The SmERF1b-like regulates tanshinone biosynthesis in Salvia miltiorrhiza hairy root

Abstract The ethylene response factor family genes are involved in the regulation of secondary metabolism in Salvia miltiorrhiza, but the mechanism underlying this regulation remains elusive. In the present study, based on the cDNA library of S. miltiorrhiza, an AP2/ERF gene was cloned and named SmERF1b-like. This gene exhibited a significant response to exogenous ethylene supply, such that ethylene remarkably upregulated SmERF1b-like expression levels in the leaves of S. miltiorrhiza. Subcellular localization showed that SmERF1b-like is located in the nucleus. Furthermore, SmERF1b-like showed a binding affinity with a GCC-box motif in the promoter region of genes associated with tanshinone biosynthesis in S. miltiorrhiza. Overexpression of SmERF1b-like in hairy roots of S. miltiorrhiza substantially upregulated SmCPS1 and SmKSL1 expression levels, resulting in increased biosynthesis of tanshinone I and cryptotanshinone contents. This finding provides valuable theoretical support for the utilization of a plant genetic engineering strategy to enhance S. miltiorrhiza resources.


Background
AP2/ERF is one of the most extensive transcription factor (TF) families in plants, consisting of APETALA2 (AP2), ethylene response factor (ERF), ethylene response element binding protein (DREB), ABI3/VPI (RAV) and Soloist (Jofuku et al. 1994;Magnani et al. 2004;Mizoi et al. 2012;Gao et al. 2017;Zhang et al. 2018;Feng et al. 2020).Among these, ERF contains only one AP2/ERF domain, which comprises about 60 amino acid residues arranged into 3 anti-parallel β-sheets and an α-helix, with alanine at the 14th position and aspartic acid at the 19th position.The GCC-box (GCCGCC) is a cis-acting element that exhibits a specific affinity for ERF binding activity (Magnani et al. 2004;Mizoi et al. 2012;Gao et al. 2017;Zhang et al. 2018;Feng et al. 2020).The ERF genes play a pivotal role in plant growth and development, such as root development, stem elongation, leaf morphology, flower formation, fruit maturation, metabolic processes as well as responses to biotic and abiotic stresses (Xu et al. 2021).Additionally, ERFs have been identified as essential factors involved in modulating the biosynthesis of secondary metabolites in medicinal plants (Xiao et al. 2020).For instance, AaERF1 and AaERF2 upregulated the expression levels of amorpha-4,11-diene synthase (ADS) and CYP71AV1, thereby enhancing the accumulation of artemisinin in Artemisia annua (Barbacka and Baer-Dubowska 2011;Yu et al. 2012;Lu et al. 2013).TcERF12 and TcERF15 regulated taxol synthase gene expression, thus modulating the taxol biosynthesis pathway in Taxus chinensis (Zhang et al. 2015;Shao et al. 2021).ORCA3 significantly improved the yield of indole alkaloids (TIA) in Catharanthus roseus (Zhu et al. 2015;Sun and Peebles 2016).
In this study, the SmERF1b-like gene was identified as a potential TF through screening the Salvia miltiorrhiza cDNA library using the yeast one-hybrid (Y1H) technique (Bai et al. 2018).The open reading frame of SmERF1b-like was successfully cloned from the S. miltiorrhiza cDNA library.The bioinformatics analysis, gene characteristic evaluations and overexpressed SmERF1b-like hairy roots were employed to investigate the role of SmERF1b-like in tanshinone biosynthesis pathways in S. miltiorrhiza.

Experimental materials and treatments
The sterile seeds of S. miltiorrhiza were cultivated in MS medium for 45 d, and then the 45 d old seedlings were used in this experiment.The S. miltiorrhiza hairy roots induced by Agrobacterium rhizogenes (ATCC15834 strain) were from resources in our laboratory that were subcultured every 30 days.Nicotiana benthamiana plants were cultivated at a temperature of 25 °C with a 12 h photoperiod.Tissue-specific expression analysis was performed on the head, stem, leaf, flower and root samples of 2-year-old S. miltiorrhiza.These samples were collected, immediately frozen in liquid nitrogen, and stored at −80 °C.For ethephon treatment, 21-day-old S. miltiorrhiza hairy roots were shaken in liquid 6,7-V medium (25 °C, 120 rpm) and treated with a final concentration of 200 μg/L ethephon.Then, the treated samples were harvested separately at 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 72 h, respectively.The untreated S. miltiorrhiza hairy roots grown under the same culture conditions were collected as controls.Each sample was subjected to three biological replicates, and after collection, the samples were frozen in liquid nitrogen, and then stored at −80 °C.

Cloning of SmERF1b-like gene
The sequence information of SmERF1b-like gene (MH006601.1)was obtained from our previous research (Bai et al. 2018).The amplification primers were designed with primer 6.0.The primers are listed in Supporting Information-Table S1.The cDNA of S. miltiorrhiza was used as the template.A 50 μL reaction system (25 μL 2xM5 HiPer plus Taq HiFi PCR mix, 2 μL upstream primer, 2 μL downstream primer, 2 μL cDNA template, 19 μL ddH 2 O) was used for amplification.The reaction conditions were: 95 °C, 5 min, 38 cycles (95 °C, 30 s, 55 °C, 30 s, 72 °C, 1 min), 72 °C, 10 min.The PCR product was separated by 1 % agarose gel electrophoresis, and the targeted gene fragment was purified and recovered using a gel recovery kit (TOLOBIO).The targeted gene fragment was ligated into pMD19-T simple cloning vector and then transformed into DH5α.Positive clones were screened on LB solid plates with 50 mg/L ampicillin at 37 °C for 12 h.Subsequently, positive clones were identified by PCR and then sequenced.

Genomic DNA extraction, total RNA extraction and cDNA synthesis
DNA extraction from S. miltiorrhiza was conducted using the CTAB method (Bai et al. 2018).Total RNA of S. miltiorrhiza was extracted using an RNA extraction kit (Tiangen, China).The integrity of RNA was assessed, and then the RNA was reversed transcribed into cDNA using a PrimeScript TM RT reagent kit with a gDNA Eraser kit (TaKaRa, China).

qRT-PCR (quantitative real-time PCR) analysis
The qRT-PCR primers of SmERF1b-like were designed using Primer 6.0 ( Supporting Information-Table S1).SmActin was used as an internal reference gene.The reaction program comprised denaturing at 95 °C for 30 s, followed by annealing at 95 °C for 5 s and 60 °C for 30 s with 40 cycles using the SYBR Premix ExTaq (TaKaRa) kit and a BioRad CFX Manager 3.1 real-time PCR system.

Subcellular localization analysis of SmERF1b-like
The full-length sequence of SmERF1b-like was recombined into PA70390 vector on the Nʹ terminal of the GFP-coding gene with the CaMV 35 S promoter downstream.Double restriction enzyme cutting sites XbaI and BamHI were used, and the primers are shown in Supporting Information-Table S1.The PA70390-SmERF1b-like plasmid was infected into Agrobacterium strain EHA105, and then transiently expressed into tobacco leaves by injection.The injected tobacco seedlings were cultured overnight in the dark at 25 °C.Afterwards, protoplasts of the injected leaves were isolated using 1.5 % Cellulase R10 and 0.4 % Macerozyme.GFP fluorescence in the protoplasts was detected at 600 nm by laser confocal microscopy (Nikon A1R inverted confocal microscope) (Bai et al. 2018).

EMSA verification
The induction and purification of MBP and SmERF1b-like protein were conducted as described in the previous study (Bai et al. 2018).SmERF1b-like protein expression was induced by 0.4 mM isopropyl-β-d-thiogalactopyranoside (IPTG).The designed and synthesized ERF protein recognition element DNA probe (CGCCGCC) was annealed, and after cooling at room temperature, the probe and purified SmERF1b-like protein were incubated at room temperature for 40 min using 6× loading buffer, and the colloid was placed in nucleic acid staining solution after completion Li et al. -SmERF1b-like was involved in tanshinone biosynthesis and staining for 30 min.After rinsing 2-3 times, the gel was then placed under a chemiluminescence imaging system (ChemiDoc MP, BioRad) for photography and preservation, using the experimental method previously reported (Bai et al. 2018).

Transgenic S. miltiorrhiza hairy roots analysis
The coding sequence of SmERF1b-like was digested and ligated with the PCAMBIA1304 vector.The overexpressed PCAMBIA1304-SmERF1b-like and PCAMBIA1304 plasmids were transformed into Agrobacterium rhizogenes ATCC15834.The positive clones were screened by YEB liquid plate with 50 mg/L kanamycin at 28 °C and 220 rpm in a shaking culture.PCAMBIA1304 and PCAMBIA1304-SmERF1b-like plasmids were used for plant transformation as previously described by our lab (Bai et al. 2018).DNA was extracted from transgenic hairy roots according to the instructions of the Plant Genomic DNA Extraction Kit (Tiangen, China).The transgene identification primers are shown in Supporting Information-Table S1.

Tanshinone determination
The S. miltiorrhiza hairy roots were collected and dried at 45 °C for 3 days, and then ground into a fine powder.A total of 0.04 g dry powder was added to 8 mL of 70 % methanol for incubation at room temperature for 8 h.Subsequently, the mixture was centrifuged at 8000 rpm for 10 min.The supernatant was used for the detection of tanshinone compounds by high-performance liquid chromatography (HPLC) as previously described by Bai et al. (2018).

Statistical analysis
Statistical analyses of HPLC data and qRT-PCR data were performed based on a one-way analysis of variance at P < 0.05 level using SPSS (version 13.0).All images were generated using EXCEL (version 2010) and TBtools software.

Cloning and bioinformatics analysis of SmERF1blike gene
The SmERF1b-like (MH006601.1)gene is composed of 540 base pairs, encoding a polypeptide chain consisting of 179 amino acids.The relative molecular mass of SmERF1b-like is 20.13 kDa (C867H1372N250O282S10), with an isoelectric point of 5.37.Its instability coefficient was calculated to be 65.15, while the average hydrophilic coefficient (GRAVY) value was −0.650, indicating that SmERF1b-like is a stable hydrophilic protein.Phylogenetic analysis revealed that SmERF1b-like exhibited the closest relationship with SsERF1B-like (XP042037389.1)(Fig. 1A and B).In terms of protein secondary structure, SmERF1b-like comprises 55.31 % α-helix, 3.35 % β-sheet, 29.05 % random coil and 12.29 % extended chain (Fig. 1C).The tertiary structure of SmERF1blike was predicted using SWISS-MODEL (Fig. 1D), which confirmed the presence of an AP2 domain.PSORT prediction indicated that SmERF1b-like possesses a nuclear localization signal peptide.Therefore, it can be inferred that SmERF1blike shares a similar function with SsERF1B-like.Notably, SmERF1b-like consists of four conserved sequences.

Characterization analysis of SmERF1b-like
SmERF1b-like is a member of the ERF family, which may play a role in ethylene signalling.To analyse the expression of the SmERF1b-like in different tissues of S. miltiorrhiza, its expression levels in root head, stem, leaf, flower and root were detected.The findings revealed that SmERF1b-like expression was considerably higher in leaves than in other tissues (P < 0.05) (Fig. 2A).The gene expression level of SmERF1blike was significantly increased at 1 h and 8 h exposure to ethylene treatment in S. miltiorrhiza hairy roots (Fig. 2B).
SmERF1b-like fused with GFP indicated a nuclear locational fluorescence signal.The GFP fluorescence of SmERF1b-like was also merged with DAPI, a nuclear marker, suggesting that SmERF1b-like localizes at the nucleus (Fig. 2C).Additionally, the GCC-box is a core cis-acting element involved in the recognition of ERF family genes.GCC-box sequence probes were used based on previous studies (Bai et al. 2018).The results of the EMSA showed clear protein and DNA binding bands when SmERF1b-like and the probe coexisted, demonstrating that SmERF1b-like protein can recognize the GCC-box (Fig. 2D).

SmERF1b-like regulates the accumulation of tanshinones in S. miltiorrhiza hairy roots
To further elucidate the regulatory role of SmERF1b-like in tanshinone biosynthesis, we generated and analysed transgenic hairy roots with overexpression of SmERF1b-like.Positive transgenic lines were identified by PCR amplification using NPT, rolb, rolc and the targeted gene as positive markers.The line exhibiting the highest expression level of SmERF1b-like was selected for subsequent experiments.There was a substantial increase in both the expression levels of SmERF1b-like and the accumulation of tanshinone compounds, particularly tanshinone I (T-I) and cryptotanshinone (CT), in the SmERF1b-like overexpressed lines (Fig. 3A and B).The content of tanshinone IIA in the SmERF1b-like overexpressed plants was also significantly increased (Fig. 3B).

Discussion
The AP2/ERF family genes are closely associated with the synthesis and regulation of active ingredients in medicinal plants (Liu et al. 2015).SmERF1b-like has 540 bp and encodes 179 amino acid residues.It contains α-helices, β-sheets and only one AP2 domain, exhibiting alanine (A) and aspartate (D) residues at positions 14 and 19 of the AP2 domain, respectively, which is consistent with the characteristics of the AP2/ ERF family genes (Nakano et al. 2006;Licausi et al. 2010;Ji et al. 2016;Xu et al. 2021).SiERF1b is associated with drought resistance in Sesamum indicum (Dossa et al. 2016).Since SmERF1b-like sequence is very similar to SiERF1b, SmERF1b-like may also be involved in plant stress resistance (Nakano et al. 2006;Ji et al. 2016).Tanshinone, one of the primary active ingredients in S. miltiorrhiza, is synthesized in leaves and transported in roots for storage (Ji et al. 2016).The expression levels of SmERF1b-like were significantly higher in leaves than in other organs, suggesting that SmERF1b-like may be involved in tanshinone biosynthesis in S. miltiorrhiza.TFs mainly participate in metabolic regulation in the nucleus (Ren and Kong 2017).SmERF1b-like contains a nuclear localization signal peptide, and transient expression in tobacco leaves confirmed that SmERF1b-like is located in the nucleus.Moreover, exogenous application of ethylene significantly induced SmERF1b-like expression, indicating that it is a typical ERF family gene.
ERF TFs mainly bind to the GCC-box in the promoters regulating the downstream gene expression.SmDXR, SmDXS2, SmHMGS, SmCPS1, SmKSL1 and SmCYP76AH1 are key genes involved in tanshinone synthesis, while SmERF1L1, SmERF6, SmERF8, SmERF128 and SmERF2 regulate tanshinone synthesis by binding with the GCC-box of their promoters (Bai et al. 2018(Bai et al. , 2020;;Huang et al. 2019;Sun et al. 2019;Zhang et al. 2019).In this study, we found that SmERF1b-like regulates tanshinone synthesis by binding with the GCC-box in the promoter region of SmKSL1, which is a crucial gene involved in tanshinone biosynthesis.Moreover, overexpression of SmERF1b-like significantly increased the content of tanshinone I and cryptotanshinone, implying that SmERF1b-like binds with the GCC-box in the promoter region of key genes involved in tanshinone synthesis.As a result, upregulation of SmERF1b-like increases tanshinone accumulation.

Conclusions
SmERF1b-like localizes in the nucleus and shows the highest expression levels in leaves.Exogenous application of ethylene remarkably induces the response of SmERF1b-like.Additionally, SmERF1b-like binds with the GCC-box motif in the promoter region of SmKSL1.The overexpression of SmERF1b-like substantially increases tanshinone I (T-I) and cryptotanshinone (CT) accumulation.Overall, SmERF1b-like is involved in promoting T-I and CT biosynthesis accumulation in S. miltiorrhiza by recognizing the GCC-box motif in the promoter region of SmKSL1 (Fig. 4).To gain further insights into the molecular mechanism of SmERF1b-like in tanshinone biosynthesis, comprehensive multi-omic approaches and in-depth molecular-level studies are needed.This study provides valuable theoretical guidance for enhancing the genetic resources of S. miltiorrhiza and serves as a reliable reference for resource selection and breeding strategies for this species.

Figure 2 .
Figure 2. The characteristics of SmERF1b-like.(A) The expression levels of SmERF1b-like in root, leaf, flower, stem and root head (the joint between the stem and root).Data are shown as cartoon heatmap.(B) The gene expression levels of SmERF1b-like under ethylene treatment along with the time course.Eth indicates ethephon.Data are shown as the mean ± SD (n = 3), and the asterisk indicates the significance (P < 0.05) according to one-way ANOVA.(C) Subcellular localization of PA70390-SmERF1b-like. Merge: the overlap of DAPI, GFP, chloroplast and DT.DAPI, the nuclear marker; GFP, green fluorescent protein; chloroplast, chloroplast autofluorescence; bright, light field observations.(D) EMSA analysis: lane 1 has only the GCC probe (+) and no proteins (−).Lane 2 has only the GCC probe (+) and no proteins (−).

Figure 3 .
Figure 3. Overexpression of SmERF1b-like in S. miltiorrhiza hairy root.WT: ATCC-induced S. miltiorrhiza hairy root.Control: the pCAMBIA1304 empty vector counterparts.OESmERF1b-like represents the positive overexpressed SmERF1b-like hairy root lines.A, the gene expression levels of SmERF1blike, SmCPS1 and SmKSL1 in WT, control and transgenic hairy root lines, respectively.(B) The tanshinone contents in WT, control and transgenic hairy root lines.DT-I, dihydrotanshinone; T-I, tanshinone I; T-IIA, tanshinone IIA; CT, cryptotanshinone.

Figure 4 .
Figure 4.The role of SmERF1b-like in the regulation of tanshinone biosynthesis.