A proteomic approach to apoplastic proteins involved in cell wall regeneration in protoplasts of Arabidopsis suspension-cultured cells

To clarify the mechanisms of cell wall construction, we used a proteomic approach to investigate the proteins secreted into cell wall spaces during cell wall regeneration from the protoplasts of Arabidopsis suspension-cultured cells. We focused on cell wall proteins loosely bound to the cell wall architecture and extractable with 1 M KCl solutions from: (i) native suspension cultured cells; (ii) protoplasts that had been allowed to regenerate their cell walls for 1 h; and (iii) protoplasts allowed to regenerate their cell walls for 3 h. We adopted a non-destructive extraction procedure without disrupting cellular integrity, thereby avoiding contamination from cytoplasmic proteins. Using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS), we separated, mapped and identified 71 proteins derived from the native cell wall, and 175 and 212 proteins derived from the 1 and 3 h regenerated protoplasts, respectively. Quite different sets of proteins with differing status of their post-translational modifications, including phosphorylation and glycosylation, were identified in the three protein fractions. This indicated dynamic in muro changes in the cell wall proteins during cell wall regeneration in the protoplasts. The analysis revealed a set of enzymes specifically involved in cell wall expansion and construction in suspension-cultured cells. This approach has also determined a set of cell wall proteins that had not been predicted to be localized in cell wall spaces.     

Cell wall analysis

The cell wall is a rigid structure essential for survival of the fungal cell. Because of its absence in mammalian cells, the cell wall is an attractive target for antifungal agents. Thus, for different reasons, it is important to know how the cell wall is synthesized and how different molecules regulate that synthesis. The Schizosaccharomyces pombe cell wall is mainly formed by glucose polysaccharides and some galactomannoproteins. Here, we describe a fast and reliable method to analyze changes in S. pombe cell wall composition by using specific enzymatic degradation and chemical treatment of purified cell walls. This approach provides a powerful means to analyze changes in (1,3)beta-glucan and (1,3)alpha-glucan, two main polysaccharides present in fungal cell walls. Analysis of cell wall polymers will be useful to search for new antifungal drugs that may inhibit cell wall biosynthesis and/or alter cell wall structure.     

The role of cell wall in plant embryogenesis

This review presents recent data about cell wall involvement in plant embryogenesis. During plant development, the cell wall is subjected to precise regulation. During this process a bidirectional information exchange between the cell wall and the protoplast is observed. The cell wall also mediates in the cell-cell (apoplastic) and cell to cell (symplastic) information flow. Especially some products derived from the hydrolysis of specific cell wall compounds can act as short distance signal transduction molecules during the development. Oligosaccharins are a group of such products. Their activity and sources focused the researchers' attention on the biochemical composition of the cell wall and the activity of some cell wall enzymes. The dramatic influence on the embryo body shape has also the cell wall synthesis machinery, including vesicular secretion pathways. Moreover, the interplay between the turgor pressure and counteracting cell walls and neighbouring cells (in higher organisms) creates the specific mechanical forces influencing the development of the whole plant. We conclude that discovering factors which can influence cell wall physiology and architecture is crucial for a better understanding of plant embryogenesis. In this review we summarize some recent experimental data reporting plant cell wall involvement in embryogenesis, putting special emphasis on somatic embryogenesis.     

Differential histone modification and protein expression associated with cell wall removal and regeneration in rice (Oryza sativa)

The cell wall is a critical extracellular structure that provides protection and structural support in plant cells. To study the biological function of the cell wall and the regulation of cell wall resynthesis, we examined cellular responses to enzymatic removal of the cell wall in rice (Oryza sativa) suspension cells using proteomic approaches. We find that removal of cell wall stimulates cell wall synthesis from multiple sites in protoplasts instead of from a single site as in cytokinesis. Nucleus DAPI stain and MNase digestion further show that removal of the cell wall is concomitant with substantial chromatin reorganization. Histone post-translational modification studies using both Western blots and isotope labeling assisted quantitative mass spectrometry analyses reveal that substantial histone modification changes, particularly H3K18(AC) and H3K23(AC), are associated with the removal and regeneration of the cell wall. Label-free quantitative proteome analyses further reveal that chromatin associated proteins undergo dramatic changes upon removal of the cell wall, along with cytoskeleton, cell wall metabolism, and stress-response proteins. This study demonstrates that cell wall removal is associated with substantial chromatin change and may lead to stimulation of cell wall synthesis using a novel mechanism.     

Cosegregation of cell wall and DNA in Bacillus subtilis.

Cosegregation of cell wall and DNA of a lysis-negative mutant of Bacillus subtilis was examined by continuously labeling (i) cell wall, (ii) DNA, and (iii) both cell wall and DNA. After four to five generations of chase in liquid media it was found by light microscope autoradiography that the numbers of wall segregation units per cell are 29 and 9 in rich and minimal medium, respectively. Under the same conditions the numbers of segregation units of DNA were almost 50% lower: 15 and 5, respectively. Simultaneous labeling of cell wall and DNA (iii) provided figures almost identical to those obtained for cell wall alone, (i), implying cosegregation of the two components. Statistical analysis ruled out their random distribution into daughter cells. Measurements of the positions of grain clusters at the end of the chase period along chains of cells, each derived from a single cell at the beginning of chase, show that cell wall units are localized according to a symmetrical pattern, whereas those of DNA are distributed in an asymmetrical but highly regular way. It appears that of two cell wall units of the same age one only has a strand of DNA attached to it. We present a simple diagrammatic model of cell wall organization and DNA-cell wall association which is compatible with our observations. Finally, we discuss previous experiments pertinent to cosegregation of cell wall and DNA obtained with cells grown on solid media as well as with germinating spores; an explanation for the independent segregation of cell wall and DNA observed in the latter case is advanced.     

Lipid and cell wall changes in an inositol-requiring mutant of Neurospora crassa.

An inositol deficiency in the inositol-requiring (inl) mutant of Neurospora crassa led to changes in the composition of the inositol-containing lipids and the cell wall. On deficient levels of inositol, phosphatidyl inositol decreased by 23-fold, di(inositolphosphoryl) ceramide decreased by 4-fold, and monoinositolphosphoryl ceramide increased slightly. The inositol deficiency also led to an aberrant hyphal morphology and changes in both the amount of cell wall and the amino sugar content of the cell wall. The glucosamine content of the cell wall decreased by 50%, the galactosamine increased by 50%, but no significant changes were found in the content of the cell wall amino sugar precursors, or in the amino acid, glucose, or total hexose content of the cell wall. Inositol-containing compounds were found associated with purified cell wall material. These compounds were bound tightly to the cell wall but could be removed by treatment with alkali, a treatment which disrupts the cell wall integrity. Possible mechanisms of how changes in lipid composition can affect cell wall biosynthesis are discussed.     

Formation of plant cell wall supramolecular structure

Plant cell wall is an example of a widespread natural supramolecular structure: its components are considered to be the most abundant organic compounds renewable by living organisms. Plant cell wall includes numerous components, mainly polysaccharidic; its formation is largely based on carbohydrate-carbohydrate interactions. In contrast to the extracellular matrix of most other organisms, the plant cell compartment located outside the plasma membrane is so structured that has been named “wall”. The present review summarizes data on the mechanisms of formation of this supramolecular structure and considers major difficulties and results of research. Existing approaches to the study of interactions between polysaccharides during plant cell wall formation have been analyzed, including: (i) characterization of the structure of natural polysaccharide complexes obtained during cell wall fractionation; (ii) analysis of the interactions between polysaccharides “at mixing in a tube”; (iii) study of the interactions between isolated individual plant cell wall matrix polysaccharides and microfibrils formed by cellulose-synthesizing microorganisms; and (iv) investigation of cell wall formation and modification directly in plant objects. The key stages in formation of plant cell wall supramolecular structure are defined and characterized as follows: (i) formation of cellulose microfibrils; (ii) interactions between matrix polysaccharides within Golgi apparatus substructures; (iii) interaction between matrix polysaccharides, newly secreted outside the plasma membrane, and cellulose microfibrils during formation of the latter; (iv) packaging of the formed complexes and individual polysaccharides in cell wall layers; and (v) modification of deposited cell wall layers.     

CELL WALL STRUCTURE AND IRON DISTRIBUTION IN THE GREEN ALGA STAURASTRUM LUETKEMUELLERI (DESMIDIACEAE)1

The cell wall of Staurastrum luetkemuelleri Donnat & Ruttner was examined with scanning electron microscope (SEM) using whole cells, in thin sections with transmission electron microscope (TEM), and in air dried whole cells and unstained thin sections with X-ray microanalysis in the scanning-transmission electron microscope (STEM). The cell wall was ornamented with spines and wartlike structures. Spines were solid structures, consisting of deposits of cell wall material between two main cell wall layers. The wart-like structures were pore organs extending through the cell wall and the mucilaginous layer outside the cell wall. The pore cylinder was surrounded by deposits of cell wall material similar to the ones in the spines. X-ray microanalysis of selected areas on whole cells from a natural population showed iron accumulation in discrete locations on the cell extensions of S. luetkemuelleri. In the unstained thin sections iron was found only in the cell wall deposits in the spines. Cells grown in laboratory cultures failed to show iron accumulation regardless of readdition of iron-EDTA (Fe-EDTA) to the culture medium.     

The Neurospora crassa dfg5 and dcw1 genes encode α-1,6-mannanases that function in the incorporation of glycoproteins into the cell wall

The covalent cross-linking of cell wall proteins into the cell wall glucan/chitin matrix is an important step in the biogenesis of the fungal cell wall. We demonstrate that the Neurospora crassa DFG5 (NCU03770) and DCW1 (NCU08127) enzymes function in vivo to cross-link glycoproteins into the cell wall. Mutants lacking DFG5 or DCW1 release slightly elevated levels of cell wall proteins into their growth medium. Mutants lacking both DFG5 and DCW1 have substantially reduced levels of cell wall proteins in their cell walls and release large amounts of known cell wall proteins into the medium. DFG5 and DCW1 are members of the GH76 family of glycosyl hydrolases, which have specificity to recognize and cleave α-1,6-mannans. A model for incorporation of glycoproteins into the cell wall through the α-1,6-mannan core of the N-linked galactomannan is presented. In this model, DFG5 and DCW1 recognize the N-linked galactomannan present on glycoproteins and cross-link it into the cell wall glucan/chitin matrix.     

Differences in osmotolerant and cell-wall properties of two<Emphasis Type="Italic">zygosaccharomyces rouxii</Emphasis> strains

The osmotolerant and cell wall properties of the two most studied wild-type Zygosaccharomyces rouxii strains (CBS 732 and ATCC 42981) were examined. Differences in their (1) tolerance to high salt content in the medium, (2) resistance to the lysing enzymes Lyticase and Zymolyase, (3) cell-wall polymer content and (4) cell wall micromorphology suggested that the less osmotolerant CBS 732 strain possesses a more rigid cell wall than the more osmotolerant ATCC 42981, whose cell wall seems to be more flexible and elastic.     

Restrictive glycosylphosphatidylinositol anchor synthesis in cwh6/gpi3 yeast cells causes aberrant biogenesis of cell wall proteins.

We previously reported that the defects in the Saccharomyces cerevisiae cwh6 Calcofluor white-hypersensitive cell wall mutant are caused by a mutation in SPT14/GPI3, a gene involved in glycosylphosphatidylinositol (GPI) anchor biosynthesis. Here we describe the effect of cwh6/spt14/gpi3 on the biogenesis of cell wall proteins. It was found that the release of precursors of cell wall proteins from the endoplasmic reticulum (ER) was retarded. This was accompanied by proliferation of ER structures. The majority of the cell wall protein precursors that eventually left the ER were not covalently incorporated into the cell wall but were secreted into the growth medium. Despite the inefficient incorporation of cell wall proteins, there was no net effect on the protein level in the cell wall. It is postulated that the availability of GPI-dependent cell wall proteins determines the rate of cell wall construction and limits growth rate.     

Regeneration and maturation of daughter cell walls in the autospore-forming green alga<Emphasis Type="Italic"> Chlorella vulgaris</Emphasis> (Chlorophyta,Trebouxiophyceae)

Cell-wall synthesis in Chlorella vulgaris, an autospore-forming alga, was observed using the cell wall-specific fluorescent dye Fluostain I. The observation suggested two clearly distinguishable stages in cell-wall synthesis: moderate synthesis during the cell-growth process and rapid synthesis at the cell-division stage. We used electron microscopy to examine the structural changes that occurred with growth in the premature daughter cell wall during the cell-growth and cell-division phases. The cell began to synthesize a new daughter cell wall shortly after its release from the autosporangium. A very thin daughter cell wall, with a thickness of about 2 nm, was formed inside the mother cell wall and completely enveloped the outer surface of the plasma membrane of the cell. The daughter cell wall gradually increased in thickness from 2 to 3.8 nm. During the protoplast-division phase in the cell-division stage, the daughter cell wall expanded on the surface of the invaginating plasma membrane of the cleavage furrow, accompanied by active synthesis of the cell wall, which increased in thickness from 3.8 to 6.1 nm. The daughter cell matured into an autospore while completely enclosed by its own thickening (from 6.1 to 17 nm) wall. Finally, the released daughter cell was enclosed by its own cell wall after the mother cell wall burst. The daughter cell with mature wall thickness (17–21 nm) emerged as a small, but complete, autospore.     

In vitro system for the synthesis of teichoic acid linked to peptidoglycan.

A crude cell wall preparation from Staphylococcus aureus H prepared by the method of Mirelman and Sharon (1972) was shown to catalyze the synthesis of polyribitol phosphate linked to the cell wall peptidoglycan. The reaction used cytidine diphosphate (CDP)-ribitol as a substrate and in addition required the presence of CDP-glycerol, uridine diphosphate (UDP)-N-acetyl-D-glucosamine, and adenosine triphosphate. Incubation of radioactive CDP-glycerol with the crude cell wall preparation resulted in the transfer of glycerol phosphate residues to the cell wall; this reaction was greatly stimulated by the presence of UDP-N-acetylglucosamine. These data suggest that polyribitol phosphate is linked to the cell wall peptidoglycan by an oligomer contaning N-acetyl-D-glucosamine and glycerol phosphate.     

Trifluoromethanesulfonic acid-based proteomic analysis of cell wall and secreted proteins of the ascomycetous fungi Neurospora crassa and Candida albicans

Cell wall proteins from purified Candida albicans and Neurospora crassa cell walls were released using trifluoromethanesulfonic acid (TFMS) which cleaves the cell wall glucan/chitin matrix and deglycosylates the proteins. The cell wall proteins were then characterized by SDS–PAGE and identified by proteomic analysis. The analyses for C. albicans identified 15 cell wall proteins and six secreted proteins. For N. crassa, the analyses identified 26 cell wall proteins and nine secreted proteins. Most of the C. albicans cell wall proteins are found in the cell walls of both yeast and hyphae cells, but some cell type-specific cell wall proteins were observed. The analyses showed that the pattern of cell wall proteins present in N. crassa vegetative hyphae and conidia (asexual spores) are quite different. Almost all of the cell wall proteins identified in N. crassa have close homologs in the sequenced fungal genomes, suggesting that these proteins have important conserved functions within the cell wall.     

Morphology of yeast cell wall as affected by genetic manipulation of beta(1 --> 6) glycosidic linkage

The morphology of yeast cells as it is affected by the glycosidic linkages of constituent glucan was studied. Four different strains of Saccharomyces cerevisiae were studied. A cell wall matrix particle representing the intact original morphology and composed entirely of beta-glucan was prepared. Using prepared cell wall glucan particles, the morphology and cell wall matrix structure were examined. Genetic modification of the cell wall structure during growth results in the alteration of the shape and hydrodnamic volume of the intact cell wall particles. The shape and hydrodynamic volume of the cell wall particles can also be modified by in vitro chemical and enzymatic treatment. The shape factor and hydrodynamic volume of the whole glucan cell wall matrix particles were evaluated quantitatively using a rheological analysis. An increased degree of beta(1 --> 6) cross-linking in the cell wall matrix induces a nearly 2-fold increase in the shape factor and a 10-fold increase in the compression modulus of the glucan particles. The disruption of beta(1 --> 6) glycosidic cross-linking causes the particles to swell by up to 18% of their original volume. This was used as a strategy to isolate a yeast mutant with a high beta(1 --> 6) glycosidic content in the cell wall glucan.     

Action of xyloglucan hydrolase within the native cell wall architecture and its effect on cell wall extensibility in azuki bean epicotyls

Xyloglucan hydrolase (XGH) has recently been purified from the cell wall of azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls as a new type of xyloglucan-degrading enzyme [Tabuchi et al. (2001) Plant Cell Physiol. 42: 154]. In the present study, the effects of XGH on the mechanical properties of the cell wall and on the level and the molecular size of xyloglucans within the native wall architecture were examined in azuki bean epicotyls. When the epidermal tissue strips from the growing regions of azuki bean epicotyls were incubated with XGH, the mechanical extensibility of the cell wall dramatically increased. XGH exogenously applied to cell wall materials (homogenates) or epidermal tissue strips decreased the amount of xyloglucans via the solubilization of the polysaccharides. Also, XGH substantially decreased the molecular mass of xyloglucans in both materials. These results indicate that XGH is capable of hydrolyzing xyloglucans within the native cell wall architecture and thereby increasing the cell wall extensibility in azuki bean epicotyls.     

Members of the Hsp70 family of proteins in the cell wall of Saccharomyces cerevisiae.

Western blot (immunoblot) analysis of cell wall and cytosolic extracts obtained from parental and ssa1 and ssa2 single- and double-mutant strains of Saccharomyces cerevisiae showed that the heat shock protein 70 (Hsp70) products of these genes, previously thought to be restricted to the cell interior, are also present in the cell wall. A cell wall location was further confirmed by indirect immunofluorescence with intact cells and biotinylation of extracellular Hsp70. Hsp70s have been implicated in translocation across the membrane and as molecular chaperones, and changes in the profile of cell wall proteins suggested that these proteins may have a similar role in the cell wall.     

Response of Paracoccidioides brasiliensis Pb01 to stressor agents and cell wall osmoregulators

In its attempt to survive, the fungal cell can change the cell wall composition and/or structure in response to environmental stress. The molecules involved in these compensatory mechanisms are a possible target for the development of effective antifungal agents. In the thermodimorphic fungus Paracoccidioides brasiliensis Pb01, the main polymers that compose the cell wall are chitin and glucans. These polymers form a primary barrier that is responsible for the structural integrity and formation of the cell wall. In this study the behaviour of P. brasiliensis was evaluated under incubation with cell wall stressor agents such as Calcofluor White (CFW), Congo Red (CR), Sodium Dodecyl Sulphate (SDS), NaCl, KCl, and Sorbitol. Use of concentrations at which the fungus is visually sensitive to those agents helped to explain some of the adaptive mechanisms used by P. brasiliensis in response to cell wall stress. Our results show that 1,3-β-D-glucan synthase (PbFKS1), glucosamine-6-phosphate synthase (PbGFA1) and β-1,3-glucanosyltransferase (PbGEL3)as well as 1,3-β-D-glucan and N-acetylglucosamine (GlcNAc) residues in the cell wall are involved in compensatory mechanisms against cell wall damage.     

Divergent selection for ester-linked diferulates in maize pith stalk tissues. Effects on cell wall composition and degradability

Cross-linking of grass cell wall components through diferulates (DFAs) has a marked impact on cell wall properties. However, results of genetic selection for DFA concentration have not been reported for any grass species. We report here the results of direct selection for ester-linked DFA concentration in maize stalk pith tissues and the associated changes in cell wall composition and biodegradability. After two cycles of divergent selection, maize populations selected for higher total DFA (DFAT) content (CHs) had 16% higher DFAT concentrations than populations selected for lower DFAT content (CLs). These significant DFA concentration gains suggest that DFA deposition in maize pith parenchyma cell walls is a highly heritable trait that is genetically regulated and can be modified trough conventional breeding. Maize populations selected for higher DFAT had 13% less glucose and 10% lower total cell wall concentration than CLs, suggesting that increased cross-linking of feruloylated arabinoxylans results in repacking of the matrix and possibly in thinner and firmer cell walls. Divergent selection affected esterified DFAT and monomeric ferulate ether cross link concentrations differently, supporting the hypothesis that the biosynthesis of these cell wall components are separately regulated. As expected, a more higher DFA ester cross-coupled arabinoxylan network had an effect on rumen cell wall degradability (CLs showed 12% higher 24-h total polysaccharide degradability than CHs). Interestingly, 8–8-coupled DFAs, previously associated with cell wall strength, were the best predictors of pith cell wall degradability (negative impact). Thus, further research on the involvement of these specific DFA regioisomers in limiting cell wall biodegradability is encouraged.