Elsevier

Phytochemistry

Volume 171, March 2020, 112236
Phytochemistry

Salinity effects on physiological and phytochemical characteristics and gene expression of two Glycyrrhiza glabra L. populations

https://doi.org/10.1016/j.phytochem.2019.112236Get rights and content

Highlights

  • Salinity caused progressive decreases in the biomass of two licorice populations.

  • Licorice could maintain high K+/Na+ homeostasis under salinity.

  • Salinity caused oxidative stress, increases in proline and antioxidant enzymes.

  • The expression of bAS, CYP88D6, and CYP72A154 were elevated under slight salinity.

  • Glycyrrhizin content of rhizome only increased under 100 mM NaCl.

Abstract

Glycyrrhiza glabra (licorice) is a medicinal plant with valuable specialised metabolites such as triterpene sweetener glycyrrhizin. Salinity stress is the main environmental stress limiting plant growth and development. The effects of six levels of NaCl (0, 100, 200, 400, 600, and 800 mM) on growth, osmolyte content, oxidative stress markers, antioxidant enzyme activities, K+/Na+ ratio, glycyrrhizin content, and gene expression of glycyrrhizin biosynthesis (bAS, CYP88D6, and CYP72A154) were investigated in licorice rhizomes of two populations. The results showed that the salt stress progressively reduced the growth parameters and increased the proline concentrations in the rhizomes. K+/Na+ ratio showed a significant decrease under salinity as compared to the controls. Salt stress resulted in oxidative stress on the rhizomes, as indicated by increased lipid peroxidation and hydrogen peroxide concentrations and elevated the activities of antioxidant enzymes (i.e., ascorbate peroxidase and superoxide dismutase). The glycyrrhizin content increased only under 100 and 200 mM NaCl treatments. The same trend was observed in the expression of bAS, CYP88D6, and CYP72A154 genes in Fars population. Fars population was found to have more glycyrrhizin content than Khorasan population. But, growth, glycyrrhizin content, and biosynthesis genes of glycyrrhizin showed more reduction in Khorasan population as compared to those of Fars population. The results indicate that the application of 100 mM NaCl up-regulated the expression of key genes involved in the biosynthesis of triterpenoid saponins and directly enhanced the production of glycyrrhizin. Accordingly, G. glabra can be introduced as a halophyte plant.

Introduction

Glycyrrhiza glabra L. (Licorice) is one of the most widely used herbs, which belongs to the Fabaceae family (Rizzato et al., 2017). Approximately 30 Glycyrrhiza species distributed throughout Southern Europe, Asia, and the Americas (Liao et al., 2012; Hosseini et al., 2018). Licorice is a popular and important herbal drug in Iran (Hosseini et al., 2018; Esmaeili et al., 2019). The root and rhizome of licorice have a large amount the oleanane-type triterpene saponin glycyrrhizin (2–8% of the dry weight), which is 50-fold sweeter than sucrose (Seki et al., 2008, 2011; Pastorino et al., 2018).

Glycyrrhizin has a wide range of pharmacological activities, including anti-inflammatory, antibacterial, antiulcer, insecticide, anti-hepatotoxic, and antiallergy, as well as involvement in the immune system activation (Karkanis et al., 2018; Pastorino et al., 2018). Besides its medicinal applications, licorice extracts are used as cosmetics, food additives, and confectionery foods (Rizzato et al., 2017).

Salinity is a severe environmental condition that can dramatically limit plant growth and production (Belkheiri and Mulas, 2013; Çoban and Baydar, 2016). Insufficient precipitation and high temperature coupled with high evaporation and irrigation of cultivated land with saline water have increased the salinity in the arid and semiarid regions (Egamberdieva and Mamedov, 2015). The effect of salinity depends on the soil salt concentration, as well as the species tolerance (Zrig et al., 2016).

Plants growing in saline soils are affected by osmotic stress, the toxic effect of high NaCl concentrations, and oxidative stress (Lokhande et al., 2010; Amanifar et al., 2019). Salinity can inhibit plant growth and development, result in ion imbalance, and membrane leakage, increase the lipid peroxidation and elevate the production of reactive oxygen species (ROS) (Amanifar et al., 2019). Growth of the plants in saline soils requires the osmotic adjustment to maintain a positive turgor pressure; moreover, the osmolytes play a key role in stress mitigation (Flowers et al., 2014; Gupta and Huang, 2014). Proline is an important compatible solute involved in osmotic activity regulation (Belkheiri and Mulas, 2013).

It is known that high K+/Na+ homeostasis is a crucial factor in salinity tolerance (Amini et al., 2017). K+/Na+ balance largely relies on the ability of salt exclusion, vacuolar salt compartmentation, and K+ uptake (Chen et al., 2019).

Plants contain complex mechanisms to withstand water stress, among which, osmolyte biosynthesis, intracellular compartmentalization of toxic ions, modifications in ion homeostasis, and ROS detoxifying systems can be mentioned (Munns and Tester, 2008; Rahmati et al., 2015; Meng et al., 2018). The enzymatic and non-enzymatic antioxidant mechanisms can regulate the balance between the generation and removal of ROS (Ozden et al., 2009). Superoxide dismutase (SOD) is the primary enzyme in the detoxifying process, converting the superoxide radical anions to hydrogen peroxide (H2O2) and O2 (Pan et al., 2006). Ascorbate peroxidase (APX) scavenges H2O2 to water and oxygen (Ozden et al., 2009; Mohammadi et al., 2019). Glycyrrhizin and its derivatives also contribute to ROS detoxifying (Hosseini et al., 2018).

The biosynthesis of specialised metabolites and their corresponding metabolic pathways are strongly related to the plant growth conditions (Nasrollahi et al., 2014). Effects of abiotic stress on the glycyrrhizin content of rhizomes have been studied in the growth chamber experiments and in the field experiments with G. uralensis (Pan et al., 2006) and G. glabra (Nasrollahi et al., 2014; Hosseini et al., 2018; Amanifar et al., 2019).

Several sequential enzymatic reactions are necessary for the biosynthesis of glycyrrhizin in plants (Nasrollahi et al., 2014). In licorice rhizomes, the production of triterpene saponin begins with the conversion of farnesyl pyrophosphate (FPP) with squalene synthase enzyme (SQS) into squalene (Shirazi et al., 2019). Beta-amyrin synthase (bAS) enzyme contributes to the cyclization of 2,3-oxidosqualene and leads to the biosynthesis of β-amyrin (Seki et al., 2015; Shirazi et al., 2018). Beta-amyrin 11-oxidase (CYP88D6) plays an important role in the engineering pathway of glycyrrhizin biosynthesis (Shirazi et al., 2018). Two-step oxidation reactions of β-amyrin by CYP88D6 and CYP72A154 lead to glycyrrhizin biosynthesis (Seki et al., 2008, 2015). Several Cytochrome P450s (CYP72 and CYP88 family proteins), as triterpene-oxidizing enzymes, are responsible for the production of terpenoids such as glycyrrhizin from G. glabra, taxol from Taxus species, and artemisinin from Artemisia annua with important effects on the human health (Seki et al., 2008, 2011).

Different studies have shown that Glycyrrhiza species like G. glabra, G. uralensis, and G. inflate can tolerate unfavorable environmental stresses such as salinity, drought, cold, and heat (Lu et al., 2013; Egamberdieva and Mamedov, 2015; Li et al., 2016; Jiang et al., 2019). Moreover, several reports have mentioned the use of licorice for remediation of abandoned salt soils (Kushiev et al., 2005; Dagar et al., 2015; Egamberdieva and Mamedov, 2015). On the other hand, the economic and medicinal significance of licorice and its extinction risk due to unexpected harvesting have further highlighted the necessity of its cultivation and domestication (Hosseini et al., 2018; Arora et al., 2019; Esmaeili et al., 2019).

Shirazi et al. (2018) found that the highest glycyrrhizin contents and the transcription levels of the biosynthetic genes of glycyrrhizin in G. glabra seeds under 150 mm NaCl after 72 h. Also, Amanifar et al. (2019) reported that symbiosis of the licorice seeds with arbuscular mycorrhizal fungi could alleviate salt stress damage. The vegetative propagation of licorice rhizomes mostly occurs, as the germination rate of its seeds is very low. On the other hand, G. glabra tolerates unfavorable environmental stresses. However, so far no studies have investigated the effects of salinity stress on licorice rhizomes under different NaCl treatments and tolerance mechanisms. Hence, the aim of the present study was to evaluate the effect of six levels of NaCl (0, 100, 200, 400, 600, and 800 mM) on growth, K+/Na+ ratio, osmolyte content, oxidative stress markers, antioxidant enzyme activity, glycyrrhizin content, and expression of glycyrrhizin biosynthesis genes (bAS, CYP88D6, and CYP72A154 genes) in rhizomes of Fars and Khorasan populations of licorice. The results could offer background information for the selection of G. glabra as a halophyte plant and could provide more insights into the molecular tolerance mechanisms against salinity in licorice.

Section snippets

Growth parameters

Analysis of variance showed that the levels of salinity and the populations had significant effects on the rhizome length, diameter, fresh weight (FW), and dry weight (DW) (P < 0.001). Also, the interaction of the salinity and the population on growth parameters (except length) was significant (P < 0.05) (Table 2). The rhizome length, diameter, FW, and DW decreased progressively in the two licorice populations with the increase of the salinity stress in comparison to the controls (Table 1). The

Discussion

Based on the obtained results, salt stress declined the progressive growth of the rhizomes. The aerial part of licorice could not survive under the maximum level of applied salinity (800 mM). Salinity could also limit the growth of different plant species, for instance, G. inflate with more than 200 mM NaCl (Lu et al., 2013), G. uralensis with 75 mM NaCl (Egamberdieva and Mamedov, 2015), and Thymus vulgaris with 150 mM NaCl (Zrig et al., 2016). The excessive absorption of NaCl causes osmotic

Conclusions

The rhizome, which is the main way of licorice vegetative propagation, was dramatically affected by salt stress. The results of this study revealed that G. glabra could tolerate salinity levels up to 800 mM NaCl without any damages to the rhizomes. G. glabra can resist salt stress through elevation of the osmolyte for maintaining the tissue osmolality, maintaining high K+/Na+ homeostasis, and enhancing the activities of antioxidant enzymes. Slight salinity (100 mM NaCl) up-regulated the

Plant material

G. glabra rhizomes were collected in October 2016 from local wild populations in Fars (lat. 52° 16′ N; long. 30° 28′ E) and Khorasan (lat. 59° 51′ N; long. 36° 40′ E) provinces with respective average rainfall of 261 and 350 mm and respective average temperatures of 23.13 and 21.08 °C. Plants were identified by the herbarium of Shiraz University, Shiraz, Iran, where a voucher specimen (no 55077 and 55078) of the plants was deposited. After washing the samples, the rhizomes with diameters of

Declaration of competing interest

The authors declare no conflicts of interest.

Acknowledgements

We would like to thank the Shiraz University Research Council for financial support (1952) of this research.

References (61)

  • M.S. Hosseini et al.

    Effect of drought stress on growth parameters, osmolyte contents, antioxidant enzymes and glycyrrhizin synthesis in licorice (Glycyrrhiza glabra L.) grown in the field

    Phytochemistry

    (2018)
  • W.C. Liao et al.

    Identification of two licorice species, Glycyrrhiza uralensis and Glycyrrhiza glabra, based on separation and identification of their bioactive components

    Food Chem.

    (2012)
  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method

    Methods

    (2001)
  • V. Nasrollahi et al.

    The effect of drought stress on the expression of key genes involved in the biosynthesis of triterpenoid saponins in liquorice (Glycyrrhiza glabra)

    Phytochemistry

    (2014)
  • M. Ozden et al.

    Effects of proline on antioxidant system in leaves of grapevine (Vitis vinifera L.) exposed to oxidative stress by H2O2

    Sci. Hortic. (Amst.)

    (2009)
  • A. Płażek et al.

    Tolerance of Miscanthus× giganteus to salinity depends on initial weight of rhizomes as well as high accumulation of potassium and proline in leaves

    Ind. Crops Prod.

    (2014)
  • G. Rizzato et al.

    A new exploration of licorice metabolome

    Food Chem.

    (2017)
  • M. Shafeiee et al.

    Physiological and biochemical mechanisms of salinity tolerance in several fennel genotypes: existence of clearly-expressed genotypic variations

    Ind. Crops Prod.

    (2019)
  • J.L. White et al.

    A simple method for detection of viral satellite RNAs in small plant tissue samples

    J. Virol. Methods

    (1989)
  • R. Xu et al.

    On the origins of triterpenoid skeletal diversity

    Phytochemistry

    (2004)
  • A. Zrig et al.

    Essential oils, amino acids and polyphenols changes in salt-stressed Thymus vulgaris exposed to open–field and shade enclosure

    Ind. Crops Prod.

    (2016)
  • H. Akhani et al.

    A contribution to the halophytic vegetation and flora of Iran

  • F. Amini et al.

    Biochemical and physiological response of Salsola arbuscula callus to salt stress

    Iran. J. Sci. Technol. A.

    (2017)
  • L.S. Bates et al.

    Rapid determination of free proline for water-stress studied

    Plant Soil

    (1973)
  • M. Becana et al.

    Some enzymes of hydrogen peroxide metabolism in leaves and root nodules of Medicago sativa

    Plant Physiol.

    (1986)
  • T. Chen et al.

    K+ and Na+ transport contribute to K+/Na+ homeostasis in Pyropia haitanensis under hypersaline stress

    Algal Res

    (2019)
  • J.C. Dagar et al.

    Liquorice (Glycyrrhiza glabra): a potenitial salt-tolerant, highly remunerative medicinal crop for remediation of alkali soils

    Curr. Sci.

    (2015)
  • D. Egamberdieva et al.

    Potential Use of Licorice in Phytoremediation of Salt Affected Soils, Plants, Pollutants and Remediation

    (2015)
  • T.J. Flowers et al.

    Sodium chloride toxicity and the cellular basis of salt tolerance in halophytes

    Ann. Bot.

    (2014)
  • N. Garg et al.

    ROS generation in plants: boon or bane?

    Plant Biosyst.

    (2009)
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