The arabinogalactan-proteins from pressed juice of Echinacea purpurea belong to the hybrid class of hydroxyproline-rich glycoproteins.

Pressed juices from Echinacea purpurea are used as non-specific immunostimulants, and arabinogalactan-proteins (AGPs) are part of the active principle. An AGP fraction was isolated from pressed juice of E. purpurea by precipitation with ss-glucosyl Yariv reagent, followed by gel-permeation chromatography. Polyclonal antibodies directed against the carbohydrate moiety of this AGP fraction showed a preferential specificity for E. purpurea AGPs from pressed juice over those extracted from E. purpurea suspension culture and other plant species. Native AGPs purified from this AGP fraction by RP-HPLC were then deglycosylated for N-terminal protein sequencing resulting in the identification of three major polypeptides. They show characteristic motifs of classical AGPs but also some features of extensins, suggesting these may be "hybrid" hydroxyproline-rich glycoproteins (HRGPs).

The S-locus of Nicotiana alata: genomic organization and sequence analysis of two S-RNase alleles.

Genomic clones encoding the S2- and S6-RNases of Nicotiana alata Link and Otto, which are the allelic stylar products of the self-incompatibility (S) locus, were isolated and sequenced. Analysis of genomic DNA by pulsed-field gel electrophoresis and Southern blotting indicates the presence of only a single S-RNase gene in the N. alata genome. The sequences of the open-reading frames in the genomic and corresponding cDNA clones were identical. The organization of the genes was similar to that of other S-RNase genes from solanaceous plants. No sequence similarity was found between the DNA flanking the S2- and S6-RNase genes, despite extensive similarities between the coding regions. The DNA flanking the S6-RNase gene contained sequences that were moderately abundant in the genome. These repeat sequences are also present in other members of the Nicotianae.

Molecular cloning of cDNAs encoding the protein backbones of arabinogalactan-proteins from the filtrate of suspension-cultured cells of Pyrus communis and Nicotiana alata.

This paper reports the isolation of cDNAs encoding the protein backbone of two arabinogalactan-proteins (AGPs), one from pear cell suspension cultures (AGPPc2) and the other from suspension cultures of Nicotiana alata (AGPNa2). The proteins encoded by these cDNAs are quite different from the 'classical' AGP backbones described previously for AGPs isolated from pear suspension cultures and extracts of N. alata styles. The cDNA for AGPPc2 encodes a 294 amino acid protein, of which a relatively short stretch (35 amino acids) is Hyp/Pro rich; this stretch is flanked by sequences which are dominated by Asn residues. Asn residues are not a feature of the 'classical' AGP backbones in which Hyp/Pro, Ser, Ala and Thr account for most of the amino acids. The cDNA for AGPNa2 encodes a 437 amino acid protein, which contains two distinct domains: one rich in Hyp/Pro, Ser, Ala, Thr and the other rich in Asn, Tyr and Ser. The composition and sequence of the Pro-rich domain resembles that of the 'classical' AGP backbone. The Asn-rich domains of the two cDNAs described have no sequence similarity; in both cases they are predicted to be processed to give a mature backbone with a composition similar to that of the 'classical' AGPs. The study shows that different AGPs can differ in the amino acid sequence in the protein backbone, as well as the composition and sequence of the arabinogalactan side-chains. It also shows that differential expression of genes encoding AGP protein backbones, as well as differential glycosylation, can contribute to the tissue specificity of AGPs.

Molecular cloning of a gene encoding an arabinogalactan-protein from pear (Pyrus communis) cell suspension culture.

Arabinogalactan-proteins (AGPs) are proteoglycans containing a high proportion of carbohydrate (typically > 90%) linked to a protein backbone rich in hydroxyproline (Hyp), Ala, Ser, and Thr. They are widely distributed in plants and may play a role in development. The structure of the carbohydrate of some AGPs is known in detail but information regarding the protein backbone is restricted to a few peptide sequences. Here we report isolation and partial amino acid sequencing of the protein backbone of an AGP. This AGP is a member of one of four major groups of AGPs isolated from the filtrate of pear cell suspension culture. A cDNA encoding this protein backbone (145 amino acids) was cloned; the deduced protein is rich in Hyp, Ala, Ser, and Thr, which together account for > 75% of total residues. It has three domains, an N-terminal secretion signal, a central hydrophilic domain containing all of the Pro residues, and a hydrophobic C-terminal domain that is predicted to be a transmembrane helix. Approximately 93% of the Pro residues are hydroxylated and hence are potential sites for glycosylation.

Nucleotide sequence and style-specific expression of a novel proline-rich protein gene from Nicotiana alata.

cDNA clones encoding a novel proline-rich protein (NaPRP4) have been isolated from a Nicotiana alata stylar cDNA library. The N-terminal part of the derived protein is highly rich in proline (32.2%) and contains several repeats such as Lys-Pro-Pro (7 times) and Pro-Thr-Lys-Pro-Pro-Thr-Tyr-Ser-Pro-Ser-Lys-Pro-Pro (twice); the C-terminal part, on the other hand, has a lower proline content (9.9%) and contains two potential N-glycosylation sites and all the six cysteine residues. Northern blot and in situ hybridisation analyses indicate that expression of the NaPRP4 gene is restricted to cells of the transmitting tract of the style.

Molecular and evolutionary aspects of self-incompatibility in flowering plants.

Self-incompatibility (SI) is widely distributed in flowering plants. In this review, early work on the biology, genetics and distribution of SI is summarized. Approaches to understanding the molecular genetics of SI have been made in two systems-Solanaceous species, for example Nicotiana alata, which have gametophytic systems of SI, and Brassica spp, which have sporophytic systems of SI. The information in both systems is derived from cDNAs that encode pistil glycoproteins (S-glycoproteins) that segregate with S-genotype. Comparison of the sequence data indicates that the gametophytic and sporophytic systems of SI probably arose independently during the evolution of angiosperms. The S-glycoproteins of a solanaceous plant Nicotiana alata, are ribonucleases (RNases). Whether the RNase activity is directly involved in the characteristic arrest of pollen tube growth during self-(incompatible) pollination, is not known. An alternative possibility is that the RNase was 'recruited' during evolution for a function in SI, without involvement of its catalytic function. The nature of the S-gene in pollen is not yet known for either the gametophytic or sporophytic SI systems. This is a key piece of information that will be required to progress our understanding of how the growth of a pollen tube bearing a particular S-allele is arrested within the style bearing an identical S-allele, but is not arrested within the style bearing other S-alleles.

Recent developments in the molecular genetics and biology of self-incompatibility.

A summary of recent work on molecular aspects of self-incompatibility in Nicotiana alata is presented. The amino acid sequences of style proteins corresponding to different S-alleles of N. alata have a high level of homology in some regions and are variable in other regions. The regions of homology include N-terminal sequences as well as most of the glycosylation sites and cysteine residues. The glycosyl substituents may consist of a number of 'glycoforms'. The isolated style S-glycoproteins inhibit in vitro growth of pollen tubes. The S-glycoproteins tested inhibited the growth of pollen of several S-genotypes, and there was some specificity in the interaction. Heat treatment of the isolated S-glycoproteins dramatically increased their activity as inhibitors of pollen tube growth, although the specificity in the interaction was lost. The nature of the S-allele products in pollen is not yet established.

A nuclear sequence associated with self-incompatibility in Nicotiana alata has homology with mitochondrial DNA.

A 1.0-kb nuclear fragment located 5' to a coding sequence associated with self-incompatibility in N. alata shows homology with mitochondrial chromosomal DNA on Southern blots. This sequence is also present in the mitochondrial DNA of two species of tomato, L. esculentum and L. pennellii, but shows no homology to mtDNA of Zea mays. The homologous mitochondrial fragment from N. alata was cloned and sequenced. A short region of 56 bp matches the nuclear sequence in 53/56 bp. Other matched but misaligned segments flank the 3' end. The nuclear sequence is marked at the 5' end by two 8 bp direct repeats. The function of the nuclear sequence is not known although, it is located 397 bp upstream from the site of transcription of the self-incompatibility gene. The mitochondrial sequence contains only limited open reading frames and the nuclear sequence has none. There is evidence that additional segments of the mitochondrial clone hybridize to other nuclear sequences. The exchange of sequences between the mitochondrial and nuclear genomes of plants is discussed.

Style proteins of a wild tomato (Lycopersicon peruvianum) associated with expression of self-incompatibility.

The identification, isolation and aminoterminal sequencing of two S-genotype-associated proteins from style extracts of Lycopersicon peruvianum Mill. is reported. There is a high level of homology between these two sequences and with the amino-terminal sequences of other S-allele-associated glycoproteins isolated from Nicotiana alata Link et Otto. These sequences were obtained by a new high-sensitivity method of selected twodimensional gel analysis followed by electroelution and purification of proteins by inverse-gradient high-performance liquid chromatography before sequencing.

Molecular basis of cell recognition during fertilization in higher plants.

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 Nicotianas 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)-beta-D-glucan (callose) and a (1----5)-alpha-L-arabinan. The pollen tube has two distinct layers: gold immunocytochemistry using a monoclonal antibody directed to terminal alpha-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 (greater than 9.5) and an apparent molecular weight of 32 X 10(3). 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation and partial characterization of components of Prunus avium L. styles, including an antigenic glycoprotein associated with a self-incompatibility genotype.

Several components of buffer extracts of Prunus avium L. styles (cv. Lambert, S 3 S 4) have been isolated and partially characterized: the major component is a glycoprotein (molecular weight approx. 90,000; 95% protein, 5.4% carbohydrate). A "sticky" uronic-acid-containing component and an arabinogalactan are also present. Two minor components are an antigenic glycoprotein associated with the self-incompatibility genotype (Antigen S) and a component found in styles of all Prunus species (Antigen P). The isolated glycoproteins have a substantial carbohydrate content (Antigen P 17.2%; Antigen S 16.3%), and have apparent molecular weights of 32,000 (Antigen P) and 37,000-39,000 (Antigen S). They are antigenically quite distinct. Material corresponding to Antigen S is secreted into the medium of suspension-cultured callus cells raised from both leaf and stem of P. avium.

The effect of isolated components of Prunus avium L. styles on in vitro growth of pollen tubes.

A number of components isolated from styles of P. avium cv. Napoleon (S 3 S 4) have been tested for their capacity to influence in vitro growth of pollen tubes from fresh and stored pollen (cv. Napoleon (S 3 S 4)). An antigenic glycoprotein (Antigen S) is a potent inhibitor of in-vitro pollen tube growth, causing a 65% reduction in tube length at a concentration of 20 µg/ml. None of the other style components were effective inhibitors of pollen tube growth; neither were proteins of animal origin such as histone, serum albumin, cytochrome C, and the glycoproteins ovalbumin and thyroglobulin, effective inhibitors.