Comprehensive characterization of safflower cytochrome P450 family and the role of CtCYP41 in flavonoid biosynthesis and drought response

Safflower (Carthamus tinctorius L.) is an economical crop rich in diverse flavonoids, which can be widely used in the pharmaceutical and food industries due to their therapeutic effects in promoting blood circulation and alleviating blood stasis. Although current studies have preliminarily elucidated the safflower flavonoid biosynthesis pathway, and confirmed the involvement of cytochrome P450 enzymes (CYPs), a comprehensive identification and analysis of the CYP gene family in safflower remains lacking. In this study, we systematically identified a total of 317 CtCYP genes and classified them into 8 clades and 41 families based on the chromosomal-level safflower genome. Conserved motif and gene structure analyses further validated the phylogenetic relationships. Prediction of promoter cis-acting elements revealed that these genes may be regulated by multiple environmental factors such as light and drought, as well as phytohormones including abscisic acid and methyl jasmonate. Collinearity analysis indicated that tandem duplication events likely served as the primary mechanism for the expansion of this gene family in safflower. By integrating transcriptomic and proteomic data from different floral colors and developmental stages of safflower, we identified five key candidate genes: CtCYP41, CtCYP100, CtCYP101, CtCYP119, and CtCYP296. The gene expression was further verified by qRT-PCR. Subcellular localization experiments suggested that CtCYP41 localized to the cytoplasm or around the nucleus, as well as within the endoplasmic reticulum. Functional validation demonstrated that overexpression of CtCYP41 in Arabidopsis and transient expression in tobacco significantly enhanced flavonoid content and drought tolerance, as well as the reduction of reactive oxygen species (ROS). We systematically identified and characterized the members of the CtCYP superfamily in safflower. The diversity of cis-acting elements and protein domains suggested that safflower P450 was involved in the response to various environmental factors along with functional differentiation. Molecular experiments further confirmed that one of the P450s (CtCYP41) may play a positive role in these processes of flavonoid synthesis, ROS clearance and drought response. This study provides a theoretical basis and genetic resources for the molecular breeding of safflower varieties with high flavonoid content and drought resistance.