Assessing the difference of tolerance and phytoremediation potential in mercury contaminated soil of a non-food energy crop, Helianthus tuberosus L. (Jerusalem artichoke)

Plant material and growth condition

Through previous analysis of the germplasm diversity of Helianthus tuberosus L. collected from East Asia and Europe, revealed by AFLP (Amplified Fragment Length Polymorphism) marker and phenotypic traits, three major genetic groups were identified: group I, which exhibited high above-ground biomass and nutrient content, and strongly sexual reproduction; group II, with a long period of vegetative growth; and group III, which had the highest tuber yield and total sugar content (Kou et al., 2013). We selected three representative cultivars (LZJ033, LZJ119 and LZJ047) representing, respectively, each of these three groups, as experimental materials.

This experiment was conducted in a rain-shelter at the Yuzhong experimental station of Lanzhou University, Gansu, China (35°56′N, 104°09′E, 1,750 m a.s.l.) in 2013. Five mercury treatments (control (CK), 0.15, 1, 5, 10 mg kg⁻¹Hg²⁺(in the form of HgCl₂)), with ten replicates for each, were set up and arranged in a completely randomized design. Topsoil (0–20 cm) from uncontaminated arable land (whose soil properties were as follows: soil pH 8.14; organic matter content 1.26%; available nitrogen 91.9 mg kg⁻¹; available phosphorus 23 mg kg⁻¹; available potassium 168.5 mg kg⁻¹; total mercury content 0.036 mg kg⁻¹) was put into plastic pots (35 cm in diameter and 30 cm in height), using about 15 kg (dry weight) per pot. The soil was then saturated with solution containing the required amount of HgCl₂. One week later, the soil in every pot was thoroughly mixed and then three tubers, selected as having 1–2 buds, weighing about 15 g, and exhibiting no decay, were planted in each pot on April 10. All pots were watered every week according to soil water content in order to prevent drought stress.

Measurements of agronomic traits

The germination rate was monitored every two weeks from the time of sowing until the 8th week, and the mortality rate was determined in the 20th week. Plant height, stem diameter and internode length were measured at maturity using, respectively, a tape measure, a vernier caliper and a portable leaf area system (LI-3000; Li-COR, Lincoln, NE, USA).

The stem and leaf tissue were cut off from the base of each plant on October 1 and washed with tap water followed by deionized water in order to remove any mercury adhering to the surface; meanwhile, the roots and tubers were dug up and vigorously washed with tap water and followed by deionized water and ultrasound bath in order to remove any soil particles and mercury adhering to the surface, and then the tubers were cut into sections about 0.5 cm thick using a knife. All plant materials were then dried at room temperature until constant weights were attained so as to avoid mercury loss.

Determination of mercury content

The samples of plant material were ground to powder (particle size <0.15 mm) after weighing, then sealed into plastic bags for mercury analysis. Dried powder samples from the leaf, stem, tuber and root of the Jerusalem artichoke were digested with concentrated HNO₃ and H₂O₂ in microwave about two hours and the resulting solutions were analyzed for mercury content using cold vapor atomic absorption spectrometry according to the method of Chen et al. (2009).

The bioaccumulation factor (BF), translocation factor (TF) and amounts of metal in the plants, which were used to evaluate the potential for phytoremediation of Jerusalem artichoke, were calculated from the following equations: ${\displaystyle \mathrm{BF}=\frac{\text{Metal concentration in plant tissue}}{\text{Metal concentration in soil}}}$ ${\displaystyle \mathrm{TF}=\frac{\text{Metal concentration in plant tissue}}{\text{Metal concentration in root}}}$ Amounts of metal in plant tissue = Metal concentration in plant tissue × tissue biomass.

Lipid peroxidation assay

The extent of leaf lipid peroxidation was determined by measuring the amount of malondialdehyde (MDA), an end-product of lipid peroxidation, by reaction with TBA (thiobarbituric acid) according to the method of Dhindsa, Dhindsa & Thorpe (1981), and the content of MDA was expressed as nmol g⁻¹ fresh weight (FW).

Analysis of photosynthetic characteristics

Leaf chlorophyll content was determined as follows: each leaf was homogenized in 80% ice-cold acetone in the dark and then centrifuged at 10,000 rpm for 10 min at 4 °C; the chlorophyll content of the supernatant was estimated using Arnon’s method (Arnon, 1949) and the chlorophyll content was expressed as mg g⁻¹ FW.

The net photosynthetic rate (P_n) of Jerusalem artichoke plants was measured in leaves (the sixth leaf from the top of the stem) using an Li-6400 portable photosynthesis system (Li-COR, Lincoln, NE, USA) with an Li-6400-02B LED light source in the middle of July. The light intensity, temperature, flow rate of the sample cell were kept as follows: 1,200 µmol m⁻² s⁻¹, 28 °C, 300 µmol s⁻¹, and the CO₂ concentration of reference cell was kept about 380 µmol mol⁻¹ through a buffer bottle. All the measurements were carried out from 9:00 to 12:00 in the fine weather.

Maximum quantum yield of primary photochemistry was measured with a portable fluorimeter (Handy PEA, Hansatech, UK). The leaves were dark-adapted about 30 min before taking measurements, then exposed to red light supplied by an LED at a wavelength of 650 nm with an excitation irradiance at a density of 3,000 µmol m⁻² s⁻¹ and a duration of 800 ms, according to the methods of Wang et al. (2014). The minimal (F₀) and maximal (F_m) fluorescence yield parameters were obtained and the maximum quantum yield of PSII photochemistry was then calculated as the ratio F_v∕F_m = (F_m − F₀)∕F_m.

Statistical analysis

All treatments were arranged in a completely randomized design with ten replicates per treatment. Data were processed with Microsoft Excel 2007 and the SPSS 16.0 statistical software package. One-way ANOVA and post hoc least significant difference (LSD) tests were used to compare the agronomic and physiological parameters of different varieties under mercury treatment, two-way ANOVA (general linear model) was carried out to assess the effects of mercury treatments, cultivars and their interactions on the variables examined, before these comparisons the data were tested for homogeneity.

Agronomic traits

Generally the agronomic traits of all cultivars changed in the same way when they were subjected to the different mercury treatments, and the mercury treatment, cultivar and the interaction between them had significant effects on plant height, internode length and leaf area (Table S1). As Table 1 shows, the final germination rate under mercury treatment ranged from 90% to 100%. Although the germination rate was thus little affected by mercury treatment, the seedling emergence time was delayed by this treatment. The emergence of LZJ047 and LZJ119 was delayed by about four weeks in the 10 mg kg⁻¹ mercury treatment, though LZJ033 was less affected. We also observed some plant death at higher mercury concentrations, but there were differences among the three cultivars (Table 1). The mercury treatment killed the plants only at 10 mg kg⁻¹ and the mortality rate was only 25.93% and 21.43% for, respectively, LZJ119 and LZJ033, but the mortality rate of LZJ047 was 85.19% under the 10 mg kg⁻¹ mercury treatment, and mercury treatment at 5 mg kg⁻¹ killed some LZJ047 plants; the mortality rate was 13.33%.

As shown in Fig. 1, a major inhibitory effect on all growth parameters except stem diameter was observed with increasing concentration of mercury in the medium; plant height, internode length and leaf area of LZJ047, LZJ119 and LZJ033 were significantly decreased by, respectively 13.24%, 18.66%, 31.74%; 16.80%, 22.53%, 29.25% and 13.03%, 22.53%, 38% of control values in the 1 mg kg⁻¹ mercury treatment and the values were reduced by 73.39%, 75.17%, 88.12%; 54.47%, 59.81%, 57.06% and 43.57%, 48.69%, 55.06% respectively compared with control values under 10 mg kg⁻¹ mercury treatment.

Although the plant height, internode length and leaf area were reduced, stem diameter exhibited some increase in response to mercury treatment. As shown in Fig. 1, the stem diameter of LZJ119 and LZJ033 significantly increased, by 17.64% and 18.36% respectively, in the 5 mg kg⁻¹ mercury treatment, and the increases were, respectively, 18.36% and 25.31% in the 10 mg kg⁻¹ mercury treatment compared with the control. The stem diameter of LZJ047 significantly increased, by 25.36% and 40.52% in the 1 mg kg⁻¹ and 5 mg kg⁻¹ mercury treatment respectively, but it only increased by 1.07% in the 10 mg kg⁻¹ mercury treatment and the increase was not significant.

Lipid peroxidation and photosynthetic characteristics

The effects of mercury on lipid peroxidation and photosynthetic traits are shown in Fig. 2; the MDA content, which is a good indicator of lipid peroxidation, increased concurrently with an increase in external mercury concentration. The MDA content significantly increased, by 27.54%, in LZJ047 exposed to 1 mg kg⁻¹ mercury, and by 35.44% and 23.63% in LZJ119 and LZJ033 in a mercury concentration of 5 mg kg⁻¹ compared with control plants; the increases for the three cultivars reached 82.4%, 52.83% and 53.67% when they were exposed to 10 mg kg⁻¹ mercury. In general LZJ047 had a high MDA content, which indicated that LZJ047 may be especially susceptible to mercury stress.

As shown in Fig. 2, the net photosynthetic rate increased by 16.86% and 6.57% under a 0.15 mg kg⁻¹ mercury concentration, representing significant changes, in, respectively, LZJ119 and LZJ047, and it then decreased with a further increase of external mercury concentration. The P _n value significantly decreased, by 12.89%, in LZJ119 exposed to 5 mg kg⁻¹ mercury, and by 16.52% and 8.35% in LZJ047 and LZJ033 in 1 mg kg⁻¹mercury; the decline in response to 10 mg kg⁻¹ mercury was 45.14%, 22.75%, 27.03% in, respectively, LZJ047, LZJ119, LZJ033 compared with the control .

The chlorophyll content and F_v∕F_m decreased as external mercury concentration increased (Fig. 2); chlorophyll content and F_v∕F_m significantly decreased under 5 mg kg⁻¹ and 1 mg kg⁻¹ mercury concentrations and the respective reductions were 19.85%, 20%, 15.28% and 18.73%, 12.88% 13.57% when 10 mg kg⁻¹ mercury was applied to, respectively, LZJ047, LZJ119, LZJ033.

Plant biomass

Mercury exposure induced a substantial decrease in dry weights in Jerusalem artichoke. As shown in Fig. 3, the leaf, root, tuber and total biomass of LZJ047 significantly decreased, by respectively 44.22%, 63.90%, 90.20% and 59.83% compared with the control, in response to 1 mg kg⁻¹ mercury, and the declines were 70.40%, 91.87%, 98.88%, 89.10% at a mercury concentration of 10 mg kg⁻¹. In LZJ119, tuber and total biomass significantly decreased, by 31.75 and 13.69% respectively, in response to 1 mg kg⁻¹ mercury; the leaf and root biomass were observed to decrease significantly (by 45.6% and 83.28%) in 5 mg kg⁻¹ mercury, and the reductions in these four biomass parameters reached, respectively, 64.23%, 73.08%, 53.27% and 90.18% compared with the control in the 10 mg kg⁻¹ mercury treatment. Although the root, tuber and total biomass of LZJ033 significantly decreased, by 50.66%, 55.28%, 46.03% respectively, in response to 1 mg kg⁻¹ mercury, the leaf biomass decreased significantly, by 28.55%, only when 10 mg kg⁻¹ mercury was present, and the declines were, respectively, 90.94%, 91% and 74.43% compared with the control in response to 10 mg kg⁻¹ mercury.

Unlike the decline in the biomass of other organs, the stem biomass was observed to increase to some extent when Jerusalem artichoke was subjected to mercury treatment (Fig. 4). The stem biomass of LZJ047 was significantly increased, by 62.35%, in response to 1 mg kg⁻¹ mercury; it then decreased and was at the significantly lower level of 61.95% of the control value at a mercury concentration of 10 mg kg⁻¹; and the stem biomass of LZJ119 underwent changes similar to those of LZJ047. The stem biomass of LZJ033 significantly increased (by 65.77%) in response to 5 mg kg⁻¹ mercury; no significant change was observed at other mercury concentrations.

Plant mercury contents

Mercury treatment, cultivar and the interaction between them had significant effects on the mercury concentration in plant organs, with the exception of tubers (Table S1 ); the concentration of mercury in the tissues of the three Jerusalem artichoke cultivars increased with an increase of external mercury concentration, and the mercury concentration of roots was higher than that of other organs (Table 2). As shown in Table 3, the mercury concentrations of leaf, stem, root and tuber in LZJ047 were, respectively, 10.89. 11.67, 93.98 and 16.06 times greater than those of control plants, and similar changes were observed in LZJ119; the mercury concentration of the four tissues was greater in LZJ033 than in the other two varieties, and the mercury concentration in roots of LZJ033 reached a maximum level of 1,269.8 µg g⁻¹, which was, respectively, 4.13 times and 2.7 times greater than the maximum values for LZJ047 and LZJ119.

Although the mercury treatment, cultivar and the interaction between them had significant effects on BF, only the mercury treatment had significant effects on TF (Table S1). The BF and TF of these Jerusalem artichokes both altered in response to mercury treatment, as shown in Table 3. The BF significantly decreased and its level was low (below 10 in all tissues except root) under medium-high mercury concentration conditions. The BF of roots was seen to increase at a mercury concentration of 10 mg kg⁻¹ and it reached its highest level, 126.98, in LZJ033, where it was 4.13 and 2.7 times greater than in, respectively, LZJ047 and LZJ119. The BF of shoots and tubers of LZJ119 was higher than that of the other two cultivars under medium-low mercury stress. The TF responded similarly to BF in these Jerusalem artichoke cultivars, remaining at a low level in medium-high mercury concentrations; and the TF values in all three cultivars were less than 0.1 (except in the case of tubers in LZJ047) at 10 mg kg⁻¹ mercury.

Amount of mercury in plant

The amounts of mercury absorbed by plants were determined from the mercury concentrations and biomasses of the plants. As shown in Table S1, mercury treatment, cultivar and the interaction between them had significant effects on the amounts of mercury in the plants. As Fig. 5 shows, the mercury amounts absorbed increased with an increase in external mercury concentration, reaching maximum of 580.9, 1327.7 and 3,001.92 µg. The root had greater amounts of mercury than other organs at medium-high mercury concentrations, and the highest level, 2,359.6 µg in LZJ033, was attained under treatment with 10 mg kg⁻¹ mercury. The maximum mercury content of tubers, 314.84 µg, was observed in LZJ119 at 5 mg kg⁻¹ mercury, and the largest amount in shoots, 523.9 µg, was found in LZJ033 at 10 mg kg⁻¹ mercury. The tuber and shoot are the main parts of Jerusalem artichoke to be harvested. The total mercury amounts of absorbed by tuber and shoot were reached to the highest level of 309.6, 658.5, 642.3 of LZJ119, LZJ119 and LZJ033 in 1 mg kg⁻¹, 5 mg kg⁻¹ and 10 mg kg⁻¹ mercury treatment, respectively.