Wall Patterning Research Articles

Arabinosyl Deacetylase Modulates the Arabinoxylan Acetylation Profile and Secondary Wall Formation.

Acetylation, a prevalent modification of cell-wall polymers, is a tightly controlled regulatory process that orchestrates plant growth and environmental adaptation. However, due to limited characterization of the enzymes involved, it is unclear how plants establish and dynamically regulate the acetylation pattern in response to growth requirements. In this study, we identified a rice (Oryza sativa) GDSL esterase that deacetylates the side chain of the major rice hemicellulose, arabinoxylan. Acetyl esterases involved in arabinoxylan modification were screened using enzymatic assays combined with mass spectrometry analysis. One candidate, DEACETYLASE ON ARABINOSYL SIDECHAIN OF XYLAN1 (DARX1), is specific for arabinosyl residues. Disruption of DARX1 via Tos17 insertion and CRISPR/Cas9 approaches resulted in the accumulation of acetates on the xylan arabinosyl side chains. Recombinant DARX1 abolished the excess acetyl groups on arabinoxylan-derived oligosaccharides of the darx1 mutants in vitro. Moreover, DARX1 is localized to the Golgi apparatus. Two-dimensional 13C-13C correlation spectroscopy and atomic force microscopy further revealed that the abnormal acetylation pattern observed in darx1 interrupts arabinoxylan conformation and cellulose microfibril orientation, resulting in compromised secondary wall patterning and reduced mechanical strength. This study provides insight into the mechanism controlling the acetylation pattern on arabinoxylan side chains and suggests a strategy to breed robust elite crops.

A Rho-based reaction-diffusion system governs cell wall patterning in metaxylem vessels

Rho GTPases play crucial roles in cell polarity and pattern formation. ROPs, Rho of plant GTPases, are widely involved in cell wall patterning in plants, yet the molecular mechanism underlying their action remains unknown. Arabidopsis ROP11 is locally activated to form plasma membrane domains, which direct formation of cell wall pits in metaxylem vessel cells through interaction with cortical microtubules. Here, we show that the pattern formation of cell wall pits is governed by ROP activation via a reaction-diffusion mechanism. Genetic analysis and reconstructive assays revealed that ROPGEF4/7 and ROPGAP3/4, which encode ROP activators and inactivators, respectively, regulated the formation of ROP-activated domains; these in turn determined the pattern of cell wall pits. Mathematical modelling showed that ROP-activation cycle generated ROP domains by reaction-diffusion mechanism. The model predicted that a positive feedback and slow diffusion of ROP11-ROPGEF4 complex were required to generate ROP-activated domains. ROPGEF4 formed a dimer that interacted with activated ROP11 in vivo, which could provide positive feedback for ROP activation. ROPGEF4 was highly stable on the plasma membrane and inhibited ROP11 diffusion. Our study indicated that ROP-based reaction-diffusion system self-organizes ROP-activated domains, thereby determines the pit pattern of metaxylem vessels.

Temporal analysis of Arabidopsis genes activated by Eucalyptus grandis NAC transcription factors associated with xylem fibre and vessel development

Secondary cell wall (SCW) deposition in Arabidopsis is regulated among others by NAC transcription factors, where SND1 chiefly initiates xylem fibre differentiation while VND6 controls metaxylem vessel SCW development, especially programmed cell death and wall patterning. The translational relevance of Arabidopsis SCW regulation theory and the utility of characterized transcription factors as modular synthetic biology tools for improving commercial fibre crops is unclear. We investigated inter-lineage gene activation dynamics for potential fibre and vessel differentiation regulators from the widely grown hardwood Eucalyptus grandis (Myrtales). EgrNAC26, a VND6 homolog, and EgrNAC61, an SND1 homolog, were transiently expressed in Arabidopsis mesophyll protoplasts in parallel to determine early and late (i.e. 7 and 14 hours post-transfection) gene targets. Surprisingly, across the time series EgrNAC26 activated only a subset of SCW-related transcription factors and biosynthetic genes activated by EgrNAC61, specializing instead in targeting vessel-specific wall pit and programmed cell death markers. Promoters of EgrNAC26 and EgrNAC61 both induced reporter gene expression in vessels of young Arabidopsis plants, with EgrNAC61 also conferring xylem- and cork cambium-preferential expression in Populus. Our results demonstrate partial conservation, with notable exceptions, of SND1 and VND6 homologs in Eucalyptus and a first report of cork cambium expression for EgrNAC61.

In vivo monitoring of plant small GTPase activation using a F\xf6rster resonance energy transfer biosensor

BackgroundSmall GTPases act as molecular switches that regulate various plant responses such as disease resistance, pollen tube growth, root hair development, cell wall patterning and hormone responses. Thus, to monitor their activation status within plant cells is believed to be the key step in understanding their roles.ResultsWe have established a plant version of a Förster resonance energy transfer (FRET) probe called Ras and interacting protein chimeric unit (Raichu) that can successfully monitor activation of the rice small GTPase OsRac1 during various defence responses in cells. Here, we describe the protocol for visualizing spatiotemporal activity of plant Rac/ROP GTPase in living plant cells, transfection of rice protoplasts with Raichu-OsRac1 and acquisition of FRET images.ConclusionsOur protocol should be adaptable for monitoring activation for other plant small GTPases and protein–protein interactions for other FRET sensors in various plant cells.

Taming axial dispersion in hydrodynamic chromatography columns through wall patterning

A well-known limitation of hydrodynamic chromatography arises from the synergistic interaction between transverse diffusion and streamwise convection, which enhances axial dispersion through the Taylor-Aris mechanism. We show that a periodic sequence of slip/no-slip conditions at the channel walls (e.g., representing wall indentations hosting stable air pockets) can significantly reduce axial dispersion, thus enhancing separation performance. The theoretical/numerical analysis is based on a generalization of Brenner’s macrotransport approach to solute transport, here modified to account for the finite-size of the suspended particles. The most effective dispersion-taming outcome is observed when the alternating sequence of slip/no-slip conditions yields non-vanishing cross-sectional flow components. The combination of these components with the hindering interaction between the channel boundaries and the finite-sized particles gives rise to a non-trivial solution of Brenner’s problem on the unit periodic cell, where the cross-sectional particle number density departs from the spatially homogeneous condition. In turn, this effect impacts upon the solution of the so-called b-field defining the large-scale dispersion tensor, with an overall decremental effect on the axial dispersion coefficient and on the Height Equivalent of a Theoretical Plate.

Pressure loss in channel flow resulting from a sudden change in boundary condition from no-slip to partial-slip

A semi-analytical model is presented for pressure-driven flow through a channel, where local pressure loss is incurred at a sudden change in the boundary condition: from no-slip to partial-slip. Assuming low-Reynolds-number incompressible flow and periodic stick–slip wall patterning, the problems for parallel-plate and circular channels are solved using the methods of eigenfunction expansion and point match. The present study aims to examine in detail how the flow will evolve, on passing through the cross section at which the change in the slip condition occurs, from a no-slip parabolic profile to a less sheared profile with a boundary slip. The present problem is germane to, among other applications, flow through a channel bounded by superhydrophobic surfaces, which intrinsically comprise an array of no-slip and partial-slip segments. Results are presented to show that the sudden change in the boundary condition will result in additional resistance to the flow. Near the point on the wall where a slip change occurs is a region of steep pressure gradient and intensive vorticity. The acceleration of near-wall fluid particles in combination with the no-slip boundary condition leads to a very steep velocity gradient at the wall, thereby a sharp increase in the wall shear stress, shortly before the fluid enters the channel with a slippery wall. Results are also presented to show the development of flow in the entrance region in the slippery channel. The additional pressure loss can be represented by a dimensionless loss parameter, which is a pure function of the slip length for channels much longer than the entrance length.

Two Complementary Mechanisms Underpin Cell Wall Patterning during Xylem Vessel Development.

The evolution of the plant vasculature was essential for the emergence of terrestrial life. Xylem vessels are solute-transporting elements in the vasculature that possess secondary wall thickenings deposited in intricate patterns. Evenly dispersed microtubule (MT) bands support the formation of these wall thickenings, but how the MTs direct cell wall synthesis during this process remains largely unknown. Cellulose is the major secondary wall constituent and is synthesized by plasma membrane-localized cellulose synthases (CesAs) whose catalytic activity propels them through the membrane. We show that the protein CELLULOSE SYNTHASE INTERACTING1 (CSI1)/POM2 is necessary to align the secondary wall CesAs and MTs during the initial phase of xylem vessel development in Arabidopsis thaliana and rice (Oryza sativa). Surprisingly, these MT-driven patterns successively become imprinted and sufficient to sustain the continued progression of wall thickening in the absence of MTs and CSI1/POM2 function. Hence, two complementary principles underpin wall patterning during xylem vessel development.

Reorganization of mammalian body wall patterning with cloacal septation

Septation of the cloaca is a unique mammalian adaptation that required a novel reorganization of the perineum–the caudal portion of the trunk body wall not associated with the hindlimb. Fish, the basal vertebrates, separate ventrolateral body wall musculature of the trunk into two discrete layers, while most tetrapods expand this pattern in the thorax and abdomen into four. Mammals, the only vertebrate group to divide the cloaca into urogenital and anorectal portions, exhibit complex muscle morphology in the perineum. Here we describe how perineal morphology in a broad sample of mammals fits into patterning of trunk musculature as an extension of the four-layer ventrolateral muscular patterning of the thorax and abdomen. We show that each perineal muscle layer has a specific function related to structures formed by cloacal septation. From superficial to deep, there is the subcutaneous layer, which regulates orifice closure, the external layer, which supplements both erectile and micturition function, the internal layer, which provides primary micturition and defecation regulation, and the transversus layer, which provides structural support for pelvic organs. We elucidate how the four-layer body wall pattern, restricted to the non-mammal tetrapod thorax and abdomen, is observed in the mammalian perineum to regulate function of unique perineal structures derived from cloacal septation.

Control of secondary cell wall patterning involves xylan deacetylation by a GDSL esterase.

O-acetylation, a ubiquitous modification of cell wall polymers, has striking impacts on plant growth and biomass utilization and needs to be tightly controlled. However, the mechanisms that underpin the control of cell wall acetylation remain elusive. Here, we show a rice brittle leaf sheath1 (bs1) mutant, which contains a lesion in a Golgi-localized GDSL esterase that deacetylates the prominent hemicellulose xylan. Cell wall composition, detailed xylan structure characterization and enzyme kinetics and activity assays on acetylated sugars and xylooligosaccharides demonstrate that BS1 is an esterase that cleaves acetyl moieties from the xylan backbone at O-2 and O-3 positions of xylopyranosyl residues. BS1 thus plays an important role in the maintenance of proper acetylation level on the xylan backbone, which is crucial for secondary wall formation and patterning. Our findings outline a mechanism for how plants modulate wall acetylation and endow a plethora of uncharacterized GDSL esterases with surmisable activities.

Proteomic Analysis of Microtubule Interacting Proteins over the Course of Xylem Tracheary Element Formation in Arabidopsis

Plant vascular cells, or tracheary elements (TEs), rely on circumferential secondary cell wall thickenings to maintain sap flow. The patterns in which TE thickenings are organized vary according to the underlying microtubule bundles that guide wall deposition. To identify microtubule interacting proteins present at defined stages of TE differentiation, we exploited the synchronous differentiation of TEs in Arabidopsis thaliana suspension cultures. Quantitative proteomic analysis of microtubule pull-downs, using ratiometric (14)N/(15)N labeling, revealed 605 proteins exhibiting differential accumulation during TE differentiation. Microtubule interacting proteins associated with membrane trafficking, protein synthesis, DNA/RNA binding, and signal transduction peaked during secondary cell wall formation, while proteins associated with stress peaked when approaching TE cell death. In particular, CELLULOSE SYNTHASE-INTERACTING PROTEIN1, already associated with primary wall synthesis, was enriched during secondary cell wall formation. RNAi knockdown of genes encoding several of the identified proteins showed that secondary wall formation depends on the coordinated presence of microtubule interacting proteins with nonoverlapping functions: cell wall thickness, cell wall homogeneity, and the pattern and cortical location of the wall are dependent on different proteins. Altogether, proteins linking microtubules to a range of metabolic compartments vary specifically during TE differentiation and regulate different aspects of wall patterning.

Cortical microtubule rearrangements and cell wall patterning.

Plant cortical microtubules, which form a highly ordered array beneath the plasma membrane, play essential roles in determining cell shape and function by directing the arrangement of cellulosic and non-cellulosic compounds on the cell surface. Interphase transverse arrays of cortical microtubules self-organize through their dynamic instability and inter-microtubule interactions, and by branch-form microtubule nucleation and severing. Recent studies revealed that distinct spatial signals including ROP GTPase, cellular geometry, and mechanical stress regulate the behavior of cortical microtubules at the subcellular and supercellular levels, giving rise to dramatic rearrangements in the cortical microtubule array in response to internal and external cues. Increasing evidence indicates that negative regulators of microtubules also contribute to the rearrangement of the cortical microtubule array. In this review, I summarize recent insights into how the rearrangement of the cortical microtubule array leads to proper, flexible cell wall patterning.

Polyamines in morphogenesis and development: a promising research area in seaweeds.

Polyamines in morphogenesis and development: a promising research area in seaweeds.

ROP GTPase Regulation of Auxin Transport in Arabidopsis

Plant Rho-like small GTPases, usually called ROPs or RACs, function as crucial molecular signal switches in regulating diverse cellular and developmental processes. Once activated by external signals, ROPs interact with their downstream effector proteins, and thus relay signals to downstream targets and initiate a variety of cellular responses such as cytoskeletal organization, vesicle trafficking, secondary cell wall patterning, and cell polarization and morphogenesis (Chen and Friml, 2014). Yet, the mechanisms by which ROP-based signaling participates in regulating these processes are less well known.

Floral biology and ovule and seed ontogeny of Nymphaea thermarum, a water lily at the brink of extinction with potential as a model system for basal angiosperms.

Nymphaea thermarum is a member of the Nymphaeales, of one of the most ancient lineages of flowering plants. This species was only recently described and then declared extinct in the wild, so little is known about its reproductive biology. In general, the complete ontogeny of ovules and seeds is not well documented among species of Nymphaea and has never been studied in the subgenus Brachyceras, the clade to which N. thermarum belongs. Flowers and fruits were processed for brightfield, epifluorescence and confocal microscopy. Flower morphology, with emphasis on the timing of male and female functions, was correlated with key developmental stages of the ovule and the female gametophyte. Development of the seed tissues and dynamics of polysaccharide reserves in the endosperm, perisperm and embryo were examined. Pollen release in N. thermarum starts before the flower opens. Cell walls of the micropylar nucellus show layering of callose and cellulose in a manner reminiscent of transfer cell wall patterning. Endosperm development is ab initio cellular, with micropylar and chalazal domains that embark on distinct developmental trajectories. The surrounding maternal perisperm occupies the majority of seed volume and accumulates starch centrifugally. In mature seeds, a minute but fully developed embryo is surrounded by a single, persistent layer of endosperm. Early male and female function indicate that N. thermarum is predisposed towards self-pollination, a phenomenon that is likely to have evolved multiple times within Nymphaea. While formation of distinct micropylar and chalazal developmental domains in the endosperm, along with a copious perisperm, characterize the seeds of most members of the Nymphaeales, seed ontogenies vary between and among the constituent families. Floral biology, life history traits and small genome size make N. thermarum uniquely promising as an early-diverging angiosperm model system for genetic and molecular studies.

Xylem sap in cotton contains proteins that contribute to environmental stress response and cell wall development.

The xylem sap of a plant is primarily responsible for transporting molecules from the underground root system to the aboveground parts of the plant body. In order to understand the role that roots play in cotton growth and development, the components present in xylem sap must be elucidated. In this study, we used a shotgun HPLC-ESI-MS/MS proteomics approach to identify 455 peptides from the xylem sap of field-grown cotton plants at peak blooming stage. Of these peptides, 384 (84.4%) were found to be secreted proteins and 320 (70.3%) had special molecular functions. Based on Gene Ontology (GO) analysis, 348 peptides were annotated in terms of molecular function, biological process, and cellular localization, with 46.9 and 45.1% being related to catalytic activity and binding activity, respectively. Many xylem sap-containing proteins were predicted to be involved in different phases of xylem differentiation including cell wall metabolism, secondary cell wall development and patterning, and programmed cell death. The identification of starch and sucrose hydrolyzing enzymes implicated the interaction between roots and aboveground parts on the aspect of carbohydrate metabolism. Many of the proteins identified in this study are involved in defense mechanisms including pathogen-related proteins, such as peroxidases, chitinases, and germin-like proteins, proteases involved in disease resistance, and phytoalexin phenylpropanoid synthesis-related proteins. The majority of identified signaling proteins were fasciclin-like arabinogalactan proteins and kinases. The results of this study provide useful insight into the communication mechanisms between cotton roots and the rest of the cotton plant.

Emerging roles of small GTPases in secondary cell wall development.

Regulation of plant cell wall deposition and patterning is essential for the normal growth and development of plants. Small GTPases play pivotal roles in the modulation of primary cell wall formation by controlling cytoskeletal organization and membrane trafficking. However, the functions of small GTPases in secondary cell wall development are poorly understood. Recent studies on xylem cells revealed that the Rho of plants (ROP) group of small GTPases critically participates in the spatial patterning of secondary cell walls. In differentiating xylem cells, a specific GTPase-activating protein (GAP)/guanine nucleotide exchange factor (GEF) pair facilitates local activation of ROP11 to establish de novo plasma membrane domains. The activated ROP11 then recruits a microtubule-associated protein, MIDD1, to mediate the mutual inhibition between cortical microtubules and active ROP. Furthermore, recent works suggest that certain small GTPases, including ROP and Rab GTPases, regulate membrane trafficking to establish secondary cell wall deposition and patterning. Accordingly, this mini-review assesses and summarizes the current literature regarding the emerging functions of small GTPases in the development of secondary cell walls.

ABORTED MICROSPORES Acts as a Master Regulator of Pollen Wall Formation in Arabidopsis.

Mature pollen is covered by durable cell walls, principally composed of sporopollenin, an evolutionary conserved, highly resilient, but not fully characterized, biopolymer of aliphatic and aromatic components. Here, we report that ABORTED MICROSPORES (AMS) acts as a master regulator coordinating pollen wall development and sporopollenin biosynthesis in Arabidopsis thaliana. Genome-wide coexpression analysis revealed 98 candidate genes with specific expression in the anther and 70 that showed reduced expression in ams. Among these 70 members, we showed that AMS can directly regulate 23 genes implicated in callose dissociation, fatty acids elongation, formation of phenolic compounds, and lipidic transport putatively involved in sporopollenin precursor synthesis. Consistently, ams mutants showed defective microspore release, a lack of sporopollenin deposition, and a dramatic reduction in total phenolic compounds and cutin monomers. The functional importance of the AMS pathway was further demonstrated by the observation of impaired pollen wall architecture in plant lines with reduced expression of several AMS targets: the abundant pollen coat protein extracellular lipases (EXL5 and EXL6), and CYP98A8 and CYP98A9, which are enzymes required for the production of phenolic precursors. These findings demonstrate the central role of AMS in coordinating sporopollenin biosynthesis and the secretion of materials for pollen wall patterning.

Comparative genomic analysis of the R2R3 MYB secondary cell wall regulators of Arabidopsis, poplar, rice, maize, and switchgrass

BackgroundR2R3 MYB proteins constitute one of the largest plant transcription factor clades and regulate diverse plant-specific processes. Several R2R3 MYB proteins act as regulators of secondary cell wall (SCW) biosynthesis in Arabidopsis thaliana (At), a dicotyledenous plant. Relatively few studies have examined SCW R2R3 MYB function in grasses, which may have diverged from dicots in terms of SCW regulatory mechanisms, as they have in cell wall composition and patterning. Understanding cell wall regulation is especially important for improving lignocellulosic bioenergy crops, such as switchgrass.ResultsHere, we describe the results of applying phylogenic, OrthoMCL, and sequence identity analyses to classify the R2R3 MYB family proteins from the annotated proteomes of Arabidposis, poplar, rice, maize and the initial genome (v0.0) and translated transcriptome of switchgrass (Panicum virgatum). We find that the R2R3 MYB proteins of the five species fall into 48 subgroups, including three dicot-specific, six grass-specific, and two panicoid grass-expanded subgroups. We observe four classes of phylogenetic relationships within the subgroups of known SCW-regulating MYB proteins between Arabidopsis and rice, ranging from likely one-to-one orthology (for AtMYB26, AtMYB103, AtMYB69) to no homologs identifiable (for AtMYB75). Microarray data for putative switchgrass SCW MYBs indicate that many maintain similar expression patterns with the Arabidopsis SCW regulators. However, some of the switchgrass-expanded candidate SCW MYBs exhibit differences in gene expression patterns among paralogs, consistent with subfunctionalization. Furthermore, some switchgrass representatives of grass-expanded clades have gene expression patterns consistent with regulating SCW development.ConclusionsOur analysis suggests that no single comparative genomics tool is able to provide a complete picture of the R2R3 MYB protein family without leaving ambiguities, and establishing likely false-negative and -positive relationships, but that used together a relatively clear view emerges. Generally, we find that most R2R3 MYBs that regulate SCW in Arabidopsis are likely conserved in the grasses. This comparative analysis of the R2R3 MYB family will facilitate transfer of understanding of regulatory mechanisms among species and enable control of SCW biosynthesis in switchgrass toward improving its biomass quality.

Mobility tensor of a sphere moving on a superhydrophobic wall: application to particle separation

The paper addresses the hydrodynamic behav- ior of a sphere close to a micropatterned superhydrophobic surface described in terms of alternated no-slip and perfect- slip stripes. Physically, the perfect-slip stripes model the parallel grooves where a large gas cushion forms between fluid and solid wall, giving rise to slippage at the gas-liquid interface. The potential of the boundary element method in dealing with mixed no-slip/perfect-slip boundary condi- tions is exploited to systematically calculate the mobility tensor for different particle-to-wall relative positions and for different particle radii. The particle hydrodynamics is characterized by a nontrivial mobility field which presents a distinct near-wall behavior where the wall patterning directly affects the particle motion. In the far field, the effects of the wall pattern can be accurately represented via an effective description in terms of a homogeneous wall with a suitably defined apparent slippage. The trajectory of the sphere under the action of an external force is also described in some detail. A ''resonant'' regime is found when the frequency of the transversal component of the force matches a characteristic crossing frequency imposed by the wall pattern. It is found that under resonance, the particle undergoes a mean transversal drift. Since the res- onance condition depends on the particle radius, the effect can in principle be used to conceive devices for particle sorting based on superhydrophobic surfaces.

AUXIN RESPONSE FACTOR17 Is Essential for Pollen Wall Pattern Formation in Arabidopsis

In angiosperms, pollen wall pattern formation is determined by primexine deposition on the microspores. Here, we show that AUXIN RESPONSE FACTOR17 (ARF17) is essential for primexine formation and pollen development in Arabidopsis (Arabidopsis thaliana). The arf17 mutant exhibited a male-sterile phenotype with normal vegetative growth. ARF17 was expressed in microsporocytes and microgametophytes from meiosis to the bicellular microspore stage. Transmission electron microscopy analysis showed that primexine was absent in the arf17 mutant, which leads to pollen wall-patterning defects and pollen degradation. Callose deposition was also significantly reduced in the arf17 mutant, and the expression of CALLOSE SYNTHASE5 (CalS5), the major gene for callose biosynthesis, was approximately 10% that of the wild type. Chromatin immunoprecipitation and electrophoretic mobility shift assays showed that ARF17 can directly bind to the CalS5 promoter. As indicated by the expression of DR5-driven green fluorescent protein, which is an synthetic auxin response reporter, auxin signaling appeared to be specifically impaired in arf17 anthers. Taken together, our results suggest that ARF17 is essential for pollen wall patterning in Arabidopsis by modulating primexine formation at least partially through direct regulation of CalS5 gene expression.