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.

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.

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.

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.

Differential expression of α-L-arabinofuranosidases during maize (Zea mays L.) root elongation.

MAIN CONCLUSION: Specific α- l -arabinofuranosidases are involved in the realisation of elongation growth process in cells with type II cell walls. Elongation growth in a plant cell is largely based on modification of the cell wall. In type II cell walls, the Ara/Xyl ratio is known to decrease during elongation due to the partial removal of Ara residues from glucuronoarabinoxylan. We searched within the maize genome for the genes of all predicted α-L-arabinofuranosidases that may be responsible for such a process and related their expression to the activity of the enzyme and the amount of free arabinose measured in six zones of a growing maize root. Eight genes of the GH51 family (ZmaABFs) and one gene of the GH3 family (ZmaARA-I) were identified. The abundance of ZmaABF1 and 3-6 transcripts was highly correlated with the measured enzymatic activity and free arabinose content that significantly increased during elongation. The transcript abundances also coincided with the pattern of changes in the Ara/Xyl ratio of the xylanase-extractable glucuronoarabinoxylan described in previous studies. The expression of ZmaABF3, 5 and 6 was especially up-regulated during elongation although corresponding proteins are devoid of the catalytic glutamate at the proper position. ZmaABF2 transcripts were specifically enriched in the root cap and meristem. A single ZmaARA-I gene was not expressed as a whole gene but instead as splice variants that encode the C-terminal end of the protein. Changes in the ZmaARA-I transcript level were rather moderate and had no significant correlation with free arabinose content. Thus, elongation growth of cells with type II cell walls is accompanied by the up-regulation of specific and predicted α-L-arabinofuranosidase genes, and the corresponding activity is indeed pronounced and is important for the modification of glucuronoarabinoxylan, which plays a key role in the modification of the cell wall supramolecular organisation.

The In Silico Characterization of Monocotyledonous α-l-Arabinofuranosidases on the Example of Maize.

Plant α-l-arabinofuranosidases remove terminal arabinose from arabinose-containing substrates such as plant cell wall polysaccharides, including arabinoxylans, arabinogalactans, and arabinans. In plants, de-arabinosylation of cell wall polysaccharides accompanies different physiological processes such as fruit ripening and elongation growth. In this report, we address the diversity of plant α-l-arabinofuranosidases of the glycoside hydrolase (GH) family 51 through their phylogenetic analysis as well as their structural features. The CBM4-like domain at N-terminus was found to exist only in GH51 family proteins and was detected in almost 90% of plant sequences. This domain is similar to bacterial CBM4, but due to substitutions of key amino acid residues, it does not appear to be able to bind carbohydrates. Despite isoenzymes of GH51 being abundant, in particular in cereals, almost half of the GH51 proteins in Poales have a mutation of the acid/base residue in the catalytic site, making them potentially inactive. Open-source data on the transcription and translation of GH51 isoforms in maize were analyzed to discuss possible functions of individual isoenzymes. The results of homology modeling and molecular docking showed that the substrate binding site can accurately accommodate terminal arabinofuranose and that arabinoxylan is a more favorable ligand for all maize GH51 enzymes than arabinan.

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs using Atomic Force Microscopy.

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young's modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

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.

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.

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.

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.

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.

Transcriptome Analysis of Intrusively Growing Flax Fibers Isolated by Laser Microdissection.

The intrusive growth, a type of plant cell elongation occurring in the depths of plant tissues, is characterized by the invasion of a growing cell between its neighbours due to a higher rate of elongation. In order to reveal the largely unknown molecular mechanisms of intrusive growth, we isolated primary flax phloem fibers specifically at the stage of intrusive growth by laser microdissection. The comparison of the RNA-Seq data from several flax stem parts enabled the characterization of those processes occurring specifically during the fiber intrusive elongation. The revealed molecular players are summarized as those involved in the supply of assimilates and support of turgor pressure, cell wall enlargement and modification, regulation by transcription factors and hormones, and responses to abiotic stress factors. The data obtained in this study provide a solid basis for developing approaches to manipulate fiber intrusive elongation, which is of importance both for plant biology and the yield of fiber crops.