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.

Tropical water lilies are a group of aquatic plants with high ornamental value and are widely favored in home gardening. However, most tropical water lily cultivars feature a large plant stature and vigorous spreading growth layout, making them unsuitable for cultivation in small containers. Therefore, developing miniaturization cultivation techniques for tropical water lilies in small containers is required. Plant growth retardants are effective in inducing plant dwarfing and overall miniaturization. In this study, the tropical water lily cultivar ‘Black Beauty’ was used as the experimental material to explore the effects of two plant growth retardants, chlormequat chloride (CCC) and paclobutrazol (PP₃₃₃), on its growth and development, with the objective of screening optimal retardant types, application concentrations and treatment methods. The results showed that the growth rate of ‘Black Beauty’ slowed down under either CCC or PP₃₃₃ treatment, and remained stable from day 21 to day 51. Compared with the control group, treated plants presented reduced crown spread, decreased leaf area and thickened leaves; the number of flowers and leaves declined slightly, and the key ornamental morphological traits (compact plant form, moderate leaf size, normal flowering) were maintained. PP₃₃₃ exhibited a stronger dwarfing effect than CCC. When the plant growth retardants were dissolved in water for application, a 6-day treatment interval delivered better dwarfing efficacy than a 9-day interval. Among all treatments, 30 mg·L⁻¹ PP₃₃₃ applied at 6-day intervals achieved the optimal miniaturization effect. Following this treatment, the endogenous accumulation of chlorophyll, soluble protein and total sugar, together with the activity levels of antioxidant enzymes (SOD, POD, CAT), were markedly higher than those measured in the control and all other treatment groups, whereas the endogenous accumulation of malondialdehyde (MDA) decreased substantially. The contents of endogenous hormones IAA, GA₃ and ZR in all treatment groups decreased with the increase in retardant concentration, whereas the ABA content showed an upward trend. The 30 mg·L⁻¹ PP₃₃₃ treatment applied every six days induced the minimal endogenous accumulation of IAA, GA₃ and ZR, alongside the maximum endogenous ABA accumulation within plant tissues. For potted cultivation of ‘Black Beauty’ water lily, it is recommended to apply 30 mg·L⁻¹ PP₃₃₃ at 6-day intervals and use containers with an outer diameter of 30–35 cm.

Nymphaea thermarum, the world’s smallest water lily, provides a useful system for investigating hormonal regulation of vegetative growth. This study investigated vegetative growth, hormone immunoreactive estimates, and tissue-specific transcriptional responses to exogenous 24-epibrassinolide (EBL) in N. thermarum. Plants were subjected to a concentration gradient of 24-epibrassinolide (EBL; 0, 200, 400, 600, 800, and 1000 nM) under soil-based pot culture with EBL treatment solutions, and morphological, hormonal, and transcriptomic analyses were performed. Growth responses were non-monotonic, with BR400 producing the highest mean longitudinal leaf radius, petiole length, and leaf area, while transverse leaf radius was comparable to that under BR200; all four traits under BR400 were significantly greater than in the control. Hormone immunoreactive estimates varied across treatments, with the highest mean gibberellin (GA)- and brassinosteroid (BR)-immunoreactive estimates observed under BR400; however, neither differed significantly from CK. The highest mean indole-3-acetic acid (IAA)-immunoreactive estimate occurred under BR600. Transcriptomic analysis identified 1053 DEGs in leaves and 2028 in petioles under BR400 treatment compared with the control. GO and KEGG analyses revealed enrichment of cell-wall-organization- and phenylpropanoid biosynthesis-related categories among DEGs. Photosynthesis-related genes were down-regulated in leaves, accompanied by lower chlorophyll a and total chlorophyll concentrations under BR400. These findings characterize EBL-associated vegetative growth and transcriptional responses in N. thermarum and identify candidate genes and processes for future functional studies of water lily architecture.