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Plant Molecular Biology

, Volume 95, Issue 1–2, pp 51–62 | Cite as

A MDR transporter contributes to the different extracellular production of sesquiterpene pyridine alkaloids between adventitious root and hairy root liquid cultures of Tripterygium wilfordii Hook.f.

Article

Abstract

Key message

TwMDR1 transports sesquiterpene pyridine alkaloids, wilforine and wilforgine, into the hairy roots of T. wilfordii Hook.f. resulting in low secretion ratio of alkaloids.

Abstract

Hairy roots (HRs) exhibit high growth rate and biochemical and genetic stability. However, varying secondary metabolites in HR liquid cultures mainly remain in root tissues, and this condition may affect cell growth and cause inconvenience in downstream extraction. Studies pay less attention to adventitious root (AR) liquid cultures though release ratio of some metabolites in AR liquid cultures is significantly higher than that of HR. In Tripterygium wilfordii Hook.f., release ratio of wilforine in AR liquid cultures reached 92.75 and 13.32% in HR on day 15 of culture. To explore potential roles of transporters in this phenomenon, we cloned and functionally identified a multidrug resistance (MDR) transporter, TwMDR1, which shows high expression levels in HRs and is correlated to transmembrane transportation of alkaloids. Nicotiana tabacum cells with overexpressed TwMDR1 efficiently transported wilforine and wilforgine in an inward direction. To further prove the feasibility of genetically engineered TwMDR1 and improve alkaloid production, we performed a transient RNAi experiment on TwMDR1 in T. wilfordii Hook.f. suspension cells. Results indicated that release ratios of wilforine and wilforgine increased by 1.94- and 1.64-folds compared with that of the control group, respectively. This study provides bases for future studies that aim at increasing secretion ratios of alkaloids in root liquid cultures in vitro.

Keywords

ABC transporter Hairy root Adventitious root Sesquiterpene pyridine alkaloids 

Notes

Acknowledgements

This study was supported by Educational Commission of Anhui Province of China (KJ2016A668), the National Natural Science Foundation of Anhui Province (1708085QC52), the National Natural Science Foundation of China (Grant No. 31272110) and the Key Project of the Outstanding Young Talent Support Program of the University of Anhui Province (gxyqZD2016264).

Author Contributions

XZ and CZ: conceived and designed the experiments. GM and JH: performed the experiments. JZ: analyzed the data. XZ and CZ: wrote the paper.

Compliance with ethical standards

Conflict of interest

The authors declare no competing financial interest.

Supplementary material

11103_2017_634_MOESM1_ESM.xlsx (32 kb)
Online Resource 1—Protein sequence alignment of TwMDR1 with CjMDR1 (BAB62040.1) and CjABCB2 (BAM11098.1). Two TMDs and two NBDs, arranged in TMD1-NBD1-TMD2-NBD2 direction, are indicated in black frame. Within NBDs, three characteristic motifs common to all ABC transporters, Walker A, Walker B, and ABC-signature motif close to the Walker B domain, are shown in bold. Identical residues in sequences are shadowed. Gaps are represented by dashes and introduced for optimal alignment (XLSX 31 KB)
11103_2017_634_MOESM2_ESM.tif (710 kb)
Online Resource 2—Accumulation of wilforine and reduction percentages in different kinds of media. On day 15 of HR liquid cultures, medium was separated from HR. After adding 100 µM MeJA to the above cultures for 9 h, medium (+MeJA) was filtrated and immediately supplemented with 40 µg/100 mL wilforine. Both media were maintained at 25 °C with 120 rpm rotation and monitored for wilforine contents at 0, 3, 6, 9, 12, and 24 h. Medium with HR (+MeJA) was directly collected from HR liquid cultures treated by 100 µM MeJA at corresponding time points (TIF 710 KB)

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Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  1. 1.Department of BioengineeringHuainan Normal UniversityHuainanChina
  2. 2.Research & Development Center of Biorational PesticidesNorthwest A&F UniversityYanglingChina

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