The Synergistic Effect of Co-Treatment of Methyl Jasmonate and Cyclodextrins on Pterocarpan Production in Sophora flavescens Cell Cultures

Abstract

Pterocarpans are derivatives of isoflavonoids, found in many species of the family Fabaceae. Sophora flavescens Aiton is a promising traditional Asian medicinal plant. Plant cell suspension cultures represent an excellent source for the production of valuable secondary metabolites. Herein, we found that methyl jasmonate (MJ) elicited the activation of pterocarpan biosynthetic genes in cell suspension cultures of S. flavescens and enhanced the accumulation of pterocarpans, producing mainly trifolirhizin, trifolirhizin malonate, and maackiain. MJ application stimulated the expression of structural genes (PAL, C4H, 4CL, CHS, CHR, CHI, IFS, I3'H, and IFR) of the pterocarpan biosynthetic pathway. In addition, the co-treatment of MJ and methyl-β-cyclodextrin (MeβCD) as a solubilizer exhibited a synergistic effect on the activation of the pterocarpan biosynthetic genes. The maximum level of total pterocarpan production (37.2 mg/g dry weight (DW)) was obtained on day 17 after the application of 50 μM MJ on cells. We also found that the combined treatment of cells for seven days with MJ and MeβCD synergistically induced the pterocarpan production (trifolirhizin, trifolirhizin malonate, and maackiain) in the cells (58 mg/g DW) and culture medium (222.7 mg/L). Noteworthy, the co-treatment only stimulated the elevated extracellular production of maackiain in the culture medium, indicating its extracellular secretion; however, its glycosides (trifolirhizin and trifolirhizin malonate) were not detected in any significant amounts in the culture medium. This work provides new strategies for the pterocarpan production in plant cell suspension cultures, and shows MeβCD to be an effective solubilizer for the extracellular production of maackiain in the cell cultures of S. flavescens.

Keywords: Sophora flavescens; cell cultures; elicitation; maackiain; pterocarpan; trifolirhizin.

Figure 1

Proposed pterocarpan biosynthetic pathway in S. flavescens. The step-wise activity of PAL, C4H, 4CL, CHS, CHR, CHI, IFS, I4’OMT, I3’H, IFR, IF7GT, and IF7MaT result in the conversion of phenylalanine to pterocarpans (maackiain, trifolirhizin, and trifolirhizin malonate). PAL: phenylalanine ammonia-lyase; C4H: cinnamate-4-hydroxylase; 4CL: 4-coumarate-CoA-ligase; CHS: chalcone synthase; CHR: chalcone reductase; CHI: chalcone isomerase; IFS: isoflavone synthase; I4′OMT: isoflavone 4′-O-methyltransferase; I3′H: isoflavone 3′-hydroxylase; IFR: isoflavone reductase; IF7GT: UDP-glucose:isoflavone 7-O-glucosyltransferase; IF7MaT: malonyl-CoA:isoflavone 7-O-glucoside-6-O-malonyltransferase. The pterocarpan biosynthetic pathway was proposed based on the KEGG database (http://www.genome.jp/kegg/kegg2.html).

Figure 2

Growth curve analyses of the S. flavescens cell cultures. (A) The growth of cells treated with or without methyl jasmonate (MJ) in 125 mL flask cultures containing 25 mL of MS1B2P liquid medium was measured at different time points. Data are the mean of three independent replicates ± SD. (B) The morphology of S. flavescens callus and cell suspension cultures. The cells were observed under an inverted light microscope (Carl Zeiss, Germany) equipped with a camera. Scale bar indicates 100 μm.

Figure 3

Effects of various elicitors on the production of pterocarpans (maackiain, trifolirhizin, and trifolirhizin malonate) in the S. flavescens cell cultures. Cells were pre-cultured for seven days and harvested three days after elicitation with the indicated elicitors. Samples were extracted with methanol and analyzed by HPLC. The elicitors used are as follows: UV, ultraviolet ray; MJ, methyl jasmonate; MV, methyl viologen; SA, salicylic acid; Chi, chitosan; ET, ethephon. Cells treated with 0.1% ethanol was used as a control. Chromatogram STD represents the authentic standards 1 (trifolirhizin), 2 (trifolirhizin malonate), and 3 (maackiain), with retention times of 7.88, 8.93, and 14.28 min, respectively.

Figure 4

Effects of MJ on the production of pterocarpans (maackiain, trifolirhizin, and trifolirhizin malonate) in S. flavescens cell cultures. (A) Effects of different concentrations of MJ on pterocarpan production in the S. flavescens cell cultures. Cells were pre-cultured for 7 days and harvested 3 days after elicitation with the indicated concentration of MJ. (B) Time-course of pterocarpan production in the S. flavescens cells elicited with MJ. The cells were pre-cultured for seven days and harvested at the indicated time points after elicitation with 50 μM MJ. Cells treated with 0.05% ethanol were used as a control. Samples were extracted with methanol and analyzed by HPLC. Data are the mean of three independent replicates ± SD. Statistical significance used Kruskal–Wallis test, * p < 0.05, *** p < 0.001.

Figure 5

Relative expression of the pterocarpan biosynthetic genes in the S. flavescens cells elicited by MJ, methyl-β-cyclodextrin (MeβCD), and MJ + MeβCD. Transcript levels of PAL, C4H, 4CL, CHS, CHR, CHI, IFS, I3’H, IFR, and IF7GT were analyzed by qRT-PCR, using S. flavescens cells elicited with 50 μM MJ, 50 mM MeβCD, or 50 μM MJ + 50 mM MeβCD for 24 h. The relative expression levels were normalized to that of actin (SfACT11; transcript ID: Sf128341_c1_g1_i1) as a quantitative control, and have been presented as the fold induction relative to the control. Data are the mean of three independent replicates ± SD.

Figure 6

Enhanced production of pterocarpans by MJ and MeβCD co-treatment in the S. flavescens cell cultures. (A) Synergistic effect of MJ and MeβCD on pterocarpan production in the cells. (B) Extracellular production of maackiain in the culture medium. Cells were pre-cultured for 7 days and harvested 7 days after co-treatment with 50 µM MJ and 50 mM MeβCD. Peaks 1 (7.86 min), 2 (8.94 min), and 3 (14.21 min) on the chromatograms represent the peaks identical to the retention times of the authentic standards, trifolirhizin, trifolirhizin malonate, and maackiain, respectively. The culture medium was extracted with ethyl acetate and analyzed by HPLC.