Protein elicitor isolated from Escherichia coli induced bioactive compound biosynthesis as well as gene expression in Glycyrrhiza uralensis Fisch adventitious roots

Abstract

This study explored the ability of three rhizobacterial strains (Bacillus subtilis, Penicillium fellutanum and Escherichia coli) to trigger metabolism. The protein fragment of more than 10 kDa significantly increased the metabolite contents in Glycyrrhiza uralensis adventitious roots. The results showed the highest accumulation of total flavonoids (7.59 mg g⁻¹), glycyrrhizic acid (0.29 mg g⁻¹), glycyrrhetinic acid (0.27 mg g⁻¹) and polysaccharide (93.11 mg g⁻¹) by up to 2.27-fold, 2.64-fold, 2.70-fold and 2.32-fold that of control roots, respectively. Besides, the protein fragment of more than 10 kDa significantly activated defense signaling and extremely up-regulated the expression of defense-related genes and functional genes in glycyrrhizic acid and flavonoid biosynthesis. In Glycyrrhiza uralensis adventitious roots, HPLC-ESI-MSⁿ analysis showed that the protein fragment of more than 10 kDa induced the generation of four new compounds over the control group.

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1. Introduction

Glycyrrhiza uralensis Fisch (G. uralensis) is one of the most valuable and useful Chinese herbs for the treatment of alleviating pain and relieving coughing. Moreover, G. uralensis is a highly nutritional plant used as a natural sweetener and flavoring agent in food products, such as tobacco, candies, chewing gum, toothpaste, and beverages.^1,2 In G. uralensis, the most important pharmacologically active components are glycyrrhizic acid, glycyrrhetinic acid and flavonoids, which exhibit anti-inflammation, anti-virus and anti-HIV properties.³ In the proposed glycyrrhizic acid biosynthesis pathway (Fig. 4(a)), CYP88D6 primarily catalyzed the sequential two-step oxidation reactions of β-amyrin at the C-11 position to produce 11-oxo-β-amyrin.⁴ A second P450 (CYP72A154) catalyzed three sequential oxidation steps at C-30 of 11-oxo-β-amyrin, and then producing the glycyrrhetinic acid.⁵ Flavonoids are synthesized via the phenylalanine metabolic pathway. Cinnamate 4-hydroxylase (C4H), the second enzyme in the phenylpropanoid pathway, transformed cinnamic acid into p-coumaric acid in higher plants.⁶ Chalcone isomerase (CHI) catalyzed chalcone to flavanone, which in succession turned into many other bioactive flavonoids.⁷ Some new technologies, such as cell, tissue and organ culture, have been developed to produce active constituents of G. uralensis because of limited natural sources.

However, the contents of flavonoids and glycyrrhizic acid in adventitious root of G. uralensis were lower than those in native one.⁸ The elicitors are demonstrated to effectively improve the productivity of secondary metabolites in plant tissue cultures. In the previous report, bacterial strains as pathogens have the ability to trigger a cascade of secondary metabolism involved in defense to biotic and abiotic stress.⁹ Biotic elicitors are substances with biological origin, they include polysaccharides (chitin or glucans) and proteins (glycoproteins, G-protein or intracellular proteins) whose functions are induced the overproduction of various secondary metabolites.¹⁰

Biotic elicitors are coupled to receptors and act by activating or inactivating a number of enzymes, leading to a wide variety of defense reactions, including Ca²⁺ ion fluxes across the cell membrane, activation of phospholipase A₂ (PLA₂) and protein kinases, oxidative burst.¹¹ In addition, this cascade of reactions turns to activate the synthesis of endogenous signaling molecules including nitric oxide (NO), ethylene (ET), abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA). Signaling molecules transferred the elicitor signals to defense genes including pheammonia-lyase (PAL) and pathogenesis-related protein 1 (PR-1) in the SA-mediated signalling pathways, lipoxygenase (LOX) and plant defensin gene 1.2 (PDF1.2) in the JA-mediated signalling pathways, NO-synthesis (NOS) in the NO-mediated signalling pathways and further amplify the elicitor signal to the biosynthesis of secondary metabolites (Fig. 2(a)).^12–14

To date, there is no reports about the effects of compounds (polysaccharide and proteins) derived from E. coli on bioactive compounds accumulation in G. uralensis adventitious roots. In this study, we investigated the secondary metabolites production in G. uralensis adventitious root after treatment with polysaccharide and proteins isolated from the E. coli. We also investigated the impacts of protein fragment more than 10 kDa on the generation of signal molecules, defense genes expression, functional genes expression and bioactive compounds accumulation in G. uralensis adventitious roots culture. Meantime, we identified bioactive compounds based on HPLC-ESI-MSⁿ analysis after treatment with protein fragment more than 10 kDa.

2. Materials and methods

2.1 Plant materials and culture conditions

In previous report, Yin et al. has established the cultivation system of G. uralensis adventitious roots. The adventitious roots (1 g) are cultivated in 250 mL Erlenmeyer flasks filled with 100 mL of medium containing half-strength MS (Murashige and Skoog 1962) supplemented with 1 mg L⁻¹ indole-3-butyric acid (IBA), 30 g L⁻¹ sucrose. After 35 days, adventitious roots were gained.¹⁵

2.2 Elicitation assays

The three strains (Bacillus subtilis, Penicillium fellutanum and Escherichia coli) were screened from G. uralensis rhizosphere soil and maintained in our laboratory. Fungal elicitor (P. fellutanum) was grown in potato dextrose agar medium. Bacterial elicitors (B. subtilis and E. coli) were grown in lysogeny broth medium. After centrifugation, the sediments were dried and then ground to a powder. The powder was dissolved in water, becoming a concentration of 10 g L⁻¹. The elicitor concentration was measured by the total carbohydrate content, which was determined by the anthrone–sulfuric acid method using glucose as the standard.¹⁶ For elicitation, three elicitors were added to the 28 day-old adventitious root culture at different concentrations (0, 100, 200, 400, 600 mg L⁻¹) respectively. After elicitor treatment for one week, the adventitious roots were harvested. Then the growth ratio and the content of bioactive compounds were analyzed.

2.3 Preparation of elicitors from E. coli

Polysaccharide elicitor was prepared from E. coli as per the previously described method with some changes.¹⁷ The E. coli (2 g) were extracted three times in 250 mL distilled water for 1 h at 100 °C for extraction of polysaccharides. Then, the extract was concentrated under vacuum at 60 °C by a rotary evaporator to a suitable volume and mixed with 4 vols of 95% ethanol. After centrifugation, the precipitate from ethanol dispersion was obtained, and then removing the protein with Sevag reagent.

Protein as elicitors were purified from the E. coli according to previously described method with some changes. A total of 50 g (fresh weight) bacteria were ruptured with Ultrasonic Cell Disruption System (Scienta, China) for 60 min, and the homogenate were added with 80 g of (NH₄)₂SO₄ at 4 °C overnight. After centrifugation 50 min with 4000 rpm, the precipitate was filtered by 0.2 μm filter to ensure removal of all bacterial debris. Then, the precipitate was collected and re-dissolved with 100 mL distilled water; this was fraction 1.¹⁸ Then, the solution was filtered through ultrafiltration tube (10 kDa molecular weight cut off). Separation was forced by centrifugation at 4000 rpm for 20 min at 4 °C. Two fractions resulted from this process, fraction 2 less than 10 kDa and fraction 3 more than 10 kDa.¹⁹ The total content of protein was measured by using Coomassie Brilliant blue staining method. (4) (A = 0.003C + 0.2627, r = 0.99).

2.4 Polysaccharide and protein treatment

Polysaccharide elicitor were added to the medium at the final concentrations (0, 100, 200, 400 and 600 mg L⁻¹). Three fractions protein separately as elicitors were added to the medium at the final concentrations (0, 25, 50, 100 and 200 mg L⁻¹). The growth ratio, the content of glycyrrhizic acid, glycyrrhetinic acid, total flavonoid and polysaccharide were measured. Compounds were identified by HPLC-ESI-MSⁿ analyses after treatment with protein fraction 3. Experiments were performed in three biological replicates.

2.5 Measurement of root biomass

After 35 days of culture, the harvested roots were separated from the liquid medium by filtration and washed with running water twice. Then, the fresh roots were dried in vacuum at 50 °C to attain the constant dry weight. The growth rate was figured out with the following formula (harvested dry weight − inoculated dry weight)/inoculated dry weight.

2.6 Extract preparation and determination of polysaccharides

The adventitious roots (0.2 g) were extracted three times in 25 mL distilled water for 1 h at 100 °C for extraction of polysaccharides. The extract was diluted with water to 100 mL. The content of polysaccharide was determined by the sulphuric acid–anthrone method.²⁰ Absorbance was measured at 585 nm on a UV mini-1240 spectrophotometer with glucose as the reference standard (A = 0.0042C + 0.0343, r = 0.9972).

2.7 HPLC-ESI-MSⁿ analyses and quantification of total flavonoids, glycyrrhizic acid, and glycyrrhetinic acid

Adventitious roots (0.2 g) were soaked overnight in 20 mL methanol, and then extracted twice for half an hour at 60 °C in the water bath oscillator. After filtration, the extracts were concentrated by vacuum drying using rotary evaporator, and then required constant volume to 1 mL. HPLC-ESI-MSⁿ analyses were performed by use of an Agilent HPLC-MS system equipped with a surveyor auto-sampling system, and connected ion-trap mass spectrometer through an electrospray ion source. The more detailed procedure was described in elsewhere.²¹

2.8 Change of signaling molecule, defense genes, and functional genes after treatment with protein fraction 3

At 28 days, fraction 3 elicitor was added to the medium. For the time course assay, adventitious roots were harvested from the culture medium on 0, 1, 6, 12, 24, and 48 h after the addition of the elicitor (50 mg L⁻¹). The endogenous signaling molecule, the expression level of functional genes and defense genes, the growth ratio, the content of total flavonoid, glycyrrhizic acid, glycyrrhetinic acid and polysaccharide, were performed simultaneously.

2.9 Quantification of Ca²⁺, PLA₂, NO, ET, ABA, JA and SA

For Ca²⁺, PLA₂, NO, ET, and ABA assay, adventitious roots (0.2 g) from the elicitor treated group and the control group were ground into homogenate with 1.8 mL PBS (pH 7.4). The supernatant were collect after centrifugation at 3000 rpm for 20 min. They were measured using commercially available kits (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) according to the manufacturer's directions. For JA and SA assays, the resulting powder was extracted in MeOH–H₂O–HOAc (90 : 9 : 1, v/v/v) solution and then supernatant was collected after centrifugation. The analysis was done according to the procedure of Segarra et al., gradient elution profile (t (min): % B) was: (0, 15), (3, 15), (5, 100), (6, 100), (7, 15), (8, 15) using a Kromasil C18 column (4.6 mm × 250 mm, 5 μm) at room temperature. The injected volume was 10 μl, the mobile phase was composed of 0.05% HOAc in H₂O (solvent A) and MeCN (solvent B) at a constant flow-rate of 600 μl min⁻¹.²²

2.10 RNA isolation and gene expression performed by quantitative real-time PCR analysis

Relative expression levels of functional genes and defense genes were achieved by the qRT-PCR method, and actin was taken as the reference gene. RNAs from both the elicitor treated group and control group were obtained by the Plant RNA Kit (OMEGA, USA). The first-strand cDNA were synthesized from approximately 3 μg RNAs using the HiFiScript 1st Strand cDNA Synthesis Kit (CWBIO, China) according to the manufacturer's directions. The forward and reverse sequences used for PCR amplification are shown in Table S1.† The first-strand cDNAs were the templates for RT-PCR reactions on the RT-PCR instrument (Veriti, America). Real-time PCR was performed using the real-time PCR cycling parameters: 94 °C for 2 min, then 35 cycles of 94 °C for 30 s, annealing temperature (T_m 57 °C) for 1 min, and 72 °C for 30 s, with a final 2 min extension at 72 °C. The sizes of the PCR products were determined via agarose gel (2.0%) electrophoresis. The size of the fragments was estimated using a 100 bp ladder (CWBIO, China) as a size marker. Experiments were performed three times.

2.11 Statistical analysis

The statistical analysis was carried out by following the SPSS V 17.0 system. All the data were expressed throughout as mean ± standard deviation (SD), and the statistical differences between means was assessed by means of the Duncan's multiple range tests. A probability of P < 0.05 was accepted as significant.

3. Results and discussion

3.1 The accumulation of metabolites in G. uralensis adventitious roots by elicitation

The three strains (B. subtilis, P. fellutanum and E. coli) were tested in G. uralensis adventitious root for their ability to enhance bioactive compounds content (Tables S2–S4†). Addition of E. coli significantly improved the content of total flavonoids (8.05 mg g⁻¹), glycyrrhizic acid (0.45 mg g⁻¹), glycyrrhetinic acid (0.42 mg g⁻¹) and polysaccharide (98.34 mg g⁻¹) and resulted in a 2.58-fold, 3.75-fold, 3.82-fold and 2.38-fold increase than control roots (Table S4†). Thus, among the effective strains to increase bioactive compounds content, E. coli was selected for elicitation assays in adventitious roots cultures, which may be attributed the compounds in E. coli. Polysaccharide and protein from E. coli were purified to research the reason that E. coli exerted elicitation function.

After the four E. coli fractions respectively added to the medium, the contents of metabolites were measured. The results demonstrated that polysaccharide and protein as elicitors all challenged the level of secondary metabolites compared with the control. However, root growth showed a slight decrease after elicitation. The contents of total flavonoids (7.54 mg g⁻¹), glycyrrhizic acid (0.23 mg g⁻¹), glycyrrhetinic acid (0.24 mg g⁻¹), and polysaccharide (85.09 mg g⁻¹) were achieved in the presence of polysaccharide (Table 1). Fraction 1, total protein from E. coli, significantly enhanced the contents of total flavonoids (8.34 mg g⁻¹), glycyrrhizic acid (0.33 mg g⁻¹), glycyrrhetinic acid (0.35 mg g⁻¹) and polysaccharide (109.98 mg g⁻¹) (Table 2). Treatments with fraction 2 increased the contents of total flavonoids (6.95 mg g⁻¹), glycyrrhizic acid (0.22 mg g⁻¹), glycyrrhetinic acid (0.23 mg g⁻¹) and polysaccharide (78.95 mg g⁻¹) (Table 3). The contents of total flavonoids (7.59 mg g⁻¹), glycyrrhizic acid (0.29 mg g⁻¹), glycyrrhetinic acid (0.27 mg g⁻¹) and polysaccharide (93.11 mg g⁻¹) were up to 2.27, 2.64, 2.70, 2.32-fold in response to fraction 3 (Table 4).

Table 1 Effects of polysaccharide on G. uralensis adventitious root growth and metabolites accumulation^a

Polysaccharide concentration (mg L^−1)	Growth rate	Total flavonoid content (mg g^−1)	Glycyrrhizic acid content (mg g^−1)	Glycyrrhetinic acid content (mg g^−1)	Polysaccharide content (mg g^−1)
a Mean followed by different letters within a row is significantly different at P < 0.05.
0	6.60 ± 0.20a	3.70 ± 0.27a	0.11 ± 0.01a	0.10 ± 0.02d	40.97 ± 0.70a
100	6.40 ± 0.07ac	5.16 ± 0.05b	0.17 ± 0.01ac	0.14 ± 0.02c	58.21 ± 0.57b
200	6.30 ± 0.07ad	7.54 ± 0.13c	0.23 ± 0.02bd	0.24 ± 0.02b	85.09 ± 0.10c
400	6.20 ± 0.07bcd	6.40 ± 0.13d	0.16 ± 0.03b	0.14 ± 0.02a	65.08 ± 0.74d
600	5.95 ± 0.03b	5.04 ± 0.06b	0.13 ± 0.01bce	0.12 ± 0.02	60.18 ± 0.03e

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Table 2 Effects of fraction 1 on G. uralensis adventitious root growth and metabolites accumulation^a

Fraction1 concentration (mg L^−1)	Growth rate	Total flavonoid content (mg g^−1)	Glycyrrhizic acid content (mg g^−1)	Glycyrrhetinic acid content (mg g^−1)	Polysaccharide content (mg g^−1)
a Mean followed by different letters within a row is significantly different at P < 0.05.
0	6.70 ± 0.13a	3.32 ± 0.18a	0.10 ± 0.01a	0.11 ± 0.02a	40.05 ± 0.71a
25	6.55 ± 0.03ac	5.95 ± 0.33b	0.23 ± 0.03b	0.24 ± 0.03b	77.34 ± 0.23b
50	6.45 ± 0.03bc	8.34 ± 0.19c	0.33 ± 0.03 cd	0.35 ± 0.01c	109.98 ± 0.85c
100	6.40 ± 0.07bc	6.24 ± 0.18b	0.27 ± 0.03bd	0.28 ± 0.02b	86.04 ± 0.41d
200	6.30 ± 0.07b	5.17 ± 0.20d	0.24 ± 0.02b	0.26 ± 0.02b	74.90 ± 0.64e

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Table 3 Effects of fraction 2 on G. uralensis adventitious root growth and metabolites accumulation^a

Fraction 2 concentration (mg L^−1)	Growth rate	Total flavonoid content (mg g^−1)	Glycyrrhizic acid content (mg g^−1)	Glycyrrhetinic acid content (mg g^−1)	Polysaccharide content (mg g^−1)
a Mean followed by different letters within a row is significantly different at P < 0.05.
0	6.40 ± 0.07a	3.42 ± 0.38a	0.11 ± 0.01a	0.11 ± 0.03a	39.05 ± 0.13a
25	6.25 ± 0.03ae	4.73 ± 0.21bd	0.15 ± 0.03ac	0.17 ± 0.01ac	45.98 ± 0.65b
50	6.10 ± 0.07be	6.95 ± 0.29c	0.22 ± 0.02b	0.23 ± 0.04bcd	59.09 ± 0.67c
100	5.90 ± 0.07c	4.24 ± 0.18ad	0.17 ± 0.02bc	0.16 ± 0.02ad	78.95 ± 0.04d
200	5.65 ± 0.03d	3.95 ± 0.29ad	0.14 ± 0.01ac	0.14 ± 0.02a	69.21 ± 0.71e

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Table 4 Effects of fraction 3 on G. uralensis adventitious root growth and metabolites accumulation^a

Fraction 3 concentration (mg L^−1)	Growth rate	Total flavonoid content (mg g^−1)	Glycyrrhizic acid content (mg g^−1)	Glycyrrhetinic acid content (mg g^−1)	Polysaccharide content (mg g^−1)
a Mean followed by different letters within a row is significantly different at P < 0.05.
0	6.00 ± 0.07a	3.35 ± 0.25a	0.11 ± 0.01a	0.10 ± 0.01a	40.07 ± 0.68a
25	5.85 ± 0.03b	5.95 ± 0.28b	0.19 ± 0.04bd	0.21 ± 0.06bc	55.95 ± 0.65b
50	5.75 ± 0.03bd	7.59 ± 0.24c	0.29 ± 0.03c	0.27 ± 0.01b	67.09 ± 0.80c
100	5.65 ± 0.03 cd	6.19 ± 0.25b	0.21 ± 0.01be	0.22 ± 0.02bd	93.11 ± 0.83d
200	5.60 ± 0.07c	5.75 ± 0.21b	0.15 ± 0.02ade	0.17 ± 0.01 acd	75.99 ± 0.67e

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Elicitors are compounds that are usually derived from components of fungal or plant cell walls. Zhao et al. proved that a series of defense reactions including the accumulation of a range of defensive secondary metabolites in plants and in cell cultures treated with elicitors occurs.¹² In the current study, E. coli have been widely applied as sources of biotic elicitors to improve the effect on secondary metabolite production in plant tissue cultures. For example, E. coli was used as effective elicitors to enhance the triterpenoid saponins-gymnemic acids accumulation in cultured plant cells.²³ Polysaccharides as elicitor had enhanced the secondary metabolites production. Polysaccharides elicitation of cell cultures showed a considerable improving impact on flavonoids production in Hypericum perforatum cell suspensions.²⁴ In this work, polysaccharide and protein as elicitors, compounds content was significantly enhanced by adding protein elicitor. These demonstrated that the protein component isolated from E. coli was the exact component for improving the specific metabolites biosynthesis. Similar result was also observed in the induction of triterpenoid biosynthesis. The accumulation of ganoderic acids was significantly stimulated by use of protein elicitor isolated from the culture mycelia of Tuber melanosporum during Ganoderma lucidum culture.¹⁸ In this study, the content of secondary metabolites increased significantly induced by fraction 3 elicitor. The results are similar to the previous report, in Hypericum perforatum plants, a series of proteins elicitors from rhizosphere bacteria have the significant ability to trigger secondary metabolism, resulting in the accumulation of hypericin and pseudohypericin.¹⁹

3.2 Identification of bioactive compounds by HPLC-ESI-MSⁿ analysis in the G. uralensis adventitious roots

The bioactive compounds were identified by HPLC-ESI-MSⁿ analysis in control group and fraction 3 elicitor treated group. Table 5 and Fig. 1 showed that ten compounds were identified including neoliquiritin, liquiritin, isoliquiritin, liquiritigenin, gancaonin D, yunganoside K₂, glycyrrhizic acid, uralsaponin B, uralenol and glycyrrhetinic acid. When elicitor was added to the G. uralensis adventitious root, some compounds were detected. As shown in Table 5, four kinds of compounds including saponin (yunganoside K₂ and uralsaponin B) and flavonoid (gancaonin D and uralenol) were identified.

Table 5 Compounds identified in G. uralensis adventitious root by LC-MSⁿ

Peak no.	tR (min)	Identification	MS (m/z)	ESI(+)MS^n	ESI(−)MS^n	Distribution
1	3.3	Neoliquiritin	418	418.2 [M + H]^+; 294.2 [M − Glc + K]^+	417.1 [M − H]^−	Control, fraction 3
2	20.3	Liquiritin	418	457.1 [M + K]^+; 256.9 [M − Glc + H]^+	416.8 [M − H]^−;	Control, fraction 3
3	33.2	Isoliquiritin	418	419.1 [M + H]^+; 257.0 [M − Glc + H]^+	417.2 [M − H]^−; 255.0 [M − Glc − H]^−	Control, fraction 3
4	35.1	Liquiritigenin	256	257 [M + H]^+; 239.9 [M − H2O + H]^+	254.9 [M − H]^−	Control, fraction 3
5	43.7	Gancaonin D	384	384.3 [M + H]^+; 255.3 [M − C4H8O − 2CO + H]^+	382.9 [M − H]^−	Fraction 3
6	60.5	Yunganoside K2	838	861.4 [M + Na]; 666.2 [M − GlcUA + Na]^+	802.5 [M − Cl]^−	Fraction 3
7	63.8	Glycyrrhizic acid	822	471.3 [M − 2GlcUA + H]^+; 398.2 [M − 2GlcUA − 4H2O + H]^+; 255.0 [M − 2GlcUA − Glc − 2H2O + H]^+	821.3 [M − H]^−	Control, fraction 3
8	68.1	Uralsaponin B	822	804.5 [M − H2O + H]^+; 453.3 [M − 2GlcUA + H2O + H]^+;		Fraction 3
9	79.6	Uralenol	370	338.2 [M − 3H2O + Na]^+; 238.8 [M − C4H8 − CO − 3H2O + Na]^+		Fraction 3
10	103.3	Glycyrrhetic acid	470	470.9 [M + H]^+; 425.0 [M − HCOOH + H]^+	469.1 [M − H]^−	Control, fraction 3

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Fig. 1 Chemical structures of compounds identified in G. uralensis adventitious root. G1: -β-D-glu(2-1)-β-D-glu; G2: -β-D-glu(3-1)-β-D-glu; glu: β-D-glucuronosyl.

The secondary metabolites have been identified by HPLC-ESI-MSⁿ analysis in the Salvia miltiorrhiza hairy root culture. Some compounds have been detected from rosmarinic acid to cryptotanshinone in the elicitor group, while these compounds cannot be identified in control group.²⁵ It could be said that elicitor reflects its elicitor activity through induction of multiple pathways, which by an array of signal transduction mechanism trigger multiple genes for the synthesis of defensive metabolites in the form of phytochemicals.²⁶

3.3 Signal molecules (Ca²⁺, PLA₂, NO, ET, ABA, SA, JA) and secondary metabolites accumulation

Table 6 showed the highest content of total flavonoids (5.55 mg g⁻¹), glycyrrhizic acid (0.19 mg g⁻¹), glycyrrhetinic acid (0.21 mg g⁻¹) and polysaccharide (70.37 mg g⁻¹) by up to 2.11, 2.38, 2.33, 2.02-fold than the control group at 48 h.

Table 6 Effects of fraction 3 on G. uralensis adventitious root growth and metabolites accumulation at different culture time^a

Culture time (h)	Growth rate	Total flavonoid content (mg g^−1)	Glycyrrhizic acid content (mg g^−1)	Glycyrrhetinic acid content (mg g^−1)	Polysaccharide content (mg g^−1)
a Mean followed by different letters within a row is significantly different at P < 0.05.
0	5.80 ± 0.07a	2.63 ± 0.03a	0.08 ± 0.01a	0.09 ± 0.01a	34.90 ± 0.22a
1	5.65 ± 0.03ac	2.96 ± 0.07b	0.12 ± 0.01b	0.13 ± 0.00b	45.44 ± 0.23b
6	5.60 ± 0.13bcd	3.62 ± 0.04c	0.14 ± 0.01bd	0.16 ± 0.00c	50.17 ± 0.19c
12	5.55 ± 0.03bc	4.29 ± 0.04d	0.16 ± 0.03 cd	0.18 ± 0.00d	56.89 ± 0.29d
24	5.45 ± 0.03b	4.89 ± 0.05e	0.17 ± 0.01 cd	0.20 ± 0.01e	64.09 ± 0.16e
48	5.35 ± 0.03be	5.55 ± 0.21f	0.19 ± 0.01c	0.21 ± 0.01e	70.37 ± 0.26f

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The results showed that protein elicitor (fraction 3) can induce Ca²⁺, PLA₂, NO, ET, ABA, SA and JA accumulation in G. uralensis adventitious roots. Fig. 2(b) and (c) showed the increase of Ca²⁺ (0.56 mmol g⁻¹ prot) and PLA₂ (74.80 U L⁻¹) were achieved at 1 h. The highest content of NO (0.41 μmol g⁻¹ prot), ET (113.77 ng L⁻¹) and ABA (117.55 ng mL⁻¹) were obtained at 12 h in Fig. 2(d)–(f), while the SA (3.72 μg g⁻¹ FW) and JA (4.13 μg g⁻¹ FW) content were observed and reached the highest level at 24 h Fig. 2(g) and (h).

Fig. 2 (a). Schematic illustration of the sequential signaling pathways and other oxylipins, and their mediation of elicitor signal transduction leading to accumulation of plant secondary metabolites. 12,13-EOT, 12,13(S)-epoxyoctadecatrienoic acid; 13-HPOT, (13S)-hydroperoxyoctadecatrienoic acid. Accumulation of Ca²⁺ (b), PLA₂ (c), NO (d), ET (e), ABA (f), SA (g), JA (h) in adventitious roots of G. uralensis as affected by fraction 3 concentrations.

Elicitor may bind to a specific receptor in the cell plasma membrane, and then activate the effectors, such as cytosolic Ca²⁺ spiking, signal molecules (NO, SA, JA, ET, ABA) production, defense response gene expression and secondary metabolite accumulation.¹² In Cupressus lusitanica suspension cells cultures, Ca²⁺ played a significant role in an early stage of the elicitation process. Ca²⁺ carried the elicitor signal and in turn regulates the biosynthesis of β-thujaplicin.²⁷ The activity of PLA₂ was found to enhance in plant cells after treatment with glycoprotein elicitor, leading to trigger alkaloid biosynthesis in Eschscholzia californica cell cultures.²⁸ Elicitor also induced the accumulation of NO, SA and JA in plant cell for the accumulation of puerarin in the Pueraria thomsonii Benth.²⁹ In Catharanthus roseus cell suspension cultures, elicitor triggered the ABA generation which is essential for catharanthine biosynthesis.³⁰ In Cupressus lusitanica cell cultures, elicitor stimulated ET production, resulting in ET signal pathways acting as elicitor signal transducer for β-thujaplicin accumulation.³¹ This is similar to the study, there was certain relationship between signal molecules (Ca²⁺, PLA₂, NO, ET, ABA, SA, JA) with the secondary metabolites level in plant cell.

3.4 The expression of defense genes (NOS, PR1, PAL, LOX, PDF1.2)

Fig. 3(a) and (b) shows the expression level of defense genes after protein elicitor (fraction 3) treatment in the adventitious roots of G. uralensis. Among the NO-signaling gene, NOS was extremely up-regulated and resulted in 2.12-fold increase over the control group. In SA-signaling pathway, the PR1 and PAL were extremely enhanced by 2.19 and 2.43-fold by protein elicitor. For JA signaling, the LOX and PDF1.2 had a 2.30 and 2.17-fold up-regulation in protein elicitor treatment.

Fig. 3 Effects of fraction 3 on the expression of defense genes in G. uralensis adventitious root (a and b).

The plant defense response forms a complex phytohormones-mediated signaling network, including NO, SA, JA pathways. Plasmopara viticola stimulated the expression of NOS genes as well as bioactive compounds accumulation by triggering NO-signaling pathways.¹³ In Nicotiana tabacum, applications of elicitor increased the levels of PAL, PDF1.2 and PR1 gene expression.³² Erwinia carotovora elicitor induced the expression of defense-related genes, including PR-1, LOX, PAL in Physcomitrella patens.³³ In this study, the expression of NOS, PR1, PAL, LOX, PDF1.2 up-regulated significantly, which indicated that the signal molecules (NO, SA, JA and ABA) generated after treatment with protein elicitor (fraction 3), resulting in the enhancement of secondary metabolites content. The result is consistent with previous studies. Cerato-platanin triggers SA-signaling pathways, as revealed by the expression of PR genes and induced the biosynthesis of camalexin.³⁴

3.5 The expression of functional genes

Fig. 4(a) and (b) showed the effect of protein elicitor (fraction 3) on HMGR, FPS, GPS, SQS, SE, β-AS, CYP88D6 and CYP72A154 mRNA levels in the roots. Under elicitors treatment, the expression levels of HMGR, FPS, GPS, SQS, SE, β-AS, CYP88D6 and CYP72A154 genes were elevated by 2.16, 2.63, 2.12, 2.14, 2.31, 2.37, 2.12 and 2.55-fold over the control group. The relative expression levels of C4H, CHI were dramatically enhanced by 2.17 and 2.20-fold in treatment group (Fig. 5(a) and (b)).

Fig. 4 The proposed pathways of glycyrrhizic acid biosynthesis in G. uralensis (a) and effects of fraction 3 on the expression of functional genes in the glycyrrhizic acid biosynthetic pathway in G. uralensis adventitious root (b).

Fig. 5 Effects of fraction 3 on the expression of functional genes in the phenylalanine metabolic pathway in G. uralensis adventitious root (a and b).

In this study, the glycyrrhizic acid content increased significantly, which was consistent with the increased expression levels of genes including HMGR, FPS, GPS, SQS, SE, β-AS, CYP88D6 and CYP72A154 in the G. uralensis adventitious roots. It turned out that gene expression levels up-regulation involved in glycyrrhizic acid biosynthesis lead to the increase of glycyrrhizic acid content. A similar phenomenon was observed in Panax ginseng C. A. Meyer suspension cell, genes (SQS, SE and β-AS) expression level was significantly enhanced by an elicitor, resulting in saponin accumulation.³⁵ In Medicago sativa cell cultures, elicitor enhanced the expression levels of C4H and CHI involved in the phenylalanine metabolic pathway and the bioactive compounds content.³⁶ It is consistent with the result of this study. The enhancement of flavonoids content was correlation with the increased expression levels of genes including C4H and CHI.

4. Conclusion

In conclusion, B. subtilis, P. fellutanum and E. coli enhanced the metabolites contents in G. uralensis adventitious root. Protein fragment more than 10 kDa significantly enhanced the content of total flavonoids (5.55 mg g⁻¹), glycyrrhizic acid (0.19 mg g⁻¹), glycyrrhetinic acid (0.21 mg g⁻¹) and polysaccharide (70.37 mg g⁻¹), which attributed to the enhancement of endogenous signaling activity, defense-related genes expression and functional genes expression. In addition, HPLC-ESI-MSⁿ analysis demonstrated that some compounds have been detected in elicitor group, including gancaonin D, yunganoside K₂, uralsaponin B and uralenol.

Abbreviations

E. coli	Escherichia coli
FW	Fresh weight
JA	Jasmonic acid
SA	Salicylic acid
NO	Nitric oxide
PLA2	Phospholipase A2
ET	Ethylene
ABA	Abscisic acid
HMGR	3-Hydroxy-3-methylglutaryl coenzyme A reductase
GPS	Geranyl diphosphate synthase
FPS	Farnesyl diphosphate synthase
SQS	Squalene synthase
SE	Squalene epoxidase
β-AS	β-Amyrin synthase
C4H	Cinnamate 4-hydroxylase
CHI	Chalcone isomerase

Acknowledgements

This research was funded by ministry of education new teacher fund, China [20130032120084], Tianjin Youth Fund (15JCQNJC13300).

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Footnote

† Electronic supplementary information (ESI) available: Table S1 is primers used for amplification of transcripts by RT-PCR. Tables S2–S4 is the content of metabolites. See DOI: 10.1039/c6ra16903a

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