Growth responses and physiological and biochemical changes in five ornamental plants grown in urban lead‐contaminated soils

Abstract An increasing concentration of lead (Pb) in urban contaminated soil due to anthropogenic activities has been a global issue threatening human health. The use of urban ornamental plants as phytoremediation of Pb‐contaminated soil is a new choice. In the present experiment, the physiological and biochemical response of five ornamental plants to increase in concentrations of C4H6O4Pb·H2O in the soil were measured to investigate these plans’ Pb tolerance strategies and abilities. Our results showed that Pb stress significantly inhibited the growth and the biomass of all the plants. The root activity (RA), net photosynthetic rate (P n), and chlorophyll (Chl) content in Pb‐stressed leaves were significantly decreased, whereas the leaf proline (Pro), soluble sugar (SS), and membrane stability index (MSI) were remarkable increased compared with those in the control group. By application of all‐subsets regression and linear regression, the reduction in photosynthetic capacity in the five plants is mainly due to the decrease in the leaf Chl content caused by Pb stress. The bioconcentration factor (BCF) in Canna generalis was greater than 1, while in the other plants were lower than 1, suggesting that Canna generalis had the highest Pb accumulation ability. The translocation factor (TF) in all the plants were lower than 1, suggesting that Pb preferentially accumulated in the external part of roots. By calculating the comprehensive evaluation value (CEV), Iris germanica L. was found to be the most sensitive species, and Canna generalis was the most tolerant species, to Pb stress among the five ornamental plants.

metals are not biodegradable and can remain in soil and water at high concentrations for hundreds of years (Verma et al., 2017), ultimately resulting in serious damage to the biotic communities (Auger et al., 2013;Sarma et al., 2017).
The non-biodegradable and immobile features of Pb due to its strong bond formation with soil organic and/or inorganic ligands makes its removal from contaminated soils very difficult (Cunningham et al., 1995;Dávila et al., 2011aDávila et al., ,2011b. Until now, many physical, chemical, and biological technologies have been developed to remediate metalliferous sites. However, only a few technologies are both economical and cost effective. Among those remediation methods, phytoremediation is an eco-friendly and successful approach for the remediation of toxic metals in polluted soils (Chandrasekhar & Ray, 2019;Cunningham & Berti, 1993;Noufal, Maalla, Noufal, & Hossean, 2017;Salazar & Pignata, 2014). It is a plant-based technology that utilizes specific hyper-accumulator plants to retain, remove or reduce toxic metals and metalloids in soil.

| Plant material and growth conditions
The experiment was conducted at Shandong Agriculture University (36°09′N, 117°09′E, 128 m above sea level) in Taian, Shandong Province, China. Healthy and uniformity seeds of Hemerocallis fulva, Iris germanica L., Canna generalis, Pennisetum clandestinum, and Miscanthus sinensis were purchased from a local market. All seeds were soaked in distilled water to improve the germination percentage. After soaking in distilled water for at least 24 hr, the seeds were placed in germinating beds consisting of 5 cm diameter Petri dishes, filled with disinfected peat. Each Petri dish contained one seed.
The dishes were incubated at 25°C and 75% relative humidity in an artificial climate chamber (RXZ-380, made by Ningbo Southeast Instrument Company), with light period and intensity of 14 hr/day and 1,200 mol m −2 day −1 , respectively. After three weeks of germination, each individual seedling with 4-5 leaves was transplanted into a plastic plot (18 cm in diameter and 22 cm in depth) which was filled with 5.5 kg Pb-treated soils. The soil in each plot was watered with deionized water to 60% soil moisture.
The soil samples were collected from the local surface soil (0-20 cm). After air drying inside the laboratory for two weeks, the soil samples were sieved through a 2-mm mesh. The soil filled pots were kept for 5 days for maturation followed by planting. The soil was classified as Alfisols under an Ultisol in USDA taxonomy and the soil pH was 7.5, the organic matter content 10.5 g/kg, the total nitrogen 0.65 g/kg, available phosphorus content 0.032 g/kg, available potassium content 0.032 g/kg and available Pb concentration was 9.85 mg/kg.
Altogether 90 individual pots were maintained in the study. Each of the treatment levels was prepared by dissolving the respective concentrations of Pb equivalent to C 4 H 6 O 4 Pb·H 2 O in 500 ml of distilled water. The soil in each plot was spiked with the required levels of Pb solutions. Before transferring the plant seedling to the plot, the Pb-spiked soil was left for two weeks to reach equilibration.

| Determination of chlorophyll (Chl) content
Chl a, b and total Chl contents in fresh leaves were extracted by acetone and measured according to the method described by Hiscox and Israelstam (1979).

| Determination of proline (Pro)
The extraction procedure and colorimetric determination of Pro in plant cells was carried out according to Bates, Waldren, and Teare (1973). Briefly, 1.0 g leaf sample was homogenized in 3% (w/v) aqueous sulfosalicylic acid. The homogenate was then filtered through two layers of filter paper to obtain a clear filtrate. After addition of the glacial acetic acid and acid ninhydrin mixture to 1 ml of the filtrate, the reaction mixture was heated in a in 100°C water bath for 1.0 hr. The reaction was terminated in an ice bath. The absorbance was read at a wavelength of 546 nm. The Pro concentration was calculated using a standard curve.

| Determination of soluble sugars (SS)
The SS in leaves were extracted and identified following the method of Nelson (1944). Briefly, 0.5 g of fresh leaves were ground in 80% neutral aqueous ethanol and heated in a 100°C water bath for 10 min. The extract was centrifuged at 5,000 rpm for 10 min, and 1.0 ml supernatant was added to 4 ml anthrone reagent followed by heating in a 100°C water bath for 10 min.
The reaction was stopped by incubation in a water bath at room temperature (20°C) for 5 min. The absorbance was measured at 630 nm.

| Measurements of the leaf net photosynthetic rate (P n )
Healthy and fully expanded leaves in three plants from each Pb treatment from different pots were chosen to measure the leaf P n using a portable photosynthesis system (LI-6400, Li-COR Inc.) with a red-blue LED light source as the illumination. The measurements were conducted from 9:00 to 11:00 a.m. on a sunny day.

| Membrane thermostability index (MTI)
The MTI was determined in 10 leaf discs from fully expanded young leaves by measuring electrolyte leakage according to the method of Sullivan and Ross (1979). After washing in deionized water, 20 ml deionized water was added to the leaf discs in a capped tube and incubated at 25°C for 24 hr. The values of electrical conductivity (EC1) were measured in the samples. The samples were then heated in a boiling water bath for 20 min. After the temperature of the samples reached 25°C, the electrical conductivity (EC2) was again measured.

| Determination of plant Pb content
The Pb content in plant shoots and roots was determined according to the procedure described by Wang et al. (2009). After washing with deionized water, the shoot and root samples were dried at 75°C for 24 hr. Then, 0.1 g of ground shoots and roots of dried samples were digested in 10 ml HNO 3 :HClO 4 solution and heated in an oven at 100-200°C until near dryness. Subsequently, 5 ml of 5% HNO 3 was added to dissolve the cooled residue in addition to ddH 2 O to a volume of 20 ml. ICP-MS (Agilent ICP-MS 7700ce, Agilent Technologies) was used to determine the plant Pb content.

| Determination of root metabolic activity (RA)
The RA was measured according to the method of Liu, Wei, and Li (2014). Briefly, 0.5 g fresh root sample was immersed in 10 ml of a mixed liquid of 0.4% TTC (2,3,5-triphenyitetrazolium chloride) and 66 mmol/L phosphate buffer solution. The reaction solution was maintained at 37°C for 3 hr, followed by the addition of 2 ml sulfuric acid (1 mol/L) to terminate the reaction. The root was removed and ground in 2 ml ethyl acetate to extract TTF (1,3,5-triphenylformazan).
The absorbance of the extract was measured at a wavelength of 485 nm.

| Plant growth parameters
At the end of the experiment, the plant height (PH), leaf area (LA), leaf numbers per plant (LN), and tiller numbers per plant (TN) of each species subjected to each Pb treatment were measured. The plant height, LN, and TN of each plant were determined prior to harvest.
The LA was determined based on the leaf area meter (CI 202, rea meter, CID Incorporated).

| Plant harvest
After 6 weeks of cultivation, all plants were harvested. The plant biomass was separated into two parts: the root biomass and aboveground biomass. The plant material (leaves, stems and roots) was heated in an oven at 105°C for 30 min and dried at 75°C for 48 hr.
After the weight of the samples reached a constant value, their dry weight was recorded.

| Bioconcentration factor (BCF) and translocation factor (TF)
The BCF and TF were calculated to evaluate the accumulation of Pb

Calculation of the comprehensive evaluation value
The comprehensive evaluation value was calculated using where W i is the factor weight, which is the ratio of the index weight of each extracted score to the weighted summations of all extracted scores in the ith plant.

Reorder of the comprehensive evaluation value
With the calculated comprehensive evaluation value, the responses of different ornamental genotypes to Pb stress could be reflected by a single value. The values of five plant species were arranged in descending order and labelled a, b, c, d, and e, respectively, where a denotes the most Pb-tolerant and e denotes the most Pbsensitive plant species.

| Statistical analysis
The experiment was conducted using a completely randomized block design with three replicates. All the parameters described above were measured at six weeks after the plants were subjected to soil Pb treatments. The data for Miscanthus sinensis exposed to Pb1000 treatment are not shown in the study because this plant could not survive under these severe Pb stress conditions. The SPSS 18.0 statistical software package (SPSS) was used to perform the statistical analyses. The mean with standard deviation (±SD) is shown for each treatment in the tables and figures. The parameters were analyzed by one-/two-way analysis of variance (ANOVA) followed by Duncan's multiple range test at p < .05. Figures 1, 2 and 3 were performed with the help of Origin 9.0 software (Origin Lab). The heatmap in Figure 4 was constructed by R 3.6.1 (Bell Laboratories).

| Plant growth and biomass
The changes in plant growth parameters (c.v. PH, LN, LA, and TN) and biomass (

| Chlorophyll content
The

| Leaf net photosynthetic rate and root activity
The leaf net photosynthetic rate and root activity of

| Analysis of variance on plant parameters
The

| Identification of Pb tolerant plant species
The CEV of the different plants in response to the six Pb treatments are listed in Table 4

| D ISCUSS I ON
Pb toxicity has been studied widely in many higher plants for evaluations of Pb tolerance or a high Pb uptake potential of plants (Andra et al., 2009), aiming to select candidate plants for the phytoremediation of Pb-contaminated regions (Gupta & Chandra, 1994 Pal, Banerjee, and Kundu (2013) and Zhao, Xiong, Li, and Zhu (2009), who showed that Pb accumulated mostly in roots, while a small quantity was translocated to shoots. In addition, the lower R/S, which was expressed as the root biomass/aboveground biomass for most plants in Pb-contaminated soil (Table 1) the organization and function of membrane ion channels (Aravind & Prasad, 2005). Furthermore, the highest MTI values in response to the same soil Pb treatment were found in the leaves of Iris germanica L., indicating that Pb stress posed a higher threat to Iris germanica L.
than the other ornamental plants.
When suffered environment stress, plants had a self-protection and stress tolerant ability in avoiding damage caused by ROS. In our study, two parameters, Pro and SS, were determined to identify the physicochemical mechanisms of plant resistance and tolerance to soil Pb stress. The accumulation of Pro and SS stimulated by various metal ions such as Cd 2+ , Pb 2+ , Zn 2+ , Cu 2+ , and Al 3+ has been widely reported in several studies as one of the most commonly induced adaptive responses of plants (Jia, Zhang, Zhao, Liu, & He, 2018;Li, Yang, Jia, Chen, & Wei, 2013;Nedjimi & Daoud, 2009;Sandra, Marija, Dragan, Vibor, & Branka, 2010;Shevyakova, Netronina, Aronova, & Kuznetsov, 2003), and this effect was greater in shoots than in roots (Tian, Guo, & Yan, 2007;Verma & Dubey, 2001). The accumulation of Pro in the cytosol under heavy metal exposure is not only regarded as an indicator of environmental stress but also to play an import-  Sun, Zhou, Sun, & Jin, 2007) by maintaining the osmotic equilibrium within plant cells (Szabados & Savourcb, 2010), scavenging hydroxyl radicals and singlet oxygen (Kavi & Sreenivasulu, 2014;Matysik, Bhalu, Mohanty, & Bohrweg, 2002;Pal et al., 2013), chelating metal ions in plants and forming a nontoxic metal-proline complex (Sharma, Schat, & Vooijs, 1998). In our study, the Pro contents in the leaves of  (Matysik et al., 2002). The accumulation of SS, which acts as an osmotic agent in stressed plants, has also been considered to be a resistance mechanism to the stress condition (Roitsch, 1999;Wu & Xia, 2006) and to play a pivotal role in the osmotic adjustment in plants (Sánchez, Manzanares, Andres, Tenorio, & Ayerbe, 1998;Zhou & Yu, 2009). Lehner (2008) found that SS could detoxify ROS and was related to ROS-producing metabolic pathways in wheat.
In this study, we found that the contents of SS in leaves of ornamental plants subjected to low levels of Pb (50-200 mg/kg) were significantly higher than in the control group, suggesting that mild Pb stress could enhance the synthesis of carbohydrates to eliminate ROS by reinforcing the antioxidant system and protecting the cells from damage (Nguyen, Hailstones, Wilkes, & Sutton, 2010).
Our results are in line with the findings of John, Ahmad, Gadgil, and Sharma (2008), who reported a significant increase in SS contents in Lemna polyrrhiza following exposure to low Pb concentration stress.
Conversely, high levels (500-1,000 mg/kg) of Pb toxicity reduced soluble sugar concentrations in the leaves of all ornamental plants.
The decrease of soluble sugar contents at the high levels of lead in soil might be attributed to the lead-induced detrimental effect on the structure and function of the photosynthetic apparatus, inhibited the plant carbon assimilation ability, and reduced the synthesis of leaf carbohydrate in the end (Jiang, Wang, Dong, & Yan, 2019).
Similar results have also been reported by Bhardwaj, Chaturvedi, and Pratti (2009), Sinha (2004), and Ali et al. (2014, demonstrating the dose dependence of the soluble sugar concentration in ornamental plants exposed to Pb treatment. Photosynthetic abilities of plants have been shown to be vulnerable to heavy metals (Boucher & Carpentier, 1999;Tanyolaç, Ekmekçi, & Ünalan, 2007). Several authors have reported negative effects on photosynthetic capacity in different species grown in media with high concentrations of Pb (Briat & Lebrun, 1999 TA B L E 4 (Continued) Yang, & Wei, 2017). Our study provided similar results showing that soil Pb even at low doses, and despite the small amount of translocation from root s to shoots (validated by TF < 1, Table 2), inhibited the photosynthetic rate in the leaves of all ornamental plants (Figure 2a).

| CON CLUS IONS
In this study, several physiological and biochemical parameters were determined and analyzed along with the Pb uptake and toler- but their self-protective abilities against Pb stress were downregulated at high Pb levels (500-1,000 mg/kg). By calculating the CEV, Iris germanica L. was found to be the most sensitive plant to Pb stress, and Canna generalis was the most suitable plant for use in phytoremediation.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.
[Correction added on 24 May 2021, after first online publication: Conflict of Interest statement added to provide full transparency.]

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.
F I G U R E 5 Schematic representation of the Pb toxicity on the shoot and root of ornamental plants and the plant tolerance mechanisms under Pb stress conditions