Angela Hilary Atkinson

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.

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 characterisation of a cDNA sequence encoding the backbone of a style-specific 120 kDa glycoprotein which has features of both extensins and arabinogalactan proteins.

Nicotiana alata has a style-specific hydroxyproline-rich glycoprotein (the 120 kDa glycoprotein) which has properties of both extensins and AGPs [19, 20]. The 120 kDa glycoprotein is a soluble component in the extracellular matrix of the transmitting tract of styles where it accounts for ca. 9% of the total buffer-soluble protein. Here we describe the molecular cloning of a cDNA representing the gene NaPRP5 which encodes the backbone of the 120 kDa glycoprotein. Expression of mRNA is restricted to styles, consistent with observations on the distribution of the 120 kDa glycoprotein. Levels of accumulation of the transcript encoding the 120 kDa protein backbone are not altered significantly by pollination with either compatible or incompatible pollen. The protein backbone of the 120 kDa glycoprotein, as predicted by the cDNA sequence, is composed of three distinct domains. The sequence of these domains, together with linkage analysis of the carbohydrate component of the 120 kDa glycoprotein, allows predictions of the likely distribution of substituent glycosyl chains along the protein backbone. The similarity of the C-terminal domains of the 120 kDa glycoprotein and GaRSGP, the galactose-rich style glycoprotein of N. alata, is consistent with the two molecules sharing a common antigenic domain in their backbones [31]. The sharing of domains between distinct hydroxyproline-rich glycoproteins suggests that identification of a glycoprotein of this class solely by its protein or carbohydrate epitope is not valid.