Enhancement of Macarpine Production in Eschscholzia Californica Suspension Cultures under Salicylic Acid Elicitation and Precursor Supplementation

Abstract

Macarpine is a minor benzophenanthridine alkaloid with interesting biological activities, which is produced in only a few species of the Papaveraceae family, including Eschscholzia californica. Our present study was focused on the enhancement of macarpine production in E. californica suspension cultures using three elicitation models: salicylic acid (SA) (4; 6; 8 mg/L) elicitation, and simultaneous or sequential combinations of SA and L-tyrosine (1 mmol/L). Sanguinarine production was assessed along with macarpine formation in elicited suspension cultures. Alkaloid production was evaluated after 24, 48 and 72 h of elicitation. Among the tested elicitation models, the SA (4 mg/L), supported by L-tyrosine, stimulated sanguinarine and macarpine production the most efficiently. While sequential treatment led to a peak accumulation of sanguinarine at 24 h and macarpine at 48 h, simultaneous treatment resulted in maximum sanguinarine accumulation at 48 h and macarpine at 72 h. The effect of SA elicitation and precursor supplementation was evaluated also based on the gene expression of 4′-OMT, CYP719A2, and CYP719A3. The gene expression of investigated enzymes was increased at all used elicitation models and their changes correlated with sanguinarine but not macarpine accumulation.

California poppy (Eschscholzia californica), the Papaveraceae golden girl model organism for evodevo and specialized metabolism

“…HPLC or LC-MS analyses easily reveal alkaloid composition of California poppy (Liscombe et al, 2009;Hori et al, 2018), whereas the more traditional TLC method is also still in use (Balazǒváet al, 2020). Kukula-Koch (2017) reported an optimized method for the LC-ESI-Q-TOF-MS analysis for the extracts of Papaveraceae and Berberidaceae families (genera: Papaver, Argemone, Eschscholzia, Chelidonium, Glaucium, and Berberis), providing even more sensitive and precise method for BIA characterization.…”

“…Cell culture systems proved also useful to dissect BIA induction and its role in the molecular mechanism of phytopathogen defense. Whereas jasmonate treatment is commonly used to activate BIA biosynthesis (Färber et al, 2003;Ikezawa et al, 2009), Balazǒváet al (2020 examined salicylic acid (SA), and simultaneous or sequential treatment of SA and L-tyrosine in cell cultures to enhance production of macarpine, a BIA specific to few Papaveraceae species only. Angelova et al (2010) used root-derived cell cultures to characterize the elicitation mechanism, initiated by a short contact to low concentrations of a yeast glycoprotein elicitor, which led to the transient acidification of the cytoplasm.…”

Formal total synthesis of macarpine via a Au(I)-catalyzed 6-endo-dig cycloisomerization strategy

“…According to their oxidation states, benzo[c]phenanthridine alkaloids can be divided into two types: partially hydrogenated base and fully aromatized base, in which natural fully aromatic alkaloids can be further classified into three subclasses: O 4 -base, O 5 -base, and O 6 -base [3]. Among these alkaloids macarpine is the most oxidized tetracyclic alkaloid with many bioactivities, including anesthesia, anticancer, anti-inflammatory [4][5][6][7][8], insecticidal, fungicidal, etc [9]. In addition to the above-mentioned activities, macarpine was also used as a DNA probe for flow cytometry and fluorescence microscopy due to its fluorescent properties [10].…”

Towards A Genetic Model Organism: An Efficient Method For Stable Genetic Transformation of Eschscholzia Californica (Ranunculales)

“…E. california's major BIAs are (S)-reticuline, berberine, and sanguinarine, however, it lacks the enzymes necessary to synthesize noscapine and morphine (Li et al 2020;Balažová et al 2020). Several regulators of the BIA biosynthesis (Yamada et al 2017) or parts of the biosynthesis pathway of isoquinoline alkaloids area have been described, but insights in BIA and isoquinoline alkaloid biosynthesis and their regulation were mostly obtained by analyses utilizing suspension cultures (Yamada et al 2017;Balažová et al 2020) and lack information on regulation of alkaloid biosynthesis obtained from fully grown transgenic plants of E. californica. A detailed protocol for suspension cultures of E. californica was established previously (Park und Facchini 2000a), where culture conditions were optimized by investigating the effect of different phytohormones and component.…”

“…These provided the base for the reconstruction of the evolutionary history of enzymes involved in BIA biosynthesis (Li et al 2020). E. california's major BIAs are (S)-reticuline, berberine, and sanguinarine, however, it lacks the enzymes necessary to synthesize noscapine and morphine (Li et al 2020;Balažová et al 2020). Several regulators of the BIA biosynthesis (Yamada et al 2017) or parts of the biosynthesis pathway of isoquinoline alkaloids area have been described, but insights in BIA and isoquinoline alkaloid biosynthesis and their regulation were mostly obtained by analyses utilizing suspension cultures (Yamada et al 2017;Balažová et al 2020) and lack information on regulation of alkaloid biosynthesis obtained from fully grown transgenic plants of E. californica.…”