Interaction between exogenous brassinolide, IAA and BAP in secondary metabolism of cultured Onosma paniculatum cells

Brassinolide (BL) together with IAA (indoleacetic acid) and BAP (6-benzylaminopurine) have been reported to enhance shikonin formation in cultured Onosma paniculatum cells . In this paper, we show that BL interacted significantly with both IAA and BAP to influence cell growth. In a BL/IAA interaction experiment, the optimal BL concentration for cell growth increased with IAA concentration. Thus, with IAA concentrations of 0.05, 0.1, 1.0, and 10 mg/L in the growth medium, the optimal BL concentrations for cell growth were 10, 103, 105, and 107pg/L, respectively. In a BL/BAP interaction experiment, cell growth decreased with increasing concentration of BL at any given concentration of BAP. The optimal concentrations of BL and IAA for cell growth were 10 pg/L and 0.05 mg/L, respectively, among all BL/IAA combinations, and concentrations of 10 pg/L and 0.5 mg/L for BL and BAP were optimal among all BL/BAP combinations. Shikonin formation was affected significantly by both BL/IAA and BL/BAP combinations. Shikonin content was enhanced by increasing BL concentrations with IAA concentrations in the range of 0.05–10 mg/L and with BAP concentrations in the range of 0.5–5 mg/L in BL/IAA and BL/BAP experiments, respectively. The optimal combination of BL and IAA for enhanced shikonin formation was 107pg/L and 0.05 mg/L, and BL and BAP concentrations of 105pg/L and 0.5 mg/L optimal for shikonin formation. These results indicate that BL-stimulated cell growth occurs at lower concentration (10 pg/L) and enhanced shikonin formation at higher concentration (105–107pg/L), in combination with IAA or BAP at appropriate concentrations. Furthermore, BL increased phenylalanine ammonia-lyase (PAL) and p-hydroxybenzoic acid -geranyltransferase (PHB-geranyltransferase) activities, but decreased the activity of PHB–O–glucosyltransferase. These results suggest that enhanced shikonin formation induced by BL involves regulation of these key enzyme activities.