Safflower is an important economic crop with a plethora of industrial and medicinal applications around the world. The bioactive components of safflower petals are known to have pharmacological activity that promotes blood circulation and reduces blood stasis. However, fine-tuning the genetic mechanism of flower development in safflower is still required. In this study, we report the genome-wide identification of MADS-box transcription factors in safflower and the functional characterization of a putative CtMADS24 during vegetative and reproductive growth. In total, 77 members of MADS-box-encoding genes were identified from the safflower genome. The phylogenetic analysis divided CtMADS genes into two types and 15 subfamilies. Similarly, bioinformatic analysis, such as of conserved protein motifs, gene structures, and cis-regulatory elements, also revealed structural conservation of MADS-box genes in safflower. Furthermore, the differential expression pattern of CtMADS genes by RNA-seq data indicated that type II genes might play important regulatory roles in floral development. Similarly, the qRT-PCR analysis also revealed the transcript abundance of 12 CtMADS genes exhibiting tissue-specific expression in different flower organs. The nucleus-localized CtMADS24 of the AP1 subfamily was validated by transient transformation in tobacco using GFP translational fusion. Moreover, CtMADS24-overexpressed transgenic Arabidopsis exhibited early flowering and an abnormal phenotype, suggesting that CtMADS24 mediated the expression of genes involved in floral organ development. Taken together, these findings provide valuable information on the regulatory role of CtMADS24 during flower development in safflower and for the selection of important genes for future molecular breeding programs.

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.