The functions of cell wall polysaccharides in composition and architecture revealed through mutations

The plant cell wall is a highly organized composite of many different polysaccharides, proteins and aromatic substances. These complex matrices define the shape of each individual cell, and ultimately, they are the determinants of plant morphology. The fine structures of the major angiosperm cell wall polysaccharides have been characterized, but it is not well understood how these polysaccharides are assembled into a metabolically active architecture. Cell wall biogenesis and remodeling may be partitioned into six major stages of development (precursor synthesis, polymerization, secretion, assembly, rearrangement and disassembly), and to date, a handful of mutations have been identified that affect the composition and structure in each of these stages. To greatly augment this collection, we have initiated a program to use Fourier transform infrared spectroscopy as a high through-put screen to identify a broad range of cell-wall mutants of Arabidopsis and maize. We anticipate that such mutants will be useful to probe the impact of the individual components and their metabolism on basic processes of plant growth and development. The structures of dicot and grass walls, the identification of representative cell wall mutants, and the use of a novel spectroscopic screen to identify many more cell wall mutants, are briefly reviewed.     

Overexpression of a developing xylem cDNA library in transgenic poplar generates high mutation rate specific to wood formation

We investigated feasibility of the Full‐length complementary DNA OvereXpression (FOX) system as a mutagenesis approach in poplar, using developing xylem tissue. The main goal was to assess the overall mutation rate and if the system will increase instances of mutants affected in traits linked to the xylem tissue. Indeed, we found a high mutation rate of 17.7%, whereas 80% of all mutants were significantly affected in cellulose, lignin and/or hemicellulose. Cell wall biosynthesis is a major process occurring during xylem development. Enrichment of mutants affected in cell wall composition suggests that the tissue source for the FOX library influenced the occurrence of mutants affected in a trait linked to this tissue. Additionally, we found that FLcDNAs from mutants affected in cell wall composition were homologous to genes known to be involved in cell wall biosynthesis and most recovered FLcDNAs corresponded to genes whose native expression was highest in xylem. We characterized in detail a mutant line with increased diameter. The phenotype was caused by a poplar homolog of LONELY GUY 1 (LOG1), which encodes an enzyme in cytokinin biosynthesis and significantly increased xylem proliferation. The causative role of LOG1 in the observed phenotype was further reaffirmed by elevated cytokinin concentration in the mutant and recapitulation overexpression experiment wherein multiple independent lines phenocopied the original FOX mutant. Our experiments show that the FOX approach can be efficiently used for gene discovery and molecular interrogation of traits specific to woody perennial growth and development.     

Impact of mitochondrial activity on the cell wall composition and on the resistance to tannic acid in Saccharomyces cerevisiae

In screening for resistance to tannic acid, mutants of Saccharomyces cerevisiae with an altered cell wall composition were recently isolated. Here we show that these mutants were all respiratory deficient. Cytoplasmic petite mutants isolated after ethidium bromide mutagenesis were resistant to tannic acid and had cell wall characteristics similar to the mutants isolated by screening for tannic acid resistance as shown by the lower sensitivity to zymolyase, a cell wall hydrolyzing enzyme, and by a changed sensitivity to calcofluor white, a molecule interfering with the cell wall assembly. Reintroducing active mitochondria to a tannic-acid-resistant mutant reduced the tannic acid resistance and zymolyase resistance to the wild-type level, showing that a mitochondrial mutation was responsible for the changes in cell wall composition and in tannic acid sensitivity.     

Classification and identification of Arabidopsis cell wall mutants using Fourier-Transform InfraRed (FT-IR) microspectroscopy

We have developed a novel procedure for the rapid classification and identification of Arabidopsis mutants with altered cell wall architecture based on Fourier-Transform Infrared (FT-IR) microspectroscopy. FT-IR transmission spectra were sampled from native 4-day-old dark-grown hypocotyls of 46 mutants and the wild type treated with various drugs. The Mahalanobis distance between mutants, calculated from the spectral information after compression with the Discriminant Variables Selection procedure, was used for alpha hierarchical cluster analysis. Despite the completely unsupervised nature of the classification procedure, we show that all mutants with cellulose defects appeared in the same cluster. In addition, mutant alleles of similar strength for several unrelated loci were also clustered, which demonstrates the sensitivity of the method to detect a wide array of cell wall defects. Comparing the cellulose-deficient cluster with the cluster that contained wild-type controls led to the identification of wave numbers that were diagnostic for altered cellulose content in the context of an intact cell wall. The results show that FT-IR spectra can be used to identify different classes of mutants and to characterize cell wall changes at a microscopic level in unknown mutants. This procedure significantly accelerates the identification and classification of cell wall mutants, which makes cell wall polysaccharides more accessible to functional genomics approaches.     

A novel screening method for cell wall mutants in Aspergillus niger identifies UDP-galactopyranose mutase as an important protein in fungal cell wall biosynthesis

To identify cell wall biosynthetic genes in filamentous fungi and thus potential targets for the discovery of new antifungals, we developed a novel screening method for cell wall mutants. It is based on our earlier observation that the Aspergillus niger agsA gene, which encodes a putative alpha-glucan synthase, is strongly induced in response to cell wall stress. By placing the agsA promoter region in front of a selectable marker, the acetamidase (amdS) gene of A. nidulans, we reasoned that cell wall mutants with a constitutively active cell wall stress response pathway could be identified by selecting mutants for growth on acetamide as the sole nitrogen source. For the genetic screen, a strain was constructed that contained two reporter genes controlled by the same promoter: the metabolic reporter gene PagsA-amdS and PagsA-H2B-GFP, which encodes a GFP-tagged nuclear protein. The primary screen yielded 161 mutants that were subjected to various cell wall-related secondary screens. Four calcofluor white-hypersensitive, osmotic-remediable thermosensitive mutants were selected for complementation analysis. Three mutants were complemented by the same gene, which encoded a protein with high sequence identity with eukaryotic UDP-galactopyranose mutases (UgmA). Our results indicate that galactofuranose formation is important for fungal cell wall biosynthesis and represents an attractive target for the development of antifungals.     

Cohesin dysfunction results in cell wall defects in budding yeast

Cohesin is a conserved chromatin-binding multisubunit protein complex involved in diverse chromosomal transactions such as sister-chromatid cohesion, chromosome condensation, regulation of gene expression, DNA replication, and repair. While working with a budding yeast temperature-sensitive mutant, mcd1-1, defective in a cohesin subunit, we observed that it was resistant to zymolyase, indicating an altered cell wall organization. The budding yeast cell wall is a strong but elastic structure essential for maintenance of cell shape and protection from extreme environmental challenges. Here, we show that the cohesin complex plays an important role in cell wall maintenance. Cohesin mutants showed high chitin content in the cell wall and sensitivity to multiple cell wall stress-inducing agents. Interestingly, temperature-dependent lethality of cohesin mutants was osmoremedial, in a HOG1-MAPK pathway-dependent manner, suggesting that the temperature sensitivity of these mutants may arise partially from cell wall defects. Moreover, Mpk1 hyper-phosphorylation indicated activation of the cell wall integrity (CWI) signaling pathway in cohesin mutants. Genetic interaction analysis revealed that the CWI pathway is essential for survival of mcd1-1 upon additional cell wall stress. The cell wall defect was independent of the cohesion function and accompanied by misregulation of expression of several genes having cell wall-related functions. Our findings reveal a requirement of cohesin in maintenance of CWI that is independent of the CWI pathway, and that may arise from cohesin’s role in regulating the expression of multiple genes encoding proteins involved in cell wall organization and biosynthesis.     

Hyperpolarized growth of Saccharomyces cerevisiae cak1P212S and cla4 mutants weakens cell walls and renders cells dependent on chitin synthase 3.

In a screen for cell wall defects in Saccharomyces cerevisiae, we isolated a strain carrying a mutation in the Cdc28-activating kinase CAK1. The cak1P212S mutant cells exhibit multiple, elongated and branched buds, beta(1,3)glucan-poor regions of the cell periphery and lysed upon osmotic shock after treatment with the chitin synthase III inhibitor Nikkomycin Z. Ultrastructural examination of cak1P212S mutants revealed a thin, uneven cell wall and marked abnormalities in septum formation. In all of the above aspects, the cak1P212S mutants are similar to previously described cla4 mutants, suggesting that the cell wall defects are common to mutants with hyperpolarized growth. In cak1P212S mutants, chitin accumulates all over the surface of the cells and glucan synthase activity is located preferentially to the tips of elongated buds. We conclude that the cell wall weakness in cak1P212S mutants is caused by hyperpolarized secretion of glucan synthase and lack of reinforcement of the lateral cell walls. Showing that the defect depends at least in part on Cdc28, the cak1P212S hyperpolarized growth phenotype can be suppressed by a Cak1-independent Cdc28-allele. The results underline the importance of a minor cell wall component, the chitin of lateral walls, for the integrity of the cell in a stress situation.     

Staphylococcus aureus mutants with increased lysostaphin resistance

Staphylococcus simulans secretes lysostaphin, a bacteriolytic enzyme that specifically binds to the cell wall envelope of Staphylococcus aureus and cleaves the pentaglycine cross bridges of peptidoglycan, thereby killing staphylococci. The study of S. aureus mutants with resistance to lysostaphin-mediated killing has revealed biosynthetic pathways for cell wall assembly. To identify additional genes involved in cell wall envelope biosynthesis, we have screened a collection of S. aureus strain Newman transposon mutants for lysostaphin resistance. Bursa aurealis insertion in SAV2335, encoding a polytopic membrane protein with predicted protease domain, caused a high degree of lysostaphin resistance, similar to the case for a previously described femAB promoter mutant. In contrast to the case for this femAB mutant, transposon insertion in SAV2335, herein named lyrA (lysostaphin resistance A), did not cause gross alterations of cell wall cross bridges such as truncations of pentaglycine to tri- or monoglycine. Also, inactivation of LyrA in a methicillin-resistant S. aureus strain did not precipitate a decrease in beta-lactam resistance as observed for fem (factor essential for methicillin resistance) mutants. Lysostaphin bound to the cell wall envelopes of lyrA mutants in a manner similar to that for wild-type staphylococci. Lysostaphin resistance of lyrA mutants is attributable to altered cell wall envelope properties and may in part be due to increased abundance of altered cross bridges. Other lyr mutants with intermediate lysostaphin resistance carried bursa aurealis insertions in genes specifying GTP pyrophosphokinase or enzymes of the purine biosynthetic pathway.     

A genomic approach for the identification and classification of genes involved in cell wall formation and its regulation in Saccharomyces cerevisiae

Using a hierarchical approach, 620 non-essential single-gene yeast deletants generated by EUROFAN I were systematically screened for cell-wall-related phenotypes. By analyzing for altered sensitivity to the presence of Calcofluor white or SDS in the growth medium, altered sensitivity to sonication, or abnormal morphology, 145 (23%) mutants showing at least one cell wall-related phenotype were selected. These were screened further to identify genes potentially involved in either the biosynthesis, remodeling or coupling of cell wall macromolecules or genes involved in the overall regulation of cell wall construction and to eliminate those genes with a more general, pleiotropic effect. Ninety percent of the mutants selected from the primary tests showed additional cell wall-related phenotypes. When extrapolated to the entire yeast genome, these data indicate that over 1200 genes may directly or indirectly affect cell wall formation and its regulation. Twenty-one mutants with altered levels of beta1,3-glucan synthase activity and five Calcofluor white-resistant mutants with altered levels of chitin synthase activities were found, indicating that the corresponding genes affect beta1,3-glucan or chitin synthesis. By selecting for increased levels of specific cell wall components in the growth medium, we identified 13 genes that are possibly implicated in different steps of cell wall assembly. Furthermore, 14 mutants showed a constitutive activation of the cell wall integrity pathway, suggesting that they participate in the modulation of the pathway either directly acting as signaling components or by triggering the Slt2-dependent compensatory mechanism. In conclusion, our screening approach represents a comprehensive functional analysis on a genomic scale of gene products involved in various aspects of fungal cell wall formation.     

Disruption of the <Emphasis Type="Italic"> Saccharomyces cerevisiae </Emphasis>cell-wall pathway gene <Emphasis Type="Italic"> SLG1</Emphasis> causes hypersensitivity to the antitumor drug bleomycin

Bleomycin is an antitumor drug that damages DNA via a free radical-dependent mechanism, and yeast mutants defective in DNA repair are hypersensitive to the drug. To identify possible pathways that may contribute to bleomycin resistance in yeast, we characterized a panel of bleomycin-sensitive mutants that were previously isolated by insertion mutagenesis using the transposon miniTn3::Leu2::LacZ::AMP( R). One of these mutants harbored a single insertion in the SLG1 gene, which encodes a cell membrane protein that senses cell wall stress, and functions to maintain cell wall function by activating the protein kinase C signaling pathway. Deletion of the SLG1 gene in parental strains caused hypersensitivity to bleomycin, and this correlated with an accumulation of damaged DNA. A plasmid that expresses the native SLG1 gene or that increases PKC1 gene dosage restored bleomycin resistance to the slg1Delta mutant. Two-dimensional gel electrophoresis revealed that exposure to bleomycin triggered the expression of certain proteins, presumably to maintain cell wall function, in a Slg1-dependent manner. In addition, mutants lacking cell wall function were found to be hypersensitive to bleomycin. We conclude that mutants deficient in proteins that maintain cell wall function are severely compromised in their ability to limit bleomycin entry into the cell. Therefore, these mutants are burdened with increased genotoxicity upon exposure to bleomycin in the medium. Our results show that major mechanisms other than DNA repair are operating in yeast to mediate bleomycin resistance.     

Isolation of rice dwarf mutants with ectopic deposition of phenolic components including lignin in parenchyma cell walls of internodes

Rice internodes must have the proper shape to support high-yielding panicles. The shape of internodes is controlled by various factors involved in their formation, such as developmental patterns, cell division, cell elongation, and cell wall biosynthesis. To understand the regulation of internode development, we screened dwarf mutants to identify those with a phenotype of ectopic deposits of phenolic components in parenchyma cell walls of internodes. We named these mutants ectopic deposition of phenolic components1 (edp1). Two alleles were identified, edp1-1 and edp1-2. Furthermore, these mutants showed disordered cell files in internode parenchyma. These abnormal phenotypes were very similar to that of a previously reported dwarf50 (d50) mutant. Genetic analyses of edp1 mutants revealed that the edp1 loci are distinct from d50. Our results indicate that analyses of edp1 mutants as well as the d50 mutant will be useful for understanding the molecular mechanisms behind ectopic deposition of cell wall phenolic components in internode parenchyma cells and the regulation of internode development.     

Mutations of the secondary cell wall

It has not been possible to isolate a number of crucial enzymes involved in plant cell wall synthesis. Recent progress in identifying some of these steps has been overcome by the isolation of mutants defective in various aspects of cell wall synthesis and the use of these mutants to identify the corresponding genes. Secondary cell walls offer numerous advantages for genetic analysis of plant cell walls. It is possible to recover very severe mutants since the plants remain viable. In addition, although variation in secondary cell wall composition occurs between different species and between different cell types, the composition of the walls is relatively simple compared to primary cell walls. Despite these advantages, relatively few secondary cell wall mutations have been described to date. The only secondary cell wall mutations characterised to date, in which the basis of the abnormality is known, have defects in either the control of secondary cell wall deposition or secondary cell wall cellulose or lignin biosynthesis. These mutants have, however, provided essential information on secondary cell wall biosynthesis.     

The assembly of a highly ordered component of the cell wall: The role of heritable factors and of physical structure

Summary An analysis has been made of the biogenesis of the highly ordered lattice structure which comprises an integral part of the cell wall of Chlamydomonas reinhardi. By examining mutants showing various wall defects, it has been shown that in some mutants lattice assembly will only occur in the presence of a particular kind of physical structure; in others the composition of the medium influences assembly. The role of a heritable factor in lattice assembly has also been demonstrated; after prolonged vegetative culture certain mutants lose the ability to form lattice, but regain this ability on passing through the diploid sexual phase. An attempt is made to integrate this information to provide a model for the control of assembly and inheritance of the wall.     

Uptake of cell wall peptides by Salmonella typhimurium and Escherichia coli.

During bacterial growth, cell wall peptides are released from the murein and reused for the synthesis of new cell wall material. Mutants defective in peptide transport were unable to reutilize cell wall peptides, demonstrating that these peptides are taken up intact into the cytoplasm prior to reincorporation into murein. Furthermore, cell wall peptide recycling was shown to play an important physiological role; peptide transport mutants which were unable to recycle these peptides showed growth defects under appropriate conditions. Using mutants specifically defective in each of the three peptide transport systems, we showed that the uptake of cell wall peptides was mediated solely by the oligopeptide permease (Opp) and that neither the dipeptide permease (Dpp) nor the tripeptide permease (Tpp) played a significant role in this process. Our data indicate that the periplasmic oligopeptide-binding protein has more than one substrate-binding site, each with different though overlapping specificities.     

Isolation of cell wall mutants in Aspergillus nidulans by screening for hypersensitivity to Calcofluor White

As a first step toward identifying novel genes of wall metabolism in filamentous fungi, we have screened a collection of Aspergillus nidulans mutants for strains exhibiting hypersensitivity toward the chitin binding agent Calcofluor White (CFW). This strategy has been used previously to identify cell wall mutants in Saccharomyces cerevisiae. We have identified 10 mutants representing eight loci, designated calA through calH, for Calcofluor hypersensitivity. All cal mutants are impaired for sporulation at 30 C or 42 C or both, and in eight of the 10 mutations this sporulation defect shows at least partial osmotic remediability. All cal mutants show elevated sensitivity to one or more of the following agents: Caspofungin, Nikkomycin, Tunicamycin, Congo red and SDS, which are recognized wall-compromising agents or have been shown to be inhibitory to wall integrity mutants in yeast. Seven of the 10 cal mutants show swelling at elevated temperature, which in most cases is osmotically remediable. Spore swelling also can be induced at 30 C in all but one of the cal mutants by germination in the presence of one or more of the following: Caspofungin, Nikkomycin or Tunicamycin. Analysis of wall sugars showed no major changes in mutant strains. We also report that the chitin synthase inhibitor Nikkomycin induces excessive spore swelling during germination in all tested strains that have wild type cell wall metabolism (GR5, A4, A28 and AH12) at 42 C but not at 30 C. This effect mimics that of certain temperature-sensitive swollen cell (swo) mutations.     

Isolation and proteomic analysis [corrected] of cell wall-deficient Haematococcus pluvialis mutants

The green alga Haematococcus pluvialis has a plant-like cell wall consisting of glycoproteins and cellulose that is modified during the cell cycle and under various conditions. These features allow Haematococcus to be used as a model organism for studying cell wall biology. Development of the Haematococcus model is hampered by the absence of mutants that could provide insight into the biosynthesis and assembly of wall components. Haematococcus mutants (WM#537 and WM#2978) (WM--wall mutant) with defective cell walls were obtained by chemical mutagenesis. WM#537 features a secondary wall of considerably reduced thickness, whereas WM#2978 possesses a somewhat reduced secondary wall with little intervening space between the wall and plasmalemma. 2-DE revealed that a majority of the cell wall proteins were present in the wild-type and mutant cell walls throughout the cell cycle. PMF identified 55 wall protein orthologs from these strains, including a subset of induced proteins known to be involved in wall construction, remodeling, and defense. Down-regulation of certain wall proteins in the two mutants was associated with the wall defects, whereas overexpression of other proteins may have compensated for the defective walls in the two mutants.     

Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red

The fungal cell wall is an essential organelle and represents a considerable metabolic investment. Its macromolecular composition, molecular organization and thickness can vary greatly depending on environmental conditions. Its construction is also tightly controlled in space and time. Many genes are therefore involved in building the fungal cell wall. Here we present a simple approach for detecting these genes. The method is based on the observation that cell wall mutants are generally more sensitive to two related anionic dyes, Calcofluor white (CFW) and Congo red (CR), both of which interfere with the construction and stress response of the cell wall. CFW-based and CR-based susceptibility assays identify cell wall mutants not only in ascomycetous yeasts (such as Saccharomyces cerevisiae and Candida albicans) but also in mycelial ascomycetes (such as Aspergillus fumigatus and Aspergillus niger), basidiomycetous species (Cryptococcus neoformans) and probably also zygomycetous fungi. The protocol can be completed in 4-6 h (excluding the incubation time required for fungal growth).