Ingrid Bonig

The location of (1→3)-β-glucans in the walls of pollen tubes of Nicotiana alata using a (1→3)-β-glucan-specific monoclonal antibody

The location of the (1→3)-β-glucan, callose, in the walls of pollen tubes in the style of Nicotiana alata Link et Otto was studied using specific monoclonal antibodies. The antibodies were raised against a laminarinhaemocyanin conjugate. One antibody selected for further characterization was specific for (1→3)-β-glucans and showed no binding activity against either a cellopentaose-bovine serum albumin (BSA) conjugate or a (1→3, 1→4)-β-glucan-BSA conjugate. Binding was inhibited by (1→3)-β-oligoglucosides (DP, 3–6) with maximum competition being shown by laminaripentaose and laminarihexaose, indicating that the epitope included at least five (1→3)-β-linked glucopyranose residues. The monoclonal antibody was determined to have an affinity constant for laminarihexaose of 2.7. 104M−1. When used with a second-stage gold-labelled, rabbit anti-mouse antibody, the monoclonal antibody probe specifically located the (1→3)-β-glucan in the inner wall layer of thin sections of the N. alata pollen tubes.

Developmentally controlled expression of a gene associated with self-incompatibility in Nicotiana alata

In many flowering plant species, a system of self-incompatibility, typically controlled by a single gene with multiple alleles (S-gene)1, enables individual plants to recognize and reject their own pollen. In gametophytically determined systems, the growth of self-pollen tubes is arrested in the style or occasionally the ovary. The expression of this self-incompatibility is developmentally regulated, being strongest in mature flowers and weak in immature styles1,2. We have used a complementary DNA clone, believed to encode the S2-allele of Nicotiana alata2,3, to detect S2 messenger RNA by in situ hybridization to sections. We report here that expression of the gene occurs in the stigma and throughout the secretory tissue, but not in other parts, of mature pistils. In immature flowers, S2 mRNA is confined to the proliferated epidermis of the stigma. We conclude that S2-gene expression correlates well with the expression of incompatibility.

The application of sirofluor, a chemically defined fluorochrome from aniline blue for the histochemical detection of callose

SummarySirofluor, a chemically defined fluorochrome from aniline blue in aqueous unbuffered solutions, complexes with isolated (1 → 3)-β-glucans, but not (1 → 4)-β-glucans, after embedding in JB-4 resin and sectioning. Under these conditions, callose deposits in plant tissues give a brilliant yellow fluorescence with essentially no background fluorescence.

A style-specific 120-kDa glycoprotein enters pollen tubes ofNicotiana alata in vivo

Pistils ofNicotiana alata (Link et Otto) contain an abundant, style-specific glycoprotein (120 kDa) that is rich in hydroxyproline and has both extensin-like and arabinogalactan-protein-like carbohydrate substituents. An antibody specific for the protein backbone of the glycoprotein was used to localise the glycoprotein in both unpollinated and pollinated pistils. The glycoprotein is evenly distributed in the extracellular matrix of the style transmitting tract of unpollinated pistils and, despite the presence of extensin-like carbohydrate substituents, is not associated with the walls of the transmitting tract cells. In pollinated pistils the 120-kDa glycoprotein is concentrated in the extracellular matrix adjacent to pollen tubes, and is also present in the cytoplasm and the cell walls of pollen tubes. Pollen tubes grown in vitro do not contain the 120-kDa glycoprotein unless it is added to the growth medium, suggesting that the 120kDa glycoprotein located in pistil-grown pollen tubes is derived from the extracellular matrix of the transmitting tract.

Action of the Style Product of the Self-Incompatibility Gene of Nicotiana alata (S-RNase) on in Vitro-Grown Pollen Tubes.

The products of the S-locus expressed in female tissues of Nicotiana alata are ribonucleases (S-RNases). The arrest of growth of incompatible pollen tubes in styles may result from entry of the S-RNase into the pollen tube and degradation of pollen tube RNA. We investigated the action of isolated S-RNases on pollen tubes grown in vitro and found that S-RNase is taken up by the pollen without substantial alteration. The S-RNases inhibit incorporation of exogenously added radioactive amino acids into protein by the germinated pollen. The S-RNases also inhibit in vitro translation of pollen tube RNA in a wheat germ cell-free extract. We found no evidence for a specific mRNA substrate for the S-RNases, which implies that if RNase activity is involved in the control of self-incompatibility, allelic specificity is more likely to depend on the selective uptake of S-RNases into pollen tubes or their selective activation or inactivation by pollen factors, rather than cleavage of a specific substrate. Heat treating S2-RNase largely destroys its RNase activity but increases its inhibitory effect on in vitro pollen tube growth. This effect is not due to an increased uptake of S2-RNase by the pollen but is associated with a greatly enhanced accumulation of S2-RNase on the outer surface of the pollen grains.

S-RNase gene of Nicotiana alata is expressed in developing pollen.

In the solanaceous plant Nicotiana alata, self-incompatibility is controlled by a single, multiallelic locus (S locus) expressed in both pollen and pistil. Previously, we have shown cosegregation between alleles of the S locus and alleles of a gene that encodes a glycoprotein with ribonuclease activity (S-RNase). Furthermore, expression of the S-RNase gene is apparently confined to the pistil and is correlated with the onset of self-incompatibility. In this paper, we report that the S-RNase gene is also expressed at low levels in developing pollen. A transcript in developing pollen hybridized to a cDNA encoding the S2-RNase allele of the parent plant and did not hybridize to cDNAs encoding other S-RNase alleles. Two cDNAs for the S2-RNase were cloned from a library derived from anthers of a plant homozygous for the S2 allele and both corresponded to the coding sequence of the S2-RNase. The product of the S-RNase gene was detected by immunocytochemistry in the intine of mature, hydrated pollen grains. These results are interpreted in the light of current knowledge of the structure of the S locus.

A simple fixation and embedding method for use in hybridization histochemistry on plant tissues

SummaryA simple method for fixing and embedding plant tissue for hybridization histochemistry has been developed. Material is fixed with paraformaldehyde-glutaraldehyde by the phase-partition technique and embedded in LR Gold. The method preserves nucleic acidsin situ. Double-stranded32P-labelled DNA probes hybridized to complementary RNAs in thin and semithin sections. Biotinylated probes detected with immunogold markers have been used to localize transcripts at the electron nucroscopic level.

Factors affecting the adhesion of micro-organisms to the surfaces of plant-parasitic nematodes

The influence of various agents on the adhesion of endospores of Pasteuria penetrans to the nematode Meloidogyne javanica was studied. Similarly, but to a lesser degree, we have also studied the adhesion of conidia of the fungus Dilophospora alopecuri and the coryneform bacterium Clavibacter sp. (syn. Corynebacterium rathayi ) to the nematode Anguina agrostis (syn. A. funesta ). Reduction in the degree of both spore and conidial attachment following their pre-treatment with periodate and the presence of PAS staining material on spores, conidia and bacteria implicated carbohydrate in these interactions. Tests involving both unbound and FITC-bound lectins demonstrated that wheat germ agglutinin (WGA) can inhibit the degree of attachment of P. penetrans to M. javanica and that this inhibition can be overcome by pre-treatment of the lectin with N, N′ -diacetyl chitobiose. Endospores of P. penetrans , amphid and buccal secretions of 2nd-stage larvae of M. javanica and the cuticle and excretory pore secretions of 2nd-stage dauer larvae of A. agrostis bound WGA, indicating that accessible N -acetyl-D-glucosamine residues are present on these structures. Endospores of P. penetrans also bound Con A, indicating the presence of accessible α-D-glucose/α-D-mannose residues on their surface.

Immuno-gold localization of α-L-arabinofuranosyl residues in pollen tubes of Nicotiana alata Link et Otto

Immuno-gold labelling using a monoclonal antibody (PCBC3) with a primary specificity for α-L-arabinofuranosyl residues was used to locate these residues in pollen tubes of Nicotiana alata grown in vivo. The antibody bound to the outer fibrillar layer of the pollen-tube wall: the inner, non-fibrillar wall layer was not labelled. Cytoplasmic vesicles (0.2 μm diameter) were also labelled. The antibody may bind to an arabinan in the pollen-tube wall.

Arabinogalactan-proteins are localized extracellularly in the transmitting tissue of Nicotiana alata link and otto, an ornamental tobacco

Abstract The localization of a class of proteoglycans, the arabinogalactan-proteins (AGPs), within mature styles of N. alata has been examined using three probes: (1) β-glycosyl Yariv reagent, a red dye which is used as a cytochemical stain for light microscopy, (2) monoclonal antibodies to terminal α- L -arabinofuranosyl residues which bind to the terminal α- L -arabinofuranosyl residues of AGPs, (3) monoclonal antibodies to terminal β- D - galactopyranosyl residues which bind to style glycoconjugates including the AGPs. The monoclonal antibodies were used in both immunofluorescence and gold immunocytochemical studies. Staining with all probes indicates that the AGPs are present mainly in the extracellular matrix of the transmitting tissue.