The Phenylpropanoid Gatekeeper CtPAL1 Coordinates ABA-Induced Flavonoid Biosynthesis and Oxidative Stress Tolerance in Safflower (Carthamus tinctorius L.)

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 (<i>Carthamus tinctorius</i> 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 <i>CtPAL1</i> expression and flavonoid accumulation. Upon ABA treatment, the expression of <i>CtPAL1</i> is rapidly induced, triggering early flavonoid biosynthesis. Moreover, <i>CtPAL1</i>-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 <i>CtPAL1</i> 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.