Comparison of elicitor-based effects on metabolic responses of Taxus × media hairy roots in perfluorodecalin-supported two-phase culture system

Abstract

Two lines of Taxus × media hairy roots harbouring or not the TXS transgene demonstrated diverse gene expression and taxane yield during cultivation in PFD-supported two liquid-phase culture system. Two lines of Taxus × media hairy roots were subjected to single or twice-repeated supplementation with methyl jasmonate, sodium nitroprusside, L-phenylalanine, and sucrose feeding. One line harboured transgene of taxadiene synthase (ATMA), while the second (KT) did not. Both hairy root lines were cultured in two-phase culture systems containing perfluorodecalin (PFD) in aerated or degassed form. The relationship between TXS (taxadiene synthase), BAPT (baccatin III: 3-amino, 3-phenylpropanoyltransferase), and DBTNBT (3'-N-debenzoyl-2-deoxytaxol-N-benzoyltransferase) genes and taxane production was analysed. The ATMA and KT lines differed in their potential for taxane accumulation, secretion, and taxane profile. In ATMA biomass, both paclitaxel and baccatin III were detected, while in KT roots only paclitaxel. The most suitable conditions for taxane production for ATMA roots were found in single-elicited supported with PFD-degassed cultures (2 473.29 ± 263.85 µg/g DW), whereas in KT roots in single-elicited cultures with PFD-aerated (470.08 ± 25.15 µg/g DW). The extracellular levels of paclitaxel never exceeded 10% for ATMA roots, while for KT increased up to 76%. The gene expression profile was determined in single-elicited cultures supported with PFD-degassed, where in ATMA roots, the highest taxane yield was obtained, while in KT the lowest one. The gene expression pattern in both investigated root lines differed substantially what resulted in taxane yield characterized particular lines. The highest co-expression of TXS, BAPT and DBTNBT genes noted for ATMA roots harvested 48 h after elicitation corresponded with their higher ability for taxane production in comparison with the effects observed for KT roots.

Keywords: Elicitation; Gene expression; Hairy roots; Perfluorodecalin; Taxus.

Fig. 1

Biomass growth (g/flask) in ATMA (harbouring TXS transgene) and KT (without TXS transgene) hairy root lines of Taxus × media cultivated in various culture variants: control—untreated cultures; single–elicited (SE) cultures without PFD; twice-elicited (TE) cultures without PFD; unelicited cultures with PFD-aerated; single-elicited cultures with PFD-aerated; twice-elicited cultures with PFD-aerated; unelicited cultures with PFD-degassed; single-elicited cultures with PFD-degassed; twice-elicited cultures with PFD-degassed. Data represents mean of three replicates ± SD; the same letters indicate means statistically different (p < 0.05)

Fig. 2

Relative expression levels of TXS, BAPT, and DBTNBT genes in Taxus × media two hairy root lines: ATMA (harbouring TXS transgene) and KT (without TXS transgene). Both hairy root lines were cultivated in two-phase PFD-degassed supported cultures elicited with MJ, SNP and supplemented with l-phenylalanine and additional sucrose. The relative gene expression levels in elicitor treated cultures was compared with their expression in untreated cultures on the 28th day (day of elicitors application) – time point “0”. The transcript level of each gene was normalized against reference genes: 18S rDNA and β-tubulin using ΔΔCt method with PCR efficiency correction. The reported values are an average of four biological replicates and three technical replicates ± SD