Interactions between second messengers, SA and MAPK6 signaling pathways lead to chitosan-induced lignan production in Linum album cell culture

Abstract

Lignans along with their special ecological role in plants have received much attention in recent decades due to their anti-cancer properties. However, the mechanisms regulating their biosynthesis are still largely unknown. Here, we investigated the potential role of certain signaling pathways and their relationships in regulating the biosynthesis of 6-methoxypodophyllotoxin (6MPTOX) through pharmacological approaches in chitosan-exposed Linum album cells. The HPLC results revealed that chitosan (100 mg L−1) induces 6MPTOX biosynthesis. Chitosan also elevated the content of second messengers cytosolic free Ca2+, hydrogen peroxide (H2O2) and nitric oxide (NO) by activating plasma-membrane Ca2+ channels, NADPH oxidase and nitrate reductase (NR)/nitric oxide synthase (NOS)-like enzymes, respectively. Co-application of chitosan with EGTA, W-7, imidazole, L-NAME and tungstate inhibitors diminished phenylalanine ammonia-lyase (PAL), pinoresinol-lariciresinol reductase (PLR) and 6MPTOX induction, indicating that Ca2+/CaM, H2O2 and NO are involved in regulating this biosynthetic pathway. Our data suggest that there is a reciprocal relationship between chitosan-induced cytosolic free Ca2+ and H2O2 rising , while NO production is unilaterally regulated by Ca2+ and H2O2. Besides, we found that chitosan also increases salicylic acid (SA) content and correspondingly, exogenous SA stimulates 6MPTOX production by affecting PAL and PLR activity. The level of SA was mutually associated with cytosolic free Ca2+, H2O2 and NO. Real-time PCR results suggest that mitogen-activated protein kinase 6 (MAPK6) acts downstream of second messengers and SA signaling pathways, therefore, it can be a point of integration for signals modulating lignan biosynthesis. Overall, our findings provide new insights into the lignan biosynthesis regulation, wherein Ca2+/CaM, H2O2 and NO signals are mediated, amplified and converged by SA and MAPK6, and the information is passed on to potential downstream targets.