Elicitor-induced phenolic acids accumulation in Salvia virgata Jacq. hairy root cultures


 Phenolic acids, as the predominant secondary metabolites of Salvia species, are largely used in pharmaceutical industries. The main aim of the study was to establish hairy root cultures of Salvia virgata Jacq. Also, the effects of methyl jasmonate (22.4 and 11.2 ppm), Ag+ ions (5 and 2.5 ppm), and yeast extract (100 and 50 ppm) were assessed on total phenol, total flavonoid, rosmarinic acid, salvianolic acid A, and caffeic acid contents in the hairy roots after 1, 3 and 5 days of exposure. Results showed the used Agrobacterium rhizogenes strains (A4, ATCC15834, R1000, GM1534, and C58C1) differed in their ability to induce hairy roots on leaf explants. The transformed roots were molecularly confirmed using rolC gene and the highest transformation frequency (56 %) was obtained by ATCC 15834 strain. Among the established hairy root lines, the highest amount of rosmarinic acid (0.45 ± 0.01 mg/g DW) and dry root biomass (2.29 ± 0.04 g) was obtained in AT3, the line which was induced by ATCC 15834 strain. The maximum accumulation of total phenol (123.6 ± 0.93 mg GAE/g DW), total flavonoid (5.09 ± 0.07 mg QUE/g DW), rosmarinic acid (18.45 ± 0.8 mg/g DW), salvianolic acid A (2.11 ± 0.04 mg/g DW) and caffeic acid (2.61 ± 0.02 mg/g DW) was observed in the hairy roots elicited with 22.4 ppm methyl jasmonate on day three after treatment. The results support that elicitation could be an effective procedure for the improvement of caffeic acid derivatives production in S. virgata hairy root cultures.


Introduction
Salvia virgate Jacq., an annual plant belonging to the Lamiaceae family, is native to Asia (northeast of Iran) and southeastern Europe. The plant has traditionally been used for the treatment of skin diseases, wounds, and blood cancer (Baytop 1999 Chemically, CA is the basic structural unit of phenolic acids in Salvia species. Rosmarinic acid is an ester of CA and 3, 4-dihydroxyphenyllactic acid, and Sal-B is identi ed as a dimmer of RA (Wang et  There is only one report (in Persian) available on the induction of HR cultures in S. virgata (Norouzi et al. 2017), and based on our knowledge, no report has been published regarding the elicitation of phenolic acids biosynthesis in HR cultures of S. virgata. Therefore, this study was designed to develop an e cient HR culture system for S. virgata, using different Agrobacterium rhizogenes strains, and to investigate the effects of biotic and abiotic elicitors on RA, CA, and Sal-A production in the transformed root cultures for the rst time.

Plant material
Mature seeds of S. virgata were collected from wild-grown plants in August 2013 at Reine village (Bojnoord, North Khorasan province, Iran) located at latitude 57• 2 N, 37• 23 E longitude, and an elevation of 1765 m above sea level. The species was identi ed at the Ferdowsi University of Mashhad herbarium (FUMH), where a voucher specimen (No. 38128) of the plant was deposited. Seeds were surface sterilized with ethanol (70 %) for 1 min, followed by sodium hypochlorite (5 %) for 5 min, and rinsed three times with sterile distilled water. For germination, seeds were placed into glass jars containing 25 mL of Murashige and Skoog (MS) medium supplemented with sucrose (3 %) and agar (0.7 %). The pH of media was adjusted to 5.6-5.8 before adding agar, and the MS basal medium was autoclaved at 120 ºC for 17 min. The glass jars were kept in the dark for three days at 25 ± 2 ºC, and after germination of the seeds, they were placed at 26 ± 2 ºC and 16/8 h (light/dark) photoperiod (45 M photons m − 2 s − 1 provided by cool white uorescent lamps) in a culture room. These conditions were also applied to all the experiments described below.

Establishment of hairy root cultures
Leaf explants were taken from S. virgata plants grown in vitro for 50 days. The explants were wounded with a needle dipped into A. rhizogenes cultures. Control explants were wounded with a sterile needle without bacteria. Infected and control explants were placed on hormone-free MS agar (0.7 %) medium and incubated in the dark at 25 ºC. After two days of inoculation, the explants were transferred to MS basal medium supplemented with sucrose (3 %), cefotaxime (300 ppm), and Difco Bacto agar (0.7 %) was used for solidifying medium. Root cultures were incubated under 16/8 h (light/dark) photoperiod (45 M photons m − 2 s − 1 provided by cool white uorescent lamps) at 25°C.
The experiment was repeated three times; 25 explants were used for each treatment (n = 25). The transformation frequencies (the percentages of explants forming roots after infection with A. rhizogenes strains) were determined 10-14 days after infection. From each strain, three obtained roots, longer than 1 cm, were excised from explants and transferred individually into 250 mL Erlenmeyer asks containing 50 mL 1/2 MS liquid medium without growth regulators and supplemented with 300 mg/L cefotaxime to prevent bacterial spread. The cefotaxime concentration was gradually decreased to 200 and 100 mg/L during the second and third subcultures, respectively. The cultures were maintained on an incubator shaker at 100 rpm at 25 ºC in the dark. After eight subcultures, when the antibiotic was eliminated from the medium, one fast-growth root line with the most RA content was obtained (ATCC15834 line AT3) and used for elicitors treatments.

Con rmation of transgenic hairy root lines
The genomic DNA (gDNA) from ve putative transformed root lines of each strain and untransformed roots (negative control) was extracted from 100 mg of plant tissue following the procedures described by Sharp et al. (1988) with slight modi cations. In this study, DNA from A. rhizogenes ATCC15834 strain served as the positive control, and seedling roots were used as the negative control.
The PCR was performed to amplify an internal rolC gene fragment (612 bp). The PCR analysis was on the C1000 Touch TM 96-Well Thermal Cycler (Bio-Rad, USA) and began with 15 µL reaction mixtures containing amplicon, gDNA (100 ng total DNA), and oligonucleotide primers in 10 µM nal concentration. The primers for detecting the rolC gene were 5´-ATG GCT GAA GAC GAC CTG TGT T-3´ and 5´-TTA GCC GAT TGC AAA CTT GCA C-3´. The PCR program comprised of an initial denaturing step of 4 min at 95°C and 35 cycles, each consisting of 60 s at 95°C, 30 s at 55°C and 60 s at 72°C, followed by a nal extension step of 7 min at 72°C. Approximately 10 µL of the PCR products were electrophoresed on 1 % (w/v) agarose gel, stained with GelRed, and visualized under UV on the Gel Imaging System (Bio-Doc, Germany). Predicted products of rolC were obtained apart from the control (non-induced root).

Elicitor treatment
Three elicitors, including Ag + ions, YE and MeJA, were tested at two concentrations. A stock solution of Ag + ions was prepared by dissolving AgCl in deionized water and added (2.5 and 5 ppm nal concentrations) in liquid hormone-free 1/2 MS medium. Carbohydrate fraction of YE was made from commercial yeast extract (Cat. no.Y4250, Sigma) by ethanol precipitation as described by Hahn and Albersheim (1978)  Hairy roots were cultured for 55 days (end of exponential growth phase), and then the old medium was substituted with 30 ml of fresh 1/2 MS liquid medium supplemented with Ag + ions, MeJA and YE. Half strength MS liquid medium with the same volume of water or ethanol (instead of elicitors) was added to control cultures. Hairy roots were harvested 1, 3, and 5 days after elicitor treatments. All cultures were maintained at the same mentioned conditions (part 2-3).

Extraction of phenolic compounds
Harvested hairy roots were oven-dried at 40 ºC to reach the constant dry weight and then grounded to a ne powder. Dried powders of plant materials were extracted with methanol (98 %) (500 mg root material/10 mL solvent) by sonication for 30 minutes at room temperature. The extracts were then ltered using Whatman No.1 lter papers. After vacuum evaporation, the dried extracts were maintained at -20 ºC.

Determination of total phenolic compounds
The total phenolic (TP) content of the samples was determined by using Folin-Ciocalteu reagent and gallic acid as standard (Wojdylo et al. 2007). Brie y, 1 mg of the dried extract sample was dissolved in 1 mL methanol (1000 ppm), then 100 µL of the prepared solution was thoroughly mixed in a test tube with 2 mL water and 200 µL Folin-Ciocalteu reagent for 3 min, and the mixture was incubated with 1 mL of 20 % (w/v) sodium carbonate solution at room temperature for 1 h. The absorbance of the extracts was measured at 765 nm against a blank (a solution without the extract). A stock solution of gallic acid (800 ppm) was prepared in distilled water and diluted to appropriate concentrations (200-800 ppm) for the construction of a calibration curve. The concentration of TP in samples was measured using the calibration equation (y = 0.1427 + 0.0029 x, r 2 = 0.999) and expressed as mg of gallic acid equivalent (GAE) per g dry weight.

Determination of total avonoids
Flavonoid concentration in the samples was measured spectrophotometrically, according to the procedure of Chang et al. (2002). The reaction mixture was prepared by mixing 0.5 mL of methanolic solution of dried extract (1000 ppm) with 1.5 mL methanol, 0.1 mL 10 % (w/v) aluminum chloride, 0.1 mL 1 M potassium acetate and, 2.8 mL distilled water. After 30 min of incubation at room temperature, absorbance was measured at 415 nm against a blank without the extract. For the establishment of a calibration curve, different concentrations of quercetin (20-100 ppm) were prepared in distilled water using a stock solution (100 ppm). The total avonoid (TF) content in samples was calculated by the standard curve equation (y = − 0.065 + 0.0088 x, r 2 = 0.939) and results were expressed as mg quercetin equivalent (QUE) per g dry weight.

HPLC analysis of phenolic acids
The content of phenolic acids was measured by the HPLC method. The HPLC apparatus was a Smartline model (Kenuer, Germany) with a quaternary pump and a reversed-phase column C18 Eurospher-100 (5 µm particles, 125 mm × 4 mm) coupled with a UV-VIS detector (D-14163 model). The data were processed by Software ChromGate (V 3.1). The separation was performed using a mobile phase consisted of water with 0.2 % glacial acetic acid (solvent A) and acetonitrile (solvent B). The ow rate was kept at 1 mL/min. The initial condition was 90/10 (v/v) A/B and 75/25 (v/v) at 15 min. The percentage of mobile-phase A decreased to 20 % at 40 min and reached 0 % at 45 min. This ratio remained stable until 50 min, and in the next 5 min, the percentage of mobile-phase A increased linearly to 90 %. The injection volume was 20 µL, and peaks were monitored at 280 nm. The samples were ltered through a hydrophilic PTFE membrane lter with a 0.45 µm pore size before injection. Peaks were identi ed by congruent retention times compared with those of standards. Rosmarinic acid, Sal-A, and CA ( Fig. 1) were detected and quanti ed using authentic standards obtained from Sigma. The content of each phenolic acid was calculated based on the equation, which was obtained from the corresponding standard calibration curve. The stock solutions of RA, Sal-A, and CA (400 ppm) were prepared in ethanol and diluted to appropriate concentration range for the construction of calibration curves. The concentrations of RA, Sal-A, and CA in samples were measured using calibration equations (y RA = 41606 x, r 2 = 0.996; y Sal−A = 19497 x, r 2 = 0.996; y CA = 7337.1 x, r 2 = 0.995). Each treatment was performed in three replicates.

Statistical analysis
All the tissue culture experiments were repeated at least three times in a completely randomized design.
The data were subjected to one-way ANOVA using SPSS software version 16.0. Mean values were compared by Duncan's Multiple Range Test and reported as means ± standard errors (SE). A probability of P ≤ 0.05 was considered to be signi cant.

Establishment of S. virgata hairy root cultures and con rmation of transgenic status
In this study, detached leaf explants from the in vitro 50-day-old plants were inoculated with ve strains of A. rhizogenes. Hairy roots initials appeared on the incision sites of the explants within 10 days. After four weeks of the inoculation, all of the bacterial strains used in this study successfully induced hairy roots at the wounded sites of leaf explants. No root formation was observed in the control explants ( Fig. 2). As shown in Fig. 3, all of the hairy roots, induced by ve different strains, were con rmed to have rolC gene in their genomes.
According to the results, selected strains of A. rhizogenes showed a signi cant difference in their ability to induce HRs, and the infection frequency varied from 20.4 ± 0.77 to 56 ± 2.67 percent depending on the bacterial strain. The highest infection frequency (56 %) was found in the leaf explants infected with ATCC15834 strain, while the lowest infection frequency (20.4 %) was obtained in the explants inoculated with C58C1 strain (Fig. 4).
In the next step of the present study, three fast-growing hairy root lines were selected from all ve groups of HR lines (induced by 5 different strains of A. rhizogenes), transferred to 1/2 MS liquid medium, and then analyzed for their growth and RA content after 2 months. The highest dry weight and RA content were obtained in the HR line AT3, which was induced by ATCC15834 strain, so it was selected as the best line for elicitor treatments (Table 1).

Effects of elicitors on phenolic acids content
Hydromethanolic extracts of elicited and non-elicited HRs (line L3) were utilized for quantitative analysis of phenolic acids (RA, Sal-A and, CA) using the HPLC method. The HPLC chromatograms of phenolic acids from some treatment as compared to the control are shown in Fig. 5. The results obtained from HPLC analysis revealed that RA, CA and, Sal-A were present in three elicited and non-elicited HRs of S. virgata. Rosmarinic acid was the most abundant phenolic acid in the extracts, followed by CA and Sal-A. Salvianolic acid B was not detected in the HR samples (Fig. 5).
According to the results shown in Fig. 6, the levels of three studied phenolic acids in HRs treated with both concentrations (11. 2.61 ± 0.02 mg/g DW CA, and 2.11 ± 0.04 mg/g DW Sal-A over the 3-day, nearly 1.81-, 4.35-and 3.76-fold of untreated HRs on the same day (Fig. 6).
The data collected from YE-treated HRs, presented in Table 3 or Fig. 7, revealed that the increase in the contents of RA and Sal-A, which was observed during the rst three days of elicitation with 50 and 100 ppm YE, was followed by a decrease on day ve after treatment. The application of 50 ppm YE for three days was the best treatment for the production of RA and Sal-A. Maximum amounts of RA (15.58 ± 0.01 mg/g DW) and Sal-A (1.65 ± 0.01 mg/g DW) were 1.44-and 2.42-fold of control, respectively (Fig. 6). Elicitation with YE at the concentration of 50 ppm was more effective than 100 ppm to accumulate CA in HRs. Caffeic acid accumulation in HRs treated with 50 ppm YE signi cantly increased on days 3 and 5 of the elicitation period, as compared to day 1. The highest accumulation of CA (1.03 ± 0.01 mg/g DW) was achieved on the 5th day of treatment (1.74-fold of control) (Fig. 6).
As shown in Fig. 6, during ve days, elicitation with Ag + ions (2.5 and 5 ppm), in most cases, resulted in a higher accumulation of phenolic acids in HRs than the control. Elicitation with 2.5 ppm Ag + was more effective on the production of phenolic acids in HRs cultures, as compared to 5 ppm Ag + . A progressive time-dependent enhancement in the contents of RA, Sal-A, and CA was found throughout elicitation with 2.5 ppm Ag + . The highest production of RA (16.01 ± 0.09 mg/g DW), CA (1.99 ± 0.01 mg/g DW), and Sal-A (1.52 ± 0.06 mg/g DW) were attained after a 5-day exposure of HRs to 2.5 ppm Ag + (1.54-, 2.45-and 3.37fold of control, respectively) (Fig. 6).

Discussion
This study aimed to establish HR cultures of S. virgata and, for the rst time, to increase the production of phenolic acids based on the biotic and abiotic elicitor application. The ndings revealed that all the tested strains of A. rhizogenes (ATCC15834, A4, R1000, C58C1, and GM1534) could generate HRs on the leaf segments. However, the infection frequency was signi cantly different among the ve strains. In the present survey, among three distinct HR lines induced by ATCC15834 strain, line AT3 was the bestgrown line with maximum biomass production and highest RA accumulation. It has been revealed that are the consequences of excessive biosynthesis of endogenous auxins and cytokinins and enhanced sensitivity of transformed plant cells to these hormones, and rol genes appear to play a critical role in these processes (Hashem 2009). Gene rolA has a promoter region similar to those of some auxinregulated genes, and its product is probably a DNA binding protein and stimulator of growth (Matveeva et al. 2015). Primary studies proposed that rolB protein is a β-glucosidase releasing auxin (IAA) from its inactive conjugated forms, thus increasing auxin sensitivity (Esruch et al. 1991). Moreover, the rolB geneencoded product was shown to exhibit tyrosine phosphatase activity (Filippini 1996;Dilshad et al. 2015) and to interact with 14- In the present study, signi cant enhancements in TP, RA, and Sal-A levels (1.  Silver ions, as one of the most potent abiotic elicitor, is believed to stimulate the production of plants secondary metabolites (Naik and Al-Khayri 2016b). In the current study, after the addition of 2.5 ppm Ag + (for ve days), the accumulation of TP, TF, RA, CA, and Sal-A was estimated to be 1.

Conclusion
This study illustrated, for the rst time, an e cient method for the production of phenolic acids in the well-established HR cultures of S. virgata using the elicitation technique. Among the ve different strains of A. rhizogenes (ATCC15834, R1000, A4, C58C1, and GM1534), the ATCC15834 showed the highest capability to induce HR formation and RA production. The elicitation technique utilized here revealed the inducible nature of phenolic acids production in HRs of S. virgata. All tested elicitors (MeJA, Ag + , and YE) positively in uenced the contents of TP, TF, RA, CA, and Sal-A in HR cultures, and the highest accumulation of these phenolic components was achieved at 22.4 ppm MeJA after three days. Subsequent investigations relevant to elicitation and mechanism of phenolic acids biosynthesis need to be conducted to further utilize the potency of S. virgata HR cultures to produce more bioactive compounds. Chemical structures of caffeic acid and some of its derivatives in Salvia genus Effects of different concentrations of the applied elicitors on phenolic acids production in the hairy root cultures of S. virgata during periods of treatments. The data represent mean ± SE of three replicates.