The influence of salicylic acid elicitation of shoots, callus, and cell suspension cultures on production of naphtodianthrones and phenylpropanoids in Hypericum perforatum L.
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Hypericum perforatum is a well known medicinal plant. The main pharmacological properties are due to the presence of naphtodianthrones such as hypericin and pseudohypericin. Unfortunately the levels of these compounds vary under different environmental conditions. Elicitation of in vitro cultures is a useful approach to enhance and extend production of desirable products. Therefore, the effects of salicylic acid were characterized on different explants of H. perforatum L. (cells, calli and shoots) cultured in vitro. It appears at first that salicylic acid did not affect growth and development of these explants. In addition, the production of both hypericin and pseudohypericin has doubled in elicited cell suspension cultures but not in the two other cultures. Furthermore, phenylpropanoids that are among the most frequently observed metabolites affected upon treatment of in vitro culture material with elicitors, were produced and the enzymatic activities of phenylalanine ammonia lyase and of chalcone isomerase were stimulated upon elicitation. These effects were dependant of the type of in vitro culture, the concentration of salicylic acid and the duration post-elicitation. The H. perforatum cells were globally more sensitive to salicylic acid elicitation when maintained in an undifferentiated state and particularly in cell suspension cultures. In the absence of glands considered as the sites of naphtodianthrones biosynthesis, cells and calli were capable of producing these compounds. This implies that salicylic acid could act at biosynthesis level but not for the accumulation of both hypericin and pseudohypericin. Consequently, the regulation of this process is more complex than cited in the literature involving the responsibility of only Hyp-1 gene, encoding a hypericin biosynthetic enzyme, cloned and characterized from H. perforatum.
KeywordsHypericin Phenolic compounds Pseudohypericin Salicylic acid
High-performance liquid chromatography
Phenylalanine ammonia lyase
Hypericum perforatum is a medicinal plant widely used for the treatment of mild and moderate depression, inflammation and wound healing (Di Carlo et al. 2001). Hypericum is also considered as an antiviral and anticancer drug in photodynamic therapy (Axarlis et al. 1998; Agostinis et al. 2002). The pharmacologically demonstrated that major bioactive compounds in Hypericum extracts are naphtodianthrones, for example hypericin and pseudohypericin, and their precursors: protohypericin, protopseudohypericin and cyclopseudohypericin (Falk 1999). It was currently suggested that they are localized in the small black glandular structures located on flower petals, stamens, leaves and stems (Zdunek and Alfermann 1992; Kornfeld et al. 2007). Currently, field-grown plant materials are generally used for commercial Hypericum products. However, quality controls of active ingredients, safety and environmental conservation have recently become serious issues in medicinal plant production. Southwell and Bourke (2001) reported that the contents of both naphtodianthrones in Hypericum leaves vary up to 50-fold in summer, and in winter grown plants. Therefore, 17-fold and 13-fold differences in hypericin and pseudohypericin amounts, respectively, can be found in different phytopharmaceutical preparations (Greeson et al. 2001). It has been reported that naphtodianthrone production is influenced by nitrogen availability (Briskin and Gawienowski, 2001), temperature season (Southwell and Bourke, 2001), plant environment (Zobayed et al. 2006) and UV-B radiation levels (Germ et al. 2010). Thus an increase in light intensity and a decrease of nitrogen supply resulted in a parallel increase of the number of leaf dark glands and hypericin content (Briskin and Gawienowski 2001). Recently, in vitro culture systems to have become worth studying as a useful alternative because of increased demand by the pharmaceutical industry and the unequal quality of products caused by environmental factors. Moreover, in vitro cultures play an important role in the studies of plant secondary metabolism. The available data suggests that secondary metabolite profile of Hypericum in vitro cultures can differ from those of whole plants (Dias et al. 1998). Hypericin contents are invariable and usually in lower amounts in vitro cultured cells and tissues compared to whole plant (Kirakosyan et al. 2000; Walker et al. 2002; Gadzovska et al. 2005).
Hypericin is described as a polyketide derived quinone named naphtodianthrone, alluding to the configuration of the compound constructed from two tricyclic anthrones called emodine anthrone (Katz and Donadio 1993). Little is known about the biosynthesis of hypericin other than it lays on the polyketide pathway, presumably through emodin, an anthraquinone, and with protohypericin as the penultimate precursor, and through the successive condensation of small carboxylic acids such as acetate and malonate (Zobayed et al. 2006). A gene named HYP-1 encoding for the phenolic coupling protein which is assumed to be involved in conversion of emodin to hypericin, has been cloned and characterized from Hypericumperforatum cell cultures (Bais et al. 2003; Košuth et al. 2007). However, the coordination of the production of naphtodianthrones with other secondary metabolites still remains unknown. Therefore, the use of elicitors has been suggested as an alternative for the stimulation of the production of biologically active compounds. Elicitors are considered as signal molecules that activate the signal-transduction cascade and lead to the activation and expression of genes related to the biosynthesis of secondary metabolites (Zhao et al. 2005). Few studies have been carried out to investigate the effects of different elicitors on the accumulation of secondary metabolites in Hypericum in vitro culture systems. Cell suspension cultures from H. perforatum have been tested for their ability to produce naphtodianthrones upon treatment with mannan, β-1,3-glucan, pectin, jasmonic acid, methyl jasmonate, salicylic acid (SA) and fungal elicitors from Colletotrichumgloeosporioides and Phytophtora cinnamoni (Kirakosyan et al. 2000; Sirvent and Gibson 2002; Walker et al. 2002; Conceição et al. 2006; Gadzovska et al. 2007). SA is an endogenous regulatory signal molecule that elicits plant resistance to pathogens and stimulates secondary metabolite productions (Wang et al. 2004). Although SA is not a universal inducer of plant defensive metabolite production, it induces gene expression related to biosynthesis of some classes of secondary metabolites in plants (Schenk et al. 2000; Taguchi et al. 2001). Phenylpropanoid/flavonoid biosynthetic pathways are among the most frequently observed metabolic activities that are induced upon treatment of plant tissue or cultured cells with elicitors (Barber et al. 2000). The enhanced secondary metabolite production is usually associated with a rapid, transient increase in activities of key enzymes of the phenylpropanoid/flavonoid pathway such as phenylalanine ammonia lyase (PAL, EC 18.104.22.168) and chalcone isomerase (CHI, EC 22.214.171.124) (Dixon et al. 2002). SA induces the production of alkaloids in Catharanthus roseus (Godoy-Hernández and Loyola-Vargas 1997), lubimin in Hyoscyamus muticus (Mehmetoglu and Curtis 1997), capsaicin in Capsicum frutescens (Sudha and Ravishankar 2003), taxol in Taxuschinensis cell suspension cultures (Wang et al. 2004). Moreover, as Colletotrichum lindemuthianum, SA stimulates the activities of CHI and PAL in common bean (Campos et al. 2003).
We have previously studied the overproduction of naphtodianthrones in H. perforatum callus cultures, shoots and plantlets. For this purpose, we have established these cultures on media containing different phytohormones such as N6-benzyladenine, indole-3-acetic acid and indole-3-butyric acid (Gadzovska et al. 2005). In addition, Hypericum cell suspension cultures upon elicitation with jasmonic acid have been studied for their ability to produce phenylpropanoids and naphtodianthrones upon elicitation with jasmonic acid (Gadzovska et al. 2007). However, the biosynthetic pathway and mechanisms for stimulation of hypericin and pseudohypericin production still remain unknown. Even if some biotic and abiotic factors can regulate hypericin and pseudohypericin production (Karakas et al. 2009), the role of elicitor supplementation in the culture medium need further investigation to complete those previously done (Liu et al. 2007a, b). Nevertheless, a polyketide pathway including type III polyketide synthases has been suggested to be involved (Karppinen 2010).
The present work was done to determine whether secondary metabolite production could be enhanced by exogenous application of SA in three Hypericum in vitro systems (shoots, calli and cells). This study has been focused on the effects of SA treatments on the production of naphtodianthrones (hypericin and pseudohypericin) and various phenylpropanoids (phenolic compounds, flavonols, flavanols and anthocyanins) according to the developmental stages of in vitro cultures, which seem to determine the levels of some of these compounds (Karppinen et al. 2006; Filippini et al. 2010). Activities of two key enzymes of the phenylpropanoid/flavonoid pathways, PAL and CHI were monitored to estimate channelling in the different metabolic pathways, in so far as, the conditions of culture could affect polyphenol biosynthesis in this species (Bruni and Sacchetti 2009). Finally, the expression of the Hyp-1 gene, encoding a hyperycin biosynthetic enzyme could be detected in our H. perforatum cells elicited or not with salicylic acid in equal amounts. Even if work had been carried out previously on these topics, our work provided complementary information. Sirvent and Gibson (2002) published results on the effects of 1, 2.5, and 15 mM SA on plantlets obtained from meristems. In our experiments, the analysis concerned other models of material (shoots, calli and cell suspension cultures) and a different range of SA concentrations (50, 100 and 250 μM). In the same way, Walker et al. (2002) used similar concentrations of SA to us, for elicitation of H. perforatum, but measured only their consequences after 28 days of culture on hypericin contents. Our approach consisted in the determination of hypericin, but also pseudohypericin and phenolic compounds all along the culture (1, 4, 7, 14 and 21 days after elicitation). Finally, Conceição et al. (2006) followed the influence of only 25 μM SA on contents of phenolics, flavonols and flavones. Our study referred to the effects of several SA concentrations on contents of various types of phenolic compounds (total phenolics, flavonols, but also flavanols and anthocyanins) through the culture duration. At the same time, work with other species of Hypericum (hirsutum and maculatum) was conducted on the effects of elicitors such as SA, on production of secondary metabolites in shoot cultures (Coste et al. 2011). It was the same with the species rumeliacum, tetrapterum and calycinum with which the influence of vitamins on polyphenolic content was studied (Danova et al. 2012). Futhermore, it must be noted that other data was obtained about secondary metabolism in H. perforatum but using very a particular in vitro culture system such as non-aerated liquid medium (Savio et al. 2012).
Materials and methods
As for a previous study (Gadzovska et al. 2007), seeds from a wild genotype of H. perforatum were collected at about 1,500 m on mount Bistra, Republic of Macedonia. Seeds were washed overnight, air dried, surface sterilized with 1 % NaOCl for 10 min, rinsed 3 times in sterile deionized water and cultured on MS/B5 medium. No growth regulator was added but the medium was supplemented with 3 % sucrose and its pH was adjusted to 5.6 before solidification with 0.7 % Difco Bacto Agar. Sterilization was then made by autoclaving 20 min at 120 °C. All In vitro cultures were placed in a growth chamber at 22 ± 1 °C, under a photoperiod of 16 h (photon flux density: 700 μmol m−2 s−1).
After in vitro germination of these seeds, apical segments containing 2–4 leaves were aseptically excised from 2 to 3 week-old plantlets and used as explants to establish shoots. Explants were cultured in 100 mL flasks containing 40 mL of MS/B5 medium with 3 % sucrose, 200 mg L−1 casein enzymatic hydrolysate and supplemented with 0.5 mg L−1 benzyladenine (BA). Then, after 3 weeks, shoots (20–25 mm long) were isolated from these obtained plantlets and subcultured for 3 weeks in the same conditions as previously (1.5–2 g). Through these cultures, samples of material were harvested on day 7, 14 and 21, frozen in liquid nitrogen, lyophilized and then stored at −80 °C.
The first pair of leaves from 2 weeks-old plantlets obtained after in vitro germination, were aseptically excised and used as explants to establish callus cultures. They were cultured in Petri dishes on MS/B5 medium supplemented with 1.0 mgL−1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg L−1 BA, 0.1 mg L−1, α-naphtaleneacetic acid (NAA), 3 % sucrose and 0.7 % agar (Bacto Agar Difco). Subcultures of calli (1.5–2 g) were carried out every 14 days.
Cell suspension cultures
Cell suspension cultures were established from callus cultures after two subcultures. For this, green calli (1.5–2 g) were inoculated in 250 mL Erlenmeyer flasks containing 100 mL liquid MS/B5 medium, composed of macro and oligonutriments (MS) of Murashige and Skoog (1962) and organic addenda B5 of Gamborg et al. (1968), supplemented with 1.0 mg L−1 2,4-D, 0.5 mg L−1 BA, 0.1 mg L−1 NAA and 3 % sucrose. Cultures were maintained on a rotary shaker at 100 rpm in the growth chamber. After 2 weeks, the cells released from calli were transferred into 4 volumes of fresh liquid medium and subcultured every 2 weeks.
Cells were photographed with a numeric camera (PDR-M65, Digital Still Camera, Toshiba), or with a photonic microscope Olympus BH-2 (at 400×). Cell viability was determined by vital staining with methylene blue stain as described by Bonora and Mares (1982). The cells were then harvested by vacuum filtration on days 1, 4, 7, 14 and 21 of culture, weighted for growth analysis, frozen in liquid nitrogen or lyophilized and stored at −80 °C, until analysis.
The subcultures of shoots and calli were done on medium supplemented with SA 50, 100 and 250 μM. Controls were cultivated in the same conditions but without SA as elicitor. Treatments of cell suspension cultures were performed 7 days after subculture, when the cells were in the log phase of growth. At the same time, control suspensions were inoculated with sterile double distilled water and used as reference during this study. Cell suspensions cultivated on MS/B5 medium were also used as control.
Extraction and quantification of secondary metabolites
Phenolic compounds extraction and quantification were performed as previously reported (Causevic et al. 2005; Gadzovska et al. 2007). Briefly, phenolic compounds were extracted from freeze-dried lyophilized and powdered plant material (0.2–0.5 g) with 80 % (v/v) methanol in ultrasonic bath for 30 min at 4 °C. Total phenol content was determined when methanolic extract were mixed with Folin–Ciocalteau reagent (Carlo Erba Reagenti, Rodano, Italy) and 0.7 M Na2CO3. Samples were incubated for 5 min at 50 °C and then cooled for 5 min at room temperature. Absorbance was measured spectrophotometrically at 765 nm. The concentration of total phenolic compounds was calculated using (+)-catechin (0–10 mg mL−1) as a standard.
Flavonol contents were determined in methanolic extracts with the method described by Markham (1989). The extracts were filtered through Sep-pack C18 cartridges (Waters) to exclude chlorophyll and carotenoid pigments (solid-phase extraction). Spectrophotometric measurements of the absorbance were made at 360 nm. Molar extinction coefficient of quercetin (ε360 = 13.6 mM−1 cm−1) was used to determine total flavonol contents.
Flavanol contents were determined in methanolic extracts with 4-(dimethylamino)-cinnamaldehyde reagent (Treutter et al. 1994) which was added to the diluted (1:10–1:100, v/v) extracts. The mixtures were incubated for 1 h at room temperature. Absorbance was measured at 637 nm. The content of flavanols was calculated using (+)-catechin (0–20 μg mL−1) as a standard.
Anthocyanin determination was performed as described by Giusti et al. (1999). Anthocyanins were extracted from freeze-dried lyophilized and powdered plant material (0.2–0.5 g) with 2 mL solution of 1 % HCl/CH3OH (15/85, v/v) ultrasonicated for 60 min at 4 °C and then centrifugated at 20,000×g for 30 min. The absorbance of supernatant was measured at 530 nm. The anthocyanin content was calculated using the molar extinction coefficient of cyaniding-3-glucoside (ε530 = 34,300 M−1 cm−1) in acidic methanol.
High performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry analyses of secondary metabolites
HPLC analyses of phenolic compounds in methanolic extracts were performed on an apparatus Hewlett Packard Series HP 1100 consisting of a G1311A pump equipped with G1315A photodiode-array detector (Gadzovska et al. 2005). Methanolic extracts were filtered through Sep-pack C18 cartridges before HPLC analysis. Separation of the compounds was performed on a Hypersil reversed-phase C18 column (150 × 4.6 mm, 5 μm, Interchim, France) at a flow-rate of 1 mL min−1 with 20 μL injected volume. The column was used at ambient temperature. Data was acquired and processed by the HP Kayak Navigator software. Composition of the extracts was separated by a linear gradient program with the following solvents: A, water:acetic acid (99.5:0.5, v/v) and B, methanol:acetonitrile (1:1, v/v). Linear gradient combinations were started with 100 % A (0–10 min), 90 % A and 10 % B (11–22 min), 82 % A and 18 % B (23–27 min), 75 % A and 25 % B (28–30 min), 70 % A and 30 % B (31–35 min,), 50 % A and 50 % B (36–45 min), 40 % A and 60 % B (46–55 min), 30 % A and 70 % B (56–60 min), 100 % B (61–65 min). The total run time was 65 min. Chromatograms were recorded at 280 nm. The quantification of the compounds was done by the external standard method, using a solution of (+)-catechin (10 μg mL−1) in methanol. Identification of several phenolics was also assessed by co-migration and spectra analyses with commercial standard of caffeic acid, chlorogenic acid, cinnamic acid, p-coumaric acid, ferrulic acid, hyperforin, kaempferol, luteolin, quercitrin, rutin and xanthone.
Hypericin and pseudohypericin extractions were performed as described by Gadzovska et al. (2005 and 2007). HPLC analyses were carried out at 25 °C on a Hypersil reversed-phase C18 column (150 × 4.6 mm, 5 μm, Interchim, France). Standard solutions of hypericin (1–100 μg mL−1) were prepared from pure commercially available standard of hypericin (Sigma, France). Pseudohypericin was isolated from plant extracts and purified onto semi preparative Nucleosil C18 column (250 × 10 mm, 5 μm, Interchim, France). Standard solutions of pseudohypericin were prepared in a concentration range of 0–100 μg.mL−1. Chromatograms were performed at 590 nm. All reagents were HPLC grade from Merck (Germany).
As previously described by Gadzovska et al. (2007), mass spectra of phenolic compounds and naphtodianthrones were acquired using a LCQ Deca mass spectrometer, equipped with an atmospheric pressure chemical ionization source (Thermo-Finnigan). Thus, phenolic compounds mass spectra were acquired in the negative ion mode. The following APCI inlet conditions were applied: heated vaporization temperature, 450 °C; heated capillary temperature, 300 °C; sheath gas, 80 psi; auxiliary gas, 10 psi and discharge current, 5 μA. Collision induced dissociation spectra were recorded at relative collision enegy of 30 %. Phenolic compounds mass spectra were acquired in the negative ion mode, scanning from m/z 150–600. Operating conditions were: sheath gas, 65 psi; auxiliary gas (nitrogen), 10 psi; ESI needle voltage 4.5 kV; capillary temperature, 250 °C; capillary voltage, −12 V. Compounds were introduced to the fused silica-lined ESI needle by syringe pump at 5 μL min−1 flow rates. Data acquisition and processing were performed with Xcalibur software (version 1.2).
Enzyme extraction and assays
The extraction procedure was based on the method of Causevic et al. (2005) and adapted as in Gadzovska et al. (2007). For enzyme assay, frozen samples were ground in automatic grounder (MM 200 Retch). The enzyme extract was prepared by homogenizing 1 g of sample in 2 mL 0.1 M KH2PO4/K2HPO4 buffer at pH 8.0, containing 2 mM ethylenediamine tetra-acetic acid, 1.4 mM β-mercaptoethanol, and 1 % (w/v) polyvinylpyrrolidone. The homogenate was centrifuged at 20,000×g for 20 min at 4 °C. The supernatant was collected for determination of protein content and enzyme assay. Protein contents in enzyme extracts were performed with a Bio-Rad Protein Assay Reagent. Bovine serum albumin was used as a standard (0–10 mg mL−1).
PAL assay was determined according to Latunde-Dada and Lucas (2001). The reaction mixture contained 2 % (w/v) solution of l-phenylalanine in 50 mM Tris–HCl at pH 8.8 and enzyme extract. Enzyme assay mixtures were incubated at 40 °C for 60 min. PAL activity was determined by measuring the rate of formation of trans-cinnamic acid as an increase in absorbance at 290 nm.
CHI assay was based on the method described by Liu et al. (1995). CHI was assayed in 60 mM KH2PO4/K2HPO4 buffer at pH 8.0, containing 50 mM KCN to inhibit peroxidase activity. Reaction was initiated by mixing enzyme extract and 2′,4,4′,6-tetrahydroxychalcone. Enzyme assay mixture was incubated at 30 °C for 45 min. The kinetics of the reaction was monitored by measuring the decrease in absorbance at 400 nm.
Enzymatic activities were expressed as pkat.mg−1 proteins.
Semi-quantitative RT-PCR analysis
Total RNAs were isolated using Nucleospin® RNA Plant (Macherey–Nagel, Hoerdt, France) and reverse transcribed using High capacity cDNA reverse transcription kit (Applied Biosystems). Constitutively expressed EF-1α gene was used as internal standard to normalize the amount of mRNA in PCR reaction according to Košuth et al. (2007). A control without reverse-transcriptase during the cDNA synthesis was performed to confirm the absence of genomic DNA in all our total RNAs preparations. Primers for HYP-1 gene (AY148090) were designed similar to those published by Košuth et al. (2007):. The number of PCR cycles was adjusted to avoid reaching saturation. PCR products were visualised on 8 % polyacrylamide gel after ethidium bromide staining and quantified with imaging software (ImageTool for Windows version 3.00). Three biological and two technical replicates were done for each gene and in vitro culture.
The experiments were independently repeated twice under the same conditions and all analyses were performed in triplicate. Error bars of graphs show the standard error of mean value (±SE). The statistical analyses were performed with the SPSS statistical software program (SPSS version 11.0.1 PC, USA, IL). Means were compared by one-way ANOVA (GML procedure). All statistical tests were considered significant at P ≤ 0.05. To avoid overcharging the figures, stars indicating significant differences between data, did not appear. But, in the text, a result considered different to another one, was the result of a statistical analysis (P ≤ 0.05).
Effects of SA on phenotype, viability and growth of Hypericum in vitro cultures
In the absence of SA, typical growth curves for fresh mass could be noted with control cultures of cell suspension cultures (Fig. 1d) and calli (Fig. 1e). Significant differences were observed in elicited materials at 7 or 14 days post-elicitation in calli and cell suspension cultures, respectively. However, in elicited cell suspension cultures a lower fresh mass was observed compared to control, while elicited calli showed an increase fresh mass compared to its control. Furthermore, these effects were accentuated with the increase of SA concentration for both cultures (Fig. 1d, e).
Levels of naphtodianthrones in SA elicited cultures
Linear correlations (Pearson’s coefficient) measured between secondary metabolite productions in H. perforatum in vitro cultures during the time of post elicitation
Levels of phenylpropanoid compounds and activities of PAL and CHI in SA elicited cultures
Amounts (in mAU g−1) of phenolic compounds in H. perforatum cell suspensions at day 14 non elicited or elicited by 100 μM JA or SA
1,100 ± 160
1,370 ± 180
787 ± 90
44 ± 13
48 ± 17
34 ± 2
26 ± 11
29 ± 13
20 ± 2
35 ± 14
34 ± 15
27 ± 2
9 ± 5
20 ± 10
15 ± 2
16 ± 6
33 ± 13
26 ± 3
20 ± 6
38 ± 12
27 ± 4
65 ± 22
95 ± 32
90 ± 3
60 ± 20
130 ± 43
60 ± 4
13 ± 5
27 ± 13
21 ± 1
16 ± 6
19 ± 7
15 ± 1
31 ± 3
28 ± 12
22 ± 2
88 ± 30
182 ± 63
68 ± 10
53 ± 20
62 ± 22
21 ± 3
44 ± 7
197 ± 37
153 ± 33
290 ± 70
490 ± 83
225 ± 23
48 ± 20
154 ± 55
120 ± 6
35 ± 7
27 ± 4
474 ± 70
240 ± 47
186 ± 36
97 ± 40
106 ± 27
202 ± 83
250 ± 80
194 ± 19
158 ± 63
In control calli (Fig. 4e), the amount of endogenous phenolic compounds did not significantly change during the 21 days. In SA-treated calli their levels were always higher than those of control with a maximum until 1 day post-elicitation and 50 μM SA (twofold increase). In calli, transient accumulation of flavonols (Fig. 4f), flavanols (Fig. 4g) and anthocyanins (Fig. 4h) could be noted particularly for the lowest SA concentrations tested.
In control shoots, the amounts of phenolics (Fig. 4i) were of 8 mg g−1 dry material at 1 day of post-elicitation and reached about 17 mg g−1 dry material after 21 days of elicitation. In SA-elicited shoots, levels of phenolics were stable or even lower than the corresponding controls, particularly for the lowest SA concentrations tested. In these cultures, i.e. shoots (Fig. 4j, k, l) all the analysed secondary metabolites were unchanged or decreased in comparison with control, particularly for the lowest SA concentrations tested.
mRNA accumulation of Hyp-1 gene in SA elicited cells
Effects of SA-elicitation on Hypericum in vitro cultures
First, it can be noted that, at the end of culture (21 days), the viability of control cells slightly decreased. In presence of SA in the culture medium, this viability also decreased but more than without this compound. At the same time, fresh weight of treated cells progressively decreased, probably on account of the lower number of cells. Consequently, as previously established with jasmonic acid (Gadzovska et al. 2007), SA negatively affects growth of H. perforatum cell suspension cultures.
Secondly, as also described with H. perforatum cell suspension cultures elicited by jasmonic acid (Gadzovska et al. 2007), all throughout the culture, no structure for any dark gland differentiation appeared either in control or in SA treated cells. Browning of the suspensions was also observed here at the end of the treatment for all SA concentrations tested. On the other hand, calli do not show such alterations. If dark glands differentiation does not occur, fresh weight is not significantly affected by SA whatever the used concentration (Fig. 1c). Concerning shoots, the use of SA at low (50 μM) but also at higher (250 μM) concentrations had no consequence on the development: here, dark oil glands and adventitious roots (Fig. 1d), were differentiated.
Effects of SA-elicitation on naphtodianthrones according to the cell differentiation state
As presently established, when cells were maintained in an undifferentiated state, SA grandly disturbed development of Hypericum. On the contrary, applied to organized structures (shoots), the effects of this elicitor were obviously smaller. Are these modifications of development depending on cell differentiation connected with changes in naphtodianhtrone production? For this data, the most efficiency could only result, either, in a possible more intimate contact in cell in suspension than in other systems, or in possible difficulties to penetrate organized structures (shoots for example) independently of the differentiation state. However, in differentiated cultures, stimulation of PAL and CHI by SA, suggests that this elicitor penetrates in explants. Indeed, penetration of SA could probably be easier in cells in suspension than in calli or shoot cultures. One explanation could be represented by the intimate contact between cells and elicitor in suspensions or due to the absence of agar in the corresponding liquid medium. Another one could be the differences in osmotic pressures between media.
The production of hypericin and pseudohypericin by H. perforatum is strongly dependent on genetic and environmental factors (Büter et al. 1998). Production of naphtodianthrones can be stimulated by elicitors such as mannan applied to shoot cultures, as reported for the first time by Kirakosyan et al. (2000). Generally, plant defences can be stimulated by SA (Hammerschmidt and Nicholson 1999; Hammerschmidt and Smith-Becker 1999) which induces various responses including pathogenesis-related proteins (Hammond-Kosack and Jones 1996). If SA appears as a potent inducer of plant defence responses including the synthesis of pathogenesis-related proteins, it is probably because it inhibits catalase activity as shown in tobacco (Conrath et al. 1995). Indeed, intracellular levels of reactive oxygen species increase, playing a role in the induction of defence response such as pathogenesis-related protein gene expression. Xu et al. (2008) established that production of hypericin by treated H. perforatum cell suspension cultures in response to a heat shock resulted in a synergism between H2O2 and NO. Consequently, in our experiments, inhibiting catalase, SA added in the medium could induce accumulation of H2O2, resulting in an increase of hypericin. Nevertheless, our data (Table 2) showed that undifferentiated systems i.e. cell suspension cultures (Fig. 2a, b) and calli (Fig. 2c, d), responded to SA by increasing naphtodianthrone levels. In an unexpected way, in shoots, SA did not induce such an increase. Fields et al. (1990) showed that only leaves of H. perforatum which are dotted with dark glands near the edges of these leaves or other aerial organs are able to produce naphtodianthrones. Moreover, Pasqua et al. (2003) established with a particular cultivar of H. perforatum (cv. Topas), that, at first, biosynthesis of hypericins is connected with the formation of secretory structures in regenerated vegetative buds. Additionally, a further degree of leaf development is necessary to stimulate the production of biological active metabolites. Cells in suspension and calli did not present these dark glands on the contrary to plantlets obtained from shoots. Even if cells, calli and shoots contain hypericin and pseudohypericin (Gadzovska et al. 2005), only in cells and calli did the levels of these compounds increase when SA was added to the culture medium. Shoots with dark glands did not positively respond to SA applications. To try to explain the production of hypericins in two selected H. perforatum shoot cultures, Kornfeld et al. (2007) suggested that differences in black gland structure can be considered. On the other hand, to determine the possible site(s) of hypericin biosynthesis, Košuth et al. (2007) analysed the expression level of the HYP-1 gene encoding for the phenolic coupling protein which is assumed to be involved in conversion of the precursor emodin to hypericin. Thus, these authors compared the expression of this gene in early stages of ontogeny of H. perforatum seedlings in different plant parts with regard to presence or absence of the black nodules. In agreement, we found that Hyp-1 mRNA could be detected in cells and that SA elicitation did not influence this accumulation. Their results may either indicate that the final stages of hypericin biosynthesis takes place in different parts, mainly in the roots which are not associated with the dark glands and primarily serve for hypericin accumulation. It was possible to assume that cells in suspension or cells of calli were able to synthesize naphtodianthrones which accumulate into leaves. SA should intervene at the site of biosynthesis i.e. in cells or in calli but not at the place of accumulation. Thus, the levels of naphtodianthrones would increase in cells and calli and not in shoots.
Effects of SA on the secondary metabolism channelling
SA elicitation of H. perforatum cells (Fig. 4a), calli (Fig. 4e) and shoots (Fig. 4i), increased the detected quantities of total phenolic compounds in comparison with untreated explants. Particularly in cell suspension cultures (Fig. 4b, c), but also and clearly less marked in calli (Fig. 4f, g), flavonols and flavanols largely increase, whereas anthocyanins (Fig. 4d, h) did not really change. In shoots, these different compounds (Fig. 4 j, k, l) were not really affected by SA treatment. Therefore, as for growth and development, it appeared that the level of differentiation took a part in the response to SA in terms of enrichment in phenolic compounds. Such an accumulation of phenolic compounds in H. perforatum cells has been previously reported after some elicitations, particularly with Colletotrichum gloeosporioides, the pathogen responsible of the fungal disease anthracnose (Conceição et al. 2006). Indeed, elicited by this fungal elicitor, suspension cultures showed a significant increase in xanthone accumulation. The possible importance of xanthone as a compound of defence reactions of H. perforatum against biotic stress was discussed by the authors. However, it has been recently proposed that the composition of the culture medium can modify the levels of endogenous phenolics of H. perforatum explants (Danova et al. 2010). For every particular experiment (effects of SA on cell suspension cultures for instance, or effects of SA on calli, or even, effects of SA on shoots) a single medium was used. Thus, the observed differences in phenolics could not result from artifacts generated by the composition of the medium. Our experiments were always conducted using the same medium, thus, the observed differences could not result from this factor. Therefore it is possible to consider that total phenolics and flavonoids but not anthocyanins, contribute eventually as xanthones to the defence reactions of H. perforatum. In the common bean, Campos et al. (2003) showed that PAL and CHI could be induced by SA or by the fungus Colletotrichum lindemuthianum. If such a consequence of applied SA existed in H. perforatum, it could be possible that the changes in phenolic levels would consist in a response to the enzymatic modifications.
In the presence of a pathogen, plants develop a vast array of metabolic defence responses sequentially activated in a complex multicomponent network that may be local and/or systemic (Hahlbrock et al. 2003). However, systemic acquired resistance named SAR, implicates SA. Furthermore, the defence responses include the production of several secondary metabolites such as phenolics (Dixon and Paiva 1995; Dixon 2001; Tan et al. 2004). It seemed wise to observe changes in phenolics in elicited plant material as done for example with the common bean (Campos et al. 2003). Our results (Table 2), obtained with H. perforatum treated by SA, effectively showed an increase of some phenolic compounds after elicitation. To explain all this data, it must be reminded that SA contributes to control expression of the CAD1 gene involved in the plant defence of Arabidopsis (Tsutsui et al. 2006). Does a comparable effect exist in H. perforatum?
Concerning naphtodianthrone biosynthesis, it proceeds via a polyketide pathway in which a polyketide synthase (pKS) intervenes (Karppinen and Hohtola 2008) as well as for hyperforins (Karppinen et al. 2007), other biological active substances present in H. perforatum. These enzymes, involved in hyperforin and naphtodianthrone synthesis, are respectively called HpKS1 and HpKS2. But the final stages of hypericin biosynthesis have been suggested to be conducted by HYP-1, a phenolic coupling protein belonging to a plant pathogenesis-related family (Bais et al. 2003; Radauer et al. 2008). The gene encoding for HYP-1 has been isolated from undifferentiated cell suspension cultures of H. perforatum by Bais et al. (2003). Then, Košuth et al. (2007) showed that there is no difference in hyp-1 expression between leaf margins that contained hypericin accumulating dark glands and leaf interior parts free of dark glands. Continuing the research on this material, Karppinen (2010) considers that HYP-1 protein is mobile but its target is not the dark glands. In agreement, we found that Hyp-1 mRNA could be detected in cells and that SA elicitation did not influence its accumulation. Furthermore, Michalska et al. (2010) suggested that the function of HYP-1 may also be closer to the storage or transport of hypericin than to biosynthesis. So, it may explain why tissues lacking dark glands such as undifferentiated cell cultures, from which HYP-1 cDNA was isolated, are able to accumulate hypericins. Therefore, we can think that different unknown genes are responsible for the biosynthesis of hypericins in different places.
Future studies are needed to test these hypothesises and to determinate the exact physiological function of HYP-1 in H. perforatum. The results would complete the previous yet exhaustive analyses of Zhao et al. (2005) and Vasconsuelo and Boland (2007) about elicitation signal transduction leading to the production of plant secondary metabolites.
Despite the notable effect on phenylpropanoid distribution, SA elicitation appears largely less efficient than JA one. On the other hand, with regards to naphtodiantrones, SA elicitation induced sometimes an important increase after a single day of culture or usually after only 4 days Moreover, the level thus reached, is maintained practically till the end of the culture (21 days). This data suggests that SA elicitation cell suspension cultures of H. perforatum could appear as a possible system to produce hypericins. After 4 days of such a culture, cells could be isolated and naphtodiantrones extracted.
This work was supported by the Ministère des Affaires Etrangères – France (Programme COCOP: Réseau d’Enseignement regional Postgraduate en Biologie, grant no. DSUR-NGE-4B1-505). We are very grateful to Professor C. Jay-Allemand from University of Montpellier (France) for helpful advice for secondary metabolites analyses. We thank Dr Jan Kosuth and Eva Cellarova from University in Kosice (Slovakia) for their help in the RTPCR analyses. We acknowledge to Professor F. Brignolas for his help in statistical analyses and Mrs Alison Price for carefully reading of the manuscript. We thank Dr S. Ounnar and G. Moreau for their technical help.
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