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 antiS4-serum prepared from Japanese pear (Pyrus serotina); no reaction with the antiserum was observed with the S(b)-RNase 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-S4-serum 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(d)-proteins 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(a)-protein. 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(c)-protein, which is consistent with the previous pollination study. All four S-proteins reacted with the anti-S4-serum, 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 had the same M(r) and were reactive with the anti-S4-serum, N-terminal amino acid sequence analysis of the four S-proteins revealed that they were highly homologous to each other and similar to the S-RNases of Malus, Pyrus, Scrophulariaceae, and Solanaceae. Taken together, RNases in the style are strongly suggested to be associated with the gametophytic SI of almond. This is the first report identifying and characterizing S-RNase in almond.