Peonies are globally renowned ornamental plants, and distant hybridization is a key method for breeding new varieties, though it often faces cross-incompatibility challenges. The metabolic mechanisms underlying the crossing barrier between tree peony (Paeonia sect. Moutan) and herbaceous peony (P. lactiflora) remain unclear. To identify key metabolites involved in cross-incompatibility, we performed a cross between P. ostii ‘Fengdanbai’ (female parent) and P. lactiflora ‘Red Sara’ (male parent) and analyzed metabolites in the stigma 12 h after pollination using UPLC-MS. We identified 1242 differential metabolites, with 433 up-regulated and 809 down-regulated, including sugars, nucleotides, amino acids, lipids, organic acids, benzenoids, flavonoids, and alkaloids. Most differential metabolites were down-regulated in hybrid stigmas, potentially affecting pollen germination and pollen tube growth. Cross-pollinated stigma exhibited lower levels of high-energy nutrients (such as amino acids, nucleotides, and tricarboxylic acid cycle metabolites) compared to self-pollinated stigma, which suggests that energy deficiency is a contributing factor to the crossing barrier. Additionally, cross-pollination significantly impacted KEGG pathways such as nucleotide metabolism, purine metabolism, and vitamin B6 metabolism, with most metabolites in these pathways being down-regulated. These findings provide new insights into the metabolic basis of cross-incompatibility between tree and herbaceous peonies, offering a foundation for overcoming hybridization barriers in peony breeding.

Paeonia ludlowii is a threatened and valuable germplasm in the cultivated tree peony gene pool, with distinctive traits such as tall stature, pure yellow flowers, and scarlet foliage in autumn. However, the crossability barrier limits gene transfer from P. ludlowii to cultivated tree peony. Therefore, our study investigated the reasons for the lack of crossability between P. ludlowii and Paeonia ostii ‘Fengdan’. Distant cross pollination (DH) resulted in the formation of many calloses at the ends of the pollen tubes, which grew non-polar, twisted, entangled, and often stopped in the style. Pollen tubes elongated the fastest in self-pollination (CK), and pollen tubes elongated faster and fewer pollen tube abnormalities were observed in stigmas treated with KCl solution before pollination (KH) than in DH. During pollen–pistil interactions, the absence of stigma exudates, high levels of H₂O₂, O₂⁻, MDA, ^•OH, ABA, and MeJA, and lower levels of BR and GA₃ may negatively affect pollen germination and pollen tube elongation in the pistil of P. ostii ‘Fengdan’. Pollen tubes in CK and KH penetrated the ovule into the embryo sac at 24 h after pollination, whereas only a few pollen tubes in DH penetrated the ovule at 36 h after pollination. Pre-embryo abnormalities and the inhibition of free nuclear endosperm division resulted in embryo abortion in most of the fruits of DH and many fruits of KH, which occurred between 10 and 20 days after pollination, whereas embryos in CK developed well. Early embryo abortion and endosperm abortion in most of the fruits of DH and KH led to seed abortion. Seed abortion in KH and DH was mainly due to an insufficient supply of auxins and gibberellins and lower content of soluble protein and soluble sugars. The cross failure between P. ludlowii and P. ostii ‘Fengdan’ is mostly caused by a pre-fertilization barrier. KH treatment can effectively promote pollen tube growth and facilitate normal development of hybrid embryos. These findings provide new insights into overcoming the interspecific hybridization barrier between cultivated tree peony varieties and wild species.