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.

Benzoxazinoids (BXs) are tryptophan-derived indole metabolites and play a role in various physiological processes, such as auxin metabolism. Auxin is essential in the process of somatic embryogenesis (SE) in plants. In this study, we used bioinformatics, transcriptome data, exogenous treatment experiments, and qPCR analysis to study the evolutionary pattern of Bx genes in green plants, the regulatory mechanism of DlBx genes during early SE, and the effect of 2,4-dihydroxy-7-methoxy-1,4-benzoxazine-3-one (DIMBOA) on the early SE in Dimocarpus longan Lour. The results showed that 27 putative DlBxs were identified in the longan genome; the Bx genes evolved independently in monocots and dicots, and the main way of gene duplication for the DlBx was tandem duplication (TD) and the DlBx were strongly constrained by purification selection during evolution. The transcriptome data indicated varying expression levels of DlBx during longan early SE, and most DlBxs responded to light, temperature, drought stress, and 2,4-dichlorophenoxyacetic acid (2,4-D) treatment; qRT-PCR results showed DlBx1, DlBx6g and DlBx6h were responsive to auxin, and treatment with 0.1mg/L DIMBOA for 9 days significantly upregulated the expression levels of DlBx1, DlBx3g, DlBx6c, DlBx6f, DlB6h, DlBx7d, DlBx8, and DlBx9b. The correlation analysis showed a significantly negative correlation between the expression level of DlBx1 and the endogenous IAA contents; DIMBOA significantly promoted the early SE and significantly changed the endogenous IAA content, and the IAA content increased significantly at the 9th day and decreased significantly at the 13th day. Therefore, the results suggested that DIMBOA indirectly promote the early SE by changing the endogenous IAA content via affecting the expression level of DlBx1 and hydrogen peroxide (H₂O₂) content in longan.

Background: Nymphaea prolifera employs a unique asexual reproductive strategy in which its normal flowers transform into branching flowers. These are specialized structures that develop vegetative propagules. These propagules can directly sprout into new plants. As the branching flowers mature, they detach from the parent plant and form new, independent individuals. However, the underlying molecular mechanisms that govern this distinctive reproductive phenomenon remain unclear. Results: In this study, microscopic observations were conducted on normal flowers and branching flowers of Nymphaea prolifera . The results showed that reproductive organ tissues such as the pistils (including ovaries and ovules) of branching flowers were completely transdifferentiated into vegetative propagules, which could continuously generate multiple generations of branching flowers. Analyses of endogenous hormones, transcriptomes, and metabolomes were performed on branching flowers at four developmental stages (bud stage, early flowering stage, middle flowering stage, and late flowering stage) as well as on normal flowers. The results revealed significant differences in eight hormone compounds between branching flowers and normal flowers, including 2 cytokinins, 4 auxin-related compounds, ABA, and GA₄. GO and KEGG functional enrichment analyses indicated that both differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) were significantly enriched in the flavonoid biosynthesis pathway. Furthermore, the expression levels of genes and metabolites associated with this pathway were gradually up-regulated during the development of branching flowers. Weighted gene co-expression network analysis (WGCNA) identified three core modules (MEgreen, MEblue, MEturquoise) that were highly correlated with hormone and flavonoid metabolism, and screened out potential candidate genes IAA17 , ARF6 , ABF1 that may be associated with endogenous hormone and flavonoid metabolism. Conclusions: This study reveals the morphological and molecular characteristics of branching flower development in N. prolifera . It identifies the hub genes, modules and metabolic pathways involved in this phenomenon, thereby providing a comprehensive morphological and molecular basis for elucidating this species’ unique asexual reproductive mechanism.

Waterlily (Nymphaea L.), a globally renowned aquatic ornamental plant, is prized for its aesthetic flowers and intense floral fragrance. However, the molecular mechanisms underlying floral scent biosynthesis in waterlily remain poorly characterized, and integrated analyses of dynamic volatile emission patterns and their associated biosynthetic pathways are lacking. In this study, we combined headspace solid-phase microextraction/gas chromatography–mass spectrometry (HS-SPME/GC-MS) with transcriptome sequencing (RNA-seq) to investigate the composition, emission dynamics, and biosynthesis of volatile organic compounds (VOCs) in the stamens of Nymphaea ‘Paul Stetson’ across three developmental stages. A total of 671 VOCs, classified into 14 categories, were identified. Transcriptome analysis revealed 47,951 differentially expressed genes (DEGs). Integrative omics analysis demonstrated correlated DEGs and differentially accumulated volatiles were significantly enriched in pathways related to phenylpropanoid biosynthesis, terpenoid backbone biosynthesis, diterpenoid biosynthesis, and ubiquinone/other terpenoid-quinone biosynthesis. Five candidate functional genes exhibiting strong positive correlations with VOC accumulation levels were identified, three of which are implicated in terpenoid biosynthesis. These findings provide a theoretical foundation for elucidating aroma composition and biosynthesis in waterlily and offer novel avenues for the genetic improvement of fragrance traits for ornamental, beverage, and cosmetic applications.