Strictosidine-related enzymes involved in the alkaloid biosynthesis of Uncaria tomentosa root cultures grown under oxidative stress

The activity and gene expression of strictosidine-related enzymes in Uncaria tomentosa root cultures exposed to oxidative stress were studied. Elicitation with 0.2 mM hydrogen peroxide (H₂O₂) or a combination of 0.8 mM buthionine sulfoximine and 0.2 mM jasmonic acid (BSO-JA) increased peroxidase activities by twofold at Day 8 and glutathione reductase by 1.4-fold at Day 5 in H₂O₂ elicited cultures respect to the control. Production of monoterpenoid oxindole alkaloids (MOA), 3α-dihydrocadambine, and dolichantoside was stimulated after H₂O₂ elicitation, reaching levels of 886.4±23.6, 847.7±25.4, and 87.5±7.2 μg/g DW, at Day 8 which were 1.7-, 2.1-, and 2.3-fold higher relative to control. BSO-JA elicited cultures produced about twice alkaloids than H₂O₂-treated cultures, following a biphasic pattern with maxima at 0.5 and 8 days. Alkaloid production was preceded by increase in strictosidine synthase (STR) and strictosidine glucosidase (SGD) activities. After elicitation with H₂O₂ or BSO-JA, the STR activity (pKat/mg protein) increased by 1.9-fold (93.8±17.8 at 24 h) or 2.5-fold (102.4±2.2 at 6 h) and the SGD activity (pKat/mg protein) by 2.8-fold (245.2±14.4 at 6 h) or 4.2-fold (421.2±1.8 at 18 h) relative to control. STR and SGD transcripts were upregulated after elicitation. H₂O₂-treated roots showed higher levels of STR at 48-192 h and SGD at 24-48 h, while BSO-JA treatments showed STR increased at 12 h and SGD at 24 h. Also, LC/ESI-MS confirmed the biosynthesis of dolichantoside from N-ω-methyltryptamine and secologanin by U. tomentosa protein extracts.