Adrienne E. Clarke

Form and function of arabinogalactans and arabinogalactan-proteins

Abstract The occurrence, isolation, chemistry and physico-chemistry of plant arabino-3,6-galactans and arabino-3,6-galactan-proteins is reviewed. The structural relationships between arabino-3,6-galactans from gymnosperm wood, gum exudates of Acacia and other trees, and from plant callus cells and whole tissues are discussed. The nature of these proteoglycans is compared with the arabinose and galactose containing cell wall glycoproteins. Interactions of the arabino-3,6-galactan proteoglycans with carbohydrate binding proteins and with Yariv antigens are described. The utility of these reactions for both cellular and subcellular localization of the proteoglycans is discussed. The possible biological roles of the arabinogalactans and the arabinogalactan-proteins are reviewed.

Proteinase inhibitors in Nicotiana alata stigmas are derived from a precursor protein which is processed into five homologous inhibitors.

A cDNA clone, NA-PI-II, encoding a protein with partial identity to proteinase inhibitor (PI) II of potato and tomato has been isolated from a cDNA library constructed from Nicotiana alata stigma and style mRNA. The cDNA encodes a polypeptide of 397 amino acids with a putative signal peptide of 29 amino acids and six repeated domains, each with a potential reactive site. Domains 1 and 2 have chymotrypsin-specific sites and domains 3, 4, 5, and 6 have sites specific for trypsin. In situ hybridization experiments demonstrated that expression of the gene is restricted to the stigma of both immature and mature pistils. Peptides with inhibitory activity toward chymotrypsin and trypsin have been isolated from stigmas of N. alata. The N-terminal amino acid sequence obtained from this protein preparation corresponds to six regions in the cDNA clone NA-PI-II. The purified PI protein preparation is likely to be composed of a mixture of up to five similar peptides of approximately 6 kD, produced in vivo by proteolytic processing of a 42-kD precursor. The PI may function to protect the reproductive tissue against potential pathogens.

Quantification of arabinogalactan-protein in plant extracts by single radial gel diffusion

The amount of arabinogalactan-protein in whole plant extracts can be quantified by single radial diffusion in agarose gels containing a dye known as the beta-glucosyl-Yariv reagent which specifically interacts with and precipitates arabinogalactan-proteins. The lower limit of quantification is 0.04 microgram of arabinogalactan-protein; gum arabic is used as a standard reference arabinogalactan-protein. In principle, this method can be adapted to measure levels of any dye-precipitating macromolecule; for example, acidic polysaccharides can be estimated by their binding to the cationic dye Alcian blue.

Genetic control of self-incompatibility and reproductive development in flowering plants

A: Self-Incompatibility. I. Molecular biology. II. Cellular and whole plant studies. B: Control of Sex Expression and Floral Development. C: Genes Active in Male Gametogenesis: Pollen Genes. I. Cellular and molecular biology. II. Male sterility. D: Female Gametogenesis and Fertilization. Index.

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.

Gametophytic Self-Incompatibility Systems.

is one of the mechanisms that have evolved to encourage outbreeding in flowering plants and is defined as “the inability of a fertile hermaphrodite seed plant to produce zygotes after self-pollination” (de Nettancourt, 1977). The effectiveness of SI in promoting outbreeding is believed to be one of the most important factors that ensured the evolu- tionary success of flowering plants (Whitehouse, 1951). It is a genetically controlled phenomenon, and in many cases, the control is by a single locus (known as the S locus) with a large number of alleles, up to severa1 hundred in some species (Ockendon, 1974; de Nettancourt, 1977). SI has been a favor- ite topic for botanists and geneticists since Darwin (1877) first discussed the phenomenon and suggested the idea of its cen- tral significance during the evolution of flowering plants. During the century or more of work on the subject, there have been a number of key reviews, the most significant

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.