Introduction

Licorice (Glycyrrhiza glabra L.) is one of the most important medicinal and economical plants of the Leguminosae family. It has roots and rhizomes that are a rich source of biologically active and natural compounds. An important group of these compounds is triterpene saponins, which have antimicrobial, insecticidal and anti-inflammatory activities (Fukai et al. 1998).

Any plant in a particular temperature range has the maximum growth and desirable performance and any deviation from it, especially lowering the critical temperature, causes stress and decreases vegetative and reproductive growth (Yang et al. 2011). Reactive oxygen species (ROS) are produced during normal oxidative processes in the cell such as respiration, photosynthesis and oxidative phosphorylation, but their concentration increases during aging and in response to biotic and abiotic stresses. Low temperatures also cause oxidative stress and ROS accumulation. Because under cold stress the plant's ability to remove oxygen radicals is impaired, two enzymatic and non-enzymatic defense systems are used against the extensive ROS accumulation (Hoyle and Santos 2010).

Titanium (Ti) as a beneficial element stimulates growth and increases the absorption of some nutrient elements (Kuzel et al. 2003). There is a paucity of literature on the effects of titanium dioxide nanoparticles (TiO₂ NPs) on the physiological responses of plants. Numerous reports showed that TiO₂ NPs reduced the destructive effects of stresses by increasing the activity of antioxidant enzymes and decreasing the free radicals of oxygen and malondialdehyde. TiO₂ NPs can increase dry weight, chlorophyll biosynthesis, sunlight absorption, rubisco activity, photosynthesis rate and nitrogen content (Hong et al. 2005; Gao et al. 2006).

Many researchers have pointed to the positive role of polyamines in confronting environmental stresses. Polyamines play important roles in regulating plant physiological activities and improving these activities under stress conditions. The antioxidant effect of polyamines is mainly related to their cationic properties which act to remove free radicals and thus can inhibit lipid peroxidation (Kusano et al. 2007). Kubis (2008) reported that spermidine prevented cell membrane damage under cold stress in barley plants, thereby reducing electrolyte leakage and amino acid withdrawal from the cell. To increase stress resistance and alleviate its negative effects, the application of TiO₂ NPs and a polyamine may be useful. Thus, the present study was aimed to investigate the effects of TiO₂ NPs and spermine on the antioxidative response of licorice plants under cold stress conditions.

Materials and Methods

Plant material and growth conditions

Glycyrrhiza glabra seeds were obtained from the Pakan-Bazr Seed Production Company (Isfahan, Iran). The experiment was conducted to study the effects of spermine (1 mM) alone and also together with two concentrations of TiO₂ nanoparticles (2 and 5 ppm) on biochemical and physiological properties of licorice plants under cold stress and normal conditions (4 ºC and 26 ºC, respectively). The cultures without any TiO₂ NPs and spermine were considered as the control. TiO₂ nanoparticles with the average particle size of 10-25 nm were supplied by the US Research Nanomaterials, Inc. Seeds were cultured on MS media containing 3% sucrose, 0.7% agar and were kept in a germination chamber with 16/8 h light/dark cycle at 26±2 ºC. The twenty-eight-days old plants were maintained under control condition (26 ºC) and cold treatment (4 ºC) for two days. All treatments were replicated three times. Samples were randomly selected from each treatment. Samples were harvested from all treatments 30 days after cultivation and stored at -70 ºC for further assays.

Measurement of the growth characters and chemical substances

Ti content

Titanium concentration in the seedlings was measured by the method of Gibson et al. (2006). Bioaccumulation of Ti in the digested samples was evaluated by using inductively coupled plasma mass spectrometry (ICP-MS, HP-4500, USA).

Growth characters

For determining the fresh weight, eight plants in each jar were measured 30 days after seed planting with a digital scale and then their mean was calculated. To obtain the turgid weight, the plants were cut and immersed in the distilled water for 72 h in the dark. Then, the total weight of the pieces was measured and their average was calculated. To measure the dry weight, the pre-treated plant parts were placed in the oven at 70 ºC for 48 h. Then, they were removed and their weight was measured again. RWC was determined according to the following formula:

RWC (%) = (FM-DM)/(TM-DM)100

Where FW is the fresh weight, DW is the dry weight and TW is the turgid weight.

Photosynthetic pigments content

Leaf pigments content was measured by the Lichtenthaler and Wellburn (1983) method and the pigment concentration was calculated as mg g^-1 FW.

Malondialdehyde and hydrogen peroxide content

The lipid peroxidation rate was measured based on the method of Heath and Packer (1968) using fresh leaf samples. Malondialdehyde (MDA) concentration was read at 532 nm and the amount of malondialdehyde was calculated using the extinction coefficient of 155 mM^-1 cm^-1 and expressed in terms of µmol g^-1 FW.The content of hydrogen peroxide (H₂O₂) was measured according to Velikova et al. (2000). The absorbance was read at 390 nm. Hydrogen peroxide content using its extinction coefficient of 0.28 mol^-1 cm^-1 was calculated and expressed in µmol g^-1 FW.

Free proline and glycine betaine content

Free proline content was determined according to Bates et al. (1973) and presented in mg g^-1 fresh weight. Glycine betaine was measured according to the method of Grieve and Grattan (1983) and reported in µmol g^-1 DW.

Total phenolics content

The total phenol content of the methanolic extracts was measured following the method of Plessi et al. (2007). Determination of the total flavonoid content of the extracts was done according to the aluminum chloride colorimetric method of Chang et al. (2002). Total anthocyanin content was determined as described by Wagner (1979).

Soluble sugars

The content of soluble sugars was measured using the method of Irigoyen et al. (1992). The absorbance was read at 485 nm for glucose, 480 nm for rhamnose and 490 nm for mannose. The concentration of each sugar was calculated using the standard curve.

Total soluble protein and antioxidant enzyme activity

Protein content was measured by the Bradford (1976) method. The absorbance of the samples was read at 595 nm. Total protein content was calculated using the standard curve of bovine serum albumin (BSA) and reported in mg g^-1 FW. The activity of superoxide dismutase (SOD) was determined following the method of Winterbourn et al. (1976). Peroxidase (POD) and catalase (CAT) activities were determined by the method of Chance and Maehly (1955). Determination of polyphenol oxidase (PPO) activity was assessed according to Kar and Mishra (1976). Ascorbate peroxidase (APX) activity assay was measured according to the Nakano and Asada (1981) method.

Glycyrrhizin content

Glycyrrhizin was extracted according to Orujei et al. (2013). The extract was quantified by high- performance liquid chromatography (HPLC) using a reverse-phase column (150×4.6 mm i.d.; 5 μm) SB-C8 (Agilent Zorbax, USA). The mobile phase flow rate was 1 mL min^-1 and glycyrrhizin was detected at 254 nm and 25 ºC and the glycyrrhizin content was calculated using the standard chromatogram.

Experimental design and statistical analysis

The experiment was carried out as a split-plot design based on randomized complete blocks with three replications. The main plots included normal (26 ºC) and cold (4 ºC) conditions and sub-plots included the following four treatments: control (without TiO2 NPs and spermine); 1 mM spermine; 1 mM spermine + 2 ppm TiO₂ NPs; 1 mM spermine + 5 ppm TiO₂ NPs. However, for the case of Ti content, the treatment of 1 mM spermine was excluded and only three treatments were evaluated at both temperatures. The data were subjected to analysis of variance followed by Duncan's test for comparing means at p ≤ 0.05 using the SPSS (Ver. 23) software.

Results

Ti content

To ensure the absorption of TiO2 NPs by plants, the Ti concentration was measured on the thirty-day-old seedlings incubated at both temperatures, separately. The amount of Ti accumulation was higher in seedlings under cold stress than that in control seedlings. After application of TiO₂ NPs and spermine together, Ti accumulated in the seedlings. Also with increasing TiO₂ NPs concentration, the Ti accumulation rate increased (Figure 1).

Growth characteristics

Cold stress significantly decreased fresh, dry and turgid weight and also relative water content (RWC) compared to the normal temperature. The significant decreases in fresh, dry and turgid weight were induced by spermine at both temperatures compared to the control. Comparison of the mean values showed that the combined effect of spermine and TiO₂ NPs significantly reduced the fresh, dry and turgid weight compared to the control plants, except the turgid weight at low temperature (Figure 2). However, the RWC increased under optimum conditions, whereas it decreased significantly under cold stress conditions compared to the control plants. Spermine significantly increased RWC at both temperatures (Figure 2).

Photosynthetic pigments content

Cold stress significantly reduced the content of chlorophyll a, chlorophyll b, total chlorophyll and carotenoid compared to the control seedlings at the optimum temperature (Figure 3). Spermine treatment increased the content of photosynthetic pigments at both optimum and low temperatures, except for chlorophyll b at the optimum temperature. The combined effect of spermine and TiO₂ NPs significantly increased the content of chlorophyll a, total chlorophyll and carotenoid in both thermal conditions, except total chlorophyll at 2 ppm TiO₂ under normal temperature conditions. The combined effect of TiO₂ NPs and spermine did

Figure 1. Ti content of Glycyrrhiza glabra seedlings treated with TiO2 nanoparticles and spermine (Spm) at optimum (26 ºC) and low (4 ºC) temperatures. Means with different letters above the bars indicate significant differences at p≤ 0.05.

Figure 2. Mean comparison of growth characteristics in Glycyrrhiza glabra seedlings treated with TiO2 nanoparticles and spermine (Spm) at optimum (26 ºC) and low (4 ºC) temperatures. (A): Fresh weight, (B): Dry weight, (C): Turgid weight, (D): Relative water content (RWC). Means with different letters above the bars indicate significant differences at p≤ 0.05.

Figure 3. Mean comparison of photosynthetic pigment content in Glycyrrhiza glabra seedlings treated with TiO2 nanoparticles and spermine (Spm) at optimum (26 ºC) and low (4 ºC) temperatures. (A): Chlorophyll a, (B): Chlorophyll b, (C): Chlorophyll t, (D): Carotenoid. Means with different letters above the bars indicate significant differences at p≤ 0.05.

not induce a significant change in the content of chlorophyll b under optimum temperature. However, under cold stress conditions, the content of chlorophyll b significantly increased when the TiO2 concentration was 5 ppm.

Malondialdehyde and H₂O₂ content

Results from the measurement of malondialdehyde content showed that cold stress significantly increased the amount of this compound compared to the control plants (Figure 4A). However, spermine caused a significant decrease of malondialdehyde content at both thermal conditions. The combined effect of TiO₂ NPs and spermine reduced malondialdehyde content under both temperatures. Results from the measurement of hydrogen peroxide content showed that cold stress increased the amount of this compound compared to the normal temperature, but spermine reduced the amount of hydrogen peroxide at both thermal conditions. The combined effect of TiO₂ NPs and spermine caused a significant decrease in the amount of H₂O₂(Figure 4B).

Free proline and glycine betaine content

The proline content was significantly increased by cold stress (Figure 5A). Spermine did not affect the proline content at the optimum temperature but increased it at the low temperature. The combined effect of TiO₂ NPs and spermine at both optimum and low temperatures caused a significant increase in the proline content in comparison with the control plants. Cold stress significantly increased also the content of glycine betainein the control plants (Figure 5B). Spermine treatment significantly increased glycine betaine content compared to control at both optimum and low temperatures. Application of TiO₂ NPs and spermine together at both optimum and low temperatures caused a significant increase in the content of glycine betainein comparison with the control plants.

Total phenolic content

Cold stress caused a significant increase in the content of total phenol, flavonoid and anthocyanin compared to the control plants at the optimum temperature. Besides, spermine significantly increased total phenol, flavonoid and anthocyanin contents at both optimum and low temperatures. Comparison of means showed that the combined effect of TiO₂ NPs and spermine at both temperatures increased significantly total phenol, flavonoid and anthocyanin content (Figure 6).

Soluble sugars

Results from the measurement of the soluble sugars indicated that the amount of glucose, rhamnose and mannose was significantly increased by cold stress in the control plants. Also, spermine alone or together with TiO₂ NPs at both temperatures also increased the content of these sugars compared to the control plants (Figure 7).

Total soluble protein content and antioxidant enzyme activity

Results showed that the amount of total protein was increased significantly by cold stress. Application of spermine increased the amount of protein at both temperatures compared to control. The combined effect of both concentrations of TiO₂ NPs with spermine at both temperatures significantly increased total protein content compared to the control plants (Figure 8A). A significant increase in the activity of antioxidant enzymes was observed under cold stress conditions. Spermine treatment significantly increased superoxide dismutase activity at both temperatures but increased polyphenol oxidase activity only under cold stress conditions. Spermine significantly reduced peroxidase activity at the optimum temperature but increased it at the low temperature. Exposure to spermine at the optimum temperature, significantly reduced ascorbate peroxidase activity, while it had no significant effect at the low temperature. In the presence of spermine, catalase activity significantly increased at the optimum temperature but reduced at the low temperature. Application of TiO₂ NPs and spermine together under normal and cold stress conditions significantly    increased    the   activity   of   these

Figure 4. Mean comparison of cell injury indicators in Glycyrrhiza glabra seedlings treated with TiO2 nanoparticles and spermine (Spm) at optimum (26 ºC) and low (4 ºC) temperatures. (A): MDA, (B): H₂O₂. Means with different letters above the bars indicate significant differences at p≤ 0.05

Figure 5. Mean comparison of oxidative damage in Glycyrrhiza glabra seedlings treated with TiO2 nanoparticles and spermine (Spm) at optimum (26 ºC) and low (4 ºC) temperatures. (A): Proline, (B): Glycine betaine. Means with different letters above the bars indicate significant differences at p≤ 0.05.

antioxidant enzymes (Figure 8B-F).

Glycyrrhizin content

Based on the results obtained from HPLC analysis, it was found that cold stress conditions significantly increased the glycyrrhizin content compared to the normal temperature in the control plants (Figure 9). Spermine caused a significant change in the glycyrrhizin content at the optimum temperature but did not affect it at the low temperature. The combined effect of TiO₂ NPs and spermine at both thermal treatments caused a significant increase in the glycyrrhizin content compared to the control plants.

Figure 6. Mean comparison of total phenolics content in Glycyrrhiza glabra seedlings treated with TiO2 nanoparticles and spermine (Spm) at optimum (26 ºC) and low (4 ºC) temperatures. (A): Total phenol, (B): Total flavonoid, (C): Total anthocyanin. Means with different letters above the bars indicate significant differences at p≤ 0.05.

Figure 7. Mean comparison of soluble sugars content in Glycyrrhiza glabra seedlings treated with TiO2 nanoparticles and spermine (Spm) at optimum (26 ºC) and low (4 ºC) temperatures. (A): Glucose, (B): Rhamnose, (C): Mannose. Means with different letters above the bars indicate significant differences at p≤ 0.05.

Figure 8. Mean comparison of total protein content and antioxidant enzyme activity in Glycyrrhiza glabra seedlings treated with TiO2 nanoparticles and spermine (Spm) at optimum (26 ºC) and low (4 ºC) temperatures. (A): Protein content, (B): SOD activity, (C): PPO activity, (D): POD activity, (E): APX activity, (F): CAT activity. Means with different letters above the bars indicate significant differences at p≤ 0.05.

Figure 9. Glycyrrhizin content of Glycyrrhiza glabra seedlings treated with TiO2 nanoparticles and spermine (Spm) at optimum (26 ºC) and low (4 ºC) temperatures. Means with different letters above the bars indicate significant differences at p≤ 0.05.