Isolation and characterization of a polymorphic stigma-specific class III peroxidase gene from Senecio squalidus L. (Asteraceae).

A novel stigma-specific class III peroxidase gene, SSP (Stigma-Specific Peroxidase), has been isolated from the self-incompatible daisy Senecio squalidus L. (Asteraceae). Expression of SSP in flower buds is developmentally regulated, with maximal levels of expression coinciding with anthesis, when stigmas are most receptive to pollen and when self-incompatibility is fully developed. In situ hybridization revealed SSP expression to be localized exclusively to the specialized secretory epidermal cells (papillae) of the stigma, which receive and discriminate pollen. SSP is therefore the first tissue-specific and cell-specific peroxidase gene identified in a plant. SSP belongs to a distinct clade of class III plant peroxidases that possess two introns, instead of the more normal situation of three conserved introns. The deduced amino acid sequence of SSP revealed a 27 amino acid signal peptide, suggesting that the SSP protein is secreted to the cell wall of the stigmatic papillae. In-gel peroxidase activity assays showed that SSP has relatively low peroxidase activity compared to other, as yet uncharacterized, peroxidases present in stigmatic extracts. Six SSP alleles have been cloned from different lines of S. squalidus carrying a range of self-incompatibility (S)-alleles but there was no consistent association between the presence of a particular SSP allele and S-genotype indicating that SSP is not the female determinant of SSI in S. squalidus. Nevertheless, the precise expression of SSP in stigmatic papillae suggests that it may have a more general function in pollen-stigma interactions, or alternatively in protection of stigmas from pathogen attack. Extensive database screens have identified homologues of SSP in other plant species, but available expression data for these genes indicates that none are flower-specific, suggesting that SSP represents a new functional type of class III peroxidase specific to the stigma. We discuss the possible function(s) of S. squalidus SSP in pollen-stigma interactions and in protection of stigmas from pathogen attack.

Sporophytic self-incompatibility in Senecio squalidus L (Asteraceae)--the search for S.

Senecio squalidus (Oxford Ragwort) is being used as a model species to study the genetics and molecular genetics of self-incompatibility (SI) in the Asteraceae. S. squalidus has a strong system of sporophytic SI (SSI) and populations within the UK contain very few S alleles probably due to a population bottleneck experienced on its introduction to the UK. The genetic control of SSI in S. squalidus is complex and may involve a second locus epistatic to S. Progress towards identifying the female determinant of SSI in S. squalidus is reviewed here. Research is focused on plants carrying two defined S alleles, S(1) and S(2). S(2) is dominant to S(1) in pollen and stigma. RT-PCR was used to amplify three SRK-like cDNAs from stigmas of S(1)S(2) heterozygotes, but the expression patterns of these cDNAs suggest that they are unlikely to be directly involved in SI or pollen-stigma interactions in contrast to SSI in the Brassicaceae. Stigma-specific proteins associated with the S(1) allele and the S(2) allele have been identified using isoelectric focusing and these proteins have been designated SSP1 (Stigma S-associated Protein 1) and SSP2. SSP1 and SSP2 cDNAs have been cloned by 3' and 5' RACE and shown to be allelic forms of the same gene, SSP. The expression of SSP and its linkage to the S locus are currently being investigated. Initial results show SSP to be expressed exclusively in stigmas and developmentally regulated, with maximal expression occurring at and just before anthesis when SI is fully functional, SSP expression being undetectable in immature buds. Together these data suggest that SSP is a strong candidate for a Senecio S-gene.