cell wall formation.

MAIN CONCLUSION: Transcriptome and biochemical analyses are applied to individual plant cell types to reveal potential players involved in the molecular machinery of cell wall formation in specialized cells such as collenchyma. Plant collenchyma is a mechanical tissue characterized by an irregular, thickened cell wall and the ability to support cell elongation. The composition of the collenchyma cell wall resembles that of the primary cell wall and includes cellulose, xyloglucan, and pectin; lignin is absent. Thus, the processes associated with the formation of the primary cell wall in the collenchyma can be more pronounced compared to other tissues due to its thickening. Primary cell walls intrinsic to different tissues may differ in structure and composition, which should be reflected at the transcriptomic level. For the first time, we conducted transcriptome profiling of collenchyma strands isolated from young celery petioles and compared them with other tissues, such as parenchyma and vascular bundles. Genes encoding proteins involved in the primary cell wall formation during cell elongation, such as xyloglucan endotransglucosylase/hydrolases, expansins, and leucine-rich repeat proteins, were significantly activated in the collenchyma. As the key players in the transcriptome orchestra of collenchyma, xyloglucan endotransglucosylase/hydrolase transcripts were characterized in more detail, including phylogeny and expression patterns. The comprehensive approach that included transcriptome and biochemical analyses allowed us to reveal peculiarities of collenchyma cell wall formation and modification, matching the abundance of upregulated transcripts and their potential substrates for revealed gene products. As a result, specific isoforms of multigene families were determined for further functional investigation.

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.

The Role of Pectobacterium atrosepticum Exopolysaccharides in Plant-Pathogen Interactions.

The phytopathogenic bacterium Pectobacterium atrosepticum (Pba), one of the members of the soft rot Pectobacteriaceae, forms biofilm-like structures known as bacterial emboli when colonizing the primary xylem vessels of the host plants. The initial extracellular matrix of the bacterial emboli is composed of the host plant's pectic polysaccharides, which are gradually substituted by the Pba-produced exopolysaccharides (Pba EPS) as the bacterial emboli "mature". No information about the properties of Pba EPS and their possible roles in Pba-plant interactions has so far been obtained. We have shown that Pba EPS possess physical properties that can promote the maintenance of the structural integrity of bacterial emboli. These polymers increase the viscosity of liquids and form large supramolecular aggregates. The formation of Pba EPS aggregates is provided (at least partly) by the acetyl groups of the Pba EPS molecules. Besides, Pba EPS scavenge reactive oxygen species (ROS), the accumulation of which is known to be associated with the formation of bacterial emboli. In addition, Pba EPS act as suppressors of the quantitative immunity of plants, repressing PAMP-induced reactions; this property is partly lost in the deacetylated form of Pba EPS. Overall, our study shows that Pba EPS play structural, protective, and immunosuppressive roles during Pba-plant interactions and thus should be considered as virulence factors of these bacteria.

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.

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.

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.

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.

Spatial structures of rhamnogalacturonan I in gel and colloidal solution identified by 1D and 2D-FTIR spectroscopy.

Rhamnogalacturonan I (RG-I), a polysaccharide found in different types of plant cell walls, fulfills specific functions, the structural basis of which remains unclear. Generalized 2D correlation FTIR spectroscopy with dehydration was employed to reveal the structure and interactions in flax RG-I solution and microwave treated gel. Varying water content allowed emphasizing a role of solvent in maintaining different structures. In the gel, 2D correlation maps prove the existence of a conformationally uniform highly hydrated structure. Such a structure is supposed to correspond to non-associated galactan helices stabilized by rare junctions. In colloidal solution the side chains of RG-I associate heterogeneously due to constrains imposed by stiff backbone. Galactan-enriched fraction of RG-I with enzymatically cleaved backbone revealed the tendency of galactan chains to strongly associate in solution. The obtained results shed light on the possible role of backbone and side chains in RG-I spatial organization and confirm the sensitivity and potential of 2D correlation FTIR spectroscopy to probe local ordered structures in non-crystalline polysaccharides.

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.

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.

Gelation of rhamnogalacturonan I is based on galactan side chain interaction and does not involve chemical modifications.

The article presents the structural principles of microwave-induced formation of new gel type from pectic rhamnogalacturonan I (RG-I). The backbone of gel-forming RG-I does not contain consecutive galacturonic residues and modifying groups that can be the cause of junction zone formation as it occurs in course of classical ways of pectin gelation. Microwave irradiation does not cause destruction and chemical modifications of RG-I. Removal of half of galactan chains from RG-I leads to loss of gelling capability pointing out on their leading role in this process. Rising of intensity of the bands attributed to galactose and glycosidic linkages in RG-I gel comparing to solution where this polymer exists as molecule associate indicates that the spatial organization of galactans in gel is changed. A model of the RG-I gelation is proposed: being destabilized at volumetric microwave heating RG-I associates are repacked forming network where RG-I molecules are entangled by galactan chains.

Metrics of rhamnogalacturonan I with ß-(1→4)-linked galactan side chains and structural basis for its self-aggregation.

Within the family of plant cell wall polysaccharides rhamnogalacturonans I are the most diverse and structurally complex members. In present study we characterize the 3-dimensional structures and dynamic features of the constituents of RG-I along MD trajectories. It is demonstrated that extended threefold helical structure of the rhamnogalacturonan linear backbone is the most energetically favorable motif. Branching helps to stabilize a conformer of the backbone twisted along 1→2 glycosidic linkage triggering the orientation of long side chains without altering the extended overall backbone chain conformation. Formation of anti-parallel pairing of the ß-galactan side chains allows us to suggest a novel mode of non-covalent cross-linking in pectins. Studied structural elements are organized to report the first attempt to characterize 3D structure of RG-I focusing on the special case of flax tertiary cell wall and elucidate the structural basis underlying the formation of RG-I self-associates and functional role of RG-I in planta.

Aspen Tension Wood Fibers Contain ß-(1---> 4)-Galactans and Acidic Arabinogalactans Retained by Cellulose Microfibrils in Gelatinous Walls.

Contractile cell walls are found in various plant organs and tissues such as tendrils, contractile roots, and tension wood. The tension-generating mechanism is not known but is thought to involve special cell wall architecture. We previously postulated that tension could result from the entrapment of certain matrix polymers within cellulose microfibrils. As reported here, this hypothesis was corroborated by sequential extraction and analysis of cell wall polymers that are retained by cellulose microfibrils in tension wood and normal wood of hybrid aspen (Populus tremula × Populus tremuloides). ß-(1→4)-Galactan and type II arabinogalactan were the main large matrix polymers retained by cellulose microfibrils that were specifically found in tension wood. Xyloglucan was detected mostly in oligomeric form in the alkali-labile fraction and was enriched in tension wood. ß-(1→4)-Galactan and rhamnogalacturonan I backbone epitopes were localized in the gelatinous cell wall layer. Type II arabinogalactans retained by cellulose microfibrils had a higher content of (methyl)glucuronic acid and galactose in tension wood than in normal wood. Thus, ß-(1→4)-galactan and a specialized form of type II arabinogalactan are trapped by cellulose microfibrils specifically in tension wood and, thus, are the main candidate polymers for the generation of tensional stresses by the entrapment mechanism. We also found high ß-galactosidase activity accompanying tension wood differentiation and propose a testable hypothesis that such activity might regulate galactan entrapment and, thus, mechanical properties of cell walls in tension wood.

Physicochemical properties of complex rhamnogalacturonan I from gelatinous cell walls of flax fibers.

The physicochemical properties of flax fiber cell wall rhamnogalacturonan I (RG-I) and its fragments, obtained after galactanase treatment (fraction G1), were characterized. RG-I retains its hydrodynamic volume after its molecular weight decreases by approximately half, as revealed by SEC. Two techniques, DLS and NMR, with different principles of diffusion experiment were used to establish the reasons for this property of RG-I. Three possible types of particles were revealed by DLS depending on the concentration of the RG-I and G1 solutions (2-2.5, 15-20, and 150-200 nm). It was determined by BPP-LED experiments that the backbone of the RG-I was 1.3-1.9-fold more mobile than the side chains. The obtained data suggest a novel type of pectin spatial organization-the formation of RG-I associates with the backbone at the periphery and the interaction between the side chains to form a core zone.

Structural details of pectic galactan from the secondary cell walls of flax (Linum usitatissimum L.) phloem fibres.

Details of the backbone and side chain structure of pectic ß-(1→4)-galactan from the secondary cell walls of flax phloem fibres were characterised by NMR and mass spectrometry of the fragments obtained after partial hydrolysis with specific endogalactanase and rhamnogalacturonan hydrolase. The proportions of branched and linear rhamnose in the backbone of the polymer equalled 72% and 28%, respectively. Rhamnose branched with a single galactose residue comprised 47% of the total rhamnose; thus, in the bulk of the polymer backbone, rhamnose had 0-1 galactose residues. Within the backbone, residues of rhamnose branched with long galactose chains alternated with linear rhamnose and rhamnose with a single galactose. Oligomeric galactose chains averaged 14 monomers in length. Alternative glycosidic bonds of galactosyl residues were present. The established structural details of cell wall galactan are compared to those of nascent galactan before incorporation into the fibre cell wall, and galactan modifications in muro are discussed.