Characterization of Ribonuclease Activity of Three S-Allele-Associated Proteins of Petunia inflata

Abstract

Three S-allele-associated proteins (S-proteins) of Petunia inflata, a species with gametophytic self-incompatibility, were previously found to share sequence similarity with two fungal ribonucleases, RNase T2 and RNase Rh. In this study, the S-proteins from P. inflata plants of SS2 and S2S3 genotypes were purified to homogeneity by gel filtration and cation-exchange chromatography, and their enzymatic properties were characterized. The three S-proteins (Si, S2, and S3), with pairwise sequence identity ranging from 73.1 to 80.5%, were similar in most of the enzymatic properties characterized. The ribonuclease activity had a pH optimum of 7.0 and a temperature optimum of 500C. Diethylpyrocarbonate at 1 millimolar almost completely abolished the ribonuclease activity; cupric sulfate and zinc sulfate at 1 millimolar reduced the ribonuclease activity of the three S-proteins by 50 to 75%. EDTA and RNasin had no inhibitory effect. All three Sproteins hydrolyzed polycytidylic acid preferentially, but varied in their nucleolytic activity toward polyadenylic acid and polyuridylic acid.Gametophytic self-incompatibility acts to prevent self-fertilization and consequent inbreeding in many species of the Solanaceae, including Petunia inflata. In Petunia, self-incompatibility is controlled by a single polymorphic locus, the Slocus. The S-allele products of pollen and pistil are presumed to interact in a manner that allows pistils to discriminate between self-and cross-pollen. The result of this specific selfrecognition event is that pollen bearing an S-allele identical to either one of the two alleles carried by the pistil suffers growth arrest, while pollen bearing an allele different from both alleles carried by the pistil is able to grow down the pistil to effect fertilization.S-allele-associated pistil proteins (S-proteins) have been identified in many species with gametophytic self-incompatibility (1, 7, 8, 17, 19-21, 23, 24, 29). The sequences of a number of solanaceous S-proteins have been deduced from the corresponding cDNA sequences (1-3, 10, 20, 30). All the full-length cDNA sequences predict a leader peptide that is not found in the mature protein.

Gametophytic Self-Incompatibility Systems.

“…The finding that the S-glycoproteins have RNase activity has led to their being referred to as S-RNases. Subsequently, S-glycoproteins from other solanaceous species have been shown to have RNase activity (Broothaerts et al, 1991;Kaufmann et al, 1991;Singh et al, 1991;Ai et al, 1992). In addition, RNases have been detected in the styles of Japanese pear (a member of the family Rosaceae), and it is possible that these are also S-related glycoproteins (Sassa et al, 1992).…”

“…When the S-RNases from P inflara are tested on homopolymeric substrates (Singh et al, 1991), they show a cleavage preference of poly(C) over poly(A) or poly(U). However, this is unlikely to be related to biological function because the S-RNases have no obvious differences in RNA substrate specificity and effectively degrade RNAfrom many sources.…”

Cloning and expression of a distinctive class of self-incompatibility (S) gene from Papaver rhoeas L.

“…To date, the S genes and their products (2-9) have fallen into two classes. The S genes under sporophytic control, from Brassica, consist of a complex family of genes (3,10,11), whereas the S genes under gametophytic control, from Nicotiana and other Solanaceae, encode ribonucleases (9,12,13).…”

The S-Ribonuclease Gene of Petunia hybrida Is Expressed in Nonstylar Tissue, Including Immature Anthers

“…Furthermore, the S-locus proteins have RNase activity in vitro Gray et al, 1991;Singh et al, 1991) and have been renamed S-RNases to reflect this function. According to current models (e.g.…”