Biomass of perennial ryegrass and uranium enrichment with different concentrations of citric acid
The repair efficiencies on the uranium contaminated soil with perennial ryegrass were positively correlated with the biomass and uranium enrichment in the plants. The uranium concentrations in the roots of all treatments were higher than those in shoots, which demonstrates that the translocation factor of uranium from roots to shoots was less than 0.165 (Table 1). Similar findings were found by some researchers (Newete et al. 2016, Nezami et al. 2016, Nie et al. 2014).
Table 1
Biomass changes and uranium concentrations in different tissues of perennial ryegrass in the presence of citric acid.
Treatment groups | Dry biomass of Shoots (g/plant) | Dry biomass of Roots (g/plant) | uranium in shoots (mg/kg) | uranium in roots (mg/kg) | Transfer coefficient |
Con + 0 | 0.718 ± 0.010 | 0.262 ± 0.002 | 59.043 ± 2.316 | 528.745 ± 33.033 | 0.112 ± 0.011 |
Con + 1 | 0.757 ± 0.034 | 0.273 ± 0.009 | 69.569 ± 3.004 | 539.371 ± 15.476 | 0.125 ± 0.012 |
Con + 5 | 0.843 ± 0.020* | 0.294 ± 0.015* | 87.014 ± 5.187* | 687.910 ± 4.299* | 0.165 ± 0.008* |
Con + 10 | 0.748 ± 0.010 | 0.284 ± 0.020 | 46.356 ± 2.879* | 440.630 ± 19.358* | 0.105 ± 0.010 |
Notes: Values are given as the mean ± SD, n = 3. |
*, P < 0.05: significant difference compared to the control (Con + 0) group. |
The biomass and uranium enrichment in the shoots and roots of perennial ryegrass increased when citric acid was not more than 5 mg/kg (Table 1). The highest values of biomass in the shoots and roots of perennial ryegrass were detected in the Con + 5 treatment, which increased by 17.41% (P < 0.05) and 12.21% (P < 0.05), respectively, compared to those in the control group (Con + 0). However, many researchers have indicated that most chelating agents added to soil increase the concentration of heavy metal ions in the soil solution, which will inhibit the plant growth and reduce biomass(Begum et al. 2012, Hseu et al. 2013, Xin et al. 2009). In several reports, the addition of organic acids in the phytoremediation process promoted the uptake of heavy metals and the biomass production (Han et al. 2016, Najeeb et al. 2009, Wang et al. 2019). In addition, the shoots and roots of the Con + 5 treatment had the highest uranium concentrations, which increased by 47.37% (P < 0.05) and 30.10% (P < 0.05), respectively. As a result, the transfer coefficient significantly increased in the Con + 5 treatment (p < 0.05). The reason for this may be that the applications of citric acid influenced the sorption of uranium by soil mixture, enhanced the mobility and the bioavailability of the uranium, thus increased the capability of plants to transfer the U from roots to shoots (Hu et al. 2019, Li et al. 2014). Introducing citric and oxalic acid treatments into the phytoremediation process can increase the 226Ra uptake by a factor of 1.5 compared to the control with corn (Nezami et al. 2016). Ping Wang et al. (Wang et al. 2016) noticed that citric acid promoted the absorption of 241Am by barley roots and its transport in the plants. However, a higher citric acid concentration (10 mmol/kg) will decrease the accumulation of uranium, although citric acid can substantially enhance the bioavailability of uranium by improving the solubilization of soil-bound uranium(Chen et al. 2020b). Thus, the toxic effects of citric acid may damage the physiological structure of perennial ryegrass and cause a decrease in plant biomass when the citric acid concentration reaches 10 mmol/kg(Duquène et al. 2008, Monroy-Figueroa et al. 2015).
These results suggest that 5 mmol/kg citric acid is the most effective concentration of citric acid to increase the biomass production (both in shoots and roots) and uranium enrichment in perennial ryegrass. To illustrate the strengthening mechanism of the citric acid-assisted uptake of uranium in perennial ryegrass, the effects of different concentrations of citric acid on the subcellular distribution of uranium, physiological characteristics of perennial ryegrass, activities of antioxidant system enzymes and cellular ultrastructure under uranium stress are analyzed in the following sections.
Subcellular distribution of uranium in perennial ryegrass with different concentrations of citric acid
Knowledge of the subcellular distribution of heavy metals in organisms is fundamental to understand the process of heavy-metal uptake, storage and detoxification (Nie et al. 2015). The distributions of uranium in the cell wall fraction, organelle fraction and cytosolic fraction in the shoots and roots of perennial ryegrass were further investigated. As shown in Table 2, low concentrations (1 mmol/kg and 5 mmol/kg) of citric acid could promote the enrichment of uranium in the subcellular structure of perennial ryegrass with 5 mmol/kg citric acid being the optimal concentration. In the Con + 5 treatment, the uranium contents in the cell wall, organelle fraction and cytosolic fraction increased by 51.68%, 36.25% and 42.24% in the shoots and by 32.96%, 18.36% and 34.24% in the roots, respectively. In contrast, the accumulation of uranium decreased in both roots and shoots in the Con + 10 treatment. These results further prove that 5 mmol/kg citric acid is the optimal concentration to enhance the enrichment of perennial ryegrass.
Table 2
Effect of citric acid on the uranium subcellular distribution of perennial ryegrass
| treatments | Uranium contents (mg/kg) |
cell wall fraction | organelle fraction | cytosol fraction | Total |
Shoots | Con + 0 | 35.16 ± 1.41 | 10.40 ± 0.66 | 12.95 ± 0.39 | 59.043 ± 2.316 |
Con + 1 | 42.18 ± 1.68 | 12.04 ± 0.49 | 14.92 ± 0.80 | 69.569 ± 3.004 |
Con + 5 | 53.33 ± 3.01* | 14.17 ± 0.78* | 18.415 ± 1.25* | 87.014 ± 5.187* |
Con + 10 | 27.74 ± 1.62 | 8.424 ± 0.56 | 9.787 ± 0.62 | 46.356 ± 2.879* |
roots | Con + 0 | 330.21 ± 21.43 | 105.29 ± 5.65 | 87.58 ± 7.26 | 528.75 ± 33.033 |
Con + 1 | 342.02 ± 10.77 | 102.09 ± 3.75 | 89.93 ± 2.27 | 539.37 ± 15.476 |
Con + 5 | 439.05 ± 5.81* | 124.62 ± 0.84* | 117.57 ± 1.65* | 687.91 ± 4.299* |
Con + 10 | 274.17 ± 11.99 | 86.41 ± 3.22 | 75.72 ± 4.52 | 440.63 ± 19.358 |
Notes: Values are given as the mean ± SD, n = 3. |
*, P < 0.05: significant difference compared to the control (Con + 0) group. |
The subcellular partitioning of uranium in plants reflects its internal processes during uranium accumulation, which can provide more mechanistic information about the uranium tolerance and the interaction process between uranium and perennial ryegrass (Nie et al. 2014). The distribution proportion of uranium in different parts of the same cell in roots was cell wall > organelle > cytosol, while the order in shoots was cell wall > cytosol > organelle (Table 2). These results demonstrate that a much greater part of uranium was stored in the cell wall fraction and the proportion was more than 60% in both roots and shoots, so the cell wall played an important role in the uranium tolerance. This trend is consistent with the results proposed by other researchers(Nie et al. 2015). Meanwhile, the storage capacity of uranium in the cell wall and the barrier protecting effect of cytosol improved when low concentrations (1 mmol/kg and 5 mmol/kg) of citric acid were added. Consequently, all distribution proportions of uranium in the cell wall were higher than those in the control group in both roots and shoots, and all values in organelles were lower than those in the control group. However, the proportions of uranium in the cytosol of the shoots contrasted with those in the roots when low concentrations of citric acid were added, which might be closely related to the buffer capacity of the cytosol (phosphate in the cytosol bioprecipitated with uranium ) and uranium concentrations in the cell wall of roots and shoots (Pan et al. 2015). A high concentration (10 mmol/kg) of citric acid decreased the accumulation of uranium in roots and shoots, and the distribution trends of uranium in different parts were consistent with those in the 5 mmol/kg citric acid treatment.
Physiological characteristics of perennial ryegrass with different concentrations of citric acid
Photosynthetic pigment content, which is a direct indicator of plant photosynthesis, can be used as a tolerance criterion for heavy metals in plants(Sebastian &Prasad 2018). In order to eliminate the interference from other ions, As shown in Fig. 1A, 5 mmol/kg citric acid enhanced the photosynthesis of perennial ryegrass, which indicates an elevated tolerance to uranium. This phenomenon was also reported in a previous study (Chen et al. 2020b). However, the enhanced effects on the uranium tolerance of perennial ryegrass with 1 mmol/kg or 10 mmol/kg citric acid were not significant compared to the 5-mmol/kg citric acid treatment. Compared to the control group (Con + 0), the levels of chlorophyll-a (chl-a), chlorophyll-b (chl-b), and carotenoids in the 5-mmol/kg treatment (Con + 5) increased by 28.29%, 44.16%, and 28.99% respectively. In contrast, the levels of chl-a, chl-b, and carotenoids did not significantly change in the Con + 1 and Con + 10 treatments (p > 0.05). The data indicate that the chl-b content in perennial ryegrass is significantly correlated with the citric acid concentration (p < 0.05). However, the chl-a and carotenoid levels were not significantly correlated with the citric acid concentration (p > 0.05). Further research is required to elucidate the enhanced mechanisms of the associations among the pigment contents, citric acid concentrations, and uranium tolerance of perennial ryegrass.
The permeability of the cell membrane reportedly increases when it is exposed to uranium, which causes the leakage of intracellular electrolytes and increases the relative electric conductivity (REC) (Dai et al. 2017). Simultaneously, the peroxidation of cell membrane lipids generates MDA, which reacts with proteins and nucleic acids, and the cell function is affected (Chen et al. 2020a, Khair et al. 2020). Therefore, the relative electrical conductivity, MDA content and soluble protein content which are major indices of the cell membrane permeability, appear to be closely related to the U tolerance. As shown in Figs. 1B, C and D, the root conductivity and MDA content were generally higher than those in the leaves. Thus, the degree of damage to the root cells in perennial ryegrass was more severe than that to the leaf cells. Meanwhile, the Con + 5 treatment had the lowest relative electrical conductivity and MDA. The mean electrical conductivity values of the shoots and roots in the Con + 5 treatment decreased by 20.19% (p < 0.05) and 20.26% (p < 0.05) compared to the control group (Fig. 1B). Similarly, the mean MDA values of the shoots and roots in the Con + 5 treatment decreased by 22.16% (p < 0.05) and 23.63% (p < 0.05) compared to the control group (Fig. 1C). Thus, citric acid (5 mmol/kg) can significantly decrease the electrical conductivity and MDA in the shoots and roots of plants, which are negatively correlated with the uranium tolerance (Li et al. 2019).
Furthermore, the contents of soluble proteins in plants of all treatments were investigated. As illustrated in Fig. 1D, the contents of soluble proteins in the plant shoots of the Con + 1, Con + 5, and Con + 10 treatments were 1.31-, 1.90-, and 1.49-fold higher than those in the control group. The contents of soluble proteins in the plant roots of the Con + 1, Con + 5, and Con + 10 treatments were 1.03-, 1.39-, and 1.05-fold higher than those in the control group. All contents of soluble protein in the shoots and roots of plants in the Con + 5 treatment were higher than those in the Con + 1 and Con + 10 treatments, which suggests that citric acid (5 mmol/kg) can significantly increase the contents of soluble proteins in the shoots and roots of plants (P < 0.05).
All of these results reveal that 5 mmol/kg citric acid can alleviate the cell damage of perennial ryegrass exposed to uranium stress.
Effects of different citric acid concentrations on the activities of antioxidant system enzymes
As shown in Fig. 2, all activities of POD (Fig. 2A), SOD (Fig. 2B), CAT(Fig. 2C), and GR(Fig. 2D) in the shoots and roots increased with different concentrations of citric acid in the Con + 1, Con + 5, and Con + 10 treatments compared to those in the control group. However, only the values in the Con + 5 treatment were significantly more efficiently affected than those in the control group (P < 0.05). Therefore, 5 mmol/kg was the most effective concentration of citric acid to enhance the activities of the four types of antioxidant enzymes in perennial ryegrass.
When subjected to uranium stress, plant cells may produce H2O2 to reduce the fixation efficiency of CO2 in cells, while H2O2 (Haber-Weiss) combines with superoxide anion (O2−) to generate reactive oxygen species (ROS), which is harmful to the plants. In plant cells, antioxidant enzymes such as POD, SOD, CAT, and GR can be used to resist the harmfulness of ROS. SOD can transform O2- into H2O2(Slomka et al. 2008), which effectively resists the generation of ROS in cells. CAT reacts with high concentrations of POD and POD reacts with low concentrations of H2O2. The two reactions interactively transform O2- into H2O and H2O2(Geebelen et al. 2002, Smeets et al. 2005). GR catalyzes the transformation of oxidized glutathione into glutathione to resist the generation of ROS in combination with SOD (Slomka et al. 2008).
Our results indicate that all changes in activities of POD, SOD, CAT, and GR in the shoots and roots of perennial ryegrass were significantly correlated with the citric acid concentration (P < 0.05), which suggests that the addition of the chelating agent citric acid can enhance the antioxidant enzyme activity of perennial ryegrass. Therefore, the chelating agent citric acid can contribute to plant adaptation to uranium-contaminated soil environments. Our results in this study are consistent with some previous reports (Gajewska &Sklodowska 2007, Slomka et al. 2008).
Cellular ultrastructure with different citric acid concentrations
According to the above results, the cellular ultrastructure was affected by the con + 5 treatment. As shown in Fig. 3A, the cellular ultrastructure of shoots in perennial ryegrass was unchanged with normal mitochondria and an evenly distributed matrix in the control group. Chloroplasts were obviously observed, normally surrounded by a double membrane. In contrast, the cell structure of plant shoots with 5 mg/kg uranium was changed with a significantly reduced number of mitochondria, expanded chloroplasts, damaged cell walls and disrupted double chloroplast membranes (Fig. 3B).In the treatment with 5 mmol/kg citric acid (Fig. 3C), the cell structure of plant shoots partially returned normal. Interestingly, in plant shoot cells, the mitochondrial number increased, the chloroplast was seemingly normal, and the cell wall was observable. The effects of uranium and citric acid on the cell structure in the plant roots were identical to those in the plant shoots. In the treatment without uranium, the cell structure of the plant roots was normal (Fig. 3D). The cell structure was partly disrupted by the treatment with 5 mg/kg uranium (Fig. 3E). The effect of uranium on the cell ultrastructure was partially alleviated when 5 mmol/kg citric acid was added (Fig. 3F).
In summary, these results suggest that citric acid (5 mmol/kg) can attenuate uranium-induced damage to perennial ryegrass.