Cell Wall Growth Research Articles

ITRAQ-based proteomics screen for potential regulators of wheat (Triticum aestivum L.) root cell wall component response to Al stress

ObjectiveThe inhibition of Aluminum (Al)-induced root tip cell elongation is a major cause of plant root elongation suppression. The inhibition of root tip cell elongation is caused by a disruption of cell wall component metabolism, growth signaling, or cellular damage. The aim of this study was to identify the proteins involved in the metabolism of the root cell wall components under Al stress in the Al-tolerant wheat (Triticum aestivum L.) cultivar ET8. MethodsDifferentially expressed proteins of Al-tolerant wheat roots were screened via isobaric tags for relative and absolute quantification (iTRAQ). Furthermore, their expression changes were detected via RT-PCR analysis. The contents of major components of the cell wall and their changes in metabolic enzyme activities were also investigated. ResultsA total of 97 differentially expressed proteins from Al-tolerant wheat roots were screened and nine of these 97 proteins were root cell wall component related. The known nucleic acid sequences of proteins were 14-3-3 protein, the plasm membrane (PM) H+-ATPase, phospholipase D, peroxidase, and glycosyltransferase. For 14-3-3 protein, phospholipase D and peroxidase, the protein expression and mRNA expression were consistent with Al-treatment; however, for PM H+-ATPase and glycosyltransferase, the protein expression and mRNA expression were inconsistent under Al-stress. Furthermore, both cellulase activity and callase activity were down-regulated by Al stress, while the phenylalanineammonialyase (PAL), cinnamyl alcohol dehydrogenase (CAD), and peroxidase (POD) activities were up-regulated. Furthermore, the PM H+-ATPase activity was decreased in response to Al stress. In addition, the contents of callose, cellulose, lignin, and H2O2 varied significantly. ConclusionsThe cell wall components, relative metabolism enzymes activity, and gene expression also changed followed by protein expression changed in response to Al stress. The results suggest that Al stress leads to marked variations in metabolic enzyme activity, carbohydrate content, followed by changes of root cell components in wheat roots.

Experimental measurement of endocytosis in fungal hyphae

The present study examines the notion that polarized exocytosis in the tips of growing hyphae creates an excess of plasma membrane and thus the need for its removal by endocytosis. To measure endocytosis experimentally, we developed a photobleaching (FRAP) procedure to count endocytic events in hyphae of Neurospora crassa carrying a fluorescent tag on the actin-binding protein fimbrin (FIM-1-GFP). Given 40 nm as the average diameter of endocytic vesicles, we calculated that about 12.5% of the plasma membrane discharged in the apex becomes endocytosed in the subapex. According to our calculations, the GFP-tagged hyphae of N. crassa, measured under the constrained conditions of confocal microscopic examination, needed about 8800 vesicles/min to extend their plasma membrane or about 9800/min, if we include predicted demands for cell wall growth and extracellular secretion. Our findings support the notion that exocytosis and endocytosis operate in tandem with the latter serving as a compensatory process to remove any excess of plasma membrane generated by the intense exocytosis in the hyphal tips. Presumably, this tandem arrangement evolved to support the hallmark features of fungi namely rapid cell extension and abundant secretion of hydrolytic enzymes.

Growing cell walls show a gradient of elastic strain across their layers.

The relatively thick primary walls of epidermal and collenchyma cells often form waviness on the surface that faces the protoplast when they are released from the tensile in-plane stress that operates in situ. This waviness is a manifestation of buckling that results from the heterogeneity of the elastic strain across the wall. In this study, this heterogeneity was confirmed by the spontaneous bending of isolated wall fragments that were initially flat. We combined the empirical data on the formation of waviness in growing cell walls with computations of the buckled wall shapes. We chose cylindrical-shaped organs with a high degree of longitudinal tissue stress because in such organs the surface deformation that accompanies the removal of the stress is strongly anisotropic and leads to the formation of waviness in which wrinkles on the inner wall surface are always transverse to the organ axis. The computations showed that the strain heterogeneity results from individual or overlaid gradients of pre-stress and stiffness across the wall. The computed wall shapes depend on the assumed wall thickness and mechanical gradients. Thus, a quantitative analysis of the wall waviness that forms after stress removal can be used to assess the mechanical heterogeneity of the cell wall.

COI1-dependent jasmonate signalling affects growth, metabolite production and cell wall protein composition in arabidopsis.

Background and AimsCultured cell suspensions have been the preferred model to study the apoplast as well as to monitor metabolic and cell cycle-related changes. Previous work showed that methyl jasmonate (MeJA) inhibits leaf growth in a CORONATINE INSENSITIVE 1 (COI1)-dependent manner, with COI1 being the jasmonate (JA) receptor. Here, the effect of COI1 overexpression on the growth of stably transformed arabidopsis cell cultures is described.MethodsTime-course experiments were carried out to analyse gene expression, and protein and metabolite levels.Key ResultsBoth MeJA treatment and the overexpression of COI1 modify growth, by altering cell proliferation and expansion. DNA content as well as transcript patterns of cell cycle and cell wall remodelling markers were altered. COI1 overexpression also increases the protein levels of OLIGOGALACTURONIDE OXIDASE 1, BETA-GLUCOSIDASE/ENDOGLUCANASES and POLYGALACTURONASE INHIBITING PROTEIN2, reinforcing the role of COI1 in mediating defence responses and highlighting a link between cell wall loosening and growth regulation. Moreover, changes in the levels of the primary metabolites alanine, serine and succinic acid of MeJA-treated Arabidopsis cell cultures were observed. In addition, COI1 overexpression positively affects the availability of metabolites such as β-alanine, threonic acid, putrescine, glucose and myo-inositol, thereby providing a connection between JA-inhibited growth and stress responses.ConclusionsThis study contributes to the understanding of the regulation of growth and the production of metabolic resources by JAs and COI1. This will have important implications in dissecting the complex relationships between hormonal and cell wall signalling in plants. The work also provides tools to uncover novel mechanisms co-ordinating cell division and post-mitotic cell expansion in the absence of organ developmental control.

Transcriptomic Analysis Reveals the Effects of Microgravity on Rice Calli on Board the Chinese Spaceship Shenzhou 8

Rice calli were loaded onto the stationary and rotating platforms of a biological incubator (SIMBOX) during spaceflight and ground control experiments. The calli in the SIMBOX were fixed with RNAlater in space after a 324-h spacecraft flight, as well as on the ground at the same time point in a ground control experiment. Microgravity-responsive (MR) transcripts were identified by a comparison of the spaceflight controls (F-μg) with the 1-g ground controls (G-1g) and 1-g inflight controls (F-1g). MapMan analysis was used to classify 955 MR transcripts. These transcripts mainly belonged to the following categories: cell wall modification and metabolism, glycolysis and the tricarboxylic acid (TCA) cycle, transcription factors, protein modification and degradation, hormone metabolism and signalling, calcium regulation, receptor-like kinase activity, and transporters. Here, we focused on the effects of microgravity on the plant cell wall and discussed the relationship between the variation in the cell wall and plant cell growth under microgravity.

Instability of endosperm development in amphiploids and their parental species in the genus Avena L.

Key messageThe development of oat endosperm is modified by chromatin and nuclei elimination, intrusive growth of cell walls, and polyploidisation of cell clones. The last event is correlated with somatic crossing-over.Grass endosperm is a variable tissue in terms of its cytogenetics and development. Free-nuclear syncytium and starchy and aleurone endosperm were the main focus of the research. These were studied in oat amphiploids (4x, 6x, and 8x) and parental species (2x, 4x, and 6x). What the levels of cytogenetic disorders and developmental anomalies in species versus hybrids are, and, what the factors are determining phenotypes of both tissue components, are open questions for oats. Chromosome bridges and micronuclei are the main cytogenetic disorders showing the elimination of parts of genomes. Bridges are formed by the AT-heterochromatin-rich and -free ends of chromosomes. In the starchy tissue, various sectors are separated structurally due to the elongation or intrusive growth of aleurone cells. The development of the aleurone layer is highly disturbed locally due to the amplification of aleurone cell divisions. Changes related to their structure and metabolism occur in the aleurone cells, for example, clones of small versus large aleurone cells. Somatic crossing-over (SCO) is expressed in clones of large polyploidised cells (r = 0.80***), giving rise to new aleurone phenotypes. The multivariate description of the endosperm instability showed that endospermal disorders were more frequent in amphiploids than in the oat species. Avena strigosa and the amphiploid A. fatua × A. sterilis appeared to be extreme units in an ordination space. Nuclear DNA elimination, periclinal and multidirectional cytokineses, polyploidisation, intrusive growth, and SCO appeared to be important factors determining oat endospermal variations.

Role of antibiotic therapy in bacterial disease: A mathematical study

Pathogenic bacteria in human system mature through the bio-synthesis of protective layer known as cell wall. This bacterial cell wall growth occurs in the presence of enzyme released by it. After maturation by the cell wall formation, pathogenic bacteria become harmful for human body as they are responsible for different diseases. Antibiotics or drugs are employed for curing bacterial diseases through the inhibition of this maturation process and it occurs by the binding progression of antibiotics with the released enzyme. But nowadays, drugs or antibiotics like [Formula: see text]-lactum family (Amoxcillin) which are generally used for inhibition of bio-synthesis of cell wall become ineffective due to evolution of antibiotic resistance by the bacteria. Antibiotic resistance occurs when an antibiotic has lost its ability to effectively control or kill bacterial growth. As a result, the bacteria becomes “resistant” and continue to multiply for the generation of robust pathogenic bacteria in spite of drug administration. This is due to the release of another type of enzyme by the resistant bacteria which binds with the active antibiotic or drug making it ineffective. Hence, another type of drug (Clauvanic acid) is combined to resist the activity of drug hydrolyzing enzyme so that the initial drug can act effectively. Hence a combination of drug therapy is applied to cure the bacterial diseases successfully. We developed a mathematical model based on the bacterial enzyme and bacterial cell wall proliferation mechanism and showed how we can reduce the bacterial infection in the resistant cases with application of combination drugs (Amoxcillin and Clauvanic acid) to restore normal health. Based on the enzymatic activity and individual drug dynamics we studied the overall system under the single drug and combinational drug administration through our formulated model analysis. We also demonstrated the different dosing time interval and dosing concentration to evaluate the optimized drug administration for arresting the cell wall formation completely. Sensitivity of the different kinetic rate constant also has been performed with subject to drug hydrolyzing enzyme. Our analytical and numerical studies also confirm the efficiency of the combinational drug treatment compared to single drug treatment being more effective in drug resistant cases providing recovery from bacterial disease.

Morphology, structure and ultrastructure of staminal nectary in Lamprocapnos (Fumarioideae, Papaveraceae)

Nectaries show high a degree of variations in terms of location, morphology and structure; these characteristics are critical for systematic information. In Papaveraceae, staminal nectaries at the stamen base are only found in bisymmetric and zygomorphic flowers, but with little description on the ultra-structure of nectary available. Here we investigate structural details of flowers and nectaries of Lamprocapnos spectabilis using LM, SEM and TEM. The nectaries of L. spectabilis are initiated at the base of stamen filaments in the young bud. The oval/ball-shaped staminal nectaries are composed of four major types of tissues in general. First and outmost layers are epidermis and hypodermis with large and elliptical cells. Beneath the epidermal layers are the second type of tissue consisting of 20–30 layers of parenchyma cells which are further distinguished by two kinds of cells: smaller secretory parenchyma cells having dense cytoplasm, large nucleus and abundant cellular organelles, and larger sub-nectary parenchyma cells containing a relatively thin cytoplasm. The third type of tissue is the darkly-stained elongated cells and embedded at 8- to 12-cell-layer deep in the nectaries, and is associated with sieve tube elements and cells resembling companion cells, and they presumably belong to underdeveloped sieve tube elements or “protophloem” based on ultrastructure features such as conspicuous cell wall ingrowths and plentiful plasmodesmata. The forth type of tissue in the nectary is the simplified vascular bundle forming with only phloem. It is reasonable to suggest that secretions in the spacious intercellular spaces between secretory parenchyma cells are exuded to the exterior via micro-channel or epidermis/cuticle pathway. In conclusion, the results show that epidermis, hypodermis, secretory parenchyma, “Arber suggestive tissue (protophloem)”, and phloem act coordinately in nectar production, secretion and exudation in Lamprocapnos. The structure of the staminal nectaries are compared within Papaveraceae and with other related groups in Ranunculales.

RodZ modulates geometric localization of the bacterial actin MreB to regulate cell shape

In the rod-shaped bacterium Escherichia coli, the actin-like protein MreB localizes in a curvature-dependent manner and spatially coordinates cell-wall insertion to maintain cell shape, although the molecular mechanism by which cell width is regulated remains unknown. Here we demonstrate that the membrane protein RodZ regulates the biophysical properties of MreB and alters the spatial organization of E. coli cell-wall growth. The relative expression levels of MreB and RodZ change in a manner commensurate with variations in growth rate and cell width, and RodZ systematically alters the curvature-based localization of MreB and cell width in a concentration-dependent manner. We identify MreB mutants that alter the bending properties of MreB filaments in molecular dynamics simulations similar to RodZ binding, and show that these mutants rescue rod-like shape in the absence of RodZ alone or in combination with wild-type MreB. Thus, E. coli can control its shape and dimensions by differentially regulating RodZ and MreB to alter the patterning of cell-wall insertion, highlighting the rich regulatory landscape of cytoskeletal molecular biophysics.

Characterization of Conserved and Novel Septal Factors in Mycobacterium smegmatis.

Septation in bacteria requires coordinated regulation of cell wall biosynthesis and hydrolysis enzymes so that new septal cross-wall can be appropriately constructed without compromising the integrity of the existing cell wall. Bacteria with different modes of growth and different types of cell wall require different regulators to mediate cell growth and division processes. Mycobacteria have both a cell wall structure and a mode of growth that are distinct from well-studied model organisms and use several different regulatory mechanisms. Here, using Mycobacterium smegmatis, we identify and characterize homologs of the conserved cell division regulators FtsL and FtsB, and show that they appear to function similarly to their homologs in Escherichia coli We identify a number of previously undescribed septally localized factors which could be involved in cell wall regulation. One of these, SepIVA, has a DivIVA domain, is required for mycobacterial septation, and is localized to the septum and the intracellular membrane domain. We propose that SepIVA is a regulator of cell wall precursor enzymes that contribute to construction of the septal cross-wall, similar to the putative elongation function of the other mycobacterial DivIVA homolog, Wag31.IMPORTANCE The enzymes that build bacterial cell walls are essential for cell survival but can cause cell lysis if misregulated; thus, their regulators are also essential. The number and nature of these regulators is likely to vary in bacteria that grow in different ways. The mycobacteria are a genus that have a cell wall whose composition and construction vary greatly from those of well-studied model organisms. In this work, we identify and characterize some of the proteins that regulate the mycobacterial cell wall. We find that some of these regulators appear to be functionally conserved with their structural homologs in evolutionarily distant species such as Escherichia coli, but other proteins have critical regulatory functions that may be unique to the actinomycetes.

Structural Evolution of Gossypium hirsutum Fibers Grown under Greenhouse and Hydroponic Conditions

Cotton is the leading fiber source in the textile industry and one of the world’s most important crops. Despite its economic interest, cotton culture exerts an enormous pressure on natural resources (land and water) and has a negative impact on the environment (abuse of pesticides). Thus, alternative cotton growing methods are urged to be implemented. Recently, we have demonstrated that Gossypium hirsutum (“Upland” cotton) can be grown in a greenhouse (controlled conditions) and hydroponically. Here we report on the elucidation of the structural changes of the Gossypium hirsutum fibers during maturation grown [10, 14, 17, 20, 36 and 51 days post anthesis (dpa)] under a greenhouse and hydroponically, by means of scanning electron microscopy (SEM), Fourier transform infrared spectroscopy with attenuated total reflectance (FT-IR ATR) and thermal gravimetric analysis/differential scanning calorimetry (TGA/DSC). The transition from primary to secondary cell wall growth occurs between 17 and 20 dpa—similarly to the soil-based cultures. However, this new cotton culture offers an advantageous pesticide and soil-free all year-round closed system with efficient water use yielding standardized mature fibers with improved properties (maturity, strength, length, whiteness).

Cell Size Regulation Through Tunable Geometric Localization of the Bacterial Actin Cytoskeleton

Virtually all cells have the ability to adjust their size in response to environmental cues, yet relatively little is known about the molecular factors responsible for tuning cell size or the mechanisms through which they reconfigure cellular dimensions. In the rod-shaped bacterium Escherichia coli, the actin-like protein MreB localizes in a curvaturedependent manner and spatially coordinates cell-wall insertion to maintain cell shape across changing environments. Here, we demonstrate that the MreB-binding protein RodZ regulates the biophysical properties of MreB filaments and thereby alters the spatial patterning of cell-wall growth to modulate cell size. The relative expression levels of MreB and RodZ changed in a manner commensurate with variations in growth rate and cell width, and single-cell analyses demonstrated that RodZ systematically alters the curvature-based localization of MreB and cell width in a manner dependent on the concentration of RodZ. We identified MreB mutants that we predict using molecular dynamics simulations to alter the bending properties of MreB filaments at the molecular scale in a manner similar to RodZ binding, and showed that these mutants rescue rod-like shape in the absence of RodZ alone or in combination with wild-type MreB. Together, our results show that E. coli controls its shape and dimensions by differentially regulating RodZ and MreB expression, highlighting the rich capacity for precise coordination of cell-shape changes with environmental conditions by exploiting cytoskeletal molecular biophysics.

Crosstalk between the tricarboxylic acid cycle and peptidoglycan synthesis in Caulobacter crescentus through the homeostatic control of α-ketoglutarate.

To achieve robust replication, bacteria must integrate cellular metabolism and cell wall growth. While these two processes have been well characterized, the nature and extent of cross-regulation between them is not well understood. Here, using classical genetics, CRISPRi, metabolomics, transcriptomics and chemical complementation approaches, we show that a loss of the master regulator Hfq in Caulobacter crescentus alters central metabolism and results in cell shape defects in a nutrient-dependent manner. We demonstrate that the cell morphology phenotype in the hfq deletion mutant is attributable to a disruption of α-ketoglutarate (KG) homeostasis. In addition to serving as a key intermediate of the tricarboxylic acid (TCA) cycle, KG is a by-product of an enzymatic reaction required for the synthesis of peptidoglycan, a major component of the bacterial cell wall. Accumulation of KG in the hfq deletion mutant interferes with peptidoglycan synthesis, resulting in cell morphology defects and increased susceptibility to peptidoglycan-targeting antibiotics. This work thus reveals a direct crosstalk between the TCA cycle and cell wall morphogenesis. This crosstalk highlights the importance of metabolic homeostasis in not only ensuring adequate availability of biosynthetic precursors, but also in preventing interference with cellular processes in which these intermediates arise as by-products.

Nitrate Uptake Affects Cell Wall Synthesis and Modeling.

Nowadays, the relationship(s) about N assimilation and cell wall remodeling in plants remains generally unclear. Enzymes involved in cell wall synthesis/modification, and nitrogen transporters play a critical role in plant growth, differentiation, and response to external stimuli. In this review, a co-expression analysis of nitrate and ammonium transporters of Arabidopsis thaliana was performed in order to explore the functional connection of these proteins with cell-wall related enzymes. This approach highlighted a strict relationship between inorganic nitrogen transporters and cell wall formation, identifying a number of co-expressed remodeling enzymes. The enzymes involved in pectin and xyloglucan synthesis resulted particularly co-regulated together with nitrate carriers, suggesting a connection between nitrate assimilation and cell wall growth regulation. Major Facilitator Carriers, and one chloride channel, are similarly co-expressed with pectin lyase, pectinacetylesterase, and cellulose synthase. Contrarily, ammonium transporters show little or no connection with those genes involved in cell wall synthesis. Different aspects related to plant development, embryogenesis, and abiotic stress response will be discussed, given the importance in plant growth of cell wall synthesis and nitrate uptake. Intriguingly, the improvement of abiotic stress tolerance in crops concerns both these processes indicating the importance in sensing the environmental constraints and mediating a response. These evaluations could help to identify candidate genes for breeding purposes.

Mycelium Composites: A Review of Engineering Characteristics and Growth Kinetics

Mycelium composites comprise of networks of filamentous hyphae, utilising biological growth rather than expensive energy intensive manufacturing processes to convert low-cost organic wastes into economically viable and environmentally friendly materials. Although generally characterised as polymer grade foams and used primarily for limited packaging and construction applications, the mechanical performance of these materials varies significantly and is governed by hyphal architecture, cell wall composition, composite constituents and growth kinetics which are in turn influenced by inherent and exogenous factors. A range of potential applications have been proposed including acoustic dampers, super absorbents, paper, textiles, structural and electronic parts. Limited research, inconclusive data and the proposed applications and feasibility suggest that further investigation is warranted.

Effects of mechanical stretching on average orientation of cellulose and pectin in onion epidermis cell wall: A polarized FT-IR study

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.

RNA-Seq Analysis of Diverse Rice Genotypes to Identify the Genes Controlling Coleoptile Growth during Submerged Germination.

The rate of coleoptile elongation varies between different rice varieties that are grown under water during the germination stage. Compared to sensitive varieties, submergence-tolerant rice exhibits substantial coleoptile elongation in order to uptake oxygen (O2) from the surface and thus have a better chance to survive water stress. We conducted RNA-seq analysis in order to investigate 7-day-old shoot transcriptome dynamics in six rice genotypes that exhibit different coleoptile elongation rates under water. This enabled us to identify the genes involved in photosynthesis, lipid metabolism, glycolysis, anaerobic fermentation, hormone synthesis, cell wall growth and elongation, and to demonstrate that these genes are differentially regulated within, and between, genotypes. Further, in addition to determining how allelic variation affects anaerobic germination, we compared the expression patterns and genomic sequences of six genotypes; this enabled us to discover that some genes carry small-to-large deletions in the coding region of sensitive varieties. These structural variations may explain the absence of transcripts in the dataset, as well as the failure of sensitive variety to respond to submergence. On the basis of these results, we hypothesize that transcriptional regulation enhances coleoptile elongation. Although this is an area for future research, the outcome of this study is expected to facilitate rice breeding for direct-seeding.

Emp10 encodes a mitochondrial PPR protein that affects the cis-splicing of nad2 intron 1 and seed development in maize.

In higher plants, many mitochondrial genes contain group II-type introns that are removed from RNAs by splicing to produce mature transcripts that are then translated into functional proteins. However, the factors involved in the splicing of mitochondrial introns and their biological functions are not well understood in maize. Here, we isolated an empty pericarp 10 (emp10) mutant and identified the underlying gene by map-based cloning. Emp10 encodes a P-type mitochondria-targeted pentatricopeptide repeat (PPR) protein with 10 PPR motifs. Loss of Emp10 function results in splicing defect of the first intron of nad2, a gene encoding subunit 2 of NADH dehydrogenase (also called complex I). The emp10 mutant has undetectable activity of complex I and has arrested development of embryo and endosperm, and thus defective seeds with empty pericarp. Additionally, the basal endosperm transfer layer cells were severely affected, indicating the deficiency of cell wall ingrowths in the emp10 kernels. Moreover, the alternative respiratory pathway involving alternative oxidase was significantly induced in the emp10 mutant. These results suggest that EMP10 is specifically required for the cis-splicing of mitochondrial nad2 intron 1, embryogenesis and endosperm development in maize.

Nanoscale movements of cellulose microfibrils in primary cell walls.

The growing plant cell wall is commonly considered a fiber-reinforced structure whose strength, extensibility and anisotropy depend on the orientation of crystalline cellulose microfibrils, their bonding to the polysaccharide matrix, and matrix viscoelasticity1–4. Structural reinforcement of the wall by stiff cellulose microfibrils is central to contemporary models of plant growth, mechanics, and meristem dynamics4–12. Although passive microfibril reorientation during wall extension has been inferred from theory and from bulk measurements13–15, nm-scale movements of individual microfibrils have not been directly observed. Here we combined nm-scale imaging of wet cell walls by atomic force microscopy (AFM) with a stretching device and endoglucanase treatment that induces wall stress relaxation and creep, mimicking wall behaviors during cell growth. Microfibril movements during forced mechanical extensions differ from those during creep of the enzymatically-loosened wall. In addition to passive angular reorientation, we observed a diverse repertoire of microfibril movements that reveal the spatial scale of molecular connections between microfibrils. Our results show that wall loosening alters microfibril connectivity, enabling microfibril dynamics not seen during mechanical stretch. These insights into microfibril movements and connectivities need to be incorporated into refined models of plant cell wall structure, growth and morphogenesis.

Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae.

SummaryChitin deacetylation results in the formation of chitosan, a polymer of β1,4‐linked glucosamine. Chitosan is known to have important functions in the cell walls of a number of fungal species, but its role during hyphal growth has not yet been investigated. In this study, we have characterized the role of chitin deacetylation during vegetative hyphal growth in the filamentous phytopathogen Magnaporthe oryzae. We found that chitosan localizes to the septa and lateral cell walls of vegetative hyphae and identified 2 chitin deacetylases expressed during vegetative growth—CDA1 and CDA4. Deletion strains and fluorescent protein fusions demonstrated that CDA1 is necessary for chitin deacetylation in the septa and lateral cell walls of mature hyphae in colony interiors, whereas CDA4 deacetylates chitin in the hyphae at colony margins. However, although the Δcda1 strain was more resistant to cell wall hydrolysis, growth and pathogenic development were otherwise unaffected in the deletion strains. The role of chitosan hydrolysis was also investigated. A single gene encoding a putative chitosanase (CSN) was discovered in M. oryzae and found to be expressed during vegetative growth. However, chitosan localization, vegetative growth, and pathogenic development were unaffected in a CSN deletion strain, rendering the role of this enzyme unclear.