Lignin and flavonoid content increases in Hypericum perforatum cell wall after Agrobacterium tumefaciens co-cultivation

Hypericum perforatum (HP), a high value medicinal plant used to produce several phytopharmaceuticals, remains recalcitrant to Agrobacterium tumefaciens (AT) mediated transformation. HP recognizes AT as a potential pathogen and activates its defense response [1]. Although elicitation of HP cells with AT is known to increase its xanthone content, flavonoids remained unaffected in the soluble fraction of HP [2]. However, specific analysis of HP cell wall biomass/fractions after AT elicitation revealed an increase in lignin and cell wall bound flavonoids content, possibly as mechanism of defense against AT. Briefly, cell wall biomass was isolated from control and AT elicited HP cells via consecutive extraction with hexane, acetone, methanol and distilled water. Determination of lignin content by acetyl bromide method [3] showed a significant increase in HP cells treated with AT, implying that upon elicitation by AT, HP reinforced its cell wall as a defense response. In order to analyze their phenolic profile, the cell wall biomass was subjected to extraction by 90% methanol after alkaline hydrolysis, HCl titration and ethyl acetate extraction. HPLC analysis of the cell wall bound phenolics fraction revealed an accumulation of flavonoids (e.g. epicatechin, quercetin derivatives) in the cell walls after elicitation. Upregulation of chalcone synthase (CHS), leucoanthocyanidin dioxygenase (LDOX) and Flavone 3-hydroxilase (F3 H) were found to be responsible for this accumulation. Further, DPPH reduction and TBARS assays clearly correlated the increased production of flavonoids with improved antioxidant protection of the cell wall. Collectively, our results allow us to conclude that in addition to the increase in xanthone production [2], co-cultivation of HP cells with AT also upregulates flavonoids biosynthesis as a defense mechanism. While the xanthones enrich the soluble phenolic fraction; flavonoids are incorporated into the cell wall.