Rho of plant GTPases with geranylgeranylation motif modulate monoterpene indole alkaloid biosynthesis in Catharanthus roseus

Rho Of Plant (ROP) GTPases function as molecular switches that control signaling processes essential for growth, development, and defense. However, their role in specialized metabolism is poorly understood. Previously, we demonstrated that inhibition of protein geranylgeranyl transferase (PGGT-I) negatively impacts the biosynthesis of monoterpenoid indole alkaloids (MIA) in Catharanthus roseus, indicating the involvement of prenylated proteins in signaling. Here, we show through biochemical, molecular and in planta approaches that specific geranylgeranylated ROPs modulate C. roseus MIA biosynthesis. Among the six C. roseus ROP GTPases (CrROPs), only CrROP3 and CrROP5, having a C- terminal CSIL motif, were specifically prenylated by PGGT-I. Additionally, both of their transcripts showed higher expression in most parts compared to other CrROPs. Protein- protein interaction studies revealed that both CrROP3 and CrROP5, but not CrROP2 (lacking CSIL motif), interacted with CrPGGT-I. Further, CrROP3 and CrROP5 exhibited nuclear localization, whereas CrROP2 was localized to plasma membrane. In planta functional studies revealed that silencing of CrROP3 and CrROP5 negatively affected MIA biosynthesis, while their overexpression upregulated MIA formation. In contrast, silencing and overexpression of CrROP2 had no effect on MIA biosynthesis. Moreover, overexpression of {Delta}CrROP3 and {Delta}CrROP5 mutants lacking the CSIL motif failed to enhance MIA biosynthesis. Taken together, these results implicate that CrROP3 and CrROP5 have positive regulatory role on MIA biosynthesis and thus shed light on how geranylgeranylated ROP GTPases mediate the modulation of specialized metabolism in C. roseus.