Comparative study of Cd uptake and tolerance of two Italian ryegrass (Lolium multiflorum) cultivars

Plant cultivation

Two extreme ryegrass cultivars (IdyII and Harukaze) with high and low Cd tolerance during seed germination (Fang et al., 2017), respectively, were selected for this study. Seeds were sterilized with 10% H₂O₂ for 10 min, rinsed thoroughly with distilled water, and germinated via immersion in distilled water at 25 °C in the dark. After five days, uniform seedlings were transferred to 1 L plastic pots (14 plants per pot) filled with 1/4 Hoagland’s solution. Seedlings were maintained for 10 days in a growth chamber at a 12 h light/dark cycle with 300 μmol m⁻² s⁻¹ light intensity, a day/night temperature of 25/20 °C and 65 ± 5% relative humidity. Five Cd concentrations for the treatments were as follows: 0, 5, 10, 25, 50, and 100 μM Cd as CdCl₂·2.5 H₂O (analytical reagent) was added to the nutrient solution. Each treatment had six replicates. The nutrient solution was renewed every three days, and the pH was adjusted to 6.5 with 2 M NaOH or 2.7 M HCl.

After a 12-day Cd exposure, plants were divided into two groups and collected. One group was for biomass and Cd concentration determination, and the other group was for physiological index measurements, including chlorophyll content, lipid peroxidation and NPT. Each group had three replicates.

Estimation of plant growth and Cd accumulation

The harvested plants were soaked in 20 mM Na₂-EDTA for 15 min, rinsed with distilled water to remove metals on the root surfaces, and separated into roots and shoots. Subsequently, samples were oven-dried at 70 °C overnight. The dried samples were weighed and digested with mixed acid [HNO₃ + HClO₄ (85:15, v/v)]. The concentrations of Cd, zinc (Zn), iron (Fe), manganese (Mn) and magnesium (Mg) were determined by an inductively coupled plasma optical emission spectrometer (ICP-OES, Optima 2100 DV; PerkinElmer, Inc., Waltham, MA, USA).

The tolerance indexes (TIs), translocation factors (TFs), bioconcentration factors (BCF), and Cd accumulation were determined according to the method of Chen et al. (2011):

• TIs = biomass_Cd/biomass_control

• TFs = Cd_{concentration in shoot}/Cd_{concentration in root}

• BCF = Cd_{concentration in root}/Cd_{concentration in the nutrient solution}

• Cd accumulation = [biomass]_{dry weight} × [Cd]_{concentration in plant tissues}

• Total Cd accumulation = Cd accumulation in root + Cd accumulation in shoot

• Cadmium distribution proportion of root = Cd accumulation in root/total Cd accumulation.

Estimation of photosynthetic pigment contents

A total of 100 mg of the middle portion of fresh leaf slices were taken for pigments extraction with 10 mL 95% ethanol for 24 h in the dark. The absorbances of pigment extract were measured at wavelengths of 665, 649 and 470 nm with spectrophotometry (Shimadzu UV-2450; Shimadzu, Kyoto, Japan). Chlorophyll a (Chl a), Chlorophyll b (Chl b) and carotenoids (Car) were estimated using the following equations (Knudson, Tibbitts & Edwards, 1977), respectively:

• Chl a = 13.95 × A₆₆₅ −6.88 × A₆₄₉

• Chl b = 24.96 × A₆₄₉ −7.32 × A₆₆₅

• Car = (1,000 × A₄₇₀ −2.05 × Chl a −114.8 × Chl b)/245

Estimation of lipid peroxidation

The level of lipid peroxidation in root and leaf was estimated with 2-thiobarbituric acid (TBA) reactive metabolites chiefly malondialdehyde (MDA) as described by Ali et al. (2014). Briefly, plant fresh tissues (0.1–0.3 g) were homogenized in 5 mL of 0.25% TBA made in 10% trichloroacetic acid. The extracts were heated at 95 °C for 30 min and then quickly cooled on ice. After centrifugation at 10,000g for 10 min, the absorbance of the supernatant was measured at 532 and 600 nm. MDA contents was calculated with the following formula: MDA = 6.45 × (A₅₃₂–A₆₀₀).

Determination of NPT

Non-protein thiols were assayed following Tian et al. (2011) with minor modifications. Fresh tissues (approximately 0.3 g) were homogenized in 3 mL ice-cold 5% sulfosalicylic acid solution and centrifuged at 12,000×g (4 °C) for 15 min. The resulting supernatant was used for NPT assays. First, 0.3 mL of the supernatant was mixed with 1.2 mL 0.1 M K-phosphate buffer (pH 7.6) and 50 μL 6 mM 5,5′-dithiobis-2-nitrobenzoic acid (dissolved in 5 mM EDTA and 0.1 M phosphate buffer solution at pH 7.6). The mixture was incubated at room temperature for 20 min and then measured for absorbance at 412 nm with spectrophotometry. The NPT content was estimated with a standard curve of reduced glutathione in the range of 0–100 μg mL⁻¹.

Statistical analysis

Statistical analyses were performed using a two-way analysis of variance (ANOVA) with SPSS version 20.0 (SPSS Inc., Chicago, IL, USA). Duncan’s multiple range test was employed to compare the changes among the different treatments at P < 0.05. The correlations among TIs, Cd uptake, Cd TFs, Cd accumulation, MDA content and NPT content of the two cultivars in the roots in plant roots were determined by Pearson’s correlation analysis.

Plant biomass, TIs and root/shoot ratio response to Cd stress

Increasing the Cd supply in the medium posed variable effects on plant biomass, TIs and the root/shoot ratio (Table 1). Cd treatments tended to reduce the biomass of both cultivars. The biomass of IdyII was significantly higher than that of Harukaze in the same treatment (P < 0.01), however biomass reductions with increasing Cd dose from 5 to 100 μM were not significant between IdyII and Harukaze. Similar alterations were also observed with the TIs. For example, when exposed to 25 μM Cd, root biomass was reduced by 37% in Harukaze and 22% in IdyII. A clear decline in the roots/shoot ratio was demonstrated in both cultivars with increasing Cd concentrations.

Ecotoxicological response based on the plant biomass inhibition rate

As shown in Table 2, positive correlations were observed between the inhibition of plant biomass (root and shoot) and Cd concentrations in the solution (P < 0.01), which was represented by the quadratic equation. To evaluate toxicity, the inhibitory concentration (IC50; Cd concentration when the root or shoot biomass decreased by 50% compared with the control) and lethal concentration (IC90; Cd concentration when the root or shoot biomass decreased by 90% compared with the control) were determined by the fitting equation. The IC50 values of the shoots and roots of IdyII were 1.9-fold and 3.2-fold higher than that of Harukaze, respectively. The lethal concentration of the roots and shoots (IC90) in IdyII were also higher than that of Harukaze, implying that IdyII was more tolerant to Cd compared to Harukaze.

Cd concentration, accumulation in plant tissues, and BCFs and TFs responses to Cd stress

With elevating Cd concentrations in the treatment solutions, root Cd concentration in both cultivars increased, ranging from 2.09 to 9.89 mg·g⁻¹ in Harukaze and from 1.26 to 6.89 mg·g⁻¹ in IdyII. Cd concentrations in Harukaze roots were higher than that of IdyII roots, especially at the 50–100 μM Cd treatments (Fig. 1A, P < 0.01). Similar trends were also observed in the shoots (Fig. 1B). A gradual increase of Cd TFs in Harukaze was correlated with the increasing Cd concentration in the treatment solutions, whereas no significant Cd TFs changes in IdyII were observed. The Cd TF value in Harukaze was significantly higher than that in IdyII after exposure to the highest Cd concentration (Fig. 1C, P < 0.01). Cd accumulation in the roots of Harukaze remained constant, while there was a dramatic increase in IdyII with the application of 25–100 μM Cd in the treatment solutions (Fig. 1D). An increasing trend of Cd accumulation in the shoots and total accumulation was observed with an increasing Cd supply, and their accumulation amounts in IdyII were significantly higher than that in Harukaze in the presence of higher Cd dosages (Figs. 1E and 1F, P < 0.05).

The proportion of cadmium distribution in the roots was 78.5–45.4% in Harukaze and 67.2–54.7% in IdyII under Cd stress. The distribution proportion in both Harukaze and IdyII significantly decreased with the 25 and 100 μM Cd treatments, respectively (Fig. 2A, P < 0.01). The increasing Cd supply reduced the root BCFs of the two cultivars (Fig. 2B), and the reduction was especially obvious in Harukaze (range from 3,715 to 880). At low Cd treatments (5–10 μM), the BCFs of Harukaze were markedly higher than that of IdyII (P < 0.01).

Effects of Cd on plant mineral concentrations and TFs

Cadmium treatments altered the uptake and TFs of several nutrient elements (Fig. 3). Compared with the control, the 25–100 μM Cd supply markedly increased the Zn and Fe concentrations in the roots of Harukaze (Figs. 3A and 3B), whereas the Mn concentration in the roots of Harukaze was significantly decreased with lower Cd concentrations (5 and 10 μM) (Fig. 3C). Additionally, Mg concentration was greatly increased at the highest Cd concentrations (Fig. 3D). In contrast, compared with the control, 100 μM Cd significantly promoted Zn uptake in the roots of IdyII (Fig. 3A), while the uptakes of Fe, Mn, and Mg exhibited no change in the roots of IdyII (Figs. 3B–3D). In the shoot, Cd supply did not affect Zn and Mg concentrations in both cultivars, but severely decreased Mn concentrations (Figs. 3E, 3H and 3G). Compared with the control, shoot Fe concentrations in IdyII exhibited a gentle decrease with increasing Cd concentration, while no significant change occurred in Harukaze (Fig. 3F). In both cultivars, Zn TFs were significantly inhibited at the 25–100 μM Cd treatments (Fig. 3I), and the amounts in IdyII were significantly higher than that of Harukaze. Fe TFs reached a maximum in both cultivars under 5 μM Cd and then showed a decrease with increasing Cd (Fig. 3J). Cd treatments in IdyII significantly reduced Mn TFs and had no change in Mg TFs. In contrast, the TFs of Mn and Mg in Harukaze decreased considerably only at the highest concentration of Cd (Figs. 3K and 3L). According to a two-way ANOVA analysis, significant differences in the concentrations of Fe and Mn (P < 0.01) of the roots were found between the two cultivars, while no significant interaction were observed for other parameters (Figs. 3A–3L), indicating these two cultivars only respond significantly different for the uptakes of Fe and Mn when exposed in excess Cd.

Effects of Cd on pigment content, lipid peroxidation and NPT content

The cadmium supply tended to reduce Chl a, Chl b, Chl (a + b) and Car contents in both cultivars (Table 3). For example, the Chl a, Chl b, and Car content decreased by 42.5%, 44.7% and 44.4% in Harukaze and by 11.8%, 5.9%, and 22.2% in IdyII under 25 μM Cd stress, respectively. The Chl a, Chl b and Chl (a + b) content of IdyII was significantly higher than that of Harukaze at 25–100 μM Cd concentrations (P < 0.01); a similar trend occurred in Car under 50–100 μM Cd stress. The Cd treatments did not affect the Chl a/b ratio of IdyII but significantly reduced the Chl a/b ratio of Harukaze at 100 μM Cd.

The MDA content in plant tissues was increased with elevated Cd concentrations, and the amounts in the leaves were higher than in the roots (Fig. 4A, P < 0.01). Compared with the control, when treated with high Cd concentrations (50 and 100 μM), the MDA content of the seedling roots increased by 252.5% and 610.2% in Harukaze but only by 140.7% and 291.7% in IdyII, respectively. Similarly, the MDA content of the leaves in Harukaze increased sharply with the 25–100 μM Cd treatments and was considerably higher than that of IdyII (Fig. 4B, P < 0.01).

As shown in Fig. 5, compared with the control, the NPT content in the roots of IdyII increased at least three times with the application of 5 μM Cd and reached its maximum under 50 μM Cd. No significant change occurred in Harukaze with the addition of 5–50 μM Cd in the medium (Fig. 5A). Furthermore, the NPT content in the roots of IdyII were significantly higher than that of Harukaze with the treatments of 10–100 μM Cd (P < 0.01). The NPT content in the shoots of the two cultivars were also enhanced under Cd stress. The values in IdyII were significantly higher than that in Harukaze at the 25–100 μM Cd treatments (Fig. 5B, P < 0.05). By a two-way ANOVA analysis, a significant interaction was found (Figs. 5A and 5B), demonstrating that both cultivars responded significantly different to Cd treatments for NTP production in root and shoot.

Correlation analysis

Pearson’s correlation analysis was carried out to investigate the correlations among TIs, Cd uptake, Cd TFs, Cd accumulation, MDA content and NPT content of the two cultivars in the roots (Table 4). TIs were negatively correlated to Cd concentration, MDA content and Cd TFs. Cd uptake and TFs were positively correlated to MDA content. Additionally, a positive correlation was observed between Cd accumulation and NPT content.