Elicitor-enhanced steroidal sapogenin accumulation in hairy root cultures of Trigonella foenum-graecum.

In current work, we studied hairy root induction in Trigonella foenum graecum, which is an important medicinal plant, and examined the impact of different elicitors on some phytochemical characteristics and metabolites production in hairy root cultures. Accordingly, some factors such as five strain types of Agrobacterium rhizogenes (1724, 15834, A4, A13 and MSU) and three different explants, namely leaf, cotyledon and hypocotyl were studied. The results showed that different A. rhizogenes strains exhibited different infection efficiency. MSU and 15834 had highest efficiency of hairy root induction than other strains. Also, hairy root induction frequency in leaf explants was higher than in other explants. Salicylic acid (SA), nitric oxide (NO), CaCl2 and penconazole (PEN) were used in elicitation process. Hairy roots were treated with SA (0.1 and 0.5 mM), NO (10 and 50 µM), CaCl2 (5 and 10 mM) and PEN (5 and 10 mg/L). Applied elicitors enhanced antioxidant enzymes activities and reduced oxidative stress markers; this observation might be ascribed to regulation of the oxidative status of the elicited cells. Significant increase of antioxidant metabolites (total phenol, flavonoid and anthocyanin) in PEN-treated hairy roots was associated to phenylalanine ammonia lyase activity, indicating an up-regulation of phenylpropanoid/flavonoid metabolism. PEN and CaCl2 treatment enhanced steroidal sapogenin in hairy root cultures. These results suggested that use of elicitors can enhance the production of secondary metabolites in transformed hairy roots. Among the elicitors applied, CaCl2 and PEN were the most effective in increasing secondary metabolite production in transformed hairy roots of T. foenum graecum.

Disruption of a licorice cellulose synthase-derived glycosyltransferase gene demonstrates its in planta role in soyasaponin biosynthesis.

KEY MESSAGE: CRISPR-Cas9-mediated disruption of a licorice cellulose synthase-derived glycosyltransferase gene, GuCSyGT, demonstrated the in planta role of GuCSyGT as the enzyme catalyzing 3-O-glucuronosylation of triterpenoid aglycones in soyasaponin biosynthesis. Triterpenoid glycosides (saponins) are a large, structurally diverse group of specialized metabolites in plants, including the sweet saponin glycyrrhizin produced by licorice (Glycyrrhiza uralensis) and soyasaponins that occur widely in legumes, with various bioactivities. The triterpenoid saponin biosynthetic pathway involves the glycosylation of triterpenoid sapogenins (the non-sugar part of triterpenoid saponins) by glycosyltransferases (GTs), leading to diverse saponin structures. Previously, we identified a cellulose synthase-derived GT (CSyGT), as a newly discovered class of triterpenoid GT from G. uralensis. GuCSyGT expressed in yeast, which could transfer the sugar glucuronic acid to the C3 position of glycyrrhetinic acid and soyasapogenol B, which are the sapogenins of glycyrrhizin and soyasaponin I, respectively. This suggested that GuCSyGT is involved in the biosynthesis of glycyrrhizin and soyasaponin I. However, the in planta role of GuCSyGT in saponin biosynthesis remains unclear. In this study, we generated GuCSyGT-disrupted licorice hairy roots using CRISPR-Cas9-mediated genome editing and analyzed the saponin content. This revealed that soyasaponin I was completely absent in GuCSyGT-disrupted lines, demonstrating the in planta role of GuCSyGT in saponin biosynthesis.