Abstract
Recent studies have shown that loss of pollen-S function in S4' pollen from sweet cherry (Prunus avium) is associated with a mutation in an S haplotype-specific F-box4 (SFB4) gene. However, how this mutation leads to self-compatibility is unclear. Here, we examined this mechanism by analyzing several self-compatible sweet cherry varieties. We determined that mutated SFB4 (SFB4') in S4' pollen (pollen harboring the SFB4' gene) is approximately 6 kD shorter than wild-type SFB4 due to a premature termination caused by a four-nucleotide deletion. SFB4' did not interact with S-RNase. However, a protein in S4' pollen ubiquitinated S-RNase, resulting in its degradation via the 26S proteasome pathway, indicating that factors in S4' pollen other than SFB4 participate in S-RNase recognition and degradation. To identify these factors, we used S4-RNase as a bait to screen S4' pollen proteins. Our screen identified the protein encoded by S 4 -SLFL2, a low-polymorphic gene that is closely linked to the S-locus. Further investigations indicate that SLFL2 ubiquitinates S-RNase, leading to its degradation. Subcellular localization analysis showed that SFB4 is primarily localized to the pollen tube tip, whereas SLFL2 is not. When S 4 -SLFL2 expression was suppressed by antisense oligonucleotide treatment in wild-type pollen tubes, pollen still had the capacity to ubiquitinate S-RNase; however, this ubiquitin-labeled S-RNase was not degraded via the 26S proteasome pathway, suggesting that SFB4 does not participate in the degradation of S-RNase. When SFB4 loses its function, S4-SLFL2 might mediate the ubiquitination and degradation of S-RNase, which is consistent with the self-compatibility of S4' pollen.