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

Stylar proteins of 13 almond (Prunus dulcis) cultivars withknown S-genotypes were surveyed by IEF and 2D-PAGE combinedwith immunoblot and N-terminal amino acid sequence analysesto identify S-RNases associated with gametophytic self-incompatibility(SI) in this plant species. RNase activities corresponding toS_a and S_b, two of the four S-alleles tested, were identifiedby IEF and RNase activity staining. The S_a-RNase band reactedwith the anti-S₄serum prepared from Japanese pear (Pyrus serotina);no reaction with the antiserum was observed with the s_bRNaseband. When the s_a-RNase band was excised from an IEF gel stainedfor RNase activity, subjected to SDS-PAGE, and detected by immunoblotting,it appeared that this band consisted of a single protein thatreacted with the anti-s₄serum with M_r of about 28 kDa. With2D-PAGE and silver staining of the stylar extracts, all fourS-proteins could be successfully distinguished from each otherin the highly basic zone of the gel. Although S_b-, S_c-, andS_dproteins had roughly the same M_r of about 30 kDa, the S_c-proteinseemed to be slightly smaller than the S_b-protein and slightlylarger than the S_aprotein. In 2D-PAGE profiles as well, theS_a-protein had M_r of about 28 kDa, apparently smaller than theother three proteins. A bud sport, in which one of the two S-allelesof the original cultivar is impaired, was visualized as a lossof S_cprotein, which is consistent with the previous pollinationstudy. All four S-proteins reacted with the anti-S₄serum, probablybecause of the differing conformations of these S-proteins inthe IEF and 2D-PAGE gels. The S_a-protein in 2D-PAGE appearedto be identical to S_a-RNase in IEF; both bad the same M_r andwere reactive with the anti-S₄-serum. N-terminal amino acidsequence analysis of the four 5-proteins revealed that theywere highly homologous to each other and similar to the 5-RNasesof Malus, Pyrus, Scrophulariaceae, and Solanaceae. Taken together,RNases in the style are strongly suggested to be associatedwith the gametophytic SI of al- mond. This is the first reportidentofiying and characterizing S-RNase in almond. (Received July 11, 1996; Accepted December 26, 1996)