CtARF4 Regulates Inflorescence Development Through Transcriptional Regulation of CtMADS24 in Safflower

Safflower (Carthamus tinctorius L.) is a traditional economic crop in China valued for its medicinal petals and high-quality seed oil. Despite the importance of floret number and capitulum architecture for petal yield in safflower, the molecular regulators linking auxin signaling to inflorescence development in this species remain poorly understood. Auxin response factors (ARFs) are key transcriptional regulators mediating auxin-responsive gene expression and developmental processes, yet their functions in safflower inflorescence development have not been systematically investigated. In this study, we identified 25 CtARF genes from the safflower genome and classified them into five phylogenetic subfamilies. Cis-regulatory analysis predicted the presence of hormone- and development-related elements in CtARF-associated promoter regions. Expression profiling revealed that CtARF4, a member of the CtARF III subfamily, exhibits preferential expression during flower development. CtARF4 was localized to the nucleus and shown to interact with the CtMADS24 promoter and the Aux/IAA protein CtIAA9 in heterologous systems. Transient overexpression of CtARF4 increased floret number and length and promoted flowering, whereas virus-induced gene silencing resulted in opposite phenotypes. In addition, CtARF4 perturbation was associated with a reduction in IAA content as measured by a kit-based assay. Collectively, these findings suggest that CtARF4 functions as an auxin-responsive transcriptional regulator contributing to inflorescence development in safflower.