Pre-fertilization hybridization barriers are the main causes of inter-subgeneric hybridization challenges in water lily. However, the mechanism underlying low compatibility between pollen and stigma of water lily remains unclear. This study demonstrates that CBL-interacting protein kinase 6 (CIPK6) responded to the signaling exchange between incompatible pollen and stigma through interactions with SNF1-related kinase 1 (SnRK1) and promotes the accumulation of SnRK1 protein. Activated SnRK1 interacted with 9-cis-epoxycarotenoid dioxygenase 2 (NCED2) to promote its degradation, thereby inhibiting abscisic acid (ABA) synthesis. A decrease in ABA content in the stigma impaired the ABA-mediated removal of reactive oxygen species (ROS), ultimately resulting in the rejection of the incompatible pollen by the stigma. Our results highlight the essential role of the NpCIPK6–NpSnRK1–NpNCED2 module in conferring inter-subgeneric hybridization barriers in water lily by interfering with ABA synthesis and promoting ROS accumulation. This study offers valuable mechanistic insights into cellular signaling and reproductive barriers in water lily as well as across other biological contexts.

The brassinazole-resistant (BZR) transcription factors (TFs) are key components of brassinosteroid (BR) signaling, which play an important role in regulating plant growth and development as well as responding to abiotic stress. However, a functional study of BZR transcription factors in lotuses has not been reported. A total 10 BZR1 genes (four NnBZR1 and six NlBZR1) were identified from the genomes of two lotus species (Nelumbo nucifera and Nelumbo lutea). The construction of the phylogenetic tree showed that the 10 BZR1 genes of the lotus were divided into four groups; the NnBZR1s and NlBZR1s were unevenly distributed on three and four chromosomes, respectively. Gene structure analysis showed that motif 1 and motif 9 are highly conserved in the lotus BZR1 protein, which might be related to the conserved domain BES_N of BZR1. The analysis of promoter cis-acting elements showed that the promoters of most of the BZR1 genes in the lotus contained elements related to light-responsive, ABA-responsive and abiotic stress-responsive factors, indicating that the BZR1 gene of the lotus played an important role in its response to abiotic stress. The responses of BZR1 genes to BR, ABA and four abiotic stresses (Cold, PEG6000, Cd and NaCl) were analyzed by qRT-PCR. The qRT-PCR results further verified that the lotus BZR1 genes play an important role in responding to hormone signals and resisting abiotic stress. This study laid the foundation for further research on the function of lotus BZR1 genes and provided a theoretical basis for future breeding and horticultural applications.

Flower morphology is a critical ornamental trait in flowering plants. Elucidating the molecular mechanisms underlying flower development is essential for the breeding of diverse ornamental plant cultivars. The ABCE model genes are pivotal in regulating floral development in angiosperms. In order to understand the molecular mechanism of flower development in Clematis, nine ABCE model genes were identified from the transcriptome data of Clematis cv. ‘Amethyst Beauty’. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed tissue-specific expression patterns among these genes. ClPI, ClSEP1, and ClSEP3 were predominantly expressed in sepals. ClAP3, ClPI, ClAG2, ClSEP1, and ClSEP3 were highly expressed in stamens. ClAG1 was exclusively expressed in pistils. These findings suggested that ABCE genes may play an important role in regulating the formation of flower morphology in the Clematis. The open reading frame (ORF) of ClAG2 was cloned and overexpression of ClAG2 in tobacco resulted in shorter corolla tube, reduced crown area, and stunted stamen. ClAG2 may have a negative effect on the formation of double-tepal flowers of Clematis and play a specific role in stamen and pistil development. Yeast two-hybrid assays demonstrated that ClAG2 could interact with class E proteins ClSEP3 and ClSEP4 but not with class B proteins ClAP3 and ClPI. Our results will lay a theoretical foundation for further research on the mechanism of flower development regulation in Clematis.