Hybridization barriers in Brassicaceae play a pivotal role in governing reproductive success and maintaining speciation. In this perspective, we highlight recent advances revealing the intricate molecular mechanisms and the interplay among key players governing these barriers. Recent studies have shed light on the molecular mechanisms that govern hybridization barriers in Brassicaceae. The interplay between pollen coat proteins, stigmatic receptors, and signaling peptides plays a crucial role in determining the success of pollination. At the core of this system, autocrine stigmatic RALF peptides (sRALF) maintain the stigmatic barrier by activating the FERONIA (FER) and ANJEA (ANJ) receptor complex, triggering the RAC/ROP-RBOHD pathway and subsequent reactive oxygen species (ROS) production. It is now established that incompatible pollen rejection is mediated by two parallel pathways: the FER-RAC/ROP-RBOHD pathway, which generates ROS, and the ARC1-mediated pathway, which degrades compatible factors required for pollen growth. Conversely, compatible pollen overcomes the stigmatic barrier through the action of pollen coat proteins (PCP-B) and paracrine pollen-derived RALF peptides (pRALF), which compete with autocrine sRALF for receptor binding, enabling successful pollen hydration and tube penetration. The "lock-and-key" mechanism involving sRALF and pRALF provides species-specific recognition of compatible pollen. These findings offer valuable insights into the molecular basis of hybridization barriers and open new possibilities for overcoming these barriers in interspecific and intergeneric crosses within Brassicaceae, with potential applications in plant breeding and crop improvement. Future research should focus on elucidating the evolutionary dynamics of these signaling pathways and exploring their manipulation for crop breeding purposes.
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