Abstract
C2H2-type zinc-finger transcription factors (ZFPs) play essential roles in plant stress signaling and development; however, their putative functions in safflower have not been systematically characterized. Leveraging the reference genome of the safflower cultivar 'Jihong-1' (Carthamus tinctorius L.), we investigated the C2H2 family and identified 62 CtC2H2 genes. Comparative phylogeny with Arabidopsis revealed six subfamilies characterized by shared features such as exon-intron organization and conserved QALGGH motif. Promoter analysis identified multiple light- and hormone-responsive cis-elements (e.g., G-box, Box 4, GT1-motif, ABRE, CGTCA/TGACG), suggesting potential multi-layered regulation. RNA-seq and qRT-PCR analysis identified tissue-specific candidate genes, with CtC2H2-22 emerging as the most petal-specific (6-fold upregulation), alongside CtC2H2-02, CtC2H2-23, and CtC2H2-24 in seeds (~3-fold), and CtC2H2-21 in roots (3-fold). Under abiotic stresses, CtC2H2 genes also demonstrated rapid and dynamic responses. Under cold stress, CtC2H2 genes showed a rapid temporal pattern of expression, with early increase for genes like CtC2H2-45 (>4-fold at 3-6 h) and a delayed increase for CtC2H2-23 at 9 h. A majority of CtC2H2 genes (8/12) were upregulated by ABA treatment, with CtC2H2-47 suggesting 3.5-fold induction. ABA treatment also led to a significant increase (2.5-fold) in total leaf flavonoid content at 24h, which is associated with the significant upregulation of flavonoid pathway genes CtANS (5-fold) and CtCHS (3.3-fold). Simultaneously, UV-B radiation induced two distinct expression patterns: a significant suppression of four genes (CtC2H2-23 decreased to 30% of control) and a complex fluctuating pattern, with CtC2H2-02 upregulated at 48 h (2.8-fold). MeJA elicitation revealed four complex expression profiles, from transient induction (CtC2H2-02, 2.5-fold at 3 h) to multi-phasic oscillations, demonstrating the functional diversity of CtC2H2-ZFPs in jasmonate signaling. Together, these results suggest stress and hormone-responsive expression modules of C2H2 ZFPs for future functional studies aimed at improving stress adaptation and modulating specialized metabolism in safflower.