Identification of Stylar RNases Associated with Gametophytic Self-Incompatibility in Almond (Prunus dulcis)

Abstract

Stylar proteins of 13 almond (Prunus dulcis) cultivars with known S-genotypes were surveyed by IEF and 2D-PAGE combined with immunoblot and N-terminal amino acid sequence analyses to identify S-RNases associated with gametophytic self-incompatibility (SI) in this plant species. RNase activities corresponding to S_a and S_b, two of the four S-alleles tested, were identified by IEF and RNase activity staining. The S_a-RNase band reacted with the anti-S₄serum prepared from Japanese pear (Pyrus serotina); no reaction with the antiserum was observed with the s_bRNase band. When the s_a-RNase band was excised from an IEF gel stained for RNase activity, subjected to SDS-PAGE, and detected by immunoblotting, it appeared that this band consisted of a single protein that reacted with the anti-s₄serum with M_r of about 28 kDa. With 2D-PAGE and silver staining of the stylar extracts, all four S-proteins could be successfully distinguished from each other in the highly basic zone of the gel. Although S_b-, S_c-, and S_dproteins had roughly the same M_r of about 30 kDa, the S_c-protein seemed to be slightly smaller than the S_b-protein and slightly larger than the S_aprotein. In 2D-PAGE profiles as well, the S_a-protein had M_r of about 28 kDa, apparently smaller than the other three proteins. A bud sport, in which one of the two S-alleles of the original cultivar is impaired, was visualized as a loss of S_cprotein, which is consistent with the previous pollination study. All four S-proteins reacted with the anti-S₄serum, probably because of the differing conformations of these S-proteins in the IEF and 2D-PAGE gels. The S_a-protein in 2D-PAGE appeared to be identical to S_a-RNase in IEF; both bad the same M_r and were reactive with the anti-S₄-serum. N-terminal amino acid sequence analysis of the four 5-proteins revealed that they were highly homologous to each other and similar to the 5-RNases of Malus, Pyrus, Scrophulariaceae, and Solanaceae. Taken together, RNases in the style are strongly suggested to be associated with the gametophytic SI of al- mond. This is the first report identofiying and characterizing S-RNase in almond.