Identification and verification of key metabolites in polyamine-regulated flavonoid production in longan embryogenic calli using widely targeted metabolomics

Polyamines, emerging as pivotal regulators, modulate a myriad of fundamental cellular processes and orchestrate key physiological and biochemical pathways, including putrescine, spermidine, spermine. This study explores the mechanisms by which exogenous polyamines regulate the growth and flavonoid accumulation in longan (Dimocarpus longan Lour.) embryogenic calli (EC). Here, we screened the effects of different types of polyamines and their inhibitor D-arginine in longan EC. Exogenous 10 μmol/L spermine demonstrated superior efficacy, enhancing the fresh weight of longan EC by 12.65 % and increasing its flavonoids production rate by 26.11 % versus control. Exogenous spermine also up-regulated the expression of polyamines and flavonoids metabolism genes. Widely targeted metabolomics indicated flavonoids as predominant differential metabolites under spermine treatment, with vitexin-2′-O-glucoside and quercetin-3-O-glucoside-7-O-rhamnoside showing the most pronounced differential accumulation. D-arginine acts mainly as inhibition. Intriguingly, exogenous 10 μmol/L quercetin outperformed Spm and vitexin in promoting both longan EC growth (by 36.58 %) and flavonoids production rate (by 65.08 %), while concomitantly upregulating the expression of polyamines metabolism genes, suggesting a possible synergistic interaction between spermine and quercetin. Furthermore, exogenous quercetin and vitexin facilitated the development of longan EC into globular embryo. Cultivation of longan EC in a stirred bioreactor (5 L working volume) supplemented with 10 μmol/L quercetin achieved 196.15 % higher flavonoid yield than that in solid medium, while reducing the culture duration from 30 to 9 days. This study establish a foundation for understanding the intrinsic mechanism by which polyamines regulate the growth and flavonoids accumulation in longan EC.