Characterization of the sexual reproductive process in the perennial tree Robinia pseudoacacia

Sexual reproduction in perennial plants, especially woody species, remains poorly understood, and most studies have focused on annuals such as Arabidopsis thaliana. This gap hinders understanding of perennial reproductive processes and limits tree breeding. Here we characterized the sexual reproductive process of Robinia pseudoacacia, a perennial leguminous tree, and established standardized methods for studying its reproduction. We optimized cytological assays for pollen viability, in vitro germination, and pollen tube dynamics, achieving over 90% pollen viability and germination efficiency within 8 h. R. pseudoacacia pollen exhibited reproductive thermotolerance and maintained over 20% germination at 37 °C, a temperature lethal to A. thaliana pollen. This thermotolerance was associated with maintenance of stable plasma membrane integrity and an organized F-actin cytoskeleton. R. pseudoacacia pollen also germinated on A. thaliana stigmas, penetrated the styles, and targeted 3.6% of A. thaliana ovules, although post-zygotic incompatibility prevented seed formation. The ANX-RALF signaling module is conserved in R. pseudoacacia, and antisense oligodeoxynucleotide-mediated suppression of RpANX2 disrupted pollen tube growth. Moreover, aberrant pollen tube guidance was linked to its low seed set. These findings provide a systematic characterization of sexual reproduction in a perennial tree and establish methodological foundations for reproductive studies in woody perennials, with implications for tree breeding.