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 Sa and Sb, two of the four S-alleles tested, were identified by IEF and RNase activity staining. The Sa-RNase band reacted with the anti-S4-serum prepared from Japanese pear (Pyrus serotina); no reaction with the antiserum was observed with the Sb-RNase band. When the Sa-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 Sb-, Sc-, and Sd-proteins had roughly the same M(r) of about 30 kDa, the Sc-protein seemed to be slightly smaller than the Sb-protein and slightly larger than the Sd-protein. In 2D-PAGE profiles as well, the Sa-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 Sc-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 Sa-protein in 2D-PAGE appeared to be identical to Sa-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.

Identification and cDNA cloning for S-RNases in self-incompatible Japanese plum (Prunus salicina Lindl. cv. Sordum).

“…1A). These proteins showed similar Mr and pI to those of other S-RNases in Rosaceae (Ishimizu et al, 1996;Sassa et al, 1993Sassa et al, , 1994Tao et al, 1997Tao et al, , 1999. As with other rosaceous S-RNases, these stylar proteins were also shown to be glycoproteins that contained sugar chains reacting with concanavalin A (Fig.…”

“…1C). The N-terminal amino acid sequence of Sa-protein was highly homologous to those of S-RNases of other Prunus spp., such as almond (Tao et al, 1997) and sweet cherry (Tao et al, 1999) (Fig. 2).…”

“…As is the case with Solanaceae and Scrophulariaceae (for reviews, Kao and McCubbin, 1996;McCubbin and Kao, 1999), S-gene products in pistil of apple and pear were shown to be SRNases (Ishimizu et al, 1996;Sassa et al, 1992Sassa et al, ,1993Sassa et al, ,1994Tomimoto et al,1996). (Tao et al, 1997(Tao et al, , 1999Ushijima et al, 1998). Based on the cDNA sequences, molecular typing system for S-alleles has been developed for sweet cherry (Tao et al,1999) .…”

Identification of an S-RNase binding protein1 (SBP1) homolog of apple (Malus^|^times;domestica)

“…The S-RNase-based gametophytic self-incompatibility (GSI) of Rosaceae, Solanaceae and Plantaginaceae is controlled by a single multiallelic S locus which contains separate genes for pistil and pollen specificities. When the S haplotype of a pollen matches one of the two S haplotypes of the diploid pistil, the pollen is recognized as self and rejected (de et al 1986;McClure et al 1989;Sassa et al 1996Sassa et al , 1997Tao et al 1997;Xue et al 1996) which is thought to be taken up by pollen tubes and act as a cytotoxin to self pollen (Goldraij et al 2006;Luu et al 2000). In Petunia of Solanaceae and species of Rosaceae tribe Pyreae, i.e., apples (Malus×domestica) and pears (Pyrus spp.…”

“…The specificities of pistil and pollen are controlled by separate but tightly linked genes located at the S locus, S-RNase and the F-box gene called SLF/SFB/SFBB, respectively (Franklin-Tong 2008; Kao and Tsukamoto 2004;Meng et al 2011;Sassa et al 2010;Tao and Iezzoni 2010). The pistil S determinant, S-RNase, encodes extracellular ribonuclease (Anderson et al 1986;McClure et al 1989;Sassa et al 1996Sassa et al , 1997Tao et al 1997;Xue et al 1996) which is thought to be taken up by pollen tubes and act as a cytotoxin to self pollen (Goldraij et al 2006;Luu et al 2000). In Petunia of Solanaceae and species of Rosaceae tribe Pyreae, i.e., apples (Malus×domestica) and pears (Pyrus spp.…”