Safflower (Carthamus tinctorius L.) is a traditional Chinese medicinal herb renowned for its high flavonoid content and significant medicinal value. However, the dynamic changes in safflower petal flavonoid profiles across different flowering phases present a challenge in optimizing harvest timing and medicinal use. To enhance the utilization of safflower, this study conducted an integrated transcriptomic and metabolomic analysis of safflower petals at different flowering stages. Our findings revealed that certain flavonoids were more abundant during the fading stage, while others peaked during full bloom. Specifically, seven metabolites, including p-coumaric acid, naringenin chalcone, naringenin, dihydrokaempferol, apigenin, kaempferol, and quercetin, accumulated significantly during the fading stage. In contrast, dihydromyricetin and delphinidin levels were notably reduced. Furthermore, key genes in the flavonoid biosynthesis pathway, such as 4CL, DFR, and ANR, exhibited up-regulated expression with safflower’s flowering progression, whereas CHI, F3H, and FLS were down-regulated. Additionally, exposure to UV-B stress at full bloom led to an up-regulation of flavonoid content and altered the expression of key flavonoid biosynthetic genes over time. This study not only elucidates the regulatory mechanisms underlying flavonoid metabolism in safflower but also provides insights for maximizing its medicinal and industrial applications.

Phenylalanine ammonia-lyase (PAL) catalyzes the first committed step in the phenylpropanoid pathway that governs the entry of carbon flux into flavonoid biosynthesis and stress-responsive metabolism. However, how PAL explicitly mediates hormonal-induced flavonoid biosynthesis and promotes the antioxidant defense system in safflower (Carthamus tinctorius L.) remains largely unknown. Here, we functionally characterize CtPAL1 and demonstrated its regulatory role in abscisic acid (ABA)-induced flavonoid biosynthesis and antioxidant defense. Using phylogenetic and structural predictions, we found that CtPAL1 was placed within a conserved branch of Asteraceae PAL proteins. A promoter analysis indicated multiple hormone- and stress-responsive cis-elements, and exposure to abiotic and hormonal treatments elicited complex, stimulus-dependent dynamics of CtPAL1 expression and flavonoid accumulation. Upon ABA treatment, the expression of CtPAL1 is rapidly induced, triggering early flavonoid biosynthesis. Moreover, CtPAL1-overexpressing Arabidopsis lines exhibited enhanced tolerance to ABA-induced stress by lower lipid peroxidation and higher antioxidant enzyme activities, accompanied with increased flavonoid production. Importantly, the transgenic overexpression of CtPAL1 in Arabidopsis led to the upregulation of the upstream flavonoid pathway genes (At4CL, AtCHI) and elevated total flavonoid levels (1.07–1.27-fold versus wild type), while silencing in safflower caused a reduced flavonoid content (0.52–0.77× controls) and the downregulation of pathway genes. A biochemical assay also confirms that recombinant CtPAL1 efficiently converts L-phenylalanine to trans-cinnamic acid, validating its catalytic function. Together, our results demonstrate that CtPAL1 functions as a highly conserved and functionally active PAL enzyme in safflower and acts as an ABA-responsive modulator of flavonoid biosynthesis and antioxidant defense.

Salinity is a major constraint that compromises safflower performance by disrupting redox balance and metabolic homeostasis. Although hormonal mechanisms for improving plant resilience to abiotic stresses have been reported, the mechanistic role of gibberellic acid (GA₃)-induced regulation of safflower tolerance to salinity remains unclear. This study aimed to investigate the impact of exogenous GA₃ application under normal and saline conditions to evaluate its effects on growth, physiology, redox regulation, and flavonoid biosynthesis in safflower. Using phenotypic, physiological, biochemical, and gene expression analysis, it is suggested that GA₃ significantly alleviates salt stress by integrating antioxidant defense and flavonoid biosynthesis. The results of phenotypic and physiological assessments showed that GA₃ at 400 mg/L GA₃ in safflower seedlings suggests enhanced vegetative growth and photosynthetic performance. Under salt stress, GA₃ significantly alleviated oxidative damage by reducing H₂O₂, ${\mathrm{O}}_2^{-}$, and malondialdehyde (MDA) levels, while enhancing osmoprotective compounds such as proline, soluble sugars, proteins, and chlorophyll. GA₃ also significantly increased the activity of antioxidant enzymes (SOD, POD, CAT, APX, GST, DHAR, and Prx), accompanied by the transcriptional upregulation of their corresponding genes, indicating GA₃-mediated regulation of redox homeostasis at both biochemical and molecular levels. In parallel, GA₃ enhanced the accumulation of major flavonoids, particularly hydroxy safflor yellow A (HSYA), with strong induction of key HSYA biosynthetic genes (CtF6H, CtCGT, Ct2OGD1), whereas salinity alone suppressed their expression. In contrast, the quercetin branch displayed a regulatory bottleneck at CtF3H, which remained suppressed under all treatments, although upstream genes were GA₃-responsive. Together, these findings demonstrate that GA₃ enhances salinity tolerance in safflower by simultaneously activating antioxidant defenses and stimulating flavonoid biosynthesis, providing mechanistic insight with practical implications for developing salt-resilient safflower varieties.