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The AP2/ERF transcription factor SmERF128 positively regulates diterpenoid biosynthesis in Salvia miltiorrhiza

  • Yu Zhang
  • Aijia Ji
  • Zhichao Xu
  • Hongmei Luo
  • Jingyuan SongEmail author
Article
  • 103 Downloads

Abstract

Key message

The novel AP2/ERF transcription factor SmERF128 positively regulates diterpenoid tanshinone biosynthesis by activating the expression of SmCPS1, SmKSL1, and SmCYP76AH1 in Salvia miltiorrhiza.

Abstract

Certain members of the APETALA2/ethylene-responsive factor (AP2/ERF) family regulate plant secondary metabolism. Although it is clearly documented that AP2/ERF transcription factors (TFs) are involved in sesquiterpenoid biosynthesis, the regulation of diterpenoid biosynthesis by AP2/ERF TFs remains elusive. Here, we report that the novel AP2/ERF TF SmERF128 positively regulates diterpenoid tanshinone biosynthesis in Salvia miltiorrhiza. Overexpression of SmERF128 increased the expression levels of copalyl diphosphate synthase 1 (SmCPS1), kaurene synthase-like 1 (SmKSL1) and cytochrome P450 monooxygenase 76AH1 (SmCYP76AH1), whereas their expression levels were decreased when SmERF128 was silenced. Accordingly, the content of tanshinone was reduced in SmERF128 RNA interference (RNAi) hairy roots and dramatically increased in SmERF128 overexpression hairy roots, as demonstrated through Ultra Performance Liquid Chromatography (UPLC) and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) analysis. Furthermore, SmERF128 activated the expression of SmCPS1, SmKSL1, and SmCYP76AH1 by binding to the GCC box, and to the CRTDREHVCBF2 (CBF2) and RAV1AAT (RAA) motifs within their promoters during in vivo and in vitro assays. Our findings not only reveal the molecular basis of how the AP2/ERF transcription factor SmERF128 regulates diterpenoid biosynthesis, but also provide useful information for improving tanshinone production through genetic engineering.

Keywords

Salvia miltiorrhiza Diterpenoid Tanshinones AP2/ERF transcription factor Herbgenomics 

Abbreviations

AP2/ERF

APETALA2/ethylene-responsive factor

CMK

4-(Cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase

CPS1

Copalyl diphosphate synthase 1

CYP76AH1

Cytochrome P450 monooxygenase 76AH1

DMAPP

Dimethylallyl diphosphate

DXR

1-Deoxy-D-xylulose-5-phosphate reductoisomerase

DXS2

1-Deoxy-D-xylulose-5-phosphate synthase 2

GFP

Green fluorescent protein

GGPP

Geranylgeranyl diphosphate

GGPPS

Geranylgeranyl diphosphate synthase

GUS

β-Glucuronidase

HDR1

4-Hydroxy-3-methylbut-2-enyl diphosphate reductase 1

HDS

4-Hydroxy-3-methylbut-2-enyl diphosphate synthase

IPP

Isopentenyl diphosphate

IPTG

Isopropyl β-D-thiogalactoside

KSL1

Kaurene synthase-like 1

MCT

2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase

MDS

2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase

NADPH

Nicotinamide adenine dinucleotide phosphate

OX

Overexpression

RNAi

RNA interference

TIA

Terpenoid indole alkaloid

TF

Transcription factor

Notes

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 81573398), CAMS Innovation Fund for Medical Sciences (CIFMS) (2017-I2M-1-009) and Foundation of Educational Department of Guangdong Province (E1-KFD015181K31).

Author contributions

JS designed the study. YZ and AJ performed experiments. YZ, AJ, ZX and HL analyzed the data. YZ, AJ, JS, and ZX wrote the manuscript. All authors approved the final manuscript.

Compliance with ethical standards

Conflict of interest

All the authors declare that they have no conflict of interests.

Supplementary material

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Supplementary material 1 (DOCX 3893 KB)
11103_2019_845_MOESM2_ESM.docx (34 kb)
Supplementary material 2 (DOCX 28 KB)

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Yu Zhang
    • 1
    • 2
  • Aijia Ji
    • 1
    • 3
  • Zhichao Xu
    • 1
  • Hongmei Luo
    • 1
    • 5
  • Jingyuan Song
    • 1
    • 4
    • 5
    Email author
  1. 1.Key Lab of Chinese Medicine Resources Conservation, State Administration of Traditional Chinese Medicine of the People’s Republic of China, Institute of Medicinal Plant Development, Chinese Academy of Medical SciencesPeking Union Medical CollegeBeijingChina
  2. 2.College of Chinese Materia MedicaShanxi University of Chinese MedicineJinzhongChina
  3. 3.School of Pharmaceutical SciencesGuangzhou University of Chinese MedicineGuangzhouChina
  4. 4.Yunnan Branch, Institute of Medicinal Plant Development, Chinese Academy of Medical SciencesPeking Union Medical CollegeJinghongChina
  5. 5.Engineering Research Center of Chinese Medicine ResourceMinistry of EducationBeijingChina

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