Regulation of lignan biosynthesis through signaling pathways mediated by H2O2 in Linum album cell culture in response to exogenous putrescine

Polyamines are small aliphatic amines whose metabolic reprogramming is involved in the regulation of various plant cellular reactions. Our previous study showed that polyamines increased lignan production in Linum album; however, little is known about the underlying mechanisms. This study aimed to provide more details on how putrescine (Put) regulates lignan biosynthesis in L. album cell culture. Our results showed that Put leads to podophyllotoxin (PTOX) and 6-methoxy podophyllotoxin (6MPTOX) accumulation by increasing the expression levels of phenylalanine ammonia-lyase (PAL) and pinoresinol-lariciresinol reductase (PLR) genes, encoding lignan biosynthesis regulatory enzymes. Put also increased hydrogen peroxide (H₂O₂) content, while its level decreased in the presence of aminoguanidine (AG) and imidazole, inhibitors of diamine oxidase (DAO) and NADPH oxidase (NOX), respectively. Elevated levels of nitric oxide (NO) and cytosolic free Ca²⁺ caused by Put treatment were reduced after using inhibitors of nitrate reductase (NR) and nitric oxide synthesis-like (NOS-like) enzymes, as well as Ca²⁺ influx. Besides, pre-treatment of cells with AG, imidazole, ethylene glycol-bis (β-aminoethyl ether)-N, N,N',N'-tetraacetic acid (EGTA) (Ca²⁺ chelator), Nɷ-nitro-L-arginine methyl ester (L-NAME), and Sodium tungstate (TUN) (NO generation inhibitors) diminished PAL and PLR transcript levels and PTOX and 6MPTOX accumulation, indicating the involvement of H₂O₂, NO, and Ca²⁺ in regulating lignan biosynthesis in L. album cells. Put also stimulated salicylic acid (SA) accumulation, being sensitive to all inhibitors used. Overall, this study suggests that Put-induced H₂O₂ generation in combination with NO and Ca²⁺ signals can regulate PAL and PLR genes expression and lignan production, likely in a SA-dependent manner.