Wall Architecture Research Articles

Ex-chitin-g news on drug-induced fungal epitope unmasking.

The microbial cell wall is an essential cellular organelle commonly targeted by antimicrobials. It is also a battleground of innate immune recognition where microbes can evade immune recognition by masking essential cell wall components. A recent study (A. S. Wagner, S. W. Lumsdaine, M. M. Mangrum, and T. B. Reynolds, mBio https://doi.org/10.1128/mbio.00074-23, 2023) provides insight into how echinocandin antifungals cause exposure of proinflammatory β(1,3)-glucan by driving excess chitin production in the weakened cell wall. Although many environmental and biological activities perturb cell wall integrity and regulate β(1,3)-glucan exposure, we still know little about which intracellular signaling components regulate the cell wall changes that result in disrupted cell wall architecture. Wagner et al. showed that calcineurin and the Mkc1p kinase regulate chitin deposition and β(1,3)-glucan unmasking. They further identified chitin synthesis as a key driving force in cell wall structure disruption leading to epitope exposure. Their findings highlight how fungal cell wall dynamics have important implications for antifungal immunity and future drug development.

Polypeptides Targeting Paracoccidioides brasiliensis Drk1.

Considering the toxicity of conventional therapeutic approaches and the importance of precise mechanistic targets, it is important to explore signaling pathways implicated in fungal pathobiology. Moreover, treatment of paracoccidioidomycosis, a systemic mycosis caused by a dimorphic fungus, requires prolonged therapeutic regimens. Among the numerous factors underpinning the establishment of Paracoccidioides spp. infection, the capacity to transition from the mycelial to the yeast form is of pivotal importance. The Drk1 protein of Paracoccidioides brasiliensis likely plays a decisive role in this morphological shift and subsequent virulence. We identified peptides with affinity for the PbDrk1 protein using the phage-display method and assessed the effects of these peptides on P. brasiliensis. The peptides were found to inhibit the phase transition of P. brasiliensis. Furthermore, a substantial proportion of these peptides prevented adhesion to pneumocytes. Although these peptides may not possess inherent antifungal properties, they can augment the effects of certain antifungal agents. Notably, the cell wall architecture of P. brasiliensis appears to be modulated by peptide intervention, resulting in a reduced abundance of glycosylated proteins and lipids. These peptides were also evaluated for their efficacy in a Galleria mellonella model and shown to contribute to enhanced larval survival rates. The role of PbDrk1, which is notably absent in mammals, should be further investigated to improve the understanding of its functional role in P. brasiliensis, which may be helpful for designing novel therapeutic modalities.

Regiospecific Cellulose Orientation and Anisotropic Mechanical Property in Plant Cell Walls.

Cellulose microfibrils (CMFs) are a major load-bearing component in plant cell walls. Thus, their structures have been studied extensively with spectroscopic and microscopic characterization methods, but the findings from these two approaches were inconsistent, which hampers the mechanistic understanding of cell wall mechanics. Here, we report the regiospecific assembly of CMFs in the periclinal wall of plant epidermal cells. Using sum frequency generation spectroscopic imaging, we found that CMFs are highly aligned in the cell edge region where two cells form a junction, whereas they are mostly isotropic on average throughout the wall thickness in the flat face region of the epidermal cell. This subcellular-level heterogeneity in the CMF alignment provided a new perspective on tissue-level anisotropy in the tensile modulus of cell wall materials. This finding also has resolved a previous contradiction between the spectroscopic and microscopic imaging studies, which paves a foundation for better understanding of the cell wall architecture, especially structure-geometry relationships.

Stomatal opening efficiency is controlled by cell wall organization in Arabidopsis thaliana.

Stomatal function in plants is regulated by the nanoscale architecture of the cell wall and turgor pressure, which together control stomatal pore size to facilitate gas exchange and photosynthesis. The mechanical properties of the cell wall and cell geometry are critical determinants of stomatal dynamics. However, the specific biomechanical functions of wall constituents, for example, cellulose and pectins, and their impact on the work required to open or close the stomatal pore are unclear. Here, we use nanoindentation in normal and lateral directions, computational modeling, and microscopic imaging of cells from the model plant Arabidopsis thaliana to investigate the precise influences of wall architecture and turgor pressure on stomatal biomechanics. This approach allows us to quantify and compare the unique anisotropic properties of guard cells with normal composition, lower cellulose content, or alterations in pectin molecular weight. Using these data to calculate the work required to open the stomata reveals that the wild type, with a circumferential-to-longitudinal modulus ratio of 3:1, is the most energy-efficient of those studied. In addition, the tested genotypes displayed similar changes in their pore size despite large differences in wall thickness and biomechanical properties. These findings imply that homeostasis in stomatal function is maintained in the face of varying wall compositions and biomechanics by tuning wall thickness.

Morphological and cellular organization of green microalgae to cope with cold stress in subarctic environment

Microalgae are one of the most widely dispersed living organisms on Earth and can be found even in extreme environments. Especially in such habitats, the microalgal cell wall plays an essential role as it is the first barrier in continuous contact with the surrounding and changing environment. In cold conditions microalgae can show changes in their morphology, also known as phenotypic plasticity which is the ability of an organism to show different phenotypes when exposed to different environmental conditions. In addition, presence or absence of algaenan is thought to be responsible for increase/decrease permeability and stiffness of the cell wall. The aim of this work was to evaluate and compare how microalgae cells can modify their cell wall components and cellular morphology under low-temperature conditions (5 °C) and differ during the exponential and stationary phases. Four microalgae species were studied: Coelastrella sp. 3–4, Chlorella vulgaris sp. 13–1, Haematococcus pluvialis, and Scenedesmus sp. B2–2, which were isolated from subarctic locations.Using a histochemical approach in conjunction with light microscopy, cell features such as size, organization and cell wall ornamentation were evaluated. Staining procedures showed changes in biochemical components such as pectins and presence or absence of exopolysaccharides and lipids. Results showed that Coelastrella cultures did not grow under low-temperature conditions. However, Chlorella vulgaris, Haematococcus pluvialis and Scenedesmus species demonstrated a slower growth rate, bigger and rounded cell-shape during cold condition. Furthermore, the latter microalgal strain also showed modification in algaenan presence as one of the main components in cell wall architecture, which can be related to the permeability of cell wall. Changes in other features such as cell organization and cell wall ornamentation were investigated.

In vitro fermentation characteristics of fruit and vegetable juicing wastes using human fecal inoculum are determined by cell wall architecture

Fruit and vegetable juicing wastes are a promising source of hydrocolloids with potential gut fermentation benefits for applications in foods. The aim of this study was to investigate the fermentation characteristics of juicing wastes from apple, beetroot, carrot, celery, orange and pineapple to characterise fermentation rates, gas production and end-products. All juicing wastes had high dietary fibre content (70–90%) and high water-holding capacity (9–12 g H2O per gram dry matter) consistent with food application potential but had diverse fermentation rates and extents over a 48h period. Apple, carrot and orange waste were well-fermented, beetroot and celery waste were less well fermented and pineapple waste was the least fermented as indicated by gas production and short chain fatty acid concentrations. Variations in fermentability were determined by cell wall architecture more than polymer chemistry. Apple, carrot and orange waste were primarily parenchymal fleshy tissue with loosely packed cell walls and readily fermentable. Beetroot and celery waste had more compact cell walls, while pineapple waste had the most densely packed cell walls and the highest cellulose content. The molecular composition of unfermented material also changed during fermentation, with pectin the fastest fermenting, followed by hemicelluloses such as (arabino)xylan, xyloglucan, and mannan while cellulose was the slowest. The results show the potential for unrefined juicing wastes to provide sustained fermentation throughout the large intestine.

The changes in the maize root cell walls after exogenous application of auxin in the presence of cadmium.

Cadmium (Cd) is a transition metal and hazardous pollutant that has many toxic effects on plants. This heavy metal poses a health risk for both humans and animals. The cell wall is the first structure of a plant cell that is in contact with Cd; therefore, it can change its composition and/or ratio of wall components accordingly. This paper investigates the changes in the anatomy and cell wall architecture of maize (Zea mays L.) roots grown for 10 days in the presence of auxin indole-3-butyric acid (IBA) and Cd. The application of IBA in the concentration 10-9 M delayed the development of apoplastic barriers, decreased the content of lignin in the cell wall, increased the content of Ca2+ and phenols, and influenced the composition of monosaccharides in polysaccharide fractions when compared to the Cd treatment. Application of IBA improved the Cd2+ fixation to the cell wall and increased the endogenous concentration of auxin depleted by Cd treatment. The proposed scheme from obtained results may explain the possible mechanisms of the exogenously applied IBA and its effects on the changes in the binding of Cd2+ within the cell wall, and on the stimulation of growth that resulted in the amelioration of Cd stress.

Extensive remodelling of the cell wall during the development of Staphylococcus aureus bacteraemia

The bloodstream represents a hostile environment that bacteria must overcome to cause bacteraemia. To understand how the major human pathogen Staphylococcus aureus manages this we have utilised a functional genomics approach to identify a number of new loci that affect the ability of the bacteria to survive exposure to serum, the critical first step in the development of bacteraemia. The expression of one of these genes, tcaA, was found to be induced upon exposure to serum, and we show that it is involved in the elaboration of a critical virulence factor, the wall teichoic acids (WTA), within the cell envelope. The activity of the TcaA protein alters the sensitivity of the bacteria to cell wall attacking agents, including antimicrobial peptides, human defence fatty acids, and several antibiotics. This protein also affects the autolytic activity and lysostaphin sensitivity of the bacteria, suggesting that in addition to changing WTA abundance in the cell envelope, it also plays a role in peptidoglycan crosslinking. With TcaA rendering the bacteria more susceptible to serum killing, while simultaneously increasing the abundance of WTA in the cell envelope, it was unclear what effect this protein may have during infection. To explore this, we examined human data and performed murine experimental infections. Collectively, our data suggests that whilst mutations in tcaA are selected for during bacteraemia, this protein positively contributes to the virulence of S. aureus through its involvement in altering the cell wall architecture of the bacteria, a process that appears to play a key role in the development of bacteraemia.

Extensive remodelling of the cell wall during the development of Staphylococcus aureus bacteraemia.

The bloodstream represents a hostile environment that bacteria must overcome to cause bacteraemia. To understand how the major human pathogen Staphylococcus aureus manages this we have utilised a functional genomics approach to identify a number of new loci that affect the ability of the bacteria to survive exposure to serum, the critical first step in the development of bacteraemia. The expression of one of these genes, tcaA, was found to be induced upon exposure to serum, and we show that it is involved in the elaboration of a critical virulence factor, the wall teichoic acids (WTA), within the cell envelope. The activity of the TcaA protein alters the sensitivity of the bacteria to cell wall attacking agents, including antimicrobial peptides, human defence fatty acids, and several antibiotics. This protein also affects the autolytic activity and lysostaphin sensitivity of the bacteria, suggesting that in addition to changing WTA abundance in the cell envelope, it also plays a role in peptidoglycan crosslinking. With TcaA rendering the bacteria more susceptible to serum killing, while simultaneously increasing the abundance of WTA in the cell envelope, it was unclear what effect this protein may have during infection. To explore this, we examined human data and performed murine experimental infections. Collectively, our data suggests that whilst mutations in tcaA are selected for during bacteraemia, this protein positively contributes to the virulence of S. aureus through its involvement in altering the cell wall architecture of the bacteria, a process that appears to play a key role in the development of bacteraemia.

Late Decrease in Cerebral Blood Flow in Bacterial Meningitis: More than a Simple Normalization of Acute Inflammatory Vessel Wall Architecture?

Acute bacterial meningitis is a disease with an overwhelmingly high mortality rate and high incidence of adverse neurological sequelae and poor neurological recovery amongst survivors. Amongst the numerous complications of bacterial meningitis, the presence of cerebrovascular disease represents a severe disease form. Vascular involvement during bacterial meningitis has long been established by numerous pathological and angiographic studies. Cerebrovascular changes known to occur in bacterial meningitis ranging from narrowing of large arteries by vasospasm to critical stenosis/obliteration of small to medium sized arteries/arterioles by vasculitis. Not surprisingly, alterations in CBF velocities have commonly been described during the inflammatory process and may represent an important component of brain injury during meningitis. In accordance with previous studies observing a biphasic cerebral flow pattern characterized by an early but transient increase in flow velocity, mostly due to reflexive vasospasm, and later by a sustained decrease in flow velocity, likely attributable to stenotic vasculitis, cerebral ischemia is a notable complication of bacterial meningitis during the advanced disease phase. Impaired cerebral perfusion during the late stages of disease may result from a variety of factors that contribute to a vital component of cerebral injury in bacterial meningitis. The pathogenesis of cerebral ischemia with progression of disease course is less clearly understood but may involve a complex interaction between inflammatory processes, systemic dysfunction, energy impairment, neuronal damage and intracranial pressure, factors of which we aim to more precisely understand and assign a more definite contributory role in the development of cerebrovascular ischemic consequences with advanced stages of bacterial meningitis.

Cell wall composition in Cryptococcus neoformans is media dependent and alters host response, inducing protective immunity.

Cryptococcus neoformans is a basidiomycete fungus that can cause meningoencephalitis, especially in immunocompromised patients. Cryptococcus grows in many different media, although little attention has been paid to the role of growth conditions on the cryptococcal cell wall or on virulence. The purpose of this study was to determine how different media influenced the amount of chitin and chitosan in the cell wall, which in turn impacted the cell wall architecture and host response. Yeast extract, peptone, and dextrose (YPD) and yeast nitrogen base (YNB) are two commonly used media for growing Cryptococcus before use in in vitro or in vivo experiments. As a result, C. neoformans was grown in either YPD or YNB, which were either left unbuffered or buffered to pH 7 with MOPS. These cells were then labeled with cell wall-specific fluorescent probes to determine the amounts of various cell wall components. In addition, these cells were employed in animal virulence studies using the murine inhalation model of infection. We observed that the growth of wild-type C. neoformans KN99 significantly changes the pH of unbuffered media during growth. It raises the pH to 8.0 when grown in unbuffered YPD but lowers the pH to 2.0 when grown in unbuffered YNB (YNB-U). Importantly, the composition of the cell wall was substantially impacted by growth in different media. Cells grown in YNB-U exhibited a 90% reduction in chitosan, the deacetylated form of chitin, compared with cells grown in YPD. The decrease in pH and chitosan in the YNB-U-grown cells was associated with a significant increase in some pathogen-associated molecular patterns on the surface of cells compared with cells grown in YPD or YNB, pH 7. This altered cell wall architecture resulted in a significant reduction in virulence when tested using a murine model of infection. Furthermore, when heat-killed cells were used as the inoculum, KN99 cells grown in YNB-U caused an aberrant hyper-inflammatory response in the lungs, resulting in rapid animal death. In contrast, heat-killed KN99 cells grown in YNB, pH 7, caused little to no inflammatory response in the host lung, but, when used as a vaccine, they conferred a robust protective response against a subsequent challenge infection with the virulent KN99 cells. These findings emphasize the importance of culture media and pH during growth in shaping the content and organization of the C. neoformans cell wall, as well as their impact on fungal virulence and the host response.

Variable Tandem Glycine-Rich Repeats Contribute to Cell Death-Inducing Activity of a Glycosylphosphatidylinositol-Anchored Cell Wall Protein That Is Associated with the Pathogenicity of Sclerotinia sclerotiorum.

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a conserved posttranslational modification in eukaryotes. GPI-anchored proteins are widely distributed in fungal plant pathogens, but the specific roles of the GPI-anchored proteins in the pathogenicity of Sclerotinia sclerotiorum, a devastating necrotrophic plant pathogen with a worldwide distribution, remain largely unknown. This research addresses SsGSR1, which encodes an S. sclerotiorum glycine- and serine-rich protein named SsGsr1 with an N-terminal secretory signal and a C-terminal GPI-anchor signal. SsGsr1 is located at the cell wall of hyphae, and deletion of SsGSR1 leads to abnormal cell wall architecture and impaired cell wall integrity of hyphae. The transcription levels of SsGSR1 were maximal in the initial stage of infection, and SsGSR1-deletion strains showed impaired virulence in multiple hosts, indicating that SsGSR1 is critical for the pathogenicity. Interestingly, SsGsr1 targeted the apoplast of host plants to induce cell death that relies on the glycine-rich 11-amino-acid repeats arranged in tandem. The homologs of SsGsr1 in Sclerotinia, Botrytis, and Monilinia species contain fewer repeat units and have lost their cell death activity. Moreover, allelic variants of SsGSR1 exist in field isolates of S. sclerotiorum from rapeseed, and one of the variants lacking one repeat unit results in a protein that exhibits loss of function relative to the cell death-inducing activity and the virulence of S. sclerotiorum. Taken together, our results demonstrate that a variation in tandem repeats provides the functional diversity of GPI-anchored cell wall protein that, in S. sclerotiorum and other necrotrophic pathogens, allows successful colonization of the host plants. IMPORTANCE Sclerotinia sclerotiorum is an economically important necrotrophic plant pathogen and mainly applies cell wall-degrading enzymes and oxalic acid to kill plant cells before colonization. In this research, we characterized a glycosylphosphatidylinositol (GPI)-anchored cell wall protein named SsGsr1, which is critical for the cell wall architecture and the pathogenicity of S. sclerotiorum. Additionally, SsGsr1 induces rapid cell death of host plants that is dependent on glycine-rich tandem repeats. Interestingly, the number of repeat units varies among homologs and alleles of SsGsr1, and such a variation creates alterations in the cell death-inducing activity and the role in pathogenicity. This work advances our understanding of the variation of tandem repeats in accelerating the evolution of a GPI-anchored cell wall protein associated with the pathogenicity of necrotrophic fungal pathogens and prepares the way toward a fuller understanding of the interaction between S. sclerotiorum and host plants.

PO-02-003 IDENTIFICATION OF AREAS OF ANISOTROPIC CONDUCTION USING OMNIPOLAR TECHNOLOGY TO PREDICT FIRST-PASS ISOLATION AND RECONNECTION OF THE POSTERIOR WALL

Posterior wall isolation (PWI) can be a useful adjunct to pulmonary vein isolation (PVI) for the management of persistent atrial fibrillation (AF). Successful isolation of the posterior wall is challenging, with first-pass isolation rates under 40%. Difficulties in achieving block may be due to inability to create transmural lesions due to complex posterior wall architecture from septopulmonary (SPB) and septoatrial (SAB) bundle insertion. Activation mapping using omnipolar technology (OT) may allow identification of areas of anisotropic conduction that represent atrial fiber and bundle insertion points to target for additional ablation. Correlate areas of anisotropy to first-pass isolation of the posterior wall and determine whether anisotropy can predict sites requiring additional ablation to achieve bidirectional block We retrospectively analyzed patients with persistent AF who underwent first-time PVI with PWI from November 2020 to December 2022 at a single tertiary referral center with using the EnSite X mapping system with OT (Abbott, IL). Demographic and procedural information was collected. The posterior wall was divided into 6 regions and areas of anisotropy, defined as an abrupt pivot or collision in OT vectors, along with mean and maximum bipolar voltage were recorded. Areas requiring additional ablation to achieve bidirectional block were recorded. 17 patients were included in our cohort, of which 5 had first-pass isolation (29%). Final isolation of the posterior wall was achieved in all cases. There were no significant differences in patient demographics. Total ablation times were significantly less in the first-pass group (2174 versus 1721 seconds, p = 0.035). Anisotropic areas were present in 80% of first-pass cases versus 75% of cases without first-pass. While overall PW voltage was similar in both groups, voltage at sites of anisotropy was significantly higher in patients without first-pass (6.8 vs 2.6, p = 0.024). In patients without first-pass, reconnection was at site of anisotropy in 50% of cases with mean bipolar voltage 7.4mV. Activation mapping using OT may help identify areas of anisotropic conduction that are common sites of epicardial connections. Targeting these areas with additional ablation may improve rates of first-pass isolation and reduce rates of chronic reconnection. Further investigation and a larger cohort is needed to validate the predictive utility of OT.

Elongated galactan side chains mediate cellulose-pectin interactions in engineered Arabidopsis secondary cell walls.

The plant secondary cell wall is a thickened matrix of polysaccharides and lignin deposited at the cessation of growth in some cells. It forms the majority of carbon in lignocellulosic biomass, and it is an abundant and renewable source for forage, fiber, materials, fuels, and bioproducts. The complex structure and arrangement of the cell wall polymers mean that the carbon is difficult to access in an economical and sustainable way. One solution is to alter the cell wall polymer structure so that it is more suited to downstream processing. However, it remains difficult to predict what the effects of this engineering will be on the assembly, architecture, and properties of the cell wall. Here, we make use of Arabidopsis plants expressing a suite of genes to increase pectic galactan chain length in the secondary cell wall. Using multi-dimensional solid-state nuclear magnetic resonance, we show that increasing galactan chain length enhances pectin-cellulose spatial contacts and increases cellulose crystallinity. We also found that the increased galactan content leads to fewer spatial contacts of cellulose with xyloglucan and the backbone of pectin. Hence, we propose that the elongated galactan side chains compete with xyloglucan and the pectic backbone for cellulose interactions. Due to the galactan topology, this may result in comparatively weak interactions and disrupt the cell wall architecture. Therefore, introduction of this strategy into trees or other bioenergy crops would benefit from cell-specific expression strategies to avoid negative effects on plant growth.

Thin wire segmentation and reconstruction based on a novel image overlap-partitioning and stitching algorithm in apple fruiting wall architecture for robotic picking

The layout of orchards usually requires the use of wires, to provide sturdy support. Such is the case of apple trees in fruiting wall architecture, where wires are conducive for mechanical harvesting and especially robotic picking. However, wires may cause damage to the robotic gripper, especially when direct picking occluded apple fruits. Hence, the importance of identifying the wires of a fruit wall architecture. In this study, a pixel-level segmentation network, BlendMask, was adopted to segment wires. The wires are thin and normally behind the branches or leaves, making difficult for their identification. Therefore, a novel data processing algorithm called image overlap-partitioning and stitching (IOS) is proposed for BlendMask to segment the wires. A total of 82 RGB (Red, Green, and Blue) images registered to create a raw dataset. The dataset was augmented and then overlap-partitioned into 12,736 images with a resolution of 800 × 1024 pixels and corresponding annotation files based on input size of BlendMask to make an overlap-partitioned dataset. Then BlendMask was trained with the overlap-partitioned dataset and tested on the other images, where additional stitching for the overlap-partitioned images was needed in the image testing dataset. Results showed that BlendMask with IOS obtained Intersection over Union (IoU) and pixel accuracy of 43.86 % and 61.01 %, respectively. It achieved a better average precision of 38.75 % with IoU of 0.5 on the overlap-partitioned dataset, which was 38.42 % higher than the full image dataset. Moreover, a reconstruction method based on feature point extraction and fitting was proposed to estimate wire skeletons, which achieved a reconstruction accuracy of 90.70 %. These results showed a promising potential using segmentation and reconstruction methods for identifying wires and thus providing a basis for robotic picking in modern orchards.

Signals and Their Perception for Remodelling, Adjustment and Repair of the Plant Cell Wall.

The integrity of the cell wall is important for plant cells. Mechanical or chemical distortions, tension, pH changes in the apoplast, disturbance of the ion homeostasis, leakage of cell compounds into the apoplastic space or breakdown of cell wall polysaccharides activate cellular responses which often occur via plasma membrane-localized receptors. Breakdown products of the cell wall polysaccharides function as damage-associated molecular patterns and derive from cellulose (cello-oligomers), hemicelluloses (mainly xyloglucans and mixed-linkage glucans as well as glucuronoarabinoglucans in Poaceae) and pectins (oligogalacturonides). In addition, several types of channels participate in mechanosensing and convert physical into chemical signals. To establish a proper response, the cell has to integrate information about apoplastic alterations and disturbance of its wall with cell-internal programs which require modifications in the wall architecture due to growth, differentiation or cell division. We summarize recent progress in pattern recognition receptors for plant-derived oligosaccharides, with a focus on malectin domain-containing receptor kinases and their crosstalk with other perception systems and intracellular signaling events.

Combining AFM, XPS and chemical hydrolysis to understand the complexity and dynamics of C. vulgaris cell wall composition and architecture

The microalgae cell wall represents its interface with its environment, and is a dynamic and complex structure that evolves depending on the growth phase or culture conditions. To apprehend this complexity, an experimental approach combining AFM, XPS, and chemical hydrolysis followed by HPAEC-PAD was developed to understand the cell wall of Chlorella vulgaris, a biotechnologically-relevant green microalgae species. Exponential and stationary growth stages were investigated, as well as saline stress condition inducing lipid production. Results showed that both the cell wall composition and architecture changes in stationary phase, with an increase of the lipidic fraction at the expense of the proteic fraction, changes in the polysaccharidic composition, and a significant increase of its rigidity. Under saline stress, cell wall architecture seems to be affected as importantly, its rigidity is reduced. Altogether, this study demonstrates the power of combining these three techniques to give new insights into C. vulgaris cell wall, in terms of composition and architecture, and of its dynamics in different conditions.

Visualization of Wall Teichoic Acid Decoration in Bacillus subtilis.

Teichoic acids are important for the maintenance of cell shape and growth in Gram-positive bacteria. Bacillus subtilis produces major and minor forms of wall teichoic acid (WTA) and lipoteichoic acid during vegetative growth. We found that newly synthesized WTA attachment to peptidoglycan occurs in a patch-like manner on the sidewall with the fluorescent labeling compound of the concanavalin A lectin. Similarly, WTA biosynthesis enzymes fused to the epitope tags were localized in similar patch-like patterns on the cylindrical part of the cell, and WTA transporter TagH was frequently colocalized with WTA polymerase TagF, WTA ligase TagT, and actin homolog MreB, respectively. Moreover, we found that the nascent cell wall patches, decorated with the newly glucosylated WTA, were colocalized with TagH and WTA ligase TagV. In the cylindrical part, the newly glucosylated WTA patchily inserted into the bottom of the cell wall layer and finally reached the outermost layer of the cell wall after approximately half an hour. Incorporation of newly glucosylated WTA was arrested with the addition of vancomycin but restored with the removal of the antibiotic. These results are consistent with the prevailing model that WTA precursors are attached to newly synthesized peptidoglycan. IMPORTANCE In Gram-positive bacteria, the cell wall is composed of mesh-like peptidoglycan and covalently linked wall teichoic acid (WTA). It is unclear where WTA decorates peptidoglycan to create a cell wall architecture. Here, we demonstrate that nascent WTA decoration occurred in a patch-like manner at the peptidoglycan synthesis sites on the cytoplasmic membrane. The incorporated cell wall with newly glucosylated WTA in the cell wall layer then reached the outermost layer of the cell wall after approximately half an hour. Incorporation of newly glucosylated WTA was arrested with the addition of vancomycin but restored with the removal of the antibiotic. These results are consistent with the prevailing model that WTA precursors are attached to newly synthesized peptidoglycan.

Cell Wall Integrity Signaling in Fruit Ripening

Plant cell walls are essential structures for plant growth and development as well as plant adaptation to environmental stresses. Thus, plants have evolved signaling mechanisms to monitor the changes in the cell wall structure, triggering compensatory changes to sustain cell wall integrity (CWI). CWI signaling can be initiated in response to environmental and developmental signals. However, while environmental stress-associated CWI signaling has been extensively studied and reviewed, less attention has been paid to CWI signaling in relation to plant growth and development under normal conditions. Fleshy fruit development and ripening is a unique process in which dramatic alternations occur in cell wall architecture. Emerging evidence suggests that CWI signaling plays a pivotal role in fruit ripening. In this review, we summarize and discuss the CWI signaling in relation to fruit ripening, which will include cell wall fragment signaling, calcium signaling, and NO signaling, as well as Receptor-Like Protein Kinase (RLKs) signaling with an emphasis on the signaling of FERONIA and THESEUS, two members of RLKs that may act as potential CWI sensors in the modulation of hormonal signal origination and transduction in fruit development and ripening.

Role of cell wall anisotropy on guard cell mechanics in Arabidopsis thaliana.

The function of plant stomata is regulated by nanoscale cellular components and turgor pressure in flanking guard cells that control stomatal pore size to facilitate gas exchange and photosynthesis. Cell wall nanostructure plays a critical role in stomatal behavior. Stomatal aperture is regulated by tuning the mechanical properties of guard cell walls and by water uptake or release by the cells. However, the specific mechanical roles of different wall components, especially in newly maturing guard cells, are unclear. Here, we used a combination of molecular genetics, nanoindentation in normal and lateral directions, computational modeling, and microscopy to investigate the influences of wall components and maturation on wall mechanics and turgor pressure in stomatal guard cells. Lateral indentation measurements allow the unique anisotropic properties of each guard cell to be quantified with respect to age and genotype, including mutants with lower cellulose content and with lower or higher pectin molecular weight. Our results highlight the significance of cellulose in modulating the dynamics of guard cells by affecting wall anisotropy and emphasize the role of pectin modification for tuning stomatal responses to environmental stimuli. Our nanoindentation measurements when combined with finite element modeling allow the wall modulus, anisotropy, and turgor pressure to be measured. We also found that both cell wall mechanics and turgor pressure undergo significant changes during stomatal maturation. The role of cell wall architecture in determining anisotropic wall mechanics is also discussed. Together, these results provide new insights of wall mechanics and their effect on stomatal function and growth in plants.