Lysine and cadaverine differentially regulate huperzine biosynthesis in axenic gametophyte cultures of Huperzia selago (L.) Bernh. ex Schrank et Mart.

Specialized metabolites represent a major component of plant adaptive and chemical diversity and constitute an important source of pharmacologically relevant compounds. Among them, Lycopodiaceae alkaloids form a distinctive group of lysine-derived metabolites characteristic of club mosses (Lycopodiaceae), a relict lineage of early-diverging lycophytes sister to the remaining extant vascular plants. Huperzia selago produces huperzine A (HupA) and huperzine B (HupB), potent acetylcholinesterase inhibitors, yet the regulation of their biosynthesis remains poorly understood, particularly in the gametophyte generation. Because lysine and its decarboxylation product cadaverine represent key entry metabolites into the Lycopodiaceae alkaloid network, precursor supplementation provides a tractable approach for probing regulatory nodes of this pathway under controlled conditions. Axenic gametophyte cultures of H. selago were maintained for 24 weeks on Whittier–Storchová medium supplemented with L-lysine (Lys), cadaverine (Cad), or combined Lys + Cad (5 + 5, 10 + 10, or 20 + 20 mg L⁻¹) under plant-growth-regulator-free conditions or in the presence of indole-3-butyric acid (IBA) + kinetin. Two-way ANOVA revealed significant effects of culture time, medium composition, and their interaction on both alkaloids. HupA showed its strongest response during the early to intermediate culture phase, reaching a maximum at week 12 in Lys + Cad-10 + 10 (5.70 ± 0.01 mg g⁻¹ DW), whereas the highest late-stage HupA levels were observed under cadaverine supplementation at week 24 (6.79 ± 0.10 mg g⁻¹ DW). In contrast, HupB exhibited delayed accumulation, with peak levels at week 24 in Lys + Cad-5 + 5 (0.97 ± 0.05 mg g⁻¹ DW). Plant growth regulators modified the feeding response in a time- and variant-dependent manner rather than acting as a uniform stimulator. LC–MS/IT–TOF profiling detected 27 signals corresponding to Lycopodiaceae alkaloids, with HupA and HupB confirmed using authentic standards. Precursor supplementation with lysine and cadaverine effectively modulates huperzine biosynthesis in H. selago gametophytes and reveals distinct temporal regulation patterns for HupA and HupB. The results support the view that the lysine-derived entry node represents an important regulatory node within the Lycopodiaceae alkaloid network. Axenic gametophyte cultures therefore provide a tractable experimental system for investigating specialized metabolism in early-diverging vascular plants and for refining strategies for the controlled production of pharmaceutically relevant alkaloids.