Putrescine Promotes Betulin Accumulation in Suspension Cell Cultures of Betula platyphylla by Regulating NO and NH4+ Production

Putrescine (Put) can enhance secondary metabolite production, but its intrinsic regulatory mechanism remains unclear. In this study, Put treatment promoted betulin production and gene expression of lupeol synthase (LUS), one of betulin synthetic enzymes. The maximum betulin content and gene expression level of LUS was 4.25 mg·g−1 DW and 8.25 at 12 h after 1 mmol·L−1 Put treatment, approximately two- and four-times that in the control, respectively. Put treatment increased the content of nitric oxide (NO) and its biosynthetic enzyme activity of nitrate reductase (NR) and NO synthase (NOS). Pretreatment of the birch suspension cells with NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline- 1-oxyl-3-oxide (cPTIO), NR inhibitor sodium azide (NaN3), and NOS inhibitor NG-nitro-L-Arg methyl ester (L-NAME) decreased Put-triggered NO generation and blocked Put-induced betulin production. Put treatment improved the content of NH4+ and its assimilation enzyme activity of glutamate synthase and glutamate dehydrogenase. NH4+ supplementation also promoted NO and betulin production. Thus, the above data indicated that Put-induced NO was essential for betulin production. NO derived from NR, NOS, and NH4+ mediated betulin production in birch suspension cell cultures under Put treatment.