Ag+ ions are effective elicitors for enhancing the production of phenolic acids and tanshinones in Salvia aristata Aucher ex Benth. hairy roots

doi:10.21203/rs.3.rs-4303897/v1

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Ag+ ions are effective elicitors for enhancing the production of phenolic acids and tanshinones in Salvia aristata Aucher ex Benth. hairy roots

https://doi.org/10.21203/rs.3.rs-4303897/v1

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published 17 Aug, 2024

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Salvia aristata Aucher ex Benth., due to containing tanshinones and phenolic acids, two main groups of bioactive metabolites, is one of the most valuable medicinal plant species in Iran. In this study, for the first time, hairy root (HR) cultures were established from 14-day-old seedlings inoculated with Agrobacterium rhizogenes strain ATCC 15834. Additionally, the effects of elicitation with Ag⁺ ions (15 and 25 µM) were investigated on the growth indices and content of phenolic acids and tanshinones of HRs in a time-course experiment. The findings of this work showed that exposure of HRs to Ag⁺ at both concentrations caused significant increases in the levels of rosmarinic acid (1.34- to 1.43-fold of control) and salvianolic acid B (1.71- to 1.82-fold of control). Specifically, 7.25-, 7.78-, 6.47-, and 3.9-fold increases were attained in total tanshinone, tanshinone I, tanshinone II-A, and cryptotanshinone contents of HRs, respectively, after seven days of exposure to 25 µM Ag⁺ ions compared to the control groups. The analysis revealed that exposure to Ag⁺ ions significantly enhanced the secretion of tanshinones, notably tanshinone II-A (31.49 ± 0.65 µg mL^− 1) by HRs into the culture medium. The changes in transcript levels of crucial genes in the biosynthesis pathways of phenolic acids (PAL, TAT, and RAS) and tanshinones (CPS and CYP76AH1) were associated with their contents in HRs under elicitation with Ag⁺ ions. Our findings confirmed the effectiveness of an elicitation strategy to improve metabolite production in HR cultures of S. aristata as potent natural sources of phenolic acids and tanshinones.

Salvia aristata Aucher ex Benth.

Hairy root culture

Ag+ ions

Phenolic acids

Tanshinones

Gene expression

Salvia aristata Aucher ex Benth. (Lamiaceae family) is an Irano-Turanian endemic medicinal plant species whose distribution is limited to the north, northwest, and central regions of Iran (Moein et al., 2019b). Recently several reports have mentioned the presence of this species in the east of Turkey (Behçet and Avlamaz 2009). There are only a few scientific reports about S. aristata; in some of them, the antioxidant and pharmacological effects are attributed to the content of compounds such as triterpenic acids (Abdollahi-Ghehi et al. 2019), essential oil compounds (Farshid et al. 2015; Emadipoor et al. 2016), phenolics, and flavonoids (Emadipoor et al. 2016). Phenolic acids, predominantly rosmarinic acid (RA), salvianolic acid A (Sal-A), salvianolic acid B (Sal-B), caffeic acid (CA), vanillic acid (VA), and chlorogenic acid (CGA) (Shi et al. 2019; Wang et al. 2019), along with tanshinones including tanshinone I )T-I(, tanshinone IIA (T-IIA), cryptotanshinone (CT), and dihydrotanshinone I (DT-I) are the most valuable metabolites in Salvia L. species (Bisio et al. 2019; Wang and Peters 2022). Phenolic acids are used for the treatment of atherogenic dyslipidemia, and cholestatic liver damage, and have antioxidant and antidepressant effects (Tan et al. 2016; Ingole et al. 2021; Xie et al. 2021; He et al. 2023; Caylak 2024). Tanshinones belong to a natural group of abietane-type norditerpenoid quinones that manifest remarkable pharmacological properties such as anti-cancer, antibacterial, antiviral, and neuroprotective effects (Fu et al. 2020; Wang et al. 2020; Lai et al. 2021; Sudha and Singh 2024).

The biosynthetic pathways of phenolic acids have been identified in Salvia miltiorrhiza Bunge (Shi et al. 2019). Phenolic acids, especially RA, are generally produced from two pathways: the phenylalanine and tyrosine-derived pathways. The first-point reactions in these pathways are catalyzed by phenylalanine ammonia-lyase (PAL)

and tyrosine aminotransferase (TAT), respectively.

The biosynthetic pathway of tanshinones proceeds in three steps: formation of terpenoid precursors, the creation of tanshinone skeleton, and subsequent skeletal modification such as oxidation, methylation, decarboxylation, or cyclization which produce diverse tanshinones. Recent studies have demonstrated that the S. miltiorrhiza HR cultures accumulate tanshinones in a manner that is consistent with the transcript levels of certain key enzymes. These enzymes include 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), which are situated upstream of the methylerythritol 4-phosphate (MEP) pathway, as well as geranylgeranyl diphosphate (GGPP), copalyl diphosphate synthetase (CPS), kaurene synthase-like (KSL(, and cytochrome P450 monooxygenases) CYP76AH1, CYP76AH3, and CYP76AK1(, which are located downstream of the MEP pathway (Jiang et al. 2019; Hu et al. 2021; Wang and Peters 2022).

Elicitation is a suitable method to increase the production of valuable compounds from medicinal plants, which can influence the gene expression of regulatory enzymes in the biosynthetic pathways and the accumulation of secondary metabolites (Bhaskar et al. 2022; Grzegorczyk-Karolak et al. 2024). Heavy metals are effective stimuli in inducing secondary metabolite production. Among the metal ions, Ag⁺ ions have been widely used to induce the production of secondary compounds in plants. Recent studies have demonstrated the effectiveness of this elicitor in increasing the content of tanshinones and phenolic acids (Xing et al. 2014; Dowom et al. 2017; Bayesteh et al. 2021; Pesaraklu et al. 2021; Attaran Dowom et al. 2022).

Salvia aristata is considered a rare, vulnerable, or endangered species due to its limited occupation level and the small population in Iran (Moein et al. 2019a). Consequently, it is necessary to employ biotechnological methods to exploit and protect the genetic preservation of this medicinal species. Furthermore, because phenolic acids and terpenoids exist in the shoots and roots of this species, HR culture could serve as a potent tool for in vitro production of these metabolites (Gantait and Mukherjee 2021; Roy 2021; Atabaki et al. 2024).

In this study, HR cultures of S. aristata were established for the first time. Moreover, the accumulation of phenolic acids and tanshinones, as well as the gene expression of the crucial enzymes involved in their biosynthesis pathways, were investigated in the HRs under elicitation with Ag⁺ ions.

Explants preparing using the seed culture method

The mature seeds of S. aristata Aucher ex Benth. were collected in September 2018 from a naturally occurring population in Gardane Kaman, Qazvain province, located in the northwest of Iran, situated at latitude 36° 27' 49'' N, longitude 50° 07' 59'' E, with an altitude of 2034 m above sea level. A voucher specimen was deposited at the herbarium of Bu-Ali Sina University of Hamedan (BASU 34046).

The collected seeds were soaked in sterile water for 10 min, followed by sterilization with 70% ethanol (v/v) for 1 min and sodium hypochlorite solution (10%) for 10 min. Since the mucilaginous coat on S. aristata seeds reduces their germination rate, the seed coats were cut and removed using a sterilized scalpel. After the removal of the seed coats, they were placed on solid MS media (Classic Murashige and Skoog 1962), solidified with 7 g^-1 L agar, and incubated at 25 ± 1°C under a light/dark cycle of 8/16 h for a period of 14 days. The 14-day-old seedlings were selected for inoculation with the bacterial strain.

Preparation of bacterial strain

The A. rhizogenes ATCC 15834 strain (American-type culture collection) was obtained from the microbial collection of the Biotechnology Research Center, Karaj, Iran. Initially, bacterial cells were cultured on Luria-Bertani medium (LB) agar medium supplemented with 50 mg^− 1 L (Sezonov et al. 2007). The cultures were then transferred to a rotary shaker and incubated at 28°C with a speed of 180 rpm for 48 h under dark conditions.

Hairy root culture

The nodal explants obtained from the 14-day-old seedlings were inoculated with a needle previously immersed in an A. rhizogenes bacterial suspension. Subsequently, the explants were transferred to MS medium supplemented with 6 g^− 1 L agar and maintained in darkness at 25°C. Control explants were inoculated using a sterile needle dipped in sterile water. After an incubation period of 48 h, the explants were rinsed with a cefotaxime solution at a concentration of 500 mg^− 1 L and then placed on an MS culture medium containing the same antibiotic concentration along with 150 µM acetosyringone. The cultures were incubated under a 16/8 hour (light/dark) photoperiod (45 µmol m^− 2 s^− 1, provided by cool white fluorescent lamps), and incubated at 25°C. The experiment was repeated three times with five explants. Twelve days after infection with A. rhizogenes, HRs began to appear on the explants, and ten days after their emergence, six fast-growing lines were selected and transferred to MS media, which solidified with 6 g^− 1 L agar and maintained at 25°C in dark condition. One week later, the selected HR lines were moved to 150 mL Erlenmeyer flasks containing 50 mL of hormone-free liquid MS medium and incubated at 25°C in the dark on a rotary shaker at 120 rpm. The HRs were subcultured into the same fresh medium every four weeks. After two cycles of subculturing, (approximately after two months of culture), the dry weight (DW) and tanshinone content of the HR lines were determined. Due to the significant levels of tanshinones along with high biomass, HR line 2 was selected for elicitor treatments.

Confirmation of transgenic hairy root lines

Genomic DNA (gDNA) from six HR lines and non-transformed roots of S. aristata was extracted from 100 mg plant tissue using the CTAB method (Khan et al. 2007). In this work, DNA from the ATCC 15834 strain of A. rhizogenes and the extracted DNA from the 50-day-old non-infected roots served as the positive and negative controls, respectively. The polymerase chain reaction (PCR) was performed to confirm the transformation of HR lines using the C1000 Touch TM 96-Well Thermal Cycler (Bio-Rad, USA), with gene-specific primers designed to detect an internal rolC gene fragment (612 bp) were F: 5' -CTCCTGACATCAAACTCGTC-3' and R:5´ -TGCTTCGAGTTATGGGTACA-3'. The PCR reaction contained Taq PCR Master Mix Kit (Qiagen), gDNA (100 ng total DNA), and oligonucleotide primers (10 µM final concentration in the total volume of 15 µL). The total volume of the reaction was achieved by mixing 0.5 µL DNA Template, 0.5 µL Premix, 5 µL 2X Blue Load Master Mix, and 4 µL of dH₂O. The PCR procedure was performed with an initial pre-denaturation step at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 40 s, and extension at 72°C for 40 s, with a final extention step was then carried out at 72°C for 7 min. For analysis, 5 µL of the PCR products were run on a 1% (w/v) agarose gel, stained with GelRed, and visualized under UV light by a Gel Doc System (Farjin Poish-Doc, Iran).

Elicitation of hairy roots

In this study, Ag⁺ ions were utilized to elicit HR cultures of line 2. A stock solution of Ag⁺ ions was prepared by dissolving AgNO₃ (Sigma-Aldrich) in deionized water. The solution was then added to a liquid hormone-free basal MS medium to achieve specified concentrations (15 and 25 µM) for 50-day-old HR cultures. Different concentrations of AgNO₃ solutions were added to the freshly prepared culture media after sterilization through 0.2 µm pore-size filters. HR samples were collected from the culture media on days 0, 1, 3, 5, and 7 following elicitation with Ag⁺ ions. To determine the optimal time for elicitor treatments, the growth curve of HR line 2 was plotted. Accordingly, root tips (0.6 g) were extracted from one-month-old HRs and transferred to 150 mL flasks containing 50 mL of liquid MS medium. The cultures were shaken (100 rpm) at 25°C under continuous darkness. Fresh weight (FW) of HR samples was measured at 4-day intervals for 70 days after cultivation, and a growth curve was constructed based on the obtained values. Treatments of the HRs with the elicitor were carried out at the end of the exponential phase, on day 50 of the HR growth curve. The same volume of water, as the solvent of AgNO3, was added to 50 mL of liquid MS medium for the control group.

Measurement of total phenolic content

The total phenolic content (TPC) of the elicited HRs was determined using the Folin-Ciocalteu method (Singleton et al. 1999). Initially, 150 µL of each HR extract was mixed with the Folin-Cicalteu reagent, and after 5 min, 1500 µL of 7% sodium carbonate solution was added to the mixture. The total volume of the solution was then adjusted to 3000 µL with distilled water. After 90 min of incubation, the absorbance was measured at 750 nm using a spectrophotometer. A standard curve was plotted with different concentrations of gallic acid (GAE), and the results were expressed as mg gallic acid per gram DW (GAE g^− 1 DW).

Extraction and HPLC analysis of phenolic acids and tanshinones

Initially, 100 mg of dried HR powder was extracted after soaking in 1 mL of methanol for 24 h. Then, the extracts were centrifugated at 10,000 rpm for 10 min, and the supernatant was filtered through a 0.45 µm Millipore filter. Phenolic acids and tanshinones present in the extracts were quantified using a Smartline HPLC system (Kenuer, Germany) equipped with a quadruple pump and a C18 Eurospher-100 reversed-phase column (5 µm particle, 250 mm × 4.6 mm). For the detection and quantification of phenolic acids, the solvent system consisted of 0.1% (v/v) phosphoric acid in water (solvent A) and acetonitrile (solvent B). The elution gradient program was set as follows: 90 − 10% A (v/v) in 0–15 min, 75 − 25% A (v/v) in 15–40 min, 20–80% A (v/v) in 40–45 min, 0-100% A (v/v) in 45–50 min, 0-100% A (v/v) in 50–55 min, 10–90% A (v/v) in 55–60 min. Tanshinones in the extracts were identified using a specific mobile phase composed of 0.02% (v/v) phosphoric acid in water (solvent C) and acetonitrile (solvent D) and a gradient washing program with the following parameters: 39–61% A (v/v) in 0–5 minutes, 90 − 10% A (v/v) 22 − 5 in min, 61 − 39% A (v/v) in 22–30 min, 61 − 39% A (v/v) in 30–35 min. The detection of phenolic acids and tanshinones was performed at 280 and 270 nm, respectively. The flow rate through the column was maintained at 1 mL min^− 1. Calibration curves, generated using standard solutions of phenolic acids (y_CA=222904x-302560, r² = 0.9957; y_RA= 41606x, r² = 0.988; y_Sal−B=2055x, r² = 0.997; y_Sal−A = 19497x, r² = 0.996; y_VA = 40885x, r² = 0.903; and tanshinones (y_T−I = 22523x, r² = 0.996; y_T−IIA =2055x, r² = 0.995; y_CT = 109103x, r² = 0.993) were utilized to estimate the contents of phenolic acids and tanshinones present in the HRs extracts. All analyses were conducted three times, and the contents of the examined compounds were expressed as mg g^− 1 DW.

RNA extraction, cDNA synthesis, and gene expression analysis

Total RNA was extracted from frozen HR tissues in liquid nitrogen using an RNX-Plus kit (RN7713C, CinnaGen, Iran), following the manufacturer's protocol with slight modifications. Before using in cDNA synthesis, the integrity of RNA molecules was assessed through 1% agarose gel electrophoresis. The purity and quantity of the RNA were determined using a SPECTROstar Nano microplate reader (BMG LABTECH, Germany). For each RNA sample, 1 µg of total RNA with a final concentration of 10 U µL^− 1 was prepared.

cDNA was synthesized from the prepared RNA samples using a 2-step Revert Aid Reverse Transcriptase kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Specific primers for the studied genes (PAL, TAT, RAS, CPS, and CYP76AH1) were designed based on the conserved regions within the Lamiaceae family sequences, using Gene Runner software (Table 1). These primers then were analyzed using BLAST software on NCBI.

Table 1

Nucleotide sequences of the designed primers for the examined genes
Gene	Forward primer (5ʹ→ 3ʹ)	Reverse primer (5ʹ→ 3ʹ)
5.8S rRNA	GATATCTCGGCTCTCGCATC	GGCTTCGGGCGCAACTTG
TAT	ATGGGAGGTTGATCTCGATG	TCTTTAGGTGCTGATATGAGTAGA
RAS	ATCGCCACGTGCGGCGA	TCAAACGGCGCCGCCCA
PAL	ATGTGCAGAGCGCGGAG	AGTAGGTTGAAGACATGAGTTTAA
CYP76AH1	CTGTCGGAGCATTTCTGGAC	ATGCGCAAGATATGCAAGGAG
CPS	GGAGCACATGACTTGTGG	GGAGGGAGACGTGAGGAA

To quantify gene expression levels, qRT-PCR assays were performed using a Corbett Rotor-Gene 6000 Real-Time PCR system (Qiagen, USA) with SYBR®Green Master Mix 2 × (Ampliqon, Denmark), following the manufacturer’s protocol. 5.8S ribosomal rRNA was used as the reference gene in the PCR analyses. The amplification reactions (10 µL final volume) consisted of 5 µL of 2× SYBR Green Real-Time PCR Master Mix (Parstous, Iran), 0.5 µL each of forward and reverse primers, 0.5 µL of cDNA, and 4 µL of nuclease-free PCR-grade water. The amplification program was as follows: one cycle of initial denaturation at 94°C for 10 min, followed by 30 cycles of 15 s at 95°C, 30 s at 60°C, and 40 s at 72°C, and finally, one cycle of 72°C for 5 min. The relative transcript levels of the examined genes were calculated using the comparative cycle threshold (Ct) method, employing the 2^−ΔΔCT formula.

Statistical analysis

Experiments were conducted using a randomized factorial design, and analyses were repeated three times per treatment. Data were analyzed using SPSS software, version 24.0. A one-way analysis of variance (ANOVA) was performed to identify any statistical significance between the treatment groups. The Duncan test was applied to compare the differences between mean values, with statistical significance set at P < 0.05. All data were presented as mean ± standard deviation (SD).

Induction of hairy roots and confirmation of transgenic genome

In this study, 14-day-old sterile seedlings obtained from seed culture (Fig. 1) were inoculated with five A. rhizogenes strains (ATCC 15834, A4, R1000, C58C1, and GM1534). The explants inoculated with the bacterial strain ATCC 15834 showed only HR formation after 12 days at the node wound site (Fig. 2). The formation of HR was not observed on the explants inoculated with other bacterial strains or in the control group. A 47% infection frequency was achieved for the explants infected with the ATCC 15834 strain. Among the HR lines induced by the ATCC 15834 strain, six lines that continued to grow were confirmed for the presence of the rolC gene in their genomes (Fig. 3). Hairy root line 2, with the highest tanshinone content and biomass, was selected for further experiments with Ag⁺ ions. Furthermore, a growth curve was plotted to find the optimum timing for elicitor treatment (Fig. 4).

Effects of Ag ⁺ ions on the growth of S. aristata hairy roots

Among the lines induced by the ATCC 15843 strain, S. aristata HR line 2, distinguished by the highest biomass and tanshinone content, was chosen for elicitor treatment. Our findings revealed that exposing HR line 2 to Ag⁺ ions on the fifth day of growth enhanced its biomass in shake-flask cultures in a concentration- and time-dependent manner. Exposure to 25 µM Ag⁺ ions led to significant increases in the FW of HRs on all days post-exposure, compared to groups treated with 15 µM elicitor and the control. However, enhancements in the DW of HR cultures were observed only after 5 and 7 days of Ag⁺ ions exposure at both concentrations compared to the control. Nevertheless, the rise in the DW of elicited HRs with 25 µM Ag⁺ ions was significant only after 7 days of exposure compared to HRs exposed to a 15 µM concentration of the elicitor. Seven days post-elicitation with 25 µM Ag⁺ ions, both DW (1.86 ± 0.01 g) and FW (7.74 ± 0.00 g) of HR cultures reached maximum values, which were 1.30- and 1.66–fold higher than those of the untreated cultures, respectively (Fig. 5).

Effects of Ag ⁺ ions on the total phenolic content in S. aristata hairy roots

The TPC of HR line 2 cultures were measured at intervals of 0, 1, 3, 5, and 7 days following elicitation with Ag⁺ ions. As illustrated in Table 2, HRs treated with 25 µM Ag⁺ ions exhibited elevated levels of TPC on days 1, 3, 5, and 7 days after treatment compared to their corresponding control groups at the same exposure times; however, the increase in TPC was significant only on the fifth day after treatment. Conversely, no significant increases were found in TPC of elicited HRs with 15 µM Ag⁺ ions at most times of the experiment, except on the initial day of exposure, compared to controls. The highest TPC (49.09 ± 0.31 mg GAE g^− 1 DW, 1.60-fold of the control group) was attained in HR cultures treated with 25 µM Ag⁺ ions for 5 days. Subsequently, the TPC of HRs dropped 7 days after treatment with both concentrations of Ag⁺ ions. The observed decrease was statistically significant only in HRs exposed to 15 µM of the elicitor compared to the control.

Table 2

Effect of elicitation with two concentrations of Ag⁺ ions on total phenol content of *S. aristata* hairy roots
Ag⁺ ions (µM)	Time after treatment (day)	Total phenol content (mg GAE g^− 1 DW)
Control	0	21.31 ± 1.15^de
Control	1	22.41 ± 3.44^ef
Control	3	30.77 ± 0.01^b − e
Control	5	30.66 ± 0.33^b − e
Control	7	22.57 ± 1.39^d − f
Ag⁺ (15)	0	20.63 ± 1.47^f
Ag⁺ (15)	1	31.49 ± 5.77^b − d
Ag⁺ (15)	3	31.74 ± 0.25^bc
Ag⁺ (15)	5	35.90 ± 0.00^b
Ag⁺ (15)	7	18.31 ± 5.66^f
Ag⁺ (25)	0	25.74 ± 0.49^c − f
Ag⁺ (25)	1	25.54 ± 0.08^c − f
Ag⁺ (25)	3	37.10 ± 0.03^b
Ag⁺ (25)	5	49.09 ± 0.31^a
Ag⁺ (25)	7	25.78 ± 4.61^c − f
The values represent the mean ± SD, which are labeled following Duncan’s Multiple Range Test. Means signed with the same letter in each column are not significantly different (P ≤ 0.05)

Effects of Ag ⁺ ions on the content of phenolic acids and tanshinones in S. aristata hairy roots

In this study, methanolic extracts from both elicited and non-elicited HRs of line 2 with Ag⁺ ions were quantitatively analyzed for their phenolic acid and tanshinone content using the HPLC method. In the chromatograms of the elicited HR extracts, four distinct phenolic acids (RA, Sal-A, Sal-B, and VA) and three tanshinones (T-I, T-IIA, and CT), were identified and their contents were subsequently quantified. The HPLC chromatograms of phenolic acids and tanshinones from the HRs elicited with 15 µM Ag⁺ ions for 5 days, are shown alongside their corresponding standards in Fig. 6.

The variable capacities of elicited S. aristata HR cultures to accumulate phenolic acids are depicted in Fig. 7. The presence of Ag⁺ ions in the culture media at both concentrations effectively increased the accumulation of CA in HRs, depending on the duration of exposure. Consequently, 1.26- and 1.37–fold increases were recorded in the content of phenolic acids after 3 and 5 days of exposure to 25 and 15 µM elicitors, respectively. Exposure to both concentrations of Ag⁺ ions significantly enhanced the RA content in HRs on days 1, 5, and 7 post-treatment. HRs treated with 15 and 25 µM Ag⁺ ions accumulated up to 0.66 ± 0.06 and 0.62 ± 0.04 mg g^− 1 DW of RA, respectively, 7 days after treatment, representing nearly 1.43- and 1.34-fold increases compared to the control groups at the same exposure time, respectively. However, the stimulatory effect of Ag⁺ ions on Sal-B accumulation in HRs was significant only one day after exposure. The highest contents of Sal-B were recorded at 0.51 ± 0.07 and 0.48 ± 0.06 mg g^− 1 DW when HRs were exposed to 15 and 25 µM Ag⁺ ions for one day, respectively. These values were 1.82- and 1.71-fold higher than those in the control groups at the same exposure time. Data analysis revealed that Ag⁺ ions had no stimulatory effects on the accumulation of Sal-A and VA in HRs; thus, treatment with both concentrations of the elicitor slightly, but not significantly, reduced the content of Sal-A and VA in the HRs at most exposure times compared to controls. Based on the results of this research, chlorogenic acid was not detected in the HR extracts.

Based on the obtained results, exposure to Ag⁺ ions at both concentrations efficiently stimulated the accumulation of tanshinones in the treated HRs. Consequently, the levels of total tanshinones were consistently higher than those in the control groups across all harvesting times (Fig. 8). After seven days of exposure to the elicitor, Ag⁺ ions at a concentration of 25 µM exhibited the most significant stimulatory effect on the accumulation of all tested tanshinones. In comparison with the controls, there was a remarkable increase of 7.78-, 6.47-, and 3.9-fold in the content of T-I, T-IIA, and CT of HRs, respectively. Additionally, under this treatment condition, the total amount of tanshinones (1360 ± 0.01 µg g^− 1 DW) in the elicited leaves was enhanced by 7.25-fold compared to the control group. Additionally, under this treatment condition, the total amount of tanshinones (1360 ± 0.01 µg g^− 1 DW) in the elicited HRs was enhanced by 7.25-fold compared to the control group. Among the three tanshinones, only the content of T- IIA showed a significant increase in the HRs treated with 15 µM Ag⁺ ions, reaching nearly 3.16- to 6.8-fold of the control group during the one to 5 days after elicitation.

Effect of Ag ⁺ ions on the metabolites released by S. aristata hairy roots into the culture medium

The metabolite contents in methanolic extract derived from ethyl acetate extraction of the culture media were quantified using the HPLC method. The analyses indicated that the quantity of phenolic acids released into the culture media was not significant compared to the content of those produced in the elicited HRs (data not provided). In contrast, the findings demonstrated that exposure to Ag⁺ ions significantly enhanced the production of tanshinones in the HRs, followed by their release into the culture medium. As detailed in Table 3, after a 5-day treatment with 25 µM of the elicitor, the HRs secreted the highest levels of CT (1.87 ± 0.06 µg mL^− 1), T-I (30.08 ± 3.27 µg mL^− 1), and T-IIA (31.49 ± 0.65 µg mL^− 1) into the culture medium. These levels were approximately 23.37-, 32.69-, and 15.58-fold higher, respectively, than those levels released by the control group during the same period of exposure.

Table 3

Effect of Ag⁺ ions on the content of tanshinones released by *S. aristata* hairy roots into the culture medium
Elicitor (µM)	Exposure time (day)	Cryptotanshinone (µg mL^− 1)	Tanshinone I (µg mL^− 1)	Tanshinone IIA (µg mL^− 1)	Total Tanshinone (µg mL¹)
Control	0	0.06 ± 0.00^d	8.24 ± 0.25^b	10.57 ± 0.00^c	18.88 ± 0.25^cd
Control	1	0.11 ± 0.00^bc	1.35 ± 0.00^c	2.60 ± 0.00^gh	4.07 ± 0.00^h
Control	3	013 ± 0.00^bc	1.26 ± 0.00^c	7.78 ± 0.00^d	9.18 ± 0.00^e − h
Control	5	0.08 ± 0.00^bc	0.92 ± 0.02^c	2.02 ± 0.00^h	3.03 ± 0.02^h
Control	7	0.10 ± 1.03^bc	7.97 ± 0.18^b	1.99 ± 0.04^h	10.07 ± 0.20^e − h
Ag⁺ 15	0	0.14 ± 0.00^bc	8.14 ± 0.00^b	11.04 ± 0.00^c	19.33 ± 0.00^cd
Ag⁺ 15	1	0.09 ± 0.00^bc	1.18 ± 0.91^c	3.67 ± 0.02^fg	4.96 ± 0.94^h
Ag⁺ 15	3	0.09 ± 0.01^bc	1.74 ± 0.15^c	5.39 ± 0.25^ef	7.23 ± 0.42^gh
Ag⁺ 15	5	0.13 ± 0.02^bc	2.07 ± 0.21^c	4.70 ± 0.23^e − g	6.91 ± 0.04^gh
Ag⁺ 15	7	0.45 ± 0.12^b	5.99 ± 1.25^cd	6.00 ± 0.00^de	12.00 ± 1.12^d − f
Ag⁺ 25	0	0.12 ± 0.00^bc	4.14 ± 0.00^bc	10.21 ± 0.00^c	14.48 ± 0.01^c − e
Ag⁺ 25	1	0.09 ± 0.01^bc	0.91 ± 0.53^c	3.10 ± 0.00^f − h	4.11 ± 0.53^h
Ag⁺ 25	3	0.16 ± 0.01^c	4.67 ± 0.06^bc	16.00 ± 0.23^b	20.84 ± 0.18^c
Ag⁺ 25	5	1.87 ± 0.063^a	30.08 ± 3.27^a	31.49 ± 0.65^a	63.45 ± 3.99^a
Ag⁺ 25	7	0.12 ± 0.02^bc	29.02 ± 7.63^a	14.64 ± 3.44^b	43.79 ± 11.10^b
The values represent the average of three replicates ± SD. According to Duncan’s Multiple Range Test, means labeled with the same letter within a column are not significantly different (P ≤ 0.05)

Effects of Ag ⁺ ions on the gene expression of enzymes involved in metabolites biosynthetic pathways in S. aristata hairy roots

We evaluated the gene expression levels of three pivotal enzymes (PAL, TAT, and RAS) in the phenolic acid biosynthesis pathways in S. aristata HRs elicited with Ag⁺ ions (Fig. 9), and statistical analysis underlined substantial changes in the transcript levels of the assessed genes following exposure to both elicitor concentrations. Our results demonstrated significant increases in the relative expression levels of PAL in HRs exposed to 15 µM Ag⁺ ions for 1, 3, and 7 days. The maximum expression level of PAL attained 35.01-fold higher than the control one day after elicitation with 15 µM Ag⁺ ions. The highest expression level of TAT, nearly 11.08-fold of the control group, was detected in HRs after three days of treatment with the same concentration of the elicitor. Compared to PAL and TAT, the gene expression levels of RAS in HRs were affected by both elicitor concentrations. In particular, the transcript levels of RAS increased in the initial days (0, 1, and 3) after treatment with 25 µM of Ag⁺ ions, attaining its maximum level (3.23-fold higher than the control) on the third day after elicitation. Moreover, the stimulatory effect of 15 µM Ag⁺ ions on the induction of RAS persisted up to 7 days after exposure in the HRs. Consequently, the highest increase in the gene expression level (2.77-fold compared to the untreated group at the same exposure time) was observed seven days after treatment of HRs with the elicitor.

In this assay, the relative expression levels of two vital genes, CPS and CYP76AH1, involved in the biosynthesis pathways of tanshinones, were estimated in Ag⁺ ions-elicited S. aristata HRs using RT-qPCR (Fig. 10). The results indicate that both concentrations of Ag⁺ ions induced significant increases in the expression level of gene CPS for up to three days post-treatment, compared with the control at similar exposure times. The maximum relative gene expression level was estimated to be 15.59-fold higher than that of the control for CPS in HRs treated for three days with a 25 µM elicitor. Similarly, treatment of HRs with 15 µM of the elicitor for only one day efficiently enhanced transcript levels of CYP76AH1 up to 38.26-fold compared to the control at the same exposure time. It was notable that prolonging the treatment time over one and three days led to statistical decreases in the transcript levels of CYP76AH1 and CPS, respectively, in HRs after exposure to both concentrations of Ag⁺ ions.

Hairy root establishment

The utilization of fast-growing HRs regenerated from A. rhizogenes transformation represents an efficient in vitro culture system to produce significant quantities of plant bioactive metabolites. In our current study, we conducted an original experiment to establish HRs in S. aristata. Additionally, we investigated the effects of Ag⁺ ions on HR growth and the accumulation of phenolic acids and tanshinones, as well as the expression patterns of some principal genes in related biosynthetic pathways. Our findings approve the successful establishment of S. aristata HRs through

the A. rhizogenes ATCC 15834 strain-mediated transformation. PCR amplification of the rolC gene fragment confirmed the transformation of HRs by the used strain. In previous research (Yang et al. 2018; Attaran Dowom et al. 2022; Wojciechowska et al. 2020; Li et al. 2016), leaves and shoot tips of Salvia species have been the primary explants for transformation. However, our results indicate that wounding at the node sites before infection was a more efficient approach for HR generation (achieving a 47% infection frequency) in S. aristata. Li et al. (2016) documented that infection of leaf explants of Salvia castanea Diels f. tomentosa Stib., a medicinal plant endemic to China with A. rhizogenes ATCC 15834 strain, led to HR regeneration with frequencies ranging from 3.92 to 8%. Additionally, Khoshsokhan et al. (2022) induced HRs in Salvia nemorosa L. using A. rhizogenes ATCC 15834, A4, R1000, and GM1534 strains. Their results indicated that the maximum transformation efficiency (76%) was related to the ATCC 15834 strain. In a study conducted by Attaran Dowom et al. (2022) on Salvia virgata Jacq., the highest transformation frequency (56%) for HR induction was obtained by infection of leaf explants with A. rhizogenes ATCC 15834 strain.

Among the developed HR lines, the AT3, generated by the ATCC 15834 strain, was notable for producing the highest biomass (2.29 ± 0.04 g) and RA content (5.47 ± 0.16 mg g^− 1 DW) in this species. Our findings are consistent with previously mentioned studies demonstrating successful HR induction using the A. rhizogenes ATCC 15834 strain in various plant species. However, the effectiveness of A. rhizogenes strains may vary depending on the specific plant species (Srivastava et al. 2019).

Phenolic acids production and gene expression patterns after elicitation

Elicitation, as a technique, has been widely adopted to improve the content of bioactive compounds in plants. However, as highlighted by Halder et al. (2019) and Guru et al. (2022), the effects of elicitation are multifaceted and influenced by factors such as the type and concentration of elicitor, harvesting time, and the plant species involved. Salvia species are well known for their rich repertoire of phenolic compounds, including RA and salvianolic acids, and their pharmacological significance provides insights into elicitation strategies for enhancing their synthesis (Krzemińska et al. 2022). Furthermore, as noted in existing literature, Ag⁺ ions have emerged as potent abiotic elicitors, especially in augmenting secondary metabolite production across diverse plant species (Halder et al. 2019).

In the current study, we observed a concentration-dependent stimulatory impact of Ag⁺ ions on the growth of S. aristata HRs and significant increases in their biomass after exposure to a 25 µM concentration. Several studies, including those by Xing et al. (2014) and Li et al. (2016), have illustrated the stimulatory effects of Ag⁺ ions on biomass and secondary metabolite production and associated gene expression in S. miltiorrhiza. Xing et al. (2014) reported that DW/FW significantly increased on days 1, 2, 6, and 9 after stimulation with Ag⁺ in HRs of S. miltiorrhiza. Consistent with these findings, our study demonstrated the effective influence of Ag⁺ ions at a 15 µM concentration in enhancing the synthesis of phenolic acids and tanshinones in S. aristata HRs. As a result, treating HRs with 15 µM Ag⁺ ions led to significant increases in the content of RA and Sal-B by 1.43 and 1.28 times, respectively. However, the elicitor had no significant effect on the contents of VA and Sal-A. An increase in the production of phenolic acids in Salvia species is correlated with the overexpression of regulatory genes, specifically PAL, TAT, and RAS, in the phenylpropanoid pathway following exposure to the elicitors (Xing et al. 2014; Dowom et al. 2017). In their study on two Iranian Salvia species, Salvia officinalis L. and Salvia verticillata L., Pesaraklu et al. (2021) showed that elicitation of both species with methyl jasmonate and Ag⁺ ions led to significantly higher expression of crucial genes (such as PAL, TAT, HPPR (4-hydroxyphenylpyruvate reductase), RAS, and CYP98A14) involved in both the phenylpropanoid and tyrosine pathways, which are responsible for the production of phenolic acids (RA, CA, Sal-A and Sal-B). However, the study found that Ag⁺ ions were more effective in enhancing the production of phenolic acids.

The results of Xing et al. (2014) on phenolic acid production in HRs of S. miltiorrhiza showed that elicitation with Ag⁺ ions (15 µM) significantly enhanced the expression levels of genes in the biosynthesis pathway of RA. Among these genes, the upstream genes (PAL, C4H (cinnamate 4-hydroxylase), 4CL (hydroxycinnamate coenzyme A ligase), TAT, and HPPR) were notably upregulated during the earlier stages of exposure, while the downstream genes (RAS, and CYP98A14) were more sensitive to Ag⁺ ions at later stages. According to the findings of Xing et al. (2018), the accumulation of phenolic acids in HR cultures of S. miltiorrhiza following treatment with methyl jasmonate was significantly correlated with increases in transcript levels of PAL, 4CL, C4H, TAT, HPPR, and RAS in a time-course manner. Kwon et al. (2021) reported a 5-fold increase in the accumulation of RA in HRs of green basil (Ocimum basilicum L.) compared to natural roots, which was found to be associated with higher transcript levels of TAT, PAL, and C4H. Comparable to the previous studies, our findings indicated that the expression of upstream genes in the phenolic acid production pathways, namely PAL and TAT, increased on the first and third days after exposure of HRs to 15 µM Ag⁺ ions by 35.01- and 11.08-fold of the control, respectively. The up-regulation of these essential genes indicated the involvement of two phenylalanine and tyrosine-derived pathways in phenolic acid production in S. aristata HRs under the elicitation of Ag⁺ ions. Compared with the findings of Xing et al. (2014), the results of this investigation revealed that the expression of RAS, one of the downstream genes in the phenolic acids biosynthesis pathway, increased in elicited- HRs after the three initial exposure days, reaching its maximum relative expression level (3.46-fold of the control) on the third day after exposing HRs to 15 µM Ag⁺ ions.

Tanshinone production and gene expression patterns after elicitation

Tanshinones, a class of abietane diterpenes, are predominantly found in certain Salvia species, with S. miltiorrhiza roots being well-known as a rich natural source of these metabolites. Tanshinones, including DT-I, T-I, CT, and T-IIA, have been identified as the principal bioactive constituents found in certain Salvia species and they are attributed to the pharmacological properties exhibited by these plants (Zhang et al. 2012). Ozyigit et al. (2023) reviewed a comprehensive range of biotechnological approaches for producing secondary metabolites in plants. They emphasized the use of HR cultures, citing the production of CT and T-II in Salvia abrotanoides (Kar.) Sytsma (formerly Perovskia abrotanoides Kar.). Su et al. (2022) developed a novel technique for the one-step induction of high tanshinones production by optimizing parameters associated with A. rhizogenes transformation in S. miltiorrhiza. According to their results, the contents of T-IIA and CT significantly increased in transgenic HRs. Li et al. (2016) showed a significant increase in T-IIA accumulation (1.80-fold compared with the control) in transgenic HRs of S. castanea after elicitation with 15µM Ag⁺ ions. Ma et al. (2020) confirmed the elicitation effects of Ag⁺ ions released from silver nanoparticles on tanshinone production by S. miltiorrhiza HRs. They reported a 1.80-fold increase in tanshinone accumulation compared to the control. In the present work, we observed significant concentration- and time-dependent increases in tanshinone contents of S. aristata HRs in response to Ag⁺ ions elicitation. Specifically, T-I, T-IIA, and CT accumulated 7.78-, 6.47-, and 3.90-fold, respectively, in HRs after exposure to the elicitor.

The production of tanshinones in Salvia species could be enhanced by the upregulation of central biosynthetic enzymes, such as CPS, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MDS), GGPPS, DXS, CYP76AH3, and CYP76AH1. The activation of these genes leads to the buildup of tanshinones, hence contributing to the therapeutic effects of plants. (Kai et al. 2011; Yang et al. 2013; Xing et al. 2014: Yang et al. 2018; Deng et al. 2019; Wei et al. 2019). Kai et al. (2011) demonstrated that overexpression of SmDXS, SmGGPPS, and SmHMGR led to enhancement in tanshinones production in S. miltiorrhiza HR lines; additionally, co-expression of SmGGPPS and SmHMGR caused a 4.74-fold increase in their tanshinones content compared to control. Inhibitor-mediated changes in the expression and activity of rate-limiting enzymes (HMGR, DXS, and DXR) in HRs of S. miltiorrhiza indicated that tanshinone synthesis is primarily affected by the mevalonate (MVA) pathway; however, the MEP pathway was more effective on HR growth (Yang et al. 2013). According to the findings of Deng et al. (2019), overexpression of the transcription factor of SmWRKY2, known as a positive regulator of SmDXS2 and SmCPS, was associated with increased tanshinones accumulation in HRs of S. miltiorrhiza. Moreover, the co-overexpression of SmDXR and SmHMGR genes in S. miltiorrhiza HRs led to a higher accumulation of tanshinones. The results of Zhou et al. (2020) confirmed the pivotal roles of CPS and CYP76AH1 in the biosynthesis of tanshinones; they showed that the upregulation of these crucial genes occurred concurrently with the increased concentrations of tanshinones in S. miltiorrhiza roots. In this work, concentration-dependent increases were observed in transcript levels of genes in the tanshinones biosynthesis pathway in response to elicitor. Specifically, the highest transcript level of CPS (15.59-fold increase compared to control) was recorded in HRs exposed to 25 µM of Ag⁺ ions, and the expression of CYP76AH1 reached its maximum level (38.26-fold increase compared to control) after exposure to HRs to 15µM elicitor.

Xing et al. (2014) reported that Ag⁺ ions, as heavy metal elicitors, could trigger the accumulation of T-I, T-IIA, CT, and DT-I and total tanshinones in S. miltiorrhiza HRs through the induction of crucial genes in their biosynthesis pathway. Their findings also revealed that upstream genes (HMGR, DXS, DXR, and GGPPS) responded to the elicitor earlier than the downstream genes (CPS, KSL, and CYP76AH1) in the pathway. Yang et al. (2018) investigated the changes in tanshinone contents and transcript levels of related genes (DXS2, GGPPS1, CPS1, CYP76AH1, and CYP76AH3) in HR cultures of two Salvia species under elicitation with Ag⁺ ions. Their findings indicated that exposure to the elicitor was more effective in inducing gene expression and consequently enhanced the accumulation of DT-I, T-I, and T-IIA in S. miltiorrhiza HRs compared to S. castanea HRs. Wei et al. (2019) also observed that the application of Ag⁺ ions on transgenic HRs expressing, a key gene in the tanshinone biosynthesis pathway, caused a 2.50-fold increase in total tanshinone content compared to non-transgenic ones.

In this work, concentration-dependent increases were observed in transcript levels of genes in the tanshinones biosynthesis pathway in response to elicitor. Specifically, the highest transcript level of CPS (15.59-fold increase compared to control) was recorded in HRs exposed to 25 µM of Ag⁺ ions, and the expression of CYP76AH1 reached its maximum level (38.26-fold increase compared to control) after exposure to HRs to 15µM elicitor.

Release of tanshinones by S. aristata hairy roots into culture medium

Large-scale production of bioactive substances in HRs depends on the efficient secretion of secondary metabolites into the culture medium. This approach eliminates the need for repetitive plant cultivation and harvesting, reducing time-consuming downstream processing and enabling continuous or intermittent extraction of bioactive compounds (Xu et al. 2021). Yang et al. (2012) investigated the effects of MEP and MVA pathway inhibitors on the production and secretion of tanshinones from S. miltiorrhiza HRs into the culture medium. Their findings exhibited that the concentration of tanshinones in the medium was generally low, and among the examined ones, HRs released higher amounts of T-IIA. Our results also supported their findings, with the highest value recorded for T-IIA (31.49 ± 0.65 µg mL^− 1) in the medium after five days of elicitation of HRs with 25 µM of Ag⁺ ions. Additionally, the findings confirmed that Ag⁺ ions were potent stimulants, causing the secretion of all three tested tanshinones by S. aristata HRs into the medium. At a concentration of 25 µM, the elicitor significantly increased the secretion of T-I, T-IIA, and CT in culture media, up to 3.64-, 15.53-, and 23.95- fold of the control, respectively. During the treatment period, it was evident that although the effectiveness of the elicitor was comparable in the accumulation of phenolic acids in S. aristata HRs at two applied concentrations, Ag⁺ ions were more influential on tanshinones production and their secretion into the culture media at 25 µM.

In this study, for the first time, we introduced the successful establishment of HR cultures as an effective method for improving the production of secondary metabolites in S. aristata. Among the different examined A. rhizogenese strains, the ATCC 15834 strain was considered the most suitable for HR induction and the production of secondary metabolites, mainly tanshinones. Our findings also underscored that elicitation with Ag⁺ ions effectively encouraged the growth and production of secondary metabolites in S. aristata HRs. The stimulatory effects of Ag⁺ ions on metabolite production in HRs relied on their used concentration and the harvesting time. Furthermore, our findings showed that elicitation of HRs with Ag⁺ ion could enhance the release of tanshinones, especially T-IIA, into the culture medium. Collectively, the findings of this research provide an opportunity for future research on S. aristata and ascertain the potential of elicitors and transgenic HRs to improve the production of valuable bioactive metabolites in this species as an alternative natural source of phenolic acids and tanshinones.

HR Hairy root

RA Rosmarinic acid

Sal-A Salvianolic acid A

Sal-B Salvianolic acid B

CA Caffeic acid

VA Vanillic acid

CGA Chlorogenic acid

T-I Tanshinone I

T-IIA Tanshinone IIA

CT Cryptotanshinone

DT-I Dihydrotanshinone I

PAL Phenylalanine ammonia-lyase

TAT Tyrosine aminotransferase

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

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

MEP Methylerythritol 4-phosphate

GGPP Geranylgeranyl diphosphate

CPS Copalyl diphosphate synthetase

KSL Kaurene synthase-like

CYP76AH1 Cytochrome P450 monooxygenases

ATCC American-type culture collection

DW Dry weight

FW Fresh weight

HPPR 4-Hydroxyphenylpyruvate reductase

C4H Cinnamate 4-hydroxylase

4CL Hydroxycinnamate coenzyme A ligase

PCR The polymerase chain reaction

TPC The total phenolic content

HMGR 3-Hydroxy-3-methylglutaryl coenzyme A reductase

MDS 2-C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase

MVA Mevalonate

Acknowledgments

This work was supported by a research grant funded by Shahed University of Tehran for the plant physiology PhD thesis.

Author contributions

This research paper was accomplished through collaboration among the authors. Raziey Rahchamani conducted the experiments analyses, and interpretation of the data and wrote the manuscript. Tayebeh Radjabian designed and supervised the study; and contributed to the conceptualization, interpretation of data, and participated in the review, and editing. Parvaneh Abrishamchi participated in the revisions of the manuscript. All authors read and approved the final manuscript.

Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Any ethical rights have been observed in writing this article, and the scientific contribution of all individuals in this research has been fully clarified and agreed upon by all.

Data Availability The data that support the findings of this study are included in the article.

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published 17 Aug, 2024

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Ag+ ions are effective elicitors for enhancing the production of phenolic acids and tanshinones in Salvia aristata Aucher ex Benth. hairy roots

Status:

Journal Publication

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Abstract

Figures

Introduction

Materials and Methods

Explants preparing using the seed culture method

Preparation of bacterial strain

Hairy root culture

Confirmation of transgenic hairy root lines

Elicitation of hairy roots

Measurement of total phenolic content

Extraction and HPLC analysis of phenolic acids and tanshinones

RNA extraction, cDNA synthesis, and gene expression analysis

Statistical analysis

Results

Induction of hairy roots and confirmation of transgenic genome

Discussion

Hairy root establishment

Phenolic acids production and gene expression patterns after elicitation

Tanshinone production and gene expression patterns after elicitation

Conclusion

Abbreviations

Declarations

References

Status:

Journal Publication

Version 1