Exogenous Brassinosteroid Promotes Vegetative Growth in the World’s Smallest Water Lily (Nymphaea thermarum)

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.