Studies on the cell surface of zoospores and cysts of the fungus Phytophthora cinnamomi: nature of the surface saccharides as determined by quantitative lectin binding studies

The nature of the surface saccharides of zoospores, "partially encysted zoospores" and cysts of the root-rotting fungus Phytophthora cinnamomi, has been examined by quantitative lectin binding studies. Zoospores bound concanavalin A (Con A), but did not bind any of a variety of other lectins tested. In contrast, both cysts and "partially encysted zoospores" bound soybean agglutinin (SBA) as well as Con A. This indicates that accessible alpha-D-glucosyl/alpha-D-mannosyl-containing glycoconjugates predominate at the zoospore surface, whereas both alpha-D-glucosyl/alpha-D-mannosyl and galactosyl and/or N-acetyl-D-galactosaminosyl residues are accessible at the surface of cysts and "partially encysted zoospores." Neither Ulex europeus lectin nor wheat germ agglutinin (WGA) bound to any of the three cell preparations, indicating the absence of accessible alpha-L-fucosyl and N-acetyl-D-glucosaminosyl residues.     

Stimulation of growth of culturedNicotiana tabacum W 38 pollen tubes by poly(ethylene glycol) and Cu(II) salts

Summary Growth of pollen tubes ofNicotiana tabacum W 38 in a defined liquid medium buffered at pH 5.9 and containing sucrose, amino-acids, boric acid, salts and an antibacterial agent was stimulated by the addition of poly(ethylene glycol) 6000 (PEG-6000) and Cu_(II) salts. In the absence of both these supplements, up to 50% of the hydrated pollen grains did not develop further, and the germinated tubes were slow-growing and abnormal, with thickened walls, kinked growth, and fragile, swollen tips containing granular cytoplasm. Addition of 10–15% (w/v) purified PEG-6000 increased germination to 80–90% and prevented the progressive bursting of pollen grains and tube tips, but growth was still slow and kinked and tips remained swollen. Addition of 30 M CuSO₄ did not stimulate germination or prevent tip bursting, but produced straight-growing tubes with smooth-sided tips resembling the tips of tubes growing through stylar tissue; the free Cu²⁺ concentration under these conditions was about 1.0 M due to chelation by amino-acids, and similar tube morphologies were obtained with 1.0–1.5 M added CuSO₄ when NH₄Cl replaced the amino-acids. When the medium containing amino-acids was supplemented with both 12.5% PEG-6000 and 30 M CuSO₄, long-term (48 h) growth of straight pollen tubes with smooth-sided tips, thin walls and long ladders of callose plugs was observed; growth occurred at 250 m/h, approximately 30–40% of the rate observed in the style. Although omission of CuSO₄ from this complete medium severely affected tube growth and callose plug deposition, it did not alter the timing of generative-nucleus division, and thus the different parameters associated with the second phase of pollen-tube growth can be uncoupled in culture. High levels of FeSO₄ (300 M) had a similar morphogenetic effect to CuSO₄, but addition of 300 M L-ascorbate or D-iso-ascorbate was required to prevent precipitation of Fe_(III) oxide and prolong the stimulation of pollen-tube growth; EDTA removed the morphogenetic effect of both CuSO₄ and FeSO₄. Further, an impure grade of PEG-4000 was contaminated with an organic morphogen that allowed continued slow growth of pollen tubes with smooth, straight-sided tips in the absence of added CuSO₄ or FeSO₄, with tube morphology unaffected by ascorbate or EDTA. However, the long-term morphogenetic effect of trace levels of CuSO₄ suggests that Cu_(II) salts play an important role in pollen-tube development in at least this species ofNicotiana.Abbreviations A₄₇₅ absorbance at 475 nm - DAPI 4,6-diamidino-2-phenylindole - EDTA ethylene-diamine N,N,N,N-tetraacetic acid - MES 2-(N-morpholino)-ethane sulphonic acid - OG ordinary grade of poly(ethylene glycol) - PEG poly(ethylene glycol) - SP Specially Purified for Biochemistry grade of poly(ethylene glycol)     

Arabinogalactan-Proteins of the Female Sexual Tissue of Nicotiana alata: I. Changes during Flower Development and Pollination

Arabinogalactan-proteins (AGPs), isolated from the pistils of Nicotiana alata, an ornamental tobacco, are developmentally regulated. Both the total amount and concentration of AGP in the stigma increase during flower development, reaching 10 micrograms AGP/stigma at maturity. In contrast, AGP concentration in the style remains constant throughout the maturation period reaching 12 micrograms AGP/style at maturity. The classes of AGP present in the stigma and style during flower development, separated according to their charge by crossed-electrophoresis, are different and change during development. Pollination of flowers of N. alata with compatible or incompatible pollen results in a significant and reproducible increase in the amount of AGPs in the stigma, but not the style, compared with control unpollinated pistils. Pollination with ethanol vapor inactivated pollen also results in an increase in the amount of AGP in the stigma, but this is less than half that observed following pollination with viable pollen. There are no significant differences in the classes of AGP, based on crossed-electrophoresis, present in the pistil following pollination.     

High-throughput mapping of cell-wall polymers within and between plants using novel microarrays

We describe here a methodology that enables the occurrence of cell-wall glycans to be systematically mapped throughout plants in a semi-quantitative high-throughput fashion. The technique (comprehensive microarray polymer profiling, or CoMPP) integrates the sequential extraction of glycans from multiple organs or tissues with the generation of microarrays, which are probed with monoclonal antibodies (mAbs) or carbohydrate-binding modules (CBMs) with specificities for cell-wall components. The profiles generated provide a global snapshot of cell-wall composition, and also allow comparative analysis of mutant and wild-type plants, as demonstrated here for the Arabidopsis thaliana mutants fra8, mur1 and mur3. CoMPP was also applied to Physcomitrella patens cell walls and was validated by carbohydrate linkage analysis. These data provide new insights into the structure and functions of plant cell walls, and demonstrate the potential of CoMPP as a component of systems-based approaches to cell-wall biology.     

Structures of the glycoinositolphospholipids from Leishmania major. A family of novel galactofuranose-containing glycolipids

Structures of the major glycolipids isolated from the protozoan parasite Leishmania major (strains V121 and LRC-L119), were elucidated by fast atom bombardment-mass spectrometry, two-dimensional proton NMR, methylation analysis, exoglycosidase digestions and mild acid hydrolysis. These glycolipids belong to a family of glycoinositolphospholipids (GIPLs), which contain 4-6 saccharide residues linked to alkylacylphosphatidylinositol (alkylacyl-PI) or lyso alkyl-PI. The general structure of the elucidated GIPLs can be expressed as follows: R-3Galf(alpha 1-3)Manp(alpha 1-3)Manp(alpha 1-4)GlcNp(alpha 1-6) alkylacyl-PI or lyso alkyl-PI where R = OH for GIPL-1; R = Galp(alpha 1- for GIPL-2; R = Galp(alpha 1-6)Galp (alpha 1- for GIPL-3 and R = Galp(alpha 1-3)Galf(alpha 1- for GIPL-A. The alkylacyl-PI lipid moieties are unusual in containing predominantly 18:0, 22:0, 24:0, or 26:0 alkyl chains and 12:0, 14:0, or 16:0 acyl chains. Remodeling of the lipid moieties may occur based on the finding that 1) lyso derivatives account for approximately 35% of the GIPL-3 fraction in strain V121 and 2) there is an increase in the proportion of 24:0 and 26:0 alkyl chains with elongation of the carbohydrate chain. Together with the elucidated structures, these properties are consistent with some of the GIPLs having a role as biosynthetic precursors to the major cell surface glycoconjugate, lipophosphoglycan. In particular, the saccharide sequences of GIPL-3, lyso-GIPL-3, and the glycan core of lipophosphoglycan (Turco, S. J., Orlandi, P. A., Homans, S. W., Ferguson, M. A. J., Dwek, R. A., and Rademacher, T. W. (1989) J. Biol. Chem. 264, 6711-6715) are identical. Finally, immunostaining of thin layer chromatograms with antibodies from patients with cutaneous leishmaniasis suggests that the major GIPLs are highly immunogenic and that the elevated anti-Gal antibodies, commonly seen in leishmaniasis patients, may be directed against terminal Galp(alpha 1-3)Galf residues.     

Composition of the cell walls of Nicotiana alata Link et Otto pollen tubes

Cell walls isolated from pollen of Nicotiana alata germinated in vitro contain glucose and arabinose as the predominant monosaccharides. Methylation analysis and cytochemical studies are consistent with the major polysaccharides being a (13)--D-glucan (callose) and an arabinan together with small amounts of cellulose. The cell walls contain 2.8% uronic acids. Alcian blue stains the pollen-tube walls intensely at the tip, indicating that acidic polysaccharides are concentrated in the tip. Synthetic aniline-blue fluorochrome is specific primarily for (13)--D-glucans and stains the pollen-tube walls, except at the tip. Protein (1.5%), containing hydroxyproline (2.4%), is present in the cell wall.     

Location of cellulose and callose in pollen tubes and grains of Nicotiana tabacum

The distribution of cellulose and callose in the walls of pollen tubes and grains of Nicotiana tabacum L. was examined by electron microscopy using gold-labelled cellobiohydrolase for cellulose and a (1,3)-β-D-glucan-specific monoclonal antibody for callose. These probes provided the first direct evidence that cellulose co-locates with callose in the inner, electron-lucent layer of the pollen-tube wall, while both polymers are absent from the outer, fibrillar layer. Neither cellulose nor callose are present in the wall at the pollen-tube tip or in cytoplasmic vesicles. Cellulose is first detected approximately 5–15 μm behind the growing tube tip, just before a visible inner wall layer commences, whereas callose is first observed in the inner wall layer approximately 30 μm behind the tip. Callose was present throughout transverse plugs, whereas cellulose was most abundant towards the outer regions of these plugs. This same distribution of cellulose and callose was also observed in pollen-tube walls of N. alata Link et Otto, Brassica campestris L. and Lilium longiflorum Thunb. In pollen grains of N. tabacum, cellulose is present in the intine layer of the wall throughout germination, but no callose is present. Callose appears in grains by 4 h after germination, increasing in amount over at least the first 18 h, and is located at the interface between the intine and the plasma membrane. This differential distribution of cellulose and callose in both pollen tubes and grains has implications for the nature of the β-glucan biosynthetic machinery. Received: 20 February 1988 / Accepted: 25 March 1998     

Analysis of the zinc finger domain of TnpA, a DNA targeting protein encoded by mobilizable transposon Tn4555

The mobilizable transposon Tn4555, found in Bacteroides spp., is an important antibiotic resistance element encoding a broad spectrum beta-lactamase. Tn4555 is mobilized by conjugative transposons such as CTn341 which can transfer the transposon to a wide range of bacterial species where it integrates into preferred sites on the host chromosome. Selection of the preferred target sites is mediated by a DNA-binding protein TnpA which has a prominent zinc finger motif at the N-terminus of the protein. In this report the zinc finger motif was disrupted by site directed mutagenesis in which two cysteine residues were changed to serine residues. Elemental analysis indicated that the wild-type protein but not the mutated protein was able to coordinate zinc at a molar ration of 1/1. DNA binding electrophoretic mobility shift assays showed that the ability to bind the target site DNA was not significantly affected by the mutation but there was about a 50% decrease in the ability to bind single stranded DNA. Consistent with these results, electrophoretic mobility shift assays incorporating zinc chelators did not have a significant on affect the binding of DNA target. In vivo, the zinc finger mutation completely prevented transposition/integration as measured in a conjugation assay. This was in contrast to results in which a TnpA knockout was still able to insert into host genomes but there was no preferred target site selection. The phenotype of the zinc finger mutation was not effectively rescued by providing wild-type TnpA in trans. Taken together these results indicated that the zinc finger is not required for DNA binding activity of TnpA but that it does have an important role in transposition and it may mediate protein/protein interactions with integrase or other Tn4555 proteins to facilitate insertion into the preferred sites.