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Aluminum toxicity decreases the phytoextraction capability by cadmium/zinc

hyperaccumulator Sedum plumbizincicola in acid soils

Jiawen Zhou, Zhu Li, Tong Zhou, Zaijun Xin, Longhua Wu, Yongming Luo,
Peter Christie

PII: S0048-9697(19)34582-6
DOI: https://doi.org/10.1016/j.scitotenv.2019.134591
Reference: STOTEN 134591

To appear in: Science of the Total Environment

Received Date: 27 July 2019

Revised Date: 19 September 2019
Accepted Date: 20 September 2019

Please cite this article as: J. Zhou, Z. Li, T. Zhou, Z. Xin, L. Wu, Y. Luo, P. Christie, Aluminum toxicity decreases
the phytoextraction capability by cadmium/zinc hyperaccumulator Sedum plumbizincicola in acid soils, Science of
the Total Environment (2019), doi: https://doi.org/10.1016/j.scitotenv.2019.134591

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Aluminum toxicity decreases the phytoextraction capability by cadmium/zinc

hyperaccumulator Sedum plumbizincicola in acid soils

Jiawen Zhou a, b, Zhu Li a, *, Tong Zhou a, Zaijun Xin c, Longhua Wu a, *, Yongming Luo a,

Peter Christie a

a Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science,

Chinese Academy of Sciences, Nanjing 210008, China.

b University of the Chinese Academy of Sciences, Beijing 100049, China

c Institute of Watershed Ecology, Jiangxi Academy of Sciences, Nanchang 330096, China

*Corresponding author, Tel.: +86 25 86881128; fax: +86 25 86881126; E-mail:

lhwu@issas.ac.cn;

Tel.: +86 25 86881108; fax: +86 25 86881126; E-mail: lizhu@issas.ac.cn

1
ABSTRACT: Excessive aluminum (Al) in acid soils or Al released due to acidification

during repeated phytoextraction might impair the phytoextraction efficiency of

hyperaccumulators but this is often neglected. Here, we investigate for the first time the

toxicity of Al to the cadmium (Cd) and zinc (Zn) hyperaccumulator Sedum plumbizincicola

with hydroponics experiments both in the long (7 weeks) and short terms (72 hours), and in

soil conditions in a pot experiment. In the long-term hydroponics experiment, observable

toxic effects of Al were found even at < 100 μM Al at pH 5.00 (soluble Al: 8.74 μM) which

lowered shoot Cd uptake by 39.3% compared with the Al-free treatment. The scanning

ion-selective electrode technique shows that root Cd2+ influx was significantly inhibited after

treatment with 200 μM Al at pH 4.00 after 48 hours. The pot experiment confirms that Al

toxicity induced inhibition of plant growth and metal uptake in the acid soil with an

exchangeable Al of 0.33 cmolc kg−1. Decreasing Cd adsorption at root surfaces induced by Al

stress may be an important factor in declining shoot Cd uptake. Analysis of the chemical

forms of metals shows that Al addition significantly influenced the chemical forms of Cd and

Zn in stems, made them less mobile and thus restrained Cd and Zn translocation. Aluminum

toxicity that potentially occurs in acid soils and in soils during repeated phytoextraction

would be a primary factor limiting metal removal efficiency from contaminated soils using

hyperaccumulators.

Key words: Aluminum toxicity, chemical forms, ion flux, low pH, metals

2
1. Introduction

Remediation of metal-contaminated soils is urgently required in areas where the soils

are acid and thus potentially toxic elements (PTEs) can be more soluble and bioavailable

(Muhammad et al., 2012; Wu et al., 2018). Hyperaccumulator plant species which have a

superior ability to extract metals from soils and accumulate them in the aboveground parts

are sometimes used in phytoextraction schemes to clean up agricultural soils contaminated

with PTEs (Tang et al., 2016; Jacobs et al., 2017). Generally, phytoextraction with

hyperaccumulators is a practical and feasible approach for the clean-up of contaminated acid

soils due to the greater bioavailability of most trace metals in acid soils than in neutral or

calcareous soils.

However, there are a number of detrimental environmental constraints to plant growth

including proton, aluminum (Al) or manganese (Mn) toxicities and deficiencies of mineral

elements such as phosphorus (P), potassium (K), calcium (Ca) and magnesium (Mg) in acid

soils and Al toxicity may be the most deleterious limiting factor at pH < 5.0 (Singh et al.,

2017). Excessive Al in growth media can interfere with a wide range of physiological and

cellular processes of plants such as nutrient uptake, enzyme activities, DNA replication, and

cell division, resulting in the inhibition of root growth and function which finally affects

other plant parts and related processes, thus depressing crop yields (Singh et al., 2017;

Bojórquez-Quintal et al., 2017).

Few studies have investigated Al toxicity associated with metal hyperaccumulating

plants although numerous studies on Al toxicity have been conducted either in hydroponics

or in the field focusing on crop plants (e.g. rice, wheat, barley, buckwheat, maize and

soybean), forage plants (e.g. ryegrass and alfalfa) and trees (Prijanbada and

3
Proklamasiningsih, 2010; Silva et al., 2012; Brunner and Sperisen, 2013; Rehmus et al.,

2014; Reyno et al., 2015; Xu et al., 2016). Hyperaccumulators of PTEs have developed

effective mechanisms such as chelation of metals with organic ligands and moving metals

into inactive compartments such as vacuoles and cell walls to tolerate metals in the aerial

parts (Rascio and Navari-Izzo, 2011). However, it remains unknown whether these

mechanisms may help hyperaccumulators to tolerate Al in soils to some extent. Growth

inhibition of hyperaccumulators in soils with low pH (pH < 5.0) have been found in previous

studies investigating the influence of soil pH on metal uptake by hyperaccumulators (e.g. the

Cd and Zn hyperaccumulators Noccaea caerulescens and Sedum alfredii) (Wang et al., 2006;

Liao et al., 2014). However, it is still not clear whether Al toxicity or other factors in acid

soils have influenced the phytoextraction performance of hyperaccumulators in these studies.

Optimum field use of phytoextraction can only be achieved by studying the effects of Al on

the growth and metal uptake of hyperaccumulators in acid soils and determining the range of

threshold soil Al concentrations above which Al phytotoxicity will affect hyperaccumulators.

The mobility and toxicity of metals in plant tissues vary with their chemical forms.

Metals exist in plants in different chemical forms such as water-soluble ions and

metal-organic complexes. When metal ions are adsorbed on the root surfaces and transported

into root cells, they will firstly bind with cell walls, then chelate with organic ligands

including proteins, polypeptide and organic acids or precipitate with phosphate or sulphate in

the cytoplasm (Zeng et al., 2011a). Usually the fraction of soluble metals which is not

immobilized in plant cells can be transferred and redistributed among different plant parts.

Therefore, determining the chemical forms of metals in plants may help us to understand

metal translocation and tolerance mechanisms in plants (Li et al., 2013).

4
 Sedum plumbizincicola is a native Cd- and Zn-hyperaccumulator found in east China

(Wu et al., 2013b) that has shown considerable capacity to accumulate shoot Cd and Zn from

soils. It has a promising potential for the remediation of Cd- and Zn-polluted soils. Several

field experiments on phytoextraction with S. plumbizincicola have been conducted in

Zhejiang province, southeast China (Deng et al., 2016). Our previous study found an

apparent decrease in the shoot biomass of S. plumbizincicola in soils with low pH (pH < 4.0)

and this was supposed to be induced by Al phytotoxicity (Wu et al., 2018). However,

detailed experiments are required to confirm this.

Here, S. plumbizincicola was grown in hydroponics and in soils spiked with a series of

Al gradients. The aims were to study the influence of Al exposure on metal uptake in both

the short and long term, to analyze the chemical forms of metals in S. plumbizincicola to

understand the inhibition mechanism of Cd and Zn transport in plants under Al exposure,

and to assess whether or not Al toxicity occurs during phytoextraction of acid soils with S.

plumbizincicola.

2. Materials and methods

2.1. Hydroponics experiments

2.1.1. Seedling preparation

Tissue cultured seedlings of S. plumbizincicola were provided by the Institute of Botany,

Chinese Academy of Sciences. Plant shoots were cut and cultured in tap water using an 8-L

plastic pot for one week and then using 1/4 Hoagland nutrient solution for a further week to

germinate. The nutrient composition of the quarter-strength Hoagland solution was:

5
Ca(NO3)2·4H2O 0.25, MgSO4·7H2O 0.125, K2HPO4 0.05 and KCl 0.025 mM; H3BO3 2.5,

Na2MoO4·2H2O 0.05, MnSO4·4H2O 0.45, CuSO4·5H2O 0.078, NiSO4·6H2O 0.125 and

Fe-EDTA 25 μM. Solution pH was maintained at 5.80 with 0.1 M NaOH and 0.1 M HCl

solutions. Plants were grown at an average temperature regime of 25/15 °C (day/night) and

without supplementary illumination. Seedlings of uniform size were then selected for the

hydroponics experiments.

2.1.2. Plant treatment with Al in the long term

A hydroponics experiment was conducted to study the cumulative effect of long-term

Al exposure on plant growth and metal uptake in S. plumbizincicola. Four seedlings were

transferred to black plastic containers containing 0.8 L 1/4 Hoagland nutrient solution. Each

container had a polystyrene cover with five evenly spaced holes. As S. plumbizincicola has a

superior uptake rate of Cd and Zn, 50 μM Cd and 500 μM Zn (as the sulphates) were added

to each container to meet the demand. There were six treatments with different pH and Al

levels (μM), namely pH 4.75 + Al 0, pH 5.80 + Al 0, pH 5.00 + Al 0, pH 5.00 + Al 20, pH

5.00 + Al 100 and pH 5.00 + Al 500. Aluminum was added in the form of AlCl3·6H2O. The

soluble Al concentration in the nutrient solution under each treatment before experiment was

determined by inductively coupled plasma emission spectroscopy (ICP-AES, Optima 8000,

Perkin Elmer, Waltham, MA) after filtration through a 0.22-µm pore size filter (Table S1).

Each treatment had four replicates. During the experiment all pots were arranged randomly

and plants were grown in the same conditions as described above for plant preparation.

Solutions were renewed once a week and the pH was controlled daily within ± 0.10 units

with 0.1 M NaOH or 0.1 M HCl solution at 9:00 and 21:00.

All plants were harvested after Al exposure for seven weeks and divided into roots,

stems and leaves, washed three times with tap water and rinsed twice with deionized water.
6
The root samples were dried with tissue paper, immersed in 25 mL 0.01 M Na2EDTA

solution for one minute to elute the metals adsorbed on the root surfaces (Zhang et al., 2016)

and Cd and Zn concentrations in the eluent were determined by AAS (SpectrAA 220FS and

240Z, Varian, Palo Alto, CA) and Al was determined by ICP-AES. The roots were then

rinsed twice with deionized water and all root, stem and leaf samples were oven dried at 75

°C, weighed and ground for metal determination.

2.1.3. Root Cd2+ flux measurements in S. plumbizincicola in the short term

A Cd-selective microelectrode combined with the scanning ion-selective electrode

technique (BIO-001A; Younger) was used to study the effect of Al exposure on the root Cd2+

fluxes of S. plumbizincicola in the short term (72 h). Eight seedlings were transferred to a

plastic container containing 0.8 L quarter-strength Hoagland solution with 50 μM Cd added.

Plants were exposed to different levels of Al, namely 0, 200 and 400 μM. Our preliminary

experiment indicated no significant influence of Al addition (200 μM) on root Cd2+ influxes

at pH 5.00 within 72 h; a lower solution pH (pH 4.00) was used throughout the exposure

period. The Cd2+ flux measurement method was described in detail by Li et al. (2017).

Briefly, after exposure to Al for 2, 12, 24, 48 and 72 h, one of the primary roots of each plant

was chosen and placed in a Petri dish that contained 40 mL measuring solution (50 μM

CdCl2, 0.1 mM CaCl2, MgCl2 and KCl, 1.0 mM NaCl, pH 4.00) and equilibrated for 30 min.

The root was then used to record the net Cd2+ flux for 3 min at the maximum Cd2+ influx

position of the root surface. A preliminary experiment had shown that the largest Cd2+ influx

rate at the root surface of S. plumbizincicola was found at positions about 200 μm from the

root tip. At least eight successive flux measurements were made for each treatment with

eight plants.

2.2. Greenhouse pot experiment

7
 A pot experiment was carried out to study Al toxicity to S. plumbizincicola in soil

conditions. Two surface (0–20cm depth) agricultural soils were used with one (Hydragric

Anthrosol) from Shaoguan city, Guangdong province, south China and the other (Haplic

Acrisol) from Guiyang city, Guizhou province, southwest China. Selected soil

physicochemical properties were determined according to the protocols described by Lu

(2000) and are shown in Table 1. The Anthrosol and Acrisol had pH values of 5.52 and 4.86,

respectively, and exchangeable Al concentrations of 0.048 and 0.93 cmol kg−1. Aluminum

toxicity may occur in the Acrisol. To obtain soil samples with similar physicochemical

properties but different Al concentration gradients, aliquots of the two soils were therefore

spiked with AlCl3·6H2O at five Al levels, namely 0, 50, 200, 500 and 1000 mg Al kg−1 based

on the fact that maximum soil exchangeable Al concentrations are > 7.00 cmolc kg−1 (630 mg

kg−1) in south China (Wilson et al., 2004). Soil water content was adjusted to and maintained

at 70% water holding capacity (WHC) and the spiked soils were pre-incubated for one month

at room temperature. The soils were then air-dried, sieved (< 4 mm) and used for the pot

experiment. The pH (1:2.5, w/v) and available (0.01 M CaCl2 (1:10, w/v)-extractable) Cd

and Zn concentrations of the pre-incubated soils were determined prior to the experiment.

Soil exchangeable Al was extracted with 1.0 M KCl (1:10, w/v, and shaken for 1 h) and

determined by titration with 0.01 M NaOH solution (Prijambada and Proklamasiningsih,

2010). In addition, soil solution was collected from each pot before the pot experiment using

soil moisture samplers which included a 5 cm porous membrane with a pore size of 0.15 µm

(Rhizon Flex, Eijkelkamp Agrisearch Equipment, Giesbeek, Netherlands) after adjustment of

the soil moisture content to 80% WHC for 24 h. Soil solution pH was measured and

elemental (K, Ca, Mg, Fe, Mn, Al, Zn and Cd) concentrations were determined using

ICP-AES after appropriate dilution.

8
 The pot experiment with S. plumbizincicola was conducted in a greenhouse at the

Institute of Soil Science, Chinese Academy of Sciences, Nanjing from 4 September 2017 to

22 January 2018. Four plant cuttings (~ 4 cm in height) were transplanted to each plastic pot

(diameter 15 cm, height 20 cm) containing 1.5 kg soil (oven-dried basis). Soil moisture was

maintained at around 70% WHC. The plants grew at average temperatures of 20–25/ 10–15

°C (day/ night) without supplementary illumination. The pots were fully randomized and

there were four replicate pots of each treatment. Harvested plants were separated into leaves,

stems and roots. The roots were washed thoroughly with tap water until there were no soil

particles attached, immersed in a solution of 20 mM Na2EDTA for 30 min and rinsed twice

with deionized water. The separated leaves and stems were washed with tap water three

times and rinsed twice with deionized water and dried with paper tissue, then cut into 1–2

mm pieces and 2.00-g sub-samples were weighed accurately and stored at −20 °C for

sequential extraction of plant metals. The roots and the remainder of the leaf and stem

samples were oven dried at 75 °C.

2.3. Sequential extraction of plant metals

Sequential extraction of Al, Cd and Zn in plant samples was conducted using the

methods of Li et al. (2013). Different extracting solutions were used to successively extract

different chemical forms of the metals in the following sequence: (1) 80% ethanol, extracting

the inorganic metal fraction including metal nitrate, chloride, and aminophenol, F1; (2)

deionized water, extracting the water soluble fraction including metals associated with

organic acids and M(H2PO4)2, F2; (3) 1 M NaCl, extracting metals integrated with pectates

and proteins, F3; (4) 2% acetic acid (HAc), extracting insoluble MHPO4 and M3(PO4)2, F4;

(5) 0.6 M HCl, extracting metal oxalate, F5; (6) residual metal forms, F6. Metal mobility and

toxicity in plants decrease successively from F1 to F6. Fresh stem or leaf samples (0.5 g)

9
were milled thoroughly with 5 mL extractant solution in a glass mortar, shaken for 22 h at 25

°C, and centrifuged at 5000 rpm for 10 min. The supernatants were carefully transferred to

50-mL conical flasks and the residues were extracted again using the same extraction

solution (2.5 ml) for a further 2 h. After shaking and centrifuging the two supernatants were

pooled and the plant materials retained were subjected to the next extraction with the same

extraction procedures. The supernatant solutions of each extraction and the plant residues at

the end of the sequential extraction were evaporated to dryness at about 70 °C and then

digested with 2 mL HClO4 and 3 mL HNO3 for analysis of metals (Al, Cd and Zn) in each

fraction by ICP-MS (Ultramass, Varian, Palo Alto, CA).

2.4. Analysis of metals in plant and soil samples

Cadmium and Zn concentrations were determined by AAS and Al was determined by

ICP-AES after digestion of ~0.2 g oven-dried root, stem or leaf samples with 6 mL HNO3

and 2 mL H2O2 or ~0.2 g air-dried soil samples with 5 mL HNO3 and 5 mL HCl at 105 °C

for 7 h. Some of the plant samples had a total biomass < 0.2 g and all of the sample was used

for digestion. Replicates, blanks and plant (GBW10048) and soil (GBW07405) reference

materials were included for quality control. The metal recoveries were 97–107%.

2.5. Statistical analysis

Data are expressed as mean ± standard error and data from different Al treatments were

analyzed by one-way analysis of variance followed by LSD (least significant difference)

tests at P < 0.05. An exponential function was used to fit the relationship between soil

exchangeable Al concentration and soil pH while a single linear equation was used to

describe the relationship between shoot metal uptake and soil pH. Data processing was

conducted with Excel 2013 and the SPSS 21.0 for Windows software package.

10
3. Results

3.1. Hydroponics experiment treated with Al in the long term

At pH 5.00 the low concentration of Al (20 μM) significantly stimulated plant growth

with shoot biomass increasing by 31.7% but high Al addition (pH 5.00 + Al 500)

significantly inhibited plant growth with root and shoot biomass decreasing by 30.9 and

41.4%, respectively, compared to the control (Table 2). In the control treatments with no Al

at different pH levels, plant growth (distinguished by the total biomass of shoots and roots) at

pH 5.80 was greater than at pH 5.00 or 4.75, and the difference between pH 5.80 and 4.75

was significant (P < 0.05), indicating that low pH might impair plant growth.

Metal (Al, Cd and Zn) concentrations in different plant parts are presented in Fig. 1.

Within each treatment, roots had the highest Al concentrations, which were 26.5–76.8 times

as high as those in stems and 103–193 times as in leaves. At pH 5.00, Al concentrations in

different parts of S. plumbizincicola generally increased with increasing Al addition rate.

However, root, stem and leaf Cd concentrations gradually decreased with increasing Al

addition. In the three treatments with no Al addition, no significant differences were

observed among leaf Cd concentrations at pH 5.80, 5.00 and 4.75. Trends of Zn

concentrations in plants were slightly different from those of Cd. The maximum root Zn

concentration at pH 5.00 appeared to occur at 500 μM Al but was not significantly different

from treatments pH 5.00 + Al 0 and pH 5.00 + Al 20. In addition, Zn concentrations in stems

reached their maximum values at 20 μM Al addition. Leaf Zn concentrations showed no

significant differences across all treatments at pH 5.00. No significant differences were

observed in leaf Zn concentrations under the three Al-free treatments.

11
 Aluminum addition significantly suppressed Cd and Zn accumulation by S.

plumbizincicola (Table 3). Compared with the control at pH 5.00, 20 μM Al addition

increased shoot Cd and Zn uptake by 21.5 and 67.5%, respectively. However, under the 100

and 500 μM Al treatments, shoot Cd uptake decreased by 39.3 and 76.1%, while shoot Zn

uptake decreased by 6.9 and 51.0%. In the treatments with no Al, root and shoot Cd uptake

and shoot Zn uptake at pH 5.80 were significantly higher than at pH 5.00 or 4.75. It should

be noted that under high Al exposure, more Zn in the aboveground parts tended to be stored

in the leaves. For example, at pH 5.00 the percentage of Zn in leaves relative to total Zn in

shoots was 50.7% when there was no Al addition. However, it increased markedly to 61.8%

and 73.7% under 100 and 500 μM Al addition.

As shown in Table 4, at pH 5.00, Al adsorption on the root surfaces gradually increased

with increasing Al addition rate. Aluminum addition significantly decreased Cd adsorption at

the root surfaces but had little influence on Zn adsorption. There was no significant

difference in the Cd or Zn adsorption on the root surfaces among the three control treatments

at different pH levels.

3.2. Net fluxes of Cd2+ under Al exposure in the short term

As shown in Fig. 2, under the control treatment, Cd2+ influx gradually increased during

the initial 24 h, then reached a stable state (about −12.6 pmol cm-2 s-1). However, in the 200

and 400 μM Al treatments, Cd2+ influxes generally increased from −7 to −12 pmol cm-2 s-1

within 24 h but dramatically decreased by 83.3 and 90.5%, respectively, in the subsequent 48

h. Compared with the control treatment, Cd2+ uptake rates under the two Al treatments

showed no significant decrease in the first 24 h but significant inhibition after 48 and 72 h.

3.3. Pot experiment treatment with Al

12
 Soil pH and soil solution pH decreased gradually with increasing Al addition level, and

soil exchangeable Al and soluble Al concentrations increased linearly (Table 5 and Fig. S1).

Compared with the control, soil pH under the highest Al treatment (1000 mg kg−1) decreased

by 2.14 and 1.21 units in the Anthrosol and Acrisol, respectively. In addition, a significant

inverse exponential relationship was found between soil exchangeable Al and soil pH: y =

8818033 × e−3.82x in the Anthrosol with determination coefficient R2 = 0.97, and y = 2598 ×

e−1.69x in the Acrisol with R2 = 0.98 (y is soil exchangeable Al, cmolc kg−1 and x is soil pH).

Furthermore, Al treatment significantly increased soil Cd and Zn availability and soil

solution nutrient ion (K+, Ca2+, Mg2+, Fe2+ and Mn2+) concentrations (Fig. S2).

S. plumbizincicola grown in soils was inhibited to a larger extent under Al stress than in

hydroponics. In both soils, except in the 50 mg Al kg−1 treatment, the hyperaccumulator died

at the highest Al addition rate (1000 mg kg−1) and shoot biomass in the 200 and 500 mg Al

kg−1 treatments decreased by 32.1 and 85.7% in the Anthrosol and by 57.7 and 80.4% in the

Acrisol compared with the control (Fig. 3). At the same Al addition level, S. plumbizincicola

grew more in the Anthrosol than in the Acrisol, and the Anthrosol had relatively lower

exchangeable Al concentration than the Acrisol.

Root Al concentrations were much higher than shoot concentrations, ranging from 2771

to 6001 mg kg−1 (Table S2). Compared with the control, Al treatments (except 50 mg Al kg−1)

significantly increased shoot Al concentrations in both soils. At the same Al addition rate,

shoot Al concentrations in the Acrisol in most cases were significantly higher than those in

the Anthrosol. Addition of 50 mg Al kg−1 to the Anthrosol had no significant influence on

shoot Cd or Zn concentrations or uptake compared with the controls, but other Al addition

treatments (200–1000 mg kg−1) significantly decreased shoot Cd and Zn concentrations and

uptake (Fig. 4). In the Anthrosol, shoot metal uptake decreased, Cd by 48.2, 93.2 and 99.2%

13
and Zn by 46.2, 91.8 and 98.7%, respectively, at 200, 500 and 1000 mg Al kg−1. In the

Acrisol, Al additions significantly depressed shoot Cd (by 91.8 and 95.6%) and Zn (by 68.4

and 89.7%) accumulation in the 500 and 1000 mg Al kg−1 treatments compared to the

controls.

3.4. Chemical forms of metals in plants as affected by Al

In both soils, Al in the stems was present mainly in the residual (F6) and

HCl-extractable (F5) forms, followed by the fraction extracted by 2% HAc (F4) (Fig. 5a).

These three fractions with low mobility and toxicity in plant tissues accounted for 70–80% of

total stem Al. There were no significant differences among Al concentrations extracted by

80% ethanol, deionized water and 1 M NaCl (F1, F2 and F3) in the stems across all

treatments (Fig. S3). In plant stems, percentages of residual Al increased significantly from

30% (Ant-0) to 45% (Ant-200) and 49% (Ant-500), while F5 fraction increased from 19%

(Acr-0) to 26% (Acr-200) and 29% (Acr-500) (Fig. 5a).

The distributions of chemical forms of Cd and Zn in the plant shoots differed from

those of Al (Fig. 5). The F3 fraction was the predominant form of Cd in stems and leaves,

followed by F1 and F2 (Figs. 5 c and d). These three fractions comprised 93% of total Cd in

stems and 85% of total Cd in leaves. Aluminum addition significantly increased the total

proportions of less mobile Cd (F4, F5 and F6) in the stems from 6.0% (Acr-0) to 8.7%

(Acr-200) and 11.8% (Acr-500). In common with Cd, shoot Zn consisted mainly of the first

three forms, representing 51–91% of the total Zn in the stems and leaves under all treatments

in both soils (Figs. 5e and f). With increasing Al the total percentages of Zn in fractions F4,

F5 and F6 in the stems increased significantly from 16% (Acr-0) to 22% (Acr-200) and 28%

(Acr-500) (Fig. 5e). At the same Al addition rate, total percentages of the F4, F5 and F6

fractions in both stems and leaves in the Acrisol were greater than in the Anthrosol.
14
4. Discussion

4.1. Plant growth under Al stress

Aluminum is an element that is regarded as toxic to the vast majority of plant species.

A concentration of Al in solution exceeding the plant tolerance limit interferes with cell

division at the root apex and lateral roots, destroys the selective permeability of the plasma

membrane, increases the rigidity of cell walls, limits water and nutrient acquisition and thus

displays toxic effects eventually (Bojórquez-Quintal et al. 2017). For example, exposure to

30 μM AlCl3 at pH 4.5 for 12 h in hydroponics reduced the elongation of soybean (Glycine

max L.) roots by 53% (Lan et al., 2016) and 50 μM Al (at pH 4.5) for 24 h can inhibit root

elongation of rice by 25–75% (Zhao et al., 2013). In an Al-treated soil with an exchangeable

Al of 0.25 cmolc kg−1, grain yield and aboveground biomass of wheat decreased significantly

by 15–20% and 15–30% compared with the untreated soil (Valle et al., 2009). Here, toxic

effects of Al were observed when S. plumbizincicola was exposed to 100 μM Al at pH 5.00

(soluble Al: 8.74 μM) in the long-term experiment (7 weeks) and to 200 μM Al at pH 4.00

after 48 h in the short-term experiment. In soil conditions, Al toxicity occurred to S.

plumbizincicola grown in soils with exchangeable Al contents of 0.33–1.43 cmolc kg−1 (200

mg Al kg−1 treatment). These results indicate that S. plumbizincicola is not an Al-tolerant

species in common with most other plant species (Valle et al., 2009; Zhao et al., 2013; Lan et

al., 2016). However, the capacity of Al tolerance varies with plant species and even

genotypes within species. Most common cereal cultivars (e.g. rice) are susceptible to around

2 mg L−1 Al (˂ 100 μM) in solution at pH ˂ 5 (Coronel et al. 1990) but tolerant rice varieties

can grow normally even in the presence of 160 µM of Al3+ activity (Famoso et al. 2011). In

Al tolerant or accumulating plant species such as Camellia oleifera Abel., the Al

15
concentration in the leaves can reach 10000 mg kg−1 (Zeng et al., 2011b) but the leaf Al

concentrations of S. plumbizincicola were < 100 mg kg−1 in hydroponics when toxic effects

were observed (Fig. 1a). This is consistent with reports that in “normal” plants without

toxicity symptoms the maximum leaf Al concentration is usually < 200 mg kg−1 (Chen et al.,

2008; Silva et al., 2012). Judging by the poor plant growth and the high leaf Al

concentrations (360 mg kg−1 on average) of S. plumbizincicola grown in the Acrisol in the

control, Al toxicity occurred in this soil without Al spiking.

Metal hyperaccumulators have a strong ability to tolerate and detoxify internal metals in

the aboveground parts by mechanisms such as chelation with organic ligands (e.g. malate

and citrate) (Wojcik et al., 2006; Yang et al., 2006). However, these mechanisms did not

appear to help S. plumbizincicola tolerate and accumulate more Al in the leaves. Perhaps for

different metals, plants have different detoxifying mechanisms and S. plumbizincicola may

not have specific strategies to tolerate Al quickly. The majority of Al taken up by S.

plumbizincicola (81–89% in hydroponics) was retained in the roots, to avoid the injury of

above ground part and caused severe toxic effects to S. plumbizincicola at high Al treatments

in the hydroponics experiment of our study. Roots make direct contact with Al in the growth

medium and are usually the most sensitive and affected parts in which the root apex is the

primary target of Al toxicity (Barcelo and Poschenrieder, 2002). Damaged brownish roots

and small black spots at the root tips in hydroponics after one week of exposure to excessive

Al in our study indicate the susceptibility of the roots of S. plumbizincicola to Al toxicity.

However, in contrast to previous studies in which rice roots were more affected than the

shoots by Al (Bhoomika et al., 2013; de Freitas et al., 2016), larger biomass reductions in

shoots (26.6% on average) than roots (13.4% on average) under Al stress in hydroponics

were found in the present study. This was associated with the much longer Al exposure times

in our study (7 weeks). At the early stages of Al stress the roots were firstly affected. As time
16
proceeded, feedback between root and shoot signals triggered by environmental stresses

would eventually restrict the growth of the aboveground parts.

In acid soils, low pH (proton toxicity) and Al toxicity often coexist to affect plant

growth. According to our previous study (Wu et al., 2018), the appropriate pH for the growth

of S. plumbizincicola was around 5.50, and lower pH may constrain plant yields. In the

present study, lowering the pH from 5.80 to 5.00 and 4.75 resulted in 16.6% and 8.5%

decreases in shoot biomass in hydroponics, indicating the negative influence of low pH on

the growth of S. plumbizincicola. Compared with the hydroponics experiment, a much larger

decline (by 32.1%) in shoot biomass was observed in the Anthrosol when the soil pH

decreased from 5.69 to 4.73, indicating that in addition to low pH, excessive Al was an

important factor affecting shoot biomass decrease in soil conditions, especially in soils

treated with 500 and 1000 mg Al kg−1 (soluble Al > 2000 μM, Table 5). However, Al spiking

increased the availability of nutrient ions such as K+, Ca2+, Mg2+ and Fe2+ (Fig. S2) and this

would have been conducive to the accumulation of plant biomass. Furthermore, Al at low

concentrations stimulated plant growth in our study. Similar results have also been reported

in other species such as rice, maize, tea and sugar maple (Schier and McQuattie, 2002;

Famoso et al. 2011; Mukhopadyay et al. 2012; Wang et al. 2015). Low levels of Al can

promote nutrient uptake, prevent biotic and abiotic stress (e.g. proton or other metal

toxicities), increase enzyme activities and stimulate the defense reactions thus leading to a

general activation of metabolism (Barcelo and Poschenrieder, 2002; Bojórquez-Quintal et al.

2017).

4.2. Translocation and uptake of Cd and Zn in relation to Al

Aluminum exposure significantly affects ion uptake in a number of plant species

through either external (outside plants) or internal (within plants) pathways (Mariano and
17
Keltjens, 2005; Olivaresa et al. 2009; Valle et al., 2011). Firstly, Al interferes with the

processes by which metals enter roots from the external environment. The trivalent ion of Al

has stronger affinity with the negative charges on root surfaces than divalent metal ions such

as Cd2+ and Zn2+. Competition of Al with other metals for the binding sites at the root

surface will greatly affect metal adsorption. Metal uptake by plants largely depends on metal

adsorption at the root surfaces (Zhang et al., 2016). A strong correlation between shoot Cd

uptake and Cd adsorption at the root surfaces (R2 = 0.84) was also found in our study.

Decreasing Cd accumulation in shoots was related to a decrease in root Cd adsorption with

Al addition in solution (Table 4). However, Zn adsorption at the root surface was not

significantly influenced by the presence of Al. This may have been due to the higher molar

concentration of Zn than Al in the experimental solutions. Zinc concentrations in the soil

solution are often higher than or comparable to that of Al in the field, and two orders of

magnitude higher than that of Cd. Therefore, compared with Cd, root Zn adsorption by S.

plumbizincicola would be much less affected by Al. The significant decrease in shoot Zn

uptake at high Al exposure may be more associated with the decline in plant biomass and

Al-induced inhibition of metal transport across membranes and in the xylem. Secondly, Al

rapidly disrupts the membrane ion-transport systems in the root apical region during the

initial stages of A1 exposure. Inhibition of Ca2+ uptake occurs at the root apex of

Al-sensitive wheat cultivars after 30 min with 20 μM Al at pH 4.50 (Huang et al., 1992) and

changes in K+ flux from efflux to influx were found in two Al sensitive mutants of

Arabidopsis after about 20 min under the 50 μM Al treatment at pH 4.20 (Bose et al., 2010).

In line with this, net Cd2+ influx of S. plumbizincicola was significantly inhibited by 200 μM

Al at pH 4.00 after 48 h in the present study (Fig. 2). However, in the initial 24 h, Al

exposure had little effect on Cd uptake rate. Net Cd2+ influx under Al treatment was even

slightly higher than the control, indicating Al tolerance of S. plumbizincicola in the initial

18
stages (within 24 h) to some extent. A previous study suggests that Al tolerance was

associated with root H+ flux. Bose et al. (2010) found that the Al-resistant mutant of

Arabidopsis had higher H+ influx than the sensitive mutants. In agreement with this, we

found that within 24 h the root H+ influx under 200 μM Al was significantly higher than in

the control (data not shown) which induced a more alkaline rhizosphere to decrease Al

activity. However, after 48 h the root H+ influx under 200 μM Al showed no difference from

that in the control.

Aluminum also interferes with metal translocation from roots to shoots. After Al enters

the plant cells, Al chelation by organic ligands or precipitation with phosphate anions or

organic phosphates such as phytate in the cytosol and sequestration into vacuoles are

important detoxifying mechanisms in plants (Gupta et al., 2013). Aluminum forms such as

Al-citrate, Al-malate or Al-oxalate complexes have been identified in roots and leaves and

also in the transport processes from roots to shoots (Brunner and Sperisen, 2013; Gupta et al.,

2013). Moreover, similar localization of Al and P in roots was reported by Iqbal (2014) due

to the formation of Al phosphate. In the present study the Al in stems and leaves of S.

plumbizincicola occurred predominantly as Al phosphate, Al-oxalate and residual forms,

indicating the importance of organic acids and phosphates in detoxifying Al. Moreover, Al

exposure leads to significant increases in the concentrations of P in roots and organic acids

such as citrate and malate in roots and leaves compared to the control (de Carvalho

Gonçalves et al., 2005; Iqbal, 2014). The increased levels of P and organic acids in plant

tissues would both immobilize Al and also react with other metals such as Cd and Zn. In

agreement with the results of Li et al. (2013), Cd and Zn occurred in the stems and leaves of

S. plumbizincicola mainly in forms with high solubility and mobility (fractions extracted by

80% ethanol, water and 1 M NaCl), helping S. plumbizincicola to easily transport and

accumulate Cd and Zn. However, with increasing soil Al concentrations larger proportions of
19
Cd and Zn in the stems became insoluble because of the formation of metal-phosphate and

metal-oxalate complexes. Cadmium and Zn are transported predominantly in the form of free

ions among different tissues (Caldelas and Weiss, 2017; Tao et al., 2017) and this will

severely restrain their translocation among different tissues, down-regulate their uptake by

roots and ultimately lower their uptake by plants. Moreover, Al-P precipitates in the apoplast

may decrease Cd and Zn movement and transport via the apoplastic pathway which is very

important in Cd hyperaccumulation by S. alfredii, a species in the same family

(Crassulaceae) as S. plumbizincicola (Tao et al., 2017).

It is notable that Al stress also influenced Zn distribution in S. plumbizincicola as this

hyperaccumulating species tended to allocate more Zn to the leaves under Al toxicity (Table

3). Perhaps Zn played an important role in alleviating Al toxicity and protecting the

metabolically active organs from Al damage. Zinc is a critical component of the antioxidant

enzyme Cu/Zn-SOD which acts as a scavenger of reactive oxygen species formed during Al

stress (Pontigo et al., 2017). Our further experiment based on the scanning ion-selective

electrode technique shows that under 200 μM Al exposure, increasing Zn addition in the

nutrient solution significantly promoted the root Cd2+ uptake rate of S. plumbizincicola in the

short term (48 h) (Fig. S4). This indicates that Zn can relieve the inhibitory influence of Al

on Cd uptake of S. plumbizincicola to some extent. However, this requires further study to

elucidate the detail mechanisms underlying this phenomenon.

4.3. Possible occurrence of Al toxicity during phytoextraction in the field

As discussed above, Al toxicity occurred in the Acrisol (pH 4.86) without Al addition.

The occurrence of Al toxicity in soils with pH < 5.0 is highly likely in field conditions. It is

known that exchangeable Al increases exponentially with decreasing soil pH (Kariuki et al.,

2007), an effect that was also found in our study, suggesting that a slight decrease in soil pH
20
would lead to a large increase in exchangeable Al in soils with low pH. In our repeated

phytoextraction experiment (data not published), the pH of the Anthrosol decreased from

5.52 to 4.31 and exchangeable Al increased about 20 times (from 0.048 to 0.95 cmolc kg−1)

after four consecutive phytoextraction crops. Consequently, shoot Cd and Zn uptake

decreased by 90 and 79 %, respectively, from the first to the fifth crop. Based on the strong

linear relationship between shoot metal uptake and soil pH (Table S3), it may be estimated

that Al toxicity reduced shoot Cd and Zn uptake by 66.5 and 65.6% in the fifth crop relative

to the first crop. It appears that rather than a decrease in soil available metals during repeated

phytoextraction, Al toxicity induced by soil acidification was the main factor responsible for

the decline in metal uptake in the Anthrosol.

Estimating the possibility of occurrence of Al toxicity during phytoextraction is very

important in guiding soil metal removal in field conditions. Metal-contaminated soils show

very similar spatial distributions to acid soils in the tropical and subtropical regions of south

and southeast China. The red soils which are typical acid soils in south and southeast China

range in pH from 4 to 6. The mean exchangeable Al of red soils in Zhejiang province (east

China) was 4.69 cmolc kg−1 (Wilson et al., 2004). Soils collected from Yingtan city, Jiangxi

province, which are typical acid soils of south China, had an average exchangeable Al of

3.71 cmolc kg−1 (Chen et al., 2008). The levels of exchangeable Al in red soils in south China

were even higher than those in the soils spiked with 500 mg Al kg−1 in our study. In general,

phytotoxic effects are observed in soils with exchangeable Al > 2 cmolc kg−1 (Valle et al.,

2009; Wu et al., 2013a). Clearly, significant Al-induced inhibition of plant metal uptake will

occur if phytoextraction with S. plumbizincicola is used to remediate these strongly acid soils

which also have high Al saturation. In addition, due to intensive industrial activities and high

fertilizer application rates, many soils in south China have been further acidified with pH

declines of 0.23–0.30 units since the 1980s (Guo et al., 2010). This evidently aggravates the
21
severity of Al toxicity. As reported by Jiang et al. (2010), soil pH decreased by 0.24–0.45

units after six successive crops of phytoextraction with S. plumbizincicola. Li et al. (2011)

found that the rhizosphere pH was 0.60–0.80 units lower than the non-rhizosphere zone pH

of S. alfredii. Aluminum toxicity may therefore occur in soils with no initial Al threat after

repeated phytoextraction. In addition, chemical elution is often used to clean soils with toxic

metal contamination. According to Guo et al. (2016), elution using chelators combined with

FeCl3 significantly decreased the topsoil pH from 4.32 to 3.85 and significantly decreased

the yield and Cd and Zn uptake of S. alfredii. Aluminum toxicity may also occur during

chemical elution of contaminated soils.

It is therefore necessary to avoid the negative impacts of Al toxicity and control the soil

environment in phytoextraction practices using hyperaccumulators in acid soils especially

the red soils of south China with high exchangeable Al and Al saturation. Lime is an

effective amendment in ameliorating Al toxicity in acid soils. Lime application to soils

unintentionally decreases metal availability thus decreasing metal uptake by

hyperaccumulators. Soil amendments such as biochars or organic manures which

preferentially adsorb or chelate with Al over other metals are suggested for use in acid soils

to increase phytoextraction efficiency.

5. Conclusions

Aluminum toxicity significantly inhibited the biomass and metal uptake of S.

plumbizincicola. Significant toxic effects of Al were observed in solutions with 100 μM Al at

pH 5.00 in the long-term experiment and in soils with an exchangeable Al of 0.33 cmolc kg−1.

Aluminum exposure significantly depressed net root Cd2+ influx in the short term (within 48

22
h), decreased Cd adsorption at the root surfaces, influencing the chemical forms of Cd and

Zn in the stems and making the metals more difficult to transport among plant tissues, and

eventually reducing shoot metal accumulation. Considering the high likelihood of Al toxicity

occurring in the strongly acid soils or in soils experiencing significant pH decrease during

repeated phytoextraction, it is necessary to ameliorate Al toxicity during phytoextraction

with hyperaccumulators to maintain efficient removal of metals.

Acknowledgements

This work was funded by the Chinese National Key Research and Development

Program (Project 2018YFC1802600).

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Figure captions

Fig. 1 (a) Al, (b) Cd and (c) Zn concentrations in different plant parts in the hydroponics

experiment under different treatments in the long term. The error bars are SE, n = 4.

Different lowercase letters indicate significance at P < 0.05 under different Al

treatments at pH 5.00 in the same plant part. Different subscripted capital letters indicate

significance at P < 0.05 among the control treatments at different pH levels in the same

plant part.

Fig. 2 Net fluxes of Cd2+ at the root surface S. plumbizincicola under Al exposure in the short

term. The error bars are SE, n = 8. Different letters indicate significant differences under

different Al treatments at the same exposure time at P < 0.05.

Fig. 3 (a) Root and (b) shoot biomass of S. plumbizincicola under different Al treatments in

the two soils. Ant: Hydragric Anthrosol; Acr: Haplic Acrisol. In the 1000 mg kg−1 Al

treatment, no root samples were obtained in either soil. The error bars are SE, n = 4.

Different letters indicate significant difference under different Al treatments in the same

soil at P < 0.05.

Fig. 4 Shoot (a) Cd and (b) Zn concentrations, shoot (c) Cd and (d) Zn uptake of S.

plumbizincicola under different Al treatments in the two soils. Ant: Hydragric Anthrosol;

Acr: Haplic Acrisol. The error bars are SE, n = 4. Different letters indicate significant

differences in the same soil treated with Al at P < 0.05.

Fig. 5 Percentages of different chemical forms of (a) Al in stems, (b) Al in leaves, (c) Cd in

stems, (d) Cd in leaves, (e) Zn in stems, and (f) Zn in leaves under different Al

treatments in the two soils. Ant: Hydragric Anthrosol; Acr: Haplic Acrisol. F1, F2, F3,

F4, F5 and F6 represent chemical forms extracted by 80% ethanol, deionized water, 1 M

NaCl, 2% HAc, 0.6 M HCl, and residual forms, respectively.

30
Table 1 Selected physicochemical properties of the two soils used in the greenhouse pot experiment

CEC SOC Clay Total Cd Total Zn Total Al Exchangeable Al

Soil pH
cmolc kg−1 g kg−1 % mg kg−1 mg kg−1 g kg−1 cmolc kg−1
Anthrosol 5.52 11.2 17.5 22.8 4.08 1160 41.6 0.048
Acrisol 4.86 13.0 31.6 25.7 1.88 96.8 36.2 0.93

31
Table 2 Biomass of different parts of S. plumbizincicola in the hydroponics system in the long term (g pot−1 DW)
Treatment Roots Stem Leaves Shoot
pH 4.75 + Al 0 0.0605 ± 0.0043 B 0.2827 ± 0.0165 A 0.4783 ± 0.0227 AB 0.7610 ± 0.0360 AB
pH 5.80 + Al 0 0.0775 ± 0.0062 A 0.3161 ± 0.0098 A 0.5151 ± 0.0187 A 0.8313 ± 0.0273 A
pH 5.00
+ Al 0 0.0612 ± 0.0023 aB 0.2694 ± 0.0180 bA 0.4236 ± 0.0202 bB 0.6930 ± 0.0370 bB
+ Al 20 0.0626 ± 0.0040 a 0.3517 ± 0.0193 a 0.5612 ± 0.0209 a 0.9129 ± 0.0401 a
+ Al 100 0.0562 ± 0.0053 ab 0.2298 ± 0.0098 b 0.3941 ± 0.0455 b 0.6239 ± 0.0511 b
+ Al 500 0.0423 ± 0.0059 b 0.1455 ± 0.0123 c 0.2603 ± 0.0205 c 0.4058 ± 0.0322 c

Data are expressed as mean ± SE, n = 4. Shoot biomass is the sum of stem and leaf biomass. Different lowercase letters indicate significance at P
< 0.05 under different Al treatments at pH 5.00. Different subscripted capital letters indicate significance at P < 0.05 among the control
treatments at different pH.

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 Table 3 Metal uptake in different parts of S. plumbizincicola in the hydroponics system in the long term (mg pot−1)
Cd Zn
Treatment
Roots Stem Leaves Shoot Roots Stem Leaves Shoot
pH 4.75
+ Al 0 0.09 ± 0.01 B 0.91 ± 0.02 A 1.68 ± 0.08 B 2.59 ± 0.09 B 0.48 ± 0.05 A 5.99 ± 0.39 A 4.58 ± 0.23 B 10.6 ± 0.5 A
pH 5.80
+ Al 0 0.18 ± 0.03 A 0.99 ± 0.04 A 2.43 ± 0.18 A 3.42 ± 0.18 A 0.66 ± 0.08 A 4.17 ± 0.70 AB 5.83 ± 0.43 A 10.0 ± 0.3 A
pH 5.00
+ Al 0 0.10 ± 0.01 aB 0.96 ± 0.07 aA 1.64 ± 0.10 bB 2.61 ± 0.05 bB 0.61 ± 0.04 aA 3.97 ± 0.60 bB 4.09 ± 0.23 bB 8.06 ± 0.44 bB
+ Al 20 0.09 ± 0.00 a 1.13 ± 0.08 a 2.04 ± 0.07 a 3.17 ± 0.15 a 0.57 ± 0.01 ab 7.92 ± 0.48 a 5.61 ± 0.25 a 13.5 ± 0.7 a
+ Al 100 0.07 ± 0.01 ab 0.57 ± 0.04 b 1.01 ± 0.11 c 1.58 ± 0.15 c 0.40 ± 0.05 b 2.86 ± 0.49 b 4.64 ± 0.49 ab 7.50 ± 0.91 b
+ Al 500 0.04 ± 0.01 c 0.23 ± 0.04 c 0.39 ± 0.03 d 0.62 ± 0.08 d 0.44 ± 0.10 ab 1.04 ± 0.19 c 2.91 ± 0.29 c 3.95 ± 0.48 c
Data are expressed as mean ± SE, n = 4. Shoot metal uptake is the total metal uptake of stem and leaves. Different lowercase letters indicate
significance at P < 0.05 under different Al treatments at the same pH. Different subscripted capital letters indicate significance at P < 0.05
among the control treatments at different pH.

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Table 4 Metal ions adsorbed to the root surface in the hydroponics experiment in the long term (cmol kg−1)
Treatment Cd Zn Al
pH 4.75 + Al 0 1.12 ± 0.04 A 12.9 ± 0.4 A -
pH 5.80 + Al 0 1.18 ± 0.27 A 10.6 ± 2.0 A -
pH 5.00
+ Al 0 1.12 ± 0.09 aA 11.4 ± 0.5 aA 0.23 ± 0.01 d
+ Al 20 0.90 ± 0.08 a 10.1 ± 1.0 a 0.75 ± 0.03 c
+ Al 100 0.57 ± 0.01 b 12.6 ± 0.3 a 1.50 ± 0.06 b
+ Al 500 0.62 ± 0.03 b 10.6 ± 0.5 b 1.35 ± 0.04 a

Data are expressed as mean ± SE, n = 4. Different letters indicate significance at P < 0.05 under different Al treatments at pH 5.00. Different
subscripted capital letters indicate significance at P < 0.05 among the control treatments at different pH.

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 Table 5 Soil properties under different Al treatments at the start of the pot experiment

Property Ant-0 Ant-50 Ant-200 Ant-500 Ant-1000 Acr-0 Acr-50 Acr-200 Acr-500 Acr-1000
pH 5.69 ± 0.01 a 5.26 ± 0.00 b 4.73 ± 0.00 c 4.08 ± 0.02 d 3.55 ± 0.01 e 4.85 ± 0.02 a 4.75 ± 0.01 b 4.36 ± 0.02 c 3.91 ± 0.01 d 3.64 ± 0.02 e
Available Cd
0.47 ± 0.01 e 0.69 ± 0.02 d 1.74 ± 0.01 c 3.16 ± 0.01 b 3.97 ± 0.01 a 0.27 ± 0.02 e 0.33 ± 0.01 d 0.55 ± 0.00 c 0.75 ± 0.02 b 0.86 ± 0.03 a
(mg kg−1)
Available Zn
21.6 ± 0.1 e 43.1 ± 1.3 d 88.3 ± 0.2 c 165 ± 0 b 235 ± 0 a 0.66 ± 0.02 e 0.82 ± 0.00 d 1.40 ± 0.02 c 2.15 ± 0.01 b 3.33 ± 0.01 a
(mg kg−1)
Exchangeable Al 0.0019 ± 0.016 ±
0.33 ± 0.01 c 1.73 ± 0.08 b 6.60 ± 0.06 a 0.74 ± 0.05 d 0.89 ± 0.09 d 1.43 ± 0.10 c 2.97 ± 0.01 b 6.52 ± 0.19 a
(cmolc kg−1) 0.0032 e 0.003 d
Soil solution pH 6.01 ± 0.03 a 5.70 ± 0.08 b 4.54 ± 0.04 c 3.76 ± 0.04 d 3.25 ± 0.04 e 4.94 ± 0.06 a 4.79 ± 0.03 b 4.22 ± 0.01 c 3.48 ± 0.07 d 3.36 ± 0.01 d
Soil solution [Cd]
0.02 ± 0.00 d 0.11 ± 0.02 d 2.15 ± 0.18 c 6.23 ± 1.06 b 7.61 ± 1.11 a 0.05 ± 0.01 d 0.13 ± 0.02 d 0.45 ± 0.07 c 1.44 ± 0.11 b 1.29 ± 0.27 a
(mg L−1)
Soil solution [Zn]
1.87 ± 0.17 e 7.05 ± 1.06 d 78.2 ± 4.5 c 247 ± 33 b 393 ± 45 a 0.42 ± 0.06 c 0.37 ± 0.04 c 1.69 ± 0.73 b 5.01 ± 0.35 a 5.59 ± 0.50 a
(mg L−1)
Soil solution [Al]
ND ND 2.52 ± 0.27 a 58.1 ± 10.7 b 484 ± 28 c 0.43 ± 0.15 d 0.74 ± 0.07 d 7.35 ± 1.18 c 174 ± 14 b 515 ± 44 a
(mg L−1)

Ant, Hydragric Anthrosol; Acr, Haplic Acrisol.

Data are expressed as mean ± SE, n = 4. For soil samples, n represents 4 replicate analyses of one soil sample. For solution samples, n represents
4 replicate samples from different pots.
Letters for soil properties under different Al treatments in the same soil indicate significant difference at P < 0.05. ND, not detected by ICP-AES
(detection limit, 0.005 mg Al L−1).

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 Fig. 1

36
 b
b b
b

a
a

Fig. 2

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Fig. 3

38
Fig. 4

39
Fig. 5

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