Three Rho of Plant (ROP)-GTPase regulatory proteins control ROP-mediated alkaloid biosynthesis in <i>Catharanthus roseus</i>

Rho of plant (ROP)-GTPase regulatory proteins (RGRPs) have been shown to control plant morphogenesis, development, and immunity; however, their role in specialized metabolism remains unknown. Here, we demonstrate that specific RGRPs control monoterpene indole alkaloid (MIA) biosynthesis by interacting with distinct ROPs in Madagascar periwinkle (Catharanthus roseus). Among the 5 guanine nucleotide exchange factors (GEFs), 4 GTPase-activating proteins (GAPs), and 2 GDP dissociation inhibitors (GDIs) identified in the C. roseus genome, only CrGEF1, CrGAP1, and CrGDI2 specifically interacted with CrROP3 and CrROP5. These RGRPs displayed distinct cytosolic and/or membrane localization patterns, with their transcripts predominantly expressed in aerial tissues. Functional studies revealed that CrGEF1 acts as a positive regulator of MIA biosynthesis, as silencing its gene led to a reduction in MIA production, while overexpression enhanced MIA levels. Conversely, CrGAP1 and CrGDI2 function as negative regulators, with silencing resulting in increased MIA production and overexpression causing reduced MIA levels. Notably, terminal truncated forms of these RGRPs showed interaction with CrROP3 or CrROP5 but failed to influence MIA biosynthesis, underscoring the importance of these domains in their regulatory functions. Overall, our findings uncover a mechanism by which distinct RGRPs coordinate with specific ROPs to regulate transcription factors and fine-tune MIA biosynthesis in C. roseus.