Abstract

Atropa belladonna L. (Solanaceae) is a significant source of the pharmacologically important tropane alkaloids (TAs), particularly hyoscyamine and scopolamine. Although rolB and rolC from Rhizobium rhizogenes are known to enhance plant secondary metabolism, their functional specificities and combined actions in TAs biosynthesis remain unclear. Here, we generated transgenic A. belladonna overexpressing 35S::rolB , 35S::rolC , and 35S::rolB+rolC to systematically investigate their effects on TAs accumulation, plant morphology, and drought tolerance. Co-expression of rolB and rolC exhibited metabolic antagonism, with TAs levels significantly lower than in rolC single transgenic plants, further establishing rolC as the primary regulator of this pathway. Phenotypic analysis revealed divergent effects: rolB increased leaf size and promoted root growth, whereas rolC caused dwarfism, early flowering, and male sterility; double transgenic plants displayed exacerbated growth inhibition. Notably, rolC-expressing plants showed improved drought performance via combined morphological and antioxidant adjustments. Mechanistically, both genes upregulated key biosynthetic genes, with rolC regulating a broader set. Protein interaction assays revealed that RolC directly interacts with TRI, calmodulin (CaM1/CaM7), and the brassinosteroid signaling component BSK. A root-specific transcription factor, AbbHLH30 , was identified as a candidate downstream component of the RolC-CaM1 module, with the capacity to activate H6H expression in transient assays. This study provides a mechanistic basis for understanding rol gene functions and offers specific molecular targets for engineering TAs production in A. belladonna .