УДК 581.1

INDUCTION OF GLYCYRRHIZIN AND TOTAL PHENOLIC COMPOUND PRODUCTION IN LICORICE BY USING ARBUSCULAR MYCORRHIZAL FUNGI1

© 2013 Y. Orujei*, L. Shabani**, M. Sharifi-Tehrani**

* Department of Biology, Payame Noor University, Najaf-Abad

** Department of Biology, Faculty of Science, Shahrekork University, Shahrekork, I.R. Iran

Received June 4, 2012

Arbuscular mycorrhizal fungi have mutualistic symbiosis with higher plants, increasing plant resistance to environmental stresses and nutrient uptake and improving soil. During arbuscular mycorrhizal symbiosis, a range of chemical and biological factors are affected. In this study, two species of arbuscular mycorrhiza (Glomus mosseae and G. intraradices) were used to assess the effects of inoculation on licorice growth and secondary metabolite production. After successful inoculation, the increase in the growth rate, P and Zn uptake, and the accumulation of secondary metabolites in licorice (Glycyrrhiza glabra L.) roots were observed in two periods of 3 and 6 months compared to control. After 6 months, more increments in growth, secondary metabolites, and P and Zn uptake were observed compared with the first 3-month period. Two groups of secondary metabolites arising from phenolic and terpenoid metabolism obviously responded to mycorrhizal fungi colonization in licorice roots.

 

Keywords: Glumus mosseae  G. intraradices  Glycyrrhiza  arbuscular mycorrhiza  glycyrrhizin

 

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1 This text was submitted by the authors in English.

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Abbreviation: AM  arbuscular mycorhhiza.

Corresponding author: Shabani Leila. Department of Biology, Faculty of Science, Shahrekork University, Shahrekork, I.R. Iran. Fax: +9 81 38144-24419; e-mails: lshabani@gmail.com; 

shabani-l@sci.sku.ac.ir

 INTRODUCTION

Licorice (Glycyrrhiza glabra L.) is one of important medicinal plants of the family Leguminosae, which has long been the focus of herbalists and pharmaceutical researches. It is a perennial herb with pinnately compound leaves and zygomorphic flowers in racemous inflorescences. Tap roots of the plants are thickened after 12 years and accumulate a sweet yellowish secondary metabolite named glycyrrhizin. The term licorice has also been used to refer to these roots. Two main varieties of this plant in Iran are recognized by the presence (var. glandulifera) or absence (var. glabra) of short hairs on glumes. Both varieties are known to accumulate much glycyrrhizin, although var. glabra is known as the main source of this compound. Glycyrrhizin is an oleanane-type triterpene saponin, a well-known natural sweetener that is 50 times sweeter than sugar and is accumulated in the roots and rhizomes of this plant [1]. A number of physiological activities have been reported for glycyrrhizin and glycyrrhetinic acid. Most important activities include: anti-inflammatory, antiulcer, antitumor, antibacterial, and antiallergic ones. Derivatives from glycyrrhizin have also been used for treatment of chronic hepatitis and gastric ulcers [2]. The activation of the medicinal plant secondary metabolism is achieved through a number of different technologies, including elicitation by fungal and chemical agents, stresses, and symbiosis association between fungi and plant root cells.

Arbuscular mycorrhizae (AM) are the most widespread form of symbiotic associations known to occur between the roots of about 80% of higher plants and fungi that belong to the order Glomerales [3]. They have ability of conducting phosphate from the soil into the roots [4]. It has also been suggested that colonization by AM fungus may induces the production of secondary metabolites, such as flavonoids [5], pathogenesis-related proteins [6], and phenolics [7] in the roots of host plants. For instance, Khaosaad et al. [8] showed that essential oils of Origanum sp. were increased in the presence of arbuscular mycorrhizal fungi. There is a belief that more terpenoids and terpene saponins participate in plant defense systems against microbial pathogens and insects. Since many saponins have potent antifungal activities, they may serve as preformed phytoprotectants against fungal attack, as shown for avenacins, oleanane-type triterpene saponins in oats [9]. 

It is well understood that phenolic compounds (in particular, flavonoids and phenolic carboxylic acids) are associated with the chemical defense of plants and act as signal molecules in plantmicrobe interactions [10]. Ponce et al. [11] showed that infection with a mycorrhizal symbiont Glomus intraradices significantly altered the composition and patterns of flavonoids in Trifolium repens roots. Although arbuscular mycorrhizal fungi are known to play a major role in the nutrition and growth of plants in many production-orientated agricultural systems, little is known about their potential effects on secondary metabolite production in medicinal and aromatic plants [8, 12, 13]. The aim of this study was to evaluate the effects of symbiosis with arbuscular mycorrhizal fungi on the induction of glycyrrhizin and total phenolic compound production in licorice.

 

MATERIALS AND METHODS

Mycorrhizal fungal inoculum preparation. The soil needed for experiments was gathered from Zayandeh-rood River near Chelvan country (45 km to Shahrekord). The gathered soil texture was loamsand, which is poor in nutrients and more suitable for mycorrhizal experiments. The soil was collected from 0 to 30 cm depth, air-dried, passed through a 2-mm sieve, and analyzed. The main characteristics of these calcareous soils were the following: organic carbon, 1.8 mg/g; total N, 0.5 mg/g; P (Olsen’s), 4.5 mg/kg; K, 172 mg/kg; CaCO3, 170 mg/g; sand, 780 mg/g; silt, 120 mg/g; and clay, 100 mg/g. The soil was autoclaved at 121°C for 1 h to eliminate all spores and fungal propagules. Licorice seeds (Glycyrrhiza glabra L.) were obtained from “Pakan-Bazr Seed Company”. Seeds were put in 95% sulfuric acid for 30 min, then washed and placed on wet Whatman paper containing soil with fungal spores (inoculum), where contamination of plantlets with fungi occurred in early stages of germination. Seeds were then transferred to soil when 2.5-cm plantlets appeared. 

The mycorrhizal fungi (Glomus mosseae and G. intraradices) were provided by “Zist Fanavar Touran Company” (Semnan, Iran). Twenty grams of fungal inoculum containing hyphae, spores, and fungal propagules were put in 35 cm depth of each pot, and twenty plantlets (2.5-cm-long) were planted in 35 cm depth of pots. After plantlets were established, soil was added to them and watered with distilled water. The non-mycorrhizal control soil was similar, but without AM fungi. Plants were then treated once a month with 25 mL of P-free Hoagland nutrient solution. Half of samples were harvested after 3 months, and other rest half was harvested after 6 months.

Determination of root colonization. To visualize AM fungal colonization, fresh root samples were treated with 10% KOH and stained with Trypan blue (0.1%) in lactophenol [14]. Root mycorrhizal colonization was estimated using the grid-line intersect method [15].

Dried root samples were ground and digested in the concentrated HNO3 at 140160°C. After cooling, the extracts were diluted with 1 N HCl up to 25 mL. The P in the digested sample was estimated by the molybdate method [16] at 700 nm, using a UV spectrophotometer. The concentration of Zn was determined using atomic absorption spectrophotometry at the wavelength of 213.9 nm.

HPLC analysis. Freeze-dried root samples were ground with a mortar and pestle. 40 mg of each powdered sample was extracted with 1 mL of 80% methanol at 60°C for 6 h. Extracts were then spun down at 4000 rpm for 15 min at room temperature. The supernatant was transferred with a Pasteur pipette to a new tube and filtered through 0.4 µm filter. The resultant extracts were then used for subsequent HPLC analyses.

HPLC analysis of glycyrrhizin was performed according to the method reported by Hurst et al. [17]. A 20-µL aliquot of the root extract was analyzed by HPLC at 30°C. The HPLC system consisted of Waters HPLC 510 pump, a Nova-pak C18 column (3.9 × 150 mm, “Waters”, United States), a Waters 2478 detector, and a Millennium chromatography data system (“Waters”). The separation was performed with an isocratic elution, using methanolwateracetic acid (60 : 34 : 6) at a flow rate of 1 mL/min with UV absorption detection at 254 nm. 

Determination of total phenolics content. The total phenolic compound content in the root methanolic extracts was determined by spectrophotometry using the FolinCiocalteu reagent assay [18]. The extract (200 µL) was mixed with 1 mL of FolinCiocalteu reagent (diluted tenfold with water) and 0.8 mL of a 7.5% sodium carbonate solution in a test tube. After stirring and 30 min later, the absorbance was measured at 765 nm by using a Jenway 6405 UV-vis spectrophotometer. Gallic acid was used as a standard for the calibration curve. The total phenolic compound content was expressed as milligrams of gallic acid equivalents per gram of dry weight (mg GAE/g dry wt).

Statistical analysis. One-way analysis of variance was carried out for each parameter studied. Duncan’s post hoc multiple mean comparison was used to test significant differences between treatments (at 5% level). All statistical analyses were performed with SAS software (version 6).

 

RESULTS

Plant growth parameters and nutrient concentration

After inoculation of licorice plants with mycorrhizal fungi, percent of colonization and root lengths where measured at end of the growth season and compared to non-inoculated plants. Percent of colonization in 6-months plantlets (the most prolonged culture time in this study) are presented in table 1. Both the Glomus species successfully colonized the roots of Glycyrrhiza glabra forming arbuscules and vesicles-type mycorrhizae (data not shown). No AM colonization was observed in those plant roots that were not inoculated with AM fungi. Percentage of root colonization varied between the fungi, and the colonization frequency was found to be 69.1 ± 2.3% for G. mosseae and 55.66 ± 2.5% for G. intraradices.

AM fungal inoculation had a significant effect on all measured plant growth parameters (tables 2, 3). However, the level, to which plant growth was enhanced, varied between the fungal inoculants. G. mosseae-colonized plants performed consistently better than G. intraradices-inoculated plants.

Measurement of P content in the roots of 3-month licorice showed that the P concentration was increased 2.5-fold in plantlets inoculated by G. mosseae about 5-fold in plantlets inoculated by G. intraradices. These results (table 4) are in accordance with normal effects of mycorrhizal symbiosis on absorption of elements, such as P. The P concentration was also significantly (P < 0.05) different between 6-month plantlets inoculated and non-inoculated with mycorrhizal fungi (table 4). Effects of inoculation with G. mosseae were increased with growth time, in a way that, the concentration of P was increased up to more than 4-fold in 6-month plantlets compared to 3-month plantlets. Thus, the inoculation with G. mosseae was more effective compared to that with G. intraradices. Zn concentration in 3-month plantlets was significantly different in mycorrhizal and non-mycorrhizal plantlets. The concentration of Zn in roots of licorice inoculated with mycorrhizal fungi was increased by 3545% (table 4). The increase in the absorption area by longitudinal growth of fungal hyphae, which enter very fine compartments of the soil, makes it easier to get Zn from soil. Inoculation of 3-month plantlets with G. intraradices made the most increase in the concentration of Zn in underground parts of the plantlets. Increment in the Zn concentration was highly increased in 6-month plantlets compared to 3-month plantlets, and G. mosseae was the most effective (8-fold) species.

 

Effect of fungal inoculation on total phenolic compounds and glycyrrhizin 

in the roots of 3- and 6-month-old plantlets

Results obtained from measuring total phenolic contents in 3-month-old plantlets of licorice showed significant difference between mycorrhizal and control plantlets (table 5). Comparison between mean values showed increase in total phenolics in 3-month plantlets up to 8-fold for plantlets inoculated with G. mosseae, and about 2-fold for plantlets inoculated with G. intraradices, which was interpreted as a better efficiency of the former species in symbiosis relationships with licorice plants. Results obtained from measuring total phenolic compounds in 6-month-old licorice plantlets also showed significant differences between mycorrhizal and control plantlets (table 5). Comparison of mean values showed that total phenolic compound concentration was increased more by mycorrhizal fungi in 6 month-old plantlets compared to 3-month-old ones. Totally, mycorrhizal inoculation had significant effects on the increase in the total phenolic compound concentration, and this effect was increased with growth time. The increase in the glycyrrhizin content, as the most important constituent of the licorice root, under mycorrhizal inoculation treatment was the main aim of this study. Results obtained from measuring glycyrrhizin concentration in 3-month-old plantlets showed significant differences between control and plantlets inoculated with mycorrhizal fungi. G. mosseae was the most effective fungal species in increasing glycyrrhizin concentration (up to 9-fold); while the other fungal species increased the glycyrrhizin concentration up to 3-fold compared to control (table 5). These results were similar to those obtained for total phenolic compounds. Results obtained from measuring glycyrrhizin concentration in 6-month-old plantlets of licorice showed significant difference between control samples and those inoculated with mycorrhizal fungi (table 5). Again, G. mosseae was more effective than G. intraradices, and the effect was more prominent in 6-month-old plantlets compared to 3-month-old ones. It seems that G. intraradices needs more time to increase secondary metabolites in licorice plants. Mycorrhizal inoculation had prominent effect on glycyrrhizin content in roots of licorice and the effect was increased with time.

 

DISCUSSION

Plant root colonization is affected by environmental conditions and fungal species adaptation to plant tissue, which is colonized. Gavito and Miller [19] and Al-Karaki and Al-Raddad [20] showed that the root system colonization rate was an important indicator of mycorrhizal fungus activity, which is affected by morphological and structural properties of the root system, amount and quality of root secretions, chemical fertilizers, such as P, and the high concentration of essential elements. Colonization of licorice roots by G. mosseae was significantly more successful than of G. intraradices and showed that G. mosseae was more adaptive to licorice roots than the other species. The improvement of nutrient element absorption by plants would often lead to positive growth responses in aboveground plant organs [21]. Growth and absorption of nutrients in plants is reported to be increased by inoculation with mycorrhizal fungi and would increase resistance to environmental stresses, disease, and improvement of plant function [22]. Phosphorus is one of essential elements for plants. Its deficiency would disturb plant growth and metabolism. Colomb et al. [23] stated that plant growth, leaf area index, and the photosynthesis rate were affected by plant P consumption, leading to activation of plant functions. Results of this study showed that Zn concentration was increased in underground parts of 3-month-old mycorrhizal plants compared to control, but it was more prominently increased after 6 months. It could be due to increased longitudinal growth of roots and absorption surface, together with more fungal hyphae, which had been developed after 6 months. Most of mycorrhizal inoculation effects are due to theincrease of root growth in soils and their penetration in less accessible soil texture; it seems that mycorrhizal fungi are most effective in plants with the larger root system, which can absorb nutrients more effectively; our results are consistent with these conclusions (table 1). Licorice as a plant with a prominent and expanded root system could absorb significant amounts of P and Zn when symbiosis with mycorrhizal fungi was established. 

Roots of licorice contain several secondary metabolites, such as triterpene saponins, flavonoids, isoflavonoids, hydroxyl coumarins, esterols, and little amounts of essential oils. Glycyrrhizin (sodium and potassium salt of glycyrrhizic acid) is the most important effective constituent of the licorice roots. Glycyrrhizin and total phenolics in underground parts of the licorice plants were measured in this study, and the effects of mycorrhizal symbiosis on the production of these compounds by licorice were evaluated. The increase in the concentration of these compounds should conceptually be expected due to the better nutritional conditions after mycorrhizal inoculation. The results of HPLC analysis indicated that, under our experimental conditions, licorice inoculation with AM fungi affected glycyrrhizin content when compared to non-mycorrhizal plants. Colonization of plants with arbuscular mycorrhizal fungi may lead to marked systemic reactions in terpenoid metabolism. Thus, G. intraradices induced the accumulation of significant amounts of leaf sesquiterpenoids in Citrus jambkiri [24]. Maier et al. [7] have shown the G. intraradices-induced accumulation of a terpenoid glycoside (blumenin) in some cereal arbuscular mycorrhiza, including barley, wheat, rye, and oat. The lignin enrichment found in mycorrhizal plantlets of Podophyllum peltatum may be related to host defense response in preventing any infections. Ex vitro G. ramisporophora inoculated plants yielded more podophyllotoxin and related lignans than the control, non-inoculated plants [25]. Therefore, it is deduced that increased glycyrrhizin accumulation in the roots of licorice plantlets inoculated with mycorrhizal fungi was mainly due to the induction of plant defense system.

Phenolic compounds, as a group of secondary metabolites, have diverse chemical structures and vast distribution in plants. Results of this study showed that the application of mycorrhizal fungi had significant effects on the accumulation of total phenolic compounds in 6-month-old plantlets of licorice and increased them up to fourfold. Experimental evidence showed that phenolic compounds acted as signals in plant development and interactions between plants and microorganisms [26]. The accumulation of these compounds in mycorrhizal plants compared to control plants was shown by Lynn and Krishna [26, 27]. The genotype of licorice examined in this study proved to be able to establish symbiosis relationship with fungal species G. mosseae and G. intraradices and the adaptation of them to licorice roots were relatively high. Positive effects of symbiosis between licorice roots and mycorrhizal fungi were not dependent on fungus species, because the inoculation of licorice roots with every of mycorrhizal-arbuscular fungi resulted in positive morphological changes, improved nutrient absorption, and secondary metabolite accumulation in licorice plantlets. However, it seems that G. mosseae is more capable to establish symbiosis relationships and colonize roots of licorice and more effectively increased biomass of shoots and roots and production of total phenolics and glycyrrhizin. G. intraradices was more effective in increasing absorption of nutrient elements and shoot and root dry weights and the number of leaflets.

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