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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

The regulation of the cell wall by glycosylphosphatidylinositol-anchored proteins in Arabidopsis

A polysaccharides-based cell wall covers the plant cell, shaping it and protecting it from the harsh environment. Cellulose microfibrils constitute the cell wall backbone and are embedded in a matrix of pectic and hemicellulosic polysaccharides and glycoproteins. Various environmental and developmental cues can regulate the plant cell wall, and diverse glycosylphosphatidylinositol (GPI)-anchored proteins participate in these regulations. GPI is a common lipid modification on eukaryotic proteins, which covalently tethers the proteins to the membrane lipid bilayer. Catalyzed by a series of enzymic complexes, protein precursors are post-translationally modified at their hydrophobic carboxyl-terminus in the endomembrane system and anchored to the lipid bilayer through an oligosaccharidic GPI modification. Ultimately, mature proteins reach the plasma membrane via the secretory pathway facing toward the apoplast and cell wall in plants. In Arabidopsis, more than three hundred GPI-anchored proteins (GPI-APs) have been predicted, and many are reported to be involved in diverse regulations of the cell wall. In this review, we summarize GPI-APs involved in cell wall regulation. GPI-APs are proposed to act as structural components of the cell wall, organize cellulose microfibrils at the cell surface, and during cell wall integrity signaling transduction. Besides regulating protein trafficking, the GPI modification is potentially governed by a GPI shedding system that cleaves and releases the GPI-anchored proteins from the plasma membrane into the cell wall.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Matrix polysaccharides affect preferred orientation of cellulose crystals in primary cell walls

The spatial organization and interactions of constituent components influence cell growth and determine physical and chemical properties of the cell wall, including its rigidity, flexibility, and degradability. Elucidating the interactions between cell wall polysaccharides is crucial for advancing our knowledge of how cell walls are assembled and for designing approaches to efficiently break down cell walls to produce renewable energy and biomaterials. Here, we investigated the effect of defects in the biosynthesis of cell wall components on the nanoscale organization of cellulose in primary cell walls through grazing incidence wide angle X-ray scattering (GIWAXS) measurements of hypocotyls of wild type Arabidopsis thaliana and of cellulose, pectin, and xyloglucan (hemicellulose) deficient mutants. GIWAXS reveals changes in lattice spacings, coherence lengths, and relative crystalline content for cellulose between wild type and mutant plants. In addition, X-ray pole figures constructed using GIWAXS and X-ray diffraction (XRD) rocking scans quantify an emerging measure of cellulose organization, the degree of preferred orientation (texture) of cellulose crystals with respect to the cell wall plane. Comparing X-ray pole figures from pectin-deficient and xyloglucan-deficient mutants to that of wild type plants reveals that cellulose texture is disrupted in pectin-deficient mutants, but not in xyloglucan mutants. Furthermore, our results indicate that a deficiency of pectin during cell wall biosynthesis alters cellulose organization in plant cell walls.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

A core of cell wall proteins functions in wall integrity responses in Arabidopsis thaliana

Abstract Cell walls surround all plant cells, and their composition and structure are tightly regulated to maintain cellular and organismal homeostasis. In response to wall damage, the cell wall integrity (CWI) system is engaged to ameliorate effects on plant growth. Despite the central role CWI plays in plant development, our current understanding of how this system functions at the molecular level is limited. Here, we investigated the transcriptomes of etiolated seedlings of mutants of Arabidopsis thaliana with defects in three major wall polysaccharides, pectin ( quasimodo2 ), cellulose ( cellulose synthase3 je5 ), and xyloglucan ( xyloglucan xylosyltransferase1 and 2 ), to probe whether changes in the expression of cell wall‐related genes occur and are similar or different when specific wall components are reduced or missing. Many changes occurred in the transcriptomes of pectin‐ and cellulose‐deficient plants, but fewer changes occurred in the transcriptomes of xyloglucan‐deficient plants. We hypothesize that this might be because pectins interact with other wall components and/or integrity sensors, whereas cellulose forms a major load‐bearing component of the wall; defects in either appear to trigger the expression of structural proteins to maintain wall cohesion in the absence of a major polysaccharide. This core set of genes functioning in CWI in plants represents an attractive target for future genetic engineering of robust and resilient cell walls.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Grazing-incidence diffraction reveals cellulose and pectin organization in hydrated plant primary cell wall

Abstract The primary cell wall is highly hydrated in its native state, yet many structural studies have been conducted on dried samples. Here, we use grazing-incidence wide-angle X-ray scattering (GIWAXS) with a humidity chamber, which enhances scattering and the signal-to-noise ratio while keeping outer onion epidermal peels hydrated, to examine cell wall properties. GIWAXS of hydrated and dried onion reveals that the cellulose ( $$110/1\overline{1}0$$ 110 / 1 1 ¯ 0 ) lattice spacing decreases slightly upon drying, while the (200) lattice parameters are unchanged. Additionally, the ( $$110/1\overline{1}0$$ 110 / 1 1 ¯ 0 ) diffraction intensity increases relative to (200). Density functional theory models of hydrated and dry cellulose microfibrils corroborate changes in crystalline properties upon drying. GIWAXS also reveals a peak that we attribute to pectin chain aggregation. We speculate that dehydration perturbs the hydrogen bonding network within cellulose crystals and collapses the pectin network without affecting the lateral distribution of pectin chain aggregates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Current limitations of solid-state NMR in carbohydrate and cell wall research

High-resolution investigation of cell wall materials has emerged as an important application of biomolecular solid-state NMR (ssNMR). Multidimensional correlation experiments have become a standard method for obtaining sufficient spectral resolution to determine the polymorphic structure of carbohydrates and address biochemical questions regarding the supramolecular organization of cell walls. Here, using plant cellulose and matrix polysaccharides as examples, we will review how the multifaceted complexity of polysaccharide structure is impeding the resonance assignment process and assess the available biochemical and spectroscopic approaches that could circumvent this barrier. We will emphasize the ineffectiveness of the current methods in reconciling the ever-growing dataset and deriving structural information. We will evaluate the protocols for achieving efficient and homogeneous hyperpolarization across the cell wall material using magic-angle spinning dynamic nuclear polarization (MAS-DNP). Critical questions regarding the line-broadening effects of cell wall molecules at cryogenic temperature and by paramagnetic biradicals will be considered. Finally, the MAS-DNP method will be placed into a broader context with other structural characterization techniques, such as cryo-electron microscopy, to advance ssNMR research in carbohydrate and cell wall biomaterials.

09 BIOMASS FUELS↗
DOE OSTI2020

Theory and Practice in Measuring In-Vitro Extensibility of Growing Plant Cell Walls

This chapter summarizes four extensometer techniques for measuring cell wall extensibility in vitro and discusses how the results of these methods relate to the concept and ideal measurement of cell wall extensibility in the context of plant cell growth. These in-vitro techniques are particularly useful for studies of the molecular basis of cell wall extension. Measurements of breaking strength, elastic compliance and plastic compliance may be informative about changes in cell wall structure, whereas measurements of wall stress relaxation and creep are sensitive to both changes in wall structure and wall-loosening processes, such as those mediated by expansins and some lytic enzymes. As a result, a combination of methods is needed to obtain a broader view of cell wall behavior and properties connected with the concept of cell wall extensibility.

36 MATERIALS SCIENCE↗
NASA NTRS1988

Evidence against the involvement of ionically bound cell wall proteins in pea epicotyl growth

Ionically bound cell wall proteins were extracted from 7 day old etiolated pea (Pisum sativum L. cv Alaska) epicotyls with 3 molar LiCl. Polyclonal antiserum was raised in rabbits against the cell wall proteins. Growth assays showed that treatment of growing region segments (5-7 millimeters) of peas with either dialyzed serum, serum globulin fraction, affinity purified immunoglobulin, or papain-cleaved antibody fragments had no effect on growth. Immunofluorescence microscopy confirmed antibody binding to cell walls and penetration of the antibodies into the tissues. Western blot analysis, immunoassay results, and affinity chromatography utilizing Sepharose-bound antibodies confirmed recognition of the protein preparation by the antibodies. Experiments employing in vitro extension as a screening measure indicated no effect upon extension by antibodies, by 50 millimolar LiCl perfusion of the apoplast or by 3 molar LiCl extraction. Addition of cell wall protein to protease pretreated segments did not restore extension nor did addition of cell wall protein to untreated segments increase extension. It is concluded that, although evidence suggests that protein is responsible for the process of extension, the class(es) of proteins which are extracted from pea cell walls with 3 molar LiCl are probably not involved in this process.

NASA Program Space Biology↗
NASA NTRS1999

A laser microsurgical method of cell wall removal allows detection of large-conductance ion channels in the guard cell plasma membrane

Application of patch clamp techniques to higher-plant cells has been subject to the limitation that the requisite contact of the patch electrode with the cell membrane necessitates prior enzymatic removal of the plant cell wall. Because the wall is an integral component of plant cells, and because cell-wall-degrading enzymes can disrupt membrane properties, such enzymatic treatments may alter ion channel behavior. We compared ion channel activity in enzymatically isolated protoplasts of Vicia faba guard cells with that found in membranes exposed by a laser microsurgical technique in which only a tiny portion of the cell wall is removed while the rest of the cell remains intact within its tissue environment. "Laser-assisted" patch clamping reveals a new category of high-conductance (130 to 361 pS) ion channels not previously reported in patch clamp studies on plant plasma membranes. These data indicate that ion channels are present in plant membranes that are not detected by conventional patch clamp techniques involving the production of individual plant protoplasts isolated from their tissue environment by enzymatic digestion of the cell wall. Given the large conductances of the channels revealed by laser-assisted patch clamping, we hypothesize that these channels play a significant role in the regulation of ion content and electrical signalling in guard cells.

Non-NASA Center↗
DOE OSTI2014

Electron Tomography of Cryo-Immobilized Plant Tissue: A Novel Approach to Studying 3D Macromolecular Architecture of Mature Plant Cell Walls In Situ

Cost-effective production of lignocellulosic biofuel requires efficient breakdown of cell walls present in plant biomass to retrieve the wall polysaccharides for fermentation. In-depth knowledge of plant cell wall composition is therefore essential for improving the fuel production process. The precise spatial three-dimensional (3D) organization of cellulose, hemicellulose, pectin and lignin within plant cell walls remains unclear to date since the microscopy techniques used so far have been limited to two-dimensional, topographic or low-resolution imaging, or required isolation or chemical extraction of the cell walls. In this paper we demonstrate that by cryo-immobilizing fresh tissue, then either cryo-sectioning or freeze-substituting and resin embedding, followed by cryo- or room temperature (RT) electron tomography, respectively, we can visualize previously unseen details of plant cell wall architecture in 3D, at macromolecular resolution (~2 nm), and in near-native state. Qualitative and quantitative analyses showed that wall organization of cryo-immobilized samples were preserved remarkably better than conventionally prepared samples that suffer substantial extraction. Lignin-less primary cell walls were well preserved in both self-pressurized rapidly frozen (SPRF), cryo-sectioned samples as well as high-pressure frozen, freeze-substituted and resin embedded (HPF-FS-resin) samples. Lignin-rich secondary cell walls appeared featureless in HPF-FS-resin sections presumably due to poor stain penetration, but their macromolecular features could be visualized in unprecedented details in our cryo-sections. While cryo-tomography of vitreous tissue sections is currently proving to be instrumental in developing 3D models of lignin-rich secondary cell walls, here we confirm that the technically easier method of RT-tomography of HPF-FS-resin sections could be used immediately for routine study of low-lignin cell walls. As a proof of principle, we characterized the primary cell walls of a mutant (cob-6) and wild type Arabidopsis hypocotyl parenchyma cells by RT-tomography of HPF-FS-resin sections, and detected a small but significant difference in spatial organization of cellulose microfibrils in the mutant walls.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Changes in Cell Wall Properties Coincide with Overexpression of Extensin Fusion Proteins in Suspension Cultured Tobacco Cells

Extensins are one subfamily of the cell wall hydroxyproline-rich glycoproteins, containing characteristic SerHyp4 glycosylation motifs and intermolecular cross-linking motifs such as the TyrXaaTyr sequence. Extensins are believed to form a cross-linked network in the plant cell wall through the tyrosine-derivatives isodityrosine, pulcherosine, and di-isodityrosine. Overexpression of three synthetic genes encoding different elastin-arabinogalactan protein-extensin hybrids in tobacco suspension cultured cells yielded novel cross-linking glycoproteins that shared features of the extensins, arabinogalactan proteins and elastin. The cell wall properties of the three transgenic cell lines were all changed, but in different ways. One transgenic cell line showed decreased cellulose crystallinity and increased wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed xyloglucan epitope extractability. In conclusion, these data indicate that overexpression of modified extensins may be a route to engineer plants for bioenergy and biomaterial production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

A Transcriptomic Analysis of Xylan Mutants Does Not Support the Existence of a Secondary Cell Wall Integrity System in Arabidopsis

Yeast have long been known to possess a cell wall integrity (CWI) system, and recently an analogous systemhas been described for the primary walls of plants (PCWI) that leads to changes in plant growth and cell wall composition. A similar system has been proposed to exist for secondary cell walls (SCWI). However, there is little data to support this. Here, we analyzed the stem transcriptome of a set of cell wall biosynthetic mutants in order to investigate whether cell wall damage, in this case caused by aberrant xylan synthesis, activates a signaling cascade or changes in cell wall synthesis gene expression. Our data revealed remarkably few changes to the transcriptome. We hypothesize that this is because cells undergoing secondary cell wall thickening have entered a committed programme leading to cell death, and therefore a SCWI system would have limited impact. The absence of transcriptomic responses to secondary cell wall alterations may facilitate engineering of the secondary cell wall of plants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

How cell wall complexity influences saccharification efficiency in Miscanthus sinensis

The production of bioenergy from grasses has been developing quickly during the last decade, with Miscanthus being among the most important choices for production of bioethanol. However, one of the key barriers to producing bioethanol is the lack of information about cell wall structure. Cell walls are thought to display compositional differences that lead to emergence of a very high level of complexity, resulting in great diversity in cell wall architectures. In this work, a set of different techniques was used to access the complexity of cell walls of different genotypes of Miscanthus sinensis in order to understand how they interfere with saccharification efficiency. Three genotypes of M. sinensis displaying different patterns of correlation between lignin content and saccharification efficiency were subjected to cell wall analysis by quantitative/qualitative analytical techniques such as monosaccharide composition, oligosaccharide profiling, and glycome profiling. When saccharification efficiency was correlated negatively with lignin, the structural features of arabinoxylan and xyloglucan were found to contribute positively to hydrolysis. In the absence of such correlation, different types of pectins, and some mannans contributed to saccharification efficiency. In conclusion, different genotypes of M. sinensis were shown to display distinct interactions among their cell wall components, which seem to influence cell wall hydrolysis.

09 BIOMASS FUELS↗
DOE OSTI2016

The Arabidopsis domain of unknown function 1218 (DUF1218) containing proteins, MODIFYING WALL LIGNIN-1 and 2 (At1g31720/MWL-1 and At4g19370/MWL-2) function redundantly to alter secondary cell wall lignin content

DUF1218 is a land plant-specific innovation and has previously been shown to be associated with cell wall biology, vasculature patterning and abiotic/biotic stress response. The Arabidopsis genome encodes 15 members, two of which (At1g31720 and At4g27435) are preferentially expressed in the secondary cell wall depositing inflorescence stems. To further our understanding of the roles of DUF1218-containing proteins in secondary cell wall biology, we functionally characterized At1g31720 (herein referred to as MODIFYING WALL LIGNIN -1 or MWL -1). Since related gene family members may contribute to functional redundancy, we also characterized At4g19370 ( MWL -2), the most closely related gene to MWL -1 in the protein family. Subcellular localization revealed that both Arabidopsis proteins are targeted to the cell periphery. The single T-DNA knockout lines, mwl -1 and mwl -2, and independent overexpression lines showed no significant differences in plant growth or changes in total lignin content relative to wild-type (WT) control plants. However, the double homozygous mutant, mwl -1/ mwl -2, had smaller rosettes with a significant decrease in rosette fresh weight and stem height relative to the WT control at four weeks and six weeks, respectively. Moreover, mwl -1/ mwl -2 showed a significant reduction in total lignin content (by ca. 11% relative to WT) and an increase in syringyl/guaiacyl (S/G) monomer ratio relative to the control plants. Lastly, our study has identified two additional members of the DUF1218 family in Arabidopsis as novel contributors to secondary cell wall biology, specifically lignin biosynthesis, and these proteins appear to function redundantly.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Cell Wall Ultrastructure of Stem Wood, Roots, and Needles of a Conifer Varies in Response to Moisture Availability

The composition, integrity, and architecture of the macromolecular matrix of cell walls, collectively referred to as cell wall ultrastructure, exhibits variation across species and organs and among cell types within organs. Indirect approaches have suggested that modifications to cell wall ultrastructure occur in response to abiotic stress; however, modifications have not been directly observed. Glycome profiling was used to study cell wall ultrastructure by examining variation in composition and extractability of non-cellulosic glycans in cell walls of stem wood, roots, and needles of loblolly pine saplings exposed to high and low soil moisture. Soil moisture influenced physiological processes and the overall composition and extractability of cell wall components differed as a function of soil moisture treatments. The strongest response of cell wall ultrastructure to soil moisture was increased extractability of pectic backbone epitopes in the low soil moisture treatment. The higher abundance of these pectic backbone epitopes in the oxalate extract indicate that the loosening of cell wall pectic components could be associated with the release of pectic signals as a stress response. The increased extractability of pectic backbone epitopes in response to low soil moisture availability was more pronounced in stem wood than in roots or needles. Additional responses to low soil moisture availability were observed in lignin associated carbohydrates released in chlorite extracts of stem wood, including an increased abundance of pectic arabinogalactan epitopes. Overall, these results indicate that cell walls of loblolly pine organs undergo changes in their ultrastructural composition and extractability as a response to soil moisture availability and that cell walls of the stem wood are more responsive to low soil moisture availability compared to cell walls of roots and needles. In conclusion, to our knowledge, this is the first direct evidence, delineated by glycomic analyses, that abiotic stress affects cell wall ultrastructure. This study is also unique in that glycome profiling of pine needles has never before been reported.

54 ENVIRONMENTAL SCIENCES↗