Phytotoxic mechanism of allelochemical liquiritin on root growth of lettuce seedlings

ABSTRACT

As the main active ingredient of the traditional Chinese medicine Glycyrrhiza uralensis Fisch, liquiritin has multiple biological activities, including anti-inflammatory, antihepatotoxicity, immune regulation, anti-virus and anti-cancer. In addition, liquiritin has been recognized as an allelochemical that displays markedly inhibitory effects on the growth of target plants, G. uralensis and lettuce. However, its phytotoxic mechanism remains unknown. In the present study, the mode of action of liquiritin against root growth of lettuce seedling was researched. After treatments with liquiritin, the cell division in root tips of lettuce seedlings was partly arrested, and the cell viability and root vitality were obviously lost. At the same time, overproduction of reactive oxygen species (ROS), malondialdehyde (MDA) and proline (Pro) in lettuce seedlings were induced by liquiritin. The results indicated that the phytotoxic effects of liquiritin was probably dependent on the induction of ROS overproduction, resulting in membrane lipids peroxidation following with cell death and mitosis process disorder.

KEYWORDS:

• Liquiritin
• lettuce
• reactive oxygen species
• cell viability
• cell division

1. Introduction

Glycyrrhiza herbs are traditional Chinese medicines that have been widely used in China, Korea and Japan with a long history. They commonly used to treat diseases in the respiratory system, digestive system and immune system, and relieve the toxicity of other drugs.¹ Modern medicine researches showed that the medicinal function of Glycyrrhiza herbs was depend on the chemicals in their roots. Up to now, secondary metabolisms isolated from Glycyrrhiza roots include triterpene saponins, flavonoids, coumarin, alkaloids, ammonia acids, volatile substances and polysaccharides. Among them, triterpene saponins and flavonoids are the main active ingredients, which displayed various medicinal functions, such as anti-inflammatory, anti-virus, anti-tumor, anti-ulcer, anti-depression, anti-oxidation, enhance memory, protect nerves and lower cholesterol.^2,3

In addition to the medicinal value, Glycyrrhiza herbs are also used in industries such as food, cosmetics, tobacco and animal husbandry.^4–6 The usage amount of Glycyrrhiza herbs is so huge, and the wild licorice resources are unable to meet the demand. Therefore, Glycyrrhiza herbs were gradually cultured in many countries.¹ For a greater production, continuous cropping mode was used as an effective cultivation. However, Glycyrrhiza herbs were frequently hampered by replanting failure, resulting in reduction in both the yield and medicinal quality.⁷ Glycyrrhiza uralensis Fisch is one of the most common planted Glycyrrhiza herbs in China. Our previous study indicated that auto-toxicity of the allelochemicals which released by G. uralensis into the rhizosphere soils played an important role in the replanting failure. The main auto-allelochemicals were identified as liquiritin, isoliquiritigenin and glycyrrhizic acid, which displayed strong inhibitory effects on target plants and with relative high concentrations in rhizosphere soils of G. uralensis.⁸

Liquiritin is one of the main active ingredients of Glycyrrhiza herbs and showed several medicinal activities, including anti-inflammatory, antihepatotoxicity, immune regulation, anti-virus and anti-cancer.⁹ Because of the great medicinal value, the pharmacodynamic mechanism of liquiritin has been well studied. As an allelochemical, liquiritin effectively inhibited the root and shoot elongation of lettuce and G. uralensis seedlings, showed obviously phytotoxic effects.⁸ However, the mechanism related to the phytotoxicity of liquiritin has never been reported. In this study, the influences of liquiritin on ROS production, cell division, cell viability, root vitality, malondialdehyde (MDA) and Proline (Pro) contents in the root of lettuce seedlings were investigated. The aim of this study is to claim the physiological mechanisms of the allelopathic activity of liquiritin on lettuce seedlings root growth.

2. Results

2.1. The influence of liquiritin on cell division in root tips of lettuce seedlings

After treatments with liquiritin, the mitosis processes in lettuce root tips were disturbed. The mitotic index (MI) was significantly decreased under concentrations of 20 μM and more (Figure 1). The inhibitory effects of liquiritin on MI were concentration dependent. At the highest test concentration, the MI was less than half of the control group. The number of cells in each mitotic phase was also recorded, and the results showed that the trend of ratio of each phase was not strictly gone down with the increasing of liquiritin concentrations. However, in general, liquiritin reduced the ratio of each phase at high concentrations (Figure 1).

Figure 1. Mitotic index and mitosis process in root tip cells of lettuce seedlings after treatments with liquiritin. The results presented are mean of three replicates ± SE, different letters denote significant differences at p < .05 according to oneway ANOVA with an LSD test.

2.2. Cell viability in root tips of lettuce seedlings under liquiritin phytotoxicity

Fluorescein diacetate (FDA) and propidium iodide (PI) double staining were used to evaluate the cell viability in root tips of lettuce. As shown in Figure 2, red fluorescence of PI, which representing dead cells, was induced in the roots by 5 μM liquiritin. With the increasing of liquiritin concentration, the red fluorescence in the root tips was gradually enhanced.

Figure 2. Cell viability in lettuce root tips after treatments with liquiritin. Lettuce seedlings were treated with liquiritin at concentrations of (a) 0, (b) 0.1, (c) 1, (d) 10, (e) 100 and (f) 1000 μM for 48 h. Roots were stained with FDA/PI. Green fluorescence and red fluorescence indicate viable and dead cells in root tips, respectively. Bar = 500 μm.

Moreover, the ratios of dead cells in lettuce root were quantified by the determination of Evans blue uptake. Under 1 and 5 μM of liquiritin, the relative uptake of Evans blue by lettuce roots was similar with control. However, at 20 μM and more, the relative uptake of Evans blue was significantly increased (Figure 3).

Figure 3. Relative Evans blue uptake and root vitality of lettuce seedlings after treatments with liquiritin. The results presented are mean of three replicates ± SE, different letters denote significant differences at p < .05 according to oneway ANOVA with an LSD test.

2.3. Root vitality of lettuce seedlings after treatments with liquiritin

As shown in Figure 3, under the concentrations of 1–20 μM, the root vitality of lettuce seedlings treated with liquiritin was similar to that of control. When the concentrations of liquiritin reached 100 μM and higher, the root vitality was significantly decreased.

2.4. ROS production and lipid peroxidation in lettuce seedling roots after treatments with liquiritin

Superoxide radical (O²⁻), one of the main ROS in plants, was analyzed by staining with dihydroethidium (DHE). Under the lowest test concentration of liquiritin, weak bright fluorescence in the root was detected (Figure 4). Then, with the increasing of the concentration used, the fluorescence intensity in treated roots was gradually enhanced with very strong fluorescence at high concentrations of 100 and 400 μM (Figure 4).

Figure 4. ROS production in lettuce roots after treatments with liquiritin. Lettuce seedlings were treated with liquiritin at concentrations of (a) 0, (b) 0.1, (c) 1, (d) 10, (e) 100 and (f) 1000 μM for 48 h. Roots were stained with DHE. Bright fluorescence shows ROS production (presumably O₂⁻). Bar = 200 μm.

Hydrogen peroxide (H₂O₂), another main ROS in plants, was quantitated to evaluate the ROS level. As shown in Figure 5, at 1 and 5 μM, liquiritin has no significant effects on the content of H₂O₂ in lettuce roots. However, under concentration of 20 μM and more, the contents of H₂O₂ were rapidly increased by liquiritin stress.

Figure 5. H₂O₂ and MDA contents in roots of lettuce seedlings after treatments with liquiritin. The results presented are mean of three replicates ± SE, different letters denote significant differences at p < .05 according to oneway ANOVA with an LSD test.

Lipid peroxidation was measured by detecting the content of MDA, an end product of lipid peroxidation. Under low concentrations of 1 and 5 μM, the MDA level induced by liquiritin was similar with the control. At 20 μM, the content of MDA was slightly increased. At high concentrations of 100 and 400 μM, the MDA level was significantly increased, indicating strong lipid peroxide formation in lettuce roots (Figure 5).

2.5. Effect of liquiritin on proline accumulation levels in lettuce seedling roots

Free proline, which generally accumulates in plant tissues under stress, was determined to evaluate the stress strength of liquiritin. Free proline levels in lettuce roots at 1 and 10 μM of liquiritin were similar to control. However, free proline content was markedly increased at 20 μM and more (Figure 6).

Figure 6. Proline content in roots of lettuce seedlings after treatments with liquiritin. The results presented are mean of three replicates ± SE, different letters denote significant differences at p < .05 according to oneway ANOVA with an LSD test.

3. Discussion

Crude extracts of G. uralensis tissues showed significant inhibitory effects on the seed germination and seedling growth of some plant species, indicating that G. uralensis has allelopathy potential.^10–13 In our previous study, the secondary metabolites in rhizosphere soils of G. uralensis were isolated and identified. Through bioassay and content detection, the main allelochemicals released by G. uralensis were characterized as liquiritin, isoliquiritigenin and glycyrrhizic acid. Liquiritin is a flavone compound and strongly inhibited the root and shoot elongation of lettuce seedlings in a concentration-dependent manner, displaying a typical phytotoxicity feature.⁸ In addition, liquiritin is also one of the main active ingredients of Glycyrrhiza herbs, exhibiting various pharmacological activities, such as anti-tumor, anti-virus and antidepressant.⁹

Liquiritin could suppress the migration and proliferation, and induce apoptosis and ROS generation in human gastric cancer cell lines.¹⁴ Moreover, liquiritin strongly decreased viability and induced apoptosis, as well as promoted the accumulation of ROS in HepG2 cells.¹⁵ These results suggested that the anti-cancer effects of liquiritin was involved in the generation of ROS.

ROS is a class of oxygen-containing groups with high biological activity. In higher plants, several types of ROS, such as H₂O₂,²⁻ hydroxyl radical (-OH) and hydroxide ion (OH⁻), are produced as byproducts of the normal metabolism of oxygen mainly in chloroplast, peroxide body and mitochondria.^16,17 As a signal molecular, ROS play important roles in mediating plant growth and development in response to biological and abiotic stresses, protein modification, and gene expression regulation.¹⁸ However, excessive ROS with high concentrations is very harmful to plant cells, will result in oxidative damage to proteins, DNA and lipids, and induce programmed cell death (PCD).¹⁹

In this study, the production of two main ROS, O₂⁻ and H₂O₂ in lettuce roots after treatments with liquiritin was determined. The O₂⁻ was intuitively estimated by staining with DHE, a specific dye reacts in proportion to the intracellular level of O₂^−.20 Florescence of DHE in root tips of lettuce seedlings was remarkably enhanced after treatments, indicating that liquiritin could efficiently induce the production and accumulation of O₂⁻ in the target plant. The level of H₂O₂ was quantitatively determined, and the result showed that the content of H₂O₂ in lettuce roots was increased after treatments, suggesting that liquiritin induces the overproduction of H₂O₂ in lettuce. These results confirmed that liquiritin could induce the accumulation of ROS in plant cells, which is consistent with the results of ROS overproduction in cancer cells exogenous application with liquiritin.^14,15

Oxidative stress produced by excess ROS will cause lipid peroxidation in the cell membranes.¹⁹ MDA, a highly reactive compound that exists as the enol, results from lipid peroxidation of polyunsaturated fatty acids. MDA is usually considered as a marker for oxidative stress.²¹ In the present study, the level of MDA in lettuce roots increased under liquiritin treatments, indicating that the phytotoxicity of liquiritin involved in lipid peroxidation and damage of membranes. Alternatively, liquiritin could alleviate the oxidative stress induced by other medicals in animals.²²

The lipid peroxidation and the breakage of lipids will change the permeability and fluidity of the membrane lipid bilayer, resulted in dramatic alternation of cell integrity and even cell death.²³ In this study, the cell integrity was determined by three methods, including doubly staining with FDA and PI, detecting the uptake of Evans blue and 2,3,5-Triphenyltetrazolium chloride (TTC) reduction. All the results showed that the cell viability in root tips of lettuce was decreased and the number of death cells was increased. Together with the results of ROS and MDA, we proposed that liquiritin could induce ROS overproduction in lettuce roots, and then the excessive ROS caused membrane disruption and loss of cell viability, as well as cell death. Therefore, ROS might play a central role in the phytotoxic effect of liquiritin.

As an anti-cancer agent, liquiritin displayed strong anti-proliferation activities on several cancer lines, and effectively arrest cell division at G0/G1 or G2/M transition.^14,24,25 In this study, liquiritin treatments also influenced the root tip cell division. The MI and the numbers of cells in all mitotic phases were gradually decreased with the increasing of liquiritin concentration, indicating that liquiritin might interfere with a certain phase of cell cycle and prevent cells from interphase entering into mitosis in target plant. Thus, the phytotoxic effects of liquiritin on lettuce root growth were at least partly dependent on the arrest of mitosis. Oxidative stress is thought to affect cell cycle progression in plants.^26,27 Therefore, the liquiritin-induced inhibition of cell division was likely involved in ROS overproduction.

Once under the environmental stresses, free proline will rapidly accumulate in plants. As a signaling molecule, proline conducts multifunctions, including osmotic adjustment, detoxification of ROS and protection of membrane integrity.²⁸ So proline is essential for the recovery of plant from stress. Previous study showed that proline accumulation was associated with ROS production and oxidative stress under environmental stresses in plants.^29,30 In this study, a rise of proline level and accumulation of ROS appeared in lettuce roots under liquiritin-treatments, suggesting that liquiritin induces strong anti-stress response of lettuce, and ROS might be involved in the accumulation of proline to regulate liquiritin-induced stress.

Recent studies revealed that the generated ROS in receptor plants was involved in the phytotoxicity of several allelochemicals.^31–36 The results highlighted the essential role of ROS in allelochemical-induced stress in plants. In addition, allelochemicals also have the ability to arrest mitosis in plant cells. They can arrest cells in all of the mitotic phases or sometimes only in the metaphase.^31–33,37 So the influence of mitosis of allelochemicals was also an important factor for the growth inhibition of plants. However, more studies will be needed to clarify the molecular target and the signal transportation pathway of a certain allelochemical.

4. Materials and methods

4.1. Plant culture

Lettuce seeds were purchased from Henan Fengyuan Seed Company. Petri dish (Φ = 9 cm) containing 2 pieces of filter paper (Qualitative, Whatman-Xinhua, Hangzhou, China) was soaked with 2 mL distilled H₂O. Then, the seeds were transferred to the Petri dish and germinated at 25 ± 1°C in a plant growth chamber. After 24 h, the germinated seeds were used for treatments.

4.2. Liquiritin treatments

Liquiritin (≥98%, Aladdin-reagent Inc., Shanghai, China) was dissolved in DMSO (Solarbio, Beijing, China) with a concentration of 200 mM, and then serially diluted to 50, 10, 2.5 and 0.5 mM, respectively, as stock solutions. In all experiments, each well of a 6-well plate (NUNC, Shanghai, China) was supplied with 2 μL stock solution and 998 μL distilled H₂O, to make the final treated concentrations as 400, 100, 20, 5 and 1 μM, respectively. The same ratio (2‰, v/v) of the solvent DMSO was used as control. For each treated group, five germinated lettuce seeds with similar size were transferred into each well with six wells. The plates were then incubated at 25 ± 1°C in a plant growth chamber. After 48 h, roots of the seedlings were used for testing.

4.3. Mitotic index (MI)

The roots of treated lettuce seedlings were cut off and immediately transferred to Carnoy’s fluid (ethanol/acetic acid, 3/1, v/v) at 4°C for 24 h. Then, the fixed roots were hydrolyzed using 5% HCl for 5 min, following with Schiff’s reagent (Leagene, Beijing, China) staining for 30 min. Root tips (about 2 mm) were excised and gently squashed, and then observed by a digital microscope (Yetech, Beijing, China). At least 800 cells were analyzed in each treatment. The ratio between the number of cells during mitosis and the number of total observed cells was calculated as the MI. The proportions of cells in each division phase including prophase, metaphase, anaphase and telophase were also analyzed.

4.4. Cell viability

The cell viability in lettuce root tips was evaluated by double staining with FDA and PI according to Pan et al.³⁸ with minor modification. After treatments, root segments were collected and stained in a mixture of FDA (12.5 μg/mL, MP Biomedicals, CA, USA) and PI (5.0 μg/mL, MP Biomedicals, CA, USA) for 10 min. The stained roots were washed with distilled H₂O to remove excess dye in the surface of the roots, and then observed by a fluorescence microscope (Leica DMI4000B, Wetzlar, Germany) with excitation 488 nm and emission > 510 nm. At least 15 roots of each treatment were analyzed and the representative results were shown in the figures.

Root cell viability was also quantitatively evaluated by detecting the relative uptake of Evans blue.³⁹ Root segments of the treated lettuce seedlings were soaked in 0.25% (w/v) Evans blue (Solarbio, Beijing, China) for 1 h at 25°C. The stained roots were washed with distilled H₂O, and then soaked in 1 mL N,N-dimethylformamide for 24 h at 25°C in darkness to extract the Evans blue that had been absorbed into the lettuce roots. Absorbance of the released Evans blue was measured at 600 nm by a spectrophotometer (UV-1750, Shimadzu Corp., Kyoto, Japan). The relative Evans blue uptake was calculated as the ratio between the OD values of the treated group and the control.

4.5. Root vitality

Root vitality of lettuce seedlings was evaluated by the method in literature.⁴⁰ About 200 mg root sample was collected and put into a tube containing 5 mL of phosphate buffer (pH, 6.9). Then, 5 mL of 0.4% TTC solution was added to the tube and place in a 37°C water bath for 90 min. Sulfuric acid (2 mL) of 1 M was added to stop the reaction, and 10 mL of ethyl acetate was used to extract red triphenylbenzidine (TTF). The absorbance of the solution was determined spectrophotometrically at 485 nm. The root vitality was estimated by comparing with standard graph and expressed in µg/g/h.

4.6. ROS production

ROS production in root tips of the treated lettuce seedlings was estimated by staining with DHE (Solarbio, Beijing, China) according to Yamamoto et al.⁴¹ Lettuce roots were soaked in the dye solution (DHE, 10 mM; acetone, 0.01%; CaCl₂, 100 mM; pH 4.75) with gentle shake for 10 min at 25°C in the dark. After washing with 100 mM CaCl₂ for 20 min, the fluorescence was observed and photographed using a fluorescence microscope (Leica DMI4000B, Wetzlar, Germany) with excitation 488 nm and emission > 510 nm. At least 15 roots per treatment were analyzed and the representative results were shown in the figures.

4.7. Determination of H₂O₂

Endogenous H₂O₂ in roots of lettuce seedlings was estimated according to Frew et al.⁴² About 100 mg lettuce roots were homogenized in 0.1 M phosphate buffer (pH 6.9), and then centrifuged at 8000 g, 4°C for 10 min (3k15, Sigma, Niedersachsen, Germany). The supernatant (4 mL) was taken and mixed with 0.3 mL reagent solution (phenol, 2.34 g/L; 4-aminoantipyrine, 1 g/L; phosphate buffer, 1 mM, pH 6.9) to determine the absorbance at 505 nm spectrophotometrically. The content of H₂O₂ in lettuce roots is compared with standard graph and expressed in µM/g FW.

4.8. Lipid peroxidation

The content of MDA was measured to evaluate the lipid peroxidation.⁴³ Lettuce roots (about 100 mg) were homogenized in 5 mL trichloroacetic acid (TCA, 10%, w/v), and then centrifuged at 4,000 g for 10 min. The supernatant (1.0 mL) was added to 2.0 mL of thiobarbituric acid (TBA, 0.6%, w/v) in 10% TCA. The tube containing the mixture was incubated in boiling water for 15 min, following with ice bath for 10 min. After centrifugation at 9,000 g for 5 min, the absorbance of the supernatant at 440, 532 and 600 nm was measured, and the levels of MDA were calculated in the following manner:

[MDA] = [6.452 (A₅₃₂ – A₆₀₀) – 0.56 A₄₅₀] _·V_T/(V_·W)

[MDA] represents the concentration of MDA expressed in µM g⁻¹. A₄₅₀, A₅₃₂ and A₆₀₀ are the absorbance values at 450 nm, 532 nm and 600 nm, respectively. V_T and V represent total volume of the extracting solution and the volume used in measurement, respectively. W is the fresh weight of the lettuce roots used.

4.9. Free proline

Free proline was determined according to the method of Bates et al.⁴⁴ About 100 mg lettuce roots were homogenized in 3 mL sulfosalicylic acid (3%, w/v) and then centrifuged at 2,000 g for 10 min. The supernatant (1.0 mL) was mixed with acid ninhydrin and glacial acetic acid at a ratio of 1:1:1, and incubated in boiling water for 1 h, and then in an ice bath for 10 min. Toluene (4.0 mL) was added to the mixture, and after the organic and inorganic phases were separated, the organic phase was monitored at 520 nm spectrophotometrically. Proline content was read from a calibration line constructed with pure proline (Alfa Aesar, Shanghai, China) standards.

4.10. Statistical analysis

All experiments list above were repeated at least three times and the results are represented as the means ± standard error (SE). SPSS 16.0 (SPSS Inc., Chicago, USA) was used for data statistical analysis. The differences between control and liquiritin-treated samples were determined using one-way ANOVA with an LSD test.

5. Conclusion

Liquiritin showed significant inhibitory effects on lettuce growth. In the cells, it induced ROS overproduction followed by lipid peroxidation and reduction of root vitality and cell death. Simultaneously, the mitotic phases and the contents of proline in root tip cells were affected by the liquiritin application. In brief, ROS probably plays a key role in the phytotoxic effects of liquiritin.

Author contributions

ZQY and LGY designed the research. ZQY and KG performed most of the experiments. JT conducted the data analysis. ZQY and LGY wrote the manuscript. All the authors contributed to improving the paper and approved the final manuscript.

Disclosure of Potential Conflicts of Interest

The authors declare no conflict of interest.

Additional information

Funding

This work was supported by the Key R&D and Promotion Projects in Henan Province [No. 192102110049], National Natural Science Foundation of China [No. 31870917], the program for Innovative Research Team of Science and Technology in University of Henan Province [No. 20IRTSTHN024] and the High-level Talent Research Foundation of Nanyang Normal University.