Primary restriction of S‐RNase cytotoxicity by a stepwise ubiquitination and degradation pathway in Petunia hybrida

In self‐incompatible Petunia species, the pistil S‐RNase acts as cytotoxin to inhibit self‐pollination but is polyubiquitinated by the pollen‐specific nonself S‐locus F‐box (SLF) proteins and subsequently degraded by the ubiquitin‐proteasome system (UPS), allowing cross‐pollination. However, it remains unclear how S‐RNase is restricted by the UPS. Using biochemical analyses, we first show that Petunia hybrida S(3)‐RNase is largely ubiquitinated by K48‐linked polyubiquitin chains at three regions, R I, R II and R III. R I is ubiquitinated in unpollinated, self‐pollinated and cross‐pollinated pistils, indicating its occurrence before PhS(3)‐RNase uptake into pollen tubes, whereas R II and R III are exclusively ubiquitinated in cross‐pollinated pistils. Transgenic analyses showed that removal of R II ubiquitination resulted in significantly reduced seed sets from cross‐pollination and that of R I and R III to a lesser extent, indicating their increased cytotoxicity. Consistent with this, the mutated R II of PhS(3)‐RNase resulted in a marked reduction of its degradation, whereas that of R I and R III resulted in less reduction. Taken together, we demonstrate that PhS(3)‐RNase R II functions as a major ubiquitination region for its destruction and R I and R III as minor ones, revealing that its cytotoxicity is primarily restricted by a stepwise UPS mechanism for cross‐pollination in P. hybrida.