Induction of Monoterpenoid Oxindole Alkaloids Production and Related Biosynthetic Gene Expression in Response to Signaling Molecules in Hamelia patens Plant Cultures.

Hamelia patens (Rubiaceae), known as firebush, is a source of bioactive monoterpenoid oxindole alkaloids (MOAs) derived from monoterpenoid indole alkaloids (MIAs). With the aim of understanding the regulation of the biosynthesis of these specialized metabolites, micropropagated plants were elicited with jasmonic acid (JA) and salicylic acid (SA). The MOA production and MIA biosynthetic-related gene expression were evaluated over time. The production of MOAs was increased compared to the control up to 2-fold (41.3 mg g DW-1) at 72 h in JA-elicited plants and 2.5-fold (42.4 mg g DW-1) at 120 h in plants elicited with SA. The increment concurs with the increase in the expression levels of the genes HpaLAMT, HpaTDC, HpaSTR, HpaNPF2.9, HpaTHAS1, and HpaTHAS2. Interestingly, it was found that HpaSGD was downregulated in both treatments after 24 h but in the SA treatment at 120 h only was upregulated to 8-fold compared to the control. In this work, we present the results of MOA production in H. patens and discuss how JA and SA might be regulating the central biosynthetic steps that involve HpaSGD and HpaTHAS genes.

Mixed elicitation with salicylic acid and hydrogen peroxide modulates the phenolic and iridoid pathways in Castilleja tenuiflora plants.

MAIN CONCLUSION: SA and H2O2, in single and mixed elicitation stimulate specialized metabolism and activate oxidative stress in C. tenuiflora plants. Single elicitation with salicylic acid (SA at 75 µM) and, hydrogen peroxide (at 150 µM), and mixed elicitation (75 µM SA + 150 µM H2O2) were evaluated on specialized metabolism in Castilleja tenuiflora Benth. plants. Total phenolic content (TPC), phenylalanine ammonia-lyase (PAL) activity, antioxidant enzymes and specialized metabolite profiles, as well as the expression levels of eight genes involved in phenolic (Cte-TyrDC, Cte-GOT2, Cte-ADD, Cte-AO3, Cte-PAL1, Cte-CHS1) and terpene pathways (Cte-DXS1 and Cte-G10H) and their correlation with major metabolite (verbascoside and aucubin) concentrations were investigated. TPC content (three-fold) and PAL activity (11.5-fold) increased with mixed elicitation, as well as catalase and peroxidase activity (11.3-fold and 10.8-fold, respectively), compared to single elicitation. Phenylethanoid accumulation was greatest under mixed elicitation, followed by SA and H2O2. Lignan accumulation was differential, depending on the plant part and the elicitor. Flavonoids only appeared after mixed elicitation. The high concentration of verbascoside under mixed elicitation was related to a high gene expression. Single elicitation induced iridoid accumulation in specific parts (H2O2 in aerial parts and SA in roots), whereas under mixed elicitation, it accumulated in both parts. A high concentration of aucubin in the aerial part was related to a high expression level of genes of the terpene pathway Cte-DXS1 and Cte-G10H, and in the root with Cte-G10H, while Cte-DXS1 was downregulated in this tissue in all treatments. Mixed elicitation with SA and H2O2 represents an interesting tool to increase the production of specialized metabolites in plants.

Influence of spermine and nitrogen deficiency on growth and secondary metabolites accumulation in Castilleja tenuiflora Benth. cultured in a RITA® temporary immersion system.

The effect of exogenous spermine (SPM) on Castilleja tenuiflora shoots developing under nitrogen deficiency (ND) stress was evaluated. Shoots cultivated in a temporary immersion system were subjected to four experimental treatments: (1) control; (2) exogenous SPM; (3) ND; and (4) ND+SPM. Shoots were longer in the ND+SPM treatment (6.3 ± 0.5 cm) than in the ND treatment (4.2 ± 0.5 cm). The total chlorophyll content was similar in the control and SPM treatments (0.41 µg mg-1 FM) and the highest values of total phenolic content were detected at 21 days in the ND+SPM treatment (84.1 ± 0.05 GAE g-1 DM). In the ND+SPM treatment, the phenylalanine ammonia lyase activity increased earlier than in ND treatment, and reached its maximum at day 21 (3.9 ± 0.2 µmol E-CIN h-1 mg-1 protein). Compared with the control, the ND and ND+SPM treatments resulted in increased secondary metabolites contents in both root and aerial parts. The strongest effect was in the roots, where the SPM and ND+SPM treatments both resulted in increased quercetin content (4.3-fold that in the control). Our results showed that SPM partially counteract the damage caused by ND and results in increased contents of valuable bioactive compounds.