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Effects of demethylating reagent 5-aza-2′-deoxycytidine on the growth and cephalotaxine production in Cephalotaxus mannii suspension cells

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Abstract

To study the effect of DNA methylation on the growth and synthesis of cephalotaxine in Cephalotaxus mannii suspension cells, demethylating reagent 5-aza-2′-deoxycytidine (5-Aza-CdR) with different concentrations (15, 25 and 35 μmol/L) was added to the suspension cultures of C. mannii. The results obtained revealed that the effect of 5-Aza-CdR on cell growth depended on its concentration. When cells were treated with an optimum concentration of 5-Aza-CdR 15 μmol/L, the biomass of cells was 15.76 g/L (dry weight, DW), 13.75% higher than control group (13.85 g/L). Additionally, 5-Aza-CdR could improve the enzyme activity associated with the metabolic pathway of cell growth and product biosynthesis. When treated with 5-Aza-CdR, the activities of glucose-6-phosphate dehydrogenase (G6PDH), pyruvate kinase (PK) and 3-deoxy-d-arabino-heptulosonate-7-phosphate (DS) synthase was increased by 21%, 172% and 26%, respectively. At the same time, the content of cephalotaxine reached 7.735 mg/L, was 30% higher than the control group (5.97 mg/L). In addition, it was found that 5-Aza-CdR reduced the expression of two genes related to methyltransferase in cells by 87.87% (caffeic acid ortho-methyltransferase gene) and 86.19% (one hypothetical protein gene), and the level of DNA methylation treated by 5-Aza-CdR was obviously lower than the control. Interestingly, the demethylation by 5-Aza-CdR was only found in the cell growth phase (days 0–15), but not found in the late culture period (days 15–30), indicating that the action sites of 5-Aza-CdR in cells were mainly focused on the primary metabolic process.

Key message

5-Aza-CdR effected the prophase of cell growth and promoted the synthesis of cephalotaxine. The decrease of DNA methylation level by 5-Aza-CdR was only found in the cell growth phase (0–15 days). But there had no significant effect at the later stage (15–30 days) of culture.

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Abbreviations

5-Aza-CdR:

5-Aza-2′-deoxycytidine

G6PDH:

Glucose-6-phosphate dehydrogenase

PK:

Pyruvate kinase

DS:

3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase

SAM:

Methyl methionine

MBD:

Methyl binding protein

NAA:

Naphthalene acetic acid

KT:

Kinetin

PVP:

Polyvinyl pyrrolidone

FW:

Fresh weight

DW:

Dry weight

HPLC:

High performance liquid chromatography

SDS:

Sodium dodecyl sulfate

EDTA:

Ethylene diamine tetraacetic acid

LDH:

Lactic dehydrogenase (rabbit muscle)

E4P:

Erythrose-4-phosphate

PEP:

Phosphoenolpyruvate

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Acknowledgements

This work was funded by the National Natural Science Foundation of China (No. 21766006).

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Authors and Affiliations

  1. College of Food Science and Engineering, Hainan University, Haikou, 570228, People’s Republic of China

    Li-Rong Wei, Wang-Yan Qin & Yong-Cheng Li

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  1. Li-Rong Wei
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  2. Wang-Yan Qin
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Contributions

LRW and YCL conceived and designed the study. LRW conducted experiments, analyzed the results and wrote the manuscripts. YCL and WYQ revised the manuscript in part. All authors read and approved the manuscript.

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Correspondence to Yong-Cheng Li.

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Communicated by Konstantin V. Kiselev.

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Wei, LR., Qin, WY. & Li, YC. Effects of demethylating reagent 5-aza-2′-deoxycytidine on the growth and cephalotaxine production in Cephalotaxus mannii suspension cells. Plant Cell Tiss Organ Cult 139, 359–368 (2019). https://doi.org/10.1007/s11240-019-01689-1

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