Outcrossing preference in Magnolia ×soulangeana 'Hongyun' is associated with differential stigmatic ROS accumulation regulated by MsFERONIA-MsROP2-MsROBHD module.

As primitive angiosperms, bisexual Magnolia species evolved a relatively advanced mating system, namely facultative outcrossing, avoiding inbreeding depression meanwhile providing a certain reproductive assurance. Explosive advances have been made in the molecular understanding of pollen-pistil interactions in the past decades, especially emphasizing the role of FERONIA-RAC/ROP-RBOHD module. However, relevant molecular framework in primitive angiosperms remains largely unknown. In this study, our systematic investigation showed that outcrossing rather than selfing benefited pollen hydration and pollen tube growth via rapidly inhibiting stigmatic ROS accumulation in Magnolia ×soulangeana 'Hongyun'. It was attributed to the differential expression and regulation of the MsFERONIA-MsROP2-MsRBOHD module. Particularly, outcrossing strongly inhibited the expression of MsRBOHD throughout the pollen-pistil interaction. Gene function verification showed that direct overexpression of MsFERONIA conferred a rapid stigmatic ROS response and promoted compatible pollen growth in Arabidopsis. Moreover, MsRBOHD overexpression resulted in elevated stigmatic ROS, suppressing compatible pollen growth in Arabidopsis. Overall, these results emphasized that this conserved MsFERONIA-MsROP2-MsRBOHD module played a pivotal role in the regulation of pollen-pistil interactions via mediating stigmatic ROS in 'Hongyun'. Our findings could deepen understanding of the molecular basis underlying mate selection in primitive angiosperms, and provide novel insights into breeding and conservation of endangered rare species.