Marchantia polymorpha is a basal terrestrial land plant, which like most liverworts accumulates structurally diverse terpenes believed to serve in deterring disease and herbivory. Previous studies have suggested that the mevalonate and methylerythritol phosphate pathways, present in evolutionarily diverged plants, are also operative in liverworts. However, the genes and enzymes responsible for the chemical diversity of terpenes have yet to be described. In this study, we resorted to a HMMER search tool to identify 17 putative terpene synthase genes from M. polymorpha transcriptomes. Functional characterization identified four diterpene synthase genes phylogenetically related to those found in diverged plants and nine rather unusual monoterpene and sesquiterpene synthase-like genes. The presence of separate monofunctional diterpene synthases for ent-copalyl diphosphate and ent-kaurene biosynthesis is similar to orthologs found in vascular plants, pushing the date of the underlying gene duplication and neofunctionalization of the ancestral diterpene synthase gene family to >400 million years ago. By contrast, the mono-and sesquiterpene synthases represent a distinct class of enzymes, not related to previously described plant terpene synthases and only distantly so to microbial-type terpene synthases. The absence of a Mg 2+ binding, aspartate-rich, DDXXD motif places these enzymes in a noncanonical family of terpene synthases.

The effects of methyl jasmonate (MJ) dosage on terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus are correlated with the relative levels of specific MJ-responsive transcription factors. In this study, the expression of transcription factors (Orca, Zct, Gbf, Myc2, At-hook, and Wrky1), TIA pathway genes (G10h, Tdc, Str, and Sgd), and TIA metabolites (secologanin, strictosidine, and tabersonine) were investigated in C. roseus hairy root cultures elicited with a range of MJ dosages (0-1,000 µM) during mid-exponential growth. The highest production of TIA metabolites occurs at 250 μM MJ, increasing by 150-370% compared with untreated controls. At this MJ dosage, the expression of the transcriptional activators (Orca) is dramatically increased (29-40 fold) while the levels of the transcriptional repressors (Zct) remain low (2-7 fold). Simultaneously, the expression of genes coding for key enzymes involved in TIA biosynthesis increases by 8-15 fold. In contrast, high MJ dosages (1,000 µM) inhibit the production of TIA metabolites. This dosage is correlated with elevated expression levels of Zct (up to 40-fold) relative to Orca (13-19-fold) and minimal induction of the TIA biosynthetic genes (0-6 fold). The significant changes in the expression of Orca and Zct with MJ dosage do not correspond to changes in the expression of the early-response transcription factors (AT-hook, Myc2, and Wrky1) believed to regulate Orca and Zct. In summary, these observations suggest that the dependence of alkaloid production on MJ dosage in C. roseus may be partly mediated through the relative levels of Orca and Zct family transcription factors.

The production of pharmaceutically important terpenoid indole alkaloids (TIAs) from Catharanthus roseus is partly regulated at the transcriptional level. In this study, limitations in TIA biosynthesis from C. roseus hairy root cultures were assessed through gene expression profiling and precursor feeding. The transcript levels of key TIA pathway genes (G10h, Tdc, Str, and Sgd) and metabolite levels associated with the TIA pathway (tryptamine, loganin, secologanin, strictosidine, ajmalicine, serpentine, and tabersonine) were monitored using quantitative RT-PCR and HPLC, respectively. In cultures elicited with methyl jasmonate (250 microM MeJA on day 21), G10h, Tdc, Str, and Sgd expression increased by 9.1, 3.1, 6.7, and 8.3-fold, respectively, after 24 h. Up-regulation of gene expression was followed by a 160, 440, and 420% increase in strictosidine, ajmalicine, and tabersonine levels, respectively, after 5 days. Precursors loganin, tryptamine, or their combination were fed to noninduced and MeJA-induced cultures to complement the above studies. TIA production was not significantly enhanced in either noninduced or MeJA-induced cultures with precursor feeding. In noninduced cells, steps downstream of loganin and tryptamine were limiting (SLS, STR, or SGD) because either loganin or tryptamine accumulated in the cells with precursor feeding. These bottlenecks were partly overcome in MeJA-induced cultures as the expression of Str and Sgd genes and TIA production increased. However, secologanin accumulated in MeJA-induced cultures with precursor feeding, suggesting that STR was likely limiting under MeJA-induced conditions.

An Agro-mediated transformation method has been adapted in Catharanthus roseus seedlings for transient overexpression. Our results suggest that Agro-mediated methods may induce defense-related genes, which should be considered in its application. The Fast Agro-mediated Seedling Transformation (FAST) method, which involves the co-cultivation and transient transformation of young seedlings with Agrobacterium, was adapted and optimized in Catharanthus roseus. We investigated the optimal conditions for Gus expression by varying the Agrobacterium density (OD600 = 0.29 and 0.50), A. rhizogenes strain (15834 and R1000), and co-cultivation time in liquid (2, 12, or 24 h) followed by incubation time on solid media (1 or 2 days). Transformation efficiency was assessed quantitatively in terms of average GUS intensity per cotyledon surface area and percentage of cotyledons transformed. GUS staining was observed in 100% of cotyledons co-cultivated with A. rhizogenes (OD600 = 0.50) co-transformed with the Mas promoter-driven Gus and pSoup helper plasmids, in the presence of 0.01% v/v Silwet L-77 for 24 h in liquid followed by 2-days on solid media. In addition, we observed that co-cultivation with Agrobacterium strongly induced Zct1 and Orca3, two transcription factors known to regulate defense-related alkaloid biosynthesis in C. roseus. Homologous transcription factors regulate defense responses in many plant species. Therefore, possible induction of defense-related genes by Agro-mediated transformation should be a consideration in experimental design.