Investigation of Linum flavum (L.) Hairy Root Cultures for the Production of Anticancer Aryltetralin Lignans

Abstract

Linum flavum hairy root lines were established from hypocotyl pieces using Agrobacterium rhizogenes strains LBA 9402 and ATCC 15834. Both strains were effective for transformation but induction of hairy root phenotype was more stable with strain ATCC 15834. Whereas similar accumulation patterns were observed in podophyllotoxin-related compounds (6-methoxy-podophyllotoxin, podophyllotoxin and deoxypodophyllotoxin), significant quantitative variations were noted between root lines. The influence of culture medium and various treatments (hormone, elicitation and precursor feeding) were evaluated. The highest accumulation was obtained in Gamborg B5 medium. Treatment with methyl jasmonate, and feeding using ferulic acid increased the accumulation of aryltetralin lignans. These results point to the use of hairy root culture lines of Linum flavum as potential sources for these valuable metabolites as an alternative, or as a complement to Podophyllum collected from wild stands.

Keywords: 6-Methoxy-podophyllotoxin; Linum flavum; aryltetralin lignans; deoxypodophyllotoxin; hairy root; podophyllotoxin.

Figure 1

Structures of the main ATL (aryltetralin lignan) and the corresponding putative biosynthetic pathway leading from DPT to PPTG and MPTG in L. flavum. DOP6H: DPT-6-hydroxylase, DOP7H: DPT-7-hydroxylase, (M)PT7G: MPT- and/or PPT-7-glucosyltransferase, DPT: deoxypodophyllotoxin, MPT: methoxypodophyllotoxin, MPTG: methoxypodophyllotoxin-7-glucoside, PPT: podophyllotoxin, PPTG: podophyllotoxin-7-glucoside.

Figure 2

A. rhizogenes-mediated transformation and molecular characterization of the resulting L. flavum HR lines. (A) Germination of wild type Linum flavum; (B) Hairy roots of Linum flavum, line HRLF15.2; (C) Hairy roots of Linum flavum, line HRLF94-6 (C: callus, B: bud, S: shoot); (D) PCR amplified DNA fragments of ROL-B, ROL-C and VIR-D2 pro-oncogenes from the plasmidic DNA of A. rhizogenes strains or genomic DNA of L. flavum. From left to right: Ladder, DNA from the plasmids of A. rhizogenes LBA 9402 and ATCC 15834 strains respectively, used as positive control, genomic DNA of wild type roots L. flavum cultivated in vitro used as negative control, genomic DNA from two transgenic L. flavum hairy roots lines, HRLF94-2 and HRLF15-2 respectively. (E) sqRT-PCR analysis of ROL-B and ROL-C pro-oncogene expressions in L. flavum wild type roots and in two transgenic L. flavum hairy roots lines, HRLF94-2 and HRLF15-2 respectively. Total RNAs isolated from hairy roots were subjected to sqRT-PCR analysis using ACTIN-2 gene as internal control. Ten microliters of RT-PCR products were loaded on 1% (w/v) agarose gel.

Figure 3

Impact of basal medium composition on the growth characteristics and ATL accumulation of the HRLF15.2 line. (A) Growth kinetics of Linum flavum HR (in g of fresh weight) cultivated in flasks containing 100 mL of B5, WPM, MS and LS culture media; (B) Growth characteristics of the HRLF15.2 line in the B5, WPM, MS and LS culture media; (C) Intracellular accumulation of the main ATL and their glucosylated forms in HRLF15.2 line growing in the B5, WPM, MS and LS culture media; (D) Extracellular accumulation of the main ATL and their glucosylated forms in HRLF15.2 line growing in the B5, WPM, MS and LS culture media; (E) Relative intracellular accumulation kinetic of the main ATL and their glucosylated forms in HRLF15.2 line growing in the B5, WPM, MS and LS culture media. Detailed ATL accumulation kinetics are presented in Figure S1. Each point is the mean and standard deviation of the three independent experiments.

Figure 4

Influence of carbon source the ATL accumulation of the HRLF15.2 line. (A) Intracellular accumulation of the main ATL and their glucosylated forms in HRLF15.2 line growing in the B5 medium supplemented with 3% or 6% (w/v) sucrose (SUC), fructose (FRU) or glucose (GLC); (B) Extracellular accumulation of the main ATL and their glucosylated forms in HRLF15.2 line growing in the B5 medium supplemented with 3% or 6% (w/v) sucrose (SUC), fructose (FRU) or glucose (GLC). Growth kinetic are presented in Figure S2. Each point is the mean and standard deviation of the three independent experiments.

Figure 5

Impact of phytohormones and elicitors treatments on the growth and ATL accumulation profiles of HRLF15.2 line. (A) Growth curves of HRLF15.2 line treated with NAA (1 mg/mL), ABA (100 µM), MeJA (100 µM), SA (100 µM) or YE (3% w/v); (B) Relative impact of NAA (1 mg/mL), ABA (100 µM), MeJA (100 µM), SA (100 µM) or YE (3% w/v) treatments on the intracellular accumulation kinetic of the main ATL and their glucosylated forms in HRLF15.2 line. Detailed ATL accumulation kinetics are presented in Figure S3. Each point is the mean and standard deviation of the three independent experiments.

Figure 6

Impact of precursors feeding and permeation on the growth and ATL accumulation profiles of HRLF15.2 line. (A) Growth curves of HRLF15.2 line fed with l-Phe (1 mM) or ferulic acid (1 mM) or treated with Tween-20 (2% w/v); (B) Impact of l-Phe (1 mM) or ferulic acid (1 mM) or Tween-20 (2% w/v) addition on the intracellular and extracellular accumulation kinetic of DPT in HRLF15.2 line; (C) Impact of l-Phe (1 mM) or ferulic acid (1 mM) or Tween-20 (2% w/v) addition on the intracellular and extracellular accumulation kinetic of MPT in HRLF15.2 line; (D) Impact of l-Phe (1 mM) or ferulic acid (1 mM) or Tween-20 (2% w/v) addition on the intracellular and extracellular accumulation kinetic of MPTG in HRLF15.2 line; (E) Impact of l-Phe (1 mM) or ferulic acid (1 mM) or Tween-20 (2% w/v) addition on the intracellular and extracellular accumulation kinetic of PPT in HRLF15.2 line; (F) Impact of l-Phe (1 mM) or ferulic acid (1 mM) or Tween-20 (2% w/v) addition on the intracellular and extracellular accumulation kinetic of PPTG in HRLF15.2 line. Data on l-Phe or ferulic acid uptakes are presented Figure S4. Each point is the mean and standard deviation of the three independent experiments.