Cadmium Uptake and Growth Responses of Seven Urban Flowering Plants: Hyperaccumulator or Bioindicator?

The application of flowering plants is the basis of urban forest construction. A newly-found flowering hyperaccumulator is crucial for remediating urban contaminated soil sustainably by cadmium (Cd). This study evaluated growth responses, Cd uptake and bioaccumulation characteristics of seven urban flowering plants. Based on growth responses of these plants, Calendula officinalis L. showed high tolerance to at least 100 mg kg−1 Cd, in terms of significant increase in biomass and with no obvious changes in height. After 60 d exposure to 100 mg kg−1 Cd, the accumulated Cd in shoots of the plant reached 279.51 ± 13.67 μg g−1 DW, which is above the critical value defined for a hyperaccumulator (100 μg g−1 DW for Cd). Meanwhile, the plant could accumulate Cd to as much as 926.68 ± 29.11 μg g−1 DW in root and 1206.19 ± 23.06 μg g−1 DW in plant, and had higher Cd uptake and bioaccumulation values. According to these traits, it is shown that Calendula officinalis L. can become a potential Cd-hyperaccumulator for phytoremediation. By contrast, Dianthus caryophyllus L. is very sensitive to Cd stress in terms of significantly decreased biomass, height and Cd uptake, indicating the plant is considered as a Cd-bioindicator.

A great number of current technologies have been developed to clean up the contaminated soil by Cd [34][35][36]. However, several conventional technologies, such as physical and chemical treatment approaches, are more expensive, less eco-friendly and bring secondary pollution problems [37][38][39]. In comparison, phytoremediation is considered as a promising

Plant Culture and Cd Exposure
The pot-culture experiment was carried out in July 2020 at the agricultural experimental field of Shenyang Agricultural University (41 • 44 N and 123 • 27 E, 44.7 m a.s.l.), which is in the temperate zone with a continental monsoon climate. The average annual temperature is 8.0 • C, the average annual precipitation is 702.9mm and the relative air humidity is 65-75%. The soil used in the pots was collected from the topsoil (0-20 cm) of the experimental field. The physical and chemical properties of the test soil was listed in Table 1. The air-dried soil samples were sieved through a 3-mm mesh sieve and placed into plastic pots with (20 cm diameter × 15 cm height) and mixed uniformly with the specified concentration of CdCl 2 ·2.5H 2 O solution. Three Cd concentrations were applied: 0 (the control), 10 and 100 mg kg −1 . Seeds of seven flowering plants (Orychophragmus violaceus (L.) O. E. Schulz., Impatiens balsamina L., Pelargonium hortorum Bailey., Mirabilis jalapa L., Althaea rosea (Linn.) Cavan., Calendula officinalis L. and Dianthus caryophyllus L.) were obtained from Liaoning Academy of Agricultural Sciences. The natural properties of sampled plants were listed in Table 2. Thirty seeds of each flowering plant were transplanted into each pot. After one month of growth, uniform seedlings with similar size and height were transplanted into the pots (ten seedlings per pot). All the pots were randomly arranged in a constant temperature room under the controlled conditions (100-150 µmol m −2 s −1 light intensity,

Plant and Soil Analysis
When the experiment was terminated, four plants with similar growth character were chosen in each treatment, and the height of these plants was recorded. The harvested plant materials were rinsed with tap water, and the roots were immersed in 20 mM Na 2 -EDTA for 20 min to clear Cd adhered to the root surface [18,42]. Then, the plants were divided into shoots and roots. The separated plant materials were rinsed with running tap water and distilled water, wiped with tissues and weighed. They were then dried at 105 • C for 30 min, then at 70 • C until weight was constant for Cd content measurement. The biomass (dry weight) of the plant materials was recorded.
The powders (0.5 g) of the harvested plant materials were digested with a concentrated acid mixture of HNO 3 /HClO 4 (3:1, v/v). The plant samples were then incubated at 60 • C for two hours (open vessel digestion). The digested plant samples were incubated overnight at room temperature. They were diluted, filtered using filter paper (Whatman Grade 1) and cellulose acetate syringe filters. The soil materials were sieved and dried overnight at 65 • C before acid digestion in HCl/HNO 3 (3:1, v/v). The soil samples were incubated overnight at room temperature. They were then filtered twice using filter paper (Whatman Grade 1) and once using cellulose acetate syringe filters (Whatman Grade 1). The concentrations of Cd in plant tissues and soils were determined with an inductively coupled plasma mass spectrometry instrument (ICP-MS, Agilent 7900, Agilent Technologies, Tokyo, Japan) [5,25].
The physical and chemical properties of the soils were measured using the analytical methods for soils and agricultural chemistry [70]. Soil pH was determined by a glass electrode pH meter in a 1:2.5 soil-water slurry. The CEC was determined using a NH 4acetate compulsory displacement method. Soil organic matter was determined by the K 2 Cr 2 O 7 titration method after digestion. Total N of the soil was determined by H 2 SO 4 digestion followed by steam distillation with 25 mL NaOH (10 mol L −1 ) and titration with 5 mmol L −1 H 2 SO 4 , and analyzed using an element analyzer (Vario MACRO cube, Elementar Inc., Germany). Available P was extracted with 0.5 mol L −1 NaHCO 3 (pH = 8.5) and then determined by colorimetric analysis. Available K was extracted with 1 mol L −1 Sustainability 2022, 14, 619 4 of 12 CH 3 COONH 4 (pH = 7.0) solution, and measured with an inductively coupled plasma mass spectrometry instrument (ICP-MS, Agilent 7900, Japan). Soil particle size distribution was determined by pipette method. Available Ca and Mg were extracted with the Mehlich 3 extractant and measured by atomic absorption spectroscopy (AAS, Hitachi Z-8000, Hitachi High-Tech, Tokyo, Japan) [71].

Plant and Soil Analysis
When the experiment was terminated, four plants with similar growth character were chosen in each treatment, and the height of these plants was recorded. The harvested plant materials were rinsed with tap water, and the roots were immersed in 20 mM Na2-EDTA for 20 min to clear Cd adhered to the root surface [18,42]. Then, the plants were divided into shoots and roots. The separated plant materials were rinsed with running tap water and distilled water, wiped with tissues and weighed. They were then dried at 105 °C for 30 min, then at 70 °C until weight was constant for Cd content measurement. The biomass (dry weight) of the plant materials was recorded.
The powders (0.5g) of the harvested plant materials were digested with a concentrated acid mixture of HNO3/HClO4 (3:1, v/v)

Plant and Soil Analysis
When the experiment was terminated, four plants with similar growth character were chosen in each treatment, and the height of these plants was recorded. The harvested plant materials were rinsed with tap water, and the roots were immersed in 20 mM Na2-EDTA for 20 min to clear Cd adhered to the root surface [18,42]. Then, the plants were divided into shoots and roots. The separated plant materials were rinsed with running tap water and distilled water, wiped with tissues and weighed. They were then dried at 105 °C for 30 min, then at 70 °C until weight was constant for Cd content measurement. The biomass (dry weight) of the plant materials was recorded.
The powders (0.5g) of the harvested plant materials were digested with a concentrated acid mixture of HNO3/HClO4 (3:1, v/v). The plant samples were then incubated at

Data Analysis
The bioaccumulation factor (BCF) indicated the ability of plants to accumulate heavy metals from soils [51,54]. BCF was calculated as: BCF = the metal concentration in plant tissues the metal concentration in soils Heavy-metal uptake was calculated using the following formula as [41].
Where M 1 and M 2 are metal concentrations in the plant tissue and W 1 and W 2 are the plant biomass at time T 1 (initial sampling) and T 2 (final sampling).

Statistical Analyses
All measurements were replicated four times. Means and standard deviations (SD) were calculated by the Microsoft Office Excel 2016 for all the data. One-way ANOVA was carried out with SPSS 22.0. The significant difference was set between treatments at p < 0.05 or p < 0.01. Multiple comparison was also made by the least significant difference (LSD) test. When the plants were exposed to 10 mg kg −1 Cd, the root biomass DW of the four flowering plants (Orychophragmus violaceus (L.) O. E. Schulz, Impatiens balsamina L., Althaea rosea (Linn.) Cavan., Calendula officinalis L.) increased to different extents compared with the control, which indicated similar increased phenomenon with the shoot growth. The increase of Cd concentration in soil led to the root biomass DW of the three flowering plants (Pelargonium hortorum Bailey Mirabilis jalapa L. and Dianthus caryophyllus L.) showing a significant decrease compared with the control (p < 0.01). As shown as in Figure 1, the total biomass DW of the seven flowering plants showed a similar change trend with the shoot biomass DW of these plants.

Results and Discussion
On the whole, after 60 d exposure to 10 mg kg −1 Cd, the shoot, root and total biomass DW of Calendula officinalis L. had a significant increase (p < 0.01), indicating that low concentration Cd in soil could have a stimulating influence on plant growth. The similar phenomenon has been reported by other researchers [18,26,[73][74][75], which is also proposed as hormesis by   [76]. When the Cd concentration in soil reached to 100 mg kg −1 , the shoot, root and total biomass DW of Calendula officinalis L. still increased to different extents compared with the control, showing that the plant had a good tolerance to Cd stress. By contrast, under the treatment of different Cd concentrations in soil, the shoot, root and total biomass DW of Dianthus caryophyllus L. all showed a significant decrease compared with the control (p < 0.01), illustrating that Dianthus caryophyllus L. is very sensitive to Cd stress.

Differences in Height among the Seven Flowering Plants
As shown as in Figure 2, after 60 d exposure to 10 mg kg −1 Cd, the height of the four plants (Orychophragmus violaceus (L.) O. E. Schulz., Pelargonium hortorum Bailey., Mirabilis jalapa L., Althaea rosea (Linn.) Cavan.) showed no significant differences, and the height of Impatiens balsamina L. and Calendula officinalis L. showed a significant increase compared with the control (p < 0.01), which is also consistent with other studies [35,42,48,53]. The hormesis phenomenon of the plant height is similar with the above biomass changes, which may originate from the internal defense mechanism induced by oxidative free radicals or the overcompensation response of plant cells or organisms to toxic pollutants. The underlying mechanism research needs to be further studied. when the Cd concentration in soil reached 100 mg kg −1 , the height of the four plants (Orychophragmus violaceus (L.) O. E. Schulz., Impatiens balsamina L., Mirabilis jalapa L., Calendula officinalis L.) showed no significant differences compared with the control. By contrast, under the treatment of different Cd concentrations (10 and 100 mg kg −1 ) in soil, the height of Dianthus caryophyllus L. all decreased significantly by 11% and 32% compared with the control (p < 0.01), which is in agreement with the change of the shoot, root and total biomass DW of the plant, reconfirming the sensitivity of Dianthus caryophyllus L. to Cd stress.
Based on the growth responses of the seven flowering plants, results showed that Calendula officinalis L. had high tolerance to not less than 100 mg kg −1 Cd, which shows significant increases in biomass and no obvious changes in height. The growth of Calendula officinalis L. was increased at the low Cd concentration (10 mg kg −1 ), which is conducive for the plant to adapt to external stress. These growth traits indicated that Calendula officinalis L. has good potential in Cd-contaminated remediation, since the tolerance of plant to heavy metal toxicity could provide an important reference to distinguish a hyperaccumulator [26,41,42,45,50]. By contrast, Dianthus caryophyllus L. is very sensitive to Cd stress showing decreased biomass and height, which indicated the plant is considered as a Cd-bioindicator.  Differences in Cd accumulation among the seven flowering plants indicated that Calendula officinalis L. had a good accumulation ability for Cd, in terms of the high concentrations of accumulated Cd in shoots, roots and plants. By contrast, Dianthus caryophyllus L. had a poor accumulation ability for Cd. The different traits of Cd accumulation in Calendula officinalis L. and Dianthus caryophyllus L. had a good correlation with growth responses of the two plants.

Differences in Cd Uptake and BCF among the Seven Flowering Plants
As shown as Table 3, Cd uptake and BCF of seven flowering plants varied with the increase of Cd concentrations in the soil. When the concentration of Cd was 10 mg kg −1 in soil, Cd uptake of Calendula officinalis L. were 8.13 ± 0.08 µg plant −1 d −1 , which was higher than six other plants. By contrast, Cd uptake of Dianthus caryophyllus L. was only 0.69 ± 0.03. When the concentration of Cd reached 100 mg kg −1 in soil, Cd uptake of Calendula officinalis L. increased significantly and reached 80.77 ± 0.19 µg plant −1 d −1 , and Cd uptake of the six other plants were all less than 14.00 µg plant −1 d −1 , especially for Dianthus caryophyllus L., only 0.43 ± 0.02. There was a positive correlation between Cd uptake and Cd concentrations accumulated in Calendula officinalis L., indicating that the plant may accumulate larger amounts of Cd when exposed to higher Cd concentrations in soil.
The bioconcentration factor (BCF) was used to evaluate the efficacy of the plant to accumulate the toxic pollutants in plant tissues [51,53,78]. When the concentration of Cd was 10 mg kg −1 in soil, the BCF values of Impatiens balsamina L. and Calendula officinalis L. were 10.69 and 13.49, which was higher than the other five plants. When the concentration Sustainability 2022, 14, 619 9 of 12 of Cd reached 100 mg kg −1 in soil, the BCF values of Calendula officinalis L. was 12.06, which was still higher than the other six plants, indicating that the plant can accumulate Cd more efficiently and still maintain high efficiency even if the concentration of Cd in soil was above 100 mg kg −1 . On the contrary, the BCF values of Dianthus caryophyllus L. was only 0.71, which was in accordance with the poor ability for Cd uptake and Cd concentrations accumulated in the plant. Based on higher Cd uptake and BCF values, the higher concentrations of accumulated Cd in shoots, roots and plants of Calendula officinalis L. showed that the plant has the potential of hyperaccumulation used for phytoremediation of Cd-contaminated soils.

Conclusions
Firstly, Calendula officinalis L., as a popular ornamental flowering plant, has been historically grown throughout the world and produced commercially in Europe and the US, which benefits from the easy and rapid growth of the plant for flower or seed production [79,80]. Secondly, seeds of Calendula officinalis L. are rich in calendic acid, which can substitute for volatile organic compounds (VOCs) as a drying agent in many industrial chemicals [81,82]. As urban residents' demands for renewable, environmentally friendly alternatives for VOCs are growing, Calendula officinalis L. may serve as an attractive oil crop for promoting sustainable urban development. Furthermore, in our present study, it was shown that Calendula officinalis L. had a good tolerance to Cd stress; at the same time, among the seven flowering plants, Calendula officinalis L. showed a better accumulation ability of Cd, above the critical value defined for the hyperaccumulator (100 µg g −1 DW for Cd) and had higher Cd uptake and BCF values. According to these traits above, it is shown Calendula officinalis L. can become a good hyperaccumulator used for phytoremediation of environmental pollution in urban areas. The present study will provide an available reference for exploring Cd tolerant strategies in hyperaccumulator or bioindicators, and will also contribute to healthy urban residents' well-being and sustainable development of urban forests.

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Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to the restriction policy of co-authors' affiliations.