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

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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.

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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

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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).

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Author notes

  1. Yu Zhang and Aijia Ji have contributed equally to this work.

Authors and Affiliations

  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 Sciences, Peking Union Medical College, Beijing, 100193, China

    Yu Zhang, Aijia Ji, Zhichao Xu, Hongmei Luo & Jingyuan Song

  2. College of Chinese Materia Medica, Shanxi University of Chinese Medicine, Jinzhong, 030619, China

    Yu Zhang

  3. School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, 510006, China

    Aijia Ji

  4. Yunnan Branch, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Jinghong, 666100, China

    Jingyuan Song

  5. Engineering Research Center of Chinese Medicine Resource, Ministry of Education, Beijing, 100193, China

    Hongmei Luo & Jingyuan Song

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  1. Yu Zhang
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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.

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Correspondence to Jingyuan Song.

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Zhang, Y., Ji, A., Xu, Z. et al. The AP2/ERF transcription factor SmERF128 positively regulates diterpenoid biosynthesis in Salvia miltiorrhiza. Plant Mol Biol 100, 83–93 (2019). https://doi.org/10.1007/s11103-019-00845-7

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