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Nanoscale structure, mechanics and growth of epidermal cell walls
Abstract
This article briefly reviews recent advances in nano-scale and micro-scale assessments of primary cell wall structure, mechanical behaviors and expansive growth. Cellulose microfibrils have hydrophobic and hydrophilic faces which may selectively bind different matrix polysaccharides and adjacent microfibrils. These distinctive binding interactions may guide partially aligned cellulose microfibrils in primary cell walls to form a planar, load-bearing network within each lamella of polylamellate walls. Consideration of expansive growth of cross-lamellate walls leads to a surprising inference: side-by-side sliding of microfibrils may be a key rate-limiting physical step, potentially targeted by specific wall loosening agents. Atomic force microscopy shows different patterns of microfibril movement during force-driven extension versus enzymatic loosening. Consequently, simulations of cell growth as elastic deformation of isotropic cell walls may need to be augmented to incorporate the distinctive behavior of growing cell walls.
... The primary CW consists predominantly of polysaccharides-cellulose, hemicellulose, and pectins. While cellulose microfibrils are responsible for the main load-bearing characteristics of the CW, the presence of hemicellulose and pectins can alter viscoelastic properties of the wall matrix (Wolf et al. 2012;Cosgrove 2018a;Zhang et al. 2021). Once the final cell size is reached, a secondary CW can be deposited in specific cell types, e.g. ...
... CW mechanical properties are modulated by controlling the biochemical composition, as well as the degree and nature of linkages between the CW polysaccharides. Interestingly, wall extensibility may be controlled in discrete regions-so-called biomechanical hotspots-where cellulose-cellulose contacts are made, potentially mediated by trace amounts of xyloglucan (Cosgrove 2014(Cosgrove , 2018a(Cosgrove , 2018b. These relatively few contact points between cellulose microfibrils may be key sites of CW loosening-a complex process allowing targeted wall expansion. ...
... Consequently, fibril-fibril sliding seems to allow CW extension and in-plane stress release of the multi-lamellate CW structure (Zhang et al. 2019;Zhang et al. 2021). Importantly, expansin-controlled CW loosening allows in-plane stress relaxation and enlargement of CWs, but does not associate with changes in CW viscoelastic properties, as measured by tensile tests (Yuan et al. 2001;Cosgrove 2018a). Thus, the CW "stiffening" or "softening" (i.e. ...
Expansins facilitate cell expansion by mediating pH-dependent cell wall (CW) loosening. However, the role of expansins in controlling CW biomechanical properties in specific tissues and organs remains elusive. We monitored hormonal responsiveness and spatial specificity of expression and localization of expansins predicted to be the direct targets of cytokinin signaling in Arabidopsis (Arabidopsis thaliana). We found EXPANSIN1 (EXPA1) homogenously distributed throughout the CW of columella/lateral root cap, while EXPA10 and EXPA14 localized predominantly at three-cell boundaries in the epidermis/cortex in various root zones. EXPA15 revealed cell type-specific combination of homogenous vs. three-cell boundaries localization. By comparing Brillouin frequency shift and AFM-measured Young's modulus, we demonstrated Brillouin light scattering (BLS) as a tool suitable for non-invasive in vivo quantitative assessment of CW viscoelasticity. Using both BLS and AFM, we showed that EXPA1 overexpression upregulated CW stiffness in the root transition zone. The dexamethasone-controlled EXPA1 overexpression induced fast changes in the transcription of numerous CW-associated genes, including several EXPAs and XYLOGLUCAN:XYLOGLUCOSYL TRANSFERASEs (XTHs), and associated with rapid pectin methylesterification determined by in situ Fourier transform infrared spectroscopy in the root transition zone. The EXPA1-induced CW remodeling associated with shortening of the root apical meristem, leading to root growth arrest. Based on our results, we propose that expansins control root growth by a delicate orchestration of CW biomechanical properties, possibly regulating both CW loosening and CW remodeling.
... Cells control the synthesis and deposition of individual cell wall components to modulate the composition of the cell wall. Spatial heterogeneity in cell wall composition is particularly important in defining the mechanical properties of the wall (Cosgrove, 2018;Grones et al., 2019;Majda et al., 2017;Phyo et al., 2017). ...
... Cellulose is a linear polymer of β-1,4-linked glucose (Glc) that forms microfibrils which resist tensile stress and control the axis of turgor-driven cell expansion (Cosgrove, 2018). Cellulose microfibrils are believed to be mechanically coupled to each other at sites called 'biomechanical hotspots' where hemicelluloses, such as xyloglucan (XG), are thought to form tight linkages between microfibrils (Cosgrove, 2014;Park & Cosgrove, 2012). ...
... This method resulted in sections that had intact epidermis, phloem and xylem cells, but softer tissues, like the pith and cortex, were less well preserved (Supplementary Figure S1A). AFM-IR imaging was conducted on the epidermal cell wall because the epidermis is composed of mostly primary cell walls (Cosgrove, 2018). Because sections were obtained perpendicular to the periclinal cell walls, the mechanical properties detected by AFM are unaffected by turgor pressure. ...
Spatial heterogeneity in composition and organisation of the primary cell wall affects the mechanics of cellular morphogenesis. However, directly correlating cell wall composition, organisation and mechanics has been challenging. To overcome this barrier, we applied atomic force microscopy coupled with infrared (AFM-IR) spectroscopy to generate spatially correlated maps of chemical and mechanical properties for paraformaldehyde-fixed, intact Arabidopsis thaliana epidermal cell walls. AFM-IR spectra were deconvoluted by non-negative matrix factorisation (NMF) into a linear combination of IR spectral factors representing sets of chemical groups comprising different cell wall components. This approach enables quantification of chemical composition from IR spectral signatures and visualisation of chemical heterogeneity at nanometer resolution. Cross-correlation analysis of the spatial distribution of NMFs and mechanical properties suggests that the carbohydrate composition of cell wall junctions correlates with increased local stiffness. Together, our work establishes new methodology to use AFM-IR for the mechanochemical analysis of intact plant primary cell walls.
... What is the role in cellulose synthesis of the cellulase KOR, the Glycosylphosphatidylinositol-anchored protein COBRA, the chitinase-like protein POMPOM1 and several Golgi-associated proteins [16]? What controls the formation of load-bearing cellulose-cellulose crosslinks in the cell wall [28]? How are the cellulose deposition patterns regulated, for instance the successive lamellae in poly-lamellate walls [29] or the, often remarkable, secondary cell wall patterns and finally how are stress patterns sensed to orient the microtubules [30]? ...
... Alexis Peaucelle, in our group, explored this in his quest to understand the biophysical basis of cell expansion. The consensus was that the turgor pressure is the motor for cell expansion and selective wall relaxation controls the expansion rate [28]. To understand how pectin metabolism fits into this picture, he expressed a PME and a PMEI respectively in the shoot apical meristem under control of an inducible promoter ( Figure 4) [38]. ...
... The relation cell wall elasticity-cell expansion rate remains correlative however, even though both are triggered by the same HG modification. This relation is also not obvious since elasticity is a reversible deformation whereas growth corresponds to the irreversible deformation through removal and/or remodeling of load-bearing crosslinks (referred to as "creep") [28]. In conclusion, it remains to be seen whether changes in wall elasticity are causative, or just an epiphenomenon, in the process that links pectin de-methylesterification to cell expansion [36]. ...
One of the many legacies of the work of Michel Caboche is our understanding of plant cell wall synthesis and metabolism thanks to the use of Arabidopsis mutants. Here I describe how he was instrumental in initiating the genetic study of plant cell walls. I also show, with a few examples for cellulose and pectins, how this approach has led to important new insights in cell wall synthesis and how the metabolism of pectins contributes to plant growth and morphogenesis. I also illustrate the limitations of the use of mutants to explain processes at the scale of cells, organs or whole plants in terms of the physico-chemical properties of cell wall polymers. Finally, I sketch how new approaches can cope with these limitations.
... The biomechanical 'hotspot hypothesis' proposes that wall extensibility is controlled at discrete sites where microfibrils come into close contact with one another (Zhang et al., 2014) via a monolayer of xyloglucan binding the hydrophobic surfaces of the two microfibrils together (Cosgrove, 2018b). These may be the selective sites of CW loosening by expansins or by CXEG-type enzymes where the microfibrils slide or separate, perhaps at a rate that is influenced by the bulk viscoelasticity of the microfibrilmatrix network (Park & Cosgrove, 2015). ...
... In some cases, slightly more complex systems such as de-frosted Arabidopsis petioles (Park & Cosgrove, 2012a;Xin et al., 2020), cucumber and Arabidopsis hypocotyls (Boron et al., 2015;Cosgrove, 1989;Marga et al., 2005;Park & Cosgrove, 2012b) or wheat coleoptiles (Hepler & Cosgrove, 2019) have been used. The advantage of using onion epidermal peels is that the mechanical properties of isolated CW fragments can be measured, largely neglecting the contribution of neighbouring cells, cell size or shape that might possibly influence the results when using indentation-based (AFM) measurements (Cosgrove, 2018b and references therein). However, new technologies such as non-contact, optical Brillouin spectroscopy are emerging as tools to probe biomechanical properties of CWs in developing organs at the cellular (Scarcelli et al., 2015) or tissue level (Elsayad et al., 2016;Samalova et al., 2020). ...
... Expansins cause almost immediate in vitro CW extension, allowing to extend the cell length 100 times when compared to its meristematic initials (Cosgrove, 2016b and references therein). Thus, to loosen CW, expansins probably modify noncovalent bonds in the cellulose microfibril network, laterally interconnected with a possible contribution of xyloglucans bound to the hydrophobic face of the cellulose microfibrils (Cosgrove, 2018b and references therein). The consequent fibril-fibril sliding seems to allow CW extension and in-plane stress release of the multilamellate CW structure (Zhang et al., 2019a;2021a). ...
Biomechanical properties of the cell wall (CW) are important for many developmental and adaptive responses in plants. Expansins were shown to mediate pH-dependent CW enlargement via a process called CW loosening. Here, we provide a brief overview of expansin occurrence in plant and non-plant species, their structure and mode of action including the role of hormone-regulated CW acidification in the control of expansin activity. We depict the historical as well as recent CW models, discuss the role of expansins in the CW biomechanics and address the developmental importance of expansin-regulated CW loosening in cell elongation and new primordia formation. We summarise the data published so far on the role of expansins in the abiotic stress response as well as the rather scarce evidence and hypotheses on the possible mechanisms underlying expansin-mediated abiotic stress resistance. Finally, we wrap it up by highlighting possible future directions in expansin research.
... Cellulosic glucan chains assemble to form higher-order fibers with amorphous and crystalline regions. [6][7][8] The inherent propensity of cellulose fibers to bundle and their modulation, mainly by their interaction with hemicelluloses and pectins, are thought to be very important as they confer additional, higher-order mechanical properties. 9,10 The cellulose fibers are secreted into the cell wall by membrane-embedded hexameric cellulose synthase complexes (CSCs), each protomer comprising a trimer of cellulose synthases (CESAs). ...
... 23 Pectins, mainly homogalacturonans (HGs), which comprise up to 60% of the dry weight of the primary cell wall, 4 are hypothesized to surround all other components and act as a matrix. 8 Composition, methylation state, and calcium levels have been shown to change the mechanical properties of pectins by altering the level of cross-linking. 24,25 Despite our knowledge of the chemical composition of the cell wall and of the diversity of the individual components, structural understanding of their secretion and interaction in the cell wall is underexplored. ...
... 34 In the most recent models of the interactions between the different components of the primary cell wall, pectins are thought to surround and tether the cellulose fibers in a calcium-dependent manner. 8,10,24 We thus hypothesized that the meshing was at high odds of being predominantly composed of HG. The cell wall peels were treated with either BAPTA, a calcium chelator, or Aspergillus pectate lyase (PL), an enzyme that digests de-methylesterified HGs, and then processed by cryo-FIB milling and cryo-ET to see whether the previously observed meshing would be morphologically altered. ...
One hallmark of plant cells is their cell wall. They protect cells against the environment and high turgor and mediate morphogenesis through the dynamics of their mechanical and chemical properties. The walls are a complex polysaccharidic structure. Although their biochemical composition is well known, how the different components organize in the volume of the cell wall and interact with each other is not well understood and yet is key to the wall’s mechanical properties. To investigate the ultrastructure of the plant cell wall, we imaged the walls of onion (Allium cepa) bulbs in a near-native state via cryo-focused ion beam milling (cryo-FIB milling) and cryo-electron tomography (cryo-ET). This allowed the high-resolution visualization of cellulose fibers in situ. We reveal the coexistence of dense fiber fields bathed in a reticulated matrix we termed “meshing,” which is more abundant at the inner surface of the cell wall. The fibers adopted a regular bimodal angular distribution at all depths in the cell wall and bundled according to their orientation, creating layers within the cell wall. Concomitantly, employing homogalacturonan (HG)-specific enzymatic digestion, we observed changes in the meshing, suggesting that it is—at least in part—composed of HG pectins. We propose the following model for the construction of the abaxial epidermal primary cell wall: the cell deposits successive layers of cellulose fibers at −45° and +45° relative to the cell’s long axis and secretes the surrounding HG-rich meshing proximal to the plasma membrane, which then migrates to more distal regions of the cell wall.
... The secondary cell wall structure begins to form after cell growth, which can finally shape and support plants. The primary cell wall is composed of three distinctive polysaccharides (cellulose, hemicellulose, and pectin) and is usually organized into multilayer nanostructures, especially in the epidermal wall that physically protect and limit growth of leaves and stems [59,60]. In each layer, the cellulose fibrils are arranged in a common direction, forming a reticulated, noncovalent network, but the direction varies between the layers; hemicellulose bind noncovalently to cellulose, and well-hydrated pectins form a gel-like matrix hosting the stiff cellulose network [61]. ...
... Expansins were discovered in cell walls from cucumber as they responsible for wall extension [62]. Bundled regions where cellulose is in close physical contact with one another potentially function as sites of cell wall loosening and creep by expansins [60]. ...
... In the acid-growth hypothesis, auxin signaling induces expression of the gene encoding plasma membrane H + -ATpase proton pump, which pumps out H + to the wall matrix, resulting in plasma membrane acidification (pH 4.5-6) [63]. Auxin-induced acidic pH is necessary to activate expansins, which cause the cellulose fibril-fibril sliding by acting on the cellulose bundled regions in a non-enzymatic manner, promoting wall loosening, hydration, and expansion [60,63]. In this study, the expansin protein encoding gene Bna-EXPA5 was significantly downregulated in the stem of the dwarf mutant ndf-2, and its expression could also be affected by exogenous auxin. ...
Plant height is one of the most important agronomic traits of rapeseeds. In this study, we characterized a dwarf Brassica napus mutant, named ndf-2, obtained from fast neutrons and DES mutagenesis. Based on BSA-Seq and genetic properties, we identified causal mutations with a time-saving approach. The ndf-2 mutation was identified on chromosome A03 and can result in an amino acid substitution in the conserved degron motif (GWPPV to EWPPV) of the Auxin/indole-3-acetic acid protein 7 (BnaA03.IAA7) encoded by the causative gene. Aux/IAA protein is one of the core components of the auxin signaling pathway, which regulates many growth and development processes. However, the molecular mechanism of auxin signal regulating plant height is still not well understood. In the following work, we identified that BnaARF6 and BnaARF8 as interactors of BnaA03.IAA7 and BnaEXPA5 as a target of BnaARF6 and BnaARF8. The three genes BnaA03.IAA7, BnaARF6/8 and BnaEXPA5 were highly expressed in stem, suggesting that these genes were involved in stem development. The overexpression of BnaEXPA5 results in larger rosettes leaves and longer inflorescence stems in Arabidopsis thaliana. Our results indicate that BnaA03.IAA7- and BnaARF6/8-dependent auxin signal control stem elongation and plant height by regulating the transcription of BnaEXPA5 gene, which is one of the targets of this signal.
... A major enigma in plant biology is how cell wall metabolism can control cell expansion and plant growth. According to the acid growth theory, the growth hormone auxin favors turgor-driven cell expansion through promoting cell wall acidification, which enhances the extensibility of the cell wall (Cosgrove, 2018). Numerous studies have observed acid promoted "creep", i.e. long-term plastic (=irreversible) deformation of the cell wall on isolated cell walls. ...
... In the traditional creep-dependent growth models, cell wall tension, generated by the turgor pressure, is the motor for growth (Cosgrove, 2018). Cell and organ morphogenesis reflects local cell wall relaxation and the mechanical anisotropy of the cell walls, imposed by the orientation of cellulose microfibrils (Baskin, 2005) (Geitmann and Ortega, 2009). ...
... This will require more precise methods to study nano-to mesoscale architecture of intact cell walls. Solid state NMR on never dried cell walls has recently provided important insights into in situ polymer structure and interactions (Cosgrove, 2018;Simmons et al., 2016) but lacks information on the topology of these interactions within the cell wall. Super-resolution microscopy offers great potential in this area. ...
A rapidly increasing body of literature suggests that many biological processes are driven by phase separation within polymer mixtures. Liquid-liquid phase separation can lead to the formation of membrane-less organelles, which are thought to play a wide variety of roles in cell metabolism, gene regulation or signaling. One of the characteristics of these systems is that they are poised at phase transition boundaries, which makes them perfectly suited to elicit robust cellular responses to often very small changes in the cell’s “environment”. Recent observations suggest that, also in the semi-solid environment of plant cell walls, phase separation not only plays a role in wall patterning, hydration and stress relaxation during growth, but also may provide a driving force for cell wall expansion. In this context, pectins, the major polyanionic polysaccharides in the walls of growing cells, appear to play a critical role. Here, we will discuss (i) our current understanding of the structure-function relationship of pectins, (ii) in vivo evidence that pectin modification can drive critical phase transitions in the cell wall, (iii) how such phase transitions may drive cell wall expansion in addition to turgor pressure and (iv) the periodic cellular processes that may control phase transitions underlying cell wall assembly and expansion.
... All plant cells are surrounded by a stiff but extensible extracellular matrix, the cell wall (CW), that performs different crucial mechanical, biochemical, and physiological functions [1][2][3]. It is now understood that the CW is a complex and plastic structure, whose composition and architecture widely varies among species and within tissues and cells of the same organism, and is extensively re-modelled during growth and development and in response to environmental cues [2]. ...
... All plant cells are surrounded by a stiff but extensible extracellular matrix, the cell wall (CW), that performs different crucial mechanical, biochemical, and physiological functions [1][2][3]. It is now understood that the CW is a complex and plastic structure, whose composition and architecture widely varies among species and within tissues and cells of the same organism, and is extensively re-modelled during growth and development and in response to environmental cues [2]. The major load-bearing component of plant CWs is cellulose, which provides tensile strength; non-cellulosic polysaccharides, structural proteins, and other non-saccharide components, like lignin, all contribute to the specific mechanical and biochemical properties of the CW in different cell types [2,3]. ...
... It is now understood that the CW is a complex and plastic structure, whose composition and architecture widely varies among species and within tissues and cells of the same organism, and is extensively re-modelled during growth and development and in response to environmental cues [2]. The major load-bearing component of plant CWs is cellulose, which provides tensile strength; non-cellulosic polysaccharides, structural proteins, and other non-saccharide components, like lignin, all contribute to the specific mechanical and biochemical properties of the CW in different cell types [2,3]. Thanks to their tensile strength, plant CWs provide mechanical support to the cell, permitting elevated internal turgor pressures and modulating cell expansion, ultimately determining cell shape and size [4]. ...
The plant cell wall (CW) is a complex structure that acts as a mechanical barrier, restricting the access to most microbes. Phytopathogenic microorganisms can deploy an arsenal of CW-degrading enzymes (CWDEs) that are required for virulence. In turn, plants have evolved proteins able to inhibit the activity of specific microbial CWDEs, reducing CW damage and favoring the accumulation of CW-derived fragments that act as damage-associated molecular patterns (DAMPs) and trigger an immune response in the host. CW-derived DAMPs might be a component of the complex system of surveillance of CW integrity (CWI), that plants have evolved to detect changes in CW properties. Microbial CWDEs can activate the plant CWI maintenance system and induce compensatory responses to reinforce CWs during infection. Recent evidence indicates that the CWI surveillance system interacts in a complex way with the innate immune system to fine-tune downstream responses and strike a balance between defense and growth.
... Speci cally, xyloglucans consist of a β-(1,4)d-glucan backbone that is quasi-regularly substituted with β-d-xylosyl residues linked to glucose through the O-6 position (Park and Cosgrove 2015). Xyloglucan is present in the primary cell walls of plants and is involved in controlling plant elongation (Park and Cosgrove 2015;Cosgrove 2018 It has been suggested but not directly proven that NP interacts and forms complexes with water-soluble polysaccharides and BC (Tokuyasu et al. 2021(Tokuyasu et al. , 2022. In this study, NP was prepared using TG, a watersoluble polysaccharide containing xyloglucan, which interacts with BC, to verify the complex formation between BC and TG. ...
... Previous reports suggested that xyloglucan binds to the hydrophobic surface of cellulose (Zhao et al. 2014;Benselfelt et al. 2016). More speci cally, (XXXG) 3 was found to bind more favorably to the (1 0 0) and (2 0 0) hydrophobic surfaces of CMF than to the (1 1 0), (0 1 0), and (1-1 0) hydrophilic surfaces (here, each [1,4]-d-glucosyl residue in the backbone was assigned a one-letter code according to its substituents: G = β-d-Glc; X = α-d-Xyl-[1,6]-β-d-Glc). This behavior was attributed to the topography of the hydrophobic CMF surface, which stabilized (XXXG) 3 in a at conformation. ...
Nata puree (NP)—obtained by disintegrating nata de coco (bacterial cellulose [BC]) using a household blender—can be combined with tamarind seed gum (TG) to generate NPTG. In this study, BC fibrils (BC-TG) were prepared by removing free TG from NPTG and characterized. BC-TG exhibited high water dispersibility and relatively long nanofibrils (> 20 µm). We examined the distribution of xyloglucan, the main component of TG, on BC nanofibrils using immunofluorescence staining with calcofluor white, which stains the hydrophilic cellulose surface, and found that xyloglucan was adsorbed at different sites along the fibers. This indicated that BC-TG was a composite nanofibril of xyloglucan and BC. Furthermore, BC-TG showed a higher degree of adsorption on hydrophobic plastic substrates than BC did, suggesting a change in the surface properties of BC. Because the BC-TG preparation process is simple, requires only water and raw materials, and does not involve chemical reactions, it is expected to be an environmentally friendly method for the preparation and modification of BC nanofibrils.
... Studies using finite element modelling (Carter et al., 2017;Cooke et al., 2008;Marom et al., 2017;Woolfenden et al., 2017;2018;Yi et al., 2018) to simulate the mechanics of the guard cell wall and stomatal kinetics have typically used assumed turgor pressure values based on measurements from other experimental systems rather than the modelled cells per se, and the predictions of those models are inherently confounded by this assumption as well as assumptions regarding cell wall properties in guard cells, for example, anisotropy and elasticity versus viscoelasticity. More importantly, this approach of using static pressure to model guard cell deformation neglects the effects of dynamic changes in guard cell volume and turgor pressure on stomatal biomechanics and does not address the knowledge gap of how turgor pressure in intact guard cells might dynamically change during stomatal opening, although evidence for these dynamic changes has recently been uncovered (Chen et al., 2021). ...
... On the other hand, hemicelluloses and pectins are thought to interact with the surface of cellulose microfibrils and also form a hydrated matrix that exhibits properties of a liquid or gel (Chanliaud et al., 2002;Whitney et al., 1999). A combination of solid cellulose microfibrils situated in a viscous wall matrix would make the cell wall exhibit mechanical behaviours of both a solid and a viscous liquid simultaneously (Baskin, 2017;Cosgrove, 2018). Viscoelasticity models describe such materials as exhibiting delayed deformation. ...
The ability of plants to absorb CO 2 for photosynthesis and transport water from root to shoot depends on the reversible swelling of guard cells that open stomatal pores in the epidermis. Despite decades of experimental and theoretical work, the biomechanical drivers of stomatal opening and closure are still not clearly defined. We combined mechanical principles with a growing body of knowledge concerning water flux across the plant cell membrane and the biomechanical properties of plant cell walls to quantitatively test the long-standing hypothesis that increasing turgor pressure resulting from water uptake drives guard cell expansion during stomatal opening. To test the alternative hypothesis that water influx is the main motive force underlying guard cell expansion, we developed a system dynamics model accounting for water influx. This approach connects stomatal kinetics to whole plant physiology by including values for water flux arising from water status in the plant .
... Taken together, the results support the idea that reduced pectin levels in adherent and non-adherent mucilage of the AtCESA1 T166A mutant (Fig. 4) may partly result from altered cellulose biosynthesis and/or deposition, which affects cellulosic ray formation and, thus, interactions of cellulose with pectin and other polysaccharides (Cosgrove, 2018;Griffiths and North, 2017). ...
... It is possible that modulation in cellulose biosynthesis and deposition activates cell wall integrity maintenance mechanisms (Gigli-Bisceglia et al., 2020;Herger et al., 2019;Voxeur and Hofte, 2016), leading to the modulation of pectin synthesis and modification (Figs. 4 and 6) as well as PA biosynthesis (Fig. 3). This could be achieved through changes in the spatial pattern of hydrophobic and hydrophilic surfaces of cellulose microfibrils and thus their interactions with pectin and hemicelluloses (Cosgrove, 2018). ...
Cell wall biogenesis is required for the production of seeds of higher plants. However, little is known about regulatory mechanisms underlying cell wall biogenesis during seed formation. Here we show a role for the phosphorylation of Arabidopsis cellulose synthase 1 (AtCESA1) in modulating pectin synthesis and methylesterification in seed coat mucilage. A phosphor-null mutant of AtCESA1 on T166 (AtCESA1T166A) was constructed and introduced into a null mutant of AtCESA1 (Atcesa1-1). The resulting transgenic lines showed a slight but significant decrease in cellulose contents in mature seeds. Defects in cellulosic ray architecture along with reduced levels of non-adherent and adherent mucilage were observed on the seeds of the AtCESA1T166A mutant. Reduced mucilage pectin synthesis was also reflected by a decrease in the level of uronic acid. Meanwhile, an increase in the degree of pectin methylesterification was also observed in the seed coat mucilage of AtCESA1T166A mutant. Change in seed development was further reflected by a delayed germination and about 50% increase in the accumulation of proanthocyanidins, which is known to bind pectin and inhibit seed germination as revealed by previous studies. Taken together, the results suggest a role of AtCESA1 phosphorylation on T166 in modulating mucilage pectin synthesis and methylesterification as well as cellulose synthesis with a role in seed development.
... However, growth does not always occur perpendicularly to cellulose microfibrils orientation: probing the structure of the cell wall at the nanoscale revealed that cellulose microfibrils are organized in stacked layers, giving the cell wall a polylamellate architecture. Interestingly, cellulose microfibrils in onion epidermal cell wall are arranged along the same direction within a layer, but this orientation shifts between adjacent layers so that across the entire wall the orientation of cellulosic fibers is almost isotropic (Cosgrove, 2018a(Cosgrove, , 2018bKafle et al., 2017). ...
... Cellulose appears the stiffest component of the wall, whereas hemicelluloses and pectin network are softer (Mirabet et al., 2011). However, the mechanical properties that result from the interactions between those elements are still far from understood (Cosgrove, 2018a(Cosgrove, , 2018b. Haas and colleagues have used 3D-STORM super-resolution microscopy and cryoSEM to study leaf pavement cell wall (Haas et al., 2020). ...
Plant epidermis are multifunctional surfaces that directly affect how plants interact with animals or microorganisms and influence their ability to harvest or protect from abiotic factors. To do this, plants rely on minuscule structures that confer remarkable properties to their outer layer. These microscopic features emerge from the hierarchical organisation of epidermal cells with various shapes and dimensions combined with different elaborations of the cuticle, a protective film that covers plant surfaces. Understanding the properties and functions of those tridimensional elements as well as disentangling the mechanisms that control their formation and spatial distribution warrant a multidisciplinary approach. Here we show how interdisciplinary efforts coupling modern tools of experimental biology, physics and chemistry with advanced computational modelling and state-of-the art microscopy are yielding broad new insights into the seemingly arcane patterning processes that sculpt the outer layer of plants.
... Cellulose consists of β-1,4-linked glucose that coalesces into microfibrils via intermolecular hydrogen bonds and van der Waal's forces. The cellulose microfibrils have a high tensile strength and work as a scaffold, providing the load-bearing strength to the cell walls [7,8]. Cellulose is produced at the cell surface by cellulose synthase (CesA) protein complexes (CSCs), which utilize cytosolic UDPglucose as their substrate [9,10]. ...
... These polymers are made in the Golgi lumen, with the possible exception of mixedlinked glucan [11][12][13], by glycosyltransferases (GTs) that use an array of nucleotide sugars as substrates. Hemicelluloses engage with cellulose and/or lignin to regulate, depending on the developmental context, either cell wall expansion and cell growth or cell wall rigidification [8,14,15]. ...
Plant cell wall-derived biomass serves as a renewable source of energy and materials with increasing importance. The cell walls are biomacromolecular assemblies defined by a fine arrangement of different classes of polysaccharides, proteoglycans, and aromatic polymers and are one of the most complex structures in Nature. One of the most challenging tasks of cell biology and biomass biotechnology research is to image the structure and organization of this complex matrix, as well as to visualize the compartmentalized, multiplayer biosynthetic machineries that build the elaborate cell wall architecture. Better knowledge of the plant cells, cell walls, and whole tissue is essential for bioengineering efforts and for designing efficient strategies of industrial deconstruction of the cell wall-derived biomass and its saccharification. Cell wall-directed molecular probes and analysis by light microscopy, which is capable of imaging with a high level of specificity, little sample processing, and often in real time, are important tools to understand cell wall assemblies. This review provides a comprehensive overview about the possibilities for fluorescence label-based imaging techniques and a variety of probing methods, discussing both well-established and emerging tools. Examples of applications of these tools are provided. We also list and discuss the advantages and limitations of the methods. Specifically, we elaborate on what are the most important considerations when applying a particular technique for plants, the potential for future development, and how the plant cell wall field might be inspired by advances in the biomedical and general cell biology fields.
... The outer epidermal wall of stems and leaves has distinctive physical and structural properties connected with its role to limit growth of these organs and to protect them [1][2][3][4][5]. Typically the outer epidermal wall is a multi-lamellate structure impregnated with hydrophobic substances (cutin, waxes) and sometimes lignified [5][6][7][8]. ...
... We also thank Dr. Sarah Kiemle for initial trials with the methanolysis with TFA protocol, and Edward Wagner for the Driselase protocol and protein determination. 1 ...
Background
Epidermal cell walls have special structural and biological roles in the life of the plant. Typically they are multi-ply structures encrusted with waxes and cutin which protect the plant from dehydration and pathogen attack. These characteristics may also reduce chemical and enzymatic deconstruction of the wall for sugar analysis and conversion to biofuels. We have assessed the saccharide composition of the outer epidermal wall of onion scales with different analytical methods. This wall is a particularly useful model for cell wall imaging and mechanics.
Results
Epidermal walls were depolymerized by acidic methanolysis combined with 2M trifluoracetic acid hydrolysis and the resultant sugars were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Total sugar yields based on wall dry weight were low (53%). Removal of waxes with chloroform increased the sugar yields to 73% and enzymatic digestion did not improve these yields. Analysis by gas chromatography/mass spectrometry (GC/MS) of per- O -trimethylsilyl (TMS) derivatives of the sugar methyl glycosides produced by acidic methanolysis gave a high yield for galacturonic acid (GalA) but glucose (Glc) was severely reduced. In a complementary fashion, GC/MS analysis of methyl alditols produced by permethylation gave substantial yields for glucose and other neutral sugars, but GalA was severely reduced. Analysis of the walls by ¹³ C solid-state NMR confirmed and extended these results and revealed 15% lipid content after chloroform extraction (potentially cutin and unextractable waxes).
Conclusions
Although exact values vary with the analytical method, our best estimate is that polysaccharide in the outer epidermal wall of onion scales is comprised of homogalacturonan (~ 50%), cellulose (~ 20%), galactan (~ 10%), xyloglucan (~ 10%) and smaller amounts of other polysaccharides. Low yields of specific monosaccharides by some methods may be exaggerated in epidermal walls impregnated with waxes and cutin and call for cautious interpretation of the results.
... In plants, cells grow through turgor-driven expansion that is constrained by the cell wall (Cosgrove, 2018). Cell-wall extensibility is under dynamic control in plant cells, and expansins play a key role by inducing the relaxation of the stress that is generated in the cell wall through the action of turgor pressure (McQueen-Mason et al., 1992). ...
... These cell-wall proteins appear to act by disrupting noncovalent associations between cellulose and matrix polysaccharides in the plant cell wall, allowing the polymers to slip relative to one another, relaxing stress in the wall and allowing it to extend (McQueen-Mason & Cosgrove, 1994). Expansins fall into two well-separated groups, designated as a and b-expansins, based on sequence homology and activity; with a-expansins more clearly associated with cell expansion and growth (Cosgrove, 2018). ...
Wheat is the most widely grown crop globally, providing 20% of all human calories and protein. Achieving step changes in genetic yield potential is crucial to ensure food security, but efforts are thwarted by an apparent trade‐off between grain size and number. Expansins are proteins that play important roles in plant growth by enhancing stress relaxation in the cell wall, which constrains cell expansion.
Here, we describe how targeted overexpression of an α‐expansin in early developing wheat seeds leads to a significant increase in grain size without a negative effect on grain number, resulting in a yield boost under field conditions.
The best‐performing transgenic line yielded 12.3% higher average grain weight than the control, and this translated to an increase in grain yield of 11.3% in field experiments using an agronomically appropriate plant density.
This targeted transgenic approach provides an opportunity to overcome a common bottleneck to yield improvement across many crops.
... The bottom-up multiscale modelling approach allows us to implement different material models for cell wall components. These can include a solid mechanics model for cellulose microfibrils behaving as an elastic material following Hooke's law and viscoelastic models for matrix components, and modelling the bonds between cellulose microfibrils and matrix components with relative deformation energetics equivalent to their hypothesized biochemical interactions (Cosgrove 2018). ...
Stomata are dynamic pores on plant surfaces that regulate photosynthesis and are thus of critical importance for understanding and leveraging the carbon-capturing and food-producing capabilities of plants. However, our understanding of the molecular underpinnings of stomatal kinetics and the biomechanical properties of the cell walls of stomatal guard cells that enable their dynamic responses to environmental and intrinsic stimuli is limited. Here, we built multiscale models that simulate regions of the guard cell wall, representing cellulose fibrils and matrix polysaccharides as discrete, interacting units, and used these models to help explain how molecular changes in wall composition and underlying architecture alter guard wall biomechanics that gives rise to stomatal responses in mutants with altered wall synthesis and modification. These results point to strategies for engineering guard cell walls to enhance stomatal response times and efficiency.
... The cell wall (CW) of land plants has been depicted as a highly intertwining architecture consisting of cellulose microfibrils, hemicellulose and pectin (Carpita & Gibeaut, 1993), which compose the three major components of the primary CW. Cellulose microfibrils are the stiffest component, playing a load-bearing role (Bashline et al., 2014;Bidhendi & Geitmann, 2016;Cosgrove, 2005Cosgrove, , 2018. ...
The plasticity and growth of plant cell walls (CWs) remain poorly understood at the molecular level. In this work, we used atomic force microscopy (AFM) to observe elastic responses of the root transition zone of 4‐day‐old Arabidopsis thaliana wild‐type and almt1‐ mutant seedlings grown under Fe or Al stresses. Elastic parameters were deduced from force‐distance curve measurements using the trimechanic‐3PCS framework. The presence of single metal species Fe ²⁺ or Al ³⁺ at 10 µM exerts no noticeable effect on the root growth compared with the control conditions. On the contrary, a mix of both the metal ions produced a strong root‐extension arrest concomitant with significant increase of CW stiffness. Raising the concentration of either Fe ²⁺ or Al ³⁺ to 20 µM, no root‐extension arrest was observed; nevertheless, an increase in root stiffness occurred. In the presence of both the metal ions at 10 µM, root‐extension arrest was not observed in the almt1 mutant, which substantially abolishes the ability to exude malate. Our results indicate that the combination of Fe ²⁺ and Al ³⁺ with exuded malate is crucial for both CW stiffening and root‐extension arrest. However, stiffness increase induced by single Fe ²⁺ or Al ³⁺ is not sufficient for arresting root growth in our experimental conditions.
... Increased interconnectivity of xylan lead to densification of the network by wrapping it with xylan chain strands, associated with an enlarged network of hydrogen bonds. At a low substitution degree of xylose backbone of xylan (approximately one substitution per eight xylose units), which was used in current study, the xylan chains adsorb to the cellulose surface via stiffer substituted domains of chain (Cosgrove, 2018), while the unsubstituted flexible domains of the xylan chain can physically bind cellulose fibres. Very similar considerations were reported by Long et al., in which the authors reported that the maximum modulus and maximum stress of cellulose-xylan composite films increase at low xylan content, which can be attributed to fibre reinforcement and increased hydrogen bonding (Long et al., 2019). ...
Bacterial cellulose (BC) is a natural biopolymer of β-(1,4)-linked β-D-glucopyranose that forms a homogeneous network of cellulose fibres with high chemical purity and modifiability. Plasticizers of the BC fibre network allow to reduce its brittleness and improve flexibility by reducing the intermolecular forces and increasing the chain mobility, while the opposite effect was observed for the antiplasticizing additives. Therefore, the aim of the current study was to investigate the effect of hemicellulose addition on the elasto-plastic response of BC-hemicellulose composite films depending on the specific type of hemicellulose and its concentration. BC-hemicellulose composite films were produced by K.xylinum bacteria strain in the hemicellulose-modified medium. The presence of xylan and arabinoxylan in the culturing medium resulted in an increase in the plastic deformation of the composite films, its maximum stress and maximum strain, which was opposite to the effects of glucomannan and xyloglucan. Spectral studies revealed that the deformation of the hemicellulose polysaccharides played a minor role in opposing of applied loads. The contribution of the hydrogen bond network in opposing of applied loads was comparable to that of the glycosidic bonds. Stretching vibrations of the structural bonds of BC-hemicellulose composite films determined elastic deformation most, compared to other bonds.
... We speculate that the different effects of dehydration on the ( 110/110 ) and (200) lattice spacings are due to differences in hydrophilicity of the faces of the CMF. Hydroxymethyl groups on (110) and ( 110 ) surfaces lead to more hydrophilicity than on (100) surfaces 56 . Water likely interacts more strongly with (110) and ( 110 ) surfaces and may influence the crystalline structure. ...
The primary cell wall is highly hydrated in its native state, yet many structural studies have been conducted on dried samples. Here, we use grazing-incidence wide-angle X-ray scattering (GIWAXS) with a humidity chamber, which enhances scattering and the signal-to-noise ratio while keeping outer onion epidermal peels hydrated, to examine cell wall properties. GIWAXS of hydrated and dried onion reveals that the cellulose (110/11¯0\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$110/1\overline{1}0$$\end{document}) lattice spacing decreases slightly upon drying, while the (200) lattice parameters are unchanged. Additionally, the (110/11¯0\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$110/1\overline{1}0$$\end{document}) diffraction intensity increases relative to (200). Density functional theory models of hydrated and dry cellulose microfibrils corroborate changes in crystalline properties upon drying. GIWAXS also reveals a peak that we attribute to pectin chain aggregation. We speculate that dehydration perturbs the hydrogen bonding network within cellulose crystals and collapses the pectin network without affecting the lateral distribution of pectin chain aggregates.
... It is only recently recognized that the crystalline arrangement of glucan chains in an elementary MF can generate surfaces with both hydrophilic and hydrophobic faces exposed facilitating selective interaction with different matrix polysaccharides and adjacent MFs 34,65,66,73 . Furthermore, recent work shows that xylan adopts a two-fold screw conformation as well as evenly spaced substitution (i.e. ...
Lignocellulose biomass has a tremendous potential as renewable biomaterials for fostering the “bio-based society” and circular bioeconomy paradigm. It requires efficient use and breakdown of fiber cell walls containing mainly cellulose, hemicellulose and lignin biopolymers. Despite their great importance, there is an extensive debate on the true structure of fiber walls and knowledge on the macromolecular nano-organization is limited and remains elusive in 3D. We employed dual-axis electron tomography that allows visualization of previously unseen 3D macromolecular organization/biopolymeric nano-architecture of the secondary S2 layer of Norway spruce fiber wall. Unprecedented 3D nano-structural details with novel insights into cellulose microfibrils (~ 2 nm diameter), macrofibrils, nano-pore network and cell wall chemistry (volume %) across the S2 were explored and quantified including simulation of structure related permeability. Matrix polymer association with cellulose varied between microfibrils and macrofibrils with lignin directly associated with MFs. Simulated bio-nano-mechanical properties revealed stress distribution within the S2 and showed similar properties between the idealized 3D model and the native S2 (actual tomogram). Present work has great potential for significant advancements in lignocellulose research on nano-scale understanding of cell wall assembly/disassembly processes leading to more efficient industrial processes of functionalization, valorization and target modification technologies.
... These proteins belong to complex multigenic families including polygalacturonases (PG), pectin lyases (PL), pectin methyl (or acetyl) esterases (PME, PAE), expansins or arabinogalactan proteins (AGP). They may remodel cell wall stiffness, thickness or cell-to-cell adhesion in a concerted way (Cosgrove, 2005(Cosgrove, , 2018. PME and PME inhibitors (PMEI) are also important factors during infection by different microbial pathogens. ...
Les hydathodes sont décrits pour la première fois à la fin du XIXe siècle, par le botaniste chirurgien microbiologiste Prussien Anton de Bary. Depuis, ces organes, pourtant présents sur les feuilles de la majorité des plantes vasculaires, ne sont étudiés que sporadiquement par la communauté scientifique. Sites de la guttation, les hydathodes se retrouvent à la pointe de la dentelure des feuilles. Constitués de pores dans l'épiderme et d'un parenchyme distinctif lié au système vasculaire, les hydathodes font le lien direct entre le milieu extérieur et les vaisseaux du xylème des feuilles. De façon surprenante, cette voie d'entrée dans les tissus végétaux n'est utilisée que par une poignée d'agents pathogènes. Parmi eux, Xanthomonas campestris pathovar campetris (Xcc) est l'agent responsable de la nervation noire de Brassicacées. Cette bactérie constitue une menace pour l'agriculture, puisqu'elle est capable d'infecter de nombreuses espèces cultivées, telles que les choux et choux-fleurs, le navet, la moutarde, le radis, etc. Si les facteurs de virulence de Xcc sont bien connus, peu d'études se sont attelées à décrypter le rôle des hydathodes dans l'immunité des plantes, alors qu'ils sont le premier lieu de contact entre Xcc et la plante. Au cours de ce projet de thèse, j'ai étudié les spécificités transcriptomiques des hydathodes par rapport au mésophylle et les particularités du métabolome du fluide de guttation en comparaison de celui de la sève brute. J'ai ensuite établi les premières bases génétiques de l'immunité post-invasive des hydathodes de chou-fleur (cultivar Clovis) en comparant leur réponse à une souche virulente et avirulente de Xcc, durant les étapes précoces de l'infection. Dans la deuxième partie du projet, j'ai mis en place un système permettant de cribler un grand nombre de mutants d'Arabidopsis thaliana en réponse à Xcc, qui sera utilisé au sein de l'équipe. Grâce à cette nouvelle méthode, j'ai entamé le criblage d'une collection de mutant d'A. thaliana et identifié plus d'une cinquantaine de lignées dont la sensibilité à Xcc est altérée. Pour finir, dans la troisième partie réalisée dans le cadre d'une collaboration, j'ai exploré le lien entre le nombre d'hydathodes, l'état hydrique des tissus et le comportement infectieux de Xcc. Ce projet apporte des données originales et des outils qui permettront de mieux comprendre le fonctionnement des hydathodes en tant qu'organes ainsi que leur rôle au cours des interactions plantes-pathogènes.
... The hemicellulose polysaccharides xyloglucan (XyG), xylan, and glucomannan can bind tightly to cellulose (Cavalier et al., 2008;Cosgrove, 2014;Simmons et al., 2016;Terrett et al., 2019). For many years, a cellulose-XyG network was proposed to be the principal load-bearing structure of the PCW in dicots (Cosgrove, 2018). However, experimental data are now more consistent with a view where cellulose fibril interactions largely determine wall extensibility (Zhang et al., 2021). ...
Hemicelluose 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 to 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 PC formation has been proposed to involve local elongation of lobes, local restriction of dimples, or a combination of both (Fu et al., 2002(Fu et al., , 2005(Fu et al., , 2009Xu et al., 2010;Zhang et al., 2011;Abley et al., 2013;Lin et al., 2013;Sampathkumar et al., 2014;Armour et al., 2015;Higaki et al., 2016;Majda et al., 2017;Sapala et al., 2018). In addition, although Roh of Plants (ROP) signaling and phytohormonal regulation of PC interdigitation play central roles in the determination of plant cell shape, the mechanical effects of the PC wall, and the mechanical interactions between the adjacent cells may be involved (Sampathkumar et al., 2014;Cosgrove, 2018;Sapala et al., 2018;Bidhendi et al., 2019;Cosgrove and Anderson, 2020;Haas et al., 2020;Liu et al., 2021). ...
The level of inorganic pyrophosphate (PPi) must be tightly regulated in all kingdoms for proper execution of cellular functions. In plants, the vacuolar proton pyrophosphatase (H+-PPase) has a pivotal role in PPi homeostasis. We previously demonstrated that excess cytosolic PPi in the H+-PPase loss-of-function fugu5 mutant inhibits gluconeogenesis from seed storage lipids, arrests cell division in cotyledonary palisade tissue, and triggers compensated cell enlargement (CCE). Moreover, PPi alters pavement cell (PC) shape, stomatal patterning, and functioning, supporting specific yet broad inhibitory effects of PPi on leaf morphogenesis. Whereas these developmental defects were totally rescued by expression of the yeast soluble pyrophosphatase IPP1, sucrose supply alone cancelled CCE in the palisade tissue but not the epidermal developmental defects. Hence, we postulated that the latter are likely triggered by excess PPi rather than a sucrose deficit. To formally test this hypothesis, we adopted a spatiotemporal approach by constructing and analyzing fugu5-1 PDF1pro::IPP1, fugu5-1 CLV1pro::IPP1, and fugu5-1 ICLpro::IPP1, whereby PPi was removed specifically from the epidermis, palisade tissue cells, or during the 4 days following seed imbibition, respectively. It is important to note that whereas PC defects in fugu5-1 PDF1pro::IPP1 were completely recovered, those in fugu5-1 CLV1pro::IPP1 were not. In addition, phenotypic analyses of fugu5-1 ICLpro::IPP1 lines demonstrated that immediate removal of PPi after seed imbibition markedly improved overall plant growth, abolished CCE, but only partially restored the epidermal developmental defects. Next, the impact of spatial and temporal removal of PPi was investigated by capillary electrophoresis time-of-flight mass spectrometry (CE-TOF MS). Our analysis revealed that metabolic profiles are differentially affected among all the above transgenic lines, and consistent with an axial role of central metabolism of gluconeogenesis in CCE. Taken together, this study provides a conceptual framework to unveil metabolic fluctuations within leaf tissues with high spatio-temporal resolution. Finally, our findings suggest that excess PPi exerts its inhibitory effect in planta in the early stages of seedling establishment in a tissue- and cell-autonomous manner.
... In all these processes, the inherent underlying mechanism at the physical level may be viewed as a form of vibration. Sophisticated tools, including atomic force microscopy (AFM) [1][2][3][4], scanning tunneling microscopy (STM) [5,6], terahertz near-field microscopy (THz-NFM) [7], and hyperspectral imaging (HSI) [8][9][10], are now helping to offer a glimpse of vibrational patterning at the subcellular and cellular levels. ...
We discuss emerging views on the complexity of signals controlling the onset of biological shapes and functions, from the nanoarchitectonics arising from supramolecular interactions, to the cellular/multicellular tissue level, and up to the unfolding of complex anatomy. We highlight the fundamental role of physical forces in cellular decisions, stressing the intriguing similarities in early morphogenesis, tissue regeneration, and oncogenic drift. Compelling evidence is presented, showing that biological patterns are strongly embedded in the vibrational nature of the physical energies that permeate the entire universe. We describe biological dynamics as informational processes at which physics and chemistry converge, with nanomechanical motions, and electromagnetic waves, including light, forming an ensemble of vibrations, acting as a sort of control software for molecular patterning. Biomolecular recognition is approached within the establishment of coherent synchronizations among signaling players, whose physical nature can be equated to oscillators tending to the coherent synchronization of their vibrational modes. Cytoskeletal elements are now emerging as senders and receivers of physical signals, “shaping” biological identity from the cellular to the tissue/organ levels. We finally discuss the perspective of exploiting the diffusive features of physical energies to afford in situ stem/somatic cell reprogramming, and tissue regeneration, without stem cell transplantation.
... Plant cells divide, expand and change their shape during development but do generally not migrate. Moreover, the growth of plant organs is physically limited by the epidermis [8]. Hence, any change to the timing of differentiation of the epidermis, when compared to reproductive maturity, should yield heterochronic variation in the morphology of plant organs. ...
Background
Understanding the relationship between macroevolutionary diversity and variation in organism development is an important goal of evolutionary biology. Variation in the morphology of several plant and animal lineages is attributed to pedomorphosis, a case of heterochrony, where an ancestral juvenile shape is retained in an adult descendant. Pedomorphosis facilitated morphological adaptation in different plant lineages, but its cellular and molecular basis needs further exploration. Plant development differs from animal development in that cells are enclosed by cell walls and do not migrate. Moreover, in many plant lineages, the differentiated epidermis of leaves, and leaf-derived structures, such as petals, limits organ growth. We, therefore, proposed that pedomorphosis in leaves, and in leaf-derived structures, results from delayed differentiation of epidermal cells with respect to reproductive maturity. This idea was explored for petal evolution, given the importance of corolla morphology for angiosperm reproductive success.
Results
By comparing cell morphology and transcriptional profiles between 5 mm flower buds and mature flowers of an entomophile and an ornitophile Loasoideae species (a lineage that experienced transitions from bee- to hummingbird-pollination), we show that evolution of pedomorphic petals of the ornithophile species likely involved delayed differentiation of epidermal cells with respect to flower maturity. We also found that developmental mechanisms other than pedomorphosis might have contributed to evolution of corolla morphology.
Conclusions
Our results highlight a need for considering alternatives to the flower-centric perspective when studying the origin of variation in flower morphology, as this can be generated by developmental processes that are also shared with leaves.
Graphical Abstract
... Plant cell wall is an important limiting factor of cell expansion. In cells, the extensibility of the cell wall is dynamically controlled [46]. Expansins play a key role in inducing stress relaxation of the cell wall under expansion pressure, thus limiting cell expansion [47,48]. ...
The plant leaf, the main organ of photosynthesis, is an important regulator of growth. To explore the difference between leaf size of Populusdeltoides ‘Danhong’ (Pd) and Populus simonii ‘Tongliao1’ (Ps), we investigated the leaf length, leaf width, leaf thickness, leaf area, leaf mass per area (LMA), and cell size of leaves from two genotypes and profiled the transcriptome-wide gene expression patterns through RNA sequencing. Our results show that the leaf area of Pd was significantly larger than that of Ps, but the epidermal cell area was significantly smaller than that of Ps. The difference of leaf size was caused by cell numbers. Transcriptome analysis also revealed that genes related to chromosome replication and DNA repair were highly expressed in Pd, while genes such as the EXPANSIN (EXPA) family which promoted cell expansion were highly expressed in Ps. Further, we revealed that the growth-regulating factors (GRFs) played a key role in the difference of leaf size between two genotypes through regulation of cell proliferation. These data provide a valuable resource for understanding the leaf development of the Populus genus.
... Attachment of plant cells to hydrophobic scaffolds in our work is similar to the interaction of plant cells to hydrophobic PET microfiber infused with PLA nanofiber scaffolds (38). While the physiological relevance of scaffold hydrophobicity is unclear, it is possible that this property mimics hydrophobic surfaces of cellulose microfibrils and other cell wall materials (56). ...
Mechanistic studies of plant development would benefit from an in vitro model that mimics the endogenous physical interactions between cells and their microenvironment. Here, we present artificial scaffolds to which both solid- and liquid-cultured tobacco BY-2 cells adhere without perturbing cell morphology, division, and cortical microtubule organization. Scaffolds consisting of polyvinylidene tri-fluoroethylene (PVDF-TrFE) were prepared to mimic the cell wall's fibrillar structure and its relative hydrophobicity and piezoelectric property. We found that cells adhered best to scaffolds consisting of nanosized aligned fibers. In addition, poling of PVDF-TrFE, which orients the fiber dipoles and renders the scaffold more piezoelectric, increased cell adhesion. Enzymatic treatments revealed that the plant cell wall polysaccharide, pectin, is largely responsible for cell adhesion to scaffolds, analogous to pectin-mediated cell adhesion in plant tissues. Together, this work establishes the first plant biomimetic scaffolds that will enable studies of how cell-cell and cell-matrix interactions affect plant developmental pathways. Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science.
... Microtubules have been shown to align preferentially along the maximal tensile stress direction at potential stress hotspots in the periclinal neck regions of Arabidopsis cotyledon pavement cells (18,114) (Figure 2b,c). In this way, microtubules are thought to align cellulose microfibrils parallel to stress directions to resist strain and maintain a growth pattern (33,125). As cellulose microfibrils can promote local growth anisotropy (118), a positive feedback loop has been proposed. ...
The plant epidermis serves many essential functions, including interactions with the environment, protection, mechanical strength, and regulation of tissue and organ growth. To achieve these functions, specialized epidermal cells develop into particular shapes. These include the intriguing interdigitated jigsaw puzzle shape of cotyledon and leaf pavement cells seen in many species, the precise functions of which remain rather obscure. Although pavement cell shape regulation is complex and still a long way from being fully understood, the roles of the cell wall, mechanical stresses, cytoskeleton, cytoskeletal regulatory proteins, and phytohormones are becoming clearer. Here, we provide a review of this current knowledge of pavement cell morphogenesis, generated from a wealth of experimental evidence and assisted by computational modeling approaches. We also discuss the evolution and potential functions of pavement cell interdigitation. Throughout the review, we highlight some of the thought-provoking controversies and creative theories surrounding the formation of the curious puzzle shape of these cells.
... T he primary cell wall physically controls many key features of growing plant cells, including size, shape, growth, water/ turgor relations, mechanical strength, and defense against pathogens (1). Comprising three distinctive polysaccharides (cellulose, hemicelluloses, and pectins), the wall is often organized as a multilayer nanostructure, particularly conspicuous in epidermal walls that physically protect and limit growth of leaves and stems (2,3). Within individual layers (lamellae), stiff cellulose fibrils (~3 nm wide, traditionally called microfibrils) form a reticulated, noncovalent network aligned in a common direction that varies among lamellae, while hemicelluloses bind noncovalently to cellulose and well-hydrated pectins form a gellike matrix hosting the stiff cellulose network. ...
Computational analysis of cell walls
Layers of intertwined fibers make up plant cell walls. The various types of fibers respond differently to deformation. Cellulose microfibrils, for example, can stretch or curve, changing their end-to-end length, and can also slide past each other, reorient relative directions, and bundle with neighboring microfibrils. Zhang et al. developed a computational model based on observations of onion skin epidermis that describes how these complex changes in space govern cell wall mechanics. The results inform ways to engineer multifunctional fibrous materials.
Science , this issue p. 706
... For a long time XGs were thought to cover most of the cellulose microfibrils, preventing their association with one another and simultaneously acting as the major load-bearing tether between adjacent microfibrils (Cosgrove, 2001;Fry, 2004;Scheller and Ulvskov, 2010). Recent studies report that only a minority of XGs interact with cellulose, forming coilshaped fine spots resembling 'biomechanical hot spots' that control wall extension (Park and Cosgrove, 2012;Wang et al., 2013), and that binding preferentially occurs at the hydrophobic surface of cellulose (Cosgrove, 2018;Zheng et al., 2018). ...
The xyloglucan endotransglucosylase/hydrolases (XTHs) are enzymes involved in cell wall assembly and growth regulation, cleaving and re-joining hemicellulose chains in the xyloglucan-cellulose network. Here, in a homologous system, we compare the secretion patterns of XTH11, XTH33 and XTH29, three members of the Arabidopsis thaliana XTH family, selected for the presence (XTH11, XTH33) or the absence (XTH29) of a signal peptide, and the presence of a transmembrane domain (XTH33). We show that XTH11 and XTH33 reached, respectively, the cell wall and plasma membrane through a conventional protein secretion (CPS) pathway, while XTH29 moves towards the apoplast following an unconventional protein secretion (UPS) mediated by exocyst-positive organelles (EXPOs). All XTHs share a common C-terminal functional domain (XET-C) that, for XTH29 and a restricted number of other XTHs (27, 28, 30), continues with an extra-terminal region (ETR) of 45 amino acids. We suggest that this region is necessary for the correct cell wall targeting of XTH29 as the ETR-truncated protein never reaches its final destination nor is recruited by EXPOs. Furthermore, RT-qPCR-analyses performed on 4-week-old Arabidopsis seedlings exposed to drought and heat stress, suggest a different involvement of the three XTHs in cell wall remodeling under abiotic stress, evidencing stress-, organ- and time-dependent variations in the expression levels. Significantly, XTH29, codifying for the only XTH following an UPS pathway, is highly up-regulated with respect to XTH11 and XTH33 codifying for CPS secreted proteins.
... Pavement cell are tightly packed in plant epidermis, with many lobes (Cosgrove, 2018;Cosgrove and Anderson, 2020). The lobes formation would be related to the dynamics of the cytoskeleton (Panteris and Galatis, 2005;Cosgrove and Anderson, 2020). ...
IQ67-domain (IQD) proteins, first identified in Arabidopsis and rice, are plant-specific calmodulin-binding proteins containing highly conserved motifs. They play a critical role in plant defenses, organ development and shape, and drought tolerance. Driven by comprehensive genome identification and analysis efforts, IQDs have now been characterized in several species and have been shown to act as microtubule-associated proteins, participating in microtubule-related signaling pathways. However, the precise molecular mechanisms underpinning their biological functions remain incompletely understood. Here we review current knowledge on how IQD family members are thought to regulate plant growth and development by affecting microtubule dynamics or participating in microtubule-related signaling pathways in different plant species and propose some new insights.
... Briefly, cell wall can be classified into primary (composed primarily by polysaccharides such as cellulose, hemicelluloses, and pectins) and secondary cell wall (composed primarily by cellulose, hemicellulose, and lignin) (Baba 2006;Somerville et al. 2004;McCahill and Hazen 2019). While in the primary wall is deposited during cell growth, it must be mechanically stable and flexible enough to allow the cells to expand, preventing their rupture; in the secondary, deposition occurs after cell growth ceases and provides mechanical stability to the plant (Cosgrove 2005(Cosgrove , 2014(Cosgrove , 2018a. Hence, cell walls are fundamental to plant growth and survival (Fry 2001;McCann and Knox 2011). ...
Pfaffia glomerata, popularly known as Brazilian-ginseng, stands out as a species of medicinal interest that has a high photoautotrophic potential for in vitro cultivation. This study aimed to analyze cell wall components of P. glomerata during in vitro cultivation in a CO2-enriched atmosphere. For this, P. glomerata plants were grown in MS medium without sucrose, in acrylic chambers with continuous forced air ventilation at 400 and 1000 μL L−1 CO2, and a control treatment with flasks put outside the chambers, without forced ventilation. The experiment was evaluated at 20, 30 and 40 days of cultivation, totaling nine treatments in a 3 × 3 factorial scheme (CO2 concentration × days), with 4 replications. Analyses of growth, photosynthesis and cell wall immunohistochemistry (using monoclonal antibodies JIM7, JIM13 and LM10) were done. The CO2 enrichment at the concentration of 1000 μL L−1 induced greater growth and accumulation of dry mass, in addition to increasing the photosynthetic rate. Immunohistochemistry analyses showed that the presence of homogalacturonan pectins detected by the JIM7 antibody decreased from 20 to 40 days, regardless of CO2 treatment. The deposition of heteroxylan and the JIM13 AGP epitope was detected exclusively in the secondary wall regions, with higher intensity in the treatment of 1000 µL L−1 CO2. This work opens new perspectives to understanding the dynamics between photoautotrophy and cell wall deposition in P. glomerata.
... CW can be described as a composite material made of cellulose microfibrils (CMFs) tethered by hemicelluloses and embedded within a pectin matrix gel and structural proteins. In this viscoelastic material, CMFs are considered as the main load-bearing components [25][26][27] . This model has been refined recently -hemicelluloses called xyloglucans can generate hybrid molecules between CMFs and hemicelluloses, leading to the formation of 'mechanical hotspots' 28 ( Figure 1), with limited access for wall-modifying enzymes, and where wall mechanics and extensibility are controlled. ...
Plants produce organs of various shapes and sizes. While much has been learned about genetic regulation of organogenesis, the integration of mechanics in the process is also gaining attention. Here, we consider the role of forces as instructive signals in organ morphogenesis. Turgor pressure is the primary cause of mechanical signals in developing organs. Because plant cells are glued to each other, mechanical signals act, in essence, at multiple scales, through cell wall contiguity and water flux. In turn, cells use such signals to resist mechanical stress, for instance, by reinforcing their cell walls. We show that the three elemental shapes behind plant organs — spheres, cylinders and lamina — can be actively maintained by such a mechanical feedback. Combinations of this 3-letter alphabet can generate more complex shapes. Furthermore, mechanical conflicts emerge at the boundary between domains exhibiting different growth rates or directions. These secondary mechanical signals contribute to three other organ shape features — folds, shape reproducibility and growth arrest. The further integration of mechanical signals with the molecular network offers many fruitful prospects for the scientific community, including the role of proprioception in organ shape robustness or the definition of cell and organ identities as a result of an interplay between biochemical and mechanical signals.
... www.nature.com/scientificreports/ Without ruling out matrix shear, it seems worthwhile to look at the possibility that some of the attachment points comprise direct contact between two microfbrils 53 , contacts capable of sliding under shear stress or breaking and re-forming in a different place 17 as has been suggested for primary cell walls 54 . The hydrated noncellulosic polymers might then have a role in modulating such direct microfibril-microfibril contacts: that is, in keeping microfibrils apart rather than joining them together. ...
Conifer wood is an exceptionally stiff and strong material when its cellulose microfibrils are well aligned. However, it is not well understood how the polymer components cellulose, hemicelluloses and lignin co-operate to resist tensile stress in wood. From X-ray scattering, neutron scattering and spectroscopic data, collected under tension and processed by novel methods, the ordered, disordered and hemicellulose-coated cellulose components comprising each microfibril were shown to stretch together and demonstrated concerted, viscous stress relaxation facilitated by water. Different cellulose microfibrils did not all stretch to the same degree. Attempts were made to distinguish between microfibrils showing large and small elongation but these domains were shown to be similar with respect to orientation, crystalline disorder, hydration and the presence of bound xylan. These observations are consistent with a major stress transfer process between microfibrils being shear at interfaces in direct, hydrogen-bonded contact, as demonstrated by small-angle neutron scattering. If stress were transmitted between microfibrils by bridging hemicelluloses these might have been expected to show divergent stretching and relaxation behaviour, which was not observed. However lignin and hemicellulosic glucomannans may contribute to stress transfer on a larger length scale between microfibril bundles (macrofibrils).
... The cell wall is a thin layer composed of many polysaccharides (cellulose, pectins, hemicellulose) and structural proteins with variable chemistry and composition, all of which are ultimately regulated by genes. The mechanical properties of the cell wall differ according to direction (in-plane, perpendicular to the plane) and may vary in space and in time, according to cell type and/or to cell status [26]. The relevance to growth of these properties is unclear, as they are measured at timescales much shorter than those of growth. ...
How interactions between mechanics and biochemistry lead to the emergence of complex cell and tissue organization is an old question that has recently attracted renewed interest from biologists, physicists, mathematicians and computer scientists. Rapid advances in optical physics, microscopy and computational image analysis have greatly enhanced our ability to observe and quantify spatiotemporal patterns of signalling, force generation, deformation and flow in living cells and tissues. Powerful new tools for genetic, biophysical and optogenetic manipulation are allowing us to perturb the underlying machinery that generates these patterns in increasingly sophisticated ways. Rapid advances in theory and computing have made it possible to construct predictive models for how cell and tissue organization and dynamics emerge from the local coupling of biochemistry and mechanics. Together, these advances have opened up a wealth of new opportunities to explore how mechanochemical patterning shapes organismal development. In this Roadmap, we present a series of forward-looking case studies on mechanochemical patterning in development, written by scientists working at the interface of the physical and biological sciences, and covering a wide range of spatial and temporal scales, organisms and modes of development. Together, these contributions highlight the many ways in which dynamic coupling of mechanics and biochemistry shape biological dynamics: from mechanoenzymes that sense force to tune their activity and motor output, to collectives of cells in tissues that flow and redistribute biochemical signals during development.
Plant cells and organs grow into a remarkable diversity of shapes, as directed by cell walls composed primarily of polysaccharides such as cellulose and multiple structurally distinct pectins. The properties of the cell wall that allow for precise control of morphogenesis are distinct from those of the individual polysaccharide components. For example, cellulose, the primary determinant of cell morphology, is a chiral macromolecule that can self-assemble in vitro into larger-scale structures of consistent chirality, and yet most plant cells do not display consistent chirality in their growth. One interesting exception is the Arabidopsis thaliana rhm1 mutant, which has decreased levels of the pectin rhamnogalacturonan-I and causes conical petal epidermal cells to grow with a left-handed helical twist. Here, we show that in rhm1 the cellulose is bundled into large macrofibrils, unlike the evenly distributed microfibrils of the wild type. This cellulose bundling becomes increasingly severe over time, consistent with cellulose being synthesized normally and then self-associating into macrofibrils. We also show that in the wild type, cellulose is oriented transversely, whereas in rhm1 mutants, the cellulose forms right-handed helices that can account for the helical morphology of the petal cells. Our results indicate that when the composition of pectin is altered, cellulose can form cellular-scale chiral structures in vivo, analogous to the helicoids formed in vitro by cellulose nano-crystals. We propose that an important emergent property of the interplay between rhamnogalacturonan-I and cellulose is to permit the assembly of nonbundled cellulose structures, providing plants flexibility to orient cellulose and direct morphogenesis.
Life evolved in the presence of reactive oxygen species (ROS) which resulted in an early integration of ROS signals and properties for cellular physiology. Originally ROS were viewed merely as metabolic by-products, but now they are viewed as conserved regulators of physiological and developmental processes throughout all kingdoms of life. Plants monitor constantly the fluctuating environmental conditions in which they grow, for which ROS signaling is essential. However, in recent years it has become clear that plants also use ROS homeostasis to guide their development. Here, an overview is presented for the role of ROS gradients and homeostasis in the development of leaves from the shoot apical meristem based mainly on the model plant species Arabidopsis thaliana. Furthermore, this chapter summarizes current knowledge on the involvement of different ROS species and molecular components in the regulation of leaf development. Although our current understanding of how plants employ ROS to guide their development is far from complete, it is clear that ROS is utilized during many developmental phases of leaf growth.
Cellulose microfibrils (CMFs) in plant cell walls are a major load-bearing component in plant primary cell walls, and their collective orientational alignment is known to be a key factor to determine the mechanical properties of the cell wall. Plant epidermis has been widely used as a model system for the primary cell wall to study the cellulose structure and tissue mechanics because of its ease of access for characterization. However, the structural information of CMFs in epidermal walls and their mechanics have often been interpreted assuming that CMFs are uniformly distributed in the whole tissue. Here, we report distinct CMF assembly patterns in the flat face region of the epidermal cell and the edge region of the cell where two cells meet. The vibrational sum frequency generation (SFG) imaging analysis found that the CMF orientation in the cell edges is preferentially aligned perpendicular to the anticlinal walls. Finite element analysis (FEA) was employed to test if the cell geometry and the discovered inhomogeneous CMF assemblies could explain the previously observed anisotropic mechanical properties of epidermal cell walls. Our study resolves discrepancies in microfibril structure obtained with different techniques and suggests that the distinct CMF assemblies in the edge region may contribute to tissue-level mechanical anisotropy of epidermal cell walls.
A growth spin model is proposed for the modelling of the expansive growth and fibre remodelling of cell wall. The introduction of the growth spin allows to relax the perfectly bonding assumption in the kinematical growth, which provides a more sophisticated and flexible kinematical description for modelling the selective control and flexible regulation of anisotropic growth. Extended hardening laws are proposed for the growth and remodelling, respectively, aiming at further clarification of the dynamic balance between hardening and softening in a growing cell wall. The proposed model may shed a new light into the micro-structural interpretation of the “fictitious” intermediate (growth) configuration in the kinematical growth modelling of soft matter. A case study of the cell wall as a growing cylindrical wall is presented to demonstrate the proposed model. Spencer’s deviatoric stress tensor and its rate format are shown to play a key role in the modelling of the cell wall as a fibre-reinforced soft tissue.
Cell wall ultrastructure has previously been assessed by thin-section transmission electron microscopy and by surface-based methods, such as atomic force microscopy. A new study uses electron tomography to image cellulose and pectin organization deep inside a thick epidermal cell wall.
NMR spectroscopy has been applied to cells and tissues analysis since its beginnings, as early as 1950. We have attempted to gather here in a didactic fashion the broad diversity of data and ideas that emerged from NMR investigations on living cells. Covering a large proportion of the periodic table, NMR spectroscopy permits scrutiny of a great variety of atomic nuclei in all living organisms non-invasively. It has thus provided quantitative information on cellular atoms and their chemical environment, dynamics, or interactions. We will show that NMR studies have generated valuable knowledge on a vast array of cellular molecules and events, from water, salts, metabolites, cell walls, proteins, nucleic acids, drugs and drug targets, to pH, redox equilibria and chemical reactions. The characterization of such a multitude of objects at the atomic scale has thus shaped our mental representation of cellular life at multiple levels, together with major techniques like mass-spectrometry or microscopies.
NMR studies on cells has accompanied the developments of MRI and metabolomics, and various subfields have flourished, coined with appealing names: fluxomics, foodomics, MRI and MRS (i.e. imaging and localized spectroscopy of living tissues, respectively), whole-cell NMR, on-cell ligand-based NMR, systems NMR, cellular structural biology, in-cell NMR… All these have not grown separately, but rather by reinforcing each other like a braided trunk. Hence, we try here to provide an analytical account of a large ensemble of intricately linked approaches, whose integration has been and will be key to their success.
We present extensive overviews, firstly on the various types of information provided by NMR in a cellular environment (the “why”, oriented towards a broad readership), and secondly on the employed NMR techniques and setups (the “how”, where we discuss the past, current and future methods). Each subsection is constructed as a historical anthology, showing how the intrinsic properties of NMR spectroscopy and its developments structured the accessible knowledge on cellular phenomena. Using this systematic approach, we sought i) to make this review accessible to the broadest audience and ii) to highlight some early techniques that may find renewed interest. Finally, we present a brief discussion on what may be potential and desirable developments in the context of integrative studies in biology.
The plant cell wall, a dynamic and complex structure surrounding every plant cell, serves not only as a passive structural barrier but also as an active monitoring system for protection against biotic and abiotic stresses. Plants monitor and maintain the status of their cell wall integrity (CWI) through the perception of signal molecules derived from pathogens or the plant cell walls themselves via pattern recognition receptors (PRRs) localised on the plasma membrane. PRRs in turn trigger a cascade of responses, including cell wall remodelling, to adapt to environmental stimuli. Pectin is the most abundant and structurally complex polysaccharide in the plant cell wall. Emerging evidence has revealed new features of pectin and challenged the classic primary cell wall model, providing an opportunity to revisit the role of pectin and CWI during plant–pathogen interactions. Here we present an overview of recent findings on the relationships between pectin metabolism and cell wall‐mediated immunity along with the underlying regulatory mechanisms. We propose future studies to reveal the fine structural and dynamic changes of specific wall polymers with improved temporal‐spatial resolution for a better understanding of pectin‐mediated plant immunity at the cellular and molecular level. Finally, we conclude that manipulating pectin composition and structure using advanced molecular breeding strategies, such as CRISPR editing technologies, can be a viable approach to develop novel crop genotypes with improved disease resistance.
One hallmark of plant cells is their pecto-cellulosic cell walls. They protect cells against the environment and high turgor and mediate morphogenesis through the dynamics of their mechanical and chemical properties. The walls are a complex polysaccharidic structure. Although their biochemical composition is well known, how the different components organize in the volume of the cell wall and interact with each other is not well understood and yet is key to the wall mechanical properties. To investigate the ultrastructure of the plant cell wall, we imaged the walls of onion (Allium cepa) bulbs in a near-native state via cryo-Focused Ion Beam milling (cryo-FIB-milling) and cryo-Electron Tomography (cryo-ET). This allowed the high-resolution visualization of cellulose fibers in situ (in muro). We reveal the coexistence of dense fiber fields bathed in a reticulated matrix we termed meshing, which is more abundant at the inner surface of the cell wall. The fibers adopted a regular bimodal angular distribution at all depths in the cell wall and bundled according to their orientation, creating layers within the cell wall. Concomitantly, employing homogalacturonan (HG)-specific enzymatic digestion, we observed changes in the meshing, suggesting that it is at least in part composed of HG pectins. We propose the following model for the construction of the abaxial epidermal primary cell wall: The cell deposits successive layers of cellulose fibers at -45 degrees and +45 degrees relative to the cell long axis and secretes the surrounding HG-rich meshing proximal to the plasma membrane, which then migrates to more distal regions of the cell wall.
Plants possess an outer cell layer called the cell wall. This matrix comprises various molecules, such as polysaccharides and proteins, and serves a wide array of physiologically important functions. This structure is not static but rather flexible in response to the environment. One of the factors responsible for this plasticity is the xyloglucan endotransglucosylase/hydrolase (XTH) family, which cleaves and reconnects xyloglucan molecules. Since xyloglucan molecules have been hypothesised to tether cellulose microfibrils forming the main load-bearing network in the primary cell wall, XTHs have been thought to play a central role in cell wall loosening for plant cell expansion. However, multiple lines of recent evidence have questioned this classic model. Nevertheless, reverse genetic analyses have proven the biological importance of XTHs; therefore, a major challenge at present is to reconsider the role of XTHs in planta. Recent advances in analytical techniques have allowed for gathering rich information on the structure of the primary cell wall. Thus, the integration of accumulated knowledge in current XTH studies may offer a turning point for unveiling the precise functions of XTHs. In the present review, we redefine the biological function of the XTH family based on the recent architectural model of the cell wall. We highlight three key findings regarding this enzyme family: (1) XTHs are not strictly required for cell wall loosening during plant cell expansion but play vital roles in response to specific biotic or abiotic stresses; (2) in addition to their transglycosylase activity, the hydrolase activity of XTHs is involved in physiological benefits; and (3) XTHs can recognise a wide range of polysaccharides other than xyloglucans.
In this glossary of plant cell structures, we asked experts to summarize a present-day view of plant organelles and structures, including a discussion of outstanding questions. In the following short reviews, the authors discuss the complexities of the plant cell endomembrane system, exciting connections between organelles, novel insights into peroxisome structure and function, dynamics of mitochondria, and the mysteries that need to be unlocked from the plant cell wall. These discussions are focused through a lens of new microscopy techniques. Advanced imaging has uncovered unexpected shapes, dynamics, and intricate membrane formations. With a continued focus in the next decade, these imaging modalities coupled with functional studies are sure to begin to unravel mysteries of the plant cell.
Plant leaves display considerable variation in shape. Here, we introduce key aspects of leaf development, focusing on the morphogenetic basis of leaf shape diversity. We discuss the importance of the genetic control of the amount, duration, and direction of cellular growth for the emergence of leaf form. We highlight how the combined use of live imaging and computational frameworks can help conceptualize how regulated cellular growth is translated into different leaf shapes. In particular, we focus on the morphogenetic differences between simple and complex leaves and how carnivorous plants form three-dimensional insect traps. We discuss how evolution has shaped leaf diversity in the case of complex leaves, by tinkering with organ-wide growth and local growth repression, and in carnivorous plants, by modifying the relative growth of the lower and upper sides of the leaf primordium to create insect-digesting traps.
Natural and largely abundant macromolecules such as carbohydrates have become a center point of interest for the polymer community, mostly due to their more environment-friendly nature and excellent capacity to bind to proteins found in the plant and animal reigns alike. The binding between saccharide units and proteins is key to plethora of biological events, therefore a fundamental understanding of this mechanism could open doors towards a new age of biomedical advances. Synthetic macromolecules bearing saccharide units (i.e., glycopolymers) are of particular interest because they can be produced on a controlled fashion with tailored molecular weight, structure, functionality and even sequencing. Vast improvements have been made for the fabrication of sequence-controlled glycopolymers, thanks in part to new monomer synthesis routes and to the recent developments in controlled polymerization techniques. This review article aims at providing the reader with a comprehensive guide on the synthesis of glycomonomers as well as on polymerization techniques for the production of block-type glycopolymers.
Background
Epidermal cell walls have special structural and biological roles in the life of the plant. Typically they are multi-ply structures encrusted with waxes and cutin which protect the plant from dehydration and pathogen attack. These characteristics may also reduce chemical and enzymatic deconstruction of the wall for sugar analysis and conversion to biofuels. We have assessed the saccharide composition of the outer epidermal wall of onion scales with different analytical methods. This wall is a particularly useful model for cell wall imaging and mechanics.
Results
Epidermal walls were depolymerized by acidic methanolysis combined with 2 M trifluoracetic acid hydrolysis and the resultant sugars were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Total sugar yields based on wall dry weight were low (53%). Removal of waxes with chloroform increased the sugar yields to 73% and enzymatic digestion did not improve these yields. Analysis by gas chromatography/mass spectrometry (GC/MS) of per- O -trimethylsilyl (TMS) derivatives of the sugar methyl glycosides produced by acidic methanolysis gave a high yield for galacturonic acid (GalA) but glucose (Glc) was severely reduced. In a complementary fashion, GC/MS analysis of methyl alditols produced by permethylation gave substantial yields for glucose and other neutral sugars, but GalA was severely reduced. Analysis of the walls by ¹³ C solid-state NMR confirmed and extended these results and revealed 15% lipid content after chloroform extraction (potentially cutin and unextractable waxes).
Conclusions
Although exact values vary with the analytical method, our best estimate is that polysaccharide in the outer epidermal wall of onion scales is comprised of homogalacturonan (~50%), cellulose (~20%), galactan (~10%), xyloglucan (~10%) and smaller amounts of other polysaccharides. Low yields of specific monosaccharides by some methods may be exaggerated in epidermal walls impregnated with waxes and cutin and call for cautious interpretation of the results.
Although it is a central question in biology, how cell shape controls intracellular dynamics largely remains an open question. Here, we show that the shape of Arabidopsis pavement cells creates a stress pattern that controls microtubule orientation, which then guides cell wall reinforcement. Live- imaging, combined with modeling of cell mechanics, shows that microtubules align along the maximal tensile stress direction within the cells, and atomic force microscopy demonstrates that this leads to reinforcement of the cell wall parallel to the microtubules. This feedback loop is regulated: cell-shape derived stresses could be overridden by imposed tissue level stresses, showing how competition between subcellular and supracellular cues control microtubule behavior. Furthermore, at the microtubule level, we identified an amplification mechanism in which mechanical stress promotes the microtubule response to stress by increasing severing activity. These multiscale feedbacks likely contribute to the robustness of microtubule behavior in plant epidermis.
The shape and function of plant cells are often highly interdependent. The puzzle-shaped cells that appear in the epidermis of many plants are a striking example of a complex cell shape, however their functional benefit has remained elusive. We propose that these intricate forms provide an effective strategy to reduce mechanical stress in the cell wall of the epidermis. When tissue-level growth is isotropic, we hypothesize that lobes emerge at the cellular level to prevent formation of large isodiametric cells that would bulge under the stress produced by turgor pressure. Data from various plant organs and species support the relationship between lobes and growth isotropy, which we test with mutants where growth direction is perturbed. Using simulation models we show that a mechanism actively regulating cellular stress plausibly reproduces the development of epidermal cell shape. Together, our results suggest that mechanical stress is a key driver of cell-shape morphogenesis.
Many cell functions rely on the ability of microtubules to self-organize as complex networks. In plants, cortical microtubules are essential to determine cell shape as they guide the deposition of cellulose microfibrils, and thus control mechanical anisotropy of the cell wall. Here we analyze how, in turn, cell shape may influence microtubule behavior. Buidling upon previous models that confined microtubules to the cell surface, we introduce an agent model of microtubules enclosed in a three-dimensional volume. We show that the microtubule network has spontaneous aligned configurations that could explain many experimental observations without resorting to specific regulation. In particular, we find that the preferred cortical localization of microtubules emerges from directional persistence of the microtubules, and their interactions with each other and with the stiff wall. We also identify microtubule parameters that seem relatively insensitive to cell shape, such as length or number. In contrast, microtubule array anisotropy depends on local curvature of the cell surface and global orientation follows robustly the longest axis of the cell. Lastly, we find that geometric cues may be overcome, as network is capable of reorienting toward weak external directional cues. Altogether our simulations show that the microtubule network is a good transducer of weak external polarity, while at the same time, easily reaching stable global configurations.
There is an emerging consensus that higher plants synthesize cellulose microfibrils that initially comprise 18 chains. However, the mean number of chains per microfibril in situ is usually greater than 18, sometimes much greater. Microfibrils from woody tissues of conifers, grasses and dicotyledonous plants, and from organs like cotton hairs, all differ in detailed structure and mean diameter. Diameters increase further when aggregated microfibrils are isolated. Because surface chains differ, the tensile properties of the cellulose may be augmented by increasing microfibril diameter. Association of microfibrils with anionic polysaccharides in primary cell walls and mucilages leads to in vivo mechanisms of disaggregation that may be relevant to the preparation of nanofibrillar cellulose products. For the preparation of nanocrystalline celluloses, the key issue is the nature and axial spacing of disordered domains at which axial scission can be initiated. These disordered domains do not, as has often been suggested, take the form of large blocks occupying much of the length of the microfibril. They are more likely to be located at chain ends or at places where the microfibril has been mechanically damaged, but their structure and the reasons for their sensitivity to acid hydrolysis need better characterization.
This article is part of a discussion meeting issue ‘New horizons for cellulose nanotechnology’.
(Full-text Open Access! www.plantphysiol.org/content/176/1/41):
Plant cells come in a striking variety of different shapes. Shape formation in plant cells is controlled through modulation of the cell wall polymers and propelled by the turgor pressure. Understanding the shaping aspects of plant cells requires knowledge of the molecular players and the biophysical conditions under which they operate. Mechanical modeling has emerged as a useful tool to correlate cell wall structure, composition, and mechanics with cell and organ shape. The finite element method is a powerful numerical approach employed to solve problems in continuum mechanics. This Update critically analyzes studies that have used finite element analysis for the mechanical modeling of plant cells. Focus is on models involving single cell morphogenesis or motion. Model design, validation, and predictive power are analyzed in detail to open future avenues in the field.
The leaf epidermis is a biomechanical shell that influences the size and shape of the organ. Its morphogenesis is a multiscale process in which nanometer-scale cytoskeletal protein complexes, individual cells, and groups of cells pattern growth and define macroscopic leaf traits. Interdigitated growth of neighboring cells is an evolutionarily conserved developmental strategy. Understanding how signaling pathways and cytoskeletal proteins pattern cell walls during this form of tissue morphogenesis is an important research challenge. The cellular and molecular control of a lobed cell morphology is currently thought to involve PIN-FORMED (PIN)-type plasma membrane efflux carriers that generate subcellular auxin gradients. Auxin gradients were proposed to function across cell boundaries to encode stable offset patterns of cortical microtubules and actin filaments between adjacent cells. Many models suggest that long-lived microtubules along the anticlinal cell wall generate local cell wall heterogeneities that restrict local growth and specify the timing and location of lobe formation. Here we used Arabidopsis reverse genetics and multivariate long-term time-lapse imaging to test current cell shape control models. We found that neither PIN proteins nor microtubules along the anticlinal wall predict the patterns of lobe formation. In fields of lobing cells, anticlinal microtubules are not correlated with cell shape and are unstable at the time scales of cell expansion. Our analyses indicate that anticlinal microtubules have multiple functions in pavement cells, and that lobe initiation is likely controlled by complex interactions among cell geometry, cell wall stress patterns, and transient microtubule networks that span the anticlinal and periclinal walls.
ELife digest
Plant and animal organs come in many different shapes, from pitcher-shaped leaves and butterfly wings, to orchid flowers and the convoluted shape of the brain. Unlike pottery or sculpture, no external hand guides the formation of these biological structures; they arise on their own, through sheets of cells developing into particular three-dimensional shapes. But how does this process of self-making operate? We know that patterns of gene activity are important, because mutations that disrupt these patterns change the shape of the organ. But it is not clear how these patterns lead to sheets of cells curving and bending themselves into their characteristic three-dimensional shapes.
Plants are particularly useful tools for studying how three-dimensional organs form because, unlike animals, their cells do not slide relative to each other, which makes the analysis simpler. Rebocho et al. used a combination of computational modelling and cell analysis to study how the intricately shaped flowers of plants known as Snapdragons form. The experiments show that genes control the shape of Snapdragon flowers by causing groups of cells to grow at different rates and in different directions. This pattern of growth creates internal conflicts that are resolved by sheets of cells curving in particular ways, accounting for the three-dimensional shape.
Rebocho et al. propose that the principles of tissue conflict resolution described in this work may also underlie the development and evolution of many other plant and animal organ shapes. A future challenge is to identify the cellular mechanisms that link patterns of gene activity to the generation and resolution of conflicting cell behaviours.
DOI: http://dx.doi.org/10.7554/eLife.20156.002
The mechanisms by which organisms acquire their sizes and shapes through growth was a major focus of D'Arcy Thompson's book On Growth and Form. By applying mathematical and physical principles to a range of biological forms, Thompson achieved fresh insights, such as the notion that diverse biological shapes could be related through simple deformations of a coordinate system. However, Thompson considered genetics to lie outside the scope of his work, even though genetics was a growing discipline at the time the book was published. Here, we review how recent advances in cell, developmental, evolutionary and computational biology allow Thompson's ideas to be integrated with genes and the processes they influence to provide a deeper understanding of growth and morphogenesis. We consider how genes interact with subcellular-, cellular- and tissue-level processes in plants to yield patterns of growth that underlie the developmental and evolutionary shape transformations Thompson so eloquently described.
Xyloglucan has been hypothesized to bind extensively to cellulose microfibril surfaces and to tether microfibrils into a load-bearing network, thereby playing a central role in wall mechanics and growth, but this view is challenged by newer results. Here we combined high-resolution imaging by Field-Emission Scanning Electron Microscopy (FESEM) with nanogold affinity tags and selective endoglucanase treatments to assess the spatial location and conformation of xyloglucan in onion cell walls. FESEM imaging of xyloglucanase-digested cell walls revealed an altered microfibril organization but did not yield clear evidence of xyloglucan conformations. Backscattered electron detection provided excellent detection of nanogold affinity tags in the context of wall fibrillar organization. Labeling with xyloglucan-specific CBM76 conjugated with nanogold showed that xyloglucans were associated with fibril surfaces in both extended and coiled conformations, but tethered configurations were not observed. Labeling with nanogold-conjugated CBM3, which binds the hydrophobic surface of crystalline cellulose, was infrequent until the wall was predigested with xyloglucanase, whereupon microfibril labeling was extensive. When tamarind xyloglucan was allowed to bind to xyloglucan-depleted onion walls, CBM76 labeling gave positive evidence for xyloglucans in both extended and coiled conformations, yet xyloglucan chains were not directly visible by FESEM. These results indicate that an appreciable, but still small, surface of cellulose microfibrils in the onion wall is tightly bound with extended xyloglucan chains and that some of the xyloglucan has a coiled conformation.
The matrix polysaccharides of plant cell walls are diverse and variable sets of polymers influencing cell wall, tissue and organ properties. Focusing on the relatively simple parenchyma tissues of four fruits – tomato, aubergine, strawberry and apple - we have dissected cell wall matrix polysaccharide contents using sequential solubilisation and antibody-based approaches with a focus on pectic homogalacturonan (HG) and rhamnogalacturonan-I (RG-I). Epitope detection in association with anion-exchange chromatography analysis indicates that in all cases solubilized polymers include spectra of HG molecules with unesterified regions that are separable from methylesterified HG domains. In highly soluble fractions, RG-I domains exist in both HG-associated and non-HG-associated forms. Soluble xyloglucan and pectin-associated xyloglucan components were detected in all fruits. Aubergine glycans contain abundant heteroxylan epitopes, some of which are associated with both pectin and xyloglucan. These profiles of polysaccharide heterogeneity provide a basis for future studies of more complex cell and tissue systems.
The doublet C4 peaks at ~ 85 and ~ 89 ppm in solid-state ¹³C NMR spectra of native cellulose have been attributed to signals of C4 atoms on the surface (solvent-exposed) and in the interior of microfibrils, designated as sC4 and iC4, respectively. The relative intensity ratios of sC4 and iC4 observed in NMR spectra of cellulose have been used to estimate the degree of crystallinity of cellulose and the number of glucan chains in cellulose microfibrils. However, the molecular structures of cellulose responsible for the specific surface and interior C4 peaks have not been positively confirmed. Using density functional theory (DFT) methods and structures produced from classical molecular dynamics simulations, we investigated how the following four factors affect ¹³C NMR chemical shifts in cellulose: conformations of exocyclic groups at C6 (tg, gt and gg), H2O molecules H-bonded on the surface of the microfibril, glycosidic bond angles (Φ, Ψ) and the distances between H4 and HO3 atoms. We focus on changes in the δ¹³C4 value because it is the most significant observable for the same C atom within the cellulose structure. DFT results indicate that different conformations of the exocyclic groups at C6 have the greatest influence on δ¹³C4 peak separation, while the other three factors have secondary effects that increase the spread of the calculated C4 interior and surface peaks.
At early stages of Arabidopsis flowering, the inflorescence stem undergoes rapid growth, with elongation occurring predominantly in the apical ~4 cm of the stem. We measured the spatial gradients for elongation rate, osmotic pressure, cell wall thickness and wall mechanical compliances, and coupled these macroscopic measurements with molecular-level characterization of the polysaccharide composition, mobility, hydration and intermolecular interactions of the inflorescence cell wall using solid-state NMR spectroscopy and small-angle neutron scattering. Force-extension curves revealed a gradient, from high to low, in the plastic and elastic compliances of cell walls along the elongation zone, but plots of growth rate versus wall compliances were strikingly nonlinear. Neutron scattering curves showed only subtle changes in wall structure, including a slight increase in cellulose microfibril alignment along the growing stem. In contrast, solid-state NMR spectra showed substantial decreases in pectin amount, esterification, branching, hydration, and mobility in an apical-to-basal pattern, while the cellulose content increased modestly. These results suggest that pectin structural changes are connected with increases in pectin-cellulose interaction and reductions in wall compliances along the apical-to-basal gradient in growth rate. These pectin structural changes may lessen the ability of the cell wall to undergo stress relaxation and irreversible expansion (e.g. induced by expansins), thus contributing to the growth kinematics of the growing stem.
Xylan and cellulose are abundant polysaccharides in vascular plants and essential for secondary cell wall strength. Acetate or glucuronic acid decorations are exclusively found on even-numbered residues in most of the glucuronoxylan polymer. It has been proposed that this even-specific positioning of the decorations might permit docking of xylan onto the hydrophilic face of a cellulose microfibril (1-3) . Consequently, xylan adopts a flattened ribbon-like twofold screw conformation when bound to cellulose in the cell wall (4) . Here we show that ESKIMO1/XOAT1/TBL29, a xylan-specific O-acetyltransferase, is necessary for generation of the even pattern of acetyl esters on xylan in Arabidopsis. The reduced acetylation in the esk1 mutant deregulates the position-specific activity of the xylan glucuronosyltransferase GUX1, and so the even pattern of glucuronic acid on the xylan is lost. Solid-state NMR of intact cell walls shows that, without the even-patterned xylan decorations, xylan does not interact normally with cellulose fibrils. We conclude that the even pattern of xylan substitutions seen across vascular plants enables the interaction of xylan with hydrophilic faces of cellulose fibrils, and is essential for development of normal plant secondary cell walls.Plant cell wall consists of multiple components and complex structure. Here, ssNMR was used to investigate the physical interactions between two principle cell wall components, cellulose and xylan, and demonstrate the mechanism for their interactions.
Interactions of water with cellulose are of both fundamental and technological importance. Here, we characterize the properties of water associated with cellulose using deuterium labeling, neutron scattering and molecular dynamics simulation. Quasi-elastic neutron scattering provided quantitative details about the dynamical relaxation processes that occur and was supported by structural characterization using small-angle neutron scattering and X-ray diffraction. We can unambiguously detect two populations of water associated with cellulose. The first is “non-freezing bound” water that gradually becomes mobile with increasing temperature and can be related to surface water. The second population is consistent with confined water that abruptly becomes mobile at ~260 K, and can be attributed to water that accumulates in the narrow spaces between the microfibrils. Quantitative analysis of the QENS data showed that, at 250 K, the water diffusion coefficient was 0.85 ± 0.04 × 10⁻¹⁰ m²sec⁻¹ and increased to 1.77 ± 0.09 × 10⁻¹⁰ m²sec⁻¹ at 265 K. MD simulations are in excellent agreement with the experiments and support the interpretation that water associated with cellulose exists in two dynamical populations. Our results provide clarity to previous work investigating the states of bound water and provide a new approach for probing water interactions with lignocellulose materials.
The organization of polysaccharides in plant cell walls is important for the mechanics of plant cells. Spectral analysis of cell walls by polarized IR can reveal polysaccharide organization, but may be complicated by dipoles not aligned with the backbone. For instance, analysis of uniaxially-aligned cellulose Iβ film revealed that the dipole transition vector of the 1160 cm⁻¹ band involving stretch vibrations of glycosidic C1–O–C4 linkages is approximately at 30° with respect to the backbone of the cellulose chain, because of coupling with C5–O–C1 bonds in the six-membered rings. In the case of homogalacturonan, the dipole transition vector of the ester carbonyl group vibration (νC=O, 1745 cm⁻¹) is expected to be nearly normal to the homogalacturonan backbone. Using this information and the dichroism equation, the change in net orientation of cell wall polymers upon mechanical stretch was determined by polarized IR analysis. Never-dried abaxial outer epidermal cell walls of the second scale of onion bulb were mechanically stretched along longitudinal or transverse directions with respect to the long axis of the cells and then dried while under mechanical stretch. The average orientations of both 1160 and 1745 cm⁻¹ vibration transition dipoles were rotated by ~5° and ~4°, respectively, along the stretch direction from their initial random distributions upon longitudinal strain by 14%; and by ~4° and ~3°, respectively, upon transverse strain by 12%. These results imply that both cellulose microfibrils and pectins in the cell wall are passively realigned along the stretch direction by external mechanical force. The analytical methodology developed here will be useful to study how cell wall polymers might reorganize during cell wall growth and development.
The shoot apical meristem (SAM) is a small population of stem cells that continuously generates organs and tissues. We will discuss here flower formation at the SAM, which involves a complex network of regulatory genes and signalling molecules. A major downstream target of this network is the extracellular matrix or cell wall, which is a local determinant for both growth rates and growth directions. We will discuss here a number of recent studies aimed at analysing the link between cell wall structure and molecular regulation. This has involved multidisciplinary approaches including quantitative imaging, molecular genetics, computational biology and biophysics. A scenario emerges where molecular networks impact on both cell wall anisotropy and synthesis, thus causing the rapid outgrowth of organs at specific locations. More specifically, this involves two interdependent processes: the activation of wall remodelling enzymes and changes in microtubule dynamics.
This article is part of the themed issue ‘Systems morphodynamics: understanding the development of tissue hardware’.
The effect of geometry on cell stiffness measured with micro-indentation techniques has been explored in single cells, however it is unclear if results on single cells can be readily transferred to indentation experiments performed on a tissue in vivo. Here we explored this question by using simulation models of osmotic treatments and micro-indentation experiments on 3D multicellular tissues with the finite element method. We found that the cellular context does affect measured cell stiffness, and that several cells of context in each direction are required for optimal results. We applied the model to micro-indentation data obtained with cellular force microscopy on the sepal of A. thaliana, and found that differences in measured stiffness could be explained by cellular geometry, and do not necessarily indicate differences in cell wall material properties or turgor pressure.
Exploitation of plant lignocellulosic biomass is hampered by our ignorance of the molecular basis for its properties such as strength and digestibility. Xylan, the most prevalent non-cellulosic polysaccharide, binds to cellulose microfibrils. The nature of this interaction remains unclear, despite its importance. Here we show that the majority of xylan, which forms a threefold helical screw in solution, flattens into a twofold helical screw ribbon to bind intimately to cellulose microfibrils in the cell wall. ¹³C solid-state magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, supported by in silico predictions of chemical shifts, shows both two- and threefold screw xylan conformations are present in fresh Arabidopsis stems. The twofold screw xylan is spatially close to cellulose, and has similar rigidity to the cellulose microfibrils, but reverts to the threefold screw conformation in the cellulose-deficient irx3 mutant. The discovery that induced polysaccharide conformation underlies cell wall assembly provides new principles to understand biomass properties.
The wall-loosening actions of β-expansins are known primarily from studies of EXPB1 extracted from maize pollen. EXPB1 selectively loosens cell walls (CWs) of grasses, but its specific binding target is unknown. We characterized EXPB1 binding to sequentially extracted maize CWs, finding that the protein primarily binds glucuronoarabinoxylan (GAX), the major matrix polysaccharide in grass CWs. This binding is strongly reduced by salts, indicating that it is predominantly electrostatic in nature. For direct molecular evidence of EXPB1 binding, we conducted solid-state NMR experiments using paramagnetic relaxation enhancement (PRE), which is sensitive to distances between unpaired electrons and nuclei. By mixing 13C-enriched maize CWs with EXPB1 functionalized with a Mn2+ tag, we measured Mn2+-induced PRE. Strong 1H and 13C PREs were observed for the carboxyls of GAX, followed by more moderate PREs for carboxyl groups in homogalacturonan and rhamnogalacturonan-I, indicating that EXPB1 preferentially binds GAX. In contrast, no PRE was observed for cellulose, indicating very weak interaction of EXPB1 with cellulose. Dynamics experiments show that EXPB1 changes GAX mobility in a complex manner: the rigid fraction of GAX became more rigid upon EXPB1 binding while the dynamic fraction became more mobile. Combining these data with previous results, we propose that EXPB1 loosens grass CWs by disrupting noncovalent junctions between highly-substituted GAX and GAX of low substitution, which binds cellulose. This study provides the first molecular evidence of β-expansin's target in grass CWs and demonstrates a new strategy for investigating ligand binding for proteins that are difficult to express heterologously.
Significance
The control of cell division plane orientation is crucial in biology and most particularly in plants, in which cells cannot rearrange their positions, as they are glued to each other by their cell walls. Cell geometry has long been proposed to determine cell division plane orientation. Here, using statistical analysis, modeling, and live imaging in the Arabidopsis shoot apex, we show that plant cells instead divide along maximal tension.
The growing cell wall in plants has conflicting requirements to be strong enough to withstand the high tensile forces generated by cell turgor pressure while selectively yielding to those forces to induce wall stress relaxation, leading to water uptake and polymer movements underlying cell wall expansion. In this article, I review emerging concepts of plant primary cell wall structure, the nature of wall extensibility and the action of expansins, family-9 and -12 endoglucanases, family-16 xyloglucan endotransglycosylase/hydrolase (XTH), and pectin methylesterases, and offer a critical assessment of their wall-loosening activity.
In plants, the shoot apical meristem contains the stem cells and is responsible for the generation of all aerial organs. Mechanistically, organogenesis is associated with an auxin-dependent local softening of the epidermis. This has been proposed to be sufficient to trigger outgrowth, because the epidermis is thought to be under tension and stiffer than internal tissues in all the aerial part of the plant. However, this has not been directly demonstrated in the shoot apical meristem. Here we tested this hypothesis in Arabidopsis using indentation methods and modeling. We considered two possible scenarios: either the epidermis does not have unique properties and the meristem behaves as a homogeneous linearly-elastic tissue, or the epidermis is under tension and the meristem exhibits the response of a shell under pressure. Large indentation depths measurements with a large tip (~size of the meristem) were consistent with a shell-like behavior. This also allowed us to deduce a value of turgor pressure, estimated at 0.82±0.16 MPa. Indentation with atomic force microscopy provided local measurements of pressure in the epidermis, further confirming the range of values obtained from large deformations. Altogether, our data demonstrate that the Arabidopsis shoot apical meristem behaves like a shell under a MPa range pressure and support a key role for the epidermis in shaping the shoot apex.
One of the most important interactions within the paracrystalline matrix of the plant cell wall occurs between cellulose microfibrils to allow for the formation of larger diameter macrofibrils. Here, we have used computational techniques to investigate how different microfibril surfaces might adsorb onto one another. Molecular dynamics simulations show that limited direct adsorption occurs between non-polar surfaces and free energy of desorption calculations suggest this is due to a high energy barrier for the removal of a single layer of water between these surfaces. Further, it is predicted that when microfibril aggregation occurs, significant conformational changes take place at the surfaces of interaction involving O2 dihedral angles, exocyclic C6 conformation, and microfibril chain tilt. It is more likely that direct interactions initially take place between polar (110) surfaces, and that surface interactions occur between the same types of surface, such as 110 to 110, 1–10 to 1–10 or 200 to 100, where hydrogen bonds can be formed, to stabilise the aggregate. Additionally, we have identified that for the exocyclic group of a glucose residue to change conformation in origin layers, the O2 dihedral in residues before and adjacent to the glucose must rotate to a more cis-like conformation, compared to the trans-like conformation observed in crystalline cellulose. This change in exocyclic conformation occurs due to a slight shift in adjacent chains that preferentially stabilises the exocyclic conformation change in a specific glucose residue of each cellobiose repeat.
Xyloglucan constitutes most of the hemicellulose in eudicot primary cell walls and functions in cell wall structure and mechanics. Although Arabidopsis thaliana xxt1 xxt2 mutants lacking detectable xyloglucan are viable, they display growth defects that are suggestive of alterations in wall integrity. To probe the mechanisms underlying these defects, we analyzed cellulose arrangement, microtubule patterning and dynamics, microtubule- and wall integrity-related gene expression, and cellulose biosynthesis in xxt1 xxt2 plants. We found that cellulose is highly aligned in xxt1 xxt2 cell walls, that its three-dimensional distribution is altered, and that microtubule patterning and stability are aberrant in etiolated xxt1 xxt2 hypocotyls. We also found that the expression levels of microtubule-associated genes such as MAP70-5 and CLASP, and receptor genes such as HERK1 and WAK1 were changed in xxt1 xxt2 plants, and that cellulose synthase motility is reduced in xxt1 xxt2 cells, corresponding with a reduction in cellulose content. Our results indicate that loss of xyloglucan affects both the stability of the microtubule cytoskeleton and the production and patterning of cellulose in primary cell walls. These findings establish new links between wall integrity, cytoskeletal dynamics, and wall synthesis in the regulation of plant morphogenesis.