Abstract
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.