Synthesis Of Cell Wall Material Research Articles

Elevated levels of sphingolipid MIPC in the plasma membrane disrupt the coordination of cell growth with cell wall formation in fission yeast.

Coupling cell wall expansion with cell growth is a universal challenge faced by walled organisms. Mutations in Schizosaccharomyces pombe css1, which encodes a PM inositol phosphosphingolipid phospholipase C, prevent cell wall expansion but not synthesis of cell wall material. To probe how Css1 modulates cell wall formation we used classical and chemical genetics coupled with quantitative mass spectrometry. We found that elevated levels of the sphingolipid biosynthetic pathway's final product, mannosylinositol phosphorylceramide (MIPC), specifically correlated with the css1-3 phenotype. We also found that an apparent indicator of sphingolipids and a sterol biosensor accumulated at the cytosolic face of the PM at cell tips and the division site of css1-3 cells and, in accord, the PM in css1-3 was less dynamic than in wildtype cells. Interestingly, disrupting the protein glycosylation machinery recapitulated the css1-3 phenotype and led us to investigate Ghs2, a glycosylated PM protein predicted to modify cell wall material. Disrupting Ghs2 function led to aberrant cell wall material accumulation suggesting Ghs2 is dysfunctional in css1-3. We conclude that preventing an excess of MIPC in the S. pombe PM is critical to the function of key PM-localized proteins necessary for coupling growth with cell wall formation.

Hypothesis of cell wall metabolism disorder in segment drying: Evidence from vesicle collapse in ‘Dayagan’ hybrid citrus fruit

Segment drying is a common physiological disorder in citrus fruit during ripening and postharvest storage. The underlying mechanisms of this disorder are beginning to be understood, with the "Disorder of cell wall metabolism" hypothesis proposed as a major driving factor for nutrient loss and vesicle disorder during granulation-type segment drying. However, the extent to which this hypothesis applies to vesicle collapse-type segment drying is not well established. Thus, this study aimed to investigate whether the data on vesicle collapse support this hypothesis using 'Dayagan' fruit as material. The results showed that the collapsed vesicles exhibited deterioration of sugars and acids, while accumulating cell wall components such as protopectin and cellulose. Metabolic pathways altered during segment drying included the activation of sucrose and citrate degradation and synthesis of cell wall materials. Enzyme activity analyses revealed that the glutamine synthesis and acetyl-CoA pathways, but not the GABA shunt, were responsible for citrate depletion and that pectin de-methylesterification might be activated in vesicles during collapse. Systematic changes in gene expression patterns further revealed the complex regulation of metabolic pathways at the transcription level, where sucrose degradation and pectin metabolism may be regulated but not the acetyl-CoA and lignin synthesis pathways. These findings provide important insights into metabolic pathway changes that are conserved during vesicle collapse and granulation, supporting the hypothesis that disorder of cell wall metabolism may result in nutrient depletion and segment drying. This study provides a conceptual foundation for further research on vesicle collapse.

GhRabA4c coordinates cell elongation via regulating actin filament-dependent vesicle transport.

Plant cell expands via a tip growth or diffuse growth mode. In plants, RabA is the largest group of Rab GTPases that regulate vesicle trafficking. The functions of RabA protein in modulating polarized expansion in tip growth cells have been demonstrated. However, whether and how RabA protein functions in diffuse growth plant cells have never been explored. Here, we addressed this question by examining the role of GhRabA4c in cotton fibers. GhRabA4c was preferentially expressed in elongating fibers with its protein localized to endoplasmic reticulum and Golgi apparatus. Over- and down-expression of GhRabA4c in cotton lead to longer and shorter fibers, respectively. GhRabA4c interacted with GhACT4 to promote the assembly of actin filament to facilitate vesicle transport for cell wall synthesis. Consistently, GhRabA4c-overexpressed fibers exhibited increased content of wall components and the transcript levels of the genes responsible for the synthesis of cell wall materials. We further identified two MYB proteins that directly regulate the transcription of GhRabA4c Collectively, our data showed that GhRabA4c promotes diffused cell expansion by supporting vesicle trafficking and cell wall synthesis.

Genetic dissection of the signaling pathway required for the cell wall integrity checkpoint.

The cell wall integrity checkpoint monitors synthesis of cell wall materials during the Saccharomyces cerevisiae cell cycle. Upon perturbation of cell wall synthesis, the cell wall integrity checkpoint is activated, downregulating Clb2 transcription. Here, we identified genes involved in this checkpoint by genetic screening of deletion mutants. In addition to the previously identified dynactin complex, the Las17 complex, in particular the Bzz1 and Vrp1 components, plays a role in this checkpoint. We also revealed that the high osmolarity glycerol (HOG) and cell wall integrity mitogen-activated protein kinase (MAPK) signaling pathways are essential for checkpoint function. The defective checkpoint caused by the deficient dynactin and Las17 complexes was rescued by hyperactivation of the cell wall integrity MAPK pathway, but not by the activated form of Hog1, suggesting an order to these signaling pathways. Mutation of Fkh2, a transcription factor important for Clb2 expression, suppressed the checkpoint-defective phenotype of Las17, HOG MAPK and cell wall integrity MAPK mutations. These results provide genetic evidence that signaling from the cell surface regulates the downstream transcriptional machinery to activate the cell wall integrity checkpoint.

Functional redundancy of division specific penicillin-binding proteins in Bacillus subtilis.

SummaryBacterial cell division involves the dynamic assembly of a diverse set of proteins that coordinate the invagination of the cell membrane and synthesis of cell wall material to create the new cell poles of the separated daughter cells. Penicillin‐binding protein PBP 2B is a key cell division protein in Bacillus subtilis proposed to have a specific catalytic role in septal wall synthesis. Unexpectedly, we find that a catalytically inactive mutant of PBP 2B supports cell division, but in this background the normally dispensable PBP 3 becomes essential. Phenotypic analysis of pbpC mutants (encoding PBP 3) shows that PBP 2B has a crucial structural role in assembly of the division complex, independent of catalysis, and that its biochemical activity in septum formation can be provided by PBP 3. Bioinformatic analysis revealed a close sequence relationship between PBP 3 and Staphylococcus aureus PBP 2A, which is responsible for methicillin resistance. These findings suggest that mechanisms for rescuing cell division when the biochemical activity of PBP 2B is perturbed evolved prior to the clinical use of β‐lactams.

Membrane Organization and Cell Fusion During Mating in Fission Yeast Requires Multipass Membrane Protein Prm1

The involvement of Schizosaccharomyces pombe prm1(+) in cell fusion during mating and its relationship with other genes required for this process have been addressed. S. pombe prm1Δ mutant exhibits an almost complete blockade in cell fusion and an abnormal distribution of the plasma membrane and cell wall in the area of cell-cell interaction. The distribution of cellular envelopes is similar to that described for mutants devoid of the Fig1-related claudin-like Dni proteins; however, prm1(+) and the dni(+) genes act in different subpathways. Time-lapse analyses show that in the wild-type S. pombe strain, the distribution of phosphatidylserine in the cytoplasmic leaflet of the plasma membrane undergoes some modification before an opening is observed in the cross wall at the cell-cell contact region. In the prm1Δ mutant, this membrane modification does not take place, and the cross wall between the mating partners is not extensively degraded; plasma membrane forms invaginations and fingers that sometimes collapse/retract and that are sometimes strengthened by the synthesis of cell-wall material. Neither prm1Δ nor prm1Δ dniΔ zygotes lyse after cell-cell contact in medium containing and lacking calcium. Response to drugs that inhibit lipid synthesis or interfere with lipids is different in wild-type, prm1Δ, and dni1Δ strains, suggesting that membrane structure/organization/dynamics is different in all these strains and that Prm1p and the Dni proteins exert some functions required to guarantee correct membrane organization that are critical for cell fusion.

Changes in the ultrastructure of meristematic root cells of Allium sativum L. treated with selenium

Ultrastructure of meristematic cells of garlic (<em>Allium sativum</em> L.) roots treated with sodium selenate and sodium selenite was assessed using transmission electron microscopy. Both selenium compounds applied at the concentrations: 80, 160 and 320 µM caused many malformations in the ultrastructure of mitochondria, plastids, endoplasmic reticulum and Golgi apparatus such as deformation in shape and size, disturbances in inner membranes organization, appearance of concentric or parallel arrangement of ER cisternae. Moreover, in the presence of selenium, beside uneven thickening of cell wall, many vacuoles of different dimensions filled with wall-like material even in the vicinity of nucleus were visible. The latter results suggest that selenium not only intensified the synthesis of cell wall material but also inhibited the process of cell wall material deposition. The similarity of all observed ultrastructural changes in garlic root cells after selenium treatment with those appearing after action of other stress factors are discussed.

Flumorph Is a Novel Fungicide That Disrupts Microfilament Organization in Phytophthora melonis

ABSTRACT The mechanism of the effects of flumorph (a novel fungicide) was investigated by analyzing alterations of hyphal morphology, cell wall deposition patterns, F-actin organization, and other organelles in Phytophthora melonis. Calcofluor white staining suggested that flumorph did not inhibit the synthesis of cell wall materials, but disturbed the polar deposition of newly synthesized cell wall materials during cystospore germination and hyphal growth. After exposure to flumorph, zoospores were able to switch into cystospores accompanied with the formation of a cell wall, whereas cystospores failed to induce the isotropic-polar switch and did not produce germ tubes but continued the isotropic growth phase. In flumorph-treated hyphae, the most characteristic change was the development of periodic swelling ("beaded" morphology) and the disruption of tip growth. Newly synthesized cell wall materials were deposited uniformly throughout the diffuse expanded region of hyphae, in contrast to their normal polarized patterns of deposition. These alterations were the result of F-actin disruption, identified with the fluorescein isothiocynate (FITC)-phalloidin staining. The disruption of F-actin also was accompanied by disorganized organelles: each swelling of subapical hyphae was associated with a nucleus. Vesicles did not undergo polarized secretion to the apical hyphae, but diffused around nuclei for the subapical growth; thus, the cell wall was thickened with periodic expansion along the hyphae. Upon removing flumorph, normal tip growth and organized F-actin were observed again. These data, as well as data published earlier, suggest that flumorph may be involved in the impairment of cell polar growth through directly or indirectly disrupting the organization of F-actin. The primary site of action by flumorph in the disruption of the F-actin organization is under investigation.

Dispersed mode of Staphylococcus aureus cell wall synthesis in the absence of the division machinery.

We have developed several new fluorescent staining procedures that enabled us to study the synthesis of cell wall material in the spherical Gram-positive bacterium Staphylococcus aureus. The results obtained support previous proposals that these cells synthesize new wall material specifically at cell division sites, in the form of a flat circular plate that is subsequently cleaved and remodelled to produce the new hemispherical poles of the daughter cells. We have shown that formation of the septal peptidoglycan is dependent on the key cell division protein FtsZ, which recruits penicillin-binding protein (PBP) 2. Unexpectedly, in FtsZ-depleted cells, the cell wall synthetic machinery becomes dispersed and new wall material is made in dispersed patches over the entire surface of the cells, which increase in volume by up to eightfold before lysing. The results have implications for understanding the nature of S. aureus morphogenesis and for inhibitors of cell division proteins as drug targets.

Putative biological action of oligosaccharides on enzymes involved in cell-wall development

Introduction Primary plant cell wall is synthesized around protoplast and its architecture is determined by a complex balance in the synthesis of several polysaccharides and glycoproteins which form an interwoven network organized around cellulose microfibrils [ 13. Therefore deposition and synthesis of cell-wall material must accompany cell progressive enlargement, and this necessitates a permanent rearrangement of the already deposited material and of the newly synthesized polymers. The chemical and biological nature of the rearrangements that occur within a given constituent polymer or between polymers, the site of their occurrence within the cell wall, and the physical, physiological and biochemical factors that control them, are still not clearly understood. Among the regulatory processes that control cell expansion, auxin-induced growth [2] involves a proton extrusion into the apoplast which may selectively activate wall-associated hydrolases with low pH optima [3]. Cleavage of hemicelluloses, especially xyloglucans (XGs), which are the predominant hemicelluloses in the primary wall of higher plants, has been observed in auxin-treated and acid-treated pea stems [4,5]. This degradation by wall-bound enzymes may have several consequences that serve cell-wall rearrangement. It may result in the breakage of XG interconnections between microfibrils [ 51, thus accounting for wall loosening, and it may at the same time result in the release of biologically active fragments with various lengths and structures. These biologically active oligosaccharides belong to the so-called oligosaccharins, which all possess signalling and regulatory activities [6].

Laser microsurgery: a versatile tool in plant (electro) physiology

In plant cells the cell wall is a formidable obstacle in many physiological studies such as patch-clamp measurements and cell labelling with antibodies. Enzymatic digestion of the cell wall, in order to release a protoplast, has a number of disadvantages; therefore we worked out an alternative method to gain access to the plasma membrane. The wall of specialized cells from three higher plant species and one unicellular alga were perforated using the focussed UV light of a nitrogen laser. In order to enhance the absorption of the UV light by the walls, a dye was used that binds specifically to cell wall components. Extrusion of the protoplast or parts thereof was controlled by a regulated gradual decrease of the osmolarity of the solution surrounding the cells. Cytoplasmic streaming and chloroplast circulation were maintained in the protoplasts, demonstrating their viability after the wall perforation with the laser. Continuous deposition of new cell wall material by the polar tip of pollen tubes after surgical removal of the wall at the tip is another demonstration of the viability of the cells. Formation of high resistance seals between the plasma membrane and a patch pipet was surprisingly difficult. The role of ‘Hechtian strands’ and continuing synthesis of cell wall material in seal formation is further investigated. Other applications for the surgical laser are: fusion of two cells or vacuoles, analysis of the composition of specific parts of the cell wall, and release of the vacuole from an identified cell type for patchclamp studies.

Effects of tensile force and nutrient availability on carbon uptake and cell wall synthesis in blades of juvenile Egregia menziesii (Turn.) Aresch. (Phaeophyta)

Although field studies have demonstrated that algal morphology and biomechanics are correlated with water motion, the physical cue(s) eliciting those whole plant responses are unknown, as are the physiological changes that underlie the responses. Because changes in morphology and biomechanics involve changes in C metabolism, juvenile feather boa kelp Egregia menziesii Turn. (Aresch.) were incubated in the presence of H 14CO 3 − to identify experimentally synthesized materials. Four combinations of nutrient availability and tensile force (both varying with water motion) were employed to investigate the incorporation of 14C into whole tissue and cell wall compounds. Force significantly increased 14C incorporation into whole tissue, while nutrient availability and tissue age had no significant effect. The synthesis of cell wall materials was significantly increased by force application, was greater at low nutrient levels, and greater in younger tissue adjacent to the meristem. Tissue age and nutrient availability had significant interactive effects on cell wall synthesis.

Formation of competent Bacillus subtilis cells.

The process of competent cell formation for transformation has been studied with early-stationary-phase (T1) cells of Bacillus subtilis which had been grown in an enriched Spizizen minimal medium and transferred to a second synthetic medium. Rifampin, chloramphenicol, and tunicamycin were strong inhibitors of competent cell formation, as well as vegetative growth. After formation, competent cells were no longer sensitive to the above agents. Methicillin and an inhibitor of chromosomal replication, hydroxyphenylazouracil, did not inhibit the development of competence. A D-alanine-requiring mutant strain developed competence even in the absence of D-alanine in the second medium. A T1-stage culture showed the activity of extracellular serine protease which is necessary for sporulation. Competent cell formation was completely blocked by 0.7 M ethanol, which is a specific inhibitor of early events during sporulation, including forespore septum formation. Competent cells were formed even in media which supported sporulation. The development of competence was also studied with spo0 mutants at 10 different loci. Most spo0 mutations repressed the development of competence except for spo0C, spo0G, and spo0J. These results suggest that competent cells are formed from early sporulating cells with the synthesis of cell wall materials and by factors whose genes are activated by the supply of nutrients. It is suggested that common steps are involved both in forespore septation and in competent cell formation.

Regulation of Glucose Metabolism and Cell Wall Synthesis in Avena Stem Segments by Gibberellic Acid

Gibberellic acid (GA) stimulated both the elongation of Avena sativa stem segments and increased synthesis of cell wall material. The effects of GA on glucose metabolism, as related to cell wall synthesis, have been investigated in order to find specific events regulated by GA. GA caused a decline in the levels of glucose, glucose 6-phosphate, and fructose 6-phosphate if exogenous sugar was not supplied to the segments, whereas the hormone caused no change in the levels of glucose 6-phosphate, fructose 6-phosphate, UDP-glucose, or the adenylate energy charge if the segments were incubated in 0.1 m glucose. No GA-induced change could be demonstrated in the activities of hexokinase, phosphoglucomutase, UDP-glucose pyrophosphorylase, or polysaccharide synthetases using UDP-glucose, UDP-galactose, UDP-xylose, and UDP-arabinose as substrates. GA stimulated the activity of GDP-glucose-dependent beta-glucan synthetase by 2- to 4-fold over the control. When glucan synthetase was assayed using UDP-glucose as substrate, only beta-1,3-linked glucan was synthesized in vitro, whereas with GDP-glucose, only beta-1,4-linked glucan was synthesized. These results suggest that one part of the mechanism by which GA stimulates cell wall synthesis concurrently with elongation in Avena stem segments may be through a stimulation of cell wall polysaccharide synthetase activity.

Intracellular Outgrowth of the Cell Wall as a Possible Cause of Senescence in <i>Taphrina maculans</i> Butler

Taphrina maculans Butler, a causal agent of leaf spot of turmeric (Curcuma longa L.) forms two types of colonies namely “salmon-red” and “creamy-white”. The former strain dies abnormally under optimal nutritional conditions while the latter does not behave so. Electron microscopic observations of both the strains manifest for the first time, an extra intracellular outgrowth from an undetermined site of cell wall that grows further traversing the entire cellular part resulting into the death of the cells. The dead cells become white. The plasmamembrane pro-bably extends further during the synthesis of the extra outgrowth material serving as the boundary wall between the extra outgrowth and the cytoplasm. Such phenomenon of extra synthesis of cell wall material that causes the death of the cell has been observed in the salmon-red strain only.

The amounts and rates of export of polysaccharides found within the membrane system of maize root cells.

1. Maize seedling roots were incubated in vivo with d-[U-(14)C]glucose for 2, 5, 10, 15, 30 and 45min. The total incorporation of radioactivity into polysaccharide components in isolated fractions was investigated, and the pattern of incorporation into different polysaccharide components within the rough endoplasmic reticulum, Golgi apparatus and exported material was analysed. 2. The membrane compartments reached a saturation value of radioactivity in polysaccharide components by 30min incubation. Radioactivity in exported polysaccharide continued to increase after that time. The latter was formed and maintained by a steady-state turnover of polysaccharide synthesis and transport from the membrane system. 3. If the only access of the slime polysaccharide to the cell surface is via dictyosome-derived vesicles, the amount of slime components in the Golgi apparatus would have to be displaced every 0.3min in order to maintain the observed rates of increase in slime. This is in contrast with a displacement time of about 2.5min that is necessary for polysaccharide components in the Golgi apparatus to produce the observed increase in cell-wall material. The activity of the membrane system in the production of maize root slime is 8 times as great as that of the membrane system involved in cell-wall synthesis. 4. If the amount of polysaccharide material in the Golgi apparatus is maintained only by inflow of polymeric material from the rough endoplasmic reticulum the total amount of slime components in the rough endoplasmic reticulum would have to be displaced every 7min to maintain a constant amount in the Golgi apparatus. If the endoplasmic reticulum contributed directly to the cell surface in the synthesis of cell-wall material, displacement times necessary to maintain the observed rate of polymer production would be very slow.

The initiation of cell wall synthesis in parasynchronous cultures of Schizosaccharomyces pombe

Schizosaccharomyces pombe has been grown in parasynchronous culture to study the synthesis of cell wall material. After a lag period of 2.5h following inoculation the cells began to grow, as measured by optical density, dry weight and cell size. The cell number remained constant until 4.5h after inoculation when approximately 70% of the population divided synchronously. Immunofluorescence studies of the growing cells have shown that new wall material is inserted at the cell apices from 2.5 h after inoculation; this result is supported by radio-isotope labelling data which indicated that synthesis of new cell wall material also commenced 2.5 h after inoculation. The incorporation experiments also demonstrated an interruption in cell wall synthesis during the cell separation stage. The composition of the cell wall material varied during the growth cycle, with maximum nitrogen levels at inoculation and following cell division. No serological differences could be detected in the cell walls during the growth cycle.

Effect of Intramuscular Injection of Penicillin on Bacterial Spoilage

The use of penicillin and various other antibiotics for therapeutic purposes is well established. Antibiotics have also been used in nutrition (Jukes, 1955). The bacteriostatic and bacteriolytic effect of various penicillins has been reported to be due to the interference of the synthesis of cell wall mucopeptides (Park and Strominger, 1957). However, the site of interference of cell wall synthesis is unknown.The importance of egg shell structures in resisting microbial spoilage has been amply demonstrated by several workers. Baker and Balch (1962), reported that the egg shell membranes are made up of N-acetylhexosamine blocks. Since penicillin affects the synthesis of cell wall material which is made up of similar building blocks, a study was undertaken to study the effect of therapeutic doses of penicillin on the spoilage of eggs laid by such birds.The eggs from a single strain of White Leghorns, 22 weeks of age, were used. Six…

Rasch ablaufende �nderungen im Gehalt an l�slichen Zuckern und Zellwandkohlenhydraten bei der phytochrominduzierten Photomorphogenese des Senfkeimlings (Sinapis alba L.)

Short term changes in the soluble sugar, starch, and cell-wall carbohydrate content of the mustard seedling have been studied in the different organs during phytochrome induced photomorphogenesis in continuous far-red. The program was: imbibition of seeds →36 hrs dark → far-red irradiation. Kinetics have been followed up to 12 hrs after the onset of irradiation. There are no substantial changes in carbohydrate content in the cotyledons and the radicle. In the cotyledons in far-red after a lag-phase of 3 hrs, there is a decrease in oligosaccharide content, and after a lag-phase of 6 hrs, an increase in cell-wall synthesis. The reducing sugar and starch content is not altered upon irradiation. In the radicle immediately after the onset of far-red, there is a temporary rise in the reducing sugar and cell-wall carbohydrate content. However, 6 hrs later the values in far-red again parallel those of the dark control. The important phytochrome dependent changes take place in the hypocotyl. In far-red after a lag-phase of 3 hrs the glucose accumulation is markedly retarded, the sucrose and starch content no longer increased, and the fructose content even decreases below the 3 hrs value. The glucose: fructose ratio, which is constant in dark, is shifted in favour of glucose. The lag-phase of phytochrome controlled hypocotyl elongation is about 1 hr, the lag-phase of the inhibition of cell-wall carbohydrate synthesis is in about the same order of magnitude. There seems to be neither any immediate connection between sugar content and cell-wall carbohydrate synthesis, as shown by the difference in lag-phases, nor does there seem to be any direct relationship between hypocotyl inhibition and overall synthesis of cell-wall material. The relative inhibition of cell-wall synthesis is less than one third of that of hypocotyl elongation (Figs. 5,6). Apparently phytochrome controls hypocotyl elongation by influencing the cell-wall structure. In spite of the fact that fat degradation is higher in far-red than in dark and respiration higher in dark than in far-red (Friederich, 1968), 6 hrs after the onset of far-red the increase of total carbohydrate content declines compared with that in dark. This finding leads to the conclusion that the efficiency of the fat-carbohydrate-transformation is higher in dark than in far-red.

Resemblance of Lomasomes of Pythium debaryanum to Structures recently described in Chara and Nitella

CYTOPLASMIC organelles associated with the cell walls of Chara and Nitella were illustrated in glutaraldehyde-fixed material by Crawley1. Similar structures were demonstrated in Chara by Barton2. These organelles consist of a network of fine tubules. They are bounded on the inner side by a membrane continuous with the cell membrane or plasmalemma and on the outer side are in contact with the cell wall. It was suggested that they might play a part in the synthesis of cell wall material.