PbrRALF5/10 prevents incompatible pollen tube death by reconstructing the methyl-esterified pectin and reactive oxygen species metabolism of pear in vitro.

Rapid alkalinization factors (RALFs) are short-chain polypeptides that regulate methyl-esterified pectin accumulation and reactive oxygen species (ROS) metabolism in pollen tubes across diverse plant species. In pear (Pyrus) self-incompatibility (SI), pollen tube polar growth is inhibited by increased apical methyl-esterified pectin content and disrupted apical ROS gradients, while pear RALF family members show no expression response to SI, indicating they are not inherently involved in the SI regulatory pathway. We investigated pollen tube-highly expressed pear RALFs (PbrRALF2/5/6/7/9/10), among which PbrRALF5/10 interact with pollen tube-expressed PbrLRX7/8/10/11 and negatively regulate apical methyl-esterified pectin content (in contrast to PbrRALF6, which competitively binds PbrLRX8 with PbrRALF10 and exerts opposite pectin-regulatory effects) and positively regulate ROS accumulation via the PbrANX/PbrBUPS receptor kinase pathway. Exogenous application of recombinant PbrRALF5/10 (rPbrRALF5/10) during pear SI responses achieved phenotypic rescue in vitro: it significantly reduced apical methyl-esterified pectin content (not to self-compatible levels), re-established the ROS polarity gradient, alleviated SI-induced nuclear DNA degradation, and alleviated incompatible pollen tube growth inhibition. These findings, based on exclusive in vitro experiments, clarify that PbrRALF5/10, while not participating in the SI pathway, mitigate SI-induced pollen tube defects by regulating pectin and ROS, providing insights into their potential for improving pear reproductive success. Notably, in vivo validation remains critical to fully support these conclusions, as no in vivo evidence was obtained to confirm the function of PbrRALF5/10 in alleviating SI under natural pollination conditions.