Philip J. Harris

Linkage of p-coumaroyl and feruloyl groups to cell-wall polysaccharides of barley straw

Abstract Treatment of cell walls of barley straw with Oxyporus “cellulase” (a mixture of polysaccharide hydrolases) released compounds containing p-coumaroyl and feruloyl groups bound to carbohydrates, two of which were identified as O-[5-O-(trans-p-coumaroyl)-α- l -arabinofuranosyl]-(1→3)-O-β- d -xylopyranosyl-(1→4)- d -xylopyranose (PAXX) and O-[5-O-(trans-feruloyl)-α- l -arabinofuranosyl]-(1→3)-O-β- d -xylopyranosyl-(1→4)- d -xylopyranose (FAXX).

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 (1→3)-β-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 (1→3)-β-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.

4,4′-Dihydroxytruxillic acid as a component of cell walls of Lolium multiflorum

Abstract 4,4′-Dihydroxytruxillic acid was released from the cell walls of Lolium multiflorum by treatment with sodium hydroxide. Combined gas chromatography-mass spectrometry indicated that other similar dimers, probably resulting from the photodimerization of p-coumaric acid or ferulic acid, were also released from the cell walls. The possible role of the dimers in cross-linking cell wall heteroxylans is discussed.

Molecular Basis of Cell Recognition During Fertilization in Higher Plants

SUMMARY The molecular basis of recognition between plant cells is incompletely understood. Some principles established for recognition between animal cells may well apply to plant cell recognition, although, in contrast to animal cells, plant cells are encased by cell walls that play an active role in plant cell–cell recognition. The interaction that controls fertilization in flowering plants involves recognition between pollen or pollen tubes and the female sexual tissues. In many flowering plant families, self-incompatibility (S) genes operate to prevent inbreeding. In plants that have gametophytically controlled self-incompatibility, recognition of common S alleles in pollen tube and style results in arrest of pollen tube growth within the style. Self-incompatibility therefore provides a model cell–cell recognition system that is genetically defined. We have taken two approaches to defining cell recognition involved in gametophytic self-incompatibility in Nicotiana alata. Firstly, we have established the major features of the pollen tube wall and the matrix of the style transmitting tissue that are in contact with the growing pollen tube. Secondly, we have established the nature of style glycoproteins that are associated with the S genotype and have initiated a program to clone the genes coding for the protein component of these glycoproteins. Analyses of the pollen tube are consistent with the major polymers being a (1→3)-β-d-glucan (callose) and a (1→5)-α-l-arabinan. The pollen tube has two distinct layers: gold immunocytochemistry using a monoclonal antibody directed to terminal α-l-arabinosyl residues shows the binding is confined to the outer layers. The major component of the extracellular matrix of the style transmitting tissue is a family of proteoglycans, the arabinogalactan-proteins. A major glycoprotein that segregates with the S2 allele is present in extracts of mature styles. This component has a high pI (>9.5) and an apparent molecular weight of 32 × 103. It is not present in extracts of immature styles of N. alata genotypes bearing the S2 allele, or in extracts from other organs of N. alata or styles of other members of the Solanaceae. The isolated glycoprotein is an effective inhibitor of in vitro pollen tube growth. This evidence suggests that the S2-associated glycoprotein is either the product of the S2 allele, or a gene closely associated with the S gene. We have prepared a cDNA library from styles of one genotype and are screening this library with mRNA from mature and immature styles. We have also prepared synthetic oligonucleotide probes to N-terminal sequences obtained from the isolated S2-associated glycoprotein for use in screening the library. This dual approach for establishing the detailed structures of the interacting components and the genetic basis of the interaction will give us a better understanding of the recognition events involved in self-incompatibility.

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.

Histochemistry and composition of the cell walls of styles of Nicotiana alata Link et Otto

The cell walls of styles of Nicotiana alata Link et Otto (ornamental tobacco; Solanaceae) were analysed chemically and examined histochemically. Cell-wall preparations were obtained from whole styles and from isolated transmitting-tissue cells. The style epidermal cells were shown histochemically to have thick, lignified secondary walls. These walls probably constituted a large proportion of the cell-wall preparation from whole styles as analysis of whole-style walls indicated that the major polysaccharides were xylans and cellulose, which are typical of lignified secondary walls of Magnoliopsida (dicotyledons). Lignification of the style epidermal walls was also demonstrated histochemically in 10 other species (5 genera including Nicotiana) of the sub-family Cestroideae of the Solanaceae, but not in 15 species (9 genera) of the sub-family Solanoideae of the Solanaceae, nor in 3 other species of dicotyledons and 2 species of Liliopsida (monocotyledons). Analysis of the cell-wall preparation from isolated transmitting-tissue cells of N. alata indicated that these contained cellulose, xyloglucans, and pectic polysaccharides, which is typical of primary cell walls of dicotyledons. However, the analysis indicated that the walls also contained an unusually high proportion of Type II arabinogalactans. Staining of the transmitting-tissue cell-wall preparation with β-glucosyl Yariv reagent, a histochemical reagent specific for arabinogalactan proteins, confirmed their presence, which may be related to the role of these cells in secreting the stylar extracellular matrix.

Composition of the walls of pollen grains of the seagrass Amphibolis antarctica

The composition of walls isolated from pollen grains of the seagrass Amphibolis antarctica was determined. Glucose, galactose, and rhamnose were the major neutral monosaccharides in the wall polysaccharides, and fucose, arabinose, xylose, and mannose were present in minor proportions. No apiose, a monosaccharide present in the wall polysaccharides of the vegetative parts of the seagrass Heterozostera tasmanica, was found. Large amounts of uronic acid (mainly as galacturonic acid) were found in the walls. The monosaccharides were probably present in cellulose and pectic polysaccharides, the latter comprising neutral pectic galactans, and rhamnogalacturonans containing high proportions of rhamnose. The walls contained a small amount of protein; glycine and lysine were the amino acids present in the highest proportions. Histochemical examination of isolated walls confirmed the presence of polyanionic components (pectic polysaccharides), β-glucans (cellulose), and protein. The composition of the walls is discussed in relation to analyses of the walls of pollen grains and vegetative organs of other plants.

The detection and quantification of apiose by capillary gas chromatography of its alditol acetates

The branched-chain sugar, apiose [3-C-(hydroxymethyl)-D-glycero-aldotetrose] occurs widely in plants, in low molecular weight glycosides’, and as a component of cell-wall polysaccharides’-s. Problems have been encountered in the analysis of apiose as its alditol acetate derivatives. For example, when pyridine was used as the acetylation catalyst6, the alditol gave two peaks by g.1.c.‘. Similar results were obtained when sodium acetate was used as the catalyst*, and underacetylation was suggested*. We have reported’ the application to apiose of the method of Blakeney et al.“, which uses reduction with sodium borohydride in methyl sulphoxide and acetylation catalysed by IV-methylimidazole. The two peaks detected were assigned to apiitol tetraand penta-acetate and reflected the slow acetylation “g’* of the tertiary hydroxyl group at C-3. Complete acetylation requires elevated temperatures and extended times (90 min)‘. Kindel and Cheng13 obtained similar results, but reported that substantial proportions of 3-C-(acetoxymethyl)-1,2,4tri-O-acetyl-3-O-(methylthiomethyl)-D-glycero-tetritol were formed as a side product. We now present additional evidence that application of the method of Blakeney et al.” to apiose gives apiitol 1,2,3,4,4’-penta-acetate and 1,2,4,4’-tetra-acetate, and propose a method for quantification based on the latter derivative.

Calcium-Dependent Protein Phosphorylation in Germinated Pollen

Ca2+-dependent protein phosphorylation represents a major mechanism for stimulus-response coupling in animal systems1–3. Soluble4–6 and membrane-associated7,8 Ca2+-dependent protein kinases are present in higher plants and are likely to be involved in Ca2+-mediated signal transduction in plant systems. We have initiated investigations into the possible involvement of Ca2+-dependent protein phosphorylation in pollen tube growth.