Overexpression of Ath-MIR160b increased the biomass while reduced the content of tanshinones in Salvia miltiorrhiza hairy roots by targeting ARFs genes

Abstract

MicroRNAs (miRNAs) have attracted increasing attention in the field of medicinal plant research. Our previous analyses indicated that the mature miR160a level was related to the accumulation of tanshinones in Salvia miltiorrhiza (S. miltiorrhiza). Therefore, the aim of this research was to reveal how the miR160a regulates the tanshinones biosynthesis. The overexpression of miR160a in hairy roots of S. miltiorrhiza down-regulated the levels of the target genes ARF10, 16, and 17, and inhibited tanshinones biosynthesis but increased hairy root biomass. In overexpression line 1, the content of four major tanshinones dropped sharply to 9.06% (dihydrotanshinone I), 12.56% (cryptotanshinone), 12.02% (tanshinone I), and 8.52% (tanshinone IIA) compared with that of the wildtype (WT). The fresh weight (FW) of hairy roots increased to 1.72-fold of the WT, and in overexpression lines 4, tanshinones were not detected. However, the FW of hairy roots in overexpression line 4 increased to 1.90-fold of the WT. We also observed the down regulated genes in the tanshinones biosynthesis pathway. In particular, the transcription level of KSL2 dropped to 3% as compared to the WT, which was consistent with the decrease in tanshinones content. We further analyzed hormone changes and found an increase in indole acetic acid accumulation, while the accumulation of salicylic acid (SA) and jasmonic acid (JA) decreased. These findings indicate that miR160a may negatively regulate the biosynthesis of tanshinones, while promote the growth of hairy roots by targeting ARF10, 16, and 17. Our findings demonstrate that miRNAs could be used as an alternative strategy to dissect the complex network of secondary metabolism in medicinal plants.

Key message

MiR160a overexpression promoted the IAA level and the growth of hairy roots of Salvia miltiorrhiza, but reduced the GH3-like level and production of tanshinones by targeting ARF10, ARF16, and ARF17.

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Abbreviations

AACT:

Acetyl-CoAC-acetyltransferase

HMGR:

3-Hydroxy-3-methylglutaryl-CoA reductase

GGPPS:

Geranylgeranyl diphosphate synthase

DXS:

1-Deoxy-d-xylulose 5-phosphate synthase

DXR:

1-Deoxy-d-xylulose 5-phosphate reductoisomerase

CPS:

Copalyl diphosphate synthase

KSL:

Ent-kaurene synthase-like

CYP76AH1:

Cytochrome P450-dependent monooxygenase

KSL:

Kaurene synthase-like

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Acknowledgements

This work was supported by a research program sponsored by the National Natural Science Foundation of China (Nos. 81778385, 31800255 and 31871694), the Natural Science Foundation of Zhejiang Province (Nos. LY18C150010 and LQ16C020002), and Science Foundation of Zhejiang Sci-Tech University (ZSTU) (No. 19042403-Y). The funding agencies were not involved in the design of the study, collection, analysis, and interpretation of data or in writing the manuscript.

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HZ conducted the laboratory work and drafted the manuscript. HC and ZH contributed advice and revised the manuscript. WJ assisted with the bioinformatics analysis and interpretation of results. ZL conceived and designed the experiment. Ling Xu revised the manuscript. All the authors have read and approved the final manuscript.

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Correspondence to Weibo Jin or Zongsuo Liang.

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Zhang, H., Chen, H., Hou, Z. et al. Overexpression of Ath-MIR160b increased the biomass while reduced the content of tanshinones in Salvia miltiorrhiza hairy roots by targeting ARFs genes. Plant Cell Tiss Organ Cult 142, 327–338 (2020). https://doi.org/10.1007/s11240-020-01865-8

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Keywords

  • Regulation mechanism
  • Secondary metabolism
  • miR160a
  • Transgenic