N-Glycyl-glucosamine: A novel constituent in the cell wall of Halococcus morrhuae

The dinitrophenyl-derivative of N-glycylglucosamine was isolated from partially hydrolyzed dinitrophenylated cell walls of Halococcus morrhuae CCM 859. To increase the yield of amino-terminal glycine residues, halococcal cell walls were treated with alkali or acid prior to dinitrophenylation. Authentic N-glycyl-glucosamine was used as a reference substance. A substitution of the amino group of glucosamine by an amino acid has so far not been found in any other wall of a pro- or eucaryotic cell. Since only 5% of the glycine residues reveal an unsubstituted carboxyl group within intact cell walls, glycine may play a role in connecting glycan strands through peptidic linkages between the amino group of glucosamine and the carboxyl group of an uronic acid or gulosaminuronic acid.Abbreviations DNP dinitrophenyl     

Deposition and reorientation of cellulose microfibrils in elongating cells of Petunia stylar tissue

According to Roelofsen and Houwink's (1953, Acta Bot. Neerl. 2, 218–225) multinet growth hypothesis, microfibrils originally deposited transversely in the cell wall become gradually reoriented towards more axial orientations during cell elongation. To establish the extent of reorientation, microfibrils were studied during their deposition and elongation, using stylar parenchyma and transmitting tissue cells of Petunia hybrida L. At the inner surface of very young cells, microfibrils were deposited in alternating Z- and S-helical orientations. The following sequence in deposition, from the exterior to the interior side of the wall, could be inferred: Axial: 150°–180° (Z-helical), 0°–30° (S-helical); oblique: 110°–150° (Z-helical), 30°–70° (S-helical); transverse: 90°–110° (Z-helical), 70°–90° (S-helical). With the increasing pitch, the density of the deposited microfibrils increased as well, giving rise to an alternating helical texture. During elongation, only transversely S- and Z-helically oriented microfibrils were deposited and all microfibrils underwent a certain reorientation as described in the multinet growth hypothesis. The texture resembled that of young cells and the wall maintained its thickness. The extent of passive reorientation was in agreement with the theoretical calculations made by Preston.Dedicated to Professor Dr. A.B. Wardrop, Melbourne, on the occasion of his 70th birthday     

Cell elongation and cell division in elongating lettuce hypocotyl sections

The roles of cell division and cell elongation in the growth of sections excised from hypocotyls of lettuce (Lactuca sativa L. cv. Arctic) were investigated. Elongation of sections incubated in the light is inhibited compared to dark-grown sections and this inhibition is reversed by gibberellic acid (GA₃). The elongation of both dark-grown and GA₃-treated, light-grown sections can be enhanced by 10mM KCl. Under all conditions of incubation, elongation growth is greatest in the uppermost quarter of the hypocotyl section while the basal quarter does not elongate. In darkness the two apical segments of sections marked into four equal parts grow at the same rate, while in light, growth of the apical segment exceeds that of the second segment. Cell division in cortical or epidermal cells, as measured by mitotic index or cell number, is not affected by illumination conditions nor by GA₃ or KCl treatments. Although -irradiation and FUDR pretreatment eliminate or cause a marked reduction in cell division in the excised hypocotyl, sections from seeds irradiated with -rays or incubated in 5-fluorodeoxyuridine elongate in response to GA₃ and KCl treatment as do sections from non-pretreated controls. Therefore, since neither GA₃ nor darkness affect celldivision activity and since treatments which eliminate or significantly reduce cell division do not affect growth, we conclude that the effect of GA₃ and darkness in this material is to increase cell elongation.Abbreviations FUDR 5-fluorodeoxyuridine - GA(s) gibberellin(s) - GA₃ gibberellic acid     

Composition of the cell wall of Chlorella fusca

Isolated cell walls of mature Chlorella fusca consisted of about 80% carbohydrate, 7% protein, and 13% unidentified material. Mannose and glucose were present in a ratio of about 2.7:1 and accounted for most of the carbohydrate. Minor components were glucuronic acid, rhamnose, and traces of other sugars; galactose was absent. After treatment with 2 M trifluoroacetic acid or with 80% acetic acid/HNO₃ (10/1, v/v), a residue with a mannose/glucose ratio of 0.3:1 was obtained, probably representing a structural polysaccharide. An X-ray diffraction diagram of the walls showed one diffuse reflection at 0.44 nm and no reflections characteristic of cellulose. Walls from young cells contained about 51% carbohydrate, 12% protein, and 37% unidentified material. Mannose and glucose were also the main sugars; their absolute amounts per wall increased 6–7 fold during cell growth. Walls isolated with omission of a dodecylsulphate/mercaptoethanol/urea extraction step had a higher protein content and, with young walls, a significantly higher glucose and fucose content. These data and other published cell wall analyses show a wide variability in cell wall composition of the members of the genus Chlorella.Abbreviations GLC gas liquid chromatography - TFA trifluoroacetic acid     

A helicoidal cell wall texture in root hairs ofLimnobium stoloniferum

Summary The cell wall of root hairs ofLimnobium stoloniferum is composed of two fibrillar layers: an outer layer with a dispersed texture and an inner layer with a helicoidal texture. In stained oblique sections the helicoidal layer appears as a series of bow-shaped structures. In sections which were shadow-casted after the embedding medium was removed, the following properties of the helicoidal layer can be directly observed. (1) It is build up of superimposed lamellae. (2) Each lamella consists of parallel oriented microfibrils. (3) Going into the helicoidal layer, there is a counter-clockwise discontinuous rotation of the microfibril orientation in successive lamellae. (4) Between adjacent lamellae the average angular displacement of the microfibril orientation is about 23 degrees. The dispersed outer layer is also polylamellated, but with randomly arranged microfibrils in each lamella. Both layers are present in the lateral wall as well as in the apical wall of the root hairs. Observations indicate that in the cell wall of the tip the parallel oriented microfibrils of the outermost helicoidal lamellae become distorted towards a dispersed arrangement. The suggestion is made that the dispersed outer layer is derived from the helicoidal layer.     

A small trypsin inhibitor from the frog of Odorrana grahami

A novel peptide inhibitor (OGTI) of serine protease with a molecular weight of 1949.8, was purified from the skin secretion of the frog, Odorrana grahami. Of the tested serine proteases, OGTI only inhibited the hydrolysis activity of trypsin on synthetic chromogenic substrate. This precursor deduced from the cDNA sequence is composed of 70 amino acid residues. The mature OGTI contains 17 amino acid residues including a six-residue loop disulfided by two half-cysteines (AVNIPFKVHFRCKAAFC). In addition to its unique six-residue loop, the overall structure and precursor of OGTI are different from those of other serine protease inhibitors. It is also one of the smallest serine protease inhibitors ever found.     

Inheritance of qualitative and quantitative trypsin inhibitor variants in Pisum

A trypsin inhibitor locus (Tri) has been mapped close to Vc-2 on Pisum (pea) linkage group 5 using recombinant inbred lines derived from crosses of genotypes showing qualitative variation in seed trypsin inhibitors. F₂ seed populations derived from crosses between lines showing qualitative variation in trypsin inhibitors as well as quantitative variation in inhibitor activity showed an association between the segregation of the structural variation and relative activity levels. Clones complementary to Pisum trypsin inhibitor mRNA were used in hybridization analyses which showed that the segregation of protein polymorphisms reflected directly the segregation of polymorphisms associated with the structural genes.     

Occurrence of cell surface arabinogalactan-protein and extensin epitopes in relation to pericycle and vascular tissue development in the root apex of four species

Monoclonal antibodies recognizing two classes of developmentally regulated plant cell surface components – arabinogalactan-proteins (AGPs) and extensins – have been used to immunolabel cells at the root apices of four species with different characteristics of pericycle and vascular tissue development. Root apices of pea (Pisum sativum L.), radish (Raphanus sativus L.), carrot (Daucus carota L.) and onion (Allium cepa L.) were immunolabelled with the anti-AGP monoclonal antibodies JIM4 and JIM13 and anti-extensin monoclonal antibodies JIM11, JIM12, JIM19 and JIM20. All of these antibodies recognized subsets of pericycle cells in at least one, but never all, of these species. The restricted patterns of epitope occurrence also reflected vascular cell development. The differences in patterns of antibody recognition in the four species are discussed in relation to the possible roles of these cell surface molecules in cell differentiation and root patterning events. Received: 11 March 1997 / Accepted: 20 May 1997     

Regeneration of vascular tissue in wounded pea roots

Severance of the stele of young main roots of pea (Pisum sativum L.) results in formation of a bridge of vascular tissue in the remaining cortex. Cell divisions occur close to the severed vascular tissues on both the proximal and distal sides of the cut within 24 h. Differentiation of new vascular strands subsequently begins in the same locations and progresses from both sides of the wound into the remaining cortex and also back along the original vascular strands. Most of the vascular tissue which forms the bridge through the cortex differentiates in the acropetal direction. Continuous strands composed of single sieve elements bypass the wound somewhat sooner than the first complete xylem strands; the latter in 60–70% of the cases, are present by 3 d. Cambial activity subsequently adds more xylem and phloem. Vascular regeneration is not affected by removal of the epicotyl or the root tip; it is greatly reduced but not prevented by removal of the cotyledons.     

A Kunitz trypsin inhibitor from chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) that exerts anti-metabolic effect on podborer (<Emphasis Type="Italic">Helicoverpa armigera</Emphasis>) larvae

Chickpea (Cicer arietinum L.) seeds contain Bowman–Birk proteinase inhibitors, which are ineffective against the digestive proteinases of larvae of the insect pest Helicoverpa armigera. We have identified and purified a low expressing proteinase inhibitor (PI), distinct from the Bowman–Birk Inhibitors and active against H. armigera gut proteinases (HGP), from chickpea seeds. N-terminal sequencing of this HGP inhibitor revealed a sequence similar to reported pea (Pisum sativum) and chickpea -l-fucosidases and also homologous to legume Kunitz inhibitors. The identity was confirmed by matrix assisted laser desorption ionization – time of flight analysis of tryptic peptides and isolation of DNA sequence coding for the mature protein. Available sequence data showed that this protein forms a distinct phylogenetic cluster with Kunitz inhibitors from Glycine max, Medicago truncatula, P. sativum and Canavalia lineata. The isolated coding sequence was cloned into a yeast expression vector and produced as a recombinant protein in Pichia pastoris. -l-fucosidase activity was not detectable in purified or recombinant protein, by solution assays. The recombinant protein did not inhibit chymotrypsin or subtilisin activity but did exhibit stoichiometric inhibition of trypsin, comparable to soybean Kunitz trypsin inhibitor. The recombinant protein exhibited higher inhibition of total HGP activity as compared to soybean kunitz inhibitor, even though it preferentially inhibited HGP-trypsins. H. armigera larvae fed on inhibitor-incorporated artificial diet showed significant reduction in average larval weight after 18 days of feeding demonstrating potent antimetabolic activity. The over-expression of this gene in chickpea could act as an endogenous source of resistance to H. armigera.     

Absorption and fluorescence spectra ofS-quinolylethylated Kunitz soybean trypsin inhibitor

Disulfide bonds in soybean trypsin inhibitor (Kunitz) were simultaneously reduced and alkylated using tri-n-butylphosphine and 2-vinylquinoline at pH 7.6 in 0.11 M Tris-4.4 M urea, 41% ethanol. The resulting S--2-quinolylethylated protein (2-QE-STI) has a new absorption peak at 315–318 nm. Its quinoline fluorescence can be excited above 310 nm independently of intrinsic protein fluorescence. Free 2-quinolylethylcysteine (2-QEC) shows unexpectedly weak fluorescence. Quinoline absorption in 2-QEC and 2-QE-STI changes with pH. The apparentpK values determined spectrophotometrically are near 5 for 2-QEC and 3 for 2-QE-STI. Fluorescence decreased with increasing pH and in the presence of chloride ions. Both structural and charge effects thus appear to influence the absorption and fluorescence of the quinoline group. Corrected fluorescence emission (excited at 316 nm) of neutral 2-QE-STI diluted in 0.1 N H₂SO₄ was directly proportional to concentration in the range 0.4–8 m 2-QEC. The 2-QEC content of the protein derivative determined by UV absorption at pH 1.5 was in agreement with the expected value of four residues per mole. Fluorescence measurements ofS-2-quinolylethylated proteins may be especially useful as a sensitive, specific assay for cyst(e)ine residues.Reference to a company or product name does not imply approval or recommendation of the product by the U.S. Department of Agriculture to the exclusion of others that may be suitable.Abbreviations used are Mops: 3-(N-morpholino)propanesulfonic acid; STI: soybean trypsin inhibitor (Kunitz); 2-PE-STI:S--2-pyridylethylated STI; 2-QEC:S--(2-quinolylethyl)-l-cysteine; 2-QE-STI:S--2-quinolylethylated STI; TosPheCH₂-trypsin: bovine trypsin treated withp-toluenesulfonyl phenylalanine chloromethyl ketone.     

A comparative study on cell wall antigens and cell surface hydrophobicity in clinical isolates ofCandida albicans

Characterization of common cell surface-bound antigens inCandida albicans strains, particularly those expressed in the walls of mycelial cells might be useful in the diagnosis of systemic candidiasis. Hence, antigenic similarities among wall proteins and mannoproteins fromC. albicans clinical serotype A and B isolates, were studied using polyclonal (mPAbs) and monoclonal (MAb 4C12) antibodies raised against wall antigens from the mycelial form of a commonC. albicans serotype A laboratory strain (ATCC 26555). Zymolyase digestion of walls isolated from cells of the different strains studied grown at 37°C (germination conditions), released, in all cases, numerous protein and mannoprotein components larger than 100 kDa, along with a 33–34 kDa species. The pattern of major antigens exhibiting reactivity towards the mPAbs preparation was basically similar for all the serotype A and B isolates, though minor strain-specific bands were also observed. The immunodeterminant recognized by MAb 4C12 was found to be absent or present in very low amounts inC. albicans isolates other than the ATCC 26555 strain, yet high molecular weight species similar in size (e.g., 260 kDa) to the wall antigen against which MAb 4C12 was raised, were observed, particularly in wall digests from serotype A strains. Cell surface hydrophobicity, an apparently important virulence factor inC. albicans, of the cell population of each serotype B strain was lower than that of the corresponding serotype A counterparts, which is possibly due to the fact that the former strains exhibited a reduced ability to form mycelial filaments under the experimental conditions used.Abbreviations CSH cell surface hydrophobicity - IIF indirect immunofluorescence     

Attachment of bean rust cell wall material to host and non-host plant tissue

A preparation of bean rust (Uromyces phaseoli) germ tube walls, consisting of short, filamentous particles, was labeled with fluorescein iso-thiocyanate. Freeze sections of host and non-host tissue were incubated in the labeled preparation. Maximum staining was observed in host plant tissue (Phaseolus vulgaris), in which bean rust regularly forms haustoria. In tissue of the non-host plantsVigna sinensis andPhaseolus lunatus, where fewer haustoria were formed, staining was only weak. However, no staining was observed in the non-host tissue ofPhaseolus aureus, Helianthus annuus, Brassica oleracea andHordeum vulgare in which the infection hypha did not form haustoria. This would appear to indicate that formation of haustoria is induced by a specific attachment of the hyphal wall to the host wall. The possibility that elicitors attach in a similar way, is discussed.Abbreviations FITC Fluorescein iso-thiocyanate - cv cultivar     

Phenotype analysis of Saccharomyces cerevisiae mutants with deletions in Pir cell wall glycoproteins

Proteins with internal repeats (Pir) belong to a minor group of covalently linked yeast cell wall proteins. They are not essential for viability but important for cell wall strength, reduced permeability against plant antifungal enzymes and maintenance of osmotic stability. Here we show the importance of Pir proteins of Saccharomyces cerevisiae for growth at low pH and in presence of various inhibitors. Cell wall analysis of Deltapir1,2,3,4 deletion strain revealed slightly increased chitin content and changes in relative proportion of alkali-soluble and insoluble glucan and chitin fractions. Activation of the cell wall integrity pathway was indicated by increased levels of double phosphorylated Mpk1p/Slt2p in the pir deletants.     

Effects of colchicine on cell shape and on microfibril arrangement in the cell wall of Closterium acerosum

Cell morphogenesis in Closterium acerosum (Schrank) Ehrenberg was greatly influenced by colchicine. Addition of colchicine to the medium led to production of tadpole-shaped cells, by decreasing the length and increasing the thickness of the new semicells. Transversely oriented wall microtubules and microfibrils, characteristic of normally elongating semicells, were not observed in colchicine-treated semicells, randomly oriented microfibrils being present instead. About 3.5 h after septum formation, the randomly oriented microfibrils began to be overlaid by bundles of microfibrils as seen in normal semicells at the later stage of elongation. When colchicine treatment was terminated 1 h after septum formation, cell elongation was partially restored and microfibrils were deposited parallel to each other and transversely to the cell axis, indicating that the effect of colchicine on microfibril arrangement in growing semicells is reversible.     

Characterization of a Kunitz trypsin inhibitor with one disulfide bridge purified from Swartzia pickellii

Swartzia pickellii is a Leguminosae that belongs to the Caesalpinioideae sub-family the Swartzia pickellii Trypsin Inhibitor (SWTI), a serine proteinase inhibitor was isolated from its seeds. SWTI is a single polypeptide chain protein and it's structure has 174 amino acid residues, it homologous to other Kunitz plant inhibitors, however shows some major differences: it contains only one disulfide bridge, instead two which are usually found in plant Kunitz inhibitors, and the SWTI reactive site does not contain the usual Arg or Lys residues at the putative reactive site (position 65). A glycosylation site was detected at Asn38 with 1188 kDa carbohydrate portion. The primary structure micro heterogeneity was found combining the sequence determination and mass spectrometry. Three forms of SWTI were actually defined: two glycosylated forms a 20,204 kDa (Arg 165) and 20,185 kDa (His 165) and one deglycosylated form 19,016 kDa (Arg 165), all of them contain a Met residue at position 130.     

A novel type of teichoic acid from the cell wall of Bacillus subtilis VKM B-762

The cell wall of Bacillus subtilis VKM B-762 contains, along with 1,5-poly[4-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)ribitol phosphate], a novel type of glycopolymer involving three types of inter-monomeric bonds in the repeating unit, viz., amide, glycosidic and phosphodiester:Such a structural pattern of natural glycopolymers has been hitherto unknown. This polymer represents a novel type of teichoic acids.     

Proline-rich cell wall proteins accumulate in growing regions and phloem tissue in response to water deficit in common bean seedlings

Plant cell walls undergo dynamic changes in response to different environmental stress conditions. In response to water deficit, two related proline-rich glycoproteins, called p33 and p36, accumulate in the soluble fraction of the cell walls in Phaseolus vulgaris (Covarrubias et al. in Plant Physiol 107:1119–1128, 1995). In this work, we show that p33 and p36 are able to form a 240 kDa oligomer, which is found in the cell wall soluble fraction. We present evidence indicating that the highest accumulation of these proteins in response to water deficit occurs in the growing regions of common bean seedlings, particularly in the phloem tissues. These proteins were detected in P. vulgaris cell suspension cultures, where the p33/p36 ratio was higher under hyperosmotic conditions than in bean seedlings subjected to the same treatment. The results support a role for these proteins during the plant cell response to changes in its water status, and suggest that cell wall modifications are induced in active growing cells of common bean in response to water limitation. Marina Battaglia and Rosa M. Solórzano contributed equally to this work.