Cell Wall Properties Research Articles

An Arabidopsis PTH2 Gene Is Responsible for Gravity Resistance Supporting Plant Growth under Different Gravity Conditions.

Terrestrial plants respond to and resist gravitational force. The response is termed “gravity resistance”, and centrifugal hypergravity conditions are efficient for investigating its nature and mechanism. A functional screening of Arabidopsis T-DNA insertion lines for the suppression rate of elongation growth of hypocotyls under hypergravity conditions was performed in this study to identify the genes required for gravity resistance. As a result, we identified PEPTIDYL-tRNA HYDROLASE II (PTH2). In the wild type, elongation growth was suppressed by hypergravity, but this did not happen in the pth2 mutant. Lateral growth, dynamics of cortical microtubules, mechanical properties of cell walls, or cell wall thickness were also not affected by hypergravity in the pth2 mutant. In other words, the pth2 mutant did not show any significant hypergravity responses. However, the gravitropic curvature of hypocotyls of the pth2 mutant was almost equal to that of the wild type, indicating that the PTH2 gene is not required for gravitropism. It is suggested by these results that PTH2 is responsible for the critical processes of gravity resistance in Arabidopsis hypocotyls.

Rootstock effects on bitter pit incidence in 'Honeycrisp' apples are associated with changes in fruit's cell wall chemical properties.

Bitter pit (BP) is a physiological disorder of apples that often appears during or after cold storage. Despite being defined as a calcium deficiency disorder, BP is a complex process that is not only affected by the total Ca2+ content in the fruit but also by the proper cellular Ca2+ homeostasis and partitioning. Early investigations have also suggested that rootstocks could affect BP development and severity. In the present study, rootstock effects on BP development were assessed on ‘Honeycrisp’ trees that were grafted on 14 different rootstocks (B.10, G.11, G.202, G.214, G.30, G.41, G.935, G.969, M.26 EMLA, M.9, V.1, V.5, V.6, and V.7). We evaluated BP incidence at harvest, and three months after cold storage for four, and three growing seasons, respectively. BP incidence was significantly reduced in ‘Honeycrisp’ trees on B.10 compared to other rootstocks, whereas trees on V.6 showed the highest percentage of BP at harvest and after cold storage. ‘Honeycrisp’ apples were collected from three different rootstocks (B.10, G.41, and V.6) two months after cold storage and evaluated for mineral nutrient composition, Ca2+ homeostasis, and cell wall properties, e.g., pectin content, pectin de-esterification rate and pectin methylesterase (PME) activity. Water-soluble and insoluble pectin content was markedly higher in fruits from B.10 than in G.41 and V.6. We also observed increased PME enzyme activity and a greater degree of water-insoluble pectin de-esterification in ‘Honeycrisp’ apples from V.6 compared to those from B.10. A significantly higher Ca2+ was found in the fruits from B.10 than G.41 and V.6. Higher Ca2+ and lower Mg2+ levels were also observed in the cell wall and water-insoluble pectin fractions of the fruits from B.10 compared to G.41 and V.6. However, the ratio of cell wall-bound Ca2+ to total Ca2+ was lower in B.10 compared to G.41 and V.6. Together, our results indicate that the tolerance of B.10 to BP could be attributed to a reduced PME activity and lower pectin de-esterification level, which in turn reduced the amount of Ca2+ cross-linked with pectin, and probably increased the apoplastic free calcium concentrations that is essential for maintaining cell membrane integrity and reducing BP development.

Differential near-infrared imaging of heterocysts using single-walled carbon nanotubes

The internalization of near-infrared (NIR) optical nanoprobes in photosynthetic microbes can be exploited for applications ranging from energy conversion to biomolecule delivery. However, the intrinsic, species-dependent properties of microbial cell walls, including their surface charge density, composition, thickness, and elasticity, can severely impact nanoprobe uptake and affect the cellular response. An examination of the interaction of the optical nanoprobe in various species and its impact on cell viability is, therefore, imperative for the development of new imaging technologies. Herein, we extend the technology recently developed for internalizing fluorescent single-walled carbon nanotubes (SWCNTs) in prokaryotes, specifically unicellular Synechocystis sp. PCC 6803, to a filamentous cyanobacterial strain, Nostoc punctiforme. Using a combination of NIR fluorescence, scanning electron microscopy (SEM), and Raman spectroscopy, we investigate uptake in vegetative cells as well as differentiated heterocysts. We demonstrate a strong dependence of long-term cell integrity, activity, and viability on SWCNT surface functionalization. We further show differential uptake of SWCNTs across a single filament, with positively charged functionalized SWCNTs preferentially localizing within the heterocysts of the filament. This cell dependency of the nanoparticle internalization motivates the use of SWCNTs as a NIR stain for monitoring cell differentiation.Graphical abstract

Protective effects of peptides on the cell wall structure of yeast under osmotic stress.

Three peptides (LL, LML, and LLL) were used to examine their influences on the osmotic stress tolerance and cell wall properties of brewer's yeast. Results suggested that peptide supplementation improved the osmotic stress tolerance of yeast through enhancing the integrity and stability of the cell wall. Transmission electron micrographs showed that the thickness of yeast cell wall was increased by peptide addition under osmotic stress. Additionally, quantitative analysis of cell wall polysaccharide components in the LL and LLL groups revealed that they had 27.34% and 24.41% higher chitin levels, 25.73% and 22.59% higher mannan levels, and 17.86% and 21.35% higher β-1,3-glucan levels, respectively, than the control. Furthermore, peptide supplementation could positively modulate the cell wall integrity pathway and up-regulate the expressions of cell wall remodeling-related genes, including FKS1, FKS2, KRE6, MNN9, and CRH1. Thus, these results demonstrated that peptides improved the osmotic stress tolerance of yeast via remodeling the yeast cell wall and reinforcing the structure of the cell wall. KEY POINTS: • Peptide supplementation improved yeast osmotic stress tolerance via cell wall remodeling. • Peptide supplementation enhanced cell wall thickness and stability under osmotic stress. • Peptide supplementation positively modulated the CWI pathway under osmotic stress.

Mechanics of Reversible Deformation during Leaf Movement and Regulation of Pulvinus Development in Legumes.

Plant cell deformation is a mechanical process that is driven by differences in the osmotic pressure inside and outside of the cell and is influenced by cell wall properties. Legume leaf movements result from reversible deformation of pulvinar motor cells. Reversible cell deformation is an elastic process distinct from the irreversible cell growth of developing organs. Here, we begin with a review of the basic mathematics of cell volume changes, cell wall function, and the mechanics of bending deformation at a macro scale. Next, we summarize the findings of recent molecular genetic studies of pulvinar development. We then review the mechanisms of the adaxial/abaxial patterning because pulvinar bending deformation depends on the differences in mechanical properties and physiological responses of motor cells on the adaxial versus abaxial sides of the pulvinus. Intriguingly, pulvini simultaneously encompass morphological symmetry and functional asymmetry along the adaxial/abaxial axis. This review provides an introduction to leaf movement and reversible deformation from the perspective of mechanics and molecular genetics.

Deposition patterns of feruloylarabinoxylan during cell wall formation in moso bamboo.

The feruloylarabinoxylan deposition was initiated at the formation of the secondary cell wall, especially S2 layer in moso bamboo, which may affect crosslinking between cell wall components and plant growth. Hemicelluloses, major components of plant cell walls that are hydrogen bonded to cellulose and covalently bound to lignin, are crucial determinants of cell wall properties. Especially in commelinid monocotyledons, arabinoxylan is often esterified with ferulic acid, which is essential to crosslinking with cell wall components. However, the deposition patterns and localization of ferulic acid during cell wall formation remain unclear. In this study, developing moso bamboo (Phyllostachys pubescens) culms were used to elucidate deposition patterns of hemicelluloses including feruloylarabinoxylan. Ferulic acid content peaked with cessation of elongation growth, and thereafter decreased and remained stable as culm development proceeded. During primary cell wall (PCW) formation, xyloglucan and (1,3;1,4)-β-glucan signals were detected in all tissues. Along with culm development, arabinoxylan and feruloylarabinoxylan signals were sequentially observed in the protoxylem, vascular fibers and metaxylem, and parenchyma. Feruloylarabinoxylan signals were observed slightly later than arabinoxylan signals. Arabinoxylan signals were observed throughout the compound middle lamella and secondary cell wall (SCW), whereas the feruloylarabinoxylan signal was localized to the S2 layer of the SCW. These results indicate that the biosynthesis of hemicelluloses is regulated in accordance with cell wall layers. Feruloylarabinoxylan deposition may be initiated at the formation of SCW, especially S2 layer formation. Ferulic acid-mediated linkages of arabinoxylan-arabinoxylan and arabinoxylan-lignin would arise during SCW formation with the cessation of elongation growth.

Eudicot primary cell wall glucomannan is related in synthesis, structure, and function to xyloglucan.

Hemicellulose polysaccharides influence assembly and properties of the plant primary cell wall (PCW), perhaps by interacting with cellulose to affect the deposition and bundling of cellulose fibrils. However, the functional differences between plant cell wall hemicelluloses such as glucomannan, xylan, and xyloglucan (XyG) remain unclear. As the most abundant hemicellulose, XyG is considered important in eudicot PCWs, but plants devoid of XyG show relatively mild phenotypes. We report here that a patterned β-galactoglucomannan (β-GGM) is widespread in eudicot PCWs and shows remarkable similarities to XyG. The sugar linkages forming the backbone and side chains of β-GGM are analogous to those that make up XyG, and moreover, these linkages are formed by glycosyltransferases from the same CAZy families. Solid-state nuclear magnetic resonance indicated that β-GGM shows low mobility in the cell wall, consistent with interaction with cellulose. Although Arabidopsis β-GGM synthesis mutants show no obvious growth defects, genetic crosses between β-GGM and XyG mutants produce exacerbated phenotypes compared with XyG mutants. These findings demonstrate a related role of these two similar but distinct classes of hemicelluloses in PCWs. This work opens avenues to study the roles of β-GGM and XyG in PCWs.

The Impact of Cell Wall Feruloylation on Plant Growth, Responses to Environmental Stress, Plant Pathogens and Cell Wall Degradability

This article summarizes evolving concepts and scientific findings on cell wall feruloylation and ferulate oxidative coupling processes in grasses, and the effects these have on the wide range of cell wall properties and consequent plant responses to biotic and abiotic stress and tissue degradability. Updates of the different strategies that have been applied to genetically modifying cell wall feruloylation are presented. Special emphasis is given to the modification of cell wall feruloylation by heterologous expression of cell wall ferulic acid esterase, as this strategy has provided insights into the impact of feruloylation on the changes in the physicochemical properties of the cell wall with consequent effects on different plant processes. Emerging feruloyl transferase candidate genes codifying enzymes accounting for ferulate incorporation into grass arabinoxylans are also highlighted.

Detailed Characterization of the Cell Wall Structure and Composition of Nordic Green Microalgae.

Green microalgae are attractive to food, pharmaceutical, and biofuel industries due to the promising and diverse properties of their intracellular components. In current biotechnological applications, however, clear bottlenecks are the cell disruption and cell harvesting steps. Challenges in both of these processes are directly linked to the properties of the microalgal cell wall. The aim of this study was to explore the cell wall compositions and morphologies of four Nordic microalgal strains (Chlorella vulgaris (13-1), Scenedesmus sp. (B2-2), Haematococcus pluvialis, and Coelastrella sp. (3-4)) and their changes in relation to logarithmic and stationary growth phases. Transmission electron microscopy imaging enabled us to visualize the cell walls and to observe structural elements such as spines, microfibrillar hairs, or layers. Using cryogenic X-ray photoelectron spectroscopy, we quantified lipid, protein, and polysaccharide content of the outer surface of the microalgal cell wall in cultures. Fourier transform infrared spectroscopy highlighted changes between growth phases within the polysaccharide and protein fractions of the cell wall. Very prominent differences were observed in sugar and protein composition of the Scenedesmus sp. (B2-2) cell wall compared to the cell walls of the other three Nordic strains using trimethylsilyl derivatization.

Genome-wide identification of the pectin methylesterase inhibitor genes in Brassica napus and expression analysis of selected members.

Pectin methylesterase inhibitors (PMEIs) modulate the status of pectin methylesterification by inhibiting the activity of pectin methylesterase (PME). Recent advances indicate PMEIs play an important role in regulating plant cell wall properties and defense responses. In this study, a genome-wide analysis of PMEI gene family in Brassica napus (B. napus) was conducted and the expression patterns of PMEI genes in response to Sclerotinia sclerotiorum (S. sclerotiorum) was investigated. A total of 190 PMEI proteins were identified from the genome of B. napus. Chromosomal location, gene structure and properties of the PMEI family were analyzed, and these features were compared with Arabidopsis thaliana (A. thaliana). A total of 123 syntenic ortholog pairs were detected from BnPMEI family by synteny analysis. Results showed the expansion of BnPMEI genes was likely predominately from whole-genome duplication (WGD) or segmental duplications. Multiple cis-elements related to plant growth and development, environmental stress responses, hormone responses were detected in the promoters of BnPMEI genes, implying they were regulated by both internal and external factors. Furthermore, expression analysis of transcriptome data combined with quantitative RT-PCR (qRT-PCR) validation identified several candidates that were strongly responsive to S. sclerotiorum infection. These BnPMEI genes are candidates for manipulation to breed novel and improved genotypes that are more resistant to sclerotinia stem rot (SSR). Extensive interactions were detected among 30 BnPMEI proteins, forming complex protein-protein interaction networks. Besides, 48 BnPMEIs showed interactions with other proteins including a range of cell wall structure-related enzymes. This study provides new insights into the evolution and function of PMEIs in B. napus and lays a foundation for breeding novel genotypes for crop improvement.

Properties and durability of wood impregnated with high melting point polyethylene wax for outdoor use

In order to develop a new wood product suitable for outdoor applications, a high melting point polyethylene wax was used for high-pressure impregnation treatment of wood. Properties of impregnated wood were studied. Wax in molten state was stable with low viscosity, good fluidity, and easily penetrate into the wood under high pressure. Full-cell process included vacuum (-0.06 MPa for 30 min), high-pressure impregnation (0.75 MPa for 3 h) and pressure release (30 min). Wax contents of treated lodgepole pine (Pinus contorta Douglas ex Loudon.) and eucalyptus (Eucalyptus saligna) were 0.205 g/cm3 and 0.321 g/cm3, respectively. Cell gaps and cell cavities of wood were filled with polyethylene wax. Nanoindentation tests showed that longitudinal mechanical properties of wood cell walls were weakened after impregnation. After a 3 h high-pressure impregnation, hardness of lodgepole pine wood cell walls was reduced by 35.1%, and the modulus of elasticity was reduced by 4.9%. Treated wood had a much lower water absorption and swelling rate than untreated wood, its weathering resistance as well as insect resistance were better.

Bamboo flattening technology ebables efficient and value-added utilization of bamboo in the manufacture of furniture and engineered composites

Bamboo flattening, a novel and efficient bamboo processing and utilization technology, can effectively flatten bamboo culms into crack-free board. The technology enables the efficient and value-added utilization of bamboo in the manufacture of furniture and engineered composites. In this study, we compared the effects of three types of flattening technologies on the macro-mechanical, macro-physical, micro-morphology, and micro-mechanical properties of untreated, softened a flattened bamboo samples. As expected, the hemicellulose and cellulose concentrations of the saturated steam treated sample (18.05% and 38.9%, respectively) were less than those the untreated sample (21.3% and 40.1%, respectively). Moreover, scanning electron microscopy (SEM) analysis showed that the notched flattening technology (NFT) inserted indents and grooves on the bamboo surface. However, the “non-notched flattening technology (NNFT)” only compressed the bamboo tissue. The flattening process increased the equilibrium moisture content and density of bamboo. The enhanced density, lignin content, and cellulose crystallinity index enhanced the bamboo cell wall properties. The non-notched-flattened bamboo board exhibited a higher cell wall modulus of elasticity and hardness (20.1 and 0.89 GPa, respectively) than the untreated bamboo samples (13.7 and 0.49 GPa, respectively). The enhanced bamboo cell wall mechanical properties of the non-notched-flattened bamboo board. Finally, we explored the practical applications, economic benefits, and limitations of the current “Non-notched bamboo flattening technology”.

Growth fitness, heme uptake and genomic variants in mutants of oxygen-tolerant Lacticaseibacillus casei and Lactiplantibacillus plantarum strains

Adaptive Laboratory Evolution (ALE) is a powerful tool to improve the fitness of industrially relevant microorganisms, because it circumvents some of the problems related to the use of genetically modified strains. In this study, we used an ALE strategy involving serial batch cultivations in aerobic and respiratory conditions to generate spontaneous mutants from the respiration-competent strain Lacticaseibacillus casei N87. Genotypic changes in selected mutants were investigated using whole genome sequencing (WGS). The O2-tolerant Lactiplantibacillus plantarum C17 and its mutant C17-m58 (obtained from a previous ALE study) were included in heme uptake experiments and in WGS and variant calling analyses. Several Lcb. casei N87 mutants cultivated under aerobic and respiratory conditions showed improved biomass production, O2 uptake and oxidative stress tolerance compared to the parental strain. Mutants of Lcb. casei and Lpb. plantarum differed from the parental strains in the ability to use heme and menaquinone. High heme concentrations (> 10 mg/L), however, were toxic for all strains. Single nucleotide modifications (SNPs) were detected in some genes encoding for proteins and transcriptional regulators involved in carbon metabolism, oxidative stress, redox balance, and cell wall properties, but their role in the evolved phenotypes needs further investigations. We conclude that prolonged adaptation to aerobic and respiratory life-style may be used as natural strategy to generate strains with improved O2-consuming ability and oxidative stress tolerance, two important features to develop robust cultures and to reduce oxidative processes in foods.

YEAST CELL WALLS ARE AN EFFECTIVE MYCOTOXIN ADSORBENT IN FEED

In the world, serious attention is paid to the development of modern and effective methods for disinfecting feed from mycotoxins. Due to the characteristic weather conditions in the Republic of Belarus, almost all large livestock complexes and private farms are faced with the problem of mycotoxicosis in animals and birds, and this, in turn, has a huge impact on feed safety and, subsequently, on food safety. The problem
 of mycotoxins in feed can be solved by using various adsorbents. A mycotoxin adsorbent based on yeast cell walls is suitable for the role of such adsorbents. This article presents the results of studies on the physicochemical properties of yeast cell walls and their adsorption capacity by high performance liquid chromatography, using a reference sample of Trylogy multitoxins, in relation to such mycotoxins as aflatoxin B1, ochratoxin A,
 zearalenone, deoxynivalenol. The obtained results show that yeast cell walls have good adsorption capacity for high molecular weight mycotoxins and poorly adsorb low molecular weight mycotoxins.

Altering arabinans increases Arabidopsis guard cell flexibility and stomatal opening

Stomata regulate plant water use and photosynthesis by controlling leaf gas exchange. They do this by reversibly opening the pore formed by two adjacent guard cells, with the limits of this movement ultimately set by the mechanical properties of the guard cell walls and surrounding epidermis.1,2 A body of evidence demonstrates that the methylation status and cellular patterning of pectin wall polymers play a core role in setting the guard cell mechanical properties, with disruption of the system leading to poorer stomatal performance.3-6 Here we present genetic and biochemical data showing that wall arabinans modulate guard cell flexibility and can be used to engineer stomata with improved performance. Specifically, we show that a short-chain linear arabinan epitope associated with the presence of rhamnogalacturonan I in the guard cell wall is required for full opening of the stomatal pore. Manipulations leading to the novel accumulation of longer-chain arabinan epitopes in guard cell walls led to an increase in the maximal pore aperture. Using computational modeling combined with atomic force microscopy, we show that this phenotype reflected a decrease in wall matrix stiffness and, consequently, increased flexing of the guard cells under turgor pressure, generating larger, rounder stomatal pores. Our results provide theoretical and experimental support for the conclusion that arabinan side chains of pectin modulate guard cell wall stiffness, setting the limits for cell flexing and, consequently, pore aperture, gas exchange, and photosynthetic assimilation.

In vitro experiments and kinetic models of Arabidopsis pollen hydration mechanics show that MSL8 is not a simple tension-gated osmoregulator

Pollen, a neighbor-less cell containing the male gametes, undergoes mechanical challenges during plant sexual reproduction, including desiccation and rehydration. It was previously shown that the pollen-specific mechanosensitive ion channel MscS-like (MSL)8 is essential for pollen survival during hydration and proposed that it functions as a tension-gated osmoregulator. Here, we test this hypothesis with a combination of mathematical modeling and laboratory experiments. Time-lapse imaging revealed that wild-type pollen grains swell, and then they stabilize in volume rapidly during hydration. msl8 mutant pollen grains, however, continue to expand and eventually burst. We found that a mathematical model, wherein MSL8 acts as a simple-tension-gated osmoregulator, does not replicate this behavior. A better fit was obtained from variations of the model, wherein MSL8 inactivates independent of its membrane tension gating threshold or MSL8 strengthens the cell wall without osmotic regulation. Experimental and computational testing of several perturbations, including hydration in an osmolyte-rich solution, hyper-desiccation of the grains, and MSL8-YFP overexpression, indicated that the cell wall strengthening model best simulated experimental responses. Finally, the expression of a nonconducting MSL8 variant did not complement the msl8 overexpansion phenotype. These data indicate that contrary to our hypothesis and to the current understanding of MS ion channel function in bacteria, MSL8 does not act as a simple membrane tension-gated osmoregulator. Instead, they support a model wherein ion flux through MSL8 is required to alter pollen cell wall properties. These results demonstrate the utility of pollen as a cellular scale model system and illustrate how mathematical models can correct intuitive hypotheses.

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).

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

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

Investigation of the relationship between surface colour, contact angle and chemical properties of heat-treated bamboo

ABSTRACT The inherent relationships between the surface phenomena and chemical structures of thermally modified bamboo were investigated to predict the intrinsic properties of bamboo during the thermal modification through the surface phenomena effectively. Moso bamboo was firstly treated by vacuum treatment at 150°C, 170°C, 190°C and 210°C for 1, 2, 3 and 4 h, respectively. And then the surface colour, contact angle and chemical compositions of heat-treated bamboo and in-situ chemical properties of the heat-treated cell wall were analysed by using the colour metre, contact angle metre and confocal scanning microscope. Results showed that the surface colour became darker and the hydrophobicity was increased after heat treatment. As compared to the control, the lightness (L*) value carried out in accordance with CIELAB of heat-treated bamboo decreased by almost 54% and the maximum increase of contact angle was 160%. The surface colour and contact angle of bamboo were closely related to the lignin content. The darkened colour and increased hydrophobicity of the bamboo surface were caused by the crack of C–O–C and the decrease of C=O in lignin. Furthermore, fluorescence analysis indicated that the lignin distribution in the cell wall increased gradually after heat treatment, especially in the thick-walled cells and the corners of cells.

Class III Peroxidases PRX01, PRX44, and PRX73 Control Root Hair Growth in Arabidopsis thaliana.

Root hair cells are important sensors of soil conditions. They grow towards and absorb water-soluble nutrients. This fast and oscillatory growth is mediated by continuous remodeling of the cell wall. Root hair cell walls contain polysaccharides and hydroxyproline-rich glycoproteins, including extensins (EXTs). Class-III peroxidases (PRXs) are secreted into the apoplastic space and are thought to trigger either cell wall loosening or polymerization of cell wall components, such as Tyr-mediated assembly of EXT networks (EXT-PRXs). The precise role of these EXT-PRXs is unknown. Using genetic, biochemical, and modeling approaches, we identified and characterized three root-hair-specific putative EXT-PRXs, PRX01, PRX44, and PRX73. prx01,44,73 triple mutation and PRX44 and PRX73 overexpression had opposite effects on root hair growth, peroxidase activity, and ROS production, with a clear impact on cell wall thickness. We use an EXT fluorescent reporter with contrasting levels of cell wall insolubilization in prx01,44,73 and PRX44-overexpressing background plants. In this study, we propose that PRX01, PRX44, and PRX73 control EXT-mediated cell wall properties during polar expansion of root hair cells.