Journal of Plant Physiology 244 (2020) 153006

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Journal of Plant Physiology

journal homepage: www.elsevier.com/locate/jplph

The ethylene response factor SmERF8 regulates the expression of SmKSL1 T

and is involved in tanshinone biosynthesis in Saliva miltiorrhiza hairy roots
Zhenqing Baia,b,e,f, Jiawen Wua,e, Wenli Huangb, Jie Jiaob, Chenlu Zhangb, Zhuoni Houc,
Kaijing Yand, Xuemin Zhangd, Ruilian Hanc, Zongsuo Liangb,c,*, Xiujuan Zhangf
a
College of Life Science, Yan′an University, Yan′an, China
b
College of Life Science, Northwest A&F University, Yangling, China
c
College of Life Science, Zhejiang Sci-Tech University, Hangzhou, China
d
Tasly R&D Institute, Tasly Holding Group Co., Ltd., Tianjin, China
e
Shaanxi Key Laboratory of Chinese Jujube (Yan′an, University), Yan′an, China
f
Inner Mongolia Autonomous Region Institute of Biotechnology, Hohhot, China

ARTICLE INFO ABSTRACT

Keywords: Saliva miltiorrhiza ethylene response factor (SmERF), predicted to be expressed genome-wide, is the potential
S. miltiorrhiza regulator of tanshinone biosynthesis. However, few studies have investigated its transcriptional regulation
SmERF8 pathways in tanshinone biosynthesis. Here, we report an ethylene response factor (SmERF8), which was screened
GCC-box by the SmKSL1 (a key gene in tanshinone biosynthesis) promoter from the S. miltiorrhiza cDNA library. The
Tanshinone
SmERF8, highly expressed in S. miltiorrhiza root head, is sensitive to Eth stress, and its protein was enriched in
Artificial microRNA
the nucleus. The SmERF8 recognizes the GCC-box in the SmKSL1 promoter. Overexpression and RNAi of SmERF8
in S. miltiorrhiza transgenic hairy roots showed that the tanshinone contents were significantly increased in the
overexpression transgenic lines and decreased in RNAi lines. These results suggest that the SmERF8 may be a
central activator that regulates the expression of SmKSL1 by binding the GCC-box and then promoting tan-
shinone biosynthesis. Thus, the SmERF8 may functionally accelerate tanshinone biosynthesis by the transcrip-
tional regulation of its key gene.

1. Introduction been reported. Tanshinone is synthesized via two distinct pathways:

ERF, a member of the APETALA2 (AP2) family, is a functional factor
in plant growth and development. There have been many reports on the
function of ERF in Arabidopsis. In Arabidopsis, there are a total of 147
genes encoding AP2/ERF transcription factors, and 122 of them encode
ERF transcription factors (Toshitsugu et al., 2006). ERF transcription
factors in Arabidopsis can be classified into 12 different groups; namely,
groups I–X, VI-L, and XbM C-L (Toshitsugu et al., 2006). In S. miltior-
rhiza, 94 AP2/ERF putative transcription factors were identified by de
novo transcriptome sequencing in S. miltiorrhiza (Hua et al., 2011).
AP2/ERF superfamily TFs, with ERF domains, can bind to GCC-box
elements or DRE motifs, thereby regulating gene expression in response
to both biotic or abiotic stress (Cao et al., 2001; Fujimoto et al., 2000).
Tanshinone is a compound with mainly medicinal properties found
in Saliva miltiorrhiza (Wang et al., 2007). Its biosynthesis pathway has
mevalonate (MVA) or 2-C-methyl-D-erythritol 4-phosphate (MEP)
pathway (Wang and Wu, 2010). Salvia miltiorrhiza kaurene synthase
like 1(SmKSL1) was reported to be the key gene in the final steps of the
tanshinone biosynthesis pathway (Zhou et al., 2012; Bai et al., 2018b).
SmKSL1 catalyzes copalyl diphosphate (CPP) to form miltiradiene,
which is the precursor of tanshinone (Gao et al., 2015b).
The tanshinone biosynthesis pathway is regulated by many TFs.
There are 171 putative APETALA2 (AP2) family TFs in S. miltiorrhiza
(Xu et al., 2016, 2015). Moreover, a relationship between AP2/ERFs
TFs and tanshinone biosynthesis has previously been reported (Hua
et al., 2011). Recently, the genome-wide identification of the AP2/ERF
genes showed that they might enhance the biosynthesis of the bioactive
compounds of S. miltiorrhiza (Li et al., 2016b). Our previous study
showed that SmERF6 regulated the expression of Salvia miltiorrhiza
copalyl diphosphate synthases 1 (SmCPS1) as well as the binding of the

Abbreviations: SmKSL1, Salvia miltiorrhiza kaurene synthase like 1; ERF, ethylene response factor; Eth, ethephon; TFs, transcriptional factor; DT-I, dihydrotan-
shinone; T-IIA, tanshinone IIA; CT, cryptotanshinone; T-I, tanshinone I
⁎
Corresponding author at: College of Life Science, Northwest A&F University, Yangling, 712100, China.
E-mail address: liangzs@ms.iswc.ac.cn (Z. Liang).

https://doi.org/10.1016/j.jplph.2019.153006
Received 5 March 2019; Received in revised form 27 June 2019; Accepted 30 June 2019
Available online 09 July 2019
0176-1617/ © 2019 Published by Elsevier GmbH.
Z. Bai, et al.
GCC-box in the promoters of SmKSL1, and was therefore involved in
tanshinone biosynthesis in S. miltiorrhiza hairy roots (Bai et al., 2018a).
ERF family members have drawn increasingly more attention on
bioactive compounds of S. miltiorrhiza, and their function has remained
largely elusive.
Here, we report that an ethylene response factor, SmERF8, regulates
the transcription of SmKSL1 by recognizing the GCC-box to promote
tanshinone biosynthesis. Thus, these regulations shift available re-
sources to tanshinone accumulation.
2. Materials and methods
2.1. Plant materials and growth conditions
The S. miltiorrhiza sterile plantlet seedlings, preserved in our lab,
were cultured on an MS medium containing 7% agarose (pH 5.8). The
hairy roots of S. miltiorrhiza were derived from these plantlets, infected
with Agrobacterium rhizogenes (ATCC15834 strain), and sub-cultured
every 30 days.
To analyze tissue expression, five kinds of tissues, including leaves,
roots, stems, flowers, and root heads, were collected when the S. mil-
tiorrhiza seedlings were at the flowering stage. Then, the expression
level of SmERF8 was assessed in these tissues.
For ethephon treatment, 18-day-old S. miltiorrhiza hairy roots were
shaken in liquid 6,7-V medium (25℃, 120 rpm) and treated to a final
concentration of 200 μg/L Eth. Then, the treated samples were har-
vested separately at 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 72 h, and after
eight days. S. miltiorrhiza hairy roots without ethephon were used as a
control. All of these collected samples were immediately frozen in li-
quid nitrogen and stored at −80 °C prior to analysis.
2.2. Total RNA and DNA extraction
Total complete RNA was isolated from the frozen S. miltiorrhiza
samples using the RNAprep Pure Plant Kit (TIANGEN, China). The RNA
was then reversely transcribed to generate the cDNA, using the
PrimeScriptTM RT reagent Kit with gDNA Eraser (Takara, Japan) ac-
cording to the manufacturer’s instructions. To isolate the genomic DNA,
an improved cetyltrimethylammonium bromide (CTAB) method was
used.
2.3. Quantitative RT-PCR (qRT-PCR) analysis
Real-time quantitative PCR was performed on an optical 96-well
plate with an ABI PRISM 7500 real-time PCR system (Applied
Biosystems) using SYBR Premix ExTaq (TaKaRa). The PCR thermal
cycles were as follows: 95℃ for 30 s, followed by 40 cycles at 95℃ for
5 s and 60℃ for 30 s. The SmActin gene was used as the endogenous
control, and relative expression levels were determined as described
previously (Livak and Schmittgen, 2001).
2.4. The gene clone and bioinformatics analyses of SmERF8
The nucleotide sequences and amino acids of SmERF8 were obtained
from the S. miltiorrhiza Information Resource (http://bi.sky.zstu.edu.
cn/DsTRD/home.php). SmERF8 was predicted by tBLASTn search of
AtERF homologs against the current S. miltiorrhiza transcriptome as-
sembly. An e-value cut-off of 1e-10 was applied to the homologue re-
cognition. The retrieved sequences were used for gene model prediction
on the SWISS-MODEL web server (https://www.swissmodel.expasy.
org/). Full-length CDSs of SmERF8 were amplified by reverse tran-
scription PCR using the primers listed in Table S1. PCR products were
gel-purified, cloned, and then sequenced. Phylogenetic trees were
constructed using the neighbor-joining (NJ) method in MEGA6.
The prediction of nucleus positioning signal peptide use online
web: http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi.
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Journal of Plant Physiology 244 (2020) 153006
The theoretical isoelectric point (pI) and molecular weight (Mw) were
predicted using the Compute pI/Mw tool on the ExPASy server (http://
web.expasy.org/compute_pi/).
2.5. Y1H verifing
The Y1H-pAbAi-SmKSL1-649 (PYK649) yeast strains were con-
structed in our previous research (Bai et al., 2018a). The primer for the
PGADT7-SmERF8 vector is listed in Table S1. The PGADT7-SmERF8 was
verified by interactions with the Y1H-pAbAi-SmKSL1-649 (PYK649)
yeast strains which recombined SmKSL1 promoters in SD/-Leu/AbA.
The PYK649 yeast strains were constructed in our previous study (Bai
et al., 2018a). The following step and ß-X gal staining were described in
the yeast protocol handbook (Clontech Laboratories, Inc., PT3024-1)
2.6. The subcellular location of SmERF8
2.6.1. Vector construction and Agrobacterium injection
S. miltiorrhiza hairy root cDNA was used to amplify the SmERF8
gene. The SmERF8 genes was fused with green fluorescent protein
(GFP) proteins into the pA7-0390-SmERF8 vector by enzyme cutting
(XhoI and SpeI restriction endonuclease) and ligase reaction, and vec-
tors were confirmed by sequencing. The primer sequences used in this
experiment are listed in Table S1. The recombination plasmid was then
extracted by TIANprep Mini Plasmid Kit (TianGen, DP103, Beijing,
China), and transformed into EHA105 Agrobacterium. Agrobacterium
was cultured to an OD600 of 1.0 at 28℃ and resuspended in injection
solution to an OD600 ≈ 0.8. The injection solution consisted of 0.5 M 4-
morpholineethanesulfonic acid (MES, pH 5.7; Sigma, cat. no. M8250),
100 mM MgCl2, and 150 mM Acetosyringone. Agrobacterium was cul-
tured in the dark for 2 h. Then, the pA7-0390-SmERF8 injection solu-
tion was resuspend by vortex instrument. Bacterial suspension mixtures
were infiltrated into the leaves of four-week-old N. benthamiana plants
using a needleless syringe. Then, the plants were co-cultured for two
days, and the protoplast prepared as previously described (Li, 2011).
2.6.2. Protoplast composition
Leaves were quickly cut into small pieces using a sharp knife. The
pieces were immediately put into prepared enzyme solution consisting
of 0.02 M MES acid, 0.4 M mannitol, 0.2 M KCl, 15 g/L cellulase R-10,
4 g/L macerozyme R-10, 0.01 M CaCl2, 0.1% (w/v) bovine serum al-
bumin, and 2 mM β-mercaptoethanol, and then shaken in a 25 °C in-
cubator at 30 rpm until the pieces were digested completely.
Afterwards, the mixture was filtered using a strainer and centrifuged at
1000 rpm for 2 min. The supernatant was removed and the pellet re-
suspended in WI solution. The solution consisted of 4 mM MES, 10 mM
KCl, and 0.5 M mannitol. The protoplast was examined using confocal
laser scanning microscopy.
2.6.3. Confocal laser scanning microscopy
Confocal laser scanning microscopy was performed with a Nikon
A1R inverted confocal microscope. GFP fusions were excited with a
488-nm argon laser and detected using a 505–530-nm band-pass
emission filter.
DAPI were excited with a 408-nm argon laser and detected using a
512–550-nm band-pass emission filter in another laser channel.
Chlorophyll was excited with a 640-nm argon laser and detected using a
660–740-nm band-pass emission filter.
2.6.4. Protein extraction and immunoblot analysis
The sequence encoding the SmERF8 protein was cloned into the
PMAL-2A vector (Novagen) (the primer is listed in Table S1) and
overexpressed protein in the Escherichia coli strain Rosetta induced by
0.4 mM Isopropyl-β-D-Thiogalactoside (IPTG). Bacterial cells were
lysed by Low-Temperature Ultra-high Pressure Continuous Flow Cell
Disrupter, centrifuged at 10,000 rpm, 4℃, for 30 min, and the
Z. Bai, et al.
supernatant was saved. The MBPTrap HP affinity resin eluted in 20 mM
Tris-HCl, 200 mM NaCl, 1 Mm EDTA, 1 mM DTT (pH 7.4). Soluble N-
terminally MBP-tagged SmERF8 was bound to MBPTrap HP affinity
resin. The eluted protein was purified by size-exclusion chromato-
graphy in 20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 1 mM DTT (pH
7.4), and 10 mM maltose. SDS-PAGE of MBP and SmERF8 purified
protein showed one major band at an approximate molecular mass of
42 kD and 66 kD, indicating pure full-length protein.
For detection of MBP tagged (malE) SmERF8 proteins, SmERF8
proteins were mixed with SDS loading buffer with bromophenol blue
dye. Then, SDS-PAGE electrophoresis assessed the size and purity of
SmERF8 proteins. The concentration of protein was determined using
the Bio-Rad protein assay kit with BSA as the standard. Immunoblot
detection of tagged proteins was performed as previously described (Liu
and Zhang, 2004). The anti MBP-Tag Mouse Monoclonal antibody
(CW0288S,1:3000 dilution) and Goat Anti-Mouse IgG, AP Conjugated
(CW0110S, 1:3000 dilution) were purchased from Kwbio company. The
BCIP/NBT Alkaline Phosphatase Color Development Kit (C3206) were
purchased from Beyotime company.
2.6.5. Electrophoretic mobility shift assay (EMSA) analysis
The proteins were purified and the quantity of purified protein es-
timated by a BSA protein quantification kit (Viagene Biotech, Yangling,
China). The oligonucleotide probes were synthesised (listed in Table
S1) and annealed for duplex self-assembly by high temperature fol-
lowed by cooling at room temperature. EMSAs were performed using
the Light Shift Chemiluminescent EMSA kit (Invitrogen, E33075,
California, USA) according to the manufacturer’s instructions. The
binding reactions were performed using 631,022 nM (2.5 mg) for each
protein sample incubated with 8 nM probe in binding buffer (10 mM
Tris- HCl, pH 7.5, 50 mM KCl, 1 mM DTT, and 5% glycerol) with a
protein-free sample as the blank control. After 20 min of incubation at
room temperature, the reactions were resolved by 6% native poly-
acrylamide gels with 0.5 × TBE buffer. The gels were imaged at 490 nm
SYBR photographic filter using a Polaroid camera.
2.6.6. The acquisition and Identification of SmERF8 overexpression
transgene hairy root
SmERF8 gene was integrated into the pCMBIA1304+ vector (stored
in our laboratory). The primer of PCAMBIA1304+-.SmERF8 is listed in
Table S2. Agrobacterium rhizogenes strain ATCC15834 (ATCC, Catalogue
15834, Manassas, VA, USA) was used in the induction of S. miltiorrhiza
hairy root. It was previously used for plant transformation in our lab (Li
et al., 2016a). Then, the transgene positive hairy root verified by
identification primer of rolb, rolc, hptII and 35S + SmERF8-1304-R, the
primers were listed in Table S1. Then, the positive hairy roots were
subculture cultivation.
2.6.7. The design and synthesis of the artificial microRNA (amiR) and the
acquisition of amiRSmERF8 overexpression transgene hairy root
The 21-nt-long artificial miRNA sequences against SmERF8 were
designed using the web miRNA designer WMD3 (http://wmd3.
weigelworld.org/cgi-bin/webapp.cgi) following the instructions given
on the website. Only green flagged sequences were selected and
checked against their target sequence and the S. miltiorrhiza Bunge
genome using psRNAtarget (http://plantgrn.noble.org/psRNATarget/)
(Devers et al., 2013). The sequence showing no or the least possible off
targets was chosen for further construction of the artificial miRNA.
Then, we chose ath-MIR159b (MI0000218) as the basic backbone.
Thus, the miR159b-SmERF8 backbone was synthesized by the gene
biosynthesis company. This molecule was cloned into the SpeI restric-
tion site of pCMBIA1304. Then, the plant was transformed and identi-
fied using the previously described method, and the positive plantlets
were verified by PCR. The identification primer is listed in Table S1.
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Journal of Plant Physiology 244 (2020) 153006
2.7. MiRNA assay, first-strand cDNA synthesis, and qRT-PCR detection
The detailed methods of MiRNA assay, first-strand cDNA synthesis,
and qRT-PCR detection were the same as in our previous report (Bai
et al., 2018a).
2.7.1. The tanshinone content analysis
The tanshinone contents were measured by High-Performance
Liquid Chromatography (HPLC). The HPLC method was performed
according to an already established method in our laboratory (Yang
et al., 2012). For each sample, 8 ml 70% methanol was added to 0.04 g
dry sample, soaked overnight, and then sonicated for 45 min, followed
by 8000-rpm centrifugation for 10 min. The sample was then filtered
through a 0.45-μm filter. HPLC analysis was performed using a Waters
(Milford, MA, USA) system with a binary pump and photodiode array
detector. A Sun Fire C18 column (250 × 4.6 mm, 5 μm; Waters) was
used. The flow rate was 1 ml/min, the column temperature was 30 °C,
and the sample size was 10 μl.
2.7.2. Data statistics and analysis
Experiments were performed with three biology and technology
replicates, respectively. The gene relative quantification method (delta-
delta Ct) was used to evaluate quantitative variation. The statistical
analysis of HPLC data and qRT-PCR data was performed using one-way
ANOVA followed by Student’s t-test in SPSS (version 13.0), and con-
sidered statistically different at p < 0.05 or p < 0.01. The figures were
drawn using Origin 9.0 software.
3. Results
3.1. Molecular cloning, expression pattern, and subcellular localization of
SmERF8
SmERF8 was demonstrated to bind to the promoter of SmKSL1
based on our Y1H results, as demonstrated previously (Bai et al.,
2018a). On the basis of sequence information, we blasted and searched
the transcriptome database. Then, the ORF of SmERF8 was amplified
and sequenced. The gene named SmERF8 (MH006600) was similarly
found on the AtERF8. SmERF8 is a member of subgroup VIIIa in the B1
family (Fig. S1a, Table S2). The nucleus positioning signal peptide
prediction of SmERF8 showed that one monopartite NLS (RGVRKRP-
WGRYAAEIRDPGKKSRV W) existed in its 25th position amino acid
(Score = 7.1). The theoretical pI/Mw of SmERF8 is 5.08/52576.28. The
predicted 3D-structure of SmERF8 showed that the protein structure
included one α-helix and three ß-pleated sheets (Fig. S1b). The SmERF8
has high nucleotide sequence identity (52.5%) with SmERF6. The
SmERF8 is highly expressed in the root head (Fig. 1a), and responded to
endogenous Eth treatment (Fig. 1b). The subcellular location results
showed that the SmERF8-GFP fused protein was enriched in the nucleus
(Fig. 1c).
3.2. SmERF8 bound to the GCC-box in the SmKSL1 promoter
To verify in vivo binding of SmERF8 to the promoter of SmKSL1, we
conducted yeast one-hybrid (Y1H) system assays. The SmERF8 was
confirmed by homologous recombination with Y1H-pAbAi-SmKSL1-
649 (PYK649) in vivo (Fig. 2a). These results provided in vivo evidence
for binding of SmERF8 to the SmKSL1 promoter.
To further verify the binding of SmERF8 to the promoters of
SmKSL1 in vitro, we performed an electrophoretic mobility shift assay
(EMSA) using a prokaryote-expressed and purified SmERF8-MBP
(malE) fusion protein (Fig. S2a–c). When we used an ERF-binding cis-
element containing a GCC-box as a labelled probe, a specific
DNA–SmERF8 protein complex was strongly detected (Fig. 2b–d).
Furthermore, SmERF8 bound to the mini GCC-box element probe, but it
could not bind to the mutated GCC-box element (Fig. 2b, d). These
Z. Bai, et al.
results confirmed that the GCC-box is the binding site of SmERF8 in the
SmKSL1 promoter.
3.3. Overexpression of SmERF8 gene positively modulated tanshinone
accumulation through activating the transcription of SmKSL1
The gene expression levels of SmERF8 and SmKSL1 were sig-
nificantly increased (P < 0.05) in four independent SmERF8 over-
expression transgenic hairy root lines (SmERF8 I, II, III, and IV)
(Fig. 3a–c). Since SmERF8 is a transcriptional activator, its over-
expression increased the levels of the target gene (SmKSL1). As a result,
the tanshinone contents (DT-I, CT, T-I, and T-IIA) in each transgenic
line were significantly increased (Fig. 3e, P < 0.05). However, we
found that the fresh weight of the transgenic hairy roots (SmERF8 I, II,
III, and IV) was significantly decreased (P < 0.05) (Fig. 3d, f). The fresh
weight of the hairy root reflects its growth state. Thus, SmERF8, a
transcriptional activator, enhances tanshinone content accumulation by
activating the transcription of SmKSL1 and negatively affects root
growth.
3.4. SmERF8 gene silencing depressed tanshinone accumulation by down-
regulating the transcription of SmKSL1
The AtMir159b-SmERF8 overexpression in positive hairy roots
(amiRSmERF8 I, II, II, IV) was identified by PCR and were used in gene
expression and tanshinone determination (Fig. 4a). The gene expression
level of amiRSmERF8 was up-regulated in amiRSmERF8 I, II, II, and IV
4
Journal of Plant Physiology 244 (2020) 153006
Fig. 1. The characteristic analysis of SmERF8.
a: the tissue-specific expression of SmERF8 in
root, leaf, flower, stem, and root head (the joint
between the stem and root). Data are shown as
the mean ± SD (n = 3), and the lowercase
letters indicate the significance (P < 0.05) ac-
cording to one-way ANOVA tests. b: the gene
expression level of SmERF8 under ethylene
treatment over time. Eth indicates ethephon.
Data are shown as the mean ± SD (n = 3),
and the asterisk indicates the significance
(P < 0.05 or 0.01) according to one-way
ANOVA tests. c: The subcellular location of
PA70390-SmERF8. Merge: the overlap of DAPI,
GFP, cyt5, and DT. DAPI: the nuclear dyes.
GFP: green fluorescent protein. Cyt5: chlor-
oplast autofluorescence. DT: light-field ob-
servations.
(Fig. 4b). However, the gene expression level of SmERF8 significantly
decreased compared to the control (P < 0.01) (Fig. 4c). Moreover, the
tanshinone accumulation content of amiRSmERF8 I, II, II, and IV de-
creased (Fig. 4e). However, the growth of amiRSmERF8 I, II, II, and IV
was not affected by the overexpression of AtMir159bSmERF8. There-
fore, we conclude that the SmERF8 is a key gene involved in the reg-
ulation of tanshinone accumulation in S. miltiorrhiza.
4. Discussion
The ERF family is an important TFs family in plant. The conserved
amino acid sequences and tertiary structure of ERF have been identified
before, and assist its binding with DNA (Nakano et al., 2006; Toshitsugu
et al., 2006). In the present study, the SmERF8 also exhibited these basic
structures, and had high nucleotide sequence identity (52.5%) with
SmERF6, which has a role in tanshinone biosynthesis (Bai et al., 2018a).
However, they belong to different ERF family subgroup. The SmERF8
belongs to the VIIIa subgroup of the ERF family. The VIIIa subgroup of
the ERF family gene has functional roles in many stress responses (Yao
et al., 2017). For example, AtERF74 and AtERF75, involved in con-
trolling an RbohD-dependent mechanism, showed responses to different
stresses, therefore maintaining H2O2 homeostasis in Arabidopsis (Yao
et al., 2017). As is characteristic of ERF family genes, SmERF8 also
showed a response to endogenous Eth stress. Meanwhile, SmERF8, a
nucleus gene, was highly expressed in the S. miltiorrhiza root head.
Tanshinone is one of the bioactive compounds in S. miltiorrhiza (Hao
et al., 2015). Recently, the AP2/ERF genes have been shown to enhance
Z. Bai, et al. Journal of Plant Physiology 244 (2020) 153006

Fig. 2. Verification of the interaction between the SmERF8 protein and SmKSL1 promoter. a: pGADT7-SmERF8: the PGADT7 vector with the SmERF8 insert.
Verification of the pGADT7-SmERF8 interaction with the bait strains with SmKSL1 (PYK649) promoter; 1×, 10×, and 100×: the dilution ratio of the yeast solution.
X-gal staining: the yeast recombinant strains (pGADT7-SmERF8- PYK649) stained by ß-X-gal. b: the GCC-box site in the promoters of SmKSL1 and the sense probes for
EMSA of the in vitro interaction verification between the SmERF8 protein and GCC-box. Mutation 1–3: the point mutation probes of the GCC-box sense element. c:
EMSA analysis: lane 1 has only the GCC-box probe (+) and no proteins (-). Lane 2 has only the SmKSL1 probe (+) and no proteins (-). Lane 3 has the GCC-box probe
and MBP proteins (+). Lane 4 has the SmKSL1 probe and MBP proteins (+). d: EMSA analysis: Lane 1 has the SmKSL1 probe and SmERF8 proteins (+). Lane 2 has
the GCC-box probe and SmERF8 proteins (+). Lanes 3–5 represent the reaction of three mutation probes (1–3) with the SmERF8 protein. With probe (+) and no
probe (-).

the biosynthesis of the bioactive compounds of S. miltiorrhiza (Li et al.,
2016b; Sun et al., 2018; Bai et al., 2018a). SmKSL1 is a key gene in the
last step of the tanshinone biosynthesis pathway (Gao et al., 2015a, b).
Its promoter contains a GCC-box and responds to exogenous Eth sig-
naling (Bai et al., 2018a). AP2/ERF superfamily transcription factors,
with ERF domains, can bind to GCC-box elements, thereby regulating
gene expression (Fujimoto et al., 2000; Cao et al., 2001). In addition,
DT-I, CT, and T-I contents were enhanced by Eth stress (Bai et al.,
2017). In our previous research, SmERF8 was screened from an S. mil-
tiorrhiza cDNA library by the SmKSL1 promoter (Bai et al., 2018a).
Thus, SmERF8 might be involved in regulating SmKSL1 to influence
tanshinone biosynthesis in S. miltiorrhiza.
ERF proteins can bind to the GCC-box and either positively or ne-
gatively regulate transcription (Ohme-Takagi and Shinshi, 1995). For
example, AtERF1, ERF2, and ERF5 all function as activators of GCC-box-
mediated transcription, but ERF3, ERF4, and ERF7 act as repressors in
Arabidopsis (Fujimoto et al., 2000; Song et al., 2005). In our previous
study, the GCC-box in the SmCPS1 and SmKSL1 promoters was the key
target and specifically combined with the SmERF6 protein (Bai et al.,
2018a). In this study, the GCC-box, which contained the promoters of
SmKSL1, was recognized by SmERF8 in vitro (Fig. 2c, d). This finding
suggests that SmERF8 might have the same function as SmERF6 in
tanshinone biosynthesis.
Our previous study revealed that SmERF6 co-regulates the

5
Z. Bai, et al. Journal of Plant Physiology 244 (2020) 153006

Fig. 3. Overexpression of SmERF8 in S. miltiorrhiza hairy root. a: PCR verification of the positive transgenic lines by specific primers (hptII, rolb, rolc, and
35S + SmERF81304+R). Control: the pCAMBIA1304+ empty vector counterparts. b and c: respectively represent the gene expression levels of SmERF8 and SmKSL1
in the control. d: transgenic hairy root lines (SmERF8 I–IV). e: The tanshinone contents in control and transgenic hairy root lines (SmERF8 I–IV). DT-I: dihy-
drotanshinone, T-I: tanshinone I, T-IIA: tanshinone IIA, CT: cryptotanshinone. f: the growth phenotypes of control and transgenic hairy root lines (SmERF8 I–IV). g:
the fresh weight difference between the control and transgenic hairy root lines (SmERF8 I– IV). Data are shown as the mean ± SD (n = 3), and the lowercase letters
indicate the significance (P < 0.05) according to one-way ANOVA tests. SmERF8 I–IV represent the positive hairy root lines overexpressing SmERF8.

6
Z. Bai, et al. Journal of Plant Physiology 244 (2020) 153006

Fig. 4. Overexpression of amiR-SmERF8 in S. miltiorrhiza hairy root. A：PCR verification of positive transgenic lines by specific primers (hptII, rolb, rolc, and
35S + amiR-SmERF8-1304R). b–d： respectively represent the gene expression levels of amiRSmERF8, SmERF8, and SmKSL1 in control e: the tanshinone contents in
control and transgenic hairy root lines (amiRSmERF8 I–IV). DT-I: dihydrotanshinone, T-I: tanshinone I, T-IIA: tanshinone IIA, CT: cryptotanshinone. f: The fresh
weight difference between the control and transgenic hairy root lines (amiRSmERF8 I–IV). Data are shown as the mean ± SD (n = 3), and the lowercase letters
indicate the significance (P < 0.05) according to one-way ANOVA tests. Control: pCAMBIA1304 empty vector counterparts. amiRSmERF8 I–IV: positive hairy root
lines overexpressing amiRSmERF8.

7
Z. Bai, et al.
transcription of SmCPS1 and SmKSL1, and it was involved in tanshinone
biosynthesis in S. miltiorrhiza hairy roots (Bai et al. 2018a). ERFs are
involved in the regulation of primary and secondary metabolism
(Francesco et al., 2013). Given our interest in tanshinone biosynthesis,
the up-regulation of SmERF8 expression triggered the increase of
SmKSL1 expression in the SmERF8-overexpressed transgenic hairy root
lines. The higher SmKSL1 expression level promoted the accumulation
of tanshinone and induced the decrease in phenolic acid and flavonoid
contents. The gene expression levels of SmERF8 and SmKSL1 sig-
nificantly declined while amiRSmERF8 was overexpressed (p < 0.05),
(Fig. 4b–e). SmKSL1 is the key gene in the tanshinone biosynthesis
pathway. The decreasing expression of SmKSL1 was followed by the
decreasing of tanshinone contents (DT-I, CT, T-I, and T-IIA). Thus,
SmERF8, regulated expression level of SmKSL1 in the nucleus, this
regulation further influence tanshinone biosynthesis of S. miltiorrhiza
(Fig. 5). These results put forward a metabolic regulation model per-
taining to tanshinone biosynthesis. The ERF group IX (B3) ancestors
were recruited early in the evolution of distinct plant lineages to reg-
ulate the synthesis of jasmonate-inducible metabolites (Kathleen et al.,
2011). However, little is known about how the VIII subgroup family
genes, which SmERF8 belongs to, regulate between plant primary and
secondary metabolism.
For further research, the advanced technology and in-depth study
will be applied to explore the function of SmERF8. For example, the
Crispr-cas9 has been developed in S. miltiorrhiza (Li et al., 2017), and
the SmERF8 knocked-out mutant was also produced by this technology;
thus, it will represent an improved experimental method to confirm the
SmERF8 function of tanshinone biosynthesis in S. miltiorrhiza. More
analyses on S. miltiorrhiza hairy transgenic roots are necessary, such as
gene response and component analysis to exogenous ethephon treat-
ment. Overall, more experiments are needed to explore the function of
SmERF8 in S. miltiorrhiza hairy transgenic roots, which will provide a
better understanding of the role of SmERF8 in the secondary metabo-
lism of S. miltiorrhiza.
Above all, SmERF8 regulated the SmKSL1 expression level to ad-
justing tanshinone biosynthesis, and it influenced hairy root growth.
Tanshinone yield is an important evaluation index of the pharmaceu-
tical value of S. miltiorrhiza. Our current research shows that SmERF8 is
a positive regulator of tanshinone biosynthesis, and may be a potential
target for further metabolic engineering of tanshinone biosynthesis in S.
8
Journal of Plant Physiology 244 (2020) 153006
Fig. 5. The role of SmERF8 in tanshinone me-
tabolic regulation. The red arrow represents
the enhanced level, and the blue arrow re-
presents the decreased level. The dotted line
represents the omitted middle steps of tan-
shinone biosynthesis. Silence represents
amiRSmERF8 down-regulation of the SmERF8
gene expression level. Acceleration represents
the up-regulation of the SmKSL1 gene expres-
sion level that promotes tanshinone biosynth-
esis. The green dot-and-dashed circle re-
presents the nucleus area. (For interpretation
of the references to colour in this figure legend,
the reader is referred to the web version of this
article.)
miltiorrhiza. In conclusion, these findings shed light on the functional
support of the SmERF gene in S. miltiorrhiza. SmERF8 has a potential
role in the development of biotechnological strategies for the genera-
tion of new S. miltiorrhiza germplasm with improved tanshinone pro-
duction in S. miltiorrhiza, as well as in improving its medicinal value.
Author contributions
BZQ conceived the research and wrote the manuscript; BZQ, JJ, and
HWL performed the experiments; ZCL and HZN analyzed data; YKJ and
ZXM revised the manuscript. HRL and LZS supported the project and
provided guidance for the experimental design. All authors commented
on the manuscript and approved the contents.
Declaration of Competing Interest
The authors have declared no conflicts of interest.
Acknowledgments
We are grateful to professors Chao Sun, Shilin Chen, and Weibo Jin
for kindly providing the genome resources and transcriptome data of S.
miltiorrhiza used in this study respectively. We thank the support from
the National Natural Science Foundation of China (81773835 and
81703646) and Natural Science Foundation of Zhejiang province
(LQ16C020002) and Natural Science Foundation of Inner Mongolia
autonomous region (2015MS0385).
References
Bai, Z., Li, W., Jia, Y., Yue, Z., Jiao, J., Huang, W., Xia, P., Liang, Z., 2018a. The ethylene
response factor SmERF6 co-regulates the transcription of SmCPS1 and SmKSL1 and is
involved in tanshinone biosynthesis in Salvia miltiorrhiza hairy roots. Planta 1–13.
Bai, Z., Liu, J., Zhang, C., Huang, W., Liang, Z., Yan, X., Liu, Y., Zhu, Y., 2018b. Coding
single nucleotide polymorphisms and SmCPS1 and SmKSL1 subcellular localization
are associated with tanshinone biosynthesis in Salvia miltiorrhiza Bunge roots. Acta
Physiol. Plant. 40 (1), 6. https://doi.org/10.1007/s11738-11017-12563-x. https://
doi.org/10.1007/s11738-017-2563-x.
Bai, Z., Xia, P., Wang, R., Jiao, J., Ru, M., Liu, J., Liang, Z., 2017. Molecular cloning and
characterization of five SmGRAS genes associated with tanshinone biosynthesis in
Salvia miltiorrhiza hairy roots. PLoS One 12 (9), e0185322. https://doi.org/10.1371/
journal.pone.0185322.
Z. Bai, et al.
Cao, Z.F., Li, J., Chen, F., Li, Y.Q., Zhou, H.M., Liu, Q., 2001. Effect of two conserved
amino acid residues on DREB1A function. Biochem. Mosc. 66 (6), 623–627.
Devers, E.A., Teply, J., Reinert, A., Gaude, N., Krajinski, F., 2013. An endogenous arti-
ficial microRNA system for unraveling the function of root endosymbioses related
genes in Medicago truncatula. BMC Plant Biol. 13, 82. https://doi.org/10.1186/
1471-2229-1113-1182.
Francesco, L., Masaru, O.T., Pierdomenico, P., 2013. APETALA2/Ethylene Responsive
Factor (AP2/ERF) transcription factors: mediators of stress responses and develop-
mental programs. New Phytol. 199 (3), 639–649.
Fujimoto, S.Y., Ohta, M., Usui, A., Shinshi, H., Ohme-Takagi, M., 2000. Arabidopsis
ethylene-responsive element binding factors act as transcriptional activators or re-
pressors of GCC box-mediated gene expression. Plant Cell 12 (3), 393–404.
Gao, W., Hillwig, M.L., Huang, L., Cui, G., Wang, X., Kong, J., Yang, B., Peters, R.J.,
2015a. A functional genomics approach to tanshinone biosynthesis provides stereo-
chemical insights. Org. Lett. 11 (22), 5170–5173.
Gao, W., Hu, T.Y., Guo, J., Lv, D.M., Dai, Z.B., Zhou, Y.J., Huang, L.Q., 2015b. Research
progress of synthetic biology for tanshinones. China J. Chin. Mater. Med. 40 (13),
2486–2491.
Hao, X., Shi, M., Cui, L., Xu, C., Zhang, Y., Kai, G., 2015. Effects of methyl jasmonate and
salicylic acid on tanshinone production and biosynthetic gene expression in trans-
genic Salvia miltiorrhiza hairy roots. Biotechnol. Appl. Biochem. 62 (1), 24–31.
Hua, W., Zhang, Y., Song, J., Zhao, L., Wang, Z., 2011. De novo transcriptome sequencing
in Salvia miltiorrhiza to identify genes involved in the biosynthesis of active in-
gredients. Genomics 98 (4), 272–279.
Kathleen, D.B., Sofie, T., Laurens, P., Robin, V.B., Valerie, D.S., Rudy, V., Pierre, H.,
Hamill, J.D., Alain, G., 2011. APETALA2/ETHYLENE RESPONSE FACTOR and basic
helix-loop-helix tobacco transcription factors cooperatively mediate jasmonate-eli-
cited nicotine biosynthesis. Plant J. 66 (6), 1053–1065.
Li, B., Cui, G., Shen, G., Zhan, Z., Huang, L., Chen, J., Qi, X., 2017. Targeted mutagenesis
in the medicinal plant Salvia miltiorrhiza. Sci. Rep. 7, 43320.
Li, B., Wang, B., Li, H., Liang, P., Mei, R., Liang, Z., Yan, X., Zhu, Y., 2016a. Establishment
of Salvia castanea Diels f. tomentosa Stib. hairy root cultures and the promotion of
tanshinone accumulation and gene expression with Ag +, methyl jasmonate, and
yeast extract elicitation. Protoplasma 253 (1), 87–100.
Li, X., 2011. A transient expression assay using Arabidopsis mesophyll protoplasts. Bio-
protocol 1 (10), 1–5. https://doi.org/10.21769/BioProtoc.70.
Li, X., Wang, J., Lei, M., Li, L., Fu, Y., Wang, Z., Ao, M., Li, Z., 2016b. Transcriptome
analysis of storage roots and fibrous roots of the traditional medicinal HerbCallerya
speciosa(Champ.) ScHot. PLoS One 11 (8). https://doi.org/10.1371/journal.pone.
0160338.
Journal of Plant Physiology 244 (2020) 153006
Liu, Y., Zhang, S., 2004. Phosphorylation of 1-aminocyclopropane-1-carboxylic acid
synthase by MPK6, a stress-responsive mitogen-activated protein kinase, induces
ethylene biosynthesis in Arabidopsis. Plant Cell 16 (12), 3386–3399.
Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-
time quantitative PCR and the 2(-delta delta C(T)) method. Methods 25 (4), 402–408.
Nakano, T., Suzuki, K., Fujimura, T., Shinshi, H., 2006. Genome-wide analysis of the ERF
gene family in Arabidopsis and rice. Plant Physiol. 140 (2), 411–432.
Ohme-Takagi, M., Shinshi, H., 1995. Ethylene-inducible DNA binding proteins that in-
teract with an ethylene-responsive element. Plant Cell 7 (2), 173–182.
Song, C.P., Agarwal, M., Ohta, M., Guo, Y., Halfter, U., Wang, P., Zhu, J.K., 2005. Role of
an Arabidopsis AP2/EREBP-type transcriptional repressor in abscisic acid and
drought stress responses. Plant Cell 17 (8), 2384–2396.
Sun, M., Shi, M., Wang, Y., Huang, Q., Yuan, T., Wang, Q., Wang, C., Zhou, W., Kai, G.,
2018. The biosynthesis of phenolic acids is positively regulated by the JA-responsive
transcription factor ERF115 in Salvia miltiorrhiza. J. Exp. Bot. 70 (1), 243–254.
https://doi.org/10.1093/jxb/ery349.
Toshitsugu, Nakano, Suzuki, Kaoru, Fujimura, Tatsuhito, Shinshi, Hideaki, 2006.
Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiol.
140 (2), 411–432.
Wang, J.W., Wu, J.Y., 2010. Tanshinone biosynthesis in Salvia miltiorrhiza and produc-
tion in plant tissue cultures. Appl. Microbiol. Biotechnol. 88 (2), 437–449.
Wang, X., Morrisnatschke, S.L., Lee, K.H., 2007. New developments in the chemistry and
biology of the bioactive constituents of Tanshen. Med. Res. Rev. 27 (1), 133–148.
Xu, H., Song, J., Luo, H., Zhang, Y., Li, Q., Zhu, Y., Xu, J., Li, Y., Song, C., Wang, B., 2016.
Analysis of the genome sequence of the medicinal plant Salvia miltiorrhiza. Mol.
Plant 9 (6), 949–952.
Xu, Z., Peters, R.J., Weirather, J., Luo, H., Liao, B., Zhang, X., Zhu, Y., Ji, A., Zhang, B.,
Hu, S., 2015. Full-length transcriptome sequences and splice variants obtained by a
combination of sequencing platforms applied to different root tissues of Salvia mil-
tiorrhiza and tanshinone biosynthesis. Plant J. Cell Mol. Biol. 82 (6), 951–961.
Yang, D., Ma, P., Liang, X., Wei, Z., Liang, Z., Liu, Y., Liu, F., 2012. PEG and ABA trigger
methyl jasmonate accumulation to induce the MEP pathway and increase tanshinone
production in Salvia miltiorrhiza hairy roots. Physiol. Plant. 146 (2), 173–183.
Yao, Y., Chen, X., Wu, A.M., 2017. ERF-VII members exhibit synergistic and separate roles
in Arabidopsis. Plant Signal. Behav. 12 (6), e1329073. https://doi.org/10.1080/
15592324.2017.1329073.
Zhou, Y.J., Gao, W., Rong, Q., Jin, G., Chu, H., Liu, W., Yang, W., Zhu, Z., Li, G., Zhu, G.,
2012. Modular pathway engineering of diterpenoid synthases and the mevalonic acid
pathway for miltiradiene production. J. Am. Chem. Soc. 134 (6), 3234–3241.